Increased tryptophan, but not increased glucose metabolism, predict resistance of pembrolizumab in stage III/IV melanoma.

Clinical trials of combined IDO/PD1 blockade in metastatic melanoma (MM) failed to show additional clinical benefit compared to PD1-alone inhibition. We reasoned that a tryptophan-metabolizing pathway other than the kynurenine one is essential. We immunohistochemically stained tissues along the nevus-to-MM progression pathway for tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1. We assessed tryptophan and glucose metabolism by performing baseline C11-labeled α-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imaging of tumor lesions in a prospective clinical trial of pembrolizumab in MM (clinicaltrials.gov, NCT03089606). We found higher protein expression of all TMEs and LAT1 in melanoma cells than tumor-infiltrating lymphocytes (TILs) within MM tumors (n = 68). Melanoma cell-specific TPH1 and LAT1 expressions were significantly anti-correlated with TIL presence in MM. High melanoma cell-specific LAT1 and low IDO1 expression were associated with worse overall survival (OS) in MM. Exploratory optimal cutpoint survival analysis of pretreatment 'high' vs. 'low' C11-AMT SUVmax of the hottest tumor lesion per patient revealed that the 'low' C11-AMT SUVmax was associated with longer progression-free survival in our clinical trial (n = 26). We saw no such trends with pretreatment FDG PET SUVmax. Treatment of melanoma cell lines with telotristat, a TPH1 inhibitor, increased IDO expression and kynurenine production in addition to suppression of serotonin production. High melanoma tryptophan metabolism is a poor predictor of pembrolizumab response and an adverse prognostic factor. Serotoninergic but not kynurenine pathway activation may be significant. Melanoma cells outcompete adjacent TILs, eventually depriving the latter of an essential amino acid.

Direct arene C-H fluorination with 18F- via organic photoredox catalysis.

Positron emission tomography (PET) plays key roles in drug discovery and development, as well as medical imaging. However, there is a dearth of efficient and simple radiolabeling methods for aromatic C-H bonds, which limits advancements in PET radiotracer development. Here, we disclose a mild method for the fluorine-18 (18F)-fluorination of aromatic C-H bonds by an [18F]F- salt via organic photoredox catalysis under blue light illumination. This strategy was applied to the synthesis of a wide range of 18F-labeled arenes and heteroaromatics, including pharmaceutical compounds. These products can serve as diagnostic agents or provide key information about the in vivo fate of the labeled substrates, as showcased in preliminary tracer studies in mice.

Synthesis and initial evaluation of radioactive 5-I-α-methyl-tryptophan: a Trp based agent targeting IDO-1.

A synthetic approach is established to achieve radioactive 5-I-α-methyl-tryptophan (5-I-AMT). An enzyme based assay demonstrated that 5-I-AMT is a substrate of IDO-1. The in vivo distribution and imaging potential of 5-I-AMT were explored with a B16F10 tumor model using I-124 as the radiotag.

Hydrophilic 18F-labeled trans-5-oxocene (oxoTCO) for efficient construction of PET agents with improved tumor-to-background ratios in neurotensin receptor (NTR) imaging.

An 18F-labeled trans-5-oxocene (oxoTCO) that is used to construct a PET probe for neurotensin receptor (NTR) imaging through tetrazine ligation is described here. PET probe construction proceeds with 70% RCY based on 18F-oxoTCO and is completed within seconds. The in vivo behaviour of the oxoTCO based PET probe was compared with those of analogous probes that were prepared from 18F-labeled s-TCO and d-TCO tracers. The hydrophilic 18F-oxoTCO probe showed a significantly higher tumor-to-background ratio while displaying comparable tumor uptake relative to the 18F-dTCO and 18F-sTCO derived probes.

Synthesis of 5-[18F]Fluoro-α-methyl Tryptophan: New Trp Based PET Agents.

Indoleamine 2,3-dioxygenase (IDO1) plays a special role in the biology of various cancer types, because it breaks down the essential amino acid tryptophan for immune cell activation. Upregulation of IDO1 significantly correlates with the number of various T cell types in tumor tissues in melanoma and other cancers, suggesting that IDO expression is linked with effective and ineffective ('exhausted') immune response in cancer. Based on the reported IDO inhibitors (α-Methylated and indole-N-methylated tryptophan (Trp)), here we report the synthesis of potential IDO1 imaging agents through direct introduction of 18F into the tryptophan aromatic ring. Overall, the resulting PET agents could be obtained in high radiochemical purity (>97%) with labeling yield ranges from 4.2-14.9% decay corrected yield. Using Trp as the model compound, our results also demonstrate that 18F could be directly introduced to the Trp backbone at the 4, 5, 6, and 7 position. Moreover, our initial imaging study suggests that 5-[18F]F-L-α-methyl tryptophan (5-[18F]F-AMT) holds great potential for cancer imaging. The success of this approach will provide researchers easy access to a library of Trp/Trp-derivative based PET agents for biomedical research, including potential IDO1 targeted imaging.

Synthesis and Evaluation of [(18) F]-Ammonium BODIPY Dyes as Potential Positron Emission Tomography Agents for Myocardial Perfusion Imaging.

