Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D475/00—Heterocyclic compounds containing pteridine ring systems
  • C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
  • C07D475/04—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
• • 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
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0459—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two nitrogen atoms as the only ring hetero atoms, e.g. piperazine
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/002—Heterocyclic compounds

Definitions

• the present invention is directed towards a new method of synthesis of 18 F-labeled pteroate or folate radiopharmaceuticals, wherein fluorine-18 is attached to a pteroate (or folate) or derivative thereof, through direct radiolabeling with 18 [F]fluoride, as well as 18 F-folate radiopharmaceuticals obtained by such method of synthesis and their use in diagnosis and monitoring of cancer therapy and therapy of inflammatory and autoimmune disease.
• Radioactive materials emitting electromagnetic radiations as ⁇ -rays or photons or particle emitting radiation selective localization of these radioactive materials in targeted cells or tissues is required to achieve either high signal intensity for visualization of specific tissues, assessing a disease and/or monitoring effects of therapeutic treatments, or high radiation dose, for delivering adequate doses of ionizing radiation to a specified diseased site, without the risk of radiation injury in other e.g. healthy tissues.
• tumors i.e. cancer
• inflammatory and autoimmune diseases such as receptors, antigens, haptens and the like which can be specifically targeted by the respective biological vehicles.
• FR-alpha The folate receptor (FR) has been identified as one of these structures.
• FR-expression is highly restricted to only a few organs (e.g. kidney, lungs, choroids plexus, and placenta).
• the FR-alpha is frequently overexpressed on a wide variety of specific cell types, such as epithelial tumours (e.g. ovarian, cervical, endometrial, breast, colorectal, kidney, lung, nasopharyngeal), and the FR-beta is frequently overexpressed in leukaemia cells (approx. 70% of acute myelogenous leukaemia (AML) are FR-beta positive).
• AML acute myelogenous leukaemia
• PET uses isotopes with short half lives, which are either covalently linked to its carrier or via a chelating moiety. Suitable isotopes include for example 11 C (ca. 20 min), 13 N (ca. 10 min), 15 O (ca. 2 min), and 18 F (ca. 110 min) as covalently bound nuclides and for example 68 Ga (ca. 68 min) which is usually linked by a chelating system.
• a folate radiopharmaceutical having a covalently linked isotope would be of great interest.
• a 18 F-labeled folate radiopharmaceutical would be most suitable for PET Imaging because of its excellent imaging characteristics which would fulfil all of the above considerations.
• 18 F is very useful because of its long half-life of approximately 110 minutes and because it decays be emitting positrons having the lowest positron energy, which allows for the sharpest images with a high-resolution PET.
• the longer half-life of 18 F also allows for syntheses that are more complex and satellite distribution to PET centers with no radiochemistry facilities.
• folic acid does not lend itself to direct radiolabeling with 18 F.
• mainly chelate-based folate radiopharmaceuticals have been synthesized and successfully evaluated as diagnostic agents for imaging folate receptor-positive tumors.
• the most widely studied derivatives were labeled either with 111 In and 99m Tc for SPECT (Siegel et al., J. Nucl. Med. 2003, 44:700; Müller et al., J. Organomet. Chem. 2004, 689:4712) or with 68 Ga for PET (Mathias et al., Nucl. Med. Biol. 2003, 30(7):725).
• the present method is a time-saving and convenient direct 18 F-labelling method, wherein no prosthetic groups are necessary and suitable precursors which carry only amide bounded activated groups as moieties for direct 18 F-labelling are easy accessible.
• the present method allows regioselective preparation and labelling of the ⁇ - or ⁇ -isomer with no need for separation, which is known to be difficult and time-consuming.
• the present invention is in a first aspect directed to a new method of synthesis of 18 F-labeled pteroate or folate radiopharmaceuticals (hereinafter also called method of the invention), wherein fluorine-18 is attached to a pteroate (or folate) or derivative thereof through direct radiolabeling with 18 [F]fluoride.
• Z is a leaving group
• X 1 to X 5 , R 1 to R 5 , S 1 , p, and q are defined as hereinabove, and (b) subjecting said precursor to direct radiolabeling with 18 [F]fluoride.
