Improving the (18)F-fluoromethylcholine ((18)F-FCH) radiochemical yield via optimising the azeotropic drying of non-carrier-added (18)F-fluorine.

(18)F-Fluoromethylcholine ((18)F-FCH) has been suggested as one of the reputable imaging tracers for diagnosis of prostate tumour in PET/CT examination. Nevertheless, it has never been synthesised in Malaysia. We acknowledged the major problem with (18)F-FCH is due to its relatively low radiochemical yield at the end of synthesis (EOS). Therefore, this technical note presents improved (18)F-FCH radiochemical yields after carrying out optimisation on azeotropic drying of non-carrier-added (18)F-Fluorine.

Synthetic approach for unsaturated precursors for parahydrogen induced polarization of choline and its analogs.

Reported here are (i) a new synthetic approach for preparation of (ii) a new compound class, of -OH, for example, an -OH group is replaced with acetyl protecting group, protected 1,2-dehydrocholine analogs and (iii) a new synthetic route for betaine aldehyde. The CC bond of 1,2-dehydrocholine moiety can be used for molecular addition of parahydrogen producing -OH protected hyperpolarized choline by parahydrogen-induced polarization (PHIP). The reported synthetic approach allows for incorporation of (15) N and deuterium labels, which are necessary for preparation of highly polarized PHIP contrast agents. Isotope labeling with (15) N and/or deuterium was conducted. Hyperpolarized (15) N-choline enabled by the reported synthetic approach can be potentially used as an imaging biomarker of cancer similar to choline positron emission tomography tracers.

15N magnetic resonance hyperpolarization via the reaction of parahydrogen with 15N-propargylcholine.

(15)N-Propargylcholine has been synthesized and hydrogenated with para-H(2). Through the application of a field cycling procedure, parahydrogen spin order is transferred to the (15)N resonance. Among the different isomers formed upon hydrogenation of (15)N-propargylcholine, only the nontransposed derivative contributes to the observed N-15 enhanced emission signal. The parahydrogen-induced polarization factor is about 3000. The precise identification of the isomer responsible for the observed (15)N enhancement has been attained through a retro-INEPT ((15)N-(1)H) experiment. T(1) of the hyperpolarized (15)N resonance has been estimated to be ca. 150 s, i.e., similar to that reported for the parent propargylcholine (144 s). Experimental results are accompanied by theoretical calculations that stress the role of scalar coupling constants (J(HN) and J(HH)) and of the field dependence in the formation of the observed (15)N polarized signal. Insights into the good cellular uptake of the compound have been gained.

Design, synthesis and biological evaluation of choline based SPECT imaging agent: Ga(III)-DO3A-EA-Choline.

The enhanced choline uptake and phosphorylation in tumor cells has motivated the development of radiolabeled choline derivatives as diagnostic markers for imaging cell membrane proliferation and noninvasive detection of prostate, brain and breast tumors. In the present work, we report a facile strategy for the synthesis of choline functionalized macrocyclic chelating agent (DO3A-EA-choline) and its radiocomplexation with (67)Ga for potential tumor imaging applications. The synthesis of the desired compound featured quaternization of N,N-dimethylaminoethanol with 1,2-dibromoethane followed by subsequent alkylation with trisubstituted cyclen (DO3A). All intermediates and final compounds have been fully characterized by spectroscopic techniques, namely, (1)H, (13)C NMR and mass spectroscopy. The compound has been successively labeled with (67)Ga-citrate in ammonium acetate buffer (pH 6.5) at 80 °C. MTT assays have been performed on the HEK cell line to determine the cytotoxicity of the compound. Cell uptake studies carried out on the U-87 MG cell line exhibited saturable binding of the radioconjugate in picomolar range with a K(d) value of 0.528 pM. The in vivo biodistribution and blood kinetics studies exhibited rapid clearance of the radiolabeled complex and excretion through the renal and hepatobiliary route. The present studies demonstrate the potential applications of (67)Ga-DO3A-EA-choline as a radiopharmaceutical for molecular imaging using ((67/68)Ga) SPECT and PET modalities.

[Synthesis of seven sinapine analogs and their effects on the tension of smooth muscle of intestines isolated from rabbit].

