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RIGIDIFIED PENTADENTATE CHELATING AGENTS USEFUL FOR THE [AL18F]2+ LABELLING OF BIOMOLECULES

20220153697 · 2022-05-19

    Inventors

    Cpc classification

    International classification

    Abstract

    Rigidified pentadentate chelating agents of Formulae (I) and (II), which are useful for the [Al.sup.18F].sup.2+ labelling of biomolecules are provided. The rigidified pentadentate chelating agents are used to form coordination complexes with [Al.sup.18F].sup.2+, which are particularly advantageous for use as tracers in molecular imaging techniques.

    ##STR00001##

    Claims

    1. A compound of Formula (I) or Formula (II): ##STR00019## wherein n is an integer selected from 0, 1 and 2; m is an integer selected from 0, 1 and 2; R.sub.1, R.sub.2 and R.sub.3 are selected from the group consisting of CH.sub.2CO—Z and CH.sub.2Ar, wherein Z is selected from the group consisting of OH, NH—OH and N(CH.sub.3)(OH), and wherein Ar is an aromatic substituent of formula ##STR00020## wherein p is an integer from 0 to 6 and R.sub.5 is selected from the group consisting of —H, —COOH, —NH.sub.2, —NCS, —SH, ##STR00021## wherein n is an integer selected from 0, 1 and 2; m is an integer selected from 0, 1 and 2; one of R.sub.1, R.sub.2 and R.sub.3 has the formula —CH.sub.2Ar, wherein Ar is an aromatic substituent of formula ##STR00022## wherein p is an integer from 0 to 6 and R.sub.5 is selected from the group consisting of —H, —COOH, —NH.sub.2, —NCS, —SH, ##STR00023## and wherein the other two of R.sub.1, R.sub.2 and R.sub.3 are independently of one another —CH.sub.2CO—Z, wherein Z is selected from the group consisting of —OH, —NH—OH, and —N(CH.sub.3)(OH); R.sub.4 is selected from the group consisting of: H, cyclic aliphatic substituents having from 3 to 8 carbon atoms optionally bearing a functional group selected from —COOH, —NH.sub.2, —NCS, —SH, ##STR00024## non-cyclic aliphatic substituents having from 1 to 10 carbon atoms optionally terminated with a functional group selected from —COOH, —NH.sub.2, —NCS, —SH, ##STR00025## and aromatic groups Ar as defined above; wherein formulae (I) and (II) include both R/S isomers and cis/trans isomers.

    2. The compound of claim 1, wherein said compound is selected from the group consisting of: ##STR00026##

    3. A coordination complex comprising a compound according to claim 1 and [AlF].sup.2+ ((18F)fluoranylaluminum(2+)).

    4. A labeled biomolecule, conjugated with a coordination complex according to claim 3.

    5. The labeled biomolecule of claim 4, wherein said labeled biomolecule is selected from antibodies, antibody fragments, nanobodies and affibodies.

    6. A Positron Emission Tomography (PET) imaging method for the diagnosis of a disease in a subject, comprising administering to said subject the coordination complex of claim 3.

    7. The PET imaging method of claim 6, wherein said disease is a tumor.

    8. A method of labelling a compound according to claim 1 with .sup.18F, comprising contacting said compound with [AlF].sup.2+ ((18F)fluoranylaluminum(2+)) in an aqueous medium.

    9. The method of claim 8, wherein said compound is conjugated with a biomolecule.

    10. The method of claim 9, wherein said biomolecule is selected from antibodies, antibody fragments, nanobodies and affibodies.

    11. The method of claim 8, wherein the aqueous medium has a pH comprised between 3.5 and 6.

    12. The method of claim 8, wherein the aqueous medium has a temperature comprised between 20° and 80° C.

    13. The labeled biomolecule of claim 4, wherein said labeled biomolecule is selected from proteins, peptides, carbohydrates, phospholipids, fatty acids, nucleosides, deoxynucleosides, nucleotides and deoxynucleotides.

    14. A Positron Emission Tomography (PET) imaging method for the diagnosis of a disease in a subject, comprising administering to said subject the labeled biomolecule of claim 5.

    15. The PET imaging method of claim 14, wherein said disease is a tumor.

    16. The method of claim 9, wherein said biomolecule is selected from proteins, peptides, carbohydrates, phospholipids, fatty acids, nucleosides, deoxynucleosides, nucleotides and deoxynucleotides.

    Description

    EXPERIMENTAL PART

    [0033] ##STR00010##

    [0034] Synthesis of 2-AMPTA(OtBu).sub.3. 2-aminomethyl piperidine (200 μl, 1.56 mmol), tert-butyl bromoacetate (687 mL, 4.68 mmol) and K.sub.2CO.sub.3 (65 mg, 4.84 mmol) were allowed to react while stirring for one night in 15 mL of CH.sub.3CN at ambient temperature; the solvent was then evaporated at reduced pressure and the mixture was redissolved in 50 mL of ethyl acetate, washed with 25 mL of H.sub.2O and 2×25 mL of a saturated NaCl solution and finally anhydrified with Na.sub.2SO.sub.4. The organic solution was then filtered and the solvent removed at reduced pressure; the crude product was purified by means of column chromatography (SiO.sub.2, PetEt/EtOAC 90:10-80:20, R.sub.f.sup.90:10=0.22), thus obtaining 2-AMPTA(OtBu).sub.3 as a solid compound (0.55 g, 77% yield). .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 3.39 (s, 6H, CH.sub.2CO), 2.95 (dd, .sup.2J.sub.HH=13.2 Hz, .sup.3J.sub.HH=5.1 Hz, 1H, CHCHH′N), 2.80 (m, 1H, NCHH′CH.sub.2), 2.69 (m, 1H, CH), 2.60 (dd, .sup.2J.sub.HH=13.2 Hz, .sup.3J.sub.HH=5.1 Hz, 1H, CHCHH′N), 2.52 (m, 1H, NCHH′CH.sub.2), 1.81 (m, 1H, NCHCHH′CH.sub.2), 1.66 (m, 1H, NCH.sub.2CHH′), 1.54 (m, 2H, NCHCH.sub.2CH.sub.2), 1.27 (m, 2H, NCH.sub.2CHH′+NCHCHH′CH.sub.2). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 170.6 (CO), 80.8 (C.sup.tBu), 57.6 (CH), 57.5 (NCHCH.sub.2N), 56.4 (CH.sub.2CO), 53.4 (NCH.sub.2CH.sub.2), 30.5 (NCHCH.sub.2CH.sub.2), 28.1 (CH.sub.3), 25.5 (NCHCH.sub.2CH.sub.2), 23.6 (NCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 457.5 [M+H.sup.+], calc. for [C.sub.24H.sub.45N.sub.2O.sub.6].sup.+=457.33 g/mol.

