CA2266298A1 - Synthesis and evaluation of two technetium-99m-labeled peptidic 2-nitroimidazoles for imaging hypoxia - Google Patents

Abstract

The presence of hypoxic cells in solid tumors is a marker for therapy resistant, aggressive disease. The non-invasive detection of hypoxic cells in tumors by radiolabeled 2-nitroimidazoles is a diagnostic technique under current evaluation. Two peptidic chelating agents, dimethylglycyl-L-seryl-L-cysteinyl-lysyl{N E-[1-(2-nitro-1H-imidazolyl)acetamido]}-glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinylglycine-[2-(2-nitro-1H-imidazolyl)ethyl]amide (RP535) have been synthesized.
Both chelating agents contain an N3S class chelator for 99m Tc and Re, and a 2-nitroimidazole group which can be enzymatically reduced and trapped in cells under hypoxic conditions. Two isomers of 99m TcO-RP435, which are assumed to be syn and anti conformations, have been observed on HPLC analysis. The interconversion of the two isomers in aqueous solution was investigated. In contrast, RP535 chelates 99m Tc to form a single isomer and no conversion to its counterpart has been observed on HPLC
analysis. The tert-butyl group on the chelator may inhibit the formation and interconversion of the syn and anti isomers of 99m TcO-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with 99m TcO-RP535 showing higher selectivity for hypoxic cells than 99m TcO-RP435.
These results suggest that 99m TcO-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.

Description

Su et al., page 2 INTRODUCTION
Detection of regions of hypoxia is important in several medical conditions. It is important to delineate hypoxic but viable tissue following interruption of blood flow in cerebral or myocardial infarction {Nunn 1995 } . Furthermore, it has recently been recognised that hypoxia in tumors plays a role not only in response to radiation, through the oxygen effect in fixation of DNA damage, but also in metastatic potential and response to other forms of therapy {Hockel 1993 } {Graeber 1996} {Brizel 1996}.
However, hypoxia is heterogeneous and tumors which appear identical by all other clinical measures can vary greatly in their proportion of hypoxic cells. At present, hypoxia is measured clinically with a polarographic oxygen electrode inserted into tumors; however, this is invasive, operator-dependent, limited to accessible tumors, and not readily repeatable {Hockel 1993}{Brizel 1996}. In recent years several radiopharmaceuticals have been developed which are targeted to hypoxic tissues via a 2-nitroimidazole (2-NI) moiety {Nunn 1995}{Chapman 1998}. 2-NIs are electron-affinic compounds which undergo an enzyme-mediated one-electron reduction; in normoxic tissue the resultant radical anion is immediately oxidized back to the starting compound, whereas under hypoxic conditions there is further reduction to products which are trapped by binding to macromolecules {Rauth 1984}. A radiolabeled 2-NI would therefore be selectively trapped in hypoxic tissues.
Clinical studies have been performed with several radiolabeled 2-NIs, including '8F-fluoromisonidazole (FMISO) {Grierson 1989} {Koh 1992} and '23I-iodoazomycin arabinoside (IAZA) {Mannan 1991 } {Parliament 1992}. However, the search continues for a 99mTc-labeled analog that would be more widely applicable. BMS181321, a

2-NI

Su et al., page 3 linked to a PnAO chelator, has been evaluated in several model systems and shows promise, but its high lipophilicity and metabolic instability result in high background levels {binder 1994} {DiRocco 1997} {Barron 1996} {Ballinger 1996}. BRU59-21 (previously known as BMS 194796) appears to possess improved characteristics but only limited information is available about it {Johnson 1998} {Melo 1998}. In addition, butyleneamine oxime (BnAO or HL91; Prognox, Nycomed-Amersham) is a non-nitro 99mTc complex that shows localization in hypoxic myocardium and tumors via an undetermined mechanism {Archer 1995} {Okada 1997} {Cook 1998} {Zhang 1998}.
Recent work involving the radioiodinated sugar derivatives of 2-NI has suggested that an oil/water partition coefficient (PC) of ~ 1 confers optimal biodistribution properties for tumor imaging {Chapman 1998}. BMS181321 and BRU59-21 have PCs which are much higher (40 and 12, respectively) and cannot be readily modified to achieve a lower PC. As part of our radiopharmaceutical development program, we designed two compounds in which peptidic chelators for 99"'Tc were linked to 2-NI and evaluated the complexes in an in vitro model of tumor hypoxia. Dimethylglycyl-L-seryl-L-cysteinylglycinamide (RP294, Figure 1 ), which contains a sulfur atom (protected by an acetoamidomethyl group), one amine nitrogen atom, and two amide nitrogen atoms is an N3S class chelator for 99mTc0" and Re0" in a distorted square pyramidal geometry with the oxo moiety in the apical position. The metal complexes exist in syn and anti isomers with respect to the position of the oxo bond and serine CHZOH side chain.
Interconversion of the two isomers in aqueous solution at room temperature has been observed { Wong 1997 } .

