UNIVERSAL BUILDING BLOCKS FOR RADIOLABELING

Abstract

The present invention describes novel chelators (multidentate ligands) and precompounds for complexation of radiometals and non-radioactive counterparts, for use in radiopharmacy. The invention includes a process and a kit involving such chelators.

Active moieties directing to a pharmaceutical target (such as peptides or proteins) can be attached to the chelator very easily via the so called click-chemistry forming a triazole-ring moiety. The aromatic triazole-nitrogen itself acts as a new and soft nucleophilic site enabling for complexation of various radiometals or non-radioactive counterparts. The chelators are capable of fast complexation at low temperature.

##STR00001##

Claims

1. A complex comprising a metal M, and a chelator, wherein the chelator is a compound of formula (III) ##STR00028## or salts thereof, wherein R.sup.1 is -alky, -aryl or -heteroaryl, or wherein R.sup.1 is a linker attached either to an active moiety or a group for functionalization.

2. A complex according to claim 1, wherein M is selected from Al, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.99mTc, .sup.186Re, and .sup.188Re.

3. A complex according to claim 1 further comprising CO, RNC, .sup.18F and O as ligands, wherein R is alkyl or aryl.

4. (canceled)

5. A compound of formula (II) or formula (III), or salts thereof ##STR00029## wherein each R is a carboxyl protecting group, wherein R.sup.1 is -alky, -aryl or -heteroaryl, or a linker attached either to an active moiety or a group for functionalization.

6. Use of one or more of compounds of claim 5 for the preparation of a complex comprising a metal M, and a chelator, wherein the chelator is a compound of formula (III) ##STR00030## or salts thereof, wherein R.sup.1 is -alky, -aryl or -heteroaryl, or wherein R.sup.1 is a linker attached either to an active moiety or a group for functionalization.

7. A compound of formula (I) or salts thereof: ##STR00031## wherein each R is a carboxyl protecting group.

8. Use of a compound according to claim 7 in radiopharmacy.

9. Use of a compound according to claim 7 for preparation of: a compound of formula (II) or formula (III), or salts thereof ##STR00032## wherein each R is a carboxyl protecting group, wherein R.sup.1 is -alky, -aryl or -heteroaryl, or a linker attached either to an active moiety or a group for functionalization, or a complex comprising a metal M, and a chelator, wherein the chelator is a compound of formula (III).

10. A non-radioactive kit for use in radiopharmacy comprising at least one container, wherein the container contains: (i) a compound of formula (III) according to claim 5.

11. A non-radioactive kit according to claim 10, wherein the kit additionally comprises one or more of the following: (ii) preservative, (iii) agent for pH adjustment and (iv) filler.

12. A complex according to claim 2 further comprising CO, RNC, .sup.18F and O as ligands, wherein R is alkyl or aryl.

13. Use according to claim 6 wherein M is selected from Al, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.99mTc, .sup.186Re, and .sup.188Re.

14. Use according to claim 13 wherein the complex further comprises CO, RNC, .sup.18F and O as ligands, wherein R is alkyl or aryl.

15. Use according to claim 6 wherein the complex further comprises CO, RNC, .sup.18F and O as ligands, wherein R is alkyl or aryl.

16. Use according to claim 9 for preparation of a complex comprising a metal M, and a chelator, wherein the chelator is a compound of formula (III), wherein M is selected from Al, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.99mTc, .sup.186Re, and .sup.188Re.

17. Use according to claim 16 wherein the complex further comprises CO, RNC, .sup.18F and O as ligands, wherein R is alkyl or aryl.

18. Use according to claim 9 wherein the complex further comprises CO, RNC, .sup.18F and O as ligands, wherein R is alkyl or aryl.

Description

General Methods for Preparation of Compound (I), Compound (II) and Compound (III)

Preparation of tert-Butyl 2-[[2-[bis(2-tert-butoxy-2-oxo-ethyl)amino]cyclohexyl]-prop-2-ynyl-amino]acetate (1)

[0078] The synthesis of the mono alkyne substituted cyclohexane compound 1 as starting material is carried out by a standard alkylation method using tert-butyl 2-[[2-[bis(2-tert-butoxy-2-oxo-ethyl)amino]cyclohexyl]amino]acetate (Mohamadi et al. (2017) and Gale et al. (2015)) as amine and propargyl bromide as alkylation agent. Additionally, were used trimethylamine (TEA) as a base and acetonitrile as solvent.

