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(R)-[(18)F]MH.MZ ([(18)F]MH.MZ) is a promising positron emission tomography (PET) radiotracer for in vivo study of the 5-HT(2A) receptor. To facilitate clinical trials a fully automated radiosynthesis procedure for [(18)F]MH.MZ was developed using commercially available materials on the iPhase Flexlab module. The overall synthesis time was 100 minutes with a radiochemical yield of 7 ± 0.9% (n=3). The radiochemical purity was greater than 99% for [(18)F]MH.MZ with a molar activity of 361 ± 57 GBq/μmol (n=3). The protocol described herein reliably provides [(18)F]MH.MZ that meets all relevant release criteria for a GMP radiopharmaceutical.

PDF file - 90KB, Scheme 1. 19FFB-VAD-FMK (3) (A) and 127IIZ-VAD-FMK (5) (B) synthetic schemes.

Purpose: Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid–specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [18F]4-fluorobenzylcarbonyl–Val–Ala–Asp(OMe)–fluoromethylketone ([18F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET).Experimental Design: Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [18F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [18F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer.Results: Accumulation of [18F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in V600EBRAF colon cancer. In the latter setting, [18F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size.Conclusions: These studies illuminate [18F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine. Clin Cancer Res; 20(8); 2126–35. ©2014 AACR.

PDF file - 79KB, 19FFB-VAD-FMK and parent peptide enzyme selectivity. Biochemistry, fluorescence measured IC50 values, collected as duplicates, of the parent peptide, VAD-FMK, and 19FFB-VAD-FMK against caspases-3/6/7/8.

PDF file - 165KB, Supplemental Materials and Methods, Figure Legends.

PDF file - 137KB, IHC semi-quantification. Semi-quantitative analysis of cleaved caspase-3 histological samples of DLD-1 and SW620 xenograft tumors treated with vehicle or AZD-1152 (A). Evaluation of multiple low field (2.5x) micrographs revealed a statistical significant difference between vehicle- and drug-treated SW620 (p < 0.0001) tumors but not for analogously treated DLD-1 tumors (p = 0.524). Similar analysis of cleaved caspase-3 histological samples of COLO-205 and LIM-2405 treatment cohorts revealed a statistical significant difference between vehicle-treated and combination-treated COLO-205 (p = 0.025) tumors but not for analogously treated LIM-2405 tumors (p = 0.325) (B).

PDF file - 93KB, Scheme 2. 18FSFB (9) (A) and 18FFB-VAD-FMK (11) (B) synthetic schemes. Compound (9) radiosynthesis was performed systematically using an automated radiochemistry module.

PDF file - 43KB, Docking scores are reported as ranked by the Crash (steric repulsion) score from the protein flexibility (PF-score) scoring function of the Tripos SYBYL-X v2.0 software suite that also features (among other scoring parameters) comparison with SurflexDock Total_Score (statistical estimate of binding affinity, -log Kd), Polar (measure of polar contacts), Strain (measure of internal ligand energetic strain) and Similarity (index of shape-based morphological similarity to reference covalent inhibitor compound) term score parameters. Proposed N-terminal modifications to the VAD-FMK peptide scaffold all scored comparably using these criteria indicating minimal predicted perturbation to interactions with the caspase-3 protein target.

A novel synthesis of the translocator protein (TSPO) ligand 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575, 3) was achieved in four steps from commercially available starting materials. Focused structure-activity relationship development about the pyridazinoindole ring at the N3 position led to the discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (14), a novel ligand of comparable affinity. Radiolabeling with fluorine-18 ((18)F) yielded 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[(18)F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide ([(18)F]-14) in high radiochemical yield and specific activity. In vivo studies of [(18)F]-14 revealed this agent as a promising probe for molecular imaging of glioma.

A novel and highly efficient synthetic method leveraging microwave-assisted organic synthesis (MAOS) to yield di-7-azaindolylmethanes (DAIMs) is reported. Under MAOS conditions, reaction of 7-azaindole with aldehydes resulted predominantly in DAIMs, as opposed to the expected 7-azaindole addition products that form at ambient temperature. Based upon studies of different indoles and azaindoles with various aromatic and aliphatic aldehydes, we herein propose a mechanism where rapid and efficient microwave heating promotes nucleophilicity of 7-azaindoles towards the corresponding alkylidene-azaindolene intermediate to form the DAIM. This sequence provides a versatile approach to efficiently synthesize novel DAIMs that may be useful pharmaceuticals.

