A Multifunctional HBED-Type Chelator with Dual Conjugation Capabilities for Radiopharmaceutical Development
Ata Makarem∗
a
German Cancer Research Center (DKFZ) Heidelberg, Division of Radiopharmaceutical Chemistry, INF 223, d-69120 Heidelberg, Germany eMail: a.makarem@dkfz.de
b
The Institute of Cancer Research, Division of Radiotherapy and Imaging, 123 Old Brompton Road, SW7 3RP, London, UK
,
Mohammadreza Kamali Sarvestani
a
German Cancer Research Center (DKFZ) Heidelberg, Division of Radiopharmaceutical Chemistry, INF 223, d-69120 Heidelberg, Germany eMail: a.makarem@dkfz.de
c
Ruprecht-Karls-Universität-Heidelberg, Institute of Inorganic Chemistry, INF 270, DE-69120 Heidelberg, Germany
,
Karel D. Klika
d
German Cancer Research Center (DKFZ) Heidelberg, Molecular Structure Analysis, INF 280, d-69120 Heidelberg, Germany
,
Klaus Kopka
a
German Cancer Research Center (DKFZ) Heidelberg, Division of Radiopharmaceutical Chemistry, INF 223, d-69120 Heidelberg, Germany eMail: a.makarem@dkfz.de
e
German Cancer Consortium (DKTK),
d-69120 Heidelberg, Germany
› InstitutsangabenThis work was partly funded by a grant from German Cancer Aid (Deutsche Krebshilfe), project number 70112043.
Bifunctional HBED chelators are hexadentate complexing ligands (chelators) that tightly coordinate to trivalent gallium and, additionally, are able to bind to bioactive molecules. In nuclear medicine, HBED-based radiopharmaceuticals are used as powerful radiotracers for tumor imaging. Among variants of bifunctional HBED chelators, HBED-CC is the most well-known; it possesses two terminal carboxylic acid groups that are able to undergo bioconjugation by amide-bond formation. However, to permit bioconjugation through click coupling, we previously modified the structure of HBED-CC and introduced HBED-NN chelator bearing two azide functions. We have now combined the conjugation capabilities of HBED-CC and HBED-NN chelators in one molecule and have created HBED-NC, which possesses both azide and carboxylic acid groups. The advantage of HBED-NC is that it provides options for constructing either monomeric or heterodimeric radiolabeling precursors. This work describes the synthesis of HBED-NC by either of two pathways.
1 New address: K. Kopka, Helmholtz-Zentrum Dresden-Rossendorf e.V., Institute of Radiopharmaceutical Cancer Research, Bautzner Landstraße 400, 01328 Dresden, Germany.
2a
Hope TA,
Truillet C,
Ehman EC,
Afshar-Oromieh A,
Aggarwal R,
Ryan CJ,
Carroll PR,
Small EJ,
Evans MJ.
J. Nucl. Med. 2017; 58: 81
9a
Baranski AC,
Schäfer M,
Bauder-Wüst U,
Wacker A,
Schmidt J,
Liolios C,
Mier W,
Haberkorn U,
Eisenhut M,
Kopka K,
Eder M.
Bioconjugate Chem. 2017; 28: 2485
10a
Afshar-Oromieh A,
Zechmann CM,
Malcher A,
Eder M,
Eisenhut M,
Linhart HG,
Holland-Letz T,
Hadaschik BA,
Giesel FL,
Debus JJ,
Haberkorn U.
Eur. J. Nucl. Med. Mol. Imaging 2014; 41: 11
17 Full name for HBED-CC: 3,3′-[{1,4-bis[(carboxymethyl)amino]-1,4-butanediyl}bis(4-hydroxy-3,1-phenylene)]dipropanoic acid.
18a
Trencsényi G,
Dénes N,
Nagy G,
Kis A,
Vida A,
Farkas F,
Szabó JP,
Kovács T,
Berényi E,
Garai I,
Bai P,
Hunyadi J,
Kertész I.
J. Pharm. Biomed. Anal. 2017; 139: 54
19 Full name for HBED-NN (ester-protected): di-tert-butyl 2,2′-(ethane-1,2-diylbis{[5-(2-azidoethyl)-2-hydroxybenzyl]azanediyl})diacetate.
20 Full name for HBED-NC (ester-protected): 3-(3-{[{2-[[5-(2-azidoethyl)-2-hydroxybenzyl](2-tert-butoxy-2-oxoethyl)amino]ethyl}(tert-butoxycarbonyl)amino]methyl}-4-hydroxyphenyl)propanoic acid.
21a
Rostovtsev VV,
Green LG,
Fokin VV,
Sharpless KB.
