Ligand-drug-conjugates containing single molecular weight polysarcosine

Abstract

To provide a single molecular weight homopolymer, using polysarcosine (poly-N-methylglycine or PSAR) which can be an alternative to PEG and can be used to design novel protein conjugates with improved properties, a method for preparing such a homopolymer, and a ligand-drug-conjugate comprising a single molecular weight homopolymer.SOLUTION: The present invention provides a ligand-drug-conjugate (LDC) with improved drug loadability, pharmacokinetics and therapeutic efficacy, using discrete monodisperse PSAR homopolymers with a defined chain length, obtained using a step-wise on-resin sub-monomer approach.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] The present invention relates to single molecular weight homopolymers, to a process for preparing such homopolymers, and to their use, particularly in composite technology.

[0002] The present invention also relates to Ligand-Drug-Conjugates (LDCs) that comprise single molecular weight homopolymers, in particular single molecular weight polysarcosine.

[0003] A Ligand-Drug-Conjugate (LDC) is a polypeptide or protein that contains at least one Ligand unit, which is covalently linked to at least one therapeutic, diagnostic or labeling molecule (hereinafter referred to as Drug or D) via a synthetic linker. This synthetic linker may contain one or several bivalent arms for linking the Ligand unit and the Drug unit, which may be selected from spacers, connectors and cleavable moieties. The linker may also have any monovalent moiety that can improve the LDC performance, such as storage stability, plasma stability or pharmacokinetic properties. Proteins or polypeptides are usually targeting units, but may have intrinsic therapeutic properties. When the Ligand unit of the conjugate is an antibody or an antibody fragment and is associated with a cytotoxic or chemotherapeutic drug, the term "antibody-drug-conjugate (ADC)" is commonly used.

[0004] The design of ADCs (antibody-drug-conjugates) involves the consideration of a number of diverse factors: (i) the nature, number, overall hydrophobicity and location of the synthetic linkers used for conjugation on the ligand; (ii) the nature and mechanism of action of the drug; (iii) the structural elements involved in drug release after cellular internalization and during intracellular trafficking; (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 variants with different pharmacological properties), limited mAb-linker or drug-linker stability, and suboptimal pharmacokinetic properties (Beck et al. Nat. Rev. Drug. Disco., 2017, 16(5), 315-337).

[0005] Another important factor to consider when designing conjugates is the drug ratio (or drug-antibody-ratio (DAR) for ADCs), which is the average number of drug units attached to the antibody. As a result of recent studies, the actual trend in the ADC field is to generate and bring to the clinic homogenous conjugates with low to moderate DAR (usually 2-4). Nevertheless, more recently, new linker-drug technologies have emerged that aim to overcome the shortcomings of highly loaded ADCs (unfavorable pharmacokinetic properties and tendency to form aggregates, thus complicating conjugate formulations). Such technologies have the potential to bring to the clinic next-generation ADCs with improved efficacy, improved pharmacokinetic properties, improved therapeutic index, and can target tumors with low target expression, slow internalization or inefficient intracellular processing. To achieve such high payload loadings without sacrificing pharmacokinetic properties and formulation stability, it is necessary to develop new linker-drug design approaches that aim to mask the apparent hydrophobicity of the cytotoxic payload.

[0006] In WO2014 / 093394A1, a protein-polymer-drug conjugate exhibiting high drug loading and strong binding to the target antigen is reported. The conjugate comprises a biodegradable and biocompatible poly-[1-hydroxymethylethylenehydroxylmethylformal] polymeric entity, which allows the conjugation of about 12-25 cytotoxic molecules per mAb with good pharmacokinetic properties. The main drawback of this approach is the extreme polydispersity of the final conjugate, resulting from (i) the polydispersity of the linker, (ii) the heterogeneous number of cytotoxic molecules per polymeric arm, and (iii) the heterogeneous number of grafted polymeric arms per mAb.

[0007] WO2015 / 057699A2 and WO2016 / 059377A1 report the formulation of 8-36 drug-loaded ADCs by including orthogonal polyethylene glycol (PEG) moieties in the linker design. PEG is well known to 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 is subject to drawbacks such as non-biodegradability, possible complement activation leading to hypersensitivity and unclear pharmacokinetics due to anti-PEG antibodies expressed by some healthy individuals.

[0008] There is a need for ligand-drug-conjugates that combine: (i) high drug loading while maintaining favorable pharmacokinetic properties and stability; (ii) complete homogeneity of the conjugates at the drug-linker level (chemically monodisperse drug-linkers) and at the conjugate level (homogeneously loaded conjugates); and (iii) based on biodegradable hydrophilic homopolymers that act as hydrophobic masking moieties.

[0009] Polysarcosine (poly-N-methylglycine or PSAR) can be an alternative to PEG and can be used to design novel protein conjugates with improved properties. PSAR is a highly hydrophilic, biodegradable, non-immunogenic, water-soluble polymer that is used in several delivery systems for drugs or diagnostics. Until now, PSAR is available only in polydisperse form, since it is obtained via a condensative ring-opening polymerization reaction of sarcosine N-carboxyanhydride (NCA) or sarcosine N-thiocarboxyanhydride (NTA). These polydisperse PSAR cannot be used in certain applications that require the use of shorter homopolymeric compounds that are relatively well-defined with acceptable dispersity (Gaussian distribution of molecular weights, with a polydispersity index >1), but have a certain length (unique and unique molecular weight) and therefore absolute homogeneity.

[0010] The use of discrete, monodisperse PSARs for macromolecular modification is a prerequisite for developing conjugates with absolute chemical homogeneity. Such homogeneous conjugates have the advantage of sharing exactly the same pharmacological properties (pharmacokinetics and efficacy), are easier to characterize, allow greater control over the reproducibility of the manufacturing process, and meet the increasingly stringent regulatory requirements for bioconjugates.

[0011] According to the present invention, discrete monodisperse PSAR homopolymers with defined chain lengths were obtained using a stepwise on-resin submonomer approach. This method is inexpensive, easily scalable, and gives final products with acceptable yields and excellent monomer purity. These monodisperse PSAR homopolymers were used in protein conjugation techniques to provide ligand-drug-conjugates (LDCs) with improved drug loading capacity, pharmacokinetics, and therapeutic efficacy.

[0012] The present invention therefore provides single molecular weight monofunctional homopolymers that meet the above requirements for use in composite technology, particularly LDC.

[0013] The homopolymer has the following formula (I):

[0014] [ka]

[0015] In the formula, R 1 and R 2 Unlike, R 1 and R 2 is H or an inert group, and R 1 and R 2 the other is a functionalized reactive group, said group being reactive to covalently bond with a bondable group under reaction conditions in which said inert group is not reactive; Z 1 and Z 2 are, either together or separately, any spacer, n is 1 or more, and k is 2 or more.

[0016] Before describing the invention in detail, definitions of terms used herein are provided below.

[0017] [Definition] According to the present invention, any compounds, such as reactants, products, monomers, homopolymers, units, etc., may be in the form of a salt, including acid addition salts, base addition salts, metal salts, ammonium and alkylated ammonium salts. Such salts are well known to those skilled in the art. Considering the intended use of the homopolymers of the present invention, they are preferably in the form of a pharma-ceutically acceptable salt.

[0018] A single molecular weight homopolymer refers to a homopolymer having a unique and specific molecular weight, as opposed to a mixture of homopolymers of the same nature but with a size distribution and a molecular weight distribution centered around an average molecular weight. A single molecular weight homopolymer can be defined by one absolute molecular formula with an absolute number of atoms.

[0019] In contrast to polydisperse homopolymers traditionally obtained by one-pot polymerization processes and having a PDI>1, single molecular weight homopolymers may also be called "monodisperse" having a polydispersity index (PDI) equal to 1. It is generally recognized herein that both the terms "monodisperse" and "discrete" are interchangeable, defining a homopolymer having a unique and absolute molecular weight, molecular formula and molecular structure, despite the fact that the term "monodisperse" does not accurately reflect the manufacturing procedure of the product.

[0020] Inert group or capping group refers to any chemically unreactive group that terminates one end of a homopolymer, said group being unreactive under a given reaction condition when compared to the functionalized reactive group that terminates the other end of the homopolymer. The resulting homopolymer is, in a sense, end-capped by this inert group and is not intended to be covalently bonded, particularly when used in LDC technology. In one embodiment, said group may only be inactivated after covalent attachment to one end of the homopolymer.

[0021] A non-exhaustive list of inert groups includes acyl groups, especially acetyl groups, amide groups, alkyl groups, especially C 1-20 These include alkyl groups, alkyl ether groups, alkyl ester groups, alkyl orthoester groups, alkenyl groups, alkynyl groups, aryl groups, aryl ester groups, tertiary amine groups, hydroxyl groups, aldehyde groups. The inert groups may also be selected from the same list of groups that define the functionalized reactive groups (see definition of functionalized reactive groups below).

[0022] A functionalized reactive group refers to any chemical moiety that is reactive to covalently bond with a bondable group and is reactive when compared to an inactive group under a given reaction condition. In particular, it can be bonded to the following groups: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl; halogen; activated esters such as N-hydroxysuccinimide esters, perfluoroesters, nitrophenyl esters, aza-benzotriazole and benzotriazole activated esters, acylureas, etc.; alkynyl; alkenyl; azide; isocyanate; isothiocyanate; aldehyde; thiol reactive moieties such as maleimide, halomaleimide, haloacetyl, pyridyl disulfide, etc.; thiol; acrylate; mesylate; tosylate; triflate, hydroxylamine; chlorosulfonyl; boronic acid-B(OR'). 2 Derivatives, where R' is hydrogen or an alkyl group.

[0023] A non-exhaustive list of functionalization reactive groups includes: carboxylic acids; primary amines; secondary amines; tertiary amines; hydroxyl; halogens; activated esters such as N-hydroxysuccinimide esters, perfluoroesters, nitrophenyl esters, aza-benzotriazole and benzotriazole activated esters, acylureas, etc.; alkynyls; alkenyls; azides; isocyanates; isothiocyanates; aldehydes; thiol reactive moieties such as maleimides, halomaleimides, haloacetyls, pyridyl disulfides, etc.; thiols; acrylates; mesylates; tosylates; triflates, hydroxylamines; chlorosulfonyls; boronic acids -B(OR'). 2 Derivatives, where R' is hydrogen or an alkyl group.

[0024] It should be mentioned that the terms "inert" and "functionalized reactive" for inert group and functionalized reactive group, respectively, are interdependent. This means that in a given reaction condition of the homopolymer of the present invention defined in any one of formulas (I), (II) and (III), the inert group does not react, and the functionalized reactive group reacts to form a covalent bond with the reactant. Thus, although the inert group and the functionalized reactive group in the homopolymer of any one of formulas (I), (II) and (III) are different, they may be selected overall from the same list of groups.

[0025] The term "group" in the functionalized reactive group or inert group according to the present invention should be understood as a group that does not exhibit any function other than being capable of covalently bonding with a reactant or being inert, respectively, under the given reaction conditions.

[0026] Alkyl, used alone or as part of an alkyl ether or alkyl ester, refers to a saturated, straight or branched chain hydrocarbon group having, for example, 1 to 20 carbon atoms, preferably 1 to 12, more preferably 1 to 6, especially 1 to 4 carbon atoms.

[0027] Alkenyl and alkynyl refer to at least partially unsaturated straight or branched chain hydrocarbon groups having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, especially 2 to 4 carbon atoms.

[0028] Aryl, used alone or as part of an aryl ester, refers to an aromatic group having one or more rings, for example containing from 6 to 14 ring carbon atoms, preferably 6 to 10, especially 6.

[0029] Alkylene, used alone or as part of an alkylene glycol, refers to a divalent saturated, linear or branched hydrocarbon group having, for example, 1 to 20 carbon atoms, preferably 1 to 12, more preferably 1 to 6, especially 1 to 4 carbon atoms.

[0030] Arylene refers to a divalent aryl group as defined above.

[0031] Heteroalkyl refers to a straight 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, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom O, N and S may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.

[0032] Heteroalkylene refers to a divalent heteroalkyl as defined above. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.

[0033] C 3 -C 8 Carbocycle refers to a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbon ring.

[0034] C 3 -C 8 Carbocyclo is a divalent C as defined above. 3 -C 8 This refers to a carbocycle.

[0035] C 3 -C 8 Heterocycle refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also called 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 may be oxidized. The ring containing a heteroatom may be aromatic or non-aromatic. Unless otherwise specified, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.

[0036] C 3 -C 8 Heterocyclo means a divalent C 3 -C8 This refers to a heterocycle.

[0037] Further, alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heteroalkyl, heteroalkylene, C 3 -C 8 carbocycle, C 3 -C 8 Carbocyclo, C 3 -C 8 Heterocycle, C 3 -C 8 The term heterocyclo refers to the -X, -R', -O - , -OR', =O, -SR', -S - , -NR' 2 , -NR' 3 , =NR', -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NRC(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 - , -PO 3 H 2 , -C(=O)R', -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -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, -CI, -H, 1 -C 20Alkyl, -C 6 -C 20 Aryl or -C 3 -C 14 It is a heterocycle.

[0038] An acyl group refers to a -CO-alkyl group, where alkyl is defined above.

[0039] A monofunctional homopolymer contains a single type of monomer (e.g., an N-methylglycine monomer for polysarcosine) having one end bearing a functionalized reactive group as defined above and another end bearing an H or an inert group as defined above.

[0040] Support for solid-phase peptide synthesis (SPPS) refers to a support that is commonly used in the well-known process of SPPS, in which peptides and insoluble polymers immobilized on a support are deprotected, washed, and coupled. The method is assembled by successive addition of Fmoc- or Boc-protected amino acids through repeated coupling-washing cycles. Each amino acid addition is called a cycle of (i) cleavage of the Nα-protecting group, (ii) a washing step, (iii) coupling of a fluorenylmethoxycarbonyl- (Fmoc)- or tert-butyloxycarbonyl- (Boc)-protected amino acid with a coupling reagent and a non-nucleophilic base, and (iv) a washing step. Since the growing chain is bound to the support, excess reagents and soluble by-products can be removed by simple filtration. Since the 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 resin, PAM resin, PAL resin, Sieber amide resin, MBHA resin, HMPB resin, HMBA resin, which are commercially available, to which peptides can be directly or indirectly coupled.

[0041] The term orthogonal connector refers to a branched linker unit component that connects a ligand to a homopolymer unit and a drug unit such that the homopolymer unit is in a parallel (as opposed to serial) arrangement relative to the drug unit. An orthogonal connector is a scaffold that has attachment sites for the components of the Ligand-Drug-Conjugate, i.e., the ligand, the homopolymer, and the drug unit. The term "parallel" is used to indicate the branching of the two components of the Ligand-Drug-Conjugate (LDC), but is not used to indicate that the two components are necessarily in close proximity in space or have the same distance between them.

[0042] An exemplary schematic representation of an LDC having a homopolymer (e.g., polysarcosine) unit in a parallel (i.e., branched) orientation to the Drug unit is as follows:

[0043] [ka]

[0044] where (L) is an orthogonal connector unit and w is 1 or more, typically 1-5, preferably 1-4, more preferably 1-3, or even 1-2. This orthogonal structure should not be confused with a linear structure. An exemplary schematic representation of an LDC having homopolymer (e.g., polysarcosine) units in a tandem (i.e., linear) orientation relative to the drug unit is as follows:

[0045] [ka]

[0046] A non-exhaustive list of orthogonal connectors 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. From his knowledge, the skilled artisan can select the appropriate orthogonal connector for the expected LDC compound. Advantageously, L is one or more natural or unnatural amino acids. In one embodiment, L is selected from glutamic acid, lysine and glycine.

