Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors.

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

Antiinflammation Derived Suzuki-Coupled Fenbufens as COX-2 Inhibitors: Minilibrary Construction and Bioassay.

A small fenbufen library comprising 18 compounds was prepared via Suzuki Miyara coupling. The five-step preparations deliver 9-17% biphenyl compounds in total yield. These fenbufen analogs exert insignificant activity against the IL-1 release as well as inhibiting cyclooxygenase 2 considerably. Both the para-amino and para-hydroxy mono substituents display the most substantial COX-2 inhibition, particularly the latter one showing a comparable activity as celecoxib. The most COX-2 selective and bioactive disubstituted compound encompasses one electron-withdrawing methyl and one electron-donating fluoro groups in one arene. COX-2 is selective but not COX-2 to bioactive compounds that contain both two electron-withdrawing groups; disubstituted analogs with both resonance-formable electron-donating dihydroxy groups display high COX-2 activity but inferior COX-2 selectivity. In silico simulation and modeling for three COX-2 active-p-fluoro, p-hydroxy and p-amino-fenbufens show a preferable docking to COX-2 than COX-1. The most stabilization by the p-hydroxy fenbufen with COX-2 predicted by theoretical simulation is consistent with its prominent COX-2 inhibition resulting from experiments.

Fluorine-18 isotope labeling for positron emission tomography imaging. Direct evidence for DBPR211 as a peripherally restricted CB1 inverse agonist.

The [18F] isotope-labelled CB1 inverse agonist 3 was elaborated and synthesized for positron emission tomography scanning studies. After immediate purification and calibration with its unlabeled counterpart, compound 3 was intravenously injected in mice and revealed that its distribution percentage in brain over 90-min scans among five region of interests, including brain, liver, heart, thigh muscle and kidney was lower than 1%, thus providing direct evidence to justify itself as a peripherally restricted CB1 antagonist.

Synthesis and Biological Evaluation of an (18)Fluorine-Labeled COX Inhibitor--[(18)F]Fluorooctyl Fenbufen Amide--For Imaging of Brain Tumors.

Molecular imaging of brain tumors remains a great challenge, despite the advances made in imaging technology. An anti-inflammatory compound may be a useful tool for this purpose because there is evidence of inflammatory processes in brain tumor micro-environments. Fluorooctylfenbufen amide (FOFA) was prepared from 8-chlorooctanol via treatment with potassium phthalimide, tosylation with Ts2O, fluorination with KF under phase transfer catalyzed conditions, deprotection using aqueous hydrazine, and coupling with fenbufen. The corresponding radiofluoro product [(18)F]FOFA, had a final radiochemical yield of 2.81 mCi and was prepared from activated [(18)F]F(-) (212 mCi) via HPLC purification and concentration. The radiochemical purity was determined to be 99%, and the specific activity was shown to exceed 22 GBq/µmol (EOS) based on decay-corrected calculations. Ex-vivo analysis of [(18)F]FOFA in plasma using HPLC showed that the agent had a half-life of 15 min. PET scanning showed significant accumulation of [(18)F]FOFA over tumor loci with reasonable contrast in C6-glioma bearing rats. These results suggest that this molecule is a promising agent for the visualization of brain tumors. Further investigations should focus on tumor micro-environments.

Recent Development of Radiofluorination of Boron Agents for Boron Neutron Capture Therapy of Tumor: Creation of 18F-Labeled C-F and B-F Linkages.

Boron neutron capture therapy (BNCT) is a binary therapeutic technique employing a boron agent to be delivered to the tumor site followed by the irradiation of neutrons. Biofunctional molecules/nanoparticles labeled with F-18 can provide an initial pharmacokinetic profile of patients to guide the subsequent treatment planning procedure of BNCT. Borono phenylalanine (BPA), recognized by the l-type amino acid transporter, can cross the blood-brain barrier and be accumulated in gliomas. The radiofluoro BNCT agents are reviewed by considering (1) less cytotoxicity, (2) diagnosing and therapeutic purposes, (3) aqueous solubility and extraction route, as well as (4), the trifluoroborate effect. A trifluoroborate-containing amino acid such as fluoroboronotyrosine (FBY) represents an example with both functionalities of imaging and therapeutics. Comparing with the insignificant cytotoxicity of clinical BPA with IC50 > 500 µM, FBY also shows minute toxicity with IC50 > 500 µM. [18F]FBY is a potential diagnostic agent for its tumor to normal accumulation (T/N) ratio, which ranges from 2.3 to 24.5 from positron emission tomography, whereas the T/N ratio of FBPA is greater than 2.5. Additionally, in serving as a BNCT therapeutic agent, the boron concentration of FBY accumulated in gliomas remains uncertain. The solubility of 3-BPA is better than that of BPA, as evidenced by the cerebral dose of 3.4%ID/g vs. 2.2%ID/g, respectively. While the extraction route of d-BPA differs from that of BPA, an impressive T/N ratio of 6.9 vs. 1.5 is noted. [18F]FBPA, the most common clinical boron agent, facilitates the application of BPA in clinical BNCT. In addition to [18F]FBY, [18F] trifluoroborated nucleoside analog obtained through 1,3-dipolar cycloaddition shows marked tumoral uptake of 1.5%ID/g. Other examples using electrophilic and nucleophilic fluorination on the boron compounds are also reviewed, including diboronopinacolone phenylalanine and nonsteroidal anti-inflammatory agents.

