WO2018234483A1 - Sondes accordables pour marquage sélectif de protéines et inhibition enzymatique - Google Patents

Sondes accordables pour marquage sélectif de protéines et inhibition enzymatique Download PDF

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WO2018234483A1
WO2018234483A1 PCT/EP2018/066633 EP2018066633W WO2018234483A1 WO 2018234483 A1 WO2018234483 A1 WO 2018234483A1 EP 2018066633 W EP2018066633 W EP 2018066633W WO 2018234483 A1 WO2018234483 A1 WO 2018234483A1
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group
compound
formula
polypeptide
peptide
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PCT/EP2018/066633
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Frederik Diness
Morten Meldal
Ahmed EMBABY
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Aaa Chemistry Aps
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the present invention relates to the field of methods of covalently reacting cysteine residues with compound as well as to the compounds per se.
  • the methods may be employed in the fields of labelling polypeptides, detecting polypeptides, modulation of enzyme activity, isolation of polypeptides, methods of diagnostics, methods of prevention or treatment of clinical conditions or methods of transportation of
  • cysteines may be attributed to the unique properties of the thiol group.
  • the S-H bond has a low dissociation energy, which facilitates the ability of the thiol to act as a nucleophile in hydrolysis and in redox reactions.
  • acidity of cysteine in proteins greatly depend on the local protein environment. At the surface of proteins, cysteine thiols have a pKa of ⁇ 8.5; whereas, it may be as low as 2.5 for a catalytic thiol in an active site.
  • Reactive electrophiles designed to probe cysteine residues have been described, including
  • CMK chloromethylketone
  • ACMK acylomethylketone
  • epoxides epoxides
  • sulfonate esters epoxides
  • sulfonate esters epoxides
  • sulfonate esters epoxides
  • sulfonate esters epoxides
  • sulfonate esters epoxides
  • halocetamides as well as a range of Michael acceptors, including maleimides, acrylamides, vinyl sulfonamides, amino methyl acrylate, and methyl vinyl sulfones.
  • aryl halides such as chloronitrobenzenes and dichlorotriazines, have been investigated for cysteine labeling. However, these have not been explored with regards to promiscuity towards other amino acid nucleophiles, or selective reactivity among different cysteine residues.
  • the present invention provides selective reactive probes comprising a benzene core.
  • the benzene core provides six positions for spatial orientation of an electrophilic carbon, tuning of reactivity, and attachment of linkers with other functionalities of interests. This is in contrast to the other reactive electrophiles listed above, which have no or little room for attenuation of reactivity by structural variation in close proximity to the carbon attacked by the thiol of cysteine ( Figure 1 ).
  • the present invention provides methods for reacting a compound of Formula I with a cysteine residue, thereby forming a covalent bond, wherein the cysteine is contained in the sequence of a polypeptide, wherein said compound has the following structure:
  • R 3 is a leaving group
  • R 1 is R 7 ;
  • R 7 is selected from the group consisting of -X-Z-R 8 , -Z-T-R 8 , and -Z-T-(R 8 ) 2 ;
  • X is selected from the group consisting of a bond, -CH 2 -N(R 9 )-, and -CH 2 -0-;
  • Z is selected from the group consisting of a bond, .
  • T is selected from the group consisting of a bond, R 9 , R 9 R 9 ,
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y, -linker-Y and -linker-R 14 , wherein said peptide optionally may be N- and/or C-terminally modified;
  • Y is a labelling molecule, a drug molecule or a prodrug
  • R 9 is individually selected from the group consisting of -H, alkyl-COOH and an amino acid side chain;
  • R 10 is selected from the group consisting of -H, -OH and R 8 ;
  • R 14 is a reactive group
  • R 2 , R 4 , R 5 , R 6 are individually selected from the group consisting of -H, R 7 , an amino acid side chain and an electron withdrawing group;
  • the present invention concerns a method of modulating the activity of a protein, said method comprising performing the method described herein. In one aspect, the present invention concerns a method for labelling a polypeptide with a labelling molecule, said method comprising performing the method described herein.
  • the present invention concerns a method of transportation of a drug molecule or a prodrug, said method comprising performing the method as described herein, using a compound of formula I, wherein Y is a drug molecule or a prodrug.
  • the present invention concerns a method for detecting a polypeptide, said method comprises the steps of a) performing the method described herein using a compound of formula I, wherein R 8 is -linker-Y or -NH-linker-Y; and b) detecting Y.
  • the present invention concerns a method for diagnosis of a clinical condition associated with a polypeptide in an individual at risk of acquiring said clinical condition, said method comprising the steps of a) performing the method as described herein on a sample from said individual using a compound of formula I, wherein R 8 is - linker-Y or -NH-linker-Y, and wherein the polypeptide is associated with said clinical condition; and b) detecting Y; thereby determining the presence, absence and/or level of said polypeptide in said sample.
  • the current invention concerns a method for treatment or prevention of a clinical condition associated with a polypeptide in an individual in need thereof, said method comprising performing the method described herein.
  • the current invention concerns a compound of Formula I:
  • R 3 is a leaving group
  • R 1 is R 7 ;
  • R 7 is selected from the group consisting of -X-Z-R 8 , -Z-T-R 8 , and -Z-T-(R 8 ) 2 ;
  • X is selected from the group consisting of a bond, -CH 2 -N(R 9 )-, and ected from the group consisting of a bond, , and
  • T is selected from the group consisting of a bond, R 9 , R 9 R 9 ,
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y and -linker-Y, wherein said peptide optionally may be N- and/or C-terminally modified;
  • Y is a labelling molecule, a drug molecule or a prodrug
  • R 9 is individually selected from the group consisting of -H, alkyl-COOH and an amino acid side chain;
  • R 10 is selected from the group consisting of -H, -OH and R 8 ;
  • R 2 , R 4 , R 5 , R 6 are individually selected from the group consisting of -H, R 7 , an amino acid side chain and an electron withdrawing group;
  • R 2 , R 4 , R 5 , and R 6 is an electron withdrawing group, and that no more than four of R 2 , R 3 , R 4 , R 5 , and R 6 are -F.
  • Figure 1 A) General approach for selective cysteine modification by tuning the reactivity of fluoroaryls in comparison to non-tunable unselective traditional reactive groups as iodacetamide. B) General scheme for protein modification through a nucleophilic aromatic substitution reaction between cysteine and fluoroaryls.
  • Figure 5 Labeling of protein sulfhydryl groups.
  • Figure 6. The ability of compounds 2a and 2g to inhibit papain (cysteine protease) while in B) no such effect was observed with subtilisin (serine protease) after incubation for 60 min at 37 °C.
  • TEV protease The activity of TEV protease was measured by FRET-substrate cleavage. Shown in red is the average from triplet determinations of FRET-substrate incubated with TEV protease. In blue is shown the average of triplet determinations of sample without enzyme (negative control).
  • ABPP Activity-based protein profiling
  • Compound 8b was able to label TEV protease (spiked) and a few other proteins from which chloramphenicol acetyl transferase enzyme was identified by HRMS.
  • Figure 9. Shows inhibition assay of caspase-1 by compounds 23-25 in comparison to AcYVAD-CMK. B) TEV inhibition assay shows that compounds 23-25 has no effect on TEV activity at the same concentration.
  • Figure 10. A) Labelling of cysteine-containing Human Serum Albumin with 27 by Cu- catalyzed click chemistry as described in Example 9.
  • a waved line ( ) indicates the point of attachment of the substituent.
  • a substituent or moiety indicated by a general formula comprising two waved lines may be linked to the compounds of the invention in any direction. Accordingly, a substituent moiety of the general formula ⁇ I ⁇ A— B- ⁇ - or ⁇ ⁇ may equally well be denoted with the
  • a substituent or moiety indicated by a general formula comprising two free bonds may be linked to the compounds of the invention in any direction. Accordingly, a substituent or moiety of the general formula -A-B- may equally well be denoted with the general formula -B-A-.
  • active site refers to the region of an enzyme where substrate molecules bind and undergo a chemical reaction.
  • acetylated means linked to an acetyl group:
  • alkane refers to saturated linear or branched carbohydrides of the general formula C n H 2n+2 .
  • alkenyl refers to a substituent derived from an alkene by removal of one -H.
  • An alkene may be any acyclic carbonhydride comprising at least one double bond. Frequently, alkenyl will have the general formula -C n H 2 n-i .
  • alkyl refers to a substituent derived from an alkane by removal of one -H.
  • amine refers to a compound comprising an -N-.
  • Primary amines comprises - NH 2 .
  • alkynyl refers to a substituent derived from an alkyne by removal of one -H.
  • An alkyne may be any acyclic carbonhydride comprising at least one triple bond. Frequently, alkynyl will have the general formula -C n H 2n-3 .
  • amino acid refers to a compound of the following general
  • R indicates the amino acid side chain.
  • R may be -H in which case the amino acid is glycine.
  • amino acid as used herein also covers amino acids linked to other amino acids in a peptide or polypeptide. Thus, amino acids may be bound to each other by peptide bonds to form peptides or R
  • polypeptides of the following general structure n , wherein n is an integer and * indicates the point of attachment to the next amino acid residue.
  • Amino acids may be standard amino acids, but also includes other amino acids of
  • Amino acids may be D-stereo-isomers (referred to as D-amino acids herein) or may be L-stereo-isomers (referred to as L-amino acids herein).
  • amino acid derivative refers to a compound which may be synthesised from an amino acid.
  • the compound has the following general structure: , wherein R indicates the amino acid side chain.
  • R may be -H in which case of derivatives of the amino acid glycine.
  • R x are individually selected from the group consisting of -H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, heteroalkyl and heteroalkenyl.