3 Su et al., page 4 In the present work, dimethylglycyl-1.-seryl-1.-cysteinyl-lysyl{NE-[1-(2-nitro-imidazolyl)acetamido]}-glycine (RP435, Figure 1) was prepared by attaching 2-NI-acetic acid to a lysine linker connected to the RP294 chelator. RP435 chelates 99mTc at room temperature and two peaks, assumed to be the syn and anti isomers, were detected by HPLC. However, the PC of 99"'Tc0-RP435 is far lower than what is believed to be the optimum value. Accordingly, a new N3S class peptide, dimethylglycyl-1.-tert-butylglycyl-L-cysteinylglycin[2-(2-nitro-1H imidazolyl)ethyl]amide (RP535, Figure 1) was designed and synthesized. The chelator (RP455, Figure I) contains a tent-butyl group, which should increase the lipophilicity of the corresponding 99"'Tc complex and inhibit the interconversion of syn and anti isomers. The labeling, stability, and in vitro cellular accumulation of these compounds was studied.
EXPERIMENTAL PROCEDURES
2-Nitroimidazole, 2-bromoethylphthalimide, N,N-dimethylformamide (DMF), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DECD, 2,3,5,6-tetrafluorophenol (TFP), and trifluoroacetic acid (TFA) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Dimethylglycyl-tent-butylglycyl-1.-cysteinylglycine (RP455) was custom synthesized by Bachem Bioscience Inc. (Philadelphia, PA).
Dimethylglycyl-1.-Beryl-1.-cysteinylglycinamide (RP294) and Re0-RP455 precursor were provided by Resolution Pharmaceuticals Inc. (Mississauga, ON). Sodium 99mTCO4 In saline was obtained from a 99Mo~'9mTc generator (DuPont Pharma, Billerica, MA). a-MEM and fetal bovine serum were purchased from Sigma Chemical Co. (St. Louis, MO).

4 Su et al., page 5 NMR data ('H, '3C, COSY, HSQC, HMQC, TOCSY) were recorded on a Varian UNITYpIus-500 spectrometer (500 Mhz) with TMS as external standard. Mass spectra (electrospray) were obtained on a Sciex API#3 mass spectrometer in the positive ion mode. Reversed phase high pressure liquid chromatography (HPLC) analyses of peptide and 99'"Tc-peptide complexes were carried out on a Beckman model 125 System Gold (Fullerton, CA), with Zorbax SB or Beckman ODS 4.6x250 mm 5-~m C,g columns.
Purification of peptides was accomplished by HPLC with a Waters C,8 RCM 8x10 semi-preparative column. UV and radiometric detectors were connected in series. Two mobile phases were used for HPLC analysis of 99'"TC tracers. Mobile phase I was composed of Hz0/ACN containing 0.1 % TFA, whereas mobile phase II was 0.02 M pH 4.6 NH40Ac buffer/MeOH; both were run as gradients. The flow rate was set at 1.0 mL/min for analysis and 2.0 mL/min for purification.
Synthesis of dimethylglycyl-L-Beryl-L-cysteinyl-lysyl{N~ [1-(2-vitro-1H
imidazolyl)acetamido]}-glycine (RP435): The peptide backbone was prepared on an automated peptide synthesizer (Applied Biosystems Inc., model 433A, Foster City, CA) using Sasrin resin and FMOC-protected amino acids. When the backbone was complete, the Dde protecting group on the s-amine on lysine was cleaved with 2%
hydrazine and the free amine was coupled with 2-nitroimidazole acetic acid (synthesized from nitroimidazole and bromoethyl acetate). The peptide was cleaved from the resin by stirring with 95% aqueous TFA at 0 °C for 30 min, then at room temperature for 60 min.
The resin was filtered and the filtrate was evaporated under vacuum. The residue was washed with a minimum amount of tent-butyl methyl ether, which caused the Su et al., page 6 precipitation of the peptide. The ether was removed and the residue was dissolved in water/ACN and then lyophilized to yield 190 mg of crude product, of which 110 mg was further purified by HPLC, yielding 35 mg (31%) pure RP435. 'H NMR (600 Mhz, db-DMSO): b 1.33 (m, 2H, H(18)), 1.43 (m, 2H, H(19)), 1.59 (m, 1H, H(17a)), 1.72 (m, 1H, H(17b)), 1.86 (s, 3H, H(30)), 2.58 (broad, 6H, H(1,2)), 2.68 (dd, 1H, H(13a), JH~3a-HI1-9.89 Hz, JH~3a-HI3b 14.04 Hz), 2.95 (dd, 1H, H(13b), JH,3b-H»- 3.91 Hz, JH,sb-Hl3a 13.92 Hz), 3.05 (m, 2H, H(20)), 3.61 (m, 2H, H(7)), 4.13 (m, 1 H, H( 15)), 4.20 (dd, 1 H, H(27a), JH27a-H28- 5.86 Hz, JHZ7a-H27b 13.67 Hz), 4.32 (dd, 1H, H(27b), JHZ7b-HZS-

