As a result of MMP by flow cytometry,compound 6g showed 80.93% mitochondrial membrane potential.According to the results of the obtained EGFR inhibitory assay, compound 6g shows inhibitory activity on the EGFR enzyme with a valueof IC50 = 0.024 ± 0.002 μM.Lung cancer is one of the most common cancer types ofcancer withthe highest mortality rates. However, while epidermal growth factorreceptor (EGFR) is an important parameter for lung cancer, EGFR inhibitorsalso show great promise in the treatment of the disease. Therefore,a series of new EGFR inhibitor candidates containing thiadiazole andpyrazole rings have been developed. The activities of the synthesizedcompounds were elucidated by in vitro MTT, -2,5-diphenyltetrazoliumbromide), cytotoxicity assay, analysis of mitochondrial membrane potential(MMP) by flow cytometry, and EGFR inhibition experiments. Moleculardocking and molecular dynamics simulations were performed as in silicostudies. Compounds  Cytoplasmic tyrosine kinases, a subgroup of kinases, arecritical for extracellular signals. These extracellular signals havebeen shown to occur in a variety of oncogenic conditions. Naturally,the development of tyrosine kinase enzyme inhibitors is very importantin cancer treatment.4Cancer is one of the mostserious and deadly health problems. Lungcancer, on the other hand, is quite deadly compared to other cancersubtypes.6Epidermal growth factorreceptors (EGFRs), one of the tyrosinekinase receptors, are responsible for cell growth and proliferation.However, abnormal conditions in EGFR functions cause cancer. Therefore,EGFR inhibitors are very important for new drug discovery in the treatmentof certain types of cancer, such as lung cancer and triple-negativebreast cancer.9 An important exampleof the heterocyclic compounds is Erlotinib.Erlotinib is an FDA-approved inhibitor of EGFR.10 Pyrazolines, which contain a double bond and two adjacentnitrogen atoms in the ring, are five-membered heterocyclic rings andshow anticancer activity.12 Furthermore, the EGFRinhibition values shown by the compounds containing the 1,3,5-trisubstitutedpyrazoline structure have been a guide in the design process of thecompounds.14 In previous studies, it was alsoobserved that compounds containing a pyrazole ring had very good inhibitionvalues on EGFR.16 Just like the pyrazole ring,the thiadiazole ring has anticancer activity. Studies have shown thatcompounds containing a thiadiazole ring are pretentious about anticanceractivity.19 Thiadiazoles also showed anticancer activity by providingEGFR inhibition.21Heterocyclic compounds areknown to have anticancer activity.26 Lazertinib, a third-generation EGFR inhibitor, has had promisingresults in patients who have not yet started treatment or in patientswith Osimertinib resistance. Lazertinib is unique and different fromother third-generation EGFR inhibitors in that it contains a pyrazolering, heterocyclic rings, hydrophobic phenyl, and hydrophilic aminestructures. As seen in 28In the studies on severalcompounds containing thiazole, a thiadiazole-likeheterocyclic ring, and a pyrazole ring, it has been determined thatthe compounds are anticancer drug candidates with a very high EGFRinhibition potential.In light of all this information, the hybrid compoundsincludingpyrazole–thiadiazol rings were designed as EGFR inhibitors.22.11H-NMR Bruker DPX 300 FT-NMR spectrometerand 13C-NMR, Bruker DPX 75 MHz spectrometer  were used for NMR analysis. Mass spectra wererecorded on an LCMS-IT-TOF  using ESI.All the chemicals wereobtained from industrial vendors and used without additional purification.Melting points (mp) were determined on the Mettler Toledo-MP90 MeltingPoint System. 2.1.12.1.1.129KOH solution (40%) was prepared in MeOH(>99.5%). Acetophenone  and the prepared KOHsolutionwere stirred at room temperature for 30 min. Then, 4-nitrobenzaldehyde was added to the reaction and mixed. After thereaction was complete, the precipitated product was filtered, washedwith MeOH, and dried.2.1.1.2303-(4-Nitrophenyl)-1-phenylprop-2-en-1-one was dissolved in ethanol (30 mL) and refluxedwith hydrazine hydrate  for 8 h. After the reactionwas complete, the reaction mixture was cooled and refrigerated overnight.The resulting solid was filtered, and the crude pyrazoline was driedand recrystallized from ethanol.2.1.1.3H-pyrazole was dissolved in 100 mL of tetrahydrofuran (THF),and triethylamine (TEA) was added to the solution. Then, chloroacetylchloride dissolved