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WO2002040060A2 - Conjugates of an antioxidants with metal chelating ligands - Google Patents

Conjugates of an antioxidants with metal chelating ligands Download PDF

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Publication number
WO2002040060A2
WO2002040060A2 PCT/US2001/046002 US0146002W WO0240060A2 WO 2002040060 A2 WO2002040060 A2 WO 2002040060A2 US 0146002 W US0146002 W US 0146002W WO 0240060 A2 WO0240060 A2 WO 0240060A2
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Prior art keywords
compound
produce
iii
stracture
contacting
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PCT/US2001/046002
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French (fr)
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WO2002040060A3 (en
Inventor
Ramachandra S. Ranganathan
Helen Fan
Michael F. Tweedle
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Bracco International Bv
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Application filed by Bracco International Bv filed Critical Bracco International Bv
Priority to EP01987230A priority Critical patent/EP1406669A2/en
Priority to AU2002239468A priority patent/AU2002239468A1/en
Priority to JP2002542432A priority patent/JP2004526671A/en
Priority to CA002421182A priority patent/CA2421182A1/en
Priority to US10/399,265 priority patent/US7160535B2/en
Publication of WO2002040060A2 publication Critical patent/WO2002040060A2/en
Publication of WO2002040060A3 publication Critical patent/WO2002040060A3/en
Priority to US11/561,638 priority patent/US7407644B2/en
Priority to US12/169,105 priority patent/US7582280B2/en

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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
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • 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
    • A61K51/041—Heterocyclic compounds
    • A61K51/0412—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K51/0419—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • 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
    • A61K51/088—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 diagnostic and therapeutic compositions, methods of their use, and processes of their preparation.
  • vitamin C vitamin C
  • 2-O-octadecylascorbic acid an ascorbic acid derivative with antioxidant properties, 2-O-octadecylascorbic acid, has been prepared and has been shown to markedly inhibit the myocardial lesions induced by ischemia- reperfusion treatment in rats.
  • Ascorbic acid may also bind to the human serum albumin (HS A) weakly with a binding constant of about 3.5 x 10 4 M "1 .
  • Ascorbic acid and other antioxidants have been used to stabilize radiopharmaceuticals by decreasing the oxidation of substituents due to radical reactions induced by the decay of the radionuclide.
  • Metal chelating ligands are designed for use in Nuclear Medicine, Magnetic Resonance Imaging (MRI), and neutron capture therapy applications.
  • Magnetic resonance (hereinafter sometimes referred to as MR) imaging is widely used for obtaining spatial images of parts of a patient for clinical diagnosis.
  • the image is obtained by placing the patient in a strong external magnetic field and observing the effect of this field on the magnetic properties of protons contained in and surrounding the organ or tissue of the patient.
  • the proton relaxation times, called Ti or spin-lattice or longitudinal relaxation time, and T 2 or spin-spin'or transverse relaxation time depend on the chemical and physical environment of the organ or tissue being imaged.
  • a diagnostic agent is administered intravenously (hereinafter sometimes referred to as IV.) and is taken up by the organs, such as the liver, spleen, and lymph nodes to enhance the contrast between healthy and diseased tissues.
  • contrast agents used in MR imaging derive their signal-enhancing effect from the inclusion of a material exhibiting paramagnetic, ferrimagnetic, ferromagnetic or superparamagnetic behavior. These materials affect the characteristic relaxation times of the imaging nuclei in the body regions into which they distribute causing an increase or decrease in MR signal intensity.
  • contrast agents such as those of the present invention, that selectively enhance signal intensity in particular tissue types, as most MR contrast agents are relatively non-specific in their distribution.
  • Nuclear medicine procedures and treatments are based on internally distributed radioactive materials, such as radiopharmaceuticals or radionuclides, which emit electromagnetic radiations as alpha or beta particles or as gamma rays or photons.
  • radioactive materials such as radiopharmaceuticals or radionuclides, which emit electromagnetic radiations as alpha or beta particles or as gamma rays or photons.
  • gamma rays are readily detected and quantified within the body using instrumentation such as scintillation and gamma cameras.
  • Compounds derivatized with alpha or beta emitters may be used for radiotherapeutic applications, providing an internal dose of cytotoxic radiation at their target tissues(s).
  • the invention relates to conjugates of an antioxidant and one or more metal chelating ligands that may be chelated to radioactive or non-radioactive metals and use of such conjugates chelated to such metals as, for example: a) Magnetic resonance diagnostic compositions for visualization of tissues and compartments that bind or utilize an antioxidant conjugated to metal chelates; b) Radiodiagnostic compositions for visualization of tissues, comprising ligands chelated to radioactive gamma-emitting metals and coupled to said antioxidant conjugates; and c) Compositions for radiotherapy or for neutron capture therapy, comprising ligands chelated to radioactive alpha or beta-emitting metals or to metals suitable for neutron capture therapy and coupled to said antioxidant conjugates.
  • the invention provides novel conjugates of antioxidants and metal chelating ligands.
  • the invention also provides novel intermediates, methods of making the conjugates and intermediates, methods of stabilizing radiopharmaceutical ligands, and kits for preparing radiopharmaceuticals.
  • Antioxidants which may be used in the present invention include ascorbic acid, » ⁇ r -aminobenzoic acid (PABA), cysteine, monothioglycerol, and gentisic acid. Ascorbic acid is a preferred antioxidant of the invention.
  • the invention provides a compound having the following chemical structure:
  • M is 9 y 9 y m m Tpccountry 6 Ho, 165 Dy, 64 Cu, 67 Cu, 97 Ru, 103 Ru. °Re, 100 Re, 3 Pb, Bi, 21 l 2Bi, 21 l 3 J ⁇ Bi, 21 l 4 ⁇ Bi,
  • X is CH 2 , an amino acid, a peptide, a protein, or an antibody.
  • X is CH 2 .
  • X is the amino acid represented by the chemical structure:
  • the metal (M) is 99m Tc, 186 Re, 188 Re, 90 Y, 88 Y, 86 Y, 177 Lu, or gadolinium (III).
  • kits for preparing a radiopharmaceutical include an oxidant covalenffy bound to a complexing (or radiopharmaceutical ligand).
  • the kit includes a targeting molecule bound to the antioxidant, the ligand, or, most preferably, both, hi certain of these embodiments, the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
  • the invention provides methods of stabilizing a radiopharmaceutical ligand, which optionally includes a targeting molecule, by conjugating the radiopharmaceutical ligand with an antioxidant.
  • DESCRIPTION OF THE INVENTION The following abbreviations are used in this specification: "DOTA” means 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid; "HATU” means
  • DLEA means diisopropylethylamine
  • DMF means N,N-dimethylformamide
  • TsCl means p-toluenesulfbnyl chloride
  • THF means tetrahydrofuran
  • TFA trifluoroacetic acid
  • RT room temperature
  • radiopharmaceutical ligand are used interchangeably throughout this specification, except where the context requires otherwise.
  • the present invention is directed, in part, to conjugates of an antioxidant, such as ascorbic acid, and one or more polydentate macrocyclic or non- macrocyclic metal-chelating ligand residues that are optionally chelated to radioactive or non-radioactive metals capable of either being detected by imaging means for diagnosis or capable of providing a therapeutic or radiotherapeutic effect.
  • the metal chelating groups can be either macrocyclic or non-macrocyclic multidentate metal chelating ligands, and the structure of these ligands and the metals that are chelated to them may be varied depending on the use envisioned for them.
  • conjugates of the present invention may be used for radiodiagnostic or radiotherapeutic purposes.
  • an antioxidant such as ascorbic acid, is conjugated to a chelating ligand, which form stable complexes with radioactive metals.
  • the chelating ligands that may be used in the practice of the present invention are not particularly limited and are well known to those skilled in the art.
  • Such ligands include, for example, Oxa-PnAO ligands and peptide analogue chelators, such as those with an N 3 S configuration.
  • Radioactive metals include the elements having atomic numbers of 22 to 29, 42, 44 and 58-70.
  • radioactive isotopes include: 99m Tc, 51 Cr, 67 Ga, 68 Ga, m In, 168 Yb, 140 La, 90 Y, 88 Y, 86 Y, 153 Sm, 166 Ho, 165 Dy, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 215 Bi, and 177 Lu.
  • Oxa-PnAO ligands or N,N-Me 2 -Gly-Ser-Cys-Gly are preferably used to form conjugates with an antioxidant, such as ascorbic acid.
  • the antioxidants used in the invention are not particularly limited, provided that the antioxidant can be conjugated to a ligand and/or targeting molecule, as described herein.
  • antioxidants which may be used in the present invention include ascorbic acid, ⁇ r ⁇ -aminobenzoic acid (PABA), cysteine, monothioglycerol, and gentisic acid. Of these, ascorbic acid is preferred.
  • the conjugates may further comprise targeting molecules such as, for example, proteins, peptides and antibodies that localize to desired areas of the body.
  • Preferred targeting molecules are peptides or analogues thereof and may include a monomer or multimer of one or more peptides.
  • suitable targeting molecules include gastrin releasing peptide (GRP) agonists, such as those disclosed in U.S. Patent No. 6,200,546, incorporated herein by reference in its entirety.
  • GRP gastrin releasing peptide
  • Other useful targeting molecules include those disclosed in U.S. Patent Nos. 5,662,885; 5,780,006; and 5,976,495, incorporated herein by reference in their entirety, and particularly monomers or multimers of TKPPR or analogues thereof.
  • Analogues of a peptide include molecules that target the peptide's receptor with the same or greater avidity, as well as muteins, retropeptides and retroinversion peptides.
  • these analogues may also contain modifications such as substitutions, deletions and/or additions of one or several amino acids, insofar as these modifications do not alter the biological activity of the peptide in a significantly negative manner.
  • General structures of Oxa-PnAO ligands are detailed in U.S. Patent No.
  • N 3 S radionuclide chelators Structures and preparation of peptide-derived N 3 S radionuclide chelators are discussed in U.S. Patent Nos. 5,662,885; 5,780,006 and 5,976,495, each of which is incorporated herein by reference in its entirety.
  • Particularly preferred N 3 S chelators are N,N-dimethyl-Gly-Ser-Cys-Gly and N,N-dimethyl-Gly-t- butylGly-Cys-Gly.
  • Radiopharmaceutical conjugates (either diagnostic or therapeutic) of the present invention confer the added benefit of introducing an antioxidant (such as ascorbic acid) in close proximity to the oxidizable groups on the radiopharmaceutical (either diagnostic or therapeutic).
  • an antioxidant such as ascorbic acid
  • Such oxidizable groups may include, for example, peptides containing methionine or free thiols. Further, such oxidizable groups may be located on either the ligand or a targeting molecule. This covalent attachment of an antioxidant and the chelator (optionally coupled to a targeting ligand) provides additional stability due to the close proximity of the antioxidant to substituents on the radiopharmaceutical that are susceptible to oxidation induced by the decay of the radionuclide.
  • the ester of 6-hydroxy ascorbic acid retains a number of useful antioxidant properties.
  • the amide bond introduced into 6-hydroxy ascorbic acid in the compounds disclosed herein is expected to have greater serum stability than the ester compounds previously disclosed, and thus, to exhibit antioxidant behavior. Therefore, the ascorbic acid or other antioxidant derivatives of the invention are expected to retain their antioxidant properties when conjugated to the chelator and/or targeting molecules, thereby improving the stability of the conjugates.
  • the antioxidant may be attached to the targeting molecule, which is attached to the chelating ligand.
  • the antioxidant may be attached to the C-terminus of a peptide targeting molecule or via the beta or gamma carboxyl group of an aspartic or glutamic acid in the peptide.
  • the 6-amino ascorbic acid could be attached to the N-terminus of the peptide via a di-carboxylic acid such as succinic acid.
  • the antioxidant may be attached to the chelating ligand as shown herein, or using methods known to those skilled in the art.
  • paramagnetic metals include the elements having atomic numbers of 22 to 29, 42, 44 and 58-70.
  • Examples of such metals are chromium (TIT), manganese (II), iron (II), iron (III), cobalt (LI), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium, (LU), dysprosium (111), holmium (Hi), erbium (111) and ytterbium (III). Chromium (III), manganese (II), iron (III) and gadolinium (111) are preferred.
