MDH-based adducts were also identified by X-ray structural analysis. TCEKs and MDH, as well as TCEK based on cyclohexanone and MDEA, form bi- and tricyclic structures: pyrrolo -quinolines (using TCEKs based on cyclohexanone and 4-propylcyclohexanone), epiminomethanoquinoline-3,4-dicarbonitrile (using TCEK based on 2-methylcyclohexanone) and cyclododec[b]pyran-3,4-dicarbonitrile (using TCEK based on cyclododecanone). MDH and TCNEH2 formed a pyrrole derivative. Thus, we synthesized the structures that are of interest for molecular design and pharmaceutical chemistry.1,1-Dimethylhydrazine ) is characterized by extremely high toxicity, teratogenicity and the ability to constantly absorb water from the atmosphere, losing its energy characteristics. In this regard, as well as due to the alternative fuel (“Angara”) transition, there is a need for UDMH utilization in huge amounts. A more benign approach involves its immediate reaction with a formalin solution to form 1,1–dimethyl-2-methylene hydrazone (MDH), which is significantly less toxic by an order of magnitude. MDH can then be polymerized under acidic conditions, and the resulting product can be burned, yielding a substantial amount of nitrogen oxides. We propose an alternative to incineration by involving MDH in organic synthesis. We studied the reactions of MDH and its analog N,N-dimethyl-2-(methylenamino)ethane-1-amine (MDEA) with available CH-acids: tetracyanoethylated ketones (TCEKs) based on cyclohexanone, 4-propylcyclohexanone, 2-methylcyclohexanone, cyclododecanone and tetracyanoethane. The structures synthesized were confirmed by IR,  Heptil  is actively used worldwide as a fuel for rocket engines. At the same time, it constantly loses its properties due to uncontrolled absorption of moisture that cannot be separated by rectification. UDMH is an extremely toxic compound (Hazard class 1), which complicates its transportation for treatment. During disposal by incineration, some oxidation products that are much more toxic than the original substance are produced. The safest way to neutralize it is its instantaneous interaction with a formalin solution to form a less toxic 1,1-dimethyl-2-methylene hydrazone (MDH) ). The maThis fragment represents a group of inhibitors belonging to various enzyme classes, including HIV-1 integrase  an enzy.The methylene derivative of N,N-dimethyl-2-(methylenamino)ethane-1-amine (MDEA), known as N,N-dimethylethane-1,2-diamine (DMEDA), possesses spaced reaction centers and undergoes reactions with TCEKs similar to MDH. Furthermore, this fragment is integral to medications employed in the treatment of acute pancreatitis  and the The compounds synthesized in this study, derived from TCEKs, MDH, and MDEA, exhibit structural similarities to well-known drugs due to the presence of hydroquinoline moieties (highlighted by the green circle in c]-quinolines (obtained through MDH and TCEK reactions using cyclohexanone and 4-propylcyclohexanone) and epiminomethanoquinoline-3,4-dicarbonitrile (synthesized via MDH and TCEK reactions using 2-methylcyclohexanone) exhibit structural similarities to compounds utilized in the treatment of neurodegenerative diseases  is expected to find application as an antiviral agent, owing to the presence of the pyran ring, which is a common structural motif in biologically active antiviral compounds  Figure .c]-quinolines 9a,b    quinoline-3,4-dicarbonitrile b]pyran-3,4-dicarbonitrile 8 (TCEK 2 with MDH) 3-Amino-5-(dimethylamino)-1-oxo-4,5,6,7,8,9-hexahydro-1. Found: C 60.28; H 6.31; N 28.05. C15H18N6O. Calculated %: C 60.39; H 6.08; N 28.17.H-pyrroloquinoline-3a,9b- dicarbonitrile (9b)3-Amino-5-(dimethylamino)-1-oxo-8-(propyl)-4,5,6,7,8,9-hexahydro-1. Found: C 63.48; H 7.13; N 24.65. C18H24N6O. Calculated %: C 63.51; H 7.11; N 24.69.H-pyrroloquinoline-3a,9b-dicarbonitrile (9′)3-Amino-5-(2-(dimethylamino)ethyl-1-oxo-4,5,6,7,8,9-hexahydro-1 Found: C 62.53 H 6.75 N 25.80. C17H22N6O. Calculated %: C 62.56; H 6.79; N 25.75.H-8a,4-(epimino)quinoline-3,4(4aH)-dicarbonitrile (12c)2-(Dimethylamino)-4a-methyl-10-oxo-5,6,7,8-tetrahydro-1. Found: C 63.08; H 6.69; N 24.55. C15H19N5O. Calculated %: C 63.14; H 6.71; N 24.54.H-cyclododeca [b]pyran-3,4-dicarbonitrile (8)(E)2-Amino-6,7,8,9,10,11,12,13-octahydro-5. Found: C 72.03; H 7.42; N 14.80. C17H21N3O. Calculated %: C 72.06; H 7.47; N 14.83.H-pyrrole-3,3,4-tricarbonitrile (12)5-Amino-1-(dimethylamino)-1,2-dihydro-3. Found, %: C 53.61; H 4.72; N 41.67. C9H10N6. Calculated %: C 53.46; H 4.98; N 41.56.Crystal Data for 9a: C15H18N6O (M =298.35 g/mol): triclinic, space group P-1 (no. 2), a = 13.9756(12) Å, b = 15.0773(15) Å, c = 18.0923(13) Å, α = 112.010(8)°, β = 93.942(7)°, γ = 112.380(9)°, V = 3167.3(5) Å3, Z = 8, T = 104(7) K, μ(Cu Kα) = 0.683 mm−1, Dcalc = 1.251 g/cm3, 34121 reflections measured (5.436° ≤ 2Θ ≤ 156.656°), 12415 unique  which were used in all calculations. The final R1 was 0.0845 (I > 2σ(I)) and wR2 was 0.2282 . CCDC number 2152262.Crystal Data for 9b: C22H32N6O3 (M =428.53 g/mol): triclinic, space group P-1 (no. 2), a = 9.42370(10) Å, b = 11.97650(10) Å, c = 11.99720(10) Å, α = 111.2460(10)°, β = 111.1820(10)°, γ = 93.9690(10)°, V = 1145.11(2) Å3, Z = 2, T = 102.2(9) K, μ(Cu Kα) = 0.690 mm−1, Dcalc = 1.243 g/cm3, 33743 reflections measured (8.14° ≤ 2Θ ≤ 153.026°), 4548 unique  which were used in all calculations. The final R1 was 0.0422 (I > 2σ(I)) and wR2 was 0.1101 . CCDC number 2152263.Crystal Data for 12c: C15H19N5O (M =285.35 g/mol): triclinic, space group P-1 (no. 2), a = 7.5867(2) Å, b = 9.1339(3) Å, c = 10.8470(4) Å, α = 76.621(3)°, β = 82.305(3)°, γ = 81.452(3)°, V= 719.26(4) Å3, Z = 2, T = 100(1) K, μ(Cu Kα) = 0.703 mm−1, Dcalc = 1.318 g/cm3, 17589 reflections measured (8.424° ≤ 2Θ ≤ 152.906°), 2865 unique  which were used in all calculations. The final R1 was 0.0399 (I > 2σ(I)) and wR2 was 0.0993 . CCDC number 2152264.Crystal Data for 8: for C17H21N3O (M =283.37 g/mol): triclinic, space group P-1 (no. 2), a = 8.5574(5) Å, b = 9.7008(6) Å, c = 9.7930(4) Å, α = 102.032(4)°, β = 103.106(4)°, γ = 101.505(5)°, V = 747.97(7) Å3, Z = 2, T = 99.9(10) K, μ(Cu Kα) = 0.632 mm−1, Dcalc = 1.258 g/cm3, 7141 reflections measured (9.618° ≤ 2Θ ≤ 153.2°), 2981 unique  which were used in all calculations. The final R1 was 0.0821 (I > 2σ(I)) and wR2 was 0.2326 . CCDC number 2152265.9a, 9b, 12c and 8 in the table below  ensures cyclization to pyrrolidin-2-one (F) and the dimethylamine fragment (B). We found out that MDH decomposed during its interactions, forming original compounds (E) and degraded further into dimethylamine and formaldoxime (C). In turn, the intermediate in the C-direction was able to produce an appropriative salt ethane-1,1,2,2-tetracarbonitrile 1b with MDH (1′a)  and cyclohexanone derivative 1a with MDEA (1′b) (intermediates 2′–8′) interacted according to the same scheme (1c with MDH (1′a) undergoes chemical transformations identical to TCEKs 1a and 1b (intermediates 2c–5c) until the moment of rearrangement.Thus, 1-(2-oxocyclohexyl)ethane-1,1,2,2-tetracarbonitrile e scheme . The derTCEKs 1a–c and is a Michael-type attachment of the CH-acid center 1a–c to 1′a,b. The resulting Michael-type adducts 2a–c, 2′ are likely to undergo intermolecular cyclization transformations catalyzed by alkali. Zwitterions 3a–c, 3′ via intermediates 4a–c, 4′ transform into heterocycles 5a–c, 5′. The rearrangement of the 2-methylcyclohexanone derivative 5c is carried out according to another scheme  (see 9b (m/z 341.2090) (see MDEA-based 9′ (m/z: 326.0000)  see , 9b (m/z090) see  and MDEA000) see   see .The substituents in the structures mentioned above are presented in the table below .TCEK 1c causes a different direction of the chemical process. Therefore, after the Thorpe–Ziegler type cyclization tetrahydropyridine 12c by analogy with the publication quinoline-3a,9b-dicarbonitrile (9b), 3-amino-5- (2-(dimethylamino)ethyl-1-oxo-4,5,6,7,8,9-hexahydro-1H-pyrroloquinoline-3a,9b-dicarbonitrile (9′), 2-(dimethylamino)-4a-methyl-10-oxo-5,6,7,8-tetrahydro-1H-8a,4-(epimino)quinoline-3,4(4aH)-dicarbonitrile (12c), (E)-2-amino-6,7,8,9,10,11,12,13-octahydro-5H-cyclododeca [b] pyran-3,4-dicarbonitrile (8)3-Amino-5-(dimethylamino)-1-oxo-4,5,6,7,8,9-hexahydro-1.Synthesis of TCEK in 5 mL of ethyl acetate was added to 8 mmol of MDH or MDEA, along with a catalytic amount of sodium hydroxide in 5 mL of ethyl acetate. The reaction progress was monitored using TLC, with N,N-dimethyl-N’-methylene hydrazine serving as the reference sample in terms of syntheses with TCEKs 1a,b,c, 2, MDEA, used as a reference sample in terms of synthesis with TCEK 1a. Then, 0.5–5 h later, sand-colored crystals precipitated. The desired product was separated by filtration through a Schott filter, followed by washing with cold ethyl acetate. Subsequently, recrystallization was performed using isopropanol as the solvent.The compounds were synthesized following a standard procedure, yielding 84% (0.21 g), 75% (0.18 g), 48% (0.12 g), 64% (0.14 g) and 62% (0.18 g), respectively. Initially, 8 mmol of H-8a,4-(epimino)quinoline-3,4(4aH)-dicarbonitrile (12c), (E)-2-amino-6,7,8,9,10,11,12,13-octahydro-5H-cyclododeca [b] pyran-3,4-dicarbonitrile (8)2-(dimethylamino)-4a-methyl-10-oxo-5,6,7,8-tetrahydro-1 without MDH catalysis.Synthesis of TCEK 2 was dissolved in 5 mL of ethyl acetate and allowed to react at room temperature for 24 h. Subsequently, the desired product precipitated out. The isolation and purification procedures were carried out following the same protocol as described above. The yield of the desired product was 54% (0.16 g).Firstly, 8 mmol of In the specified quantities, the liberation of hydrogen cyanide does not present any danger. However, when dealing with larger quantities, it is imperative to utilize the aforementioned apparatus.5-Amino-1-(dimethylamino)-1,2-dihydro-3H-pyrrole-3,3,4-tricarbonitrile (12).Synthesis of MDH was added to a solution of 1 mmol of 2TCNEH in 2 mL of ethyl acetate. The mixture was kept at room temperature for 14 h (TLC control) and cooled. Precipitate was filtered off and washed with 2 mL of cold ethyl acetate. Yield: 81%.Firstly, 1.1 mmol of –Double bond (addition to the methylene-active position of tetracyanoketones via a Michael-type reaction);–Dimethylamine fragment (rearrangement in the 2-methylcyclohexanone derivative);–Labile hydrogen (addition to the double bond of tetracyanoethylene);–Serving as a catalyst (accelerating the cyclization of cyclohexanone into a pyran derivative).Thus, 1,1-dimethylhydrazine can engage in reactions with various structural elements:This makes it an indispensable starting compound for the synthesis of various organic structures. It should be noted that the exceptional availability of this reagent is of great significance.