[0047] The spacer is a bivalent linear arm that covalently bonds the two components of the ligand-drug-conjugate, such as: - a ligand unit and an orthogonal connector unit; - Orthogonal connector units and homopolymer units, - Orthogonal connectors and disconnectable parts; - a cleavable moiety and a drug, or - Orthogonal connectors and agents.

[0048] For example, the spacer may be a divalent linear alkylene group, preferably (CH 2 ) 4 It is.

[0049] A non-exhaustive list of spacer units includes alkylene, heteroalkylene (alkylene interrupted by at least one heteroatom selected from Si, N, O and S); alkoxy; polyethers such as polyalkylene glycols and typically polyethylene glycols; one or more natural or unnatural amino acids such as glycine, alanine, proline, valine, N-methylglycine; 3 -C 8 Heterocyclo;C 3 -C 8 carbocyclo; arylene, and any combination thereof. When the spacer is present between the cleavable moiety and the drug unit, or between the orthogonal connector and the drug unit, it can be linked to one or more drug units. For example, the spacer can be linked to 1 to 4 drug units, preferably 1 to 2 drug units. In one embodiment, the spacer between the cleavable moiety and the drug unit is (4-amino-1,3-phenylene)dimethanol.

[0050] In one embodiment, the spacer unit is of formula (XVII), formula (XVIII), formula (XIX), formula (XX), formula (XXI) or formula (XXII):

[0051] [ka]

[0052] The wave form bond represents the bond point, and R 6 -C 1 -C 10 Alkylene-, -C 1 -C 10 Heteroalkylene-, -C 3 -C 8 Carbocyclo-, -O-(C 1 -C 8 Alkyl)-, -arylene-, -C 1 -C 10 Alkylene-arylene-, -arylene-C 1 -C 10 Alkylene-, -C1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-, -C 3 -C 8 Heterocyclo-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Heteroalkylene-C(=O)-, -C 3 -C 8 Carbocyclo-C(=O)-, -O-(C 1 -C 8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1 -C 10 Alkylene-arylene-C(=O)-, -arylene-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-C(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 3 -C 8 Heterocyclo-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-C(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C1 -C 10 Alkylene-NH-, -C 1 -C 10 Heteroalkylene-NH-, -C 3 -C 8 Carbocyclo-NH-, -O-(C 1 -C 8 Alkyl)-NH-, -Arylene-NH-, -C 1 -C 10 Alkylene-arylene-NH-, -arylene-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-NH-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-NH-, -C 3 -C 8 Heterocyclo-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-NH-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-S-, -C 1 -C 10 Heteroalkylene-S-, -C 3 -C 8 Carbocyclo-S-, -O-(C 1 -C 8 Alkyl)-)-S-, -Arylene-S-, -C 1 -C 10 Alkylene-arylene-S-, -arylene-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-S-, -(C 3 -C 8 Carbocyclo)-C1 -C 10 Alkylene-S-, -C 3 -C 8 Heterocyclo-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-S-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Carbocyclo-OC(=O)-, -O-(C 1 -C 8 Alkyl)-OC(=O)-, -Arylene-OC(=O)-, -C 1 -C 10 Alkylene-arylene-OC(=O)-, -arylene-C 1 -C 10 Alkylene-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-OC(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Heterocyclo-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-OC(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene -OC(=O)-.

[0053] R 6 Any of the groups -X, -R', -O - , -OR', =O, -SR', -S - , -NR' 2, -NR' 3 + , =NR', -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NR'C(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 - , -PO 3 H 2 , -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -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; 1 -C 20 Alkyl, -C 6 -C 20 Aryl or -C 3 -C 14 It is a heterocycle.

[0054] Advantageously, the spacer unit is of formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII):

[0055] [ka]

[0056] The wave form bond represents the bond point, and R6 -C 1 -C 10 Alkylene-, -C 1 -C 10 Heteroalkylene-, -C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Heteroalkylene-C(=O)-, -arylene-C 1 -C 10 Alkylene-C(=O)-, -Arylene-C 1 -C 10 Alkylene -OC(=O)-.

[0057] R 6 Any of the groups are optionally substituted with one or more =O.

[0058] Ligands refer to any macromolecule (polypeptide, protein, peptide, typically an antibody) or small molecule, such as a folate or an aptamer, as typically used in LDC (e.g., antibody-drug conjugate) technology, that may be covalently attached to the synthetic or drug linkers of this study using bioconjugate techniques (see Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). Ligands are traditionally compounds that are selected for their targeting capabilities. A non-exhaustive list of ligands is given below: The term "antibody" as used herein 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 prepare the conjugates are antibodies. As used herein, the term "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, modified monoclonal and modified 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.

[0059] As referred to herein, the term "antibody" includes whole antibodies and any antigen binding fragment (ie, "antigen-binding portion") or single chains thereof.

[0060] A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as V H The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as V L The light chain constant region is composed of one domain, C L It consists of V H and V L The regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V Lis 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 region of the antibody may 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.

[0061] The term "antigen-binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to a full-length or one or more fragments of an antibody that retain 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 by the term "antigen-binding portion" of an antibody are V L , V H , C L and a CH1 domain; F(ab) is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. 2 Fragment;V H and an Fd fragment consisting of the CH1 domain; L and V H Fv fragment consisting of domains; V H domains (Ward et al., 1989 Nature 341:544-546); and isolated complementarity determining regions (CDRs), or any fusion proteins containing such antigen-binding portions.

[0062] In addition, two domains of the Fv fragment, V L and V H Although the V and VL are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that allows them to be made as a single chain protein in which the V L and V HThe domains pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc Natl Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are known to those of skill in the art. The fragments are obtained using conventional techniques, and the fragments are screened for utility in the same manner as are intact antibodies.

[0063] In certain embodiments, the ligand of the LDC is a chimeric, humanized, or human antibody.

[0064] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a human sequence, e.g., a human germline sequence, or a variant version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al. (2000. J Mol Biol 296, 57-86)).

[0065] Human antibodies may 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.

[0066] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.

[0067] As used herein, "isotype" refers to the antibody class (eg, IgM, IgE, IgG such as IgG1 or IgG4) that is provided by the heavy chain constant region genes.

[0068] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0069] The cleavable group (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. This unit can thus form a cleavable bond for the release of the drug unit, for example, by enzymatic processing or disulfide elimination mechanisms. The recognition site for enzymatic processing is usually a dipeptide cleavage site (e.g., Val-Cit, Val-Ala, or Phe-Lys), or a sugar cleavage site (e.g., a glucuronide cleavage site). For example, the cleavable group is a glucuronide group. This technology is well known to the skilled artisan, and from his knowledge, he can select a cleavable group appropriate for the drug of the LDC (e.g., ADC) compound. For example, the cleavable group includes disulfide-containing linkers cleavable by disulfide exchange, acid-labile linkers cleavable at acidic pH, and linkers cleavable by hydrolases (e.g., peptidases, esterases, and glucuronidases). The cleavable group can be in a form selected from the following: - a cleavable peptide comprising one or more natural or unnatural amino acids, for example 2 to 12 amino acids, - a sugar moiety linked to a self-immolative group via an oxygen glycosidic bond, - a disulfide linker, and - Acid-labile linkers that are hydrolyzable in the lysosome.

[0070] Advantageously, the cleavable group may be in a form selected from the following: - a cleavable peptide comprising one or more natural or unnatural amino acids, for example 2 to 12 amino acids, and - A sugar moiety linked to a self-immolative group via an oxygen glycosidic bond.

[0071] When a sugar moiety is used, the self-immolative group is considered to be part of the cleavable group. A "self-immolative group" is a trifunctional chemical moiety that can covalently link together three spaced apart chemical moieties: a sugar moiety (via a glycosidic bond), a drug D (directly or indirectly via a spacer Z), and an orthogonal connector L (directly or indirectly via a spacer Z). The glycosidic bond can be cleavable at the target site to initiate a self-immolative reaction sequence that results in release of the drug.

[0072] When a disulfide linker is used, cleavage occurs between the two sulfur atoms of the disulfide. A variety of disulfide linkers are known in the art and can be adapted for use in the present disclosure, including those that can be formed using, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), and SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate). See, for example, U.S. Patent No. 4,880,935.

[0073] In some embodiments, the cleavable unit is pH sensitive and may include, for example, an acid-labile linker (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, or ketal group) that is hydrolyzable in the lysosome (see, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; and 5,622,929). Such linkers are relatively stable under neutral pH conditions (e.g., blood pH conditions), but are unstable at pH 5.5 or 5.0 (approximate pH of the lysosome).

[0074] Ligand drug conjugate (LDC) refers to any conjugate that is linked to a ligand and a drug as defined above, including any mean as defined above, and as described in the Examples herein. When the ligand is an antibody, it may also be referred to as an antibody drug conjugate (ADC), which is a preferred embodiment of the present disclosure.

[0075] A bondable group refers to a group that can react with a functionalized reactive group to form a covalent bond. A bondable group thus includes a reactive group that reacts with a functionalized reactive group under a given reaction condition. Specifically, the bondable group can include one of the following groups: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl; halogen; activated esters such as N-hydroxysuccinimide esters, perfluoroesters, nitrophenyl esters, aza-benzotriazole and benzotriazole activated esters, acylureas, etc.; alkynyl; alkenyl; azide; isocyanate; isothiocyanate; aldehyde; thiol reactive moieties such as maleimide, halomaleimide, haloacetyl, pyridyl disulfide, etc.; thiol; acrylate; mesylate; tosylate; triflate, hydroxylamine; chlorosulfonyl; boronic acid-B(OR'). 2 Derivatives, where R' is hydrogen or an alkyl group.

[0076] Drug refers to any type of drug or compound, for example, a cytotoxic, cytostatic, immunosuppressive, anti-inflammatory or anti-infective compound. Among the cytotoxic compounds, calicheamicin; uncialamicin; auristatins (such as monomethylauristatin E, known as MMAE); tubulysin analogues; maytansines; cryptophysins; benzodiazepine dimers (including pyrrole[2,1-c][1,4]benzodiazepines, known as PDB's); indolinobenzodiazepine pseudodimers (IGNs); duocarmycins; anthracyclines (such as doxorubicin or PNU159682); camptothecin analogues (such as 7-ethyl-10-hydroxy-camptothecin, known as SN38 or exatecan); Bcl2 and Bcl-xl inhibitors; tylanstatin; amatoxins (including α-amanitin); kinesin spindle protein (KSP) inhibitors; vinorelbine; cyclin-dependent kinase (CDK) inhibitors; bleomycin; dactinomycin or radionuclides and their complexing agents (DOTA / 177 Among the anti-inflammatory drugs, mention may be made of corticosteroids, such as dexamethasone or fluticasone. Among the anti-infective drugs, mention may be made of antibiotics, such as rifampicin or vancomycin.

[0077] The present invention will now be described in more detail. It will be described more specifically with respect to the single molecular weight of polysarcosine homopolymer, but it should be appreciated that its scope extends to any single molecular weight encompassed within formula (I) above. Moreover, the benefits of the present invention are specifically demonstrated in LDC technology. Of course, its advantages are not limited to such technology, and it can show similar or better performance in any area where a single molecular weight, biocompatible, biodegradable homopolymer is required.

[0078] Thus, the present invention more particularly relates to a single molecular weight homopolymer of sarcosine having formula (II):

[0079] [ka]

[0080] (In the formula, R 1 and R 2 Unlike, R 1 and R 2 is H or an inert group, and R 1 and R 2 the other is a functionalized reactive group, which is reactive to covalently bond with a bondable group under reaction conditions in which the inert group is not reactive; Z 1 and Z 2 are, either together or separately, any spacer, k is 2 or greater).

[0081] Further characteristics of the homopolymers of formula (I), and in particular of the homopolymers of formula (II), taken alone or in any combination, are given below.

[0082] k is an integer of at least 2, preferably at most 100, more preferably at most 50, specifically 2-30, and more specifically 2-24, 6-24, or 12-24.

[0083] In either formula (I) or formula (II), the functionalizing reactive group R 1 or R 2 may be selected from the following group: - a carboxylic acid group, - Amino group NRR'' (wherein R and R″ are independently H and optionally interrupted by at least one heteroatom selected from among O, N, and S (C 1 -C 6 ) alkyl; - hydroxyl group, - halogen atoms, - Hydrazine (-NH 2 -NH 2 ) group, - a nitro group, - hydroxylamine groups, - an azide group, - (C 2 -C 6 ) an alkynyl group, - (C 2 -C 6 ) an alkenyl group, - thiol groups, - activated ester groups such as N-hydroxysuccinimide esters, perfluoroesters, nitrophenyl esters, aza-benzotriazole and benzotriazole activated esters, acylureas, - Boronic acid-B(OR'''') 2 base (wherein R"" is a hydrogen atom or C 1 -C 6 alkyl group), - thiol-reactive groups such as maleimides, halomaleimides, haloacetyls, pyridyl disulfides, etc. - mesylate group, - tosylate group, - a triflate group, - aldehyde group, - isocyanate or isothiocyanate groups, - chlorosulfonyl group, - Acrylate groups.

[0084] As mentioned above, the spacer Z is optional, and Z 1 and Z 2 may be present, and Z 1 and Z 2 may be present, or they may be absent. In this latter case, and when the homopolymer of the invention is a homopolymer of sarcosine, it has the formula (III):

[0085] [ka]

[0086] (In the formula, R 1 , R 2 and k are as defined above).

[0087] In formula (I), formula (II) or formula (III), R 1 is H or an inert group, R 2 may be a functionalized reactive group, or R 1 is the functionalizing reactive group, R 2 may be H or an inert group.

[0088] According to a preferred embodiment, the functionalized reactive group R 1 or R 2 is a secondary amine and the inert group R 1 or R 2 is the carboxylic acid that remains unreacted and not attached onto the final LDC structure.

[0089] In a preferred embodiment, in formula (I), formula (II) or formula (III), R 1 OH and NH 2 is selected from R 1 When is OH, R 2 COCH 3 and R 1 NH 2 Then, R 2 is CO-G-COOH, where G is CH 2 CH 2 , C.H. 2 CH 2 CH 2 , C.H. 2 CH 2 CH 2 CH 2 , C.H. 2 OCH 2 , C.H. 2 SCH 2 , C.H. 2 CH(CH 3 )CH 2 , C.H. 2 C(CH 3 ) 2 CH 2 or CH 2 N(CH 3 )CH 2 It is.

[0090] The present invention also relates to a method for preparing a single molecular weight homopolymer of either formula (I), formula (II) or formula (III). In general, each N-methylglycine monomer is assembled on a solid support from two submonomers, a haloacetic acid and a methylamine. Each monomer addition is referred to as a cycle of (i) acylation of the resin-bound secondary amine with a haloacetic acid and a carbodiimide, or acylation by other suitable carboxylate activation methods, (ii) a washing step, (iii) nucleophilic displacement of the resin-bound halogen with methylamine, and (iv) a washing step.

