Synthesis and Radiopharmacological Characterisation of a Fluorine‐18‐Labelled Azadipeptide Nitrile as a Potential PET Tracer for in vivo Imaging of Cysteine Cathepsins

A fluorinated cathepsin inhibitor based on the azadipeptide nitrile chemotype was prepared and selected for positron emission tomography (PET) tracer development owing to its high affinity for the oncologically relevant cathepsins L, S, K and B. Labelling with fluorine‐18 was accomplished in an efficient and reliable two‐step, one‐pot radiosynthesis by using 2‐[¹⁸F]fluoroethylnosylate as a prosthetic agent. The pharmacokinetic properties of the resulting radiotracer compound were studied in vitro, ex vivo and in vivo in normal rats by radiometabolite analysis and small‐animal positron emission tomography. These investigations revealed rapid conjugate formation of the tracer with glutathione in the blood, which is associated with slow blood clearance. The potential of the developed ¹⁸F‐labelled probe to image tumour‐associated cathepsin activity was investigated by dynamic small‐animal PET imaging in nude mice bearing tumours derived from the human NCI‐H292 lung carcinoma cell line. Computational analysis of the obtained image data indicated the time‐dependent accumulation of the radiotracer in the tumours. The expression of the target enzymes in the tumours was confirmed by immunohistochemistry with specific antibodies. This indicates that azadipeptide nitriles have the potential to target thiol‐dependent cathepsins in vivo despite their disadvantageous pharmacokinetics.     

Design,Synthesis, and Evaluation of an 18F‐Labeled Radiotracer Based on Celecoxib–NBD for Positron Emission Tomography (PET) Imaging of Cyclooxygenase‐2 (COX‐2)

A series of novel fluorine‐containing cyclooxygenase‐2 (COX‐2) inhibitors was designed and synthesized based on the previously reported fluorescent COX‐2 imaging agent celecoxib–NBD ( 3 ; NBD=7‐nitrobenzofurazan). In vitro COX‐1/COX‐2 inhibitory data show that N‐(4‐fluorobenzyl)‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide ( 5 ; IC₅₀=0.36 μM , SI>277) and N‐fluoromethyl‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide ( 6 ; IC₅₀=0.24 μM , SI>416) are potent and selective COX‐2 inhibitors. Compound 5 was selected for radiolabeling with the short‐lived positron emitter fluorine‐18 (¹⁸F) and evaluated as a positron emission tomography (PET) imaging agent. Radiotracer [¹⁸F] 5 was analyzed in vitro and in vivo using human colorectal cancer model HCA‐7. Although radiotracer uptake into COX‐2‐expressing HCA‐7 cells was high, no evidence for COX‐2‐specific binding was found. Radiotracer uptake into HCA‐7 tumors in vivo was low and similar to that of muscle, used as reference tissue.     

Probing the S1′ Site for the Identification of Non‐Zinc‐Binding MMP‐2 Inhibitors

Matrix metalloproteinases (MMPs) are zinc‐dependent enzymes involved in several pathological states. Among them, MMP‐2 is a relevant therapeutic target because of its role in cancer development and progression. Many MMP inhibitors (MMPIs) have been discovered over the last 30 years, and the majority of them contain a functional group that binds the zinc ion (zinc‐binding group; ZBG). Unfortunately, no MMPIs have reached the market yet, owing to toxic effects due to unselective interactions of the ZBG. The new generation of MMPIs that do not bind the zinc ion could overcome problems of selectivity and toxicity, but have so far been developed only for MMP‐8, ‐12, and ‐13. In this work, a virtual screening protocol was established by combining ligand‐ and structure‐based methods to identify non‐zinc‐binding MMP‐2 inhibitors using a new‐generation MMP‐8 inhibitor as a probe to find unexplored interactions in the MMP‐2 S1′ site. The screening allowed the identification of micromolar MMP‐2 inhibitors that putatively avoid binding the zinc ion, as demonstrated by docking calculations. The LIA model, built to correlate predicted and experimental binding energies of the identified non‐zinc‐binding MMP‐2 hits, underpins the reliability of the predicted docking poses.     

