WO2023247966A1 - Réactions de cycloaddition - Google Patents

Réactions de cycloaddition Download PDF

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Publication number
WO2023247966A1
WO2023247966A1 PCT/GB2023/051643 GB2023051643W WO2023247966A1 WO 2023247966 A1 WO2023247966 A1 WO 2023247966A1 GB 2023051643 W GB2023051643 W GB 2023051643W WO 2023247966 A1 WO2023247966 A1 WO 2023247966A1
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reactant
moiety
copper
reaction
biomolecule
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PCT/GB2023/051643
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English (en)
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Glenn Burley
Frederik PESCHKE
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University Of Strathclyde
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

  • the present disclosure relates to copper-catalysed cycloaddition reactions.
  • the use of copper-catalysed cycloaddition reactions in bioconjugation reactions e.g. bioconjugation reactions involving the use of an ynamine as a substrate.
  • Orthogonal control of chemical reactivity is an essential requirement for the selective modification of biomolecules.
  • the ‘bio-orthogonality’ of these reactions - either produced in nature or synthetically - is contingent on reagent pairs reacting with each other whilst minimizing or avoiding any cross-reactivity with other natural and non-natural of functional groups.
  • One example class of bio-orthogonal reaction available for chemoselective modification of biomolecules is the copper-catalysed azide-alkyne [3+2] cycloaddition (which is often abbreviated to CuAAC) or “click” reaction. This involves the use of a Cu (I) catalyst to promote a [3+2]cycloaddition with an azide to exclusively form a 1 ,4-disubstituted triazole.
  • CuAAC Copper-based addition
  • SPAAC strain- promoted azide-alkyne cycloaddition
  • iEDDA inverse electron demand Diels-Alder
  • Efforts to mitigate oxidative damage and improve CuAAC reaction kinetics have primarily focused on developing water-soluble Cu(l)-stabilizing ligands, Cu nanoparticles, and Cu-chelating azide groups.
  • Aromatic ynamines have recently been identified as alkyne surrogates for CuAAC reactions, which undergo chemoselective formation of 1 ,4-triazoles in the presence of other terminal alkynes. 131 In such reactions, ynamine chemoselectivity provides a shift in the rate determining step (RDS) away from acetylide formation towards the azide ligation step using a Cu(ll) pre-catalyst. [4] This results in reducing the Cu dependency of the ynamine activation step, and thereby affording a kinetic advantage relative to regular terminal alkynes in low [Cu] conditions ( Figure 1 b).
  • RDS rate determining step
  • the present disclosure is based on identification of a bioconjugation reaction between a reactant comprising an ynamine moiety and a reactant comprising an azide moiety, wherein at least one of the reactants is a biomolecule.
  • a method of forming a triazole moiety in a bioconjugation reaction to link a first and a second reactant comprises: contacting a first reactant comprising an ynamine moiety with a second reactant comprising an azide moiety in the presence of a copper catalyst and a reducing agent, wherein at least one of the first and second reactants is a biomolecule.
  • the first and second reactants may be contacted together under suitable conditions which allow them to react to form the triazole moiety.
  • the reaction may be modulated, controlled, promoted and/or catalysed by a copper catalyst in the presence of reducing agent.
  • the reaction may be modulated, controlled, promoted and/or catalysed by adding the copper catalyst and reducing agent to the first and second reactants.
  • the reaction may take place in any suitable solvent.
  • the reducing agent may be selected from glutathione and sodium ascorbate.
  • the biomolecule may be selected from proteins, carbohydrates, lipids, nucleic acids, polypeptides, peptides, amino acids, polysaccharides, oligosaccharides, monosaccharides, polynucleotides, oligonucleotides, nucleotides, and antibodies. In some examples, the biomolecule may be selected from a peptide and an oligonucleotide.
  • the present disclosure relates to the identification that a reaction between a reactant comprising an ynamine moiety and a reactant comprising an azide moiety to provide a triazole-containing product can be modulated, controlled and/or promoted by the use of glutathione (GSH).
  • GSH glutathione
  • the conditions described herein have been shown to be highly chemoselective and to proceed in good yields even at low concentrations of copper catalyst.
  • the reactions and conditions described herein may be compatible with more complex systems, tolerating the presence of many different functional groups. Consequently, this class of reaction may find particular utility in orthogonal reaction systems (e.g. as a bio-orthogonal tool in the sequential ligation of biomolecules such as peptides and oligonucleotides).
  • Glutathione is the principle redox mediator in live cells, minimizing the production of deleterious reactive oxygen species (ROS) within a cellular environment.
  • ROS reactive oxygen species
  • GSH can modulate the oxidation state of Cu ions by chelating Cu(ll) as well as acting as a reducing agent to form GSH-Cu(l) complexes.
  • Complexes can also be formed between Cu ions and glutathione disulfide (GSSG), which is influenced by the ratio of GSH/GSSG present.
  • GSSG glutathione disulfide
  • One of the primary functions of GSH within a cell is to mediate the cell’s oxidation state and to deactivate electrophilic agents.
  • a method of promoting and/or catalysing a reaction between a first reactant comprising an ynamine moiety and a second reactant comprising an azide moiety to provide a product comprising a triazole moiety may be modulated, controlled, promoted and/or catalysed by a copper catalyst in the presence of glutathione.
  • the reaction may be modulated, controlled, promoted and/or catalysed by adding the copper catalyst and glutathione to the first and second reactants.
  • the reaction may take place in any suitable solvent.
  • a method of forming a triazole-containing product comprising reacting a first reactant comprising an ynamine moiety together with a second reactant comprising an azide moiety in the presence of a copper catalyst and glutathione.
  • the step of reacting together the first reactant and second reactant may comprise contacting the first and second reactants in the presence of a copper catalyst and reducing agent (e.g. glutathione).
  • a copper catalyst and reducing agent e.g. glutathione
  • the ynamine and azide moieties may react together in the presence of the copper catalyst and the reducing agent (e.g. glutathione) to produce the triazole- containing product.
  • the method may be a click reaction.
  • the reaction may be a click ligation reaction.
  • the method may comprise a cycloaddition reaction.
  • a cycloaddition reaction may refer to a reaction in which two or more unsaturated moieties react together to form a cyclic product.
  • the two or more unsaturated moieties may generally belong to different reactants but can also be present on the same reactant. There may be a net reduction in bond multiplicity following the cycloaddition reaction.
  • the cyclic product of the cycloaddition reaction may comprise an increased number of sigma (o) bonds and a decreased number of pi (TT) bonds relative to the number of these bonds in the reactants.
  • a cycloaddition reaction may be pre-fixed with a notation to denote the number of atoms involved in the reaction.
  • a cycloaddition reaction may be denoted as an [x+y] cycloaddition, wherein x is the number of atoms comprised on the first unsaturated moiety and y is the number of atoms comprises on the second unsaturated moiety.
  • the reaction may be a [3+2] cycloaddition reaction. That is to say, three atoms from a first reactant (e.g. the azide moiety) and two atoms from a second reactant (e.g. the ynamine moiety) may be involved in the cycloaddition reaction.
  • a first reactant e.g. the azide moiety
  • a second reactant e.g. the ynamine moiety
  • the [3+2] cycloaddition may provide a five-membered ring product, such as a triazole.
  • the reaction may provide a product comprising a triazole moiety.
  • the triazole may sometimes be referred to as a 1 ,2,3-triazole, wherein the three nitrogen atoms are at adjacent positions within the five-membered ring.
  • the cycloaddition reaction may produce one or more regioisomers.
  • the cycloaddition reaction may produce a product comprising a 1 ,4-substituted triazole or a 1 ,5-substituted triazole moiety (illustrated in Figure 1 ).
  • the reaction may be regioselective or regiospecific and/or may proceed regioselectively or reg iospecif ical ly .
  • the reaction may favour the formation of one or more particular regioisomers over one or more other regioisomers.
  • the reaction may favour the formation of a 1 ,4-substituted triazole.
  • the reaction may favour the formation of the 1 ,4-substituted triazole over the 1 ,5-substituted triazole.
  • the molar amount of the 1 ,4-substituted triazole product obtained following the reaction may be greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, greater than about 99% or greater than about 99.9% of the total molar amount of triazole product obtained.
  • a method of forming a 1 ,4- substituted triazole-containing product comprising the step of: contacting or reacting together a first reactant comprising an ynamine moiety and a second reactant comprising an azide moiety in the presence of a copper catalyst and glutathione.
  • the reaction may proceed chemoselectively.
  • the present inventors have identified a set of conditions under which a cycloaddition reaction between an ynamine and an azide will proceed, but under which alkyne reagents have shown no, or substantially no, reaction.
  • the first reactant comprising an ynamine moiety may preferentially react with the second reactant comprising the azide moiety even when the reaction is conducted in the presence of an alkyne moiety.