[0091] A method according to the invention comprising the steps of: a) Formula (IV)

[0092] [ka]

[0093] (In the formula, R 3 is a peptide synthesis solid phase support, and m is equal to or greater than 1 and less than k,

[0094] [ka]

[0095] (wherein Hal is a halogen) with an acid of formula (VI)

[0096] [ka]

[0097] (In the formula, R 3 , m and Hal are as defined above. obtaining a compound of b) reacting the compound of formula (VI) with methylamine to obtain a compound of formula (VII)

[0098] [ka]

[0099] (In the formula, R 3 and m is as defined above. obtaining a compound of c) Formula (VIII)

[0100] [ka]

[0101] (In the formula, R 3 is defined above and k is as defined above) repeating steps a) and b) until a compound of formula (I) is obtained; d) reacting said compound (VIII) to obtain a compound of formula (IX)

[0102] [ka]

[0103] (In the formula, R 2 is an inert group, R 3 is defined above and k is as defined above) obtaining a compound of e) A cleavage reaction to give a single molecular weight homopolymer of formula (III) as defined above.

[0104] According to an embodiment of this method, in step a), a compound of formula (IV), 3 is a peptide synthesis solid phase support, m is 3), said compound being obtained by the Fmoc-solid phase peptide synthesis method. They are well known to the person skilled in the art, and from his knowledge he can select any suitable coupling reagent, such as N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU).

[0105] In an alternative embodiment of the method of the present invention, preparing a single molecular weight homopolymer comprises the steps of: a) Formula (X)

[0106] [ka]

[0107] (In the formula, R 3 is a peptide synthesis solid phase, and m is equal to or greater than 1 and less than k. With a compound of formula (V)

[0108] [ka]

[0109] (wherein Hal is a halogen) with an acid of formula (XI)

[0110] [ka]

[0111] (In the formula, R 3 , m and Hal are as defined above. obtaining a compound of b) reacting the compound of formula (XI) with methylamine to obtain a compound of formula (XII)

[0112] [ka]

[0113] (In the formula, R 3 and m is as defined above. obtaining a compound of c) Formula (XIII)

[0114] [ka]

[0115] (In the formula, R 3 and k is as defined above). repeating steps a) and b) until a compound of formula (I) is obtained; d) Formula (XIV)

[0116] [ka]

[0117] where k is as defined above. a cleavage reaction to give a compound of formula (I), 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 single molecular weight homopolymer of formula (III) as defined above.

[0118] As stated above, the homopolymers of the present invention are useful in LDC technology, without being limited to this technology.

[0119] Thus, the present invention also relates to a Ligand-Drug-Conjugate compound (LDC) having the following formula (XV):

[0120] [ka]

[0121] (Wherein, L is (HP SMW ) is oriented orthogonally to (XD), HP SMW results from the covalent attachment of a single molecular weight homopolymer of the invention, as described above, to said orthogonal connector L, D is a drug, in particular a cytotoxic drug such as monomethylauristatin E (MMAE) or SN38; X is any cleavable moiety to release D; Z is an optional spacer, a is 1 or greater, b is 1 or greater, and m is 1 or greater.

[0122] Single molecular weight homopolymers, particularly single molecular weight polysarcosines, when grafted in a parallel (i.e., orthogonal) orientation to the drug unit, provide efficient hydrophobic masking properties, reduced apparent hydrophobicity, better pharmacokinetic properties, and improved in vivo activity of the conjugate compared to ligand-drug-conjugates that do not contain a single molecular weight homopolymer grafted in parallel.

[0123] In alternative embodiments, D is selected from the group consisting of a biologically active molecule, a therapeutic molecule such as an anti-cancer agent, an imaging agent, and a fluorophore.

[0124] According to an alternative embodiment of the present invention, a is an integer number which is at least 1, preferably at most 6, more preferably at most 3, in particular 2, more in particular 1; and / or b is an integer number which is at least 1, preferably at most 6, more preferably at most 3, in particular 2, more in particular 1; and / or m is an integer and is at least 1, preferably at most 30, more preferably at most 15, particularly 8, more particularly 4.

[0125] Advantageously, the single molecular weight homopolymer is polysarcosine.

[0126] In one embodiment, the intercalation between L and the ligand and / or between L and HP SMW and / or between L and X, and / or between X and D, there is a spacer Z.

[0127] Typically, an orthogonal connector connects the releasable assembly-drug unit (XD) or drug unit (D) via one or more linker unit components such that the (XD) or (D) unit is in a parallel (rather than series) configuration relative to the homopolymer unit.

[0128] The present invention also relates to an intermediate compound having formula (XVI):

[0129] [ka]

[0130] where L is an orthogonal connector, HP SMW results from the covalent attachment of a single molecular weight homopolymer of the invention to said orthogonal connector L, D is a cytotoxic drug; X is any cleavable moiety to release D; Z is an optional spacer capable of binding to a ligand; a is 1 or more, and b is 0 or 1 or more.

[0131] The present disclosure also relates to a compound having the formula (XXIII):

[0132] [ka]

[0133] In the formula, R 6 -C 1 -C 10 Alkylene-, -C 1 -C 10 Heteroalkylene-, -C 3 -C 8 Carbocyclo-, -O-(C 1 -C 8 Alkyl)-, -arylene-, -C 1 -C 10 Alkylene-arylene-, -arylene-C1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-, -C 3 -C 8 Heterocyclo-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Heteroalkylene-C(=O)-, -C 3 -C 8 Carbocyclo-C(=O)-, -O-(C 1 -C 8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1 -C 10 Alkylene-arylene-C(=O)-, -arylene-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-C(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 3 -C 8 Heterocyclo-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-C(=O)-, -(C 3 -C 8 Heterocyclo)-C 1-C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Heteroalkylene-NH-, -C 3 -C 8 Carbocyclo-NH-, -O-(C 1 -C 8 Alkyl)-NH-, -Arylene-NH-, -C 1 -C 10 Alkylene-arylene-NH-, -arylene-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-NH-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-NH-, -C 3 -C 8 Heterocyclo-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-NH-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-S-, -C 1 -C 10 Heteroalkylene-S-, -C 3 -C 8 Carbocyclo-S-, -O-(C 1 -C 8 Alkyl)-)-S-, -Arylene-S-, -C 1 -C 10 Alkylene-arylene-S-, -arylene-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-S-, -(C3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-S-, -C 3 -C 8 Heterocyclo-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-S-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Carbocyclo-OC(=O)-, -O-(C 1 -C 8 Alkyl)-OC(=O)-, -Arylene-OC(=O)-, -C 1 -C 10 Alkylene-arylene-OC(=O)-, -arylene-C 1 -C 10 Alkylene-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-OC(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Heterocyclo-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-OC(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 alkylene -OC(=O)-, R 6 Any of the groups -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2 , -NR' 3 + , =NR', -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NR'C(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 - , -PO 3 H 2 , -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -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, -C 1 -C 20 Alkyl, -C 6 -C 20 Aryl or -C 3 -C 14 is a heterocyclic ring, Z is an optional spacer, L is an orthogonal connector, X is any cleavable moiety to release D; D is a cytotoxic drug; a is 1 or more, and b is 0 or 1 or more; HP SMW results from the covalent attachment of a single molecular weight homopolymer of the invention to said orthogonal connector L as defined above.

[0134] According to a preferred embodiment, HP SMW results from the covalent attachment of the polysarcosine homopolymer of the present invention to the orthogonal connector L. In this case, in formula (XV), formula (XVI) and formula (XXIII), HP SMW represents:

[0135] [ka]

[0136] where the wavy bond, if present, represents the point of attachment to L or to the spacer Z; k is 2 or more, preferably, k is 2 to 50; R 4 represents a capping group).

[0137] Advantageously, R 4 -R', -O - , -OR', -SR', -S - , -NR' 2 , -NR' 3 + , =NR', -CX 3 , -CN, -NRC(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 - , -PO 3 H 2 , -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' 2 , C(=S)NR' 2 , or C(=NR')NR'2 where each X is independently a halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C 1 -C 20 Alkyl, -C 6 -C 20 Aryl or -C 3 -C 14 Typically, R 4 -OR', -NR' 2 or -C(=O)R'.

[0138] In one embodiment, the present disclosure also relates to a Ligand-Drug-Conjugate compound (LDC) having the following formula (XV):

[0139] [ka]

[0140] wherein the ligand is an antibody; L is HP SMW is an orthogonal connector that allows (XD) to be in an orthogonal orientation to (XD), and is selected from natural or unnatural amino acids, amino alcohols, amino aldehydes, polyamines, and combinations thereof; HP SMW represents the following:

[0141] [ka]

[0142] where the wavy bond, if present, represents the point of attachment to L or to the spacer Z; k is 2 or more, preferably, k is 2 to 50; R 4 represents a capping group), D is a drug, particularly a cytotoxic drug such as monomethylauristatin E (MMAE) or SN38; X is any cleavable moiety to release D; a cleavable peptide comprising one or more natural or unnatural amino acids, e.g., 2 to 12 amino acids; A sugar moiety linked to a self-immolative group via an oxygen glycosidic bond, Disulfide linkers, and Acid-labile linkers that are hydrolyzable in the lysosome A form selected from Z is an optional spacer between L and X, and / or between X and D, and / or between L and HP SMW between and, alkylene, heteroalkylene; alkoxy; polyether; one or more natural or unnatural amino acids; C 3 -C 8 Heterocyclo;C 3 -C 8 carbocyclo; arylene, and any combination thereof; a is 1 or greater, b is 1 or greater, and m is 1 or greater.

[0143] The present invention also relates to pharmaceutical compositions comprising at least one LDC compound of the invention and a pharma- ceutically acceptable carrier.

[0144] The present disclosure also relates to LDC compounds as described above for use as medicaments.

[0145] The compound of formula (XXIII) can be used directly without a ligand, since the maleimide moiety can react in vivo with a protein such as serum albumin, which then becomes a ligand. Thus, the present disclosure also relates to a compound of formula (XXIII) as described above, for use as a medicament.

[0146] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 represents a hydrophobic interaction chromatogram according to Example 12.

[0147] FIG. 2 shows the hydrophobic interaction chromatogram according to Example 13.

[0148] FIG. 3 shows the pharmacokinetic profile in mice according to Example 14.

[0149] 4A shows the tumor volume as a function of time according to Example 15. FIG. 4B shows the survival rate of mice according to Example 15.

[0150] FIG. 5 shows the pharmacokinetic profile in mice according to Example 16.

[0151] FIG. 6 shows the tumor volume as a function of time according to Example 17.

[0152] [Example] Materials and General Methods All solvents and reagents were obtained from commercial sources (Sigma-Aldrich, Alfa Aesar, Fluorochem, Thermo Fisher, Carbosynth) and used without further purification unless otherwise noted. 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-hydroxycamptothecin (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# A3782) was purchased from Sigma-Aldrich. Anti-CD19 and anti-CD22 antibodies were purchased from Euromedex. Trastuzumab (Herceptin®) was purchased from Roche. An empty SPE plate equipped with a 20 μm polyethylene frit (Sigma-Aldrich) was used. On-resin synthesis was carried out in plastic tubes. A Titramax 101 platform shaker (Heidolph) was used for agitation. All chemical reactions were carried out at room temperature under an inert argon atmosphere unless otherwise noted.

[0153] Liquid nuclear magnetic resonance spectra were recorded on a Bruker Fourier 300HD spectrometer using the residual solvent peak for calibration. Mass spectrometry was performed by the Centre Commun de Spectrometrie de Masse (CCSM), CNRS laboratory, UMR 5246, University Claude Bernard Lyon 1.

[0154] Normal phase flash chromatography was performed on a Teledyne Isco CombiFlash® Companion® instrument or a Teledyne Isco CombiFlash® Rf200 instrument using Int Reverse phase chromatography was performed using either Biotage® SNAP Ultra C18 (25 μm) cartridges or Interchim PuriFlash RP-AQ (30 μm) cartridges. Chemical reactions and compound characterization were performed using either Biotage® SNAP Ultra C18 (25 μm) cartridges or Interchim PuriFlash RP-AQ (30 μm) cartridges. , respectively, were monitored and analyzed by thin-layer chromatography using precoated 40-63 μm silica gel (Macherey-Nagel) and HPLC-UV (Agilent 1050) or UHPLC-UV / MS (Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact II™ Q-ToF mass spectrometer, or Agilent 1260 HPLC system equipped with a Bruker MicrOTOF-QII mass spectrometer).

[0155] HPLC Method 1: Agilent 1050 with DAD detection. Mobile phase A was water and mobile phase B was acetonitrile. The column was an Agilent Zorbax SB-Aq 4.6 x 150 mm 5 μm (room temperature). The gradient was 5% B to 95% B in 20 min, followed by a 5 min hold at 95% B. The flow rate was 1.5 mL / min. UV detection was monitored at 214 nm.

[0156] HPLC Method 2: Agilent 1050 with DAD detection. Mobile phase A was water and mobile phase B was acetonitrile. The column was an Agilent Zorbax SB-Aq 4.6×150 mm 5 μm (room temperature). The gradient was 0% B to 50% B in 30 min, followed by a 5 min hold at 50% B. The flow rate was 1.0 mL / min. UV detection was monitored at 214 nm.

[0157] HPLC Method 3: Same as HPLC Method 1, but containing 0.1% TFA in mobile phase A.

[0158] HPLC Method 4: Same as HPLC Method 2, but containing 0.1% TFA in mobile phase A.

[0159] 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 column was an Agilent PLRP-S 1000Å 2.1×150 mm 8 μm (80° C.). The gradient was 10% B to 50% B in 25 min. The flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. The Q-ToF mass spectrometer was run with a 1000 nm elution column for the m / z range 500-3500 (ESI + ) Data were deconvoluted using the MaxEnt algorithm included in the Bruker Compass® software.

[0160] HPLC Method 6: Agilent 1050 with DAD detection. Mobile phase A was water + 5 mM ammonium formate and mobile phase B was acetonitrile. The column was an Agilent Poroshell 120EC-C18 3.0 x 50 mm 2.7 μm (room temperature). The gradient was 5% B to 90% B in 10 min, followed by a 2 min hold at 90% B. The flow rate was 0.8 mL / min. UV detection was monitored at 214 nm.

[0161] The following Examples 1-4 illustrate the synthesis of the monomolecular weight polysarcosines of the present invention, which are part of the present invention that includes various solid phase synthesis methods.

[0162] Example 1: Synthesis of polysarcosine compound (on-resin synthesis method 1) The reaction scheme is set forth below.

[0163]

number

[0164] 1.1) General method On-resin syntheses were performed in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for agitation. All reported synthetic yields are based on an initial theoretical resin loading of 0.63 mmol / g (the labeling degree indicated by the manufacturer). All reactions were carried out at room temperature unless otherwise stated.

[0165] 1.2) Resin Loading Typically, 500 mg of NovaGEL™ Rink amide beads (0.63 mmol / g, Novabiochem) were swollen in 5 mL of DMF for 15 min. The first monomer was added by reacting 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide (Sigma-Aldrich) in 5 mL of DMF for 60 min at room temperature, followed by extensive washing with DMF (5 times 5 mL). The bromoacetylated resin was incubated with 5 mL of 40% (wt) aqueous methylamine (Sigma-Aldrich) for 30 min on a shaker platform, followed by extensive washing with DMF (5 times 5 mL) and DCM (5 times 5 mL). The resulting resin was ready for elongation.

[0166] 1.3) Elongation of polysarcosine compounds The polysarcosine oligomers were elongated 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 5 mL of DMF. The mixture was stirred for 30 min, drained, and washed with DMF (4 times 5 mL). For the amine displacement step, 5 mL of 40% (by weight) aqueous methylamine (Sigma-Aldrich) was added, and the vessel was shaken for 30 min, drained, and washed with DMF (4 times 5 mL) and DCM (4 times 5 mL).

