DIPHOSPHINE COMPOUNDS AND COMPLEXES
20250215027 ยท 2025-07-03
Assignee
Inventors
- Michelle Therese Ma (London, GB)
- Ingebjorg Narvestad Hungnes (London, GB)
- Charlotte Rivas (London, GB)
- Truc Thuy Pham (London, GB)
- Paul Gerard Pringle (Bristol, GB)
- Rachel Elizabeth Nuttall (Bristol, GB)
Cpc classification
A61K51/0489
HUMAN NECESSITIES
International classification
Abstract
A diphosphine precursor compounds of Formula (I), conjugates thereof of Formula (H) and radionuclide conjugate complexes thereof are disclosed herein. The compounds are advantageous at least because they enable the easy one-step extemporaneous preparation of the corresponding complexes in the clinic in high radiochemical yields and under mild conditions. Also disclosed are the methods of making the compounds and complexes herein along with their uses. The complexes are particularly useful in the field of medicine and diagnosis, such as in medical imaging and targeted payload delivery.
##STR00001##
Claims
1-26. (canceled)
27. A conjugated diphosphine precursor compound according to Formula (II) that is suitable for preparing a conjugated radiolabelled agent: ##STR00026## wherein; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group or a substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl group wherein each substituent is selected from the group consisting of a C.sub.1-C.sub.4alkyl group, C.sub.5-C.sub.12aryl or heteroaryl group, a C.sub.1-C.sub.4 acylamido group, a sulfylhydro group, a C.sub.1-C.sub.4 alkylthio group, a C.sub.1-C.sub.4(di)alkylphosphino group, a hydroxy group, a C.sub.1-C.sub.4alkoxy group, a carboxyl group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. the shortest linear chain of carbon atoms between the two Z groups is 4 to 7; LIG comprises a ligand with a binding motif corresponding to a biological target; and the compound is not ##STR00027##
28. A conjugated diphosphine precursor compound of claim 27, according to Formula (IIa) that is suitable for preparing a conjugated radiolabelled agent: ##STR00028## wherein; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8aryl group wherein each substituent is selected from the group consisting of a C.sub.1-C.sub.4alkyl group, a C.sub.1-C.sub.4alkoxy group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt), cyclic(Arg-Gly-Asp-dPhe-Lys) (RGD), pentixafor peptide, a minigastrin peptide analogue for targeting cholecystokinin-2 receptor, a c-Met-targeting peptide, an alpha-MSH peptide, a bisphosphonate, a folate or a carbohydrate.
29. The conjugated diphosphine precursor compound of claim 28, wherein the conjugated diphosphine precursor is according to Formula (IIb) and/or Formula (IIc): ##STR00029## wherein; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently p-tolyl, m-tolyl, o-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, 2,6-xylyl, 3,4-xylyl, 3,5-xylyl,r p-methoxyphenyl, o-methoxyphenyl, 4-(MeO(CH.sub.2CH.sub.2O))phenyl, 4-(MeO(CH.sub.2CH.sub.2O).sub.2)phenyl, 4-(MeO(CH.sub.2CH.sub.2O).sub.3)phenyl or 4-dimethylaminophenyl; and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt) or cyclic(Arg-Gly-Asp-dPhe-Lys) (RGD).
30. A compound of claim 27, that is: ##STR00030##
31. A diphosphine precursor compound according to Formula (I) that is suitable for preparing a conjugated radiolabelled agent: ##STR00031## wherein ring A is a 5, 6, 7 or 8 membered ring; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group wherein any substituents selected from the group consisting of a C.sub.1-C.sub.4alkyl group, C.sub.5-C.sub.12aryl or heteroaryl group, a C.sub.1-C.sub.4 acylamido group, a sulfylhydro group, a C.sub.1-C.sub.4 alkylthio group, a C.sub.1-C.sub.4(di)alkylphosphino group, a hydroxy group, a C.sub.1-C.sub.4alkoxy group, a carboxyl group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and the compound is not ##STR00032##
32. A diphosphine precursor compound of claim 30, according to Formula (Ia) that is suitable for preparing a conjugated radiolabelled agent: ##STR00033## wherein each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted C.sub.5-C.sub.8aryl group having one or more substituents selected from the group consisting of a C.sub.1-C.sub.4alkyl group, a C.sub.1-C.sub.4alkoxy group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
33. The diphosphine precursor compound of claim 32 wherein the diphosphine precursor is according to Formula (Ib) and/or (Ic): ##STR00034## wherein; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently p-tolyl, m-tolyl, o-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, 2,6-xylyl, 3,4-xylyl, 3,5-xylyl, p-methoxyphenyl, o-methoxyphenyl, 4-(MeO(CH.sub.2CH.sub.2O))phenyl, 4-(MeO(CH.sub.2CH.sub.2O).sub.2)phenyl, 4-(MeO(CH.sub.2CH.sub.2O).sub.3)phenyl or 4-dimethylaminophenyl.
34. A compound of claim 27 wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are the same.
35. A compound of claim 31 wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are the same.
36. A radiolabelled diphosphine complex comprising at least two compounds of claim 27 as ligands that are co-ordinated with one or more radionuclides selected from .sup.99mTc, .sup.212Pb, .sup.212Bi .sup.213Bi, .sup.186Re, .sup.188Re, .sup.89Zr, .sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.64Cu, .sup.62Cu, .sup.61Cu, .sup.60Cu, .sup.62Zn and .sup.52Mn; and the complex is not; ##STR00035## wherein in compound (Tc-III-1-RGD) Tc is .sup.99mTc, and in compound (Re-III-1-RGD) Re is selected from .sup.186Re and .sup.188Re.
37. A radiolabelled conjugated diphosphine complex of claim 36 that is either: (a) according to Formula (M-IIIa-trans) or Formula (M-IIIa-cis) or a mixture thereof ##STR00036## wherein M is a radionuclide selected from one or more of .sup.99mTc, .sup.186Re and .sup.188Re; or (b) according to Formula (M-IIIb-trans) or Formula (M-IIIb-cis) or a mixture thereof ##STR00037## wherein M is a radionuclide selected from one or more of .sup.99mTc, .sup.186Re and .sup.188Re; or (c) according to Formula (Cu-IIIc-A) or Formula (Cu-IIIc-B) or a mixture thereof; ##STR00038## wherein Cu is selected from .sup.67Cu, .sup.64Cu, .sup.62Cu, .sup.61Cu and .sup.60Cu; or (d) according to Formula (Cu-IIId-A) or Formula (Cu-IIId-B) or a mixture thereof; ##STR00039## wherein Cu is selected from .sup.67Cu, .sup.64Cu, .sup.62Cu, .sup.61Cu and .sup.60Cu; and wherein; X is a phenyl group having one or more substituents selected from the group consisting of a C.sub.1-C.sub.4alkyl group and a C.sub.1-C.sub.4alkoxy group; and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt), cyclic(Arg-Gly-Asp-dPhe-Lys) (RGD), pentixafor peptide, a minigastrin peptide analogue for targeting cholecystokinin-2 receptor, a c-Met-targeting peptide, an alpha-MSH peptide, a bisphosphonate, a folate or a carbohydrate optionally, wherein the radiolabelled conjugated diphosphine complex is either: (a) according to Formula (M-IIIa-trans) or Formula (M-IIIa-cis) or a mixture thereof ##STR00040## wherein M is a radionuclide selected from one or more of .sup.99mTc, .sup.186Re and .sup.188Re; or (b) according to Formula (M-IIIb-trans) or Formula (M-IIIb-cis) or a mixture thereof ##STR00041## wherein M is a radionuclide selected from one or more of .sup.99mTc, .sup.186Re and .sup.188Re; and wherein; X is a phenyl group having one or more substituents selected from the group consisting of a C.sub.1-C.sub.4alkyl group and a C.sub.1-C.sub.4alkoxy group; and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt), cyclic(Arg-Gly-Asp-dPhe-Lys) (RGD), pentixafor peptide, a minigastrin peptide analogue for targeting cholecystokinin-2 receptor, a c-Met-targeting peptide, an alpha-MSH peptide, a bisphosphonate, a folate or a carbohydrate; optionally, wherein; X is p-tolyl, m-tolyl, o-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, 2,6-xylyl, 3,4-xylyl, 3,5-xylyl, p-methoxyphenyl, o-methoxyphenyl, 4-(MeO(CH.sub.2CH.sub.2O))phenyl, 4-(MeO(CH.sub.2CH.sub.2O).sub.2)phenyl, 4-(MeO(CH.sub.2CH.sub.2O).sub.3)phenyl or 4-dimethylaminophenyl; and LIG comprises a prostate specific membrane antigen targeting ligand (PSMAt) or cyclic(Arg-Gly-Asp-dPhe-Lys) (RGD).
38. A complex according to claim 36 according to Formula (M-IIIa-trans) or Formula (M-IIIa-cis) or a mixture thereof; optionally, wherein the complex is an approximate 1:1 mixture of the cis/trans isomers.
39. A method of making a conjugated diphosphine precursor compound of claim 27, or Compound (II-1-RGD), comprising a step of mixing a diphosphine precursor compound or Compound (I-1) and LIG-H in the presence of a base, wherein LIG comprises a peptide or carbohydrate ligand with a binding motif corresponding to a biological target; optionally, wherein the base is N,N-diisopropylethylamine which is added dropwise and the reaction is conducted in N,N-dimethylformamide at room temperature; wherein the diphosphine precursor compound has a structure according to Formula (I): ##STR00042## where ring A is a 5, 6, 7 or 8 membered ring; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group wherein any substituents selected from the group consisting of a C.sub.1-C.sub.4alkyl group, C.sub.5-C.sub.12aryl or heteroaryl group, a C.sub.1-C.sub.4 acylamido group, a sulfylhydro group, a C.sub.1-C.sub.4 alkylthio group, a C.sub.1-C.sub.4(di)alkylphosphino group, a hydroxy group, a C.sub.1-C.sub.4alkoxy group, a carboxyl group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and the compound is not ##STR00043##
40. A method of making the radiolabelled conjugated diphosphine complex of claim 36, Compound (Tc-III-1-RGD), Compound (.sup.186Re-III-1-RGD) or Compound (Re-III-1-RGD), comprising the step of mixing a conjugated diphosphine precursor compound or Compound (II-1-RGD) with a radionuclide, in the presence of an intermediate ligand, a reducing agent, a buffer and a solvent optionally, wherein the radionuclide is selected from one of more of .sup.99mTc, .sup.212Bi, .sup.213Bi, .sup.186Re, .sup.188Re, .sup.89Zr, .sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.64Cu, .sup.62Cu, .sup.61Cu, .sup.60Cu and .sup.52Mn, the intermediate ligand is sodium tartrate, the reducing agent is tin(II) chloride, the buffer sodium hydrogen carbonate and the solvent is preferably selected from one or more of water, a saline solution, methanol, ethanol, propanol and isopropanol; wherein the conjugated diphosphine precursor compound has a structure according to Formula (II): ##STR00044## wherein; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8 aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group or a substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl group wherein each substituent is selected from the group consisting of a C.sub.1-C.sub.4alkyl group, C.sub.5-C.sub.12aryl or heteroaryl group, a C.sub.1-C.sub.4 acylamido group, a sulfylhydro group, a C.sub.1-C.sub.4 alkylthio group, a C.sub.1-C.sub.4(di)alkylphosphino group, a hydroxy group, a C.sub.1-C.sub.4alkoxy group, a carboxyl group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. the shortest linear chain of carbon atoms between the two Z groups is 4 to 7; LIG comprises a ligand with a binding motif corresponding to a biological target; and the compound is not ##STR00045##
41. A pharmaceutical composition comprising a compound or complex of claim 27 in combination with a pharmaceutically acceptable carrier.
42. A kit for preparing a complex according to claim 38, Compound (Tc-III-1-RGD), Compound (.sup.186Re-III-1-RGD) or Compound (Re-III-1-RGD) comprising a mixture of a reducing agent, a buffering agent, an intermediate co-ligand and a conjugated diphosphine precursor compound or Compound (II-1-RGD); wherein the conjugated diphosphine precursor compound has a structure according to option i) or ii): i) the conjugated diphosphine precursor compound has a structure according to Formula (II) ##STR00046## wherein; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group or a substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl group wherein each substituent is selected from the group consisting of a C.sub.1-C.sub.4alkyl group, C.sub.5-C.sub.12aryl or heteroaryl group, a C.sub.1-C.sub.4 acylamido group, a sulfylhydro group, a C.sub.1-C.sub.4 alkylthio group, a C.sub.1-C.sub.4(di)alkylphosphino group, a hydroxy group, a C.sub.1-C.sub.4alkoxy group, a carboxyl group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. the shortest linear chain of carbon atoms between the two Z groups is 4 to 7; LIG comprises a ligand with a binding motif corresponding to a biological target; and the compound is not ##STR00047## ii) the diphosphine precursor compound has a structure according to Formula (I): ##STR00048## wherein ring A is a 5, 6, 7 or 8 membered ring; each Z is independently O or S; Y is NH or O; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently a substituted or unsubstituted C.sub.5-C.sub.8aryl group, a substituted or unsubstituted 5- to 8-membered heteroaryl group wherein any substituents selected from the group consisting of a C.sub.1-C.sub.4alkyl group, C.sub.5-C.sub.12aryl or heteroaryl group, a C.sub.1-C.sub.4 acylamido group, a sulfylhydro group, a C.sub.1-C.sub.4 alkylthio group, a C.sub.1-C.sub.4(di)alkylphosphino group, a hydroxy group, a C.sub.1-C.sub.4alkoxy group, a carboxyl group, a C.sub.1-C.sub.4(di)alkylamino group and a C.sub.1-C.sub.4alkoxy-(CH.sub.2CH.sub.2O).sub.n group wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and the compound is not ##STR00049##
43. The kit of claim 42, wherein the kit further comprising a radionuclide selected from .sup.99mTc, .sup.212Bi, .sup.213Bi, .sup.186Re, .sup.188Re, .sup.89Zr, .sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.64Cu, .sup.62Cu, .sup.61Cu, .sup.60Cu and .sup.52Mn.
44. The kit according to claim 43, wherein the radionuclide is selected from .sup.99mTc, .sup.186Re, .sup.188Re, and .sup.52Mn.
45. A method of treating or diagnosing a disease comprising administering a compound or complex of claim 27, Compound (I-1), Compound (II-1-RGD), Compound (Tc-III-1-RGD), Compound (.sup.186Re-III-1-RGD) or Compound (Re-III-1-RGD) to a subject.
46. Non-therapeutic use of a compound or complex of claim 27, Compound (I-1), Compound (II-1-RGD), Compound (Tc-III-1-RGD), Compound (.sup.186Re-III-1-RGD) or Compound (Re-III-1-RGD) in imaging or cell labelling.
Description
SUMMARY OF THE FIGURES
[0124] So that the invention may be understood, and so that further aspects and features thereof may be appreciated, embodiments illustrating the principles of the invention will now be discussed in further detail with reference to the accompanying figures, in which:
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DETAILED DESCRIPTION
[0156] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
[0157] While the invention has been described in conjunction with the exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the invention.
[0158] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.
[0159] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0160] Throughout this specification, including the claims which follow, unless the context requires otherwise, the words have, comprise, and include, and variations such as having, comprises, comprising, and including will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. However, each disclosure herein also includes the option of excluding any other integer or step or group of integers or steps.