Synthesis and evaluation of [18F]Fluorobutyl ethacrynic amide: a potential PET tracer for studying glutathione transferase.

[(18)F]Flurobutyl ethacrynic amide ([(18)F]FBuEA) was prepared from the precursor tosylate N-Boc-N-[4-(toluenesulfonyloxy)butyl]ethacrynic amide with a radiochemical yield of 3%, a specific activity of 48 GBq/µmol and radiochemical purity of 98%. Chemical conjugation of [(18)F]FBuEA with glutathione (GSH) via a self-coupling reaction and enzymatic conjugation under catalysis of glutathiontransferase alpha (GST-α) and π provided about 41% yields of radiochemical conjugated product [(18)F]FBuEA-GSH, 85% and 5-16%, respectively. The catalytic selectivity of this tracer toward GST-alpha was addressed. Positron emission tomography (PET) imaging of [(18)F]FBuEA in normal rats showed that a homogeneous pattern of radioactivity was distributed in the liver, suggesting a catalytic role of GST. By contrast, PET images of [(18)F]FBuEA in rats with thioacetamide-induced cholangiocarcinoma displayed a heterogeneous pattern of radioactive accumulation with cold spots in tumor lesions. PET imaging with [(18)F]FBuEA could be used for early diagnosis of hepatic tumor with a low GST activity as well as liver function.

Synthesis of amino core compounds of galactosyl phytosyl ceramide analogs for developing iNKT-cell inducers.

1-Aminophytosphingosine and 6-aminogalactosyl phytosphingosine were prepared in 61% and 40% yield libraries with 44 carboxylic acids showed that a 4-butylbenzoic acid-derived product exe, respectively. Glycosylation using benzoyl-protected lipid resulted in better a-selectivity for ceramide analogs, but the yield was less than that obtained with benzyl moieties. Screening the amide rted less cytotoxicity. These analogs were purified for validation of immunological potencies and the a-GalCer analog but not the sphingosine analog stimulated human iNKT cell population.

Solution-phase parallel synthesis and screening of anti-tumor activities from fenbufen and ethacrynic acid libraries.

The derivatives with fenbufen and ethacrynic acid core compounds was synthesized through a facial preparation of 1-amino-4-azidobutane. The subsequent coupling with 102 members of carboxylic acids afforded amide products. The in situ screening using colorimetric assay with 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide showed that fenbufen but not ethacrynic acid butyl amide members displayed the cytotoxicities to tumor cells substantially, including two human cell lines (MCF7 and A549) and two murine cell lines (C26 and TRAMP-C1). Three fenbufen analogs were found to have a good anti-tumor activity comparable to cisplatin.

Synthesis, Radiolabeling, and Preliminary in vivo Evaluation of [68Ga] IPCAT-NOTA as an Imaging Agent for Dopamine Transporter.

