Drug-linker reagents for conjugation

Abstract

The present invention provides novel drug-linker reagent compounds of formula (I) designed to react with a suitable functional group of a carrier for the preparation of conjugates. Synthetic process for the preparation of such drug-linker reagents is also described.

Description

[0001] DRUG-LINKER REAGENTS FOR CONJUGATION

[0002] FIELD OF THE INVENTION

[0003] The present invention provides novel drug-linker reagents of formula (I) that, reacting with a carrier moiety, such as aminoacids, proteins, peptides, antibodies, aptamers, polymers or nanoparticles, are suitable for the preparation of conjugates (drug-linker-carrier).

[0004] Synthetic process for the preparation of such drug-linker reagents is also object of the present invention.

[0005] BACKGROUND OF THE INVENTION

[0006] Conjugates are attractive selective chemo-therapeutic molecules, as they combine ideal properties of selectivity to a target cell and cytotoxic drugs. By directing potent cytotoxic drugs to a target cell, the desired therapeutic effect may be enhanced in the target cell while minimizing the effect on non-targeted cells.

[0007] Conjugation of drugs with carriers, through suitable linkers, is also an approach largely used to improve characteristics such as solubility, permeability into the cell, in vivo therapeutic window and ability to reach the target according to the nature of the specific molecule conjugated with the drug.

[0008] Drug-linker-carrier conjugates are stable in the bloodstream but, under the effect of specific intracellular stimuli or conditions, efficiently release the cytotoxic payloads. Therefore, they are useful for the treatment and / or prevention of proliferative diseases and cancer.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows the chemical structure of the Conjugate A1 obtained by reaction of Comp. 1 with cysteine as carrier.

[0011] Figure 2 shows the HPLC chromatogram of the Conjugate A1 @ 254 nm and its m / z value.

[0012] Figure 3 shows the HPLC chromatogram of the Conjugate A1 after 16 hrs treatment with lysosome extract.

[0013] Figure 4 shows representative antiproliferation dose-response curves of Conjugate A1 and of Conjugate A1 previously treated with lysosome extract in SW48 cell line (144 hours treatment).

[0014] DEFINITIONS

[0015] Carrier or carrier moiety” refer to polyclonal and monoclonal antibodies, proteins or peptides of natural or synthetic origin, aptamers or nanoparticles. In some embodiments the carrier is an antibody having binding affinity for the target expressing an antigen. Carrier moieties are suitable for conjugation with drug-linker reagents of formula (I). Carrier moieties may be derived from polyclonal antibodies raised against tumor associated antigens; or from monoclonal antibodies binding to antigens preferentially or selectively expressed on tumor cell populations; or from natural or recombinant peptides or proteins or growth factors preferentially or selectively binding to tumor cells; or from natural or synthetic polymeric carriers such as polylysine, polyglutamic acid, polyaspartic acid and their analogues and derivatives, or such as dextran or other polymeric carbohydrate analogues and their derivatives; or from synthetic copolymers such as those derived from N-(2-hydroxypropyl)methacrylamide (HPMA) see: J. Kopecek, Macromolecules. H. Benoit & P. Rempp, Ed.: 505-520 (1982) Pergamon Press. Oxford, England; or from poly(aminoacid) copolymers such as poly(Glu Na, Ala, Tyr) which are useful as targetable drug-carriers for lung tissue R. Duncan et al., Journal of Bioactive and Compatible Polymers, Vol 4, July 1989.

[0016] The term “payload / s”, as used herein, refers to any chemical structure deriving from chemical or biochemical degradation of the conjugate and comprising a benzamidopyridine derivative affecting the cellular activity. In one particular embodiment preferred payloads are compound of formula (la).

[0017] The term “aminoacid” includes those groups found in: (i) naturally occurring aminoacids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (ii) minor aminoacids such as ornithine and citrulline; (iii) unnatural D-aminoacids, beta-aminoacids, synthetic analogs and derivatives of naturally occurring aminoacids; and (iv) all enantiomers, diastereomers, isomerically enriched, isotopically labelled, protected forms, and racemic mixtures thereof.

[0018] With the term “linear or branched C1-C8 alkyl”, hence comprehensive of C1 -C6 alkyl and C1-C4 alkyl, we intend any of the groups such as, for instance, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, sec-butyl, tertbutyl.

[0019] With the term "linear or branched C2-C8 alkenyl", we intend any of the groups such as, for instance, vinyl, allyl, 1- propenyl, isopropenyl, 1 -butenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 1 -hexenyl, and the like.

[0020] With the terms cyclic C3-C8 alkyl, hence comprehensive of cyclic C3-C6 alkyl, we refer to 3- to 8-membered allcarbon monocyclic ring. Examples of cyclic C3-C8 alkyl, without limitation, are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.

[0021] With the term C3-C8 cycloalkenyl, we refer to 3- to 8-membered all-carbon monocyclic ring which contain one or more double bonds but does not have a completely conjugated ir-electron system. Examples of C3-C8 cycloalkenyl, without limitation, are cyclopentenyl, cyclohexenyl, 1 ,3-cyclohexadienyl, cycloheptenyl, cyclooctenyl, 1 ,3- cycloheptadienyl.

[0022] With the term “linear or branched C1-C4 hydroxyalkyl”, we intend any of the groups such as, for instance, 2- hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, 4-hydroxybutyl, 3-hydroxybutyl, 2-hydroxybutyl.

[0023] With the term “halogen”, we intend a fluorine, chlorine, bromine or iodine.

[0024] With the term “straight or branched C1 -C6 haloalkyl”, we intend any of the above defined C1 -C6 alkyl groups which are substituted by one or more than one halogen atom. Examples of straight or branched C1 -C6 haloalkyl, without limitation, are for instance, fluoromethyl, chloromethyl, bromomethyl, difluoromethyl, dichloromethy, dibromomethy, 1- fluoroethyl, 1 -chloroethyl, 1 -bromoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, trifluoroethyl, trichloroethyl, tribromoethyl, trifluoropropyl, 2.difluoropropyl, trifluorobutyl; also included are C1 -C6 alkyl groups wherein all hydrogen atoms are replaced byhalogen atoms, in particular fluoro atoms, for instance, trifluoromethyl, CF3CF2-, CF3CF2CF2-, CF3CF2CF2 CF2- and like. The term “leaving group” refers to a group that can be substituted by another group in a substitution reaction. Such leaving groups are well-known in the art and examples include, but are not limited to, halides (fluoride, chloride, bromide and iodide), azides, sulfonates (e.g., an optionally substituted CrCe alkanesulfonate, such as methanesulfonate and trifluoromethanesulfonate, or an optionally substituted C7-C12 alkylbenzenesulfonate, such as p-toluenesulfonate), succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, carboxylates, aminocarboxylates (carbamates) and alkoxycarboxylates (carbonates). For substitutions at saturated carbon, halides and sulfonates are preferred leaving groups. For substitutions at a carbonyl carbon a halide, succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, a carboxylate, or an alkoxycarboxylate (carbonate) may for example be used as a leaving group. The term "leaving group" also refers to a group that is eliminated as a consequence of an elimination reaction, e.g., an electronic cascade reaction or a spirocyclization reaction. In this instance, a halide, a sulfonate, an azide, an aminocarboxylate (carbamate) or an alkoxycarboxylate (carbonate) may for example be used as a leaving group.

[0025] Therefore, the term “protecting group” refers to a group used to protect such reactive centers in a chemical synthesis, for example, a hydroxyl group (— OH) , an amino group (-NH), a thiol group (-SH), a carbonyl group (-C=O) , a carboxylic group (-C00H). Examples of protecting groups are those reported in the literature (see, for instance, ibidem).

[0026] The term “nitrogen protecting group” refers to a group that with the nitrogen atom form carbamates, amides, cyclic imides, A / -alkyl and / V-aryl amines. Such protecting groups are well-known in the art (see e.g. ibidem). Non limiting examples of carbamate protecting groups are, for instance, methyl and ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 2,2,2-trichloroethylcarbamate (Troc), t-butyl carbamate (BOC), vinyl carbamate (Voc), allyl carbamate (Alloc), benzyl carbamate (Cbz), p-nitrobenzyl and the like. Non limiting examples of amides are, for instance N- trichloroacetamide, A / -trifluoroacetamide and the like. Non limiting examples of cyclic imide protecting groups are, for instance, A / -phthalimide, A / -dithiasuccinoylimide (Dts) and the like. Non limiting examples of A / -alkyl and / V-aryl protecting groups are, for instance, / V-allylamine, N-benzylamine and the like.

[0027] The term “hydroxyl protecting group” refers to a group that with the oxygen atom form ethers, esters, cyclic acetals or ketals. Such protecting groups are well-known in the art (see e.g. ibidem). Non limiting examples of ethers protecting groups are, for instance, alkyl ethers and benzyl ethers, such as methoxymethyl ether (M0M-0R), tetrahydropyranyl ether (THP-OR), allyl ether (Allyl-OR), benzyl ether (Bn-OR), triphenylmethyl ether (Tr-OR) and the like, or silyl ethers, such as trimethylsilyl ether (TMS-OR), t-butyldimethylsilyl ether (TBS-OR or TBDMS-OR), t-butyldiphenylsilyl ether (TBDPS-OR) diphenylmethylsilyl ether (DPMS-OR) and the like. Non limiting examples of esters protecting groups are, for instance, trifluoroacetate, benzoate (Bz-OR) and carbonates, such as ethylcarbonate and the like. Non limiting examples of cyclic acetals or ketals protecting groups are, for instance, methylene acetal, ethylidene acetal, methoxymethylene acetal and the like.

[0028] The term "active ester" refers to a functional group in which the alkoxy group of the ester moiety is a good leaving group. Examples of such alkoxy groups include, but are not limited to, succinimide-A / -oxide (NHS esters), p- nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, 1 -hydroxybenzotriazole and 1 -hydroxy-7- azabenzotriazole, and groups with comparable leaving capability. Unsubstituted alkyl-based alkoxy groups such as methoxy, ethoxy, isopropoxy, and t-butoxy do not qualify as good leaving groups and methyl, ethyl, isopropyl, and t- butyl esters are therefore not considered to be active esters.

[0029] The term “electron withdrawing group” refers to an atom group that draws electron density from neighboring atoms towards itself, usually by resonance or inductive effects. Non limiting examples are halogens, trifluoromethyl group, nitro (NO2) group and nitrile (CN).

[0030] With the term “reactive moiety”, we refer to a group that is able to react with a counterpart, namely the carrier moiety, to generate a stable covalent bound.

[0031] With the term “salts” or “pharmaceutically acceptable salts” we refer to the salts of the compounds of formula (I) with inorganic or organic acids, e.g. nitric, hydrochloric, hydrobromic, hydroiodic, sulfuric, perchloric, phosphoric, formic, acetic, trifluoroacetic, propionic, glycolic, lactic, oxalic, fumaric, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulphonic, p-toluensulfonic, isethionic and salicylic acid. Pharmaceutically acceptable salts of the compounds of formula (I) also include the salts with inorganic or organic bases, e.g. alkali or alkaline-earth metals, especially sodium, potassium, calcium, ammonium or magnesium hydroxides, carbonates or bicarbonates, acyclic or cyclic amines.

[0032] SUMMARY OF THE INVENTION

[0033] The present invention provides novel drug-linker reagent compounds of formula (I) designed to react with a suitable functional group of a carrier for the preparation of conjugates.

