[18F]Fluoroethyltriazolyl Monocyclam Derivatives as Imaging Probes for the Chemokine Receptor CXCR4

Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis. [68Ga]Pentixafor is a promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of 68Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of [18F]RPS-534 (7.2 ± 0.3 %ID/g) and [18F]RPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background, [18F]RPS-547, and especially [18F]RPS-534, are promising 18F-labeled candidates for imaging CXCR4 expression.


General
All commercially available materials were used as received unless otherwise indicated. 4-(bromomethyl)-3-iodobenzoic acid [1] and 2-fluoroethyl azide [2] were synthesized according to literature procedures. All reactions were carried out under an atmosphere of argon in an oven-dried round bottom flask with magnetic stirring, unless otherwise noted. Reactions were monitored by UPLC. HPLC purifications were performed using a Waters AutoPure HPLC/MS system equipped with XBridge OBD prep C18 5m (19 x 150 mm) column and SQD2 mass spectrometer. A 10-minute gradient of 5% -95% acetonitrile (0.1% formic acid) in water (0.1% formic acid) was used as mobile phase. All NMR spectra were recorded on Bruker DRX-500 spectrometer (500 MHz for 1 H and 125 MHz for 13 C). Chemical shifts, , are reported in ppm, with the residual solvent resonance as internal standard. NMR data are reported as following: chemical shift (multiplicity s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad, coupling constant in Hz, and integration). PKS8163: 4-(bromomethyl)-3-iodo-benzoic acid (8.00 g, 23.5 mmol) was dissolved in methanol (50.0 mL) and H2SO4 (2.50 mL, 46.93 mmol) was added to the solution. The mixture was refluxed under overnight. The solvent was removed, and the mixture was purified by Combi-Flash (silica gel; slow 0 -10% ethyl acetate in hexanes gradient) to give product (3.4 g, 54%) as white solid. 1  , transdichlorobis(triphenylphosphine)palladium (II) (74.9 mg, 107 mol) and triethylamine (1.5 eq.) were dissolved in THF (2 mL) and stirred at ambient temperature for 1 h in a microwave vessel flushed with argon to give a yellow suspension. The copper acetylide solution was added to the microwave vessel to give a clear solution. The mixture was heated to 120 °C under microwave for 1 h. The mixture was cooled, the solvent was evaporated, and the crude residue was purified by Combi-Flash (silica gel; 0 -10 % gradient of methanol in dichloromethane) to give the silyl phenylacetylide (130 mg, 67%) as a white solid. The solid (125 mg, 137 mol) was dissolved in THF (3 mL). The solution was cooled to 10 °C and tetrabutylammonium fluoride (1.0 M in THF) (138 mol, 140 L) was added. The solution was warmed to ambient temperature and stirred for 1 h. The solvent was evaporated, and crude residue was purified by HPLC to give p-RPS-533 (57 mg, 50%) as a white solid. 1

RPS-533:
Copper sulfate pentahydrate (0.5 M, 66 L) and sodium ascorbate (1.5 M, 22 L) were mixed under argon atmosphere and stirred for 20 min until the solution turned dark orange. In another round bottom flask p-RPS-533 (14.0 mg, 17 mol) and 1-azido-2-fluoro-ethane (0.33 M, 102 L) were dissolved in DMF (1 mL). To this mixture was added the Cu(I) reagent, and the resulting reaction was stirred at ambient temperature overnight. The mixture was diluted with dichloromethane and passed through a basic alumina plug. The filtrate was evaporated, and crude product was purified by HPLC to give a white solid (8.7 mg, 56%). The solid (6.0 mg, 6.5 mol) was dissolved in DCM (0.5 mL) and the solution was cooled to 0 °C. Trifluoroacetic acid (0.5 mL) was added to the solution and the mixture was slowly warmed to ambient temperature. After completion of reaction, excess solvent and TFA were evaporated and crude was purified by HPLC to give RPS-533 (5.8 mg, 74%) as the TFA salt and a white solid. 1   PKS8199: A mixture of PKS8168 (206 mg, 277 mol) and triBoc-cyclam (138 mg, 277 µmol) in DCM (5 mL) was stirred at ambient temperature for 2 h and then sodium triacetoxyborohydride (176 mg, 830 mol) was added. The mixture was stirred at ambient temperature overnight. Excess reagent was quenched with aqueous NaHCO3, the layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated, and the crude residue was purified by Combi-Flash (silica gel; ethyl acetate in hexane) to give PKS8199 (230 mg, 67%) as a colorless gum which turned into a fluffy solid under vacuum. 1

