Classifications

• • 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
  • C07D213/79—Acids; Esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

• the present invention relates to bifunctional chelating agents, to complexes of these chelating agents with metal ions, and to conjugates of these complexes with biological carriers. More particularly, the present invention relates to chelates for radiometals useful in molecular imaging and therapy, in particular, to radioisotopes of gallium such as 66 Ga, 67 Ga, and 68 Ga.
• Chelates are widely employed to isolate metal ions from environmental factors that would interfere with the intended use of the metal ions. This is commonly seen in the field of nuclear medicine, where radioactive isotopes of metals, i.e., radiometals, are used for molecular imaging and therapy due to their decay characteristics such as half-life and emission profile and due to their chemical properties such as lipophilicity and coordination behaviour.
• Gallium is a main group metal that comprises three radioactive isotopes useful in nuclear medicine.
• 66 Ga is a positron-emitter with a half-life of 9.5 h;
• 67 Ga a gamma-emitter with a half-life of 3.26 d;
• 68 Ga a positron-emitter with a half-life of 68 min.
• Positron-emitters are useful for positron-emission tomography (PET) imaging; gamma-emitters, for single-photon-emission computed tomography (SPECT) imaging.
• PET positron-emission tomography
• SPECT single-photon-emission computed tomography
• the chelates that are currently commonly used to bind gallium radiometals to biological targeting molecules are dominated by 1 ,4,7, 10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA) and l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) and their derivatives.
• DOTA 10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid
• NOTA l,4,7-triazacyclononane-l,4,7-triacetic acid
• Chelates comprising picolinyl groups attached to a nitrogen atom 6 ' 7 , ethylenediamine (en) 8 - 9,10 ' 1 1 ' 12 " 13 ' 14 , cyclohexane-l,2-diamine 15 ' 16 , and 1,4,7- triazacyclononane 17 ' 18 ' 19 0 ' 21 have been reported in the scientific literature and have been the subject of patent applications. 22 ' 23 These chelates have been designed to coordinate lanthanide ions, which are relatively large metal ions that in some instances possess large magnetic moments and are useful as magnetic-resonance- imaging contrast agents.
• the chelate comprising en has four picolinyl groups attached to each of the two en nitrogen atoms to give a decadentate (10-coordinate) chelate whereas the chelate comprising 1 ,4,7-triazacyclononane has three picolinyl groups attached one each to the three 1 ,4,7-triazacyclononane nitrogen atoms to give a nonadentate (9-coordinate) chelate.
• Classes of chelates comprising these particular chelates have been the subject of patent applications.
• a chelate comprising two picolinyl groups attached one each to the two nitrogen atoms of en, hereafter called dedpa, and also of cyclohexane-l,2-diamine has been reported in the scientific literature 24 .
• Complexes of said chelate dedpa with the metal ions Zn 2+ , Cd 2+ , and Pb 2+ were synthesized and found to comprise a hexadentate chelate bound to the metal ions to form an octahedral coordination environment.
• the present invention relates to chelates based on dedpa that can form complexes with radiometals useful in molecular imaging and therapy, more specifically for forming complexes with gallium radiometals for molecular imaging, because gallium radiometals prefer an octahedral coordination environment.
• An added advantage of using chelates based on dedpa for forming complexes with gallium radiometals is that gallium radiometals exist under physiological conditions as tripositive ions (Ga 3+ ) that form stronger coordination complexes than Zn + , Cd 2+ , or Pb 2+ because of the increased charge of the ion.
• the present invention relates to bifunctional chelating agents, to complexes of these chelating agents with metal ions, and to conjugates of these complexes with a biological carrier. More particularly, the present invention relates to chelates for radiometals useful in molecular imaging and therapy, in particular, to radioisotopes of gallium such as Ga, Ga, and Ga.
• the present invention provides a bifunctional chelating agent of the formula
• Q 1 , Q 2 and Q 4 are independently H or R;
• Q 3 is H, -(CHR 2 ) w COR 3 or -(CHR 2 ) w P0 2 R 4 R 5 ;
• Q 7 is H or R
• a 1 and A 2 form together with the atoms to which they are attached a Ce-Cio-aryl, Ce-Cio-heteroaryl, C 3 -Ci 0 -cycloalkyl or C 3 -Ci 0 - heterocyclyl group; -(CHR 2 ) p P0 2 R 4 R 5 , provided that at least one of Q l , Q 2 , Q 4 , Q 5 and Q 7 is R;
• R 1 and R 1 are independently
• X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon—carbon bond;
• Z and Z are independently CH or N;
• n is an integer from 0 to 10 inclusive
• n 0 or 1 ;
• p 1 or 2;
• r is 0 or 1 ;
• w is 0 or 1 ;
• z is 1, 2 or 3;
• L is a linker/spacer group covalently bonded to, and replaces one hydrogen atom of the carbon atom to which it is joined, said
• s is an integer of 0 or 1 ;
• t is an integer of 0 to 20 inclusive;
• R 7 , R 8 and R 9 are independently H; an electrophilic, nucleophilic or electron-rich moiety that allows for covalent attachment to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor; a protected form or a precursor of the electrophilic, nucleophilic or electron-rich moiety; or a synthetic linker having an electrophilic, nucleophilic or electron-rich moiety that allows for covalent attachment to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor, or a protected form or a precursor of the electrophilic, nucleophilic or electron-rich moiety of the synthetic linker, and
• Cyc represents a cyclic aliphatic moiety, aromatic moiety, aliphatic heterocyclic moiety, or aromatic heterocyclic moiety, each of said moieties optionally substituted with one or more groups, which do not interfere with binding to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor;
• the chelating agent is not 6,6',6",6"'-((ethane-l,2- diylbis(azanetriyl))tetrakis(methylene))tetrapicolinic acid, 6,6'-((ethane- 1 ,2- diylbis((phosphonomethyl)azanediyl))bis(methylene))dipicolinic acid, 6,6'-((ethane- l,2-diylbis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid or 6,6'- ((ethane- 1 ,2-diylbis((pyridin-2-ylmethyl)azanediyl))bis(methylene))dipicolinic acid.
• the bifunctional chelating agent is of the formula (la):
• Q 1 and Q 2 are each H and O is VY/m wherein L, X, Y, m, n and r are as defined above.
• Q 1 and Q 2 are and Q 4 is H, wherein R 6 and n are as defined above.
• Q 1 and Q 2 are -(CHR 2 ) p COR 3 and Q 4 is R, wherein R 2 , R 3 , R and p are as defined above.
• the present invention also pertains to the bifunctional chelating agents defined above, wherein R 6 is N0 2 , NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl.
• the present invention provides a complex comprising the bifunctional chelating agent defined above or a pharmaceutically acceptable salt thereof, and an ion of a stable or radioactive form of a metal selected from a group consisting of Ga, In, Tl, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, Ti, Zr, Cr, Mn, Tc, Re, Fe, Ru, Co, Rh, Ir, Ni, Pd, Pt, Cu, Zn, Cd, Hg, Al, Ge, Sn, Pb, Sb, Bi, Te, Po, Mg, Ca, Sr, Ba, Ra, Ac, Th and U.
• a metal selected from a group consisting of Ga, In, Tl, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, Ti, Zr, Cr,
• the present invention provides a complex comprising the bifunctional chelating agent defined above or a pharmaceutically acceptable salt thereof, and an ion of a stable or radioactive form of a metal selected from a group consisting of 66 Ga, 67 Ga, 68 Ga, " 'in, 20 I T1, l4 Pr, l49 Pm, 153 Sm, 153 Gd, 159 Gd, ,66 Ho, 175 Yb, 177 Lu, 47 Sc, 90 Y, 89 Zr, 51 Cr, 99m Tc, l88 Re, 186 Re, 57 Co, 101m Rh, 62 Cu, 64 Cu, 67 Cu, 1 , 7m Sn, 203 Pb, 2,2 Pb, 2,2 Bi, 13 Bi, 223 Ra, and 225 Ac.
• a metal selected from a group consisting of 66 Ga, 67 Ga, 68 Ga, " 'in, 20 I T1, l4 Pr, l49 Pm, 153 Sm, 153 Gd, 159 G
• the present invention provides a conjugate of a bifunctional chelating agent of the formula (I) or (la) defined above with a carrier comprising a biotargeting moiety, a lipophilic group or a biosensor.