[0034] Accordingly, the first object of the present invention is to provide drug-linker reagent compound of formula (I): wherein:

[0035] Z is N or CH;

[0036] Y is selected from the group consisting of -SO2CH3, -SO2CF3, -NO2, -CF3and -CN;

[0037] X is selected from the group consisting of hydrogen, halogen, -CN, linear or branched C1-C8 alkyl and cyclic C3-C8 alkyl;

[0038] R1 is selected from the group consisting of linear or branched C1-C8 alkyl, cyclic C3-C8 alkyl, -(CH2CH2O)nCH3, R3R4N-C1 -C8 alkyl and R3O-C1 -C8 alkyl; wherein:

[0039] R3 and R4 are, each independently, selected from the group consisting of hydrogen, linear or branched C1- C8 alkyl and cyclic C3-C8 alkyl; n is an integer selected from 1 to 24; R2 is selected from the group consisting of linear or branched C1-C8 alkyl, cyclic C3-C8 alkyl, linear or branched C2-

[0040] C8 alkenyl, C3-C8 cycloalkenyl and linear or branched C1 -C6 haloalkyl and

[0041] L is a linker of formula (II):

[0042] W-P-G-RM (II) wherein:

[0043] W is a self immolative system selected from group (Illa), (I lib), (I I Ic) or (Hid):

[0044] (I lla) (l llb) (l llc) (H id) wherein:

[0045] R5 and R6 are, each independently, hydrogen, halogen, linear or branched C1-C4 alkyl, linear or branched C1 -C4 hydroxyalkyl, -O(CH2-CH2O)mCH3or -(CH2-CH2O)mCH3; m is an integer selected from 1 to 24;

[0046] Su is a group selected from (IVa)-(IVc):

[0047] * is the attachment point of the self immolative system W to the adjacent group P, G or RM;

[0048] P is independently null or a dipeptidic, tripeptidic or tetrapeptidic moiety, consisting of any combination of natural L-aminoacids and unnatural D-aminoacids wherein, the C-terminal aminoacid residue is linked to W moiety and the N-terminal aminoacid residue is linked to G moiety or to the RM moiety, when G is null;

[0049] G is independently null or selected from the group consisting of (Va)-(Vg):

[0050] wherein: y is an integer selected from 0 to 24 k is an integer from 0 to 8 and

[0051] * is the attachment point to the RM group;

[0052] RM is a reactive moiety selected from the group consisting of (Vla)-(Vlh): wherein:

[0053] R7 and R8 are, each independently, hydrogen, linear or branched C1 -C4 alkyl, straight or branched C1-C4 hydroxyalkyl or, R7 and R8 taken together form a cyclic C3-C6 alkyl;

[0054] R9 is hydrogen or an electron-withdrawing group selected from -NO2 and -CN; r is an integer selected from 0 to 7;

[0055] LG is an halogen; j is an integer from 1 to 8 or a salt thereof with a counterion [A’]. In the compound of formula (I) the self-immolative system W tethers in a stable way the pyridinium salt moiety to the P-G-RM residue, or to the G-RM residue when P is null or to the P-RM residue when G is null or to the RM residue when both P and G are null.

[0056] In the drug-linker reagent of formula (I) the drug moiety is a benzamidopyridine derivative of formula (la): wherein:

[0057] Y, X, Z, R1 and R2 are defined above.

[0058] Preferred compounds of formula (I) are the compounds wherein:

[0059] Y is selected from the group consisting of -SO2CF3 and -NO2;

[0060] X is selected from the group consisting of hydrogen, fluorine, -ON and linear or branched C1 -C4 alkyl;

[0061] R1 is selected from the group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, -(CH2CH2O)nCH3 and R3O-C1 -C8 alkyl, wherein:

[0062] R3 is selected from hydrogen and linear or branched C1 -C4 alkyl; n is an integer selected from 1 to 8;

[0063] R2 is selected from the group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, C3-C8 cycloalkenyl and linear or branched C1-C6 haloalkyl;

[0064] L is a linker of formula (II): wherein:

[0065] W is a self immolative system selected from group (llla)-(llld), wherein:

[0066] R5 and R6 are, each independently, hydrogen, linear C1 -C4 hydroxyalkyl, -O(CH2-CH2O)mCH3 or - (CH2-CH2O)mCH3; m is an integer selected from 1 to 8;

[0067] Su is group selected from (IVa)-(IVc)

[0068] * is attachment point of the self immolative system W to the adjacent group P, G or RM;

[0069] P is independently null or a dipeptidic, tripeptidic or tetrapeptidic moiety, consisting of any combination of natural or unnatural aminoacids selecting from glycine, alanine, leucine, valine, citrulline, phenylalanine, glutamic acid and aspartic acid wherein, the C-terminal aminoacid residue is linked to W moiety and the N- terminal aminoacid residue is linked to G moiety or to the RM moiety, when G is null; RM is a reactive moiety selected from the group consisting of (Vla)-(Vlh), wherein:

[0070] R7 and R8 are, each independently, hydrogen or linear C1 -C4 alkyl;

[0071] R9 is hydrogen or -NO2; r is an integer selected from 0 to 7;

[0072] LG is bromine, j is an integer from 1 to 8; and

[0073] G and Z are as defined above; or a salt thereof with a counterion [A’].

[0074] More preferred compounds of formula (I) are the compounds wherein:

[0075] Z is N;

[0076] R1 is selected from a group consisting of linear or branched C1 -C4 alkyl, -(CH2CH2O)nCH3 and R3O-C1 -C6 alkyl, R2 is selected from the group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, C3-C8 cycloalkenyl, - CF3 and -CF2CF2CF3;

[0077] L is a linker of formula (II), wherein:

[0078] W is a self immolative system of formula (Illa);

[0079] P is independently null or a dipeptidic, tripeptidic or tetrapeptidic moiety, selecting from the combinations consisting of: valine-alanine, valine-citrulline, glutamic acid-valine-alanine, glutamic acid-valine-citrulline, glycine-glycine-phenyl alanine-glycine, phenyl alanine-leucine-glycine, aspartic acid-valine-glycine and aspartic acid-valine-citrulline wherein, the C-terminal aminoacid residue is linked to W and the N-terminal aminoacid residue is linked to G moity or to the RM moiety when G is null;

[0080] G is independently null or a group of formula (Va) or (Vg);

[0081] RM is a reactive moiety of formula (Via) or (Vlh); and

[0082] X and Y are as defined above; or salt thereof with a counterion [A-].

[0083] Preferred specific compounds of formula (I), or a pharmaceutically acceptable salt thereof, are the compounds listed below:

[0084] N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 1);

[0085] N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]-5-ureido- pentanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 2);

[0086] (4S)-5-[[(1S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl]amino] pyridin- 1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-[6-(2,5-dioxopyrrol-1 -yl) hexanoylamino]-5-oxo-pentanoic acid (comp. 3); N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy] ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 4);

[0087] N-[5-cyclopentyl-1-[[4-[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]acetyl]amino]acetyl]amino]-3-phenyl- propanoyl]amino]acetyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro- benzamide (comp. 5);

[0088] N-[1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl] methyl]-3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-1-ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5- nitro-benzamide (comp. 6) N-[1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl] amino]phenyl]methyl]-3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-1-ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5- yl]sulfanyl-5-nitro-benzamide (comp. 7);

[0089] (4S)-4-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-5-[[(1S)-1 -[[(1 S)-2-[4-[[3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)-2- [[2-[1 -(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]amino]-5-oxo-pentanoic acid (comp. 8);

[0090] N-[1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy] propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-5-(1 ,1 , 2, 2, 3,3,3- heptaf luorop ropy l)py ridi n- 1 -ium-2-yl]-2-[1 -(2-hyd roxyethy l)tetrazol -5-yl]su Ifany I -5-nitro-benzamide (comp. 9); N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl) benzamide (comp. 10);

[0091] (4S)-5-[[(1S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl) benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-[6-(2,5- dioxopyrrol-1 -yl)hexanoylamino]-5-oxo-pentanoic acid (comp. 11);

[0092] N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl)benzamide (comp. 12);

[0093] N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 13); N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 14);

[0094] N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-[1 -(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzamide (comp. 15); N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-[1 - (2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzamide (comp. 16);

[0095] N-[3-cyano-5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoyl]amino]phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 17); N-[3-cyano-5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy] ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 18);

[0096] (2S)-N'-[(1 S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1-methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl]amino]pyridin- 1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl) propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy ]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]pentanediamide (comp. 19).

[0097] In one preferred embodiment the counter ion [A’] of the pyridinium compound of formula (I) is selected from Cl’, Br, I’, CH3CO2’ (acetate), CF3CO2(trifluoroacetate), HCO2- (formate), CH3PhSO2- (tosylate) and CH3SO3(mesylate).

[0098] In one more preferred embodiment the counter ion [A’] is Cl’, Br, HCO2’.

[0099] For sake of clarity, since a compound of formula (I) comprises the pyridinium cation and other chemical residues that can be further salified, di o poly pharmaceutically acceptable salts of compound of formula (I) are also object of this invention, as well as tautomers, hydrates, solvates and N-oxides thereof.

[0100] Accordingly to a second object of the present invention is to provide a process for the preparation of a compound of formula (I) as defined above, by using the reaction routes and synthetic schemes described below, employing the techniques available in the art and starting materials readily available. The preparation of certain embodiments of the present invention is described in the examples that follow, but those of ordinary skill in the art will recognize that the preparations described may be readily adapted to prepare other embodiments of the present invention. For example, the synthesis of non-exemplified compounds according to the invention may be performed by modifications apparent to those skilled in the art, for instance, by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. Alternatively, other reactions referred to herein or known in the art will be recognized as having adaptability for preparing other compounds of the invention.

[0101] The compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. Unless otherwise indicated, the starting materials are known compounds or may be prepared from known compounds according to well-known procedures. It will be appreciated that, where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures) are described, different process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary depending upon the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Compounds of formula (I) can be prepared as reported in the following Scheme 1 :

[0102] Scheme 1

[0103] Accordingly, a process of the present invention comprises the following steps:

[0104] Step 1) reacting the compound of formula (VII): wherein X is selected from a group consisting of hydrogen, halogen, -CN, linear or branched C1-C8 alkyl and cyclic C3-C8 alkyl; R2 is selected from a group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, linear or branched C2-C8 alkenyl, C3-C8 cycloalkenyl and linear or branched C1 -C6 haloalkyl; with a compound of formula (XI):

[0105] LG1 -W-P-G-PG (XI) wherein LG 1 is a leaving group preferably selected from mesylate, tosylate and halogen; W is a self immolative system selected from (llla)-(llld), wherein R5 and R6 are, each independently, hydrogen, halogen, linear or branched C1-C4 alkyl, linear or branched C1 -C4 hydroxyalkyl, -O(CH2-CH2O)mCH3 or -(CH2-CH2O)mCH3; m is an integer selected from 1 to 24, Su is group selected from (IVa)-(IVc); P is independently null or a dipeptidic, tripeptidic ortetrapeptidic moiety, consisting of any combination of natural L-aminoacids and unnatural D-aminoacids; G-PG is independently null or selected from the group consisting of (Xlla)-(Xllg) :

[0106] wherein y is an integer selected from 0 to 24; k is an integer from 0 to 8 and PG is an amino protecting group preferably selected from methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 2,2,2-trichloroethylcarbamate (Troc), t-butyl carbamate (BOC), vinyl carbamate (Voc), allyl carbamate (Alloc) and benzyl carbamate (Cbz), thus obtaining a compound of formula (VIII): wherein X, R2, W, P, G and PG are as defined above.