Angew. Chem. Int. Ed. 2002; 41: 2596
23 Compounds 1, 2, and 4 were prepared by the reported methods.15,16 Characterization data for these compounds, general experimental methods, and all NMR spectra are provided in the Supporting Information.
242-{[(2-Aminoethyl)amino]methyl}-4-(2-azidoethyl)phenol (3)
To a solution of compound 1 (1.0 g, 5.3 mmol, 1.0 equiv) in CH2Cl2 (5 mL) was added tert-butyl (2-aminoethyl)carbamate (1.0 g, 6.3 mmol, 1.2 equiv). The mixture was stirred for 1 h at r.t. then diluted with CH2Cl2 and washed with 0.5 M aq NaHSO3. The organic phase was dried (Na2SO4), filtered, and concentrated under reduced pressure. The resulting orange solid was dissolved in warm F3CCH2OH (40 mL) at 40–50 °C, then cooled in an ice bath. NaBH4(0.47 g, 12.45 mmol, 2.5 equiv) was added in portions, and the mixture was allowed to warm to r.t. and stirred overnight under an inert atmosphere. The reaction was quenched with H2O (100 mL) and the resulting mixture was extracted with CH2Cl2. The organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was dissolved in a 4 M solution of HCl in 1,4-dioxane (10 mL) and the mixture was stirred for 5 min at r.t. Et2O (40 mL) was added and the mixture was stirred for another 10 min. The precipitate was collected by filtration, washed with Et2O, and dried under vacuum to give salt 3 as white solid; yield: 1.4 g (4.54 mmol, 87%).
1H NMR (400.13 MHz, D2O, 25 °C): δ = 7.29 (m, 2 H, CHCHCCH), 6.98 (d, 3JH–H = 8.3 Hz, 1 H, CHCHCCH), 4.33 (s, 2 H, BnCH2), 3.56 (t, 3JH–H = 6.7 Hz, 2 H, CH2CH2N3), 3.45 (m, 4 H, CH2CH2NH), 2.87 (t, 3JH–H = 6.7 Hz, 2 H, CH2CH2N3). 13C{1H} NMR (100.61 MHz, D2O, 25 °C): δ = 153.81 (COH), 132.06 (CHCHCCH), 130.92 (CHCHCCH), 116.88 (CCH2N), 115.81 (CHCHCCH), 52.22 (CH2N3), 47.38 (BnCH2), 43.44 (BnCH2NHCH2), 35.39 (CH2NH2), 33.44 (CH2CH2N3). HRMS (ESI+, MeOH): m/z [M + H]+ calcd for C11H18N5O: 236.1506; found: 236.1539 (100%).
(27) Methyl 3-(3-{[(2-{[5-(2-Azidoethyl)-2-hydroxybenzyl]amino}ethyl)amino]methyl}-4-hydroxyphenyl)propanoate (5)
Method 1: To a mixture of compound 3 (0.34 g, 1.1 mmol, 1.1 equiv) and aldehyde 2 (0.21 g, 1.0 mmol, 1.9 equiv) in dry MeOH (6 mL) was added anhyd Et3N (420 μL, 1.0 mmol, 3 equiv), and the mixture was stirred at r.t. for 30 min. The volatiles were then completely removed under reduced pressure, and the residue was dissolved in warm F3CCH2OH (8 mL) at 40–50 °C. The resulting solution was cooled in an ice bath and NaBH4 (0.19 g, 5 mmol, 5 equiv) was then added in portions. The mixture was then allowed to warm to r.t. and stirred overnight under an inert atmosphere. The reaction was quenched with H2O (~70 mL), and the mixture was extracted with CH2Cl2. The extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure to give product 5 in quantitative yield. The NMR spectrum of this product showed no significant impurities, and it was therefore used without further purification in the next step. However, for spectroscopic characterization a portion was purified by reverse-phase HPLC to give a colorless solid.
Method 2: The same procedure as described in Method 1 was applied to aldehyde 1 (0.19 g, 1.0 mmol, 1 equiv) and salt 4 (0.36 g, 1.1 mmol, 1.1 equiv). Compound 5 was again obtained in quantitative yield.
