Such cleavage occurs by treatment with hydrated alumina (Al2O3) or silica (SiO2) oxides, thus allowing the target heptanoids obtained in high yields as an amorphous powder to be filtered off directly from the reaction media. Furthermore, crystallization instead of chromatographic procedures provides a straightforward purification step. The ease and efficiency with which the present methodology can be applied to synthesizing the title compounds earns the terms “click” and “unclick” applied to describe particularly straightforward, efficient reactions. Furthermore, the methodology offers a simple, versatile, fast, and economical synthetic alternative for the obtention of curcumin (85% yield), bis-demethoxycurcumin (78% yield), and the symmetrical heterocyclic curcuminoids (80–92% yield), in pure form and excellent yields.The worldwide known and employed spice of Asian origin, turmeric, receives significant attention due to its numerous purported medicinal properties. Herein, we report an optimized synthesis of curcumin and symmetric curcuminoids of aromatic (bisdemethoxycurcumin) and heterocyclic type, with yields going from good to excellent using the cyclic difluoro-boronate derivative of acetylacetone prepared by reaction of 2,4-pentanedione with boron trifluoride in THF (ca. 95%). The subsequent cleavage of the BF Curcuma longa Asian spice from Zingiberaceae family  , also kne family ,18,19,20e family ,22,23,24e family ,29,30,31e family , antimete family , antioxie family ,35,36, fe family , anti-ine family ,39, antie family  and antie family ,43,44,45The importance of having curcumin as a pure metabolite lies in expanding its complementary studies on its pharmacokinetics, pharmacodynamics, and toxicological studies , which h21H20O6) could be assessed by Miłobȩdzka, J. et al. in 1910 ·xH2O (molecular sieves) or Al2O3 xH2O . The removal of boron reaction by-products has been reported using inorganic salts  . However, ethanol is more eco-friendly, and curcumin was obtained 72 h later in low yield (possibly due to the adsorption of curcumin to silica). Therefore, methanol was found more appropriate for removing the boron-difluoride moiety .3·THF; (2) condensation of the corresponding aromatic aldehyde catalyzing with n-butylamine; (3) cleavage of the BF2 group by means of hydrated metal oxides. Curcumin, bis-demethoxycurcumin itself, and two heterocyclic curcuminoids were obtained with very good yields and were fully characterized by spectroscopic techniques.The synthesis of curcumin and curcuminoids has been carried out with three simple reaction steps: (1) protection of keto-enol functionality of acetylacetone (acac) by BF2 group and allowing the recovery of the original b-diketone function.In a general description, this procedure consists of three basic yet simple general steps: (a) a protective step  while activating the methyl groups promoting (b) the efficient aldol condensation and (c) the deprotecting reaction step removing the BF2 group is due to an anion exchange phenomenon involving the reaction of boron and the basic OH—group or water in agreement with previous mechanistic proposals [It suggested that the mechanism for the removal of the BFroposals ,82, whicroposals ,84,85. A−)4 species are supported by previous references [The proposal mechanism in step I is supported by Venkata , and steferences ,82,83,842 generates HF [The integrity of the free curcuminoid on the aluminum oxides or under the reaction medium does not lead to decomposition since it is known that the breakdown of the BFrates HF , and therates HF . Furtherrates HF ,86.2O3, probably associated with the more significant boron adsorption in acidic pH, though other authors associate boron adsorption with the presence of hydroxide ions present in alumina [Our best yields in the obtention of curcumin were achieved using MeOH/Al alumina .1H-NMR spectrum of curcumin (2) methylene carbons; methines (CH) and methyl groups (CH3) are observed as positive signals and fit satisfactorily with data reported in the literature [1H-NMR spectra of all other symmetric curcuminoids show a consistent correlation between structure and spectral features  m/z = 308, furan-curcumin (7) m/z = 256, and thiophene-curcumin (8) m/z = 288 show a consistent peak with the chemical formulas C19H16O4, C15H12O4, and C15H12O2S2, respectively (see The mass spectrum (MS) of curcumin shows a characteristic peak at vely see .6–8) with 4-hidroxybenzaldehyde, furfural, and thiophenecarboxaldehyde. Thus, when 4-hidroxybenzaldehyde and furfural were used in the corresponding curcuminoid synthesis using a modified Pabon´s approach, the yields decreased significantly to a reported 33 and 8%, respectively [The present synthetic route was successfully extended for the obtention of other symmetrical curcuminoids  afforded excellent yields (95%) in the aldol condensation reaction, while the cleavage of the BF2 group on MeOH/alumina afforded (compound 8) in 92% yield. This overall high yield is even higher than the corresponding one observed for curcumin.Interestingly, the heterocyclic curcuminoid resulting from 2-thiophene carboxaldehyde , silica gel high-purity grade, average pore size 60 Å (52–73 Å), 70–230 mesh, 63–200 μm, for column chromatography (CAS 112926-00-8), molecular sieves 4Å beads, 8–12 mesh (CAS 70955-01-0) and Alumina Brockmann III (1344-28-1) were purchased from Sigma-Aldrich and were used without prior activation or purification.All chemicals were available commercially, and the solvents were purified with conventional methods before use .Melting points were determined on an Electrothermal Engineering IA9100 digital melting point apparatus in open capillary tubes and were uncorrected .1H and 13C NMR spectra were obtained in a Bruker Fourier 400 MHz spectrometer using TMS as an internal reference and CDCl3 or Acetone-6d, or DMSO-6d as solvents. NMR spectra were processed with MestreNova software 12.0.0 [e 12.0.0  and are −1 as KBr pellets [Spectroscopic measurements. IR absorption spectra were recorded using an FT-IR Bruker Tensor 27 spectrophotometer in the range of 4000–400 cm+, 350 °C, positive ion mode and calibration standard with PEG 600) [Mass Spectra were recorded using The MStation JMS-700 JEOL equipment  and the AccuTOF JMS-T100LC JEOL equipment  100 × 2.0 mm 3 μm; eluting with a solvent gradient  from3·THF, 98 mmol) was added to the solution, and the reaction was left overnight with magnetic stirring at room temperature. After, the organic phase was concentrated in vacuo affording the resulting product, which can be directly used for the following reaction step.In a 250 mL round flask, 10 mL of 2,4-pentanodione  was dissolved in 30 mL of dichloromethane; subsequently, 11 mL of boron trifluoride tetrahydrofuran complex (BFSynthon): yield 95%, solid amber, melting point 40 °C, 1H NMR : δ 5.96 , 2.27 ; 13C NMR : δ 192.63 (C=O), 102.12 (Cmethine-H), 24.32 (-CH3). IR (KBr) 1556 v, 1148 v, 1086 vcm−1, DART+-MS: m/z (%) = 129 (148–19), m/z calc. = 148.2,2-difluoro-4,6-dimethyl-2H-1,3,2-dioxaborinin-1-ium-2-uide  was dissolved in 25 mL of EtOAc, 6.3 mL of tributyl borate (24.5 mmol) was added, and this mixture was heated until homogenization was achieved.synthon  was dissolved in 25 mL of EtOAc, and then the homogenous product 1 was added to the solution; then, 2.7 mL of N-butylamine  was added dropwise. The reaction was left overnight with magnetic stirring at room temperature. Finally, a solid red precipitate was filtered-off and washed with a mixture of 50 mL water/acetone 90::10. This same methodology (same molar amounts) was carried out to synthesize the symmetrical curcuminoids-BF2.Mixture 2. In a 250 mL round flask, 4 g of 2 (1): 2,2-difluoro-4,6-bis((E)-4-hydroxy-3-methoxystyryl)-2H-1,3,2-dioxaborinin-1-ium-2-uide, yield 90%, red solid, melting point 230 °C, 1H NMR : δ 10.10 , 7.92 , 7.47 , 7.34 , 7.02 , 6.88 , 6.45 , 3.85 ; 13C NMR : δ 178.72 (C=O), 151.34 (C-OH), 148.17 (Caryl), 146.97 (Cvinyl-H), 125.99 (Caryl), 125.26 (Caryl-H), 117.86 (Cvinyl-H), 115.95 (Caryl-H), 112.39 (Caryl-H), 101.12 (Cmethine-H), 55.76 (-OCH3). IR (KBr) 3482 v(-OH), 1615 v(C=O), 1586 v(C=C), 1509 v, 1146 v cm−1, DART+-MS: m/z = 397 (416–19), m/z calc. = 416.Spectral data of Curcumin-BF2 (2) 2,2-difluoro-4,6-bis((E)-4-hydroxystyryl)-2H-1,3,2-dioxaborinin-1-ium-2-uide, yield 85%, red powder, melting point 225 °C, 1H NMR : δ 9.29 , 7.96 , 7.73 , 6.97 , 6.90 , 6.39 ; 13C NMR : δ 180.81 (C=O), 162.16 (C-OH), 147.35 (Cvinyl-H), 132.67 (Caryl-H), 127.27 (Caryl), 118.96 (Cvinyl-H), 117.15 (Caryl-H), 102.20 (Cmethine-H). IR (KBr) 3422 v(-OH), 1598 v(C=O), 1579 v(C=C), 1518 v, 1147 v cm−1, EI-MS: m/z = no observed, m/z calc. = 356.4-hydroxy-curcuminoid-BF2 (3) 2,2-difluoro-4,6-bis((E)-2-(furan-2-yl)vinyl)-2H-1,3,2-dioxaborinin-1-ium-2-uide, yield 80%, red powder, melting point 200 °C, 1H NMR : δ 7.64 , 7.51 , 6.75 , 6.51 , 6.47 , 5.96 ; 13C NMR : δ 178.98 (C=O), 151.01 (Caryl), 146.78 (Caryl-H), 131.91 (Cvinyl-H), 119.04 (Caryl-H), 117.95 (Cvinyl-H), 113.37 (Caryl-H),102.34 (Cmethine-H). IR (KBr) 1619 v(C=O), 1569 v, 1455 v(C-H), 1385 v(C-H), 1276 v(C-O), 1067 v cm−1, DART+-MS: m/z = 305 (304 + 1), m/z calc. = 304.Furan-curcuminoid-BF2 (4) 2,2-difluoro-4,6-bis((E)-2-(thiophen-2-yl)vinyl)-2H-1,3,2-dioxaborinin-1-ium-2-uide, yield 95%, violet powder, melting point 270 °C, 1H NMR : δ 8.20 , 7.84 , 7.73 , 7.26 , 6.79 , 6.51 ; 13C NMR : δ 180.71 (C=O), 140.92 (Caryl), 139.92 (Cvinyl-H), 135.63 (Caryl-H), 133.32 (Caryl-H), 130.14 (Caryl-H), 120.67 (Cvinyl-H), 102.81(Cmethine-H). IR (KBr) 1594 v(C=O), 1541 v(C=O), 1494 (C=C), 1411(-C=Cring), 1291 v(C-O), 1152 v cm−1, DART+-MS: m/z = 317 (336–19), m/z calc. = 336. Thiophene-curcuminoid-BF2 was dissolved in 400 mL of methanol (MeOH), 20% weight of metal oxide  was added to the solution, and the mixture was left overnight under magnetic stirring at reflux. The reaction was quenched by filtration using a sintered glass funnel packed with celite. MeOH was evaporated in vacuo, and reaction crude was extracted with 150 mL of EtOAc (ethyl acetate) and water (3 × 100 mL). The organic phase was dried with Na2SO4 and concentrated in vacuo to afford the curcuminoid product, which was purified by recrystallization using EtOAc and hexane. The yields obtained for the synthesis of curcumin with several catalyzers were as follows: silica (70%), molecular sieves (80%) and alumina (85%). This same methodology (same amounts) was carried out for the synthesis of symmetrical curcuminoids (compounds 6–8).In a 500 mL round flask, 10 g of curcuminoid-BF1H NMR : δ 16.47 , 9.66 , 7.55 , 7.32 , 7.15 , 6.82 , 6.75 , 6.06 , 3.84 ; 13C NMR : δ 183.22 (C=O), 149.36 (C-OH), 148.00 (Caryl), 140.72 (Cvinyl-H), 126.34 (Caryl), 123.14 (Caryl-H), 121.10 (Cvinyl-H), 115.70 (Caryl-H), 111.33 (Caryl-H), 100.85 (Cmethine-H), 55.69 (-OCH3). IR (KBr) 3506 v(OH), 1628 v(C=O), 1602 v(C=Cring), 1509 v, 1428 v(C-Ophenol), 1281 v(C-Oenol), 1154 v(C-O), 1028 v(=C-O-CH3) cm−1, EI-MS: m/z = 368, m/z calc. = 368.Curcumin (5) 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-1,6-heptadien-3,5-dione, yellow-orange powder, purified by recrystallization using EtOAc and hexane, purity by HPLC 99.48%, melting point 180 °C, 2Cl2 and MeOH, purity by HPLC 99.45%, melting point 215 °C, 1H NMR : δ 9.24 , 7.60 , 7.55 , 6.90 , 6.66 , 5.99 ; 13C NMR : δ 184.57 (C=O), 160.77 (C-OH), 141.19 (Cvinyl-H), 131.03 (Caryl-H), 127.58 (Caryl), 121.98 (Cvinyl-H), 116.86 (Caryl-H), 101.78 (Cmethine-H). IR (KBr) 3232 v(OH), 1622 v(C=O), 1599 v( C=O), 1513 v, 1444 v(OH), 1276 v(C-Oenol), 1140 v(C-O)cm−1, EI-MS: m/z = 308, m/z calc. = 308.Bis-demethoxycurcumin (6) 1,7-bis(4-hydroxyphenyl)-1,6-heptadien-3,5-dione, red-orange powder, purified by recrystallization using CH1H NMR : δ 16.06 , 7.87 , 7.45 , 6.96 , 6.66 , 6.57 , 6.19 ; 13C NMR : δ 182.48 (C=O), 151.02 (Caryl), 146.12 (Caryl-H), 126.94 (Cvinyl-H), 121.18 (Cvinyl-H), 116.15 (Caryl-H), 113.03 (Caryl-H), 101.98 (Cmethine-H). IR (KBr) 3124 v(C=Cring), 1628 v(C=O), 1563 v, 1468 v(C=Cring), 1262 v(C-Oenol), 1139 v(C-O), 962 v(C-C=C) cm−1, EI-MS: m/z = 256, m/z calc. = 256.Furan-curcuminoid (7) 1,7-di(furan-2-yl)-5-hydroxyhepta-1,4,6-trien-3-one, brown powder, purified by recrystallization using EtOAc and hexane, purity by HPLC 99.33%, melting point 130 °C, 1H NMR : δ 16.07 , 7.81 , 7.75 , 7.54 , 7.17 , 6.56 , 6.19 ; 13C NMR : δ 182.52 (C=O), 139.83 (Caryl), 133.23 (Cvinyl-H), 132.06 (Caryl-H), 130.06 (Caryl-H), 128.76 (Caryl-H), 122.70 (Cvinyl-H), 101.52 (Cmethine-H). IR (KBr) 3102 v(C=Cring), 1619 v(C=O), 1565 , 1505 v(C-O), 1418 v(C-OH), 964 (C-C=C) cm−1, EI-MS: m/z = 288, m/z calc. = 288.Thiophene-curcuminoid (8) 1,7-di(thiophen-2-yl)-5-hydroxyhepta-1,4,6-trien-3-one, yellow powder, purified by recrystallization using acetone and hexane, purity by HPLC 98.68%, melting point 184 °C, Using simple high-yield steps, we contribute with a laboratory-scale strategy to obtain curcumin and symmetric curcuminoids. As a result, it can provide significant quantities of these compounds for physicochemical, analytical, and biological assay studies. This synthetic route is appropriate for using different aldehydes to obtain the corresponding symmetric curcuminoids. Due to the accessibility of this simple three-step synthetic approach, the method offers excellent potential for making available curcumin and symmetric curcuminoids on a large scale. The benefits of the present synthesis widen the perspectives for expanding the scientific studies concerning the fascinating molecular structures of curcuminoids and their widely recognized biological effects. An application for a patent is underway in the country of the authors.