Seven sinapine analogs (6a-6g) were synthesized using cinnamon acid or benzoic acid and their derivatives as starting materials, which obtained from substituted benzaldehyde and malonate. The structures of target compounds were characterized by IR, 1H NMR and elemental analysis. The effects of compounds 6a-6g on the smooth muscle of intestine isolated from rabbit were studied, and the experimental results showed that compounds 6a, 6d and 6g had diastolic action, while 6f had contractile action.

Reduced dimethylaminoethanol in [(18)F]fluoromethylcholine: an important step towards enhanced tumour visualization.

PURPOSE: [(18)F]Fluoromethylcholine ([(18)F]FCho) is a radiotracer generally used for tumour visualization in patients. Due to high levels of dimethylaminoethanol (DMAE) remaining in [(18)F]FCho solutions synthesized by currently available methods, tumour visualization might be compromised. METHODS: An improved purification method involving an optimized purification step for reducing the levels of DMAE was conceived. The physiological explanation for the interference of residual DMAE in [(18)F]FCho pharmacokinetics was further elaborated in a xenograft mouse model. RESULTS: The use of a series of polymer solid-phase extraction cartridges (Oasis HLB/WCX), instead of the commonly used combination of tC18 and Accell CM cartridges, reduced DMAE levels from 402.2±49.6 ppm to 3.0±0.5 ppm. Subsequent in vitro tests proved that (1) [(18)F]FCho uptake was reduced in the presence of DMAE at concentrations above 0.5 µM and (2) DMAE is a competitive inhibitor of [(18)F]FCho transport. In vivo experiments in xenograft mouse models corroborated reduced tumour uptake at DMAE plasma levels of about 2.5 µM as found in patients injected with contaminated [(18)F]FCho. CONCLUSION: Residual DMAE, even at levels below choline plasma concentrations found during fasting, compromises [(18)F]FCho uptake in vivo and care should be taken to avoid its interference in molecular imaging with [(18)F]FCho.

Sustainable Straightforward Synthesis and Evaluation of the Antioxidant and Antimicrobial Activity of Sinapine and Analogues.

Naturally occurring sinapine was successfully synthesized through a proline-mediated Knoevenagel-Doebner condensation in ethanol. This synthetic process involving biobased syringaldehyde, Meldrum's acid, and choline chloride offers a sustainable alternative to the existing low-yield pathways. This two-step strategy gives access to sinapine in a 52% overall yield and has been implemented in the synthesis of sinapine analogues, using 4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, and vanillin as precursors, giving target molecules with 34-61% overall isolated yields. The purity of synthetic sinapine and its analogues (ca. 95%) was assessed by NMR and high-performance liquid chromatography-mass spectrometry analyses. Furthermore, the antioxidant and antimicrobial activities were assessed, and the potential of this series of molecules was confirmed.

Assessment of false transmitters as treatments for nerve agent poisoning.

Nerve agents inhibit acetylcholinesterase (AChE), leading to a build-up of acetylcholine (ACh) and overstimulation at cholinergic synapses. Current post-exposure nerve agent treatment includes atropine to treat overstimulation at muscarinic synapses, a benzodiazepine anti-convulsant, and an oxime to restore the function of AChE. Aside from the oxime, the components do not act directly to reduce the overstimulation at nicotinic synapses. The false transmitters acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) are analogs of ACh, synthesised similarly at synapses. AMECh and ADECh are partial agonists, with reduced activity compared to ACh, so it was hypothesised the false transmitters could reduce overstimulation. Synthetic routes to AMECh and ADECh, and their precursors, monoethylcholine (MECh) and diethylcholine (DECh), were devised, allowing them to be produced easily on a laboratory-scale. The mechanism of action of the false transmitters was investigated in vitro. AMECh acted as a partial agonist at human muscarinic (M1 and M3) and muscle-type nicotinic receptors, and ADECh was a partial agonist only at certain muscarinic subtypes. Their precursors acted as antagonists at muscle-type nicotinic, but not muscarinic receptors. Administration of MECh and DECh improved neuromuscular function in the soman-exposed guinea-pig hemi-diaphragm preparation. False transmitters may therefore help reduce nerve agent induced overstimulation at cholinergic synapses.

GO-META-TiO2 composite monolithic columns for in-tube solid-phase microextraction of phosphopeptides.