[0161] It must be noted that, as used in the specification and the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent about, it will be understood that the particular value forms another embodiment. The term about in relation to a numerical value is optional and means, for example, +/10%.
[0162] The words preferred and preferably are used herein refer to embodiments of the invention that may provide certain benefits under some circumstances. It is to be appreciated, however, that other embodiments may also be preferred under the same or different circumstances. The recitation of one or more preferred embodiments therefore does not mean or imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, or from the scope of the claims.
[0163] The compounds of the present invention include isomers, salts, solvates, and chemically protected forms thereof, as explained in more detail below.
[0164] In the present invention, alkyl groups are generally C.sub.1-C.sub.4 alkyl groups. The term C.sub.1-C.sub.4 alkyl, as used herein, includes a monovalent moiety obtained by removing a hydrogen atom from a C.sub.1-C.sub.4 hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated. The term C.sub.1-C.sub.4 alkyl includes methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, ethenyl, cis/trans-1-propenyl, 2-propenyl, cis/trans-1-butenyl, cis/trans-2-butenyl and 3-butenyl. In preferred embodiments, the C.sub.1-C.sub.4 alkyl group is a saturated alkyl group and/or an acyclic alkyl group. In even more preferred embodiments the C.sub.1-C.sub.4 alkyl group is methyl or an ethyl group as shorter chain alkyl groups tend to make the compounds of the present invention less hydrophobic.
[0165] In the present invention, alkoxy groups are generally C.sub.1-C.sub.4 alkoxy groups. The term C.sub.1-C.sub.4 alkoxy, as used herein, includes a monovalent moiety obtained by removing the hydrogen atom from the oxygen atom of a C.sub.1-C.sub.4 alcohol compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated. The term C.sub.1-C.sub.4 alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, t-butoxy, cyclobutoxy, ethenoxy, cis/trans-1-propenoxy, 2-propenoxy, cis/trans-1-butenoxy, cis/trans-2-butenoxy and 3-butenoxy. In preferred embodiments, the C.sub.1-C.sub.4 alkoxy group is a saturated alkoxy group and/or an acyclic alkoxy group. In even more preferred embodiments the C.sub.I-C.sub.4 alkoxy group is methoxy or an ethoxy group as shorter chain alkoxy groups tend to make the compounds of the present invention less hydrophobic.
[0166] In the present invention, a heteroaryl group is generally a C.sub.5-C.sub.12 heteroaryl group, and is preferably a 5 or 6 membered heteroaryl group and as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a C.sub.3-C.sub.12 heterocyclic compound. The heteroaryl groups may be partially or fully unsaturated. The present invention provides example of compounds in which one or more pyridyl groups (e.g. one or more 2-pyridyl groups) are present. However, examples of heteroaryl compounds that could be employed in accordance with the present invention include:
[0167] Imidazole: a five membered aromatic ring having two nitrogen atoms and three carbon atom.
[0168] Triazole: a five membered aromatic ring having three nitrogen atoms and two carbon atoms, with two ring isomers 1,2,3,triazole, 1,2,4 triazole.
[0169] Tetrazole: a five membered aromatic ring having four nitrogen atoms and one carbon atom.
[0170] Pyridine: a six membered aromatic ring having one nitrogen atom and 5 carbon atoms.
[0171] Diazine: a six membered aromatic ring having two nitrogen atoms and four carbon atoms, with three ring isomers, 1,2-diazine, 1,3-diazine and 1,4-diazine.
[0172] Triazine: a six membered aromatic ring having three nitrogen atoms and three carbon atoms, with three ring isomers, 1,2,3-triazine, 1,2,4-triazine and 1,3,5-triazine.
[0173] Tetrazine: a six membered aromatic ring having four nitrogen atoms and two carbon atoms, with three ring isomers 1,2,3,4-tetrazine, 1,2,3,5-tetrazine and 1,2,4,5-tetrazine.
[0174] Fused ring systems such as quinoline, isoquinoline and indole.
[0175] It is generally preferred that the sp.sup.2 nitrogen containing heterocyclic group has a donor electron pair in the ortho position relative to the methylene bridge of the bisphosphonate compound in order to facilitate chelation of the radionuclide by the heteroatom. A preferred heteroatom is nitrogen, i.e. providing pyridyl heteroaryl groups.
[0176] In the present invention, Re and .sup.188Re refer to rhenium-188 (.sup.188Re), whereas .sup.186Re refers to rhenium-186, and .sup.natRe refers to naturally abundant rhenium. Tc and .sup.99mTc refer to technetium-99m (.sup.99mTc), whereas .sup.99gTc refers to techniutium-99g. .sup.natCu refers to naturally abundant copper.
Other Forms of the Substituents
[0177] Included in the above are the well-known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (COO), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (N.sup.+HR.sup.1R.sup.2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (O), a salt or solvate thereof, as well as conventional protected forms of a hydroxyl group.
Isomers, Salts, Solvates, Protected Forms, and Prodrugs
[0178] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1-forms; (+) and () forms; keto, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; - and -forms; axial and equatorial forms; boat-, chair-, twist-envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as isomers (or isomeric forms).
[0179] Note that, except as discussed below for tautomeric forms, specifically excluded from the term isomers, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, OCH.sub.3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, CH.sub.2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures or to a general formula includes structurally isomeric forms falling within that class or formula and, except where specifically stated or indicated, all possible conformations and configurations of the compound(s) herein are intended to be included in the general formula(e).
[0180] The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
[0181] Note that specifically included in the term isomer are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including .sup.1H, .sup.2H(D), and .sup.3H(T); C may be in any isotopic form, including .sup.12C, .sup.13C, and .sup.14C; O may be in any isotopic form, including .sup.16O and .sup.18O; and the like.
[0182] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
[0183] Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.
[0184] It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
[0185] For example, if the compound is anionic, or has a functional group which may be anionic (e.g., COOH may be COO.sup.), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na.sup.+ and K.sup.+, alkaline earth cations such as Ca.sup.2+ and Mg.sup.2+, and other cations such as Al.sup.3+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH.sub.4.sup.+) and substituted ammonium ions (e.g., NH.sub.3R.sup.+, NH.sub.2R.sub.2.sup.+, NHR.sub.3.sup.+, NR.sub.4.sup.+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH.sub.3).sup.4+.
[0186] If the compound is cationic, or has a functional group which may be cationic (e.g., NH.sub.2 may be NH.sup.3+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric, phenylsulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, and gluconic. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
[0187] It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term solvate is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
[0188] It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form. The term chemically protected form, as used herein, includes a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts, Wiley, 1999).
[0189] For example, a hydroxy group may be protected as an ether (OR) or an ester (OC(O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (OC(O)CH.sub.3, OAc).
[0190] For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (>CO) is converted to a diether (>C(OR).sub.2), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.
[0191] For example, an amine group may be protected, for example, as an amide or a urethane, for example, as: a methyl amide (NHCOCH.sub.3); a benzyloxy amide (NHCOOCH.sub.2C.sub.6H.sub.5, NH-Cbz); as a t-butoxy amide (NHCOOC(CH.sub.3).sub.3, NHBoc); a 2-biphenyl-2-propoxy amide (NHCOOC(CH.sub.3).sub.2C.sub.6H.sub.4C.sub.6H.sub.5, NH-Bpoc), as a 9-fluorenylmethoxy amide (NHFmoc), as a 6-nitroveratryloxy amide (NHNvoc), as a 2-trimethylsilylethyloxy amide (NHTeoc), as a 2,2,2-trichloroethyloxy amide (NH-Troc), as an allyloxy amide (NHAlloc), as a 2(-phenylsulphonyl)ethyloxy amide (NHPsec); or, in suitable cases, as an N-oxide (>NO).
[0192] For example, a carboxylic acid group may be protected as an ester for example, as: an C.sub.1-C.sub.7 alkyl ester (e.g. a methyl ester; a t-butyl ester); a C.sub.1-C.sub.7 haloalkyl ester (e.g., a C.sub.1-C.sub.7-trihaloalkyl ester); a tri-C.sub.1-C.sub.7-alkylsilyl-C.sub.1-C.sub.7-alkyl ester; or a C.sub.5-C.sub.20 aryl-C.sub.1-C.sub.7-alkyl ester (e.g. a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.
[0193] It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term prodrug, as used herein, includes a compound which, when metabolised (e.g. in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.
[0194] For example, some prodrugs are esters of the active compound (e.g. a physiologically acceptable metabolically labile ester). During metabolism, the ester group (C(O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Examples of such metabolically labile esters include those wherein R is C.sub.1-7 alkyl (e.g. Me, -Et); C.sub.1-7 aminoalkyl (e.g. aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4 morpholino)ethyl); and acyloxy-C.sub.1-C.sub.7 alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl; 1-(1-methoxy-1-methyl)ethyl-carbonxyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1-(4-tetrahydropyranyloxy)carbonyloxyethyl; (4-tetrahydropyranyl)carbonyloxymethyl; and 1-(4 tetrahydropyranyl)carbonyloxyethyl).
[0195] Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
Complexes of the Compounds and Their Uses
[0196] The compounds of the present invention may be used for therapy, in particular the treatment of arthritis and cancer. In addition, the compounds of the present invention may be used to chelate radionuclides, for example to enable them to be employed in imaging studies or for therapeutic purposes. Examples of radionuclides that are chelatable by the compounds of the present invention include technetium, rhenium and copper isotopes such as .sup.99mTc, .sup.186Re, .sup.188Re, .sup.67Cu, .sup.64Cu, .sup.62Cu, .sup.61Cu, .sup.60Cu. The present invention may employ the radionuclides alone or in combinations. For example, one commonly used combination is .sup.186/188Re. Other combinations are .sup.99mTc/.sup.188Re or .sup.99mTc/.sup.186Re. In general, technetium isotopes are employed for imaging purposes, rhenium isotopes for therapeutic purposes and copper isotopes for both imaging and therapy. Where a specific isotope is not shown for an atom, it may be selected as any of the known isotopes or a mixture thereof.
[0197] The present invention provides active compounds for use in a method of treatment of the human or animal body. Such a method may comprise administering to such a subject a therapeutically-effective amount of an active compound, preferably in the form of a pharmaceutical composition.
[0198] The term treatment, as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, relief of pain, and cure of the condition. Treatment as a preventative measure, i.e. prophylaxis, is also included. By way of example, the compounds and complexes of the present invention may be used for the treatment of arthritis and for the treatment of cancer. The treatment of cancer may involve palliative and/or therapeutic treatment.
[0199] The term therapeutically-effective amount as used herein, includes that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
Formulations and Dosage
[0200] While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.
[0201] Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein.
[0202] The term pharmaceutically acceptable as used herein includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be acceptable in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
[0203] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution or suspension which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
[0204] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
[0205] It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
[0206] Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
EXAMPLES
Materials and Methods
[0207] All chemicals were supplied by Sigma-Aldrich or Fisher Scientific if not otherwise specified. Sodium (pertechnetate) (Na[.sup.99mTcO.sub.4]) in saline was supplied by Guy's and St Thomas' Hospital Nuclear Medicine Services. Cyclic RGD peptide (Arg-Gly-Asp-D-Phe-Lys, cyclised via the peptide backbone) and PSMAt peptide were purchased from Peptide Synthetics (Hampshire, UK).
[0208] NMR data (.sup.1H, .sup.13C{H}and .sup.31P{H}1D spectra and COSY, TOCSY and HSQC spectra) were acquired on a Bruker Avance III 400 spectrometer equipped with a QNP probe or a Bruker Avance III 700 spectrometer equipped with an AVIII console and a quadruple-resonance QCI cryoprobe. High resolution mass spectrometry (MS) was performed by the King's College London Mass Spectrometry Facilities, using a high resolution Thermo Exactive mass spectrometer in positive electrospray mode. Samples were infused to the ion source at a rate of 10 l/min using a syringe pump. High performance liquid chromatography (HPLC) was carried out on an Agilent 1200 LC system with the Laura software, a Rheodyne sample loop (200 L) and UV spectroscopic detection at 220 nm or 254 nm. The HPLC was attached to a LabLogic Flow-Count detector with a sodium iodide probe (B-FC-3200) for radiation detection. Semi-preparative (9.4250 mm, 5 m) and analytical (4.6150 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 columns were used with purified water (A) and acetonitrile (B) containing 0.005% and 0.1% TFA as mobile phases for semi-preparative and analytical runs, respectively.
[0209] General HPLC methods used herein include; HPLC Method 1 (semi-preparative): 100 minutes, 1% min-1 linear increase from 100% A to 100% B, flow rate=3 ml min.sup.1. HPLC Method 2 (analytical): 20 minutes, 5% min.sup.1 linear increase from 100% A to 100% B (flow rate of 1 ml/min). HPLC Method 3 (semi-preparative): 200 minutes, 0.5% min.sup.1 linear increase from 95% A to 100% B (flow rate of 3 ml/min). HPLC Method 4 (analytical): 55 minutes, 2.5% min.sup.1 linear increase from 100% A to 25% B over 10 min, followed by 0.33% min.sup.1 linear increase from 25% A to 40% B over 45 min (flow rate of 1 mL min.sup.1).
[0210] Instant thin layer chromatography (iTLC) used iTLC SGIO001 strips (Varian Medical Systems, Crawley, UK). The iTLC plates were scanned with a Perkin Elmer Storage Phosphor System (Cyclone) or a LabLogic miniScan TLC reader equipped with Laura software.
[0211] High performance liquid chromatography (HPLC) was carried out on an Agilent 1200 HPLC system with Laura software, a Rheodyne sample loop (200 L) and ultraviolet (UV) spectroscopic detection at 214 nm, 220 nm, 254 nm or 280 nm.
Example 1Synthesis of Compound (I-1); 3,4-bis(bisphenylphosphanyl)furan-2,5-dione (Compound (I-1)
##STR00020##
[0212] Diphenylphosphine (2.2 equiv., 5.04 mmol, 0.88 mL) was added to a solution of dichloromaleic anhydride (1 equiv., 2.42 mmol, 404.0 mg) in diethyl ether (15 mL) to give a pale-yellow solution. Triethylamine (2.2 equiv. 5.04 mmol, 0.7 mL) was added dropwise and the dark yellow suspension stirred (rt, 2 h) until a compact sludge had formed. The solids, which contained product, were isolated by filter cannula and washed with ice cold diethyl ether (310 mL). The crude product was re-dissolved and passed through a silica plug in dichloromethane, after which the solvent was removed under reduced pressure to yield a yellow solid. This product was recrystallised from chloroform/diethyl ether, furnishing crystalline yellow needles (390.7 mg, 837.7 mol, 34.6%).
