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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
  • C07D309/32—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
  • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D309/30—Oxygen atoms, e.g. delta-lactones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
  • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
  • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D495/04—Ortho-condensed systems

Definitions

• the present invention is directed to novel natural product-derived ratjadone-based compounds useful as payloads (or toxins) in drug-conjugates constructs with cell target binding moieties (CTBM) and payload-linker compounds useful in connection with drug conjugates.
• CTBM cell target binding moieties
• the present invention further relates to new ratjadone compositions including the aforementioned payloads, payload-linkers and drug conjugates, and methods for using these payloads, payload-linkers and drug conjugates, to treat pathological conditions including cancer, inflammatory and infectious diseases.
• Chemotherapy functions on the basis of a cytotoxic effect: a toxin kills cancer cells, thus halting tumor growth.
• Chemotherapeutic agents primarily damage and destroy cells with a high level of cell-division activity.
• these therapeutics have long struggled with the need to target and destroy malignant cells while minimizing undesired collateral toxicity to normal tissue. Since such drugs also damage healthy cells, patients suffer severe side effects.
• Numerous highly cytotoxic drugs are of limited clinical utility because they are equally aggressive against both normal and malignant tumoral cells. Healthy tissue can be heavily affected by cytotoxins. Since these drugs do not explicitly discriminate between tumor cells and normal cells, leading to side effects, drugs are often dosed at minimum levels, which may be not effective. This is the reason why it is important to find a way to specifically target tumor cells.
• Extracellular targeted carrier-drug conjugates seek to overcome these limitations of both nonspecific cytotoxic drugs and specific CTBM.
• EDCs are composed of three key elements: an CTBM (antibody, small molecule, antibody fragment, antibody mimic, etc. (see below), designed to selectively target the tumor of interest), a toxic payload (a cytotoxic compound that will kill the tumor) and a linker (used to conjugate the toxic payload to the antibody) and are an extensively investigated research field for the development of novel selective cancer therapies.
• cytotoxic drug is attached via an intracellularly cleavable linker to a carrier molecule with a high affinity for a specific biomarker[1 1-13] that is overexpressed on the surface of cancer cells.
• the drug can selectively be delivered to those cells.
• the linker is enzymatically cleaved and the cytotoxic payload is released and kills the cancer cell.
• MED minimal- effective dose
• MTD maximal-tolerated dose
• Carrier molecules designed to selectively target the tumor of interest, which can be utilized for such EDCs, can be antibodies, [1 ,3,9] probodies, antibody fragments, peptides with high affinity for receptors on cancer cell surfaces such as octreotide,[14-17] gonadotropins[18-22] or anticalins[23-25] and vitamins such as folic acid, [26-29] vitamin B9, essential for growth of cancer cell and taken up by specific high-affinity receptor.
• ADCs Antibody Drug Conjugates
• an antibody designed to selectively target the tumor of interest
• a toxic payioad a cytotoxic compound that will kill the tumor
• the linker used to conjugate the toxic payioad to the antibody.
• the benefits of such constructs are the significant improvement of the therapeutic window: increased half-life and specificity of the toxic payioad, reducing off-target effects and toxicity.
• the use of ADCs has been extensively investigated for the last three decades ( ooiten et ai. (1972), J Natl. Cancer Inst. 49(4):1057-62, Chari et ai, (2014) Angew. Chem. Int.
• the cytotoxic payloads utilized in the far majority of these approaches are all derived from natural products; which can be classified according to their mode of action in two groups: 1 ) antimitotic, microtubule binding agents, 2) agents causing DNA damage by alkylation or intercalation.
• payloads (or toxins) used in ADCs include bacterial toxins such as diphtheria toxin (Levy et ai (1975) Cancer Res. 35(5):1 182-6), plant toxins such as ricin, small molecule toxins such as maytansinoids (EP 1391213; Liu et ai, (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode et ai (1998) Cancer Res. 58:2925-2928; Upeslacis et ai, (1993) Cancer Res.
• bacterial toxins such as diphtheria toxin (Levy et ai (1975) Cancer Res. 35(5):1 182-6)
• plant toxins such as ricin
• small molecule toxins such as maytansinoids
• calicheamicin Liu et ai (1998) Cancer Res. 58:2925-2928
• cancer is an extremely complex disease with a multitude of different origins as well as forms of occurrence in various human tissues - genetically, no two tumors are the same. This high heterogeneity of tumors often causes cancer relapse after putatively successful chemotherapeutic treatment. [13,30-32] Furthermore, tumors not responding to established chemotherapeutics and multi-drug resistant tumors are still a big problem in cancer therapy.
• Ratjadone A is the most abundant member of the small class of polyketidic natural products, the ratjadones (see Figure 1 ), which have been isolated from cultures of the myxobacterium Sorangium cellulosum. [35-37] The ratjadones have been found to be highly potent anti-proliferating agents showing sub-nanomolar IC 5 o values for the growth inhibition of several cancer cell lines. [37]
• the anti-proliferating activity of the ratjadones is based on the covalent inhibition of Crm1 ,[37-39] an evolutionary highly conserved protein, which is responsible for the nuclear export of several proteins as well as mRNA into the cytoplasm.
• the present invention relates to a compound according to Formula
• R 1 and R 2 is NHR 9 and one is selected from H and OH;
• R 3 , R 4 and R 5 are independently of one another selected from the group that consists of H, CH 3 and C2H5;
• R 6 is CH 3 or C 2 H 5 ;
• R 7 and R 8 are independently of one another selected from the group that consists of H, CH 3 , C2H5, n-C3H 7 ,
• R is H.
• the present invention relates to a compound according to Formula I; wherein:
• R 1 to R 8 are as defined above;
• R 9 is L-RM * , wherein L is a linker, particularly a self-immolative linker, RM * is selected from RM and RM', wherein RM is a reactive moiety being able to form a covalent bond with a targeting moiety, particularly a target-binding antibody or functional fragment thereof, and wherein RM' is a moiety RM carrying a protecting group.
• the present invention relates to a compound according to Formula I, wherein
• R 1 to R 8 are as defined above;
• R 9 is L-TM, wherein L is a linker, particularly a self-immolative linker, and TM is a targeting moiety.
• the present invention relates to a method of synthesizing a toxic compound-linker-reactive moiety compound of the present invention, comprising the step of reacting a free toxic compound of the present invention via the amino group R 1 or R 2 with a compound X-L'-RM * , wherein
• X is a group that is (i) able to react with an amine, or (ii) can be replaced by an amine;
• L' is a linker
• the present invention relates to a method of synthesizing a toxic compound-linker-targeting moiety compound of the present invention, comprising the step of reacting a toxic compound-linker-reactive moiety compound of the present invention with a targeting moiety.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the toxic compound-linker-targeting moiety compound of the present invention or a toxic compound-linker-targeting moiety compound synthesized according to the present invention.
• the present invention relates to a pharmaceutical composition of the present invention for use in the treatment of cancer.
• the present invention relates to a method for the treatment of cancer comprising the step of administering a toxic compound-linker- targeting moiety compound of the present invention or the pharmaceutical composition of the present invention to a patient in need of such treatment.
• Figure 2 shows the design of folate-ratjadone conjugates.
• Figure 3 shows the determination of the absolute stereo configuration at C16 for the 16-amino-ratjadones via the Mosher amide 18.
• Figure 4 and Figure 5 show the results of the assay determining the export inhibitory ability.
• Figure 9 shows the results of cell imaging of KB3.1 cells treated with 1 ⁇ FA-1 a-FITC (AR280) in different media at different time points.
• RPMI +/+ folate containing RPMI medium with 10% folate containing FCS
• RPMI +/- folate containing RPMI medium with 10% dialyzed folate-free FCS
• RPMI -/+ folate-free RPMI medium with 10% folate containing FCS
• RPMI -/- folate-free RPMI medium with 10% dialyzed folate-free FCS.
• Figure 10 shows the results of cell imaging of KB 3.1 cells in presence and absence of folates and A549 cells treated with 8.6 ⁇ FA-1 a-FITC (AR280) in in folate-free RPMI medium with 10% folate-containing FCS.
• Figure 11 shows the gel of a-streptavidin pulldown experiment: identification of protein band in the eluate of biotin-PEG3-16S-aminoratjadone.