    [0035] Synthesis of 2-AMPTA. 2-AMPTA(OtBu).sub.3 (3.1 g, 0.69 mmol) was dissolved in a 1:1 mixture of TFA and CH.sub.2Cl.sub.2 (4 mL) and reacted while stirring for 5 hours at ambient temperature. After removing the solvent at reduced pressure, the species was dissolved in CH.sub.3CN (1 mL) and precipitated in diethyl ether. The solid was centrifuged (4000 rpm, 15 min) and washed/centrifuged 3 times with diethyl ether, thereby obtaining the white solid 2-AMPTA (0.18 g, 92% yield). .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 7.93 (m, 2H, CHCHCNO.sub.2+CCHCNO.sub.2), 6.88 (d, .sup.2J.sub.HH=8.8 Hz, 1H, CHCOH), 3.90 (s, 2H, NCH.sub.2Ar), 3.71 (m, 3H, NCH+NCHCH.sub.2N), 3.51 (m, 5H, NCHCH.sub.2N+NCH.sub.2CO), 3.32 (m, 1H, NCH.sub.2CH.sub.2), 3.09 (m, 1H, NCH.sub.2CH.sub.2), 2.00 (m, 1H, NCH.sub.2CH.sub.2), 1.9-1.7 (m, 4H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.57 (m, 1H, NCHCH.sub.2CH.sub.2). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 173.2 (COOH), 161.8 (COH), 141.0 (CNO.sub.2), 128.8 (CCHCNO.sub.2), 127.0 (NCH.sub.2C), 125.8 (CHCHCNO.sub.2), 116.4 (CHCOH), 66.7 (NCH.sub.2CO), 60.6 (NCH), 51.8 (NCH.sub.2CH.sub.2), 39.5 (NCHCH.sub.2N), 30.8 (NCH.sub.2Ar), 25.1 (NCH.sub.2CH.sub.2), 20.3 (NCHCH.sub.2CH.sub.2+NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 289.4 [M+H.sup.+], calc. for [C.sub.12H.sub.21N.sub.2O.sub.6].sup.+=289.14 g/mol. ESI.sup.+ MS: m/z. 289.2 (M+H.sup.+); calc. for [C.sub.12H.sub.21N.sub.2O.sub.6].sup.+=289.14 g/mol.

    ##STR00011##

    [0036] Synthesis of 2-(N-(o-hydroxybenzyl)aminomethyl) piperidine. 2-aminomethyl piperidine (100 mg, 0.88 mmol) was dissolved in anhydrous THF (5 mL). A drop of acetic acid and then salicylaldehyde (0.094 mL, 0.88 mmol) were subsequently added at 0° C. and the yellow mixture was allowed to react while stirring for 1 hour at ambient temperature. NaBH.sub.4 (333 mg, 8.80 mmol) was then added in portions at 0° C. and the suspension stirred at rt for a further 2 hours. H.sub.2O (5 mL) was then added and stirred for another 30 min at rt. 10 mL of AcOEt was then added to the mixture and the organic phase was washed with 25 mL of H.sub.2O and 2×25 mL of a saturated NaCl solution, anhydrified with Na.sub.2SO.sub.4 and filtered. Finally, the solvent was evaporated, thus obtaining N-(o-hydroxybenzyl)-2-aminomethyl piperidine (193 mg) which was used without further purification. ESI+ MS: m/z. 221.2 (M+H.sup.+); calc. for [C.sub.13H.sub.21N.sub.2O].sup.+=221.32 g/mol.

    [0037] Synthesis of 2-AMPDA-HB(OtBu).sub.2. 2-(N-o-hydroxybenzyl-aminomethyl) piperidine (193 mg, 0.88 mmol, theoretical), tert-butyl bromoacetate (0.258 mL, 1.76 mmol), K.sub.2CO.sub.3 (486 mg, 1.88 mmol) in CH.sub.3CN (10 mL) were reacted for 2 hours while stirring at ambient temperature. After removing the solvent at reduced pressure, the product was redissolved in AcOEt (10 mL) and washed with 5 mL of H.sub.2O and 2×5 mL of a saturated NaCl solution. The organic phase was then anhydrified with Na.sub.2SO.sub.4, filtered and the solvent removed in the rotavapor. The crude product was purified by means of column chromatography (SiO.sub.2, PetEt/EtOAc 90:10>70:30, R.sub.f(80:20)=0.17), thus obtaining 2-AMPDA-HB(OtBu).sub.2 (165 mg, 42%). .sup.1H NMR (500 MHz, 25° C., MeOD), δ (ppm): 7.17 (t, .sup.3J.sub.HH=7.2 Hz, 1H, CHCHCOH), 6.95 (d, .sup.3J.sub.HH=7.1 Hz, 1H, CHCCH.sub.2), 6.83 (d, .sup.3J.sub.HH=8.0 Hz, 1H, CHCOH), 6.76 (t, .sup.3J.sub.HH=7.4 Hz, 1H, CHCHCCH.sub.2), 3.90 (s, 2H, NCH.sub.2Ar), 3.71 (m, 3H, NCH+NCHCH.sub.2N), 3.51 (m, 5H, NCHCH.sub.2N+NCH.sub.2CO), 3.32 (m, 1H, NCH.sub.2CH.sub.2), 3.09 (m, 1H, NCH.sub.2CH.sub.2), 2.08 (m, 1H, NCH.sub.2CH.sub.2), 1.9-1.7 (m, 4H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.58 (m, 1H, NCHCH.sub.2CH.sub.2), 1.48 (s, 9H, 3×CH.sub.3), 1.46 (s, 9H, 3×CH.sub.3). .sup.13C NMR (125 MHz, 25° C., MeOD), δ (ppm): 170.3 (CO), 157.5 (COH), 129.5 (CHCCH.sub.2), 129.2 (CHCHCOH), 122.0 (CCH.sub.2), 119.2 (CHCHCCH.sub.2), 116.4 (CHCOH), 82.3 (C.sup.tBu), 59.4 (NCH.sub.2CO), 58.8 (NCH.sub.2Ar), 60.6 (NCH), 51.8 (NCH.sub.2CH.sub.2), 39.5 (NCHCH.sub.2N), 28.2 (CH.sub.3), 25.1 (NCH.sub.2CH.sub.2), 20.3 (NCHCH.sub.2CH.sub.2+NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z. 449.4 (M+H.sup.+); calc. for [C.sub.25H.sub.41N.sub.2O.sub.5].sup.+=449.30 g/mol.

    [0038] Synthesis of 2-AMPDA-HB. 2-AMPDA-HB(OtBu).sub.2 (88 mg, 0.20 mmol) was reacted with TFA (2 mL), CH.sub.2Cl.sub.2 (2 mL) and a drop of triisopropylsilane for 15 hours while stirring at ambient temperature. The solvents were then evaporated at reduced pressure and the residue was dissolved in CH.sub.3CN (1 mL) and precipitated in diethyl ether. The solid was centrifuged and washed 3 times with diethyl ether, thus obtaining 2-AMPDA-HB (61 mg, 90%). .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 7.34 (m, 2H, CHCHCNO.sub.2+CCHCNO.sub.2), 6.88 (d, .sup.2J.sub.HH=8.8 Hz, 1H, CHCOH), 3.90 (s, 2H, NCH.sub.2Ar), 3.71 (m, 3H, NCH+NCHCH.sub.2N), 3.51 (m, 5H, NCHCH.sub.2N+NCH.sub.2CO), 3.32 (m, 1H, NCH.sub.2CH.sub.2), 3.09 (m, 1H, NCH.sub.2CH.sub.2), 2.08 (m, 1H, NCH.sub.2CH.sub.2), 1.9-1.7 (m, 4H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.58 (m, 1H, NCHCH.sub.2CH.sub.2). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 173.2 (COOH), 161.8 (COH), 141.0 (CNO.sub.2), 128.8 (CCHCNO.sub.2), 127.0 (NCH.sub.2C), 125.8 (CHCHCNO.sub.2), 116.4 (CHCOH), 66.7 (NCH.sub.2CO), 60.6 (NCH), 51.8 (NCH.sub.2CH.sub.2), 39.5 (NCHCH.sub.2N), 30.8 (NCH.sub.2Ar), 25.1 (NCH.sub.2CH.sub.2), 20.3 (NCHCH.sub.2CH.sub.2+NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 337.4 [M+H.sup.+], calc. for [C.sub.17H.sub.25N.sub.2O.sub.5].sup.+=337.18 g/mol.