in 10 mL of THF was added dropwise to the solutionplaced in the ice bath because of the obtained 2-chloro-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-yl)ethan-1-one (3).First,5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-12.1.1.431An excess of hydrazine hydrate was added to the mixtureof alkyl/aryl isothiocyanates (0.010 mol) in ethanol and stirred in an ice bath for 2 h. After the reaction was finished,the precipitated product was filtered and washed with cold EtOH. Theproduct was then dried and recrystallized from EtOH.2.1.1.54a–4j (0.005 mol) wereadded. Then, carbon disulfide  was added to themixture and refluxed for 4 h. After the reaction was complete, themixture was poured into ice water and acidified to pH 4–5 with20% HCl. The precipitated product was filtered and washed with water.The product was then dried and recrystallized from EtOH.31First, NaOH  was dissolved in 40 mLof ethanol, and compounds 2.1.1.65a–5j) (0.001 mol) and compound 3  were dissolved in 20 mL of acetone. 0.001 mol of K2CO3 was then added, and the mixture was refluxedovernight. After the reaction was complete, the mixture was cooled,and the precipitated product was filtered and purified by recrystallizationwith ethanol.32First, thiadiazole derivatives (H-pyrazol-1-yl)ethan-1-one (6a): yield: 81%,mp: 161.7–162.3 °C. 1H-NMR : δ = 1.17 , 3.26 , 3.24 , 3.32 ,4.01 , 4.46 , 5.79 , 7.52 , 7.60 , 7.83  7.85 , 8.25 . 13C-NMR : δ = 14.60 (ethyl-C), 37.29(−CH2−), 39.83 (ethyl-C), 42.29 (pyrazole-C),59.87 (pyrazole-C), 124.37 (phenyl-C), 127.40 (phenyl-C), 127.56 (phenyl-C),129.27 (phenyl-C), 130.94 (phenyl-C), 131.19 (phenyl-C), 147.18 (phenyl-C),148.96 (phenyl-C), 149.47 (pyrazole-C), 155.71 (thiadiazole-C), 165.34(thiadiazole-C), 170.32 (C=O). HRMS (m/z): [M + H]+ calcd for C21H20N6O3S2, 469.1111; found, 469.1106.2-((5-(Ethylamino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-((5-(propylamino)-1,3,4-thiadiazol-2-yl)thio)ethan-1-one(6b): yield: 72%, mp: 120.6–122.8 °C. 1H-NMR : δ= 0.87 , 1.52 , 3.26 , 3.94 , 4.40 , 5.73, 7.46 , 7.54 , 7.78 , 7.82 , 8.19 . 13C-NMR : δ = 11.80 (propyl-C), 22.18 (propyl-C),37.26 (−CH2), 42.29 (pyrazole-C), 46.82 (propyl-C),59.87 (pyrazole-C), 124.36 (phenyl-C), 127.40 (phenyl-C), 127.56 (phenyl-C),129.27 (phenyl-C), 130.94 (phenyl-C), 131.19 (phenyl-C), 147.18 (phenyl-C),149.47 (phenyl-C), 155.70 (pyrazole-C), 165.35 (thiadiazole), 170.07(thiadiazole-C), 170.50 (C=O). HRMS (m/z): [M + H]+ calcd for C22H22N6O3S2, 483.1268; found, 483.1265.1-(5-(4-Nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6c): yield: 82%,mp: 105.2–107.4 °C. 1H-NMR : δ = 1.13 , 3.24 ,3.72 , 3.95 ,4.41 , 5.73 , 7.46 , 7.54 , 7.72 , 7.81 , 8.19 . 13C-NMR : δ = 22.51 (isopropyl-C), 37.20 (−CH2−), 42.28 (pyrazole-C), 47.02 (isopropyl-C), 59.88 (pyrazole-C),124.36 (phenyl-C), 127.40 (phenyl-C), 127.57 (phenyl-C), 129.28 (phenyl-C),130.94 (phenyl-C), 131.20 (phenyl-C), 147.18 (phenyl-C), 148.85 (phenyl-C),149.48 (phenyl-C), 155.70 (pyrazole-C), 165.34 (thiadiazole-C), 169.45(C=O). HRMS (m/z): [M + H]+ calcd for C22H22N6O3S2, 483.1268; found, 483.1250.2-((5-(Isopropylamino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6d): yield: 82%,mp: 118.5–120.6 °C. 1H-NMR : δ = 1.16 , 1.83, 3.02 , 3.23 , 3.72 ,3.91 , 4.40 , 5.73 , 7.45 , 7.54 , 7.78 , 7.85 , 8.18 . 13C-NMR : δ= 20.48 (−CH2−), 27.93 (pyrazole-C), 52.66, 59.84 (pyrazole-C), 124.35 , 127.39 (phenyl-C),127.55 (phenyl-C), 128.99 (phenyl-C), 129.27 (phenyl-C), 130.40 ,130.94 (phenyl-C), 131.19 (phenyl-C), 147.18 (phenyl-C), 148.83 (phenyl-C),149.47 (phenyl-C), 155.69 (pyrazole-C), 165.35 (thiadiazole-C), 170.63(C=O). HRMS (m/z): [M + H]+ calcd for C22H20N6O3S2, 481.1111; found, 481.1113.2-((5-(Allylamino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6e): yield: 73%,mp: 123.7–126.4 °C. 1H-NMR : δ = 0.86 , 1.16 , 1.31 , 1.49 , 3.17 , 3.94 , 4.40 , 5.73, 7.46 , 7.54 , 7.77 ,7.83 , 8.19 . 13C-NMR : δ = 14.07 (butyl-C), 19.98 (butyl-C),30.95(−CH2−), 42.30 (pyrazole-C), 44.71 (butyl-C),46.12 (butyl-C), 59.83 (pyrazole-C), 124.37 (phenyl-C), 127.40 (phenyl-C),127.56 (phenyl-C), 129.27 (phenyl-C), 130.97 (phenyl-C), 131.19 (phenyl-C),133.96 (phenyl-C), 149.48 (phenyl-C), 155.69 (pyrazole-C), 165.35(thiadiazole-C), 170.47 (C=O). HRMS (m/z): [M + H]+ calcd for C23H24N6O3S2, 497.1424; found, 497.1416.2-((5-(Isopropylamino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6f): yield: 74%,mp: 136.0–137.4 °C. 1H-NMR : δ = 1.61 , 1.92, 3.23 , 3.90 , 4.40, 5.73 ,7.46 , 7.54 , 7.77 ,7.82 , 8.19 . 