  • the azide moiety reacts preferentially with the ynamine moiety over an alkyne moiety. Consequently, the chemoselectivity of this method may be of particular use in sequential bioconjugation reactions.
  • the various reactions disclosed herein may be catalysed and/or promoted by a copper catalyst.
  • the catalytically active species in the cycloaddition reactions described herein is typically Cu(l). Therefore, as used herein “copper catalyst” may embrace any copper species that is comprises, consists essentially of, or consists of a Cu(l) salt (such as a copper halide (e.g. copper iodide or copper bromide)). Additionally or alternatively, as used herein, “copper catalyst” may be generated in situ, e.g. by reduction of a Cu(ll) species or by oxidation of Cu(0) to produce the catalytically active species.
  • the copper catalyst is a copper (II) salt.
  • Representative examples include, but are not limited to, copper (II) acetate (Cu(OAc) 2 ), copper (II) halides, copper (II) oxide, copper (II) carbonate, copper (II) acetylacetonate, copper (II) sulfate, copper (II) carboxylate (e.g. copper benzoate, copper malonate, copper (II) pyrrole carboxylates), copper (II) proline, copper (II) triflate and the like.
  • the copper catalyst may be copper (II) acetate.
  • the copper catalyst may comprise metallic copper.
  • Representative examples include, but are not limited to, copper nanoparticles and copper tubing (e.g. copper derived from copper tubing used in the process and/or copper tubing used to house the reaction).
  • the copper catalyst may not be metallic copper.
  • copper ligands may be added, e.g. ligands which may coordinate to Cu ( I) and/or stabilise Cu ( I) .
  • Suitable ligands will be known to those skilled in the art. Representative examples include, but are not limited to, THPTA (tris-hydroxypropyltriazolylmethylamine) and tris(benzyltriazolylmethyl)amine (TBTA).
  • the reaction may take place under batch conditions. In some examples, the reaction may not take place under flow conditions.
  • the reactions as disclosed herein may be modulated, controlled, promoted and/or catalysed by a relatively low concentration of copper catalyst (e.g. a relatively low concentration of copper catalyst in the reaction mixture).
  • a relatively low concentration of copper catalyst e.g. a relatively low concentration of copper catalyst in the reaction mixture.
  • a reducing agent e.g. glutathione
  • the reaction between the ynamine moiety and the azide moiety may proceed relatively quickly and/or in good yield at relatively low concentrations of copper catalyst. This may be beneficial in certain bioconjugation applications as high levels of copper can lead to oxidative damage of cells.
  • the present inventors have observed that under the equivalent low concentrations of copper catalyst, no cycloaddition reaction was observed if the ynamine substrate were replaced with a conventional alkyne substrate.
  • the present inventors hypothesise that the relatively higher reactivity of an ynamine moiety under the copper catalysed conditions can facilitate the use of the disclosed methods in bioconjugation reactions, as the lower levels of copper lead to less degradation of the highly functionalized and/or relatively sensitive biomolecules (e.g. oligonucleotides and peptides).
  • the concentration of the copper catalyst may be less than or equal to about 1 mM, less than or equal to about 750 pM, less than or equal to about 500 pM, less than or equal to about 400 pM, less than or equal to about 350 pM, less than or equal to about 250 pM, less than or equal to about 100 pM, or less than or equal to about 50 pM.
  • the concentration of the copper catalyst may be greater than or equal to about 0.1 pM, greater than or equal to about 1 pM, or greater than or equal to about 10 pM.
  • the concentration of the copper catalyst may be between about 0.1 pM and about 1 mM, between about 1 pM and about 750 pM, or between about 10 pM and about 500 pM. In some examples, the concentration of the copper catalyst may be between about 50 pM and about 250 pM, e.g. about 100 pM.
  • the concentration of the reducing agent may be less than or equal to about 10 mM, less than or equal to about 5 mM, less than or equal to about 1 mM, less than or equal to about 750 pM, less than or equal to about 500 pM, less than or equal to about 400 pM, less than or equal to about 350 pM, less than or equal to about 250 pM, less than or equal to about 100 pM, or less than or equal to about 50 pM.
  • the concentration of the reducing agent e.g. glutathione or sodium ascorbate
  • the concentration of the reducing agent may be between about 0.1 pM and about 1 mM, between about 1 pM and about 750 pM, or between about 10 pM and about 500 pM. In some examples, the concentration of the reducing agent (e.g. glutathione or sodium ascorbate) may be between about 50 pM and about 250 pM, e.g. about 100 pM.
  • the molar ratio of the copper catalyst and the reducing agent may modulate and/or control the cycloaddition reactions described herein.
  • the molar ratio of the copper catalyst and the reducing agent e.g. glutathione or sodium ascorbate
  • the molar ratio of the copper catalyst and the reducing agent e.g. glutathione or sodium ascorbate
  • the present inventors have also identified that the reaction between the ynamine moiety and the azide moiety can proceed at relatively low concentrations of copper catalyst even in the absence of glutathione or other reductant (e.g. sodium ascorbate).
  • glutathione or other reductant e.g. sodium ascorbate
  • a method of promoting and/or catalysing a reaction between a first reactant comprising an ynamine moiety and a second reactant comprising an azide moiety to provide a product comprising a triazole moiety wherein the copper catalyst is present at a concentration of less than or equal to about less than or equal to about 500 pM, less than or equal to about 400 pM, less than or equal to about 350 pM, less than or equal to about 250 pM, less than or equal to about 100 pM, or less than or equal to about 50 pM.
  • the copper catalyst may be present at a concentration between about 10 pM and about 500 pM.
  • the various reactions according to the present disclosure may be carried out or conducted in a solvent.
  • the solvent may be any solvent that is compatible with the reactants and products and/or any solvent that allows, facilitates or enables the formation of the triazole-containing product.
  • the solvent may be any solvent that promotes the formation of the triazole-containing product.
  • the reaction may be carried out in any solvent that is capable of promoting the cycloaddition reaction.
  • the solvent may be or comprise a polar solvent, a non-polar solvent and/or an aqueous solvent.
  • the solvent may be or comprise a water miscible solvent. Thus, the reaction may proceed in an aqueous solution.
  • the solvent may comprise a single solvent or mixture of different solvents.
  • Suitable solvents may include, but are not limited to, an alcohol, water, a fluorinated solvent, acetonitrile (MeCN), dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), or any combination thereof.
  • Representative examples of an alcohol solvent used in the reaction may include, but are not limited to, methanol (MeOH), ethanol (EtOH), propanol (such as iso-propanol (iPrOH)), butanol (such as iso-butanol or tert-butanol), or ethylene glycol.
  • a fluorinated solvent used in the reaction may include a Ci-C 6 alcohol in which one or more hydrogen atoms have been replaced with a fluorine atom, such as 2,2,2-Trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), hexafluoro-2-methyl-2-propanol (HFMP), 2- (nonafluorobut-1 -yl)ethan-1 -ol, 2-(perfluorobut-1 -yl)ethan-1 -ol (PFH), 1 ,1 ,1 - trifluoropropan-2-ol (TFP), perfluoro-tert-butanol (PTB), 1 ,6-dihydroxy-2,2,3,3,4,4,5,5- octafluorohexane (DHP), 2,2,3,3,3-pentafluoro-1 -propanol (PFP) and 2,4,6- trifluorophenol.
  • TFE 2,2,2-Tri
  • the solvent may be an aqueous buffered solution such as a phosphate buffered saline solution (e.g. Dulbecco’s phosphate -buffered saline (DPBS)).
  • a phosphate buffered saline solution e.g. Dulbecco’s phosphate -buffered saline (DPBS)
  • certain co-solvents have been observed to promote and/or facilitate the formation of the triazole-containing product (e.g. when added to water or an aqueous buffered solution).
  • certain co-solvents may increase the rate of reaction and/or conversion to the triazole-containing product.
  • protic fluorinated solvents such as TFE or HFIP
  • enhanced polarity and hydrogen-bonding character of these solvents may act to facilitate and/or promote the cycloaddition reaction between the ynamine and azide moieties.
  • a Ci-C 6 alcohol such as methanol
  • the solvent may comprise an aqueous solution (e.g. an aqueous buffered solution) and a co-solvent which is a protic fluorinated solvent (such as trifluoroethanol or hexafluoropropanol).
  • the aqueous solution may comprise the co-solvent in an amount of between about 1 % and about 50% by volume, between about 2% and about 30% by volume, or between about 5% and about 20% by volume. In some examples, the aqueous solution may comprise about 10% by volume of the co-solvent. By way of further example, the aqueous buffered solution may comprise approximately 10% by volume of a protic fluorinated solvent (such as trifluoroethanol or hexafluoropropanol).