  • conjugates 22a and 22b are prepared, conjugates 22a and 22b. These conjugates have the following structure:
  • DOTA-G-tri- t-butyl ester 4 an intermediate compound in the synthesis of the conjugate 22, was synthesized starting with glycine benzylester hydrochloride (Aldrich), as described in Scheme 1. Chloroacetylchloride (Aldrich) was added to glycine benzylester hydrochloride in the presence of K 2 CO 3 to produce N-(chloroacetyl)-glycine benzyl ester 1. The ester 1 was added to a suspension of DO3A-tri-t-butyl ester hydrochloride 2 (see U.S. Patent No. 5,573,752) in K 2 CO 3 to produce DOTA-G-tri-t-butyl-benzyl ester 3.
  • Relaxivity of a paramagnetic material in the presence of a large protein such as human seram albumin may be used to study the ability of the compounds of the present invention to bind the target protein.
  • a large protein such as human seram albumin
  • the relaxivity of the former will increase because of an increase in its rotational correlation time. This increase in relaxivity may be used not only to measure the extent of binding but also to evaluate the viability of the paramagnetic agent as a blood pool contrast medium.
  • the relaxivity of the conjugates 22a and 22b in water as well as in water containing a known amount (20%, v/v) of a HSA preparation, known as, seronom (Table 1) were studied.
  • the relaxation time of the samples (22a or 22b) in seronom (TlGd-iigandin s eronom) and in water (Tlod-iigand in water) were measured at 38°C using an IBM PC/20 multispec relaxometer.
  • the sample in aqueous seronom was placed in a centrifree micropartition device (Millipore, Beverly, MA). The device was centrifuged at 500xg for 45 min in a fixed angle rotor (Beckman Model J2-21M, JA-20 rotor). The solution (0.5 mL) was taken from below the filter (unbound Gd-ligand) for [Gd] ICP measurement. The [Gd] of the uncentrifuged sample was also measured as a control. The Fraction Bound was calculated by the following equation:
  • Fraction Bound ([Gd-ligand] contro ⁇ - [Gd-ligand] un 0 und)/ [Gd- ligand]controi- Table 1 details the results of the tests.
  • Table 1 shows that the increase in relaxivity in the presence of seronom is 32% and 14% in the case of 22a and 22b, respectively. Based on these results, it is believed that the binding of these conjugates with HSA may not be strong enough for commercial blood pool MRI applications. However, the described conjugates 22a and 22b may be useful as extravascular MRI contrast agents. Moreover, the chemistry described and the conjugates made herein may be used in other applications such as those discussed herein and may be particularly useful where antioxidant properties of ascorbic acid may be required. It is understood that, for radiopharmaceutical or radiotherapy applications, it is convenient to prepare the complexes of the present invention at, or near, the site where they are to be used.
  • the amount administered may be selected based on the desired use, such as to produce a diagnostic image of an organ or other site of a subject or a desired radiotherapeutic effect, by methods known in the art. Exemplary dosages are those employing about 2-200 mCi rhenium, lutetium, or yttrium (for radiotherapy), or about 10-60 mCi technetium (for imaging).
  • Kits of the present invention comprise one or more vials containing the sterile formulation of a predetermined amount of a complexing ligand, an oxidant and optionally other components such as reducing agents, transfer ligands, buffers, lyophilization aids or bulking agents, stabilization aids, solubilization aids and bacteriostats.
  • the inclusion of one or more optional components in the formulation will frequently improve the ease of synthesis of the radiopharmaceutical by the practicing end user, the ease of manufacturing the kit, the shelf-life of the kit, or the stability and shelf-life of the radiopharmaceutical.
  • the improvement achieved by the inclusion of an optional component in the formulation must be weighed against the added complexity of the formulation and added cost to manufacture the kit.
  • the one or more vials that contain all or part of the formulation can independently be in the form of a sterile solution or a lyophilized solid.
  • Buffers useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of the radiopharmaceuticals include but are not limited to phosphate, citrate, sulfosalicylate, and acetate. A more complete list can be found in the United States Pharmacopeia. Lyophilization aids or bulking agents useful in the preparation of diagnostic kits useful for the preparation of radiopharmaceuticals are known in the art and include lactose, sodium chloride, maltose, sucrose, PEG 8000, cyclodextrins, such as hydroxypropyl- ⁇ -cyclodextrin (HP- ⁇ -CD), dextran, Ficoll, and polyvinylpyrrolidine (PVP).
  • Lyophilization aids or bulking agents useful in the preparation of diagnostic kits useful for the preparation of radiopharmaceuticals are known in the art and include lactose, sodium chloride, maltose, sucrose, PEG 8000, cyclodextrins, such as hydroxypropyl-
  • Stabilization aids such as antioxidants, useful in the preparation of radiopharmaceuticals and in diagnostic kits for the preparation of radiopharmaceuticals include but are not limited to ascorbic acid, para- aminobenzoic acid (PABA), cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, and inositol.
  • PABA para- aminobenzoic acid
  • cysteine monothioglycerol
  • sodium bisulfite sodium metabisulfite
  • gentisic acid sodium metabisulfite
  • inositol inositol.
  • conjugates of ascorbic acid are exemplified, the invention includes conjugates of other antioxidants.
  • one or more additional stabilzation aids may be added to formulations of the conjugates of the invention.
  • Solubilization aids useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of the radiopharmaceuticals include but are not limited to ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monooloeate, polysorbates, poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers
  • Preferred solubilizing aids are polyethylene glycol, and
  • Bacteriostats useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of the radiopharmaceuticals include but are not limited to benzyl alcohol, benzalkonium chloride, chlorbutanol, and methyl, propyl or butyl paraben.
  • a component in a diagnostic kit can also serve more than one function.
  • a reducing agent can also serve as a stabilization aid
  • a buffer can also serve as a transfer ligand
  • a lyophilization aid can also serve as a transfer, ancillary or co- ligand and so forth.
  • the predetermined amounts of each component in the formulation are determined by a variety of considerations familiar to those skilled in the art. These considerations are in some cases specific for that component and in other cases dependent on the amount of another component or the presence and amount of an optional component. In general, the minimal amount of each component is used that will give the desired effect of the formulation.
  • the desired effect of the formulation is that the practicing end user can synthesize the radiopharmaceutical and have a high degree of certainty that the radiopharmaceutical can be safely injected into a patient and will provide diagnostic information about the disease state of that patient.
  • EXAMPLE 1 The synthesis of methyl (3aS,3bRJaS,8aR)-2,2,5,5- tetramethyltefrahydro-8aH-[l,3]dioxolo[4,5]furo[3,2-d][l,3]dioxine-8a- carboxylate 10 is detailed.
  • Example 3A From sulfite 12 and sulfate 13 (Scheme 2) i) Methyl (4aS,5aR,8aS,8bR)-7J-dimethyltetrahydro-5aH-
  • Example 3B From the tosylate 17 (Scheme 3) i) Methyl (3aS,5R,6S,6aR)-6-hydroxy-2,2-dimethyl-5-( ⁇ [(4- methylphenyl)sulfonyl]oxy ⁇ methyl)dihydrofuro[2,3-d][l,3]dioxole-
  • Ci 0 Hi 5 N 3 O 6 C 43.96, H 5.53, N 15.38, O 35.13%.
  • EXAMPLE 6 The synthesis of (3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5- ⁇ [(N- ⁇ [4,7, 10-tris(2-tert-butoxy-2-oxoethyl)- 1 ,4,1, 10-tet ⁇ aazacyclododecan- 1 - yl]acetyl ⁇ glycyl)amino]methyl ⁇ dihydrofuro[2,3-d][l,3]dioxole-3a(5H)- carboxylic acid 20a is detailed.
  • EXAMPLE 7 The synthesis of 1,4,7, 10-tetraazacyclododecane-l,4J-triacetic acid, 10- [2-[[2-[[(2R)-2-[(2S)-2, 5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2- hy ⁇ roxyethyl]amino]-2-oxoethyl]amino]2-oxoetl yl]- 21a is detailed.
  • TLC Silica gel, R f 0.50, MeOH/CHCl 3 1/20.
  • TLC Silica gel, R f 0.75, MeOH/CHCl 3 1/10.
  • NCH7CH7 CH7CONHCH7CONH. CH7CONHCH7CONH & CH 2 adjacent to cyclohexyl ring); 3.82 (s, 3H, OCH 3 ); 3.95 (m, 2H, CH 2 adjacent to gulonic ring); 4.19, 4.90, 5.12 (m, 3H, CH's on the gulonic ring); 6.32, 6.48, 6.80 (t, 3H, NH's).
  • EXAMPLE 11 The synthesis of 1,4,7, 10-tetraazacyclododecane-l,4J-triacetic acid, 10- [2-[[[4-[[[[(2R)-2-[(2S)-2, 5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2- hydroxyethyl] amino] carbonyl] cyclohexyljmethyl] amino] -2-oxoethyl] amino]2- oxoethyl]- 21b is detailed.

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Abstract

The invention provides radiopharmaceuticals for diagnostic and therapeutic applications, conjugates of antioxidants with metal chelating ligands, intermediate compounds, methods of making such radiopharmaceuticals, ligands, and intermediate compounds, and kits for preparing the radiopharmaceutical complexes.

Description

CONJUGATES OF ANTIOXLDANTS WITH METAL CHELATING LIGANDS FOR USE IN DIAGNOSTIC AND THERAPEUTIC
APPLICATIONS
FIELD OF INVENTION The present invention relates to diagnostic and therapeutic compositions, methods of their use, and processes of their preparation.
BACKGROUND OF INVENTION Ascorbic acid (vitamin C) and other antioxidants, such as α-tocopherol, minimize tissue damage caused by oxidative metabolic processes and also have acceptable biological tolerance. Recently, an ascorbic acid derivative with antioxidant properties, 2-O-octadecylascorbic acid, has been prepared and has been shown to markedly inhibit the myocardial lesions induced by ischemia- reperfusion treatment in rats. Ascorbic acid may also bind to the human serum albumin (HS A) weakly with a binding constant of about 3.5 x 104 M"1. Ascorbic acid and other antioxidants have been used to stabilize radiopharmaceuticals by decreasing the oxidation of substituents due to radical reactions induced by the decay of the radionuclide.
Metal chelating ligands are designed for use in Nuclear Medicine, Magnetic Resonance Imaging (MRI), and neutron capture therapy applications. Magnetic resonance (hereinafter sometimes referred to as MR) imaging is widely used for obtaining spatial images of parts of a patient for clinical diagnosis. Typically, the image is obtained by placing the patient in a strong external magnetic field and observing the effect of this field on the magnetic properties of protons contained in and surrounding the organ or tissue of the patient. The proton relaxation times, called Ti or spin-lattice or longitudinal relaxation time, and T2 or spin-spin'or transverse relaxation time depend on the chemical and physical environment of the organ or tissue being imaged. In order to improve the clarity of the image, a diagnostic agent is administered intravenously (hereinafter sometimes referred to as IV.) and is taken up by the organs, such as the liver, spleen, and lymph nodes to enhance the contrast between healthy and diseased tissues.
The contrast agents used in MR imaging derive their signal-enhancing effect from the inclusion of a material exhibiting paramagnetic, ferrimagnetic, ferromagnetic or superparamagnetic behavior. These materials affect the characteristic relaxation times of the imaging nuclei in the body regions into which they distribute causing an increase or decrease in MR signal intensity. There is a need for contrast agents such as those of the present invention, that selectively enhance signal intensity in particular tissue types, as most MR contrast agents are relatively non-specific in their distribution.
Nuclear medicine procedures and treatments are based on internally distributed radioactive materials, such as radiopharmaceuticals or radionuclides, which emit electromagnetic radiations as alpha or beta particles or as gamma rays or photons. Following I.V., oral or inhalation administration, gamma rays are readily detected and quantified within the body using instrumentation such as scintillation and gamma cameras. Compounds derivatized with alpha or beta emitters may be used for radiotherapeutic applications, providing an internal dose of cytotoxic radiation at their target tissues(s).
SUMMARY OF THE INVENTION The invention relates to conjugates of an antioxidant and one or more metal chelating ligands that may be chelated to radioactive or non-radioactive metals and use of such conjugates chelated to such metals as, for example: a) Magnetic resonance diagnostic compositions for visualization of tissues and compartments that bind or utilize an antioxidant conjugated to metal chelates; b) Radiodiagnostic compositions for visualization of tissues, comprising ligands chelated to radioactive gamma-emitting metals and coupled to said antioxidant conjugates; and c) Compositions for radiotherapy or for neutron capture therapy, comprising ligands chelated to radioactive alpha or beta-emitting metals or to metals suitable for neutron capture therapy and coupled to said antioxidant conjugates.
