Synthesis, Radiolabeling and Biological Evaluation of Propylene Amine Oxime Complexes Containing Nitrotriazoles as Hypoxia Markers

Two propylene amine oxime (PnAO) complexes, 1, containing a 3-nitro-1,2,4-triazole and 2, containing two 3-nitro-1,2,4-triazoles, were synthesized and radiolabeled with 99mTc in high labeling yields. Cellular uptakes of 99mTc-1 and 99mTc-2 were tested using a S180 cells line. Under anoxic conditions, the cellular uptakes of 99mTc-1 and 99mTc-2 were 33.7 ± 0.2% and 35.0 ± 0.7% at 4 h, whereas the normoxic uptakes of the two complexes were 6.0 ± 1.6% and 4.6 ± 0.9%, respectively. Both 99mTc-1 and 99mTc-2 displayed significant anoxic/normoxic differentials. The cellular uptakes were highly dependent on oxygen and temperature. Biodistribution studies revealed that both 99mTc-1 and 99mTc-2 showed a selective localization in tumor and slow clearance from it. At 4 h, the tumor-to-muscle ratios (T/M) were 3.79 for 99mTc-1 and 4.58 for 99mTc-2. These results suggested that 99mTc-labeled PnAO complexes 99mTc-1 and 99mTc-2 might serve as novel hypoxia markers. By introducing a second nitrotriazole redox center, the hypoxic accumulation of the marker was slightly enhanced.


Introduction
Owing to the vascular deficiencies, hypoxic cells exist in solid tumors and are resistant to radiation [1]. To improve the efficacy of radiotherapy, numerous radiosensitizers have been developed. One important strategy to develop hypoxia markers is the use of hypoxic radiosensitizers. A variety of nitroimidazole derivatives had been synthesized and evaluated as hypoxia markers [2], especially 2-nitroimidazole derivatives. For instance, the 18 [3], stroke [4] and ischemic myocardium [5]. Recently, 4-and 5-nitroimidazolyl compounds had also been developed as hypoxia markers [6,7].
In our previous work, several 99m Tc-labeled PnAO complexes containing nitroimidazoles were prepared, and the effect of a second nitroimidazole redox center on the hypoxic accumulation was investigated. It was very interesting that by introducing a second 2-nitroimidazole redox center to BMS181321, the hypoxic accumulation increased significantly. However, a second 4-nitroimidazole showed little effect [19].
To study the effect of a second nitrotriazole, another redox center on the hypoxic accumulation, in this study, using nitrotriazole as redox center and propylene amine oxime (PnAO) as chelating group, we synthesized PnAO complexes 1, containing a 3-nitro-1,2,4-triazole and 2, containing two 3-nitro-1,2,4-triazoles ( Figure 1). Compounds 1 and 2 were labeled with 99m Tc. The in vitro and in vivo evaluation was also investigated.

Synthesis
The synthesis routes of PnAO complexes 1 and 2 are shown in Scheme 1. Alkylation of 3-nitro-1H-1,2,4-triazole with bromo olefin provided compound 3. Chloronitroso derivative 4 was obtained by the addition of conc. HCl to the mixture of isoamyl nitrite and olefin 3, and then the reaction between chloronitroso derivative 4 and diamine mono oxime 5 afforded precursor 1. Precursor 2 was achieved via the reaction of chloronitroso derivative 4 with 1,3-diaminopropane

