Neopentyl glycol-based radiohalogen-labeled amino acid derivatives for cancer radiotheranostics

Background L-type amino acid transporter 1 (LAT1) is overexpressed in various cancers; therefore, radiohalogen-labeled amino acid derivatives targeting LAT1 have emerged as promising candidates for cancer radiotheranostics. However, 211At-labeled amino acid derivatives exhibit instability against deastatination in vivo, making it challenging to use 211At for radiotherapy. In this study, radiohalogen-labeled amino acid derivatives with high dehalogenation stability were developed. Results We designed and synthesized new radiohalogen-labeled amino acid derivatives ([211At]At-NpGT, [125I]I-NpGT, and [18F]F-NpGT) in which L-tyrosine was introduced into the neopentyl glycol (NpG) structure. The radiolabeled amino acid derivatives were recognized as substrates of LAT1 in the in vitro studies using C6 glioma cells. In a biodistribution study using C6 glioma-bearing mice, these agents exhibited high stability against in vivo dehalogenation and similar biodistributions. The similarity of [211At]At-NpGT and [18F]F-NpGT indicated that these pairs of radiolabeled compounds would be helpful in radiotheranostics. Moreover, [211At]At-NpGT exhibited a dose-dependent inhibitory effect on the growth of C6 glioma-bearing mice. Conclusions [211At]At-NpGT exhibited a dose-dependent inhibitory effect on the tumor growth of glioma-bearing mice, and its biodistribution was similar to that of other radiohalogen-labeled amino acid derivatives. These findings suggest that radiotheranostics using [18F]F-NpGT and [123/131I]I-NpGT for diagnostic applications and [211At]At-NpGT and [131I]I-NpGT for therapeutic applications are promising. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-024-00244-4.

[ 125 I]NaI (ca.3.7 MBq/μL) was purchased from PerkinElmer (Waltham, MA, USA). 211At was supplied by the Research Center for Nuclear Physics at Osaka University through the Supply Platform of Short-lived Radioisotopes and from Fukushima Medical University. 211At was produced by the 209 Bi(α, 2n) 211 At reaction followed by separation and purification by a dry distillation method. 18F was supplied by the Chiba University Hospital. 1 H-NMR, 13 C-NMR and 19 F-NMR spectra were recorded on a JEOL JNM-ECS 400 spectrometer (JEOL, Tokyo, Japan).Mass spectrometry was carried out using an AccuTOF LC-plus (JMS-T100LP, JEOL, Tokyo).HPLC purification was performed using a Hitachi L-2400 system coupled to a NaI(Tl) radioactivity detector (Gabi star, Raytest, Strubenhardt, Germany).The radioactivity counts were determined with an automated gamma well counter (Wizard 3, PerkinElmer Japan, Yokohama, Japan).N αtert-butoxycarbonyl-L-tyrosine tert-butyl ester was prepared according to a previously described method (1).The other chemicals obtained from the commercial sources were of reagent grade or higher and were used without purification.

HPLC or TLC methods
Analytical reversed-phase HPLC (RP-HPLC) was performed with a Unison US-C18 column (4.6 × 150 mm, Imtakt, Kyoto, Japan) at a flow rate of 1 mL/min using a linear gradient mobile phase starting from 90% A (0.1% aqueous trifluoroacetic acid (TFA)) and 10% B (acetonitrile with 0.1% TFA) to 50% A and 5% B in 20 min, followed by 0% A and 100% B in 30 min (System A) or from 95% A and 5% B to 0% A and 100% B in 30 min (System B).In System C, the mobile phase was maintained 100% A and 0% B for 5 min, then changed to 70% A and 30% B, followed by a change 0% A and 100% B. In System D, the mobile phase was maintained 95% A (0.01 M acetate buffer (pH 6.0)) and 0% B (acetonitrile) for 5 min, then changed gradiently to 70% A and 30% B in 20 min, followed by a change 0% A and 100% B in 30 min.RP-TLC was performed using Silica gel 60 RP-18 F254S (Merck, Darmstadt, Germany) and was

S5
developed in a mixture of tert-butyl alcohol : acetic acid : H2O (4:1:1, v/v).Preparative RP-HPLC was performed with a Unison US-C18 column (20 × 150 mm, Imtact, Kyoto) at a flow rate of 5 mL/min using a linear gradient mobile phase starting from 90% A and 10% B to 50% A and 50% B in 30 min (System D).

