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WO2024263030A1 - Couplage peptidique à l'aide de dérivés de thiocycloheptyne - Google Patents

Couplage peptidique à l'aide de dérivés de thiocycloheptyne Download PDF

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Publication number
WO2024263030A1
WO2024263030A1 PCT/NL2024/050322 NL2024050322W WO2024263030A1 WO 2024263030 A1 WO2024263030 A1 WO 2024263030A1 NL 2024050322 W NL2024050322 W NL 2024050322W WO 2024263030 A1 WO2024263030 A1 WO 2024263030A1
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group
compound
hetero
molecule
formula
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Cristianne Johanna Ferdinand Rijcken
Mattijs TIMMERS
Robertus Matthias Joseph Liskamp
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Cristal Delivery BV
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Cristal Delivery BV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the invention relates to peptidic and proteinaceous molecules.
  • the invention relates to compounds and methods for coupling of peptidic and proteinaceous molecules to other molecules, such as drugs, drug delivery systems, targeting and imaging ligands, in particular using copper-free click chemistry.
  • Chemical methods for site-specific functionalization of peptides and proteins are useful in a variety of synthesis, research and biomedical applications. Examples include functionalization of peptides and proteins with affinity tags to facilitate isolation, purification, and characterization, with imaging labels to enable detection, with several molecules such as polymers, ligands, small molecules, etc.
  • N-Hydroxysuccinimide (NHS) chemistry to introduce a chelator, fluorescent label or another small molecule such as biotin to the 2PCA molecule prior to N-terminal functionalization. Coupling of these molecules to the 2PCA is not possible after N-terminal functionalization of the peptide/protein with the 2PCA moiety. Further, MacDonald et al. showed a variance in the percentage modification which could be achieved per protein, ranging from 43-95%.
  • the compounds and methods of the invention allow virtually any peptidic or proteinaceous molecule to be specifically modified at the N-terminus followed by a strain promoted azide alkyne cycloaddition (SPAAC) reaction for coupling to virtually any molecule including another peptide protein in a site-specific manner, that is non-toxic in biological systems.
  • SPAAC strain promoted azide alkyne cycloaddition
  • the invention therefore provides a compound of Formula (I), wherein n and m are independently 0, 1, or 2 with the proviso that n + m is 2; R 1 -R 8 are independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group wherein the (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (
  • the invention provides a compound comprising a compound of formula (I) according to the invention coupled to a peptidic or proteinaceous molecule, in particular to the N-terminus of the peptidic or proteinaceous molecule.
  • the compound comprising a compound of formula (I) according to the invention is site specifically coupled to the N-terminus of a peptidic or proteinaceous molecule.
  • the invention provides a compound comprising a compound of formula (I), (II) or (III) according to the invention, wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to a compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, 1,3-dipole or 1,3-(hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound.
  • the invention provides a use of a compound of formula (I), (II) or (III) according to the invention, in a bio-orthogonal, copper-free, click reaction. Such reaction results in the formation of a bioconjugate.
  • the invention provides a use of a compound of formula (I), (II) or (III) according to the invention for bioconjugation, in particular for coupling a molecule to a peptidic or proteinaceous molecule, preferably wherein the molecule is selected from the group consisting of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl.
  • the invention provides a method for coupling a molecule to a peptidic or proteinaceous molecule comprising reacting a compound of formula (I), (II) or (III) according to the invention with said molecule, wherein said molecule comprises a thiol, a 1,3-dipole or a 1,3-(hetero)diene, and wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to the compound comprising a thiol or a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)d
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise.
  • the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer.
  • the compounds may occur in different tautomeric forms.
  • the compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise.
  • the compounds disclosed in this description and in the claims may further exist as exo and endo stereoisomers.
  • any compound in the description and in the claims is meant to include both the individual exo and the individual endo stereoisomer of a compound, as well as mixtures thereof.
  • the compounds disclosed in this description and in the claims may exist as cis and trans isomers.
  • the description of any compound in the description and in the claims is meant to include both the individual cis and the individual trans isomer of a compound, as well as mixtures thereof.
  • the structure of a compound is depicted as a cis isomer, it is to be understood that the corresponding trans isomer or mixtures of the cis and trans isomer are not excluded from the invention of the present application.
  • halogen refers to fluoro, chloro, bromo, or iodo. Preferred halogen atoms are fluoro and chloro.
  • a (Cx-y)alkyl refers to a branched or unbranched alkyl group having x-y carbon atoms.
  • (C1-6)alkyl means a branched or unbranched alkyl group having 1-6 carbon atoms, for example methyl, ethyl, propyl, isopropyl or butyl.