Knowledge about cyanobacteria diversity in the Azores is spread over several publications, dating from 1874, with some of them not generally available to the scientific community due to their restricted access. The dispersion and sometimes inaccessibility of this information hinder a deeper analysis and a better understanding of the biodiversity of the Azores Islands and more general ecological processes in oceanic islands. Here we present the first checklist of cyanobacteria for the Azores Archipelago with updated taxonomy of all recorded taxa.Chroococcales, Nostocales, Oscillatoriales, Pleurocapsales, Spirulinales and Synechococcales). Our literature review reveals that the Azores Archipelago hosts a high diversity of cyanobacteria, despite several overlooked habitats that may present great potential regarding cyanobacteria diversity. Increasing efforts to study these neglected habitats could contribute to the knowledge of cyanobacteria taxonomy. This checklist provides the basis for future works on the taxonomy and taxa richness of cyanobacteria in the Azores and the Atlantic Islands, as also for understanding and monitoring non-indigenous and invasive species.This work provides a compiled and annotated checklist of all known cyanobacteria from the Azores Archipelago with morphological identification from preserved samples and cultures, based on published literature. All records of taxa known to occur in the Azores were taxonomically updated. The present checklist comprises 225 taxa distributed by six orders ( Cyanobacteria are gram-negative photosynthetic prokaryotes that developed around 3500 million years ago , thermal (pool)Frémy, 19300D2AD3D2-D028-58BE-A590-48FA64D17004Flores Freshwater (lake)Schmidle, 1900A08E012F-C9DA-5AF1-BB70-C235C1D41E16Flores Freshwater (lake)Lemmermann, 18964111C797-D7D8-5993-AFA8-0D5E4BD049C8São Miguel Freshwater (lake)Bornet & Flahault, 18861FAF86B2-90A8-535C-A933-4D1A13F04C1BFlores , Pico L, São MigFreshwater (lake)Lagerheim ex Bornet & Flahault, 1886C1B36671-29CE-56F0-8A6A-081ECE6D1BBFCorvo FreshwaterV.V.Miller, 1923B645D965-F87A-5BF0-A39E-AEEF5BA728D8Corvo , Flores Freshwater (lake)(G.S.West) Woloszynska & V.Miller in V.Miller, 19236B3731E2-A5FF-5165-9ECF-1BEBC72481B2Flores Freshwater (lake)(C.Agardh ex Gomont) Strunecký, Bohunická, J.R.Johansen & J.Komárek, 20173346370C-7D38-5534-A00C-B9D4226E09EBFlores , São MigBrackish (lake), freshwater (lake)(W.West & G.S.West) Komárek, Kastovsky & Jezberová, 2011D865FE01-3303-5AB1-8080-DB04348ACF89São Miguel Freshwater (lake)(West) Komárek, Kastovsky & Jezberová, 2011CB0697C9-5FF4-5FD6-B3AB-DE2875CED4A4São Miguel Freshwater (lake)A.Morren ex É.Bornet & C.Flahault, 1886F2B889F6-8C7A-5CC4-9D65-27039A0847C5São Miguel Freshwater (lake)Ralfs ex Bornet & Flahault, 1886746F96B3-B418-564F-A851-BC6EC5320709Corvo , Flores Freshwater (lake)Lemmermann, 1907B4275013-C555-5491-A49B-7FA2E31BA156Flores , Pico L, São MigFreshwater (lake)Bourrelly in Bourrelly & Manguin, 19528EBDF4DD-1806-5534-8691-5E77650B43E5Pico Freshwater (lake)Nägeli, 18491E05D72B-365C-5C3C-BFE2-9569B08DBC37São Miguel Freshwater (lake)West & G.S.West, 191287DBC12D-68F6-5815-AA71-6FD82440167FPico , São MigFreshwater (lake)West & G.S.West, 18948FD0214C-D1F9-5BCC-B9E7-DCF30A00BFA1São Jorge , São MigFreshwater (lake), terrestrial(Berkeley) Rabenhorst, 186539825678-DC02-5F4D-AAE9-7A5D6C688C1DSão Jorge Freshwater(Lemmermann) G.Cronberg & Komárek, 199454E2F8B0-827F-526B-863B-2652DB96E871São Miguel Freshwater (lake)Nägeli, 1849BA39489C-B663-5554-A246-5D4BEC62B1EAFlores , São MigFreshwater (lake)(Kützing) Rabenhorst, 1865497B852B-F6D3-5A40-9BF8-65C6038B0BFFFlores TerrestrialNägeli, 1849E5C56523-31FD-5A31-91B1-3EF75A823619São Miguel Freshwater (lake), terrestrialWartmann in Rabenhorst, 18656DCFDD27-026B-589E-A429-349A6ED23A87São Miguel FreshwaterP.Richter, 1884DC09DA52-E952-5BBD-BFCB-DB537B81C2A3São Miguel Freshwater (lake)(Kützing) Rabenhorst, 18632E7D654E-447C-5597-8F66-C5D97900A1C2São Miguel Freshwater (lake)Nägeli, 18498411CDF5-C0CE-5678-BB2F-697D7C02E1E8São Miguel Terrestrial(Sprengel) A.Braun in Rabenhorst, 186392387C66-DD74-547C-AC27-D22A52C37C46Pico FreshwaterSitzenberger ex Gomont, 18923D71333A-E7E2-5B03-B290-633181D35210São Miguel Freshwater (lake)C.Agardh ex Bornet & Flahault, 18867EE8080E-33F4-5D0D-B13F-F56B3F7F90D5Flores , São JorFreshwater West & G.S.West, 18977F2A9670-72FA-5282-A8A1-657C0CB5CA08Flores Freshwater (lake)Bornet & Flahault, 1886EEB25B65-A92B-5B2A-A6A6-07FB71990651Pico , São MigFreshwater (lake)Thuret ex Bornet & Flahault, 18867421F633-1861-5DE5-99F0-3F466F984AC4São Miguel Terrestrial(A.K.Mitra) A.K.Mitra & D.C.Pandey, 196761D91FBD-E047-5ACB-A99A-516EF09A6401São Miguel Thermal (stream)Nägeli, 18496EA6AF4F-B38D-53ED-B0E8-4BAE0D8371ABSão Miguel Freshwater (lake), thermal (spring)(Keissler) Lemmermann, 1904E13877D8-7240-563C-961C-FB00B97DEBD4Pico , São MigFreshwater (lake)(Meneghini) Nägeli, 1849D0F29FFA-1B90-5FF8-BEA8-6331700F7430São Miguel Thermal (stream)(Kützing) Nägeli, 18496FCC9259-D6DF-5AC4-8A82-A2004AFACA42São Miguel Freshwater (lake)(Kützing) Nägeli, 18494F1AED92-AD9C-5A81-B4E8-4FC65E7E21A8Flores , Pico J, São MigFreshwater (lake)(Kirchner) Hieronymus, 1892C0A6C736-2475-581E-9B95-A841D97CBEA0São Miguel Freshwater (lake)(Kützing) Nägeli, 184951399019-4C66-5844-9523-0BFE6A161DFCCorvo , Faial , Flores Freshwater (lake), terrestrial(Nägeli) Hansgirg, 1887F5B2773F-B33D-5A1B-802A-071C23288BE3Flores FreshwaterJ.B.Petersen, 192345271513-58C3-51E7-BBD4-2E5F56FDCC4DSão Miguel TerrestrialNägeli, 1849327A108E-1D20-5FF7-A77F-34556379954CSão Miguel Freshwater (lake)Nägeli, 1849732E25B0-EA89-59F3-8178-3D047DF4DF90São Miguel Freshwater (lake)Kirchner ex Frank, 1886ADD1E5D6-BA23-5557-8C20-25144AF9EADDFlores , Pico L, São MigFreshwater (lake), thermal Kirchner ex Frank, 18869ACACF4D-5D2C-5DA9-B3E0-C89AF2C18D73São Miguel Freshwater (lake)Komárek 19994D270238-0B1E-5853-9CBB-2AFCEC985639São Miguel Freshwater (lake)R.Rippka & G.Cohen-Bazire, 1983BC7204EE-5549-518E-B23E-F522E5A9E588São Miguel Freshwater (lake) Komárek, J.Kopecký & Cepák, 19996510DFA6-A64B-5CAF-8366-EF20536A93DBSão Miguel Freshwater (lake)K.J.Lukas & S.Golubic, 1981490DA872-E818-5B86-86C6-4DFE8D7740ECFaial Marine Kützing ex Bornet & Flahault, 188676D98B8A-1394-5587-B60D-DE3B1F3481A3Pico , São MigFreshwater Kützing ex Bornet & Flahault, 1886CD0072DE-A493-58B1-B413-2BF2DD2535A2São Miguel FreshwaterKützing ex Bornet & Flahault, 1886CBB9E118-DC1A-5F6A-9EDF-A06F2BD09469Faial , Flores Freshwater (lake)Bornet & Flahault, 1886605545D7-3ADB-54CE-ACCB-E1ED74D5C226Corvo FreshwaterFrémy, 19244520628B-ECB1-566B-8BAA-9415AAF283FAFlores Freshwater (lake) P.Wacklin, L.Hoffmann & J.Komárek, 2009C646515E-D412-5E81-A619-B70D9690425BSão Miguel Freshwater (lake)(Lemmermann) Wacklin, L.Hoffmann & Komárek, 2009608983D2-24B1-5FB3-AEC7-518B93CCE7A8Pico , São MigFreshwater (lake)(Rabenhorst ex Bornet & Flahault) Wacklin, L.Hoffmann & Komárek, 2009B91AFB58-208C-5199-834C-428612019756São Miguel Freshwater (lake)(Lemmermann) Wacklin, L.Hoffmann & Komárek, 2009DCB702F8-E4B4-51D9-9E3C-BB0BA69AA83FCorvo , Flores Freshwater (lake)(Brébisson ex Bornet & Flahault) Wacklin, L.Hoffmann & Komárek, 20099AAD69CE-9507-5D0A-BF8D-4DCEEC859E4FSão Miguel Freshwater (lake) Wacklin, L.Hoffmann & Komárek, 2009BD58B186-0E34-5D9C-AC6E-6456585EBFD1Pico , São MigFreshwater (lake)(Elenkin) Wacklin, L.Hoffmann & Komárek, 20091221A41B-8B2A-5882-83A8-EC38D2C68492Corvo , Flores Freshwater (lake)(Nygaard) Wacklin, L.Hoffmann & Komárek, 2009D088D6B9-11F2-5244-BDE2-4DABC9EDEC1EPico , São MigFreshwater (lake)(Klebahn) Wacklin, L.Hoffmann & Komárek, 2009E4A9E0C6-7AC7-5C97-A7A3-C9BF9608CE35Flores , Pico L, São MigFreshwater (lake)(Klebhan) Wacklin, L.Hoffmann & Komárek, 20095456697E-14D1-5872-9A51-463C92738403São Miguel Freshwater (lake)F.E.Clements & H.L.Schantz, 1909754423E1-3256-547E-B07F-C607CCC76555Corvo , Flores Freshwater (lake)F.E.Fritsch, 1912CB370973-6823-52D0-97DC-D726C2358396São Miguel Freshwater (lake)(Bornet & Flahault) Gomont, 189540F27740-A289-594B-909C-D13CFFACF8F2São Miguel Thermal (pool)De Toni, 1936D306D1CC-AAAE-5C46-B132-4F957B23002BPico Freshwater (lake)(F.C.Hy) De Toni, 1936B6B3BC2F-0524-52FA-819A-1166708AEBF1Pico Freshwater (lake)(Skuja) Anagnostidis, 1989FC04E65F-29F9-5D3D-8396-2652EDB8D803Santa Maria Terrestrial(Greville ex Gomont) Anagnostidis, 19898390719B-3AA3-5717-8F40-68E97BD9EC77Pico , São MigFreshwater (lake)Kützing, 1843A37A7C2F-E744-5BFF-BE9D-BB661590FE41São Miguel Freshwater (lake)Kützing, 18437BE6EE57-67E8-514B-914F-859004B4C25DSão Miguel , TerceirFreshwater, terrestrialRabenhorst, 1865D4BAF589-2FDC-5142-8E17-AFA299A8F4ABTerceira FreshwaterKützing, 184730649D81-E503-5960-BC44-04FEA3407185Flores Freshwater (lake)Kützing, 1843D3D4627F-5A99-5CCE-AC62-8B448724F8E3São Jorge , TerceirFreshwaterKützing, 1846CFEC0C70-C9A3-5820-9EF4-D4149633E7F7São Miguel FreshwaterKützing, 184798995BAA-76E2-5DD9-97FE-2A19E17DB7FCSão Jorge , TerceirFreshwaterKützing, 1843C4DB2CC3-FED3-5ED7-A966-F96D11B9E93DSão Miguel Thermal(Novácek) Komárek & Anagnostidis ex Komárek 19930D8C7E2E-A879-5D1F-A7BA-4C4A5ECAAC3ESão Miguel Thermal (pool)(Brébisson) Komárek & Anagnostidis ex Komárek, 1993DFC47F20-3A6B-553C-9036-7665DF7A6678São Miguel Terrestrial Rabenhorst, 1865A7C4A689-2C1C-57CB-AAC5-CBA2D7318CC6São Miguel Terrestrial(Lyngbye) Bornet in Wittrock & Nordstedt, 1880DC2E50FA-F6D1-5806-B78F-E6B8FDD54616Flores , São MigFreshwater (lake)Thuret ex Bornet & Flahault, 188609E5ADCA-D25A-5C28-AFEC-37D61E8AA44DSão Miguel TerrestrialMiscoe & J.R.Johansen, 2016E2CF2F03-F5E2-5849-A65E-A8F40B75C549Flores Freshwater (lake)Kützing, 1836349C4809-0663-5289-BBAB-6BDF0F1F9C77São Miguel Freshwater (lake)West & G.S.West, 1896C653B3E8-D5BE-54F8-ABB2-4ACB7EB84967Corvo , Flores Freshwater (lake)West & G.S.West, 189432DF9264-1C08-5D22-A516-FC43697EDAFESão Jorge , TerceirFreshwater Kirchner ex Bornet & Flahault, 188747DFBC51-FC9C-56AB-9315-55CB3780F404Flores , Santa MFreshwater (stream), terrestrialNägeli ex É.Bornet & C.Flahault, 188698E15AF8-C4EF-5A52-AC93-EB2168A2EB78São Miguel Terrestrial(Schmidle) Compère, 1985803EECD2-B18B-510A-89DE-7524AED43D2ESão Jorge FreshwaterG.S.West, 1912C5EB2BAA-E51F-589A-B07F-B43890A79542São Jorge FreshwaterSchwabe ex Bornet & Flahault 188703DF7F50-C3A3-5112-94B5-AF68ADD1DAF0São Miguel Freshwater (lake)É.Bornet & C.Flahault, 18886588D41C-AF0E-5D30-BACF-2FFFB3C665ACFaial Marine Bornet & Flahault, 1888AFDB46F8-C22A-5062-BE0B-912F4391D908Faial Marine Lukas & Golubic, 198385CAA134-6978-55EB-A7B6-BD600287FE89Faial Marine Starmach 196292F4F0E2-A517-5298-8771-8F6BCA36A2B0Flores Freshwater (lake)O.Strunecký, J.Komárek & J.Smarda, 20148BED7D41-3DC7-5DA1-B982-E08CB245EEF8Flores Freshwater (lake)(Bory ex Gomont) Strunecký, Komárek & J.Smarda, 20149939C7C1-000A-537D-9296-D8C2CFAA4EBESanta Maria , São MigFreshwater, brackish (lake), thermal (stream)Ercegovic, 1929FC2C266E-CC53-529F-A8B7-E8A794B80998Faial Marine Raabová, Kovacik & Strunecký, 2019416BE2FB-5390-5BF0-A207-6DC44FBF4BCBSão Miguel Freshwater (lake)Anagnostidis & Komárek, 1988541015A2-AB83-51AD-81AC-793EA01FDF72São Jorge , São MigFreshwater (lake), marine (lake), thermal (Woronichin) Anagnostidis & Komárek, 19884B830626-D8B2-53B7-8938-4135F4D35596São Miguel Thermal (stream)(J.J.Copeland) Anagnostidis 1936C2EBE813-7B38-5D80-B1C1-703CFBD03695São Miguel Thermal (Gomont ex Gomont) Anagnostidis & Komárek, 19884E808AF6-FFC4-5188-85E6-FA70E21E70C7São Miguel Thermal (spring)(Bornet ex Gomont) Anagnostidis & Komárek, 198881E64AEC-EA8C-56D1-B716-42ABE80F86D4São Miguel Freshwater (lake)(Thuret ex Gomont) Anagnostidis & Komárek, 1988FA3EA5D5-6F68-5FD1-ACEA-21B97685FB6BSão Miguel Thermal (pool)(Gomont) Anagnostidis & Komárek, 1988AFDF8AF7-C5A4-5379-89A8-27FED5A8133BSão Miguel Freshwater (lake)(Gomont) Anagnostidis 2001039FE232-94C8-5E4F-833C-5DCE69EB496ASão Miguel Thermal (spring)(Gomont) Anagnostidis & Komárek, 198842D201A5-4778-5DD8-AFD1-2BDDB89BD077São Miguel TerrestrialM.