5.13 Hz, JH2sb-H28a 13.67 Hz), 4.44 (m, 1 H, H(6)), 4.56 (m, 1 H, H( 11 )), 5.07 (s, 2H, H(23)), 5.09 (broad, 1 H, H(8)), 7.18 (d, H(25), JHZS-H24- 0.97 Hz), 7.61 (d, 1 H, H(24), JHZa-HZS= 0.98 Hz), 8.0 (d, 1 H, H(14), JH14-15- 757 Hz), 8.31 (m, 2H, H(10, 21)), 8.56 (t, 1H, H(28), JHZS-H27-

6.47 Hz).
Electrospray mass spectrum: mle= 646.15 ([M+H]+, Cz4H4°N9O,°S, calculated: 646.69).
2-(2-Nitro-1H imidazolyl)ethylamine Hydrochloride {Hay 1994}. 0.56 g (5.0 mmol) 2-nitroimidazole, 1.33 g (5.2 mmol) N (2-bromoethyl)phthalimide, and 0.72 g (5.2 mmol) potassium carbonate were mixed in 15 mL DMF, and heated at 110 °C
for 2 h.
After removing the solvents, water was added to the residue to dissolve the salts. The precipitate was collected, washed by water, and dried under vacuum to yield 0.8 g (2.8 mmol, yield 56%) of raw product. The raw product was refluxed with 0.28 g (5.6 mmol) hydrazine monohydrate in 20 mL EtOH for 2 h, and then cooled to 4 °C, filtered, and evaporated to dryness. The residue was dissolved in 20 mL 1 N HCI, filtered, and brought to dryness again. The residue was recrystallized from MeOH/EtOAc to yield 240 mg (1.25 mmol, yield 44%) of product.

Su et al. , page 7 Dimethylglycyl-tert-butylglycyl-L-cysteinylglycine-[2-(2-nitro-1H imidazolyl)-ethyl]amide (RP535): 80 mg (0.18 mmol) RP455, 55.4 mg (0.29 mmol) DECI, 48 mg (0.29 mmol) TFP were dissolved in 1 mL DMF. The mixture was stirred in an ice-bath for 10 min, followed by 30 min at room temperature, and then in a 45°C
oil bath for 60 min. 37.2 mg (0.19 mmol) 2-(2-nitro-1H imidazolyl)ethylamine hydrochloride was added to the mixture which was set in an ice bath. To the mixture was added dropwise 74.7 mg (0.58 mmol) DIEA (di-isopropyl ethylamine) in 1 mL DMF. The mixture was stirred at 45°C for 1 h. The solvent was removed after the reaction was completed.
About 2 mL of water was added and then removed. The crude product was purified by HPLC, yielding 50 mg (0.085 mmol, 29%) pure RP535. 'H-NMR (500 MHz, db-DMSO): b 0.93 (s, 9H, H(8,9,10)), 1.86 (s, 3H, H(28)), 2.71 (dd, 1H, H(14a), JH~4a-H13-9.3 Hz, JH~4a-Hl4b 13.9 Hz), 2.79 (t, 6H, H(1,2), J=4.0 Hz), 2.90 (dd, 1H, H(14b), JH,ab-H13-4.6 Hz, JH~46-Hl4a 13.9 Hz), 4.25 (d, 1H, H(25a), JHZSa-HZ6- 6.59 Hz), 4.27 (d, 1H, H(25b), JH256-H26- 6.59 Hz), 4.37 (d, 1 H, H(6), JH6-HS- 9.27 Hz), 4.47 (m, 1 H, H( 13)), 7.16 (d, 1 H, H(23), JH23-H22- 1.2), 7.51 (d, 1H, H(22), JH22-HZS- 0.98 Hz), 7.89 (t, 1H, H(19), JH~g_H20 5.98 Hz), 8.14 (t, 1H, H(16), JH~6-H17- 5.6 Hz), 8.39 (d, 1H, H(12), JH~2-H13-

7.57 Hz),

8.45 (t, 1H, H(26), JH26-H25- 6.59 Hz), 8.61 (d, 1H, H(5), JHS-H6 9.27 Hz).
Electrospray mass spectrum: mle = 586.11 ([M+H]+, C23H40N907S, calculated: 586.69).
Synthesis of 99"'Tc0-RP294 and 99"'Tc0-RP455: 100 to 200 ~g of RP294 or RP455 was added to Na99mTcO4 (2 to 10 mCi) in 200 pL of saline. To the mixture was added 100 pL of stannous gluconate which contained 40 ~g of SnClz and 0.25 to 1.0 mg of sodium gluconate. The mixture was incubated in a 95°C water bath for 15 min.

