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

Abstract

The present invention relates to enzyme-triggered self-reactive arm compounds, chemical intermediates used to prepare such compounds, and their uses, particularly in prodrug design and conjugation techniques.The present invention also relates to ligand-drug-conjugates (LDCs) that contain such enzyme-triggered self-reactive arms.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to compounds containing enzyme-triggered self-reactive arms, chemical intermediates used to prepare such compounds, and their uses, particularly in prodrug design and conjugation techniques.

[0002] The present invention also relates to Ligand-Drug-Conjugates (LDCs) which contain such enzyme-triggered self-reactive arms.

[0003] 〔background〕 Ligand-drug-conjugates (LDCs) are designed to specifically deliver active compounds to target tissues while sparing healthy tissues. For example, in the case of LDCs aimed at delivering cytotoxic anticancer drugs to tumor cells, the LDC format can be used to improve the toxicity profile and enhance the tolerability of the treatment.

[0004] LDCs contain at least one ligand unit, which is usually a polypeptide, protein or targeting small molecule, covalently linked to at least one therapeutic, diagnostic or labeling compound (hereinafter referred to as drug or D) via a synthetic linker. The synthetic linker may contain one or more monovalent or divalent arms for linking the ligand unit(s) with the drug unit(s), which may be selected from spacers, connectors and enzyme-sensitive cleavable moieties. The linker may also orthogonally carry any moiety that can improve the LDC performance (such as storage stability, phenotypic stability or pharmacokinetic properties). When the ligand unit of the conjugate is an antibody or antibody fragment and is linked to an immunostimulant, cytotoxic or chemotherapeutic drug, the term antibody-drug-conjugate (ADC) is commonly used.

[0005] When designing LDCs, it is necessary to covalently link the final active drug to the ligand targeting unit while allowing the eventual release of the drug unit by selective enzymatic mechanisms after cellular internalization or in the microenvironment of diseased tissues. From this perspective, several peptidase-sensitive cleavable linker chemistry strategies and glycosidase-sensitive cleavable linker chemistry strategies (associated with self-sacrificial chemistry) have been developed. These cleavable linkers and the 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).

[0006] For example, WO2011145068 discloses the use of glycosidase-sensitive cleavable drug linkers based on the 4-(1-hydroxybut-3-yn-1-yl)phenol self-immolative chemical spacer, which provides a terminal alkyne handle for click chemistry performance (see formula below).

[0007] [ka]

[0008] WO2017089895 describes the use of glycosidase-sensitive cleavable drug linkers based on the 2-hydroxy-5-(hydroxymethyl)benzoic acid self-immolative chemical spacer (see formula below).

[0009] [ka]

[0010] However, there is a continuing desire to develop enzyme-sensitive self-immolative linkers that would improve the physicochemical and pharmacological properties of LDCs.

[0011] In this context, therefore, one of the objectives of the present disclosure is to provide enzyme-sensitive self-immolative linkers that can be used to prepare LDCs.

[0012] Another object of the present disclosure is an enzyme-sensitive self-immolative linker that can improve the physicochemical and / or pharmacological properties of LDCs.

[0013] Another object of the present disclosure is to provide compounds that can be used to prepare LDCs.

[0014] 〔summary〕 In a first aspect, the present disclosure relates to a Ligand-Drug-Conjugate Compound (LDC) having the following formula (I):

[0015] [ka]

[0016] During the ceremony, L is a ligand; X1 is the connector unit; Z is an optional spacer; X2 is the connector unit; K is any hydrophobic masking material, preferably selected from polysarcosine and polyethylene glycol; R1 is H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; D is an active agent, preferably selected from the group consisting of a drug, an imaging agent and a fluorescent agent; Each R2 is an electron withdrawing group and a C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y is O when T is a sugar cleavable unit or NR3 when T is a polypeptide cleavable unit; R3 is H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0017] It has been found that the use of 2-amino-1-(4-aminophenyl)ethan-1-ol-based self-immolative linkers or 2-amino-1-(4-oxophenyl)ethan-1-ol-based self-immolative linkers as in formula (I) surprisingly results in LDCs with improved hydrophilicity and improved in vivo pharmacokinetic profile. This self-immolative linker also facilitates the manufacture, purification and formulation of the compound of formula (I), thus allowing for a higher yield. The compound of formula (I) also has a lower possibility of aggregation due to the presence of this specific linker.

[0018] The present disclosure also relates to a pharmaceutical composition comprising a compound of the present disclosure, preferably a compound of formula (I), and a pharma- ceutically acceptable carrier.

[0019] The present disclosure also relates to compounds of the present disclosure, preferably compounds of formula (I), for use as a medicament.

[0020] The present disclosure also relates to an intermediate compound of formula (II):

[0021] [ka]

[0022] During the ceremony, X1' is a group capable of reacting with a ligand to form a connector unit; Z is an optional spacer; X2 is the connector unit; K is any hydrophobic masking material, preferably selected from polysarcosine and polyethylene glycol; R1' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; D is an active agent, preferably selected from the group consisting of a drug, an imaging agent and a fluorescent agent; Each R2 is an electron withdrawing group and a C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0023] The present disclosure also relates to an intermediate compound of formula (III)

[0024] [ka]

[0025] During the ceremony, R1' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; D is an active agent, preferably selected from the group consisting of a drug, an imaging agent and a fluorescent agent; Each R2 is an electron withdrawing group and a C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0026] The present disclosure also relates to an intermediate compound of formula (IV)

[0027] [ka]

[0028] During the ceremony, R1' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; Each R2 is an electron withdrawing group and a C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS 1 and 2 show the hydrophobic interaction chromatograms according to Example 3.

[0030] 3 and 4 represent the in vitro cytotoxicity assay of the conjugate according to Example 4.

[0031] FIG. 5 represents the in vitro cytotoxicity assay of the conjugate according to Example 5.

[0032] 6, 7, 8 and 9 represent the in vivo pharmacokinetic profile in rats and the pharmacokinetic parameters according to Example 6.

[0033] FIG. 10 depicts tumor volume over time in a mouse xenograft model of gastric cancer according to Example 7.

[0034] Detailed Description [Definition] Various embodiments of the present disclosure are described herein. It will be recognized that the features specified in each embodiment may be combined with other specified features to provide further embodiments.

[0035] The present disclosure encompasses the compounds of the present disclosure, their tautomers, enantiomers, diastereomers, racemates or mixtures thereof, as well as hydrates, esters, solvates or pharma- ceutically acceptable salts thereof.

[0036] Any of the formulas shown herein may be used with deuterium labeled compounds or 14 It is intended to represent unlabeled forms of the compounds as well as isotopically labeled forms of the compounds, such as C-labeled compounds.

[0037] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the disclosed compounds and is typically not biologically or otherwise undesirable. In many cases, the disclosed compounds can form acid salts and / or base salts by virtue of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with organic and / or inorganic acids. Pharmaceutically acceptable base addition salts can be formed with organic and / or inorganic bases. Such salts are known to those skilled in the art.

[0038] As used herein, "C 1 -C 24 The term "alkyl" by itself or as part of another substituent refers to a straight chain or branched alkyl functional group having 1 to 24 carbon atoms, preferably 1 to 20 carbon atoms, and more preferably 1 to 12 or 1 to 6 carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and t-butyl, pentyl and its isomers (e.g., n-pentyl, iso-pentyl), and hexyl and its isomers (e.g., n-hexyl, iso-hexyl).

[0039] Alkylene, used alone or as part of, for example, alkylene glycol, refers to a divalent saturated, straight chain or branched alkyl group as defined herein.

[0040] Alkenyl and alkynyl are at least partially unsaturated alkyl groups having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, especially 2 to 4 carbon atoms. It refers to a straight-chain or branched hydrocarbon group. An alkenyl group contains at least one C=C double bond; an alkynyl group contains at least one C≡C triple bond.

[0041] As used herein, "C 3 -C 8 The term "cycloalkyl" or "carbocycle" refers to a saturated or unsaturated cyclic group having 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms. Cycloalkyls can have a single ring or multiple rings fused together. Cycloalkyls can also include spirocycles. Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0042] As used herein, "C 3 -C 8 The term "cycloalkylene" or "carbocyclo" refers to a divalent cycloalkyl as defined herein.

[0043] As used herein, the term "halogen" refers to a fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I) group.

[0044] As used herein, "C 1 -C 6 The term "haloalkyl" refers to any C, as defined herein, substituted by one or more halogen groups, as defined herein. 1 -C 6 Refers to alkyl. Preferred C 1 -C 6 Haloalkyl groups include trifluoromethyl and dichloromethyl.

[0045] As used herein, the term "heteroalkyl" refers to a straight or branched hydrocarbon chain consisting of 1 to 12 carbon atoms, preferably 1 to 10, more preferably 1 to 6 carbon atoms, and 1 to 3 heteroatoms selected from the group consisting of O, N, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized (e.g., sulfoxide or sulfone) and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, and S may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.

[0046] Heteroalkylene refers to a divalent heteroalkyl as defined above. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.

[0047] As used herein, "C 1 -C 6 The term "alkoxy" refers to an -O-alkyl group, where the alkyl group is a C alkyl group as defined herein. 1 -C 6 Alkyl. Preferred C 1 -C 6 Alkoxy groups include methoxy, ethoxy and propoxy.

[0048] As used herein, "C 1 -C 6 The term "haloalkoxy" refers to any of the C, C-, C-alkyl, C-alkylamino groups, as defined herein, substituted with one or more halogen groups, as defined herein. 1 -C 6 It refers to an alkoxy group. Suitable haloalkoxy groups include trifluoromethoxy.

[0049] As used herein, the term "aryl having 6 to 10 ring atoms" refers to a polyunsaturated aromatic hydrocarbyl group having a single ring or multiple aromatic rings fused together, containing 6 to 10 ring atoms, where at least one ring is aromatic. The aromatic ring may optionally contain 1 to 2 additional rings (cycloalkyl, heterocyclyl, or heteroaryl, as defined herein) fused to the aromatic ring. Suitable aryl groups include phenyl, naphthyl, and a phenyl ring fused to a heterocyclyl (such as benzopyranyl, benzodioxolyl, benzodioxanyl, etc.).

[0050] Arylene refers to a divalent aryl group as defined above.

[0051] As used herein, the term "heteroaryl having 5-10 ring atoms" refers to a single ring containing 5-10 atoms or a polyunsaturated aromatic ring system having multiple aromatic rings fused together or covalently bonded together containing 5-10 atoms, where at least one ring is aromatic and at least one ring atom is a heteroatom selected from N, O and S. The nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. Such rings may be fused to an aryl ring, a cycloalkyl ring or a heterocyclyl ring. Non-limiting examples of such heteroaryls include: furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl and quinoxalinyl.

[0052] 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, in which at least one ring atom is a heteroatom selected from N, O and S. The nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocycles may contain fused or bridged rings, as well as 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.

[0053] As used herein, the term "heterocycle" or "heterocycloalkylene" refers to a divalent heterocycle, as defined herein.

[0054] Further, alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heteroalkyl, heteroalkylene, C 3 -C 8 carbocycle, C 3 -C 8 Carbocyclo, C 3 -C 8 Heterocycle, C 3 -C 8 The term heterocyclo, polyether refers to a group that is optionally substituted with one or more substituents selected from: -X, -R', -O - , -OR', =O, -SR', -S - , -NR' 2 , -NR' 3 , =NR', -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NRC(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3- , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 - , -PO 3 H 2 , -C(=O)R', -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' 2 , C(=S)NR' 2 , and C(=NR')NR' 2 where each X is independently a halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C 1- C 20 Alkyl, -C 6 -C 10 Aryl, or -C 3 -C 10 It is a heterocycle.

[0055] An acyl group refers to a -CO-alkyl group, where alkyl has the above definition.

[0056] As used herein, the term "polyether" refers to a polymer containing ether bonds. The number of ether moieties in a polyether can be comprised between 2 and 100, preferably between 2 and 25, and especially between 2 and 10. Examples of polyethers include polyethylene glycols, such as polyethylene glycols having between 2 and 100, preferably between 2 and 25, and especially between 2 and 10 ether moieties.

[0057] The term "optionally substituted polyether" can refer to polyethers, particularly polyethylene glycols, optionally substituted with one or more substituents selected from the following: halogen, oxo, -OH, -NO 2 , -CN,C 1 -C6 Alkyl, C 3 -C 6 Cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 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 H and C 1 -C 6 alkyl.

[0058] Solid-phase peptide synthesis (SPPS) refers to a known process in which peptides anchored to an insoluble polymer support are assembled by successive addition of Fmoc- or Boc-protected amino acids with repeated cycles of deprotection-wash-coupling-wash. Each amino acid addition is referred to as a cycle: (i) cleavage of the Nα-protecting group, (ii) a washing step, (iii) coupling of a fluorenylmethoxycarbonyl- (Fmoc-) or tert-butyloxycarbonyl- (Boc-) protected amino acid with a coupling reagent and a non-nucleophilic base, (iv) a washing step. As the growing chain is attached to the support, excess reagents and soluble by-products can be removed by simple filtration. Repeated coupling reactions with hindered Fmoc- or Boc-protected N-methylated amino acids are difficult and often suboptimal, resulting in low crude purity, difficult purification, and low expected yields with this technique. Examples of such supports include Wang resin, Rink amide resin, trityl- and 2-chlorotrityl resins, PAM resin, PAL resin, Sieber amide resin, MBHA resin, HMPB resin, and HMBA resin, which are commercially available and to which peptides are directly or indirectly bound.

