N-Methylpropargylamine-Conjugated Hydroxamic Acids as Dual Inhibitors of Monoamine Oxidase A and Histone Deacetylase for Glioma Treatment.

Glioma treatment remains a challenge with a low survival rate due to the lack of effective therapeutics. Monoamine oxidase A (MAO A) plays a role in glioma development, and MAO A inhibitors reduce glioma growth. Histone deacetylase (HDAC) inhibition has emerged as a promising therapy for various malignancies including gliomas. We have synthesized and evaluated N-methylpropargylamine-conjugated hydroxamic acids as dual inhibitors of MAO A and HDAC. Compounds display potent MAO A inhibition with IC50 from 0.03 to <0.0001 µM and inhibit HDAC isoforms and cell growth in the micromolar to nanomolar IC50 range. These selective MAO A inhibitors increase histone H3 and α-tubulin acetylation and induce cell death via nonapoptotic mechanisms. Treatment with 15 reduced tumor size, reduced MAO A activity in brain and tumor tissues, and prolonged the survival. This first report on dual inhibitors of MAO A and HDAC establishes the basis of translational research for an improved treatment of glioma.

Reconstitution and Structural Analysis of a HECT Ligase-Ubiquitin Complex via an Activity-Based Probe.

Ubiquitin activity-based probes have proven invaluable in elucidating structural mechanisms in the ubiquitin system by stabilizing transient macromolecular complexes of deubiquitinases, ubiquitin-activating enzymes, and the assemblies of ubiquitin-conjugating enzymes with ubiquitin ligases of the RING-Between-RING and RING-Cysteine-Relay families. Here, we demonstrate that an activity-based probe, ubiquitin-propargylamine, allows for the preparative reconstitution and structural analysis of the interactions between ubiquitin and certain HECT ligases. We present a crystal structure of the ubiquitin-linked HECT domain of HUWE1 that defines a catalytically critical conformation of the C-terminal tail of the ligase for the transfer of ubiquitin to an acceptor protein. Moreover, we observe that ubiquitin-propargylamine displays selectivity among HECT domains, thus corroborating the notion that activity-based probes may provide entry points for the development of specific, active site-directed inhibitors and reporters of HECT ligase activities.

Synthesis of propargylamine mycophenolate analogues and their selective cytotoxic activity towards neuroblastoma SH-SY5Y cell line.

Twenty six propargylamine mycophenolate analogues were designed and synthesized from mycophenolic acid 1 employing a key step A3-coupling reaction. Their cytotoxic activity was examined against six cancer cell lines. Compounds 6a, 6j, 6t, 6u, and 6z exhibited selective cytotoxicity towards neuroblastoma (SH-SY5Y) cancer cells and were less toxic to normal cells in comparison to the lead compound, MPA 1 and a standard drug, ellipticine. Molecular docking results suggested that compound 6a is fit well in the key amino acid of three proteins (CDK9, EGFR, and VEGFR-2) as targets in cancer therapy. The propargylamine mycophenolate scaffold might be a valuable starting point for development of new neuroblastoma anticancer drugs.

Exploring the Versatility of the Covalent Thiol-Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease.

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CDWT and catalytically inactive mutant USP16CDC205A. Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.

Immobilized Ag NPs on chitosan-biguanidine coated magnetic nanoparticles for synthesis of propargylamines and treatment of human lung cancer.

The magnetically isolable nanobiocomposites have significant impact as the modified new generation catalysts in recent days. This has persuaded us to design and synthesis of a novel Ag NPs decorated biguanidine-chitosan (Bigua-CS) dual biomolecular functionalized core-shell type magnetic nanocomposite (Ag/Bigua-CS@Fe3O4). Bigua-CS could be introducing polysaccharide materials as potential coating agent to immobilizing and stabilizing metal nanoparticles. The material was characterized using several advanced techniques like fourier transformed infrared spectroscopy (FT-IR), inductively coupled plasma (ICP), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX), atomic mapping, high resolution transmission electron microscopy (HR-TEM), vibrating sample magnetometer (VSM) and X-ray diffraction (XRD). Towards the chemical applications of the material, we headed the multicomponent synthesis of diverse propargylamines by A3 coupling in water, which ended up with excellent yields. Due to strong paramagnetism, the catalyst was easily isolable and reused in 9cycles without any leaching and considerable change in reactivity. In addition, the catalyst was engaged in biological assays like study of anti-oxidant properties by DPPH mediated free radical scavenging test using BHT as a reference molecule. Thereafter, on having a significant IC50 value in radical scavenging assay, we extended the bio-application of the catalyst in anticancer study of adenocarcinoma cells of human lungs. The three different cancer cell lines, PC-14, LC-2/ad and HLC-1 were used in this regard. The best result was achieved in the case of PC-14 cell line with strong IC50 values.

