Patent Abstract:
porphyrin compounds useful in the field of magnetic resonance imaging as contrast agents . the compounds are relatively lipophilic porphyrins , include one or more enzyme - reactive functional groups , and are cell membrane permeable . relatively lipophilic group can be enzymatically released within a cell to produce a relatively hydrophilic porphyrin compound .

Detailed Description:
certain mnps can reach unusual high relaxivity , especially at high magnetic fields of clinical scanners . in 1984 , chen et al first reported the relaxivity of mntpps as ˜ 12 mm − 1 s − 1 at 0 . 47 t ( 20 mhz for 1 h larmor frequency ), 37 ° c . 45 a measurement of the t 1 nuclear magnetic resonance dispersion ( nmrd ) profile of mntpps was subsequently reported by konieg et al . 46 the nmrd profile ( field - dependent relaxivity ) for mntpps is unique among the known small molecule t 1 agents , because the relaxivity increases with magnetic fields above 1 mhz . the opposite trend has been shown for most small gd - based as well as mn ( ii )- based cas where the nmrd profile decreases with increasing field strength . these results were surprising because gd ( iii ) has seven unpaired electrons ( s = 7 / 2 ) and mn ( ii ) has five unpaired electrons ( s = 5 / 2 ) compared to mn ( iii ) with only four unpaired electrons ( s = 4 / 2 ). therefore , mntpps was described as having “ anomalous high relaxivity ”. 46 this uniquely high relaxivity at high fields is beneficial for the development of new cas for mr cell imaging . mnps are highly stable against mn - dissociation and free mn is less toxic than gd . porphyrins have a fully conjugated 7 - system leading to a conformationally rigid macrocycle with a predefined , metal binding pocket compatible for mn ( iii ) ions leading to a thermodynamically and kinetically stable complex . this also results in the correct geometry and molecular orbital energy levels for mn ( d ) to ligand ( π *) back - bonding , further increasing the bond strength as evident from spectroscopic studies of mnps , thereby further reducing the likelihood of metal leakage . 27 while mn , an endogenous micronutrient , is less toxic than gd ( iii ), there are still concerns about high concentrations of free mn ( ii ) in vivo , and a highly stable chelate would be desirable to eliminate any concerns about toxicity especially for longitudinal tracking of labeled cells in vivo . mnps are amenable to structural modifications . while the mn is bound in the middle of the porphyrin core , different functional groups can be introduced at peripheral sites on the porphyrin ring , including meso - and beta - positions , without significantly influencing the mn - binding . these possibilities for structural modifications allow systematic engineering of the molecular parameters of mnps , including lipophilicity , water - solubility , charge , size , etc , thereby controlling the cell - permeability , intracellular and in vivo distribution , and biocompatibility . in the current invention , we introduced enzyme - reactive groups attached on the porphyrin ring , leading to cell - permeable and trappable mnps . in general , porphyrins can be synthesized from pyrrole and a chosen aldehyde via the lindsey method . 47 functional group modifications , manganese insertion and addition of esterase labile bonds lead to cell permeable and trappable mri contrast agents . alternatively , monomeric porphyrin building blocks can be subjected to dimerization , oligomerization or polymerization followed by functional group modification manganese insertion and installation of esterase labile groups . sequence of these reactions steps may vary depending on the structure of the substrates . porphyrin monomers can be synthesized by the lindsey reaction . different aldehydes condensed with pyrrole followed by oxidation give access to a variety of symmetric ( r 1 = r 2 = r 3 = r 4 ) or non - symmetric ( at least two r groups are different ) porphyrins , as exemplified in scheme 2 . the symmetric tetrakis ( ethoxycarbonyl ) porphyrin ( r 1 = r 2 = r 3 = r 4 =— cooet ), 1 , was prepared using this method . scheme 2 : general method for synthesis of symmetric or non - symmetric porphyrin monomers a ) 1 . bf 3 . oet 2 , dcm ; 2 . ddq . the porphyrin monomers were subjected to manganese insertion , to give several contrast agents . all other mnps were deprotected and a varying number of acetoxymethyl ester groups were installed at the meso