2,11-diaza-[3.3](2,6)pyridinophane compounds and their application as ligands of essential metal ion based MRI contrast agents and 52MN based PET contrast agents

Abstract

The one subject of the invention is the compounds of general formula (I), their isomers, their physiologically acceptable salts and/or Mn(II), Fe(II), Fe(III), Co(II) and Ni(II) complexes. The other subject of the invention is the application of the above compounds. The compounds of general formula (I): wherein —NRR.sub.1 group may refer to: a) —NRR.sub.1 with N atom in the ring means a ring of 4 to 7, that in certain cases may contain another heteroatom, and in specific cases the ring may be replaced with an aryl group (of 5 to 7 carbon atoms) substituted with —COOH, —OH, —OCH.sub.3, —NO.sub.2, —NH.sub.2, —NCS, —NHS-activated ester, aryl (of 5 to 7 carbon atoms), or nitro-, amino- or isothiocyanate group, or b) in the —NRR.sub.1 group R means a H atom, alkyl, aryl, nitroaryl, aminoaryl or isothiocyanate-aryl group (of 1 to 6 carbon atoms) and R.sub.4 is a H atom, alkyl (of 1 to 6 carbon atoms) or —(CH.sub.2).sub.n—COOH group, whereas n=1 to 10 integer, or c) —NRR.sub.1 group is one of the following groups: (formula II) whereas R.sub.2 is a H atom, carboxyl- or alkyl-carbonyl group (of 1 to 4 carbon atoms); (formula III) and R.sub.2 is a H atom or alkyl or aryl group (of 1 to 6 carbon atoms), and X means independently from one another H atom, —CH.sub.3, —COOH, —OH, —OCH.sub.3, alkoxy- (of 2 to 6 carbon atoms), —NO.sub.2, —NH.sub.2, —NCS, —NHS-activated ester, alkyl (of 2 to 12 carbon atoms) or aryl (of 5 to 7 carbon atoms) group, in certain cases the latter may be substituted with hydroxyl, hydroxyalkyl (of 1 to 6 carbon atoms), nitro, amino or isothiocyanate group. ##STR00001##

Claims

1. A compound selected from: ##STR00026## ##STR00027## ##STR00028## ##STR00029##

2. A complex comprising the compound of claim 1 and a metal selected from Mn(II), Fe(II), Fe(III), Co(II), and Ni(II).

3. A method of imaging, comprising: administering a compound according to claim 1 as a contrast agent in diagnostic imaging, and performing diagnostic imaging.

4. A kit for use in diagnostic imaging, comprising: a compound of claim 1.

5. A method of imaging, comprising: administering a complex according to claim 2 as a contrast agent in diagnostic imaging, and performing diagnostic imaging.

6. A kit for use in diagnostic imaging, comprising: a complex of claim 2.

Description

EXAMPLE 1

Synthesis of BP2AM.SUP.Pyp

a.) 2-bromo-1-(piperidine-1-yl)ethanone

(1) Bromoacetyl bromide (3.56 g, 17.6 mmol, 1.55 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (50 ml) and K.sub.3PO.sub.4 (6.41 g, 30.2 mmol, 2.5 equ.) was mixed in a flask of 250 ml under N.sub.2 atmosphere. Piperidine (1.00 g, 11.7 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 30 ml) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×15 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×30 ml, 10 m/m %) and saturated NaCl solution (1×30 ml). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 1.73 g (70%).

(2) ##STR00005##

(3) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.59 (2H, m, (CH.sub.2) ring), 1.67 (4H, m, (CH.sub.2) ring), 3.45 (2H, t, (CH.sub.2) ring), 3.59 (2H, t, (CH.sub.2) ring), 3.87 (2H, s, (CH.sub.2)),

(4) .sup.13C NMR [100 MHz, CDCl.sub.3] δ 25.4 (2 pcs CH.sub.2 ring); 26.0 (CH.sub.2 ring); 27.2 (CH.sub.2Br); 44.2 (2 pcs CH.sub.2 ring); 169.5 (C(═O));

b.) Synthesis of BP2AM.SUP.Pyp

(5) The 2-bromo-1-(piperidin-1-yl)ethanone obtained as described above (0.22 g, 1.05 mmol, 2.5 equivalent) was dissolved in dry acetonitrile and added dropwise to the acetonitrile solution (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) obtained form commercial sources and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. The received compound is 2,11-bis[2-oxo-2-(piperidine-1-il)-ethyl]-2,11-diaza[3.3](2,6)-pyridinophane. Yield: 0.11 g (52%)