[0107] Step 2) reacting the compound of formula (VIII) with the acid derivative of formula (XIII): wherein Z is N or CH; Y is selected from a group consisting of -SO2CH3, -SO2CF3, -NO2, -CF3 and -CN; R1 is selected from a group consisting of linear or branched C1-C8 alkyl, cyclic C3-C8 alkyl, -(CH2CH2O)nCH3, R3R4N-C1 -C8 alkyl and R3O-C1-C8 alkyl, wherein R3 and R4 are, each independently, selected from the group consisting of hydrogen, linear or branched C1 -C8 alkyl and cyclic C3-C8 alkyl and n is an integer selected from 1 to 24, in the presence of suitable coupling agents or after activation of acid of formula (XIII) in the corresponding acid halides, thus obtaining a compound of formula (IX): wherein X, Y, Z, R1 , R2, W, P, G and PG are as defined above.

[0108] Step 3) removing from the resulting compound of formula (IX) the PG protecting group, so obtaining a compound of formula (X): wherein X, Y, Z, R1 , R2, W, P and G are as defined above.

[0109] Step 4) reacting the compound of formula (X) with the acid compound of formula (XIV):

[0110] HO— RM

[0111] (XIV) wherein RM is a reactive moiety selected from the group consisting of (Vla)-(Vlh): wherein R7 and R8 are, each independently, hydrogen, linear or branched C1 -C4 alkyl, straight or branched C1-C4 hydroxyalkyl or, R7 and R8 taken together form a cyclic C3-C6 alkyl; R9 is hydrogen or an electron-withdrawing group selected from -NOsor -ON; r is an integer selected from 0 to 7, LG is a halogen and j is an integer from 1 to 8, in the presence of suitable coupling agents or after activation of acid (XIV) in the corresponding acid halides, so obtaining a compound of formula (I) wherein X, Y, Z, R1 , R2, W, P,G and RM are as defined above.

[0112] According to Step 1 , the reaction of a compound of formula (VII) with a compound of formula (XI) can be accomplished in a variety of ways. This reaction may be carried out in a suitable organic solvent, preferably AON, DCM, acetone or TH F, at a temperature ranging from room temperature to 80 C° and for a time varying from about 30 minutes to about 48 hours.

[0113] According to Step 2 the coupling reaction of a compound of formula (VIII) with a compound of formula (XIII) can be accomplished in a variety of ways and experimental conditions, which are widely known in the art for the preparation of amide. This reaction may be carried out in a suitable organic solvent, preferably pyridine, DCM, THF or DMF, in presence of coupling agent preferably POCI3, HATU or TBTU at a temperature ranging from 0 °C to 80 °C and for a time varying from about 30 minutes to about 24 hours.

[0114] According to Step 3 the removal of the protecting group from a compound of formula (IX) can be accomplished in a variety of ways and experimental conditions, depending on the nature of protecting group, which are widely known in the art (see e.g. Protective Groups in Organic Synthesis; Theodora W. Greeen, Peter G. M. Wuts 4th edition). Preferably, when the protecting group is a BOC protecting group, the reaction is performed under acidic conditions, such as for instance TFA, HCI and the like, in a solvent such as DCM, 1 ,4-dioxane.

[0115] According to Step 4 the coupling reaction of a compound of formula (X) with a compound of formula (XIV) can be accomplished in a variety of ways and experimental conditions, which are widely known in the art for the preparation of amide. This reaction may be carried out in a suitable organic solvent, preferably pyridine, DCM, THF, ACN or DMF, in presence of a suitable coupling agent preferably DCC, HATU, EDCI or TBTU optionally in presence of a suitable additive like HOBT or N-Hydroxysuccinimide, at a temperature ranging from 0° C to 80 °C and for a time varying from about 30 minutes to about 24 hours.

[0116] Alternatively, the preparation of compounds (I) can be also carried out starting from compound of formula (VII) by introducing the W-P-G-PG residue step by step, by using the appropriate protecting groups (as shown for example in the experimental section for compound 19). For example, intermediate (VII) can be bond first to the W-P group and then to the G-PG group or, alternatively, intermediate (VII) can be bond first to W group and then to the P-G-PG group.

[0117] From all of the above, it is clear to the skilled person that the conversion of a compound of formula (I) into a pharmaceutically acceptable salt thereof or, alternatively, the conversion from a salt of a compound of formula (I) into a compound (I), according to procedures well-known in the art, is still within the scope of the invention. When preparing the compounds of formula (I) according to any variant of the process, which are all to be intended as within the scope of the invention, optional functional groups within the starting materials, the reagents or the intermediates thereof, and which could give rise to unwanted side reactions, need to be properly protected according to conventional techniques. Protection of such reactive centers, and subsequent deprotection at the end of the synthetic transformations, can be accomplished following standard procedures described, for instance, in: Protective Groups in Organic Synthesis; Theodora W. Greeen, Peter G. M. Wuts 4th edition.

[0118] According to any variant of the process for preparing the compounds of the formula (I), the starting materials and any other reactants are known or easily prepared according to known methods.

[0119] The compounds of the formula (XI), formula (XIII) and formula (XIV) are either commercially available or can be prepared with known methods. The final compounds may be isolated and purified using conventional procedures, for example chromatography and / or crystallization and salt formation.

[0120] The synthesis of a compound of general formula (I), according to the synthetic processes described above, can be conducted in a stepwise manner, whereby each intermediate is isolated and purified if needed by standard purification techniques, like, for example, column chromatography, before carrying out the subsequent reaction. Alternatively, two or more steps of the synthetic sequence can be carried out in a so-called “one-pot” procedure, as known in the art, whereby only the compound resultant from the two or more steps is isolated and purified.

[0121] In cases where a compound of general formula (I) contains one or more asymmetric centers, said compound can be separated into the single stereoisomers by procedures known to those skilled in the art. Such procedures comprise standard chromatographic techniques, including chromatography using a chiral stationary phase, or crystallization. General methods for separation of compounds containing one or more asymmetric centers are reported, for instance, in Jacques, Jean; Collet, Andre; Wilen, Samuel H., Enantiomers, Racemates, and Resolutions, John Wiley & Sons Inc., New York (NY), 1981.

[0122] In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.

[0123] In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form. Another object of the invention is the use of the compound of formula (I) for the preparation of conjugates.

[0124] In the preparation of the conjugate the reactive moiety (RM) of the compound (I) reacts with a suitable functional group of the carrier, so to obtain a new covalent bond that tether in a stable way the compound of formula (I) to the carrier. In one preferred embodiment the functional group of the carrier is thiol (-SH) or amino (-NH2) group. Therefore, the new covalent bond can be a thioether bond, a disulfide bond or an amide bond as depicted in the scheme 2 below. Scheme 2

[0125]

[0126] Upon administration in a subject, release of the payload from the conjugate in a target cell, by appropriate chemical or biochemical stimuli, is believed to result from braking of the covalent bond RM-carrier and / or one or more of the bound / s of the linker L, as depicted below:

[0127] The released payload is endowed with inhibitory activity on cell viability, thus useful in the therapy or treatment of cellular disorders, especially in the therapy or treatment of cancer.

[0128] In one preferred embodiment the released payload is a compound of formula (la).

[0129] EXPERIMENTAL PART

[0130] The short forms and abbreviations used herein have the following meaning: g gram mg milligram mL milliliter jxL microliter mM millimolar mmol millimole jxM (micromolar) MHz (Mega-Hertz) h hour(s) Hz (Hertz) mm (millimetres) min (minutes) pm (micron) M (molar)

[0131] BSA bovine serum albumine DTT dithiothreitol

[0132] NADPH Nicotinamide adenine dinucleotide phosphate Rt retention time 2-HG 2-Hydroxy glutaric acid KOtBu (potassium tert-butoxide) rt (room temperature) TEA (triethylamine)

[0133] DMAP (4-dimethylaminopy ridine) DME (1 ,2-dimethoxyethane)

[0134] TFA (trifluoroacetic acid) NasSC (sodium sulphate) AcOH (acetic acid) ESI (electrospray ionization)

[0135] Na2CO3(sodium carbonate) K2CO3 (potassium carbonate)

[0136] Cs2CO3(caesium carbonate) K3PO4 (potassium phosphate)

[0137] LiOH (lithium hydroxide) NaOH (sodium hydroxide)

[0138] KOH (potassium hydroxide) p-TsOH (p-toluensulfonic acid)

[0139] EtOAc (ethyl acetate) LiHMDS (lithium bis(trimethylsilyl)amide)

[0140] NMP (N-methyl-2-pyrrolidone) NaH (sodium hydride)

[0141] DMA (N,N-dimethylacetamide) KH (potassium hydride) DMF (N,N-dimethylformamide) DCM (dichloromethane)

[0142] DIPEA (N,N-diisopropyl-N-ethylamine) hex (hexane)

[0143] THF (tetrahydrofuran) DMSO (dimethylsulfoxide)

[0144] MeOH (methanol) ACN (acetonitrile)

[0145] EtOH (ethanol) Bn (benzyl)

[0146] -OMs (mesylate) -OTs (tosylate)

[0147] HOBT (N-hydroxy-benzotriazole) DCC (1 ,3-dicyclohexylcarbodi i m ide)

[0148] NMR Nuclear magnetic resonance MS Mass spectroscopy m / z mass to charge ratio LC Liquid chromatography

[0149] MgCls Magnesium chloride Tris (tris-hydroxymethil-aminomethane) NBS (N-Bromosuccinimide) HCI Hydrochloric acid

[0150] Cys Cysteine TLC (Thin-layer chromatography)

[0151] Rf (retention factor) PPh3(triphenylphosphine)

[0152] POCI3 (Phosphorus oxychloride) FA (formic acid)

[0153] EEDQ (2-Ethoxy-1 -ethoxycarbonyl-1 ,2-dihydroqui noline) nm (nanometers)

[0154] EDCI (1 -ethyl -3-(3-dimethy lami nopropyl)carbodiimide hydrochloride) TBTU (N,N,N’,N’-tetramethyl-O-(benzotriazol-1 -yl)uronium-tetrafluoroborate)

[0155] RP-HPLC (reverse phase high performance liquid chromatography) HATU (Hexafluorophosphate azabenzotriazole tetramethyl uranium) EDTA (Ethylenediaminetetraacetic acid)

[0156] T4P (1 ,3,5,2,4,6-Trioxatriphosphorinane, 2,4,6-tributyl-, 2,4,6-trioxide)

[0157] PREPARATION OF A CONJUGATE

[0158] As an example, that is not intended to limit the scope of the invention, of the preparation of a conjugate between the drug-linker reagent of formula (I), i.e. comp. 1 with a carrier, i.e cysteine, is shown in Figure 1.

