Synthesis and Cellular Studies of Porphyrin− Cobaltacarborane

Bioconjugate Chem. 2005, 16, 1495−1502

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Synthesis and Cellular Studies of Porphyrin-Cobaltacarborane Conjugates Erhong Hao, Timothy J. Jensen, Brandy H. Courtney, and M. Grac¸ a H. Vicente* Department of Chemistry, Louisiana State University, Baton Rouge LA, 70803. Received July 15, 2005; Revised Manuscript Received August 25, 2005

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′-C2B9H11)]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH2CH2O)2 chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.

INTRODUCTION

Boron neutron capture therapy (BNCT), a binary modality for cancer treatment based on the ability of 10B nuclides to capture low-energy neutrons with subsequent production of high linear energy transfer particles (4He2+ and 7Li3+), has attracted much interest in the past decades because it can selectively target and destroy malignant cells in the presence of normal cells (1-3). The success of BNCT depends on the availability of nontoxic boron-containing compounds that can selectively target and accumulate in tumors with high tumor:normal tissues and tumor:blood boron concentration ratios. Among all BNCT agents reported to date, porphyrins are particularly promising due to their known ability to selectively incorporate into tumor cells in high amounts and their subsequent persistence within tumors (3-5). Furthermore, porphyrins are highly fluorescent, thus enabling tumor diagnosis and facilitating treatment planning. The timely discovery of borane clusters in the late 1950s (6) has made possible the syntheses of porphyrins with high percentages of boron by weight, able to deliver therapeutic concentrations of boron (∼20 µg 10 B/g tumor) to target tumors. Several porphyrins carrying multiple boron clusters have been synthesized and evaluated in both cellular and animal studies (4, 5, 7-10), but to date there is still limited availability of boroncontaining porphyrins because of lengthy synthetic routes and overall low reaction yields. Consequently, there is a lack of structure/activity relationships conducted for this type of compound that could allow the identification of the best candidates for application in BNCT (11). Such studies have been much more extensively reported in the development of porphyrins for application in the photodynamic therapy (PDT) of tumors, another binary therapy that uses red light for the activation of a tumor-localized photosensitizer (4, 12, 13). These studies show that the uptake of porphyrins by tumors is highly dependent upon their physicochemical properties, such as structural * Corresponding author. Phone: (225) 578 7405. Fax: (225) 578 3458. E-mail: [email protected].

features (nature of peripheral side chains), charge and charge distribution, hydrophobic character, and aggregation state. Metallacarboranes, first reported in 1965 (14), belong to the large family of metallocene-type complexes. Among these, the cobaltabisdicarbollide anion [3,3′-Co(1,2C2B9H11)2]- was among the first to be synthesized, and its chemistry has been extensively investigated (15, 16). In the 1980s Spryshkova et al. showed that the potassium salt of [3,3′-Co(1,2-C2B9H11)2]- had low toxicity (LD50 ) 0.08750 mg/kg) in tumor-bearing rats (17, 18). This property, along with the remarkable thermal, kinetic, and photochemical stabilities of cobaltacarborane and some of its derivatives, has led to their investigation as radiotherapeutic (19, 20) and as BNCT agents (21-23). The bisdicarbollide complexes contain a larger number of boron atoms than the carboranes and the closododecaborate anions and are water-soluble in the form of sodium or potassium salts while still maintaining hydrophobic character needed for crossing cellular membranes. Since the discovery of a convenient synthesis of the zwitterionic [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′C2B9H11)] (24, 25), several new cobaltacarborane-substituted molecules have been reported (26-30). The dioxane ring of [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′-C2B9H11)] readily undergoes a ring-opening reaction in the presence of a variety of nucleophilic reagents, including halide and hydroxide anions (26), phthalimide ions and amines (27), phenolate ions and phosphines (28), pyrrolyl salts (29), and pyridine and phenol (30). Using this methodology, a nucleoside-cobaltacarborane conjugate was recently synthesized and found to have low cytotoxicity toward Vero and A549 cells (IC50 > 80 µM), using an MTT-based assay (23). With the aim to investigate structure/activity relationships in porphyrin-carborane conjugates, we report herein the synthesis and in vitro evaluation of a series of five conjugates (1-5) containing one to four cobaltabisdicarbollide anions linked to the porphyrin macrocycle via (CH2CH2O)2 chains. An efficient one-step ringopening reaction between cobaltacarborane 6 and five pyridylporphyrins (7-11) afforded the target conjugates

