CN117157102A - Enzyme-triggered self-reactive linkers with improved physicochemical and pharmacological properties - Google Patents

Abstract

The present invention relates to enzyme-triggered self-reacting arm compounds, chemical intermediates for the preparation of such compounds and their use, in particular in prodrug design and coupling techniques. The invention also relates to ligand-drug-conjugates (LDCs) comprising such enzyme-triggered self-reactive arms.

Description

Enzyme-triggered self-reactive linkers with improved physicochemical and pharmacological properties

Technical Field

The present invention relates to compounds comprising an enzyme-triggered self-reactive arm, chemical intermediates for preparing such compounds and their use, in particular in prodrug design and conjugation technology.

The present invention also relates to ligand-drug-conjugates (LDCs) comprising such an enzyme-triggered self-reactive arm.

Background Art

Ligand-drug-conjugates (LDCs) are designed to deliver active compounds specifically to target tissues while sparing healthy tissues. For example, for LDCs designed to deliver cytotoxic anticancer agents to tumor cells, the LDC format can be used to improve the toxicity profile and improve the tolerability of the treatment.

LDC comprises at least one ligand unit, which is usually a polypeptide, protein or targeted small molecule, and the ligand unit is covalently linked to at least one treatment, diagnosis or labeling compound (hereinafter referred to as drug or D) through a synthetic linker. The synthetic linker may include one or more monovalent or bivalent arms for connecting the ligand unit and the drug unit, which may be selected from a spacer, a connector and an enzyme-sensitive cleavable portion. The linker may also orthogonally carry any portion that can improve LDC performance (such as storage stability, plasma stability or pharmacokinetic properties). When the ligand unit of the conjugate is an antibody or antibody fragment and is coupled to an immunostimulatory drug, a cytotoxic drug or a chemotherapeutic drug, the term antibody-drug-conjugate (ADC) is generally used.

When designing LDC, it is necessary to covalently connect the final active drug to the ligand targeting unit, while allowing the drug unit to be finally released by a selective enzyme mechanism after cell internalization or in the diseased tissue microenvironment. To this end, several peptidase and glycosidase sensitive cleavable linker chemical strategies (related to self-degradable chemistry) have been developed. These cleavable linkers and their corresponding cleavage mechanisms are well known and have been described in several publications (e.g., Bargh JG et al., Chem. Soc. Rev., 2019, 48, 4361, Toki et al. J. Org. Chem. 2002, 67, 6, 1866–1872, Scott et al. Bioconjugate Chem. 2006, 17, 3, 831–840).

For example, WO2011145068 discloses the use of glycosidase-sensitive cleavable drug-linkers based on a 4-(1-hydroxybut-3-yn-1-yl)phenol self-immolative chemical spacer, thereby conferring the ability of the chemical terminal alkyne to control click chemistry (see the formula below).

WO2017089895 describes the use of glycosidase-sensitive cleavable drug-linkers based on a 2-hydroxy-5-(hydroxymethyl)benzoic acid self-immolative chemical spacer (see the formula below).

However, there is an ongoing need to develop alternative enzyme-sensitive self-immolative linkers to improve the physicochemical and pharmacological properties of LDCs.

Therefore, in this context, one of the objects of the present disclosure is to provide enzyme-sensitive self-immolative linkers that can be used to prepare LDCs.

Another object of the present disclosure is an enzyme-sensitive self-immolative linker that can improve the physicochemical and/or pharmacological properties of LDC.

Another object of the present disclosure is to provide compounds that can be used to prepare LDC.

Brief description of the invention

In a first aspect, the present disclosure relates to a ligand-drug-conjugate compound (LDC) having the following formula (I):

in

L is a ligand;

X1 is the connector unit;

Z is an optional spacer;

X2 is the joint unit;

K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

R1 is selected from the group consisting of H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl having 6-10 ring atoms, C ₃ -C ₈ cycloalkyl, heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores;

Each R2 is independently selected from the group consisting of an electron withdrawing group and a C ₁ -C ₄ alkyl group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y is O, or when T is a polypeptide cleavable unit, Y is NR3;

R3 is selected from the group consisting of H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl having 6-10 ring atoms, C ₃ -C ₈ cycloalkyl, heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms, and any combination thereof;

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

Surprisingly, the use of a self-immolative linker based on 2-amino-1-(4-aminophenyl)ethane-1-ol or based on 2-amino-1-(4-oxophenyl)ethane-1-ol as in formula (I) produces an LDC with improved hydrophilicity and improved in vivo pharmacokinetic characteristics. This self-immolative linker also makes the preparation, purification and formulation of the compound of formula (I) easier and therefore more productive. Due to the presence of this particular linker, the compound of formula (I) also has a smaller aggregation potential.

The present disclosure also relates to a pharmaceutical composition comprising a compound of the present disclosure, preferably a compound of formula (I), and a pharmaceutically acceptable carrier.

The present disclosure also relates to compounds of the present disclosure, preferably compounds of formula (I), for use as medicaments.

The present disclosure also relates to intermediate compounds of formula (II):

in

X1′ is a group that can react with a ligand to form a linking unit;

Z is an optional spacer;

X2 is the joint unit;

K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

R1′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof;

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores;

Each R2 is independently selected from the group consisting of an electron withdrawing group and a C ₁ -C ₄ alkyl group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y′ is O, or when T is a polypeptide cleavable unit, Y′ is NR3′;

R3′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

The present disclosure also relates to an intermediate compound of formula (III)

in

R1′ is selected from the group consisting of an amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, an aryl group having 6-10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3-10 ring atoms, a heteroaryl group having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores;

Each R2 is independently selected from the group consisting of an electron withdrawing group and a C ₁ -C ₄ alkyl group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a Sugar Cleavable Unit, Y' is O, or when T is a Polypeptide Cleavable Unit, Y' is NR3';

R3′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

The present disclosure also relates to an intermediate compound of formula (IV)

in

R1′ is selected from the group consisting of an amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, an aryl group having 6-10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3-10 ring atoms, a heteroaryl group having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

Each R2 is independently selected from the group consisting of: an electron withdrawing group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y′ is O, or when T is a polypeptide cleavable unit, Y′ is NR3′;

R3′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

1 and 2 show hydrophobic interaction chromatograms according to Example 3.

3 and 4 show in vitro cytotoxicity assays of the conjugates according to Example 4.

FIG. 5 shows an in vitro cytotoxicity assay of the conjugate according to Example 5.

6 , 7 , 8 and 9 show the in vivo pharmacokinetic curves and pharmacokinetic parameters in rats according to Example 6.

FIG. 10 shows the changes in tumor volume over time in the gastric cancer mouse xenograft model according to Example 7.

DETAILED DESCRIPTION OF THE INVENTION

definition

Various embodiments of the present disclosure are described herein. It will be appreciated that features specified in each embodiment may be combined with other specified features to provide further embodiments.

The present disclosure encompasses the compounds of the present disclosure, their tautomers, enantiomers, diastereomers, racemates or mixtures thereof, and hydrates, esters, solvates or pharmaceutically acceptable salts thereof.

Any chemical formula given herein is also intended to represent unlabeled as well as isotopically labeled forms of the compounds, such as a deuterium-labeled compound or ^a14C -labeled compound.

The term " pharmaceutically acceptable salt " refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure, and which are typically not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with organic acids and/or inorganic acids. Pharmaceutically acceptable base addition salts can be formed with organic bases and/or inorganic bases. These salts are well known to those skilled in the art.

As used herein, the term " C ₁ -C ₂₄ alkyl " by itself or as part of another substituent refers to a straight or branched chain alkyl functional group having 1 to 24 carbon atoms, preferably 1 to 20 carbon atoms, more preferably 1 to 12 or 1 to 6 carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl and its isomers (e.g., n-pentyl, isopentyl) and hexyl and its isomers (e.g., n-hexyl, isohexyl).

For example, alkylene, used alone or as part of an alkylene glycol, refers to a divalent saturated, straight or branched chain alkyl group as defined herein.

Alkenyl and alkynyl refer to at least partially unsaturated straight-chain or branched hydrocarbon radicals having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, and especially 2 to 4 carbon atoms. Alkenyl contains at least one C=C double bond; alkynyl contains at least one C≡C triple bond.

As used herein, the term " C ₃ -C ₈ cycloalkyl " or " carbocycle " refers to a saturated or unsaturated cyclic group having 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms. The cycloalkyl group may have a single ring or multiple rings fused together. The cycloalkyl group may also include a spiro ring. Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, the term " C ₃ -C ₈ cycloalkylene " or " carbocycle " refers to a divalent cycloalkyl group as defined herein.

As used herein, the term " halogen " refers to a fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I) group.

As used herein, the term " C ₁ -C ₆ haloalkyl " refers to a C ₁ -C ₆ alkyl group as defined herein, substituted by one or more halogen groups as defined herein. Suitable C ₁ -C ₆ haloalkyl groups include trifluoromethyl and dichloromethyl.

As used herein, the term " heteroalkyl " refers to a straight or branched hydrocarbon chain consisting of 1-12 carbon atoms, preferably 1-10, more preferably 1-6 carbon atoms and 1-3 heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized (e.g., sulfoxide or sulfone) and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N and S may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule.

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

As used herein, the term " C ₁ -C ₆ alkoxy " refers to -O-alkyl, wherein the alkyl is a C ₁ -C ₆ alkyl as defined herein. Suitable C ₁ -C ₆ alkoxy groups include methoxy, ethoxy, propoxy.

As used herein, the term " C ₁ -C ₆ haloalkoxy " refers to a C ₁ -C ₆ alkoxy group as defined herein, substituted by one or more halo groups as defined herein. Suitable haloalkoxy groups include trifluoromethoxy.

As used herein, the term " aryl having 6-10 ring atoms " refers to a polyunsaturated aromatic hydrocarbon group having a single ring or multiple aromatic rings fused together, containing 6-10 ring atoms, wherein at least one ring is aromatic. The aromatic ring may optionally include 1-2 other rings (such as cycloalkyl, heterocyclyl or heteroaryl as defined herein) fused thereto. Suitable aryl groups include phenyl, naphthyl and benzene rings fused to heterocyclyls, such as benzopyranyl, benzodioxolyl, benzodioxanyl, and the like.

Arylene refers to a divalent aromatic group as defined above.

As used herein, the term " heteroaryl having 5-10 ring atoms " refers to a polyunsaturated aromatic ring system having a single ring or multiple aromatic rings fused together or covalently linked, containing 5-10 atoms, wherein at least one ring is aromatic and at least one ring atom is a heteroatom selected from N, O and S. The nitrogen heteroatom and sulfur heteroatom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Such rings may be fused to an aryl, cycloalkyl or heterocyclyl ring. Non-limiting examples of such heteroaryl groups include furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, and quinoxalinyl.

As used herein, the term " heterocyclyl having 3-10 ring atoms ", " heterocycloalkyl having 3-10 ring atoms " or " heterocyclyl " refers to a saturated or unsaturated cyclic group having 3-10 ring atoms, preferably 3-8 ring atoms, wherein at least one ring atom is a heteroatom selected from N, O and S. Nitrogen heteroatoms and sulfur heteroatoms may be optionally oxidized, and nitrogen heteroatoms may be optionally quaternized. The heterocycle may include fused or bridged rings and spirocycles. Examples of heterocycles include, but are not limited to, tetrahydropyridyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperazinyl, 1-azepanyl, imidazolinyl, 1,4-dioxanyl, and the like.

As used herein, the term " heterocyclyl " or " heterocycloalkylene " refers to a divalent heterocycle as defined herein.

Furthermore, the terms alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heteroalkyl, heteroalkylene, C ₃ -C ₈ carbocyclyl, C ₃ -C ₈ carbocycle, C ₃ -C ₈ heterocyclyl, C ₃ -C ₈ heterocycle, polyether refer to substituted groups optionally having one or more substituents selected from the group consisting of -X, -R', -O-, -OR', =O, -SR', -S-, -NR'2, _-NR'3 , =NR' _{, -CX3 ,} -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, _-NO2 , = _N2 , _-N3 , -NRC(=O)R', _-C (=O)R', -C(=O) _NR'2 , -SO3-, _-SO3H , -S(=O) _2R ', -OS(=O) ₂ OR', -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 , wherein each X is independently halogen: -F, -Cl, -Br or -I; and each R' is independently -H, _-C1 - _C20 alkyl, _{-C6 - C10} aryl or -C3- _C10 heterocycle.

Acyl refers to -CO-alkyl, wherein alkyl has the above definition.

As used herein, the term "polyether" refers to a polymer containing ether bonds. The number of ether moieties in the polyether may be 2-100, preferably 2-25, and particularly 2-10. Examples of polyethers include polyethylene glycols, such as polyethylene glycols having 2-100, preferably 2-25, and particularly 2-10 ether moieties.

The term "optionally substituted polyether" may refer to a polyether (particularly polyethylene glycol) optionally substituted with one or more substituents selected from the group consisting of halogen, oxo, -OH, _-NO2 , -CN, _C1 - _C6 alkyl, _{C3- C6} cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, _C1 - _C6 alkoxy, _C1 - _C6 haloalkyl, _C1 - _C6 haloalkoxy, -(CO)-R', -O-(CO)-R', -(CO)-OR', -(CO)-NR"R"', -NR"-(CO)-R' and -NR"R"';R',R" and R"' are independently selected from H and _C1 - _C6 alkyl.

Solid phase peptide synthesis (SPPS) refers to a well-known method in which peptides anchored to a support (insoluble polymer) are assembled by successive additions of Fmoc- or Boc-protected amino acids through repeated cycles of deprotection-wash-coupling-wash. Each amino acid addition refers to the following cycle: (i) cleavage of the Nα-protecting group, (ii) a washing step, (iii) coupling of the fluorenylmethoxycarbonyl protecting group-(Fmoc-) or tert-butyloxycarbonyl-(Boc-) protected amino acid using a coupling agent and a non-nucleophilic base, (iv) a washing step. When the growing chain is bound to the support, excess reagents and soluble byproducts can be removed by simple filtration. Because repeated coupling reactions with hindered Fmoc- or Boc-protected N-methylated amino acids are difficult and generally suboptimal, low crude purity, difficult purification, and low yields are expected using 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 bind directly or indirectly to the peptide.

Ligand drug conjugate (LDC) refers to any conjugate of a ligand and a drug covalently linked as defined herein, and relates to any means as described herein, and will be illustrated in the examples of the specification. When the ligand is an antibody, it may refer to an antibody drug conjugate (ADC), which is a preferred embodiment of the present disclosure.

The term linker unit refers to a component that connects different parts of a compound together, for example, a linker can connect a ligand to a spacer, or a spacer to an amide functional group -CO-NR1-. A linker is a scaffold with attachment sites for the components of a ligand-drug-conjugate (i.e., a ligand, a spacer, a hydrophobic masking entity, and/or an amide functional group -CO-NR1-).