[0213] .sup.1H NMR (399 MHz, acetonitrile-d.sub.3, 298 K): (ppm) 7.38-7.42 (m, 12H, H.sub.meta and H.sub.para), 7.34-7.30 (m, 8H, H.sub.ortho);
[0214] .sup.13C NMR (100 MHz, acetonitrile-d.sub.3, 298 K): (ppm) 163.22 (m, C.sub.carbonyl), 153.50 (m, C.sub.alkene), 134.12 (m, C.sub.ortho), 133.00 (m, C.sub.subset), 129.84 (m, C.sub.para), 128.73 (m, C.sub.meta);
[0215] .sup.31P{.sup.1H}NMR (162 MHz, acetonitrile-d.sub.3, 298 K): (ppm) 18.37;
[0216] .sup.31P{.sup.1H}NMR (162 MHz, dimethylformamide-d.sub.7, 298 K): (ppm) 19.07;
[0217] .sup.31P{.sup.1H}NMR (162 MHz, chloroform-d.sub.3, 298 K): (ppm) 20.53;
[0218] HR-MS-ESI m/z: [M+H].sup.+ 467.0954 (Calculated for C.sub.28H.sub.21O.sub.3P.sub.2 467.0960);
[0219] IR (solid) .sub.max (cm.sup.1) 3054 (w), 1834 (m), 1811 (m), 1757 (s), 1496 (w), 1484(w), 1435 (m), 1244 (s), 913 (s);
[0220] m.p. 149.6 C.
Example 2Synthesis of Compound (I-2); 3,4-bis(bis-p-tolylphosphanyl)furan-2,5-dione (Compound (I-2))
##STR00021##
[0221] STEP 1: Bis(p-tolyl)chlorophosphine (1 equiv., 4.02 mmol, 0.9 mL) in diethyl ether (5 mL) was added dropwise to a slurry of lithium aluminium hydride (3.2 equiv., 13.01 mmol, 493.8 mg) in diethyl ether (20 mL) at 0 C. The grey suspension was stirred at 0 C. (30 min) and then at room temperature until reaction completion (22 h), determined by in situ .sup.31P{.sup.1H}NMR. The reaction was quenched by dropwise addition of i) degassed water (0.5 mL), ii) 15% NaOH.sub.(aq) (0.5 mL) and iii) degassed water (1.5 mL) at 0 C.
[0222] The white precipitate was removed from the filtrate (that contained the product) by filter cannula. The precipitate was then washed with diethyl ether (210 mL) and these washes were combined with the filtrate. The resulting solution was dried on magnesium sulfate and re-isolated by filter cannula, washing the magnesium sulfate with diethyl ether (210 mL) and combining the filtrate and washes. The solvent was removed under reduced pressure to yield the product as a clear liquid (593.4 mg, 2.77 mmol, 68.9%) that crystallized below 20 C. When the reaction scale was doubled, the crude product was purified by distillation at 200 C. and 2.510-1 mbar.
[0223] 1H NMR (400 MHz, Chloroform-d) 7.48-7.36 (m, 4H, Hb), 7.22-7.12 (m, 4H, He), 5.25 (d, JH-P=174.9 Hz, 1H, PH), 2.38 (s, 6H, He);
[0224] .sup.31P{.sup.1H}NMR (162 MHz, Chloroform-d) 41.93;
[0225] .sup.31P NMR (162 MHz, Chloroform-d) P 41.92 (d, J=174.9 Hz).
[0226] STEP 2: 3,4-bis(bis-o-tolylphosphanyl)furan-2,5-dione was prepared from (Tol).sub.2PH by the following method: A solution of ditolylphosphine (1.9 equiv., 0.36 mmol, 77.0 mg) in diethylether (0.2 mL) was added dropwise to a solution of dichloromaleic anhydride (1 equiv., 0.19 mmol, 31.0 mg) in tetrahydrofuran (1.3 mL) to give a clear orange solution. Triethylamine (3 equiv. 0.58 mmol, 0.08 mL) was added dropwise and the dark orange suspension stirred (rt, 2 h). The solids were removed by filter cannula, washing with tetrahydrofuran (32 mL). The filtrate and washes (that contained the product) were combined and the solvent removed from the resulting product solution under reduced pressure. The crude product was re-dissolved and passed through a silica plug in dichloromethane and the solvent removed under reduced pressure. The product was dissolved in a minimal amount of chloroform and the solution layered with diethyl ether. The precipitate was collected by filtration and dried to yield the product as yellow needle crystals (30.2 mg, 0.06 mmol, 16.1%).
[0227] .sup.1H NMR (500 MHz, Chloroform-d) H 7.21 (dt, J=8.6, 4.4 Hz, 8H, H.sub.ortho), 7.08 (d, J=7.7 Hz, 8H, H.sub.meta), 2.34 (s, 12H, H.sub.para-Methyl);
[0228] .sup.13C{.sup.1H}NMR (125 MHz, Chloroform-d): C (ppm) 162.84 (t, J=2.86, C.sub.carbonyl), 155.03 (m, C.sub.alkene), 140.06 (s, C.sub.para), 134.24 (m, C.sub.ortho), 129.60 (t, J=4.40, C.sub.meta), 129.04 (m, C.sub.subst), 21.55 (s, C.sub.para-Methyl);
[0229] .sup.31P{.sup.1H}NMR (162 MHz, Chloroform-d) P 23.08 (s);
[0230] HR-MS-ESI m/z: [M+H].sup.+ 523.1602 (calculated for C.sub.32H.sub.28O.sub.3P.sub.2 523.1592).
Example 3aSynthesis of bis(para-methoxyphenyl )phosphine((p-MeOC.SUB.6.H.SUB.4.).SUB.2.PH)
##STR00022##
[0231] A solution of bis(4-methoxyphenyl)chlorophosphine (1 g, 3.56 mmol) in Et.sub.2O (4.5 mL) was added dropwise to a suspension of LiAlH.sub.4 (1.24 g, 11.4 mmol, 3.2 eq) in Et.sub.2O (18 mL) at 0 C. The solution was stirred for a further 30 min at 0 C. before allowing to warm to RT and then stirred overnight. The reaction mixture was cooled to 0 C. and quenched by the careful addition of H.sub.2O (0.5 mL), 15% NaOH (0.5 mL) and H.sub.2O (2.5 mL). After stirring for 1 hr, the solution was isolated by filtration and then concentrated in vacuo to give the title compound (744 mg, 3.02 mmol, 85%) as a white solid.
[0232] .sup.1H NMR (400 MHz, CDCl.sub.3): .sub.H (ppm) 7.46-7.28 (m, 4H, ArH), 6.90-6.82 (m, 4H, ArH), 5.38-4.98 (br. s, PH), 3.80 (s, 6H, OMe).
[0233] .sup.31P{.sup.1H}NMR (162 MHz, CDCl.sub.3): .sub.P (ppm) 44.2 (s). The spectroscopic data is in accordance with the literature (Y. Y. Yan and T. V. RajanBabu, Org. Lett., 2000, 2, 4137-4140).
Example 3bSynthesis of Compound (I-11); 3,4-bis[bis(4-methoxyphenyl)phosphanyl]furan-2,5-dione
##STR00023##
[0234] NEt.sub.3 (30.4 L, 2.18 mmol, 2.2 eq) was added to a solution of (p-MeOC.sub.6H.sub.4).sub.2PH (50.0 mg, 0.203 mmol, 2.05 eq) in Et.sub.2O (0.5 mL). A solution of 2,3-dichloromaleic anhydride (16.5 mg, 98.8 mol) in Et.sub.2O (0.5 mL) was added dropwise, which resulted in an immediate colour change from colourless to deep red solution. Once the reaction had reached completion, as monitored by .sup.31P NMR spectroscopy, the volatiles were removed in vacuo. The crude product was dissolved in DCM and passed through a silica plug (2% MeOH in DCM) and concentrated to dryness. Residual (p-MeOC.sub.6H.sub.4).sub.2PH was removed under high vacuum (c.a. 10.sup.7 Torr) to give the title compound (51.4 mg, 87.7 mol, 89%) as an orange solid.
[0235] .sup.31P{.sup.1H}NMR (162 MHz, CDCl.sub.3): .sub.P (ppm) 22.3 (s).
[0236] .sup.1H NMR (400 MHz, CDCl.sub.3): .sub.H (ppm) 7.28-7.21 (m, 8H, ArH), 6.83-6.78 (m, 8H, ArH), 3.80 (s, 12H, OMe).
[0237] .sup.13C NMR (101 MHz, CDCl.sub.3): .sub.C (ppm) 163.0 (m, CO), 161.1 (s, p-ArC), 154.2 (m, CC), 135.9 (t, .sup.2J.sub.P,C=12.2, o-ArCH), 123.4 (s, ArC), 114.5 (t, .sup.3J.sub.P,C=4.9 Hz, m-ArCH), 55.3 (s, OMe).
[0238] HR-MS (Nanospray): m/z calcd. for C.sub.32H.sub.29O.sub.7P.sub.2 [M+H].sup.+=587.1389; obs.=587.1395.
Example 4Synthesis of PEG-PSMA Peptide Conjugates (II-1-PSMAt1), (II-2-PSMAt1) and (II-11-PSMAt1)
##STR00024## ##STR00025##
[0239] Under a stream of nitrogen, Compound (I-1), Compound (I-2) or Compound (I-11) (5-10 mg, 1 equiv.) in DMF (100 L, dry, degassed) and Lys-((PEG).sub.4NH.sub.2)-uredo-Glu, 5-10 mg, 1 equiv.) in DMF (100 L, dry, degassed) were combined and N,N-diisopropylethylamine (DIPEA, 6 L) added. The tube was sealed and the solution agitated at room temperature (15-20 min). The product was isolated by semi-preparative C.sub.18-HPLC (mobile phases: 0.01% acetic acid in water (A) and acetonitrile (B); method starting at 95% A and increasing to 100% B; unreacted compound elutes at 100% acetonitrile). Product-containing fractions were neutralised with aqueous ammonium bicarbonate buffer (0.125 M, 15 L/mL elute) and freeze-dried to yield the PSMAt1 peptide conjugate (>60.0%) as a solid.
[0240] The reaction is reversible under acidic conditions, but simple addition of ammonium bicarbonate to solutions of isolated material prevents this.
Characterization of Compound (II-1-PSMAt1)
[0241] (700 MHz, DMF-d.sub.7, 298 K): (ppm) 1.382-1.436 (m, 2H, Lys, H.sub.), 1.445-1.504 (m, 2H, Lys, H.sub.), 1.608-1.660 (m, 1H, Lys, H.sub.), 1.742-1.795 (m, 1H, Lys, H.sub.), 1.842-1.893 (m, 1H, Glu, H.sub.), 1.986-2.039 (m, 1H, Glu, H.sub.), 2.324-2.364 (m, 1H, Glu, H.sub.), 2.386 (t, J=6.24 Hz, 2H, PEG, H.sub.o), 2.455 (dt, J.sub.1=14.68 Hz, J.sub.2=8.39 Hz, 1H, Glu, H.sub.), 2.955-2.983 (m, 2H, PEG, H.sub.h), 3.014-3.045 (m, 2H, PEG, H.sub.i), 3.128 (dd, J.sub.1=12.81 Hz, J.sub.2=6.46 Hz, 2H, Lys, H.sub.), 3.417-3.431 (m, 2H, PEG, H.sub.j-o), 3.520-3.591 (m, 10H, PEG, H.sub.j-o), 3.683 (t, J=6.24 Hz, 2H, PEG, H.sub.p), 4.204-4.233 (m, 1H, Lys, H.sub.), 4.261-4.285 (m, 1H, Glu, H.sub.), 6.543 (m, 1H, Glu, NH), 6.639 (d, J=7.48 Hz, 1H, Lys, NH), 7.208-7.257 (m, 12H, DP.sup.Ph, H.sub.e/f), 7.431-7.466 (m, 4H, DP.sup.Ph, CH.sub.d/d), 7.574-7.601 (m, 4H, DP.sup.Ph, H.sub.d/d), 7.806 (t, J=5.44 Hz, 1H, PEG, NH), 7.828 (t, J=5.67 Hz, 1H, Lys, NH.sub.); .sup.13C NMR (176 MHz, DMF-d.sub.7, 298 K): (ppm), 23.063 (s, Lys, C.sub.), 29.320 (Lys, C.sub.), 29.840 (Glu, C.sub.), 32.208 (s, Glu, C.sub.), 32.673 (s, Lys, C.sub.), 36.718 (s, PEG, C.sub.q), 38.739 (s, PEG, C.sub.h), 38.834 (s, Lys, C.sub.), 53.364 (s, Glu, C.sub.), 53.498 (s, Lys, C.sub.), 67.398 (s, PEG, C.sub.p), 69.051 (s, PEG, C.sub.i), 70.096 (s, PEG, C.sub.j-o), 70.217 (s, PEG, C.sub.j-o), 70.292 (s, PEG, C.sub.j-o), 70.419 (s, PEG, C.sub.j-o), 70.430 (s, PEG, C.sub.j-o), 127.814 (d, J=6.78 Hz, DP.sup.Ph, C.sub.e/e), 127.870 (d, J=7.35 Hz, DP.sup.Ph, C.sub.e/e), 128.150 (s, DP.sup.Ph, C.sub.f/f), 128.379 (s, DP.sup.Ph, C.sub.f/f), 134.035 (dd, J.sub.1=19.47 Hz, J.sub.2=5.82 Hz, DP.sup.Ph, C.sub.d/d), 134.653 (d, J=20.35 Hz, DP.sup.Ph, C.sub.d/d), 136.936 (m, DP.sup.Ph, C.sub.c/c), 137.650 (m, DP.sup.Ph, C.sub.c/c), quaternary carbons: 157.065 (s), 170.452 (s), 174.729 (s), 175.006 (s), 175.232 (s), remaining signals corresponding to quaternary carbons could not be distinguished from noise; .sup.31P{.sup.1H}NMR (283 MHz, DMF-d.sub.7, 298 K): (ppm) 13.18 (d, J=162.7 Hz), 12.15 (d, J=162.7 Hz).
[0242] HR-MS-ESI m/z: [M+H].sup.+ 1033.3759 (calculated for C.sub.51H.sub.63O.sub.15N.sub.4P.sub.2 1033.3760), [M+Na].sup.+ 1055.3579 (calculated for C.sub.51H.sub.62O.sub.15N.sub.4P.sub.2Na 1055.3579).
