phthalocyanine derivatives have attracted attention as functional chromophores for 
 applications , especially organic charge carriers in photocopiers , as laser 
 light absorbers in data storage systems , as photoconductors in photovoltaic 
 cells , and in electrochromic displays [ 13 ] . moreover , phthalocyanine derivatives can 
 be utilized as sensitizers in photodynamic therapy of cancer ( pdt ) . sensitizers for pdt require high photostability , high selectivity to 
 tumors , no dark cytotoxity , strong absorption in the region between 600 and 800 nm where penetration of tissue is good , a long triplet lifetime , and 
 satisfactory photosensitization of singlet oxygen . we previously synthesized the nonperipherally substituted phthalocyanine 
 derivatives , zinc alkylbenzopyridoporphyrazines , which possessed didecylbenzenoid and pyridinoid 
 moieties in the molecule and described regio isomer separation of one of the 
 alkylbenzopyridoporphyrazines . we reported a fundamental study on pdt by 
 measuring for the triplet state lifetime of the alkylbenzopyridoporphyrazins 
 and regio isomers [ 10 , 11 ] . as alkylbenzopyridoporphyrazine exhibited 
 solubility in organic solvents and was expected to have a higher tumor 
 affinity , quaternation of the pyridine nitrogen in the alkylbenzopyridoporphyrazine 
 was done to give solubility in an aqueous media , and to have bioavailability 
 and in vivo distribution . then , nyokong et al . reported that 
 phthalocyanine analogues , tetra-2 , 3-pyridoporphyrazine and its quaternized 
 compounds have excellent properties compared to zinc phthalocyanine - type 
 photosensitizer . the amphiphilic phthalocyanine derivatives were concluded the best 
 compound for a new generation of photosensitizers for pdt . in our previous 
 papers [ 912 ] , the reported zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido ) porphyrazine 
 and its regio isomers were prepared by 
 1 : 1 mixture of 3,6-didecylphthalonitrile 
 and 3,4-pyridine dicarbonitrile . in the present study , another type , novel nonperipheral , substituted phthalocyanine 
 derivative , zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine was synthesized . in the case of related compounds , 2,3-pyridoporphyrazines are known to have not 
 only longer wavelength but stronger absorption intensity than corresponding 
 phthalocyanines and 3,4-pyridoporphyrazines . in accordance with , it 
 is expected that zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine and its 
 quaternation compounds have stronger absorption intensities than that of zinc 
 bis(1,4-didecylbenzo)-bis(3,4-pyrido ) porphyrazines reported before [ 912 ] . 
 therefore , the novel compound , zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine and its 
 quaternation compounds are expected to be excellent photosensitizer for pdt . the synthetic procedure used to prepare the 
 novel nonperipheral - substituted phthalocyanine derivative , zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine , was the same as that used 
 for the preparation of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido ) porphyrazine 
 [ 912 ] . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine was synthesized in 80% yield 
 using equimolar amounts of 3,6-didecylphthalodinitrile and 2,3-pyridine 
 carbonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) as basic catalyst ( see figure 1 ) . the target 
 compound , zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine , and its 
 intermediates were studied using fourier transformation infrared ( ir ) , proton 
 nuclear magnetic resonance ( h - nmr ) , ultraviolet - visible 
 ( uv - vis ) spectroscopy , and elemental analysis . the analytical data of the compound were 
 in good agreement with the proposed structure . the synthesized zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine was anticipated to be a 
 mixture of products , with different numbers of pyridine rings in the molecule . however , the target compound comprised only the proposed constituent as 
 confirmed by thin layer 
 chromatography ( tlc ) . as the target compound had been purified by tlc using benzene as 
 eluent , only one blue - colored constituent was obtained . it is thought that the 
 desired compound was obtained in accordance with the mole ratio of the raw 
 materials used . the same phenomenon has been observed in the case of synthesis 
 of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido ) porphyrazine [ 912 ] . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) 
 porphyrazine has two alkylbenzenoid and two pyridinoid rings in different 
 locations ; thus , it has five regio isomers , three of which have rings adjacent 
 to the pyridinoido rings while the other two have opposed pyridinoid rings . 