• a carrier comprising a biotargeting moiety, a lipophilic group or a biosensor, wherein:
• Q , Q and Q are independently H or R;
• Q 3 is H, -(CHR 2 ) w COR 3 or -(CHR 2 ) w P0 2 R 4 R 5 ;
• Q 5 is H, R or R 1 ;
• Q 6 is H or R 1 " ;
• Q 7 is H or R
• a 1 and A 2 form together with the atoms to which they are attached a C 6 -Cio-aryl, C 6 -C
• R is -C(0)-L, , -(CHR 2 ) p COR 3 or
• R r and R 1" are independently
• X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon—carbon bond;
• n is an integer from 0 to 10 inclusive
• Z and Z are independently CH or N;
• n 0 or 1 ;
• p 1 or 2;
• r is 0 or 1;
• w is 0 or 1 ;
• z is 1, 2 or 3;
• L is a linker/spacer group covalently bonded to, and replaces one hydrogen atom of the carbon atom to which it is joined, said
• linker/spacer group being represented by the formula (II) or (III):
• s is an integer of 0 or 1 ;
• t is an integer of 0 to 20 inclusive
• R 7 , R 8 and R 9 are independently H; an electrophilic,
• nucleophilic or electron-rich moiety that allows for covalent
• a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor; a protected form or a precursor of the
• electrophilic, nucleophilic or electron-rich moiety or a synthetic linker having an electrophilic, nucleophilic or electron-rich moiety that allows for covalent attachment to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor, or a protected form or a precursor of the electrophilic, nucleophilic or electron-rich moiety of the synthetic linker, and
• Cyc represents a cyclic aliphatic moiety, aromatic moiety, aliphatic heterocyclic moiety, or aromatic heterocyclic moiety, each of said moieties optionally substituted with one or more groups, which do not interfere with binding to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor; is a moiety comprising a biotargeting group, a lipophilic group or a biosensor, and
• the biotargeting group of the conjugate defined above may be a protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen or hapten.
• the present invention provides a complex comprising (i) a conjugate of a bifunctional chelating agent of the formula (I):
• a carrier comprising a biotargeting moiety, a lipophilic group or a biosensor, wherein:
• Q 1 , Q 2 and Q 4 are independently H or R;
• Q 3 is H, -(CHR 2 ) w COR 3 or -(CHR 2 ) w P0 2 R 4 R 5 ;
• Q 5 is H, R or R 1 " ;
• Q 6 is H or R 1 " ;
• Q 7 is H or R
• a 1 and A 2 form together with the atoms to which they are attached a C6-Cio-aryl, C 6 -Cio-heteroaryl, C3-Cio-cycloalkyl or C 3 -C 10 - heteroc clyl group;
• R', R' and R 1 are independently-
• each R is independently hydrogen; C 1 -C4 alkyl or (Ci-C 2 alkyl)phenyl; each R 3 , R 4 and R 5 are independently OH, an -O-protecting group, such as -0-(Ci-C 2 alkyl) phenyl or -0-Cr 4 alkyl, or a leaving group; R 6 is H; OH; an alkyl-LG or alkoxy-LG, wherein LG is a leaving group; a boronate ester or a leaving group;
• X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon— carbon bond;
• n is an integer from 0 to 10 inclusive
• Z and Z are independently CH or N; n is 0 or 1 ;
• p 1 or 2;
• r is 0 or 1 ;
• w is 0 or 1 ;
• z is 1, 2 or 3;
• L is a linker/spacer group covalently bonded to, and replaces one hydrogen atom of the carbon atom to which it is joined, said linker/ spacer group being represented by the formula (II) or (III):
• s is an integer of 0 or 1 ;
• t is an integer of 0 to 20 inclusive
• R 7 , R 8 and R 9 are independently H; an electrophilic, nucleophilic or electron-rich moiety that allows for covalent attachment to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor; a protected form or a precursor of the electrophilic, nucleophilic or electron-rich moiety; or a synthetic linker having an electrophilic, nucleophilic or electron-rich moiety that allows for covalent attachment to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor, or a protected form or a precursor of the electrophilic, nucleophilic or electron-rich moiety of the synthetic linker, and
• Cyc represents a cyclic aliphatic moiety, aromatic moiety, aliphatic heterocyclic moiety, or aromatic heterocyclic moiety, each of said moieties optionally substituted with one or more groups, which do not interfere with binding to a carrier comprising a biotargeting group, a lipophilic moiety or a biosensor; ( Carrier ) . . . ⁇ , . . , ⁇ , ⁇ , ⁇ , ⁇
• ⁇ ⁇ is a moiety comprising a biotargeting group, a lipophili group or a biosensor, and
• a metal selected from a group consisting of Ga, In, Tl, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, Ti, Zr, Cr, Mn, Tc, Re, Fe, Ru, Co, Rh, Ir, Ni, Pd, Pt, Cu, Zn, Cd, Hg, Al, Ge, Sn, Pb, Sb, Bi, Te, Po, Mg, Ca, Sr, Ba, Ra, Ac, Th and U.
• a metal selected from a group consisting of Ga, In, Tl, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, Ti, Zr, Cr, Mn, Tc, Re, Fe, Ru, Co, Rh, Ir, Ni, Pd, Pt, Cu, Zn, Cd, Hg, Al, Ge
• the present invention also relates to the complex defined above, wherein the ion is an ion of a radioactive metal selected from a group consisting of 66 Ga, 67 Ga,
• the present invention also relates to the complex defined above, wherein the biotargeting moiety is a protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen or hapten.
• the present invention provides a conjugate comprising ⁇ of the complexes defined above covalently attached to a biological carrier.
• the biological carrier is a protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen or hapten.
• the present invention provides a conjugate comprising one of the complexes defined above covalently attached to a biological carrier, such as a protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen or hapten.
• a biological carrier such as a protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen or hapten.
• the present invention provides a pharmaceutical composition comprising the conjugate defined above, and a pharmaceutically acceptable carrier.
• the present invention provides a method of therapeutic treatment of a mammal having cancer which comprises administering to said mammal a therapeutically effective amount of the pharmaceutical composition defined above.
• the present invention also relates to the complex and conjugate defined above, wherein the chelating agent is not 6,6',6",6"'-((ethane-l ,2- diylbis(azanetriyl))tetrakis(methylene))tetrapicolinic acid, 6,6'-((ethane- 1 ,2- diylbis((phosphonomethyl)azanediyl))bis(methylene))dipicolinic acid, 6,6'-((ethane- 1 ,2-diylbis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid or 6,6'- ((ethane- 1 ,2-diylbis((pyridin-2-ylmethyl)azanediyl))bis(methylene))dipicolinic acid.
• the chelating agent is not 6,6',6",6"'-((ethane-l ,2- diylbis(azanetri
• FIG. 1 illustrates HPLC traces of 67 Ga(dedpa) + vs. apo-transferrin in competition; for reference the trace of 67 Ga-transferrin is shown (gradient: A: NaOAc buffer, pH 4.5, B: MeOH. 0-100% B linear gradient 20 min).
• FIG. 2 illustrates solid-state structure of the cation in Ga(dedpa)C10 4 .
• FIG. 3 illustrates the solid-state structure of the cation in Ga(66)C10 4 .
• FIG. 4 illustrates biodistribution over 4h of 67 Ga(dedpa) + in female ICR mice.
• FIG. 5 illustrates biodistribution of 67 Ga(66) + (upper) and 67 Ga(69) + (lower) in female ICR mice over 4h; complete data for urine is shown also in separate diagrams to the right.
• FIG. 6A illustrates the ⁇ -NMR of H 2 dedpa-2HC1 in DMSO-d 6 .
• FIG. 6B illustrates the ⁇ -NMR (MeOD-d 4 , 300 MHz) of Ga(dedpa)N0 3 .
• FIG. 7A illustrates the ⁇ -NMR of H 2 66 in MeOD-d 4 .
• FIG. 7B illustrates the ⁇ -NMR of Ga(66)N0 3 in MeOD-d 4 .
• FIG. 8A illustrates the ⁇ -NMR of H 2 69 in MeOD-d 4 .
• FIG. 8B illustrates the ⁇ -NMR of Ga(69)N0 3 in MeOD-d 4 .
• FIG. 9 illustrates the labelling trace of 67 Ga(dedpa) + on HPLC.
• FIG. 10 illustrates the labelling trace of 67 Ga(66) + on HPLC.
