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  • 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
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  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
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• • 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
  • A61K51/0402—Organic compounds carboxylic acid carriers, fatty acids
• • 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
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  • 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
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  • C07D—HETEROCYCLIC COMPOUNDS
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  • C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
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  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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  • C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic Table
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Definitions

• the present invention relates to novel folate-conjugates and the corresponding metal-chelate complexes as well as pharmaceutical compositions thereof, their method of production and their use in diagnostic and therapeutic medical applications, such as di- agnostic imaging and radiotherapy.
• the present invention relates in a first aspect to novel folate- conjugates, hereinafter also called compounds of the invention, and their complexes with at least one radionuclide, which can overcome one or more of the disadvantages associated with the related art as discussed hereinabove .
• R 3 and R 4 are independently of each other H, formyl, iminomethyl , nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosub- stituted C1-C12 alkanoyl,
• R c is H, CO 2 R' , COR' , -SO 3 R' , -NHR' , or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , wherein R' represents H, C1-C6 alkyl, m is 0, 1, 2. 3 or 4, n is 1 or 2 , p has a value of 0 , 1 or 2 , q has a value of 1 to 7 , and r is 0 or 1.
• the present invention is directed to a compound of formula I, wherein F is represented by a folic acid (i.e. a pteroyl-glutamic acid) derivative as shown in formula ill
• Xi, X 2 , X 3 , X 4 and X 5 are independently of each other C or N;
• the present invention provides methods for synthesizing a compound of the invention and the corresponding metal-chelate complex thereof.
• the invention provides pharmaceutical compositions comprising a diagnostic imaging amount or a therapeutically effective amount of at least one complex of the present invention and a pharmaceutically acceptable carrier therefor.
• the pharmaceutical compositions contain at least one complex that contains Tc-99m, Re- 186 or Re-188.
• the present invention provides uses of complexes and/or pharmaceutical compositions of the present invention for convenient and effective administration to a subject in need for diagnostic imaging or radiotherapy.
• the subject of the methods of the present invention is preferably a mammal, such as an animal or a human, preferably a human.
• the present invention provides a single or multi-vial kit containing all of the components needed to prepare the compounds of this invention, other than the radionu- elide ion itself.
• the present invention relates in a first aspect to novel folate- conjugates, hereinafter also called compounds of the invention, and their complexes with a radionuclide, which can overcome one or more of the disadvantages associated with the related art.
• the present invention is directed to a compound of formula I
• F is a folate or derivative thereof
• Z 2 , Z 3 are independently of each other C or N,
• R a/ Ra ' / R b are independently of each other H, -OR', -COOR', - NHR' , -CONHR' , -SR' , a phosphine or a heterocyclic group, wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above, and wherein of groups R a , R a » and R b at least two adjacent groups are a donor group -OH, -COOH, -NHR' , -CONH 2 , -SH, a phosphine or a heterocyclic group
• R c is H, COOR', COR', -SO 3 R', -NHR', wherein R' represents H, C1-C6 alkyl, or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a F as defined hereinabove , m is 0 , 1, 2, 3, or 4, and n is 1 or 2.
• permutations of compounds of formula I in combination with one or more groups F in posi- tion(s) R a , R a ' # R b (and R c ) can be part of this invention (as schematically illustrated in Scheme 1 with DG representing donor group) :
• folates are based on a folate (pteroyl-glutamic acid) skeleton and include optionally- substituted folic acid, folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropter- ins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.
• Folic acid is the preferred conjugate-forming ligands used for the compounds of this invention.
• 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.
• Preferred deaza analogs compounds include N- [4- [2- [ (6R) -2-amino-l,4,5,6,7,8-hexahydro-4-oxopyrido[2,3- d] pyrimidin-6-yl] ethyl] benzoyl] -L-glutamic acid (Lometrexol) and N- [4- [1- [ (2 , 4-diamino-6-pteridinyl) methyl] propyl] benzoyl] -L- glutamic acid (Edatrexate) .
• Xi, X 2 , X 3 , X 4 and X 5 are independently of each other C or N,
• R 1 and 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) carbony1 , and (C1-C12 al- kylamino) carbonyl, wherein R' is H or C1-C6 alkyl,
• R 3 and R 4 are independently of each other H, formyl, iminomethyl , nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosub- stituted 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 , and (C1-C12 alkylamino) carbonyl,
• R a , R a -, R b are independently of each other H, -OR', -COOR', - NHR' , -CONHR' , -SR' , a phosphine or a heterocyclic group, wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above, and wherein of groups R a/ R a - and R b at least two adjacent groups are a donor group -OH, -COOH, -NHR' , -CONH 2 , -SH, a phosphine or a heterocyclic group
• these permutations further include a compound of formula II or II' having two further groups F.
• These include a compound of formula II or II', wherein (i) R a and R a ⁇ are a group F, or (ii) R a and R b are a group F, or (iii) R a ⁇ and R b are a group F.