  • a protic fluorinated solvent such as trifluoroethanol or hexafluoropropanol
  • the solvent may be water and the co-solvent may be a Ci-C 6 alcohol (such as methanol).
  • the co-solvent may be present in an amount between about 1 % and 20% by volume.
  • the use of an alcohol as a co-solvent may assist in limiting degradation of the oligonucleotide.
  • the reactions of the present disclosure may be carried out at temperatures above 0 °C, above 5 °C, above 10 °C, above 15 °C or above 30°C. In some cases, the reaction may be carried out at a temperature between about 0 °C and about 100 °C. For example, the reaction may be carried out at a temperature between about 5 °C and 50 °C, between about 10 °C and 30 °C, or between about 15 °C and 25 °C. In some cases, the reaction may be carried out at about 20 °C.
  • the reactions of the present disclosure may be carried out under atmospheric pressure.
  • atmospheric pressure for example, at a pressure in the region of 101.325 kPa or 1 atmosphere.
  • the first and second reactants may be present in any molar amount relative to one another that allows, facilitates or enables the formation of the triazole-containing product.
  • the first and second reactants may be present in a molar ratio of between 5:1 to 1 :5.
  • the first and second reactants may be present in a molar ratio of 2:1 , 1 .5:1 , 1 .2:1 , 1 .1 :1 , 1 :1 , 1 :1.1 , 1 :1 .2, 1 :1 .5, 1 :2.
  • the first and second reactants may be present in an equimolar amount.
  • the first and second reactants may be present in a 1 :1 molar ratio.
  • the reaction proceeds in the presence of a magnesium salt, such as a magnesium halide salt.
  • a magnesium salt such as a magnesium halide salt.
  • Representative examples include, but are not limited to, magnesium fluoride, magnesium chloride, magnesium bromide, and magnesium iodide.
  • the addition of a magnesium salt may be particularly useful as it can displace any copper species bound to the phosphate backbone of the oligonucleotide, ensuring that the catalytic species is available to promote the reaction between the ynamine and the azide.
  • the magnesium salt may be present in the reaction at a concentration between about 0.1 mM and about 100 mM, between about 1 mM and about 50 mM, or between about 5 mM and about 30 mM, such as about 20 mM.
  • the reactions described herein may proceed in the absence of sodium ascorbate.
  • the absence of sodium ascorbate may mean that there is substantially no sodium ascorbate present in the reaction mixture.
  • the amount of sodium ascorbate may be negligible or trace.
  • the first and second reactants may both be comprised on the same molecule.
  • a molecule may comprise both an ynamine and an azide moiety that are spatially arranged such that they have the ability to react together.
  • the first reactant comprises an ynamine moiety.
  • the ynamine may be an aliphatic or aromatic ynamine.
  • the ynamine may be an aromatic ynamine.
  • the nitrogen of the ynamine group may be substituted with an aromatic group or may form part of an aromatic ring (e.g. the nitrogen atom of the ynamine group may be a ring atom comprised within an aromatic ring).
  • the first reactant comprising an ynamine moiety may be represented according to formula (I):
  • R 1 R 2 N — — R 3 (I) wherein R 1 and R 2 may each be independently selected from H, optionally substituted aryl, optionally substituted heteroaryl and Ci-C 6 alkyl, on the proviso that at least one of R 1 and R 2 is an optionally substituted aryl or optionally substituted heteroaryl group; or wherein R 1 and R 2 may together form an optionally substituted heteroaryl ring; and wherein R 3 may be H or Ci-Ce alkyl.
  • the structure shown in formula (I) is covalently bound to the biomolecule by way of a substitution on one of the aryl or heteroaryl rings.
  • a covalent bond to an atom on the biomolecule may replace a hydrogen bond at any chemically suitable position on the aryl or heteroaryl rings on the structure shown in formula (I) providing valencies are satisfied.
  • R 1 and R 2 may together form an optionally substituted heteroaryl ring; and R 3 may be H, or Ci-C 6 alkyl.
  • R 1 and R 2 together form a heteroaryl ring
  • the nitrogen atom of the ynamine is incorporated into the heteroaryl ring.
  • R 1 and R 2 may together form a five- to fourteen-membered heteroaryl ring containing at least N ring heteroatom (i.e. incorporating the nitrogen of the ynamine group as shown in formula (I)) and optionally containing an additional one to three ring heteroatoms each independently selected from N, O and S.
  • the heteroaryl may be optionally substituted with from one to five substituents each independently selected from Ci-C 6 alkyl, Ci-C 6 haloalkyl, halo, Ci- C 6 alkoxy, aryl and heteroaryl, wherein the aryl and heteroaryl substituents are each optionally substituted with one to three substituents each independently selected from Ci-C 6 alkyl, Ci-C 6 haloalkyl, halo, and Ci-C 6 alkoxy.
  • R 1 and R 2 may together form an imidazole ring or a benzimidazole ring incorporating the nitrogen of the ynamine group.
  • the imidazole or benzimidazole ring may comprise one or more (e.g. one to five or one to three) substituents.
  • the imidazole or benzimidazole ring may comprise one or more Ci-C 6 alkyl substituents, such as a methyl group, one or more halo substituents, one or more Ci-C 6 alkoxy substituents, or a combination thereof.
  • the first reactant may be referred to as comprising a terminal ynamine moiety.
  • the first reactant comprising an ynamine moiety may be represented according to formula (la): wherein R 6 and R 7 are each independently selected from the group consisting of H, optionally substituted aryl, and optionally substituted heteroaryl; or wherein R 6 and R 7 together form an optionally substituted aryl or an optionally substituted heteroaryl ring; and wherein R 8 is selected from H and Ci-Ce alkyl.
  • the structure shown in formula (la) is covalently bound to the biomolecule by way of a substitution on one of the aryl or heteroaryl rings.
  • a covalent bond to an atom on the biomolecule may replace a hydrogen bond at any chemically suitable position on the aryl or heteroaryl rings on the structure shown in formula (la) providing valencies are satisfied.
  • R 8 is H.
  • R 6 and R 7 are each independently selected from the group consisting of H, optionally substituted aryl and optionally substituted heteroaryl, there may be a proviso that at least one of R 6 and R 7 is optionally substituted aryl or optionally substituted heteroaryl.
  • R 6 and R 7 are each independently selected from the group consisting of H, a six- to ten-membered ring aryl, and five- to ten-membered ring heteroaryl containing from one to three heteroatoms each independently selected from N, O and S, wherein the aryl and heteroaryl are optionally substituted with from one to five substituents each independently selected from Ci-C 6 alkyl, Ci-C 6 haloalkyl, halo, and Ci-C 6 alkoxy.
  • the two N ring heteroatoms of the imidazolyl core of formula (la) are comprised within the fused ring system.
  • the R 6 and R 7 groups may together form an aryl ring (i.e. a ring formed of carbon atoms)
  • the overall ring system would be considered heteroaromatic as the rings are fused together.
  • this ring formed by the R 6 and R 7 groups of formula (la) may contain may contain up to three additional heteroatoms (e.g. one or two additional heteroatoms) each independently selected from N, O and S.
  • R 6 and R 7 may together form a six- to ten-membered aryl ring which is optionally substituted with from one to five substituents each independently selected from Ci-C 6 alkyl, Ci-C 6 haloalkyl, halo, and Ci-C 6 alkoxy.
  • substituents each independently selected from Ci-C 6 alkyl, Ci-C 6 haloalkyl, halo, and Ci-C 6 alkoxy.
  • R 6 and R 7 may together form a five- to ten-membered heteroaryl ring containing from one to three heteroatoms each independently selected from N, O and S, which is optionally substituted with from one to five substituents each independently selected from Ci-C 6 alkyl, Ci-C 6 haloalkyl, halo, and Ci-C 6 alkoxy.
  • a heteroaryl ring would be fused with the imidazolyl core of formula (la) and so the resulting ring system would be an eight- to fourteen-membered heteroaromatic group).
  • the first reactant comprising the ynamine moiety comprises a structure according to formula (Ic): wherein p is 0, 1 , 2 or 3;
  • R 13 is selected from Ci-C 6 alkyl, halo, Ci-C 6 haloalkyl, and Ci-C 6 alkoxy.
  • the structure shown above is covalently bound to the biomolecule by way of a substitution on the heteroaryl ring.
  • the ynamine moiety may sometimes be appended, conjugated or linked (e.g. covalently linked) to a biomolecule or a tagging moiety.
  • compounds of formulae (I) and (la) may be covalently bonded to the biomolecule or tagging moiety by way of a substitution to the aryl or heteroaryl ring(s) defined in formulae (I) and (la).
  • any hydrogen atom(s) on the aryl or heteroaryl ring may be replaced with the covalent bond to the biomolecule or tagging moiety, providing valencies are satisfied.
