RADIODRUG FOR DIAGNOSTIC/THERAPEUTIC USE IN NUCLEAR MEDICINE AND RADIO-GUIDED MEDICINE

Abstract

Radio-drug suitable for performing radio-guided surgery, imaging diagnostics, radio-metabolic therapy.

Claims

1. A compound of Formula I ##STR00009## wherein A is an anchoring portion chosen from the group consisting of amide, anilide, ether, amine and sulfonamide, linked to the meta or para position of benzylguanidine (BG); L is a linker portion chosen from the group consisting of diaminoalkyl chains with lengths ranging from 2 to 6 methylene units and diaminopolyethylene glycol chains with lengths ranging from 2 to 6 ethylene glycol units; BFC is a bifunctional chelator chosen from the group consisting of DOTA, NOTA, TETA and DTPA; and Me is a radiation-emitting or non-radiation-emitting metal cation chosen from the group consisting of .sup.90Y.sup.3+ 177Lu.sup.3+, .sup.86Y.sup.3+, .sup.89Y.sup.3+, .sup.68Ga.sup.3+ and .sup.111In.sup.3+.

2. The compound according to claim 1, wherein said metal cation is a pure β.sup.− radiation-emitting metal cation.

3. The compound according to claim 1, wherein said bifunctional chelator BFC is DOTA.

4. The compound according to claim 1, wherein that that said linker portion L is an ethylenediamine group.

5. The compound according to claim 1, wherein said anchoring portion A is an amide group.

6. The compound according to claim 1 of Formula II: ##STR00010## wherein Me is a radiation-emitting or non-radiation-emitting metal cation chosen from the group consisting of .sup.86Y.sup.3+, .sup.89Y.sup.3+, .sup.68Ga.sup.3+, .sup.111In.sup.3+, .sup.177Lu.sup.3+ and .sup.90Y.sup.3+, preferably it is .sup.90Y.sup.3+ or .sup.89Y.sup.3+.

7. The compound according to claim 1, having the following Formula III: ##STR00011## wherein n is equal to 1 and wherein Me is a radiation-emitting or non-radiation-emitting metal cation selected from the group consisting of .sup.86Y.sup.3+, .sup.89Y.sup.3+, .sup.68Ga.sup.3+, .sup.111In.sup.3+, .sup.177Lu.sup.3+ and .sup.90Y.sup.3+.

8. The compound according to claim 1 having the following formula IV: ##STR00012## wherein n is equal to 2 and wherein Me is a radiation-emitting or non-radiation-emitting metal cation selected from the group consisting of .sup.86Y.sup.3+, .sup.89Y.sup.3+, .sup.68Ga.sup.3+, .sup.111In.sup.3+, .sup.177Lu.sup.3+ and .sup.90Y.sup.3+.

9. A medicament comprising the compound according to claim 1.

10. Method of β radio-tracing with the compound according to claim 1 in a subject having a tumor, said method comprising administering to said subject an effective amount of said compound.

11. A method of treating or diagnosing a subject having a tumor with the medicament according to claim 9, wherein said tumors are selected from the group consisting of neuroendocrine tumors which overexpress the norepinephrine transporter (NET), pheochromocytoma, paraganglioma, carcinoid tumor and neuroblastoma.

12. Method of radio-guided surgery (RGS) with the medicament according to claim 9.

13. The method according to claim 12, wherein the tumor surgery is radio-guided by the β.sup.− particle detection (β.sup.−-RGS).

14. A pharmaceutical composition comprising the compound according to claim 1 and pharmaceutically suitable excipients.

15. The compound according to claim 7, wherein Me is .sup.90Y.sup.3+ or .sup.89Y.sup.3+.

16. The compound according to claim 8, wherein Me is .sup.90Y.sup.3+ or .sup.89Y.sup.3+.

Description

EXPERIMENTAL SECTION

[0046] Preparation of the Compound 1 of Formula II

[0047] The preparation of the compound 1 of Formula II (also denoted as .sup.90Y-DOTA-BG chelate, compound 1 or MC4324) has been carried out as depicted in Scheme 1. The commercial cyclen 2 has been converted to the mono-alkylated intermediate 3 by reaction with ethyl bromoacetate in dichloromethane (DCM) at room temperature. The alkylation reaction of the compound 3 with tert-butyl bromoacetate in the presence of anhydrous potassium carbonate in anhydrous acetonitrile then provided tri-tert-butyl 2,2′,2″-(10-(2-ethoxy-2-oxoethyl)-cyclen-1,4,7-tri-yl)triacetate 4 which has then been treated with an excess of pure ethylenediamine at room temperature to give the synthon A (5) (Scheme 1A). The synthesis of the synthon B (7) instead has been carried out to start from the commercial 3-(aminomethyl)-benzoic acid 6 by guanidination reaction with N,N′-bis-tert-butoxycarbonyl-1H-pyrazol-1-carboxamidine in anhydrous tetrahydrofuran (THF) in the presence of triethylamine (TEA) at room temperature (Scheme 1B). The coupling between the synthon A (5) and the synthon B (7) in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI), hydrated 1-hydroxybenzotriazole (HOBt) and TEA in anhydrous DCM at room temperature, followed by a total deprotection reaction by using a mixture of trifluoroacetic acid (TFA):tri-isopropylsilane (TIS):water 95/2.5/2.5 (vol/vol/vol) then led to the free binder 9 (MC4325) (Scheme 1C). The DOTA-BG conjugate 9 has then been hot complexed with a slight excess of .sup.89Y(NO.sub.3).sub.3 in the presence of an ammonium acetate buffer at pH 5.5-6 in closed vial to give the non-radioactive analogue, also called “cold tracer”, of the compound of Formula II (1a, MC4324). The DOTA-BG conjugate 9 has been finally used in large excess for the hot radio-marking with .sup.111YCl.sub.3 still in the presence of the ammonium acetate buffer at pH 5.5-6 (Scheme 1C) to give the compound of Formula II (1, MC4324).

##STR00005## ##STR00006## ##STR00007##

[0048] The .sup.1H-NMR and .sup.13C-NMR spectra have been recorded at 400 MHz and 100 MHz, respectively, by using a Bruker AC 400 spectrometer; the chemical shifts are reported in δ units (ppm) with respect to tetramethylsilane used as internal reference (Me.sub.4Si). The .sup.1H-NMR and .sup.13C-NMR spectra of the compound of Formula II (.sup.89Y-DOTA-BG, compound 1a or MC4324) have been recorded at 600 MHz and 150 MHz, respectively, by using a Bruker AC 600 spectrometer. The low-resolution mass spectra have been recorded on an API-TOF Mariner Perspective Biosystem (Stratford, Tex., USA), the samples have been injected by a Harvard pump by using a flow rate of 5-10 μl/min, infused in the Electrospray system. The high-resolution mass spectra (HR-MS) have been recorded on Orbitrap Exactive spectrometer (Thermo Fisher Scientific, Austin, Tex. USA). The analytical HPLC analyses of the compounds 8, 9 and .sup.89Y-DOTA-BG as non-radioactive analogue of the compound of Formula II (1a or MC4324) have been carried out on: [0049] liquid chromatograph UHPLC Accela equipped with pumping system, autosampler and PDA Accela detector coupled to mass spectrometer LTQ ion trap equipped with an ESI interface (Thermo Fisher Scientific, Austin, Tex. USA); the experimental data have been acquired, processed and reworked by Xcalibur software (Thermo Fisher Scientific, Austin, Tex., USA); [0050] Shimadzu Nexera liquid chromatograph equipped with CBM-20A controller, four LC-30AD pumps, a DGU-20 A5R degasser, a SPD-M20A PDA detector (Standard Analytical 2.50 μl Semimicro cell), sampling speed: 100 Hertz; resolution: 4.0 nm), a thermostated column housing CTO-20AC and an autosampler SIL-30AC; an ELSD detector (Sedex 90, SEDERE, France) is connected in series to the system; the experimental data have been acquired, processed and reworked by LabSolution software. (Shimadzu Italia S.r.l., Milan, Italy).