A novel composite monolithic column based on graphene oxide-trimethyl-2-methacroyloxyethylammonium chloride-titania (GO-META-TiO2) was developed for the enrichment of phosphopeptides. META was proposed as a "bridge" to connect GO and TiO2 species to prepare GO-META-TiO2 composite. This high surface area composite (surface area = 196.93 m2 g-1) was fixed in the monolithic column via an in situ UV polymerization process. In-tube solid phase microextraction (IT-SPME) using this composite was coupled with matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) for the enrichment and detection of phosphorylated peptides from a digestion mixture of α-casein, ß-casein, and bovine serum albumin (BSA) in molar ratios of 1:1:1, 1:1:10, and 1:1:100. The key factors affecting the IT-SPME of the phosphopeptides, such as the elution solution concentrations, the extraction flow rate, and the elution flow rate were comprehensively investigated. For further demonstration, this method was employed for the enrichment and detection of phosphorylated peptides from digested chicken egg white. The obtained results indicated that the GO-META-TiO2 composite monolithic column rapidly and efficiently captured the phosphopeptides present in these complex biological samples, even in the 10 fmol ß-casein tryptic digest. We therefore propose that the reported GO-META-TiO2 composite monolithic column possesses a suitable affinity for the selective extraction of phosphopeptides from biological samples. This method paves a way in extending the application of nanomaterials.

Fully automated [18F]fluorocholine synthesis in the TracerLab MX FDG Coincidence synthesizer.

INTRODUCTION: We developed a new fully automated method for the radiosynthesis of [18F]fluorocholine by modifying the commercial 2-[18F]fluoro-2-d-deoxy-glucose ([18F]FDG) synthesizer module (GE TracerLab MX, formerly Coincidence). METHODS: [18F]Flurocholine was synthesized by (18)F-fluoroalkylation of N,N-dimethylaminoethanol using [18F]fluorobromomethane as fluoromethylating agent. [18F]Fluorobromomethane was produced by reaction of dibromomethane with [18F]fluoride, assisted by Kryptofix 2.2.2. RESULTS: After purification on solid-phase extraction cartridges, the [18F]fluorocholine was obtained in 15-25% radiochemical yields (decay not corrected), with more than 99% radiochemical purity. Specific activity was more than 37 GBq/micromol. Synthesis time was less than 35 min. CONCLUSION: This new automated synthesis technique provides high and reproducible yields that could be dedicated for routine use with the same [18F]FDG disposable cassette system.

Simplified fast and high yielding automated synthesis of [18F]fluoroethylcholine for prostate cancer imaging.

(11)C- and (18)F-labeled choline analogues are successful tracers for prostate cancer imaging using positron emission tomography (PET). Due to the practical advantages of the longer-living radioisotope (18)F (t(1/2)=110 min) instead of (11)C (t(1/2)=20 min), [(18)F]fluoroethylcholine has been introduced to increase the opportunity of widespread clinical application. Nevertheless, the various known synthetic methods provide [(18)F]fluoroethylcholine for human use only in moderate overall yields of up to 30% so far. Here, a new simplified and high yield two-step-synthesis for [(18)F]fluoroethylcholine is described for potential clinical applications starting from 2-bromoethyl triflate (BETfO) using a modified, commercially available fully automated synthesis module. All synthesis parameters were subsequently optimized resulting in a total yield of 47+/-5% (not decay corrected) in only 40min. [(18)F]fluoroethylcholine could be obtained ready for human use as physiological solution after fixation on Sep-Pak Accell Light cartridges (waters((R))) and formulation with saline without the need of GC or HPLC purification. Radiochemical purity was >99.9% and no contamination of the sterile solution with chemicals used during the synthesis was detected.

alpha,beta-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase.

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84-93%. The potency of the amino acid ester derivatives was tested by measuring K(i) values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH(3)O(C(6)H(4))CONHC(CHC(6)H(5))COOCH(2)CH(2)N(+)(CH(3))(3). This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K(i) values of 10 microM and 0.08 microM, respectively. These K(i) values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 A from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.

A simple Tracerlab module modification for automated on-column [11C]methylation and [11C]carboxylation.

A modification of commercial [11C]methylation module which can be implemented for both on-column [11C]methylation and [11C]carboxylation in the same automated system is described. This module configuration was applied to the solid-phase synthesis of N-[11C]methyl-choline ([11C]choline) and L-(S-methyl-[11C])methionine ([11C]methionine), using [11C]CH(3)I as methylating agent, as well as to the synthesis of [11C]acetate by [11C]carboxylation with [11C]CO2 of methylmagnesium chloride with high and reproducible radiochemical yields in short reaction time, demonstrating to be a fast and reliable tool for the production of these [11C]radiopharmaceuticals for clinical use.