[0059] Ligand drug conjugates (LDCs), as defined herein, refer to conjugates that covalently link a ligand and a drug, including any method as described herein and as illustrated in the examples herein. When the ligand is an antibody, reference can be made to antibody drug conjugates (ADCs), which are a preferred embodiment of the present disclosure.

[0060] The term connector unit refers to a component that links together moieties of different compounds, for example, a connector can link a ligand to a spacer or a spacer to an amide function -CO-NR1-. A connector is a scaffold that has attachment sites for the components of a ligand-drug-conjugate, i.e., a ligand, a spacer, a hydrophobic masking agent and / or an amide function -CO-NR1-.

[0061] The skilled person can use his knowledge to select a suitable connector for the expected LDC compound. A non-exhaustive list of connectors includes amino acids (e.g., lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, homoalanine; amino alcohols; amino aldehydes; polyamines or any combination thereof). Advantageously, the connector units X1 and / or X2 are one or more natural or unnatural amino acids. In one embodiment, the connector units X1 and X2 are 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 having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C 2 -C 10 alkenylene, and any combination thereof, wherein the alkylene and the alkenylene are optionally interrupted by one or more heteroatoms or one or more 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, the arylene, the cycloalkylene, the heterocycloalkylene, the heteroarylene, and the alkenylene are optionally substituted with one or more substituents selected from the following: halogen, oxo, -OH, -NO2 , -CN,C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 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 H and C 1 -C 6 alkyl.

[0062] Examples of connector units are optionally substituted polyethers, amino acids, benzyl groups, amines, ketones,

[0063] [ka]

[0064] Includes.

[0065] In particular, the connector unit may be divalent or trivalent, for example, when a hydrophobic masking substance K is present, X2 may be a trivalent connector unit.

[0066] The term "amino acid" refers to a natural or unnatural amino acid. The CO moiety of the -CONR1- or -CONR1'- group can be considered as part of the X2 connector unit when X2 consists of one or more amino acids. A non-exhaustive list of amino acids includes lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine and homoalanine.

[0067] The spacer is a bivalent arm (such as two connector units) that covalently links the two components of the ligand-drug-conjugate. The spacer Z may or may not be present.

[0068] A non-exhaustive list of spacer units includes: alkylene, heteroalkylene (alkylene interrupted by at least one heteroatom selected from Si, N, O and S); alkoxy; polyether (such as polyalkylene glycol and typically polyethylene glycol); one or more natural or unnatural amino acids (such as glycine, alanine, proline, valine, N-methylglycine); C 3 -C 8 Heterocyclo;C 3 -C 8 carbocyclo; arylene, and any combination thereof. For example, the spacer can be a divalent linear alkylene group, preferably (CH 2 ) 4 It is.

[0069] For example, the spacer is -C 1 -C 10 Alkylene-, -C 1 -C 10 Heteroalkylene-, -C 3 -C 8 Carbocyclo-, -O-(C 1 C 8 Alkyl)-, -arylene-, -C 1 -C 10 Alkylene-arylene-, -arylene-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-, -C 3 -C 8 Heterocyclo-, -C 1 -C 10 Alkylene-(C3 -C 8 Heterocyclo)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Heteroalkylene-C(=O)-, -C 3 -C 8 Carbocyclo-C(=O)-, -O-(C 1 -C 8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1 -C 10 Alkylene-arylene-C(=O)-, -arylene-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-C(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 3 -C 8 Heterocyclo-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-C(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Heteroalkylene-NH-, -C 3 -C 8 Carbocyclo-NH-, -O-(C 1 -C 8 Alkyl)-NH-, -Arylene-NH-, -C 1 -C 10 Alkylene-arylene-NH-, -arylene-C1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-NH-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-NH-, -C 3 -C 8 Heterocyclo-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-NH-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-S-, -C 1 -C 10 Heteroalkylene-S-, -C 3 -C 8 Carbocyclo-S-, -O-(C 1 -C 8 Alkyl)-)-S-, -Arylene-S-, -C 1 -C 10 Alkylene-arylene-S-, -arylene-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-S-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-S-, -C 3 -C 8 Heterocyclo-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-S-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10Alkylene-S-, -C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Carbocyclo-OC(=O)-, -O-(C 1 -C 8 Alkyl)-OC(=O)-, -Arylene-OC(=O)-, -C 1 -C 10 Alkylene-arylene-OC(=O)-, -arylene-C 1 -C 10 Alkylene-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-OC(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Heterocyclo-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-OC(=O)-, and -(C 3 -C 8 Heterocyclo)-C 1 -C 10 alkylene -OC(=O)-.

[0070] Any of the above groups may be optionally substituted with one or more substituents selected from the following: -X, -R', -O - , -OR', =O, -SR', -S - , -NR' 2 , -NR' 3 + , =NR', -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NR'C(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3- , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 - , -PO 3 H 2 , -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' 2 , C(=S)NR' 2 , and C(=NR')NR' 2 where each X is independently a halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C 1- C 20 Alkyl, -C 6 -C 10 Aryl, or -C 3 -C 10 It is a heterocycle.

[0071] Ligand refers to any macromolecule (polypeptide, protein, peptide, typically an antibody) or small molecule (such as folate or an aptamer) that is typically employed in LDC (e.g., antibody-drug conjugate) technology and can be covalently attached to the synthetic linker or drug linker of this study using bioconjugate technology (see Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). Ligands are traditionally compounds that are selected for their targeting capabilities. A non-exhaustive list of ligands includes proteins, polypeptides, peptides, antibodies, full-length antibodies and their antigen-binding fragments, interferons, lymphokines, hormones, growth factors, vitamins, transferrin, or other cell-binding molecules or substances. The main type of ligand used in the preparation of conjugates is an antibody. An example of a protein is human serum albumin.

[0072] 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 (Affibody®, Affilin®, Affimer®, Nanofitin®, Cell Penetrating Alphabody®, Anticalin®, Avimer®, Fynomer®, Monobodies or nanoCLAMP®). An example of an antibody is trastuzumab.

[0073] The term "antibody" referred to herein includes whole antibodies and antigen-binding fragments (ie, "antigen-binding portions") or single chains thereof.

[0074] A naturally occurring "antibody" is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as V H Each light chain comprises a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V L The light chain constant region comprises one domain, C L Includes: V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions of greater conservedness, called framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0075] The term "antigen-binding portion" (or simply "antigen portion") of an antibody, as used herein, refers to a full-length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody are Fab fragments, V L Domain, V H Domain, C L A monovalent fragment consisting of the F(ab) domain and the CH1 domain; 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; V H Fd fragment consisting of the V domain and the CH1 domain of a single arm of an antibody;L Domains and V H Fv fragment consisting of domains; V H domains (Ward et al., 1989 Nature 341:544-546); as well as isolated complementarity determining regions (CDRs), or any fusion proteins containing such antigen-binding portions.

[0076] In addition, two domains of the Fv fragment, V L and V H are encoded by separate genes, but using recombinant methods, V L Area and V H The domains can be linked by a synthetic linker that allows them to be produced as a single-chain protein that pairs to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0077] In certain embodiments, the ligand of the LDC is a chimeric, humanized or human antibody.

[0078] The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a human sequence (e.g., human germline sequence), or a variant of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al. (2000. J Mol Biol 296, 57-86)).

[0079] Human antibodies may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, have been grafted onto human framework sequences.

[0080] 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.

[0081] As used herein, "isotype" refers to the antibody class (e.g., IgM, IgE, IgG (such as IgG1 or IgG4)) that is provided by the heavy chain constant region genes.

[0082] As used herein, the expressions "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably with the term "an antibody that specifically binds to an antigen."

[0083] An active agent refers to a bioactive or therapeutic molecule. Examples of active agents include drugs, imaging agents, and fluorescent agents. Imaging agents can include fluorescent agents such as indocyanine green.

[0084] Drug refers to any type of drug or compound having unique pharmacological or diagnostic properties (e.g., a cytotoxic compound, a cytostatic compound, an immunomodulatory compound, an immunosuppressant compound, an immunostimulatory compound, an anti-inflammatory compound, an ionizing compound, or an anti-infective compound). Cytotoxic compounds include calicheamicins; uncialamycins; auristatins (such as monomethylauristatin E, known as MMAE); halichondrin derivatives (such as eribulin), tubulysin analogs; maytansines; cryptophycins; benzodiazepine dimers (including pyrrolo[2,1-c][1,4]benzodiazepines, known as PBDs); indolinobenzodiazepine pseudodimers (IGNs); duocarmycins; anthracyclines (such as doxorubicin or PNU159682); camptothecin analogs (such as 7-ethyl-10-hydroxy-camptothecin, known as SN38 or exatecan); Bcl2 and Bcl-xl inhibitors; tylanstatin; amatoxins (including α-amanitin); kinesin spindle protein (KSP) inhibitors; vinorelbine; cyclin-dependent kinase (CDK) inhibitors; molecular adhesive degraders, bleomycin; dactinomycin or radionuclides and their complexing agents (DOTA / 177 Anti-inflammatory drugs can include corticosteroids, such as dexamethasone or fluticasone. Anti-infective drugs can include antibiotics, such as rifampicin or vancomycin. The drug may be an anti-cancer drug or an immunomodulatory drug. Examples of anti-cancer drugs are cytotoxic and cytostatic compounds, preferably cytotoxic compounds.

[0085] Hydrophobic masking substances refer to groups capable of reducing the apparent hydrophobicity of a compound. The hydrophobic masking substances can be selected from polysarcosine, polyethylene glycol, and chitooligosaccharides. The number of ethylene glycol or sarcosine moieties may vary in a wide range. For example, the number of ethylene glycol or sarcosine moieties in the hydrophobic masking substance can be comprised between 2 and 500, preferably between 5 and 100, in particular between 5 and 25. In one embodiment, the hydrophobic masking substance is polysarcosine comprising between 6 and 24 sarcosine moieties, preferably between 10 and 12 sarcosine moieties. The number of chitosans in the chitooligosaccharides can be comprised between 2 and 20, for example between 2 and 8.

[0086] An electron-withdrawing group refers to an atom or group that attracts electron density from neighboring atoms toward itself, usually by resonance or inductive effects. Electron-withdrawing groups include halogens, (-CF 3 haloalkyl, -CN, -SO 3 H group, -NO 2 groups, and -C(O)R groups, with 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 to the YT substituent on the phenyl ring.

[0087] 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 the regenerated functional group 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, for example, in Wutz et al. ("Greene's Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). Protecting groups for protecting amino groups as described in Wutz et al. (pages 696-927) are used in certain embodiments. Representative examples of amino protecting groups include, but are not limited to, t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), carboxybenzyl group (Cbz), benzyl group (Bn), allyl, trifluoroacetyl, allyloxycarbonyl (Alloc) group, and 2,2,2-trichloroethoxycarbonyl (Troc).

[0088] A group capable of reacting with a ligand to form a connector unit refers to any chemical moiety that is reactive to covalently attach a ligand, in particular, that can react with a thiol group present on the ligand.

[0089] A non-exhaustive list of chemical moieties that are reactive for covalently attaching ligands includes: carboxylic acids; primary amines; secondary amines; tertiary amines; hydroxyls; halogens; activated esters (such as N-hydroxysuccinimide esters, perfluoroesters, nitrophenyl esters, azabenzotriazole and benzotriazole activated esters), acylureas; alkynyls; alkenyls; azides; isocyanates; isothiocyanates; aldehydes; thiols; reactive moieties (such as maleimides, halomaleimides, haloacetyls, pyridyl disulfides); thiols; acrylates; mesylates; tosylates; triflates, hydroxylamines; chlorosulfonyls; boronic acids-B(OR'). 2Derivatives, where R' is hydrogen or an alkyl group.

[0090] "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 either 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. Cleavage of the cleavable unit preferably triggers the self-immolation of the phenyl-containing linker of the compound of the present invention and the release of the active agent D. In the present disclosure, the "cleavable sugar unit" can refer to a sugar moiety, preferably a glucuronide or a galactoside. In the present disclosure, the "polypeptide cleavable unit" can refer to a polypeptide, preferably a dipeptide or a tripeptide.

[0091] As used herein, the term "one or more" may be understood to refer to a number from 1 to 20, or from 1 to 10, or from 1 to 5.

[0092] Compounds of formula (I) The present disclosure relates to compounds of formula (I):

[0093] [ka]

[0094] During the ceremony, L is a ligand; X1 is the connector unit; Z is an optional spacer; X2 is the connector unit; K is any hydrophobic masking material, preferably selected from polysarcosine and polyethylene glycol; R1 is H, C 1 -C 12 Alkyl, C 2 -C 6Alkenyl; optionally substituted polyethers, polysarcosine, aryls having 6 to 10 ring atoms, C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; D is an active agent, preferably selected from the group consisting of a drug, an imaging agent and a fluorescent agent; Each R2 is an electron withdrawing group and C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y is O when T is a sugar cleavable unit or NR3 when T is a polypeptide cleavable unit; R3 is H, C 1-C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0095] 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.

[0096] According to one embodiment, D is selected from the group consisting of drugs, preferably, D is an anticancer drug or an immunomodulatory drug. According to one embodiment, D is exatecan or monomethylauristatin E (MMAE).

[0097] According to one embodiment, X1 and X2 are 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 8Cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C 2 -C 10 alkenylene, and any combination thereof; said alkylene and said alkenylene are optionally interrupted by one or more heteroatoms or one or more 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 with one or more substituents selected from the following: halogen, oxo, -OH, -NO 2 , -CN,C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 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 H and C 1 -C 6 alkyl.

[0098] 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, C 1 -C 12It is selected from the group consisting of alkylene, arylene having 6 to 10 ring atoms, and any combination thereof.