Based on their knowledge, the skilled person is able to select a linker suitable for the intended LDC compound. A non-exhaustive list of linkers includes: amino acids, such as lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, homoalanine; amino alcohols; aminoaldehydes; polyamines or any combination thereof. Advantageously, the linker units X1 and/or X2 are one or more natural or non-natural amino acids. In one embodiment, the linker units X1 and/or X2 are selected from glutamic acid, lysine and glycine. The linking units X1 and X2 may be independently selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, _C1 - _C12 alkylene, arylene with 6-10 ring atoms, _C3 - _C8 cycloalkylene, heterocycloalkylene with 5-10 ring atoms, heteroarylene with 5-10 ring atoms, _C2 - _C10 alkenylene, and any combination thereof, wherein the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -OC(O)NR"-, and triazole,

and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted by one or more substituents selected from the following: halogen, oxo, -OH, _-NO2 , -CN, _C1 - _C6 alkyl, _C3 - _C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, C1- _C6 alkoxy, C1 _{- C6 haloalkyl, C1-C6 haloalkoxy ,} -(CO)-R′, -O-(CO)-R′, -(CO)-OR′, -(CO)-NR"R"′, -NR"-(CO)-R′ and -NR"R"′ _; R′, R" and R"′ are independently selected from H and _C1 - _C6 alkyl.

Examples of linker units include optionally substituted polyethers, amino acids, benzyl groups, amines, ketones,

as well as

In particular, the linker unit may be divalent or trivalent. For example, when a hydrophobic masking entity K is present, X2 may be a trivalent linker unit.

The term "amino acid" refers to natural or unnatural amino acids. When X2 consists of one or more amino acids, the CO portion of the -CONR1- or -CONR1'- group can be considered as part of the X2 linker unit. A non-exhaustive list of amino acids includes lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine and homoalanine.

A spacer arm is a bivalent arm that covalently binds two components of a Ligand-Drug-Conjugate (such as two Linker units). A spacer group Z may be present or absent.

A non-exhaustive list of spacer units includes: alkylene, heteroalkylene (e.g., alkylene interrupted by at least one heteroatom selected from Si, N, O, and S); alkoxy; polyethers such as polyalkylene glycols and typically, polyethylene glycol; one or more natural or unnatural amino acids, such as glycine, alanine, proline, valine, N-methylglycine; C ₃ -C ₈ heterocycle; C ₃ -C ₈ carbocycle; arylene, and any combination thereof. For example, the spacer is a divalent linear alkylene, preferably (CH ₂ ) ₄ .

For example, the spacer can be selected from the group consisting of _{-C1- C10} alkylene-, _-C1 - _C10 heteroalkylene-, _-C3 - _C8 carbocycle-, -O-( _C1 - _C8 alkyl)-, -arylene-, _-C1 - _C10 alkylene-arylene-, _{-arylene -} C1- _C10 alkylene, -C1- _C10 alkylene-( _C3 - _C8 carbocycle)-, -( _C3 - _C8 carbocycle) _-C1 - _C10 alkylene, -( _C3 - _C8 heterocycle)-, -C1-C10 alkylene-( _C3 - _C8 heterocycle)-, -( _{C3 - C8 heterocycle} )-C1- _C10 alkylene-, _{-C1 -C10 alkylene} -C(＝O)-, _-C1 -C _-C 1 -C 8 heteroalkylene-C(＝O)-, -C ₃ -C ₈ carbocycle-C(＝O)-, -O-(C ₁ -C ₈ alkyl)-C(＝O)-, -arylene-C(＝O)-, -C ₁ -C ₁₀ alkylene-arylene-C(＝O)-, -arylene-C ₁ -C ₁₀ alkylene-C(＝O)-, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ carbocycle)-C(＝O)-, -(C ₃ -C ₈ carbocycle)-C ₁ -C ₁₀ alkylene-C(＝O)-, -C ₃ -C ₈ heterocycle-C(＝O)-, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ heterocycle)-C(＝O)-, -(C ₃ -C ₈ heterocycle)-C ₁ -C -C ₁ -C ₁₀ alkylene-C(═O)—, -C ₁ -C ₁₀ alkylene-NH—, -C ₁ -C 10 heteroalkylene-NH—, -C ₃ -C ₈ carbocycle-NH—, -O-(C ₁ -C ₈ alkyl) _-NH— , -arylene-NH—, -C ₁ -C ₁₀ alkylene-arylene-NH—, -arylene-C 1 -C ₁₀ alkylene-NH—, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ carbocycle ₎ -NH—, -(C ₃ -C ₈ carbocycle)-C ₁ -C ₁₀ alkylene-NH—, -C ₃ -C ₈ heterocycle-NH—, -C ₁ -C 10 alkylene-(C ₃ -C ₈ heterocycle)-NH—, -(C ₃ -C ₈ heterocycle)-C ₁ -C ₁₀ alkylene-NH—, -C -C ₁ -C ₁₀ alkylene-S-, -C ₁ -C ₁₀ heteroalkylene-S-, -C ₃ -C ₈ carbocycle-S-, -O-(C ₁ -C ₈ alkyl)-)-S-, -arylene-S-, -C ₁ -C ₁₀ alkylene-arylene-S-, -arylene-C ₁ -C ₁₀ alkylene-S-, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ carbocycle)-S-, -(C ₃ -C ₈ carbocycle)-C ₁ -C ₁₀ alkylene-S-, -C ₃ -C ₈ heterocycle-S-, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ heterocycle)-S-, -(C ₃ -C ₈ heterocycle)-C ₁ -C 10 alkylene-S-, -C ₁ -C ₁₀ -OC(＝O)-, -C ₃ -C ₈ carbocycle-OC(＝O)-, -O-(C ₁ -C ₈ alkyl)-OC(＝O)-, -arylene-OC(＝O)-, -C ₁ -C ₁₀ alkylene-arylene-OC(＝O)-, -arylene-C ₁ -C ₁₀ alkylene-OC(＝O)-, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ carbocycle)-OC(＝O)-, -(C ₃ -C ₈ carbocycle)-C ₁ -C ₁₀ alkylene-OC(＝O)-, -C _{3 -C 8} heterocycle-OC(＝O)-, -C ₁ -C ₁₀ alkylene-(C ₃ -C ₈ heterocycle)-OC(＝O)-, and -(C ₃ -C ₈ heterocycle)-C ₁ -C 10 _10Alkylene -OC(=O)-.

Any of the above groups may be optionally substituted by one or more substituents selected from the group consisting of -X, -R', -O-, -OR', =O, -SR', _-S- , -NR'2, _-NR'3 ⁺ , =NR', _-CX3 , -CN, -OCN, -SCN, -N=C=O _{, -NCS} , -NO, -NO2, = _N2 , _-N3 , -NR'C(=O)R', -C(=O)R _' , -C(=O)NR'2, -SO3-, -SO3H, -S(=O) _2R ', -OS(=O)2OR', _-S (=O)2NR _' , -S(=O)R', -OP(=O)(OR _' ) _{2 ,} -P(=O)(OR ^' ) ₂ , _-PO3- , _-PO3H2 , -C(＝O)X, -C(＝S)R', _-CO2R ', _-CO2 , -C(＝S)OR', C(＝O)SR', C(＝S)SR', C(＝O) _NR'2 , C(＝S) _NR'2 and C(＝NR') _NR'2 , wherein each X is independently a halogen: -F, -Cl, -Br or -I; and each R' is independently -H, _-C1 - _C20 alkyl, _-C6 - _C10 aryl or _-C3 - _C10 heterocycle.

A ligand refers to any macromolecule (polypeptide, protein, peptide, typically an antibody) typically used in LDC (e.g., antibody-drug conjugate) technology, or to a small molecule, such as folic acid or an aptamer, which can be covalently coupled to a synthetic linker or drug-linker of the invention using bioconjugation technology (see Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). The ligand is typically a compound selected for its targeting ability. A non-exhaustive list of ligands includes: proteins, polypeptides, peptides, antibodies, full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors, vitamins, transferrin, or any other cell-binding molecule or substance. The main class of ligands used to prepare conjugates is antibodies. An example of a protein is human serum albumin.

As used herein, the term " antibody " is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, modified monoclonal and polyclonal antibodies, monospecific antibodies, multispecific antibodies such as bispecific antibodies, antibody fragments, and antibody mimetics ( Cell Penetrating Monobodies or ). An example of an antibody is trastuzumab.

The term " antibody " as referred to herein includes whole antibodies and any antigen binding fragment (ie, "antigen binding portion") or single chains thereof.

Naturally occurring "antibodies" are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region consists of one domain, _CL . The _VH and _VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

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

In addition, although the two domains _VL and _VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be made into single-chain proteins, wherein the _VL and _VH regions are paired to form monovalent molecules (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 encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as with intact antibodies.

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

As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. 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 human framework sequence analysis, such as, for example, as described in Knappik, et al. (2000. J Mol Biol 296, 57-86).

Human antibodies may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) are grafted onto human framework sequences.

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

As used herein, "isotype" refers to the antibody class determined by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl or IgG4).

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

Active agent refers to a biologically active molecule or a therapeutic molecule. Examples of active agents include drugs, imaging agents, and fluorophores. Among imaging agents, fluorophores such as indocyanine green can be cited.

Drug refers to any type of drug or compound with intrinsic pharmacological or diagnostic properties, such as cytotoxic, cytostatic, immunomodulatory, immunosuppressive, immunostimulant, anti-inflammatory, ionizing or anti-infective compounds. Examples of cytotoxic compounds include calicheamicin; uncialamycin; auristatins (such as monomethyl auristatin E, known as MMAE); halichondrin derivatives (such as eribulin), tubulysin analogs; maytansine; cryptophycin; benzodiazepine dimers (including pyrrolo[2,1-c][1,4]benzodiazepines known as PBD); indolinobenzodiazepine pseudodimer (IGN); duocarmycin; anthracyclines; in) (such as doxorubicin or PNU159682); camptothecin analogs (such as 7-ethyl-10-hydroxy-camptothecin known as SN38 or exatecan); Bcl2 and Bcl-xl inhibitors; thailanstatins; amatoxins (including α-amanitin); kinesin spindle protein (KSP) inhibitors; vinorelbine; cyclin-dependent kinase (CDK) inhibitors; molecular glue degraders, bleomycin; dactinomycin or radionuclides and their complexing agents (such as DOTA/ ¹⁷⁷ Lu). Anti-inflammatory drugs can include corticosteroids such as dexamethasone or fluticasone. Anti-infective drugs can include antibiotics such as rifampicin or vancomycin. The drug can be an anticancer drug or an immunomodulator. Examples of anticancer agents are cytotoxic and cytostatic compounds, with cytotoxic compounds being preferred.

Hydrophobic masking entities refer to groups that can reduce the apparent hydrophobicity of a compound. Hydrophobic masking entities can be selected from polysarcosine, polyethylene glycol and chitosan. The number of ethylene glycol or sarcosine moieties can vary over a wide range. For example, the number of ethylene glycol or sarcosine moieties in the hydrophobic masking entity can be 2-500, preferably 5-100, particularly 5-25. In one embodiment, the hydrophobic masking entity is a polysarcosine comprising 6-24 sarcosine moieties, preferably 10-12 sarcosine moieties. The number of chitosan in chitooligosaccharide can be 2-20, for example 2-8.

An electron withdrawing group refers to an atom or group that attracts electron density from neighboring atoms to itself, usually by resonance or inductive effect. Electron withdrawing groups include halogen, haloalkyl (such as -CF ₃ ), -CN, -SO ₃ H, -NO ₂ , and -C(O)R groups, where R＝H, OH or alkoxy. Advantageously, the electron withdrawing group is -NO _2. In one embodiment, the electron withdrawing group is in the ortho position relative to the YT substituent of the benzene ring.

As used herein, the term " protecting group " refers to a chemical substituent that can be selectively removed by readily available reagents that do not attack regenerated functional groups or other functional groups in the molecule. Suitable protecting groups are known in the art and continue to be developed. Suitable protecting groups can be found in, for example, Wutz, etc. ("Greene's Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). Protecting groups for protecting amino groups are described in Wutz, etc. (pages 696-927), and are used in certain embodiments. Representative examples of amino protecting groups include, but are not limited to, tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), carboxybenzyl (Cbz), benzyl (Bn), allyl, trifluoroacetyl, allyloxycarbonyl (Alloc) and 2,2,2-trichloroethoxycarbonyl (Troc).

A group that can react with a ligand to form a linker unit refers to any chemical moiety that is reactive toward covalently binding a ligand. In particular, it can react with a thiol group present on a ligand.

A non-exhaustive list of chemical moieties reactive toward covalently bound ligands includes: carboxylic acids; primary amines; secondary amines; tertiary amines; hydroxyls; halogens; activated esters such as N-hydroxysuccinimide esters, perfluorinated esters, nitrophenyl esters, azabenzotriazole and benzotriazole activated esters, acylureas; alkynyls; alkenyls; azides; isocyanates; isothiocyanates; aldehydes; thiol-reactive moieties such as maleimide, halomaleimide, haloacetyl, pyridyl disulfide; thiols; acrylates; mesylates; tosylates; triflates, hydroxylamines; chlorosulfonyls; boronic acid-B(OR′) ₂ derivatives, wherein R′ is hydrogen or alkyl.

"Cleavable unit" refers to a chemical group that can be cleaved by the action of an internal or external (preferably external) stimulus. In the present disclosure, the cleavable unit is a polypeptide cleavable unit or a sugar cleavable unit. The stimulus that triggers the cleavage of the cleavable unit can be, for example, pH or temperature conditions, or the presence of an enzyme. The cleavage of the cleavable unit preferably triggers the self-decomposition of the phenyl-containing linker of the compound of the present invention and the release of the active agent D. In the present disclosure, a "cleavable sugar unit" may refer to a sugar moiety, preferably a glucuronide or a galactoside. In the present disclosure, a "polypeptide cleavable unit" may refer to a polypeptide, preferably a dipeptide or a tripeptide.

As used herein, the term "one or more" may be understood to refer to a number from 1-20, or from 1-10, or from 1-5.

Compounds of formula (I)

The present disclosure relates to compounds of formula (I):

in

L is a ligand;

X1 is the connector unit;

Z is an optional spacer;

X2 is the joint unit;

K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

R1 is selected from the group consisting of H, C ₁ -C ₁₂ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, polysarcosine, aryl having 6-10 ring atoms, C ₃ -C ₈ cycloalkyl, heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores;

Each R2 is independently selected from the group consisting of an electron withdrawing group and a C ₁ -C ₄ alkyl group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y is O, or when T is a polypeptide cleavable unit, Y is NR3;

R3 is selected from the group consisting of H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl having 6-10 ring atoms, C ₃ -C ₈ cycloalkyl, heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

According to one embodiment, L is a ligand selected from the group consisting of a polypeptide, a protein, an antibody and an antigen-binding fragment thereof, preferably L is an antibody or an antigen-binding fragment thereof, more preferably L is an antibody. According to one embodiment, the antibody is trastuzumab.