In one embodiment, the invention provides novel conjugates of antioxidants and metal chelating ligands. The invention also provides novel intermediates, methods of making the conjugates and intermediates, methods of stabilizing radiopharmaceutical ligands, and kits for preparing radiopharmaceuticals. Antioxidants which may be used in the present invention include ascorbic acid, »αr -aminobenzoic acid (PABA), cysteine, monothioglycerol, and gentisic acid. Ascorbic acid is a preferred antioxidant of the invention.
In a preferred embodiment, the invention provides a compound having the following chemical structure:
wherein M is 9 y9ymmTpc„
Figure imgf000004_0001
6Ho, 165Dy, 64Cu, 67Cu, 97Ru, 103Ru. °Re, 100Re, 3Pb, Bi, 21l2Bi, 21l3J τBi, 21l4τBi,
215Bi, 177Lu, cliromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (11), praseodymium (III), neodymium (El), samarium (III), gadolinium (III), terbium, (ϋl), dysprosium (III), holmium (m), erbium (III) or ytterbium (III); and X is CH2, an amino acid, a peptide, a protein, or an antibody.
In one preferred embodiment, X is CH2. h another preferred embodiment, X is the amino acid represented by the chemical structure:
Figure imgf000004_0002
hi certain preferred embodiments, the metal (M) is 99mTc, 186Re, 188Re, 90Y, 88Y, 86Y, 177Lu, or gadolinium (III).
In another embodiment, the invention provides kits for preparing a radiopharmaceutical. Kits of the invention include an oxidant covalenffy bound to a complexing (or radiopharmaceutical ligand). In preferred embodiments, the kit includes a targeting molecule bound to the antioxidant, the ligand, or, most preferably, both, hi certain of these embodiments, the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
In yet another embodiment, the invention provides methods of stabilizing a radiopharmaceutical ligand, which optionally includes a targeting molecule, by conjugating the radiopharmaceutical ligand with an antioxidant.
DETAILED DESCRIPTION OF THE INVENTION The following abbreviations are used in this specification: "DOTA" means 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid; "HATU" means
O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate;
"DLEA" means diisopropylethylamine; "DMF" means N,N-dimethylformamide;
"TsCl" means p-toluenesulfbnyl chloride; "THF" means tetrahydrofuran;
"TFA" means trifluoroacetic acid; and "RT" means room temperature, h addition, the terms "chelating ligand," "complexing ligand," and
"radiopharmaceutical ligand" are used interchangeably throughout this specification, except where the context requires otherwise.
The present invention is directed, in part, to conjugates of an antioxidant, such as ascorbic acid, and one or more polydentate macrocyclic or non- macrocyclic metal-chelating ligand residues that are optionally chelated to radioactive or non-radioactive metals capable of either being detected by imaging means for diagnosis or capable of providing a therapeutic or radiotherapeutic effect. The metal chelating groups can be either macrocyclic or non-macrocyclic multidentate metal chelating ligands, and the structure of these ligands and the metals that are chelated to them may be varied depending on the use envisioned for them. For example, for compounds of the present application that are used for Magnetic Resonance Imaging applications, chelating polyaza macrocyclic ligands that form stable compounds with superparamagnetic or paramagnetic metals, and chelating ligands that provide enhanced relaxivity properties (vide infra) are preferred. For such applications, gadolinium is the preferred metal. hi a further embodiment, conjugates of the present invention may be used for radiodiagnostic or radiotherapeutic purposes. In this application, an antioxidant, such as ascorbic acid, is conjugated to a chelating ligand, which form stable complexes with radioactive metals. The chelating ligands that may be used in the practice of the present invention are not particularly limited and are well known to those skilled in the art. Such ligands include, for example, Oxa-PnAO ligands and peptide analogue chelators, such as those with an N3S configuration. Radioactive metals include the elements having atomic numbers of 22 to 29, 42, 44 and 58-70. For example, radioactive isotopes include: 99mTc, 51Cr, 67Ga, 68Ga, mIn, 168Yb, 140La, 90Y, 88Y, 86Y, 153Sm, 166Ho, 165Dy, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 215Bi, and 177Lu. The choice of metal ion will be determined based on the desired therapeutic or diagnostic application. Where 99mTc is the radioactive metal used, Oxa-PnAO ligands or N,N-Me2-Gly-Ser-Cys-Gly are preferably used to form conjugates with an antioxidant, such as ascorbic acid. The antioxidants used in the invention are not particularly limited, provided that the antioxidant can be conjugated to a ligand and/or targeting molecule, as described herein. For example, antioxidants which may be used in the present invention include ascorbic acid,^αrα-aminobenzoic acid (PABA), cysteine, monothioglycerol, and gentisic acid. Of these, ascorbic acid is preferred.
The conjugates may further comprise targeting molecules such as, for example, proteins, peptides and antibodies that localize to desired areas of the body. Preferred targeting molecules are peptides or analogues thereof and may include a monomer or multimer of one or more peptides. Examples of suitable targeting molecules include gastrin releasing peptide (GRP) agonists, such as those disclosed in U.S. Patent No. 6,200,546, incorporated herein by reference in its entirety. Other useful targeting molecules include those disclosed in U.S. Patent Nos. 5,662,885; 5,780,006; and 5,976,495, incorporated herein by reference in their entirety, and particularly monomers or multimers of TKPPR or analogues thereof. Analogues of a peptide include molecules that target the peptide's receptor with the same or greater avidity, as well as muteins, retropeptides and retroinversion peptides. One of ordinary skill will appreciate that these analogues may also contain modifications such as substitutions, deletions and/or additions of one or several amino acids, insofar as these modifications do not alter the biological activity of the peptide in a significantly negative manner. General structures of Oxa-PnAO ligands are detailed in U.S. Patent No.
6,093,382, which is incorporated by reference herein. These conjugates are intended for preparation of compounds for use in nuclear medicine and radiotherapy applications and are based on the general oxa-PnAO ligand class described in U.S. Pat. No. 5,608,110, which are incorporated by reference herein. For diagnostic applications, 99mTc is the preferred metal.
Structures and preparation of peptide-derived N3S radionuclide chelators are discussed in U.S. Patent Nos. 5,662,885; 5,780,006 and 5,976,495, each of which is incorporated herein by reference in its entirety. Particularly preferred N3S chelators are N,N-dimethyl-Gly-Ser-Cys-Gly and N,N-dimethyl-Gly-t- butylGly-Cys-Gly.
Radiopharmaceutical conjugates (either diagnostic or therapeutic) of the present invention confer the added benefit of introducing an antioxidant (such as ascorbic acid) in close proximity to the oxidizable groups on the radiopharmaceutical (either diagnostic or therapeutic). Such oxidizable groups may include, for example, peptides containing methionine or free thiols. Further, such oxidizable groups may be located on either the ligand or a targeting molecule. This covalent attachment of an antioxidant and the chelator (optionally coupled to a targeting ligand) provides additional stability due to the close proximity of the antioxidant to substituents on the radiopharmaceutical that are susceptible to oxidation induced by the decay of the radionuclide. Indeed, it has been reported that the ester of 6-hydroxy ascorbic acid retains a number of useful antioxidant properties. The amide bond introduced into 6-hydroxy ascorbic acid in the compounds disclosed herein is expected to have greater serum stability than the ester compounds previously disclosed, and thus, to exhibit antioxidant behavior. Therefore, the ascorbic acid or other antioxidant derivatives of the invention are expected to retain their antioxidant properties when conjugated to the chelator and/or targeting molecules, thereby improving the stability of the conjugates.
Specifically, where a targeting ligand is used, the antioxidant may be attached to the targeting molecule, which is attached to the chelating ligand. For example, where ascorbic acid is the antioxidant used, 6-amino ascorbic acid may be attached to the C-terminus of a peptide targeting molecule or via the beta or gamma carboxyl group of an aspartic or glutamic acid in the peptide. Similarly, the 6-amino ascorbic acid could be attached to the N-terminus of the peptide via a di-carboxylic acid such as succinic acid.
Alternatively, in the absence of a targeting molecule, the antioxidant may be attached to the chelating ligand as shown herein, or using methods known to those skilled in the art.
Examples of paramagnetic metals include the elements having atomic numbers of 22 to 29, 42, 44 and 58-70. Examples of such metals are chromium (TIT), manganese (II), iron (II), iron (III), cobalt (LI), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium, (LU), dysprosium (111), holmium (Hi), erbium (111) and ytterbium (III). Chromium (III), manganese (II), iron (III) and gadolinium (111) are preferred.
In one embodiment of the invention, two conjugates of ascorbic acid with DOTA were prepared, conjugates 22a and 22b. These conjugates have the following structure:
Figure imgf000009_0001
22a 22b
These two conjugates were synthesized as described below.
Scheme 1
Figure imgf000009_0002
DOTA-G-tri- t-butyl ester 4, an intermediate compound in the synthesis of the conjugate 22, was synthesized starting with glycine benzylester hydrochloride (Aldrich), as described in Scheme 1. Chloroacetylchloride (Aldrich) was added to glycine benzylester hydrochloride in the presence of K2CO3 to produce N-(chloroacetyl)-glycine benzyl ester 1. The ester 1 was added to a suspension of DO3A-tri-t-butyl ester hydrochloride 2 (see U.S. Patent No. 5,573,752) in K2CO3 to produce DOTA-G-tri-t-butyl-benzyl ester 3. Subsequent catalytic hydrogenation produced the DOTA-G-tri-t-butyl ester 4. Methyl 6-amino-6-deoxy-2,3-O-isopropylidene-2-keto-L-gulonate 16, which was a key protected intermediate for the synthesis of the conjugate 22, was synthesized starting from (2S,8S,lR,6R)-4,4,l l,l l-tetramethyl-3,5J,10,12- pentaoxatricyclo[6.4.0.0<2,6>]dodecane-6-carboxylic acid 9. The compound was synthesized as described in Scheme 2. The commercially available acid 9 was methylated by Mel in the presence of K CO3. Mono-deprotection of the methyl ester 10 by Cu(OAc)2 in H2O produced the diol 11. The sulfite 12 was prepared from the diol 11 using thionyl chloride in the presence of Et3N. Oxidation of the sulfite 12 with NaIO4/RuCl3 gave the cyclic sulfate 13. Treatment of the sulfate 13 with NaN3 in CH3CN (acetonitrile), in the presence
Figure imgf000010_0001
as a phase transfer catalyst, effected the ring opening with N3 " substitution to provide the azide 14. Subsequent hydrolysis and catalytic hydrogenation of the azide 14 produced the desired amine 16 in an overall yield of 6.9%.
Scheme 2
XJOOH
£ Xrx
Figure imgf000011_0001
Figure imgf000011_0002
11 12
e
Figure imgf000011_0003
Figure imgf000011_0004
15 16
In an alternative embodiment for synthesizing compound 16, displacement of the -OTs group by N3 ~ in 6-O-p-toluenesulfonyl-4-hydroxy- gulonate 17 was achieved and is illustrated in Scheme 3. The mono-tosylate 17 was prepared by treatment of the diol 11 with one equivalent of TsCl in pyridine in 63% yield. Tosylate 17 was treated with NaN3 in DMF at 100 °C for 16 hours that produced the substituted azide 15 as a clean product by TLC. The pure material 15 was isolated by silica column chromatography providing a 52% yield. Subsequent catalytic hydrogenation produced the desired amine 16 in an overall yield of 16.8%
The method of synthesis of the ascorbic conjugates from methyl (5S,7S,lR,6R)-7-(arninomethyl)-6-hydroxy-3,3-dimethyl-2,4,8- trioxabicyclo[3.3.0]octanecarboxlate 16 is shown in Scheme 4. To obtain the desired conjugate 22a, the amine 16 was first coupled with DOTA-G-tri-t-butyl ester 4 (synthesized as in Scheme 1) in the presence of HATU/Et3N to obtain compound 19a. Basic hydrolysis of compound 19a produced the tris-t-Bu- DOTA-G-CH2-gulonic acid conjugate 20a. Further deprotection of compound 20a was investigated under several conditions as follows: 1) 6N HCl/THF 1/1, v/v, 45°C for 7h; 2) 4.5N H2SO4/THF (1/1, v/v), RT for 16h; 3) TFA/H2O (7/1, v/v), RT for 16h. Of these methods, method 1 gave the highest yield of 30% for the conjugate 21a, after purification by HPLC. Chelation with gadolinium produced the ascorbic-Gd chelate conjugate 22a in an overall yield of 13% from compound 16.