10.1021/bc0502098 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/01/2005

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Chart 1. Structures of Porphyrin-Cobaltacarborane Conjugates 2-5

in 88-98% yields. We have previously shown that this reaction also proceeds with phenol-substituted porphyrins as the nucleophilic species (30). Since cobaltacarborane-porphyrins contain twice as many boron atoms as nido-carboranylporphyrins while still bearing a delocalized negative charge, we anticipate that the conjugates reported herein will be very promising boron carriers for BNCT. EXPERIMENTAL PROCEDURES

Syntheses. All reactions were monitored by TLC using 0.25 mm silica gel plates with or without UV indicator (Merck 60F-254). Silica gel from Sorbent Technologies 32-63 µm was used for flash column chromatography. 1 H and 13C NMR were obtained on either a DPX-250 or a ARX-300 Bruker spectrometer. Chemical shifts (δ) are given in ppm relative to acetone-d6 (2.05 ppm, 1H; 207.07 ppm, 13C) unless otherwise indicated. Electronic absorption spectra were measured on a Perkin-Elmer Lambda 35 UV-Vis spectrophotometer, and fluorescence spectra were measured on a Perkin-Elmer LS55 spectrometer. Mass spectra were obtained on an Applied Biosystems QSTAR XL. All solvents were purchased from Fisher Scientific (HPLC grade) and used without further purification. Zwitterionic [3,3′-Co(8-C4H8O2-1,2-C2B9H11)(1′,2′C2B9H11)] (6) was prepared from the cesium salt of cobaltabisdicarbollide, obtained from Katchem Ltd (Czech Republic), as described in the literature (25). The 5-(4′pyridyl)-10,15,20-triphenylporphyrin (7), trans-5,15-di(4′-pyridyl)-10,20-diphenylporphyrin (8), cis-5,10-di(4′pyridyl)-15,20-diphenylporphyrin (9), and 5,10,15-tri(4′pyridyl)-20-phenylporphyrin (10) were prepared according to the literature (31). 5,10,15,20-Tetra(4′-pyridyl)porphyrin (11) was obtained from Sigma-Aldrich and recrystallized from chloroform/methanol before use. General Procedure for Conjugate Synthesis. Cobaltacarborane 6 and the 4-pyridylporphyrin were dis-

