ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Antioxidants are ineffective at quenching reactive oxygen species inside bacteria and should not be used to diagnose oxidative stress.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.

Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Promoting the Anticancer Activity with Multidentate Furan-2-Carboxamide Functionalized Aroyl Thiourea Chelation in Binuclear Half-Sandwich Ruthenium(II) Complexes.

A new set of binuclear arene ruthenium complexes [Ru2(p-cymene)2(k4-N2OS)(L1-L3)Cl2] (Ru2L1-Ru2L3) encompassing furan-2-carboxamide-based aroylthiourea derivatives (H2L1-H2L3) was synthesized and characterized by various spectral and analytical techniques. Single-crystal XRD analysis unveils the N^O and N^S mixed monobasic bidentate coordination of the ligands constructing N, S, Cl/N, O, and Cl legged piano stool octahedral geometry. DFT analysis demonstrates the predilection for the formation of stable arene ruthenium complexes. In vitro antiproliferative activity of the complexes was examined against human cervical (HeLa), breast (MCF-7), and lung (A549) cancerous and noncancerous monkey kidney epithelial (Vero) cells. All the complexes are more efficacious against HeLa and MCF-7 cells with low inhibitory doses (3.86-11.02 µM). Specifically, Ru2L3 incorporating p-cymene and -OCH3 fragments exhibits high lipophilicity, significant cytotoxicity against cancer cells, and lower toxicity on noncancerous cells. Staining analysis indicates the apoptosis-associated cell morphological changes expressively in MCF-7 cells. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) analyses reveal that Ru2L3 can raise ROS levels, reduce MMP, and trigger mitochondrial dysfunction-mediated apoptosis. The catalytic oxidation of glutathione (GSH) to its disulfide form (GSSG) by the complexes may simultaneously increase the ROS levels, alluding to their observed cytotoxicity and apoptosis induction. Flow cytometry determined the quantitative classification of late apoptosis and S-phase arrest in MCF-7 and HeLa cells. Western blotting analysis confirmed that the complexes promote apoptosis by upregulating Caspase-3 and Caspase-9 and downregulating BCL-2. Molecular docking studies unfolded the strong binding affinities of the complexes with VEGFR2, an angiogenic signaling receptor, and BCL2, Cyclin D1, and HER2 proteins typically overexpressed on tumor cells.

Identification of 7-aminourea or 7-aminothiourea derivatives of camptothecin as selective topoisomerase I inhibitors with anti-colorectal cancer activities.

Colorectal cancer (CRC) remains one of the most prevalent malignant tumors of the digestive system, yet the availability of safe and effective chemotherapeutic agents for clinical use remains limited. Camptothecin (CPT) and its derivatives, though approved for cancer treatment, have encountered significant challenges in clinical application due to their low bioavailability and high systemic toxicity. Strategic modification at the 7-position of CPT enables the development of novel CPT derivatives with high activity. In the present study, a series of compounds incorporating aminoureas, amino thioureas, and acylamino thioureas as substituents at the 7-position were screened. These compounds were subsequently evaluated for their cytotoxicity against the human gastric cancer (GC) cell line AGS and the CRC cell line HCT116. Two derivatives, XSJ05 (IC50 = 0.006 ± 0.003 µM) and XSJ07 (IC50 = 0.013 ± 0.003 µM), exhibited remarkably effective anti-CRC activity, being better than TPT. In addition, they have a better safety profile. In vitro mechanistic studies revealed that XSJ05 and XSJ07 exerted their inhibitory effects on CRC cell proliferation by suppressing the activity of topoisomerase I (Topo I). This suppression triggers DNA double-strand breaks, leads to DNA damage and subsequently causes CRC cells to arrest in the G2/M phase. Ultimately, the cells undergo apoptosis. Collectively, these findings indicate that XSJ05 and XSJ07 possess superior activity coupled with favorable safety profiles, suggesting their potential as lead compounds for the development of CRC therapeutics.

Discovery of novel pyrazole based Urea/Thiourea derivatives as multiple targeting VEGFR-2, EGFRWT, EGFRT790M tyrosine kinases and COX-2 Inhibitors, with anti-cancer and anti-inflammatory activities.

