photodynamic therapy ( pdt ) has emerged as a promising 
 modality for the treatment of malignant tumors and wet age - related macular 
 degeneration [ 13 ] . it is a binary therapy that involves the combination of visible light 
 and a photosensitizer . each component is 
 harmless by itself , but in combination with molecular oxygen , they result in 
 the generation of reactive oxygen species ( ros ) causing oxidative cellular and 
 tissue damage . this treatment has 
 several potential advantages including its minimally invasive nature , tolerance 
 of repeated doses , and high specificity that can be achieved through precise application of the light with modern fiber - optic systems and various types of endoscopy . currently , only a few porphyrin derivatives including porfimer sodium , temoporfin , and verteporfin are clinically approved for systemic administration . these compounds , though giving a positive 
 response in a high percentage of patients , still have various deficiencies that demand a further development of better candidates 
 . owing to the desirable electronic absorption and photophysical properties , phthalocyanines are one of the most promising classes of candidates for this application . over the last few years , we have been interested in rational 
 modification of this class of functional dyes with the goal of enhancing their pdt efficiency . several new series of silicon(iv ) and zinc(ii ) phthalocyanines have been synthesized 
 and evaluated for their photo - physical and biological properties ; see 
  and the references . as the amphiphilicity of photosensitizers is believed to have a beneficial effect on their photodynamic activity , 
 amphiphilic phthalocyanines have been our targets . in this paper , we report the synthesis , photophysical properties , and in vitro photodynamic 
 activity of three novel zinc(ii ) 
 phthalocyanines bearing one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy 
 substituent(s ) . having three or six triethylene glycol chains on one side 
 of the macrocycle , tetrahydrofuran ( thf ) , n - pentanol , dichloromethane , and n , n - dimethylformamide 
 ( dmf ) were distilled from sodium benzophenone ketyl , sodium , calcium 
 hydride , and barium oxide , respectively . chromatographic purifications were performed on silica gel ( macherey - nagel , 70230 mesh ) columns with the 
 indicated eluents . size exclusion chromatography 
 was carried out on bio - rad bio - beads s - x1 beads ( 200400 mesh ) . h and c{h } nmr spectra were 
 recorded on a bruker dpx 300 spectrometer ( h , 300 ; c , 
 75.4 mhz ) in cdcl3 or dmso - d6 . spectra were referenced internally using the 
 residual solvent [ h : cdcl3 (  7.26 ) ; dmso - d6 (  
 2.50 ) ] or solvent [ c : cdcl3 (  77.0 ) ; 
 dmso - d6 (  39.7 ) ] resonances relative to sime4 . electrospray ionization ( esi ) mass spectra 
 were measured on a thermo finnigan mat 95 xl mass spectrometer . uv - vis and 
 steady - state fluorescence spectra were taken on a cary 5 g uv - vis - nir 