INTRODUCTION: Novel radiotracer development for imaging dopamine transporters is a subject of interest because although [99mTc]TRODAT-1, [123I]ß-CIT, and [123I]FP-CIT are commercially available; 99Mo/99mTc generator is in short supply and 123I production is highly dependent on compact cyclotron. Therefore, we designed a novel positron emission tomography (PET) tracer based on a tropane derivative through C-2 modification to conjugate NOTA for chelating 68Ga, a radioisotope derived from a 68Ge/68Ga generator. METHODS: IPCAT-NOTA 22 was synthesized and labeled with [68Ga]GaCl4 - at room temperature. Biological studies on serum stability, LogP, and in vitro autoradiography (binding assay and competitive assay) were performed. Furthermore, ex vivo autoradiography, biodistribution, and dynamic PET imaging studies were performed in Sprague Dawley rats. RESULTS: [68Ga]IPCAT-NOTA 24 obtained had a radiochemical yield of ≥90% and a specific activity of 4.25 MBq/nmol. [68Ga]IPCAT-NOTA 24 of 85% radiochemical purity (RCP%) was stable at 37°C for up to 60 minutes in serum with a lipophilicity of 0.88. The specific binding ratio (SBR%) reached 15.8 ± 6.7 at 60 minutes, and the 85% specific uptake could be blocked through co-injection at 100- and 1000-fold of the cold precursor in in vitro binding studies. Tissue regional distribution studies in rats with [68Ga]IPCAT-NOTA 24 showed striatal uptake (0.02% at 5 minutes and 0.007% at 60 minutes) with SBR% of 6%, 25%, and 62% at 5-15, 30-40, and 60-70 minutes, respectively, in NanoPET studies. The RCP% of [68Ga]IPCAT-NOTA 24 at 30 minutes in vivo remained 67.65%. CONCLUSION: Data described here provide new information on the design of PET probe of conjugate/pendent approach for DAT imaging. Another chelator or another direct method of intracranial injection must be used to prove the relation between [68Ga]IPCAT-NOTA 24 uptake and transporter localization.

Synthesis and structure-activity relationships of fenbufen amide analogs.

The previous discoveries of butyl fenbufen amide analogs with antitumor effects were further examined. The amide analogs with 1, 3, 4 and 8 carbons chains were prepared in 70-80% yield. Fenbufen had no cytotoxic effects at concentrations ranging from 10 to 100 µM. Methyl fenbufen amide had significant cytotoxic effects at a concentration of 100 µM. As the length of the alkyl amide side chain increased, the cytotoxic effects increased, and the octyl fenbufen amide had the greatest cytotoxic effect. After treatment with 30 µM octyl fenbufen amide, nearly seventy percent of the cells lost their viability. At the concentration of 10 µM, fenbufen amide analogs did not show cytotoxicity according to the MTT assay results. The NO scavenging activities of the fenbufen amide analogs were not significantly different from those of fenbufen.

Boron Accumulation in Brain Tumor Cells through Boc-Protected Tryptophan as a Carrier for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a binary therapeutic approach. Nonradioactive boron-10 atoms accumulated in tumor cells combining with the neutron beams produce two highly energetic particles that could eradicate the cell that takes it and the neighboring cells. Small molecules that carry boron atom, e.g. 5- and 6-boronated and 2,7-diboronated tryptophans, were assessed for their boron accumulation in U87-MG, LN229, and 3T3 for BNCT. TriBoc tryptophan, TB-6-BT, shows boron-10 at 300 ppm in both types of tumor cells with a tumor to normal ratio (T/N) of 5.19-5.25 (4 h). TB-5-BT and DBA-5-BT show boron-10 at 300 ppm (2 h) in U87-MG cells. TB-5-BT exerts a T/N of >9.66 (1 h) in LN229 compared with the current clinical boronophenyl alanine with a highest T/N of 2.3 (1 h) and accumulation concentration of <50 ppm. TB-5-BT and TB-6-BT warrant further animal study.

Synthesis and evaluation of ortho-[18F] fluorocelecoxib for COX-2 cholangiocarcinoma imaging.

BACKGROUND: An 18F-tagged NSAID analog was prepared for use as a probe for COX-2 expression, which is associated with tumor development. METHODS: The in vivo uptake of celecoxib was monitored with ortho-[18F]fluorocelecoxib using positron emission tomography (PET). The binding affinity of ortho-[18F]fluorocelecoxib to COX-1 and COX-2 enzymes were assessed using the competitor celecoxib. RESULTS: The IC50 values were 0.039 µM and 0.024 µM, respectively. A selectivity index of 1.63 was obtained (COX-2 vs COX-1). COX-2 overexpressed cholangiocarcinoma (CCA) murine cells took up more ortho-[18F]fluorocelecoxib than that by usual CCA cells from 10 to 60 minutes post incubation. Competitive inhibition (blocking) of the tracer uptake of ortho-[18F]fluorocelecoxib in the presence of celecoxib by the COX-2 overexpressed CCA cells and the usual CCA cells gave the IC50 values of 0.5 µM and 46.5 µM, respectively. Based on the in vitro accumulation data and in vivo metabolism half-life (30 min), PET scanning was performed 30-60 min after the administration of ortho-[18F]fluorocelecoxib through the tail vein. Study of ortho-[18F]F-celecoxib in the CCA rats showed a tumor to normal ratio (T/N) of 1.38±0.23 and uptake dose of 1.14±0.25 (%ID/g). CONCLUSION: The inferior in vivo blocking results of 1.48±0.20 (T/N) and 1.18±0.22 (%ID/g) suggests that the nonspecificity is associated with the complex role of peroxidase or the binding to carbonic anhydrase.

Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM.

[123I]IBZM is used widely for in vivo imaging of D2 receptors in human brain and shows relatively fast kinetics and a greater susceptibility to synaptic dopamine release than other single-photon emission computed tomography (SPECT) radioligands. A reliable and reversed-phase HPLC method using UV/VIS and radiometric detectors has been developed for qualitative analysis of BZM and IBZM and radiochemical purity in [123I]IBZM preparations. The method uses gradient elution on a Zorbax XDB C-18 column with a mobile phase that consists of 10mM 3,3-dimethylglutaric acid (DMGA), pH 7.0 and acetonitrile (ACN). The flow rate was 1.0mL/min with detection at λ=254nm. The method was validated for system suitability, precision, accuracy, specificity, linearity, robustness, limit of detection (LOD) and limit of quantification (LOQ), as described in ICH guidelines. The results are described as follows: (1) The system suitability includes the tailing factor, theoretical plate number and resolution, which are 0.962, 10656.11 and 9.367, respectively. (2) For specificity, the BZM and [123I]NH4I did not interfere with the retention time of the [123I]IBZM. (3) The percentage coefficient of variation for analysis of precision, including repeatability and intermediate precision, is less than 2.0%. (4) Accuracy of the method is within the range of 85-100%. (5) The range of linearity is from 100% to 70% radiochemical purity (%RCP) of [123I]IBZM, with the correlation coefficient (R) always being above 0.995. (6) The data of method robustness are within acceptance criteria. (7) The LOD and LOQ for impurity (BZM) are 0.145 and 0.50µg/mL, respectively. All of the analysis results demonstrate that this method is sensitive, specific and suitable for routine analysis of the radiochemical purity in [123I]IBZM preparations.

Synthesis and characterization of boron fenbufen and its F-18 labeled homolog for boron neutron capture therapy of COX-2 overexpressed cholangiocarcinoma.

Boron neutron capture therapy (BNCT) is a binary therapy that employs neutron irradiation on the boron agents to release high-energy helium and alpha particles to kill cancer cells. An optimal response to BNCT depends critically on the time point of maximal 10B accumulation and highest tumor to normal ratio (T/N) for performing the neutron irradiation. The aggressive cholangiocarcinoma (CCA) representing a liver cancer that overexpresses COX-2 enzyme is aimed to be targeted by COX-2 selective boron carrier, fenbufen boronopinacol (FBPin). Two main works were performed including: 1) chemical synthesis of FBPin as the boron carrier and 2) radiochemical labeling with F-18 to provide the radiofluoro congener, m-[18F]fluorofenbufen ester boronopinacol (m-[18F]FFBPin), to assess the binding affinity, cellular accumulation level and distribution profile in CCA rats. FBPin was prepared from bromofenbufen via 3 steps with 82% yield. The binding assay employed [18F]FFBPin to compete FBPin for binding to COX-1 (IC50=0.91±0.68µM) and COX-2 (IC50=0.33±0.24µM). [18F]FFBPin-derived 60-min dynamic PET scans predict the 10B-accumulation of 0.8-1.2ppm in liver and 1.2-1.8ppm in tumor and tumor to normal ratio=1.38±0.12. BNCT was performed 40-55min post intravenous administration of FBPin (20-30mg) in the CCA rats. CCA rats treated with BNCT display more tumor reduction than that by NCT with respect of 2-[18F]fluoro-2-deoxy glucose uptake in the tumor region of interest, 20.83±3.00% (n=12) vs. 12.83±3.79% (n=10), P=0.05. The visualizing agent [18F]FFBPin resembles FBPin to generate the time-dependent boron concentration profile. Optimal neutron irradiation period is thus determinable for BNCT. A boron-substituted agent based on COX-2-binding features has been prepared. The moderate COX-2/COX-1 selectivity index of 2.78 allows a fair tumor selectivity index of 1.38 with a mild cardiovascular effect. The therapeutic effect from FBPin with BNCT warrants a proper COX-2 targeting of boron NSAIDs.