[0051] The purification of the compound .sup.89Y-DOTA-BG (1a or MC4324) as non-radioactive analogue of the compound of Formula II (1 or MC4324) has been carried out on Waters semi-preparative liquid chromatograph (RP-HPLC) equipped with Waters 590 model pump, 250 μl injector and UV spectrophotometric detector with variable wavelength and Omniscribe recorder on paper.

[0052] All the compounds have been regularly controlled by TLC and .sup.1H-NMR. The TLC has been carried out on silica gel plates supported by aluminum (Merck DC, Alufolien Kieselgel 60 F254) with spots displayed by UV light or by using an alkaline solution of KMnO.sub.4. The concentration of the solutions after the reactions and extractions has been carried out by using a rotary evaporator operating under a reduced pressure of 20 Torr. The organic solutions have been dried on anhydrous sodium anhydrous sulphate or magnesium sulphate. All the chemical reactants have been purchased from Sigma Aldrich s.r.l., Milan (Italy), TCI Europe N.V., Zwijndrecht (Belgium) or Perkin Elmer (USA) and were of the utmost purity. Normally, the samples prepared for physical and biological tests have been dried under high vacuum on P.sub.2O.sub.5 for 20 hours at temperatures between 25 and 40° C., depending on the melting point of the sample. Herein below the experimental procedures for the preparation and chemical-physical characterization of the compounds 1-9 are described.

[0053] Synthesis of the compound ethyl 2-(1,4,7,10-tetraazacyclododecan-1-yl)acetate (3). A solution of ethyl bromoacetate (1.0 g; 6.0 mmol) in DCM (10 ml) is added dropwise over 10 minutes to a solution of commercial cyclen 2 (1.36 g, 7.9 mmol) in DCM (16 ml) cooled in an ice bath. After 2 hours, the reaction mixture is brought to room temperature and left to react for additional 22 hours. The resulting suspension is filtered and the filtrate is evaporated under reduced pressure. The crude, oily yellow, reaction product is finally purified by flash chromatography (from 100% DCM to DCM/methanol/ammonia/water 70:30:5:5) thus obtaining the compound 3 (1.61 g, 79%) in form of white viscous oil. .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.27 (t, J=7.2 Hz, 3H; CH.sub.2CH.sub.3), 2.84 (n, 8H, CH), 2.95 (n, 8H, CH), 3.49 (n, 2H, NCH.sub.2COOCH.sub.2CH.sub.3), 4.16 (q, J=7.2 Hz, 3H, C(O)OCH.sub.2CH.sub.3). MS (ESI): 259 [M+H]+.

[0054] Synthesis of tri-tert-butyl 2,2′,2″-(10-(2-ethoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tri-yl)triacetate (4) To a solution of mono-alkylated cyclen 3 (0.98 g, 3.8 mmoles) in acetonitrile (35 ml), anhydrous potassium carbonate (3.14 g, 22.8 mmoles) is added. To this suspension a solution of tert-butyl bromoacetate (2.22 g, 11.4 mmoles) has been added dropwise in acetonitrile (10 ml) over 20 minutes. The suspension is left under stirring at room temperature for 4 hours. The suspended solid has then been removed by filtration, the solvent evaporated under reduced pressure and the residue has been finally purified by flash chromatography (from 100% DCM to 20% methanol/DCM) to give the compound 4 (1.90 g, 84%) in form of white foam. .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.15 (t, J=7.2 Hz, 3H, C(O)OCH.sub.2CH.sub.3), 1.33 (s, 9H, tBu), 1.34 (s, 9H, tBu), 1.35 (s, 9H, tBu), 1.80-3.70 (very broad multiplet set with integration corresponding to 24H, CH.sub.2), 4.04 (q, J=7.2 Hz, 3H, C(O)OCH.sub.2CH.sub.3). MS (ESI): 623 [M+Na]+.

[0055] Synthesis of tri-tert-butyl 2,2′,2″-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (5, Synthon A). The compound 4 (1.31 g, 2.18 mmoles) has been dissolved in pure ethylenediamine (2 ml, 29.9 mmoles) and the resulting solution has been left under stirring at room temperature for 65 hours. At the end of the reaction, ethylenediamine has been removed under reduced pressure and the residue dried under vacuum to give a light yellow foam which finally has been purified by flash chromatography (from 100% DCM to DCM/methanol 1:1) to provide the compound 5 (1.04 g, 72%) in form of white foam. .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.38 (s, 27H, 3×tBu), 1.48 (m, 2H, NH.sub.2), 2.45 (m, 4H, 2×CH.sub.2), 2.63 (m, 4H, 2×CH.sub.2), 2.72 (t, 2H, CH.sub.2), 2.77-2.80 (m, 8H, 4×CH.sub.2), 2.99 (s, 2H, CH.sub.2), 3.17 (br s, 4H, 2×CH.sub.2), 3.20 (s, 2H, CH.sub.2), 3.21-3.27 (q, 2H, CH.sub.2), 8.68 (t, 1H, CONH); 13C-NMR (CDCl.sub.3) δ ppm: 27.68, 27.84, 36.60, 40.03, 55.39, 55.51, 55.90, 81.61, 81.70, 172.17, 172.27. MS (ESI): 637 [M+Na]+.

[0056] Synthesis of N,N′-bis-tert-butoxycarbonyl-3-guanidinomethyl-benzoic acid (7, Synthon B). To a suspension of commercial 3-(aminomethyl)-benzoic acid 6 (1.66 mmol, 250.0 mg) in a mixture of methanol anhydrous (16.5 ml) and anhydrous THF (16.5 ml), TEA (6.61 mmol, 669.4 mg, 0.92 ml) and N,N-bis-tert-butoxycarbonyl-1H-pyrazole-1-carboxamidine (1.98 mmol, 615.9 mg) have been sequentially added and the resulting mixture has been left under stirring for 24 hours at room temperature. At the completion of the reaction, the solvent has been evaporated under reduced pressure and the residue has been dissolved in ethyl acetate and washed in succession with 0.1 N potassium bisulphate and sodium chloride saturated solution. Thus, the organic phase has been dried over magnesium sulphate and the solvent removed under reduced pressure. The crude reaction product has been finally purified by flash chromatography on silica gel (Biotage Isolera One®) by eluting with a chloroform/methanol 0.fwdarw.6% mixture to provide the intermediate 7 as a white solid. .sup.1H-NMR (DMSO-d6) δ ppm: 1.38 (s, 9H, C(CH3)3), 1.49 (s, 9H, C(CH.sub.3).sub.3), 4.58 (d, 2H, NHCH.sub.2Ph-COOH), 7.45-7.51 (t, 1H, CH benzene ring), 7.54-7.56 (m, 1H, CH benzene ring), 7.83-7.85 (m, 1H, CH benzene ring), 7.89 (s, 1H, CH benzene ring), 8.76-8.79 (t, 1H, NHCH.sub.2Ph-COOH), 11.53 (s, 1H, Boc-NH), 12.98 (br s, 1H, COOH). MS (ESI): 392 [M−H]−.