  • (C1-2) alkyl refers to an alkyl group having 1 or 2 carbon atoms.
  • Preferred alkyl groups are methyl and ethyl.
  • the term (Cx-y)alkoxy refers to an alkoxy group having x-y carbon atoms, wherein the alkyl moiety is as defined above.
  • the term (C2-6)alkoxy means an alkoxy group having 2-6 carbon atoms.
  • Preferred alkoxy groups are methoxy and ethoxy.
  • (Cx-y)alkenyl refers to a branched or unbranched alkenyl group, i.e. having at least one double bond, having x-y carbon atoms.
  • (C2-6)alkylene means a saturated alkylene group having 2-6 carbon atoms.
  • suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl and pentenyl. Unsubstituted alkenyl groups may also contain a cyclic moiety.
  • (Cx-y)alkynyl refers to a branched or unbranched alkynyl group having x-y carbon atoms, wherein the triple bond may be present at different positions in the group, for example ethynyl, propanyl, 1-butynyl, 2- butynyl.
  • (C2-6)alkynyl refers to a branched or unbranched alkynyl group having 2-6 carbon atoms.
  • (Cx-y)cycloalkyl group refers to a cyclic alkyl group having x-y carbon atoms, which can be a single ring or multiple rings, such as a fused bicyclic ring.
  • (C3-6)cycloalkyl refers to a cyclic alkyl group having 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, which are preferred cycloalkyl groups.
  • (Cx-y)heterocycloalkyl group refers to a cyclic group comprising at least one heteroatom, preferably O, S or N, in the cyclic backbone, i.e. having x-y carbon atoms + heteroatoms.
  • the group can be a single ring or multiple rings, such as a fused bicyclic ring.
  • (C3-12) heterocycloalkyl group refers to a cyclic alkyl group having 3-6 carbon atoms, such as pyrrolidine, pyrazolidine, piperidine, piperazine, tetrahydrothiophene, thiane, dithiane, tetrahydrofuran, thiomorpholine.
  • (Cx-y)aryl group refers to cyclic aromatic groups with x-y carbon atoms and may include monocyclic, bicyclic and polycyclic structures. Optionally, the aryl groups may be substituted by one or more substituents further specified.
  • aryl groups include phenyl, naphthyl, anthracyl.
  • a preferred aryl group is phenyl.
  • (Cx-y)heteroaryl group refers to cyclic aromatic groups with x-y carbon atoms + heteroatoms and may include monocyclic, bicyclic and polycyclic structures.
  • the aryl groups may be substituted by one or more substituents further specified.
  • Preferred examples of heteroaryl groups include furan, pyridine, pyrazine, pyrrole, imidazole, pyrazole, oxazole and thiophene.
  • (Cx-y)alkyl(hetero)aryl group encompasses (Cx- y)alkylheteroaryl groups and (Cx-y)alkylaryl group having x-y atoms carbon atoms (in the case of (Cx-y)alkylaryl) or x-y carbon atoms + heteroatoms (in the case of (Cx-y)alkylheteroaryl groups in the alkyl and (hetero)aryl groups taken together.
  • (Cx-y)(hetero)arylalkyl encompasses (Cx-y)heteroarylalkyl groups and (Cx-y)arylalkyl group having x-y atoms carbon atoms (in the case of (Cx-y)arylalkyl) or x-y carbon atoms + heteroatoms (in the case of (Cx- y)heteroarylalkyl groups in the (hetero)aryl and alkyl groups taken together.
  • substituents the term “optionally substituted” indicates a group may be unsubstituted or substituted with the indicated number and type of the substituent(s).
  • independently substituted means that if a group that is substituted with more than one substituent, these substituents may be the same or different from each other.
  • 2PCA can be introduced in a thiocycloheptyne derivative and used to site-specifically functionalize peptides and proteins at the N-terminus, with the aim to provide a wide range of bioconjugates.
  • the synthesis of the alternative click reagent 2PCA-DBCO would lead to a relatively hydrophobic derivative, which has an adverse effect on solubility and hence reactivity of peptides and proteins.
  • the resulting 2PCA-DBCO reagent might not be sufficiently stable for N- terminal introduction.
  • the alternative click reagent 2PCA-BCN would also be relatively hydrophobic and less reactive as compared to the reagent of the present invention.
  • N-Hydroxysuccinimide (NHS) chemistry as required by e.g. MacDonald et al., a chelator, fluorescent label or other small molecule needs to be introduced to the 2PCA molecule prior to N-terminal functionalization of the peptide/protein.
  • thiocycloheptyne- 2PCA compound of the present invention allows for the clicking of any kind of small or large compound after N-terminal functionalization of peptides/proteins.