Scheme 4
Figure imgf000014_0001
Following a similar approach, but by starting from 6-[trans-4- (aminomethyl)-cyclohexyl-l-carbony]-amino-6-deoxy-2,3-isopropylidene-2- keto-gulonate 18, the ascorbic-Gd chelate conjugate 22b was obtained, containing a longer linker residue in an overall yield of 4.8%. The final products and intermediates were characterized by mass spectra, elemental analysis, and NMR, as detailed in the Example section below. The 6-[trans-4-(aminomethyl)- cyclohexyl-l-carbony]-amino-6-deoxy-2,3-isopropylidene-2-keto-gulonate 18 was synthesized as shown in Scheme 5.
Scheme 5
Figure imgf000015_0001
81% 24
Figure imgf000015_0002
Relaxivity of a paramagnetic material in the presence of a large protein such as human seram albumin may be used to study the ability of the compounds of the present invention to bind the target protein. As such, when a small molecule binds a large protein, the relaxivity of the former will increase because of an increase in its rotational correlation time. This increase in relaxivity may be used not only to measure the extent of binding but also to evaluate the viability of the paramagnetic agent as a blood pool contrast medium.
The relaxivity of the conjugates 22a and 22b in water as well as in water containing a known amount (20%, v/v) of a HSA preparation, known as, seronom (Table 1) were studied. The relaxation time of the samples (22a or 22b) in seronom (TlGd-iigandin seronom) and in water (Tlod-iigand in water) were measured at 38°C using an IBM PC/20 multispec relaxometer. The relaxivity of the samples in seronom (rlod- Hgand in seronom) and in water (rlod-iigand in water) were calculated by the following equations: rlGd-ligand in seronom = (1 1 iGd-ligand in seronom — 1 1 seronom)' LGu-llganαJ rlGd-ligand in water = (1/ Tlαd-ligand in water ~ l/Tlwater)/[Gd-ligand], where [Gd-ligand] is the concentration of the chelate 22a or 22b, which was determined by ICP11; Tlseronom is the relaxation time for pure aqueous seronom; TI water is the relaxation time for pure water.
After the measurement, the sample in aqueous seronom was placed in a centrifree micropartition device (Millipore, Beverly, MA). The device was centrifuged at 500xg for 45 min in a fixed angle rotor (Beckman Model J2-21M, JA-20 rotor). The solution (0.5 mL) was taken from below the filter (unbound Gd-ligand) for [Gd] ICP measurement. The [Gd] of the uncentrifuged sample was also measured as a control. The Fraction Bound was calculated by the following equation:
Fraction Bound = ([Gd-ligand]controι - [Gd-ligand]un 0und)/ [Gd- ligand]controi- Table 1 details the results of the tests.
Table 1
Figure imgf000016_0001
Table 1 shows that the increase in relaxivity in the presence of seronom is 32% and 14% in the case of 22a and 22b, respectively. Based on these results, it is believed that the binding of these conjugates with HSA may not be strong enough for commercial blood pool MRI applications. However, the described conjugates 22a and 22b may be useful as extravascular MRI contrast agents. Moreover, the chemistry described and the conjugates made herein may be used in other applications such as those discussed herein and may be particularly useful where antioxidant properties of ascorbic acid may be required. It is understood that, for radiopharmaceutical or radiotherapy applications, it is convenient to prepare the complexes of the present invention at, or near, the site where they are to be used. A single, or multi-vial kit that contains all of the components needed to prepare the complexes of this invention, other than the radionuclide ion itself, is an integral part of this invention. The amount administered may be selected based on the desired use, such as to produce a diagnostic image of an organ or other site of a subject or a desired radiotherapeutic effect, by methods known in the art. Exemplary dosages are those employing about 2-200 mCi rhenium, lutetium, or yttrium (for radiotherapy), or about 10-60 mCi technetium (for imaging).
Kits of the present invention comprise one or more vials containing the sterile formulation of a predetermined amount of a complexing ligand, an oxidant and optionally other components such as reducing agents, transfer ligands, buffers, lyophilization aids or bulking agents, stabilization aids, solubilization aids and bacteriostats. The inclusion of one or more optional components in the formulation will frequently improve the ease of synthesis of the radiopharmaceutical by the practicing end user, the ease of manufacturing the kit, the shelf-life of the kit, or the stability and shelf-life of the radiopharmaceutical. The improvement achieved by the inclusion of an optional component in the formulation must be weighed against the added complexity of the formulation and added cost to manufacture the kit. The one or more vials that contain all or part of the formulation can independently be in the form of a sterile solution or a lyophilized solid.
Buffers useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of the radiopharmaceuticals include but are not limited to phosphate, citrate, sulfosalicylate, and acetate. A more complete list can be found in the United States Pharmacopeia. Lyophilization aids or bulking agents useful in the preparation of diagnostic kits useful for the preparation of radiopharmaceuticals are known in the art and include lactose, sodium chloride, maltose, sucrose, PEG 8000, cyclodextrins, such as hydroxypropyl-γ-cyclodextrin (HP-γ-CD), dextran, Ficoll, and polyvinylpyrrolidine (PVP).
Stabilization aids, such as antioxidants, useful in the preparation of radiopharmaceuticals and in diagnostic kits for the preparation of radiopharmaceuticals include but are not limited to ascorbic acid, para- aminobenzoic acid (PABA), cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, and inositol. One skilled in the art will appreciate that while conjugates of ascorbic acid are exemplified, the invention includes conjugates of other antioxidants. Also, in addition to the covalent attachment of an antioxidant to a complexing ligand discussed herein, one or more additional stabilzation aids may be added to formulations of the conjugates of the invention.
Solubilization aids useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of the radiopharmaceuticals include but are not limited to ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monooloeate, polysorbates, poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers
(Pluronics) and lecithin. Preferred solubilizing aids are polyethylene glycol, and
Pluronics. Bacteriostats useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of the radiopharmaceuticals include but are not limited to benzyl alcohol, benzalkonium chloride, chlorbutanol, and methyl, propyl or butyl paraben.
A component in a diagnostic kit can also serve more than one function. A reducing agent can also serve as a stabilization aid, a buffer can also serve as a transfer ligand, a lyophilization aid can also serve as a transfer, ancillary or co- ligand and so forth.
The predetermined amounts of each component in the formulation are determined by a variety of considerations familiar to those skilled in the art. These considerations are in some cases specific for that component and in other cases dependent on the amount of another component or the presence and amount of an optional component. In general, the minimal amount of each component is used that will give the desired effect of the formulation. The desired effect of the formulation is that the practicing end user can synthesize the radiopharmaceutical and have a high degree of certainty that the radiopharmaceutical can be safely injected into a patient and will provide diagnostic information about the disease state of that patient.
The present invention will be illustrated in greater detail by the following specific examples. It is understood that these examples are given by way of illustration and are not meant to limit the disclosure or claims. Moreover, these examples are meant to further demonstrate that the synthesis of conjugates of ascorbic acid with macrocyclic polyaminopolycarboxylates chelates. All percentages in the examples or elsewhere in the specification are by weight unless otherwise specified.
EXAMPLE 1 The synthesis of methyl (3aS,3bRJaS,8aR)-2,2,5,5- tetramethyltefrahydro-8aH-[l,3]dioxolo[4,5]furo[3,2-d][l,3]dioxine-8a- carboxylate 10 is detailed.
To a solution of (3aS,3bRJaS,8aR)-2,2,5,5-tetramethyltetrahydro-8aH- [l,3]dioxolo[4,5]furo[3,2-d][l,3]dioxine-8a-carboxylic acid monohydrate 9 (10 g, 34.2 mmol, Aldrich) in DMF (anhydrous, 32 mL, Aldrich) was mixed with K2CO3 (3.4 g, 24.8 mmol, Aldrich). Mel (7.1 g, 50 mmol, Aldrich) was added dropwise through a dropping funnel. The resulting mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was dissolved in EtOAc (100 mL). It was washed with H2O (2x60 mL), brine (1x50 mL) and dried over MgSO4. EtOAc was evaporated. The crude material was purified by silica gel chromatography using EtOAc/Hexane to obtain product 10 (5.6g; yield 57%).
TLC: Silica gel, Rf 0.45, EtOAc/hexane 1/4.
1HNMR (CDC13, ppm): 1.35, 1.40, 1.50 (s, 12H, CH3's on the two isopropylidine rings); 3.85 (s, 3H, OCH3); 4.08, (m, 2H, CH2); 4.15, 4.30,
. 4.85 (s, 3H, CH's on C-3, C-4, C-5).
Mass spectrum: 311.3 (M + Na)+; 289.3 (M + H)+. EXAMPLE 2 The synthesis of Methyl (3aS,5R,6S,6aR)-6-hydroxy-5-(hydroxymethyl)- 2,2-dimethyldihydrofuro[2,3-d][l,3]dioxole-3a(5H)-carboxylate 11 is detailed. To a suspension of compound 10 (5.6 g, 18.3 mmol) in H O (65 mL) was added a solution of Cu(OAc) 'H O (25 mg, Aldrich) in H2O (5mL). It was refluxed (oil bath) for 15 min. The solution became clear. This solution was cooled and evaporated to dryness. Purification of the residue by silica gel chromatography using EtOAc/Hexane afforded product 11 as a waxy solid (4.1g; yield 90.3%).
TLC: Silica gel, Rf 0.70, EtOAc.
1HNMR (CDC13, ppm): 1.40, 1.50 (s, 6H, CH3's on the isopropylidine ring); 3.85 (s, 3H, OCH3); 3.98-4.00 (m, 1H, CH on C-4); 4.10-4.13 (m, 1H, CH on C-5); 4.80 (s, 2H, CH2); 4.75 (m, 1H, CH on C-3). Mass spectrum: 271.2 (M + Na)+; 249.3 (M + H)+.
EXAMPLE 3 Two different synthesis processes of Methyl (3aR,5S,6R)-5- (azidomethyl)-6-hydroxy-2,2-dimethyldihydrofuro[2,3-d][l,3]dioxole-3a(5H)- carboxylate 15 are detailed.
Example 3A: From sulfite 12 and sulfate 13 (Scheme 2) i) Methyl (4aS,5aR,8aS,8bR)-7J-dimethyltetrahydro-5aH-
[ 1 ,3] dioxolo [4,5] furo[3 ,2-d] [ 1 ,3 ,2]dioxathiine-5a-carboxylate 2-oxide 12 To a solution of the diol 11 (3.4g, 13.7 mmol) and triethylamine (0.58 mol, 80 mL) in CH2C12 (40 mL) was added dropwise a solution of SOCl2 (2.3g, 19.2 mmol, Aldrich) in CH2C12 (2 mL) at 0 °C. It was stirred at 0 °C for 15 min. It was then diluted with cold ether (80 mL), washed with cold water (150 mL x 2), dried over MgSO4 and evaporated in vacuo. Purification by silica column chromatography afforded product 12 (lg, yield 25%). TLC: Silica gel, Rf 0.70, EtOAc/hexane 1/1.
1HNMR (CDC13, ppm): 1.40, 1.50 (s, 6H, CH3's on the isopropylidine ring); 3.90 (s, 3H, OCH3); 4.2 (d, 1H, CH on C-3); 4.31 (m, 1H, CH on C- 5); 4.92 (m, 1H, CH on C-4); 4.98 (m, 2H, CH on C-6). Mass spectrum: 317.1 (M + Na)+. ii) Methyl (4aS,5aR,8aS,8bR)-7J-dimethyltetrahydro-5aH-
[ 1 ,3]dioxolo[4,5]furo[3,2-d] [ 1 ,3,2]dioxathiine-5a-carboxylate 2,2- dioxide 13 To a solution of the sulfite 12 (lg, 3.4 mmol) in CC14/CH3CN (10 mL, 1/1 v/v) was added a suspension of RuCl3'xH2O (1 mg) and NaIO4 (2.9 g, 13.6 mmol) in H2O (10 mL) at 0 °C. The mixture was stirred vigorously at RT for 7 h. It was diluted with ether (20 mL), washed with water (30 mL x 2), sat. NaHCO3 (30 mL x 1), and dried over MgSO . Evaporation of the solution afforded product 13 as a white crystalline solid (lg, yield 95%). The material was directly used without purification.
