Bioconjugate Chem. 2007, 18, 731−735
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N,N-Dimethylsphingosine-Coumarin: Synthesis, Chemical Characterization, and Biological Evaluation Sukhen C. Ghosh,† Edmond Auzenne,‡ David Farquhar,† and Jim Klostergaard*,‡ Departments of Experimental Therapeutics and Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030.. Received September 11, 2006; Revised Manuscript Received February 1, 2007
Coumarin derivatives of N,N-dimethylsphingosine (DMSP) were prepared and chemically characterized. They were apparently biologically equivalent to DMSP in terms of tumor cell cytotoxicity and were used to establish the rapid mitochondrial localization of this sphingolipid in tumor cells, followed closely by its marked reduction of mitochondrial membrane potential.
INTRODUCTION
Scheme 1
Sphingolipids (SLs) and their metabolic pathways are increasingly recognized as promising targets for cancer therapy. Of particular focus in these pathways is the conversion of proapoptotic sphingosine (Sp) to anti-apoptotic sphingosine-1phosphate (Sp-1-P) by the enzyme sphingosine kinase (SpK) (1). Since Sp is derived by deacylation of ceramide (Cer), a SL or Cer/Sp-1-P rheostat has been hypothesized to determine cellular fate; the relative cellular concentrations of Cer and Sp vs Sp-1-P determine whether a cell proliferates or undergoes apoptosis (2). SpK is itself oncogenic (3), governed by growth factors (4) and protein kinases (5). Dimethylsphingosine (DMSP) is a competitive inhibitor of SpK (6) and is a useful tool to elucidate the role of Sp-1-P as an intracellular second messenger. DMSP is also a promising compound for the chemotherapeutic management of various types of malignancies (7). Aside from inhibition of SpK, some evidence indicates that mitochondria are a target of Sp, the parent compound (8). Since DMSP lacks a significant spectroscopic signature and in order to facilitate mechanistic studies of this SL, we incorporated a fluorescent probe into it and conducted chemical and biological characterization of the fluorescenated DMSP. 7-(Diethylamino)coumarin-3-carboxylic acid, 1, was converted to the corresponding acid chloride, 2, by reaction with oxalyl chloride in dichloromethane (Scheme 1). Compound 2 was then reacted with DMSP, 3, to give a mixture of the isomeric esters, 4 and 5, together with a trace amount of the diester 6 (Scheme 2). These products were separated by column chromatography on silica gel. No differences in cytotoxic activity against three tumor cell lines were observed among compounds 4, 5, and DMSP, indicating that these substitutions were without significant effect on the biological activity of DMSP. Time-lapse fluorescence microscopy studies with 4 and 5 indicated that they were rapidly accumulated intracellularly and localized in mitochondria. This localization was associated with a rapid and marked reduction in mitochondrial membrane potential.
EXPERIMENTAL PROCEDURES Chemistry. All chemicals and reagents were purchased either from Sigma-Aldrich Company (St. Louis, MO) or from Mo*
Author to whom correspondence should be addressed. Department of Experimental Therapeutics. ‡ Department of Molecular and Cellular Oncology. †
a
a Reagents and conditions: (a) oxalyl chloride, CH Cl , catalytic 2 2 amount of DMF, rt for 12 h.
Scheme 2
a
a Reagents and conditions: (a) 2,4,6-collidine, CH Cl , -40 °C for 2 2 4 h, then 25 °C for 12 h.
lecular Probes, Inc. (Carlsbad, CA). Nuclear magnetic resonance spectra (1H NMR and 13C NMR) were recorded at ambient temperature on Bruker Advance model 300 or 500 MHz spectrometers. Samples were dissolved in an appropriate deuterated solvent (CDCl3 or D2O). The 1H chemical shifts are
10.1021/bc060285q CCC: $37.00 © 2007 American Chemical Society Published on Web 04/14/2007
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Figure 1. Absorption spectrum of compound 1 in methanol solution (UV cutoff 205 nm).