[0099] According to one embodiment, X1 is as follows:

[0100] [ka]

[0101] According to one embodiment, X2 is one or more amino acids, one or more N-substituted amino acids, C 1 -C 12 It is selected from the group consisting of alkylene, optionally substituted polyether, and any combination thereof.

[0102] According to one embodiment, Z is one or more amino acids, one or more N-substituted amino acids, an optionally substituted polyether, C 1 -C 12 Alkylene, arylene with 6 to 10 ring atoms, C 3 -C 8 Cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C 2 -C 10 alkenylene, and any combination thereof; said alkylene and said alkenylene are optionally interrupted by one or more heteroatoms or one or more 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 with one or more substituents selected from the following: halogen, oxo, -OH, -NO 2 , -CN,C 1 -C6 Alkyl, C 3 -C 6 Cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 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 H and C 1 -C 6 alkyl.

[0103] According to one embodiment, Z is selected from the group consisting of one or more amino acid(s), one or more N-substituted amino acids, and polyethylene glycol.

[0104] According to one embodiment, Z is absent and X1 and X2 are directly bonded to each other via a single bond.

[0105] According to one embodiment, K is a polysarcosine, preferably a monodisperse polysarcosine. The polysarcosine may have from 1 to 50 repeat units.

[0106] In certain embodiments, K is a polysarcosine, preferably a polysarcosine of formula (V):

[0107] [ka]

[0108] In the formula, k is an integer of 2 to 50, preferably 4 to 30. and R6 corresponds to OH or NH2.

[0109] According to one embodiment, T is a sugar cleavable unit that is a glucuronide or a galactoside. The glycosidic bond linking T to the aryl group may be one that can be cleaved to initiate a self-immolative reaction sequence that results in release of the drug.

[0110] According to one embodiment, T is a dipeptide, preferably selected from Val-Cit, Val-Ala and Phe-Lys.

[0111] According to one embodiment, R1 is selected from the group consisting of H and C 1 -C 6 alkyl, preferably H.

[0112] According to one embodiment, R3 is selected from the group consisting of H and C 1 -C 6 alkyl, preferably H.

[0113] According to one embodiment, R4 is selected from the group consisting of H and C 1 -C 6 alkyl.

[0114] According to one embodiment, R5 is selected from the group consisting of H and C 1 -C 6 alkyl.

[0115] According to one embodiment, both R4 and R5 are H.

[0116] According to one embodiment, n is 0, Y is NR3, and T is a polypeptide cleavable unit. According to another embodiment, n is 1 and R2 is -NO 2 where Y is O and T is a sugar cleavable unit.

[0117] According to a preferred embodiment, L is an antibody or an antigen-binding fragment thereof.

[0118] According to one embodiment, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group including or consisting of CD19, CD20, CD22, CD30, CD37, CD79b, HER2, and PSMA.

[0119] According to one embodiment, the antibody or antigen-binding fragment thereof binds to HER2 / neu.

[0120] Some examples of antibodies suitable as ligand L in the compounds of formula (I) described herein include, but are not limited to, trastuzumab, brentuximab, roncatuximab, rosopatamab, rituximab, pinatuzumab, polatuzumab, and naratuximab.

[0121] According to one embodiment, the antibody is trastuzumab.

[0122] The compounds of formula (I) have at least one asymmetric carbon and therefore may exist in different stereoisomers. For example, the compounds of formula (I) may exist in the (R) or (S) form, as shown below:

[0123] [ka]

[0124] According to one embodiment, the compound of formula (I) is (S).

[0125] In certain embodiments, the compound of formula (I) is a compound of formula (VI):

[0126] [ka]

[0127] wherein k is an integer from 2 to 50, preferably from 4 to 30; and T is a polypeptide cleavable unit, preferably a dipeptide.

[0128] In certain embodiments, the compound of formula (I) is a compound of formula (VII)

[0129] [ka]

[0130] In the formula, k is an integer of 2 to 50, preferably 4 to 30.

[0131] In certain embodiments, the compound of formula (I) is a compound of formula (VIII)

[0132] [ka]

[0133] In the formula, k is an integer from 2 to 50, preferably from 4 to 30; and T is a sugar cleavable unit, preferably a glucuronide or a galactoside.

[0134] In certain embodiments, the compound of formula (I) is a compound of formula (IX)

[0135] [ka]

[0136] In the formula, k is an integer of 2 to 50, preferably 4 to 30.

[0137] The compounds of formula (I) according to the present invention may be synthesised by any suitable process known in the art, such as the processes disclosed in the Examples section.

[0138] The embodiments described for compounds of formula (I) also apply to compounds of formula (II), formula (III) and formula (IV).

[0139] Compound of formula (II) The present disclosure also relates to a compound of formula (II):

[0140] [ka]

[0141] During the ceremony, X1' is a group capable of reacting with a ligand to form a connector unit; Z is an optional spacer; X2 is the connector unit; K is any hydrophobic masking material, preferably selected from polysarcosine and polyethylene glycol; R1' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; D is an active agent, preferably selected from the group consisting of a drug, an imaging agent and a fluorescent agent; Each R2 is an electron withdrawing group and C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0142] According to one embodiment, X1' is maleimide.

[0143] According to one embodiment, R1' is H.

[0144] According to one embodiment, R3' is H.

[0145] The compound of formula (II) can be used to synthesize the compound of formula (I) and thus the compound of formula (II) is an intermediate in the synthesis of the compound of formula (I).

[0146] Compound of formula (III) The present disclosure also relates to a compound of formula (III)

[0147] [ka]

[0148] During the ceremony, R1' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; D is an active agent, preferably selected from the group consisting of a drug, an imaging agent and a fluorescent agent; Each R2 is independently selected from the group consisting of electron withdrawing groups; n is 0, 1 or 2; R4 is H, C1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0149] The compound of formula (III) can be used to synthesize a compound of formula (II) and thus the compound of formula (III) is an intermediate in the synthesis of a compound of formula (I).

[0150] Compound of formula (IV) The present disclosure also relates to a compound of formula (IV)

[0151] [ka]

[0152] During the ceremony, R1' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; Each R2 is an electron withdrawing group and C 1 -C 4 independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C 1 -C 6 Alkyl and C 2 -C 6alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C 1 -C 6 Alkyl and C 2 -C 6 alkenyl, wherein said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleavable unit or a polypeptide cleavable unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C 1 -C 24 Alkyl, C 2 -C 6 Alkenyl; optionally substituted polyethers; aryl having 6 to 10 ring atoms; C 3 -C 8 selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl are optionally interrupted by one or more heteroatoms or one or more 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; R'' and R''' are H and C 1 -C 6 independently selected from alkyl; and pharma- ceutically acceptable salts thereof.

[0153] According to one embodiment, R4 and R5 are H, Y' is O and T is a sugar cleavable unit.

[0154] According to another embodiment, Y' is NR3' and T is a polypeptide cleavable unit.

[0155] The compound of formula (IV) can be used to synthesize a compound of formula (III) and thus the compound of formula (IV) is an intermediate in the synthesis of a compound of formula (I).

[0156] Pharmaceutical Compositions The present disclosure also relates to a pharmaceutical composition comprising the compound of the present disclosure and at least one pharma- ceutically acceptable carrier.In particular, the present disclosure relates to a pharmaceutical composition comprising the compound of formula (I) and at least one pharma- ceutically acceptable carrier.

[0157] As used herein, "pharmaceutical acceptable carrier" includes any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like. 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 route or intratumoral injection. Depending on the route of administration, the active compound may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The formulation may further include one or more excipients, preservatives, solubilizers, buffers, and the like.

[0158] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will, of course, depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

[0159] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, nasal, intraocular, intravenous, intramuscular, or subcutaneous administration, and the like.

[0160] Preferably, the pharmaceutical composition comprises a pharma- ceutically acceptable vehicle for injectable preparations.These may in particular be isotonic, sterile, saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of these salts), or a dry, in particular lyophilized, composition, which may be made up into an injection solution by adding sterile water or saline, if necessary.

[0161] The dose used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, as a function of the pathology involved, or alternatively as a function of the desired duration of treatment.

[0162] To prepare pharmaceutical compositions, an effective amount of a compound according to the present disclosure may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0163] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders or lyophilisates for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms (such as bacteria and fungi).

[0164] Solutions of the active compounds as free bases 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, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0165] The compounds of the present disclosure can be formulated in composition in neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups), which are formed with, for example, inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.).The salts formed with free carboxyl groups can also be derived from, for example, inorganic bases (such as sodium, potassium, ammonium, calcium or ferric hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.).

[0166] The carrier can also be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coating agents (such as lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, it is preferable to include an isotonic agent (for example, sugars or sodium chloride). Prolonged absorption of the injectable composition can be achieved by the use of absorption-delaying agents (for example, aluminum monostearate and gelatin) in the composition.

[0167] Sterile injectable solution is prepared by incorporating the required amount of active compound in a suitable solvent with some other components listed above, and then sterilizing by filtration as required.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains basic dispersion medium and other components listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying technology and freeze-drying technology, which adds powder of active component to desired additional component from the solution that has been previously sterile-filtered.

[0168] Highly concentrated solutions for further preparation or direct injection are also envisioned using DMSO as a solvent to deliver high concentrations of active agents to small tumor areas resulting in extremely rapid penetration.

[0169] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like may also be employed.

[0170] When administered parenterally in an aqueous solution, for example, the solution is suitably buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous medium that can be employed will be known to those skilled in the art in light of this disclosure. For example, one dose may be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection fluid or injected at the intended site of injection (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Dosages will necessarily vary somewhat depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject.

[0171] In addition to compounds formulated for parenteral administration (such as intravenous or intramuscular injection), other pharma- ceutically acceptable forms include, for example, tablets or other solids for oral administration; time-release capsules; and any other form currently in use.

[0172] In certain embodiments, the use of liposomes and / or nanoparticles for the introduction of antibodies into host cells is contemplated. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0173] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can degrade in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet such requirements are contemplated for use in the present disclosure, and such particles can be easily manufactured.

[0174] Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters ranging from 25 nm to 4 μm. When MLVs are sonicated, small unilamellar vesicles (SUVs) are formed, which contain aqueous solution in their core and have diameters in the range of 200-500 A. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0175] The dose used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, as a function of the pathology involved, or alternatively as a function of the desired duration of treatment. It is understood that the appropriate dose of the compounds and compositions containing the compounds may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit against the risk, or adverse side effects, of any treatment described herein. The selected dose level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, as well as the age, sex, weight, condition, general health, and medical history of the patient. The amount of the compound and the route of administration are ultimately at the discretion of the physician, but will generally be an amount to achieve a local concentration at the site of action that achieves the desired effect without causing substantial adverse or harmful side effects. For example, the dose used for administration can be about 0.1 to 1000 mg of the compound of the present disclosure for a subject weighing about 50 to 70 kg.

[0176] [How to use] The compounds of the present disclosure show valuable pharmaceutical properties, as shown in the in vitro and in vivo tests provided in the examples, and are therefore indicated for treatment.The present disclosure also relates to the compounds of the present disclosure for use as drugs.In particular, the present disclosure relates to the ligand-drug conjugate compound of formula (I), more particularly the antibody-drug conjugate compound of formula (1), in which L is antibody or its antigen-binding portion, for use as drugs.

[0177] In particular, the compounds of the present disclosure, more particularly, the antibody-drug conjugates of formula (I) of the present disclosure, are useful for the prevention or treatment of cancer, inflammatory disease and / or infectious disease. In a preferred embodiment, the compounds of formula (I) for use in the prevention or treatment of cancer are ligand-drug conjugates (LDC) of formula (I), where L is an antibody or an antigen-binding portion thereof, and more preferably, D is a cytotoxic compound.

[0178] The present disclosure also relates to the compounds of the present disclosure for use in a method for treating cancer. As used herein, the term "cancer" has its general meaning in the art and includes an abnormal state or condition characterized by rapidly proliferating cell proliferation. The term is meant to include all types of cancerous growths or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or invasive stage. The term cancer includes cancers of various organ systems, such as those affecting the skin, lung, breast, thyroid, lymphatic system, gastrointestinal tract, and genitourinary tract, as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate cancer, and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.

[0179] The present disclosure relates to a method for treating cancer, 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 The method includes administering

[0180] In a preferred embodiment, the present disclosure relates to a method for treating or preventing cancer in a subject in need thereof, said 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, D is a cytotoxic compound.

[0181] The present disclosure also relates to compounds of the present disclosure for use in a method for treating an inflammatory disease.

[0182] As used herein, the term "inflammatory disease" is used to define any disease caused by or leading to inflammation in a subject. This 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 undesirable inflammatory responses are inhibited.

[0183] The present disclosure relates to a method for treating an inflammatory 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 The method includes administering

[0184] The present disclosure also relates to a compound of the present disclosure for use in a method for treating an infectious disease.

[0185] As used herein, the term "infectious disease" is used to define any disease caused by the invasion of a subject by an infectious agent (or pathogen), its proliferation, and the response of the subject's tissues to these infectious agents and the toxins they produce. This term includes, but is not limited to, (1) bacterial infections, (2) viral infections, (3) fungal infections, and (4) parasitic infections.

[0186] 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 The method includes administering

[0187] In a preferred embodiment, the present disclosure relates to a method for treating or preventing an infectious disease in a subject in need thereof, said 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, D is an anti-infective agent (e.g., an antibiotic or an antiviral agent).

[0188] As used herein, the term "treating" includes reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which the term applies, or one or more symptoms or manifestations of the disease, disorder, or condition. Preventing refers to keeping the disease, disorder, condition, or such symptoms or manifestations, or the worsening of such severity, from occurring. Thus, the presently disclosed compounds can be administered prophylactically to prevent or reduce the occurrence or recurrence of a disease, disorder, or condition.

