Intramolecular Hydrogen Bonding Based CEST MRI Contrast Agents As an Emerging Design Strategy: A Mini-Review.

Intramolecular hydrogen bonding-based chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) contrast agents represent an innovative design strategy aiming to overcome limitations in diamagnetic CEST (diaCEST) MRI contrast agent specificity and also those associated with traditional metal-based MRI contrast agents. Ward and Balaban's proposal of small diamagnetic compounds marked a paradigm shift in contrast-based radiologic research, inspiring extensive investigations since 2000. These contrast agents leverage labile hydrogen bonds, serving as chemical exchange sites to induce saturation of water. The selective manipulation of radiofrequency (RF) allows for optimized signal contrast in soft tissue, with a significant signal amplification even at low probe concentrations, mitigating concerns about dose-dependent toxicities. This mini-review delves into the evolution of CEST MRI, its classification, and the strategic design principles of synthetic small molecules containing intramolecular hydrogen bonds. With a focus on applications and potential clinical relevance, the authors highlight the promising role of intramolecular hydrogen bonding-based CEST MRI in diverse medical contexts, especially renal imaging and pH mapping, paving the way for enhanced molecular imaging capabilities. Ongoing research endeavors aim to further optimize and expand the utility of these contrast agents, underscoring their transformative potential in clinical diagnostics and imaging.

Discovery of tert-Butyl Ester Based 6-Diazo-5-oxo-l-norleucine Prodrugs for Enhanced Metabolic Stability and Tumor Delivery.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) exhibits remarkable anticancer efficacy; however, its therapeutic potential is hindered by its toxicity to gastrointestinal (GI) tissues. We recently reported the discovery of DRP-104, a tumor-targeted DON prodrug with excellent efficacy and tolerability, which is currently in clinical trials. However, DRP-104 exhibits limited aqueous solubility, and the instability of its isopropyl ester promoiety leads to the formation of an inactive M1-metabolite, reducing overall systemic prodrug exposure. Herein, we aimed to synthesize DON prodrugs with various ester and amide promoieties with improved solubility, GI stability, and DON tumor delivery. Twenty-one prodrugs were synthesized and characterized in stability and pharmacokinetics studies. Of these, P11, tert-butyl-(S)-6-diazo-2-((S)-2-(2-(dimethylamino)acetamido)-3-phenylpropanamido)-5-oxo-hexanoate, showed excellent metabolic stability in plasma and intestinal homogenate, high aqueous solubility, and high tumor DON exposures and preserved the ideal tumor-targeting profile of DRP-104. In conclusion, we report a new generation of glutamine antagonist prodrugs with improved physicochemical and pharmacokinetic attributes.

Synthesis and Pharmacological Characterization of a Difluorinated Analogue of Reduced Haloperidol as a Sigma-1 Receptor Ligand.

Reduced haloperidol (1) was previously reported to act as a potent sigma-1 receptor (S1R) ligand with substantially lower affinity to the dopamine D2 receptor (D2R) compared to haloperidol. It was also found to facilitate brain-derived neurotrophic factor (BDNF) secretion from astrocytic glial cell lines in a sigma-1 receptor (S1R)-dependent manner. Although an increase in BDNF secretion may have beneficial effects in some neurological conditions, the therapeutic utility of reduced haloperidol (1) is limited because it can be oxidized back to haloperidol in the body, a potent dopamine D2 receptor antagonist associated with well-documented adverse effects. A difluorinated analogue of reduced haloperidol, (±)-4-(4-chlorophenyl)-1-(3,3-difluoro-4-(4-fluorophenyl)-4-hydroxybutyl)piperidin-4-ol (2), was synthesized in an attempt to minimize the oxidation. Compound (±)-2 was found to exhibit high affinity to S1R and facilitate BDNF release from mouse brain astrocytes. It was also confirmed that compound 2 cannot be oxidized back to the corresponding haloperidol analogue in liver microsomes. Furthermore, compound 2 was distributed to the brain following intraperitoneal administration in mice and reversed the learning deficits in active avoidance tasks. These findings suggest that compound 2 could serve as a promising S1R ligand with therapeutic potential for the treatment of cognitive impairments.

Topical SCD-153, a 4-methyl itaconate prodrug, for the treatment of alopecia areata.

Alopecia areata is a chronic hair loss disorder that involves autoimmune disruption of hair follicles by CD8+  T cells. Most patients present with patchy hair loss on the scalp that improves spontaneously or with topical and intralesional steroids, topical minoxidil, or topical immunotherapy. However, recurrence of hair loss is common, and patients with extensive disease may require treatment with oral corticosteroids or oral Janus kinase (JAK) inhibitors, both of which may cause systemic toxicities with long-term use. Itaconate is an endogenous molecule synthesized in macrophages that exerts anti-inflammatory effects. To investigate the use of itaconate derivatives for treating alopecia areata, we designed a prodrug of 4-methyl itaconate (4-MI), termed SCD-153, with increased lipophilicity compared to 4-MI (CLogP 1.159 vs. 0.1442) to enhance skin and cell penetration. Topical SCD-153 formed 4-MI upon penetrating the stratum corneum in C57BL/6 mice and showed low systemic absorption. When added to human epidermal keratinocytes stimulated with polyinosinic-polycytidylic acid (poly I:C) or interferon (IFN)γ, SCD-153 significantly attenuated poly I:C-induced interleukin (IL)-6, Toll-like receptor 3, IL-1ß, and IFNß expression, as well as IFNγ-induced IL-6 expression. Topical application of SCD-153 to C57BL/6 mice in the resting (telogen) phase of the hair cycle induced significant hair growth that was statistically superior to vehicle (dimethyl sulfoxide), the less cell-permeable itaconate analogues 4-MI and dimethyl itaconate, and the JAK inhibitor tofacitinib. Our results suggest that SCD-153 is a promising topical candidate for treating alopecia areata.