  • amino acid side chain refers to -H or a substituent of the
  • R 11 , R 12 , R 13 individually are selected from the group consisting of -H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, heteroalkyl and heteroalkenyl.
  • amino acid side chains are the side chains of the standard amino acids, for example the side chains of the 21 proteinogenic oarmino acids found in eukaryotes.
  • arene refers to aromatic mono- or polycyclic carbonhydrides.
  • aromatic refers to a chemical substituent characterised by the following: • contains a delocalized conjugated ⁇ system, most commonly an arrangement of alternating single and double bonds
  • aryl refers to a substituent derived from an arene by removal of one -H from a C in the ring.
  • Examples of useful aryls to be used with the present invention comprise phenyl, napthyl, anthracenyl, phenanthrenyl, and pyrenyl.
  • C-terminally modified refers to a peptide being covalently linked to a moiety at the C-terminus.
  • a non-limiting example of a C-terminal modification may be a C- terminal amidation or esterification, e.g. the C-terminal OH group of the peptide may be exchanged with an amine group or an alkoxy group.
  • recognition sequence for peptide cleaving enzyme refers to an amino acid sequence recognised and hydrolysed by a peptide cleaving enzyme, such as a protease.
  • a “recognition sequence” may comprise or consists of a
  • the recognition sequence for the peptide cleaving enzyme comprises the amino acid sequence ENLYFQGY.
  • the recognition sequence for the peptide cleaving enzyme comprises the amino acid sequence E N L Y F Q G K. This may in particular be the case when the peptide cleaving enzyme is TEV protease.
  • detectable label refers to any label, which can be detected, i.e. capable of generating detectable signals. Detectable labels are also commonly known in the art as “detectable moieties”. Examples of detectable labels include tags and probes. The detectable label may for example be selected from the group consisting of radiolabels, biotin, fluorescent labels, luminescent labels and coloured labels. Examples of detectable labels include chromogenic moieties, fluorescent moieties, radioactive moieties and electrochemically active moieties.
  • a chromogenic moiety is a moiety which is coloured, which becomes coloured when it is incorporated into a conjugate, or which becomes coloured when it is incorporated into a conjugate and the conjugate subsequently interacts with a secondary target species (for example, where the conjugate comprises a protein which then interacts with another target molecule).
  • the term "chromogenic moiety” refers to a group of associated atoms which can exist in at least two states of energy, a ground state of relatively low energy and an excited state to which it may be raised by the absorption of light energy from a specified region of the radiation spectrum. Often, the group of associated atoms contains delocalised electrons. Examples include porphyrins, polyenes, polyynes and polyaryls.
  • a fluorescent moiety is a moiety which comprises a fluorophore, which is a fluorescent chemical moiety.
  • fluorescent compounds include: the Alexa Fluor ® dye family available from Invitrogen; cyanine and merocyanine; the BODIPY (boron- dipyrromethene) dye family, available from Invitrogen; the ATTO dye family manufactured by ATTO-TEC GmbH; fluorescein and its derivatives; rhodamine and its derivatives; naphthalene derivatives such as its dansyl and prodan derivatives;
  • pyridyloxazole nitrobenzoxadiazole and benzoxadiazole derivatives
  • coumarin and its derivatives pyrene derivatives
  • Oregon green eosin, Texas red, Cascade blue and Nile red, available from Invitrogen.
  • a radioactive moiety is a moiety that comprises a radionuclide.
  • radionuclides include iodine-131 , iodine-125, bismuth-212, yttrium-90, yttrium-88, technetium-99m, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-1 1 1 , indium-1 14m, indium-1 14, boron-10, tritium (hydrogen-3), carbon-14, sulfur-35, fluorine-18 and carbon-1 1. Fluorine-18 and carbon-1 1 , for example, are frequently used in positron emission tomography.
  • the radioactive moiety may consist of the radionuclide alone.
  • the radionuclide may be incorporated into a larger radioactive moiety, for example by direct covalent bonding to a linker group (such as a linker containing a thiol group) or by forming a co- ordination complex with a chelating agent.
  • a linker group such as a linker containing a thiol group
  • a co- ordination complex with a chelating agent.
  • Suitable chelating agents known in the art include DTPA (diethylenetriamine- pentaacetic anhydride), NOTA (1,4,7- triazacyclononane-N,N',N"-triacetic acid), DOTA (1 , 4,7,10-tetraazacyclododecane- N,N',N",N"'-tetraacetic acid), TETA (1 ,4,8,1 l-tetraazacyclotetra-decane-N,N',N",N"'- tetraacetic acid), DTTA ( ⁇ '- ⁇ - isothiocyanatobenzyl)-diethylene-triamine-N 1 ,N 2 ,N 3 - tetraacetic acid) and DFA ( ⁇ '- [5 - [[5- [ [5-acetylhydroxyamino)pentyl] amino] - 1 ,4- dioxobutyl]hydroxyamino]pentyl] - N-
  • An electrochemically active moiety is a moiety that comprises a group that is capable of generating an electrochemical signal in an electrochemical method such as an amperometric or voltammetric method.
  • an electrochemically active moiety is capable of existing in at least two distinct redox states.
  • electro withdrawing group refers to a substituent which has a positive Hammett Meta Substituent Constant (o m ) and a positive Hammett Para Substituent Constant (o p ). (As listed in table I in Chemical Reviews 1991 , 91 , 165-195)
  • Preferred "electron withdrawing groups” X, SX 5 , S0 2 NR 2 , CX 3 , OCX 3 , SCX 3 , SOCX 3 , S0 2 CX 3, SOR , S0 2 R , CN, CHX 2 , COR, CONR 2 and COOR , wherein X is a halogen and R is alkyl.
  • halogen refers to a substituent selected from the group consisting of -F, -CI, -Br and -I.
  • heteroalkenyl refers to a straight- or branched-chain alkenyl group, of which one or more carbon has been replaced by a heteroatom selected from S, O and N.
  • exemplary heteroalkenyls include alkyl esters, ketones, aldehydes, amides, carbamates, ureas, guanidines, and sulfoxides.
  • heteroalkyl refers to a straight- or branched-chain alkyl group, of which one or more carbon has been replaced by a heteroatom selected from S, O and N.
  • heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.
  • heteroaryl refers to a substituent derived from an heteroarene by removal of one -H from an atom in the ring structure of said heteroarene.
  • Heteroarenes are mono- or polycyclic aromatic compounds comprising one or more heteroatoms in the ring structure. Said heteroatoms are preferably selected from the group consisting of S, N and O.
  • Non limiting examples of useful heteroaryls to be used with the present invention comprise azolyl, pyridinyl, pyrimidinyl, furanyl, and thiophenyl.
  • the term "individual” as used herein refers to any individual, preferably a mammal, and more preferably a human being.
  • the term “Labelling molecule” refers to any moiety, which can be used to label a compound.
  • a labelling molecule is a detectable label.
  • the labelling molecule may be selected from the group consisting of a fluorescent labelling molecule, a radioisotope labelling molecule, an affinity molecule, an azide, a terminal alkyne, a spin label, a prodrug, a mass tag, and a photoreactive group.
  • linker refers to a chemical moiety linking two other chemical moieties. Preferred linkers to be used with the invention are described herein elsewhere.
  • N-terminally modified refers to a peptide being covalently linked to a moiety at the N-terminus.
  • a non-limiting example of an N-terminal modification may be that said peptide may contain an N-terminal acetylation, carboxylation, or alkylation, e.g. the N-terminal amine may be acetylated, benzoylated, carbobenzoxylated, methylated or benzylated.
  • peptide refers to a shorter sequence of amino acid residues linked by peptide bonds.
  • a peptide may consist of in the range of 2 to 40 amino acids.
  • Peptides may be N- and/or C-terminally modified.
  • polypeptide refers to a sequence of amino acids linked by peptide bonds. In general a polypeptide comprises at least 4 amino acid residues.
  • K a also called the acidity constant, is defined as: [A IfSH ⁇ l
  • small molecule refers to organic molecules with molecular weights of no more than 900 g/mol.
  • standard amino acid refers to the 20 amino acids encoded by the standard genetic code.
  • the amino acids are referred to herein using standard lUPAC
  • Standard amino acids are all L-amino acids.
  • substituted refers to hydrogen group(s) being substituted with another moiety.
  • substituted with X refers to hydrogen group(s) being substituted with X.
  • substituted X refers to X, wherein one hydrogen group has been substituted with another moiety.
  • substituted alkyl refers to alkyl-R, wherein R is any moiety but -H.
  • substituted refers to atom or group of atoms substituted in place of a hydrogen atom.
  • the present invention concerns a method for reacting a compound of Formula I with a cysteine residue, thereby forming a covalent bond, wherein the cysteine is contained in the sequence of a polypeptide.
  • the compound of formula I may be any of the compounds of formula I described herein below in the section
  • Compound of formula I may be any compound of one of the more specific formulas II, III, IV, VI, VII, VIII, IX or X described herein below in the section compound of formulas II to X.
  • the methods of the invention may be performed in any manner allowing for reaction between the compound of formula I and the cysteine residue.
  • the skilled person will be able to select appropriate conditions.
  • the methods of the invention are performed in an aqueous environment, for example in an aqueous solvent.
  • the methods of the invention are performed in vivo. This may in particular be the case in embodiments, where the cysteine residue is contained in the sequence of a polypeptide present in vivo. In such embodiments, the methods are performed under in vivo conditions.
  • reaction of compound of Formula I with a cysteine residue is conducted at the surface of or inside a living organism.
  • the methods may be performed in or on a sample obtained from an individual.
  • the methods are performed in vitro.
  • the methods may comprise the steps of:
  • the steps of said method may be performed in any order.