According to one embodiment, D is selected from the group consisting of drugs, preferably D is an anticancer drug or an immunomodulator. According to one embodiment, D is exatecan or monomethyl auristatin E (MMAE).

According to one embodiment, X1 and X2 are independently selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, _C1 - _C12 alkylene, arylene with 6-10 ring atoms, _C3 - _C8 cycloalkylene, heterocycloalkylene with 5-10 ring atoms, heteroarylene with 5-10 ring atoms, _C2 - _C10 alkenylene, and any combination thereof,

The alkylene and alkenylene groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"- and triazole,

and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted by one or more substituents selected from the group consisting of halogen, oxo, -OH, _-NO2 , -CN, _C1 - _C6 alkyl, _C3 - _C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, _C1 - _C6 alkoxy, C1- _{C6 haloalkyl, C1-C6 haloalkoxy , -} (CO)-R', -O-(CO)-R', -(CO)-OR', -(CO)-NR"R"', -NR"-(CO)-R' and -NR"R"';

R', R" and R'" are independently selected from H and _C1 - _C6 alkyl.

According to one embodiment, X1 is selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, _C1 - _C12 alkylene groups, arylene groups having 6-10 ring atoms, and any combination thereof.

According to one embodiment, X1 is

According to one embodiment, X2 is selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, C ₁ -C ₁₂ alkylene, optionally substituted polyethers, and any combination thereof.

According to one embodiment, Z is selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C ₁ -C ₁₂ alkylene, arylene with 6-10 ring atoms, C ₃ -C ₈ cycloalkylene, heterocycloalkylene with 5-10 ring atoms, heteroarylene with 5-10 ring atoms, C ₂ -C ₁₀ alkenylene, and any combination thereof,

The alkylene and alkenylene groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"- and triazole,

and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted by one or more substituents selected from the group consisting of halogen, oxo, -OH, _-NO2 , -CN, _C1 - _C6 alkyl, _C3 - _C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, _C1 - _C6 alkoxy, C1- _{C6 haloalkyl, C1-C6 haloalkoxy , -} (CO)-R', -O-(CO)-R', -(CO)-OR', -(CO)-NR"R"', -NR"-(CO)-R' and -NR"R"';

R', R" and R'" are independently selected from H and _C1 - _C6 alkyl.

According to one embodiment, Z is selected from the group consisting of one or more amino acids, one or more N-substituted amino acids and polyethylene glycol.

According to one embodiment, Z is absent and X1 and X2 are directly linked to each other via a single bond.

According to one embodiment, K is polysarcosine, preferably monodisperse polysarcosine. Polysarcosine may have 1 to 50 repeating units.

In a particular embodiment, K is polysarcosine, preferably having the following formula (V):

wherein k is an integer of 2-50, preferably 4-30, and R6 corresponds to OH or NH2.

According to one embodiment, T is a sugar cleavable unit which is a glucuronide or a galactoside. The glycosidic bond connecting T to the aromatic group may be a glycosidic bond which may be cleaved to initiate a self-degrading reaction sequence leading to drug release.

According to one embodiment, T is a dipeptide, preferably selected from Val-Cit, Val-Ala and Phe-Lys.

According to one embodiment, R1 is selected from the group consisting of H and C ₁ -C ₆ alkyl, preferably H.

According to one embodiment, R3 is selected from the group consisting of H and C ₁ -C ₆ alkyl, preferably H.

According to one embodiment, R4 is selected from the group consisting of H and C ₁ -C ₆ alkyl.

According to one embodiment, R5 is selected from the group consisting of H and C ₁ -C ₆ alkyl.

According to one embodiment, R4 and R5 are both H.

According to one embodiment, n is 0, Y is NR3, and T is a polypeptide cleavable unit. According to another embodiment, n is 1, R2 is _-NO2 , Y is O, and T is a sugar cleavable unit.

According to a preferred embodiment, L is an antibody or an antigen-binding fragment thereof.

According to one embodiment, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD37, CD79b, HER2 and PSMA.

According to one embodiment, the antibody or antigen-binding fragment thereof binds HER2/neu.

Some examples of antibodies suitable as ligand L in the compounds of Formula (I) described herein include, but are not limited to, trastuzumab, brentuximab, loncastuximab, rosopatamab, rituximab, pinatuzumab, polatuzumab, and naratuximab.

According to one embodiment, the antibody is trastuzumab.

The compound of formula (I) has at least one asymmetric carbon and therefore may exist in different stereoisomers. For example, the compound of formula (I) may exist in the (R) form or the (S) form shown below:

According to one embodiment, the compound of formula (I) is (S).

In a particular embodiment, the compound of formula (I) is a compound of formula (VI):

wherein k is an integer of 2-50, preferably 4-30; and T is a polypeptide cleavable unit, preferably a dipeptide.

In a particular embodiment, the compound of formula (I) is a compound of formula (VII)

wherein k is an integer of 2-50, preferably 4-30.

In a particular embodiment, the compound of formula (I) is a compound of formula (VIII)

wherein k is an integer of 2-50, preferably 4-30; and T is a sugar cleavable unit, preferably a glucuronide or a galactoside.

In a particular embodiment, the compound of formula (I) is a compound of formula (IX)

wherein k is an integer of 2-50, preferably 4-30.

The compounds of formula (I) according to the present invention can be synthesized by any suitable method known in the art, such as the methods disclosed in the Examples section.

The embodiments described for compounds of formula (I) also apply to compounds of formula (II), (III) and (IV).

Compound of formula (II)

The present disclosure also relates to compounds of formula (II):

in

X1′ is a group that can react with a ligand to form a linker unit;

Z is an optional spacer;

X2 is the joint unit;

K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

R1′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores;

Each R2 is independently selected from the group consisting of an electron withdrawing group and a C ₁ -C ₄ alkyl group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y′ is O, or when T is a polypeptide cleavable unit, Y′ is NR3′;

R3′ is selected from the group consisting of an amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl, optionally substituted polyether, an aryl group having 6-10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3-10 ring atoms, a heteroaryl group having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

According to one embodiment, X1' is maleimide.

According to one embodiment, R1' is H.

According to one embodiment, R3′ is H.

The compound of formula (II) can be used to synthesize the compound of formula (I), and is therefore an intermediate in the synthesis of the compound of formula (I).

Compound of formula (III)

The present disclosure also relates to compounds of formula (III)

in

R1′ is selected from the group consisting of an amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, an aryl group having 6-10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3-10 ring atoms, a heteroaryl group having 5-10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores;

Each R2 is independently selected from the group consisting of: an electron withdrawing group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y′ is O, or when T is a polypeptide cleavable unit, Y′ is NR3′;

R3′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

The compound of formula (III) can be used to synthesize the compound of formula (II) and is therefore an intermediate in the synthesis of the compound of formula (I).

Compound of formula (IV)

The present disclosure also relates to compounds of formula (IV)

in

R1′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

Each R2 is independently selected from the group consisting of an electron withdrawing group and a C ₁ -C ₄ alkyl group;

n is 0, 1, or 2;

R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-;

T is a carbohydrate cleavable unit or a polypeptide cleavable unit;

When T is a carbohydrate cleavable unit, Y′ is O, or when T is a polypeptide cleavable unit, Y′ is NR3′;

R3′ is selected from the group consisting of an amino protecting group, H, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₆ alkenyl group, an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C ₃ -C ₈ cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof,

The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C(O)NR"-, -NR"-C(O)-, -NR"-C(O)-NR"'-, -NR"-C(O)-O-, -O-C(O)NR"-, and triazole;

R" and R"' are independently selected from H and C ₁ -C ₆ alkyl;

and pharmaceutically acceptable salts thereof.

According to one embodiment, R4 and R5 are H, Y' is O and T is a sugar cleavable unit.

According to another embodiment, Y' is NR3' and T is a polypeptide cleavable unit.

The compound of formula (IV) can be used to synthesize the compound of formula (III) and is therefore an intermediate in the synthesis of the compound of formula (I).

Pharmaceutical composition

The present disclosure also relates to pharmaceutical compositions comprising a compound of the present disclosure and at least one pharmaceutically acceptable carrier. In particular, the present disclosure relates to pharmaceutical compositions comprising a compound of formula (I) and at least one pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous routes or intratumoral injection. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the effects of acid and other natural conditions that may inactivate the compound. The formulation may further include one or more excipients, preservatives, solubilizers, buffers, etc.

The form of the pharmaceutical composition, route of administration, dosage and regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, and the like.

The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, intranasal, intraocular, intravenous, intramuscular or subcutaneous administration, among others.

Preferably, the pharmaceutical composition contains a carrier which is pharmaceutically acceptable for an injectable formulation. These may be in particular isotonic, sterile saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry, in particular lyophilized compositions which, after addition of sterile water or physiological saline, can be formulated into injectable solutions.

The dosage for administration can be adjusted according to various parameters, in particular according to the mode of administration used, the pathology concerned or the desired duration of treatment.

To prepare a pharmaceutical composition, an effective amount of a compound according to the present disclosure may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol formulations; and sterile powders or lyophilizates for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid for easy injection. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compound as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

The compounds of the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions and by using a surfactant, suitable fluidity can be maintained. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, such as sugar or sodium chloride. The extended absorption of the injectable composition can be achieved by using an agent (such as aluminum monostearate and gelatin) that delays absorption in the composition.

Sterile injectable solutions are prepared by incorporating the desired amount of the active compound into an appropriate solvent along with several other ingredients listed above (if necessary), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any other required ingredients from its previously sterile filtered solution.

The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, with the use of DMSO as a solvent envisioned to achieve extremely rapid penetration, delivering high concentrations of active agents to small tumor areas.

When formulated, the solution will be administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulation is readily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like may also be used.

For parenteral administration in aqueous solution, for example, if necessary, the solution should be properly buffered, and first the liquid diluent is isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, according to the present disclosure, the sterile aqueous medium that can be used is known to those skilled in the art. For example, a dose can be dissolved in 1mL isotonic NaCl solution and added to 1000mL subcutaneous infusion or injected at the intended infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038and 1570-1580). Depending on the condition of the subject being treated, some changes will inevitably occur in the dosage. In any case, the person responsible for administration will determine the appropriate dose for individual subjects.

In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; sustained release capsules; and any other forms currently used.

In certain embodiments, it is contemplated that liposomes and/or nanoparticles are used to introduce antibodies into host cells. The preparation and use of liposomes and/or nanoparticles are known to those skilled in the art.

Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) are generally designed using polymers that can degrade in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure, and such particles can be easily prepared.

Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Ultrasonication of MLVs results in the formation of Small unilamellar vesicles (SUVs) of size 1.4 to 1.5 mm in diameter, the core of which contains an aqueous solution. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

The dosage for administration can be adjusted according to various parameters, in particular according to the mode of administration used, the relevant pathology or the required duration of treatment. It should be understood that the appropriate dosage of the compound and the composition comprising the compound may vary from patient to patient. Determining the optimal dosage generally includes balancing the level of therapeutic benefit with any risk or harmful side effects of the treatment described herein. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, the route of administration, the time of administration, the discharge rate of the compound, the duration of treatment, other drugs, compounds and/or materials used in combination, and the age, sex, weight, condition, general health and previous medical history of the patient. The amount of the compound and the route of administration will ultimately be determined by the physician, although the dosage will usually reach a local concentration at the site of action, thereby achieving the desired effect without causing substantial harm or harmful side effects. For example, for a subject of about 50-70 kg, the dosage for administration may be about 0.1-1000 mg of the compound of the present disclosure.

How to use

The compounds of the present disclosure exhibit valuable pharmaceutical properties as shown in the in vitro and in vivo tests provided in the examples, and are therefore suitable for treatment. The present disclosure also relates to compounds of the present disclosure, which are used as drugs. In particular, the present disclosure relates to ligand-drug conjugate compounds of formula (I), more specifically, antibody-drug conjugate compounds of formula (I), wherein L is an antibody or an antigen-binding portion thereof, which are used as drugs.

In particular, the compounds of the present disclosure, more specifically the antibody-drug conjugates of formula (I) of the present disclosure can be used to prevent or treat cancer, inflammatory diseases and/or infectious diseases. In a preferred embodiment, the compound of formula (I) for preventing or treating cancer is a ligand-drug conjugate (LDC) of formula (I), wherein L is an antibody or an antigen-binding portion thereof, more preferably wherein D is a cytotoxic compound.

The disclosure also relates to compounds of the disclosure, which are used for methods of treating cancer. As used herein, the term "cancer" has its general meaning in the art and includes abnormal states or conditions characterized by rapid proliferation of cell growth. The term includes all types of cancerous growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the histopathological type or invasive stage. The term cancer includes malignant tumors of various organ systems, such as malignant tumors affecting the skin, lung, breast, thyroid, lymph, gastrointestinal and urogenital tracts, and adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancers and/or testicular tumors, non-small cell lung cancers, small intestinal cancers and esophageal cancers.

The present disclosure relates to a method for treating cancer, comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of

(i) a compound of the present disclosure, or

(ii) a pharmaceutical composition as described herein.

In a preferred embodiment, the present disclosure relates to a method for treating or preventing cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of a ligand-drug conjugate (LDC) of formula (I), wherein L is an antibody or an antigen-binding portion thereof, and more preferably, wherein D is a cytotoxic compound.

The present disclosure also relates to compounds of the present disclosure for use in methods of treating inflammatory diseases.

As used herein, the term "inflammatory disease" is used to define any disease caused by or resulting in inflammation in a subject. The term may include, but is not limited to, (1) inflammatory and/or allergic diseases, (2) autoimmune diseases, (3) transplant rejection, and (4) other diseases in which an undesirable inflammatory response is suppressed.

The present disclosure relates to methods for treating inflammatory diseases, the methods comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of

(i) a compound of the present disclosure, or

(ii) a pharmaceutical composition as described herein.

The present disclosure also relates to compounds of the present disclosure for use in methods of treating infectious diseases.

As used herein, the term "infectious disease" is used to define any disease caused by the invasion of an infectious agent (or pathogen) into a subject, their proliferation, and the response of the subject's tissues to these infectious agents and the toxins they produce. The term may include, but is not limited to, (1) bacterial infection, (2) viral infection, (3) fungal infection, and (4) parasitic infection.

The present disclosure relates to a method for treating an infectious disease, the method comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of

(i) a compound of the present disclosure, or

(ii) a pharmaceutical composition as described herein.

In a preferred embodiment, the present disclosure relates to a method for treating or preventing an infectious disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a ligand-drug conjugate (LDC) of formula (I), wherein L is an antibody or an antigen-binding portion thereof, and more preferably, wherein D is an anti-infective agent, such as an antibiotic or an antiviral agent.

As used herein, the term "treat" includes reversing, alleviating, inhibiting the progression of the disease, disorder, or condition to which such term applies, preventing or reducing the likelihood of the disease, disorder, or condition, or one or more symptoms or manifestations of such disease, disorder, or condition. Prevention refers to preventing the disease, disorder, condition, or its symptoms or manifestations, or its severity from worsening. Therefore, the compounds disclosed in the present invention can be administered prophylactically to prevent or reduce the incidence or recurrence of a disease, disorder, or condition.