The combined toxicological effects of airborne particulate matter (PM), such as PM2.5, and Asian sand dust (ASD), with surrounding chemicals, particularly quinones, on human airway epithelial cells remain underexplored. In this study, we established an in vitro combination exposure model using 1,2-naphthoquinones (NQ) and 9,10-phenanthroquinones (PQ) along with heated PM (h-PM2.5 and h-ASD) to investigate their potential synergistic effects. The impacts of quinones and heated PM on tetrazolium dye (WST-1) reduction, cell death, and cytokine and reactive oxygen species (ROS) production were examined. Results revealed that exposure to 9,10-PQ with h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction at 1 μM compared to the corresponding control while markedly decreasing it at 10 μM. Higher early apoptotic, late apoptotic, or necrotic cell numbers were detected in 9,10-PQ + h-PM2.5 exposure than in 9,10-PQ + h-ASD or 9,10-PQ + h-PM2.5 + h-ASD. Additionally, 1,2-NQ + h-PM2.5 exposure also resulted in an increase in cell death compared to 1,2-NQ + h-ASD and 1,2-NQ + h-PM2.5 + h-ASD. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD significantly increased ROS production, especially with h-PM2.5. Our findings suggest that quinones, at relatively low concentrations, induce cell death synergistically in the presence of h-PM2.5 rather than h-ASD and h-PM2.5 + h-ASD, partially through the induction of apoptosis with increased ROS generation. Particulate matter (PM) induces adverse health effects, including respiratory toxicity. PM is a complex mixture of carbon particles in which various organic compounds, including polycyclic aromatic hydrocarbons (PAHs), are adsorbed. PM2.5 is a complex mixture of solid and liquid airborne particles, and different PM2.5 components have different effects on cell inflammatory responses . In ChinQuinones are by-products of the partial combustion of biomass and fossil fuels, petroleum products , PAHs, and polychlorinated biphenyls. These quinones are ubiquitous environmental pollutants, and humans are increasingly getting exposed to them, posing health risks, including cancer. Toxicological studies of environmentally occurring quinones, such as naphthoquinones  and phenanthroquinones 9,10-PQ), have reported that these compounds can cause mutations, DNA damage, chromosomal aberrations, and altered signal transduction pathways. These toxic effects have been attributed to their electrophilic and redox-cycling properties and have been linked to various cancers, including leukemias ,9,10,11.,10-PQ, hp < 0.01: 9,10-PQ at 0.1 and 1 μM; 9,10-PQ + h-PM2.5 at 0.1 and 1 μM; 9,10-PQ + h-ASD at 1 μM; and 9,10-PQ + h-PM2.5 + h-ASD at 0.1 and 1 μM). However, the WST-1 reduction was markedly decreased at 10 μM (p < 0.01) when compared to the corresponding control for all groups.BEAS-2B cells were treated with varying concentrations of 9,10-PQ alone, 9,10-PQ + h-PM2.5, and/or h-ASD, and their WST-1 reduction was studied. 9,10-PQ with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD dose-dependently increased WST-1 reduction vs. corresponding controls (p < 0.01). At 0.1 µM, a lower WST-1 reduction was seen after 9,10-PQ + h-PM2.5 and/or + h-ASD exposure than after 9,10-PQ exposure (p < 0.01). At 1 µM, a lower WST-1 reduction was observed after 9,10-PQ + h-ASD exposure than after 9,10-PQ exposure (p < 0.01)  A.p < 0.01 for 1,2-NQ at 1 and 10 μM, 1,2-NQ + h-PM2.5 at 1 and 10 μM, 1,2-NQ + h-ASD at 10 μM, and 1,2-NQ + h-PM2.5 + h-ASD at 10 μM).We treated BEAS-2B cells with varying concentrations of 1,2-NQ alone, 1,2-NQ + h-PM2.5, and/or h-ASD and studied WST-1 reduction. 1,2-NQ with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD dose-dependently increased WST-1 reduction vs. corresponding controls (p < 0.01)  B.At 1 µM of 9,10-PQ, 9,10-PQ + h-PM2.5 increased the number of early apoptotic cells, late apoptotic or necrotic cells, and necrotic cells compared with h-PM2.5, whereas the numbers of early apoptotic cells and late apoptotic or necrotic cells after exposure to 9,10-PQ, 9,10-PQ + h-ASD, were lower than those after vehicle and h-ASD, respectively A. ExposuAt 10 µM of 1,2-NQ, 1,2-NQ + h-PM2.5 increased the number of late apoptotic or necrotic cells, and necrotic cells compared with h-PM2.5, whereas the numbers of early apoptotic cells or late apoptotic or necrotic cells after exposure to 1,2-NQ + h-ASD and 1,2-NO + h-PM2.5 + h-ASD were lower than those after h-ASD and h-PM2.5 + h-ASD, respectively B. Exposup < 0.01). A relative comparison between compounds showed that at 0.1, 1, and 10 µM, the production was comparable between 9,10-PQ and combined exposure at every dose  compared to the corresponding control. At 10 µM of 9,10-PQ, the IL-8 levels decreased in the 9,10-PQ, 9,10-PQ + h-PM2.5, 9,10-PQ + h-ASD, and 9,10-PQ + h-PM2.5 + h-ASD-treated cells (p < 0.01) vs. the corresponding control. Relative comparisons between compounds showed that at 0.1, 1, and 10 µM, the production was comparable between 9,10-PQ and the combined exposure at every dose . Relative comparison between compounds showed that at 0.1, 1, and 10 µM of 1,2-NQ, the production was similar between 1,2-NQ and the combined exposure at every dose . Relative comparison between compounds showed that at 10 µM of 1,2-NQ, the production was higher following 1,2-NQ + h-PM2.5 exposure than following 1,2-NQ exposure  compared to the corresponding control. However, no significant differences in ROS production were found between the groups treated with 9,10-PQ with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD  at concentrations of 1 and 10 μM of 9,10-PQ + h-PM2.5 and 0.1, 1, and 10 μM of 9,10-PQ + h-PM2.5 + h-ASD compared to the corresponding control . At 0.1 μM, ROS levels increased in 9,10-PQ + h-PM2.5 (p < 0.01), 9,10-PQ + h-ASD (p < 0.05), or 9,10-PQ + h-PM2.5 + h-ASD (p < 0.01) compared with those in 9,10-PQ alone. At 1 μM and 10 μM, ROS levels increased in 9,10-PQ + h-PM2.5, 9,10-PQ + h-ASD, or 9,10-PQ + h-PM2.5 + h-ASD compared with those in 9,10-PQ alone .When comparing h-PM2.5, h-ASD, and h-PM2.5 + h-ASD in the absence of 9,10-PQ, higher ROS production was observed in h-PM2.5 or h-ASD than in vehicles  compared to the corresponding control. In the comparison at 1 and 10 µM of 1,2-NQ, ROS levels increased significantly after exposure to 1,2-NQ + h-PM2.5 compared to exposure to 1,2-NQ alone . This effect was observed at 10 µM of 1,2-NQ, 10 µM of 1,2-NQ + h-PM2.5, and 1 and 10 µM of 1,2-NQ + h-ASD and 1,2-NQ + h-PM2.5 + h-ASD (p< 0.01) compared to the corresponding control , h-ASD (p < 0.05), or h-PM2.5 + h-ASD (p< 0.05) than in vehicle. At 0.1 μM, ROS levels increased significantly in 1,2-NQ + h-PM2.5 (p < 0.01), 1,2-NQ + h-ASD (p < 0.01), or 1,2-NQ + h-PM2.5 + h-ASD (p < 0.01) compared to 1,2-NQ alone. At 10 μM, ROS levels increased significantly in 1,2-NQ + h-PM2.5, 1,2-NQ + h-ASD, or 1,2-NQ + h-PM2.5 + ASD compared to 1,2-NQ alone  > V (IV) > Cr (III) > Cu (I) > Co (II)> Ni (II) > Pb (II) > Cd (II) [In this study, the levels of Pb (780 ng/mg), As (150 ng/mg), Cu (180 ng/mg), Cr (91 ng/mg), Ni (98 ng/mg), and Cd (9.5 ng/mg) in h-PM2.5 were 3.12-fold, 9.4-fold, 4.4-fold, 1.5-fold, 1.8-fold, and 24.4-fold higher than those in h-ASD, respectively. It has been reported that metals with H Cd (II) . Therefo Cd (II) . Higher  Cd (II) . Enhance Cd (II) ,36,37,38 Cd (II) . These rIn conclusion, this study demonstrated that exposure to two types of quinones, quinones + h-PM2.5 and/or h-ASD, dose-dependently increased WST-1 reduction at lower concentrations compared to the corresponding control, whereas it markedly decreased at higher concentrations. At a lower concentration of 9,10-PQ, exposure to h-ASD or h-PM2.5 + h-ASD increased the number of late apoptotic or necrotic cells, whereas exposure to 9,10-PQ + h-PM2.5 markedly increased the number. At a higher concentration of 1,2-NQ, exposure to h-ASD or h-PM2.5 + h-ASD also increased the number of late apoptotic or necrotic cells, whereas exposure to 1,2-NQ + h-PM2.5 especially increased their number. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD dose-dependently increased ROS production compared to the corresponding control. In the cell-free condition, quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD also dose-dependently increased ROS production compared to the corresponding control. Specifically, quinones + h-PM2.5 significantly increased the value.These results suggest that exposure to quinone chemicals at low concentrations combined with h-PM2.5 induces apoptosis or necrosis of bronchial epithelial cells, which is concomitant with intracellular or extracellular ROS overproduction. In contrast, h-ASD alone or combined with quinones induces apoptosis or necrosis. The different cell death patterns resulting from the coexistence of quinone chemicals may lead to different lung inflammations and injuries.2.The BEAS-2B cell line, derived from human bronchial epithelial cells transformed by an adenovirus 12-SV40 hybrid virus, was purchased from the European Collection of Cell Cultures . Airway epithelial cells were seeded in collagen-I-coated 96- or 12-well plates and incubated for 72 h to reach semiconfluence. The cells were incubated in serum-free medium LHC-9  at 37 °C in a humidified atmosphere containing 5% CO42− (16 μg/mg), Cl- (4.0 μg/mg), and NO3− (0.89 μg/mg). Additionally, the concentrations of water-soluble components were SO42− (16 μg/mg), NO3− (0.89 μg/mg), Ca2+ (78 μg/mg), Mg2+ (4.5 μg/mg), Na+ (3.5 μg/mg), and K+ (3.4 μg/mg). The levels of elements were analyzed by inductively coupled plasma atomic emission spectroscopy . The ions were analyzed by an ion chromatograph . The median diameter of h-PM2.5 was 1.26 ± 0.73 μm [PM reference material (CRM No. 28) is atmospheric dust collected by a ventilation filter of a building in Beijing from 1996–2005  and was  0.73 μm . 42- (0.68 μg/mg), NO3− (0.71 μg/mg), Ca2+ (21 μg/mg), Mg2+ (4.9 μg/mg), Na+ (2.9 μg/mg), and K+ (1.2 μg/mg). The median diameter of h-ASD was 2.87 ± 2.42 μm [ASD was purchased as a reference material  from the National Institute for Environmental Studies, Japan, and was purified. To exclude substances that are sensitive to heat, such as PAH, the ASD was treated by heating at 360 °C for 30 min, resulting in heated-ASD (h-ASD) . The maj 2.42 μm .After airway epithelial cells were grown to semiconfluence in LHC-9, we exposed the cells to two types of quinones  as an external electron acceptor for reduction using the Premix WST-1 Cell Proliferation Assay System . Briefly, after treatment for 21 h, the WST-1 reagent was added to each well of a 96-well plate and mixed by gentle shaking. After incubating BEAS-2B cells with the WST-1 reagent at 37 °C for 3 h, absorbance was measured at a wavelength and a reference wavelength of 450 nm and 630 nm, respectively, using an iMarkMicroplate Absorbance Reader . The results were expressed as the percentage of exposed cells relative to untreated cells .Cell death through apoptosis and necrosis was measured using annexin V (FITC) and Ethidium Homodimer III in an Apoptotic/Necrotic/Healthy Cells Detection Kit  and a fluorescence-activated cell sorter .n = 4), the cells were pooled together for measurement of (1) non-stain, (2) FITC-Annexin V stain, (3) Ethidium Homodimer III stain, and (4) FITC-Annexin V+ Ethidium Homodimer III stain. These (1)–(3) were used to determine autofluorescence and AnnexinV and Ethidium homodimer positive areas for each group, and each gating area  was defined. Cells were exposed to quinones + h-PM2.5 (50 μg/mL), quinones + h-ASD (50 μg/mL), and quinones + h-PM2.5 (25 μg/mL) + h-ASD (25 μg/mL). After treatment for 24 h (g for 5 min to remove floating cells. The resulting supernatant was stored at −80 °C until further use. The levels of interleukin (IL)-6 and IL-8 in the culture medium were quantified using an ELISA kit , following the manufacturer’s instructions. The absorbance was measured on the iMark microplate absorbance reader at 450 nm with a reference wavelength of 550 nm.After cell treatment for 24 h, the cell culture medium was collected and centrifuged at 300× 2DCFDA), was used. For intracellular ROS measurements, cells were incubated with 5 μM CM-H2DCFDA for 30 min to allow the dye to enter the cells. The cells were then washed to remove the extracellular dye, and the cells were exposed to two types of quinones  at a concentration of 0.1, 1, and 10 μM: quinones + h-PM2.5 (50 μg/mL), quinones + h-ASD (50 μg/mL), and quinones + h-PM2.5 (25 μg/mL) + h-ASD (25 μg/mL). Fluorescence intensity during 0–3 h was measured .To measure intracellular and cell-free ROS production, a fluorescent probe, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester  at concentrations of 0.1, 1, and 10 μM with quinones + h-PM2.5 (50 μg/mL), quinones + h-ASD (50 μg/mL), and quinones + h-PM2.5 (25 μg/mL) + h-ASD (25 μg/mL). We then added 5 μM CM-H2DCFDA, and fluorescence intensity was measured for 3 h using excitation and emission wavelengths of 485 nm and 530 nm, respectively.To measure cell-free ROS, 1 mM CM-Hn = 4). The significance of variation among different groups was determined by a one-way analysis of variance. Differences among groups were analyzed using Tukey’s multiple comparison tests . A p-value < 0.05 was considered statistically significant. For the detection of cell death via apoptosis and necrosis, the cells were pooled together after treatment for 24 h (n = 4).The data were presented as the mean ± standard deviation for each experimental group (


