CYCLEN BASED COMPOUNDS, COORDINATION COMPOUNDS, PEPTIDES, PHARMACEUTICAL PREPARATION, AND USE THEREOF

Abstract

Cyclen based compounds of general formula (I) are disclosed. X is nitrogen and Y, Z are —CH—, or X, Z are —CH— and Y is nitrogen, or X, Y are —CH— and Z is nitrogen. R.sup.1 is independently selected from H; COOH; benzyloxycarbonyl; fluorenylmethyloxycarbonyl; tert-butoxycarbonyl; methylcarbonyl; trifluoromethylcarbonyl; benzyl; triphenylmethyl; tosyl; mesyl; benzyloxymethyl; phenylsulfonyl; ethoxycarbonyl; 2,2,2-trichloroethyloxycarbonyl; methoxycarbonyl; methoxymethyloxycarbonyl; R.sup.2 is selected from H; methylcarbonyl; tert-butyldimethylsilyl; (C1-C4)alkyl; R.sub.3 is independently selected from H; (C1-C6)alkyl.

Claims

1: A cyclen based compound of general formula (I), ##STR00045## wherein X is nitrogen and Y, Z are —CH—, or X, Z are —CH— and Y is nitrogen, or X, Y are —CH— and Z is nitrogen; R.sup.1 is independently selected from H; COOH; benzyloxycarbonyl; fluorenylmethyloxycarbonyl; tertbutoxycarbonyl; methylcarbonyl; trifluoromethylcarbonyl; benzyl; triphenylmethyl; tosyl; mesyl; benzyloxymethyl; phenylsulfonyl; ethoxycarbonyl; 2,2,2-trichloroethyloxycarbonyl; methoxycarbonyl; methoxymethyloxycarbonyl; R.sup.2 is selected from H; methylcarbonyl; tert-butyldimethylsilyl; (C1-C4)alkyl, which can be linear or branched, and which can optionally be substituted with CH.sub.3O—, CH.sub.3S—; oxacyclohexyl; allyl; tert-butyldiphenylsilyl; tertbutylcarbonyl; phenylcarbonyl; nitrobenzyl; benzyloxymethyl, which can optionally be substituted with CH.sub.3O—, —NO.sub.2; fluorenylmethyloxycarbonyl; trichlorocarbonyl; trifluorocarbonyl; benzyl; tosyl; mesyl; phenylsulfonyl; allylsulphonyl; ethoxycarbonyl; 2,2,2-trichloroethyloxycarbonyl; methoxycarbonyl; methoxymethyloxycarbonyl; R.sup.3 is independently selected from H; (C1-C6)alkyl, which can be linear or branched, and which can optionally be substituted with —CH.sub.3, —Cl, —F, —CN, tosyl, triisopropylsilyl, CH.sub.3O—, CH.sub.3S—; (C5-C6)cycloalkyl, which can optionally be substituted with —CH.sub.3, —Cl, —F, —CN; (C6-C10)aryl, which can optionally be substituted with —CH.sub.3, —Cl, —F, —CN; allyl, propargyl; fluorenylmethyl; benzoylmethyl; phenyloxymethyl; oxacyclopentyl; 2-oxo-1,2-diphenylethyl; with the proviso that where R.sup.1 is bound to nitrogen, then R.sup.1 is not COOH; with the proviso that where R.sup.1 is bound to —CH—, then R.sup.1 is independently H or COOH; with the proviso that one R.sup.1 is COOH; and with the proviso that one —CH—R.sup.1 group is —CH.sub.2—.

2: The cyclen based compound of general formula (I) according to claim 1, wherein Y is nitrogen, X—R.sup.1 is —CH.sub.2—, and Z—R.sup.1 is —CH(COOH)—.

3: The cyclen based compound of general formula (I) according to claim 1, wherein Y is nitrogen, X—R.sup.1 is —CH(COOH)—, and Z—R.sup.1 is —CH.sub.2—.

4: The cyclen based compound of general formula (I) according to claim 1, wherein X is nitrogen, Y—R.sup.1 is —CH(COOH)—, and Z—R.sup.1 is —CH.sub.2—.

5: The cyclen based compound of general formula (I) according to claim 1, wherein X—R.sup.1 is —CH.sub.2—, Y—R.sup.1 is —CH(COOH)—, and Z is nitrogen.

6: The cyclen based compound of general formula (I) according to claim 1, wherein R.sup.1 is selected from H, COOH, benzyloxy carbonyl and fluorenylmethyloxycarbonyl.

7: The cyclen based compound of general formula (I) according to claim 1, wherein R.sup.2 is selected from H, methylcarbonyl and tert-butyldimethylsilyl.

8: The cyclen based compound of general formula (I) according to claim 1, wherein R.sup.3 is selected from H, methyl and tert-butyl.

9: The cyclen based compound of general formula (1) according to claim 1, wherein the compound of general formula (I) is selected from the group consisting of: (2S,3S,4S)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3R,4R)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; 2,2′,2″-(10-((2S,3S,4S)-1-(2-(9H-fluoren-9-yl)acetoxy)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; (2S,3S,4S)-4-acetoxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3R,4R)-3-acetoxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-3-(4,740-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid: 2,2′,2″-(10-((2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-2-carboxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; (2S,3S,4S)-4-acetoxy-3-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3R,4R)-3-acetoxy-4-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-3-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3S,4S)-4-((tert-butyldimethylsilyl)oxy)-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3R,4R)-3-((tert-butyldimethylsilyl)oxy)-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((tert-butyldimethylsilyl)oxy)-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2R,3S,4S)-4-hydroxy-3-(4,7,10-tis(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2R,3R,4R)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2R,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; (2R,3S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid; 2,2′,2″-(10-((2R,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; 2,2′,2″-(10-((3R,4S,5R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-5-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid.

10: A coordination compound of general formula (1a), ##STR00046## wherein X is nitrogen and Y, Z are —CH—, or X, Z are —CH— and Y is nitrogen, or X, Y are —CH— and Z is nitrogen; R.sup.1 is independently selected from H; COOH; benzyloxycarbonyl; fluorenylmethyloxycarbonyl; tert-butoxycarbonyl; methylcarbonyl; trifluoromethylcarbonyl; benzyl; triphenylmethyl; tosyl; mesyl; benzyloxymethyl; phenylsulfonyl; ethoxycarbonyl; 2,2,2-trichloroethyloxycarbonyl; methoxycarbonyl; methoxymethyloxycarbonyl; R.sup.2 is selected from H; methylcarbonyl; tert-butyldimethylsilyl; (C1-C4)alkyl, which can be linear or branched, and which can optionally be substituted with CH.sub.3O—, CH.sub.3S—; oxacyclohexyl; allyl; tert-butyldiphenylsilyl; tert-butylcarbonyl; phenylcarbonyl; nitrobenzyl; benzyloxymethyl, which can optionally be substituted with CH.sub.3O—, —NO.sub.2; fluorenylmethyloxycarbonyl; trichlorocarbonyl; trifluorocarbonyl; benzyl; tosyl; mesyl; phenylsulfonyl; allylsulphonyl; ethoxycarbonyl; 2,2,2-trichloroethyloxycarbonyl; methoxycarbonyl; methoxymethyloxycarbonyl; M.sup.3+ is a metal cation selected from die group consisting of In.sup.3+, Ga.sup.3+, trivalent cations of rare earth elements, selected from lanthanide(III) cations, Y(III) and Sc(III), preferably the metal cation is selected from Gd.sup.3+ and Lu.sup.3+; with the proviso that where R.sup.1 is bound to nitrogen, then R.sup.1 is not COOH; with the proviso that where R.sup.1 is bound to —CH—, then R.sup.1 is independently H or COOH; with the proviso that one R.sup.1 is COOH; and with the proviso that one —CH—R.sup.1 group is —CH.sub.2—.