[0189] As used herein, the term "therapeutically effective amount" of a compound refers to an amount of a compound that elicits a biological or medical response in a subject (e.g., an amount of a compound that ameliorates symptoms, alleviates a condition, slows the progression of a disease, or prevents a disease).

[0190] The present disclosure also relates to the use of the compounds of the present disclosure, preferably compounds of formula (I), for the manufacture of a medicament for the treatment of cancer, inflammatory diseases and / or infectious diseases, preferably for the treatment of cancer.

[0191] The compound of formula (II) can be used directly without ligand.For example, when X1' is a maleimide moiety, the compound of formula (II) can react with protein such as serum albumin in vivo, and then become ligand.Therefore, the present disclosure also relates to the compound of formula (II) as described above for use as drug.

[0192] [Example] Materials and General Methods All solvents and reagents were obtained from reliable commercial sources (Sigma-Aldrich, Fluorochem, TCI Chemicals, Acros Organics, Alfa Aesar, Enamine, Thermo Fisher, Carbosynth, WuXi AppTec, Iris Biotech) and used without further purification unless otherwise noted. Anhydrous solvents were purchased from Sigma-Aldrich. Fmoc-amino acids, 2-chlorotrityl, Wang and Rink Amide polystyrene 1% DVB 100-200 mesh resin (preloaded 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 (cat#A3782) was purchased from Sigma-Aldrich. Trastuzumab (Herceptin® IV) was purchased from Roche. Non-commercially available monoclonal antibodies were custom produced by transient transfection into CHO cell lines and protein-A / SEC purified by GTP Technologies (Toulouse, France).

[0193] On-resin synthesis was carried out in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for mixing. All chemical reactions were carried out at room temperature under an inert argon atmosphere unless otherwise noted.

[0194] Liquid nuclear magnetic resonance spectra were recorded on a Bruker Fourier 300HD or Bruker AVANCE III HD400 mass spectrometer using the residual solvent peak for calibration. Mass spectrometry was performed by the Centre Commun de Spectrometrie de Masse (CCSM) of the CNRS laboratory UMR5246, University Claude Bernard Lyon 1.

[0195] Normal phase flash chromatography was performed on a Teledyne Isco CombiFlash® Rf200 instrument using Macherey-Nagel Chromabond® flash cartridges (40-63 μm). Reverse phase chromatography was performed on a Teledyne Isco Combiflash® Rf200 instrument using Biotage® Sfar C18 Duo 100A 30 μm cartridges or Interchim PuriFlash RP-AQ (30 μm) cartridges; or on an Agilent 1100 preparative binary HPLC system.

[0196] Chemical reactions and compound properties were monitored and analyzed by thin-layer chromatography using precoated 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.

[0197] HPLC method 1: Agilent 1100 HPLC system equipped with a DAD detector. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile. The column was an Agilent Zorbax SB-Aq 4.6 × 150 mm 5 μm (room temperature). The linear gradient was 0% B to 50% B for 30 min, followed by a hold at 50% B for 5 min. The flow rate was 1.0 mL / min.

[0198] HPLC method 2: Agilent 1100 HPLC system equipped with a DAD detector. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile. The column was an Agilent Poroshell 120 EC-C18 3.0 × 50 mm 2.7 μm (room temperature). The linear gradient was 5% B to 80% B in 9 min, followed by a hold at 80% B for 1 min. The flow rate was 0.8 mL / min.

[0199] HPLC method 3: Agilent 1100 HPLC system equipped with a DAD detector. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile. The column was an Agilent Poroshell 120 EC-C18 3.0 × 50 mm 2.7 μm (room temperature). The linear gradient was 5% B to 80% B for 20 min, followed by a 2 min hold at 80% B. The flow rate was 0.8 mL / min.

[0200] HPLC method 4: Thermo UltiMate 3000 UHPLC system + Bruker Impact II™ Q-ToF mass spectrometer. Mobile phase A was water + 0.1% formic acid and mobile phase B was acetonitrile + 0.1% formic acid. The column was an Agilent PLRP-S 1000A 2.1×150 mm 8 μm (80° C.). The linear gradient was 10% B to 50% B in 25 min. The flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. A Q-ToF mass spectrometer was used with an m / z range of 500-3500 (ESI + ). Data were deconvoluted using the MaxEnt algorithm included in the Bruker Compass® software.

[0201] HPLC Method 5 (Preparative): Teledyne Isco CombiFlash® Rf200 binary MPLC system equipped with a DAD detector. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile. The reusable cartridge was a Biotage® Sfar C18 Duo 100A 30 μm (30 g). The linear gradient was 10% B to 50% B in 35 min followed by a hold at 50% B for 5 min. The flow rate was 25 mL / min.

[0202] HPLC method 6 (preparative method): Agilent 1100 preparative binary HPLC system equipped with a dual loop autoinjector, DAD detector, and fraction collector. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The column was a Waters SunFire C18 OBD Prep Column, 100A, 5 μm, 19 mm × 250 mm (room temperature). The linear gradient was 10% B to 60% B for 40 min, followed by a hold at 60% B for 5 min. The flow rate was 25 mL / min.

[0203] 1) Preparation of the Chemical Compounds of the Invention 1.1) Monodisperse polysarcosine intermediate 1.1.1) General method On-resin synthesis of monodisperse polysarcosines was achieved using an iterative submonomer synthesis procedure (described in patent WO2019081455) on Rink amide and 2-chlorotrityl resins, and following the classical Fmoc / SPPS method using the commercially available Fmoc-Sar-Sar-OH dipeptoid building block (CAS#2313534-20-0) on Wang resin. It should be noted that peptoid submonomer synthesis on Wang resin was unsuccessful. In all cases, the on-resin dimer stage (n=2) was avoided due to diketopiperazine formation. All synthesis yields are reported based on the initial Fmoc-sarcosine loading as indicated by the manufacturer. All reactions were performed at room temperature unless otherwise noted. Rink amide, 2-chlorotrityl or Wang polystyrene 1% DVB 100-200 mesh resins preloaded with the first Fmoc-sarcosine residue (Christof Senn Laboratories) were used (typical initial loadings of 0.6-1 mmol / g).

[0204] 1.1.2) Polysarcosine elongation Rink amide, 2-chlorotrityl or Wang resin preloaded with Fmoc-sarcosine was treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 2×15 min at room temperature. The resin was then washed with DMF (4×) and DCM (4×). A solution of Fmoc-Sar-Sar-OH (3 eq), HATU (2.9 eq) and DIPEA (6 eq) in DMF (1 mL per 100 mg resin) was added to the resin. The reaction vessel was agitated for 2 h and the resin was washed with DMF (4×) and DCM (4×). The resin was treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 2×15 min at room temperature. The resin was then washed with DMF (4×) and DCM (4×).

[0205] For synthesis on Rink Amide or 2-chlorotrityl resin, classical submonomer synthesis procedures were used as described in WO2019081455. Elongation of n=3 polysarcosine oligomers was performed by alternating bromoacetylation and amine displacement steps until the desired length was obtained. The bromoacetylation step was performed by adding 10 eq bromoacetic acid and 13 eq diisopropylcarbodiimide in DMF (2 mL per 100 mg resin). The mixture was stirred for 30 min, drained and washed with DMF (4 times). For the amine displacement step, 40% (wt) methylamine in water was added (1.5 mL per 100 mg resin) and the vessel was shaken for 30 min, drained and washed with DMF (4 times) and DCM (4 times).

[0206] The synthesis on Wang resin used a classical Fmoc / SPPS procedure. The elongation of the n=3 polysarcosine oligomer was achieved by iterative coupling of the Fmoc-Sar-Sar-OH dipeptoid building block (CAS#2313534-20-0). To the resin was added a DMF solution of Fmoc-Sar-Sar-OH (3 eq), HATU (2.9 eq) and DIPEA (6 eq) (1 mL per 100 mg of resin). The reaction vessel was agitated for 90 min and the resin was washed extensively with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2×15 min 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 length of polysarcosine was obtained. If necessary, the final coupling is carried out using commercially available Fmoc-Sar-OH amino acid in place of the Fmoc-Sar-Sar-OH dipeptoid unit to obtain polysarcosines of equal length.

[0207] 1.1.3) Final on-resin side functionalization of polysarcosine, optional capping, resin cleavage and purification Once the desired polysarcosine monodisperse oligomer length on resin is reached, orthogonal chemical functionalization is performed, optionally followed by final capping with Fmoc-amino acids (e.g., Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-amino-3,6-dioxaoctanoic acid, Fmoc-9-amino-4,7-dioxanoic acid). The Fmoc protecting group capping the N-terminus of the final compound can be removed before or after resin cleavage, depending on the orthogonal functionalization chemistry used (described below).

[0208] 1.1.3.1) 2-Azidoethane-1-amine functionalized polysarcosine To the Rink or 2-chlorotrityl resin, 10 eq bromoacetic acid and 13 eq diisopropylcarbodiimide in DMF were added (2 mL per 100 mg resin). The mixture was stirred for 30 min, drained and washed with DMF (4 times). A 3 molar solution of 2-azidoethan-1-amine in DMF (1 mL per 100 mg resin) was added and the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times). This was followed by 1 h of Fmoc-Gly-OH coupling (5 eq Fmoc-Gly-OH, 4.9 eq HATU, 10 eq DIPEA (1 mL per 100 mg resin) in DMF) and Fmoc-deprotection with 20% piperidine in DMF (1 mL per 100 mg resin) for 2 x 15 min at room temperature. The resin was washed with DMF (4 times) and DCM (4 times).

[0209] The final polysarcosine compounds were cleaved from the resin (Rink and Wang resins: 100% TFA x 2 x 30 min, 2-chlorotrityl resin: 20% TFA in DCM x 15 min). The resin was filtered and the volatiles removed under reduced pressure to give the crude product, which was purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0210] 1.1.3.2) β-Alanine-functionalized polysarcosine To the Rink resin or 2-chlorotrityl resin, 10 eq bromoacetic acid and 13 eq diisopropylcarbodiimide in DMF are added (2 mL per 100 mg resin). The mixture is stirred for 30 min, drained, and washed with DMF (4 times). A DMF solution of 3 molar allyl 3-aminopropanoate (synthesized as described in Schroer et al., J. Org. Chem. 1997, 62, 10, 3220-3229) and 3 eq Na in THF is added. 2 CO 3 (freebased for 20 min at 50 °C with 0.01% NaOH) was added (1 mL per 100 mg of resin) and the vessel was shaken for 45 min, drained and washed with DMF (4 times) and DCM (4 times). This was followed by 1 h of Fmoc-Gly-OH coupling (5 eq Fmoc-Gly-OH, 4.9 eq HATU, 10 eq DIPEA (1 mL per 100 mg of resin) in DMF). The resin was washed with DMF (4 times) and DCM (4 times). 0.25 eq Pd(PPh 3 ) 4 The alloc protecting groups were removed by treatment with a solution of 50 eq DIC and 20 eq phenylsilane in DCM for 2×30 min (gently agitated under argon). The resin was then washed with DMF (5×) and DCM (5×). Optionally, N-hydroxysuccinimide (NHS) esters were introduced at the carboxylic acid side chains of the final polysarcosine compounds by treatment with a solution of 50 eq DIC and 60 eq N-hydroxysuccinimide in DMF (1.5 mL per 100 mg of resin) for 90 min. The resin was then washed with DMF (4×) and DCM (4×).

[0211] The final polysarcosine compounds were cleaved from the resin (Rink and Wang resins: 100% TFA x 2 x 30 min, 2-chlorotrityl resin: 20% TFA in DCM x 15 min). The resin was filtered and the volatiles removed under reduced pressure to give the crude product, which was purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0212] 1.1.3.3) Polysarcosine functionalized with glutamic acid side groups To the 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 per 100 mg of resin). The reaction vessel was agitated for 90 min and the resin was washed extensively with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2 x 15 min at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). This was followed by coupling with Fmoc-amino-3,6-dioxaoctanoic acid (3 eq), HATU (2.9 eq), DIPEA (6 eq) (1 mL per 100 mg of resin) in DMF for 1 h. The resin was washed with DMF (4 times) and DCM (4 times). 0.25 eq of Pd(PPh 3 ) 4 The alloc protecting groups were removed by treatment with a solution of 50 eq DIC and 20 eq phenylsilane in DCM for 2×30 min (gently agitated under argon). The resin was then washed with DMF (5×) and DCM (5×). Optionally, N-hydroxysuccinimide (NHS) esters were introduced at the carboxylic acid side chains of the final polysarcosine compounds by treatment with a solution of 50 eq DIC and 60 eq N-hydroxysuccinimide in DMF (1.5 mL per 100 mg of resin) for 90 min. The resin was then washed with DMF (4×) and DCM (4×).

[0213] The final polysarcosine compounds were cleaved from the resin (100% TFA x 2 x 30 min for Rink and Wang resins, 2-chlorotrityl resin 20% TFA in DCM x 15 min for 2 x 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles removed under reduced pressure to give the crude material, which was purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0214] 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 per 100 mg resin). The reaction vessel was agitated for 90 min and the resin was washed extensively with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 2×15 min at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). This was followed by coupling with Fmoc-amino-3,6-dioxaoctanoic acid (3 eq), HATU (2.9 eq), DIPEA (6 eq) (1 mL per 100 mg resin) in DMF for 1 h. The resin was washed with DMF (4 times) and DCM (4 times).

[0215] The final polysarcosine compounds were cleaved from the resin (Rink and Wang resins: 100% TFA x 2 x 30 min, 2-chlorotrityl resin: 20% TFA in DCM x 15 min). The resin was filtered and the volatiles removed under reduced pressure to give the crude product, which was purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0216] 1.1.4) Final polysarcosine intermediate The following Table 1 lists the compounds obtained.