• FIG. 1 1 illustrates the labelling trace of 67 Ga(69) + on HPLC.
• FIG. 12 illustrates the HPLC chromatogram for 67 Ga-transferrin .
• FIG. 13 illustrates stacked labelling traces of 67 Ga(dedpa) + of 2-h stability experiment against apotransferrin.
• FIG. 14 illustrates the labelling trace of competition between 3 ⁇ 4dedpa and NOTA for coordination of 67 Ga. on HPLC.
• FIG. 15 illustrates the HPLC chromatogram for 67 Ga-transferrin (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 CN. 0-100% B linear gradient 20 min; reference for stability measurements of 67 Ga(66) + and 67 Ga(69) + ).
• FIG. 16 illustrates stacked labelling traces of 67 Ga(66) + of 2-h stability experiment against apotransferrin (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 CN. 0- 100% B linear gradient 20 min) .
• FIG.17 illustrates stacked labelling traces of 67 Ga(69) + of 2-h stability experiment against apotransferrin (gradient: A: NaOAc buffer, pH 4.5, B: CH3CN. 0- 100%) B linear gradient 20 min).
• FIG. 18 illustrates ⁇ -NMR and l3 C-NMR spectra of compound 104 in D 2 0.
• FIG. 19 illustrates ⁇ -NMR and 13 C-NMR spectra of compound 108 in D 2 0. DETAILED DESCRIPTION OF THE INVENTION
• the present invention relates to bifunctional chelating agents, to complexes of these chelating agents with metal ions, and to conjugates of these complexes with a biological carrier. More particularly, the present invention relates to chelates for radiometals useful in molecular imaging and therapy, in particular, to radioisotopes of gallium such as 66 Ga, 67 Ga, and 68 Ga.
• complex refers to a complex of the compound of the invention, e.g. Formula (I), complexed with a metal ion, where at least one metal atom is chelated or sequestered.
• the complexes of the present invention can be prepared by methods well known in the art. Thus, for example, see Chelating Agents and Metal Chelates, Dwyer & Mellor, Academic Press (1964), Chapter 7. See also methods for making amino acids in Synthetic Production and Utilization of Amino Acids, (edited by Kameko, et al.) John Wiley & Sons (1974).
• An example of the preparation of a complex involves reacting a bicyclopolyazamacrocyclophosphonic acid with a paramagnetic metal ion under aqueous conditions at a pH from 5 to 7.
• the complex formed is by a chemical bond and results in a stable paramagnetic nuclide composition, e.g. stable to the disassociation of the paramagnetic nuclide from the ligand.
• a “conjugate” refers to a metal-ion chelate that is covalently attached to a carrier, such as a carrier comprising a biotargeting group, a lipohilic group or a biosensor.
• alkyl refers to a straight- or branched chain saturated hydrocarbon group having from 1 to about 20 carbon atoms, including groups having from 1 to about 7 carbon atoms.
• alkyl groups include, without limitation, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl and s-pentyl.
• aliphatic heterocyclic moiety means a non-aromatic mono- or bi- cyclic radicals of three to eight ring atoms in which one or two ring atoms are heteroatoms selected from N, O, or S(0) n (where n is an integer from 0 to 2), the remaining ring atoms being C.
• cyclic aliphatic moiety means a non-aromatic mono- or bi-cyclic radical of three to eight ring atoms.
• aryl or "aromatic moiety” means a monovalent cyclic aromatic moiety consisting of a mono-, bi- or tricyclic aromatic ring wherein each member of the ring is carbon.
• aryl moieties include, but are not limited to, optionally substituted phenyl, naphthyl, phenanthryl, fluorenyl, indenyl, pentalenyl, azulenyl, oxydiphenyl, biphenyl, methylenediphenyl, aminodiphenyl, diphenylsulfidyl, diphenylsulfonyl, and diphenylisopropylidenyl.
• heteroaryl or "aromatic heterocyclic moiety” means a monocyclic or bicyclic radical having at least one aromatic ring containing one, two, three or four ring heteroatoms, the remaining ring atoms being carbon, with the understanding that the attachment point of the radical to the remainder of the molecule is on the aromatic ring moiety containing the heteroatom(s).
• heteroaryl moieties include, but are not limited to, optionally substituted imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, thienyl, benzothienyl, furanyl, pyridyl, pyrrolyl, pyrazolyl, pyrimidyl, quinolinyl, isoquinolinyl, benzofurylbenzimidazolyl, benzooxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isoindolyl, triazolyl, triazinyl, quinoxalinyl, purinyl, quinazolinyl, quinolizinyl, naphthyridinyl, pteridinyl, carbazolyl
• heteroatom means an atom selected from the group consisting of N, O, P and S.
• biological targeting group refers to any biological targeting vector, such as a protein, peptide, peptidomimetic, an antibody, an antibody fragment, a hormone, an aptamer, an affibody molecule, a morpholino compound, a growth factor, an antigen, a hapten or any other carrier, which functions in this invention to recognize a specific biological target site.
• Antibody and antibody fragment refers to any polyclonal, monoclonal, chimeric, human, mammalian, single chain, dimeric and tetrameric antibody or antibody fragment.
• Such biological carrier when attached to a functionalized complex, serves to carry the attached ion to specific targeted tissues.
• bifunctional chelating agent or "bifunctional chelator” refers to compounds that have a chelant moiety capable of chelating a metal ion and a moiety covalently bonded to the chelant moiety that is capable of serving as a means to covalently attach to a biological carrier for example, a molecule having specificity for tumor cell epitopes or antigens, such as an antibody or antibody fragment. Such compounds are of great utility for therapeutic and diagnostic applications when they are, for example, complexed with radioactive metal ions and covalently attached to a specific antibody.
• the bifunctional chelating agents described herein can be used to chelate or sequester a metal ion to form metal-ion chelates (also referred to herein as "complexes", as defined above).
• the complexes because of the presence of the functionalizing moiety (represented by R 6 in Formula I), can be covalently attached to a biologically active material, such as dextran, molecules that have specific affinity for a receptor, affibody molecules, morpholino compounds or antibodies or antibody fragments.
• a biologically active material such as dextran, molecules that have specific affinity for a receptor, affibody molecules, morpholino compounds or antibodies or antibody fragments.
• the term "antibody” refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a heteroantibody, or a fragment thereof.
• Antibodies used in the present invention may be directed against, for example, cancer, tumors, bacteria, fungi, leukemias, lymphomas, autoimune disorders involving cells of the immune system, normal cells that need to be ablated such as bone marrow and prostate tissue, virus infected cells including HIV, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules.
• antibodies are HuM195 (anti-CD33), CC-11, CC-46, CC-49, CC- 49 F(ab') 2 , CC-83, CC-83 F(ab') 2 , and B72.3, 1 1 16-NS-19-9 (anti-colorectal carcinoma), 1 1 16-NS-3d (anti-CEA), 703D4 (anti-human lung cancer), and 704A1 (anti-human lung cancer).
• the hybridoma cell lines 1116-NS-19-9, 11 16-NS-3d, 703D4, 704A1, CC49, CC83 and B72.3 are deposited with the American Type
• Antibody fragment includes Fab fragments and F(ab') 2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes.
• Complexes of the present invention which include a radioisotopic metal ion having a relatively short half-life, such as Ga-68, can be conjugated with biological carriers having relatively short or relatively long biological clearance times from a subject.
• a radioisotopic metal ion having a relatively short half-life such as Ga-68
• complexes are typically conjugated to biological carriers having a biological clearance time that is within the lifetime of the short-lived radioisotope so that the systemic background signal produced by unbound conjugated complex can be sufficiently reduced in time to permit imaging of the conjugated complex bound to the target site of the biological carrier.
• biological carriers or “biotargeting carriers”, which can be conjugated to complexes of the present invention, include peptides or molecular constructs, such as mini-bodies, nano-bodies or affi-bodies. Specific examples of peptides having relatively short clearance times are described in Maecke HR and Reubi JC 2008 Peptide based probes for cancer imaging. J. Nucl. Med. 49: 1735-38; Kren ing, EP, de Jong M, Kooij PP, Breeman, WA, Bakker WH et. al. 1999 Radiolabelled somatostatin analogue(s) for peptide receptor scintigraphy and radionuclide therapy. Ann. Oncol.