A novel series of pyrazole derivatives with urea/thiourea scaffolds 16a-l as hybrid sorafenib/erlotinib/celecoxib analogs was designed, synthesized and tested for its VEGFR-2, EGFRWT, EGFRT790M tyrosine kinases and COX-2, pro-inflammatory cytokines TNF-α and IL-6 inhibitory activities. All the tested compounds showed excellent COX-2 selectivity index in range of 18.04-47.87 compared to celecoxib (S.I. = 26.17) and TNF-α and IL-6 inhibitory activities (IC50 = 5.0-7.50, 6.23-8.93 respectively, compared to celecoxib IC50 = 8.40 and 8.50, respectively). Screening was carried out against 60 human cancer cell lines by National Cancer Institute (NCI), compounds 16a, 16c, 16d and 16 g were the most potent inhibitors with GI% ranges of 100 %, 99.63-87.02 %, 98.98-43.10 % and 98.68-23.62 % respectively, and with GI50 values of 1.76-15.50 µM, 1.60-5.38 µM, 1.68-7.39 µM and 1.81-11.0 µM respectively, in addition, of showing good safety profile against normal cell line (F180). Moreover, compounds 16a, 16c, 16d and 16 g had cell cycle arrest at G2/M phase with induced necrotic percentage compared to sorafenib of 2.06 %, 2.47 %, 1.57 %, 0.88 % and 1.83 % respectively. Amusingly, compounds 16a, 16c, 16d and 16 g inhibited VEGFR-2 with IC50 of 25 nM, 52 nM, 324 nM and 110 nM respectively, compared to sorafenib (IC50 = 85 nM), and had excellent EGFRWT and EGFRT790M kinase inhibitory activities (IC50 = 94 nM, 128 nM, 160 nM, 297 nM), (10 nM, 25 nM, 36 nM and 48 nM) respectively, compared to both erlotinib and osimertinib (IC50 = 114 nM, 56 nM) and (70 nM, 37 nM) respectively and showed (EGFRwt/EGFRT790M S.I.) of (range: 4.44-9.40) compared to erlotinib (2.03) and osmertinib (1.89).

Reactive Oxygen Species Mediate the Bactericidal Activity of Chlorine Against Salmonella.

Chlorine and its derivatives have been used as an antibacterial agent to reduce Salmonella contamination in poultry meat during processing. We evaluated the survival of 4 different Salmonella serotypes (Typhimurium, Enteritidis, Heidelberg, and Gaminara) in the presence of 50 ppm sodium hypochlorite (NaOCl) alone or with the addition of thiourea (radical scavenger) or Dip (iron chelator) to determine the contribution of reactive oxygen species (ROS) in the bactericidal activity of NaOCl. The result showed that for all four serotypes the addition of thiourea or Dip significantly increased the % survival as compared to the respective NaOCl treatment groups, while it was significantly higher with thiourea as compared to Dip (P < 0.05). We also evaluated the survival of 11 deletion mutants of S. Typhimurium, which were demonstrated to increase (∆atpC, ∆cyoA, ∆gnd, ∆nuoG, ∆pta, ∆sdhC, and ∆zwf) or decrease the production of ROS (∆edd, ∆fumB, ∆pykA, and ∆tktB) in Escherichia coli (E. coli), in the presence of 50 ppm. The results showed that only two (∆sdhC and ∆zwf) out of 7 ROS-increasing mutants showed reduced % survival as compared to the wild-type (P < 0.05), while all four deletion ROS-decreasing mutants showed significantly higher % survival as compared to the wild-type (P < 0.05). This work suggests that the production of ROS is a major component of the bactericidal activity of NaOCl against Salmonella serotypes and there might be a significant difference in the metabolic pathways involved in ROS production between Salmonella and E. coli.

Exposure to thiourea during the early stages of development impedes the formation of the swim bladder in zebrafish larvae.