 spectrophotometer and a hitachi f-4500 spectrofluorometer , respectively . / fref)(aref / asample)(nsample2/nref2)f(ref ) , where f , a , and n are the measured fluorescence ( area under the emission peak ) , the absorbance at the excitation 
 position ( 610 nm ) , and the refractive index of the solvent , respectively . the unsubstituted zinc(ii ) phthalocyanine ( znpc ) 
 in dmf was used as the reference [ f(ref)=0.28 ] . to minimize reabsorption of radiation by the ground - state species , the 
 emission spectra were obtained in very dilute solutions where the absorbance at 
 610 nm was less than 0.03 . singlet oxygen 
 quantum yields (  ) were measured in dmf by 
 the method of chemical quenching of 1,3-diphenylisobenzofuran ( dpbf ) using znpc 
 as reference ( =0.56 ) . to a mixture of 3-nitrophthalonitrile 
 ( 1.73 g , 10 mmol ) and compound 1 ( 2.97 g , 5 mmol ) in dmf ( 30 ml ) was added anhydrous k2co3 ( 6.90 g , 50 mmol ) . the solvent was then evaporated under reduced pressure and the residue 
 was mixed with chcl3 ( 60 ml ) and water ( 60 ml ) . the aqueous layer was separated and extracted 
 with chcl3 ( 60 ml 3 ) . the 
 combined organic layers was dried over anhydrous mgso4 , then 
 evaporated to dryness . the residue was 
 purified by silica - gel column chromatography using chcl3/meoh ( 60 : 1 v / v ) as eluent to give compound 2 as 
 a colorless liquid ( 1.41 g , 39% ) . h 
 nmr :  7.61 ( vt , j = 8.7 hz , 1 h , arh ) , 7.37 ( d , j = 7.5 hz , 1 h , arh ) , 7.25 ( d , j = 
 7.5 hz , 1 h , arh ) , 6.66 ( s , 2 h , arh ) , 5.17 ( s , 2 h , arch2 ) , 4.134.18 
 ( m , 6 h , ch2 ) , 3.85 ( t , j = 4.8 hz , 4 h , 
 ch2 ) , 3.79 ( t , j = 5.1 hz , 2 h , ch2 ) , 3.703.74 ( m , 6 h , ch2 ) , 3.623.68 ( m , 12 h , ch2 ) , 
 3.533.57 ( m , 6 h , ch2 ) , 3.37 ( two partially overlapping s , 9 h , ch3 ) ; c{h } nmr ( dmso - d6 ) :  160.9 , 152.4 , 137.7 , 
 136.0 , 130.7 , 126.2 , 119.3 , 116.0 , 115.6 , 113.9 , 107.1 , 103.6 , 72.0 , 71.5 , 
 71.2 , 70.2 , 70.0 , 69.9 , 69.8 , 69.1 , 68.5 , 58.2 ( some of the ch2 signals are overlapped ) ; ms ( esi ) : an isotopic cluster peaking at m / z 743 100% , [ m + na ] ; hrms ( esi ) : m / z calcd for c36h52n2nao13[m + na ] : 743.3362 , found 743.3365 . according to the above procedure 
 using 4- instead of 3-nitrophthalonitrile as a starting material , compound 3 was obtained as a colorless liquid 
 ( 1.71 g , 47% ) . h nmr ( cdcl3 ) :  7.70 
 ( d , j = 8.7 hz , 1 h , arh ) , 7.31 ( d , j = 2.4 hz , 1 h , arh ) , 7.22 ( dd , j = 2.4 , 8.7 hz , 1 h , arh ) , 6.60 ( s , 2 h , arh ) , 5.02 
 ( s , 2 h , arch2 ) , 4.114.15 ( m , 6 h , ch2 ) , 3.82 ( t , j = 4.8 hz , 4 h , ch2 ) , 3.76 ( t , j = 5.1 hz , 2 h , ch2 ) , 3.683.71 ( m , 6 h , 
 ch2 ) , 3.603.65 ( m , 12 h , ch2 ) , 3.503.53 ( m , 6 h , ch2 ) , 
 3.34 ( s , 9 h , ch3 ) ; c{h } nmr ( cdcl3 ) : 
  161.5 , 152.9 , 138.7 , 135.2 , 129.7 , 120.0 , 119.6 , 117.3 , 115.5 , 115.1 , 107.5 , 
