Synthesis and Biological Evaluation of Pyrazolo[1,5-a]-pyrimidine-containing 99mTc Nitrido Radiopharmaceuticals as Imaging Agents for Tumors

The compound 5-((2-aminoethylamino)methyl)-7-(4-bromoanilino)-3-cyano-pyrazolo[1,5-a]pyrimidine (ABCPP) was synthesized and conjugated with N-mercapto-acetylglycine (MAG), N-mercaptoacetylphenylalanine (MAF) and N-mercaptoacetylvaline (MAA), respectively. These three compounds were labeled successfully with [99mTcN]2+ intermediate in high radiochemical purities. Biodistribution in tumor-bearing mice demonstrated that the three new complexes showed tumor accumulation, high tumor-to-muscle (T/M) ratios and fast clearance from blood and muscle. Among them, the 99mTcN-MAG-ABCPP showed the most favorable characteristics, with tumor/blood and tumor/ muscle ratios reaching 1.51 and 2.97 at 30 min post-injection, 1.84 and 2.49 at 60 min post-injection, suggesting it could be further studied as potential tumor imaging agent for single photon emission computed tomography (SPECT).

The novel nitrido core [ 99m TcN] 2+ is isoelectronic with the [ 99m TcO] 3+ core. The nitrido ligand is a powerful -electron donor and shows a high capacity to stabilize the Tc(V) oxidation state [9]. The [ 99m TcN] 2+ core exhibits a very high chemical stability over a wide range of experimental conditions (such as oxidation-reduction reactions and pH variations), and high affinity toward chelating ligands containing sulfur atoms. The presence of the [ 99m TcN] 2+ core in the molecular structure of a radiopharmaceutical may dramatically affect its physical and biological behaviour [10]. A convenient preparation of 99m Tc nitrido complexes at the tracer level and in sterile and pyrogen-free conditions in which an intermediate 99m TcN complex of succinic dihydrazide (SDH) is initially formed in the presence of stannous chloride as reducing agent, has been extensively investigated [11]. So far, the use of the 3-cyano-5,7-disubstistituted pyrazolo [1,5-a]pyrimidine moiety in the preparation of 99m TcN complexes as targeted agents for tumor imaging has not been reported. Therefore, it may be of great interest to probe the 99m Tc nitrido chemistry with some ligands involving the ABCPP molecular structure to explore the biological behaviour of a potential new class of diagnostic agents.
The purpose of this study was to conjugate ABCPP with three chelating agents (MAG, MAF and MAA), and to evaluate the feasibility of the 99m Tc-labeled 3-cyanopyrazolo[1,5-a]pyrimidine derivatives as useful candidates for tumor imaging.

Chemistry
The preparation of N-[(Tritylmercapto)acetyl]glycine (Tr-MAG, 4a) was carried out using the procedure shown in Scheme 1. After protecting the thiol group with trityl chloride, the resulting compound 2 was reacted with N-hydroxysuccinimide (NHS) using dicyclohexylcarbodiimide (DCC) as condensation reagent to obtain the active ester 3. The active ester 3 was reacted with the amine group of glycine to provide the Tr-MAG (4a). N-[(Tritylmercapto)acetyl]phenylalanine (Tr-MAF, 4b) and N-[(tritylmercapto)acetyl]valine (Tr-MAA, 4c) were synthesized by the same procedure as 4a. Scheme 1. Synthesis of Tr-MAG, Tr-MAF and Tr-MAA.
The compound Tr-MAG-ABCPP was synthesized by conjugating Tr-MAG with ABCPP using 1hydroxybenzotriazole (HOBt) as nucleophilic catalyst and DCC as condensation agent. The reaction is shown schematically in Scheme 3. The thiol group was deprotected in trifluoroacetic acid (TFA) to give 13a. For labeling, 99m TcN-MAG-ABCPP was prepared through a SDH kit. [ 99m TcO 4 ]was reacted with SDH in the presence of stannous chloride as reducing agent to form a technetium-99m nitrido intermediate. The [ 99m TcN] 2+ is a suitable substrate for the substitution reaction with MAG-ABCPP at 100 ºC for 15 min to give the final complex 99m TcN-MAG-ABCPP (14a). 99m TcN-MAF-ABCPP (14b) and 99m TcN-MAA-ABCPP (14c) were prepared using the same method.  Figure 1. It was observed that the retention time of [ 99m TcN] 2+ int was 1.5 min, while those of 99m TcN-MAG-ABCPP, 99m TcN-MAF-ABCPP and 99m TcN-MAA-ABCPP were found to be 2.7 min, 3.6 min and 3.4 min, respectively. The radiochemical purity of the three products was over 90% immediately after the preparation.

Measurement of partition coefficients
The partition coefficients were determined by mixing the complex with an equal volume of 1octanol and phosphate buffer (0.025 mol/L, pH 7.4) in a centrifuge tube. The partition coefficients (logP) of 99m TcN-MAG-ABCPP, 99m TcN-MAF-ABCPP and 99m TcN-MAA-ABCPP are shown in Table 1. All of them were hydrophilic. 99m TcN-MAG-ABCPP was the most hydrophilic one and 99m TcN-MAA-ABCPP was more hydrophilic than 99m TcN-MAF-ABCPP.

In vitro stability study
As the HPLC analysis results for the three complexes indicate, they were all stable in PBS after incubation for 2 h.