Propargylamine-selective dual fluorescence turn-on method for post-synthetic labeling of DNA.

We have developed a propargylamine-selective dual fluorescence turn-on system, using ylidenemalononitrile enamines, for post-synthetic DNA labeling, allowing the direct monitoring of DNA using dual emission in living cells.

Targeted delivery and enhanced gene-silencing activity of centrally modified folic acid-siRNA conjugates.

One of the major hurdles in RNAi research has been the development of safe and effective delivery systems for siRNAs. Although various chemical modifications have been proposed to improve their pharmacokinetic behaviour, their delivery to target cells and tissues presents many challenges. In this work, we implemented a receptor-targeting strategy to selectively deliver siRNAs to cancer cells using folic acid as a ligand. Folic acid is capable of binding to cell-surface folate receptors with high affinity. These receptors have become important molecular targets for cancer research as they are overexpressed in numerous cancers despite being expressed at low levels in normal tissues. Employing a post-column copper-catalyzed alkyne-azide cycloaddition (CuAAC), we report the synthesis of siRNAs bearing folic acid modifications at different positions within the sense strand. In the absence of a transfection carrier, these siRNAs were selectively taken up by cancer cells expressing folate receptors. We show that centrally modified folic acid-siRNAs display enhanced gene-silencing activity against an exogenous gene target (∼80% knockdown after 0.75 µM treatment) and low cytotoxicity. In addition, these siRNAs achieved potent dose-dependent knockdown of endogenous Bcl-2, an important anti-apoptotic gene.

One-pot synthesis of propargylamines using magnetic mesoporous polymelamine formaldehyde/zinc oxide nanocomposite as highly efficient, eco-friendly and durable nanocatalyst: optimization by DOE approach.

Magnetic mesoporous polymelamine formaldehyde nanocomposite-incorporating ZnO nanoparticles were successfully synthesized using solvothermal and sol-gel methods. Fourier-transform infrared spectrometry (FT-IR), X-ray diffraction, Brunauer-Emmett-Teller, vibrating sample magnetometer, thermogravimetric analysis, elemental analysis, transmission electron microscopy and field emission scanning electron microscopy techniques were then utilized for evaluation of nanocomposites. The as-prepared nanocomposite can be used as heterogeneous nanocatalyst with remarkable performance for A3 coupling reaction toward one-pot synthesis of propargylamine and its derivatives under solvent-less condition. In order to maximize the product yield, the variables, i.e., reaction time, temperature and catalyst amount, were optimized by using a statistical approach. The synthesized nanocomposite can be easily separated from the reaction medium and reused over and over, without significant changes in its catalytic activity.

Evaluation of drug candidature: In silico ADMET, binding interactions with CDK7 and normal cell line studies of potentially anti-breast cancer enamidines.

We have recently explored novel class of potentially anti-breast cancer active enamidines in which four molecules 4a-c and 4h showed higher anticancer activity compared to standard drug doxorubicin. As a part of extension of this work, we have further evaluated in silico cheminformatic studies on bioactivity prediction of synthesized series of enamidines using mole information. The normal cell line study of four lead compounds 4a-c and 4h against African green monkey kidney vero strain further revealed that the compounds complemented good selectivity in inhibition of cancer cells. The in silico bioactivity and molecular docking studies also revealed that the compounds have significant interactions with the drug targets. The results reveal that enamidine moieties are vital for anti-breast cancer activity as they possess excellent drug-like characteristics, being potentially good inhibitors of cyclin dependent kinases7 (CDK7).

