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1002

Bioconjugate Chem. 2009, 20, 1002–1009

Design, Synthesis, and Imaging of Small Amphiphilic Rhenium and 99m Technetium Tricarbonyl Complexes Eszter Boros,†,§ Urs O. Ha¨feli,‡ Brian O. Patrick,† Michael J. Adam,*,§ and Chris Orvig*,† Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146, East Mall, Vancouver, British Columbia, V6T 1Z3, Canada, and TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, V6T 2A3, Canada. Received January 20, 2009; Revised Manuscript Received March 9, 2009

The design, synthesis, radiolabeling, evaluation of stability, and in vivo investigation are presented of three variably charged, novel, short C8 chain derivatized chelators for the [M(CO)3]+ core (M ) 99mTc or Re). Labeling with [99mTc(CO)3]+ showed complex formation in under 1 h reaction time and high stability toward 24 h histidine and cysteine challenges, as well as distinctive log P values for each complex. Distinct localization of small amphiphilic molecules in in vivo systems depends on the charge of the polar moiety and was studied via biodistribution (4 h) and imaging (15 min, 1, 2, and 4 h) in female C57Bl/6 mice.

INTRODUCTION Because of its low cost and availability, 99mTc has been a radionuclide of interest in radiopharmaceutical chemistry and nuclear medicine for the past four decades (1). Resulting from intensive research, the vast majority of nuclear medicine scans utilize this isotope, which displays the ideal physical properties t1/2 ) 6 h and γ ) 141 keV for imaging with single photon emission computed tomography (SPECT) (2). While today’s imaging facilities still rely on high oxidation state Tc cores (3-5), much research has turned its focus toward the versatile and highly stable fac-[99mTc(CO)3(H2O)3]+ core developed by Alberto et al. (6). The fac-[99mTc(CO)3(H2O)3]+ complex has the metal center in a 99mTc(I)-d6 low-spin electron configuration (inert toward a broad range of experimental conditions) and can be prepared in high yields via a commercially available kit (Isolink, Covidien, Mansfield, MA) under aqueous conditions using boranocarbonate anion as the reducing agent and in situ CO source (6). Simple exchange of the three water molecules can be achieved with suitable chelates in a fac coordination mode (7, 8), shown previously on examples of bioconjugates such as derivatized amino acids, carbohydrates, peptides, and nucleosides (9-11). Over the past decade, synthesis of novel imaging agents based on 99mTc has shifted toward target-specific peptide derivatives with one type of chelating entity for the [99mTc(CO)3]+ core, following the so-called “technetium-tagged” approach (12), In contrast to this, it was our aim in this work to obtain a simple, small, and nonspecific “technetium-based” complex with potential uptake in a large range of different tissues and also to explore the in vivo properties of novel, amphiphilic metal complexes of the [Tc(CO)3]+ core. In an attempt to achieve this, the synthesis incorporated a mimic of the charge distribution of molecules highly abundant and essential to cell membranes such as sphingosine (neutral), lysophosphatidic acid (negatively charged headgroup), and sphingomyelin (zwitterionic headgroup), aiming for an in vivo distribution of the resulting * To whom correspondence should be addressed: [email protected] (M.J.A.); [email protected] (C.O.). † Department of Chemistry, University of British Columbia. ‡ Faculty of Pharmaceutical Sciences, University of British Columbia. § TRIUMF.

Figure 1. Envisioned biological pathway of the “integrated approach molecules”.

complexes comparable to other small amphiphilic molecules such as cocamidopropyl betaine (CAPB, zwitterionic), sodium dodecylsulfate (SDS, anionic), and octanol (neutral). These compounds, also known as surfactants, are known to integrate into the lipid bilayer, before their concentration (much larger than the concentration range imaging agents usually operate at) exceeds the saturation concentration and solubilizes the membrane (13). The synthesis was achieved by varying the tridentate chelator but attaching a short, saturated C8-chain in a consistent fashion via a simple ester bond; this differs strongly from the previously used tagged approach with long-chain fatty acids (14). The goal was also to investigate and compare the heartspecific uptake of these new compounds with strictly cationic conjugates (15, 16). Therefore, the design of these molecules was such that the size of the polar and lipophilic entities are in the same size range but of strongly differing polarity (Figure 1). Exploring the in vivo behavior of these compounds, following the less investigated approach of nontarget-specific uptake of small amphiphilic molecules, was of major interest.

EXPERIMENTAL DETAILS Instruments and Materials. All solvents and reagents were used as received. Re(CO)5Br is commercially available (Strem). The analytical thin-layer chromatography (TLC) plates were aluminum-backed ultrapure silica gel 60, 250 µm; the flash column silica gel (standard grade, 60 Å, 32-63 mm) was from

10.1021/bc900022c CCC: $40.75  2009 American Chemical Society Published on Web 04/02/2009

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Bioconjugate Chem., Vol. 20, No. 5, 2009 1003