• Xi, X 2 , X 3 , X 4 and X 5 are independently of each other C or N,
• Z 1 , Z 2 , Z 3 are independently of each other C or N
• Ri and 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 , and (C1-C12 al- kylamino) carbonyl, wherein R' is H or C1-C6 alkyl,
• R 3 and R 4 are independently of each other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosub- stituted C1-C12 alkanoyl,
• R 5 is H, CN, Hal, NO 2 , C1-C12 alkyl, C1-C12 alkoxy, C1-C12 al ⁇ kanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy) carbonyl , and (C1-C12 alkylamino) carbonyl,
• R a/ R a' , R b are independently of each other -OH, -COOH, -NHR', -CONH 2 , -SH, a phosphine or a heterocyclic group, wherein R' represents H, C1-C6 alkyl,
• R c is H, CO 2 R', COR', -SO 3 R', -NHR', or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , wherein R' represents H, C1-C6 alkyl, n is 1 or 2, p has a value of 0 , 1 or 2, q has a value of 1 to 7, and r is 0 or 1.
• Z 1 is N
• Z 3 is C
• Z 2 is C or N.
• Z 1 is C and Z 2 and Z 3 are N.
• S 1 is a single bond or 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 non-adjacent CH 2 groups may independently be replaced by -0-, -CO-, -C0-0-, -NR'-, -NR' -CO-, -CO-NR'-, wherein R' represents H or C1-C6 alkyl.
• S 2 , S 3 are independently of each other straight-chain or branched C1-C8 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 re ⁇ placed by -0-, -CO-, -C0-0-, -NR'-, -NR' -CO-, -CO-NR'-, wherein R' represents H or C1-C6 alkyl, most preferably S 2 , S 3 are inde ⁇ pendently of each other straight-chain or branched C1-C6 alkyl, which is unsubstituted or substituted by at least one CN, Hal, OH, or NO 2 .
• R c is H, CO 2 R' , COR' , -NHR' or unsubstituted C1-C6 alkyl, wherein R' represents H or C1-C6 alkyl.
• Si and R c include amino acids, short peptides, sugar molecules. A person skilled in the art would know how to choose .
• the present invention is directed to a compound of formula II, wherein S x is an amino acid moiety, i.e. wherein F represents a folate structure comprising a pteroyl moiety linked to an amino acid moiety.
• amino acid includes 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.
• 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 herein- above are covalently linked, i.e. linked through conventional peptide or other bonds.
• polyamino acid also termed polypeptide
• Preferred amino acids include for example glutamic acid, aspar- tic acid, glutamine, aspartine, lysine, arginine, cystein, and derivatives thereof and preferred polyamino acids include ho- mopolymers the respective homopolymers thereof (i.e. polyglu- tamic acid, polyaspartic acid, etc) . Most preferred are optionally substituted aspartic and glutamic acid.
• the present invention is directed to a compound of formula II, wherein F represents a pteroyl glutamic acid (or folic acid) skeleton having two attachment sites as represented by compound of formula III,
• Xi, X 2 , X 3 , X 4 and X 5 are independently of each other C or N;
• Yi, Y 2 are independently of each other C, 0 or N,
• R 1 and 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 , and (C1-C12 alkylamino) carbonyl , wherein R' is H or C1-C6 alkyl,
• R 3 and R 4 are independently of each other H, formyl, iminomethyl , nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosub- stituted 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 , and (C1-C12 alkylamino) carbonyl, p has a value of 0 , 1 or 2, q has a value of 1 to 7, r is 0 or 1,
• R a/ R a . , R b are independently of each other H, -OR', -COOR', - NHR' , -CONHR' , -SR' , a phosphine or a heterocyclic group, wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above, and wherein of groups R a , R a . and R b at least two adjacent groups are a donor group -OH, -COOH, -NHR', -CONH 2 , -SH, a phosphine or a heterocyclic group.
• R c is H, CO 2 R', COR', -SO 3 R', -NHR', wherein R' represents H, C1-C6 alkyl, or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a F as defined hereinabove,
• n 1 or 2
• S 2 , S 3 , S 4 are independently of each other straight-chain or branched C1-C8 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 re- placed by -0-, -CO-, -C0-0-, -NR'-, -NR' -CO-, -CO-NR'-, wherein R' represents H or C1-C6 alkyl.
• S 2 , S 3 , S 4 are independently of each other straight-chain or branched C1-C6 alkyl, which is unsubstituted or substituted by at least one CN, Hal, OH, or NO 2 .
• R c is H, CO 2 R' , COR' , -SO 3 R' , - NHR' or C1-C12 alkyl, wherein R' represents H or C1-C6 alkyl.
• the present invention is directed to a compound of formula III, wherein (a) R 6 is H, straight chain or branched Ci-Ci 2 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 , and R 7 is a group of formula IV, (b) R 6 is a group of formula IV, and R 7 is H, straight chain or branched C x -Ci 2 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 , or (c) both R 6 and R 7 are a group of formula IV.
• m is 0 or 1.
• Z 2 , Z 3 are independently of each other C or N,
• A represents independently of each other -COOH, -NH 2 , -CONH 2 , or -SH,
• R a/ R b are independently of each other H, -OR' , -COOR' ,
• NHR' , -CONHR' , -SR' , a phosphine or a heterocyclic group wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above , R ⁇ is H, CO 2 R' , COR' , -SO 3 R' , -NHR' , wherein R' represents H, C1-C6 alkyl, or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a F as defined hereinabove, and n is 1 or 2.