reported as parts per million (δ ppm) relative to tetramethylsilane (Me4Si, δ ) 0.00) as an internal standard. Chemical shifts for 13C are reported as δ relative to deuterated chloroform (CDCl , 3 central peak δ ) 77.0 ppm) as an internal standard. Column chromatography was performed on silica gel (Merck, 230-400 mesh). UV spectra were obtained on a Perkin-Elmer spectrophotometer. Mass spectra analyses were performed by the analytical services section of the Department of Experimental Therapeutics, M. D. Anderson Cancer Center, Houston, TX. All reactions were carried out in dry glassware and protected from atmospheric moisture. Solvents were dried over freshly activated (300 °C, 1 h) molecular sieves (type 4A). Evaporations were carried out on a rotary evaporator under aspirator vacuum at a bath temperature of 30-40 °C. Synthesis of 7-(Diethylamino)-2-oxo-2H-chromene-3-carboxylic Acid Chloride (2). Oxalyl chloride (150 µL, 240 mg, 1.9 mmol) was added, under a dry argon atmosphere, to a solution of 7-(diethylamino)coumarin-3-carboxylic acid, 1 (100 mg, 0.38 mmol), in anhydrous CH2Cl2 (20 mL), followed by DMF (20 µL) as a catalyst. Gas evolution ensued for about 5 min during which time the solution darkened. After 12 h at room temperature, the solvent was evaporated under vacuum to give a red solid. 1H NMR (CDCl3): δ 8.68 (s, 1H), 7.45 (d, 1H, J ) 9.0 Hz), 6.69 (dd, 1H, J1 ) 9.0 Hz and J2 ) 3.9 Hz), 6.48 (d, 1H, J ) 3.0 Hz), 3.50 (q, 4H), 1.35 (t, 6H, J ) 6.0 Hz). The UV spectrum of the starting compound 1 is shown in Figure 1. Syntheses of 2-N,N-Dimethylamino-3-[7-(diethylamino)2-oxo-2H-chromone-3-carboxyl]-octadec-4-ene-1-ol (4), 2-N,NDimethylamino-1-[7-(diethylamino)-2-oxo-2H-chromone-3carboxyl]-octadec-4-ene-3-ol (5), and 2-N,N-Dimethylamino1,3-bis[7-(diethylamino)-2-oxo-2H-chromone-3-carboxyl]octadec-4-ene (6). N,N-Dimethylsphingosine (3, 100 mg, 0.30 mmol) was added under a dry argon atmosphere to a solution of 2 (80 mg, 0.30 mmol) in anhydrous CH2Cl2 maintained at -40 °C. 2,4,6-Collidine (75 mg, 0.60 mmol) was added, and the reaction mixture was maintained at -40 °C for 4 h and then at 25 °C for a further 12 h. It was washed several times with an equal volume of saturated sodium bicarbonate solution, dried, and evaporated. The crude reaction product was separated
Ghosh et al.
Figure 2. Absorption spectrum of compound 5 in methanol solution (UV cutoff 205 nm).