Scheme 1. Synthesis Pathway for Proligands HL1 and H2L3

Scheme 2. Synthesis Pathway for Proligand HL2

Scheme 3

Silicycle. 1H and 13C NMR spectra were recorded on Bruker AV300 or AV400 instruments at ambient temperature; the NMR spectra are expressed on the δ scale and referenced to residual solvent peaks or internal tetramethylsilane. Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded on a Micromass LCT instrument at the Department of Chemistry, University of British Columbia. High-performance liquid chromatography (HPLC) analysis of cold compounds was done on a Phenomenex Synergi 4 µm Hydro-RP 80A column (250 × 4.6 mm) in a Waters WE 600 HPLC system equipped with a 2478 dual-wavelength absorbance UV detector run using the Empower software package. IR spectra were collected neat in the solid or liquid state on a Thermo Nicolet 6700 FT-IR spectrometer. TLC analysis of radiolabeled compounds was performed on a Bioscan-System 200 Imaging scanner, equipped with a Bioscan-Autochanger 100. Radio-TLCs were prepared with acetonitrile as the mobile phase. HPLC analyses of radiolabeled complexes were performed on a Knauer Wellchrom K-1001 HPLC equipped with a K-2501 absorption detector and a Capintec radiometric well counter. A Phenomenex HydroSynergi 4 µm C18 RP analytical column with dimensions 250 × 4.6 mm was used. HPLC solvents consisted of 0.1% trifluoroacetic acid in water (solvent A) and methanol (solvent B). Samples were analyzed and purified with a linear gradient

method (100% solvent A to 100% solvent B over 30 min). For biodistributions, organ samples were counted in a Packard Cobra II autogamma counter one to two days after administration. µSPECT and CT images were recorded on an XSPECT instrument from Gamma Medica (Northridge, CA). Syntheses (Schemes 1 and 2). N-Boc-Ethanolamine. Boc2O (2.18 g, 10 mmol) was added to ethanolamine (610 mg, 10 mmol) at 0 °C and allowed to stir for 1 h. A colorless precipitate formed immediately with the evolution of heat. Column chromatography (hexanes/ethyl acetate, silica, 6:1) with subsequent evaporation of the solvent yields 1.45 g of the desired product as a colorless oil (9.9 mmol, yield 90%, Rf ) 0.1). ESIMS (MeOH, 30 V): 184.1 [M + Na]+. 1H NMR (CDCl3, 300 MHz, δ): 3.55 (t, CH2-OH, 2H), 3.14 (t, CH2-NH-Boc, 2H), 1.33 (s, (CH3)3, 9H), 13C NMR (CDCl3, 300 MHz, δ): 155.6 (carbonyl-C), 79.34 (C-(CH3)3), 61.6 (CH2-OH), 42.98 (CH2NH-Boc), 28.17 ((CH3)3). IR (cm-1): 1682 (m), 1517 (m), 1164 (m). N-Boc-2-Aminoethyl Ester Octanoic Acid. N-Boc-Ethanolamine (1.45 g, 9.9 mmol) was dissolved in toluene (50 mL) to afford a clear solution. EDMA (ethyldimethylamine, 20 mL) and octanoyl chloride (1.908 g,11.78 mmol, 1.3 equiv) were added dropwise simultaneously. A cloudy precipitate formed immediately, and the reaction solution was stirred at room