Characterization of Compound (II-2-PSMAt1)
[0243] .sup.1H NMR (700 MHz, DMF-d.sub.7, 298 K): (ppm) 1.389-1.444 (m, 2H, Lys, H.sub.), 1.453-1.503 (m, 2H, Lys, H.sub.), 1.618-1.670 (m, 1H, Lys, H.sub.), 1.752-1.801 (m, 1H, Lys, H.sub.), 1.899-1.949 (m, 1H, Glu, H.sub.), 1.983-2.035 (m, 1H, Glu, H.sub.), 2.262 (s, 6H, DP.sup.Tol, H.sub.g/g), 2.293 (s, 6H, DP.sup.Tol, H.sub.g/g), 2.345-2.384 (m, 1H, Glu, H.sub.), 2.389 (t, J=6.25 Hz, 2H, PEG, H.sub.o), 2.451 (dt, J.sub.1=15.00 Hz, J.sub.2=8.17 Hz, 1H, Glu, H.sub.), 2.962-3.008 (m, 4H, PEG, H.sub.h/i), 3.139 (hidden, 2H, Lys, H.sub.), 3.407-3.421 (m, 2H, PEG, H.sub.j-o), 3.524-3.591 (m, 10H, PEG, H.sub.j-o), 3.685 (t, J=6.25 Hz, 2H, PEG, H.sub.p), 4.217-4.247 (m, 1H, Lys, H.sub.), 4.268-4.294 (m, 1H, Glu, H.sub.), 6.559 (d, J=4.84 Hz, 1H, Glu, NH), 6.631 (d, J=7.72 Hz, 1H, Lys, NH), 7.005 (d, J=7.63 Hz, 4H, DP.sup.Ph, H.sub.e), 7.036 (d, J=7.62 Hz, 4H, DP.sup.Ph, H.sub.e), 7.325 (dd, J.sub.1=14.22 Hz, J.sub.2=7.36 Hz, 4H, DP.sup.Ph, CH.sub.d/d), 7.427 (t, J=7.68 Hz, 4H, DP.sup.Ph, H.sub.d/d), 7.799-7.824 (m, 1H, PEG, NH), 7.799-7.824 (m, 1H, Lys, NH.sub.); .sup.13C NMR (176 MHz, DMF-d.sub.7, 298 K): (ppm) 20.667 (s, DP.sup.Tol, C.sub.g/g), 20.686 (s, DP.sup.Tol, C.sub.g/g), 23.064 (s, Lys, C.sub.), 29.306 (hidden, Lys, C.sub.), 29.680 (hidden, Glu, C.sub.), 31.929 (s, Glu, C.sub.), 32.656 (s, Lys, C.sub.), 36.726 (s, PEG, C.sub.q), 38.703 (s, PEG, C.sub.h), 38.840 (s, Lys, C.sub.), 53.328 (s, Glu, C.sub.), 53.453 (s, Lys, C.sub.), 67.389 (s, PEG, C.sub.p), 69.058 (s, PEG, C.sub.i), 70.176 (s, PEG, C.sub.j-o), 70.223 (s, PEG, C.sub.j-o), 70.308 (s, PEG, C.sub.j-o), 70.436 (s, PEG, C.sub.j-o), 70.454 (s, PEG, C.sub.j-o), 128.505 (d, J=7.07 Hz, DP.sup.Tol, C.sub.e/e), 128.568 (d, J=7.35 Hz, DP.sup.Tol, C.sub.e/e), 134.197 (d, J=19.96 Hz, DP.sup.Tol, C.sub.d/d), 134.675 (d, J=20.96 Hz, DP.sup.Tol, C.sub.d/d), 137.682 (s, DP.sup.Tol, C.sub.c/c), 137.949 (s, DP.sup.Tol, C.sub.f/f), quaternary carbons: 158.097 (s), 170.435 (s), 174.665 (s), 175.003 (s), 175.179 (s), remaining signals corresponding to quaternary carbons could not be distinguished from noise; .sup.31P{.sup.1H}NMR (283 MHz, DMF-d.sub.7, 298 K): (ppm) 15.776 (d, J=151.40 Hz), 14.392 (d, J=151.40 Hz).
[0244] HR-MS-ESI m/z: [M+H].sup.+ 1089.4373 (calculated for C.sub.55H.sub.71O.sub.15N.sub.4P.sub.2 1089.4386), [M+Na].sup.+ 1111.4193 (calculated for C.sub.55H.sub.70O.sub.15N.sub.4P.sub.2Na 1111.4205), [M+MeOH+H].sup.+ 1121.4275 (calculated for C.sub.56H.sub.75O.sub.16N.sub.4P.sub.2 1121.4648).
Characterization of Compound (II-11-PSMAt1)
[0245] .sup.31P NMR 283 MHz, DMF-d.sub.7, 298 K): (ppm) 17.21 (d, J=139.3 Hz), 19.24 (d, 139.3 Hz).
[0246] HRMS: [M+H].sup.+ 1153.4196 (observed), 1153.4182 (calculated)
Example 5Preparation and Characterisation of the Cis/Trans Re-Complex of the Conjugated Peptide RGD Using Compound (.SUP.nat.Re-III-1-RGD)/Compound (II-1-RGD)
[0247] The chemistry of Re and Tc is similar. As Tc has no stable isotopes, it was convenient to prepare Compound (.sup.natRe-III-1-RGD)/[.sup.natReO.sub.2(II-1-RGD).sub.2].sup.+ to obtain full characterisation.
[0248] A solution of [.sup.natReO.sub.2I(PPh.sub.3).sub.2](3.0 mg, 3.45 mol) in DMF (100 L) was combined with a solution of Compound (II-1-RGD) (3.7 mg, 3.45 mol) and DIPEA (6 L) in DMF (200 L). The resulting dark brown/black solution was agitated at room temperature for 10 min. Upon addition of ice-cold diethyl ether, a precipitate formed. The supernatant was removed, and the precipitate was dissolved in DMF (200 L) and applied to a semi-preparative HPLC column. Reaction components were separated using HPLC method 3. A solution of aqueous ammonium bicarbonate (0.125 M) was added to each fraction containing cis/trans-[.sup.natReO.sub.2(II-1-RGD).sub.2].sup.+ at a ratio of 10 L of ammonium acetate solution: 1 mL of HPLC eluate. Solutions containing cis/trans-[.sup.natReO.sub.2(II-1-RGD).sub.2].sup.+ were lyophilised. The lyophilised fractions that eluted at 65-67 min and 68-70 min were identified as trans-[.sup.natReO.sub.2(II-1-RGD).sub.2].sup.+ (0.8 mg, 0.34 mol, 9.9%) and cis-[.sup.natReO.sub.2(II-1-RGD).sub.2].sup.+ (0.9 mg, 0.38 mol, 11.0%), respectively.
Characterising Data for trans-[.SUP.nat.ReO.SUB.2.(II-1-RGD).SUB.2.].SUP.+
[0249] .sup.1H NMR (700 MHz, DMF-d.sub.7, 298 K): (ppm) 1.184 (m, 4H, Lys, CH.sub.2), 1.300 (m, 4H, Lys, CH.sub.2), 1.511 (m, 2H, Arg, CH), 1.602 (m, 2H, Lys, CH), 1.612 (m, 2H, Arg, CH), 1.681 (m, 2H, Lys, CH), 1.769 (m, 4H, Arg, CH.sub.2), 2.261 (m, hidden, Asp, CH), 2.601 (dd, J1=14.51 Hz, J.sub.2=9.26 Hz, 2H, Phe, CH), 2.940 (m, hidden, Asp, CH), 2.943 (hidden, Lys, CH.sub.2), 3.067 (m, 2H, Arg, CH), 3.144 (m, 2H, Arg, CH), 3.33 (dd, J1=9.26 Hz, J.sub.2=5.00 Hz, 2H, Phe, CH), 3.416 (dd, J.sub.1=16.45 Hz, J.sub.2=9.05 Hz, 2H, Gly, CH), 4.307 (dd, J1=16.24 Hz, J.sub.2=2.67 Hz, 2H, Gly, CH), 4.358 (m, 2H, Asp, CH), 4.365 (m, 2H, Lys, CH), 4.543 (m, 2H, Arg, CH), 4.796(m, 2H, Phe, CH), 7.111 (m, PPh.sub.2, aromatic CH.sub.m), 7.161 (m, Phe, aromatic CH.sub.p), 7.170 (m, PPh.sub.2, aromatic CH.sub.m), 7.204 (m, PPh.sub.2, aromatic CH.sub.o), 7.232 (m, Phe, aromatic CH.sub.o and CH.sub.m), 7.281 (m, PPh.sub.2, aromatic CH.sub.o), 7.436 (m, PPh.sub.2, aromatic CH.sub.p), 7.477 (m, PPh.sub.2, aromatic CH.sub.p), 7.787 (d, J=8.98 Hz, 2H, Phe, NH), 8.089 (m, 2H, Gly, NH), 8.196 (m, 2H, Lys, NH), 8.294 (m, 2H, Lys, NH), 8.358 (d, J=9.32 Hz, 2H, Asp, NH), 8.629 (d, J=8.98 Hz, 2H, Arg, NH).
[0250] .sup.13C NMR (176 MHz, CD.sub.3CN, 298 K): (ppm) 15.56 (s, Lys, CH.sub.2), 24.66 (s, Arg, CH.sub.2), 27.14 (s, Arg, CH.sub.2), 29.47 (s, Lys, CH.sub.2), 32.87 (s, Lys, CH.sub.2), 36.86 (s, Phe, CH.sub.2), 38.77 (s, Asp, CH.sub.2), 38.98 (s, Lys, CH.sub.2), 41.04 (s, Arg, CH.sub.2), 43.11 (s, Gly, CH.sub.2), 49.12 (s, Asp, CH), 51.32 (s, Arg, CH), 53.36 (s, Phe, CH), 55.59 (s, Lys, CH), 118.09 (m, PPh.sub.2, aromatic C.sub.s), 125.99 (s, Phe, aromatic C.sub.p), 127.55 (m, PPh.sub.2, aromatic C.sub.m), 128.02 (s, Phe, aromatic C.sub.o or C.sub.m), 129.31 (s, Phe, aromatic C.sub.o or C.sub.m), 131.130 (m, PPh.sub.2, aromatic C.sub.p), 134.63 (m, PPh.sub.2, aromatic C.sub.o), 158.21-172.63 (>9 signals for X=CR.sub.2; where X is N, O or C).
[0251] .sup.31P NMR (283 MHz, DMF-d.sub.7, 298 K): (ppm) 23.781 (m), 24.506 (m).
[0252] HR-MS-ESI m/z: [M+H].sup.2+ 1179.3826 (Calculated for C.sub.110H.sub.122N.sub.18O.sub.22P.sub.4Re.sup.+ 1179.3773), [M+2H].sup.3+ 786.5921 (Calculated for C.sub.110H.sub.122N.sub.18O.sub.22P.sub.4Re.sup.+ 786.5906).
Characterising Data for Cis-[.SUP.nat.ReO.SUB.2.(II-1-RGD).SUB.2.].SUP.+
[0253] .sup.1H NMR (700 MHz, DMF-d.sub.7, 298 K): (ppm) 1.188 (m, 4H, Lys, CH.sub.2), 1.300 (m, 4H, Lys, CH.sub.2), 1.528 (m, 2H, Arg, CH), 1.599 (m, 2H, Lys, CH), 1.608 (m, 2H, Arg, CH), 1.664 (m, 2H, Lys, CH), 1.785 (m, 4H, Arg, CH.sub.2), 2.283 (m, 2H, Asp, CH), 2.606 (m, 2H, Phe, CH), 2.876 (m, 4H, Lys, CH.sub.2), 2.924 (hidden, Asp, CH), 3.109 (m, 2H, Arg, CH), 3.148 (m, 2H, Arg, CH), 3.319 (dd, J.sub.1=14.51 Hz, J.sub.2=5.14 Hz, 2H, Phe, CH), 3.415 (hidden, Gly, CH), 4.308 (dd, J.sub.1=16.64 Hz, J.sub.2 =9.05 Hz, 2H, Gly, CH), 4.367 (m, 2H, Lys, CH), 4.379 (m, 2H, Asp, CH), 4.525 (m, 2H, Arg, CH), 4.789 (m, 2H, Phe, CH), 7.151 (m, PPh.sub.2, aromatic CH.sub.m), 7.158 (m, Phe, aromatic CH.sub.p), 7.175 (m, PPh.sub.2, aromatic CH.sub.o), 7.220 (m, Phe, aromatic CH.sub.o and CH.sub.m), 7.305 (m, PPh.sub.2, aromatic CH.sub.o), 7.439 (m, PPh.sub.2, aromatic CH.sub.p), 7.798 (d, J=9.37 Hz, 2H, Phe, NH), 8.087 (m, 2H, Gly, NH), 8.088 (m, 2H, Lys, NH), 8.253 (d, J=7.46 Hz, 2H, Lys, NH), 8.335 (d, J=8.85 Hz, 2H, Asp, NH), 8.621 (d, J=8.85 Hz, 2H, Arg, NH).
[0254] .sup.13C NMR (176 MHz, CD.sub.3CN, 298 K): (ppm) 15.56 (s, Lys, CH.sub.2), 24.32 (s, Arg, CH.sub.2), 27.09 (s, Arg, CH.sub.2), 29.54 (hidden, Lys, CH.sub.2), 32.84 (s, Lys, CH.sub.2), 36.81 (s, Phe, CH.sub.2), 38.63 (s, Asp, CH.sub.2), 38.91 (s, Lys, CH.sub.2), 41.04 (s, Arg, CH.sub.2), 43.09 (s, Gly, CH.sub.2), 49.21 (s, Asp, CH), 51.37 (s, Arg, CH), 53.37 (s, Phe, CH), 55.52 (s, Lys, CH), 118.05 (m, PPh.sub.2, aromatic C.sub.s), 125.99 (s, Phe, aromatic C.sub.p), 127.55 (m, PPh.sub.2, aromatic C.sub.m), 128.02 (s, Phe, aromatic C.sub.o or C.sub.m), 129.31 (s, Phe, aromatic C.sub.o or C.sub.m), 131.04 (m, PPh.sub.2, aromatic C.sub.p), 134.27 (m, PPh.sub.2, aromatic C.sub.o), 143.80 (m, PPh.sub.2, aromatic C.sub.o), 158.03-185.17 (several signals for X=CR.sub.2; where X is N, O or C; too weak to characterise).
[0255] .sup.31P NMR (283 MHz, DMF-d.sub.7, 298 K): (ppm) 21.848 (dm, J.sub.1=356.1 Hz), 26.335 (dm, J.sub.1=356.1 Hz).
[0256] HRMS-ESI m/z: [M+H].sup.2+ 1179.3826 (Calculated for C.sub.110H.sub.122N.sub.18O.sub.22P.sub.4Re.sup.+ 1179.3773), [M+2H].sup.3+ 786.5921 (Calculated for C.sub.110H.sub.122N.sub.18O.sub.22P.sub.4Re.sup.+ 786.5906).
Example 6Preparation and Characterisation of Compound (Tc-III-1-RGD)/[.SUP.99m.TcO.SUB.2.(II-1-RGD).SUB.2.]+
Preparation of Radiolabelling Kits
[0257] An aqueous stock solution was prepared containing the required amounts of sodium bicarbonate, tin(II) chloride dihydrate, sodium gluconate or sodium tartrate dibasic dihydrate. The pH of this solution was adjusted to 8.5 by dropwise addition of an aqueous solution of sodium hydroxide (0.1M). Aliquots of the stock solution were mixed with the required amount of Compound (II-1-RGD) (in ethanol), and the resulting solutions were frozen and lyophilised. The lyophilised kits were stored at 18 C. prior to use.
.SUP.99m.Tc Radiolabelling
[0258] Compound (II-1-RGD) was radiolabelled with generator-produced .sup.99mTcO.sub.4.sup. in saline solution (0.9% NaCl in water, w/v). For each radiolabelling, a radiolabelling kit was thawed and reconstituted with a total of 300 L of saline, .sup.99mTcO.sub.4.sup. in saline solution and ethanol. The reconstituted kit was heated at 60 C. for 30 min, and then analysed by analytical HPLC (method 2) and instant thin layer chromatography (iTLC) using iTLC SGI0001 strips (9 or 10 cm length; Varian Medical Systems, Crawley, UK). The iTLC plates were scanned with a Perkin Elmer Storage Phosphor System (Cyclone) or a LabLogic miniScan TLC reader equipped with Laura software.
[0259] Two separate iTLC analyses were undertaken, to enable quantification of .sup.99mTc-colloids, unreacted .sup.99mTcO.sub.4.sup. and Compound (Tc-III-1-RGD).