solved into a 1:1 (v/v) mixture of chloroform and acetonitrile (Scheme 1). The reaction was stirred at 60 °C under an argon atmosphere until the reaction was complete (monitored by TLC and 1H NMR). The reaction mixture was cooled to room temperature and the solvent evaporated under vacuum. The remaining residue was washed with diethyl ether (3 × 5 mL) and with methanol (3 × 5 mL). Finally, the product was dried overnight under vacuum to afford the targeted conjugate. 5-(4′-Cobaltacarboranepyridyl)-10,15,20-triphenylporphyrin (1). Porphyrin 7 (61.6 mg, 0.10 mmol) and compound 6 (61.5 mg, 0.15 mmol) were heated in 40 mL of chloroform/acetonitrile 1:1 for 12 h. The title conjugate was obtained in 98.4% yield (101.0 mg) as a purple solid. UV-Vis (acetone) λmax (/M-1 cm-1) 418 (157 600), 515 (10 100), 551(5300), 590 (3400), 646 (2600). 1H NMR (acetone-d6): δ 9.73 (d, 2H, J ) 6.7 Hz, o-PyrH), 9.109.07 (m, 4H, β-H), 9.00 (d, 2H, J ) 4.8 Hz, m-PyrH), 8.90 (s, 4H, β-H), 8.28-8.24 (m, 6H, o-PhH), 7.86-7.76 (m, 9H, m,p-PhH), 5.34-5.30 (m, 2H, NCH2), 4.51-4.42 (m, 2H, OCH2), 4.06-4.03 (m, 4H, OCH2), 3.84-3.82 (br s, 4H, carborane-H), 1.6-3.0 (br, 17H, BH), -2.74 (s, 2H, NH). 13C NMR (acetone-d6): δ 160.1, 144.8, 142.2, 135.1, 133.6, 128.9, 127.7, 123.0, 122.1, 113.3, 73.0, 69.7, 61.8, 52.5, 46.9. HRMS (MALDI-TOF) m/z 1026.5713, calculated for C51B18H58N5O2Co 1026.5739. trans-5,15-Di(4′-cobaltacarboranepyridyl)-10,20diphenylporphyrin (2). Porphyrin 8 (16.0 mg, 0.026 mmol) and compound 6 (43 mg, 0.10 mmol) were heated in 40 mL of chloroform/acetonitrile 1:1 for 2 days, affording 34.1 mg (91.1%) of the title conjugate. UV-Vis (acetone) λmax (/M-1 cm-1) 422 (129 400), 516 (8400), 554 (5200), 590 (3400), 651 (2800). 1H NMR (acetone-d6): δ 9.76 (d, 4H, J ) 6.7 Hz, o-PyrH), 9.13-9.09 (m, 8H, β-H), 9.04 (d, 4H, J ) 4.9 Hz, m-PyrH), 8.29-8.26 (m, 4H, o-PhH), 7.91-7.83 (m, 6H, m,p-PhH), 5.34-5.32 (br m, 4H, NCH2), 4.44-4.42 (br m, 4H, OCH2), 4.06 (br s, 4H,

Porphyrin−Cobaltacarborane Conjugates Scheme 1

a

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a

Conditions: (a) CHCl3/CH3CN 1:1, 60 °C, 12 h (98.4%).