PKS8201
: Ethynyl(trimethyl)silane (52 L, 366 mol) and triethyl amine (1.5 eq.) were added to a suspension of copper(I) iodide (23 mg, 122 mol) in THF (1 mL) under argon atmosphere and the mixture was stirred at ambient temperature for 1 h. PKS8199 (150 mg, 122 mol), transdichlorobis(triphenylphosphine)palladium (II) (43 mg, 61 mol) and triethylamine (1.5 eq.) were dissolved in THF (2 mL) and stirred at ambient temperature for 1 h in a microwave vessel flushed with argon to give a yellow suspension. The copper acetylide reagent was added to the microwave vessel to give a clear solution. The mixture was heated to 120 °C under microwave for 1 h. The mixture was cooled, the solvent was evaporated, and the crude residue was purified by Combi-Flash (silica gel; 0 -10% gradient of methanol in dichloromethane) to give PKS8201 (120 mg, 82%). 1  p-RPS-545: PKS8201 (120 mg, 100 mol) was dissolved in THF (3 mL) and cooled to 10 °C. Tetrabutylammonium fluoride (1.0 M in THF) (100 L, 100 mol) was added, and the solution was warmed to ambient temperature and stirred for 1 h. The solvent was evaporated, and the crude residue was purified by HPLC to give p-RPS-545 (35 mg, 31%) as white solid. 1 (1 mL). The Cu(I) reagent was added to the mixture, resulting in a dull green color, and the reaction was stirred at ambient temperature overnight. The mixture was diluted with dichloromethane and passed through a basic alumina plug. The filtrate was evaporated, and crude residue was purified by HPLC to give PKS8239 (10 mg, 95%) as a white solid. 1

PKS8179:
To a solution of PKS8178 (96 mg, 211 mol) in DCM (5 mL) was added manganese dioxide (86 mg, 845 mol, 85% purity). The mixture was stirred at ambient temperature for 24 h. The reaction was then filtered through Celite and evaporated to give PKS8179 (87 mg, 91%) as a white solid that was used without further purification. 1

PKS8181:
A mixture of PKS8179 (85 mg, 188 mol) and triBoc-cyclam (94 mg, 188 mol) in DCM (4 mL) was stirred at ambient temperature for 2 h and then sodium triacetoxyborohydride (120 mg, 564 mol) was added. The resulting mixture was stirred at ambient temperature overnight. Excess reagent was quenched with aqueous NaHCO3, the layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated, and the crude residue was purified by Combi-Flash (silica gel; ethyl acetate in hexane) to give PKS8181 (140 mg, 80%) as a colorless gum which turned into a fluffy solid under vacuum. 1  PKS8183: Ethynyl(trimethyl)silane (25 mg, 256 mol) and triethyl amine (1.5 eq.) were added to a suspension of copper iodide (16 mg, 85 mol) in THF (1 mL) under argon atmosphere and the mixture was stirred at ambient temperature for 1 h. In parallel, PKS8181 (80 mg, 85 mol), transdichlorobis(triphenylphosphine)palladium (II) (30 mg, 43 mol) and triethylamine (1.5 eq.) in THF (1 mL) were stirred at ambient temperature for 1 h in a microwave vessel flushed with argon to give a yellow suspension. The copper acetylide reagent was added to the microwave vessel to give a clear solution.
The mixture was heated at 120 °C under microwave for 1 h. The mixture was cooled, the solvent was evaporated, and the crude residue was purified by Combi-Flash (silica gel; 0 -10 % gradient of methanol in dichloromethane) to give PKS8183 (42 mg, 54%) as a white solid. 1  p-RPS-534: PKS8183 (30 mg, 33 mol) was dissolved in THF (2 mL) and cooled to 10 °C. Then tetrabutylammonium fluoride (1.0 M in THF, 33 L, 33 mol) was added, the solution was warmed to ambient temperature, and stirred for 1 h. The solvent was evaporated, and crude residue was purified by HPLC to give p-RPS-534 (22.5 mg, 81%) as a white solid. 1