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II.

Glutamate carboxypeptidase-II (GCPII) is a zinc-dependent metalloenzyme implicated in numerous neurological disorders. The pharmacophoric requirements of active-site GCPII inhibitors makes them highly charged, manifesting poor pharmacokinetic (PK) properties. Herein, we describe the discovery and characterization of catechol-based inhibitors including L-DOPA, D-DOPA, and caffeic acid, with sub-micromolar potencies. Of these, D-DOPA emerged as the most promising compound, with good metabolic stability, and excellent PK properties. Orally administered D-DOPA yielded high plasma exposures (AUCplasma = 72.7 nmol·h/mL) and an absolute oral bioavailability of 47.7%. Unfortunately, D-DOPA brain exposures were low with AUCbrain = 2.42 nmol/g and AUCbrain/plasma ratio of 0.03. Given reports of isomeric inversion of D-DOPA to L-DOPA via D-amino acid oxidase (DAAO), we evaluated D-DOPA PK in combination with the DAAO inhibitor sodium benzoate and observed a >200% enhancement in both plasma and brain exposures (AUCplasma = 185 nmol·h/mL; AUCbrain = 5.48 nmol·h/g). Further, we demonstrated GCPII target engagement; orally administered D-DOPA with or without sodium benzoate caused significant inhibition of GCPII activity. Lastly, mode of inhibition studies revealed D-DOPA to be a noncompetitive, allosteric inhibitor of GCPII. To our knowledge, this is the first report of D-DOPA as a distinct scaffold for GCPII inhibition, laying the groundwork for future optimization to obtain clinically viable candidates.

Discovery of Orally Bioavailable and Brain-Penetrable Prodrugs of the Potent nSMase2 Inhibitor DPTIP.

Extracellular vesicles (EVs) can carry pathological cargo and play an active role in disease progression. Neutral sphingomyelinase-2 (nSMase2) is a critical regulator of EV biogenesis, and its inhibition has shown protective effects in multiple disease states. 2,6-Dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazol-2-yl)phenol (DPTIP) is one of the most potent (IC50 = 30 nM) inhibitors of nSMase2 discovered to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), limiting its clinical development. To overcome DPTIP's PK limitations, we synthesized a series of prodrugs by masking its phenolic hydroxyl group. When administered orally, the best prodrug (P18) with a 2',6'-diethyl-1,4'-bipiperidinyl promoiety exhibited >fourfold higher plasma (AUC0-t = 1047 pmol·h/mL) and brain exposures (AUC0-t = 247 pmol·h/g) versus DPTIP and a significant enhancement of DPTIP half-life (2 h vs ∼0.5 h). In a mouse model of acute brain injury, DPTIP released from P18 significantly inhibited IL-1ß-induced EV release into plasma and attenuated nSMase2 activity. These studies report the discovery of a DPTIP prodrug with potential for clinical translation.

Lipid Peroxidation Produces a Diverse Mixture of Saturated and Unsaturated Aldehydes in Exhaled Breath That Can Serve as Biomarkers of Lung Cancer-A Review.

The peroxidation of unsaturated fatty acids is a widely recognized metabolic process that creates a complex mixture of volatile organic compounds including aldehydes. Elevated levels of reactive oxygen species in cancer cells promote random lipid peroxidation, which leads to a variety of aldehydes. In the case of lung cancer, many of these volatile aldehydes are exhaled and are of interest as potential markers of the disease. Relevant studies reporting aldehydes in the exhaled breath of lung cancer patients were collected for this review by searching the PubMed and SciFindern databases until 25 May 2022. Information on breath test results, including the biomarker collection, preconcentration, and quantification methods, was extracted and tabulated. Overall, 44 studies were included spanning a period of 34 years. The data show that, as a class, aldehydes are significantly elevated in the breath of lung cancer patients at all stages of the disease relative to healthy control subjects. The type of aldehyde detected and/or deemed to be a biomarker is highly dependent on the method of exhaled breath sampling and analysis. Unsaturated aldehydes, detected primarily when derivatized during preconcentration, are underrepresented as biomarkers given that they are also likely products of lipid peroxidation. Pentanal, hexanal, and heptanal were the most reported aldehydes in studies of exhaled breath from lung cancer patients.

Clinical development of metabolic inhibitors for oncology.

Metabolic inhibitors have been used in oncology for decades, dating back to antimetabolites developed in the 1940s. In the past 25 years, there has been increased recognition of metabolic derangements in tumor cells leading to a resurgence of interest in targeting metabolism. More recently there has been recognition that drugs targeting tumor metabolism also affect the often acidic, hypoxic, immunosuppressive tumor microenvironment (TME) and non-tumor cell populations within it, including immune cells. Here we review small-molecule metabolic inhibitors currently in clinical development for oncology applications. For each agent, we evaluate the preclinical studies demonstrating antitumor and TME effects and review ongoing clinical trials. The goal of this Review is to provide an overview of the landscape of metabolic inhibitors in clinical development for oncology.

Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement.

BACKGROUND: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. OBJECTIVE: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. METHODS: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. RESULTS: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. CONCLUSION: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry.

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4 ± 11.5 and 10.1 ± 4.0 nmol/mg protein, respectively.

A chemoselective, one-pot transformation of aldehydes to nitriles.

This paper describes a procedure for direct conversion of aldehydes to nitriles using O-(diphenylphosphinyl)hydroxylamine (DPPH). Aldehydes are smoothly transformed to their corresponding nitriles by heating with DPPH in toluene. The reaction can be accomplished in the presence of alcohol, ketone, ester, or amine functionality.