Development of [ 18 F]Fluoro-C-glycosides to radiolabel peptides for PET application

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1 Development of [ ]luoro-c-glycosides to radiolabel peptides for PET application Dr Charlotte Collet, Petry., Chrétien., Karcher G., Pellegrini-Moïse., Lamandé-Langle. ancyclotep, Plateforme d imagerie moléculaire Université de Lorraine CR, UMR 7565 CHRU de ancy-brabois ancy-rance 1

2 Biomolecules Labelling Biomolecules antibodies, nanobodies, peptides : RGD, omatostine ew radiopharmaceutic PET imaging 68 Ga, ensitivity of the biomolecules direct labelling with is not possible Using prosthetic group (small molecule easy radiolabelled) coupling with the biomolecule LG Prosthetic Group Prosthetic Biomolecule group Prosthetic group radiolabelled biomolecule 1

3 Biomolecules labelling umerous coupling methods: Acylation : -C-H- -Alkylation : -- xime : -CH=-- Prosthetic group link Hydrazone : -CH=-H- Click : -triazol- oft conditions, excellent yields Glycosylation : DG Increase the biodistribution Kuhnast B., Dollé. Curr Radiopharm., 2010, 3, C. ; C. Wängler et al. Curr. Med. Chem., 2010, 17,

4 Biomolecules labelling umerous coupling methods: Acylation : -C-H- -Alkylation : -- xime : -CH=-- Prosthetic group link Hydrazone : -CH=-H- Click : -triazol- oft conditions, excellent yields Glycosylation : DG Increase the biodistribution Click glycosylation. Maschauer et al. Mol. Pharmaceutics, 2014, 11, ;. Maschauer at al. Carbohydr. Res., 2009, 344, ;. Prante et al. Bioconjugate Chem. 2007,, ;. Maschauer et al. Angew. Chem. Int. Ed., 2010, 49, ;. Prante et al. J. Label. Compd. Radiopharm., 2007, 50, ;. Boutureira et al. Chem. Commun., 2011, 47,

5 Previous works -Glycosidic Prosthetic group H H H Good incorporation yields on the position 6 Easy to conjuguate with peptide via propargylated cysteine H H H CuAAC + peptide 55 to 97% H H H Peptide = gluthation, "RGD" peptide αvβ3 A high uptake of -glyco-c(rgdfc) was shown by PET in a subcutaneous abscess model in the rat, revealing the potential of this tracer to monitor integrin erum stability 60 min Hydrolytic lability of glycosidic bound Collet C., et al. [2016], J. Label. Compd. Radiopharm. ; Lamandé-Langle., et al. [2014], Bioorg. Med. Chem. 22: ; Lamandé-Langle.,et al. [2014], W A ; Chapleur et al. [2013]Toward imaging glycotools by click coupling; Witczak, Z. J.,Bielski, R. Eds,Click Chemistry in Glycoscience: ew Develpments and strategies, Wiley,

6 bjectives Develop new carbohydrate prosthetic group to label peptide by click glycosylation H H H stability H H H Glucose derivative -Glycoside C-Glycoside on-radioactive synthesis Precursor of radiolabelling on radioactive references Radiochemistry Radiolabelling Automation of [ ]Glycopeptide radiosynthesis 4

7 ynthesis of C-glycosidic prosthetic groups tereoselective allylation Bn Bn Bn Bn Tetrabenzyl glucose Commercial H Ac 2, pyridine Bn Bn Bn 1 2 Bn TMI, CH 2 Cl 2 AllylMgBr Ac AllylTM, B 3.Et 2, CH 3 C Bn Bn Bn Bn Bn Bn Bn 3 Bn 3 : 90/10 : 5/95 Parallel synthesis for alpha and beta configuration same synthetic strategy Bn Bn Bn 1) 9-BB, TH then EtH, ah, H 2 2 Bn 1) H 2 (30 psi), Pd(H) 2, dioxane Bn Ac Bn Bn 2) PPh 3, CBr 4, CH 2 Cl 2 Bn Br Ac 2) isopropenyl acetate, Ac p-tsh Ac Br 1) a, DM 2) a 0, MeH H H H H 1) TrCl, Pyr, DMAP then Ac 2 2) HCH, Et 2 Ac Ac H Ac 6 7 5