(6) ##STR00006##

(7) .sup.1H NMR [360 MHz, D.sub.2O] δ 1.30-1.55 (12H, m, (6 pcs CH.sub.2)), 3.30 (4H, t, (2 pcs CH.sub.2)), 3.4 (4H, t, (2 pcs CH.sub.2)), 4.59 (8H, s, (4 pcs CH.sub.2)), 4.84 (4H, s, (2 pcs CH.sub.2)), 7.08 (4H, d, (4 pcs CH) aromatic), 7.56 (1H, t, (2 pcs CH) aromatic);

(8) .sup.13C NMR [100 MHz, D.sub.2O] δ 23.4 (2 pcs CH.sub.2); 24.9 (2 pcs CH.sub.2); 25.5 (2 pcs CH.sub.2); 43.7 (4 pcs CH.sub.2); 45.6 (2 pcs CH.sub.2); 58.5 (2 pcs CH.sub.2); 60.8 (2 pcs CH.sub.2); 124.1 (4 pcs CH aromatic); 140.5 (2 pcs CH aromatic); 149.6 (4 pcs C aromatic); 164.0 (2 pcs C(═O));

(9) MS (ESI) m/z 491.500 (M+H).sup.+100%; 513.500 (M+Na).sup.+10%;

(10) IR: 1624 cm.sup.−1 (>C═O); 2164, 2024 (aromatic >C═C) és 1093 cm.sup.−1 (≥C—O—C≤);

EXAMPLE 2

Synthesis of BP2AM.SUP.Morf

(11) The commercially available 4-(bromoacetyl)morpholine (0.22 g, 1.06 mmol, 2.5 equivalent) was dissolved in dry acetonitrile (5 ml), then added dropwise to the acetonitrile suspension (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and the solvent was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.09 g (43%).

(12) ##STR00007##

(13) .sup.1H NMR [360 MHz, D.sub.2O] δ 3.35 (4H, s, (2 pcs CH.sub.2)), 3.51-3.71 (16H, m, (8 pcs CH.sub.2)), 4.33 (8H, br, (4 pcs CH.sub.2)), 7.18 (4H, d, (4 pcs CH) aromatic), 7.74 (2H, t, (2 pcs CH) aromatic); .sup.13C NMR [100 MHz, D.sub.2O] δ 45.5 (4 pcs CH.sub.2); 56.1 (2 pcs CH.sub.2); 59.9 (4 pcs CH.sub.2); 69.1 (4 pcs CH.sub.2); 123.7 (4 pcs CH aromatic); 133.4 (2 pcs CH aromatic); 150.2 (4 pcs C aromatic); 166.1 (2 pcs C(═O)

EXAMPLE 3

Synthesis of BP2AMP.SUP.PipAc

(14) The commercially available 1-acetyl-4-(bromoacetyl) piperazine (0.265 g, 1.06 mmol, 2.5 equivalent) was dissolved in dry acetonitrile (5 ml), then added dropwise to the acetonitrile suspension (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and the filtrate was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.13 g (54%).

(15) ##STR00008##

(16) .sup.1H NMR [360 MHz, D.sub.2O] δ 2.23 (6H, s, 2 pcs CH.sub.3), 3.33 (4H, s, (2 pcs CH.sub.2)), 3.50-3.74 (16H, m, (8 pcs CH.sub.2)), 4.29 (8H, br, (4 pcs CH.sub.2)), 7.21 (4H, d, (4 pcs CH) aromatic), 7.77 (2H, t, (2 pcs CH) aromatic);

(17) .sup.13C NMR [100 MHz, D.sub.2O] δ 23.5 (2 pcs CH.sub.3); 54.3 (8 pcs CH.sub.2); 58.8 (2 pcs CH.sub.2); 65.3 (4 pcs CH.sub.2); 124.4 (4 pcs CH aromatic); 131.7 (2 pcs CH aromatic); 151.1 (4 pcs C aromatic); 167.8 (2 pcs C(═O)); 173.3 (2 pcs C(═O))

EXAMPLE 4

Synthesis of diOH—BP2AM.SUP.Pyp

(18) The 2-bromo-1-(piperidine-1-yl)ethanone manufactured as per Example 1 (0.19 g, 0.93 mmol, 2.5 equivalent) was dissolved in dry acetonitrile, then added dropwise to the acetonitrile solution (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane-7,15-diol (0.10 g, 0.37 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.16 g, 1.11 mmol, 3 equivalent) at room temperature within 30 minutes (the diOH—BP2AM macrocycle was synthesized by using literature procedure K. M. Lincoln, P. Gonzalez, K. N. Green, US. Pat. Appl. 2014, US 20140206862 A1 20140724 patent). Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and the mother liquor was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.11 g (57%).