[0217] [Table 1] JPEG2024512986000020.jpg234169JPEG2024512986000021.jpg234169

[0218] 1.2) Synthesis of intermediate compounds 1.2.1) Synthesis of Compound B1 and Compound B2

[0219] [ka]

[0220] These compounds were synthesized according to the procedures described in patent WO20199081455. The compounds are equimolar diastereoisomeric mixtures (stereoisomeric centers are indicated by asterisks).

[0221] 1.2.2) Synthesis of Compound B3 and Compound B4

[0222] [ka]

[0223] 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 into a round-bottom flask and suspended in 4 mL of distilled water. The flask was chilled at 0 °C and 4 mL of pre-chilled 65% nitric acid solution was slowly added. The reaction was held at 0 °C for 20 min and showed complete mononitration of the starting material as assessed by HPLC. The contents of the flask were transferred to a 250 mL pre-chilled Erlenmeyer flask and diluted with saturated NaHCO at 0 °C until the pH value reached 8-9. 3 The solution (approximately 50 mL) was slowly used to neutralize the mixture. Then, 30 mL of dioxane was added, followed by Boc 2 O (7202 mg / 13.2 mmol) was added. The reaction was then allowed to come to room temperature and stirred overnight. The reaction was then diluted with EtOAc and washed three times with saturated aqueous citric acid and once with saturated aqueous NaCl. The organic phase was washed with MgSO 4 Drying over, filtration and evaporation in vacuo gave a crude material which was purified by chromatography on silica gel (petroleum ether / EtOAc, gradient from 70:30 to 20:80) to afford the title compound (1320 mg / 40%) as a thick yellow to brown oil. 1 H NMR (300MHz, DMSO-d 6)δ10.79(s,1H), 7.79(d,J=2.1Hz,1H), 7.47(dd,J=8.6,2.1Hz,1H), 7.08(d,J=8.6Hz,1H ), 6.74(t,J=5.9Hz,1H), 4.56(t,J=6.3Hz,1H), 3.07(td,J=6.1,1.6Hz,2H), 1.31(s,9H). MSm / z(ESI + ):Calc[M+H] + =299.1;Exp[M+H] + = 299.1. HPLC Method 2 Retention Time = 5.5 min.

[0224] 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 Round bottom flash, Ag 2 CO 3 (1500mg / 5.4mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (251mg / 1.1mmol) were taken up in 4mL of anhydrous acetonitrile and stirred at room temperature for 2h. The precursor compounds tert-butyl(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (292mg / 0.98mmol) and 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronide methyl ester (583mg / 1.46mmol) were added at 0°C and the solution mixture was stirred at room temperature for 4h. The reaction was then filtered over Celite, diluted with EtOAc, washed three times with saturated aqueous citric acid and once with saturated aqueous NaCl. The organic phase was removed using MgSO 4 Drying over, filtration and evaporation in vacuo gave a crude material which was purified by chromatography on silica gel (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 (300MHz, DMSO-d 6)δ7.76(dd,J=3.3,2.1Hz,1H),7.60(t,J=7.6Hz,1H),7.36(dd,J=8.7,2.6Hz,1H), 6.77(s,1H),5.71(d,J=7.8Hz,1H),5.61(s,1H),5.46(td,J=9.5,1.1Hz,1H),5.21 -5.02(m,3H),4.75(dd,J=9.9,1.4Hz,1H),4.62(s,1H),3.65(s,3H),3.17(s,2H), 3.10(t,J=6.1Hz,2H),2.81-2.59(m,6H),2.05-1.96(m,9H),1.30(d,J=1.7Hz,9H). MSm / z(ESI + ):Calc[M+Na] + =637.2;Exp[M+Na] + = 637.2. HPLC Method 2 Retention Time = 6.75 min.

[0225] 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 precursor compound (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 (334 mg / 0.54 mmol) and 4-nitrophenyl chloroformate (219 mg / 1.09 mmol) were dissolved in 6 mL of dry 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 was filtered through a 0.45 μm PTFE filter and purified by chromatography on silica gel (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 (300MHz, 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=9.5Hz,1H),5.11(q,J=9.6Hz,2H),4.77(d,J=9.9Hz,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). MSm / z(ESI + ):Calc[M+Na] + =802.15;Exp[M+Na] + = 802.15. HPLC Method 2 Retention Time = 8.5 min.

[0226] 1.2.2.4) Synthesis of compound B3 101 mg (0.13 mmol) of precursor 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-triyl triacetate, 98 mg (0.14 mmol) of MMAE and 18 mg (0.13 mmol) of HOBt were dissolved in 1 mL of anhydrous DMF / pyridine mixture 85:15 (v / v). 16.7 mg (0.13 mmol) of DIPEA was added. The reaction was stirred at room temperature for 16 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by chromatography on silica gel (DCM / MeOH 99:1 to 95:5 gradient) to give 147 mg (84%) of the intermediate compound (white solid) directly involved in the deprotection step. ESI + [M+H] + = 1358.7. HPLC Method 2 Retention Time = 8.8 min.

[0227] 144 mg (0.106 mmol) of this intermediate compound was dissolved in 3 mL of MeOH at 0° C. LiOH monohydrate (44.5 mg / 1.06 mmol) was dissolved in water (0.4 mL) and added to the reaction vessel. After stirring at 0° C. for 30 min (post reaction, HPLC), the mixture was neutralized with acetic acid (83 mg / 1.4 mmol) and concentrated under reduced pressure. The resulting crude was redissolved at 0° C. using a TFA / DCM (30:70 v / v) solution and stirred at room temperature for 15 min. The volatiles were evaporated under reduced pressure and the crude residue was taken up in a water / ACN (1:1 v / v) solution and purified using HPLC preparative method 5 to give 85 mg (72%) of compound B3 as a white solid. HRMS m / z (ESI + ):Calc[M+H] + =1118.5867;Exp[M+H] + = 1118.5842; Error = 2.2 ppm. HPLC Method 3 Retention Time = 9.36 min.

[0228] 1.2.2.5) Synthesis of compound B4 Compound B4 was synthesized using the same procedure used to synthesize compound B3 with some 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.

[0229] 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]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate was obtained as a yellow / green solid. ESI + [M+Na] + = 1098.3. HPLC Method 3 retention times = 14.7 min and 14.9 min (diastereoisomeric mixture).

[0230] Deprotection of the glucuronide moiety was carried out using MeOH / THF (75:25 v / v) instead of pure MeOH. Boc-deprotection was carried out as described for compound B3.

[0231] Purification using HPLC preparative method 5 afforded 65 mg (67%) of compound B4 as a yellow solid. ESI + [M+H] + = 836.2. HPLC Method 3 retention time = 7.3 min and 7.8 min (diastereoisomeric mixture).

[0232] 1.2.2.6) Synthesis of stereopure compounds B4-S and B4-R

[0233] [ka]

[0234] 1.2.2.6.1) Chiral separation of the racemic mixture of tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate Chiral separation of racemic tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (synthesized as described above) was performed using a Chiralflash® IC MPLC column 30×100 mm, 20 μm (Daicel cat#83M73) on a Teledyne Isco CombiFlash® Rf200 system. The mobile phase was DCM+0.2% (v / v) EtOH (isocratic gradient). The flow rate was 12 mL / min. The sample solvent was DCM+0.2% (v / v) EtOH. The mass recovery of the two enantiomers after separation was greater than 80%.

[0235] The retention time of tert-butyl (S)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 15 min, whereas the retention time of tert-butyl (R)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 25 min.

[0236] To determine the absolute configuration, the phenolic position of both 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 chromatography on silica gel (petroleum ether / EtOAc, gradient 90:10 to 10:90) to give 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenyl 4-nitrobenzoic acid. 1 H NMR (300MHz, DMSO-d 6 )δ8.51(d,J=9.1Hz,2H),8.44(d,J=9.1Hz,2H),8.19(d,J=1.9Hz,1H),7.88(d,J=10.2Hz,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 enantiomers (previously dissolved in a 1:1 mixture of heptane / dichloromethane and allowed to slowly evaporate for 3 weeks to induce crystal formation) was confirmed by X-ray crystallography. Block crystals were mounted on nylon loops in perfluoroether oil. Data were collected using Xcalibur, Atlas, and Gemini ultradiffractometers equipped with Oxford Cryosystems cryostats operating at T = 150.00(5) K. Data were analyzed using a CuK α The structure was determined using a radioactive ω-scan. The structure was solved using the ShelXT solution program with the dual method, 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 in ShelXL 2018 / 3 (Sheldrick, GM, Crystal structure refinement with ShelXL, Acta Cryst., 2015, C71, 3-8) and the F 2 was refined using full matrix least squares minimization.

[0237] 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 in the previous section 1.2.2 without significant changes in reaction conditions, reactivity, or overall yields.

[0238] Final purification using HPLC preparative method 5 afforded 33 mg of compound B4-S as a yellow solid. ESI + [M+H] + = 836.2. HPLC Method 3 Retention Time = 7.3 min.

[0239] Final purification using HPLC preparative method 5 afforded 21 mg of compound B4-R as a yellow solid. ESI+ [M+H] + = 836.2. HPLC Method 3 Retention Time = 7.8 min.

[0240] 1.2.3) Synthesis of Compound B5 and Compound B6

[0241] [ka]

[0242] 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 sequentially. The mixture was then cooled to 0° C., and EDC-HCl (530 mg / 2.75 mmol) was added. The resulting mixture was stirred at 0° C. overnight. The reaction was diluted with 20 mL of 2 M HCl, and the layers were separated. The organic phase was washed twice with 2 M HCl and twice with saturated NaHCO 3 The organic phase was washed twice with saturated NaCl solution and once with MgSO 4 The mixture was dried over ice, filtered and evaporated in vacuo to give 714 mg (89%) of benzylacetyl-L-valyl-L-alaninate as a white solid intermediate.

[0243] This intermediate was dissolved in 10 mL of 1:1 (v / v) EtOAc / MeOH and transferred to a stainless steel hydrogenation reactor. After an initial argon purge, a catalytic amount of 5 wt% Pd / C was added. The reactor was then filled with H 2 Purge twice with 10 bar H 2 The reaction mixture was kept under pressure at room temperature overnight. After filtering the reaction mixture through a 0.45 μm PTFE filter and removing the solvent under vacuum, pure acetyl-L-valyl-L-alanine was quantitatively obtained as a white solid. 1 H NMR (300MHz, DMSO-d 6)δ12.45(s,1H),8.23(d,J=6.9Hz,1H),7.85(d,J=9.0Hz,1H),4.25-4.11(m,2H),1.94(d t,J=13.6,6.8Hz,1H),1.85(s,3H),1.26(d,J=7.3Hz,3H),0.85(dd,J=12.1,6.8Hz,6H).

[0244] 1.2.3.2) Synthesis of (2S)-2-acetamido-N-((2S)-1-((4-(1-hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide In a round-bottom flash, 420 mg (2.60 mmol) of 1-(4-aminophenyl)but-3-yn-1-ol (synthesized according to the procedure described in Sharma A. et al., Chem 2018, 4 (10), 2370-2383) and 600 mg (2.60 mmol) of the precursor 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), previously dissolved in 5 mL of anhydrous DMF, were placed in the flask and the cloudy reaction mixture was stirred overnight at room temperature. Afterwards, the volatiles were evaporated under reduced pressure and the crude residue was taken to dryness and purified by chromatography on silica gel (gradient from DCM / MeOH 99:1 to 85:15) to give 809 mg (83%) of the title compound as a white solid. 1 H NMR (300MHz, DMSO-d 6 )δ9.84(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= 7.6,7.2Hz,1H),4.17(dd,J=8.5,6.8Hz,1H),2.70(t,J=2.6Hz,1H),1.96(dt,J=1 3.2,6.6Hz,1H),1.88(s,3H),1.30(d,J=7.1Hz,3H),0.86(dd,J=10.9,6.8Hz,6H). ESI +[M+H] + = 374.2. HPLC Method 2 Retention Time = 3.95 min.

[0245] 1.2.3.3) Synthesis of 1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate 94 mg (0.25 mmol) of (2S)-2-acetamido-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 chromatography on silica gel (DCM / MeOH 99:1 to 90:10 gradient) to give 118 mg (87%) of the title compound as a yellow solid. 1 H NMR (300MHz, 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=7.2Hz,1H),4.17(dd,J=8.5,6.9Hz,1H),3.01-2.84(m,3H),1.9 9-1.91(m,1H),1.88(s,3H),1.31(d,J=7.1Hz,3H),0.86(dd,J=11.2,6.8Hz,6H). ESI + [M+H] + = 539.1. HPLC Method 2 Retention Time = 6.48 min.

[0246] 1.2.3.4) Synthesis of compound B5 44 mg (0.082 mmol) of precursor compound 1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate, 53 mg (0.074) of MMAE and 11.1 mg (0.082 mmol) of HOBt were dissolved in 1 mL of anhydrous DMF / pyridine mixture 85:15 (v / v). 10.6 mg (0.082 mmol) of DIPEA was added. The reaction was stirred at room temperature for 16 h and directly purified using HPLC preparative method 5 to give compound B5 as a white solid, 41 mg (50%). ESI + [M+Na] + = 1139.7. HPLC Method 2 Retention Time = 7.40 min.

