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WO2013181697A1 - Composés de bicyclo[6.1.0]non-4-yne conçus pour être utilisés comme lieurs dans des applications biologiques - Google Patents

Composés de bicyclo[6.1.0]non-4-yne conçus pour être utilisés comme lieurs dans des applications biologiques Download PDF

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WO2013181697A1
WO2013181697A1 PCT/AU2013/000591 AU2013000591W WO2013181697A1 WO 2013181697 A1 WO2013181697 A1 WO 2013181697A1 AU 2013000591 W AU2013000591 W AU 2013000591W WO 2013181697 A1 WO2013181697 A1 WO 2013181697A1
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substituted
optionally
group
alkyl
formula
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Spencer John Williams
Paul Stephen Donnelly
Paul Mulvaney
Christine SCHIEBER
Isaac Ojea JIMENEZ
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The University Of Melbourne
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C07D213/82—Amides; Imides in position 3
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/04—Systems containing only non-condensed rings with a four-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

  • the invention relates to compounds to link substrates to molecules, typically for biological applications.
  • the invention relates to compounds that can be used to link nanoparticles to biological molecules to allow for applications such as imaging and the like.
  • Dye-labelling used in biological systems typically involves the process of attaching a fluorophore or other photodetectable dye to a molecule, such as a protein or nucleic acid. Generally this is achieved by using a reactive derivative of the dye that selectively binds to a functional group contained in the target molecule.
  • fluorescently-labelled molecules are antibodies, which are then used as highly-specific probes for optical detection of a particular target. Fluorescent dye labelling is typically accomplished using a chemically-reactive derivative of a fluorophore.
  • Common reactive groups include amine-reactive isothiocyanate derivatives such as fluorescein isocyanate (FITC) and tetramethylrhodamine (TRITC); amine-reactive succinimidyl esters such as NHS- fluorescein; and sulfhydryl-reactive maleimide-activated fluorophores such as fluorescein-5-maleimide or iodoacetamide-derivatives such as 5-(iodoacetamido) fluorescein (5-IAF). Reaction of any of these reactive dyes with another molecule results in a covalent bond being formed between the fluorophore and the labelled molecule.
  • FITC fluorescein isocyanate
  • TRITC tetramethylrhodamine
  • amine-reactive succinimidyl esters such as NHS- fluorescein
  • sulfhydryl-reactive maleimide-activated fluorophores such as fluorescein-5-maleimi
  • Reactive fluorescent dyes are available from many sources and can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in labelling kits that contain all of the components necessary to carry out a labelling reaction.
  • Fluorescent labels are generally used for detection of a protein or other labelled molecule via fluorescence microscopy, flow cytometry (FCM), or some other fluorescence measuring technique.
  • FCM flow cytometry
  • High resolution optical microscopy, western blot assays, and other immunoanalytical methods may also be useful in localization of a target within a cell. More flexibility is typically required of these dyes, and the traditional dyes are often unable to meet expectations.
  • QDs semiconducting nanoparticles, hereinafter referred to as quantum dots (QDs), typically provide distinct advantages over traditional organic dyes, which include:
  • Semiconductor quantum dots have been employed for in vitro imaging of pre-labelled cells.
  • the ability to image single-cell migration in real time is important to several research areas such as fluorescence microscopy, histology, flow cytometry, fluorescence in-situ hybridization, DNA sequencing, immuno-assays, binding assays, separation, etc.
  • Core-shell CdSe/ZnS quantum dots are semiconducting nanocrystals that exhibit unique optical and physical features.
  • the size of the QDs can be adjusted to tune the optical properties to give substances with tunable, narrow emission profiles, high quantum yields and excellent photostability.
  • QDs are therefore an attractive alternative to organic fluorescent dyes and QDs tethered to molecules such as antibodies or peptides have great promise for use in biological imaging applications.
  • the linker molecules or conjugation strategy may destabilize the nanocrystals leading to unwanted aggregation or non-specific binding of the nanocrystals to the biological target or surfaces.
  • a conjugation strategy using molecules that use stable reactive group chemistry can be stored for long periods in solution or as pure reagents, will work under a wide range of buffer and pH conditions, and are bio- orthogonal, i.e. cross reactions with other chemical groups present in biological systems is minimal.
  • the present invention relates to a series of compounds that meets the above requirements and enable bioconjugations to be carried out between a wide range of substrates and a wide range of molecules. It is an aspect of the current invention to disclose how such compounds may be prepared and purified.
  • the present invention relates to a compound, which can be used to tether substrates to molecules.
  • the molecules that can be tethered to the substrates include any molecule suitable for conjugation, including polyatomic ions.
  • Molecules used in the present invention for linking to the substrates include, but are not limited to, biological molecules.
  • Some examples of the biological molecules for use in the present invention include large polymeric molecules such as proteins, peptides, polysaccharides, lipids, hormones and nucleic acids as well as small molecules such as natural products and derivatives thereof.
  • the substrates may include any surface or material that may be conjugated by the methods disclosed in this invention.
  • Some substrates may be particles. A skilled addressee would appreciate that the particles may have different shapes and sizes.
  • Typical particles for conjugation may include microparticles, mesoparticles, nanoparticles or any other particle suitable for conjugation.
  • Other examples include semiconducting, metallic and magnetic nanocrystals (or mixtures thereof); particles of non-spherical shapes such as nanorods and nanowires; complex particles consisting of a core and shell or alloy structure; silica or silicic acid based particles; or organic semiconducting polymer-based particles that have useful labelling properties may also be conjugated through the methods outlined herein.
  • the substrates may be proteins or peptides.
  • the current invention discloses a method of conjugation, which can be applied to dendrimers, various polymers such as PEG-polymers and dextran polymers, carbon structures such as C60, C70 and carbon nanotubes and their derivatives, and also to particles carrying further functional molecules such as dyes, surface-enhanced Raman-scattering labels or stabilizing molecules.
  • nanocrystals also termed nanoparticles or quantum dots
  • protocols may be suitably varied to enable any particle to be conjugated.
  • the invention relates to a compound, which contains a cyclooctyne moiety and a moiety for conjugation to an amine-, aldehyde- or ketone-functionalised substrate or molecule.
  • the following moieties may form covalent bonds on exposure to amine, aldehyde or ketone groups: squarate, hydrazide, semicarbazide, carbohydrazide, aminooxy and amine groups.
  • the amine, aldehyde and ketone groups on the substrate or molecule of interest may be native to the substrate or molecule or they can be created via oxidation or reduction of suitable reactive groups.
  • the cyclooctyne moiety acts as the coupling partner to an azide-functionalised substrate such as a QD or biological molecule, via a [3+2] cycloaddition, whilst the squarate, hydrazide, semicarbazide, carbohydrazide, aminooxy or amine moiety acts as the coupling partner to an amine-, aldehyde- or ketone-functionalised substrate such as QD or biological molecule.
  • the cyclooctyne and aldehyde moieties are joined via a linking group.
  • the present invention relates to solution-stable linker molecules that can be used to conjugate particles and other structures to each other or to biological molecules and substrates of interest (e.g. cells.).
  • this invention discloses a variety of novel molecules.
  • hydrazide, semicarbazide, carbohydrazide, aminooxy and amine derivatives can be used for conjugation to aldehyde or ketone groups.
  • squarate derivatives can be employed.
  • the substrates for conjugation are particles.
  • the particles are microparticles, mesoparticles and/or nanoparticles.
  • the particles are QDs.
  • the molecules for conjugation to a substrate are biological molecules.
  • the molecules are synthetic molecules or naturally occurring molecules.
  • the molecules are large polymeric molecules such as proteins, peptides, polysaccharides, lipids, hormones and/or nucleic acids as well as small molecules such as natural products
  • the invention relates to a compound which can be used to tether QDs to biological molecules.
  • the compound contains a cyclooctyne moiety and a second moiety for conjugation to QDs and biological molecules.
  • the cyclooctyne moiety acts as the coupling partner to an azide-functionalised QD or biological molecule, via a [3+2] cycloaddition, whilst the second moiety acts as the coupling partner to an amine- aldehyde- or ketone-functionalised QD or biological molecule.
  • Typical moieties which react with aldehyde or ketone functional groups may include N-containing moieties such as an amine, aminooxy, hydrazide, semicarbazide and carbohydrazide.
  • Typical moieties which react with amine functional groups may include a squarate. The cyclooctyne moiety and the second moiety are joined via a linking group.
  • the present invention provides a compound of the Formula (I):
  • each R a and R b is independently-selected from the group consisting of: H, OH, halogen, optionally-substituted alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally- substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally- substituted OC 2- i2 alkenyl, optionally-substituted OC 2- i2 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl;
  • each R 1 is independently selected from the group consisting of: H, halogen, OH, NO 2 , CN, SH, NH 2 , CF 3 , OCHF 2 , OCF 3 , optionally-substituted C1.12 alkyl, optionally-substituted haloalkyl, optionally-substituted C 2- 12 alkenyl, optionally- substituted 02-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted 03-12 cycloalkenyl, optionally-substituted 02-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted C 6- 18 aryl, optionally-substituted CM S heteroaryl, optionally-substituted C ⁇ . ⁇ 2 alkyloxy, optionally-substituted
  • L 1 is a bond or linking group
  • R 2 is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C3-12 cycloalkyl, optionally substituted C3-12 cycloalkenyl, NH 2 , ONH 2 , NHNH 2 , NHNR a R p , NH(CO)(optionally-substituted Ci_ 6 alkyl)ONH 2 , NH(CO)(optionally-substituted Ci_ 6 alkyl)NH 2 , NH(CO)(optionally-substituted Ci_ 6 alkyl)NHNH 2 , NH(CO)(optionally-substituted Ci_ 6 alkyl)NHNR a R p , (CO)NH(optionally- substituted Ci_ 6 alkyl)ONH 2 , (CO)NH(optionally-substituted Ci -6 alkyl)NH 2
  • each of R X and R 5 is independently selected from the group consisting of H , optionally-substituted alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8;
  • R C , R D and R E are each independently-selected from the group consisting of: H , OH , halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted OC6-18 aryl, and optionally-substituted OCM S heteroaryl;
  • stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo.
