Ligand-drug-conjugates comprising single molecular weight poly(sarcosine)

Abstract

The present invention relates to ligand-drug-conjugates (LDCs) comprising single molecular weight homopolymers, in particular single molecular weight poly(sarcosine).

Description

Field of the Invention

[0001] The present invention relates to single molecular weight homopolymers, methods for preparing such homopolymers and their use, in particular in conjugation techniques.

[0002] The present invention also relates to ligand-drug-conjugates (LDCs) comprising single molecular weight homopolymers, in particular single molecular weight poly(sarcosine). Background Art

[0003] A ligand-drug-conjugate (LDC) consists of at least one ligand unit which is a polypeptide or protein covalently linked via a synthetic linker to at least one therapeutic, diagnostic or labeling molecule (hereinafter referred to as drug or D). The synthetic linker may comprise one or several divalent arms for connecting the ligand unit and the drug unit, which may be selected from spacers, linkers and cleavable moieties. The linker may also carry any monovalent moiety which may improve the properties of the LDC, such as storage stability, plasma stability or pharmacokinetic properties. The protein or polypeptide is usually the targeting unit, but 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-conjugates (ADCs) is commonly used.

[0004] The design of ADCs involves the consideration of numerous different factors: (i) the nature, number, overall hydrophobicity and position of the synthetic linker for conjugation on the ligand; (ii) the nature and mechanism of action of the drug; (iii) the structural elements responsible for drug release after 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 drawbacks of available ADCs, such as heterogeneous drug loading (subspecies of ADCs with different pharmacological properties), limited mAb-linker or drug-linker stability and suboptimal pharmacokinetic properties (Beck et al., Nat. Rev. Drug Discov., 2017, 16(5), 315-337).

[0005] Another important factor to consider when designing conjugates is the drug ratio (or drug-to-antibody ratio (DAR) for ADCs), which is the average number of drug units conjugated to an antibody. The latest findings show that the actual trend in the ADC field is to generate homogeneous conjugates with low to moderate DAR (usually 2-4) and introduce them into the clinic. However, new linker-drug technologies have recently emerged, aiming to overcome the drawbacks of high-loaded ADCs (unfavorable pharmacokinetic properties and the tendency to form aggregates, which complicates the preparation of conjugates). Such technologies have the potential to introduce next-generation ADCs with improved efficacy, improved pharmacokinetic properties, improved therapeutic indices, and the ability to target tumors with low target expression, slow internalization, or ineffective intracellular processing. To achieve such a high payload loading without sacrificing pharmacokinetic properties and formulation stability, new linker-drug design methods aimed at masking the apparent hydrophobicity of cytotoxic payloads need to be developed.

[0006] In WO2014 / 093394A1, protein-polymer-drug conjugates showing high drug loading and strong binding to the target antigen were reported. The conjugate involves a biodegradable and biocompatible poly[1-hydroxymethylvinyl formal] polymer entity that allows the conjugation of approximately 12-25 cytotoxic molecules per mAb and has good pharmacokinetic properties. The main drawback of this approach is the extreme polydispersity of the final conjugate, which is due to (i) the polydispersity characteristics of the linker, (ii) the heterogeneous number of cytotoxic molecules per polymer arm, and (iii) the heterogeneous number of polymer arms grafted per mAb.

[0007] In WO2015 / 057699A2 and WO2016 / 059377A1, ADCs loaded with 8-36 drugs were formulated by including orthogonal polyethylene glycol (PEG) moieties in the linker design. It is well known that PEG can improve the hydrophilicity, stability, and circulation time of small drugs, proteins, bioconjugates, and nanoparticles due to its hydrophilicity, biocompatibility, and high hydration shell. However, due to the anti-PEG antibodies expressed by some healthy individuals, PEG cannot avoid drawbacks such as non-biodegradability, possible complement activation leading to hypersensitivity, and unclear pharmacokinetics.

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

[0009] Poly(sarcosine) (poly-N-methylglycine or PSAR) can replace PEG and can be used to design novel protein conjugates with improved properties. PSAR is a highly hydrophilic, biodegradable, non-immunogenic and water-soluble polymer that has been used in several delivery systems for drugs or diagnostics. To date, PSAR has only been available in polydisperse form as it is obtained via the condensation ring-opening polymerization of sarcosine N-carboxyanhydride (NCA) or sarcosine N-thiocarboxyanhydride (NTA). Although the acceptable dispersity is relatively well-defined (a Gaussian molecular weight distribution with a polydispersity index > 1), these polydisperse PSARs cannot be used in some application areas that require the use of shorter homopolymer compounds with a consistent length (unique and specific molecular weight) and thus absolute homogeneity.

[0010] The use of discrete monodisperse PSAR for macromolecular modification is a requirement for the development of conjugates with absolute chemical homogeneity. Such homogeneous conjugates have the advantages of sharing exactly the same pharmacological properties (pharmacokinetics and pharmacodynamics), more straightforward characterization, allowing better control of the reproducibility of the preparation process, and meeting the increasingly stringent regulatory requirements for bioconjugates. Summary of the Invention

[0011] According to the present invention, discrete monodisperse PSAR homopolymers with a defined chain length have been obtained using a stepwise on-resin submonomer method. This method is inexpensive, easy to scale up, and can give an acceptable yield and excellent monomer purity to the final product. These monodisperse PSAR homopolymers are used in protein conjugate technology, thereby providing improved drug-loading capacity, pharmacokinetics, and therapeutic efficacy for ligand-drug-conjugates (LDCs).

[0012] Therefore, the present invention provides single-molecular-weight monofunctional homopolymers that meet the above requirements for use in conjugate technology, particularly in LDCs.

[0013] Such a homopolymer has the following formula (I)

[0014]

[0015] wherein

[0016] R1 and R2 are different, and

[0017] one of R1 and R2 is H or an inert group, and the other of R1 and R2 is a functionalized reactive group that is reactive towards covalently binding a bindable group under reaction conditions where the inert group is non-reactive,

[0018] Z1 and Z2, which may be the same or different, are optional spacers, and

[0019] n is 1 or greater and k is 2 or greater.

[0020] Before disclosing the present invention in detail, definitions of terms used herein are given below.

[0021] Definitions

[0022] According to the present invention, any compound (such as reactants, products, monomers, homopolymers, units) can be in the form of a salt, including acid addition salts, base addition salts, metal salts, and ammonium and alkylated ammonium salts. Such salts are well known to those skilled in the art. Considering the intended use of the homopolymers of the present invention, they are preferably in the form of pharmaceutically acceptable salts.

[0023] Single molecular weight homopolymer Refers to a homopolymer having a unique and specific molecular weight concentrated around the average molecular weight, as opposed to a mixture of homopolymers of the same nature but having a size and molecular weight distribution. The single molecular weight homopolymer can be defined by an absolute molecular formula having an absolute number of atoms.

[0024] As opposed to polydisperse homopolymers obtained conventionally by one-pot polymerization methods and having a PDI > 1, this single molecular weight homopolymer can also be referred to as "monodisperse" with a polydispersity index (PDI) equal to 1. In the present specification, it is generally recognized that the terms "monodisperse" and "discrete" are interchangeable, both defining a homopolymer having a unique and absolute molecular weight, molecular formula, and molecular structure, although the term "monodisperse" does not accurately reflect the preparation process of the product.

[0025] Inert group Or End-capping group Refers to any chemically non-reactive group that terminates one end of a homopolymer and is non-reactive compared to the functionalized reactive group that terminates the other end of the homopolymer under defined reaction conditions. The resulting homopolymer is capped by this inert group in a certain way and is not intended to be covalently linked during use, especially in LDC technology. In one embodiment, the group becomes inert only after it is covalently bound to one end of the homopolymer.

[0026] A non-exhaustive list of inert groups includes: acyl groups, especially acetyl groups, amide groups, alkyl groups, especially C 1-20 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 group can also be selected from the same list of groups that define the functionalized reactive groups (see the definition of functionalized reactive groups below).

[0027] Functionalized reactive groupRefers to any chemical moiety that is reactive towards covalently bindable groups and is reactive under defined reaction conditions as compared to inert groups. In particular, it can bind to the following groups: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl group; halogen; activated esters such as N-hydroxysuccinimide ester, perfluorinated ester, nitrobenzene ester, azido-benzotriazole and benzotriazole activated esters, acylurea; alkynyl group; alkenyl group; azide; isocyanate; isothiocyanate; aldehyde; thiol-reactive moieties such as maleimide, halo-maleimide, haloacetyl, pyridyl disulfide; thiol; acrylate; mesylate; tosylate; triflate; hydroxylamine; chlorosulfonyl group; boronic acid -B(OR’)2 derivatives, where R’ is hydrogen or alkyl.

[0028] A non-exhaustive list of functionalized reactive groups includes: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl group; halogen; activated esters such as N-hydroxysuccinimide ester, perfluorinated ester, nitrobenzene ester, azido-benzotriazole and benzotriazole activated esters, acylurea; alkynyl group; alkenyl group; azide; isocyanate; isothiocyanate; aldehyde; thiol-reactive moieties such as maleimide, halo-maleimide, haloacetyl, pyridyl disulfide; thiol; acrylate; mesylate; tosylate; triflate; hydroxylamine; chlorosulfonyl group; boronic acid -B(OR’)2 derivatives, where R’ is hydrogen or alkyl.

[0029] It should be noted that the terms “inert” and “functionalized reactive” for inert groups and functionalized reactive groups respectively are interdependent. This means that under the defined reaction conditions of the homopolymers of the present invention defined by any one of formulas (I), (II) and (III), the inert groups will not react while the functionalized reactive groups will react to covalently bind reactants. Thus, the inert groups and functionalized reactive groups in the homopolymers of any one of formulas (I), (II) and (III) are different, but they can be selected from the same list of groups as a whole.

[0030] The term “group” in the functionalized reactive group or inert group according to the present invention should be understood as a group that has no other function except being able to covalently bind reactants or remain inert respectively under defined reaction conditions.

[0031] For example, used alone or as part of an alkyl ether or alkyl ester Alkyl group Refers to a saturated straight-chain or branched-chain hydrocarbon group having 1 - 20 carbon atoms, preferably 1 - 12 carbon atoms, more preferably 1 - 6 carbon atoms, especially 1 - 4 carbon atoms.

[0032] Alkenyl and alkynyl groups Refers to an at least partially unsaturated straight-chain or branched-chain hydrocarbon group having 2 - 20 carbon atoms, preferably 2 - 12 carbon atoms, more preferably 2 - 6 carbon atoms, especially 2 - 4 carbon atoms.

[0033] For example, when used alone or as part of an aryl ester, Aryl group refers to an aromatic group having one or more rings containing 6-14, preferably 6-10, especially 6 ring carbon atoms.

[0034] For example, when used alone or as part of an alkylene glycol, Alkylene group refers to a divalent saturated straight-chain or branched hydrocarbon group having 1-20, preferably 1-12, more preferably 1-6, especially 1-4 carbon atoms.

[0035] Arylene group refers to a divalent aryl as defined above.

[0036] Heteroalkyl group refers to a straight-chain or branched hydrocarbon chain composed of 1-20 or 1-10 carbon atoms and 1-10, preferably 1-3 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 heteroatoms O, N, and S may be located at any internal position of the heteroalkyl or at the position where the alkyl is connected to the rest of the molecule.

[0037] Heteroalkylene group refers to a divalent heteroalkyl as defined above. For a heteroalkylene group, the heteroatom may also occupy one or both of the chain ends.

[0038] C3-C8 carbocyclic group refers to a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocyclic ring.

[0039] C3-C8 carbocyclic group refers to a divalent C3-C8 carbocyclic group as defined above.

[0040] C3-C8 heterocyclic group refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic system having 3-8 carbon atoms (also called ring members) and 1-4 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 the heteroatom may be aromatic or non-aromatic. Unless otherwise specified, the heterocycle is attached to its side group at any heteroatom or carbon atom where a stable structure is formed.

[0041] C3-C8 heterocyclic group refers to a divalent C3-C8 heterocyclic group as defined above.

[0042] In addition, the terms alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heteroalkyl, heteroalkylene, C3-C8 carbocyclic group, C3-C8 carbocycle, C3-C8 heterocyclic group, C3-C8 heterocycle refer to groups optionally substituted with one or more substituents selected from the following: -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2, -NR'3, =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)R', -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2 and C(=NR')NR'2, where each X is independently a halogen: -F, -Cl, -Br or -I; and each R' is independently -H, -C1C 20 alkyl, -C6-C 20 aryl or -C3-C 14 heterocyclic group.

[0043] Acyl group The group refers to -CO-alkyl, where the alkyl is as defined above.

[0044] Mono-functional homopolymer Include a single type of monomer (e.g., the N-methylglycine monomer for poly(sarcosine)), which has a functionalized reactive group as defined above at one end and H or an inert group as defined above at the other end.

[0045] Solid-phase peptide synthesis (SPPS) supportRefers to a support commonly used in SPPS, which is a well-known method where peptides anchored to the support (an insoluble polymer) are assembled via repeated deprotection-washing-coupling-washing cycles by successive addition of Fmoc- or Boc-protected amino acids. Each amino acid addition refers to the following cycle: (i) cleavage of the Nα-protecting group, (ii) washing step, (iii) coupling of a fluorenylmethoxycarbonyl- (Fmoc-) or tert-butoxycarbonyl- (Boc-) protected amino acid using a coupling agent and a non-nucleophilic base, (iv) washing step. Since the growing chain is attached to the support, excess reagents and soluble by-products can be removed by simple filtration. Because repeated coupling reactions with sterically hindered Fmoc- or Boc-protected N-methylated amino acids are difficult and usually suboptimal, low crude purity, difficult purification, and low yields are expected with this technique. Examples of such supports are Wang resin, Rink amide resin, trityl and 2-chlorotrityl resins, PAM resin, PAL resin, Sieber amide resin, MBHA resin, HMPB resin, HMBA resin, which are commercially available and to which peptides are bound either directly or indirectly.

[0046] The term " Orthogonal linker " refers to a linker unit assembly that attaches a ligand to a homopolymer unit and a drug unit such that the homopolymer unit is in a parallel configuration (as opposed to a tandem configuration) relative to the drug unit. An orthogonal linker is a scaffold that bears attachment sites for the components of a ligand-drug-conjugate (i.e., the ligand, homopolymer, and drug units). The term "parallel" is used to denote the branching of two components of a ligand-drug-conjugate (LDC), but not to imply that the two components must be very close in space or have the same distance between them.

[0047] An exemplary graphical representation of an LDC having a homopolymer (e.g., poly(sarcosine)) unit with a parallel (i.e., branched) orientation relative to the drug unit is as follows:

[0048]

[0049] where (L) is an orthogonal linker unit, w is 1 or greater, typically 1 - 5, preferably 1 - 4, more preferably 1 - 3, and even 1 and 2. This orthogonal structure should not be confused with a linear structure. An exemplary graphical representation of an LDC having a homopolymer (e.g., poly(sarcosine)) unit with a consecutive (i.e., linear) orientation relative to the drug unit is as follows:

[0050] Ligand—Homopolymer—Drug

[0051] Non-exhaustive lists of orthogonal linkers include: 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. Based on their knowledge, those skilled in the art can select an orthogonal linker suitable for the intended 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.

[0052] Spacer is a bivalent linear arm that covalently binds two components of a ligand-drug-conjugate, such as:

[0053] - a ligand unit and an orthogonal linker unit,

[0054] - an orthogonal linker unit and a homopolymer unit,

[0055] - an orthogonal linker and a cleavable moiety,

[0056] - a cleavable moiety and a drug, or

[0057] - an orthogonal linker and a drug.

[0058] For example, the spacer is a bivalent linear alkylene group, preferably (CH2)4.

[0059] Non-exhaustive lists of spacer units include: alkylene, heteroalkylene (i.e., alkylene interrupted by at least one heteroatom selected from Si, N, O, and S); alkoxy; polyethers such as polyalkylene glycols, typically polyethylene glycol; one or more natural or unnatural amino acids such as glycine, alanine, proline, valine, N-methylglycine; C3-C8 heterocyclic groups; C3-C8 carbocyclic groups; arylene and any combination thereof. When present between the cleavable moiety and the drug unit or between the orthogonal linker and the drug unit, the spacer can be linked to one or more drug units. For example, the spacer can be linked to 1-4 drug units, preferably 1-2 drug units. In one embodiment, the spacer between the cleavable moiety and the drug unit is (4-amino-1,3-phenylene)dimethanol.