11: A peptide, characterized in that it has a chain length of from 2 to 20 amino acids, wherein at least one amino acid is replaced by the coordination compound according to claim 10.

12: A peptide, characterized in that it has a chain length of from 2 to 20 amino acids, wherein at least one amino acid is replaced by the coordination compound according to claim 10, characterized in that it contains at least two coordination compounds according to claim 10, wherein at least two of the coordination compounds contain different metal ions.

13: A pharmaceutical preparation, characterized in that it contains at least one coordination compound according to claim 10, and a pharmaceutically acceptable auxiliary substance.

14: A method of treatment, comprising the step of proving a medicine comprising the coordination compound according to claim 10 to a subject in need thereof.

15: A method of radiotherapy, comprising the step of administering the coordination compound according to claim 10 as MRI contrast agent and/or PET contrast agent and/or MRI-SPECT contrast agent and/or combined MRI contrast agent and radiopharmaceutical agent and/or combined PET contrast agent and radiopharmaceutical agent, to a subject in need thereof.

Description

EXAMPLES

[0064] The numerical values of chemical shift in NMR spectra are given in ppm. Notation used in the NMR spectra: s (singlet), d (dublet), t (triplet), m (multiplet), bs (broad singlet). The reference was set to the following values: .sup.1H: 7.26 ppm (CDCl.sub.3); 1.94 (CD.sub.3CN); 2.5 ppm (DMSO-d6). .sup.13C: 77.16 ppm (CDCl.sub.3); 118.26 ppm (CD.sub.3CN); 39.52 ppm (DMSO-d6).

List of Abbreviations

[0065] Bn (benzyl); Cbz (benzyloxycarbonyl); DCM (dichloromethane); DIPEA (N,N-diisopropylethylamine); DMF (dimethylformamide); ESI (electrospray ionization); FA (formic acid); Fmoc (fluorenylmethyloxycarbonyl); HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate); HPLC (high performance liquid chromatography); HRMS (high resolution mass spectrometry); ICP-AES (inductively coupled plasma-atomic emission spectroscopy); LC-MS (liquid chromatography-mass spectrometry); MOPS (3-morpholinopropane-1-sulfonic acid); NMP (N-Methyl-2-pyrrolidone); TFA (trifluoroacetic acid); UV (ultraviolet).

I. Synthesis of Compounds

Example 1

Preparation of dibenzyl (1R,2S,5S)-6-oxa-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (1a) and dibenzyl (1S,2S,5R)-6-oxa-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (1b)

[0066] Dibenzyl (S)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylate (6.80 g, 20.16 mmol) was dissolved in chloroform (200 mL) at room temperature and m-chloroperoxybenzoic acid (77%, 6.78 g, 30.26 mmol) was added. The reaction mixture was heated up to 85° C. on oil bath for 24 h. The volume of the solvent was reduced on rotary evaporator. DCM (70 mL) was added to the precipitate and reaction mixture was put into freezer. After 24 h the white precipitate was removed by filtration, the filtrate was evaporated and the residue was purified by FLASH chromatography on 120 g silica gel column using petroleum ether:ethyl acetate gradient from (100:0) to (60:40). The chromatography provided separation of the two isomers. Fractions containing pure compounds were evaporated, giving 3.3 g of 1a as transparent oil (9.32 mmol, 46% yield) and 1.5 g of 1b as white solid (4.23 mmol, 21% yield).

##STR00003##

[0067] .sup.1H NMR (CDCl.sub.3, 25° C., 400 MHz): δ.sub.H 3.56 (ddd, 1H); δ.sub.H 3.68 (ddd, 1H); δ.sub.H 3.77 (d, 1H); 3.91-3.99 (m, 1H); 4.74 (d, 1H); 5.05-5.28 (m, 4H); 7.23-7.40 (arom., in, 10H).

[0068] HRMS (ESI) m/z: [(M+Na).sup.+] (C.sub.20H.sub.19O.sub.5NNa) calculated: 376.11554. found: 376.11523.

##STR00004##

[0069] .sup.1H NMR (CDCl.sub.3, 25° C., 400 MHz): δ.sub.H 3.61 (ddd, 1H); δ.sub.H 3.77-3.81 (m, 1H); δ.sub.H 3.79 (d, 1H); 3.88-3.98 (m, 1H); 4.49 (dd, 1H); 4.94-5.31 (m, 4H); 7.21-7.39 (arom., m, 10H).

[0070] HRMS (ESI) m/z: [(M+Na).sup.+] (C.sub.20H.sub.19O.sub.5NNa) calculated: 376.11554. found: 376.11514.

Preparation of dibenzyl (2S,3S,4S)-3-(1,4,7,10-tetraazacyclododecan-1-yl)-4-hydroxypyrrolidine-1,2-dicarboxylate (1c) and dibenzyl (2S,3S,4R)-4-(1,4,7,10-tetraazacyclododecan-1-yl)-3-hydroxypyrrolidine-1,2-dicarboxylate (1d)

[0071] ##STR00005##

[0072] Dibenzyl (1R,2S,5S)-6-oxa-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (1a) (2 g, 5.66 mmol) and 1,4,7,10-tetraazacyclododecane (3.9 g, 22.64 mmol) in 117 mL of t-BuOH were placed into a 250 mL round bottom flask and the mixture was stirred for 24 hours and heated under reflux. After cooling to room temperature the reaction mixture was neutralized with TFA (3 mL, 39.20 mmol). Reaction mixture was concentrated on rotary evaporator. Resulting oil was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Fractions containing mixture of the two products were pooled, evaporated and dried in high vacuum. The residue was dissolved in water (5 ml) and lyophilized giving 2.3 g of the product as a brown solid in form of TFA salt (30.40 mmol, 54% yield relative to 1a). Based on .sup.1H NMR the product contained mixture of isomers (2S,3S,4S) (1c)/(2S,3S,4R) (1d) in ratio 9/1.

[0073] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.28H.sub.40O.sub.5N.sub.5) calculated: 526.30240. found: 526.30100.

[0074] Elem. analysis: M.2.1TFA.0.5H.sub.2O, calculated: C; (50.0), H; (5.5), N; (9.0), F; (15.5). found: C; (49.5), H; (5.1), N; (8.8), F; (16.3).

Preparation of dibenzyl (2S,3S,4S)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (1e) and dibenzyl (2S,3S,4R)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxyethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (1f)

[0075] ##STR00006##

[0076] A mixture of isomers 1c and 1d in ratio 9/1 (1 g, 1.32 mmol), t-Butyl bromoacetate (799 uL, 5.41 mmol), anhydrous cesium carbonate (2.58 g, 7.92 mmol) and acetonitrile (50 mL) were placed into a 100 mL round bottom flask and the mixture was stirred for 1 hour at room temperature. The solids were filtered off and the filtrate was concentrated on rotary evaporator. Resulting oil was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Fractions containing mixture of the two products were pooled, evaporated and dried in high vacuum giving 1.19 g of yellow glass-like solid. The product contained mixture of isomers (2S,3S,4S) (1e)/(2S,3S,4R) (1f) in ratio 9/1 (based on LC-MS) in the form of salt with TFA (83% yield, assuming composition M.2TFA).

[0077] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.46H.sub.70O.sub.11N.sub.5) calculated: 868.50663. found: 868.50631.