[0260] To quantify amounts of unreacted .sup.99mTcO.sub.4.sup., acetone was used as a mobile phase: R.sub.f values: .sup.99mTcO.sub.4.sup.>0.9, .sup.99mTc colloids<0.1, Compound (Tc-III-1-RGD)<0.1.
[0261] To quantify .sup.99mTc-colloid formation, a 1:1 mixture of methanol and 2M aqueous ammonium acetate solution was used as a mobile phase: .sup.99mTcO.sub.4.sup.>0.9, .sup.99mTc colloids<0.1, Compound (.sup.99mTc-III-1-RGD)>0.9.
[0262] Co-elution of (Tc-III-1-RGD) with cis/trans-(.sup.natRe-III-1-RGD): [.sup.99mTcO.sub.2(II-1-RGD).sub.2].sup.+ was prepared in >90% RCY as described above, and co-injected with cis-[.sup.natReO.sub.2(II-1-RGD).sub.1].sup.+ and separately, trans-[.sup.natReO.sub.2(II-1-RGD).sub.2].sup.+, onto a reverse-phase analytical HPLC column (method 4). Retention times: trans/cis-(Tc-III-1-RGD) 41.0 min and 44.1 min (NaI scintillator detection); trans-(.sup.natRe-III-1-RGD) 38.3 min and cis-(.sup.natRe-III-1-RGD) 42.6 min.
Log D (pH 7.4)
[0263] The following procedure was carried out in triplicate. A solution containing (.sup.99mTc-III-1-RGD) (1 MBq in 7.5 L) was combined with phosphate buffered saline (pH 7.4, 500 L) and octanol (500 L), and the mixture was agitated for 30 min. The mixture was then centrifuged (10 000 rpm, 10 minutes), and aliquots of octanol and aqueous PBS were analysed for radioactive using a gamma counter. log D.sub.OCT/PBS=1.640.04.
Serum Stability:
[0264] A solution containing Compound (Tc-III-1-RGD) (100 L, 79 MBq) was added to filtered human serum (Sigma-Aldrich, 900 L) and incubated at 37 C. for 4 h. At 1 and 4 h, aliquots were taken. Each aliquot (300 L) was treated with ice-cold acetonitrile (300 L) to precipitate and remove serum proteins. Acetonitrile in the supernatant was then removed by evaporation under a stream of N.sub.2 gas (40 C., 30 min). The final solution was then analysed by reverse-phase analytical HPLC (method 2).
v3-Integrin Solid-Phase Competitive Binding Assay:
[0265] The affinity of Compound (Tc-III-1-RGD) for v3 integrin was determined in a solid-phase competitive binding assay. In brief, wells of a 96 well plate were coated with 150 ng/mL integrin v3 in 100 L coating buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM CaCl.sub.2, 0.5 mM MgCl.sub.2, and 1 mM MnCl.sub.2) overnight at 4 C. Wells were then washed twice in binding buffer (coating buffer plus 0.1% bovine serum albumin (BSA)) before being blocked for 2 hours at room temperature with blocking buffer (coating buffer plus 1% BSA). After a further two washes in binding buffer, both (Tc-III-1-RGD) (RCY >96%, 1-2 kBq in 50 L binding buffer, containing 1.2 mol Compound (II-1-RGD) peptide) and RGD peptide (10.0 M to 10,000 nM, 50 L in binding buffer) were added simultaneously to wells, and left to incubate for 1 h at room temperature, before being washed twice as before. Finally, the amount of activity bound to the wells was counted.
[0266] Binding of Compound (Tc-III-1-RGD) to v3 integrin was displaced by RGD peptide in a concentration-dependent manner. The pseudo-IC.sub.50 value of 8.543.45 nM (95% CI: 1.67-15.41 nM) was calculated using a non-linear regression model (Binding/Saturation, one sitetotal) in GraphPad Prism (n=6 from one experiment).
Example 7Pre-clinical imaging and in vivo biodistribution studies of Compound (Tc-III-1-RGD)/[.SUP.99m.TcO.SUB.2.(II-1-RGD).SUB.2.].SUP.+
[0267] Animal imaging studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 (ASPA) UK Home Office regulations governing animal experimentation. SPECT/CT imaging was accomplished using a pre-clinical nanoScan SPECT/CT Silver Upgrade instrument (Mediso) calibrated for technetium-99m. All scans were acquired by helical SPECT (4-head scanner with 49 [1.4 mm] pinhole collimators), and helical CT with 1.4 mm aperture collimators. All acquired images were reconstructed using a full 3D Monte Carlo-based iterative algorithm (Tera-Tomo; Mediso) and further processed and analysed using VivoQuant software (inviCRO, USA).
SPECT/CT Imaging and Biodistribution in Healthy Mice
[0268] A female, balb/c mouse (2 months old) was anaesthetised (2-3% v/v isofluorane in oxygen), scanned by CT and injected intravenously (tail vein) with Compound (Tc-III-1-RGD) (21 MBq containing 22 g of Compound (II-1-RGD) peptide). SPECT images (830 min images) were acquired over 4 h. At the end of the imaging procedure, the mouse was culled by cervical dislocation and a sample of the urine analysed by reverse-phase HPLC (analytical, method 2).
[0269] Female balb/c mice (2 months old) were anaesthetised (2-3% v/v isofluorane in oxygen) and injected intravenously (tail vein) with (.sup.99mTc-III-1-RGD) (2.7-5.3 MBq containing 5 g of Compound (II-1-RGD)). For blocking studies, animals were co-injected with RGD peptide (400 g). Mice remained under anaesthetic for 1 h, after which they were culled (pentabarbitone by i.v. injection). Tissues and organs were harvested and weighed, and radioactivity counted using a Gamma Counter (Wallac 1282 CompuGamma Universal Gamma Counter).
SPECT/CT Imaging and Biodistribution in Mice Induced with Rheumatoid Arthritis
[0270] An AK/BxN serum transfer arthritis (STA) model of rheumatoid arthritis was used (P. A. Monach, et al, Curr. Protoc. Immunol., 2008, 81, 15.22.1-15.22.12 and C. Imberti et al, Bioconjugate Chem., 2017, 28, 481-495). On day 0 and 2, female C57Bl/6J mice (2 months old) were injected intraperitoneally with arthritogenic serum in sterile filtered PBS (150 L, 50% v/v, serum obtained from arthritic K/BxN transgenic mice). Disease severity was evaluated in mice throughout the induction period, by measuring weight, thickness of swollen paws using microcallipers, and visual scoring on a scale of 0-3 per paw. SPECT/CT imaging and biodistribution was undertaken on day 7.
[0271] Mice were anesthetised (2.5-3% v/v isofluorane) and their paws were measured using microcallipers. Mice were then injected intravenously with Compound (Tc-III-1-RGD) (approx. 5 MBq containing 5 g of Compound (II-1-RGD)) and allowed to recover from anaesthetic administration. At 1 h post-injection of radiotracer, mice were culled (sodium pentabarbitone), and underwent SPECT/CT scanning post-mortem for 60-180 min. Finally, tissues and organs were harvested and weighed, and radioactivity counted using a Gamma Counter (Wallac 1282 CompuGamma Universal Gamma Counter). The acquired images were processed to units of % ID and the regions of interest (ROIs) delineated by CT using VivoQuant software (inviCRO, USA). Radioactivity in ankle and wrist ROIs were obtained in units of % ID and % ID/cm.sup.3. Each ankle ROI was defined as the area between the tibiofibula joint and the base of phalanx V. Each wrist ROI was defined as the area between the narrowest point of the wrist (ulna and radius) and the end of the forepaw.
Example 8Preparation and characterisation of Compound (Tc-III-1-PSMAt1)/[.SUP.99m.TcO.SUB.2.(II-1-PSMAt1).SUB.2.].SUP.+ Compound (Tc-III-2-PSMAt1)/.SUP.99m.TcO.SUB.2.(II-2-PSMAt1).SUB.2.].SUP.+ and Compound (Tc-III-11-PSMAt1)/(.SUP.99m.TcO.SUB.2.(II-11-PSMAt1).SUB.2.]+
[0272] Kit preparation: An aqueous stock solution was prepared containing the required amounts of sodium bicarbonate, tin chloride and sodium tartrate. The pH was adjusted to either 7.5 or 8-8.5 by dropwise addition of an aqueous solution of either sodium hydroxide (0.1 M) or hydrochloric acid (0.1 M). Aliquots of the stock solution were mixed with the required amount of (II-1-PSMAt1), (II-2-PSMAt1), or (II-11-PSMAt1) (dissolved in a mixture of water/ethanol (50%/50%)) to form the kit solutions outlined in the table below, which were immediately frozen and lyophilised using a freeze dryer. The lyophilised kits were stored in a freezer prior to use.
TABLE-US-00005 TABLE 5 Lyophilised kit formulations for (II-1-PSMAt1), (II-2-PSMAt1) and (II-11-PSMAt1) for radiolabelling. Kit Compositions (II-1-PSMAt1) (II-2-PSMAt1) (II-11-PSMAt1) Kit Kit Kit moles/ weight/ moles/ weight/ moles/ weight/ Components: mol mg mol mg mol mg (II-1-PSMAt1) 0.11 0.11 (II-2-PSMAt1) 0.11 0.12 (II-11- 0.11 0.12 PSMAt1) SnCl.sub.22H.sub.2O 0.11 0.03 0.11 0.03 0.11 0.03 Sodium 1.15 0.26 1.15 0.26 1.15 0.26 tartrate NaHCO.sub.3 10.71 0.90 10.71 0.90 10.71 0.90
The kits may be scaled to, for example, two or three times the amounts shown in the table above.
Radiolabelling of (II-1-PSMAt1), (II-2-PSMAt1), or (II-11-PSMAt1) with .sup.99mTcO.sub.4.sup.
[0273] (II-1-PSMAt1) or (II-2-PSMAt1) were radiolabelled with generator-produced .sup.99mTcO.sub.4.sup.+ in saline solution (0.9% NaCl in water, w/v), using the lyophilised kits described. The radiolabelling reaction mixtures were either left to react at ambient temperature (22 C.) for 5 min, or heated at 100 C. for 5 min. Aliquots were analysed by iTLC and analytical C.sub.18-HPLC to determine radiochemical yields. The species attributed as (Tc-III-1-PSMAt1) eluted at 11.0-12.5 min; (Tc-III-2-PSMAt1) eluted at 12.5-14.0 min. Analytical HPLC conditions: 20 min, 5% min.sup.1 linear increase from 100% A to 100% B (flow rate of 1 ml/min, A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6150 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 column).
[0274] (II-11-PSMAt1) was radiolabelled with generator-produced .sup.99mTcO.sub.4.sup. (200 MBq, 300 uL) in saline solution (0.9% NaCl in water, w/v), using the lyophilised kit described above. The radiolabelling reaction mixture was heated at 100 C. for 5 min. Aliquots were analysed by iTLC and analytical C.sub.18-HPLC to determine radiochemical yields. The species attributed as (Tc-III-11-PSMAt1) eluted at 9.7-11.7 min. Analytical HPLC conditions: 20 min, 5% min.sup.1 linear increase from 100% A to 100% B (flow rate of 1 ml/min, A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6150 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 column).
[0275] Two separate iTLC analyses were undertaken, to enable quantification of .sup.99mTc-colloids, unreacted .sup.99mTcO.sub.4.sup. and the complex.
[0276] To quantify amounts of unreacted .sup.99mTcO.sub.4.sup., acetone was used as a mobile phase: R.sub.f values: .sup.99mTcO.sub.4.sup.>0.9, .sup.99mTc colloids<0.1, complex <0.1.
[0277] To quantify .sup.99mTc-colloid formation, a 1:1 mixture of methanol and 2M aqueous ammonium acetate solution was used as a mobile phase: .sup.99mTcO.sub.4.sup.>0.9, .sup.99mTc colloids<0.1, the complex >0.9.
[0278] For in vitro and in vivo studies, these kit-based reaction solutions were further purified. Solutions of either (Tc-III-1-PSMAt1), (Tc-III-2-PSMAt1), or (Tc-III-11-PSMAt1) prepared from kits were applied to a SE-HPLC column, using an aqueous mobile phase of phosphate buffered saline. Fractions containing either (Tc-III-1-PSMAt1), (Tc-III-2-PSMAt1), or (Tc-III-11-PSMAt1) (>95% radiochemical purity) eluted at 10-12 mins. Other reaction components, including unreacted starting materials and impurities also eluted at distinct retention times: unlabelled (II-1-PSMAt1) ligand eluted at 16-17 min, unlabelled (II-2-PSMAt1) eluted at 27-28 min, .sup.99mTcO.sub.4.sup. eluted at 14-15 min and .sup.99mTc-colloid was trapped on the column.
Preparation of Compounds (.SUP.99g.Tc-III-1-PSMAt1) and (.SUP.99g.Tc-III-2-PSMAt1)
[0279] The .sup.99gTc(V) precursor N.sup.tBu.sub.4[.sup.99gTcOCl.sub.4] was prepared following a previously described method (A. Davison, C. Orvig, H. S. Trop, M. Sohn, B. V. Depamphilis and A. G. Jones, Inorg. Chem., 1980, 19, 1988-1992). A solution of either (II-1-PSMAt1) or (II-2-PSMAt1) (1.0 mg, 1 mol, 2 equiv.) dissolved in methanol (300 L, degassed) was combined with a solution of N.sup.tBu.sub.4[.sup.99gTcOCl.sub.4](0.25 mg, 0.46 mol, 1 equiv.) in methanol (50 L). The resulting pale yellow solution was left to react at ambient temperature for 15 min.
[0280] (.sup.99gTc-III-1-PSMAt1): HR-MS-ESI m/z: [M+H].sup.2+ 1098.8183 (calculated for C.sub.102H.sub.125N.sub.8O.sub.32P.sub.4Tc 1098.8221 (100% abundance peak)), [M+Na].sup.2+ 1109.8091 (calculated for C.sub.102H.sub.124N.sub.8O.sub.2P.sub.4TcNa 1109.8130 (100% abundance peak)); LR-MS-ESI m/z: [M+H].sup.2+ 1099.0 (calculated for C.sub.102H.sub.125N.sub.8O.sub.32P.sub.4Tc 1098.5), [M+Na].sup.2+ 1110.0 (calculated for C.sub.102H.sub.124N.sub.8O.sub.32P.sub.4TcNa 1109.5), [M+K].sup.2+ 1117.7 (calculated for C.sub.102H.sub.124N.sub.8O.sub.32P.sub.4TcK 1117.5), [M+2H].sup.3+ 732.7 (calculated for C.sub.102H.sub.126N.sub.8O.sub.32P.sub.4Tc 732.7), [M+H+K].sup.3+ 745.2 (calculated for C.sub.102H.sub.126N.sub.8O.sub.32P.sub.4TcK 745.3).