  • the invention provides a process for the preparation of a compound of Formula (V):
  • each R 1 is independently selected from the group consisting of: H, halogen, OH, NO 2 , CN, SH, NH 2 , CF 3 , OCHF 2 , OCF 3 , optionally-substituted C1-12 alkyl, optionally-substituted Ci-12 haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C 3- 12 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally-substituted C 2- 12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-MS heteroaryl, optionally-substituted C-M2 alkyloxy, optionally-sub
  • R c , R d and R e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted C ⁇ . ⁇ 2 alkyl, optionally-substituted C 2- 12 heteroalkyl, optionally-substituted C 3- 12 cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OCM S heteroaryl;
  • stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo;
  • L is of the Formula (III): X 3 (CR 8 R 9 ) p [X 4 (CR 10 R 1 1 ) q ] r (CR 12 R 13 ) S X 5
  • X 3 is selected from the group consisting of: O, NH and NR 14 ;
  • X 4 is independently selected from the group consisting of: a bond, O, NH, NR 14 , S and CR 15 R 16 ;
  • X 5 is selected from the group consisting of: O, NH, NR 14 , optionally-substituted
  • R 2 is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C 3- i 2 cycloalkyl, optionally substituted C 3- i 2 cycloalkenyl, NHR 17 , ONHR 17 , NHNHR 17 , NHNR a R p , NH(CO)(optionally-substituted C 1-6 alkyl)ONHR 17 , NH(CO)(optionally-substituted C 1-6 alkyl)NHR 17 , NH(CO)(optionally-substituted C 1-6 alkyl)NHNHR 17 , NH(CO)(optionally-substituted C 1-6 alkyl)NHNR a R p , (CO)NH(optionally-substituted Ci -6 alkyl)ONHR 17 , (CO)NH(optionally-substituted
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted C1-12 alkyl, optionally-substituted C 2- i 2 alkenyl, optionally- substituted C 2- i 2 alkynyl, optionally-substituted C3-i 2 cycloalkyl, optionally-substituted C3-i 2 cycloalkenyl, optionally-substituted OCi-i 2 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC 3- i 2 cycloalkyl and optionally-substituted OC 3- cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a): wherein each of R x and R 5 is independently selected from the group consisting of H,
  • each R 8 R 9 , R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C 2- 12 alkenyl, optionally-substituted C 2- 12 alkynyl, optionally- substituted C3-i 2 cycloalkyl, optionally-substituted C3-i 2 cycloalkenyl, optionally- substituted OCi-i 2 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3-i 2 cycloalkyl and optionally-substituted OC3- 2 cycloalkenyl;
  • each R 14 and R 17 is independently selected from the group consisting of: H, optionally-substituted Ci-i 2 alkyl and N-protecting group;
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8;
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, the process comprising:
  • R 1 and n are as defined above and LG is a leaving group; in a coupling reaction with a compound of Formula (Vll-a): X 3A (CR 8 R 9 ) P [X 4 (CR 1 0 R 1 1 ) Q ] R (CR 12 R 1 3 )s X 5A
  • X 3a is independently selected from the group consisting of: OH, NH 2 and NHR 14 ;
  • X 4 is independently selected from the group consisting of: a bond, O, NH, NR 14 , S and CR 15 R 16 ;
  • X 5a is selected from the group consisting of: OH, NH 2 , NHR 14 , OR 18 CO 2 R 19 ; each R 8 R 9 , R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted alkyl, optionally-substituted C 2- 12 alkenyl, optionally-substituted C 2- 12 alkynyl, optionally- substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally- substituted OCi-12 alkyl, optionally-substituted OC 2- i2 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3- 12 cycloalkenyl;
  • R 14 is independently selected from the group consisting of: optionally- substituted C-i-12 alkyl and N-protecting group;
  • R 18 is selected from the group consisting of: optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally- substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl and O-protecting group;
  • R 19 is selected from the group consisting of: H, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl optionally-substituted C 6- 18 aryl, optionally-substituted C 6- 18 alkylaryl, optionally- substituted C-i-18 heteroaryl, optionally-substituted CM S alkylheteroaryl, optionally- substituted Ci-12 alkyloxy, optionally-substituted C2-12 alkenyloxy, optionally- substituted C2-12 alkynyloxy, optionally-substituted C2-12 heteroalkyloxy, optionally- substituted C3-12 cycloalkyloxy, optionally-substituted C3-12 cycloalkeny
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; to provide a compound of Formula (VIII):
  • B is selected from the group consisting of: a bond, an optionally-substituted d.
  • each R 2 and R 20 is selected independently from the group consisting of: OH, CO2R 22 , optionally-substituted OC1-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl, optionally-substituted OC3-12 cycloalkenyl, NHR 17 , NHNHR 17 , NHNR a R p , ONHR 17 , NH(CO)(optionally-substituted Ci_ 6 alkyl)ONHR 17
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted Ci-12 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or FT and R p when combined together provide the group of Formula (l-a):
  • H optionally-substituted C-.- 2 alkyl, optionally-substituted C 2- 12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;
  • each R 17 and R 21 is independently selected from the group consisting of: H, optionally-substituted Ci-i 2 alkyl and N-protecting group;
  • R 22 is selected from the group consisting of: H, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally-substituted C 2- 12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted Ce-18 alkylaryl, optionally-substituted CM S heteroaryl, optionally-substituted C-M S alkylheteroaryl,
  • the invention provides a process for the preparation of a compound of Formula (V):
  • each R 1 is independently selected from the group consisting of: H, halogen, OH, NO 2 , CN, SH, NH 2 , CF 3 , OCHF 2 , OCF 3 , optionally-substituted C1-12 alkyl, optionally-substituted C-i- 12 haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C 3- 12 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally-substituted C 2- 12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-MS heteroaryl, optionally-substituted C-M2 alkyloxy, optional
  • R c , R d and R e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted C ⁇ . ⁇ 2 alkyl, optionally-substituted C 2- 12 heteroalkyl, optionally-substituted C 3- 12 cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OCM S heteroaryl;
  • stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo;
  • L is of the Formula (III): X 3 (C R 8 R 9 ) P [X 4 (C R 1 0 R 1 1 ) Q ] R (C R 1 2 R 1 3 ) S X 5
  • X 3 is selected from the group consisting of: OH, NH and NR 14 ;
  • X 4 is independently selected from the group consisting of: a bond, O, NH, NR 14 , S and CR 15 R 16 ;
  • X 5 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 , optionally-substituted C-i- 12 alkyl, HN-(optionally-substituted C-i- 12 alkyl)-, R 14 N- (optionally-substituted alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted 05-12 aryl)-, R 14 N-(optionally- substituted 05-12 aryl)-, O-(optionally-substituted Cs-12 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)- R 14 N(CO)-(optionally-substituted C5-12 aryl)- O(CO)- (optionally-substituted Cs-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)
  • R 2 is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR 17 , ONHR 17 , NHNHR 17 , NHNR a R p , NH(CO)(optionally-substituted Ci -6 alkyl)ONHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNR a R p , (CO)NH(optionally-substituted Ci -6 alkyl)ONHR 17 , (CO)NH(optionally-substituted Ci -6 alkyl
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted Ci_i 2 alkyl, optionally-substituted C 2- i 2 alkenyl, optionally- substituted C 2- i 2 alkynyl, optionally-substituted C3-i 2 cycloalkyl, optionally-substituted C3-i 2 cycloalkenyl, optionally-substituted OCi-i 2 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3-i 2 cycloalkyl and optionally-substituted OC3-i 2 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a):
  • each of R x and R 5 is independently selected from the group consisting of H, optionally-substituted Ci_i 2 alkyl, optionally-substituted C 2- i 2 alkenyl, optionally- substituted C 2- i 2 alkynyl, optionally-substituted C3-i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl; each R 3 , R 4 R 6 R 7 , R 8 R 9 , R 10 R 1 1 , R 12 R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted C1-12 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2 -12 alkynyl, optionally-substituted 03-12 cycloalkyl, optionally-substituted C 3- 12 cycloalkenyl, optionally-substituted
  • each R 14 and R 17 is independently selected from the group consisting of: H, optionally-substituted C ⁇ . ⁇ 2 alkyl and N-protecting group; and
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8;
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, the process comprising:
  • X 3a is selected from the group consisting of: OH, NH 2 and NHR 14 ;
  • X 4 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 ;
  • X 5 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 , optionally-substituted alkyl, HN-(optionally-substituted alkyl)-, R 14 N- (optionally-substituted alkyl)-, O-(optionally-substituted Ci-12 alkyl)-, optionally- substituted C 5- i 2 aryl, HN-(optionally-substituted C 5- i 2 aryl)-, R 14 N-(optionally- substituted 05-12 aryl)-, O-(optionally-substituted 05-12 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)-, R 14 N(CO)-(optionally-substituted C
  • R 2 is selected from the group consisting of: optionally substituted C-M2 alkyl, optionally substituted C 2- 12 alkenyl, optionally substituted C 2- 12 alkynyl, optionally substituted C3-12 cycloalkyl, optionally substituted C3-12 cycloalkenyl, NHR 17 , ONHR 17 , NHNHR 17 , NHNR a R p , NH(CO)(optionally-substituted Ci -6 alkyl)ONHR 17 , NH(CO) (optionally-substituted Ci -6 alkyl)NHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNR a R p , (CO)NH (optionally-substituted Ci -6 alkyl)ONHR 17 , (CO)NH(optionally-substituted Ci -6
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted Ci-12 alkyl, optionally-substituted C 2 -12 alkenyl, optionally- substituted C 2 -12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C 3- 12 cycloalkenyl, optionally-substituted OCi-i 2 alkyl, optionally-substituted OC 2- i2 alkenyl, optionally-substituted OC 2 -12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC 3 -12 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a):
  • each of R x and R 5 is independently selected from the group consisting of H, optionally-substituted Ci- 12 alkyl, optionally-substituted C 2 - 12 alkenyl, optionally- substituted C 2 - 12 alkynyl, optionally-substituted C3- 12 cycloalkyl, optionally-substituted C 3-12 cycloalkenyl;
  • each R 8 R 9 , R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci- 12 alkyl, optionally-substituted C 2 - 12 alkenyl, optionally-substituted C 2 - 12 alkynyl, optionally- substituted C 3- i 2 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally- substituted OC-i- 12 alkyl, optionally-substituted OC 2 - 12 alkenyl, optionally-substituted 0C 2-12 alkynyl, optionally-substituted OC 3- i 2 cycloalkyl and optionally-substituted OC 3- 12 cycloalkenyl;
  • each R 14 and R 17 is independently selected from the group consisting of: H, optionally-substituted alkyl and N-protecting group; and
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; coupling reaction with a compound of Formula (VI):
  • R 1 and n are as defined above and LG is a leaving group; to provide the compound of Formula (V).