Su et al. , page 8 99mTc0-RP294 and 99mTc0-RP455 were analyzed by HPLC (Zorbax SB column, HZO/ACN containing 0.1 % TFA; gradient 100% to 90% HZO over 45 min for 99mTc0-RP294, and 100% to 50% HZO over 45 min for 99mTc0-RP455). The labeling efficiency for 99'"Tc0-RP294 was >97%, while that for 99"'Tc0-RP455 was >99%.
Synthesis of 99"'Tc0-RP435 and 99"'Tc0-RP535: To a 3-mL tube were added 100 to 200 ~,g of RP435 or RP535, Na99mTcO4 (2 to 10 mCi) in 200 ~.L saline, and 100 ~L of stannous gluconate solution which contained 10 to 40 ~g of SnClz and 1.0 mg of sodium gluconate. The labeling of 99"'Tc0-RP435 was carried out at room temperature, while that of 99mTcO-RP535 was incubated in a boiling water bath for 30 min. The products were analyzed by HPLC (Zorbax SB column, H20/ACN containing 0.1 % TFA; gradient for 99mTc0-RP435 was 100% to 70% Hz0 over 45 min, while that for 99mTc0-RP535 was 100% to 50% H20 over 45 min). The yield of 99'"Tc0-RP435 was >68%, while that for 99mTcO-RP535 was >78%. The two compounds were purified for further experiments by HPLC in the same conditions stated above.
Synthesis of Re0-RP535: 10 mg (0.017 mmol) of Re0-RP455 and 13.3 mg (0.070 mmol) of DECI were dissolved in 0.6 mL ACN solution which contained 20%
0.01 M NaOAc/HOAc. To the mixture was added 2.89 mg (0.017 mmol) of TFP
dissolved in 0.3 mL ACN-acetate solution. The mixture was stirred at room temperature for 30 min, and then at 45°C for 1 h. To the mixture was added 3.34 mg (0.017 mmol) of 2-(2-nitro-1H imidazolyl)ethylamine HCl in 0.5 mL ACN-acetate solution, followed by 2.6 mg (0.20 mmol) of DIEA. After stirring at 45 to 50°C for 2 h, the solvent was removed, and the crude product was purified by HPLC (Zorbax SB column, HZO/ACN

Su et al., page 9 containing 0.1 % TFA, flow rate 1.0 mL/min; gradient 100% to 50% H20 in 45 min). The fraction with a retention time of 11.62 min was collected and lyophilised. 'H
NMR (500 MHz, d6-DMSO): 8 1.03 (s, 9H, H(8,9,10)), 2.44 (s, 3H, H(1)), 3.55 (s, 3H, H(2)), 3.83 (d, 1H, H(14a), JH,4a_H,3-12.2 Hz), 4.02 (d, 1H, H(3a), JH3a-H3b 14.9 Hz), 4.40 (t, 1H, H(21 a), J,jzla-H20 5.62 Hz), 4.41 (t, 1 H, H(21 b), JH216-H20 5.6 Hz), 4.46 (s, 1 H, H(6)], 4.82 [d, 1H, H(3b), J,~3b-H3a 14.9 Hz), 5.08 (d, 1H, H(14b), JH,4b-H13-6~84 Hz), 6.99 (d, 1H, H(23), JH~3_H22 1.22 Hz), 7.44 (d, 1H, H(22), JHZZ-HZS-1.22 Hz), 7.72 (t, 1H, H(19)), JH,9_ Hzo 5.86 Hz), 8.19 (t, 1H, H(16)), JH,6_H"=5.62 Hz). ES-MS: mle 712.93 ([M+H]+, Cz°H3zNg0 ,'85ReS, calculated: 712.53) and mle 714.94 ([M+H]+, CzoH3zNg0,'g'ReS, calculated: 714.54), confirming a 1:1 ratio of ligand to metal ion.
Partition Coefficient (n-OctanoUPBS) Determination for 99'"Tc0-RP435 and 99m.1.c0-RP535: 10 pL of purified 99'"Tc0-RP435 or 99mTc0-RP535 was added to a tube which contained 1.0 mL of n-octanol and 1.0 mL of 0.1 M pH 7.4 PBS. The tube was shaken for 1 min. After partial separation of the phases by gravity, 0.7 mL of each phase was transferred to a tube and centrifuged at 12,000 g for 5 min. Duplicate 0.2-mL
aliquots of each phase were taken for y-radioactivity counting.
Cellular Accumulation in an In Vitro Model: Each complex was evaluated in an in vitro model which has been used in the study of other tracers of hypoxia {Ballinger 1996} {Melo 1998} {Zhang 1998} {Ballinger 1993}. Suspensions of Chinese hamster ovary (CHO) cells were incubated at 37°C with stirring under an atmosphere of air or nitrogen (both containing 5% COz) to generate aerobic or hypoxic conditions, respectively. The tracer was added and aliquots were removed over the course of 4 h,