Cyperus sexangularis (CS) is a plant in the sedges family (Cyperaceae) that grows abundantly in swampy areas. The leaf sheath of plants in the Cyperus genus are mostly used domestically for mat making, while they are implicated for skin treatment in traditional medicine. The plant was investigated for its phytochemical contents as well as its antioxidant, anti-inflammatory and anti-elastase properties. The n-hexane and dichloromethane leaf extracts were chromatographed on a silica gel column to afford compounds 1–6. The compounds were characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. The inhibitory effect of each compound against 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and ferric ion radicals were determined by standard in vitro antioxidant methods. The in vitro anti-inflammatory response was measured using egg albumin denaturation (EAD) assay, while the anti-elastase activity of each compound in human keratinocyte (HaCaT) cells was also monitored. The compounds were characterized as three steroidal derivatives, stigmasterol (1), 17--hexadecahydro-cyclopenta[a]phenanthrene (2) and β-sitosterol (3), dodecanoic acid (4) and two fatty acid esters, ethyl nonadecanoate (5) and ethyl stearate (6). Stigmasterol (1) exhibited the best biological properties, with IC50 of 38.18 ± 2.30 µg/mL against DPPH, 68.56 ± 4.03 µg/mL against NO and 303.58 ± 10.33 µAAE/mg against Fe3+. At 6.25 µg/mL, stigmasterol inhibited EAD by 50%. This activity was lower when compared to diclofenac (standard), which demonstrated 75% inhibition of the protein at the same concentration. Compounds 1, 3, 4 and 5 showed comparable anti-elastase activity with an IC50 ≥ 50 µg/mL, whereas the activity of ursolic acid (standard) was double fold with an IC50 of 24.80 ± 2.60 µg/mL when compared to each of the compounds. In conclusion, this study has identified three steroids (1–3), one fatty acid (4), and two fatty acid esters (5 and 6) in C. sexangularis leaf for the first time. The compounds showed considerable antioxidant, anti-inflammatory and anti-elastase properties. Thus, the findings may serve as a justification for the folkloric use of the plant as a local skin ingredient. It may also serve to validate the biological role of steroids and fatty acid compounds in cosmeceutical formulations. Skin illnesses are prevalent in all human age groups and may be due to exposure towards radiation, biological toxins, bacteria, natural chemical agents found in the environment and, to some extent, due to starvation . Skin alOxidative stress is one of the main mechanisms that leads to skin aging and other skin problems, while sunlight ultraviolet (UV) radiation is one of the most known harmful factors that causes skin problems . AlteratThe dermal layer is structurally a flexible and reliable component of the skin . ElastinSkin normally requires elastase to remove non-functional proteins that remain in the extracellular matrix; for example, skin wound portion reconstruction needs dysfunctional proteins to be removed before the repair can occur ,13. An iIn recent times, more attention has been paid to the application of herbal cosmetics when compared to drug/synthetic cosmetics, because the herbal cosmetic ingredients have been found to be more potent and biocompatible than the synthetic ones, with little or no side effects . As a maCyperus sexangularis Nees (Family Cyperaceae) is a tufted, long-lasting  sedge that thrives in the wetland habitat +, m/z 355.2563 (40.0%) [M-57]+, m/z 204.3010 (88.0%) [M-108]+, m/z 157.0350  [M-255]+; 1H NMR (600 MHz in CDCl3), δ ppm: 0.68 , 0.80 , 0.82 , 0.84 , 0.92 , 1.00 , 3.53 , 5.35 ; COSY: 2.25 (H-4) and 3.53 (H-3), 5.01  and 5.15 ; 13C NMR (150 MHz in CDCl3), δ ppm: 37.40 (C-1), 34.08 (C-2), 71.95 (C-3), 42.42 (C-4), 140.89 (C-5), 121.87 (C-6), 31.78 (C-7), 32.04 (C-8), 50.27 (C-9), 36.29 (C-10), 21.23 (C-11), 39.92 (C-12), 42.45 (C-13), 56.91 (C-14), 24.45 (C-15), 29.29 (C-16), 56.20 (C-17), 12.12 (C-18), 18.93 (C-19), 40.63 (C-20), 19.97 (C-21), 138.46 (C-22), 129.41 (C-23), 45.97 (C-24), 26.21 (C-25), 12.00 (C-26), 29.85 (C-27), 19.54 (C-28), 19.18 (C-29). The full NMR assignments (Compound ignments  were com2 (21 mg): 17--hexadecahydro-cyclopenta[a] phenanthrene; C21H34 (exact mass = 286.15 g/mol); 1H NMR (400 MHz in CDCl3), δ ppm: 0.87 , 4.96 , 5.82 ; 13C NMR (100 MHz in CDCl3), δ ppm: 28.74 (C-1), 26.45 (C-2), 24.18 (C-3), 29.40 (C-4), 31.70 (C-5), 29.07 (C-6), 25.83 (C-7), 31.39 (C-8), 29.74 (C-9), 32.33 (C-10), 28.42 (C-11), 26.79 (C-12), 32.96 (C-13), 33.36 (C-14), 22.24 (C-15), 19.26 (C-16), 36.88 (C-17), 32.02 (C-18), 13.81 (C-19), 138.72 (C-20), 113.65 (C-21). The full NMR assignments (Compound ignments  were com3 (10 mg): β-sitosterol; C29H50O (exact mass = 414.71 g/mol); 1H NMR (400 MHz in C6D6), δ ppm: 0.66 , 0.78 , 0.80 , 0.82 , 0.91 , 0.99 , 2.25 , 3.52 , 5.32 ; 13C NMR (100 MHz in CDCl3), δ ppm: 37.48 (C-1), 31.86 (C-2), 71.78 (C-3), 42.28 (C-4), 140.66 (C-5), 121.39 (C-6), 30.36 (C-7), 29.73 (C-8), 50.02 (C-9), 36.86 (C-10), 22.93 (C-11), 39.52 (C-12), 42.28 (C-13), 56.60 (C-14), 28.09 (C-15), 29.73 (C-16), 55.82 (C-17), 11.84 (C-18), 17.98 (C-19), 36.24 (C-20), 19.23 (C-21), 35.61 (C-22), 25.74 (C-23), 45.71 (C-24), 24.33 (C-25), 11.96 (C-26), 29.69 (C-27), 20.88 (C-28), 19.28 (C-29). The full NMR assignments (Compound ignments  were com4 (22 mg): dodecanoic acid; C12H24O2 (exact mass = 200.32 g/mol); 1H NMR (400 MHz in C6D6), δ ppm: 0.86 , 1.23–1.28 , 1.58 ; 13C NMR (150 MHz in C6D6/TFT), δ ppm: 179.84 (C-1), 31.92 (C-2), 29.36 (C-3), 29.85 , 29.89 (C-10), 22.89 (C-11), 14.12 (C-12). The full NMR assignments (Compound ignments  were com5 (19 mg): ethyl nonadecanoate; C21H42O2 (exact mass = 326.56 g/mol); TOF ES+/MS (% rel. abundance): m/z 327.2211 (19.0%) [M+H]+, m/z 290.1702 (38.5%), m/z 157.0352  [M-169]+; 1H NMR (600 MHz in C6D6), δ ppm: 0.91 , 1.26 , 2.21 , 4.07 ; COSY: 1.61 (H-3) and 2.21 (H-2), 1.26 (H-18) and 0.91 (H-19); 13C NMR (150 MHz in C6D6/TFT), δ ppm: 173.01 (C-1), 34.59 (C-2), 25.49 (C-3), 26.41 (C-4), 29.27 (C-5), 29.60 (C-6), 29.69 (C-7), 29.74 (C-8), 29.95 (C-9), 29.81 (C-10), 30.05 (C-11), 30.00 (C-12), 30.13 (C-13), 30.09 (C-14), 30.15 (C-15), 30.21 (C-16), 32.36 (C-17), 23.10 (C-18), 14.00 (C-19), 64.31 (C-1′), 14.30 (C-2′).Compound 6 (21 mg): ethyl stearate; C20H40O2 (exact mass = 312.51 g/mol); TOF ES+/MS (% rel. abundance): m/z 313.4124 (16%) [M+H]+, m/z 273.1667 (70.0%) [M-39]+, m/z 217.0994 (34.5%) [M-95]+, 157.0376  [M-155]+. 1H NMR (600 MHz in C6D6), δ ppm: 0.91 , 1.26 , 2.21 , 4.08 ; COSY: 0.91 (H-18) and 1.24 (H-17), 1.26 (H-2′) and 4.08 (H-1′); 13C NMR (150 MHz in C6D6), δ ppm: 173.01 (C-1), 34.59 (C-2), 25.49 (C-3), 26.41 (C-4), 29.27 (C-5), 29.59 (C-6), 29.69 (C-7), 29.73 (C-8), 29.95 (C-9), 29.82 (C-10), 30.05 (C-11), 29.99 (C-12), 30.14 (C-13), 30.08 (C-14), 30.20 (C-15), 32.35 (C-16), 23.10 (C-17), 14.29 (C-18), 64.31 (C-1′), 14.29 (C-2′). The full NMR assignments of compounds 5 and 6 (Compound  5 and 6  were comC. sexangularis are presented in 1), 17--hexadecahydro-cyclopenta[a]phenanthrene (2), β-sitosterol (3), dodecanoic acid (4), ethyl nonadecanoate (5) and ethyl stearate (6) are reported in this plant for the first time.The structures of the six compounds isolated from the leaf of 4 and, in comparison, with the spectra data of undecanoic acid (Lauric acid) reported by Abe et al.  (1% w/v). Standard compounds used were L (+)-Ascorbic acid. The resulting mixture was incubated at 50 °C for 20 min, followed by the addition of 0.25 mL of CCl3COOH (10% w/v) and then centrifuged at 3000 rpm for 10 min. The upper layer of the solution was mixed with 1 mL of 2% DMSO and the same amount and 0.5 mL of FeCl3 . The experiment was conducted in triplicate, and absorbance was measured at 700 nm on a microplate reader against a blank sample of only phosphate buffer. Thus, the ferric reducing power of the isolated compounds at 100.00, 50.00, 25.00, 12.50 and 6.25 µg/mL concentration in methanol were determined as ascorbic acid equivalent (AAE) from the calibration curve of the positive control (L-ascorbic acid).The reducing antioxidant capacity of each isolated compound was evaluated according to the method of Kumar and Pandey , with soEAD = egg albumin denaturation, ABSsample = absorbance of compounds/Diclofenac, ABScontrol = absorbance of negative control (vehicle), i.e., 2% DMSO).The test was carried out using the egg albumin denaturation assay method reported by Chatterjee et al. . Here, a2 and 37 °C until a confluent monolayer formed. Trypsin was used to detach and sub-culture cells once a confluent monolayer had formed.The method as described by Lall et al.  was foll50, or concentration at which 50% of the enzyme activity is inhibited was calculated using GraphPad Prism 4 .The ability of each isolated compound to inhibit porcine pancreatic elastase (PPE) (Sigma) was determined by measuring the release of p-nitroaniline from N-succinyl-ala-ala-ala-p-nitroanilide spectrophotometrically at 405nm according to the method of Bieth et al.  and modit-test, using an Excel statistical application .The antioxidant and anti-inflammatory data were analyzed using a one-way analysis of variance (ANOVA), followed by Student’s 1), phenanthrene derivative (2), β-sitosterol, dodecanoic acid (4), ethyl nonadecanoate (5) and ethyl stearate (6), from C. sexangularis leaf for the first time. The compounds showed considerable in vitro antioxidant, anti-inflammatory and anti-elastase properties, with stigmasterol exhibiting the best bioactivity. Thus, this report may serve as a justification for the ethnomedicinal use of the plant for local skin treatment. It may also serve to validate the biological role of steroids and fatty acid compounds in cosmeceutical formulations.This study isolated and characterized six compounds, stigmasterol (


In this study, using a multi-step approach, it was possible to synthesize new pyrazinoquinazoline-3,6-diones focusing on compounds with fluorine substituents since, to the best of our knowledge, the synthesis of fluorinated fumiquinazoline derivatives had not been attempted before. The new synthesized derivatives were screened for antibacterial activity and, along with previously synthetized pyrazinoquinazoline-3,6-diones, were characterized for their antibiofilm and efflux-pump-inhibiting effects against representative bacterial species and relevant resistant clinical strains. Several compounds showed relevant antibacterial activity against the tested Gram-positive bacterial species with MIC values in the range of 12.5–77 μM. Furthermore, some derivatives showed promising results as antibiofilm agents in a crystal violet assay. The results of the ethidium bromide accumulation assay suggested that some compounds could potentially inhibit bacterial efflux pumps.Drug resistance is rising to alarming levels, constituting one of the major threats to global health. The overexpression of efflux pumps and the formation of biofilms constitute two of the most common resistance mechanisms, favoring the virulence of bacteria. Therefore, the research and development of effective antimicrobial agents that can also counteract resistance mechanisms are extremely important. Pyrazino[2,1- Antimicrobial resistance has been rising and it is already considered one of the major threats to global health ,2, with Escherichia coli is one of the most studied multidrug resistance efflux pumps quinazoline-3,6-dione core linked to an indole moiety  [Vibrio harveyi [minimum inhibitory concentration (MIC) = 32 µg/mL]. Among the alanine-derived alkaloids, fumiquinazoline F , as well as against sensitive and vancomycin-resistant strains of Enterococcus faecalis (MIC = 8 µg/mL) . It. It8a–8ees 8b–8e .11a–11f were synthesized from their respective 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids by treatment with SOCl2 in dry CH2Cl2 (10a), Fmoc-L-Ala-Cl (10b), and Fmoc-L-Leu-Cl (10c). Afterwards, the synthesis of the linear tripeptides 11a–11f (8a–8e with the respective Fmoc-protected acid chlorides (10a–10c) . It. It11a–1 11a–11f  was achi10a–10c) . This re11b and 11c were isolated and it was possible to purify and characterize them. However, in the case of tripeptides 11a, 11d, 11e, and 11f, they were used without further purification to make the synthetic pathway faster and to avoid yield losses. Three different methods were employed concerning the cyclization of the linear tripeptides, which are summarized in 1). It was possible to verify that the intermediate oxazine was not stable during the purification process, and therefore the three reactions were performed sequentially. Purification was carried out by preparative thin layer chromatography (TLC) followed by crystallization in MeOH that allowed the obtainment of glyantrypine .The obtained tripeptides ypine 1,  as white2). With Method B, the 1H-NMR spectrum of the obtained compound suggested that it corresponded to fumiquinazoline F , that to the best of our knowledge, had never been synthesized before. The employed multi-step synthetic pathway proved to be effective for the synthesis of fumiquinazoline-related derivatives. Nevertheless, it comprised some difficulties, such as the purification of the final compounds, which was hindered by the presence of a significant amount of secondary products formed in the last step, and the instability of some of the intermediates.Methods B  and C 3 Scheme  were theine F 2, , but sho F 2, 2,  in a 35% 12a–12d  were obt1H-NMR, 13C-NMR, heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC), and these confirmed the predicted structures. For the final derivatives, high-resolution mass spectrometry (HRMS) analysis  were lower than those obtained for the syn-enantiomers 12c and 12d (δ = 4.39 and 4.24 ppm). As is shown in anti-enantiomers. On the contrary, in the syn-enantiomers, this shielding effect was observed in the signals associated with H-25 and H-26 from the isobutyl substituent (δ 2.04–1.29 ppm for the anti-enantiomers and δ 1.23–0.66 ppm for the syn-enantiomers). This effect had already been reported by Hernandez et al. quinazoline-3,6-diones  were screened for their antibacterial, antibiofilm, and efflux-pump-inhibiting activities. Since, to the best of our knowledge, there has been no report to date of the screening of these type of fumiquinazoline derivatives for efflux pump inhibition, we decided to extend the screening of the antibiofilm activity and efflux-pump-inhibiting capacity to the related compounds 4 and 13a–13d, synthesized by our group in previous works quinazoline-3,6(4H)-dione : White crystals . m.p. 159.7–162.3 °C   , CHCl3) ). 