 although we previously reported the separation of regio isomers in 
 alkylbenzopyridoporphyrazine [ 912 ] , no attempt 
 was made in this work to isolate the isomers of zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine . of course , the obtained blue - colored 
 constituent will be further 
 separated into five regio isomers 
 by using toluene - pyridine 
 7 : 3 eluent in accordance with [ 912 ] . the zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine reacted with quaternizing 
 agents such as monochloroacetic acid ( mcaa ) , diethyl sulfate ( des ) , and 
 dimethyl sulfate ( dms ) in n , n - dimethylformamide ( dmf ) as a 
 solvent at 140c for 2 hours . the respective products obtained were greenish - blue - colored 
 powders of which the yields 
 were 24 , 21 , and 25% for mcaa , des , and dms , respectively ( see figure 3 ) . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) 
 porphyrazine was dissolved in toluene , chloroform , pyridine , and methanol but 
 not in water . after reacting with quaternizing agents , in the cases of mcaa and dms , analysis 
 revealed that the structures of the products were in good agreement with those 
 having n - ch2cooh and n - ch3 groups , respectively . whereas when des was used as a quaternizing agent , no   n - ch2ch3 singlet peak was present in the h - nmr spectrum , s = o stretching in the ir spectrum was observed . the uv - vis spectrum of zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines around 700 nm is 
 characteristic of phthalocyanine analogues , with the q band attributable to the 
 difference between the highest occupied molecular orbital ( homo ) energy level 
 and the lowest unoccupied molecular orbital ( lumo ) energy , that is , the - * transition of the phthalocyanine ring . the quarternized derivatives of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines showed strongest absorption 
 at 676 , 687 , and 687 nm in water after reaction with dms , des , and mcaa , 
 respectively ( table 1 ) ; these q bands were bathochromic compared to the nonquaternized 
 parent compound . as the uv - vis spectra of the quaternized compounds in water 
 showed very broad peaks , the amphiphilic compounds had excellent molecular 
 association tendency . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine 
 and its quaternized compounds fluoresced on exposure to ultraviolet light . although fluorescence 
 spectra generally were known to be mirror images of uv - vis spectra at the longer 
 wavelengths , the q bands nearly overlapped with the wavelengths at which fluorescence 
 occurs in the case of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine 
 and its quaternized compounds , thus , the 
 differences between 
 max of uv - vis and the fmax of fluorescence spectra , called the storkes shift , were very small between 10 to 20 nm . these observations 
 are similar to that seen with the phthalocyanines zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) 
 porphyrazine and its quaternized derivatives . these compounds are molecules 
 with high planarity which can not change their configuration after quaternization . the important parameters of a cyclic 
 voltammetry ( cv ) are the reduction ( epc ) and oxidation ( epa ) potential , the difference between reduction and oxidation potentials , ( epc ) , and formal reduction potential ( ) of the observed waves . the potential difference in cvs between the 
 reduction and oxidation correspond to the homo - lumo energy gaps of the compound 
 . just as chemical reactions occur during the electron transfer between 
 homo and lumo energy levels , photochemical reactions are also based on similar 
 phenomena of energy transfer . before and after the quaternization , the homo - lumo 
 energy gap of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine was 
 unchanged . the shapes of cvs clearly showed that quaternized zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines had increased electron 
 responsibility . after incubation for 3 hours , cellular quaternized zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines was observed with a fluorescence microscope . a fluorescent substance was 
 noted when the uptake of dms quaternized 
 zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines in iu-002 cells was 
 carried out . intact cells selectively concentrated fluorescence . after exposure to halogen light for 10 minutes 
 showed damage and loss of fluorescence although fluorescence in cells occurred in 
 perinuclear area ( see figure 4 ) . concequently , the light exposed dms 
 quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines in cells 
 produces cell disruption that can be detected as a decrease as 
 fluorescence . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines were synthesized from an 
 equimolar mixture of 3,6-didecylphthalonitrile and 2,3-pyridine carbonitrile in 
 the presence of basic catalyst . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines having two pyridine and two 
 alkyl - substituted benzene rings reacted with dms , des , and mcaa as quaternizing agents . when mcaa and dms were employed as 
 quaternizing agents , zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines however , when des was employed , 
 we showed that sulfonation but not quaternization was achieved . electrochmeical characterization of zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine and its quaternized 
 derivatives were estimated by cv technique . the shapes of cvs clearly showed that quaternized 
 zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines had increased electron 
 responsibility . the uptake of dms - quaternized zinc 
 bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines was done in iu-002 cells . the light - exposed dms quaternized 
 zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines in cells produces cell 
 disruption that can be detected as a decrease in fluorescence . ir spectra were recorded on a shimadzu ft - ir 
 8100a spectrometer using potassium bromide ( kbr ) pellets . uv - vis spectra were 
 measured on a shimadzu uv-2400pc spectrometer ; each sample was prepared at 
 5.0  105 mol dm in pyridine , toluene , and water . fluorescence spectra were 
 recorded in toluene , pyridine , and water using either a hitachi f-4500 
 fluorescence spectrometer or a jasco ( nihon bunko ) fp-6600 spectrofluorometer . h - nmr 
 spectra were measured at 400 mhz on a bruker avance 400s in benzene - d6 ( c6h6-d6 ) or chloroform - d ( chcl3-d ) 
 using tetramethylsilane ( tms ) as an internal standard . samples for elemental 
 analysis were purified by repeated sublimation ; the instrument was calibrated 
 with copper phthalocyanine . cvs were recorded on a bas cv-50 w voltammetric 
 analyzer at room temperature in1  10 mol dm acetonitrile 
 solution containing a 0.01 mol dm tetrabutylammonium perchlorate ( tbap ) . cvs were recorded by scanning the potential at a rate of 50 mv s. 