Thiourea, a widely used agrochemical, is known to inhibit the activity of thyroid peroxidase, a key enzyme in the biosynthetic pathway of thyroid hormones. Thyroid insufficiency compromises the basal metabolic rate in warm-blooded organisms and embryonic development in vertebrates. In this study, we looked for developmental defects by exposing the zebrafish embryos to an environmentally relevant dose of thiourea (3 mg/mL). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to validate thiourea's presence in the treated zebrafish embryos. Structural anomalies like bent tail and pericardial edema were noticed in 96-h post-fertilization (hpf) larvae. On histological examination, underdeveloped swim bladder was noticed in 96 hpf larvae exposed to 3 mg/mL thiourea. The treated larvae also failed to follow the characteristic swimming behavior in response to stimuli due to defective swim bladder. Swim bladder being homologous to the lung of tetrapod, the role of Bmp4, a major regulator of lung development, was studied along with the associated regulatory genes. Gene expression analysis revealed that thiourea administration led to the downregulation of bmp4, shh, pcna, anxa5, acta2, and the downstream effector snail3 but the upregulation of caspase3. The protein expression showed a similar trend, wherein Bmp4, Shh, and Pcna were downregulated, but Cleaved Caspase3 showed an increased expression in the treated group. Therefore, it is prudent to presume that exposure to thiourea significantly reduces the expression of Bmp4 and other key regulators; hence, the larvae fail to develop a swim bladder, a vital organ that regulates buoyancy.

Design, synthesis, and biological evaluation of (thio)urea derivatives as potent Escherichia coli ß-glucuronidase inhibitors.

EcGUS has drawn considerable attention for its role as a target in alleviating serious GIAEs. In this study, a series of 72 (thio)urea derivatives were designed, synthesised, and biologically assayed. The bioassay results revealed that E-9 (IC50 = 2.68 µM) exhibited a promising inhibitory effect on EcGUS, surpassing EcGUS inhibitor D-saccharic acid-1,4-lactone (DSL, IC50 = 45.8 µM). Additionally, the inhibitory kinetic study indicated that E-9 (Ki = 1.64 µM) acted as an uncompetitive inhibitor against EcGUS. The structure-activity relationship revealed that introducing an electron-withdrawing group into the benzene ring at the para-position is beneficial for enhancing inhibitory activity against EcGUS. Furthermore, molecular docking analysis indicated that E-9 has a strong affinity to EcGUS by forming interactions with residues Asp 163, Tyr 472, and Glu 504. Overall, these results suggested that E-9 could be a potent EcGUS inhibitor, providing valuable insights and guidelines for the development of future inhibitors targeting EcGUS.

Sulfonyl thioureas with a benzo[d]thiazole ring as dual acetylcholinesterase/butyrylcholinesterase and human monoamine oxidase A and B inhibitors: An in vitro and in silico study.

A series of sulfonyl thioureas 6a-q containing a benzo[d]thiazole ring with an ester functional group was synthesized from corresponding substituted 2-aminobenzo[d]thiazoles 3a-q and p-toluenesulfonyl isothiocyanate. They had remarkable inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), monoamine oxidase (MAO)-A, and MAO-B. Among thioureas, several compounds had notable activity in the order of 6k > 6 h > 6c (AChE), 6j > 6g > 6k (BChE), 6k > 6g > 6f (MAO-A), and 6i > 6k > 6h (MAO-B). Compound 6k was an inhibitor of interest due to its potent or good activity against all studied enzymes, with IC50 values of 0.027 ± 0.008 µM (AChE), 0.043 ± 0.004 µM (BChE), 0.353 ± 0.01 µM (MAO-A), and 0.716 ± 0.02 µM (MAO-B). This inhibitory capacity was comparable to that of the reference drugs for each enzyme. Kinetic studies of two compounds with potential activity, 6k (against AChE) and 6j (against BChE), had shown that both 6k and 6j followed competitive-type enzyme inhibition, with Ki constants of 24.49 and 12.16 nM, respectively. Induced fit docking studies for enzymes 4EY7, 7BO4, 2BXR, and 2BYB showed active interactions between sulfonyl thioureas of benzo[d]thiazoles and the residues in the active pocket with ligands 6k, 6i, and 6j, respectively. The stability of the ligand-protein complexes while each ligand entered the active site of each enzyme (4EY7, 7BO4, 2BXR, or 2BYB) was confirmed by molecular dynamics simulations.

