Biological activity and computational analysis of novel acrylonitrile derived benzazoles as potent antiproliferative agents for pancreatic adenocarcinoma with antioxidative properties.

Continuing our research into the anticancer properties of acrylonitriles, we present a study involving the design, synthesis, computational analysis, and biological assessment of novel acrylonitriles derived from methoxy, hydroxy, and N-substituted benzazole. Our aim was to examine how varying the number of methoxy and hydroxy groups, as well as the N-substituents on the benzimidazole core, influences their biological activity. The newly synthesized acrylonitriles exhibited strong and selective antiproliferative effects against the Capan-1 pancreatic adenocarcinoma cell line, with IC50 values ranging from 1.2 to 5.3 µM. Consequently, these compounds were further evaluated in three other pancreatic adenocarcinoma cell lines, while their impact on normal PBMC cells was also investigated to determine selectivity. Among these compounds, the monohydroxy-substituted benzimidazole derivative 27 emerged with the most profound and broad-spectrum anticancer antiproliferative activity being emerged as a promising lead candidate. Moreover, a majority of the acrylonitriles in this series exhibited significant antioxidative activity, surpassing that of the reference molecule BHT, as demonstrated by the FRAP assay (ranging from 3200 to 5235 mmolFe2+/mmolC). Computational analysis highlighted the prevalence of electron ionization in conferring antioxidant properties, with computed ionization energies correlating well with observed activities.

Novel acrylonitrile derived imidazo[4,5-b]pyridines as antioxidants and potent antiproliferative agents for pancreatic adenocarcinoma.

We present the design, synthesis, computational analysis, and biological assessment of several acrylonitrile derived imidazo[4,5-b]pyridines, which were evaluated for their anticancer and antioxidant properties. Our aim was to explore how the number of hydroxy groups and the nature of nitrogen substituents influence their biological activity. The prepared derivatives exhibited robust and selective antiproliferative effects against several pancreatic adenocarcinoma cells, most markedly targeting Capan-1 cells (IC50 1.2-5.3 µM), while their selectivity was probed relative to normal PBMC cells. Notably, compound 55, featuring dihydroxy and bromo substituents, emerged as a promising lead molecule. It displayed the most prominent antiproliferative activity without any adverse impact on the viability of normal cells. Furthermore, the majority of studied derivatives also exhibited significant antioxidative activity within the FRAP assay, even surpassing the reference molecule BHT. Computational analysis rationalized the results by highlighting the dominance of the electron ionization for the antioxidant features with the trend in the computed ionization energies well matching the observed activities. Still, in trihydroxy derivatives, their ability to release hydrogen atoms and form a stable O-H⋯O•⋯H-O fragment upon the H• abstraction prevails, promoting them as excellent antioxidants in DPPH• assays as well.

Synthesis and Biological Evaluation of Novel Amino and Amido Substituted Pentacyclic Benzimidazole Derivatives as Antiproliferative Agents.

Newly designed pentacyclic benzimidazole derivatives featuring amino or amido side chains were synthesized to assess their in vitro antiproliferative activity. Additionally, we investigated their direct interaction with nucleic acids, aiming to uncover potential mechanisms of biological action. These compounds were prepared using conventional organic synthesis methodologies alongside photochemical and microwave-assisted reactions. Upon synthesis, the newly derived compounds underwent in vitro testing for their antiproliferative effects on various human cancer cell lines. Notably, derivatives 6 and 9 exhibited significant antiproliferative activity within the submicromolar concentration range. The biological activity was strongly influenced by the N atom's position on the quinoline moiety and the position and nature of the side chain on the pentacyclic skeleton. Findings from fluorescence, circular dichroism spectroscopy, and thermal melting assays pointed toward a mixed binding mode-comprising intercalation and the binding of aggregated compounds along the polynucleotide backbone-of these pentacyclic benzimidazoles with DNA and RNA.

