Synthesis and evaluation of novel potent TSPO PET ligands with 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide

Translocator protein (TSPO) expression is closely related with neuroinflammation and neuronal damage which might cause several central nervous system diseases. Herein, a series of TSPO ligands (11a–c and 13a–d) with a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide structure were prepared and evaluated via an in vitro binding assay. Most of the novel ligands exhibited a nano-molar affinity for TSPO, which was better than that of DPA-714. Particularly, 11a exhibited a subnano-molar TSPO binding affinity with suitable lipophilicity for in vivo brain studies. After radiolabeling with fluorine-18, [¹⁸F]11a was used for a dynamic positron emission tomography (PET) study in a rat LPS-induced neuroinflammation model; the inflammatory lesion was clearly visualized with a superior target-to-background ratio compared to [¹⁸F]DPA-714. An immunohistochemical examination of the dissected brains confirmed that the uptake location of [¹⁸F]11a in the PET study was consistent with a positively activated microglia region. This study proved that [¹⁸F]11a could be employed as a potential PET tracer for detecting neuroinflammation and could give possibility for diagnosis of other diseases, such as cancers related with TSPO expression.     

[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-dimethyl phenylamino)pyrazin-2(1H)-one: Introduction of N3-phenylpyrazinones as potential CRF-R1 PET imaging agents

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [¹⁸F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

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.     

Structure-activity relationship study towards non-peptidic positron emission tomography (PET) radiotracer for gastrin releasing peptide receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [¹⁸F](S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.     

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[¹⁸F]fluoroethoxy)benzothiazole ([¹⁸F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [¹⁸F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [¹⁸F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35 GBq/μmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [¹⁸F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.     

Synthesis and preliminary evaluation of 4-hydroxy-6-(3-[11C]methoxyphenethyl)pyridazin-3(2H)-one,a 11C-labeled d-amino acid oxidase (DAAO) inhibitor for PET imaging

Selective DAAO inhibitors have demonstrated promising therapeutic effects in clinical studies, including clinically alleviating symptoms of schizophrenic patients and ameliorating cognitive function in Alzheimer’s patients with early phase. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography ligand based on a DAAO inhibitor, DAO-1903 (8). ¹¹C-Isotopologue of 8 was prepared in high radiochemical yield with high radiochemical purity (>99%) and high molar activity (>37 GBq/µmol). In vitro autoradiography studies indicated that the ligand possessed high in vitro specific binding to DAAO, while in vivo dynamic PET studies demonstrated that [¹¹C]8 failed to cross the blood–brain barrier possibly due to moderate brain efflux mechanism. Further chemical scaffold optimization is necessary to overcome limited brain permeability and improve specific binding.     

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 of (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one as a new potential PET probe for imaging of the enzyme PIM1

(Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one is a potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor with an IC₅₀ value of 3 nM. (Z)-2-((1H-Indazol-3-yl)methylene)-6-[¹¹C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one, a new potential PET probe for imaging of the enzyme PIM1, was first designed and synthesized in 20–30% decay corrected radiochemical yield and 370–740 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled Boc-protected intermediate followed by a quick acidic de-protection.     

2-aryl-8-chloro-1,2,4-triazolo[1,5-a]quinoxalin-4-amines as highly potent A1 and A3 adenosine receptor antagonists

Some 2-aryl-8-chloro-1,2,4-triazolo[1,5-a]quinoxaline derivatives 2-18, obtained by introducing different substituents on either the 4-amino moiety (acyl or carbamoyl groups) or the 2-phenyl ring (4-OCH3) of previously reported 8-chloro-2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-4-amine (1), have been synthesized and tested in radioligand binding assays at bovine A1 and A(2A) and at cloned human A1 and A3 adenosine receptors. The rationally designed 8-chloro-2-(4-methoxy-phenyl)-1,2,4-triazolo[1,5-a]quinoxalin-4-acetylamine (14) can be considered one of the most potent and hA3 versus hA1 selective AR antagonists reported till now. The structure-activity relationships of compounds 2-18 are in agreement with those of previously reported 2-aryl-1,2,4-triazolo[4,3-a]quinoxalines (series A) and 2-arylpyrazolo[3,4-c]quinolines (series B), thus suggesting a similar AR binding mode. In fact, the importance for the A3 receptor-ligand interaction of both a strong acidic NH proton donor and a C=O proton acceptor at position-4, able to engage hydrogen-bonding interactions with specific sites on the A3 AR, has been confirmed. Using our recently published hA3 receptor model, to better elucidate our experimental results, we decided to theoretically depict the putative TM binding motif of the herein reported 1,2,4-triazolo[1,5-a]quinoxaline derivatives on human A3 receptor. Structure-activity relationships have been explained analyzing the three-dimensional structure of the antagonist-receptor models obtained by molecular docking simulation.     

Potential CRF1R PET imaging agents: 1-Fluoroalkylsubstituted 5-halo-3-(arylamino)pyrazin-2(1H)-ones

A series of pyrazinones were prepared and evaluated as potential CRF₁R PET imaging agents. Optimization of their CRF₁R binding potencies and octanol–phosphate buffer phase distribution coefficients are discussed herein.     

