Replacement of the hydroxamic acid group in the selective HDAC8 inhibitor PCI-34051.

PCI-34051 is a valuable tool to interrogate the therapeutic effects of selective inhibition of HDAC8. However, it has not advanced to clinical trials, perhaps due to poor PK or off-target effects. We hypothesized that the presence of a hydroxamic acid (HA) group in PCI-34051 contributed to its lack of advancement. Therefore, we replaced the HA in the PCI-34051 scaffold with a series of moieties that have the potential to bind to Zn and evaluated their activity in a HDAC8 assay. Surprisingly, none of the replacements effectively mimicked the HA, and analogs lost significant potency. Evaluation of the analogs' affinity to Zn indicated that none had affinity for Zn within the same range as the HA. These studies point to the difficulty in the application of bioisosteric replacements for Zn binding motifs.

Anticancer efficacy triggered by synergistically modulating the homeostasis of anions and iron: Design, synthesis and biological evaluation of dual-functional squaramide-hydroxamic acid conjugates.

Targeting the homeostasis of anions and iron has emerged as a promising therapeutic approach for the treatment of cancers. However, single-targeted agents often fall short of achieving optimal treatment efficacy. Herein we designed and synthesized a series of novel dual-functional squaramide-hydroxamic acid conjugates that are capable of synergistically modulating the homeostasis of anions and iron. Among them, compound 16 exhibited the most potent antiproliferative activity against a panel of selected cancer cell lines, and strong in vivo anti-tumor efficacy. This compound effectively elevated lysosomal pH through anion transport, and reduced the levels of intracellular iron. Compound 16 could disturb autophagy in A549 cells and trigger robust apoptosis. This compound caused cell cycle arrest at the G1/S phase, altered the mitochondrial function and elevated ROS levels. The present findings clearly demonstrated that synergistic modulation of anion and iron homeostasis has high potentials in the development of promising chemotherapeutic agents with dual action against cancers.

Synthesis and biological evaluation of ortho-phenyl phenylhydroxamic acids containing phenothiazine with improved selectivity for class IIa histone deacetylases.

Class IIa histone deacetylases (HDACs) have been linked to tumorigenesis in various cancers. Previously, we designed phenylhydroxamic acid LH4f as a potent class IIa HDAC inhibitor. However, it also unselectively inhibited class I and class IIb HDACs. To enhance the compound's selectivity towards class IIa HDACs, the ortho-phenyl group from the selective HDAC7 inhibitor 1 is incorporated into ortho position of the phenylhydroxamic acid in LH4f. Compared to LH4f, most resulting compounds displayed substantially improved selectivity towards the class IIa HDACs. Notably, compound 7 g exhibited the strongest HDAC9 inhibition with an IC50 value of 40 nM. Molecular modelling further identified the key interactions of compound 7 g bound to HDAC9. Compound 7 g significantly inhibited several human cancer cells, induced apoptosis, modulated caspase-related proteins as well as p38, and caused DNA damage. These findings suggest the potential of class IIa HDAC inhibitors as lead compounds for the development of cancer therapeutics.

Synthesis and Biological Evaluation of Novel 2-Aroyl Benzofuran-Based Hydroxamic Acids as Antimicrotubule Agents.

Because of synergism between tubulin and HDAC inhibitors, we used the pharmacophore fusion strategy to generate potential tubulin-HDAC dual inhibitors. Drug design was based on the introduction of a N-hydroxyacrylamide or a N-hydroxypropiolamide at the 5-position of the 2-aroylbenzo[b]furan skeleton, to produce compounds 6a-i and 11a-h, respectively. Among the synthesized compounds, derivatives 6a, 6c, 6e, 6g, 11a, and 11c showed excellent antiproliferative activity, with IC50 values at single- or double-digit nanomolar levels, against the A549, HT-29, and MCF-7 cells resistant towards the control compound combretastatin A-4 (CA-4). Compounds 11a and 6g were also 10-fold more active than CA-4 against the Hela cell line. When comparing the inhibition of tubulin polymerization versus the HDAC6 inhibitory activity, we found that 6a-g, 6i, 11a, 11c, and 11e, although very potent as inhibitors of tubulin assembly, did not have significant inhibitory activity against HDAC6.

Synthesis and Evaluation of the First 68Ga-Labeled C-Terminal Hydroxamate-Derived Gastrin-Releasing Peptide Receptor-Targeted Tracers for Cancer Imaging with Positron Emission Tomography.