• Complexes of the present invention can also be conjugated to non-biological carriers, such as carriers comprising lipophilic groups, such as phenyl groups having one, two or three alkoxy groups, for example, one, or more than one 1 - methoxyphenyl, 1 ,3-dimethoxyphenyl, or 1, 3, 5-trimethoxyphenyl group, to increase the lipophilicity of the complexes to which they are conjugated.
• non-biological carriers such as carriers comprising lipophilic groups, such as phenyl groups having one, two or three alkoxy groups, for example, one, or more than one 1 - methoxyphenyl, 1 ,3-dimethoxyphenyl, or 1, 3, 5-trimethoxyphenyl group, to increase the lipophilicity of the complexes to which they are conjugated.
• lipophilic groups include Ci-C6-alkyl, C i-C6-alkoxy-Ci-C6-alkyl, acetal and Ci-C 6 -alkyl-crown ether groups (e.g., -CH 2 -12-crown-4, -CH 2 -15-crown-5 and - CH 2 -18-crown-6 groups).
• Ci-C6-alkyl C i-C6-alkoxy-Ci-C6-alkyl
• acetal and Ci-C 6 -alkyl-crown ether groups e.g., -CH 2 -12-crown-4, -CH 2 -15-crown-5 and - CH 2 -18-crown-6 groups.
• the resulting conjugates are useful for imaging myocardial blood flow.
• complexes of the present invention can be conjugated to comprising a biosensor, such as a nitroimidazole group for targeting hypoxia.
• Conjugated carriers comprising nitroimidazole derivatives are reduced to a chemical form that is trapped in hypoxic tissue so that a radio-tracer complexed to the chelator of the conjugate builds in concentration selectively compared to normal tissue.
• the "antibody” is meant to include whole antibodies and/or antibody fragments, including semisynthetic or genetically engineered variants thereof. Such antibodies normally have a highly specific reactivity.
• the antibodies or antibody fragments which may be used in the conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see for example, Kohler and Milstein Nature, 256, 495-497 (1975); and Eur. J.
• antibodies normally have a highly specific reactivity in the antibody targeted conjugates, antibodies directed against any desired antigen or hapten may be used.
• the antibodies which are used in the conjugates are monoclonal antibodies, or fragments thereof having high specificity for a desired epitope(s).
• salts means any salt or mixture of salts of a complex or conjugate of formula (I) which is sufficiently non-toxic to be useful in therapy or diagnosis of animals, preferably mammals. Thus, the salts are useful in accordance with this invention.
• salts formed by standard reactions from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, gluconic, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids.
• salts formed by standard reactions from both organic and inorganic sources such as ammonium or 1 -deoxy- 1 -(methylamino)-D-glucitol, alkali metal ions, alkaline earth metal ions, and other similar ions.
• Particularly preferred are the salts of the complexes or conjugates of formula (I) where the salt is potassium, sodium or ammonium.
• mixtures of the above salts are also included.
• the present invention may be used with a physiologically acceptable carrier, excipient or vehicle therefor.
• a physiologically acceptable carrier excipient or vehicle therefor.
• the methods for preparing such formulations are well known.
• the formulations may be in the form of a suspension, injectable solution or other suitable formulations.
• Physiologically acceptable suspending media, with or without adjuvants, may be used.
• an "effective amount" of the formulation is used for diagnosis or for therapeutic treatments of diseases.
• the dose will vary depending on the disease and physical parameters of the animal, such as weight.
• In vivo diagnostics are also contemplated using formulations of this invention.
• the chelates of the present invention are useful for binding radioisotopes to biological targeting molecules in order to produce constructs for molecular imaging and therapy, more specifically to produce constructs comprising gallium radioisotopes for molecular imaging.
• chelates of the present invention may include the removal of undesirable metals (i.e. iron) from the body, attachment to polymeric supports for various purposes, e.g. as diagnostic agents, and removal of metal ion by selective extraction.
• undesirable metals i.e. iron
• the free acid of the compounds of formula (I) may be used, also the protonated form of the compounds, for example when the carboxylate is protonated and/or the nitrogen atoms, i.e. when the HC1 salt is formed.
• the complexes so formed can be attached (covalently bonded) to an antibody or fragment thereof and used for therapeutic and/or diagnostic purposes.
• the complexes and/or conjugates can be formulated for in vivo or in vitro uses.
• a preferred use of the formulated conjugates is the diagnosis of diseased states (e.g., cancer) in animals, especially humans.
• Biotargeted radiopharmaceuticals that employ the chelating agent (ligand) of the present invention to secure a metal radionuclide can be prepared by two methods: 1) Pre-complexation - the metal ligand complex (chelate) can first be prepared followed by covalent attachment of the chelate to a biotargeting group, for example a monoclonal antibody; 2) Post-complexation - a covalent conjugate between the ligand and the biotargeting molecule can be prepared in a first step followed by introduction and complexation of the metal radionuclide. Both methods have merits and shortcomings.
• Method 1 is appealing from the standpoint that forcing conditions can be utilized to facilitate complexation however subsequent attachment of the complex to a targeting vector requires more elaborate chemical transformation that can be difficult to perform rapidly in a hospital setting.
• method 2 is desirable since it allows the more intricate chemistry required for conjugation of the ligand and targeting vector to be performed in controlled environment without time constraints introduced by the radionuclide.
• the complexation step can then be conducted onsite at the hospital pharmacy by clinical technicians however this step can be problematic since the ligand bound conjugate is much more sensitive to rigorous conditions that favor rapid and complete complexation.
• the post-complexation strategy is clearly the most desirable if appropriate ligands and/or conditions can be devised that facilitate rapid and complete incorporation of the radionuclide.
• structural and conformational components can be introduced that can minimize kinetic barriers to complexation. For example, molecular architecture which can enhance pre-organization of the ligand binding site toward the necessary conformational requirements of the metal ion should produce faster complexation kinetics.
• the bifunctional chelating agents described herein are designed to form thermodynamically stable and kinetically inert complexes with the main group series of metals. Complexation kinetics can be modulated by altering backbone structural rigidity, electronic character of the coordinate donor atoms, and conformational accessibility of the metal binding site. [0082] While not wishing to be bound by theory, it is believed that kinetic advantages associated with the present invention are a function of structural modifications that lead to preferred molecular geometries (pre-organization) which match ligating requirements of the metal. In this manner the ligand-metal binding event is accelerated without the need for harsh reaction conditions.
• the generation of optimal pre- organized ligand structures conducive to rapid complexation kinetics is significantly influenced by the judicious placement of the linking group.
• the linking group can be engineered to assume a position distant from the metal binding site during the initial stages of the metal docking process followed by the adoption of a secondary conformation induced by complexation that effectively shields the metal from reversible dissociation pathways.
• the positional orientation of the linking group also affects the electronic nature of the coordinate donor atoms and their juxtaposed lone pair electrons which are critical for satisfying the geometric requirements of the metal ion.
• the present invention also includes formulations comprising the conjugates of this invention and a pharmaceutically acceptable carrier, especially formulations where the pharmaceutically acceptable carrier is a liquid.
• the present invention is also directed to a method of therapeutic treatment of a mammal having cancer which comprises administering to said mammal a
• the present invention may be practiced with the conjugate of the present invention being provided in a pharmaceutical formulation, both for veterinary and for human medical use.
• Such pharmaceutical formulations comprise the active agent (the conjugate) together with a physiologically acceptable carrier, excipient or vehicle therefore.
• the methods for preparing such formulations are well known.
• the carrier(s) must be physiologically acceptable in the sense of being compatible with the other ingredient(s) in the formulation and not unsuitably deleterious to the recipient thereof.
• the conjugate is provided in a therapeutically effective amount, as described above, and in a quantity appropriate to achieve the desired dose.
• Complexes of the chelating agents of the present invention with a suitable metal ion, and conjugates of these complexes can be used in diagnostic medical imaging procedures.
• complexes of the present invention formed with a positron-emitter and the corresponding conjugates of these complexes are useful for positron-emission tomography (PET) imaging.
• PET positron-emission tomography
• complexes of the present invention formed with a gamma-emitter and the corresponding conjugates are useful for single-photon-emission computed tomography (SPECT) imaging.
• SPECT single-photon-emission computed tomography
• complexes of the present invention formed with a paramagnetic metal ion, such as Gd +3 , Mn +2 or Fe +3 , and corresponding conjugates of these complexes can act as contrast agents in magnetic resonance imaging (MRI), and complexes of the present invention formed with a lanthanide metal ion such as, Tb 3+ , Eu 3+ , Sm 3+ or Dy + , and the corresponding conjugates can be used in fluorescent imaging.