As used herein, the term "therapeutically effective amount" of a compound refers to an amount of the compound that will elicit a biological or medical response in a subject, such as improving symptoms, alleviating a condition, slowing or delaying disease progression, or preventing a disease.

The present disclosure also relates to the use of the compounds of the present disclosure, preferably the compounds of formula (I), in the preparation of medicaments for treating cancer, inflammatory diseases and/or infectious diseases, preferably for treating cancer.

The compound of formula (II) can be used without a ligand. For example, when X1' is a maleimide moiety, the compound of formula (II) can react with a protein (such as serum albumin) in vivo, which then becomes a ligand. Therefore, the present disclosure also relates to a compound of formula (II) as described above, which is used as a drug.

DETAILED DESCRIPTION

Materials and general methods

Unless otherwise stated, all solvents and reagents were obtained from reputable commercial sources (Sigma-Aldrich, Fluorochem, TCI Chemicals, Acros Organics, Alfa Aesar, Enamine, Thermo Fisher, Carbosynth, WuXi AppTec, Iris Biotech) and used without further purification. Anhydrous solvents were purchased from Sigma-Aldrich. Fmoc-amino acids, 2-chlorotrityl, Wang and Rink amide polystyrene 1% DVB 100-200 mesh resin (pre-loaded with the first Fmoc-sarcosine amino acid) were purchased from Christof Senn Laboratories and Sigma-Aldrich. Monomethyl auristatin E (MMAE), exatecan mesylate, and 7-ethyl-10-hydroxycamptothecin (SN38) were purchased from DCChemicals, MedchemExpress, or Abzena. Human albumin (Catalog No. A3782) was purchased from Sigma-Aldrich. Trastuzumab ( IV) was purchased from Roche. Non-commercially available monoclonal antibodies were custom produced by transient transfection on a CHO cell line and protein-A/SEC purified by GTP Technologies (Toulouse, France).

On-resin synthesis was performed in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). Agitation was performed using a Titramax 101 platform shaker (Heidolph). Unless otherwise stated, all chemical reactions were performed at room temperature under an inert argon atmosphere.

Liquid NMR spectra were recorded on a Bruker Fourier 300HD or Bruker AVANCE III HD400 spectrometer using the residual solvent peak for calibration. Mass spectrometric analyses have been performed by the Centre commune de Spectrométrie de Masse (CCSM), UMR5246 CNRS Faculty, University Claude Bernard Lyon 1.

At Teledyne Isco Macherey-Nagel on RF200 Normal phase flash chromatography was performed using a flash column (40-63 μm). C18 Duo 30μm column or Interchim PuriFlash RP-AQ (30μm) column in Teledyne Isco Reverse phase chromatography was performed on an Rf200 instrument; or using an Agilent 1100 preparative binary HPLC system.

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 1100 system), or UHPLC-UV/MS (Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact II ^™ Q-ToF mass spectrometer or Agilent 1260 HPLC system equipped with a Bruker MicroTOF-QII mass spectrometer), respectively.

HPLC Method 1: Agilent 1100 HPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, mobile phase B was acetonitrile. Column was Agilent Zorbax SB-Aq 4.6x150mm 5μm (room temperature). Linear gradient was 0% B to 50% B in 30 min, then held at 50% B for 5 min. Flow rate was 1.0 mL/min.

HPLC method 2: Agilent 1100 HPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, mobile phase B was acetonitrile. Column was Agilent Poroshell 120EC-C18 3.0×50 mm 2.7 μm (room temperature). Linear gradient was 5% B to 80% B in 9 min, then held at 80% B for 1 min. Flow rate was 0.8 mL/min.

HPLC method 3: Agilent 1100 HPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, mobile phase B was acetonitrile. Column was Agilent Poroshell 120EC-C18 3.0×50 mm 2.7 μm (room temperature). Linear gradient was 5% B to 80% B in 20 min, then held at 80% B for 2 min. Flow rate was 0.8 mL/min.

HPLC method 4: Thermo UltiMate 3000UHPLC 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-S1000A2.1x150mm 8μm (80°C). The linear gradient is 10% B to 50% B in 25min. The flow rate is 0.4mL/min. UV detector monitoring is performed at 280nnm. Q-ToF mass spectrometer is used in the m/z range of 500-3500 (ESI ⁺ ). Using Bruker The MaxEnt algorithm included in the software deconvolves the data.

HPLC method 5 (preparative method): Teledyne Isco Combi Rf200 binary MPLC system with DAD detection. Mobile phase A is water + 0.1% TFA, mobile phase B is acetonitrile. Reusable columns are C18 Duo 100A 30 μm (30 g). Linear gradient was 10% B to 50% B in 35 min, then held at 50% B for 5 min. Flow rate was 25 mL/min.

HPLC method 6 (preparative method): Agilent 1100 preparative binary HPLC system equipped with dual loop autoinjector, DAD detection and fraction collector. Mobile phase A is water + 0.1% TFA, mobile phase B is acetonitrile. Column is Waters SunFire C18 OBD Prep column, 5 μm, 19 mm×250 mm (room temperature). Linear gradient: 10% B to 60% B in 40 min, then hold at 60% B for 5 min. Flow rate: 25 mL/min.

1) Preparation of the compounds of the present invention

1.1) Monodisperse polysarcosine intermediate

1.1.1) General Methods

An iterative procedure for submonomer synthesis using Rink amide and 2-chlorotrityl resin (described in patent WO2019081455) or following the classical Fmoc/SPPS method with commercially available Fmoc-Sar-Sar-OH dipeptide building blocks (CAS#2313534-20-0) for Wang resin was used to achieve the on-resin synthesis of monodisperse polysarcosine. Please note that the peptide submonomer synthesis on Wang resin was unsuccessful. In all cases, the on-resin dimerization stage (n=2) was avoided due to diketopiperazine formation. All synthesis yields are provided based on the initial Fmoc-sarcosine loading given by the manufacturer. Unless otherwise stated, all reactions were performed at room temperature. Rink amide, 2-chlorotrityl or Wang polystyrene 1% DVB 100-200 mesh resin (Christof Senn Laboratories) pre-loaded with the first Fmoc-sarcosine residue was used (typically, the initial loading was 0.6-1 mmol/g).

1.1.2) Extension of polysarcosine

Rink amide, 2-chlorotrityl or Wang resin preloaded with Fmoc-sarcosine was treated with 20% piperidine in DMF (1 mL/100 mg resin) twice for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times). A solution of Fmoc-Sar-Sar-OH (3 eq.), HATU (2.9 eq.) and DIPEA (6 eq.) in DMF (1 mL/100 mg resin) was added to the resin. The reaction vessel was stirred for 2 hours and the resin was washed with DMF (4 times) and DCM (4 times). It was treated with 20% piperidine in DMF (1 mL/100 mg resin) twice for 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times).

For the synthesis on Rink amide or 2-chlorotrityl resin, the classic submonomer synthesis procedure is used as described in WO2019081455. By alternating bromoacetylation and amine displacement steps, the extension of n=3 polysarcosine oligomers is carried out until the desired length is obtained. The bromoacetylation step is carried out by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2mL/100mg resin). The mixture is stirred for 30min, drained and washed with DMF (4 times). For the amine displacement step, 40% (wt) methylamine aqueous solution (1.5mL/100mg resin) is added and the container is shaken for 30min, drained and washed with DMF (4 times) and DCM (4 times).

For the synthesis on Wang resin, the classic Fmoc/SPPS procedure was used. Extension of n=3 polysarcosine oligomers was performed by iterative coupling of Fmoc-Sar-Sar-OH dipeptide building blocks (CAS#2313534-20-0). A solution of Fmoc-Sar-Sar-OH (3 equiv.), HATU (2.9 equiv.) and DIPEA (6 equiv.) in DMF (1 mL/100 mg resin) was added to the resin. The reaction vessel was stirred for 90 min, and the resin was washed thoroughly with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL/100 mg resin) twice for 15 min each at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). This coupling/Fmoc-deprotection cycle was repeated until the desired polysarcosine length was obtained. If desired, the final coupling is performed using commercially available Fmoc-Sar-OH amino acids instead of Fmoc-Sar-Sar-OH dipeptide units to obtain final polysarcosine of uniform length.

1.1.3) Final resin side functionalization of polysarcosine, optional end-capping, resin cleavage and purification

When the desired length of the polysarcosine monodisperse oligomer on the resin is reached, orthogonal chemical functionalization is performed. Optionally, final end-capping is performed subsequently with an Fmoc-amino acid (e.g., Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-amino-3,6-dioxaoctanoic acid, Fmoc-9-amino-4,7-dioxanononanoic acid). The Fmoc protecting group at the N-terminus of the end-capped final compound can be removed before or after resin cleavage, depending on the orthogonal functionalization chemistry used (see below).

1.1.3.1) Polysarcosine functionalized with 2-azidoethyl-1-amine side

To Rink or 2-chlorotrityl resin was added 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL/100 mg resin). The mixture was stirred for 30 min, drained and washed with DMF (4 times). 3 mol of 2-azidoethyl-1-amine in DMF (1 mL/100 mg resin) was added, the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times). Then, Fmoc-Gly-OH coupling (5 equivalents of Fmoc-Gly-OH, 4.9 equivalents of HATU, 10 equivalents of DIPEA in DMF (1 mL/100 mg resin) was performed at room temperature for 1 hour and Fmoc-deprotection was performed twice with 20% piperidine in DMF (1 mL/100 mg resin) for 15 min each time. The resin was washed with DMF (4 times) and DCM (4 times).

The final polysarcosine compound was cleaved from the resin (100% TFA 2 times 30 min for Rink and Wang resins, 20% TFA in DCM 2 times 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles were removed under reduced pressure to give the crude product. Purification was performed on a RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile.

1.1.3.2) Polysarcosine functionalized with β-alanine side

To Rink or 2-chlorotrityl resin, add 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL/100 mg resin). Stir the mixture for 30 min, drain and wash with DMF (4 times). Add allyl 3-aminopropionate (such as et al., J. Org. Chem. 1997, 62, 10, 3220-3229 and free-based with 3 equivalents of _Na2CO3 in _THF at 50°C for 20 min) in a 3 mol solution in DMF (1 mL/100 mg resin) and the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times). Then, Fmoc-Gly-OH coupling (5 equivalents of Fmoc-Gly-OH, 4.9 equivalents of HATU, 10 equivalents of DIPEA in DMF (1 mL/100 mg of resin) was carried out for 1 hour. The resin was washed with DMF (4 times), DCM (4 times). The Alloc-protecting group was removed by treating with a DCM solution containing 0.25 equivalents of Pd(PPh3) ₄ and 20 equivalents of phenylsilane twice for 30 min (gently stirring under an argon stream). Then, the resin was washed with DMF (5 times) and DCM (5 times). Optionally, N-hydroxysuccinimide (NHS) ester was introduced into the carboxylic acid side chain of the final polysarcosine compound by treating with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mL/100 mg of resin) for 90 min. Then, the resin was washed with DMF (4 times) and DCM (4 times).

The final polysarcosine compound was cleaved from the resin (100% TFA 2 times 30 min for Rink and Wang resins, 20% TFA in DCM 2 times 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles were removed under reduced pressure to give the crude product. Purification was performed on a RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile.

1.1.3.3) Polysarcosine functionalized with glutamic acid side

To Rink, Wang or 2-chlorotrityl resin was added a solution of Fmoc-Glu(OAll)-OH (3 eq.), HATU (2.9 eq.), and DIPEA (6 eq.) in DMF (1 mL/100 mg resin). The reaction vessel was stirred for 90 min, and the resin was washed thoroughly with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL/100 mg resin) twice for 15 min each at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). Then, coupled with Fmoc-amino-3,6-dioxanoic acid (3 eq.), HATU (2.9 eq.), DIPEA (6 eq.) in DMF (1 mL/100 mg resin) for 1 hour. The resin was washed with DMF (4 times), DCM (4 times). The Alloc-protecting group was removed by treating with a DCM solution containing 0.25 equivalents of Pd(PPh3) ₄ and 20 equivalents of phenylsilane for 2 times for 30 min (gentle stirring under an argon stream). Then, the resin was washed with DMF (5 times) and DCM (5 times). Optionally, N-hydroxysuccinimide (NHS) ester was introduced into the carboxylic acid side chain of the final polysarcosine compound by treating with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mL/100 mg resin) for 90 min. Then, the resin was washed with DMF (4 times) and DCM (4 times).

The final polysarcosine compound was cleaved from the resin (100% TFA 2 times 30 min for Rink and Wang resins, 20% TFA in DCM 2 times 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles were removed under reduced pressure to give the crude product. Purification was performed on a RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile.

1.1.3.4) γ-Azidohomoalanine-functionalized polysarcosine

To Rink, Wang or 2-chlorotrityl resin was added a solution of Fmoc-γ-azidohomoalanine (3 eq.), HATU (2.9 eq.), and DIPEA (6 eq.) in DMF (1 mL/100 mg resin). The reaction vessel was stirred for 90 min and the resin was washed thoroughly with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL/100 mg resin) twice for 15 min each at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). Then, coupled with Fmoc-amino-3,6-dioxaoctanoic acid (3 eq.), HATU (2.9 eq.), DIPEA (6 eq.) in DMF (1 mL/100 mg resin) for 1 hour. The resin was washed with DMF (4 times), DCM (4 times).

The final polysarcosine compound was cleaved from the resin (100% TFA 2 times 30 min for Rink and Wang resins, 20% TFA in DCM 2 times 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles were removed under reduced pressure to give the crude product. Purification was performed on a RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile.

1.1.4) Final polysarcosine intermediate

The obtained compounds are listed in Table 1 below.

1.2) Synthesis of intermediate compounds

1.2.1) Synthesis of Compound B1 and Compound B2

These compounds were synthesized according to the method described in patent WO2019081455. These compounds are equimolar diastereoisomer mixtures (* indicates stereocenter).

1.2.2.1 Synthesis of tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate

(±)-Octopamine hydrochloride (1690 mg/11 mmol) was weighed in a round bottom flask and suspended in 4 mL of distilled water. The flask was cooled at 0°C and 4 mL of pre-cooled 65% nitric acid solution was slowly added. The reaction was kept at 0°C for 20 min, as assessed by HPLC, showing complete mononitration of the starting material. The contents of the flask were transferred to a 250 mL pre-cooled Erlenmeyer and slowly neutralized with saturated NaHCO ₃ solution (about 50 mL) at 0°C until a pH of 8-9 was reached. Then, 30 mL of dioxane was added, followed by Boc ₂ O (7202 mg/13.2 mmol). Then, the reaction was allowed to reach room temperature and stirred overnight. Then, the reactant was diluted with EtOAc, washed 3 times with saturated citric acid solution, and once with saturated NaCl solution. The organic phase was dried over _MgSO4 , filtered and evaporated under vacuum to give the crude product, which was purified by silica gel chromatography (petroleum ether/EtOAc, gradient from 70:30 to 20:80) to give the title compound (1320 mg/40%) as a viscous yellow to brown oil.1H ^NMR (300 MHz, DMSO- _d6 ) δ 10.79 (s, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.47 (dd, J = 8.6, 2.1 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.74 (t, J = 5.9 Hz, 1H), 4.56 (t, J = 6.3 Hz, 1H), 3.07 (td, J = 6.1, 1.6 Hz, 2H), 1.31 (s, 9H). MS m/z (ESI ⁺ ): calcd. [M+H] ⁺ = 299.1; found [M+H] ⁺ = 299.1. HPLC method 2 retention time = 5.5 min.