OCH2), 4.02 (br s, 4H, OCH2), 3.87-3.83 (m, 8H, carborane-H), 1.6-3.0 (br, 34H, BH), -2.80 (s, 2H, NH). 13C NMR (acetone-d6): δ 160.0, 145.3, 142.2, 135.5, 133.8, 129.5, 128.2, 123.2, 115.5, 73.7, 70.4, 62.6, 53.1, 47.6. HRMS (MALDI-TOF) m/z 1438.9027, calculated for C58B36H86N6O4Co2 1438.8987. cis-5,10-Di(4′-cobaltacarboranepyridyl)-15,20diphenylporphyrin (3). Porphyrin 9 (16.0 mg, 0.026 mmol) and compound 6 (43 mg, 0.10 mmol) were heated in 40 mL of chloroform/acetonitrile 1:1 for 2 days, affording 33.5 mg (91.0%) of the title conjugate. UV-Vis (acetone) λmax (/M-1 cm-1) 423 (141 000), 518 (10 600), 553 (5000), 590 (4000), 645 (1600). 1H NMR (acetoned6): δ 9.75 (d, 4H, J ) 6.7 Hz, o-PyrH), 9.18 (s, 2H, m-PyrH), 9.11 (d, 6H, J ) 6.7 Hz, β-H), 9.04 (d, 2H, J ) 4.9 Hz, β-H), 8.92 (s, 2H, m-PyrH), 8.27-8.23 (m, 4H, o-PhH), 7.85-7.82 (m, 6H, m,p-PhH), 5.33-5.31 (br m, 4H, NCH2), 4.50-4.48 (br m, 4H, OCH2), 4.09-4.03 (m, 8H, OCH2), 3.86-3.81 (m, 8H, carborane-H), 1.6-3.0 (br, 34H, BH), -2.75 (s, 2H, NH). 13C NMR (acetone-d6): δ 159.9, 145.3, 142.1, 135.4, 133.8, 129.4, 128.0, 124.2, 114.6, 73.6, 70.3, 70.2, 62.5, 53.0, 47.5. HRMS (MALDITOF) m/z 1438.8960, calculated for C58B36H86N6O4Co2 1438.8987. 5,10,15-Tri(4′-cobaltacarboranepyridyl)-20-phenylporphyrin (4). Porphyrin 10 (31.0 mg, 0.05 mmol) and compound 6 (100.3 mg, 0.24 mmol) were heated in 40 mL of chloroform/acetonitrile 1:1 for 2 days to afford 83.0 mg (90.0%) of the title conjugate. UV-Vis (acetone) λmax (/M-1 cm-1) 425 (168 000), 516 (12 600), 553 (5600), 591 (4300), 647 (1900). 1H NMR (acetone-d6): δ 9.799.75 (m, 6H, o-PyrH), 9.21-9.19 (m, 6H, m-PyrH), 9.149.11 (m, 6H, β-H), 9.07 (d, 2H, J ) 5.8 Hz, β-H), 8.298.25 (m, 2H, o-PhH), 7.91-7.86 (m, 3H, m,p-PhH), 5.34 (br s, 6H, NCH2), 4.43 (br s, 6H, OCH2), 4.04 (s, 6H, OCH2), 3.99 (s, 6H, OCH2), 3.84 (s, 12H, carborane-H), 1.6-3.0 (br, 51H, BH), -2.83 (s, 2H, NH). 13C NMR (acetone-d6): δ 159.2, 145.2, 142.1, 135.2, 133.5, 129.4, 127.9, 124.2, 116.1, 73.4, 70.0, 62.3, 52.7, 47.3. HRMS (MALDI-TOF) m/z 1850.2320, calculated for C65B54H114N7O6Co3 1850.2263. 5,10,15,20-Tetra(4′-cobaltacarboranepyridyl)porphyrin (5). Porphyrin 11 (17.0 mg, 0.027 mmol) and compound 6 (62.0 mg, 0.15 mmol) were heated in 40 mL of chloroform/acetonitrile 1:1 for 3 days, affording 53.3 mg (87.7%) of the title conjugate. UV-Vis (acetone) λmax (/M-1 cm-1) 427 (210 000), 517 (15 600), 553 (5880), 590 (5630), 645 (1500). 1H NMR (acetone-d6): δ 9.80 (d, 8H, J ) 6.3 Hz, o-PyrH), 9.26 (s, 8H, β-H), 9.15 (d, 8H, J )

6.3 Hz, m-PyrH), 5.36 (br s, 8H, NCH2), 4.43 (br s, 8H, OCH2), 4.05 (br s, 8H, OCH2), 4.00 (br s, 8H, OCH2), 3.86-3.84 (m, 16H, carborane-H), 1.6-3.0 (br, 68H, BH), -2.89 (s, 2H, NH). 13C NMR (acetone-d6): δ 159.4, 145.9, 134.1, 117.4, 74.0, 70.6, 70.5, 62.9, 53.3, 47.9. HRMS (MALDI-TOF) m/z 2262.5566, calculated for C72H142N8O8B72Co4 2262.5509. Molecular Structure. The crystal structure of porphyrin-cobaltacarborane conjugate 1 was determined using data collected at T ) 110 K with Mo KR radiation on a Nonius KappaCCD diffractometer. Crystal data: C51H58B18CoN5O2, triclinic space group P1 h , a ) 6.941(5), b ) 10.982(7), c ) 35.48(3) Å, R ) 89.46(2), β ) 86.86(3), γ ) 75.03(4)°, V ) 2609(3) Å3, Z ) 2, R ) 0.125 (F2 > 2σ), Rw ) 0.359 (all F2) for 7181 unique data and 324 refined parameters. Due to the limited quality of the crystal, anisotropic refinement was not possible, except for the Co atom. Disorder was present in the chain connecting the porphyrin to the cobaltacarborane, and two atoms were modeled as pairs of half-populated sites. The N-H hydrogen atoms could not be located. Cell Culture. Human HEp2 cells were obtained from the ATCC and maintained in a 50:50 mixture of DMEM: Advanced MEM (Gibco) supplemented with 5% FBS (Gibco). Phosphate buffered saline (PBS), FBS and trypsin were purchased from Gibco. All conjugate solutions were filter sterilized using a 0.22 µm Pal syringe filter. Cellular Uptake. HEp2 cells were seeded at 10 000 cells per well in a Costar 96 well plate and incubated overnight. Solutions of the conjugates 10 mM in DMSO (Sigma) were prepared and further diluted to 1 mM by adding Cremophor EL (Fluka) to give final concentrations of 90% DMSO and 10% Cremophor EL. These solutions were then diluted 50-fold into medium and filter-sterilized to give 2× stock solutions. Equal volumes of conjugate stocks were added to the wells containing cells, giving a final concentration of 10 µM, and incubated for 0, 1, 2, 4, 8, and 24 h. The conjugate uptake was stopped by removing the loading medium and washing the cells with 200 µL of PBS. The cells were solubilized in 0.25% Triton X-100 (Calbiochem) in PBS. Conjugate concentration was determined by measuring the conjugate fluorescence using a BMG FLUOstar Optima plate reader at 410/650 nm excitation/emission. Cell counts were performed using the CyQuant cell proliferation assay reagent (Molecular Probes) as described by the manufacturer. Dark Cytotoxicity. Cells were plated in 96-well plates and incubated overnight. Conjugate was added to

