Carbon-11 N-methyl alkylation of L-368,899 and in vivo PET imaging investigations for neural oxytocin receptors

Compound L-368,899 was successfully alkylated with [¹¹C]iodomethane to generate the oxytocin receptor selective (2R)-2-amino-N-((2S)-7,7-dimethyl-1-(((4-(o-tolyl)piperazin-1-yl)sulfonyl)methyl)bicyclo[2.2.1]heptan-2-yl)-N-[¹¹C]methyl-3-(methylsulfonyl)propanamide ([¹¹C]1) with very high radiochemical purity and high specific activity. PET imaging studies were performed with [¹¹C]1 to investigate brain penetration and oxytocin receptor uptake using rat and cynomolgus monkey models. For rat baseline scans, brain penetration was observed with [¹¹C]1, but no specific uptake could be distinguished in the brain region. By administering a peptide oxytocin receptor selective antagonist for peripheral blocking of oxytocin receptors, the uptake of [¹¹C]1 was amplified in the rat brain temporarily to enable some visual uptake within the rat brain. A baseline scan of [¹¹C]1 in a cynomolgus monkey model resulted in no detectable specific uptake in anticipated regions, but activity did accumulate in the choroid plexus.     

Trichodiene biosynthesis and the stereochemistry of the enzymatic cyclization of farnesyl pyrophosphate

Cyclization of trans,trans-[1-³H₂,12,13-¹⁴C]farnesyl pyrophosphate (2a) by a preparation of trichodiene synthetase isolated from the fungus, Trichothecium roseum, gave trichodiene (5a), which was shown by chemical degradation to retain both tritium atoms of the precursor at C-11. Incubation of 1S-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2b) and 1R-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2c) with trichodiene synthetase and degradation of the resulting labeled trichodienes, 5b and 5c, established that the displacement of the pyrophosphate moiety from C-1 of the precursor and formation of the new C-C bond in the formation of trichodiene takes place with net retention of configuration. These results are accounted for by an isomerization-cyclization mechanism involving the intermediacy of nerolidyl pyrophosphate (4).     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Synthesis and biological evaluation of 11C-labeled β-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled β-galactosyl triazoles 1-(β-d-galactopyranosyl)-4-(p-[¹¹C]methoxyphenyl)-1,2,3-triazole ([¹¹C]-6) and 1-(β-d-galactopyranosyl)-4-(6-[¹¹C]methoxynaphthyl)-1,2,3-triazole ([¹¹C]-13). The precursors for the radiolabeling and the non-radioactive analogues (6 and 13) were synthesized using straightforward ‘click’ chemistry. In vitro incubation experiments of 6 with β-galactosidase in the presence of o-nitrophenyl β-d-galactopyranoside (ONPG) showed that the triazolic compound was an inhibitor of β-galactosidase activity. Radiolabeling of both precursors was performed using [¹¹C]methyl iodide as alkylating agent at 70 °C in DMF in the presence of a small amount of base. The log P values were ?0.1 and 1.4, respectively, for [¹¹C]-6 and [¹¹C]-13, the latter therefore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in normal mice showed a good clearance from blood for both tracers. [¹¹C]-6 was mainly cleared via the renal pathway, while the more lipophilic [¹¹C]-13 was excreted almost exclusively via the hepatobiliary system. Despite the lipophilicity of [¹¹C]-13, no brain uptake was observed. Reversed phase HPLC analysis of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-293T cells showed an increased cell uptake for the more lipophilic [¹¹C]-13, however, there was no statistically higher uptake in LacZ expressing cells compared to control cells.     