  • n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) ;
  • X 1 represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to 15) nucleotide residues;
  • L is a covalent linker; p is 0, 1 , 2 or 3; and R 13 is selected from Ci-C 6 alkyl, halo, Ci-C 6 haloalkyl, and Ci-C 6 alkoxy.
  • the first reactant may be selected from one of the following examples. wherein X 1 represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to
  • first reactants or compounds of the present disclosure may exist in different stereoisomeric forms.
  • the present disclosure includes within its scope the use of all stereoisomeric forms, or the use of a mixture of stereoisomers of the first reactants or compounds.
  • the present disclosure encompasses each individual enantiomer of the first reactant or compound as well as mixtures of enantiomers including racemic mixtures of such enantiomers.
  • the present disclosure encompasses each individual diastereomer of the first reactant or compound, as well as mixtures of the various diastereomers.
  • the first reactant may be represented as: wherein n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10).
  • the first reactant may be represented as:
  • the second reactant comprising an azide moiety may be any organic molecule comprising an azide group.
  • the second reactant comprising an azide moiety may be represented as formula (II):
  • R 4 C1-C4 alkyl — N 3 (II) wherein R 4 may be optionally substituted aryl, optionally substituted heteroaryl, or -XR 5 ; wherein X may be selected from NR 9 , O and S;
  • R 5 may be selected from H, Ci-C 6 alkyl, Ci-C 6 haloalkyl, and SO 2 R 10 ;
  • R 9 may be selected from H and Ci-C 6 alkyl
  • R 10 may be selected from optionally substituted aryl, optionally substituted heteroaryl and Ci-C 6 alkyl.
  • R 4 may be -OH.
  • R 4 may be a six- to ten-membered aryl ring optionally substituted with from one to five substituents selected from halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, Ci-Ce haloalkyl, hydroxyl, amino and -NR 11 R 12 , wherein R 11 and R 12 are each independently selected from H and Ci-C 6 alkyl.
  • R 4 may be a five- to ten-membered heteroaryl ring containing from one to three ring heteroatoms each independently selected from N, O and S, and being optionally substituted with from one to five substituents selected from halo, Ci-C 6 alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, hydroxyl, amino and -NR 11 R 12 , wherein R 11 and R 12 are each independently selected from H and Ci-C 6 alkyl.
  • R 4 may be NHSO2R 10 ’ wherein R 10 is six- to ten-membered aryl ring which is optionally substituted with from one to five substituents selected from halo, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, hydroxyl, amino and -NR 11 R 12 , wherein R 11 and R 12 are each independently selected from H and Ci-C 6 alkyl.
  • R 4 may be -OH, optionally substituted phenyl or optionally substituted pyridyl.
  • R 4 — C1-C4 alkyl may be selected from any of the structures a, b, c, d or shown below. a b e d e
  • the second reactant may be selected from the group consisting of benzyl azide, azidoethanol and picolyl azide.
  • the azide moiety may sometimes be appended, conjugated or linked (e.g. covalently linked) to a biomolecule or a tag.
  • compounds of formula (II) may be covalently bonded to the biomolecule or tag moiety at any suitable position.
  • any hydrogen atom(s) on the second reactant may be replaced with the covalent bond to the biomolecule or tag moiety, providing valencies are satisfied.
  • the second reactant may be a peptide (e.g. an oligopeptide or a cell penetrating peptide) which has been derivatised with one or more azide groups.
  • a peptide e.g. an oligopeptide or a cell penetrating peptide
  • the reactions and/or catalytic systems described herein may show particular utility not only in the synthesis of compounds but also in bioconjugation reactions, such as the preparation of biomolecule-containing compounds and the efficient labelling and/or tagging of biomolecules.
  • the reactions and/or catalytic systems described herein have been shown to be highly chemoselective and may find use in site-specific and/or sequential modification of organic compounds and biomolecules.
  • the reactions and/or catalytic systems described herein may show particular utility in the preparation of heterobifunctional molecules (such as proteolysis targeting chimeric (PROTAC) molecules), peptide-oligonucleotide or peptide- antibody-drug conjugates.
  • heterobifunctional molecules such as proteolysis targeting chimeric (PROTAC) molecules
  • peptide-oligonucleotide or peptide- antibody-drug conjugates may show utility in the fluorescent- or biotin-tagging of azide- modified biomolecules (e.g. glycoproteins, RNA, DNA and the like).
  • a method of forming a triazole moiety in a bioconjugation reaction to link (e.g. covalently link) a first and second reactant comprises: contacting a first reactant comprising an ynamine moiety with a second reactant comprising an azide moiety in the presence of a copper catalyst and a reducing agent, which may preferably be glutathione, wherein at least one of the first and second reactants is a biomolecule.
  • the other one of the first and second reactants is a tagging moiety (such as a labelling or binding tag).
  • the first reactant comprising the ynamine moiety may be or comprise a biomolecule.
  • the first reactant may be or comprise the ynamine moiety appended, conjugated or linked (e.g. covalently linked) to the biomolecule.
  • the second reactant comprising the azide moiety may be or comprise a tagging moiety (such as a labelling or binding tag).
  • the second reactant may be or comprise the azide moiety appended, conjugated or linked (e.g. covalently linked) to the tagging moiety.
  • the ynamine may react with the azide group to form the triazole product and link (e.g. covalently link) the biomolecule to the tag.
  • the first reactant comprising the ynamine moiety may be or comprise a tagging moiety (such as a labelling or binding tag).
  • the first reactant may be or comprise the ynamine moiety appended, conjugated or linked (e.g. covalently linked) to the tagging moiety.
  • the second reactant comprising the azide moiety may be or comprise a biomolecule.
  • the second reactant may be or comprise the azide moiety appended, conjugated or linked (e.g. covalently linked) to the biomolecule.
  • the ynamine may react with the azide group to form the triazole product and link (e.g. covalently link) the biomolecule to the tagging moiety.
  • the method further comprises forming the triazole moiety in a site-specific and/or sequential manner.
  • the biomolecule may comprise at least two sites able to participate in the cycloaddition reaction (such as two azide groups, or an ynamine and alkyne group).
  • the present inventors have identified that it is possible to effect site-specific and/or sequential modification of the biomolecule.
  • the method may comprise forming (e.g. selectively and/or sequentially forming) a first triazole moiety and a second triazole moiety in the biomolecule.
  • the method may comprise reacting a first ynamine (under the reaction conditions described herein) preferentially (or specifically) with the first azide moiety.
  • the method may then comprise reacting an alkyne reagent preferentially (or specifically) with the second azide moiety. In this way, the method may provide a dual-functionalized biomolecule with good selectivity.
  • the biomolecule may comprise an ynamine moiety and an alkyne moiety.
  • the method may comprise reacting a first azide- containing reactant preferentially (or specifically) with one or other of the ynamine and alkyne on the biomolecule.
  • the method may then further comprise preferentially (or specifically) reacting a second azide-containing reactant with the other of the ynamine and alkyne on the biomolecule.
  • the method may provide a dual-functionalized biomolecule with good selectivity.
  • the biomolecule may be any biological molecule typically found in a living organism.
  • the term “biomolecules” may encompass at least proteins, carbohydrates, lipids and nucleic acids, and the like.
  • biomolecules may also embrace polypeptides, peptides, amino acids, polysaccharides, oligosaccharides, monosaccharides, polynucleotides, oligonucleotides, nucleotides, antibodies and the like.
  • the biomolecule may be a peptide (such as a cell penetrating peptide) or an oligonucleotide.
  • a bioconjugation reaction may refer to a method of forming a covalent bond between two molecules, one of which is a biomolecule.
  • Suitable tags may include those that are typically used in the art to label compounds (e.g. labelling tags) and also those tags that are used to bind compounds (e.g. binding tags).
  • Suitable labelling tags may include, but are not limited to, fluorophores, selflabelling protein tags (such as HaloTag or SNAP-tag®), sulfonyl(VI) fluorides (SuFEx reagents) or the like.
  • SuFEx (sulfonyl (VI) fluoride reagents are electrophilic reactive groups that can modify nucleophilic side chains (e.g. lysine, tyrosine, serine, and the like). SuFEx reagents may therefore be considered as labelling tags.
  • a representative example of a labelling tag includes Sulfo-Cy3 azide.
  • Suitable binding tags may include, but are not limited to, those typically used in pull-down assays or the like. Representative examples include biotin and derivatives thereof e.g. d-desthiobiotin.
  • the present disclosure is further directed to any one or more novel compounds such as are described herein.
  • the present disclosure is also directed to a number of novel compounds that find particular applications in the methods described herein.
  • R 6 and R 7 may be any of those described in relation to the first reactant as shown and described in relation to formula (la) above).
  • R 8 may be any of those described in relation to the first reactant (e.g. as shown and described in relation to formula (la)).
