Branched linker for protein drug conjugates

1. A method (MI) for connecting a ligand LI with a drug DR, the method comprising

covalently connecting the ligand LI to the drug DR using a linker LIN,

wherein the connecting step forms a ligand-linker-drug conjugate, wherein

LI is selected from the group consisting of amino acids LI-AA, mono- or polyclonal antibodies LI-Ab, antibody fragments LI-AbFrag, proteins LI-Prot and peptides LI-Pep;

DR is a pharmaceutically active drug;

LIN comprises a connecting group CG2; and

CG2 is a connecting group of formula (CG2-1)

wherein

(***) denotes the connecting site which is used to connect LI;

(****) denotes the connecting site which is used to connect DR;

(******) denotes the connecting, site to which a linear peptide is connected, said peptide having, 2 to 8 amino acid residues.

2. The method (MI) according to claim 1 , wherein the ligand-linker-drug conjugate is in the form of a compound of formula (I),

wherein

T1 is —S—, —O— or —NH—;

T2 is —N(R4)-, —O— or —S—;

R4 H or C 1-4 alkyl;

T4 —O—;

n1 is 0 or 1;

n2 is 0 or 1;

n3 is 2, 3, 4, 5, 6, 7 or 8;

n4 is an integer from 1 to n3;

CG1 is a connecting group selected from the group consisting of a connecting group of formula (CG1-I) and spacer group of formula (CG1-II), a connecting group of formula (CG1-III) and a connecting group of formula (CG1-IV);

wherein

m30 and m32 are identical or different and independently from each other 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

(*) in the formulae of CG1 denotes the bond between T1 and CG1, the covalently connected LI forms in compound of formula (I) a ligand residue LIRes, which is covalently connected to CG1 via T1;

LI is a compound of formula (LIRes-T1-H);

LIRes-T1-H  (LIRes-H)

LIRes is selected from the group consisting of amino acid residue LIRes-AA, mono- or polyclonal antibody residue LIRes-Ab, antibody fragment residue LIRes-AbFrag, protein residue LIRes-Prot and peptide residue LIRes-Pep;

LI has a functional group selected from the group consisting of SH, OH or NH 2 , which forms in formula (I) the T1, the T1 is bonded to CG1 via the bond (*);

SG is a spacer group selected from the group consisting of a spacer group of formula (SG-II) and a spacer group of formula (SG-III);

wherein

m1 and m2 are identical or different and independently from each other 0 or 1;

m10, m11 and m12 are identical or different and independently from each other 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

with the proviso, that m2 and m10 are not simultaneously 0 and that m1 , m11 and m12 are not simultaneously 0;

SGPEG is a connecting group of formula (SGPEG-I);

wherein

 m20 is 1, 2, 3, 4, 5 or 6;

CG3 is selected from the group consisting of connecting group of formula (CG3-I) and connecting group of formula (CG3-II);

wherein

R5 and R6 are identical or different and independently from each other C 1-4 alkyl;

(******) in the formulae of CG2 denote the bond between to CG2 and AA n4 ;

the covalently connected DR forms in compound of formula (I) a drug residue DRRes, which is covalently connected to CG2 via T2;

DR is a compound of formula (DDRes-T2-H);

H-T2-DRRes  (DRRes-T2-H)

DRRes is a drug residue derived from DR;

DR has a functional group selected from the group consisting of —N(R4)H, —OH or —SH, which forms in formuls (I) the T2;

AA n4 is an amino acid residue and (AA n4 ) n3 is a linear peptide with n3 amino acid residues wherein n4 denotes the position of the amino acid residue in the peptide starting from CG2 with AA 1 being the first amino acid residue in the chain and being connected to CG2 via the bond (******), with the bond (******) being an amide bond of the carboxylic acid group of AA 1 with the amino group of CG2, and the AA n3 being the last amino acid residue in the chain, and with the individual amino acid residues being independently from each other identical or different, wherein the individual amino acid residures are connected to each other via a peptide bond;

(3) denotes the N-terminal amino group of said linear peptide, which is the amino group of AA n3 ;

R1 and R2 are identical or different and independently from each other selected from the group consisting of hydrogen, C 1-4 alkyl, C(O)—(CH 2 —O—) m5 -(GRPEG) m4 -R3 and PGN;

R3 C 1-4 alkyl;

m4 is 0 or 1;

m5 is 0 or 1;

PGN is a protecting group;