Synthesis of carbon-11-labeled 5-HT6R antagonists as new candidate PET radioligands for imaging of Alzheimer’s disease

Carbon-11-labeled serotonin (5-hydroxytryptamine) 6 receptor (5-HT₆R) antagonists, 1-[(2-bromophenyl)sulfonyl]-5-[¹¹C]methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (O-[¹¹C]2a) and 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-[¹¹C]methyl-1-piperazinyl)methyl]-1H-indole (N-[¹¹C]2a), 5-[¹¹C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1-(phenylsulfonyl)-1H-indole (O-[¹¹C]2b) and 5-methoxy-3-((4-[¹¹C]methylpiperazin-1-yl)methyl)-1-(phenylsulfonyl)-1H-indole (N-[¹¹C]2b), 1-((4-isopropylphenyl)sulfonyl)-5-[¹¹C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1H-indole (O-[¹¹C]2c) and 1-((4-isopropylphenyl)sulfonyl)-5-methoxy-3-((4-[¹¹C]methylpiperazin-1-yl)methyl)-1H-indole (N-[¹¹C]2c), 1-((4-fluorophenyl)sulfonyl)-5-[¹¹C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1H-indole (O-[¹¹C]2d) and 1-((4-fluorophenyl)sulfonyl)-5-methoxy-3-((4-[¹¹C]methylpiperazin-1-yl)methyl)-1H-indole (N-[¹¹C]2d), were prepared from their O- or N-desmethylated precursors with [¹¹C]CH₃OTf through O- or N-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370–740?GBq/μmol with a total synthesis time of ～40-min from EOB.     

Radiosynthesis of a carbon-11-labeled AMPAR allosteric modulator as a new PET radioligand candidate for imaging of Alzheimer’s disease

To develop PET tracers for imaging of Alzheimer’s disease, a new carbon-11-labeled AMPAR allosteric modulator 4-cyclopropyl-7-(3-[¹¹C]methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide ([¹¹C]8) has been synthesized. The reference standard 4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (8) and its corresponding desmethylated precursor 4-cyclopropyl-7-(3-hydroxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (9) were synthesized from 4-methoxyabiline and chlorosulfonyl isocyanate in eight and nine steps with 3% and 1% overall chemical yield, respectively. The target tracer [¹¹C]8 was prepared from the precursor 9 with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 10–15% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (A_M) at EOB was 370–740?GBq/μmol with a total synthesis time of 35–40-minutes from EOB.     

Facile synthesis of carbon-11-labeled sEH/PDE4 dual inhibitors as new potential PET agents for imaging of sEH/PDE4 enzymes in neuroinflammation

To develop PET tracers for imaging of neuroinflammation, new carbon-11-labeled sEH/PDE4 dual inhibitors have been synthesized. The reference standard N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (1) and its corresponding desmethylated precursor N-(4-hydroxy-2-(trifluoromethyl)benzyl)benzamide (2) were synthesized from (4-methoxy-2-(trifluoromethyl)phenyl)methanamine and benzoic acid in one and two steps with 84% and 49% overall chemical yield, respectively. The standard N-(4-methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (MPPA, 4) and its precursor N-(4-hydroxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (5) were synthesized from methyl 4-piperidinecarboxylate, propionyl chloride and (4-methoxy-2-(trifluoromethyl)phenyl)methanamine in two and three steps with 62% and 34% overall chemical yield, respectively. The target tracers N-(4-[¹¹C]methoxy-2-(trifluoromethyl)benzyl)benzamide ([¹¹C]1) and N-(4-[¹¹C]methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide ([¹¹C]MPPA, [¹¹C]4) were prepared from their corresponding precursors 2 and 5 with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 25–35% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (A_M) at EOB was 370–740 GBq/μmol with a total synthesis time of 35–40-minutes from EOB.     