  • R 8 may be a silyl protecting group.
  • R 8 may be any silyl group that acts to protect the acetylenic hydrogen of the ynamine.
  • the silyl protecting group may be derived from an organosilane, e.g. an alkylsilane, which in some cases may be a trialkylsilane.
  • TIPS triisopropylsilyl
  • TDMS tert-butyldimethylsilyl
  • TMS trimethylsilyl
  • TDS thexyldimethylsilyl
  • R 8 in the example compounds above is H or silyl protecting group (e.g. triisopropylsilyl).
  • R 8 is H or silyl protecting group (e.g. triisopropylsilyl); n is 0 or any number between 1 and 10; p is 0, 1 , 2 or 3; and
  • R 13 is selected from Ci-C 6 alkyl, halo, Ci-C 6 haloalkyl, and Ci-C 6 alkoxy.
  • n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) ;
  • R 8 in the example compounds above is H or silyl protecting group (e.g. triisopropylsilyl);
  • X 1 represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to 15) nucleotide residues;
  • L is a covalent linker
  • R 13 is selected from Ci-C 6 alkyl, halo, Ci-C 6 haloalkyl, and Ci-C 6 alkoxy.
  • novel compounds of the disclosure may be selected from one of the following examples.
  • R 8 in the example compounds above is H or silyl protecting group (e.g. triisopropylsilyl); and wherein X 1 represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to 15) nucleotide residues.
  • the compounds of the present disclosure may exist in different stereoisomeric forms.
  • the present disclosure includes within its scope the use of all stereoisomeric forms, or the use of a mixture of stereoisomers of the compounds.
  • the present disclosure encompasses each individual enantiomer of the compound as well as mixtures of enantiomers including racemic mixtures of such enantiomers.
  • the present disclosure encompasses each individual diastereomer of the compound, as well as mixtures of the various diastereomers.
  • the compounds provided by the present disclosure may be represented as: wherein n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) ; wherein R 8 in the example compound above is H or silyl protecting group (e.g. triisopropylsilyl).
  • a compound provided by the present disclosure may be represented as: wherein R 8 in the example compound above is H or silyl protecting group (e.g. triisopropylsilyl).
  • kits for use in cycloaddition e.g. Click
  • the kit may be of particular use in bioconjugation reactions, such as the tagging and/or labelling of biomolecules.
  • the kit may comprise:
  • one or more first reactants comprising an ynamine moiety (as described herein, such as those of formulae (la), (lb) and (Ic) and those example compounds described herein); and (ii) one or more second reactants comprising an azide moiety (as described herein, such as those of formula (II) and those example compounds described herein).
  • the kit may further comprise one or more additional components selected from the following list:
  • a reducing agent e.g. glutathione or sodium ascorbate
  • a copper catalyst as described herein e.g. Cu(OAc) 2 );
  • a solvent as described herein such as an aqueous buffered solution
  • a co-solvent as described herein such as a protic fluorinated solvent
  • alkyl refers to a straight or branched chain hydrocarbyl group.
  • the chain may be saturated or unsaturated, e.g. in some cases the chain may contain one or more double or triple bonds.
  • Ci-C n alkyl may be selected from straight or branched chain hydrocarbyl groups containing from 1 to n carbon atoms.
  • Ci-Cealkyl may be selected from straight or branched chain hydrocarbyl groups containing from 1 to 6 carbon atoms
  • Ci-C 3 alkyl may be selected from straight or branched chain hydrocarbyl groups containing from 1 to 3 carbon atoms.
  • a Ci-C 6 alkyl group any hydrogen atom(s), CH 3 , CH 2 or CH group(s) may be replaced with the substituent(s), providing valencies are satisfied.
  • Ci-C 6 alkyl comprises a divalent hydrocarbon radical (containing from 1 to 6 carbon atoms)
  • this moiety may sometimes be referred to herein as a Ci-C 6 alkylene.
  • aryl may be a single or fused ring system comprising one or more aromatic rings.
  • the term “aryl” may refer to a mono- or polycyclic aromatic hydrocarbon system having 6 to 14 carbon ring atoms, in some cases 6 to 10 carbon ring atoms. Where the aryl is a fused ring system, at least one of the rings is aromatic and the other ring(s) may be aromatic or aliphatic.
  • aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, 1 -naphthyl, 2- naphthyl and anthracenyl.
  • substituted aryl refers to an aryl group as defined herein which comprises one or more substituents on the aromatic ring. When an aryl group is substituted, any hydrogen atom(s) may be replaced with the substituent(s), providing valencies are satisfied.
  • heteroaryl may be a single or fused ring system comprising one or more aromatic rings, wherein the one or more aromatic rings comprise one or more O, N and/or S atoms.
  • heteroaryl may refer to a mono- or polycyclic heteroaromatic system having 5 to 14 ring atoms, in some cases 5 to 10 ring atoms. Where the heteroaryl is a fused ring system, at least one of the rings is aromatic and the other ring(s) may be aromatic or aliphatic.
  • heteroaryl groups may include, but are not limited to, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, benzofuranyl, benzothiazolyl, benzimidazolyl, indazolyl, benzoxazolyl, benzisoxazolyl, benzodioxanyl etc.
  • substituted heteroaryl refers to a heteroaryl group as defined herein which comprises one or more substituents on the heteroaromatic ring.
  • any hydrogen atom(s) may be replaced with the substituent(s), providing valencies are satisfied.
  • substituted means that the moiety comprises one or more substituents.
  • optionally substituted means that the moiety may comprise one or more substituents.
  • a “substituent” may include, but is not limited to, hydroxyl, thiol, carboxyl, cyano (CN), nitro (NO 2 ), halo, haloalkyl (e.g. a Ci to C 6 haloalkyl), an alkyl group (e.g. Ci to C or Ci to C 6 ), aryl (e.g. phenyl and substituted phenyl for example benzyl or benzoyl), alkoxy group (e.g. Ci to C 6 alkyl) or aryloxy (e.g. phenoxy and substituted phenoxy), thioether (e.g. Ci to C 6 alkyl or aryl), keto (e.g.
  • Ci to C 6 keto e.g. Ci to C 6 alkyl or aryl, which may be present as an oxyester or carbonylester on the substituted moiety
  • thioester e.g. Ci to C 6 alkyl or aryl
  • alkylene ester such that attachment is on the alkylene group, rather than at the ester function which is optionally substituted with a Ci to C 6 alkyl or aryl group
  • amine including a five- or six-membered cyclic alkylene amine, further including a Ci to C 6 alkyl amine or a Ci to C 6 dialkyl amine which alkyl groups may be substituted with one or two hydroxyl groups
  • amido e.g.
  • Ci to C 6 alkyl groups including a carboxamide which is optionally substituted with one or two Ci to C 6 alkyl groups
  • alkanol e.g. Ci to C 6 alkyl or aryl
  • carboxylic acid e.g. Ci to C 6 alkyl or aryl
  • sulfoxide e.g. sulfone, sulfonamide
  • urethane such as -O-C(O)-NR 2 or-N(R)-C(0)-0-R, wherein each R in this context is independently selected from Ci to C 6 alkyl or aryl).
  • a “substituent” may include, but is not limited to, halo, Ci to C 6 alkyl, Ci to C 6 haloalkyl and Ci to C 6 alkoxy.
  • halo group may be F, Cl, Br, or I. In some examples, halo may be F.
  • haloalkyl may be an alkyl group in which one or more hydrogen atoms thereon have been replaced with a halogen atom
  • a Ci-C 6 haloalkyl may be a Ci to C 6 alkyl in which one or more hydrogen atoms thereon have been replaced with a halogen atom.
  • a Ci-C 6 haloalkyl may be a fluoroalkyl, such as trifluoromethyl (-CF 3 ) or 1 ,1 -difluoroethyl (-CH 2 CHF 2 ).
  • alkoxy may refers to an alkyl group, as defined above, appended to the parent molecular moiety through an oxy group, -O-.
  • a Ci-C 6 alkoxy refers to a Ci-C 6 alkyl group (as defined above), appended to the parent molecular moiety through a oxy group, -O-.
  • Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy etc.
  • Ci-C 6 alcohol refers to a Ci-C 6 alkyl group (as defined above) which is appended to an -OH group.
  • a silyl protecting group may refer to any silyl group that acts to protect the acetylenic hydrogen of the ynamine.
  • the silyl protecting group may be derived from an organosilane, e.g. an alkylsilane, which in some cases may be a trialkylsilane.
  • TIPS triisopropylsilyl
  • TDMS tert-butyldimethylsilyl
  • TMS trimethylsilyl
  • TDS thexyldimethylsilyl
  • BDMS benzyldimethylsilyl
  • BDMS biphenyldimethylsilyl
  • BDIPS biphenyldiisopropylsilyl
  • TBPS tris(biphenyl-4-yl)silyl
  • linker or “covalently linked” may be a covalent linker.