Synthesis and Biological Evaluation of 2-Substituted Quinazolin-4(3H)-Ones with Antiproliferative Activities.

Sixteen new 2-substituted quinazolines were synthesized using a straightforward methodology starting from 2-methoxybezoic acid or 3-methoxy-2-naphthoic acid. The anti-proliferative activity of the target compounds was evaluated against nine cancer cell lines. Additionally, all the compounds were screened for their potency and selectivity against a panel of 109 kinases and four bromodomains, using Differential Scanning Fluorimetry (DSF). Compound 17 bearing a 2-methoxyphenyl substitution along with a basic side chain displayed a remarkable profile against the majority of the tested cell lines.

Synthesis and Antiproliferative Activity of 2,6-Disubstituted Imidazo[4,5-b]pyridines Prepared by Suzuki Cross Coupling.

A series of novel 2,6-diphenyl substituted imidazo[4,5-b]pyridines was designed and synthesized using optimized Suzuki cross coupling to evaluate their biological activity in vitro. The conditions of the Suzuki coupling were evaluated and optimized using a model reaction. To study the influence of the substituents on the biological activity, we prepared N-unsubstituted and N-methyl substituted imidazo[4,5-b]pyridines with different substituents at the para position on the phenyl ring placed at position 6 on the heterocyclic scaffold. Antiproliferative activity was determined on diverse human cancer cell lines, and the selectivity of compounds with promising antiproliferative activity was determined on normal peripheral blood mononuclear cells (PBMC). Pronounced antiproliferative activity was observed for p-hydroxy substituted derivatives 13 and 19, both displaying strong activity against most of the tested cell lines (IC50 1.45-4.25 µM). The unsubstituted N-methyl derivative 19 proved to be the most active derivative. There was a dose-dependent accumulation of G2/M arrested cells in several cancer cell lines after exposure to compound 19, implying a cell cycle-phase-specific mechanism of action. Additionally, the novel series of derivatives was evaluated for antiviral activity against a broad panel of viruses, yet the majority of tested compounds did not show antiviral activity.

4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1.

IMPORTANCE: Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics.

Novel hydroxy- and amidino-substituted benzimidazoles and benzothiazoles as antibacterial and antiproliferative agents.

Aim: The aim was synthesis of novel benzazoles bearing amidino and 2-hydroxyphenyl substituents to explore their biological activity. Methods: Condensation of 5-substituted salicylaldehydes and intermediates gave new benzazoles by previously published and developed procedures, which were tested for antibacterial and antiproliferative activity in vitro. Results: The best antibacterial activity showed benzimidazole with 2-imidazolinyl group 27 and benzothiazole with an unsubstituted amidine 48 (minimum inhibitory concentration 8 µg/ml). Benzothiazole 53 proved most potent at inhibiting proliferation of all cancer cells (IC50: 1.2-2.0 µM). Conclusion: Most active compounds have been recognized as lead compounds for additional optimization to improve their biological activity. The type of amidine moiety markedly influenced the biological activity. Benzothiazoles showed improved antiproliferative activity in comparison to benzimidazoles.

TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry.

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

Novel Biologically Active N-Substituted Benzimidazole Derived Schiff Bases: Design, Synthesis, and Biological Evaluation.

Herein, we present the design and synthesis of novel N-substituted benzimidazole-derived Schiff bases, and the evaluation of their antiviral, antibacterial, and antiproliferative activity. The impact on the biological activity of substituents placed at the N atom of the benzimidazole nuclei and the type of substituents attached at the phenyl ring were examined. All of the synthesized Schiff bases were evaluated in vitro for their antiviral activity against different viruses, antibacterial activity against a panel of bacterial strains, and antiproliferative activity on several human cancer cell lines, thus enabling the study of the structure-activity relationships. Some mild antiviral effects were noted, although at higher concentrations in comparison with the included reference drugs. Additionally, some derivatives showed a moderate antibacterial activity, with precursor 23 being broadly active against most of the tested bacterial strains. Lastly, Schiff base 40, a 4-N,N-diethylamino-2-hydroxy-substituted derivative bearing a phenyl ring at the N atom on the benzimidazole nuclei, displayed a strong antiproliferative activity against several cancer cell lines (IC50 1.1-4.4 µM). The strongest antitumoral effect was observed towards acute myeloid leukemia (HL-60).