Preparation of (2S,3S,4S)-4-hydroxy-3-(4,7,1-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (1g) and (2S,3R,4R)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (1h)

[0078] A mixture of isomers 1e and 1f in ratio 9/1 (500 mg, 0.46 mmol) in 100 mL round bottom flask with septum

##STR00007##

was dissolved in MeOH (32 mL). 10% Pd/C (41 mg, 0.038 mmol) was added to the reaction mixture. The mixture was stirred for 30 minutes under argon atmosphere at room temperature after which the reaction mixture was filtered through celite pad. The filtrate was concentrated on rotary evaporator and dried in high vacuum. The residue was dissolved in water (2 mL) and lyophilized to give 390 mg of white solid. The product contained mixture of isomers (2S,3S,4S) (1g)/(2S,3R,4R) (1h) in ratio 9/1 in the form of salt with TFA (97% yield, assuming composition M.2TFA).

[0079] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.31H.sub.58O.sub.9N.sub.5) calculated: 644.42290. found: 644.42270.

Example 2: Preparation of (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (2)

[0080] ##STR00008##

[0081] A mixture of isomers 1g and 1h in ratio 9/1 (390 mg, 0.45 mmol) was dissolved in mixture of acetonitrile (23.4 mL) and borate buffer (22 mL, 0.2 M, pH=9). Fmoc chloride (0.114 g, 0.44 mmol) was added to reaction mixture and reaction mixture was stirred for 30 minutes at room temperature. The reaction mixture was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Chromatography provided separation of (2S,3S,4S) and (2S,3S,4R). Fractions containing pure isomer (2S,3S,4S) were pooled, evaporated and dried in high vacuum giving 286 mg of product as white crystalline solid in the form of salt with TFA (58% yield, assuming composition M.2TFA).

[0082] HRMS (ESI) m/z: [(M−H).sup.−] (C.sub.46H.sub.66O.sub.11N.sub.5) calculated: 864.47643. found: 864.47490.

Example 3: Preparation of 2,2′,2″-(10-((2S,3S,4S)-1-(2-(9H-fluoren-9-yl)acetoxy)-2-carboxy-4-hydroxypyrrolidin-3-yl-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (3)

[0083] ##STR00009##

[0084] Compound 2 prepared in Example 2 (286 mg, 0.26 mmol) was dissolved in TFA (5 mL, 65.34 mmol). The mixture was stirred for 30 minutes at 70° C. after which the reaction mixture was concentrated on rotary evaporator and dried in high vacuum. The residue was dissolved in water (2 ml) and lyophilized to give 200 mg of brown solid in the form of salt with TFA (95% yield, assuming composition M.2TFA).

[0085] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.34H.sub.44O.sub.11N.sub.5) calculated: 698.30318. found: 698.30337.

[0086] .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ.sub.H 2.67-3.29 (cycle+proline arm, m, 17H); 3.42-3.93 (proline arm+acetates, m, 8H); 4.07-4.33 (proline arm+fmoc, m, 5H); 7.29-7.37 (arom., m, 2H); 7.40-7.45 (arom., m, 2H); 7.62-7.67 (arom., m, 2H); 7.87-7.92 (arom., m, 2H); .sup.13C{.sup.1H} NMR (DMSO-d.sub.6, 125 MHz): 45.68-54.03 (cycle+carboxylates, m); 46.82, 46.89 (fmoc, s); 50.91, 51.4 (proline arm, s); 57.01, 56.84 (proline arm, s); 66.89, 67.33 (proline arm, s); 69.13, 69.24 (proline arm, s); 70.20, 71.43 (proline arm, s); 120.33-120.44 (arom., m); 125.30, 125.38, 125.45, 125.54 (arom., s); 127.35-127.43 (arom., m); 127.97 (arom., s); 140.86, 140.92, 140.99, 141.02 (arom., s); 143.85, 143.88, 143.95, 143.99 (arom., s); 153.84, 154.05 (N—COO); 170.25, 172.11 (CH2-COO); 173.48, 173.73 (CO).

Example 4: Preparation of Gd(III) Complex of 2,2′,2″-(10-((2S,3S,4S)-1-(2-(9H-fluoren-9-yl)acetoxy)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Gd-3)

[0087] ##STR00010##

[0088] Fmoc Compound 3 prepared in Example 3 (200 mg, 0.22 mmol) was dissolved in a mixture of methanol (20 mL) and water (20 mL). Aqueous solution of GdCl.sub.3 OH 25 (2.167 mL, 0.1 M) and aqueous solution of N-methyl morpholine (15.166 mL, 0.1 M) was added to the reaction mixture. The reaction mixture was stirred for 1 hour at room temperature. Then, reaction mixture was concentrated on rotary evaporator. The residue was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient). Fractions containing pure product were pooled, evaporated and dried in high vacuum. The residue was dissolved in water (2 ml) and lyophilized giving 170 mg of the product as a white solid (87% yield relative to 3).

[0089] HRMS (ESI) m/z: [(M−H).sup.−] (C.sub.34H.sub.39O.sub.11N.sub.5Gd) calculated: 851.18926. found: 851.18776.

[0090] Elem. analysis: M.2H.sub.2O, calculated: C; (46.0), H; (5.0), N; (7.9), Gd; (17.7). found: C; (46.5), H; (5.5), N; (8.2), Gd; (14.8).

Example 5: Preparation of Lu(III) Complex of 2,2′,2″-(10-((2S,3S,4S)-1-(2-(9H-fluoren-9-yl)acetoxy)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Lu-3)

[0091] ##STR00011##

[0092] According to procedure in Example 4, reaction of compound 3 (83 mg, 0.083 mmol), aqueous solution of LuCl.sub.3 (838 uL, 0.1 M) and aqueous solution of N—OH methyl morpholine (5.867 mL, 0.1 M) analogously provided 31 mg of the product as a white solid (43% yield relative to 3).

[0093] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.34H.sub.41O.sub.11N.sub.5Lu) calculated: 870.22048. found: 870.21970.

Example 6: Preparation of Gd(III) Complex of 2,2′,2″-(10-((2S,3S,4S)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Gd-6)

[0094] ##STR00012##

[0095] Compound Gd-3 (7 mg, 0.008 mmol) was dissolved in water (0.5 mL). Aqueous solution of LiOH (0.5 mL, 1 M) was added. The reaction mixture was stirred for 1 hour at room temperature. Then, reaction mixture was concentrated on rotary evaporator. The residue was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% formic acid in the mobile phase). Fractions were pooled, evaporated and dried in high vacuum giving 3 mg of the product as a white solid. The product was in the form of salt with formic acid (54% yield, assuming composition M.1FA).

[0096] LCMS (ESI) m/z: [(M+H).sup.+] (C.sub.19H.sub.31O.sub.9N.sub.5) calculated: 631.1. found: 631.4.

Example 7: Preparation of dibenzyl (2S,3S,4S)-4-acetoxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (7a) and dibenzyl (2S,3S,4R)-3-acetoxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (7b)

[0097] ##STR00013##

[0098] A mixture of isomers 1e and 1f in ratio 9/1 prepared in Example 1 (100 mg, 0.091 mmol), acetic anhydride (15 uL, 0.015 mmol), triethyl amine (64 uL, 0.046 mmol) and dimethyl aminopyridine (0.2 mg, 0.0002 mmol) was dissolved in acetonitrile (50 mL). The mixture was stirred overnight at room temperature after which the reaction mixture was concentrated on rotary evaporator and dried in high vacuum. Resulting oil was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Fractions containing mixture of the two products were pooled, evaporated and dried in high vacuum giving 103 mg of colorless solid. The product contained mixture of isomers (2S,3S,4S) (7a)/(2S,3S,4R) (7b) in ratio 9/1 in the form of salt with TFA (99% yield, assuming composition M.2TFA).

[0099] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.48H.sub.72O.sub.12N.sub.5) calculated: 910.51720. found: 910.51711.