Synthesis and in vivo evaluation of [11C]tariquidar,a positron emission tomography radiotracer based on a third-generation P-glycoprotein inhibitor

The aim of this study was to develop a positron emission tomography (PET) tracer based on the dual P-glycoprotein (P-gp) breast cancer resistance protein (BCRP) inhibitor tariquidar (1) to study the interaction of 1 with P-gp and BCRP in the blood–brain barrier (BBB) in vivo. O-Desmethyl-1 was synthesized and reacted with [¹¹C]methyl triflate to afford [¹¹C]-1. Small-animal PET imaging of [¹¹C]-1 was performed in naïve rats, before and after administration of unlabeled 1 (15 mg/kg, n = 3) or the dual P-gp/BCRP inhibitor elacridar (5 mg/kg, n = 2), as well as in wild-type, Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice (n = 3). In vitro autoradiography was performed with [¹¹C]-1 using brain sections of all four mouse types, with and without co-incubation with unlabeled 1 or elacridar (1 μM). In PET experiments in rats, administration of unlabeled 1 or elacridar increased brain activity uptake by a factor of 3–4, whereas blood activity levels remained unchanged. In Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice, brain-to-blood ratios of activity at 25 min after tracer injection were 3.4, 1.8 and 14.5 times higher, respectively, as compared to wild-type animals. Autoradiography showed approximately 50% less [¹¹C]-1 binding in transporter knockout mice compared to wild-type mice and significant displacement by unlabeled elacridar in wild-type and Mdr1a/b^(?/?) mouse brains. Our data suggest that [¹¹C]-1 interacts specifically with P-gp and BCRP in the BBB. However, further investigations are needed to assess if [¹¹C]-1 behaves in vivo as a transported or a non-transported inhibitor.     

Four analogues of spiroleptosphol isolated from Leptosphaeria doliolum

Spiroleptosphol B (2), spiroleptosphol C (3), norleptosphol C (4) and hydroleptosphol (5) were isolated from ascomycete Leptosphaeria doliolum. Detailed ¹H and ¹³C NMR spectral analyses revealed these were structural analogues of spiroleptosphol (1) which we have recently isolated from the same fungi. Spiroleptosphol B (2) carried an unprecedent 5,3-dioxatricyclo[4.4.0.1^1.4]undecane framework in place of the spirobicyclo ring system of 1. Spiroleptosphol C (3) was a 17-(R)-hydroxy derivative of 1. Norleptosphol C (4) was deduced to be the monocyclic structure biosynthetically resulted by decarboxylation from 3. Although 5 gave broaden ¹H NMR spectrum, it was gradually transformed to 2 which suggested being a hydrolysate of 1.     

Synthesis,in vitro and in vivo evaluation of [11C]MMTP: A potential PET ligand for mGluR1 receptors

Synthesis, in vitro and in vivo evaluation of [O-methyl-¹¹C]dimethylamino-3(4-methoxyphenyl)-3H-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-one (1), a potential imaging agent for mGluR1 receptors using PET are described. Synthesis of the corresponding desmethyl precursor 2 was achieved by demethylation of the methoxyphenyl compound 1 in 90% yield. Methylation using [¹¹C]MeOTf in presence of NaOH afforded [¹¹C]1 in 30% yield (EOS) with >99% chemical and radiochemical purities and with a specific activity of 3–5 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. The radiotracer selectively labeled mGluR1 receptors in slide-mounted sections of postmortem human brain containing cerebellum, hippocampus, prefrontal cortex and striatum as demonstrated by in vitro autoradiography using phosphor-imaging. PET studies in anesthetized baboon show that [¹¹C]1 penetrates the BBB and accumulates in cerebellum, a region reported to have higher expression of mGluR1. These findings suggest [¹¹C]1 is a promising PET radiotracer candidate for mGluR1.     

Radiosynthesis of novel carbon-11-labeled triaryl ligands for cannabinoid-type 2 receptor

Two novel triaryl ligands 2 and 5 with potent in vitro binding affinities for the cannabinoid subtype-2 (CB2) receptor were labeled with a positron-emitting radioactive nuclide ¹¹C. Radioligands [¹¹C]2, [¹¹C]5, and their analogs [¹¹C]3 and [¹¹C]4 were synthesized by O-[¹¹C]methylation of their corresponding phenol precursors with [¹¹C]CH₃I. [¹¹C]2–5 had relatively high uptakes (>1.2% injected dose/g tissue) in mouse brains.     