##STR00013##

[0079] .sup.1H NMR (400 MHz, CDCl.sub.3) 3.69, 3.50, 3.39, 2.69, 2.17, 2.03, 1.67, 1.46, 1.45, 1.25, 1.13. .sup.13C NMR (101 MHz, CDCl.sub.3) 171.74, 171.21, 81.87, 80.55, 80.28, 77.20, 72.21, 62.88, 62.82, 53.71, 52.32, 40.01, 29.92, 28.27, 28.14, 28.12, 25.65, 25.59.

[0080] Chemical Formula: C.sub.27H.sub.46N.sub.2O.sub.6, Molecular Weight: 494.66 g/mol, ESI.sup.+ m/z: 495 [M+H].sup.+.

Preparation of tert-Butyl 2-[[2-[bis(2-tert-butoxy-2-oxo-ethyl)amino]cyclohexyl]-hex-2-ynyl-amino]acetate (1)

[0081] The synthesis of the mono alkyne substituted cyclohexane compound 1 as starting material is carried out by a standard alkylation method using tert-butyl 2-[[2-[bis(2-tert-butoxy-2-oxo-ethyl)amino]cyclohexyl]amino]acetate (Mohamadi et al. (2017) and Gale et al. (2015)) as amine and 6-chloro-1-hexyn as alkylation agent. Additionally, were used trimethylamine (TEA) as a base and acetonitrile as solvent.

##STR00014##

Preparation of 5-[4-[[[2-[bis(2-tert-butoxy-2-oxo-ethyl)amino]cyclohexyl]-(2-tert-butoxy-2-oxo-ethyl)amino]methyl]triazol-1-yl]pentanoic acid (2)

[0082] To a solution of 20 mg alkyne 1 (40 mol) and 11 mg 5-azidopentanoic acid (80 mol) in tert-butyl alcohol (750 l) 1.3 mg TBTA (2.5 mol) was added. After brief stirring, 1 M sodium ascorbate (50 l) and 0.1 M CuSO.sub.4 (100 l) solutions were added and the gentle mixing was continued at room temperature for 24 h. The reaction mixture was then diluted with ethyl acetate (10 ml) and extracted three times with aqueous 0.05 M EDTA (35 ml) and then 1 M NH.sub.4OH (35 ml) solutions to remove copper salts. The organic phase was dried (Na.sub.2SO.sub.4), filtered and evaporated under reduced pressure to afford the crude product, which was used without further purification. Yield: 20.1 mg (31 mol; 77%)

##STR00015##

[0083] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.01, 5.20, 4.34, 3.95, 3.79, 3.70, 3.38, 3.30, 2.61, 2.38, 1.99, 1.97, 1.69, 1.45, 1.43, 1.41, 1.11. .sup.13C NMR (101 MHz, CDCl.sub.3) 171.85, 171.69, 124.20, 80.77, 77.20, 67.06, 63.00, 52.97, 52.88, 49.82, 45.39, 29.55, 28.11, 28.06, 25.77, 25.53.

[0084] Chemical Formula: C.sub.32H.sub.55N.sub.5O.sub.8, Molecular Weight: 637.81 g/mol, ESI.sup.+ m/z: 638 [M+H].sup.+.

Preparation of [2-[bis(carboxymethyl)amino]cyclohexyl]-[[1-(4-carboxybutyl)triazol-4-yl]methyl]-(carboxymethyl)ammonium trifluoroacetate (3)

[0085] 10 mg (15 mol) of the tert-butyl ester 2 was dissolved in a mixture of TFA (450 l) and water (50 l) and stirred for 5 h at rt. Then the reaction mixture was added dropwise to ice cooled Et.sub.2O (10 ml). The supernatant was decanted and the precipitate was washed twice with cold Et.sub.2O. Yield: 5.4 mg (9 mol; 60%),

##STR00016##

[0086] .sup.1H NMR (400 MHz, D.sub.2O) 8.12, 4.47, 4.36, 3.81, 3.44, 3.42, 3.30, 2.29, 2.14, 1.96, 1.83, 1.76, 1.72, 1.43, 1.19, 1.05. .sup.13C NMR (101 MHz, D.sub.2O) 178.09, 162.67, 127.71, 117.97, 114.76, 65.93, 64.09, 50.27, 32.80, 28.49, 24.16, 23.62, 20.95, 13.99.