Radiolabeling
The 99m Tc-complexes were prepared using SnCl 2 as a reducing agent. Precursors 1 and 2 were labeled with 99m Tc by ligand exchange from 99m Tc-DTPA. The labeling mixture was analyzed by high performance liquid chromatography (HPLC) [19]. The retention times of 99m Tc-DTPA and 99m TcO 4 − were 1.8 and 2.7 min, whereas the radioactivity signals of 99m Tc-1 and 99m Tc-2 showed a single peak at 13.7 and 13.8 min, respectively ( Figure 2). The initial radiochemical purities of 99m Tc-1 and 99m Tc-2 were above 95%. The radiochemical purities remained above 90% after being kept at room temperature for 8 h. The stability of 99m Tc-2 containing two nitrotriazoles was better than that of the PnAO analogue containing two 2-nitroimidazoles, for which the purity remained above 80% after 4 h [19]. Tc-DTPA (A), 99m TcO4 − (B), 99m Tc-1 (C) and 99m Tc-2 (D).
BMS181321 was found to selectively accumulate in anoxic CHO cells [17]. In our previous work [19], BMS181321 also accumulated in S180 cells under anoxic conditions, and the anoxic uptake was 24.4 ± 0.7% at 4 h. In the cells, nitroazoles including nitroimidazole and nitrotriazole undergo a single electron reduction catalyzed by xanthine oxidases. In the presence of sufficient oxygen, the free radical anion can be reoxidized by oxygen, and diffuse out of the cells. However, under hypoxic condition, it can be further reduced and bind to cellular macromolecules [22]. By changing 2-nitroimidazole to 3-nitro-1,2,4-triazole, the anoxic uptake increased to 33.7 ± 0.2% at 4 h. The reduction potential of 3-nitro-1, 2,4-triazole (−0.55 V) is more negative than 2-nitroimidazole (−0.40 V) [23]. Thus 2-nitroimidazole was believed to be a more efficient electron acceptor, and it can accept electrons more easily to form the radical anion. However, 99m Tc-1 containing a 3-nitro-1,2,4-triazole showed higher anoxic cellular accumulation compared with BMS181321 containing a 2-nitroimidazole [19].
Both 99m Tc-1 and 99m Tc-2 containing nitrotriazoles showed significant anoxic/normoxic differentials. The uptake ratios (anoxic/normoxic) at 30 min, 1, 2 and 4 h were 2.3, 3.0, 4.7 and 5.6 for 99m Tc-1, and 3.0, 3.3, 6.2 and 7.5 for 99m Tc-2. 99m Tc-2 showed higher anoxic/normoxic uptake ratios compared with 99m Tc-1, indicating that the anoxic/normoxic differentials increased by introducing a second 3-nitro-1, 2,4-triazole moiety into the molecule. Both 99m Tc-1 and 99m Tc-2 exhibited higher anoxic/normoxic uptake ratios compared with BMS181321 [19]. Thus 3-nitro-1,2,4-triazole moiety can work as a redox center with appropriate reduction potential. In another series of cellular uptake studies, the anoxic exposure was switched to the normoxic exposure at 2 h. The cellular uptakes of both 99m Tc-1 and 99m Tc-2 declined slightly in the next two hours (Figure 3). Oxygen can reoxidize the free radical anion to the original drug. The entry of oxygen restrained the cellular uptakes of nitrotriazole derivatives. Therefore, there was no further accumulation after 2 h. All the metabolism products of markers would be bound to the cellular components irreversibly under ideal conditions. However, incomplete retention of metabolized markers had been reported in previous studies. For instance, BMS181321 showed 60% of the counts were retained after washing, and these counts were continually lost in the later incubation [17]. The radiolabeled metabolites of BRU59-21 appeared in the supernatants in hypoxic cells [18]. In this study, the supernatants were also analyzed with radio-HPLC during the cellular studies. The initial radiochemical purity of 99m Tc-1 was above 95%. After 4 h, there was little change in the radio chromatographic signals under normoxic conditions, the radiochemical purity remained 94%. In contrast, after 4 h of incubation under anoxic condition, the radiochemical purity of 99m Tc-1 was only 44%. The main metabolites showed the same retention time as 99m TcO4 − . These results suggested that the metabolites of 99m Tc-1 might be released from the anoxic cells into the medium. Therefore, the cellular uptakes of 99m Tc-1 and 99m Tc-2 declined slightly after switching to the normoxic exposure.
To study the effect of temperature, the cellular uptake studies were also performed at 4 C and 25 C (Figure 4). Under anoxic conditions, the cellular uptakes of 99m Tc-1 and 99m Tc-2 were increased dramatically by rising the temperature. At 4 h, the anoxic uptakes of 99m Tc-1 at 4 C, 25 C and 37 C were 3.5 ± 0.4%, 23.1 ± 0.6% and 33.7 ± 0.2%, respectively. In contrast, the anoxic uptakes of 99m Tc-2 at 4 C, 25 C and 37 C were 2.3 ± 0.3%, 14.1 ± 1.2% and 35.0 ± 0.7% at 4 h. The anoxic uptakes of 99m Tc-1 and 99m Tc-2 strongly depended on temperature. Under normoxic conditions, the cellular uptakes were low and the measurement errors were relatively high, so the normoxic uptakes showed no significant difference at various temperatures. The differences in anoxic accumulation at 4 C, 25 C and 37 C might be due to the enzyme activity and membrane fluidity. The reduction of nitrotriazole in the cell is an enzymatic process. At low temperature, enzymes (xanthine oxidases) are inactive. Enzyme activity increases gradually with rising temperature until the optimum temperature (35-45 C for xanthine oxidases). On the other hand, the markers enter the cells by diffusion. High temperatures increase the fluidity of membranes, and the markers enter the cells more readily. Both of 99m Tc-1 (log P o/w = 1.74) and 99m Tc-2 (log P o/w = 1.42) showed lipophilic nature, and the high partition coefficients allowed them cross the membrane readily by diffusion. 99m Tc-1 was more lipophilic than 99m Tc-2, however, 99m Tc-2 exhibited higher anoxic/normoxic uptake ratios. These results indicated that the lipophilicity may be an important factor in cellular accumulation, but other factors are of greater importance in this study.