1004 Bioconjugate Chem., Vol. 20, No. 5, 2009

temperature for another 1.5 h, after which time the solvent and a large fraction of the added EDMA were evaporated under reduced pressure. The residue was redissolved in H2O (30 mL) and extracted into ether (30 mL) twice. The organic phase was collected and the solvent evaporated under reduced pressure to afford the crude product as a yellow oil which was purified by column chromatography (hexanes/ethyl acetate, 1:1, Rf ) 0.6) to yield N-Boc-2-aminoethyl ester octanoic acid (1.505 g, 5.45 mmol, yield 53%) after solvent evaporation. ESI-MS (MeOH, 30 V): 326.2 [M + K]+. 1H NMR (CDCl3, 400 MHz, δ): 4.07 (t, CH2-OCOR, 2H), 3.33 (t, CH2-NHCOR, 2H), 2.27 (t, ROCOCH2, 2H), 1.57 (m, ROCOCH2-CH2, 2H), 1.40 (s, (CH3)3, 9H), 1.24 (m, CH2-CH2-CH2, 8H), 0.83 (t, CH3, 3H). 13C NMR (CDCl3, 400 MHz, δ): 173.54 (carbonyl-C), 155.69 (carbamateC), 79.55 (C-(CH3)3), 63.22 (CH2-OCOR), 39.55 (CH2-NHBoc), 33.98, (ROCO-CH2), 31.48, (ROCOCH2-CH2), 28.75 (ROCO(CH2)2-CH2), 28.17 ((CH3)3), 24.72 (CH2), 22.42 (CH2CH3), 13.86 (CH3). IR (cm-1): 1684 (m), 1631 (m), 1482 (w). 2-Aminoethyl Ester Octanoic Acid. N-Boc-2-Aminoethyl ester octanoic acid (1.505 g, 5.45 mmol) was dissolved in dichloromethane (4 mL). Trifluoroacetic acid (4 mL) was added, and the reaction solution was stirred for 20 min after which time a slow change of color to light yellow was observed. TFA and DCM were removed in vacuo, and the oily residue was purified by column chromatography (DCM/MeOH, 4:1, Rf ) 0.15) yielding 2-aminoethyl ester octanoic acid (444 mg, 2.37 mmol, yield 43%) after rotary evaporation as a colorless oil which solidifies upon standing. ESI-MS (MeOH, 30 V): 188.2 [M + H]+, 210 [M + Na]+. 1H NMR (MeOD, 300 MHz, δ): 4.47 (s, NH2, 2H), 3.55 (t, CH2-OCOR, 2H), 3.25 (t, CH2-NH2, 2H), 2.15 (t, ROCO-CH2, 2H), 1.57 (m, ROCOCH2-CH2, 2H), 1.28 (m, CH2-CH2-CH2, 8H), 0.86 (t, CH3, 3H). 13C NMR (MeOD, 300 MHz, δ): 173.7 (carbonyl-C), 62.27 (CH2-OCOR), 43.42(CH2-NH2), 37.61-23.95 (six signals, CH2), 14.66 (CH3). IR (cm-1): 3312 (w), 2954 (s), 2924 (s), 2855 (s), 1637 (s), 1552 (m), 1464 (m). N-Pyridinemethyl-2-aminoethyl Ester Octanoic Acid. 2-Aminoethyl ester octanoic acid (0.51 g, 2.7 mmol) was dissolved in dry ethanol (15 mL). Na2CO3 (∼10 equiv, 2.86 g) was added to afford a turbid solution. Pyridine-2-aldehyde (0.27 g, 237 µL, 0.95 equiv) was added, and the reaction mixture was stirred overnight at room temperature. After the formation of the intermediate imine was monitored by TLC, NaBH4 (95 mg, 2.51 mmol, 0.98 equiv) was added and the reaction mixture was subsequently stirred for another 1 h. DCM (20 mL) was then added and extracted twice with a saturated solution of NaHCO3 (20 mL). The organic fractions were collected, and the solvent was evaporated. The crude product was purified by column chromatography (DCM/MeOH, 85:15, Rf ) 0.55) yielding N-pyridinemethyl-2-aminoethyl ester octanoic acid (361 mg, 1.3 mmol, yield 48%) as a colorless oil with low viscosity, after evaporation of the solvent. ESI-MS (MeOH, 30 V): 279 [M + H]+, 301.2 [M + Na]+. 1H NMR (CDCl3, 300 MHz, δ): 8.48 (d, o-pyridine-H, 1H), 7.66 (t, p-pyridine-H, 1H), 7.32 (d, m-pyridine-H, 1H), 7.17 (t, m-pyridine-H, 1H), 4.72 (s, NH, 1H), 3.91 (s, pyr-CH2, 2H), 3.63 (t, CH2-OCOR, 2H), 3.37 (t, CH2-NH, 2H), 2.14 (t, ROCO-CH2, 2H), 1.56 (m, ROCOCH2CH2, 2H), 1.22 (m, CH2-CH2-CH2, 8H), 0.82 (t, CH3, 3H). 13C NMR (CDCl3, 300 MHz, δ): 174.54 (carbonyl-C), 149.03 (opyridine-C), 148.27 (o-pyridine-C), 136.84 (m-pyridine-C), 122.26 (m-pyridine-C), 120.71 (p-pyridine-C), 64.21 (pyr-CH2), 61.60 (CH2-NH2), 54.06 (CH2-OCOR), 42.12-22.46 (six signals, CH2), 13.93 (CH3). IR (cm-1): 3294 (br), 2925 (s), 2855 (s), 1632 (s), 1595 (s), 1549 (m). N-Bis(tert-Butylcarboxyethyl)-2-aminoethyl Ester Octanoic Acid. 2-Aminoethyl ester octanoic acid (0.1 g, 0.53 mmol) was dissolved in dry DCM (10 mL). Na2CO3 (1 g) was added to

Boros et al.