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a final concentration of 10 µM as described above and incubated overnight. The conjugate was then removed and replaced with medium containing Cell Titer Blue (Promega) viability reagent. The cytotoxicity was assayed as per the manufacturer’s instructions. The controls consist of cells not treated with either conjugate or vehicle, but incubated under the same conditions to provide a 100% viable reference point. Cells were also treated with 0.2% saponin overnight to provide a 100% dead control, and with vehicle alone (0.9%DMSO/0.1% Cremophor EL) to test residual toxic effects of the delivery vehicle. Microscopy. Cells were trypsinized and plated on LabTek 2 chamber coverslips (Nunc) and allowed to attach overnight. Lipofectamine liposomes were used as a delivery vehicle. The conjugate stocks were first prepared in DMSO to a concentration of 1 mM, and 1 µL of this stock was mixed with 5 µL of Lipofectamine (Invitrogen), vortexed, and incubated for 15 min. Serum free AMEM (100 µL) was then added, and the mixture was vortexed and incubated for 30 min at room temperature. During this incubation, the cells were washed once with serum free AMEM. The liposome complex was then diluted with 900 µL of serum free AMEM and the mixture transferred to the cells and incubated for 4 h. At the end of the incubation period, 1 mL of medium containing 2 × FBS was added, and the cells were incubated overnight. The cells were then washed five times with medium supplemented with 50 mM HEPES pH 7.4 and viewed using a Zeiss Axiovert 200M inverted microscope fitted with standard FITC and Texas Red fluorescent filter sets (Chroma). For the colocalization experiments, LysoSensor Green and MitoTracker Green (Molecular Probes) were diluted into medium to a final concentration of 50 nM (LysoSensor Green) and 250 nM (MitoTracker Green). The cells were incubated concurrently with conjugate for 30 min before being washed and viewed by microscopy as described above.

Hao et al.

Figure 1. Molecular structure of porphyrin-cobaltacarborane 1.

Figure 2. Optical spectra of conjugates 1 (black), 2 (purple), 3 (green), 4 (red), and 5 (blue) at 1 µM in acetone solution.