• 18 [F]fluoride in step (b) is activated by phase transfer catalysts such as tetrabutylammonium carbonate or aminopolyethers (e.g. Kryptofix ⁇ 2.2.2) in combination with potassium carbonate or oxalate.
• phase transfer catalysts such as tetrabutylammonium carbonate or aminopolyethers (e.g. Kryptofix ⁇ 2.2.2) in combination with potassium carbonate or oxalate.
• Z is a leaving group such as Hal, NO 2 , diazonium salts, sulfonate esters, including mesylate CH 3 SO 2 O—, tosylate CH 3 C 6 H 4 SO 2 O—, pentafluorobenzoate, triflateCF 3 SO 2 O—, iodonium salts —I + R′′′ 2 , dialkyl/-aryl silanes —SiOHR′′′ 2 , and silanols —SiHR′′′ 2 , wherein R′′′ is independently a straight-chain or branched C (1-24) alkyl group or an optionally substituted carbocyclic and heterocyclic group comprising five-, six- or ten-membered ring systems and the like.
• R′′′ is independently a straight-chain or branched C (1-24) alkyl group or an optionally substituted carbocyclic and heterocyclic group comprising five-, six- or ten-membered ring systems and the like.
• S 1 is a straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , and wherein one or more of the non-adjacent CH 2 groups may independently be replaced by —O—, —CO—, —CO—O—, —O—CO—, —NR′—, —N ⁇ , —NR′—CO—, —CO—NR′—, —NR′—CO—O—, —O—CO—NR′—, —NR′—CO—NR′—, —CH ⁇ CH—, —C ⁇ C—, —S—, —SO 3 R′—, —PR′—, or a five- or six-membered aromatic ring having 0, 1 or 2 heteroatoms, which is unsubstituted or substituted with CN, Hal, NO 2 , COR′, or COOR′, wherein R′ represents H or C1-C6 alkyl
• the present invention is directed to new 18 F-folate radiopharmaceuticals (hereinafter also called compounds of the invention), such as compounds of formulae I through V, obtained by the new method of direct radiofluorination of the invention, as well as pharmaceutical compositions and uses thereof, in particular uses in diagnosis and monitoring of cancer therapy and therapy of inflammatory and autoimmune diseases in vitro or in vivo.
• compounds of the invention such as compounds of formulae I through V
• the present invention is directed towards uses of the compounds of the invention for diagnostic imaging of a cell or population of cells expressing a folate-receptor.
• the present invention includes methods for diagnostic imaging of a cell or population of cells expressing a folate-receptor, which includes for example methods for in vitro detection of a cell expressing the folate receptor, for example a tumor cell, in a tissue sample. Such methods may also be performed in vivo.
• the present invention is directed towards uses of the compounds of the invention for convenient and effective administration to a subject in need for diagnostic imaging and/or monitoring of cancer therapy and therapy of inflammatory and autoimmune diseases.
• the subject of the methods of the present invention is preferably a mammal, such as an animal or a human, preferably a human.
• Such methods may be performed in combination with any other methods of diagnosis or therapy of cancer and inflammatory and autoimmune diseases including methods using other already developed diagnostic and/or therapeutic agents and utilizing x-ray computed tomography (CT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), single photon emission computed tomography (SPECT), optical imaging, and ultrasound.
• CT computed tomography
• MRI magnetic resonance imaging
• fMRI functional magnetic resonance imaging
• SPECT single photon emission computed tomography
• ultrasound ultrasound.
• the present invention is in a first aspect directed to a new method of synthesis of 18 F-labeled pteroate or folate radiopharmaceuticals (hereinafter also called method of the invention), wherein fluorine-18 is attached through direct radiolabeling with 18 [F]fluoride (hereinafter also abbreviated by “ 18 F”).
• a “condensed pyrimidine heterocycle” includes a pyrimidine fused with a further 5- or 6-membered heterocycle, such as a pteridine or a pyrrolopyrimidine bicycle.
• the term “pteroate” also includes folates, which as used herein are compounds based on a folate skeleton (i.e. based on pteroyl-glutamic acid or N-[4(pteridin-6-ylmethylamino)benzoyl]-glutamic acid), and derivatives thereof. These include optionally substituted folic acid, folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.