-E.Meffert, 198801B69F67-4BB1-583A-979C-6098D2CFA6F1Flores , São MigFreshwater (lake)(Woloszynska) Meffert, 1988422E794B-09DC-5C26-8098-9E60113DFD28São Miguel Freshwater (lake)C.Agardh ex Gomont, 18921394AE00-73BF-5720-9E8D-4C50AB36D3C5São Miguel Freshwater (lake)Gomont ex Gomont, 1892F36215C1-C130-51A9-BF4A-56A2B165F739Terceira MarineMeneghini ex Gomont, 1892B2589F32-A84E-5CAE-8803-BA40FA5DB33FSão Miguel , TerceirFreshwater, thermal, terrestrialCohn ex Kirchner, 1898ADD76FAA-B7B9-51CC-AC79-70D1CA76489ESão Miguel Thermal Cohn ex Kirchner, 1898DC1E7734-3205-5857-BE27-D9A58E52D9CCSão Miguel Thermal Lagerheim ex Bornet & Flahault, 18867D733C69-72DF-58F1-8E82-624DD0DAEBC8Faial Marine Meyen, 18393664D887-87A5-5ACF-8D81-350E4491F435São Miguel , Pico LFreshwater (lake)(Ehrenberg) Kützing, 184506B94FB9-EA53-5880-A2EC-11F2DF98BB7ASão Miguel , TerceirFreshwater (lake)Lemmermann, 18988D51F50A-BD13-5ED3-92DA-76C84678A48DGraciosa , São MigFreshwater (lake)J.Copeland, 1936CEA81422-B88D-507A-964C-D6F4BF0C8342São Miguel Thermal (spring)Thuret ex Bornet & Flahault, 18861C3B2104-118D-5F9F-8BF2-DF992186E19DPico , São MigFreshwater (lake), terrestrial(Gomont) Strunecky, Komárek & J.R.Johansen, 20138EB10850-E428-50B4-97E0-31EA76A3BCDDFlores , São MigFreshwater (lake)(Gomont) Strunecky, Komárek & J.R.Johansen, 20131BD74790-908A-5C96-8B7E-AEA77CCD7732Flores , São MigFreshwater (stream), terrestrialKützing ex Forti, 19072D1AAA68-E16F-54D3-A17F-5CAE600F154ATerceira MarineLemmermann, 1907D0BD10AF-5680-54F2-8D50-79431D9E342ASão Miguel Freshwater (lake)(Kützing) Kützing, 18462DEBF0D2-749E-5B17-8DA6-A07B3DCE594BCorvo , Flores Freshwater (lake)(Wittrock) Kirchner, 1898861390ED-F70D-574A-A12D-8469593AC2BFFlores , São MigFreshwater (lake)(H.C.Wood) Forti, 1907C73B7D99-7A17-5D65-AE82-8E4E3B389155Pico , São MigFreshwater (lake)(H.W.Clark) Nygaard, 1925A32BC5F3-66D5-54FC-87A8-41A428764EB6São Miguel , TerceirFreshwater (lake)Thuret ex Bornet & Flahault, 1886C9A5F77F-12BC-5F0D-8FA2-9846A59B9BF1Corvo , TerceirBrackish (lake), marineVaucher ex Bornet & Flahault, 18860D64A689-B759-5FD5-8F43-E84FDC561634Corvo , Flores Freshwater , terrestrialC.Agardh ex Bornet & Flahault, 18863504816D-643E-5E8A-BC52-BA57396FBA60Faial FreshwaterVaucher ex Bornet & Flahault, 18862ABBBB94-EA1F-5222-8F7C-6CE439EE2578Pico Freshwater (lake)Rabenhorst ex Bornet & Flahault, 1886FE19FC1D-AD73-5DE3-B0DE-5B54BC182A77Corvo , São MigFreshwaterKützing ex Bornet & Flahault, 1886EA708117-914D-55F6-B68B-0AA4D4836EA2Corvo , Santa MFreshwater (lake)Hariot, 1891E1749B75-D37F-5E43-9D4F-764E93EA3339São Miguel Freshwater (lake), terrestrialVaucher ex Bornet & Flahault, 1886C7A90830-003D-5439-BA48-09318FE82DB4Faial , Flores Freshwater (lake), terrestrialKützing ex Bornet & Flahault, 1886BF7A8666-B84A-5336-9C50-A8562CE303E7Faial , São JorFreshwaterVaucher ex Bornet & Flahault, 18869081A85F-45F8-52FF-8A28-2CE46A1BB116São Miguel , TerceirFreshwater (stream)H.C.Wood ex Bornet & Flahault, 18868A5D8CAA-A884-5E5E-8181-EDD72490185ESão Miguel TerrestrialVaucher ex Gomont, 1892DCBF69E6-84DD-51C8-BD2F-AD4D08A29F46Flores , Pico I, São MigFreshwater (lake), marineBourrelly in Bourrelly & Manguin, 1946624C3A67-32CB-5903-9830-A1F1B72051A6Flores Freshwater (stream)Woloszynska, 1912F448449E-96CF-5022-AB0C-4B4696B4C372São Miguel Freshwater (lake)Vaucher ex Gomont, 1892DC340E66-4A89-5F9A-8D52-AAE724E171D7Terceira MarineKützing ex Gomont, 18920EBB4815-E988-5846-AE6D-F85788925D50São Miguel TerrestrialC.Agardh ex Gomont, 189250EEEE63-B5C9-5190-8602-C0D3B894A7D4Corvo , Faial , Flores Freshwater , terrestrialMai, J.R.Johansen & Bohunická, 2018A2B01D08-5109-5AAA-8195-C13EA19C31A2São Miguel Freshwater (lake)Migula, 19071FDA5E34-D314-5044-A091-E398FB8682D1Flores FreshwaterKützing ex Gomont, 18926D106293-F8FA-5AD2-9EAA-0BCA8DCCBF7ECorvo , Faial , Pico L, São JorFreshwater (lake), marine (lake), thermal(Gomont) Anagnostidis & Komárek, 1988170CD658-E933-5F78-8EED-5BBDA9A07FC5São Miguel Freshwater (lake)(Frémy) Anagnostidis & Komárek, 19888745AAB7-6F51-581A-86C6-791A2403BC9BTerceira Freshwater(Kützing ex Gomont) Anagnostidis & Komárek, 1988F1939C00-6610-5FCC-993F-E00C5F8BA537São Miguel Marine, thermalN.L.Gardner, 1927976B2EF6-DAE6-5169-AC4B-D2F22ACA0425São Jorge Freshwater(Kützing ex Gomont) Anagnostidis & Komárek, 1988F1217E6A-93E8-56C4-BD3A-35B2E2702A22São Jorge , TerceirFreshwater (stream)Frémy, 1930542492AB-F5B9-571F-8D6F-77150A2458DDSão Jorge , TerceirFreshwaterKützing ex Gomont, 18925F2C1CBB-3DF1-5E29-9F40-213BE887C7D8Faial , São JorFreshwaterItzigsohn in Rabenhorst, 1865EEEAF295-3AEF-5C6F-8EA4-5ECF20493ED5São Jorge Freshwater(C.Agardh ex Gomont) Anagnostidis & Komárek, 198891BE4B1D-9F31-5A08-824C-1BE87AA1C733São Miguel ThermalAnagnostidis & Komárek, 1988424EF739-A621-5534-B535-EF66951A6898Flores , Pico L, São MigFreshwater (lake)(Lemmermann) Komárková-Legnerová & Cronberg, 1992081964B8-8A08-5C9E-8267-DB5602DA1713Pico , São MigFreshwater (lake)(Gomont) Anagnostidis & Komárek, 198839DA6936-415F-5ADD-A16A-3AB4B772982DSão Miguel Freshwater (lake)Ercegovic, 1932BECB3156-D1B4-57DF-80EF-A214C58D8FE2Faial Marine Bornet & Flahault ex Gomont, 18926A2CAE44-AE45-5789-B5C1-9EBCB17D6BA1Faial Marine Lauterborn, 1915D7A53FFE-E282-5537-890B-7A77C1F7165FCorvo , Flores Freshwater (lake)Lauterborn, 1915C84CFF8A-311B-5950-A45C-3DD3C2489636São Miguel Freshwater (lake)(Lemmermann) Komárek, 1974DDD3F482-CBB4-57C6-87EF-B67D916046CDCorvo , Flores Freshwater (lake)(G.S.An) Anagnostidis, 200137631284-F293-59FC-9CE5-C0729E4C9404Pico Freshwater (lake) Schwabe, 196431E35EB1-8639-5C50-A0AC-553E39D0D570São Miguel Freshwater (lake)(Anissimova) Anagnostidis, 200187385094-7C2F-5121-9349-7C77CA85F825Santa Maria BrackishF.E.Fritsch & M.F.Rich, 19303A722206-9AB1-5D6E-9A17-CF13C825CC19Corvo , Flores Freshwater (lake)C.Agardh ex Bornet & Flahault, 188642B20208-B89E-5807-94D2-3FBA4452FD97São Miguel , TerceirFreshwater, marineMeneghini ex Bornet & Flahault 18863160A454-4A04-59F0-AFEE-655A8B61CE3ESão Miguel Freshwater (lake)Berkeley ex Bornet & Flahault, 18869D4D8EE8-D694-5307-8DC9-9F3728882850São Miguel FreshwaterC.Agardh ex Bornet & Flahault, 1886A46388FB-F8D7-5479-BB64-B958C5C201BAFlores FreshwaterKützing ex M.Gomont, 189231F968A0-515F-5248-B3BC-8D059332E39ATerceira Freshwater(West & G.S.West) West & G.S.West, 189608E0E674-48CD-5B5C-9F5B-ACCBD9BB343CFaial TerrestrialKutzing ex Gomont, 1892047A30CD-2CFD-5872-A650-870B836EE18DTerceira FreshwaterA.Braun ex Gomont, 18927A9DF08F-78C6-5FAA-83B7-65DEFCB50236São Jorge Freshwater(Kützing ex Forti) Geitler, 193272497F8B-92AA-56AA-B265-6EAFFAABA2C0Terceira Marine(N.L.Gardner) Geitler, 1932FBABED4B-739C-50F5-A2AC-9272B7C92A36Terceira FreshwaterGomont, 18906E3A16CB-DF26-5765-A68D-117120FDDF50Faial FreshwaterGomont, 18905DCA9FDD-7E6D-56BC-AD98-E97330CBDFC2São Jorge FreshwaterC.Agardh ex É.Bornet & C.Flahault, 188670A0C04A-3976-5165-8064-FD9C492C6892Flores TerrestrialWest & G.S.West, 1895299294E7-25B1-5DD5-8C14-8E0E2B7418CDSão Miguel TerrestrialBharadwaja, 19343FC47D9F-74F5-501F-93D2-16148B94C535Terceira FreshwaterBornet & Flahault, 18884D41D234-53B5-582B-BB8F-5AE76A05ABDEFlores , São MigTerrestrialC.Agardh ex Bornet & Flahault, 1886EE7ACCD6-0733-5846-A65D-84F072549D31São Jorge , São MigFreshwater (lake)Bornet ex Bornet & Flahault, 188682B473F8-3801-57BC-8C6B-80DB19B69118Flores Freshwater (lake)Bornet, 188925239CE2-7AFC-5963-85D4-408D7A91CDEAPico , São JorFreshwater , terrestrialBornet ex Bornet & Flahault, 188764736D9B-8EFE-53DB-9CD5-BD27C912F2C4Flores Freshwater (lake)E.I.Kiseleva, 193010ABBF2D-A791-5A8C-8518-8A784FEC0455Flores , Pico CFreshwater (lake)A.A.Elenkin, 1938DBE1C8D2-C455-537E-9828-C3AB301CE9DCSão Miguel Freshwater (lake)(Chodat) Komárek & Hindák, 198863FC5AEE-E9C9-55CA-94C0-E9C24E7C50A7São Miguel Freshwater (lake)Zapomelová, Jezberová, Hrouzek, Hisem, Reháková & Komárková, 2010137EBCE7-E44A-5419-A1D3-ADF0E71501D8Pico Freshwater (lake)(Forti) Zapomelová, Jezberová, Hrouzek, Hisem, Reháková & Komárková, 201065F558B6-ED44-5180-B435-CF311BE91388Pico Freshwater (lake)Oersted ex Gomont, 18923EE7C9AC-5043-5CC6-9045-6AE70547876CSão Jorge Marine (lake)Miscoe & J.R.Johansen, 2016B32D9844-A013-54F3-B033-E57ED65D674DPico Freshwater (lake)Bornet & Flahault, 18866BD919A7-E6FF-5A3A-B580-1D0D03ADD80FFlores , São JorFreshwater (lake), terrestrialKützing ex Bornet & Flahault, 18866F5FAA7E-D4FE-5EC1-A7E6-8418FB59DE6CSão Jorge , TerceirFreshwaterC.Agardh ex Bornet & Flahault, 188622169D47-4EEB-5C16-ABCD-76F02AB91E49São Jorge , São MigFreshwater, terrestrialHassall ex Bornet & Flahault, 1886754A2D0E-CA33-5F36-8B5C-C44ED15B7BB0Flores , São JorFreshwater (lake)N.L.Gardner, 1927277B6A92-D8C9-512F-9388-6E4A5D9C0288São Jorge FreshwaterThuret ex Bornet & Flahault, 1886B9F5BB81-4CFC-5CD5-8B5E-C85CD2BD0696Flores , São MigFreshwater (lake)Bornet & Flahault, 18861330F108-17FC-5C05-B141-A525244AD341São Miguel Freshwater (stream)N.L.Gardner, 19278A74E630-C1AF-58C7-B0E9-22CBED7AACF1São Jorge FreshwaterHieronymus, 18951045DD00-12D4-53AA-825D-C66527124414São Jorge , São MigFreshwater, terrestrialGomont, 1892A43E25D7-0839-504E-80F4-D75E2F3E3FC9São Miguel Thermal (pool)Gomont, 18922E16802F-02EF-5943-8166-25744AD2B127São Miguel ThermalNägeli, 1849D0B2F7AA-BF3C-5370-8A8F-7CBAD85CAAF6São Miguel , TerceirFreshwater (lake)(Pringsheim) Komárek, 1970C77F166C-9212-5512-A98A-58F52E052F9ESão Miguel Freshwater (lake)C.Sauvageau, 1892056B3AAE-A756-5693-BE6B-956F18AA1215São Miguel , Pico LFreshwater (lake)Mai, J.R.Johansen & Pietrasiak, 20189B23D44E-FAC3-501A-818D-EAB3F9F691E1São Miguel Freshwater (lake)Kützing ex É.Bornet & C.Flahault, 18867520D80D-06F6-5E30-B71E-9004D6CB5EE0São Miguel FreshwaterKützing ex Bornet & Flahault, 18862D14EF00-E006-5AE9-8B5F-C1CC1C660B3CSão Miguel FreshwaterLemmermann, 191006F802E4-E334-50CB-AB11-E3583746C1BBPico Freshwater (lake)Wartmann ex Bornet & Flahault, 18862DB9AB26-62EF-5163-9B99-0A2FEDD0EFAFSão Miguel Freshwater (lake)Kützing ex Bornet & Flahault, 18860649CE44-C607-5C1E-A860-CA690555C2F8Flores Freshwater(Kützing ex Bornet & Flahault) Komárek & Anagnostidis, 19896CC32149-43D9-50EE-A5D7-E394851983EASão Miguel Thermal (pool)K.Anagnostidis & J.Komárek, 1988E422F595-49ED-5FCA-98EB-F0AABA8BCE92São Miguel Freshwater (lake)Janet, 19410ADAA7EE-9A01-588E-9516-525C8CC18002São Miguel Freshwater (lake), thermal (stream)(Unger) Elenkin, 19334FBC75DF-F1C4-5FB8-B0A8-E83DA94C0A07Corvo Freshwater (lake)Chroococcales, Nostocales, Oscillatoriales, Pleurocapsales, Spirulinales and Synechococcales), 30 families and 79 genera  and Synechococcales (21.2%) orders. Chroococcales and Oscillatoriales orders contributed almost with the same number of species , despite their different genera contributions.The present work comprises 225 taxa, 179 identified species and 11 only to genus level, distributed by six orders  was evident between species richness (S) and island area. This correlation is best described by a linear relationship Fig. , where PChroococcus Nägeli, 1849, Nostoc Vaucher ex Bornet & Flahault, 1886 and Oscillatoria Vaucher ex Gomont, 1892. After that, as seen in Fig. Cyanobacteria flora were sporadic, but significant, mainly with th century and the 21st century and presently stands at 225 taxa with 179 identified species , probably due to low sampling efforts. The availability of freshwater habitats in the Azores favours the establishment of incoming cyanobacteria in São Miguel, Flores, Terceira and São Jorge. These Islands have permanent streams, lakes, peat bogs and wetlands, providing highly diverse habitats for incomers, while these are absent in Faial, Graciosa and Santa Maria.Several islands of the Azores have active volcanoes and present high numbers of fumarolic fields, geysers and hot springs  creatingThe cyanobacteria diversity and distribution in lotic systems in the Azores are much less known compared to other sites . Only a One of the most accepted explanations for regional biodiversity is the species-area relationship (SAR), according to which the number of species along the spatial scale increases with the area e.g. . This paNostocales and Oscillatoriales being the richest orders. However, Nostocales represents a much higher contribution to the regional species richness in the Azores and Madeira Archipelagos, which could reflect their longer dispersion capabilities  of cyanobacteria taxa and occurrence data used for the presented and analysed checklist.Brief description10.15468/bfktqo) about the reports of cyanobacteria in the Azores Archipelago. The taxon (core) data table contains 229 records of cyanobacteria (from class to species level). One extension data table exists, with a total of 2838 occurrence records of cyanobacteria found in literature. The taxon data table is constructed, based on the occurrence data table.Published Darwin Core Archive with two data tables in GBIF (doi: File: oo_694835.ziphttps://binary.pensoft.net/file/694835Luz R, Cordeiro R, Fonseca A, Gonçalves V


Interestingly, computational studies for the 6-O-butynyloxy (10) and 6-O-pentynyloxy (11) derivatives identified an alternative access channel lined by Phe221, Trp224, Gln225 and Leu477, providing further insight into the potential binding mode and interactions of the non-steroidal aromatase inhibitors.Aromatase (CYP19A1) inhibitors are the mainstay therapeutics for the treatment of hormone dependant breast cancer, which accounts for approximately 70% of all breast cancer cases. However, increased resistance to the clinically used aromatase inhibitors, including letrozole and anastrazole, and off target effects, necessitates the development of aromatase inhibitors with improved drug profiles. The development of extended 4th generation pyridine based aromatase inhibitors with dual binding (haem and access channel) is therefore of interest and here we describe the design, synthesis and computational studies. Cytotoxicity and selectivity studies identified the pyridine derivative (4-bromophenyl)(6-(but-2-yn-1-yloxy)benzofuran-2-yl)(pyridin-3-yl)methanol (10c) as optimal with CYP19A1 IC 4th generation pyridine based dual site aromatase (CYP19A1) inhibitors were synthesised and evaluated for CYP19A1 inhibitory activity, cytotoxicity and CYP selectivity. Computational studies provided insight into the potential binding mode. Non-steroidal aromatase inhibitors (e.g. letrozole and anastrazole) bind to the enzyme active site through coordination of a heterocyclic nitrogen lone pair with the haem iron. Imidazole was reported to be more efficient in respect to the coordination potential followed by triazole then tetrazole, however, triazole compounds were found to be more selective.5 Pyridine nitrogen has availability of a lone pair with the potential to coordinate the haem iron, and the AI activity was found to fall between triazole and tetrazole moieties making pyridine an interesting heterocycle for the aromatase inhibition due to the increased size, which may lead to a closer interaction with the haem.6 Several studies have focused on the ability of pyridine compounds to bind to and inhibit the aromatase (CYP19A1) enzyme.7–9 In a previous study reported by us on benzofuran derivatives, the aromatase inhibitory activity was found to be directly proportional to the heterocyclic nitrogen basicity ,6 however we also observed improved cytotoxicity profile (LC50/IC50 > 2000) of pyridine derivatives compared with triazole derivatives.11Aromatase inhibitors (AIs) have proved to be very successful clinically in the treatment and prophylaxis of hormone dependent breast cancer,10 Inclusion of a long alkynyloxy chain at the 6-position of the benzofuran ring resulted in these AIs having dual CYP19A1 binding properties by binding in both the haem and the front door access channel sites , which used a human placental microsomal assay to determine aromatase inhibition.11 The halides were the most promising derivatives , therefore the first modification was to prepare and evaluate the 4-bromo and 2,4-dichloro as well as the parent 4-fluoro and 4-chlorophenyl benzofuran pyridines for evaluation in the placental choriocarcinoma JEG-3 cell aromatase assay, producing four different derivatives to provide better understanding of the SAR. The second modification was achieved by changing the methoxy group on the benzofuran ring with longer chain substituents, namely but-2-ynyloxy and pent-2-ynyloxy, to investigate the binding potential in the front door access channel of the enzyme to provide dual binding aromatase inhibitors.A series of pyridine-based benzofuran compounds was designed based on the parent scaffold previously reported by our research group . The yields varied from 25% to 92%  with the 6-methoxy substitution on the benzofuran ring11 . The fir% to 92% , which c10,11 from pyran protected salicylaldehyde (5) and the substituted bromoacetophenones (2), followed by removal of the pyran group under acidic conditions to give the phenols (7). The phenol derivatives were then deprotonated using K2CO3 and treated with either 1-bromobut-2-yne or 1-bromopent-2-yne to give the corresponding alkyne ethers (8 and 9). Grignard reaction of 8 and 9 with pyridine 3-magnesium bromide then provided the final pyridyl products 10 in yields of 34–90% and 11 in yields of 15–91% . The distinct new C-1 quaternary carbon was observed in 13C NMR at δ 75.9–77.2 for methoxy derivatives 4, ∼δ 84.1 for the but-2-ynyl derivatives (10) and ∼δ 89.9 for the pent-2-ynyl derivatives (11)  were confirmed by the pyridine signals in the aromatic region of the ves (11) .12 at a single concentration (10 nM). Briefly, placental choriocarcinoma JEG-3 cells, known to have high aromatase activity, were grown to approximately 80% confluence in six-well culture plates. Once established, cells were treated with androst-4-ene-3,17-dione[1β-3H] as aromatase substrate. Aromatase activity was measured in the absence and presence of inhibitors  were tested for their aromatase inhibitory activity using a modified tritiated water assay previously reportedhibitors .50 determination using a range of concentrations (0.001–10 nM). Aromatase activity results were determined as a concentration of product formed per mg of protein per hour. Each data point was measured in triplicates and the error in the IC50 calculations represented as 95% confidence interval  than the respective extended compounds (10 and 11). However, for the 6-O-but-2-yne derivatives the 4-Cl (10b IC50 1.05 nM), and 4-Br (10c IC50 0.83 nM) were comparable with the standard, letrozole (IC50 0.70 nM), and for the 6-O-pent-2-yne derivatives the 4-Cl (11b IC50 0.92 nM) was also comparable with letrozole. The least favourable substitution was 2,4-dichloro  followed by 4-F , which mirrored the trend observed for the simpler methoxy derivatives .The methoxy derivatives (4) were more potent , oestrogen-dependent breast cancer cells (MCF-7), and non-cancerous breast epithelial cells (MCF-10A).Statistics using one-way ANOVA followed by a Tukey's Multiple Comparison test comparing all compounds against control showed no significant difference between the tested compounds and the negative control indicating that the compounds had no impact on MDA-MB-231 , MCF-7  or MCF-113 Compound 10c displayed excellent selectivity for CYP19A1 compared with CYPs 1A2 and 2D6 (>30 000) and very good selectivity compared with CYPs 2C9, 2C19 and 3A4  (6-(but-2-yn-1-yloxy)benzofuran-2-yl)(pyridin-3-yl)methanol (10c), was tested for inhibitory activity against a CYP panel  by Cyprotex Discovery Limited using a human liver microsomal assay with a CYP isoform specific probe substrate.ctively) .R- and S-enantiomers of compounds 4, 10 and 11 were prepared by docking the compounds with the X-ray crystal structure of CYP19A1 (PDB 3S79)14 using molecular operating environment (MOE) software.15 The prepared protein–ligand complexes were then subject to 200 ns molecular dynamics simulations using the Desmond programme of Schrödinger Maestro software.16,17 The methoxy derivatives 4a, 4c and 4d were consistent in the positioning of the R- and S-enantiomers in the haem active site, with the R-enantiomers positioned with the benzofuran moiety in the pocket lined by Arg115, Ile133, Phe134 and Met374 and the phenyl halide moiety in the pocket lined by Asp309, Thr310 and Ser478, while this