  • the steps of said method are performed in the above mentioned order.
  • the pH may optionally be adjusted to any pH useful for performing the method, for example the pH may be adjusted to in the range of 6 to 9.
  • the reacting step is performed in the absence of a catalyst.
  • the method may be performed in a solvent, which is a protic solvent mixture.
  • the solvent is an aqueous solvent mixture.
  • the solvent may be a solvent containing material from living organisms e.g. body fluids and/or tissues.
  • the body fluid may for example be plasma. Accordingly, said polypeptide may be provided in the form of a sample from an individual.
  • the solvent may be a solvent compatible with hosting living organisms, e.g. a culture medium or a buffer with physiological salinity and pH.
  • Example 1 Preferred examples of general reaction schemes for reacting compounds of formula I with a polypeptide are shown in Example 1 in Method A and Method B.
  • the scheme shows reaction of a protected cysteine with fluoro-benzene, however, the scheme can easily be adapted to a reaction between any polypeptide comprising cysteine and any compound of formula I.
  • Example 1 Another example of a general reaction schemes for reacting compounds of formula I with a polypeptide are shown in Example 1 in the general procedure (1 ) for S-arylation. This scheme can also be adapted as described above.
  • the invention relates to methods of reacting a compound of formula I with a cysteine residue. Furthermore, the invention relates to compounds of formula I per se.
  • the compound of Formula I may be any compound having the following structure:
  • R 3 is a leaving group
  • R 1 is R 7 ;
  • R 7 is selected from the group consisting of -X-Z-R 8 , -Z-T-R 8 , and -Z-T-(R 8 ) 2 ;
  • X is selected from the group consisting of a bond, -CH 2 -N(R 9 )-, and -CH 2 -0-;
  • Z is selected from the group consisting of a bond, .
  • T is selected from the group consisting of a bond, R 9 , R 9 R 9 ,
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y, -linker-Y, and -linker-R 14 , wherein said peptide optionally may be N- and/or C-terminally modified;
  • Y is a labelling molecule, a drug molecule or a prodrug
  • R 9 is individually selected from the group consisting of -H, alkyl-COOH and an amino acid side chain;
  • R 10 is selected from the group consisting of-OH and R 8 ;
  • R 14 is a reactive group
  • R 2 , R 4 , R 5 , R 6 are individually selected from the group consisting of -H, R 7 , an amino acid side chain and an electron withdrawing group;
  • R 2 , R 4 , R 5 , and R 6 is an electron withdrawing group.
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y, and -linker-Y, wherein said peptide optionally may be N- and/or C-terminally modified.
  • no more than four of R 2 , R 3 , R 4 , R 5 , R 6 are -F.
  • R 2 , R 4 , R 5 , R 6 are individually selected from the group consisting of -H, R 7 , an amino acid side chain and an electron withdrawing group with the proviso that at least one of R 2 , R 4 , R 5 , and R 6 is an electron withdrawing group, and that no more than four of R 2 , R 3 , R 4 , R 5 , and R 6 are -F.
  • moieties comprising two waved lines or two free bonds may be linked to the compounds of the invention in any direction. Accordingly, individual X, Z and T described above may be placed in the compounds of the invention in any direction.
  • R 1 is para to R 3 . In another embodiment, R 1 is ortho to R 3 . In some embodiments, R 3 is selected from the group consisting of -F, -CI, -Br, -I, -N0 2 and S0 2 -alkyl. Preferably, R 3 is selected from the group consisting of -F and -CI. In a preferred embodiment, R 3 is -F.
  • the electron withdrawing groups individually are selected from the group consisting of -F, -CI, -CF 3 , -CCI 3 , S0 2 -alkyl and -C ⁇ N.
  • the electron withdrawing group may be selected from the group consisting of -F and -CI.
  • At least one, such as at least two of R 2 , R 4 , R 5 , and R 6 are -F or - CI. In one embodiment, at least one, such as at least two, but at the most three of R 2 , R 4 , R 5 , and R 6 are -F or -CI. In another embodiment, all of R 2 , R 4 , R 5 and R 6 are -H, - F or -CI, with the proviso that at least one, such as at least two of R 2 , R 4 , R 5 , and R 6 are -F or -CI.
  • all of R 2 , R 4 , R 5 and R 6 are -H or -F, with the proviso that at least two of R 2 , R 3 , R 4 , R 5 , and R 6 are -F. In one embodiment, at the most three of R 2 , R 4 , R 5 , and R 6 are -F or -CI. In another embodiment, all of R 2 , R 4 , R 5 and R 6 are -H, -F or -CI, with the proviso that at the most three of R 2 , R 4 , R 5 , and R 6 are -F or -CI.
  • all of R 2 , R 4 , R 5 and R 6 are -H or -F, with the proviso that at the most three of R 2 , R 3 , R 4 , R 5 , and R 6 are -F.
  • R 3 , R 4 , R 5 and R 6 are -F, and R 2 is -F or R 9 .
  • R 3 , R 4 , R 5 and R 6 are -F, one of R 3 , R 4 , R 5 and R 6 is -H and R 2 is -F or R 9 .
  • at least one R 9 is -H.
  • all R 9 is -H.
  • R 8 may individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, -NH-linker-Y and -linker-Y, wherein said peptide optionally may be N- and/or C-terminally modified.
  • R 8 may individually selected from the group consisting of amino acids, peptides, alkylamines, -NH-linker-Y and -linker-Y, wherein said peptide optionally may be N- and/or C-terminally modified.
  • R 8 may individually be selected from the group consisting of -NH- linker-Y and -linker-Y.
  • the linker may be an alkyl wherein one or more -C(H 2 )- have been replaced with - C(O)-, -N(H)-, -0-, or -S-.
  • the linker is selected from the group consisting of peptides, oligosaccharides and steroids.
  • the linker is or comprises -N(H)-(PEG) n , wherein n is an integer from 0 to 10.
  • the linker is or comprises -N(H)-(CH2-CH2-0-) n -(CH2) m -, wherein n is an integer from 0 to 20 and m is an integer from 0 to 5.
  • the linker is or comprises -(CH 2 -CH2-0-) n -(CH2)m--
  • n is an integer from 4 to 20.
  • n is an integer from 3 to 10, such as from 3 to 5, such as 3.
  • m is 2.
  • the linker comprises a triazole moiety.
  • R 8 is -linker-R 14 , wherein R 14 is a first reactive group.
  • first reactive group refers to groups that are capable, under suitable conditions, of reacting with a second reactive group. Said first reactive group may be reacted with any compound comprising the second reactive group.
  • a compound comprising a second reactive group further comprises Y. In such embodiments Y may be linked to the compound of formula I following a reaction between the first reactive group and the second reactive group. The reaction between a compound of Formula I wherein R 8 is -linker-R 14 , and a compound comprising Y and a second reactive group, may occur before or after the compound of Formula I has reacted with said cysteine residue.
  • the reactive group is an azide group or an alkyne.
  • said first reactive group is an azide group
  • said second reactive group is an alkyne.
  • Said azide group may be reacted with the alkyne group to form a triazole moiety through a 1 ,3-dipolar cycloaddition, optional in the presence of a transition metal catalyst.
  • the reaction between a compound of Formula I, wherein R 8 is a linker with a terminal azide group, and a compound comprising Y and an alkyne group may occur before or after the compound of Formula I has reacted with said cysteine residue, see e.g. Examples 3 and 9.
  • Z is ° . This may in particular be the case in embodiments, wherein at the most 4, such as at the most 3 of R 2 , R 3 , R 4 , R 5 , and R 6 are -F.
  • Z is 0 , and at the most 4, such as at the most 3, of R 2 , R 3 , R 4 , R 5 , and R 6 are -F.
  • the labelling molecule Y may be any labelling molecule.
  • the labelling molecule may be a detectable label.
  • the labelling molecule Y may be selected from the group consisting of a fluorescent labelling molecule, a radioisotope labelling molecule, an affinity molecule, an azide, a terminal alkyne, a spin label, a prodrug, a mass tag, and a photoreactive group.
  • Y is a protective group, for example a protective group selected from the group consisting of Boc and Fmoc.
  • Y is a reactive group, which can be detected by reacting the reactive group with a directly detectable compound.
  • the labelling molecule may be azide, which can be detected by reacting the azide moiety with a directly detectable compound by click chemistry.
  • the directly detectable compound may for example be a fluorescent molecule or a dye, such as cyanine dye.
  • the labelling molecule Y is biotin.
  • the fluorescent labelling molecule may be any fluorescent molecule, e.g. rhodamine or sulforhodamine.
  • the Y is a prodrug or a drug molecule.
  • said prodrug or drug molecule is a small molecule.
  • the person of skill in the art is well aware of prodrugs and drug molecules that are suitable as Y of the present invention.
  • such a small molecule may be ibuprofen, as in Example 9.
  • R 8 may individually be selected from the group consisting of amino acids and peptides.
  • R 8 is a peptide.
  • said peptide is linked to the benzene, Z or T via the N- terminus or the C-terminus.
  • the compound of Formula I is:
  • p is an integer from 0 to 10, such as 1 , such as 2, such as 3, such as 4, such as 5, such as 6, such as 7, such as 8, such as 9, such as 10.
  • the compound of Formula I is:
  • the compound of Formula I is selected from the group consisting of:
  • the compound of Formula I is selected from the group consisting of compounds 2g, 8a, 8b, 8c, 8d, 10, and 11 described in the Examples below. In one embodiment of the invention, the compound of formula I is selected from the group consisting of compounds 22c, 22d, 22e, 22f, 23 and 24 described in the Examples below.