positions for cell permeability and hydrolysis by intracellular esterase . scheme 3 : examples of mn - insertion , deprotection and installation of esterase labile groups ( in this example acetoxymethyl ester groups ). b ) mn insertion : mncl 2 , dmf , reflux c ) 2 m naoh , thf , etoh , reflux , 12 h d ) 1 m hcl ( aq ) e ) dbu , ambr , 55 ° c ., 30 hours . all reagents and solvents were of commercial reagent grade and were used without further purification except where noted . all reactions were carried out with oven dried glassware , anhydrous solvents and under argon atmosphere unless stated otherwise . column chromatography was carried out using caledon silica gel 60 ; 50 - 200 microns 70 - 300 mesh , or using sephadex ™ lh - 20 with dry bead size of 18 - 111 □ m from ge health care . dialysis was performed with spectrumlabs float - a - lyzer ™ g2 500 mwco . cation ion exchange was performed using an amberlite ® ir120 , h resin . phosphate buffer saline was purchased from sigma ® life science , sterile filtered and endotoxin tested . the cell line was obtained from atcc ( american tissue culture collection manassas , va ., usa ). 1640 - rpmi medium was purchased from sigma - aldrich , trypsin edta was purchased from gibco ( carlsbad , calif ., usa ). all the spectroscopy data for structural characterizations were obtained using the research facilities at university of toronto scarborough campus ( traces center ) or at st . george campus ( chemistry department ). nmr spectra were recorded on brucker - 500 mhz or varian unity 500 mhz spectrometer . uv - vis spectra were recorded on an agilent 8453 uv - visible spectroscopy system . infrared spectra were recorded on a bruker alpha ft - ir spectrometer . high resolution mass spectra were obtained from an abi / sciex qstar mass spectrometer ( esi ). synthesis of 5 , 10 , 15 , 20 - tetrakis ( ethoxycarbonyl ) porphyrin , 1 was performed with a modification of a literature method . 48 ethyl glyoxalate ( 50 % in toluene , 1 . 88 ml , 9 . 4 mmol ) in dichloromethane and pyrrole ( 0 . 65 ml , 9 . 4 mmol ) were stirred at room temperature , in the dark and under an argon atmosphere . after 10 minutes bf 3 . oet 2 ( 42 ml , 3 . 10 mmol ) was added drop wise . the reaction was stirred at room temperature for 1 . 25 hours followed by the addition of ddq ( 1 . 5999 g , 7 . 05 mmol ). after a stirring period of 2 . 25 hours net 3 ( 0 . 43 ml , 3 . 06 mmol ) was added via syringe and the reaction mixture was concentrated on a rotary evaporator . the crude solution was suction filtered over sealite using dcm as an elution solvent . the solution was concentrated on a rotary evaporator . purification by column chromatography ( dcm ) on silica gel gave 169 . 2 mg ( 12 %) of compound 1 as a black - purple solid . 1 h nmr ( cdcl 3 ) 9 . 52 ( 8h , s , por - β ), 5 . 11 ( 8h , q , j = 7 . 2 ), 1 . 81 ( 12h , t , j = 7 . 2 ), − 3 . 33 ( 2h , s , nh ). uv - vis ( dcm ) λ max = 409 nm . the synthesis of [ 5 , 10 , 15 , 20 - tetrakis ( ethoxycarbonyl ) porphyrinato ] manganese ( iii ) chloride 1 - mn was performed according to a literature method . 48 compound 1 ( 17 . 8 mg , 29 . 7 μmol ) was dissolved in 2 ml of dmf . mncl 2 . 4h 2 o ( 17 . 7 mg , 89 . 2 μmol ) was added and the reaction was refluxed open to air for 5 hours . the reaction was stirred at room temperature open to air for a further 11 . 5 hours . distillation of dmf resulted in a black - purple solid . purification by stepped gradient column chromatography ( eluting with dcm to 7 % meoh in dcm ) on silica gel gave 16 . 5 mg ( 85 %) of compound 1 - mn as a black - purple solid . esi ms found m / z = 651 . 1 [ m +], calcd for c 32 h 28 mnn 4 o 8 + , m / z = 651 . 1 . uv - vis ( meoh ) λ abs = 328 , 366 , 387 , 413 , 456 , 552 nm . synthesis of [ 5 , 10 , 15 , 20 - tetrakis ( carboxy ) porphyrinato ] manganese ( iii ) chloride 1 - mn tcp was carried out as described in canadian patent application no . 2 , 805 , 543 . ethanol ( 10 ml ) and 2 m naoh ( aq ) ( 10 ml ) were added to a solution of 1 - mn ( 14 . 3 mg , 21 . 9 μmol ) in 6 ml of thf . the reaction was refluxed for 12 hours followed by neutralization with 3 m h 2 so 4 ( aq ) . purification first by sephadex lh - 20 chromatography with ultrapure water followed by dialysis with ultrapure water gave the desired product as a red - brown solid in 85 % yield . esi ms found m / z = 539 . 0 [ m +], calcd for c 24 h 12 mnn 4 o 8 + , m / z = 539 . 0 . uv - vis ( hepes buffer , ph = 7 . 