Developing Allosteric Inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase.

The use of Fpocket and virtual screening techniques enabled us to identify potential allosteric druggable pockets within the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Of the compounds screened, compound 1 was identified as a promising inhibitor, lowering a SARS-CoV-2 RdRp activity to 57% in an enzymatic assay at 10 µM concentration. The structure of compound 1 was subsequently optimized in order to preserve or enhance inhibitory activity. This involved the substitution of problematic ester and aromatic nitro groups with more inert functionalities. The N,N'-diphenylurea scaffold with two NH groups was identified as essential for the compound's activity but also exhibited high toxicity in Calu-3 cells. To address this issue, a scaffold hopping approach was employed to replace the urea core with potentially less toxic urea isosteres. This approach yielded several structural analogues with notable activity, specifically 2,2'-bisimidazol (in compound 55 with residual activity RA = 42%) and (1H-imidazol-2-yl)urea (in compounds 59 and 60, with RA = 50 and 28%, respectively). Despite these advances, toxicity remained a major concern. These compounds represent a promising starting point for further structure-activity relationship studies of allosteric inhibitors of SARS-CoV-2 RdRp, with the goal of reducing their cytotoxicity and improving aqueous solubility.

Synthesis, in vitro, and in silico study of novel pyridine based 1,3-diphenylurea derivatives as tyrosinase inhibitors.

Tyrosinase inhibitors are studied in the cosmetics and pharmaceutical sectors as tyrosinase enzyme is involved in the biosynthesis and regulation of melanin, hence these inhibitors are beneficial for the management of melanogenesis and hyperpigmentation-related disorders. In the current work, a novel series of diphenyl urea derivatives containing a halo-pyridine moiety (5a-t) was synthesized via a multi-step synthesis. In vitro, tyrosinase inhibitory assay results showed that, except for two compounds, the derivatives were excellent inhibitors of human tyrosinase. The average IC50 value of the inhibitors (15.78 µM) is lower than that of kojic acid (17.3 µM) used as the reference compound, indicating that, on average, these molecules are more potent than the reference. Derivative 5a was identified as the most potent human tyrosinase inhibitor of the series, with an IC50 value of 3.5 ± 1.2  µM, approximately 5 times more potent than kojic acid. To get further insights into the nature of binding site interactions, molecular docking and molecular dynamics simulation studies were carried out. Moreover, the evaluation of in silico ADME properties showed a highly favorable profile for the synthesized compounds. These findings suggested that the further development of this class of compounds could be useful to get potent drug-like compounds that can target hyperpigmentation-related disorders.

Cytokinin oxidase/dehydrogenase inhibitors: progress towards agricultural practice.

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The most active compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (identified as 82) activated a cytokinin primary response gene, ARR5:GUS, and a cytokinin sensor, TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the up-regulation of cytokinin signalling. Overall, this study identifies highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

Synthesis and activity study of novel N,N-diphenylurea derivatives as IDO1 inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) has attracted much attention in the field of cancer immunotherapy as an immunomodulatory enzyme. To identify potential IDO1 inhibitors, a novel series of compounds with N,N-diphenylurea and triazole structures were synthesized. The designed compounds underwent organic synthesis, and subsequent enzymatic activity experiments targeting IDO1 confirmed their activity at the molecular level. These experiments provided validation for the efficacy of the designed compounds in inhibiting IDO1, compound 3g exhibited an IC50 value of 1.73 ± 0.97 µM. Further molecular docking study further explained the binding mode and reaction potential of compound 3g with IDO1. Our research has resulted in a series of novel IDO1 inhibitors, which is beneficial to the development of drugs targeting IDO1 in numerous cancer diseases.

Cytokinin oxidase/dehydrogenase inhibitors: outlook for selectivity and high efficiency.

Inhibitors of cytokinin oxidase/dehydrogenase (CKX) reduce the degradation of cytokinins in plants, and this effect can be exploited in agriculture and in plant tissue culture. In this study, we examine the structure-activity relationship of two series of CKX inhibitors based on diphenylurea. The compounds of Series I were derived from the recently published CKX inhibitors 3TFM-2HM and 3TFM-2HE, and we identified key substituents with increased selectivity for maize ZmCKX1 and ZmCKX4a over AtCKX2 from Arabidopsis. Series II contained compounds that further exceled in CKX inhibitory activity as well as in the ease of their synthesis. The best inhibitors exhibited half-maximal inhibitory concentration (IC50) values in low nanomolar ranges with ZmCKX1 and especially with ZmCKX4a, which is generally more resistant to inhibition. The activity of the key compounds was verified in tobacco and lobelia leaf-disk assays, where N6-isopentenyladenine was protected from degradation and promoted shoot regeneration. All the prepared compounds were further tested for toxicity against Caenorhabditis elegans, and the assays revealed clear differences in toxicity between compounds with and without a hydroxyalkyl group. In a broader perspective, this work increases our understanding of CKX inhibition and provides a more extensive portfolio of compounds suitable for agricultural and biotechnological research.