    ##STR00012##

    [0039] 2-Boc-aminomethyl)-N-(2-hydroxybenzyl-4-nitrophenylmethyl) piperidine. 2-(Boc-aminomethyl) piperidine (322 mg, 1.50 mmol) was dissolved in DMF (10 mL). K.sub.2CO.sub.3 (415 mg, 3.00 mmol) was then added, followed by 2-chloromethyl-4-nitrophenol (563 mg, 3.00 mmol), and the mixture was stirred under reflux overnight. After removing the solvent at reduced pressure, the product was redissolved in AcOEt (40 mL) and washed with 2×50 mL of H.sub.2O and 50 mL of a saturated NaCl solution. The organic phase was then anhydrified with Na.sub.2SO.sub.4, filtered and the solvent removed in the rotavapor. The crude product was purified by means of column chromatography (SiO.sub.2, cyclohexane/acetone 95:5>80:20, R.sub.f(90:10)=0.34) to obtain 2-Boc-aminomethyl)-N-(2-hydroxy-4-nitrophenylmethyl) piperidine as a white solid (262 mg, 48%).

    [0040] .sup.1H NMR (500 MHz, 25° C., CDCl.sub.3), δ (ppm): 7.94 (d, .sup.2J.sub.HH=8.9 Hz, 1H, CHCHCNO.sub.2), 7.82 (s, 1H, CCHCNO.sub.2), 6.70 (d, .sup.2J.sub.HH=8.9 Hz, 1H, CHCOH), 4.37 (d, .sup.2J.sub.HH=12.1 Hz, 1H, NCH.sub.2Ar), 3.61 (d, .sup.2J.sub.HH=12.1 Hz, 1H, NCH.sub.2Ar), 3.40 (m, 1H, CH.sub.2NH), 3.33 (m, 1H, CH.sub.2NH), 2.85 (m, 1H, NCH), 2.60 (m, 1H, NCH.sub.2CH.sub.2), 2.27 (m, 1H, NCH.sub.2CH.sub.2), 1.69 (m, 1H, NCHCH.sub.2CH.sub.2), 1.68 (m, 1H, NCHCH.sub.2CH.sub.2), 1.55 (m, 1H, NCHCH.sub.2CH.sub.2), 1.49 (m, 2H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.39 (m, 1H, NCH.sub.2CH.sub.2), 1.32 (s, 9H, CH.sub.3). .sup.13C NMR (125 MHz, 25° C., CDCl.sub.3), δ (ppm): 165.2 (COH), 156.0 (COO), 139.5 (CNO.sub.2), 125.0 (CCHCNO.sub.2), 124.6 (NCH.sub.2C), 121.5 (CHCHCNO.sub.2), 116.3 (CHCOH), 79.5 (C.sup.tBu), 60.5 (NCH), 60.3 (NCH.sub.2CH.sub.2), 56.3 (NCH.sub.2Ar), 50.8 (CH.sub.2NH), 40.9 (NCHCH.sub.2CH.sub.2), 28.2 (CH.sub.3.sup.tBu), 24.1 (NCH.sub.2CH.sub.2), 20.9 (NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 366.6 [M+H.sup.+], calc. for [C.sub.18H.sub.28N.sub.3O.sub.5].sup.+=366.20 g/mol.

    [0041] 2-aminomethyl-N-(2-hydroxy-4-nitrophenylmethyl) piperidine. 2-Boc-aminomethyl)-N-(2-hydroxy-4-nitrophenylmethyl) piperidine (235 mg, 0.64 mmol) was dissolved in DCM (5 mL). TFA (5 mL) was added and the mixture was stirred at rt for 3 hours. The solvents were then evaporated at reduced pressure and the residue redissolved in TFA (1 mL) and precipitated while adding Et.sub.2O (10 mL). The suspension was then centrifuged (4000 rpm, 15 min, 10° C.) and the precipitate washed/centrifuged with Et.sub.2O (3×10 mL). The product was obtained as a salt of trifluoroacetate (216 mg, 89%).

    [0042] .sup.1H NMR (500 MHz, 25° C., MeOD), δ (ppm): 8.31 (s, 1H, CCHCNO.sub.2), 8.17 (d, .sup.2J.sub.HH=9.0 Hz, 1H, CHCHCNO.sub.2), 7.02 (d, .sup.2J.sub.HH=8.9 Hz, 1H, CHCOH), 4.54 (d, .sup.2J.sub.HH=13.2 Hz, 1H, NCH.sub.2Ar), 4.30 (d, .sup.2J.sub.HH=13.2 Hz, 1H, NCH.sub.2Ar), 3.72 (m, 1H, CH.sub.2NH), 3.68 (m, 1H, NCH), 3.46 (m, 1H, CH.sub.2NH), 3.37 (m, 1H, NCH.sub.2CH.sub.2), 3.07 (m, 1H, NCH.sub.2CH.sub.2), 2.12 (m, 1H, NCH.sub.2CH.sub.2), 1.86-1.83 (m, 4H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.62 (m, 1H, NCHCH.sub.2CH.sub.2). .sup.13C NMR (125 MHz, 25° C., CDCl.sub.3), δ (ppm): 162.6 (COH), 140.4 (CNO.sub.2), 128.8 (CCHCNO.sub.2+CCHCNO.sub.2), 127.8 (CHCHCNO.sub.2), 116.1 (CHCOH), 60.5 (NCH), 51.2 (NCH.sub.2CH.sub.2), 48.7 (NCH.sub.2Ar), 38.9 (CH.sub.2NH.sub.2), 24.8 (NCH.sub.2CH.sub.2), 20.3 (NCHCH.sub.2CH.sub.2+NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 266.2 [M+H.sup.+], calc. for [C.sub.13H.sub.20N.sub.3O.sub.3].sup.+=266.15 g/mol.

    [0043] 2-AMPDA-NHB(OtBu).sub.2. 2-aminomethyl-N-(2-hydroxy-4-nitrophenylmethyl) piperidine (216 mg, 0.57 mmol) was dissolved in DMF (2 mL). K.sub.2CO.sub.3 (157 mg, 1.14 mmol) was added, followed by t-butyl bromoacetate (0.167 mL, 1.14 mmol), and the mixture was stirred at rt for 18 hours. After removing the solvent at reduced pressure, the product was redissolved in AcOEt (10 mL) and washed with 2×5 mL of H.sub.2O and 5 mL of a saturated NaCl solution. The organic phase was then anhydrified with Na.sub.2SO.sub.4, filtered and the solvent removed in the rotavapor. The crude product was purified by means of flash chromatography (SiO.sub.2, PetEt/EtOAc 60:40.fwdarw.20:80, R.sub.f.sup.30:70=0.24), thus obtaining the product 2-AMPDA-NHB(OtBu).sub.2 as a yellow solid (194 mg, 69%).

    [0044] .sup.1H NMR (500 MHz, 25° C., CDCl.sub.3), δ (ppm): 8.31 (d, .sup.2J.sub.HH=8.9 Hz, 1H, CHCHCNO.sub.2), 7.85 (s, 1H, CCHCNO.sub.2), 6.75 (d, .sup.2J.sub.HH=8.9 Hz, 1H, CHCOH), 3.93 (d, .sup.2J.sub.HH=12.1 Hz, 1H, NCH.sub.2Ar), 3.79 (d, .sup.2J.sub.HH=12.1 Hz, 1H, NCH.sub.2Ar), 3.5-3.3 (m, 5H, NCH+NCH.sub.2CO), 3.30 (m, 1H, CHCH.sub.2N), 3.08 (m, 1H, NCH.sub.2CH.sub.2), 2.88 (m, 1H, CHCH.sub.2N), 2.75 (m, 1H, NCH.sub.2CH.sub.2), 2.04 (m, 1H, NCH.sub.2CH.sub.2), 1.8-1.7 (m, 4H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.63 (m, 1H, NCHCH.sub.2CH.sub.2). 1.42 (s, 18H, CH.sub.3). .sup.13C NMR (125 MHz, 25° C., CDCl.sub.3), δ (ppm): 169.8 (COO), 163.8 (COH), 140.6 (CNO.sub.2), 129.4 (CCHCNO.sub.2), 127.3 (NCH.sub.2C), 125.3 (CHCHCNO.sub.2), 118.0 (CHCOH), 83.0 (C.sup.tBu), 60.6 (NCH), 57.9 (NCH.sub.2CO), 56.1 (NCHCH.sub.2N), 50.6 (NCH.sub.2CH.sub.2), 39.9 (NCH.sub.2Ar), 28.8 (CH.sub.3.sup.tBu), 26.2 (NCH.sub.2CH.sub.2), 22.4 (NCHCH.sub.2CH.sub.2), 21.4 (NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 494.4 [M+H.sup.+], calc. for [C.sub.25H.sub.40N.sub.3O.sub.7].sup.+=494.29 g/mol.