Biodistribution study
The biodistribution results are summarized in Tables 2-4. There were significant similarities in the biodistribution patterns of these three complexes, which all demonstrated tumor accumulation and high tumor-to-muscle (T/M) ratios. The blood and muscle clearance was faster than that of the tumor so that the tumor/blood and tumor/muscle ratios increased with time. For 99m TcN-MAG-ABCPP, the tumor/blood and tumor/muscle ratios reached 1.51 and 2.97 at 30 min post-injection, 1.84 and 2.49 at 60 min post-injection. For 99m TcN-MAF-ABCPP, the tumor/blood and tumor/muscle ratios reached 0.51 and 2.56 at 30 min post-injection. For 99m TcN-MAA-ABCPP, the tumor/blood and tumor/muscle ratios reached 0.72 and 2.32 at 30 min post-injection. Early hepatic and renal activity reflected the fact that the three complexes were excreted through the hepatobiliary as well as the renal system. The three complexes were hydrophilic, so that they were unable to cross the blood brain barrier, thus making their brain uptake much lower. Among them, 99m TcN-MAG-ABCPP showed the most favorable characteristics with highest uptake at the tumor site and fast clearance from blood and muscle. T/M = tumor-to-muscle, T/B = tumor-to-blood; a All data are the mean percentage (n = 3) of the injected dose of 99m TcN-MAG-ABCPP; per gram of tissue, ± the standard deviation of the mean.

General
99 Mo/ 99m Tc generator was obtained from the China Institute of Atomic Energy (CIAE). All other chemicals were of analytical grade and were used without further purification. 1 H-and 13 C-NMR spectra were recorded in CDCl 3 or DMSO-d 6 on a Bruker spectrometer operating at 400 and 100 MHz, respectively. The IR spectra were recorded on a Nicolet-AVATAR 360 FT-IR spectrometer using KBr pellets in the 4,000-400 cm -1 region. ESI-MS were performed on Waters LCT Premier XE. HPLC analyses were performed on a Shimadzu SCL-10 AVP equipped with a Packard 500 TR series flow scintillation analyzer. A C-18 reversed-phase Alltima column (5um, 150 mm × 4.6 mm) was used for radiochemical purity analysis.

Radiochemistry
The tritylated compound 12a (5 mg) was treated with trifluoroacetic acid under cation trapping conditions (5% triethylsilane) at room temperature. After removing the solvent under a stream of nitrogen, the residue was neutralized with 0.1 M NaOH. The solution was extracted with dichloromethane, and the aqueous phase was under nitrogen protection. Saline containing [ 99m TcO 4 ] -(1 mL, 15MBq) was added to a kit containing stannous chloride dihydrate(0.05 mg), succinic dihydrazide (SDH, 5.0 mg) and propylenediamine tetraacetic acid (PDTA, 5.0 mg). The mixture was kept at room temperature for 15 min. Next, the MAG-ABPP solution (1 mL) was added and the reaction allowed to stand for 15 min at 100 ºC to give the final complex 99m TcN-MAG-ABCPP (14a). 99m TcN-MAF-ABCPP (14b) and 99m TcN-MAA-ABCPP (14c) were prepared using the same method. Formation of the complexes and nature of the species formed was determined by Radio-HPLC analysis. Solvent system used for elution was water (solvent A) and acetonitrile (solvent B). The HPLC gradient system for analysis of the product started with 100% A/0% B with a linear gradient to 0% A/100% B from 0 to 30 min. The flow rate was 1.0 mL/min. Five microliters of the sample was used for analysis. Recovery was determined by summing the total counts in all fractions and comparing them to the total injected activity.

Measurement of partition coefficients
The partition coefficients of the three complexes were determined according to the published method [16] by mixing each complex with an equal-volume of 1-octanol and phosphate buffer (0.025 M, pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5,000 rpm for 5 min. From each phase, 0.1 mL of the aliquot was pipetted and counted in a well-type NaI(Tl) detector. The measurement was repeated three times. Care was taken to avoid cross contamination between the phases. Usually the final partition coefficient value was expressed as log P, where the partition coefficient, P, was calculated using the following equation: P = (cps in octanol-cps in background)/(cps in buffer-cps in background)

In vitro stability study
The stability of the complexes was studied by measuring the radiochemical purity using HPLC analysis at different time intervals after preparation. The complexes were added to test tubes containing PBS solution (0.025 M, pH 7.4). The mixtures were incubated by shaking them at 37 ºC in a thermomixer. The radiochemical purity was measured at 15 min, 30 min, 60 min and 120 min by Radio-HPLC.

Biodistribution study
Biodistribution studies were performed in Kunming female mice (weighing 18-20 g) bearing S 180 tumors, which grew to a leg diameter of 10-15 mm. 99m TcN-MAG-ABCPP, 99m TcN-MAF-ABCPP and 99m TcN-MAA-ABCPP (0.1 mL, 7.4 × 10 5 Bq) were injected via a tail vein and the injected radioactivity was measured with a well-type NaI(Tl) detector, respectively. The mice were sacrificed at 5 min, 30 min, 60 min and 120 min post-injection. The tumor, other organs of interest and blood were collected, weighed and measured for radioactivity. The results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g). All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation.

Conclusion
In summary, the new 3-cyanopyrazolo[1,5-a]pyrimidine ABCPP was successfully synthesized and conjugated with MAG, MAF and MAA, respectively. The three compounds were labeled with 99m Tc in high radiochemical purities through a ligand exchange reaction, which can be easily used for the preparation of a radiopharmaceutical through a freeze-dried kit formulation. The three complexes demonstrated tumor accumulation, high tumor-to-muscle (T/M) ratios and fast clearance from blood and muscle. Among them, 99m TcN-MAG-ABCPP showed the most favorable characteristics and could be further studied as potential tumor imaging agent.