PKS8233:
Copper sulfate pentahydrate (0.5 M, 18 L) and sodium ascorbate (1.5 M, 12 L) were mixed in an argon atmosphere and stirred for 20 min. The solution turned black and then brown. In another round bottom flask, p-RPS-534 (7.4 mg, 9 mol) and 1-azido-2-fluoro-ethane (1.6 mg, 18 mol) were dissolved in DMF (1 mL). To this mixture was added the Cu(I) reagent, resulting in a dull green solution. The reaction was stirred at ambient temperature overnight, diluted with dichloromethane and passed through a basic alumina plug. The filtrate was evaporated, and the crude residue was purified by HPLC to give PKS8233 (5.8 mg, 71%) as a white solid. 1  RPS-534: PKS8233 (5.8 mg, 6 mol) was dissolved in DCM (0.5 mL) and the solution was cooled to 0 °C. Trifluoroacetic acid (0.5 mL) was added to the solution and the mixture was allowed to warm to ambient temperature slowly. After completion of reaction, excess solvent and TFA were evaporated and the crude residue was purified by HPLC to give RPS-534 (3.2 mg, 42%) as a colorless gum. 1 1,145.9,137.4,137.2,132.7,130.6,130.5,130.0,128.0,124.3,123.7,123.3,81.8 (d,J = 168.2 Hz), 53.7, 50.6 (d, J = 19.1 Hz), 49.8, 49.6,49.4, 48.8, 48.4, 47.7, 45.1, 44.2, 43.9, 43.7, 24.8, 22.0. 19   PKS8204: A mixture of terephthalaldehyde (134 mg, 1.0 mmol) and triBoc-cyclam (250 mg, 500 mol) in DCM (10 mL) was stirred at ambient temperature for 2 h and then sodium triacetoxyborohydride (318 mg, 1.5 mmol) was added. The mixture was stirred overnight at ambient temperature. The reaction was quenched with aqueous NaHCO3, the layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated. The crude residue was purified by Combi-Flash (silica gel; ethyl acetate in hexanes) to give PKS8204 (198 mg, 64%) as a colorless gum, which turned into a fluffy solid under vacuum. 1  p-RPS-547: A mixture of PKS8204 (90 mg, 145 mol), propargyl amine (12 L, 189 mol) and acetic acid (4.2 L, 73 mol,) in DCM (4 mL) was stirred at ambient temperature for 2 h, then sodium triacetoxyborohydride (37 mg, 175 mol) was added. The mixture was stirred overnight at ambient temperature, but the reaction did not go to completion. Therefore additional propargyl amine (12 L) and sodium triacetoxyborohydride (37 mg) were added and the mixture was stirred for an additional 24 h. The reaction was quenched with aqueous NaHCO3, the layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated. The crude residue was purified by Combi-Flash (silica gel; ethyl acetate in hexane) and p-RPS-547 (76 mg, 79%) was isolated as a colorless gum that turned into a fluffy solid under vacuum. 1 (500 L). The Cu(I) reagent was added to the reaction mixture, resulting in a dull green color. The mixture was stirred at ambient temperature overnight. It was then diluted with dichloromethane and passed through a short plug of basic alumina. The filtrate was evaporated, and the crude residue was purified by HPLC to give PKS8256 (12 mg, 72%). 1

Figure S6. Synthesis of p-RPS-546 and RPS-546.
p-RPS-546: A mixture of 4-ethynylbenzaldehyde (65 mg, 500 mol) and triBoc-cyclam (250 mg, 500 mol) in dichloromethane (4 mL) was stirred at ambient temperature for 2 h, and then sodium triacetoxyborohydride (318 mg, 1.5 mmol) was added. The mixture was stirred at ambient temperature overnight. The reaction was quenched with aqueous NaHCO3, the layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated, and the crude residue was purified by Combi-Flash (silica gel; ethyl acetate in hexanes) to give p-RPS-546 (185 mg 60%) as a white solid. 1

A.
B. Table S2. Hill slopes determined from the competition assay vs [ 68 Ga]Pentixafor in PC3-CXCR4 cells and the saturation binding assays in PC3-CXCR4 cells. Slopes are expressed as value ± SD. AMD-3465 was not included in the saturation binding assays.