1.2.2.2 Synthesis of (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

In a round-bottom flask, Ag ₂ CO ₃ (1500 mg/5.4 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (251 mg/1.1 mmol) were dissolved in 4 mL of anhydrous acetonitrile and stirred at room temperature for 2 hours. The aforementioned compound (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamic acid tert-butyl ester (292 mg/0.98 mmol) and 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronide methyl ester (583 mg/1.46 mmol) were added at 0°C, and the solution mixture was stirred at room temperature for 4 hours. Then, the reactant was filtered on celite, diluted with ETOAc, washed 3 times with saturated citric acid solution, and washed once with saturated NaCl solution. The organic phase was dried over MgSO ₄ , filtered and evaporated under vacuum to give the crude product, which was purified by silica gel chromatography (petroleum ether/EtOAc, gradient from 70:30 to 30:70) to give the title compound (244 mg/48%) as a yellow foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.76 (dd, J=3.3, 2.1 Hz, 1H), 7.60 (t, J=7.6 Hz, 1H), 7.36 (dd, J=8.7, 2.6 Hz, 1H), 6.77 (s, 1H), 5.71 (d, J=7.8 Hz, 1H), 5.61 (s, 1H), 5.46 (td, J=9.5, 1.1 Hz, 1H), 5.21 -5.02 (m, 3H), 4.75 (dd, J = 9.9, 1.4 Hz, 1H), 4.62 (s, 1H), 3.65 (s, 3H), 3.17 (s, 2H), 3.10 (t, J = 6.1 Hz, 2H), 2.81-2.59 (m, 6H), 2.05-1.96 (m, 9H), 1.30 (d, J = 1.7 Hz, 9H). MS m/z (ESI ⁺ ): calculated value [M+Na] ⁺ = 637.2; found value [M+Na] ⁺ = 637.2. HPLC method 2 retention time = 6.75 min.

1.2.2.3 Synthesis of (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

The aforementioned compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (334 mg/0.54 mmol) and 4-nitrophenyl chloroformate (219 mg/1.09 mmol) were dissolved in 6 mL of anhydrous DCM at 0°C. Anhydrous pyridine (112 mg/1.41 mmol) was added and the mixture was stirred at room temperature for 30 min. The reaction mixture was filtered through a 0.45 μm PTFE filter and purified by silica gel chromatography (petroleum ether/EtOAc, gradient from 85:15 to 30:70) to give the title compound (380 mg/90%) as a yellow foam. ¹ H NMR (300 MHz, DMSO-d ₆ )δ8.39-8.26 (m, 2H), 7.93 (d, J = 2.2Hz, 1H), 7.75 (d, J = 8.8Hz, 1H), 7.55 (dd, J = 9.2, 1.2Hz, 2H), 7.46 (d, J = 8.8Hz, 1H), 7.20 (d, J = 4.8Hz, 1H), 5.77 (dd, J = 7 .7, 3.7Hz , 1H), 5.47 (t, J = 9.5 Hz, 1H), 5.11 (q, J = 9.6 Hz, 2H), 4.77 (d, J = 9.9 Hz, 1H), 3.73-3.59 (m, 3H), 3.59-3.37 (m, 2H), 2.05-1.96 (m, 9H), 1.48-1.35 (m, 1H), 1.32 (s, 9H). MS m/z (ESI ⁺ ): calculated value [M+Na] ⁺ = 802.15; found value [M+Na] ⁺ = 802.15. HPLC method 2 retention time = 8.5 min.

1.2.2.4) Synthesis of Compound B3

101 mg (0.13 mmol) of the aforementioned compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate, 98 mg (0.14 mmol) of MMAE and 18 mg (0.13 mmol) of HOBt were dissolved in 1 mL of an 85:15 (v/v) anhydrous DMF/pyridine mixture. 16.7 mg (0.13 mmol) of DIPEA was added. The reaction was stirred at room temperature for 16 hours and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM/MeOH gradient from 99:1 to 95:5) to give 147 mg (84%) of the intermediate compound (white solid) which was used directly in the deprotection step. ESI ⁺ [M+H] ⁺ = 1358.7. HPLC method 2 retention time = 8.8 min.

144mg (0.106mmol) of this intermediate compound is dissolved in 3mL methanol at 0 ℃.LiOH monohydrate (44.5mg/1.06mmol) is dissolved in water (0.4mL) and added in reaction vessel.After stirring 30min (HPLC after reaction) at 0 ℃, mixture is neutralized with acetic acid (83mg/1.4mmol) and concentrated under reduced pressure.The crude product obtained is redissolved at 0 ℃ with TFA/DCM (30:70v/v) solution and stirred at room temperature for 15min.Volatiles are evaporated under reduced pressure, crude residue is dissolved in water/ACN (1:1v/v) solution and purified using HPLC preparation method 5 to obtain 85mg (72%) of compound B3, which is a white solid. HRMS m/z (ESI ⁺ ): Calculated value [M+H] ⁺ =1118.5867; Found value [M+H] ⁺ =1118.5842; Error = 2.2 ppm. HPLC method 3 Retention time = 9.36 min.

1.2.2.5) Synthesis of Compound B4

Compound B4 was synthesized by the same method used to synthesize compound B3 with slight modifications. The coupling reaction of (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate with exatecan mesylate was carried out at 40°C for 2 hours instead of overnight at room temperature.

125 mg (87%) of the intermediate compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate were obtained as a yellow/green solid. ESI ⁺ [M+Na] ⁺ = 1098.3. HPLC method 3 retention time = 14.7 and 14.9 min (mixture of diastereomers).

Deprotection of the glucuronide moiety was performed with MeOH/THF (75:25 v/v) instead of pure MeOH. Boc-deprotection was performed as described for compound B3.

Purification using HPLC preparative method 5 gave 65 mg (67%) of compound B4 as a yellow solid. ESI ⁺ [M+H] ⁺ = 836.2. HPLC method 3 retention time = 7.3 and 7.8 min (mixture of diastereomers).

1.2.2.6.1 Chiral Separation of a Racemic Mixture of tert-Butyl (2-Hydroxy-2-(4-Hydroxy-3-nitrophenyl)ethyl)carbamate

use IC MPLC column 30×100 mm, 20 μm (Daicel cat#83M73) was purchased from Teledyne Isco Com The chiral separation of racemic tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (synthesized as described above) was performed on the Rf200 system. The mobile phase was DCM + 0.2% (v/v) ethanol (isocratic gradient). The flow rate was 12 mL/min. The sample solvent was DCM + 0.2% (v/v) ethanol. The mass recovery of the two enantiomers after separation was greater than 80%.

The retention time of (S)-tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 15 min, and the retention time of (R)-tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 25 min.

To determine the absolute configuration, the phenol position of the two enantiomers was esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine in anhydrous THF. The compound was purified by silica gel chromatography (petroleum ether/ETOAc, gradient from 90:10 to 10:90) to give 4-nitrobenzoic acid 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenyl ester. ¹ H NMR (300MHz, DMSO-d ₆ ) δ 8.51 (d, J = 9.1 Hz, 2H), 8.44 (d, J = 9.1 Hz, 2H), 8.19 (d, J = 1.9 Hz, 1H), 7.88 (d, J = 10.2 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 6.93 (t, J = 5. 9Hz, 1H), 5.84 (d, J = 4.7Hz, 1H), 4.86-4.76 (m, 1H), 3.24 (t, J = 6.1Hz, 2H), 1.38 (s, 9H). ESI ⁺ [M+Na] ⁺ =470.1. The absolute configuration of the enantiomer (pre-dissolved in a 1:1 mixture of heptane/dichloromethane and allowed to evaporate slowly for 3 weeks to induce the formation of crystals) was confirmed by x-ray crystallography. The bulk crystals were mounted on a nylon ring in perfluoroether oil. Data were collected using Xcalibur, Atlas, Gemini superdiffractometers equipped with an Oxford Cryosystems cryogenic device operating at T = 150.00 (5) K. Cu K _α radiation was used to measure the data with ω scanning. The structure was solved by the ShelXT solver using a dual method and by using Olex2 (OV Dolomanov et al., Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., 2009, 42, 339-341) as a graphical interface. The model was refined using ShelXL 2018/3 (Sheldrick, GM, Crystal structure refinement with ShelXL, Acta Cryst., 2015, C71, 3-8) using full-matrix least squares minimization on ^F2 .

1.2.2.6.2) Synthesis of stereopure B4-S and B4-R compounds

Stereopure compounds B4-S and B4-R were synthesized as described above in Section 1.2.2 without any significant changes in reaction conditions, reactivity or overall yield.

Final purification using HPLC preparative method 5 gave 33 mg of compound B4-S as a yellow solid. ESI ⁺ [M+H] ⁺ = 836.2. HPLC method 3 retention time = 7.3 min.

Final purification using HPLC preparative method 5 gave 21 mg of compound B4-R as a yellow solid. ESI ⁺ [M+H] ⁺ = 836.2. HPLC method 3 retention time = 7.8 min.

1.2.3.1) Synthesis of Ac-Val-Ala-OH (acetyl-L-valyl-L-alanine)

To a solution of L-alanine benzyl ester hydrochloride (542 mg/2.5 mmol) in 30 mL of DCM, triethylamine (254 mg/2.5 mmol), distilled water (30 mL), N-α-acetyl-L-valine (400 mg/2.5 mmol) and HOBt (339 mg/2.5 mmol) were added in sequence. Then, the mixture was cooled to 0°C and EDC-HCl (530 mg/2.75 mmol) was added. The resulting mixture was stirred at 0°C overnight. The reactants were diluted with 20 mL of 2M HCl and the layers were separated. The organic phase was washed twice with 2M HCl, twice with saturated NaHCO ₃ solution, and once with saturated NaCl solution. The organic phase was dried over MgSO ₄ , filtered, and evaporated in vacuo to give 714 mg (89%) of acetyl-L-valyl-L-alanine benzyl ester as a white solid intermediate.

This intermediate is dissolved in 10mL EtOAc/MeOH1:1 (v/v) and transferred in the stainless steel hydrogenation reactor.After the first argon purge, add the 5wt%Pd/C of catalytic amount.Then, with reactor H ₂ purge twice and at room temperature at the H of 10bar ₂ pressure, keep overnight.After filtering the reactant with 0.45μm PTFE filter and removing solvent under vacuum, obtain quantitative pure acetyl-L-valyl-L-Alanine, be white solid. ¹ H NMR (300MHz, DMSO-d ₆ ) δ 12.45 (s, 1H), 8.23 (d, J = 6.9Hz, 1H), 7.85 (d, J = 9.0Hz, 1H), 4.25-4.11 (m, 2H), 1.94 (dt, J = 13.6, 6.8Hz, 1H), 1.85 (s, 3H), 1.2 6 (d, J=7.3Hz, 3H), 0.85 (dd, J=12.1, 6.8Hz, 6H).

1.2.3.2 Synthesis of (2S)-2-acetamido-N-((2S)-1-((4-(1-hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutanamide

In a round bottom flask, 420 mg (2.60 mmol) of 1-(4-aminophenyl)but-3-yn-1-ol (synthesized according to the following method described in Sharma A. et al., Chem 2018, 4 (10), 2370–2383) and 600 mg (2.60 mmol) of the aforementioned compound Ac-Val-Ala-OH were suspended in 20 mL of anhydrous THF. Then, 676 mg (2.74 mmol) of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) pre-dissolved in 5 mL of anhydrous DMF was added to the flask, and the turbid reaction mixture was stirred at room temperature overnight. Then, the volatiles were evaporated under reduced pressure, and the crude residue was dry loaded and purified by silica gel chromatography (DCM/MeOH gradient from 99:1 to 85:15) to give 809 mg (83%) of the title compound as a white solid. ¹ HNMR (300MHz, DMSO-d ₆ ) δ 9.84 (s, 1H), 8.18 (d, J = 7.0Hz, 1H), 7.90 (d, J = 8.6Hz, 1H), 7.53 (d, J = 8.6Hz, 2H), 7.28 (d, J = 8.6Hz, 2H), 5.44 (d, J = 4.4Hz, 1H), 4. 62(q,J＝6.2Hz,1H), 4.39(p,J＝ 7.6,7.2 Hz,1H), 4.17 (dd, J＝8.5,6.8 Hz,1H), 2.70 (t, J＝2.6 Hz,1H), 1.96 (dt, J＝13.2,6.6 Hz,1H), 1.88 (s,3H), 1.30 (d, J＝7.1 Hz,3H), 0.86 (dd, J＝10.9,6.8 Hz,6H). ESI ⁺ [M+H] ⁺ =374.2. HPLC method 2 retention time =3.95 min.

1.2.3.3) Synthesis of 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate

94 mg (0.25 mmol) of (2S)-2-acetylamino-N-((2S)-1-((4-(1-hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide and 153 mg (0.50 mmol) of bis(4-nitrophenyl)carbonate were dissolved in 2 mL of anhydrous DMF. 98 mg (0.76 mmol) of DIPEA were added and the reaction mixture was stirred at room temperature overnight. The volatiles were removed under reduced pressure and the crude residue was purified by silica gel chromatography (DCM/MeOH gradient from 99:1 to 90:10) to give 118 mg (87%) of the title compound as a yellow solid. ¹ H NMR (300 MHz, DMSO-d ₆ )δ9.99(s,1H), 8.35-8.26(m,2H), 8.22(d,J＝7.0Hz,1H), 7.89(d,J＝8.6Hz,1H), 7.63(d,J＝8.7Hz,2H), 7.58-7.48(m,2H), 7.43(d,J＝8.7Hz,2H), 5.74( d,J＝7.5Hz , 1H), 4.39 (p, J = 7.2 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 3.01-2.84 (m, 3H), 1.99-1.91 (m, 1H), 1.88 (s, 3H), 1.31 (d, J = 7.1 Hz, 3H), 0.86 (dd, J = 11.2, 6.8 Hz, 6H). ESI ⁺ [M+H] ⁺ = 539.1. HPLC method 2 retention time = 6.48 min.