2-Thioxo-3,4-dihydropyrimidine and thiourea endowed with sulfonamide moieties as dual EGFRT790M and VEGFR-2 inhibitors: Design, synthesis, docking, and anticancer evaluations.

The effectiveness of a new series of thiopyrimidine and thiourea containing sulfonamides moieties was tested on HCT-116, MCF-7, HepG2, and A549. HepG2 cell line was the one that all the new derivatives affected the most. The greatest potent compounds against the four HepG2, HCT116, MCF-7, and A549 cell lines were 8f and 8g with IC50 = 4.13, 6.64, 5.74, 6.85 µM and 4.09, 4.36, 4.22, 7.25 µM correspondingly. Compound 8g exhibited higher activity than sorafenib against HCT116 and MCF-7 but exhibited lower activity against HepG2 and A549. Moreover, compounds 8f and 8g exhibited higher activities than erlotinib on HepG2, HCT116, and MCF-7 but demonstrated lower activity on A549. The most potent cytotoxic derivatives 6f, 6g, 8c, 8d, 8e, 8f, and 8g were examined on normal VERO cell lines. Our derivatives have low toxicity on VERO cells with IC50 values ranging from 32.05 to 53.15 µM. Additionally, all compounds were assessed for dual VEGFR-2 and EGFRT790M inhibition effects. Compounds 8f and 8g were the most potent derivatives inhibited VEGFR-2 at IC50 value of 0.88 and 0.90 µM, correspondingly. As well, derivatives 8f and 8g could inhibit EGFRT790M demonstrating strongest effects with IC50 = 0.32 and 0.33 µM sequentially. Additionally, the greatest active derivatives ADMET profile was evaluated in relationship with sorafenib and erlotinib as reference agents. The data attained from docking were greatly related to that achieved from the biological testing.

In Vitro Cytotoxic Potential and In Vivo Antitumor Effects of NOS/PDK-Inhibitor T1084.

Previously, we showed the antitumor activity of the new NOS/PDK inhibitor T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). The present study included an assessment of in vitro cytotoxicity against human malignant and normal cells according to the MTT-test and in vivo antitumor effects in solid tumor models in comparison with precursor compounds T1023 (NOS inhibitor; 1-isobutanoyl-2-isopropylisothiourea hydrobromide) and Na-DCA (PDK inhibitor; sodium dichloroacetate), using morphological, histological, and immunohistochemical methods. The effects of T1084 and T1023 on the in vitro survival of normal (MRC-5) and most malignant cells (A375, MFC-7, K562, OAW42, and PC-3) were similar and quantitatively equal. At the same time, melanoma A375 cells showed 2-2.5 times higher sensitivity (IC50: 0.39-0.41 mM) to the cytotoxicity of T1023 and T1084 than other cells. And only HeLa cells showed significantly higher sensitivity to the cytotoxicity of T1084 compared to T1023 (IC50: 0.54 ± 0.03 and 0.81 ± 0.02 mM). Comparative studies of the in vivo antitumor effects of Na-DCA, T1023, and T1084 on CC-5 cervical cancer and B-16 melanoma in mice were conducted with subchronic daily i.p. administration of these agents at an equimolar dose of 0.22 mmol/kg (33.6, 60.0, and 70.7 mg/kg, respectively). Cervical cancer CC-5 fairly quickly evaded the effects of both Na-DCA and T1023. So, from the end of the first week of Na-DCA or T1023 treatment, the tumor growth inhibition (TGI) began to decrease from 40% to an insignificant level by the end of the observation. In contrast, in two independent experiments, CC-5 showed consistently high sensitivity to the action of T1084: a significant antitumor effect with high TGI (43-58%) was registered throughout the observation, without any signs of neoplasia adaptation. The effect of precursor compounds on melanoma B-16 was either minimal (for Na-DCA) or moderate (for T1023) with TGI only 33%, which subsequently decreased by the end of the experiment. In contrast, the effect of T1084 on B-16 was qualitatively more pronounced and steadily increasing; it was accompanied by a 3-fold expansion of necrosis and dystrophy areas, a decrease in proliferation, and increased apoptosis of tumor cells. Morphologically, the T1084 effect was 2-fold superior to the effects of T1023-the TGI index reached 59-62%. This study suggests that the antitumor effects of T1084 develop through the interaction of NOS-dependent and PDK-dependent pathophysiological effects of this NOS/PDK inhibitor. The NOS inhibitory activity of T1084 exerts an anti-angiogenic effect on neoplasia. At the same time, the PDK inhibitory activity of T1084 enhances the cytotoxicity of induced intratumoral hypoxia and suppresses the development of neoplasia adaptation to anti-angiogenic stress. Such properties allow T1084 to overcome tumor resistance and realize a stable synergistic antitumor effect.