afford a turbid solution. tert-Butyl bromoacetate (209 mg, 179 µL, 1.06 mmol, 2 equiv) was added, and the mixture was stirred for 2 days. After the presence of the product was confirmed by ESI-MS, the solvent was evaporated and the crude mixture was purified by column chromatography (silica, hexanes/ethyl acetate, 1:1, Rf ) 0.8) to yield N-bis(tert-butylcarboxyethyl)2-aminoethyl ester octanoic acid (60 mg, 0.144 mmol, yield 27%) after rotary evaporation of all solvents. ESI-MS (MeOH, 30 V): 438.3 [M + Na]+. 1H NMR (CDCl3, 300 MHz, δ): 4.13 (t, CH2-OCOR, 2H), 3.69 (s, tBu-carbonyl-C-CH2-N, 4H), 3.45 (t, CH2-N, 2H), 2.95 (t, ROCO-CH2, 2H), 1.55 (m, ROCOCH2CH2, 2H),, 1.41 (s, (CH3)3, 18 H), 1.21 (m, CH2-CH2-CH2, 8H), 0.84 (t, CH3, 3H). 13C NMR (CDCl3, 300 MHz, δ): 170.18 (carbonyl-C), 166.03 (tBu-carbonyl-C), 62.68 (CH2-OCOR), 58.19 (tBu-carbonyl-C-CH2-N), 52.42 (CH2-N), 34.06-22.40 (six signals, CH2), 27.61 ((CH3)3), 13.88 (CH3). N-N-(tert-Butylcarboxyethyl)-pyridinemethyl-2-aminoethyl Ester Octanoic Acid. N-Pyridinemethyl-2-aminoethyl ester octanoic acid (361 mg, 1.3 mmol) was dissolved in dry DCM (20 mL). Na2CO3 (∼10 equiv, 2.86 g) was added to afford a turbid solution. tert-Butyl bromoacetate (1.4 equiv, 354 mg, 1.8 mmol) was added, and the mixture was stirred for 2 days. The solvent was evaporated after confirmation of product presence by ESI-MS, and the crude product was purified by column chromatography (silica, DCM/MeOH, 95:5, Rf ) 0.8) yielding N-N-(tert-butylcarboxyethyl)-pyridinemethyl-2-aminoethyl ester octanoic acid (109 mg, 0.27 mmol, yield 21%), after rotary evaporation of all solvents, as a light yellow oil. ESI-MS (MeOH, 30 V): 415.3 [M + Na]+. 1H NMR (CDCl3, 300 MHz, δ): 8.52 (d, o-pyridine-H, 1H), 7.68 (t, p-pyridine-H, 1H), 7.56 (d, m-pyridine-H, 1H), 7.17 (t, m-pyridine-H, 1H), 4.16 (t, CH2OCOR, 2H), 4.03 (s, pyr-CH2, 2H), 3.40 (s, tBu-carbonyl-CCH2-N, 2H), 3.00 (t, CH2-N, 2H), 2.26 (t, ROCO-CH2, 2H), 1.56 (m, ROCOCH2-CH2, 2H), 1.46 (s, (CH3)3, 9 H), 1.26 (m, CH2-CH2-CH2, 8H), 0.87 (t, CH3, 3H). 13C NMR (CDCl3, 300 MHz, δ): 184.65 (tBu-carbonyl-C), 173.66 (carbonyl-C), 148.64 (o-pyridine-C), 136.82 (o-pyridine-C), 122.91 (m-pyridine-C), 122.11(m-pyridine-C), 62.52 (pyr-CH2), 60.30 (CH2-N), 56.21 (tBu-carbonyl-C-CH2-N), 52.53 (CH2-OCOR), 34.24-22.54 (six signals, CH2) 28.15 ((CH3)3), 14.02 (CH3). N-N-Bis(tert-Butylcarboxyethyl)-pyridinemethyl-2-aminoethyl Ester Octanoic Acid. N-N-Bis(tert-Butylcarboxyethyl)pyridinemethyl-2-aminoethyl ester octanoic acid is afforded as a side product of the synthesis of N-N-(tert-butylcarboxyethyl)pyridinemethyl-2-aminoethyl ester octanoic acid and separated from the main product by column chromatography (silica, DCM/ MeOH, 95:5, Rf of product: 0.35) yielding N-N-bis(tertbutylcarboxyethyl)-pyridinemethyl-2-aminoethyl ester octanoic acid (37 mg, 0.072 mmol, yield 5.6%) after solvent evaporation as a light orange oil. ESI-MS (MeOH, 30 V): 507.3 [M]+. 1H NMR (CDCl3, 300 MHz, δ): 9.59 (d, o-pyridine-H, 1H), 8.48 (t, p-pyridine-H, 1H), 8.21 (d, m-pyridine-H, 1H), 8.05 (t, m-pyridine-H, 1H), 4.38 (t, CH2-OCOR, 2H), 4.10 (s, pyr-CH2, 2H), 3.71 (s, tBu-carbonyl-C-CH2-N, 2H), 3.92 (t, CH2-N, 2H), 2.26 (t, ROCO-CH2, 2H), 1.56 (m, ROCOCH2-CH2, 2H), 1.47 (s, (CH3)3, 18 H), 1.23 (m, CH2-CH2-CH2, 8H), 0.83 (t, CH3, 3H). 13C NMR (CDCl3, 300 MHz, δ): 169.48 (o-pyridine-C), 164.73 (o-pyridine-C), 155.39 (m-pyridine-C), 128.75 (mpyridine-C), 64.74 (CH2-N), 61.74, (pyr-CH2) 55.21 (tBucarbonyl-C-CH2-N), 52.65 (CH2-OCOR), 34.04-22.42 (six signals, CH2) 27.25 ((CH3)3), 13.91 (CH3). N-N-Carboxyethyl-pyridinemethyl-2-aminoethyl Ester Octanoic Acid (HL1). N-N-(tert-Butylcarboxyethyl)-pyridinemethyl-2-aminoethyl ester octanoic acid (109 mg, 0.27 mmol) was dissolved in dry DCM (1 mL). TFA (1 mL) was added, and the reaction mixture was stirred for 30 min at room temperature. Xylene (1 mL) was added, and all the solvents

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Technetium Tricarbonyl Complexes