• Y 1 , Y 2 are independently of each other C. 0 or N,
• Z 1 , Z 2 , Z 3 are independently of each other C or N;
• Ri and 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 , and (C1-C12 al- kylamino) carbonyl, wherein R' is H or C1-C6 alkyl ,
• R 3 and R 4 are independently of each other H, formyl, iminomethyl , nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl , halosub- stituted 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 , and (C1-C12 alkylamino) carbonyl,
• R 6 , R 7 are independently of each other H, straight chain or branched Ci-C 12 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 ,
• R a , R a' , R b are independently of each other H, -OR', -COOR', - NHR' , -CONHR' , -SR' , a phosphine or a heterocyclic group, wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above, and wherein of groups R a , R a , and R b at least two adjacent groups are a donor group -OH, -COOH, -NHR', -CONH 2 , -SH, a phosphine or a heterocyclic group
• R c is H, CO 2 R' , COR' , -SO 3 R' , -NHR' , wherein R' represents H,
• C1-C6 alkyl or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a F as defined hereinabove,
• n 0, 1, 2, 3, or 4
• p has a value of 0 , 1 or 2
• q has a value of 1 to 7
• r is 0 or 1.
• the present invention contem ⁇ plates compounds wherein S 2 , S 3 , S 4 are independently of each other straight-chain or branched C1-C8 alkyl, which is unsubsti- tuted or substituted by at least one CN, Hal, OH, or NO 2 and wherein one or more of non-adjacent CH 2 groups may independently be replaced by -0-, -CO-, -CO-O-, -NR'-, -NR' -CO-, -CO-NR'-, wherein R' represents H or C1-C6 alkyl.
• n is 0 or 1.
• the present invention is for example directed to a compound of formulas VI and VI', Via and Via 1 , and VIb and VIb',
• Xi, X 2 , X 3 , X 4 and X 5 are independently of each other N or C,
• Z 2 , Z 3 are independently of each other C or N,
• Yi, Y 2 are independently of each other C, 0 or N,
• Ri and 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 , and (C1-C12 al- kylamino) carbonyl, wherein R' is H or C1-C6 alkyl,
• R 3 and R 4 are independently of each other H, formyl, iminomethyl , nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl , halosub- stituted 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 , and (C1-C12 alkylamino) carbonyl,
• R 6 , R 7 are independently of each other H or straight chain or branched Ci-Ci 2 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 ,
• R a/ R b are independently of each other a donor group such as -OH, -COOH, -NHR', -CONH 2 , -SH, or a heterocyclic group selected from pyridyl, pyrrolyl, and thiazolyl, wherein R' represents H or Cl- C6 alkyl
• R c is H, CO 2 R' , COR' , -SO 3 R' , -NHR' , wherein R' represents H or C1-C6 alkyl, or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 ( p has a value of 0, 1 or 2 , q has a value of 1 to 7, s is 1 to 8, and o is 1 to 6.
• Z 1 is N
• Z 3 is C and Z 2 is C or N
• Z 1 is C and Z 2 and Z 3 are N.
• the present inven- tion is directed to a compound of formulae VII and VII' , Vila and Vila', and VIIb and VIIb'
• Xi, X 2 , X3, X4 and X 5 are independently of each other N or C, Y 1 , Y 2 are independently of each other C, 0 or N,
• Ri and 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 , and (C1-C12 al- kylamino) carbonyl , wherein R' is H or C1-C6 alkyl,
• R 3 and R 4 are independently of each other H, formyl, iminomethyl , nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosub- stituted C1-C12 alkanoyl,
• R 5 is H, CN, Hal, NO 2 , C1-C12 alkyl, C1-C12 alkoxy, C1-C12 al- kanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy) carbonyl , and (C1-C12 alkylaraino) carbonyl,
• R 6 is H or straight chain or branched Ci-Ci 2 alkyl, which is un- substituted or substituted by at least one CN, Hal or NO 2 ,
• R a and R b are independently of each other a donor group such as - OH, -COOH, -NHR', -CONH 2 , -SH, or a heterocyclic group selected from pyridyl, pyrrolyl, and thiazolyl, wherein R' represents H or C1-C6 alkyl
• R c is H, CO 2 R' , COR' , -SO 3 R' , -NHR' , wherein R' represents H or C1-C6 alkyl, or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or N0 2/
• p has a value of 0, 1 or 2
• q has a value of 1 to 7
• s is 1 to 8
• o is 1 to 6.
• R c is H, CO 2 R' , COR' , -SO 3 R' , - NHR', wherein R' represents H or C1-C6 alkyl, or C1-C12 alkyl.
• R a is -NH 2
• R b is -OH
• R c is H.
• o 1, 2, 3 or 4.
• H H substituents on the indicated ring (i.e. on X 3 , C 6 , C 7 and X 4 ) .