[0159] 8 jxL of 1.5 mM solution of the comp 1 in DMSO were incubated with 2 jxL Cys 100 mM and 1 jxL of 1 M Tris-HCI pH 7.4 in a total volume of 25 jxL aqueous solution, for 1 hr at 25°C. The obtained conjugate A1 (m / z = 1033.38 (M+H)+; Figure 2); then was analyzed by HPLC ESI-MS using a reversed phase HPLC method (PLRP-S column 1000A 8uM 150 X 2.1 mm) on a 1100 Agilent HPLC instrument coupled with an Agilent 1946 single quadrupole mass spectrometry detector with an orthogonal ESI source.

[0160] RELEASE OF THE PAYLOAD FROM A CONJUGATE

[0161] As an example, that is not intended to limit the scope of the invention, the release of the payload (comp. 1a) from the conjugate A1 was performed in presence of lysosome extract as reported below.

[0162] Two aliquots of 10 jxL of the conjugate were incubated with 40 jxL of 200 mM sodium acetate buffer pH 5.5 and 1 mM EDTA. Then one aliquot was treated with 1 jxL 0.8 mg / mL lysosome extract, while the other was used as reference. The reaction was incubated for 16 hours at 37 °C with gentle shaking, then the samples were analyzed by HPLC-MS with UV detection at 254 nm and MS detection in the 400-3000 m / z range performed with ESI-ToF MS (Agilent).

[0163] In these experimental conditions, about 57% of comp. 1a (m / z = 444.13) was released from the conjugate A1 (m / z = 1033.38) by the degradating action of lysosome extract (Figure 3).

[0164] BIOLOGICAL ASSAY

[0165] The antiproliferative activity of Conjugate A1 and of Conjugate A1 previously treated with lysosome extract, as described above, was tested in human cancer cell lines including SW48, HCT-116 and KM12 colorectal cancer cells. Exponentially growing cells were seeded in 384-well (Microclear black, Greiner, Frickenhausen, Germany) at a final density ranging from 3,000 to 30,000 cells per cm2in appropriate culture medium + 10% Fetal Calf Serum (FCS, Gibco, Thermo Fisher Scientific). After 24 hours, compound solutions were serially diluted in 100% DMSO and then added to the cell cultures to obtain final compound concentrations in the range 0.002 - 1 p M. The final DMSO concentration in test wells was adjusted to 0.1% (v / v); control wells were mock-treated and contained the same final concentration of DMSO as test wells. All treatments were performed in duplicate. Cell viability was determined after 144 hours by ATP determination in each test well with a luciferase-based detection system (CellTiterGlo, Promega, Madison, Wl, USA) following manufacturer’s instructions. The intensity of emitted light (RLU) was measured using an EnVision reader (PerkinEI me r, Waltham, MA USA) and expressed as percentage of untreated controls. Inhibitory activity was evaluated comparing treated versus control data using Assay Explorer software (Symix Technologies Inc.). For some cell lines, IC50 values were calculated by sigmoidal fitting interpolation using a quadriparametric Hill equation (Graphpad Prism). Table 1 shows the percentage of cell proliferation inhibition (compared with untreated controls) in SW48, HCT-115 and KM12 cell lines treated for 144 hours with 0.5 p M of Conjugate A1 or with 0.5 p M of Conjugate A1 previously treated with lysosome extract. The data indicate that the Conjugate A1 previously treated with lysosome extract, but not the Conjugate A1 itself, significantly inhibits the proliferation of all cell lines tested at 0.5 p M with percentages between 36.1% and 66.6%. Representative antiproliferation dose-response curves of SW48 cells treated for 144 hours with Conjugate A1 or with Conjugate A1 previously treated with lysosome extract are shown in Figure 4. The Conjugate A1 showed no cytotoxic activity up to 1 p M (IC50 > 1 p M) while, after treatment with lysosome extract, it inhibited the proliferation of SW48 cells with an IC50 of 0.303 ± 0.040 p M.

[0166] Table 1

[0167] PREPARATION OF COMPOUNDS OF FORMULA (I)

[0168] For a reference to any specific compound of formula (I) of the invention, optionally in the form of a pharmaceutically acceptable salt, see the experimental section and claims. Referring to the examples that follow, compounds of the present invention were synthesized using the methods described herein, or other methods, which are well known in the art.

[0169] With the aim at better illustrating the present invention, without posing any limitation to it, the following examples are given.

[0170] As used herein the symbols and conventions used in the processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry.

[0171] Compound names are IUPAC names, generated by using Biovia Draw 2021 (by Dassault Systemes).

[0172] Unless otherwise noted, all materials, including anhydrous solvent such as DMF, THF, DCM, were obtained from commercial suppliers, of the best grade and used without further purification. All reactions involving air- or moisturesensitive compounds were performed under nitrogen or argon atmosphere.

[0173] GENERAL PURIFICATION AND ANALYTICAL METHODS

[0174] Flash Chromatography was performed on silica gel (Merck grade 9395, 60A).

[0175] The HPLC equipment consisted of a SHIMADZU LC-20AD system and LCMS-2020 mass spectrometer equipped with an electrospray (ESI) ion source. HPLC was carried out at 50°C at a flow rate of 1.0 mL / min using Agilent PoroShell 120 EC-C18 (2.7jxm 3.0*50mm) column. Mobile phase was 0.037% TFA in a mixture of water and acetonitrile (19:1) (solvent A) and 0.018% TFA in acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 3 minutes and holding at 95% for 0.5 minutes at a flow rate of 1 .0 ml / min , return back to 5% acetonitrile (solvent B) in water and hold for 0.5 min.

[0176] HPLC and High-Resolution Mass Spectra (HRMS)

[0177] HPLC-MS / UV analyses and High-Resolution Mass Spectra (HRMS) were performed on a Waters Alliance LC 2795 equipped with a Waters PDA UV detector 2996 and a TOF Waters LCT Premier XE mass detector (ESI interface) supported by a Waters Reagent Manager liquid pump. The assay is based on generic gradient reversed-phase chromatography that allows complementing an identity-purity assay with determination and confirmation of the expected exact mass of the compounds in the same run. Compound identity is accomplished by on-line serial ESI(+) Full Scan MS detection, sample purity is obtained as relative "Area Percent" of the integrated LC / UV trace at 216-400 nm. The liquid chromatograph is equipped with a Waters XBridge CSH C18 column (3.0x30 mm, 3.5 gm particle size) thermostated at 50 °C. Alternatively a Supelco column Ascentis Express C18 (2.7x 30mm x 3um) was used.

[0178] Mobile phases A was 0.05% w / v formic acid in highly purified water and mobile phase B was a 70 / 25 / 5 (v / v / v) mixture of MeOH / iPrOH / H2O containing 0.035% w / v of formic acid. Gradient from 0 to 100 % B in 17.5 minutes, hold 100% B 5 minutes. Flow rate 0.8 mL / min, Injection volume 4 jxL. The ESI source operated at 100 °C, 2.5 kV capillary voltage, 60 V cone, 600 L / hr nitrogen desolvation flow at 350 °C and 10 L / hr nitrogen cone flow. The "Normal" Zfocus is set at 140. The analyzer is normally optimized at 7200 V flight tube.

[0179] In order to obtaining high resolution mass spectra, the eluent from the HPLC column was split and 25 y.L / min were mixed with a 100 y.L / min stream of a 30 / 10 / 60 (v / v / v) mixture of MeOH / iPrOH / H2O containing 0.01% w / v of formic acid and 80 nM Trimethoprim coming from a Waters Reagent Manager pump before entering the MS source. Trimethoprim was chosen as stable, soluble and appropriate reference compound for real-time single-point mass correction. ES(+) full scan 80-1200 amu centroided data acquisition was carried out at 2 Hz sampling rate in the "W" mode. The LCT embedded PC provided both real-time data centroiding and real-time mass correction based on the Trimethoprim. H+ reference mass of 291 .1452 Da. Proper intensity MS spectra (40 to 2000 analyte counts) were averaged to obtain the final result.

[0180] 1H-NMR spectra were recorded Bruker Avance in 400MHz NMR spectrometer. Chemical shifts were referenced with respect to the residual solvent signals (DMSO-cfe: 2.50 ppm CDCh: 7.26 ppm for1H). Data are reported as follows: chemical shift (5), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br. s = broad singlet, dd = doublet of doublets, ddd = doublet of doublets of doublets, m = multiplet), coupling constants {J, Hz) and number of protons.

[0181] Example 1

[0182] Preparation of tert-butyl N-[( 1 S) - 1 -[[(1 S) -2-[4-(bromomethyl)an ilino]- 1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl- propyl]carbamate

[0183] Step 1 tert-butylN-[(1 S)-1 -[[(1 S)-2-[4-(hydroxymethyl)anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl] carbamate

[0184] A mixture of the commercially available (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl- butanoyl]amino]propanoic acid (7.00 g, 24.3 mmol), 4-aminobenzyl alcohol (4.48 g, 36.4 mmol), EEDQ (12.0 g, 48.6 mmol) in DCM (70 mL) and MeOH (35 mL) was degassed and purged with N2 for 3 times, then the mixture was stirred at 25 °C for 16 hrs under N2atmosphere. TLC (petroleum ether: EtOAc = 1 : 1 , Rf = 0.34) showed the reaction was complete. The reaction mixture was filtered, the solvent evaporated to dryness and the residue was purified by column chromatography (silica gel, petroleum ether: EtOAc = from 3: 1 to 1 : 1) to give the tert-butyl N-[(1 S)-1-[[(1 S)-2-[4- (hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (9.11 g, 85.6% yield) as a yellow solid.

[0185] Step2 tert-butyl N-[(1 S)-1 -[[(1 S) -2-[4-(bromomethyl)an i li no]- 1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate

[0186] A mixture of compound tert-butyl N-[(1 S)-1 -[[(1 S)-2-[4-(hydroxymethyl)anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2- methyl-propyl]carbamate (3.00 g, 7.62 mmol), NBS (2.04 g, 11.4 mmol), PPh3(3.00 g, 11.44 mmol) in THF (30 mL) was degassed and purged with N2for 3 times, then the mixture was stirred at 25 °C for 16 hrs under N2atmosphere. TLC (petroleum ether: EtOAc = 1 : 1 , Rf = 0.63) showed the reaction was complete. The reaction mixture extracted with EtOAc (30 mL x 2). The combined organic layers were washed with NaCI (30 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, petroleum ether: EtOAc = 3: 1 to 1 : 1) to give the title intermediate (4.00 g, 50.9% yield) as a yellow solid.