11C‐ and 18F‐Labeled Radioligands for P‐Glycoprotein Imaging by Positron Emission Tomography

P‐Glycoprotein (P‐gp) is an efflux transporter widely expressed at the human blood–brain barrier. It is involved in xenobiotics efflux and in onset and progression of neurodegenerative disorders. For these reasons, there is great interest in the assessment of P‐gp expression and function by noninvasive techniques such as positron emission tomography (PET). Three radiolabeled aryloxazole derivatives: 2‐[2‐(2‐methyl‐(¹¹C)‐5‐methoxyphenyl)oxazol‐4‐ylmethyl]‐6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline ([¹¹C]‐ 5 ); 2‐[2‐(2‐fluoromethyl‐(¹⁸F)‐5‐methoxyphenyl)oxazol‐4‐ylmethyl]‐6,7‐dimethoxy‐1,2,3,4‐tetra‐hydroisoquinoline ([¹⁸F]‐ 6 ); and 2‐[2‐(2‐fluoroethyl‐(¹⁸F)‐5‐methoxyphenyl)oxazol‐4‐ylmethyl]‐6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline ([¹⁸F]‐ 7 ), were tested in several in vitro biological assays to assess the effect of the aryl substituent in terms of potency and mechanism of action toward P‐gp. Methyl derivative [¹¹C]‐ 5 is a potent P‐gp substrate, whereas the corresponding fluoroethyl derivative [¹⁸F]‐ 7 is a P‐gp inhibitor. Fluoromethyl compound [¹⁸F]‐ 6 is classified as a non‐transported P‐gp substrate, because its efflux increases after cyclosporine A modulation. These studies revealed a promising substrate and inhibitor, [¹¹C]‐ 5 and [¹⁸F]‐ 7 , respectively, for in vivo imaging of P‐gp by using PET.     

[18F]SiFA‐isothiocyanate: A New Highly Effective Radioactive Labeling Agent for Lysine‐Containing Proteins

A highly efficient ¹⁸F‐labeling synthon for universal protein labeling is reported. Diverse ¹⁸F‐labeled proteins of 66–144 kDa were prepared with [¹⁸F]SiFA‐isothiocyanate synthesized by an isotopic ¹⁹F for ¹⁸F exchange at the silicon atom. Overall preparative radiochemical yields were 20–40 % after 40–50 min. No bone uptake of ¹⁸F radioactivity was detected until 90 min post‐injection of ¹⁸F‐SiFA‐RSA; this demonstrates the metabolic stability of the [¹⁸F]SiFA moiety.

     

Imaging Memory T-Cells Stratifies Response to Adjuvant Metformin Combined with αPD-1 Therapy

The low response rates associated with immune checkpoint inhibitor (ICI) use has led to a surge in research investigating adjuvant combination strategies in an attempt to enhance efficacy. Repurposing existing drugs as adjuvants accelerates the pace of cancer immune therapy research; however, many combinations exacerbate the immunogenic response elicited by ICIs and can lead to adverse immune-related events. Metformin, a widely used type 2 diabetes drug is an ideal candidate to repurpose as it has a good safety profile and studies suggest that metformin can modulate the tumour microenvironment, promoting a favourable environment for T cell activation but has no direct action on T cell activation on its own. In the current study we used PET imaging with [¹⁸F]AlF-NOTA-KCNA3P, a radiopharmaceutical specifically targeting K_V1.3 the potassium channel over-expressed on active effector memory T-cells, to determine whether combining PD1 with metformin leads to an enhanced immunological memory response in a preclinical colorectal cancer model. Flow cytometry was used to assess which immune cell populations infiltrate the tumours in response to the treatment combination. Imaging with [¹⁸F]AlF-NOTA-KCNA3P demonstrated that adjuvant metformin significantly improved anti-PD1 efficacy and led to a robust anti-tumour immunological memory response in a syngeneic colon cancer model through changes in tumour infiltrating effector memory T-cells.     

Exploration of Sulfur‐Containing Analogues for Imaging Vesicular Acetylcholine Transporter in the Brain