Dataset on the synthesis and characterization of boron fenbufen and its F-18 labeled homolog.

The data presented in this article are related to the research article entitled "Synthesis and Characterization of Boron Fenbufen and its F-18 Labeled Homolog for Boron Neutron Capture Therapy of COX-2 Overexpressed Cholangiocarcinoma". The contents of the data article include 1) the set up for performing in vitro binding assay, 2) 1H-, 13C- and 19F-NMR of compounds described in main text, 3) HPLC chromatogram of the fluorination mixtures, 4) data of in vitro stability test, cell survival assay, western blot and PCR analysis, 5) the modules for fixing the two CCA rats for BNCT, and 6) bar diagram for tumor reduction using [18F]FDG-PET 24 h post treatment with BNCT.

Synthesis of 5-radioiodoarabinosyl uridine analog for probing HSV-1 thymidine kinase gene: an unexpected chelating effect.

Tumor cells transduced with herpes simplex virus thymidine kinase gene has been intensively applied to the field of positron emission tomography via imaging of its substrate. As a pilot synthesis approach, a facial preparation of 5-[125I]iodoarabinosyl uridine starting from commercial available uridine is reported herein. Interestingly, the tin group in 5-trimethylstannyl arabinosyluridine was easily removed during purification. The destannylation through the formation of a six-ligand coordination involving 2'-hydroxyl and tin was thereby proposed.

Investigation of brain tumors using (18)F-fluorobutyl ethacrynic amide and its metabolite with positron emission tomography.

To date, imaging of malignant glioma remains challenging. In positron emission tomography-related diagnostic imaging, differential tumor uptake of 3'-deoxy-3'-[(18)F] fluorothymidine ([(18)F]FLT) has been shown to reflect the levels of cell proliferation and DNA synthesis. However, additional biomarkers for tumors are urgently required. Aberrant levels of glutathione transferase (GST) activity have been hypothesized to constitute such a novel diagnostic marker. Here, a C6 rat glioma tumor model was used to assess the ability of the positron emission tomography tracers, [(18)F]FLT and (18)F-fluorobutyl ethacrynic amide ([(18)F]FBuEA), to indicate reactive oxygen species-induced stress responses as well as detoxification-related processes in tumors. Using a GST activity assay, we were able to demonstrate that FBuEA is more readily catalyzed by GST-π than by GST-α. Furthermore, we showed that FBuEA-GS, a metabolite of FBuEA, elicits greater cytotoxicity in tumor cells than in normal fibroblast cells. Finally, in vitro and in vivo investigation of radiotracer distribution of [(18)F]FBuEA and [(18)F] FBuEA-GS revealed preferential accumulation in C6 glioma tumor cells over normal fibroblast cells for [(18)F]FBuEA-GS but not for [(18)F]FBuEA.

Identification of SPHK1 as a therapeutic target and marker of poor prognosis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by a uniquely aggressive behavior and lack of effective targeted therapies. After analyzing the gene expression profiles of seven paired intrahepatic CCA microarrays, a novel sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) pathway and a novel target gene, SPHK1, were identified. We hypothesized that therapeutic targeting of this pathway can be used to kill intrahepatic cholangiocarcinoma (CCA) cells. High levels of SPHK1 protein expression, which was evaluated by immunohistochemical staining of samples from 96 patients with intrahepatic CCA, correlated with poor overall survival. The SPHK1 inhibitor SK1-I demonstrated potent antiproliferative activity in vitro and in vivo. SK1-I modulated the balance of ceramide-sphinogosine-S1P and induced CCA apoptosis. Furthermore, SK1-I combined with JTE013, an antagonist of the predominant S1P receptor S1PR2, inhibited the AKT and ERK signaling pathways in CCA cells. Our preclinical data suggest SPHK1/S1P pathway targeting may be an effective treatment option for patients with CCA.

Synthesis of a novel aldehyde: 4-O-methyl-5-formylmethyl-2'-deoxyuridine.

The synthesis of the blocked nucleoside 3',5'-di-O-p-toluoyl-4-O-methyl-5-formylmethyl-2'-deoxyuridine was accomplishied in eleven steps from gamma-butyrolactone. This aldehyde, which should facilitate the synthesis of nucleosides containing 18F. was converted to the corresponding blocked dithianyl nucleoside, and also to 5-(2,2-difluoroethyl)-substituted derivatives of 2'-deoxyuridine and 2'-deoxycytidine.