[0167] 1.4) Cutting from resin Cleavage of the polysarcosine oligomer was carried out with 5 mL of TFA / triisopropylsilane (95:5) solution at room temperature under stirring. The resin was filtered and the resulting solution was evaporated under reduced pressure to give a clear oily material.

[0168] At this stage, PSARn-N(CH 3 )H was either dissolved in water for purification (see below) or involved in final functionalization.

[0169] 1.5) Final Functionalization PSARn-CH 2 -CH 2 To obtain the -COOH compound, the N-terminus of the oligomer was functionalized with 2.5 equivalents of succinic anhydride and 10 equivalents of DIPEA in anhydrous acetonitrile. The mixture was stirred at room temperature for 1 h and the volatiles were removed under reduced pressure.

[0170] 1.6) Purification PSAR compounds were purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 0 to 30% B.

[0171] 1.7) Single molecular weight polysarcosine compounds The resulting PSAR compounds are listed in Table 1 below.

[0172] [Table 1]

[0173] Example 2: Synthesis of polysarcosine compound (on-resin synthesis method 2) The reaction scheme is set forth below.

[0174]

number

[0175] 2.1) General method On-resin syntheses were carried out in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for agitation. All reported synthetic yields are based on an initial resin loading of 1.1 mmol / g (the degree of label indicated by the manufacturer). All reactions were carried out at room temperature unless otherwise stated.

[0176] 2.2) Synthesis of Fmoc-Sar-Sar-OH

[0177]

number

[0178] 2.2.1) Synthesis of Fmoc-Sar-Sar-OtBu Fmoc-Sar-OH (2000 mg / 6.42 mmol) and HATU (2443 mg / 6.42 mmol) were dissolved in 28 mL of anhydrous DMF in a round-bottom flask. DIPEA (2491 mg / 19.27 mmol) was added and the mixture was stirred at room temperature for 3 min. Sarcosine hydrochloride tert-butyl ester (1167 mg / 6.42 mmol) was then added and the reaction mixture was stirred at room temperature for 90 min. The volatiles were removed under vacuum and the residue was diluted with water and extracted three times with EtOAc. The organic phase was washed with MgSO 4The crude product was dried at 40° C., filtered and evaporated in vacuo to give a solid crude product. The crude product was dissolved 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 60:40 to 20:80) to give Fmoc-Sar-Sar-OtBu (2310 mg / 82%) as a white solid. HRMS m / z (ESI + ):Calc[M+H] + =439.2227;Exp[M+H] + = 439.2234; Error = -1.5 ppm. HPLC Method 1 Retention Time = 13.3 min. TLC eluted with 100% EtOAc: Rf = 0.8.

[0179] 2.2.2) tert-Butyl ester removal Fmoc-Sar-Sar-OtBu (2310 mg / 5.27 mmol) was dissolved in 20 mL of DCM and 8.5 mL of TFA was slowly added. The aqueous solution was stirred at room temperature until complete tert-butyl ester deprotection was observed by HPLC (approximately 2 h). Volatiles were then removed under vacuum and the residue was triturated with diethyl ether to give Fmoc-Sar-Sar-OH (1690 mg / 84%) as a white solid. 1 H NMR (500 MHz, DMSO-d 6 , 100℃) δ(ppm)2.84(s, 3H), 2.93(s, 3H), 4.01(s, 2H), 4.05(s, 2H), 4.25(t, J=4.3Hz, 1H), 4.34(d, J=6 .4Hz, 2H), 7.33(t, J=7.4Hz, 2H), 7.41(t, J=7.4Hz, 2H), 7.63(d, J=7.4Hz, 2H), 7.85(d, J=7.5Hz, 2H). HRMS m / z(ESI + ):Calc[M+H] + =383.1601;Exp[M+H] + = 383.1602; Error = 0.0 ppm. HPLC Method 1 Retention time = 6.2 min. TLC eluted with DCM / MeOH 85:15 (v / v): Rf = 0.65.

[0180] 2.3) Resin Loading Typically, 1000 mg of 2-chlorotrityl chloride resin beads (100-200 mesh, 1% DVB, 1.1 mmol / g, Novabiochem) were swollen in 10 mL of DCM for 10 min. Fmoc-Sar-OH (1.2 eq.), previously dissolved in 10 mL of dry DCM, was added onto the resin. DIPEA (5 eq.) was added and the reaction vessel was stirred at room temperature for 2 h. 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 substitution level was assessed from the weight gain of the resin and / or Fmoc cleavage test (absorbance measurement at 301 nm) and was found to be semi-quantitative (usually 0.95-1.1 mmol / g). The resin was stored at -20 °C until further use.

[0181] 2.4) Fmoc-Sar-Sar-OH Coupling Method The resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times). A solution of Fmoc-Sar-Sar-OH (3 eq.), HATU (2.85 eq.), and DIPEA (6 eq.) in DMF was added to the resin (1 mL per 100 mg of resin). The reaction vessel was agitated for 2 h and the resin was washed extensively with DMF (5 times) and DCM (5 times). The resin was dried under vacuum and stored at -20°C until further use.

[0182] 2.5) Elongation of polysarcosine compounds The resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature, then the resin was washed with DMF (4 times) and DCM (4 times).

[0183] The polysarcosine oligomers were elongated by alternating bromoacetylation and amine displacement steps until the desired length was obtained. The bromoacetylation step was carried out 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% (by weight) aqueous methylamine 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).

[0184] 2.6) Final acetylation When the desired oligomer length was obtained, the N-terminus was acetylated using a capping solution consisting of acetic anhydride / DIPEA / DMF (1:2:3 v / v) (the vessel was shaken for 30 min). 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).

[0185] 2.7) Resin cutting Cleavage of the polysarcosine oligomer from the resin was carried out with HFIP / DCM (20:80 v / v) solution under stirring for 30 min. The resin was filtered and the volatiles were removed under reduced pressure to give a solid crude product.

[0186] 2.8) Purification PSAR compounds were purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA.

[0187] 2.9) Single molecular weight polysarcosine compounds The resulting PSAR compounds are listed in Table 2 below.

[0188] [Table 2]

[0189] Example 3: Synthesis of polysarcosine compounds with one or several azide-functionalized orthogonal connectors (on-resin synthesis method 3) The reaction scheme is set forth below.

[0190]

number

[0191] 3.1) General Methodology On-resin syntheses were carried out in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for agitation. All reported synthesis yields are based on an initial resin loading of 1.1 mmol / g (as labeled by the manufacturer). All reactions were carried out at room temperature unless otherwise stated. Starting materials were obtained as described in Example 2 above.

[0192] 3.2) Process (1) A 3M solution of 2-azidoethan-1-amine in DMF was added (1 mL per 100 mg of resin) and the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times).

[0193] 3.3) Process (2) 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 agitated for 90 min and the resin was washed with DMF (3 times) and DCM (3 times).

[0194] 3.4) Process (3) Cleavage of the target compound from the resin was carried out with 1% TFA in DCM (v / v) solution under stirring for 5 min (repeated twice). The resin was filtered and the volatiles were removed under reduced pressure to give a solid crude product, which was purified using the protocol described in Example 2 above.

[0195] 3.5) Process (4) The bromoacetylation step was carried out 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 substitution step, 30% (wt) aqueous ammonia was added (2 mL per 100 mg of resin), the vessel was shaken for 30 min, 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 in Example 2 above.

[0196] 3.6) Process (5) The final bromoacetylation step was carried out 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) 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 in Example 2 above.

[0197] 3.7) Process (6) The bromoacetylation step was carried out 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% (by weight) aqueous methylamine was added (1.5 mL per 100 mg of resin) and the vessel was shaken for 30 min, drained and washed with DMF (4 times) and DCM (4 times).

[0198] 3.8) Process (7) The bromoacetylation step was carried out 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, a 3M solution of 2-azidoethan-1-amine in DMF was added (1 mL per 100 mg of resin) and the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times).

[0199] 3.9) Process (8) 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 agitated for 90 min and the resin was 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 in Example 2 above.

[0200] 3.10) Single molecular weight polysarcosine compounds The resulting PSAR compounds are listed in Table 3 below.

[0201] [Table 3]

[0202] Example 4: Synthesis of polysarcosine compounds with terminal non-orthogonal azide-functionalized connectors (On-resin synthesis method 4) The reaction scheme is set forth below.

[0203]

number

[0204] 4.1) General Methodology On-resin syntheses were carried out in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for agitation. All reported synthetic yields are based on an initial theoretical resin loading of 0.47 mmol / g (the labeling degree indicated by the manufacturer). All reactions were carried out at room temperature unless otherwise stated.

[0205] 4.2) Resin Loading Typically, 500 mg of Ramage ChemMatrix® beads (0.47 mmol / g, Sigma-Aldrich) were swollen in 5 mL of DCM for 15 min. The resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times). To the resin was added a solution of Fmoc-L-γ-azidohomoalanine OH (3 eq.), HATU (2.9 eq.), and DIPEA (6 eq.) in DMF (1 ml per 100 mg of resin). The reaction vessel was agitated for 1.5 h and the resin was washed extensively with DMF (5 times) and DCM (5 times). Unreacted sites were acetylated using a capping solution consisting of acetic anhydride / DIPEA / DMF (1:2:3 v / v) (the vessel was 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 twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times).

[0206] 4.3) Fmoc-Sar-Sar-OH Coupling Method To the resin was added a solution of Fmoc-Sar-Sar-OH (4 eq.), HATU (3.9 eq.), and DIPEA (8 eq.) in DMF (1 ml per 100 mg of resin). The reaction vessel was agitated for 2 h and the resin was washed extensively with DMF (4 times) and DCM (4 times). The resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times).

[0207] 4.4) Elongation of polysarcosine compounds The polysarcosine oligomers were elongated 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% (by weight) 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).

[0208] 4.5) Process (6) 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 agitated for 90 min and the resin was washed with DMF (3 times) and DCM (3 times).

[0209] 4.5) Cleavage from the resin and purification Cleavage of the oligomer from the resin was carried out with 5 mL of TFA / DCM (50:50) solution at room temperature under stirring for 30 min. This process was repeated once and the pooled filtrates were evaporated under reduced pressure to give a solid crude product, which was purified as described in Example 2 above.

[0210] 4.6) Single molecular weight polysarcosine compounds Table 4 below lists the resulting PSAR compounds.

[0211] [Table 4]

[0212] Example 5: Synthesis of Polyethylene Glycol (PEG) Compounds with Azide-Functionalized Orthogonal Connectors (On-Resin Synthesis Method 5) The reaction scheme is set forth below.

[0213]

number

[0214] 5.1) General Methodology On-resin syntheses were performed in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for agitation. All reported synthetic yields are based on an initial resin loading of 1.1 mmol / g (the labeling degree indicated by the manufacturer). All reactions were carried out at room temperature unless otherwise stated.

[0215] 5.2) Resin Loading Typically, 200 mg of 2-chlorotrityl chloride resin beads (100-200 mesh, 1% DVB, 1.1 mmol / g, Novabiochem) were swollen in 4 mL of DCM for 10 min. Fmoc-PEG 12 -CH 2 CH 2 COOH (PurePEG™, 1.2 eq.) was predissolved in 2 mL of dry DCM and added to the resin. DIPEA (3 eq.) was added and the reaction vessel was agitated at room temperature for 1 h. 300 μL of MeOH was added to quench the unreacted resin. After shaking for 10 min, the solution was drained and the resin was washed with DMF (3 times) and DCM (3 times). The resin was dried under high vacuum.

[0216] 5.2) Process (1) The resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times). The bromoacetylation step was carried out 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, a solution of 3 M 2-azidoethan-1-amine in DMF was added (1 mL per 100 mg of resin) and the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times).

[0217] 5.3) Step (2) The 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]acetic acid coupling procedure and cleavage from the resin were carried out as described in Example 3 above, and the compound was purified as described in Example 2 above.

[0218] 5.4) Single molecular weight PEG compounds The resulting PEG compounds are listed in Table 5 below.

[0219] [Table 5]

[0220] Example 6: Synthesis of MMAE, SN38, Exatecan and PNU159682-Based Intermediate Compounds 6.1) Synthesis of the compound alkyne-glucuronide-MMAE

[0221]

number

[0222] Starting material 110.8 mg (0.087 mmol) (Renoux et al., Chem. Sci., 2017, 8(5), 3427-3433) was dissolved in MeOH (10 mL) at 0 °C. LiOH monohydrate (36.7 mg / 0.87 mmol) was dissolved in water (1 mL) and slowly added 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 dissolved in water / MeOH / DMF aqueous solution (1:1:1 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. 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.

[0223] The compound alkyne-glucuronide-MMAE (mixture of two diastereoisomers) was obtained as a white solid (95 mg / 96%). LC-HRMS m / z (ESI + ):Calc[M+H] + =1127.5758;Exp[M+H] + =1127.5757;Error=0.1ppm. HPLC method 3 retention time = 10.3 minutes.

[0224] 6.2) Compound alkyne r-glucuronide-b (MMAE) 2 Synthesis of

[0225]

number

[0226] 6.2.1) Compound alkyne-glucuronide-(PNP) 2 Synthesis of 165 mg (0.240 mmol) of starting material (Renoux et al., Chem. Sci., 2017, 8 (5), 3427-3433), 64.2 mg (0.419 mmol) of commercially available 4-amino-3-(hydroxyphenyl)- (hydroxymethyl)phenylmethanol and 40.7 mg (0.300 mmol) of HOBt were dissolved in anhydrous DMF. After stirring at 50° C. for 3 h, the volatiles were evaporated and the residue was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 40:60 to 0:100) to give the intermediate diol compound as a yellow foam.

[0227] Anhydrous pyridine (4 molar equivalents) was added dropwise to a cooled solution (0° C.) of 4-nitrophenyl chloroformate (4 molar equivalents) in anhydrous DCM. The mixture was stirred at 0° C. for 15 min. An aqueous solution of the 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 solution of NaCl and extracted three times with DCM. The organic phase was washed with MgSO 4 The mixture was dried at 40° C., filtered, and evaporated under reduced pressure to give a solid crude product, which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 60:40 to 30:70) to give the compound alkyne-glucuronide-(PNP). 2 (52 mg / 21% over two steps) was obtained as a white solid. 1 H NMR (300 MHz, CDCl 3 )δ(ppm)2.04(s, 3H), 2.06(s, 3H), 2.11(d, J=2.0Hz, 3H), 2.71-2.92(m, 2H), 3.72(s, 3H), 4.11(q, J=7.1Hz, 1H), 4.22(d, J=8.6Hz, 1H), 5.18-5.4 1(m, 8H), 5.87(t, J=6.5Hz, 1H), 7.31-7.41(m, 5H), 7.45-7.55(m, 2H), 7 .56-7.71(m, 2H), 7.83(d, J=8.2Hz, 1H), 7.89(s, 1H), 8.21-8.32(m, 4H). HRMS m / z(ESI + ):Calc[M+Na] + =1055.1925;Exp[M+Na] + =1055.1955;Error=-2.9ppm.