A fragment-like approach to PYCR1 inhibition.

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays.

Building and Breaking Bonds via a Compact S-Propargyl-Cysteine to Chemically Control Enzymes and Modify Proteins.

Analogous to reversible post-translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S-propargyl-cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol-yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd-cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.

Structure-Activity Relationship of Propargylamine-Based HDAC Inhibitors.

As histone deacetylases (HDACs) play an important role in the treatment of cancer, their selective inhibition has been the subject of various studies. These continuous investigations have given rise to a large collection of pan- and selective HDAC inhibitors, containing diverse US Food and Drug Administration (FDA)-approved representatives. In previous studies, a class of alkyne-based HDAC inhibitors was presented. We modified this scaffold in two previously neglected regions and compared their cytotoxicity and affinity toward HDAC1, HDAC6, and HDAC8. We were able to show that R-configured propargylamines contribute to increased selectivity for HDAC6. Docking studies on available HDAC crystal structures were carried out to rationalize the observed selectivity of the compounds. Substitution of the aromatic portion by a thiophene derivative results in high affinity and low cytotoxicity, indicating an improved drug tolerance.

Design, synthesis and evaluation of coumarin-pargyline hybrids as novel dual inhibitors of monoamine oxidases and amyloid-ß aggregation for the treatment of Alzheimer's disease.

A series of coumarin-pargyline hybrids (4a-x) have been designed, synthesized and evaluated as novel dual inhibitors of Alzheimer's disease (AD). Most of the compounds exhibited a potent ability to inhibit amyloid-ß (Aß) aggregation and monoamine oxidases. In particular, compound 4x exhibited remarkable inhibitory activities against monoamine oxidases (IC50, 0.027 ± 0.004 µM for MAO-B; 3.275 ± 0.040 µM for MAO-A) and Aß1-42 aggregation (54.0 ± 1.1%, 25 µM). Moreover, compound 4x showed low toxicity according to in vitro cell toxicity test. The results of the parallel artificial membrane permeability assay for blood-brain barrier indicated that compound 4x would be potent to cross the blood-brain barrier. Collectively, these findings demonstrate that compound 4x was an effective and promising candidate for AD therapy.

A bioorthogonal nanosystem for imaging and in vivo tumor inhibition.

Bioorthogonal bond-cleavage reactions have emerged as promising tools for manipulating biological processes, still the therapeutic effect of these reactions in vivo needs to be explored. Herein a bioorthogonal-activated prodrug has been developed for bioimaging and therapy, which is composed of a Pd-mediated cleavable propargyl, a coumarin fluorophore and a potent anticancer drug. In vitro investigations show that, the presence of a Pd complex induces the cleavage of propargyl and subsequently trigger the cascade of reactions, thereby activating the coumarin fluorophore for imaging and releasing the anticancer drug for therapy. Both the prodrug and Pd complex were then separately encapsulated into phospholipid liposomes to form a two-component bioorthogonal nanosystem. The lyposomal nanosystem can be readily internalized by HeLa cells and displays strong intracellular fluorescence under one- or two-photon excitation, indicating the release of the active drug in cells as a result of the Pd-mediated bioorthogonal bond-cleavage reaction. More importantly, the nanosystem shows considerable high activity and exerts efficient inhibition towards tumor growth in a mouse model. This work demonstrates that, if properly formulated, a bioorthogonal system can perform well in vivo. This strategy may offer a new approach for designing bioorthogonal prodrugs with imaging and therapeutic capability.

Propargyl-Assisted Selective Amidation Applied in C-terminal Glycine Peptide Conjugation.