  • the linker acts to tether ynamine or azide moiety to a biomolecule or tagging moiety whilst also allowing the biomolecule to interact with the cellular environment, allowing the tagging moiety to perform its function (e.g. a labelling function), and allowing the ynamine or azide moiety to participate in the reaction to form a triazole. In many cases, a broad range of linkers will be tolerated.
  • the selection of linker may depend upon the nature of the biomolecule the ynamine or azide-containing reactant is being tethered to. The linker may be selected to provide a particular length and/or flexibility.
  • the linker may be a covalent bond.
  • the linker may comprise any number of atoms between 1 and 30 or between 1 and 10.
  • the linker may comprise any number of atoms in a single linear chain of between 1 and 30 or between 1 and 10.
  • the ynamine or azide-containing reactant and the biomolecule may be covalently linked to L through any group which is appropriate and stable to the chemistry of the linker.
  • the linker may be covalently bonded to these moeities via a carboncarbon bond, keto, amino, amide, ester or ether linkage.
  • the disclosure also encompasses various deuterated forms of the compounds of any of the Formulae disclosed herein, including Formulae (I), and (II) (inc. corresponding subgeneric formulae defined herein), respectively, or a pharmaceutically acceptable salt and/or a corresponding tautomer form thereof (including subgeneric formulae, as defined above) of the present disclosure.
  • Each available hydrogen atom attached to a carbon atom may be independently be present as a deuterium atom.
  • a person of ordinary skill in the art will know how to synthesize deuterated forms of the compounds of any of the Formulae disclosed herein, including Formulae (I) and (II), (inc.
  • deuterated materials such as alkyl groups may be prepared by conventional techniques (see for example: methyl-c/ 3 -amine available from Aldrich Chemical Co., Milwaukee, Wl, Cat. No.489, 689-2).
  • the disclosure also includes isotopically-labelled compounds which are identical to those recited in any of the Formulae disclosed herein, including Formulae (I) and (II) (inc. corresponding subgeneric formulae defined herein), respectively, or a pharmaceutically acceptable salt and/or a corresponding tautomer form thereof (including subgeneric formulae, as defined above) of the present disclosure but for the fact that one or more atoms may be present as an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
  • isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as 3 H, 11 C, 14 C, 18 F, 123 l or 125 L
  • isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as 3 H, 11 C, 14 C, 18 F, 123 l or 125 L
  • Compounds of the present disclosure and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present disclosure.
  • Figure 1 shows (a) CuAAC as a bio-orthogonal tool in chemical biology; (b) GSH acts as a Cu ligand and redox mediator; and (c) Design concept: sequential labelling of biomolecules exploiting conditional ynamine reactivity mediated by GSH:Cu.
  • Figure 2 shows RP-HPLC susceptibility study of ynamine (1 a) in the presence of GSH. Reaction conditions: (a) 1a (200 pM), GSH (1 - 10 mM), room temperature (rt); and (b) 1a (200 pM), Cu(OAc) 2 (350 pM),GSH (1 - 10 mM), room temperature (rt).
  • Figure 3a shows the influence of [GSH] on the formation of 5a; Reaction conditions: 1a (200 pM), 4a (500 pM), Cu(OAc) 2 (350 pM), GSH (1 -4 mM), rt.
  • Figure 3b shows the influence of [GSH] (glutathione concentration) and sodium ascorbate (NaAsc) on the formation of 5a; Reaction conditions (all): 1a (200 pM), 4a (500 pM), Cu(OAc) 2 (1.75 equiv., 350 pM), rt; black line (no GSH or NaAsc); red line GSH (1 mM), blue line NaAsc (1 mM).
  • GSH glutthione concentration
  • NaAsc sodium ascorbate
  • Figure 4 shows reaction kinetics as a function of azide. Reaction conditions: lari (200 pM), 4a-c (500 pM), Cu(OAc) 2 (350 pM), GSH (1 mM), 10% MeOH, DPBS, rt. (The asterix (*) indicates that addition of sodium ascorbate (NaAsc) (1 mM) was required).
  • Figure 5 shows investigation into the reaction conditions of the ynamine-azide [3+2]cycloaddition.
  • Reaction conditions 1a (200 pM), 4a (500 pM) CU(OAC) 2 (100 pM), GSH (100 pM), DPBS in 10% organic co-solvent.
  • Figure 6 shows exemplary structures of azide-modified cell penetrating peptides (CPPs) used in the present disclosure.
  • Figure 7a and 7b shows structures of triazole products.
  • Figure 7c shows reaction profile of the formation of 20 (red) and 21 (grey). Reaction conditions: 10 (200 pM), 18 or 20 (200 pM), Cu(OAc) 2 (100 pM), GSH (100 pM), 10% HFIP in 1X DPBS, rt, 1 - 4 h.
  • Figure 7d shows comparative analyses of the reaction profile of the formation of 20 using different co-solvents (10% co-solvent) in 1 X DPFS.
  • Figure 8 shows: (a) Competition experiments highlighting the conditional reactivity of ynamine (10); (b) Reaction profile for the formation of 24a/b.
  • Reaction conditions 10 (200 M), 22 (200 pM), 18 (200 pM), Cu(OAc) 2 (500 pM),10% HFIP in 1 X DPBS, rt, 1 h;
  • Reaction profile for the formation of 23 Reaction conditions: 10 (200 M), 22 (200 pM), 18 (200 pM), Cu(OAc) 2 (500 pM), GSH (500 pM), 10% HFIP in 1 X DPS, rt, 1 h.
  • Figure 9 shows (a) Competition experiments highlighting the conditional reactivity of ynamine (10) with peptide (19); (b) Reaction profile for the formation of 26a/b.
  • Reaction conditions 10 (200 M), 22 (200 pM), 19 (200 pM), Cu(OAc) 2 (500 pM),10% HFIP in 1 X DPBS, rt, 1 h;
  • Reaction conditions 10 (200 M), 22 (200 pM), 18 (200 pM), Cu(OAc) 2 (500 pM), GSH (500 pM), 10% HFIP in 1 X DPBS, rt, 1 h.
  • Figure 10 shows structure of bifunctional peptide (27) containing two azide functional groups.
  • Figure 1 1 shows: (a) Competition experiments highlighting the conditional reactivity of ynamine (10) and DBCO (22) with peptide (27); (b) Reaction profile for the formation of 28: Reaction conditions: (i) 10 (220 pM), 22 (220 pM), 27 (200 pM), CU(GAC) 2 (500 pM), 10% HFIP in 1 X DPBS, rt, 2 h; (ii) EDTA (5 mM) shaken for 4 h.
  • Figure 12 shows (a) Reaction of ODN-1 to form ODN-2. (b) Influence of the [Cu/GSH] on the formation of ODN-2. Conditions: ODN-1 (50 pM), 30 (100 pM) Cu(OAc) 2 (50 (grey), 100 (red) or 500 (blue) pM), GSH (50 (grey), 100 (red) or 500 (blue) pM), 10% HFIP in 1 X DBPS, rt, 16 - 20 h.
  • Figure 13 shows (a) Sequential modification of a dual-tagged oligonucleotide 31 .
  • Reaction conditions (i) 31 (20 pM), 30 (20 pM), 5% HFIP in 1X DPBS (20 mM MgCI 2 ), rt, 4 h; (ii) 32 (30 pM), Cu(OAc) 2 (50 pM), GSH (50 pM), rt, 2 h.
  • Figure 14 shows: (a) Reaction of ODN 3 with sulfo-Cy3-azide using Cu(OAc) 2 and GSH. (b) % Total area of triazole product ODN 4 after 2 hours as a function of cosolvent. (c) % Total area of triazole product ODN 4 after 2 hours as a function of buffer, (c) Time course of the reaction of ODN 3 with sulfo-Cy3-azide as a function of copper source.
  • Figure 15 shows (a) Degradation of ODN 3 in the presence of Cu/GSH and Cu/THPTA/NaAsc. (b) Stability of ODN 3 in the presence of Cu/GSH. Conditions (Cu/GSH): ODN 3 (10 pM), Cu(OAc) 2 (50 pM), GSH (50 pM) in buffer containing 10% MeOH unless otherwise indicated.
  • Figure 16 shows (a) Reaction of ODN 3 with sulfo-Cy3-azide using Cu(OAc) 2 , THPTA and NaAsc at different concentrations, (b) HPLC time courses of DoE results, (c) Further HPLC screening of THPTA and NaAsc concentrations to improve reactivity.
  • Figure 17 shows a) Reaction of ODN 3 with different azides using the optimised conditions for the Cu/THPTA/NaAsc system, (b) Percentage total area (HPLC) of triazole products after the indicated reaction time.