• folates which as used herein are compounds based on a folate skeleton (i.e. based on pteroyl-glutamic acid or N-[4(pteridin-6-ylmethylamino)benzoyl]-glutamic acid), and derivatives thereof. These include optionally substituted folic acid, folinic acid,
• the terms “deaza” and “dideaza” analogs refers to the art recognized analogs having a carbon atom substituted for one or two nitrogen atoms in the naturally occurring folic acid structure.
• the deaza analogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs.
• the dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs.
• a pteroic (or folic) acid moiety of general formulae I or IV, respectively may be 18 F-radiolabeled easily and efficiently in a direct manner and thus with no need for synthesis and purification of an intermediate 18 F-labelling agent.
• the direct radiolabeling of precursor IV is performed with 18 F activated by phase transfer catalysts such as tetrabutylammonium carbonate or aminopolyethers (e.g. Kryptofix ⁇ 2.2.2) in combination with potassium carbonate or oxalate.
• phase transfer catalysts such as tetrabutylammonium carbonate or aminopolyethers (e.g. Kryptofix ⁇ 2.2.2) in combination with potassium carbonate or oxalate.
• 18 F is activated with Kryptofix, in a polar aprotic solvent selected from acetonitrile, acetone, 1,4-dioxane, tetrahydrofuran (THF), N-methylpyrrolidinone (NMP), dimethoxyethane (DME), dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) and hexamethylphosphoramide (HMPA) and mixtures thereof.
• a polar aprotic solvent selected from acetonitrile, acetone, 1,4-dioxane, tetrahydrofuran (THF), N-methylpyrrolidinone (NMP), dimethoxyethane (DME), dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) and hexamethylphosphoramide (HMPA) and mixtures thereof.
• the leaving group Z may be any common leaving group known in the art and includes for example Hal, NO 2 , diazonium salts, sulfonate esters, including mesylate CH 3 SO 2 O—, tosylate CH 3 C 6 H 4 SO 2 O—, pentafluorobenzoate, triflateCF 3 SO 2 O—, iodonium salts —I + R′′′ 2 , dialkyl/-aryl silanes —SiOHR′′′ 2 , and silanols —SiHR′′′ 2 , wherein R′′′ is independently a straight-chain or branched C (1-24) alkyl group or an optionally substituted carbocyclic and heterocyclic group comprising five-, six- or ten-membered ring systems and the like.
• R′′′ is independently a straight-chain or branched C (1-24) alkyl group or an optionally substituted carbocyclic and heterocyclic group comprising five-, six- or ten-membered
• S 1 is preferably a straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , and wherein one or more of the non-adjacent CH 2 groups may independently be replaced by —O—, —CO—, —CO—O—, —O—CO—, —NR′—, —N ⁇ , —NR′—CO—, —CO—NR′—, —NR′—CO—O—, —O—CO—NR′—, —NR′—CO—NR′—, —CH ⁇ CH—, —C ⁇ C—, —S—, —SO 3 R′—, —PR′—, or a five- or six-membered aromatic ring having 0, 1 or 2 heteroatoms, which is unsubstituted or substituted with CN, Hal, NO 2 , COR′, or COOR′, wherein R′ represents H or C1-C6 alkyl,
• S 1 is straight-chain or branched C1-C8 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a five- or six-membered aromatic ring having 0, 1 or 2 heteroatoms, which is unsubstituted or substituted with CN, Hal, NO 2 , COR′, or COOR′, wherein R′ represents H or C1-C6 alkyl, or a combination thereof.
• S 1 may specifically include amino acids, which as used herein are compounds with both an amino group (e.g., NH 2 or NH 3 + ) and a carboxylic acid group (e.g., COOH or COO ⁇ ).
• the amino acid may be an ⁇ -amino acid, a ⁇ -amino acid, a D-amino acid or an L-amino acid.
• the amino acid may be a naturally occurring amino acid (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, or histidine, etc.) or it may be a derivative thereof. Examples of derivatives include optionally substituted amino acids, e.g. having one or more substituents selected from CN, Hal, and/or NO 2 .
• the amino acid may also include any other non-naturally occurring amino acids, such as e.g.
• the amino acid may also be part of a polyamino acid (also termed polypeptide), wherein a plurality of same or different amino acids as defined hereinabove are covalently linked, i.e. linked through conventional peptide or other bonds.
