f - fluoro - deoxy - glucose ( fdg ) , an f - labeled glucose analog , is the most common radiotracer for positron emission tomography ( pet ) in cancer diagnosis . however , fdg - pet has several limitations in practice , for example , fdg can not distinguish tumor tissues from inflammatory or normal brain tissues . therefore , f - labeled amino acid - based radiotracers have been reported as an alternative , which is based on the fact that tumor cells take up and consume more amino acids to maintain their sustained fast growth . among those radiotracers , f - labeled alpha - methyl tyrosine ( amt ) has shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma     . f - amt enters the tumor cells via l - type amino acid transporters ( lat ) , which is the only system that can transport large neutral amino acids with aromatic rings . lat , especially its subtype lat1 , was reported to be highly expressed in many cancer cell lines and positively correlates with tumor growth     [ 4 , 5 ] . so far , f - amt is the most suitable amino acid transporter - targeting radiotracer for tumor imaging , regardless of low synthesis yield and requirement of an on - site cyclotron to produce f.   although pet has emerged as an advanced imaging tool for cancer diagnosis , only limited facilities around the world can afford complete armamentarium of pet and cyclotron for the local production of short - lived positron - emitting radionuclides such as c and f. therefore , mature technologies , that is , single photon emission computed tomography ( spect ) or its combination with computed tomography ( ct ) , still play important and irreplaceable roles in nuclear imaging area . ( tc ,   t1/2 = 6.02  h ) , which is produced by in - house generator and does not require the cyclotron . due to their similar chemistry , the diagnostic radioisotope tc and the therapeutic radioisotope rhenium-188 ( re ) could be labeled to the same ligand , which leads to the diagnostic / therapeutic matched pair . unlike most of the cyclotron - produced radionuclides that utilize the covalent chemistry for labeling , tc requires a the nitrogen , oxygen , and sulfur combinations have been shown to be stable chelators for tc such as n4 ( e.g. , dota , cyclam-14 ) , n3s ( e.g. , mag-3 ) , n2s2 ( e.g. , ecd ) , ns3 , s4 ( e.g. , sulfur colloid ) , diethylenetriamine pentaacetic acid ( dtpa ) , o2s2 ( e.g. , dmsa ) , and hydrazinonicotinamide ( hynic )     [ 813 ] .    here , we report the synthesis of precursor o-[3-(1,4,8,11-tetraazacyclotetradecan)-propyl]--methyl tyrosine ( n4-amt ) and its radiolabeling with tc . in   vitro cellular uptake kinetics and planar scintigraphic imaging of tc - n4-amt all chemicals of analytical grade and solvents of hplc grade for compound synthesis were purchased from sigma - aldrich ( st . louis , mo ) . h- , c - nmr spectra were performed on bruker 300  mhz spectrometer in cdcl3 , cd3od , and d2o . sodium pertechnetate ( natco4 ) was obtained from mo / tc generator in mallinckrodt ( houston , tx ) . thionyl chloride ( 10  ml ; 137.42  mmol ) was added to a solution of -methyltyrosine 1 ( 10.00  g ; 51.22  mmol ) in anhydrous ethanol ( 60  ml ) at 0c , and then heated at 78c for 4  h while stirring . after cooling , the reaction mixture was reduced to 20  ml , and then 10  ml of triethylamine was added into it . h - nmr ( cdcl3 )  = 7.02 ( d , 2h , j = 8.4  hz ) , 6.70 ( d , 2h , j = 8.4  hz ) , 4.22 ( dd , 2h , j = 7.2  hz , j = 7.8  hz ) , 3.14 ( dd , 2h , j = 13.5  hz , j = 13.5 ) , 1.42 ( s , 3h ) , 1.33 ( t , 3h , j = 16.2  hz )  ppm . c - nmr  = 175.47 , 156.36 , 130.66 , 126.39 , 114.87 , 61.05 ,   58.65 , 45.29 , 24.08 , 13.07  ppm , ms : m / z = 224.23 [ m ] . compound 2 ( 2.09  g ; 9.36  mmol ) was dissolved in 40  ml of anhydrous dmf under nitrogen and treated with triethylamine ( 2.78  ml ; 