were evaporated under reduced pressure to yield the crude product, which was purified by preparative HPLC to afford N-Ncarboxyethyl-pyridinemethyl-2-aminoethyl ester octanoic acid (45 mg, 0.133 mmol, yield 49%) after solvent evaporation. ESIMS (MeOH, 30 V): 337.2 [M + H]+. 1H NMR (MeOD, 400 MHz, δ): 8.76 (d, o-pyridine-H, 1H), 8.41 (t, p-pyridine-H, 1H), 7.93 (d, m-pyridine-H, 1H), 7.86 (t, m-pyridine-H, 1H), 4.48 (s, HOOC-CH2-N, 2H), 4.22 (t, CH2-OCOR, 2H), 3.78 (s, pyrCH2, 2H),, 3.23 (t, CH2-N, 2H), 2.28 (t, ROCO-CH2, 2H), 1.56 (m, ROCOCH2-CH2, 2H), 1.29 (m, CH2-CH2-CH2, 8H), 0.9 (t, CH3, 3H). 13C NMR (MeOD, 400 MHz, δ): 184.22 (carboxylC), 171.35 (carbonyl-C), 156.08 (o-pyridine-C), 146.14 (opyridine-C), 144.42 (m-pyridine-C), 127.00 (m-pyridine-C), 84.15 (HOOC-CH2-N), 63.06 (pyr-CH2), 57.75 (CH2-N), 55.30 (CH2-OCOR), 35.22-23.97 (six signals, CH2), 13.93 (CH3). IR (cm-1): 2929 (w), 2858 (w), 1727 (m), 1668 (m). N-Bis(carboxyethyl)-2-aminoethyl Ester Octanoic Acid (H2L2). The exact same procedure as for HL1 was followed with N-bis(tert-butylcarboxyethyl)-2-aminoethyl ester octanoic acid (120 mg, 0.288 mmol) to yield N-bis(carboxyethyl)-2aminoethyl ester octanoic acid (56 mg, 0.184 mmol, yield 64%). ESI-MS (MeOH, 30 V): 304.2 [M + H]+. 1H NMR (MeOD, 400 MHz, δ): 4.60 (s, HOOC-CH2-N, 4H), 3.86 (t, CH2-OCOR, 2H), 3.44 (t, CH2-N, 2H), 2.35 (t, ROCO-CH2, 2H), 1.61 (m, ROCOCH2-CH2, 2H), 1.32 (m, CH2-CH2-CH2, 8H), 0.91 (t, CH3, 3H). 13C NMR (MeOD, 400 MHz, δ): 183.61 (carboxylC), 177.18 (carbonyl-C), 62.45 (CH2-OCOR), 57.55 (carboxylC-CH2-N), 55.64 (CH2-N), 38.33-24.10 (six signals, CH2), 16.22 (CH3). IR (cm-1): 2928 (w), 2857 (w), 1737(m), 1682 (m). N-N-Bis(carboxyethyl)-pyridinemethyl-2-aminoethyl Ester Octanoic Acid (H2L3). The exact same procedure as for HL1 was followed with N-N-bis(tert-butylcarboxyethyl)-pyridinemethyl-2-aminoethyl ester octanoic acid (37 mg, 0.072 mmol) to afford N-N-bis(carboxyethyl)-pyridinemethyl-2-aminoethyl ester octanoic acid (12 mg, 0.0303 mmol, yield 42%). ESI-MS (MeOH, 30 V): 395.2 [M]+. 1H NMR (MeOD, 400 MHz, δ): 8.95 (d, o-pyridine-H, 1H), 8.65 (t, p-pyridine-H, 1H), 8.30 (d, m-pyridine-H, 1H), 8.11 (t, m-pyridine-H, 1H), 5.77 (s, HOOCCH2-N, 4H), 4.32 (t, CH2-OCOR, 2H), 4.07 (s, pyr-CH2, 2H), 3.91 (t, CH2-N, 2H), 2.19 (t, ROCO-CH2, 2H), 1.60 (m, ROCOCH2-CH2, 2H), 1.31 (m, CH2-CH2-CH2, 8H), 0.90 (t, CH3, 3H). 13C NMR (MeOD, 400 MHz, δ): 186.22 (carboxylC), 169.48 (o-pyridine-C), 163.94 (p-pyridine-C), 128.20 (mpyridine-C), 65.31 (CH2-N), 61.79 (pyr-CH2) 56.65 (HOOCCH2-N), 52.65 (CH2-OCOR), 37.26-23.81 (six signals, CH2), 14.54 (CH3). IR (cm-1): 2927(w), 2857(w), 1731(m), 1630 (m). N-N-Carboxyethyl-pyridinemethyl-2-aminoethyl Ester Octanoic Acid Tricarbonylrhenium(I) [Re(L1)(CO)3]. A solution of [NEt4]2[ReBr3(CO)3] (0.040 g, 0.052 mmol) and HL1 (0.017 g, 0.052 mmol) in MeOH (5 mL) was allowed to reflux overnight. The solution flask was placed into the freezer for 2 h to afford the product as a white solid (0.021 g, 0.034 mmol, yield 66%) after filtration. Small, colorless crystals were obtained by slow diffusion of the product dissolved in DCM into diethyl ether. ESI-MS (MeOH, 30 V): 605.1 [185Re + H]+, 607.1 [187Re + H]+. 1H NMR (CDCl3, 300 MHz, δ): 8.84 (d, o-pyridine-H, 1H), 7.79 (t, p-pyridine-H, 1H), 7.56 (d, mpyridine-H, 1H), 7.46 (t, m-pyridine-H, 1H), 4.55 (s, HOOCCH2-N, 2H), 4.48 (t, CH2-OCOR, 2H), 3.89 (s, pyr-CH2, 2H), 3.78 (t, CH2-N, 2H), 2.40 (t, ROCO-CH2, 2H), 1.54 (m, ROCOCH2-CH2, 2H), 1.30 (m, CH2-CH2-CH2, 8H), 0.89 (t, CH3, 3H). IR (cm-1): 2020 (m), 1905 (w), 1870 (m) (νCdO). Sodium N-Bis(carboxyethyl)-2-aminoethyl Ester Octanoic Acid Tricarbonylrhenate Na[Re(L2)(CO)3]. A solution of [NEt4]2[ReBr3(CO)3] (0.040 g, 0.052 mmol) and L2 (0.016 g, 0.052 mmol) in MeOH (5 mL) was allowed to reflux overnight.

Bioconjugate Chem., Vol. 20, No. 5, 2009 1005

Figure 2. ORTEP view of complex Re(CO)3L1 (50% thermal probability ellipsoids).