  • Another advantage of the thiocycloheptyne compound is that it is resistant to strong acidic conditions, which opens up possibilities for its introduction under peptide synthesis conditions.
  • the compounds and methods of the invention in particular allow for a wider range of applications than known so far because introducing an azide in the molecule or coupling partner is easier than e.g. introducing a strained alkyne.
  • TMTHSI-like-2PCA derivative of the invention show a good biological and chemical stability. They remain stable in the presence of human serum (see Example 2 and figure 7). In addition, the stability over time under acidic conditions (pH 2) was maintained for at least 56 hours and at pH 7 for prolonged period of time (see Example 3 and figure 8). Since the derivatives of the invention are for use under physiological conditions, the acid sensitivity is only of importance during synthesis, work-up, and formulation steps.
  • TMTHSI-like-2PCA conjugates Since the conjugation of TMTHSI-like-2PCA to biomolecules works best at a pH between 7-8, TMTHSI-like-2PCA conjugates would only be exposed to an acidic environment during work-up and/or analysis. As the amount of the 2PCA conjugate in solution stays above 95% for about 56 hours, it will remain stable for the duration of work-up and analysis steps. Finally, peptide or protein functionality is not compromised when functionalized in accordance with the present invention.
  • the invention therefore provides a compound of Formula (I), wherein n and m are independently 0, 1, or 2 with the proviso that n + m is 2; R 1 -R 8 are independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group wherein the (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group wherein
  • a preferred compound of formula (I) is a compound of formula (IA):
  • a preferred compound of formula (II) is a compound of formula (IIA):
  • a preferred compound of formula (III) is a compound of formula (IIIA):
  • R 1 -R 8 are preferably independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-4)alkyl and (C1- 4)alkoxy, wherein the (C1-4)alkyl and (C1-4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy.
  • R 1 -R 8 are independently selected from the group consisting of hydrogen, halogen and C1-C4 alkyl, preferably methyl or ethyl. More preferably, R 1 -R 8 are independently selected from the group consisting of hydrogen, methyl and ethyl, more preferably hydrogen and methyl. In one particularly preferred embodiment, R 1 -R 4 are methyl and R 5 -R 8 are hydrogen. It is further preferred that m and n are both 1.
  • a preferred compound of formula (I) is a compound of formula (1B):
  • a preferred compound of formula (II) is a compound of formula (IIB):
  • a preferred compound of formula (III) is a compound of formula (IIIB):
  • L is a linking group.
  • linking group and “linker” are used interchangeably and refer to a chemical group having functionality to connect different parts of a molecule or compound, typically two parts. In the present disclosure, the linking group connects the thiocycloheptyne moiety to the 2-pyridinecarboxaldehyde moiety (2PCA).
  • L is a degradable linker.
  • degradable linker refers to a linker that over time and/or under specific circumstances such as physiological conditions is cleavable.
  • the linker is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions or by the enzymatic activity.
  • Suitable degradable linkers are selected from linkers comprising an ester, orthoester, amide, carbonate, carbamate, anhydride, ketal, acetal and hydrazone.
  • Another example of a suitable degradable linker is a linker comprising a valine–citrulline (VCit) dipeptide linker or a glutamic acid–valine–citrulline tripeptide linker.
  • Such linkers are commonly used as enzymatically cleavable linkers.
  • the linking group L comprise or is selected from linear or branched C1 - C24 alkylene groups, C2 - C24 alkenylene groups, C2 - C24 alkynylene groups, C3 - C24 cycloalkylene groups, C5 - C24 cycloalkenylene groups, C5 - C24 cycloalkynylene groups, C7 -C24 alkyl(hetero)arylene groups, C7 - C24 (hetero)arylalkylene groups, C5 - C24 (hetero)arylalkenylene groups, C9 - C24 (hetero)arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, (hetero)arylalkeny
  • L is a linear or branched carbon atom chain with a length of 1 - 50 atoms, whereby said chain length is determined by the number of atoms in the longest linear chain of atoms, and whereby said longest linear chain may comprise one or more heteroatoms and/or one or more saturated or unsaturated cyclic or heterocyclic moieties.
  • L is preferably minimally a CH2 group.
  • the number of atoms in the longest linear chain of atoms of linking group L is 6.
  • the chain has a length of 1 – 40 atoms in the longest linear chain of atoms, more preferably 1 – 30 atoms, more preferably 1 – 25 atoms, more preferably 1 – 20 atoms, more preferably 1 – 15 atoms.
  • Said longest linear chain may comprise one or more heteroatoms such as O, N, S and P.
  • it may comprise one or more N and/or O atoms, more preferably one or more O and one or more N atoms.