20  mmol ) while stirring . ditertiarbutyl dicarbonate ( 3.27  g ; 15  mmol ) was added to the reaction mixture and stirred over night at room temperature . the solvent was removed under reduced pressure to yield a residue , which was extracted with ethyl acetate and dried with anhydrous mgso4 . the extraction was filtered and evaporated to give yellow oil which was purified by column chromatography on silica gel and eluted with hexane : ethyl acetate ( 5  :  1.5  v / v ) . h - nmr ( cdcl3 )  = 6.97 ( d , 2h , j = 8.4  hz ) , 6.75 ( d , 2h , j =   8.7  hz ) , 4.22 ( dd , 2h , j = 2.7  hz , j = 7.2  hz ) , 3.32 ( dd , 2h , j = 15.0  hz , j = 13.5 ) , 1.55 ( s , 3h ) , 1.48 ( s , 9h ) , 1.32 ( t , 3h , j = 18.0  hz )  ppm . c - nmr  = 174.16 , 155.04 , 154.51 , 131.16 , 128.50 , 115.14 , 77.25 , 61.64 , 60.54 , 40.99 ,   28.39 , 23.55 , 14.77  ppm , ms : m / z = 324.36 [ m ] . sodium metal ( 0.09  g ; 14.02  mmol ) was dissolved in 30  ml of anhydrous ethanol with stirring under nitrogen . compound 3 ( 1.00  g ; 3.09  mmol ) was dissolved in 50  ml of anhydrous ethanol and treated with sodium ethoxide solution and refluxed for 2.5  h at 70c . 3-bromopropanol ( 0.56  ml ; 6.18  mmol ) was added and continued heating over night . the ethanol was removed under reduced pressure and replaced with 40  ml of ethyl acetate , washed with water , and dried over mgso4 . after removal of the solvent , it was purified by silica gel column chromatography ( hexane : ethylacetate 2  :  1 ) , giving 0.83  g ( 2.17  mmol , 71% yield ) of the product as clear yellow oil . h - nmr ( cdcl3 )  = 7.01 ( d , 2h , j = 6.3  hz ) , 6.81 ( d , 2h , j =   6.6  hz ) , 4.20 ( dd , 2h , j = 5.1  hz , j = 5.1  hz ) , 4.12 ( t , 2h , j = 15.0  hz ) , 3.86 ( t , 2h , j = 10.2  hz ) , 3.16 ( dd , j = 13.5  hz , j = 12.9  hz ) , 2.06 ( m , 2h ) , 1.54 ( s , 3h ) , 1.47 ( s , 9h ) , 1.31 ( t , 3h , j = 12.3  hz )  ppm . c - nmr  = 174.01 , 171.91 , 157.73 , 154.35 , 131.07 , 128.65 , 114.14 , 79.39 , 65.65 , 61.54 , 60.40 , 60.24 , 40.79 , 32.01 , 28.39 , 23.58 , 14.15  ppm , ms : m / z = 381.033 [ m ] . a solution of 4 ( 5.44  g ; 16.82  mmol ) and 1,3-dibromopropane ( 136.74  g ;   677.30  mmol ) in 100  ml acetone was purged with nitrogen for 15  min . potassium carbonate ( 22.62  g ; 163.69  mmol ) was added to the reaction mixture , the mixture was then refluxed for 12  h at 75c . after the removal of solvents and excessive reagents under reduced pressure , the residue was dissolved in chloroform , washed with water , and dried in anhydrous magnesium sulfate . a yellow liquid was purified by silica gel flash chromatography ( hexane : ethylacetate , 2  :  1 ) to furnish   compound 5 as pale yellow liquid . h - nmr ( cdcl3 )  = 7.02 ( d , 2h , j = 9.00  hz ) , 6.82 ( d , 2h , j = 9.00  hz ) ,   4.23 ( dd , 2h , j = 27.00 ) , 4.09 ( t , 2h , j = 12.00  hz ) , 3.61 ( t , 2h , j = 12.00  hz ) , 3.32 , 3.16 ( d , 2h , j = 15.00 , 12.00  hz ) , 2.34 ( dd , j = 24.00  hz ) , 1.54 ( s , 3h ) , 1.47 ( s , 9h ) , 1.32 ( t , 3h , j = 15.0  hz )  ppm . c - nmr  = 173.96 , 157.63 , 154.32 , 131.09 , 128.78 , 114.18 , 77.48 , 65.24 , 61.51 , 60.35 , 40.80 , 30.00 , 28.40 , 23.60 , 14.19  ppm . ms : m / z = 446.3 [ m ] . 1,4,8,11-tetraazacyclotetradecane ( cyclam ) ( 15.00  g ; 74.88  mmol ) was dissolved in 150  ml of anhydrous ethanol , and diethyl oxalate ( 10.94  g ; 74.88  mmol ) was added . the solvent was rotary evaporated , and the crude product was recrystallized in acetone : ethanol to yield white crystals of n , n - dioxylyl-1,4,8,11 - 1,5,8,12-tetraazabicyclotetradecane ( n , n - cyclooxamide ) 6 . h - nmr ( cdcl3 )  = 4.35 ( m , 2h ) , 375 ( m , 2h ) , 3.40 ( m , 2h ) , 2.77 ( m , 2h ) , 2.68 ( m , 2h ) , 2.54 ( m , 2h ) , 2.43 ( m , 4h ) , 1.75 ( m , 2h ) , 1.24 ( m , 2h ) )   ppm . c - nmr  = 158.55 , 49.92 , 49.38 , 47.73 , 44.13 , 25.42  ppm . ms : m / z = 255.33 [ m ] . compound 6 ( 1.00  g ; 3.93  mmol ) was dissolved in 20  ml of anhydrous dmf and treated with a solution of n - t - butoxycarbonyl - o-[3-br - propyl]--methyl tyrosine   ethylester 5 ( 1.74  g ; 3.93  mmol ) in 40  ml of dmf under nitrogen atmosphere . the reaction mixture was heated to reflux for 18  h at 75c and then allowed to cool down to room temperature . the residue was dissolved in chloroform ( 30  ml ) , washed with water and 1  m na2co3 , ( 12  ml ) , and then separated for the organic layer . the crude compound was purified by silica gel column chromatograph ( chloroform : methanol 9  :  1 ) . h - nmr ( cdcl3 )  = 7.00 ( d , 2h , j = 9.00  hz ) , 6.79 ( d , 2h , j = 9.00  hz ) , 4.56 ( m , 2h ) , 4.22 ( t , 2h , j = 12.0  hz ) , 3.95 ( m , 2h ) , 3.67 ( m , 2h ) , 3.55 ( t , 2h , j = 15.0  hz ) , 3.30 ( m , 4h ) , 2.81 ( m , 12h ) , 1.88 ( m , 6h ) , 1.54 ( s , 3h ) , 1.47 ( s , 9h ) , 1.32 ( t , 3h , j=   15.0  hz ) ppm . c - nmr  = 173.97 , 158.65 , 158.06 , 157.91 , 154.31 , 131.01 , 128.34 , 114.04 , 79.38 , 77.27 , 66.01 , 61.50 , 60.26 , 53.76 , 52.76 , 49.20 , 48.41 , 47.54 , 46.15 , 45.83 , 44.50 , 42.60 , 40.84 , 28.40 , 26.39 , 23.57 , 23.36 , 23.07 , 14.14  ppm . ms : m / z = 618.38 [ m ] . compound 7 ( 0.25  g ; 0.40  mmol ) was dissolved in dichloromethane ( 10  ml ) , and trifluoroacetic acid ( 1.0  ml ) was added to it . the solution was stirred over night at room   temperature , and volatiles were removed in   vacuo . the crude compound was purified by chromatography over silica gel ( chloroform : methanol 9  :  1 ) as white solid . h - nmr ( cd3od )  = 7.14 ( d , 2h , j = 9.0  hz ) , 6.94 ( d , 2h ) , 4.33 ( m , 4h ) , 4.05 ( t , 2h , j = 9.0  hz ) , 3.85 ( m , 2h ) , 3.71 ( m , 2 h ) , 3.26 ( m , 13h ) , 2.07 ( m , 6h ) , 1.58 ( s , 3h ) , 1.33 ( t , 3h , j = 15.0  hz ) ppm . c - nmr  = 170.65 , 161.87 , 161.42 , 159.70 , 159.02 , 158.67 , 131.01 , 125.14 , 118.79 , 114.65 , 65.34 , 62.47 , 60.33 , 56.92 ,   52.16 , 51.26 , 45.64 , 45.31 , 44.50 , 43.20 , 42.05 , 24.31 , 22.00 , 21.39 , 21.03 , 16.96 , 12.90  ppm , ms : m / z = 517.63 [ m ] . to a solution of compound 8 ( 0.20  g ; 0.39  mmol )   in a 5  ml of water ,   10  n naoh ( 2  ml ) was added , stirred , and refluxed over night at 90c . the solvent was evaporated under vacuum , giving white solid , which was dissolved in 5  ml of water and neutralized with 5  n hcl solution to ph = 7 . the solid was stirred in 25  ml anhydrous methanol , filtered , and evaporated to afford an off white solid . h - nmr ( d2o )  = 7.21 ( d , 2h , j = 9.00  hz ) , 7.00 ( d , 2h , j = 9.00  hz ) , 4.14 ( t , 2h , j = 12.0  hz ) , 3.10   ( m , 5h ) , 2.94 ( m , 2h ) , 2.84 ( m , 13h ) , 1.95 ( m , 6h ) , 1.33 ( s , 3h ) ppm.c - nmr  = 180.69 , 163.42 , 163.16 , 162.88 , 162.60 , 157.06 , 131.40 , 129.19 , 117.57 , 115.26 , 115.10 , 112.93 , 66.25 , 60.73 , 53.69 , 51.46 , 49.88 , 49.26 , 48.04 , 47.84 , 46.60 , 45.42 , 45.07 , 43.94 , 24.71 , 24.32 , 23.62 , 22.55 ppm . ms : m / z = 436.327 [ m ] . briefly , radiosynthesis of tc - n4-amt was achieved by adding a required amount of sodium pertechnetate into a vial containing precursor n4-amt and sncl2 ( 100  g ) . radiochemical purity was assessed by high - performance liquid chromatography ( hplc ) , equipped with nai and uv detector ( 274  nm ) , and was performed using a c-18 reverse column with a mobile phase of acetonitrile  :  water ( 7  :  3 ) at a flow rate of 0.5  ml / min . rat mammary tumor cell line 13762 was obtained from american type culture collection ( rockville , md ) . the same cell line was used to create the animal model for in   vivo evaluation . cells were maintained at 37c in a humidified atmosphere containing 5% co2 in dulbecco 's modified eagle 's medium and nutrient mixture f-12 ham ( dmem / f12 ; gibco , grand