[0057] Synthesis of tri-tert-butyl 2,2′,2″-(10-(2-((2-(3-((2,3-bis(tert-butoxycarbonyl)guanidino) methyl)benzamido)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triyl) triacetate (8). To a solution of N,N-bis-tert-butoxycarbonyl-3-guanidinomethyl-benzoic acid 7 (0.16 mmol, 64.0 mg) in anhydrous DCM (1.5 ml), HOBt (0.21 mmol, 28.6 mg), EDCI (0.21 mmol, 40.5 mg), tri-tert-butyl 2,2′,2″-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-cyclen-1,4,7-triyl)triacetate 5 (100.0 mg, 0.16 mmol) and finally TEA (0.58 mmol, 59.2 mg, 81.6 μl) are added in succession by cooling in ice bath. After about 25 hours at room temperature, the solvent is removed by evaporation and the resulting crude product has been purified by flash chromatography on silica gel (Biotage Isolera One®) by eluting with a chloroform/methanol 0.fwdarw.8% mixture to provide the compound 8 (70.5 mg, yield=44%) in form of white solid. .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.35 (s, 9H, tBu), 1.39 (s, 27H, 3×tBu), 1.44 (s, 9H, tBu), 1.85-3.65 (very broad multiplet set with integration corresponding to 28H, 14×CH.sub.2), 4.60 (d, 2H, NHCH.sub.2), 7.33-7.36 (in, 2H, CH benzene ring), 7.99 (s, 1H, CH benzene ring), 8.15 (d, 1H, CH benzene ring), 8.49 (m, 1H, NHCH.sub.2), 8.81 (m, 1H, CONH), 9.03 (m, 1H, CONH), 11.5 (s, 1H, BocNH). MS (ESI): 990 [M+H]+.

[0058] Synthesis of 2,2′,2″-(10-(2-((2-(3-(guanidinomethyl)benzamido)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (9, also denoted as MC4325 or DOTA-BG conjugate) A mixture of TFA: tri-isopropylsilane:water 95:2.5:2.5 v:v:v (6.0 ml) has been added to the intermediate 8 (0.091 mmol, 90.0 mg) by cooling at 0° C. The deprotection reaction has been checked by RP-HPLC in the following analytical conditions: column: Sunfire C18, 3.5 μm (150*4.6 mm ID); Eluents: A) H2O/ACN 95/5+0.1% TFA, B) ACN+0.1% TFA. Gradient elution (start A/B 100/0; 1 min A/B 100/0; 15 min A/B 0/100, 20 min A/B 0/100). Flow: 1.0 ml/min. Detection: PDA (200-400 nm), ELSD (T: 80° C., P: 4 bar), T col=30° C.; samples dissolved in MeOH. After about 22 hours at room temperature the reaction proved to be completed. Thus, the solvent has been evaporated and the residue co-evaporated several times with anhydrous DCM (6×3 ml) and anhydrous diethyl ether (5×3 ml). The crude product thus obtained has then been triturated with anhydrous diethyl ether, filtered and washed on filter with anhydrous diethyl ether to provide the desired compound 9 (88.2 mg, yield=81%) in form of white solid. .sup.1H-NMR (D.sub.2O) δ ppm: 2.90-3.75 (very broad multiplet set with integration corresponding to 28H, 14×CH.sub.2), 4.41 (d, 2H, NHCH.sub.2), 7.43 (m, 2H, CH benzene ring), 7.62 (m, 2H, CH benzene ring).

[0059] HR-MS (ESI): calculated mass for [C.sub.27H.sub.43N.sub.9O.sub.8+H].sup.+=622.3307 m/z; determined mass: 622.3294 m/z. Source parameters: capillary temperature 275° C.; spray voltage 3.70 kV; sheath gas 6; tube lens voltage 125 V. Resolution input: 100000. Samples dissolved in MeOH (10.sup.0M).

[0060] Preparation of the chelate .sup.89Y-DOTA-BG as non-radioactive analogue of the compound of Formula II (1a, MC4324). To the binder 9 (36.0 mg, 30.2 μmol) dissolved in 1 ml of 1M ammonium acetate buffer (pH=5.5-6), the commercial salt 89Y(NO.sub.3).sub.3.4H.sub.2O (15.7 mg, 45.3 μmol) has been added. The resulting solution has then been hot stirred (90° C.) in closed vial for 3 hours. At the end of the reaction, the mixture has been left to cool at room temperature and then the crude chelate has been purified from the excess salts by semi-preparative HPLC (conditions: column Hypersil ODS GOLD 5 μm; 250×10 mm; eluent: MeOH/H.sub.2O 5:95 v/v+0.02% TFA, flow: 4.0 ml/min; UV detection 254 nm) until providing, after lyophilization of the clean fractions, the “cold” chelate 89Y-DOTA-BG 1a (MC4324) as a white powdered solid (19 mg, yield=90%). .sup.1H-NMR (D.sub.2O, 600 MHz, T=5° C.) δ ppm: 2.08 (d, 1H, CHH), 2.22-2.33 (m, 5H, 2×CH, and CHH), 2.53-2.67 (m, 7H, 3×CH, and CHH), 2.98 (d, 1H, CH), 3.08 (d, 2H, CH.sub.2), 3.18-3.40 (m, 6H, 3×CH.sub.2), 3.43-3.56 (m, 4H, 2×CH.sub.2), 3.64 (m, 2H, CH.sub.2), 4.38 (d, 2H, —CH.sub.2-guanidine), 7.43 (m, 2H, CH benzene ring), 7.60 (m, 2H, CH benzene ring). 13C-NMR (D.sub.2O, 150 MHz, T=5° C.) δ ppm: 180.29, 179.71, 175.80, 169.98, 156.53, 136.86, 133.23, 130.63, 129.15, 129.13, 126.19, 125.21, 65.46, 65.32, 65.22, 63.04, 55.64, 55.47, 55.46, 55.35, 54.73, 54.72, 54.59, 54.18, 43.75, 39.20, 38.40.

[0061] HR-MS (ESI): calculated mass for [C.sub.27H.sub.40N.sub.9O.sub.8Y+H].sup.+=708.2131 m/z; determined mass: 708.2124 m/z. Source parameters: capillary temperature 275° C.; spray voltage 3.70 kV; sheath gas 6; tube lens voltage 125 V. Resolution input: 100000. Samples dissolved in MeOH (10.sup.−5M).

[0062] Preparation of the compound of Formula II (1, .sup.90Y-DOTA-BG, MC4324). The chelate .sup.90Y-DOTA-BG of Formula II (1) has been prepared by adding 30 μl of a commercial solution of 90YCl3 in 0.05 M HCl (1.07 mCi, 22.1 μmol) to 132 μl of a 0.1 mM binder 9 solution (13.2 nmol) in 1M ammonium acetate buffer (pH=5.5-6) and bringing to the final volume of 500 μl by the addition of 338 μl of the same 1M ammonium acetate buffer (pH=5.5-6). The resulting solution has then been incubated in closed vial placed on a heating block under shielded hood at 90° C. for 30 minutes. At the end of the reaction, after cooling to room temperature, different aliquots of the solution (2-10 μl) have been collected without dilution to evaluate the radio-marking yield and to carry out the quality control by ITLC. The ITLC methods used ITLC-SG and ITLC-SA Agilent plates and different eluent systems such as 1M ammonium acetate buffer (pH=5.5-6):methanol (50:50 v/v), 1M ammonium acetate buffer (pH=5.5-6):methanol:ammonia 33% (50:50:5 v/v/v) and 50 mM EDTA in 0.1 M ammonium acetate buffer (pH=6). The results of different chromatographic runs showed in agreement >99% radio-marking yield and radiochemical purity (FIG. 1). FIG. 1 depicts the ITLC-SG runs relative to the radio-marking of the binder 9 with .sup.90YCl.sub.b for the preparation of the chelate .sup.90Y-DOTA-BG of Formula II (1, or MC4324).