  • Figure 1 is a schematic of a linker of the invention showing its reactive ends available to form bonds with an azide functionalised material (a Quantum dot in this case) and an amino functionalised biological molecule (transferrin in this case).
  • Figure 2 shows the idealised structure of the linker shown in figure 1 after binding.
  • Figures 3 shows the mass spectral analysis of the reaction of the linker of example 3 with transferrin at a ratio of linker to transferrin of 2:1 .
  • Figure 4 shows the mass spectral analysis of the reaction of the linker of example 3 with transferrin at a ratio of linker to transferrin of 4:1 .
  • Figure 5 shows the reaction product of the linker bound to transferrin with a small molecule azide, azidoacetanilide.
  • Figure 6 shows the mass spectral analysis of the reaction product of the linker with transferrin.
  • Figure 7 shows the product of reaction of the mixture analysed in figure 6 with an azide showing reactivity of the transferrin-bound cyclo-octyne moiety with an azide.
  • Figure 8 shows the stylised product of the linker of example 3 bound to an amino- Alexa dye and an azide-modified Quantum dot.
  • Figure 9 shows the normalised absorbance of separate quantum dot and the dye.
  • Figure 10 shows the fluorescent intensity with differing amounts of dye bound through the linker to the quantum dot.
  • Figure 1 1 shows an agarose gel electrophoresis of QD's and QD plus transferrin under UV lamp.
  • Figure 12 shows the agarose gel electrophoresis of QD's and QD plus transferrin after staining with Coomassie blue.
  • Figure 13 shows Fe 2 -transferrin uptake into HeLa cells using A568-Fe 2 Tf (A1 -A3), QD100-Fe 2 Tf (B1 -B3) and QD100 (C1 -C3).
  • Figure 14 shows time dependent QD uptake into cells and co-staining with early and late endosomal markers, a) QD100-Fe 2 Tf uptake into HeLa cells were performed for 15 min at 37°C. Cells were fixed in 4% paraformaldehyde and stained with rabbit anti-EEA1 (early endosome antigen 1 ) followed by A568-conjugated anti-rabbit IgG, and mouse anti-CD63 followed by A647-conjugated mouse IgG. (A, D) EEA1 has been pseudo-colored green; (B, E) QD100-Fe2Tf has been pseudo-colored red. D-H show 2x magnification of the boxed region.
  • G is overlay of D and E; H is overlay of E and Fb) QD100-Fe2Tf uptake into HeLa cells was performed for 2 h at 37°C. Cells were fixed in 4% paraformaldehyde and stained and pseudo-colored as for a). D-H show 2x magnification of the boxed region. G is overlay of D and E; H is overlay of E and F. Representative regions of overlap are indicated by arrows.
  • Figure 16 and figure 17 show mass spectral analysis of Herceptin conjugated to the linker of example 3 using a ratio of antibody to linker of 1 : 10 (figure 16) and a ration of 1 :20 (figure 17).
  • Figure 18 is an absorption spectrum of Herceptin-linker-azido-Fluor 585 dye conjugates. After oxidation of glycan residues on an antibody with Nal0 4 , a linker of example 8 was reacted in a ratio of 4: 1 , 10:1 and 100: 1 of linker to antibody in an aniline buffer, and then finally with azide-AlexaFluor585. DETAILED DESCRIPTION OF THE INVENTION
  • substrate may include any surface or material for conjugation by the methods disclosed in this invention. Some substrates include deposited or adhered particles, which may include coatings or layers. In some embodiments of the invention, the term substrate refers to particles. A skilled addressee would appreciate that the term “particle” encompasses particles of different shapes and sizes. Typical particles for conjugation may include microparticles, mesoparticles, nanoparticles or any other particle suitable for conjugation.
  • Other examples include semiconducting, metallic and magnetic nanocrystals (or mixtures thereof); particles of non-spherical shapes such as nanorods and nanowires; complex particles consisting of a core and shell or alloy structure; silica or silicic acid based particles; or organic semiconducting polymer-based particles.
  • Quantum dots are semiconductors whose electronic characteristics are closely related to the size and shape of the individual crystal.
  • Quantum dot is readily understood by the skilled person in the art.
  • an “azide-functionalised” substrate or an “azide-functionalised” molecule refers to a substrate or a molecule bearing one or more azide (-N 3 ) groups.
  • the azide groups are present at the surface of the quantum dot or biological molecule such that the azide group can react with other molecules or particles, which possess other reactive functional groups.
  • the azide group can react with other molecules or particles, which possess an alkyne group or cycloalkyne group.
  • an "amine-functionalised” substrate or an “amine-functionalised” molecule refers to a substrate or molecule, which has one or more reactive amine groups.
  • the amine groups are present at the surface of the substrate or molecule such that the amine group can react with molecules or particles, which possess other reactive functional groups.
  • the amine group can react with other molecules or particles, which possess a squarate group, a carboxylic acid group, or an activated equivalent such as an NHS ester.
  • an "aldehyde-functionalised” substrate or an “aldehyde-functionalised” molecule refers to a substrate or molecule, which has one or more reactive aldehyde groups.
  • the aldehyde moiety may be present in free or hydrated form.
  • the aldehyde groups are present at the surface of the substrate or molecule such that the aldehyde group can react with molecules or particles, which possess other reactive functional groups.
  • the aldehyde group can react with other molecules or particles, which possess an amine, aminooxy, hydrazide, semicarbazide or carbohydrazide group.
  • ketone-functionalised substrate or a “ketone -functionalised” molecule refers to a substrate or molecule, which has one or more reactive ketone groups.
  • the ketone moiety may be present in free or hydrated form.
  • the ketone groups are present at the surface of the substrate or molecule such that the ketone group can react with molecules or particles, which possess other reactive functional groups.
  • the ketone group can react with other molecules or particles, which possess an amine, aminooxy, hydrazide, semicarbazide or carbohydrazide group.
  • molecule is readily understood by a skilled addressee to include polyatomic compounds held together by covalent bonds.
  • the molecules of the present invention may also include polyatomic ions.
  • biological molecule refers to a synthetic or naturally occurring molecule produced or used by an organism, cell or cellular fraction. This includes, but is not limited to, large polymeric molecules such as proteins, peptides, polysaccharides, lipids, hormones and nucleic acids as well as small molecules such as natural products and derivatives thereof.
  • the term "[3+2] cycloaddition" reactions is readily understood by the skilled addressee.
  • the [3+2] cycloaddition reactions refer to the reaction of a dipolarophile with a 1 ,3-dipolar compound that leads to 5-membered (hetero)cycles.
  • dipolarophiles are alkenes and alkynes and molecules that possess related heteroatom functional groups (such as carbonyls and nitriles).
  • 1 ,3-Dipolar compounds typically contain one or more heteroatoms and can be described as having at least one mesomeric structure that represents a charged dipole.
  • Examples of 1 ,3-dipolar compounds are azides, nitrile oxides, nitrones and diazoalkanes.
  • a “leaving group” is a chemical group that is readily displaced by a nucleophilic incoming chemical moiety. Accordingly in any situation the choice of leaving group will depend upon the ability of the particular group to be displaced by the incoming chemical moiety. Suitable leaving groups are well known in the art, see for example “Advanced Organic Chemistry” Jerry March 4 th Edn. pp 351 -357, Oak Wick and Sons NY (1997). Examples of suitable leaving groups include, but are not limited to, halogen, alkoxy (such as ethoxy, methoxy), sulfonyloxy, optionally-substituted arylsulfonyl. Specific examples include chloro, iodo, bromo, fluoro, ethoxy, methoxy, methansulphonyl, triflate and the like.
  • normal chain refers to the direct chain joining the two ends of a linking moiety.
  • the group may be a terminal group or a bridging group. This is intended to signify that the use of the term is intended to encompass the situation where the group is a linker between two other portions of the molecule as well as where it is a terminal moiety.
  • alkyl alkyl
  • alkylene alkylene
  • examples of acyl include acetyl and benzoyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.
  • Alkenyl as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain.
  • the group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z.
  • the alkenyl group is preferably a 1 -alkenyl group.
  • Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.
  • the group may be a terminal group or a bridging group.
  • alkenyloxy refers to an alkenyl-O- group in which alkenyl is as defined herein.
  • Preferred alkenyloxy groups are C1-C6 alkenyloxy groups.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Alkyl as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C1-C12 alkyl, more preferably a C1-C10 alkyl, most preferably CrC 6 unless otherwise noted.
  • suitable straight and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t- butyl, hexyl, and the like.
  • the group may be a terminal group or a bridging group.
  • Alkylamino includes both mono-alkylamino and dialkylamino, unless specified.
  • “Mono-alkylamino” means an Alkyl-NH- group, in which alkyl is as defined herein.
  • “Dialkylamino” means a (alkyl) 2 N- group, in which each alkyl may be the same or different and are each as defined herein for alkyl.
  • the alkyl group is preferably a Ci- C 6 alkyl group.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.
  • Alkyloxy refers to an alkyl-O- group in which alkyl is as defined herein.
  • the alkyloxy is a C1-C6 alkyloxy. Examples include, but are not limited to, methoxy and ethoxy.
  • the group may be a terminal group or a bridging group.
  • Alkyloxyalkyl refers to an alkyloxy-alkyl- group in which the alkyloxy and alkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • Alkyloxyaryl refers to an alkyloxy-aryl- group in which the alkyloxy and aryl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the aryl group.
  • the alkyl group is preferably a C1-C6 alkyl group. Examples include, but are not limited to, methoxycarbonyl and ethoxycarbonyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.
  • Alkyloxycycloalkyl refers to an alkyloxy-cycloalkyl- group in which the alkyloxy and cycloalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the cycloalkyl group.
  • Alkyloxyheteroaryl refers to an alkyloxy-heteroaryl- group in which the alkyloxy and heteroaryl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroaryl group.