[0247] 1.2.3.5) Synthesis of compound B6 Compound B6 was synthesized using the same procedure used to synthesize compound B5 with some modifications. The coupling reaction of 1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)but-3-yn-1-yl (4-nitrophenyl)carbonate with exatecan mesylate was carried out at 40° C. for 3 h instead of overnight at room temperature. After removing the volatiles under reduced pressure, the reaction mixture was purified by chromatography on silica gel (DCM / MeOH 99:1 to 95:5 gradient) 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 min and 6.60 min (diastereoisomeric mixture).

[0248] 1.2.4) Synthesis of Compound B7 and Compound B8

[0249] [ka]

[0250] 1.2.4.1) Synthesis of tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate A solution of 911 mg (3.23 mmol) of commercially available tert-butyl (2-hydroxy-2-(4-nitrophenyl)ethyl)carbamate (CAS#939757-25-2) in MeOH was transferred to a stainless steel hydrogenation reactor. After an initial argon purge, a catalytic amount of 5 wt. % Pd / C was added. The reactor was then heated with H 2 Purge twice with 10 bar H 2 The reaction was kept under stirring at room temperature for 5 hours under pressure of 1000 psi. The reaction was filtered through a 0.45 μm PTFE filter and after removing MeOH under vacuum, 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.

[0251] 1.2.4.2) Synthesis of tert-butyl (2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-hydroxyethyl)carbamate 150 mg (0.60 mmol) of precursor compound tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate, 222 mg (0.71 mmol) of Fmoc-Ala-OH and 81 mg (0.62 mmol) of DIPEA were dissolved in 5 mL of anhydrous DMF. 181 mg (0.71 mmol) of HATU were added and the reaction was stirred at room temperature overnight. The volatiles were then removed under reduced pressure and the crude residue was purified by chromatography on silica gel (gradient DCM / MeOH 100:0 to 90:10) to quantitatively obtain the first intermediate tert-butyl (2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)phenyl)-2-hydroxyethyl)carbamate, which is directly involved in the Fmoc-deprotection. Retention time HPLC method 2 = 7.7 min.

[0252] tert-Butyl (2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)phenyl)-2-hydroxyethyl)carbamate was dissolved in 5 mL of 9:1 (v / v) DMF / piperidine and stirred at room temperature for 15 min. The volatiles were then removed under reduced pressure and the crude residue was purified by chromatography on silica gel (DCM / MeOH 99:1 to 80:20 gradient) to give 130 mg (68% over two steps) of the second intermediate tert-butyl (2-(4-((S)-2-aminopropanamido)phenyl)-2-hydroxyethyl)carbamate as a white foamy solid. HPLC Method 2 retention time = 3.75 min.

[0253] 130 mg (0.40 mmol) of tert-butyl (2-(4-((S)-2-aminopropanamido)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. The volatiles were then 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 (300MHz, 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=8.4H z,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=7.1Hz,1H),4.17(dd,J=8.4,6.8Hz,1H),3.15-2.90 (m,2H),1.88(s,4H),1.35(s,9H),1.30(d,J=7.1Hz,3H),0.92-0.73(m,6H). ESI + [M+H] + = 487.3. HPLC Method 2 Retention Time = 4.50 min.

[0254] 1.2.4.3) Synthesis of tert-butyl (2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate 286 mg (0.62 mmol) of tert-butyl (2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)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 h. The volatiles were removed under reduced pressure and the crude residue was purified by chromatography on silica gel (DCM / MeOH 99:1 to 90:10 gradient) to give 336 mg (87%) of the title compound as a yellow solid. 1 H NMR (300MHz, 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 .0Hz,1H),4.38(p,J=7.1Hz,1H),4.17(dd,J=8.5,6.9Hz,1H),3.49-3.34(m,2H),2.01-1. 90(m,1H),1.87(s,3H),1.37(s,9H),1.30(d,J=7.1Hz,3H),0.86(dd,J=11.1,6.8Hz,6H). ESI + [M+Na] + = 652.2. HPLC Method 2 Retention Time = 7.13 min.

[0255] 1.2.4.4) Synthesis of compound B7 43.2 mg (0.069 mmol) of the precursor compound tert-butyl (2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate, 41 mg (0.057) of MMAE and 6.7 mg (0.057 mmol) of HOBt were dissolved in 1 mL of anhydrous DMF / pyridine mixture 85:15 (v / v). 11.0 mg (0.086 mmol) of DIPEA was added. The reaction was stirred at room temperature for 16 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by chromatography on silica gel (gradient DCM / MeOH 99:1 to 90:10) to give 47 mg (55%) of the intermediate compound (pale yellow solid) directly involved in the Boc-deprotection step. ESI + [M+Na] + = 1230.7. HPLC Method 2 Retention Time = 7.55 min.

[0256] The resulting solid was redissolved in 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 and the crude residue was taken up in water / ACN (1:1 v / v) solution and purified using HPLC preparative method 5 to give compound B7 as a white solid, 15 mg (30%). ESI + [M+H] + = 1108.7. HPLC Method 2 retention time = 5.8 min and 5.9 min (diastereoisomeric mixture).

[0257] 1.2.4.5) Synthesis of compound B8 Compound B8 was synthesized using the same procedure used to synthesize compound B7 with a slight modification: the coupling reaction of tert-butyl(2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)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.

[0258] The intermediate compound 1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)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]indolizino[1,2-b]quinolin-1-yl)carbamate was obtained as a brownish yellow solid. ESI + [M+H] + = 926.4. HPLC Method 2 retention time = 6.7 min and 6.8 min (diastereoisomeric mixture).

[0259] Boc-deprotection was carried out as described for compound B7. Purification using HPLC preparative method 5 afforded 47 mg (70%) of compound B8 as a yellow solid. ESI + [M+H] + = 826.4. HPLC Method 3 retention times = 8.30 min and 8.75 min (diastereoisomeric mixture).

[0260] 1.2.4.6) Synthesis of stereopure compounds B8-S and B8-R

[0261] [ka]

[0262] 1.2.4.6.1) Chiral separation of the racemic mixture of tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate Chiral separation of racemic tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate was performed using a Chiralflash® IC MPLC column 30×100 mm, 20 μm (Daicel cat#83M73) on a Teledyne Isco CombiFlash® Rf200 system. The mobile phase was DCM+0.2% (v / v) EtOH (isocratic gradient). The flow rate was 12 mL / min. The sample solvent was DCM+0.2% (v / v) EtOH. The mass recovery of the two enantiomers after separation was greater than 75%.

[0263] The retention time of tert-butyl (S)-(2-(4-aminophenyl)-2-hydroxyethyl)carbamate was 21 min, whereas the retention time of tert-butyl (R)-(2-(4-aminophenyl)-2-hydroxyethyl)carbamate was 29 min. The absolute configuration of the enantiomers (previously dissolved in a 1:1 mixture of heptane / ethanol and allowed to slowly evaporate for a week to induce crystal formation) was confirmed by X-ray crystallography. Block crystals were mounted on nylon loops in perfluoroether oil. Data were collected using Xcalibur, Atlas, and Gemini ultradiffractometers equipped with Oxford Cryosystems cryostats operating at T = 150.00(10) K. Data were analyzed by NMR spectroscopy using a CuK α The structure was determined using ω-scans using radiation. The structure was solved using the ShelXT solution program using the dual method, 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) to obtain the F2 was refined using full matrix least squares minimization.

[0264] 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 in the previous section 1.2.4 without significant changes in reaction conditions, reactivity, or overall yields.

[0265] Final purification using HPLC preparative method 5 afforded 54 mg of compound B8-S as a yellow solid. ESI + [M+H] + = 826.4. HPLC Method 3 Retention Time = 8.45 min.

[0266] Final purification using HPLC preparative method 5 afforded 46 mg of compound B8-R as a yellow solid. ESI + [M+H] + = 826.4. HPLC Method 3 Retention Time = 8.90 min.

[0267] 1.2.5) Synthesis of Compounds B9, B10 and B11 1.2.5.1) Synthesis of compound B9

[0268] [ka]

[0269] 20.0 mg (0.036 mmol) of Boc-Val-Ala-PAB-PNP (CAS#1884578-00-0, Iris Biotech), 23.1 mg (0.032) of MMAE and 5 mg (0.036 mmol) of HOBt were dissolved in 1 mL of anhydrous DMF / pyridine mixture 85:15 (v / v). 4.6 mg (0.036 mmol) of DIPEA was added. The reaction was stirred at room temperature for 16 h and the volatiles were evaporated under reduced pressure. The crude residue was dissolved in 2 mL of TFA / DCM (30:70 v / v) solution and stirred at room temperature for 1 h. The volatiles were evaporated under reduced pressure and the crude residue was taken up in water / ACN (1:1 v / v) solution and purified using HPLC preparative method 5 to give 11 mg (26%) of compound B9 as a white solid. ESI + [M+H] + = 1037.7. HPLC Method 2 retention time = 7.9 min.

[0270] 1.2.5.2) Synthesis of compound B10

[0271] [ka]

[0272] Compound B10 was synthesized using the same procedure used to synthesize compound B9 with some 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.

[0273] Boc-deprotection was carried out as described above for compound B9. Purification using HPLC Prep Method 5 afforded 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.

[0274] 1.2.5.3) Synthesis of compound B11

[0275] [ka]

[0276] The compound (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-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 synthesized according to the method of Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831-840.

[0277] 1.3) Drug Linker Synthesis 1.3.1) Synthesis of Glucuronide-Based Drug Linkers 1.3.1.1) Synthesis of compound DL1

[0278] [ka]

[0279] 100 mg (0.081 mmol) of compound A1 (NHS-activated polysarcosine intermediate) and 51 mg (0.061 mmol) of compound B4 (NH 2-payload) was dissolved in anhydrous DMF in a small vial (concentration of compound B4 is 0.080M). 41 mg (0.405 mmol) of triethylamine was added and the reaction was stirred at room temperature for 30 min. After total conversion of the reaction observed by HPLC, piperidine was added directly to the reaction vial to make 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 slowly neutralized with a solution of 10% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 5 to give 74 mg (70% yield based on starting compound B4) of the intermediate compound (2S,3S,4S,5R,6S)-6-(4-(41-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]indolizino[ 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-tridecaoxo-2-oxa-5,9,12,15,18,21,24,27,30,33,36,39-dodecaazahenetetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid was obtained as a yellow solid. ESI + [M+H] + = 1731.7. HPLC Method 3 retention times = 7.40 min and 7.70 min (equimolar diastereoisomeric mixture).

[0280] 17.8 mg (0.010 mmol) of this compound and 2.85 mg (0.011 mmol) of maleimidoacetic acid N-hydroxysuccinimide ester were dissolved in anhydrous DMF (0.1 M concentration of maleimido compound). 1.56 mg (0.015) of triethylamine was added and stirred for 2 h until total conversion of the reaction was observed by HPLC. The reaction mixture was then diluted with a solution of 1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 6 to give 13.7 mg (72%) of compound DL1 as a yellow solid. HRMS m / z (ESI + ):Calc[M+2H] 2+ =934.8559;Exp[M+2H] 2+ = 934.8544; Error = 1.7 ppm. HPLC Method 3 retention times = 7.73 min and 8.08 min (equimolar diastereoisomeric mixture).

[0281] 1.3.1.2) Synthesis of compound DL2

[0282] [ka]

[0283] 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 100 mM PBS (pH=7.5) and DMSO (1:1 v / v) to a concentration of compound B2 of 0.060 M. Freshly prepared CuSO was then added to the reaction mixture to give 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). 4The pentahydrate and sodium ascorbate solution (approximately 250 mg / mL) were added sequentially to the reaction vial. The reaction was purged with argon and stirred at 40° C. The reaction was monitored by HPLC and was complete within 2 hours. The reaction mixture was diluted with a solution of 0.1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 5 to give 44 mg (77% based on 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-undeca ... Azapentatriacontyl)-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-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid was obtained as a yellow solid. ESI + [M+H] + = 1755.7. HPLC Method 3 retention times = 7.51 min and 7.82 min (diastereoisomeric mixture).

[0284] 26.0 mg (0.015 mmol) of this compound and 4.11 mg (0.016 mmol) of maleimidoacetic acid N-hydroxysuccinimide ester were dissolved in anhydrous DMF (0.1 M concentration of maleimido compound). 2.25 mg (0.022 mmol) of triethylamine was added and stirred for 2 h until total conversion of the reaction was observed by HPLC. The reaction mixture was diluted with a solution of 1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 6 to give 16.0 mg (57%) of compound DL2 as a yellow solid. ESI + [M+H] += 1892.7. HPLC Method 3 retention times = 7.95 min and 8.20 min (equimolar diastereoisomeric mixture).

[0285] 1.3.1.3) Synthesis of compound DL3

[0286] [ka]

[0287] 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 was added. The reaction was pre-incubated at room temperature for 2 min and transferred onto 9.8 mg (0.012 mmol) of compound B4 (pre-weighed in a reaction vial). The reaction was stirred for 30 min until total conversion of the reaction was observed by HPLC. The reaction mixture was diluted with a solution of 1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 5 to give 4.7 mg (40%) of compound DL3 as a yellow solid. HRMS m / z (ESI + ):Calc[M+H] + =1049.2847;Exp[M+H] + = 1049.2873; Error = -2.5 ppm. HPLC Method 3 retention time = 9.80 min (equimolar diastereoisomeric mixture).