GRPEG is a connecting group of formula (GRPEG-I);

wherein

m21 is 1, 2, 3, 4, 5 or 6

wherein

if n1 being 1, the (**) in the formulae of CG1 and the (**) in the formula of SG denote the bond between CG1 and SG and the (***) in the formula of SG and the (***) in the formula of CG2 denote the bond between SG and CG2; and further wherein if n1 and CG1 is a connecting group of formula (CG1I), the nitrogen atom denoted with (**) in SG forms an endocyclic nitrogen atom thereby replacing the hydrogen atom of said nitrogen atom by an endocyclic bond;

if n1 is 0, the (**) in the formulae of CG1 and the (***) in the formula of CG2 denote the bond between CG1 and CG2; and further with the proviso that if n1 is 0 then CG1 is not a connecting group of formula (CG1-I);

if n2 is 1, the (****) in the formula of CG2 denotes the bond between CG2 and T4 and the (****) in the formula of CG3 denote the bonds between CG3 and T4; and further wherein n2 is 1, the (*****) in the formula of CG3 denotes the bond between CG3 and T2; and

if n2 being 0, the (****) in the formula of CG2 denotes the bond between CG2 and T2.

3. The method (MI) according to claim 2 , the method comprising reacting a compound of formula (II) with a compound of formula (LIRes-T1H);

wherein

CG1M is a connecting group selected from the group consisting of connecting group of formula (CG1M-I), connecting group of formula (CG1M-II), connecting group of formula (CG1M-III) and connecting group of formula (CG1M-IV);

wherein

X1 is Cl, Br or I.

4. A method (MII) for the preparation of compound of formula (II), with the compound of formula (II) as defined in claim 3 ,

wherein

if n2 is 1 and CG3 is a connecting group of formula (CG3-I), then method (MII) comprises a step (MIIa) and a step (MIIb);

if n2 is 1 and CG3 is a connecting group of formula (CG3-II), then method (MII) comprises the step (MIIa), a step (MIIc), a step (MIId) and a step (MIIe);

if n2 is 0 and CG1M is a connecting group of formula (CG1M-IV) then method (MII) comprises one step, a step (MII0-IV), or two steps, a step (MII0-I-IVa) and a step (MII0-1-IVb);

if n2 is 0 and CG1M is a connecting group of formula (CG1M-III) then method (MII) comprises a step (MII0-III);

if n2 is 0 and CG1M is a connecting group of formula (CG1M-II) then method (MII) comprises two steps, a step (MII0-IIa) and a step (MII0-IIb), or one step, a step (MII0-IIc);

if n2 is 0 and CG1M is a connecting group of formula (CG1M-I) then method (MII) comprises one step, a step (MII0-I), or two steps, a step (MII0-I-IVa) and a step (MII0-I-IVb);

wherein

step (MIIa) comprises a reaction a compound of formula (III) with a compound of formula (II-I) to form a reaction product (MIIa);

the compound of formula (II-I) is selected from the group consisting of a compound of formula (II-1), 1,1′-carbonyldiimidazole, 4-nitrophenylchloroformate, phosgene, diphosgene, triphosgene and mixtures thereof;

wherein

step (MIIb) comprises a reaction product (MIIa) with a compound of formula (DRRes-T2-H);

wherein

step (MIIc) comprises a reaction (MIIa) with a compound of formula (CG3M-II) to provide a compound of formula (IIc);

wherein

step (MIId) comprises reacting compound of formula (IIc) with the compound of formula (II-I) to form a reaction product (MIId;

step (MIIe) comprises a reaction product (MIId) with a compound of formula (DRRes-T2-H);

wherein

step (MII0-I-IVa) comprises a reacting a compound of formula (III) with a compound (II0-I-IVa) to provide a compound of formula (III0-I-IVa);

wherein

n2 is 0 in formula (III0-I-IVa);

compound (II0-I-IVa) is selected from the group consisting of p-toluenesulfonyl chloride, p-toluenesulfonic anhydride, methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride and mixtures thereof;

X2 is selected from the group consisting of —OTs, —OMs and —OTf;

wherein

step (MII0-1-IVb) comprises a reacting the compound of formula (III0-I-IVa) with a compound of formula (DRRes-T2-H);

wherein

step (MII0-I-IVb) comprises reacting the compound of formula (III0-I-Iva) with a compound formula (DRRes-T2-H);

the compound of formula (III) is prepared

in a step (MIII-IV) for the case that CG1M is a connecting group of formula (CG1M-IV); or

in a step (MIII-III) for the case that CG1M is a connecting group of formula (CG1M-III); or

in two steps, a step (MIII-IIa) and a step (MIII-IIb), or in one step, a step (MIII-IIc), for the case that CG1M is a connecting group of formula (CG1M-II) or a step (MIII-I) for the case that CG1 M is a connecting group of formula (CG1 M-I);