[0187] MS (ESI) : [M+H]+456.1

[0188] Operating in an analogous way, but employing suitably substituted starting materials, the following compounds were obtained: tert-butylN-[(1 S)-1 -[[(1 S)-1 -[[4-(bromomethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl] carbamate

[0189] MS (ESI) : [M+H]+542.2 allyl(4S)-5-[[(1S)-1 -[[(1 S)-2-[4-(bromomethyl)anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-(tert- butoxycarbonylamino)-5-oxo-pentanoate

[0190] MS (ESI) : [M+H]+625.2 tert-butyl N-[2-[2-[2-[2-[3-[[( 1 S) -1 -[[(1 S) -2-[4-(bromomethyl)an ilino]- 1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl] amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate

[0191] MS (ESI) : [M+H]+703.3 tert-butylN-[2-[[2-[[(1 S)-1 -benzyl-2-[[2-[4-(bromomethyl)anilino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl] amino]-2-oxo-ethyl]carbamate

[0192] MS (ESI) : [M+H]+604.2

[0193] Example 2

[0194] Synthesis of N-[5-cyclopentyl- 1 -[[4-[[(2S)-2-[[(2S) -2-[6-(2,5-dioxopy rrol- 1 -yl)hexanoylamino]-3-methyl-butanoyl] amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro- benzamide; chloride (comp. 1 ; chloride); tert-butyl N-[(1 S)-1-[[(1S)-2-[4-[(2-amino-5-cyclopentyl-3-fluoro-pyridin-1 -ium-1 -yl)methyl]anilino]-1 -methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]carbamate;bromide.(VIII)

[0195] To a solution of 5-cyclopentyl-3-fluoro-py ridin-2-amine (900 mg, 4.99 mmol) in ACN (60 mL) was added tert-butyl N- [(1 S) -1 -[[(1 S)-2-[4-(b romomethyl)an i I i no]- 1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (6.50 g, 5.98 mmol). The mixture was stirred at 25 °C for 16 hrs. TLC (DCM: MeOH = 10: 1 , Rf = 0.18) showed the reaction was completed. The reaction mixture was concentrated under vacuum. The residue was purified by column chromatography (silica gel, DCM: MeOH = 100: 1 to 10: 1) to give tert-butyl N-[(1S)-1 -[[(1 S)-2-[4-[(2-amino-5- cyclopentyl-3-fluoro-pyridin-1 -ium-1 -yl)methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate; bromide. (VI 11) (1.75 g, 59.1 % yield) as a yellow solid.

[0196] 1 H NMR: 400 MHz, DMSO-d6

[0197] 5 10.13 (s, 1 H), 8.70 (s, 2H), 8.01 -8.15 (m, 2H) 7.96 (s, 1 H), 7.62 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.69 (d, J = 8.4 Hz, 1 H), 5.75 (s, 1 H), 5.48 (s, 2H), 4.36-4.47 (m, 1 H), 3.77-3.87 (m, 1 H), 2.86-2.97 (m, 1 H), 1 .92-1.99 (m, 2H), 1.73 (s, 2H), 1.56-1 .66 (m, 2H), 1 .43-1 .53 (m, 2H), 1.32-1.42 (m, 9H), 1 .29 (d, J = 6.6 Hz, 3H), 0.74-0.92 (m, 6H). Step 2 N-[5-cyclopentyl-3-fluoro-1 -[[4-[[rac-(2S)-2-[[rac-(2S)-3-methyl-2-(methylamino)butanoyl]amino]propanoyl]amino] phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide;chloride (IX)

[0198] To a solution of compound tert-butyl N-[(1 S)-1 -[[(1 S)-2-[4-[(2-amino-5-cyclopentyl-3-fluoro-pyridin-1 -ium-1 - yl)methyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate;bromide (1.00 g, 1.80 mmol) and compound 2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoic acid (459 mg, 1.63 mmol) in pyridine (10 mL) was added POCI3 (381 pL, 4.08 mmol). The mixture was stirred at 0 °C for 1 hr. TLC (DCM: MeOH = 10: 1 , Rf = 0.52) showed the reaction was complete. The reaction mixture was quenched by MeOH (10 mL) at 25°C and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C18 250 x 50mm x 10um; mobile phase: [water (FA)-ACN]; gradient: 35%- 75% B over 20 min) to give N-[5-cyclopentyl-3-fluoro-1 -[[4- [[rac-(2S)-2-[[rac-(2S)-3-methyl-2-(methylamino)butanoyl]amino]propanoyl]amino] phenyl]methyl]pyridin-1 -ium-2-yl]- 2-(1 -methyltetrazol-5-y l)sulfanyl-5-n itro-benzam ide ;ch loride (IX) (800 mg, 51 .98% yield) as a yellow solid. 1 H NMR: 400 MHz, CDCI3

[0199] 5 9.07 (s, 1 H), 8.88 (s, 1 H), 8.00-8.10 (m, 1 H), 7.65 (d, J = 7.6 Hz, 2H), 7.40-7.54 (m, 2H), 7.30 (d, J = 7.9 Hz, 2H), 6.83 (d, J = 9.0 Hz, 1 H), 6.66 (d, J = 6.8 Hz, 1 H), 5.54 (s, 2H), 5.02 (d, J = 6.3 Hz, 1 H), 4.55-4.68 (m, 1 H), 4.05 (d, J = 0.6 Hz, 3H), 3.91 -3.99 (m, 1 H), 2.84-2.97 (m, 1 H), 2.14-2.26 (m, 1 H), 2.02 (d, J = 0.8 Hz, 4H), 1 .65-1 .86 (m, 5H), 1 .39- 1.50 (m, 13H), 0.87-1.05 (m, 6H).

[0200] Step 3

[0201] N-[1 -[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-5-cyclopentyl-3-fluoro- pyridin-1 -ium-2-yl]-2-( 1 -methyltetrazol-5-y l)sulfanyl-5-n itro-benzamide;chloride (X)

[0202] To a solution of compound N-[5-cyclopentyl-3-fluoro-1-[[4-[[rac-(2S)-2-[[rac-(2S)-3-methyl-2- (methylamino)butanoyl]amino]propanoyl]amino]phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5- nitro-benzamide;chloride (400 mg, 7.75 mmol) in DCM (5 mL), TFA (500 pL, 6.73 mmol) was added. The mixture was stirred at 25 °C for 1 hr. LC-MS showed the reaction was completed. The reaction mixture was concentrated under reduced pressure to give the compound N-[1 -[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl]-5-cyclopentyl-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl) su If anyl -5-nit ro-benzam ide ;ch loride (X) (450 mg, crude) as a yellow oil which was used into the next step without further purification.

[0203] MS (ESI): [M+H] + 719.3

[0204] Step 4

[0205] The title compound (compd 1 : chloride)

[0206] To a solution of N-[1 -[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-5- cyclopentyl -3-fluo ro-py ridi n -1 -ium-2-yl]-2-( 1 -methy ltetrazol-5-yl)sulfanyl-5-n itro-benzamide;chloride (450 mg, 8.03 mmol) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (225 mg, 729 pmol) in DMF (5 mL) was added DIPEA (254 pL, 1 .46 mmol). The mixture was stirred at 25 °C for 2 hs. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (column: Xtimate C18 150 x 40mm x 10um; mobile phase: [water (FA) - ACN]; gradient: 28%- 68% B over 25 min) to give compound 1 (240 mg, 52.6% yield, 97.2% purity) as a yellow solid.

[0207] HRMS (ESI) calcd for C44H51 FN11O8S [M+H]+912.3621 , found 912.3632. 1 H NMR: 400 MHz, DMSO-d6

[0208] 6 9.94-10.00 (m, 1 H), 8.89-8.93 (m, 1H), 8.31 (s, 1 H), 8.08-8.17 (m, 2H), 8.02 (dd, J = 11.2, 1.5 Hz, 1 H), 7.79 (d, J = 8.8 Hz, 1 H), 7.58 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 6.99 (s, 2H), 6.72 (d, J = 8.8 Hz, 1 H), 5.58 (s, 2H), 4.31 - 4.40 (m, 1 H), 4.11 -4.20 (m, 1 H), 4.02 (s, 3H), 3.33-3.37 (m, 2H), 3.00 (s, 1 H), 1.98-2.22 (m, 4H), 1.88-1.97 (m, 1 H), 1.74-1.84 (m, 2H), 1.41 -1.69 (m, 8H), 1.28 (d, J = 7.2 Hz, 3H), 1.16 (s, 2H), 0.82 (dd, J = 15.6, 6.8 Hz, 6H).

[0209] Operating in an analogous way, but employing suitably substituted starting materials, the following compounds were obtained:

[0210] N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]-5-ureido- pentanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide; chloride (comp 2; chloride)

[0211] HRMS (ESI) calcd for C47H57FN13O9S [M+H]+998.4102, found 998.4115.

[0212] (4S)-5-[[(1S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl] amino]pyridin- 1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-[6-(2,5-dioxopyrrol-1 -yl) hexanoylamino]-5-oxo-pentanoic acid; chloride (comp 3; chloride)

[0213] HRMS (ESI) calcd for C49H58FN12011 S [M+H] + 1041 .4047, found 1041 .4042.

[0214] N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy] ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide; chloride (comp 4; chloride)

[0215] HRMS (ESI) calcd for C52H66FN12O13S [M+H]+1117.4572, found 1117.4561.

[0216] N-[5-cyclopentyl-1-[[4-[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]acetyl]amino]acetyl]amino]-3-phenyl- propanoyl]amino]acetyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro- benzamide; chloride (comp 5; chloride)

[0217] HRMS (ESI) calcd for C51 H55FN1301 OS [M+H]+1060.3894, found 1060.3910.

[0218] N-[1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl] methyl]-3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-1-ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5- nitro-benzamide; chloride (comp 6; chloride)

[0219] HRMS (ESI) calcd for C43H44F8N11O9S [M+H] + 1042.2911 , found 1042.2900.

[0220] N-[1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl] amino]phenyl]methyl]-3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-1-ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5- yl]sulfanyl-5-nitro-benzamide; chloride (comp 7; chloride)

[0221]

[0222] HRMS (ESI) calcd for C46H50F8N13010S [M+H]+1128.3391 , found 1128.3382.

[0223] (4S)-4-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-5-[[(1S)-1-[[(1 S)-2-[4-[[3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)-2-

[0224] [[2-[1 -(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]amino]-5-oxo-pentanoic acid; chloride (comp 8; chloride)

[0225] HRMS (ESI) calcd for C48H51 F8N 12012S [M+H]+1171 .3337, found 1171 .3355.

[0226] N-[1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy] propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-5-(1 ,1 , 2, 2, 3,3,3- heptaf luorop ropy l)py ridi n- 1 -ium-2-yl]-2-[1 -(2-hyd roxyethy IJtetrazol -5-yl]su Ifany I -5-nitro-benzamide; chloride (comp 9; chloride)

[0227] HRMS (ESI) calcd for C51 H59F8N12O14S [M+H]+1247.3861 , found 1247.3845. N-[5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-

[0228] (trifluoromethylsulfonyl)benzamide; chloride (comp 10; chloride)

[0229] HRMS (ESI) calcd for C45H51 F4N10O8S2 [M+H]+999.3264, found 999.3278.

[0230] (4S)-5-[[(1S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl) benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-[6-(2,5- dioxopy rrol- 1 -yl)hexanoylamino]-5-oxo-pentanoic acid; chloride (comp 11 ; chloride)

[0231] HRMS (ESI) calcd for C50H58F4N11011S2 [M+H]+1128.3689, found 1128.3691 .

[0232] N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl)benzamide; chloride (comp 12; chloride)

[0233] HRMS (ESI) calcd for C53H66F4N11013S2 [M+H]+1204.4214, found 1204.4244. N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide; chloride (comp 13; chloride)

[0234] HRMS (ESI) calcd for C45H53FN11O8S [M+H]+926.3778, found 926.3789.

[0235] N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide; chloride (comp 14; chloride)

[0236] HRMS (ESI) calcd for C53H68FN12O13S [M+H] + 1131.4728, found 1131.4701.

[0237] N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzamide; chloride (comp 15; chloride)

[0238] HRMS (ESI) calcd for C46H55FN11O9S [M+H] + 956.3884, found 956.3910.

[0239] N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-[1 - (2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzamide; chloride (comp 16; chloride)

[0240] HRMS (ESI) calcd for C54H70FN12O14S [M+H]+1 161.4834, found 1 161.4839.