• This invention is used with a physiologically acceptable carrier, excipient or vehicle therefore.
• the formulations may be in the form of a suspension, injectable solution or other suitable formulations. Physiologically acceptable suspending media, with or without adjuvants, may be used.
• compositions include those suitable for parenteral (including
• Formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the conjugate into association with a carrier, excipient or vehicle therefore. In general, the formulation may be prepared by uniformly and intimately bringing the conjugate into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into desired formulation. In addition, the formulations of this invention may further include one or more accessory
• a treatment regime might include pretreatment with non-radioactive carrier.
• compositions of the present invention may be either in suspension or solution form.
• suitable formulations it will be recognized that, in general, the water solubility of the salt is greater than the acid form.
• the complex (or when desired the separate components) is dissolved in a physiologically acceptable carrier.
• suitable solvent include, for example, water, aqueous alcohols, glycols, and phosphonate or carbonate esters.
• aqueous solutions contain no more than 50 percent of the organic solvent by volume.
• Injectable suspensions are compositions of the present invention that require a liquid suspending medium, with or without adjuvants, as a carrier.
• the suspending medium can be, for example, aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, polyols, or aqueous
• Suitable physiologically acceptable adjuvants may be chosen from among thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates.
• thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates.
• surfactants are also useful as suspending agents, for example, lecithin, alkylphenol, polyethyleneoxide adducts, naphthalenesulfonates, alkylbenzenesulfonates, and polyoxyethylene sorbitan esters.
• diamino compound 5 is first alkylated with two equivalents of tert-butyl 6- (bromomethyl)picolinate 2 to form dipicolinate ester 6, which can be either deprotected to form dipicolinic acid derivative 8 or alkylated with an alkyl halide or allowed to react with an acyl halide to form the alkylated or amidated derivative 7, which is then deprotected to form alkylated (amidated), dipicolinic acid derivative 9.
• Intermediate 6 can alternatively be formed by reacting diamino compound 5 with two equivalents of methyl 6-formylpicolinate 4 to form a diimine intermediate, which is reduced using NaBH 4 .
• Reactant 2 can be formed by bromination of tert-butyl 6- methylpicolinate 1.
• Reactant 4 can be prepared by reducing dimethyl pyridine-2,6- dicarboxylate 3 to the corresponding monoalcohol using NaBH 4 followed by oxidation of the alcohol to an aldehyde using SeC ⁇ .
• Scheme 2 illustrates examples of methods for preparing the chelators of the present invention having picolinic acid groups and derived from 2-(aminomethyl)-2- methylpropane- 1 ,3 -diamine (14a) and Nl,Nl-bis(2-aminoethyl)ethane-l,2-diamine (14b).
• Diamino compound 14 is first alkylated with two to three equivalents of tert- butyl 6-(bromomethyl)picolinate 2 to form di- or tri-picolinate ester 15, which can be either deprotected to form di- or tri -picolinic acid derivative 17 or further alkylated with an alkyl halide or allowed to react with an acyl halide to form an alkylated or amidated derivative, which is subsequently deprotected to form alkylated (amidated), - or tri-picolinic acid derivative 16.
• the chelates of the present invention having picolinic acid moieties and derived from alkylene diamine groups can be prepared by the synthetic approach illustrated in Scheme 3, which initially involves selective single alkylation of the primary amines of an alkylene diamine (18f), a backbone-substituted alkylene diamine (18a), a mono- or di-, N-alkylated alkylene diamine (18d-e) or a backbone- substituted and mono- or di-, N-alkylated alkylene diamine (18b-c) with a 6- (bromomethyl)picolinate derivative protected with a base stable, acid labile carboxy- protecting group, such as tert-butyl 6-(bromomethyl)picolinate (2), which can be formed by bromination of tert-butyl 6-methylpicolinate (1), to form alkylated product (19a-f) (See T.
• Scheme 3 initially involves selective single alkylation of the primary amines of an alkylene diamine (18f), a back
• the chelates of the present invention having pyrimidine-2-carboxylic acid groups, pyrimidine-4-carboxylic acid groups or l,3,5-triazine-2-carboxylic acid groups can be prepared starting from tert-butyl 4- (bromomethyl)pyrimidine-2-carboxylate, tert-butyl 2-(bromomethyl)pyrimidine-4- carboxylate, and tert-butyl 4-(bromomethyl)-l,3,5-triazine-2-carboxylate,
• the chelates of the present invention having quinolinyl-8-ol groups or quinoline-2-carboxylic acid groups can be prepared starting from 2- (bromomethyl)-8-(tert-butoxy)quinoline and tert-butyl 7-bromoquinoline-2- carboxylate, respectively.
• Compounds (18b) and (18c) may be prepared by selective single alkylation of the primary amines of diamine (18a) using 1 or 2 equivalents of an alkylating agent R-L, where L is a leaving group.
• Compounds (18e) and (18d) may be similarly prepared starting from diamine (18f).
• the functional group of the R group of diamine (18a) may need to be protected prior to the alkylation reaction used to prepare compound (18b) or (18c) to prevent any undesirable side reactions taking place between the functional groups of the substituent R of diamine (18a) and the alkylating agent R-L.
• the protecting group can be removed after a later conjugation step involving a carrier, such as a carrier comprising a biological targeting moiety, which is used to form the conjugated bifunctional chelate of the present invention.
• suitable protecting groups for use in the method of the present invention may be found in ocienski, P. J. Protecting Groups, 3rd ed.; Georg Thieme Verlag: New York, 2005, the disclosure of which is incorporated by reference herein.
• any free secondary amines in the resulting dipicolinic acid derivative (19a, 19b or 19e) may then be protected with a suitable protecting group, for example, an acid-labile protecting group, such as a t-butyl carbamate (BOC) protecting group, to prevent undesired couplings taking place in a subsequent conjugation reaction involving a carrier (See Tarbell et al., Procl. Natl. Acad.
• a suitable protecting group for example, an acid-labile protecting group, such as a t-butyl carbamate (BOC) protecting group
• the synthetic method of the present invention for forming a conjugated bifunctional chelator would include a step of hydrolysing these ester groups under basic conditions, using a suitable base such as LiOH, to form the corresponding acids (See Corey et al., Tetrahedron Lett., 1977, 3529, the disclosure of which is incorporated by reference herein.)
• Coupling of (19a-e) or the corresponding acids with a biological carrier of interest may then be carried out to form a stable linkage, such as an amide linkage, between the chelating agent (19a-e) and the carrier.
• a stable linkage such as an amide linkage
• Scheme 3 Synthetic scheme for forming the bifunctional chelators of the present invention derived from an alkylene diamine moiety
• the primary amino groups of Compound 19 are first protected with benzyl groups and then the resulting dibenzylated compound 20 is allowed to react with 3-4 equivalents of an alkylating group containing a picolinic acid ester group (R la -Br) to produce protected intermediate 21.
• the benzyl groups of this compound are then removed using 10 mol% Pd/C to produce intermediate 22 having protected picolinic acid groups.
• ester groups of intermediate 22 can either be hydrolyzed to produce chelate 22a or intermediate 22 may be allowed to react with 1 -4 equivalents of an alkyl halide or acyl halide to produce the protected bifunctional chelate 23, which can subsequently be deprotected to produce bifunctional chelate 23a.
• Intermediate 19a may then be deprotected to produce chelate 19b or reacted with 1-4 equivalents of an alkyl halide or an acyl halide and the resulting poly-alkylated
• Scheme 5 shows examples of synthetic schemes for forming chelators of the present invention, which include pendant 2-methylquinolin-8-ol and quinoline-2- carboxylic acid groups for chelation.
• the alcohol group of methyl 8-hydroxyquinoline-2-carboxylate 24 is first protected using TBSC to produce protected intermediate methyl 8-((tert-butyldimethylsilyl)oxy)quinoline-2-carboxylate 25.
• the ester group of Compound 25 is then converted to an aldehyde group through a reductive step using NaBH4 to produce an alcohol, which is oxidized to aldehyde 26 using Se0 2 .
• chelate 27 which may be further reacted, if necessary, to form a bifunctional chelator.
• Chelators of the present invention having pendant quinoline-2-carboxylic acid groups can be formed by first converting alcohol 24 to bromide 28 using NBS/PPh . Alkylation of diamino compound 5 with bromide 28 is then conducted under Buchwald/Hartwig reaction conditions to afford chelator 29, which may be further alkylated, if necessary, to form a bifunctional chelator of the present invention.