Characteristic lindenane sesquiterpenoid dimers and labdane diterpenoids from Chloranthus serratus

Chemical investigation on the roots of Chloranthus serratus (Chloranthaceae) afforded 11 terpenoids, including four lindenane-type sesquiterpenoid dimers (1–4), and seven labdane diterpenoids (5–11). The structures of these terpenoids were established by spectroscopic analysis (MS, ¹H, and ¹³C NMR). Compounds 4–11 were isolated from C. serratus for the first time. The chemotaxonomic significance of the isolated compounds was summarized herein.     

Synthesis of l-(4-2H)erythrose,l-)1-13C, 5-2H)arabinose and l-(2-13C, 5-2H)arabinose and identification of the intermediates by 2H and 13C-N.M.R. spectroscopy

l-(1-¹³C, 5-²H)Arabinose (6D) and l-(2-¹³C, 5-²H)arabinose (8D) have been synthesized by degradation of 2,3-O-isopropylidene-α-l-rhamnofuranose (2) to l-(4-²H)erythrose (5β,5αD), with subsequent chain elongation to 6D plus l-(1-¹³C, 5-²H)ribose (7D), the latter being converted into 8D. Intermediates were identified by complete assignment of the ¹³C chemical shifts employing carbon-carbon and carbon-deuterium coupling constants, deuteration shifts, differential isotope-shifts, and deuterium spectra. The anomeric carbon atoms of 2 and 2,3-O-isopropylidene-l-(1-²H) erythrose (4D) gave only single ¹³C resonances, suggesting that these two compounds exists in only one major anomeric configuration, clarifying previously reported work. The synthesis of 2,3-O-isopropylidene-l-(1-²H)rhmanitol (3D) facilitated the assignment of the signals in the ¹³C spectra of the nondeuterated analog. Specific deuterium-enrichment and the observed carbon-deuterium coupling (¹J_C,D ∼22 Hz) not only served to identify the deuterated carbon atom unambiguously in 3 but also permitted assignment of closely spaced resonances. The deuterium spectrum of 2,3-O-isopropylidene-l-(1-²H)erythrofuranose (4D) showed only a single resonance, indicating preponderance of one anomer, in accord with the observation of a single C-1 resonance in the ¹³C spectrum.     

Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide molecule with twin-nonyl chain

A series of cationic lipo-benzamide compounds with varying lengths of hydrocarbon chains (C2M–C18M) were evaluated for anti-Candida activity. Four compounds harbouring 8–11 hydrocarbon chains demonstrated concentration-dependent inhibition of fungal cell growth with Minimum Inhibitory Concentration (MIC) of ≤6.2?µg?ml^?1. The most active compound (C9M) inhibited growth of both Candida albicans and non-albicans strains and is equally active against pairs of azole sensitive and resistant clinical isolates of C. albicans. Compound C9M also inhibited different stages of Candida biofilms. Scanning Electron Microscopy (SEM) of Candida cells after C9M treatment was also done and no significant cell lysis was observed. Hemolysis assay was performed and only 2.5% haemolysis was observed at MIC concentration.     

N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide: A promising positron emission tomography ligand for fatty acid amide hydrolase

To visualize fatty acid amide hydrolase (FAAH) in brain in vivo, we developed a novel positron emission tomography (PET) ligand N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[¹¹C]methylphenyl)thiazol-2-yl]-1-carboxamide ([¹¹C]DFMC, [¹¹C]1). DFMC (1) was shown to have high binding affinity (IC₅₀: 6.1 nM) for FAAH. [¹¹C]1 was synthesized by C–¹¹C coupling reaction of arylboronic ester 2 with [¹¹C]methyl iodide in the presence of Pd catalyst. At the end of synthesis, [¹¹C]1 was obtained with a radiochemical yield of 20 ± 10% (based on [¹¹C]CO₂, decay-corrected, n = 5) and specific activity of 48–166 GBq/μmol. After the injection of [¹¹C]1 in mice, high uptake of radioactivity (>2% ID/g) was distributed in the lung, liver, kidney, and brain, organs with high FAAH expression. PET images of rat brains for [¹¹C]1 revealed high uptakes in the cerebellar nucleus (SUV = 2.4) and frontal cortex (SUV = 2.0), two known brain regions with high FAAH expression. Pretreatment with the FAAH-selective inhibitor URB597 reduced the brain uptake. Higher than 90% of the total radioactivity in the rat brain was irreversible at 30 min after the radioligand injection. The present results indicate that [¹¹C]1 is a promising PET ligand for imaging of FAAH in living brain.     