  • Alkyloxyheterocycloalkyl refers to an alkyloxy-heterocycloalkyi- group in which the alkyloxy and heterocycloalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heterocycloalkyl group.
  • the alkyl group is preferably a CrC 6 alkyl group.
  • Exemplary alkylsulfinyl groups include, but not limited to, methylsulfinyl and ethylsulfinyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.
  • the alkyl group is preferably a C1-C6 alkyl group. Examples include, but not limited to methylsulfonyl and ethylsulfonyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.
  • Alkynyl as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl.
  • the group may be a terminal group or a bridging group.
  • Alkynyloxy refers to an alkynyl-O- group in which alkynyl is as defined herein. Preferred alkynyloxy groups are C1-C6 alkynyloxy groups.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Aminoalkyl means an NH 2 -alkyl- group in which the alkyl group is as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.
  • Aryl as a group or part of a group denotes (i) an optionally-substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring.
  • aryl groups include phenyl, naphthyl, and the like; (ii) an optionally-substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a Cs-7 cycloalkyl or Cs-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.
  • the group may be a terminal group or a bridging group.
  • an aryl group is a C6-C18 aryl group.
  • Arylalkenyl means an aryl-alkenyl- group in which the aryl and alkenyl are as defined herein.
  • exemplary arylalkenyl groups include phenylallyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.
  • Arylalkyl means an aryl-alkyl- group in which the aryl and alkyl moieties are as defined herein. Preferred arylalkyl groups contain a d-salkyl moiety. Exemplary arylalkyl groups include benzyl, phenethyl, 1 -naphthalenemethyl and 2- naphthalenemethyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • “Arylalkyloxy” refers to an aryl-alkyl-O- group in which the alkyl and aryl are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Arylamino includes both mono-arylamino and di-arylamino unless specified.
  • Mono-arylamino means a group of formula arylNH-, in which aryl is as defined herein.
  • Di-arylamino means a group of formula (aryl) 2 N- where each aryl may be the same or different and are each as defined herein for aryl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.
  • Arylheteroalkyl means an aryl-heteroalkyl- group in which the aryl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Aryloxy refers to an aryl-O- group in which the aryl is as defined herein.
  • the aryloxy is a Ce-Cisaryloxy, more preferably a C6-Cioaryloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.
  • a “bond” is a linkage between atoms in a compound or molecule.
  • the bond may be a single bond, a double bond, or a triple bond.
  • Cycloalkenyl means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 4-10 carbon atoms per ring.
  • Exemplary monocyclic cycloalkenyl rings include cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl.
  • the cycloalkenyl group may be substituted by one or more substituent groups.
  • a cycloalkenyl group typically is a C3-C12 alkenyl group. The group may be a terminal group or a bridging group.
  • Cycloalkyi refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified.
  • a cycloalkyi group typically is a C3-C12 alkyl group.
  • the group may be a terminal group or a bridging group.
  • Cycloalkylalkyl means a cycloalkyl-alkyl- group in which the cycloalkyi and alkyl moieties are as defined herein.
  • Exemplary monocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • Cycloalkylalkenyl means a cycloalkyl-alkenyl- group in which the cycloalkyi and alkenyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.
  • Cycloalkylheteroalkyl means a cycloalkyl-heteroalkyl- group in which the cycloalkyi and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Cycloalkyloxy refers to a cycloalkyl-O- group in which cycloalkyi is as defined herein.
  • the cycloalkyloxy is a Ci-C6cycloalkyloxy. Examples include, but are not limited to, cyclopropanoxy and cyclobutanoxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Cycloalkenyloxy refers to a cycloalkenyl-O- group in which the cycloalkenyl is as defined herein.
  • the cycloalkenyloxy is a Ci-C 6 cycloalkenyloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Haloalkyl refers to an alkyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine.
  • a haloalkyl group typically has the formula C n H(2n+i -m ) m wherein each X is independently selected from the group consisting of F, CI, Br and I. In groups of this type n is typically from 1 to 10, more preferably from 1 to 6, most preferably 1 to 3.
  • m is typically 1 to 6, more preferably 1 to 3.
  • Examples of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.
  • Haloalkenyl refers to an alkenyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom independently selected from the group consisting of F, CI, Br and I.
  • Haloalkynyl refers to an alkynyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom independently selected from the group consisting of F, CI, Br and I.
  • Halogen represents chlorine, fluorine, bromine or iodine.
  • Heteroalkyl refers to a straight- or branched-chain alkyl group preferably having from 2 to 12 carbons, more preferably 2 to 6 carbons in the chain, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced by a heteroatomic group selected from S, 0, P and NR' where R' is selected from the group consisting of H, optionally-substituted C1 -C12 alkyl, optionally- substituted C3-C12 cycloalkyl, optionally-substituted C6-C18 aryl, and optionally- substituted Ci-Ci 8 heteroaryl.
  • heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like.
  • heteroalkyl also include hydroxyCi-C6 alkyl, Ci-C6-alkyloxyCi-C6 alkyl, amino-Ci-C6 alkyl, C1 -C6- alkylamino C1 -C6 alkyl, and di(Ci-C6-alkyl)amino C1 -C6 alkyl.
  • the group may be a terminal group or a bridging group.
  • Heteroalkyloxy refers to a heteroalkyl-O- group in which heteroalkyl is as defined herein.
  • the heteroalkyloxy is a C2-C6 heteroalkyloxy.
  • the group may be a terminal group or a bridging group.
  • Heteroaryl either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur.
  • heteroaryl examples include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, pheno
  • Heteroarylalkyl means a heteroaryl-alkyl group in which the heteroaryl and alkyl moieties are as defined herein. Preferred heteroarylalkyl groups contain a lower alkyl moiety. Exemplary heteroarylalkyl groups include pyridylmethyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • Heteroarylalkenyl means a heteroaryl-alkenyl- group in which the heteroaryl and alkenyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.
  • Heteroarylheteroalkyl means a heteroaryl-heteroalkyl- group in which the heteroaryl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Heteroaryloxy refers to a heteroaryl-O- group in which the heteroaryl is as defined herein.
  • the heteroaryloxy is a Ci -Ci 8 heteroaryloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Heterocyclic refers to saturated, partially unsaturated or fully unsaturated monocyclic, bicyclic or polycyclic ring system containing at least one heteroatom selected from the group consisting of nitrogen, sulfur and oxygen as a ring atom.
  • heterocyclic moieties include heterocycloalkyi, heterocycloalkenyl and heteroaryl.
  • Heterocycloalkenyl refers to a heterocycloalkyi group as defined herein but containing at least one double bond.
  • a heterocycloalkenyl group typically is a C2-C12 heterocycloalkenyl group. The group may be a terminal group or a bridging group.
  • Heterocycloalkyi refers to a saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered.
  • heterocycloalkyi substituents examples include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1 ,3-diazapane, 1 ,4-diazapane, 1 ,4- oxazepane, and 1 ,4-oxathiapane.
  • a heterocycloalkyi group typically is a C2-C12 heterocycloalkyi group. The group may be a terminal group or a bridging group.
  • Heterocycloalkylalkyl refers to a heterocycloalkyl-alkyl- group in which the heterocycloalkyi and alkyl moieties are as defined herein.
  • exemplary heterocycloalkylalkyl groups include (2-tetrahydrofuryl)methyl,
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • "Heterocycloalkylalkenyl” refers to a heterocycloalkyl-alkenyl- group in which the heterocycloalkyl and alkenyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.
  • Heterocycloalkylheteroalkyl means a heterocycloalkyl-heteroalkyl- group in which the heterocycloalkyl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Heterocycloalkyloxy refers to a heterocycloalkyl-O- group in which the heterocycloalkyl is as defined herein.
  • the heterocycloalkyloxy is a C1-C6 heterocycloalkyloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Heterocycloalkenyloxy refers to a heterocycloalkenyl-O- group in which heterocycloalkenyl is as defined herein.
  • the heterocycloalkenyloxy is a Ci- C6 heterocycloalkenyloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.
  • Hydroalkyi refers to an alkyl group as defined herein in which one or more of the hydrogen atoms has been replaced with an OH group.
  • a hydroxyalkyi group typically has the formula C n H(2n+i-x ) (OH) x .
  • n is typically from 1 to 10, more preferably from 1 to 6, most preferably 1 to 3.
  • x is typically 1 to 6, more preferably 1 to 3.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.
  • “N-protecting group” means a group that can prevent the nitrogen atom reacting during further derivatisation of the protected compound and which can be readily removed when desired. Examples of N-protecting groups include alkyl amines, benzyl amines, t-Boc, Alloc, CBz and Fmoc. Further examples of these groups are found in: Greene, T. W. and Wuts, P. G.
  • O-protecting group means a group that can prevent the oxygen atom reacting during further derivatisation of the protected compound and which can be readily removed when desired.
  • O-protecting groups include silyl ethers (e.g. trimethylsilyl ether, tert-butyldimethylsilyl ether), acetyl, benzoyl, benzyl trityl. Further examples of these groups are found in: Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, Second edition; Wiley-lnterscience: 1991 ; Chapter 7; McOmie, J. F. W. (ed.), Protective Groups in Organic Chemistry, Plenum Press, 1973; and Kocienski, P. J., Protecting Groups, Second Edition, Theime Medical Pub., 2000.
  • the compounds of the invention include a linking moiety (L 1 ) which links the moiety containing the cyclooctyne to the second moiety (R 2 or X 5 -R 2 ).
  • L 1 is a linking group that serves to act as a spacer between the cyclooctyne moiety and the second moiety.
  • the linking group separates the cyclooctyne moiety and second moiety, which can individually react with a functionalised substrate and a functionalised molecule, tethering the substrate and molecule.
  • L 1 is a linking moiety having from 1 to 25 atoms in the normal chain. In some embodiments L 1 is a linking moiety having from 1 to 20 atoms in the normal chain. In some embodiments L 1 is a linking moiety having from 1 to 15 atoms in the normal chain. In some embodiments L 1 is a linking moiety having from 1 to 12 atoms in the normal chain. In some embodiments L 1 is a linking moiety having from 1 to 10 atoms in the normal chain. In some embodiments L 1 is a linking moiety having from 1 to 8 atoms in the normal chain. In some embodiments L 1 has 8 atoms in the normal chain. In some embodiments L 1 has 7 atoms in the normal chain.