• Scheme 5 Example of synthetic schemes for forming chelators having pendant 2- methylquinolin-8-ol and quinoline-2-carboxylic acid groups.
• Scheme 6 illustrates examples of forming conjugates from chelators of the present invention.
• the amino groups of a chelator 6a are alkylated with 2 equivalents of a carrier molecule having a pendant bromide group (30) and the picolinate ester moities of the resulting intermediate are then hydrolyzed to form di-N-alkylated conjugate 31.
• a chelator having a pendant carboxylic acid group (32) is coupled with a carrier having a pendant amino group (33) to form an amide linkage and the picolinate ester moities of the resulting intermediate are subsequently hydrolyzed to form conjugate 34.
• a chelator having a pendant isothiocyanate group (37) can be coupled to carrier 33 to form a conjugate having a thiourea linkage (38).
• Chelator 37 can be produced starting from a di-N-benzylated chelator having a Ph-N0 2 group moiety disposed along the alkylene diamine backbone (35a).
• chelator 35a Hydrogenation of chelator 35a using a Pd(OH) 2 catalyst results in the reduction of the Ph-N0 2 group and deprotection of the secondary amino groups of that compound.
• the ester groups of the resultant intermediate are then hydrolyzed under basic conditions to afford a molecule having a Ph-NH 2 group and picolinic acid groups (36), which is subsequently converted to Compound 37 by reaction with SCC1 2 .
• Compound 35a is produced by alkylating dibenzylated alkylenediamine 35 with 2 equivalents of methyl 6- (bromomethyl)picolinate.
• bifunctional chelators of the present invention are bifunctional chelators of the present invention.
• Scheme 7 illustrates examples of synthetic schemes for forming chelator molecules of the present invention, which have substituted picolinic acid groups that can be conjugated with a carrier.
• These chelator molecules are formed by first esterifying 4- oxo-l,4-dihydropyridine-2,6-dicarboxylic acid hydrate (39) with MeOH to form diester 40, which is in equilibrium with its tautomeric form 41.
• Tautomer 41 may be reacted with bromoalcohol 42 to form hydroxyalkoxylated derivative 43.
• the alcohol group of derivative 43 can then be converted to a bromide using PBr 3 to afford reactive intermediate 44.
• One of the ester groups of intermediate 44 is then converted to an aldehyde group using successive steps of reduction and oxidation involving NaBH 4 and Se0 2 to produce aldehyde 45.
• the alcohol group of tautomer 41 may alternatively be converted to a bromide using NBS/PPh 3 to produce dimethyl 4-bromopyridine-2,6-dicarboxylate 47.
• Intermediate diester 47 is converted to aldehyde 48 using sucessive steps of reduction and oxidation involving NaBH 4 and Se0 2 . Reaction of intermediate 48 with
• Scheme 7 Examples of synthetic schemes for forming chelators of the present invention, which include picolinic acid groups having pendant groups useful for conjugation with a carrier comprising a targeting group.
• Scheme 8 illustrates the synthetic steps involved in conjugating the chelators shown in Scheme 7 to a carrier molecule.
• Chelator 46 which has a pendant alkyl bromide group, is directly coupled to a carrier having a free amino group by way of a nucleophilic displacement reaction. The resulting intermediate conjugate is deprotected under basic conditions to afford conjugate 52.
• Chelator 51 having picolinyl moieties substituted with boronate ester moieties can be coupled to a carrier molecule having a pendant bromide group (53) using a Pd(PPh 3 ) 4 catalyst. The resulting intermediate conjugate is deprotected under basic conditions to produce conjugate 54.
• conjugate 49 which has picolinyl moieties substituted with bromide groups, can be linked to a carrier having a free amino group under Buchwald-Hartwig reaction conditions. Deprotection of the intermediate conjugate under basic conditions affords conjugate 56.
• carrier-NH 2 LiOH, THF/H 2 0
• Scheme 9 Example of synthetic scheme for forming conjugated, di-, N- derivatized bifunctional dedpa.
• Scheme 10 illustrates an example of a method for forming a conjugated, backbone-substituted bifunctional chelator of the present invention. The synthetic method involves:
• the terms "degree of complexation” and “percent complexation” are used interchangeably and are defined to mean the percentage of the ion that is successfully complexed with the bifunctional chelant.
• percent complexation is expressed as radiochemical yield, which is the yield of radiolabeled complex expressed as a fraction of the radioactivity originally present.
• the value of radiochemical yield obtained when making the ion complexes of the present reaction can be greater than 90% or greater than 95%, as measured by reverse phase chromatography (HPLC).
• the conjugates of the present invention can be prepared by first forming the complex and then attaching to the biological carrier.
• the process involves preparing or obtaining the ligand, forming the complex with an ion and then adding the biological carrier.
• the process may involve first conjugation of the ligand to the biological carrier and then the formation of the complex with an ion. Any suitable process that results in the formation of the ion-conjugates of this invention is within the scope of the present invention.
• the complexes, bifunctional chelates and conjugates of the present invention are useful as diagnostic agents in the manner described.
• These formulations may be in kit form such that the two components (i.e., ligand and metal, complex and antibody, or ligand/antibody and metal) are mixed at the appropriate time prior to use. Whether premixed or as a kit, the formulations usually require a pharmaceutically acceptable carrier.
• Tissue specificity may also be realized by ionic or covalent attachment of the chelate of formula (I) (where R 6 is NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl group) to a naturally occurring or synthetic molecule having specificity for a desired target tissue.
• R 6 is NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl group
• chelate conjugated monoclonal antibodies which would transport the chelate to diseased tissue enabling visualization. The surgeon could then illuminate soft tissue with a UV light source coupled with an appropriate detector, if necessary, and surgically remove the indicated tissue.
• H 2 dedpa (originally named 3 ⁇ 4bpce) have been previously reported with divalent metals showing reasonable chelation properties. 26 Under mild reaction conditions (room temperature, aqueous buffer, pH 4), 3 ⁇ 4dedpa coordinated 67 Ga (a longer-lived Ga isotope that can serve as a model for 68 Ga) quantitatively within 10 minutes (as does NOTA 27 ). DOTA however, requires heating for quantitative reaction yields. 28 Concentration dependent coordination of H 2 dedpa at concentrations as low as 10 "7 M to both 68 Ga and 67 Ga showed quantitative conversion to the desired product.
• FIG. 9 illustrates the labelling trace of 67 Ga(dedpa) + on HPLC. (t R : 6.1 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 OH. 0-5% B linear gradient 20 min). Yield: 99%)
• FIG. 9 illustrates the labelling trace of 67 Ga(dedpa) + on HPLC. (t R : 6.1 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 OH. 0-5% B linear gradient 20 min). Yield: 99%)
• FIG. 9 illustrates the labelling trace of 67 Ga(dedpa) + on HPLC. (t R : 6.1 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 OH. 0-5% B linear gradient 20 min). Yield: 99%)
• FIG. 9 illustrates the labelling trace of 67 Ga(dedpa) + on HPLC.
• FIG. 12 illustrates the HPLC chromatogram for 67 Ga-transferrin (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 OH. 0-100% B linear gradient 20 min); reference for stability measurements of 67 Ga(dedpa) + .
• FIG. 13 illustrates stacked labelling traces of 67 Ga(dedpa) + of 2 h stability experiment against apo-transferrin (gradient: A: NaOAc buffer, pH 4.5, B: MeOH. 0-100% B linear gradient 20 min).
• FIG. 14 illustrates the labelling trace of competition between
• Compound 66 displays derivatization through the two aliphatic nitrogens, affording a scaffold capable of carrying two targeting molecules, while compound 69 is derivatized through the backbone of the ethylenediamine (en) component of the basic ligand structure, retaining the original coordination environment more closely, but only capable of carrying one targeting molecule.
• Both 66 and 69 incorporate the nitrobenzyl functionality which can be converted easily into the corresponding amino- or isothiocyanato-benzyl, coupling moieties frequently employed for conjugation to target molecules via a free carboxylate or primary amine, respectively.
• Compound 66 was synthesized from l,2- ⁇ 6-(methoxycarbonyl)pyridin-2- yl ⁇ methylaminoethane, 39 which is then subsequently alkylated with 4-nitrobenzyl bromide.