positioning was flipped in the S-enantiomers   that were more sterically restrained within the CYP19A1 active site, with the ) Fig. S2.R-enantiomer of the dichloro derivative (R-10d) were positioned in the haem active site and the front door access channel  were positioned in the haem active site, the benzofuran but-2-ynyloxy portion was positioned in a different site  and dichloro derivative (10d) did not bind to the haem via the pyridine N, however they did bind with the haem iron through the hydroxy group either directly  (Both enantiomers of the fluoro derivative (10a) and the y S-10d,  or water, S-10c) .3 CH2, allowed better fit within the binding sites. With the exception of the S-enantiomer of 11d, all the pent-2-ynyloxy derivatives (11) bind with the haem through the N of pyridine  does not extend fully into the access channel and has the largest N–Fe3+ binding (2.97 Å) (With the exception of channels . The chl(2.97 Å) .50 > 10 nM) and pent-2-ynyloxy  derivatives. The but-2-ynyloxy derivatives (10) were the most sterically restricted, in particular the S-enantiomers (50 0.83 nM) found to occupy the alternative access channel lined by Phe221, Trp224, Gln225 and Leu477 (The methoxy derivatives (4) were all positioned within the haem binding site with the dichloro derivative (4d) least optimal with respect to binding and CYP19A1 inhibitory activity and this trend was also observed for the but-2-ynyloxy  was found to bind in both access channels  and had the optimal N–Fe3+ binding distance , with the addition of a CH channel  and S3†  channel .10 The m50 < 10 nM were found to be non-toxic against MDA-MB-231, MCF-7 and MCF-10A cells  displayed excellent selectivity against a human CYP panel (50 0.83 nM) and 11b (IC50 0.92 nM) were also found to have a profile similar to the standard AI letrozole, potently inhibiting CYP19A1 (IC50 0.70 nM). Further research, including resolution of enantiomers to investigate individual R- and S-enantiomers with respect to inhibitory activity, cytotoxicity and selectivity, as well as establishing whether these extended pyridine dual binding site AIs are effective against AI resistant breast cancer cell lines are therefore warranted.The pyridine AIs with IC0A cells . The curYP panel , improve254) with visualisation via UV light (254 nm). Melting points were determined on an electrothermal instrument  and were uncorrected. 1H, 13C, and 19F NMR spectra were recorded on a Bruker Advance DP500 spectrometer operating at 500, 125 and 470 MHz, respectively. Chemical shifts are given in parts per million (ppm) relative to the internal standard tetramethylsilane (Me4Si). Elemental analysis was performed by MEDAC Ltd  and HPLC-HRMS was performed at the Department of Pharmacy & Pharmacology, University of Bath, Bath, UK. on a Zorbax Eclipse Plus C18 Rapid Resolution 2.1 × 50 mm, 1.8 μm particle size using a 7.5 minute gradient method 5 : 95 v/v water : methanol with 0.1% formic acid as additive. Experimental details for intermediates 3 and 6–9 are provided in the ESI.†All chemicals, reagents and solvents were purchased from Sigma-Aldrich, Alfa Aesar, VWR, Acros and Fluka. Solvents were dried prior to use over molecular sieves (4 Å). For column chromatography, a glass column was slurry packed in the appropriate eluent with silica gel (Fluka Kieselgel 60). TLC was performed on pre-coated silica plates (dimension 20 × 20 cm) (ALUGRAM® SIL G/UV−1) was added pyridin-3-yl magnesium bromide (7 m.eq.) and the reaction mixture was heated at 70 °C overnight. The reaction mixture was concentrated under reduced pressure and the resulting residue extracted between H2O (50 mL) and EtOAc (2 × 100 mL). The combined organic layers were combined, dried (MgSO4) and concentrated under vacuum. The product was purified by gradient column chromatography to give (phenyl)(pyridin-3-yl)methanol derivatives  at 60% EtOAc in petroleum ether (v/v) as a colourless oil.To a solution of (6-methoxybenzofuran-2-yl)(phenyl)methanone derivatives  (1 m.eq.) in THF (10 mL mmolRf = 0.42 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (DMSO-d6): δ 8.52 , 7.71 , 7.49 , 7.41 , 7.21 , 7.16 , 6.88 , 6.47 , 3.77 . 13C NMR (DMSO-d6): δ 162.86 , 156.63 (C), 158.23 (C), 156.17 (C), 148.93 (CH), 148.61 (CH), 141.22 , 141.00 (C), 135.00 (CH), 129.52 , 123.56 (CH), 122.05 (CH), 120.96 (C), 115.36 , 112.50 (CH), 106.10 (CH), 96.44 (CH), 75.86 (C), 56.02 (CH3). HPLC: 100% at R.T. = 4.61 min. HRMS (ESI) calculated 350.1187 [M + H]+, found 350.1189 [M + H]+.Prepared from (4-fluorophenyl)(6-methoxybenzofuran-2-yl)methanone (3a) .Yield: 0.2 g (83%); Rf = 0.4 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.57 , 8.52 , 7.78 , 7.38 , 7.34 , 6.96 , 6.90 , 6.27 , 3.84 , 2.37 . 13C NMR (CDCl3): δ 158.42 (C), 157.61 (C), 156.28 (C), 148.32 (CH), 148.11 (CH), 141.89 (C), 139.97 (C), 135.55 (CH), 134.22 (C), 128.67 (2× CH), 128.51 (2× CH), 123.21 (CH), 121.58 (CH), 120.69 (C), 112.37 (CH), 106.82 (CH), 96.04 (CH), 76.51 (C), 55.72 (CH3). HPLC: 100% at R.T. = 4.73 min. HRMS (ESI) calculated (35Cl) 366.0896 [M + H]+, found 366.0891 [M + H]+; calculated (37Cl) 368.0868 [M + H]+, found 368.0872 [M + H]+.Prepared from (4-chlorophenyl)(6-methoxybenzofuran-2-yl)methanone (3b) .Yield: 0.5 g (92%); Rf = 0.47 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (DMSO-d6): δ 8.52 , 7.71 , 7.58 , 7.49 , 7.41 , 7.30 , 7.16 , 6.88 , 6.48 , 3.77 . 13C NMR (DMSO-d6): δ 159.26 (C), 158.26 (C), 156.18 (C), 149.01 (CH), 148.58 (CH), 144.38 (C), 140.71 (C), 135.01 (CH), 131.46 (2× CH), 129.63 (2× CH), 123.60 (CH), 122.08 (CH), 121.36 (C), 120.94 (C), 112.53 (CH), 106.25 (CH), 96.44 (CH), 75.92 (C), 56.03 (CH3). HPLC: 98.9% at R.T. = 4.76 min. HRMS (ESI) calculated (79Br) 410.0391 [M + H]+, found 410.0387 [M + H]+; calculated (81Br) 412.0372 [M + H]+, found 412.0369 [M + H]+.Prepared from (4-bromophenyl)(6-methoxybenzofuran-2-yl)methanone (3c) .Yield: 0.13 g (25%); Rf = 0.5 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.49 , 7.69 , 7.34 , 7.30 , 7.23 , 7.14 , 7.01 , 6.91 , 6.81 , 6.22 , 4.39 , 3.75 . 13C NMR (CDCl3): δ 158.42 (C), 156.35 (C), 156.19 (C), 148.99 (CH), 148.56 (CH), 139.06 (C), 138.38 (C), 135.21 (C), 134.82 (CH), 133.89 (C), 131.43 (CH), 131.09 (CH), 127.02 (CH), 123.02 (CH), 121.63 (CH), 120.76 (C), 112.48 (CH), 106.99 (CH), 96.09 (CH), 77.18 (C), 55.72 (CH3). HPLC: 100% at R.T. = 4.78 min. HRMS (ESI) calculated (35Cl) 400.0508 [M + H]+, found 400.0532 [M + H]+; calculated (37Cl) 402.0478 [M + H]+, found 402.0494 [M + H]+.Prepared from (6-methoxybenzofuran-2-yl)methanone (3d) . Yield: 0.18 g (32%); Rf = 0.37 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.48 , 7.65 , 7.30 , 7.26 , 7.20 , 6.98 , 6.85 , 6.15 , 4.59 , 3.90 , 1.78 . 13C NMR (CDCl3): δ 163.46 , 158.23 (C), 156.52 (C), 156.03 (C), 148.92 (CH), 148.73 (CH), 139.68 (C), 139.24 , 134.98 (CH), 129.16 , 123.02 (CH), 121.55 (CH), 121.29 (C), 115.29 , 112.96 (CH), 106.72 (CH), 97.41 (CH), 84.06 (C), 76.60 (C), 73.83 (C), 57.02 (CH2), 3.74 (CH3). HPLC: 100% at R.T. = 4.70 min. HRMS (ESI) calculated 388.1348 [M + H]+, found 388.1351 [M + H]+.Prepared from (6-(but-2-yn-1-yloxy)benzofuran-2-yl)(4-fluorophenyl)methanone (8a) . Yield: 0.1 g (80%); Rf = 0.4 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.60 , 7.74 , 7.40 , 7.36 , 7.08 , 6.95 , 6.26 , 4.69 , 3.74 , 1.88 . 13C NMR (CDCl3): δ 157.84 (C), 156.55 (C), 156.05 (C), 149.12 (CH), 148.76 (CH), 141.81 (C), 139.40 (C), 134.92 (CH), 134.27 (C), 128.68 (2× CH), 128.52 (2× CH), 123.05 (CH), 121.59 (CH), 121.24 (C), 113.04 (CH), 106.89 (CH), 97.40 (CH), 84.08 (C), 76.65 (C), 73.80 (C), 57.03 (CH2), 3.74 (CH3). HPLC: 100% at R.T. = 4.80 min. HRMS (ESI) calculated (35Cl) 404.1009 [M + H]+, found 404.1053 [M + H]+; calculated (37Cl) 406.0979 [M + H]+, found 406.1030 [M + H]+.Prepared from (6-(but-2-yn-1-yloxy)benzofuran-2-yl)(4-chlorophenyl)methanone (8b) . Yield: 0.16 g (94%); Rf = 0.42 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.47 , 7.64 , 7.40 , 7.30 , 7.20 , 6.97 , 6.85 , 6.16 ,), 4.59 , 3.97 , 1.78 . 13C NMR (CDCl3): δ 157.87 (C), 156.56 (C), 156.04 (C), 148.98 (CH), 148.66 (CH), 142.43 (C), 139.36 (C), 134.98 (CH), 131.46 (2× CH), 129.00 (2× CH), 123.05 (CH), 122.44 (C), 121.58 (CH), 121.24 (C), 113.01 (CH), 106.85 (CH), 97.39 (CH), 84.07 (C), 76.64 (C), 73.82 (C), 57.02 (CH2), 3.75 (CH3). HPLC: 100% at R.T. = 4.83 min. HRMS (ESI) calculated (79Br) 448.0504 [M + H]+, found 448.0543 [M + H]+; calculated (81Br) 450.0483 [M + H]+, found 450.0527 [M + H]+.Prepared from (6-(but-2-yn-1-yloxy)benzofuran-2-yl)(4-bromophenyl)methanone (8c) . Yield: 0.07 g (70%); Rf = 0.4 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.52 , 7.69 , 7.36 , 7.32 , 7.25 , 7.15 , 7.03 , 7.00 , 6.87 , 6.23 , 4.61 , 4.06 , 1.80 . 13C NMR (CDCl3): δ 156.60 (C), 156.47 (C), 155.97 (C), 149.16 (CH), 148.63 (CH), 138.92 (C), 138.24 (C), 135.29 (C), 134.78 (CH), 133.84 (C), 131.45 (CH), 131.13 (CH), 127.07 (CH), 123.06 (CH), 121.65 (CH), 121.28 (C), 113.13 (CH), 107.09 (CH), 97.47 (CH), 84.09 (C), 76.77 , 73.80 (C), 57.04 (CH2), 3.75 (CH3). HPLC: 100% at R.T. = 4.84 min. HRMS (ESI) calculated (35Cl) 438.0619 [M + H]+, found 438.0659 [M + H]+; calculated (37Cl) 440.0590 [M + H]+, found 440.06354 [M + H]+.Prepared from (6-(but-2-yn-1-yloxy)benzofuran-2-yl) methanone (8d) . Yield: 0.08 g (34%); Rf = 0.45 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.53 , 7.66 , 7.31 , 7.28 , 7.19 , 7.00 , 6.99 , 6.86 , 6.16 , 4.62 , 3.32 , 2.19 , 1.08 . 13C NMR (CDCl3): δ 176.59 (C), 163.49 , 158.11 (C), 156.58 (C), 156.00 (C), 139.15 , 134.83 (CH), 129.17 , 121.54 (CH), 121.26 (C), 115.33 , 113.05 (CH), 106.83 (CH), 97.48 (CH), 89.83 (C), 76.76 , 73.94 (C), 57.13 (CH2), 13.58 (CH3), 12.51 (CH2). *three CH pyridine peaks too small to be detected. HPLC: 100% at R.T. = 4.78 min. HRMS (ESI) calculated 402.1505 [M + H]+, found 402.1501 [M + H]+.Prepared from (4-fluorophenyl)(6-(pent-2-yn-1-yloxy)benzofuran-2-yl)methanone (9a) . Yield: 0.04 g (20%); Rf = 0.32 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.49 , 7.64 , 7.30 , 7.26 , 6.98 , 6.86 , 6.16 , 4.61 , 3.79 , 2.18 , 1.07 . 13C NMR (CDCl3): δ 157.90 (C), 156.61 (C), 156.04 (C), 149.02 (CH), 148.70 (CH), 141.87 (C), 139.46 (C), 134.96 (CH), 134.23 (C), 128.68 (2× CH), 128.51 (2× CH), 123.05 (CH), 121.55 (CH), 121.24 (C), 113.05 (CH), 106.86 (CH), 97.48 (CH), 89.87 (C), 76.61 (C), 73.97 (C), 57.13 (CH2), 13.58 (CH3), 12.51 (CH2). HPLC: 100% at R.T. = 4.87 min. HRMS (ESI) calculated (35Cl) 418.1165 [M + H]+, found 418.1204 [M + H]+; calculated (37Cl) 420.1136 [M + H]+, found 420.1187 [M + H]+.Prepared from (4-chlorophenyl)(6-(pent-2-yn-1-yloxy)benzofuran-2-yl)methanone (9b) . Yield: 0.18 g (91%); Rf = 0.50 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.48 , 7.64 , 7.41 , 7.30 , 7.20 , 6.98 , 6.86 , 6.16 , 4.61 , 3.68 , 2.19 , 1.07 . 