[0228] 6.2.2) Compound alkyne-glucuronide-(MMAE) 2 Synthesis of 52 mg (0.050 mmol) of the above compound alkyne-glucuronide (PNP) 2 , 13.7 mg (0.100 mmol) of HOBt and 74.1 mg (0.103 mmol) of monomethylauristatin 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 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient 97:3 to 90:10) to give 77 mg (70%) of the intermediate compound, which was directly taken on to the deprotection step without extensive characterization. 77 mg (0.035 mmol) of this compound was dissolved in MeOH (7 mL) at 0 °C. 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 min, the mixture was neutralized with acetic acid (27.4 mg / 0.457 mmol) and concentrated under reduced pressure. The resulting material was dissolved in a water / MeOH / DMF solution (1:1:1 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA.

[0229] Compound Alkyne-Glucuronide-(MMAE) 2 (a mixture of two diastereoisomers) was obtained as a white solid (40 mg / 56%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1025.5623;Exp[M+2H] 2+ =1025.5599;Error=2.4ppm. HPLC method 3 retention time = 13.0 minutes.

[0230] 6.3) Synthesis of the compound alkyne-val-cit-PAB-MMAE

[0231]

number

[0232] 58 mg (0.052 mmol) of starting material (synthesized as described in Tang et al., Org. Biomol. Chem., 2016, 14(40), 9501-9518) and 15 mg (0.077 mmol) of 1H-TETRA-3-(2-methyl-1,2-dihydro-2,4-trimethylphenyl)-2-propanediol were added. l) 4-Pentynoic acid succinimidyl ester was dissolved in 3 mL of anhydrous DCM. 16.7 mg (0.129 mmol) DIPEA was added and the reaction was stirred at room temperature under argon atmosphere for 16 h. The volatiles were then removed under reduced pressure and the resulting material was dissolved in DMF solution and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. 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.

[0233] 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 minutes.

[0234] 6.4) Synthesis of compound alkyne-SN38

[0235]

number

[0236] 156 mg (0.308 mmol) of 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 dimethylaminopyridine. ) of 4-nitrophenyl chloroformate was dissolved in 8 mL of anhydrous DCM. The solution was stirred at room temperature for 90 min, diluted with 5% acetic acid in water and extracted three times with DCM. The organic phase was washed with MgSO 4 It was dried over water, filtered and evaporated under reduced pressure to give a yellow solid which was used in the next step without further purification.

[0237] 175 mg (0.261 mmol) of this yellow solid was dissolved in 4 mL of anhydrous DMF and 43 mg (0.782 mmol) of propargylamine was added slowly. The reaction was stirred at room temperature under argon for 16 h. The volatiles were removed in vacuo and the residue was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 40:60 to 0:100) to give compound alkyne-SN38 (71 mg / 56%) as a light yellow solid. HRMS m / z (ESI + ):Calc[M+H] + =474.1660;Exp[M+H] + = 474.1664; Error = -0.9 ppm. HPLC Method 3 Retention Time = 9.7 min. TLC eluted with 100% EtOAc: Rf = 0.15.

[0238] 6.5) Synthesis of compound alkyl glucuronide-SN38

[0239]

number

[0240] 50 mg (0.074 mmol) of starting material TBDMS-SN38-OPNP (synthesized as described in section 6.4 above) and 5 mg (0.037 mmol) of HOBt were weighed into a reaction vessel. 58.5 mg (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, was added to the reaction vessel. The reaction was stirred at room temperature for 16 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient 98:2 to 90:10) to give 48 mg (95%) of the intermediate compound (yellow solid), which was used directly in the deprotection step. HRMS m / z (ESI + ):Calc[M+H] + =681.3130;Exp[M+H] +=681.3113;Error=2.5ppm.

[0241] 48 mg (0.071 mmol) of this compound was dissolved in 2 mL of DCM and 500 μL of TFA was added. The solution was stirred at room temperature for 90 min and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient 94:6 to 80:20) to give 39.8 mg (96%) of compound SN38-methylamine as a yellow solid. HRMS m / z (ESI + ):Calc[M+H] + =581.2606;Exp[M+H] + =581.2601;Error=0.8ppm. HPLC method 3 retention time = 7.7 minutes.

[0242] 48 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 chemical The 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 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient 98:2 to 95:5) to give 57 mg (74%) of the intermediate compound (yellow solid), which was used directly in the deprotection step. ESI + [M+H] + =1130.4.

[0243] 57 mg (0.050 mmol) of this compound was dissolved in MeOH (6 mL) at 0 °C. LiOH monohydrate (21.2 mg / 0.504 mmol) was dissolved in water (0.6 mL) and slowly added to the reaction vessel. After stirring for 70 min 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 dissolved in a water / MeOH / DMF solution (1:1:1 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. 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.

[0244] The compound alkyne-glucuronide-SN38 was obtained as a yellow solid (20.5 mg / 42%). LC-HRMS m / z (ESI + ):Calc[M+H] + =990.3251;Exp[M+H] + =990.3210;Error=4.1ppm. HPLC method 3 retention time = 8.2 minutes.

[0245] 6.6) Synthesis of the compound alkyne-glucuronide-exatecan

[0246]

number

[0247] 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 ethylenediaminetetraacetate (EtOH), 10 ... Xatecan mesylate and 26 mg (0.192 mmol) of HOBt were dissolved in 1 mL of anhydrous DMF / pyridine 8:2 (v / v) mixture. The reaction was stirred at room temperature for 16 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient 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.

[0248] 165 mg (0.168 mmol) of this compound was dissolved in MeOH / THF 1:1 v / v (16 mL) at 0 °C. LiOH monohydrate (70.3 mg / 1.675 mmol) was dissolved in water (1.6 mL) and slowly added to the reaction vessel. After stirring for 70 min at 0 °C, the mixture was neutralized with acetic acid (131 mg / 2.18 mmol) and concentrated under reduced pressure. The resulting material was dissolved in a water / MeOH / DMF solution (1:1:1 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10 to 50% B.

[0249] The compound alkyne-glucuronide-exatecan was obtained as a yellow solid (98 mg / 69%). LC-HRMS m / z (ESI + ):Calc[M+H] + =845.2312;Exp[M+H] + =845.2360;Error=-4.8ppm. HPLC method 3 retention time = 8.0 minutes.

[0250] 6.7) Synthesis of compound alkyne-PNU159682

[0251]

number

[0252] 6.7.1) Synthesis of N-(2-((2-aminoethyl)amino)-2-oxoethyl)propiolamide 762 mg (4.54 mmol) of glycine tert-butyl ester hydrochloride, 318.3 mg (4.54 mmol) of propiolic acid, and 61.4 mg (0.454 mmol) of HOBt were dissolved in 5 mL of anhydrous DMF. 1103 mg (10.9 mmol) of DIPEA were added and the solution was stirred on ice (0° C.). 871 mg (4.54 mmol) of EDC hydrochloride was suspended in 12 mL of anhydrous DMF and added to the reaction vessel. The mixture was stirred in the dark at room temperature for 16 hours. The volatiles were then removed under reduced pressure. NH 4 A saturated solution of Cl was added and extracted three times with DCM. The organic phase was washed with MgSO 4 The mixture was dried at 70° C., filtered and evaporated. The crude residue was purified by silica gel chromatography (petroleum ether / EtOAc gradient 80:20 to 50:50) to give 255 mg (31%) of tert-butyl propioloyl glycinate as a clear oil. MS (ESI + ):[M+H] + = 184.0; eluted with petroleum ether / EtOAc (40:60 v / v), KMnO 4 Stained with: Rf=0.75.

[0253] 255 mg (1.39 mmol) of tert-butyl propioloyl glycinate was dissolved in 5 mL of DCM / TFA (1:1 v / v) solution. The deprotection reaction was complete after stirring at room temperature for 1 h as assessed by TLC analysis. Volatiles were removed under reduced pressure to give 188 mg (105%) of propioloyl glycine as an oily residue, which was used in the next step without purification.

[0254] 178 mg (1.40 mmol) of propioloylglycine and 504.8 mg (1.33 mmol) of HATU were dissolved in 3 mL of anhydrous DMF. 180.6 mg (1.40 mmol) of DIPEA was added and the reaction was stirred at room temperature for 5 min. Then, 291 mg (1.82 mmol) of N-Boc-ethylenediamine, previously dissolved in 1 ml of anhydrous DMF, was added and the reaction mixture was stirred in the dark at room temperature for 30 min. The volatiles were then removed under reduced pressure. NH 4 A saturated solution of Cl was added and extracted three times with DCM. The organic phase was washed with MgSO 4 The mixture was dried at 70° C., filtered and evaporated. The crude residue was purified by silica gel chromatography (petroleum ether / EtOAc gradient 20:80 to 0:100) to give 204 mg (54%) of tert-butyl (2-(2-propiolamidoacetamido)ethyl)carbamate as a slightly yellow oil. MS (ESI + ):[M+H] + = 270.1, eluted with 100% EtOAc, KMnO 4 TLC stained with: Rf=0.35.

[0255] 204 mg (0.758 mmol) of tert-butyl (2-(2-propiolamidoacetamido)ethyl)carbamate was dissolved in 5 mL of DCM / TFA (7:3 v / v) solution. The deprotection reaction was complete after stirring at room temperature for 45 min as assessed by TLC analysis. Volatiles were removed under high vacuum overnight to give 196 mg (92%) of N-(2-((2-aminoethyl)amino)-2-oxoethyl)propiolamido TFA salt as a slightly yellow thick wax. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm)2.84(q, J=6.2Hz, 2H), 3.29(q, J=6.3Hz, 2H), 3.72(d, J=6.0Hz, 2H) , 4.20(s, 1H), 7.78(br.s, 3H), 8.12(t, J=5.6Hz, 1H), 8.94(t, J=5.9Hz, 1H). MS(ESI + ):[M+H] + =170.0.

[0256] 6.7.2) Synthesis of alkyne-PNU159682 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 anhydrous DMF in a round-bottom flask. 10.2 mg (0.080 mmol) of DIPEA was 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)propiolamide TFA salt (previously dissolved in 500 μL of anhydrous DMF) was 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 99:1 to 90:10) to give 14.7 mg (49%) of alkyne-PNU159682 as a red solid. HRMS m / z(ESI + ):Calc[M+H] + =779.2770;Exp[M+H] + =779.2758;Error=1.5ppm. HPLC method 6 retention time = 5.4 minutes.

[0257] Example 7: Synthesis of polysarcosine-based drug conjugate linkers using glutamic acid as the orthogonal moiety The reaction scheme is set forth below.

[0258]

number

[0259] 7.1) Resin Loading with Ethylenediamine 500 mg of 2-chlorotrityl chloride resin beads (100-200 mesh, 1% DVB, 1.1 mmol / g, 0.55 mmol scale, Novabiochem) were swollen in 5 mL of DCM for 10 min, 5 equivalents (2.75 mmol, 165.3 mg) of ethylenediamine (Sigma-Aldrich) were added, and the mixture was shaken at room temperature for 4 h, followed by extensive washing 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 min treatment). The resin was washed extensively with DCM (5 times 5 mL) and MeOH (5 times 5 mL), dried under vacuum, and stored at -20 °C until further use.

[0260] 7.2) Fmoc-Glu(OAll)-OH Coupling To the resin containing the deprotected N-terminus (1 eq.) was added a solution of Fmoc-Glu(OAll)-OH (3 eq.), HATU (2.85 eq.), and DIPEA (6 eq.) in DMF (1 mL per 100 mg of resin). The reaction vessel was agitated for 2 h and the resin was washed extensively with DMF (5 times 3 mL) and DCM (5 times 3 mL). The completeness of the reaction was confirmed by a negative Kaiser test. The resin was dried under vacuum and stored at -20 °C until further use.

[0261] 7.3) Removal of the Allok Protecting Group The resin was suspended in DCM (4 mL per 100 mg of resin) and the mixture was gently stirred with a stream of argon introduced from under a fritted disk. Phenylsilane (20 equiv.) was added and stirring was continued for 5 min, after which Pd(PPh 3 ) 4 (0.25 equiv.) was added. Stirring of the mixture at room temperature under argon flow and protection from light was continued for 30 min, after which the solution was drained. Phenylsilane and Pd(PPh 3 ) 4The treatment with was repeated once and the resin was washed extensively with DCM (5 times 5 mL), DMF (5 times 5 mL), and MeOH (5 times 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.

[0262] 7.4) (2R,3R,4R,5S,6R)-6-(2-(3-aminopropanamido)-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 (NH 2 -Glucuronide-MMAE) conjugation method To the resin containing the deprotected carboxylic acid group (1 equiv.) was added a solution of HATU (4 equiv.) and DIPEA (4.2 equiv.) in DMF. The reaction vessel was agitated for 25 min, drained, and the resin was washed with DMF (4 times 5 mL). The resin was then treated with 1.5 equivalents of the compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropanamido)-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 ("NH 2-glucuronide-MMAE"; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831-840) and DIPEA (3.2 equiv.) were added. The reaction vessel was stirred for 3 h, 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.

[0263] 7.5) Fmoc deprotection method The resin containing the Fmoc-protected amino acid was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) at room temperature for 2 times 15 min. The resin was then 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.

[0264] 7.6) Polysarcosine coupling method The resin containing the deprotected primary amine group (1 equivalent) was treated with polysarcosine-CH in DMF. 2 -CH 2 A solution of -COOH (2.2 eq.), HATU (2 eq.), and DIPEA (6 eq.) was added. The reaction vessel was agitated for 2.5 h, drained, and the resin was 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.

[0265] 7.7) Cutting from resin The final cleavage from the 2-chlorotrityl resin was carried out with a 20% (v / v) HFIP in DCM solution (2 mL per 100 mg of resin) at room temperature under stirring. The reaction time was 60 min. The resin was filtered and the resulting solution was evaporated under a stream of argon gas. The final residue was dried under high vacuum and used as is in the following step.

[0266] 7.8) 3-(Maleimido)propionic acid N-hydroxysuccinimide ester coupling method To the residue dissolved in anhydrous DMF was added 3-(maleimido)propionic acid N-hydroxysuccinimide ester (8 equiv.). DIPEA (10 equiv.) was 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 μm) cartridge. 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.

[0267] The compound MAL-Glu (glucuronide MMAE)-CH 2 -CH 2 -PSAR6 was obtained as a clear oil (5.8 mg / 14% yield based on initial resin loading). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =989.5064;Exp[M+2H] 2+ 989.5023;Error=4.2ppm. HPLC method 1 retention time = 6.7 minutes.

[0268] The compound MAL-Glu (glucuronide MMAE)-CH 2 -CH 2 -PSAR12 was obtained as a clear oil (4.6 mg / 20% yield based on initial resin loading). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1202.6178;Exp[M+2H] 2+ 1202.6178;Error=0.0ppm. HPLC method 1 retention time = 6.9 minutes.

[0269] The compound MAL-Glu (glucuronide MMAE)-CH 2 -CH 2 -PSAR18 was obtained as a clear oil (14% yield based on 2.4 mg / initial resin loading). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1415.7291;Exp[M+2H] 2+1415.7282;Error=0.7ppm. HPLC method 1 retention time = 6.8 minutes.

[0270] Example 8: Synthesis of polysarcosine-based drug conjugate linkers using lysine as the orthogonal moiety The reaction scheme is set forth below.