    [0045] 2-AMPDA-NHB. 2-AMPDA-NHB(OtBu).sub.2 (27 mg, 0.055 mmol) was reacted with TFA (2 mL), CH.sub.2Cl.sub.2 (2 mL) and a drop of triisopropylsilane for 15 hours while stirring at ambient temperature. The solvents were then evaporated at reduced pressure and the residue was dissolved in concentrated HCl (5 mL) and, after stirring for 30 min, the solvent was removed in the rotavapor. The residue was then redissolved with TFA (1 mL) and precipitated in diethyl ether (10 mL). The solid was centrifuged and washed 3 times with diethyl ether, thus obtaining 2-AMPDA-HB hydrochloride (14 mg, 61%) after drying under vacuum.

    [0046] .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 7.93 (m, 2H, CHCHCNO.sub.2+CCHCNO.sub.2), 6.88 (d, .sup.2J.sub.HH=8.8 Hz, 1H, CHCOH), 3.90 (s, 2H, NCH.sub.2Ar), 3.71 (m, 3H, NCH+NCHCH.sub.2N), 3.51 (m, 5H, NCHCH.sub.2N+NCH.sub.2CO), 3.32 (m, 1H, NCH.sub.2CH.sub.2), 3.09 (m, 1H, NCH.sub.2CH.sub.2), 2.08 (m, 1H, NCH.sub.2CH.sub.2), 1.9-1.7 (m, 4H, NCHCH.sub.2CH.sub.2+NCH.sub.2CH.sub.2), 1.58 (m, 1H, NCHCH.sub.2CH.sub.2). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 173.2 (COOH), 161.8 (COH), 141.0 (CNO.sub.2), 128.8 (CCHCNO.sub.2), 127.0 (NCH.sub.2C), 125.8 (CHCHCNO.sub.2), 116.4 (CHCOH), 66.7 (NCH.sub.2CO), 60.6 (NCH), 51.8 (NCH.sub.2CH.sub.2), 39.5 (NCHCH.sub.2N), 30.8 (NCH.sub.2Ar), 25.1 (NCH.sub.2CH.sub.2), 20.3 (NCHCH.sub.2CH.sub.2+NCHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 382.2 [M+H.sup.+], calc. for [C.sub.17H.sub.24N.sub.3O.sub.7].sup.+=382.16 g/mol.

    ##STR00013##

    [0047] Synthesis of 3-AMPTA(OtBu).sub.3. 3-aminomethyl piperidine (200 μl, 1.56 mmol), tert-butyl bromoacetate (687 ml, 4.68 mmol) and K.sub.2CO.sub.3 (65 mg, 4.84 mmol) were allowed to react while stirring for one night in 15 mL of CH.sub.3CN at ambient temperature; the solvent was then evaporated at reduced pressure and the mixture was redissolved in 50 mL of CHCl.sub.3, washed with 3×25 mL of water and finally anhydrified with Na.sub.2SO.sub.4. The organic solution was filtered and the solvent removed at reduced pressure; the crude product was purified by means of column chromatography (SiO.sub.2, acetone/hexane 5:95>20:80), thus obtaining 3-AMPTA(OtBu).sub.3 as a solid compound (0.51 g, 71.5% yield), ESI.sup.+ MS, m/z: 457.4 (M+H.sup.+); calc. for [C.sub.24H.sub.45N.sub.2O.sub.6].sup.+=457.33 g/mol. .sup.1H NMR (D.sub.2O, 500 MHz), δ(ppm): 3.96 (s, 4H, 2×CH.sub.2), 3.84 (s, 2H, CH.sub.2), 3.67 (d, 2H, CHCH.sub.2N), 3.26 (m, 2H, CH.sub.2.sup.pip), 3.04 (m, 2H, CH.sub.2.sup.pip), 2.14 (m, 3H, CH+ CH.sub.2.sup.pip), 1.64 (m, 2H, CH.sub.2.sup.pip), 1.18 (s, 27H, CH.sub.3). .sup.13C NMR (D.sub.2O, 500 MHz) δ(ppm): 170.3 (CO), 169.5 (CO), 66.7 (C), 61.6 (CH.sub.2), 58.3 (CH.sub.2), 58.1 (CH.sub.2), 53.5 (CH.sub.2), 30.3 (CH.sub.2), 29.8 (CH.sub.2), 27.6 (CH.sub.2), 14.8 (CH.sub.3).

    [0048] Synthesis of 3-AMPTA. 3-AMPTA(OtBu).sub.3 (74 mg) was dissolved in a 1:1 mixture of TFA and CH.sub.2Cl.sub.2 (4 mL) and reacted while stirring for 5 hours at ambient temperature. After removing the solvent at reduced pressure, the species was dissolved in CH.sub.3CN (1 mL) and precipitated in diethyl ether. The solid was centrifuged and washed 3 times with diethyl ether, thus obtaining the white solid 3-AMPTA. The final quantity is equal to 49 mg and the yield is quantitative. .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 3.93 (s, 4H, 2×CH.sub.2), 3.82 (s, 2H, CH.sub.2), 3.54 (d, 2H, CHCH.sub.2N), 2.46 (m, 2H, CH.sub.2.sup.pip), 2.42 (m, 2H, CH.sub.2.sup.pip), 1.73 (m, 1H, CH), 1.72 (m, 2H, CH.sub.2.sup.pip), 1.68 (m, 2H, CH.sub.2.sup.pip). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 173.9 (CO), 173.1 (CO), 63.2 (CH.sub.2), 59.9 (2×CH.sub.2), 58.2 (CH.sub.2), 54.5 (CH.sub.2), 30.7 (CH.sub.2), 30.1 (CH.sub.2), 28.6 (CH.sub.2), 26.7 (CH). ESI.sup.+ MS: m/z. 289.2 (M+H.sup.+); calc. for [C.sub.12H.sub.21N.sub.2O.sub.6].sup.+=289.14 g/mol.

