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Synthesis, Radiolabeling and Biological Evaluation of (99m)Tc-labeled Deoxyglucose Derivatives for Molecular Imaging.

Two deoxyglucose (DG) derivatives, (α,β)-2-deoxy-2-amino(ethylcarbamate)-D-glucose (ECB-DG) and (α,β)-2-deoxy-2-amino(1,2-dihydroxypropyl)-D-glucose (DHP-DG), were synthesized and radiolabeled successfully with [99mTc(H2O)3(CO)3]+ complex. [99mTc]-ECB-DG and [99mTc]-DHP-DG complexes were prepared (96% and 93% radiochemical purities respectively) by using 46 mCi of Na99mTcO4 in 1 mL saline. Radio-HPLC analysis of [99mTc]- ECB-DG at pH = 7.4, revealed that labeling with 99mTc leads to formation of one radiochemical species with tR = 381 second. Three radiochemical species, Na99mTcO4, [99mTc]-DHP-DG and [99mTc(H2O)3(CO)3]+ complexes with tR = 342 sec, tR = 567 sec and tR = 1586 sec respectively, were obtained when [99mTc]-DHP-DG complex evaluated by HPLC. Biodistribution of two complexes were studied on normal mice at 10, 30 and 60 min post-injections. Compared to the 18F-FDG, [99mTc]-ECB-DG displayed a 2.8-fold reduction in brain uptake (1.7 ± 0.2 versus 0.61% ± 0.09) ,whereas [99mTc]-DHP-DG just showed 1.9-fold reduction in heart uptake (2.2 ± 0.05 towards 1.16±0.10) at 1 h post-injection. On the basis of our results, it seems that ECB-DG and DHP-DG analogues could be used as brain and heart imaging agent respectively.


Introduction
Carbohydrates are of primary importance, because they are sources of energy for living organisms. Therefore, the design of carbohydrateappended 99m Tc complexes for potential use in nuclear medicine is of considerable interest (1). The accurate and early non-invasive detection of malignant disease is an important factor in the treatment and prognosis of a patient with cancer. Improvements in tumor radionuclide imaging depend on the development of more tumor-specific radiopharmaceuticals (2). One compound that is very useful for the detection of tumors and metastatic tissue is 2-deoxy-2-[ 18 F]fluoro-D-glucose (FDG), which is imaged by positron emission tomography (PET) (1). Because the production of Flourine requires a cyclotron, and the isotope has a short halflife (110 min) , its utility is somewhat limited. Hence, this problem led to the search for alternatives to FDG by utilizing radionuclide that decay by a process that can be imaged by single photon emission computed tomography (SPECT). SPECT is much more prevalent and enabling the use of 99m Tc, which has ideal nuclear properties (t 1/2 = 6.01 h, γ = 140 keV). It is also the most widely used radioisotope in nuclear medicine (3).
that N-functionalized glucosamines show activity with GLUTs (glucose transporters) and hexokinases, the enzymes that are most closely associated with the metabolism of FDG, even when the functional group is large (2,14). For example, Yang et al. reported that ethylenedicysteine-deoxyglucose (EC-DG) as an N-functionalized glucosamine, shows activity with hexokinases. Their findings suggested that hexokinase-catalyzed phosphorylation had occurred with EC-DG, 2-deoxy-D-glucose, 2-fluorodeoxyglucose (FDG), and glucose. Whether EC-DG and 2-deoxy-D-glucose use different glucose transporters,both of them need to be further investigated (5). In addition, it has been confirmed that there are at least two mechanisms for the cellular processes of glucosamine. The first mechanism resembles the cellular process mechanism of a glucose transporter system. In the second mechanism, glucosamine enters cells and forms glucosamine-6-phosphate directly in the additional transcriptional pathways (15)(16)(17)(18)(19). Lots of 99m Tc-labeled glucose and D-glucosamine derivatives have been synthesized in order to develop one subrogate in SPECT for [ 18 F]-FDG in PET (2,4,5,11,12,[20][21][22][23][24].