Preparation of (2S,3S,4S)-4-acetoxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (7c) and (2S,3R,4R)-3-acetoxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (7d)

[0100] ##STR00014##

[0101] The procedure was analogous to preparation of compounds 1g and 1h in Example 1. Reaction of the mixture of isomers 7a and 7b in ratio 9/1 (93 mg, 0.082 mmol), 10% Pd/C (8 mg, 0.008 mmol) in MeOH (32 mL) gave analogously 65.5 mg of the product as a white solid. The product contained mixture of isomers (2S,3S,4S) (7a)/(2S,3R,4R) (7b) in ratio 9/1 in the form of salt with TFA (88% yield, assuming composition M.2TFA).

[0102] LCMS (ESI) m/z: [(M+H).sup.+] (C.sub.33H.sub.60O.sub.10N.sub.5) calculated: 686.43. found: 686.6.

Example 8: Preparation of (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (8)

[0103] ##STR00015##

[0104] According to procedure in Example 2, reaction of a mixture of isomers 7a and 7b in ratio 9/1 (60 mg), Fmoc chloride (18 mg, 0.07 mmol) in acetonitrile (3.6 mL) and borate buffer (3.4, 0.2 M, pH=9) was carried out. Chromatography analogously to Example 2 provided separation of the isomers (2S,3S,4S) and (2S,3S,4R), yielding 41 mg of pure isomer (2S,3S,4S) as a colorless solid in the form of salt with TFA (55% yield, assuming composition M.2TFA).

[0105] LCMS (ESI) m/z: [(M+H).sup.+] (C.sub.48H.sub.70O.sub.12N.sub.5) calculated: 908.5. found: 908.5.

Example 9: Preparation of 2,2′,2″-(10-((2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-2-carboxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9)

[0106] ##STR00016##

[0107] According to procedure in Example 3, reaction of starting compound 8 prepared in Example 8 (41 mg) in TFA (2 mL, 26.14 mmol) gave analogously 35 mg of the product as a brownish solid in the form of salt with TFA (100% yield, assuming composition M.2TFA).

[0108] HRMS (EST) m/z: [(M+H).sup.+] (C.sub.36H.sub.46O.sub.12N.sub.5) calculated: 740.31375. found: 740.31301.

Example 10: Preparation of Gd(III) Complex of 2,2′,2″-(10-((2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-2-carboxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Gd-9)

[0109] ##STR00017##

[0110] According to procedure in Example 4, reaction of starting compound 9 prepared in Example 9 (25 mg, 0.026 mmol) in methanol (2.5 mL) and water (2.5 mL) with aqueous solution of GdCl.sub.3 (263 uL, 0.1 M) and aqueous solution of N-methyl morpholine (1.842 mL, 0.1 M) gave analogously 14 mg of the product as a white solid (57% yield relative to compound 9).

[0111] HRMS (ESI) m/z: [(M−H).sup.−] (C.sub.36H.sub.41O.sub.12N.sub.5Gd) calculated: 893.19982. found: 893.19873.

[0112] Elem. analysis: M.3H.sub.2O, calculated: C; (45.6), H; (5.1), N; (7.4), Gd; (16.6), found: C; (46.0), H; (5.4), N; (7.6), Gd; (13.7).

Example 11: Preparation of dibenzyl (2S,3S,4S)-4-hydroxy-3-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (11a) and dibenzyl (2S,3S,4R)-3-hydroxy-4-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate

[0113] ##STR00018##

[0114] A mixture of isomers 1c and 1d in ratio 9/1 (200 mg, 0.26 mmol), methyl bromoacetate (143 uL, 1.23 mmol), anhydrous cesium carbonate (516 mg, 1.59 mmol) and acetonitrile (10 mL) were placed into a 100 mL round bottom flask and the mixture was stirred for 2 h at room temperature. The solids were filtered off and the filtrate was concentrated on rotary evaporator. Resulting oil was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Fractions containing mixture of the two products were pooled, evaporated and dried in high vacuum giving 197.5 mg of a colourless solid. The product contained mixture of isomers (2S,3S,4S) (11a)/(2S,3S,4R) (11b) isomers in ratio 9/1 in the form of salt with TFA (77% yield, assuming composition M.2TFA).

[0115] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.37H.sub.52O.sub.11N.sub.5) calculated: 742.36578. found: 742.36566.

Preparation of dibenzyl (2S,3S,4S)-4-acetoxy-3-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (11c) and dibenzyl (2S,3S,4R)-3-acetoxy-4-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate

[0116] ##STR00019##

[0117] The reaction was carried out according to procedure in Example 7 for preparation of 7a and 7b. Analogously, a mixture of isomers 11a and 11b in ratio 9/1 prepared in Example 11 (158 mg, 0.16 mmol), acetic anhydride (31.5 uL, 0.034 mmol), triethyl amine (64 uL, 0.081 mmol) and dimethyl aminopyridine (0.3 mg, 0.0003 mmol) was dissolved in acetonitrile (7.563 mL) gave analogously 100 mg of the product as a colorless solid. The product contained mixture of isomers (2S,3S,4S) (11c)/(2S,3S,4R) (11d) in ratio 9/1 in the form of salt with TFA (81% yield, assuming composition M.2TFA).

[0118] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.39H.sub.54O.sub.12N.sub.5) calculated: 784.37635. found: 784.37567.

Preparation of (2S,3S,4S)-4-acetoxy-3-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (11e) and (2S,3R,4R)-3-acetoxy-4-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (11f)

[0119] ##STR00020##

[0120] The procedure was analogous to preparation of compounds 1g and 1h in Example 1. Reaction of the mixture of isomers 11c and 11d in ratio 9/1 (100 mg, 0.13 mmol), 10% Pd/C (11 mg, 0.011 mmol) in MeOH (10 mL) gave analogously 76 mg of the product as a colorless solid. The product contained mixture of isomers (2S,3S,4S) (11e)/(2S,3R,4R) (11f) in ratio 9/1 in the form of salt with TFA (74% yield, assuming composition M.2TFA).

[0121] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.24H.sub.42O.sub.10N.sub.5) calculated: 560.29262. found: 560.29214.

Example 12: Preparation of (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-acetoxy-3-(4,7,10-tris(2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (12)

[0122] ##STR00021##

[0123] According to procedure in Example 2, reaction of a mixture of isomers 11e and 11f in ratio 9/1 (70 mg, 0.089 mmol), Fmoc chloride (18.5 mg, 0.072 mmol) in acetonitrile (4.3 mL) and borate buffer (4 mL, 0.2M, pH=9) was carried out. Chromatography analogously to Example 2 provided separation of the isomers (2S,3S,4S) and (2S,3S,4R), yielding 74 mg of pure isomer (2S,3S,4S) as a colorless solid in the form of salt with TFA (82% yield, assuming composition M.2TFA).

[0124] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.39H.sub.52O.sub.12N.sub.5) calculated: 782.36070. found: 782.35995.

[0125] .sup.1H NMR (CD.sub.3CN, 500 MHz): δ.sub.H 1.99, 2.02 (acetate, bs, 3H); 3.71 (methyl ester, s, 3H), 3.77 (Methyl ester, m, 6H); 2.66-4.12 (proline arm, cycle, cycle carboxylate, m, 25H); 4.13-4.32 (proline arm, fmoc, m, 2H); 4.37-4.50 (fmoc, m, 2H); 4.96, 5.05 (proline arm, bs, 1H) 7.33-7.37 (arom., m, 2H); 7.41-7.44 (arom., m, 2H); 7.61-7.67 (arom., m, 2H); 7.82-7.86 (arom., m, 2H); .sup.13C{.sup.1H} NMR (CD.sub.3CN, 125 MHz): 21.31 (acetate, s); 48.24 (fmoc, s); 52.99, 53.62 (methyl ester, s); 46.89-55.14 (proline arm, cycle, carboxylates, m); 58.77, 59.93 (proline arm, s); 68.43 (fmoc, s); 69.05, 70.43 (proline arm, s); 74.03 (proline arm, s); 121.12, 126.22, 128.30, 128.90 (arom., s); 142.25-142.41 (arom., m); 145.06-145.28 (arom., m); 154.69, 155.47 (N—COO); 169.48-172.59 (COO, m).