  • L 1 has 6 atoms in the normal chain. In some embodiments L 1 has 5 atoms in the normal chain. In some embodiments L 1 has 4 atoms in the normal chain. In some embodiments L 1 has 3 atoms in the normal chain. In some embodiments L 1 has 2 atoms in the normal chain. In some embodiments L 1 has 1 atom in the normal chain.
  • a wide range of possible moieties may be used to create a linking moiety of this type.
  • moieties that may be used in the creation of L 1 include optionally-substituted C1-C12 alkyl, optionally-substituted C1 -C12 heteroalkyl, optionally-substituted C3-C12 cycloalkyl, optionally-substituted C6-C18 aryl, and optionally-substituted C1-C18 heteroaryl.
  • L 1 is of the Formula (II):
  • A is bonded to the cyclopropane group of Formula (I) and A is selected from the group consisting of: a bond and (CR 3 R 4 ) m ;
  • X 1 is selected from the group consisting of: a bond, 0, NH, NR 5 , S and CR 6 R 7 ;
  • X 2 is selected from the group consisting of: 0 and S;
  • L 2 is bonded to the R 2 group and is of the Formula X 3 (CR 8 R 9 ) p [X 4 (CR 10 R 1 1 ) q ] r (CR 12 R 13 ) S X 5
  • X 3 is selected from the group consisting of: a bond, 0, NH, NR 14 , S and
  • X 4 is selected from the group consisting of: a bond, 0, NH, NR 14 , S and CR 15 R 16 ;
  • X 5 is selected from the group consisting of: a bond, 0, NH, NR 14 , S, CR 15 R 16 , optionally-substituted alkyl, HN-(optionally-substitute alkyl)-, R 14 N- (optionally-substituted alkyl)-, 0-(optionally-substituted kyl)-, optionally- substituted C 5- 12 aryl, HN-(optionally-substituted C 5- i 2 aryl)-, R 14 N-(optionally- substituted C 5- i 2 aryl)-, 0-(optionally-substituted C 5- i 2 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)-, R 14 N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)- (optionally-substituted 05-12 aryl)-, (CO)NH-(optionally
  • each R 3 , R 4 , R 6 , R 7 , R 8 R 9 R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: H, OH, halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C 2- 12 alkenyl, optionally-substituted C 2- 12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally- substituted OC 3- i2 cycloalkenyl;
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8
  • m is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6.
  • each R is independently selected from the group consisting of: H, halogen, OH, NO 2 , CN, SH, NH 2 , CF 3 , OCHF 2 , OCF 3 , optionally-substituted C1-12 alkyl, optionally-substituted haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 2- 12 heteroalkyl, optionally-substituted C 3- 12 cycloalkyl, optionally-substituted C 3- 12 cycloalkenyl, optionally-substituted 02-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-M S heteroaryl, optionally-substituted Ci-12 alkyloxy, optionally-substituted C 2- 12
  • A is selected from the group consisting of: a bond and (CR 3 R 4 ) m ;
  • X 1 is selected from the group consisting of: a bond, O, N H, NR 5 , S and CR 6 R 7 ;
  • X 2 is selected from the group consisting of: O and S;
  • L 2 is of the Formula (III): X 3 (CR 8 R 9 ) p [X 4 (CR 1 0 R 1 1 ) q ] r (CR 12 R 1 3 ) S X 5
  • R 2 is selected from the group consisting of: optionally substituted C-M2 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C 3- i 2 cycloalkyl, optionally substituted C 3- i 2 cycloalkenyl, NH 2 , ONH 2 , NHNH 2 , NHNR a R p , NH(CO) (optionally-substituted C 1 -6 alkyl)ONH 2 , NH(CO) (optionally-substituted Ci -6 alkyl)NH 2 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNH 2 , NH(CO) (optionally-substituted C 1 -6 alkyl)NHNR a R p , (CO)NH (optionally-substituted Ci -6 alkyl)ONH 2 , (CO)NH (optionally-substituted
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C 3- 12 cycloalkyl, optionally-substituted C 3- 12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC 2- i2 alkynyl, optionally-substituted OC 3- i 2 cycloalkyl and optionally-substituted OC 3- i 2 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a):
  • each of R x and R 5 is independently selected from the group consisting of H, optionally-substituted alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;
  • X 3 is selected from the group consisting of: a bond, O, NH, NR 14 , S and
  • X 4 is selected from the group consisting of: a bond, O, NH, NR 14 , S and CR 15 R 16 ;
  • X 5 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 , optionally-substituted alkyl, HN-(optionally-substituted Ci-12 alkyl)-, R 14 N- (optionally-substituted Ci-i 2 alkyl)-, O-(optionally-substituted Ci-i 2 alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted Cs-12 aryl)-, R 14 N-(optionally- substituted Cs-12 aryl)-, O-(optionally-substituted Cs-12 aryl)-, HN(CO)-(optionally- substituted C 5- i 2 aryl)-, R 14 N(CO)-(optionally-substituted C 5- i 2 aryl)-, O(CO)- (optionally-substituted C 5- i 2 ary
  • each R 5 and R 14 is independently selected from the group consisting of: optionally-substituted Ci-12 alkyl and N-protecting group;
  • each R 3 , R 4 , R 6 , R 7 , R 8 R 9 R 10 , R 1 1 , R 12 , R 1 3 , R 15 and R 16 is independently selected from the group consisting of: H, OH, halogen, optionally-substituted Ci-i 2 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted 0C 2- 12 alkynyl, optionally-substituted OC 3- i 2 cycloalkyl and optionally- substituted OC3-12 cycloalkenyl;
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; m is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; and each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6.
  • the present invention includes mixtures of compounds wherein the value of p may vary within the mixture of compounds. In some embodiments the present invention includes mixtures of compounds wherein the value of q may vary within the mixture of compounds. In some embodiments the present invention includes mixtures of compounds wherein the value of r may vary within the mixture of compounds. In some embodiments the present invention includes mixtures of compounds wherein the value of s may vary within the mixture of compounds.
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-a):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-c):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-d):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-e):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-f):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-h):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-i):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-l):
  • A, X 1 , X 2 and L 2 are chosen and combined such that L 1 is of Formula (X-n):
  • n is 0.
  • R a and R b are independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted alkyl, and optionally-substituted OCi-12 alkyl.
  • R a and R b are independently selected from the group consisting of: hydrogen, OH, alkyl, and OC-i-12 alkyl.
  • R a and R b are hydrogen.
  • A is a bond. In some embodiments A is (CR 3 R 4 ) m . In some embodiments m is 1 , in some embodiments m is 2, in some embodiments m is 3, in some embodiments m is 4, in some embodiments m is 5, in some embodiments m is 6. In some embodiments R 3 and R 4 are each H.
  • A is CH 2 .
  • A is CH 2 and X 1 is O. This provides a compound of Formula (ll-c):
  • A is CH 2 and X 1 is O and X 2 is O.
  • X 3 is O or NH.
  • A is CH 2 and X 1 is 0 and X 2 is 0 and X 3 is NH such that L 2 has the Formula (lll-b): NH(CR 8 R 9 ) p [X 4 (CR 10 R 11 ) q ] r (CR 12 R 3 ) S X 5
  • R 8 , R 9 , R 12 , and R 13 are H. In some embodiments R 10 and R 11 are H.
  • p is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments p is 0, in some embodiments p is 1 , in some embodiments p is 2, in some embodiments p is 3, in some embodiments p is 4, in some embodiments p is 5, in some embodiments p is 6.
  • q is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments q is 0, in some embodiments q is 1 , in some embodiments q is 2, in some embodiments q is 3, in some embodiments q is 4, in some embodiments q is 5, in some embodiments q is 6. In some embodiments r is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments r is 0, in some embodiments r is 1 , in some embodiments r is 2, in some embodiments r is 3, in some embodiments r is 4, in some embodiments r is 5, in some embodiments r is 6.
  • s is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments s is 0, in some embodiments s is 1 , in some embodiments s is 2, in some embodiments s is 3, in some embodiments s is 4, in some embodiments s is 5, in some embodiments s is 6.
  • X 4 is O. In some embodiments X 4 is NH.
  • X 5 is 0.
  • X 5 is NH
  • X 5 is optionally-substituted Ci--i 2 alkyl.
  • X 5 is HN- (optionally-substituted Ci--i 2 alkyl)-.
  • X 5 is R 14 N-(optionally- substituted Ci-12 alkyl)-.
  • X 5 is 0-(optionally-substituted Ci-12 alkyl)-.
  • X 5 is HN(CO)-(optionally-substituted C 5- i 2 aryl). In some embodiments X 5 is HN(CO)-(optionally-substituted C2-12 heteroaryl)-.
  • each R 1 is independently selected from the group consisting of H, OH, C 1-6 alkyl and OC 1-6 Alkyl.
  • R 2 is
  • the compound is of the Formula (IV):
  • each R a and R b is independently selected from the group consisting of: H, OH, halogen, optionally-substituted alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally- substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally- substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl;
  • each R 1 is independently selected from the group consisting of: H, halogen,
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8;
  • each R C , R D and R E is independently selected from the group consisting of: H, OH, halogen, optionally-substituted Ci-i 2 alkyl, optionally-substituted C 2- 12 heteroalkyl, optionally-substituted C3- 12 cycloalkyl, optionally-substituted OC6-18 aryl, and optionally-substituted OCM S heteroaryl.
  • R 2A is selected from the group consisting of: OH, optionally-substituted OCi- 12 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3- 12 cycloalkyl and optionally-substituted OC3- 12 cycloalkenyl.
  • L 1 is of the Formula (II):
  • A is bonded to the cyclopropane group of Formula (I) and A is selected from the group consisting of: a bond and (CR 3 R 4 ) m ;
  • X 1 is selected from the group consisting of: a bond, O, NH, NR 5 , S and CR 6 R 7 ;
  • X 2 is selected from the group consisting of: O and S;
  • L 2 is bonded to the squarate group and is of the Formula (III): X 3 (CR 8 R 9 ) p [X 4 (CR 10 R 1 1 ) q ] r (CR 12 R 13 ) S X 5
  • each formula includes compounds having the indicated structure, including the hydrated as well as the non- hydrated forms.