Miltoncostaea marina gen. nov. sp. nov., and Miltoncostaea oceani sp. nov., a novel deep branching phylogenetic lineage within the class Thermoleophilia isolated from marine environments,and proposal of Miltoncostaeaceae fam. nov. and Miltoncostaeales ord. nov

Two novel marine actinobacteria, designated as SCSIO 60955^T and SCSIO 61214^T, were isolated from deep-sea sediment samples collected from the South China Sea. The cells of these organisms stained Gram-negative and were rod shaped. These strains were aerobic, and catalase- and oxidase-positive. Optimal growth occurred at 28 °C and pH 7 over 14 days of cultivation. Both strains possessed phospholipids and phosphoglycolipids. The main menaquinone was MK-7. The major fatty acid was C_16:0. The peptidoglycan structure was type A1γ′ (meso-Dpm). Analysis of genome sequences revealed that the genome size of SCSIO 60955^T was 3.37 Mbp with G + C content of 76.1%, while the genome size of SCSIO 61214^T was 3.67 Mbp with a G + C content of 74.8%. The ANI and 16S rRNA gene analysis results showed that the pairwise similarities between the two strains were 73.4% and 97.7% and that with other recognized Thermoleophilia species were less than 69.1% and 87.8%, respectively. Phylogenetic analysis of the 16S rRNA gene sequences showed that strains SCSIO 60955^T and SCSIO 61214^T were separately clustered together and formed a well-separated phylogenetic branch distinct from their most related neighbor Gaiella occulta. Based on the data presented here, these two strains are proposed to represent two novel species of a novel genus, for which the name Miltoncostaea marina gen. nov., sp. nov., with the type strain SCSIO 60955^T (=DSM 110281^T =CGMCC 1.18757^T), and Miltoncostaea oceani sp. nov., with the type strain SCSIO 61214^T (=KCTC 49527^T =CGMCC 1.18758^T) are proposed. We also propose that these organisms represent a novel family named Miltoncostaeaceae fam. nov. of a novel order Miltoncostaeales ord. nov.     

Trichloroamine complexes of platinum: preparation,crystal structure and solution behavior of cytosinium trichlorocytosineplatinate(II)

A complex containing a protonated and N3-platinated cytosine (C), [CH][Cl₃Pt(C)] (1a) has been prepared, converted into its K[Cl₃Pt(C)] (1b) and NH₄[Cl₃]Pt(C)]·H₂O (1c) analogs, and structurally characterized (X-ray, Raman, NMR). Reaction of 1b with L = 1-methylcytosine and with L = Me₂SO gave the neutral mixed-ligand complexes cis-Cl₂Pt(C)L. Excess NH₃ was used to convert the anion of 1b into the cation [(NH₃)₃Pt(C)]²⁺ (3a). The pK_a of the N(1)H proton in 3a is 9.4, as determined by UV spectroscopy. The N(1)H is displaced by Pt(II) electrophiles even at neutral pH to give N3,N1-diplatinated cytosinato complexes, as shown by ¹H NMR (³J coupling or ¹⁹⁵Pt at N(1) with H6, 29 Hz, and ⁴J coupling of ¹⁹⁵Pt at N(3) with H5, 14Hz). The results of the X-ray structure determination of 1a (R = 0.031, R_w = 0.034) are of relevance in that they permit a direct comparison of the effect of a proton as opposed to that of a Pt electrophile on the nucleobase geometry. Moreover, the expected decrease in CO(2) bond length as a consequence of Pt binding is observed.     