13C-NMR : δ = 9.03 (cyclohexyl-C), 24.67 (cyclohexyl-C),25.66 (cyclohexyl-C), 32.46 (−CH2−), 37.23(pyrazole-C), 45.82 (cyclohexyl-C), 53.91 (pyrazole-C), 124.35 (phenyl-C),127.55 (phenyl-C), 129.27 (phenyl-C), 130.94 (phenyl-C), 131.18 (phenyl-C),147.17 (phenyl-C), 148.68 (phenyl-C), 149.48 (phenyl-C), 155.66 (pyrazole-C),165.36 (thiadiazole-C), 169.01, 169.45 (thiadiazole-C), 170.08 (C=O).HRMS (m/z): [M + H]+ calcdfor C25H26N6O3S2, 523.1581; found, 523.1561.2-((5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)ethan-1-one(6g): yield: 86%, mp: 225.9–226.8 °C. 1H-NMR : δ= 3.25 , 4.03 ,4.62 , 5.82 , 7.06 , 7.39 , 7.52 , 7.54 , 7.64 , 7.85 , 8.25 , 10.44 . 13C-NMR : δ = 37.01 (−CH2−), 42.33 (pyrazole-C),59.92 (pyrazole-C), 117.80 (phenyl-C), 122.47 (phenyl-C), 124.36 (phenyl-C),127.41 (phenyl-C), 127.56 (phenyl-C), 129.27 (phenyl-C), 129.56 (phenyl-C),130.92 (phenyl-C), 131.22 (phenyl-C), 140.78 (phenyl-C), 147.20 (phenyl-C),149.45 (phenyl-C), 152.41 (thiadiazole-C), 155.85 (pyrazole-C), 165.14(thiadiazole-C), 165.60 (C=O). HRMS (m/z): [M + H]+ calcd for C25H20N6O3S2, 517.1111; found, 517.1109.1-(5-(4-Nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-((5-(p-tolylamino)-1,3,4-thiadiazol-2-yl)thio)ethan-1-one (6h): yield: 83%, mp: 184.5–186.7 °C. 1H-NMR : δ = 2.25, 3.25 ,3.97 , 4.54 , 5.75 , 7.13 , 7.41 , 7.48 , 7.55 , 7.79 , 8.19 , 10.30 . 13C-NMR : δ = 20.85 (−CH3), 37.04 (−CH2−), 42.33 (pyrazole-C), 59.91 (pyrazole-C), 117.94(phenyl-C), 124.37 (phenyl-C), 127.41 (phenyl-C), 127.56 (phenyl-C),129.28 (phenyl-C), 129.95 (phenyl-C), 130.92 (phenyl-C), 131.23 (phenyl-C),131.44 (phenyl-C), 138.42 (phenyl-C), 147.19 (phenyl-C), 149.46 (phenyl-C),151.89 (thiadiazole-C), 155.84 (pyrazole-C), 165.15 (thiadiazole-C),165.84 (C=O). HRMS (m/z):[M + H]+ calcd for C26H22N6O3S2, 531.1268; found, 531.1251.1-(5-(4-Nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6i): yield: 88%,mp: 180.9–181.6 °C. 1H-NMR : δ = 3.25 , 3.79 , 4.03 ,4.58 , 5.81 , 6.97 , 7.48 , 7.53 , 7.61 , 7.85 , 8.25 , 10.24 . 13C-NMR : δ = 37.08 (−CH2−), 42.32 (pyrazole-C),55.70 (methoxy-C), 59.91 (pyrazole-C), 114.74 (phenyl-C), 119.68 (phenyl-C),124.37 (phenyl-C), 127.41 (phenyl-C), 127.56 (phenyl-C), 129.28 (phenyl-C),130.93 (phenyl-C), 131.22 (phenyl-C), 134.28 (phenyl-C), 147.20 (phenyl-C),149.45 (phenyl-C), 151.35 (phenyl-C), 155.08 (thiadiazole-C), 155.83(pyrazole-C), 165.19 (thiadiazole-C), 166.34 (C=O). HRMS (m/z): [M + H]+ calcd for C26H22N6O4S2, 547.1217;found, 547.1220.2-((5-((4-Methoxyphenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6j): yield: 88%,mp: 162.5–163.4 °C. 1H-NMR : δ = 3.23 , 3.96 , 4.75 , 5.74 , 6.97 , 7.48 , 7.53 , 7.61 , 7.85 , 8.25 , 10.52 . 13C-NMR : δ = 37.00 (−CH2−), 42.87 (pyrazole-C), 60.02 (pyrazole-C), 119.33(phenyl-C), 124.36 (phenyl-C), 124.47 (phenyl-C), 125.82 (phenyl-C),127.48 (phenyl-C), 127.62 (phenyl-C), 129.29 (phenyl-C), 129.36 (phenyl-C),130.88 (phenyl-C), 131.32 (phenyl-C), 139.66 (phenyl-C), 147.29 (phenyl-C),149.33 (phenyl-C), 149.44 (thiadiazole-C), 155.86 (pyrazole-C), 165.10(thiadiazole-C), 165.23(C=O). HRMS (m/z): [M + H]+ calcd for C25H19N6O3S2Cl, 551.0721; found, 551.0723.2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-12.22.2.13336The principleof the MTT test used to determine the metabolic activity of livingcells is based on the reduction of colorless 3--2,5diphenyltetrazolium salt to the purple-colored formazan product. Thanksto this color change, the cell viability rate can be determined spectrometrically.2.2.22 incubator.At the end of the 24 h incubation period, the medium in the flaskswas removed, and compound 6g and the reference drug wereadded to the flasks (at IC50 concentration). Cells werecollected and centrifuged in line with the instructions in the kitat the conclusion of this period. The upper part was removed, JC-1dye was added, and the mixture was incubated for 10–15 minat 37 °C. The CytoFLEX flow cytometer  and CytExpert for CytoFLEX Acquisition and Analysis SoftwareVersion 2.2.0.97 were used to read it after two washings with washingsolution at the conclusion of the period.The BD MitoScreen Mitochondrial MembranePotential Detection JC-1 Kit (available from ref ) was use2.2.3The EGFRKinase Assay Kit (available from ref ) was use2.36g in the activeregions of the crystal structures of EGFR (PDB ID: 1M17),39 which were retrievedfrom the Protein Data Bank server (www.pdb.org). The Schrödinger Maestro docking mode was