  • the compound may find a multiple number of applications in which the compound can be used to link nanoparticles to biological molecules.
  • a quantum dot QD
  • QD quantum dot
  • the compounds of the present invention possess a cyclooctyne functional group at one end and another functional group at the other end, which is capable of reacting with an amine, aldehyde or ketone group.
  • functional groups that are capable of reacting with an aldehyde or ketone groups may include amine, aminooxy, hydrazide, semicarbazide and carbohydrazide functional groups.
  • Examples of functional groups that are capable of reacting with an amine may include a squarate. These functional groups provide means to link the functionalised QD and the functionalised biological molecule by reacting the functional groups of the compound with the functionalised QD and/or functionalised biological molecule.
  • the invention relates to a process of linking a quantum dot (QD) to a biological molecule, the process comprising the step: i) reacting an end of the compound as previously described, with either a functionalised QD or a functionalised biological molecule, linking either the functionalised QD or the functionalised biological molecule to a first end of the compound; and step ii) reacting the remaining end group from the product of step i), with either one of the functionalised QD or the functionalised biological molecule which was not used in step i) to provide a QD linked to a biological molecule.
  • QD quantum dot
  • step i) comprises reacting the end of the compound which contains the alkyne group, with a functionalised QD, linking the QD to a first end of the compound; and step ii) comprises reacting the end of the product from step i) which does not contain an alkyne group with a functionalised biological molecule, linking the biological molecule to a second end.
  • step i) comprises reacting the end of the compound which does not contain the alkyne functional group, with a functionalised biological molecule, linking the biological molecule to a first end of the compound; and step ii) comprises reacting the end of the product from step i) which contains the alkyne group with a functionalised QD, linking the QD to a second end.
  • the functionalised QD is an azide-functionalised QD.
  • the functionalised biological molecule is an amine-, aldehyde- or ketone-functionalised biological molecule.
  • a QD can be linked to the biological molecule by reacting a functionalised QD with one end of the compound. This results in a QD which is linked to the compound at one end.
  • the product can be reacted with a functionalised biological molecule, linking the biological molecule to the opposite end of the compound.
  • the QD can be linked to the biological molecule by reacting a functionalised biological molecule with one end of the compound. This results in a biological molecule which is linked to the compound at one end.
  • the product can be reacted with a functionalised QD, linking the QD to the opposite end of the compound.
  • the cyclooctyne moiety can react with an either an azide-functionalised QD or an azide-functionalised biological molecule.
  • the cyclooctyne moiety will react with the azide-functionalised QD or azide-functionalised biological molecule via a [3+2] cycloaddition reaction.
  • the amine, aminooxy, hydrazide, semicarbazide or carbohydrazide moiety of the compound can react with an aldehyde- or ketone- functionalised QD or an aldehyde- or ketone-functionalised biological molecule.
  • a squarate moiety of the compound can react with an amine-functionalised QD or an amine-functionalised biological molecule.
  • the compound therefore, acts as a tether, covalently linking the QD to the biological molecule.
  • the cyclooctyne moiety reacts with an azide-functionalised QD and in some embodiments the squarate, amine, aminooxy, hydrazide, semicarbazide or carbohydrazide moiety reacts with an amine-, aldehyde- or ketone-functionalised biological molecule.
  • Scheme 5 Another example is illustrated below in Scheme 5.
  • the embodiment shown in Scheme 5 the cyclooctyne moiety reacts with an azide-functionalised biological molecule and the squarate moiety reacts with an amino-functionalised quantum dot.
  • the compounds of Formula (V) as used in the process of the present invention may be synthesized using a number of synthetic routes.
  • the invention provides a process for the preparation of a compound of Formula (V).
  • the process comprises coupling the compound of Formula (VI):
  • LG is a leaving group, R 1 and n are as defined above, in a coupling reaction with a compound of Formula (Vll-a):
  • X 3a is selected from the group consisting of: OH, NH 2 and NHR 14 ;
  • X 4 is independently selected from the group consisting of: a bond, O, NH, NR 14 , S and CR 15 R 16 ;
  • X 5a is selected from the group consisting of: OH, NH 2 , NHR 14 , OR 18 CO 2 R 19 ; each R 8 R 9 , R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci--i 2 alkyl, optionally-substituted C 2- i 2 alkenyl, optionally-substituted C 2- i 2 alkynyl, optionally- substituted C 3- i 2 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally- substituted OCi-i 2 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3-i 2 cycloalkyl and optionally-substit
  • R 14 is independently selected from the group consisting of: optionally- substituted C1-12 alkyl and N-protecting group;
  • R 18 is selected from the group consisting of: optionally-substituted C1-12 alkyl, optionally-substituted C 2- i 2 alkenyl, optionally-substituted C 2- i 2 alkynyl, optionally- substituted C 3- i 2 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl and O-protecting group;
  • R 19 is selected from the group consisting of: H, optionally-substituted C ⁇ . ⁇ 2 alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl and COOH-protecting group; and
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6;
  • the compound of Formula (VI) is typically reacted, in the presence of a base, with the compound of Formula (Vll-a).
  • the solvent is chosen so as not to be reactive with the base.
  • suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents. Specific examples of suitable solvents include dichloromethane (CH2CI2), chloroform (CCI3), ethanol, methanol, DMSO and DMF. In general a base is used. In some instances the amine of Formula (Vll-a) may also be used as a base.
  • suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides.
  • suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine. In some instances the base may also act as a solvent.
  • the amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents
  • the reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C.
  • the reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR.
  • R 2 and R 20 is selected independently from the group consisting of: OH, CO2R 22 , optionally-substituted OC1-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl, optionally-substituted OC3-12 cycloalkenyl, NHR 17 , NHNHR 17 , NHNR a R p , ONHR 17
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C 3- 12 cycloalkenyl, optionally-substituted OCi-i 2 alkyl, optionally-substituted OC 2- i2 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a):
  • each of R x and R 5 is independently selected from the group consisting of H, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C 3- 12 cycloalkenyl;
  • each R 17 and R 21 is independently selected from the group consisting of: H, optionally-substituted Ci-12 alkyl and N-protecting group;
  • R is selected from the group consisting of: H, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted C 2- 12 alkenyl, optionally-substituted C 2- 12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted C2-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted Ce-18 alkylaryl, optionally-substituted heteroaryl, optionally-substituted Ci-i 8 alkylheteroaryl to provide a compound of Formula (V):
  • each R 1 is independently selected from the group consisting of: H, halogen, OH, NO 2 , CN, SH, NH 2 , CF 3 , OCHF 2 , OCF 3 , optionally-substituted C1-12 alkyl, optionally-substituted Ci--i 2 haloalkyl, optionally-substituted C 2- i 2 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 2- 12 heteroalkyl, optionally-substituted C3-i 2 cycloalkyl, optionally-substituted C3-i 2 cycloalkenyl, optionally-substituted C 2- i 2 heterocycloalkyl, optionally-substituted C 2- i 2 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-MS heteroaryl, optionally-substituted
  • R c , R d and R e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted Ci_i 2 alkyl, optionally-substituted C 2- i 2 heteroalkyl, optionally-substituted C3-i 2 cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OCM S heteroaryl;
  • stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo;
  • X 4 is independently selected from the group consisting of: a bond, O, NH, NR 14 , S and CR 15 R 16 ;
  • X 5 is selected from the group consisting of: O, NH, NR 14 , optionally-substituted C-i-12 alkyl, HN-(optionally-substituted C-i-12 alkyl)-, R 14 N-(optionally-substituted C-i-12 alkyl)- O-(optionally-substituted Ci-i 2 alkyl)-, optionally-substituted C 5- i 2 aryl, HN- (optionally-substituted Cs-12 aryl)-, R 14 N-(optionally-substituted Cs-12 aryl)-, O- (optionally-substituted Cs-12 aryl)-, HN(CO)-(optionally-substituted Cs-12 aryl)-, R 14 N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)-(optionally-substituted C5-12 aryl)-
  • R 2 is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR 17 , ONHR 17 , NHNHR 17 , NHNR a R p , NH(CO)(optionally-substituted Ci_ 6 alkyl)ONHR 17 , NH(CO)(optionally-substituted Ci_ 6 alkyl)NHR 17 , NH(CO)(optionally-substituted Ci_ 6 alkyl)NHNHR 17 , NH(CO)(optionally-substituted Ci_ 6 alkyl)NHNR a R p , (CO)NH(optionally-substituted Ci -6 alkyl)ONHR 17 , (CO)NH(optionally-substituted Ci -6 alkyl
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted C-i-12 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a):
  • each of R x and R 5 is independently selected from the group consisting of H, optionally-substituted alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl; each R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally
  • each R 14 and R 17 is independently selected from the group consisting of: H, optionally-substituted alkyl and N-protecting group;
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8;
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6.
  • the compound of Formula (VIII) is reacted with a compound of Formula (IX) in the presence of a base.
  • the solvent is chosen so as not to be reactive with the base.
  • suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents.
  • Specific examples of suitable solvents include dichloromethane (CH2CI2), chloroform (CCI3), ethanol, methanol, DMSO and DMF.
  • a base is used.
  • suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides.
  • suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine.
  • the base may also act as a solvent.
  • the amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used.
  • the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents
  • the reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C.
  • the reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR. In some embodiments of the invention the compound of Formula (IX) is of Formula (IX)
  • R 17 is selected from the group consisting of: optionally-substituted Ci-i 2 alkyl and N-protecting group.
  • the compound of Formula (IX) is of Formula (IX-b):
  • R 17 is selected from the group consisting of: optionally-substituted alkyl and N-protecting group.
  • the compound of Formula (IX) is of Formula (IX-c):
  • R 17 is selected from the group consisting of: optionally-substituted Ci-i 2 alkyl and N-protecting group.
  • the compound of Formula (IX) is of Formi. (IX-d):
  • R 2a is selected from the group consisting of: OH, optionally-substituted OCi- 12 alkyl, optionally-substituted OC 2- i2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3- 12 cycloalkyl and optionally-substituted OC3- 12 cycloalkenyl.
  • the compound is of Formula (IX-d), wherein R 2a is OMe. In some embodiments of the invention the compound is of Formula (IX- d), wherein R 2a is OMEt. In some embodiments of the invention the compound of Formula (IX) is of Formula (IX-e):
  • the compound produced in step b) is further N-deprotected or N-dealkylated.