Example 13: Preparation of dibenzyl (2S,3S,4S)-4-((tert-butyldimethylsilyl)oxy)-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (13a) and dibenzyl (2S,3S,4R)-3-((tert-butyldimethylsilyl)oxy)-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (13b)

[0126] ##STR00022##

[0127] A mixture of isomers 1e and 1f in ratio 9/1 prepared in Example 1 (24 mg, 0.022 mmol), t-Butyl dimethyl silyl chloride (62.5 mg, 0.43 mmol), 1,8-diazabicyklo (5.4.0)undec-7-en (62.5 mg, 0.43 mmol) and dimethyl aminopyridine (62.5 mg, 0.43 mmol) was dissolved in acetonitrile (1 mL). The mixture was stirred for 7.5 hours at 70° C., after which the reaction mixture was diluted with methanolic solution of triethylammonium acetate (1 mL, 1M). Reaction mixture was purified on preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% acetic acid in the mobile phase). Fractions mixture of the two products were pooled, evaporated and dried in high vacuum giving 7 mg of the product as a colorless solid. The product contained mixture of isomers (2S,3S,4S) (13a)/(2S,3S,4R) (13b) in ratio 9/1 in the form of salt with acetic acid (29% yield, assuming composition M.2AcOH).

[0128] LCMS (ESI) m/z: [(M+H).sup.+] (C.sub.52H.sub.84O.sub.11N.sub.5Si) calculated: 982.6. found: 982.7.

Preparation of (2S,3S,4S)-4((tert-butyldimethylsilyl)oxy)-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (13c) and (2S,3R,4R)-3-((tert-butyldimethylsilyl)oxy)-4(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (13d)

[0129] ##STR00023##

[0130] The procedure was analogous to preparation of compounds 1g and 1h in Example 1. Reaction of the mixture of isomers 13a and 13b in ratio 9/1 (7 mg, 0.006 mmol), 10% Pd/C (11 mg, 0.001 mmol) in MeOH/DCM (1/1) (3 mL) gave analogously 4 mg of the product as a colorless solid. The product contained mixture of isomers (2S,3S,4S) (13c)/13c 13d (2S,3R,4R)(13d) in ratio 9/1 in the form of salt with acetic acid (77% yield, assuming composition M.2AcOH).

[0131] LCMS (ESI) m/z: [(M+H).sup.+] (C.sub.37H.sub.72O.sub.9N.sub.5Si) calculated: 758.5. found: 758.5.

Example 14: Preparation of (2S,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((tert-butyldimethylsilyl)oxy)-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (14)

[0132] ##STR00024##

[0133] According to procedure in Example 2, reaction of a mixture of isomers 13c and 13d in ratio 9/1 prepared in Example 13 (4 mg, 0.004 mmol), Fmoc chloride (0.8 mg, 0.003 mmol) in acetonitrile (560 uL) and borate buffer (520 uL, 0.2M, pH=9) was carried out. Reaction mixture was purified on preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% acetic acid in the mobile phase). The chromatography provided separation of the isomers (2S,3S,4S) and (2S,3S,4R), yielding 2 mg of pure isomer (2S,3S,4S) as a colorless solid in the form of salt with acetic acid (45% yield, assuming composition M.2AcOH).

[0134] LCMS (ESI) m/z: [M+2H.sup.+].sup.2+ (C.sub.52H.sub.82O.sub.11N.sub.5Si) calculated: 490.8. found: 490.9.

Example 15: Preparation of dibenzyl (2R,3S,4S)-3-(1,4,7,10-tetraazacyclododecan-1-yl)-4-hydroxypyrrolidine-1,2-dicarboxylate (15a) and dibenzyl (2R,3S,4R)-4-(1,4,7,10-tetraazacyclododecan-1-yl)-3-hydroxypyrrolidine-1,2-dicarboxylate (15b)

[0135] ##STR00025##

[0136] Dibenzyl (1S,2S,5R)-6-oxa-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (1b) (500 mg, 1.41 mmol) and 1,4,7,10-tetraazacyclododecane (975 mg, 5.66 mmol) in 29.25 mL of t-BuOH were placed into a 100 mL round bottom flask and the mixture was stirred for 18 hours and heated under reflux. After cooling to room temperature the reaction mixture was neutralized with TFA (600 uL, 7.84 mmol). Reaction mixture was concentrated on rotary evaporator. Resulting oil was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Fractions containing mixture of the two products were pooled, evaporated and dried in high vacuum. The residue was dissolved in water (5 ml) and lyophilized giving 526 mg of the product as a brown solid in form of TFA salt (49% yield relative to epoxide). Based on .sup.1H NMR the product contained mixture of isomers (2R,3S,4S) (15a)/(2R,3S,4R) (15b) in ratio 1/1.

[0137] Elem. analysis: M.2.1TFA.0.5H.sub.2O, calculated: C; (50.0), H; (5.5), N; (9.0), F; (15.5). found: C; (49.5), H; (5.3), N; (8.8), F; (16.0).

[0138] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.28H.sub.40O.sub.5N.sub.5) calculated: 526.30240. found: 526.30161.

Preparation of dibenzyl (2R,3S,4S)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (15c) and dibenzyl (2R,3S,4R)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-1,2-dicarboxylate (15d)

[0139] ##STR00026##

[0140] A mixture of isomers 15a and 15b in ratio 1/1 (426 mg, 0.563 mmol), t-butyl bromoacetate (341 uL, 2.31 mmol), anhydrous cesium carbonate (1.1 g, 3.34 mmol) and acetonitrile (21.3 mL) were placed into a 100 mL round bottom flask and the mixture was stirred for 1 hour at room temperature. The solids were filtered off and the filtrate was concentrated on rotary evaporator. Resulting oil was purified on reversed-phase flash chromatography (C18 column, acetonitrile/water gradient with 0.1% trifluoroacetic acid in the mobile phase). Fractions containing mixture of the two products were pooled, evaporated and dried in high vacuum giving 535 mg of white solid. The product contained mixture of isomers (2R,3S,4S) (15c)/(2R,3S,4R) (15d) in ratio 1/1 (based on LC-MS) in the form of salt with TFA (87% yield, assuming composition M.2TFA).

[0141] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.46H.sub.70O.sub.11N.sub.5) calculated: 868.50663. found: 868.50611.

Preparation of (2R,3S,4S)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (15e) and (2R,3R,4R)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (15f)

[0142] ##STR00027##

[0143] The procedure was analogous to preparation of compounds 1g and 1h in Example 1. Reaction of the mixture of isomers 15c and 15d in ratio 1/1 (450 mg, 0.41 mmol), 10% Pd/C (37 mg, 0.346 mmol) in MeOH (34.6 mL) gave analogously 337 mg of the product as a white solid. The product contained mixture of isomers (2R,3S,4S) (15e)/(2R,3R,4R) (15f) in ratio 1/1 in the form of salt with TFA (95% yield, assuming composition M.2TFA).

[0144] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.32H.sub.58O.sub.9N.sub.5) calculated: 644.42290. found: 644.42197.

Example 16: Preparation of (2R,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-hydroxy-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (16)

[0145] ##STR00028##

[0146] According to procedure in Example 2, reaction of a mixture of isomers 15e and 15f in ratio 1/1 (284 mg, 0.26 mmol), Fmoc chloride (78.0 mg, 0.30 mmol) in acetonitrile (16 mL) and borate buffer (15 mL, 0.2M, pH=9) was carried out. Chromatography analogously to Example 2 provided separation of the isomers (2R,3S,4S) and (2R,3S,4R). Fractions containing pure isomer (2S,3S,45) were processed according to procedure in Example 2, giving analogously 57 mg of the product as a colorless solid in the form of salt with TFA (20% yield, assuming composition M.2TFA).