island , ny ) . cells were plated to 6-well tissue culture plates ( 2  10 cells / well ) for two days before the study , and incubated with tc - n4-amt ( 0.05  mg / well , 8  uci / well ) or tc - n4 chelator itself ( 0.025  mg / well , 8  uci / well ) for 15  min4  h. after incubation , cells were washed with ice - cold pbs twice and detached by adding 0.5  ml of trypsin . cells were then collected and the radioactivity was measured with gamma counter ( cobra quantum ; packard , mn ) . all animal work was carried out in the small animal imaging facility ( saif ) at university of texas md anderson cancer center under the protocol approved by institutional animal care and use committee ( iacuc ) . tissue distribution studies of tc - n4-amt ( study i , n = 9 ) or tc - n4 ( study ii , n = 9 ) were conducted by using normal female fischer 344 rats ( 150  25 g , n = 18 ) ( harlan sprague - dawley , indianapolis , in ) . for each radiotracer , nine rats were divided into three groups for three time intervals ( 0.5 , 2 , 4  h ) . the injection activity was 25  0.5  ci / rat intravenously . at each time interval , the rats were sacrificed , and the selected tissues were excised , weighed , and measured for radioactivity by gamma counter . data from each sample were represented as the percentage of the injected dose per gram of tissue wet weight ( % id / g ) . female fischer 344 rats were inoculated subcutaneously with 0.1  ml of 13762 rat mammary tumor cell suspension ( 10 cells / rat ) into the right posterior legs using 22-gauge needles . imaging studies were performed 14 to 17 days after inoculation when tumors reached approximately 1  cm in diameter . the anesthetized rats were injected intravenously with tc - n4-amt ( 0.3  mg / rat , 300  ci / rat ; n = 3 ) or with tc - n4 ( 0.15  mg / rat , 300  ci / rat ; n = 3 ) , respectively . planar scintigraphic images were obtained using m - cam ( siemens medical solutions , hoffman estates , il ) equipped with a low - energy high - resolution collimator at 30120  min . the intrinsic spatial resolution was 3.2  mm and the pixel size was from 19.18  mm ( 32  32 , zoom = 1 ) to 0.187  mm ( 1024  1024 , zoom = 3.2 ) . computer outlined regions of interest ( roi ) ( counts per pixel ) of tumors , and normal muscle tissues at symmetric sites were used to calculate tumor - to - muscle ( t / m ) ratios . commercially available -methyl tyrosine 1 was converted into corresponding acid chloride , then to ethyl ester by reacting with thionyl chloride in ethanol . the amine in -methyl tyrosine ethyl ester 2 was protected as its boc - derivative n - t - butoxycarbonyl--methyl tyrosine ethylester 3 with triethylamine and di - t - butyldicarbonate in dmf . the chain at the oh group was extended when compound 3 was treated with 1 , 3-dibromopropane and obtained as n - t - butoxycarbonyl - o-[3-bromopropyl]--methyl tyrosine ethylester 4 . acylation of 1,4,8,11-tetraazacyclo - tetradecane ( cyclam )   with diethyloxalate led to n , n - dioxalyl 1,4,8,11-tetraazacyclotetradecane   ( n , n - cyclamoxamide ) 5 . under sn condition 4 and 5 were   efficiently converted to alkylated compound n - t - butoxycarbonyl - o-[3-(n , n - dioxalyl-1,4,8,11-tetraazacyclotetradecane)-propyl]--methyl tyrosine ethylester 6 . exposure of 6 to trifluoroacetic acid in ch2cl2 caused qualitative de - t - butoxycarboxylation to yield o-[3-(n , n - dioxalyl 1,4,8,11-tetraazacyclotetradecane)-propyl]--methyl tyrosine ethylester 7 . 