Herein we report the synthesis and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as novel positive allosteric modulators of mGlu4. We detail our work towards finding phenyl replacements for the core scaffold of previously reported phenyl sulfonamides and phenyl sulfone compounds. Our efforts culminated in the identification of N-(1-((3,4-dimethylphenyl)sulfonyl)-1H-pyrrol-3-yl)picolinamide as a potent PAM of mGlu4.

The efficacy of positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 4 (mGlu4) in preclinical rodent models of Parkinson’s disease has been established by a number of groups. Here, we report an advanced preclinically characterized mGlu4 PAM, N-(3-chloro-4-fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (VU0418506). We detail the discovery of VU0418506 starting from a common picolinamide core scaffold and evaluation of a number of amide bioisosteres leading to the novel pyrazolo[4,3-b]pyridine head group. VU0418506 has been characterized as a potent and selective mGlu4 PAM with suitable in vivo pharmacokinetic properties in three preclinical safety species.

A potent and selective inhibitor of KCNQ2, (S)-5 (ML252, IC50 = 69 nM), was discovered after a high-throughput screen of the MLPCN library was performed. SAR studies revealed a small structural change (ethyl group to hydrogen) caused a functional shift from antagonist to agonist activity (37, EC50 = 170 nM), suggesting an interaction at a critical site for controlling gating of KCNQ2 channels.

The enantioselective total synthesis of Resolvin E1 (RvE1), a naturally occurring small molecule mediator of inflammation resolution, is reported. Two routes are presented, both modular and convergent in nature, with an excellent control of all stereocenters. The C12- and C18-hydroxy groups are derived from (S)-glycidol while the C5-hydroxy group is installed via enantioselective reduction of a ketone precursor. Both the cis-alkenes are introduced with excellent control by the reduction of a late-stage bis-alkyne intermediate. The synthetic disconnections are very amenable to analog preparation, and further modifications to the chemistry have allowed for scale-up and First in Man testing of this novel pro-resolution molecule.

Using a functional high-throughput screening (HTS) and subsequent solution-phase parallel synthesis approach, we have discovered a novel series of positive allosteric modulators for mGlu₄, a G-protein coupled receptor. This series is comprised of a homopiperazine central core. The solution-phase parallel synthesis and SAR of analogs derived from this series will be presented. This series of positive allosteric modulators of mGlu₄ provide critical research tools to further probe the mGlu₄-mediated effects in Parkinson's disease.

A novel and highly efficient microwave promoted reaction of 7-azaindoles with aldehydes to give di-7-azaindolylmethanes (III) is developed.

The synthesis of (+)-trans-2-mercaptocyclohexanol and (-)-trans-2-mercaptocyclohexanol was described. Ring opening of cyclohexene oxide with (-)-4-methoxybenzyl-3(R)-nopanethiol followed by oxidn. gives two readily separable diastereomeric sulfoxides. These sulfoxides display very different thermal stability but both undergo regio-specific syn-elimination to give 1-cyclohexanol-2-sulfenic acid that can be treated in situ with 3,5-dimethylthiophenol to give a mixed disulfide. Redn. of these disulfides with lithium aluminum hydride gives the title compds. in enantiomerically pure form.

Purpose: Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid–specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [18F]4-fluorobenzylcarbonyl–Val–Ala–Asp(OMe)–fluoromethylketone ([18F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET). Experimental Design: Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [18F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [18F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer. Results: Accumulation of [18F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in V600EBRAF colon cancer. In the latter setting, [18F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size. Conclusions: These studies illuminate [18F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine. Clin Cancer Res; 20(8); 2126–35. ©2014 AACR.

High-yielding, automated production of a PET tracer that reflects proliferation, 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), is reported using a modified Bioscan Coincidence FDG reaction module.Production of [(18)F]FLT was implemented through: (1) modification of an original FDG manifold; (2) application of an alternate time sequence; and (3) altered solid-phase extraction (SPE) purification. Quality control testing, including standard radiochemical figures of merit and preclinical positron emission tomography (PET) imaging, was carried out.High decay-corrected yields of [(18)F]FLT (16-39%) were reproducibly obtained. The product exhibited very high specific activity (4586.9TBq/mmol; 123,969Ci/mmol) and radiochemical purity (99%). Overall, the [(18)F]FLT produced in this manner was superior to typical productions that utilized a GE TRACERlab FXF-N reaction module. Additionally, purification with SPE cartridges, followed by manual elution, accelerated overall run time and resulted in a two-fold increase in [(18)F]FLT concentration. PET imaging showed the [(18)F]FLT produced by this method was highly suitable for non-invasive tumor imaging in mice.The Bioscan Coincidence GE FDG Reaction Module was readily adapted to reproducibly provide [(18)F]FLT in high yield, specific activity, and radiochemical purity. The approach was suitable to provide sufficient amounts of material for preclinical studies.