TLC: Silica gel, Rf 0.60, EtOAc/hexane 1/1.
1HNMR (CDC13, ppm): 1.41, 1.51 (s, 6H, CH3's on the isopropylidine ring); 3.88 (s, 3H, OCH3); 4.38 (s, 1H, CH on C-5); 4.85-4.98 (m, 2H, CH on C-6); 5.06 (s, 1H, CH on C-3); 5.20 (s, 1H, CH on C-4). Mass spectrum: 333.0 (M + Na)+. iii) Conversion of sulfate 13 into azide 15
To a solution of the sulfate 13 (2g, 6.45 mmol) in CH3CN (10 mL) was added Me^Cl" (35 mg) and NaN3 (2.1g, 32.3 mmol). It was refluxed for 4 h. The reaction was monitored for the disappearance of the starting sulfate by TLC. At the end of the reaction, the solvent was evaporated. To this residue was added dioxane (28 mL) and diluted H2SO4 (cone. H2SO4/H2O 1/5 v/v, 2 mL). The mixture was stirred at RT for 16 h. Solvents were evaporated. The residue was dissolved in EtOAc (80 mL), washed with water (80 mL x 2), dried over MgSO4 and solvent evaporated to dryness to obtain 15 (lg, yield 57%). TLC: Silica gel, Rf OJO, EtOAc/hexane 3/7.
1HNMR (CDC13, ppm): 1.40, 1.50 (s, 6H, CH3's on the isopropylidine ring); 3.20 (d, 1H, OH); 3.50 (q, 2H, CH2); 3.75 (s, 3H, OCH3); 4.18 (d, 1H, CH on C-4); 4.30 (m, 1H, CH on C-5); 4.65 (s, 1H, CH on C-3). Mass spectrum: 296.1 (M + Na)+. Example 3B: From the tosylate 17 (Scheme 3) i) Methyl (3aS,5R,6S,6aR)-6-hydroxy-2,2-dimethyl-5-({[(4- methylphenyl)sulfonyl]oxy}methyl)dihydrofuro[2,3-d][l,3]dioxole-
3a(5H)-carboxylate_17 To a solution of 11 (1.0 g, 4.0 mmol) in pyridine (13 mL, Aldrich) at 0°C (ice water bath) was added TsCl (0.76 g, 4.0 mmol, Aldrich) in portions. The resulting mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was partitioned between EtOAc/H2O. The EtOAc layer was washed with H2O (2x40 mL), brine (1x40 mL) and dried over MgSO4. EtOAc was evaporated and the crade material was purified by silica gel chromatography using EtOAc/Hexane. Product 17 was obtained as a white solid (l.Og; yield 63%). TLC: Silica gel, Rf 0.85, EtOAc/hexane 7/3.
1HNMR (CDC13, ppm): 1.40, 1.50 (s, 6H, CH3's on the isopropylidine ring); 2.44 (CH3 on phenyl ring); 2.88 (d, 1H, OH); 3.85 (s, 3H, OCH3); 4.19, 4.38 (m, 2H, CH2); 4.27 (d, 1H, CH on C-4); 4.50 (m, 1H, CH on C- 5); 4.69 (s, 1H, CH on C-3); 7.35, 7.80 (d, 4H, CH's on phenyl ring). Mass spectrum: 425.2 (M + Na)+; 403.2 (M + H)+. ii) Conversion of tosylate 17 into azide 15
To a solution of 17 (400 mg, 1.0 mmol) in DMF (2 mL) was added NaN3 (97 mg, 1.5 mmol). The reaction mixture was heated at 100°C (oil bath) for 20 h. The solvent was removed in vacuo and the residue was partitioned between EtOAc/H2O. The EtOAc layer was washed with H2O (2x20 mL), brine (1x20 mL) and dried over MgSO4. EtOAc was evaporated and the crade material was purified by silica gel chromatography using EtOAc/Hexane. Product 15 was obtained as a white solid (140mg; yield 52%).
TLC: Silica gel, Rf 0.70, EtOAc/hexane 3/7. 1HNMR (CDCI3, ppm): 1.40, 1.50 (s, 6H, CH3's on the isopropylidine ring); 3.20 (d, 1H, OH); 3.50 (q, 2H, CH2); 3.75 (s, 3H, OCH3); 4.18 (d, 1H, CH on C-4); 4.30 (m, 1H, CH on C-5); 4.65 (s, 1H, CH on C-3). 13CNMR (CDCI3, ppm): 25.0, 26.0 (CH3's on the isopropylidine ring); 49.0 (CH2); 53.5 (OCH3); 74.0, 81.3, 88.0 (CH's); 109.5, 114.5 (C on C-l & C on the isopropylidine ring); 168.2 (CO). Mass spectrum: 296.1 (M + Na)+. Elemental Analysis: Found: C 44.34, H 5.42, N 15.43%. Calculated for
Ci0Hi5N3O6: C 43.96, H 5.53, N 15.38, O 35.13%.
EXAMPLE 4 The synthesis of Methyl (3aR,5S,6R)-5-(aminomethyl)-6-hydroxy-2,2- dimethyldihydrofuro[2,3-d][l,3]dioxole-3a(5H)-carboxylate 16 is detailed. To a solution ofl5 (1.0 g, 3.7 mmol) in MeOH (30 mL) were added aqueous HC1 (cone, 0.32 mL) and palladium, 10 wt. % (dry basis) on activated carbon (wet, Degussa type ElOl NE/W, 307 mg, Aldrich). It was hydrogenated at 22 psi for 0.5 h. The mixture was filtered through a Celite cake and the solvent evaporated to dryness. Product 16 was obtained as an off white solid (l.Og; yield 91.0%).
1HNMR (CDC13, ppm): 1.40, 1.50 (s, 6H, CH3's on the isopropylidine ring); 3.48-3.55 (m, 2H, CH2); 3.80 (s, 3H, OCH3); 4.48 (m, 1H, CH on C-4); 4.65 (m, 1H, CH on C-5); 4.80 (m, 1H, CH on C-3). 13CNMR (CDC13, ppm): 25.5, 27.0 (CH3's on the isopropylidine ring);
38.0 (CH2); 53.5 (OCH3); 74.0, 77.5, 88.0 (CH's); 109.5, 114.5 (C on C-l & C on the isopropylidine ring); 167.0 (CO). Mass spectrum: 248.2 (M + Na)+.
EXAMPLE 5
The synthesis of methyl (3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5- { [(N- {[4,7, 10-tris(2-tert-butoxy-2-oxoethyl)- 1 ,4,1, 10-tetraazacyclododecan- 1 - yl]acetyl}glycyl)amino]methyl}dihydrofuro[2,3-d][l,3]dioxole-3a(5H)- carboxylate 19a is detailed. i) Synthesis of DOTA-G-tri- t-butyl ester (4)
Preparation of N-(chloroacetyl)-glvcine benzyl ester (1) To a suspension of glycine benzylester hydrochloride (25.2 g, 12.5 mmol, Aldrich) in CH2C12 (200 ml) was added a solution of K2CO3 (77 g, 55.8 mmol) in H2O (200 ml). The mixture was cooled to 0°C and chloroacetylchloride (21.0 g, 18.6 mol, Aldrich) was added dropwise in 15 min. The mixture was warmed to room temperature and stirred for 2 h. The two layers were separated and the aqueous layer was extracted with CH2C12 (100 ml). The organic layers were combined and washed with H2O (100 ml), brine (100 ml), dried (MgSO4). Evaporation of solvent afforded a light yellowish glassy solid. This was triturated with 250 ml of hexane. The colorless solid was collected and dried to give 30.0 g of the material 1. Yield: 100 %
TLC : Rf 0.8 (silica gel, 30 % EtOAc/hexane).
1HNMR (CDC13): δ 4.09 and 4.05 (2s, 6H, NCH2 and COCH2Cl); 5.18
(s, 2H, benzylic CH2); 7.15 (s, 1H, NH); 7.34 (s, 5H, ArH). Preparation of DOTA-G-tri- t-butyl-benzyl ester (3) To a suspension of DO3A tri-t-butyl ester hydrochloride 2 (55.0 g, 100 mmol) in acetonitrile (250 ml) was added anhydrous K CO3(50.0 g, 360 mmol). After 15 min. of stirring at room temperature a solution of 1 (29.0 g, 120 mmol) in 50 ml of acetonitrile was added in 15 min. The mixture was stirred at room temperature for 24 h. K2CO3 was filtered off and solvent evaporated. The residue was dissolved in EtOAc (300 ml) and washed with H2O (150 ml), brine (100 ml) and dried (MgSO4). The EtOAc solution was concentrated to 150 ml in vacuo and 50 ml of hexane was added. The product crystallized out on keeping the solution at room temperature for 2 h. The crystals were filtered and dried. 51.5 g of the material 3 was obtained as a colorless crystalline solid. Yield: 73 %.
TLC : Rf 0.55 (silica gel, 5 % MeOH/CHCl3).
'HNMR CDCD): δ 1.41(s,27 H, CH3); 1.70- 4.42 (26 H, CH2); 5.10 (s,
2H, benzylic CH2); 7.34 (m, 5H, ArH); 9.53 (s, 1H, NH).
Mass Spectrum: 720.4 (M+H) +, 742.3 (M+Na)+, 664, 608, 552 Preparation of DOTA-G-tri- t-butyl ester (4)
To a solution of 3 (10.8 g, 15 mol) in MeOH (50 ml) was added palladium, 10 wt. % (dry basis) on activated carbon (wet, Degussa type ElOl NE/W, 2.0 g). The mixture was hydrogenated at 50 psi for 4 h. The catalyst was filtered through a celite cake and evaporation of solvent afforded 8.8 g of 4 as a colorless solid.
Yield: 94 %
TLC : Rf 0.2 (silica gel, 5 % MeOH/CHCl3). HPLC system : Retention Time 18.63 min; Assay: >100% (area %); Column: YMC, C18; 0.46 x 25 cm; solvent: Water(0.1%TFA)-
Acetonitrile(0.1 %TFA),
Initial condition, 15 % ACN, Linear gradient to 55 % ACN in 20 min and then to 90% CH3CN in 40 min; Flow rate: 1.0 mL/min; Detection UN λ =220. 1HΝMR(CDC13): δ 4.07(s, 27 H, CH3); 1.80- 4.35 (bm, 28 H, CH2), 8.35
(s, 1H, NH).
Mass Spectrum: 630.4 (M+H) +, 652.4 (M+Na)+, 574, 518, 462. ii) Synthesis of methyl (3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5-{[(N- { [4,7, 10-tris(2-tert-butoxy-2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecan- 1 - yl] acetyl} glycyl)amino]methyl} dihydrofuro [2,3-d] [ 1 ,3] dioxole-3a(5H)- carboxylate 19a
To a solution of 16 (0.9 g, 3.2 mmol) in CH2C12 (6 mL) was added 4 (2.0 g, 3.2 mmol), HATU [O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate] (1.2 g, 3.2 mmol, PerSeptive Biosystems), and triethylamine (0.64 g, 6.4 mmol, Aldrich). The clear solution was stirred at room temperature for 4 h. Solvents were evaporated and it was dissolved in EtOAc (50 mL). It was washed with 5 % NaHCO3 (2 x 30 mL), 0.05 N HCl (2 x 30 mL), H O (1 x 30 mL), and dried (MgSO4). Evaporation of solvent afforded product 19a (2.3g; yield 84%). TLC: Silica gel, Rf 0.70, MeOH/CHCl3 1/4.
1HNMR (CDC13, ppm): 1.38-1.50 (m, 33H, CH3's on t-Bu's & on isopropylidene ring); 1.90-3.60 (m, 28H, NCH2COOtBu, NCH2CH2, CH7CONHCH7CONBL CH2CONHCH2CONH & CONHCH?CONHCHτ); 3.70 (s, 3H, OCH3); 4.02, 4.25, 4.80 (m, 3H, CH's on the gulonic ring); 6.75, 6.95 (t, 2H, NH's). Mass spectrum: 859.6 (M + H)+.
EXAMPLE 6 The synthesis of (3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5-{[(N- { [4,7, 10-tris(2-tert-butoxy-2-oxoethyl)- 1 ,4,1, 10-tetτaazacyclododecan- 1 - yl]acetyl}glycyl)amino]methyl}dihydrofuro[2,3-d][l,3]dioxole-3a(5H)- carboxylic acid 20a is detailed.