used for docking runs.Moleculardocking studies were performed using an in silico procedure to definethe binding modes of active compound r Maestro interfac2.46g within the scope of this study. MD studieswere performed for 100 ns as previously reported.46 Following the completion of thesystem setup, the MD simulation was run using the settings. The Desmondapplication calculated the values for Rg (radius of gyration), root mean square fluctuation (RMSF), and rootmean square deviation (RMSD).48Molecular dynamics (MD) simulations, which are considered an importantcomputational tool to evaluate the time-dependent stability of a ligandat an active site for a drug–receptor complex, were performedfor compound 33.16a–6j) were prepared in a total of six steps. The syntheticroute to the obtained compounds 6a–6j is presentedin 1) was obtained from acetophenone and 4-nitrobenzaldehyde using Claisen–Schmidtreaction. Compound 1 was refluxed with hydrazine hydrate,and the ring closure reaction was performed to obtain 5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2).Target compounds (2 was acetylated usingan ice bath. Compounds 4a–4j were obtained withthe reaction between isothiocyanatesand hydrazine hydrate. The thiadiazol derivatives (5a–5j) were gained using a ring closure reaction in basic conditions.The thiadiazoles (5a–5j) obtained because of thiswere reacted with compound 3 to obtain target compounds(6a–6j). The structures of the compounds obtainedwere established by spectroscopic methods, namely, 1H-NMR, 13C-NMR, and HRMS (Supporting Information).Compound 1H-NMR results. The peaks of the pyrazoline were obtained in the rangeof 3.17–3.32, 3.90–4.03, and 5.73–5.82 ppm. Thecharacteristic signals of the ethylene −CH2 protonswere found at 4.40–4.78 ppm. In the secondary amine peaks,if the bonding group is aliphatic, the peaks in the range of 7.72–7.85ppm were obtained, but if the bonded group is aromatic, the peaksin the range of 10.24–10.52 ppm were obtained. In 13C-NMR analyses, the peaks of carbon atoms with carbonyl groups wereconsistently around 165 ppm.Characteristic peaks of the protons belonging to the pyrazolinering, ethylene group, and secondary amine were observed in 3.23.2.16a–6j were performed withthe 24 h MTT procedure. The results are presented in 50 < 10 μM. These compounds include allyl (6d), phenyl (6g), and 4-chlorophenyl (6j)moieties. In contrast, no activity was observed in the compounds containingisobutyl (6e) and cyclohexyl (6f) moieties.Compound 6d showed activity against the A549 cell linewith an IC50 = 5.176 ± 0.164 μM. Compound 6g showed activity against the A549 cell line with an IC50 = 1.537 ± 0.097 μM. Compound 6j showedactivity against the A549 cell line with an IC50 = 8.493± 0.667 μM. Against the MCF-7 cell line, all compoundsshowed less activity than IC50 = 10 μM. But compounds 6d, 6g, and 6j were the most activecompounds with their IC50 = 35.724 ± 2.237 μM,IC50 = 15.925 ± 0.054 μM, and IC50 = 67.246 ± 3.627 μM activity values against the MCF-7cell line, respectively. In this case, it is possible to say thatthe compounds exhibited a selective activity against the A549 cellline. Compound 6d showed cytotoxicity with an IC50 = 11.907 ± 0.486 μM. When the healthy cell linewas examined, compound 6g showed cytotoxicity with anIC50 = 11.115 ± 0.205 μM. Compound 6j showed cytotoxicity with an IC50 = 48.260 ± 1.717μM. The selectivity indexes of compounds 6d, 6g, and 6j were calculated as 2.3, 7.23, and5.68, respectively. In this case, compound 6g was boththe most effective derivative against the A549 cell line and the derivativewith the highest selectivity index. It can be predicted that the sideeffect and toxic effect profile of compound 6g will beless than that of compounds 6d and 6j.Cytotoxicityassays of compounds 3.2.249 Mitochondria are an important target in thecase of cancer cells that are resistant to drug therapy. Mitochondriaplay a key regulatory role in the apoptotic pathway. Therefore, theyare identified as an important target for cancer therapy.50 Mitochondria generate a membrane potential usingoxidizable substrates. The provision of these substrates is associatedwith the growth factor. Therefore, growth factor reduction or extracellularglucose loss reduces the MMP. If growth factor or glucose deprivationpersists, the cells ultimately undergo apoptosis that is initiatedby cytochrome c release from mitochondria.51 For this purpose, the mitochondrial membranepotential