 107.2 , 72.2 , 71.8 , 71.0 , 70.7 , 70.6 , 70.4 , 69.6 , 68.9 , 58.9 ( some of the ch2 signals are overlapped ) ; ms ( esi ) : an isotopic cluster peaking at m / z 743 100% , [ m + na ] ; hrms ( esi ) : m / z calcd for c36h52n2nao13[m + na ] : 743.3362 , found 743.3361 . a mixture of phthalonitrile 2 ( 0.26 g , 0.36 mmol ) , unsubstituted phthalonitrile ( 0.46 g , 3.59 mmol ) , and zn(oac)2 2h2o 
 ( 0.22 g , 1.00 mmol ) in n - pentanol ( 15 ml ) was heated to 100c , then a small 
 amount of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) ( 0.5 ml ) was added . the mixture was stirred at 140150c 
 for 24 hours after a brief cooling , the 
 volatiles were removed under reduced pressure . the residue was dissolved in chcl3 ( 120 ml ) , then filtered to 
 remove part of the unsubstituted zinc(ii ) phthalocyanine formed . the residue was 
 purified by silica - gel column chromatography using chcl3/ch3oh 
 ( 30 : 1 v / v ) as eluent , followed by size exclusion chromatography using thf as 
 eluent . the crude product was further 
 purified by recrystallization from a mixture of thf and hexane ( 0.11 g , 26% ) . h 
 nmr ( cdcl3 with a trace amount of 
 pyridine - d5 ) :  9.419.46 ( m , 5 h , pc - h ) , 9.16 ( d , j = 7.5 hz , 1 h , pc - h ) , 9.13 
 ( d , j = 6.9 hz , 1 h , pc - h ) , 
 8.078.15 ( m , 7 h , pc - h ) , 7.69 ( d , j = 7.8 hz , 1 h , pc - h ) , 7.30 ( s , 2 h , arh ) , 5.80 ( s , 2 h , arch2 ) , 4.31 ( t , j = 5.1 hz , 2 h , ch2 ) , 4.19 ( t , j = 5.1 hz , 4 h , 
 ch2 ) , 3.90 ( t , j = 5.1 hz , 2 h , ch2 ) , 3.783.82 ( m , 2h , ch2 ) , 3.663.75 ( m , 8 h , ch2 ) , 
 3.553.59 ( m , 6 h , ch2 ) , 3.473.51 ( m , 8 h , ch2 ) , 3.383.41 
 ( m , 7 h , ch2 and ch3 ) , 3.24 ( s , 6 h , ch3 ) ; c{h } 
 nmr ( dmso - d6 ) :  155.6 , 152.7 , 152.6 , 152.3 , 152.2 , 140.3 , 138.4 , 
 137.9 , 137.7 , 137.6 , 133.5 , 130.6 , 129.1 , 125.3 , 122.5 , 122.4 , 122.1 , 115.3 , 
 114.0 , 107.8 , 72.3 , 71.6 , 71.5 , 71.0 , 70.2 , 70.0 , 69.9 , 69.8 , 69.3 , 68.8 , 58.3 , 
 58.2 ( some of the signals are overlapped ) ; ms ( esi ) : 
 an isotopic cluster peaking at m / z 
 1191 100% , [ m + na ] ; hrms ( esi ) : m / z calcd for c60h64n8nao13zn 
 [ m + na ] : 1191.3777 , found 1191.3783 . according to the above procedure , phthalonitrile 3 ( 0.26 g , 0.36 mmol ) was treated with 
 unsubstituted phthalonitrile ( 0.46 g , 3.59 mmol ) and zn(oac)22h2o ( 0.22 g , 1.00 mmol ) to give phthalocyanine 5 as a blue solid ( 0.09 g , 21% ) . h 
 nmr ( cdcl3 with a trace amount of 
 pyridine - d5 ) :  9.209.35 ( m , 6 h , pc - h ) , 9.05 ( d , j = 7.8 hz , 1 h , pc - h ) , 8.66 ( s , 1 h , pc - h ) , 8.068.12 ( m , 6 h , pc - h ) , 7.62 ( d , j = 8.4 hz , 1 h , pc - h ) , 6.99 ( s , 2 h , arh ) , 5.46 ( s , 2 h , arch2 ) , 4.33 ( t , j = 4.8 hz , 4 h , ch2 ) , 4.24 ( t , j = 5.1 hz , 2 h , ch2 ) , 3.94 
 ( t , j = 4.8 hz , 4 h , ch2 ) , 
 3.86 ( t , j = 4.8 hz , 2 h , ch2 ) , 3.763.80 ( m , 6 h , ch2 ) , 
 3.623.71 ( m , 12 h , ch2 ) , 3.533.57 ( m , 6 h , ch2 ) , 3.38 
 ( s , 3 h , ch3 ) , 3.36 ( s , 6 h , ch3 ) ; c{h } 
 nmr ( dmso - d6 ) :  159.9 , 152.6 , 152.4 , 152.3 , 152.2 , 152.0 , 151.7 , 