10  n naoh in water at 75c promoted   the hydrolysis of ester to acid and a   simultaneous deoxalation of   7 to yield the final compound o-[3-(1,4,8,11-tetraazacyclotetradecan)-propyl]--methyl tyrosine ( n4-amt ) 8 . the structure and purity of the compounds at each step were validated by h- and c - nmr , mass spectra , and hplc . as shown in figure 2 , precursor n4-amt could be labeled with tc successfully in a high radiochemical purity ( > 96% ) . the retention time of tc - n4-amt was 6.899  min . given that tc - n4-amt is a kit - product and labeled without any further purification , the radiochemical yield was assumed to be identical to its radiochemical purity . in this study , we labeled amino acid -methyltyrosine amt with tc using cyclam n4 as the chelator because of its stable chelating ability and fast renal clearance . tc was selected as the radionuclide due to its favorable physical characteristics , suitable half - life , and cost - effectiveness . the cellular uptake kinetics of tc - n4-amt and tc - n4 in rat breast tumor cell line 13762 is shown in figure 3 . there was a drastically increased uptake for tc - n4-amt in the tumor cells at 15240  min , but not for tc - n4 chelator . these findings suggest that by adding the amino acid amt , tc - n4-amt can enter and accumulate into tumor cells effectively and rapidly . to further investigate the transport mechanisms of tc - n4-amt , the competitive inhibition study using various types of transporter inhibitors will be conducted in the future . the result of the in   vivo biodistribution studies in the normal fischer rats at 0.5 , 2 , and 4 hours after intravenous administration of tc - n4-amt is shown in table 1 . planar scintigraphic images of tc - n4-amt and tc - n4 at 30 , 60 , and 120  min in breast tumor - bearing rats are shown in figure 4 . t / m ratios of tc - n4-amt were 2.34.0 , whereas those of tc - n4 were 1.92.5 , respectively . tumors could be clearly visualized by tc - n4-amt , but not by tc - n4 . in addition , the rat kidneys showed intense activity of tc - n4-amt in the planar images , which was consistent with the results from the in   vivo biodistribution studies in the normal rats . this may be due to the nature of amt , an inhibitor of tyrosine hydroxylase that can not be excreted from kidneys and hence crystallized in the proximal tubules because of its poor solubility at the hydrogen ion concentrations of body fluids ( ph 58 ) . in the future , in   vivo uptake blocking study using the unlabeled amt will be performed to ascertain whether accumulation of tc - n4-amt in the kidney is attributed to amt module . in conclusion , efficient synthesis of n4-amt was achieved .   in   vitro cellular uptake and in vivo imaging findings collectively suggest that tc - n4-amt is a potential radiotracer for breast cancer imaging . in compliance with the chelating capability of n4 , n4-amt could be labeled with positron emitting radionuclides such as gallium-68 or with short - ranged beta emitters for internal radiotherapeutic purposes hereafter .
purpose . this study was to develop an efficient synthesis of 99mtc - o-[3-(1,4,8,11-tetraazabicyclohexadecane)-propyl]--methyl tyrosine ( 99mtc - n4-amt ) and evaluate its potential in cancer imaging . methods . 
 n4-amt was synthesized by reacting n4-oxalate and 3-bromopropyl amt ( n - boc , ethyl ester ) . in vitro cellular uptake kinetics of 99mtc - n4-amt 
 was assessed in rat mammary tumor cells . 
 tissue distribution of the radiotracer was determined in normal rats at 0.54  h , while planar imaging was performed in mammary tumor - bearing rats at 30120 min . 
 results . 
 the total synthesis yield of n4-amt was 14% . 
 cellular uptake of 99mtc - n4-amt was significantly higher than that of 99mtc - n4 . 
 planar imaging revealed that 99mtc - n4-amt rendered greater tumor / muscle ratios than 99mtc - n4 . 
 conclusions . 
 n4-amt could be synthesized with a considerably high yield . 
 our in vitro and in vivo data suggest that 99mtc - n4-amt , a novel amino acid - based radiotracer , efficiently enters breast cancer cells , effectively distinguishes mammary tumors from normal tissues , and thus holds the promise for breast cancer imaging .