• Preferred amino acids include for example glutamic acid, aspartic acid, glutamine, aspartine, lysine, arginine, cystein, and derivatives thereof and preferred polyamino acids include homopolymers the respective homopolymers thereof (i.e. polyglutamic acid, polyaspartic acid, etc). Most preferred are optionally substituted aspartic and glutamic acid.
• Groups X 1 to X 5 , R 1 to R 5 , p and q are defining the nature of group P in more detailed manner. A person skilled in the art would though know the range of these groups within the pteroic and/or folic acid skeleton.
• X 1 to X 5 as used herein are independently of each other C or N.
• R 1 and R 2 are independently of each other represent H, alkyl, —OR′, —NHR′, more preferably —OR′, NHR′.
• R 3 is H, C1-C12 alkyl or C1-C12 alkanoyl.
• R 4 is H, nitroso, C1-C12 alkoxy, or C1-C12 alkanoyl.
• R 5 is H, CN, Hal, NO 2 , C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, (C1-C12 alkylamino)carbonyl. More preferably, R 5 is H, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl.
• (H) q represents all H substituents on the indicated ring (i.e. on X3, C6, C7 and X4), thus q may have a value of 1 to 7.
• p depends on the nature of X and the aromaticity of the ring and thus may be 0, 1 or 2.
• the present invention is directed towards a method of synthesis in accordance with scheme 1, wherein the glutamate moiety of a folic acid or derivative thereof is directly radiolabeled with 18 F and the obtained compound has thus the formula V
• X 6 , X 7 are independently of each other C, N or O, R 6 , R 7 are independently of each other H, straight chain or branched C 1 -C 12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 or a group —S 2 — 18 F, wherein S 2 is straight chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , and wherein one or more of the non-adjacent CH 2 groups may independently be replaced by —O—, —CO—, —C0-O—, —O—CO—, —NR′—, —N ⁇ , —NR′—CO—, —CO—NR′—, —NR′—CO—O—, —O—CO—NR′—, —NR′—CO—NR′—, —CH ⁇ CH—, —C ⁇ C—, —S—,
• the obtained compound of formula V may be represented by compounds having the formulae VI or VIa,
• the present invention contemplates in a specific embodiment a method of synthesis of an 18 F-labeled compound of general formulae VI or VIa in accordance with scheme 1,
• X 1 to X 5 are independently of each other C or N
• X 6 , X 7 are independently of each other C, O or N
• R 1 , R 2 are independently of each other H, Hal, —OR′, —NHR′, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, or (C1-C12 alkylamino)carbonyl, wherein R′ is H or C1-C6 alkyl, R 3 , R 4 are independently of each other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, R 5 is H, CN, Hal, NO 2 , C1-C12 alky
• Z is a leaving group
• X 1 to X 7 , R 1 to R 7 , S 2 , m, p, and q are defined as hereinabove, and (b) subjecting said precursor to direct radiolabeling with 18 F.
• S 2 is preferably straight chain or branched C1-C8 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a five- or six-membered aromatic ring, which is unsubstituted or substituted with CN, Hal, NO 2 , COR′, or COOR′, wherein R′ represents H or C1-C6 alkyl, or a combination thereof, more preferably straight-chain or branched C1-C6 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 .
• a further specific embodiment of the compounds of the invention includes for example compounds wherein X 1 to X 5 are N, R 1 is NY 1 Y 2 , R 2 is O, R 4 is Y 3 , p is 0 and q is 1.
• the present invention is directed to a compounds of formulae VIII or VIIIa,
• X 6 , X 7 are independently of each other C, N or O
• Y 1 , Y 2 are independently of each other selected from H, formyl, straight chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2
• Y 3 is selected from H, formyl, nitroso, straight chain or branched C 1 -C 12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2
• R 6 , R 7 are independently of each other H or straight chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2
• S 2 is straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2
• S 2 is straight-chain or branched C1-C12 alkyl
• alkyl when used singly or in combination, refers to straight chain or branched alkyl groups containing 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, secbutyl, isobutyl, t-butyl, pentyl isopentyl, neopentyl, hexyl and the like.