• q 5 for a fully saturated unsubstituted analog
• q 7 for a fully saturated unsubstituted 5,8-dideaza analog
• R 1 and R 2 may independently of each other represent H, alkyl , -OR', -NHR', more preferably -OR', - NHR' .
• R 3 is H, formyl, C1-C12 alkyl or Cl- C12 alkanoyl .
• R 4 is H, nitroso, C1-C12 alkoxy, or C1-C12 alkanoyl.
• R 6 is H or straight chain or branched Ci-C 12 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 , more preferably R 6 is H or straight chain or branched Ci-Ci 2 alkyl. In a most preferred embodiment, R 6 is H.
• R a , R a > , R b are independently of each other H, -OR', -COOR', - NHR', -CONHR', -SR', or a heterocyclic group selected from pyridyl, pyrrolyl, and thiazolyl wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above. More preferably R a , R a - , R b are independently of each other H, -OR', -COOR', - NHR', -CONHR', -SR', wherein R' represents H or C1-C6 alkyl, or a F as defined hereinabove.
• Preferred donor groups for R a , R a # , R b are -OH, -COOH, -NHR', - CONH 2 , -SH, or a heterocyclic group selected from pyridyl, pyrrolyl, and thiazolyl, wherein R' represents H or C1-C6 alkyl. More preferred donor groups for R a/ R a ⁇ , R b are independently of each other -OH, -COOH, -NHR', -CONH 2 , -SH, wherein R' represents H or C1-C6 alkyl.
• X 1 to X 5 are N, R 1 is NH 2 , R 2 is 0, R 4 is H, s is 1, 3 or 5, and all the other parameters are as defined in formulae VII, Vila or VIIb
• X 1 to X 5 are N, Y is 0, R 1 is NH 2 , R 2 is 0, R 3 is H, methyl or formyl, R 4 is H, methyl or formyl, R 6 is H, methyl or ethyl, s is 1, 3 or 5 , and all the other parameters are as defined in formulae VII, Vila or VIIb
• X 1 to X 5 are N, Y is 0, R 1 is NH 2 , R 2 is 0, R 3 is H, methyl or formyl, R 4 is H, R 6 is H, s is 1, 3 or 5, R a and R b are -OH, and all the other parameters are as de- fined in formulae VII, Vila or VIIb
• the present invention is directed to a compound of formula VIII,
• Yi, Y 2 are independently of each other C, N or 0,
• R 8 , R- 9 are independently of each other H, formyl, straight chain or branched Ci-Ci 2 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 ,
• R 4 is selected from H, nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, and
• 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 , and (C1-C12 alkylamino) carbonyl,
• R 6 and R 7 are independently of each other H, straight chain or branched C x -Ci 2 alkyl, which is unsubstituted or substituted by at least one CN, Hal or NO 2 , or a group of formula IVa, IVb and/or a group of formula IVb'
• Z 2 , Z 3 are independently of each other C or N,
• A represents independently of each other -COOH, -NH 2 , -CONH 2 , or -SH,
• R a/ R b are independently of each other H, -OR' , -COOR' ,
• NHR' NHR' , -CONHR' , -SR' , a phosphine or a heterocyclic group , wherein R' represents H or C1-C6 alkyl, or a F as defined herein- above,
• R c is H, CO 2 R', COR', -SO 3 R', -NHR', wherein R' represents H, C1-C6 alkyl, or straight-chain or branched C1-C12 alkyl, which is unsubstituted or substituted by at least one CN, Hal, or NO 2 , or a F as defined hereinabove, and n is 1 or 2.
• Z 1 is N
• Z 3 is C and Z 2 is C or N
• Z 1 is C and Z 2 and Z 3 are N.
• alkyl when used singly or in combination, refers to straight chain or branched alkyl groups containing 1 to 12 car ⁇ bon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec- butyl, 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 alkylene groups containing 2 to 12 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, t-butylene, sec- butylene, isobutylene, amylene, isoamylene, pentylene, isopenty- lene, hexylene and the like.
• alkenyl groups con- tain 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 pref- erably 2 to 6 carbon atoms.
• alkoxy refers to alkyl, as defined above, substituted with oxygen, such as methoxy, ethoxy, pro- poxy, 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, propylamine tert-butylamino, and the like, and dialkylamino such as dimethylamino, diethyl- amino, methylpropylamino, and the like.
• halo refers to any Group 7 element and includes fluoro, chloro, bromo, iodo, and astatine (o).
• five- or six-membered aromatic carbocyclic or hetero- cyclic ring refers to five- or six-membered aromatic carbocyclic rings such as phenyl, cycloheptyl, cyclohexyl, and cyclopen- tyl, and five- or six-membered aromatic heterocyclic rings containing at least one heteroatom selected from N, S, 0, and P , such as pyridyl , piperidino, piperazino, morpholino, imidazolyl , triazolyl, tetrazolyl, oxazolyl, thiazolyl, pyrrolidinyl, and pyrazolyl .
• heterocyclic group refers to a saturated heterocyclic group or unsaturated heterocyclic group hav- ing at least one heteroatom selected from N, S, 0, and P, preferably N or S .