[0271]

number

[0272] 8.1) Fmoc-D-Lys(glucuronide MMAE)-NH 2 Synthesis of

[0273] [ka]

[0274] The compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropanamido)-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 ("NH 2Fmoc-D-Lys(Boc)-OSu (35 mg / 0.062 mmol) and Fmoc-D-Lys(Boc)-OSu (69.9 mg / 0.062 mmol) were dissolved in 1.2 mL of anhydrous DMF. DIPEA (24.0 mg / 0.186 mmol) was added and the mixture was stirred at room temperature for 20 h. The volatiles were removed under vacuum. The flask containing the slightly yellow crude product was placed on an ice bath (0° C.) and 7 mL of DCM / TFA (7:3 v / v) solution was added slowly. The solution was stirred on ice until complete Boc deprotection was observed by HPLC (approximately 2 h). Volatiles were then removed under reduced pressure and the residue was dissolved in DMF for purification on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient was 10-60% B, eluting with the compound Fmoc-D-Lys(glucuronide MMAE)-NH 2 was obtained as a white solid (59 mg / 65%). LC-HRMS m / z (ESI + ):Calc[M+H] + =1480.7862;Exp[M+H] + =1480.7890;Error=-1.9ppm. HPLC method 1 retention time = 10.5 minutes.

[0275] 8.2) Synthesis of Fmoc-D-Lys(glucuronide MMAE)-PSARn

[0276] [ka]

[0277] The compound PSARn-COOH (2 eq.; obtained as described in Example 2 and predissolved as a 0.15 M stock solution in anhydrous DMF) 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. The compound Fmoc-D-Lys(glucuronide MMAE)-NH 2 (1 equiv.; pre-dissolved as a 0.05M stock in anhydrous DMF) was added. Stirred at room temperature for 1 hour and purified by injection onto a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10-60% B.

[0278] The compound Fmoc-D-Lys(glucuronide MMAE)-PSAR6 was obtained as a white solid (9.8 mg / 38%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =975.0133;Exp[M+2H] 2+ =975.0088;Error=4.6ppm. HPLC method 1 retention time = 7.5 minutes.

[0279] The compound Fmoc-D-Lys(glucuronide MMAE)-PSAR12 was obtained as a white solid (3.6 mg / 28%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1188.1247;Exp[M+2H] 2+ =1188.1233;Error=1.1ppm. HPLC method 1 retention time = 7.6 minutes.

[0280] 8.3) 6-(Maleimido)hexanoic acid N-hydroxysuccinimide ester coupling method The compound Fmoc-D-Lys(glucuronide MMAE)-PSARn from the 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. 6-(maleimido)hexanoic acid N-hydroxysuccinimide ester (8 eq.) and DIPEA (10 eq.) were then added and the mixture was stirred at room temperature for 30 min. Quenched with water / TFA (99.5:0.5 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. After a 10 min isocratic hold (5% B), the compound of interest was eluted isocratically at 40% B.

[0281] [ka]

[0282] The compound MAL-Lys(glucuronide MMAE)-PSAR6 was obtained as a clear oil (5.0 mg / 52%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =960.5163;Exp[M+2H] 2+ =960.5167;Error=-0.5ppm. HPLC method 1 retention time = 7.1 min.

[0283] The compound MAL-Lys(glucuronide MMAE)-PSAR12 was obtained as a clear oil (1.8 mg / 51%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1173.6276;Exp[M+2H] 2+ 1173.6229;Error=4.0ppm. HPLC method 1 retention time = 7.0 minutes.

[0284] Example 9: Synthesis of polyalkosine- or polyethylene glycol-based drug conjugate linkers using glycine as the orthogonal moiety 9.1) The compound Bromoacetamide-Ngly (triazole-) glucuronide (MMAE)-PSARn

[0285]

number

[0286] Alkyne-glucuronide-MMAE (1 equivalent; obtained as described in Example 6), PSARn-N 3 -Bromoacetamide (1.1 equiv.; obtained as described in Example 3), and tetrakis(acetonitrile)copper(I) hexafluorophosphate (3 equiv.) were combined in a reaction vessel. A 1:1 (v / v) solution of DCM / acetonitrile was added to reach a final alkyne-glucuronide-MMAE concentration of 12 μmol / mL. The reaction was stirred in the dark under argon at room temperature for 16-20 h. After removal of volatiles under reduced pressure, the residue was dissolved in DMF and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 10-50% B.

[0287] The compound bromoacetamide-Ngly(triazole-glucuronide MMAE)-PSAR12 was obtained as a white solid (8.5 mg / 51%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1151.0179;Exp[M+2H] 2+ =1151.0188;Error=-0.8ppm. HPLC method 3 retention time = 8.5 minutes.

[0288] 9.2) The compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSARn

[0289]

number

[0290] Alkyne-glucuronide-MMAE (obtained as described in Example 6) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in section 9.1 using DCM as the reaction solvent.

[0291] The compound MAL-Phenyl-Ngly(triazole-glucuronide MMAE)-PSAR6 was obtained as a white solid (3.3 mg / 20%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =955.9566;Exp[M+2H] 2+ =955.9533;Error=3.4ppm. HPLC method 3 retention time = 9.2 minutes.

[0292] The compound MAL-Phenyl-Ngly(triazole-glucuronide MMAE)-PSAR12 was obtained as a white solid (9.0 mg / 33%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1169.0679;Exp[M+2H] 2+ =1169.0621;Error=4.9ppm. HPLC method 3 retention time = 8.7 minutes.

[0293] The compound MAL-Phenyl-Ngly(triazole-glucuronide MMAE)-PSAR18 was obtained as a white solid (11.5 mg / 40%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1382.1792;Exp[M+2H] 2+ =1382.1803;Error=-0.7ppm. HPLC method 3 retention time = 8.6 minutes.

[0294] The compound MAL-Phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 was obtained as a white solid (15 mg / 44%). LC-HRMS m / z (ESI + ):Calc[M+4Na] 4+ =820.1309;Exp[M+4Na] 4+=820.1324;Error=-1.8ppm. HPLC method 3 retention time = 8.4 minutes.

[0295] 9.3) The compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG

[0296]

number

[0297] Alkyne-glucuronide-MMAE (obtained as described in Example 6) and PEG-N 3 -Phenyl-MAL (obtained as described in Example 5) was reacted and purified as described above in section 9.1 using NMP / DCM 2:1 (v / v) as the reaction solvent.

[0298] The compound MAL-Phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 was obtained as a slightly yellow oil (10.4 mg / 45%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1042.5211;Exp[M+2H] 2+ =1042.5218;Error=-0.7ppm. HPLC method 3 retention time = 8.0 minutes.

[0299] 9.4) The compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)-PSARn

[0300]

number

[0301] Alkyne-glucuronide-MMAE (3 equivalents; obtained as described in Example 6), PSARn-N 3 -N 3-Phenyl-MAL (1 eq.; obtained as described in Example 3), and tetrakis(acetonitrile)copper(I) hexafluorophosphate (5 eq.) were combined in a reaction vessel. DCM was added and the reaction was stirred in the dark under argon at room temperature for 16-20 hours. After removal of volatiles under reduced pressure, the residue was dissolved in DMF and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 10-50% B.

[0302] The 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 + ):Calc[M+4H] 4+ =1022.5082;Exp[M+4H] 4+ =1022.5093;Error=-1.0ppm. HPLC method 3 retention time = 9.5 minutes.

[0303] 9.5) The compound MAL-Phenyl-Ngly [Triazole-Glucuronide (MMAE) 2 ]-PSARn

[0304]

number

[0305] Alkyne glucuronide (MMAE) 2 (obtained as described in Example 6) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in section 9.1 using DCM as the reaction solvent.

[0306] The compound MAL-Phenyl-Ngly [Triazole-Glucuronide (MMAE) 2]-PSAR24 was obtained as a white solid (12.5 mg / 39%). LC-HRMS m / z (ESI + ):Calc[M+3H] 3+ =1371.3767;Exp[M+3H] 3+ =1371.3818;Error=-3.8ppm. HPLC method 3 retention time = 10.0 minutes.

[0307] 9.6) The compound MAL-Phenyl-Ngly [Triazole-galactoside (MMAE) 2 ]-PSARn

[0308]

number

[0309] Alkyne-galactoside (MMAE) 2 (synthesized as described in Alsarraf et al., Chem. Commun., 2015, 51(87), 15792-15795) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described in Section 9.1 using DCM as the reaction solvent.

[0310] The compound MAL-phenyl-Ngly [triazole-galactoside (MMAE) 2 ]-PSAR24 was obtained as a white solid (6.5 mg / 55%). LC-HRMS m / z (ESI + ):Calc[M+4H] 4+ =1022.7895;Exp[M+4H] 4+ =1022.7903;Error=0.8ppm. HPLC method 3 retention time = 9.2 minutes.

[0311] 9.7) Compound MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSARn

[0312]

number

[0313] Alkyne-val-cit-PAB-MMAE (obtained as described in Example 6) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in Section 9.1 using NMP as the reaction solvent.

[0314] The compound MAL-Phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSAR12 was obtained as a white solid (4.5 mg / 12%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1207.1494;Exp[M+2H] 2+ =1207.1535;Error=-3.5ppm. HPLC method 3 retention time = 8.6 minutes.

[0315] 9.8) Compound MAL-phenyl-Ngly(triazole-SN38)-PSARn

[0316]

number

[0317] Alkyne-SN38 (obtained as described in Example 6) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described in Section 9.1 using DCM / DMF 2:1 (v / v) as the reaction solvent.

[0318] The compound MAL-Phenyl-Ngly(Triazole-SN38)-PSAR18 was obtained as a light yellow solid (4.0 mg / 20%). LC-HRMS m / z (ESI + ):Calc[M+2Na] 2+ =1077.4562;Exp[M+2Na] 2+ =1077.4588;Error=-2.4ppm. HPLC method 3 retention time = 7.5 minutes.

[0319] 9.9) Compound MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSARn

[0320]

number

[0321] Alkyne-SN38 (obtained as described in Example 6) and PSARn-N 3 -N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in Section 9.4.

[0322] The compound MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18 was obtained as a yellow solid (5.1 mg / 43%). LC-HRMS m / z (ESI + ):Calc[M+2Na] 2+ =1412.5812;Exp[M+2Na] 2+ =1412.5852;Error=-3.8ppm. HPLC method 3 retention time = 8.4 minutes.

[0323] 9.10) The compound MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSARn

[0324]

number

[0325] Alkyne-glucuronide-SN38 (obtained as described in Example 6) and PSARn-N 3 -N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in section 9.4 using DCM / MeOH 8:2 (v / v) as the reaction solvent.

[0326] The 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 + ):Calc[M+2H] 2+ =1271.5080;Exp[M+2H] 2+ =1271.5103;Error=-1.8ppm. HPLC method 3 retention time = 7.8 minutes.

[0327] 9.11) The compound MAL-phenyl-Ngly(triazole-glucuronide-exatecan)-PSARn

[0328]

number

[0329] Alkyne-glucuronide-exatecan (obtained as described in Example 6) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in section 9.1 using DCM as the reaction solvent.

[0330] The compound MAL-Phenyl-Ngly(triazole-glucuronide-exatecan)-PSAR18 was obtained as a yellow solid (13.2 mg / 64%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1241.0069;Exp[M+2H] 2+ =1241.0088;Error=-1.1ppm. HPLC method 3 retention time = 7.1 minutes.

[0331] 9.12) The compound MAL-phenyl-Ngly(triazole-PNU159682)-PSARn

[0332]

number

[0333] Alkyne-PNU159682 (obtained as described in Example 6) and PSARn-N 3 -Phenyl-MAL (obtained as described in Example 3) was reacted and purified as described above in Section 9.1, using DCM as the reaction solvent and replacing the 0.1% TFA additive in the mobile phase with 0.1% formic acid during reverse phase purification.

[0334] The compound MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12 was obtained as a red solid (4.8 mg / 40%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =994.9185;Exp[M+2H] 2+ =994.9184;Error=0.1ppm. HPLC method 6 retention time = 5.0 minutes.

[0335] The compound MAL-Phenyl-Ngly(triazole-PNU159682)-PSAR18 was obtained as a red solid (7.2 mg / 33%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1208.0298;Exp[M+2H] 2+ =1208.0295;Error=0.3ppm. HPLC method 6 retention time = 4.9 minutes.

[0336] Example 10: Synthesis of negative control drug conjugate linkers MAL-glucuronide MMAE, MAL-phenyl-triazole-glucuronide MMAE, and MAL-phenyl-PSARn-triazole-glucuronide MMAE 10.1) Synthesis of compound MAL-glucuronide MMAE

[0337]

number

[0338] The starting compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropanamido)-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 ("NH 2 3-(maleimido)propionic acid N-hydroxysuccinimide ester (14.6 mg / 55 μmol) was weighed and dissolved in 200 μL of anhydrous DMF. DIPEA (8.5 mg / 66 μmol) was added and the mixture was stirred at room temperature for 30 min. The mixture was quenched with 1.5 mL of water / TFA (99:1 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. 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.

[0339] The title compound MAL-glucuronide MMAE was obtained as a white solid (4.1 mg / 59%). LC-HRMS m / z (ESI + ):Calc[M+H] + =1281.6501;Exp[M+H] + =1281.6489;Error=0.9ppm. HPLC method 1 retention time = 7.1 min.

[0340] 10.2) Synthesis of the compound MAL-phenyl-triazole-glucuronide MMAE 10.2.1) Synthesis of perfluorophenyl 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetate

[0341] [ka]

[0342] 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 anhydrous 1,2-dimethoxyethane in a reaction vessel. After stirring at room temperature for 2 h, the insoluble material was removed by filtration and the filtrate was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 80:20 to 20:80) to give the title compound (400 mg / 78%) as a white solid. 1 H NMR (300 MHz, CDCl 3 )δ(ppm)4.01(s, 2H), 6.87(s, 2H), 7.40(d, J=8.7Hz, 2H), 7.47(d, J=8.7Hz, 2H). HRMS m / z(ESI + ):Calc[M+H] + =398.0446;Exp[M+H] + =398.0448;Error=-0.4ppm.

[0343] 10.2.2) Synthesis of N-(2-azidoethyl)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide

[0344] [ka]

[0345] 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 anhydrous DCM in a reaction vessel. 2-Azidoethan-1-amine (33.8 mg / 0.40 mmol) was added and the reaction was stirred at room temperature for 1 h. 1N HCl solution was then added and the mixture was extracted three times with DCM. The organic phase was washed with MgSO 4 The mixture was dried at 40° C., filtered and evaporated under reduced pressure to give a crude solid product which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 60:40 to 0:100) to give the title compound (18 mg / 31%) as a white solid. MS (ESI + ):[M+H] + = 300.1; HPLC Method 1 Retention Time = 8.4 min. TLC eluted with 100% EtOAc: Rf = 0.65.

[0346] 10.2.3) Synthesis of the compound MAL-phenyl-triazole-glucuronide MMAE

[0347]

number

[0348] The compound alkyne-glucuronide MMAE (17 mg / 15.1 μmol) from Example 6, 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 combined 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 under argon at room temperature for 16 hours. After removal of volatiles under reduced pressure, the residue was dissolved in DMF and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 10 to 60%B.

[0349] The title compound MAL-phenyl-triazole-glucuronide MMAE was obtained as an off-white solid (10.3 mg / 48%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =713.8425;Exp[M+2H] 2+ =713.8415;Error=1.3ppm. HPLC method 3 retention time = 10.2 minutes.