Recently, we demonstrated the potential of a [(18) F]-trimethylammonium BODIPY dye for cardiac imaging. This is the first example of the use of the [(18) F]-ammonium BODIPY dye for positron emission tomography (PET) myocardial perfusion imaging (MPI). In this report, we extend our study to other ammonium BODIPY dyes with different nitrogen substituents. These novel ammonium BODIPY dyes were successfully prepared and radiolabeled by the SnCl4 -assisted (18) F-(19) F isotopic exchange method. The microPET results and the biodistribution data reveal that nitrogen substituent changes have a significant effect on the in vivo and pharmacological properties of the tracers. Of the novel [(18) F]-ammonium BODIPY dyes prepared in this work, the [(18) F]-dimethylethylammonium BODIPY is superior in terms of myocardium uptake and PET imaging contrast. These results support our hypothesis that the ammonium BODIPY dyes have a great potential for use as PET/optical dual-modality MPI probes.

PET Imaging of Dll4 Expression in Glioblastoma and Colorectal Cancer Xenografts Using (64)Cu-Labeled Monoclonal Antibody 61B.

Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel antihuman Dll4 monoclonal antibody (61B), we developed a (64)Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the (64)Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-(64)Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2%ID/g at 24 and 48 h postinjection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-(64)Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3%ID/g at 24 and 48 h postinjection, respectively). The tumor accumulation of 61B-DOTA-(64)Cu was significantly higher than that of hIgG-DOTA-(64)Cu in both xenografts models. Immunofluorescence staining of the tumor tissues further confirmed that tumor accumulation of 61B-Cy5.5 was correlated well with in vivo PET imaging data using 61B-DOTA-(64)Cu. In conclusion, 61B-DOTA-(64)Cu PET probe was successfully synthesized and demonstrated prominent tumor uptake by targeting Dll4. 61B-DOTA-(64)Cu has great potential to be used for noninvasive Dll4 imaging, which could be valuable for tumor detection, Dll4 expression level evaluation, and Dll4-based treatment monitoring.

Improved metabolic stability for 18F PET probes rapidly constructed via tetrazine trans-cyclooctene ligation.

The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an (18)F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a (19)F-labeled TCO derivative to give HPLC standards. The analogous (18)F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at µM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an (18)F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the (18)F-labeled TCO (concentration calculated based on product's specific activity). The radiochemical purity of the (18)F-RGD peptides was >95% and the specific activity was 111 GBq/µmol. Noninvasive microPET experiments demonstrated that (18)F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of (18)F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in (18)F-PET probe construction, providing nearly quantitative (18)F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.

Alkene hydrofunctionalization using hydroxamic acids: a radical-mediated approach to alkene hydration.

A radical-mediated approach to alkene hydration is described. The present strategy capitalizes on the unique radical reactivity of hydroxamic acids, which are capable of functioning as both synthetically useful oxygen-centered radical species and suitable hydrogen atom-based donors. This reaction manifold has been applied to both alkene hydrations and tandem alkene-alkene carbocyclization processes.

Metal-free, aerobic dioxygenation of alkenes using simple hydroxamic acid derivatives.

The dioxygenation of alkenes using molecular oxygen and a simple hydroxamic acid derivative has been achieved. The reaction system consists of readily prepared methyl N-hydroxy-N-phenylcarbamate and molecular oxygen with a radical initiator, offering an alternative to common dioxygenation processes catalyzed by precious transition metals. This transformation capitalizes on the unique reactivity profile of hydroxamic acid derivatives in radical-mediated alkene addition processes.

The application of (Z)-3-aryl-3-haloenoic acids to the synthesis of (Z)-5-benzylidene-4-arylpyrrol-2(5H)-ones.

Studies directed at the synthesis of (Z)-5-benzylidene-4-arylpyrrol-2(5H)-ones from (Z)-3-aryl-3-haloenoic acids are described. The successful strategy relies on the preparation of (Z)-3-aryl-3-haloenoic acids from acetophenones through the corresponding (Z)-3-aryl-3-haloenals and the conversion of the (Z)-3-aryl-3-haloenoic acids to (Z)-5-benzylidene-4-aryl-5H-furan-2-ones. The furanones were subsequently treated with primary amines and dehydrated to the corresponding (Z)-5-benzylidene-4-arylpyrrol-2(5H)-ones.

Further studies on vinamidinium salt amine exchange reactions, borohydride reductions and subsequent transformations.

Studies directed at the amine exchange reaction of vinamidinium salts followed by sodium borohydride reduction to secondary and tertiary allylic amines are described. The tertiary allylic amines were alkylated and subjected to base mediated rearrangement to yield a variety of highly functionalized tertiary homoallylic amines.

The application of vinylogous iminium salt derivatives to efficient formal syntheses of the marine akaloids lamellarin G trimethyl ether and ningalin B.

Studies directed at the synthesis of lamellarin G trimethyl ether and ningalin B via vinylogous iminium salt derivatives are described. The successful strategy relies on the formation of a 2,4-disubstituted pyrrole or a 1,2,3,4-tetrasubstituted pyrrole from a vinylogous iminium salt or vinylogous iminium salt derivative. Subsequent transformations of these highly substituted pyrroles lead to efficient and regiocontrolled formal syntheses of the respective pyrrole containing natural products.