Biological evaluation of novel amidino substituted coumarin-benzazole hybrids as promising therapeutic agents.

Herein we present the design and the synthesis of novel substituted coumarin-benzimidazole/benzothiazole hybrids bearing a cyclic amidino group on the benzazole core as biologically active agents. All prepared compounds were evaluated for their in vitro antiviral and antioxidative activity as well as for their in vitro antiproliferative activity against a panel of several human cancer cell lines. Coumarin-benzimidazole hybrid 10 (EC50 9.0-43.8 µM) displayed the most promising broad spectrum antiviral activity, while two other coumarin-benzimidazole hybrids 13 and 14 showed the highest antioxidative capacity in the ABTS assay, superior to the reference standard BHT (IC50 0.17 and 0.11 mM, respectively). Computational analysis supported these results and demonstrated that these hybrids benefit from the high C-H hydrogen atom releasing tendency of the cationic amidine unit, and the pronounced ease with which they can liberate an electron, promoted by the electron-donating diethylamine group on the coumarin core. The coumarin ring substitution at position 7 with a N,N-diethylamino group also caused a significant enhancement of the antiproliferative activity, with the most active compounds being derivatives with a 2-imidazolinyl amidine group 13 (IC50 0.3-1.9 µM) and benzothiazole derivative with a hexacyclic amidine group 18 (IC50 1.3-2.0 µM).

Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H+ -ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.

Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections.

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.

Imidazo[4,5-b]pyridine derived tubulin polymerization inhibitors: Design, synthesis, biological activity in vitro and computational analysis.

Imidazo[4,5-b]pyridine derived acrylonitriles were synthesized and explored for their in vitro antiproliferative effect on a diverse human cancer cell line panel. Three compounds, 20, 21 and 33, showed strong activity in the submicromolar range (IC50 0.2-0.6 µM), and were chosen for further biological experiments. Immunofluorescence staining and tubulin polymerization assays confirmed tubulin as the main target, but excluded its colchicine-binding site as a potential interacting unit. This was supported by the computational analysis, which revealed that the most potent ligands act on the extended colchicine site on the surface between interacting tubulin subunits, where they interfere with their polymerization and reveal pronounced antitumor properties. In addition, lead molecule 21 potently inhibited cancer cell migration, while it did not affect the viability of normal cells even at the highest concentration tested (100 µM).

Hybridization Approach to Identify Salicylanilides as Inhibitors of Tubulin Polymerization and Signal Transducers and Activators of Transcription 3 (STAT3).

The superimposition of the X-ray complexes of cyclohexanediones (i.e., TUB015), described by our research group, and nocodazole, within the colchicine binding site of tubulin provided an almost perfect overlap of both ligands. This structural information led us to propose hybrids of TUB015 and nocodazole using a salicylanilide core structure. Interestingly, salicylanilides, such as niclosamide, are well-established signal transducers and activators of transcription (STAT3) inhibitors with anticancer properties. Thus, different compounds with this new scaffold have been synthesized with the aim to identify compounds inhibiting tubulin polymerization and/or STAT3 signaling. As a result, we have identified new salicylanilides (6 and 16) that showed significant antiproliferative activity against a panel of cancer cells. Both compounds were able to reduce the levels of p-STAT3Tyr705 without affecting the total expression of STAT3. While compound 6 inhibited tubulin polymerization and arrested the cell cycle of DU145 cells at G2/M, similar to TUB015, compound 16 showed a more potent effect on inhibiting STAT3 phosphorylation and arrested the cell cycle at G1/G0, similar to niclosamide. In both cases, no toxicity towards PBMC cells was detected. Thus, the salicylanilides described here represent a new class of antiproliferative agents affecting tubulin polymerization and/or STAT3 phosphorylation.