[0087] Chemical Formula: [C.sub.20H.sub.32N.sub.5O.sub.8].sup.+[C.sub.2F.sub.3O.sub.2].sup., Molecular Weight(cation): 470.50 g/mol, ESI.sup.+ m/z: 471 [M+H].sup.+.

[0088] Preparation of Other Chelators (4-6) of General Formula (III):

[0089] Other chelator compounds were synthesized in the same way by copper click reaction of the alkyne 1 with different azido derivatives and subsequent acid deprotection with TFA.

N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(bis(carboxymethyl)amino)-N-(carboxymethyl)cyclohexan-ammonium trifluoroacetate (4)

[0090] ##STR00017##

[0091] Starting material: Alkyne 1 and (azidomethyl)benzene. .sup.1H NMR (400 MHz, CD.sub.3CN) 8.06, 7.42, 7.40, 7.38, 7.36, 7.32, 7.30, 5.59, 4.56, 4.53, 4.47, 4.13, 4.06, 3.91, 3.87, 3.58, 3.34, 2.87, 2.06, 1.95, 1.95, 1.94, 1.78, 1.56, 1.42, 1.28. .sup.13C NMR (101 MHz, CD.sub.3CN) 171.67, 169.51, 167.23, 159.29, 159.08, 158.69, 158.13, 135.27, 128.96, 128.50, 127.98, 127.24, 119.35, 117.29, 114.08, 112.10, 65.24, 60.15, 54.30, 53.80, 49.34, 48.36, 24.73, 24.10, 23.79, 23.71. Chemical Formula (cation): C.sub.22H.sub.30N.sub.5O.sub.6.sup.+ Molecular Weight: 460.51 g/mol, ESI.sup.+ m/z: 461 [M+H].sup.+

2-(bis(carboxymethyl)amino)-N-((1-(2-((5-carboxy-5-(3-(1,3-dicarboxypropyl)ureido)pentyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-N-(carboxymethyl)cyclohexan-ammonium trifluoroacetate (5)

[0092] ##STR00018##

[0093] Starting material: Alkyne 1 and di-tert-butyl ((6-(2-azidoacetamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)glutamate. .sup.1H NMR (400 MHz, D.sub.2O) 8.10, 4.65, 4.37, 4.34, 4.18, 4.17, 4.16, 4.14, 4.10, 4.09, 4.08, 4.07, 3.60, 3.56, 3.17, 3.15, 3.14, 2.43, 2.41, 2.39, 2.12, 2.09, 2.06, 2.05, 1.90, 1.88, 1.75, 1.61, 1.45, 1.44, 1.30, 1.20, 1.07. .sup.13C NMR (101 MHz, D.sub.2O) 177.23, 177.12, 176.27, 167.32, 159.30, 62.55, 53.10, 52.56, 52.27, 39.24, 30.44, 30.03, 27.51, 26.17, 24.18, 23.74, 22.12. Chemical Formula(cation): C.sub.29H.sub.45N.sub.8O.sub.14.sup.+ Molecular Weight: 728.72 g/mol, ESI.sup.+ m/z: 729 [M+H].sup.+

N-((1-TATE-1H-1,2,3-triazol-4-yl)methyl)-2-(bis(carboxymethyl)amino)-N-(carboxymethyl)cyclohexan-ammonium trifluoroacetate (6)

[0094] ##STR00019##

[0095] Starting material: Alkyne 1 and azidopentanamido [Tyr3]octreotate (TATE-derivative: (2S,3R)-2-[[(4R7S,10S,13R,16S,19R)-10-(4-aminobutyl)-19-[[(2R)-2-amino-3-phenylpropanoyl]amino]-7-[(1R)-1-hydroxyethyl]-16-[(4-hydroxyphenyl)methyl]-13-(1H-indol-3-ylmethyl)-(3,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]amino]-3-hydroxybutanoic acid).