• Figure 12 shows the results of a LC-MS proteomic approach for target identification.
• Figure 13 and Figure 14 show the results of the plasma stability assay. DETAILED DESCRIPTION OF THE INVENTION
• the present invention is based on a combination of different advantageous elements and features and in particular on the unexpected observation that amino- substituted derivatives of ratjadone are particularly stable, while simultaneously being highly toxic in target cells.
• the present invention relates to a compound according to Formula
• R 1 and R 2 is NHR 9 and one is selected from H and OH;
• R 3 , R 4 and R 5 are independently of one another selected from the group that consists of H, CH 3 and C2H5;
• R 6 is CH 3 or C 2 H 5 ;
• R 7 and R 8 are independently of one another selected from the group that c
• R is H.
• ratjadone A derivatives for example starting from ratjadone A produced by a fermentation procedure or by chemical synthesis[45; 72], ratjadone B or C [37; 72], or from derivatives thereof synthesized according to published procedures [44 to 48], and demonstrated their use as cytotoxic payload within extracellular-targeted drug conjugates.
• the present invention relates to a compound according to Formula I; wherein:
• R 1 to R 8 are as defined above;
• R 9 is L-RM * , wherein L is a linker, particularly a self-immolative linker, RM * is selected from RM and RM', wherein RM is a reactive moiety being able to form a covalent bond with a targeting moiety, particularly a target-binding antibody or functional fragment thereof, and wherein RM' is a moiety RM carrying a protecting group.
• the synthesized derivatives of ratjadone present different RM functionalities such as ketones, amino groups, terminal alkynes, cyclooctyne moieties or reactive disulfides, which allow bio-orthogonal or orthogonal coupling to biologically active carrier molecules. Therefore, these compounds enable the development and construction of extracellular-targeted drug conjugates using ratjadone derivatives as cytotoxic payloads with new mode of action for the targeted therapy of cancer.
• protecting group refers to a group that is attached to a functional group present in RM in order to block said functional group from reacting with other reactants used to synthesize and/or to further functionalize compounds according to Formula I.
• One of ordinary skill in the art is well familiar with the different protecting groups that are available in the art and that can be attached to the corresponding functional group when needed to protect that functional group, and that can be cleaved off subsequently, when such protection is no longer needed.
• the functional group present in RM is an amino group
• that amino group may be N-protected by using an N-acylating reagent.
• RM' is an amino group- containing moiety RM carrying an N-acyl group as protecting group.
• the N-protection uses an N-alkylating reagent.
• RM' is an amino group-containing moiety RM carrying an N-alkyl group as protecting group.
• the present invention relates to a compound according to Formula I, wherein
• R 1 to R 8 are as defined above;
• R 9 is L-TM, wherein L is a linker, particularly a self-immolative linker, and TM is a targeting moiety.
• one of R 1 and R 2 is NHR 9 and one is OH.
• R 1 is OH and R 2 is NHR 9 .
• R 2 is 16R-NHR 9 .
• R 2 is 16S-NHR 9 .
• R 1 is NHR 9 and R 2 is OH.
• target-binding moiety refers to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope.
• Preferred target-binding moieties in the context of the present application are (i) antibodies or antigen-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers.
• “Target-binding moieties” suitable for use in the present invention typically have a molecular mass of 40 000 Da (40 kDa) or more.
• a first compound e.g. an antibody
• a second compound e.g.
• an antigen such as a target protein
• a dissociation constant KD to said second compound of 100 ⁇ or less, particularly 50 ⁇ or less, particularly 30 ⁇ or less, particularly 20 ⁇ or less, particularly 10 ⁇ or less, particularly 5 ⁇ or less, more particularly 1 ⁇ or less, more particularly 900 nM or less, more particularly 800 nM or less, more particularly 700 nM or less, more particularly 600 nM or less, more particularly 500 nM or less, more particularly 400 nM or less, more particularly 300 nM or less, more particularly 200 nM or less, even more particularly 100 nM or less, even more particularly 90 nM or less, even more particularly 80 nM or less, even more particularly 70 nM or less, even more particularly 60 nM or less, even more particularly 50 nM or less, even more particularly 40 nM or less, even more particularly 30 nM or less, even more particularly 20 nM or less, and even more particularly 10 nM or less.
• target molecule and “target epitope”, respectively, refers to an antigen and an epitope of an antigen, respectively, that is specifically bound by a target-binding moiety.
• target molecule is a tumor-associated antigen, in particular an antigen or an epitope which is present on the surface of one or more tumor cell types in an increased concentration and/or in a different steric configuration as compared to the surface of non-tumor cells.
• said antigen or epitope is present on the surface of one or more tumor cell types, but not on the surface of non-tumor cells.
• antibody or antigen binding fragment thereof refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen-binding site that immunospecifically binds an antigen.
• antigen-binding fragments thereof refers to a fragment of an antibody comprising at least a functional antigen- binding domain.
• immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to a target molecule.
• the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule.
• Antibodies and antigen-binding fragments thereof suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (in particular CDR-grafted), deimmunized, or chimeric antibodies, single chain antibodies (e.g.
• scFv fragments
• F(ab')2 fragments fragments produced by a Fab expression library, diabodies or tetrabodies (Holliger P. et al., Proc Natl Acad Sci USA. 90 (1993) 6444-8), nanobodies, antiidiotype (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
• the antigen-binding fragments are human antigen- binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH domain.
• Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable domain(s) alone or in combination with the entirety or a portion of the following: hinge region, CL, CH1 , CH2, and CH3 domains.
• antigen-binding fragments also comprising any combination of variable domain(s) with a hinge region, CL, CH1 , CH2, and CH3 domains.
• Antibodies usable in the invention may be from any animal origin including birds and mammals.
• the antibodies are from human, rodent (e.g. mouse, rat, guinea pig, or rabbit), chicken, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog origin. It is particularly preferred that the antibodies are of human or murine origin.
• "human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described for example in U.S. Patent No. 5,939,598 by Kucherlapati & Jakobovits.
• antibody-like protein refers to a protein that has been engineered (e.g. by mutagenesis of loops) to specifically bind to a target molecule.
• an antibody-like protein comprises at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the antibody-like protein to levels comparable to that of an antibody.
• the length of the variable peptide loop typically consists of 10 to 20 amino acids.
• the scaffold protein may be any protein having good solubility properties. Particularly, the scaffold protein is a small globular protein.
• Antibody-like proteins include without limitation affibodies, anticalins, and designed ankyrin repeat proteins (for review see: Binz et al.
• Antibody-like proteins can be derived from large libraries of mutants, e.g. be panned from large phage display libraries and can be isolated in analogy to regular antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.
• nucleic acid aptamer refers to a nucleic acid molecule that has been engineered through repeated rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a target molecule (for a review see: Brody and Gold, (2000) J Biotechnol. 74:5-13).
• the nucleic acid aptamer may be a DNA or RNA molecule.
• the aptamers may contain modifications, e.g. modified nucleotides such as 2'-fluorine-substituted pyrimidines.
• a "linker” in the context of the present invention refers to a structure that is connecting two components, each being attached to one end of the linker.
• a direct linkage of the toxic compound to the antibody may decrease the ability of the toxic compound to interact with its molecular target inside the cell.
• the linker increases the distance between two components and alleviates steric interference between these components, such as in the present case between the antibody and the toxic compound.
• the linker has a continuous chain of between 1 and 30 atoms (e.g.
• the length of the linker is defined as the shortest connection as measured by the number of atoms or bonds between the toxic compound moiety and the antibody, wherein one side of the linker backbone has been reacted with the toxic compound and, the other side is available for reaction, or has been reacted, with an antibody.
• a linker particularly is a Ci-20-alkylene, Ci-20-heteroalkylene, C2-20- alkenylene, C2-2o-heteroalkenylene, C2-2o-alkynylene, C2-2o-heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or a heteroaralkylene group, optionally substituted.
• the linker may contain one or more structural elements such as carboxamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties and the like.
• the linker may also contain combinations of two or more of these structural elements.
• each one of these structural elements may be present in the linker more than once, e.g. twice, three times, four times, five times, or six times.
