Chemical Synthesis

18 kDa Translocator protein (TSPO) ligand synthesis

MBa-144-2 (50 mg, 0.11 mmole), Pd[P(t-Bu)₃]₂ (1.0 mg, 2.0 µmole), hexamethylenediamine (40 mg, 0.34 mmole), potassium hydroxide (85%, 10 mg, 0.15 mmole), cetyltrimethylammonium bromide (0.4 mg, 1.1 µmole), water (3 µL) and dry toluene (0.20 mL) were mixed under Argon. The stirring mixture was heated at 90 °C for 16 hours (h). After the reaction, the mixture was minimized under vacuum and the residue was purified with column chromatography using dichloromethane/methanol/ammonia = 100 : 10 : 0.1 to yield the compound as colorless oil (29 mg, 53%).

N-Fmoc-DOX-14-O-hemiglutarate synthesis

Doxorubicin hydrochloride salt (100 mg, 0.17 mmol) and Fmoc-OSu (60 mg, 90 µmol) was dissolved in Dimethylformamide (DMF) (2.0 mL). N,N-Diisopropylethylamine (DIPEA) (60 µL) was added to the stirring mixture in room temperature. After 3 h, the solvent was removed in vacuo, and the residue was triturated from 0.1% aqueous trifluoroacetic acid (vol/vol). The precipitates were collected by filtration and washed with cold diethyl ether to yield the protected doxorubicin as red orange powder (115 mg, 87%). The fluorenylmethoxycarbonyl (Fmoc)-protected doxorubicin was subsequently dissolved in DMF (2.0 mL). Glutaric anhydride (23 mg, 0.20 mmole) and DIPEA (52 µL) was added to the solution. The resulting mixture was stirred overnight at room temperature. The solvent was removed in vacuo, and the residue was triturated from 0.1% aqueous trifluoroacetic acid (vol/vol). The precipitates were collected by filtration to yield the ester compound as a red orange powder (107 mg, 81%).

Dox-TSPO synthesis

N-Fmoc-DOX-14-O-hemiglutarate (20 mg, 23 µmol) and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU) (20 mg, 53 µmol) were dissolved in DMF (1.0 mL) under Argon. The orange solution turned into a dark red color after addition of DIPEA (10 µL). After stirring at 0 °C for 10 minutes, a solution of the TSPO ligand (10 mg, 21 mmol) in DMF (0.50 mL) was introduced. The resulting mixture was continually stirred for 18 h at room temperature. The volatiles were removed via rotary evaporation and the residue was subjected to column chromatography using dichloromethane/methanol = 100 : 1.5 to 100 : 3 to 100 : 4.5. The combined fraction containing the desired product was dried to yield a red paste (23 mg, 82%).

The Fmoc-protected product (10 mg) was subsequently dissolved in dry DMF (1.0 mL). Piperidine (50 µL) was added and the solution turned dark blue instantly. The reaction was monitored using electrospray ionization mass spectroscopy (ESI-MS) until the starting materials were consumed. The reaction mixture was titrated with trifluoroacetic acid in DMF until the mixture turned red-orange color. The solvent was removed under reduced pressure. The product was purified by semi-preparative reverse-phase high-performance liquid chromatography (HPLC) using gradient eluents from 60% A to 40% A over 40 min, held at 5% A for an additional 5 min, and finally return to 60% A in 1 minute. The low boiling solvent of the collected fractions (retention time = 8.9 min) was removed using rotary evaporation. The remaining solution was lyophilized to give the titled compound as a red solid (2.3 mg, 28%).

