Branched linker for protein drug conjugates
The present invention relates to method for connecting a protein and a drug to a protein drug
conjugate, wherein the drug is linked to the protein through a specific branched linker, said
branched linker comprises a peptide chain and is derived from o-hydroxy p-amino benzylic
alcohol, wherein the peptide chain is connected to the phenyl ring via the p-amino group, the
drug is connected to the phenyl ring via the benzylic alcohol moiety, and the protein is
connected to the phenyl ring via the o-hydroxy group;
further to a process for the preparation of said protein-drug-conjugates via various
intermediates, to the pharmaceutical use of such protein drug conjugates, such as methods of
controlling the growth of undesirable cells, to pharmaceutical compositions comprising such
protein drug conjugates, and to intermediates of the preparation of the protein drug
conjugates.
Most drugs used for chemotherapy have severe side-effects which limit their efficacy and use.
Linking such payloads, i.e. pharmaceutically active compounds, such as drugs, to targeting
agents, in particular monoclonal antibodies, affords novel antibody drug conjugates (ADC)
for e.g. cancer therapy. Tissue-specificity is typically governed by the monoclonal antibody
(mAb) component, while the drug provides the therapeutic effect. The efficiency and
tolerability of ADCs is dependent on the interplay between the target antigen, drug potency
and conjugation technology. In particular, linker chemistry strongly influences the ADC
specificity and safety.
Instead of chemically labile linkers, which have limited stability in physiological extracellular
conditions, such as hydrazone- and disulfide-based linkers, linkers, which are stable in
physiological extracellular conditions, especially which have high plasma stability, are
desired for improving the therapeutic applicability, because the drug should be released only
within the cell, which is targeted by the protein, to which the drug is linked, and not outside of
the cell.
Non cleavable linkers have disadvantages: ADC internalization followed by complete
hydrolysis of the polypeptide backbone of the mAb is required for payload release, and
reduced efficacy may be encountered when ADC internalization is poor. Thus, ADCs bearing
non-cleavable linkers are highly dependent on the biology of the target cell. Also, not all
payloads retain their biological activity when attached to the last amino acid of the mAb as is
the case after mAb degradation.
In order to allow the ADC to deliver the payload not only to tumor cells but also to adjacent
antigen-negative cells, i.e. bystander effect, the released payload must readily diffuse through
hydrophobic cell membranes, which is not the case, when ADCs with non-cleavable linkers
release their payloads in form of an amino acid bearing drug with the amino acid being in a
zwitterionic state, i.e. having a positively charged ammonium and a negatively charged
carboxylate.
Therefore it is desired to have a linker which shows high plasma stability, and which releases
the drug without the drug being chemically modified after its release.
Furthermore it is desired to have protein drug conjugates, which show reduced agglomeration
or aggregation, which would impair their performance.
EP 624377 A discloses a drug ligand conjugate, wherein the linker comprises a linear peptide.
Fanny Guzman et al, Electronic Journal of Biotechnology, 2007, 10, 279-314; Yoshio Okada,
Current Organic Chemistry, 2001, 5, 1-43; US 6897289 B and the text books "Houben-Weyl
Synthesis of Peptides and Peptidomimetics (Methods in Organic Chemistry)", Murray
Goodman et al, Thieme Publishing Group, 2001, in particular Volumes E22a and E22b;
disclose protecting groups, peptide bond formation, the synthesis of peptides and proteins
detailing general and specific methods, and analytical techniques used to determine the
structure and composition of peptides.
Known linkers comprising linear peptide chains still show deficiencies. There was a need for
linkers and for protein drug conjugates based on such linkers, which show improved
performance. Surprisingly, specific branched linkers derived from o-hydroxy p-amino
benzylic alcohol show the desired performance.
In the following text, the following abbreviations are used, if not otherwise stated:
DCC N,N'-dicyclohexylcarbodiimide
EDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
Fmoc 9-fluorenylmethoxycarbonyl
Boc tert-butoxycarbonyl
Boc20 di-tert-butyl dicarbonate
Cit citrulline
BS N-bromosuccinimide
NHS compound of formula (HOSu), N-hydroxysuccinimide
NIS N-iodosuccinimide
-OTs tosylate
-OMs mesylate
-OTf triflate
PBS phosphate buffered saline
Red-Al sodium bis(2-methoxyethoxy)aluminium hydride
TCEP tris(2-carboxyethyl)phosphine hydrochloride
Tos or Ts Tosyl or p-toluenesulfonyl
TsCl Tosyl chloride or p-toluenesulfonyl chloride
Z or Cbz benzyloxycarbonyl
Subject of the invention is a method (MI) for connecting a ligand LI with a drug DR,
LI is selected from the group consisting of amino acids LI-AA, mono- or polyclonal
antibodies LI-Ab, antibody fragments LI-AbFrag, proteins Ll-Prot and peptides LIPep;
DR is a pharmaceutically active drug;
characterized that a linker LIN is used to covalently connect LI with DR;
LIN comprises a connecting group CG2;
CG2 is derived from o-hydroxy p-amino benzylic alcohol and is a connecting group of
formula (CG2-1);
(CG2-1)
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
has 2 to 8 amino acid residues;
(4) denotes the p-amino group of the o-hydroxy p-amino benzylic alcohol from
which CG2 is derived.
Further subject of the invention is a method (MI), with the method (MI) as defined herein,
also with all its preferred embodiments,
wherein LI is covalently connected with DR in form of a compound of formula (I);
CGI SG -CG2— T4 -CG3 -T2 DRRes
nl n2 n2
Tl
LIRes
Rl R2
CG2 is as defined herein, also with all its preferred embodiments;
CGI is a connecting group selected from the group consisting of connecting group of
formula (CGI -I), connecting group of formula (CGI -II), connecting group of formula
(CGI -III) and connecting group of formula (CGI -IV);
CGI -I) O (CGI -II)
(CG1-III) (CG1-IV)
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 CGI denotes the bond between Tl and CGI,
the covalently connected LI forms in compound of formula (I) a ligand residue LIRes, which
is covalently connected to CGI via Tl;
LI is as defined herein, also with all its preferred embodiments, and is a compound of
formula (LIRes-Tl-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 H2, which forms
in formula (I) the Tl, the Tl is bonded to CGI via the bond (*);
Tl is -S-, -O- or - H-;
nl is 0 or 1;
is a spacer group selected from the group consisting of spacer group of formula (SGII)
and spacer group of formula (SG-III);
HN— HCH- SGPEG CH -NH
ml l ml m\2 (SG-III)
o
ml and m2 are identical or different and independently from each other 0 or 1;
mlO, ml 1 and ml2 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 mlO are not simultaneously 0;
with the proviso, that ml, mi l and ml2 are not simultaneously 0;
SGPEG is a connecting group of formula (SGPEG-I);
CH2 CH2 O- CH H (SGPEG-I)
m20
m20 is 1, 2, 3, 4, 5 or 6,
n2 is 0 or 1;
T4 is -0-;
CG3 is selected from the group consisting of connecting group of formula (CG3-I) and
connecting group of formula (CG3-II);
R5 and R6 are identical or different and independently from each other C1-4 alkyl;
nl being 1, the (**) in the formulae of CGI and in the formulae of SG denote the bond
between to CGI and SG, and the (***) in the formula of SG and in the formula of
CG2 denote the bond between SG and CG2; in case that CGI is a connecting group of
formula (CGI -I), 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;
nl being 0, the (**) in the formulae of CGI and the (***) in the formula of CG2 denote
the bond between CGI and CG2;
with the proviso, that in case that nl is 0, then CGI is not a connecting group of formula
(CG1-I);
for n2 being 1, the (****) in the formula of CG2 and in the formula of CG3 denote the bonds,
with which CG2 and CG3 are bonded to T4; and the (*****) in the formula
of CG3 denotes the bond between CG3 and T2:
for n2 being 0, the (****) in the formula of CG2 denotes the bond between CG2 and T2;
the (******) in the formula of CG2 denotes the bond between CG2 and AA 4 ;
the covalently connected DR forms in compound of formula (I) a drug residue DRRes, which
is covalently connected to CG2 via T2;
DR is as defined herein, also with all its preferred embodiments, and is a compound of
formula (DRRes-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 formula (I) the T2;
T2 is -N(R4)-, -O- or -S-;
R4 is H or C alkyl;
n3 is 2, 3, 4, 5, 6, 7 or 8;
n4 is an integer from 1 to n3;
AA 4 is an amino acid residue, with n4 being the index of said amino acid residue, and
(AA 4) 3 is a linear peptide with n3 amino acid residues AA 4 and with n4 denoting the
position of the amino acid residue AA 4 in the peptide starting from CG2, in which
peptide the individual amino acid residues are connected to each other via a peptide
bond, with AA1 being the first amino acid residue in the chain and being connected to
CG2 via the bond (******) with the bond (******) being an amid bond of the
carboxylic acid group of AA1 with the amino group denoted with (4) of CG2, and with
AA being the last amino acid residue in the chain, and with the individual AA 4
being independently from each other identical or different;
(3) denotes the N-terminal amino group of AA ;
Rl and R2 are identical or different and independently from each other selected from the
group consisting of hydrogen, C1-4 alkyl, C(0)-(CH 2-0-) M5-(GRPEG)M4-R3 and PGN;
R3 is Ci-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);
(GRPEG-I)
m21 is 1, 2, 3, 4, 5 or 6;
LI, DR and CG2 are as defined herein, also with all their preferred embodiments.
Preferably, LIN is compound of formula (LIN).
Rl R2
Preferably, the drug DR is selected from group consisting of cytotoxic agents, other
chemotherapeutic and antimetastatic agents.
Preferably, the other chemotherapeutic and antimetastatic agents are selected from the group
consisting of tyrosine kinase inhibitors and Racl inhibitors.
Preferably, tyrosine kinase inhibitors are selected from the group of active pharmaceutical
ingredients (API) consisting of Imatinib, Lapatimib, Sunitimib, Nilotimib and Dasatimib.
Preferably, Racl inhibitors is NSC 23766.
Preferable cytotoxic agents are those used for cancer therapy.
Preferable classes of cytotoxic agents include, for example, the enzyme inhibitors such
anthracycline family of drugs, the bleomycins, the cytotoxic nucleosides,
dihydrofolate reductase inhibitors, differentiation inducers, DNA cleavers, DNA
intercalators, diynenes, the mitomycins, the podophyllotoxins, the pteridine family of
drugs, taxols, thymidylate synthase inhibitors, topoisomerase inhibitors, and the vinca
drugs.
Preferable useful members of various classes of cytotoxic agents are selected from the group
consisting of N8-acetyl spermidine, actinomycin, 9-amino camptothecin, aminopterin,
anguidine, anthracycline, auristatin, bleomycin, calicheamycin, camptothecin (lactone
or ring-opened form of the lactone), carminomycin, CC-1065, clofaribine, l-(2-
chloroethyl)-l,2-dimethanesulfonyl hydrazide, cyclopropabenzindol-4-one (CBI),
cytarabine, cytosine arabinoside, daunorubicin, dichloromethotrexate, n-(5,5-
diacetoxy-pentyl) doxorubicin,
l,8-dihydroxy-bicyclo[7.3. 1] trideca-4-9-diene-2,6-diyne-13-one, difluoronucleosides,
doxorubicin, duocarmycin, epirubicin, esperamicin, etoposide, 5-fluorouracil,
irinotecan, leurosideine, leurosine, maytansine, melphalan, 6-mercaptopurine,
methopterin, methotrexate, mitomycin A, mitomycin C, morpholine-doxorubicin,
nemorubicin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or
etoposide phosphate, retinoic acid, saporin, tallysomycin, vinblastine, vincristine,
vindesine, taxane, such as taxol or paclitaxel, taxotere or docetaxel, and taxotere
retinoic acid, and analogues and derivatives thereof.
More preferable cytotoxic agents are selected from the group consisting of anthracycline,
auristatin, calicheamycin, cyclopropabenzindol-4-one (CBI), doxorubicin,
duocarmycin, maytansine, mitomycin C, taxol, and analogues and derivatives of these
substances.
Preferably, R4 is H.
Preferably, Tl is -NH- or -S-.
Preferably, n2 is 1 and T2 is - H-, -O- or -S- and is connected via the bond (*****) with
CG3; more preferably, T2 is -NH- or -S-.
Doxorubicin has the CAS number 23214-92-8 and is the compound of formula (DOXO).
OH
(d2)
Compound of formula (DOXO) is also used in form of its hydrochloride salt.
Doxorubicin can be connected to CG3 or CG2 respectively via one of its functional groups,
e.g. via one of the functional groups denoted with (dl), (d2), (d3) and (d4) in formula
(DOXO).
The functional groups denoted with (dl), (d2), (d3) and (d4) function then as the T2 in
formula (I).
Preferably doxorubicin is connected via the amino group denoted with (dl) in formula
(DOXO) with CG3 via the bond (*****).
Maytansine has the CAS number 35846-53-8 and the formula (MAYT).
Maytansine can be connected to CG3 or CG2 respectively via the -OH denoted with (m2) in
formula (MAYT).
Or the -C(0)-CH 3 group denoted with (ml) in the formula (MAYT) is exchanged against an
acyl group, said acyl group has a nucleophilic group -SH, -NH2 or -OH, which again is
the connected to CG3 or CG2 respectively.
The -OH denoted with (m2) or said nucleophilic group of the acyl group function then as the
T2 in formula (I).
A preferred taxane is taxol with the CAS number 33069-62-4 and the formula (TAXO).
Taxol can be connected to CG3 or CG2 respectively via one of the -OH denoted with (tl), (t2)
and (t3) in formula (TAXO). Said -OH functions then as the T2 in formula (I).
In one preferred embodiment, n2 is 0, T2 is -O- and is connected via the bond (****) with
CG2, and DRRes is derived from DR, with DR being compound of formula (TAXO).
In another preferred embodiment, n2 is 1, T2 is -O- and is connected via the bond (*****)
with CG3, CG3 is the connecting group of formula (CG3-II) and DRRes is derived
from DR, with DR being compound of formula (TAXO), one possible Taxol
intermediate for this embodiment is the compound of formula (TAXO-tl-1).
A Camptothecin is (S)-(+)-camptothecin, which has the CAS number 7689-03-4 and is the
compound of formula (CAMPTO).
(CAMPTO)
Camptothecin can be connected to CG3 or the CG2 respectively via the functional
denoted with (cl) in formula (CAMPTO).
The functional group denoted with (cl) functions then as the T2 in formula (I).
In one preferred embodiment, n2 is 0, T2 is -O- and is connected via the bond (****) with
CG2, and DRRes is derived from DR, with DR being compound of formula
(CAMPTO).
In another preferred embodiment, n2 is 1, T2 is -O- and is connected via the bond (*****)
with CG3, CG3 is the connecting group of formula (CG3-II) and DRRes is derived
from DR, with DR being compound of formula (CAMPTO).
In one preferred embodiment, CGI is the connecting group of formula (CGI -I), (CGI -III) or
(CG1-IV) and the sulphur atom of the side chain of a Cys residue of LIRes is Tl and is
connected via the bond (*) to CGI .
In another preferred embodiment, CGI is the connecting group of formula (CG1-II), and Tl is
-N- or -O- of LIRes and is connected to CGI via the bond (*). This amino or hydroxyl
group of LIRes connected to CGI is preferably the N-terminal amino group of LIRes
or an amino or hydroxy group of a side chain of an amino acid residue of LIRes.