13C NMR (CDCl3): δ 157.79 (C), 156.62 (C), 156.04 (C), 148.97 (CH), 148.63 (CH), 142.39 (C), 139.49 (C), 135.00 (CH), 131.48 (2× CH), 129.00 (2× CH), 123.02 (CH), 122.47 (C), 121.55 (CH), 121.22 (C), 113.06 (CH), 106.90 (CH), 97.47 (CH), 89.87 (C), 76.67 (C), 73.97 (C), 57.13 (CH2), 13.58 (CH3), 12.51 (CH2). HPLC: 100% at R.T. = 4.90 min. HRMS (ESI) calculated (79Br) 462.0660 [M + H]+, found 462.0703 [M + H]+; calculated (81Br) 464.0640 [M + H]+, found 464.0686 [M + H]+.Prepared from (4-bromophenyl)(6-(pent-2-yn-1-yloxy)benzofuran-2-yl)methanone (9c) . Yield: 0.05 g (50%); Rf = 0.37 (petroleum ether–EtOAc 1 : 1 v/v). 1H NMR (CDCl3): δ 8.54 , 7.69 , 7.36 , 7.32 , 7.26 , 7.15 , 7.03 , 7.00 , 6.87 , 6.22 , 4.62 , 4.06 , 2.19 , 1.08 . 13C NMR (CDCl3): δ 156.63 (C), 156.48 (C), 155.96 (C), 149.14 (CH), 148.61 (CH), 138.94 (C), 137.56 (C), 135.29 (C), 134.71 (CH), 133.84 (C), 131.45 (CH), 131.13 (CH), 130.68 (CH), 127.06 (CH), 121.62 (CH), 121.27 (C), 113.17 (CH), 107.10 (CH), 97.55 (CH), 89.89 (C), 76.77 , 73.96 (C), 57.14 (CH2), 13.58 (CH3), 12.52 (CH2). HPLC: 100% at R.T. = 4.92 min. HRMS (ESI) calculated (35Cl) 452.0776 [M + H]+, found 452.0816 [M + H]+; calculated (37Cl) 454.0746 [M + H]+, found 454.0791 [M + H]+.Prepared from (6-(pent-2-yn-1-yloxy)benzofuran-2-yl)methanone (9d) . Yield: 0.03 g (15%); −1 epidermal growth factor (EGF), 100 ng mL−1 cholera toxin, 0.01 mg mL−1 insulin, 500 ng mL−1 hydrocortisone, and 5% horse serum (Sigma). MDA-MB-231 cells were purchased from ATCC and grown in Roswell Park Memorial Institute medium (RPMI1690) supplemented with 10% FCS. All cells were cultured at 37 °C under 5% CO2 in a humidified incubator.JEG-3 cells were purchased from ATCC and grown in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal calf serum (FCS). MCF-10A cells were a gift from Prof. Christopher McCabe (University of Birmingham) and were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 20 ng mL12 JEG-3 cells were grown in 1 mL EMEM to approximately 80% confluence in six-well cell culture plates. Androst-4-ene-3,17-dione[1β-3H] was dissolved in serum-free cell culture medium and added into each well. Aromatase activity was measured in the absence and presence of inhibitor (0.001–10 nM). After a 1 h incubation at 37 °C followed by a 5 min incubation on ice, 500 μL of culture medium was taken from each well. Medium was vortexed with 2% dextran-treated charcoal (Sigma-Aldrich) in PBS and centrifuged at 4000 rpm. The supernatant containing the product, [3H] H2O, was quantified by scintillation counting. Cell protein concentrations were determined using Pierce BCA assay kit (Thermo Fisher Scientific). Aromatase activity results were determined as a concentration of product formed per mg of protein per hour (pmol mg−1 h−1). Results were shown as a % change in activity compared to control. Each data point was measured in triplicates and the error in the IC50 calculations represented as 95% confidence interval.Aromatase activity was assayed using a modified tritiated water assay as previously reported.3 cells per well (for MCF-10A) or 5 × 103 cells per well  as a vehicle control, or at a dose of 1 μM of inhibitor or doxorubicin control, for 48 h. Effects of drug treatment on cell growth were detected using the BrdU cell proliferation assay (Roche) according to the manufacturer's recommendations. The BrdU colorimetric immunoassay is a quantitative cell proliferation assay based on the measurement of BrdU incorporation during DNA synthesis. After treatments 20 μL per well of BrdU were added to each well, followed by an incubation of 2 h at 37 °C. The cells were subsequently fixed, and the DNA denatured. Anti-BrdU–peroxidase immune complexes were detected by substrate reaction and quantified in an ELISA reader at 370 nm.MCF-10A and MDA-MB-231 cells were plated onto 96-well microtiter tissue culture plates in RPMI1690 medium at a density of 8 × 103S79),14 was downloaded from the protein data bank (https://www.rcsb.org). Missing hydrogens were added, and the charge and geometry of the iron atom were adjusted as previously described.10 Using the site finder tool in molecular operating environment (MOE) 2015-10 software,15 the active site was chosen to contain the main amino acid residues and the haem molecule. The amino acids constituting the wall of the active site contained Arg115, Ile133, Phe134, Phe221, Trp224, Ile305, Ala306, Asp309, Thr310, Val370, Leu372, Val373, Met374, Leu477, Ser478. The 3D structures of the ligands (R- and S-enantiomers) were generated using MOE builder, energy minimised and saved in a dataset ready for docking studies. The complexes for molecular dynamics (MD) studies were prepared by docking the compounds using MOE.The crystal structure of human placental aromatase (CYP19A1) refined at 2.75 Å  were performed using OPLS_2005 forcefield at 300 K and constant pressure (1 bar).Molecular dynamics simulations were performed using Schrödinger 2020-1 Desmond programmeResearch concept designed by CS, PAF and JG. AGE performed the chemistry and analysis of all compounds supervised by CS. PAF and LEP, supervised by PAF, performed the cell proliferation and aromatase inhibition assays. Computational studies and visualisation performed by CS. Manuscript reviewed by all authors.There are no conflicts to declare.MD-014-D2MD00352J-s001


Aspidopterys obcordata Hemsl, five new polyoxypregnane glycosides, named obcordatas J–N (1–5), were obtained. Their structures were fully elucidated and characterized by HRESIMS and extensive spectroscopic data. In addition, all of the new compounds were screened for their antinephrolithiasis activity in vitro. The results showed that compounds 1–3 have prominent protective effects on calcium oxalate crystal-induced human kidney 2 (HK-2) cells, with EC50 values ranging from 6.72 to 14.00 μM, which is consistent with the application value of A. obcordata in folk medicine for kidney stones.From the dried vines of  Pregnane glycosides are substances with a basic steroidal skeleton and at least one glycosidic bond structure ,2,3. SucA. obcordata, a wood liana of the family Malpighiaceae, is distributed mainly in Xishuangbanna, Yunnan Province, China. The vines of this plant have a long history as a “Dai Medicine” for the treatment of urinary tract infections, chronic nephritis, rheumatic bone pain, cystitis and kidney stones + . Its IR spectrum showed absorption bands ascribed to the hydroxyl (3390 cm−1) and carbonyl (1718 cm−1) groups. With the assistance of the HSQC spectrum  and 1.14 ], one acetyl methyl proton signal [δH 2.06 ] and one olefic proton signal [δH 5.29 ], as well as three downfield carbon signals , which suggests the presence of a C21 steroidal pregen-5-en-20-one skeleton in the molecular structure , a tigloyl group  and three anomeric methines .Compound spectrum , the 1H-ata of 1  displayetructure . In addi1H-1H COSY spectrum  and Bz-C-1 (δC 164.8), H-12 (δH 5.01) and Tig-C-1 (δC 166.8). Meanwhile, the key HMBC correlations between 8-OH (δH 3.98) and C-8 (δC 75.1) and between 14-OH (δH 4.75) and C-8 (δC 75.1), C-13 (δC 54.6) and C-14(δC 84.5) indicate that two hydroxyl groups are substituted at C-8 and C-14, respectively. Afterwards, according to careful analysis of the 2D NMR spectral data (δH 4.21) and Allo-C-4 (δC 81.6), between Allo-H-1 (δH 4.42) and Oli-C-4 (δC 87.2), and between Oli-H-1 (δH 4.60) and C-3 (δC 76.8). Based on the above analysis, the planar construction of 1 was determined.The spectrum  showed fral data , three s1. The β configurations of the three sugars were each confirmed by their large coupling constants . Moreover, the coupling constant (J = 10.8 Hz) between H-11 and H-12 suggests that both protons are in different directions, which was further verified in the NOESY experiment. Subsequently, the NOE correlations . The 1H-NMR and 13C-APT spectral data  and 11-O-Tig-C-1 (δC 165.9), H-12 and 12-O-Tig-C-1 (δC 166.9) illustrate that two tigloyl groups are located at C-11 and C-12. Thus, the complete structure of 2 was established and named obcordata K.Compound ral data  of 2 werspectrum  of 2, th3, obtained as a white amorphous powder, was established to have the molecular formula C47H72O19 by the HRESIMS ion peak at m/z 963.4518 [M + Na]+ . Its 1D-NMR data ; δC 169.6 (Ac-C-1), 21.1 (Ac-C-2)] in 3. The HMBC correlations between H-11 (δH 5.25) and Ac-C-1 (δC 169.6) indicate that the acetoxyl group is replaced at C-11. Based on the HMBC correlations between 14-OH (δH 4.48) and C-8 (δC 36.8), C-13 (δC 54.0), C-14 (δC 82.9) and C-15 (δC 33.3), the remaining hydroxyl group was identified to be substituted at C-14. Together with its NOESY spectral data , which showed the molecular formula C47H74O19 and two more mass units than obcordata L (3). By detailed comparison of the 1D-NMR spectral data  and one additional methylene δC 29.6 (C-6), which indicates that 4 is the reduction product of 3. In the NOESY spectrum, correlations between H-3 and H-5, H-5 and H-9 suggest that H-5 is α-oriented. Finally, compound 4 was illustrated and given the name obcordata M.Compound ral data  with tho5 was suggested to be C45H70O14 based on the HRESIMS pseudomolecular ion peak at m/z 857.4640 [M + Na]+ . The overall consideration of 1D- -20-one-3β,11α,12β,14β-tetradroxypregnane aglycone moiety in 5. With the aid of the HSQC spectrum  and two sugar units (two anomeric carbons at δC 100.8 (Allo-C-1) and 96.4 (Ole-C-1)). Two tigloyl groups were confirmed to be located at C-11 and C-12 by the HMBC correlations between H-11 (δH 5.21) and 11-O-Tig-C-1 (δC 166.4) and between H-12 (δH 4.75) and 12-O-Tig-C-1 (δC 166.8). With the assistance of 2D-NMR spectral data, two sugar units were fully assigned as D-oleandrose (Ole) and 6-deoxy-3-O-methyl-D-allopyranose (Allo) after hydrolysis, which are consistent with the sugars of obcordata A previously reported from A. obcordata 25D +37 ; UV λmax (methanol) (log ε): 275 (3.62) nm; IR (KBr) νmax: 3,423, 1718, 1660, 1512, 1420 and 1210 cm−1; HRESIMS m/z 1041.4652 [M + Na]+ ; 1H- and 13C-NMR spectral  data: see Obcordata J (2): White amorphous powder; [α]25D +40.5 ; UV λmax (methanol) (log ε): 275 (3.35) nm; IR (KBr) νmax: 3390, 1715, 1658, 1503, 1427 and 1215 cm−1; HRESIMS m/z 1019.4803 [M + Na]+ ; 1H- and 13C-NMR spectral  data: see Obcordata K (3): White amorphous powder; [α]25D +48.2 ; UV λmax (methanol) (log ε): 272 (3.04) nm; IR (KBr) νmax: 3403 and 1720 cm−1; HRESIMS m/z 963.4518 [M + Na]+ ; 1H- and 13C-NMR spectral  data: see Obcordata L (4): White amorphous powder; [α]25D +52.2 ; UV λmax (MeOH) (log ε): 225 (2.84) nm; IR (KBr) νmax: 3410, 1718; 1636 and 1524 cm−1; HRESIMS m/z 