  • Compounds of formulas II to X are selected from the group consisting of compounds 2g, 8a, 8b, 8c, 8d, 10, and 11 described in the Examples below. In one embodiment of the invention, the compound of formula I is selected from the group consisting of compounds 22c, 22d, 22e, 22f, 23 and 24 described in the Examples below.
  • the compound of formula I may in preferred embodiments be any of the compounds of one of the more specific formulas II, III, IV, VI, VII, VIII, IX or X described in this section.
  • the benzene of the compound of Formula I is integrated into the backbone of a peptide, which may optionally be C- or N-terminally modified.
  • the benzene may be integrated into the backbone of a peptide, wherein the peptide may comprise a recognition sequence for a peptide cleaving enzyme in a manner, wherein said benzene replaces at least part of said recognition sequence.
  • Said recognition sequence may for example be represented by the general formula RSI-RSII-RSIII.
  • RSii may for example comprise the cleavage site.
  • the benzene of the compound of Formula I may in some embodiments be incorporated into a peptide comprising or consisting of the sequence of the sequence RSi-RSn-RSin, wherein the benzene replaces RS N , or RS r RSn or RSn-RSin.
  • the peptide RSi-RSn-RSin is a substrate for a peptide cleaving enzyme.
  • R 1 of the compound of Formula I is RSi and R 2 of the compound of Formula I is RSm, wherein RSi and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C-terminally modified.
  • RSi and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C-terminally modified.
  • RSi is linked to said RSm via RSn, which is selected from the group consisting of a bond, amino acids and peptides, they together form the peptide RSi-RSn-RSin, which is a substrate for an enzyme.
  • said enzyme is a peptide cleaving enzyme.
  • the compound of Formula I may be a compound of the general formula II
  • RSi when linked to RSm via RS M together forms the peptide RSi-RSn-RSin , which is a substrate for a peptide cleaving enzyme; and RS
  • RSii is selected from the group consisting of a bond, amino acids and peptides; and R 3 , R 4 , R 5 and R 6 are as defined in item 1.
  • Z-T-R is In one embodiment, Z-T-R is In another embodiment, Z-T-R is
  • R 10 is -OH.
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y and -linker-Y.
  • R 8 is individually selected from the group consisting of amino acids, peptides and alkylamines, wherein said amino acid or peptide optionally may be N- and/or C-terminally modified.
  • R 8 is an amino acid or a peptide.
  • Said peptide may consist of 2 to 5 amino acids, such as 2 to 3 amino acids.
  • R 8 is a protecting group, which may be selected from the group consisting of Boc and Fmoc.
  • T is and one R 9 is -H and the other R 9 is alkyl- COOH, preferably, -CH 2 -COOH.
  • the compound of Formula I is bonded to a nitrogen of the peptide backbone as in Formula X:
  • R 1 of the compound of Formula I is RSi or RSm, wherein RSi and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C-terminally modified.
  • RSi and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C-terminally modified.
  • RSi is linked to said RSm via RSn, which is selected from the group consisting of a bond, amino acids and peptides, they together form the peptide RSi-RSn-RSin, which is a substrate for an enzyme.
  • said enzyme is a peptide cleaving enzyme.
  • the benzene of the compound of Formula I is linked to the N- or C-terminal of a peptide, such as depicted in the general formula III or IV: wherein
  • RSi when linked to RSn-RSm or RSm when linked to RS r RSn together forms the peptide RSi-RSn-RSm , which is a substrate for a peptide cleaving enzyme; and RSi and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide may be N- or C-terminally modified; and
  • RSn is selected from the group consisting of a bond, amino acids and peptides; and R 2 , R 3 , R 4 , R 5 and R 6 are as defined in item 1 .
  • RSI-RSII-RSIII denotes a peptide sequence, which is a substrate for a peptide cleaving enzyme.
  • RSi-RSn-RSin may be a substrate for any peptide cleaving enzyme, for example it may be a substrate for any of the hydrolases containing a cysteine in its active site described herein below in the section "Polypeptides".
  • the compound of Formula I is a compound of any one of Formulas VI to VIII:
  • R aa is individually selected from the group consisting of amino acid side chains.
  • R aa is individually selected from the group consisting of the side chains of the 20 standard amino acid.
  • the waved lines indicate points of attachment.
  • the waved line may indicate a point of attachment to an amino acid or a peptide, wherein said amino acids of peptides optionally may be C- or N-terminally modified.
  • the present invention provides methods for reacting a compound of Formula I with a cysteine residue, thereby forming a covalent bond, wherein the cysteine is contained in the sequence of a polypeptide.
  • Said polypeptide may be any polypeptide comprising at least one cysteine in its sequence.
  • the polypeptide may also be referred to as a "polypeptide comprising the cysteine residue in its sequence".
  • the polypeptide comprising the cysteine residue in its sequence is an enzyme.
  • the polypeptide comprising the cysteine residue in its sequence may be an enzyme.
  • said cysteine residue is positioned within the active site of the enzyme, e.g. a cysteine protease.
  • the polypeptide comprising the cysteine residue in its sequence may be a hydrolase containing a cysteine it its active site.
  • the polypeptide comprising the cysteine residue in its sequence may be a cysteine protease.
  • the methods may preferably comprise reacting the cysteine residue positioned in the active site of said hydrolase (e.g. said protease) with a compound of Formula I.
  • the hydrolase containing a cysteine in its active site may be selected from the group consisting of cathepsin B (EC 3.4.22.1 ), papain (EC 3.4.22.2), ficain (EC 3.4.22.3), chymopapain (EC 3.4.22.6), asclepain (EC 3.4.22.7), clostripain (EC 3.4.22.8), cerevisin (EC 3.4.21.48), streptopain (EC 3.4.22.10), insulysin (EC 3.4.24.56), Y-glutamyl hydrolase (EC 3.4.19.9), actinidain (EC 3.4.22.14), cathepsin L (EC 3.4.22.15), cathepsin H (EC 3.4.22.16), prolyl oligopeptidase (EC 3.4.21.26), thimet oligopeptidase (EC 3.4.24.15), proteasome endopeptidase complex (EC 3.4.25.1 ), saccharolysin (EC 3.4.24.
  • cathepsin O EC 3.4.22.42
  • cathepsin V EC 3.4.22.43
  • TEV nuclear- inclusion-a endopeptidase EC 3.4.22.44
  • helper-component proteinase EC
  • the polypeptide comprising the cysteine residue in its sequence is a human protein.
  • the polypeptide comprising the cysteine residue in its sequence is an albumin, for example human serum albumin.
  • the polypeptide comprising the cysteine residue in its sequence is an antibody or an antigen-binding fragment, which are capable of binding to a specific antigen via an epitope on the antigen.
  • an antibody or an antigen-binding fragment according to the invention is a polypeptide or protein capable of recognising and binding an antigen, said polypeptide comprising at least one antigen binding site. Said antigen binding site preferably comprises at least one CDR.
  • the antibody may be a naturally occurring antibody, a fragment of a naturally occurring antibody or a synthetic antibody.
  • Naturally occurring antibody refers to heterotetrameric glycoproteins capable of recognising and binding an antigen and comprising two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region (abbreviated herein as C H ).
  • Each light chain comprises a light chain variable region (abbreviated herein as V L ) and a light chain constant region
  • C L complementarity determining regions
  • FRs framework regions
  • Antibodies may comprise several identical heterotetramers.
  • the antibody may in particular be a monoclonal antibody.
  • the antibody may also be a humanised antibody or a human antibody.
  • the antibody may be a monoclonal, humanised or human antibody.
  • Antibodies according to the invention may for example be monoclonal antibodies, chimeric antibodies, humanised antibodies, isolated human antibodies, single chain antibodies, bi-epitopic antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen-binding fragments and derivatives of the same.
  • Suitable antigenic) binding fragments and derivatives include, but are not necessarily limited to, Fv
  • fragments e.g. single chain Fv and disulphide-bonded Fv
  • Fab-like fragments e.g. Fab fragments, Fab' fragments and F(ab)2 fragments
  • single variable domains e.g. VH and VL domains
  • domain antibodies dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
  • the compound of Formula I may comprise at least part of the recognition sequence for said peptide cleaving enzyme.
  • the compound of Formula I may be a compound of any one of Formulas II, III or IV, wherein RS
  • the polypeptide is a peptide cleaving enzyme.
  • the polypeptide may be papain.
  • the compound of formula I may for example be compound 2g described in the Examples below.
  • the polypeptide may be TEV protease.
  • the compound of formula I may for example be selected from the group consisting of compounds 2g, 2h, 8a, 8b and 8c described in the Examples below.
  • the polypeptide may be caspase-1 .
  • the compound of formula I may for example be selected from the group consisting of compounds 22c, 22e, 22f, 23 and 24 described in the Examples below.
  • the compound of formula I may be selected from the group consisting of compounds 23 and 24 described in the Examples below.
  • the current invention concerns a method of modulating the activity of a protein.
  • the methods of modulating the activity of a protein in general involve reacting a compound of Formula I (e.g. any of the compounds described herein above in the sections "Compound of Formula I” and “Compounds of Formula II to X) with a cysteine residue contained in the sequence of a polypeptide constituting at least part of said protein.
  • said protein may comprise one or more polypeptides, and the methods may involve reacting at least one cysteine residue contained in at least one of said polypeptides, with a compound of Formula I.
  • the protein is an enzyme.
  • said enzyme is an enzyme with a cysteine residue in the active site.
  • the enzyme is a hydrolase comprising a cysteine in the active site, for example the enzyme may be a cysteine hydrolase.
  • the enzyme is a cysteine protease.
  • the methods may preferably comprise reacting the cysteine residue positioned in the active site of said hydrolase (e.g. said protease) with a compound of Formula I.
  • the compound of Formula I is capable of binding to the active site of said enzyme, for example said cysteine hydrolase, such as said cysteine protease.