1H NMR3P (5 equiv.) and I2 (4.9 equiv.) in dry CH2Cl2 (40 mL) N,N-diisopropylethylamine (10.1 equiv.) were added. The reaction mixture was stirred at room temperature for 5 h under a nitrogen atmosphere. Then, an aqueous solution of Na2CO3 (1M) was added to quench the reaction. The aqueous phase was extracted with CH2Cl2 (3×) and the organic phase was washed with brine (1×). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. Hexane (3×) was added to remove excess Ph3P. The precipitate was filtered under vacuum and dissolved in EtOAc. Piperidine (25.84 equiv.) was added and the mixture was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc:MeOH and stirred with Acros Organics silica gel 60 (0.2–0.5 mm) at room temperature overnight.To a solution of the appropriate tripeptide (1 equiv.), Ph11b  was dehydrated for 5 h with Ph3P , I2 , and N,N-diisopropylethylamine  in dry CH2Cl2 (40 mL). Deprotection with piperidine  in EtOAc (6.7 mL) for 1 h, was followed by stirring with silica gel (3.82 g) overnight in EtOAc:MeOH . After filtration, the compound was purified by preparative TLC using EtOAc as the mobile phase. The compound was further purified by crystallization in MeOH and fumiquinazoline F (2) was obtained as a white solid in a 35% yield.Tripeptide S,4R)-4-((1H-indol-3-yl)methyl)-1-methyl-1,2-dihydro-6H-pyrazinoquinazoline-3,6(4H)-dione : White solid . m.p. 138.5–140.5 °C (lit. mp 137 °C (foam) [D25] = −334   , CHCl3) ). 1H NMR11c  was dehydrated with Ph3P  and I2  in the presence of N,N-diisopropylethylamine  in dry CH2Cl2 (26.1 mL). Deprotection with piperidine  in EtOAc (4.42 mL) for 1 h, was followed by stirring with silica gel (2.5 g) overnight in EtOAc:MeOH . Purification by preparative TLC with silica plates and EtOAc:CH2Cl2 (6:4) as the mobile phase gave 12a as a very-light-pink solid in a 22% yield.Tripeptide S,4R)-4-((1H-indol-3-yl)methyl)-8-fluoro-1-isobutyl-1,2-dihydro-6H-pyrazinoquinazoline-3,6(4H)-dione (12a): Light-pink solid . m.p. 107.3–111.7 °C. [αD25] = −464 . 1H NMR  δ 8.11 , 7.99 , 7.60 , 7.52–7.44 , 7.29 , 7.13 , 6.98 , 6.66 , 5.75 , 5.65 , 3.77 , 3.65 , 2.73 , 2.04–1.92 , 1.41–1.29 , 0.77 , 0.29 . 13C NMR  δ 169.20 (C-3), 160.97 , 160.17 , 150.85 (C-14), 143.67 (C-12), 136.11 (C-19), 129.81 , 127.11 (C-24), 123.48 (C-17), 123.32 , 122.85 (C-21), 121.40  120.35 (C-22), 118.72 (C-23), 111.66 , 111.15 (C-20), 109.65 (C-16), 57.49 (C-4), 50.69 (C-1), 40.15 (C-25), 27.06 (C-15), 24.14 (C-26), 23.30 (CH3), 19.71 (CH3). HRMS (ESI−): m/z [C24H22FN4O2-H]− calcd. for [C24H22FN4O2]: 417.17213; found 417.17342. Peak purity = 99.9%; Chromatographic Purity = 97.9% was dehydrated with Ph3P  and I2  in the presence of N,N-diisopropylethylamine  in dry CH2Cl2 (14.7 mL), followed by deprotection with piperidine  in EtOAc (2.43 mL) for 1 h, and by stirring with silica gel (1.38 g) overnight in EtOAc:MeOH . Purification by preparative TLC with silica plates and EtOAc:CH2Cl2 (6:4) as the mobile phase gave 12b as a light-yellow solid in a 20% yield.Tripeptide S,4R)-4-((1H-indol-3-yl)methyl)-9-fluoro-1-isobutyl-1,2-dihydro-6H-pyrazinoquinazoline-3,6(4H)-dione (12b): Light-yellow solid . m.p. 112.0–114.7 °C. [αD25] = −273 . 1H-NMR  δ 8.37 , 8.16 , 7.46 , 7.30 , 7.30–7.21 , 7.22 , 7.14 , 6.98 , 6.66 , 5.80 , 5.65 , 3.76 , 3.63 , 2.71 , 1.97 , 1.40–1.29 , 0.76 , 0.29 . 13C NMR  δ 169.22 (C-3), 166.65 , 160.10 (C-6), 152.95 (C-14), 149.14 , 136.12 (C-19), 129.64 , 127.10 (C-24), 123.52 (C-17), 122.84 (C-21), 120.33 (C-22), 118.68 (C-23), 116.94 , 115.97 , 112.62 , 111.18 (C-20), 109.58 (C-16), 57.30 (C-4), 50.86 (C-1), 40.17 (C-25), 27.13 (C-15), 24.14 (C-26), 23.26 (CH3), 19.74 (CH3). HRMS (ESI−): m/z [C24H22FN4O2-H]− calcd. for [C24H22FN4O2]: 417.17213; found 417.17347. HPLC-DAD: Peak purity = 99.9%; Chromatographic Purity = 94.27% was dehydrated with Ph3P  and I2  in the presence of N,N-diisopropylethylamine  in dry CH2Cl2 (7.16 mL), followed by deprotection with piperidine  in EtOAc (1.17 mL) for 1 h, and by stirring with silica gel (0.661 g) overnight in EtOAc:MeOH . Purification by preparative TLC with silica plates using EtOAc:CH2Cl2 (6:4) as the mobile phase gave 12c as a light-yellow solid in a 8.2% yield.Tripeptide S,4S)-8,10-dichloro-4-((6-fluoro-1H-indol-3-yl)methyl)-1-isobutyl-1,2-dihydro-6H-pyrazinoquinazoline-3,6(4H)-dione (12c): Light-yellow solid . m.p. 163.0–168 °C. [αD25] = +450 . 1H-NMR  δ 8.24 , 8.19 , 7.83 , 7.33 , 6.97 , 6.74 , 6.65 , 6.58 , 5.47 , 4.39 , 3.75–3.70 , 1.23–1.16 , 1.05–0.98 , 0.74 , 0.60 . 13C NMR  δ 166.95 (C-3), 159.91, , 159.57 (C-6), 151.99 (C-14), 142.68 (C-12), 135.73 , 135.11 (C-10), 132.20 (C-9), 132.16 (C-11), 124.99 (C-8), 124.38 (C-24), 123.86 , 121.98 (C-7), 119.80 , 109.32 , 108.88 (C-16), 97.51 , 57.00 (C-4), 54.25 (C-1), 45.88 (C-25), 24.10 (C-26), 22.61 (CH3), 20.75 (CH3′). HRMS (ESI−): m/z [C24H20Cl2FN4O2-H]− calcd. for [C24H20Cl2FN4O2]: 485.09419; found 485. 09547. Peak purity = 94.2%; Chromatographic Purity = 100% was dehydrated with Ph3P  and I2  in the presence of N,N-diisopropylethylamine  in dry CH2Cl2 (15.9 mL), followed by deprotection with piperidine  in EtOAc (2.59 mL) for 1 h, and stirring with silica gel (1.49 g) overnight in EtOAc:MeOH . Purification by preparative TLC with silica plates and EtOAc:CH2Cl2 (6:4) as the mobile phase gave 12d as a light-yellow solid in a 4.2% yield.Tripeptide S,4S)-8,10-dichloro-4-((7-chloro-1H-indol-3-yl)methyl)-1-isobutyl-1,2-dihydro-6H-pyrazinoquinazoline-3,6(4H)-dione (12d): Light-yellow solid  m.p. 196.0–199.3 °C. [αD25] = +510 . 1H-NMR  δ 10.21 , 8.13 , 7.85 , 7.75 , 7.21 , 7.00 , 6.75 , 6.69 , 5.31 , 4.24 , 3.64 , 3.60 , 1.24–1.18 , 0.73–0.66 , 0.62 , 0.50 . 13C NMR  δ 166.32 (C-3), 159.49 (C-6), 152.66 (C-14), 142.66 (C-12), 134.68 (C-10), 133.06 (C-19), 132.49 (C-11), 131.59 (C-9), 129.45 (C-24), 125.03 (C-17), 124.71 (C-8), 121.80 (C-7), 121.14 (C-21), 119.99 (C-22), 117.22 (C-23), 116.64 (C-20), 109.51 (C-16), 57.06 (C-4), 53.91 (C-1), 46.34 (C-25), 26.41 (C-15), 23.71 (C-26), 22.23 (CH3), 20.98 (CH3′). HRMS (ESI−): m/z [C24H20Cl3N4O2-H]− calcd. for [C24H20Cl3N4O2]: 501.06464; found 501.06607. Peak purity = 97.4%; Chromatographic Purity = 100%, CCCP, crystal violet (CV), DMSO, Luria-Bertani (LB) broth, and LB agar, tryptic soy broth (TSB), and tryptic soy agar (TSA) purchased from Sigma-Aldrich Chemie GmbH ; phosphate-buffered saline , reserpine (RES), and ethanol purchased from Sigma-Aldrich Chemie GmbH ; Mueller–Hinton (MH) agar purchased from Sigma-Aldrich Chemie GmbH  and from BioKar Diagnostics ; dimethylsulfoxide (DMSO) purchased from Sigma-Aldrich Chemie GmbH  and Alfa Aesar, ; Dulbecco’s modified Eagle’s medium , containing 4.5 g/L glucose, supplemented with 10% heat-inactivated fetal bovine serum (FBS); and doxorubicin .Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212; and Gram-negative: Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, as well as clinically relevant strains, which included methicillin-resistant S. aureus , vancomycin-resistant Enterococcus  and a wild-type E. coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl Δ supE44], expressing the AcrAB–TolC efflux pump (EP) at its basal level and the AcrAB–TolC-deleted mutant E. coli AG100 A. For the compounds that showed relevant activity against E. faecalis B3/101, two VRE strains (#PSi/107 and #PFi/2) isolated from raw sewage were also tested ) was added to each wells and incubated for 15 min at room temperature. CV was discarded from the wells and the plate was washed with water again. Then, 200 μL of 70% ethanol was added to each well and the biofilm formation was determined by measuring the OD at 600 nm using a Multiscan EX ELISA plate reader . The antibiofilm effect of the compounds was expressed as the percentage (%) decrease in biofilm formation.The biofilm-forming ability of the E. coli SA/2, oxacillin  for S. aureus 66/1, and vancomycin  for E. faecalis B3/101. Antibiotic disks alone (controls) and antibiotic disks impregnated with 15 mg of each compound were also placed on the MH agar plates. Sterile 6 mm blank papers impregnated with 15 mg of each compound alone were also tested. A blank disk with DMSO was used as a negative control. MH inoculated plates were incubated for 18–20 h at 37 °C. Potential synergism was recorded when the halo of an antibiotic disk, impregnated with a compound, was greater than the halo of the antibiotic- or compound-impregnated blank disk alone. The most relevant compounds were also further tested for synergism using the broth microdilution method, using half of the MIC concentration of each compound and varied concentrations of each antibiotic, following the same procedure previously described for the MIC determination. Potential synergism was recorded when the MIC of the antibiotic was reduced to at least half the original value.To evaluate the combined effect of the compounds and clinically relevant antimicrobial drugs, initial screening was conducted using the Kirby–Bauer disk diffusion method, as recommended by the CLSI . A set o4 cells in 100 μL of medium were added to each well, with the exception of the medium control. In the case of NIH/3T3 cells, the compounds were added after seeding the cells at 37 °C overnight. The plates were incubated at 37 °C for 24 h; after the incubation period, 20 μL of MTT solution (from a 5 mg/mL stock) was added to each well. After 4 h of incubation at 37 °C, 100 μL of SDS solution (10% in 0.01 M HCl) was added to each well and the plates were incubated at 37 °C overnight. Cell growth was determined by measuring the optical density (OD) at 540 nm (ref. 630 nm) with a Multiscan EX ELISA reader. Inhibition of cell growth was determined as IC50 values, defined as the inhibitory dose that reduced the growth of the cells exposed to the tested compounds by 50%. IC50 values and the SD of triplicate experiments were calculated by using GraphPad Prism software version 5.00 for Windows with a nonlinear regression curve fit . Doxorubicin  was used as the positive control. The solvent (DMSO) did not affect the cell growth in the tested concentration (2%).The effects of increasing concentrations of compounds on cell growth were tested in 96-well flat-bottomed microtiter plates. The compounds were diluted in a volume of 100 μL of medium. Afterwards, 1 × 10E. coli (PDB: 2GFP) as the homolog, as described in quinazoline-3,6-diones and reinforces the idea that halogenated fumiquinazoline-related derivatives constitute a novel and relevant approach in the development of new antibacterial agents as well as in the development of compounds capable of fighting common resistance mechanisms. This study also allowed a more thorough study of the structure–activity relationship of the fumiquinazoline derivatives, that can provide useful insight for future optimization of the antibacterial potential of these compounds.In conclusion, this work allowed the synthesis of a small library of new indole-containing pyrazino[2,1-Future work will involve further studies to evaluate the biological activities of the synthesized compounds. Studies to investigate the mechanism of action of the compounds would also be interesting. Furthermore, molecular modifications to reduce the cytotoxicity of some of the most potent compounds might be required in the future.