• saturated heterocyclic group include tetrahydrofuryl , pyrrolidinyl, pyrazolidinyl, imidazolid- inyl, piperidyl, rnorpholinyl, thiamorpholinyl and piperazinyl.
• Examples of a unsaturated heterocyclic group include furyl, pyr- rolyl, thienyl, thiazolyl, isothiazolyl , oxazolyl, isooxazolyl and pyrazolyl, pyridyl, pyrimidinyl , pyrazinyl and pyridazinyl .
• These heterocyclic groups may be substituted by alkyl such as methyl or ethyl, halogen atom or phenyl. When the heterocyclic group is substituted by phenyl, it may form a condensed ring combining the two adjacent carbon atoms in the heterocyclic group with the phenyl group.
• condensed rings examples include benzothiazolyl , benzofuryl, quinazolinyl and quinoxalinyl .
• Preferred heterocyclic groups are pyridyl, pyrrolyl, und thia- zolyl .
• phosphine includes, for example, tri- arylphosphines, trialkylphosphines and tris (dialkylamino)phosphines, which may have a substituent, and the like. Specific examples thereof include, for example, 1,2- bis (dimethylphosphino) ethane and tris-hydroxymethylenephosphine.
• the term "optionally substituted” includes C (1-6) alkyl, C (1-6) alkenyl, C(l-6)acyl, aryl C (1-6) alkylaryl, cyano, nitro and halo, preferably C(l-6) alkyl, cyano, nitro and halo, most preferably C (1-4) alkyl .
• the invention provides complexes comprising compounds of the present invention and 99m Tc, 186/188 Re, 111 In +3 , 67/ ⁇ 8 Ga +3 , 9 V 3 , 109 Pd +2 , 105 Rh +3 , 177 Lu, 64/67 Cu 166 Ho, 213 Bi hereinaf- ter also called complexes of the present invention.
• the complexes of the present invention comprise 99m Tc, 186 Re or 186 Re.
• Technetium which is particularly useful as a diagnostic imaging agent, is preferably one or more of the radionuclides
• the preferred radioisotope for medical imaging is 99m Tc. Its 140 keV .gamma. - photon is ideal for use with widely-available gamma cameras. It has short (6 hour) half-life, which is desirable when consider- ing patient dosimetry.
• Rhenium which is particularly useful as a radiotherapy agent, is preferably one of the radionuclides 186 Re or 188 Re, or a mixture thereof.
• the present invention also provides methods of synthesizing a compound of the invention.
• the heterocyclic ligand site for the radionuclide is synthesized first and subsequently linked through a suitable linker to a suitably protected pteroic or folic acid derivative to obtain the final compound of choice (see for example Figure 1) .
• this method of synthesis for example includes in a first step coupling of a histidine fragment (compound 1 in Fig 1) , which is suitably protected at the amino- and carboxy-terminus to a linker S 3 having a suitable leaving group LG. Subsequent couplings to first a glutamic acid residue and then to a folic acid residue results in the final folic acid conjugate which upon deprotection can be complexed with a suitable radionuclide.
• a histidine fragment compound 1 in Fig 1
• LG linker S 3 having a suitable leaving group LG
• a folic or pteroic acid moiety and a ligand site moiety are synthesized first, wherein the folic or pteroic acid moiety carries an azide group and the ligand site moiety carries an alkyne group (or vice versa) and subsequently coupled in a cycloaddition under thermal conditions or in the presence of a catalyst to obtain the final compound of choice (KoIb and Sharpless, Drug Discovery Today 2003, 8, 1128; KoIb et al. Angew. Chem. Int. Ed. 2001, 40, 2004; Rostovtsev, V. V. et al. Angew. Chem. Int. Ed.2002, 41, 2596; US 2005/02222427; WO 06/116629) .
• the modular and versatile nature of the reaction allows to employ a wide variety of linkers to couple the radioisotope to folic acid.
• the cycloaddition is performed under thermal conditions, i.e. at temperatures ranging from 10 to 200 0 C, preferably from 10 to 100 0 C.
• the cycloaddition is performed in the presence of a catalyst, such as a transition metal complex, such as Ru and Cu(I).
• a catalyst such as a transition metal complex, such as Ru and Cu(I).
• Preferred catalysts are Cu(I) salts, such as Cu(I) chloride, bromide, iodide.
• Cu(I) can be obtained by in situ reduction of a Cu(II) salt.
• This reaction can be performed in a variety of protic or aprotic solvents, such as for example methanol, ethanol, 2-propanol, tertiary-butanol, n- butanol and/or water or buffered solutions thereof, at a wide range of temperatures (such as between 10 and 100 0 C, preferably room temperature) and varying pH (such as from 4 to 12) , under oxidative or reducing conditions and in the presence of other functional groups with no need for protecting groups.
• protic or aprotic solvents such as for example methanol, ethanol, 2-propanol, tertiary-butanol, n- butanol and/or water or buffered solutions thereof, at a wide range of temperatures (such as between 10 and 100 0 C, preferably room temperature) and varying pH (such as from 4 to 12) , under oxidative or reducing conditions and in the presence of other functional groups with no need for protecting groups.