  • said cysteine hydrolase such as said cysteine protease.
  • the compound of Formula I may comprise at least part of the recognition sequence for said cysteine protease.
  • the compound of Formula I may be a compound of any one of Formulas II, III or IV, wherein RSi-RSn-RSin together forms a recognition sequence for said peptide cleaving enzyme.
  • the method of modulating the activity of a protein may modulate the activity in any manner, however, in particular, the method may be a method of reducing or even inhibiting the activity of said protein, e.g. a method reducing or even inhibiting the activity of said enzyme, such as a method of reducing or even inhibiting the activity of said cysteine hydrolase.
  • the present invention concerns a method for treatment or prevention of a clinical condition associated with a polypeptide comprising a cysteine residue in an individual in need thereof.
  • Such methods in general involve reacting a compound of Formula I (e.g. any of the compounds described herein above in the sections
  • the method may comprise administering a compound of Formula I capable of reacting with said polypeptide to said individual in a therapeutically effective amount.
  • the clinical condition may be associated with a polypeptide in various manners.
  • the clinical condition may be associated with the presence of said polypeptide.
  • the clinical condition may also be associated with elevated levels of said polypeptide.
  • the clinical condition may also be associated with aberrant activity of said polypeptide.
  • the polypeptide may be a protein or may constitute part of a protein, in which case, the clinical condition may be associated with aberrant activity of said protein, e.g. of an enzyme.
  • the clinical condition may be associated with elevated activity of said protein, e.g. of an enzyme.
  • the methods of treatment or prevention of a clinical condition may comprise modulating the activity of said protein, for example by any of the methods of modulating activity of a protein described herein above in the section "Method of modulating the activity of a protein”.
  • the clinical condition may be any clinical condition associated with a polypeptide.
  • the polypeptide may for example be any of the polypeptides described herein above in the section "Polypeptides".
  • the clinical condition is selected from the group consisting of osteoporosis, cancer and arthritis.
  • the polypeptide may for example be a cysteine protease, such as cathepsin B, K or L or caspase 1 .
  • the method of treatment or prevention of a clinical condition involves transporting a prodrug to the site of the clinical condition.
  • the methods may involve use of a compound of Formula I, wherein Y is a prodrug.
  • Said prodrug preferably is a prodrug of a drug useful in the treatment of prevention of said clinical condition.
  • the present invention concerns a method for labelling a polypeptide with a label, e.g. with a labelling molecule.
  • the methods of labelling a polypeptide in general involve reacting a compound of Formula I with a cysteine residue contained in the sequence of said polypeptide, wherein the compound of Formula I comprises a labelling molecule.
  • the compound of Formula I may for example be any of the compounds of Formula I described herein above in the sections "Compound of Formula I” and “Compounds of Formula II to X", wherein R 8 is selected from the group consisting of -NH-linker-Y and - linker-Y, wherein Y is a labelling molecule.
  • the molecule Y may for example be selected from the group consisting of a fluorescent labelling molecule, a radioisotope labelling molecule, an affinity molecule, an azide, a terminal alkyne, a spin label, a prodrug, a mass tag, and a photoreactive group.
  • the labelling molecule may be a detectable label.
  • the methods may be used for labelling any polypeptide comprising a cysteine residue.
  • the methods may be used for labelling any of the polypeptides described herein above in the section "Polypeptides”.
  • the current invention concerns a method for detecting a polypeptide, said method comprises the steps of a. Reacting said polypeptide with a compound of Formula I, wherein R 8 is -linker-Y or -NH-linker Y; and
  • the labelling molecule may be used in a method of isolation of said polypeptide.
  • said polypeptide may for example be isolated using an isolation method based on affinity isolation using a moiety with affinity for said labelling molecule.
  • the labelling molecule is tag
  • the method of isolation may be based on use of antibodies specifically binding said tag.
  • the labelling molecule is biotin said method of isolation may be based on use of streptavidin and/or avidin.
  • Compounds of formula I useful for methods of labelling include compounds of Formula I, wherein R 8 is -linker-Y or -NH-linker Y.
  • R 8 is -linker-Y or -NH-linker Y.
  • Non-limiting examples of such compounds includes compounds 8a, 8b, 8c, 8d, 10, 11 , 17 and 18 described in the Examples below, and in particular compounds 8a, 8b, 8c, 8d, 10 or 11.
  • the present invention concerns a method for diagnosis of a clinical condition associated with a polypeptide in an individual at risk of acquiring said clinical condition.
  • said method comprises the steps of:
  • Step a. of said method may for example be performed under conditions allowing for reaction between the compound of Formula I and a cysteine residue in said
  • the clinical condition may be associated with a polypeptide in various manners.
  • the clinical condition may be associated with the presence of said polypeptide, in which case the method may determine the presence of said polypeptide in said sample.
  • the presence of said polypeptide in the sample may be indicative of said individual suffering from said clinical condition.
  • the clinical condition may also be associated with elevated levels of said polypeptide in which case the method may determine the level of said polypeptide in said sample.
  • An elevated level of said polypeptide in the sample may be indicative of said individual suffering from said clinical condition.
  • the clinical condition may also be associated with the absence of said polypeptide, in which case the method may determine the presence of said polypeptide in said sample.
  • the absence of said polypeptide in the sample may be indicative of said individual suffering from said clinical condition.
  • the clinical condition may also be associated with the reduced levels of said polypeptide, in which case the method may determine the levels of said polypeptide in said sample.
  • Reduced levels of said polypeptide in the sample may be indicative of said individual suffering from said clinical condition.
  • the methods for determining the presence, absence or level of said polypeptide may for example comprise performing on a sample from said individual any of the methods of labelling said polypeptide described herein above in the section "Method of labelling" followed by detecting the labelling molecule.
  • Method of labelling e.g., Method of labelling
  • the compounds of formula I may be prepared by any useful method available to the skilled person.
  • the methods may comprise preparing a benzene substituted with one or more electron withdrawing groups, e.g. a fluoro-benzene. This may also be referred to as the aryl part of the compound.
  • the aryl part of the compound i.e. the benzene part of a compound of Formula I including T, Z, X and/or non-peptide R 1 -R 6 , can be synthesized according to conventional methods.
  • the fluoro-benzene may be prepared by conventional methods or they may be purchased.
  • a fluorophenyl acid or a fluorophenyl acid chloride may be used as starting compound.
  • Such compounds are commercially available.
  • the starting compound may be reacted with additional substituents R 1 to R 6 according to methods known to the skilled person.
  • the compound of formula I comprises an amide bond
  • this may be prepared according to the general procedure (2) for amide bond formation described in Example 1 below.
  • the peptide part(s) of the compound and the aryl part of the compound can be synthesized separately, and then coupled together.
  • the peptide part of the compounds can be prepared by conventional solid-phase peptide synthesis. Such methods are well known to the skilled person and may for example be standard SPPS synthesis or it may be conducted essentially as described in Examples 2 or 8 herein below.
  • the aryl part may be prepared using substituted a phenyl acid or a phenyl acid chloride substituted with the appropriate electron withdrawing groups, e.g. fluorophenyl acid or fluorophenyl acid chloride as starting compound.
  • the starting compounds may be reacted with e.g. substituent T, wherein T may be protected.
  • T may be protected.
  • the starting compounds may be reacted with a protected amino-amino compound yielding an intermediate compound.
  • the protected compound may comprise one or more protecting groups, e.g. a benzyl and/or Boc This may be done as described in the General Procedure A described in Example 8.
  • the intermediate compound be deprotected by removal of one or more of the protecting groups, e.g. by removal of the benzyl. This may be done as described in general procedure B in Example 8.
  • the aryl part thus prepared may be useful as a compound of formula I or it may further be coupled to a peptide part.
  • the coupling between the peptide part and the aryl part may be done by esterification of the aryl part to a HMBA linker followed by deprotection, e.g. deprotection of the Boc group. Then reductive amination of the terminal amine with an boc protected amino acid derivative may be performed followed by standard SPPS protocol.
  • the individual amino acids of the peptide may be added one after the other to the aryl part by standard SPPS protocol.
  • the amino acid derivative may be an amine, e.g. an amino acid lacking the carboxyl group.
  • each of 26 selected fluorobenzene derivatives (compounds 2a-z, see Table 1 ) were tested for reaction with Boc-L-cysteine in DMF using diisopropylethyl amine (DIPEA) as base (method A). The reactions were followed by LC-MS. Only 13 compounds displayed reactivity (groups I and II of Table 1 ). The remaining derivatives did not react under the same conditions.
  • DIPEA diisopropylethyl amine
  • the 13 reactive fluorobenzenes (groups I and II of Table 1 ) were then tested for reactivity in an aqueous mixture of AcN/water (1 :1 ) at pH 8.5 (method B). Only five candidates (2a, 2d, 2g , 2j and 2w) showed partial reactivity that increased with increased pH (see Figure 2).
  • the reactivity of the pentafluorobenzene derivatives could be directly linked to the electron withdrawing capacity of the last substituent.
  • the nitro-, cyano-, sulfonamide, and trifluoromethyl-substituents all promoted reactivity both in water and DMF, and reactivity correlated to a Hammett o p -constant above 0.5.
  • the sulfonylamide- and trifluoromethyl- substituted trifluorobenzenes were not reactive with unactivated sulfhydryl groups in water opposite to their corresponding pentafluorobenzene derivatives (2h and 2k).
  • the amidoyl-substituted trifluorobenzene did not react with unactivated sulfhydryl groups under any of the tested conditions while the corresponding pentafluorobenzene reacted in DMF (2b and 2c).
  • the reactivity may be reduced even further by omitting additional fluoro-substituents.