Specific and spatial labeling of choline-containing teichoic acids in Streptococcus pneumoniae by click chemistry.

Propargyl-choline was efficiently incorporated into teichoic acid (TA) polymers on the surface of Streptococcus pneumoniae. The introduction of a fluorophore by click chemistry enabled sufficient labeling of the pneumococcus, as well as its specific detection when mixed with other bacterial species. The labeling is localized at the septal site, suggesting a similar location of the TA and peptidoglycan (PG) synthetic machineries. This method is a unique opportunity to improve our understanding of the spatial location of pneumococcal TA biosynthesis.

Double-Mannich annulation of cyclic ketones using N,N-Bis(ethoxymethyl)alkylamine reagents.

[Structure: see text] N,N-Bis(ethoxymethyl)alkylamines function as effective reagents in the double-Mannich annulation of cyclic ketone substrates, providing efficient access to a series of azabicyclic ketones. These ring systems are a useful scaffold for the four-step synthesis of novel constrained homocholine analogues.

Simple automated system for simultaneous production of 11C-labeled tracers by solid supported methylation.

We herein describe a simple setup for the automated simultaneous synthesis of L-[methyl-11C]methionine and N-[methyl-11C]choline by solid-supported methylation. The setup is extremely simple and easy to adapt to other automated systems and due to its versatility, the method can be utilized for the production of other radiopharmaceuticals requiring a simple [11C]methylation step. Furthermore, it can be used for multiple simultaneous synthesis.

Convenient and Efficient Method for Quality Control Analysis of 18F-Fluorocholine: For a Small Scale GMP-based Radiopharmaceuticals Laboratory Set-up.

BACKGROUND AND OBJECTIVE: Prostate cancer continues to be the most prevalent cancer in men in Malaysia. As time progresses, the prospect of PET imaging modality in diagnosis of prostate cancer is promising, with on-going improvement on novel tracers. Among all tracers, 18F-Fluorocholine is reported to be a reputable tracer and reliable diagnostic technique for prostate imaging. Nonetheless, only 18F-Fluorodeoxyglucose (18F-FDG) is available and used in most oncology cases in Malaysia. With a small scale GMP-based radiopharmaceuticals laboratory set-up, initial efforts have been taken to put Malaysia on 18F-Fluorocholine map. This article presents a convenient, efficient and reliable method for quality control analysis of 18F-Fluorocholine. Besides, the aim of this research work is to assist local GMP radiopharmaceuticals laboratories and local authority in Malaysia for quality control analysis of 18F-Fluorocholine guideline. METHODS: In this study, prior to synthesis, quality control analysis method for 18F-Fluorocholine was developed and validated, by adapting the equipment set-up used in 18F-Fluorodeoxyglucose (18FFDG) routine production. Quality control on the 18F-Fluorocholine was performed by means of pH, radionuclidic identity, radio-high performance liquid chromatography equipped with ultraviolet, radio- thin layer chromatography, gas chromatography and filter integrity test. RESULTS: Post-synthesis; the pH of 18F-Fluorocholine was 6.42 ± 0.04, with half-life of 109.5 minutes (n = 12). The radiochemical purity was consistently higher than 99%, both in radio-high performance liquid chromatography equipped with ultraviolet (r-HPLC; SCX column, 0.25 M NaH2PO4: acetonitrile) and radio-thin layer chromatography method (r-TLC). The calculated relative retention time (RRT) in r-HPLC was 1.02, whereas the retention factor (Rf) in r-TLC was 0.64. Potential impurities from 18F-Fluorocholine synthesis such as ethanol, acetonitrile, dimethylethanolamine and dibromomethane were determined in gas chromatography. Using our parameters, (capillary column: DB-200, 30 m x 0.53 mm x 1 um) and oven temperature of 35°C (isothermal), all compounds were well resolved and eluted within 3 minutes. Level of ethanol and acetonitrile in 18F-Fluorocholine were detected below threshold limit; less than 5 mg/ml and 0.41 mg/ml respectively. Meanwhile, dimethylethanolamine and dibromomethane were undetectable. CONCLUSION: A convenient, efficient and reliable quality control analysis work-up procedure for 18FFluorocholine has been established and validated to comply all the release criteria. The convenient method of quality control analysis may provide a guideline to local GMP radiopharmaceutical laboratories to start producing 18F-Fluorocholine as a tracer for prostate cancer imaging.