    ##STR00014##

    [0049] Synthesis of 3-(N-o-hydroxybenzyl-aminomethyl) piperidine. 3-aminomethyl piperidine (200 mL, 1.57 mmol) and salicylaldehyde (104 mL, 1.57 mmol) in CH.sub.3OH (5 ml) were reacted while stirring for 3 hours at ambient temperature, and then NaBH.sub.4 (175 mg, 4.71 mmol) was added in an ice bath and the reaction continued while stirring for another 3 hours. The solvent was then evaporated at reduced pressure. The mixture was dissolved in 25 mL of AcOEt and washed with 3×10 mL of H.sub.2O. The organic solution was then anhydrified with Na.sub.2SO.sub.4, filtered through a pleated filter and the solvent was evaporated, thus obtaining N-(o-hydroxybenzyl)-3-aminomethyl piperidine (107 mg, 31%). .sup.1H NMR (500 MHz, 25° C., MeOD), δ (ppm): 7.21 (t, .sup.3J.sub.HH=7.6 Hz, 1H, CHCHCOH), 7.13 (d, .sup.3J.sub.HH=7.4 Hz, 1H, CHCCH.sub.2), 6.85 (d, .sup.3J.sub.HH=8.1 Hz, 1H, CHCOH), 6.82 (t, .sup.3J.sub.HH=7.4 Hz, 1H, CHCHCCH.sub.2), 4.10 (m, 2H, CH.sub.2Ar), 3.51 (m, 1H, NHCHH′.sup.PiCH), 3.26 (m, 1H, NHCHH′CH.sub.2), 2.84 (m, 2H, NHCH.sub.2CH), 2.77 (m, 1H, NHCHH′CH.sub.2), 2.61 (m, 1H, NHCHH′.sup.PiCH), 2.32 (m, 1H, NHCH.sub.2CH), 1.9-1.8 (m, 3H, NHCH.sub.2CH.sub.2+CHCHH′CH.sub.2), 1.21 (m, 1H, CHCHH′CH.sub.2). .sup.13C NMR (125 MHz, 25° C., MeOD), δ (ppm): 155.9 (COH), 141.0 (CNO.sub.2), 131.5 (CHCHCOH+CHC), 126.9 (CCH.sub.2), 120.3 (CHCHC), 115.9 (CHCOH), 49.1 (NHCH.sub.2CH), 48.3 (NHCH.sub.2C), 46.3 (NHCH.sub.2.sup.PiCH), 43.8 (NHCH.sub.2CH.sub.2), 31.2 (NHCH.sub.2CH), 26.5 (CHCH.sub.2CH.sub.2), 21.5 (NHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 221.1 [M+H.sup.+], calc. for [C.sub.13H.sub.21N.sub.2O].sup.+=221.16 g/mol.

    [0050] Synthesis of 3-AMPDA-HB(OtBu).sub.2. 3-(N-o-hydroxybenzyl-aminomethyl) piperidine (107 mg, 0.49 mmol), tert-butyl bromoacetate (144 ml, 0.98 mmol) and K.sub.2CO.sub.3 (148 mg, 1.07 mmol) in CH.sub.3CN (10 mL) were reacted for 2 hours while stirring at ambient temperature; after removing the solvent at reduced pressure, the crude product was purified by means of column chromatography (SiO.sub.2, acetone/hexane 10:90>40-60), thus obtaining 3-AMPDA-HB(OtBu).sub.2 (101 mg, 46%). ESI+ MS: m/z. 449.4 (M+H.sup.+). .sup.1H NMR (500 MHz, 25° C., MeOD), δ (ppm): 7.17 (t, .sup.3J.sub.HH=7.2 Hz, 1H, CHCHCOH), 6.95 (d, .sup.3J.sub.HH=7.1 Hz, 1H, CHCCH.sub.2), 6.83 (d, .sup.3J.sub.HH=8.0 Hz, 1H, CHCOH), 6.76 (t, .sup.3J.sub.HH=7.4 Hz, 1H, CHCHCCH.sub.2), 3.77 (d, .sup.2J.sub.HH=13.5 Hz, 1H, NCHH′Ar), 3.68 (d, .sup.2J.sub.HH=13.5 Hz, 1H, NCHH′Ar), 3.28 (d, .sup.2J.sub.HH=13.5 Hz, 1H, NCHH′CO), 3.19 (d, .sup.2J.sub.HH=13.5 Hz, 1H, NCHH′CO), 3.21 (bs, 2H, NCH.sub.2CO), 2.97 (m, 2H, NCH.sub.2.sup.PipCH), 2.37 (d, .sup.3J.sub.HH=7.0 Hz, 2H, NCH.sub.2CH), 2.03 (m, 1H, CH.sup.Pip), 1.96 (m, 1H, CH.sup.Pip), 1.76 (m, 2H, 2×CH.sup.Pip), 1.68 (m, 1H, CH.sup.Pip), 1.48 (s, 9H, 3×CH.sub.3), 1.46 (s, 9H, 3×CH.sub.3), 0.84 (m, 2H, 2×CH.sup.Pip). .sup.13C NMR (125 MHz, 25° C., MeOD), δ (ppm): 170.3 (CO), 157.5 (COH), 129.5 (CHCCH.sub.2), 129.2 (CHCHCOH), 122.0 (CCH.sub.2), 119.2 (CHCHCCH.sub.2), 116.4 (CHCOH), 82.3 (C.sup.tBu), 59.4 (NCH.sub.2CO), 58.8 (NCH.sub.2Ar), 57.4 (NCH.sub.2CH), 55.6 (NCH.sub.2CO), 53.4 (CH.sub.2.sup.Pip), 33.6 (CH.sub.2.sup.Pip), 28.2 (CH.sub.3+2×CH.sub.2.sup.Pip), 24.5 (CH.sub.2.sup.Pip). ESI.sup.+ MS: m/z 449.5 [M+H.sup.+], calc. for [C.sub.25H.sub.41N.sub.2O.sub.5].sup.+=449.30 g/mol.

    [0051] Synthesis of 3-AMPDA-HB. 3-AMPDA-HB(OtBu).sub.2 (101 mg) was reacted with TFA (6 mL) and CH.sub.2Cl.sub.2 (6 mL) for 3 hours while stirring at ambient temperature, and the product was then dissolved in CH.sub.3CN (1 mL) and precipitated in diethyl ether. The solid was centrifuged and washed 3 times with diethyl ether, thus obtaining 3-AMPDA-HB (75 mg, 98%). ESI+ MS: m/z. 337.2 (M+H.sup.+). .sup.1H NMR (D.sub.2O, 500 MHz), δ(ppm): 7.34 (t, 1H, CH.sup.Ar), 7.30 (d, 1H, CH.sup.Ar), 6.96 (m, 1H, 2×CH.sup.Ar), 4.44 (m, 2H, CH.sub.2), 3.84 (s, 2H, CH.sub.2), 3.80 (s, 2H, CH.sub.2), 3.68 (m, 1H, CH.sup.pip), 3.57 (m, 1H, CH.sup.pip), 3.19 (m, 2H, CH.sub.2), 2.93 (m, 1H, CH.sup.pip), 2.74 (m, 1H, CH.sub.piP), 2.49 (m, 1H, CH.sup.pip), 2.00 (m, 1H, CH.sub.2.sup.pip), 1.81 (m, 1H, CH.sup.pip), 1.25 (m, 1H, CH.sup.pip). .sup.13C NMR (D.sub.2O, 500 MHz) δ(ppm): 170.3 (CO), 169.4 (CO), 156.0 (C.sup.ArOH), 133.3 (CH.sup.Ar), 133.0 (CH.sup.Ar), 121.6 (CH.sup.Ar), 116.4 (CH.sup.Ar), 116.2 (C.sup.Ar), 66.7 (CH.sub.2), 58.4 (CH.sub.2), 56.8 (CH.sub.2), 55.7 (CH.sub.2), 54.3 (CH.sub.2), 31.4 (CH), 26.0 (CH.sub.2), 22.6 (CH.sub.2).