• the linker may comprise a disulfide bond. It is understood that the linker has to be attached either in a single step or in two or more subsequent steps to the toxic compound and the antibody. To that end the linker to be will carry two groups, particularly at a proximal and distal end, which can (i) form a covalent bond to a group present in one of the components to be linked, particularly an activated group on an toxic compound or the target binding-peptide or (ii) which is or can be activated to form a covalent bond with a group on an toxic compound.
• chemical groups are at the distal and proximal end of the linker, which are the result of such a coupling reaction, e.g. an ester, an ether, a urethane, a peptide bond etc.
• the linker L is a linear chain of between 1 and 20 atoms independently selected from C, O, N and S, particularly between 2 and 18 atoms, more particularly between 5 and 16 atoms, and even more particularly between 6 and 15 atoms.
• at least 60% of the atoms in the linear chain are C atoms.
• the atoms in the linear chain are linked by single bonds.
• the linker L is an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or a heteroaralkylene group, comprising from 1 to 4 heteroatoms selected from N, O, and S, wherein said linker is optionally substituted.
• alkylene refers to a bivalent straight chain saturated hydrocarbon groups having from 1 to 20 carbon atoms, including groups having from 1 to 10 carbon atoms. In certain embodiments, alkylene groups may be lower alkylene groups.
• lower alkylene refers to alkylene groups having from 1 to 6 carbon atoms, and in certain embodiments from 1 to 5 or 1 to 4 carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH 2 -), ethylene (-CH 2 -CH 2 -), n-propylene, n-butylene, n-pentylene, and n-hexylene.
• alkenylene refers to bivalent straight chain groups having 2 to 20 carbon atoms, wherein at least one of the carbon-carbon bonds is a double bond, while other bonds may be single bonds or further double bonds.
• alkynylene herein refers to groups having 2 to 20 carbon atoms, wherein at least one of the carbon-carbon bonds is a triple bond, while other bonds may be single, double or further triple bonds.
• alkynylene groups include ethynylene, 1 -propynylene, 2- propynylene, and so forth.
• cycloalkylene is intended to refer to a bivalent ring being part of any stable monocyclic or polycyclic system, where such ring has between 3 and 12 carbon atoms, but no heteroatom, and where such ring is fully saturated
• cydoalkenylene is intended to refer to a bivalent ring being part of any stable monocyclic or polycyclic system, where such ring has between 3 and 12 carbon atoms, but no heteroatom, and where such ring is at least partially unsaturated (but excluding any arylene ring).
• cycloalkylenes include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene.
• cycloalkenylenes include, but are not limited to, cyclopentenylene and cyclohexenylene.
• heterocycloalkylene and “heterocycloalkenylene” are intended to refer to a bivalent ring being part of any stable monocyclic or polycyclic ring system, where such ring has between 3 and about 12 atoms, and where such ring consists of carbon atoms and at least one heteroatom, particularly at least one heteroatom independently selected from the group consisting of N, O and S, with heterocycloalkylene referring to such a ring that is fully saturated, and heterocycloalkenylene referring to a ring that is at least partially unsaturated (but excluding any arylene or heteroarylene ring).
• arylene is intended to mean a bivalent ring or ring system being part of any stable monocyclic or polycyclic system, where such ring or ring system has between 3 and 20 carbon atoms, but has no heteroatom, which ring or ring system consists of an aromatic moiety as defined by the "4n+2" ⁇ electron rule, including phenylene.
• heteroarylene refers to a bivalent ring or ring system being part of any stable mono- or polycyclic system, where such ring or ring system has between 3 and 20 atoms, which ring or ring system consists of an aromatic moiety as defined by the "4n+2" ⁇ electron rule and contains carbon atoms and one or more nitrogen, sulfur, and/or oxygen heteroatoms.
• substituted is intended to indicate that one or more hydrogens present in the backbone of a linker is replaced with a selection from the indicated group(s), provided that the indicated atom's normal valency, or that of the appropriate atom of the group that is substituted, is not exceeded, and that the substitution results in a stable compound.
• optionally substituted is intended to mean that the linker is either unsubstituted or substituted, as defined herein, with one or more substituents, as defined herein. When a substituent is a keto (or oxo, i.e.
• substituents include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, aroyl, heteroaroyl, carboxyl, alkoxy, aryloxy, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, halogen, (thio)ester, cyano, phosphoryl, amino, imino, (thio)amido, sulfhydryl, alkylthio, acylthio, sulfonyl, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, nitro, azido, haloal
• n is 2 and n is 1 , or m is 3 and n is 2.
• the linker comprises 2 or 3 unsubstituted alkylene groups, and 1 or 2, respectively, disulfide, ether, thioether, amine, ester, carboxamide, urethane or urea moieties linking the unsubstituted alkylene groups.
• the C atoms in the linear chain are independently part of optionally substituted methylene groups (-CH 2 -).
• the optional substituents are independently selected from halogen and Ci-6-alkyl, particularly methyl.
• the linker L is a stable linker.
• stable linker refers to a linker that is stable (i) in the presence of enzymes, and (ii) in an intracellular reducing environment.
• the stable linker does not contain (i) an enzyme- cleavable substructure, and/or (ii) a disulfide group.
• the linker has a length of up to 12 atoms, particularly from 2 to 10, more particularly from 4 to 9, and most particularly from 6 to 8 atoms.
• the linker is a cleavable linker.
• cleavable linker refers to a linker that is (i) cleavable by chemical cleavage, or (ii) a reducible linker.
• the linker is cleavable by reduction.
• the term "cleavable by reduction” refers to a linker that can be cleaved in the intracellular reducing environment, particularly a linker that contains a disulfide groups, resulting in the intracellular release of the toxin cargo conjugated to the target-binding moiety after internalization by the intracellular reducing environment (see Shen et ai, (1985) J. Biol. Chem. 260:10905-10908).
• the linker comprises a disulfide bond, particularly a -CMe 2 -S-S-CMe 2 - moiety.
• the linker is attached to the thiol group of the targeting moiety via a disulfide bond.
• the linker is cleavable by chemical cleavage, particularly by hydrolysis or proteolysis, particularly wherein such chemical cleavage is catalyzed by an enzyme.
• the term "chemical cleavage is catalyzed by an enzyme” refers to a linker that can be cleaved by an enzyme, particularly by a lysosomal peptidase, such as Cathepsin B, resulting in the intracellular release of the toxin cargo conjugated to the targeting antibody after internalization (see Dubowchik et ai, (2002) Bioconjug Chem. 13:855-69).
• the cleavable linker comprises a dipeptide selected from: Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit, in particular Val-Cit.
• the linker comprises a hydrazone group.
• cleavage occurs by hydrolysis in the lysosome.
• the linker is a self-immolative linker.
• the term "self-immolative linker” refers to a linker that comprises a cleavable bond, wherein after cleavage a fragmentation takes place that removes that part of the linker that is still attached to the toxin after said cleavage.
• the self-immolative linker comprises a p-aminobenzyl (PAB) spacer between a cleavable dipeptide and the toxic payload.
• PAB p-aminobenzyl
• the cleavable linker comprises a structure L 1 -L * -L 2 , wherein L * is p-aminobenzyl dipeptide moiety, L 1 is a part of the linker that connects L * to the toxic payload, in particular, wherein L 1 is connected to L * via a - NH- group, and wherein L 2 is a part of the linker that connects L * to the target- binding moiety, in particular wherein L 2 is connected to L * via a -(CH 2 ) m - moiety, with m being an integer selected from 1 to 8, in particular from 1 to 5, or via a -(CH 2 CH 2 O) n - moiety, with n being an integer selected from 1 to 3, in particular from 1 to 2.
• the structure of L 1 -L * -L 2 is as follows:
• L * comprises the dipeptide Val-Lys and has the following structure:
• the linker L 1 is a linear chain of between 1 and 4 atoms independently selected from C, O, N and S, particularly between 1 and 3 atoms, more particularly between 1 and 2 atoms, and even more just 1 atom.
• at least 50% of the atoms in the linear chain are C atoms.
• the atoms in the linear chain are linked by single bonds.
• L 1 is the -NH- group that is part of substituent R 1 or R 2 of the toxic payload according to the present invention.
• the present invention relates to a method of synthesizing a toxic compound-linker-reactive moiety compound of the present invention, comprising the step of reacting a compound according to the present invention via the amino group R 1 or R 2 with a compound X-L'-RM * , wherein
• X is a group that is (i) able to react with an amine, or (ii) can be replaced by an amine; and L' is a linker;
• the method further comprises the step of deprotecting the moiety RM' to result in RM.