[0063] Chemical Stability of the Chelate .sup.89Y-DOTA-BG (1a, MC4324) as Non-Radioactive Analogue of the Compound of Formula II (1, MC4324) in Physiological Conditions.

[0064] The stability of the chelate .sup.89Y-DOTA-BG (1a, MC4324) (non-radioactive analogue of the compound of Formula II, 1 or MC4324) in physiological conditions has been evaluated by analysis with analytical HPLC (Shimadzu Nexera chromatograph equipped with a SPD-M20A PDA detector; Hypersil ODS GOLD 250×4.6 mm column; eluent: MeOH/H.sub.2O 5:95 v/v+0.02% TFA, flow: 1.0 ml/min; UV detection 214 nm) repeated at regular time intervals after having solubilized it in PBS buffer (c=0.9 mg/ml) and kept at T=37° C. After 5 days, the chelate was found to be still perfectly intact, with a chemical purity constantly higher than 99.5% (see Table 1). The chelate .sup.89Y-DOTA-BG (peak 1 in the chromatogram in FIG. 2A-C) immediately revealed the presence of a peak 2 (lower than 0.1%) which has also remained constant over time.

[0065] FIG. 2 depicts the HPLC plot relative to the compound .sup.89Y-DOTA-BG (1a) solubilized in PBS buffer (c=0.9 mg/ml) and at T=37° C. time t=0 min (A), t=21 hours (B) and t=5 days (C).

TABLE-US-00001 TABLE 1 Trend over time of the areas of the chromatographic peaks relative to the compound 1a .sup.89Y-DOTA-BG (1) and impurity 2. Time Area 1 Area (1 + 2) 0 min 34.78 35.13 30 min 35.09 35.29 1 hour 30 min 35.07 35.30 5 hours 35.13 35.40 21 hours 37.15 37.45 5 days 38.34 38.65

[0066] Biological Validation of the .sup.89Y-DOTA-BG Chelate (1a, MC4324) (Non-Radioactive Analogue of the Compound of Formula II, 1 or MC4324) as Substrate of NET in the Human Neuroblastoma Line SK-N-SH.

[0067] The ability of the .sup.89Y-DOTA-BG chelate (1a, MC4324) (non-radioactive analogue of the compound of Formula II, 1 or MC4324) to act as substrate of NET has been evaluated in competition experiments with the tritiated endogenous substrate .sup.3H-NE for the uptake by the human neuroblastoma cells SK-N-SH, which are known to express the transporter NET in large quantity. In this cell line, the .sup.89Y-DOTA-BG chelate showed dose-dependent inhibition of the uptake/internalization of .sup.3H-NE (IC.sub.50˜10 μM), showing to be a substrate of NET analogously to MIBG, even though with lower power (FIG. 3) and without dose-dependent cytotoxic effects which are evident up to the maximum concentration tested (100 μM). It is interesting to note how the analogous of the .sup.89Y-DOTA-BG chelate not containing yttrium, the free binder 9 (MC4325), was completely unable to compete with .sup.3H-NE for the internalization (FIG. 3). This provides the ability to prepare radio-marked versions of the chelate of Formula II (or MC4324) with an even low specific activity, because the absence of competition for NET between the chelate (MC4324) and the free binder 9 (MC4325) doesn't compromise the bonding ability to NET of the chelate (compound of Formula II, MC4324) also in the presence of an excess of free binder 9 (MC4325).

[0068] FIG. 3 depicts the competition of the .sup.89Y-DOTA-BG chelate (1a, MC4324) (non-radioactive analogue of the compound of Formula II, 1 or MC4324) and of free binder 9 (MC4325) with .sup.3H-NE (50 nM, 1 hour incubation, T=37° C.) for the uptake by the SK-N-SH cells. .sup.1H-NE a 20 μM and MIBG @ 2 μM have been used as positive controls.

[0069] Materials and Methods Relative to the Cell Tests

[0070] Cell Lines and Culture Conditions

[0071] The human neuroblastoma cell line SK-N-SH has been purchased from ATCC. The cells have been kept in E-MEM medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics in humidified atmosphere with 5% CO.sub.2 at 37° C.

[0072] Cell Uptake of .sup.3H-NE

[0073] .sup.3H-NE has been purchased from Perkin Elmer. To measure the initial cell uptake speeds of .sup.3H-NE, the cells have been grown for 16 hours in the presence of serum only, then incubated with binder medium (EMEM containing 0.2% BSA and 20 mM Hepes, pH 7.5) for 10 minutes by heating in a water bath at 37° C. The bond has been initiated by adding 0.5 ml per well of 50 nM .sup.3H-NE to the binder medium for a long time, the plates have then been placed on ice and washed three times with frozen PBS. Thus, the cell monolayers have been dried and lysed with 2% NaOH 1N SDS. The cpm (“counts” per minute) have been determined separately in the different wells in triple for each time. By cpm is meant “counts” or “hits” per minute, i.e. the nuclear disintegrations that occur in one minute. These disintegrations come from inside the cells after these have been, following the order below: 1) incubated with .sup.3H-NE, 2) washed with PBS to eliminate .sup.3H-NE remained outside the cells, 3) lysed for better detecting the radioactivity due to the internalized .sup.3H-NE only. The cpm are thus due to .sup.3H-NE which is internalized by the cell and are maximum when no competition occurs (control/non-treated or better treated cells with .sup.3H-NE only) whereas cpm decrease in a more marked way the more effective the competition with .sup.3H-NE of the substance tested each time is.

[0074] The background bond has been determined in parallel in a fourth well containing a 400 times molar excess of non-marked .sup.1H-NE (“cold”). For the competition study, the cells have been plated as above, and thus incubated for 1 hour with 50 nM .sup.3H-NE in binder medium in the absence and presence of the .sup.89Y-DOTA-BG chelate (compound of Formula II, 1a or MC4324) (non-radioactive analogue of the compound of Formula II, 1 or MC4324) and free binder 9 (MC4325) at different concentrations. The radioactivity has been determined separately in the wells in triple for each concentration value. The uptake of .sup.3H-NE has been expressed as total radioactivity percentage normalized for mg of protein.

[0075] Stability in Serum of the Chelate .sup.90Y-DOTA-BG of Formula II (1 or MC4324).

[0076] The stability in serum of the .sup.111Y-DOTA-BG chelate of Formula II (1 or MC4324) has been evaluated by measuring the release of the metal cation .sup.90Y.sup.3+ from the chelate to the serum proteins during 14 days in which it has been kept in physiological conditions. Briefly, the .sup.90Y-DOTA-BG chelate of Formula II (1 or MC4324) has been incubated with human serum (32 MBq for 16 ml of serum) at T=37° C. At regular time intervals serum aliquots have been collected and, by using centrifuge filter tubes (Amicon® Ultra-4 3K, Merck Millipore) and by centrifuging at 5500 g, the serum proteins have been separated from the non-protein fraction of serum and the radioactivity of both fractions has been measured with a liquid scintillation β counter (scintillation liquid used is Perkin Elmer ULTIMA GOLD). During the 14 days of observation no measurable loss of radioactivity (.sup.90Y.sup.3+) in favor of the protein fraction of serum has been detected.