Their structures, including absolute configurations, were identified by comprehensive spectroscopic analyses and computational methods. Inspired by our previous study on the same kinds of skeletons, we speculated that the new compounds have anticancer and anti-inflammatory activities. The results did not show any activity, but they revealed the structure–activity relationships (SAR).Five new eudesmane-type sesquiterpenoids (aquisinenoids F–J ( Aquilaria species of the Thymelaeaceae family + , 13C NMR, and DEPT spectra, indicating 5 degrees of unsaturation. The 1H NMR spectrum of 1 , two olefinic protons , and an oxygenated methylene at δH 4.06 . The 13C NMR and DEPT (2), one methine, and four nonprotonated carbons (including three sp2 and one sp3). The planar structure of 1 was mainly constructed by 2D NMR analysis. First, the 1H,1H-COSY spectrum (C 148.3), H-3/C-4 (δC 126.8), C-5 and H3-15/C-1 (δC 40.6), C-5, and C-10 (δC 36.2) indicate the presence of a six-membered ring. Additional HMBC correlations of H-6/C-4, C-5, H-7/C-5, and H3-15/C-9 (δC 43.0) allowed us to assign another six-membered ring, as shown in 3-15/C-1, C-5, C-9, and C-10, respectively. In addition, the HMBC correlations of H-3/C-14 (δC 174.9) and H-6/C-4 indicates the presence of a carboxyl group at C-4. Finally, the HMBC correlations of H-13/C-7 (δC 43.9), C-11 (δC 154.7), and C-12 (δC 65.0) demonstrate that C-7 is connected with acryl alcohol. Thus, the planar structure of 1 was assigned  D20 +33.33 ; CD (MeOH) Δε201 −2.01, Δε208 −3.21; UV (MeOH) λmax (log ε) 200 (2.89) nm, 222 (2.73) nm; HRESIMS (m/z): [M + Na]+ calcd for C15H22O3Na, 273.1461; found, 273.1464; 1H and 13C NMR data, see Compound 2: Pale yellow gum. [α]D20 +18.25 ; CD (MeOH) Δε200 +2.99, Δε211 −5.12, Δε239 +0.53; UV(MeOH) λmax (log ε) 200 (3.02) nm, 246 (2.11) nm; HRESIMS (m/z): [M + Na]+ calcd for C15H24O2Na, 259.1669; found, 259.1671; 1H and 13C NMR data, see Compound 3: Pale yellow gum. [α]D20 +26.00 ; CD (MeOH) Δε202 −0.16, Δε224 +1.31; UV(MeOH) λmax (log ε) 223 (2.34) nm; HRESIMS (m/z): [M + Na]+ calcd for C15H24O3Na, 275.1618; found, 275.1622; 1H and 13C NMR data, see Compound 4: White powder. [α]D20 +20.99 ; CD (MeOH) Δε203 −0.10, Δε211 −1.43, Δε238 −7.89; UV (MeOH) λmax (log ε) 218 (2.85) nm; HRESIMS (m/z): [M + Na]+ calcd for C16H24O3Na, 287.1618; found, 287.1614; 1H and 13C NMR data, see Compound 5: Colorless gum. [α]D20 +74.00 ; CD (MeOH) Δε201 +2.69, Δε227 −0.05; UV (MeOH) λmax (log ε) 200 (2.82) nm; HRESIMS (m/z): [M + Na]+ calcd for C15H26O2Na, 261.1825; found, 261.1825; 1H and 13C NMR data, see Compound 1H and 13C NMR chemical shifts using the PCM solvent model in Gaussian 09 software [13C NMR chemical shift was analyzed by regression with the experimental one. Boltzmann weighing of the predicted chemical shift of each isomer and the DP4+ parameters were calculated using the Excel file provided by Ariel M. Sarotti [Using density functional theory (DFT) and B3LYP/6-31G  levels isoftware . The NMR Sarotti .Using the Spartan'14 software package  and the Gaussian 09 software package, the conformational search was performed using the molecular Merck force field (MMFF)  with CONFile 11–5, NMR and ECD calculations, and bioactivity assay data.MS, UV, and NMR spectra of compounds


The reaction of 4,8-dibromobenzobis with thiophenes in the presence of palladium acetate as a catalyst and potassium pivalate as a base, depending on the conditions used, selectively gave both mono- and bis-thienylated benzo-bis-thiadiazoles in low to moderate yields; arenes were found to be inactive in these reactions. It was discovered that direct C–H arylation of benzobisarenes and -thiophenes in the presence of Pd(OAc)2 and di-tert-butyl(methyl)phosphonium tetrafluoroborate salt is a powerful tool for the selective formation of 4-mono- and 4,8-di(het)arylated benzo-bis-thiadiazoles. Oxidative double C–H hetarylation of benzobis Th. Thc:4,5operties . It was mponents .isoBBT derivatives bis 1 with halogen (het)aryls; and 3. double oxidative arylation of benzobis with C-H (het)aryl. These three routes were investigated for 2,1,3-benzothiadiazole (BTD) derivatives , sodium tert-butoxide and neodecanoic acid was effective for the synthesis of 4,7-bis(5-hexyl-2-thienyl)benzo[c]thiadiazole thiadiazole [o-(di-o-tolylphosphino)-benzyl]dipalladium(II)), cesium pivalate as base and tris(o-methoxyphenyl)phosphine as ligand thiadiazoles [Unsubstituted benzo[c]thiadiazole reacted with bromoarenes or hetarenes , path 2 diazoles ,35.2) thienylation of benzo[c]thiadiazoles with thiophenes 2 gave higher yields of dithienylated benzo[c]thiadiazoles bis in low yield upon treatment of isoBBT 1 with palladium (II) acetate in the presence of potassium pivalate and di-tert-butyl(methyl)phosphonium tetrafluoroborate salt (PtBu2Me·HBF4) in toluene at 120 °C thiophen-2-yl)benzo..d:4,5-d′d:4,5-d′]bis 1 and its 4,8-dibromo derivative 2 with aromatic and heterocyclic compounds.To elucidate the applicability of direct C–H (het)arylation reactions of tricyclic benzo-bis-thiadiazoles, this paper describes the study of the reaction of benzobis 2 with (2-ethylhexyl)thiophene 3a in the presence of various palladium catalysts and organic ligands. The results of this study are summarized in 2 with potassium pivalate as a base in toluene, both mono-4a- and bis-aryl derivatives 5a can be obtained. The nature of the solvent and ligand, the temperature of the chemical transformation, and the excess of the reagent significantly affected the results of the reactions (2 (2 with the formation of the target product 4a in moderate yield (5a in a yield close to that of mono-product 4a (tert-butylphosphine (But3P), bis(diphenylphosphino)ferrocene (dppf) or XPhos, PBut2MeHBF4, both in toluene and in DMA, stopped the formation of products 4a and 5a; in these cases, the starting heterocycle 2 decomposed slowly under the reaction conditions  as a halogen-containing substrate are summarized in 2) and potassium pivalate (PivOK) resulted in partial decomposition of the starting bicycle 1 without the formation of target products 7a and 5a (tert-butylphosphine (But3P) or bis(diphenylphosphino)ferrocene (dppf) did not activate the cross-coupling reaction (7a with a low yield (3)4), tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), and bis(triphenylphosphine)palladium chloride (PdCl2(PPh3)2) also did not run the cross-coupling reaction (2) and di-tert-butyl(methyl)phosphonium tetrafluoroborate salt (P(But)2MeHBF4) bis 1 was carried out in refluxing toluene in the presence of potassium pivalate, then the bis-coupling product 7a was formed (2 and P(But)2MeHBF4 led to the formation of compound 7a in 45% yield thiophene in the presence of Pd(OAc)2) and P(But)2MeHBF4) led to the formation of a mixture of mono-7a and bis-5a substituted products in a ratio of 2:1 (6a(I) to two equivalents and replacing toluene with xylene resulted in the selective formation of the bis-coupling product 5a in 54% yield thiophene a and 5a , entry 1ow yield , entry 22MeHBF4) . If the s formed , entry 92 and PBut2MeHBF4 catalytic system in refluxing toluene at 110 °C or in xylene at 130 °C) were extended to halogenated derivatives of thiophene and benzene 6b-j. If for 2-bromothiophenes 6a-c,e(Br) the hetarylation reactions proceeded selectively and with moderate yields of both mono-7 and bis-5 products, then for bromoarenes the chemical transformation led to a lower yield of mono- and bis-coupling products (6f(Br) by the more reactive iodobenzene 6f(I) made it possible to significantly increase the yield of both mono-coupling 7f and bis-coupling 5f products (6(I) in the reaction with tricycle 1 gave the target products 7 and 5 in moderate yields 1 with thiophene derivatives were studied using (2-ethylhexyl)thiophene 3a, palladium trifluoroacetate and acetate as catalysts under the action of silver (I) oxide (Ag2O) as an oxidizing agent in dimethyl sulfoxide as described for BTD derivatives (see 7a and bis-5a coupling products (3), silver tetrafluoroborate (AgBF4), and silver perchlorate (AgClO4). It was shown that in the case of silver acetate, the total yield of the mixture of products 7a and 5a was only 25%, while in the case of silver nitrate, compound 5a was isolated in 4% yield, and the use of silver tetrafluoroborate and silver perchlorate did not lead to the formation of thienylated products  1 and its dibromo derivative 2 with classical cross-coupling reactions, we analyzed the results obtained in this work using data on the Suzuki and Stille reactions of dibromo derivative 2 described in bis 2 gave good yields of bis-arylated heterocycles 4 , and the Suzuki–Miyaura reaction led to the selective formation of both mono- 4  and bis-(het)arylated 5  benzobis bis 1 bis 2 bis 2 , thiophene 3a-d (0.28 mmol) in anhydrous toluene (8 mL). The resulting mixture was degassed by argon in a sealed vial. The resulting mixture was then stirred at 110 °C for the time shown in 2Cl2 (3 × 35 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography.Pd(OAc)2 , pivalic acid  and K2CO3  were added to a solution of 4,8-dibromobenzobis 2 , thiophene 3a-d (0.56 mmol) in anhydrous xylene (8 mL). The resulting mixture was degassed by argon in a sealed vial. The resulting mixture was then stirred at 130 °C for the time shown in 2Cl2 (3 × 35 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography.Pd(OAc)2 , (P(But)2MeHBF4) , pivalic acid  and K2CO3  were added to a solution of benzobis 1 , bromide or iodide 6a-d,f-j(X) (1.03 mmol) in anhydrous toluene (8 mL). The resulting mixture was degassed by argon in a sealed vial. The resulting yellow mixture was then stirred at 110 °C for the time shown in 2Cl2 (3 × 35 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography.Pd(OAc)2 , (P(But)2MeHBF4) , pivalic acid  and K2CO3  were added to a solution of benzobis 1 , bromide or iodide 6a-d,f-j(X) (2.06 mmol) in anhydrous xylene (8 mL). The resulting mixture was stirred and degassed by argon in a sealed vial. The resulting yellow mixture was then stirred at 130 °C for the time shown in 2Cl2 (3 × 35 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography.Pd(OAc)2O  and Pd(OAc)2  were added to a solution of benzobis 1  and thiophene 3a-c,e (1.53 mmol) in dry DMSO (5 mL). The resulting mixture was degassed by argon in a sealed vial. The resulting yellow mixture was then stirred at 90 °C for the time shown in 2Cl2 (3 × 35 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography.Ag2O  and Pd(OAc)2  were added to a solution of benzobis 1  and 2-(2-ethylhexyl)thiophene 3a (0.51 mmol) in dry DMSO (5 mL). The resulting mixture was degassed by argon in a sealed vial. The resulting yellow mixture was then stirred at 90 °C for the time shown in 2Cl2 (3 × 35 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography.Agd:4,5-d′]bis (4a).4-Bromo-8-(5-(2-ethylhexyl)thiophen-2-yl)benzobis (4b).4-Bromo-8-(thiophen-2-yl)benzobis (4c).4-Bromo-8-(4-hexylthiophen-2-yl)benzobis (4d).4--8-bromobenzobis (5a).4,8-Bis(5-(2-ethylhexyl)thiophen-2-yl)benzobis (5b).4,8-Di(thiophen-2-yl)benzobis (5c).4,8-Bis(4-hexylthiophen-2-yl)benzobis (5d).4,8-Dibenzobis (5e).4,8-Bis(5′-(trimethylsilyl)--5-yl)benzobis (5f).4,8-Diphenylbenzobis (5g).4,8-Di-2Cl2:hexane, 1:2 (v/v). Rf = 0.4 (CH2Cl2:hexane 1:1 (v/v)). Mp > 250 °C. The data of the 1H and 13C NMR spectra correspond to the literature data bis (5h).4,8-Bis(4-methoxyphenyl)benzobis-4,8-diyl)dibenzoate (5i).Dimethyl 4,4′-. Rf = 0.1 ). Mp > 250 °C. IR νmax : 2954, 2925, 2854, 1724, 1642, 1608, 1430, 1413, 1317, 1287, 1209, 1189, 1112, 1012, 960, 860, 825, 766, 695, 646, 567. 