 the working and counter electrodes were platinum wires and the reference 
 electrode was a silver / silver chloride- ( ag / agcl ) saturated sodium chloride 
 electrode . merck silica 
 gel 60 , particle size 0.0630.200 nm 7734 grade was used in chromatographic 
 separations . reagents were purchased from sigma - aldrich chemicals ( miss , usa ) and 
 were used as received without further purification . 3,6-didecylphthalonitrile was synthesized from thiophene via 
 2,5-didecylthiophene and 2,5-didecylthiophene-1,1-dioxide , in accordance with 
 our previous reports [ 912 ] . h nmr (  400 mhz , chcl3-d/ 
 ppm ) 0.88 ( t , 6h ) , 1.26 ( m , 32h ) , 2.85 ( t , 4h ) , 7.46 ( s , 2h ) ; ir( kbr / cm ) 2960 ( c  h ) , 2240 ( cn ) , 1560 ( c = c ) , 1460 ( c  c ) , 1410 ( c  c ) , 1230 ( c  h ) , 730 ( c  h ) ; anal calcd . for c28h4n2 : 
 c. 82.69 : h. 10.85 : n.6.86 pyridine-2,3-dicarboxylic acid ( 15 g , 0.09 mol ) 
 in 200 cm of ethanol was refluxed in 7.5 cm of 
 concentrated sulfonic acid for 48 hours . after the solvent was removed and 
 neutralized with 3 m naoh solution , the organic layer was extracted with three 
 times of 75 cm of diethyl ether . the ester was dissolved in concentrated 
 aqueous ammonia and the solution was stirred for 48 hours to afford a white - colored 
 precipitate . 179181c . thionyl chloride ( 9.6 g , 0.08 mol ) was added 
 dropwise to the diamide ( 6.6 g , 0.04 mmol ) in 56 g of dmf at 10c . this mixture was allowed to warm to room 
 temperature and was stirred for 24 hours . the solvent was removed by 
 evaporation and water was added to make the thionyl chloride ineffective and 
 then filtered . the product was purified by chromatography over silica gel with 
 benzene - petroleum ether 
 1 : 1 as eluent , giving a colorless solid ( 1.97 g , 17% ) , 
 m.p . h nmr (  400 mhz , chcl3-d / ppm ) 7.26 ( s , 1h ) , 
 7.75 ( s , 1h ) , 9.09 ( s , 1h ) ; ir( kbr / cm ) 
 3090 ( c  h ) , 2240 ( c  n ) , 1600 ( c  c ) , 1550 ( c  c ) , 1470 ( c  c ) , 1220 ( c  h ) , 750 ( c  h ) ; anal calcd . 