[0060] In one embodiment, the spacer unit is of formula (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII),

[0061]

[0062] where the wavy bond represents the point of attachment and R6 is –C1-C 10 alkylene-, –C1-C 10Heteroalkylene-, -C3-C8 cycloalkyl-, -O-(C1-C8 alkyl)-, -arylene-, –C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, –C1-C 10 Alkylene-(C3-C8 cycloalkyl)-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-, -C3-C8 heterocyclic group-, –C1-C 10 Alkylene-(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)–C1-C 10 Alkylene-, –C1-C 10 Alkylene-C(=O)-, –C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 cycloalkyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 cycloalkyl)-C(=O)-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic group-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-C(=O)-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10杂 Alkylene-NH-, -C3-C8 cycloalkyl-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 cycloalkyl)-NH-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic group-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-NH-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 cycloalkyl-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-S-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic group-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-S-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-S-, –C1-C 10 Alkylene-O-C(=O)-, -C3-C8 carbocyclic group-O-C(=O)-, -O-(C1-C8 alkyl)-O-C(=O)-, -arylene-O-C(=O)-, -C1-C 10 Alkylene-arylene-O-C(=O)-, -arylene-C1-C 10 Alkylene-O-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-O-C(=O)-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-O-C(=O)-, -C3-C8 heterocyclic group-O-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-O-C(=O)-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-O-C(=O)-.

[0063] Any one of the R6 groups is optionally substituted by one or more substituents selected from the following: -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2, -NR'3 + , =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 -, -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2 and C(=NR')NR'2, where each X is independently a halogen: -F, -Cl, -Br or -I; each R' is independently -H, -C1C 20 alkyl, -C6-C 20 aryl or -C3-C 14 heterocyclic group.

[0064] Advantageously, the spacer unit is of formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII),

[0065]

[0066] where the wavy bond represents the point of attachment and R6 is –C1-C 10 alkylene-, –C1-C 10 heteroalkylene-, –C1-C 10 alkylene-C(=O)-, –C1-C 10 heteroalkylene-C(=O)-, -arylene-C1-C 10 alkylene-C(=O)-, -arylene-C1-C 10 alkylene-O-C(=O)-.

[0067] Any one of the R6 groups is optionally substituted by one or more =O.

[0068] Ligand refers to any macromolecule (polypeptide, protein, peptide, typically an antibody) commonly used in LDC (such as antibody-drug conjugate) technology, or a small molecule (such as folic acid or aptamer) that can be covalently conjugated to the synthetic linker or drug-linker of this work using bioconjugation techniques (Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). Ligands are traditionally compounds selected based on their targeting ability. A non-exhaustive list of ligands includes: proteins, polypeptides, peptides, antibodies, full-length antibodies and their antigen-binding fragments, interferons, lymphokines, hormones, growth factors, vitamins, transferrin or any other cell-binding molecule or substance. The main class of ligands used for preparing conjugates is antibodies. As used herein, the term "antibody" is used in the broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, modified monoclonal and polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), antibody fragments and antibody mimetics (Affibody , Affilin , Affimer , Nanofitin , CellPenetrating Alphabody , Anticalin , Avimer , Fynomer , Monobodies or nanoCLAMP ). An example of an antibody is trastuzumab. An example of a protein is human serum albumin.

[0069] As used herein, the term “antibody” includes intact antibodies and any antigen-binding fragment (i.e., “antigen-binding portion”) thereof or single chains thereof.

[0070] A naturally occurring “antibody” is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains that are inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. 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 ) and a light chain constant region. The light chain constant region is composed of one domain, C L . V H and V L regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs, which are arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0071] As used herein, the term “antigen-binding portion” (or simply “antigen portion”) of an antibody refers to the full-length antibody or one or more fragments thereof that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include Fab fragments, which are composed of V L , V H , C LA monovalent fragment consisting of the CH1 domain; an F(ab)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; an Fd fragment, which consists of V H and the CH1 domain; an Fv fragment, which consists of the V L and V H domains of a single arm of the antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of the V H domain; and an isolated complementarity determining region (CDR) or any fusion protein containing such an antigen-binding portion.

[0072] In addition, although the two domains V L and V H of the Fv fragment are encoded by different genes, they can be joined using recombinant methods by a synthetic linker so that they form a single-chain protein, in which the V L and V H regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, 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 obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0073] In certain embodiments, the ligand of the LDC is a chimeric antibody, a humanized antibody, or a human antibody.

[0074] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework regions and the CDR regions are derived from human sequences. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant forms of human germline sequences, or antibodies containing consensus framework sequences derived from an analysis of human framework sequences, for example, as described by Knappik et al. (2000. J Mol Biol 296, 57-86).

[0075] A human antibody may include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, as used herein, the term "human antibody" 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.

[0076] The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable regions in which both the framework regions and the CDR regions are derived from human sequences.

[0077] As used herein, "isotype" refers to the antibody class provided by the heavy chain constant region gene (e.g., IgM, IgE, IgG, such as IgG1 or IgG4).

[0078] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds an antigen".

[0079] Cleavable group (X), also referred to as the "releasable assembly unit", links the drug unit to the remainder of the ligand-drug-conjugate. The function of the cleavable group is to release the drug at the site targeted by the ligand. Thus, this unit is capable of forming a cleavable bond for the release of the drug unit, for example, upon enzymatic treatment or via a disulfide elimination mechanism. Recognition sites for enzymatic treatment are typically dipeptide cleavage sites (e.g., Val-Cit, Val-Ala or Phe-Lys) or sugar cleavage sites (e.g., glucuronide cleavage sites). For example, the cleavable group is a glucuronide group. This technology is well known to those skilled in the art, and based on their knowledge, they are able to select a cleavable group suitable for the drug of an LDC (e.g., ADC) compound. For example, cleavable groups include 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 selected from the following

[0080] - one or more natural or unnatural amino acids, such as a cleavable peptide containing 2-12 amino acids,

[0081] - a sugar moiety linked via an O-glycosidic bond to a self-eliminating group,

[0082] - a disulfide linker, and

[0083] - an acid-labile linker hydrolyzable in lysosomes.

[0084] Advantageously, the cleavable group can be selected from the following

[0085] - one or more natural or unnatural amino acids, such as a cleavable peptide containing 2-12 amino acids, and

[0086] - a sugar moiety linked via an O-glycosidic bond to a self-eliminating group,

[0087] When a sugar moiety is used, the self-eliminating group is considered to be part of the cleavable group. A "self-eliminating group" is a trifunctional chemical moiety capable of covalently linking three spaced chemical moieties, namely a sugar moiety (via a glycosidic bond), a drug D (directly or indirectly via a spacer Z), and an orthogonal linker L (directly or indirectly via a spacer Z). The glycosidic bond can be a glycosidic bond cleavable at the target site to initiate a self-eliminating reaction sequence leading to drug release.

[0088] 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 applicable to the present disclosure, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT (N-succinimidyl-oxycarbonyl)-α-methyl-α-(2-pyridyl-dithio)toluene), and SPP (N-succinimidyl 4-(2-pyridyl-dithio)valerate). See, for example, U.S. Patent No. 4,880,935.

[0089] In some embodiments, the cleavable unit is pH-sensitive and, for example, will comprise an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitate, orthoester, acetal, or ketal group) that is hydrolyzable in lysosomes that can be used. (See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929). Such linkers are relatively stable at neutral pH conditions (such as in blood), but are unstable at pH 5.5 or 5.0 (approximate pH of lysosomes).

[0090] Ligand-drug conjugate (LDC) Refers to any conjugate that binds a ligand and a drug as defined above and involves any of the means described above, and will be illustrated in the examples of the specification. When the ligand is an antibody, it can refer to an antibody-drug conjugate (ADC), which is a preferred embodiment of the present disclosure.

[0091] Bindable groupRefers to a group that can react with a functionalized reactive group to form a covalent bond. Thus, the bindable group contains a reactive group that reacts with the functionalized reactive group under defined reaction conditions. In particular, the bindable group can contain one of the following groups: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl group; halogen; activated ester, such as N-hydroxysuccinimide ester, perfluorinated ester, nitrobenzene ester, azido-benzotriazole and benzotriazole activated ester, acylurea; alkynyl group; alkenyl group; azide; isocyanate; isothiocyanate; aldehyde; thiol-reactive moiety, such as maleimide, halo-maleimide, haloacetyl, pyridyl disulfide; thiol; acrylate; mesylate; tosylate; triflate; hydroxylamine; chlorosulfonyl group; boronic acid -B(OR’)2 derivative, where R’ is hydrogen or alkyl.

[0092] Drug Refers to any type of drug or compound, such as a cytotoxic compound, a cell growth inhibitory compound, an immunosuppressive compound, an anti-inflammatory compound or an anti-infective compound. Among cytotoxic compounds, calicheamicin can be cited; uncialamycin; auristatins (such as monomethyl auristatin E called MMAE); tubulin analogs; maytansine; cryptophycin; benzodiazepine dimers (including pyrrolo[2,1-c][1,4]benzodiazepines called PBD's); indolobenzodiazepine pseudodimers (IGN); duocarmycin; anthracyclines (such as doxorubicin or PNU159682); camptothecin analogs (such as 7-ethyl-10-hydroxycamptothecin called SN38 or irinotecan); Bcl2 and Bcl-xl inhibitors; Thailanstatins; amatoxins (including α-amanitin); kinesin spindle protein (KSP) inhibitors; vinorelbine; cyclin-dependent kinase (CDK) inhibitors; bleomycin; actinomycin or radionuclides and their complexing agents (such as DOTA / 177 Lu). Among anti-inflammatory drugs, corticosteroids such as dexamethasone or fluticasone can be cited. Among anti-infective drugs, antibiotics such as rifampicin or vancomycin can be cited.

[0093] The present invention is now disclosed in more detail. Although it has been more specifically described with reference to poly(sarcosine) homopolymers of single molecular weight, it should be recognized that its scope extends to any single molecular weight covered by the above formula (I). Moreover, the benefits of the present invention have been clearly demonstrated in LDC technology. Of course, its advantages are not limited to this technology, and in any field where single molecular weight, biocompatible, biodegradable homopolymers are required, it can exhibit similar or better performance.

[0094] Therefore, the present invention more particularly relates to single molecular weight homopolymers of sarcosine having the formula (II)

[0095]

[0096] wherein

[0097] R1 and R2 are different, and

[0098] one of R1 and R2 is H or an inert group, and the other of R1 and R2 is a functionalized reactive group which is reactive towards a covalently bindable group under reaction conditions under which the inert group is non-reactive,

[0099] Z1 and Z2, which may be the same or different, are optional spacers, and

[0100] k is 2 or greater.

[0101] The homopolymers of formula (I), in particular those of formula (II), are further characterized below and may be used alone or in any combination.

[0102] k is an integer of at least 2, which is preferably at most 100, more preferably at most 50, especially 2 - 30, and more especially 2 - 24, 6 - 24 or 12 - 24.

[0103] In formula (I) or formula (II), the functionalized reactive group R1 or R2 may be selected from the following groups:

[0104] - carboxylic acid group,

[0105] - amino group NRR”, where R and R” are each independently selected from H, (C1 - C6) alkyl optionally interrupted by at least one heteroatom selected from O, N and S,

[0106] - hydroxy group,

[0107] - halogen atom,

[0108] - hydrazine (-NH2 - NH2) group,

[0109] - nitro group,

[0110] - hydroxylamine group,

[0111] - azide group,

[0112] - (C2 - C6) alkynyl group,

[0113] - (C2 - C6) alkenyl group,

[0114] - thiol group,

[0115] - activated ester groups such as N - hydroxysuccinimide ester, perfluoroester, nitrobenzene ester, azido - benzotriazole and benzotriazole activated esters, acylurea,

[0116] - a boronic acid–B(OR””)2 group, where R”” is a hydrogen atom or a C1-C6 alkyl group,

[0117] - a thiol-reactive group such as maleimide, halomaleimide, haloacetyl, pyridyldisulfide,

[0118] - a mesylate group,

[0119] - a tosylate group,

[0120] - a triflate group,

[0121] - an aldehyde group,

[0122] - an isocyanate or isothiocyanate group,

[0123] - a chlorosulfonyl group,

[0124] - an acrylate group.

[0125] As described above, the spacer Z is optional, Z1 and Z2 can both be present, only one of Z1 and Z2 can be present, or neither Z1 nor Z2 can be present. In the latter case, and when the homopolymer of the present invention is a homopolymer of sarcosine, it has the formula (III)

[0126]

[0127] where R1, R2 and k are as defined above.

[0128] In formula (I), formula (II) or formula (III), R1 can be H or an inert group, and R2 can be a functionalized reactive group, or R1 can be a functionalized reactive group, and R2 can be H or an inert group.

[0129] According to a preferred embodiment, the functionalized reactive group R1 or R2 is a secondary amine, and the inert group R1 or R2 is a carboxylic acid that remains unreacted and unbonded on the final LDC structure.

[0130] In a preferred embodiment, in formula (I), formula (II) or formula (III), R1 is selected from OH and NH2, and

[0131] when R1 is OH, R2 is COCH3 and

[0132] when R1 is NH2, R2 is CO—G—COOH, where G is CH2CH 2、 CH2CH2CH2, CH2CH2CH2CH2, CH2OCH2, CH2SCH2, CH2CH(CH3)CH2, CH2C(CH3)2CH2 or CH2N(CH3)CH2.

[0133] The present invention also relates to a method for preparing a single molecular weight homopolymer of formula (I), formula (II) or formula (III). Generally, each N-methylglycine monomer is assembled from two sub-monomers (i.e., haloacetic acid and methylamine) on a solid phase support. Each monomer addition refers to the following cycle: (i) acylating the resin-bound secondary amine with haloacetic acid and carbodiimide or other suitable carboxylic acid ester activation methods, (ii) a washing step, (iii) nucleophilic substitution of the resin-bound halogen with methylamine, (iv) a washing step.

[0134] The method according to the present invention comprises the following steps:

[0135] a) Reacting a compound of formula (IV) with an acid of formula (V) to obtain a compound of formula (VI)

[0136]

[0137] wherein R3 is a solid phase support for peptide synthesis, and m is 1 or greater and less than k,

[0138]

[0139] wherein Hal is a halogen,

[0140]

[0141] wherein R3, m and Hal are as defined above,

[0142] b) Reacting the compound of formula (VI) with methylamine

[0143] to obtain a compound of formula (VII)

[0144]

[0145] wherein R3 and m are as defined above,

[0146] c) Repeating steps a) and b) until a compound of formula (VIII) is obtained

[0147]

[0148] wherein R3 and m are as defined above,

[0149] d) Reacting the compound (VIII) to obtain a compound of formula (IX),

[0150]

[0151] wherein R2 is an inert group, R3 is as defined above and k is as defined above,

[0152] e) A cleavage reaction to obtain a single-molecular-weight homopolymer of formula (III) as defined above.

[0153] According to one embodiment of the method, it includes, in step a), reacting a compound of formula (IV) in which R3 is a solid-phase carrier for peptide synthesis and m is 3, said compound being obtained by the Fmoc-solid-phase peptide synthesis method. They are well known to those skilled in the art, and based on their knowledge, he can select any suitable coupling agent, such as N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide).

[0154] In an alternative embodiment of the method of the present invention, the preparation of the single-molecular-weight homopolymer comprises the following steps:

[0155] a) Reacting a compound of formula (X) with an acid of formula (V) to obtain a compound of formula (XI)

[0156]

[0157] wherein R3 is a solid phase for peptide synthesis, and m is 1 or greater and less than k,

[0158]

[0159] wherein Hal is a halogen,

[0160]

[0161] wherein R3, m and Hal are as defined above,

[0162] b) Reacting the compound of formula (XI) with methylamine to obtain a compound of formula (XII)

[0163]

[0164] wherein R3 and m are as defined above,

[0165] c) Repeating steps a) and b) until a compound of formula (XIII) is obtained,

[0166]

[0167] wherein R3 and k are as defined above,

[0168] d) A cleavage reaction to obtain a compound of formula (XIV)

[0169]

[0170] wherein k is as defined above,

[0171] e) Reacting the 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.