Gastrin-releasing peptide receptor (GRPR), overexpressed in many solid tumors, is a promising imaging marker and therapeutic target. Most reported GRPR-targeted radioligands contain a C-terminal amide. Based on the reported potent antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, we synthesized C-terminal hydroxamate-derived [68Ga]Ga-LW02075 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) and [68Ga]Ga-LW02050 ([68Ga]Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), and compared them with the closely related and clinically validated [68Ga]Ga-SB3 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt). Binding affinities (Ki) of Ga-SB3, Ga-LW02075, and Ga-LW02050 were 1.20 ± 0.31, 1.39 ± 0.54, and 8.53 ± 1.52 nM, respectively. Both Ga-LW02075 and Ga-LW02050 were confirmed to be GRPR antagonists by calcium release assay. Imaging studies showed that PC-3 prostate cancer tumor xenografts were clearly visualized at 1 h post injection by [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 in PET images, but not by [68Ga]Ga-LW02075. Ex vivo biodistribution studies conducted at 1 h post injection showed that the tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 ± 1.00 vs. 6.98 ± 1.36 %ID/g), followed by [68Ga]Ga-LW02075 (3.97 ± 1.71 %ID/g). [68Ga]Ga-SB3 had the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [68Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 ± 0.11 %ID/g. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing cancer lesions.

Structure-activity relationship and mechanistic studies for a series of cinnamyl hydroxamate histone deacetylase inhibitors.

Histone deacetylases (HDACs) are a family of enzymes that modulate the acetylation status histones and non-histone proteins. Histone deacetylase inhibitors (HDACis) have emerged as an alternative therapeutic approach for the treatment of several malignancies. Herein, a series of urea-based cinnamyl hydroxamate derivatives is presented as potential anticancer HDACis. In addition, structure-activity relationship (SAR) studies have been performed in order to verify the influence of the linker on the biological profile of the compounds. All tested compounds demonstrated significant antiproliferative effects against solid and hematological human tumor cell lines. Among them, 11b exhibited nanomolar potency against hematological tumor cells including Jurkat and Namalwa, with IC50 values of 40 and 200 nM, respectively. Cellular and molecular proliferation studies, in presence of compounds 11a-d, showed significant cell growth arrest, apoptosis induction, and up to 43-fold selective cytotoxicity for leukemia cells versus non-tumorigenic cells. Moreover, compounds 11a-d increased acetylated α-tubulin expression levels, which is phenotypically consistent with HDAC inhibition, and indirectly induced DNA damage. In vitro enzymatic assays performed for 11b revealed a potent HDAC6 inhibitory activity (IC50: 8.1 nM) and 402-fold selectivity over HDAC1. Regarding SAR analysis, the distance between the hydroxamate moiety and the aromatic ring as well as the presence of the double bond in the cinnamyl linker were the most relevant chemical feature for the antiproliferative activity of the series. Molecular modeling studies suggest that cinnamyl hydroxamate is the best moiety of the series for binding HDAC6 catalytic pocket whereas exploration of Ser568 by the urea connecting unity (CU) might be related with the selectivity observed for the cinnamyl derivatives. In summary, cinnamyl hydroxamate derived compounds with HDAC6 inhibitory activity exhibited cell growth arrest and increased apoptosis, as well as selectivity to acute lymphoblastic leukemia cells. This study explores interesting compounds to fight against neoplastic hematological cells.

Effect of phenylurea hydroxamic acids on histone deacetylase and VEGFR-2.

A series of phenylurea hydroxamic acids incorporating pharmacophores of inhibitors of HDAC inhibitors and VEGFR-2 has been designed. Most of the compounds show antiproliferative activity comparable to that of Vorinostat and Sorafenib, and better EPC inhibitory activity. Enzymatic assays and Western blotting results indicated that compound 14 not only inhibits HDAC but also has slight VEGFR-2 inhibitory activity. A docking study revealed that the polar hydroxamic acid retains the interaction with HDAC through a zinc ion and also interacts with some residues of the active site of VEGFR-2. Despite 14 displaying a weaker VEGFR-2 activity, a possible route to develop potent HDAC/VEGFR-2 inhibitors is suggested.

Discovery of Novel Dihydropyrimidine and hydroxamic acid hybrids as potent Helicobacter pylori Urease inhibitors.