    ##STR00015##

    [0052] Synthesis of 4-AMPTA(OtBu).sub.3. 4-aminomethyl piperidine (500 mL, 4.17 mmol), tert-butyl bromoacetate (1.895 mL, 12.92 mmol) and K.sub.2CO.sub.3 (1.79 g, 12.93 mmol) in CH.sub.3CN (15 ml) were stirred for 5 hours at ambient temperature. After removing the solvent at reduced pressure, the crude product was purified by means of column chromatography (SiO.sub.2, acetone/hexane 10:90>30:70), thus obtaining 4-AMPTA tri(OtBu) as a solid compound (1.26 g, 66%). ESI.sup.+ MS, m/z: 457.4 (M+H.sup.+). .sup.1H NMR (CDCl.sub.3, 500 MHz), δ(ppm): 3.38 (s, 4H, CH.sub.2), 3.13 (s, 2H, CH.sub.2), 2.21-2.97 (d, 2H, CH.sub.2, cycle), 2.57-2.55 (d, 2H, CH.sub.2), 1.67 (s, 2H, CH.sub.2, cycle), 1.80-1.77 (d, 2H, CH.sub.2, cycle), 1.35-1.30 (m, 3H, CH.sub.2—CH). .sup.13C NMR (CDCl.sub.3, 500 MHz), δ(ppm): 171.6 (CO), 81.6 (C(CH.sub.3).sub.3), 61.4 (CH.sub.2, cycle), 60.9 (NCH.sub.2COOt-Bu), 57.4 (N(CH.sub.2COOt-Bu).sub.2), 54.0 (CH.sub.2, —NCH.sub.2, cycle), 34.9 (CH, cycle), 31.0 (CH.sub.2, CH, cycle), 28.9 (CH.sub.3, t-Bu).

    [0053] Synthesis of 4-AMPTA. 4-AMPTA(OtBu).sub.3 (1.26 g) was dissolved in a 1:1 mixture of TFA and CH.sub.2Cl.sub.2 (60 mL) and stirred for 5 hours at ambient temperature. After removing the solvent at reduced pressure, the residue was dissolved in CH.sub.3CN (1 mL) and precipitated in diethyl ether. The solid was centrifuged and washed 3 times with diethyl ether, thus obtaining the white solid 4-AMPTA. The final quantity is equal to 686 mg and the yield is 86%. ESI+ MS: m/z. 289.2 (M+H.sup.+). ESI+ MS: m/z. 289.2 (M+H.sup.+). .sup.1H NMR (D.sub.2O, 500 MHz), δ(ppm): 4.00 (s, 4H, 2×CH.sub.2), 3.88 (s, 2H, CH.sub.2), 3.68 (d, 2H, CHCH.sub.2N), 3.28 (m, 2H, CH.sub.2.sup.pip), 3.05 (m, 2H, CH.sub.2.sup.pip), 2.14 (m, 3H, CH+CH.sub.2.sup.pip), 1.64 (m, 2H, CH.sub.2.sup.pip). .sup.13C NMR (D.sub.2O, 500 MHz) δ(ppm): 169.0 (N(CH.sub.2COOH).sub.2), 168.4 (NCH.sub.2COOH), 61.9 (CHCH.sub.2), 57.1 (NCH.sub.2COOH), 56.5 (N(CH.sub.2COOH).sub.2), 53.59 (CH.sub.2, —N(CH.sub.2).sub.2 cycle), 28.6 (CH, cycle) 27.6, (CH.sub.2, —CH(CH.sub.2).sub.2, cycle).

    ##STR00016##

    [0054] Synthesis of 4-(N-o-hydroxybenzyl-aminomethyl) piperidine. 4-aminomethyl piperidine (500 ml, 4.17 mmol) and salicylaldehyde (435 ml, 4.17 mmol) in CH.sub.3OH (5 mL) were stirred for 3 hours at ambient temperature, and then NaBH.sub.4 (347 mg, 8.34 mmol) was added in an ice bath for 2 hours and the solvent was evaporated at reduced pressure. The solution was redissolved with 25 mL of AcOEt and washed with 3×10 mL of H.sub.2O, then anhydrified with Na.sub.2SO.sub.4. The organic solution was then filtered through a pleated filter and the solvent evaporated in the rotavapor, thus obtaining N-(o-hydroxybenzyl)-4-aminomethyl piperidine (328 mg, 45%). ESI+ MS: m/z. 221.2 (M+H.sup.+). .sup.1H NMR (CDCl.sub.3, 500 MHz), δ(ppm): 7.16, 7.17, 7.19, (t, 1H, ring), 6.82-7.00 (m, 1H, ring), 6.77-7.00 (m, 1H, ring), 7.19 (s, 1H, ring), 3.10, 3.12, (d, 2H, cycle), 2.62, 2.64, (m, 2H, cycle), 2.56, 2.60, (m, 2H, CH.sub.2—NH.sub.2), 1.20, 1.23 (m, 1H, cycle). .sup.13C NMR (CDCl.sub.3, 500 MHz), δ(ppm): 159.0 (C, ring), 129.5 (CH, ring), 129.0 (CH, ring), 123.3 (C, ring), 119.2 (CH, ring), 117.1 (CH, ring), 53.7, 55.9, (CH.sub.2, CH.sub.2—NH), 47.0, 32.0, (CH.sub.2, ring), 37.0, (CH, ring).

    [0055] Synthesis of 4-AMPDA-HB-(OtBu).sub.2. K.sub.2CO.sub.3 (622 mg, 4.5 mmol) was added to a solution of 4-(N-o-hydroxybenzyl-aminomethyl) piperidine (328 mg, 1.49 mmol) and tert-butyl bromoacetate (437 ml, 2.98 mmol) in CH.sub.3CN (10 mL). The mixture was allowed to react while stirring for 2 hours at ambient temperature; after removing the solvent at reduced pressure, the crude product was purified by means of column chromatography (SiO.sub.2, acetone/hexane 1:99>20:80), thus obtaining the product at 40% yield. ESI+ MS: m/z. 449 (M+H.sup.+). .sup.1H NMR (CDCl.sub.3, 500 MHz), δ(ppm): 7.18 (t, 1H, CH.sup.Ar), 6.96 (d, 1H, CH.sup.Ar), 6.85 (d, 1H, CH.sup.Ar), 6.78 (t, 1H, CH.sup.Ar), 3.79 (s, 2H, CH.sub.2), 3.22 (s, 2H, CH.sub.2), 3.12 (s, 2H, CH.sub.2), 2.93 (d, 2H, CHCH.sub.2N), 2.44 (m, 2H, CH.sub.2.sup.pip), 2.18 (m, 3H, CH+ CH.sub.2.sup.pip), 1.74 (m, 2H, CH.sub.2.sup.pip), 1.47 (s, 18H, CH.sub.3). .sup.13C NMR (CDCl.sub.3, 500 MHz) δ(ppm): 170.7 (CO), 158.2 (C.sup.ArOH), 122.6 (CH.sup.Ar), 122.1 (CH.sup.Ar), 119.9 (CH.sup.Ar), 117.1 (CH.sup.Ar), 112.1 (C.sup.Ar), 82.7 (C), 66.8 (CH.sub.2), 60.8 (CH.sub.2), 59.4 (CH.sub.2), 56.7 (CH.sub.2), 53.7 (CH.sub.2), 33.9 (CH), 30.9 (CH.sub.2), 28.9 (CH.sub.3).

    [0056] Synthesis of 4-AMPDA-HB. 4-AMPDA-HB(Ot-Bu).sub.2 (247 mg) was reacted with TFA (10 mL) in CH.sub.2Cl.sub.2 (10 mL) for one night at ambient temperature. After removing the solvent at reduced pressure, the species was dissolved in CH.sub.3CN (1 mL) and precipitated in diethyl ether. The solid was centrifuged and washed 3 times with diethyl ether, thus obtaining a crude reaction product which was purified by means of semi-preparative HPLC-MS using CH.sub.3OH 0.1% TFA and H.sub.2O 0.1% TFA as eluents. The 4-AMPDA-HB peak came out at minute 1.8 (32 mg, 17%).