  • the compound of Formula (IX) is hydrazine (NH 2 NH 2 ).
  • the compound of Formula (Vll-a) is 4,7,10-trioxa- 1 , 13-tridecanediamine of Formula (Vll-c):
  • the compound of Formula (Vll-a) is the compound of Formula (Vll-d):
  • the compound of Formula (Vll-a) is the compound of Formula (Vll-e):
  • the compound of Formula (VI) is selected from: wherein LG is a leaving group, selected independently from the group consisting of:
  • the compound of Formula (VI) is selected from:
  • the solvent is chosen so as not to be reactive with the base.
  • suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents.
  • suitable solvents include dichloromethane (CH 2 CI 2 ), chloroform (CCI 3 ), ethanol, methanol, DMSO and DMF.
  • the reaction of 4-nitrophenyl chloroformate can be conducted in the presence of a base.
  • suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides.
  • suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine.
  • the base may also act as a solvent.
  • the amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents
  • the reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However, the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C.
  • the reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR.
  • a process for the preparation of a compound of the present invention, endo-5 may include the process illustrated in Scheme 6:
  • the invention provides another process for the preparation of a compound of Formula (V).
  • the process comprises coupling a compound of Formula (Vll-b):
  • X 3a is selected from the group consisting of: OH, NH 2 and NHR 14 ;
  • X 4 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 ;
  • X 5 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 , optionally-substituted alkyl, HN-(optionally-substituted Ci-12 alkyl)-, R 14 N- (optionally-substituted Ci-i 2 alkyl)-, O-(optionally-substituted Ci-i 2 alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted Cs-12 aryl)-, R 14 N-(optionally- substituted C 5- i 2 aryl)-, O-(optionally-substituted C 5- i 2 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)- R 14 N(CO)-(optionally-substituted C5-12 aryl)- O(CO)- (optionally-substituted Cs-12 aryl)-,
  • R 2 is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C 2- i 2 alkenyl, optionally substituted C 2- i 2 alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR 17 , ONHR 17 , NHNHR 17 , NHNR a R p , NH(CO)(optionally-substituted Ci -6 alkyl)ONHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNHR 17 , NH(CO)(optionally-substituted Ci -6 alkyl)NHNR a R p , (CO)NH(optionally-substituted Ci -6 alkyl)ONHR 17 , (CO)NH(optionally-substituted Ci
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted C1- 2 alkyl, optionally-substituted C 2- i 2 alkenyl, optionally- substituted C 2- i 2 alkynyl, optionally-substituted C3-i 2 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally-substituted OCi_i 2 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3-i 2 cycloalkyl and optionally-substituted OC3-i 2 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a): wherein each of R x and R 5 is independently selected from the group consisting
  • each R 8 R 9 , R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C 2- 12 alkenyl, optionally-substituted C 2- 12 alkynyl, optionally- substituted C3-i 2 cycloalkyl, optionally-substituted C3-i 2 cycloalkenyl, optionally- substituted OCi-i 2 alkyl, optionally-substituted OC 2- i 2 alkenyl, optionally-substituted OC 2- i 2 alkynyl, optionally-substituted OC3-i 2 cycloalkyl and optionally-substituted OC3- 2 cycloalkenyl;
  • each R 14 and R 17 is independently selected from the group consisting of: H, optionally-substituted Ci-i 2 alkyl and N-protecting group; and
  • each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, and in a coupling reaction with a compound of Formula of Formula (VI):
  • R 1 and n are as defined above and LG is a leaving group;
  • R 1 is selected from the group consisting of: H, halogen, OH, NO 2 , CN, SH, NH 2 , CF 3 , OCHF 2 , OCF3, optionally-substituted C1-12 alkyl, optionally-substituted Ci-12 haloalkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted 03-12 cycloalkyl, optionally-substituted C 3- i 2 cycloalkenyl, optionally-substituted C 2- 12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted CM S heteroaryl, optionally-substituted Ci-12 alkyloxy, optionally- substituted C2-12 alky
  • R c , R d and R e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted C1-12 alkyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OC1-18 heteroaryl;
  • L is of the Formula (III): X 3 (CR 8 R 9 )p [X 4 (CR 10 R 1 1 ) q ] r (CR 12 R 13 )s X 5
  • X 3 is selected from the group consisting of: O, NH and NR 14 ;
  • X 5 is selected from the group consisting of: a bond, O, NH, NR 14 , S, CR 15 R 16 , optionally-substituted C1-12 alkyl, HN-(optionally-substituted C1-12 alkyl)-, R 14 N- (optionally-substituted C1-12 alkyl)-, O-(optionally-substituted C1-12 alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted C5-12 aryl)-, R 14 N-(optionally- substituted C5-12 aryl)-, O-(optionally-substituted C5-12 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)-, R 14 N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)- (optionally-substituted C5-12 aryl)-,
  • each R a and R p is independently selected from the group consisting of: H, OH, optionally-substituted C-i-i2 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R a and R p when combined together provide the group of Formula (l-a):
  • each of R x and R 5 is independently selected from the group consisting of H, optionally-substituted C-i-i2 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C 2- 12 alkynyl, optionally-substituted C 3- i 2 cycloalkyl, optionally-substituted C3-12 cycloalkenyl; each R 3 , R 4 , R 6 , R 7 , R 8 R 9 R 10 , R 11 , R 12 , R 13 , R 15 and R 16 is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-
  • each R 14 and R 17 is independently selected from the group consisting of: H, optionally-substituted Ci-i 2 alkyl and N-protecting group; and
  • n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6.
  • the compound of Formula (VI) is typically reacted with the compound of Formula (VII- a) or Formula (Vll-b) in the presence of a base.
  • the solvent is chosen so as not to be reactive with the base.
  • suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents.
  • suitable solvents include dichloromethane (CH 2 CI 2 ), chloroform (CCI 3 ), ethanol, methanol, DMSO and DMF.
  • a base is used.
  • the compound of Formula (Vll-a) or Formula (Vll-b) may also be used as a base.
  • suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides.
  • suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine.
  • the base may also act as a solvent.
  • the amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents
  • the reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C.
  • the reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR.
  • the product can then be optionally deprotected or dealkylated to provide the product of Formula (V).
  • a process for the preparation of a compound of the present invention, endo-11 may include the process illustrated in Scheme 10:
  • the agents of the various embodiments may be prepared using the reaction routes and synthesis schemes as described below, employing the techniques available in the art using starting materials that are readily available.
  • the preparation of particular compounds of the embodiments is described in detail in the following examples, but the artisan will recognize that the chemical reactions described may be readily adapted to prepare a number of other agents of the various embodiments.
  • the synthesis of non-exemplified compounds may be successfully performed by modifications apparent to those skilled in the art, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions.
  • a list of suitable protecting groups in organic synthesis can be found in T.W.
  • UV-vis spectra were recorded in water with a Shimadzu UV-1650PC UV-vis spectrophotometer using the UVPC c3.9 software program.
  • Rabbit polyclonal antibodies to human EEA1 were purchased from Cell Signalling Technology (USA).
  • Mouse monoclonal anti-human CD63 was from Santa Cruz Biotechnology (USA).
  • Alexa Fluor 568-conjugated human transferrin, goat anti-rabbit IgG Alexa Fluor 568 and goat anti-mouse IgG Alexa Fluor 647 were purchased from Life Technologies (USA).
  • exo-12 and endo-12 are known in the literature. As such the exo-12 and endo-12 compounds can be synthesised as described by Dommerholt J. et al. (see Scheme 12 which depicts synthesis of the exo-isomer). Alternative methodologies to provide either exo-12 or endo-12 can also be employed.
  • 6-Hydrazinonicotinic Acid Acetone Hydrazone 16 was synthesised following published procedures and activated as the NHS-ester 17 (Bioconjugate Chem, 20, 10, 2009, 1950).
  • CdSe/ZnS core/shell quantum dots were synthesized using high-temperature reaction of organometallic precursors according to previously reported methods (J Van Embden, P Mulvaney, Langmuir 2005, 21 , 10226).
  • the quality of the nanocrystals was assessed by optical analysis: absorption spectra were recorded on a Cary 5 UV- Vis-NIR spectrophotometer. Steady-state photoluminescence spectra were measured on a Horiba Jobin Yvon Fluorolog-3 spectrofluorometer, with slit widths of 1 nm and integration times of 0.1 -0.5 s.
  • the Gaussian full-width-at-half maximum (FWHM) was found to range between 25 and 33 nm, depending on the emission peak position of the QDs.
  • Water-soluble QDs were generated following the polymer encapsulation technique described in Lees et al (E lees, T Nguyen, A Clayton, P Mulvaney, ACS Nano 2009, 3, 1 121 ) Briefly, 25-50 nmol of freshly synthesized QDs in ODE were placed in trioctyl phosphine/trioctyl phosphine oxide (TOP/TOPO 20 times excess with respect to the total number of surface atoms) overnight at 60°C to assure uniform coating. The TOP/TOPO-coated QDs were then washed free of excess ligand using a mixture of chloroform and methanol and precipitated with acetone.
  • TOP/TOPO 20 trioctyl phosphine/trioctyl phosphine oxide
  • polystyrene-co-maleic anhydride polymer PSMA, Mn 1600, 200 mM in CHCI3
  • PSMA polystyrene-co-maleic anhydride polymer
  • the reaction mixture was then stirred at rt for 3 h to allow the formation of the PSMA shell.
  • a 5- fold molar excess of Jeffamine M1000 or amino-PEG-azide H2N-PEG-N3 (200 mM in CHCI3) was added and the sample was left to stir at room temperature overnight.
  • QDs functionalized with different amounts of azide groups on their surface were obtained by varying the ratio of Jeffamine M1000 and H2N-PEG-N3 used.
  • QD10 were obtained using 10% H2N-PEG-N3 mixed with 90% M1000 (v/v).
  • water (2-3 ml_) containing ethanolamine (20 ⁇ _) was added to ring-open remaining maleic anhydride groups.
  • the CHCI3 was removed using a rotary evaporator.
  • Additional CHCI3 (2-3 ml_) was added to the resulting water solution of QDs to extract any hydrophobic ligands.