[0096] Chemical Formula(cation): C.sub.77H.sub.109N.sub.22O.sub.15S.sub.2.sup.+ Molecular Weight: 1658.91 g/mol, MALDI-TOF MS.sup.+ m/z: 1659 [M+H].sup.+

General Method for Preparation of the Radiolabeled Complexes of Al.sup.18F, .sup.68Ga and .sup.64Cu:

[0097] Radiolabeling of chelator, e.g. [2-[bis(carboxymethyl)amino]cyclohexyl]-[[1-(4-carboxybutyl)triazol-4-yl]methyl]-(carboxymethyl)ammonium trifluoroacetate, with F-18 as Al-Fluorid, Ga-68 and Cu-64 is performed as follows:

[0098] To 50-100 g of above-named chelator dissolved in 200 l 0.1 M MES buffer pH 5.5 the prepared solution of the radionuclide was added and mixed for 30 min at room temperature.

[0099] Before, Al.sup.18F is freshly prepared by mixing from F-18 sodium fluoride solution in saline and 20 l of a 0.002 M AlCl.sub.3 solution.

[0100] Ga-68 gallium(III) chloride diluted in 0.5 M HCl was used directly after elution from the Ge.sup.68/Ga.sup.68 generator.

[0101] Cu-64 copper(II) chloride was used as solution in 0.01 M HCl after the copper separation.

[0102] The completion of the reaction was confirmed by TLC control (2 M NH.sub.4OAc/Methanol 1:1 (v/v) RP18 material). For all of the tested radionuclides (AlF-18, Ga-68, Cu-64) unreacted radiometal species remain on the start of the TLC stripes (Rf=0), whereas the complexes move with Rf=0.7-1 under these conditions.

[0103] M=mol/l.

[0104] Radio-TLC examples using 2 M NH.sub.4OAc/Methanol 1:1 (v/v) as eluent and RP18 material:

[0105] Complex of F-18 Aluminum fluoride (Rf=0) with above named chelator (3):

[0106] (Rf=0.7, Yield 97%):

##STR00020##

[0107] FIG. 1 shows Radio-TLC of the Al.sup.18F-complex.

[0108] Complex of Ga-68 with Above Named Chelator (3):

[0109] (Rf=1, Yield: 80%):

##STR00021##

[0110] FIG. 2 shows the Radio-TLC of the .sup.68Ga-complex.

[0111] Complex of Cu-64 with Above Named Chelator (3):

[0112] (Rf=0.9, Yield: 99%):

##STR00022##

[0113] FIG. 3 shows the Radio-TLC of the .sup.64Cu-complex.

[0114] Additional ligands 4-6 were tested exemplarily only for F-18 labeling as AlFluorid complexes:

[0115] Complex Comprising Metal Al and Further Ligand .sup.18F and Chelator (4):

[0116] (Rf=0.7, Yield: 99%):

##STR00023##

[0117] FIG. 4 shows the Radio-TLC of the complex of chelator (4), wherein the metal is Al and the complex further comprises .sup.18F as a ligand.

[0118] Complex Comprising Metal Al and Further Ligand .sup.18F and Chelator (5):

[0119] (Rf=0.9, Yield: 99%):

##STR00024##

[0120] FIG. 5 shows the Radio-TLC of the complex of chelator (5), wherein the metal is Al and the complex further comprises .sup.18F as a ligand.

[0121] Complex Comprising Metal Al and Further Ligand .sup.18F and Chelator (6):

[0122] (Rf=0.8, Yield: 98%):

##STR00025##

[0123] FIG. 6 shows the Radio-TLC of the complex of chelator (6), wherein the metal is Al and the complex further comprises .sup.18F as a ligand.

[0124] Preparation of a Non-Radioactive Kit

[0125] The formulation of a non-radioactive kit is produced by mixing all ingredients in an aqueous solution. The formulation may then be sterile filtered, e.g. through a sterile 0.2 m filter. The formulation is preferably filled into sterile containers. The containers are subsequently sealed and optionally lyophilized. This is preferably performed by first partially sealing the containers, followed by lyophilization and subsequent sealing and capping.

[0126] Preparation of a Complex from a Non-Radioactive Kit:

[0127] The complex is preferably formed by first adding to the first container, containing chelator of formula (III) or a salt thereof, and optionally a filler, the content of the second container which contains an agent for pH adjustment. In case of a powdery buffer or agent for pH adjustment, a diluent comprising water, preferably water for injections or a saline solution (sterile solution of sodium chloride) is added to the second vial prior to adding the content to the first vial. Subsequently, as an example for Tc-complexes, the pertechnetate solution is added to the mixture of the first and the second vial, resulting in the formation of a pharmaceutical formulation for intravenous administration.