Alkyl esters, such as propargyl esters, typically lack the electron-withdrawing inductive effects needed to participate in nucleophilic acyl substitution reactions. Herein, we report an unusual observation in which glycine propargyl ester derivatives displayed selective, base-independent reactivity towards linear alkylamines under mild, metal-free conditions. Through global reaction route mapping (GRRM) modeling calculations, it is predicted that these observations may be governed by factors related to hydrogen-bonding and intermolecular interactions, rather than electron-withdrawing inductive effects. Based on this concept of propargyl-assisted selective amidation, a direct application was made to develop a novel site-specific C-terminal glycine peptide bioconjugation technique as a proof-of-concept, which relies upon the selective reactivity of glycine propargyl esters over that of aspartate and glutamate side-chain-linked propargyl esters.

Synthesis and biological evaluation of novel propargylquinobenzothiazines and their derivatives as potential antiproliferative, anti-inflammatory, and anticancer agents.

Azaphenothiazines containing the quinoline ring, 8-10-substituted 6H-quinobenzothiazines and 6H-diquinothiazine were transformed into new 6-propargyl and 6-dialkylaminobutynyl derivatives containing the triple bond. Most of them displayed strong antiproliferative actions against human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin A (PHA), strongly suppressed lipopolysaccharide (LPS)-induced TNF-α production by whole blood human cell cultures, and exhibited low cytotoxicity. Three propargylquinobenzothiazines with the bromine, trifluoromethyl, and methylthio groups at position 9 and propargyldiquinothiazine exhibited comparable actions to cisplatin against the L-1210 and SW-948 tumor lines. 6-Propargyl-9-trifluoromethylquinobenzothiazine was shown to block caspase 3 expression and inhibit expression of caspase 8 and 9 in Jurkat cells indicating its possible mechanism of action. These derivatives could be promising, potential therapeutics for treatment of neoplastic diseases and autoimmune disorders.

Base-Promoted N-Pyridylation of Heteroarenes Using N-Propargyl Enaminones as Equivalents of Pyridine Scaffolds.

N-Pyridylation of various N-heteroarenes, including N-heteroarene-containing peptides, was achieved using N-propargyl enaminones (isolated or generated in situ from propargyl amine and propynones) as masked polysubstituted pyridine cores. This metal-free procedure proceeds under mild reaction conditions and generates 1 equiv of H2O as the sole byproduct.

Visual detection of formaldehyde by highly selective fluorophore labeling via gold(III) complex-mediated three-component coupling reaction.

A novel method for visual detection of formaldehyde with excellent selectivity via a gold(iii) complex-mediated three-component coupling reaction of resin-linked sterically bulky amines and fluorescent alkynes has been developed.

Spectroscopic studies of the equilibrium between complexes of lasalocid acid with propargylamine and metal cations.

The molecular structure of 1:1 complex formed between the naturally occurring polyether ionophore, called lasalocid acid (LAS) and propargylamine (PROP) is studied by X-ray, FT-IR, (1)H NMR, (13)C NMR and ESI-MS methods. The complex formed between deprotonated LAS acid and protonated PROP molecule is stabilized by intra- and inter-molecular hydrogen bonds. The protons of the protonated amine group are hydrogen bonded to etheric and hydroxyl oxygen atoms of the LAS anion. The similarity of the FT-IR spectra of the LAS-PROP complex in solid state and in solution demonstrated that the molecular structures of the complex in both states are comparable. It is shown that LAS in solution can form concurrent complexes with metal cations (M=Li(+), Na(+), K(+)) and amine existing in equilibrium. Analysis of the structures of lasalocid complexes is important for a better understanding of the antibacterial and anticancer properties of lasalocid acid.

Propargylamine as functional moiety in the design of multifunctional drugs for neurodegenerative disorders: MAO inhibition and beyond.

Much progress has been made in designing analogues that can potentially confer neuroprotection against debilitating neurodegenerative disorders, yet the multifactorial pathogenesis of this cluster of diseases remains a stumbling block for the successful design of an 'ultimate' drug. However, with the growing popularity of the "one drug, multiple targets" paradigm, many researchers have successfully synthesized and evaluated drug-like molecules incorporating a propargylamine function that shows potential to serve as multifunctional drugs or multitarget-directed ligands. It is the aim of this review to highlight the reported activities of these propargylamine derivatives and their prospect to serve as drug candidates for the treatment of neurodegenerative disorders.