• the preferred alkyl groups contain 1 to 8, more preferably 1 to 4 carbon atoms.
• alkenyl refers to straight chain or branched alkyl groups containing 2 to 12 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, t-butylene, sec-butylene, isobutylene, amylene, isoamylene, pentylene, isopentylene, hexylene and the like.
• the preferred alkenyl groups contain 2 to 6 carbon atoms.
• alkynyl refers to a linear or branched chain of carbon atoms with one or more carbon-carbon triple bonds.
• the preferred alkynyl groups contain 2 to 12, more preferably 2 to 6 carbon atoms.
• alkoxy refers to alkyl, as defined above, substituted with oxygen, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy and the like.
• alkanoyl refers to formyl, or alkyl, as defined above, terminally-substituted with a carbonyl such as acetyl, propanoyl, butanoyl, pentanoyl and the like.
• alkylamino refers to alkyl, as defined above, substituted with nitrogen, including both monoalkylamino such as methylamino, ethylamino, propylamino, tert-butylamino, and the like, and dialkylamino such as dimethylamino, diethylamino, methylpropylamino, and the like.
• halo refers to any Group 7 element and includes fluoro, chloro, bromo, iodo, and astatine(O).
• the present invention is directed towards compounds obtained by the methods of the invention, such as compounds of formulae I to VIII.
• the present invention provides uses of the new folate radiopharmaceuticals obtained by the method of the invention for convenient and effective administration to a subject in need for diagnostic imaging.
• the present invention provides a method for diagnostic imaging of a cell or population of cells expressing a folate-receptor, said method comprising the steps of administering at least one folate radiopharmaceutical of the invention in a diagnostic imaging amount, and obtaining a diagnostic image of said cell or population of cells.
• Such imaging may be performed on a cell or population of cells expressing a folate-receptor in vitro or in vivo.
• the present invention provides a method for in vitro detection of a cell expressing the folate receptor in a tissue sample which includes contacting said tissue sample with at least one folate radiopharmaceutical of the invention in effective amounts and for sufficient time and conditions to allow binding to occur and detecting such binding by PET imaging.
• the present invention provides uses of folate radiopharmaceuticals of the present invention for convenient and effective administration to a subject in need for diagnostic imaging or monitoring of cancer therapy and therapy of inflammatory and autoimmune diseases.
• the present invention provides a method for simultaneous diagnosis and therapy, comprising the steps of administering to a subject in need thereof at least one folate radiopharmaceutical of the present invention in a diagnostically effective amount in combination with a therapeutically active, and obtaining a diagnostic image of said tissues to follow the course of treatment.
• the subject of the methods of the present invention is preferably a mammal, such as an animal or a human, preferably a human.
• the dosage depends on the nature of the effect desired, such as the form of diagnosis or therapy, on the kind and frequency of treatment, on the diagnostic instrumentation, on the form of application of the preparation, and on the age, weight, nutrition and condition of the recipient, kind of concurrent treatment, if any.
• the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.
• Treatment can commence with a smaller amount, below the optimum amount, which can be increased in order to achieve the optimum effect.
• the imaging procedure in the PET scanner takes place from within minutes to 2-4 hours after administration of the radiotracer.
• the schedule depends on the imaging target and kinetics of the radiotracer as well as the desired information.
• the preferred route of administration of the folate radiopharmaceuticals of the present invention is by intravenous injection.
• the suitable forms for injection include sterile aqueous solutions or dispersions of the above mentioned folate radiopharmaceuticals of the present invention.
• the radiopharmaceutical will be formulated in physiological buffer solutions.
• the folate radiopharmaceuticals undergo sterilization by any art recognized technique, including but not limited to, addition of antibacterial of antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Preferably they undergo a sterile filtration before administration eliminating the need of additional sterilisation agents.
• a preferred unit dosage is from about 0.01 mL to about 10 mL.
• imaging of the organ or tumor in vivo can take place, if desired, from within minutes to 2 to 4 hours after the radiolabeled reagent has been administered to a subject to allow a sufficient amount of the administered dose to accumulate in the targeted area of choice.
• the folate radiopharmaceuticals of the invention may also be used for in vitro detection of a cell expressing the folate receptor in a tissue biopsy taken from a subject.