1H NMR (400.13 MHz, CDCl3, 25 °C): δ = 7.00 (m, 2 H, COHCHCH), 6.79 (m, 4 H, CCHC and COHCHCH), 3.97 (d, 4 H, BnCH2), 3.66 (s, 3 H, CH3), 3.44 (t, 3JH–H = 7.2 Hz, 2 H, CH2CH2N3), 2.80 (m, 8 H, NH–C2H4–NH, CH2CH2CO2 and CH2CH2N3), 2.57 (t, 3JH–H = 8.0 Hz, 2 H, CH2CH2CO2). 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C): δ = 173.62 (CO2CH3), 156.91 (COH–ArCO2 or COH–ArN3), 156.42 (COH–ArCO2 or COH–ArN3), 131.31 (C–C2H4–CO2), 129.25 (CCHC–C2H4–CO2), 128.99 (CCHC–C2H4–N3), 128.73 (CHCHC–C2H4–CO2), 128.50 (CHCHC–C2H4–N3), 122.36 (CCHC–C2H4–CO2), 122.19 (CCHC–C2H4–N3), 116.76 (CHCHC–C2H4–CO2 or CHCHC–C2H4–N3), 116.56 (CHCHC–C2H4–CO2 or CHCHC–C2H4–N3), 52.82 (CH2CH2N3), 52.66 (d, BnCH2), 51.62 (CO2CH3), 48.02 (d, NHCH2CH2NH), 36.12 (CH2CH2CO2), 34.60 (CH2CH2N3), 30.16 (CH2CH2CO2). HRMS (ESI+, MeCN–MeOH): m/z [M + H]+ Calcd for C22H30N5O4: 428.2292; Found: 428.2294 (59%).
3-[3-({[3-[[5-(2-Azidoethyl)-2-hydroxybenzyl](2-tert-butoxy-2-oxoethyl)amino]-1-(tert-butoxycarbonyl)propyl]amino}methyl)-4-hydroxyphenyl]propanoic Acid (HBED-NC)
A mixture of compound 5 (0.89 g, 2.03 mmol, 1 equiv), Na2CO3 (0.86 g, 8.13 mmol, 4 equiv), and tert-butyl 2-bromoacetate (630 μL, 4.23 mmol, 2.1 equiv) in anhyd MeCN (40 mL) was stirred under reflux overnight. The mixture was cooled to r.t., filtered, and concentrated under reduced pressure. The residue was dissolved in warm MeOH (30 mL, ~50 °C), and 4 M aq NaOH (10 mL) was slowly added. The resulting mixture was stirred for 2 h then the pH was adjusted to 4–5 with 1 M aq HCl. The crude product was extracted with EtOAc and the extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The product was isolated by reverse-phase HPLC as a colorless solid; yield: 0.07 g (0.11 mmol, 5.4%)
1H NMR (400.13 MHz, CDCl3, 25 °C): δ = 7.00 (m, 2 H, COHCHCH), 6.76 (m, 4 H, CCHC, COHCHCH and COHCHCH), 3.69 (s, 2 H, BnCH2–ArN3), 3.65 (s, 2 H, BnCH2–ArCO2Me), 3.40 (t, 3JH–H = 7.2 Hz, 2 H, CH2CH2N3), 3.14 (d, 4 H, NCH2-t-BuO2), 2.82 (t, 3JH–H = 7.6 Hz, 2 H, CH2CH2CO2H), 2.75 (t, 3JH–H = 7.2 Hz, 2 H, CH2CH2N3), 2.65 [br s, 4 H, N(CH2)2N], 2.59 (t, 3JH–H = 7.6 Hz, 2 H, CH2CH2CO2H), 1.44 (d, 18 H, CH3). 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C): δ = 176.16 (CO2H), 170.22 (d, CO2t-Bu), 156.31 (COH–ArCO2 or COH–ArN3), 155.89 (COH–ArCO2 or COH–ArN3), 130.92 (C–C2H4–CO2H), 129.69 (C–C2H4–N3), 129.53 (CCHC–C2H4–CO2H), 129.33 (CCHC–C2H4–N3), 129.05 (CHCHC–C2H4–CO2H), 128.66 (CHCHC–C2H4–N3), 121.80 (CCHC–C2H4–CO2H), 121.67 (CCHC–C2H4–N3), 116.73 (CHCHC–C2H4–CO2H), 116.58 (CHCHC–C2H4–N3), 82.33 [(CO2CCH3)–ArCO2H], 82.24 [(CO2CCH3)–ArN3], 58.04 (BnC), 55.73 [(NCH2CO2)–ArCO2H], 55.60 [(NCH2CO2)–ArN3], 52.84 (CH2CH2N3), 50.35 (NCH2CH2N), 36.13 (CH2CH2CO2H), 34.67 (CH2CH2N3), 29.99 (CH2CH2CO2H), 28.16 (CH3). HRMS (ESI+, MeCN–MeOH): m/z [M + H]+ Calcd for C33H48N5O8+: 642.3497; Found: 642.3503 (100%).