  • the linear or branched carbon atom chain with a length of 1 - 50 atoms, preferably 1 – 40, 1 – 30, 1 – 25, 1 – 20 or 1 – 15 atoms comprises one or more moieties independently selected from the group consisting of -S(O)2-, -S-, -S-S-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, (C6-12)heteroaryl and combinations thereof in the longest linear chain, preferably 1 – 10 of said moieties.
  • the linear or branched carbon atom chain comprises 1, 2 or 3 of said moieties.
  • the linear or branched carbon atom chain comprises a (C3-12)heterocycloalkyl, preferably a (C3-8)heterocycloalkyl, more preferably a (C5-6)heterocycloalkyl, most preferably a C6-heterocycloalkyl.
  • Preferred examples of (C3-12)heterocycloalkyl are pyrrolidine, pyrazolidine, piperidine, piperazine, tetrahydrothiophene, thiane, dithiane, tetrahydrofuran and thiomorpholine.
  • said chain with a length of 1 - 50 atoms preferably 1 – 40, 1 – 30, 1 – 25, 1 – 20 or 1 – 15 atoms, is not branched, i.e.
  • the chain is linear, optionally with the exception of the atoms or heteroatoms in moiety or moieties -S(O)2-, -S-, -S-S-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3- 12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, (C6-12)heteroaryl that are not comprised in the longest linear chain.
  • L comprises a piperazine or polyethylene glycol (PEG) moiety.
  • L in particular the linear or branched carbon atom chain, comprises a piperazine, more preferably the moiety .
  • the linking group L has the structure , whereby the -C(O)- is attached to the nitrogen atom of the thiocycloheptyne.
  • linking group L comprises or is a polyethylene glycol (PEG) moiety, preferably ⁇ (O ⁇ CH2 ⁇ CH2)n ⁇ wherein n is up to 13, more preferably n is an integer from 2 to 10.
  • the linking group L has the , whereby the -C(O)- is attached to the nitrogen atom of the thiocycloheptyne.
  • the compound of formula (I) is selected from: .
  • the invention also provides methods for the preparation of compounds of formula (I), (IA) and (IB). Compounds of formula (I) can be prepared by methods known in the art.
  • TTHSI 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7- tetrahydro-1H-1 ⁇ 6-thiepine 1-oxide
  • the compound comprising a compound of formula (I) according to the invention is site specifically coupled to the N-terminus of a peptidic or proteinaceous molecule.
  • the compounds are compounds of formula (IIA) or (IIIA):
  • X is a peptidic or proteinaceous molecule.
  • peptidic molecule and “proteinaceous molecule” refer to a molecule comprising at least one amino acid residue, preferably comprising a plurality of amino acids residues.
  • peptidic molecule is used for molecules containing relatively short amino acid chains, e.g. up to about 50 amino acids and the term “proteinaceous molecule” is used for molecules containing larger amino acids chains, e.g. from about 50 amino acids.
  • X is a peptide or protein.
  • the peptidic or proteinaceous molecule comprises at least two amino acid residues bound to each other via a peptide bond, i.e. the peptidic or proteinaceous molecule comprises at least a dipeptide.
  • the N-terminal amino acid of this peptidic or proteinaceous molecule reacts with the 2PCA and forms a cyclic structure, in particular the 5- membered ring in compounds (II), (IIA), (IIB) or the 6-membered ring in compounds (III), (IIIA) and (IIIB).
  • X is the peptidic or proteinaceous molecule without the N- terminal amino acid that is reacted with 2PCA.
  • Any peptidic or proteinaceous molecule as described herein preferably comprises one or more of a peptide and a protein, and optionally one or more of a non-amino acid moiety and a cleavable moiety.
  • the peptidic or proteinaceous molecule comprises or is a peptide or protein.
  • Non-limiting examples are therapeutic peptides and proteins, including antibodies, antigens and ligands, targeting and imaging ligands, and any other biologically active peptide or protein.
  • biologically active peptide or protein refers to a peptide or protein that exerts an activity when administered to a subject.
  • the activity can be any activity, including, but not limited to, a therapeutic or prophylactic activity, a binding activity, a diagnostic activity or a targeting activity.
  • the peptidic or proteinaceous molecule is a peptide or protein, meaning that it consists entirely of amino acid residues.
  • the peptidic or proteinaceous molecule comprises one or more non-amino acid moieties.
  • any molecule can be coupled to at least a dipeptide to form a peptidic or proteinaceous molecule as defined herein.
  • Non-limiting examples or non-amino acid moieties are oligonucleotides, peptidomimetics, targeting or imaging labels, saccharide, and any other biologically active molecule. These non-amino acids are attached to the at least one amino acid via a secondary amine.