wherein

step (MIII-IV) comprises a reaction (MIII-IV) of a compound of formula (IV),

with a compound of formula (CG1MR-IV);

wherein

R20 is a residue of formula (R20-1);

wherein

step (MIII-III) comprises reacting a compound of formula (IV) with a compound of formula (CG1MR-III);

wherein

step (MIII-IIa) comprises a reacting the compound of formula (IV) with a compound of formula (CG1MR-IIa) to provide a compound of formula (IV-IIa);

wherein

step (MIII-IIb) comprises a reacting the compound of formula (IV-IIa) with a compound of formula (HOSu);

wherein

step (MIII-IIc) comprises a reacting the compound of formula (IV) with a compound of formula (CG1MR-IIc);

wherein

step (MIII-I) comprises a reacting a compound of formula (IV) with a compound of formula (MA);

wherein

the compound of formula (IV) is prepared in a step (MIV); wherein step (MIV) comprises reducing a compound of formula (V) with a compound (IV);

 wherein

compound (IV) is selected from the group consisting of NaBH 4 , BH 3 , DIBAL-H, sodium bis(2-methoxyethoxy)aluminium hydride and mixtures thereof;

 wherein the compound of formula (V) is prepared in a step (MVb);

wherein step (MVb) comprises cleaving R30 from a compound of formula (Va) with HCl;

wherein

 R30 is connected to SG via the bond denoted with (**) in the formulae of SG and is Boc;

wherein

step (MII0-IV) comprises a reacting a compound of formula (III0) with a compound of formula (CG1MR-IV);

wherein

n2 is 0 in formula (III0);

wherein

step (MII0-III) comprises a reacting the compound of formula (III0) with a compound of formula (CG1MR-III);

wherein

step (MII0-IIa) comprises a reacting the compound of formula (III0) with a compound of formula (CG1MR-IIa) to provide a compound of formula (III0-IIa);

wherein

n2 is 0 in formula (III0-IIa);

wherein

step (MII0-IIb) comprises reacting the compound of formula (III0-IIa) with a compound of formula (HOSu);

wherein

step (MII0-IIc) comprises reacting the compound of formula (III0) with a compound of formula (CG1MR-IIc);

wherein

step (MII0-I) comprises reacting a compound of formula (III0) with a compound of formula (MA);

wherein

compound of formula (III0) is prepared in a step (MIII0), wherein step (MIII0) comprises cleaving R30 from compound of formula (IV0) with HCl;

wherein

n2 is 0 in formula (IV0);

wherein

the compound of formula (IV0) is prepared in a step (MIV0a) and a step (MIV0b),

wherein

step (MIV0a) comprises a reacting a compound of formula (V0) with a compound (RIV0a) to provide a compound of formula (IV0a);

wherein

compound (RIV0a) is selected from the group consisting of p-toluenesulfonyl chloride, p-toluenesulfonic anhydride, methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride, SOCl 2 , (COCl) 2 , POCl 3 , PCl 3 , PCl 5 , POBr 3 , PBr 3 , PBr 5 , N-bromosuccinimide, N-iodosuccinimide, HCl, HBr, HI and mixtures thereof;

wherein

X3 is selected from the group consisting of —OTs, —OMs, —OTf, —Cl, —Br and —I;

wherein

step (MIV0b) comprises a reacting the compound of formula (IV0a), with the compound of formula (DRRes-T2-H);

wherein

the compound of formula (V0) is prepared in a step (MV0), step (MV0) comprises reducing a compound of formula (Va) with compound (IV).

5. The method (MII) according to claim 4 , wherein the compound of formula (Va) is prepared in a step (MVa), wherein step (MVa) comprises reacting a compound of formula (VI) with a compound of formula (SGM);

wherein

R31 is connected to SG via the bond denoted with (***) in the formulae of SG and is —OTs, —OMs, —OTf, —Br, —Cl or —I.

6. The method (MII) according to claim 5 , wherein the compound of formula (VI) is prepared in a step (MVIa) and optionally in a further step (MVIb);

wherein

step (MVIa) comprises coupling the n3 AA n4 to a compound of formula (VII-1) and then to the respective products of the preceding peptide coupling reactions;

wherein

step (MVIb) comprises a reacting the N-terminal amino group of AA n3 denoted with (3) in formula (VI) is reacted with a compound NTermProt;

wherein

NTermProt is selected from the group consisting of C 1-4 alkyl iodide, C 1-4 alkyl bromide, Cl—C(O)-(GRPEG) m4 -R3, R3-C(O)—O—C(O)—R3 and PGNPrec;

wherein

PGNPrec is a reagent which provides for the introduction of PGN on the N-terminal amino group of AA n3 denoted with (3) in formula (VI).