To a solution of 19a (1.0 g, 1.17 mmol) in MeOH (2 mL) was added a solution of LiOH'H2O (48.5 mg, 1.17 mmol, Aldrich). The resulting solution was stirred at room temperature for 4 h. Solvents were evaporated and the residue was purified by silica column chromatography. Product 20a was obtained as a white solid (0.8g; yield 81%).
TLC: Silica gel, Rf 0.25, MeOH/CHCl3 1/9. !HNMR (CDC13, ppm): 1.38-1.50 (m, 33H, CH3's on t-Bu's & on isopropylidene ring); 1.90-3.60 (m, 28H, NCH2COOtBu, NCH2CH2, CH7CONHCH7CONH. CHϊCONHCTLCONH & CONHCH CONHCH2 ; 4.02, 4.25, 4.80 (m, 3H, CH's on the gulonic ring.
Mass spectrum: 845.5 (M + H)+.
EXAMPLE 7 The synthesis of 1,4,7, 10-tetraazacyclododecane-l,4J-triacetic acid, 10- [2-[[2-[[(2R)-2-[(2S)-2, 5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2- hyάroxyethyl]amino]-2-oxoethyl]amino]2-oxoetl yl]- 21a is detailed.
20a (1.3 g, 1.58 mmol) was dissolved in 6N HCl/THF (1/1, v/v, 30 mL). The solution was stirred at 45 °C for 6.5 h. The solvents were evaporated to dryness. The residue was dissolved in H2O (30 mL) and purified by preparative HPLC employing a YMC C-l 8 column. The column was eluted at 15 mL/min., 0% CH3CN/H2O (both containing 0.1 % TFA). Fractions were lyophilized to give pure 21a as a white fluffy solid (0.3 g; yield 30.2%). 1HNMR (D2O, ppm): 2.90-4.05 (m, NCH2COOH, NCH2CH2, CHϊCONHCHϊCONH, CH7CONHCH2CONH. CONHCT CONHCH?. CHOH & OCH on ascorbic ring). Mass spectrum: 619.3 (M + H)+. HPLC: Column: YMC C-l 8. Conditions: 3% CH3CN/H2O (both containing 0.1% TFA), UN at 254 nm; flow rate 1.0 mL/min.; tR: 5.14 min. Elemental Analysis: Found: C 36.91, H 4.56, Ν 9.36%. Calculated for C24H38Ν6Oι3 '2.4TFA2H2O: C 37.26, H 4.83, N 9.06, O 34.13, F 14.74%.
EXAMPLE 8
The synthesis of l,4,7,10-tefraazacyclododecane-l,4,7-triacetic acid, 10- [2-[[2-[[(2R)-2-[(2S)-2, 5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2- hydroxyethyl]amino]-2-oxoethyl]amino]2-oxoethyl]-, gadolinium salt 22a is detailed.
To a suspension of 21a (86 mg, 0.093 mmol) in H2O (30 mL) was added IN NaOH (Aldrich) solution to adjust the pH to 5. A solution of Gd(OAc)3 (62.2 mg, 0.15 mmol, Aldrich) in H2O (5 mL) was added and the pH of the mixture was maintained at pH 6 by adding IN NaOH. The cloudy solution was stirred at room temperature for 16 h, then was warmed to 50 °C for 4 h. The suspension was filtered and purified by preparative HPLC employing a YMC C- 18 column. The column was eluted at 15 mL/min., 0% CH CN/H2O. Fractions were lyophilized to give pure material 22a as a white fluffy solid (50 mg; yield 63.3%). Mass spectrum: 774.2 (M + H)+.
HPLC: Column: YMC C-l 8. Conditions: 3% CH3CN/H2O (both containing 0.1% TFA), UN at 254 nm; flow rate 1.0 mL/min.; tR: 7.86 min.
Elemental Analysis: Found: C 33.81, H 4.87, Ν 9.47, Gd 18.50%. Calculated for C24H35Ν6O13G 4.5H2O: C 33.76, H 5.19, N 9.84, Gd
18.42, O 32.79%. EXAMPLE 9 The synthesis of methyl (3aS,5S,6S,6aR)-6-hydroxy-2,2-dimethyl-5- ({[(4-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-l,4J,10- tetraazacyclododecan- 1 - yl]acetyl}glycyl)amino]methyl}cyclohexyl)carbonyl]ammo}methyl)dihydrofuro [2,3-d][l,3]dioxole-3a(5H)-carboxylate 19b is detailed. i) Synthesis of 6-[trans-4-(aminomethyl)-cyclohexyl-l-carbony]-amino- 6-deoxy-2,3-isopropylidene-2-keto-gulonate l8 Preparation of 4- {[(phenylmethoxy carbonylamino]methyl}cvclohexanecarboxylic acid 23
To a solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (10 g, 63.6 mmol, Aldrich) in 2N aqueous NaOH (65 ml) at 0 °C was added benzyl chloroformate (11.9 g, 70 mmol, Aldrich) and the reaction temperature was maintained below 10 °C. The cloudy mixture was stirred at RT for 0.5 h. It was then diluted with H2O (100 ml). The clear solution was washed with ether (3 x 80 ml). The pH of the aqueous layer was adjusted to 2 by adding 6N HCl. The precipitates were filtered and dried in vacuo. 17.8 g of 23 was obtained as a white solid.
* Yield: 96%. 1HNMR (CDC13, ppm): 0.90-2.30 (m, 9H, CH2's & CH's on cyclohexyl); 2.20-2.30 (m, 1H, CHCOOH); 2.95-3.05 (m, 2H, NCH2); 4.80-4.85 ( , 1H, NH); 5.10 (s, 2H, PhCH?); 7.30-7.40 (m, 5H, CH's on phenyl ring).
Mass spectrum: (M.+ H)+ at 292.2. Preparation of methyl(lS.5SJS.6RV6-hvdroxy-3.3-dimethyl-2.4.8-trioxa-7-
{[(phenylmethoxy carbonylamino1methyl>cvclohexyl carbonylamino]methyl bi cvclo[3.3.0]octanecarboxylate 24
To a solution of 16 (2.0 g, 7.1 mmol) in CH2C12 (40 ml) was added 23 (2.1 g, 7.1 mmol), HATU [O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate] (2.7 g, 7.1 mmol, PerSeptive Biosystems). It was cooled to 0 °C by ice-water bath. To this mixture triethylamine (1.43 g, 14.2 mmol, Aldrich) was added and the mixture was stirred at 0 °C for 4 h. Solvents were evaporated and it was dissolved in EtOAc (100 ml). It was washed with 5 %
NaHCO3 (2 x 50 ml), 0.05 N HCl (2 x 50 ml), H2O (1 x 50 ml), and dried
(MgSO4). Evaporation of the solvents and silica gel chromatography purification using MeOH/CHCl3 afforded 3 g of 24 as a white solid.
Yield: 81%
TLC: Silica gel, Rf 0.50, MeOH/CHCl3 1/20.
1HNMR (CDC13, ppm): 1.36, 1.46 (s, 6H, CH3's on isopropylidene ring); 0.85-
0.95 (m, 10H, CH2's and CH's on cyclohexyl); 2.98-3.02 (m, 2H, OCONHCHTV. 3.80 (s, 3H, OCH3); 3.95-4.15 (m, 2H, CONHCHzV. 4.52-4.85 (m, 3H, CH's on the gulonic ring); 5.03 (s, 2H, CH2Ph); 7.21-7.35 (m, 5H, CH's on phenyl ring).
Mass spectrum: (M + H)+ at 521.3.
Preparation of 6-[trans-4-(aminomethyl -cvclohexyl-l-carbonv]-amino-6-deoxy-
2,3-isopropylidene-2-keto-gulonate 18 To a solution of 24 (2.9 g, 5.6 mmol) in MeOH (60 mL) was added palladium,
10 wt. % (dry basis) on activated carbon (wet, Degussa type ElOl NE/W, 1.0 g,
Aldrich). The mixture was hydrogenated at 50 psi for 16 h. Pd/C was filtered through a celite cake and solvent evaporated. 2.0 g of the material 18 was obtained. Yield: 93%.
1HNMR (MeOH, ppm): 1.36, 1.46 (s, 6H, CH3's on isopropylidene ring); 0.95-2.15 (m, 10H, CH2's and CH's on cyclohexyl); 2.65 (m, 2H, NH7CH7 : 3.20-3.30 (m, 2H, NCH2); 3.60, 4.0, 4.25 (m, 3H, CH's on the gulonic ring); 3.75 (s, 3H, OCH3). Mass spectrum: (M + H)+ at 387.2. ii) Synthesis of methyl (3aS,5S,6S,6aR)-6-hydroxy-2,2-dimethyl-5-
( { [(4- {[(N- { [4,1, 10-tris(2-tert-butoxy-2-oxoethyl)- 1 ,4,7, 10- tetraazacyclododecan- 1 - yl]acetyl}glycyl)amino]methyl}cyclohexyl)carbonyl]amino}methyl)dihydrofuro [2,3-d][l,3]dioxole-3a(5H)-carboxylate 19b
To a solution of 18 (2.0 g, 5.2 mmol) in CH2C12 (30 mL) was added 6
(3.3 g, 5.2 mmol), HATU (2.0 g, 5.2 mmol, PerSeptive Biosystems). The mixture was cooled to 0 °C by ice-water bath and triethylamine (0.53 g, 5.2 mmol, Aldrich) was added. The clear solution was stirred at 0 °C for 4 h. Solvents were evaporated and the residue was dissolved in EtOAc (100 mL). It was washed with 5 % NaHCO3 (2 x 50 mL), 0.05 N HCl (2 x 50 mL), H2O (1 x 50 mL), and dried (MgSO ). Evaporation of solvent and purification by silica gel chromatography using MeOH/CHCl3 afforded product 19b (1.5g; yield 29%).
TLC: Silica gel, Rf 0.75, MeOH/CHCl3 1/10.
1HNMR (CDC13, ppm): 1.0-2.13 (m, 10H, CH2's & CH's on cyclohexyl ring); 1.43 (m, 27 H, CH3's on t-Bu's); 1.40, 1.50 (s, 6H, CH3's on isopropylidene ring); 1.90-3.60 (m, 28H, NCH2COOfBu, NCH7CH7.
NCH7CH7. CH7CONHCH7CONH. CH7CONHCH7CONH & CH2 adjacent to cyclohexyl ring); 3.82 (s, 3H, OCH3); 3.95 (m, 2H, CH2 adjacent to gulonic ring); 4.19, 4.90, 5.12 (m, 3H, CH's on the gulonic ring); 6.32, 6.48, 6.80 (t, 3H, NH's).
Mass spectrum: 998.6 (M + H)+.
EXAMPLE 10 The synthesis of (3aS,5S,6S,6aR)-6-hydroxy-2,2-dimethyl-5-({[(4-{[(N- { [4,7, 10-tris(2-tert-butoxy-2-oxoethyl)- 1 ,4,1, 10-tetraazacyclododecan- 1 - yl]acetyl}glycyl)amino]methyl}cyclohexyl)carbonyl]amino}methyl)dihydrofuro [2,3-d][l,3]dioxole-3a(5H)-carboxylic acid 20b is detailed.
To a solution of 19b (1.5 g, 1.5 mmol) in MeOH (4 mL) was added a solution of LiOH'H2O (63 mg, 1.5 mmol, Aldrich). The resulting solution was stirred at room temperature for 4 h. 30 mg of LiOH H2O was added and it was stirred at RT for 20 more hours. Solvents were evaporated to obtain crude product 20b (1.45g; crude yield 99%). The material was used without further purification.
TLC: Silica gel, Rf 0.20, MeOH/CHCl3 15/100. 1HNMR (MeOH, ppm): 0.85-2.05 (m, 43H, CH2's & CH's on cyclohexyl ring, CH3's on t-Bu's & on isopropylidene ring); 1.90-3.60 (m, 28H, NCH2COOtBu, NCH7CH7. NCTLCHT,. CH7CONHCH2CONH. CH7CONHCH7CONH & CH2 adjacent to cyclohexyl ring); 3.48 (m, 2H, NCH2 adjacent to gulonic ring); 3.90, 4.19, 4.40 (m, 3H, CH's on the gulonic ring).
Mass spectrum: 984.6 (M + H)+.