Diphenylurea-derived cytokinin oxidase/dehydrogenase inhibitors for biotechnology and agriculture.

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10-8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.

Spontaneous and Direct Transformation of N,O-Diaryl Carbamates into N,N'-Diarylureas.

We have discovered a spontaneous reaction of N,O-diaryl carbamates to afford symmetrical N,N'-diarylureas. Optimization of the conditions indicated that N,N-dimethylformamide (DMF) was the best solvent and triethylamine (Et3N) was the best additive for this transformation. The reaction requires the presence of aryl groups on the nitrogen and oxygen atoms of carbamates. Substrates bearing an electron-donating methoxy group on either of the aryl groups reacted slowly under these conditions.

Concomitant polymorphs of 1,3-bis(3-fluorophenyl)urea.

Hydrogen bonding between urea functionalities is a common structural motif employed in crystal-engineering studies. Crystallization of 1,3-bis(3-fluorophenyl)urea, C13H10F2N2O, from many solvents yielded concomitant mixtures of at least two polymorphs. In the monoclinic form, one-dimensional chains of hydrogen-bonded urea molecules align in an antiparallel orientation, as is typical of many diphenylureas. In the orthorhombic form, one-dimensional chains of hydrogen-bonded urea molecules have a parallel orientation rarely observed in symmetrically substituted diphenylureas.

Synthesis and assay of retro-α4ß1 integrin-targeting motifs.

In recent years, several research groups proposed new peptidomimetic antagonists of integrins αvß3, α5ß1, αIIbß3, αvß6, αvß5, etc. based on retro sequences of the classic integrin-binding motif RGD. The retro strategy is still largely ignored for the non-RGD-binding α4ß1 integrin. Herein we present the first examples of retro sequences for targeting this integrin, composed of Asp or isoAsp equipped with an aromatic cap at the N-terminus, (S)-pyrrolidine-3-carboxylic acid (ß(2)-Pro) as a constrained core, and the amino variant (AMPUMP) of the well-known α4-targeting diphenylurea MPUPA. We discuss α4ß1 receptor affinity (SPA), cell adhesion assays, stability in mouse serum, and conformational analysis. For their significant ability to inhibit cell adhesion and remarkable stability, the retro-peptide mimetics BnCO-Asp-ß-Pro-AMPUMP (3) and BnCO-isoAsp-ß-Pro-AMPUMP (4) represent promising candidates for designing small molecules as potential anti-inflammatory agents.

Dispersive liquid-liquid microextraction for the determination of three cytokinin compounds in fruits and vegetables by liquid chromatography with time-of-flight mass spectrometry.

The paper presents a novel approach for the determination of three cytokinin compounds, thidiazuron (TDZ), 1,3-diphenylurea (1,3-DPU) and forchlorfenuron (CPPU), in fruit and vegetables samples using liquid chromatography with electrospray ionization and time-of-flight mass spectrometry (LC-ESI-TOFMS). Analytes were extracted from the sample matrix with ethanol, and the extract, after dilution with water, was submitted to dispersive liquid-liquid microextraction (DLLME). Once acetonitrile and 1,2-dichloroethane had been selected as extraction and disperser solvents, respectively, the influence of the following experimental parameters was studied using a Plackett-Burman design: volume of extraction and disperser solvents, sample mass and time and speed of centrifugation. The best analytical conditions were 250 µL 1,2-dichloroethane, 1.5 mL acetonitrile, 5 g sample mass, and centrifugation at 3000 rpm for 3 min. The optimized method provided DLs in the range 0.02-0.05 ng g(-1), depending on the compound. Satisfactory recovery values between 89 and 106% were obtained for spiked samples (kiwifruit, watermelon, grape and tomato) in the 0.2-1.0 ng g(-1) concentration range, depending on the compound. None of the target analytes was detected in any of the samples analyzed.