Discovery of Indole-Thiourea Derivatives as Tyrosinase Inhibitors: Synthesis, Biological Evaluation, Kinetic Studies, and In Silico Analysis.

Tyrosinase, a key enzyme in melanin synthesis, represents a crucial therapeutic target for hyperpigmentation disorders due to excessive melanin production. This study aimed to design and evaluate a series of indole-thiourea derivatives by conjugating thiosemicarbazones with strong tyrosinase inhibitory activity to indole. Among these derivatives, compound 4b demonstrated tyrosinase inhibitory activity with an IC50 of 5.9 ± 2.47 µM, outperforming kojic acid (IC50 = 16.4 ± 3.53 µM). Kinetic studies using Lineweaver-Burk plots confirmed competitive inhibition by compound 4b. Its favorable ADMET and drug-likeness properties make compound 4b a promising therapeutic candidate with a reduced risk of toxicity. Molecular docking revealed that the compounds bind strongly to mushroom tyrosinase (mTYR) and human tyrosinase-related protein 1 (TYRP1), with compound 4b showing superior binding energies of -7.0 kcal/mol (mTYR) and -6.5 kcal/mol (TYRP1), surpassing both kojic acid and tropolone. Molecular dynamics simulations demonstrated the stability of the mTYR-4b complex with low RMSD and RMSF and consistent Rg and SASA values. Persistent strong hydrogen bonds with mTYR, along with favorable Gibbs free energy and MM/PBSA calculations (-19.37 kcal/mol), further support stable protein-ligand interactions. Overall, compound 4b demonstrated strong tyrosinase inhibition and favorable pharmacokinetics, highlighting its potential for treating pigmentary disorders.

Design, Synthesis, Computational Studies, and Anti-Proliferative Evaluation of Novel Ethacrynic Acid Derivatives Containing Nitrogen Heterocycle, Urea, and Thiourea Moieties as Anticancer Agents.

In the present work, the synthesis of new ethacrynic acid (EA) derivatives containing nitrogen heterocyclic, urea, or thiourea moieties via efficient and practical synthetic procedures was reported. The synthesised compounds were screened for their anti-proliferative activity against two different cancer cell lines, namely, HL60 (promyelocytic leukaemia) and HCT116 (human colon carcinoma). The results of the in vitro tests reveal that compounds 1-3, 10, 16(a-c), and 17 exhibit potent anti-proliferative activity against the HL60 cell line, with values of the percentage of cell viability ranging from 20 to 35% at 1 µM of the drug and IC50 values between 2.37 µM and 0.86 µM. Compounds 2 and 10 showed a very interesting anti-proliferative activity of 28 and 48% at 1 µM, respectively, against HCT116. Two PyTAP-based fluorescent EA analogues were also synthesised and tested, showing good anti-proliferative activity. A test on the drug-likeness properties in silico of all the synthetised compounds was performed in order to understand the mechanism of action of the most active compounds. A molecular docking study was conducted on two human proteins, namely, glutathione S-transferase P1-1 (pdb:2GSS) and caspase-3 (pdb:4AU8) as target enzymes. The docking results show that compounds 2 and 3 exhibit significant binding modes with these enzymes. This finding provides a potential strategy towards developing anticancer agents, and most of the synthesised and newly designed compounds show good drug-like properties.