Experimental
All chemicals were purchased from Aldrich and Merck companies and used without further purification. Carbon monoxide was obtained in the form of refillable canisters (0.5 L) from M/s Alchemie Gases & Chemicals. 99m TcO 4 was eluted from an in-house 99 Mo/ 99m Tc column generator using normal saline. HPLC analyses were performed on a JASCO 880-PU HPLC (Tokyo, Japan) equipped with a Ray test-Gabi gama ray detector. A Polygosil 5 µm RP-C18 analytical column (reverse phase) with dimensions of 250×4.6 mm was used. IR spectra were taken films KBr pellets on a Bomem spectrometer (Bgrams). 1 H NMR and 13 C NMR spectra were run on Bruker (DRX-500 Advance) spectrometer at 500 ( 1 H NMR) and 125 ( 13 C NMR) MHz, in D 2 O as solvent. Chemical shift has been expressed in ppm rel. to Me 4 Si as internal standard. Mass spectra were recorded on Instrument Mass HP (Agilent technologies) 5937 mass selective detector using For these reasons, we seek a 99m Tc SPECT tracer that will mimic the biodistribution of FDG for molecular imaging. Our approach is to attach to glucose a chelating ligand that, in a subsequent reaction, will bind the radioisotope 99m Tc. A metal-chelate could be preformed and then attached to glucose. To mimic the properties of FDG, it is imperative that the effects of the tracer group on the properties of the glucose molecule be minimized. Existing 99m Tc labeled glucose derivatives fail this criterion: they are either ionic or have relatively high molecular weight (i.e. carry two glucose moieties) (4, 5).
Stable core structures have found utility in the chemistry of Tc due to the wide range of accessible oxidation states (Tc(-1) to Tc(+7)). For example, the {M (CO) 3 } + (M = Tc/Re) core has garnered significant interest since its development by Jaouen (6) and Alberto and co-workers (7). This organometallic core offers advantages in terms of stability, kinetic inertness, and size. The lowspin d 6 -electron configuration and the stability of CO ligands to substitution protect the metal center from further ligand substitution and/or oxidation. For the above reasons there has been widespread interest in the development of targetspecific radiopharmaceuticals exploiting the {M (CO) 3 } + (Tc/Re) core. In neutral complexes with simple N, O donors the fac-[ 99m Tc (I)-(CO) 3 ] core possesses intermediate lipophilicity, an advantage in living systems (8). The [ 99m Tc (CO) 3 ] + carbonyl moiety is extremely interesting due to its high in-vitro and in-vivo stability when it is connected to various biomolecules. It has been reviewed in detail for its use in the second generation of single photon emission computed tomography (SPECT) radiopharmaceuticals (4,(8)(9)(10)(11)(12). Introduction of the Tc (I) can be achieved by convenient use of the [ 99m Tc(H 2 O) 3 (CO) 3 ] + complex ,which can be synthesized easily from a commercially available kit formulation (Isolink1, Mallinckrodt) following Alberto's method of synthesizing [ 99m Tc(CO) 3 (H 2 O) 3 ] + from [ 99m TcO 4 ]in aqueous solution (13).
Glucosamine (2-amino-2-deoxy-D-glucose) is a highly attractive scaffold for a glucosyl ligand, because the amine acts both as a potential coordination site and as a useful target for further functionalization. Furthermore, there is much evidence in the literature which suggest electron ionization (EI) in-positive mode. All radioactivity measurements were carried out using NaI (Tl) scintillation counter.
Ethylchloroformate (0.33 mL, 3 mmol) was added dropwise to an ice-cooled solution of (α,β)-2-amino-2-deoxy-D-glucose hydrochloride (0.5 g, 3.07 mmol) and sodium hydrogen carbonate (0.49 g, 5.83 mmol) in water (5 mL). The mixture was stirred in an ice bath for 2 h and then at room temperature overnight then dioxane (20-30 mL) was added to the mixture. The reaction mixture was filtered, and the solvent was removed. The product was purified by flash column chromatography on silica gel using CH 3 OH/ethyl acetate (2:8)

Synthesis of (α,β)-2-deoxy-2-amino(1,2dihydroxypropyl)-D-glucose (2)
3-Chloro-1,2-propandiol (0.049 mL, 0.5 mmol) was added to a solution of (α,β)-2amino-2-deoxy-D-glucose hydrochloride (1.0 g, 6.13 mmol) and powdered sodium carbonate (0.0934 g, 0.9 mmol) in water (10 mL), and then the mixture was heated at 110 ºC with stirring. After 10 h, the mixture was cooled to reach the room temperature, and ethanol (20-30 mL) was added. Then the reaction mixture was filtered, and ethanol was removed. The residue was extracted with ethyl acetate, and recrystallized in water yielded DHP-DG as a colorless crystal; Preparation of 99m Tc tricarbonyl precursor (3) Synthesis of [ 99m Tc (H 2 O) 3 (CO) 3 ] + complex was carried out according to the previously reported method (10). Complex 3 was prepared using a modification of the procedure described by Alberto et al. A 10 mL vial containing Na 2 CO 3 (5 mg), NaBH 4 (4 mg) and sodium potassium tartarate (10 mg) was capped with a rubber stopper and then flushed with a stream of CO gas (99.5%) at room temperature for 10 min. One milliliter of sodium pertechnetate (Na 99m TcO 4 ) with up to 20-100 mCi were added by a syringe and then heated to 75 °C for 30 min. After rapid cooling down to room temperature, 0.3 mL of 0.1 M HCl was added to decrease the pH (pH = 9.5-10). The radiochemical purity was > 98% which was determined by HPLC.