    [0057] ESI+ MS: m/z. 337.3 (M+H.sup.+). .sup.1H NMR (D.sub.2O, 500 MHz), δ(ppm): 7.41 (t, 1H, CH), 7.35 (d, 1H, CH.sup.Ar), 7.01 (m, 1H, 2×CH.sup.Ar), 3.88 (s, 2H, CH.sub.2), 3.81 (s, 2H, CH.sub.2), 3.57 (d, 2H, CHCH.sub.2N), 3.34 (s, 2H, CH.sub.2), 3.26 (m, 2H, CH.sub.2.sup.pip), 2.28 (m, 1H, CH), 2.15 (m, 2H, CH.sub.2.sup.pip), 1.65 (m, 2H, CH.sub.2.sup.pip). .sup.13C NMR (D.sub.2O, 500 MHz) δ(ppm): 170.5, 169.5, (C, COOH), 133.3, 130.0, 123.1, 121.7, 116.5, 113.3, (CH, ring), 61.0, 58.4, (CH.sub.2, CH.sub.2COOH), 56.8, (CH.sub.2, CH.sub.2N), 53.5, (CH.sub.2, cycle), 49.7 (CH.sub.2, CH.sub.2N), 30.0, (CH, cycle), 27.4 (CH.sub.2, cycle).

    ##STR00017##

    [0058] trans-diaminocyclohexane-N-trifluoroacetamide. trans-diaminocyclohexane (500 mg, 4.38 mmol) was dissolved in dioxane (20 mL). The solution was cooled to 5° C. and ethyl trifluoroacetate (0.52 mL, 4.38 mmol) was progressively added. After removing the ice bath, the mixture was stirred at ambient temperature for one night. The solvent was then evaporated at reduced pressure and the residue was suspended in H.sub.2O (50 mL) and extracted with EtOAc (5×10 mL). The joined organic phases were dried with anhydrous MgSO.sub.4, filtered and evaporated. The crude product (584 mg) was used for the next step without further purification. .sup.1H NMR (500 MHz, 25° C., CDCl.sub.3), δ (ppm): 6.62 (bs, 1H, NH), 3.39 (m, 1H, CHNH), 2.43 (m, 1H, CHNH.sub.2), 2.08 (m, 1H, NHCHCHH′), 1.91 (1H, NH.sub.2CHCHH′), 1.68 (m, 2H, NHCHCH.sub.2CH.sub.2), 1.4-1.0 (m, 6H, NHCHCHH′+NH.sub.2CHCHH′+NH.sub.2CHCH.sub.2CH.sub.2+NH.sub.2). .sup.13C NMR (125 MHz, 25° C., CDCl.sub.3), δ (ppm): 157.3 (q, .sup.2J.sub.CF=36.4 Hz, CO), 115.9 (q, .sup.1J.sub.CF=286.7 Hz, CF.sub.3), 57.0 (CHNH), 54.7 (CHNH.sub.2), 36.2 (NH.sub.2CHCH.sub.2), 31.6 (NHCHCH.sub.2), 24.9 (NHCHCH.sub.2CH.sub.2), 24.7 (NH.sub.2CHCH.sub.2CH.sub.2). ESI.sup.+ MS: m/z 211.2 [M+H.sup.+], calc. for [C.sub.8H.sub.14F.sub.3N.sub.2O].sup.+=211.11 g/mol.

    [0059] trans-diaminocyclohexane-N-trifluoroacetamide-N′-bis(ethyl acetate). trans-diaminocyclohexane-N-trifluoroacetamide (580 mg, 2.76 mmol) was solubilized in MeCN (20 mL). K.sub.2CO.sub.3 (1.14 g, 8.28 mmol) was added, followed by ethyl bromoacetate (1.46 mL, 13.24 mmol), and the reaction mixture was stirred at reflux temperature for one night. The solvent was evaporated at reduced pressure, and the residue was suspended in EtOAc (40 mL) and washed with H.sub.2O (2×20 mL) and saturated aqueous NaCl (20 mL). The organic phase was dried on anhydrous Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product (731 mg) was used in the next reaction without further purification. ESI.sup.+ MS: m/z 383.2 [M+H.sup.+], calc. for [C.sub.16H.sub.26F.sub.3N.sub.2O.sub.5].sup.+=383.18 g/mol.

    [0060] trans-diaminocyclohexane-N-bis(acetate). trans-diaminocyclohexane-N-trifluoroacetamide-N′-bis(ethyl acetate) (50 mg, 0.13 mmol) was dissolved in THF (4 mL). An aqueous solution 1 M of LiOH (4 mL) was added and the biphasic mixture obtained was heated to 50° C. and stirred vigorously for 2 hours. The solvents were evaporated at reduced pressure, the residue was redissolved in H.sub.2O (5 mL) and the pH corrected to approximately 7 using HCl 1 M. The resulting solution was lyophilized and the crude product thus obtained was used for the subsequent synthesis without further purification. ESI.sup.+ MS: m/z 231.3 [M+H.sup.+], calc. for [C.sub.10H.sub.19N.sub.2O.sub.4].sup.+=231.13 g/mol.

    [0061] trans-diaminocyclohexane-N-bis(acetate)-N′-o-hydrozybenzyl (CDHB-DA). trans-diaminocyclohexane-N-bis(acetate) (50 mg, 0.22 mmol) was dissolved in anhydrous MeOH (2 mL). Salicylaldehyde (0.050 mL, 0.47 mmol) was then added dropwise and the reaction mixture was stirred at ambient temperature for 1 h. The solution was then cooled to 0° C. and NaBH.sub.4 (83 mg, 2.20 mmol) was added in small portions. The resulting suspension was stirred for one night at ambient temperature, after which H.sub.2O (5 mL) was added at 0° C. and the mixture was stirred for a further 30 min. The solvents were evaporated at reduced pressure and the residue was resuspended in H.sub.2O (10 mL), filtered using a membrane filter and the filtrate was finally lyophilized. The product was purified by means of preparative HPLC-MS and obtained as a white solid (34 mg, 78%). .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 7.30 (m, 2H, CHCHCOH+CHCCH.sub.2), 6.92 (m, 2H, CHCOH+CHCHCCH.sub.2), 4.34 (d, .sup.2J.sub.HH=13.2 Hz, 1H, NCHH′Ar), 4.05 (d, .sup.2J.sub.HH=13.2 Hz, 1H, NCHH′Ar), 3.6-3.1 (bm, 4H, CH.sub.2CO), 2.90 (m, 1H, CHNH), 2.53 (m, 1H, CHN), 2.32 (m, 1H, CHH′.sup.CyHex), 1.92 (m, 1H, CHH′.sup.CyHex), 1.73 (m, 2H, 2×CHH′.sup.CyHex), 1.38 (m, 1H, CHH′CyHex), 1.17 (m, 3H, 3×CHH′.sup.CyHex). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 175.7 (COOH), 155.0 (COH), 131.6 (CHCCH.sub.2), 131.5 (CHCHCOH), 120.6 (CHCHCCH.sub.2), 117.5 (CCH.sub.2), 115.2 (CHCOH), 63.2 (CHN), 57.0 (CHNH), 44.2 (NCH.sub.2Ar), 27.1 (CH.sub.2.sup.CyHex), 24.5 (CH.sub.2.sup.CyHex) 24.1 (CH.sub.2.sup.CyHex), 23.3 (CH.sub.2.sup.CyHex). ESI.sup.+ MS: m/z 337.3 [M+H.sup.+], calc. for [C.sub.17H.sub.25N.sub.2O.sub.5].sup.+=337.18 g/mol.