(19) ##STR00009##

(20) .sup.1H NMR [360 MHz, D.sub.2O] δ 1.54 (12H, m, (6 pcs CH.sub.2)), 3.32 (4H, s, (2 pcs CH.sub.2)) 3.41 (8H, m, (4 pcs CH.sub.2)) 3.8-4.1 (8H, m, (4 pcs CH.sub.2)), 6.41 (4H, s, (CH) aromatic);

(21) .sup.13C NMR [100 MHz, D.sub.2O] δ 23.3 (2 pcs CH.sub.2); 25.1 (2 pcs CH.sub.2); 25.4 (2 pcs CH.sub.2); 43.7 (4 pcs CH.sub.2); 57.9 (2 pcs CH.sub.2); 61.9 (4 pcs CH.sub.2); 112.8 (4 pcs CH aromatic); 154.3 (2 pcs C(OH) aromatic); 157.3 (4 pcs C aromatic); 169.1 (2 pcs C(═O));

EXAMPLE 5

Synthesis of diOMe-BP2AM.SUP.Pyp

(22) The 2-bromo-1-(piperidine-1-yl)ethanone manufactured as per Example 1 (0.17 g, 0.83 mmol, 2.5 equivalent) was dissolved in dry acetonitrile, then added dropwise to the acetonitrile solution (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane-7,15-dimethoxi (0.10 g, 0.33 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.14 g, 0.99 mmol, 3 equivalent) at room temperature within 30 minutes (the diOMe-BP2AM macrocycle was prepared according to the following literature description: F. Banse, R. Carina, M. Delroisse, J.-J. Girerd, R. Hage, J. A. Simaan, D. Tetard, WO 1999065905 A1 Number of patent submission: PCT/GB1999/001850 patent). Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and the mother liquor was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 m) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.09 g (49%).

(23) ##STR00010##

(24) .sup.1H NMR [360 MHz, D.sub.2O] δ 1.54 (12H, m, (6 pcs CH.sub.2)), 3.29 (4H, s, (2 pcs CH.sub.2)) 3.44 (8H, m, (4 pcs CH.sub.2)) 3.8-4.3 (8H, m, (4 pcs CH.sub.2)), 4.12 (6H, s, 2 pcs CH.sub.3), 6.68 (4H, s, (CH) aromatic);

(25) .sup.13C NMR [100 MHz, D.sub.2O] δ 24.1 (2 pcs CH.sub.2); 25.3 (2 pcs CH.sub.2); 25.6 (2 pcs CH.sub.2); 44.1 (4 pcs CH.sub.2); 56.6 (2 pcs CH.sub.3); 58.6 (2 pcs CH.sub.2); 61.6 (4 pcs CH.sub.2); 110.1 (4 pcs CH aromatic); 153.2 (4 pcs C aromatic); 157.2 (2 pcs C(OCH.sub.3) aromatic); 172.3 (2 pcs C(═O));

EXAMPLE 6

Synthesis of BP2AM.SUP.Pro

a.) Tert-butyl 1-(2-bromoacetyl)pyrrolidine-2-carboxylate

(26) Bromoacetyl bromide (1.44 g, 7.2 mmol, 0.63 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (30 ml) and K.sub.3PO.sub.4 (2.55 g, 12.0 mmol, 2.5 equ.) was mixed in a flask of 250 ml and stirred under N.sub.2 atmosphere. D-proline tert-butyl ester hydrochloride (1.00 g, 4.8 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 20 ml) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×10 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×20 ml, 10 m/m %) and saturated NaCl solution (1×20 ml). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 1.05 g (75%).

(27) ##STR00011##

(28) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.60 (9H, s, (CH.sub.3)), 2.15 (2H, m, (CH.sub.2) ring), 2.43 (2H, m, (CH.sub.2)ring), 3.72 (2H, m, (CH.sub.2) ring), 4.00 (2H, s, (CH.sub.2)), 4.55 (1H, m, (CH) ring);

(29) .sup.13C NMR [100 MHz, CDCl.sub.3] δ 25.0 CH.sub.2 ring; 27.0 CH.sub.2Br; 28.0 (3C CH.sub.3); 29.2 CH.sub.2 ring; 47.5 CH.sub.2 ring; 60.2 CH ring; 81.8 CH t-butyl; 165.2 C(═O); 170.9 C(═O);

b.) BP2AM.SUP.Pro .Synthesis

(30) The tert-butyl 1-(2-bromoacetyl)pyrrolidine-2-carboxylate obtained as described above (0.31 g, 1.05 mmol, 2.5 equivalent) was dissolved in dry acetonitrile (20 ml) and added dropwise to the acetonitrile solution of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained yellowish oil was dissolved in CH.sub.2Cl.sub.2 (10 ml), then trifluoroacetic acid is added to it (0.20 ml, 6 equivalent) and the reaction mixture was refluxed for 24 hours. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.13 g (57%).