9 Su et al., page 10 centrifuged, and the radioactivity associated with the cell pellet was measured in a y well counter. The results were expressed as the ratio of concentration of radioactivity in the cell pellet to that in an equivalent volume of supernatant medium (C;"/Co",) as a function of time, as described previously {Ballinger 1996}. In experiments in which 5 mM
misonidazole or 8 mM metronidazole were added, the competitor was added 30 min before the tracer, as described previously {Melo 1997}.
RESULTS
Synthesis of RP435 and RP535. RP435 was prepared by attaching 2-NI-acetic acid to a lysine connected to a peptidic N3S chelator (Figure 1). After being cleaved from the resin, RP435 was purified by reversed phase HPLC. The purity was shown to be greater than 98%. RP535 was prepared by coupling 2-(2-nitro-1H
imidazolyl)ethylamine to RP455 (Figure 1). The purity of RP535 was analyzed by two HPLC gradients and shown to be greater than 97%.
The molecular weight of RP435 was determined by electrospray mass spectrometry (ES-MS) with mle 646.15 ([M+H]+, C24H39N9010S requires 645.69).
The'H
NMR (500 MHz) spectrum of RP435 revealed two protons of the 2-nitroimidazole at 7.19 and 7.61 ppm with a weak mutual coupling (J= 0.97 Hz). The proton of the hydroxyl, H(8), appeared at 5.09 ppm as a broad peak. Four of the five amide protons of RP43 5, H( 11 ), H( 14), H(21 ), and H(28), were identified. H( 10) and H(21 ) overlapped each other at 8.31 ppm. The six protons of H( 1 ) and H(2) formed a broad peak at 2.57 Su et al. , page 11 ppm, while the two protons of H(3) were merged in the solvent peak ranging from 3.26 to 3.36 ppm.
The molecular weight of RP535 was determined by ES-MS which showed the molecular ion signal at mle 586.11 ([M+H]+, C23H40N907S requires 585.69). The 'H
NMR spectrum of RP535 exhibited signals of all five amide protons (Figure 1), with H(5) at 8.61 ppm, H(26) at 8.45 ppm, H(12) at 8.39 ppm, H(16) at 8.13 ppm, and H(19) at 7.89 ppm, respectively. Similar to RP435, the two protons of the 2-nitroimidazole of RP535 showed a weak mutual coupling (JHZZ-HZS- 1.1 Hz). The nine protons of the tent-butyl group, H(8,9,10), appeared as a singlet peak at 0.91 ppm. It is interesting to note that the six protons, H(1,2), of the two methyl groups of dimethylglycine produced triplet peaks, instead of a singlet peak, at 2.80 ppm. The signals of H(17) and H(20) were partly merged in the solvent peak ranging from 3.50 to 3.66 ppm, while H(13) and H(21) overlapped each other at 4.45 ppm (m, 3H). These data indicated that RP535 contained the N3S chelator, a 2-nitroimidazole group, and the acetamidomethyl protecting group for the sulfur atom of cysteine.
99m~j~C labeling of RP294, RP455, and RP435. 99"'TC labeling of RP294 and RP435, which have the same N3S chelator, can be carried out at room temperature via transchelation from 99mTc-gluconate. However, 99'"Tc labeling of RP455, which has a tent-butyl group on the chelator, require heating in a boiling water bath for

10 to 15 min.
99mTc0-RP294 showed two peaks on HPLC in 0.1 % aqueous TFA/ACN in a total yield >97%. In contrast, 99"'Tc0-RP455 showed one single peak (yield >99%). When analyzed in acetate/MeOH, 99'"Tc-RP294 showed two major peaks, at 33.69 min (39.6%) and 36.81

11 Su et al., page 12 min (59.3%), with a gradient of 100% to 10% acetate over 45 min, while 99mTc-showed a major peak at 35.55 min (84.8%) with the same gradient.
Stannous gluconate solution was used as reducing agent for Na99mTc04 in the presence of 100 to 300 ~g of RP435. Two main peaks (peak A: 32.42 min, yield 26.4%;
peak B: 35.59 min, yield 42.5%) were detected on HPLC (Figure 2) with aqueous TFA/ACN (gradient: 100% to 70% water over 45 min). The ratio of peaks A and B
in the reaction mixture varied with time, while their retention times remained unchanged.
The difference in retention times of peaks A and B of 99"'Tc0-RP435 was 3.1 min when measured by HPLC with elution gradient of 100% to 70% water over 45 min, while that of the two peaks from 99'"Tc0-RP294 was 0.7 min.
Stability of 99'"Tc0-RP435. Peaks A and B of 99'"Tc0-RP435, after being separated, were reanalyzed by HPLC after different time intervals. A slow interconversion of the two isomers in aqueous solution containing 0.1 % TFA
was observed. For example, 4.2% of B, which came from the conversion of A, was detected 103 minutes after the isolation of A from the labeling mixture; and 4.0% of A, which came from the conversion of B 118 minutes after isolation, was detected. The interconversion of A and B could be greatly accelerated when they were mixed with an equal volume of 0.25 M pH 7.4 phosphate buffer (PB). Figure 3 shows the interconversion of purified A to B and of purified B to A, and it is evident that an approximate 1:1 equilibrium was reached within 1 hour.
99mTC labeling of RP535. RP535 (100 to 300 fig) was labeled with 2 to 20 mCi 99"'Tc with stannous gluconate as reducing agent. Higher temperatures (60 to 100°C)