• Intermediate 65 is purified and the carboxylates are deprotected under standard conditions to afford the clean product 66 as a white solid.
• the complex with cold (non-radioactive) gallium(III) is formed within 2 h at pH 4-5 under gentle heating and again the C 2 rotational axis was confirmed in both the solid-state structure and the solution NMR spectrum (see FIGS. 6-8). Coordination to 67 Ga or 68 Ga forms the complex within 10 minutes at room temperature in 98% radiochemical yield.
• FIG. 15 illustrates the HPLC chromatogram for 67 Ga-transferrin (reference for stability measurements of 67 Ga(66) + and 67 Ga(69) + ), and FIGS.
• 16-17 illustrate stacked labelling traces of 67 Ga(66) + and 67 Ga(69) + of 2 h stability experiment against apo- transferrin (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 CN. 0-100% B linear gradient 20 min).
• 67 Ga(dedpa) + was supported by the low uptake in bone, which is known to be a site of increasing accumulation for weakly chelated 67 Ga. 41
• the overall biodistribution profile compares well to macrocyclic chelators evaluated in a similar study, also exhibiting the low uptake in liver and intestines characteristic of ionic compounds.
• the persistent high uptake in the blood serum was not confirmed with the derivatized compounds, suggesting that added functionality influences biodistribution.
• Serum stability studies done in vitro confirmed that 67 Ga(dedpa) + was stable to transchelation by serum proteins.
• H 2 dedpa complexes quickly with Ga, and forms complexes of very high stability, comparing well to the widely used macrocyclic chelator NOTA and exceeding the properties of DOTA.
• H 2 dedpa and its derivatives can be coordinated to Ga isotopes under mild room temperature conditions at high specific activities in short reaction times, making it an ideal scaffold for further elaboration and applications such as peptide labeling.
• the high radiochemical yield and high specific activity of the products could obviate the need for time consuming HPLC purification, a major advantage for the short lived isotope 68 Ga.
• Ga or 6? Ga can be formed at a ligand concentration of 10 7 M.
• a specific radioactivity of up to 9.8 mCi/ nmol can be achieved when preparing a complex of dedpa with 68 Ga with a radiolabeling yield of above 97%.
• IR spectra were collected neat in the solid state on a Thermo Nicolet 6700 FT-IR spectrometer.
• HPLC analysis or purification of non-radioactive compounds was done on a Phenomenex Synergi 4 mm Hydro-RP 80 A column (250 x 4.6 mm) in a Waters WE 600 HPLC system equipped with a 2478 dual wavelength absorbance UV detector run controlled by Empower software package.
• the HPLC system used for analysis of the radiochemical complexes consists of a Waters Alliance HT 2795 separation module equipped with a Raytest Gabbistar Nal detector and a Waters 996 photodiode array (PDA) detector.
• PDA Waters 996 photodiode array
• 67 Ga was obtained as a 0.1 M HC1 solution
• 68 Ga (5-10 mCi/mL) (both MDS Nordion Inc.) was obtained from a generator constructed of titanium dioxide sorbent that was charged with 68 Ge and eluted with aqueous HC1 (0.1M). 42
• the generator has been previously used for radiolabeling NOT A- and DOTA-based chelate systems and the resulting radiochemical yields and specific activities achievable for these chelates using this generator have been reported.
• FIG. 6A illustrates the ⁇ -NMR (DMSO-d 6 , 300 MHz) of H 2 dedpa-2HC1.
• Ga(dedpa) CIO4 [00145] Ga(dedpa)CI0 4 .
• H 2 dedpa-2HC1 (21 mg, 0.052 mmol) was dissolved in a CH 3 OH- water mixture (1 :2).
• Ga(C10 4 ) 3 -6H 2 0 (24mg, 0.052 mmol) was added and the pH was adjusted to 4.5 by addition of 0.1 M NaOH.
• the reaction mixture was heated for 30 minutes and then set aside in the fume hood for slow evaporation. After 72 h, rhombic colorless crystals suitable for X-ray diffraction had precipitated in
• radiolabeled products were identified by comparison of the radiation detector trace and the UV/visible detector trace of a cold complex as a standard, where the cold complex is the non-radioactive gallium complex that has been prepared and characterized to confirm its chemical identity. Results from radiolabelling are summarized in Table 1.
• Entry 5 in Table 1 shows the fast, high radiochemical yield incorporation of 6 7 Ga into the dedpa chelate.
• the radiochemical yield after only 10 minutes under mild room temperature aqueous conditions was 99% as determined by HPLC.
• Chelates with small structural differences do not show the same rapid, high yield, radiolabeling under mild conditions as dedpa.
• entry 6 which differs from dedpa by one extra carbon between the primary amines
• entry 2 which differs from dedpa by the carboxylate coordinating groups attached to the alkyl amine rather than the pyridyl ring, do not achieve >95%> radiochemical yield in 10 minutes even at increased temperatures.
• Dedpa shows high affinity for Ga and fast radiolabeling kinetics, even when compared to similar chelates.
• Entry 8 which contains two extra carboxylate groups compared to dedpa also gave high radiochemical yields (95%) under mild conditions in 10 minutes.
• This chelate is the basis for a bifunctional chelate that retains the chelate structure of dedpa, while allowing conjugation to biomolecules via the additional two carboxylate arms.
• the chelators 1-6 (Table 2) were made up as stock solutions (1 mg/mL, -10 " 3 M), which were then diluted into buffered labeling solutions (100 ⁇ ⁇ ⁇ "3 M chelate stock solution into 900 ⁇ of pH 4.5. 10 mM NaOAc buffer, final working solution of -10 "4 M). A 7 ⁇ , aliquot of the 1 ' l In stock solution (7 mCi / 10 ⁇ ) was diluted with 93 of deionized water to make a working 6.5 mCi / 100 ⁇ , 11 ⁇ solution.
• the HPLC system used for analysis consisted of a Waters Alliance HT 2795 separation module equipped with a Raytest Gabi Star Nal ( 11) detector and a Waters 996 photodiode array (PDA) detector.
• PDA photodiode array
• a Phenomenex Hydrosynergy RP CI 8 4.6 mm * 150 mm analytical column was used for all radiolabeled chelate complexes. Elution conditions used were gradient: A: 10 mM NaOAc buffer pH 4.5, B: CH 3 CN. 0-100% B linear gradient 20 min).
• PD-10 columns were discarded after a single use.
• the vial containing the eluent was then measured with a Capintec scintillation counter to determine the amount of 1 1 'in bound to transferrin.
• the difference in radioactivity between the competition experiment solutions and the eluent from the PD-10 columns was used to determine the percent 1 1 1 In bound to transferrin, and therefore the percent stability of the complexes.
• the dedpa chelate gave the same high radiochemical yields under mild room temperature conditions within 10 minutes with 68 Ga as was shown in example 1 with 6 7 Ga.
• serial dilutions of the chelate solution used in labelling it was determined that a 0.1 ⁇ chelate concentration was needed for efficient radiolabeling.
• the maximum specific activity, defined here as mCi of isotope / nmol of chelate, achieved was 10 mCi/nmol without post labelling purification.
• FIG. 9 illustrates the labelling trace of 67 Ga(dedpa) + on HPLC. (t R : 6.1 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 OH. 0-5% B linear gradient 20 min). Yield: 99%).
• Transferrin is an iron transport protein found in high concentrations in the blood that has an affinity for Ga due to the similar ionic size and charge of Ga 3+ and Fe 3+ .
• Direct competition of Ga(dedpa) + with apo-transferrin gives an indication of the radiolabeled complexes' expected stability in vivo. As no decomposition was detected within 2 h, Ga(dedpa) + is expected to have in vivo stability appropriate for imaging with 68 Ga.
• 67 GaCl 3 was added into 10 "4 M solution of NOTA in 10 mM NaOAc solution (pH 4.5). Complete complexation of the 67 Ga 3+ by NOTA was confirmed by HPLC. An equivalent amount of the dedpa ligand was then added to the 67 Ga-NOTA product. After 10 minutes at room temperature the reaction mixture was analyzed by HPLC. Over 98% of the 67 Ga-dedpa complex was formed as opposed to only 0.2 % Ga-NOTA, confirming that the acyclic dedpa chelate has faster complexation kinetics than the macrocyclic NOTA chelate, as would be expected.