[0128] Alternatively, the technetium complex is formed by adding the pertechnetate solution to the first container and immediately of after labeling is finished transferring the content of the second container to the first container.

[0129] Biodistribution in SKH1 Mice

[0130] The compounds were prepared and complexed with Al.sup.18F as described above.

[0131] For the biodistribution two groups of SKH1 mice (n=4) were sacrificed under desflurane anaesthesia at 60 and 240 min, respectively, after injection of 0.30 MBq each. Organs and tissues of interest were removed, weighed, and the activity was measured in a cross-calibrated well counter and dose calibrator. The decay corrected data were normalized to the amount of injected activity calculated from the activity of injection syringes before and after injection and expressed as percentage of injected activity per gram tissue (% ID/g tissue). Values are quoted as meanstandard deviation (mean+/SD) for one group of animals.

##STR00026##

TABLE-US-00001 60 min SD 240 min SD % ID/g blood 1.19 1.01 0.23 0.42 brown adipose tissue 0.05 0.01 0.02 0.01 skin 0.12 0.04 0.04 0.03 brain 0.01 0.00 0.00 0.00 ovaries 0.15 0.08 0.01 0.01 uterus 0.13 0.03 0.02 0.01 pancreas 0.12 0.05 0.01 0.01 spleen 0.14 0.04 0.01 0.01 adrenals 0.22 0.11 0.03 0.03 sum kidneys 0.37 0.08 0.03 0.01 fat 0.21 0.29 0.01 0.01 muscle 0.06 0.01 0.02 0.02 heart 0.09 0.01 0.02 0.01 sum lung 0.23 0.09 0.03 0.01 thyroid 0.25 0.09 0.38 0.49 gall bladder sum liver 0.25 0.13 0.12 0.07 femur 0.36 0.05 0.37 0.09 % ID intestine 13.51 1.09 20.90 9.94 urine 79.26 1.31 73.24 11.75

##STR00027##

TABLE-US-00002 60 min SD 240 min SD % ID/g blood 1.99 3.91 0.02 0.01 brown adipose tissue 0.13 0.03 0.02 skin 0.07 0.06 0.09 0.09 brain 0.01 0.00 0.01 0.00 ovaries 0.18 0.17 0.06 0.07 uterus 0.26 0.30 0.04 0.02 pancreas 0.20 0.33 0.04 0.02 spleen 0.14 0.25 0.02 0.02 adrenals 0.25 0.45 0.14 0.06 sum kidneys 0.14 0.04 0.02 fat 0.16 0.28 0.10 0.09 muscle 0.07 0.05 0.02 0.01 heart 0.07 0.10 0.01 0.01 sum lung 0.30 0.02 0.01 thyroid 0.35 0.40 0.18 0.10 gall bladder sum liver 1.84 0.22 0.21 femur 0.40 0.12 0.46 0.18 % ID intestine 51.28 1.65 54.06 7.77 urine 39.20 2.12 32.68 1.81

LITERATURE

[0132] Gale, E. M.; Atanasova, I. P.; Blasi, F.; Ay, I.; Caravan, P.

A Manganese Alternative to Gadolinium for MRI Contrast

[0133] J. Am. Chem. Soc. 137 (2015), 15548-15557 [0134] Cleeren, F.; Lecina, J.; Billaud, E. M. F.; Ahamed, M.; Verbruggen, A.; Bormans, G. M.

New Chelators for Low Temperature Al.SUP.18.F-Labeling of Biomolecules

Bioconjugate Chem. 27 (2016), 790-798

[0135] Mohamadi, A.; Miller, L. W.
Efficient route to pre-organized and linear polyaminopolycarboxylates: Cy-TTHA, Cy-DTPA and mono/di-reactive, tert-butyl protected TTHA/Cy-TTHA

Tetrahedron Letters 58 (2017), 1441-1444

[0136] Vanasschen, C.; Molnr, E.; Tircs, G.; Klmn, F. K.; Tth, .; Brandt, M.; Coenen, H. H.; Neumaier, B.

Novel CDTA-based, Bifunctional Chelators for Stable and Inert Mn.SUP.II .Complexation:

Synthesis and Physicochemical Characterization

[0137] Inorg. Chem. 56 (2017), 7746-7760