1.2.3.4) Synthesis of Compound B5

44 mg (0.082 mmol) of the aforementioned compound 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate, 53 mg (0.074) MMAE and 11.1 mg (0.082 mmol) HOBt were dissolved in 1 mL of an 85:15 (v/v) anhydrous DMF/pyridine mixture. 10.6 mg (0.082 mmol) DIPEA was added. The reaction was stirred at room temperature for 16 hours and directly purified using HPLC preparative method 5 to give 41 mg (50%) of compound B5 as a white solid. ESI ⁺ [M+Na] ⁺ = 1139.7. HPLC method 2 retention time = 7.40 min.

1.2.3.5) Synthesis of Compound B6

Compound B6 was synthesized by the same method used to synthesize compound B5 with slight modifications. The coupling reaction of 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate with exatecan mesylate was carried out at 40°C for 3 hours instead of overnight at room temperature. After removing the volatiles under reduced pressure, the reaction mixture was purified by silica gel chromatography (DCM/MeOH gradient from 99:1 to 95:5) to give 53.3 mg (78%) of compound B6 as a yellow/green solid. ESI ⁺ [M+H] ⁺ = 835.3. HPLC Method 2 Retention time = 6.50 and 6.60 min (diastereoisomer mixture).

1.2.4.1 Synthesis of tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate

A MeOH solution containing 911 mg (3.23 mmol) of commercially available tert-butyl (2-hydroxy-2-(4-nitrophenyl)ethyl)carbamate (CAS# 939757-25-2) was transferred to a stainless steel hydrogenation reactor. After the first argon purge, a catalytic amount of 5 wt% Pd/C was added. The reactor was then purged twice with _H2 and maintained at room temperature under 10 bar _H2 pressure for 5 hours while the reaction was stirred. After filtering the reactants through a 0.45 μm PTFE filter and removing the MeOH in vacuo, 749 mg (92%) of the title compound was obtained as a white solid. ESI ⁺ [M+H] ⁺ = 253.2. HPLC Method 2 Retention time = 2.73 min.

1.2.4.2 Synthesis of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-hydroxyethyl)carbamate

150mg (0.60mmol) of the aforementioned compound (tert-butyl 2-(4-aminophenyl)-2-hydroxyethyl)carbamate, 222mg (0.71mmol) Fmoc-Ala-OH and 81mg (0.62mmol) DIPEA were dissolved in 5mL anhydrous DMF. 181mg (0.71mmol) HATU was added and the reactant was stirred at room temperature overnight. Then, the volatiles were removed under reduced pressure and the crude residue was purified by silica gel chromatography (DCM/MeOH gradient from 100:0 to 90:10) to quantitatively provide the first intermediate (tert-butyl 2-(4-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionylamino)phenyl)-2-hydroxyethyl)carbamate, which is directly involved in Fmoc-deprotection. HPLC method 2 retention time = 7.7min.

T-butyl (2-(4-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)phenyl)-2-hydroxyethyl)carbamate is dissolved in 5mL DMF/piperidines 9:1 (v/v) and stirred at room temperature for 15min.Then, volatile matter is removed under reduced pressure, and the crude residue is purified by silica gel chromatography (DCM/methanol, gradient from 99:1 to 80:20), 130mg (68%, through two steps) second intermediate (2-(4-((S)-2-aminopropionamido)phenyl)-2-hydroxyethyl)carbamate is obtained, which is a white foamy solid. HPLC method 2 retention time=3.75min.

130 mg (0.40 mmol) of tert-butyl (2-(4-((S)-2-aminopropionamido)phenyl)-2-hydroxyethyl)carbamate and 124 mg (0.48 mmol) of commercially available Ac-Val-Osu (CAS# 56186-37-9) were dissolved in 3 mL of anhydrous DMF and the reaction mixture was stirred at room temperature overnight. Then, the volatiles were removed under reduced pressure and the crude residue was triturated with 3 mL of DCM to give 95 mg (51%) of the title compound as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ )δ9.82(s,1H), 8.16(d,J＝7.1Hz,1H), 7.88(d,J＝8.6Hz,1H), 7.53(d,J＝8.4Hz,2H), 7.22(d,J＝8.5Hz,2H), 6.73-6.58(m,1H), 5.27(d,J＝4.4Hz,1H), 4.58 -4.47 (m, 1H), 4.40 (q, J = 7.1 Hz, 1H), 4.17 (dd, J = 8.4, 6.8 Hz, 1H), 3.15-2.90 (m, 2H), 1.88 (s, 4H), 1.35 (s, 9H), 1.30 (d, J = 7.1 Hz, 3H), 0.92-0.73 (m, 6H). ESI ⁺ [M+H] ⁺ = 487.3. HPLC method 2 retention time = 4.50 min.

1.2.4.3 Synthesis of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate

286 mg (0.62 mmol) of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-hydroxyethyl)carbamate and 375 mg (1.23 mmol) of bis(4-nitrophenyl)carbonate were dissolved in 3 mL of anhydrous DMF. 318 mg (2.46 mmol) of DIPEA were added and the reaction mixture was stirred at room temperature for 3 hours. The volatiles were removed under reduced pressure and the crude residue was purified by silica gel chromatography (DCM/MeOH gradient from 99:1 to 90:10) to give 336 mg (87%) of the title compound as a yellow solid. ¹ H NMR (300 MHz, DMSO-d ₆ )δ9.99(s,1H), 8.36-8.26(m,2H), 8.22(d,J＝6.9Hz,1H), 7.89(d,J＝8.6Hz,1H), 7.63(d,J＝8.6Hz,2H), 7.56-7.46(m,2H), 7.34(d,J＝8.6Hz,2H), 7.22(t ,J=5.6Hz,1H),5. 68 (t, J = 6.0 Hz, 1H), 4.38 (p, J = 7.1 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 3.49-3.34 (m, 2H), 1.87 (s, 3H), 1.37 (s, 9H), 1.30 (d, J = 7.1 Hz, 3H), 0.86 (dd, J = 11.1, 6.8 Hz, 6H). ESI ⁺ [M+Na] ⁺ = 652.2. HPLC method 2 retention time = 7.13 min.

1.2.4.4) Synthesis of Compound B7

43.2 mg (0.069 mmol) of the aforementioned compound (tert-butyl 2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate, 41 mg (0.057) MMAE and 6.7 mg (0.057 mmol) HOBt were dissolved in 1 mL 85:15 (v/v) anhydrous DMF/pyridine mixture. 11.0 mg (0.086 mmol) DIPEA was added. The reaction was stirred at room temperature for 16 hours and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM/MeOH, gradient from 99:1 to 90:10) to give 47 mg (55%) of the intermediate compound (yellowish solid) which was used directly in the Boc-deprotection step. ESI ⁺ [M+Na] ⁺ =1230.7. HPLC Method 2 Retention time = 7.55 min.

The obtained solid was redissolved with TFA/DCM (30:70 v/v) solution at 0°C and stirred at room temperature for 15 min. The volatiles were evaporated under reduced pressure, the crude residue was dissolved in water/ACN (1:1 v/v) solution and purified using HPLC preparative method 5 to give 15 mg (30%) of compound B7 as a white solid. ESI ⁺ [M+H] ⁺ = 1108.7. HPLC method 2 retention time = 5.8 and 5.9 min (diastereoisomer mixture).

1.2.4.5)4 Synthesis of compound B8

Compound B8 was synthesized by the same method used to synthesize compound B7 with slight modifications. The coupling reaction of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate with exatecan mesylate was carried out at 40°C for 3 hours instead of overnight at room temperature.

69 mg (95%) of the intermediate compound 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-((tert-butoxycarbonyl)amino)ethyl((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizinyl[1,2-b]quinolin-1-yl)-1-yl)carbamate were obtained as a brownish yellow solid. ESI ⁺ [M+H] ⁺ =926.4. HPLC method 2 retention time=6.7 and 6.8 min (mixture of diastereomers).

Boc-deprotection was performed as described for compound B7. Purification using HPLC preparative method 5 gave 47 mg (70%) of compound B8 as a yellow solid. ESI ⁺ [M+H] ⁺ = 826.4. HPLC method 3 retention time = 8.30 and 8.75 min (mixture of diastereomers).

1.2.4.6.1 Chiral Separation of a Racemic Mixture of tert-Butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate

use IC MPLC column 30x100mm, 20μm (Daicel cat#83M73) in TeledyneIsco Chiral separation of racemic tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate was performed on an Rf200 system. The mobile phase was DCM + 0.2% (v/v) ethanol (isocratic gradient). The flow rate was 12 mL/min. The sample solvent was DCM + 0.2% (v/v) ethanol. The mass recovery of the two enantiomers after separation was greater than 75%.

The retention time of (S)-tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate was 21 min, while the retention time of (R)-tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate was 29 min. The absolute configuration of the enantiomers (pre-dissolved in a 1:1 mixture of heptane/ethanol and allowed to evaporate slowly for 1 week to induce the formation of crystals) was confirmed by x-ray crystallography. The bulk crystals were mounted on a nylon loop in perfluoroether oil. Data were collected using an Xcalibur, Atlas, Gemini superdiffractometer equipped with an Oxford Cryosystems cryogenic device operating at T = 150.00 (10) K. Data were measured with ω scanning using Cu K _α radiation. The structure was solved with the ShelXT solver using a dual approach and by using Olex2 (OVDolomanov et al., Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., 2009, 42, 339-341) as a graphical interface. The model was refined using ShelXL2018/3 (Sheldrick, GM, Crystal structure refinement with ShelXL, Acta Cryst., 2015, C71, 3-8) on ^F2 using full matrix least squares minimization.

1.2.4.6.2) Synthesis of stereopure B8-S and B8-R compounds

Stereopure compounds B8-S and B8-R were synthesized as described above in Section 1.2.4 without any significant changes in reaction conditions, reactivity or overall yield.

Final purification using HPLC preparative method 5 gave 54 mg of compound B8-S as a yellow solid. ESI ⁺ [M+H] ⁺ = 826.4. HPLC method 3 retention time = 8.45 min.

Final purification using HPLC preparative method 5 gave 46 mg of compound B8-R as a yellow solid. ESI ⁺ [M+H] ⁺ = 826.4. HPLC method 3 retention time = 8.90 min.

1.2.5) Synthesis of Compound B9, Compound B10 and Compound B11

1.2.5.1) Synthesis of Compound B9

20.0 mg (0.036 mmol) Boc-Val-Ala-Pab-PNP (CAS#1884578-00-0, IrisBiotech), 23.1 mg (0.032 mmol) MMAE and 5 mg (0.036 mmol) HOBt were dissolved in 1 mL of 85:15 (v/v) anhydrous DMF/pyridine mixture. 4.6 mg (0.036 mmol) DIPEA was added. The reaction was stirred at room temperature for 16 hours and the volatiles were evaporated under reduced pressure. The crude residue was dissolved in 2 mL TFA/DCM (30:70 v/v) solution and stirred at room temperature for 1 hour. The volatiles were evaporated under reduced pressure, the crude residue was dissolved in water/ACN (1:1 v/v) solution and purified using HPLC preparative method 5 to obtain 11 mg (26%) of compound B9 as a white solid. ESI ⁺ [M+H] ⁺ =1037.7. HPLC Method 2 Retention time = 7.9 min.

1.2.5.2) Synthesis of compound B10

Compound B10 The same procedure as used for the synthesis of compound B9 was followed with slight modifications. The coupling reaction of Boc-Val-Ala-Pab-PNP (CAS# 1884578-00-0) with exatecan mesylate was carried out at 40°C for 3 hours instead of overnight at room temperature.

Boc-deprotection was performed as described above for compound B9. Purification using HPLC preparative method 5 gave 52 mg (90% over two steps) of compound B10 as a yellow solid. ESI ⁺ [M+H] ⁺ = 755.3. HPLC method 2 retention time = 5.70 min.

1.2.5.3) Synthesis of Compound B11

Compound (2S,3S,4S,5R,6S)-6-(2-(3-aminopropionamido)-4-((5R,8R,11R,12S)-11-((R)-sec-butyl)-12-(2-(2-((1R,2S)-3-(((1R,2S)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Compound B11) was prepared according to Jeffrey SC et al. al., Bioconjug. Chem., 2006, 17(3), 831–840).

1.3) Synthesis of drug-linker

1.3.1) Synthesis of glucuronide-based drug-linkers

1.3.1.1) Synthesis of compound DL1

100 mg (0.081 mmol) of Compound A1 (NHS-activated polysarcosine intermediate) and 51 mg (0.061 mmol) of Compound B4 ( _NH2 -payload) were dissolved in anhydrous DMF (0.080 M concentration of Compound B4) in a vial. 41 mg (0.405 mmol) of triethylamine was added and the reaction was stirred at room temperature for 30 min. After complete conversion of the reaction was observed by HPLC, piperidine was added directly to the reaction vial to achieve an 8% (v/v) piperidine solution in DMF. The reaction was then stirred at room temperature for 5-10 min until complete Fmoc deprotection was observed by HPLC. The reaction was heated to 40 ℃ for 1 h with 10% TFA in water/ACN. The 1:1 (v/v) solution was slowly neutralized and purified using HPLC preparation method 5 to obtain 74 mg (70% yield, based on the starting compound B4) of the intermediate compound (2S,3S,4S,5R,6S)-6-(4-(4-1-amino-1-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizinyl[1,2-b ]quinolin-1-yl)amino)-9-glycyl-12,15,18,21,24,27,30,33,36,39-decamethyl-1,6,11,14,17,20,23,26,29,32,35,38,41-trideca-2-oxa-5,9,12,15,18,21,24,27,30,33,36,39-dodeca-tetradecane-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid as a yellow solid. ESI ⁺ [M+H] ⁺ = 1731.7. HPLC Method 3 Retention time = 7.40 min and 7.70 min (equimolar diastereoisomer mixture).

17.8 mg (0.010 mmol) of the compound and 2.85 mg (0.011 mmol) of maleimidoacetic acid N-hydroxysuccinimide ester were dissolved in anhydrous DMF (0.1 M concentration of maleimide compound). 1.56 mg (0.015) of triethylamine were added and the reaction was stirred for 2 hours until complete conversion of the reaction was observed by HPLC. The reaction mixture was then diluted with 1% TFA in water/ACN 1:1 (v/v) and purified using HPLC preparative method 6 to give 13.7 mg (72%) of compound DL1 as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+2H] ²⁺ = 934.8559; found value [M+2H] ²⁺ = 934.8544; error = 1.7 ppm. HPLC Method 3 Retention time = 7.73 min and 8.08 min (equimolar mixture of diastereomers).