[0147] HRMS (ESI) m/z: [(M+H).sup.+] (C46H68O11N5) calculated: 866.49098. found: 866.49067.

[0148] .sup.1H NMR (CD.sub.3CN, 500 MHz, 318 K): δ.sub.H 1.47-1.50 (tBu, m, 27H; 2.76-3.57 (proline arm, cycle, cycle carboxylate, m, 19H); 3.74-3.91 (proline arm, cycle carboxylate, m, 6H); 3.98-4.06 (proline arm, m, 1H) 4.13-4.17 (proline arm, m, 1H); 4.22-4.49 (fmoc, m, 3H); 7.32-7.37 (arom., m, 2H); 7.40-7.45 (arom., m, 2H); 7.62-7.68 (arom., m, 2H); 7.82-7.85 (arom., m, 2H). .sup.13C{.sup.1H} NMR (CD.sub.3CN, 125 MHz, 318 K): 45.74-51.87 (cycle, m); 47.11 (fmoc, s); 50.64, 50.87 (proline arm, s); 54.41-54.85 (carboxylates, m); 56.67, 57.22 (proline arm, s); 67.27 (fmoc, s); 69.09-70.68 (proline arm, m); 82.75-84.41 (tBu, m) 119.97-120.03 (arom., m); 125.11-125.16 (arom., m); 127.19-127.24 (arom., m); 127.75-127.80 (arom., m); 141.08-141.26 (arom., m); 143.99, 144.06, 144.16, 144.26 (arom., s); 153.89, 154.57 (N—COO); 167.10, 170.33 (COOtBu); 172.43 (COOH, s)

Example 17: Preparation of (2R,3S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-hydroxy-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pyrrolidine-2-carboxylic acid (17)

[0149] Compound was prepared as the second isomer (2S,3S,4R) by procedure in Example 16, obtaining 40 mg of product as a colorless solid in the form of salt with TFA (14% yield, assuming composition M.2TFA).

##STR00029##

[0150] HRMS (ESI) m/z: [(M+H).sup.+] (C46H68O11N5) calculated: 866.49098. found: 866.49086.

Example 18: Preparation of 2,2′,2″-(10-((2R,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (18)

[0151] According to procedure in Example 3, reaction of starting compound 16 prepared in Example 16 (57 mg, 0.052 mmol) in TFA (2 mL, 26.14 mmol) gave analogously 35 mg of the product as a white fluffy solid in the form of salt with TFA (73% yield, assuming composition M.2TFA).

##STR00030##

[0152] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.34H.sub.44O.sub.11N.sub.5) calculated: 698.30318. found: 698.30267.

Example 19: Preparation of 2,2′,2″-(1-((3R,4S,5R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-5-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (19)

[0153] According to procedure in Example 3, reaction of starting compound 17 prepared in Example 17 (40 mg, 0.036 mmol) in TFA (2 mL, 26.14 mmol) gave analogously 22 mg of the product as a white fluffy solid in the form of salt with TFA (66% yield, assuming composition M.2TFA).

##STR00031##

[0154] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.34H.sub.44O.sub.11N.sub.5) calculated: 698.30318. found: 698.30269.

[0155] .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ.sub.H 2.85-3.52 (cycle+proline arm, m, 17H); 3.55-4.10 (proline arm+carboxylates, m, 8H); 4.11-4.40 (proline arm+fmoc, m, 4H); 4.60-4.74 (proline arm, m, 1H) 7.31-7.37 (arom., m, 2H); 7.41-7.44 (arom., m, 2H); 7.64-7.68 (arom., m, 2H); 7.88-7.91 (arom., m, 2H). .sup.13C{.sup.1H} NMR (DMSO-d.sub.6, 125 MHz): 45.68-54.03 (cycle+carboxylates, m); 46.71, 46.79 (fmoc, s); 53.66, 53.91 (carboxylates, s); 62.32, 62.58 (proline arm, s); 67.36, 67.50 (fmoc, s); 68.97, 69.60 (proline arm, s); 120.36-120.47 (arom., m); 125.52-125.73 (arom., m); 127.40-127.59 (arom., m); 127.97-128.05 (arom., m); 140.86, 140.88, 140.92, 140.95 (arom., s); 143.73, 143.77, 143.88, 144.00 (arom., s); 153.84, 154.16 (N—COO); 170.52 (CH.sub.2—COO); 171.36, 171.65 (COO).

Example 20: Preparation of Gd(III) complex of 2,2′,2″-(10-((2R,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Gd-18)

[0156] According to procedure in Example 4, reaction of starting compound 18 prepared in Example 18 (5 mg, 0.005 mmol) in methanol (500 uL) and water (500 uL) with aqueous solution of GdCl.sub.3 (50 uL, 0.1 M) and aqueous solution of N-methyl morpholine (353 uL, 0.1 M) gave analogously 3 mg of the product as a white solid (70% yield relative to 18).

[0157] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.34H.sub.41O.sub.11N.sub.5GdNa) calculated: 853.20381. found: 853.20420.

##STR00032##

Example 21: Preparation of Gd(III) Complex of 2,2′,2″-(10-((3R,4S,5R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-5-carboxy-4-hydroxypyrrolidin-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Gd-19)

[0158] According to procedure in Example 4, reaction of starting compound 19 prepared in Example 19 (5 mg, 0.005 mmol) in methanol (500 uL) and water (500 uL) with aqueous solution of GdCl.sub.3 (50 uL, 0.1 M) and aqueous solution of N-methyl morpholine (353 uL, 0.1 M) gave analogously 2 mg of the product as a white solid (47% yield relative to 19).

[0159] HRMS (ESI) m/z: [(M+Na).sup.+] (C.sub.34H.sub.40O.sub.11N.sub.5GdNa) calculated: 875.18575, found: 875.18740.

##STR00033##

[0160] General Procedure for Peptide Couplings

[0161] Fmoc protected Rink Amide resin (TentaGel® R RAM, Rapp Polymere) resin (5 mg, 1 umol) was swelled for 30 minutes in 500 ul DMF. DMF was then removed. Then, 20% piperidine in DMF (500 ul) was used for deprotection for 2 minutes. After that the resin was washed with DMF (3×300 uL), DCM (3×300 uL) and DMF (3×300 uL).

[0162] For coupling of commercial amino acids, the procedure was as follows: Stock solutions were prepared in DMF of the amino acid (100 mg/mL) and of HATU (100 mg/mL). Coupling reaction was carried out by mixing the stock solution of amino acid, stock solution of HATU, neat DIPEA and DMF with the deprotected resin to reach the following final concentrations: 100 mM amino acid, 90 mM HATU and 300 mM DIPEA. The reaction mixture was let shaking for 30 minutes at room temperature after which the liquids were separated from the resin and the resin was washed with DMF (3×300 uL), DCM (3×300 uL) and DMF (3×300 uL).

[0163] For coupling of chelator or chelate building blocks, the procedure was as follows: Stock solutions were prepared in DMF of the chelator or chelate building block (100 mg/mL) and of HATU (100 mg/mL). Coupling reaction was carried out by mixing the stock solution of the building block, stock solution of HATU, neat DIPEA and NMP with the deprotected resin to reach the following final concentrations: 50 mM amino acid, 45 mM HATU and 150 mM DIPEA. The reaction mixture was let shaking for 60 minutes at room temperature after which the liquids were separated from the resin and the resin was washed with DMF (3×300 uL), DCM (3×300 uL), DMF (3×300 uL), MeOH (3×300 uL), DMF (3×300 uL), DCM (3×300 uL), DMF (3×300 uL).