(31) ##STR00012##

(32) .sup.1H NMR [360 MHz, D.sub.2O] δ 2.1 (8H, m, (CH.sub.2)), 2.34 (4H, m, (CH.sub.2)), 4.48 (4H, m, (CH.sub.2)), 4.55 (2H, m, (CH)), 4.61 (8H, s, (CH.sub.2)), 7.35 (4H, d, (CH) aromatic), 7.88 (2H, t, (CH) aromatic);

(33) .sup.13C NMR [100 MHz, D.sub.2O] δ 22.6 2 pcs CH.sub.2; 29.0 (2 pcs CH.sub.2); 46.1 (2 pcs CH.sub.2); 54.5 (2 pcs CH.sub.2); 59.1 (4 pcs CH.sub.2); 61.2 (2 pcs CH); 121.3 (4 pcs CH aromatic); 136.8 (2 pcs CH aromatic); 156.2 (4 pcs C aromatic); 167.9 (2 pcs C(═O)); 175.2 (2 pcs C (COOH));

(34) MS (ESI) m/z 551.46 (M+H).sup.+ 100%; 573.500 (M+Na).sup.+ 10%;

(35) IR: 1729, 1654 cm.sup.−1 (>C═O); 2163,1996 (aromatic >C═C) és 1147 cm.sup.−1 (≥C—O—CS≤;

EXAMPLE 7

Synthesis of BP2AM.SUP.Sar

a.) N-(bromoacetyl)sarcosine tert-butyl ester

(36) Bromoacetyl bromide (3.56 g, 17.6 mmol, 1.55 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (50 ml) and K.sub.3PO.sub.4 (6.41 g, 30.2 mmol, 2.5 equ.) was mixed in a flask of 250 ml under N.sub.2 atmosphere. Sarcosine tert-butyl ester (1.7 g, 11.7 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 30 mil) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×15 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×30 ml, 10 m/m %) and saturated NaCl solution (1×30 ml). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 2.01 g (65%).

(37) ##STR00013##

(38) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.6 (9H, s, CH.sub.3) 2.8 (3H, s, CH.sub.3), 4.01 (2H, s, CH.sub.2), 4.4 (2H, s, CH.sub.2)

b). Synthesis of BP2AM.SUP.Sar

(39) The N-(bromoacetyl)sarcosine tert-butyl ester obtained as described above (0.28 g, 1.05 mmol, 2.5 equivalent) was dissolved in dry acetonitrile (20 ml) and added dropwise to the acetonitrile solution of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained yellowish oil was dissolved in CH.sub.2Cl.sub.2 (10 ml), then trifluoroacetic acid is added to it (0.25 ml, 6 equivalent) and the reaction mixture was refluxed for 24 hours. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.12 g (57%). .sup.1H NMR [360 MHz, D.sub.2O] δ 7.91 (2H, t, aromatic), 7.10 (4H, d, aromatic) 4.96 (4H, s, CH.sub.2) 4.11 (8H, s, CH.sub.2) 3.67 (6H, s, CH.sub.3) 3.49 (4H, CH.sub.2).

(40) ##STR00014##

EXAMPLE 8

Synthesis of BP2AM.SUP.PypCOOH

a.) N-(bromoacetyl)piperidine-4-carboxylic acid tert-butyl ester

(41) Bromoacetyl bromide (3.56 g, 17.6 mmol, 1.55 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (50 ml) and K.sub.3PO.sub.4 (6.41 g, 30.2 mmol, 2.5 equivalent) was mixed in a flask of 250 ml under N.sub.2 atmosphere. Piperidine tert-butyl ester (2.2 g, 11.7 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 30 ml) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×15 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×30 ml, 10 m/m %) and saturated NaCl solution (1×30 ml). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 2.1 g (59%).

(42) ##STR00015##

(43) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.50 (9H, s, CH.sub.3) 2.50 (1H, s, CH), 4.01 (2H, s, (CH.sub.2), 3.5-1.6 (8H, m, CH.sub.2), 4.31 (2H, s, CH.sub.2)

b.) Synthesis of BP2AM.SUP.PypCOOH

(44) The N-(bromoacetyl)piperidine tert-butyl ester obtained as described above (0.32 g, 1.05 mmol, 2.5 equivalent) was dissolved in dry acetonitrile (20 ml) and added dropwise to the acetonitrile solution of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained yellowish oil was dissolved in CH.sub.2Cl.sub.2 (10 ml), then trifluoroacetic acid is added to it (0.25 ml, 6 equivalent) and the reaction mixture was refluxed for 24 hours. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.14 g (60%). 1H NMR [360 MHz, D.sub.2O] δ 7.91 (2H, t, aromatic), 7.33 (4H, d, aromatic) 4.10 (8H, s, CH.sub.2) 3.51-3.18 (8H, m, CH.sub.2) 3.40 (4H, CH.sub.2) 2.45 (2H, m, CH) 1.91-1.50 (8H, m, CH.sub.2)