1.3.1.2) Synthesis of compound DL2

44.5 mg (0.049 mmol) of compound A2 (azide-polysarcosine intermediate) and 27.5 mg (0.033 mmol) of compound B2 (alkyne-payload) were dissolved in a 1:1 (v/v) solution of 100 mM PBS (pH=7.5) and DMSO to obtain a 0.060 M concentration of compound B2. Then, freshly prepared CuSO ₄ pentahydrate and sodium ascorbate solution (about 250 mg/mL) were added to the reaction vial in sequence to obtain 0.08 molar equivalents of Cu and 1 molar equivalent of sodium ascorbate (based on the molar equivalents of compound B2 in the reaction mixture). The reactants were purged with argon and stirred at 40 ° C. The reaction was monitored by HPLC and completed in less than 2 hours. Then, the reaction mixture was treated with 0.1% TFA in water/ACN The mixture was diluted with 1:1 (v/v) solution in 4% paraformaldehyde and purified using HPLC preparation method 5 to give 44 mg (77%, based on the starting compound B2) of the intermediate compound (2S,3S,4S,5R,6S)-6-(4-(2-(1-(35-amino-3-glycyl-6,9,12,15,18,21,24,27,30,33-decamethyl-5,8,11,14,17,20,23,26,29,32,35-undecaoxo-3,6,9,12,15,18,21,24, 27,30,33-Undecazatricosane-1H-1,2,3-triazol-4-yl)-1-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-pentahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid as a yellow solid. ESI ⁺ [M+H] ⁺ = 1755.7. HPLC Method 3 Retention time = 7.51 min and 7.82 min (diastereoisomer mixture).

26.0mg (0.015mmol) of the compound and 4.11mg (0.016mmol) of maleimidoacetic acid N-hydroxysuccinimide ester were dissolved in anhydrous DMF (0.1M concentration of maleimide compound). 2.25mg (0.022mmol) of triethylamine was added and the reaction was stirred for 2 hours until the complete conversion of the reaction was observed by HPLC. Then, the reaction mixture was diluted with a solution of 1% TFA in water/ACN 1:1 (v/v) and purified using HPLC preparation method 6 to obtain 16.0mg (57%) of compound DL2 as a yellow solid. ESI ⁺ [M+H] ⁺ = 1892.7. HPLC method 3 retention time = 7.95min and 8.20min (equimolar diastereoisomer mixture).

1.3.1.3) Synthesis of compound DL3

5.4 mg (0.023 mmol) of commercially available 2-(4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)phenyl)acetic acid (CAS#91574-45-7) and 9.04 mg (0.021 mmol) of COMU were dissolved in anhydrous DMF (0.1 M concentration of maleimide compound). 4.75 mg (0.067 mmol) of triethylamine were added. The reactants were pre-incubated at room temperature for 2 min and transferred to 9.8 mg (0.012 mmol) of compound B4 (pre-weighed in a reaction vial). The reactants were stirred for 30 min until complete conversion of the reaction was observed by HPLC. The reaction mixture was then diluted with a solution of 1% TFA in water/ACN 1:1 (v/v) and purified using HPLC preparative method 5 to give 4.7 mg (40%) of compound DL3 as a yellow solid. HRMS m/z (ESI ⁺ ): Calculated [M+H] ⁺ =1049.2847; Found [M+H] ⁺ =1049.2873; Error = -2.5 ppm. HPLC Method 3 Retention time = 9.80 min (equimolar mixture of diastereomers).

1.3.1.4) Synthesis of compound DL4

16.0 mg (0.054 mmol) of N-(2-azidoethyl)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide (synthesized according to the procedure described in patent WO2019081455 - a very unstable compound that can only be kept for a few days after storage at -20°C) and 22.6 mg (0.027 mmol) of compound B2 were dissolved in a 1:1 (v/v) solution of 100 mM PBS (pH = 7.5) and DMSO to obtain a 0.060 M concentration of compound B2. Then, freshly prepared CuSO ₄ pentahydrate and sodium ascorbate solution (about 250 mg/mL) were added sequentially to the reaction vial to obtain 0.08 molar equivalents of Cu and 1 molar equivalent of sodium ascorbate (based on the molar equivalents of compound B2 in the reaction mixture). The reactants were purged with argon and stirred at room temperature. The reaction was monitored by HPLC and completed in less than 1 hour. The reaction mixture was then diluted with 0.1% TFA in water/ACN 1:1 (v/v) and purified using HPLC preparative method 6 to give 16 mg (53%) of compound DL4 as a yellow solid. HRMS m/z (ESI ⁺ ): Calculated value [M+H] ⁺ = 1144.3331; Found value [M+H] ⁺ = 1144.3351; Error = -1.8 ppm. HPLC method 3 retention time = 9.61 min (equimolar diastereoisomer mixture).

1.3.1.5) Synthesis of compound DL5

Compound DL5 was synthesized as described above using the same procedure as for compound DL1. The starting materials were compound A4 (NHS-activated polysarcosine intermediate) and compound B3 ( _NH2 -payload).

10.8 mg (48% for two steps) of compound DL5 were obtained as a light yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+3H] ³⁺ =765.0460; found value [M+3H] ³⁺ =765.0443; error = 2.2 ppm. HPLC method 3 retention time = 9.14 min (equimolar mixture of diastereomers).

1.3.1.6) Synthesis of compound DL6

Compound DL6 was synthesized as described above using the same procedure as for compound DL2. The starting materials were compound A5 (azide-polysarcosine intermediate) and compound Bl (alkyne-payload).

16.3 mg (41%, two steps) of compound DL6 were obtained as a light yellow solid. ESI ⁺ [M+2H] ²⁺ = 1159.1. HPLC method 3 retention time = 9.32 min and 9.45 min (equimolar mixture of diastereomers).

1.3.1.7) Synthesis of compound DL7

Compound DL7 was synthesized as described above using the same procedure as for compound DL3. The starting materials were the commercially available compound Maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and compound B3 (NH ₂ -payload).

15.6 mg (69%) of compound DL7 were obtained as a white solid. ESI ⁺ [M+2Na] ²⁺ = 701.3. HPLC method 3 retention time = 10.94 min (equimolar mixture of diastereomers).

1.3.1.8) Synthesis of compound DL8

15.0 mg (0.013 mmol) of compound B1 (alkyne-payload) and 3.44 mg (0.040 mmol) of 2-azidoethylamine were dissolved in a 1:1 (v/v) solution of 100 mM PBS (pH=7.5) and DMSO to obtain a 0.060 M concentration of compound B1. Then, freshly prepared CuSO ₄ pentahydrate and sodium ascorbate solution (about 250 mg/mL) were added to the reaction vial in sequence to obtain 0.08 molar equivalents of Cu and 1 molar equivalent of sodium ascorbate (based on the molar equivalents of compound B1 in the reaction mixture). The reactants were purged with argon and stirred at room temperature. The reaction was monitored by HPLC and completed in less than 1 hour. Then, the reaction mixture was heated to 40 °C with 0.1% TFA in water/ACN. The mixture was diluted with a solution in 1:1 (v/v) and purified using HPLC preparation method 5 to give 18.2 mg (110%, based on the starting compound B1) of the intermediate compound (2S,3S,4S,5R,6S)-6-(4-((3S,4R,7R,10R)-15-(1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)-4-((R)-sec-butyl)-3-(2-(2-((1S,2S)-3-( ((1R,2S)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecan-14-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid as a white solid. ESI ⁺ [M+H] ⁺ = 1213.6. HPLC method 2 retention time = 5.55 min and 5.65 min (diastereoisomer mixture).

5.79 mg (0.022 mmol) of commercially available maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and 9.00 mg (0.021 mmol) of COMU were dissolved in anhydrous DMF (0.1 M concentration of maleimide compound). 6.1 mg (0.060 mmol) of triethylamine were added. The reaction was pre-incubated at room temperature for 2 min and transferred to 18.2 mg (0.012 mmol) of the aforementioned compound (pre-weighed in a reaction vial). The reaction was stirred for 30 min until complete conversion of the reaction was observed by HPLC. The reaction mixture was then diluted with a solution of 1% TFA in water/ACN 1:1 (v/v) and purified using HPLC preparative method 5 to give 16.0 mg (73%) of compound DL8 as a white solid. HRMS m/z (ESI ⁺ ): Calculated [M+2H] ²⁺ =726.8609; Found [M+2H] ²⁺ =726.8631; Error = -3.1 ppm. HPLC Method 3 Retention time = 10.40 min and 10.56 min (equimolar mixture of diastereomers).

1.3.1.9) Synthesis of compound DL9

Compound DL9 was synthesized as described above using the same procedure as for compound DL3. The starting materials were the commercially available compound Maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and compound B11 (NH ₂ -payload).

10.0 mg (41%) of compound DL9 was obtained as a white solid. HRMS m/z (ESI ⁺ ): calculated value [M+2H] ²⁺ =685.3549; found value [M+2H] ²⁺ =685.3554; error = -0.8 ppm. HPLC method 3 retention time = 11.38 min.

1.3.1.10) Synthesis of compound DL10

Compound DL10 was synthesized as described above using the same procedure as for compound DL1. The starting materials were compound A6 (NHS-activated polysarcosine intermediate) and compound B4 ( _NH2 -payload).

54.1 mg (44% for two steps) of compound DL10 were obtained as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+2H] ²⁺ = 934.8559; found value [M+2H] ²⁺ = 934.8545; error = 1.5 ppm. HPLC method 3 retention time = 7.68 min and 8.01 min (equimolar diastereomeric mixture).

1.2.1.11) Synthesis of compounds DL10-S and DL10-R

Compounds DL10-S and DL10-R were synthesized as described above using the same procedure as for compound DL1. The starting materials were compounds B4-S and B4-R ( _NH2 -payload) and compound A6 (NHS-activated polysarcosine intermediate), respectively.

4.7 mg of compound DL10-S were obtained as a yellow solid. ESI ⁺ [M+Na] ⁺ = 1890.7. HPLC method 3 retention time = 7.62 min.

4.0 mg of compound DL10-R were obtained as a yellow solid. ESI ⁺ [M+Na] ⁺ = 1890.7. HPLC method 3 retention time = 8.06 min.

1.2.1.12) Synthesis of compound DL11

Compound DL11 was synthesized as described above using the same procedure as for compound DL2. The starting materials were compound A3 (azide-polysarcosine intermediate) and compound B2 (alkyne-payload).

14.3 mg (43% for two steps) of compound DL11 were obtained as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+2H] ²⁺ =947.3536; found value [M+2H] ²⁺ =947.3540; error = -0.5 ppm. HPLC method 3 retention time = 8.13 min and 8.36 min (equimolar mixture of diastereomers).

1.3.2) Synthesis of dipeptide-based drug-linkers

1.3.2.1) Synthesis of compound DL12

Compound DL12 was synthesized as described above using the same procedure as for compound DL1. The starting materials were compound A7 (NHS-activated polysarcosine intermediate) and compound B8 ( _NH2 -payload).

34.9 mg (46% for two steps) of compound DL12 were obtained as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+2H] ²⁺ = 981.4394; found value [M+2H] ²⁺ = 981.4398; error = -0.4 ppm. HPLC method 3 retention time = 8.81 min and 8.94 min (equimolar mixture of diastereomers).

1.3.2.2) Synthesis of compounds DL12-S and DL12-R

Compounds DL12-S and DL12-R were synthesized as described above using the same procedure as for compound DL1. The starting materials were compounds B8-S and B8-R ( _NH2 -payload) and compound A7 (NHS-activated polysarcosine intermediate), respectively.

18.1 mg of compound DL12-S was obtained as a yellow solid. ESI ⁺ [m+2H] ²⁺ = 981.4. HPLC method 3 retention time = 8.78 min.

16.3 mg of compound DL12-R were obtained as a yellow solid. ESI ⁺ [m+2H] ²⁺ = 981.4. HPLC method 3 retention time = 8.96 min.

1.3.2.3) Synthesis of compound DL13

Compound DL13 was synthesized as described above using the same procedure as for compound DL2. The starting materials were compound A8 (azide-polysarcosine intermediate) and compound B6 (alkyne-payload).

4.5 mg (24%, two steps) of compound DL13 were obtained as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+2H] ²⁺ = 993.4451; found value [M+2H] ²⁺ = 993.4416; error = 3.5 ppm. HPLC method 3 retention time = 8.88 min and 9.11 min (equimolar diastereoisomer mixture).

1.3.2.4) Synthesis of compound DL14

Compound DL14 was synthesized as described above using the same procedure as for compound DL3. The starting materials were the commercially available compound maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and compound B8 (NH ₂ -payload).

7.0 mg (59%) of compound DL14 were obtained as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+H] ⁺ = 1065.4364; found value [M+H] ⁺ = 1065.4370; error = -0.5 ppm. HPLC method 3 retention time = 10.20 min and 10.44 min (equimolar mixture of diastereomers).

1.3.2.5) Synthesis of compound DL15

Compound DL15 was synthesized as described above using the same procedure as for compound DL8.

3.1 mg (20% for two steps) of compound DL15 were obtained as a yellow solid. HRMS m/z (ESI ⁺ ): calculated value [M+H] ⁺ = 1160.4848; found value [M+H] ⁺ = 1160.4854; error = -0.6 ppm. HPLC method 3 retention time = 9.96 min and 10.12 min (equimolar mixture of diastereomers).

1.3.2.6) Synthesis of compound DL16

Compound DL16 was synthesized as described above using the same procedure as for compound DL3. The starting materials were the commercially available compound Maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and compound B7 (NH ₂ -payload).

5.3 mg (41%) of compound DL16 were obtained as a white solid. HRMS m/z (ESI ⁺ ): calculated value [M+Na] ⁺ =1369.7618; found value [M+Na] ⁺ =1369.7621; error = -0.3 ppm. HPLC method 3 retention time = 11.38 min (equimolar mixture of diastereomers).

1.3.2.7) Synthesis of compound DL17

Compound DL17 was synthesized as described above using the same procedure as for compound DL8.

8.9 mg (24% for two steps) of compound DL17 were obtained as a white solid. HRMS m/z (ESI ⁺ ): calculated value [M+H] ⁺ =1442.8294; found value [M+H] ⁺ =1442.8284; error = 0.7 ppm. HPLC method 3 retention time = 11.81 min (equimolar mixture of diastereomers).

1.3.2.8) Synthesis of compound DL18

Compound DL18 was synthesized as described above using the same procedure as for compound DL3. The starting materials were the commercially available compound Maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and compound B10 (NH ₂ -payload).

14.5 mg (62%) of compound DL18 were obtained as a yellow solid. ESI ⁺ [M+H] ⁺ = 994.4. HPLC method 3 retention time = 11.62 min.

1.3.2.9) Synthesis of compound DL19

Compound DL19 was synthesized as described above using the same procedure as for compound DL3. The starting materials were the commercially available compound Maleimide-PEG ₂ -acid (CAS# 1374666-32-6) and compound B9 (NH ₂ -payload).

4.7 mg (35%) of compound DL19 were obtained as a white solid. HRMS m/z (ESI ⁺ ): calculated value [M+H] ⁺ =1276.7439; found value [M+H] ⁺ =1276.7441; error = -0.1 ppm. HPLC method 3 retention time = 13.28 min.

1.3.2.10) Synthesis of compound DL20

Compound DL20 was synthesized as described above using the same procedure for compound DL2. The starting materials were compound A3 (azide-polysarcosine intermediate) and compound B6 (alkyne-payload).

14.0 mg (36% for two steps) of compound DL20 were obtained as a yellow solid. ESI ⁺ [M+H] ⁺ = 1883.8. HPLC method 3 retention time = 8.82 min and 9.07 min (equimolar mixture of diastereomers).