 151.6 , 139.5 , 137.8 , 137.5 , 132.7 , 130.7 , 129.0 , 128.9 , 123.0 , 122.2 , 117.8 , 107.2 , 
 105.6 , 72.1 , 71.5 , 70.3 , 70.1 , 70.0 , 69.9 , 69.4 , 68.8 , 58.3 , 55.1 ( some of the 
 signals are overlapped ) ; ms ( esi ) : an isotopic 
 cluster peaking at m / z 1191 95% , [ m + na ] ; hrms ( esi ) : m / z calcd for c60h64n8nao13zn 
 [ m + na ] : 1191.3777 , found 1191.3771 . a mixture of compound 6 ( 2.06 g , 3.36 mmol ) , 2,3-dicyanohydroquinone ( 0.27 g , 1.69 mmol ) , and k2co3 ( 1.17 g , 8.47 mmol ) in dmf ( 10 ml ) was stirred at 100c 
 for 24 hours the volatiles were then removed under reduced 
 pressure . the residue was mixed with 
 water ( 50 ml ) and the mixture was extracted with chcl3 ( 50 ml  3 ) . the combined organic extracts was 
 dried over anhydrous mgso4 and evaporated under reduced pressure . the residue was then purified by silica - gel column chromatography using chcl3/meoh 
 ( 20:1 v / v ) as eluent to give the product as a pale yellow transparent liquid 
 ( 1.95 g , 88% ) . h nmr ( cdcl3 ) :  7.13 ( s , 2 h , arh ) , 6.64 ( s , 4 h , 
 arh ) , 5.07 ( s , 4 h , arch2 ) , 4.124.17 ( m , 12 h , ch2 ) , 
 3.84 ( t , j = 4.8 hz , 8 h , ch2 ) , 3.78 ( t , j = 5.1 hz , 4 h , ch2 ) , 3.713.74 ( m , 12 h , 
 ch2 ) , 3.623.67 ( m , 24 h , ch2 ) , 3.523.57 ( m , 12 h , ch2 ) , 
 3.37 ( two partially overlapping s , 18 h , ch3 ) ; c{h } 
 nmr ( cdcl3 ) :  154.7 , 152.8 , 138.2 , 130.3 , 119.4 , 112.9 , 106.4 , 
 105.7 , 72.2 , 71.7 , 71.6 , 70.6 , 70.5 , 70.3 , 69.5 , 68.7 , 58.8 ( some of the ch2 signals are overlapped ) ; ms ( esi ) : an isotopic cluster peaking at m / z 1336 100% , [ m + na ] ; hrms ( esi ) : m / z calcd for c64h100n2nao26 [ m + na ] : 1335.6457 , found 1335.6462 . according to the procedure described for 4 , phthalonitrile 7 ( 0.50 g , 0.38 mmol ) was treated with unsubstituted 
 phthalonitrile ( 0.49 g , 3.82 mmol ) and zn(oac)22h2o 
 ( 0.23 g , 1.05 mmol ) to give phthalocyanine 8 as a blue - green oil ( 54 mg , 8% ) . h 
 nmr ( cdcl3 ) :  9.439.47 ( m , 4 h , pc - h ) , 
 9.20 ( d , j = 7.5 hz , 2 h , pc - h ) , 8.038.15 ( m , 6 h , pc - h ) , 7.68 ( s , 2 h , pc - h ) , 7.37 ( s , 4 h , arh ) , 5.90 ( s , 4 h , arch2 ) , 4.05 ( t , j = 5.1 hz , 4 h , ch2 ) , 3.80 ( t , j = 5.1 hz , 8 h , ch2 ) , 3.70 ( t , j = 5.1 hz , 4 h , ch2 ) , 3.613.65 ( m , 4 h , 
 ch2 ) , 3.553.58 ( m , 8 h , ch2 ) , 
 3.443.48 ( m , 12 h , ch2 ) , 3.343.37 ( m , 
 12 h , ch2 ) , 3.30 ( s , 6 h , ch3 ) , 3.183.21 ( m , 8 h , ch2 ) , 
 3.12 ( s , 12 h , ch2 ) , 2.90 ( s , 12 h , ch3 ) ; c{h } 
 nmr ( cdcl3 ) : 153.6 , 153.4 , 153.3 , 152.5 , 152.2 , 150.2 , 138.6 , 138.3 , 
 138.1 , 137.5 , 133.6 , 129.0 , 128.8 , 128.6 , 128.5 , 122.6 , 122.3 , 122.1 , 116.5 , 
 107.0 , 72.3 , 72.2 , 71.8 , 71.3 , 70.5 , 70.4 , 69.9 , 69.7 , 69.2 , 68.1 , 58.8 , 58.4 
 ( some of the ch2 signals are overlapped ) ; ms 
 ( esi ) : an isotopic cluster peaking at m / z 1784 20% , [ m + na ] ; hrms 
 ( esi ) : m / z calcd for c88h112n8nao26zn 
 [ m + na ] : 1783.6871 , found 1783.6862 . the ht29 human colorectal carcinoma 
 cells ( from atcc , no . 