[0172] As described above, the homopolymers of the present invention can be used in LDC technology, but are not limited to this technology.

[0173] Accordingly, the present invention also relates to a ligand-drug-conjugate (LDC) having the following formula (XV)

[0174]

[0175] wherein,

[0176] L is an orthogonal linker that allows (HP SMW ) to be in an orthogonal orientation relative to (X-D),

[0177] HP SMW is produced by covalently binding the single molecular weight homopolymer of the present invention as described above to the orthogonal linker L,

[0178] D is a drug, particularly a cytotoxic drug such as monomethyl auristatin E (MMAE) or SN38,

[0179] X is an optional cleavable moiety for releasing D,

[0180] Z is an optional spacer, and

[0181] a is 1 or greater, b is 1 or greater and m is 1 or greater.

[0182] Compared with ligand-drug-conjugates that do not contain a single molecular weight homopolymer with parallel grafting, when grafted in a parallel (i.e., orthogonal) orientation relative to the drug unit, the single molecular weight homopolymer, particularly single molecular weight poly(sarcosine), provides effective hydrophobic masking properties of the conjugate, reduced apparent hydrophobicity, better pharmacokinetic properties and improved in vivo activity.

[0183] In an alternative embodiment, D is selected from the group consisting of: bioactive molecules, therapeutic molecules (such as anti-cancer drugs), imaging agents and fluorophores.

[0184] Alternative embodiments according to the present invention:

[0185] a is an integer of at least 1, preferably at most 6, more preferably at most 3, specifically 2, and more specifically 1, and / or

[0186] b is an integer of at least 1, preferably at most 6, more preferably at most 3, specifically 2, and more specifically 1, and / or

[0187] m is an integer of at least 1, preferably at most 30, more preferably at most 15, specifically 8, and more specifically 4.

[0188] Advantageously, the single molecular weight homopolymer is poly(sarcosine).

[0189] In one embodiment, between L and the ligand, and / or between L and HP SMW between, and / or between L and X, and / or between X and D, there is a spacer Z.

[0190] Typically, the orthogonal linker connects the releasable assembled drug unit (XD) or drug unit (D) through one or more linker unit assemblies in such a way that the (XD) or (D) unit is in a parallel configuration (opposite to the tandem configuration) with respect to the homopolymer unit.

[0191] The present invention also relates to an intermediate compound having formula (XVI)

[0192]

[0193] wherein

[0194] L is an orthogonal linker,

[0195] HP SMW is produced by covalently binding the single molecular weight homopolymer of the present invention to the orthogonal linker L,

[0196] D is a cytotoxic drug,

[0197] X is an optional cleavable moiety for releasing D,

[0198] Z is an optional spacer that is capable of binding a ligand, and

[0199] a is 1 or greater and b is 0, 1 or greater.

[0200] The present disclosure also relates to a compound having formula (XXIII)

[0201]

[0202] wherein, as defined above,

[0203] R6 is –C1-C10 Alkylene-, –C1-C 10 Heteroalkylene-, -C3-C8 cycloalkyl-, -O-(C1-C8 alkyl)-, -Arylene-, –C1-C 10 Alkylene-arylene-, -Arylene-C1-C 10 Alkylene-, –C1-C 10 Alkylene-(C3-C8 cycloalkyl)-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-, -C3-C8 heterocyclic-, –C1-C 10 Alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)–C1-C 10 Alkylene-, –C1-C 10 Alkylene-C(=O)-, –C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 cycloalkyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -Arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -Arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 cycloalkyl)-C(=O)-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 cycloalkyl-NH-, -O-(C1-C8 alkyl)-NH-, -Arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -Arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 cycloalkyl)-NH-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10Heteroalkylene-S-, -C3-C8 cycloalkyl-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 alkylene-S-, -C1-C 10 alkylene-(C3-C8 cycloalkyl)-S-, -(C3-C8 cycloalkyl)-C1-C 10 alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-S-, –C1-C 10 alkylene-O-C(=O)-, -C3-C8 cycloalkyl-O-C(=O)-, -O-(C1-C8 alkyl)-O-C(=O)-, -arylene-O-C(=O)-, -C1-C 10 alkylene-arylene-O-C(=O)-, -arylene-C1-C 10 alkylene-O-C(=O)-, -C1-C 10 alkylene-(C3-C8 cycloalkyl)-O-C(=O)-, -(C3-C8 cycloalkyl)-C1-C 10 alkylene-O-C(=O)-, -C3-C8 heterocyclic-O-C(=O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-O-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-O-C(=O)-,

[0204] Any one of the R6 groups is optionally substituted by one or more substituents selected from the following: -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2, -NR'3 + , =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 -, -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2, and C(=NR')NR'2, where each X is independently a halogen: -F, -Cl, -Br, or -I; and each R' is independently -H, -C1C 20 alkyl, -C6-C 20 aryl, or -C3-C 14 heterocyclic group,

[0205] Z is an optional spacer,

[0206] L is an orthogonal linker,

[0207] X is an optional cleavable moiety for releasing D,

[0208] D is a cytotoxic drug,

[0209] a is 1 or greater and b is 0, 1 or greater, and

[0210] HP SMW is produced by covalently binding a single molecular weight homopolymer of the present invention to the orthogonal linker L.

[0211] According to a preferred embodiment, HP SMW is produced by covalently binding a poly(sarcosine) homopolymer of the present invention to the orthogonal linker L. In this case, in formulas (XV), (XVI), and (XXIII), HP SMW represents

[0212]

[0213] wherein the wavy bond represents the point of attachment to L or the spacer Z (if present),

[0214] k is 2 or greater, preferably k is 2 - 50, and

[0215] R4 represents a capping group.

[0216] Advantageously, R4 represents -R', -O - , -OR', -SR', -S - , -NR'2, -NR'3 + , =NR', -CX3, -CN, -NRC(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 -, -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2 or C(=NR')NR'2, where each X is independently a halogen: -F, -Cl, -Br, or -I; and each R' is independently -H, -C1C 20 alkyl, -C6-C 20 aryl, or -C3-C 14 heterocyclic group. Typically, R4 is -OR', -NR'2 or -C(=O)R'.

[0217] In one embodiment, the present invention also relates to a ligand-drug-conjugate (LDC) having the following formula (XV)

[0218]

[0219] where

[0220] the ligand is an antibody,

[0221] L is an orthogonal linker that allows HP SMW to be in an orthogonal orientation with respect to (X-D), and is selected from natural or unnatural amino acids; amino alcohols; amino aldehydes; polyamines or any combination thereof,

[0222] HP SMW represents

[0223]

[0224] where the wavy bond represents the point of attachment to L or the spacer Z (if present),

[0225] k is 2 or greater, preferably k is 2-50, and

[0226] R4 represents a capping group.

[0227] D is a drug, particularly a cytotoxic drug such as monomethyl auristatin E (MMAE) or SN38,

[0228] X is an optional cleavable moiety for releasing D, and is selected from

[0229] one or more natural or unnatural amino acids, such as a cleavable peptide containing 2-12 amino acids,

[0230] o A sugar moiety linked to a self - eliminating group via an O - glycosidic bond,

[0231] o A disulfide linker, and

[0232] o An acid - labile linker that is hydrolyzable in lysosomes,

[0233] Z is an optional spacer that can also be present between L and X, and / or between X and D, and / or between L and HP SMW and is selected from alkylene, heteroalkylene; alkoxy; polyether; one or more natural or unnatural amino acids; C3 - C8 heterocyclic group; C 3- C8 carbocyclic group; arylene and any combination thereof,

[0234] a is 1 or greater, b is 1 or greater, and m is 1 or greater.

[0235] The present invention also relates to a pharmaceutical composition comprising at least one LDC compound of the present invention and a pharmaceutically acceptable carrier.

[0236] The present disclosure also relates to the use of the LDC compound as described above as a drug.

[0237] The compound of formula (XXIII) can be used as it is without a ligand because the maleimide moiety can react with a protein (such as serum albumin) in vivo and then become a ligand. Therefore, the present disclosure also relates to the use of the compound of formula (XXIII) as described above as a drug. Brief Description of the Drawings

[0238] Figure 1 Represents the hydrophobic interaction chromatogram according to Example 12.

[0239] Figure 2 Represents the hydrophobic interaction chromatogram according to Example 13.

[0240] Figure 3 Represents the pharmacokinetic curve in mice according to Example 14.

[0241] Figure 4A Represents the tumor volume over time according to Example 15. Figure 4B Represents the percentage of survival of mice according to Example 15.

[0242] Figure 5 Represents the pharmacokinetic curve in mice according to Example 16.

[0243] Figure 6 Represents the tumor volume over time according to Example 17. Detailed Description

[0244] Materials and General Methods

[0245] Unless otherwise stated, all solvents and reagents were obtained from commercial sources (Sigma - Aldrich, Alfa Aesar, Fluorochem, Thermo Fisher, Carbosynth) and used without further purification. 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 DCChemicals. PNU159682 was purchased from Kerui Biotechnology Co., Ltd. and irinotecan mesylate was purchased from Angene Chemical. Human albumin (catalog number A3782) was purchased from Sigma - Aldrich. Anti - CD19 and anti - CD22 antibodies were purchased from Euromedex. Trastuzumab (Herceptin IV) was purchased from Roche. Solid - phase synthesis was carried out in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma - Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). Unless otherwise stated, all chemical reactions were carried out at room temperature under an inert argon atmosphere.

[0246] Liquid nuclear magnetic resonance spectra were recorded on a Bruker Fourier 300HD spectrometer and calibrated using the residual solvent peak. Mass spectrometry analysis was performed by the Commun de Spectrométrie de Masse center (CCSM) of the UMR5246 CNRS Institute at the Claude Bernard Lyon 1 University.

[0247] Normal - phase flash chromatography was performed using an Interchim (spherical HP 50 μm) or Biotage ZIP (50 μm) silica column on a Teledyne Isco CombiFlash Companion equipment or a Teledyne Isco CombiFlash Rf200 equipment. Reverse-phase chromatography was performed using a SNAPUltra C18 (25 μm) column or an Interchim PuriFlash RP-AQ (30 μm) column. Chemical reactions and compound characterization were monitored and analyzed by thin-layer chromatography using pre-coated 40-63 μm silica gel (Macherey-Nagel), HPLC-UV (Agilent 1050), or UHPLC-UV / MS (Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact II TM Q-ToF mass spectrometer or Agilent 1260 HPLC system equipped with a Bruker MicrOTOF-QII mass spectrometer).

[0248] HPLC Method 1: Agilent 1050 equipped with a DAD detector. Mobile phase A is water and mobile phase B is acetonitrile. The column is Agilent Zorbax SB-Aq 4.6 x 150 mm 5 μm (room temperature). The gradient is from 5% B to 95% B in 20 minutes, then held at 95% B for 5 minutes. The flow rate is 1.5 mL / min. UV detection is monitored at 214 nm.

[0249] HPLC Method 2: Agilent 1050 equipped with a DAD detector. Mobile phase A is water and mobile phase B is acetonitrile. The column is Agilent Zorbax SB-Aq 4.6 x 150 mm 5 μm (room temperature). The gradient is from 0% B to 50% B in 30 minutes, then held at 50% B for 5 minutes. The flow rate is 1.0 mL / min. UV detection is monitored at 214 nm.

[0250] HPLC Method 3: The same as HPLC Method 1, but containing 0.1% TFA in mobile phase A.

[0251] HPLC Method 4: The same as HPLC Method 2, but containing 0.1% TFA in mobile phase A.

[0252] UHPLC Method 5: Thermo UltiMate 3000 UHPLC system + Bruker Impact II TM Q-ToF mass spectrometer. Mobile phase A is water + 0.1% formic acid and mobile phase B is acetonitrile + 0.1% formic acid. The column is Agilent PLRP-S 1000 2.1 x 150 mm 8 μm (80 °C). The gradient is from 10% B to 50% B in 25 minutes. The flow rate is 0.4 mL / min. UV detection is monitored at 280 nm. The m / z range of the Q-ToF mass spectrometer is 500 - 3500 (ESI+ )。Use the MaxEnt algorithm included in Bruker Compass software to deconvolute the data.

[0253] HPLC method 6: Agilent 1050 equipped with a DAD detector. Mobile phase A is water + 5 mM ammonium formate, and mobile phase B is acetonitrile. The chromatographic column is Agilent Poroshell 120 EC-C18 3.0 x 50 mm 2.7 μm (room temperature). The gradient is from 5% B to 90% B within 10 minutes, and then held at 90% B for 2 minutes. The flow rate is 0.8 mL / min. UV detection is monitored at 214 nm.

[0254] The following Examples 1-4 illustrate the synthesis of single molecular weight poly(sarcosine) of the present invention, which is part of the present invention and relates to different solid-phase synthesis methods.

[0255] Example 1: Synthesis of poly(sarcosine) compounds (Synthesis method 1 on resin)

[0256] The reaction scheme is as follows.

[0257]

[0258] 1.1) General method

[0259] Synthesis on the resin was carried out in an empty SPE plastic tube equipped with 20 μm polyethylene frit (Sigma-Aldrich). Stirring was carried out using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial theoretical resin loading of 0.63 mmol / g (the marked range indicated by the manufacturer). Unless otherwise stated, all reactions were carried out at room temperature.

[0260] 1.2) Resin loading

[0261] Typically, 500 mg of NovaGEL TM Rink Amide beads (0.63 mmol / g, Novabiochem) were swollen in 5 mL of DMF for 15 minutes. The first monomer was added by reacting 10 equivalents of bromoacetic acid with 13 equivalents of diisopropylcarbodiimide (Sigma-Aldrich) in 5 mL of DMF at room temperature for 60 minutes, and then washed thoroughly with DMF (5 times 5 mL). The bromoacetylated resin was incubated with 5 mL of 40% (wt) aqueous methylamine solution (Sigma-Aldrich) on a horizontal shaker for 30 minutes, and then washed thoroughly with DMF (5 times 5 mL) and DCM (5 times 5 mL). The obtained resin was ready for extension.

[0262] 1.3) Extension of sarcosine compounds

[0263] The poly(sarcosine) oligomers were extended until the desired length was obtained by alternately performing bromoacetylation and amine displacement steps. The bromoacetylation step was carried out by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in 5 mL of DMF. The mixture was stirred for 30 minutes, drained, and washed with DMF (4 times 5 mL). For the amine displacement step, 5 mL of 40% (wt) aqueous methylamine solution (Sigma - Aldrich) was added, the container was shaken for 30 minutes, drained, and washed with DMF (4 times 5 mL) and DCM (4 times 5 mL).

[0264] 1.4) Cleavage from resin

[0265] The poly(sarcosine) oligomers were cleaved using a 5 mL solution of TFA / triisopropylsilane (95:5) under stirring at room temperature. The resin was filtered, and the obtained solution was evaporated under reduced pressure to give an oily transparent material.

[0266] At this stage, PSARn - N(CH3)H was dissolved in water for purification (see below) or for final functionalization.

[0267] 1.5) Final functionalization

[0268] To obtain the PSARn - CH2 - CH2 - COOH compound, the N - terminus of the oligomer was functionalized using 2.5 equivalents of succinic anhydride and 10 equivalents of DIPEA in anhydrous acetonitrile. The mixture was stirred at room temperature for 1 hour, and the volatiles were removed under reduced pressure.

[0269] 1.6 Purification

[0270] The PSAR compounds were purified on an Interchim RP - AQ (30 μm) column. 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.

[0271] 1.7) Single - molecular - weight poly(sarcosine) compounds

[0272] The obtained PSAR compounds are listed in Table 1 below.

[0273] Table 1

[0274]

[0275] Example 2: Synthesis of poly(sarcosine) compounds (Synthesis method 2 on resin)

[0276] The reaction scheme is as follows.

[0277]

[0278] 2.1) General method

[0279] Synthesis on the resin was carried out in an empty SPE plastic tube equipped with 20 μm polyethylene fritted disk (Sigma - Aldrich). Stirring was carried out using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields were based on an initial resin loading of 1.1 mmol / g (the marked range indicated by the manufacturer). Unless otherwise stated, all reactions were carried out at room temperature.