Preferably, in case Tl is an amino group of a side chain of an amino acid residue of
LIRes connected to CGI, said amino acid residue of LIRes is preferably a Lys; in case
Tl is an hydroxyl group of a side chain of an amino acid residue of LIRes connected
to CGI, said amino acid residue of LIRes is preferably a Tyr, Ser or Thr.
Preferably, LI is selected from the group consisting of mono- or polyclonal antibodies LI-Ab,
antibody fragments LI-AbFrag, proteins Ll-Prot and peptides Li-Pep; and
LIRes is selected from the group consisting of mono- or polyclonal antibody residue LIRes-
Ab, antibody fragment residue LIRes-AbFrag, protein residue LIRes-Prot and peptide
residue LIRes-Pep.
In the case that LIRes is LIRes-AA, LIRes is preferably an alpha amino acid residue.
LIRes can be preferably connected to CGI via one of two possible functional groups of
LIRes: via a N-terminal amino group or via a functional group of a side chain of LIRes, if
LIRes has such a side chain with a functional group. This functional group, which connects
LIRes with CGI, is the Tl. In case that LIRes is connected via a functional group of a side
chain of LIRes, said side chain is preferably a side chain of a Cys, Lys, Tyr, Ser or Thr
residue of LIRes.
In case of LIRes being LIRes-AA, the remaining functional groups of LIAARes-AA, which
are not connected to CGI, can be protected by a protecting group commonly used in peptide
chemistry, e.g. a non-connected amino group can carry a acetate, a non-connected carboxy
group can be esterified with a C1-4 alkohol, a non-connected functional group of a side chain
can carry a side chain protecting group conventionally used in peptide chmistry.
More preferably, LIRes-AA is an alpha amino acid residue with a side chain having a
functional group and is connected via this functional group with CGI, even more preferably
LIRes-AA is derived from Cys, Lys, Tyr, Ser or Thr.
Preferably, in the case, that LIRes is connected via the side chain of a Cys residue of LIRes,
Tl is formed by the sulfur atom of the side chain of said Cys residue and is connected
through the bond (*) with CGI, preferably with CGI being the connecting group of
formula (CGI -I), (CGI -III) or (CGI -IV); or
in the case, that LIRes is connected via the side chain of a Lys, Tyr, Ser or Thr residue of
LIRes, Tl is formed by the nitrogen or oxygen atom of the side chain of said Lys, Tyr,
Ser or Thr residue and is connected through the bond (*) with CGI, preferably with
CGI being the connecting group of formula (CGI -II).
LIRes-Pep can be derived from a cell-penetrating peptide.
LIRes-AB and LIRes-ABFrag are preferably derived from antibodies and antibody fragments
used in treatment of dieases, preferably in cancer treatment.
Preferably, m30 and m32 are identical or different and independently from each other 2, 3, 4,
5 or 6 .
Preferably, m30 is 2 when CGI is a connecting group of formula (CGl-IV).
Preferably, m32 is 2 when CGI is a connecting group of formula (CGI -II).
Preferably, nl is 1.
Preferably, ml and m2 are 0 or 1.
Preferably, mlO, ml 1 and ml2 are identical or different and independently from each other 0,
1, 2, 3, 4, 5 or 6 .
Preferably, m20 is 1, 2, 3 or 4 .
More preferably,
mlO is 6 and m2 is 0; or
m2 is 1, mlO is 0 and m20 is 1; or
ml is 1, mi l is 1, ml2 is 1 and m20 is 3; or
ml is 0, mi l is 3, ml2 is 0 .
Preferably, R5 and R6 are CH3.
In a particular embodiment, CGI is a connecting group of formula (CGI -I), SG is a spacer
group of formula (SG-II), ml 0 is 6 and m2 is 0 .
In another particular embodiment, CGI is a connecting group of formula (CGI -II) with m32
being 2 or a connecting group of formula (CGI -III), or a connecting group of formula
(CGl-IV) with m30 being 2, and SG is a spacer group of formula (SG-II), mlO is 0,
m2 is 1 and m20 is 1.
In another particular embodiment, CGI is a connecting group of formula (CG1-II) with m32
being 2 or a connecting group of formula (CGI -III), or a connecting group of formula
(CGl-IV) with m30 being 2, and SG is a spacer group of formula (SG-III) with ml
being 1, mi l being 1, ml2 being 1 and m20 being 3, or SG is a spacer group of
formula (SG-III) with ml being 0, ml 1 being 3 and ml2 being 0 .
Preferably, n3 is 2, 3, 4, 5 or 6; more preferably 2, 3 or 4; even more preferably, n3 is 2 or 3 .
Preferably, AA 4 is an alpha amino acid residue.
More preferably, AA 4 is selected from the group consisting of alanine, valine, leucine,
isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine side chain
protected with acetyl or formyl, arginine, arginine side chain protected, preferably
protected with tosyl or nitro groups, histidine, ornithine, ornithine side chain protected,
preferably protected with acetyl or formyl, and citrulline.
Even more preferably, AA 4 is alanine, glycine, phenylalanine, valine, lysine, leucine,
tryptophan, arginine, side-chain protected arginine or citrulline, especially alanine,
glycine, phenylalanine, valine, lysine or citrulline.
In case that AA 4 has a side chain with a functional group, this functional group can be
protected by a protecting group commonly used for protecting functional groups of side
chains of amino acids.
In case of Lys, the side chain is preferably protected with acetyl or formyl
Examples of peptides for the (AA 4) 3 peptide chain are Phe-Lys, Val-Lys, Phe-Phe-Lys, DPhe-
Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Trp-Cit, Phe-Ala, Gly-Phe-
Leu-Gly, Ala-Leu-Ala-Leu, Phe-N9-tosyl-Arg and Phe-N9-Nitro-Arg, preferably Phe-Lys,
Val-Lys, Val-Cit and D-Phe-L-Phe-Lys; any Lys side chain being optionally protected,
preferably with acetyl .
Especially preferably, n3 is 2 or 3, and AA 4 is alanine, glycine, valine or citrulline;
more especially,
n3 is 2 and AA1 is citrulline and AA2 is valine or alanine; or
n3 is 3 and AA1 is citrulline, AA2 is valine and AA3 is glycine.
Preferably, m4 is 1.
Preferably, R3 is methyl.
Preferably, Rl and R2 are identical or different and independently from each other selected
from the group consisting of hydrogen, methyl, C(0)-(CH 2-0-) m5-(GRPEG)m4-CH3
and PGN.
Preferably, m21 is 2, 3 or 4 .
Preferably, PGN is a protecting group commonly used in peptide chemistry for protecting the
N-terminus of a peptide or for protecting the alpha amino group of an alpha amino
acid used as building block in peptide synthesis.
More preferably, PGN is selected from group consisting of Boc, Fmoc and Z.
Even more preferably, Rl is hydrogen, methyl, acetyl or C(0)-(CH 2-0-) m5-(GRPEG)m4-CH 3
with m4 being 1 and m21 being 3, and R2 is hydrogen or methyl.
Especially,
Rl is acetyl and R2 is hydrogen; or
Rl and R2 are methyl; or
Rl is C(0)-(CH 2-0-) m5-(GRPEG)m4-CH3 with m5 being 0, m4 being 1 and m21 being
3, and R2 is hydrogen; or
Rl is C(0)-(CH 2-0-) m -(GRPEG)m4-CH3 with m5 being 1, m4 being 1 and m21 being
2, and R2 is hydrogen.
Further subject of the invention is a method (MI);
wherein compound of formula (I) is prepared in a step (MI);
step (MI) comprises a reaction (MI), wherein a compound of formula (II) is reacted with a
compound of formula (LIRes-Tl-H);
DRRes
Rl (3) R2
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);
(CG1M-III) (CGIM-IV)
XI is CI, Br or I ;
SG, nl, n4, n3, AA 4, (3), Rl, R2, T4, CG3, n2, T2, DRRes, m30 and m32 are as defined
herein, also with all their preferred embodiments;
CG2 is as defined herein, also with all its preferred embodiments.
Preferably, XI is CI or Br, more preferably XI is Br.
Preferably, CG1M is a connecting group of formula (CGIM-IV).
More preferably, CG1M is a connecting group of formula (CGIM-IV) and m30 is 2 .
In a particular embodiment, CG1M is a connecting group of formula (CG1M-I), SG is a
spacer group of formula (SG-II), mlO is 6 and m2 is 0 .
In another particular embodiment, CG1M is a connecting group of formula (CG1M-II) with
m32 being 2 or a connecting group of formula (CG1M-III) or a connecting group of
formula (CGIM-IV) with m30 being 2, and SG is a spacer group of formula (SG-II),
mlO is 0, m2 is 1 and m20 is 1.
In another particular embodiment, CG1M is a connecting group of formula (CGI -II) with
m32 being 2 or a connecting group of formula (CGI -III), or a connecting group of
formula (CGIM-IV) with m30 being 2, and SG is a spacer group of formula (SG-III)
with ml being 1, ml 1 being 1, ml2 being 1 and m20 being 3, or SG is a spacer group
of formula (SG-III) with ml being 0, ml 1 being 3 and ml2 being 0 .
Preferably, the reaction temperature of reaction (MI) is from 0 to 150 °C, more preferably
from 5 to 50 °C, even more preferably from 10 to 40 °C.
Preferably, the reaction time of reaction (MI) is from 1 min to 168 h, more preferably from 10
min to 24 h, even more preferably from 15 min to 3 h .
Reaction (MI) is usually done in a solvent (MI).
Preferably, solvent (MI) is selected from the group consisting of water, N,Ndimethylacetamide,
N,N-dimethylformamide, N,N-dimethylsulfoxide and mixtures thereof.
In case of water, the water can comprise a buffer (MI), preferably the buffer (MI) is a buffer
conventionally used in protein chemistry, more preferably buffer (MI) is derived from a
buffering substance selected from the group consisting of acetic acid, citric acid, dithiothreitol
(DTT), ethylenediaminetetraacetic acid (EDTA), glycine, histidine, phosphoric acid (incl.
phosphate buffered saline, PBS), polysorbate 20, polysorbate 80, saccharose, sodium chloride,
succinic acid, trehalose, tris-(hydroxymethyl)-aminomethane, mixtures thereof and salts
thereof.
The salts of said buffering substance are preferably sodium salt, potassium salts or HC1 salts.
Preferably, the amount of solvent (MI) is from 5 to 10000 fold, more preferably from 10 to
5000 fold, even more preferably from 50 to 500 fold, of the weight of compound of (LIRes-
Tl-H).
Preferably, in reaction (MI), from 1 to 100 mol equivalents, more preferably from 2 to 20 mol
equivalents, even more preferably from 3 to 10 mol equivalents, of compound of formula (II)
are used, the mol equivalents being based the mol of compound of formula (LIRes-Tl-H).
Reaction (MI) can be done in the presence of TCEP.
TCEP is preferably used, when LI is a mono- or polyclonal antibodies LI-Ab or an antibody
fragments LI-AbFrag.
Preferably, in reaction (MI), from 0.5 to 20 mol equivalents, more preferably from 1 to 10
mol equivalents, even more preferably from 1.5 to 5 mol equivalents, of TCEP are used, the
mol equivalents being based the mol of compound of formula (LIRes-Tl-H)
After the reaction (MI), compound of formula (I) can be isolated by standard methods such as
washing, extraction, filtration, concentration and drying. The compound of formula (I) can be
purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Alternatively, compound of formula (I) can be purified by standard methods such as filtration,
ultrafiltration, diafiltration and chromatography, and ca be stored or further used as a solution.