The solvent was evaporated, and the oily residue was purified by preparative HPLC to afford the product as a white solid (0.005 g, 0.0087 mmol, yield 15%) after rotary evaporation of all solvents. ESI-MS (MeOH, 30 V): 570.1 [185Re]-, 572.1 [187Re]-. 1H NMR (MeOD, 300 MHz, δ): 4.80 (s, HOOC-CH2N, 4H) 3.97 (t, CH2-OCOR, 2H), 3.78 (t, CH2-N, 2H), 2.35 (t, ROCO-CH2, 2H), 1.62 (m, ROCOCH2-CH2, 2H), 1.31 (m, CH2CH2-CH2, 8H), 0.90 (t, CH3, 3H). IR (cm-1): 2017 (m), 1881 (m, br) (νCdO), 1606 (w). N-N-Bis(carboxyethyl)-pyridinemethyl-2-aminoethyl Ester Octanoic Acid Tricarbonylrhenium [Re(L3)(CO)3]. A solution of [NEt4]2[ReBr3(CO)3] (0.040 g, 0.052 mmol) and H2L3 (0.016 g, 0.052 mmol) in MeOH (5 mL) was allowed to reflux overnight. The solvent was evaporated, and the oily residue was purified by preparative HPLC to afford the product as a yellow solid (0.003 g, 0.0045 mmol, yield 8%). MALDI-MS: 663.1 [185Re + H]+, 665.1 [187Re + H]+. 1H NMR (MeOD, 400 MHz, δ): 8.96 (d, o-pyridine-H, 1H), 8.66 (t, p-pyridine-H, 1H), 8.30 (d, m-pyridine-H, 1H), 8.08 (t, m-pyridine-H, 1H), 5.71 (s, HOOC-CH2-N, 4H), 4.36 (t, CH2-OCOR, 2H), 4.14 (s, pyrCH2, 2H),, 3.97 (t, CH2-N, 2H), 2.32 (t, ROCO-CH2, 2H), 1.54 (m, ROCOCH2-CH2, 2H), 1.30 (m, CH2-CH2-CH2, 8H), 0.90 (t, CH3, 3H). IR (cm-1): 2018 (s), 1894 (m), 1874 (m) (νCdO), 1661 (w). [99mTc(H2O)3(CO)3]+ Labeling Studies. The organometallic precursor [99mTc(H2O)3(CO)3]+ was prepared from a saline solution of Na[99mTcO4] (1 mL, 200 MBq) using the Isolink kit. A solution of Na[99mTcO4] (1 mL) was added to the Isolink kit, and the vial was heated to reflux for 25 min. Upon cooling, 0.1 M HCl solution (1 mL) was added to adjust the pH to 7-8. Labeling was achieved by mixing an aliquot (0.2 mL) of the [99mTc(H2O)3(CO)3]+ precursor with a 0.1 mM solution of L1-L3 (with the solution pH previously adjusted to 7-7.5 with an aqueous 0.1 M NaHCO3 solution) at 90 °C for 25 (L1), 35 (L2), or 45 (L3) min. Analysis was performed by HPLC and TLC. HPLC solvents consisted of 0.1% trifluoroacetic acid in water (solvent A) or neat methanol (solvent B). Samples were analyzed with a linear gradient method (100% solvent A to 100% solvent B over 30 min). TLCs were analyzed with acetonitrile as mobile phase. Cysteine and Histidine Challenge Experiments. To a solution of either cysteine (0.1 M, 0.9 mL) or histidine (0.1 M, 0.9 mL) in PBS (phosphate-buffered saline, 1 mM, pH 7.4) was added a solution of the 99mTc complex (final ligand concentration 10-5 M). The samples were incubated at 37 °C and aliquots analyzed after 24 h by analytical TLC and HPLC. Octanol-Water-Distribution Coefficients. The log Po/w values of complexes [99mTc(CO)3L1], Na[99mTc(CO)3L2], and [99mTc(CO)3L3] were determined by extraction with equivalent amounts of octanol and aqueous solutions of the 99mTc complex

1006 Bioconjugate Chem., Vol. 20, No. 5, 2009

Boros et al.

Table 1. Experimental Conditions and log Po/w Data for All Complexes complex

radio-chemical yield (%)

labeling time (min)

T (°C)

ligand concn

tR (min)

log Po/w

Rf

Tc(CO)3L1 [99mTc(CO)3L2]99m Tc(CO)3L3

>95 >95 ∼ 30

25 35 45

90 90 90

3 × 10-4 3.3 × 10-4 1 × 10-3

20.5 (21.3)a 15.6 (14.01) a 18.1 (18.7) a

-0.034 -1.014 -0.38

0.49 0.39 0.46

99m

a

Retention time for the analogous cold-chemical Re complexes.

barbital, and samples of blood, liver, kidney, muscle, spleen, heart, brain, lung, gallbladder, intestine, stomach, bladder and tumor were removed, weighed, and counted in a gamma counter. Results are expressed as the percentage of the injected dose per gram of tissue (%ID/g). Tumor-to-organ ratios were calculated from %ID/g of the tumor and relevant organs.

RESULTS AND DISCUSSION

Figure 3. Biodistribution of 99mTc-labeled ligands at the 4 h time point (n ) 4).