of compound 6g (the most active compound) wasdetermined against the A549 cell line by in vitro flow cytometricmethods. Both the inhibitory compound and the reference drug wereadministered at IC50 concentrations. After a 24 h incubationperiod (5% CO2), mitochondrial membrane potential was determinedby flow cytometry using JC-1 dye. The resulting flow cytometry diagramsare presented in 6g showed 80.93% mitochondrialmembrane potential. This rate is promising. The high MMP potentialof compound 6g provides both the restriction of resistancedevelopment and a safer treatment with the apoptotic pathway.The membrane potential of mitochondria, which have a central rolein apoptosis, is a measurable change in flow cytometry.3.2.352 Inhibitionof this member of the tyrosine kinase family, which is a new approachin cancer chemotherapy, is important. Therefore, the EGFR inhibitionpotential of compound 6g, which is the most active compound,was evaluated by the in vitro kit method. The IC50 valuefor compound 6g was calculated using the kit procedure.According to the results obtained, compound 6g showsinhibitory activity on the EGFR enzyme with a value of IC50 = 0.024 ± 0.002 μM. For Erlotinib, the IC50 value was found to be 0.002 ± 0.001 μM.EGFR kinase,which belongs to the ErbB family of tyrosine kinases, is known toregulate the signaling pathways of cell survival, migration, proliferation,differentiation, and adhesion.3.36g had the highest activity among the synthesized compounds. Dockingstudies were carried out to examine the binding modes of compound 6g with the enzyme active site. The docking studies were performed,with the EGFR enzyme active site. Crystal structure of the EGFR enzyme(PDB code: 1M17)39 was retrieved from the PDB data bank.In the studies, the docking technique was performed with the Glide7.1.42As a resultof the activity studies, it was seen that compound 6g with the EGFR enzyme active site. The benzene ring near the thiadiazolering formed a cation−π interaction with the amine ofLys721. The amine group between the benzene and thiadiazole ringsestablished a hydrogen bond with the carbonyl of Asp831. Another hydrogenbond was noted between the carbonyl near the pyrazole ring and theamine of Cys773. The last hydrogen bond was detected between the nitromoiety and the amine of Met769. Also, the benzene ring to which thisnitro moiety was attached formed an aromatic hydrogen bond with thecarbonyl of Met769.3.46g and EGFR (PDB ID: 1M17), were taken into consideration for the100 ns MD simulation study in an explicit hydration environment.The MD simulationmethod is commonly used to investigate the dynamic behavior of proteinsor protein–ligand complexes. In the current study, to evaluatethe stability of the docking complex formed between the promisingmolecule 6g–EGFR enzyme complexare shown in X-axisshow how contacting residues between each protein chain and its ligandcontributed to the overall result.The results for the compound 6g contacted20 aminoacids of EGFR protein, namely, Leu694, Phe699, Val702, Lys704, Val718,Lys721, Glu738, Met742, Leu764, Gln767, Met769, Pro770, Phe771, Gly772,Cys773, Asp776, Arg817, Leu820, Thr830, and Asp831. Major fluctuationswere observed in the C-terminal and loop regions, which are positionedaway from the binding pocket. On the other hand, in the 1M17–6g complex, the fluctuations of the simulated compound haveno substantial changes.As per the RMSF plot, compound 6g and the carbonyl of Met769; between the phenylring near the pyrazole ring of compound 6g and the carbonylof Leu694; between the phenyl ring near the secondary amine groupof compound 6g and the carbonyl of Ala719; between thephenyl ring near the secondary amine group of compound 6g and the carbonyl of Ile720; between the phenyl ring near the secondaryamine group of compound 6g and the carbonyl of Leu764;between the phenyl ring near the secondary amine group of compound 6g and the carbonyl of Asp831; between the phenyl ring nearto the secondary amine group of compound 6g and the hydroxylof Thr766; and between the phenyl ring near the secondary amine groupof compound 6g and the hydroxyl of Thr830.By watching the MD simulation video,aromatic hydrogen bonds weredetermined for 100 ns s. Accordingly, the aromatic hydrogen bondsformed can be listed as follows: between the 4-nitrophenyl ring ofcompound As seenin 6g providesthe stability in the enzyme active site of EGFR by interacting especiallyMet769, Asp831, Asp776, and