Biodistribution Study
The in vivo studies were performed in Kunming mice bearing S180 tumor to determine the tumor specificity. The biodistribution results are tabulated in Tables 1 and 2. For both 99m Tc-1 and 99m Tc-2, the liver was the tissue with highest radioactivity, and the radioactivity in the kidneys was low. These results suggested lipophilic complexes 99m Tc-1 and 99m Tc-2 were largely cleared through the hepatobiliary pathway. The tumor uptakes decreased slowly after injection, from 0.47 ± 0.08 %ID/g at 0.5 h to 0.35 ± 0.03 %ID/g at 4 h for 99m Tc-1 and 0.39 ± 0.06 %ID/g at 0.5 h to 0.25 ± 0.06 %ID/g at 4 h for 99m Tc-2. The tumor-to-blood ratios (T/B) were 0.39 for 99m Tc-1 and 0.31 for 99m Tc-2 at 4 h. Both 99m Tc-1 and 99m Tc-2 showed a slow blood clearance, similar to that observed in BMS181321 [17]. This might be due to the high octanol/water partition coefficients of these complexes. However, the uptakes in muscle were low and cleared relatively quickly. The muscle uptakes decreased from 0.19 ± 0.03 %ID/g at 0.5 h to 0.09 ± 0.01 %ID/g at 4 h for 99m Tc-1, and from 0.22 ± 0.04 %ID/g at 0.5 h to 0.05 ± 0.01 %ID/g at 4 h for 99m Tc-2. The tumor-to-muscle ratios (T/M) increased with time after injection, and the ratios were 3.79 for 99m Tc-1 and 4.58 for 99m Tc-2 at 4 h. Compared with BMS181321 at 4 h after injection (3.53 in KHT tumor, 4.21 in SCC tumor and 3.54 in RIF-1 tumor) [17], 99m Tc-1 and 99m Tc-2 showed similar or higher tumor specificity. By introducing a second nitrotriazole, 99m Tc-2 was a better tumor hypoxia marker compared with 99m Tc-1.