2) Preparation and characterization of conjugates

2.1) Preparation of Antibody-Drug Conjugates

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. The fully reduced antibody was buffer exchanged with 100 mM potassium phosphate + 1 mM EDTA pH 7.4 using an Amicon 30K centrifugal filter unit (Millipore) by three rounds of dilution/centrifugation. 10-12 molar equivalents of drug-linker (from 12 mM DMSO stock solution) were added to the antibody (residual DMSO <10% v/v) to obtain a drug-antibody ratio (DAR) of 8. The solution was incubated at room temperature for 30 min. The conjugate was buffer exchanged/purified with PBS pH 7.4 by four rounds of dilution/centrifugation using an Amicon 30K centrifugal filter unit and sterile filtered (0.20 μm PES filter).

Conjugates containing self-hydrolyzable maleimides (maleimide-phenyl and maleimide-glycine) were incubated at 5 mg/mL in PBS 8.0 at 37°C for 24 hours to ensure complete hydrolysis of the succinimidyl moiety, buffer exchanged with PBS pH 7.4 using an Amicon 30K centrifugal filter device and sterile filtered (0.20 μm PES filter).

Final protein concentration was assessed spectrophotometrically at 280 nm using a Colibri microspectrometer apparatus (Titertek Berthold).

2.2) Characterization of conjugates

The obtained conjugates were characterized as follows:

Reverse Phase Liquid Chromatography-Mass Spectrometry (RPLC-MS):

Denaturing RPLC-QToF analysis was performed using the UHPLC method 4 described above. Briefly, a mobile phase gradient of water/acetonitrile + 0.1% formic acid (0.4 mL/min) was used on an Agilent PLRP-S The conjugate was eluted on a 2.1x150 mm 8μm (80°C) and detected using a Bruker Impact II ^™ Q-ToF mass spectrometer scanning the m/z range of 500-3500 (ESI ⁺ ). The MaxEnt algorithm included in the software deconvolves the data.

Size Exclusion Chromatography (SEC):

SEC was performed on an Agilent 1100 HPLC system with an extra-column volume of less than 15 μL (equipped with a short section of 0.12 mm id peek tubing and a microvolume UV flow cell). The column was an Agilent Advance BioSEC 4.6×150 mm 2.7 μm (kept at 30° C.). The mobile phase was 100 mM sodium phosphate and 200 mM sodium chloride (pH 6.8). 10% acetonitrile (v/v) was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and to prevent bacterial growth. The flow rate was 0.35 mL/min. UV detector monitoring was performed at 280 nm.

Hydrophobic Interaction Chromatography (HIC):

Hydrophobic interaction chromatography (HIC) was performed on an Agilent 1100 HPLC system. The column was a Tosoh TSK-GELBUTYL-NPR 4.6x35mm 2.5μm (25°C). Mobile phase A was 1.5M ( _NH4 ) _2SO4 + 25mM potassium phosphate pH 7.0. Mobile phase B was 25mM potassium phosphate pH 7.0 + 15% isopropanol (v/v). The linear gradient was 0% _B to 100% B in 10 min, then held at 100% B for 3 min. The flow rate was 0.75mL/min. UV detector monitoring was performed at 220 and 280nm.

3) Hydrophobic interaction chromatography (HIC) profile of antibody-drug conjugates (ADCs)

The apparent hydrophobicity of the conjugates was assessed by hydrophobic interaction chromatography (HIC) using a Tosoh TSK-GEL BUTYL-NPR column as described in Section 2.

The results for the glycosidase-based drug-linkers of the present invention are shown in Figure 1 (octopamine structure), and the results for the dipeptidase-based drug-linkers of the present invention are shown in Figure 2 (2-amino-1-(4-aminophenyl)ethan-1-ol structure). The conjugates of the present invention are systematically more hydrophilic (shorter retention time in HIC chromatography) than the known corresponding structures. This effect is observed for glycosidase-based drug-linkers and dipeptidase-based drug-linkers. This effect is observed with or without the presence of the hydrophobic masking entity polysarcosine in the drug-linker structure. This effect is observed with drug payloads of different nature and different levels of inherent hydrophobicity.

4) In vitro cytotoxicity assay of drug-linker conjugates based on the present invention and drug-linker conjugates based on known structures

The in vitro cytotoxicity of the conjugate was evaluated on several antigen-positive cancer cell lines. Depending on the cell line, the cells were seeded in 96-well plates at an appropriate density (1000-10000 cells/well in 100 μL of appropriate culture medium) and incubated at 37 ° C for 24 hours. A serial dilution of the test compound pre-dissolved in the culture medium (50 μL) was added, and for MMAE-based conjugates, incubated at 37 ° C for 72 hours, and for exatecan-based conjugates, incubated for 144 hours. MTT (5 mg/mL, 20 μL, Sigma-Aldrich) was added to the wells, and incubated for 1-2 hours at 37 ° C. Then, the culture medium was carefully removed and the well contents were evenly dissolved with acidified isopropanol. The absorbance value was measured using a wavelength of 570 nm (reference wavelength 690 nm) on a Multiskan ^TM Sky microplate reader (Thermo Scientific). _IC50 concentration values compared to untreated control cells were determined using inhibitory dose-response curve fitting (GraphPad Prism 9).

The results of the glycosidase-sensitive drug-linker based conjugates are shown in Figure 3, and the results of the dipeptidase-sensitive drug-linker based conjugates are shown in Figure 4. When compared with the known corresponding structures, the conjugates of the present invention (octopamine and 2-amino-1-(4-aminophenyl)ethan-1-ol structures) systematically show similar potency.

5) In vitro cytotoxicity assay of conjugates based on the DL10 drug-linker (equimolar diastereoisomer mixture) of the present invention, the stereo-defined DL10-S drug-linker of the present invention, and the stereo-defined DL10-R drug-linker of the present invention

The in vitro cytotoxicity of the conjugates was evaluated on several antigen-positive cancer cell lines according to the experimental protocol described in section 4) above.

The results are shown in Figure 5. No difference in in vitro potency was observed between stereopure drug-linkers and equimolar diastereomeric mixtures of the same drug-linkers.

6) Pharmacokinetic curves of the conjugate after a single intravenous administration of 3 mg/kg to rats (total antibody-drug conjugate concentration over time)

ADC was injected into female Sprague-Dawley rats (4-6 weeks old-Charles River) at 3 mg/kg via the tail vein (three animals per group, randomly assigned). Blood was drawn into citrate tubes by retro-orbital bleeding at different time points, processed into plasma and stored at -80°C until analysis. ADC concentration was assessed using a human IgG ELISA kit (Stemcell ^TM Technologies) according to the manufacturer's protocol. Quantification was performed using a standard curve of the corresponding monoclonal antibody. Pharmacokinetic parameters (clearance, half-life and AUC) were measured using a PK function Software (plug-in developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA), calculated by two-compartment analysis.

The results for the glycosidase-sensitive drug-linker based conjugates are shown in Figure 6 (PK curves) and Figure 7 (PK parameters), and the results for the dipeptidase-sensitive drug-linker based conjugates are shown in Figure 8 (PK curves) and Figure 9 (PK parameters). When compared to the known corresponding structures, the conjugates of the present invention (octopamine and 2-amino-1-(4-aminophenyl)ethan-1-ol structures) systematically produce improved pharmacokinetic curves and pharmacokinetic parameters (improved exposure, increased half-life and reduced clearance).

7) Changes in tumor volume (mm 3 ) over time in the NCI-N87 HER2+ gastric cancer xenograft model after a single intravenous administration of ¹ mg/kg ADC110 conjugate (octopamine structure of the present invention with glucuronide-exatecan payload) and ADC112 conjugate (triazole structure with glucuronide-exatecan payload)

NCI-N87 gastric cancer cells were implanted subcutaneously into female SCID mice (4 weeks old). When the tumor grew to about 150 mm ³ , ADC was intravenously administered once at a single therapeutic dose of 1 mg/kg (6 animals per group, grouped to minimize the difference in initial tumor volume between groups). Tumor volume was measured by caliper every 3-5 days and calculated using the formula (L×W ² )/2. Mice were sacrificed when the tumor volume exceeded 1000 mm ³ .

The results are shown in Figure 10. When compared with the triazole structure-based conjugate ADC112, the octopamine structure-based conjugate ADC110 of the present invention showed improved in vivo activity. No significant weight loss was observed in all treated mice.

Claims (17)

1. Ligand-drug-conjugate compound (LDC) having the following formula (I) in L is the ligand; X1 is the joint unit; Z is an optional spacer; X2 is the joint unit; K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol; R1 is selected from the following group: H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ -C ₈ cycloalkyl , heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores; Each R2 is independently selected from the group consisting of electron-withdrawing groups and C ₁ -C ₄ alkyl groups; n is 0, 1 or 2; R4 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally interrupted by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; R5 is selected from the group consisting of H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a sugar cleavable unit, Y is O, or when T is a polypeptide cleavable unit, Y is NR3; R3 is selected from the following group: H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ -C ₈ cycloalkyl , heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O), -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”-and triazole; R" and R"' are independently selected from H and C ₁ -C ₆ alkyl; and pharmaceutically acceptable salts thereof. 2. The LDC compound according to claim 1, wherein L is a ligand selected from the group consisting of polypeptides, proteins, antibodies and antibody fragments, preferably L is an antibody. 3. The LDC compound according to claim 1 or 2, wherein D is selected from the group consisting of drugs, preferably D is an anticancer drug or an immunomodulator. 4. The LDC compound according to any one of the preceding claims, wherein X1 and X2 are independently selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C ₁ -C ₁₂ alkylene, arylene with 6 to 10 ring atoms, C ₃ -C ₈ cycloalkylene, heterocycloalkylene with 5 to 10 ring atoms, 5 to 10 ring atoms heteroarylene, C ₂ -C ₁₀ alkenylene and any combination thereof of ring atoms, The alkylene and alkenylene groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, - C(O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR "-and triazole, And the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene groups are optionally substituted by one or more substituents selected from the following: halogen, oxo , -OH, -NO ₂ , -CN, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, heterocyclic group with 5-10 ring atoms, aryl group with 6-10 ring atoms , heteroaryl with 5-10 ring atoms, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy, -(CO)-R', -O- (CO)-R', -(CO)-O-R', -(CO)-NR"R"', -NR"-(CO)-R' and -NR"R"'; R', R" and R"' are independently selected from H and C ₁ -C ₆ alkyl. 5. The LDC compound according to any one of the preceding claims, wherein Z is independently selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C ₁ -C ₁₂ alkylene, arylene having 6 to 10 ring atoms, C ₃ -C ₈ cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, having 5 to 10 ring atoms heteroarylene, C ₂ -C ₁₀ alkenylene and any combination thereof, The alkylene and alkenylene groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, - C(O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR "-and triazole, And the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene groups are optionally substituted by one or more substituents selected from the following: halogen, oxo , -OH, -NO ₂ , -CN, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, heterocyclic group with 5-10 ring atoms, aryl group with 6-10 ring atoms , heteroaryl with 5-10 ring atoms, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy, -(CO)-R', -O- (CO)-R', -(CO)-O-R', -(CO)-NR"R"', -NR"-(CO)-R' and -NR"R"'; R', R" and R"' are independently selected from H and C ₁ -C ₆ alkyl. 6. The LDC compound of any one of the preceding claims, wherein K is polysarcosine. 7. The LDC compound of any one of the preceding claims, wherein T is a sugar cleavable unit which is a glucuronide. 8. The LDC compound according to any one of claims 1 to 6, wherein T is a dipeptide, preferably selected from: Val-Cit, Val-Ala and Phe-Lys. 9. The LDC compound according to any one of claims 1-6, wherein the compound is a compound of formula (VI) wherein k is an integer from 2 to 50, preferably 4 to 30; and T is a polypeptide cleavable unit, preferably a dipeptide. 10. The LDC compound according to any one of claims 1-6, wherein the compound is a compound of formula (VIII) wherein k is an integer from 2 to 50, preferably 4 to 30; and T is a sugar cleavable unit, preferably glucuronide or galactoside. 11. A pharmaceutical composition comprising an LDC compound according to any one of the preceding claims and a pharmaceutically acceptable carrier. 12. The LDC compound according to any one of claims 1-10 for use as a medicament. 13. Intermediate compound of formula (II) in X1' is a group that can react with a ligand to form a linker unit; Z is an optional spacer; X2 is the joint unit; K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol; R1' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores; Each R2 is independently selected from the group consisting of electron-withdrawing groups and C ₁ -C ₄ alkyl groups; n is 0, 1 or 2; R4 is selected from the group consisting of: H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; R5 is selected from the group consisting of: H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a sugar cleavable unit, Y' is O, or when T is a polypeptide cleavable unit, Y' is NR3'; R3' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally separated by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; R" and R"' are independently selected from H and C ₁ -C ₆ alkyl; and pharmaceutically acceptable salts thereof. 14. The intermediate compound of formula (II) according to claim 11, wherein K is polysarcosine. 15. Intermediate compound of formula (III) in R1' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; D is an active agent, preferably selected from the group consisting of drugs, imaging agents and fluorophores; Each R2 is independently selected from the group consisting of electron-withdrawing groups and C ₁ -C ₄ alkyl groups; n is 0, 1 or 2; R4 is selected from the group consisting of: H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; R5 is selected from the group consisting of: H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a sugar cleavable unit, Y' is O, or when T is a polypeptide cleavable unit, Y' is NR3'; R3' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; R" and R"' are independently selected from H and C ₁ -C ₆ alkyl; and pharmaceutically acceptable salts thereof. 16. Intermediate compounds of formula (IV) in R1' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; Each R2 is independently selected from the group consisting of electron-withdrawing groups and C ₁ -C ₄ alkyl groups; n is 0, 1 or 2; R4 is H; R5 is H; T is a sugar cleavable unit; Y' is O; and pharmaceutically acceptable salts thereof. 17. Intermediate compounds of formula (IV) in R1' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; Each R2 is independently selected from the group consisting of electron-withdrawing groups and C ₁ -C ₄ alkyl groups; n is 0, 1 or 2; R4 is selected from the group consisting of: H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; R5 is selected from the group consisting of: H, C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl, the alkyl and alkenyl groups being optionally separated by one or more heteroatoms or chemical groups selected from : -O-, -S-, -C(O)- and -NR”-; Y' is NR3'; T is the cleavable unit of the polypeptide; R3' is selected from the following group: amino protecting group, H, C ₁ -C ₂₄ alkyl, C ₂ -C ₆ alkenyl; optionally substituted polyether, aryl group with 6-10 ring atoms, C ₃ - C ₈ cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)-, -NR"-, -C( O)NR”-, -NR”-C(O)-, -NR”-C(O)-NR”’-, -NR”-C(O)-O-, -O-C(O)NR”- and triazole; R" and R"' are independently selected from H and C ₁ -C ₆ alkyl; and pharmaceutically acceptable salts thereof.