  • Figure 18 shows (a) Reaction of ODN 3 with sulfo-Cy3-azide using Cu(OAc) 2 and GSH. (b) Increasing the Cu/GSH ratio increases the reaction rate.
  • the objective of this study was to determine the optimal conditions required to undergo a chemoselective ynamine-azide tagging of biomolecules within biological media.
  • the present inventors hypothesized that the structural and mechanistic differences of ynamines relative to conventional alkynes would manifest in a divergence in their reactivity with an azide in the presence of biologically-relevant concentrations of GSH.
  • GSH a chemoselective ynamine-azide tagging of biomolecules within biological media.
  • GSH adducts 2 and 3 In the presence of 1 and 5mM of GSH, only ⁇ 10% of ynamine (1a) formed GSH adducts 2 and 3 ( Figure 2a). These adducts could arise from either a radical-based thiolyne addition to 1a to form 2, [91 or via a step-wise activation and subsequent nucleophilic attack of the thiol(ate) to form 3. 1101 Although an increase in the formation of 2 was only observed at 10mM [GSH], at least 75% of 1a still remained after 24 hours, which confirmed the potential utility of ynamines as an alkyne surrogate for bio-orthogonal CuAAC ligations.
  • Electron paramagnetic resonance (EPR) spectroscopy was then used to investigate the influence of the GSH:Cu ratio on the Cu oxidation state. Complete reduction of Cu(ll) to Cu(l) was observed when GSH:Cu >3:1 , whereas only partial reduction was observed when GSH:Cu ⁇ 3:1 was used. At a 1 :1 GSH:Cu, only partial reduction occurred in the first 1 min of the reaction in both HFIP and MeOH solvents, which suggests a mixed Cu oxidation state may be favourable in some cases. The residual Cu(ll) signal after 60 min resembled the EPR spectrum of GSSG + Cu(OAc) 2 suggesting formation of a Cu(ll)-GSSG complex.
  • a solvent screen of polar protic and non -protic co-solvents (10% co-solvent in 1X DPBS buffer) revealed a striking increase in the reaction rate and conversion to 5a using either TFE or HFIP as the co-solvent relative to other polar solvents, reaching maximum conversion after 10min relative to 2.5 hr required when DMSO was used as the corresponding co-solvent (Figure 5b).
  • a pertinent comparator is the observed differences in conversion to 5a using TFE (full conversion in 10 min) relative to EtOH (75 % conversion after 2.5 hr), thus highlighting the importance of the enhanced polarity and hydrogen-bond donating character of these fluorinated solvents on the CuAAC even in the presence of the GSH additive.
  • ynamine analogues tagged with a desthiobiotin (10) and phosphoramidite (17) were prepared.
  • the desthiobiotin group is used extensively for pull-down assays, whereas the phosphoramidite is an important group for solid phase nucleic acid synthesis.
  • Peptide (27) contained a regular aliphatic azide on the C-terminus and a picolyl azide on the AZ-terminus.
  • the picolyl azide functionality is known to react faster in the presence of an aliphatic azide [3c ’ 121 by virtue of its capacity to chelate a Cu atom within proximity to the benzylic azide group. It was therefore hypothesised that a sequential click reaction, first at the picolyl azide site, then at the regular azide would be possible using 10 and 22. Furthermore, the modulation of ynamine (10) reactivity would enable positional and sequential control of each triazole formed.
  • Dual azide-labelled peptide (27) was prepared by solid phase synthesis ( Figure 10) and used in competition experiments in the presence of equimolar amounts of ynamine (10) and DBCO analogue (22).
  • Previous work by Hosoya et al. has shown that transient protection of the internal alkyne of DBCO occurs in the presence of Cu(l).
  • Cu(l) can form and perform two roles: first, activate the ynamine-based [3+2]cycloaddition preferably at the picolyl azide site, and second, act as a transient protecting group on the DBCO, thereby suppressing competitive reactivity.
  • the ynamine functional group exhibits conditional reactivity in the presence of the GSH. This phenomenon is not observed with conventional terminal alkynes, thereby rendering the ynamine functionality as an environmentally sensitive bio-orthogonal reagent.
  • the ability to alter the reactivity of ynamines expands the arsenal of the bio-orthogonal toolkit by providing step-efficient approaches to prepare discrete mono- and dual-functionalized biomolecules.
  • ODN-3 has the sequence shown in Figure 14a.
  • the third difference is the degradation profiles for the NaAsc and GSH systems.
  • the NaAsc system degradation is coupled to NaAsc consumption and once the antioxidant depleted (-4 hours in H 2 O) then degradation stopped.
  • the GSH system led to steady degradation over time, which is less influence by buffer and co-solvent parameters.
  • the product ratios obtained with the Cu/GSH system were consistently higher than the Cu/NaAsc system ( ⁇ 95%+ vs -90%). It can be concluded that the advantage of the Cu/GSH system is less oligonucleotide degradation while the Cu/NaAsc gives faster reaction rates at the cost of oligonucleotide degradation.
  • ODN-1 and ODN-3 were synthesized using standard solid phase oligonucleotide synthesis protocols on an ABI 392 synthesizer.
  • Phosphoramidites and CPG supports loaded with standard nucleosides were purchased from LINK Technologies Ltd (Bellshill, UK).
  • 2-cyanoethyl 6-(1 -((triisopropylsilyl)ethynyl)-1 H- benzo[cf]imidazol-6-yl)hexyl
  • Pd(PPh 3 )2CI 2 (0.1 g, 0.2 mmol, 0.09 equiv.), Cui (0.09 g, 0.3 mmol, 0.16 equiv.) and 1 - ((2fi,4S,5fi)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxytetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1 /-/,3/-/)-dione (1.3 g, 2.0 mmol, 1 equiv.) were placed in a dried flask filled with argon.
  • A/,A/-Diisopropylethylamine (0.4 mL, 2.4 mmol, 6 equiv.) and 2-cyanoethyl diisopropylchlorophosphoramidite (0.1 mL, 0.5 mmol, 1.2 equiv.) were added to a solution of 7-(1 -((2fi,4S,5fi)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxytetrahydro-furan-2-yl)-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-5-yl)-/V-(1 - ((triisopropylsilyl)ethynyl)- 1 H-benzo[c]imidazol-6-yl)hept-6-ynamide (0.4 g, 0.4 mmol, 1 equiv.) in THF (2.7 mL).
  • reaction mixture was stirred at room temperature for 1 .5 h, and then partitioned between CHCI 3 (50 mL) and aq. sat. solution of NaHCO 3 (10 mL). The organic layer was washed with brine (2 x 50 mL), dried over Na 2 SO 4 , and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, n-hexane/EtOAc, 1/1 to 0/1 +2% NEt 3 ) to provide the desired product as a white solid (0.2 g, 47%).