1H NMR : δ 8.33 , 8.10 , 4.01 . 13C NMR : δ 166.1, 155.3, 141.7, 140.9, 131.7, 130.3, 129.6, 127.5, 52.2. HRMS (ESI-TOF), m/z: calcd for C22H15N4O4S2 [M + H]+, 463.0529, found, 463.0521. MS , m/z : 462 , 431 (4), 406 (12), 375 (11), 347 (60), 332 (4), 303 (7), 288 (25), 203 (8), 144 (40), 59 (100), 15 (25).Yellow solid, 233 mg , eluent–CHd:4,5-d′]bis-4,8-diyl)bis (5j).4,4′-. Rf = 0.5 ). Mp > 250 °C. : δ 7.85 , 7.35–7.30 , 7.24–7.08 .Red solid, 175 mg , eluent-CH> 250 °C ). The daure data ). 1H NMRd:4,5-d′]bis (7a).4-(5-(2-Ethylhexyl)thiophen-2-yl)benzo+, 389.0923, found, 389.0921. MS , m/z : 390 , 389 , 388 , 360 (80), 332 (15), 261 (18), 248 (38), 233 (100), 69 (28), 57 (60), 41 (45), 29 (37).Orange solid, 179 mg , 69 mg , eluent-CHd:4,5-d′]bis (7b).4-(Thiophen-2-yl)benzo+, 276.9671, found, 276.9663. MS , m/z : 276 , 248 (75), 220 (10), 176 (11), 151 (100), 93 (25), 69 (95), 45 (12), 28 (5).Orange solid, 85 mg , or 50 mg , eluent–CHd:4,5-d′]bis (7c).4-(4-Hexylthiophen-2-yl)benzo+, 361.0610, found, 361.0606. MS , m/z : 362 , 361 , 360 , 332 (100), 248 (20), 235 (19), 220 (12), 165 (18), 120 (13), 69 (60), 43 (57), 29 (48).Yellow solid, 140 mg , eluent-CHd:4,5-d′]bis (7e).4-(5′-(Trimethylsilyl)--5-yl)benzo+, 430.9943, found, 430.9928. MS , m/z : 432 , 431 , 430 , 402 (7), 305 (6), 200 (10), 175 (12), 93 (45), 69 (100), 45 (30).Red solid, 140 mg , eluent-CHd:4,5-d′]bis (7f).4-Phenylbenzo+, 271.0107, found, 271.0109. MS , m/z : 270 , 242 (58), 214 (26), 170 (23), 145 (90), 93 (20), 69 (100), 28 (40).Yellow solid, 125 mg , eluent-CHp-Tolyl)benzobis (7g).4-. Rf = 0.3 ). Mp = 229–232 °C. IR νmax : 2925, 1639, 1609, 1507, 1427, 1379, 1331, 1317, 1291, 1275, 1192, 1147, 1120, 895, 865, 828, 804, 763, 716, 670, 609, 556, 536, 488. 1H NMR : δ 9.24 , 7.89 , 7.46 , 2.51 . 13C NMR : δ 157.8, 155.5, 140.6, 140.4, 139.8, 134.0, 130.0, 129.8, 129.4, 112. 1, 21.3. HRMS (ESI-TOF), m/z: calcd for C13H8BrN4S2 [M + H]+, 285.0263, found, 285.0266. MS , m/z : 284 , 256 (8), 227 (5), 159 (25), 139 (5), 93 (7), 69 (100), 63 (7), 51 (10), 39 (30), 28 (45), 18 (70).Green solid, 131 mg , eluent-CHd:4,5-d′]bis (7h).4-(4-Methoxyphenyl)benzo+, 301.0212, found, 301.0215. MS , m/z : 302 , 301 , 300 , 272(50), 229 (45), 201 (25), 175 (80), 132 (65), 93 (35), 69 (100), 28 (30).Orange solid, 185 mg , eluentCHd:4,5-d′]bis-4-yl)benzoate (7i).Methyl 4-. Rf = 0.1 ). Mp = 235–237 °C. IR νmax : 2956, 2925, 2854, 1724, 1608, 1463, 1431, 1377, 1277, 1189, 1110, 1084, 1018, 965, 895, 867, 839, 811, 754, 702, 632. 1H NMR : δ 9.33 , 8.31 , 8.07 , 4.01 . 13C NMR : δ 166.4, 158.0, 155.5, 141.0, 140.8, 140.1, 131.7, 130.5, 129.8, 128.6, 113.6, 52.5. HRMS (ESI-TOF), m/z: calcd for C14H9N4O2S2 [M + H]+, 329.0161, found, 329.0151. MS , m/z : 329 , 328 , 300 (100), 256 (10), 227 (12), 213 (30), 203 (65), 144 (45), 93 (10), 69 (80), 59 (8).Green solid, 152 mg , eluent-CHd:4,5-d′]bis-4-yl)-N,N-diphenylaniline (7j).4-. Rf = 0.25 ). Mp = 213–215 °C. IR νmax : 1727, 1590, 1487, 1428, 1321, 1276, 1195, 1125, 1073, 894, 865, 835, 808, 748, 696, 624, 512. 1H NMR : δ 9.18 , 7.88 , 7.35 , 7.28–7.12 . 13C NMR : δ 158.0, 155.3, 150.0, 146.9, 140.8, 139.3, 130.6, 129.8, 129.6, 129.0, 125.7, 124.3, 121.4, 111.5. HRMS (ESI-TOF), m/z: calcd for C24H15N5S2 [M]+, 437.0763, found, 437.0757. MS , m/z : 438 , 437 , 409 (6), 381 (4), 312 (12), 168 (3), 69 (15), 18 (100).Orange solid, 180 mg , eluent-CHd:4,5-d′]bis with thiophenes catalyzed by palladium acetate in the presence of potassium pivalate as a base, and no reaction occurred for substituted arenes. The catalytic system, containing Pd(OAc)2 and di-tert-butyl(methyl)phosphonium tetrafluoroborate salt, proved to be the best for the synthesis of (het)arylated benzo-bis-thiadiazoles from unsubstituted benzobis (het)arenes. Bis(thienyl)benzo-bis-thiadiazoles were successfully prepared by oxidative hetarylation of benzobis acetate and silver (I) oxide in DMSO.The study of direct palladium-catalyzed (het)arylation reactions of strong electron-withdrawing benzo[1,2-


In 2008 Uruguay included pneumococcal conjugate vaccine (PCV) 7valent in children´s vaccination program (2+1). In 2010 PCV13 replaced PCV7. Since 2015, adults at risk of invasive pneumococcal disease (IPD) may receive 23 valent polysaccharide vaccine (PV23) or combined schedule PCV13+PV23.Objective: to assess the impact of this strategy on IPD hospitalization.Rates per 10,000 discharges , diseases and serotypes (ST) are described before PCV7 vaccination (2006-2007), the year of vaccine implementation (2008), early after implementation (2009-2015) and late after implementation (2016-2022), the last 3 years during COVID-19 pandemic.In 2006-2022 were 33 IPD cases in children and 88 in adults over 15 years old. Children´s hospitalization median annual rates were as follow 2006-2007: 50 (25-75); 2008: 18,5 (-2-39); 2009-2015: 8,5 (3-14); 2016-2022: 4 (0-8); percentages of rate reduction were 82,5% and 92% (p< 0.0000). In adults 2006-2007: 17 (10-25); 2008: 13,4 ; 2009-2015: 6,6 ; 2016-2022: 6 ; percentages of rate reduction were 63,5% and 66 % .Pneumococcal pneumonia (PP) was the most frequent disease in children 22/33 , followed by bacteremia 8/32. PP was also in adults 68/88 , followed by meningitis (7/88). One non vaccinated child died in 2008. Case fatality rate in adults was 9% (8/88), 3 died in 2006-2007, 2 in 2009-20215 and 3 in 2016-2022.24 different ST were isolated in adults. Most frequent ST were 12F(15), 7F(11), 1(8), 8(7), 3(6) and 5(6). PCV13 would cover 38/88 (43%). A significant decline in PCV13 serotypes (PCV13-ST) was observed comparing the period prior PCV 17/22 (77%) with the late period 8/24 (34%) (p< 0.0000).15 different ST were isolated in children: 26 PCV13-ST. 16 prior 2008: most frequent ST 1(5), 14(4), 3(2) and 5(2). In 2009-2022 13 hospitalizations; PCV13-ST: 1(2), 19A(2), 14(1), 6B< (1), 9V(1), 3(1) and non PCV13-ST: 7B/C(1), 24F(1), 13(1), 15A(1), 15B(1). A significant decline of PCV13-ST was observed comparing the period prior PCV  with the late period  (p< 0.0000).There was a reduction in children’s discharge rates and in adults´ probably due to herd effect. Adult vaccination will improve individual immunization.María Catalina Pírez, Pediatrician, Pediatric infectologist, microbiologist Professor of pediatric, Degree V, Merck, Pfizer: Expert Testimony|Merck, Pfizer: Honoraria


A series of carbamothioyl-furan-2-carboxamide derivatives were synthesized using a one-pot strategy. Compounds were obtained in moderate to excellent yields (56–85%). Synthesized derivatives were evaluated for their anti-cancer  and anti-microbial potential. Compound p-tolylcarbamothioyl)furan-2-carboxamide showed the highest anti-cancer activity at a concentration of 20 μg/mL against hepatocellular carcinoma, with a cell viability of 33.29%. All compounds showed significant anti-cancer activity against HepG2, Huh-7, and MCF-7, while indazole and 2,4-dinitrophenyl containing carboxamide derivatives were found to be less potent against all tested cell lines. Results were compared with the standard drug doxorubicin. Carboxamide derivatives possessing 2,4-dinitrophenyl showed significant inhibition against all bacterial and fungal strains with inhibition zones (I.Z) in the range of 9–17 and MICs were found to be 150.7–295 μg/mL. All carboxamide derivatives showed significant anti-fungal activity against all tested fungal strains. Gentamicin was used as the standard drug. The results showed that carbamothioyl-furan-2-carboxamide derivatives could be a potential source of anti-cancer and anti-microbial agents. Carbamothioyl-aryl-2-carboxamide has promising pharmacological properties ,2,3,4. TCarbamothioyl-aryl-2-carboxamide derivatives including furane, thiazole, benzothiozole, and thiophene moieties showed active anti-fungal (i), anti-bacterial (ii) and anti-cancer potential (iii) ,6 Figur. 4a–f), and these derivatives were screened for their anti-cancer and anti-microbial activities.Scientists are attempting to incorporate furan-based building blocks into drug molecules to give them exceptional medicinal properties. The furan moiety is physiologically active, and it has the potential to modify protein-based molecules ,8. Polym2 were removed under reduced pressure, a further reaction mixture was concentrated in the vacuum, and furan-2-carbonyl chloride in a colorless liquid was obtained (2). Furan-2-carbonyl chloride (2) was treated with anhydrous KSCN in the presence of dry acetone to synthesize furoyl iso-thiocyanate (3) in a quantitative yield. Iso-thiocyanate is a useful precursor in synthetic chemistry for the synthesis of different substituted carbamothioyl-furan-2-carboxamides  = 290.0. Anal. Calcd. (%) for C12H9N3O4S: C, 49.48; H, 3.11C; Found: C, 49.45; H, 3.14. EI-HR-MS C12H9N3O4S [M]•+ calcd = 291.0314; found = 291.0297.(4b): N-(3-nitrophenylcarbamothioyl)furan-2-carboxamide, , yellowish white solid, m.p: 201 °C; IR ; 3295 (N-H), 1664 (C=O), 1588 (C=C), 1442 (NO), 1343 (C-N), 1266 (C=S), 1H NMR : δ (ppm): 12.62 , 9.30 , 8.76 , 8.16 , 8.08 , 7.69–7.60 , 7.47–7.44 , 6.70 , 13C-NMR  δ: 178.5 (C=S), 156.9 (C=O), 148.4, 146.2, 144.6, 138.8, 129.8, 129.7, 121.4, 119.6, 118.9, 113.6. LC/MS m/z; [M − H] = 290.08. Anal. calcd. (%) for C12H9N3O4S: C, 49.48; H, 3.11; Found: C, 49.45; H, 3.14. EI-HR-MS C12H9N3O4S [M]•+ calcd = 291.0314; found = 291.0326.(4c):N-(4-nitrophenylcarbamothioyl)furan-2-carboxamide, , yellowish white solid, m.p.: 212 °C; IR ; 3321 (N-H), 1664 (C=O), 1568 (C=C), 1504 (NO), 1346 (C-N), 1210 (C=S), 1H NMR : δ (ppm): 12.82 , 9.32 , 8.30 , 8.05 , 7.68–7.63 , 7.46 , 6.69 , 13C-NMR  δ: 180.0 (C=S), 157.9 (C=O), 146.9, 145.2, 130.9, 128.8, 124.6, 123.2, 119.7, 113.6. LC/MS m/z; [M − H] = 290.08. Anal. calcd. (%) for C12H9N3O4S: C, 49.48; H, 3.11; Found: C, 49.45; H, 3.14. EI-HR-MS C12H9N3O4S [M]•+ calcd = 291.0314; found = 291.0297.(4d):N-(p-tolylcarbamothioyl)furan-2-carboxamide, , off white solid, m.p. 136 °C; IR : 3389 (N-H), 1667 (C=O), 1581 (C=C), 1329 (C-N), 1251 (C=S), 1H NMR : δ (ppm): 10.46 , 8.76 , 7.62 , 7.45 , 7.41 , 7.17 , 6.63 , 2.35 , 13C-NMR  δ: 178.1 (C=S), 158.1 (C=O), 146.2, 145.6, 134.5, 134.2, 129.6, 120.5, 118.1, 113.1, 20.9. LC/MS m/z; [M − H] = 259.08. Anal. calcd. (%) for C13H12N2O2S: C, 59.98; H, 4.65; Found: C, 59.93; H, 4.61. EI-HR-MS C13H12N2O2S [M]•+ calcd = 260.0619; found = 291.0624.(4e): N-(1H-indazol-3-ylcarbamothioyl)furan-2-carboxamide, , off white solid, m.p. 210 °C; IR : 3189 (NH), 1678 (C=O), 1577 (C=C), 1338 (C-N), 1205 (C=S), 1095 (N-N), 1H NMR : δ (ppm): 12.98 , 12.53  9.56 , 7.82 , 7.69–7.62 , 7.53–7.47 , 6.65 , 13C-NMR  δ: 176.1 C=S, 157.4 C=O, 153.6, 146.5, 143.3, 140.5, 127.3, 119.2, 118.0, 116.4, 114.0, 113.1, 110.6. LC/MS m/z; [M − H] = 285.08. Anal. calcd. (%) for C13H10N4O2S: C, 54.54; H, 3.52; Found: C, 54.52; H, 3.49. EI-HR-MS C13H10N4O2S [M]•+ calcd = 286.0524; found = 286.0531.