Further subject of the invention is a method (Mil) for the preparation of compound of formula
(II), with the compound of formula (II) as defined herein, also with all its preferred
embodiments,
wherein
in case that n2 is 1 and CG3 is a connecting group of formula (CG3-I), then method (Mil)
comprises a step (Mlla) and a step (Mllb);
in case that n2 is 1 and CG3 is a connecting group of formula (CG3-II), then method (Mil)
comprises the step (Mlla), a step (Mile), a step (Mild) and a step (Mile);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-IV) then method
(Mil) comprises one step, a step (MIIO-IV), or two steps, a step (MII0-I-IVa) and a
step (MII0-I-IVb);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-III) then method
(Mil) comprises a step (MIIO-III);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-II) then method
(Mil) comprises two steps, a step (MII0-IIa) and a step (MII0-IIb), or one step, a
step (MIIO-IIc);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-I) then method (Mil)
comprises one step, a step (MIIO-I), or two steps, a step (MII0-I-IVa) and a step
(MII0-I-IVb);
step (Mlla) comprises a reaction (Mlla), wherein a compound of formula (III) is reacted with
a compound (II-I);
Rl R 2
formula (III) is as defined herein, also with all its preferred embodiment;
compound (II-I) is selected from the group consisting of compound of formula (II-l), 1,1'-
carbonyldiimidazole, 4-nitrophenylchloroformate, phosgene, diphosgene, triphosgene
and mixtures thereof;
step (Mllb) comprises a reaction (Mllb), wherein the reaction product from the reaction
(Mlla) is reacted with a compound of formula (DRRes-T2-H);
step (MIIc) comprises a reaction (MIIc), wherein the reaction product from the reaction
(Mlla) is reacted with a compound of formula (CG3M-II) to provide a compound of
formula (He);
Rl ) R2 (lie)
n2 in formula (lie) is as defined in claim 2;
step (Mild) comprises a reaction (Mild), wherein compound of formula (He), prepared in
reaction (Mile), is reacted with the compound (II-I);
step (Mile) comprises a reaction (Mile), wherein the reaction product from the reaction
(Mild) is reacted with a compound of formula (DRRes-T2-H);
step (MII0-I-IVa) comprises a reaction (MllO-I-IVa), wherein a compound of formula (III) is
reacted with a compound (II0-I-IVa) to provide a compound of formula (III0-I-IVa);
(AA 4 )
n3 (III0-I-IVa)
Rl (3) R2
n2 is 0 in formula (III0-I-IVa);
compound (II0-I-IVa) is selected from the group consisting of p-toluenesulfonyl chloride, ptoluenesulfonic
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;
step (MII0-I-IVb) comprises a reaction (MII0-I-IVb), wherein compound of formula
(III0-I-IVa), prepared in reaction (MII0-I-IVa), is reacted with compound of formula
(DRRes-T2-H);
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 CG1M is a connecting group of formula (CG1M-I);
step (MIII-IV) comprises a reaction (MIII-IV) of a compound of formula (IV),
Rl (3) R2
n2 in formula (IV) is as defined herein, also with all its preferred embodiments;
with a compound of formula (CGIMR-IV);
(CG1MR-IV)
m30 is as defined herein, also with all its preferred embodiments;
R20 is a residue of formula (R20-1);
(R20-1)
step (MIII-III) comprises a reaction (MIII-III) of a compound of formula (IV) with a
compound of formula (CG1MR-III);
(CG1MR-III)
with XI as defined herein, also with all its preferred embodiments;
step (MIII-IIa) comprises a reaction (MIII-IIa) of the compound of formula (IV) with a
compound of formula (CGlMR-IIa) to provide a compound of formula (IV-IIa);
(CGlMR-IIa)
Rl ( ) R2
with m32 as defined herein, also with all its preferred embodiments;
step (MIII-IIb) comprises a reaction (MIII-IIb) of compound of formula (IV-IIa) prepared in
step (MIII-IIa) with a compound of formula (HOSu);
(HOSu)
step (MIII-IIc) comprises a reaction (MIII-IIc) of the compound of formula (IV) with a
compound of formula (CGlMR-IIc);
(CGlMR-IIc)
with m32 as defined herein, also with all its preferred embodiments;
step (MIII-I) comprises a reaction (MIII-I) of a compound of formula (IV) with a compound
of formula (MA);
(MA)
compound of formula (IV) is prepared in a step (MIV);
step (MIV) comprises a reaction (MIV), reaction (MIV) is a reduction of a compound of
formula (V) with a compound (IV);
compound (IV) is selected from the group consisting of NaBH 4, BH3, DIBAL-H, sodium
bis(2-methoxyethoxy)aluminium hydride and mixtures thereof;
SG, nl, AA, n4, n3, (3), Rl and R2 have the same definition as above, also with all their
preferred embodiments;
compound of formula (V) is prepared in a step (MVb);
step (MVb) comprises a reaction (MVb), wherein R30 is cleaved off from compound of
formula (Va) with HC1;
R30 is connected to SG via the bond denoted with (**) in the formulae of SG and is Boc;
step (MIIO-IV) comprises a reaction (MIIO-IV) of a compound of formula (IIIO) with a
compound of formula (CG1MR-IV);
H- SG -CG2 — T4 -CG3 -T2 DRRes
n l n2 n2
(AA 4 )
n3
(IIIO)
Rl R 2
with
SG, CG2, nl, AA, n4, n3, (3), Rl, R2, T2 and DRRes as defined herein, also with all their
preferred embodiments, and n2 is 0 in formula (IIIO);
step (MIIO-III) comprises a reaction (MIIO-III) of a compound of formula (IIIO) with a
compound of formula (CGIMR-III);
step (MII0-IIa) comprises a reaction (MII0-IIa) of the compound of formula (IIIO) with a
compound of formula (CGlMR-IIa) to provide a compound of formula (III0-IIa);
with
SG, CG2, nl, AA, n4, n3, (3), Rl, R2, T2 and DRRes and m32 as defined herein, also with all
their preferred embodiments, and n2 is 0 in formula (III0-IIa);
step (MII0-IIb) comprises a reaction (MII0-IIb) of compound of formula (III0-IIa) prepared
step (MII0-IIa) with a compound of formula (HOSu);
step (MIIO-IIc) comprises a reaction (MIIO-IIc) of the compound of formula (IIIO) with a
compound of formula (CGlMR-IIc);
step (MIIO-I) comprises a reaction (MIIO-I) of a compound of formula (IIIO) with a compound
of formula (MA);
compound of formula (IIIO) is prepared in a step (MIIIO),
step (MIIIO) comprises a reaction (MIIIO), wherein R30 is cleaved off from compound of
formula (IVO) with HC1;
R30— - SG CG2 -CG3 -T2 DRRes
n l i n2 n2
(AA 4 )
n3
(IVO)
R R2
n2 is 0 in formula (IVO);
compound of formula (IVO) is prepared in a step (MlVOa) and a step (MlVOb),
step (MlVOa) comprises a reaction (MlVOa), wherein a compound of formula (VO)
with a compound (RIVOa) to provide a compound of formula (IVOa);
(AA 4 ) (VO)
n3
(3) R2
(AA 4 ) (IVOa)
n3
R 1 R2
with
R30, SG, CG2, nl, AA, n4, n3, (3), R l and R2 as defined herein, also with all their preferred
embodiments;
compound (RIVOa) is selected from the group consisting of p-toluenesulfonyl chloride, ptoluenesulfonic
anhydride, methanesulfonyl chloride, methanesulfonic anhydride,
trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride, SOCl2,
(COCl) 2, POCl3, PCI3, PCI5, POBr 3, PBr3, PBr , N-bromosuccinimide,
N-iodosuccinimide, HC1, HBr, HI and mixtures thereof;
X3 is selected from the group consisting of -OTs, -OMs, -OTf, -CI, -Br and -I;
step (MlVOb) comprises a reaction (MlVOb), wherein compound of formula (IVOa), prepared
in reaction (MIVO), is reacted with compound of formula (DRRes-T2-H);
compound of formula (V0) is prepared in a step (MVO),
step (MVO) comprises a reaction (MVO), reaction (MVO) is a reduction of a compound of
formula (Va) with a compound (IV);
CG1M and XI are as defined herein, also with all its preferred embodiments;
SG, nl, n4, n3, AA 4, (3), Rl, R2, T4, CG3, n2, T2, DRRes, compound of formula (DRRes-
T2-H), m30, m32, R5 and R6 are as defined herein, also with all its preferred embodiments;
CG2 is as defined herein, also with all its preferred embodiments.
The compounds (II-I) and of formula (CG3M-II) are known compounds and can be prepared
according to known methods, often they are even commercially available.
Reaction (Mlla) and reaction (Mild) are similar reactions and can be done under similar
reaction parameters ranges as defined herein, with the individual reaction parameters for each
of said two reactions being selected independently from each other.
Preferably, compound (II-I) is compound of formula (II-l).
Reaction (Mlla) and reaction (Mild) are usually done in a solvent (Mlla).
Preferably, solvent (Mlla) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF and
mixtures thereof.
Preferably, the amount of solvent (Mlla) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (III).
Preferably, in the reaction (Mlla) and in the reaction (Mild), from 0.5 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 2 mol
equivalents, of compound (II-I) are used, the mol equivalents being based the mol of
compound of formula (III).
Reaction (Mlla) and reaction (Mild) can be done in the presence of a base (Mlla).
Preferably, the base (Mlla) is selected from the group consisting of K2C0 3, Na2C0 3,
diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures
thereof.
Preferably, in the reaction (Mlla) and in the reaction (Mild), from 0.5 to 50 mol equivalents,
more preferably from 1 to 20 mol equivalents, even more preferably from 2 to 10 mol
equivalents, of base (Mlla) are used, the mol equivalents being based the mol of compound of
formula (III).
Preferably, the reaction (Mlla) and the reaction (Mild) are done under inert atmosphere.
After the reaction (Mlla) and the reaction (Mild), the reaction product of reaction (Mlla) and
of reaction (Mild) can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. Any of the compounds can be purified before or after isolation,
preferably by chromatography or crystallization from an appropriate solvent.
The crude reaction mixture from reaction (Mlla) and from reaction (Mild) can also be directly
used in reaction (Mllb) or reaction (Mile).
More preferably, reaction (Mlla) and reaction (Mllb) as well as reaction (Mild) and reaction
(Mile) are done consecutively in the same solvent and in one pot.
Reaction (Mllb) and reaction (Mile) are similar reactions and can be done under similar
reaction parameters ranges as defined herein, with the individual reaction parameters for each
of said two reactions being selected independently from each other.
Reaction (Mllb) and reaction (Mile) are usually done in a solvent (Mllb).
Preferably, solvent (Mllb) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF,
water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (Mllb) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of the reaction product of
reaction (Mlla) or of reaction product of reaction (Mild) respectively.
Preferably, in reaction (Mllb) and in reaction (Mile), from 0.2 to 10 mol equivalents, more
preferably from 0.5 to 5 mol equivalents, even more preferably from 0.8 to 2 mol equivalents,
of compound of formula (DRRes-T2-H) are used, the mol equivalents being based the mol of
the reaction product of reaction (Mlla) or of the reaction product of reaction (Mild)
respectively.
Preferably, reaction (Mllb) and reaction (Mile) are done under inert atmosphere.
Reaction (MII0-I-IVb) and reaction (MlVOb) are similar reactions and can be done under
similar reaction parameters ranges as defined herein, with the individual reaction parameters
for each of said two reactions being selected independently from each other.
Reaction (MII0-I-IVb) and reaction (MlVOb) are usually done in a solvent (MlVOb).
Preferably, solvent (MlVOb) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF and mixtures thereof.
Preferably, the amount of solvent (MlVOb) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (III0-
I-IVb) or of formula (IVOa) respectively.
Preferably, in reaction (MII0-I-IVb) and in reaction (MlVOb), from 0.2 to 10 mol equivalents,
more preferably from 0.5 to 5 mol equivalents, even more preferably from 0.8 to 2 mol
equivalents, of compound of formula (DRRes-T2-H) are used, the mol equivalents being
based the mol of compound of formula (III0-I-IVb) or of formula (IVOa) respectively.
Preferably, reaction (MII0-I-IVb) and reaction (MlVOb) are done under inert atmosphere.
Reaction (Mile) is usually done in a solvent (Mile).
Preferably, solvent (Mile) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethyl sulfoxide, acetonitrile, acetone, 1,4-dioxane, THF,
water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (Mile) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of reaction product of reaction
(Mlla).
Preferably, in the reaction (Mile) from 0.2 to 20 mol equivalents, more preferably from 0.5 to
10 mol equivalents, even more preferably from 0.8 to 5 mol equivalents, of compound of
formula (CG3M-II) are used, the mol equivalents being based the mol of the reaction product
of reaction (Mlla).
Preferably, the reaction (Mile) is done under inert atmosphere.
After the reaction (Mile), the reaction product of the reaction (Mile) can be isolated by
standard methods such as washing, extraction, filtration, concentration and drying. Any of the
compounds can be purified before or after isolation, preferably by chromatography or
crystallization from an appropriate solvent.
After reaction (Mllb), reaction (Mile), reaction (MII0-I-IVb) and reaction (MlVOb),
compound of formula (II) or compound of formula (IVO) respectively can be isolated by
standard methods such as washing, extraction, filtration, concentration and drying. Any of the
compounds can be purified before or after isolation, preferably by chromatography or
crystallization from an appropriate solvent.
Preferably, CG1M is a connecting group of formula (CG1M-IV);
more preferably, CG1M is a connecting group of formula (CG1M-IV) and m30 is 2 .
Compound of formula (V) and compound of formula (IIIO) can be used in unprotonated form
or in protonated form as a salt in reaction (MIV), reaction (MIIO-I), reaction (MII0-IIa),
reaction (MIIO-III) and reaction (MIIO-IV)
Reaction (MIII-IV) and reaction (MIIO-IV) are similar reactions and can be done under
similar reaction parameters ranges as defined herein, with the individual reaction parameters
for each of said two reactions being selected independently from each other.
Reaction (MIII-IV) and reaction (MIIO-IV) are usually done in a solvent (MIII-IV).
Preferably, solvent (MIII-IV) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF, water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MIII-IV) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (IV).
Preferably, in the reaction (MIII-IV) and in the reaction (MIIO-IV), from 1 to 20 mol
equivalents, more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5
mol equivalents, of compound (CG1MR-IV) are used, the mol equivalents being based the
mol of compound of formula (IV) or of formula (IIIO) respectively.
Preferably, reaction (MIII-IV) and reaction (MIIO-IV) are done under inert atmosphere.
Reaction (MIII-IV) and reaction (MIIO-IV) is usually done in the presence of a base (Mill-
IV).
Preferably, the base (MIII-IV) is selected from the group consisting of K2C0 3, Na2C0 3,
diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures
thereof.
Preferably, in the reaction (MIII-IV) and in reaction (MIIO-IV), from 0.5 to 50 mol
equivalents, more preferably from 1 to 20 mol equivalents, even more preferably from 2 to 10
mol equivalents, of base (MIII-IV) are used, the mol equivalents being based the mol of
compound of formula (IV) or of formula (IIIO) respectively.
After reaction (MIII-IV) and reaction (MIIO-IV), the compound of formula (III) or of formula
(II) respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. The compound of formula (III) or of formula (II) respectively can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MIII-III) and reaction (MIIO-III) are similar reactions and can be done under similar
reaction parameters ranges as defined herein, with the individual reaction parameters for each
of said two reactions being selected independently from each other.
Reaction (MIII-III) and reaction (MIIO-III) are usually done in a solvent (MIII-III).
Preferably, solvent (MIII-III) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF, water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MIII-III) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (IV).
Preferably, in reaction (MIII-III) and in reaction (MIIO-III), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound (CGIMR-III) are used, the mol equivalents being based the mol of
compound of formula (IV) or of formula (IIIO) respectively.
Preferably, reaction (MIII-III) and reaction (MIIO-III) are done under inert atmosphere.
After reaction (MIII-III) and reaction (MIIO-III), the compound of formula (III) or of formula
(II) respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. The compound of formula (III) or of formula (II) respectively can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MIII-IIa) and reaction (MII0-IIa) are similar reactions and can be done under
similar reaction parameters ranges as defined herein, with the individual reaction parameters
for each of said two reactions being selected independently from each other.
Reaction (MIII-IIa) and reaction (MII0-IIa) are usually done in a solvent (MIII-IIa).
Preferably, solvent (MIII-IIa) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF, water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MIII-IIa) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (IV).
Preferably, in reaction (MIII-IIa) and in reaction (MII0-IIa), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound (CGlMR-IIa) are used, the mol equivalents being based the mol of
compound of formula (IV) or of formula (IIIO).
Preferably, reaction (MIII-IIa) and reaction (MII0-IIa) done under inert atmosphere.
After reaction (MIII-IIa) and reaction (MII0-IIa), the compound of formula (IV-IIa) or
formula (III0-IIa) respectively can be isolated by standard methods such as washing,
extraction, filtration, concentration and drying. The compound of formula (IV-IIa) or of
formula (III0-IIa) respectively can be purified before or after isolation, preferably by
chromatography or crystallization from an appropriate solvent.
Reaction (MIII-IIb) and reaction (MII0-IIb) are similar reactions and can be done under
similar reaction parameters ranges as defined herein, with the individual reaction parameters
for each of said two reactions being selected independently from each other.
Preferably, reaction (MIII-IIb) and reaction (MllO-IIb) are done in the presence of a
compound (COUPADD).
Compound (COUPADD) is a coupling additive conventionally used in peptide chemistry for
the coupling reaction of amino acid to peptides by amide bond formation. Preferably,
compound (COUPADD) is selected from the group consisting of DCC, EDC and mixtures
thereof.
Preferably, in reaction (MIII-IIb) and in reaction (MII0-IIb), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound (COUPADD) are used, the mol equivalents being based the mol of
compound of formula (IV-IIa) or of formula (III0-IIa) respectively.
Reaction (MIII-IIb) and reaction (MII0-IIb) are usually done in a solvent (MIII-IIb).
Preferably, solvent (MIII-IIb) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF and mixtures thereof.
Preferably, the amount of solvent (MIII-IIb) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (IVIIa).
Preferably, in reaction (MIII-IIb) and in reaction (MII0-IIb), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound of formula (HOSu) are used, the mol equivalents being based the
mol of compound of formula (IV-IIa) or of formula (III0-IIa) respectively.
Preferably, reaction (MIII-IIb) and reaction (MII0-IIb) are done under inert atmosphere.
After reaction (MIII-IIb) and reaction (MllO-IIb), the compound of formula (III) or of formula
(II) respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. The compound of formula (III) or of formula (II) respectively can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MIII-IIc) and reaction (MIIO-IIc) are similar reactions and can be done under
similar reaction parameters ranges as defined herein, with the individual reaction parameters
for each of said two reactions being selected independently from each other.
Reaction (MIII-IIc) and reaction (MIIO-IIc) are usually done in a solvent (MIII-IIc).
Preferably, solvent (MIII-IIc) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF, water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MIII-IIc) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (IV).
Preferably, in reaction (MIII-IIc) and in reaction (MIIO-IIc), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound (CGlMR-IIa) are used, the mol equivalents being based the mol of
compound of formula (IV) or of formula (IIIO) respectively.
Preferably, reaction (MIII-IIc) and reaction (MIIO-IIc) are done under inert atmosphere.
Reaction (MIII-IIc) and reaction (MIIO-IIc) are usually done in the presence of a base (MIIIlie).
Preferably, the base (MIII-IIc) is selected from the group consisting of K2C0 3, Na2C0 3,
diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures
thereof.