• Ga-NOTA complex When the Ga-NOTA complex is pre-formed and then an equivalent amount of dedpa is added, Ga is transchelated from the Ga-NOTA complex to form Ga- dedpa. About 10% of Ga-NOTA was converted to Ga-dedpa in 10 minutes. As Ga- NOTA is known to be very stable, this result was unpredicted and indicates greater affinity of Ga for dedpa.
• 6-methylpicolinic acid (4.86 g, 35.5 mmol), boron trifluoride etherate (0.709 mL, 20 ⁇ 7 ⁇ 1 6-methylpicolinic acid), and t-butyl 2,2,2-trichloroacetimidate (12.7 mL, 70.91 mmol, 2 eq.) in DCM (200 mL) were added to a round-bottomed flask and refluxed for two days. NaHC0 3 was added to quench residual BF 3 . The mixture was stirred for 10 minutes. The solid was filtered off, the filtrate was collected and the solvent was removed in vacuo. Hexane was added to the milky oil to precipitate residual starting materials, which could be filtered off.
• N-bromosuccinimide 1.95 g, 10.99 mmol
• 6-methylpicolinic acid tert-butyl ester 2.1 175 g, 10.96 mmol
• benzoyl peroxide 0.27 g, 1.10 mmol, 0.1 eq.
• FIG. 7A illustrates the ⁇ -NMR (MeOD-d 4 , 300 MHz) of H 2 66.
• FIG. 7B illustrates the ⁇ -NMR (MeOD-d 4 , 300 MHz) of Ga(66)N0 3 .
• 67/ 8 Ga(66) + 100 of 67 GaCl 3 or 68 Ga + (1 mCi) in a 0.1 M HC1 solution was added into 10 "4 M solution of ligand in 10 mM NaOAc solution (pH 4) and left to react for 10 minutes at room temperature. Reaction control was performed by analytical HPLC which showed that the reaction had proceeded to 98%. Product on HPLC: 10.8 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 OH. 0-100% B linear gradient 20 min). For the apo-transferrin competition, 67 GaCl 3 was added to 10 " 4 M solution of 66 in 10 mM NaOAc solution (pH 4.5).
• FIG. 10 illustrates the labelling trace of 67 Ga(66) + on HPLC. (t R : 10.8 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 CN. 0-100% B linear gradient 20 min). Yield: 98%) [00167] 2-(p-Nitrobenzyl)-N, N'-6- ⁇ methoxycarbonyl ⁇ -pyridin-2-yl
• methylamino)ethane (68).
• 67 and 4 were synthesized according to the literature. 39 ' 40 To a mixture of 67 (0.46 g, 2.36 mmol) in methanol (50 mL), 4 (0.78 g, 4.72 mmol) was added. The mixture was refluxed for 2 h and then cooled to 0 °C in an ice bath. After cooling, NaBH 4 (0.139 g, 3.67 mmol) was added slowly and stirred at 0 °C for 2 h. Saturated aqueous NaHC0 3 was then added (150 mL) and the mixture stirred for 15 min, followed by extraction with DCM (5 x 80 mL).
• FIG. 8A illustrates the ⁇ -NMR (MeOD-d 4 , 300 MHz) of H 2 69.
• Ga(N0 3 ) 3 -6H 2 0 (2 mg, 5.5 ⁇ ) was added and the pH was adjusted to 5 by addition of 0.1 M NaOH .
• the reaction mixture was stirred at 60 °C for 2 h.
• the solvent was removed in vacuo to afford an off-white solid in quantitative yield.
• FIG. 8B illustrates the ⁇ -NMR (MeOD-d 4 , 300 MHz) of Ga(69)N0 3 .
• FIG. 11 illustrates the labelling trace of 67 Ga(69) + on HPLC. (t R : 7.7 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 CN.
• Tables 4-6 show the biodistibution data of 67 Ga(dedpa) + , 67 Ga(66) + and 67 Ga(69) + , respectively, in female ICR mice.
• FIG.4 illustrates biodistribution over 4h of 67 Ga(dedpa) + in female ICR mice
• FIG.5 illustrates biodistribution of 7 Ga(66) + (upper) and 67 Ga(69) + (lower) in female ICR mice over 4h; complete data for urine are shown also in separate diagrams to the right.
• Results are expressed as the mean ⁇ standard deviation of %ID/g (4 animals used per time point)
• the SQUEEZE 5 1 program was used to generate a data set free of residual electron density in that region. All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in calculated positions and refined using a riding model. Tables 8 and 9 provide summaries of the relevant crystal lographic data for both compounds. Table 8. Relevant solid state crystallographic data for the complex Ga(dedpa)C10 4
• FIG.3 illustrates the solid state structure of the cation in Ga(66)C10 4 (relevant bond lengths A: Nl-Ga: 1.992(5); N2-Ga: 1.981(5); N3-Ga: 2.188(5); N4- Ga: 2.159(5); Ol-Ga: 1.967(4); 02-Ga: 1.976(4)).
• the Ga complex of 75 was synthesized according to a standard procedure.
• Serum stability challenge 10 min 96 % 95 %
• Retention time was measured using a Waters XBridge BEH130 4.6 * 150 mm (gradient: A: NaOAc buffer, pH 4.5, B: CH 3 CN, 0-100% B linear gradient 20 min) * Retention time was measured using a Waters XBridge BEH130 4.6 ⁇ 150 mm (gradient: A: H 2 0, 0.1 % TFA, B: CH 3 CN, 5-100% B linear gradient 30 min)
• Ga 68 labeling 10 "6 M ligand cone, 97 %, 0.45 mCi/ nmol
• Ga 68 labeling 10 "6 M ligand cone, 96.5 %, 0.45 mCi/ rrmol
• Ga 68 labeling 10 "6 M ligand cone., 95 %, 0.45 mCi/ nmol
• Ga 68 labeling 10 "6 M ligand cone, 94 %, 0.45 mCi/ nmol
• the first two steps include a two-step methylation of the two carboxylates and the alcohol according to reference 52 , followed by a standard partial reduction according to the literature 53 , followed by bromination of the alcohol 54 to afford 94.
• N, N"-(benzyl)diethylenetriamine (101) Diethylenetriamine 100 (420 ⁇ , 3.877 mmol) and benzaldehyde (830 ⁇ , 8.142 mmol) were dissolved in methanol (30 mL) and refluxed overnight. The solvent was evaporated and then diethyl ether was added to try to precipitate the crude diimine product, but was unsuccessful. The crude diimine product (0.247 g, -0.621 mmol) was dissolved in methanol (20 mL), cooled to 0°C in an ice bath, and NaBH 4 (43 mg, 1.1 18 mmol) was slowly added. After 2.5 hours saturated NaHC0 3 (20 mL) was added.
• N, N"-(benzyl)diethylenetriamine (0.140 g crude, 0.434 mmol, 101) was dissolved in acetonitrile (30 mL) with Na 2 C0 3 (600 mg). 6-bromomethylpyridine-2-methoxycarbonyl 102 (0.302 g, 1.304 mmol), was added, and the mixture was refluxed overnight under argon gas. Excess Na 2 C0 3 was filtered and discarded, and deionized water (30 mL) was added.
• the crude product was subsequently extracted with chloroform (5 x 30 mL). The organic extracts were combined and dried over anhydrous MgSC>4, filtered, and then evaporated to dryness.
• the crude product (0.2932 g) was purified by column chromatography twice; the first time starting the elution with CH 2 Cl 2 :triethylamine (95:5), and then switching to CH2Cl 2 :MeOH:triethylamine (92.5:5:2.5), and a second time with hexanes:ethyl acetate (80:20). The product still contained impurities and was used without further purification. Rf. 0.60, 90: 10 CH 2 Cl 2 :MeOH + 1 % triethylamine). HR-ESI-MS calcd. for C42H47N6O6: 731.3557: found M + H + 731.3547.
• Diethylenetriamine (66 ⁇ iL, 0.606 mmol) and methyl-6-formylpyridine-2-carboxylate (0.200 g, 1.21 mmol, 4) were dissolved in methanol (20 mL) and refluxed overnight. The solvent was evaporated and then diethyl ether was added to try to precipitate the crude diimine product, but was unsuccessful.
• the crude diimine product (0.247 g, -0.621 mmol) was dissolved in methanol (20 mL), cooled to 0°C in an ice bath, and NaBH 4 (43 mg, 1.1 18 mmol) was slowly added. After 2.5 hours saturated NaHC0 3 (20 mL) was added.

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