into its individual components, 4, 5, and 6, by column chromatography on silica gel using CH2Cl2 and CH3OH (9:1) as eluent. Compound 4. Yield 18%. Rf ) 0.5 (CH2Cl2/MeOH, 9:1). 1H NMR (CDCl3): δ 8.42 (s, 1H), 7.36 (d, 1H, J ) 8.7 Hz), 6.61 (dd,1H, J1 ) 9.0 Hz and J2 ) 2.4 Hz), 6.45(d,1H, J) 2.4 Hz), 5.86-5.76 (m,1H), 5.71 (t, 1H, J ) 6.6 Hz), 5.53 (dd, 1H, J1 ) 15.3 Hz and J2 ) 6.9 Hz), 3.81 (m, 2H), 3.48 (q, 4H), 2.80 (m, 1H), 2.41 (s, 6H), 2.02 (q, 2H), 1.26 (m, 28H), 0.9(t, 3H, J ) 6.0 Hz). 13 C NMR (CDCl3): δ 163.44, 158.59, 158.49, 153.06,149.77, 135.57, 131.20, 126.98, 109.64, 108.46, 107.79, 96.67, 72.87, 68.19, 58.22, 45.13, 41.44, 32.28, 31.93, 29.68, 29.66, 29.61, 29.45, 29.36, 29.21, 28.80, 22.69, 14.13, 12.43. Compound 5. Yield 50%. Rf ) 0.4 (CH2Cl2/MeOH, 9:1). 1H NMR (CDCl3): δ 8.44 (s, 1H), 7.37 (d, 1H, J ) 8.7 Hz), 6.62 (dd, 1H, J1 ) 9.0 Hz and J2 ) 2.4 Hz), 6.45 (d, 1H, J ) 2.1 Hz), 5.79 (m, 1H), 5.54 (dd, 1H, J1 ) 15.45 Hz and J2 ) 6.0 Hz), 4.51 (m, 2H), 4.38 (t, 1H, J ) 5.4 Hz), 3.66 (d, 1H, J ) 5.7 Hz), 3.47 (q, 4H), 2.88 (q, 1H), 2.50 (s, 6H), 2.03 (q, 2H), 1.26 (m, 28 H), 0.9 (t, 3H, J ) 6.3 Hz). 13 C NMR (CDCl3): δ 164.40, 158.61, 158.36, 153.06, 149.71, 132.59, 131.21, 129.78, 109.63, 108.28, 107.76, 96.70, 70.76, 66.26, 62.06, 45.13, 43.05, 32.37, 31.93, 29.69, 29.66, 29.64, 29.52, 29.36, 29.25, 29.22, 22.69, 14.13, 12.43. The UV spectrum and the MALDI-TOF mass spectrogram of compound 5 are shown in Figures 2 and 3, respectively. Compound 6. Yield 2% (Rf ) 0.7, CH2Cl2/MeOH, 9:1). 1 H NMR (CDCl3): δ 8.42 (s, 1H), 8.41 (s, 1H), 7.38 (d, 1H, J ) 4.2 Hz), 7.36 (d, 1H, J ) 4.2 Hz), 6.60 (t, 1H, J ) 2.4 Hz), 6.57 (t, 1H, J ) 2.4 Hz), 6.41 (t, 2H, J ) 3.0 Hz), 5.8 (m, 2H), 5.64 (dd, 1H, J1 ) 4.2 Hz and J2 ) 8.7 Hz), 4.64 (m, 2H), 3.45 (q, 8H), 3.20 (q, 1H), 2.43 (s, 3H), 2.07 (q, 2H), 1.61 (s, 3H), 1.23 (m, 28 H), 0.88 (t, 3H, J ) 6.0 Hz). 13 C NMR: δ 164.02, 163.12, 158.86, 158.81, 158.60, 158.44, 153.28, 149.68, 149.52, 135.88, 131.69, 126.97, 109.86, 109.04, 108.07, 97.01, 73.34, 65.80, 61.79, 45.46, 42.28, 32.75, 32.31, 30.08, 30.05, 29.85, 29.74, 29.61, 29.27, 23.07, 14.51, 12.82. Cytotoxicity Assays. Human PC-3 prostatic carcinoma cells were cultured in DMEM/F-12 medium (GIBCO, Grand Island,
N,N-Dimethylsphingosine−Coumarin
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Figure 4. Dose-response effects of DMSP and DMSP-coumarin on viability (24 h MTT assay) of human PC-3 prostatic carcinoma, human HL60 acute myelogenous leukemia, and mouse P388 multidrug-resistant lymphocyte leumemia cell lines. Figure 3. ESI mass spectrogram of compound 5.