Preferably, in the reaction (MIII-IIc) and reaction (MIIO-IIc), from 0.5 to 50 mol equivalents,
more preferably from 1 to 20 mol equivalents, even more preferably from 2 to 10 mol
equivalents, of base (MIII-IIc) are used, the mol equivalents being based the mol of
compound of formula (IV) or of formula (IIIO) respectively.
After reaction (MIII-IIc) and reaction (MIIO-IIc), the compound of formula (III) or of formula
(II) respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. The compound of formula (III) or of formula (II) respectively can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MIII-I) and reaction (MIIO-I) are similar reactions and can be done under similar
reaction parameters ranges as defined herein, with the individual reaction parameters for each
of said two reactions being selected independently from each other.
Preferably, the reaction time of reaction (MIII-I) and of reaction (MIIO-I) is from 1 min to 168
h, more preferably from 2 to 144 h, even more preferably from 12 to 120 h .
Reaction (MIII-I) and reaction (MIIO-I) are usually done in a solvent (MIII-I).
Preferably, solvent (MIII-I) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF and
mixtures thereof.
Preferably, the amount of solvent (MIII-I) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (IV) or
of formula (IIIO) respectively.
Preferably, in reaction (MIII-I) and in reaction (MIIO-I), from 1 to 20 mol equivalents, more
preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol equivalents, of
compound of formula (MA) are used, the mol equivalents being based the mol of compound
of formula (IV) or of formula (IIIO) respectively.
Preferably, reaction (MIII-I) and reaction (MIIO-I) are done under inert atmosphere.
Preferably, reaction (MIII-I) and reaction (MIIO-I) are done in the presence of compound
(COUPADD).
Preferably, in reaction (MIII-I) and reaction (MIIO-I), from 1 to 20 mol equivalents, more
preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol equivalents, of
compound (COUPADD) are used, the mol equivalents being based the mol of compound of
formula (IV) or of formula (IIIO) respectively.
Preferably, reaction (MIII-I) and reaction (MIIO-I) are done in the presence of compound of
formula (HOSu).
Preferably, reaction (MIII-I) and reaction (MIIO-I) are done in the presence of compound
(COUPADD) and compound of formula (HOSu).
Preferably, in reaction (MIII-I) and reaction (MIIO-I), from 1 to 20 mol equivalents, more
preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol equivalents, of
compound of formula (HOSu) are used, the mol equivalents being based the mol of
compound of formula (IV) or of formula (IIIO) respectively.
After reaction (MIII-I) and reaction (MIIO-I), the compound of formula (III) or of formula (II)
respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. The compound of formula (III) or of formula (II) respectively can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MIV) and reaction (MVO) are similar reactions and can be done under similar
reaction parameters ranges as defined herein, with the individual reaction parameters for each
of said two reactions being selected independently from each other.
Preferably, the reaction time of reaction (MIV) and of reaction (MVO) is from 1 min to 168 h,
more preferably from 1 to 120 h, even more preferably from 6 to 48 h .
Reaction (MIV) and reaction (MVO) are usually done in a solvent (MIV).
Preferably, solvent (MIV) is selected from the group consisting of water, methanol, ethanol,
N,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile,
acetone, 1,4-dioxane, THF, water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MIV) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (V) or
of formula (Va) respectively.
Preferably, reaction (MIV) and reaction (MVO) are done under inert atmosphere.
Preferably, compound (IV) is selected from the group consisting of NaBH 4, DIBAL-H and
mixtures thereof.
Compound (IV) used in reaction (MIV) and compound (IV) used in reaction (MVO) can be
independently from each identical or different.
Preferably, in the reaction (MIV) and in reaction (MVO), from 1 to 50 mol equivalents, more
preferably from 1 to 20 mol equivalents, even more preferably from 2 to 10 mol equivalents,
of compound (IV) are used, the mol equivalents being based the mol of compound of formula
(V) or of formula (Va).
Reaction (MIV) and reaction (MVO) can be done in the presence of a salt (MIV), salt (MIV)
is selected from the group consisting of LiCl, CaCl 2, A1C13, ZnCl 2 and mixtures thereof.
Preferably, if salt (MIV) is used in the reaction (MIV) and in reaction (MVO), from 1 to 20
mol equivalents, more preferably from 1 to 10 mol equivalents, even more preferably from
1.5 to 5 mol equivalents, of salt (MIV) are used, the mol equivalents being based the mol of
compound of formula (V) or of formula (Va).
After reaction (MIV) and reaction (MVO), the compound of formula (IV) or of formula (V0)
respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. The compound of formula (IV) or of formula (V0) respectively can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MII0-I-IVa) and reaction (MlVOa) are similar reactions and can be done under
similar reaction parameters ranges as defined herein, with the individual reaction parameters
for each of said two reactions being selected independently from each other.
Preferably, the reaction time of reaction (MII0-I-IVa) and of reaction (MlVOa) is from 1 min
to 168 h, more preferably from 2 to 144 h, even more preferably from 12 to 120 h .
Reaction (MII0-I-IVa) and reaction (MlVOa) are usually done in a solvent (MlVOa).
Preferably, solvent (MlVOa) is selected from the group consisting of N,Ndimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone,
1,4-dioxane, THF, water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MlVOa) is from 1 to 500 fold, more preferably from 5 to
50 fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (V0).
Preferably, in reaction (MII0-I-IVa) and in reaction (MlVOa), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound (II0-I-IVa) or of compound (RIVOa) respectively are used, the mol
equivalents being based the mol of compound of formula (V0).
Preferably, reaction (MII0-I-IVa) and reaction (MlVOa) are done under inert atmosphere.
Preferably, reaction (MII0-I-IVa) and reaction (MlVOa) are done in the presence of compound
of formula (HOSu).
Preferably, in reaction (MII0-I-IVa) and reaction (MlVOa), from 1 to 20 mol equivalents,
more preferably from 1 to 10 mol equivalents, even more preferably from 1 to 5 mol
equivalents, of compound of formula (HOSu) are used, the mol equivalents being based the
mol of compound (II0-I-IVa) or of compound (RIVOa) respectively.
After reaction (MII0-I-IVa) and reaction (MlVOa), compound of formula (III0-I-IVa) or of
formula (IVOa) respectively can be isolated by standard methods such as washing, extraction,
filtration, concentration and drying. Any of the compounds can be purified before or after
isolation, preferably by chromatography or crystallization from an appropriate solvent.
Further subject of the invention is a method (MVa) for the preparation of compound of
formula (Va), with the compound of formula (Va) as defined herein, also with all its
preferred embodiments;
method (MVa) comprises a step (MVa);
step (MVa) comprises a reaction (MVa), wherein a compound of formula (VI) is reacted with
a compound of formula (SGM);
R30 SG R31 (SGM)
R31 is connected to SG via the bond denoted with (***) in the formulae of SG and is -OTs,
-OMs, -OTf, -Br, -CI or -I;
R30 is as defined herein, also with all its preferred embodiments;
SG, n4, n3, AA 4, (3), Rl, R2 are as defined herein, also with all their preferred embodiments.
Preferably, method (Mil) comprises as a further step the step (MVa), wherein compound of
formula (Va) is prepared.
Preferably, the reaction temperature of reaction (MVa) is from 0 to 150 °C, more preferably
from 20 to 100 °C, even more preferably from 30 to 60 °C.
Preferably, the reaction time of reaction (MVa) is from 1 min to 168 h, more preferably from
1 to 144 h, even more preferably from 12 to 120 h .
Reaction (MVa) is usually done in a solvent (MVa).
Preferably, solvent (MVa) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitnle, acetone, 1,4-dioxane, THF and
mixtures thereof.
Preferably, the amount of solvent (MVa) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (VI).
Preferably, in the reaction (MVa), from 1 to 20 mol equivalents, more preferably from 1 to 10
mol equivalents, even more preferably from 1.5 to 5 mol equivalents, of compound (SGM)
are used, the mol equivalents being based the mol of compound of formula (VI).
Preferably, the reaction (MVa) is done under inert atmosphere.
Reaction (MVa) are usually done in the presence of a base (MVa).
Preferably, base (MVa) is selected from the group consisting of K2C0 3, Na2C0 3,
diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures
thereof.
Preferably, in the reaction (MVa), from 1 to 20 mol equivalents, more preferably from 1 to 10
mol equivalents, even more preferably from 1.5 to 5 mol equivalents, of base (MVa) are used,
the mol equivalents being based the mol of compound of formula (VI).
After the reaction (MVa), compound of formula (Va) can be isolated by standard methods
such as washing, extraction, filtration, concentration and drying. Any of the compounds can
be purified before or after isolation, preferably by chromatography or crystallization from an
appropriate solvent.
Reaction (MVb) and reaction (MIIIO) are similar reactions and can be done under similar
reaction parameters ranges as defined herein, with the individual reaction parameters for each
of said two reactions being selected independently from each other.
Preferably, in reaction (MVb) and in reaction (MIIIO), from 1 to 500 mol equivalents, more
preferably from 5 to 100 mol equivalents, even more preferably from 10 to 50 mol
equivalents, of HC1 are used, the mol equivalents being based the mol of compound of
formula (Va) or of formula (IV0) respectively.
Preferably, the reaction time of reaction (MVb) and of reaction (MIIIO) is from 1 min to 168
h, more preferably from 1 to 48 h, even more preferably from 2 to 24 h .
Reaction (MVb) and reaction (MIIIO) is usually done in a solvent (MVb).
Preferably, solvent (MVb) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF,
water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MVb) is from 1 to 500 fold, more preferably from 5 to 50
fold, even more preferably from 10 to 30 fold, of the weight of compound of formula (Va) or
of formula (IV0).
Preferably, the reaction (MVb) and reaction (MIIIO) is done under inert atmosphere.
After reaction (MVb) and reaction (MIIIO), compound of formula (V) or of formula (III0)
respectively can be isolated by standard methods such as washing, extraction, filtration,
concentration and drying. They can be isolated in protonated form of their salts or in
unprotonated form.
Any of the compounds can be purified before or after isolation, preferably by chromatography
or crystallization from an appropriate solvent.
Further subject of the invention is a method (MVI) for the preparation of compound of
formula (VI), with the compound of formula (VI) being as defined herein, also with all
its preferred embodiments;
method (MVI) comprises a step (MVIa) and optionally a step (MVIb);
in step (MVIa) the n3 AA are consecutively connected to a compound of formula (VII-1) by
peptide coupling reactions and then to the respective products of the preceding peptide
coupling reactions;
step (MVIb) comprises a reaction (MVIb), wherein the N-terminal amino group of AA
denoted with (3) in formula (VI) is reacted with a compound NTermProt;
NTermProt is selected from the group consisting of C1-4 alkyl iodide, C1-4 alkyl bromide,
Cl-C(0)-(GRPEG) m4-R3, R3-C(0)-0-C(0)-R3 and PGNPrec;
PGNPrec is a reagent which provides for the introduction of PGN on the N-terminal amino
group of AA denoted with (3) in formula (VI);
GRPEG, m4, R3 and PGN have the same definition as above, also with all their preferred
embodiments;
n4, n3, AA 4 and (3) are as defined herein, also with all their preferred embodiments.
Preferably, method (Mil) comprises as further steps the step (MVa), wherein compound of
formula (Va) is prepared, and the step (MVIa) and optionally the step (MVIb),
wherein compound of formula (VI) is prepared.
Compound of formula (VII-1) is a known compound and can be prepared by known methods.
For simplicity sake, AA 4 in this text is used both for the covalently bonded amino acid
residue, e.g. in formula (II), and for the amino acid used in method (MVIa).
In case that AA has a side chain with a functional group, this functional group can be
protected by a protecting group commonly used for protecting functional groups of side
chains of amino acids.
Preferably, NTermProt is Ac20 .
Preferably, PG Prec is Boc20 , FmocCl or CbzCl.
Method (MVIa) is done using methodology, parameters and reagents commonly used in
peptide synthesis, and which are known to the skilled person. Above cited references give the
necessary information.
Preferably, the reaction time of reaction (MVIb) is from 1 min to 168 h, more preferably from
1 to 48 h, even more preferably from 2 to 24 h .
Reaction (MVIb) is usually done in a solvent (MVIb).
Preferably, solvent (MVIb) is selected from the group consisting of N,N-dimethylformamide,
N,N-dimethylacetamide, N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF,
water, methanol, ethanol and mixtures thereof.
Preferably, the amount of solvent (MVIb) is from 1 to 500 fold, more preferably from 2 to 50
fold, even more preferably from 5 to 20 fold, of the weight of compound of formula (VII-1).
More preferably, method (MVIa) and reaction (MVIb) are done consecutively in the same
solvent.
More preferably, method (MVIa) and reaction (MVIb) are done consecutively in the same
solvent and in one pot.
Preferably, in the method (MVIa), from 1 to 20 mol equivalents, more preferably from 1 to 10
mol equivalents, even more preferably from 1 to 5 mol equivalents, of NTermProt are used,
the mol equivalents being based the mol of compound of formula (VII-1).
Preferably, the reaction (MVIb) is done under inert atmosphere.
After the method (MVIa) or reaction (MVIb), the reaction product of the method (MVIa) or
of the reation (MVIb), each of which is a respective compound of formula (VI), can be
isolated by standard methods such as washing, extraction, filtration, concentration and drying.
Any of the compounds can be purified before or after isolation, preferably by chromatography
or crystallization from an appropriate solvent.
Compound of formula (SGM) is a known compound and can be prepared according to known
methods.
Preferably, compound of formula (SGM) is selected from the group consisting of compound
SGM-II and compound SGM-III.
Preferably, compound SGM-II is prepared by reacting a compound of formula (HSGH-II)
first with Boc20 and then with compound (SGM-II-R3 1).
Compound (SGM-II-R31) is selected from the group consisting of p-toluenesulfonyl chloride,
p-toluenesulfonic anhydride, methanesulfonyl chloride, methanesulfonic anhydride,
trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride, SOCl2,
(COCl)2, POCI3, PCI3, PCI5, POBr 3, PBr3, PBr , N-bromosuccinimide,
N-iodosuccinimide, HC1, HBr, HI and mixtures thereof.
Preferably, compound (SGM-II-R31) is TsCl.
H2N CH- SGPEG OH (HSGH-II)
mlO ml
Preferably, compound SGM-III is prepared by reacting a compound of formula (HSGH-III)
first with Boc20 and then with a compound of formula (HSGHReac-1).
(HSGHReac-1)
Compound of formula (HSGH-II) is preferably a compound of formula (HSGH-II- 1) or a
compound of formula (HSGH-II-2).
H2 , OH
O' (HSGH-II- 1)
Compound SGM-II is preferably a compound of formula (SGM-II-1) or a compound of
formula (SGM-II-2).
,NH ,OTs (SGM-II-1)
Boc
Bo ,OTs (SGM-II-2)
Compound of formula (HSGH-III) is preferably a compound of formula (HSGH-III- 1) or a
compound of formula (HSGH-III-2).
0
H2N ' (HSGH-III-1)
H2N NH (HSGH-III-2)
Compound SGM-III is preferably a compound of formula (SGM-III-1) or a compound of
formula (SGM-III-2).
Compound of formula (HSGH-II) and compound of formula (HSGH-III) are a known
compound, can be prepared according to known methods, and are often even commercially
available.
Compounds of formulae (CGIMR-IV), (CGIMR-III), (CGlMR-IIa), (HOSu) and compound
(COUPADD) are known compounds, can be prepared by known methods and are
often even commercially available.