[0164] General Procedure for Cleavage of Peptides from Resin Support

[0165] Peptides were cleaved from solid support by 500 uL of 47.5% TFA, 50% DCM, 2.5% triisopropyl silane mixture at room temperature for 1 hour. After that liquids were filtered off and the resin was washed with 40% solution of water in acetonitrile.

Example 22: Preparation of Dipeptide (Gd-3)-Gly-NH.SUB.2

[0166] According to the general procedure for peptide couplings, the synthesis was carried out in these steps: [0167] 1. Coupling: resin (5 mg, 1 umol), Fmoc-Gly-OH (2.3 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol). [0168] 2. Coupling: Compound Gd-3 (3.4 mg, 3.8 umol), HATU (1.3 mg, 3.4 umol), DIPEA (2 ul, 11.4 umol), NMP (54 uL).

##STR00034##

[0169] LCMS (ESI) m/z: [M+2H.sup.+].sup.2+ (C.sub.36H.sub.45O.sub.11N.sub.7Gd) calculated: 455.1. found: 454.7.

Example 23: Preparation of Dipeptide (Gd-3)-Pro-NH.SUB.2

[0170] According to the general procedure for peptide couplings, the synthesis was carried out in these steps: [0171] 1. Coupling: resin (5 mg, 1 umol), Fmoc-Pro-OH (2.7 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol). [0172] 2. Coupling: Compound Gd-3 (3.4 mg, 3.8 umol), HATU (1.3 mg, 3.4 umol), DIPEA (2 ul, 11.4 umol), NMP (54 uL).

##STR00035##

[0173] LCMS (ESI) m/z: [(M−H).sup.−] (C.sub.39H.sub.47O.sub.11N.sub.7Gd) calculated: 947.3. found: 947.3.

Example 24: Preparation of Dipeptide (Gd-3)-Phe-NH.SUB.2

[0174] According to the general procedure for peptide couplings, the synthesis was carried out in these steps: [0175] 1. Coupling: resin (5 mg, 1 umol), Fmoc-Phe-OH (2.9 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol). [0176] 2. Coupling: Compound Gd-3 (3.4 mg, 3.8 umol), HATU (1.3 mg, 3.4 umol), DIPEA (2 ul, 11.4 umol), NMP (54 uL).

##STR00036##

[0177] LCMS (ESI) m/z: [(M−H).sup.−] (C.sub.43H.sub.49O.sub.11N.sub.7Gd) calculated: 997.3. found: 997.2.

Example 25: Preparation of Dipeptide (Gd-3)-Trp-NH.SUB.2

[0178] According to the general procedure for peptide couplings, the synthesis was carried out in these steps: [0179] 1. Coupling: resin (5 mg, 1 umol), Fmoc-Trp-OH (4.0 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4.0 g, 22.8 umol). [0180] 2. Coupling: Compound Gd-3 (3.4 mg, 3.8 umol), HATU (1.3 mg, 3.4 umol), DIPEA (2 ul, 11.4 umol), NMP (54 uL).

##STR00037##

[0181] LCMS (ESI) m/z: [(M−H).sup.−] (C.sub.45H.sub.50O.sub.11N.sub.8Gd) calculated: 1036.3. found: 1036.2.

Example 26: Preparation of Dipeptide (2)-Phe-OH

[0182] WANG resin preloaded with phenylalanine (3.8 mg, 0.8 umol) was treated with solution (1 mg/30 uL in DMF) of compound 14 (1 mg, 1 umol). After that solution (1 mg/20 uL in DMF) of HATU (0.4 mg, 1 umol) and 100 uL of 20% solution of N-methylmorpholine was added. The reaction mixture was let shaking for 1 h at room temperature after which the liquids were separated from the resin and the resin was washed with DMF (3×300 uL), NO DCM (3×300 uL) and DMF (3×300 uL). The product was cleaved from resin by mixture of 47.5% TFA, 50% DCM, 2.5% triisopropyl silane at room temperature for 1 hour. Although compound 14 was used in the synthesis, the removal of tert-butyldimethylsilyl protective group during cleavage from the resin makes it a derivative of compound 2 in the final product.

##STR00038##

[0183] LCMS (ESI) m/z: [(M−H).sup.−] (C.sub.55H.sub.75O.sub.12N.sub.6) calculated: 1011.6. found: 1011.5.

Example 27: Preparation of Dipeptide (8)-Phe-OH

[0184] WANG resin preloaded with phenylalanine (3.8 mg, 0.8 umol) was treated with solution (1 mg/30 uL in DMF) of compound 8 (1 mg, 1 umol). After that solution (mg/20 uL in DMF) of HATU (0.4 mg, 1 umol) and 100 uL of 20% solution of N-methylmorpholine was added. The reaction mixture was let shaking for 1 h at room temperature after which the liquids were separated from the resin and the resin was washed with DMF (3×300 uL), DCM (3×300 uL) and DMF (3×300 uL). The product was cleaved from resin by mixture of 47.5% TFA, 50% DCM, 2.5% triisopropyl silane at room temperature for 1 hour.

##STR00039##

[0185] LCMS (ESI) m/z: [(M−H).sup.−] (C.sub.57H.sub.77O.sub.13N.sub.6) calculated: 1053.6. found: 1053.5.

Example 28: Preparation of Pentapeptide (12)-Gly-Phe-(Gd-6)-Gly-NH.SUB.2

[0186] According to the general procedure for peptide couplings, the synthesis was carried out in these steps:

##STR00040## [0187] 1. Coupling: resin (10 mg, 2 umol), Fmoc-Gly-OH (4.5 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol). [0188] 2. Coupling: Gd-3 (6.75 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol), NMP (54 uL). [0189] 3. Coupling: Gd-3 (6.75 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol), NMP (54 uL). [0190] 4. Coupling: Fmoc-Phe-OH (5.9 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol). [0191] 5. Coupling: Fmoc-Gly-OH (4.5 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol). [0192] 6. Resin was treated with 0.1 M solution of LiOH in MeOH (500 uL) for 2 hours at room temperature, followed by wash with MeOH (3×300 uL) and wash sequence in general procedure. [0193] 7. Coupling: Compound 12 (7.7 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol), NMP (54 uL). [0194] 8. The product was cleaved from resin by mixture of 47.5% TFA, 50% DCM, 2.5% triisopropyl silane at room temperature for 1 hour.

[0195] Product was purified by preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% formic acid in the mobile phase). Fractions containing pure product were pooled, evaporated, the residue was dissolved in water (1 mL) and lyophilized to give 0.3 mg of product as white fluffy solid. Although compound Gd-3 was used in the synthesis, the removal of Fmoc protective group makes it a derivative of compound Gd-6 in the final product.

[0196] HRMS (ESI) m/z: [z=2 (M+H+Na).sup.2+] (C.sub.71H.sub.96O.sub.22N.sub.14GdNa) calculated: 838.79757. found: 838.79781.

Example 29: Preparation of Pentapeptide (6)-Gly-Phe-(Gd-6)-Gly-NH.SUB.2

[0197] Pentapeptide (12)-Gly-Phe-(Gd-6)-Gly-NH.sub.2 prepared in Example 28 was dissolved in water (1 mL) and aqueous solution of LiOH (500 uL, 1M) was added. Reaction mixture was stirred for 30 minutes at room temperature. Then, neat formic acid (19 uL) was added. Product was purified by preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% formic acid in the mobile phase). Fractions containing pure product were pooled and evaporated. The reaction removed methyl ester groups and Fmoc simultaneously, thus converting the N-terminal chelator amino acid in the product to a derivative of compound 6.