  • Compound 3b was synthesized by general procedure (1) using Boc-L- cysteine (110.5 mg, 0.5 mmol) in DMF (5.0 mL), DIPEA (350 ⁇ _, 2.4
  • Boc-L-cysteine (1 10.5 mg, 0.5 mmol) in DMF (5.0 mL), DIPEA (350 ⁇ _,
  • the 2g was found most interesting as potential general compound for chemo-selective labeling of cysteines in proteins under aqueous conditions.
  • This fluorobenzene can be linked to other molecules through the sulfonamide bond and is hydrophilic compared to e.g. a trifluoromethyl substitution.
  • peptide 4 (scheme 2) containing all nucleophilic protein functionalities, was synthesized. The peptide was reacted with an excess (3 eq.) of 2g under aqueous condition (see Scheme 2). Only the monosubstituted adduct 5 could be observed and was identified by LC-MS. The structure of 5 was confirmed by MS/MS sequencing and only the target cysteine was modified by the compound (see Figure 3). Under the same conditions, compound 2b was found not to react (see Figure 4).
  • Fmoc deprotection was achieved by two treatments of 5 min with 20% piperidine in DMF (v/v), followed by washing of the resin with DMF (x10).
  • the free amine content was analyzed using the Kaiser test.
  • Pentafluorophenylsulfonylchloride, 7 was reacted with amino-functionalized PEG linkers containing a tag, such as an azide group for click chemistry (CuAAC or SPACC) (8a), the sulforhodamine B fluorophore (10) or biotin affinity probe (11 ) (see Figure 5).
  • the generated tag-containing compounds (8a, 10, or 11 ) were reacted with enhanced green fluorescent protein (eGFP) comprising two cysteines, not involved in disulfide bonds; and with bovine serum albumin, which has 17 conserved disulfide bonds and one free thiol (Cys 34). All three types of reporter-linked compounds were capable of labeling eGFP and albumin in a concentration-dependent manner (see Figure 5).
  • the reactive fluoroaryl compounds did not interfer with the functional click, fluorescence or affinity tags under the protein labeling conditions.
  • the reaction mixture (15 ⁇ _) was analyzed by SDS-PAGE electrophoresis followed by in-gel fluorescence scanning at Aex 650 nm and Aem 680 nm on a Typhoon FLA 7000 laser scanner, and by staining with Coomassie Brilliant Blue.
  • reaction mixture was analyzed by SDS-PAGE electrophoresis followed by in-gel fluorescence scanning at Aex 530 nm and Aem 580 nm on a Typhoon FLA 7000 laser scanner, and by staining with Coomassie Brilliant Blue.
  • Example 4 Selective cysteine protease inhibition
  • the compounds may also be applied for selective inhibition of cysteine proteases over other classes of proteases including the closely related serine proteases.
  • an on-bead inhibition screening assay towards the cysteine protease papain and the serine protease subtilisin was conducted using compounds 2a, 2b, 2g, 2h and 2f.
  • FRET substrates for the two proteases were synthesized attached to polymer beads of
  • TEV protease Three fluorobenzene derivatives with different reactivities (2g, 2h and 2c) were investigated for their ability to react selectively with one among several cysteine residues in the same protein, as well as their ability to modify cysteine proteases in an activity-based manner (see Figure 7A).
  • Tobacco Etch Virus (TEV) protease was chosen as a model protease as it contains unpaired surface exposed cysteines in addition to the cysteine (Cys170) in the catalytic site.
  • TEV protease has a sequence-specific activity and is an important tool for the removal of fusion tags or the cleavage of fusion proteins in vitro and in vivo.
  • the activity of the protease was confirmed using a FRET peptide with the sequence Y(3-N0 2 ) E N L Y F Q G K(Abz) G-OH (12), as substrate (see Figure 7B).
  • the enzyme was incubated with fluoroaryl compounds 2g, 2h and 2c at a final concentration of 100 ⁇ in the assay buffer. As negative control only assay buffer was added. After 2 h of incubation with the most reactive compound 2g, the mono- (13a) and the disubstituted (13b) adducts of TEV protease were identified; the latter (13b) being the major product (see Table 2).
  • substituted adduct mean TEV protease covalently liked to fluoroaryl.
  • TEV 13e After 10 h of incubation, the mono- (13a), the di- (13b), the tri- (13c), and the tetrasubstituted (13d) adducts were detected; at this time point the tetrasubstituted (13d) was the major product (see Table 3). Table 3. LCMS with ESI + -TOF analysis of reaction mixture (TEV + 2g). After incubation for 10 h at 37 °C: TEV peak at 28,843 completely disappeared and five major peaks that correspond to the di, tri and tetra- adduct of
  • the selective arylation of the cysteine in the catalytic site by compound 2h was confirmed by in-solution and in- gel tryptic digestion followed by high-resolution mass spectrometry.
  • the peptide fragment containing the unmodified cysteine in the active site (Cys170) was found at 1219.59 (red) in the native TEV protease (negative control). This signal did not appear after the treatment with both 2g (green) and 2h (blue), but new mass peaks appeared at 1514.62 and 1478.65, respectively, corresponding to modified peptides fragments (see Figure 7C).
  • the peptide fragment with the unmodified surface-exposed cysteine was found at 1267.68.
  • BL21 (DE3) competent cells 50 ⁇ _ were transformed with 1 ⁇ _ pET15 plasmid carrying the gene for TEV protease with a N-terminal His6-tag.
  • Cells were plated on LB-agar plates supplemented with ampicillin (100 ⁇ g mL).
  • LB medium supplemented with ampicillin (100 g/mL) was inoculated with a single colony and grown overnight at 37 °C.
  • a 200 mL expression culture was prepared in LB medium supplemented with ampicillin (100 g/mL) and the addition of overnight culture to an absorbance at 600 nm of 0.1 . When the culture reached an absorbance at 600 nm of 0.6, it was induced with IPTG to a final concentration of 1 mM and expression was allowed to proceed at 37 °C for 4 h. The culture was thereafter harvested and stored at 20 °C.
  • the cell pellet from a 200 mL cell culture was suspended and incubated on ice in 10 mL 50 mM NaH2P04, 300 mM NaCI, 1 mg/mL lysozyme, pH 7.5.
  • the suspension was then sonicated twice on ice with a sequence of 6 x 10 s at 50% amplitude with 20 s pauses.
  • the lysate was centrifuged for 10 min at 20,000 x g, 4 °C and the cleared lysate isolated. DTT and imidazole were added to a final concentration of 5 mM and 20 mM, respectively.
  • the solution was loaded onto a 1-mL HisTrap HP column (GE Healthcare), and after washing with wash buffer (50 mM NaH 2 P0 4 , 500 mM NaCI, 20 mM imidazole, pH 7.5) until the absorbance signal did not further decrease, the protein was eluted in 50 mM NaH 2 P0 4 , 500 mM NaCI, 250 mM imidazole, pH 7.5. Fractions were stored at 20 °C. Relevant HisTrap fractions were pooled and DTT added to a final concentration of 5 mM.
  • the solution was then centrifuged for 15 min at 11 ,000 x g, 4 °C and the resulting supernatant loaded onto a HiLoad 16/600 Superdex 75 pg (GE Healthcare) with 20 mM NaH 2 P0 4 , 150 mM NaCI, pH 7.5 as buffer.
  • Relevant fractions were concentrated using an Amicon ultracentrifugation unit with a molecular weight cutoff of 10 kDa (Millipore) and the concentrated samples were diluted to 0.3 mg/mL in storage buffer containing 20 mM NaH2P04, 300 mM NaCI, 5 mM DTT, 50% glycerol, pH 7.5.
  • Portions of 10 ⁇ purified TEV protease were mixed with 2 ⁇ 0.5 ⁇ g/ ⁇ L trypsin (sequencing-grade, Roche) in 40 ⁇ 0.1 M (NH4)2C03 and incubated overnight at 37 °C. To the solution was then added formic acid to 1 % final concentration. The tryptic peptides were desalted using C18 ZipTips according to the manufacturer's protocol (Millipore) and analyzed by MALDI-TOF using aCHCA as matrix.
  • tryptic peptides were desalted using C18 ZipTips according to the manufacturer's protocol (Millipore) and analyzed by MALDI-TOF using aCHCA as matrix.
  • MALDI MS were recorded on a Bruker Solaris XR ICR-instrument. TLC plates used were Fluka silica gel F254 on aluminum. Column chromatography was performed with Merck silica 60 (0.015 - 0.040 mm). Chromatographic purifications (flash) were performed with Silica Gel 60 from Fluka (0.015 - 0.040 mm). Preparative RP-HPLC was performed on a Gilson 215 semi-prep HPLC-system using an XTerra® Prep column (10 ⁇ 19 x 150 mm, flow rate 15 mL min "1 ).
  • Analytical HPLC was performed on an Agilent HP1 100 instrument using an XBridge® column (10 ⁇ 4.6 x 100 mm, flow rate 1 .0 mL min "1 ). Compounds were detected by UV absorption at 215 and 254 nm. Eluents for both the semi-prep and analytical RP-HPLC were: A: water with 0.05 % TFA, B:
  • Trifluoroacetic acid (1 .0 mL) was added to a solution
  • the fluoroaryl ligation concept also was investigated for activity-based protein profiling (ABPP).
  • a series of azide-functionalized derivatives (8a, 8b, 8c and 8d, Figure 8A) was tested.
  • Native or heat-denatured TEV protease was treated with compound 8a, 8b, 8c or 8d at 10 ⁇ and subsequently, after gel electrophoresis, exposed to a cyanine alkyne "click" dye for visualization.