Stability studies
The radioactive [ 99m Tc]-ECB-DG and [ 99m Tc]-DHP-DG complexes were tested in-vitro in 1N physiological phosphate buffer (PBS, pH=7.4) and human serum at 37 °C for 3, 7, 10 and 24 h. The stability was assayed by monitoring the HPLC elution profile and determined the radiochemical purity after incubation at 37 until 24 h. To determine in-vitro serum stability, 150 μL of radiolabeled complexes were incubated with 1 mL human serum at 37 ºC for mentioned times. A 1 mL ethanol was added to the aforementioned solutions. The precipitates were separated by centrifugation and the supernatants were injected in HPLC to determine the stability of these complexes.

Results and Discussions
In the context of our general interest for the synthesis of glucosamine derivatives for molecular imaging (17)   with ethylchloroformate and 1, 2-propandiol moieties were accomplished in one step ( Figure 1). [ 99m Tc (H 2 O) 3 (CO) 3 ] + has proved to be an excellent agent for labeling different kind of ligands. It can be formed in high yield directly from generator eluted pertechnetate in aqueous solution. As three coordinated water are labile, they could be exchanged readily with a variety of mono-, bi-and tridentate ligands forming complexes. The major advantage of using the carbonyl precursor is that high specific activity labeling of biomolecules can be obtained. Labeling with 99m Tc 99m Tc (I) tricarbonyl precursor, complex 3, was successfully prepared with 98% radiolabeling using a modification of the procedure described by Alberto et al. The complex was stable (> 62%) for approximately    Maximum yields of labeling (~93%) could be achieved when the reactions were carried out at biological pH, but cleavage of the glucosamine moiety was observed when the labeling was carried out in alkaline condition. Although, we reached to better resolutions, but the expanding of the peaks may be took place because of the presence of α,β-anomeric carbons in compounds 1 and 2. However, these complexes were stabled for 7 hours in 1 N of PBS as well as human serum at 37 ºC (Table 1).

Biodistribution in mice
The animals were housed at The Animal Center of Nuclear Science & Technology Research Institute in Iran. Biodistribution experiments of injected radiolabeled sugar were performed on normal mice (25-35 g). Each animal was cited in specific container and then was anesthetized with ketamine/xylazine and injected intravenously with [ 99m Tc]-ECB-DG and [ 99m Tc]-DHP-DG complexes (0.3 mCi) in 0.1-0.3 mL of saline into a lateral tail vein. At 10, 30, and 60 min post-injection, blood samples were drawn by cardiac puncture, and the mice were sacrificed thereafter by cardiectomy while under ketamine/xylazine anesthesia. The organs of interest were then excised, blotted with tissue paper, weighed, and the radioactivity was counted. The percent of injected dose/ organ was determined by comparison of the tissue radioactivity with suitably diluted, known quantity aliquots of the injected dose.
Results of the biodistribution studies of these complexes are summarized in tables 2 and 3. [ 99m Tc]-ECB-DG and [ 99m Tc]-DHP-DG complexes showed brain and heart uptake (0.61%±0.09 and 1.16±0.10) at 60 min post-  injection respectively, an efficient clearance from the blood, a rapid excretion to the urine and a low retention in the liver and kidneys. Compared to the 18 F-FDG (25,26), [ 99m Tc]-ECB-DG displayed a 2.8-fold reduction in brain uptake (1.7 ± 0.2 versus 0.61% ± 0.09) whereas ,[ 99m Tc]-DHP-DG just showed 1.9-fold reduction in heart uptake (2.2 ± 0.05 towards 1.16±0.10) at 1 h postinjection. Therefore, these results indicate that ECB-DG and DHP-DG analogues could be used as brain and heart imaging agent respectively. Planar SPECT imaging of the [ 99m Tc]-ECB-DG complex was obtained at 2 h after administration of 4 in normal mouse (Figure 4). On the basis of SPECT image, it seems that this complex have a moderate brain uptake.

Conclusion
In summary, two new DG derivatives were synthesized and labeled with technetium-99mcarbonyl successfully. In our studies, [ 99m Tc]-ECB-DG and [ 99m Tc]-DHP-DG were synthesized and their labeling took place at biological pH. Low molecular weight accelerated clearance from blood, and different linkers and chelate cores changed the excretion path. [ 99m Tc]-ECB-DG shows moderate brain uptake (0.61%±0.09) and [ 99m Tc]-DHP-DG shows good heart uptake (1.16±0.10) at 1 h post-injection respectively. On the basis of our results, it seems that ECB-DG and DHP-DG analogues could be used as brain and heart imaging agents respectively. Related work is underway in our laboratory and will be reported in due course.