(17). Octanol (1 mL) was mixed with an equivalent amount of the aqueous reaction mixture and stirred for 1 min. Subsequently, the two phases were partitioned, and the activity of both was determined by the measurement of activity of 5 µL droplets of each solution on a radio-TLC-plate. X-ray Diffraction Analysis of Re(CO)3L1. The sample was mounted on a glass fiber and cooled to 173 K. X-ray data were collected and processed using a Bruker X8 APEX II diffractometer using graphite-monochromated Mo KR radiation (λ ) 0.71073 Å) to a maximum 2θ value of 48.0°. Data were collected and integrated using the Bruker SAINT software package (18) and corrected for Lorentz and polarization effects, as well as for absorption effects, using the multiscan technique (SADABS) (19). The X-ray structure was solved using direct methods (SIR92) (20) and expanded using Fourier techniques (21). All calculations were performed using the SHELXTL crystallographic software package (22). Imaging and Biodistribution. Animals were used in accordance with the regulations on the protection of animals in Canada. Female C57Bl/6 mice were obtained from Sarah Reid, McMaster University. Each mouse had a 4 mm tumor in its neck, grown from a primary cell line at passage six derived from mammary tumors of a PyVMT transgenic mouse. 99mTc complexes were prepared as described above. While complexes [99mTc(CO)3L1] and [99mTc(CO)3L2]- were injected without any further purification because of their high yield in synthesis, [99mTc(CO)3L3] was purified by preparative HPLC. The solvent was removed from the collected fraction in vacuo, and [99mTc(CO)3L3] was redissolved in 0.9 M saline solution. Aliquots (100 µL) of complex solutions of [99mTc(CO)3L1], [99mTc(CO)3L2]-, and [99mTc(CO)3L3] were injected into groups of four mice via their tail vein. After injection of [99mTc(CO)3L1] and [99mTc(CO)3L2]-, dynamic imaging was carried out during the first 15 min. All animals were imaged under isoflurane anesthesia in the supine position during the first 15 min, and then 1, 2, and 4 h after injection for 15 min each. After the last time point, animals were sacrificed by an overdose of pento-

Proligand Synthesis. The tridentate chelator moieties of HL1 and H2L2 were selected for this study since they were previously known for forming highly stable complexes with the metals of interest (8, 23). The chelation entity of H2L3 is of a novel type arising from the synthesis of HL1 where its tert-butyl-protected form appears as a byproduct in the synthesis of tert-butylprotected HL1. All three ligand systems were synthesized via the free, C8-chain-derivatized amine, which was subsequently converted by reductive amination and/or alkylation into the corresponding protected ligand system and in a final step was deprotected and purified by preparative HPLC. The identity of all ligands was confirmed by characterization through 1H and 13 C NMR spectroscopy, as well as mass spectrometry and IR spectrophotometry. Elemental analysis was uninformative because of the oily consistency of the synthesized compounds. Re Complexes. Re(CO)3 complexes were synthesized as macroscopic models for the 99mTc(CO)3 complexes by mixing a methanolic solution of ligand with [Re(CO)3Br3][NEt4]2 previously dissolved in H2O and stirring the mixture at elevated temperatures overnight (24). While the neutral complex [Re(CO)3L1] precipitates from the reaction solution, the solutions of the complexes Na[Re(CO)3L2] or [Re(CO)3L3] turn slightly yellow in color without precipitation of the product. Na[Re(CO)3L2] and [Re(CO)3L3] were isolated and purified via preparative HPLC. All complexes were characterized by 1 H and 13C NMR spectroscopy, as well as mass spectrometry and IR spectrophotometry, where intense, diagnostic CO bands between 2020 and 1870 cm-1 can be assigned to the fac[Re(CO)3L]-products. Solid-State Structure of [Re(CO)3L1]. Colorless crystals of [Re(CO)3L1] were obtained by slow diffusion of the product dissolved in DCM into diethyl ether. An ORTEP diagram is shown in Figure 2. The complex displays a nearly perfect octahedral coordination sphere. The Re-C bond lengths are all close to 1.9 Å and therefore shorter than the Re-O bond length (2.142 Å) and the Re-N bond lengths (2.184 Å, 2.227 Å). The Re-N2 bond proves to be the longest because of the nondirected lone pair on the tertiary amine nature of the nitrogen atom N2. Synthesis, Stability, and Lipophilicity of 99mTc Complexes. The complexes [99mTc(CO)3L1] and [99mTc(CO)3L2]- were prepared in high radiochemical yield through reaction of fac[99mTc(CO)3(H2O)3]+ with the ligands HL1 and H2L2. The ligands were initially deprotonated with NaHCO3 (0.1 M solution, 2 equiv) and added to the technetium tricarbonyl solution with final concentrations of approximately 3 × 10-4 M. After a reaction time of 25-35 min at 90 °C, the chelation reaction was 95% complete as verified by HPLC and radioTLC analysis. No major byproducts were detected by HPLC. 99m Tc(CO)3L3 was produced in only 30% yield after a 45 min reaction time. Several byproducts were visible in the γ-trace of

Rhenium and

99m

Technetium Tricarbonyl Complexes

Bioconjugate Chem., Vol. 20, No. 5, 2009 1007

Figure 4. Representative µSPECT/CT pictures taken 15 min after injection of (A) [99mTc(CO)3L1], (B) [99mTc(CO)3L2]-, and (C) [99mTc(CO)3L3]. Abbreviations of organs: li: liver, ki:kidney, bl: bladder, gb: gallbladder.

Figure 5. Logarithmic table of injected dose per gram of tissue (%ID/ g) of 99mTc-labeled ligands at the 4 h time point (n ) 4).

Figure 6. Logarithmic table of organ/blood ratio of ligands at the 4 h time point (n ) 4).