• the present invention relates to a method of synthesizing a toxic compound-linker-targeting moiety compound of the present invention, comprising the step of reacting a toxic compound-linker-reactive moiety compound of the present invention with a targeting moiety.
• the method further comprises the step of first deprotecting the moiety RM' to result in RM.
• Conjugation to the targeting moiety may be achieved via coupling of the toxin-L-RM construct to free amino groups present in the targeting moiety.
• the group RM may be selected from an activated carboxylic acid derivative, such as an N-hydroxy succinimide ester; or an activated carbonic acid derivative, such as an isothiocyanate.
• Conjugation to the targeting moiety may further be achieved via coupling of the toxin-L-RM construct to free thiol groups present in the targeting moiety.
• the group RM may be selected from a haloacetyl group; an RM group comprising an acceptor-substituted alkene (Michael system), particularly a maleimide group or propenoyl group (see Badescu et al., (2014) Bioconjugate Chem. 25:460-469); a maleimide group substituted in 3-position or disubstituted in 3,4-positions with a leaving group X, particularly wherein X is selected from CI, Br, and aryl-S-, particularly Ph-S-.
• the thiol group is part of a single, uncoupled cysteine residue present in the wildtype targeting moiety.
• the thiol group is part of a single, uncoupled cysteine residue that has been generated from a wildtype targeting moiety, particularly by recombinant genetic engineering, for example by insertion into the wildtype sequence, by removing a second cysteine that is forming a disulfide bridge with the first cysteine residue in the wildtype targeting moiety, or by replacing a non-cysteine residue.
• the thiol group is generated by reduction of a disulfide linkage between two cysteines present in the wildtype targeting moiety.
• Conjugation to the targeting moiety may further be achieved via coupling of the toxin-L-RM construct to two free thiol groups present in the targeting moiety.
• the group RM may be a maleimide group disubstituted in 3,4-positions with leaving groups X, particularly wherein X is selected from CI, Br, and aryl-S-, particularly Ph-S-.
• the two thiol groups are each part of a single, uncoupled cysteine residue present in the wildtype targeting moiety.
• the thiol groups are part of two single, uncoupled cysteine residues that have been generated from a wildtype targeting moiety, particularly by recombinant genetic engineering, for example by insertion into the wildtype sequence, by removing a second cysteine that is forming a disulfide bridge with the first cysteine residue in the wildtype targeting moiety, or by replacing a non-cysteine residue.
• the two thiol groups are generated by reduction of a disulfide linkage between two cysteines present in the wildtype targeting moiety. By reaction of such two thiol groups with a disubstituted maleimide, the thiol groups are bridged, thus mimicking the originally present disulfide bridge.
• Conjugation to the targeting moiety may further be achieved by coupling to unnatural amino acids introduced by genetic engineering, for example by introducing p-acetyl phenylalanine and subsequent oxime ligation (see Kazane et al., (2012) Proc. Natl. Acad. Sci. U.S.A, 109:3731-3736).
• Conjugation to the targeting moiety may further be achieved by coupling of cyclic diazodicarboxamides to the phenyl ring of tyrosine residues in the targeting moiety (see Ban et al., (2010) J Am. Chem. Soc. 13:1523-5).
• Conjugation to the targeting moiety may be achieved via 1 ,3-dipolar cycloaddition (click chemistry).
• the targeting moiety comprises a double or triple bond and the toxin-L-RM construct comprises a 1 ,3-dipole, particularly an azide group.
• the targeting moiety is first reacted with dibenzocyclooctyne-N-hydroxysuccinimide ester or azadibenzocyclooctyne-N- hydroxysuccinimide ester (see, for example, Zhou et al., (2013) J Am Chem Soc. 135:12994-7).
• the targeting moiety comprises a 1 ,3- dipole, particularly an azide group and the toxin-L-RM construct comprises a double or triple bond.
• the targeting moiety is a glycosylated antibody that is first coupled to an azide-containing molecule by an enzyme- catalyzed reaction (tradename SiteClick; see Zeglis et al., (2013) Bioconjug Chem. 24:1057-67).
• an azido group is incorporated via the unnatural amino acid p-azido-phenylalanine (see Kazane et al., (2012) Proc. Natl. Acad. Sci. U.S.A, 109:3731-3736).
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the toxic compound-linker-targeting moiety compound of the present invention or a toxic compound-linker-targeting moiety compound synthesized according to the present invention.
• the present invention relates to a pharmaceutical composition of the present invention for use in the treatment of cancer.
• "treat", “treating” or “treatment” of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).
• the treatment may comprise administering a conjugate or a pharmaceutical composition according to the present invention to a patient, wherein "administering" includes in vivo administration, as well as administration directly to tissue ex vivo, such as vein grafts.
• a "therapeutically effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose.
• the effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration.
• the effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
• the pharmaceutical composition is used in the form of a systemically administered medicament.
• injectables are formulated either in the form of ampoules or as so called ready-for-use injectables, e.g. ready-to-use syringes or single-use syringes and aside from this in puncturable flasks for multiple withdrawal.
• injectables can be in the form of subcutaneous (s.c), intramuscular (i.m.), intravenous (i.v.) or intracutaneous (i.e.) application.
• s.c subcutaneous
• i.m. intramuscular
• i.v. intravenous
• i.e. intracutaneous
• Injectable formulations can further be produced as concentrates, which can be dissolved or dispersed with aqueous isotonic diluents.
• the infusion can also be prepared in form of isotonic solutions, fatty emulsions, liposomal formulations and micro-emulsions. Similar to injectables, infusion formulations can also be prepared in the form of concentrates for dilution. Injectable formulations can also be applied in the form of permanent infusions both in in-patient and ambulant therapy, e.g. by way of mini-pumps.
• parenteral drug formulations for example, albumin, plasma, expander, surface-active substances, organic diluents, pH-influencing substances, complexing substances or polymeric substances, in particular as substances to influence the adsorption of the target-binding moiety toxic compound conjugates of the invention to proteins or polymers or they can also be added with the aim to reduce the adsorption of the target-binding moiety toxic compound conjugates of the invention to materials like injection instruments or packaging- materials, for example, plastic or glass.
• parenteral drug formulations for example, albumin, plasma, expander, surface-active substances, organic diluents, pH-influencing substances, complexing substances or polymeric substances, in particular as substances to influence the adsorption of the target-binding moiety toxic compound conjugates of the invention to proteins or polymers or they can also be added with the aim to reduce the adsorption of the target-binding moiety toxic compound conjugates of the invention to materials like injection instruments or packaging- materials, for example, plastic or glass.
• the toxic compounds (payloads) of the present invention comprising a target-binding moiety can be bound to microcarriers or nanoparticles in parenterals like, for example, to finely dispersed particles based on poly(meth)acrylates, polylactates, polyglycolates, polyamino acids or polyether urethanes.
• Parenteral formulations can also be modified as depot preparations, e.g. based on the "multiple unit principle", if the conjugates of the present invention are introduced in finely dispersed, dispersed and suspended form, respectively, or as a suspension of crystals in the medicament or based on the "single unit principle” if the conjugate of the invention is enclosed in a formulation, e.g.
• implants or depot medicaments in single unit and multiple unit formulations often consist of so called biodegradable polymers like e.g. polyesters of lactic acid and glycolic acid, polyether urethanes, polyamino acids, poly(meth)acrylates or polysaccharides.
• Adjuvants and carriers added during the production of the pharmaceutical compositions of the present invention formulated as parenterals are particularly aqua sterilisata (sterilized water), pH value influencing substances like, e.g. organic or inorganic acids or bases as well as salts thereof, buffering substances for adjusting pH values, substances for isotonization like e.g. sodium chloride, sodium hydrogen carbonate, glucose and fructose, tensides and surfactants, respectively, and emulsifiers like, e.g. partial esters of fatty acids of polyoxyethylene sorbitans (for example, Tween ® ) or, e.g.
• fatty acid esters of polyoxyethylenes for example, Cremophor ®
• fatty oils like, e.g. peanut oil, soybean oil or castor oil
• synthetic esters of fatty acids like, e.g. ethyl oleate, isopropyl myristate and neutral oil (for example, Miglyol ® ) as well as polymeric adjuvants like, e.g. gelatin, dextran, polyvinylpyrrolidone, additives which increase the solubility of organic solvents like, e.g. propylene glycol, ethanol, ⁇ , ⁇ -dimethylacetamide, propylene glycol or complex forming substances like, e.g.