[0077] Preparation of the Compounds of Formula III and IV

[0078] The preparation of the compounds of Formula III and IV (also denoted as compounds 10a, 10a′ and 10b, 10b′ or MC4801 and MC4803, respectively) has been carried out as depicted in Scheme 2.

[0079] In particular, the compounds 10a and 10a′ correspond to the general formula III when n is equal to 1, and Me is chosen from .sup.90Y.sup.3+ or .sup.89Y.sup.3+, respectively. The compounds 10b and 10b′ correspond instead to the general formula IV when n is equal to 2, and Me is chosen from .sup.89Y.sup.3+ or .sup.89Y.sup.3+, respectively.

[0080] Tri-tert-butyl 2,2′,2″-(10-(2-ethoxy-2-oxoethyl)-cyclen-1,4,7-tri-yl)triacetate 4, prepared as shown in Scheme 1, has been treated with an excess of the suitable commercial diamine (1,3-propanediamine or 1,4-butanediamine) at room temperature to give the respective intermediates of the synthon A′ 11a and 11b. These have then been conjugated with the synthon B (7) prepared as reported in Scheme 1, in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI), hydrated 1-hydroxybenzotriazole (HOBt) and TEA in anhydrous DCM at room temperature to give the completely protected derivatives 12a and 12b. These have then been subjected to total deprotection reaction by using a trifluoroacetic acid (TFA):tri-isopropylsilane (TIS):water 95/2.5/2.5 (vol/vol/vol) mixture leading to the respective free binders 13a (MC4802) and 13b (MC4804). Thus the free binders 13a,b have been hot complexed with a slight excess of .sup.89Y(NO.sub.3); in the presence of an ammonium acetate buffer at pH 5.5-6 in closed vial to give the non-radioactive analogues, also called “cold tracers”, of the compounds of Formula III (10a′, MC4801) and IV (10b′, MC4803). The free binders 13a,b have finally been used in large excess for the hot radio-marking with .sup.90YCl.sub.3 still in the presence of the ammonium acetate buffer at pH 5.5-6, to give the compounds of Formula III (10a, MC4801) and IV (10b, MC4803).

##STR00008##

[0081] The .sup.1H-NMR spectra of synthetic intermediates 11-13 have been recorded at 400 MHz by using a Bruker AC 400 spectrometer, whereas the .sup.1H-NMR and .sup.13C-NMR spectra of the compounds of Formula III and IV 10a′ (MC4801) and 10b′ (MC4803) have been recorded at 600 MHz and 150 MHz, respectively, by using a Bruker AC 600 spectrometer; the chemical shifts are reported in δ units (ppm) with respect to tetramethylsilane used as internal reference (Me.sub.4Si). The low-resolution mass spectra have been recorded on an API-TOF Mariner Perspective Biosystem (Stratford, Tex., USA), the samples have been injected by a Harvard pump by using a flow rate of 5-10 μl/min, infused in the Electrospray system. The high-resolution mass spectra (HR-MS) have been recorded on Orbitrap Exactive spectrometer (Thermo Fisher Scientific, Austin, Tex. USA). The analytical HPLC analyses of the compounds 12a,b, and 13a,b and compounds 10a′,b′ as non-radioactive analogues of the compounds of Formula III (10a, MC4801) and IV (10b, MC4803) have been carried out on: [0082] liquid chromatograph UHPLC Accela equipped with pumping system, autosampler and PDA Accela detector coupled to mass spectrometer LTQ ion trap equipped with an ESI interface (Thermo Fisher Scientific, Austin, Tex. USA); the experimental data have been acquired, processed and reworked by Xcalibur software (Thermo Fisher Scientific, Austin, Tex., USA); [0083] Shimadzu Nexera liquid chromatograph equipped with CBM-20A controller, four LC-30AD pumps, a DGU-20 A5R degasser, a SPD-M20A PDA detector (Standard Analytical 2.50 μl Semimicro cell), sampling speed: 100 Hertz; resolution: 4.0 nm), a thermostated column housing CTO-20AC and an autosampler SIL-30AC; an ELSD detector (Sedex 90, SEDERE, France) is connected in series to the system; the experimental data have been acquired, processed and reworked by LabSolution software. (Shimadzu Italia S.r.l., Milan, Italy).

[0084] The purification of the compounds containing .sup.89Y.sup.3+ [compounds of Formula III (10a′, MC4801) and IV (10b′, MC4803)] as non-radioactive analogues of the compounds of Formula III (10a, MC4801) and IV (10b, MC4803) has been carried out on Waters semi-preparative liquid chromatograph (RP-HPLC) equipped with Waters 590 model pump, 250 μl injector and UV spectrophotometric detector with variable wavelength and Omniscribe recorder on paper.

[0085] All the compounds have been regularly controlled by TLC and .sup.1H-NMR. The TLC has been carried out on silica gel plates supported by aluminum (Merck DC, Alufolien Kieselgel 60 F254) with spots displayed by UV light or by using an alkaline solution of KMnO.sub.4. The concentration of the solutions after the reactions and extractions has been carried out by using a rotary evaporator operating under a reduced pressure of 20 Torr. The organic solutions have been dried on anhydrous sodium anhydrous sulphate or magnesium sulphate. All the chemical reagents have been purchased from Sigma Aldrich s.r.l., Milan (Italy), TCI Europe N.V., Zwijndrecht (Belgium) or Perkin Elmer (USA) and were of the utmost purity. Normally, the samples prepared for physical and biological tests have been dried under high vacuum on P.sub.2O.sub.5 for 20 hours at temperatures between 25 and 40° C., depending on the melting point of the sample.

[0086] Herein below the experimental procedures for the preparation and chemical-physical characterization of the compounds 10-13 are described.

[0087] General procedure for the preparation of the intermediates 11a,b. Example: tri-tert-butyl 2,2′,2″-(10-(2-((3-aminopropyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triyl)triacetate (11a). The compound 4 (1.0 g, 1.66 mmoles) has been dissolved in commercial, pure 1,3-diamino propane (1.5 ml, 17.9 mmoles) and the resulting solution has been left under stirring at room temperature for 72 hours. At the end of the reaction, the propylenediamine has been removed under reduced pressure and the residue dried under vacuum to give a dark yellow foam which finally has been purified by flash chromatography (from 100% DCM to DCM/methanol 40:60 v/v) to provide the compound 11a (0.78 g, 75%) in form of white foam. .sup.1H-NMR (CDCl3) δ ppm: 1.39 (s, 27H, 3×tBu), 1.47 (m, 2H, NH.sub.2), 1.85 (n, 2H, CONHCH.sub.2CH.sub.2CH.sub.2NH), 2.44 (m, 4H, 2×CH.sub.2), 2.63 (m, 4H, 2×CH.sub.2), 2.66 (m, 2H, CONHCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2.76-2.81 (m, 8H, 4×CH.sub.2), 2.98 (s, 2H, NCH.sub.2CONH), 3.16 (br s, 4H, 2×NCH.sub.2COO/Bu), 3.20 (s, 2H, NCH.sub.2COOtBu), 3.15-3.18 (m, 2H, COHCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 8.68 (t, 1H, CONH); MS (ESI): 629 [M+H].sup.+.