 htb-38 ) were maintained in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen , cat no . 10313 - 021 ) supplemented with 
 fetal calf serum ( 10% ) , penicillin - streptomycin ( 100 units ml and 
 100 mgml , resp . ) , l - glutamine ( 2 mm ) , and transferrin ( 10 mgml ) . the hepg2 human hepatocarcinoma cells ( from 
 atcc , no . 23400 - 021 ) supplemented with fetal calf serum ( 10% ) and penicillin - streptomycin 
 ( 100 units ml and 100 mgml , resp . ) . approximately 3104 ( for ht29 ) 
 or 4104 ( for hepg2 ) cells per well in these media were inoculated 
 in 96-multiwell plates and incubated overnight at 37c in a humidified 5% co2 atmosphere . phthalocyanines 4 , 5 , and 8 were first dissolved in dmf to give 1.5 mm solutions , which 
 were diluted to 80 m with an aqueous solution of cremophor el ( sigma , 0.47 g in 
 100 ml of water ) . the solutions were 
 filtered with a 0.2 m filter , then diluted with the culture medium to appropriate 
 concentrations ( two - fold dilutions from 8 m ) . the cells , after being rinsed with phosphate buffered saline ( pbs ) , were 
 incubated with 100 l of these phthalocyanine solutions for 2 hours at 37c under 
 5% co2 . the cells were then 
 rinsed again with pbs and refed with 100 l of the culture medium before being 
 illuminated at ambient temperature . the 
 light source consisted of a 300 w halogen lamp , a water tank for cooling , and a 
 color glass filter ( newport ) cut - on 610 nm . an illumination of 20 minutes led to a total 
 fluence of 48 j cm . an mtt ( sigma ) solution in pbs ( 3 mgml , 
 50 l ) was added to each well followed by incubation for 2 hours under the same 
 environment . a solution of sodium 
 dodecyl sulfate ( sds , sigma ) ( 10% by weight , 50 l ) was then added to each 
 well . the plate was incubated in an oven 
 at 60c for 30 minutes , then 80 l of iso - propanol was added to each 
 well . the plate was agitated on a 
 bio - rad microplate reader at ambient temperature for 10 sec before the 
 absorbance at 540 nm at each well was taken . the average absorbance of the blank wells , which 
 did not contain the cells , was subtracted from the readings of the other 
 wells . the cell viability was then 
 determined by the equation : % viability = [ (ai / acontrol100)]/n , 
 where ai is the absorbance of the ith data ( i=1,2,  ,n ) , acontrol is the average absorbance of the control 
 wells , in which the phthalocyanine was absent , and n (= 4 ) is the number of the data points . for the detection of the intracellular 
 fluorescence intensity of compounds 4 , 5 , and 8 , approximately 
 1.2105 ht29 cells in the culture medium ( 2 ml ) were seeded on a 
 coverslip ( diameter = 25 mm ) and incubated overnight at 37c under 5% co2 . the medium was removed , then the cells were 
 incubated with 2 ml of an 8 m phthalocyanine dilution in the medium for 2 h 
 under the same conditions . the cells 
 were then rinsed with pbs and viewed with an olympus ix 70 inverted 
 microscope . the excitation light source 
 ( at 630 nm ) was provided by a multiwavelength illuminator ( polychrome iv , till 
 photonics ) . the emitted fluorescence 
 ( > 660 nm ) was collected using a digital cooled ccd camera ( quantix , 
 photometrics ) . in addition to 
 their relatively high stability , the closed - shell zinc(ii ) center imparts desirable photophysical characteristics to the 
 macrocycles . introduction of substituents 
 at the peripheral positions can also tailor the properties of the macrocycles such as their 
 solubility in biological media , aggregation behavior , and targeting properties . as a 
 result , zinc(ii ) we 
 describe herein three novel zinc(ii ) phthalocyanines ( compounds 4 , 5 , and 8) which contain the 
 bulky and hydrophilic 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy 