• organic solvents e.g. propylene glycol, ethanol, ⁇ , ⁇ -dimethylacetamide, propylene glycol or complex forming substances like, e.g.
• citrate and urea preservatives like, e.g. benzoic acid hydroxypropyl ester and methyl ester, benzyl alcohol, antioxidants like e.g. sodium sulfite and stabilizers like e.g. EDTA.
• preservatives like, e.g. benzoic acid hydroxypropyl ester and methyl ester, benzyl alcohol, antioxidants like e.g. sodium sulfite and stabilizers like e.g. EDTA.
• compositions of the present invention when formulating the pharmaceutical compositions of the present invention as suspensions in a preferred embodiment thickening agents to prevent the setting of the conjugates of the invention or, tensides and polyelectrolytes to assure the resuspendability of sediments and/or complex forming agents like, for example, EDTA are added. It is also possible to achieve complexes of the active ingredient with various polymers. Examples of such polymers are polyethylene glycol, polystyrene, carboxymethyl cellulose, Pluronics ® or polyethylene glycol sorbit fatty acid ester.
• the conjugates of the invention can also be incorporated in liquid formulations in the form of inclusion compounds e.g. with cyclodextrins. In particular embodiments dispersing agents can be added as further adjuvants. For the production of lyophilisates scaffolding agents like mannite, dextran, saccharose, human albumin, lactose, PVP or varieties of gelatin can be used.
• the present invention relates to a method for the treatment of cancer comprising the step of administering a toxic compound-linker- targeting moiety compound of the present invention or the pharmaceutical composition of the present invention to a patient in need of such treatment.
• Example 1 Generation and synthesis of the toxin unit ICQO-1
• Flash chromatography was done either manually using appropriate glass columns filled with silicagel (Merck, Silicagel 60, 1 .151 1 1 .1000, 15-40 ⁇ ) or using the Reveleris® X2 flash chromatography system and prepacked cartridges (Reveleris® Flash Cartridges Silica 40 ⁇ ) from the company Buchi.
• Preparative reversed phase high pressure liquid chromatography prep.
• HPLC RP HPLC RP
• Phenomenex Gemini C18 RP-column 00G- 4436-NO 10 ⁇ , 1 10 A, 250x 1 0.00 mm (5 mL/min) or a Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm (9 mL/min) or a Thermo Fisher Scientific BDS Hypersil C18 RP-column 28105-259370, 5 ⁇ , 250x30 mm, (25 mL/min) or a Macherey-Nagel Nucleosil 100-7 VP C18 RP column715691 - 1 1 16949, 250x40 mm (45 mL/min) using a Thermo Fisher Scientific Dionex Ultimate 3000 HPLC system. Eluents, gradients and additives are given in parentheses. Product containing fractions were combined diluted with dest. H 2
• TLC Thin-layer chromatography
• KMnQ 4 staining solution [KMnO 4 1: 1 .5 g KMnO 4 , 10 g K 2 CO 3 , and 1 .25 mL 10% NaOH in 200 mL H 2 O.
• PMA staining solution [PMA1: 10 g phosphomolybdic acid in 100 mL abs. EtOH.
• CAM staining solution [CAM]: 1 g Ce(IV)(SO 4 ) 2 , 2.5 g (NH 4 ) 6 Mo 4 O 7 in 100 mL 10% H 2 SO 4
• Vanillin staining solution [Van]: 15 g vanillin in 250 mL abs. EtOH and 2.5 mL cone. H 2 SO 4 .
• NMR spectra were recorded on a Bruker Avance III or a Bruker Avance III HD with cryoprobe system.
• 1 H NMR spectra were recorded at 500 MHz and 700 MHz.
• 13 C NMR spectra were recorded at 126 MHz and 176 MHz. Chemical shifts are reported in ppm relative to solvent signal. Multiplicity is indicated as follows: s (singlet); bs (broad singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of doublets), etc.
• IR spectra were recorded on a Bruker ALPHA FT IR spectrometer with ATR-technique. Only the wave numbers of observed absorption peaks are given.
• HRMS High resolution mass spectrometry
• Ratjadone A (1) - (R)-6-((1 E,3Z,5R,7E,9E,1 1 R)-1 1 -hydroxy- 1 1 -((2S,4R,5S,6S)-4-hydroxy-5-methyl-6-((E)-prop-1 -en-1 -yl)tetrahydro-2H-pyran-2- yl)-3,5,7-trimethylundeca-1 ,3,7,9-tetraen-1 -yl)-5,6-dihydro-2H-pyran-2-one
• Ratjadone A TCL (CH 2 CI 2 :MeOH/95:5) R f : 0.35 [UV 254 , CAM], IR (ATR) [cm "1 ]: 3417, 2962, 2921 , 2871 , 1715, 1653, 1625, 1451 , 1438, 1380, 1344, 1295, 1245, 1 149, 1 121 , 1056, 101 1 , 963, 916, 881 , 814, 732, 660.
• 16-Oxo-Ratjadone (8) TCL (CH 2 CI 2 :MeOH/98:2) R f : 0.26 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3456, 2970, 2922, 2883, 1723, 1682, 1620, 1581 , 1451 , 1437, 1380, 1362, 1307, 1247, 1218, 1 126, 1084, 1056, 1013, 968, 914, 884, 783, 732, 700.
• IBX was freshly prepare from 2-iodobenzoic acid according to the procedure of Santagostino and coworkers. 1681
• 16R-Amino-Ratjadone (11) IR (ATR) [cm "1 ]: 3403, 2961, 2923, 1674, 1522, 1452, 1436, 1382, 1345, 1253, 1201, 1160, 1134, 1058, 1014, 967, 918, 883, 816, 800, 722, 661.
• the reaction mixture was diluted with 200 ⁇ _ CH2CI2 and directly purified by preparative thin-layer chromatography (CH2CI2: MeOH/98:2, 1 x Development), yielding 2.3 mg (3.42 ⁇ , 52%) of Mosher amide 15 as a yellow solid foam.
• Both compounds 15 and 17 slightly contained DCU and traces of silicon grease as impurities.
• Mosher amide 15 TCL (CH 2 CI 2 :MeOH/98:2) R f : 0.46 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3421 , 2961 , 2924, 2854, 1722, 1693, 1625, 1506, 1451 , 1380, 1260, 1 164, 1080, 1056, 1015, 966, 918, 880, 802, 767, 735, 717, 698, 661 .
• Mosher amide 17 TCL (CH 2 CI 2 :MeOH/98:2) R f : 0.41 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3418, 2961 , 2925, 2854, 1721 , 1696, 1625, 1506, 1451 , 1360, 1260, 1 164, 1081 , 1054, 1017, 965, 918, 877, 802, 767, 734, 718, 698, 668.
• the reaction mixture was diluted with 200 ⁇ _ CH2CI2 and directly purified by preparative thin-layer chromatography (CH2CI2: MeOH/98:2, 1 x Development), yielding 2.2 mg (3.27 ⁇ , 50%) of Mosher amide 16 as a yellow solid foam.
• Both compounds 16 and 18 slightly contained DCU and traces of silicon grease as impurities.
• Mosher amide 16 TCL (CH 2 CI 2 :MeOH/98:2) R f : 0.42 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3417, 2960, 2925, 2854, 1721 , 1693, 1552, 151 1 , 1451 , 1380, 1335, 1321 , 1260, 1 102, 1061 , 1056, 1015, 966, 918, 880, 866, 801 , 767, 734, 717, 698, 668.
• Mosher amide 18 TCL (CH 2 CI 2 :MeOH/98:2) R f : 0.42 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3419, 3328, 2926, 2852, 1722, 1654, 1624, 1577, 1534, 1503, 1450, 1380, 1343, 1260, 1245, 1164, 1104, 1088, 1056, 1020, 966, 917, 905, 892, 814, 801, 767, 733, 713, 697, 662. 19 F-NMR (471 MHz, CDCI 3 ) ⁇ [ppm]: -68.80 (s, 3F).
• Figure 3 shows the determination of the configuration at position 16 via the Mosher amide 18.