Inhibition of sterol biosynthesis and accumulation of 2,3-oxidosqualene in bramble cell suspension cultures treated with 2-aza-2,3-dihydro-squalene and 2-aza-2,3-dihydrosqualene-N-oxide

2-Aza-2,3-dihydrosqualene (1), 2-aza-2,3-dihydro-squalene-N-oxide (2) and derivatives are new compounds designed to inhibit the 2,3-oxidosqualene-cycloartenol (lanosterol) cyclase. The effects of these compounds were studied on sterol biosynthesis in suspension cultures of bramble cells. Both 1 and 2 inhibited the growth of cells with an IC₅₀ of 11 μM for 1 and 21 μM for 2. When the cells grown in the presence of the two drugs were analysed, accumulation of squalene and 2,3-oxidosqualene was observed but no significant decrease of the total sterol content per g of dry weight of cells was noticed. Pulse experiments with [2-¹⁴C]acetate on 15-day-old cells treated with 1 resulted in a strong decrease of the incorporation of radioactivity into the 4-desmethyl sterol fraction. An IC₅₀ of 7.5 μM was determined when the cells were preincubated for a period of two hr with 1 or 2. This inhibition was correlated with an accumulation of [¹⁴C]-2,3-oxidosqualene and of [¹⁴C]-squalene. No [¹⁴C]-2,3:22(23)-dioxidosqualene was detectable in these conditions. Derivatives of 1 and 2 or similar compounds were also assayed; N-lauryl-dimethylamino-N-oxide (LDAO) was shown to be particularly effective and produced accumulation of enormous amounts of [¹⁴C]-2,3-oxidosqualene. Compound 1 (but not 2 or LDAO) leads also to the accumulation of a red pigment identified as lycopene. Our work confirms studies with enzymatic systems in demonstrating that 1, 2 and LDAO inhibit the 2,3-oxidosqualene-cycloartenol cyclase and provides evidence that the squalene synthetase and the Δ⁸ → Δ⁷-sterol isomerase are also inhibited.     

Radiosynthesis and evaluation of a novel monoacylglycerol lipase radiotracer: 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[11C]carboxylate

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, the inhibition of MAGL is an attractive therapeutic target for neurodegenerative diseases. In this study, to visualize MAGL via positron emission tomography (PET), we report a new carbon-11-labeled radiotracer, namely 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[¹¹C]carboxylate ([¹¹C]6). Compound 6 exhibited high in vitro binding affinity (IC₅₀ = 0.41 nM) to MAGL in the brain with a suitable lipophilicity (cLogD = 3.29). [¹¹C]6 was synthesized by reacting 1,1,1,3,3,3-hexafluoropropanol (7) with [¹¹C]phosgene ([¹¹C]COCl₂), followed by a reaction with 3-(1-benzyl-1H-pyrazol-3-yl)azetidine hydrochloride (8), which resulted in a 15.0 ± 6.8% radiochemical yield (decay-corrected, n = 7) based on [¹¹C]CO₂ and a 45 min synthesis time from the end of bombardment. A biodistribution study in mice showed high uptake of radioactivity in MAGL-rich organs, including the lungs, heart, and kidneys. More than 90% of the total radioactivity was irreversibly bound in the brain homogenate of rats 5 min and 30 min after the radiotracer injection. PET summation images of rat brains showed high radioactivity in all brain regions. Pretreatment with 6 or MAGL-selective inhibitor JW642 significantly reduced the uptake of radioactivity in the brain. [¹¹C]6 is a promising PET tracer which offers in vivo specific binding and selectivity for MAGL in rodent brains.     

Synthesis and preliminary biological evaluation of [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for the fractalkine receptor (CX3CR1)

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate (5) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucine (6) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [¹¹C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate ([¹¹C]5) was prepared from the acid precursor with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ～40-min from EOB. The radioligand depletion experiment of [¹¹C]5 did not display specific binding to CX₃CR1, and the competitive binding assay of ligand 5 found much lower CX₃CR1 binding affinity.