 moiety . having one or two of these substituents , the - stacking tendency is reduced and the macrocycles 
 become amphiphilic in nature . these 
 properties should be advantageous for singlet oxygen generation and cellular 
 uptake , by which the photodynamic activity can be enhanced . the relatively rare 1,4-disubstituted phthalocyanine 8 also has a longer q - band maximum 
 compared with the - 
 and -monosubstituted counterparts , which is also an advantage that can 
 increase the light penetration depth . scheme 1 
 shows the synthetic route used to prepare the monosubstituted phthalocyanines 4 and 5 . reaction of benzyl alcohol 1 with 3- or 4-nitrophthalonitrile in the presence of k2co3 in dmf gave the substituted phthalonitrile 2 or 3 , respectively . these 
 compounds then underwent a mixed cyclization with an excess of the 
 unsubstituted phthalonitrile ( 10 equiv . ) in the presence of zn(oac)22h2o 
 and dbu in n - pentanol to afford the 
 corresponding  3 + 1  products 4 and 5 . these reactions also produced the unsubstituted znpc as a major 
 side - product , which could be separated readily by filtration and chromatography 
 as a result of its lower solubility and slower mobility in the silica gel 
 column . during the chromatographic 
 purification , a trace amount of some other blue products was also separated , 
 but no attempt was made to characterize these minor side - products . similarly , treatment of 2,3-dicyanohydroquinone with benzyl chloride 6 and k2co3 afforded dinitrile 7 , 
 which was then cyclized with the unsubstituted phthalonitrile in the presence of zn(oac)22h2o 
 to give 8 ( scheme 2 ) . all these zinc(ii ) 
 phthalocyanines were soluble in common organic solvents and possessed high 
 stability , which facilitated the purification by silica - gel column chromatography , size exclusion 
 chromatography , followed by recrystallization . the nmr signals for the 
 phthalocyanine ring protons of 4 , 5 , and 8 are very distinct in cdcl3 ( with a trace amount of 
 pyridine - d5 for the former two complexes to reduce their 
 aggregation ) , which provide a useful means for characterization . as shown in figure 1 , the h nmr spectrum of the -substituted phthalocyanine 4 shows a multiplet at  9.419.46 ( 5 h ) and two doublets at  9.16 ( 1 h ) and 9.13 ( 1 h ) for the 7 phthalocyanine  protons . the 8  protons resonate as a multiplet at  8.078.15 ( 7 h ) and a doublet at  7.69 ( 1 h ) . for the -analogue 5 , a multiplet at  9.209.35 ( 6 h ) , a doublet at  9.05 ( 1 h ) , and a 
 singlet at  8.66 ( 1 h ) are seen for the 8 phthalocyanine  protons , while the 
 signals for the 7  protons appear as a multiplet at  8.068.12 ( 6 h ) and a doublet at  7.62 ( 1 h ) . the doublet at  9.20 can be assigned 
 to the two phthalocyanine  ring protons close to the benzoxy groups , 
 while the multiplet at  9.439.47 is due to the remaining four  protons . the singlet at  7.68 can be readily 
 assigned to the two  protons adjacent to the benzoxy groups , while 
 the multiplet at  8.038.15 is attributed to the remaining six  protons . the c nmr data of these compounds were 
 also in accord with the structures though some of the phthalocyanine ring 
 carbon signals ( for 4 and 5 ) and the chain ch2 signals 
 were overlapped . for compound 8 , a total of 20 signals were observed 
 in the aromatic region (  107.0153.6 ) for the 16 phthalocyanine 
 and 4 benzene ring carbons , which is consistent with the c2v symmetry . the molecular ion [ m + na ] isotopic cluster could be detected 
 in all the cases . the identity of these species was also 
 confirmed by accurate mass measurements . the electronic absorption and basic photophysical 
 data of phthalocyanines 4 , 5 , and 8 were measured in dmf and are summarized in table 1 . all the three compounds gave very similar 