[0241] N-[3-cyano-5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoy l]ami no]pheny l]methyl]py ridi n - 1 -i u m -2-y l]-2-( 1 -methy Itet razol -5-y l)su If any I -5-nit ro-benzam ide; chloride (comp 17; chloride)

[0242] HRMS (ESI) calcd for C45H51 N12O8S [M+H]+919.3668, found 919.3652.

[0243] N-[3-cyano-5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino] ethoxy]ethoxy] ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide; chloride (comp 18; chloride)

[0244] HRMS (ESI) calcd for C53H66N13O13S [M+H] + 1 124.4618, found 1124.4633.

[0245] Example 3

[0246] Synthesis of (2S)-N'-[( 1 S) - 1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-( 1 -methyltetrazol -5-yl)su lfanyl-5-nitro- benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-2-[3-[2-[2-[2-[2-[3- (2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]pentanediamide; chloride (comp 19; chloride)

[0247] Step 1

[0248] 9H-fluoren-9-ylmethyl N-[(1S)-4-[[(1 S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5- nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-1 -[2-

[0249] [2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethylcarbamoyl]-4-oxo-butyl]carbamate;chloride

[0250] To a solution of (4S)-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-5-oxo-pentanoic acid (250 mg, 0.111 mmol, HCI) and N-[1 -[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino] phenyl]methyl]-5-cyclopentyl-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide;chloride (84.4 mg, 0.111 mmol, prepared as reported in example 2) in DCM (5 mL), T4P (120 mg, 0.167 mmol, 50% purity) and DIEA (43.3 mg, 0.335 mmol, 58.4 pL) were added. The mixture was stirred at 25 °C for 12 h under N2 atmosphere. TLC (DCM: MeOH = 10: 1 , Rf = 0.24) showed the reaction was completed. The reaction mixture was diluted with water (20 mL) and extracted with DCM (20 mL x 2). The combined organic layers were washed with brine (20 mL), dried over anhydrous NasSC , filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0~7% MeOH / CH2CI2 gradient @ 15 mL / min) to give the 9H-fluoren-9-ylmethyl N-[(1 S)-4-[[(1 S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3- fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]amino]-1-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]-4-oxo-butyl]carbamate; chloride (110 mg, 36.4% yield) as yellow oil.

[0251] HRMS (ESI) : [M+3H]3+714,4

[0252] Step 2

[0253] (2S)-2-amino-N'-[(1 S)-1-[[(1S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl] amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-N-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]pentaned iamide;chloride

[0254] A mixture of 9H-fluoren-9-ylmethyl N-[(1 S)-4-[[(1 S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5- yl)sulfanyl-5-nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl] amino]-1 -[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethylcarbamoyl]-4-oxo-butyl]carbamate;chloride (60 mg, 27.5 pmol) in DMF (1 mL) and TEA (214 mg, 2.12 mmol, 295 pL) was stirred at 25 °C for 12 hrs. LC-MS showed the reaction was completed. The reaction mixture was concentrated under reduced pressure to give (2S)-2-amino-N'-[(1 S)-1-[[(1S)-2-[4-[[5-cyclopentyl-3-fluoro- 2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl] carbamoyl]-2-methyl-propyl]-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy) ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]pentanediamide;chloride (53 mg, crude) as a yellow oil. Product was used without further purification un next step.

[0255] MS (ESI) : [M+3H]3+640.5

[0256] Step 3

[0257] The title compound (comp. 19: chloride)

[0258] To a solution of (2S)-2-amino-N'-[(1 S)-1-[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5- nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-N-[2-[2-[2-[2- [2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethyl]pentanediamide;chloride (53.0 mg, 27.1 pmol) in DCM (1 mL), DIEA (7.01 mg, 54.2 pmol, 9.5 pL) and (2,5- dioxopyrrolidin-1 -yl) 3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (13.9 mg, 27.1 pmol) were added. The mixture was stirred at 25 °C for 12 hrs. LC-MS showed the reaction was completed. The reaction mixture was concentrated under reduced pressure to give. The crude product was purified by Pre-HPLC (column: F-Prepulite XP tC 18 40 x 200mm x 7pm; mobile phase: [water (NFUHCC^-ACN]; gradient: 24%-64% B over 14 min) to give title compound 19 (30.0 mg, 44.7% yield) as yellow oil.

[0259] 1 H NMR (400 MHz, CD3OD)

[0260] 58.96 (d, J = 2.4 Hz, 1 H), 8.10-8.22 (m, 2H), 7.87-7.94 (m, 1 H), 7.60 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 6.69- 6.85 (m, 2H), 5.66 (s, 2H), 4.54-4.63 (m, 4H), 4.35-4.50 (m, 2H), 4.23-4.31 (m, 1 H), 4.13-4.20 (m, 1 H), 4.03 (s, 3H), 3.48-3.72 (m,118 H), 3.35-3.37 (m, 3H), 3.03-3.17 (m, 2H), 2.47 (dd, J = 3.2, 6.4 Hz, 4H), 2.37 (t, J = 7.2 Hz, 2H), 2.02- 2.19 (m, 4H), 1.84-1.98 (m, 3H), 1.57-1.80 (m, 4H), 1.43 (d, J = 7.2 Hz, 3H), 0.92-1.02 (m, 6H). F NMR (400 MHz, CD30D) -116.85 (s, 1 F)

[0261] MS (ESI) : [M+2H]2+1159.0

[0262] Synthesis of intermediates

[0263] Preparation of: 2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoic acid

[0264] Methyl 2-fluoro-5-nitrobenzoate (4.0 g; 19.1 mmol; 1.0 eq.) and 5-mercapto-1 -methyltetrazole (3.4 g; 28.6 mmol; 1.5 eq.) were suspended in triethylamine (8 mL; 57.4 mmol; 3.0 eq.). The reaction mixture was stirred overnight at 110 °C and evaporated. The remaining dark residue was diluted with aqueous 5% NaHCO3solution and extracted three times with EtOAc. The combined aqueous phases were acidified with 25% HCI to pH ~2 and extracted three times with EtOAc. The combined organic phases were dried over Na2SO4, filtered and evaporated. The solid residue was triturated with diethyl ether, filtered off and dried in air to give 2-[(1 -methyl-1 H-1 ,2,3,4-tetrazol-5-yl)sulfanyl]-5-nitrobenzoic acid (3.1 g; 10.8 mmol; 56% yield; 99% purity) as a beige solid.

[0265] MS (ESI) : [M+H]+282.1.

[0266] Preparation of: lithium 5-nitro-2-({1 -[2-(oxan-2-yloxy)ethyl]-1 H-1 ,2,3,4-tetrazol-5-y l}sulfanyl)benzoate

[0267] Step 1 methyl 2-{[1 -(2-hyd roxyethyl)- 1 H-1 ,2,3,4-tetrazol-5-yl]sulfanyl}-5-nitrobenzoate

[0268] In a microwave vial under argon atmosphere, methyl 2-iodo-5-nitrobenzoate (0.2 g, 0.65 mmol, 1 eq.), 2-(5-sulfanyl- 1H-1 ,2,3,4-tetrazol-1-yl)ethan-1-ol (0.107 g, 0.72 mmol, 1.1 eq.), Pd2(dba)3(0.03 g, 0.03 mmol, 0.05 eq.), xantphos (0.039 g, 0.07 mmol, 0.1 eq.), DIPEA (0.258 g, 1.95 mmol, 3 eq.) and degassed dry 1 ,4-dioxane (7 mL) were added. The mixture was heated (microwave irradiation) at 110 °C for 1 h. HPLC-MS analysis showed the presence of the expected product. After cooling the mixture to room temperature, it was diluted with AcOEt, washed with H2O three times and once with brine. The organic layer was dried on Na2SO4 and evaporated. The product (beige solid) was precipitated with DCM (0.139 mg, 0.43 mmol, 66% yield).

[0269] MS (ESI) : [M+H]+326.1.

[0270] Step 2 methyl 5-nitro-2-({ 1 -[2-(oxan-2-yloxy)ethyl]-1 H-1 ,2,3,4-tetrazol-5-yl}sulfany I) benzoate To a solution of methyl 2-{[1 -(2-hyd roxyethy I) - 1 H-1 ,2,3,4-tetrazol-5-y l]sulfany l}-5-nitrobenzoate (0.087 g, 0.27 mmol, 1 eq.) in THF dry (5 mL), 3,4-dihydro-2H-pyran (0.046 g, 0.53 mmol, 2 eq.) and pyridinium p-toluenesulfonate (0.014 g, 0.05 mmol, 0.2 eq.) were added. The reaction mixture was stirred at room temperature overnight. The HPLC-MS shows the presence of the planned compound. Water was added to the mixture and the product was extracted with AcOEt 3 times, organic layer was washed with brine, dried on NasSC and evaporated. The product was purified by chromatographic column hexane / acetone (9 / 1 to 6 / 4) (0.103 g, 0.25 mmol, 94% yield).

[0271] MS (ESI) : [M+H]+410.1.

[0272] Step 3 lithium 5-nitro-2-({1 -[2-(oxan-2-yloxy)ethyl]-1 H-1 ,2,3 ,4-tetrazol -5-yl}su Ifany I) benzoate

[0273] In a reactor methyl 5-nitro-2-({1 -[2-(oxan-2-yloxy)ethyl]-1 H-1 ,2,3,4-tetrazol-5-yl}sulfanyl)benzoate (0.100 g, 0.24 mmol, 1 eq.), LiOH. H2O (0.012 g, 0.29 mmol, 1.2 eq.), THF (3.6 mL) and water (0.27 mL) were added. The reaction mixture was stirred at room temperature for 4 h. HPLC-MS analysis had shown the presence of planned product. The reaction mixture was evaporated without heating bath, the residue was suspended in acetone. The solid was filtrated and the solvent was evaporated to dryness to achieve the product (0.096 g, 0.24 mmol, 98% yield,).

[0274] MS (ESI) : [M+H]+396.1.

[0275] Preparation of: 2-[(1 -methyl-1 H-1 ,2 ,3 ,4-tetrazol -5-yl)su lfanyl]-5-trif luoromethanesu Ifonylbenzoic acid

[0276] To a solution of 2-fluoro-5-trifluoromethanesulfonylbenzoic acid (0.100 g, 0.368 mmol, 1 eq.) in ACN (4 mL), (1- methyltetrazol-5-yl)sulfanylpotassium (0.057 g, 0.368 mmol, 1 eq.) was added. The reaction mixture was stirred in microwave at 100 °C for 1 .5 h. HPLC-MS analysis had shown the presence of planned product. Solvent was removed under reduced pressure and the compound was used in the next step without further purification (0.08 g, 0.217 mmol, 59% yield, 70% purity).

[0277] MS (ESI) : [M+H]+ 369.1.