  • compound 8b and 8c demonstrated ability of labeling TEV protease in an activity-dependent manner, as only the native TEV protease and not the preheated sample displayed fluorescence.
  • TEV protease The activity of TEV protease was measured by proteolytic cleavage of the Abz-E N L Y F Q G Y(N02) G-OH FRET substrate.
  • TEV protease (170 nM) was incubated with the substrate (160 ⁇ ) in aqueous buffer containing 50 mM Tris-HCI, 0.5 mM EDTA, 1 mM DTT at pH 8.3. Hydrolysis was monitored in triplo at 25 °C using a microtiter plate reader (Synergy H4 hybrid reader). A sample without enzyme and one with buffer only were included as negative controls. Labeling of TEV protease with fluoroaryl probes 2g and 2h followed by trypsin digestion and MALDI-TOF analysis
  • Active TEV protease (10 ⁇ _, ⁇ 4 ⁇ ) was incubated with iodoacetamide, 2g or 2h (10 ⁇ , 200 ⁇ ) in TEV assay buffer (50 mM Tris, 0.5 mM EDTA and 1 mM TCEP, pH 8.5) at 37 °C for 2-16 h. Excess DTT (5 ⁇ _, 100 mM) was added for 30 minutes followed by in-solution trypsin digestion. That is, 2 ⁇ _ 0.5 ⁇ g ⁇ L trypsin in 40 ⁇ _ 0.1 M (NH4)2C03 was added to the solution and incubated overnight at 37 °C.
  • TEV assay buffer 50 mM Tris, 0.5 mM EDTA and 1 mM TCEP, pH 8.5
  • the tryptic digested peptides were desalted using C18 ZipTips according to the manufacturer's protocol (Millipore). Desalted samples were analyzed by MALDI-TOF using aCHCA as matrix. Labeling of active and heat-inactivated TEV protease with azide probes 8a, 8b, 8c and 8d, followed by click chemistry-mediated fluorescent labeling (in-gel fluorescence scanning)
  • TEV protease (10 ⁇ _, ⁇ 4 ⁇ ) were incubated with fluoroaryl azide probes 8a, 8b, 8c and 8d (10 ⁇ _, 20 ⁇ ) in TEV assay buffer (50 mM Tris, 0.5 mM EDTA and 1 mM TCEP) at 37 °C for 16 h.
  • click mixture consisting of CuS04 (1 ⁇ _, 45 mM), THPTA (1 ⁇ _, 90 mM) and sodium ascorbate (1 ⁇ _, 60 mM) as well as alkyne cyanine dye 718 (Sigma Aldrich 30154) (3 ⁇ _, 1 mM) were added at 22 oC and left for 2 h.
  • the reaction mixture was analyzed by SDS-PAGE electrophoresis followed by in-gel fluorescence scanning at Aex 650 nm and Aem 680 nm on a Typhoon FLA 7000 laser scanner, and staining with Coomassie Brilliant Blue.
  • Preparation and labeling of a bacterial cell lysate spiked with TEV protease with probe 8b in-gel fluorescence scanning
  • lysis buffer 50 mM NaH2P04, 300 mM NaCI, pH 8.0
  • TEV protease 5 ⁇ _, ⁇ 4 ⁇
  • fluoroaryl azide probe 8b 10 ⁇ _, 20 ⁇
  • TEV buffer 50 mM Tris, 0.5 mM EDTA and 1 mM TCEP
  • click mixture consisting of CuS04 (1 ⁇ _, 45 mM), THPTA (1 ⁇ _, 90 mM) and sodium ascorbate (1 ⁇ _, 60 mM) as well as alkyne cyanine dye 718 (Sigma Aldrich 30154) (3 ⁇ _, 1 mM) were added followed by incubation at 22 oC for 2 h.
  • the reaction mixture was analyzed by SDS-PAGE electrophoresis followed by in-gel fluorescence scanning at Aex 650 nm and Aem 680 nm on a Typhoon FLA 7000 laser scanner, and staining with Coomassie Brilliant Blue.
  • the intense band observed just below the 25-kDa protein marker band was excised from the destained gel and cut into small pieces.
  • the pieces were further destained by soaking them in 0.1 M aq. sodium bicarbonate/AcN (1 :1 ). Subsequently, the pieces were incubated in 100 ⁇ trypsin digestion buffer (10 ng/ ⁇ trypsin in 0.1 M aq. sodium bicarbonate) at 37 °C overnight.
  • the tryptic digested peptides were desalted using C18 ZipTips according to the manufacturer's protocol (Millipore). Desalted samples were analyzed by MALDI-TOF using aCHCA as matrix.
  • An in-gel digestion after reduction and treatment with iodoacetamide was performed and analyzed by MALDI-TOF MS. The mass list was generated from the MALDI-TOF data.
  • OA Osteoarthritis
  • RA Rheumatoid arthritis
  • the peptide synthesis is performed according to standard SPPS synthesis.
  • PEGA 8 oo was coupled with 4-hydroxymethyl benzoic acid (HMBA) using the TBTU activation.
  • HMBA 4-hydroxymethyl benzoic acid
  • L-Boc- amino acids were coupled using the general procedure for peptide couplings described below and finally the free amine was acetylated using acetic acid.
  • the peptide was cleaved from the resin using 5% TEA/water for 2 hours.
  • TBTU couplings were performed by dissolving the amino acid (3 equiv.) in DMF with 4-ethylmorpholine (NEM) (3 equiv.), followed by addition of 1 H-benzotriazoyl tetramethyluronium tetrafluoroborate (TBTU) (3 equiv.). The resulting solution was left to pre-activate for 3 min before being added to the resin. Coupling reactions were allowed a reaction time of 2-3 h). Peptide couplings were generally performed in an amount of solvent just enough to cover the resin and washings were conducted slowly and without back mixing. After reaction, the resin was washed with DMF (x6) and finally checked using the Kaiser test.
  • NEM 4-ethylmorpholine
  • TBTU 1 H-benzotriazoyl tetramethyluronium tetrafluoroborate
  • the mixture was shaken at 31 °C for 4 days.
  • the mixture was transferred to an Amicon R Ultra-0.5 filter device and centrifuged at 14000 G for 12 minutes at 4 ° C.
  • the invention may also be defined by the following items.
  • R 3 is a leaving group
  • R 1 is R 7 ;
  • R 7 is selected from the group consisting of -X-Z-R 8 , -Z-T-R 8 , and -Z-T-(R 8 ) 2 ;
  • X is selected from the group consisting of a bond, -CH 2 -N(R 9 )-, and -CH 2 -0-;
  • Z is selected from the group consisting of a bond, .
  • T is selected from the group consisting of a bond, R 9 , R 9 R 9 ,
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y, -linker-Y and -linker-R 14 , wherein said peptide optionally may be N- and/or C-terminally modified;
  • Y is a labelling molecule, a drug molecule or a prodrug
  • R 9 is individually selected from the group consisting of -H, alkyl-COOH and an amino acid side chain;
  • R 10 is selected from the group consisting of-OH and R 8 ;
  • R 14 is a reactive group
  • R 2 , R 4 , R 5 , R 6 are individually selected from the group consisting of -H, R 7 , an amino acid side chain and an electron withdrawing group;
  • R 2 , R 4 , R 5 , and R 6 is an electron withdrawing group.
  • R 1 is para to R 3 .
  • R 1 is ortho to R 3 .
  • R is a peptide, wherein said peptide optionally may be N- and/or C-terminally modified.
  • and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C- terminally modified;
  • RSn is selected from the group consisting of a bond, amino acids and peptides; and R 3 , R 4 , R 5 and R 6 are as defined in item 1.
  • R 2 is RSm;
  • RSi and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C- terminally modified;
  • RSi when linked to RSm via RS M together forms the peptide RSi-RSn-RSm, which is a substrate for a peptide cleaving enzyme
  • RSn is selected from the group consisting of a bond, amino acids and peptides.
  • when linked to RSn-RSm or RSm when linked to RS r RSn together forms the peptide RSi-RSn-RSm , which is a substrate for a peptide cleaving enzyme, ; and RS
  • RSii is selected from the group consisting of a bond, amino acids and peptides; and R 2 , R 3 , R 4 , R 5 and R 6 are as defined in item 1.
  • and RSm individually are selected from the group consisting of amino acids and peptides, wherein said amino acid or peptide optionally may be N- or C- terminally modified;
  • RSi when linked to RSm via RS M together forms the peptide RSi-RSn-RSm, which is a substrate for a peptide cleaving enzyme
  • RSII is selected from the group consisting of a bond, amino acids and peptides.
  • the substrate for a peptide cleaving enzyme is the recognition sequence for a cysteine protease selected from the group consisting of cathepsin B (EC 3.4.22.1 ), papain (EC 3.4.22.2), ficain (EC 3.4.22.3), chymopapain (EC 3.4.22.6), asclepain (EC 3.4.22.7), clostripain (EC 3.4.22.8), cerevisin (EC 3.4.21 .48), streptopain (EC 3.4.22.10), insulysin (EC 3.4.24.56), ⁇ -glutamyl hydrolase (EC 3.4.19.9), actinidain (EC 3.4.22.14), cathepsin L (EC 3.4.22.15), cathepsin H (EC
  • prolyl oligopeptidase EC 3.4.21 .26
  • thimet oligopeptidase EC 3.4.24.15
  • proteasome endopeptidase complex EC 3.4.25.1
  • saccharolysin EC 3.4.24.37
  • kexin EC 3.4.21 .61
  • Cathepsin T EC 3.4.22.24
  • Glycyl endopeptidase EC 3.4.22.25)
  • Cancer procoagulant EC 3.4.22.26)
  • cathepsin S EC 3.4.22.27)
  • picornain 3C EC 3.4.22.28)
  • picornain 2A EC 3.4.22.29
  • Caricain EC 3.4.22.30
  • Ananain EC 3.4.22.31
  • Stem bromelain EC
  • R aa is individually selected from the group consisting of amino acid side chains.