  • the sample was then centrifuged for 5 min at 6000 rpm and the QD water solution was separated and passed through a 0.22 ⁇ filter.
  • the filtrate was concentrated using a 50 kDa MWCO Vivaspin filter unit (Sartorius Stedim Biotech). Quantum Yield Measurements
  • the quantum yield (QY) for the QDs was measured relative to the dye Rhodamine 6G (QY 95% in EtOH) with excitation at 400 nm. Fluorescence spectra of QDs and dye were measured under identical conditions and care was taken that the optical density of each sample did not exceed 0.1 at the excitation wavelength.
  • the QY of QDs in chloroform was typically approximately 40% whereas for the samples in water after polymer encapsulation the QY was measured to be 16-26%.
  • linker (L) of example 3 was performed in 0.5 M borate buffer at pH 9.
  • Stock solutions of linker in DMSO (4 mM) and Fe 2 Tf in MilliQ deionized water (0.6 mM) were prepared and both were added to the borate buffer at a final concentration of 0.2-0.8 mM, depending on the ratio of linker to Fe 2 Tf required.
  • the resulting mixture was allowed to react for 6 h at RT and analyzed by ESI-TOF mass spectroscopy (Agilent 6220 Accurate-Mass TOF LC/MS fitted with a C4 desalting column).
  • the reaction between dye (ALEXA594) and linker (L) of example 3 was carried out as described in example 10 at a 2: 1 final concentration of linker (0.8 mM) and dye Alexa Fluor 594 cadaverine (Invitrogen) (0.4 mM).
  • the resulting L-A594 conjugate was reacted with QDs functionalized with different percentages of azide groups (QD0, QD1 , QD10, QD100).
  • the reaction mixture containing the L-A594 conjugate in a 50- fold molar excess was added to QDs in water, except for the sample QD100 for which the excess was 100-fold. The mixture was allowed to react for 6 h at rt.
  • the L-Fe 2 Tf conjugate was prepared as outlined in example 10 at a ratio 2:1 linker to protein and final concentration of 0.2 mM of Fe2Tf. The mixture was then added to QDs functionalized with different percentages of azide groups in a 10-fold molar excess and allowed to react for 6 h at RT.
  • HeLa cells were seeded onto glass coverslips at 0.6 ⁇ 10 5 cells/ml and grown for 48 hr. Cells were then serum starved for 3 h followed by 10 min incubation on ice to stop endocytosis. Chilled cells were then incubated with pre-chilled 100 ⁇ / ⁇ ⁇ (A) Alexa Fluor 568-conjugated human transferrin (Life Technologies, USA), (C) QDs, or (B) QD100-Fe2Tf (diluted in serum-free DMEM) on ice for 30 min. Unbound transferrin was removed using cold PBS washes and internalisation of bound conjugated transferrin performed for the indicated duration at 37 °C in serum-free DMEM.
  • Cells were fixed in 4% (w/v) paraformaldehyde for 15 min and then incubated with 50 mM NH 4 CI/PBS for 10 min to quench free aldehydes. Cells were washed in PBS, permeabilized in 0.1 % Triton X-100/PBS for 4 min and again washed in PBS. Blocking was achieved by incubating cells in 5% (v/v) FCS/PBS at room temperature for 30 min. Cells were incubated in primary antibodies diluted in 5% (v/v) FCS/PBS for 30 min at room temperature. Excess antibodies were removed by PBS washes.
  • Fluorochrome-conjugated secondary antibodies were added and incubated with cells for 30 min at room temperature followed by further PBS washes. Nuclei of cells were stained with 4,6-diamino-2-phenylindole (DAPI; Sigma-Aldrich, USA) where indicated and cells were washed as before. Cells were mounted in Mowiol (10% (w/v) Hopval 5- 88 (Hoechst, Australia), 25% (w/v) glycerol, 0.1 M Tris). Images of cells were captured using a Leica TCS SP2 laser confocal unit and Leica Confocal Software version 2.61 . For multi-colour labelling, images were collected independently.
  • FIG. 13 shows Fe 2 -transferrin uptake into HeLa cells performed using (A1 -A3) A568-Fe 2 Tf, (B1 -B3) QD100-Fe 2 Tf and (C1 -C3) QD100 for the indicated times at 37°C.
  • HERCEPTIN is a recombinant DNA-derived humanized monoclonal antibody that selectively targets the extracellular domain of the human epidermal growth factor receptor 2 protein (HER2).
  • the antibody is an lgG1 kappa that contains human framework regions with the complementarity-determining regions of a murine anti-p185 HER2 antibody that binds to HER2.
  • Trastuzumab is composed of 1 ,328 amino acids and has a molecular weight of ⁇ 148 kDa.
  • Figure 15 shows the MS of Herceptin showing variations in the number of bound hexoses.
  • the conjugation buffer was Na 3 P0 4 (100 mM), NaCI (150 mM) at pH6.
  • the catalyst buffer was a solution of aniline (10 mM).
  • Azide Fluor 585 dye was purchased from Jena Bioscience.
  • trastuzumab-ox (1 15 ⁇ _) was added to aniline buffer (10 ⁇ _) followed by the linker of example 8 (5.4 ⁇ _ of 0.5 mM solution in DMF) and the mixture was incubated at room temperature for 1 hour. To this mixture was added Azide Fluor 585 dye (1 .5 ⁇ _ of 3.4 mM solution) and the mixture was left at room temperature overnight. The mixture was purified by spin filtration (Amicon Ultra 10 kDa MWCO) until no more dye was visible in the filtrate. The conjugate was resuspended in buffer and the amount of dye conjugated to the trastuzumab-ox was measured by electronic spectroscopy
  • the conjugation was repeated with 10:1 and 100:1 ratios of linker to antibody.

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Abstract

La présente invention concerne des composés qui peuvent être utilisés pour lier des substrats à des molécules, généralement dans le cadre d'applications biologiques telles que l'imagerie et analogue(s). Les composés contiennent un cyclooctyne qui est lié, par un groupe de liaison, à un fragment de type squarate, amine, aminooxy, hydrazide, semi-carbazide ou carbohydrazide. Le fragment cyclooctyne sert de partenaire de couplage à un substrat ou à une molécule à fonctionnalité azide, alors que l'autre fragment sert de partenaire de couplage à une molécule ou à un substrat à fonctionnalité amine, aldéhyde ou cétone. L'invention concerne ainsi également des procédés de synthèse desdits composés et une nouvelle utilisation des composés pour lier un substrat à une molécule.
PCT/AU2013/000591 2012-06-05 2013-06-05 Composés de bicyclo[6.1.0]non-4-yne conçus pour être utilisés comme lieurs dans des applications biologiques WO2013181697A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
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CN104744293A (zh) * 2013-12-31 2015-07-01 深圳先进技术研究院 胆碱类似物及其制备方法和应用、由染料分子标记的四嗪探针及其制备方法和应用
WO2017001374A1 (fr) * 2015-06-30 2017-01-05 Imec Vzw Immobilisation en surface d'une molécule de reconnaissance d'analyte
KR20170008257A (ko) * 2014-12-19 2017-01-23 보에 테크놀로지 그룹 컴퍼니 리미티드 개질된 양자점 및 그의 제조 방법, 착색제, 감광성 수지 조성물, 컬러 필터 및 디스플레이 디바이스
WO2017013004A1 (fr) * 2015-07-17 2017-01-26 Orphidia Limited Molécule de liaison destinée à traiter une surface de substrat
CZ307452B6 (cs) * 2016-11-03 2018-09-05 Vysoká škola chemicko-technologická v Praze Aminooxylipidy pro konstrukci samoskladných liposomálních systémů umožňujících jejich následnou modifikaci biologicky funkčními molekulami
US10758623B2 (en) 2013-12-09 2020-09-01 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same
JP2022126716A (ja) * 2018-06-14 2022-08-30 持田製薬株式会社 新規な架橋アルギン酸

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US11529420B2 (en) 2013-12-09 2022-12-20 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same
US10758623B2 (en) 2013-12-09 2020-09-01 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same
CN104744293A (zh) * 2013-12-31 2015-07-01 深圳先进技术研究院 胆碱类似物及其制备方法和应用、由染料分子标记的四嗪探针及其制备方法和应用
KR101886948B1 (ko) 2014-12-19 2018-08-08 보에 테크놀로지 그룹 컴퍼니 리미티드 개질된 양자점 및 그의 제조 방법, 착색제, 감광성 수지 조성물, 컬러 필터 및 디스플레이 디바이스
KR20170008257A (ko) * 2014-12-19 2017-01-23 보에 테크놀로지 그룹 컴퍼니 리미티드 개질된 양자점 및 그의 제조 방법, 착색제, 감광성 수지 조성물, 컬러 필터 및 디스플레이 디바이스
EP3235893A4 (fr) * 2014-12-19 2018-07-25 Boe Technology Group Co. Ltd. Point quantique modifié et procédé de préparation associé, colorant, composition de résine photosensible, filtre coloré et dispositif d'affichage
US10746731B2 (en) 2015-06-30 2020-08-18 Imec Vzw Surface immobilization of an analyte-recognizing molecule
AU2016287197B2 (en) * 2015-06-30 2020-07-09 Imec Vzw Surface immobilization of an analyte-recognizing molecule
WO2017001374A1 (fr) * 2015-06-30 2017-01-05 Imec Vzw Immobilisation en surface d'une molécule de reconnaissance d'analyte
CN108369229A (zh) * 2015-07-17 2018-08-03 奥菲迪亚有限公司 用于处理基底表面的连接分子
WO2017013004A1 (fr) * 2015-07-17 2017-01-26 Orphidia Limited Molécule de liaison destinée à traiter une surface de substrat
CZ307452B6 (cs) * 2016-11-03 2018-09-05 Vysoká škola chemicko-technologická v Praze Aminooxylipidy pro konstrukci samoskladných liposomálních systémů umožňujících jejich následnou modifikaci biologicky funkčními molekulami
JP2022126716A (ja) * 2018-06-14 2022-08-30 持田製薬株式会社 新規な架橋アルギン酸
JP7350940B2 (ja) 2018-06-14 2023-09-26 持田製薬株式会社 新規な架橋アルギン酸
US11932708B2 (en) 2018-06-14 2024-03-19 Mochida Pharmaceutical Co., Ltd. Crosslinked alginic acid

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