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Abstract

La présente divulgation concerne des réactions de cycloaddition catalysées par du cuivre. En particulier, l'utilisation de réactions de cycloaddition catalysées par du cuivre dans des réactions de bioconjugaison, par exemple des réactions de bioconjugaison impliquant l'utilisation d'une ynamine en tant que substrat. Les procédés et les réactifs présentement divulgués peuvent trouver une application particulière dans des réactions de bioconjugaison impliquant des biomolécules telles que des peptides et des oligonucléotides.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0026908A1 (fr) * 1979-10-08 1981-04-15 BASF Aktiengesellschaft Procédé de préparation d'imidazoles substitués en position 1
EP2743266A2 (fr) * 2011-06-16 2014-06-18 Obshchestvo S Ogranichennoy Otvetstvennostyou "Fusion Pharma" Inhibiteurs de protéines kinases (variantes), leur utilisation dans le traitement de maladies oncologiques et composition pharmaceutique obtenue à partir de ceux-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0026908A1 (fr) * 1979-10-08 1981-04-15 BASF Aktiengesellschaft Procédé de préparation d'imidazoles substitués en position 1
EP2743266A2 (fr) * 2011-06-16 2014-06-18 Obshchestvo S Ogranichennoy Otvetstvennostyou "Fusion Pharma" Inhibiteurs de protéines kinases (variantes), leur utilisation dans le traitement de maladies oncologiques et composition pharmaceutique obtenue à partir de ceux-ci

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
A. D. JENKINS ET AL., PURE & APPL. CHEM., vol. 68, 1996, pages 2287 - 2311
A. MEISTERM. E. ANDERSON, ANN. REV. BIOCHEM., vol. 52, 1983, pages 711 - 760
BURLEY GLENN A. ET AL: "Cu-Catalyzed N - Alkynylation of Imidazoles, Benzimidazoles, Indazoles, and Pyrazoles Using PEG as Solvent Medium", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 75, no. 3, 4 January 2010 (2010-01-04), pages 980 - 983, XP093073872, ISSN: 0022-3263, DOI: 10.1021/jo902466f *
C. CATIVIELAJ. I. GARCIAJ. A. MAYORALL. SALVATELLA, CAN. J. CHEM., vol. 72, 1994, pages 308 - 311
C. J. PICKENSS. N. JOHNSONM. M. PRESSNALLM. A. LEONC. J. BERKLAND, BIOCONJUGATE CHEMISTRY, vol. 29, 2018, pages 686 - 701
C. P. SEATHG. A. BURLEYA. J. WATSON, ANGEW. CHEM. INT. ED., vol. 56, 2017, pages 3314 - 3318
C. SADLERL. A. MCLEANB. C. WHITEHURSTC. P. SEATHL. D. HUMPHREYSR. J. YOUNGA. J. B. WATSONG. A. BURLEY, ORG. LETT., vol. 18, 2016, pages 1694 - 1697
C. WORCHC. J. STUBBSM. J. PRICEA. P. DOVE, CHEM. REV., 2021
D. L. DAVISE. K. PRICES. O. ADERIBIGBEM. X. H. LARKINE. D. BARLOWR. CHENL. C. FORDZ. T. GRAYS. H. GRENY. JIN, J. ORG. CHEM., vol. 81, 2016, pages 6816 - 6819
D. VULUGAJ. LEGROSB. CROUSSEA. M. Z. SLAWINC. LAURENCEP. NICOLETD. BONNET-DELPON, J. ORG. CHEM., vol. 76, 2011, pages 1126 - 1133
F. DENESM. PICHOWICZG. POVIEP. RENAUD, CHEM. REV., vol. 114, 2014, pages 2587 - 2693
G. R. ABELZ. A. CALABRESEJ. AYCOJ. E. HEINT. YE, BIOCONJUG. CHEM., vol. 27, 2016, pages 698 - 704
H. CHENS. LAMT. CAIZ. ZHUS. J. BARKC. CAI, BIOCONJUGATE CHEM., vol. 27, 2016, pages 2315 - 2322
HATIT MARINE Z. C. ET AL: "Chemoselective Sequential Click Ligations Directed by Enhanced Reactivity of an Aromatic Ynamine", ORGANIC LETTERS, vol. 18, no. 7, 22 March 2016 (2016-03-22), US, pages 1694 - 1697, XP093073868, ISSN: 1523-7060, DOI: 10.1021/acs.orglett.6b00635 *
HATIT MARINE Z. C. ET AL: "Strategy for Conditional Orthogonal Sequential CuAAC Reactions Using a Protected Aromatic Ynamine", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 82, no. 10, 5 May 2017 (2017-05-05), pages 5461 - 5468, XP093073853, ISSN: 0022-3263, DOI: 10.1021/acs.joc.7b00545 *
J. LIUL. WANGX. WANGL. XUZ. HAOJ. XIAO, ORG. BIOMOL. CHEM., vol. 14, 2016, pages 11510 - 11517
J. SECKUTEN. K. DEVARAJ, CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 17, 2013, pages 761 - 767
JACOBSEN, M. F.RAVNSBAEK, J. B.GOTHELF, K. V.: "Small Molecule Induced Control in Duplex and Triplex DNA-Directed Chemical Reactions", ORG BIOMOL CHEM, vol. 8, no. 1, 2010, pages 50 - 52, Retrieved from the Internet <URL:https://doi.org/10.1039/B919387A>
KOLLASCHINSKI, M.SOBOTTA, J.SCHALK, A.FRISCHMUTH, T.GRAF, B.SERDJUKOW, S.: "Efficient DNA Click Reaction Replaces Enzymatic Ligation", BIOCONJUG. CHEM., vol. 31, no. 3, 2020, pages 507 - 512, Retrieved from the Internet <URL:https://doi.org/10.1021/acs.bioconjchem.9b00805>
LAROCHE C ET AL: "Lithiation and functionalization of 1-alkynylimidazoles at the 2-position", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 50, no. 37, 16 September 2009 (2009-09-16), pages 5194 - 5197, XP026418873, ISSN: 0040-4039, [retrieved on 20090704], DOI: 10.1016/J.TETLET.2009.06.133 *
LAROCHE CHRISTOPHE ET AL: "Cytotoxic 1,2-Dialkynylimidazole-Based Aza-Enediynes: Aza-Bergman Rearrangement Rates Do Not Predict Cytotoxicity", JOURNAL OF MEDICINAL CHEMISTRY, vol. 54, no. 14, 28 June 2011 (2011-06-28), US, pages 5059 - 5069, XP093082329, ISSN: 0022-2623, DOI: 10.1021/jm200289j *
LI J ET AL: "Synthesis and biological evaluation of p38@a kinase-targeting dialkynylimidazoles", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 19, no. 22, 15 November 2009 (2009-11-15), pages 6293 - 6297, XP026703793, ISSN: 0960-894X, [retrieved on 20090927], DOI: 10.1016/J.BMCL.2009.09.094 *
M. J. MEREDITHD. J. REED, J. BIOL. CHEM., vol. 257, 1982, pages 3747 - 3753
M. T. TAYLORM. L. BLACKMANO. DMITRENKOJ. M. FOX, J. AM. CHEM. SOC., vol. 133, 2011, pages 13984 - 14001
M. Z. C. HATITC. P. SEATHA. J. B. WATSONG. A. BURLEY, J. ORG. CHEM., vol. 82, 2017, pages 5461 - 5468
M. Z. C. HATITL. F. REICHENBACHJ. M. TOBINF. VILELAG. A. BURLEYA. J. B. WATSON, NAT. COMMUN., vol. 9, 2018, pages 4021
MEDZIUNE, J.KAPUSTINA, Z.ZEIMYTE, S.JAKUBOVSKA, J.SINDIKEVICIENE, R.CIKOTIENE, I.LUBYS, A.: "Advanced Preparation of Fragment Libraries Enabled by Oligonucleotide-Modified 2',3'-Dideoxynucleotides", COMMUN. CHEM., vol. 5, no. 1, 2022, pages 34, Retrieved from the Internet <URL:https://doi.org/10.1038/s42004-022-00649-9>
P. M. FISCHERN. Z. ZHELEVS. WANGJ. E. MELVILLER. FAHRAEUSD. P. LANE, J. PEPTIDE RES., vol. 55, 2000, pages 163 - 172
R. VAN GEELG. J. M. PRUIJNF. L. VAN DELFTW. C. BOELENS, BIOCONJUGATE CHEMISTRY, vol. 23, 2012, pages 392 - 398
S. YOSHIDAT. KURIBARAH. ITOT. MEGUROY. NISHIYAMAF. KARAKIY. HATAKEYAMAY. KOIKEI. KIIT. HOSOYA, CHEM. COMMON., vol. 55, 2019, pages 3556 - 3559
S. YOSHIDAY. HATAKEYAMAK. JOHMOTOH. UEKUSAT. HOSOYA, J. AM. CHEM. SOC., vol. 136, 2014, pages 13590 - 13593
SCHWEIGERT, N.ACERO, J. L.VON GUNTEN, U.CANONICA, S.ZEHNDER, A. J. B.EGGEN, R. I. L.: "DNA Degradation by the Mixture of Copper and Catechol Is Caused by DNA-Copper-Hydroperoxo Complexes, Probably DNA-Cu(I)OOH", ENVIRON. MOL. MUTAGEN., vol. 36, no. 1, 2000, pages 5 - 12, XP071735707, Retrieved from the Internet <URL:https://doi.org/10.1002/1098-2280(2000)36:1<5::AID-EM2>3.0.CO;2-4> DOI: 10.1002/1098-2280(2000)36:1<5::AID-EM2>3.0.CO;2-4
T. V. TRANG. COUTUREL. H. DO, DALTON TRANSACTIONS, vol. 48, 2019, pages 9751 - 9758
V. HONGN. F. STEINMETZM. MANCHESTERM. G. FINN, BIOCONJUGATE CHEM, vol. 21, 2010, pages 1912 - 1916
WEI-SHENG HUANG ET AL: "Discovery of 3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{ 4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a Potent, Orally Active Pan-Inhibitor of Breakpoint Cluster Region-Abelson (BCR-ABL) Kinase Including the T315I Gatekeeper Mutant", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 53, no. 12, 24 June 2010 (2010-06-24), pages 4701 - 4719, XP002615777, ISSN: 0022-2623, [retrieved on 20100601], DOI: 10.1021/JM100395Q *
ZHAO LEI ET AL: "Synthesis of Sulfonamide-Based Ynamides and Ynamines in Water", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 86, no. 2, 23 December 2020 (2020-12-23), pages 1938 - 1947, XP093073865, ISSN: 0022-3263, DOI: 10.1021/acs.joc.0c02326 *

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