• an alkynyl derivatized chelating moiety or precursor thereof e.g. propargyl glycine
• azido folic acid under standard conditions (for example Na-ascorbate, Cu(OAc) 2 , ⁇ uOHZH 2 O (1:1), rt) .
• a chelating moiety or precursor thereof functionalized with an azido group is coupled to an alkyne substituted folic acid or derivative of choice in a Cu (I) -catalyzed cycloaddition under standard conditions. Both click product are then labelled with e.g. ["" 1 Tc(CO) 3 (H 2 O) 3 I + to provide the SPECT tracer.
• both routes allow the incorporation of a wide variets of linkers to couple a different chealting moieties (and hence radiometals) to folic acid.
• the present invention further provides a method of synthesizing a complex of the invention, which comprises labeling a compound of the invention, which includes the steps of first obtaining a compound of the invention, and reacting the compound with a radionuclide as specified hereinabove, preferably Tc-99m, Re-186 or Re-188, generally in the presence of a reducing agent to form a metal chelate complex between the compound of the invention and the radionuclide.
• the reducing agent may be any known reducing agent, but will preferably be a dithionite ion or a stannous ion.
• preparation of a complex of the present invention containing rhenium as the metal may be accom- pushed using rhenium in the +5 or +7 oxidation state.
• rhenium examples of compounds in which rhenium is in the Re(VII) state are NaReO 4 or KReO 4 .
• Re(V) is available as Re-gluconate, Re-glucoheptonate, Re-tartrate, Re-citrate.
• Other rhenium reagents capable of forming a rhenium complex may also be used.
• the invention provides pharmaceutical compositions comprising a diagnostic imaging amount or a therapeutically effective amount of at least one complex of the present invention and a pharmaceutically acceptable carrier therefor.
• the pharmaceutical compositions contain at least one complex that contains Tc-99m, Re-186 or Re-188.
• a pharmaceutically acceptable carrier which is present in an appropriate dosage, includes solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like which are physiologically acceptable.
• solvents dispersion media, antibacterial and antifungal agents, isotonic agents, and the like which are physiologically acceptable.
• the use of such media and agents are well-known in the art.
• the present invention provides uses of complexes and/or pharmaceutical compositions of the present invention for convenient and effective administration to a subject in need for diagnostic imaging or radiotherapy.
• the subject of the methods of the present invention is preferably a mammal, such as an animal or a human, preferably a human.
• 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 complex or composition of the present invention in a diagnostic imaging amount, and obtaining a diagnostic image of said cell or population of cells.
• the complexes and/or compositions of the present invention may also be used as radioptherapy agents useful for the treatment of a subject in need thereof.
• the present invention provides a method for radiotherapy comprising the steps of administering to a subject in need thereof at least one complex or composition of the present invention in therapeutically effective amounts, and after localization of said at least one complex or composition in the desired tissues, subjecting the tissues to irradiation to achieve the desired therapeutic effect.
• the present invention provides a method for simultaneous diagnosis and radiotherapy comprising the steps of administering to a subject in need thereof at least one complex or composition of the present invention in a diag- nostically and therapeutically effective amount, and after localization of said at least one complex or composition in the desired tissues, subjecting the tissues to irradiation, and ob- taining a diagnostic image of said tissues to follow the course of treatment .
• An image of a cell or tissue expressing the folate receptor, i.e. a tumor cell or tissue, labeled with one or more of the complexes or compositions of the present invention can be de- tected using a radiation detector, e.g. a ⁇ -radiation detector.
• a radiation detector e.g. a ⁇ -radiation detector.
• radionuclide of choice e.g. 99m Tc, 185/188 Re, 111 In +3 , 67768 Ga +3 , 90 Y +3 , 109 Pd +2 , 105 Rh +3 , 177 Lu, 64/67 Cu 166 Ho, 213 Bi, preferably Tc-99m, Re-186 or Re-188, will be taken into consideration in determining a dosage for diagnostic imaging or radio- therapy.
• the unit dose to be administered has a radioactivity of about 0.01 mCi to about 300 mCi, preferably 10 mCi to about 200 mCi .
• 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 hours or even longer, after the radiolabeled reagent has been administered to a subject.
• a sufficient amount of the administered dose will accumulate in the targeted area to be imaged within about 0.1 to 1 of an hour .
• the complexes and/or compositions of the present invention may be administered by an appropriate route such as parentally (for example, intravenously) , intramuscularly or intraperitoneally or by any other suitable method.
• the complexes and/or compositions of this invention may be administered to a subject by bolus or slow infusion intravenous injection.
• the suitable forms for injection include sterile aqueous solutions or disper- sions and sterile powders of the above mentioned complexes and/or compositions of the present invention.
• the complexes or pharmaceutical compositions are preferably sterile. Sterilization can be accomplished by any art recognized technique, including but not limited to, addition of antibacte ⁇ rial of antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, thimerosal , and the like.
• the complexes and/or compositions 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 complex or composition 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 con- tain 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 of bound complex. Samples can also be fixed with an appropriate fixative either before or after incubation with one of the complexes or compositions of the present invention to improve the histological quality of sample tissues.