• Compound 24 TCL (CH 2 CI 2 :MeOH/98:2) R f : 0.15 [UV 254 , CAM], IR (ATR) [cm "1 ]: 3442, 3309, 3034, 2963, 2923, 1718, 1653, 1605, 1566, 1497, 1437, 1361, 1335, 1291, 1246, 1136, 1081,.1056, 1013, 967, 937, 918, 883, 816, 776, 722, 683, 651.
• Fmoc-Cit-PABOH (26) 5 TCL (CH 2 CI 2 :MeOH/95:5) R f : 0.10 [UV 254 , CAM], IR (ATR) [cm "1 ]: 3280, 3128, 3063, 2923, 2864, 1689, 1653, 1600, 1568, 1532, 1478, 1450, 1414, 1387, 1334, 1281 , 12551 , 1231 , 1 164, 1 153, 1 1 16, 1 103, 1085, 1044, 1033, 1016, 989, 938, 823, 797, 778, 756, 737, 701 , 674, 662, 640, 610.
• BCN-0(CO)HN-Val-Cit-PABOH (29) TCL (CH 2 CI 2 :MeOH/9:1 ) R f : 0.21 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3444, 3271 , 2925, 2853, 2604, 2468, 1694, 1637, 1516, 1439, 1417, 1381 , 1335, 1301 , 1238, 1 172, 1 139, 1 120, 1090, 1027, 91 1 , 864, 825, 770, 734, 696, 670, 582, 554.
• reaction mixture was diluted with 300 ⁇ _ CH2CI2 and directly purified by flash chromatography through silicagel (CH2CI2: MeOH/95:5), yielding 78.1 mg (0.108 mmol, 86%) of BCN-O(CO)HN-Val-Cit-PABO(CO)O(4-NO 2 -Ph) 30 as an amorphous, white solid.
• reaction mixture was diluted with 1.0 ml_ CH2CI2 and directly purified by preparative thin-layer chromatography (CH 2 Cl 2 :MeOH/95:5, 3x Development), yielding 28.0 mg (26.99 ⁇ , 76%) of BCN-O(CO)HN-Val-Cit-PABO(CO)NH-Ratja 31 as an amorphous, white solid.
• Homopropargyl-0(CO)HN-Val-Cit-PABOH (32) TCL (CH 2 CI 2 :MeOH/9:1 ) R f : 0.17 [UV 254 , CAM], IR (ATR) [cm “1 ]: 3267, 2960, 2925, 2871 , 1690, 1639, 1602, 1535, 1465, 1445, 1415, 1386, 1339, 1295, 1248, 1 184, 1 136, 1 1 17, 1094, 1075, 1035, 1015, 924, 823, 803, 774, 697, 661 , 651 , 606.
• the reaction mixture was diluted with 300 ⁇ _ CH2CI2 and directly purified by flash chromatography through silicagel (CH2CI2: MeOH/95:5), yielding 43 mg (0.067 mmol, 64%) of Homopropargyl-O(CO)HN-Val-Cit-PABO(CO)O(4-NO 2 -Ph) 33 as an amorphous, pale-yellow solid.
• reaction mixture was diluted with 1 .0 ml_ CH2CI2 and directly purified by preparative thin-layer chromatography (CH 2 CI 2 :MeOH:EtOAc/9:1:1, 2x Development), yielding 7.8 mg (8.104 ⁇ , 66%) of Homopropargyl-O(CO)HN-Val-Cit-PABO(CO)NH-Ratja 34 as an amorphous, white solid.
• reaction mixture was diluted with toluene (20 mL), concentrated and co-evaporated with toluene (3x 10 mL) under reduced pressure and dried in HV.
• the residue containing the free amine was dissolved together with 83 ⁇ (0.749 mmol, 5.0 eq) NMM in 499 ⁇ dry DMF and added to a preactivated 7 solution of 26.9 mg (0.165 mmol, 1 .0 eq) 4-azidobenzoic acid, 83 ⁇ (0.749 mmol, 5.0 eq) NMM, 22.4 mg (0.165 mmol, 1 .0 eq) HOAt and 62.6 mg (0.165 mmol, 1 .0 eq) HATU in 499 ⁇ dry DMF.
• the mixture was diluted with 1 .2 mL H 2 O, filtered through a Whatman ® filter (45 ⁇ ) and directly purified RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + TFA/ 10:90 + 0.1 % ⁇ 95:5 + 0.1 % in 60 min) yielding after lyophilization 35.3 mg (41 .1 ⁇ , 45%) of Fa-N 3 -1 as a deep-yellow solid.
• Fa-Ns-1 LRMS (ESI-Quad) [m/z]: 858.3 [M+H] + , HRMS (ESI-IT) [m/z]: 858.327991 , calculated 858.327776 for 0 38 ⁇ 44 ⁇ 3 ⁇ ⁇ [M+H] + , err [ppm] -0.25 Synthesis of methyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4- azidobenzoyl)-L-lysinate (46)
• the solvent was removed from the resin and the resin was reacted shaking it for 1 h at 23°C with a solution of 1 ml_ MeOH and 220 ⁇ _ (2.0 mmol, 20 eq) NMM in 4 ml_ dry DMF.
• the resin was washed with DMF, CH2CI2 and DMF (each 3x 4 ml_, 2 min). Fmoc cleavage achieved by repeated reaction of the resin shaking it with a mixture of piperidine in DMF (Pip:DMF/1 :4, 3x 4 ml_, 10 min) and subsequent washing of the resin with DMF, CH2CI2 and DMF (each 3x 4 ml_, 2 min).
• the coupling of the amino acids was performed shaking with preactivated solutions of 84.4 mg (200 ⁇ , 2.0 eq) Fmoc-Asp(OtBu)-OH, 27.2 (200 ⁇ , 2.0 eq) HOAt, 55 ⁇ _ (600 ⁇ , 6.0 eq) NMM and 165 mg (200 ⁇ , 2.0 eq) HATU in 4 ml_ of dry DMF with coupling times of 4 h at 23°C. Subsequently the resin was washed with DMF, CH 2 CI 2 and DMF (each 3x 4 ml_, 2 min).
• the cleavage of the tripeptide from the resin was managed by repeated treatment of the resin with a mixture of CH 2 Cl2:HFIP/4:1 (3x 4 ml_, 10 min).
• the combined cleavage solutions were concentrated under reduced pressure and the residue was purified by flash-chromatography through silicagel (PE:EtOAc/8:2 - 1 :1 ) yielding Fmoc-(Asp(OtBu)) 3 -OH 47 as a white, amorphous solid.
• the reaction was diluted with toluene (5 ml_), concentrated and coevaporated with toluene (3x 5 ml_) under reduced pressure.
• the resulting residue was dried in HV, re-dissolved in 466 ⁇ _ CH2CI2 and 75 ⁇ _ (3.2 mL/mmol) diethylamine was added and the reactions mixture was stirred for 20 h at 23°C.
• the mixture was diluted with toluene (5 ml_), concentrated and coevaporated with toluene (3x 5 ml_) under reduced pressure.
• the mixture was diluted with 600 ⁇ _ H 2 O:ACN/70:30 + 0.05% TFA, filtered through a Whatman ® filter (45 ⁇ ) and directly purified RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mUmin, ACN:H 2 O + TFA/ 10:90 + 0.1 % ⁇ 95:5 + 0.1 % in 60 min) yielding after lyophilization 8.0 mg (7.45 ⁇ , 32%) of FA-N 3 -2 as a deep- yellow solid.
• Fa-Ns-2 LRMS (ESI-Quad) [m/z]: 1074.4 [M+H] + , HRMS (ESI-IT) [m/z]: 1074.36278, calculated 1074.36601 for C 45 H 5 i Ni 5 Oi 7 [M+H] + , err [ppm] -3.006.
• Fmoc cleavage was achieved by repeated reaction of the resin shaking it with a mixture of piperidine in DMF (Pip:DMF/1 :4, 3x 5 mL, 10 min) and subsequent washing of the resin with DMF, CH2CI2 and DMF (each 3x 4 mL, 2 min).