EXAMPLE 11 The synthesis of 1,4,7, 10-tetraazacyclododecane-l,4J-triacetic acid, 10- [2-[[2-[[[4-[[[(2R)-2-[(2S)-2, 5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2- hydroxyethyl] amino] carbonyl] cyclohexyljmethyl] amino] -2-oxoethyl] amino]2- oxoethyl]- 21b is detailed.
20b (1.41 g, 1.43 mmol) was dissolved in 6N HCl/THF (1/1, v/v, 30 mL). The solution was stirred at 45 °C for 6.5 h. The solvents were evaporated to dryness. The residue was dissolved in H2O (30 mL) and purified by preparative HPLC employing a YMC C-l 8 column (250 x 30 mm). The column was eluted at 25 mL/min., 0% CH3CN/H2O (both containing 0.1 % TFA) for 15 min., then 0%-20% in 60 min. Fractions were lyophilized to give pure 21b as a white fluffy solid (325 mg; yield 30%).
1HNMR (D2O, ppm): 0.78-2.20 (m, 10H, CH2's & CH's on cyclohexyl ring); 2.90-3.95 (m, 32 H, NCH2COOH, NCH7CH7, NCH7CH7, CH7CONHCH7CONH. CH7CONHCH7CONΗ, CONHCH7CONHCH7. CONHCHTCHOH, CHOH & OCH on ascorbic ring).
13CNMR (D2O, ppm): 31.9, 32.0. and 32.9 (four Cyclohexyl methylene carbons), 39.5 and 48.0 (two Cyclohexyl methine carbons), 45.5, 46.3, and 48.0 (fifteen N-CH2- carbons), 70.0 and 79.5 (two ascorbic ring methine carbons), 121.0 and 158.5 (two olefmic carbons), 176.5, 183.5, and 184.6 (seven carbonyl carbons). Mass spectrum: 758.3 (M + H)+.
HPLC: Column: YMC C-l 8. Conditions: 7% CH3CN/H2O (both containing 0.1% TFA), UN at 254 nm; flow rate 1.0 mL/min.; tR: 7.65 min.
Elemental Analysis: Found: C 42.81, H 5.77, Ν 9.82%. Calculated for C32H5ιΝ7Oι42.0TFAΗ2O: C 43.06, H 5.53, N 9.77%. EXAMPLE 12 The synthesis of 1,4,7, 10-tetraazacyclododecane-l,4,7-triacetic acid, 10- [2-[[2-[[[4-[[[(2R)-2-[(2S)-2, 5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2- hydroxyethyl]amino]carbonyl]cyclohexyl]methyl]amino]-2-oxoethyl]amino]2- oxoethyl]-, gadolinium salt 22b is detailed.
To a suspension of 21b (100 mg, 0.132 mmol) in H2O (20 mL) was added IN NaOH (Aldrich) solution to adjust the pH to 5. A solution of Gd(OAc)3 (58.99 mg, 0.15 mmol, Aldrich) in H2O (5 mL) was added and the pH of the mixture was maintained at pH 6 by adding IN NaOH. The cloudy solution was stirred at room temperature for 16 h. The suspension was filtered and purified by preparative HPLC employing a YMC C-l 8 column (250 x 20 mm). The column was eluted at 20 mL/min., 0% CH3CN/H2O, then 0%-20% in 60 min. Fractions were lyophilized to give pure material 22b as a white fluffy solid (68 mg; yield 56.4%).
Mass spectrum: 913.3 (M + H)+. tøLC: Column: YMC C-l 8. Conditions: 7% CH3CN/H2O (both containing 0.1% TFA), UN at 254 nm; flow rate 1.0 mL/min.; tR: 10.72 min. Elemental Analysis: Found: C 36.81, H 5.40, Ν 9.11, Gd 15.09%;
Calculated for C32H47Ν7Oι4GaΝa'6.0H2O: C 36.88, H 5.71, N 9.41, Gd 15.09%.
As previously stated, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. It will be appreciated that many modifications and other variations that will be appreciated by those skilled in the art are within the intended scope of this invention as claimed below without departing from the teachings, spirit and intended scope of the invention. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. What is claimed is:

Claims

A compound having the following chemical structure:
Figure imgf000034_0001
wherein M is 99raTc, 51Cr, 67Ga, 68Ga, lπIn, 168Yb, 140La, 90Y, 88Y, 86Y, 153Sm, 166Ho, 165Dy, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 215Bi, 177Lu, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (TTT), samarium (111), gadolinium (III), terbium, (III), dysprosium (III), holmium (HI), erbium (III) or ytterbium (III); and X is CH2, an amino acid, a peptide, a protein, or an antibody.
2. The compound of claim 1 , wherein X is CH2.
3. The compound of claim 1, wherein X is
Figure imgf000034_0002
4. The compound of claim 2 or 3, wherein M is 99mTc, 186Re, 188Re, 90Y,
88Y, 86Y, 177Lu, or gadolinium (TTT). .
5. A method of synthesizing a compound of claim 1 , the method comprising contacting a compound having the following structure:
Figure imgf000035_0001
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody, with a metal selected from 9 y9ymmrTr,c, 5311,C-.r, lll
Figure imgf000035_0002
Tτ„n, 1 l608Υx b La, 90Y, 88Y,
86Y, 153Sm, 166Ho, Dy, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi 215Bi, 177Lu, chromium (III), manganese (II), iron (11), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium, (III), dysprosium (III), holmium (III), erbium (III) and ytterbium (III).
6. The method of claim 5, wherein the metal is gadolinium (TTT).
7. The method of claim 6, wherein the gadolinium (III) is provided as Gd(OAc)3.
8. The method of claim 6, wherein X is CH2.
The method of claim 6, wherein X is
Figure imgf000035_0003
10. The method of claim 5, further comprising deprotecting a compound having the structure:
Figure imgf000036_0001
to produce the compound having the structure:
Figure imgf000036_0002
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody.
11. The method of claim 10, wherein the deprotecting step comprises adding 6N HCl/THF (1/1, v/v) and stirring for about seven hours at about 45 °C.
12. The method of claim 10, further comprising the step of hydrolyzing a compound having the structure:
Figure imgf000036_0003
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody,
with a base to produce the compound having the structure:
Figure imgf000037_0001
13. The method of claim 12, wherein the base is lithium hydroxide.
14. The method of claim 12, further comprising the step of contacting a compound having the structure :
0 .COOMe
RHN X"X Y-o
HO^ wherein R is hydrogen, an amino acid, a peptide, a protein, an antibody, or
Figure imgf000037_0002
with a compound having the structure:
Figure imgf000037_0003
to produce the compound having the structure:
Figure imgf000038_0001
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody.
15. The method of claim 14, wherein R is hydrogen.
16. The method of claim 14, wherein R is
Figure imgf000038_0002
17. The method of claim 16, further comprising the step of hydro genating a compound having the structure:
Figure imgf000038_0003
to produce the compound having the structure:
Figure imgf000038_0004
18. The method of claim 17, further comprising the step of contacting a compound having the structure:
O
^-- "~NHBz with a compound having the structure:
Figure imgf000039_0001
and with O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate, to produce the compound having the structure:
Figure imgf000039_0002
19. The method of claim 18 , further comprising the step of contacting trans- 4-(aminomethyl)cyclohexanecarboxylic acid with benzyl chloroformate to produce the compound having the structure:
Figure imgf000039_0003
20. The method of claim 15 or claim 18, further comprising the step of hydrOgenating a compound having the structure:
Figure imgf000039_0004
HO
to produce the compound having the structure:
Figure imgf000039_0005
21. The method of claim 20, wherein the step of hydro genating comprises catalytic hydrogenation.
22. The method of claim 20, further comprising the step of contacting a compound having the structure:
Figure imgf000040_0001
with sodium azide in acetonitrile, and with (CH3)4N+Cr to form an azide compound having the structure:
Figure imgf000040_0002
and hydrolyzing the azide compound to produce the compound having the structure:
Figure imgf000040_0003
HO
23. The method of claim 22, further comprising the step of oxidizing a compound having the structure:
Figure imgf000040_0004
to produce a compound having the structure:
Figure imgf000040_0005
24. The method of claim 23, further comprising the step of contacting a compound having the structure:
Figure imgf000041_0001
with triethylamine and thionyl chloride to produce the compound having the structure:
Figure imgf000041_0002
25. The method of claim 24, further comprising the step of deprotecting an ester compound having the structure:
O .COOMe
to produce the compound having the structure:
Figure imgf000041_0003
26. The method of claim 25, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
27. The method of claim 25, further comprising the step of methylating a compound having the structure:
Figure imgf000041_0004
to produce the compound having the structure:
Figure imgf000042_0001
28. The method of claim 20, further comprising the step of contacting a compound having the structure:
Figure imgf000042_0002
with sodium azide to produce the compound having the structure:
Figure imgf000042_0003
HO
29. The method of claim 28, further comprising the step of contacting a compound having the structure :
Figure imgf000042_0004
with p-toluenesulfonyl chloride to produce the compound having the structure:
Figure imgf000042_0005
30. The method of claim 29, further comprising the step of deprotecting an ester compound having the structure:
Figure imgf000043_0001
to produce the compound having the structure:
Figure imgf000043_0002
31. The method of claim 30, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
32. The method of claim 30, further comprising the step of methylating a compound having the structure:
Figure imgf000043_0003
to produce the compound having the structure:
Figure imgf000043_0004
33. A compound having the structure :
Figure imgf000044_0001
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody.
34. The compound of claim 33, wherein X is CH2.
35. The compound of claim 33 , wherein X is
Figure imgf000044_0002
36. A method of synthesizing a compound of claim 33 , comprising deprotecting a compound having the structure:
Figure imgf000044_0003
to produce the compound having the structure:
Figure imgf000045_0001
wherein X is CH , an amino acid, a peptide, a protein, or an antibody.
37. The method of claim 36, wherein X is CH2.
38. The method of claim 36, wherein X is
Figure imgf000045_0002
39. A compound having the structure:
Figure imgf000045_0003
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody.
40. The compound of claim 39, wherein X is CH2.
41. The compound of claim 39, wherein X is
Figure imgf000045_0004
42. A method of synthesizing the compound of claim 39, comprising hydrolyzing a compound having the structure:
Figure imgf000046_0001
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody, with a base to produce the compound having the structure:
Figure imgf000046_0002
43. The method of claim 42, wherein X is CH2.
44. The method of claim 42, wherein X is
Figure imgf000046_0003
45. A compound having the structure:
Figure imgf000046_0004
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody.
46. The compound of claim 45, wherein X is CH2.
47. The compound of claim 45, wherein X is
Figure imgf000047_0001
48. A method of synthesizing a compound of claim 45, comprising the step of contacting a compound having the stracture:
O COOMe
RHN /"•'■( Y-o
HO^ wherein R is hydrogen or
Figure imgf000047_0002
with a compound having the stracture:
Figure imgf000047_0003
to produce the compound having the structure:
Figure imgf000047_0004
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody.