Sixteen new sulfur‐containing compounds targeting the vesicular acetylcholine transporter (VAChT) were synthesized and assessed for in vitro binding affinities. Enantiomers (?)‐(1‐(3‐hydroxy‐1,2,3,4‐tetrahydronaphthalen‐2‐yl)piperidin‐4‐yl)(4‐(methylthio)phenyl)methanone [(?)‐ 8 ] and (?)‐(4‐((2‐fluoroethyl)thio)phenyl)(1‐(3‐hydroxy‐1,2,3,4‐tetrahydronaph‐thalen‐2‐yl)piperidin‐4‐yl)methanone [(?)‐ 14 a ] displayed high binding affinities, with respective K_i values of 1.4 and 2.2 nm for human VAChT, moderate and high selectivity for human VAChT over σ₁ (≈13‐fold) and σ₂ receptors (>420‐fold). Radiosyntheses of (?)‐[¹¹C] 8 and (?)‐[¹⁸F] 14 a were achieved using conventional methods. Ex vivo autoradiography and biodistribution studies in Sprague–Dawley rats indicated that both radiotracers have the capacity to penetrate the blood–brain barrier, with high initial brain uptake at 5 min and rapid washout. The striatal region had the highest accumulation for both radiotracers. Pretreating the rats with the VAChT ligand (?)‐vesamicol decreased brain uptake for both radiotracers. Pretreating the rats with the σ₁ ligand YUN‐122 (N‐(4‐benzylcyclohexyl)‐2‐(2‐fluorophenyl)acetamide) also decreased brain uptake, suggesting these two radiotracers also bind to the σ₁ receptor in vivo. The microPET study of (?)‐[¹¹C] 8 in the brain of a non‐human primate showed high striatal accumulation that peaked quickly and washed out rapidly. Although preliminary results indicated these two sulfur‐containing radiotracers have high binding affinities for VAChT with rapid washout kinetics from the striatum, their σ₁ receptor binding properties limit their potential as radiotracers for quantifying VAChT in vivo.     

A Series of 18F‐Labelled Pyridinylphenyl Amides as Subtype‐Selective Radioligands for the Dopamine D3 Receptor

Synthesis, biological activity, and structure–selectivity relationship (SSR) studies of a novel series of potential dopamine D3 receptor radioligands as imaging agents for positron emission tomography (PET) are reported. Considering a structurally diverse library of D3 ligands, SSR studies were performed for a new series of fluorinated pyridinylphenyl amides using CoMFA and CoMSIA methods. The in vitro D3 affinities of the predicted series of biphenyl amide ligands 9 a – d revealed single‐digit to sub‐nanomolar potencies (K_i=0.52–1.6 nM ), displaying excellent D3 selectivity over the D2 subtype of 110‐ to 210‐fold for the test compounds 9 a – c . Radiofluorination by nucleophilic substitution of Br or NO₂ by ¹⁸F led to radiochemical yields of 66–92 % for [¹⁸F] 9 a – d . However, the specific activities of [¹⁸F] 9 b and [¹⁸F] 9 d were insufficient, rendering their use for in vivo studies impossible. Biodistribution studies of [¹⁸F] 9 a and [¹⁸F] 9 c using rat brain autoradiography revealed accumulation in the ventricles, thus indicating insufficient biokinetic properties of [¹⁸F] 9 a and [¹⁸F] 9 c for D3 receptor imaging in vivo.     

Synthesis,Biological Evaluation and Molecular Modeling Studies of Propargyl‐Containing 2,4,6‐Trisubstituted Pyrimidine Derivatives as Potential Anti‐Parkinson Agents

Monoamine oxidase B (MAO‐B) inhibitors are potential drug candidates for the treatment of various neurological disorders including Parkinson's disease. A total of 20 new propargyl‐containing 2,4,6‐trisubstituted pyrimidine derivatives were synthesized and screened for MAO inhibition using Amplex Red assays. All the synthesized compounds were found to be reversible and selective inhibitors of the MAO‐B isoform at sub‐micromolar concentrations. MVB3 was the most potent MAO‐B inhibitor with an IC₅₀ value of 0.38±0.02 μμ , whereas MVB6 (IC₅₀=0.51±0.04 μμ ) and MVB16 (IC₅₀=0.48±0.06 μμ ) were the most selective for MAO‐B with a selectivity index of more than 100‐fold. In cytotoxic studies, these compounds were found to be nontoxic to human neuroblastoma SH‐SY5Y cells at concentrations of 25 μm . MVB6 was found to decrease the intracellular level of reactive oxygen species to 68 % at 10 μm concentration, whereas other compounds did not produce significant changes in reactive oxygen species levels. In molecular modeling studies, MVB3 displayed strong binding affinity for the MAO‐B isoform with a dock score of ?10.45, in agreement with the observed activity. All the compounds fitted well in the hydrophobic cavity of MAO‐B. Thus, propargyl‐substituted pyrimidine derivatives can be promising leads in the development of potent, selective and reversible MAO‐B inhibitors for the treatment of Parkinson's disease.     