7. A compound of formula (I) as defined in claim 2 .

8. Compound of formula (I) according to claim 7 , wherein compound of formula (I) is selected from the group consisting of a compound of formula (10), compound of formula (II), compound of formula (12), a compound of formula (12-101), compound of formula (13), compound of formula (14), a compound of formula (15), compound of formula (15-102) and compound of formula (16);

wherein

doxorubicin is the compound of formula (DOXO),

wherein

(d1), (d2), (d3) and (d4) in formula (DOXO) denote functional groups;

wherein

the compound of formula (DOXO) is connected via the amino group denoted with (d1) in formulae (10), (11), (12), (12-101), (13), (14), (15), (15-102) and (16) respectively and in formula (DOXO).

9. A compound of formula (II) as defined in claim 3 .

10. Compound of formula (II) according to claim 9 , wherein compound of formula (II) is selected from the group consisting of a compound of formula (20), compound of formula (21), a compound of formula (22), a compound of formula (23), compound of formula (24), a compound of formula (25), a compound of formula (26), compound of formula (20-CAMPTO), a compound of formula (21-CAMPTO), a compound of formula (22-CAMPTO), a compound of formula (23-CAMPTO) and a compound of formula (21-TAXO-t1-1);

wherein

doxorubicin is a compound of formula (DOXO),

wherein

(d1), (d2), (d3) and (d4) in formula (DOXO) denote functional groups;

wherein

the compound of formula (DOXO) is connected via the amino group denoted with (d1) in formula (20), (21), (22), (23), (24), (25) and (26) respectively and in formula (DOXO);

wherein

camptothecin is the compound of formula (CAMPTO),

wherein

(c1) in formula (CAMPTO) denotes a functional group;

wherein

the compound of formula (CAMPTO) is connected via the hydroxy group denoted with (c1) in formula (20-CAMPTO), (21-CAMPTO), (22-CAMPTO) and (23-CAMPTO) respectively and in formula (CAMPTO);

wherein

taxo-t1-1 is the compound of formula (TAXO),

wherein

the compound of formula (TAXO) is connected via the hydroxy group denoted with (t1) in formula (21-TAXO-t1-1) and in formula (TAXO).

11. A compound selected from the group consisting of the compound of formula (IIc), the compound of formula (III), the compound of formula (IV), the compound of formula (IV-IIa), the compound of formula (V), the compound of formula (Va), the compound of formula (III0-IIa), the compound of formula (III0), the compound of formula (IV0), the compound of formula (IV0a), the compound of formula (V0) and compound of formula (III0-I-IVa) as defined in claim 4 .

12. Compound according to claim 11 ,

wherein

compound of formula (IIc) is compound of formula (20c);

wherein

compound of formula (III) is selected from the group consisting of formula (30), a compound of formula (31), a compound of frmula (32), compound of formula (33), a compound of formula (34), a compound of formula (35) and compound of formula (36);

wherein

the compound of formula (III0) is compound of formula (300);

wherein

camptothecin is the compound of formula (CAMPTO),

wherein

(c1) in formula (CAMPTO) denotes a functional group;

wherein

the compound of formula (CAMPTO) is connected via the hydroxy group denoted with (c1) in formula (300) respectively and in formula (CAMPTO);

wherein

compound of formula (III0-I-IVa) is compound of formula (320);

wherein

the compound of formula (IV) is selected from the group consisting of compound of formula (40), a compound of formula (41), a compound of formula (42), a compound of formula (43), a compound of formula (44), a compound of formula (45) and compound of formula (46);

wherein

the compound of formula (IV0) is compound of formula (400);

wherein

camptothecin is the compound of formula (CAMPTO),

wherein

compound of formula (IV0a) is compound of formula (400a);

wherein

compound of formula (V) is selected from the group consisting of compound of formula (50), a compound of formula (50-1), a compound of formula (51), a compound of formula (51-1), a compound of formula (52), a compound of formula (52-1), a compound of formula (53), a compound of formula (53-1), a compound of formula (54), a compound of formula (54-1), a compound of formula (54-2), a compound of formula (54-3), a compound of formula (55), a compound of formula (55-1), a compound of formula (56) and a compound of formula (56-1);

wherein

the compound of formula (V0) is compound of formula (500);

13. A compound of formula (VI) as defined in claim 5 .

14. The compound of formula (VI) according to claim 13 , wherein the compound of formula (VI) is selected from the group consisting of a compound of formula (6), a compound of formula (6-1), a compound of formula (6-2),a compound of formula (6b), a compound of formula (6b-1), a compound of formula (6b-2), a compound of formula (6c) and compound of formula (6-5);

15. A pharmaceutical composition or of a drug, wherein the pharmaceutical composition and the drug comprise the compound of formula (I) as defined in claim 2 .