Towards tropomyosin-related kinase B (TrkB) receptor ligands for brain imaging with PET: Radiosynthesis and evaluation of 2-(4-[18F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one and 2-(4-([N-methyl-11C]-dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-one

The interaction of tropomyosin-related kinase B (TrkB) with the cognate ligand brain-derived neurotrophic factor (BDNF) mediates fundamental pathways in the development of the nervous system. TrkB signaling alterations are linked to numerous neurodegenerative diseases and conditions. Herein we report the synthesis, biological evaluation and radiosynthesis of the first TrkB radioligands based on the recently identified 7,8-dihydroxyflavone chemotype. 2-(4-[¹⁸F]fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one ([¹⁸F]10b) was synthesized in high radiochemical yields via an efficient S_NAr radiofluorination involving a para-Michael acceptor substituted aryl followed by BBr₃-promoted double demethylation. Selective N-[¹¹C]methylation afforded 2-(4-([N-methyl-¹¹C]-dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-one ([¹¹C]10c) from the fully deprotected catechol-bearing normethyl precursor 13 with [¹¹C]MeOTf. In vitro autoradiography of [¹⁸F]10b with transverse rat brain sections revealed high specific binding in the cortex, striatum, hippocampus and thalamus in accordance with expected TrkB distribution. Blockade experiments with both 7,8-dihydroxyflavone (1a) and TrkB cognate ligand, BDNF, led to decreases of 80% and 85% of radioligand binding strongly supporting the hypothesis that 7,8-dihydroxyflavones exert their effect on TrkB phosphorylation via direct TrkB extracellular domain (ECD) binding. Positron emission tomography (PET) studies revealed that [¹⁸F]10b and [¹¹C]10c brain uptake is minimal and that they are rapidly eliminated from the plasma (effective plasma half-life 5–10 min) via hepatic secretion. Nevertheless, the high specific binding and TrkB specificity derived from in vitro experiments suggests that the 7,8-disubstituted flavone chemotype represents a promising scaffold for the development of TrkB radiotracers for PET.     

Synthesis of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones as potential cytotoxic agents

The new derivatives based on (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-one scaffold was synthesized and evaluated for their in vitro cytotoxic potential against a panel of cancer cell lines, viz., A549 (human lung cancer), HCT-116 (human colorectal cancer), B16F10 (murine melanoma cancer), BT-474 (human breast cancer), and MDA-MB-231 (human triple-negative breast cancer). Among them, many of the synthesized compounds exhibited promising cytotoxic potential against the panel of tested cancer cell lines with IC₅₀ <30 µM. Based on the preliminary screening results, the structure-activity relationship (SAR) of the compounds was established. Among the synthesized compounds, 15i displayed a potential anti-proliferative activity against HCT-116 cancer cell line with an IC₅₀ value of 1.21 ± 0.14 µM. Flow cytometric analysis revealed that compound 15i arrested the G0/G1 phase of the cell cycle. Moreover, increased reactive oxygen species (ROS) generation, clonogenic assay, acridine orange staining, DAPI nuclear staining, measurement of mitochondrial membrane potential (ΔΨm), and annexin V-FITC assays revealed that compound 15i promoted cell death through apoptosis.     

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.     

Discovery of N-benzyl-2-[(4S)-4-(1H-indol-3-ylmethyl)-5-oxo-1-phenyl-4,5-dihydro-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-6-yl]-N-isopropylacetamide,an orally active,gut-selective CCK1 receptor agonist for the potential treatment of obesity

We describe the design, synthesis, and structure–activity relationships of triazolobenzodiazepinone CCK1 receptor agonists. Analogs in this series demonstrate potent agonist activity as measured by in vitro and in vivo assays for CCK1 agonism. Our efforts resulted in the identification of compound 4a which significantly reduced food intake with minimal systemic exposure in rodents.     

The first radiosynthesis of [11C]AZD8931 as a new potential PET agent for imaging of EGFR,HER2 and HER3 signaling

The reference standard AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-methylacetamide} (11a) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-chloro-N-methylacetamide in 11 steps with 2–5% overall chemical yield. The precursor N-desmethyl-AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)acetamide} (11b) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-bromoacetamide in 11 steps with 2–4% overall chemical yield. The target tracer [¹¹C]AZD8931 {2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-[¹¹C]methylacetamide} ([¹¹C]11a) was prepared from N-desmethyl-AZD8931 (11b) with [¹¹C]CH₃OTf under basic condition (NaH) through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB) with 370–1110 GBq/μmol specific activity at EOB.     

Radiosynthesis of [11C]Vandetanib and [11C]chloro-Vandetanib as new potential PET agents for imaging of VEGFR in cancer

Vandetanib (ZD6474) and its chlorine analogue chloro-Vandetanib are potent and selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors with low nanomolar IC₅₀ values. [¹¹C]Vandetanib and [¹¹C]chloro-Vandetanib, new potential PET agents for imaging of VEGFR in cancer, were first designed, synthesized and labeled at nitrogen and oxygen positions from their corresponding N- and O-des-methylated precursors, in 40-50% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB).     

Synthesis and antibacterial activity of novel 6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-4-oxoquinoline-3-carboxylic acids bearing cyclopropane-fused 2-amino-8-azabicyclo[4.3.0]nonan-8-yl substituents at the C-7 position

A series of novel 6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-4-oxoquinoline-3-carboxylic acids bearing cyclopropane-fused 2-amino-8-azabicyclo[4.3.0]nonan-8-yl substituents at the C-7 position were synthesized to obtain potent drugs for the treatment of Gram-positive infections. Some compounds exhibited excellent antibacterial activity, and potent inhibitory activity against bacterial DNA topoisomerase IV. In addition, some of the potent compounds showed reduced inhibitory activity against human DNA topoisomerase II compared with the corresponding noncyclopropane-fused compounds.