(45) ##STR00016##

EXAMPLE 9

Synthesis of BP2AM.SUP.PypCOONHS

(46) The BP2AM.sup.PypCOOH (0.20 g, 0.35 mmol, 1.0 equivalent) obtained as described above was dissolved in dry DMF, then DCC (0.15 g, 0.70 mmol, 2 equ.) was added at room temperature and the reaction mixture was stirred at room temperature for 2 hours. Then NHS (N-Hydroxysuccinimide) (0.08 g, 0.70 mmol, 2 equ.) was added and the reaction mixture was stirred for additional 20 hours. When the reaction time was elapsed, the precipitate was filtered, and the filtrate was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.13 g (48%).

(47) .sup.1H NMR [360 MHz, D.sub.2O] δ 7.9 (2H, t, aromatic), 7.30 (4H, d, aromatic) 4.11 (8H, s, CH.sub.2) 3.61-3.17 (8H, m, CH.sub.2) 3.49 (4H, CH.sub.2) 2.55 (8H, s, CH.sub.2) 2.44 (2H, m, CH) 1.96-1.50 (8H, m, CH.sub.2).

(48) ##STR00017##

EXAMPLE 10

Synthesis of BP2AM.SUP.PypCH2OH

a.) N-bromoacetyl-4-hydroxymethyl piperidine

(49) Bromoacetyl bromide (3.56 g, 17.6 mmol, 1.55 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (50 ml) and K.sub.3PO.sub.4 (6.41 g, 30.2 mmol, 2.5 equ.) was mixed in a flask of 250 ml under N.sub.2 atmosphere. 4-hydroxymethyl piperidine (1.34 g, 11.7 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 30 ml) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×15 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×30 ml, 10 m/m %) and saturated NaCl solution (1×30 mil). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 1.85 g (67%).

(50) ##STR00018##

(51) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.61 (1H, m, CH) 1.71-1.30 (8H, m, CH.sub.2), 3.52 (2H, m, (CH.sub.2), 4.21 (2H, s, CH.sub.2).

b.) Synthesis of BP2AM.SUP.PypCH2OH

(52) The N-bromoacetyl-4-hydroymethyl piperidine obtained as described above (0.25 g, 1.05 mmol, 2.5 equivalent) was dissolved in dry acetonitrile and added dropwise to the acetonitrile solution (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.15 g (66%).

(53) .sup.1H NMR [360 MHz, D.sub.2O] δ 7.92 (2H, t, aromatic), 7.34 (4H, d, aromatic) 4.00 (8H, s, CH.sub.2) 3.70 (4H, d, CH.sub.2) 3.51-3.24 (8H, m, CH.sub.2) 3.39 (4H, CH.sub.2) 1.68 (2H, m, piperidin CH) 1.69-1.24 (8H, m, CH.sub.2)

(54) ##STR00019##

EXAMPLE 11

Synthesis of BP2AM.SUP.PypBn

a.) N-bromoacetyl-4-benzylpiperidine

(55) Bromoacetyl bromide (3.56 g, 17.6 mmol, 1.55 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (50 ml) and K.sub.3PO.sub.4 (6.41 g, 30.2 mmol, 2.5 equ.) was mixed in a flask of 250 ml under N.sub.2 atmosphere. 4-benzylpiperidine (2.05 g, 11.7 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 30 ml) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×15 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×30 ml, 10 m/m %) and saturated NaCl solution (1×30 ml). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 1.85 g (67%).

(56) ##STR00020##

(57) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.91 (1H, m, CH) 1.69-1.24 (8H, m, CH.sub.2) 2.66 (2H, m, CH.sub.2) 4.23 (2H, s, (CH.sub.2), 7.05-7.33 (5H, m, aromatic)

b.) Synthesis of BP2AM.SUP.PypBn

(58) The N-bromoacetyl-4-benzylpiperidine obtained as described above (0.31 g, 1.05 mmol, 2.5 equivalent) was dissolved in dry acetonitrile and added dropwise to the acetonitrile solution (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.19 g (66%).