Cys773. It was concluded by analyzingthe MD simulation video where the phenyl ring near the pyrazole actsin a flexible conformation. It was thought that the placement of chemicalstructures of larger volume instead of the phenyl ring may contributeto the stability positively and therefore may obtain a stronger bindingprofile.Consequently, it was seen that compound 46d, 6g, and 6j. These compounds had IC50 = 5.176 ± 0.164μM, IC50 = 1.537 ± 0.097 μM, and IC50 = 8.493 ± 0.667 μM activity values against theA549 cell line, respectively. The compound with the highest activity, 6g, showed 80.93% mitochondrial membrane potential. Compound 6g shows an inhibitory activity on the EGFR enzyme with avalue of IC50 = 0.024 ± 0.002 μM. Moleculardocking and MD simulations showed that the compound fully fits intothe 6g EGFR active site.Today, EGFR inhibitorsare used in the treatment of cancer typeswith high mortality rates such as lung cancer. New EGFR inhibitorsare a great source of hope for the treatment of diseases such as lungcancer. Therefore, in this study, it was desired to synthesize newEGFR inhibitors with improved effects. It is expected that the newlydesigned compounds containing pyrazole and thiadiazole in their structureswill reach high activity values through these structures they contain.After the six-step synthesis and characterization studies, activitystudies were started. Cytotoxicity experiments showed that the mostactive compounds were


The State of Arizona in the south-western United States supports a high diversity of insects. Digitised occurrence records, especially from preserved specimens in natural history collections, are an important and growing resource to understand biodiversity and biogeography. Underlying bias in how insects are collected and what that means for interpreting patterns of insect diversity is largely untested. To explore the effects of insect collecting bias in Arizona, the State was regionalised into specific areas. First, the entire State was divided into broad biogeographic areas by ecoregion. Second, the 81 tallest mountain ranges were mapped on to the State. The distribution of digitised records across these areas were then examined.Insecta, Coleoptera) of the Sand Tank Mountains is presented. The Sand Tanks are a low-elevation range in the Lower Colorado River Basin subregion of the Sonoran Desert from which a single beetle record was published before this study.A case study of surveying the beetles (Coleoptera from the Sand Tank Mountains, based on 914 digitised voucher specimens. These specimens add important new records for taxa that were previously unavailable in digitised data and highlight important biogeographic ranges.The number of occurrence records and collecting events are very unevenly distributed throughout Arizona and do not strongly correlate with the geographic size of areas. Species richness is estimated for regions in Arizona using rarefaction and extrapolation. Digitised records from the disproportionately highly collected areas in Arizona represent at best 70% the total insect diversity within them. We report a total of 141 species of Possible underlying mechanisms causing bias are discussed and recommendations are made for future targeted collecting of under-sampled regions. Insect species diversity is apparently at best 70% documented for the State of Arizona with many thousands of species not yet recorded. The Chiricahua Mountains are the most densely sampled region of Arizona and likely contain at least 2,000 species not yet vouchered in online data. Preliminary estimates for species richness of Arizona are at least 21,000 and likely much higher. Limitations to analyses are discussed which highlight the strong need for more insect occurrence data. Insects represent over half of all described species  and perhiNaturalist which have their own slightly different biases, limitations and strengths. We broadly consider fine-scale documentation of individual insects to be "collecting" for the purposes of this paper, though most of our recommendations are focused on traditional preserved-specimen-based collections.Biodiversity occurrence records represent an enormously important, invaluable and irreplaceable data source for understanding biodiversity, evolution and ecology . The vasThe goals of this study are twofold. First, we present an analysis of digitised insect occurrence data from the State of Arizona and compare the relative levels of sampling