This Paper describes the continued optimization of an MLPCN probe molecule M(1) antagonist (ML012) through an iterative parallel synthesis approach. After several rounds of modifications of the parent compound, we arrived at a new azetidine scaffold that displayed improved potency while maintaining a desirable level of selectivity over other muscarinic receptor subtypes. Data for representative molecules 7w (VU0452865) and 12a (VU0455691) are presented.

Two closely related diaryl acylsulfonamides were recently reported as potent antitumor agents against a broad spectrum of human tumor xenografts (colon, lung, breast, ovary, and prostate) in nude mice. Especially intriguing was their activity against colorectal cancer xenografts. In this paper, rapid parallel synthesis along with traditional medicinal chemistry techniques were used to quickly delineate the structure-activity relationships of the substitution patterns in both phenyl rings of the acylsufonamide anti-proliferative scaffold. Although the molecular target of the compounds remains unclear, we determined that the vascular endothelial growth factor-dependent human umbilical vein endothelial cells assay in combination with a soft agar disk diffusion assay allowed for optimization of potency in the series. The pharmacokinetic properties and in vivo activity in an HCT116 xenograft model are reported for representative compounds.

The anti-proliferative activity of acylated heterocyclic sulfonamides is described in Vascular Endothelial Growth Factor-dependent Human Umbilical Vascular Endothelial Cells (VEGF-HUVEC) and in HCT116 tumor cells in a soft agar diffusion assay.

There exists a continued need for the development of improved positron emission tomography (PET) biomarkers for glioma imaging to aid in tumor diagnosis, inform clinical outcome, and quantify response to therapeutic intervention. Translocator protein (TSPO) expression is elevated in cancer and has been linked with disease progression and diminished survival and tends to be a hallmark of aggressive tumors. Our laboratory has pursued development of TSPO PET ligands as candidates for molecular imaging of glioma, as TSPO ligand binding correlates with tumor grade in this setting. We report the first exploration of the structure-activity relationship (SAR) around the pyridazinoindole ring and subsequent radiofluorination of a potent and novel pyridazinoindole-based TSPO-selective ligand for cancer imaging. Library and SAR development around the N3 position of the pyridazinoindole scaffold led to the discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (VUIIS8310), a novel TSPO ligand exhibiting a binding affinity comparable to SSR180575, yet bearing a fluorine atom for subsequent radiolabeling with fluorine-18 (18F) to give 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (18F-VUIIS8310). Initial production feasibility and radiochemical development were evaluated with microfluidics and subsequently translated to traditional box-based methods. Subsequently, quantitative, preclinical elevation of 18F-VUIIS8310 was conducted in a rat glioma model, given our previous experience in this setting. Glioma-bearing rats were imaged in a microPET system, with dynamic PET acquisitions acquired simultaneously upon tracer injection. PET ligand reversibility and specificity were evaluated by ligand displacement studies that utilized VUIIS8310. 18F-VUIIS8310 exhibited elevated uptake and specific, displaceable binding in tumor, in contrast with normal brain, which exhibited very low uptake. 18F-VUIIS8310 uptake in the tumor, relative to normal brain, reached a tumor-to-normal brain ratio of greater than 10:1. Ex vivo histological analysis of resected tissue correlated well with PET imaging data. These preclinical studies illuminate 18F-VUIIS8310 as a promising, novel TSPO PET ligand for imaging glioma and potentially other solid tumors. Importantly, the in vivo stability and high signal-to-noise achieved between tumor and surrounding normal brain suggest the potential of this PET ligand for early tumor detection within this setting. Future head-to-head comparisons between this tracer and emerging, potent TSPO ligands (J. Med. Chem. 2013, 56, 3429) are underway. Citation Format: Yiu-Yin Cheung, Jason R. Buck, Michael L. Nickels, Dewei Tang, H. Charles Manning. Preclinical evaluation of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide: A novel pyridazinoindole ligand for PET imaging of TSPO in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 110. doi:10.1158/1538-7445.AM2014-110