(4f):N-hydrazinecarbamothioyl)furan-2-carboxamide, , yellow solid, m.p. 124 °C; IR ; 3214 (N-H), 1669 (C=O), 1573 (C=C), 1530 (NO), 1336 (C-N), 1259 (C=S), 1094 (N-N), 1H NMR : δ (ppm):10.99 , 9.09 , 8.25 , 8.14 , 7.92 , 7.67–7.58 , 7.47–7.36 , 4.62–3.86 , 2.65 , 13C-NMR  δ: 180.0 (C=S), 158.2 (C=O), 153.1, 146.3, 144.6, 137.2, 133.6, 129.3, 122.4, 119.1, 119.5, 113.2. LC/MS m/z; [M − H] = 350.08. Anal. calcd. (%) for C12H9N5O6S: C, 41.03; H, 2.58; Found: C, 41.05; H, 2.55. EI-HR-MS C12H9N5O6S [M]•+ calcd = 351.0413; found = 351.0419.(2 (5%). Cancer cells were treated with carbamothioyl-furan-2-carboxamide derivatives dissolved in solvent (DMSO) with a final concentration of 0.05%The cancer cell lining was grown in Dulbecco’s Modified Eagle medium which provided fetal bovine serum (10%) and gentamicin (100 µg/mL) and was maintained at normal human body temperature (37 °C) with COCell viability was calculated by MTT assay . Cancer Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) and 2 fungal strains (Fusarium bracchygibossum and Aspergillus niger). Pure microbes (bacteria and fungi) were collected from the zoology department at the Government College University Faisalabad, Pakistan. Synthesized derivatives were dissolved in 10 mg/mL of DMSO to prepare the stock solution [Anti-microbial activity of synthesized carbamothioyl-furan-2-carboxamide derivatives was checked using the well diffusion method against 2 bacterial strains, solution . In a Pesolution . The dru4a–f) [v/v) was placed in first row of 96-well plates. Then, 50 µL each of Muller-Hinton-broth for the bacteria and thefungus and nutrition broth were added to all other wells. The test substance was serially diluted twice so that each well contained 50 µL at progressively lower concentrations.A modified resazurin microtiter plate test was used to determine the minimum inhibitory concentration (MIC) of carbamothioyl-furan-2-carboxamide derivatives (4a–f) . Every cIndicator resazurin solution was formed by dissolving 270 mg resazurin tablet into 40 ml of water and 10 µL indicator solution was added in each well. Then 10 µL of bacterial or fungal suspensions were incorporated into the each well. 96-well plates have been covered with aluminum foil and had a set of controls: a column with respective solvents as the negative control, a column with broad spectrum antibiotics as positive control, a column with all solutions with the exception of the test samples, a column with all solutions without the bacterial/fungal solution (with 10 µL of broth instead). Plates have been formed in triplicate and further incubation was carried out at 37 °C for 20 to 24 h and 28 °C for 40 to 48 h for bacteria and fungi respectively. Growth have been showed by colour changes from purple-pink or may be colorless. The minimum concentration at which the color change appeared was considered as the MIC value. g for 10 min; following erythrocyte isolation, they were rinsed 3 times with a 5 mL cold-sterilized PBS solution with a pH of 7.4. A total of 20 µL of sample solution (400 µg/mL) in DMSO was combined with the blood suspension and incubated at 37 °C for 30 min. As positive and negative controls, respectively, ABTS and DMSO were employed. At 576 nm, the sample’s absorbance was noticed, and the percentage of hemolysis was computed.To determine their hemolytic potential, all of the synthetic compounds were tested in accordance with the literature . Bovine 4a, 4b, 4c, and 4d showed significant anti-cancer potential, and results were compared with standard doxorubicin. Compounds 4b, 4a, and 4f were found to be more potent  against E.coli, S. aureus, and B. cereus, respectively. The anti-fungal activity of these compounds against all fungal strains is highly appreciable compared to standard doxorubicin. The results of biological activity tests reveal that these carbamothioyl-furan-2-carboxamide derivatives could be a good starting point for drug-discovery-related research. The modification of these molecules could introduce lead molecules for therapeutics in the future.We designed and synthetized a series of carbamothioyl-furan-2-carboxamide derivatives and evaluated their anti-cancer and anti-microbial activities. Synthesized compounds showed considerable anti-cancer activity against tested cell lines but


The mesoionic compounds were generated in situ by action of acetic anhydride on 3(2H)pyridazinone acids obtained from corresponding esters by alkaline hydrolysis followed by acidification. The structures of the compounds were confirmed by elemental analyses and IR, 1H-NMR, 13C-NMR, and X-ray diffraction data. The regioselectivity of cycloaddition was evidenced by NMR spectroscopy and confirmed by X-ray analysis. The compounds were evaluated for their cytotoxicity on plant cells (Triticum aestivum L.) and crustacean animal cells (Artemia franciscana Kellogg and Daphnia magna Straus). The results indicated that the tested compounds exhibited low toxicity on the plant cell , while on Artemia nauplii no lethality was observed. Daphnia magna assay showed that pyrrolopyridazines 5a and 5c could exhibit toxic effects, whereas, for the other compounds, toxicity was low to moderate. Also, the cytotoxic effects of the compounds were tested on three human adenocarcinoma-derived adherent cell lines . The in vitro compound-mediated cytotoxicity assays, performed by the MTS technique, demonstrated dose- and time-dependent cytotoxic activity for several compounds, the highest anti-tumor activity being observed for 5a, 2c, and 5f, especially against colon cancer cells.New pyrrolo[1,2- Five- and six-membered heterocyclic compounds present a special role in drug discovery design due to their remarkable biological properties ,2,3. Frob]pyridazines, have particular interest owing to their biological activities and significant optical properties [b]pyridazines have been reviewed extensively in 1977 [b]pyridazines regarding their pharmacological potentials such as antitumor pyridazine derivatives, starting from 3(2H)pyridazinone acids. The pyridazinone skeleton is also present in many compounds with significant biological activities including anticancer, anti-inflammatory, analgesic, antimicrobial, antidiabetic, anticonvulsant, anxiolytic, antidepressant, antihypertensive pyridazines start from pyridazines or pyrroles. Two of the most productive methods starting from pyridazine, which involved the in situ generation of some N-ylides or mesoionic 1,3-oxazole-5-ones, are presented in The main methods leading to pyrrolopyridazines by 1,3-dipolar cycloaddition reaction between mesoionic bicyclic oxazolo-pyridazinones and non-symmetrical activated alkyne dipolarophiles. New pyrrolopyridazines and their acid precursors were investigated for their cytotoxic activity on plant cells (Triticum aestivum L.), crustacean cells (Artemia franciscana Kellogg and Daphnia magna Straus) and several human adherent cell lines derived from human solid tumors such as LoVo (colon adenocarcinoma), SK-OV-3 (ovary carcinoma), and MCF-7 (breast adenocarcinoma). The antiproliferative activity of the compounds under study was compared with the effects induced by several oncolytic drugs such as cisplatin (CisPt), doxorubicin (DOX), or 5-fluorouracil (5-FU), which were used as positive controls of the assays.Taking into account the literature data, herein we report the regioselective synthesis of new pyrrolopyridazine derivatives [b]pyridazine derivatives by 3 + 2 cycloaddition between bicyclic oxazolo-pyridazinone münchnones and marginal acetylenic dipolarophiles such as methyl or ethyl propiolate. The starting material for in situ generation of bicyclic mesoionic münchnones 3a–c was the 3(2H)pyridazinone-butanoic acid precursors 2a–c that have been obtained by alkaline hydrolysis of corresponding esters 1a–c followed by acidification, with good yields (87% for 2a and 90% for 2b and 2c). The corresponding esters were synthesized from 6-aryl-3(2H)pyridazinone and ethyl 2-bromobutanoate by a procedure described in the literature for compounds with similar structure pyridazine derivatives 5a–f were obtained by 1,3-dipolar cycloaddition reactions between the bicyclic mesoionic 1,3-dipoles 3a–c and methyl or ethyl propiolate as non-symmetrical acetylenic dipolarophiles, with yields between 41–52%. The mesoionic oxazolopyridazinones 3a–c were in situ generated by the action of acetic anhydride on the acids 2a–c. The generation of mesoionic 1,3-dipoles and cycloaddition reaction to form pyrrolopyridazines 5a–f was performed in acetic anhydride at 90 °C for 3–4 h. The acetic anhydride was used both as reaction solvent and reagent which allows simultaneous dehydration and cyclization of pyridazinone acids 2a–c to mesoionic compounds 3a–c . The multiplicity of pyrrolic proton indicated that the only possible position for the hydrogen atom is in the 6 position of the pyrrolopyridazine ring. In the case when the cycloaddition had reversed regiochemistry, the pyrrolic proton would be in the 5 position and the coupling would not occur. Also, the absence in the spectra of these new compounds of the CH proton signals from the butanoic acid fragment in the intermediate acids (5.53–5.66 ppm) confirms the cycloaddition reaction. The 13C-NMR spectra present the expected signals, the main feature being the chemical shifts attributed to the carbonyl carbon of the ester groups which are in the range 164.5–165.1 ppm. The signal for C-6 from the pyrrole ring chemical has chemical shifts of 112.6–112.8 ppm. Compared to the precursor acids, the IR spectra of pyrrolopyridazines reveal a single absorption band, in the region 1688–1666 cm−1, due to the stretching vibration of the C=O ester group.The 5a crystallizes in the P-1 space group of the triclinic system with two chemically identic, but crystallographic independent molecules (denoted as A and B) in the asymmetric part of the unit cell. As an example, the structure of molecule A is depicted in 1c and 2b the aromatic fragment in two independent molecules A and B is also non-planar with the dihedral angle of 26.82(6)° and 16.42(6)°, respectively. It is to note, that the main crystal packing motif is determined by a system of C-H···O hydrogen bonding and essentially arises from the parallel packing of a one-dimensional supramolecular array running along the b axis, as depicted in According to X-ray crystallography, compound 1c, 2b, and 5a are provided in Selected crystallographic data and structure refinement details for compounds p < 0.001) and there was generally a concentration-dependent inhibitory effect of the three first compounds (2a–c). There was no statistically significant difference between 2a and 2b, whereas for 2c there were significant interactions between this derivative and concentration . The sense of the interactions is shown in b]pyridazines 5a–c and 5f, the root length also increased with time (day of measurement) (p < 0.001) (p = 1.00), and for most concentrations, but for 5b and 5c there were several significant compound–concentration interactions , whereas 5f did not differ significantly from 5a (p = 0.121). The compound 5e was analyzed alone; therefore, it was only compared with the control group. It showed a concentration-dependent inhibitory effect, statistically significant at the first two levels , approaching the conventional significance threshold at 100 μM (p = 0.059) and not significant at the lower concentrations (p > 0.145)  . Conside> 0.145) g.50 values for those four compounds for which they could be estimated (monotonic concentration-dependent