NY) supplemented with 5% fetal calf serum (GIBCO) at 37 °C in an incubator supplied with 5% CO2/95% air, as previously described (9). The murine P388 acute myelogenous leukemia (AML) MDR-expressing subline P388MDR, and HL60, a human promyelocytic-like AML line, were cultured similarly. The P388MDR line had been selected from the parental cells by growth in the presence of increasing concentrations of doxorubicin (Dox) and displayed a stable resistance phenotype in the absence of further selection pressure. To ensure that the leukemia cells were in the logarithmic phase of growth for the cytotoxicity assays, they were allowed to grow to a density of 106 cells/mL before experimental manipulation. Tumor cells were plated at (1.5-2.0) × 104 cells in 100 µL of medium per triplicate cell in flat-bottom 96-well microwell plates (Corning Glass Works, Corning, NY). Thereafter, they were incubated with known concentrations of DMSP or coumarin-tagged DMSP for 24 h. Since preliminary studies had revealed that the cytotoxic activity of compounds 4 and 5 were indistinguishable, their admixture was used in these cytotoxicity assays, as well as in the time-lapse fluorescence studies (see below). Remaining viable cells were stained with MTT. Resolubilized dye (MTT-formazan) was quantified by measurement of optical density at 570 nm in a microplate reader. The viability of treated cells was determined as a percentage of the optical density of dye in control cultures. Time-Lapse Fluorescence Microscopy. Digital time lapse acquisition of PC-3 cells was performed using the Cell Observer function of a Zeiss Axiovert 200M motorized inverted microscope. Cells growing in a 12-well tissue culture plate were placed in a chamber on a temperature-controlled stage and maintained in a heated, humidified, 5% CO2 atmosphere. The Axiovision Software was used to program the motorized stage to move sequentially from well-to-well and to acquire phase contrast as well as fluorescent color images at preselected time points. Phase images and fluorescent images, using appropriate excitation and emission settings for DMSP-coumarin (490/515 nm), were acquired. Mitochondrial Membrane Potential (MMP) Plate-Based Fluorescence Assay. PC-3 cells were plated in triplicate in flatbottom 96-well microwell plates (CoStar), essentially as described above for the cytotoxicity assays. After overnight incubation, DMSP (20 µM), DMSP-coumarin (20 µM), or control media was added and incubation was continued for 2 h. Thereafter, the protocol of the vendor (Cell Technology, Mountain View, CA) was followed for the addition of the fluorescent cationic dye to detect MMP. MMP-linked fluores-
cence in untreated control and treated (DMSP or DMSPcoumarin) cells was detected by excitation at 530 nm and emission at 590 nm. Background fluorescence was determined in untreated control cells that did not have the cationic dye added. The percentage of MMP in treated cells was compared with that in untreated control cells by the expression Percentage of control MMP ) (fluorescence in treated cells - background fluorescence) ÷ (fluorescence in untreated control cells background fluorescence).