  • the peptidic or proteinaceous molecule is a peptide, protein or molecule coupled to at least a dipeptide, preferably the molecule is selected from the group consisting of oligonucleotides, peptidomimetics, targeting or imaging labels, saccharide, and any other biologically active molecule.
  • the peptidic or proteinaceous molecule comprises a cleavable moiety or linker, preferably between the at least dipeptide that is coupled to a compound of formula (I) of the invention and the peptide, protein or other molecule of interest.
  • cleavable moiety or linker provides for release of the peptide, protein or other molecule of interest under the appropriate circumstances.
  • the cleavable moiety or linker is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions or by the enzymatic activity.
  • Suitable cleavable moieties or linkers are selected from the group consisting of an ester, orthoester, amide, carbonate, carbamate, anhydride, ketal, acetal and hydrazone.
  • Another example of a suitable cleavable moiety or linker is a linker comprising a valine–citrulline (VCit) dipeptide linker or a glutamic acid–valine–citrulline tripeptide linker.
  • the peptide or proteinaceous molecule may comprise any number of amino acids and any non-amino acid moiety as the ability of the molecule to be coupled to a compound according to the invention is not limited by the size of peptidic or proteinaceous molecule and the nature or size of the non-amino acid moiety or moieties.
  • the molecule may comprises a peptide of between 2 and 50 amino acids, between 2 and 100 amino acids, between 2 and 500 amino acids or between 2 and 1000 amino acids.
  • the peptidic or proteinaceous molecule may comprise a single, amino acid chain.
  • the peptidic or proteinaceous molecule may comprise two or more amino acid chains that are connected via non- peptide bonds.
  • the at least two amino acid residues bound to each other via a peptide bond in the peptidic or proteinaceous molecule before coupling to the compounds of formula (I), (IA) or (IB) must have a free N-terminus. This free N-terminus is available for coupling to the 2PCA moiety of a compound of the invention of formula (I), (IA) or (IB).
  • the N-terminal amino acid of the peptidic or proteinaceous molecule before coupling to the compounds of formula (I), (IA) or (IB) can be any amino acid, either a naturally occurring amino acid or non-natural amino acid, and either a L-amino acid or a D-amino acid.
  • the amino acid is an alpha amino acid or a beta amino acid.
  • Coupling of peptidic or proteinaceous molecule comprising an N-terminal alpha amino acid to a compound of formula (I), (IA) and (IB) results in a compound of formula (II).
  • Coupling of peptidic or proteinaceous molecule comprising an N-terminal beta amino acid to a compound of formula (I), (IA) and (IB) results in a compound of formula (III).
  • any peptidic or proteinaceous molecule that is coupled to a compound of formula (I), (IA) or (IB) of the present invention is not proline, more preferably not proline or another amino acid containing a tertiary amine.
  • R 9 is an amino acid side chain.
  • one of R 9 and R 10 is an amino acid side chain and the other of R 9 and R 10 is hydrogen or both of R 9 and R 10 are an amino acid side chain.
  • amino acid side chain is well known in the art and refers to the characterizing substituent of the relevant amino acid. This term refers to the substituent bound to the alpha-carbon of the amino acid.
  • the side chain can be any amino acid, either a natural or non-natural a-amino acid.
  • Coupling of a peptidic or proteinaceous molecule to a compound of formula (I), (IA) or (IB) can be achieved by methods well known in the art. A suitable method for such coupling is described in the examples herein. In brief, (I), (1A) or (1B) is dissolved in phosphate buffer pH 7.4, with 10% ACN.
  • the solution with concentration of 10 mM is added to the peptidic or proteinaceous molecule which is also dissolved in phosphate buffer pH 7.4, at 0.1mM concentration. This is reacted overnight at room temperature.
  • the reaction mixture was purified using dialysis against 1 kDa MWCO membrane, PD Sephadex column G10 or G25, depending on size of the peptidic molecule. In all cases using 20 mM phosphate buffer pH 7.4 as eluent.
  • excess of the thiocycloheptyne-2PCA reagent can be removed by other known techniques, for instance by incubation with hydroxylamine-containing beads, which capture the aldehyde of the reagent by formation of an oxime.
  • the alkyne group of the compounds of the invention is reactive and can be functionalised using for instance cycloaddition-type reactions, in particular to provide bioconjugates.
  • bioconjugate refers to the coupling of two molecules, at least one of which is a biological molecule, in particular a peptide or protein.
  • the alkyne group can be reacted with a compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene.
  • the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene is an azide- comprising compound, a nitrone-comprising compound or a nitrile oxide comprising compound. Most preferred is an azide-comprising compound.