RESULTS

Synthesis and Characterization. Cobaltacarborane 6 was prepared from the reaction of cobaltabisdicarbollide anion [Co(C2B9H11)2]- with 1,4-dioxane in the presence of BF3‚Et2O, according to the literature procedure (25). Pyridylporphyrins 7-11, synthesized using an AdlerLongo mixed condensation in boiling propionic acid (31), were used as the nucleophilic species in the dioxane ringopening of 6. Conjugates 1-5 were prepared in 88-98% yield in a single-step reaction between the corresponding 4-pyridylphenylporphyrin and an excess of cobaltacarborane 6, in a mixture of chloroform and acetonitrile at 60 °C (30). At higher temperatures (e.g. at 80 °C or in refluxing chloroform) small amounts of green byproducts were also obtained, due to substitution reactions at the porphyrin core (32). The targeted conjugates were isolated in pure form upon washing the resulting reaction residues with ethyl ether to remove the excess of compound 6, without need of chromatography. The molecular structure of conjugate 1 is shown in Figure 1. The porphyrin core is reasonably planar, having mean deviation of 24 atoms from their best plane of only 0.07 Å and maximum deviation 0.18(2) Å. The two dicarbollide moieties coordinate the Co atom with their five-membered rings parallel. The Co atom is equidistant from the two five-membered rings, having a perpendicular distance of 1.463(2) Å to the untethered dicarbollide and 1.464(2) Å to the tethered one. The centroid-Co-centroid angle is 178.3(1)°.

Figure 3. Fluoresence emission spectra of conjugates 1 (black), 2 (purple), 3 (green), 4 (red), and 5 (blue) at 1 µM in acetone solution upon excitation at 416 nm at room temperature.

The zwiterionic porphyrin conjugates 1-5 are soluble in polar aprotic solvents, such as acetonitrile, acetone, ethyl acetate, THF, DMF, and DMSO but are not soluble in methanol or water. Their observed low extinction coefficients in acetone solution compared with the corresponding starting porphyrins (Figure 2) indicates the formation of small aggregates even at micromolar concentrations and may explain their poor solubility in protic solvents. Interestingly, 2-3 nm red-shifts of the conjugate Soret bands were observed with increasing number

Porphyrin−Cobaltacarborane Conjugates

Figure 4. Time-dependent uptake of conjugates 1 (black), 2 (purple), 3 (green), 4 (red), and 5 (blue) at 10 µM by HEp2 cells.

Figure 5. Dark cytotoxicity of conjugates 1-5 and controls toward HEp2 cells using the Cell Titer Blue assay.

of cobaltacarborane moieties linked to the porphyrin macrocycle; for example, the Soret bands of conjugates 1 and 5 appear at 418 and 427 nm, respectively. The fluorescence spectra of conjugates 1-5 (Figure 3) all display emissions at ∼653 nm in acetone solution, upon

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excitation at 512 nm. The most intense fluorescence band was observed for conjugate 1, containing only one cobaltacarborane substituent on the porphyrin macrocycle. Cellular Studies. The time-dependent uptake of conjugates 1-5 was evaluated in HEp2 cells at a concentration of 10 µM (Figure 4). Since the conjugates were poorly soluble in water, DMSO and Cremophore EL were used as the delivery vehicles (0.9%DMSO/0.1% Cremophor EL). At 10 µM concentration all conjugates were found to be nontoxic to the cells, as evaluated using the Cell Titer Blue assay (Figure 5). Higher concentrations were not investigated due to the poor solubility of the conjugates in aqueous solutions. At the low concentration used, the DMSO/Cremophor EL vehicle was nontoxic to cells (Figure 5, vehicle control), while 0.2% saponin provided 100% cell death, and the control cells (in the absence of both conjugate and vehicle) showed 100% cell viability (Figure 5). The cellular uptake of conjugates 1-5 significantly increased with the number of cobaltacarborane moieties linked to the porphyrin macrocycle, as shown in Figure 4. Conjugate 5, bearing four cobaltacarboranes, accumulated the most within cells, approximately 16 times more than 1, bearing only one cobaltacarborane, at all time points. Conjugate 3, with two cobaltacarboranes linked to adjacent meso-pyridyl rings, was taken up by HEp2 cells to a significantly higher extent (∼25%) than 2, bearing two cobaltacarboranes linked to opposite pyridyls. The uptake kinetics were similar for all conjugates, proceeding rapidly in the first 1-2 h after which a plateau was reached. The intracellular localization of conjugates 1-5 in HEp2 cells was investigated by fluorescence microscopy, using Lipofectamine liposomes as the delivery vehicle (Figure 6). In the absence of liposomes the fluorescent signal for the conjugates was too weak to be clearly detected above the background autofluorescence, due to their poor water solubility. Using liposomes as the delivery vehicle, we observed a very punctate fluorescence for all conjugates, as seen in Figure 6, indicating that they all localize within vesicles. Colocalization experiments with LysoSensor Green and Mitotracker Green, as seen in Figure 7 for conjugate 3 (similar figures are available for conjugates 1, 2, 4, and 5 in the Supporting Information), indicate that some of the conjugates’ fluorescence correlates with the cell lysosomes.