[0350] 10.3) Synthesis of the compound MAL-phenyl-PSARn-triazole-glucuronide MMAE

[0351]

number

[0352] The compound alkyne-glucuronide MMAE from Example 6 (20 mg / 17.7 μmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (13.2 mg / 35 μmol), and N 3 -PSARn-phenyl-MAL (34.3 mg / 28 μmol) were combined in an HPLC vial. 900 μL of NMP / DCM 2:1 (v / v) solution was added and the reaction was stirred under argon at room temperature for 16 h. After removal of volatiles under reduced pressure, the residue was dissolved in DMF and purified on a 30 g Biotage® SNAP Ultra C18 (25 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 10 to 60% B.

[0353] The title compound MAL-phenyl-PSARn-triazole-glucuronide MMAE was obtained as a white solid (16.0 mg / 39%). LC-HRMS m / z (ESI + ):Calc[M+2H] 2+ =1168.5759;Exp[M+2H] 2+=1168.5792;Error=-2.8ppm. HPLC method 3 retention time = 6.8 minutes.

[0354] Example 11: Preparation of LDC Compounds of the Invention The following LDC compounds were prepared and characterized: Trastuzumab-Glu(Glucuronide MMAE)-CH 2 -CH 2 -PSAR6 Trastuzumab-Glu(Glucuronide MMAE)-CH 2 -CH 2 -PSAR12 Trastuzumab-Glu(Glucuronide MMAE)-CH 2 -CH 2 -PSAR18 Trastuzumab-Lys(glucuronide MMAE)-PSAR6 Trastuzumab-Lys(glucuronide MMAE)-PSAR12 Trastuzumab-BAC-Ngly (triazole-glucuronide MMAE)-PSAR12 Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR6 Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR12 Trastuzumab-MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSAR12 Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR18 Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-PSAR18 Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide-exatecan)-PSAR18 Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12 Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18 Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR24 CD19-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR24 CD22-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)-PSAR18 Trastuzumab-MAL-phenyl-Ngly [triazole glucuronide (MMAE) 2 ]-PSAR24 Trastuzumab-MAL-phenyl-Ngly [triazole-galactoside (MMAE) 2 ]-PSAR24 Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18 Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSAR18 Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 Trastuzumab-glucuronide MMAE Trastuzumab-MAL-phenyl-triazole-glucuronide MMAE Trastuzumab-MAL-phenyl-PSAR12-triazole-glucuronide MMAE Human Albumin-MAL-Phenyl-Ngly (Triazole-Glucuronide MMAE)-PSAR24 Their structures are set out in Table 6 below.

[0355] [Table 6] JPEG0007644173000067.jpg251170JPEG0007644173000068.jpg186170JPEG0007644173000069.jpg233170JPEG00076441730 00070.jpg247170JPEG0007644173000071.jpg177170JPEG0007644173000072.jpg238170JPEG0007644173000073.jpg206170

[0356] 11.1) Preparation of the Complex 11.1.1) Preparation of Antibody-Drug-Conjugates A solution of antibody (10 mg / mL in PBS 7.4 + 1 mM EDTA) was treated with 14 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 2 h at 37 °C. For maleimide-based coupling, the fully reduced antibody was buffer-exchanged with potassium phosphate 100 mM pH 7.4 + 1 mM EDTA by three rounds of dilution / centrifugation using Amicon 30K centrifugal filter devices (Merck Millipore). 10-12 equivalents of drug-linker (from a 12 mM DMSO stock solution) were added to the antibody (residual DMSO < 10% v / v). The solution was incubated for 30 min at room temperature. For bromoacetamide-based coupling, the fully reduced antibody was buffer-exchanged with borate buffer 50 mM pH 8.1 + 1 mM EDTA and conjugation was carried out with 16 molar equivalents of drug-linker for 24 h at 37 °C in the dark. The complexes were buffer exchanged / purified in PBS7.4 by four rounds of dilution / centrifugation using Amicon 30K centrifugal filter devices. Alternatively, the complexes were buffer exchanged / purified using PD MiniTrap G-25 columns (GE Healthcare) and sterile filtered (0.20 μm PES filters). Complexes incorporating autohydrolyzable maleimide (MAL-phenyl) groups were incubated at 5 mg / mL for 48 h at 37 °C in PBS7.4 to ensure complete hydrolysis of the succinimidyl moieties. Final protein concentrations were assessed spectrophotometrically at 280 nm using a Colibri microvolume spectrometer device (Titertek Berthold).

[0357] 11.1.2) Preparation of human albumin-drug-conjugates To a solution of human albumin (10 mg / mL in potassium phosphate 100 mM pH 7.4 + 1 mM EDTA) were added 2 molar equivalents of drug-linker (from a 12 mM DMSO stock solution in water). Residual DMSO was <10% (v / v). The solution was incubated for 4 hours at room temperature. The conjugate was buffer exchanged / purified in PBS7.4 by 4 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 5 mg / mL in PBS7.4 for 48 hours at 37 °C to ensure complete hydrolysis of the succinimidyl sites. The final protein concentration was assessed spectrophotometrically at 280 nm using a Colibri microvolume spectrometer device (Titertek Berthold).

[0358] 11.2) Characterization of the Complex The resulting complex was characterized as follows: Reversed-phase liquid chromatography-mass spectrometry (RPLC-MS): Modified RPLC-QToF analysis was performed using the UHPLC method described above. Briefly, the conjugates were eluted on an Agilent PLRP-S 1000 Å 2.1 × 150 mm 8 μm (80 °C) using a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) and analyzed in the 500–3500 m / z range (ESI + ) was used for mass detection on a Bruker Impact II™ Q-ToF mass spectrometer. Data were deconvoluted using the MaxEnt algorithm included in the Bruker Compass® software.

[0359] Size Exclusion Chromatography (SEC): SEC was performed on an Agilent 1050 HPLC system (equipped with a short section of 0.12 mm ID peek tubing and a microvolume UV flow cell) with an extra-column volume of less than 15 μL. The columns were Waters Acquity UPLC® Protein BEH SEC 200 Å 4.6×150 mm 1.7 μm (kept at room temperature) or Agilent AdvanceBio SEC 300 Å 4.6×150 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 prevent bacterial growth. The flow rate was 0.35 mL / min. UV detection was monitored at 280 nm.

[0360] Hydrophobic Interaction Chromatography (HIC): Hydrophobic interaction chromatography (HIC) was performed on an Agilent 1050 HPLC system. The column was a Tosoh TSK-GEL BUTYL-NPR 4.6 × 35 mm 2.5 μm (25 °C). Mobile phase A was 1.5 M (NH 4 ) 2 SO 4 Mobile phase A was 25 mM potassium phosphate pH 7.0 + 25% isopropanol (v / v). The linear gradient was 0% B to 100% B in 10 min, followed by a 3 min hold at 100% B. The flow rate was 0.75 mL / min. UV detection was monitored at 220 and 280 nm.

[0361] 11.3) Overview of Complex Characterization The conjugates showed one LC-1d (light chain with one drug-linker attached) and one HC-3d (heavy chain with three drug-linkers attached) absorbance peaks on their denaturing RPLC chromatograms (DAR8 conjugate). For mass spectrometry of the heavy chain, the major glycoform was reported (G0F for trastuzumab). The conjugates showed a single absorbance peak on their HIC chromatograms.

[0362] Trastuzumab-Glu(Glucuronide MMAE)-CH 2 -CH 2 -PSAR6(DAR8): Deconvoluted LC-1d Calc:25416;Obs:25417 / Deconvoluted HC-3d Calc:56529;Obs:56528 Monomer purity: 97.2% HIC retention time: 8.8 minutes Trastuzumab-Glu(Glucuronide MMAE)-CH 2 -CH 2 -PSAR12(DAR8): Deconvoluted LC-1d Calc:25844;Obs:25844 / Deconvoluted HC-3d Calc:57805;Obs:57805 Monomer purity: 99.0% HIC retention time: 8.8 minutes Trastuzumab-Glu(Glucuronide MMAE)-CH 2 -CH 2 -PSAR18(DAR8): Deconvoluted LC-1d Calc:26270;Obs:26270 / Deconvoluted HC-3d Calc:59086;Obs:59086 Monomer purity: 96.5% HIC retention time: 8.8 minutes Trastuzumab-Lys(glucuronide MMAE)-PSAR6(DAR8): Deconvoluted LC-1d Calc:25360;Obs:25360 / Deconvoluted HC-3d Calc:56352;Obs:56353 Monomer Purity: 99+% HIC retention time: 7.6 minutes Trastuzumab-Lys(glucuronide MMAE)-PSAR12(DAR8): Deconvoluted LC-1d Calc:25786;Obs:25786 / Deconvoluted HC-3d Calc:57634;Obs:57632 Monomer Purity: 99+% HIC retention time: 7.5 minutes Trastuzumab-BAC-Ngly (triazole-glucuronide MMAE)-PSAR12 (DAR8) Deconvoluted LC-1d Calc:25661;Obs:25662 / Deconvoluted HC-3d Calc:57264;Obs:57262 Monomer Purity: 99+% HIC retention time: 7.0 min (DAR8 conjugate). This ADC is a heterogeneous mixture containing -20% DAR6; -20% DAR7, and -60% DAR8 conjugates as observed in the HIC chromatogram.

[0363] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR6(DAR8)(=ADC-PSAR6) Deconvoluted LC-1d Calc:25368;Obs:25368 / Deconvoluted HC-3d Calc:56382;Obs:56380 Monomer Purity: 99+% HIC retention time: 7.5 minutes Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR12 (DAR8) (=ADC-PSAR12) Deconvoluted LC-1d Calc:25794;Obs:25794 / Deconvoluted HC-3d Calc:57660;Obs:57660 Monomer Purity: 99+% HIC retention time: 7.1 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSAR12(DAR8) Deconvoluted LC-1d Calc:25871;Obs:25870 / Deconvoluted HC-3d Calc:57889;Obs:57888 Monomer Purity: 99+% HIC retention time: 9.2 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR18 (DAR8) (=ADC-PSAR18) Deconvoluted LC-1d Calc:26221;Obs:26221 / Deconvoluted HC-3d Calc:58939;Obs:58939 Monomer Purity: 99+% HIC retention time: 6.8 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-PSAR18(DAR8) Deconvoluted LC-1d Calc:25567;Obs:25567 / Deconvoluted HC-3d Calc:56979;Obs:56977 Monomer Purity: 99+% HIC retention time: 5.1 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide-exatecan)-PSAR18 (DAR8) Deconvoluted LC-1d Calc:25938;Obs:25937 / Deconvoluted HC-3d Calc:58092;Obs:58089 Monomer Purity: 99+% HIC retention time: 5.7 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12(DAR8) Deconvoluted LC-1d Calc:25446;Obs:25446 / Deconvoluted HC-3d Calc:56614;Obs:56612 Monomer Purity: 99+% HIC retention time: 5.2 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18(DAR8) Deconvoluted LC-1d Calc:25872;Obs:25872 / Deconvoluted HC-3d Calc:57894;Obs:57892 Monomer Purity: 99+% HIC retention time: 5.1 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24(DAR8)(=ADC-PSAR24) Deconvoluted LC-1d Calc:26647;Obs:26674 / Deconvoluted HC-3d Calc:60218;Obs:60218 Monomer Purity: 99+% HIC retention time: 6.7 minutes CD19-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR24 (DAR8) Deconvoluted LC-1d Calc:27347;Obs:27347 / Deconvoluted HC-3d Calc:60137;Obs:60132 Monomer purity: 92.6% HIC retention time: 6.8 minutes CD22-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR24 (DAR8) Deconvoluted LC-1d Calc:27341;Obs:27341 / Deconvoluted HC-3d Calc:60314;Obs:60311 Monomer purity: 97.4% HIC retention time: 6.8 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)Ngly(triazole-glucuronide MMAE)-PSAR18(DAR16) Deconvoluted LC-1d Calc:27544;Obs:27545 / Deconvoluted HC-3d Calc:62910;Obs:62907 Monomer purity: 98.5% HIC retention time: 8.7 minutes Trastuzumab-MAL-phenyl-Ngly [triazole glucuronide (MMAE) 2 ]-PSAR24(DAR16) Deconvoluted LC-1d Calc:27569;Obs:27570 / Deconvoluted HC-3d Calc:62985;Obs:62983 Monomer purity: 98.2% HIC retention time: 9.9 minutes Trastuzumab-MAL-phenyl-Ngly [triazole-galactoside (MMAE) 2 ]-PSAR24(DAR16) Deconvoluted LC-1d Calc:27555;Obs:27554 / Deconvoluted HC-3d Calc:62943;Obs:62940 Monomer Purity: 99+% HIC retention time: 10.2 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18(DAR16) Deconvoluted LC-1d Calc:26237;Obs:26237 / Deconvoluted HC-3d Calc:58989;Obs:58987 Monomer Purity: 99+% HIC retention time: 6.6 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSAR18(DAR16) Deconvoluted LC-1d Calc:27270;Obs:27270 / Deconvoluted HC-3d Calc:62086;Obs:62087 Monomer Purity: 99+% HIC retention time: 5.7 minutes Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 (DAR8) (=ADC-PEG12) Deconvoluted LC-1d Calc:25541;Obs:25541 / Deconvoluted HC-3d Calc:56901;Obs:56900 Monomer Purity: 99+% HIC retention time: 7.4 minutes Trastuzumab-glucuronide MMAE (DAR8): Deconvoluted LC-1d Calc:24721;Obs:24720 / Deconvoluted HC-3d Calc:54439;Obs:54438 Monomer purity: 95.2% HIC retention time: 9.2 minutes Trastuzumab-MAL-phenyl-triazole-glucuronide MMAE (DAR8) (=ADC-PSAR0): Deconvoluted LC-1d Calc:24884;Obs:24884 / Deconvoluted HC-3d Calc:54926;Obs:54926 Monomer purity: 98.5% HIC retention time: 8.4 minutes Trastuzumab-MAL-phenyl-PSAR12-triazole-glucuronide MMAE (DAR8) (=ADC-PSAR12L): Deconvoluted LC-1d Calc:25793;Obs:25793 / Deconvoluted HC-3d Calc:57657;Obs:57657 Monomer Purity: 99+% HIC retention time: 8.5 minutes Human Albumin-MAL-Phenyl-Ngly(Triazole-Glucuronide MMAE)-PSAR24(DAR1): Deconvoluted Calc:69765;Obs:69645 Monomer purity: 90.8% HIC retention time: 3.9 minutes Trastuzumab: Deconvoluted LC Obs: 23439 / Deconvoluted HC Obs: 50595 Monomer Purity: 99+% HIC retention time: 4.7 minutes Anti-CD19 antibody: Deconvoluted LC Obs: 24139 / Deconvoluted HC Obs: 50517 Monomer purity: 93.2% HIC retention time: 4.7 minutes Anti-CD22 antibody: Deconvoluted LC Obs: 24133 / Deconvoluted HC Obs: 50692 Monomer Purity: 99+% HIC retention time: 4.8 minutes Human Albumin: Deconvoluted Obs: 66556 Monomer purity: 92.4% HIC retention time: 2.5 minutes.

[0364] Example 12: Hydrophobic interaction chromatography (HIC) characteristics of non-polysarcosine-based antibody-drug-conjugates (ADC-PSAR0), polysarcosine-based antibody-drug-conjugates with orthogonal configuration (ADC-PSAR12), and polysarcosine-based antibody-drug-conjugates with linear configuration (ADC-PSAR12L) The relative exposure of the conjugated payload to bulk solvent and the apparent hydrophobicity of the trastuzumab-based DAR8 ADC were assessed by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR column, as described in Example 11. The results are shown in Figure 1. When polysarcosine is grafted in a parallel (i.e., orthogonal) orientation relative to the drug unit, it provides efficient hydrophobic masking properties and reduces the apparent hydrophobicity of the conjugate (ADC-PSAR12). However, when polysarcosine is in a linear (i.e., serial) configuration, no reduction in the apparent hydrophobicity of the conjugate is observed (ADC-PSAR12L).