[0281] (.sup.99gTc-III-2-PSMAt1): HR-MS-ESI m/z: [M+H].sup.2+ 1154.8811 (calculated for C.sub.110H.sub.141N.sub.8O.sub.32P.sub.4Tc 1154.8847 (100% abundance peak)), [M+Na].sup.2+ 1165.8718 (calculated for C.sub.110H.sub.140N.sub.8O.sub.32P.sub.4TcNa 1165.8756 (100% abundance peak)); LR-MS-ESI m/z: [M+H].sup.2+ 1155.0 (calculated for C.sub.110H.sub.141N.sub.8O.sub.32P.sub.4Tc 1154.5), [M+Na].sup.2+ 1165.8 (calculated for C.sub.110H.sub.140N.sub.8O.sub.32P.sub.4TcNa 1165.5), [M+K].sup.2+ 1173.8 (calculated for C.sub.110H.sub.140N.sub.8O.sub.32P.sub.4TcK 1173.5), [M+2H].sup.3+ 770.3 (calculated for C.sub.100H.sub.142N.sub.8O.sub.32P.sub.4Tc 770.0), [M+H+K].sup.3+ 782.8 (calculated for C.sub.110H.sub.141N.sub.8O.sub.32P.sub.4TcK 782.7).
Example 9Biological evaluation of (Tc-III-1-PSMAt1), (Tc-III-2-PSMAt1), and (Tc-III-11-PSMAt1)
[0282] Compound (Tc-III-1-PSMAt1)/[.sup.99mTcO.sub.2(II-1-PSMAt1).sub.2].sup.+ and Compound (Tc-III-2-PSMAt1)/[.sup.99mTcO.sub.2(II-2-PSMAt1).sub.2].sup.+ were isolated and purified to evaluate stability, affinity for PSMA in vitro and in vivo and pharmacokinetics.
TABLE-US-00006 TABLE 6 Dissociated .sup.99mTc (measured by analytical HPLC) Compound (Tc-III-1-PSMAt1) Compound (Tc-III-2-PSMAt1) Time ([.sup.99mTcO.sub.2(II-1-PSMAt1).sub.2].sup.+) ([.sup.99mTCO.sub.2(II-2-PSMAt1).sub.2].sup.+) 1 h 0% 0.1% 4 h 0.7% 1.6% 24 h 4.2% 6.5%
[0283] Table 6 shows the amount of dissociated .sup.99mTc after incubation of Compound (Tc-III-1-PSMAt1) and Compound (Tc-III-2-PSMAt1) in serum.
[0284] The stability of Compound (Tc-III-1-PSMAt1) and Compound (Tc-III-2-PSMAt1) were assessed in serum over 24 hours. Both tracers exhibit high stability, with over 90% intact over 24 hours, as determined by analytical C18 radio-HPLC. With the exception of free .sup.99mTc, no other degradation products are observed in HPLC chromatograms. The log D.sub.OCT/PBS of (Tc-III-1-PSMAt1) is 2.45 and the log D.sub.OCT/PBS of (Tc-III-2-PSMAt1) is 2.08, suggesting that both are hydrophilic and are likely to clear via a renal pathway.
[0285] A solution containing Compound (Tc-III-11-PSMAt1) (20 L, 13 MBq) was added to filtered human serum (180 L) and incubated at 37 C. At 1, 4 and 24 h, samples were taken and treated with an equal volume of ice-cold acetonitrile to precipitate and remove serum proteins. Acetonitrile in the supernatant was then removed by evaporation under a stream of N.sub.2 gas. The final solution was then analysed by reverse-phase analytical HPLC (
[0286] .sup.99mTc-DP-peptide radiotracers contain two different isomers. Such isomers are known as geometric cis/trans isomers. To show that the isomers have equivalent biological behaviour, the cis and trans geometric isomers of (Tc-III-1-PSMAt1) were separated: both have near identical uptake in PSMA-positive cells (
(Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) uptake in DU145, DU145-PSMA, LNCaP, and PC-3 cells
[0287] The following experiment was performed in biological triplicate.
[0288] A panel of cell lines were selected that either expressed GCP(II)/PSMA (DU145-PSMA (genetically modified to express PSMA) (see F. Kampmeier, J. D. Williams, J. Maher, G. E. Mullen and P. J. Blower, EJNMMI Res., 2014, 4, 13), and LNCaP (CRL-1740)), or had low GCP(II)/PSMA expression (DU145 (HTB-81) and PC-3 (CRL-1435)). All cell lines were cultured in RPMI 1640 medium (R0883, Sigma) containing 10% foetal bovine serum, 2 mM L-glutamine, and 100 U.Math.mL.sup.1 penicillin and 100 g.Math.mL.sup.1 streptomycin, except for PC-3 cells which were cultured in low-glucose Dulbecco's Modified Eagle Medium (DMEM, D5546, Sigma) supplemented as above. Cells were maintained at 37 C. and 5% CO.sub.2. Cells were seeded in 6-well plates at a density of 510.sup.5 cells per well in 2 mL complete media to achieve 70-80% confluency the following day. Prior to treating cells, cell medium (1 mL/well) was replaced. Solutions containing either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) (100 kBq, in 5-12 L of phosphate buffered saline, >95% radiochemical purity) were added to each well, and the cells incubated at 37 C. for 1 h. Uptake studies were also performed after a 2 min incubation with the PSMA inhibitor 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA; 30 L of 750 M PMPA solution/well). After 60 min incubation, the plates were placed on ice, the supernatant was removed and the cells were washed with ice cold phosphate buffered saline solution (30.5 mL). The cells were lysed with ice cold radioimmunoprecipitation assay buffer (RIPA buffer, 500 L; 150 mM sodium chloride, 0.1% w/w sodium dodecyl sulfate (SDS), 0.5% w/w sodium deoxycholate (NaDOC), 1% w/w Triton-X) and samples were collected for radioactivity counting. Results in
[0289] (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) exhibited uptake in DU145-PSMA+ cells (12.42.8% AR [percentage added radioactivity], and 7.81.3% AR respectively). This uptake was specific: DU145-PSMA+ cell uptake of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) could be blocked with PMPA, and there was negligible uptake in parental DU145 cells (
[0290] In LNCaP cells, uptake of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) measured 3.71.2% AR and 3.00.8% AR respectively, whilst uptake of both tracers in PC3 cells measured less than 0.3% AR. Uptake in LNCaP cells could also be blocked with PMPA (
In Vitro Time Course and Localisation of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1)
[0291] To determine the cellular uptake and localisation of each tracer over time, DU145-PSMA and LNCAP cells were seeded as above. Cells were replenished with complete medium (1 mL) 1 h prior to the addition of either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) (100 kBq, in 5-7 L of phosphate buffered saline, >95% radiochemical purity). Cells were incubated at 37 C. under 5% CO.sub.2 with three technical replicates for each condition. Following 15, 30, 60 and 120 min incubation, the supernatant was collected and cells washed three times with PBS (1 mL) to determine the unbound fraction, followed by an acid wash (0.5 M glycine, pH 2.5) to determine cell surface-bound activity. Cells were then lysed with cold RIPA buffer (500 l) to determine activity internalised by the cells. Radioactivity content was determined by gamma-counter. Results are depicted in
[0292] Uptake of both radiotracers increased over 2 hours, and the majority of .sup.99mTc-cell associated radioactivity was present in the internalised cell fraction at all measured time points, suggesting that (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) are rapidly internalised after PSMA binding, for both PSMA-expressing cell lines. (Tc-III-1-PSMAt1) uptake (both surface-bound and internalised radioactivity) was slightly higher than that for (Tc-III-2-PSMAt1).
In Vivo Imaging of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) in Healthy Mice
[0293] Animal imaging studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 (ASPA) UK Home Office regulations governing animal experimentation. Mice were purchased from Charles River (Margate, UK). A male SCID-beige mouse (approx. 3 months old, n=1) was anaesthetised (2.5% v/v isofluorane, 0.8-1.0 L/min 02 flow rate) and injected intravenously via the tail vein with (Tc-III-1-PSMAt1) (100 L, 26 MBq, >99% RCP, 0-5 g PSMAt peptide in phosphate buffered saline) or (Tc-III-2-PSMAt1) (160 L, 30 MBq, >99% RCP, 0-5 g PSMAt peptide in phosphate buffered saline), followed immediately by CT acquisition, and SPECT scanning. SPECT/CT imaging was accomplished using a pre-clinical nanoScan SPECT/CT Silver Upgrade instrument (Mediso), calibrated for technetium-99m (
Biodistribution of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) in Healthy Mice
[0294] Male SCID-beige mice (approx. 3 months old) were weighed, anaesthetised (2.0-2.5% v/v isofluorane, 1.0-1.0-1.5 L/min 02 flow rate) and injected with (Tc-III-1-PSMAt1) solution (50 L, approx. 13 MBq in phosphate buffered saline, n=4) or (Tc-III-2-PSMAt1) solution (80 L, approx. 15 MBq in phosphate buffered saline, n=4) by intravenous tail vein injection. The mice were kept under anaesthesia until they were culled by cervical dislocation 2 h post-injection. The biodistribution of the tracer was assessed by dissecting, weighing and gamma counting organs/tissues, alongside standard solutions of known .sup.99mTc radioactivity. The radioactivity measured for each organ/tissue was normalised to obtain values of percentage injected dose per gram (% ID/g) (
Example 10Biodistribution of Compound (Tc-III-1-PSMAt1)/[.SUP.99m.TcO.SUB.2.(DP.SUP.Ph.-PSMAt).SUB.2.].SUP.+ and Compound (Tc-III-2-PSMAt1)/[99m.TcO.SUB.2.(DP.SUP.Tol.-PSMAt) ).SUB.2.+ in Mice Bearing Prostate Cancer Tumours
[0295] The biodistributions of (Tc-III-1-PSMAt1) and (Tc-III-2-PSMAt1) were assessed in SCID/Beige mice bearing DU145-PSMA+ tumours (
[0296] To assess specificity of each radiotracer, separate groups of animals, also bearing DU145-PSMA+ tumours, were co-administered either (Tc-III-1-PSMAt1) and PMPA, or (Tc-III-2-PSMAt1) and PMPA, to inhibit PSMA-mediated uptake of radiotracer. Additionally, groups of mice bearing parental DU145 tumours (that do not express PSMA) were also administered these .sup.99mTc radiotracers. Animals were also euthanised 2 h post-injection, followed by organ harvesting for ex vivo radioactivity counting (
[0297] In mice bearing DU145-PSMA+ tumours, co-administration of PMPA substantially decreased uptake of both (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) in tumours (
[0298] For both radiotracers, the concentration of .sup.99mTc radioactivity in kidneys 2 h post-injection was high (
SPECT/CT Imaging
[0299] In SPECT/CT scans of animals administered either (Tc-III-1-PSMAt1) or (Tc-III-2-PSMAt1) only, tumours could be clearly delineated at both 2 h (
[0300] Preparation of tumour-bearing mice: The GCP(II)/PSMA-negative cell line used in these experiments was DU145, a human carcinoma prostate cancer cell line derived from a brain metastatic site. The GCP(II)/PSMA-expressing cell line used in these experiments was a genetically modified daughter cell line of DU145, DU145-PSMA+. This cell line had previously been transduced to express full-length human GCP(II)/PSMA, following F. Kampmeier, J. D. Williams, J. Maher, G. E. Mullen and P. J. Blower, EJNMMI Res., 2014, 4, 13. These cells were cultured in DMEM medium supplemented with 10% foetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin. To prepare for experiments, cells were grown at 37 C. in an incubator with humidified air equilibrated with 5% CO.sub.2.
[0301] Animal studies complied with the guidelines on responsibility in the use of animals in bioscience research of the U.K. Research Councils and Medical Research Charities, under U.K. Home Office project and personal licences. Subcutaneous prostate cancer xenografts were produced in SCID/beige mice (male, 7-12 weeks old) by injecting 410.sup.6 DU145-PSMA or DU145 cells suspended in PBS (100 L) on the right shoulder. Imaging was performed once a tumour had reached 5-10 mm in diameter (3-4 weeks after injection). For imaging purposes, the mice were anaesthetised, positioned on the scanner, and tail vein cannulated. For biodistribution purpose, the mice were anaesthetised, the radiotracers were injected via the tail vein.
[0302] SPECT/CT scanning: SPECT/CT scans were acquired on a dedicated small animal SPECT system, NanoSPECT/CT Silver Upgrade (Mediso Ltd., Budapest, Hungary), calibrated for .sup.99mTc. The animals (2 mice per group) were cannulated via tail vein, the radiotracers (10-26 MBq) were administered while the animals were on the scanner followed by a helical CT scan (45 kVP X-ray source, 1000 ms exposure time in 180 projections over 7.5 min). After 15 min post-injection, whole body SPECT scans were acquired (30 min4, conducted sequentially) with a frame time of 33 s (using a 4-head scanner with 49 [1.4 mm] pinhole collimators in helical scanning mode). After this, animals were allowed to recover, culled at 24 h post-injection (by cervical dislocation and tail-vein nick to confirm death), organs/tissues harvested, weighed and radioactivity counted using a gamma counter. For each radiotracer, an additional animal was administered tracer and recovered, before being anaesthetised and undergoing SPECT/CT scanning at 24 h post-injection, followed by culling and ex vivo tissue counting. SPECT/CT images were reconstructed in a 256256 matrix using HiSPECT (ScivisGmbH), a reconstruction software package and visualised and quantified using VivoQuant VivoQuant v.3.5 software (InVicro LLC., Boston, USA).
[0303] Biodistribution studies: The .sup.99mTc radiotracers (7-18 MBq) were administered via tail vein injection under isoflurane anaesthesia (5 mice per group). The animals were allowed to recover, roaming free in a gridded cage. The animals were euthanised by cervical dislocation 2 h post-injection, organs/tissues harvested, weighed and radioactivity counted using a gamma counter. Data were analysed in GraphPad Prism 9 (version 9.1.1) and expressed as meanstandard deviation (SD). Student t tests were used to determine statistical significance.
Example 11Preparation and Characterisation of (.SUP.99g.Tc-III-11-PSMAt1)
[0304] To a sample of Compound (II-11-PSMAt1) (1 mg) dissolved in DMF was added N.sup.tBu.sub.4 [.sup.99gTcOCl.sub.4](0.3 mg). The solution was analysed.
[0305] LC-MS (ESI, positive mode, low resolution) Retention time=8:04-8:17 min LRMS: [M+H].sup.2+ 1220 (observed), 1219 (calculated).
Example 12Preparation and Characterisation of Compound (.SUP.64.Cu-III-1-PSMAt1)/[.SUP.64.Cu(II-1-PSMAt1).SUB.2.+ and Compound (.SUP.64.Cu-III-2-PSMAt1)/[.SUP.64.Cu(II-2-PSMAt1).SUB.2.].SUP.+
.SUP.64.Cu Radiolabelling of (II-1-PSMAt1) and (II-2-PSMAt1)
[0306] .sup.64Cu was produced by .sup.64Ni(p,n).sup.64Cu nuclear reaction on a CTI RDS 112 11 MeV cyclotron and purified to give .sup.64Cu.sup.2+ in 0.1 M HCl solutions used for radiolabelling (see M. S. Cooper, M. T. Ma, K. Sunassee, K. P. Shaw, J. D. Williams, R. L. Paul, P. S. Donnelly and P. J. Blower, Bioconjug. Chem., 2012, 23, 1029-1039). The .sup.64Cu.sup.2+ solutions (in 0.1 M HCl) were dried under a flow of nitrogen with heating at 100 C., and the residue re-dissolved in ammonium acetate solution (0.1 M, pH 7). An aliquot of ammonium acetate solution containing .sup.64Cu.sup.2+ (10 MBq, 50-100 L) was added to either (II-1-PSMAt1) (50 g) or (II-2-PSMAt1) (50 g) dissolved in aqueous ammonium acetate (0.1 M), to give a final radiolabelling solution of 200 L volume. The radiolabelling mixtures were left to react at ambient temperature (22 C.) for 20 min. Aliquots were analysed by iTLC and analytical HPLC to determine radiochemical yield. By Cis-analytical HPLC, the species attributed as (.sup.64Cu-III-1-PSMAt1) eluted at 12.0-13.0 min; (.sup.64Cu-III-2-PSMAt1) eluted at 13.5-14.5 min; unreacted .sup.64Cu.sup.2+ eluted with the solvent front at 2.0-3.5 min.