[0088] 2,2′,2″-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triyl)triacetate (11b). .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.38 (s, 27H, 3×tBu), 1.48 (m, 2H, NH.sub.2), 1.51 (m, 4H, CONHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2.45 (m, 4H, 2×CH.sub.2), 2.63 (m, 4H, 2×CH.sub.2), 2.67 (m, 2H, CONHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH), 2.77-2.82 (m, 8H, 4×CH.sub.2), 2.99 (s, 2H, NCH.sub.2CONH), 3.02-3.05 (m, 2H, CONHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2), 3.17 (br s, 4H, 2×NCH.sub.2COO/Bu), 3.21 (s, 2H, NCH.sub.2COO/Bu), 8.67 (t, 1H, CONH); MS (ESI): 643 [M+H].sup.+.

[0089] General procedure for the preparation of the intermediates 12a,b. Example: tri-tert-butyl 2,2′,2″-(10-(2-((4-(3-((2,3-bis(tert butoxycarbonyl)guanidino)methyl)benzamido)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triyl)(Z)-triacetate (12b).

[0090] To a solution of N,N′-bis-tert-butoxycarbonyl-3-guanidinomethyl-benzoic acid 7 (0.16 mmol, 65.0 mg) in anhydrous DCM (1.5 ml), HOBt (0.21 mmol, 29.0 mg), EDCI (0.21 mmol, 41.0 mg), tri-tert-butyl 2,2′,2″-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triyl)triacetate 11b (102.9 mg, 0.16 mmol) and finally TEA (0.58 mmol, 59.2 mg, 82.0 μl) are added in succession by cooling in ice batch. After about 24 hours at room temperature, the solvent is removed by evaporation and the resulting crude product has been purified by flash chromatography on silica gel (Biotage Isolera One®) by eluting with a chloroform/methanol 0.fwdarw.9% mixture to provide the compound 12b (84.7 mg, yield=52%) in form of white solid. .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.35 (s, 9H, tBu), 1.39 (s, 27H, 3×tBu), 1.44 (s, 9H, tBu), 1.49-1.57 (m, 4H, CONHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCO), 1.92-3.58 (very broad multiplet set with integration corresponding to 28H, 14×CH.sub.2), 4.63 (d, 2H, NHCH.sub.2), 7.34-7.38 (m, 2H, CH benzene ring), 7.95 (m, 1H, CH benzene ring), 8.12 (d, 1H, CH benzene ring), 8.49 (m, 1H, NHCH.sub.2), 8.81 (m, 1H, CH.sub.2CONH), 9.03 (m, 1H, PhCONH), 11.4 (s, 1H, BocNH). MS (ESI): 1019 [M+H].sup.+.

[0091] Tri-tert-butyl 2,2′,2″-(10-(2-((3-(3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)benzamido)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraaza cyclododecan-1,4,7-triyl)(Z)-triacetate (12a). .sup.1H-NMR (CDCl.sub.3) δ ppm: 1.35 (s, 9H, tBu), 1.39 (s, 27H, 3×tBu), 1.44 (s, 9H, tBu), 1.70-1.76 (m, 2H, CONCH.sub.2CH.sub.2CH.sub.2NHCO), 1.92-3.58 (very broad multiplet set with integration corresponding to 28H, 14×CH.sub.2), 4.63-4.65 (d, 2H, NHCH.sub.2), 7.35-7.39 (m, 2H, CH benzene ring), 7.97 (m, 1H, CH benzene ring), 8.16 (d, 1H, CH benzene ring), 8.51 (m, 1H, NHCH.sub.2), 8.83 (m, 1H, CH CONH), 9.07 (m, 1H, PhCONH), 11.6 (s, 1H, BocNH). MS (ESI): 1005 [M+H]+.

[0092] General procedure for the preparation of the intermediates 13a,b. Example: 2,2′,2″-(10-(2-((3-(3-(guanidinomethyl)benzamido)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (13a, also denoted as MC4802). A mixture of TFA:tri-isopropylsilane:water 95:2.5:2.5 v:v:v (6.0 ml) has been added to the intermediate 12a (0.099 mmol, 100.0 mg) by cooling at 0° C. The deprotection reaction has been checked by RP-HPLC in the following analytical conditions: column: Sunfire C18, 3.5 μm (150*4.6 mm ID); Eluents: A) H2O/ACN 95/5+0.1% TFA, B) ACN+0.1% TFA. Gradient elution (start A/B 100/0; 1 min A/B 100/0; 15 min A/B 0/100, 20 min A/B 0/100). Flow: 1.0 ml/min. Detection: PDA (200-400 nm), ELSD (T: 80° C., P: 4 bar); T col=30° C.; samples dissolved in MeOH. After about 24 hours at room temperature the reaction proved to be completed. Thus, the solvent has been evaporated and the residue co-evaporated several times with anhydrous DCM (6×4 ml) and anhydrous diethyl ether (6×3 ml). The thus obtained crude product has then been triturated with anhydrous diethyl ether, filtered and washed on filter with anhydrous diethyl ether to provide the desired compound 13a (53.4 mg, yield=85%) in form of white solid. .sup.1H-NMR (D2O) δ ppm: 1.70-1.76 (m, 2H, CONHCH.sub.2CH.sub.2CH.sub.2NHCO), 2.92-3.73 (very broad multiplet set with integration corresponding to 28H, 14×CH.sub.2), 4.42 (d, 2H, NHCH.sub.2), 7.45 (m, 2H, CH benzene ring), 7.63 (m, 2H, CH benzene ring).

[0093] HR-MS (ESI): calculated mass for [C.sub.28H.sub.45N.sub.9O.sub.8+H].sup.+=636.3469 m/z; determined mass: 636.3456 m/z. Source parameters: capillary temperature 275° C.; spray voltage 3.70 kV; sheath gas 6; tube lens voltage 125 V. Resolution input: 100000. Samples dissolved in MeOH (10.sup.−5 M).

[0094] 2,2′,2″-(10-(2-((3-(3-(guanidinomethyl)benzamido)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) Triacetic Acid (13b, also Denoted as MC4804).

[0095] .sup.1H-NMR (D.sub.2O) δ ppm: 1.50-1.61 (m 4H, CONHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCO), 2.88-3.77 (very broad multiplet set with integration corresponding to 28H, 14×CH.sub.2), 4.39 (d, 2H, NHCH.sub.2), 7.44-7.47 (m, 2H, CH benzene ring), 7.60-7.63 (m, 2H, CH benzene ring).

[0096] HR-MS (ESI): calculated mass for [C.sub.29H.sub.47N.sub.9O.sub.8+H].sup.+=650.3626 m/z; determined mass: 650.3612 m/z. Source parameters: capillary temperature 275° C.; spray voltage 3.70 kV; sheath gas 6; tube lens voltage 125 V. Resolution input: 100000. Samples dissolved in MeOH (10.sup.−5 M).