Optimising the Azeotropic Drying of 18F-Fluorine Wayto Improve the 18F-Fluorocholine Radiochemical Yield.

BACKGROUND AND OBJECTIVE: 18F-Fluorocholine has been suggested as one of the reputable imaging tracers for diagnosis of prostate tumour in Positron Emission Tomography / Computed Tomography (PET/CT) modality. Nevertheless, it has never been synthesised in Malaysia. We acknowledged that the major problem with 18F-Fluorocholine is due to its relatively low radiochemical yield at the end of synthesis (EOS). Therefore, this article presents improved 18FFluorocholine radiochemical yields after carrying out optimisation on azeotropic drying of 18F-Fluorine. METHODS: In the previous study, the azeotropic drying of non-carrier-added (n.c.a) 18F-Fluorine in the reactor was conducted at atmospheric pressure (0 atm) and shorter duration time. In this study, however, the azeotropic drying of non-carried-added (n.c.a) 18FFluorine was made at a high vacuum pressure (- 0.65 to - 0.85 bar) with an additional time of 30 seconds. At the end of the synthesis, the mean radiochemical yield was statistically compared between the two azeotropic drying conditions so as to observe whether the improvement made was significant to the radiochemical yield. RESULTS: From the paired sample t-test analysis, the improvement done to the azeotropic drying of non-carrier-added (n.c.a) 18F-Fluorine was statistically significant (p < 0.05). With the improvement made, the 18F-Fluorcholine radiochemical yield was found to have increase by one fold. CONCLUSION: Improved 18F-Fluorocholine radiochemical yields were obtained after the improvement had been done to the azeotropic drying of non-carrier-added (n.c.a) 18F-Fluorine. It was also observed that improvement made to the azeotropic drying of non-carrier-added (n.c.a) 18F-Fluorine did not affect the 18F-Fluorocholine quality control analysis.

The quest for biocompatible phthalocyanines for molecular imaging: Photophysics, relaxometry and cytotoxicity studies.

Water soluble phthalocyanines bearing either four PEG500 or four choline substituents in the macrocyclic structure, as well as their Zn(II) and Mn(III) complexes were synthesized. The metal-free and Zn(II) complexes present relatively high fluorescence quantum yields (up to 0.30), while the Mn(III) complexes show no fluorescence as a consequence of rapid non-radiative deactivation of the Mn(III) phthalocyanine excited states through low-lying metal based or charge-transfer states. The effect of DMSO on the aggregation of the phthalocyanines was studied. It was not possible to obtain the Mn(II) complexes by reduction of the corresponding Mn(III) complexes due to the presence of electron donating substituents at the periphery of the phthalocyanines. The (1)H NMRD plots of the PEG500 and choline substituted Mn(III)-phthalocyanine complexes are typical of self-aggregated Mn(III) systems with r1 relaxivities of 4.0 and 5.7mM(-1)s(-1) at 20MHz and 25°C. The Mn(III)-phthalocyanine-PEG4 complex shows no significant cytotoxicity to HeLa cell cultures after 2h of incubation up to 2mM concentration. After 24h of cell exposure to the compound, significant toxicity was observed for all the concentrations tested with IC50 of 1.105mM.

Novel N-allyl/propargyl tetrahydroquinolines: Synthesis via Three-component Cationic Imino Diels-Alder Reaction, Binding Prediction, and Evaluation as Cholinesterase Inhibitors.

New N-allyl/propargyl 4-substituted 1,2,3,4-tetrahydroquinolines derivatives were efficiently synthesized using acid-catalyzed three components cationic imino Diels-Alder reaction (70-95%). All compounds were tested in vitro as dual acetylcholinesterase and butyryl-cholinesterase inhibitors and their potential binding modes, and affinity, were predicted by molecular docking and binding free energy calculations (∆G) respectively. The compound 4af (IC50 = 72 µm) presented the most effective inhibition against acetylcholinesterase despite its poor selectivity (SI = 2), while the best inhibitory activity on butyryl-cholinesterase was exhibited by compound 4ae (IC50 = 25.58 µm) with considerable selectivity (SI = 0.15). Molecular docking studies indicated that the most active compounds fit in the reported acetylcholinesterase and butyryl-cholinesterase active sites. Moreover, our computational data indicated a high correlation between the calculated ∆G and the experimental activity values in both targets.