Nuclear Magnetic Resonance Spectroscopy (NMR) Data

Proton Nuclear Magnetic Resonance Spectroscopy (¹H-NMR) (400 MHz, DMSO-d₆)

δ 14.04 (s, 1H), 13.27 (s, 1H), 7.94 (d, 2H, J = 4.8 Hz), 7.74 (m, 4H,), 7.68 (t, 1H, J = 4.9 Hz), 7.31 (t, 2H, J = 7.2 Hz), 7.25 (m, 1H), 7.17 (m, 1H), 7.13 (t, 1H, J = 7.2 Hz), 6.81 (m, 1H), 6.68 (m, 2H), 6.27 (br s, 1H), 5.59 (s, 1H), 5.47 (br s, 1H), 5.29 (d, 1H, J = 1.7 Hz), 5.14 (q, 2H, J = 17.7 Hz), 4.97 (m, 1H), 4.80 (d, 1H, J = 14.0 Hz), 4.64 (d, 1H, J = 14.4 Hz), 4.21 (q, 1H, J = 6.6 Hz), 4.00 (s, 3H), 3.00 (q, 2H, J = 6.6 Hz), 2.99 (ABq, 4H, Δν_AB = 53.6 Hz, J_AB = 18.5 Hz), 2.67 (quin., 1H, J = 1.7 Hz), 2.39 (t, 3H, J = 7.3 Hz), 2.33 (q, 1H, J = 1.6 Hz), 2.28 (d, 2H, J = 14.8 Hz), 2.12 (t, 3H, J = 7.4 Hz), 2.07 (d, 2H, J = 14.8 Hz), 1.87(s, 3H), 1.77 (q, 2H, J = 7.0 Hz), 1.75 (s, 1H), 1.68 (d, 2H, J = 11.9 Hz), 1.43 (m, 2H), 1.34 (quin., 2H, J = 7.0 Hz), 1.25 (q, 4H, J = 7.7 Hz), 1.16 (d, 3H, J = 6.6 Hz).

Carbon-13 Nuclear Magnetic Resonance Spectroscopy (13C-NMR) (100 MHz, CD₃OD)

δ 209.3, 188.4, 188.1, 175.5, 174.4, 162.7, 158.4, 157.5, 156.7, 137.5, 136.5, 135.7, 133.4, 131.4, 125.9, 121.7, 120.8, 120.7, 120.65, 120.63, 120.3, 119.6, 118.1 (q, J = 93 Hz), 116.4, 112.7, 112.4, 101.3, 77.6, 71.7, 68.2, 68.1, 67.2, 57.4, 56.4, 50.3, 50.0, 49.9, 40.3, 37.5, 36.2, 34.0, 34.0, 33.67, 33.66, 30.3, 29.66, 29.64, 29.63, 29.4, 29.0, 28.9, 28.04, 28.03, 28.02, 27.6, 27.4, 23.9, 22.6, 22.1, 17.2. MS (ESI): calculated for C₆₀H₆₈FN₄O₁₆ ([M+H]⁺) m/z = 1119.461, found 1119.460.

Devices

¹H and ¹³C NMR spectra were recorded at 25 ºC on the Brucker Avance III 400 megahertz (MHz). Unless otherwise specified, chemical shifts δ were expressed in parts per million (ppm) based on the residual solvent signal in dimethyl sulfoxide (DMSO)-d₆ (δ 2.50 ppm) or CD₃OD (δ 0.00 ppm), and coupling constants J are given in Hz. Coupling patterns are abbreviated as s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), ABq (AB quartet), and m (multiplet).

Electrospray mass spectra were recorded on a Waters LCT Premier mass spectrometer in positive ion mode.

Reverse phase HPLC was performed using Waters HPLC system equipped with Waters-1525 solvent delivery systems and Waters 2998 ultraviolet (UV) detector. Separation was achieved on a semi-preparative Phenomenex (Torrance, CA) Jupiter® 5µm C18 300Å column (5 micron, 250 × 10.00 mm, Part Number: 00G-4053-N0) at a flow rate maintained at 2.0 mL/min. A solvent system composed of water containing 0.1% trifluoroacetic acid (TFA) as solvent A and acetonitrile containing 0.1% TFA as solvent B was used over the stated gradient. The column effluent was monitored with a ultraviolet-visible (UV/Vis) detector set at 254 and 480 nanometer (nm).