  • peptides individually selected from the group consisting of amino acids, peptides and alkylamines, wherein said peptide optionally may be N- and/or C-terminally modified.
  • peptide wherein said peptide optionally is N- and/or C-terminally modified.
  • R 8 is an amino acid, wherein said amino acid optionally is N-terminally modified.
  • peptide consisting of in the range of 2 to 5 amino acids, such as of in the range 2 to 3 amino acids, wherein said peptide optionally is N- and/or C-terminally modified.
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, - NH-linker-Y and -linker-Y, wherein said peptide optionally may be N- and/or C- terminally modified.
  • R is a protecting group and the protecting group is selected from the group consisting of Boc, Cbz and Fmoc.
  • R aa is individually selected from the group consisting of amino acid side chains.
  • R 3 is selected from the group consisting of -F, -CI, -Br, -I, -N0 2 and S0 2 -alkyl.
  • R 3 is selected from the group consisting of -F and -CI.
  • R 3 is -F. 28.
  • the electron withdrawing group individually are selected from the group consisting of -F, -CI, -CF 3 , -CCI 3 , S0 2 -alkyl and -C ⁇ N.
  • linker is selected from the group consisting of peptides, oligosaccharides and steroids. 39. The method according to any one of items 1 to 37, wherein linker is -N(H)- (CH2-CH 2 -0-)n, wherein n is an integer from 0 to 10.
  • linker is -N(H)- (CH2-CH2-0-)n-(CH2)m-, wherein n is an integer from 0 to 10 and m is an integer from 0 to 5.
  • linker is -N(H)- (CH2-CH2-0-)n-(CH2)m-NH-, wherein n is an integer from 0 to 10 and m is 2.
  • polypeptide comprising the cysteine residue in its sequence is an enzyme, e.g. a cysteine protease. 47. The method according to any one of the preceding items, wherein the
  • polypeptide comprising the cysteine residue in its sequence is an enzyme and said cysteine is positioned within the active site of the enzyme, e.g. a cysteine protease.
  • a cysteine protease The method according to any one of items 46 to 47, wherein the enzyme is a cysteine protease.
  • the method according to any one of items 1 to 48, wherein the polypeptide comprising the cysteine residue in its sequence is a human protein.
  • the method according to any one of items 1 to 45, wherein the polypeptide comprising the cysteine residue in its sequence is an albumin.
  • the method according to item 50, wherein the albumin is human serum albumin.
  • the method according to any one of items 1 to 45, wherein the polypeptide comprising the cysteine residue in its sequence is an antibody or an antigen- binding fragment thereof.
  • the method according to item 52, wherein the antibody is a monoclonal antibody.
  • the method according to item 52, wherein the antibody is a humanised antibody or a human antibody.
  • a method of modulating the activity of a protein comprising
  • a method of modulating the activity of a protein comprising
  • polypeptide is an enzyme with a cysteine residue in the active site.
  • enzyme is a cysteine hydrolase, for example a cysteine protease.
  • RSi-RSn-RSin is a substrate for said cysteine protease.
  • a method for labelling a polypeptide with a labelling molecule comprising performing the method according to any one of items 1 to 65.
  • Y is selected from the group consisting of a fluorescent labelling molecule, a radioisotope labelling molecule, an affinity molecule and a photoreactive group.
  • the polypeptide is a protein.
  • polypeptide is an antibody or an antigen-binding fragment thereof.
  • a method of conjugating a prodrug or a drug molecule to a polypeptide comprising reacting a compound of Formula I wherein Y is a prodrug or a drug molecule with a cysteine residue of said polypeptide using the method according to any one of the preceding items.
  • Y is a prodrug or a drug molecule.
  • a method for detecting a polypeptide comprising the steps of a. performing the method according to any one of the preceding items, wherein R 8 is -linker-Y or -NH-linker-Y; and
  • a method for detecting a polypeptide comprising the steps of a. performing the method according to any one of the preceding items, wherein R 8 is -linker-R 14 ;
  • a method for diagnosis of a clinical condition associated with a polypeptide in an individual at risk of acquiring said clinical condition comprising the steps of:
  • a method for diagnosis of a clinical condition associated with a polypeptide in an individual at risk of acquiring said clinical condition comprising the steps of:
  • a method for treatment or prevention of a clinical condition associated with a polypeptide in an individual in need thereof comprising performing the method according to any one of the preceding items.
  • R 3 is a leaving group
  • R 1 is R 7 : R 7 is selected from the group consisting of -X-Z-R 8 , -Z-T-R 8 , and -Z-T-(R 8 ) 2 ; wherein
  • X is selected from the group consisting of a bond, -CH 2 -N(R 9 )-, and -CH 2 -0-;
  • Z is selected from the group consisting of a bond, .
  • T is selected from the group consisting of a bond, R 9 , R 9 R 9 ,
  • R 8 is individually selected from the group consisting of amino acids, amino acid derivatives, peptides, peptide derivatives, peptidomimetic, alkylamines, protecting groups, -NH-linker-Y and -linker-Y, wherein said peptide optionally may be N- and/or C-terminally modified;
  • Y is a labelling molecule, a drug molecule or a prodrug
  • R 9 is individually selected from the group consisting of -H, alkyl-COOH and an amino acid side chain;
  • R 10 is selected from the group consisting of -H, -OH and R 8 ;
  • R 2 , R 4 , R 5 , R 6 are individually selected from the group consisting of -H, R 7 , an amino acid side chain and an electron withdrawing group;
  • the compound according to item 93, wherein the compound of Formula I is as defined in any one of items 1 to 45.

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Abstract

La présente invention concerne des méthodes de réaction sélective d'un composé de Formule (I) avec un résidu de cystéine, qui est contenu dans la séquence d'un polypeptide. Les méthodes peuvent être utilisées dans les domaines du marquage de polypeptides, de la détection de polypeptides, de la modulation de l'activité enzymatique, de l'isolement de polypeptides, des méthodes de diagnostic, des méthodes de prévention ou de traitement d'états cliniques ou des méthodes de transport de composés, par exemple des promédicaments.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109535090A (zh) * 2019-01-29 2019-03-29 吉林大学 1,2,3-三氮唑醇类衍生化布洛芬及其制备方法
WO2021009568A1 (fr) * 2019-07-17 2021-01-21 2692372 Ontario, Inc. Dérivés de benzènesulfonamide et leurs utilisations
WO2022090724A1 (fr) * 2020-10-29 2022-05-05 Sitryx Therapeutics Limited Nouveaux composés
WO2022106897A3 (fr) * 2020-11-20 2022-06-30 2692372 Ontario, Inc. Procédés et composition pour effectuer des modifications de kras

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006114190A1 (fr) * 2005-04-25 2006-11-02 Merck Patent Gmbh Nouveaux inhibiteurs specifiques de la caspase-10

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006114190A1 (fr) * 2005-04-25 2006-11-02 Merck Patent Gmbh Nouveaux inhibiteurs specifiques de la caspase-10

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"The Antibodies", 1995, HARWOOD ACADEMIC PUBLISHERS
ALEXANDER M. SPOKOYNY ET AL: "A Perfluoroaryl-Cysteine S N Ar Chemistry Approach to Unprotected Peptide Stapling", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 135, no. 16, 24 April 2013 (2013-04-24), US, pages 5946 - 5949, XP055237282, ISSN: 0002-7863, DOI: 10.1021/ja400119t *
CHEMICAL REVIEWS, vol. 91, 1991, pages 165 - 195
D. ALEXANDER SHANNON ET AL: "Investigating the Proteome Reactivity and Selectivity of Aryl Halides", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 136, no. 9, 24 February 2014 (2014-02-24), US, pages 3330 - 3333, XP055494151, ISSN: 0002-7863, DOI: 10.1021/ja4116204 *
KAISER ET AL., ANALYTICAL BIOCHEMISTRY, vol. 34, 1970, pages 595 - 598
SONG J ET AL.: "PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites", PLOS ONE, vol. 7, no. 11, 2012, pages e50300, XP055257940, DOI: doi:10.1371/journal.pone.0050300
STEFAN G. KATHMAN ET AL: "A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases", JOURNAL OF MEDICINAL CHEMISTRY, vol. 57, no. 11, 28 May 2014 (2014-05-28), pages 4969 - 4974, XP055494263, ISSN: 0022-2623, DOI: 10.1021/jm500345q *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109535090A (zh) * 2019-01-29 2019-03-29 吉林大学 1,2,3-三氮唑醇类衍生化布洛芬及其制备方法
WO2021009568A1 (fr) * 2019-07-17 2021-01-21 2692372 Ontario, Inc. Dérivés de benzènesulfonamide et leurs utilisations
CN114502533A (zh) * 2019-07-17 2022-05-13 2692372安大略公司 苯磺酰胺衍生物及其用途
GB2606804A (en) * 2019-07-17 2022-11-23 2692372 Ontario Inc Benzenesulfonamide derivatives and uses thereof
GB2606804B (en) * 2019-07-17 2023-12-27 2692372 Ontario Inc Benzenesulfonamide derivatives and uses thereof
WO2022090724A1 (fr) * 2020-10-29 2022-05-05 Sitryx Therapeutics Limited Nouveaux composés
WO2022106897A3 (fr) * 2020-11-20 2022-06-30 2692372 Ontario, Inc. Procédés et composition pour effectuer des modifications de kras
GB2618680A (en) * 2020-11-20 2023-11-15 2692372 Ontario Inc Methods and composition for KRAS modifications

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