  • the azide-comprising compound can be coupled using a copper-free click reaction. The coupling results in a triazole-type structure.
  • the compounds of the invention are suitable for coupling of a peptide or proteinaceous molecule to another molecule.
  • the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene can be coupled to a thiocycloheptyne compound of the present invention before or after a peptidic or proteinaceous molecule is coupled thereto.
  • a compound comprising a compound according to any of the invention wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to a compound comprising a thiol, a 1,3-dipole or a 1,3- (hetero)diene.
  • the compound comprising a thiol, 1,3-dipole or 1,3- (hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound.
  • the functionalisation can be with any compound, for instance with a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl.
  • radioactive label a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate, in particular comprising a thiol, 1,3-dipole or 1,3-(hetero)diene, preferably an azide, a nitrone or a nitrile oxide, more preferably an azide.
  • SPR surface plasmon resonance
  • the compound comprising a thiol, a 1,3- dipole or a 1,3-(hetero)diene comprises a one or more of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate.
  • SPR surface plasmon resonance
  • the molecule may be attached to the compounds of formula (I), (II) or (III) via a cleavable moiety or linker.
  • the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene comprises a cleavable moiety or linker, preferably between the thiol, a 1,3-dipole or a 1,3-(hetero)diene that is coupled to a compound of formula (I), (II) or (III) of the invention and the peptide, protein or other molecule.
  • Such cleavable moiety or linker provides for release of the molecule under the appropriate circumstances.
  • the cleavable moiety or linker can be any cleavable moiety or linker described herein above. It is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions or by the enzymatic activity. Suitable cleavable moieties or linkers are selected from the group consisting of an ester, orthoester, amide, carbonate, carbamate, anhydride, ketal, acetal and hydrazone. Another example of a suitable cleavable moiety or linker is a linker comprising a valine– citrulline (VCit) dipeptide linker or a glutamic acid–valine–citrulline tripeptide linker.
  • VCit valine– citrulline
  • the invention further provides a use of a compound of any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB), in a bioorthogonal, copper-free, click reaction.
  • Copper-free click chemistry is a bioorthogonal reaction, as by eliminating cytotoxic copper catalysts, the reaction is performed without toxicity to biological systems such as cells and tissues.
  • a method for coupling a molecule to a peptidic or proteinaceous molecule comprising reacting a compound any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) with said molecule, wherein said molecule comprises a thiol a 1,3-dipole or a 1,3-(hetero)diene, and wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to the compound comprising a thiol or a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, a
  • the molecule that is coupled to a compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is a drug delivery vehicle, in particular a nanoparticle.
  • the compound any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is coupled to a nanoparticle at the alkyl group of the thiocycloheptyne of this compound.
  • Drug delivery systems are increasingly used in pharmaceutical science. The use of these drug delivery systems such as nanoparticles or liposomes is developing rapidly in the pharmaceutical sciences.
  • Nanoparticles typically having diameters of ⁇ 100nm have been made from a wide variety of materials and anticipated applications in medicine include drug delivery, both in vitro and in vivo diagnostics, nutraceuticals and production of improved biocompatible material. Nanoparticles can be customised for particular purposes ad a wide variety of materials is available. Most pharmaceutically interesting nanoparticles are based on (bio)polymeric materials that can be in a variety of forms. Source materials may be of biological origin like phospholipids, lipids, lactic acid, dextran, chitosan, or have more “chemical” characteristics like various (co)polymers.
  • nanoparticles are typically functionalised by connecting, coupling, binding (covalently) active ingredients (drugs, ligand, imaging ligands etc.).
  • the functionalisation of many of these nanoparticles can be achieved by a wired variety of chemistries such as copper free click chemistry using the thiocycloheptyne derivatives of the present invention.
  • the nanoparticle is a self-assembling polymeric micelle, preferably from thermosensitive block copolymers.
  • copolymers based on PEG-b-poly(N- hydroxyalkyl methacrylamide-oligolactates) with partially methacrylated oligolactate units are preferred, but also other (meth)acrylamide esters can be used to construct the thermosensitive block, e.g. esters, and optionally (oligo)lactate esters, of HPMAm (hydroxypropyl methacrylamide) and HEMAm (hydroxyethylmethacrylamide), and N- (meth)acryloyl amino acid esters.
  • esters e.g. esters, and optionally (oligo)lactate esters, of HPMAm (hydroxypropyl methacrylamide) and HEMAm (hydroxyethylmethacrylamide), and N- (meth)acryloyl amino acid esters.
  • thermo-sensitive block copolymers are derived from monomers containing functional groups which may be modified by derivatised and underivatised methacrylate groups, such as HPMAm-lactate polymers; that is, this modification encompassing the incorporation of linker moieties.