[0280] 2.2) Synthesis of Fmoc - Sar - Sar - OH

[0281]

[0282] 2.2.1) Synthesis of Fmoc - Sar - Sar - OtBu

[0283] In a round - bottom flask, Fmoc - Sar - OH (2000 mg / 6.42 mmol) and HATU (2443 mg / 6.42 mmol) were dissolved in 28 mL of anhydrous DMF. DIPEA (2491 mg / 19.27 mmol) was added, and the mixture was stirred at room temperature for 3 minutes. Then, sarcosine tert - butyl ester hydrochloride (1167 mg / 6.42 mmol) was added, and the reaction mixture was stirred at room temperature for 90 minutes. Volatiles were removed in vacuo, and the residue was diluted with water and extracted 3 times with EtOAc. The organic phase was dried over MgSO4, filtered, and evaporated in vacuo to give a crude solid. The crude product was taken up in EtOAc / DCM 80:20 (v / v), and white insoluble matter was removed via filtration. The filtrate was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 60:40 to 20:80) to give Fmoc - Sar - Sar - OtBu as a white solid (2310 mg / 82%). HRMS m / z (ESI + ) : Calculated value [M + H] + = 439.2227; Experimental value [M + H] + = 439.2234; Error = - 1.5 ppm. HPLC method 1 retention time = 13.3 minutes. TLC eluted with 100% EtOAc: Rf = 0.8.

[0284] 2.2.2) Removal of the tert - butyl ester

[0285] Fmoc-Sar-Sar-OtBu (2310 mg / 5.27 mmol) was dissolved in 20 mL of DCM, and 8.5 mL of TFA was added slowly. The solution was stirred at room temperature until complete deprotection of the tert-butyl ester was observed by HPLC (ca. 2 h). Then, the volatiles were removed in vacuo, and the residue was triturated with diethyl ether to afford Fmoc-Sar-Sar-OH as a white solid (1690 mg / 84%). 1 H NMR (500 MHz, DMSO-d6, 100 °C) δ (ppm) 2.84 (s, 3H), 2.93 (s, 3H), 4.01 (s, 2H), 4.05 (s, 2H), 4.25 (t, J = 4.3 Hz, 1H), 4.34 (d, J = 6.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.63 (d, J = 7.4 Hz, 2H), 7.85 (d, J = 7.5 Hz, 2H). HRMS m / z (ESI + ): calculated for [M+H] + = 383.1601; found [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.

[0286] 2.3) Resin loading

[0287] 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 equiv) pre-dissolved in 10 mL of dry DCM was added to the resin. DIPEA (5 equiv) 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 overnight under high vacuum. The substitution level was evaluated from the weight gain of the resin and / or from Fmoc cleavage tests (absorbance measurement at 301 nm) and was found to be quasi-quantitative (typically 0.95 - 1.1 mmol / g). The resin was stored at -20 °C until further use.

[0288] 2.4) Fmoc-Sar-Sar-OH coupling procedure

[0289] At room temperature, the resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg resin) for 15 minutes. Then, the resin was washed with DMF (4 times) and DCM (4 times). A DMF solution (1 mL per 100 mg resin) of Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.85 equivalents), and DIPEA (6 equivalents) was added to the resin. The reaction vessel was stirred for 2 hours, and the resin was thoroughly washed with DMF (5 times) and DCM (5 times). The resin was dried under vacuum and stored at -20 °C until further use.

[0290] 2.5) Elongation of the poly(sarcosine) compound

[0291] At room temperature, the resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg resin) for 15 minutes. Then, the resin was washed with DMF (4 times) and DCM (4 times).

[0292] The poly(sarcosine) oligomer was elongated until the desired length was obtained by alternately performing bromoacetylation and amine displacement steps. 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 resin). The mixture was stirred for 30 minutes, drained, and washed with DMF (4 times). For the amine displacement step, 40% (wt) aqueous methylamine solution (1.5 mL per 100 mg resin) was added, the container was shaken for 30 minutes, drained, and washed with DMF (4 times) and DCM (4 times).

[0293] 2.6) Final acetylation

[0294] When the desired oligomer length was obtained, the N-terminus was acetylated using a capping solution made of acetic anhydride / DIPEA / DMF (1:2:3 v / v) (the container was shaken for 30 minutes). The solution was drained, and the reaction was repeated once with fresh capping solution. The resin was washed with DMF (4 times) and DCM (4 times).

[0295] 2.7) Resin cleavage

[0296] The poly(sarcosine) oligomer was cleaved from the resin with an HFIP / DCM (20:80 v / v) solution under stirring for 30 minutes. The resin was filtered, and the volatiles were removed under reduced pressure to obtain a crude solid.

[0297] 2.8) Purification

[0298] The PSAR compound was purified on an Interchim RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile + 0.1% TFA.

[0299] 2.9) Single molecular weight poly(sarcosine) compound

[0300] Table 2 below lists the obtained PSAR compounds

[0301] Table 2

[0302]

[0303] Example 3: Synthesis of poly(sarcosine) compounds with one or more azide-functionalized orthogonal linkers (Synthesis method 3 on resin) Example 4: Synthesis of poly(sarcosine) compounds with a terminal non-orthogonal azide-functionalized linker (Synthesis method 4 on resin)

[0304] The reaction scheme is as follows.

[0305]

[0306] 3.1) General method

[0307] Synthesis on resin was carried out in an empty SPE plastic tube equipped with 20 μm polyethylene frit (Sigma - Aldrich). Stirring was carried out using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial resin loading of 1.1 mmol / g (the marked range indicated by the manufacturer). Unless otherwise stated, all reactions were carried out at room temperature. The starting materials were obtained as described in Example 2 above.

[0308] 3.2) Step (1)

[0309] Add a DMF solution of 3 moles of 2 - azidoethan - 1 - amine (1 mL per 100 mg of resin), shake the container for 45 minutes, drain and wash with DMF (4 times) and DCM (4 times).

[0310] 3.3) Step (2)

[0311] Add a DMF solution of commercially available 2 - [4 - (2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)phenyl]acetic acid (5 equivalents), COMU (4.9 equivalents) and DIPEA (4.9 equivalents) to the resin (1 mL per 100 mg of resin). Stir the reaction container for 90 minutes and wash the resin with DMF (3 times) and DCM (3 times).

[0312] 3.4) Step (3)

[0313] Liberate the target compound from the resin using a 1% TFA in DCM (v / v) solution under stirring for 5 minutes (repeat twice). Filter the resin and remove the volatiles under reduced pressure to obtain a solid crude product, which was purified using the protocol described in Example 2 above.

[0314] 3.5) Step (4)

[0315] 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 minutes, drained and washed with DMF (4 times). For the amine displacement step, 30% (wt) aqueous ammonia solution (2 mL per 100 mg of resin) was added, the container was shaken for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times). At this stage, the compound was cleaved from the resin (as described in step (3)) and purified using the protocol described in Example 2 above.

[0316] 3.6) Step (5)

[0317] 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 minutes, drained and washed with DMF (4 times) and DCM (4 times). At this stage, the compound was cleaved from the resin (as described in step (3)) and purified using the protocol described in Example 2 above.

[0318] 3.7) Step (6)

[0319] 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 minutes, drained and washed with DMF (4 times). For the amine displacement step, 40% (wt) aqueous methylamine solution (1.5 mL per 100 mg of resin) was added, the container was shaken for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0320] 3.8) Step (7)

[0321] 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 minutes, drained and washed with DMF (4 times). For the amine displacement step, a DMF solution of 3 moles of 2-azidoethan-1-amine (1 mL per 100 mg of resin) was added, the container was shaken for 45 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0322] 3.9) Step (8)

[0323] Add a DMF solution of commercially available 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]acetic acid (5 equivalents), COMU (4.9 equivalents), and DIPEA (4.9 equivalents) (1 mL per 100 mg of resin) to the resin. Stir the reaction vessel for 90 minutes and wash the resin with DMF (3 times) and DCM (3 times). At this stage, cleave the compound from the resin (as described in step (3)) and purify it using the protocol described in Example 2 above.

[0324] 3.10) Single molecular weight poly(sarcosine) compounds

[0325] The resulting PSAR compounds are listed in Table 3 below.

[0326] Table 3

[0327]

[0328]

[0329]

[0330] Example 5: Synthesis of poly(ethylene glycol) (PEG) compounds with azide-functionalized orthogonal linkers (Synthesis method 5 on resin) Example 6: Synthesis of intermediate compounds based on MMAE, SN38, irinotecan, and PNU159682

[0331] The reaction scheme is as follows.

[0332]

[0333] 4.1) General method

[0334] Synthesis on resin was carried out in an empty SPE plastic tube equipped with 20 μm polyethylene frit (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial theoretical resin loading of 0.47 mmol / g (the marked range indicated by the manufacturer). Unless otherwise stated, all reactions were carried out at room temperature.

[0335] 4.2) Resin loading

[0336] Typically, 500 mg of Ramage ChemMatrix Beads (0.47 mmol / g, Sigma - Aldrich) were swollen in 5 mL of DCM for 15 minutes. At room temperature, the resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 minutes. Then, the resin was washed with DMF (4 times) and DCM (4 times). A DMF solution (1 mL per 100 mg of resin) of Fmoc - L - γ - azido - homoalanine - OH (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) was added to the resin. The reaction vessel was stirred for 1.5 hours, and the resin was thoroughly washed with DMF (5 times) and DCM (5 times). The unreacted sites were acetylated using a capping solution made of acetic anhydride / DIPEA / DMF (1:2:3 v / v) (the container was shaken for 30 minutes). The solution was drained, and the resin was washed with DMF (4 times) and DCM (4 times). At room temperature, the resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 minutes. Then, the resin was washed with DMF (4 times) and DCM (4 times).

[0337] 4.3) Fmoc - Sar - Sar - OH Coupling Procedure

[0338] A DMF solution (1 mL per 100 mg of resin) of Fmoc - Sar - Sar - OH (4 equivalents), HATU (3.9 equivalents), and DIPEA (8 equivalents) was added to the resin. The reaction vessel was stirred for 2 hours, and the resin was thoroughly washed with DMF (4 times) and DCM (4 times). At room temperature, the resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 minutes. Then, the resin was washed with DMF (4 times) and DCM (4 times).

[0339] 4.4) Extension of Poly(sarcosine) Compounds

[0340] The extension of the poly(sarcosine) oligomer was carried out by alternately performing 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 minutes, drained, and washed with DMF (4 times). For the amine displacement step, 40% (wt) aqueous methylamine solution (1.5 mL per 100 mg of resin) was added, the container was shaken for 30 minutes, drained, and washed with DMF (4 times) and DCM (4 times).

[0341] 4.5) Step (6)

[0342] Add a DMF solution (1 mL per 100 mg of resin) of commercially available 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]acetic acid (5 equivalents), COMU (4.9 equivalents), and DIPEA (4.9 equivalents) to the resin. Stir the reaction vessel for 90 minutes and wash the resin with DMF (3 times) and DCM (3 times).

[0343] 4.5) Cleavage and purification from the resin

[0344] At room temperature and with stirring, cleave the oligomer from the resin using a 5 mL solution of TFA / DCM (50:50) for 30 minutes. Repeat the process once and evaporate the combined filtrate under reduced pressure to obtain a crude solid, which is purified as described in Example 2 above.

[0345] 4.6) Single molecular weight poly(sarcosine) compounds

[0346] The resulting PSAR compounds are listed in Table 4 below.

[0347] Table 4

[0348]

[0349] 6.1) Synthesis of compound alkynyl-glucuronide-MMAE 6.4 Synthesis of compound alkynyl-SN38

[0350] The reaction scheme is as follows.

[0351]

[0352] 5.1) General method

[0353] Synthesis on resin was carried out in an empty SPE plastic tube equipped with 20 μm polyethylene frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial resin loading of 1.1 mmol / g (the marked range indicated by the manufacturer). Unless otherwise stated, all reactions were carried out at room temperature.

[0354] 5.2) Resin loading

[0355] 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 minutes. Fmoc-PEG pre-dissolved in 2 mL of dry DCM 12-CH2CH2COOH (PurePEG™, 1.2 equivalents) was added to the resin. DIPEA (3 equivalents) was added, and the reaction vessel was stirred at room temperature for 1 hour. 300 μL of MeOH was added to quench the unreacted resin. After shaking for 10 minutes, the solution was drained, and the resin was washed with DMF (3 times) and DCM (3 times). The resin was dried under high vacuum.

[0356] 5.2) Step (1)

[0357] At room temperature, the resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) twice for 15 minutes. Then, the resin was 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 minutes, drained, and washed with DMF (4 times). For the amine displacement step, a DMF solution of 3 moles of 2-azidoethan-1-amine (1 mL per 100 mg of resin) was added, the container was shaken for 45 minutes, drained, and washed with DMF (4 times) and DCM (4 times).

[0358] 5.3) Step (2)

[0359] The coupling step of 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]acetic acid was carried out and cleavage from the resin as described in Example 3 above. Purification of the compound was carried out as described in Example 2 above.

[0360] 5.4) Single molecular weight PEG compounds

[0361] The resulting PEG compounds are listed in Table 5 below.

[0362] Table 5

[0363]

[0364]

[0365] 6.6 Synthesis of compound alkynyl-glucuronide-irinotecan

[0366] 6.7 Synthesis of compound alkynyl-PNU159682

[0367]

[0368] At 0 °C, 110.8 mg (0.087 mmol) of the starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427 - 3433) was dissolved in MeOH (10 mL). LiOH monohydrate (36.7 mg / 0.87 mmol) was dissolved in water (1 mL) and slowly added to the reaction vessel. After stirring at 0 °C for 70 minutes, the mixture was neutralized with acetic acid (68.2 mg / 1.14 mmol) and concentrated under reduced pressure. The resulting material was absorbed in a water / MeOH / DMF solution (1:1:1 v / v) and purified on a 30 g Biotage SNAP Ultra C18 (25 μm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B.

[0369] The compound alkynyl - glucuronide - MMAE (a mixture of two diastereomers) as a white solid (95 mg / 96%) was obtained. LC - HRMS m / z (ESI + ): calculated value for [M + H] + = 1127.5758; experimental value for [M + H] + = 1127.5757; error = 0.1 ppm. HPLC method 3 retention time = 10.3 minutes.

[0370] 6.2) Synthesis of the compound alkynyl - glucuronide - (MMAE)2

[0371]

[0372] 6.2.1) Synthesis of the compound alkynyl - glucuronide - (PNP)2

[0373] 165 mg (0.240 mmol) of the starting material (synthesized according to the method described in Renoux et al., Chem. Sci., 2017, 8(5), 3427 - 3433), 64.2 mg (0.419 mmol) of commercially available 4 - amino - 3 - (hydroxymethyl)phenylmethanol and 40.7 mg (0.300 mmol) of HOBt were dissolved in anhydrous DMF. After stirring at 50 °C for 3 hours, the volatiles were evaporated and the residue was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 40∶60 to 0∶100) to give a yellow foam - like intermediate diol compound.

[0374] Add anhydrous pyridine (4 molar equivalents) dropwise to a cooled anhydrous DCM solution of 4-nitrophenyl chloroformate (4 molar equivalents) at 0 °C. Stir the mixture at 0 °C for 15 minutes. Add a DCM solution of the previous intermediate diol compound (1 molar equivalent), and stir the mixture at room temperature for 1 hour. Quench the reaction with saturated NaCl solution and extract 3 times with DCM. Dry the organic phase over MgSO4, filter and evaporate in vacuo to obtain a crude solid, which is purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 60:40 to 30:70) to give the compound alkynyl-glucuronide-(PNP)2 as a white solid (52 mg / 21% over two steps). 1 H NMR (300 MHz, CDCl3) δ (ppm) 2.04 (s, 3H), 2.06 (s, 3H), 2.11 (d, J = 2.0 Hz, 3H), 2.71 - 2.92 (m, 2H), 3.72 (s, 3H), 4.11 (q, J = 7.1 Hz, 1H), 4.22 (d, J = 8.6 Hz, 1H), 5.18 - 5.41 (m, 8H), 5.87 (t, J = 6.5 Hz, 1H), 7.31 - 7.41 (m, 5H), 7.45 - 7.55 (m, 2H), 7.56 - 7.71 (m, 2H), 7.83 (d, J = 8.2 Hz, 1H), 7.89 (s, 1H), 8.21 - 8.32 (m, 4H). HRMS m / z (ESI + ): Calculated value for [M+Na] + = 1055.1925; Experimental value for [M+Na] + = 1055.1955; Error = -2.9 ppm.