Preferably, compound of formula (CGIMR-IV) is prepared by reacting a compound of
formula (CGIMR-IV-OH) with a compound of formula (HOSu);
(CGIMR-IV-OH)
with the compound of formula (HOSu) as defined herein;
preferably, compound of formula (CGIMR-IV-OH) is prepared by reacting compound of
formula (MA) with compound of formula (AC);
with compound of formula (MA) and m30 as defined herein, also with all their preferred
embodiments.
Any of the above defined reactions can be done under similar reaction conditions with the
individual reaction parameters for each of these reactions being selected independently from
each other:
with respect to pressure: any of the above defined reactions can be done under vacuum, at
atmospheric pressure or even under pressure, the pressure can for example be up to 10
bar, preferably they are done under atmospheric pressure;
with respect to temperature: preferably, the reaction temperature of any of the above defined
reactions is from -20 to 100 °C, more preferably from 0 to 75 °C, even more
preferably from 10 to 50 °C;
with respect to reaction time: the reaction time of any of the above defined reactions is from 1
min to 168 h, more preferably from 0.5 to 24 h, even more preferably from 1 to 12 h;
if not stated otherwise for any of the above defined reactions.
In particular, compound of formula (I) is selected from the group consisting of compound of
formula (10), compound of formula ( 11), compound of formula (12), compound of formula
(12-101), compound of formula (13), compound of formula (14), compound of formula (15),
compound of formula (15-102) and compound of formula (16);
53
54
55

wherein doxorubicin is the compound of formula (DOXO), which is connected via the amino
group denoted with (dl) in formula (10), ( 11), (12), (12-101), (13), (14), (15), (15-102) and
(16) respectively and in formula (DOXO).
In particular, compound of formula (II) is selected from the group consisting of compound of
formula (20), compound of formula (21), compound of formula (22), compound of formula
(23), compound of formula (24), compound of formula (25), compound of formula (26),
compound of formula (20-CAMPTO), compound of formula (21-CAMPTO), compound of
formula (22-CAMPTO), compound of formula (23-CAMPTO) and compound of formula
(21-TAXO-tl-l);
doxorubicin

60
5
wherein
doxorubicin is the compound of formula (DOXO), which is connected via the amino group
denoted with (dl) in formula (20), (21), (22), (23), (24), (25) and (26) respectively and
in formula (DOXO);
camptothecin is the compound of formula (CAMPTO), which is connected via the hydroxy
group denoted with (cl) in formula (20-CAMPTO), (21 -CAMPTO), (22-CAMPTO)
and (23-CAMPTO) respectively and in formula (CAMPTO);
taxo-tl-1 is the compound of formula (TAXO), which is connected via the hydroxy group
denoted with (tl) in formula (21-TAXO-tl-l), in formula (TAXO-tl-1) and in formula
(TAXO).
In particular, compound of formula (III) is selected from the group consisting of compound of
formula (30), compound of formula (31), compound of formula (32), compound of formula
(33), compound of formula (34), compound of formula (35) and compound of formula (36).
64
65
66
In particular, compound of formula (IIIO) is compound of formula (300);
camptothecin
wherein
camptothecin is the compound of formula (CAMPTO), which is connected via the hydroxy
group denoted with (cl) in formula (300) respectively and in formula (CAMPTO).
In particular, compound of formula (III0-I-IVa) is compound of formula (320).
In particular, compound of formula (IV) is selected from the group consisting of compound of
formula (40), compound of formula (41), compound of formula (42), compound of formula
(43), compound of formula (44), compound of formula (45) and compound of formula (46).

In particular, compound of formula (IVO) is compound of formula (400);
wherein
camptothecin is the compound of formula (CAMPTO), which is connected via the hydroxy
group denoted with (cl) in formula (400) respectively and in formula (CAMPTO).
In particular, compound of formula (IVOa) is compound of formula (400a).
In particular, compound of formula (V) is selected from the group consisting of compound of
formula (50), compound of formula (50-1), compound of formula (51), compound of formula
(51-1), compound of formula (52), compound of formula (52-1), compound of formula (53),
compound of formula (53-1), compound of formula (54), compound of formula (54-1),
compound of formula (54-2), compound of formula (54-3), compound of formula (55),
compound of formula (55-1), compound of formula (56) and compound of formula (56-1).
73
74
75
76
77
In particular, compound of formula (VI) is selected from the group consisting of compound of
formula (6), compound of formula (6-1), compound of formula (6-2), compound of formula
(6-3), compound of formula (6-4), compound of formula (6b), compound of formula (6b-l),
compound of formula (6b-2), compound of formula (6b-3), compound of formula (6b-4),
compound of formula (6c) and compound of formula (6-5).
BocHN
(6-4)
80
81
Further subject of the invention is a compound selected from the group consisting of
compound of formula (I), compound of formula (II), compound of formula (He), compound
of formula (III), compound of formula (IV), compound of formula (IV-IIa), compound of
formula (V), compound of formula (Va), compound of formula (VI), compound of formula
(III0-IIa), compound of formula (IIIO), compound of formula (IVO), compound of formula
(IVOa), compound of formula (V0) and compound of formula (III0-I-IVa); with these
compounds being as defined herein, also with all their preferred embodiments.
Further subject of the invention is a compound selected from the group consisting of
compound of formula (10), compound of formula ( 11), compound of formula (12), compound
of formula (12-101), compound of formula (13), compound of formula (14), compound of
formula (15), compound of formula (15-102), compound of formula (16), compound of
formula (20c), compound of formula (20), compound of formula (21), compound of formula
(22), compound of formula (23), compound of formula (24), compound of formula (25),
compound of formula (26), compound of formula (20-CAMPTO), compound of formula (21-
CAMPTO), compound of formula (22-CAMPTO), compound of formula (23-CAMPTO),
compound of formula (21-TAXO-tl-l), compound of formula (30), compound of formula
(31), compound of formula (32), compound of formula (33), compound of formula (34),
compound of formula (35), compound of formula (36), compound of formula (300),
compound of formula (320), compound of formula (40), compound of formula (41),
compound of formula (42), compound of formula (43), compound of formula (44), compound
of formula (45), compound of formula (46), compound of formula (400), compound of
formula (400a), compound of formula (50), compound of formula (50-1), compound of
formula (51), compound of formula (51-1), compound of formula (52), compound of formula
(52-1), compound of formula (53), compound of formula (53-1), compound of formula (54),
compound of formula (54-1), compound of formula (54-2), compound of formula (54-3),
compound of formula (55), compound of formula (55-1), compound of formula (56),
compound of formula (56-1), compound of formula (500), compound of formula (6),
compound of formula (6-1), compound of formula (6-2), compound of formula (6-3),
compound of formula (6-4), compound of formula (6b), compound of formula (6b- 1),
compound of formula (6b-2), compound of formula (6b-3), compound of formula (6b-4),
compound of formula (6c) and compound of formula (6-5); with these compounds being as
defined herein.
Further subject of the invention is the use of the compound of formula (I), the compound of
formula (I) being as defined herein, also in all its preferred embodiments, for the
preparation of a pharmaceutical composition or of a drug.
Further subject of the invention is a pharmaceutical composition or a drug, wherein the
pharmaceutical composition and the drug comprise the compound of formula (I), the
compound of formula (I) being as defined herein, also in all its preferred
embodiments.
Further subject of the invention is a compound of formula (I), a pharmaceutical composition
or a drug, wherein the pharmaceutical composition and the drug comprise the
compound of formula (I), the compound of formula (I) being as defined herein, also in
all its preferred embodiments,
for use in treatment of a disease or an illness, preferably of cancer.
Compound of formula (II) can be readily covalently attached to a ligand LI. It was surprising,
that the protein drug conjugates of the instant invention, which comprise a connecting group
CG2, CG2 being derived from o-hydrox-p-amino benzylic alcohol, and which comprise a
linear peptide residue, in particular the compounds of formula (I), show increased plasma
stability, and release the drug without the drug being chemically modified, Furtehrmore, they
show good water solubility and low aggregation.
Abbreviations
DCM dichoromethane
DIBAL-H diisobutylaluminium hydride
DIPEA N,N-diisopropylethylamine
DMA N,N-dimethylacetamide
DMAP 4-dimethylaminopyridine
DMF N,N-dimethylformamide
EDTA ethylenediaminetetraacetic acid
EEDQ 2-ethoxy- 1-ethoxycarbonyl- 1,2-dihydroquinoline
ESI-MS electrospray ionisation mass spectrometry
EtOAc ethyl acetate
HIC hydrophobic interaction chromatography
MeCN acetonitrile
NAP-25 column NAP-25 columns of GE Healthcare are disposable columns prepacked
with Sephadex™ G-25 DNA Grade and require only gravity to run
NMR nuclear magnetic resonance
PE petroleum ether
Rf retention factor in TLC
RP-HPLC reversed phase HPLC
RT room temperature
SAFC Sigma Aldrich Fine Chemicals
SEC-HPLC size exclusion chromatography HPLC
TBTU 2-(lH-benzotriazole-l-yl)-l, 1,3,3-tetramethylaminium tetrafluorob orate
THF tetrahydrofurane
TFA Trifluoroacetic acid
TLC thin layer chromatography
Raw Materials
compound of formula (DOXO) doxorubicin, commercially available as hydrochloride
salt from Beijing Zhongshuo Pharmaceutical Technology
Development Co. Ltd.
daunorubicin commercially available as hydrochloride salt from
Aldrich
PBS The PBS used in the experiments had the composition
KH2P04: 144 mg/L, NaCl: 9000 mg/L and Na2HP04:
795 mg/L
Example 1
To a mixture of p-amino salicylic acid (15.0 g) and MeOH ( 113.0 ml) at 0 °C, cone. H2SO4
(30.0 ml) was added dropwise. The resulting mixture was heated to reflux and stirred for 2
hours to form a homogeneous solution. The reaction mixture was then cooled to RT. Water
(360 ml) was added, followed by solid NaHC0 3 until pH 7 . The resulting mixture was
filtered, the wet cake was washed with water (3 times with 80 mL each) and dried under
vacuum at 55 °C to afford 14.7 g of compound of formula (VII-1) as a solid (89% yield).
1H MR (400 MHz, CDC13, 20 °C) delta 3.90 (1H, s), 4.12 (2H, brs), 6.16 to 6.19 (2H, m),
7.62 to 7.65 (1H, m).
ESI-MS: 168.0 (M+H)+
Example 2
To a mixture of H-Cit-OH (40.0 g, 1.0 eq.) and Na2C0 3 (50.0 g, 2.0 eq.) in water (300 ml)
and THF (150 ml), a solution of di-tert-butyl dicarbonate (60.0 g, 1.2 eq.) in THF (100 ml)
was added dropwise within 1 hour. The resulting mixture was allowed to stir at RT overnight.
After that, the suspension was washed with PE (2 times with 150 ml each), then the mixture
was concentrated to about 300 ml under vacuum. The mixture was acidified to pH 2 with 4.0
M aqueous KHSO 4, then extracted with EtOAc (5 times with 150 ml each). The organic
phases were combined and washed with saturated brine (100 ml), dried over anhydrous
Na2S0 4 and filtered. The filtrate was evaporated to dryness to afford 52.0 g of Boc-Cit-OH as
white solid (83 % yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 1.38 (9H, s), 1.36 to 1.43 (2H, m), 1.47 to 1.69
(2H, m), 2.93 (2H, q, J = 6.3 Hz), 3.82 to 3.87 (1H, m), 5.36 (2H, brs), 5.92 (1H, t, J = 5.6
Hz), 7.07 (1H, d, J = 8.0 Hz), 12.44 (1H, brs).
ESI-MS: 275.8 (M+H)+ , 550.8 (2M+H)+
Example 3
Boc-Cit-OH (53.0 g, 1.0 eq.), prepared according to example 2, and EEDQ (72.0 g, 1.5 eq.)
were added to THF (400 ml). To this mixture was then added compound of formula (VII-1)
(49.0 g, 1.5 eq.), prepared according to example 1. The resulting mixture was stirred at RT for
14 hours. Then the reaction mixture was diluted with water (500 ml), extracted with EtOAc (3
times with 250 ml each). The organic phases were combined and washed with 1.0 M aqueous
solution of citric acid (2 time with 150 ml each) and saturated brine (150 ml). After that, the
organic phase was dried over anhydrous Na2S0 4, then concentrated to dryness, the crude
product was purified by silica gel chromatography (eluent PE : EtOAc = 4:l to 2:l to l:l
(v/v) to pure EtOAc) and isolated to afford 62.5 g of compound of formula (6-4) as yellow
solid (76 % yield).
Analysis by silica gel TLC: EtOAc as eluent (Rf = 0.3, UV254)
1H MR (400 MHz, CDC13, 20 °C) delta 1.40 (9H, s), 1.57 to 1.83 (4H, m), 3.08 to 3.16 (2H,
m), 3.90 (3H, s), 4.43 (1H, s), 5.26 (2H, s), 5.85 (2H, s), 7.1 1 (1H, d, J = 8.8 Hz), 7.28 (1H, s),
7.68 (1H, d, J = 8.8 Hz), 9.74 (1H, s), 10.82QH, brs).
ESI-MS: 325.2 (M-tBuOCO+2H)+
Example 4
Compound of formula (6-4) (62.0 g, 1 eq.), prepared according to example 3, was suspended
in a solution of 15% (w/w) HC1 in 1,4-dioxane (100 ml), the resulting mixture was stirred at
RT for 1 hour. The reaction mixture was then concentrated under vacuum to afford 51.6 g of
compound of formula (6-3) as HC1 salt, being a white solid (98 % yield).
Example 5
Compound of formula (6-3) as HC1 salt (75.6 g, 1.0 eq.), prepared according to example 4,
Boc-L-Val (43.0g, 1.0 eq.) and TBTU (135.5 g, 2.0 eq.) were dissolved in DMF (250 ml).
Then DIPEA (71.2 g, 2.6 eq.) was added. The resulting solution was stirred at RT for 17 h .
The reaction mixture was then diluted with water (750 ml), extracted with EtOAc (5 times
with 200 ml each), the organic phases were combined and washed with 1.0 M aqueous
NaHC0 3 solution (3 times with 300 ml each) and then with saturated brine (150 ml). The
organic phase was collected and concentrated to dryness, the crude product was then purified
by silica gel chromatography (eluent DCM : MeOH = 20 : 1 to 10 : 1 to 7 : 1 (v/v) and
isolated to afford 62.0 g of compound of formula (6-2) as a solid (57% yield).
Analysis by silica gel TLC: DCM : MeOH = 6 : 1 (v/v) as eluent (Rf = 0.4, UV254)
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.84 (6H, dd, J l = 17.2 Hz, J2 = 6.8 Hz), 1.39
(9H, s), 1.42 to 1.51 (2H, m), 1.55 to 1.73 (2H, m), 1.93 to 1.98 (1H, m), 2.90 to 3.08 (2H,
m), 3.82 to 3.84 (1H, m), 3.88 (3H, s), 4.39 to 4.45 (1H, m), 5.43 (2H, s), 6.01 (1H, t, J = 5.8
Hz), 6.72 (1H, d, J = 8.8 Hz), 7.09 (1H, dd, J l = 8.8 Hz, J2 = 2.0 Hz), 7.73 (1H, d, J = 8.8
Hz), 8.08 (1H, d, J = 7.2 Hz), 10.34 (1H, s), 10.62 (1H, brs).
ESI-MS: 524.3 (M+H)+, 424.3 (M-tBuOCO+2H)+
Example 6
Compound of formula (6-2) (62.0 g, 1 eq.), prepared according to example 5, was suspended
in a solution of 15% (w/w) HC1 in 1,4-dioxane (200 ml), the resulting mixture was stirred at
RT for 1 hour. The reaction mixture was then concentrated under vacuum to afford 52.4 g of
compound of formula (6-1) as HC1 salt, being a white solid (97% yield).