Synthesis, Computational Study, and In Vitro α-Glucosidase Inhibitory Action of Thiourea Derivatives Based on 3-Aminopyridin-2(1H)-Ones.

Reactions with allyl-, acetyl-, and phenylisothiocyanate have been studied on the basis of 3-amino-4,6-dimethylpyridine-2(1H)-one, 3-amino-4-phenylpyridine-2-one, and 3-amino-4-(thiophene-2-yl)pyridine-2(1H)-one (benzoyl-)isothiocyanates, and the corresponding thioureide derivatives 8-11a-c were obtained. Twelve thiourea derivatives were obtained and studied for their anti-diabetic activity against the enzyme α-glucosidase in comparison with the standard drug acarbose. The comparison drug acarbose inhibits the activity of α-glucosidase at a concentration of 15 mM by 46.1% (IC50 for acarbose is 11.96 mM). According to the results of the conducted studies, it was shown that alkyl and phenyl thiourea derivatives 8,9a-c, in contrast to their acetyl-(benzoyl) derivatives and 10,11a-c, show high antidiabetic activity. Thus, 1-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-3-phenylthiourea 9a has the highest inhibitory activity against the enzyme α-glucosidase, exceeding the activity of the comparison drug acarbose, which inhibits the activity of α-glucosidase by 56.6% at a concentration of 15 mm (IC50 = 9,77 mM). 1-(6-methyl-2-oxo 4-(thiophen-2-yl)-1,2-dihydropyridin-3-yl)-3-phenylthiourea 9c has inhibitory activity against the enzyme α-glucosidase, comparable to the comparison drug acarbose, inhibiting the activity of α-glucosidase at a concentration of 15 mm per 41.2% (IC50 = 12,94 mM). Compounds 8a, 8b, and 9b showed inhibitory activity against the enzyme α-glucosidase, with a lower activity compared to acarbose, inhibiting the activity of α-glucosidase at a concentration of 15 mM by 23.3%, 26.9%, and 35.2%, respectively. The IC50 against α-glucosidase for compounds 8a, 8b, and 9b was found to be 16.64 mM, 19.79 mM, and 21.79 mM, respectively. The other compounds 8c, 10a, 10b, 10c, 11a, 11b, and 11c did not show inhibitory activity against α-glucosidase. Thus, the newly synthesized derivatives of thiourea based on 3-aminopyridine-2(1H)-ones are promising candidates for the further modification and study of their potential anti-diabetic activity. These positive bioanalytical results will stimulate further in-depth studies, including in vivo models.

Design and Synthesis of Thiourea-Conjugating Organic Arsenic D-Glucose with Anticancer Activities.

Organic arsenic compounds such as p-aminophenylarsine oxide (p-APAO) are easier for structural optimization to improve drug-like properties such as pharmacokinetic properties, therapeutic efficacy, and target selectivity. In order to strengthen the selectivity of 4-(1,3,2-dithiarsinan-2-yl) aniline 7 to tumor cell, a thiourea moiety was used to strengthen the anticancer activity. To avoid forming a mixture of α/ß anomers, the strategy of 2-acetyl's neighboring group participation was used to lock the configuration of 2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate from 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide. 1-(4-(1,3,2-dithiarsinan-2-yl) aniline)-2-N-(2,3,4,6-tetra-O-acetyl-ß-d-glucopyranos-1-yl)-thiourea 2 can increase the selectivity of human colon cancer cells HCT-116 (0.82 ± 0.06 µM vs. 1.82 ± 0.07 µM) to human embryonic kidney 293T cells (1.38 ± 0.01 µM vs. 1.22 ± 0.06 µM) from 0.67 to 1.68, suggesting a feasible approach to improve the therapeutic index of arsenic-containing compounds as chemotherapeutic agents.