[0288] 1.3.1.4) Synthesis of compound DL4

[0289] [ka]

[0290] 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 already described in patent WO2019081455; a rather unstable compound that can only be stored for a few days when stored at -20°C) and 22.6 mg (0.027 mmol) of compound B2 were dissolved in a solution of 100 mM PBS (pH=7.5) and DMSO 1:1 (v / v) to a concentration of 0.060 M of compound B2. Then, freshly prepared CuSO 4 The pentahydrate and sodium ascorbate solutions (approximately 250 mg / mL) were added sequentially to the reaction vial to provide 0.08 molar equivalents of Cu and 1 molar equivalent of sodium ascorbate (based on the 2 molar equivalents of compound B in the reaction mixture). The reaction was purged with argon and stirred at room temperature. The reaction was monitored by HPLC and was complete within 1 h. The reaction mixture was then diluted with a solution of 0.1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 6 to give 16 mg (53%) of compound DL4 as a yellow solid. HRMS m / z (ESI + ):Calc[M+H] + =1144.3331;Exp[M+H] + = 1144.3351; Error = -1.8 ppm. HPLC Method 3 retention time = 9.61 min (equimolar diastereoisomeric mixture).

[0291] 1.3.1.5) Synthesis of compound DL5

[0292] [ka]

[0293] Compound DL5 was synthesized as described above using the same procedure used for compound DL1. The starting materials were compound A4 (NHS-activated polysarcosine intermediate) and compound B3 (NH 2 -Payload).

[0294] Obtained 10.8 mg (48% over two steps) of compound DL5 as a slightly yellow solid. HRMS m / z (ESI + ):Calc[M+2H] 3+ =765.0460;Exp[M+3H] 3+ = 765.0443; Error = 2.2 ppm. HPLC Method 3 retention time = 9.14 min (equimolar diastereoisomeric mixture).

[0295] 1.3.1.6) Synthesis of compound DL6

[0296] [ka]

[0297] Compound DL6 was synthesized using the same procedure as used for compound DL2, as described above. The starting materials were compound A5 (azide-polysarcosine intermediate) and compound B1 (alkyne-payload).

[0298] Obtained 16.3 mg (41% over two steps) of compound DL6 as a slightly yellow solid. ESI + [M+2H] 2+ = 1159.1. HPLC Method 3 retention times = 9.32 min and 9.45 min (equimolar diastereoisomeric mixture).

[0299] 1.3.1.7) Synthesis of compound DL7

[0300] [ka]

[0301] Compound DL7 was synthesized using the same procedure used for compound DL3, as described above. The starting material was the commercially available compound maleimide-PEG. 2 -acid (CAS#1374666-32-6) and compound B3 (NH 2 -Payload).

[0302] Obtained 15.6 mg (69%) of compound DL7 as a white solid. ESI + [M+2Na] 2+ = 701.3. HPLC Method 3 retention time = 10.94 min (equimolar diastereoisomeric mixture).

[0303] 1.3.1.8) Synthesis of compound DL8

[0304] [ka]

[0305] 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 such that the concentration of compound B1 was 0.060 M. Then, freshly prepared CuSO 4The pentahydrate and sodium ascorbate solution (approximately 250 mg / mL) were added sequentially to the reaction vial to give 0.08 molar equivalents of Cu and 1 molar equivalent of sodium ascorbate (based on 1 molar equivalent of Compound B in the reaction mixture). The reaction was purged with argon and stirred at room temperature. The reaction was monitored by HPLC and was complete within 1 hour. The reaction mixture was diluted with a solution of 0.1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 5 to give 18.2 mg (110% based on 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-((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 was obtained as a white solid. ESI + [M+H] + = 1213.6. HPLC Method 2 retention times = 5.55 min and 5.65 min (diastereoisomeric mixture).

[0306] 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 was added. The reaction was pre-incubated at room temperature for 2 min and transferred onto 18.2 mg (0.012 mmol) of precursor compound (pre-weighed in a reaction vial). The reaction was stirred for 30 min until total conversion of the reactant was observed by HPLC. The reaction mixture was diluted with a solution of 1% TFA in 1:1 (v / v) water / ACN and purified using HPLC preparative method 5 to give compound DL8 as a white solid, 16.0 mg (73%). HRMS m / z (ESI + ):Calc[M+2H] 2+ =726.8609;Exp[M+2H] 2+ = 726.8631; Error = -3.1 ppm. HPLC Method 3 retention times = 10.40 min and 10.56 min (equimolar diastereoisomeric mixture).

[0307] 1.3.1.9) Synthesis of compound DL9

[0308] [ka]

[0309] Compound DL9 was synthesized as described above using the same procedure used for compound DL3. The starting material was the commercially available compound maleimide-PEG. 2 -acid (CAS#1374666-32-6) and compound B11 (NH 2 -Payload).

[0310] 10.0 mg (41%) of compound DL9 was obtained as a white solid. HRMS m / z (ESI + ):Calc[M+2H] 2+ =685.3549;Exp[M+2H] 2+ = 685.3554; Error = -0.8 ppm. HPLC Method 3 Retention Time = 11.38 min.

[0311] 1.3.1.10) Synthesis of compound DL10

[0312] [ka]

[0313] Compound DL10 was synthesized by the same procedure used for compound DL1, as described above. The starting materials were compound A6 (NHS-activated polysarcosine intermediate) and compound B4 (NH 2 -Payload).

[0314] Obtained 54.1 mg (44% over two steps) of compound DL10 as a yellow solid. HRMS m / z (ESI + ):Calc[M+2H] 2+ =934.8559;Exp[M+2H] 2+ = 934.8545; Error = 1.5 ppm. HPLC Method 3 retention times = 7.68 min and 8.01 min (equimolar diastereoisomeric mixture).

[0315] 1.2.1.11) Synthesis of compounds DL10-S and DL10-R

[0316] [ka]

[0317] Compounds DL10-S and DL10-R were synthesized as described above using the same procedure as that used for compound DL1. The starting materials were compounds B4-S and B4-R (NH 2 -payload) as well as compound A6 (NHS-activated polysarcosine intermediate).

[0318] 4.7 mg of compound DL10-S was obtained as a yellow solid. ESI + [M+Na] + = 1890.7. HPLC Method 3 retention time = 7.62 min.

[0319] 4.0 mg of compound DL10-R was obtained as a yellow solid. ESI + [M+Na] + = 1890.7. HPLC Method 3 Retention Time = 8.06 min.

[0320] 1.2.1.12) Synthesis of compound DL11

[0321] [ka]

[0322] Compound DL11 was synthesized using the same procedure as used for compound DL2, as described above. The starting materials were compound A3 (azide-polysarcosine intermediate) and compound B2 (alkyne-payload).

[0323] Obtained 14.3 mg (43% over two steps) of compound DL11 as a yellow solid. HRMS m / z (ESI + ):Calc[M+2H] 2+ =947.3536;Exp[M+2H] 2+ = 947.3540; Error = -0.5 ppm. HPLC Method 3 retention times = 8.13 min and 8.36 min (equimolar diastereoisomeric mixture).

[0324] 1.3.2) Synthesis of dipeptide-based drug linkers 1.3.2.1) Synthesis of compound DL12

[0325] [ka]

[0326] Compound DL12 was synthesized by the same procedure used for compound DL1, as described above. The starting materials were compound A7 (NHS-activated polysarcosine intermediate) and compound B8 (NH 2 -Payload).

[0327] Obtained 34.9 mg (46% over two steps) of compound DL12 as a yellow solid. HRMS m / z (ESI + ):Calc[M+2H] 2+ =981.4394;Exp[M+2H] 2+ = 981.4398; Error = -0.4 ppm. HPLC Method 3 retention times = 8.81 min and 8.94 min (equimolar diastereoisomeric mixture).

[0328] 1.3.2.2) Synthesis of compounds DL12-S and DL12-R

[0329] [ka]

[0330] Compounds DL12-S and DL12-R were synthesized as described above using the same procedure as used for compound DL1. The starting materials were compounds B8-S and B8-R (NH 2 -payload) as well as compound A7 (NHS-activated polysarcosine intermediate).

[0331] Obtained 18.1 mg of compound DL12-S as a yellow solid. ESI + [M+2H] 2+ = 981.4. HPLC Method 3 retention time = 8.78 min.

[0332] 16.3 mg of compound DL12-R was obtained as a yellow solid. ESI + [M+2H] 2+ = 981.4. HPLC Method 3 retention time = 8.96 min.

[0333] 1.3.2.3) Synthesis of compound DL13

[0334] [ka]

[0335] Compound DL13 was synthesized using the same procedure as used for compound DL2, as described above. The starting materials were compound A8 (azide-polysarcosine intermediate) and compound B6 (alkyne-payload).

[0336] 4.5 mg (24% over two steps) of compound DL13 was obtained as a yellow solid. HRMS m / z (ESI + ):Calc[M+2H] 2+ =993.4451;Exp[M+2H] 2+ = 993.4416; Error = 3.5 ppm. HPLC Method 3 retention times = 8.88 min and 9.11 min (equimolar diastereoisomeric mixture).

[0337] 1.3.2.4) Synthesis of compound DL14

[0338] [ka]

[0339] Compound DL14 was synthesized using the same procedure used for compound DL3, as described above. The starting material was the commercially available compound maleimide-PEG. 2 -acid (CAS#1374666-32-6) and compound B8 (NH 2 -Payload).

[0340] 7.0 mg (59%) of compound DL14 was obtained as a yellow solid. HRMS m / z (ESI + ):Calc[M+H] + =1065.4364;Exp[M+H] + = 1065.4370; Error = -0.5 ppm. HPLC Method 3 retention times = 10.20 min and 10.44 min (equimolar diastereoisomeric mixture).

[0341] 1.3.2.5) Synthesis of compound DL15

[0342] [ka]

[0343] Compound DL15 was synthesized as described above using the same procedure used for compound DL8.

[0344] Obtained 3.1 mg (20% over two steps) of compound DL15 as a yellow solid. HRMS m / z (ESI + ):Calc[M+H] + =1160.4848;Exp[M+H] + = 1160.4854; Error = -0.6 ppm. HPLC Method 3 retention times = 9.96 min and 10.12 min (equimolar diastereoisomeric mixture).

[0345] 1.3.2.6) Synthesis of compound DL16

[0346] [ka]

[0347] Compound DL16 was synthesized as described above using the same procedure used for compound DL3. The starting material was the commercially available compound maleimide-PEG. 2 -acid (CAS#1374666-32-6) and compound B7 (NH 2 -Payload).

[0348] 5.3 mg (41%) of compound DL16 was obtained as a white solid. HRMS m / z (ESI + ):Calc[M+Na] + =1369.7618;Exp[M+Na] + = 1369.7621; Error = -0.3 ppm. HPLC Method 3 retention time = 11.38 min (equimolar diastereoisomeric mixture).

[0349] 1.3.2.7) Synthesis of compound DL17

[0350] [ka]

[0351] Compound DL17 was synthesized as described above using the same procedure used for compound DL8.

[0352] Obtained 8.9 mg (24% over two steps) of compound DL17 as a white solid. HRMS m / z (ESI + ):Calc[M+H] + =1442.8294;Exp[M+H] + = 1442.8284; Error = 0.7 ppm. HPLC Method 3 retention time = 11.81 min (equimolar diastereoisomeric mixture).

[0353] 1.3.2.8) Synthesis of compound DL18

[0354] [ka]

[0355] Compound DL18 was synthesized as described above using the same procedure used for compound DL3. The starting material was the commercially available compound maleimide-PEG. 2 -acid (CAS#1374666-32-6) and compound B10 (NH 2 -Payload).

[0356] Obtained 14.5 mg (62%) of compound DL18 as a yellow solid. ESI + [M+H] + = 994.4. HPLC Method 3 retention time = 11.62 min.

[0357] 1.3.2.9) Synthesis of compound DL19

[0358] [ka]

[0359] Compound DL19 was synthesized as described above using the same procedure used for compound DL3. The starting material was the commercially available compound maleimide-PEG. 2-acid (CAS#1374666-32-6) and compound B9 (NH 2 -Payload).

[0360] 4.7 mg (35%) of compound DL19 was obtained as a white solid. HRMS m / z (ESI + ):Calc[M+H] + =1276.7439;Exp[M+H] + = 1276.7441; Error = -0.1 ppm. HPLC Method 3 Retention Time = 13.28 min.

[0361] 1.3.2.10) Synthesis of compound DL20

[0362] [ka]

[0363] Compound DL20 was synthesized using the same procedure as used for compound DL2, as described above. The starting materials were compound A3 (azide-polysarcosine intermediate) and compound B6 (alkyne-payload).

[0364] Obtained 14.0 mg (36% over two steps) of compound DL20 as a yellow solid. ESI + [M+H] + = 1883.8. HPLC Method 3 retention times = 8.82 min and 9.07 min (equimolar diastereoisomeric mixture).

[0365] 2) Preparation and characterization of conjugates 2.1) Preparation of antibody-drug conjugates A solution of antibody (10 mg / mL in PBS 7.4 + 1 mM EDTA) was treated with 14 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 2 h at 37 °C. The fully reduced antibody was buffer exchanged with potassium phosphate 100 mM pH 7.4 + 1 mM EDTA by dilution / centrifugation three times using an Amicon 30K centrifugal filter device (Millipore). 10-12 molar equivalents of drug linker (from a 12 mM DMSO stock solution) were added to the antibody to give a drug-antibody ratio (DAR) of 8 (residual DMSO <10% v / v). The solution was incubated for 30 min at room temperature. The conjugate was buffer exchanged / purified with PBS pH 7.4 by dilution / centrifugation four times using an Amicon 30K centrifugal filter device and sterile filtered (0.20 μm PES filter).

[0366] Conjugates incorporating autohydrolyzable maleimides (maleimide-phenyl and maleimide-glycine) were incubated at 5 mg / mL in PBS 8.0 for 24 h at 37° C. to ensure complete hydrolysis of the succinimidyl moiety, buffer exchanged into PBS pH 7.4 using an Amicon 30K centrifugal filter device, and sterile filtered (0.20 μm PES filter).