12 Su et al., page 13 were required to label the compound. One major labeled peak (yield over 78%) has been obtained when the reaction mixture was incubated in a boiling water bath for 30 min (Figure 4, lower trace). Varying the pH from 2 to 7 did not significantly affect the labeling yield. Although the nitroimidazole group of RP535 was stable under the labeling conditions, significant reduction was noted (analyzed by HPLC and monitored by the UV
detector at 214 and 320 nm) when larger amounts of SnCl2 were added, resulting in a dramatic decrease in the yield of 99'"Tc0-RP535. For example, the yield of 99"'Tc0-RP535 dropped to less than 5% of the total detected radioactivity on HPLC when 100 ~g of SnClz was used, and no intact RP535 remained when 600 ~g of SnCl2 was added.
Determination of partition coefficients (PCB of 99"'Tc0-RP435 and 99m.1.cO-RP535 between n-octanol and phosphate buffered saline (PBS). It is impossible to isolate individually the syn and anti isomers of 99'"Tc0-RP435 by HPLC with mobile phase I or II due to their rapid interconversion in aqueous solution at room temperature.
The reported PC (0.0013) of 99"'Tc0-RP435 is in fact the average PCs of the two isomers of the 99mTc0-RP435. The PC of 99'"Tc0-RP535 was 2.8~0.1, n=4. Thus, the difference in partition coefficients of 99'"Tc0-RP535 and 99'"Tc0-RP435 is over 2,000 fold.
Preparation of Re0-RP535 and co-injection with 99"'Tc0-RP535. It is difficult to determine directly the structure of a 99"'Tc complex because of the extremely small amount of the compound present. 99Tc is a long-lived radioactive isotope (t"2=2.1x105 y) which restricts its application. However, the composition and structure of 99'"Tc0-RP535 could be indirectly determined by comparison with Re0-RP535, which can be prepared in weighable quantities and characterized. Re belongs to the same group (VIIb) of the

13 Su et al., page 14 periodic table and possesses many similar chemical characteristics with Tc because of the lanthanide contraction.
Re0-RP535 was prepared by coupling 2-(2-nitro-1H imidazolyl)ethylamine with Re0-RP455. The compound was purified by HPLC. 'H NMR data showed that the two amide protons, H(5) and H(12), disappeared after the chelation of RP535 to Re03+, while those of H(16) and H(19) of the uncoordinated RP535 (Figure 1) remained. The two methyl groups and the methylene group of dimethylglycine could no longer freely rotate around the C(3)-N~",;~e bond in Re0-RP535, which made the protons of these groups become chemically non-equivalent. For example, the protons of H( 1 ) shifted from 2.80 ppm to 2.44 ppm, and H(2) shifted from 2.80 ppm to 3.55 ppm upon the formation of Re0-RP535. This indicated that the N atom of the dimethylglycine co-ordinate to Re03+
core. The two protons of H(3) of Re0-RP535 were in a chemically non-equivalent environment as well. H(3a) appeared at 4.02 ppm and H(3b) at 4.82 ppm with a coupling constant JH3a-H3b at 14.9 Hz. H(14a) coupled H(13) Wlth JHl4a-H13 for 6.8 ppm, while H(14b) coupled H(13) Wlth JH~4a-H13 for 12.2 ppm. However, no signals of H(14a) coupling with H(14b) were found. All this evidence supported the assumption that the Re03+ core was complexed with the N3S chelator. The signals of H(17) and H(20) overlapped each other at the range of 3.46 to 3.54 ppm, while that of H(13) was partly merged in solvent peak (in the range of 3.28 to 3.36 ppm). No evidence of the co-existence of the syn and anti isomers of Re0-RP535 was found in the'H NMR
spectrum.
The prepared Re0-RP535 can be either a syn or anti isomer. The electrospray mass spectrum exhibited the characteristic two molecular ions at mle 712.93 ([M+1 ]+) which corresponded to a 1:1 'g5Re0 to RP535 compound and mle 714.94 ([M+1]+) which

14 Su et al., page 15 corresponded to another 1:1 'g'Re0 to RP535 compound (the required molecular weights are 712.53 and 714.54). Moreover, the relative abundances of the two peaks (57.4% and 100%) are consistent with the natural abundances of'85Re and'8'Re (37.4% and 62.6%).
An aqueous solution of Re0-RP535 was mixed with the 99"'Tc0-RP535 labeling mixture and co-injected into the HPLC for analysis. Two gradients of aqueous TFA/ACN were used: 100% to 30% and 100% to 50% water over 45 min. Two detectors, UV and radiometric, were connected in series with the eluent entering the UV
detector first and then the radiometric detector. A time delay of 0.3 to 0.4 min for signal response between the UV and radiometric detectors existed. Figure 4 shows the chromatogram of the co-injection of Re0-RP535 with 99'"Tc0-RP535 with the 100%
to 30% water gradient. The retention time of Re0-RP535 was 33.04 min, while that of 99'"Tc0-RP535 was 33.35 min. The UV peak at 23.37 min was free RP535. The retention times of Re0-RP535 and 99"'Tc0-RP535 became 39.91 min and 40.51 min, when 100% to 50% water gradient was used. The retention times of Re0-RP535 and 99"'Tc0-RP535 are the same when the time delay in signal response is taken into account.
This implies that 99"'Tc0-RP535 has the same composition and structure as Re0-RP535;
i.e. 99mTc is coordinated by the N3S chelator of RP535, there is a 99'"Tc=O
core, and the ratio of 99'"Tc to RP535 is 1:1.
Stability of 99"'Tc0-RP535. 99mTc0-RP535 was isolated by HPLC and the collected 99mTcO-RPS3S in aqueous TFA/ACN was re-injected into the HPLC for analysis after different time intervals at room temperature. It was evident that 99'"TCO-RP535 was more stable than 99"'Tc0-RP435 in aqueous solution which contained 0.1%