for different mountain ranges and ecoregions. Second, we address one example of an underexplored region and provide the first checklist of beetle species from the Sand Tank Mountains of central Arizona. We hope that these data and analyses can inform and bolster future insect collecting to improve our understanding of Arizona's biodiversity.Arizona encompasses a wide array of habitat types ranging from extreme deserts to mesic conifer forests. To efficiently classify these regions, different levels of the hierarchical ecoregions defined by 1Arizona can also be regionalised by its many mountain ranges. The Madrean Sky Islands are a series of discrete mountain ranges that arise from surrounding grasslands and are variously forested at their higher elevations Fig. 1. Beyond 11Outlines of the 81 mountain ranges in Arizona with the highest peaks were geographically mapped for use in this study and are shown in Fig. 11The Sand Tank Mountains, located in south central Arizona Fig. 1, cover aPantheraonca , family Felidae) record from 1930 which represents the south-western known limit of the species in the State and likely the furthest documented excursion of the species into the Sonoran Desert  portal contains 612,142 records using the same search parameters and the Symbiota Collections of Arthropods (SCAN) portal contains 683,645 records, nearly all of which are overlapping between the portals. The GBIF mediated data are further enhanced by their backbone taxonomy which is a synthetic management classification for the portal , differences in survey effort (e.g. one area may have been visited by 100 researchers a year and another area by 10 researchers per year) and differences in social practices and research interests . Insect occurrence records were, therefore, analysed according to three different metrics, namely records, collecting events and species. First, the total number of occurrence records for a given ecoregion or mountain range were tallied as a sum. Second, collecting effort was approximated by pooling records into putative collecting events. All insect records from a particular ecoregion or mountain range that had an identical date  and collector (dwc:recordedBy field) were considered to belong to a single collecting event. Third, putatively unique insect taxa were totalled for each ecoregion and mountain range by counting unique scientific names (dwc:scientificName field). These names correspond to the taxonomic interpretation according to the GBIF backbone taxonomy. This count may be considered an overestimate because different individuals of the same taxon may have been identified to different ranks  and be counted multiple times. However, because so many taxa at the species level are not known to the GBIF taxonomy, many differently identified taxa are prone to being 'lumped' into a higher classification level . For stuSampling effort to geographic area relationships were explored using linear regressions of both total occurrence records and tabulated collecting events to geographic area of regions (both for ecoregions and mountain ranges). A linear fit with high correlation would indicate that insect collecting was evenly distributed throughout space, while stronger departures from such a relationship would indicate confounding factors affecting the distribution of insect sampling across the State. There is some debate about what the most appropriate models are for species-area relationships and growing evidence suggests that very small, intermediate and very large areas operate under very different scaling parameters . DifferePossible factors responsible for underlying bias within the occurrence records were assessed using the R package sampbias  to examiSpecies richness within areas was estimated using the R package iNEXT  to perfoEcdysis portal built on the Symbiota software . Uncovered pitfall traps were set here and in a wide sandy wash 556E25A5-4CC2-5842-B2D8-25EA91D5084BIdentification reference: Abdullah (1964)Abdulluh, 19649DE05AE5-8AC0-532D-BFA4-273D28B930EAIdentification reference: Abdullah (1964)Horn, 1884ADEBC8E0-FD06-5ACC-939F-0EBFFE742546Identification reference: Chandler (1982)ED0ACD7E-3616-5B56-BD4A-62C2413FAE0DIdentification reference: Werner (1961)44EE7654-4228-5276-99EA-296FC2DDCFDDD5BB4323-283D-50E5-A43C-F56B9A0AE9DBIdentification reference: Fisher (1950)Horn, 187859153EBE-F94F-5C62-A02F-12C9B7C98254Identification reference: Fisher (1950)72BD7E41-A4A6-58CC-99E3-F512873F3346Identification