965.4703 [M + Na]+ ; 1H- and 13C-NMR spectral  data: see Obcordata M (5): White amorphous powder; [α]25D +44.6 ; UV λmax (methanol) (log ε): 230 (2.90) nm; IR (KBr) νmax: 3385, 1690, 1620 and 1542 cm−1; HRESIMS m/z 857.4640 [M + Na]+ ; 1H- and 13C-NMR spectral  data: see Obcordata N (1–5 was the same as reported in our previous study [The hydrolysis method of compounds us study .4 cells/mL) and treated with compounds 1–5 in different concentrations  for 24 h. After that, the cytotoxicity of all compounds against HK-2 cells was determined via the MTT assay. Furthermore, an automatic multifunctional microplate reader was used to measure the OD values at 570 nm.In 96-well microplates, HK-2 cells were seeded  crystals was used with little modification, as described previously ,25,26. D1–5), were obtained from 95% EtOH extracts of the dried vines of A. obcordata. The complete structures of all of these new compounds were eventually elucidated by extensive spectral analysis. Their antinephrolithiasis activities were measured based on the viability of HK-2 cells exposed to COM crystals in vitro. Among all of the tested compounds, obcordata J (1) was revealed to have the most potent antinephrolithiasis activity, with an EC50 value of 6.72 μM. To date, about 60 compounds, including 17 polyoxypregnane glycosides, have been isolated from A. obcordata. Interestingly, the glycoside obcordata A was reported to exert its antinephrolithiasis activity through the NOX4/ROS/P38 MAPK pathway [A. obcordata antinephrolithiasis effect, which deserves in-depth study.In this study, five new polyoxypregnane glycosides, obcordatas J–N ( pathway ,23,24,28


H-benzimidazole-2-yl)-phenol derivatives and corresponding metal salt solutions. The compound structures were identified by FT-IR, 1H-NMR, powder X-ray diffraction and ESI-MS analyses, and the presence of the metal in the complexes was confirmed by ultraviolet-visible spectroscopy and ICP optical emission spectrometry. Electronic structure calculations were also carried out to describe the detailed structures in addition to the electronic absorption spectra of the ligands. The cytotoxic activity of the complexes was evaluated against three human cancer cell lines: lung (A549), breast (MDA-MB-231) and prostate (PC3) cancer cells. All complexes inhibited anti-proliferative cancer cells better than free ligands, especially Zn (II) and Ag (I) complexes, which are most sensitive to MDA-MB-231 cells. In addition, showing the growth inhibition of three cancer cell lines with IC50 < 10.4 µM, complexes 1C, 3C and 14C could be considered potential multi-targeted anti-cancer agents.In this study, a series of 14 Cu (II), Zn (II), Ni (II) and Ag (I) complexes containing bis-benzimidazole derivatives were successfully designed and synthesized from 2-(1 Since the serendipitous discovery of cisplatin shows anti-proliferative activity, metallodrugs have prompted an exciting research topic within medicinal chemistry as promising agents for cancer treatment . CisplatIn an attempt to replace platinum-based drugs, the transition metals, particularly copper, zinc and nickel, are the most investigated classes due to their biocompatibility and endogenous presence in the living system as cofactors in numerous enzymes . MoreoveH-benzimidazol-2-ylimino)-methyl]-6-methoxyphenol ligand exhibits a profound potential against MCF-7 human breast cancer cells using the MTT method and simultaneously releases chronic pain in cancer patients in the last stage as non-steroidal inflammatory drugs + = 211.0793, found 211.0877; UV-Vis  : 293 (9300), 319 (13 300), 334 (12 100); 1H-NMR : 13.17 , 13.14 , 8.06–8.04 , 7.72–7.71 , 7.61–7.59 , 7.40–7.37 , 7.29 , 7.05–7.00 . 13C-NMR : 157.85, 151.63, 140.80, 133.10, 131.66, 126.14, 123.21, 122.34, 119.04, 117.89, 117.11, 112.53, 111.45.The product was a light-yellow powder. Yield: 92%; M.p.: 230–231°C; IR : ν (-NH) & ν (-OH) 3237, ν (-C = N) 1639, ν (C = C) 1540, δ (-NH) 1487, ν (C-O) 1261; ESI-HRMS: calcd. for C14H12N2O: [M + H]+ = 225.1027, found [M + H]+ = 225.1039; UV-Vis  : 296 (9800), 322 (15 200), 337 (14 100); 1H-NMR : 13.18 , 13.05 , 8.04–8.02 , 7.59 , 7.511 , 7.39–7.36 , 7.12 , 7.04–6.99 , 2.46 . 13C-NMR : 157.89, 131.47, 126.11, 125.96, 123.90, 118.98, 117.64, 117.07, 112.64, 111.12, 21.20.The product was a light brown powder. Yield: 90%; M.p.: 248–249°C; IR : ν (-N-H) & ν (-OH) 3330, ν (-C = N) 1633, ν (C = C) 1585, δ (-NH) 1489, ν (C-O) 1257; ESI-HRMS: calcd. for C13H9N2OCl: [M + H]+ = 245.0481, found 245.0463; UV-Vis  : 297 (9400), 322 (16 300), 336 (15 000); 1H-NMR : 12.73 , 8.06–8.04 , 7.66 , 7.41–738 , 7.30–7.28 , 7.06–7.01 . 13C-NMR : 157.79, 152.80, 132.04, 126.56, 122.99, 119.26, 117.19, 112.51.The product was a light-yellow powder. Yield: 68%; M.p.: 240–241°C. IR : ν (-N-H) & ν (-OH) 3296, ν (-C = N) 1613, ν (C = C) 1573, δ (-NH) 1487, ν (C-O) 1259; ESI- HRMS: Exact mass of molecular ion m/z [M + H]+ = 315.1131, calcd. for C20H14N2O2: [M + H]+ = 315.1055; UV-Vis  : 337 (16 200); 1H-NMR : 13.38 , 12.68 , 8.09–8.08 , 8.02 , 7.83–7.75 , 7.70–7.67 , 7.60–7.57 , 7.44–7.40 , 7.08–7.03 . 13C-NMR : 195.45, 157.89, 137.93, 132.32, 132.12, 131.53, 129.41, 128.42, 126.80, 119.35, 117.25, 112.51.The product was a light-yellow powder. Yield: 75% M.p.: 262–263°C; IR : ν (-N-H) 3052, ν (-C = N) 1620, ν (C = C) 1583, δ (-NH) 1433; ESI-HRMS: calcd. for C20H14N2O2: [M + H]+ = 297.0406, found [M + H]+ = 297.0408; UV-Vis  : 206 (24 000), 292 (11 800); 1H-NMR d: 12.99 , 7.95 , 7.76–7.86 , 7.69 , 7.63 , 7.26–7.28 . 3C-NMR : 150.80, 132.44, 132.17, 130.46, 129.68, 129.67, 128.24, 127.75, 127.51, 126.94, 126.70, 126.66, 126.62, 126.58, 124.76, 122.58, 120.41.The product was a light-grey powder. Yield: 83%. M.p.: 173–174°C. IR  and Zn(CH3COO)2.4H2O  were completely dissolved in ethanol (15 ml) and stirred vigorously at room temperature. The colour of the solution changed, and the precipitate appeared immediately. The reaction was monitored by TLC. After a 24 h reaction, the precipitate was filtered and washed with water and cool ethanol, and the product was crystallized in DMF. The product was a white powder. Yield: 91%; M.p.: greater than 300°C; IR : ν (-NH) 3108, ν (-C = N) 1625, ν (C = C) 1531, δ (-NH) 1477, ν (C-O) 1252, ν (Zn-O) 539, ν (Zn-N) 427; ESI-HRMS: calcd. for C26H18N4O2Zn: [M + H]+ = 483.0799, found [M + H]+ = 483.0718; UV-Vis  : 212 (33 500), 291 (7100), 317 (12 900), 330 (15 000), 363 (1500); 1H-NMR : 8.00–7.98 , 7.59–7.57 , 7.29–7.22 , 7.08–7.05 , 6.82–6.80 , 6.70–6.67 . ICP: calcd. for Zn = 13.4%, found Zn = 13.5%.Ligand 2C–4C were synthesized from ligands 2L–4L and Zn(CH3COO)2.4H2O.Similar to the procedure above, complexes . 2.3.2−1): ν (-NH) 3054, ν (-C = N) 1604, ν (C = C) 1532, δ (-NH) 1477, ν (C-O) 1311, ν (Zn-O) 517, ν (Zn-N) 428; ESI- HRMS: calcd. for C28H22N4O2Zn: [M + H]+ = 511.1112, found [M + H]+ = 511.0798; UV-Vis  : 241 (47 700), 290 (7100), 318 (7000), 334 (7800), 365 (3200); 1H-NMR : 7.97 , 7.48–7.26 , 7.09–7.06 , 6.88–6.68 , 2.15–2.53 .White powder, yield: 92%; M.p.: greater than 300°C; IR : ν (-NH) 3058, ν (-C = N) 1603, ν (C = C) 1526, δ (-NH) 1477, ν (C-O) 1249, ν (Zn-O) 508, ν (Zn-N) 422; ESI- HRMS: calcd. for C26H16N4O2Cl2Zn: [M + H]+ = 551.0020, found [M + H]+ = 551.0064; UV-Vis  : 242 (42 000), 319 (8100), 335 (7800); 1H-NMR : 13.39 , 7.95 , 7.56–7.54 , 7.24 , 7.10  6.77, 6.63 .White powder, yield: 61%; M.p.: greater than 300°C; IR : ν (-NH) 3087, ν (-C = N) 1622, ν (C = C) 1526, δ (-NH) 1477, ν (C-O) 1247, ν (Zn-O) 503, ν (Zn-N) 435; ESI-HRMS: calcd. for C40H26N4NaO4Zn: [M + Na]+ = 713.1143, found [M + Na]+ = 713.1153; UV-Vis  : 237 (42 800), 375 (28 800); 1H-NMR : 7.90 , 7.71–7.38 , 7.21 , 6.70–6.61. ICP: calcd. for Zn = 9.40%, found Zn = 9.01%.White powder, yield: 78%; M.p.: greater than 300°C; IR  and Cu(CH3COO)2.H2O  in distilled ethanol (15 ml) was stirred at room temperature. The colour of the solution changed and the precipitate appeared immediately. The reaction was monitored by TLC. After a 24 h reaction, the precipitate was collected by filtration and crystallized from DMF. The given product is a brown powder, yield: 89%; M.p.: greater than 300°C; IR : ν (-NH) 3057, ν (-C = N) 1602, ν (C = C) 1554, δ (-NH) 1478, ν (C-O) 1259, ν (Cu-O) 474, ν (Cu-N) 438; ESI-HRMS: calcd. for C26H18N4O2Cu: [M + H]+ = 482.0804, found [M + H]+ = 482.0751; UV-Vis   (4.15−5M): 297 (17 100), 318 (12 100), 334 (13 500), 356 (21 800), 368 (21 100), 640 (130); ICP: calcd. for Cu = 13.27%, found Cu = 12.82%.A mixture of ligand 6C–8C were synthesized from ligands 2L–4L and Cu(CH3COO)2.H2O.Similar to the procedure above, compounds . 2.3.6−1): ν (-NH) 3049, ν (-C = N) 1604, ν (C = C) 1535, δ (-NH) 1479, ν (C-O) 1261, ν (Cu-O) 469, ν (Cu-N) 438; ESI-HRMS: calcd. for C28H22N4O2Cu: [M + H]+ = 510.1117, found [M + H]+ = 510.1112; UV-Vis  : 300 (18 900), 323 (13 200), 358 (14 900), 371 (14 200), 641(90); ICP: calcd. for Cu = 12.55%, found Cu = 12.99%.Brown powder, yield: 62%; M.p.: greater than 300°C; IR : ν (-NH) 3113, ν (-C = N) 1602, ν (C = C) 1554, δ (-NH) 1476, ν (C-O) 1255, ν (Cu-O) 523, ν (Cu-N) 439; ESI-HRMS: calcd. for C26H16N4O2Cl2Cu: [M + H]+ = 551.0049, found [M + H]+ = 551.0226; UV-Vis   (3.63−5M): 302 (15 100), 321 (10 900), 337 (19 200), 359 (24 600), 631(130); ICP: calcd. for Cu = 11.60%, found Cu = 8.90%.Brown powder, yield: 96%; M.p.: greater than 300°C; IR : ν (-NH) 3052, ν (-C = N) 1617, ν (C = C) 1550, δ (-NH) 1475, ν (C-O) 1254, ν (Cu-O) 476, ν (Cu-N) 441; ESI-HRMS: calcd. For C40H26N4NaO4Zn: [M + Na]+ = 712.1147, found [M + Na]+ = 712.1145; UV-Vis  : 380 (25 000), 656 (80); ICP: calcd. for Cu = 9.28%, found Cu = 8.10%.Brown powder, yield: 78%; M.p.: greater than 300°C; IR  was dissolved in distilled ethanol (10 ml) in a two-neck round-bottom flask. Then, a solution of Ni(CH3COO)2.4H2O  was added, and the colour of the mixture changed immediately. The mixture was stirred at room temperature. After 3 days, the precipitate was filtered and crystallized from DMF. The given product is a yellow powder, yield: 68%; M.p.: greater than 300°C; IR : ν (-NH) 3098, ν (-C = N) 1605, ν (C = C) 1564, δ (-NH) 1481, ν (C-O) 1262, ν (Ni-O) 468, ν (Ni-N) 441; ESI-HRMS: calcd. for C26H18N4O2Ni: [M + Na]+ = 499.0680, found [M + Na]+ = 499.0521; UV-Vis  : 292 (16 100), 318 (15 300), 332 (15 600), 370 (15 600), 589 (70), 695 (50); ICP calcd. for Ni = 12.18%, found Ni = 11.60%.Ligand 10C–12C were synthesized from the ligands 2L–4L and Ni(CH3COO)2.4H2O.Similar to the procedure above, compounds . 