Example 7
Compound of formula (6-1) as HC1 salt (52.4 g, 1.0 eq.), prepared according to example 6,
acetic anhydride (60.0 g, 5.0 eq.), pyridine (100.0 g, 11.0 eq.) and methanol (150 ml) were
mixed and stirred at RT for 7 days. The suspension was filtered and the resulting wet cake
was washed with MeOH (4 times with 200 ml each), then dried under vacuum to afford 32.9
g of compound of formula (6) as a white solid (62 % yield).
1H NMR (400 MHz, DMSO-d 6, 20 °C) delta 0.86 (6H, dd, J l = 12.4 Hz, J2 = 6.8 Hz), 1.35 to
1.50 (2H, m), 1.56 to 1.74 (2H, m), 1.87 (3H, s), 1.94 to 2.00 (1H, m), 2.93 to 3.06 (2H, m),
3.87 (3H, s), 4.20 (1H, t, J = 7.6 Hz), 4.34 to 4.39 (1H, m), 5.43 (2H, s), 6.00 (1H, t, J = 5.2
Hz), 7.1 1 (1H, dd, J l = 8.8 Hz, J2 = 1.6 Hz), 7.41 (1H, d, J = 1.6 Hz), 7.73 (1H, d, J = 8.8
Hz), 7.89 (1H, d, J = 8.4 Hz), 8.21 (1H, d, J = 7.2 Hz), 10.24 (1H, s), 10.62 (1H, s).
ESI-MS: 466.3 (M+H)+ , 931.3 (2M+H)+
Example 8
Compound of formula (MA) (5.5 g, 1.0 eq.) and beta-alanine (5.0 g, 1.0 eq.) in DMF (30.0
ml), were stirred under nitrogen atmosphere for 2 hours. The mixture was then cooled to 0 °C.
Compound of formula (HOSu) (8.0 g, 1.3 eq.) and DCC (24.0 g, 2.0 eq.) were added. Then
the reaction mixture was allowed to warm up to RT and stirred at RT overnight. The reaction
mixture was then filtered, the resulting wet cake was washed with DMF (40.0 ml), the organic
phases were combined and then diluted with water (120 ml) and extracted with DCM (3 times
with 50 ml each). The organic phases were combined, washed with water (50 ml), then with
5% (w/w) aqueous NaHC03 solution (50 ml) and then with saturated brine (50 ml). The
organic phase was dried over anhydrous Na2S0 4, then concentrated until solid started to
precipitate. Then PE (20 ml) was added to the mixture and the resulting mixture was stirred at
RT for 10 min. The mixture was then filtered, the wet cake was washed with PE (20 ml) and
then dried under vaccum at 40 °C overnight to afford 4.0 g of compound of formula
(CGlMR-IV-1) as a white solid (27 % yield).
(CGlMR-IV-1)
1H MR (400 MHz, CDC13, 20 °C) delta 2.84 (4H, s), 3.04 (2H, t, J = 7.0 Hz), 3.95 (2H, t, J
= 7.0 Hz), 6.75 (2H, s).
ESI-MS: 267.2 (M+H)+ , 289.4 (M+Na)+
Example 9
To a mixture of compound of formula (HSGH-II-1) (10.0 g, 1.0 eq.), MeOH (50 ml) and
Et3N (10.7 g, 1.1 eq.), a solution of Boc20 in MeOH (22.8 g, 1.1 eq. Boc20 in 50 ml MeOH)
was added dropwise. Then the resulting mixture was stirred at RT for 15 hours and then dried
under vacuum to afford 20.0 g of compound of formula (Boc-HSGH-II-1) as a slightly yellow
oil (quantitative yield).
1H NMR (400 MHz, CDC13, 20 °C) delta 1.44 (9H, s), 2.73 (1H, brs), 3.32 (2H, q, J = 5.2
Hz), 3.54 to 3.58 (4H, m), 3.72 to 3.74 (2H, m), 5.15 (1H, brs).
Example 10
To a mixture of compound of formula (Boc-HSGH-II-1) (6.17 g, 1 eq.), prepared according to
example 9, pyridine (2.86 g, 1.2 eq) and DCM (30 ml) at 0 °C, DMAP (0.366 g, 0.1 eq) were
added. A mixture of tosyl chloride (6.31 g, 1.1 eq) and DCM (45 ml) was added dropwise.
The resulting mixture was stirred at RT for 8 days. Then the resulting mixture was poured
onto DCM (100 ml), the resulting mixture was washed with water ( 1 time with 100 ml), the
organic phase was then dried over anhydrous Na2S0 4. The resulting solution was further
concentrated and purified by silica gel chromatography (PE : EtOAc = 6 : 1 to PE : EtOAc =
1 : 1 (v/v)) to afford 8.1 g of compound of formula (SGM-II-1) as a colorless oil (75% yield).
1H NMR (400 MHz, DMSO-d 6, 20 °C) delta 0.86 (6H, dd, J l = 12.4 Hz, J2 = 6.8 Hz), 1.35 to
1.50 (2H, m), 1.56 to 1.74 (2H, m), 1.87 (3H, s), 1.94 to 2.00 (1H, m), 2.93 to 3.06 (2H, m),
3.87 (3H, s), 4.20 (1H, t, J = 7.6 Hz), 4.34 to 4.39 (1H, m), 5.43 (2H, s), 6.00 (1H, t, J = 5.2
Hz), 7.1 1 (1H, dd, J l = 8.8 Hz, J2 = 1.6 Hz), 7.41 (1H, d, J = 1.6 Hz), 7.73 (1H, d, J = 8.8
Hz), 7.89 (1H, d, J = 8.4 Hz), 8.21 (1H, d, J = 7.2 Hz), 10.24 (1H, s), 10.62 (1H, s).
Example 11
A mixture of compound of formula (6) (3.00 g, 1 eq), prepared according to example 7,
compound of formula (SGM-II-1) (4.65 g, 2 eq), prepared according to example 10, K2C0 3
(1.82 g, 2 eq) and anhydrous DMF (30 ml) was heated to 50 °C and stirred under nitrogen
atmosphere for 7 days. Then the reaction mixture was concentrated to dryness under vacuum.
To the residue methanol (30 ml) was added, the resulting mixture was stirred for 10 min and
then filtered. The wet cake was washed with methanol (3 times with 10 ml each). The organic
phase were combined and then concentrated. The residue was purified by silica gel column
chromatography (DCM : MeOH = 7 : 1 (v/v)) to afford 3.40 g of compound of formula (50-1)
as a slightly yellow solid (81% yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) d 0.86 (6H, dd, J l = 12.8 Hz, J2 = 6.8 Hz), 1.38 to
1.51 (2H, m), 1.56 to 1.80 (2H, m), 1.89 (3H, s), 1.92 to 2.01 (1H, m), 2.92 to 3.05 (2H, m),
3.07 to 3.12 (2H, m), 3.50 (2H, t, J = 6.0 Hz), 3.75 to 3.77 (5H, m), 4.08 to 4.10 (2H, m), 4.20
(1H, dd, J l = 8.4 Hz, J2 = 6.8 Hz), 4.34 to 4.40 (1H, m), 5.42 (2H, s), 5.99 (1H, t, J = 5.8 Hz),
6.74 (1H, t, J = 5.6 Hz), 7.26 (1H, dd, J l = 8.4 Hz, J2 = 1.6 Hz), 7.49 (1H, d, J = 2.0 Hz), 7.68
(1H, d, J = 8.8 Hz), 7.91 (1H, d, J = 8.4 Hz), 8.19 (1H, d, J = 7.6 Hz),10.20 (1H, s).
ESI-MS: 653.4 (M+H)+ , 675.5 (M+Na)+, 553.5 (M-tBuOCO+2H)+
Example 12
A mixture of compound of formula (50-1) (500 mg), prepared according to example 11, and a
solution of 10% (w/w) HC1 in 1,4-dioxane (10 ml) was stirred at RT for 15 hours. The solvent
was removed under vacuum to afford 43 1mg of compound of formula (50) as HC1 salt, being
a slightly yellow solid (quantitative yield).
ESI-MS: 553.5 (M+H)+ , 1105.2 (2M+H)+
Example 13
To a mixture of compound of formula (50) as HC1 salt (1.10 g, 1.0 eq), prepared according to
example 12, and anhydrous THF (20 ml) at -30 °C was added a solution of DIBAL-H in
hexanes ( 1 M, 11.9 ml, 6.0 eq). The resulting mixture was then heated to 0 °C and stirred
under nitrogen atmosphere for 15 hours. Then methanol (2.0 mL) was added. Then saturated
aqueous potassium sodium tartrate solution (10 ml) was added to the mixture and the mixture
was stirred for 30 min at RT. The resulting mixture was evaporated to dryness to generate a
white residue which was washed with methanol (5 times with 10 ml each). The combined
organic phases were concentrated and purified by silica gel column chromatography (DCM :
MeOH : Et3N = 65 : 33 : 2 (v/v)) to afford 0.65 g of compound of formula (40) as a white
solid (72% yield).
1H MR (400 MHz, DMSO-d 6, 60 °C) delta 0.86 (6H, t, J = 6.6 Hz), 1.37 to 1.53 (2H, m),
1.63 to 1.79 (2H, m), 1.92 (3H, s), 1.99 to 2.07 (1H, m), 2.70 (2H, brs), 2.99 to 3.02 (2H, m),
3.47 (2H, t, J = 5.6 Hz), 4.07 (2H, t, J = 5.8 Hz), 4.18 (1H, t, J = 7.6 Hz), 4.34 to 4.41 (1H,
m), 5.25 (2H, s), 5.90 (1H, brs), 7.16 (1H, d, J = 8.0 Hz), 7.26 (2H, d, J = 8.0 Hz), 7.32 (1H,
s), 7.76 (1H, d, J = 7.6 Hz), 7.91 (1H, d, J = 4.4 Hz), 9.69 (1H, s).
ESI-MS: 525.6 (M+H)+
Example 14
To a mixture of compound of formula (40) (500.2 mg, 1.0 eq.), prepared according to
example 13, compound of formula (CGlMR-IV-1) (281.0 mg, 1.1 eq), prepared according to
example 8, and DMF (9.5 ml) at RT, DIPEA (140.3 mg, 1.1 eq) was added. The resulting
mixture was stirred at RT for 17 hours. Then the DMF was removed under vacuum to afford a
slightly yellow residue. The residue was then mixed with acetone (10 ml) and stirred at RT
for 18 h . The mixture was filtered, the wet cake was washed with acetone (2 times with 5 ml
each), then dried under vacuum to afford 515.0 mg of compound of formula (30) as a slightly
yellow solid (80% yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.86 (6H, dd, J l = 11.2 Hz, J2 = 6.8 Hz), 1.32 to
1.48 (2H, m), 1.55 to 1.76 (2H, m), 1.89 (3H, s), 1.94 to 2.02 (1H, m), 2.33 (2H, t, J = 7.2
Hz), 2.93 to 3.04 (2H, m), 3.16 to 3.20 (2H, q, J = 5.6 Hz), 3.46 (2H, t, J = 5.8 Hz), 3.60 (2H,
t, J = 7.8 Hz), 3.73 (2H, t, J = 4.6 Hz), 4.04 (2H, t, J = 4.6 Hz), 4.18 (1H, dd, J l = 8.4 Hz, J2 =
6.8 Hz), 4.34 to 4.39 (1H, m), 4.44 (2H, s), 4.88 (1H, brs), 5.42 (2H, s), 6.00 (1H, t, J = 5.4
Hz), 7.00 (2H, s), 7.16 (1H, dd, J l = 8.4 Hz, J2 = 2.0 Hz), 7.26 (1H, d, J = 8.4 Hz), 7.32 (1H,
d, J = 1.6 Hz), 7.92 (1H, d, J = 8.4 Hz), 8.05 (1H, t, J = 5.6 Hz), 8 .12 (1H, d, J = 7.6 Hz), 9.88
(1H, s).
Example 15
A mixture of compound of formula (30) (400 mg, 1.0 eq), prepared according to example 14,
DIPEA (231.0 mg, 3.0 eq), 4 angstrom molecular sieves (800 mg) and dry DMF (8.0 ml) was
stirred for 5 min. Then compound of formula (II- 1) (361.2 mg, 2 eq) was added. The resulting
mixture was stirred for 3 h at RT. Then compound of formula (DOXO) as HC1 salt (342.8 mg,
1.0 eq) was added and the mixture was stirred for 4 hours. Then MeCN (40.0 ml) was added.
A precipitate had formed and was filtered and washed with a mixture of MeCN and DMF (5 :
1 (v/v), 2 times with 5 ml each). The filtrates were combined and dried under vacuum at 45
°C to get a dark red residue. The residue was washed with MeCN (2 times with 10 ml each),
then the residue was dissolved in a mixture of acetone and water (20 : 1 (v/v)) and purified by
preparative silica gel TLC (DCM :MeOH = 5 : 1 (v/v), Rf = 0.15). The product was extrated
from the silica-gel by a mixture of acetone and water (20 : 1 (v/v), 6 times with 20 ml each),
the combined extraction solutions were dried under vacuum to afford a crude product as a red
solid. The crude product was then mixed with acetonitrile (5.0 ml), the mixture was stirred at
RT for 5 hours and then filtered. The presscake was mixed with acetonitrile (5.0 ml) and the
mixture stirred for 3 hours at RT. The mixture was then filtered. The presscake was dried
under vacuum at RT to afford 48.0 mg of compound of formula (20) as a red solid (7% yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.85 (6H, dd, J l = 11.2 Hz, J2 = 6.8 Hz), 1.13
(3H, d, J = 6.4 Hz), 1.34 to 1.47 (3H, m), 1.57 to 1.72 (2H, m), 1.82 to 1.90 (1H, m), 1.90
(3H, s), 1.93 to 1.98 (1H, m), 2.08 to 2.22 (2H, m), 2.29 (2H, t, J = 7.2 Hz), 2.91 to 3.01 (4H,
m), 3.1 1 to 3.16 (2H, m), 3.41 (2H, t, J = 5.6 Hz), 3.45 to 3.46 (1H, m), 3.54 (2H, t, J = 7.2
Hz), 3.68 to 3.74 (3H, m), 3.98 (3H, s), 4.03 (2H, t, J = 4.0 Hz), 4.17 (2H, t, J = 7.6 Hz), 4.32
to 4.38 (1H, m), 4.58 (2H, d, J = 5.2 Hz), 4.69 (2H, d, J = 5.6 Hz), 4.85 to 4.89 (2H, m), 4.94
(1H, brs), 5.22 (1H, brs), 5.40 (2H, s), 5.46 (1H, brs), 5.98 (1H, t, J = 4.8 Hz), 6.80 (1H, d, J =
8.0 Hz), 6.95 (2H, s), 7 .12 (1H, d, J = 8.4 Hz), 7.19 (1H, d, J = 8.4 Hz), 7.36 (1H, s), 7.64
(1H, brs), 7.89 to 7.91 (3H, m), 7.99 (1H, t, J = 5.2 Hz), 8.10 (1H, d, J = 7.2 Hz), 9.92 (1H, s),
13.26 (1H, s), 14.02 (1H, s).