RESULTS AND DISCUSSION Cytotoxicity Assays. The results are shown in Figure 4. PC-3 cells were essentially all killed by the highest concentration (40 µM) of either DMSP or DMSP-coumarin, whereas the IC50 was reached between 10 and 20 µM of either agent, indicating that substitution with coumarin did not cause significant effects on DMSP functions associated with induction of cell death. Similar results were obtained with these reagents on HL-60 cells, although both of the IC50’s were lower: ∼5-10 µM for DMSP and 5 µM slightly exceeded 50% killing of HL60 cells for DMSP-coumarin. With the P388MDR targets, again there was minimal distinction between DMSP and DMSP-coumarin, and the IC50’s were ∼10 µM for DMSP and ∼5-10 µM for DMSP-coumarin. Overall, based on the cytotoxicity assays in the prostatic as well as the leukemia models, substitution of DMSP with coumarin was well-tolerated. Time-Lapse Fluorescence Microscopy. The results in Figure 5 demonstrate the uptake of DMSP-coumarin in PC-3 cells, tracking its green fluorescence. PC-3 cells viewed prior to or immediately after introduction of DMSP-coumarin (20 µM) were clearly evident by phase contrast but not by fluorescence microscopy (data not shown). However, already after 60 min of further incubation (panel A), initial intracellular accumulation and localization to perinuclear mitochondria was apparent. This localization became increasingly pronounced and more clearly punctate after 2 h (panel B) and 4 h (panel C). (In related studies, we have shown that this accumulation is closely linked with loss of inner mitochondrial membrane potential, release of cytochrome c, and caspase activation). Effects of DMSP-Coumarin on MMP. PC-3 cells were treated with DMSP or DMSP-coumarin (20 µM) for 2 h prior to assaying mitochondrial membrane potential in control and treated cells. The results are shown in Table 1. DMSP rapidly and dramatically reduced the MMP of PC-3 cells to only background levels; similarly, the effect of DMSP-coumarin was
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for example, SKI II (10, 11). In our hands, SKI II causes a much more protracted effect against human prostatic cancer and leukemic target cell lines than does DMSP and, particularly, lacks the rapid effect on MMP observed with DMSP (Klostergaard et al., in preparation). The fungal metabolite FTY720 has been shown to down-regulate G-protein-coupled receptors through which extracellular Sp-1-P exerts its effects; further, FTY720 is endowed with a modified sphingoid backbone and also displays rapid homing to mitochondria and the ability to disrupt MMP (12, 13), suggesting that the latter property might be inherent in this backbone structure. The identification of mitochondrial domains with which the sphingoid backbone interacts might be illuminating with regard to its mechanism of induction of the intrinsic apoptotic pathway: for example, by conducting experiments using fluorescenated sphingosine(s) and mitochondria prelabeled with suitable fluorescent dyes appropriate for a donor-acceptor interaction for fluorescence resonance energy transfer (14). In summary, the availability of a fluorescent, biologically equivalent DMSP will advance studies both in Vitro and in ViVo, such as of its subcellular localization, mechanism of action, biodistribution, and pharmacology.
ACKNOWLEDGMENT We thank Mr. William Spohn for assistance with the timelapse fluorescence microscopy studies and Dr. Jaw-Ching Liu for assistance with manuscript preparation.
LITERATURE CITED
Figure 5. Time-lapse fluorescence microscopy of uptake of DMSPcoumarin in PC-3 cells. Green fluorescence is evident after 60 min of incubation (A) as intracellular accumulation and localization to perinuclear mitochondria; localization became increasingly pronounced after 2 h (B) and 4 h (C). Table 1. Effect of DMSP and DMSP-coumarin on MMP in PC-3 Cellsa treatment
% MMPb,c
DMSP DMSP-coumarin
-3.0 ( 8.8 7.1 ( 6.4
a PC-3 cells were treated with DMSP or DMSP-coumarin (both at 20 µM) for 2 h prior to addition of fluorescent cationic dye to detect MMP. MMP-linked fluorescence was detected by excitation at 530 nm and emission at 590 nm. b Percentage of MMP in DMSP- or DMSP-coumarintreated cells compared with that in untreated control PC-3 cells. Calculated as (fluorescence in treated cells - background fluorescence) ÷ (fluorescence in untreated control cells - background fluorescence). c Mean ( SEM from six determinations.
also pronounced, causing reduction to 7.1% ( 6.4% of control MMP. Thus, when considered together, the kinetics of localization and subsequent loss of MMP indicate that a very rapid depolarization is induced by DMSP in PC-3 targets once it reaches the mitochondria. Recent studies have revealed pharmacological tools with which to perturb the SL rheostat, in Vitro and in ViVo. For example, chemical library screening has defined inhibitors of SpK that are reportedly more specific and potent than DMSP,
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