Figure 6. Subcellular localization of conjugates 1-5 in HEp2 cells at 1 µM in liposomes for 18 h. (a) Phase contrast, (b) 1 fluorescence, (c) 2 fluorescence, (d) 3 fluorescence, (e) 4 fluorescence, (f) 5 fluorescence. Scale bar: 10 µm.

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Figure 7. Subcellular localization of conjugate 3 in HEp2 cells at 1 µM in liposomes, for 18 h. (a) Phase contrast, (b) overlay of 3 fluorescence and phase contrast, (c) LysoSensor Green fluorescence, (e) MitoTracker Green fluorescence, (d), (f) overlays of organelle tracers with 3 fluorescence. Scale bar: 10 µm. DISCUSSION

Cobaltacarborane-porphyrin conjugates 1-5 were synthesized in 88-98% yields in a single-step reaction between cobaltacarborane 6 and pyridylporphyrins 7-11. This methodology allows the easy and expeditious preparation of the target porphyrin conjugates, in gram amounts. N-Alkylated pyridylporphyrins, in particular meso-tetra(N-methylpyridyl)porphyrin, have been extensively studied as DNA and RNA binding probes (33-37) and as antibacterial and antiviral agents (38-41). The biological activity of this type of compound depends on the size and distribution of the macrocycle periphery groups, the molecule charge and its distribution, and on the nature of the centrally chelated metal ion and associated axial ligand(s) (42). The new zwiterionic porphyrin conjugates 1-5 bearing one to four N-cobaltacarboranepyridyl moieties were synthesized in order to study their biological activity and to correlate their structure and charge distribution with their cellular uptake and preferential sites of intracellular localization. All conjugates display absorption spectra characteristic of porphyrin macrocycles (Figure 2) and fluorescence emissions in the red region of the optical spectrum (Figure 3). The cellular uptake of all conjugates in the presence of a DMSO/Cremophor EL delivery vehicle was studied in HEp2 cells at a concentration of 10 µM. The amount of conjugate accumulated by cells depended significantly on the number of cobaltacarborane residues linked to the porphyrin macrocycle, and on its distribution about the ring (conjugates 2 and 3). Compound 5, bearing four cobaltacarboranes, accumulated the most within cells,

while conjugate 1, bearing a single cobaltacarborane, accumulated the least (Figure 4). Although conjugate 1, with only one positive and one negative charges, is expected to be more hydrophobic than 5, which bears four positive and four negative charges, the latter showed decreased solubility in polar solvents and increased tendency for aggregation, as indicated by its lower emission intensity of all conjugates studied (Figure 3). Porphyrin aggregates are potentially taken up to a higher extent by cells in culture, via an endocytic pathway (43). We have previously observed that free-base carboranylporphyrins accumulated within cells to a higher extent than their Zn(II) counterparts, as a result of their higher tendency for forming aggregates and consequently higher hydrophobicity (10, 44). We have also observed that a porphyrin bearing eight nido-carborane groups was taken up by T98G cells to a lower extent than a related tetra(nido-carboranyl)porphyrin, although in this case the overall molecule charge changed from -8 to -4 and the octa-anionic porphyrin was more hydrophilic than the tetra-anionic derivative (10). On the other hand, the overall molecule charge for all conjugates 1-5 is zero, and their tendency for aggregation likely increases with the number of charges. The charge distribution about the porphyrin ring was also found to affect cellular uptake; conjugate 3, bearing two cobaltacarboranes on adjacent pyridyl rings, accumulated to a higher extent within cells compared with 2, bearing the charges and substituents on opposite pyridyl rings. It is expected that conjugate 3, with two distinctive charged/hydrophobic moieties, will have different aggregation behavior from 2. In agreement with these results, we have recently discovered that two