[0278] Preparation of 5-cyclopentyl-3-fluoropy ridin-2-amine 5-(cyclopent-1 -en-1 -yl)-3-fluoro-2-nitropyridine

[0279] 5-Bromo-3-fluoro-2-nitropyridine (1 g; 4.39 mmol; 1 eq.) and 1 -cyclopenteneboronic acid pinacol ester were dissolved in 1 mL dried methanol, 10 mL dried 1 ,4-dioxane and 2.5 mL demineralized water. Then potassium carbonate (1.8 g; 13,17 mmol; 3 eq.) and [1 , 1”-bis(diphenylphosphino)ferrocene]dichloropalladium(ll), complex with DCM (181.4 mg; 0.22 mmol; 0.050 eq.) were added, flushed with argon, closed with the septum and stirred in a microwave for 30 min at 100 °C. The mixture was filtered over Celite, and the filtrate was reduced to dryness. The residue was purified by flash chromatography (40 g silica gel, heptane / 0 - 40% AcOEt) to get 5-(cyclopent-1 -en-1 -yl)-3-fluoro-2-nitropyridine (770 mg; 3.69 mmol; 84% yield; 99% purity) as a yellow solid.

[0280] MS (ESI) : [M+H]+209.1.

[0281] Step 2

[0282] 5-cyclopentyl-3-fluoropyridin-2-amine

[0283] 5-(cyclopent-1 -en-1 -yl)-3-fluoro-2-nitropyridine (770 mg, 3.69 mmol) was dissolved in 10 mL THF and hydrogenated at room temperature and atmospheric pressure overnight using 0.400 g Pd / C-5% (3.76 mmol, 54.8% water) as catalyst. After consumption of 325 mL hydrogen gas the reaction was stopped. The reaction mixture was filtered over celite, the celite was washed with THF and MeOH and the resulting filtrate was evaporated to dryness to yield 5-cyclopentyl-3- fluoropyridin-2-amine (648 mg, 3.38 mmol, 91.6% yield, 94% purity) as brown solid which was used without further purification.

[0284] MS (ESI) : [M+H]+ 181.1.

[0285] Preparation of: 3-fluoro-5-( 1 ,1 ,2,2,3,3,3-heptafluoropropyl)py ridin-2-amine

[0286] 3-Fluoro-5-iodopyridin-2-amine (500 mg; 2.04 mmol) was dissolved in 10 mL dried DMSO, copper powder (324 mg; 5.1 mmol; 2.5 eq.) and perfluoropropyl iodide (1.5 mL; 10.2 mmol; 5 eq.) were added under argon atmosphere. The dark orange reaction suspension was stirred at 120 °C for 4 h. The reaction suspension was diluted with water and extracted 3x with DCM. The organic phase was filtrated over celite, washed with brine, dried over NasSC , filtrated over celite and evaporated. The residue was purified by flash chromatographie (40 g silica gel; heptane / 0 - 50% AcOEt) to get 3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-2-amine (324 mg; 1.14 mmol; 56% yield; 99% purity) a light beige solid.

[0287] MS (ESI) : [M+H]+280.9.

[0288] Preparation of: 5-cyclohexyl-3-fluoropy ridin-2-amine

[0289] Step 1

[0290] 5-(cyclohex-1 -en-1 -yl)-3-fluoro-2-nitropyridine

[0291] Under argon atmosphere 5-bromo-3-fluoro-2-nitropyridine (2.15 mmol; 500 mg), 1 -cyclohexenylboronic acid (3.22 mmol; 150 mol%; 406 mg) and potassium carbonate (6.45 mmol; 300 mol%; 891 mg) were suspended in 0.5 mL methanol, 1 mL demineralized water and 4 mL 1 ,4-dioxane in a microwave vessel (2-5 mL). Then [1,1”- bis(diphenylphosphino)ferrocene]dichloropalladium(ll), complex with DCM (0.11 mmol; 5 mol%; 89 mg) was added and heated by microwave (100 °C; 4 h). The reaction mixture was filtered over celite. The filtrate was reduced by vacuum and then absorbed onto diatomaceous earth (Isolute HM-N) and purified by flash chromatography (silica gel; n-heptane / 0% - 50% AcOEt to get 5-(cyclohex-1 -en-1 -y I) -3-f luoro-2-nitropy ridi ne (236 mg; 1 .04 mmol; 48% yield; 98% purity) as yellow crystals.

[0292] MS (ESI) : [M+H]+223.1.

[0293] Step 2

[0294] 5-cyclohexyl-3-fluoropyridin-2-amine

[0295] 5-(Cyclohex-1 -en-1 -yl)-3-fluoro-2-nitropyridine (1.04 mmol; 236 mg) was dissolved in 10 mL THF and hydrogenated at room temperature and atmospheric pressure overnight using 200 mg Pd / C-5% as catalyst. After consumption of 105 mL of hydrogen the reaction was stopped. The reaction mixture was filtered over celite. The celite was washed with THF and MeOH and the resulting filtrate was evaporated to dryness. The residue was dissolved in THF, absorbed onto diatomaceous earth (Isolute HM-N) and purified by flash chromatography (silica gel; n-heptane / O - 20% AcOEt to get 5-cyclohexyl-3-fluoropyridin-2-amine (178mg; 0.88 mmol; 84.4% yield; 96% purity) as a yellow solid.

[0296] MS (ESI) : [M+H]+195.1.

[0297] Preparation of: 2-amino-5-cyclopentylpy ridine-3-carbonitrile

[0298] Step 1

[0299] 2-amino-5-(cyclopent-1 -en-1 -yl) py ridine-3-carbonitrile

[0300] 2-Amino-5-bromopyridine-3-carbonitrile (1.00 g; 5.05 mmol; 1.0 eq.) and (cyclopent-1 -en-1 -yl)boronic acid (840 mg; 7.12 mmol; 1.4 eq.) were dissolved in 40 mL 1 ,4-dioxane and 8 mL water. K3PO4 (2.14 g, 9.58 mmol; 1.9 eq.), Pd(OAc>2 (224 mg; 0.95 mmol; 0.2 eq.) and X-Phos (480 mg, 0.96 mmol; 0.2 eq.) were added under nitrogen atmosphere. The resulting mixture was stirred for 16 h at 100 °C under nitrogen atmosphere. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc: 10 / 1) giving 2-amino-5-(cyclopent-1 -en-1 -yl)pyridine-3-carbonitrile (850 mg; 4.57 mmol; 91% yield; 100% purity).

[0301] MS (ESI) : [M+H]+186.0.

[0302] Step 2

[0303] 2-amino-5-cyclopentylpyridine-3-carbonitrile

[0304] 2-Amino-5-(cyclopent-1 -en-1 -yl)pyridine-3-carbonitrile (777 mg; 4.18 mmol; 1.0 eq.) was dissolved in 78 mL MeOH. 10% Pd / C (311 mg; 0.29 mmol; 0.07 eq.) was added under nitrogen atmosphere. The resulting solution was hydrogenated using a hydrogen balloon for 2 h at rt. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure giving 2-amino-5-cyclopentylpyridine-3-carbonitrile (373 mg; 1.97 mmol; 47% yield; 99% purity) as a white solid.

[0305] MS (ESI) : [M+H]+ 188.0.

[0306] Preparation of (4S)-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-5-oxo-pentanoic acid

[0307] Step 1 tert-butyl (4S)-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-

[0308] [2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-5-oxo-pentanoate

[0309] To a solution of (2S)-5-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (136 mg, 320 pmol) and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethanamine (250 mg, 320 pmol) in DCM (5 mL) was added DIEA (207 mg, 1.60 mmol, 279 pL) and T4P (462 mg, 641 pmol, 50% purity). The mixture was stirred at 25 °C for 12 hrs. TLC (Dichloromethane: Methanol = 10: 1 , Rf = 0.28) showed the reaction was completed. The reaction mixture was concentrated under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0~7% MeOH / CH2CI2 gradient @ 15 mbmin) to give tert-butyl (4S)-4-(9H-fluoren-9-ylmethoxy carbonylamino)-5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy) ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-5-oxo-pentanoate (260 mg, 54.21% yield) as colorless oil.

[0310] 1 H NMR (400 MHz, CDCI3) 57.77 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.2 Hz, 2H), 7.38-7.45 (m, 2H), 7.30-7.35 (m, 2H), 6.76-6.86 (m, 1 H), 5.69-5.85 (m, 1 H), 4.33-4.45 (m, 2H), 4.16-4.30 (m, 2H), 3.58-3.70 (m, 90H), 3.53-3.57 (m, 3H), 3.44-3.52 (m, 3H), 3.39 (s, 3H), 2.27-2.46 (m, 2H), 1.89-2.16 (m, 2H), 1.46 (s, 9H) Step 2

[0311] Title compound

[0312] A mixture of tert-butyl (4S)-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-5-oxo-pentanoate (260 mg, 173 pmol) in DCM (3 mL) was added HCI / dioxane (1 mL, 2M) at O °C. The mixture was stirred at 25 °C for 12 hrs. LC-MS showed the reaction was completed. The reaction mixture was concentrated under vacuum to give compound 5 (250 mg, 97.4% yield, HCI) as yellow oil MS (ESI) : [M+2H]2+720.7.

[0313] Preparation of: (2,5-dioxopyrrolidin-1 -yl)3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy] ethoxy] ethoxy] propanoate

[0314] Step 1 tert-butyl 3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0315] To a solution of tert-butyl 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoate (5.20 g, 16.2 mmol ) in DCM (80 mL) was added DIPEA (4.18 g, 32.4 mmol, 5.64 mL), then added (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl) propanoate (4.31 g, 16.2 mmol), the mixture was stirred at 25 °C for 1 hr. TLC (DCM / EtOAc = 0 / 1 , Rf = 0.1) showed the reaction was complete. The reaction mixture was diluted with DCM (100mL x 2) and extracted with H2O (100 mL x 2), washed with brine (80mL x 2). The combined organic layers were dried over NasSC , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, DCM / EtOAc =1 / 0 to 50 / 1) to give tert-butyl3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino] ethoxy]ethoxy]ethoxy]ethoxy]propanoate (6.0 g, 78.5% yield) as colorless oil.

[0316] 1 H NMR (400 MHz, CDCh) 56.70 (s, 2H), 6.45 (br s, 1 H) , 3.85 (t, J = 8.0 Hz, 2H), 3.71 (t, J = 6.40 Hz, 2H), 3.58-3.68 (m, 12H), 3.51-3.57 (m, 2H),

[0317] 3.43 (q, J = 5.20 Hz, 2H), 2.51 (q, J = 7.20 Hz, 4H), 1 .45 (s, 9H).

[0318] Step 2

[0319] 1 -(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid

[0320] To a solution of tert-butyl 3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino] ethoxy]ethoxy]ethoxy]ethoxy]propanoate (2.20 g, 4.66 mmol) in DCM (25 mL) was added TFA (6.78 g, 59.4 mmol, 4.40 mL), the mixture was stirred at 25 °C for 1 hr. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure to give 1 -(2, 5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3-oxo-7, 10,13, 16-tetraoxa-4- azanonadecan- 19-oic acid (1 .90 g, 98.0% yield) as a brown oil.

[0321] Step 3

[0322] Title compound

[0323] To a solution of 3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino] ethoxy]ethoxy] ethoxy]ethoxy]propanoic acid (1.85 g, 4.44 mmol) in DCM (30 mL), bis(2,5-dioxopyrrolidin-1 -yl) carbonate (1.71 g, 6.66 mmol) and TEA (1.35 g, 13.3 mmol, 1 .86 mL) were added. The mixture was stirrred at 25 °C for 1 hr. The reaction mixture was diluted with DCM (20 mL x 2) and extracted with H2O (20 mL x 2), washed with brine (10mL x 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give (2,5-dioxopyrrolidin-1 -yl)3-[2-[2-[2-[2-[3-(2,5- dioxopyrrol-1 -yl)propanoylamino]ethoxy] ethoxy]ethoxy]ethoxy]propanoate (2.20 g, 95.7% yield, 99.2% purity) as yellow oil.