Su et al., page 16 TFA, because neither decomplexation nor change in the radioactive peak was seen over 26 hours.
Theoretically, the formation of syn and anti isomers of 99"'Tc0-RP535 is possible, when the tent-butyl side chain on the N3S chelator is in the syn and anti conformations with respect to the 99'"Tc=O core. When the isolated 99mTc0-RP535 was mixed with an equal volume of 0.25 M pH 7.4 PB and reinjected after different time intervals, a decomplexation of the complex, instead of interconversion of syn and anti isomers, was observed (Figure SA). 99"'Tc0-RP535 (retention time 40.27 min) decomplexed in PB and formed three products which appeared in the region of 22.1 to 23.7 min on the HPLC.
These decomplexed compounds cannot be the isomers of 99'"Tc0-RP535 because they did not exist in the labeling mixture (Figure 4, lower trace) and they did not reach any fixed equilibrium with 99"'Tc0-RP535 over time. The decomplexation might occur when one or more coordination bonds of the 99"'Tc0-RP535 complex broke. No free pertechnetate was detected from the decomplexation of 99"'Tc0-RP535. The half time for decomplexation was about 200 min (Figure SB). Interestingly, 99mTc0-RP535 decomposed immediately with release of pertechnetate when mixed with 0.01 N
NaOH.
These results indicate that the tent-butyl group on the N3S chelator acts as an important steric hindrant that inhibits the co-existence and interconversion of syn and anti isomers of 99"'Tc0-RP535. However, the configuration of 99mTc0-RP535 remains to be determined.
In Vitro Tests of 99'"Tc0-RP294, 99"'Tc0-RP455, 99"'Tc0-RP435, and 99",TcO-RP535. The ability of these 99mTc labeled compounds to be taken up by mammalian cells Su et al. , page 17 under aerobic or hypoxic conditions was tested using CHO cells in suspension culture in equilibrium with 95%air/5% COZ or 95% Nz/5% COz gas mixtures. Samples were removed as a function of time and ratios of cell associated radioactivity (C;n) to that in the medium (Co~,) were calculated. The control compounds, 99"'Tc0-RP294 and 99"'TCO-RP455, which do not contain 2-NI groups, did not show selective accumulation in hypoxic CHO cells. In contrast, 99'"Tc0-RP435 showed differential accumulation between hypoxic and aerobic cells. The C;~/CoU, for aerobic cells at 4 hours was 0.3, while that of hypoxic cells was 0.8 (Figure 6A), for a hypoxic/aerobic differential of 2.1 ~ 1.0 (n=8). In comparison, 99"'Tc0-RP535 revealed a greater differential accumulation between hypoxic and aerobic cells than 99"'Tc0-RP435. When 99mTc0-RP535 was added to a suspension of CHO cells in vitro, there was a modest accumulation of radioactivity in the cells under aerobic conditions, reaching a C;n/CoU, value of 0.7 at 4 hours (Figure 6B).
In contrast, under hypoxic conditions there was a further increase to a C;n/Cout value of 2.1, for a 3.3 ~ 1.2 (n=9) fold hypoxic/aerobic differential.
Previous work {Melo 1997} with the 99"'Tc-labeled 2-NI BMS181321, using the same cell line and in vitro system, showed that a large molar excess of the 2-NI
compound misonidazole prevented selective accumulation in hypoxic cells while the 5-nitroimidazole metronidazole stimulated it. In the present work, addition of 5 mM
misonidazole abolished the hypoxia-specific accumulation of 99'"Tc0-RP535, whereas 8 mM metronidazole selectively increased hypoxic accumulation to 130% of the control value.