[0097] General procedure for the preparation of the chelates of Formula III (10a′, MC4801) and IV (10b′, MC4803) in the “cold” versions. Example: chelate 10a′ (MC4801). To the binder 13a (19.1 mg, 30.2 μmol) dissolved in 1 ml of 1M ammonium acetate buffer (pH=5.5-6), the commercial salt 89Y(NO3)3.4H2O (15.7 mg, 45.3 μmol) has been added. The resulting solution has then been hot stirred (90° C.) in closed vial for 3 hours. At the end of the reaction, the mixture has been left to cool at room temperature and then the crude chelate has been purified from the excess salts by semi-preparative HPLC (conditions: column Hypersil ODS GOLD 5 μm: 250×10 mm; eluent: MeOH/H2O 5:95 v/v+0.02% TFA, flow: 4.0 ml/min; UV detection 254 nm) until providing, after lyophilization of the clean fractions, the “cold” chelate 10a′ (MC4801) as a white powdered solid (19.9 mg, yield=92%). .sup.1H-NMR (D2O, 600 MHz, T=5° C.) δ ppm: 1.68 (m, 2H, CONHCH.sub.2CH.sub.2CH.sub.2NHCO), 2.09 (d, 1H, CHH), 2.21-2.34 (m, 5H, 2×CH, and CHH), 2.54-2.65 (m, 7H, 3×CH.sub.2 and CHH), 2.97 (d, 1H, CH), 3.09 (d, 2H, CH.sub.2), 3.17-3.42 (m, 6H, 3×CH.sub.2), 3.44-3.58 (m, 4H, 2×CH.sub.2), 3.63 (m, 2H, CH.sub.2), 4.37 (d, 2H, —CH.sub.2-guanidine), 7.44 (m, 2H, CH benzene ring), 7.61 (m, 2H, CH benzene ring). 13C-NMR (D2O, 150 MHz, T=5° C.) δ ppm: 180.25, 179.70, 175.82, 169.96, 156.56, 136.84, 133.21, 130.62, 129.15, 129.13, 126.17, 125.22, 65.45, 65.30, 65.21, 63.02, 55.63, 55.45, 55.43, 55.35, 54.74, 54.71, 54.57, 54.16, 43.75, 39.18, 38.39, 29.95.

[0098] HR-MS (ESI): calculated mass for [C.sub.28H.sub.42N.sub.9O.sub.8Y+H].sup.+=722.2293 m/z; determined mass: 722.2284 m/z. Source parameters: capillary temperature 275° C.; spray voltage 3.70 kV; sheath gas 6; tube lens voltage 125 V. Resolution input: 100000. Samples dissolved in MeOH (10.sup.−5 M).

[0099] Chelate 10b′ (MC4803).

[0100] .sup.1H-NMR (D.sub.2O, 600 MHz, T=5° C.) δ ppm: 1.56 (m, 4H, CONHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCO), 2.07 (d, 1H, CHH), 2.21-2.32 (m, 5H, 2×CH, and CHH), 2.54-2.65 (m, 7H, 3×CH, and CHH), 2.98 (d, 1H, CHH), 3.07 (d, 2H, CH.sub.2), 3.16-3.42 (m, 6H, 3×CH.sub.2), 3.44-3.59 (m, 4H, 2×CH.sub.2), 3.65 (m, 2H, CH.sub.2), 4.37 (d, 2H, —CH.sub.2-guanidine), 7.44 (m, 2H, CH benzene ring), 7.62 (m, 2H, CH benzene ring). 13C-NMR (D.sub.2O, 150 MHz, T=5° C.) δ ppm: 180.30, 179.70, 175.82, 169.97, 156.53, 136.85, 133.22, 130.65, 129.12, 129.10, 126.21, 125.20, 65.46, 65.31, 65.23, 63.05, 55.64, 55.44, 55.42, 55.36, 54.71, 54.70, 54.59, 54.19, 43.77, 39.21, 38.42, 27.17, 27.10.

[0101] HR-MS (ESI): calculated mass for [C.sub.29H.sub.44N.sub.9O.sub.8Y+H].sup.+=736.2450 m/z; determined mass: 736.2442 m/z. Source parameters: capillary temperature 275° C.; spray voltage 3.70 kV; sheath gas 6; tube lens voltage 125 V. Resolution input: 100000. Samples dissolved in MeOH (10.sup.−5 M).

[0102] General procedure for the preparation of the chelates (radio-marking) of Formula III (10a, MC4801) and IV (10b, MC4803). Example: chelate 10a (MC4801). The chelate of Formula III (10a) has been prepared by adding 30 μl of a commercial solution of 90YCl.sub.b in 0.05 M HCl (1.07 mCi, 22.1 μmol) a 132 μl of a 0.1 mM binder 13a solution (13.2 nmol) in 1M ammonium acetate buffer (pH=5.5-6) and bringing to the final volume of 500 μl by the addition of 338 μl of the same 1M ammonium acetate buffer (pH=5.5-6). The resulting solution has then been incubated in closed vial placed on a heating block under shielded hood at 90° C. for 30 minutes. At the end of the reaction, after cooling to room temperature, different aliquots of the solution (2-10 μl) have been collected without dilution to evaluate the radio-marking yield and to carry out the quality control by ITLC. The ITLC methods used ITLC-SG and ITLC-SA Agilent plates and different eluent systems such as 1M ammonium acetate buffer (pH=5.5-6):methanol (50:50 v/v), 1M ammonium acetate buffer (pH=5.5-6):methanol:ammonia 33% (50:50:5 v/v/v) and 50 mM EDTA in 0.1 M ammonium acetate buffer (pH=6). The results of different chromatographic runs showed in agreement >99% radio-marking yield and radiochemical purity.

[0103] Chemical Stability of the Chelates Containing .sup.89Y.sup.3+ [Compounds of Formula III (10a′, MC4801) and IV (10b′, MC4803)] as Non-Radioactive Analogues of the Chelates of Formula III and IV (10a,b or MC4801 and MC4803) in Physiological Conditions.

[0104] The chemical stability of the non-radioactive analogues of the chelates of Formula III and IV, (10a′,b′ or MC4801 and MC4803) in physiological conditions has been evaluated by analysis with analytical HPLC (Shimadzu Nexera chromatograph equipped with a SPD-M20A PDA detector; Hypersil ODS GOLD 250×4.6 mm column; eluent: MeOH/H.sub.2O 5:95 v/v+0.02% TFA, flow: 1.0 ml/min; UV detection 214 nm) repeated at regular time intervals after having solubilized it in PBS buffer (c=0.9 mg/ml) and kept at T=37° C. After 5 days, both chelates were found to be still perfectly intact, with a chemical purity constantly higher than 99.5%.

[0105] Biological Validation of the Chelates Containing .sup.89Y.sup.3+ [Compounds of Formula III (10a′, MC4801) and IV (10b′, MC4803)] as Non-Radioactive Analogues of the Chelates of Formula III (10a, MC4801) and IV (10b, MC4803) as Substrates of NET in the Human Neuroblastoma Line SK-N-SH.

[0106] The ability of the “cold” chelates of Formula III (10a′, MC4801) and IV (10b′, MC4803) to act as substrates of NET has been evaluated in competition experiments with the tritiated endogenous substrate .sup.3H-NE for the uptake by the human neuroblastoma cells SK-N-SH, which are known to express the transporter NET in large quantity. In this cell line, both chelates 10a′,b′ (MC4801 and MC4803) showed dose-dependent inhibition of the uptake/internalization of .sup.3H-NE, showing to be a substrate of NET analogously to MIBG, even though with lower power (FIG. 4) and without dose-dependent cytotoxic effects which are evident up to the maximum concentration tested (100 μM). It is interesting to note how, also in this case, analogously to what already observed with the free binder 9 (MC4325) of the compound of Formula II (MC4324), the free binders 13a,b (denoted as MC4802 and MC4804, respectively) of the compounds of general formula III (MC4801) and IV (MC4803) were found to be completely unable to compete with .sup.3H-NE for the internalization up to the higher concentration tested (FIG. 4). Therefore also in this case it is possible to prepare radio-marked versions of the chelates of Formula III (10a, MC4801) and IV (10b, MC4803) with low specific activity, as the absence of competition for NET between the chelates 10a,b (MC4801 and MC4803) and the respective free binders 13a,b (MC4802 and MC4804) doesn't compromise the bonding ability to NET of the chelates [compounds of Formula III (10a, MC4801) and IV (10b, MC4803)] also in the presence of an excess of free binders 13a,b (MC4802 and MC4804).