 for c7h3n3 : c. 65.11 : h. 2.34 : n.32.55 . found : 
 c.65.12 : h. 2.34 : n. 32.56 . 3,6-didecylphthalonitrile ( 0.12 g , 0.29 mmol ) 
 and 2,3-dicyanopyridine ( 0.04 g , 0.29 mmol ) ( see figure 1 ) were dissolved in 
 pentanol ( 7 cm ) and zinc chloride ( 0.05 g ) was added ; the ensuing 
 mixture was heated for 4 hours in the presence of dbu as catalyst . after 
 cooling , the reaction mixture was dissolved in toluene ( 50 cm ) and 
 filtered ; the solvent was removed by evaporation . the product was purified by tlc ( eluent : toluene ) yielding a blue solid ( 0.13 g ; yield 80% ) . h nmr (  400 mhz , c6h6-d6/ppm ) 0.9 ( m , 12h , ch3 ) , 1.612.61 ( m , 64h , ch2 ) , 
 4.184.36 ( m , 8h , -ch2 ) , 
 7.45 ( m , 4h , arom ) , 8.26 ( m , 6h , py ) ; 
  ir( kbr / cm ) 2960 ( c  h ) , 1600 ( c  c ) , 1500 ( c  c ) , 1420 ( c  c ) , 1200 ( c  h ) , 1100 ( c  h ) , 750 ( c  h ) ; uv - vis [ max toluene / nm 
 ( log max ) ] 665 ( 5.494 ) ; 
 anal calcd . for c70h94n10zn : c. 73.68 : h. 
 8.30 : n.12.28 . zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) 
 porphyrazine ( 0.17 g , 0.15 mmol ) reacted with mcaa ( 0.57 g , 6 mmol ) , des ( 0.1 g , 
 0.6 mmol ) and dms ( 0.2 g , 1.5 mmol ) , respectively , in n , n - dimetylformamide ( dmf ) 140c for 2 hours the reaction mixture was dissolved in acetone ( 20 cm ) , cooled 
 to room temperature , and the resulting solution was filtered . the solvent was 
 removed and the product was purified by tlc ( eluent : thf - toluene , 8 : 2 ) ; the 
 product was recovered from the tlc plate via dissolution in pyridine followed 
 by filtration and solvent removal . mcaa : yield 25% , h nmr (  400 mhz , c6h6-d6/ 
 ppm ) 0.87(m , 12h , ch3 ) , 1.131.70(m , 56h , -ch2 ) , 1.822.61(m , 8h , -ch2 ) , 4.114.38(m , 4h , -ch2 ) , 6.20(s , 2h , ch2 ) , 
 7.147.27(m , 4h , arom ) , 8.73.16(m , 6h , py ) : ir( kbr / cm ) 3480(o  h ) , 3050 , 2970(c h ) , 1740(c = o ) , 1600 , 1500 , 1400(c = c ) , 1210 , 1100 , 940 , 790 , 
 690(c  h ) ; des yield 21% , 
 (  400 mhz , c6h6-d6/ppm ) 0.86(m , 12h , ch3 ) , 1.021.63(m , 56h , -ch2 ) , 1.882.61(m , 8h , -ch2 ) , 4.264.50(m , 4h , -ch2 ) , 7.37(m , 4h , arom ) , 
 8.22(m , 4h , py ) : 
 ir( kbr / cm ) 
 3480(o  h ) , 3050 , 2960(c  h ) , 1600 , 1460 , 1400(c = c ) , 1350 , 1150(s = o ) , 1250 , 920 , 770(c h ) , 580(cs ) ; dms yield 25% , (  400 mhz , c6h6-d6/ppm ) 0.90(m , 12h , 
 ch3 ) , 0.951.45(m , 56h , -ch2 ) , 1.602.41(m , 8h , -ch2 ) , 4.05(s , 6h , ch3 ) , 4.254.42(m , 4h , -ch2 ) , 7.45(m , 4h , arom ) , 
 8.02(m , 6h , py ) : ir( kbr / cm ) 
 3070 , 2980(c h ) , 1500 , 1400(c = c ) , 1250 , 1100 , 950 , 810 , 
 660(c  h ) . cells seeded into 96-well tissue culture plates and incubated to allow 
 attachment to the plates . the 
 sensitizer was added to the medium at concentration ranging from 
 0 to 2mgcm . the medium was removed , the cells were washed with 
 phosphate - buffered saline ( pbs ) , and fresh medium was added .
the phthalocyanine analogue containing nonperipheral long alkyl - substituted benzenoid rings and pyridine rings , zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine , was synthesized . 
 zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine reacted   with dimethyl sulfate and monochloroacetic acid to produce their quaternized products and   diethyl sulfate to produce the sulfo - substituted products . 
 all quaternized and sulfo - substituted showed amphiphilic character . 
 identical peaks in cyclic voltammograms appeared for these products before and after quaternization . during   the evaluation of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazine for its photodynamic therapy of cancer ( pdt ) efficacy by cancer cell culture , the light exposed dimethyl sulfate quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido ) porphyrazines in iu-002 cells produce cell disruption that can be detected as a decrease in fluorescence .