Discovery of 2-Phenylquinolines with Broad-Spectrum Anti-coronavirus Activity.

A selection of compounds from a proprietary library, based on chemical diversity and various biological activities, was evaluated as potential inhibitors of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a phenotypic-based screening assay. A compound based on a 2-phenylquinoline scaffold emerged as the most promising hit, with EC50 and CC50 values of 6 and 18 µM, respectively. The subsequent selection of additional analogues, along with the synthesis of ad hoc derivatives, led to compounds that maintained low µM activity as inhibitors of SARS-CoV-2 replication and lacked cytotoxicity at 100 µM. In addition, the most promising congeners also show pronounced antiviral activity against the human coronaviruses HCoV-229E and HCoV-OC43, with EC50 values ranging from 0.2 to 9.4 µM. The presence of a 6,7-dimethoxytetrahydroisoquinoline group at the C-4 position of the 2-phenylquinoline core gave compound 6g that showed potent activity against SARS-CoV-2 helicase (nsp13), a highly conserved enzyme, highlighting a potentiality against emerging HCoVs outbreaks.

Quinoline-Dihydropyrimidin-2(1H)-one Hybrids: Synthesis, Biological Activity, and Mechanistic Studies.

A novel class of quinoline-dihydropyrimidin-2(1H)-one (DHPM) hybrids was synthesized and in vitro antiplasmodial activity was evaluated against chloroquine sensitive (D10) and chloroquine resistant (Dd2) strains of Plasmodium falciparum, the human malaria parasite. The antiplasmodial activity was compared to previously reported DHPM based molecular hybrids. Dual mode of antiplasmodial action of the most active member has been evaluated through heme binding study and in silico docking in the active site of dihydrofolate enzymes (wild-type as well as mutant). Favourable pharmacokinetic parameters were predicted in the ADMET evaluation. The new hybrids were also tested against a number of DNA and RNA viruses. No antiviral activity was found, except for one hybrid that showed mild inhibitory activity against two strains of cytomegalovirus (AD-169 and Davis), The most active hybrid was found to be a selective inhibitor of the growth of P. falciparum as well as a modest inhibitor of varicella zoster virus in HEL cells. Cytotoxicity of all hybrids was assessed in HEL, HeLa, Vero, MDCK, and CRFK cell cultures.

Anthranilamides with quinoline and ß-carboline scaffolds: design, synthesis, and biological activity.

In the present study, we report the design and synthesis of novel amide-type hybrid molecules based on anthranilic acid and quinoline or ß-carboline heterocyclic scaffolds. Three types of biological screenings were performed: (i) in vitro antiproliferative screening against a panel of solid tumor and leukemia cell lines, (ii) antiviral screening against several RNA viruses, and (iii) anti-quorum sensing screening using gram-negative Chromobacterium violaceum as the reporter strain. Antiproliferative screening revealed a high activity of several compounds. Anthranilamides 12 and 13 with chloroquine core and halogenated anthranilic acid were the most active agents toward diverse cancer cell lines such as glioblastoma, pancreatic adenocarcinoma, colorectal carcinoma, lung carcinoma, acute lymphoblastic, acute myeloid, chronic myeloid leukemia, and non-Hodgkin lymphoma, but also against noncancerous cell lines. Boc-protected analogs 2 and 3 showed moderate activities against the tested cancer cells without toxic effects against noncancerous cells. A nonhalogenated quinoline derivative 10 with N-benzylanthranilic acid residue was equally active as 12 and 13 and selective toward tumor cells. Chloroquine and quinoline anthranilamides 10-13 exerted pronounced antiviral effect against human coronaviruses 229E and OC43, whereas 12 and 13 against coronavirus OC43 (EC50 values in low micromolar range; selectivity indices from 4.6 to > 10.4). Anthranilamides 14 and 16 with PQ core inhibited HIV-1 with EC50 values of 9.3 and 14.1 µM, respectively. Compound 13 displayed significant anti-quorum/biofilm effect against the quorum sensing reporter strain (IC50 of 3.7 µM) with no apparent bactericidal effect.