[0365] Example 13: Hydrophobic Interaction Chromatography (HIC) Characteristics of Polysarcosine and Polyethylene Glycol-Based Antibody-Drug-Conjugates The relative exposure of the conjugated payload to bulk solvent and the apparent hydrophobicity of the trastuzumab-based DAR8 ADC was assessed by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR column according to the method described in Example 11. The results are shown in Figure 2. At equal length (n=12 monomer units), polysarcosine confers better hydrophobic masking properties than polyethylene glycol (lower retention time).

[0366] Example 14: Pharmacokinetic properties (total antibody concentration over time) in mice after a single intravenous administration 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) Male SCID mice (4-6 weeks old) were injected with ADC at 3 mg / kg via the tail vein (five per treatment group, randomly assigned). Blood was collected at various time points via retro-orbital bleeding into citrate tubes and processed to plasma. Total ADC concentrations were assessed using a human IgG ELISA kit (Stemcell™ 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 with PK functionality (an add-in developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA). The results are shown in Figure 3. ADCs containing polysarcosine showed a significant increase in the clearance of polysarcosine compared to the control. The ADCs exhibit favorable pharmacokinetics when compared to those that do not contain

[0367] Example 15: Tumor volume (mm) in a BT-474 breast cancer xenograft model following a single intravenous dose of 3 mg / kg of a non-polysarcosine-based ADC (ADC-PSAR0) and a polysarcosine-based ADC (ADC-PSAR12) 3 ) and survival curves BT-474 breast cancer cells were implanted subcutaneously into female SCID mice (4 weeks old). The ADC from Example 14 above was used to treat tumors growing to approximately 150 mm 3 When the tumors had grown to 100 mg / kg, they were administered a single dose of 3 mg / kg intravenously (day 20, 5 animals per group, assigned to minimize differences in initial tumor volume between groups). The results are shown in Figures 4A and 4B. Tumor volumes were measured every 3 to 5 days using a caliper device and calculated using the formula (L × W 2 ) / 2. 3 Mice were sacrificed when the ADCs containing polysarcosine showed improved in vivo activity when compared to ADCs without polysarcosine. No significant weight change was observed in treated mice.

[0368] Example 16: Pharmacokinetic properties in mice (total antibody concentration over time) following a single intravenous administration of a 3 mg / kg dose of polysarcosine-based antibody-drug-conjugate (ADC-PSAR12) and poly(ethylene glycol)-based antibody-drug-conjugate (ADC-PEG12) The experiment was performed in male CD-1 mice (4-6 weeks old) according to the procedure described in Example 14. The results are shown in Figure 5. ADCs containing polysarcosine have improved pharmacokinetic parameters when compared to ADCs containing poly(ethylene glycol).

[0369] Example 17: Tumor volume (mm) in a BT-474 breast cancer xenograft model following a single intravenous administration of 2.5 mg / kg polysarcosine-based antibody-drug conjugate with orthogonal orientation and different PSAR lengths (ADC-PSAR6, ADC-PSAR12, ADC-PSAR18, ADC-PSAR24); an orthogonal poly(ethylene glycol)-based antibody-drug conjugate (ADC-PEG12) and a linear polysarcosine-based antibody-drug conjugate (ADC-PSAR12L). 3 ) The experiment was carried out as described in Example 15. BT-474 breast cancer cells were implanted subcutaneously in female SCID mice (4 weeks old). Tumors grew to approximately 150 mm 3When the tumors had grown to 100% tumor volume, ADC was administered intravenously once at a dose of 2.5 mg / kg (day 13, 6 animals per group, assigned to minimize differences in initial tumor volume between groups). The results are shown in Figure 6. No significant weight changes were observed in treated mice. [Brief description of the drawings]

[0370] [Figure 1] FIG. 1 represents a hydrophobic interaction chromatogram according to Example 12. [Diagram 2] FIG. 2 shows the hydrophobic interaction chromatogram according to Example 13. [Diagram 3] FIG. 3 shows the pharmacokinetic profile in mice according to Example 14. [Figure 4A] FIG. 4A depicts tumor volume as a function of time according to Example 15. [Figure 4B] FIG. 4B shows the survival rate of mice according to Example 15. [Diagram 5] FIG. 5 shows the pharmacokinetic profile in mice according to Example 16. [Figure 6] FIG. 6 shows the tumor volume as a function of time according to Example 17.

Claims

1. A ligand-drug-conjugate compound (LDC) having the formula (XV): 【Chemistry 1】 In the formula, L is (HP ＳＭＷ ) is oriented orthogonally relative to (X-D), L is selected from natural or unnatural amino acids, amino alcohols, amino aldehydes, polyamines, and combinations thereof; HP ＳＭＷ results from the covalent attachment of a polysarcosine homopolymer to said orthogonal connector L, HP ＳＭＷ represents the following: 【Chemistry 2】 where the wavy bond, if present, represents the point of attachment to L or the spacer Z; k is 6 or more; R ４ is -R', -O － , -OR', -SR', -S － , -NR' ２ , -NR' ３ ＋ , =NR', -CX ３ , -CN, -NRC(=O)R', -C(=O)R', -C(=O)NR' ２ , -SO ３ － , -SO ３ H, -S(=O) ２ R', -OS(=O) ２ OR', -S(=O) ２ NR', -S(=O)R', -OP(=O)(OR') ２ , -P(=O)(OR') ２ , -PO ３ － , -PO ３ H ２ , -C(=O)X, -C(=S)R', -CO ２ R', -CO ２ , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' ２ , C(=S)NR' ２ or C(=NR')NR' ２ represents a capping group selected from wherein each X is independently a halogen: -F, -CI, -Br, or -I; Each R' is independently -H, -C １ -C ２０ Alkyl, -C ６ -C ２０ Aryl, or -C ３ -C １４ heterocyclic ring); D is a cytotoxic drug; X is a cleavable moiety to release D and is capable of forming a cleavable bond to release D; Z is a spacer; a is 1 or greater, b is 1 or greater, and m is 1 or greater; and The ligand is selected from a protein, a polypeptide, a peptide and an aptamer.

2. 2. The LDC compound of claim 1, wherein L is one or more natural or unnatural amino acids.

3. 3. The LDC compound of claim 1, wherein L is selected from glutamic acid, lysine and glycine.

4. X is one or more natural or unnatural amino acids, - a sugar moiety linked to a self-immolative group via an oxygen glycosidic bond, - a disulfide linker, and - Acid-labile linker that is hydrolyzable in the lysosome 4. The LDC compound according to claim 1 , wherein the LDC compound is selected from the group consisting of

5. Z is an alkylene, heteroalkylene; an alkoxy; a polyether; one or more natural or unnatural amino acids; ３ -C ８ Heterocyclo; C ３ -C ８ 5. The LDC compound of claim 1, which is selected from the group consisting of carbocyclo; arylene; and any combination thereof.

6. The LDC compound according to any one of claims 1 to 5, wherein Z is formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII): 【Chemistry 3】 where the wavy bond represents the point of attachment, and R ６ is -C １ -C １０ Alkylene-, -C １ -C １０ Heteroalkylene-, -C １ -C １０ Alkylene-C(=O)-, -C １ -C １０ Heteroalkylene-C(=O)-, -arylene-C １ -C １０ Alkylene-C(=O)-, -arylene-C １ -C １０ alkylene -O-C(=O)-, R ６ Any of the groups are optionally substituted with one or more =O.

7. An intermediate compound having the formula (XVI): 【Chemistry 4】 where L is an orthogonal connector; L is selected from amino alcohols, amino aldehydes, polyamines, and combinations thereof; HP ＳＭＷ results from the covalent attachment of a polysarcosine homopolymer to said orthogonal connector L, The above website ＳＭＷ represents the following: 【Chemistry 5】 where the wavy bond, if present, represents the point of attachment to L or the spacer Z; k is 6 or more; R ４ is -R', -O － , -OR', -SR', -S － , -NR' ２ , -NR' ３ ＋ , =NR', -CX ３ , -CN, -NRC(=O)R', -C(=O)R', -C(=O)NR' ２ , -SO ３ － , -SO ３ H, -S(=O) ２ R', -OS(=O) ２ OR', -S(=O) ２ NR', -S(=O)R', -OP(=O)(OR') ２ , -P(=O)(OR') ２ , -PO ３ － , -PO ３ H ２ , -C(=O)X, -C(=S)R', -CO ２ R', -CO ２ , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' ２ , C(=S)NR' ２ or C(=NR')NR' ２ represents a capping group selected from wherein each X is independently a halogen: -F, -CI, -Br, or -I; Each R' is independently -H, -C １ -C ２０ Alkyl, -C ６ -C ２０ Aryl, or -C ３ -C １４ heterocyclic ring); D is a cytotoxic drug; X is a cleavable moiety to release D and is capable of forming a cleavable bond to release D; Z is a spacer; and a is 1 or more and b is 1 or more.

8. 7. The LDC according to any one of claims 1 to 6 for use as a medicament.

9. Compounds having formula (XXIII): 【Chemistry 6】 In the formula, R ６ is -C １ -C １０ Alkylene-, -C １ -C １０ Heteroalkylene-, -C ３ -C ８ Carbocyclo-, -O-(C １ -C ８ Alkyl)-, -arylene-, -C １ -C １０ Alkylene-arylene-, -arylene-C １ -C １０ Alkylene-, -C １ -C １０ Alkylene-(C ３ -C ８ Carbocyclo)-, -(C ３ -C ８ Carbocyclo)-C １ -C １０ Alkylene-, -C ３ -C ８ Heterocyclo-, -C １ -C １０ Alkylene-(C ３ -C ８ Heterocyclo)-, -(C ３ -C ８ Heterocyclo)-C １ -C １０ Alkylene-, -C １ -C １０ Alkylene-C(=O)-, -C １ -C １０ Heteroalkylene -C(=O)-, -C ３ -C ８ Carbocyclo-C(=O)-, -O-(C １ -C ８ Alkyl)-C(=O)-, -arylene-C(=O)-, -C １ -C １０ Alkylene-arylene-C(=O)-, -arylene-C １ -C １０ Alkylene-C(=O)-, -C １ -C １０ Alkylene-(C ３ -C ８ Carbocyclo)-C(=O)-, -(C ３ -C ８ Carbosiloxane)-C １ -C １０ Alkylene-C(=O)-, -C ３ -C ８ Heterocyclo-C(=O)-, -C １ -C １０ Alkylene-(C ３ -C ８ Heterocyclo)-C(=O)-, -(C ３ -C ８ Heterocyclo)-C １ -C １０ Alkylene-C(=O)-, -C １ -C １０ Alkylene -NH-, -C １ -C １０ Heteroalkylene -NH-, -C ３ -C ８ Carbocyclo-NH-, -O-(C １ -C ８ alkyl)-NH-, -arylene-NH-, -C １ -C １０ Alkylene-arylene-NH-, -arylene-C １ -C １０ Alkylene -NH-, -C １ -C １０ Alkylene-(C ３ -C ８ Carbocyclo)-NH-, -(C ３ -C ８ Carbocyclo)-C １ -C １０ Alkylene -NH-, -C ３ -C ８ Heterocyclo-NH-, -C １ -C １０ Alkylene-(C ３ -C ８ Heterocyclo)-NH-, -(C ３ -C ８ Heterocyclo)-C １ -C １０ Alkylene -NH-, -C １ -C １０ Alkylene-S-, -C １ -C １０ Heteroalkylene -S-, -C ３ -C ８ Carbocyclo-S-, -O-(C １ -C ８ Alkyl)-S-, -arylene-S-, -C １ -C １０ Alkylene-arylene-S-, -arylene-C １ -C １０ Alkylene-S-, -C １ -C １０ Alkylene-(C ３ -C ８ Carbocyclo)-S-, -(C ３ -C ８ Carbocyclo)-C １ -C １０ Alkylene-S-, -C ３ -C ８ Heterocyclo-S-, -C １ -C １０ Alkylene-(C ３ -C ８ Heterocyclo)-S-, -(C ３ -C ８ Heterocyclo)-C １ -C １０ Alkylene-S-, -C １ -C １０ Alkylene -O-C(=O)-, -C ３ -C ８ Carbocyclo-O-C(=O)-, -O-(C １ -C ８ Alkyl)-O-C(=O)-, -arylene-O-C(=O)-, -C １ -C １０ Alkylene-arylene-O-C(=O)-, -arylene-C １ -C １０ Alkylene -O-C(=O)-, -C １ -C １０ Alkylene-(C ３ -C ８ Carbocyclo)-O-C(=O)-, -(C ３ -C ８ Carbocyclo)-C １ -C １０ Alkylene -O-C(=O)-, -C ３ -C ８ Heterocyclo-O-C(=O)-, -C １ -C １０ Alkylene-(C ３ -C ８ Heterocyclo)-O-C(=O)-, -(C ３ -C ８ Heterocyclo)-C １ -C １０ alkylene -O-C(=O)-; R ６ Any of the groups may be -X, -R', -O － , -OR', =O, -SR', -S － , -NR' ２ , -NR' ３ ＋ , =NR', -CX ３ , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO ２ , =N ２ , -N ３ , -NR'C(=O)R', -C(=O)R', -C(=O)NR' ２ , -SO ３ － , -SO ３ H, -S(=O) ２ R', -OS(=O) ２ OR', -S(=O) ２ NR', -S(=O)R', -OP(=O)(OR') ２ , -P(=O)(OR') ２ , -PO ３ － , -PO ３ H ２ , -C(=O)X, -C(=S)R', -CO ２ R', -CO ２ , -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, —C １ -C ２０ Alkyl, -C ６ -C ２０ Aryl, or -C ３ -C １４ heterocyclic ring); Z is a spacer; L is an orthogonal connector; L is selected from amino alcohols, amino aldehydes, polyamines, and combinations thereof; X is a cleavable moiety to release D and is capable of forming a cleavable bond to release D; D is a cytotoxic drug; a is 1 or greater and b is 1 or greater; and HP ＳＭＷ results from the covalent attachment of a polysarcosine homopolymer to said orthogonal connector L, The above website ＳＭＷ represents the following: 【Chemistry 7】 where the wavy bond, if present, represents the point of attachment to L or the spacer Z; k is 6 or more; R ４ is -R', -O － , -OR', -SR', -S － , -NR' ２ , -NR' ３ ＋ , =NR', -CX ３ , -CN, -NRC(=O)R', -C(=O)R', -C(=O)NR' ２ , -SO ３ － , -SO ３ H, -S(=O) ２ R', -OS(=O) ２ OR', -S(=O) ２ NR', -S(=O)R', -OP(=O)(OR') ２ , -P(=O)(OR') ２ , -PO ３ － , -PO ３ H ２ , -C(=O)X, -C(=S)R', -CO ２ R', -CO ２ , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' ２ , C(=S)NR' ２ or C(=NR')NR' ２ represents a capping group selected from wherein each X is independently a halogen: -F, -CI, -Br, or -I; Each R' is independently -H, -C １ -C ２０ Alkyl, -C ６ -C ２０ Aryl, or -C ３ -C １４ Heterocyclic ring)

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