(59) ##STR00021##

(60) .sup.1H NMR [360 MHz, D2O] δ 7.92 (2H, t, aromatic), 7.51 (4H, m, aromatic) 7.33-7.17 (6H, m, aromatic) 7.41 (4H, d, aromatic) 4.15 (8H, s, CH.sub.2) 3.55-3.11 (8H, m, CH.sub.2) 3.34 (4H, m, CH.sub.2) 2.66 (4H, d, CH.sub.2) 1.92 (2H, m, CH) 1.70-1.36 (8H, m, CH.sub.2)

EXAMPLE 12

Synthesis of BP2AMP.SUP.PypBnNO2

a.) N-bromoacetyl-4-(4′-nitrobenzyl)piperidine

(61) Bromoacetyl bromide (3.56 g, 17.6 mmol, 1.55 ml, 1.5 equivalent), dry CH.sub.2Cl.sub.2 (50 ml) and K.sub.3PO.sub.4 (6.41 g, 30.2 mmol, 2.5 equ.) was mixed in a flask of 250 ml under N.sub.2 atmosphere. 4-(4′-nitrobenzyl)piperidine (2.57 g, 11.7 mmol, 1.0 equivalent) was dissolved in dry CH.sub.2Cl.sub.2 (20 ml) and was added dropwise to dichloromethane solution of bromoacetyl bromide at 0° C. in 30 minutes, then the reaction mixture was stirred for additional 12 hours at room temperature under N.sub.2 atmosphere before the aqueous HCl solution (0.5 M, 30 ml) was added to the reaction mixture. After the addition of HCl solution, the reaction mixture was stirred for additional 5 minutes, then the two phases were separated using a separatory funnel. The aqueous phase was washed with CH.sub.2Cl.sub.2 (1×15 ml) and then the unified organic phases were washed with KHCO.sub.3 solution (2×30 ml, 10 m/m %) and saturated NaCl solution (1×30 ml). The organic phase was dried with MgSO.sub.4, then dichloromethane was evaporated at reduced pressure, and the crude product was stored at −20° C. until further use. Yield: 2.43 g (61%).

(62) ##STR00022##

(63) .sup.1H NMR [360 MHz, CDCl.sub.3] δ 1.90 (1H, m, CH) 1.62-1.33 (8H, m, CH.sub.2) 2.56 (2H, m, CH.sub.2) 4.21 (2H, s, (CH.sub.2), 7.40-8.3 (4H, m, aromatic)

b.) Synthesis of BP2AM.SUP.PypBnNO2

(64) The N-bromoacetyl-4-(4′-nitrobenzyl)piperidine obtained as described above (0.36 g, 1.05 mmol, 2.5 equivalent)) was dissolved in dry acetonitrile and added dropwise to the acetonitrile solution (30 ml) of 2,11-diaza[3.3](2,6)-pyridinophane (0.10 g, 0.42 mmol, 1 equivalent) and K.sub.2CO.sub.3 (0.17 g, 1.26 mmol, 3 equivalent) at room temperature within 30 minutes. Then the reaction mixture was refluxed in N.sub.2 atmosphere for 24 hours. After 24 hours, K.sub.2CO.sub.3 was filtered from the hot solution, and acetonitrile was evaporated at reduced pressure. The obtained crude product was purified with HPLC (Luna 10u-Prep C18(2) 100A (250×21.20 mm; 10 μm) column), ACN:H.sub.2O/TFA was applied as eluent [ACN: acetonitrile; TFA: trifluoroacetic acid]. TFA was contained only in water in 0.005 M concentration. Yield: 0.22 g (70%).

(65) ##STR00023##

(66) .sup.1H NMR [360 MHz, D2O] δ 8.41 (4H, m, aromatic) 7.95 (2H, t, aromatic), 7.75 (4H, m, aromatic) 7.32 (4H, d, aromatic) 3.84 (8H, s, CH.sub.2) 3.49-3.19 (8H, m, CH.sub.2) 3.49 (4H, CH.sub.2) 2.44 (4H, d, CH.sub.2) 1.98 (2H, m, CH) 1.33-1.24 (8H, m, CH.sub.2)

c.) Synthesis of BP2A.SUP.PypBnNH

(67) The above obtained BP2A.sup.PypBnNO2 (0.4 g, 0.26 mmol) was dissolved in dry methanol, 0.04 g Pd-carbon catalyst was added, and then the mixture was reduced under 1 bar hydrogen pressure at room temperature for 2 hours. The catalyst was removed by filtration, the filtrate was evaporated at reduced pressure. Yield: 0.15 g (83%). .sup.1H NMR [360 MHz, D.sub.2O] δ 8.41 (4H, m, aromatic) 7.95 (2H, t, aromatic), 7.75 (4H, m, aromatic) 7.32 (4H, d, aromatic) 3.84 (8H, s, CH.sub.2) 3.49-3.19 (8H, m, CH.sub.2) 3.49 (4H, CH.sub.2) 2.44 (4H, d, CH.sub.2) 1.98 (2H, m, CH) 1.33-1.24 (8H, m, CH.sub.2)

(68) ##STR00024##

EXAMPLE 13

Efficacy Data

(69) During the physico-chemical studies of BP2AM.sup.Pyp and BP2AM.sup.Pro compounds prepared according to Example 1 and 2, their protonation constants, as well as equilibrium behaviour and kinetic inertness of their Mn(II) complexes was studied in detail, and the characteristic relaxivity values of the complexes were determined in the presence and absence of HSA (Human Serum Albumin), at 25 and 37° C. and physiological pH. All studies were performed in the presence of 0.15 M NaCl, the same concentration as that of the electrolyte under physiological conditions.