[0307] iTLC analysis was undertaken to enable quantification of unreacted .sup.64Cu.sup.2+ and the complex. Citrate buffer (0.1 M, pH 5) was used as a mobile phase: R.sub.f values: unreacted .sup.64Cu.sup.2+>0.9, complex <0.1.
[0308] Log D.sub.OCT/PBS D of (.sup.64Cu-III-1-PSMAt1) and (.sup.64Cu-III-2-PSMAt1)
[0309] The following procedure was carried out in triplicate. A solution containing either (.sup.64Cu-III-1-PSMAt1) or (.sup.64Cu-III-2-PSMAt1) (0.5 MBq in 20 L) was combined with phosphate buffered saline (pH 7.4, 480 L) and octanol (500 L), and the mixture was agitated for 30 min. The mixture was then centrifuged (10 000 rpm, 10 min), and aliquots of octanol and aqueous phosphate buffered saline were analysed for radioactive using a gamma counter. log D.sub.OCT/PBS (.sup.64Cu-III-1-PSMAt1): 3.300.03; log D.sub.OCT/PBS (.sup.64Cu-III-2-PSMAt1): 3.010.06.
Serum Stability of (.sup.64Cu-III-1-PSMAt1) and (.sup.64Cu-III-2-PSMAt1)
[0310] A sample of (.sup.64Cu-III-1-PSMAt1) (>99.0% RCP, 1.7 MBq, 5 g DP.sup.Ph-PSMAt ligand) or (.sup.64Cu-III-2-PSMAt1) (>99.0% RCP, 1.7 MBq, 5 g DP.sup.Tol-PSMAt ligand) in an aqueous solution of ammonium acetate (20 L, 0.1 M) was added to filtered human serum from a healthy volunteer (180 L), and incubated at 37 C. At 1, 4 and 24 h, aliquots were taken. Each aliquot (300 L) was treated with ice-cold acetonitrile (300 L) to precipitate and remove serum proteins. Acetonitrile in the supernatant was then removed by evaporation under a stream of N.sub.2 gas (40 C., 30 min). The final solution was then analysed by reverse-phase analytical HPLC (method 2). Radiochromatograms of serum samples showed that (.sup.64Cu-III-1-PSMAt1) and (.sup.64Cu-III-2-PSMAt1) were still present, even after 24 h incubation in serum, with no other degradation products detectable.
Preparation of (.SUP.nat.Cu-III-1-PSMAt1) and (.SUP.nat.Cu-III-2-PSMAt1)
[0311] A solution of either (II-1-PSMAt1) or (II-2-PSMAt1) (1.0 mg, 1 mol, 2 equiv.) in saline (500 L) was added to a solution of [Cu.sup.I(MeCN).sub.4] PF.sub.6 (170-180 g, 0.5 mol, 1 equiv.) in acetonitrile (dry, deoxygenated, 500 L). The reaction mixture was left to react at ambient temperature for 60 min. The product was isolated by semi-preparative HPLC (method 6), lyophilising the product fractions eluting at either 46-47 min ((.sup.natCu-III-1-PSMAt1)) or 56-57 min ((.sup.64Cu-III-2-PSMAt1)). Yield=30-40%.
[0312] (.sup.natCu-III-1-PSMAt1): HR-MS-ESI m/z: [M+H].sup.2+ 1064.3338 (calculated for C.sub.102H.sub.125O.sub.30N.sub.8P.sub.4Cu 1064.3369); LR-MS-ESI+m/z: [M+H].sup.2+ 1065.8 (calculated for C.sub.102H.sub.125O.sub.30N.sub.8P.sub.4Cu 1065.3), [M+Na].sup.2+ 1077.2 (calculated for C.sub.102H.sub.124O.sub.30N.sub.8P.sub.4CuNa 1076.3), [M+K].sup.2+ 1084.6 (calculated for C.sub.102H.sub.124O.sub.30N.sub.8P.sub.4CuK 1084.3), [M+2H].sup.3+ 711.0 (calculated for C.sub.102H.sub.126O.sub.30N.sub.8P.sub.4Cu 710.5).
[0313] (.sup.natCu-III-2-PSMAt1): HR-MS-ESI m/z: [M+H].sup.2+ 1120.3973 (calculated for C.sub.100H.sub.141O.sub.30N.sub.8P.sub.4Cu 1120.3995); LR-MS-ESI+m/z: [M+H].sup.2+ 1121.3 (calculated for C.sub.110H.sub.141O.sub.30N.sub.8P.sub.4Cu 1121.4), [M+Na].sup.2+ 1132.6 (calculated for C.sub.110H.sub.140O.sub.30N.sub.8P.sub.4CuNa 1132.4), [M+2H].sup.3+ 748.0 (calculated for C.sub.100H.sub.142O.sub.30N.sub.8P.sub.4Cu 747.9), [M+H+K].sup.3+ 761.3 (calculated for C.sub.110H.sub.141O.sub.30N.sub.8P.sub.4CuK 760.6).
Example 13Preparation and Characterisation of Compound (.SUP.nat.Re-III-1-PSMAt1)/[.SUP.nat.ReO.SUB.2.(II-1-PSMAt1).SUB.2.].SUP.+ and Compound (.SUP.nat.Re-III-2-PSMAt1)/[.SUP.nat.ReO.SUB.2.(II-2-PSMAt1).SUB.2.]+
Preparation of (.SUP.nat.Re-III-1-PSMAt1) and (.SUP.nat.Re-III-2-PSMAt1)
[0314] A solution of either (II-1-PSMAt1) (5.1 mg, 4.9 mol, 1 equiv.) or (II-2-PSMAt1) (2.6 mg, 2.4 gmol, 1 equiv.) and DIPEA (6 L) in DMF was combined with a solution of [.sup.natReO.sub.2I(PPh.sub.3).sub.2](2.2 mg, 2.5 mol, 0.5 or 1 equiv., respectively) in DMF. The resulting dark brown solution was left to react at room temperature for 2-3 h. The reaction solution was applied to a reverse phase C.sub.18 semi-preparative HPLC column, and purified by HPLC (C.sub.18 semi-preparative HPLC (9.4250 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 column: 90 min, isocratic flow at 95% A for 5 min, then 0.93% min.sup.1 linear increase from 95% A/5% B to 25% A/75% B, followed by 2.5% min.sup.1 linear increase from 25% A to 0% A, flow rate of 3 mL min.sup.1; A=water with 0.005% acetic acid, B=acetonitrile with 0.005% acetic acid; Detection at 214 and 254 nm). The fractions containing the desired product were lyophilised to yield (.sup.natRe-III-1-PSMAt1) (1-2 mg, 0.4-0.8 mol, 15-30% yield) and (.sup.natRe-III-2-PSMAt1) (-1-1.5 mg, 0.5 mol, 20% yield) as solids.
[0315] Using a relatively long HPLC method (gradient mobile phase for 60 min; 1 ml min.sup.1 flow rate; 1% min.sup.1 linear increase from 100% A/0% B to 40% A/60% B; A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6150 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 column) to separate out cis and trans isomers, the species attributed as (.sup.natRe-III-1-PSMAt1) eluted at 38.11 and 38.51 min; (.sup.natRe-III-2-PSMAt1) eluted at 45.37 and 46.07 min.
[0316] (.sup.natRe-III-1-PSMAt1): HR-MS-ESI m/z: [M+2H].sup.3+ 761.9001 (calculated for C.sub.102H.sub.126O.sub.32N.sub.8P.sub.4Re 761.8990); [M+H+Na].sup.3+ 769.2274 (calculated for C.sub.102H.sub.125O.sub.32N.sub.8P.sub.4ReNa 769.2263). (.sup.natRe-III-2-PSMAt1): HR-MS-ESI m/z: [M+2H].sup.3+ 799.2757 (calculated for C.sub.100H.sub.142O.sub.32N.sub.8P.sub.4Re 799.2741); [M+H+Na].sup.3+ 806.6023 (calculated for C.sub.110H.sub.141O.sub.32N.sub.8P.sub.4ReNa 806.6014).
Example 14Preparation and Use of Radiolabelling Kits and Effect on Radiochemical Yield
[0317] To assess the feasibility of .sup.99mTc radiolabelling of (II-1-RGD), (II-1-PSMAt1), (II-2-PSMAt1), and (II-11-PSMAt1) with a kit formulation, lyophilised mixtures of (II-1-RGD), (II-1-PSMAt1), (II-2-PSMAt1), or (II-11-PSMAt1), tin(II) chloride, sodium bicarbonate, and sodium gluconate or sodium tartrate were prepared.
[0318] An aqueous stock solution was prepared containing the required amounts of sodium bicarbonate, tin chloride and sodium gluconate or sodium tartrate. The pH was adjusted to either 7.5 or 8-8.5 by dropwise addition of an aqueous solution of either hydrochloric acid (0.1 M) or sodium hydroxide (0.1 M). Aliquots of the stock solution were mixed with the required amount of (II-1-RGD), (II-1-PSMAt1), (II-2-PSMAt1), and (II-11-PSMAt1) (dissolved in a mixture of water/ethanol (70%/30%) to form the kit solutions outlined in Table 7, which were immediately frozen and lyophilised using a freeze dryer. The lyophilised kits were stored in a freezer prior to use.
[0319] Generator-produced .sup.99mTcO.sub.4.sup. (200 MBq) in saline solution was then added to these kits, and the mixtures left to react at ambient temperature (around 22 C.) for 5 min, or heated at 100 C. for 5 min, prior to analysis by radio-iTLC and radio-HPLC.
[0320] Using a relatively short HPLC method (gradient mobile phase for 20 min; 1 ml min.sup.1 flow rate; linear increase from 100% A/0% B to 0% A/100% B; A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6150 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 column)), the species attributed as (Tc-III-1-PSMAt1) eluted at 11.0-12.5 min; (Tc-III-2-PSMAt1) eluted at 12.5-14.0 min.
[0321] Using a relatively long HPLC method (gradient mobile phase for 60 min; 1 ml min.sup.1 flow rate; 1% min.sup.1 linear increase from 100% A/0% B to 40% A/60% B; A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, analytical (4.6150 mm, 5 m) Agilent Zorbax Eclipse XDB-C18 column) to separate out cis and trans isomers, the species attributed as (Tc-III-1-PSMAt1) eluted at 38.89 and 39.25 min; (Tc-III-2-PSMAt1) eluted at 46.21 and 46.83 min.
[0322] Two separate iTLC analyses were undertaken, to enable quantification of .sup.99mTc-colloids, unreacted .sup.99mTcO.sub.4.sup. and (Tc-III-2-PSMAt1)/(Tc-III-2-PSMAt1).
[0323] To quantify amounts of unreacted .sup.99mTcO.sub.4.sup., acetone was used as a mobile phase: R.sub.f values: .sup.99mTcO.sub.4.sup.>0.9, .sup.99mTc colloids<0.1 (Tc-III-1-PSMAt1)/(Tc-III-2-PSMAt1)<0.1.
[0324] To quantify .sup.99mTc-colloid formation, a 1:1 mixture of methanol and 2M aqueous ammonium acetate solution was used as a mobile phase: .sup.99mTcO.sub.4.sup.>0.9, .sup.99mTc colloids<0.1, (Tc-III-1-PSMAt1)/(Tc-III-2-PSMAt1)>0.9.
TABLE-US-00007 TABLE 7 Kit Components Radiochemical Yield 1 (II-1-RGD): 1 mg (0.93 mol); (Tc-III-1-RGD) 34% Sodium gluconate (NaC.sub.6H.sub.11O.sub.7): 1 mg (4.6 mol); SnCl.sub.22H.sub.2O: 50 g (0.22 mol); NaHCO.sub.3: 1.8 mg (21.4 mol); pH 8-8.5 then added .sup.99mTcO.sub.4.sup. in 150 L saline/150 L EtOH and heated at 60 C. for 30 min. 2 (II-1-RGD): 500 g (0.47 mol); (Tc-III-1-RGD) 85% Sodium tartrate (Na.sub.2C.sub.4H.sub.4O.sub.6): 1.05 mg (4.6 mol); SnCl.sub.22H.sub.2O: 50 g (0.22 mol); NaHCO.sub.3: 1.8 mg (21.4 mol); pH 8-8.5 then added .sup.99mTcO.sub.4.sup. in 150 L saline/150 L EtOH and heated at 60 C. for 30 min. 3 (II-1-RGD): 125 g (0.12 mol); (Tc-III-1-RGD) 90% Sodium tartrate: 0.26 mg (1.15 mol); SnCl.sub.22H.sub.2O: 25 g (0.11 mol); NaHCO.sub.3: 0.9 mg (10.7 mol); pH 8-8.5 then added .sup.99mTcO.sub.4.sup. in 250 L saline/50 L EtOH and heated at 60 C. for 30 min. 4 (II-1-RGD): 64 g (0.06 mol); (Tc-III-1-RGD) 65% Sodium tartrate: 0.26 mg (1.15 mol); SnCl.sub.22H.sub.2O: 25 g (0.11 mol); NaHCO.sub.3: 0.9 mg (10.7 mol); pH 8-8.5 then added .sup.99mTcO.sub.4.sup. in 260 L saline/40 L EtOH and heated at 60 C. for 30 min. 5 (II-1-PSMAt1) 113 g (0.11 mol); (Tc-III-1-PSMAt1) sodium tartrate: 0.26 mg (1.15 mol); 75.33 3.0% SnCl.sub.22H.sub.2O: 25 g (0.11 mol); at 22 C. NaHCO.sub.3: 0.9 mg (10.71 mol); 81.2 1.8% pH 8-8.5 at 100 C. then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at either 22 C. or 100 C. and incubated for 5 min. 6 (II-2-PSMAt1) 119 g (0.11 mol); (Tc-III-2-PSMAt1) sodium tartrate: 0.26 mg (1.15 mol); 83.53 1.5% SnCl.sub.22H.sub.2O: 25 g (0.11 mol); at 22 C. NaHCO.sub.3: 0.9 mg (10.71 mol); 88.0 0.6% pH 8-8.5 at 100 C. then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at either 22 C. or 100 C. and incubated for 5 min. 7 (II-1-PSMAt1): 85 g (0.08 mol) (Tc-III-1-PSMAt1) Sodium tartrate: 0.20 mg (0.87 mol) 81.1 3.1% SnCl.sub.22H.sub.2O: 19.0 g (0.08 mol) at 100 C. NaHCO.sub.3: 0.68 mg (8.13 mol) pH 8.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min 8 (II-1-PSMAt1): 85 g (0.08 mol) (Tc-III-1-PSMAt1) Sodium tartrate: 0.20 mg (0.87 mol) 83.5 5.8% SnCl.sub.22H.sub.2O: 19.0 g (0.08 mol) at 100 C. NaHCO.sub.3: 0.68 mg (8.13 mol) pH 7.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min 9 (II-1-PSMAt1): 85 g (0.08 mol) (Tc-III-1-PSMAt1) Sodium tartrate: 0.26 mg (1.15 mol) 89.9 3.8% SnCl.sub.22H.sub.2O: 25.0 g (0.11 mol) at 100 C. NaHCO.sub.3: 0.90 mg (10.71 mol) pH 7.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min 10 (II-1-PSMAt1): 85 g (0.08 mol) (Tc-III-1-PSMAt1) Sodium tartrate: 0.26 mg (1.15 mol) 94.0 2.7% SnCl.sub.22H.sub.2O: 19.0 g (0.08 mol) at 100 C. NaHCO.sub.3: 0.90 mg (10.71 mol) pH 7.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min 11 (II-1-PSMAt1): 85 g (0.08 mol) (Tc-III-1-PSMAt1) Sodium tartrate: 0.53 mg (2.29 mol) 98.0 0.5% SnCl.sub.22H.sub.2O: 19.0 g (0.08 mol) at 100 C. NaHCO.sub.3: 0.90 mg (10.71 mol) pH 7.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min 12 (II-1-PSMAt1): 85 g (0.08 mol) (Tc-III-1-PSMAt1) Sodium tartrate: 0.53 mg (2.29 mol) 97.0 1.0% SnCl.sub.22H.sub.2O: 19.0 g (0.08 mol) at 100 C. NaHCO.sub.3: 0.90 mg (10.71 mol) pH 8.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min 13 (II-11-PSMAt1) 119 g (0.11 mol); (Tc-III-11-PSMAt1) sodium tartrate: 0.26 mg (1.15 mol); 90% at 100 C. SnCl.sub.22H.sub.2O: 25 g (0.11 mol); NaHCO.sub.3: 0.9 mg (10.71 mol); pH 8-8.5 then added 200 MBq .sup.99mTcO.sub.4.sup. in saline solution at 100 C. and incubated for 5 min.