##STR00041##

[0198] HRMS (ESI) m/z: [z=2 (M+2H).sup.2+] (C.sub.51H.sub.79O.sub.19N.sub.14Gd) calculated: 674.74380. found: 674.74361.

Example 30: Preparation of Pentapeptide (Lu-6)-Gly-Phe-(Gd-6)-Gly-NH.SUB.2

[0199] Pentapeptide (6)-Gly-Phe-(Gd-6)-Gly-NH.sub.2 prepared in Example 29 was dissolved in water (2 mL), aqueous solution of LuCl.sub.3 (8 uL, 0.04683 M) and aqueous solution of N-methyl morpholine (216 uL, 0.1 M) were added and the reaction mixture was stirred at room temperature for 1 hour. Product was purified by preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% formic acid in the mobile phase). Fractions containing pure product were pooled, evaporated, the residue was dissolved in water (1 mL) and lyophilized to give 0.1 mg of product as white fluffy solid. Complexation of Lu(III) converted the N-terminal chelator amino acid in the product to a derivative of Lu-6, which is analogous to Gd-6 from Example 6, with the exception that Gd is replaced with Lu.

##STR00042##

[0200] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.51H.sub.75O.sub.19N.sub.14GdLu) calculated: 1520.39761. found: 1520.39719.

Example 31: Preparation of tetrapeptide Gly-Phe-(Gd-6)-Gly-NH.SUB.2

[0201] According to the general procedure for peptide couplings, the synthesis was carried out in these steps:

##STR00043## [0202] 1. Coupling: resin (20 mg, 4 umol), Fmoc-Gly-OH (9 mg, 30.4 umol), HATU (10.4 mg, 27.4 umol), DIPEA (16 ul, 91.2 umol). [0203] 2. Coupling: Gd-3 (13.5 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol), NMP (109 uL). [0204] 3. Coupling: Gd-3 (13.5 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol), NMP (109 uL). [0205] 4. Coupling: Fmoc-Phe-OH (11.8 mg, 30.4 umol), HATU (10.4 mg, 27.4 umol), DIPEA (16 ul, 91.2 umol). [0206] 5. Coupling: Fmoc-Gly-OH (9 mg, 30.4 umol), HATU (10.4 mg, 27.4 umol), DIPEA (16 ul, 91.2 umol). [0207] 6. The product was cleaved from resin by mixture of 47.5% TFA, 50% DCM, 2.5% triisopropyl silane at room temperature for 1 hour.

[0208] Product was purified by preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% formic acid in the mobile phase). Fractions containing pure product were pooled, evaporated, the residue was dissolved in water (1 mL) and lyophilized to give 0.1 mg of product as white fluffy solid. Although compound Gd-3 was used in the synthesis, the removal of Fmoc protective group makes it a derivative of compound Gd-6 in the final product.

[0209] HRMS (ESI) m/z: [(M+H).sup.+] (C.sub.33H.sub.47O.sub.11N.sub.9Gd) calculated: 891.26306. found: 891.26321.

Example 32: Preparation of Tetrapeptide Gly-Phe-(Lu-6)-Gly-NH.SUB.2

[0210] According to the general procedure for peptide couplings, the synthesis was carried out in these steps:

##STR00044## [0211] 1. Coupling: resin (10 mg, 2 umol), Fmoc-Gly-OH (4.5 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol). [0212] 2. Coupling: Lu-3 (6.79 mg, 7.6 umol), HATU (2.6 mg, 6.8 umol), DIPEA (4 ul, 22.8 umol), NMP (54 uL). [0213] 3. Coupling: Fmoc-Phe-OH (5.9 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol). [0214] 4. Coupling: Fmoc-Gly-OH (4.5 mg, 15.2 umol), HATU (5.2 mg, 13.7 umol), DIPEA (8 ul, 45.6 umol). [0215] 5. The product was cleaved from resin by mixture of 47.5% TFA, 50% DCM, 2.5% triisopropyl silane at room temperature for 1 hour.

[0216] Product was purified by preparative HPLC (C18 column, acetonitrile/water gradient with 0.1% formic acid in the mobile phase). Fractions containing pure product were pooled, evaporated, the residue was dissolved in water (1 mL) and lyophilized. Although compound Lu-3 was used in the synthesis, the removal of Fmoc protective group makes it a derivative of compound Lu-6 in the final product, which is analogous to Gd-6 from Example 6, with the exception that Gd is replaced with Lu.

[0217] LC-MS (ESI) m/z: [(M+H).sup.+] (C.sub.32H.sub.47O.sub.11N.sub.9) calculated: 908.3. found: 908.3.

[0218] II. Properties of Gd(II) Chelates

Example 33: Relaxivity of Gd(III) Chelates

[0219] Relaxivity of Gd(III) chelate Gd-6 and of a peptide Gly-Phe-(Gd-6)-Gly-NH.sub.2 have been measured at 40° C. and 0.5 T in 10 mM MOPS buffer pH=7.0. Table 1 summarizes the results. It is apparent from the data that the relaxivity of the peptide, where the Gd-6 is incorporated within the peptide chain, is higher than the relaxivity of a standalone Gd-6. This demonstrates that covalent linkage of the chelate Gd-6 to molecular chains through peptide bonds provides the means to increase relaxivity. For comparison, relaxivity of most clinically used MRI contrast agents at comparable conditions is within the range 3-4 mM.sup.−1 s.sup.−1 (Rohrer M. (2005), Invest. Radiol. 40, 715-724). The relaxivity of peptide Gly-Phe-(Gd-6)-Gly-NH.sub.2 is above this range despite of its relatively small size.

TABLE-US-00001 TABLE 1 Gd Relaxation Relaxivity r.sub.1 concentration time (mM.sup.−1.s.sup.−1) at 40° C., Gd compound c (mM) * T.sub.1 (ms) and 0.5 T ** Gd-6 (Example 6) 0.087 1562 3.5 Peptide Gly-Phe- 0.138 1004 4.8 (Gd-6)-Gly-NH.sub.2 (Example 31) * Determined by ICP-AES. ** Calculated r.sub.1 = R.sub.1/c, where c is Gd concentration in mM, and R.sub.1 = 1/(T.sub.1/1000) − 1/(T.sub.1D/1000), where T.sub.1 is the relaxation time of the sample (in ms) and T.sub.1D is the relaxation time of the buffer without Gd.

Example 34: Kinetic Inertness of the Building Block Gd-3

[0220] Stability (kinetic inertness) of the Gd(III) building block Gd-3 prepared in Example 4 was tested by following acid-assisted decomplexation by HPLC. Isocratic elution method was used for analysis. Constant concentration of TFA (0.08%) was maintained in the mobile phase (pH ˜2.0), while methanol content was varied to tune the retention time of the analyte. With increasing retention time (i.e. time spent in the acidic medium), more decomplexation should occur and the amount of intact chelate should decrease mono-exponentially (pseudo first-order reaction conditions), while a peak of free chelator should appear in the chromatogram. The HPLC conditions were as follows: column Kinetex C18, 100×3 mm, 2.6 um; column temperature maintained at 40° C.; mobile phase: TFA (constant 0.08% vol.), MeOH (variable), water; flow-rate 0.6 mL/min; UV detection at 280 nm; injection of 2 uL of 0.5 mM sample. Table 2 summarizes the results, showing that there was no decrease in the peak area of the intact chelate Gd-3, thus demonstrating high kinetic inertness of Gd-3 under acidic conditions.

TABLE-US-00002 TABLE 2 Methanol in Retention Peak area mobile phase (%) time (min) (mAU.s) 40 4.632 681.6 39 5.044 685.5 38 5.570 688.4 37 6.072 686.6 36 6.894 690.9