• the coupling of the amino acids was performed shaking with preactivated solutions of Fmoc-protected amino acids (436 ⁇ , 2.0 eq), 59 mg (436 ⁇ , 2.0 eq) HOAt, 144 ⁇ (1 .308 mmol, 6.0 eq) NMM and 166 mg (436 ⁇ , 2.0 eq) HATU in 4 mL of dry DMF with coupling times of 4 h at 23°C. Subsequently the resin was washed with DMF, CH 2 CI 2 and DMF (each 3x 4 mL, 2 min).
• Fmoc cleavage was achieved by repeated reaction of the resin shaking it with a mixture of piperidine in DMF (Pip:DMF/1 :4, 3x 5 mL, 10 min) and subsequent washing of the resin with DMF, CH2CI2 and DMF (each 3x 4 mL, 2 min).
• the resin was washed with DMF, CH 2 CI 2 , DMF, CH 2 CI 2 (each 3x 4 mL, 2 min) and the assembled folic acid derivative was cleaved from the resin by repeated treatment with a 4:1 -mixture of CH 2 Cl 2 :HFIP (3x 5 mL, 10 min).
• the cleavage solution was concentrated under reduced pressure and the residue treated with an Argon-flow-degassed mixture of TFA:TIPS:H 2 O:nPrSH/100:3:3:3 at 23°C (2x 5 mL, 1 h).
• FA-Ns-3 LRMS (ESI-Quad) [m/z]: 1060.4 [M+H] + , HRMS (ESI-IT) [m/z]: 530.678558, calculated 530.678819 for C 44 H 5 iNi 5 Oi 7 [M+2H] 2+ , err [ppm] 0.491 FA-Ns-4 - N 2 -N 2 -((S)-4-(4-((((2-amino-4-hydroxypteridin-6- yl)methyl)amino)benzamido)-4-carboxybutanoyl)-D-aspartyl-D-aspartyl-N 6 -(4- azidobenzoyl)-L-lysyl-L-aspartyl-L-aspartyl-N 6 -(4-azidobenzoyl)-L-lysine
• FA-Ns-4 LRMS (ESI-Quad) [m/z]: 1448.7 [M+H] + , HRMS (ESI-IT) [m/z]: 724.7536, calculated 724.7536 for C61 H71 N21 O22 [M+2H] 2+ , err [ppm] 0.00.
• FA-Ns-5 LRMS (ESI-Quad) [m/z]: 1 157.3 [M+H] + , HRMS (ESI-IT) [m/z]: 579.179534, calculated 579.179380 for C 44 H 54 Ni 6 O 2 2 [M+2H] 2+ , err [ppm] -0.266.
• FA-N3-6 LRMS (ESI-Quad) [m/z]: 844.3 [M+H] + , HRMS (ESI-IT) [m/z]: 844.31 1777, calculated 844.312126 for C37H42N13O11 [M+H] + , err [ppm] 0.413
• FA-Ns-7 LRMS (ESI-Quad) [m/z]: 71 1 .3 [M+H] + , HRMS (ESI-IT) [m/z]: 71 1 .2589, calculated 71 1 .2594 for C29H35N 12O10 [M+H] + , err [ppm] 0.600.
• FA-N3-8 LRMS (ESI-Quad) [m/z]: 843.4 [M+H] + , HRMS (ESI-IT) [m/z]: 843.364120, calculated 843.364496 for C38H 47 Ni 4 O 9 [M+H] + , err [ppm] 0.445.
• FA-Ns-9 LRMS (ESI-Quad) [m/z]: 710.3 [M+H] + , HRMS (ESI-IT) [m/z]: 710.31 1272, calculated 710.31 1732 for C 3 oH 4 oNi 3 O8 [M+H] + , err [ppm] 0.647.
• FA-Ns-1 0 LRMS (ESI-Quad) [m/z]: 1285.6 [M+H] + , HRMS (ESI-IT) [m/z]: 643.226691 , calculated 643.226862 for CsoHeeN ⁇ s [M+2H] 2+ , err [ppm] 0.265.
• FA-Ns-1 1 LRMS (ESI-Quad) [m/z]: 1552.5 [M+H] + , HRMS (ESI-IT) [m/z]: 776.762091 , calculated 776.762663 for C58H75N25O27 [M+2H] 2+ , err [ppm] 0.736.
• the solvent was removed from the resin and the resin was reacted shaking it for 1 h at 23°C with a solution of 1 mL MeOH and 2.12 mL (19.36 mmol, 20 eq) NMM in 7.5 mL dry DMF.
• the resin was washed with DMF, CH 2 CI 2 and DMF (each 3x 10 mL, 2 min). Fmoc cleavage achieved by repeated reaction of the resin shaking it with a mixture of piperidine in DMF (Pip:DMF/1 :4, 3x 10 mL, 10 min) and subsequent washing of the resin with DMF, CH2CI2 and DMF (each 3x 10 mL, 2 min).
• the resin was washed with DMF, CH2CI2, DMF, CH2CI2 (each 3x 10 mL, 2 min) and the assembled folic acid derivative was cleaved from the resin by repeated treatment with an Argon-flow-degassed mixture of TFA:TIPS:H 2 O:nPrSH/100:3:3:3 at 23°C (2x 5 mL, 1 h).
• FA-SH-1 LRMS (ESI-Quad) [m/z]: 1086.5 [M+H] + , HRMS (ESI-IT) [m/z]: 1086.441323, calculated 1086.442337 for [M+H] + , err [ppm] -0.933.
• the peptide was cleaved from the resin and deprotected by a treatment with TFA:TIPS:H 2 O/95:3:2 (10 ml/g resin) over 3 h at 23°C. After precipitation with t-butylmethyl ether, the resulting crude peptide was purified by reparative HPLC (RP-18) with water/acetonitrile gradients containing 0.1 % TFA and characterized by analytical HPLC and MALDI-MS.
• LHRH LRMS (MALDI) [m/z]: 1 182.5 [M+H] + , HRMS (ESI-IT) [m/z]: 1 182.5802, calculated 1 182.5803 for C55H76N 17O13 [M+H] + , err [ppm] 0.1 .
• L-Ns-Orn-LHRH LRMS (MALDI) [m/z]: 1256.6 [M+H] + , HRMS (ESI-IT) [m/z]: 633.3182, calculated 633.3180 for C58H82N20O13 [M+2H] 2+ , err [ppm] -0.300.
• D-Ns-Orn-LHRH LRMS (MALDI) [m/z]: 1265.7 [M+H] + , HRMS (ESI-IT) [m/z]: 633.3182, calculated 633.3180 for C58H82N20O13 [M+2H] 2+ , err [ppm] -0.300.
• Rapp S RAM resin (100 ⁇ , 0.8 mmol/g) was treated for 10 min with 20% piperidine in DMF at 23°C to remove the Fmoc group, followed by washing with DMF (5x 5 mL, 2 min) and DCM (5x 5 mL, 2 min). 162 mg (1 .0 mmol, 10.0 eq) carbonyldiimidazole in dry DCM (3 mL) were added and the mixture was incubated for 3 h at 23°C with shaking, followed by washing with DCM (5x 5 mL, 2 min) and DMF (5x 5 mL, 2 min). The resin was then treated with hydrazine in DMF (5 mL, ca.
• D-Ns-Orn-Goserellin LRMS (MALDI) [m/z]: 1266.8 [M+H] + , HRMS (ESI- IT) [m/z]: 633.8156, calculated 633.8156 for C57H75N21O13 [M+ 2H] 2+ , err [ppm] 0.0.
• the mixture was diluted with toluene (5 mL), concentrated and coevaporated with toluene (3x 4 mL) under reduced pressure and the residue was dried in HV.
• the residue was then dissolved in 1 .3 mL dry DMF, 43 ⁇ (384 ⁇ , 3.0 eq) NMM and 30 mg (128 ⁇ , 1 .0 eq) 23 were added and the mixture was stirred for 48 h at 23°C.
• the reaction mixture was diluted with toluene (5 mL), concentrated and coevaporated with toluene (5x 5 mL) under reduced pressure.
• reaction mixture was diluted with 1 00 ⁇ _ H 2 O and purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 1 0 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 1 0:90 ⁇ 95:5 in 35 min) yielding after lyophilization 1 7.9 mg (20.55 ⁇ , 75%) of EC17 as a deep orange, amorphous solid.
• the reaction mixture was diluted with 100 ⁇ _ of MeOH, filtered through a Whatman ® filter (45 ⁇ ) and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding the Folate-Fluorescein Conjugates after lyophilization as a deep orange, amorphous solids.