[0375] 6.2.2) Synthesis of the compound alkynyl-glucuronide-(MMAE)2

[0376] Dissolve 52 mg (0.050 mmol) of the previous compound alkynyl - glucuronide-(PNP)₂, 13.7 mg (0.100 mmol) of HOBt, and 74.1 mg (0.103 mmol) of monomethyl auristatin E (MMAE) in 1 mL of an 8:2 (v / v) mixture of anhydrous DMF / pyridine. Stir the reaction at room temperature for 24 hours and evaporate the volatiles under reduced pressure. Purify the crude residue by silica gel chromatography (DCM / MeOH gradient from 97:3 to 90:10) to obtain 77 mg (70%) of the intermediate compound, which can be directly used in the deprotection step without extensive characterization. At 0 °C, dissolve 77 mg (0.035 mmol) of this compound in MeOH (7 mL). Dissolve LiOH monohydrate (14.7 mg / 0.350 mmol) in water (0.7 mL) and slowly add it to the reaction vessel. After stirring at 0 °C for 60 minutes, neutralize the mixture with acetic acid (27.4 mg / 0.457 mmol) and concentrate it under reduced pressure. Absorb the resulting material in a water / MeOH / DMF solution (1:1:1 v / v) and purify it on a 30 g Biotage SNAP Ultra C18 column. Mobile phase A is water + 0.05% TFA, and mobile phase B is acetonitrile + 0.05% TFA.

[0377] Obtain the compound alkynyl - glucuronide-(MMAE)₂ as a white solid (a mixture of two diastereoisomers) (40 mg / 56%). LC - HRMS m / z (ESI + ): calculated value for [M + 2H] 2+ = 1025.5623; experimental value for [M + 2H] 2+ = 1025.5599; error = 2.4 ppm. HPLC method 3 retention time = 13.0 min.

[0378] 6.3) Synthesis of the compound alkynyl - val - cit - PAB - MMAE

[0379]

[0380] Dissolve 58 mg (0.052 mmol) of the starting material (synthesized as described in Tang et al., Org. Biomol. Chem., 2016, 14(40), 9501 - 9518) and 15 mg (0.077 mmol) of 4 - pentanoic acid succinimidyl ester in 3 mL of anhydrous DCM. Add 16.7 mg (0.129 mmol) of DIPEA and stir the reaction at room temperature under an argon atmosphere for 16 hours. Then, remove the volatiles under vacuum, absorb the resulting material in a DMF solution, and purify it on a 30 g Biotage Purification on a SNAP UltraC18 (25 μm) column. Mobile phase A is water + 0.1% TFA, and mobile phase B is acetonitrile + 0.1% TFA. The gradient ranges from 25% to 70% B.

[0381] The compound propargyl-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.

[0382] 6.7.2) Synthesis of alkynyl-PNU159682

[0383]

[0384] 156 mg (0.308 mmol) of the starting material TBDMS-SN38 (synthesized as described in Moon et al., J. Med. Chem., 2008, 51(21), 6916 - 6926), 113 mg (0.924 mmol) of 4-(dimethylamino)pyridine, and 75 mg (0.369 mmol) of 4-nitrophenyl chloroformate were dissolved in 8 mL of anhydrous DCM. The solution was stirred at room temperature for 90 minutes, diluted with an aqueous solution of 5% acetic acid, and extracted 3 times with DCM. The organic phase was dried over MgSO4, filtered, and evaporated in vacuo to give a yellow solid, which was used in the next step without further purification.

[0385] 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 for 16 hours under an argon atmosphere. Volatiles were removed in vacuo, and the residue was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 40:60 to 0:100) to give the compound propargyl-SN38 as a bright yellow solid (71 mg / 56%). HRMS m / z (ESI + ): calculated for [M+H] + = 474.1660; found [M+H] + = 474.1664; error = -0.9 ppm. HPLC method 3 retention time = 9.7 minutes. TLC eluted with 100% EtOAc: Rf = 0.15.

[0386] 6.5 Synthesis of the compound propargyl-glucuronide-SN38

[0387]

[0388] Weigh 50 mg (0.074 mmol) of the starting material TBDMS-SN38-OPNP (synthesized as described in Section 6.4 previously) and 5 mg (0.037 mmol) of HOBt in a reaction vessel. Add 58.5 (0.223 mmol) of tert-butyl (2-((2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate (synthesized as described in WO2011 / 133039), which was previously dissolved in a 8:2 (v / v) mixture of 1 mL of anhydrous DMF / pyridine, to the reaction vessel. Stir the reaction at room temperature for 16 h and evaporate the volatiles under reduced pressure. Purify the crude residue by silica gel chromatography (DCM / MeOH gradient from 98:2 to 90:10) to obtain 48 mg (95%) of the intermediate compound (yellow solid), which was directly used in the deprotection step. HRMS m / z (ESI + ): Calculated [M+H] + = 681.3130; Found [M+H] + = 681.3113; Error = 2.5 ppm.

[0389] Dissolve 48 mg (0.071 mmol) of this compound in 2 mL of DCM, then add 500 μL of TFA. Stir the solution at room temperature for 90 min and remove the volatiles under reduced pressure. Purify the crude residue by silica gel chromatography (DCM / MeOH gradient from 94:6 to 80:20) to obtain 39.8 mg (96%) of the compound SN38-methylamine as a yellow solid. HRMS m / z (ESI + ): Calculated [M+H] + = 581.2606; Found [M+H] + = 581.2601; Error = 0.8 ppm. HPLC method 3 retention time = 7.7 min.

[0390] 48 mg (0.069 mmol) of the starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427 - 3433), 39.8 mg (0.069 mmol) of the previous compound SN38 - methylamine, and 9.3 mg (0.069 mmol) of HOBt were dissolved in 1.5 mL of an 8:2 (v / v) mixture of anhydrous DMF / pyridine. 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 from 98:2 to 95:5) to afford 57 mg (74%) of the intermediate compound (yellow solid), which was used directly in the deprotection step. ESI + [M+H] + = 1130.4.

[0391] At 0 °C, 57 mg (0.050 mmol) of this compound was dissolved in MeOH (6 mL). Lithium hydroxide monohydrate (21.2 mg / 0.504 mmol) was dissolved in water (0.6 mL) and added slowly to the reaction vessel. After stirring at 0 °C for 70 min, the mixture was neutralized with acetic acid (39.4 mg / 0.656 mmol) and concentrated under reduced pressure. The resulting material was taken up in a water / MeOH / DMF solution (1:1:1 v / v) and purified on a 30 g Biotage SNAP Ultra C18 (25 μm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B.

[0392] The compound alkynyl - glucuronide - SN38 (20.5 mg / 42%) was obtained as a yellow solid. LC - HRMS m / z (ESI + ): calculated [M+H] + = 990.3251; found [M+H] + = 990.3210; error = 4.1 ppm. HPLC method 3 retention time = 8.2 min.

[0393] Example 7: Synthesis of a poly(sarcosine)-based drug conjugate linker using glutamate as the orthogonal moiety

[0394]

[0395] 132.1 mg (0.192 mmol) of the starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427 - 3433), 102 mg (0.192 mmol) of irinotecan mesylate, and 26 mg (0.192 mmol) of HOBt were dissolved in 1 mL of an 8:2 (v / v) mixture of anhydrous DMF / pyridine. The reaction was stirred at room temperature for 16 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 98:2 to 90:10) to give 165 mg (87%) of the intermediate compound (yellow solid), which was used directly in the deprotection step. ESI + [M+H] + = 985.3.

[0396] At 0 °C, 165 mg (0.168 mmol) of this compound was dissolved in MeOH / THF 1:1 v / v (16 mL). Lithium hydroxide monohydrate (70.3 mg / 1.675 mmol) was dissolved in water (1.6 mL) and added slowly to the reaction vessel. After stirring at 0 °C for 70 min, the mixture was neutralized with acetic acid (131 mg / 2.18 mmol) and concentrated under reduced pressure. The resulting material was taken up in a water / MeOH / DMF solution (1:1:1 v / v) and purified on a 30 g Biotage SNAP Ultra C18 (25 μm) column. 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.

[0397] The compound propargyl - glucuronide - irinotecan was obtained as a yellow solid (98 mg / 69%). LC - HRMS m / z (ESI + ): calculated for [M+H] + = 845.2312; found [M+H] + = 845.2360; error = - 4.8 ppm. HPLC method 3 retention time = 8.0 min.

[0398] Example 8: Synthesis of a poly(sarcosine)-based drug conjugate linker using lysine as the orthogonal moiety

[0399]

[0400] 6.7.1) Synthesis of N-(2-((2-aminoethyl)amino)-2-oxoethyl)propiolamide

[0401] 762 mg (4.54 mmol) of tert-butyl glycinate hydrochloride, 318.3 mg (4.54 mmol) of propionic acid, and 61.4 mg (0.454 mmol) of HOBt were dissolved in 5 mL of anhydrous DMF. 1103 mg (10.9 mmol) of DIPEA was added, and the solution was stirred on ice (0 °C) for 10 minutes. 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 at room temperature in the dark for 16 hours. Then, the volatiles were removed under reduced pressure. Saturated NH4Cl solution was added, and the mixture was extracted 3 times with DCM. The organic phase was dried over MgSO4, filtered, and evaporated. The crude residue was purified by silica gel chromatography (petroleum ether / EtOAc gradient from 80:20 to 50:50) to give 255 mg (31%) of tert-butyl propioloyl glycinate as a clear oil. MS (ESI + ): [M+H] + = 184.0. Eluted with petroleum ether / EtOAc (40:60 v / v) and stained with KMnO4 TLC: Rf = 0.75.

[0402] 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 completed after stirring at room temperature for 1 hour as evaluated by TLC analysis. The volatiles were removed under reduced pressure to give 188 mg (105%) of propioloyl glycine as an oily residue, which was used for the next step without purification.

[0403] 178 mg (1.40 mmol) of propioloyl glycine 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 minutes. Then, 291 mg (1.82 mmol) of N-Boc-ethylenediamine, pre-dissolved in 1 mL of anhydrous DMF, was added, and the reaction mixture was stirred at room temperature in the dark for 30 minutes. Then, the volatiles were removed under reduced pressure. Saturated NH4Cl solution was added, and the mixture was extracted 3 times with DCM. The organic phase was dried over MgSO4, filtered, and evaporated. The crude residue was purified by silica gel chromatography (petroleum ether / EtOAc gradient from 20:80 to 0:100) to give 204 mg (54%) of tert-butyl (2-(2-propiolamidoacetamido)ethyl)carbamate as a pale yellow oil. MS (ESI + ): [M+H] += 270.1; TLC eluted with 100% EtOAc and stained with KMnO4: Rf = 0.35.

[0404] 204 mg (0.758 mmol) of tert-butyl (2-(2-propynamidoacetamido)ethyl)carbamate was absorbed in 5 mL of a DCM / TFA (7:3 v / v) solution. The deprotection reaction was completed after stirring at room temperature for 45 minutes as evaluated by TLC analysis. Volatiles were removed under high vacuum overnight to give 196 mg (92%) of N-(2-((2-aminoethyl)amino)-2-oxoethyl)propynamide TFA salt as a pale yellow thick wax. 1 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.84 (q, J = 6.2 Hz, 2H), 3.29 (q, J = 6.3 Hz, 2H), 3.72 (d, J = 6.0 Hz, 2H), 4.20 (s, 1H), 7.78 (br.s, 3H), 8.12 (t, J = 5.6 Hz, 1H), 8.94 (t, J = 5.9 Hz, 1H). MS (ESI + ): [M+H] + = 170.0.

[0405] Example 9: Synthesis of a poly(sarcosine)- or poly(ethylene glycol)-based drug conjugate linker using glycine as the orthogonal moiety

[0406] In a round-bottom flask, 25 mg (0.040 mmol) of the PNU159692 carboxylic acid derivative (a purple solid synthesized as described in WO / 2016 / 040825, see the chemical structure above) and 15.1 mg (0.040 mmol) of HATU were dissolved in 1 mL of anhydrous DMF. 10.2 mg (0.080 mmol) of DIPEA was added and the mixture was stirred at room temperature for 2 minutes. 13.4 mg (0.047 mmol) of N-(2-((2-aminoethyl)amino)-2-oxoethyl)propynamide TFA salt (pre-dissolved in 500 μL of anhydrous DMF) was added and the reaction mixture was stirred at room temperature for 5 minutes. Volatiles were removed under high vacuum and the residue was purified by silica gel chromatography (DCM / MeOH, gradient from 99:1 to 90:10) to give 14.7 mg (49%) of alkynyl-PNU159682 as a red solid. HRMS m / z (ESI + ): calculated [M+H] + = 779.2770; found [M+H] + = 779.2758; error = 1.5 ppm. HPLC method 6 retention time = 5.4 minutes.

[0407] Example 10: Synthesis of negative control drug conjugate linkers MAL-glucuronide MMAE, MAL-phenyl-triazole-glucuronide MMAE, and MAL-phenyl-PSARn-triazole-glucuronide MMAE

[0408] The reaction scheme is as follows.

[0409]

[0410] 7.1) Ethylenediamine-loaded resin

[0411] Swell 500 mg of 2-chlorotrityl chloride resin beads (100 - 200 mesh, 1% DVB, 1.1 mmol / g, 0.55 mmol ratio, Novabiochem) in 5 mL of DCM for 10 minutes. Add 5 equivalents (2.75 mmol, 165.3 mg) of ethylenediamine (Sigma - Aldrich), and shake the mixture at room temperature for 4 hours, then wash it thoroughly with DCM (5 times 5 mL). Block the unreacted sites on the resin using a DCM / MeOH / DIPEA (17:2:1 v / v) solution (treatment for 20 minutes). Wash the resin thoroughly with DCM (5×5 mL) and MeOH (5×5 mL), dry it under vacuum and store it at -20 °C until further use.

[0412] 7.2) Fmoc - Glu(OAll) - OH coupling

[0413] Add a DMF solution (1 mL per 100 mg of resin) of Fmoc - Glu(OAll) - OH (3 equivalents), HATU (2.85 equivalents) and DIPEA (6 equivalents) to the resin containing the deprotected N - terminus (1 equivalent). Stir the reaction vessel for 2 hours, and wash the resin thoroughly with DMF (5×3 mL) and DCM (5×3 mL). Confirm the completion of the reaction by a negative Kaiser test. Dry the resin under vacuum and store it at -20 °C until further use.

[0414] 7.3) Removal of the Alloc protecting group

[0415] Suspend the resin in DCM (4 mL per 100 mg of resin), and gently stir the mixture by introducing an argon stream from below the fritted disk. Add phenylsilane (20 equivalents), and continue stirring for 5 minutes, then add Pd(PPh3)4 (0.25 equivalent). Continue stirring the mixture under argon stream at room temperature for 30 minutes under light - protected conditions, then drain the solution. Repeat the treatment with phenylsilane and Pd(PPh3)4 once, and wash the resin thoroughly with DCM (5×5 mL), DMF (5×5 mL) and MeOH (5×5 mL). Dry the resin under vacuum and store it at -20 °C until further use. Evaluate the resin loading (Fmoc cleavage test, absorbance measurement at 301 nm), usually 0.70 - 0.80 mmol / g.

[0416] 7.4 (2R,3R,4R,5S,6R)-6-(2-(3-Aminopropionamido)-4-((5S,8S,11S,12R)-11-((S)-sec-Butyl)-12-(2-(2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (NH2-Glucuronide-MMAE) Coupling Procedure

[0417] To the resin containing the deprotected carboxylic acid group (1 equivalent), a DMF solution of HATU (4 equivalents) and DIPEA (4.2 equivalents) was added. The reaction vessel was stirred for 25 minutes, drained, and the resin was washed with DMF (4 times 5 mL). Then, 1.5 equivalents of the compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropionamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-(2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (“NH2-Glucuronide-MMAE”; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831–840) and DIPEA (3.2 equivalents) in DMF were added to the resin. The reaction vessel was stirred for 3 hours, drained, and washed with DMF (5 times 3 mL) and DCM (5 times 3 mL). The resin was dried under vacuum and stored at -20 °C until further use.