Porphyrin−Cobaltacarborane Conjugates

(N-trimethylaminophenyl)porphyrins, bearing two positive charges on either adjacent or opposite meso-phenyl rings, display different photodynamic efficacy, possibly as a result of their different interactions with biological substrates (45). All porphyrin-cobaltacarborane conjugates localized into vesicles within HEp2 cells, when delivered by Lipofectamine liposomes (Figure 6). Colocalization experiments showed that these vesicles correlated to some extent with the cell lysosomes (Figure 7). Our group and others have previously observed that carboranylporphyrins preferentially localize in the cell lysosomes (10, 44, 46). However, the use of liposomes as delivery vehicle could potentially alter the subcellular distribution of porphyrin sensitizers, as we have recently reported (47, 48). This could in part explain the vesicle localization observed in this study for conjugates 1-5 and its partial correlation with the cell lysosomes. ACKNOWLEDGMENT

The authors thank Dr. Frank Fronczek for assistance with the molecular structure determination and Dr. Martha Sibrian-Vazquez for assistance with HPLC. This work was supported by the National Institutes of Health, grant number R01 CA098902. Supporting Information Available: Additional spectroscopic data including HRMS and NMR spectra for all conjugates synthesized, HPLC conditions, and subcellular localization figures for conjugates 1, 2, 4, and 5 is available free of charge via Internet at http://pubs.acs.org. Crystallographic data (excluding structure factors) for the structure in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 272582. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK, (fax: +44(0)1223-336033 or e-mail: [email protected]). LITERATURE CITED (1) Soloway, A. H., Tjarks, W., Barnum, B. A., Rong, F. G., Barth, R. F., Codogni, I. M., and Wilson, J. G. (1998) The chemistry of neutron capture therapy. Chem. Rev. 98, 15151562. (2) Hawthorne, M. F. (1993) The role of chemistry in the development of boron neutron capture therapy of cancer. Angew. Chem., Int. Ed. Engl. 32, 950-984. (3) Barth, R. F., Coderre, J. A., Vicente, M. G. H., and Blue, T. E. (2005) Boron neutron capture therapy of cancer: Current status and future prospects. Clin. Cancer Res. 11, 3987-4002. (4) Vicente, M. G. H. (2001) Porphyrin-based sensitizers in the detection and treatment of cancer: Recent progress. Curr. Med. Chem. Anti-Cancer Agents 1, 175-194. (5) Bregadze, V. I., Sivaev, I. B., Gabel, D., and Wohrle, D. (2001) Polyhedral boron derivatives of porphyrins and phthalocyanines. J. Porphyrins Phthalocyanines 5, 767-781. (6) Hawthorne, M. F., and Pitochelli, A. R. (1959) The reaction of bis-acetonitrile decarborane with amines. J. Am. Chem. Soc. 81, 5519-5519. (7) Miura, M., Morris, G. M., Micca, P. L., Lombardo, D. T., Youngs, K. M., Kalef-Ezra, J. A., Hoch, D. A., Slatkin, D. N., Ma, R., and Coderre, J. A. (2001) Boron neutron capture therapy of a murine mammary carcinoma using a lipophilic carboranyltetraphenylporphyrin. Radiat. Res. 155, 603-610. (8) Vicente, M. G. H., Wickramasinghe, A., Nurco, D. J., Wang, H. J. H., Nawrocky, M. M., Makar, M. S., and Miura, M. (2003) Syntheses, toxicity and biodistribution of two 5,15-di[3,5-(nido-carboranylmethyl)phenyl]porphyrin in EMT-6 tumor bearing mice. Bioorg. Med. Chem. 11, 3101-3108. (9) Ozawa, T., Santos, R. A., Lambom, K. R., Bauer, W. F., Koo, M.-S., Kahl, S. B., and Deen, D. F. (2004) In vivo evaluation

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