• Time and conditions sufficient for binding of a complex of the present invention to a folate receptor on the cell include stan- dard 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 complex or composition of the present invention in an isotonic or physiological buffer.
• a typical amount of said complex of the present invention for in vitro detection of a tumor cell can range from about 1 ng/1 to about 1000 ⁇ g/1.
• a preferred amount is about 1 ⁇ g/1 to about
• Preferred complexes to be used for in vitro diagnosis of a tumor cell are the same as for in vivo applications and in- elude 99m Tc, 186 Z 188 Re, 111 In +3 , 67/68 Ga +3 , 90 Y +3 , 109 Pd +2 , 105 Rh +3 , 177 Lu, 64/67 Cu 166 Ho, 213 Bi, preferably Tc-99m, Re-186 or Re-188.
• the present invention provides a single or multi-vial kit containing all of the components needed to prepare the complexes or compositions of this invention, other than the radionuclide ion itself.
• a preferred single-vial kit of the present invention comprises a compound of the present invention, and a source of a pharmaceutically acceptable reducing agent such as a stannous salt.
• the kit comprises optionally further additives, for example the kit is buffered with a pharmaceutically acceptable acid or base to adjust the pH to a desired value for complex formation.
• Such a single vial kit may optionally contain exchange ligands such as glucoheptonate, gluconate, mannitol, maleate, citric or tartaric acid and may also contain reaction modifiers, such as diethylenetriaminepentaacetic acid or ethyl- enediamine tetraacetic acid. Additional additives, such as solu- bilizers (for example a cyclodextrin) , antioxidants (for example ascorbic acid) and/or fillers (for example, NaCl) may be employed to improve the radiochemical purity and stability of the final product, or to aid in the production of the kit.
• the radionuclide e.g. Tc or Re, will preferably be added separately in the form of a solution, e.g. a pertechnetate or perrhenate solution.
• a preferred multi-vial kit of the present invention comprises, in one vial, the components, other than the radionuclide itself, required to form a labile radionuclide complex, that is, an exchange ligand and a pharmaceutically acceptable reducing agent such as a stannous salt.
• a compound of the present invention is contained in a second vial, as well as optional additives such as buffers appropriate to adjust the pH to its optimal value.
• the radionuclide will be provided in form of a solution, e.g. for example a pertechnetate or perrhenate solution, to be added.
• kit components may be in liquid, frozen or dry form.
• kit components are provided in lyophi- lized form.
• HPLC System 1 XBridge ® column (C18, 5 ⁇ m, 4.6 x 150 mm, Waters); 0.1 % TFA aq (solvent A), acetonitrile (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; HPLC System 2: XTerra ® column (MSC18, 5 ⁇ m, 4.6 x 150 mm, Waters); 0-15 min 5 ⁇ 80 % B; 15-20 min 95 % B.
• Ci H1 ( B )H); 6,95 (s, NH); 7,40 (d, 3 J 8, ⁇ , 2 x Cp te (5 ' )H) ; 7,48 (s,
• the product was isolated by reversed phase medium pressure liquid chromatography (RP-MPLC, solid phase: Eu- roprep 60-60 C-18; 60 A; 35-70 ⁇ m, 140 g; 36 cm x 26 mm, liquid phase: 0-10 min. 99,9 % H 2 O, 0,1 % HCOOH, 10-40 min. 34,9 % MeOH, 65 % H 2 O, 0,1 % HCOOH) to give 120 mg of Pte-Glu (H-His ( ⁇ - (4-N- Butyl) ) -OH) -OH as a yellowish resin.
• RP-MPLC reversed phase medium pressure liquid chromatography
• the sealed reaction vial was heated for 60 min at 100 0 C to form the corresponding in excellent yield (> 98 %) .
• Example 4 Synthesis of Triazol-Folate a) Synthesis of 4 -azido-butane-amine .
• the Boc-protected intermediate azide (0.42 g, 2.0 mmol ; prepared according to Link et al J. Am. Chem. Soc. 2004, 126, 10598) was dissolved in CH 2 Cl 2 (5 mL) and trifluoroacetic acid (TFA, 1.0 mL) was added.
• BocGluOMe (261 mg, 1.0 mmol) in dry DMF (5 mL, over molecular sieves 4 A) and
• N2-N,N- dimethylaminomethylene-10-formyl-pteroic acid 198 mg, 0.5 mmol
• dry DMF 10 mL, over molecular sieves 4 A
• Et 3 N 104 ⁇ L, 0.75 mmol
• HBTU 380 mg, 0.5 mmol
• amine TFA salt obtained under c) (186 mg, 0.5 mmol) in dry DMF (9 mL) containing Et 3 N (210 ⁇ L, 1.5 mmol).
• Synthesis B Deprotected azido folate (36 mg, 0.05 mmol; obtained under e) ) was suspended in fc BuOH/H 2 O (1:1, 3 mL) and L- propargyl glycine (6 mg, 0.053 mmol), Cu(OAc) 2 (2 mg, 20 mol%) and sodium ascorbate (4 mg, 40 mol%) were added. The mixture was stirred at 80 0 C for 20 min after which HPLC (HPLC system 1) indicated completed conversion of the starting substrate. The brown suspension was dissolved by addition of IM NaOH and as cleared by filtration through CeliteTM. The product was precipi- tated by adjusting the pH of the solution to pH ⁇ 2 with 1 M HCl.