99m

Tc-labeled

the HPLC spectrum, perhaps from coordination with decomposition fragments of L3. The product peak was assigned by comparison of the retention time of the Re complex isolated by preparative HPLC. To

investigate the complex stability toward the naturally occurring amino acids cysteine and histidine (both can act as potential tridentate ligands), the 99mTc-complexes were kept in 10-3 M cysteine/histidine solutions in PBS (pH ) 7.4, 37 °C) for 24 h. In the subsequent HPLC analysis of the solution, no noticeable decomposition could be detected for any of the three complexes. The lipophilicity of the complexes was determined by evaluation of water/octanol partition coefficients. [99mTc(CO)3L2]- should be the most ionic compound by virtue of its full -1 charge, followed by the zwitterionic [99mTc(CO)3L3] and the nonpolar [99mTc(CO)3L1]; this was confirmed by the order of lipophilicity log P values ranging from [99mTc(CO)3L1] > [99mTc(CO)3L3] > [99mTc(CO)3L2] (listed in the order from highest to lowest lipophilicity, Table 1). Imaging and Biodistribution. Imaging of all three compounds was performed at 15 min, 1 h, 2 h, and 4 h after injection. Biodistribution studies were performed at the 4 h time point after injection (Figure 3). This tumor-bearing model was used to evaluate the imaging potential of [99mTc(CO)3L1], [99mTc(CO)3L2]-, and [99mTc(CO)3L3]. Data from the imaging is displayed in Figures S2, S3, and S4 (Supporting Information). Data from the biodistribution is displayed in Figures 5 and 6 as well as in Tables S3 and S4 (Supporting Information). Imaging screenshots of mice 15 min postinjection are shown in Figure 4. All compounds show very distinct biodistribution behavior. The in vivo distribution of [99mTc(CO)3L1] could be followed from the onset of injection. Very fast localization of the complex in the liver was observed, with clearance through the intestines, which is confirmed through the biodistribution data, showing large uptake of activity in liver, intestines, and feces. This behavior is not surprising considering that this complex has no overall charge and a very small log P value. [99mTc(CO)3L2]was also followed after injection with dynamic imaging. Here, fast processing of the compound through the kidneys, within the first 5-10 min after injection, and consequent localization in the bladder, followed by excretion was observable. The excretion of large portions of the dose is confirmed by the small fraction of the originally injected dose in the biodistribution measurements. The large log P value of this compound, plus its localized negative charge, determines its fate to be excreted through the renal tract. Finally, for [99mTc(CO)3L3] after 1 h after injection, high localization in the gall bladder was observed, paired with slow clearance through the intestines. The biodistribution profile shows some similarity to the profile of [99mTc(CO)3L1], but with faster excretion through the intestines and the renal system (high percentage of dose in the feces, elevated organ/blood ratio in kidneys), which can be a conse-

1008 Bioconjugate Chem., Vol. 20, No. 5, 2009

quence of the increased polarity of the complex (compared to [99mTc(CO)3L1]). No significant heart, brain, or tumor uptake was observed for any of the investigated compounds.

CONCLUSION For the first time, small amphiphilic 99mTc(CO)3 complexes of which one bore a localized negative charge, one was a zwitterionic complex, and one was an entirely neutral molecule, have been synthesized, fully characterized, and investigated for their lipophilicity and behavior in vivo. Among the molecules, for the zwitterionic complex, a new type of chelation entity was incorporated, forming eight-membered rings between the metal center and the ligand sphere. This chelator proves to be slower coordinating but still high in stability toward cysteine and histidine challenges. Lower reaction yields arise from instability of the ligand toward prolonged heating at 90 °C. The products afforded by this decomposition are capable of coordinating to the metal center and make an additional purification step inevitable. Unfortunately, none of the three investigated complexes showed significant brain, tumor, or heart uptake. The last confirms that negatively charged or zwitterionic complexes not do show the same potential for myocardial uptake as do other, cationic complexes for this very purpose. One way to confirm this would be the investigation of the corresponding cationic compound. Also, for a more efficient mimicking of phospholipid-type molecules, a technetium-tagged approach might be more suitable, such as for example attachment of a small neutral 99mTccomplex to the aliphatic residue of the corresponding molecule. Changing the charge of a small amphiphilic molecule results in highly contrasting but still defined biodistribution behavior in an in vivo system. This is somewhat surprising and of potential importance for future applications.

ACKNOWLEDGMENT The authors acknowledge UBC for a University Graduate Fellowship (E.B.), Troy Farncombe, Chantal Saab, and Rod Rhem for excellent imaging support at the McMaster Centre for Preclinical and Translational Imaging (MCPTI) and for use of their SPECT/CT scanner, Drs. Andrea Armstrong and John Valliant for advice and use of the radiochemistry facilities in the McMaster Institute of Applied Radiation Sciences (McIARS), MDS Nordion (Canada) for some financial support of this project, Covidien for the donation of Isolink Kits, Vancouver Coastal Health-UBC Hospital for [99mTcO4]-, and the Natural Sciences and Engineering Research Council of Canada (NSERC) for a Strategic Grant. Supporting Information Available: Crystal data for [Re(CO)3L1], radio-HPLC traces of reaction mixtures of 99mTccompounds, curves of all 99mTc-compounds’ levels in selected organs taken from the SPECT/CT images. This information is available free of charge via the Internet at http://pubs.acs.org.

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