ESI-MS: 1245.5 (M+H)+
Example 16
To a mixture of compound of formula (HSGH-III-1) ( 110.16 g, 5 eq) and dioxane (400 ml) at
0 °C, a mixture of Boc20 (22.10 g, 1 eq) in dioxane (200 ml) was added dropwise. The
resulting mixture was then heated to RT and then stirred for 20 hours. Then the solvent was
removed under vacuum. The resulting residue was added to water (300 ml), the resulting
mixture was extracted with DCM (2 times with 300 ml each). The organic layer was dried
over anhydrous Na2S0 4 and then evaporated to dryness. The residue was purified by silica gel
column chromatography (DCM : EtOAc = 1 : 1 (v/v), then with DCM :MeOH : Et3N = 89 : 9
: 2 (v/v)) to afford 17.7 g of compound of formula (Boc-HSGH-III-1) as a slightly yellow oil
(55% yield).
H O O N (Boc-HSGH-III-1)
2 H
1H MR (400 MHz, CDC13, 20 oC) d 1.44 (9H, s), 1.64 (2H, s), 1.72-1.78 (4H, m), 2.81
(2H, t, J = 6.6 Hz), 3.23 (2H, q, J = 6.0 Hz), 3.55-3.62 (8H, m), 3.64-3.66 (4H, m), 5.14 (1H,
brs).
Example 17
To a mixture of compound of formula (Boc-HSGH-III-1) (5.00 g, 1.0 eq), prepared according
to example 18, K2C0 3 (4.30 g, 2.0 eq) and DCM (40 ml) at 0 °C, a mixture of compound of
formula (HSGHReac-1) (2.17 g, 1.2 eq) in DCM (20 ml) was added dropwise within 1 hour.
The resulting mixture was warmed to RT and stirred for 20 hours. Then the solid was filtered,
the cake was washed with DCM (2 times with 5 ml each). The filtrates were combined and
evaporated to dryness. The residue was then purified by silica gel column chromatography
(eluent EtOAc : PE = 3 : 1) to afford 5.6 g of compound of formula (SGM-III-1) as a slightly
yellow oil (90 % yield).
1H MR (400 MHz, CDC13, 20 oC) d 1.44 (9H, s), 1.64 (2H, s), 1.73-1.86 (4H, m), 3.23
(2H, q, J = 6.0 Hz), 3.44 (2H, q, J = 6.0 Hz), 3.54 (2H, t, J = 6.0 Hz), 3.58-3.66 (10H, m),
4.04 (2H, s), 4.98 (1H, brs), 7.31 (1H, brs).
Example 18
Compound of formula (6) (5.80 g, 1.0 eq), prepared according to example 7, K2C0 3 (5.18 g,
2.0 eq), compound of formula (SGM-III-1) (9.95 g, 2.0 eq), prepared according to example
17, and DMF (45 ml) were mixed. The resulting mixture was then heated to 50 °C and stirred
for 7 days under nitrogen atmosphere. The resulting reaction mixture was evaporated to
dryness under vacuum. Methanol (40 ml) was added to the residue, the resulting mixture was
stirred for 10 min and then filtered. The wet cake was washed with methanol (3 times with 10
ml each). The organic filtrates were collected and combined and then evaporated. The residue
was purified by silica gel column chromatography (DCM :MeOH = 7 : 1 (v/v)) to afford 7.20
g of compound of formula (51-1) as a slightly yellow solid (70% yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.86 (6H, dd, J l = 13.6 Hz, J2 = 6.8 Hz), 1.32 to
1.49 (2H, m), 1.37 (9H, s), 1.55 to 1.78 (6H, m), 1.89 (3H, s), 1.94 to 1.99 (1H, m), 2.93 to
3.07 (4H, m), 3.26 (2H, q, J = 6.8 Hz), 3.37 (2H, t, J = 6.4 Hz), 3.43 to 3.52 (10H, m), 3.81
(3H, s), 4.20 (1H, dd, J l = 8.4 Hz, J2 = 6.8 Hz), 4.35 to 4.41 (1H, m), 4.53 (2H, s), 5.44 (2H,
s), 6.03 (1H, t, J = 5.6 Hz), 6.74 (1H, t, J = 5.6 Hz), 7.36 (1H, dd, J l = 8.4 Hz, J2 = 2.0 Hz),
7.41 (1H, d, J = 2.0 Hz), 7.80 (1H, d, J = 8.8 Hz), 7.90 (1H, d, J = 8.4 Hz ), 8.06 (1H, t, J =
5.6 Hz), 8.21 (1H, d, J = 7.2 Hz), 10.32 (1H, s).
ESI-MS: 726.6 (M-tBuOCO+2H)+, 826.3 (M+H)+, 848.5 (M+Na)+
Example 19
Compound of formula (51-1) (1.00 g), prepared according to example 18, and a solution of
15% (v/v) HC1 in 1,4-dioxane (10 ml) were mixed. The mixture was stirred at RT for 3 hours.
The solvent was removed under vacuum to afford 0.99 g of compound of formula (51) as HC1
salt, being a slightly yellow solid (quantitative yield).
ESI-MS: 726.6 (M+H)+
Example 20
A mixture of compound of formula (51) as HC1 salt (6.08 g, 1 eq), prepared according to
example 19, water (100 ml), CaCl2 (1.86 g, 2.0 eq), and NaBH 4 (1.27 g, 4.0 eq) was stirred at
RT. Further NaBH 4 was added portion wise (1.27 g, 4.0 eq. after a total of 15 hours stirring;
1.27 g, 4.0 eq. after a total of 20 hours stirring; 1.27 g, 4.0 eq. after a total of 24 hours of
stirring). After a total of 36 hours of stirring, MeOH (30 ml) was added to the mixture. The
reaction mixture was then filtered and the wet cake was washed with MeOH (3 times with 10
ml each). The liquid phase was collected, combined and then evaporated to dryness. The
residue was purified by silica gel column chromatography (eluent DCM :MeOH : Et3N = 65
: 33 : 2 (v/v)) to afford 2.70 g of compound of formula (41) as a slightly yellow solid (48%
yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.86 (6H, dd, J l = 10.8 Hz, J2= 6.8 Hz), 1.31 to
1.49 (2H, m), 1.55 to 1.82 (6H, m), 1.90 (3H, s), 1.93 to 2.02 (1H, m), 2.82 (2H, t, J = 7.4
Hz), 2.98 to 3.05 (2H, m), 3.15 to 3.20 (2H, q, J = 6.8 Hz), 3.34 to 3.51 (12H, m), 4.16 to 4.20
(1H, m), 4.34 to 4.41 (1H, m), 4.46 (2H, s), 4.50 (2H, s), 5.47 (2H, s), 6.13 (1H, t, J = 5.6 Hz),
7.25 (2H, s), 7.26 (1H, s), 7.95 (1H, d, J = 8.8 Hz), 8.10 (1H, t, J = 5.6 Hz), 8.14 (1H, d, J =
7.6 Hz), 10.01 (1H, s).
ESI-MS: 698.7 (M+H)+
Example 21
Compound of formula (41) (2.78 g, 1.0 eq.), prepared according to example 20, compound of
formula (CGlMR-IV-1) (1.18 g, 1.1 eq), prepared according to example 8, and DMF (30 ml)
were mixed at RT. Then DIPEA (0.58 g, 1.1 eq) was added. The resulting mixture was stirred
at RT for 16 hours. Then DMF was removed under vacuum to afford a slightly yellow
residue. The residue was then mixed with acetone (30 ml) and the mixture was stirred at RT
for 5 hours. Then the mixture was filtered, the wet cake was washed with acetone (2 times
with 15 ml each) and then dried under vacuum to afford 2.55 g of compound of formula (3 1)
as a slightly yellow solid (75% yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.86 (6H, dd, J l = 11.2 Hz, J2 = 6.8 Hz), 1.32 to
1.48 (2H, m), 1.55 to 1.73 (2H, m), 1.89 (3H, s), 1.93 to 2.01 (1H, m), 2.31 (2H, t, J = 7.2
Hz), 2.90 to 3.1 1 (4H, m), 3.17 (2H, q, J = 6.4 Hz), 3.36 (2H, t, J = 6.4 Hz), 3.44 to 3.60 (8H,
m), 3.60 (2H, t, J = 7.2 Hz), 4.19 (1H, dd, J l = 8.4 Hz, J2 = 6.8 Hz), 4.35 to 4.40 (1H, m),
4.46 (2H, s), 4.51 (2H, s), 5.08 (1H, brs), 5.42 (2H, s), 6.00 (1H, t, J = 5.6 Hz), 7.00 (2H, s),
7.22 (1H, s), 7.25 (1H, s), 7.89 to 7.91 (1H, m), 8.04 (1H, t, J =5.6 Hz), 8.10 (1H, d, J = 7.6
Hz), 9.92 (1H, s).
ESI-MS: 831.6 (M-OH)+ ,849.4 (M+H)+
Example 22
A mixture of compound of formula (31) (500.4 mg, 1.0 eq), prepared according to example
21, DIPEA (305.7 mg, 4.0 eq), 4 angstrom molecular sieves (500.5 mg) and dry DMF (10.0
ml) was stirred for 5 min. Then compound of formula (II- 1) (271.1 mg, 1.5 eq.) was added.
The resulting mixture was stirred for 5 hours at RT. Then compound of formula (DOXO) as
HC1 salt (342.8 mg, 1.0 eq.) was added and the resulting mixture was stirred for 3.5 hours.
Then MeCN (50.0 ml) was added. A precipitate had formed and was filtered and washed with
a mixture of MeCN with DMF (5 : 1 (v/v), 3 times with 10 ml each). The filtrates were
combined and dried under vacuum at 45 °C to afford a dark red residue. The residue was
dissolved in a mixture of DCM with MeOH (7 : 1 (v/v)) and purified by preparative silica gel
TLC (DCM :MeOH = 7 : 1 (v/v), Rf = 0 . 15). The product was extrated from the silica gel by
a mixture of acetone with water (20 : 1 (v/v), 5 times with 50 ml each), the combined extracts
were dried under vacuum to afford the crude product as a red solid. The crude product was
then mixed with acetonitrile (30 ml), the mixture was stirred at RT for 18 hours and was then
filtered. The cake was mixed with acetonitrile (10 ml) and the mixture was stirred for 3 hours
at RT. The mixture was then filtered. The cake was dried under vacuum at RT to afford 100.3
mg of compound of formula (21) as a red solid (12 % yield).
1H MR (400 MHz, DMSO-d 6, 20 °C) delta 0.85 (6H, dd, J l = 11.8 Hz, J2 = 6.8 Hz), 1.13
(3H, d, J = 6.4 Hz), 1.34 to 1.68 (9H, m), 1.82 to 1.90 (1H, m), 1.90 (3H, s), 1.93 to 2.00 (1H,
m), 2.13 to 2.24 (2H, m), 2.30 (2H, t, J = 7.2 Hz), 2.91 to 3.04 (6H, m), 3.10 to 3.15 (2H, m),
3.29 (2H, t, J = 6.0 Hz), 3.37 to 3.49 ( 11H, m), 3.59 (2H, t, J = 7.2 Hz), 3.68 to 3.76 (1H, m),
3.99 (3H, s), 4.13 to 4.19 (2H, m), 4.33 to 4.41 (3H, m), 4.58 (2H, d, J = 5.6 Hz), 4.71 (1H, d,
J = 5.2 Hz), 4.86 (1H, t, J = 5.8 Hz), 4.95 (1H, brs), 5.02 (2H, s), 5.23 (1H, brs), 5.41 (2H, s),
5.46 (1H, brs), 5.99 (1H, t, J = 4.6 Hz), 6.87 (1H, d, J = 7.2 Hz), 7.00 (2H, s), 7.21 to 7.25
(3H, m), 7.66 (1H, t, J = 4.6 Hz), 8.1 1 (1H, d, J = 7.2 Hz), 9.97 (1H, s), 13.28 (1H, s), 14.04
(1H, s).
ESI-MS: 1417.8 (M+H)+

Claims
1. A method (MI) for connecting a ligand LI with a drug DR,
LI is selected from the group consisting of amino acids LI-AA, mono- or polyclonal
antibodies LI-Ab, antibody fragments LI-AbFrag, proteins Ll-Prot and peptides Li-Pep;
DR is a pharmaceutically active drug;
characterized that a linker LIN is used to covalently connect LI with DR;
LIN comprises a connecting group CG2;
CG2 is derived from o-hydroxy p-amino benzylic alcohol and is a connecting group of formula
(CG2-1);
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 has
2 to 8 amino acid residues;
(4) denotes the p-amino group of the o-hydroxy p-amino benzylic alcohol from which
CG2 is derived.
2 . Method (MI) according to claim 1,
wherein LI is covalently connected with DR in form of a compound of formula (I);
CGI SG -CG2— T4 -CG3 -T2 DRRes
nl n2 n2
Tl
LIRes
Rl R2
CG2 is as defined in claim 1;
is a connecting group selected from the group consisting of connecting group of formula
(CGI -I), connecting group of formula (CGI -II), connecting group of formula (CGI -III)
and connecting group of formula (CGI -IV);
(CGI -III) (CGI -IV)
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 CGI denotes the bond between Tl and CGI,
the covalently connected LI forms in compound of formula (I) a ligand residue LIRes, which is
covalently connected to CGI via Tl;
LI is a compound of formula (LIRes-Tl-H);
LIRes Tl 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 NH2, which forms in
formula (I) the Tl, the Tl is bonded to CGI via the bond (*);
Tl is -S-, -O- or - H-;
nl is 0 or 1;
SG is a spacer group selected from the group consisting of spacer group of formula (SG-II)
and spacer group of formula (SG-III);
\ ***
HN- -CH SGPEG CH -NH
ml l ml m\2 (SG-III)
o
ml and m2 are identical or different and independently from each other 0 or 1;
mlO, ml 1 and ml2 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 mlO are not simultaneously 0;
with the proviso, that ml, mi l and ml2 are not simultaneously 0;
SGPEG is a connecting group of formula (SGPEG-I);
CH2 CH2 O- CH CH (SGPEG-I)
m20
m20 is 1, 2, 3, 4, 5 or 6;
n2 is 0 or 1;
T4 is -0-;
CG3 is selected from the group consisting of connecting group of formula (CG3-I) and
connecting group of formula (CG3-II);
R5 and R6 are identical or different and independently from each other C1-4 alkyl;
for nl being 1, the (**) in the formulae of CGI and in the formulae of SG denote the bond
between to CGI and SG, and the (***) in the formula of SG and in the formula of CG2
denote the bond between SG and CG2; in case that CGI is a connecting group of formula
(CGI -I), 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;
for nl being 0, the (**) in the formulae of CGI and the (***) in the formula of CG2 denote the
bond between CGI and CG2;
with the proviso, that in case that nl is 0, then CGI is not a connecting group of formula (CGIi);
for n2 being 1, the (****) in the formula of CG2 and in the formula of CG3 denote the bonds,
with which CG2 and CG3 are bonded to T4; and the (*****) in the formula of
CG3 denotes the bond between CG3 and T2;
for n2 being 0, the (****) in the formula of CG2 denotes the bond between CG2 and T2;
the (******) in the formula of CG2 denotes the bond between CG2 and AA 4 ;
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 (DRRes-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 formula (I) the T2;
T2 is -N(R4)-, -O- or -S-;
R4 is H or C alkyl;
n3 is 2, 3, 4, 5, 6, 7 or 8;
n4 is an integer from 1 to n3;
AA 4 is an amino acid residue, with n4 being the index of said amino acid residue, and (AA 4) 3
is a linear peptide with n3 amino acid residues AA 4 and with n4 denoting the position of
the amino acid residue AA 4 in the peptide starting from CG2, in which peptide the
individual amino acid residues are connected to each other via a peptide bond, with AA1
being the first amino acid residue in the chain and being connected to CG2 via the bond
(******), with the bond (******) being an amid bond of the carboxylic acid group of
AA1 with the amino group denoted with (4) of CG2, and with AA being the last amino
acid residue in the chain, and with the individual AA 4 being independently from each
other identical or different;
denotes the N-terminal amino group of said linear peptide, which is the amino group of
Rl and R2 are identical or different and independently from each other selected from the
group consisting of hydrogen, C1-4 alkyl, C(0)-(CH 2-0-) m 5-(GRPEG) m 4-R3 and PGN;
R3 is Ci-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);
CH- -CH -o- (GRPEG-I)
m21
m21 is 1, 2, 3, 4, 5 or 6
3. A method (MI) according to claim 2;
wherein compound of formula (I) is prepared in a step (MI);
step (MI) comprises a reaction (MI), wherein a compound of formula (II) is reacted with a
compound of formula (LIRes-Tl-H);
DRRes
Rl (3) R2
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);
(CG1M-III) (CG1M-IV)
XI is CI, Br or I ;
SG, nl, n4, n3, AA 4, (3), Rl, R2, T4, CG3, n2, T2, DRRes, m30 and m32 are as defined
claim 2;
CG2 is as defined in claim 1.