8 ynthesis of C-glycosidic prosthetic groups on-radioactive references and precursors of radiolabelling Ac Ac H 7 Ac 1) DAT, diglyme 100 C, 1h 2) a 0, MeH Tf 2, CH 2 Cl 2, pyridine H H Ac Ac Tf H 8 H H Ac Ac H 8 on radioactive prosthetic group Ac 9 Tf Ac 9 Precursor of radiolabelling ptimized syntheses global yields : 15 % over 14 steps 6

9 ynthesis of non radioactive glycopeptides Click coupling between C-glycosidic prosthetic groups and peptides H peptide H 2, MeH, H 4 H, ta, 3h Br (1 eq.) H H H + peptide Cu(Ac) 2,a ascorbate H 2, ta, 2h 68 to 87% H H H peptide Model peptides : RGDC and c(rgdfc) 4 Cold glyco"rgd" H H H - -Gluc-RGDC - -Gluc-RGDC H H H H - -Gluc-c(RGDfC) - -Gluc-c(RGDfC) H H H 2 H H 2 H H 2 H H H CH CH H H H H H 7

10 Radiolabelling radiolabelling performed in classical conditions (K 222 /K 2 C 3 ) in CH 3 C Tf Ac Ac Ac K[ ]-K 222 CH 3 C (2mL) H H H Both C-glycoside ( and ) configurations were studied: everal parameters were studied : - Amount of precursors (5, 7.5 and 10 mg) - Ratio of K 222 /K 2 C 3 ( ) - T C (85 and 95 C) - Reaction duration (10, 15 and 20 min) 8

11 Radiolabelling radiolabelling performed in classical conditions (K 222 /K 2 C 3 ) in CH 3 C Tf Ac Ac Ac K[ ]-K 222 CH 3 C (2mL) H H H Both C-glycoside ( and ) configurations were studied: everal parameters were studied : - Amount of precursors (5, 7.5 and 10 mg) - Ratio of K 222 /K 2 C 3 ( ) - T C (85 and 95 C) - Reaction duration (10, 15 and 20 min) Best conditions : 7,5 mg of precursor K 222 /K 2 C 3 (5.3 mg/1.3 mg) 85 C 15 min C-Glycoside : 60 ±3%* C-Glycoside : 71 ±5%* * : radiochemical yield decay corrected 8

12 Automation - preparation labelling pre-purification Tf Ac Ac Ac precursor of radiolabeling [ ]K 222 /K 2 C 3 AC Ac 10 min, 85 C Ac Ac automated semipreparative HPLC deprotection click HPLC control 9

13 Automation HPLC purification - preparation labelling pre-purification automated semipreparative HPLC deprotection automated semipreparative HPLC C8 Column, AC/H 2 50/50 MBq/mL Ac Ac Ac rt: 11 min 0 min radio click HPLC control mau 10 1, ,8 4 0,6 2 0,4 0, ,5 1 1, min Excellent radiochemical and chemical purity 10

14 Automation HPLC purification - preparation labelling pre-purification automated semipreparative HPLC deprotection MBq/mL automated semipreparative HPLC C8 Column, AC/H 2 50/50 Ac Ac Ac rt: 11 min 0 min radio Analytical HPLC C8 Column, AC/H 2 50/50 radio UV 220 nm click HPLC control mau 10 1, ,8 4 0,6 2 0,4 0, ,5 1 1, UV min ELD Excellent radiochemical and chemical purity 10

15 Automation RGD - preparation labelling pre-purification Ac Ac Ac ah 1M 30 C, 5M H H H Cu(Ac) 2,a ascorbate 55 C, 20min H H H RGD automated semipreparative HPLC deprotection click HPLC control 11

16 Automation RGD - preparation labelling pre-purification Ac Ac Ac ah 1M 30 C, 5M H H H Cu(Ac) 2,a ascorbate 55 C, 20min H H H 50-68% RGD automated semipreparative HPLC deprotection Good radiochemical yield and purity H H H - -Gluc-RGDC - -Gluc-RGDC H H H H - -Gluc-c(RGDfC) - -Gluc-c(RGDfC) H H 2 H Click H H 2 H H 2 H H H CH CH H H H H H HPLC control tability of glycopeptide in foetal bovin serum >2h 11

17 Conclusion ynthesis and radiosynthesis of 2 new C-glycosidic prosthetic groups to radiolabel peptide Good -incorporation yield up to 76% Click conjugation with RGD peptide derivatives in good yield Excellent serum stability > 2h Perspectives In vitro evaluation (integrine) In vivo PET evaluation by with tumor overexpressing integrins 12

18 Thank you for your attention