Su et al., page 18 DISCUSSION
Based on promising results obtained with 'gF-FMISO and 'z3I-IAZA {Grierson 1989} {Koh 1992} {Mannan 1991 } {Parliament 1992}, there has been great interest in the development of 99"'Tc-labeled markers of hypoxia which would be convenient to prepare and widely applicable. However, the standard 99'"Tc-chelation systems have not all proved to be useful. The first attempt using the BATO approach resulted in a complex which was not efficiently reduced and trapped, and which showed inadequate permeability to lipophilic membranes {Linder 1993}. A bis(amine-phenol) complex similarly failed to cross cell membranes {Ramalingam 1994}. Greatest success thus far has been obtained with amineoxime-type chelators, specifically BMS181321 {Linder 1994}, BRU59-21 {Wedeking 1995}, and BnAO {Archer 1995}; however, each has its limitations. BMS 181321 has a PC of 40, which results in slow clearance from the blood and background tissues, and extensive elimination via the gastrointestinal tract {Ballinger 1996}. BRU59-21 has a lower PC of 12 with resultant improved clearance from the blood, but the extent of hepatobiliary excretion remains high {Johnson 1998}
{Melo 1998}. In contrast, BnAO undergoes extensive renal elimination but, lacking a nitro group, its mechanism of hypoxia-specific localization is unclear {Zhang 1998}.
BnAO
has a low PC of 0.1, although this does not appear to limit its penetration of tissue {Zhang 1998}. Work in the radioiodinated sugar 2-NI series has suggested an optimum PC of ~ 1 { Chapman 1998 } .
The RP294 chelation system for 99"'Tc was developed by Resolution Pharmaceuticals Inc. {along 1997} and is used in RP128, a peptide for imaging Su et al., page 19 inflammation {Caveliers 1996}. The free carboxylic acid group of the molecule contributes to the hydrophilicity of the complex and RP435, the 2-NI-containing derivative of RP294, has an extremely low PC of 0.0013. Therefore, an analog containing tent-butyl glycine in place of serine was developed. In the present work, the utility of this chelator on a 2-NI compound targeted to hypoxic tumor cells was evaluated.
The partition coefficient of 99"'Tc0-RP535 was 2.8, which is similar to that reported for IAZA {Mannan 1991 }.
Although the presence of the tert-butyl group was desirable to increase the PC
of the complex, it made the chelator more difficult to label. While 99"'Tc labeling of RP294 and RP435 by transchelation from gluconate proceeded efficiently at room temperature, the tent-butyl analogs RP455 and RP535 required heating. Maximal yields obtained were 26% and 43% for the two peaks of 99mTc0-RP435 (Figure 2) and 78% for 99"'Tc0-(Figure 4, lower trace). It was shown that the nitro group was not reduced by the typical quantities of stannous chloride used in 99"'Tc labeling, but that it could be reduced by excessive amounts.
The structure of RP294 was determined by '3C and 'H NMR, except the assignments of the amide protons { Wong 1997 } . In addition, Re0-RP294 and 99TcO-RP294 have been structurally confirmed by 'H NMR and X-ray crystallography, which indicated that the metal ions were co-ordinated by the N3S chelator. RP435, containing the same N3S chelator as RP294, can be presumed to chelate the 99'"TCO3+ core in the same manner. As has been shown previously for 99mTc0-RP294 { Wong 1997 }, two complexes of 99mTc0-RP435 were formed, presumably syn and anti isomers (Figure 2).
Following separation by HPLC these were quite stable in acidic aqueous solution (4%

Su et al., page 20 interconversion in 2 hours) but equilibrated in 0.25 M pH 7.4 phosphate buffer with a half time of ~l hour (Figure 3). In contrast, 99'"Tc0-RP535 existed as a single isomer (Figure 4, lower trace), confirmed by co-injection with its Re analog (Figure 4, upper trace), which was extremely stable in acidic aqueous medium. When diluted in PB, 99"'Tc0-RP535 decomplexed with a half time of ~2 hours, resulting in the formation of several hydrophilic species but no release of free pertechnetate (Figure 5).
However, when 99'"Tc0-RP535 was diluted in 0.01 N NaOH it decomposed rapidly to pertechnetate.
Following HPLC purification, 99"'Tc0-RP435 and 99"'Tc0-RP535 were evaluated in an in vitro model of cellular hypoxia that has been used extensively in radiation biology and investigation of bioreductive drugs, including other 99mTc- and 'gF-labeled hypoxia tracers {Ballinger 1996} {Melo 1998} {Zhang 1998} {Ballinger 1993}.
Both tracers showed a modest level of uptake in aerobic CHO cells, which increased slightly over the course of four hours. In contrast, hypoxic cells accumulated 2 to 3 times as much radioactivity over that time period, a significant degree of hypoxia-specific accumulation (Figure 6). Moreover, the selective accumulation of 99mTc0-RP535 could be modulated by co-incubation with millimolar concentrations of unlabeled nitro compounds (data not shown). The 2-NI misonidazole abolished the hypoxia-specific accumulation of 99"'Tc0-RP535 while metronidazole, a 5-NI of lower electron affinity, enhanced this accumulation. The same effects have been reported previously with the 2-NI 99mTc-BMS 181321 and suggest a bioreductive mechanism of localization of the tracer {Melo 1997}.

Su et al. , page 21 CONCLUSIONS
RP435 and RP535 have a similar N3S chelator in structure, but their ability to form chelates with 99mTC 1S different. The 99"'Tc0-RP435 chelate could be prepared at room temperature, and showed interconversion of syn and anti isomers in aqueous solution. The interconversion of the two isomers was much faster in neutral than in acidic medium. In contrast, the tent-butyl group on the backbone of the N3S
chelator of RP535 hindered the chelation with 99mTc, presumably by steric effects, and blocked the formation and interconversion of the theoretically existing syn and anti isomers of 99mTc0-RP535 in acidic and neutral aqueous solution at room temperature.
However, the tent-butyl group of RP535 did have the desired effect of significantly increasing the lipophilicity of the corresponding 99mTc chelate. Both 99"'Tc0-RP435 and 99mTc0-RP535 showed selective uptake in hypoxic cells, suggesting that 99'"Tc0-peptidic complexes containing the 2-nitroimidazole group can be a new class of hypoxia imaging agents.

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Claims