4 . A method (Mil) for the preparation of compound of formula (II), with the compound of
formula (II) as defined in claim 3,
wherein
in case that n2 is 1 and CG3 is a connecting group of formula (CG3-I), then method (Mil)
comprises a step (Mlla) and a step (Mllb);
in case that n2 is 1 and CG3 is a connecting group of formula (CG3-II), then method (Mil)
comprises the step (Mlla), a step (MIIc), a step (Mild) and a step (Mile);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-IV) then method (Mil)
comprises one step, a step (MIIO-IV), or two steps, a step (MII0-I-IVa) and a step (MIIOI-
IVb);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-III) then method (Mil)
comprises a step (MIIO-III);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-II) then method (Mil)
comprises two steps, a step (MII0-IIa) and a step (MllO-IIb), or one step, a
step (MIIO-IIc);
in case that n2 is 0 and CG1M is a connecting group of formula (CG1M-I) then method (Mil)
comprises one step, a step (MIIO-I), or two steps, a step (MllO-I-IVa) and a step (MIIO-IIVb);
step (Mlla) comprises a reaction (Mlla), wherein a compound of formula (III) is reacted with a
compound of formula (II-I);
Rl R 2
formula (III) is as defined in claim 2;
compound of formula (II-I) is selected from the group consisting of compound of formula (II-l),
I , -carbonyldiimidazole, 4-nitrophenylchloroformate, phosgene, diphosgene,
triphosgene and mixtures thereof;
step (Mllb) comprises a reaction (Mllb), wherein the reaction product from the reaction (Mlla) is
reacted with a compound of formula (DRRes-T2-H);
step (MIIc) comprises a reaction (MIIc), wherein the reaction product from the reaction (Mlla) is
reacted with a compound of formula (CG3M-II) to provide a compound of formula (He);
n2 in formula (lie) is as defined in claim 2;
step (Mild) comprises a reaction (Mild), wherein compound of formula (He), prepared in
reaction (MIIc), is reacted with the compound of formula (II-I);
step (Mile) comprises a reaction (Mile), wherein the reaction product from the reaction (Mild) is
reacted with a compound of formula (DRRes-T2-H);
step (MII0-I-IVa) comprises a reaction (MllO-I-IVa), wherein a compound of formula (III) is
reacted with a compound (II0-I-IVa) to provide a compound of formula (III0-I-IVa);
(AA 4 )
n3 (III0-I-IVa)
Rl (3) R2
n2 is 0 in formula (III0-I-IVa);
compound (II0-I-IVa) is selected from the group consisting of p-toluenesulfonyl chloride, ptoluenesulfonic
anhydride, methanesulfonyl chloride, methanesulfonic anhydride,
trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride and mixtures
thereof;
selected from the group consisting of -OTs, -OMs and -OTf;
step (MII0-I-IVb) comprises a reaction (MII0-I-IVb), wherein compound of formula
(III0-I-IVa), prepared in reaction (MII0-I-IVa), is reacted with compound of formula
(DRRes-T2-H);
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 CG1M is a connecting group of formula (CG1M-I);
step (MIII-IV) comprises a reaction (MIII-IV) of a compound of formula (IV),
Rl (3) R2
formula (IV) is as defined in claim 2;
with a compound of formula (CGIMR-IV);
(CGIMR-IV)
R20 is a residue of formula (R20-1);
(R20-1)
step (MIII-III) comprises a reaction (MIII-III) of a compound of formula (IV) with a compound
of formula (CG1MR-III);
CI
XI
o
(CG1MR-III)
step (MIII-IIa) comprises a reaction (MIII-IIa) of the compound of formula (IV) with a
compound of formula (CGlMR-IIa) to provide a compound of formula (IV-IIa);
(CGlMR-IIa)
step (MIII-IIb) comprises a reaction (MIII-IIb) of compound of formula (IV-IIa) prepared
(MIII-IIa) with a compound of formula (HOSu);
(HOSu)
step (MIII-IIc) comprises a reaction (MIII-IIc) of the compound of formula (IV) with a
compound of formula (CGlMR-IIc);
(CGlMR-IIc)
step (MIII-I) comprises a reaction (MIII-I) of a compound of formula (IV) with a compound of
formula (MA);
(MA)
compound of formula (IV) is prepared in a step (MIV);
step (MIV) comprises a reaction (MIV), reaction (MIV) is a reduction of a compound of formula
(V) with a compound (IV);
compound (IV) is selected from the group consisting of NaBH 4, BH3, DIBAL-H, sodium bis(2-
methoxyethoxy)aluminium hydride and mixtures thereof;
compound of formula (V) is prepared in a step (MVb);
step (MVb) comprises a reaction (MVb), wherein R30 is cleaved off from compound of formula
(Va) with HC1;
R30 is connected to SG via the bond denoted with (**) in the formulae of SG and
step (MIIO-IV) comprises a reaction (MIIO-IV) of a compound of formula (IIIO) with a
compound of formula (CG1MR-IV);
H- SG -CG2 — T4 -CG3 -T2 DRRes
n l n2 n2
(AA 4 )
n3
(IIIO)
R l (3) R2
n2 is 0 in formula (IIIO);
step (MIIO-III) comprises a reaction (MIIO-III) of a compound of formula (IIIO) with a
compound of formula (CGIMR-III);
step (MII0-IIa) comprises a reaction (MII0-IIa) of the compound of formula (IIIO) with a
compound of formula (CGlMR-IIa) to provide a compound of formula (III0-IIa);
R l (3) R2
n2 is 0 in formula (III0-IIa);
step (MII0-IIb) comprises a reaction (MII0-IIb) of compound of formula (III0-IIa) prepared in
step (MII0-IIa) with a compound of formula (HOSu);
step (MIIO-IIc) comprises a reaction (MIIO-IIc) of the compound of formula (IIIO) with a
compound of formula (CGlMR-IIc);
step (MIIO-I) comprises a reaction (MIIO-I) of a compound of formula (IIIO) with a compound of
formula (MA);
compound of formula (IIIO) is prepared in a step (MIIIO),
step (MIIIO) comprises a reaction (MIIIO), wherein R30 is cleaved off from compound of
formula (IVO) with HC1;
R30— - SG CG2 -CG3 -T2 DRRes
n l i n2 n2
(AA 4 )
n3
(IVO)
R R2
n2 is 0 in formula (IVO);
compound of formula (IVO) is prepared in a step (MlVOa) and a step (MlVOb),
step (MlVOa) comprises a reaction (MlVOa), wherein a compound of formula (VO)
with a compound (RIVOa) to provide a compound of formula (IVOa);
(AA 4 ) (VO)
n3
(3) R2
(AA 4 ) (IVOa)
n3
R 1 R2
compound (RIVOa) is selected from the group consisting of p-toluenesulfonyl chloride, ptoluenesulfonic
anhydride, methanesulfonyl chloride, methanesulfonic anhydride,
trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride, SOCl2,
(COCl) 2, POCl3, PCI3, PCI5, POBr 3, PBr3, PBr , N-bromosuccinimide,
N-iodosuccinimide, HC1, HBr, HI and mixtures thereof;
X3 is selected from the group consisting of -OTs, -OMs, -OTf, -CI, -Br and -I;
step (MlVOb) comprises a reaction (MlVOb), wherein compound of formula (IVOa), prepared
reaction (MIVO), is reacted with compound of formula (DRRes-T2-H);
compound of formula (V0) is prepared in a step (MVO),
step (MVO) comprises a reaction (MVO), reaction (MVO) is a reduction of a compound of
formula (Va) with a compound (IV);
CG1M and XI are as defined in claim 3;
SG, nl, n4, n3, AA 4, (3), Rl, R2, T4, CG3, n2, T2, DRRes, compound of formula (DRRes-T2-
H), m30, m32, R5 and R6 are as defined in claim 2;
CG2 is as defined in claim 1.
5 . Method (Mil) according to claim 4, wherein
compound of formula (Va) is prepared in a step (MVa);
step (MVa) comprises a reaction (MVa), wherein a compound of formula (VI) is reacted with a
compound of formula (SGM);
R30 SG R31 (SGM)
R31 is connected to SG via the bond denoted with (***) in the formulae of SG and is -OTs, -
OMs, -OTf, -Br, -CI or -I;
R30 is as defined in claim 4;
SG, n4, n3, AA 4, (3), Rl, R2 are as defined in claim 2 .
6 . Method (Mil) according to claim 5, wherein
compound of formula (VI) is prepared in a step (MVIa) and optionally in a further step (MVIb);
in step (MVIa) the n3 AA 4 are consecutively connected to a compound of formula (VII- 1) by
peptide coupling reactions and then to the respective products of the preceding peptide
coupling reactions;
step (MVIb) comprises a reaction (MVIb), wherein the N-terminal amino group of AA denoted
with (3) in formula (VI) is reacted with a compound NTermProt;
NTermProt is selected from the group consisting of C1-4 alkyl iodide, C1-4 alkyl bromide,
Cl-C(0)-(GRPEG) m4-R3, R3-C(0)-0-C(0)-R3 and PGNPrec;
PGNPrec is a reagent which provides for the introduction of PGN on the N-terminal amino
group of AA denoted with (3) in formula (VI);
GRPEG, m4, R3 and PGN are as defined in claim 2;
n4, n3, AA 4 and (3) are as defined in claim 2 .
7 . A compound of formula (I), with 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 compound of formula (10), compound of formula ( 11),
compound of formula (12), compound of formula (12-101), compound of formula (13),
compound of formula (14), compound of formula (15), compound of formula (15-102) and
compound of formula (16);
149
150
151
152
wherein
(dl), (d2), (d3) and (d4) in formula (DOXO) denote functional groups;
compound of formula (DOXO) is connected via the amino group denoted with (dl) in formula
(10), ( 11), (12), (12-101), (13), (14), (15), (15-102) and (16) respectively and in formula
(DOXO).
9 . A compound of formula (II), with 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 compound of formula (20), compound of formula (21),
compound of formula (22), compound of formula (23), compound of formula (24), compound of
formula (25), compound of formula (26), compound of formula (20-CAMPTO), compound of
formula (21-CAMPTO), compound of formula (22-CAMPTO), compound of formula (23-
CAMPTO) and compound of formula (21-TAXO-tl-l);
doxorubicin
155
156
157
5
wherein
doxorubicin is the compound of formula (DOXO) as defined in claim 8,
which is connected via the amino group denoted with (dl) in formula (20), (21), (22), (23), (24),
(25) and (26) respectively and in formula (DOXO);
camptothecin is the compound of formula (CAMPTO),
(CAMPTO)
wherein
(cl) in formula (CAMPTO) denotes a functional group;
compound of formula (CAMPTO) is connected via the hydroxy group denoted with (cl)
formula (20-CAMPTO), (21-CAMPTO), (22-CAMPTO) and (23-CAMPTO)
respectively and in formula (CAMPTO);
taxo-tl-1 is the compound of formula (TAXO),
which is connected via the hydroxy group denoted with (tl) in formula (21-TAXO-tl-l) and in
formula (TAXO).
11. A compound selected from the group consisting of
compound of formula (lie), compound of formula (III), compound of formula (IV), compound of
formula (IV-IIa), compound of formula (V), compound of formula (Va), compound of formula
(III0-IIa), compound of formula (IIIO), compound of formula (IVO), compound of formula
(IVOa), compound of formula (V0) and compound of formula (III0-I-IVa);
with
compound of formula (lie), compound of formula (III), compound of formula (IV), compound of
formula (IV-IIa), compound of formula (V), compound of formula (Va), compound of formula
(III0-IIa), compound of formula (IIIO), compound of formula (IVO), compound of formula
(IVOa), compound of formula (V0) and compound of formula (III0-I-IVa) as defined in claim 4 .
12. Compound according to claim 11,
wherein
compoun f formula (lie) is compound of formula (20c);
compound of formula (III) is selected from the group consisting of compound of formula (30),
compound of formula (31), compound of frmula (32), compound of formula (33), compound of
formula (34), compound of formula (35) and compound of formula (36);
162
163
164
compound of formula (IIIO) is compound of formula (300);
camptothecin
wherein
camptothecin is the compound of formula (CAMPTO) as defined in claim 10,
which is connected via the hydroxy group denoted with (cl) in formula (300) respectively and in
formula (CAMPTO);
compound of formula (III0-I-IVa) is compound of formula (320);
compound of formula (IV) is selected from the group consisting of compound of formula (40),
compound of formula (41), compound of formula (42), compound of formula (43), compound of
formula (44), compound of formula (45) and compound of formula (46);
167
compound of formula (IVO) is compound of formula (400);
wherein
camptothecin is the compound of formula (CAMPTO) as defined in claim 10,
which is connected via the hydroxy group denoted with (cl) in formula (400) respectively and in
formula (CAMPTO);
compound of formula (IVOa) is compound of formula (400a);
compound of formula (V) is selected from the group consisting of compound of formula (50),
compound of formula (50-1), compound of formula (51), compound of formula (51-1),
compound of formula (52), compound of formula (52-1), compound of formula (53), compound
of formula (53-1), compound of formula (54), compound of formula (54-1), compound of
formula (54-2), compound of formula (54-3), compound of formula (55), compound of formula
(55-1), compound of formula (56) and compound of formula (56-1);
171
172
173
174
175
13. A compound of formula (VI), with compound of formula (VI) as defined in claim 5 .
14. Compound of formula (VI) according to claim 13, wherein
compound of formula (VI) is selected from the group consisting of compound of formula (6),
compound of formula (6-1), compound of formula (6-2), compound of formula (6-3), compound
of formula (6-4), compound of formula (6b), compound of formula (6b- 1), compound of formula
(6b-2), compound of formula (6b-3), compound of formula (6b-4), compound of formula (6c)
and compound of formula (6-5).
BocHN
(6-4)
178
179
15. Use of the compound of formula (I), the compound of formula (I) as defined in claim 2,
for the preparation of a pharmaceutical composition or of a drug.
16. A pharmaceutical composition or a drug, wherein the pharmaceutical composition and
the drug comprise the compound of formula (I), the compound of formula (I) as defined
in claim 2 .
17. Compound of formula (I), a pharmaceutical composition or a drug, wherein the
compound of formula (I) is as defined in claim 2, and the pharmaceutical composition
and the drug are as defined in claim 16,
for use in treatment of a disease or an illness.