[0324] 1 H NMR (400 MHZ,CDCI3)

[0325] 5 6.70 (s, 2H), 6.44 (br s, 1 H), 3.79-3.92 (m, 4H), 3.59-3.71 (m, 10H), 3.50-3.58 (m, 2H), 3.43 (q, J = 5.20 Hz, 2H), 2.91 (t, J = 6.40 Hz, 2H), 2.81 -2.88 (m, 4H), 2.52 (t, J = 7.20 Hz, 2H).

Claims

CLAIMS1. A drug-linker reagent compound of formula (I):wherein:Z is N or CH;Y is selected from the group consisting of -SO2CH3, -SO2CF3, -NO2, -CF3and -CN;X is selected from the group consisting of hydrogen, halogen, -CN, linear or branched C1-C8 alkyl and cyclic C3-C8 alkyl;R1 is selected from the group consisting of linear or branched C1-C8 alkyl, cyclic C3-C8 alkyl, -(CH2CH2O)nCH3, R3R4N-C1 -C8 alkyl and R3O-C1 -C8 alkyl; wherein:R3 and R4 are, each independently, selected from the group consisting of hydrogen, linear or branched C1- C8 alkyl and cyclic C3-C8 alkyl; n is an integer selected from 1 to 24;R2 is selected from the group consisting of linear or branched C1-C8 alkyl, cyclic C3-C8 alkyl, linear or branched C2-C8 alkenyl, C3-C8 cycloalkenyl and linear or branched C1 -C6 haloalkyl andL is a linker of formula (II):W-P-G-RM (II) wherein:W is a self immolative system selected from group (Illa), (I lib), (I I Ic) or (Hid):(Il la) (ll lb) (ll lc) (Hid) wherein:R5 and R6 are, each independently, hydrogen, halogen, linear or branched C1-C4 alkyl, linear or branched C1 -C4 hydroxyalkyl, -O(CH2-CH2O)mCH3 or -(CH2-CH2O)mCH3; m is an integer selected from 1 to 24;Su is a group selected from (IVa)-(IVc):* is the attachment point of the self immolative system W to the adjacent group P, G or RM;P is independently null or a dipeptidic, tripeptidic or tetrapeptidic moiety, consisting of any combination of natural L-aminoacids and unnatural D-aminoacids wherein, the C-terminal aminoacid residue is linked to W moiety and the N-terminal aminoacid residue is linked to G moiety or to the RM moiety, when G is null;G is independently null or selected from the group consisting of (Va)-(Vg):wherein: y is an integer selected from 0 to 24 k is an integer from 0 to 8 and* is the attachment point to the RM group;RM is a reactive moiety selected from the group consisting of (Vla)-(Vlh):R7 and R8 are, each independently, hydrogen, linear or branched C1 -C4 alkyl, straight or branched C1-C4 hydroxyalkyl or, R7 and R8 taken together form a cyclic C3-C6 alkyl;R9 is hydrogen or an electron-withdrawing group selected from -NO2 and -CN; r is an integer selected from 0 to 7;LG is an halogen; j is an integer from 1 to 8.

2. A compound of formula (I), according to claim 1 , wherein:Y is selected from the group consisting of -SO2CF3 and -NO2;X is selected from the group consisting of hydrogen, fluorine, -CN and linear or branched C1 -C4 alkyl;R1 is selected from the group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, -(CH2CH2O)nCH3 and R3O-C1 -C8 alkyl, wherein:R3 is selected from hydrogen and linear or branched C1 -C4 alkyl; n is an integer selected from 1 to 8;R2 is selected from the group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, C3-C8 cycloalkenyl and linear or branched C1-C6 haloalkyl;L is a linker of formula (II): wherein:W is a self immolative system selected from group (I I la)-(l I Id) , wherein:R5 and R6 are, each independently, hydrogen, linear C1 -C4 hydroxyalkyl, -O(CH2-CH2O)mCH3 or - (CH2-CH2O)mCH3; m is an integer selected from 1 to 8;Su is group selected from (IVa)-(IVc)* is attachment point of the self immolative system W to the adjacent group P, G or RM;P is independently null or a dipeptidic, tripeptidic or tetrapeptidic moiety, consisting of any combination of natural or unnatural aminoacids selecting from glycine, alanine, leucine, valine, citrulline, phenylalanine,glutamic acid and aspartic acid wherein, the C-terminal aminoacid residue is linked to W moiety and the N- terminal aminoacid residue is linked to G moiety or to the RM moiety, when G is null;RM is a reactive moiety selected from the group consisting of (Vla)-(Vlh), wherein:R7 and R8 are, each independently, hydrogen or linear C1 -C4 alkyl;R9 is hydrogen or -NO2; r is an integer selected from 0 to 7;LG is bromine, and j is an integer from 1 to 8.

3. A compound of formula (I), according to claim 2 wherein:Z is N;R1 is selected from a group consisting of linear or branched C1 -C4 alkyl, -(CH2CH2O)nCH3 and R3O-C1 -C6 alkyl, R2 is selected from the group consisting of linear or branched C1 -C8 alkyl, cyclic C3-C8 alkyl, C3-C8 cycloalkenyl, - CF3 and -CF2CF2CF3;L is a linker of formula (II), wherein:W is a self immolative system of formula (Illa);P is independently null or a dipeptidic, tripeptidic or tetrapeptidic moiety, selecting from the combinations consisting of: valine-alanine, valine-citrulline, glutamic acid-valine-alanine, glutamic acid-valine-citrulline, glycine-glycine-phenyl alanine-glycine, phenyl alanine-leucine-glycine, aspartic acid-valine-glycine and aspartic acid-valine-citrulline wherein, the C-terminal aminoacid residue is linked to W and the N-terminal aminoacid residue is linked to G moity or to the RM moiety when G is null;G is independently null or a group of formula (Va) or (Vg); andRM is a reactive moiety of formula (Via) or (Vlh).

4. A compound of formula (I), according to claim 1 , or a pharmaceutically acceptable salt thereof, selected from the group consisting of:N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 1);N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]-5-ureido- pentanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 2);(4S)-5-[[(1S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl]amino] pyridin- 1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-[6-(2,5-dioxopyrrol-1 -yl) hexanoylamino]-5-oxo-pentanoic acid (comp. 3);N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 4);N-[5-cyclopentyl-1-[[4-[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]acetyl]amino]acetyl]amino]-3-phenyl- propanoyl]amino]acetyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro- benzamide (comp. 5);N-[1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl] methyl]-3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-1-ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5- nitro-benzamide (comp. 6) N-[1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl] amino]phenyl]methyl]-3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)pyridin-1-ium-2-yl]-2-[1-(2-hydroxyethyl)tetrazol-5- yl]sulfanyl-5-nitro-benzamide (comp. 7);(4S)-4-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-5-[[(1S)-1 -[[(1 S)-2-[4-[[3-fluoro-5-(1 ,1 ,2,2,3,3,3-heptafluoropropyl)-2- [[2-[1 -(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]amino]-5-oxo-pentanoic acid (comp. 8);N-[1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy] propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-5-(1 ,1 , 2, 2, 3,3,3- heptaf luorop ropy l)py ridi n- 1 -ium-2-yl]-2-[1 -(2-hyd roxyethy l)tetrazol -5-yl]su Ifany I -5-nitro-benzamide (comp. 9); N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl) benzamide (comp. 10);(4S)-5-[[(1S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1 -methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl) benzoyl]amino]pyridin-1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-4-[6-(2,5- dioxopyrrol-1 -yl)hexanoylamino]-5-oxo-pentanoic acid (comp. 11);N-[5-cyclopentyl-1-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy] ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-(trifluoromethylsulfonyl)benzamide (comp. 12);N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 13); N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 14);N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoyl] amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-[1 -(2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzamide (comp. 15);N-[5-cyclohexyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy]ethoxy]ethoxy] ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-3-fluoro-pyridin-1 -ium-2-yl]-2-[1 - (2-hydroxyethyl)tetrazol-5-yl]sulfanyl-5-nitro-benzamide (comp. 16);N-[3-cyano-5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1 -yl)hexanoylamino]-3-methyl-butanoyl]amino] propanoyl]amino]phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 -methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 17);N-[3-cyano-5-cyclopentyl-1 -[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 -yl)propanoylamino]ethoxy] ethoxy] ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]pyridin-1 -ium-2-yl]-2-(1 - methyltetrazol-5-yl)sulfanyl-5-nitro-benzamide (comp. 18);(2S)-N'-[(1 S)-1 -[[(1 S)-2-[4-[[5-cyclopentyl-3-fluoro-2-[[2-(1-methyltetrazol-5-yl)sulfanyl-5-nitro-benzoyl]amino]pyridin- 1 -ium-1 -yl]methyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1 - yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]pentanediamide (comp. 19).

5. A salt of the compound of formula (I), according to anyone of claims from 1 to 4 with a counterion [A’].

6. Use of a compound of formula (I), as defined in anyone of claims from 1 to 5, in the preparation of conjugates.

7. A compound of formula (I) as defined in anyone of claims from 1 to 5 to be used in the preparation of conjugates.

8. Process for the preparation of compound of formula (I), or pharmaceutical acceptable salt thereof, as defined in claim 1 or 5, said process comprises the following steps:Step 1) reacting the compound of formula (VII):wherein X and R2 are as defined in claim 1 ; with a compound of formula (XI):LG1 -W-P-G-PG (XI) wherein LG1 is a leaving group preferably selected from mesylate, tosylate and halogen; G-PG is independently null or selected from the group consisting of (Xlla)-(Xllg):wherein PG is an amino protecting group preferably selected from methyl carbamate, ethyl carbamate, 9- fluorenylmethyl carbamate (Fmoc), 2,2,2-trichloroethylcarbamate (Troc), t-butyl carbamate (BOO), vinyl carbamate(Voc), allyl carbamate (Alloc) and benzyl carbamate (Cbz), and k, y, W and P are as defined in claim 1 , thus obtaining a compound of formula (wherein PG is as defined above and X, R2, W, P,and G are as defined in claim 1 ;Step 2) reacting the compound of formula (VIII) with the acid derivative of formula (XIII):wherein Z, Y and R1 are as defined in claim 1 , in the presence of suitable coupling agents or after activation of acid of formula (XIII) in the corresponding acid halides, thus obtaining a compound of formula (IX):wherein X, Y, Z, R1 , R2, W, P, G and PG are as defined in claim 1.Step 3) removing from the resulting compound of formula (IX) the PG protecting group, so obtaining a compound of formula (wherein X, Y, Z, R1 , R2, W, P and G are as defined in claim 1 ;Step 4) reacting the compound of formula (X) with the acid compound of formula (XIV):HO-RM(XIV) wherein RM is a reactive moiety selected from the group consisting of (Vla)-(Vlh):wherein R7, R8 , R9 , r, j and LG are as defined in claim 1 , in the presence of suitable coupling agents or after activation of acid (XIV) in the corresponding acid halides, so obtaining a compound of formula (I) wherein X, Y, Z, R1 , R2, W, P, G and RM are as defined in claim 1 .

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