In situ generation of hydrogen peroxide and its use for enzymatic degradation of 2,4,6‐trinitrotoluene

A novel electroenzymatic method hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase (Lip) for treatment of TNT (2,4,6‐trinitrotoluene) waste was studied. In the presence of Phanerochaete chrysosporium lignin peroxidase, TNT was degraded in an electrochemical reactor using hydrogen peroxide, produced by an electrode reaction. The effectiveness of the electroenzymatic method was examined in this study. The efficiency of removal of TNT was greater than that of biochemical methods under optimal conditions. The effects of reaction conditions on TNT degradation and denitrification, TOC removal efficiency, and power consumption were also investigated. A potential (vs Ag/AgCl) of +0.1 V was selected as being optimal for the electroenzymatic reaction. TNT degradation was significantly improved in the combined veratryl alcohol–lignin peroxidase oxidation procedure, showing complementary effects of veratryl alcohol (VA) in the TNT degradation reaction. Denitrification was found to be proportional to the amount of TNT degraded. One intermediate, 2,4‐diamino‐6‐nitrotoluene, derived from the oxidative degradation of TNT, was eluted on an HPLC chromatogram and was detected using mass spectrometry. The electroenzymatic method had a lower power requirement than electrochemical oxidation at ?0.2 and ?0.4 V. The electroenzymatic method may be easily applied to biodegradation systems and provide added benefit for highly recalcitrant chemicals since the system would not be susceptible to the toxicity of the chemical. Also H₂O₂ instability and decomposition in the samples after preparation would not be of concern. © 2001 Society of Chemical Industry     

Design of gem‐Difluoro‐bis‐Tetrahydrofuran as P2 Ligand for HIV‐1 Protease Inhibitors to Improve Brain Penetration: Synthesis,X‐ray Studies,and Biological Evaluation

The structure‐based design, synthesis, biological evaluation, and X‐ray structural studies of fluorine‐containing HIV‐1 protease inhibitors are described. The synthesis of both enantiomers of the gem‐difluoro‐bis‐THF ligands was carried out in a stereoselective manner using a Reformatskii–Claisen reaction as the key step. Optically active ligands were converted into protease inhibitors. Two of these inhibitors, (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐methoxyphenyl)sulfonamido)‐1‐phenylbutan‐2‐yl) carbamate ( 3 ) and (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐aminophenyl)sulfonamido)phenylbutan‐2‐yl) carbamate ( 4 ), exhibited HIV‐1 protease inhibitory K_i values in the picomolar range. Both 3 and 4 showed very potent antiviral activity, with respective EC₅₀ values of 0.8 and 3.1 nM against the laboratory strain HIV‐1_LAI. The two inhibitors exhibited better lipophilicity profiles than darunavir, and also showed much improved blood–brain barrier permeability in an in vitro model. A high‐resolution X‐ray structure of inhibitor 4 in complex with HIV‐1 protease was determined, revealing that the fluorinated ligand makes extensive interactions with the S2 subsite of HIV‐1 protease, including hydrogen bonding interactions with the protease backbone atoms. Moreover, both fluorine atoms on the bis‐THF ligand formed strong interactions with the flap Gly 48 carbonyl oxygen atom.     

Comparison of in Silico,Electrochemical, in Vitro and in Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography

The imaging of σ₁ receptors in the brain by fluorinated radiotracers will be used for the validation of σ₁ receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1′‐benzyl‐3‐(ω‐fluoromethyl to ω‐fluorobutyl)‐3H‐spiro[2]benzofuran‐1,4′‐piperidine] ([¹⁸F] 1 – 4 ) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [¹⁸F] 1 – 4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC–MS) analysis allowed structural identification of non‐radioactive metabolites. Radio‐HPLC and radio‐TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [¹⁸F] 2 – 4 , but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [¹⁸F] 4 reached the brain, rendering [¹⁸F] 4 less appropriate as a PET tracer than [¹⁸F] 2 and [¹⁸F] 3 . Compounds [¹⁸F] 2 and [¹⁸F] 3 possess the most promising properties for imaging of σ₁ receptors in the brain. High σ₁ affinity (K_i=0.59 nm ), low lipophilicity (logD_7.4=2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [¹⁸F] 2 slightly over the fluoropropyl derivative [¹⁸F] 3 for human use.     

Inspired by Nature: The 3‐Halo‐4,5‐dihydroisoxazole Moiety as a Novel Molecular Warhead for the Design of Covalent Inhibitors

Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re‐emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3‐chloro‐4,5‐dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3‐halo‐4,5‐dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3‐halo‐4,5‐dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors.     

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis,Fluorescent Properties,and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.