[0367] Final protein concentration was estimated spectrophotometrically at 280 nm using a Colibri microspectrometer instrument (Titertek Berthold).

[0368] 2.2) Characterization of the conjugate The resulting conjugate was characterized as follows: Reversed-phase liquid chromatography mass spectrometry (RPLC-MS): Modified RPLC-QToF analysis was performed using the UHPLC method described above. Briefly, conjugates were eluted on an Agilent PLRP-S 1000A 2.1 × 150 mm 8 μm (80 °C) using a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) and analyzed using a Bruker Impact II™ Q-ToF mass spectrometer in the 500-3500 m / z range (ESI + ) were scanned and detected. Data were deconvoluted using the MaxEnt algorithm included in the Bruker Compass® software.

[0369] Size Exclusion Chromatography (SEC): SEC was performed on an Agilent 1100 HPLC system with an extra column volume of 15 μL or less (equipped with a short section of Peak tubing with an internal diameter of 0.12 mm and a microvolume UV flow cell). The column was an Agilent AdvanceBioSEC 300A 4.6 × 150 mm 2.7 μm (maintained 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 prevent bacterial growth. The flow rate was 0.35 mL / min. UV detection was monitored at 280 nm.

[0370] Hydrophobic Interaction Chromatography (HIC): Hydrophobic interaction chromatography (HIC) was performed on an Agilent 1100 HPLC system. The column was a Tosoh TSK-GEL BUTYL-NPR 4.6 × 35 mm 2.5 μm (25 °C). Mobile phase A was 1.5 M (NH 4 ) 2 SO 4Mobile phase A was 25 mM potassium phosphate pH 7.0 + 25% isopropanol (v / v). The linear gradient was 0% B to 100% B for 10 min, followed by a hold at 100% B for 3 min. The flow rate was 0.75 mL / min. UV detection was monitored at 220 nm and 280 nm.

[0371] 2.3) Overview of the synthesized conjugates The antibody-drug conjugates showed one LC-1d (light chain with one drug linker attached) and one HC-3d (heavy chain with three drug linkers attached) absorbance peaks in the denaturing RPLC chromatogram (DAR8 conjugate). Mass spectrometry analysis of the heavy chain reported the major glycoform (G0F for trastuzumab).

[0372] [Table 2] JPEG2024512986000054.jpg231169JPEG2024512986000055.jpg231169

[0373] 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 according to the method described in section 2.

[0374] The results are shown in FIG. 1 for the glycosidase-based drug linker of the present invention (octopamine architecture) and in FIG. 2 for the dipeptidase-based drug linker of the present invention (2-amino-1-(4-aminophenyl)ethan-1-ol architecture). The conjugates of the present invention were systematically more hydrophilic (shorter retention time in HIC chromatograms) compared to the known corresponding architectures. This effect is observed with glycosidase-based drug linkers and dipeptidase-based drug linkers. This effect is observed with and without polysarcosine, a hydrophobic masking agent of the drug linker architecture. This effect is observed with drug payloads of different natures and different levels of intrinsic hydrophobicity.

[0375] 4) In vitro cytotoxicity assays of conjugates based on the drug linkers of the present invention and conjugates based on drug linkers of known architectures The in vitro cytotoxicity of the conjugates was evaluated on several antigen-positive cancer cell lines. Cells were plated in 96-well plates at appropriate densities (1000–10000 cells / well in 100 μL of the appropriate medium) depending on the cell line and incubated at 37 °C for 24 h. Serial dilutions of the test compounds (50 μL) previously dissolved in medium were added and incubated at 37 °C for 72 h for MMAE-based conjugates and 144 h for exatecan-based conjugates. MTT (5 mg / mL, 20 μL, Sigma-Aldrich) was added to the wells and incubation was continued at 37 °C for 1–2 h. Afterwards, the medium was carefully removed and the well contents were dissolved homogeneously with acidified isopropanol. Absorbance values ​​were measured using a Multiskan™ Sky microplate reader (Thermo Scientific) at a wavelength of 570 nm (reference wavelength 690 nm). IC compared to untreated control cells 50 Concentration values ​​were determined using inhibition dose-response curve fitting (GraphPad Prism 9).

[0376] The results are shown in Figure 3 for the conjugates based on glycosidase-sensitive drug linkers and in Figure 4 for the conjugates based on dipeptidase-sensitive drug linkers. The conjugates of the present invention (octopamine and 2-amino-1-(4-aminophenyl)ethan-1-ol architectures) showed systematically similar potency compared to the known corresponding architectures.

[0377] 5) In vitro cytotoxicity assays of conjugates based on the DL10 drug linker of the invention (equimolar diastereoisomeric mixture), the stereodefined DL10-S drug linker of the invention, and the stereodefined DL10-R drug linker of the invention. The in vitro cytotoxicity of the conjugates was evaluated in several antigen-positive cancer cell lines according to the experimental protocol in section 4) above.

[0378] The results are shown in Figure 5. No differences in potency were observed in vitro between stereopurified drug linkers and equimolar diastereomeric mixtures of the same drug linkers.

[0379] 6) Pharmacokinetic profile in rats after a single intravenous administration of 3 mg / kg of the conjugate (time course of total antibody-drug conjugate concentration) Female Sprague-Dawley rats (4-6 weeks old - Charles River) were injected with 3 mg / kg ADC via the tail vein (3 rats per group, randomized). Blood was collected at various time points by retro-orbital bleeding into citrate tubes, processed to plasma and stored at -80°C until analysis. ADC concentrations were assessed using a human IgG ELISA kit (Stemcell™ 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 calculated by 2-compartment analysis using Microsoft® Excel® software with PK functions (add-in developed by Usansky et al., Division of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA).

[0380] The results are shown in Figure 6 (PK profile) and Figure 7 (PK parameters) for the conjugates based on glycosidase-sensitive drug linkers and in Figure 8 (PK profile) and Figure 9 (PK parameters) for the conjugates based on dipeptidase-sensitive drug linkers. The conjugates of the present invention (octopamine and 2-amino-1-(4-aminophenyl)ethan-1-ol architectures) systematically provided improved pharmacokinetic profiles and parameters (improved exposure, increased half-life and decreased clearance rate) compared to the known corresponding architectures.

[0381] 7) The effect of a single intravenous dose of 1 mg / kg of the ADC110 conjugate (an octopamine architecture of the invention with a glucuronide-exatecan payload) and the ADC112 conjugate (a triazole architecture with a glucuronide-exatecan payload) on tumor volume (mm ) in an NCI-N87 HER2+ gastric cancer xenograft model. 3 ) over time.

[0382] NCI-N87 gastric cancer cells were subcutaneously implanted into female SCID mice (4 weeks old). ADCs were administered at 10-20°C until tumors reached approximately 150 mm 3 When the tumors had grown to 100 mm Hg, a subcritical dose of 1 mg / kg was administered intravenously once (six mice per group, assigned to minimize the difference in initial tumor volume between groups). Tumor volumes were measured every 3 to 5 days using a caliper device and calculated using the formula (L × W 2 ) / 2. 3 Mice were sacrificed at time points beyond this time.

[0383] The results are shown in Figure 10. The conjugate ADC110 based on the octopamine architecture of the present invention showed improved in vivo activity compared to the conjugate ADC112 based on the triazole architecture. No significant weight loss was observed in all treated mice. [Brief description of the drawings]

[0384] [Figure 1] FIG. 1 represents a hydrophobic interaction chromatogram according to Example 3. [Diagram 2] FIG. 2 shows the hydrophobic interaction chromatogram according to Example 3. [Figure 3-1] FIG. 3 represents an in vitro cytotoxicity assay of the conjugate according to Example 4. [Figure 3-2] Continued from Figure 3. [Figure 3-3] Continued from Figure 3. [Figure 4-1] FIG. 4 represents an in vitro cytotoxicity assay of the conjugate according to Example 4. [Figure 4-2] Continued from Figure 4. [Figure 4-3] Continued from Figure 4. [Diagram 5] FIG. 5 represents the in vitro cytotoxicity assay of the conjugate according to Example 5. [Figure 6] FIG. 6 represents the in vivo pharmacokinetic profile in rats and the pharmacokinetic parameters according to Example 6. [Figure 7] FIG. 7 depicts the in vivo pharmacokinetic profile in rats and the pharmacokinetic parameters according to Example 6. [Figure 8] FIG. 8 depicts the in vivo pharmacokinetic profile in rats and the pharmacokinetic parameters according to Example 6. [Figure 9] FIG. 9 represents the in vivo pharmacokinetic profile in rats and the pharmacokinetic parameters according to Example 6. [Figure 10] FIG. 10 depicts tumor volume over time in a mouse xenograft model of gastric cancer according to Example 7.

Claims

1. Ligand-drug-conjugate compounds (LDCs) having the following formula (I): 【Chemistry 1】 During the ceremony, L is a ligand selected from the group consisting of proteins, polypeptides, antibodies, and antibody fragments; X1 is a connector unit; Z is an optional spacer; X2 is a connector unit; K is any hydrophobic masking material selected from polysarcosine and polyethylene glycol; R1 is H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 selected from the group consisting of drugs; Each R2 is an electron withdrawing group and a C １ -C ４ independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleaving unit or a polypeptide cleaving unit; Y is O when T is a sugar cleavable unit or NR3 when T is a polypeptide cleavable unit; R3 is H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 H and C １ -C ６ independently selected from alkyl; and pharma-ceutically acceptable salts thereof.

2. The LDC compound of claim 1, wherein L is an antibody.

3. The LDC compound of claim 1 or 2, wherein D is an anticancer agent or an immunomodulatory agent.

4. X1 and X2 are each independently one or more amino acids, one or more N-substituted amino acids, an optionally substituted polyether, C １ -C １２ alkylene, arylene having 6 to 10 ring atoms, C ３ -C ８ Cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C ２ -C １０ alkenylene, and any combination thereof; said alkylene and said alkenylene may be interrupted by one or more heteroatoms or one or more 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, the arylene, the cycloalkylene, the heterocycloalkylene, the heteroarylene, and the alkenylene may be substituted with one or more substituents selected from the following: halogen, oxo, -OH, -NO ２ , -C.N., C. １ -C ６ Alkyl, C ３ -C ６ cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 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 H and C １ -C ６ 2. The LDC compound of claim 1, wherein each of the groups is independently selected from alkyl.

5. Z is one or more amino acids, one or more N-substituted amino acids, an optionally substituted polyether, C １ -C １２ alkylene, arylene having 6 to 10 ring atoms, C ３ -C ８ Cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C ２ -C １０ alkenylene, and any combination thereof; said alkylene and said alkenylene may be interrupted by one or more heteroatoms or one or more 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, the arylene, the cycloalkylene, the heterocycloalkylene, the heteroarylene, and the alkenylene may be substituted with one or more substituents selected from the following: halogen, oxo, -OH, -NO ２ , -C.N., C. １ -C ６ Alkyl, C ３ -C ６ cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 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 H and C １ -C ６ 2. The LDC compound of claim 1, wherein each of the groups is independently selected from alkyl.

6. 2. The LDC compound of claim 1, wherein K is polysarcosine.

7. A pharmaceutical composition comprising the LDC compound of claim 1 and a pharma- ceutically acceptable carrier.

8. 2. The LDC compound of claim 1 for use as a medicament.

9. Intermediate compound of formula (II) 【Chemistry 4】 During the ceremony, X1' is a group capable of reacting with a ligand to form a connector unit; Z is an optional spacer; X2 is a connector unit; K is any hydrophobic masking material selected from polysarcosine and polyethylene glycol; R1' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 selected from the group consisting of drugs; Each R2 is an electron withdrawing group and a C １ -C ４ independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleaving unit or a polypeptide cleaving unit; Y' is O when T is a sugar cleavable unit, and is NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 H and C １ -C ６ independently selected from alkyl; and pharma-ceutically acceptable salts thereof.

10. 10. The intermediate compound of formula (II) according to claim 9, wherein K is polysarcosine.

11. Intermediate compound of formula (III) 【Chemistry 5】 During the ceremony, R1' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 selected from the group consisting of drugs; Each R2 is an electron withdrawing group and a C １ -C ４ independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; T is a sugar cleaving unit or a polypeptide cleaving unit; Y' is O when T is a sugar cleavable unit, and NR3' when T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 H and C １ -C ６ independently selected from alkyl; and pharma-ceutically acceptable salts thereof.

12. Intermediate compound of formula (IV) 【Chemistry 6】 During the ceremony, R1' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 an electron withdrawing group and a C １ -C ４ alkyl, said electron withdrawing groups being selected from the group consisting of halogen, haloalkyl, -CN, -SOH, -NO, and -C(O)R groups, where R = H, OH, or alkoxy; n is 0, 1 or 2; R4 is H; R5 is H; T is a sugar cleavable unit; Y' is O; and pharma-ceutically acceptable salts thereof.

13. Intermediate compound of formula (IV) 【Chemistry 7】 During the ceremony, R1' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 an electron withdrawing group and a C １ -C ４ independently selected from the group consisting of alkyl; n is 0, 1 or 2; R4 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; R5 is H, C １ -C ６ Alkyl and C ２ -C ６ alkenyl, wherein said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more chemical groups selected from -O-, -S-, -C(O)-, and -NR''-; Y' is NR3'; T is a polypeptide cleavable unit; R3' is an amino protecting group, H, C １ -C ２４ Alkyl, C ２ -C ６ alkenyl; optionally substituted polyether; aryl having 6 to 10 ring atoms; C ３ -C ８ selected from the group consisting of cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof; said alkyl and said alkenyl may be interrupted by one or more heteroatoms or one or more 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 H and C １ -C ６ independently selected from alkyl; and pharma-ceutically acceptable salts thereof.

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