General
3-Nitro-1H-1,2,4-triazole was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). 1,3-Diaminopropane (98%) and N,N-diisopropylethylamine (98%) were supplied from Acros Organics (Geel,Belgium). All other reagents were of analytical grade. NMR spectra were obtained on Bruker (400 MHz and 500 MHz) spectrometers (Bruker, Faellanden, Switzerland). Chemical shifts are given in ppm relative to tetramethylsilane used as an internal standard, the coupling constants are expressed in hertz, and the splitting patterns are designated as s (singlet), d (doublet), t (triplet) and m (multiplet). Mass spectra were measured on a Bruker APEX IV FTMS, positive mode, ESI. Elemental analyses were carried out on an Elementar Vario MICRO CUBE (Hanau, Germany). RP-HPLC analyses were performed on a Waters 1525 binary HPLC pump and a Waters 2487 UV absorbance dual  detector (Milford, MA USA). The elution was also monitored with a Packard 500 TR flow scintillation radioactivity detector (Meriden, CT, USA). Murine sarcoma S180 cell line was provided by the

Octanol/Water Partition Coefficient
The partition coefficients were determined by the reported procedure [20,21] with minor differences. Octanol (1.0 mL), 99m Tc-labeling solution (0.5 mL) and saline (0.5 mL) were mixed in a 2 mL centrifugal tube. The tube was vigorously vortexed for 5 min and centrifuged at 3000 rpm for another 5 min. Five samples (10 µL) of each phase were removed into gamma counter tubes and counted for radioactivity. The P O/W was the ratio of the radioactivity of the octanol layer to that of the water layer. Then the octanol layer (0.8 mL) was moved to another centrifugal tube, in which octanol (0.2 mL) and saline (1.0 mL) were added. The tube was vortexed and centrifuged. Samples of each phase were removed and counted. This process was repeated for several times until consistent P O/W was obtained.

In Vitro Study
The cellular accumulation experiments were performed according to the literature methods [25,26]. Murine sarcoma S180 cells were suspended in a fresh DMEM medium plus 10% (v/v) of fetal bovine serum (FBS). The final cell concentration was 2 × 10 6 cells/mL. Aliquots of 20 mL were added to glass vials and gently stirred with magnetic spinbars. The glass vials were placed in water baths to control the temperature. The cells were incubated under normoxic conditions (95% air plus 5% carbon dioxide) or anoxic conditions (95% nitrogen plus 5% carbon dioxide, below 10 ppm O 2 ). After an equilibration for 30-45 min, The JPSJ-605 dissolved oxygen meter read 0.00 mg/L, and the 99m Tc-labeling marker (5 MBq in 0.5 mL) was added to each glass vial and the final concentration of unlabeled drug was 5 µg/mL. More than 1 mL sample was removed each 30 minutes. Five aliquots (200 µL) were pipetted from each sample removed and centrifuged at 1500 rpm for 5 minutes. 180 µL of supernatant was removed for counting (A), and the cells and medium left were also counted (B). The cellular uptakes were calculated as % Uptake = [(B − A/9) / (A + B)] × 100%. The trypan blue exclusion assay revealed that S180 cells maintained more than 90% viability for 4 h.
To imitate the physiological circumstances, the temperature in the accumulation experiments was 37 C. At 1, 2 and 4 h, aliquots of supernatant were filtrated by 0.22 µm filter. Filtered supernatant was analyzed with radio-HPLC [18]. To investigate the effect of entry of oxygen to the cellular uptakes of hypoxia markers, the anoxic exposure was switch to normoxic exposure in another series of cellular accumulation experiments. To study the effort of temperature, the cellular accumulation experiments were also conducted at 25 C (room temperature) and 4 °C [27].

Biodistribution Study
All the animal experiments were performed in accordance with the national laws related to the conduct of animal experimentation. The biodistribution of 99m Tc-1 and 99m Tc-2 was evaluated in Kunming male mice (20-25 g) bearing the S180 tumor in the left front leg (diameter of 10-15 mm). The 99m Tc-complex was (1 MBq in 0.1 mL) administered by tail vein injection. The mice were sacrificed by cervical dislocation at different times after injection. Various organs and tissues were excised, weighed and counted. The percent injected dose per gram (%ID/g) of each organ/tissue or tumor was calculated by the above data. The final results were expressed as means ± SD. of five parallel experiments.