2.3.10−1): ν (-NH) 3056, ν (-C = N) 1605, ν (C = C) 1539, δ (-NH) 1481, ν (C-O) 1262, ν (Ni-O) 469, ν (Ni-N) 441; ESI-HRMS: calcd. for C28H22N4O2Ni: [M-H]− = 503.1017, found [M-H]− = 503.0702; UV-Vis  : 295 (12 400), 303 (12 800), 321 (12 500), 338 (16 900), 370 (17 800), 586 (30), 694 (40)Yellow powder, yield: 71%; M.p.: greater than 300°C; IR : ν (- NH) 3041, ν (-C = N) 1605, ν (C = C) 1563, δ (-NH) 1479, ν (C-O) 1253, ν (Ni-O) 553, ν (Ni-N) 440; UV-Vis  : 296 (10 900), 305 (11 900), 337 (19 200), 376 (15 000), 599 (50); ESI-HRMS: calcd. for C26H16N4O2Cl2Ni: [M + H]+ = 545.0077, found [M + H]+ = 545.0068.Yellow powder, yield: 65%; M.p.: greater than 300°C; IR : ν (-NH) 3050, ν (-C = N) 1618, ν (C = C) 1527, δ (-NH) 1479, ν (C-O) 1253, ν (Ni-O) 473, ν (Ni-N) 422; ESI-HRMS: calcd. for C40H26N4O2Ni: [M + Na]+ = 707.1205, found [M + Na]+ = 707.1296; UV-Vis  : 379 (28 800), 594 (60).Yellow powder, yield: 72%; M.p.: greater than 300°C; IR  was dissolved in distilled ethanol (10 ml) in a two-neck round-bottom flask. Then, a solution of AgNO3 (0.6 mmol) was added and the colour of the mixture changed immediately. The mixture was stirred at room temperature. After 3 days, a precipitate crystallized and was filtered from DMF. The product is the pink powder, yield: 78%; M.p.: greater than 300°C; IR : ν (-NH) & ν (-OH) = 3336, ν (-C = N) 1605, ν (C = C) 1531, δ (-NH) 1469, ν (C-O) 1231, ν (Ag-N) 438; ESI-HRMS: calcd. for C26H20N4O2Ag: [M + H]+ = 527.0637, found [M + H]+ = 527.0710; UV-Vis  : 281 (6200), 306 (8000), 320 (15 000); 1H-NMR : 13.17 , 12.86 , 8.07–8.05 , 7.72–7.63 , 7.40–7.37 , 7.29 , 7.06–7.00 .Ligand . 2.3.14; IR : ν (-NH) 3064, ν (C = N) 1624, ν (C-N) 1317, ν (Ag-N) 472; ESI-HRMS: calcd. for C28H16F6Cl2Ag: [M + H]+ = 699.9707, found [M + H]+ = 699.9754. UV-Vis  : 203 (55 800), 284 (24 400); 1H-NMR : 13.46 , 7.94–7.92 , 7.82–7.74 , 7.69 , 7.36–7.34 .Grey powder, yield: 63%; M.p.: greater than 300°C. 2.4Geometry optimizations of the ligands and complexes were carried out using Gaussian 09  at the B−1.The UV-Vis absorption spectra were simulated for the ligands in the Franck–Condon region (denoted as FC-UV). The lowest excited-state geometries were optimized using time-dependent density functional theory. The harmonic vibrational frequencies of both the ground and excited states are carefully verified, for which no imagination frequency is expected. A small benchmark of different density functionals with various exchange-correlation contributions, including global hybrid functionals  and range-separated hybrid functional (CAMB3LYP and wB97XD) were chosen to study the quality of the vibrationally resolved electronic spectra, as previously recommended . The 6-3. 2.5−1 of penicillin and 100 µg ml−1 streptomycin in a 5% CO2 atmosphere for 48 h. After that, the cells were seeded in 96-well plates at a density of 104 cells/well. After 24 h, the cells were treated with a culture medium containing the tested compounds in the concentration range. After 72 h, cell viability was determined as mitochondrial succinate dehydrogenase (SDH) activity using 0.5 mg ml−1 of the 3--2,5-diphenyltetrazolium bromide (MTT) test as a marker of viable cells and incubated at 37°C and 5% CO2 for 4 h, following the procedure described by Mosmann + = 482.0751, 511.1145 and 551.0226, respectively. The Cu (II) complexes 5C and 6C experience the disappearance of one molecule of 1L and 2L, respectively, and the appearance of a new fragment obtained [M-L1 + H2O] (C13H11N2O2Cu + H2O) at m/z 290.0152 for 5C and [M-L2 + H2O] (C14H13N2O2Cu + H2O) at m/z 304.0325 for 6C. Demetallation was recorded in these complexes, leading to the isolation of the metal-free ligand 1L (m/z = 211.0918). Likewise, the Zn (II), Cu (II), Ni (II) and Ag (I) complexes 1C–3C, 7C and 9C–14C displayed two dominant ionic fragments in the data, including a central metal (II) coordinated with a bidentate ligand and a peak of free ligands. On the other hand, compounds 4C, 8C and 12C show pseudo-molecular ions, with peaks at [M + Na]+ 713.1153, 712.1142 and 707.1196, respectively. This indicated that the compounds were unstable and could open during ESI-MS analysis.The molecular ion fragments in mass spectrometry were used to elucidate the probable formulae of the complexes. These results showed that the formation of the complexes occurs through a central metal that links two benzimidazole-derived molecules. For example, the mass spectral data for the copper (II) complexes 1H-NMR and ICP-OES) are in good agreement with the correlative structures of the complexes.Finally, according to the detailed analyses, spectral data . Indexing of the diffraction patterns was displayed using X'Pert HighScore Plus 3.0 software and the Miller indices (hkl) along with observed and calculated 2θ angles, relative intensities, and d values are shown in the electronic supplementary material. The type of crystal system, cell volume and lattice parameters of these compounds were calculated from the indexed data and are listed in D = Kλ/βCos θ, where D is the particle size, K is the dimension shape factor, λ is the X-ray wavelength (0.15406 Å), β is the full-width at half-maximum of the diffraction peak and θ is the diffraction angle was used to calculate the average crystalline sizes of the complexes. The complexes have a crystalline size ranging from 25 to 58 nm, suggesting that they are in the nanocrystalline phase.PXRD studies were carried out for all complexes , MDA-MB-231 (human breast adenocarcinoma cell line) and PC3 (human prostate carcinoma cell line) by using MTT -2,5-diphenyltetrazolium bromide) assay. The cytotoxicity efficacy of the compounds was expressed as IC50 values, as shown in 50 greater than 100 µM), with the IC50 values in the range of 5.82–80.83 µM, except ligand 5L, which exhibits the highest anti-proliferation activity in the MDA-MB-231 and PC3 cell line assay, with the IC50 values 23.9 ± 0.9 and 23.4 ± 0.6 µM, respectively. Remarkably, the complexes 1C, 3C and 14C possessed anti-cancer activity better than the reference drug cisplatin against A549 cells with their IC50 values of 8.9 ± 0.6, 8.7 ± 1.0, 5.8 ± 0.8 and 16.5 ± 1.0 µM, respectively, while the IC50 values of 1C, 3C, 4C, 8C and 14C were lower than 10 µM and that of cisplatin (IC50 = 19.4 ± 1.7 µM) on MDA-MB-231 cell line.The 1C and 3C) are sensitive to all cancer cell lines, with IC50 values as low as 8.7–10.4 µM. This showed that the Cu (II) and Ni (II) complexes have less activity than the Zn (II) chelates. Cu (II) complexes (5C and 7C) exhibited the lowest anti-proliferation activity among the complexes, with IC50 values greater than 100 µM. The resemblance of the anti-tumour activity of the Ni (II) chelates was lower than that of the Zn (II) complexes, with IC50 varies in the range of 14.6–82.1 µM. In particular, Ag (I) complexes, that is, complexes 13C and 14C, coordinated by ligands 1L and 5L, respectively, have significantly increased anti-cancer activity in all cancer cell lines. While the IC50 values of 13C are in the range of 15.00–20.7 µM, the lowest IC50 values are obtained for 14C, being 5.8 ± 0.8, 6.1 ± 1.0 and 7.0 ± 0.9 µM, for A549, MDA-MB-231 and PC3 cell lines, respectively. This illustrates that an appropriate choice of the benzimidazole-derived scaffold and the metal ions plays a crucial role in designing metallodrugs with selectivity and sensitivity against A549, MDA-MB-231 and PC3 cell lines. In addition, the structure–activity relationship was determined by comparing complex 14C with 13C and others, and the substituents of chlorine and trifluoromethyl groups in benzimidazole derivatives can boost the anti-proliferative activity against cell lines. It is noteworthy that three cancer cell lines, A549, MDA-MB-231 and PC3, were concomitantly inhibited by complexes 1C, 3C and 14C, with IC50 < 10 µM. Therefore, these complexes can be considered multi-targeted anti-cancer agents.It was found that Zn (II) complexes  at a concentration of 20 µM between 200 and 750 nm for 24 h at room temperature to determine the stability in a physiological environment. This test demonstrated the possibility of transformation such as hydrolysis, reduction and precipitation of the complexes. According to electronic supplementary material, figure S85, no change was observed in the UV-Vis bands of complexes 1C and 14C . Although there is a decrease in absorbance of UV-Vis spectrum of 3C leading to the disappearance of the bands after 8 h, suggesting the decay of 3C, it was stable over the first 8 h in this buffer without significant change in the spectrum .The complexes were not insoluble in ethanol, water and chloroform and they dissolved completely in DMF and DMSO. In addition, MeCN is selectively soluble for the complexes The cytotoxic activity of the complexes is proposed by the binding ability of benzimidazole moieties to DNA grooves via the interactions such as hydrogen bonds, electrostatic interactions and π–π stacking , one of Thus, the combined effects could result in the proliferation of bioactivities or activate new mechanisms of action . On the et al. [Besides, the results of anti-tumour activities of Zn (II) complexes have recorded plenty of evidence that displays an intriguing link between zinc and malignant cells. The growth of cancer cells can be inhibited by increasing zinc levels ,64. Typiet al.  reportedet al. . Moreoveet al. .H-benzimidazol-2-yl)-phenoxy-derived scaffolds with cytotoxic activity in human cancer cell lines A549, MDA-MB-231 and PC3.From the above results, the coordinate between the transition metal and benzimidazole moieties showed a significant increase in cytotoxicity, and complexes may be candidates for multi-targeted anti-cancer agents in the future. These compounds are the first examples of metal complexes of 2-, MDA-MB-231 (human breast adenocarcinoma cell line) and PC3 (human prostate carcinoma cell line). Specifically, complexes 1C, 3C and 14C with the IC50 values of 5.8–10.4 µM showed the best anti-proliferative activity and better than the reference drug, cisplatin, whose IC50 values are 16.5 ± 1.0 and 19.4 ± 1.7 µM on A549 and MDA-MB-231 cell lines, respectively. Thus, they could be the new promising agents in chemotherapeutic applications and will be investigated on the other cancer cell lines and the particular mechanism of action in further research.Five free ligands and 14 transition metal complexes containing benzimidazole derivatives were designed, synthesized and characterized in this study. Multiple structural analyses revealed that metal M (II)  complexes adopt a distorted tetrahedral coordination geometry, while Ag (I) complexes have linear coordination. The detailed structures, together with the electronic absorption spectra of the ligands, were also calculated using electronic structure calculations to complement the experimental data. Moreover, This article does not contain any studies involving animals performed by any of the authors.