Activation of Transcription Factor Nrf2 in HeLa Cells under the Action of Nitrosyl Iron Complex with N-Ethylthiourea.

We studied the effect of an NO donor, nitrosyl iron complex with N-ethylthiourea, on Nrf2-dependent antioxidant system activation of tumor cells in vitro. The complex increased intracellular accumulation of Nrf2 transcription factor and induced its nuclear translocation. It was shown that both heme oxygenase-1 gene and protein expression increased significantly under the influence of the complex. Nrf2 activation was accompanied by a decrease in the intracellular accumulation of proinflammatory transcription factor NF-κB p65 subunit and expression of its target genes. The cytotoxic effect of N-ethylthiourea leads to induction of Nrf2/HO-1 antioxidant response and suppression of NF-κB-dependent processes in tumor cells.

Thiourea derivatives inhibit key diabetes-associated enzymes and advanced glycation end-product formation as a treatment for diabetes mellitus.

This study was designed to screen novel thiourea derivatives against different enzymes, such as α-amylase, α-glucosidase, protein tyrosine phosphatase 1 B, and advanced glycated end product (AGEs). A cytotoxicity analysis was performed using rat L6 myotubes and molecular docking analysis was performed to map the binding interactions between the active compounds and α-amylase and α-glucosidase. The data revealed the potency of five compounds, including E (1-(2,4-difluorophenyl)-3-(3,4-dimethyl phenyl) thiourea), AG (1-(2-methoxy-5-(trifluoromethyl) phenyl)-3-(3-methoxy phenyl) thiourea), AF (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), AD (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), and AH (1-(2,4-difluorophenyl)-3-(2-iodophenyl) thiourea), showed activity against α-amylase. The corresponding percentage inhibitions were found to be 85 ± 1.9, 82 ± 0.7, 75 ± 1.2, 72 ± 0.4, and 65 ± 1.1%, respectively. These compounds were then screened using in vitro assays. Among them, AH showed the highest activity against α-glucosidase, AGEs, and PTP1B, with percentage inhibitions of 86 ± 0.4% (IC50  = 47.9 µM), 85 ± 0.7% (IC50  = 49.51 µM), and 85 ± 0.5% (IC50  = 79.74 µM), respectively. Compound AH showed an increased glucose uptake at a concentration of 100 µM. Finally, an in vivo study was conducted using a streptozotocin-induced diabetic mouse model and PTP1B expression was assessed using real-time PCR. Additionally, we examined the hypoglycemic effect of compound AH in diabetic rats compared to the standard drug glibenclamide.

New thiourea and benzamide derivatives of 2-aminothiazole as multi-target agents against Alzheimer's disease: Design, synthesis, and biological evaluation.

In this study, two series of compounds were designed and synthesized, bearing thiourea and benzamide derivatives at position 2 of 4-subtituted-2-aminothiazole, respectively. Then, the inhibition potency of all final compounds for cholinesterase enzymes were evaluated. Among the thiourea derivatives, 3c (IC50 = 0.33 µM) was identified as the most potent and selective butyrylcholinesterase inhibitor. Additionally, benzamide derivative 10e (AChE IC50 = 1.47 and BChE IC50 = 11.40 µM) was found as a dual cholinesterase inhibitor. The type of inhibition for both compounds was determined by kinetic studies and the results showed that the compounds were mixed type inhibitors. Moreover, all title compounds were investigated in terms of their antioxidant (DPHH, ORAC) and metal chelator activities. In addition, the neuroprotective effects of selected compounds (3c, 3e, 6c, 6e and 10e) against H2O2-induced damage in the PC12 cell line were tested. The experimental findings demonstrated that thiourea-derived 6e (40.4 %) and benzamide-derived 10e (37.8 %) have a neuroprotective effect of about half as ferulic acid at 10 µM. Subsequently, the cytotoxicity of selected compounds was examined by the MTT assay, and the compounds were found not to have cytotoxic effect on the PC12 cell line in 24 h. Additionally, compounds 6e and 10e were also found to be more effective in inhibiting the release of IL-1ß, IL-6, TNF-α and NO compared to other selected compounds in this study.