0 ) λ abs = 325 , 377 , 397 , 421 , 465 , 561 , 592 nm . 1 - mntcp ( 23 . 2 mg , 38 . 3 μmol ) was dissolved in ultrapure water ( 5 ml ). 1 m hcl ( aq ) was added dropwise until the ph reached 2 . 2 and precipitation of a red solid occurred . after cooling in an ice - water bath for 30 minutes the solid was filtered and the clear filtrate was discarded . cold ultrapure water was used to wash the solid and drying under under reduced pressure resulted in protonated 1 - mntcp ( 12 . 3 mg , 22 . 8 μmol ). the water wash contained salt and 1 - mntcp and was subjected to dialysis . protonated 1 - mntcp ( 9 . 8 mg , 18 . 2 μmol ) was suspended in dmf ( 2 ml ). under continuous stirring , 1 , 8 - diazabicyclo [ 5 . 4 . 0 ] undec - 7 - ene ( dbu , 36 μl , 145 μmol , 8 eq .) was added dropwise . after 10 min . the first aliquot of acetoxymethyl bromide ( 6 μl , 61 μmol , 3 . 4 eq ) was added dropwise . the reaction temperature was maintained at 55 ° c . for 30 hours . the progress of the reaction was monitored by tlc . after 6 hours a second aliquot of acetoxymethyl bromide ( 6 μl , 61 μmol , 3 . 4 eq ) was added dropwise . after 24 hours a third aliquot of acetoxymethyl bromide ( 3 μl , 31 μmol , 1 . 7 eq ) was added . distillation of dmf at 60 ° c . under reduced pressure resulted in a crude dark oil . the crude material was dissolved in dcm and washed with water twice , then washed with brine twice . the organic layer was dried over sodium sulphate and filtered prior to concentration on a rotary evaporator . purification by stepped gradient column chromatography ( eluting with dcm to 10 % meoh in dcm ) on silica gel gave 6 . 8 mg ( 50 %) of compound 2 - mntriamp as a red - brown solid and 5 % of mntetraamp as a red - brown solid . characterization of mntamp : esi ms positive mode : found m / z = 827 . 0885 [ m + ], calculated for c 36 h 28 mnn 4 o 16 + , m / z = 827 . 0875 and characterization of 2 - mntriamp : esi ms found m / z = 755 . 0678 [ m + ], calculated for c 33 h 24 mnn 4 o 14 , m / z = 755 . 0664 , ir ( cm − 1 ) 1744 . 27 ( c ═ o ), 1555 . 34 ( coo − ). human breast cancer cells mda - mb - 231 were grown at 37 ° c . with 5 % co 2 in 1640 - rpmi medium supplemented with 10 % fetal bovine serum and 0 . 5 % penicillin streptomycin . contrast agents were quantified for manganese content by flame aas prior to cell labeling . mntcp was dissolved in ultrapure water and mntriamp was dissolved in dmso to give 17 mm stock solutions . the stock solutions were added to the medium with the cells resulting in 83 μm incubation of 2 - mntriamp or 1 - mntcp for 2 hours . the medium was removed and the cells were washed with fresh medium 5 × and then were harvested by washing with pbs followed by addition of 0 . 05 % trypsin edta to detach the cells . viability was assessed directly after labeling with trypan blue exclusion test and observation of cell morphology by phase microscopy . the results are listed in table 1 . for mri , cell pellets were formed by centrifugation at 440 g for 10 minutes . subsequent mr was done with a t 1 - weighted 2d spin - echo image : tr = 100 ms , te = 14 . 163 ms , 3 mm slice thickness , 0 . 5 × 0 . 5 mm in - plane resolution ( fig1 ). the signal was analyzed to give the relaxivity of the cell pellets as seen in table 2 . the intracellular contrast agent concentration was determined after cell labeling by centrifugation of a known number of cells followed by removal of the supernatant and digestion of the cells by addition of 1 m nitric acid ( 0 . 5 ml ) and 30 min of heating at 70 ° c . then 3 ml of ultrapure water was added to the digested cells , the mixture was filtered and then quantified by graphite furnace atomic absorption spectroscopy ( gfaas ) shown in table 3 . the cell labeling showed significant uptake of the hydrophobic compound 2 - mntriamp compared to the hydrophilic tetracarboxylic acid , 1 - mntcp as shown by the reduced relaxation times of the 2 - mntriamp treated cells and the quantitative gfaas data . the viability showed a statistically insignificant drop in healthy cells indicating lack of toxicity of the contrast agents after labeling . therefore this compound shows great promise as a cell permeable and trappable positive contrast agent for cellular mri applications . this invention covers the structure , preparation and applications of a series of paramagnetic porphyrins designed as mri t 1 contrast agent for cell - 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