(70) The results of equilibrium study are summarized in Table 1, in addition to the protonation constants, total basicity of ligands and stability constants of their Mn(II) complexes, the pMn value calculated for complexes are also represented in the table.

(71) TABLE-US-00001 TABLE 1 Protonation constants and total basicity of the studied ligands, stability constants of their Mn(II) complexes and calculated pMn values (25° C., 0.15M NaCl). logK.sub.1 logK.sub.2 logK.sub.3 logK.sub.4 ΣlogK.sub.i.sup.H logK.sub.MnL pMn BP2AM.sup.Pyp 9.57(2) 2.99(2) — — 12.56 13.58(1) 8.19 BP2AM.sup.Pro 8.83(1) 3.89(2) 3.05(2) 2.39(2) 18.16 11.47(5) 7.51 pMn values were calculated by using the equilibrium constants at pH = 7.4 and cMn = cL = 10.sup.−5 M
Based on the pMn values presented in Table 1 (calculated using the equilibrium constants at pH=7.4 and cMn=cL=10.sup.−5 M), it can be concluded that the studied Mn(II) complexes are formed in 100% at physiological pH, which is an essential aspect of the practical use.

(72) An important parameter of using Mn(II) containing contrast agents in vivo is the low kinetic reactivity of the complex. The kinetic reactivity is generally tested with metal ion exchange reactions, where the replacing metal ion is Zn(II) or Cu(II) in most of the cases. The application of Cu(II) is advantageous for more reasons, in one hand the complexes with ligands are of great thermodynamic stability, so relatively small excess of Cu(II) ion leads to complete replacement, on the other hand molar absorbance values of Cu(II) complexes both in UV and visible range are sufficiently high to enable spectrophotometric method for examinations even at low concentrations. Moreover, the endogenic character of the Cu(II) ion provides additional information on in vivo processes. Replacement reactions were executed with at least 10-fold excess Cu(II) ion concentration to ensure pseudo-first order conditions.

(73) Dissociation reactions of Mn(II) complexes may take place in several pathways as represented below.

(74) ##STR00025##

(75) The k.sub.0, k.sub.H, k.sub.H.sup.H, k.sub.Cu and k.sub.Cu.sup.H, rate constants indicate the spontaneous, proton associated, metal assisted and proton-metal assisted (when the replacing metal ion attacks the protonated complex) reaction pathways of the complex. The K.sub.MnHL, K.sub.MnH.sub.2.sub.L and K.sub.MnLCu are stability constants of the protonated and binuclear intermediate complexes.

(76) In case of metal complexes formed with macrocyclic ligands the above detailed mechanism involves only proton associated dissociation pathways (in some instances spontaneous dissociation may have some role), since the formation of binuclear complexes are inhibited (denticity of rigid ligands does not exceed the coordination number of the metal ion, Mn.sup.2+). Due to this reason, replacement reactions were executed in 2.0-5.0 pH range with only 10-fold Cu(II) replacement metal ion excess.

(77) In general the k.sub.obs pseudo-first order rate constants obtained in each reaction are given with the following equation, where the stability constants of each reaction pathway and that of the forming intermediate are also considered:

(78) $\begin{array}{cc}{k}_{\mathrm{obs}}=\frac{k_0+k_1\left[H^{+}\right]+{k_2\left[H^{+}\right]}^2+k_3\left[{\mathrm{Cu}}^{2+}\right]+k_4\left[{\mathrm{Cu}}^{2+}\right]\left[H^{+}\right]}{1+K_{\mathrm{MnHL}}\left[H^{+}\right]+{K_{{\mathrm{MnH}}_{2}L}\left[H^{+}\right]}^2+K_{\mathrm{MnLCu}}\left[{\mathrm{Cu}}^{2+}\right]},& \left(1\right)\end{array}$
whereas K.sub.MnHL=[Mn(HL)]/[Mn(L)][H.sup.+], K.sub.MnH.sub.2.sub.L=[Mn(H.sub.2L)]/[Mn(HL)][H.sup.+], K.sub.MnLCu=[Mn(L)M]/[Mn(L)][M], k.sub.1=k.sub.H.Math.K.sub.MnHL, k.sub.2=k.sub.H.sup.H.Math.K.sub.MnHL.Math.K.sub.MnH.sub.2.sub.L, k.sub.3=k.sub.Cu.Math.K.sub.MnLCu k.sub.4=k.sub.Cu.sup.H.Math.K.sub.MnHL