 uv - vis spectra , which are typical for nonaggregated phthalocyanines . the spectrum of compound 5 , for example , showed the b - band at 344 nm , a vibronic band at 606 nm , and an intense and sharp q - band at 672 nm , which strictly followed the 
 lambert beer 's law ( figure 2 ) . upon 
 excitation at 610 nm , the compound is emitted at 677 nm with a fluorescence 
 quantum yield of 0.19 . substitution at the  position 
 ( compound 4 ) slightly shifted the q - band and 
 fluorescence emission to the red by 4 - 5 nm . introduction of an additional -substituent ( compound 8) further shifted the q - band to 690 nm 
 and the fluorescence emission to 696 nm . the singlet oxygen quantum yields (  ) of these 
 compounds were also determined using 1,3-diphenylisobe - nzofuran ( dpbf ) as the 
 scavenger . the concentration of the quencher was 
 monitored spectroscopically at 411 nm along with time , from which the values of 
  could be determined by the method described previously . figure 3 compares the rates of decay 
 of dpbf using these compounds and znpc as the photosensitizers . it can be seen that all these phthalocyanines 
 are efficient singlet oxygen generators , particularly the 1,4-disubstituted 
 analogue 8 , of which the value of  ( 0.84 ) is significantly higher than that of znpc 
 ( 0.56 ) , which was used as the reference . the in vitro 
 photodynamic activity of photosensitizers 4 , 5 , and 8 in cremophor el emulsions was investigated against two different 
 cell lines , namely , ht29 human colorectal carcinoma 
 and hepg2 human hepatocarcinoma cells . as shown in figure 4 , the three compounds are essentially 
 noncytotoxic in the absence of light , but exhibit a very high 
 photocytotoxicity . it can 
 be seen that all these compounds are highly potent and the 
 effects on ht29 are greater than those on hepg2 . it 
 is worth noting that although phthalocyanine 4 exhibits a relatively lower singlet oxygen quantum yield than the 
 other two analogues in dmf ( table 1 ) , its photocytotoxicity is the highest 
 among the three photosensitizers ( table 2 ) . to account for the results , as shown in figure 5 , the q - band for compound 4 in the dmem medium ( for ht29 ) remains very sharp and intense , 
 while those for 5 and 8 are weaker and broadened . this is a strong 
 indication that compound 4 is 
 significantly less aggregated in these media , which should lead to a higher 
 photosensitizing efficiency . to further explain 
 the photocytotoxicity results , fluorescence microscopic studies were also 
 carried out to shed light on the cellular uptake of photosensitizers 4 , 5 , 
 and 8 . after incubation with these compounds 
 ( formulated with cremophor el ) for 2 hours and upon excitation at 630 nm , the ht29 
 cells showed intracellular fluorescence throughout 
 the cytoplasm as shown in figure 6 , 
 indicating that there 
 was a substantial uptake of the dyes . the qualitative fluorescence intensity 
 follows the order 4 > 5 > 8 , suggesting 
 that the -substituted 
 phthalocyanine 4 also has the highest uptake among the three 
 photosensitizers . this may also account 
 for the highest photocytotoxicity of this compound . in conclusion , we 
 have prepared and characterized three novel  3 + 1  zinc(ii ) phthalocyanines 
 substituted with one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy group(s ) . these compounds exhibit a high 
 photocytotoxicity against ht29 and hepg2 cells with ic50 values down 
 to 0.02 m . the mono--substituted phthalocyanine 4 is more potent than the other two analogues , which can be partly 
 explained by its lower aggregation tendency in the culture media and higher 
 cellular uptake as shown by absorption spectroscopy and fluorescence 
 microscopy , respectively .