  • functional thermosensitive (co)polymers which can be used, are hydrophobically modified poly(N-hydroxyalkyl) (meth) acrylamides, copolymer compositions of N-isopropylacrylamide (NIPAAm) with monomers containing reactive functional groups (e.g., acidic acrylamides and other moieties such as N- acryloxysuccinimide) or similar copolymers of poly(alkyl) 2-oxazalines, etc.
  • thermo-sensitive groups can be based on NIPAAm and/or alkyl-2- oxaxolines, which monomers may be reacted with monomers containing a reactive functional group such as (meth)acrylamides or (meth)acrylates containing hydroxyl, carboxyl, amine or succinimide groups.
  • Suitable thermo-sensitive polymers are described in US-B-7,425,581 and in EP-A- 1776400, which is incorporated by reference herein.
  • WO 2010/033022 and WO2013/002636 which are incorporated by reference herein.
  • drug-polymer matrix particles are described using such polymers.
  • biodegradable linker molecules are described that may be used in these known polymer matrix particles.
  • nanoparticles based on thermosensitive block-copolymers as outlined hereinabove can be linked to the compounds of the invention using azide- alkyne copper- free coupling. Examples thereof are described in WO2017/086794, which is incorporated by reference herein.
  • a nanoparticle is prepared wherein a compound of the current invention is coupled to an azide- containing nanoparticle.
  • the nanoparticle is a self- assembling polymeric micelle, preferably from thermosensitive block copolymers.
  • the molecule that is coupled to a compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is an antibody.
  • the compound any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is coupled to an azide-containing antibody at the alkyl group of the thiocycloheptyne of this compound.
  • ADCs Antibody-drug conjugates
  • Antibody ⁇ drug conjugates combine the specificity and targeting potential of monoclonal antibodies (mAbs) with e.g. the potency of cytotoxic molecules.
  • ADCs are for instance developed for the treatment of cancer, where they aim to target and kill tumor cells while sparing non-tumor cells.
  • An example of an antibody-drug conjugate in accordance with the invention is therefore a bioconjugate of a cytotoxic peptide that is functionalized with a thiocycloheptyne-2PCA compound of the invention.
  • Any cytotoxic peptide can be functionalized with a thiocycloheptyne-2PCA compound of the invention and subsequently coupled to an antibody to form an ADC.
  • Figure 3 TP10-2PCA-glycol-TMTHSI (right peak) and TP10-2PCA-glycol- TMTHSI clicked with azidoethanol (left peak indicated with arrow) resulting in a peak shift.
  • Figure 4 Reaction of Evasin-3 with 16 (top) and chromatogram of reaction mixture after 26 hours (bottom)
  • Figure 5 HPLC trace of Evasin-3-TMTHSI 20.
  • Figure 6 Reaction of Evasin-3 TMTHSI 20 with CDP-azide 21 (top) and trace of purified product with mass inserted from deconvoluted MS spectrum of peak (bottom).
  • Figure 7 Decrease of the radiochemical purity of B2 over time.
  • the first click construct (3) that we evaluated was obtained in 50% yield by combining 2PCA (2) and TMTHSI via a piperazine linker in a reaction with our earlier described TMTHSI-hydroxy succinimide derivative 1 (Scheme 1).
  • Scheme 1 Synthesis of TMTHSI-2PCA 3
  • Octreotide 4 a clinically used peptide for the treatment of acromegaly, was used for studying this new TMTHSI-2PCA reagent 3. Since octreotide is a cyclized peptide, containing several functionalities including a lysine side chain NH2 and a disulfide bridge, it is a valid model for evaluation of this potential N-specific reagent 3. After reaction with 20 eq.
  • TMTHSI-2PCA 3 Reaction of TMTHSI-2PCA 3 with LTX 6 containing several lysine residues.
  • the TMTHSI-2PCA 3 reagent is a suitable reagent for N-specific introduction of a TMTHSI-containing linker into a multifunctional peptide.
  • TMTHSI derivative with a larger distance between the aldehyde reacting with the N-terminus and the TMTHSI-moiety, that is 16 (Scheme 4).
  • the larger distance formed by an ethylene glycol spacer may be also favourable for water solubility of the adduct, in addition to the creation of a suitable distance between the strain promoted azide alkyne cycloaddition reaction and the site of attachment of the reagent in a sizable peptide or protein.
  • the TMTHSI-glycol-2PCA derivative 16 was obtained in a straightforward 7-step synthesis starting from ethylene glycol derivative 8 and pyridine bismethylene alcohol 11 (Scheme 4). Scheme 4. Synthesis of TMTHSI-glycol-2PCA 16. This TMTHSI-glycol-2PCA derivative 16 was used in the N-terminal modification of the peptide Angiopep217 (Scheme 5).