[0418] 7.5) Fmoc Deprotection Procedure

[0419] At room temperature, the resin containing the Fmoc-protected amino acid was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 minutes. Then, the resin was washed with DMF (5 times 5 mL) and DCM (5 times 5 mL). The resin was dried under vacuum and stored at -20 °C until further use.

[0420] 7.6) Poly(sarcosine) Coupling Procedure

[0421] To the resin containing a deprotected primary amine group (1 equivalent), a DMF solution of poly(sarcosine)-CH2-CH2-COOH (2.2 equivalents), HATU (2 equivalents) and DIPEA (6 equivalents) was added. The reaction vessel was stirred 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.

[0422] 7.7) Cleavage from the resin

[0423] Final cleavage from the 2-chlorotrityl resin was carried out using a DCM solution of 20% (v / v) HFIP (2 mL per 100 mg of resin) with stirring at room temperature. The reaction time was 60 min. The resin was filtered and the resulting solution was evaporated under an argon stream. The final residue was dried under high vacuum and used directly for the next step.

[0424] 7.8) Coupling procedure for N-hydroxysuccinimidyl 3-(maleimidyl)propionate

[0425] To the residue dissolved in anhydrous DMF, N-hydroxysuccinimidyl 3-(maleimidyl)propionate (8 equivalents) was added. DIPEA (10 equivalents) 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) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B.

[0426] A transparent oily compound MAL-Glu (glucuronide MMAE)-CH2-CH2-PSAR6 (5.8 mg / 14% yield, based on the initial resin loading) was obtained. LC-HRMS m / z (ESI + ): Calculated [M+2H] 2+ = 989.5064; Found [M+2H] 2+ 989.5023; Error = 4.2 ppm. HPLC method 1 retention time = 6.7 min.

[0427] A transparent oily compound MAL-Glu (glucuronide MMAE)-CH2-CH2-PSAR12 (4.6 mg / 20% yield, based on the initial resin loading) was obtained. LC-HRMS m / z (ESI + ): Calculated [M+2H] 2+ = 1202.6178; Found [M+2H] 2+1202.6178; Error = 0.0 ppm. HPLC method 1 retention time = 6.9 minutes.

[0428] Obtained a transparent oily compound MAL-Glu (glucuronide MMAE)-CH2-CH2-PSAR18 (2.4 mg / 14% yield, based on the initial resin loading). LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 1415.7291; Experimental value [M+2H] 2+ 1415.7282; Error = 0.7 ppm. HPLC method 1 retention time = 6.8 minutes.

[0429] Example 11: Preparation of the LDC compounds of the present invention

[0430] The reaction scheme is as follows.

[0431]

[0432] 8.1) Synthesis of Fmoc-D-Lys (glucuronide MMAE)-NH2

[0433]

[0434] 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-dioxatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (“NH2-glucuronide-MMAE”; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831–840) and Fmoc-D-Lys(Boc)-OSu (35 mg / 0.062 mmol) were dissolved in 1.2 mL of anhydrous DMF. DIPEA (24.0 mg / 0.186 mmol) was added and the mixture was stirred at room temperature for 20 h. Volatiles were removed in vacuo. The flask containing the pale yellow crude 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 (ca. 2 h). Then, volatiles were removed in vacuo and the residue was taken up in DMF for purification on a 30 g Biotage SNAP UltraC18 (25 μm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B. The compound Fmoc-D-Lys(glucuronide MMAE)-NH2 was obtained as a white solid (59 mg / 65%). LC-HRMS m / z (ESI + ): calculated [M+H] + = 1480.7862; found [M+H] + = 1480.7890; error = -1.9 ppm. HPLC method 1 retention time = 10.5 min

[0435] 8.2) Synthesis of Fmoc-D-Lys(glucuronide MMAE)-PSAR n Synthesis

[0436]

[0437] Compound PSARn-COOH (2 equiv.; obtained as described in Example 2 and pre-dissolved in anhydrous DMF as a 0.15 M stock solution) was added to HATU (1.8 equiv.) in a vial. DIPEA (5 equiv.) was added and the mixture was stirred at room temperature for 3 minutes. Then, compound Fmoc-D-Lys (glucuronide MMAE)-NH2 (1 equiv.; pre-dissolved in anhydrous DMF as a 0.05 M stock solution) was added. The mixture was stirred at room temperature for 1 hour and then injected into a 30 g Biotage SNAP Ultra C18 (25 μm) column for purification. 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.

[0438] Compound Fmoc-D-Lys (glucuronide MMAE)-PSAR6 was obtained as a white solid (9.8 mg / 38%). LC-HRMS m / z (ESI + ): calculated value for [M + 2H] 2+ = 975.0133; experimental value for [M + 2H] 2+ = 975.0088; error = 4.6 ppm. HPLC method 1 retention time = 7.5 minutes.

[0439] Compound Fmoc-D-Lys (glucuronide MMAE)-PSAR12 was obtained as a white solid (3.6 mg / 28%). LC-HRMS m / z (ESI + ): calculated value for [M + 2H] 2+ = 1188.1247; experimental value for [M + 2H] 2+ = 1188.1233; error = 1.1 ppm. HPLC method 1 retention time = 7.6 minutes.

[0440] 8.3) Coupling procedure for N-hydroxysuccinimide ester of 6-(maleimidyl)hexanoic acid

[0441] At room temperature, the compound Fmoc-D-Lys (glucuronide MMAE)-PSARn from the previous step was treated with 20% piperidine in DMF for 5 minutes. Volatiles were removed under high vacuum and the dried residue was dissolved in anhydrous DMF. Then, N-hydroxysuccinimide ester of 6-(maleimidyl)hexanoic acid (8 equiv.) and DIPEA (10 equiv.) were added and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with water / TFA (99.5:0.5 v / v) and applied to a 30 g Biotage Column purification on SNAPUltra C18 (25μm) cartridge. Mobile phase A is water + 0.05% TFA, and mobile phase B is acetonitrile + 0.05% TFA. After isocratic retention for 10 minutes (5% B), the target compound is eluted isocratically with 40% B.

[0442]

[0443] Obtained a transparent oily compound MAL-Lys (glucuronide MMAE)-PSAR6 (5.0 mg / 52%). LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 960.5163; Experimental value [M+2H] 2+ = 960.5167; Error = -0.5 ppm. HPLC method 1 retention time = 7.1 minutes.

[0444] Obtained a transparent oily compound MAL-Lys (glucuronide MMAE)-PSAR12 (1.8 mg / 51%). LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 1173.6276; Experimental value [M+2H] 2+ 1173.6229; Error = 4.0 ppm. HPLC method 1 retention time = 7.0 minutes.

[0445] Reverse-phase liquid chromatography-mass spectrometry (RPLC-MS): Size exclusion chromatography (SEC):

[0446] 9.1) Compound bromoacetamide-Ngly (triazole-glucuronide MMAE)-PSARn

[0447]

[0448] Mix alkynyl-glucuronide-MMAE (1 equivalent; obtained as described in Example 6), PSARn-N3-bromoacetamide (1.1 equivalents; obtained as described in Example 3), and copper(I) tetra(acetonitrile) hexafluorophosphate (3 equivalents) in a reaction vessel. Add a DCM / acetonitrile 1:1 (v / v) solution to make the final concentration of alkynyl-glucuronide-MMAE 12 μmol / mL. Stir the reaction in the dark at room temperature under argon for 16 - 20 hours. After removing volatiles under reduced pressure, absorb the residue in DMF and purify it on a 30 g Biotage SNAP Ultra C18 (25μm) column. Mobile phase A is water + 0.1% TFA, and mobile phase B is acetonitrile + 0.1% TFA. The gradient ranges from 10% to 50% B.

[0449] A white solid compound bromoacetamide-Ngly(triazole-glucuronide MMAE)-PSAR12 (8.5 mg / 51%) was obtained. LC-HRMS m / z (ESI + ): Calculated value [M + 2H] 2+ = 1151.0179; Experimental value [M + 2H] 2+ = 1151.0188; Error = -0.8 ppm. HPLC method 3 retention time = 8.5 minutes.

[0450] 9.2) Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSARn

[0451]

[0452] Using DCM as the reaction solvent, the alkyne-glucuronide-MMAE (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described in Section 9.1 above.

[0453] A white solid compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR6 (3.3 mg / 20%) was obtained. LC-HRMS m / z (ESI + ): Calculated value [M + 2H] 2+ = 955.9566; Experimental value [M + 2H] 2+ = 955.9533; Error = 3.4 ppm. HPLC method 3 retention time = 9.2 minutes.

[0454] A white solid compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR12 (9.0 mg / 33%) was obtained. LC-HRMS m / z (ESI + ): Calculated value [M + 2H] 2+ = 1169.0679; Experimental value [M + 2H] 2+ = 1169.0621; Error = 4.9 ppm. HPLC method 3 retention time = 8.7 minutes.

[0455] A white solid compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR18 (11.5 mg / 40%) was obtained. LC-HRMS m / z (ESI + ): Calculated value [M + 2H] 2+ = 1382.1792; Experimental value [M + 2H] 2+= 1382.1803; Error = -0.7 ppm. HPLC method 3 retention time = 8.6 minutes.

[0456] Obtained compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 as a white solid (15 mg / 44%). LC-HRMS m / z (ESI + ): Calculated value [M+4Na] 4+ = 820.1309; Experimental value [M+4Na] 4+ = 820.1324; Error = -1.8 ppm. HPLC method 3 retention time = 8.4 minutes.

[0457] 9.3) Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEGn

[0458]

[0459] Using NMP / DCM 2:1 (v / v) as the reaction solvent, react and purify the alkyne-glucuronide-MMAE (obtained as described in Example 6) and PEGn-N3-phenyl-MAL (obtained as described in Example 5) as described in Section 9.1 above.

[0460] Obtained compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 as a pale yellow oil (10.4 mg / 45%). LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 1042.5211; Experimental value [M+2H] 2+ = 1042.5218; Error = -0.7 ppm. HPLC method 3 retention time = 8.0 minutes.

[0461] 9.4) Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)-PSARn

[0462]

[0463] Mix the alkyne-glucuronide-MMAE (3 equivalents; obtained as described in Example 6), PSARn-N3-N3-phenyl-MAL (1 equivalent; obtained as described in Example 3), and copper(I) tetra(acetonitrile) hexafluorophosphate (5 equivalents) in a reaction vessel. Add DCM, and stir the reaction at room temperature in argon in the dark for 16 - 20 hours. After removing the volatiles under reduced pressure, absorb the residue in DMF and on 30 g Biotage Purification on a SNAP Ultra C18 (25 μm) column. Mobile phase A is water + 0.1% TFA, and mobile phase B is acetonitrile + 0.1% TFA. The gradient ranges from 10% to 50% B.

[0464] 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 + ): Calculated value for [M + 4H] 4+ = 1022.5082; experimental value for [M + 4H] 4+ = 1022.5093; error = -1.0 ppm. HPLC method 3 retention time = 9.5 minutes.

[0465] 9.5) Compound MAL-phenyl-Ngly[triazole-glucuronide (MMAE)2]-PSARn

[0466]

[0467] Using DCM as the reaction solvent, the alkyne-glucuronide-(MMAE)2 (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described in Section 9.1 above.

[0468] 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 + ): Calculated value for [M + 3H] 3+ = 1371.3767; experimental value for [M + 3H] 3+ = 1371.3818; error = -3.8 ppm. HPLC method 3 retention time = 10.0 minutes.

[0469] 9.6) Compound MAL-phenyl-Ngly[triazole-galactoside (MMAE)2]-PSARn

[0470]

[0471] Using DCM as the reaction solvent, the alkyne-galactoside-(MMAE)2 (synthesized as described in Alsarraf et al., Chem. Commun., 2015, 51(87), 15792-15795) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described in Section 9.1 above.

[0472] Obtained the compound MAL-phenyl-Ngly[triazole-galactoside (MMAE)2]-PSAR24 as a white solid (6.5 mg / 55%). LC-HRMS m / z (ESI + ): Calculated value of [M + 4H] 4+ = 1022.7895; Experimental value of [M + 4H] 4+ = 1022.7903; Error = 0.8 ppm. HPLC method 3 retention time = 9.2 minutes.

[0473] 9.7) Compound MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSARn

[0474]

[0475] Using NMP as the reaction solvent, react and purify propargyl-val-cit-PAB-MMAE (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) as described in Section 9.1 above.

[0476] Obtained the compound MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSAR12 as a white solid (4.5 mg / 12%). LC-HRMS m / z (ESI + ): Calculated value of [M + 2H] 2+ = 1207.1494; Experimental value of [M + 2H] 2+ = 1207.1535; Error = -3.5 ppm. HPLC method 3 retention time = 8.6 minutes.

[0477] 9.8) Compound MAL-phenyl-Ngly(triazole-SN38)-PSARn

[0478]

[0479] Using DCM / DMF 2:1 (v / v) as the reaction solvent, react and purify propargyl-SN38 (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) as described in Section 9.1 above.

[0480] Obtained the compound MAL-phenyl-Ngly(triazole-SN38)-PSAR18 as a bright yellow solid (4.0 mg / 20%). LC-HRMS m / z (ESI + ): Calculated value of [M + 2Na] 2+ = 1077.4562; Experimental value of [M + 2Na] 2+= 1077.4588; Error = -2.4 ppm. HPLC method 3 retention time = 7.5 minutes.

[0481] 9.9) Compound MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSARn

[0482]

[0483] As described in Section 9.4 above, alkynyl-SN38 (obtained as described in Example 6) and PSARn-N3-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified.

[0484] 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 + ): Calculated value [M+2Na] 2+ = 1412.5812; Experimental value [M+2Na] 2+ = 1412.5852; Error = -3.8 ppm. HPLC method 3 retention time = 8.4 minutes.

[0485] 9.10) Compound MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSARn

[0486]

[0487] Using DCM / MeOH 8:2 (v / v) as the reaction solvent, alkynyl-glucuronide-SN38 (obtained as described in Example 6) and PSARn-N3-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described in Section 9.4 above.

[0488] Compound MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSAR18 was obtained as a yellow solid (7.0 mg / 30%). LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 1271.5080; Experimental value [M+2H] 2+ = 1271.5103; Error = -1.8 ppm. HPLC method 3 retention time = 7.8 minutes.

[0489] 9.11) Compound MAL-phenyl-Ngly(triazole-glucuronide-irinotecan)-PSARn

[0490]

[0491] Using DCM as the reaction solvent, react and purify alkynyl-glucuronide-irinotecan (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) as described in Section 9.1 above.

[0492] Obtain the compound MAL-phenyl-Ngly(triazole-glucuronide-irinotecan)-PSAR18 (13.2 mg / 64%) as a yellow solid. LC-HRMS m / z (ESI + ): Calculated [M+2H] 2+ = 1241.0069; Observed [M+2H] 2+ = 1241.0088; Error = -1.1 ppm. HPLC method 3 retention time = 7.1 minutes.

[0493] 9.12) Compound MAL-phenyl-Ngly(triazole-PNU159682)-PSARn

[0494]

[0495] 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, react and purify alkynyl-PNU159682 (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) as described in Section 9.1 above.

[0496] Obtain the compound MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12 (4.8 mg / 40%) as a red solid. LC-HRMS m / z (ESI + ): Calculated [M+2H] 2+ = 994.9185; Observed [M+2H] 2+ = 994.9184; Error = 0.1 ppm. HPLC method 6 retention time = 5.0 min.

[0497] Obtain the compound MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18 (7.2 mg / 33%) as a red solid. LC-HRMS m / z (ESI + ): Calculated [M+2H] 2+ = 1208.0298; Observed [M+2H] 2+ = 1208.0295; Error = 0.3 ppm. HPLC method 6 retention time = 4.9 minutes.