16. The method (MI) according to claim 2 , wherein the compound of formula (I) is selected from the group consisting of a compound of formula (10), a compound of formula (11), a compound of formula (12), a compound of formula (12- 101), a compound of fornula (13), a compound of formula (14), a compound of formula (15). A compound of formula (15- 102) and a compound of formula (16);

wherein

doxorubicin is a compound of formula. (DOXO),

wherein

(d1), (d2), (d3) and (d4) in formula (DOXO) denote functional groups;

wherein

the compound of formula (DOXO) is connected via the amino group denoted with (d1) in formulae (10), (11), (12), (12-101), (13), (14), (15), (15-102) and (16) respectively and in formula. (DOXO).

17. The method (MI) according to claim 3 , wherein the compound of formula (II) is selected from the group consisting of a compound of formula (20), a compound of formula (21), a compound of formula (22), a compound of formula (23), a compound of formula (24), a compound of formula (25), a compound of formula (26), a compound of formula (20-CAMPTO), a compound of formula (21-CAMPTO), a compound of formula (22-CAMPTO), a compound of formula (23-CAMPTO) and a compound of formula (21-TAXO-t1-1);

wherein

doxorubicin is a compound of formula (DOXO),

wherein

(d1), (d2),(d3) and (d4) in formula (DOXO) denote functional groups;

wherein

the compound of formula (DOXO) is connected via the amino group denoted with (d1) in formula (20), (21), (22), (23), (24), (25) and (26) respectively and in formula (DOXO);

wherein

camptothecin is a compound of formula (CAMPTO),

wherein

(c1) in formula (CAMPTO) denotes a functional group;

wherein

the compound of formula (CAMPTO) is connected via the hydroxy group denoted with (c1) in formulae (20-CAMPTO), (21-CAMPTO), (22-CAMPTO) and (23-CAMPTO) respectively and in formula (CAMPTO);

wherein

taxo-t1-1 is a compound of formula (TAXO),

wherein

the compound of formula (TAXO) is connected via the hydroxy group denoted with (t1) in formula (21-TAXO-t1-1) and in formula (TAXO).

18. The method (MII) for the preparation of the compound of formula (II) according to claim 4 ,

wherein

the compound of formula (IIc) is a compound of formula (20c);

wherein

the compound of formula (III) is selected from the group consisting of a compound of formula (30), a compound of formula (31), a compound of formula (32), a compound of formula (33), a compound of formula (34), a compound of formula (35) and a compound of formula (36);

wherein

the compound of formula (III0) is a compound of formula (300);

wherein

camptothecin is a compound of formula (CAMPTO),

wherein

(c1) in formula (CAMPTO) denotes a functional group;

wherein

the compound of formula (CAMPTO) is connected via the hydroxy group denoted with (c1) in formula (300) respectively and in formula (CAMPTO);

wherein

the compound of formula (III0-I-IVa) is a compound of formula (320);

wherein

the compound of formula (IV) is selected from the group consisting of a compound of formula (40), a compound of formula (41), a compound of formula (42), a compound of formula (43), a compound of formula (44), a compound of formula (45) and a compound of formula (46);

wherein

the compound of formula (IV0) is a compound of formula (400);

wherein

camptothecin is the compound of formula (CAMPTO);

wherein

the compound of formula (IV0a) is a compound of formula (400a);

wherein

the compound of formula (V) is selected from the group consisting of a compound of formula (50), a compound of formula (50-1), a compound of formula (51), a compound of formula (51-1), a compound of formula (52), a compound of formula (52-1), a compound of formula (53), a compound of formula (53-1), a compound of formula (54), a compound of formula (54-1), a compound of formula (54-2), a compound of formula (54-3), a compound of formula (55), a compound of formula (55-1), a compound of formula (56) and a compound of formula (56-1);

wherein

the compound of formula (V0) is a compound of formula (500);

19. The method (MII) according to claim 5 , wherein the compound of formula (VI) is selected from the group consisting of a compound of formula (6), a compound of formula (6-1), a compound of formula (6-2), a compound of formula (6-3), a compound of formula (6-4), a compound of formula (6b), a compound of formula (6b-1), a compound of formula (6b-2), a compound of formula (6b-3), a compound of formula (6 b-4), a compound of formula (6c) and a compound of formula (6-5);