Two novel series of Dihydropyrimidine-hydroxamic acid hybrids (4a-4l and 5a-5l) were designed, synthesized and evaluated for in vitro Helicobacter pylori urease inhibition. In vitro enzyme inhibition screening led to the discovery of three potent urease inhibitors 2-[[4-(4-hydroxy phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy acetamide (4g), 2-[[4-(4-chloro phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy acetamide (4b) and 3-[[4-(3-methoxy phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy propanamide (5l). Compound 4g showed excellent urease inhibition with IC50 value of 14 ± 1 nM, indicated by its strong interactions with both metallic Ni++ ions, Gly279, His221, Ala365, Asp362, Asn168, Arg338 and His322 residues of the active site of urease. Further, compounds 4b and 5l displayed very good activity with IC50 value of 0.082 ± 0.004 µM and 0.14 ± 0.013 µM respectively compared to standard Acetohydroxamic acid (IC50 - 27.4 ± 1.2 µM). Kinetic studies revealed that a mixed inhibition with both competitive and non-competitive aspects is involved in the urease inhibition mechanism. The in vitro urease inhibition results were supported by molecular docking studies. Collectively, this study indicates that 4g could be considered as promising lead molecule that can be further developed as a potent drug molecule for the treatment of Helicobacter pylori caused gastritis for further studies.

Design, synthesis, and biological evaluation of hydroxamic acid-substituted 2,4-diaryl aminopyrimidines as potent EGFRT790M/L858R inhibitors for the treatment of NSCLC.

A series of 2,4-diarylaminopyrimidine derivatives bearing hydrophilic hydroxamic acids were designed and synthesized as potent EGFRT790M/L858R inhibitors. Among the derivatives synthesized, 10c (IC50 = 5.192 nM), 10j (IC50 = 10.35 nM), and 10o (IC50 = 0.3524 nM) exhibited higher potencies against EGFRT790/M/L858R compared to the known EGFR inhibitor AZD-9291 (IC50 = 20.80 nM). Moreover, 10j showed moderate activity against H1975 cells transfected with the EGFRT790M/L858R mutant, with an IC50 of 0.2113 µM over A431 (wild-type EGFR, SI = 47.3). In addition, 10j exhibited low toxicity in normal HBE cells (human bronchial epithelial cells, IC50 > 40 µΜ). Analysis of the mode of action indicated that 10j effectively induced apoptosis in H1975 cells by arresting the cells in the G2/M phase. Compound 10j also demonstrated efficacy in inhibiting tumor growth in a H1975 xenograft mouse model without losing body weight or killing the mice. Taken together, these results suggested that 10j might be a promising candidate for development as a potential treatment for NSCLC harboring the EGFRT790M/L858R mutation.

Investigation of the effect of different linker chemotypes on the inhibition of histone deacetylases (HDACs).

Histone Deacetylases (HDACs) are among the most attractive and interesting targets in anticancer drug discovery. The clinical relevance of HDAC inhibitors (HDACIs) is testified by four FDA-approved drugs for cancer treatment. However, one of the main drawbacks of these drugs resides in the lack of selectivity against the different HDAC isoforms, resulting in severe side effects. Thus, the identification of selective HDACIs represents an exciting challenge for medicinal chemists. HDACIs are composed of a cap group, a linker region, and a metal-binding group interacting with the catalytic zinc ion. While the cap group has been extensively investigated, less information is available about the effect of the linker on isoform selectivity. To this aim, in this work, we explored novel linker chemotypes to direct isoform selectivity. A small library of 25 hydroxamic acids with hitherto unexplored linker chemotypes was prepared. In vitro tests demonstrated that, depending on the linker type, some candidates selectively inhibit HDAC1 over HDAC6 isoform or vice versa. Docking calculations were performed to rationalize the effect of the novel linker chemotypes on biologic activity. Moreover, four compounds were able to increase the levels of acetylation of histone H3 or tubulin. These compounds were also assayed in breast cancer MCF7 cells to test their antiproliferative effect. Three compounds showed a significant reduction of cancer proliferation, representing valuable starting points for further optimization.

Effect of 3-subsitution of quinolinehydroxamic acids on selectivity of histone deacetylase isoforms.

A series of 3-subsituted quinolinehydroxamic acids has been synthesised and evaluated for their effect on human lung cancer cell line (A549), human colorectal cancer cell line (HCT116) and HDAC isoforms 1, 2, 6, and 8. The results indicated that substitution at C3 of quinoline is favoured for HDAC6 selectivity. Two compounds (25 and 26) were also found to be potent anti-proliferative compounds with IC50 values ranging from 1.29 to 2.13 µM against A549 and HCT116 cells. These compounds displayed remarkable selectivity for HDAC6 over other HDAC isoforms with nanomolar IC50 values. Western blot analysis revealed that compounds of this series activate apoptotic caspase pathway as indicated by cleavage of caspase 3, 8, and 9 and also increase phosphorylated H2AX thus inducing DNA double strand fragmentation in a concentration dependent manner. Flow cytometric analysis also displayed a dose dependent increase of cell population in sub G1 phase.