[0498] Hydrophobic interaction chromatography (HIC): Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR6 (DAR8)

[0499] 10.1) Synthesis of Compound MAL-Glucuronide MMAE

[0500]

[0501] Weigh 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-dioxatetradecanyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (“NH2-Glucuronide-MMAE”; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831–840) (6.2 mg / 5 μmol) and N-hydroxysuccinimide 3-(maleimidyl)propionate (14.6 mg / 55 μmol), and dissolve them in 200 μL of anhydrous DMF. Add DIPEA (8.5 mg / 66 μmol), and stir the mixture at room temperature for 30 minutes. Quench the reaction mixture with 1.5 mL of water / TFA (99:1 v / v), and purify it on a 30 g Biotage SNAP Ultra C18 (25 μm) column. Mobile phase A is water + 0.05% TFA, and mobile phase B is acetonitrile + 0.05% TFA. The gradient ranges from 10% to 70% B.

[0502] Obtain the title compound MAL-Glucuronide MMAE as a white solid (4.1 mg / 59%). LC-HRMS m / z (ESI + ): Calculated value for [M+H] + = 1281.6501; Experimental value for [M+H] + = 1281.6489; Error = 0.9 ppm. HPLC method 1 retention time = 7.1 minutes.

[0503] 10.2) Synthesis of Compound MAL-Phenyl-Triazole-Glucuronide MMAE

[0504] 10.2.1) Synthesis of Perfluorophenyl 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetate

[0505]

[0506] In a reaction vessel, commercially available 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]acetic acid (299 mg / 1.29 mmol), N,N'-dicyclohexylcarbodiimide (267 mg / 1.29 mmol), and pentafluorophenol (238 mg / 1.29 mmol) were dissolved in 15 mL of anhydrous 1,2-dimethoxyethane. After stirring at room temperature for 2 hours, the insoluble material was removed by filtration, and the filtrate was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 80:20 to 20:80) to obtain the title compound as a white solid (400 mg / 78%). 1 1H NMR (300 MHz, CDCl3) δ (ppm) 4.01 (s, 2H), 6.87 (s, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H). HRMS m / z (ESI + ): Calculated value for [M+H] + = 398.0446; Experimental value for [M+H] + = 398.0448; Error = -0.4 ppm.

[0507] 10.2.2) Synthesis of N-(2-azidoethyl)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide

[0508]

[0509] In a reaction vessel, the previous compound perfluorophenyl 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetate (78 mg / 0.20 mmol) was dissolved in 1 mL of anhydrous DCM. 2-Azidoethan-1-amine (33.8 mg / 0.40 mmol) was added, and the reaction was stirred at room temperature for 1 hour. Then, 1N HCl solution was added, and the mixture was extracted with DCM three times. The organic phase was dried over MgSO4, filtered, and evaporated in vacuo to obtain a crude solid, which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 60:40 to 0:100) to obtain the title compound as a white solid (18 mg / 31%). MS (ESI + ): [M+H] += 300.1; HPLC method 1 retention time = 8.4 minutes. TLC eluted with 100% EtOAc: Rf = 0.65.

[0510] 10.2.3) Synthesis of Compound MAL-Phenyl-Triazole-Glucuronide MMAE

[0511]

[0512] Mix the compound alkyne-glucuronide MMAE from Example 6 (17 mg / 15.1 μmol), copper(I) tetra(acetonitrile) hexafluorophosphate (11.2 mg / 30 μmol), and N-(2-azidoethyl)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide from the previous step (6.3 mg / 21 μmol) in an HPLC vial. Add 800 μL of anhydrous DCM / acetonitrile / NMP 1:1:1 (v / v / v) solution, and stir the reaction under argon at room temperature for 16 hours. After removing the volatiles under reduced pressure, absorb the residue in DMF and purify it on a 30 g Biotage SNAP UltraC18 (25 μm) column. Mobile phase A is water + 0.1% TFA, and mobile phase B is acetonitrile + 0.1% TFA. The gradient ranges from 10% to 60% B.

[0513] Obtain the title compound MAL-phenyl-triazole-glucuronide MMAE as an off-white solid (10.3 mg / 48%). LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 713.8425; Experimental value [M+2H] 2+ = 713.8415; Error = 1.3 ppm. HPLC method 3 retention time = 10.2 minutes.

[0514] 10.3) Synthesis of Compound MAL-Phenyl-PSARn-Triazole-Glucuronide MMAE

[0515]

[0516] Mix the compound alkyne-glucuronide MMAE from Example 6 (20 mg / 17.7 μmol), copper(I) tetra(acetonitrile) hexafluorophosphate (13.2 mg / 35 μmol), and N3-PSARn-phenyl-MAL from Example 4 (34.3 mg / 28 μmol) in an HPLC vial. Add 900 μL of NMP / DCM 2:1 (v / v) solution, and stir the reaction at room temperature under argon for 16 hours. After removing the volatiles under reduced pressure, absorb the residue in DMF and in 30 g Biotage Purification on a SNAP Ultra C18 (25 μm) column. Mobile phase A is water + 0.1% TFA, and mobile phase B is acetonitrile + 0.1% TFA. The gradient ranges from 10% to 60% B.

[0517] The title compound, MAL-phenyl-PSARn-triazole-glucuronide MMAE, was obtained as a white solid (16.0 mg / 39%). LC-HRMS m / z (ESI + ): Calculated [M + 2H] 2+ = 1168.5759; Observed [M + 2H] 2+ = 1168.5792; Error = -2.8 ppm. HPLC method 3 retention time = 6.8 minutes.

[0518] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR12 (DAR8):

[0519] The following LDC compounds were prepared and characterized:

[0520] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR6

[0521] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR12

[0522] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR18

[0523] Trastuzumab-Lys (glucuronide MMAE)-PSAR6

[0524] Trastuzumab-Lys (glucuronide MMAE)-PSAR12

[0525] Trastuzumab-BAC-Ngly (triazole-glucuronide MMAE)-PSAR12

[0526] Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR6

[0527] Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR12

[0528] Trastuzumab-MAL-phenyl-Ngly (triazole-val-cit-PAB-MMAE)-PSAR12

[0529] Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronide MMAE)-PSAR18

[0530] Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-PSAR18

[0531] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide-irinotecan)-PSAR18

[0532] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12

[0533] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18

[0534] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0535] CD19-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0536] CD22-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0537] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)-PSAR18

[0538] Trastuzumab-MAL-phenyl-Ngly[triazole-glucuronide(MMAE)2]-PSAR24

[0539] Trastuzumab-MAL-phenyl-Ngly[triazole-galactoside(MMAE)2]-PSAR24

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

[0541] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSAR18

[0542] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12

[0543] Trastuzumab-glucuronide MMAE

[0544] Trastuzumab-MAL-phenyl-triazole-glucuronide MMAE

[0545] Trastuzumab-MAL-phenyl-PSAR12-triazole-glucuronide MMAE

[0546] Human Albumin-MAL-Phenyl-Ngly(Triazole-Glucuronide MMAE)-PSAR24

[0547] Their structures are described in Table 6 below.

[0548] Table 6

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559] 11.1) Preparation of the Conjugate

[0560] 11.1.1) Preparation of the Antibody-Drug Conjugate

[0561] The antibody solution (10 mg / mL in PBS 7.4 + 1 mM EDTA) was treated with 14 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 2 hours. For maleimide-based conjugation, the fully reduced antibody was buffer-exchanged with potassium phosphate 100 mM pH 7.4 + 1 mM EDTA using an Amicon 30K centrifugal filter device (Merck Millipore) by three rounds of dilution / centrifugation. 10 - 12 molar equivalents of the drug-linker (from a 12 mM DMSO stock solution) were added to the antibody (residual DMSO < 10% v / v). The solution was incubated at room temperature for 30 minutes. For bromoacetamide-based conjugation, the fully reduced antibody was buffer-exchanged with borate buffer 50 mM pH 8.1 + 1 mM EDTA and conjugation was achieved using 16 molar equivalents of the drug-linker over 24 hours at 37 °C in the dark. The conjugate was buffer-exchanged / purified with PBS 7.4 by four rounds of dilution / centrifugation using 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 incorporating a self-hydrolyzable maleimide (MAL-phenyl) group was incubated in PBS 7.4 at 5 mg / mL at 37 °C for 48 h to ensure complete hydrolysis of the succinimide moiety. The final protein concentration was evaluated spectrophotometrically at 280 nm using a Colibri micro-spectrophotometer device (Titertek Berthold).

[0562] 11.1.2) Preparation of human albumin-drug conjugates

[0563] 2 molar equivalents of the drug-linker (from a 12 mM DMSO stock solution) were added to the human albumin solution (10 mg / mL in potassium phosphate 100 mM pH 7.4 + 1 mM EDTA). Residual DMSO < 10% (v / v). The solution was incubated at room temperature for 4 hours. The conjugate was buffer-exchanged / purified with PBS 7.4 by four rounds of dilution / centrifugation using an Amicon 30K centrifugal filter device. Alternatively, the conjugate was buffer-exchanged / purified using a PD MiniTrap G-25 column (GE Healthcare) and sterile filtered (0.20 μm PES filter). The conjugate was incubated in PBS 7.4 at 5 mg / mL at 37 °C for 48 h to ensure complete hydrolysis of the succinimide moiety. The final protein concentration was evaluated spectrophotometrically at 280 nm using a Colibri micro-spectrophotometer device (Titertek Berthold).

[0564] 11.2) Characterization of the conjugate

[0565] The resulting conjugate was characterized as follows:

[0566] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR18 (DAR8):

[0567] Denaturing RPLC-QToF analysis was performed using the above UHPLC method 5. Briefly, the conjugate was eluted on an Agilent PLRP-S 1000 2.1 x 150 mm 8 μm (80 °C) with a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min), and scanned in the range of 500 - 3500 m / z using a Bruker Impact II TM Q-ToF mass spectrometer (ESI + ) to detect the conjugate. The data was deconvoluted using the MaxEnt algorithm included in the Bruker Compass software.

[0568] Trastuzumab-Lys (glucuronide MMAE)-PSAR6 (DAR8):

[0569] SEC was performed on an Agilent 1050 HPLC system with an extra-column volume of less than 15 μL (equipped with a PEEK tube with an inner diameter of 0.12 mm and a small-volume UV flow cell). The column was a Waters Acquity UPLC Protein BEH SEC 200 4.6 x 150 mm 1.7 μm (maintained at room temperature) or an Agilent AdvanceBio SEC 300 4.6 x 150 mm 2.7 μm (maintained 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.

[0570] Trastuzumab-Lys (glucuronide MMAE)-PSAR12 (DAR8):

[0571] Hydrophobic interaction chromatography (HIC) was performed on an Agilent 1050 HPLC system. The chromatographic column was Tosoh TSK-GEL BUTYL-NPR 4.6 x 35 mm 2.5 μm (25 °C). Mobile phase A was 1.5 M (NH4)2SO4 + 25 mM potassium phosphate at pH 7.0. Mobile phase B was 25 mM potassium phosphate at pH 7.0 + 15% isopropanol (v / v). The linear gradient was from 0% B to 100% B in 10 minutes, followed by holding at 100% B for 3 minutes. The flow rate was 0.75 mL / min. UV detection was monitored at 220 and 280 nm.

[0572] 11.3) Overview of conjugate characterization

[0573] The conjugate showed an LC-1d (light chain with 1 drug-linker) and an HC-3d (heavy chain with 3 drug-linkers) absorption peak (DAR8 conjugate) on its denaturing RPLC chromatogram. For the mass spectrometry analysis of the heavy chain, the major glycoform (G0F of trastuzumab) was reported. The conjugate showed a single absorption peak on its HIC chromatogram.

[0574] Trastuzumab-BAC-Ngly (triazole-glucuronide MMAE)-PSAR12 (DAR8) :

[0575] Deconvoluted LC-1d calculated value: 25416; Observed value: 25417 / Deconvoluted HC-3d calculated value: 56529; Obs: 56528

[0576] Monomer purity: 97.2%

[0577] HIC retention time: 8.8 minutes

[0578] ​

[0579] Deconvoluted LC-1d calculated value: 25844; Observed value: 25844 / Deconvoluted HC-3d calculated value: 57805; Observed value: 57805

[0580] Monomer purity: 99.0%

[0581] HIC retention time: 8.8 minutes

[0582] ​ Deconvoluted LC-1d calculated value: 26270; Observed value: 26270 / Deconvoluted HC-3d calculated value: 59086; Observed value: 59086

[0583] Monomer purity: 96.5%

[0584] HIC retention time: 8.8 minutes

[0585] ​

[0586] Deconvolution LC-1d calculated value: 25360; Observed value: 25360 / Deconvolution HC-3d calculated value: 56352; Observed value: 56353

[0587] Monomer purity: 99+%

[0588] HIC retention time: 7.6 minutes

[0589] ​

[0590] Deconvolution LC-1d calculated value: 25786; Observed value: 25786 / Deconvolution HC-3d calculated value: 57634; Observed value: 57632

[0591] Monomer purity: 99+%

[0592] HIC retention time: 7.5 minutes

[0593] ​

[0594] Deconvolution LC-1d calculated value: 25661; Observed value: 25662 / Deconvolution HC-3d calculated value: 57264; Observed value: 57262

[0595] Monomer purity: 99+%

[0596] HIC retention time: 7.0 minutes (DAR8 conjugate). As observed in the HIC chromatogram, the ADC is a heterogeneous mixture that contains 20% of DAR6, 20% of DAR7 and 60% of DAR8 conjugate.

[0597] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR6 (DAR8)(=ADC-PSAR6)

[0598] Deconvolution LC-1d calculated value: 25368; Observed value: 25368 / Deconvolution HC-3d calculated value: 56382; Observed value: 56380

[0599] Monomer purity: 99+%

[0600] HIC retention time: 7.5 minutes

[0601] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR12 (DAR8)(=ADC- PSAR12)

[0602] Deconvolution LC-1d calculated value: 25794; Observed value: 25794 / Deconvolution HC-3d calculated value: 57660; Observed value: 57660.