[0325] The amounts of tin(II) chloride, sodium bicarbonate and sodium gluconate reagents used in Kit 1 replicate those in the tetrofosmin kit. Addition of generator-produced .sup.99mTcOin saline solution (20-55 MBq) to the contents of Kit 1, followed by heating at 60 C. for 30 min, resulted in formation of Compound (Tc-III-1-RGD) in radiochemical yields of up to 34%. Replacing sodium gluconate with sodium tartrate in the kit mixture whilst lowering the amount of Compound (II-1-RGD) conjugate from 1 mg to 0.5 mg, increased radiochemical yields to 85% (Kit 2).
[0326] In Kit 3, radiochemical yields of 90% were consistently achieved (93.01.0%, n=4), with 45-65 MBq of .sup.99mTcOand only 125 g of Compound (II-1-RGD). In Kit 3, sodium tartrate and tin(II) chloride amounts were also reduced. However, further decreasing Compound (II-1-RGD), to 63 jig in Kit 4, reduced radiochemical yields to 65%. All radiolabelling reactions were undertaken in a mixture of saline and ethanol to dissolve Compound (II-1-RGD); lower amounts of ethanol were required for kits containing lower amounts of Compound (II-1-RGD).
[0327] In Kit 5 and Kit 6, it is shown that the substitution of the aryl phosphine substituent with an electron donating group, in this case a phenyl substituted in the para position with a methyl group, improves the yield at both room temperature and 100 C. In Kit 13, it is shown that a more electron-donating group, in this case a phenyl substituted in the para position with a methoxy group, improves the yield at 100 C. even more than a methyl substituent using the same method.
Example 15aKit Radiolabelling of (II-1-PSMAt1), (II-2-PSMAt1), and (II-11-PSMAt1) with .SUP.188.ReO.SUB.4..SUP.
[0328] A sample of .sup.188ReO.sub.4.sup. in saline solution were obtained from an Oncobeta .sup.188W/.sup.188Re generator. .sup.188ReO.sub.4.sup. was pre-concentrated: a solution of .sup.188ReO.sub.4.sup. in saline was passed through a Ag.sup.+ cartridge (Dionex OnGuard II Ag; preconditioned with 10 mL water) and onto a QMA cartridge (Sep-Pak Light (46 mg) Accell Plus QMA Carbonate; preconditioned with 5 mL EtOH, then 10 mL water), where the .sup.188ReO.sub.4.sup. was trapped. The QMA cartridge was then washed with water (4 mL), before eluting the .sup.188ReO.sub.4.sup. in a small volume of saline (0.9% NaCl in water, w/v). This pre-concentration process could be combined with the generator-elution, facilitating direct concentration of the generator eluate while minimising radioactivity handling. Direct concentration of the eluate was achieved using tubing to connect the generator outlet to the two cartridges (in tandem), which was in turn attached to a vacuum pump via two or more receiver vials.
[0329] Aqueous saline solution containing .sup.188ReO.sub.4.sup. (125 L, 30-450 MBq) was added to an aqueous solution of sodium citrate (1 M, 50 L) and stannous chloride (3.75 mg), and heated at 90 C. for 30 min. An aliquot of this solution (50 L, 10-150 MBq) was then added to the contents of either two (II-1-PSMAt1) kits, two (II-2-PSMAt1) kits, or two (II-11-PSMAt1) kits (as described in Table 5), to give a solution of pH 8-8.5, which was then heated at 90 C. for 30 min. Aliquots of the reaction solution were then analysed by reverse phase C18 radio-HPLC (30 min method).
[0330] Aliquots of the reaction solution were then analysed by reverse phase C18 radio-HPLC.
[0331] Unreacted .sup.188ReO.sub.4.sup. and .sup.188Re-citrate eluted at 2.0-2.3 min. The species attributed as (Re-III-1-PSMAt1) eluted at 12.7 min in 73% radiochemical yield (
Example 15bPurification and Stability of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1)
[0332] Crude reaction mixture containing either (Re-II-1-PSMAt1) or (Re-II-2-PSMAt1), prepared as described above, were applied to a reverse phase C18 analytical HPLC column and isolated using the following linear HPLC gradient: 0 min, 100% A/0% B to 60 min, 40% A/60% B, 1 mL min.sup.1 flow rate. Fractions containing either [(Re-II-1-PSMAt1) (eluted at 38-40 min as a double peak) or (Re-II-2-PSMAt1) (eluted at 46-48 min as a double peak) were immediately frozen and lyophilized. The resulting samples of (Re-II-1-PSMAt1) or (Re-II-2-PSMAt1) were dissolved in phosphate buffered saline and measured 95% radiochemical purity (by analytical C18 radio-HPLC and radio-iTLC).
[0333] Solutions of (Re-II-1-PSMAt1) or (Re-II-2-PSMAt1) in phosphate buffered saline (20 L, 0.5-1.5 MBq) were added to samples of human serum (180 L) and incubated at 37 C. At 1 and 24 h, samples were treated with ice-cold acetonitrile (300 L) to precipitate and remove serum proteins. Acetonitrile in the supernatant was then removed by evaporation under a stream of N.sub.2 gas. The final solution was then analysed by reverse-phase analytical radioHPLC (
Example 16Uptake of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1) in Prostate Cancer Cell Lines
[0334] A panel of cell lines were selected that either expressed GCP(II)/PSMA-(DU145-PSMA (genetically modified to express PSMA) [1], or had low GCP(II)/PSMA expression (DU145 (HTB-81)). The cell lines were cultured in RPMI 1640 medium (R.sub.0883, Sigma) containing 10% foetal bovine serum, 2 mM L-glutamine, and 100 U.Math.mL.sup.1 penicillin and 100 g.Math.mL.sup.1 streptomycin. Cells were maintained at 37 C. and 5% CO.sub.2. Cells were seeded in 6-well plates at a density of 510.sup.5 cells per well in 2 mL complete media to achieve 70-80% confluency the following day. The cell medium (1 mL/well) was replaced 1 h prior to treating the cells. Solutions containing either (Re-III-1-PSMAt1) or (Re-III-2-PSMAt1) (50 kBq, in 5-10 uL of phosphate buffered saline, >95% radiochemical purity) were added to each well, and the cells incubated at 37 C. for 1 h. Uptake studies were also performed after a 2 min incubation with the PSMA inhibitor 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA; 30 L of 750 M PMPA solution/well). After 1 h incubation, the supernatant was removed and the cells were washed with cold phosphate buffered saline solution (31 mL). The cells were lysed with cold radioimmunoprecipitation assay buffer (RIPA buffer, 500 L; 150 mM sodium chloride, 0.1% w/w sodium dodecyl sulfate (SDS), 0.5% w/w sodium deoxycholate (NaDOC), 1% w/w Triton-X) and samples were collected for radioactivity counting. Results are depicted as meansSD of independent biological experiments (performed on different days with different radiotracer preparations).
[0335] (Re-III-1-PSMAt1) and (Re-III-2-PSMAt1) exhibited uptake in DU145-PSMA+ cells (14.372.25% AR [percentage added radioactivity], and 9.231.04% AR respectively). This uptake was specific: DU145-PSMA+ cell uptake of (Re-III-1-PSMAt1) and (Re-III-2-PSMAt) could be blocked with PMPA, and there was negligible uptake in parental DU145 cells (
Example 17Preparation of (.SUP.186.Re-III-1-PSMAt) and Kit Radiolabeling
[0336] .sup.186Re-III-1-PSMA was prepared in two steps from a saline solution containing .sup.186ReO.sub.4.sup..
[0337] SnCl.sub.2.Math.2H.sub.2O (15 mg) was dissolved in aqueous sodium citrate solution (100 L, 1 M). A sample of this solution (25 L) was added to an aqueous saline solution containing .sup.186ReO.sub.4.sup. (10 MBq, 65 L). The reaction mixture was heated to 90 C. for 30 min, yielding .sup.186Re(V)-citrate in 96% radiochemical yield.
[0338] Following this, .sup.186Re(V)-citrate (4-5 MBq, 50 L) was added to a pre-fabricated, lyophilized kit (Table 8, also used for .sup.188Re radiolabelling), containing sodium carbonate, sodium tartrate, tin chloride and DP.sup.Ph-PSMAt. This solution was heated at 90 C. for 30 min, resulting in formation of .sup.186Re-DP1-PSMA in 5.5% radiochemical yield as determined by radio-HPLC.
TABLE-US-00008 TABLE 8 Lyophilised kit formulations for (III- 1-PSMAt) for .sup.186Re radiolabelling. moles/ weight/ Components: mol mg (II-1-PSMAt1) 0.22 0.22 SnCl.sub.22H.sub.2O 0.22 0.05 Sodium tartrate 2.29 0.53 NaHCO.sub.3 21.42 1.80
[0339] Solutions of (.sup.186Re-III-1-PSMAt1) prepared from kits as described above were applied to a reverse phase C18 analytical HPLC column and isolated using the following linear HPLC gradient: 0 min, 100% A/0% B to 60 min, 40% A/60% B, 1 mL min.sup.1 flow rate (A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA). Fractions containing (.sup.186Re-III-1-PSMAt1) eluted at 39.8 mins, and were immediately frozen and lyophilised. Analytical reverse-phase HPLC indicated that radiochemical purity of (.sup.186Re-III-1-PSMAt1) was >95%. This radiolabeled species co-eluted with the non-radioactive (.sup.natRe-III-1-PSMAt1) standard.
Example 18Uptake of (.SUP.186.Re-III-1-PSMAt1) in Prostate Cancer Cell Lines
[0340] GCP(II)/PSMA-expressing cells, DU145-PSMA+ and LNCaP cells, were suspended in RPMI media (5 million cells, 1 mL). (.sup.186Re-III-1-PSMAt1) (10,000 cpm, in-10 L of phosphate buffered saline, >95% radiochemical purity) was added to each cell sample, and the cells incubated at 37 C. for 1 h, with constant agitation. Additionally, non-specific uptake was also determined by using non-GCP(II)/PSMA-expressing cells (DU145) or by blocking PSMA-expressing cells (DU145-PSMA+ and LNCaP cells) with the PSMA-inhibitor, PMPA (30 L of a 750 uM PMPA solution/5 million cells). After 60 min incubation, the supernatant was removed and the cells were washed three times with ice cold phosphate buffered saline solution. The cells were treated with ice cold RIPA buffer (500 L, 150 mM sodium chloride, 0.1% w/w sodium dodecyl sulfate (SDS), 0.5% w/w sodium deoxycholate (NaDOC), 1% w/w Triton-X) to lyse the cells, and samples collected for radioactivity counting. Uptake of .sup.186Re-DP1-PSMA measured 4.230.99% AR [percentage added radioactivity], in DU145-PSMA+ cells, and this decreased to 0.080.14% AR in PSMA-negative DU145 cells, and 0.210.16% AR when co-incubated with an excess of PMPA. Uptake of .sup.186Re-DP1-PSMA measured 3.980.98% AR in LNCaP cells, and this decreased to 0.550.15% AR when co-incubated with an excess of PMPA (see
Example 19Biodistributions of (.SUP.88.Re-III-1-PSMAt1) and (.SUP.88.Re-III-2-PSMAt1) in Mice Bearing Prostate Cancer Tumours
[0341] The biodistributions of (.sup.188Re-III-1-PSMAt1) and (.sup.188Re-III-2-PSMAt1) were assessed in SCID/Beige mice bearing DU145-PSMA+ tumours (
[0342] Urine was collected from mice administered either (.sup.188Re-III-1-PSMAt1) or (.sup.188Re-III-2-PSMAt1) at 2 h post-injection, and analysed by reverse-phase radio-HPLC. Radio-chromatograms showed that both (.sup.188Re-III-1-PSMAt1) and (.sup.188Re-III-2-PSMAt1) are highly stable, with >94% of radioactivity associated with either (.sup.188Re-III-1-PSMAt1) or (.sup.188Re-III-2-PSMAt1) respectively. (
[0343] The GCP(II)/PSMA-expressing cell line used in these experiments was a genetically modified daughter cell line of DU145, DU145-PSMA+. This cell line had previously been transduced to express full-length human GCP(II)/PSMA, following F. Kampmeier, J. D. Williams, J. Maher, G. E. Mullen and P. J. Blower, EJNMMI Res., 2014. 4, 13. These cells were cultured in DMEM medium supplemented with 10% foetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin. To prepare for experiments, cells were grown at 37 C. in an incubator with humidified air equilibrated with 5% CO.sub.2.
[0344] Animal studies complied with the guidelines on responsibility in the use of animals in bioscience research of the U.K. Research Councils and Medical Research Charities, under U.K. Home Office project and personal licenses. Subcutaneous prostate cancer xenografts were produced in SCID/beige mice (male, 7-12 weeks old) by injecting 410.sup.6 DU145-PSMA or DU145 cells suspended in PBS (100 L) on the right shoulder. Biodistribution studies were performed once a tumour had reached 5-10 mm in diameter (3-4 weeks after injection). For imaging purposes, the mice were anaesthetised, positioned on the scanner, and tail vein cannulated. For biodistribution, the mice were anaesthetised, the radiotracers were injected via the tail vein.