49. The method of claim 48, wherein X is CH2.
50. The method of claim 48, wherein X is
Figure imgf000048_0001
51. A compound having the structure:
Figure imgf000048_0002
52. A method of synthesizing the compound of claim 51 , comprising the step of hydro genating a compound having the structure:
Figure imgf000048_0003
to produce the compound having the structure:
Figure imgf000048_0004
53. The method of claim 52, further comprising the step of contacting a compound having the structure:
Figure imgf000048_0005
with a compound having the stracture:
π .COOMe
HO
and with O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate, to produce the compound having the stracture:
Figure imgf000049_0001
54. The method of claim 53, further comprising the step of contacting trans- 4-(aminomethyl)cyclohexanecarboxylic acid with benzyl chloroformate to produce the compound having the stracture:
Figure imgf000049_0002
55. The method of claim 53, further comprising the step of hydro genating a compound having the structure:
Figure imgf000049_0003
HO
to produce the compound having the stracture:
Figure imgf000049_0004
56. The method of claim 55, wherein the step of hydrogenatmg comprises catalytic hydrogenation.
57. The method of claim 55, further comprising the step of contacting a compound having the structure:
Figure imgf000050_0001
with sodium azide in acetonitrile, and with (CH3)4N+C1" to form an azide compound having the structure:
Figure imgf000050_0002
and hydrolyzing the azide compound to produce the compound having the structure:
Figure imgf000050_0003
HO
58. The method of claim 57, further comprising the step of oxidizing a compound having the structure:
Figure imgf000050_0004
rid having the structure
Figure imgf000050_0005
59. The method of claim 58, further comprising the step of contacting a compound having the structure:
0 .COOMe
HO N ~
HO - with triethylamine and thionyl chloride to produce the compound having the stracture:
Figure imgf000051_0001
60. The method of claim 59, further comprising the step of deprotecting an ester compound having the stracture:
Figure imgf000051_0002
to produce the compound having the structure:
n .COOMe w/ -°
HO O
61. The method of claim 60, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
62. The method of claim 60, further comprising the step of methylating a compound having the structure:
Figure imgf000051_0003
to produce the compound having the structure:
Figure imgf000052_0001
63. The method of claim 55, further comprising the step of contacting a compound having the structure:
Figure imgf000052_0002
with sodium azide to produce the compound having the stracture:
Figure imgf000052_0003
HO
64. The method of claim 63, further comprising the step of contacting a compound having the structure:
n .COOMe
HO
with p-toluenesulfonyl chloride to produce the compound having the stracture:
0 .COOMe
/""■(' ^o
TsO
H&
65. The method of claim 64, further comprising the step of deprotecting an ester compound having the structure:
Figure imgf000053_0001
to produce the compound having the stracture:
Figure imgf000053_0002
66. The method of claim 65, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
67. The method of claim 65, further comprising the step of methylating a compound having the stracture:
Figure imgf000053_0003
to produce the compound having the stracture:
Figure imgf000053_0004
68. A compound having the structure:
Figure imgf000053_0005
69. A method of synthesizing the compound of claim 68, comprising the step of hydrogenatmg a compound having the structure:
Figure imgf000054_0001
to produce the compound having the stracture:
Figure imgf000054_0002
70. The method of claim 69, wherein the step of hydrogenating comprises catalytic hydrogenation.
71. The method of claim 69, further comprising the step of contacting a compound having the structure:
Figure imgf000054_0003
with sodium azide in acetonitrile, and with (CH3)4N+CT to form an azide compound having the stracture:
O .COOMe
/ "'« Q N3
Ό "°3SO and hydrolyzing the azide compound to produce the compound having the structure:
Figure imgf000055_0001
HO
72. The method of claim 71 , further comprising the step of oxidizing a compound having the structure:
Figure imgf000055_0002
to produce a compound having the structure
Figure imgf000055_0003
73. The method of claim 72, further comprising the step of contacting a compound having the structure:
Figure imgf000055_0004
with triethylamine and thionyl chloride to produce the compound having the stracture:
74. The method of claim 73, further comprising the step of deprotecting an ester compound having the structure:
Figure imgf000056_0001
to produce the compound having the structure:
Figure imgf000056_0002
75. The method of claim 74, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
76. The method of claim 74, further comprising the step of methylating a compound having the stracture:
Figure imgf000056_0003
to produce the compound having the stracture:
Figure imgf000056_0004
77. The method of claim 69, further comprising the step of contacting a compound having the stracture:
Figure imgf000056_0005
with sodium azide to produce the compound having the structure:
Figure imgf000057_0001
HO
78. The method of claim 77, further comprising the step of contacting a compound having the structure:
Figure imgf000057_0002
with p-toluenesulfonyl chloride to produce the compound having the stracture:
Figure imgf000057_0003
79. The method of claim 78, further comprising the step of deprotecting an ester compound having the structure:
Figure imgf000057_0004
to produce the compound having the stracture:
Figure imgf000057_0005
80. The method of claim 79, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
81. The method of claim 79, further comprising the step of methylating a compound having the stracture:
Figure imgf000058_0001
to produce the compound having the structure:
Figure imgf000058_0002
82. A compound having the following structure:
Figure imgf000058_0003
HO
83. A method of synthesizing the compound of claim 82, comprising the step of contacting a compound having the structure:
Figure imgf000058_0004
with sodium azide in acetonitrile, and with (CH3)4N+C1" to form an azide compound having the stracture:
Figure imgf000058_0005
and hydrolyzing the azide compound to produce the compound having the stracture:
Figure imgf000059_0001
HO
84. The method of claim 83, further comprising the step of oxidizing a compound having the structure:
Figure imgf000059_0002
to produce a compound having the stracture:
Figure imgf000059_0003
85. The method of claim 84, further comprising the step of contacting a compound having the stracture:
n pOOMe s H<? X Nσ°
with triethylamine and thionyl chloride to produce the compound having the structure:
Figure imgf000059_0004
86. The method of claim 85, further comprising the step of deprotecting an ester compound having the stracture:
n POOMe
to produce the compound having the structure:
Figure imgf000060_0001
87. The method of claim 86, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
88. The method of claim 86, further comprising the step of methylating a compound having the structure:
Figure imgf000060_0002
to produce the compound having the structure:
Figure imgf000060_0003
89. A method of synthesizing the compound of claim 82, comprising the step of contacting a compound having the structure:
O POOMe
TsO' /'"■■( y-o
Figure imgf000060_0004
with sodium azide to produce the compound having the stracture:
Figure imgf000061_0001
HO
90. The method of claim 89, further comprising the step of contacting a compound having the structure:
Figure imgf000061_0002
with p-toluenesulfonyl chloride to produce the compound having the structure:
Figure imgf000061_0003
91. The method of claim 90, further comprising the step of deprotecting an ester compound having the stracture:
Figure imgf000061_0004
to produce the compound having the structure:
n POOMe
92. The method of claim 91, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
93. The method of claim 91, further comprising the step of methylating a compound having the structure:
Figure imgf000062_0001
to produce the compound having the stracture:
Figure imgf000062_0002
94. A compound having the structure:
Figure imgf000062_0003
95. A method of making the compound of claim 94, comprising comprising the step of contacting a compound having the structure:
Figure imgf000062_0004
with a compound having the stracture:
Figure imgf000062_0005
and with O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate, to produce the compound having the structure:
Figure imgf000063_0001
96. The method of claim 95, further comprising the step of contacting trans- 4-(aminomethyl)cyclohexanecarboxylic acid with benzyl chloroformate to produce the compound having the stracture:
Figure imgf000063_0002
97. The method of claim 95, further comprising the step of hydrogenatmg a compound having the stracture:
Figure imgf000063_0003
HO
to produce the compound having the structure:
Figure imgf000063_0004
98. The method of claim 97, wherein the step of hydrogenatmg comprises catalytic hydrogenation.
99. The method of claim 97, further comprising the step of contacting a compound having the structure:
Figure imgf000064_0001
with sodium azide in acetonitrile, and with (CH3)4N+C1" to form an azide compound having the structure:
Figure imgf000064_0002
and hydrolyzing the azide compound to produce the compound having the structure:
Figure imgf000064_0003
HO
100. The method of claim 99, further comprising the step of oxidizing a compound having the structure :
Figure imgf000064_0004
to produce the compound having the structure:
Figure imgf000064_0005
101. The method of claim 100, further comprising the step of contacting a compound having tk°; stracture:
Figure imgf000065_0001
with triethylamine and thionyl chloride to produce the compound having the structure:
Figure imgf000065_0002
102. The method of claim 101, further comprising the step of deprotecting an ester compound having the stracture:
0 POOMe χ'"'
0 i. Y-o X X • "" to produce the compound having the structure:
Figure imgf000065_0003
103. The method of claim 102, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
104. The method of claim 102, further comprising the step of methylating a compound having the stracture:
Figure imgf000066_0001
to produce the compound having the structure:
Figure imgf000066_0002
105. The method of claim 97, further comprising the step of contacting a compound having the stracture:
Figure imgf000066_0003
with sodium azide to produce the compound having the structure:
Figure imgf000066_0004
HO
106. The method of claim 105, further comprising the step of contacting a compound having the stracture:
Q POOMe
HO/ ( V
HO* with p-toluenesulfonyl chloride to produce the compound having the structure:
Figure imgf000067_0001
107. The method of claim 106, further comprising the step of deprotecting an ester compound having the stracture:
n POOMe
to produce the compound having the stracture:
n POOMe
»χ HO* Xζ .
108. The method of claim 107, wherein the step of deprotecting comprises contacting the ester compound with Cu(OAc)2.
109. The method of claim 107, further comprising the step of methylating a compound having the structure:
Figure imgf000067_0002
to produce the compound having the structure:
0 POOMe
110. A kit for the preparation of a radiopharmaceutical, the kit comprising an antioxidant covalently bound to a complexing ligand.
111. The kit of claim 110, further comprising a targeting molecule covalently bound to the complexing ligand.
112. The kit of claim 111, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
113. The kit of claim 110, further comprising a targeting molecule covalently bound to the antioxidant.
114. The kit of claim 113, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
115. The kit of claim 110, further comprising a targeting molecule bound to both the antioxidant and the complexing ligand.
116. The kit of claim 115, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
117. The kit of claim 110, wherein the antioxidant is ascorbic acid.
118. The kit of claim 110, wherein the complexing ligand is Oxa-PnAO,
N,N-dimethyl Gly-Ser-Cys-Gly, N,N-dimethyl Gly-t-butylGly-Cys-Gly, or DOTA.
119. The kit of claim 110, wherein the radiopharmaceutical comprises a metal selected from 99mTc, 51Cr, 67Ga, 68Ga, ιπln, 168Yb, 140La, 90Y, 88Y, 86Y,
153Sm, 166Ho, 165Dy, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 215Bi, 177Lu, chromium (in), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (HI), gadolinium (III), terbium, (III), dysprosium (III), holmium (III), erbium (III) and ytterbium (III).
120. The kit of claim 119, wherein the antioxidant is ascorbic acid, the complexing ligand is DOTA, and the metal is 99mTc, 186Re, 188Re, 0Y, 88Y, 86Y, 177Lu, or gadolinium (TTT).
121. The kit of claim 120, further comprising a targeting molecule bound to both the antioxidant and the complexing ligand.
122. The kit of claim 121, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
123. A kit for the preparation of a radiopharmaceutical, the kit comprising a compound having the stracture:
Figure imgf000069_0001
wherein X is CH2, an amino acid, a peptide, a protein, or an antibody
124. The kit of claim 108, wherein X is CH2.
125. The kit of claim 108, wherein X is
Figure imgf000069_0002
126. The kit of claim 108, wherein the radiopharmaceutical comprises a metal selected from 99mTc, 51Cr, 67Ga, 68Ga, Ln, 168Yb, 140La, 90Y, 88Y, 86Y, 153Sm, 166Ho, 165Dy, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 215Bi, 177Lu, chromium (in), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (ffl), gadolinium (III), terbium, (III), dysprosium (TTT), holmium (III), erbium (ID) and ytterbium (III).
127. The kit of claim 111, wherein the metal is 99mTc, 186Re, 188Re, 90Y, 88Y, 86Y, 177Lu, or gadolinium (III).
128. A method of stabilizing a radiopharmaceutical ligand, the method comprising covalently binding an antioxidant to a radiopharmaceutical ligand optionally including a targeting molecule.
129. The method of claim 128, wherein the antioxidant is ascorbic acid.
130. The method of claim 128, wherein the radiopharmaceutical ligand is Oxa-PnAO, N,N-dimethyl-Gly-Ser-Cys-Gly, N,N-dimethyl-Gly-t- butylGly-Cys-Gly, or DOTA.
131. The method of claim 128, wherein the radiopharmaceutical ligand is covalently bound to a targeting molecule.
132. The method of claim 131, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
133. The method of claim 128, wherein the antioxidant is covalently bound to a targeting molecule.
134. The method of claim 133, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
135. The method of claim 128, wherein both the antioxidant and the radiopharmaceutical ligand are covalently bound to a targeting molecule.
136. The method of claim 135, wherein the targeting molecule is an amino acid, a peptide, a protein, or an antibody.
137. The method of claim 128, wherein the radiopharmaceutical ligand further comprises a metal selected from 99mTc, 51Cr, 67Ga, 68Ga, πιIn, 168Yb, 140La, 90Y, 88Y, 86Y, 153Sm, 166Ho, 165Dy, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 215Bi, 177Lu, chromium (III), manganese (II), iron (II), iron (III), cobalt (11), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (in), gadolinium (TTT), terbium, (III), dysprosium (III), holmium (TTT), erbium (TTT) and ytterbium (III).
138. The method of claim 137, wherein the metal is 99mTc, 186Re, 188Re, 90Y, 88Y, 86Y, 177Lu, or gadolinium (TTT).
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