• the suspension was centrifuged (10 min at 3500 rpm) , the supernatant decanted and the solid product dried under reduced pressure yielding the penta-hydrochloride salt of the desired Tria- zol-folate as an orange solid (42 mg, quantitative) .
• One-Pot-Synthesis B Deprotected Azido folate (obtained under step 4e) ; 40 ⁇ L, ca . 10 ⁇ 3 M in MeOH/PBS pH 7.4 (5:1)) was mixed with L-propargyl glycine (20 ⁇ L, 10 ⁇ 2 M in water), Cu(OAc) 2 (5 ⁇ L, 10 '2 M in water) and sodium ascorbate (20 ⁇ L, 10 '2 M in water) .
• Example 6 Synthesis of Re (CO) 3 -Triazol-folate complex Triazol-folate (obtained hereinabove) ; tri-trifluoroactetate mono-hydrate, 0.7 mg, 0.7 ⁇ mol) and [Re(Br) 3 (CO) 3 ] [Et 4 N] 2 (1.0 mg, 1.4 ⁇ mol) were suspended in water (0.5 mL) . Addition of NaOH (0.1 M) to a final pH of 8 yielded a yellow solution which was stirred at 50 0 C for Ih after which HPLC (HPLC system 1) indicated complete conversion of the starting azido folate. The so- lution was cooled to rt and the pH adjusted to pH ⁇ 2 by addition of dilute aq.
• Biodistribution studies were performed with 4-5-week-old male, athymic nude mice (NMRI nu/nu; Charles River, The Netherlands) . The animals were acclimated and fed with a folate-deficient rodent diet starting 5 days prior to the tumor cell inoculation. The mice were inoculated subcutaneously with the KB-tumor cell suspension (5 x 10 6 cells) into the subcutis of each shoulder. Radiofolate biodistribution studies were carried out approx. 14 days after tumor cell inoculation when the tumor size reached a size of approx. 0.5-1.5 cm 3 . The experiments were performed in triplicate.
• the 99m Tc (CO) 3 -His-folate and 99m Tc (CO) 3 -Triazol- folate, respectively, (1.5 MBq in 100 ⁇ L) were administered via a lateral tail vein.
• pemetrexed PMX; Alimta " ; Lilly, Bad Homburg, Germany
• PMX pemetrexed
• the animals were sacrificed at 1 h, 4 h and 24 h after administration of 99ra Tc-radiofolates alone or with pre-injected PMX.
• the selected tissues were removed, weighed, and counted for radioactivity in a ⁇ -counter to determine the percentage of injected activity per gram of tissue (% IA/g) .
• Table 2b Site 4 h P . i . blood 0.05 ⁇ 0.04 heart 0.34 + 0.15 lung 0.31 + 0.15 spleen 0.08 ⁇ 0.03 kidneys 4.22 + 2.28 stomach 0.70 ⁇ 0.26 intestines 0.53 + 0.30 liver 0.49 ⁇ 0.30 muscle 0.35 ⁇ 0.08 bone 0.18 + 0.04 parotid gland - tumor right 2.79 + 0.67 tumor left 2.56 + 0.42 tumor-to-blood 68.31 ⁇ 33.15 tumor-to-liver 7.16 ⁇ 4.49 tumor-to-kidney 0.71 ⁇ 0.33
• Jn vitro Autoradiography In vitro autoradiography was performed on adjacent sections of those prepared from tumor and kidneys for ex vivo autoradiography.
• the slides with tumor sections were pre-incubated in Tris-HCL buffer 8170 mM, pH 7.6, with 5 mM MgCl 2 ) with 0.25 (w/v) BSA for 10 min at room temperature.
• the sections were incubated with a solution of 99m Tc-His-folate or 99m Tc-Triazol-folate (0.5 MBq/mL in Tris-HCl buffer, contain- ing 1 % BSA) for 60 min at RT.
• SPECT/CT imaging was performed with a four-headed multiplexing multi-pinhole NanoSPECT (Bioscan Inc., Washington D. C). Each head was outfitted with a tungsten collimator of nine 1.4 mm- diameter pinholes, imaging a cylindrical field of view that is 37 mm in diameter by 16 mm in length.
• the axial FOV is extended using a step-and-shoot helical scan of the animal, with the user defining a range from 16 to 180 mm according to the region to be imaged.
• the apertures used in this study provided a reconstructed resolution in the submillimetre range at 140 keV.
• the acquisition time per view was chosen for 1000 s.
• CT was performed with the integrated CT using a tube voltage of 45 kV and an exposure time of 1000 ms per view.
• SPECT and CT data were reconstructed iteratively with the HiSPECT software (Bioscan Inc., Washington D. C, USA) software.
• the SPECT and CT fusion was performed using the MIPtool software (version 1.20, Bioscan Inc.) .

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• 2008
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