root length) are shown in 50 value, 2b was the most phytotoxic , whereas 5e was the least phytotoxic. However, 5a–c and 5f seemed even less phytotoxic , the appearance of chromosomal bridges, or delayed chromosomes. These changes were also determined by compounds 2a–c and 5e at lower concentrations tested. Although compound 5a had no mitoinhibitory effect at 1000 μM, it did affect the cell walls, which had a wavy appearance. The same effect was observed for compound 5e, also appearing changes in the shape of the nuclei appeared. The migration of some chromosomes into the telophase was also delayed  compounds Artemia franciscana Kellogg for all compounds assessed in concentrations up to 1000 µM, indicating a lack of acute toxicity of these compounds. Artemia nauplii are generally more sensitive to toxicants than rodents pyridazine 5a (63.46%), for 2c containing a chlorine atom (78.17%) and its derivatives 5c (68.56%) and 5f (78.18%). Instead, fluorinated homologous 2b and pyrrolopyridazine 5b and 5e were demonstrated to have a lower effect, and cell viability was reduced to 90.98%, 87.77%, and 82.24%, respectively. Even lower concentrations of 5a inhibited the cell growth: both 50 and 100 μM, till 79%, and 78.1%, respectively  compared to the other tested derivatives pyridazinone acids derivatives, a PASS analysis was performed to indicate the post-probable biological targets for these. The analysis returned a number of 1789 possible targets for which the Pa values were higher than the corresponding Pi values. The Pa values were higher than 0.7 for only 19 targets. Of these targets, only the proteasome ATPase is correlated with anticancer effects. The use of proteasome inhibitors has proven to be clinically successful in treating various types of cancer, especially blood cancers. The proteasomal ATPase provides the energy for the substrate translocation and facilitates protein degradation pyridazine derivatives. The analysis of the predicted pharmacological effects revealed other ATPases as potential targets, like chloride-transporting ATPase, polyamine-transporting ATPase, phospholipid-translocating ATPase, proton-exporting ATPase, or myosin ATPase. These results indicate that the new compounds could function as a structural analog of ATP. This observation led to the search for other ATP-dependent targets and thus revealed significant Pa values for a few kinases, like sphinganine kinase, NADH kinase, and N-acylmannosamine kinase. Interestingly, the calculated Pa values were low in the case of protein kinases.In order to better evaluate the antiproliferative effect of new 2-phenylpyrrolobutanoate (1c). The compound was purified by crystallization from 2-propanol as colorless crystals with mp 49–51 °C; Yield 74%. Anal. Calcd. for C16H17ClN2O3 (320.77 g/mol): C, 59.91; H, 5.34; N, 8.73. Found C, 60.22; H, 5.72; N, 8.97. IR : 1665 (νC=O), 1738 (νC=O), 3061 (νCH). 1H-NMR  δ ppm: 0.98 , 1.24 , 2.23–2.36 , 4.17–4.27 , 5.51, 5.54 , 7.05 , 7.42 , 7.69 , 7.73 . 13C-NMR  δ ppm: 10.6, 14.0 (2CH3), 22.9 (CH2), 61.4 (CH2O), 62.5 (CH), 127.0 , 129.0 , 129.6 (C-4), 129.8 (C-5), 133.0 (C-4′), 135.5 (C-1′), 143.1 (C-3), 159.7 (C-6), 169.5 (COO).Ethyl 2-[6-oxo-3-(4-chlorophenyl)pyridazin-1-yl]butanoate  and 100 mL of 10% sodium hydroxide solution was refluxed for 2 h. The reaction mixture was subsequently acidified at pH = 2 with 10% hydrochloric acid solution and the colorless precipitate formed was filtered off, washed with water, and then dried and purified by crystallization from a suitable solvent.The acids and King ,45. A mi2a). The compound was purified by crystallization from nitromethane as colorless crystals with mp 141–142 °C; Yield 87%. Anal. Calcd. for C14H14N2O3 (258.27 g/mol): C, 65.11; H, 5.46; N, 10.85. Found C, 65.39; H, 5.72; N, 11.14. IR : 1632 (νC=O), 1708 (νC=O), 2932 (νOH), 3061 (νCH). 1H-NMR  δ ppm: 0.97 , 2.28–2.42 , 5.53, 5.56 , 7.16 ; 7.43–7.45 , 7.71 , 7.75–7.78 , 9.65 . 13C-NMR  δ ppm: 10.8 (CH3), 22.9 (CH2), 63.2 (CH), 126.2 , 129.1 , 129.8 (C-4′), 129.9 (C-4), 130.6 (C-5), 134.6 (C-1′), 145.2 (C-3), 160.9 (C-6), 173.5 (COO).2-(6-Oxo-3-phenylpyridazin-1-yl)butanoic acid (2b). The compound was purified by crystallization from nitromethane as colorless crystals with mp 174–175 °C; Yield 90%. Anal. Calcd. for C14H13FN2O3 (276.26 g/mol): C, 60.87; H, 4.74; N, 10.14. Found C, 61.18; H, 5.05; N, 10.41. IR : 1633 (νC=O), 1708 (νC=O), 2980 (νOH), 3073 (νCH). 1H-NMR  δ ppm: 1.03 , 2.38–2.49 , 5.64, 5.66 , 7.21 , 7.47 , 7.82 , 7.98 . 13C-NMR  δ ppm: 10.4 (CH3), 22.7 (CH2), 64.2 (CH), 116.4 , 128.9 (C-4), 132.3 (C-5), 128.4 , 129.5 ; 147.5 (C-3), 162.4 (C-6), 164.1 , 175.6 (COO).2-[6-Oxo-3-(4-fluorophenyl)pyridazin-1-yl]butanoic acid (2c). The compound was purified by crystallization from ethanol as colorless crystals with mp 191–193 °C; Yield 90%. Anal. Calcd. for C14H13ClN2O3 (292.72 g/mol): C, 57.44; H, 4.48; N, 9.57. Found C, 57.70; H, 4.79; N, 9.80. IR : 1631 (νC=O), 1708 (νC=O), 2931 (νOH), 3079 (νCH). 1H-NMR  δ ppm: 1.03 , 2.38–2.50 , 5.61, 5.63 , 7.45 , 7.48 , 7.77 , 7.97 . 13C-NMR  δ ppm: 10.4 (CH3), 22.7 (CH2), 64.4 (CH), 127.6 , 129.0 (C-4), 129.6 , 131.8 (C-1′), 132.3 (C-5), 137.2 (C-4′), 147.4 (C-3), 162.5 (C-6), 175.5 (COO).2-[6-Oxo-3-(4-chlorophenyl)pyridazin-1-yl]butanoic acid (2a–c (15 mmol) were dissolved in 20 mL of hot acetic anhydride. Over the solution cooled to room temperature, 19 mmol of activated alkyne was added under stirring and the reaction mixture was heated at 90 °C for 3 h. The reaction mixture was cooled and then 30 mL of ethanol was added when a precipitate was obtained. The yellow fluorescent crystals were separated by filtration and were crystallized from alcohol .The pyridazinone acids b]pyridazine-5-carboxylate (5a). The compound was purified by crystallization from methanol as fluorescent yellow crystals with mp 98–100 °C; Yield 42%. Anal. Calcd. for C17H16N2O2 (280.32 g/mol): C, 72.84; H, 5.75; N, 9.99. Found C, 73.13; H, 5.97; N, 10.28. IR : 1685 (νC=O), 3021 (νCH). 1H-NMR  δ ppm: 1.41 , 3.03 , 3.91 , 7.11 , 7.23 , 7.45–7.48 , 7.95–7.98 , 8.42 . 13C-NMR  δ ppm: 11.7 (CH3), 18.8 (CH2), 51.0 (CH3O), 103.0 (C-5), 111.7 (C-3), 112.6 (C-6), 126.8 , 127.6 (C-4), 128.3, 132.9 , 128.9 , 129.7 (C-4′), 136.2 (C-1′), 150.5 (C-2), 165.0 (COO).Methyl 7-ethyl-2-phenylpyrrolopyridazine-5-carboxylate (5b). The compound was purified by crystallization from ethanol as fluorescent yellow crystals with mp 131–133 °C; Yield 41%. Anal. Calcd. for C17H15FN2O2 (298.31 g/mol): C, 68.45; H, 5.07; N, 9.39. Found C, 68.81; H, 5.33; N, 9.67. IR : 1685 (νC=O); 3058 (νCH). 1H-NMR  δ ppm: 1.38 , 3.01 , 3.89 , 7.09–7.17 , 7.93 , 8.42 . 13C-NMR  δ ppm: 11.9 (CH3), 19.0 (CH2), 51.3 (CH3O), 103.4 (C-5), 111.6 (C-3), 112.8 (C-6), 116.1 , 128.0 (C-4), 128.4, 133.1 , 128.9 , 132.5 , 149.8 (C-2), 162.5 , 165.1 (COO).Methyl 7-ethyl-2-(4-fluorophenyl)pyrrolopyridazine-5-carboxylate (5c). The compound was purified by crystallization from ethanol as fluorescent yellow crystals with mp 143–144 °C; Yield 52%. Anal. Calcd. for C17H15ClN2O2 (314.77 g/mol): C, 64.87; H, 4.80; N, 8.90. Found C, 64.59; H, 5.07; N, 9.13. IR : 1681 (νC=O); 3105 (νCH). 1H-NMR  δ ppm: 1.40 , 3.07 , 3.90 , 7.13 , 7.20 , 7.45 , 7.91 , 8.46 . 13C-NMR  δ ppm: 11.7 (CH3), 18.9 (CH2), 51.2 (CH3O), 103.4 (C-5), 111.3 (C-3), 112.8 (C-6), 128.0 (C-4), 128.1 , 128.3, 133.1 , 129.2 , 134.7 (C-1′), 136.0 (C-4′), 149.5 (C-2), 165.0 (COO).Methyl 2-(4-chlorophenyl)-7-ethylpyrrolopyridazine-5-carboxylate (5d). The compound was purified by crystallization from ethanol as fluorescent yellow crystals with mp 99–101 °C; Yield 41%. Anal. Calcd. for C18H18N2O2 (294.35 g/mol): C, 73.45; H, 6.16; N, 9.52. Found C, 73.78; H, 6.55; N, 9.77. IR : 1666 (νC=O); 3055 (νCH). 1H-NMR  δ ppm: 1.41, 1.42 , 3.07 , 4.39 , 7.15 , 7.26 , 7.46–7.54 , 7.98–8.02 , 8.48 . 13C-NMR  δ ppm: 11.7, 14.6 (2CH3), 18.8 (CH2), 59.7 (CH2O), 103.4 (C-5), 111.7 (C-3), 112.6 (C-6), 126.8 , 127.8 (C-4), 128.3, 132.9 , 128.9 , 129.7 (C-4′), 136.4 (C-1′), 150.6 (C-2), 164.6 (COO).Ethyl 7-ethyl-2-phenylpyrrolopyridazine-5-carboxylate (5e). The compound was purified by crystallization from ethanol as fluorescent yellow crystals with mp 111–113 °C; Yield 47%. Anal. Calcd. for C18H17FN2O2 (312.34 g/mol): C, 69.22; H, 5.49; N, 8.97. Found C, 69.57; H, 5.81; N, 9.28. IR : 1688 (νC=O); 3073 (νCH). 1H-NMR  δ ppm: 1.42 , 3.07 , 4.39 , 7.14–7.26 , 7.99 , 8.49 . 13C-NMR  δ ppm: 11.7, 14.6 (2CH3), 18.8 (CH2), 59.7 (CH2O), 103.6 (C-5), 111.3 (C-3), 112.7 (C-6), 115.8 , 127.9 (C-4), 128.1, 132.5 , 128.7 , 132.9 , 149.6 (C-2), 162.2 , 164.5 (COO).Ethyl 7-ethyl-2-(4-fluorophenyl)pyrrolopyridazine-5-carboxylate (5f). The compound was purified by crystallization from ethanol as fluorescent yellow crystals with mp 102–104 °C; Yield 41%. Anal. Calcd. for C18H17ClN2O2 (328.79 g/mol): C, 65.75; H, 5.21; N, 8.52. Found C, 65.57; H, 5.50; N, 8.79. IR : 1676 (νC=O); 3060 (νCH). 1H-NMR  δ ppm: 1.42, 1.43 , 3.07 , 4.38 , 7.15 , 7.24 , 7.47 , 7.94 , 8.49 . 13C-NMR  δ ppm: 11.6, 14.6 (2CH3), 18.8 (CH2), 59.8 (CH2O), 103.5 (C-5), 111.1 (C-3), 112.7 (C-6), 127.9 (C-4), 128.0 , 128.1, 132.9 , 129.1 , 134.6 (C-1′), 135.8 (C-4′), 149.3 (C-2), 164.5 (COO).Ethyl 2-(4-chlorophenyl)-7-ethylpyrrolopyridazines were synthesized, with moderate yields, by 3 + 2 cycloaddition reaction between mesoionic oxazolo-pyridazinones and terminal activated alkyne dipolarophiles and their cytotoxicity was evaluated. The obtaining of mesoionic oxazolo-pyridazinones intermediate took place in situ from the 3(2H)pyridazinone acids in the presence of acetic anhydride. In the first stage, 3(2H)pyridazinone esters were synthesized by multi-step synthesis that led to the corresponding acids by hydrolysis. The spectral analysis , X-ray diffraction, and elemental analysis confirmed the structures of the synthesized compounds. The toxicity studies on Triticum aestivum L. cells indicated that the toxicity was low for all compounds, with IC50 higher than 200 µM, the acids 2b and 2a having the lowest values. The toxicity studies on crustaceans indicated that except for 5a and 5c could have a toxicity effect, the newly synthetized compounds showed moderate or no toxicity on Daphnia, while on Artemia nauplii no lethality was induced. The cytotoxic effects of the compounds on three human adenocarcinoma-derived adherent cell lines  and on HUVEC endothelial cells highlighted that several of these display satisfactory anticancer activities, and very low cytotoxic effects towards normal cells. The in vitro compound-mediated cytotoxicity assays demonstrated dose- and time-dependent cytotoxic activity for several newly synthesized compounds, the highest anti-tumor properties, based on the cell viability, being assessed for acid 2a and its derivative 5a, and for 2c and the derivatives 5c and 5f, especially against colon cancer cells. Among them, compounds 5a, 2c, and 5f showed the lowest IC50 values on the LoVo cell line. The obtained results prompted us to improve the anti-cancer properties of the most promising tested compounds, and further expand our studies on their biological activities, in order to modulate the chemo-sensitivity of tumor cells to innovative drug treatments that might overcome or reverse the chemo-resistance usually found in cancer patients after several cycles of chemotherapy.New pyrrolo[1,2-