    ##STR00018##

    [0062] trans-N,N′-dibenzyl diaminocyclohexane. trans-diaminocyclohexane (500 mg, 4.38 mmol) was dissolved in anhydrous MeOH (20 mL). Salicylaldehyde (1.34 mL, 13.14 mmol) was added dropwise and the reaction mixture was stirred at ambient temperature for 1 h. The solution was then cooled to 0° C. and NaBH.sub.4 (663 mg, 17.52 mmol) was added in small portions. The resulting suspension was stirred for one night at ambient temperature, after which H.sub.2O (5 mL) was added at 0° C. and the mixture was stirred for a further 30 min. The solvents were evaporated at reduced pressure and the residue was resuspended in EtOAc (20 mL) and washed with H.sub.2O (2×10 mL) and saturated aqueous NaCl (10 mL). The organic phase was dried on anhydrous Na.sub.2SO.sub.4, filtered and evaporated under vacuum, and the desired product was obtained in a sufficiently pure form as a yellow oil (658 mg, 51%). .sup.1H NMR (500 MHz, 25° C., CDCl.sub.3), δ (ppm): 7.4-7.2 (m, 10H, Ph), 3.92 (d, .sup.2J.sub.HH=13.0 Hz, 2H, NCHH′Ph), 3.78 (d, .sup.2J.sub.HH=13.0 Hz, 2H, NCHH′Ph), 2.72 (m, 2H, CHNH), 1.80 (m, 2H, NHCHCHH′), 1.55 (m, 2H, NHCHCHH′), 1.35 (m, 4H, NHCHCH.sub.2CH.sub.2). .sup.13C NMR (125 MHz, 25° C., CDCl.sub.3), δ (ppm): 142.4 (C.sup.Ph), 130.7, 130.1, 129.0 (3×CH.sup.Ph), 61.3 (CHNH), 31.7 (NHCHCH.sub.2), 27.3 (NHCHCH.sub.2CH.sub.2), ESI.sup.+ MS: m/z 295.4 [M+H.sup.+], calc. for [C.sub.20H.sub.27N.sub.2].sup.+=295.22 g/mol.

    [0063] trans-N,N′-dibenzyl diaminocyclohexane-N,N′-bis(tert-butyl acetate). trans-N,N′-dibenzyl diaminocyclohexane (500 mg, 1.70 mmol) was solubilized in MeCN (10 mL). K.sub.2CO.sub.3 (704 g, 5.09 mmol) was added, followed by tert-butyl bromoacetate (0.55 mL, 3.74 mmol), and the reaction mixture was stirred at reflux temperature for one night. The solvent was evaporated at reduced pressure, and the residue was suspended in EtOAc (20 mL) and washed with H.sub.2O (10 mL) and saturated aqueous NaCl (2×10 mL). The organic phase was dried on anhydrous MgSO.sub.4, filtered and evaporated under vacuum. The crude product (876 mg) was used in the next reaction without further purification. ESI.sup.+ MS: m/z 523.6 [M+H.sup.+], calc. for [C.sub.32H.sub.47N.sub.2O.sub.4].sup.+=523.73 g/mol.

    [0064] trans-diaminocyclohexane-N,N′-bis(tert-butyl acetate). trans-N,N′-dibenzyl diaminocyclohexane-N,N′-bis(tert-butyl acetate) (876 mg, 0.1.68 mmol) was dissolved in MeOH (40 mL). 10% Pd/C (88 mg) suspended in MeOH (1 mL) was added, the suspension was saturated with H.sub.2 by means of bubbling for 10 min, 3 vacuum/H.sub.2 cycles were applied and the mixture was stirred at ambient temperature for one night under an H.sub.2 atmosphere. The suspension was then filtered through Celite® and the filtrate was evaporated at reduced pressure. The crude product (520 mg) was obtained as a beige solid and used for the subsequent procedure without further purification steps. ESI.sup.+ MS: m/z 343.3 [M+H.sup.+], calc. for [C.sub.18H.sub.35N.sub.2O.sub.4].sup.+=343.48 g/mol.

    [0065] trans-N,N′-dibenzyl diaminocyclohexane-N,N′-bis(acetate). trans-diaminocyclohexane-N,N′-bis(tert-butyl acetate) (520 mg, 1.52 mmol) and triisopropylsilane (3 drops) were dissolved in DCM (10 mL), TFA (10 mL) was added and the mixture obtained was stirred at ambient temperature for one night. The solvents were evaporated at reduced pressure, and the residue was redissolved in TFA (1 mL) and precipitated in diethyl ether. The solid was centrifuged and washed 3 times with diethyl ether and dried under vacuum (white solid, 331 mg). The crude product thus obtained was used for the subsequent synthesis without further purification. ESI.sup.+ MS: m/z 231.4 [M+H.sup.+], calc. for [C.sub.10H.sub.19N.sub.2O.sub.4].sup.+=231.26 g/mol.

    [0066] trans-diaminocyclohexane-N—(O-acetyl-o-hydroxybenzyl)-N,N′-bis(acetate). trans-N,N′-diaminocyclohexane-N,N′-bis(acetate) (0.51 mmol theoretical) was solubilized in MeCN (10 mL). K.sub.2CO.sub.3 (70 mg, 0.51 mmol) was added, followed by O-acetyl-2-bromomethylphenyl (117 mg, 0.51 mmol), and the reaction mixture was stirred at reflux temperature for one night. The solvent was evaporated at reduced pressure, and the residue was suspended in EtOAc (20 mL) and extracted with H.sub.2O (2×10 mL). The aqueous phase was lyophilized and the crude product obtained was used immediately for the final deprotection reaction. ESI.sup.+ MS: m/z 379.3 [M+H.sup.+], calc. for [C.sub.19H.sub.27N.sub.2O.sub.6].sup.+=379.19 g/mol.

    [0067] trans-diaminocyclohexane-N-(o-hydroxybenzyl)-N,N′-bis(acetate). trans-diaminocyclohexane-N—(O-acetyl-o-hydroxybenzyl)-N,N′-bis(acetate) (0.51 mmol theoretical) was dissolved in THF (7 mL). An aqueous solution 1 M of NaOH (7 mL) was added and the mixture obtained was heated to 50° C. and stirred vigorously for 3 hours. The solvents were evaporated at reduced pressure and the residue was purified by means of preparative HPLC-MS and obtained as a white solid (5 mg). .sup.1H NMR (500 MHz, 25° C., D.sub.2O), δ (ppm): 7.46 (m, 2H, 2×CH.sup.Ar), 7.36 (m, 2H, 2×CH.sup.Ar), 4.45 (d, .sup.2J.sub.HH=12.9 Hz, 1H, NCHH′Ar), 4.08 (d, .sup.2J.sub.HH=12.9 Hz, 1H, NCHH′Ar), 3.94 (d, .sup.2J.sub.HH=16.4 Hz, 1H, NCHH′CO), 3.64 (d, .sup.2J.sub.HH=16.4 Hz, 1H, NCHH′CO), 3.34 (m, 1H, CHNH), 3.10 (m, 1H, CHN), 2.21 (m, 1H, CHH′.sup.CyHex), 1.88 (m, 1H, CHH′.sup.CyHex), 1.78 (m, 2H, 2×CHH′.sup.CyHex), 1.65 (m, 1H, CHH′.sup.CyHex), 1.2-1.0 (m, 3H, 3×CHH′.sup.CyHex). .sup.13C NMR (125 MHz, 25° C., D.sub.2O), δ (ppm): 176.2 (COOH), 155.0 (COH), 131.4 (CHCCH.sub.2), 131.2 (CHCHCOH), 128.1 (CHCHCCH.sub.2), 126.4 (CCH.sub.2), 122.1 (CHCOH), 63.9 (CHN), 57.6 (CHNH), 48.5 (NCH.sub.2Ar), 31.0 (CH.sub.2.sup.CyHex), 25.5 (CH.sub.2.sup.CyHex), 24.6 (CH.sub.2.sup.CyHex), 24.3 (CH.sub.2.sup.CyHex). ESI.sup.+ MS: m/z 337.3 [M+H.sup.+], calc. for [C.sub.17H.sub.25N.sub.2O.sub.5].sup.+=337.18 g/mol.