• the present invention provides a method for in vitro detection of a cell expressing the folate receptor, e.g. a tumor cell, in a tissue sample which includes contacting said tissue sample with a folate radiopharmaceutical of the present invention in effective amounts and for sufficient time and conditions to allow binding to occur and detecting such binding by imaging techniques.
• Samples can be collected by procedures known to the skilled person, e.g., by collecting a tissue biopsy or a body fluid, by aspirating for tracheal or pulmonary samples and the like.
• Tissue samples to be tested include any tissue suspected to contain a cell expressing a folate receptor, such as tumor cells, epithelial cells, kidneys, gastrointestinal or the hepatobiliary system, and others. Samples can be sectioned, e.g., with a microtome, to facilitate microscopic examination and observation. Samples can also be fixed with an appropriate fixative either before or after incubation with one of the folate radiopharmaceuticals of the present invention to improve the histological quality of sample tissues.
• a folate receptor such as tumor cells, epithelial cells, kidneys, gastrointestinal or the hepatobiliary system, and others.
• Samples can be sectioned, e.g., with a microtome, to facilitate microscopic examination and observation. Samples can also be fixed with an appropriate fixative either before or after incubation with one of the folate radiopharmaceuticals of the present invention to improve the histological quality of sample tissues.
• Time and conditions sufficient for binding of a folate radiopharmaceutical of the present invention to a folate receptor on the cell include standard tissue culture conditions, i.e. samples can be cultured in vitro and incubated with one of the complexes or compositions of the present invention in physiological media. Such conditions are well known to the skilled person. Alternatively, samples can be fixed and then incubated with a folate radiopharmaceutical of the present invention in an isotonic or physiological buffer.
• Infrared spectra were recorded on a Jasco FT/IR-6200 ATR-IR. Nuclear magnetic resonance spectra were recorded with a Bruker 400 MHz or 500 MHz spectrometer with the corresponding solvent signals as an internal standard. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (0.00 ppm). Values of the coupling constant, J, are given in Hertz (Hz); the following abbreviations are used in the experimental section for the description of 1 H-NMR spectra: singlet (s), doublet (d), triplet (t), multiplet (m), doublet of doublets (dd). The chemical shifts of complex multiplets are given as the range of their occurrence. Low resolution mass spectra (LR-MS) were recorded with a Micromass Quattro MicroTM API LC-ESI.
• LR-MS Low resolution mass spectra
• Analytical HPLC was performed with an XBridge® column (C18, 5 ⁇ m, 4.6 ⁇ 150 mm, Waters) using the following solvent system (1 mL/min): 0.1% TFA aq (solvent A), acetonitril (solvent B), 1 mL/min; 0-1 min, 95% A; 1-15 min, 95 ⁇ 5% A; 15-20 min, 5% A; 20 ⁇ 22 min, 5 ⁇ 95% A; 22 ⁇ 25 min, 95% A.
• Semi-prep HPLC was performed with XBridge® semiprep column (C18, 5 ⁇ m, 10 ⁇ 150 mm, Waters), 3 mL/min, isochratic NH 4 HCO 3 (10 mM, 88%)/CH 3 CN(12%). All chemicals were used as supplied unlike stated otherwise.
• ⁇ -(2-hydroxyethyl) folic acid amide (26 ⁇ mol) was dissolved in dichloromethane (10 ml) and cooled to 0° C.
• Et 3 N (10 ⁇ l, 1.5 eq.) and TsCl (7 mg, 1.4 eq.) were added and the reaction mixture was stirred for 2 h at 0° C. Then the mixture was allowed to warm to room temperature and stirred overnight.
• the reaction mixture was poured into water (15 ml), layers were separated. The aqueous phase was extracted with dichloromethane and combined organic phases were dried over MgSO 4 . The solvents were removed under vacuo. The product was purified by column chromatography using silica gel and an eluent system of pentane and ethyl acetate to give a yellow oil.
• This compound serves then as precursor for a direct aliphatic nucleophilic 18 F-labelling according to literature procedures (Coenen, H. H. PET Chemistry—The Driving Force in Molecular Imaging, Schubiger, P. A.; Lehmann, L.; Friebe, M., Eds.; Springer: Berlin, 2007, pp. 15-50).

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