  • Angiopep2 is applied as a conjugate for intracellular delivery of oligonucleotides and blood-brain barrier transport (Lei et al. 2022).
  • Excess of TMTHSI-glycol-2PCA can be removed by incubation, after completion of the reaction, with hydroxylamine containing beads (Scheme 5). These beads capture the aldehyde of TMTHSI-glycol-2PCA by formation of an oxime, which is illustrated in the work-up of the reaction of peptide TP10 (sequence) with hydroxyl amine beads.
  • Scheme 5 Capture of excess of TMTHSI-glycol-2PCA 16 using hydroxyl amine beads.
  • TMTHSI-2PCA conjugates with BSA in human serum The aim of this Example was to determine the stability of TMTHSI-2PCA BSA conjugates under physiological conditions.
  • Bovine serum albumin (BSA) was used as model protein, conjugation will occur with the 2PCA moiety -
  • DFO-N3 was attached to the TMTHSI moiety via the alkyne functionality making 89 Zr labelling possible -
  • the reaction mixture was purified using a PD10 cartridge column according to the following method: - Pipette 500 ⁇ l of the BSA modification solution onto the column - Pipette 1750 ⁇ l 0.1 M PBS onto the column - Pipette 1750 ⁇ l PBS onto the column; this fraction is caught
  • the fraction caught from the PD-10 column was further purified using the following 30 kDa spin filter method: - Fill three 30 kDa spin filters with 500 ⁇ l, and one 30 kDa spin filter with 250 ⁇ l, and centrifuge the solution at 10000 rcf for 3 minutes - Wash the spin filter with 200 ⁇ l 1.0 M PBS, and centrifuge at 10000 rcf for 3 minutes - Repeat the washing step a total of 3 times - Reverse the spin filter into a new Eppendorf vial, and centrifuge at 10000 rcf for 3 minutes - Top the caught solution to 500 ⁇ l using PBS.
  • the counting window is between 400-1100 keV, with the counting time set to one minute -
  • the full gamma-counter procedure lasts ⁇ 15 minutes and is performed at room temperature
  • the radiochemical purity of B2 is determined by looking at the radiochemical purity of the tube containing the spin filter. Monitoring the stability of B2 according to the spin filter method described above yielded the radiochemical purity results shown in Figure 7. Although the control experiment (dashed, grey line) was not monitored as long as the experiment in human serum (solid, black line), the decrease in radiochemical purity follow similar trends in both experiments. Additionally, considering the standard deviation, the difference in radiochemical purity over time between the control and serum experiments does not appear to be significant. The results indicate that the stability of B2 is not affected upon being exposed to human serum.
  • Example 3 Chemical stability of TMTHSI-2PCA with model peptides under rigorous acidic conditions The aim of this Example was to evaluate the stability of TMTHSI-2PCA conjugates at acidic pH values. - Tuftsin was used as a model peptide - Since 2PCA absorbs well at 267 nm, there was no need to use a chromophore to label the tuftsin-CliCr construct (C1) via the alkyne - The stability was determined by monitoring the decrease in C1 over time (vs.
  • TMTHSI-2PCA conjugates would only be exposed to an acidic environment during work-up and/or analysis (Timmers et al. 2023). As Figure 8 shows, the amount of C1 in solution stays above 95% for about 56 hours, meaning that the 2PCA conjugate will remain stable for the duration of work-up and analysis steps.
  • Soomets U.; Lindgren, M.; Gallet, X.; Hällbrink, M.; Elmquist, A.; Balaspiri, L.; Zorko, M.; Pooga, M.; Brasseur, R.; Langel, Ü. Deletion Analogues of Transportan. Biochim. Biophys. Acta 2000, 1467 (1), 165–176.

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Abstract

L'invention concerne de nouveaux composés de formule (I), éventuellement couplés à une molécule peptidique ou protéique, et leur synthèse. L'invention concerne également l'utilisation des nouveaux composés dans des réactions de couplage avec des lieurs et d'autres molécules. L'invention concerne en outre l'utilisation des nouveaux composés dans la cycloaddition bioorthogonale induite par la tension (sans cuivre) c'est-à-dire des réactions click.
PCT/NL2024/050322 2023-06-20 2024-06-20 Couplage peptidique à l'aide de dérivés de thiocycloheptyne Pending WO2024263030A1 (fr)

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WO2010033022A1 (fr) 2008-09-18 2010-03-25 Universiteit Utrecht Holding B.V. Procédé d'élaboration d'un système à libération contrôlée
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