211
At was dissolved in acetonitrile (20 µL), 10% sodium ascorbate solution (2.5 μL), and 1% N, Ndiisopropylethylamine (DIEA)/acetonitrile solution of compound 4 (0.3 mg/50 µL) were added.The mixture was heated at 37°C for 30 min and passed through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvent.After removing the solvent, 80% trifluoroacetic acid (TFA)/H2O was added to remove the protecting groups.The mixture was heated at 60°C for 30 min.TFA was evaporated by N2 gas, and the solution was neutralized with a 2M sodium acetate solution.The solution was purified by RP-HPLC with system A, then desalted through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvents.The solvent was removed in vacuo, diluted with Phosphate-Buffered Saline (PBS), and used for further evaluation studies.
The mixture was heated at 37°C for 60 min and passed through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvent.After removing the solvent, 80% TFA/H2O was added to remove the protecting groups.The mixture was heated at 60°C for 30 min.TFA was evaporated by N2 gas, and the solution was neutralized with a 2M sodium acetate solution.The solution was purified by RP-HPLC with system A, then desalted through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvents.The solvent was removed in vacuo, diluted with PBS, and used for further evaluation studies.

Radiosynthesis of [ 18 F]F-NpGT
To the reaction vial, an aqueous potassium carbonate solution (5.85 mg/L, 0.3 mL), a Cryptofix 222 acetonitrile solution (14 mg/0.3 mL), and a solution containing 18 F were added.The mixture was concentrated using a CEV1B evaporator (BioChromato, Kanagawa, Japan) with heating to 100°C.Subsequently, super-dehydrated acetonitrile (Wako, S10 Osaka, Japan) was added to facilitate azeotropic evaporation.An acetonitrile solution of compound 4 (1.0 mg/50 μL) was added to the reaction vial.The mixture was heated at 100°C for 30 min and passed through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvent.After removing the solvent, 80% TFA/H2O was added to remove the protecting groups.
The mixture was heated at 60°C for 30 min.TFA was evaporated by N2 gas, and the solution was neutralized with a 2M sodium acetate solution.The solution was purified by RP-HPLC with system C, then desalted through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvents.The solvent was removed in vacuo, diluted with PBS, and used for further evaluation studies.

Radiosynthesis of [ 125 I]-IMT
α-Methyl L-tyrosine (0.2 ng/35 µL in 0.4 M phosphate buffer (pH 6.2)) and [ 125 I]NaI (0.5 μL, ca. 1 MBq) were mixed.A 10 µL of Chloramine-T (0.45 mg/mL in 0.05 M phosphate buffer (pH 6.2)) was added to the mixture for 2 min at room temperature.The reaction was terminated by adding a 10% Na2SO4 solution (20 μL).The solution was purified by RP-HPLC using system B, and [ 125 I]I-IMT was desalted through a Sep-Pak C18 cartridge using H2O and acetonitrile as solvent.The product solution was concentrated in vacuo, diluted with PBS, and used for further evaluation studies.

FIGURE
FIGURE S3.RP-HPLC radiochromatograms of [ 125 I]I-NpGT and radiometabolites in the urine.[125 I]I-NpGT (185 kBq) was administered to ICR normal mice and urine was collected up to 6 hours after administration.The urine was analyzed using RP-HPLC (system D).As a result, we observed intact [ 125 I]I-NpGT and unidentified metabolites.Very little radioactivity was observed in the void volume fraction where free iodine elutes.