• FA-3a-FITC LRMS (ESI-Quad) [m/z]: 1685.5 [M+H] + , HRMS (ESI-IT) [m/z]: 841 .2571 1 , calculated 841 .25840 for C / sH / sN ⁇ [M-2H] 2" , err [ppm] -1 .53.
• FA-1a-FITC LRMS (ESI-Quad) [m/z]: 1484.6 [M+H] + , HRMS (ESI-IT) [m/z]:742.2617, calculated 742.2617 for C72H76N16O18S [M+2H] 2+ , err [ppm] 0.0.
• the reaction mixture was diluted with 100 ⁇ _ of MeOH, filtered through a Whatman ® filter (45 ⁇ ) and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding the Folate-Fluorescein Conjugates after lyophilization as a deep orange, amorphous solids.
• FA-9b-FITC LRMS (ESI-Quad) [m/z]: 1256.3 [M+H] + , HRMS (ESI-IT) [m/z]: 628.22296, calculated 628.22234 for CssH ⁇ NieOisS [M+2H] 2+ , err [ppm] 0.986.
• FA-11 b-(FITC) 3 LRMS (ESI-Quad) [m/z]: 1064.32 [M+3H] 3+ , HRMS (ESI- IT) [m/z]: 1063.9719, calculated 1063.9715 for Ci 42 Hi 45 N3 4 O 4 8S3 [M+3H] 3+ , err [ppm] 0.376.
• FA-8b-FITC LRMS (ESI-Quad) [m/z]: 694.6 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 694.7487, calculated 694.7487 for CeeH / iNuOieS [M+2H] 2+ , err [ppm] 0.0.
• FA-10b-FITC LRMS (ESI-Quad) [m/z]: 915.9 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 915.7897, calculated 915.7897 for C78H89N21O30S [M+2H] 2+ , err [ppm] 0.0.
• FA-7b-FITC LRMS (ESI-Quad) [m/z]: 915.8 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 1256.3869, calculated 1256.3850 for C57H58N15O17S [M+H] + , err [ppm] -1 .5.
• FA-6b-FITC [00287] Applying FA-N3-6 to the general procedure D, 3.8 mg (2.08 ⁇ , 57%) FA- FITC-6b were obtained as a deep-orange solid.
• FA-6b-FITC LRMS (ESI-Quad) [m/z]: 681 .3 [M+2H] 2+ .
• FA-3b-FITC LRMS (ESI-Quad) [m/z]: 803.2 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 803.2417, calculated 803.2417 for C 7 2H 7 Ni8O 2 S [M+2H] 2+ , err [ppm] 0.0.
• FA-4b-(FITC) 2 LRMS (ESI-Quad) [m/z]: 1270.3 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 1269.8793, calculated 1269.8792 for C117H 117N27O36S2 [M+H] + , err [ppm] - 0.078.
• the reaction mixture was diluted with 100 ⁇ _ of MeOH and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding after lyophilization 3.6 mg (1 .847 ⁇ , 69%) of D-Orn-LHRH-PEG 2 -CF as yellow, amorphous solid.
• reaction mixture was diluted with 100 ⁇ _ of MeOH and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding after lyophilization 3.4 mg (1 .744 ⁇ , 70%) of D-Orn-Goserellin-PEG 2 -CF as yellow, amorphous solid.
• reaction mixture was diluted with 100 ⁇ _ of MeOH and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435- PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mUmin, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding after lyophilization 4.0 mg (2.09 ⁇ , 60%) L-Orn-LHRH- CF as a deep yellow, amorphous solid.
• L-Orn-LHRH-CF LRMS (ESI-Quad) [m/z]: 840.4 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 560.5794, calculated 560.5724 for C82H97N 10O20 [M+3H] 3+ , err [ppm] 12.487.
• Biotin-PEG 3 -16S-Aminoratjadone LRMS (ESI-Quad) [m/z]: 996.5 [M+H] + , HRMS (ESI-IT) [m/z]: 996.546028, calculated 996.547454 for C51 H78N7O11S [M+H] + , err [ppm] -1 .431 1.1.1.14 Folate-Ratjadone Conjugates
• the reaction mixture was diluted with 100 ⁇ _ of MeOH, filtered through a Whatman ® filter (45 ⁇ ) and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding the Folate-Ratjadone Conjugates after lyophilization as a yellow, amorphous solids.
• FA-1 -Val-Cit-PABA-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1896.0 [M+H] + , HRMS (ESI-IT) [m/z]: 948.46350, calculated 948.46328 for C96H 125N 19O22 [M+2H] 2+ , err [ppm] 0.231 .
• FA-2-Val-Cit-PABA-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 21 12.9 [M+H] + , HRMS (ESI-IT) [m/z]: 1056.4847, calculated 1056.4825 for C103H133N21O28 [M+2H] 2+ , err [ppm] 2.082.
• FA-3-Val-Cit-PABA-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1049.8 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 1049.47480, calculated 1049.47475 for C102H131 N21O28 [M+2H] 2+ , err [ppm] 0.047.
• FA-4-(Val-Cit-PABA-16R-Aminoratjadone) 2 LRMS (ESI-Quad) [m/z]: 1 175.4 [M+3H] 3+ , HRMS (ESI-IT) [m/z]: 1 174.89793, calculated 1 174.89827 for Ci 77 H 2 32N 3 3O 44 [M+3H] 3+ , err [ppm] -0.289.
• FA-6-Val-Cit-PABA-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1882.6 [M+H] + , HRMS (ESI-IT) [m/z]: 940.9543, calculated 940.9540 for C95H 123N 19O22 [M+2H] 2+ , err [ppm] 0.318.
• FA-8-Val-Cit-PABA-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 940.6 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 940.98325, calculated 940.98163 for C96H128N20O20 [M+2H] 2+ , err [ppm] 1 .76.
• the reaction mixture was diluted with 100 ⁇ _ of MeOH, filtered through a Whatman ® filter (45 ⁇ ) and directly purified by RP prep HPLC (Phenomenex Gemini C18 RP-column 00G-4435-PO-AX, 5 ⁇ , 1 10 A, 250x21 .20 mm, Flow: 9 mL/min, ACN:H 2 O + 0.1 % TFA/ 10:90 ⁇ 95:5 in 45 min) yielding the Folate-Ratjadone Conjugates after lyophilization as yellow, amorphous solids.
• FA-3-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1612.4 [M+H] + , HRMS (ESI-IT) [m/z]: 806.3405, calculated 806.3412 for C77H96N16O23 [M+2H] 2+ , err [ppm] -0.639.
• FA-3-19-Aminoratjadone LRMS (ESI-Quad) [m/z]: 777.8 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 777.3384, calculated 777.3384 for C75H94N16O21 [M+2H] 2+ , err [ppm] 0.0.
• FA-3b-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1596.7 [M+H] + , HRMS (ESI-IT) [m/z]: 798.34173, calculated 798.34370 for C77H96N 16O22 [M+H] + , err [ppm] -2.41
• FA-5-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1709.9 [M+H] + , HRMS (ESI-IT) [m/z]: 854.8417, calculated 854.8417 for C77H99N17O28 [M+2H] 2+ , err [ppm] 0.0.
• FA-6-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1396.3 [M+H] + , HRMS (ESI-IT) [m/z]: 698.32208, calculated 698.32204 for C 7 oH 87 Ni 4 Oi 7 [M+2H] 2+ , err [ppm] 0.057
• FA-7-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1263.8 [M+H] + , HRMS (ESI-IT) [m/z]: 631 .79626, calculated 631 .79566 for C62H 8 i N i 3 Oi6 [M+2H] 2+ , err [ppm] -0.949
• FA-8-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 698.2 [M+2H] 2+ , HRMS (ESI-IT) [m/z]: 697.84802, calculated 697.84823 for C ⁇ F ⁇ NsOe [M+H] + , err [ppm] -0.288.
• FA-9-16R-Aminoratjadone LRMS (ESI-Quad) [m/z]: 1261 .9 [M+H] + , HRMS (ESI-IT) [m/z]: 613.3218, calculated 613.3218 for C 63 H 8 6Ni 4 Oi 4 [M+H] + , err [ppm] 0.0.

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