(79) Results of the kinetic study showed that in the dissociation of [Mn(BP2AM.sup.Pyp)].sup.2+- and [Mn(BP2AM.sup.Pro)] complexes, the proton associated dissociation (characterized with k.sub.1) plays an important role. Using these rate constants the half-life (t.sub.1/2) of [Mn(BP2AM.sup.Pyp)].sup.2+- and [Mn(BP2AM.sup.Pro)] complexes dissociation may be calculated at physiologic pH, being 1.37×10.sup.5 and 1.1×10.sup.4 hours, respectively.

(80) In order to estimate the quantity of complex decomposing in the body, it is useful to handle elimination and complex dissociation as parallel, primary reaction characterized by the (2) equation set for Gd.sup.3+ complexes [F. K. Kálmán and G. Tircsó, Inorg. Chem., 2012, 51, 10065]:

(81) $\begin{array}{cc}{\left[\mathrm{GdL}\right]}_d={\frac{k_d}{k_d+k_{\mathrm{ex}}}\left[\mathrm{GdL}\right]}_0\left(1-e^{-\left(k_d+k_{\mathrm{ex}}\right)t}\right)& \left(2\right)\end{array}$
The equation indicates that dissociation degree of the complex depends on the ration of rate constants. For the (renal) elimination of contrast agent 1.6 hour half life can be given in general, characterized by a k.sub.ex=0.433 h.sup.−1 rate constant. Using the k.sub.d values of Mn(II) complexes and the k.sub.ex values characteristic for elimination, one can calculate the percentile ratio of injected complex dissociated in vivo until complete elimination (12-24 hours). Calculation verified, that less than 0.8% of the complexes would dissociate before the elimination of the complex. Considering the endogenic characteristic of Mn(II) complexes and its negligible amount it cannot cause significant burden for MRI tested patients. Considering the new results, during the in vivo dissociation (37° C.) of [Gd(DTPA)].sup.2− complex (Magnevist) applied in practice, 2.2% Gd(III) ion releases being 4.4-fold of the value calculated on the basis of experiments at 25° C. (0.5%). [Sarka L. et al, Chem. Eur. J., 2000, 6, 719]. Using this finding, the quantity of in vivo releasing Mn(II) for the presented Mn(II) complexes is approximately 3%. This value is better than some of those applied for Gd containing contrast agents in practice [Baranyai Z et al, Chem. Eur. J., 2015, 21, 4789]

(82) In addition to appropriately low kinetic reactivity, complexes shall also have suitable relaxivity for the purpose of practical use (relaxivity (mM.sup.−1s.sup.−1): relaxation rate increase of 1 mM solution of the paramagnetic substance compared to the measured value under diamagnetic conditions [Tóth É., et. al., The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, Chichester: John Wiley & Sons, 2001.]). Higher complex relaxivity results in higher contrast increasing effect, meaning that the same image quality is obtained by introducing less amount of complex with higher relaxivity. The relaxivity value of both complexes were determined at pH=7.4 and 25 and 37° C. in the presence and absence of HSA (Human Serum Albumin, c=0.7 mM) to better stimulate conditions of the in vivo application. Relaxivity values of the complexes are presented in Table 2. Comparing the data in Table 2 with the relaxivity values of DOTAREM ([Gd(DOTA)].sup.− complex, r.sub.1=3.83 mM.sup.−1s.sup.−1) and MAGNEVIST ([Gd(DTPA)].sup.2− complex, r.sub.1=4.02 mM.sup.−1s.sup.−1) [Powell, D. H., Ni Dhubhghaill, O. M., Pubanz, D. et al. (1996) J. Am. Chem. Soc., 118, 9333-9346] applied in practice under the same conditions, the Mn(II) complexes presented herein obviously have higher contrast enhancing effect.

(83) TABLE-US-00002 TABLE 2 Relaxivity values (20 MHz) of the Mn.sup.2+ complexes prepared and studied (pH = 7.4). Complex T (° C.) r.sub.1 (mM.sup.−1s.sup.−1) r.sub.1 (mM.sup.−1s.sup.−1) HSA [Mn(BP2AM.sup.Pyp)].sup.2+ 25 4.68 7.94 37 3.74 6.32 [Mn(BP2AM.sup.Pro)] 25 5.67 6.94 37 4.37 5.29