Itaconic acid hybrids as potential anticancer agents.

In this paper, we report the synthesis of novel hybrids 2-14 based on itaconic acid and fluoroaniline, pyridine, indole and quinoline scaffolds. Itaconic acid is a naturally occurring compound with a Michael acceptor moiety, a key structural feature in several anticancer and antiviral drugs, responsible for the covalent binding of a drug to the cysteine residue of a specific protein. Aromatic parts of the hybrids also come from the substances reported as anticancer or antiviral agents. The synthetic route employed to access the amido-ester hybrids 2-13 used monomethyl itaconate or monomethyl itaconyl chloride and corresponding amines as the starting materials. Dimers 14 and 15 with two aminoindole or mefloquine moieties were prepared from itaconic acid and corresponding amino derivative, using standard coupling conditions (HATU/DIEA). All hybrids exerted anticancer effects in vitro against almost all the tumour cell lines that were evaluated (MCF-7, HCT 116, H460, LN-229, Capan-1, DND-41, HL-60, K-562, Z-138). Solid tumour cells were, in general, more responsive than the haematological cancer cells. The MCF-7 breast adenocarcinoma cell line appeared the most sensitive. Amido-ester 12 with chloroquine core and mefloquine homodimer 15 showed the highest activity with GI50 values between 0.7 and 8.6 µM. In addition, compound 15 also exerted antiviral activity against Zika virus and Coxsackievirus B4 in low micromolar concentrations.

Discovery of ( ±)-3-(1H-pyrazol-1-yl)-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazine derivatives with promising in vitro anticoronavirus and antitumoral activity.

A new series of ( ±)-(3-(3,5-dimethyl-1H-pyrazol-1-yl)-6-phenyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)(phenyl)methanones were efficiently synthesized starting from 4-amino-5-hydrazinyl-4H-1,2,4-triazole-3-thiol 1, acetyl acetone 2, various aromatic and heterocyclic aldehydes 3 and phenacyl bromides 4. All the newly synthesized compounds were tested for their antiviral and antitumoral activity. It was shown that subtle structural variations on the phenyl moiety allowed to tune biological properties toward antiviral or antitumoral activity. Mode-of-action studies revealed that the antitumoral activity was due to inhibition of tubulin polymerization.

Biological Activity of Amidino-Substituted Imidazo [4,5-b]pyridines.

A series of cyano- and amidino-substituted imidazo[4,5-b]pyridines were synthesized using standard methods of organic synthesis, and their biological activity was evaluated. Biological evaluation included in vitro assessment of antiproliferative effects on a diverse selection of human cancer cell lines, antibacterial activity against chosen Gram-positive and Gram-negative bacterial strains, and antiviral activity on a broad panel of DNA and RNA viruses. The most pronounced antiproliferative activity was observed for compound 10, which contained an unsubstituted amidino group, and compound 14, which contained a 2-imidazolinyl amidino group; both displayed selective and strong activity in sub-micromolar inhibitory concentration range against colon carcinoma (IC50 0.4 and 0.7 µM, respectively). All tested compounds lacked antibacterial activity, with the exception of compound 14, which showed moderate activity against E. coli (MIC 32 µM). Bromo-substituted derivative 7, which contained an unsubstituted phenyl ring (EC50 21 µM), and para-cyano-substituted derivative 17 (EC50 58 µM) showed selective but moderate activity against respiratory syncytial virus (RSV).