[0107] FIG. 4 depicts the competition of the “cold” chelates of Formula III (10a′, MC4801) and IV (10b′, MC4803) and respective free binders 13a,b (MC4802 and MC4804) with .sup.3H-NE (50 nM, 1 hour of incubation, T=37° C.) for the uptake by the SK-N-SH cells. .sup.1H-NE at 20 μM and MIBG at 2 μM have been used in analogous competition experiments as positive controls.

[0108] Materials and Methods Relative to the Cell Tests

[0109] Cell Lines and Culture Conditions

[0110] The human neuroblastoma cell line SK-N-SH has been purchased from ATCC. The cells have been kept in E-MEM medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics in humidified atmosphere with 5% CO.sub.2 at 37° C.

[0111] Cell Uptake of .sup.3H-NE

[0112] .sup.3H-NE has been purchased from Perkin Elmer. To measure the initial cell uptake speeds of .sup.3H-NE, the cells have been grown for 16 hours in the presence of serum only, then incubated with binder medium (EMEM containing 0.2% BSA and 20 mM Hepes, pH 7.5) for 10 minutes by heating in a water bath at 37° C. The bond has been initiated by adding 0.5 ml per well of 50 nM .sup.3H-NE to the binder medium for a long time, the plates have then been placed on ice and washed three times with frozen PBS. Thus, the cell monolayers have been dried and lysed with 2% NaOH 1N SDS. The internal cpm (“counts” per minute) have been determined separately in the different wells in triple for each time. The background bond has been determined in parallel in a fourth well containing a 400 times molar excess of non-marked .sup.1H-NE (“cold”). For the competition study, the cells have been plated as above, and thus incubated for 1 hour with 50 nM .sup.3H-NE in binder medium in the absence and presence of the “cold” chelates of Formula III (10a′, MC4801) and IV (10b′, MC4803) and respective free binders 13a,b (MC4802 and MC4804, respectively) at different concentrations. The radioactivity has been determined separately in the wells in triple for each concentration value. The uptake of .sup.3H-NE has been expressed as total radioactivity percentage normalized for mg of protein.

[0113] Stability in Serum of the Chelates of Formula III (10a, MC4801) and IV (10b, MC4803).

[0114] The stability in serum of the chelates of Formula III (10a, MC4801) and IV (10b, MC4803) has been evaluated by measuring the release of the metal cation .sup.90Y.sup.3+ from the chelates to the serum proteins during 14 days in which they have been kept in physiological conditions. Briefly, the chelates of Formula III (10a, MC4801) and IV (10b, MC4803) have been incubated with human serum (32 MBq per 16 ml of serum) at T=37° C. At regular time intervals serum aliquots have been collected and, by using centrifuge filter tubes (Amicon® Ultra-4 3K, Merck Millipore) and by centrifuging at 5500 g, the serum proteins have been separated from the non-protein fraction of serum and the radioactivity of both fractions has been measured with a liquid scintillation β counter (scintillation liquid used is Perkin Elmer ULTIMA GOLD). During the 14 days of observation no measurable loss of radioactivity (.sup.90Y.sup.3+) in favor of the protein fraction of serum has been detected.

BRIEF DESCRIPTION OF THE FIGURES

[0115] FIG. 1 depicts the ITLC-SG runs relative to the radio-marking of the binder 9 with .sup.90YCl.sub.3.

[0116] FIG. 2 depicts the HPLC plots relative to the evaluation of the chemical stability over time of the compound .sup.89Y-DOTA-BG (1a).

[0117] FIG. 3 depicts the competition of the chelate of Formula II (1a, MC4324) and the respective free binder 9 (MC4325) with .sup.3H-NE for the uptake by the neuroblastoma cells SK-N-SH

[0118] FIG. 4 depicts the competition of the chelates of Formula III (10a′, MC4801) and IV (10b′, MC4803) and respective free binders 13a (MC4802) and 13b (MC4804) with .sup.3H-NE for the uptake by the neuroblastoma cells SK-N-SH

DETAILED DESCRIPTION OF THE FIGURES

[0119] FIG. 1 depicts the ITLC-SG runs relative to the radio-marking of the binder 9 with .sup.90YCl.sub.3 for the preparation of the chelate .sup.90Y-DOTA-BG of Formula II (1, or MC4324).

[0120] FIG. 2 depicts the HPLC plots relative to the chemical stability over time of the .sup.89Y-DOTA-BG chelate (1a) solubilized in PBS buffer (c=0.9 mg/ml). FIG. 2A depicts the plot of .sup.89Y-DOTA-BG (1a) solubilized in PBS buffer at T=37° C. and time t=0 min. FIG. 2B depicts the plot of .sup.111Y-DOTA-BG (1a) solubilized in PBS buffer at T=37° C. and time t=21 hours. FIG. 2C depicts the plot of .sup.89Y-DOTA-BG (1a) solubilized in PBS buffer at T=37° C. and time t=5 days.

[0121] FIG. 3 depicts the results of the competition assay of the chelate of Formula II (1a, MC4324) and respective free binder 9 (MC4325) with .sup.3H-NE for the uptake/internalization through NET by the human neuroblastoma cells SK-N-SH. The first column of FIG. 3 starting from the left is relative to the non-treated control (lack of competition). The following two columns are relative to two positive controls, the .sup.1H-NE itself (20 μM) and the MIBG (2 μM) both non-marked which compete with .sup.3H-NE for the internalization at the depicted doses. From the fourth to the eighth column in FIG. 3 the results of the competition experiments with increasing doses (5 to 100 μM) between the chelate of Formula II (1a, MC4324) and .sup.3H-NE are depicted. In the ninth and tenth column in FIG. 3 the results of the competition experiments at two different concentrations (50 and 100 μM) between the free binder of the compound of Formula II (9 or MC4325) and .sup.3H-NE are reported.

[0122] FIG. 4 depicts the results of the competition assay of the chelates of Formula III (10a′, MC4801) and IV (10b′, MC4803) and the respective free binders 13a (MC4802) and 13b (MC4804) with .sup.3H-NE for the uptake/internalization through NET by the human neuroblastoma cells SK-N-SH. The first column of FIG. 4 starting from the left is relative to the non-treated control (absence of competition). The following two columns are relative to two positive controls, the .sup.1H-NE itself (20 μM) and the MIBG (2 μM) both non-marked which compete with .sup.3H-NE for the internalization at the depicted doses. The fourth and fifth column in FIG. 4 depict the results of the competition experiments between the chelate of Formula III (10a′, MC4801) at two different concentrations (50 and 100 μM) and .sup.3H-NE. The sixth and seventh column in FIG. 4 depict the results of the competition experiments between the free binder of the chelate of Formula III (13a, MC4802) at two different concentrations (50 and 100 μM) and .sup.3H-NE. The eighth and ninth column in FIG. 4 depict the results of the competition experiments between the chelate of Formula IV (10b′, MC4803) at two different concentrations (50 and 100 μM) and .sup.3H-NE. The tenth and eleventh column in FIG. 4 depict the results of the competition experiments between the free binder of the chelate of Formula IV (13b, MC4804) at two different concentrations (50 and 100 μM) and .sup.3H-NE.