[0603] Monomer purity: 99+%

[0604] HIC retention time: 7.1 minutes

[0605] Trastuzumab-MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSAR12 (DAR8)

[0606] Deconvolution LC-1d calculated value: 25871; Observed value: 25870 / Deconvolution HC-3d calculated value: 57889; Observed value: 57888

[0607] Monomer purity: 99+%

[0608] HIC retention time: 9.2 minutes

[0609] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR18 (DAR8)(=ADC- PSAR18)

[0610] Deconvolution LC-1d calculated value: 26221; Observed value: 26221 / Deconvolution HC-3d calculated value: 58939; Observed value: 58939

[0611] Monomer purity: 99+%

[0612] HIC retention time: 6.8 minutes

[0613] Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-PSAR18 (DAR8)

[0614] Deconvolution LC-1d calculated value: 25567; Observed value: 25567 / Deconvolution HC-3d calculated value: 56979; Observed value: 56977

[0615] Monomer purity: 99+%

[0616] HIC retention time: 5.1 minutes

[0617] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide- Con)-PSAR18(DAR8)

[0618] Deconvolution LC-1d calculated value: 25938; Observed value: 25937 / Deconvolution HC-3d calculated value: 58092; Observed value: 58089

[0619] Monomer purity: 99+%

[0620] HIC retention time: 5.7 minutes

[0621] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12 (DAR8)

[0622] Deconvolution LC-1d calculated value: 25446; Observed value: 25446 / Deconvolution HC-3d calculated value: 56614; Observed value: 56612

[0623] Monomer purity: 99+%

[0624] HIC retention time: 5.2 minutes

[0625] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18 (DAR8)

[0626] Deconvolution LC-1d calculated value: 25872; Observed value: 25872 / Deconvolution HC-3d calculated value: 57894; Observed value: 57892

[0627] Monomer purity: 99+%

[0628] HIC retention time: 5.1 minutes

[0629] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR8)(=ADC- PSAR24)

[0630] Deconvolution LC-1d calculated value: 26647; Observed value: 26674 / Deconvolution HC-3d calculated value: 60218; Observed value: 60218

[0631] Monomer purity: 99+%

[0632] HIC retention time: 6.7 minutes

[0633] CD19-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR8)

[0634] Deconvolution LC-1d calculated value: 27347; Observed value: 27347 / Deconvolution HC-3d calculated value: 60137; Observed value: 60132

[0635] Monomer purity: 92.6%

[0636] HIC retention time: 6.8 minutes

[0637] CD22-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR8)

[0638] Deconvolution LC-1d calculated value: 27341; Observed value: 27341 / Deconvolution HC-3d calculated value: 60314; Observed value: 60311

[0639] Monomer purity: 97.4%

[0640] HIC retention time: 6.8 minutes

[0641] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)- PSAR18 (DAR16)

[0642] Deconvolution LC-1d calculated value: 27544; Observed value: 27545 / Deconvolution HC-3d calculated value: 62910; Observed value: 62907

[0643] Monomer purity: 98.5%

[0644] HIC retention time: 8.7 minutes

[0645] Trastuzumab-MAL-phenyl-Ngly[triazole-glucuronic acid (MMAE)2 -PSAR24 (DAR16)

[0646] Deconvolution LC-1d calculated value: 27569; Observed value: 27570 / Deconvolution HC-3d calculated value: 62985; Observed value: 62983

[0647] Monomer purity: 98.2%

[0648] HIC retention time: 9.9 minutes

[0649] Trastuzumab-MAL-phenyl-Ngly[triazole-galactoside (MMAE)2 -PSAR24 (DAR16)

[0650] Deconvolution LC-1d calculated value: 27555; Observed value: 27554 / Deconvolution HC-3d calculated value: 62943; Observed value: 62940

[0651] Monomer purity: 99+%

[0652] HIC retention time: 10.2 minutes

[0653] Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18 (DAR16)

[0654] Deconvolution LC-1d calculated value: 26237; Observed value: 26237 / Deconvolution HC-3d calculated value: 58989; Observed value: 58987

[0655] Monomer purity: 99+%

[0656] HIC retention time: 6.6 minutes

[0657] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)- PSAR18 (DAR16)

[0658] Deconvolution LC-1d calculated value: 27270; Observed value: 27270 / Deconvolution HC-3d calculated value: 62086; Observed value: 62087

[0659] Monomer purity: 99+%

[0660] HIC retention time: 5.7 minutes

[0661] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 (DAR8)(=ADC-PEG12)

[0662] Deconvolution LC-1d calculated value: 25541; Observed value: 25541 / Deconvolution HC-3d calculated value: 56901; Observed value: 56900​​

[0663] Monomer purity: 99+%

[0664] HIC retention time: 7.4 minutes

[0665] Trastuzumab-glucuronide MMAE (DAR8):

[0666] Deconvolution LC-1d calculated value: 24721; Observed value: 24720 / Deconvolution HC-3d calculated value: 54439; Observed value: 54438

[0667] Monomer purity: 95.2%

[0668] HIC retention time: 9.2 minutes

[0669] Trastuzumab-MAL-phenyl-triazole-glucuronide MMAE (DAR8)(=ADC-PSAR0):

[0670] Deconvolution LC-1d calculated value: 24884; Observed value: 24884 / Deconvolution HC-3d calculated value: 54926; Observed value: 54926

[0671] Monomer purity: 98.5%

[0672] HIC retention time: 8.4 minutes

[0673] Trastuzumab-MAL-phenyl-PSAR12-triazole-glucuronide MMAE (DAR8)(=ADC-PSAR12L):

[0674] Deconvolution LC-1d calculated value: 25793; Observed value: 25793 / Deconvolution HC-3d calculated value: 57657; Observed value: 57657

[0675] Monomer purity: 99+%

[0676] HIC retention time: 8.5 minutes

[0677] Human albumin-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR1):

[0678] Deconvolution calculated value: 69765; Observed value: 69645

[0679] Monomer purity: 90.8%

[0680] HIC retention time: 3.9 minutes

[0681] Trastuzumab:

[0682] Deconvolution LC observed value: 23439 / Deconvolution HC observed value: 50595

[0683] Monomer purity: 99+%

[0684] HIC retention time: 4.7 minutes

[0685] Anti-CD19 antibody:

[0686] Deconvoluted LC Observation: 24139 / Deconvoluted HC Observation: 50517

[0687] Monomer Purity: 93.2%

[0688] HIC Retention Time: 4.7 minutes

[0689] Anti-CD22 antibody:

[0690] Deconvoluted LC Observation: 24133 / Deconvoluted HC Observation: 50692

[0691] Monomer Purity: 99+%

[0692] HIC Retention Time: 4.8 minutes

[0693] Human albumin :

[0694] Deconvoluted Observation: 66556

[0695] Monomer Purity: 92.4%

[0696] HIC Retention Time: 2.5 minutes

[0697] Example 12: Hydrophobic interaction chromatography (HIC) profiles of non-poly(sarcosine)-based antibody-drug conjugates (ADC-PSAR0), poly(sarcosine)-based antibody-drug conjugates with orthogonal configuration (ADC-PSAR12), and poly(sarcosine)-based antibody-drug conjugates with linear configuration (ADC-PSAR12L). Figure 1 Example 13: Hydrophobic interaction chromatography (HIC) profiles of poly(sarcosine)- and poly(ethylene glycol)-based antibody-drug conjugates

[0698] According to the method described in Example 11, the relative exposure of the conjugated payload to the bulk solvent and the apparent hydrophobicity of the trastuzumab-based DAR8 ADC were evaluated by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR column. The results are as Figure 2 shown. When poly(sarcosine) is grafted in a parallel (i.e., orthogonal) direction relative to the drug unit, effective hydrophobic masking properties can be provided and the apparent hydrophobicity of the conjugate (ADC-PSAR12) can be reduced. However, when poly(sarcosine) is in a linear (i.e., continuous) configuration, no reduction in the apparent hydrophobicity of the conjugate (ADC-PSAR12L) was observed.

[0699] Example 14: Pharmacokinetic curves (total antibody concentration over time) in mice after a single intravenous injection of 3 mg / kg dose of non-poly(sarcosine)-based antibody-drug conjugate (ADC-PSAR0) and poly(sarcosine)-based antibody-drug conjugate (ADC-PSAR12). Figure 3

[0700] According to the method described in Example 11, the relative exposure of the conjugated payload to the bulk solvent and the apparent hydrophobicity of the trastuzumab-based DAR8 ADC were evaluated by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR column. The results are as Example 15: Single intravenous administration of 3 mg / kg dose of non-poly(sarcosine)-based antibody-drug conjugate in BT-474 breast cancer xenograft modelAs shown, in the case of equal length (n = 12 monomer units), poly(sarcosine) has better hydrophobic masking properties (shorter retention time) than polyethylene glycol.

[0701] Figure 4A Example 16: Pharmacokinetic profiles (total antibody concentration over time) in mice after a single intravenous injection of 3 mg / kg dose of poly(sarcosine)-based antibody-drug conjugate (ADC-PSAR12) and poly(ethylene glycol)-based antibody-drug conjugate (ADC-PEG12). Figure 5

[0702] Male SCID mice (4 - 6 weeks old) were injected via the tail vein with the ADC at a dose of 3 mg / kg (five animals per dose group, randomly assigned). At different time points, blood was drawn into citrate tubes via retro-orbital bleeding and processed into plasma. The total concentration of the ADC was evaluated using a human IgG ELISA kit (Stemcell TM Technologies) according to the manufacturer's protocol. A standard curve of trastuzumab was used for quantification. Pharmacokinetic parameters (clearance and AUC) were calculated by non-compartmental analysis using software incorporating PK functions (developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA). The results are shown in Pharmacokinetics of 2.5 mg / kg of poly(sarcosine)-based antibody-drug conjugates with different PSAR lengths in orthogonal directions (ADC-PSAR6, ADC-PSAR12, ADC-PSAR18, ADC-PSAR24); poly(ethylene glycol)-based antibody-drug conjugate with orthogonal configuration (ADC-PEG12) and poly(sarcosine)-based antibody-drug conjugate with linear configuration (ADC-PSAR12L). . The ADC containing poly(sarcosine) exhibited favorable pharmacokinetics compared to the ADC without poly(sarcosine).

[0703] Figure 6 Tumor volumes (mm 3 ) and survival curves of amino acid-based ADC (ADC-PSAR0) and poly(sarcosine)-based ADC (ADC-PSAR12)

[0704] BT-474 breast cancer cells were subcutaneously implanted into female SCID mice (4 weeks old). When the tumors grew to approximately 150 mm 3 , the ADC of Example 14 above was administered as a single intravenous dose at a rate of 3 mg / kg (on day 20, 5 animals per group to minimize the difference in initial tumor volume between groups). The results are shown in ​ and 4B . The tumor volume was measured every 3 - 5 days using a caliper device and calculated using the formula (L × W 2 ) / 2. The mice were sacrificed when the tumor volume exceeded 1000 mm 3 . The ADC containing poly(sarcosine) had improved in vivo activity compared to the ADC without poly(sarcosine). No significant change in body weight was observed in the treated mice.

[0705] ​ ​ ​

[0706] Experiments were conducted in male CD-1 mice (4 - 6 weeks old) according to the method described in Example 14. The results are shown in​ Among ADCs containing poly(ethylene glycol), the ADCs containing poly(sarcosine) have improved pharmacokinetic parameters.

[0707] Example 17: Tumor volume (mm 3 ) in a BT-474 breast cancer xenograft model, where the model was administered a single intravenous dose ​ ​ ​

[0708] Experiments were conducted as described in Example 15. BT-474 breast cancer cells were subcutaneously implanted into female SCID mice (4 weeks old). When the tumors grew to approximately 150 mm 3 in size, the ADC was administered as a single intravenous dose at a dose of 2.5 mg / kg (on day 13, 6 animals per group to minimize the difference in initial tumor volume between groups). The results are shown in ​ No significant change in body weight was observed in the treated mice.

Claims

1. A ligand-drug conjugate (LDC) having the following formula (XV) wherein L is an orthogonal linker that permits (HP SMW ) to be in an orthogonal direction with respect to (X-D), and L is selected from one or more natural or unnatural amino acids, HP SMW is produced by covalently binding a poly(sarcosine) homopolymer to the orthogonal linker L, and the HP SMW represents wherein R4 represents -OR' or -NR'2, or wherein R4 represents -C(=O)R', the wavy bond represents the point of attachment to L, or, if a spacer Z is present, the point of attachment to spacer Z, k is from 2 to 50, and Each R’ is independently -H or -C1-C 20 alkyl; D is a cytotoxic drug, X is a cleavable moiety for releasing D upon enzymatic treatment, wherein the recognition site for enzymatic treatment is a dipeptide cleavage site, Z is an optional spacer selected from alkylene; heteroalkylene; alkoxy; polyether; one or more natural or unnatural amino acids; C3-C8 heterocyclic group; C3-C8 carbocyclic group; arylene; and any combination thereof, and a is 1 or greater, b is 1 or greater and m is 1 or greater, and the ligand is selected from full-length antibodies and antigen-binding fragments thereof.

2. The LDC compound of claim 1, wherein the spacer Z is present between the L and the ligand, and / or between the L and the HP SMW , and / or between the L and the X, and / or between the X and the D.

3. The LDC compound of any one of claims 1-2, wherein D is an anti-cancer drug.

4. The LDC compound of any one of claims 1-3, wherein L is selected from glutamic acid, lysine and glycine.

5. The LDC compound of any one of claims 1-4, wherein Z has formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII), wherein the tilde represents a linking point and R6 is –C1-C 10 alkylene-, –C1-C 10 heteroalkylene-, –C1-C 10 alkylene-C(=O)-, –C1-C 10 heteroalkylene-C(=O)-, -arylene-C1-C 10 alkylene-C(=O)-, -arylene-C1-C 10 alkylene-O-C(=O)-, and any one of the R6 groups is optionally substituted by one or more ═O.

6. An intermediate compound having formula (XVI) wherein L is an orthogonal linker, and L is selected from one or more natural or unnatural amino acids, HP SMW is produced by covalently binding a poly(sarcosine) homopolymer to the orthogonal linker L, And the HP SMW represents wherein R4 represents -OR' or -NR'2, or wherein R4 represents -C(=O)R', Among them, the wavy bond represents the point of attachment to L, or, if a spacer Z is present, the point of attachment to spacer Z, k is from 2 to 50, and Each R’ is independently -H or -C1-C 20 alkyl; D is a cytotoxic drug, X is a cleavable moiety for releasing D upon enzymatic treatment, wherein the recognition site for enzymatic treatment is a dipeptide cleavage site, Z is an optional spacer selected from alkylene; heteroalkylene; alkoxy; polyether; one or more natural or unnatural amino acids; C3-C8 heterocyclic group; C3-C8 carbocyclic group; arylene; and any combination thereof, and a is 1 or greater and b is 1 or greater.

7. Use of an LDC according to any one of claims 1-5 for the manufacture of a medicament.

8. A compound having formula (XXIII) wherein R6 is –C1-C 10 alkylene-, –C1-C 10 heteroalkylene-, -C3-C8 cycloalkyl-, -O-(C1-C8 alkyl)-, -arylene-, –C1-C 10 alkylene-arylene-, -arylene-C1-C 10 alkylene-, –C1-C 10 alkylene-(C3-C8 cycloalkyl)-, -(C3-C8 cycloalkyl)-C1-C 10 alkylene-, -C3-C8 heterocyclic group-, –C1-C 10 alkylene-(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)–C1-C 10 alkylene-, –C1-C 10 alkylene-C(=O)-, –C1-C 10 heteroalkylene-C(=O)-, -C3-C8 cycloalkyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 alkylene-arylene-C(=O)-, -arylene-C1-C 10 alkylene-C(=O)-, -C1-C 10 alkylene-(C3-C8 cycloalkyl)-C(=O)-, -(C3-C8 cycloalkyl)-C1-C 10 alkylene-C(=O)-, -C3-C8 heterocyclic group-C(=O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic group)-C(=O)-, -(C3-C8 heterocyclic group)-C1-C 10 alkylene-C(=O)-, -C1-C 10 alkylene-NH-, -C1-C 10 heteroalkylene-NH-, -C3-C8 cycloalkyl-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 alkylene-NH-, -C1-C 10 alkylene-(C3-C8 cycloalkyl)-NH-, -(C3-C8 cycloalkyl)-C1-C 10 alkylene-NH-, -C3-C8 heterocyclic group-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic group)-NH-, -(C3-C8 heterocyclic group)-C1-C 10 alkylene-NH-, -C1-C 10 alkylene-S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 cycloalkyl-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 cycloalkyl)-S-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-S-, –C1-C 10 Alkylene-O-C(=O)-, -C3-C8 cycloalkyl-O-C(=O)-, -O-(C1-C8 alkyl)-O-C(=O)-, -arylene-O-C(=O)-, -C1-C 10 Alkylene-arylene-O-C(=O)-, -arylene-C1-C 10 Alkylene-O-C(=O)-, -C1-C 10 Alkylene-(C3-C8 cycloalkyl)-O-C(=O)-, -(C3-C8 cycloalkyl)-C1-C 10 Alkylene-O-C(=O)-, -C3-C8 heterocyclic-O-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-O-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-O-C(=O)-, Any one of the R6 groups is optionally substituted by one or more substituents selected from the following: -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2, -NR'3 + , =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2 and C(=NR')NR'2, where each X is independently a halogen: -F, -CI, -Br, or –I; and each R' is independently -H, -C1-C 20 alkyl, -C6-C 20 aryl, or -C3-C 14 heterocyclic group, Z is an optional spacer selected from alkylene; heteroalkylene; alkoxy; polyether; one or more natural or unnatural amino acids; C3-C8 heterocyclic group; C3-C8 carbocyclic group; arylene; and any combination thereof, L is an orthogonal linker and L is selected from one or more natural or unnatural amino acids, X is a cleavable moiety for releasing D upon enzymatic treatment, wherein the recognition site for enzymatic treatment is a dipeptide cleavage site, D is a cytotoxic drug, a is 1 or greater and b is 1 or greater, and HP SMW is produced by covalently binding a poly(sarcosine) homopolymer to the orthogonal linker L, And the HP SMW represents wherein R4 represents -OR' or -NR'2, or wherein R4 represents -C(=O)R', Among them, the wavy bond represents the point of attachment to L, or, if a spacer Z is present, the point of attachment to spacer Z, k is from 2 to 50, and Each R’ is independently -H or -C1-C 20 alkyl group.

Images