Design, synthesis and biological evaluation of N-hydroxy-aminobenzyloxyarylamide analogues as novel selective κ opioid receptor antagonists.

Aminobenzyloxyarylamide derivatives 1a-i and 2a-t were designed and synthesized as novel selective κ opioid receptor (KOR) antagonists. The benzoyl amide moiety of LY2456302 was changed into N-hydroxybenzamide and benzisoxazole-3(2H)-one to investigate whether it could increase the binding affinity or selectivity for KOR. All target compounds were evaluated in radioligand binding assays for opioid receptor binding affinity. These efforts led to the identification of compound 1c (κ Ki = 179.9 nM), which exhibited high affinity for KOR. Moreover, the selectivity of KOR over MOR and DOR increased nearly 2-fold and 7-fold, respectively, compared with (±)LY2456302.

Synthesis and evaluation of novel technetium-99m-hydroxamamide complex based on imidazothiadiazole sulfonamide targeting carbonic anhydrase-IX for tumor imaging.

Carbonic anhydrase-IX (CA-IX) is an attractive target for detecting tumors associated with a poor prognosis. We previously reported a [99mTc]hydroxamamide complex based on ureidosulfonamide as a CA-IX ligand ([99mTc]URB2A), which showed a favorable affinity for CA-IX high-expressing cells in vitro and tumors in vivo; however, radioactivity retention in the blood pool suggested a high background signal on imaging. To improve the pharmacokinetics of [99mTc]URB2A, in this study, we designed and synthesized [99mTc]ISB2 based on imidazothiadiazole sulfonamide, which exhibited greater CA-IX affinity and faster clearance from the blood pool than ureidosulfonamide in studies using corresponding 111In-labeled compounds, and evaluated its utility for CA-IX imaging. In an in vitro cell binding assay, [99mTc]ISB2 markedly bound to CA-IX high-expressing (HT-29) cells; moreover, its binding was greater than that of [99mTc]URB2A. In an in vivo biodistribution assay, [99mTc]ISB2 showed faster clearance from the blood pool than [99mTc]URB2A; however, lower HT-29 tumor accumulation was observed. Further structural modification of [99mTc]ISB2 to improve its stability may lead to the development of a useful [99mTc]hydroxamamide complex for CA-IX imaging.

Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor.

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc. Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range. In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC's) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX). Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.

Synthesis and biological evaluation of triazolyl-substituted benzyloxyacetohydroxamic acids as LpxC inhibitors.

The bacterial deacetylase LpxC is a promising target for the development of antibiotics selectively combating Gram-negative bacteria. To improve the biological activity of the reported benzyloxyacetohydroxamic acid 9 ((S)-N-hydroxy-2-{2-hydroxy-1-[4-(phenylethynyl)phenyl]ethoxy}acetamide), its hydroxy group was replaced by a triazole ring. Therefore, in divergent syntheses, triazole derivatives exhibiting rigid and flexible lipophilic side chains, different configurations at their stereocenter, and various substitution patterns at the triazole ring were synthesized, tested for antibacterial and LpxC inhibitory activity, and structure-activity relationships were deduced based on docking and binding energy calculations.

Novel amidase catalysed process for the synthesis of vorinostat drug.

AIM: Presently, N-hydroxy-N'-phenyloctanediamide (vorinostat) which is an effective histone deacetylase inhibitor, is being synthesized chemically. Hence, present study aims to develop an eco-friendly approach for the synthesis of vorinostat from N'-phenyloctanediamide through biotransformation. METHODS AND RESULTS: Using the amidase of Bacillus smithii IIIMB2907 in time course conversion and organic solvent compatibility, maximum bioconversion was observed at 12 h of reaction time and in presence of ethanol, respectively. Potassium phosphate buffer of pH 7·0 supported maximum bioconversion of N'-phenyloctanediamide (10 mmol l-1 ) into N-hydroxy-N'- phenyloctanediamide at 40°C. Bench scale study was successfully carried out with 83% yield of purified vorinostat. CONCLUSION: In this study, an eco-friendly approach for the biotransformation of N'-phenyloctanediamide into vorinostat was developed by using cell free extract of thermophilic strain B. smithii IIIMB2907. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial amidase has achieved remarkable attention in the field of biotransformation for the green synthesis of hydroxamic acids. Utilization of amidase from B. smithii IIIMB2907, specifically in the synthesis of vorinostat drug is a foremost attempt in the development a novel process and can also be employed in the synthesis of its derivatives as well.

Design and synthesis of selective and blood-brain barrier-permeable hydroxamate-based gelatinase inhibitors.

Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix, make a major contribution to the progression of a vast number of diseases, such cancer or epilepsy. Although several MMP inhibitors (MMPi) have been developed to date for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and, most importantly, the presence of severe side effects. The latter can be explained by their lack of selectivity of these inhibitors. In this regard, MMPs' family members have a high structural homology, which challenge the development of selective inhibitors for a specific MMP. Here, we have used in silico calculations and in vitro data to design MMPi that selectively target gelatinases (MMP-2 and MMP-9) and have the capacity to cross the blood-brain barrier. Following this approach, we obtained compound 40 that shows high proteolytic stability and low cytotoxicity. This compound may be of particular interest for the treatment of central nervous diseases such epilepsy or Alzheimer's disease, where gelatinase activity is increased. Our data show the specificity of compound 40 for recombinant MMP-9 and MMP-2 and endogenous MMP-9 from rat hippocampal cell cultures, and reveals its permeability across the blood-brain barrier in vivo.

Synthesis and structure-activity relationships of novel 5-(hydroxamic acid)methyl oxazolidinone derivatives as 5-lipoxygenase inhibitors.

Oxazolidinone hydroxamic acid derivatives were synthesised and evaluated for inhibitory activity against leukotriene (LT) biosynthesis in three in vitro cell-based test systems and on direct inhibition of recombinant human 5-lipoxygenase (5-LO). Thirteen of the 19 compounds synthesised were considered active ((50% inhibitory concentration (IC50) ≤ 10 µM in two or more test systems)). Increasing alkyl chain length on the hydroxamic acid moiety enhanced activity and morpholinyl-containing derivatives were more active than N-acetyl-piperizinyl derivatives. The IC50 values in cell-based assay systems were comparable to those obtained by direct inhibition of 5-LO activity, confirming that the compounds are direct inhibitors of 5-LO. Particularly, compounds PH-249 and PH-251 had outstanding potencies (IC50 < 1 µM), comparable to that of the prototype 5-LO inhibitor, zileuton. Pronounced in vivo activity was demonstrated in zymosan-induced peritonitis in mice. These novel oxazolidinone hydroxamic acid derivatives are, therefore, potent 5-LO inhibitors with potential application as anti-allergic and anti-inflammatory agents.

The Development Process: from SAHA to Hydroxamate HDAC Inhibitors with Branched CAP Region and Linear Linker.

Histone deacetylases (HDACs) belong to a group of epigenetic regulatory enzymes that participate in modulating the acetylation level of histone lysine residues as well as non-histone proteins, and they play a key role in the regulation of gene expression. HDACs are potential anticancer drug targets highly expressed in various kinds of cancer cells. So far, five small molecules targeting HDACs have been approved for the therapy of cancer, and over 20 inhibitors of HDACs are under different phases of clinical trials. Among them, hydroxamate-based HDAC inhibitors (HDACis) represent a well-investigated series of chemical entities. The current review covers the recent progress in the discovery process, form SAHA to hydroxamate HDAC inhibitors with branched CAP region and linear linker. At the same time, the pharmacological and structure-activity relationship (SAR) studies of the specific derivatives from SAHA and the HDACis with branched CAP region and linear linker are also introduced.

Radiosynthesis and preliminary evaluation of an 18 F-labeled tubastatin A analog for PET imaging of histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family because of its characteristics, namely, its cytoplasmic localization and ubiquitin binding. HDAC6 has been implicated in cancer metastasis and neurodegeneration. In the present study, we performed radiosynthesis and biological evaluation of a fluorine-18-labeled ligand [18 F]3, which is an analog of the HDAC6-selective inhibitor tubastatin A, for positron emission tomography (PET) imaging. [18 F]3 was synthesized by a two-step reaction composed of 18 F-fluorination and formation of a hydroxamic acid group. IC50 values of 3 against HDAC1 and HDAC6 activities were 996 nM and 33.1 nM, respectively. A biodistribution study in mice demonstrated low brain uptake of [18 F]3. Furthermore, bone radioactivity was stable at around 2% ID/g after injection, suggesting high tolerance to defluorination. Regarding metabolic stability, 70% of the compound was observed as the unchanged form at 30 minutes post injection in mouse plasma. A small animal PET study in mice showed that pretreatment with cyclosporine A had no effect on initial brain uptake of [18 F]3, suggesting low brain uptake of [18 F]3 was not caused by the P-glycoprotein-mediated efflux. While PET imaging using [18 F]3 has a limitation with respect to neurodegenerative diseases, further studies evaluating its utility for certain cancers are worth evaluating.