reference: Arnett et al. (2002)1D8D04B9-919D-5075-9B4A-686C4CA9A001Identification reference: Fisher (1950)74D18D55-634F-5FF4-BA35-08BCBF7DCCC5Identification reference: Fisher (1950)4D1D0564-0000-5FAE-B184-2B75F488687DLeConte, 187494ADD790-C17C-5338-8CB6-B41A29EDFA4CIdentification reference: Costa et al. (2006)543D471A-7219-5B1D-8197-57B89736E24EFall, 19073F0D8D9D-FFF3-585F-9992-DEBEF358210FIdentification reference: 41295C3C-5841-595F-8BDE-14E7DFE688B4Identification reference: iNaturalistDC32ADD5-F707-5A46-B432-73F30E9F3E45Identification reference: Sloop (1937)C0590844-1327-59B3-943E-8828636BF14F12B1BF5D-B133-5685-A8F2-629D2EF9222FThis genus is in need of revision and we were unable to identify our specimens beyond genus.Casey, 1918D982D779-39A6-5F75-9167-A0707BDBE1ECIdentification reference: Lindroth (1963)LeConte, 185392B3741D-EA08-5146-8EFA-9D3F2EFD1A39Identification reference: Gidaspow (1959)Horn, 18711468E917-6936-53F4-884D-0346499AFC5DIdentification reference: Bell (1960)LeConte, 1851A1612B58-1D3C-556F-99B7-8231F0ED4232Identification reference: Horn (1882)Casey, 19145E5A1AC8-B1F7-58D3-B69F-6D9F8D14DF24Identification reference: Casey (1914)144769EF-D9E3-5942-8E6F-F0BBE0FD0E06Identification reference: Erwin (1974)D6A636DE-4F78-50C8-82FA-C9B9D0120B5EIdentification reference: Madge (1967)DB2496C0-2E7B-58F5-961B-C4B23AA1D0C7Identification reference: Noonan (1973)Van Dyke, 19259A61B35C-74B9-5D4B-814E-30B914071F1CIdentification reference: Whitehead (1972)Casey, 188496B0087C-7F02-5C20-87CC-8C29D4EA92A1Identification reference: Messer and Raber (2021)Schaeffer, 1910A9FD2932-D3E0-53C5-B4A6-CB95D81F4E81Identification reference: Messer and Raber (2021)Horn, 18686675B9A6-1DCA-5838-917D-C5DFCFE03CFEIdentification reference: Horn (1881)D2928EA4-E2BA-52D1-93A8-048B5572DA14404DC5A1-DBDE-5DDB-91C8-CE62FD95F49AIdentification reference: F.W. Skillman unpublished data.2EC2C75A-9A92-58F0-9842-8B25B4E38168Identification reference: F.W. Skillman unpublished data.5CD51E8C-788C-5582-B17F-02B8CF3B01E3Identification reference: F.W. Skillman unpublished data.Linsley, 194263BAF14E-32AB-5396-BFBD-CA02FD8322F8Identification reference: F.W. Skillman unpublished data.Fisher, 1920FD9D01F7-9B68-52AD-B9C3-86430C11E428Identification reference: F.W. Skillman unpublished data.B6D95084-AF19-5BD9-B7AB-B772E6DE9A49Identification reference: F.W. Skillman unpublished data.Fall, 1929F4FAD1C4-4CD1-5EAD-9AF8-BE8794E5B280Identification reference: F.W. Skillman unpublished data.CB2F6A6C-267B-5D9A-BBA4-10882537F753Identification reference: F.W. Skillman unpublished data.75627271-F5EF-5D7F-82D7-374F0A537F5DIdentification reference: F.W. Skillman unpublished data.FE15B802-4C44-5816-82D0-A848D899B9F5Schaeffer, 193364A8BF95-1A06-5281-A9A2-7F1174384144Identification reference: Blake (1976)68C32E26-AB47-54C1-BC50-6D70DF036AB0This genus needs revision and we were unable to identify our specimens beyond genus.9564A5AC-A180-5A3B-8A8C-E2161C641A6BIdentification reference: Moldenke (1970)084AA5D4-4C37-5422-B2AC-CFC4745B5695Identification reference: TRACY and ROBBINS (2009)ED88B8C9-384B-5385-AEBA-4D43C2ACD3DAThis genus needs revision and we were unable to identify our specimens beyond genus.Fall, 1915923E5AB1-0EEB-5E42-AEF5-D0B39D40BAA9Identification reference: Barney (2019)Bowditch, 19091C36C4C7-F759-5838-B629-786C16E0FE8FIdentification reference: Fall (1915)Fall, 1915F71EC8E7-CCE7-584B-AE83-59EB9EE19CFCIdentification reference: Barney (2019)Bowditch, 190952002F17-5CAF-5669-9B48-DF132BAF7B2CIdentification reference: Barney (2019)Crotch, 1873FD29851E-63CF-54ED-B246-3D2056B7E924Identification reference: Barney (2019)LeConte, 1857BB59A861-5348-583F-8940-F3BD4F13B4B7Identification reference: Moldenke (1970)BA894C10-BDE8-5DC5-8A0B-35C4C497C525E476C649-DCCC-5BDC-9A8B-757BA7CB732DIdentification reference: B.H. Reily unpublished data.Schaeffer, 1904BDC25CC1-656F-52BC-B8BD-01AE02E10B9BIdentification reference: B.H. Reily unpublished data.Horn, 18768A1BE50C-D93F-58FB-82A0-A32F45DE8A8CIdentification reference: B.H. Reily unpublished data.Leconte, 1852E00F2696-3D98-5A16-9958-213D33E1E496Identification reference: B.H. Reily unpublished data.1DE00017-E9EE-5A22-B2F0-5ED12C45794BIdentification reference: B.H. Reily unpublished data.Wolcott, 1927B8924B82-45EF-5898-ADAC-A9FD60F1E0CBIdentification reference: B.H. Reily unpublished data.CFE93839-BB5E-504F-BDD5-27768617EB20Identification reference: B.H. Reily unpublished data.FF55D192-DA90-5F8F-B3CC-8F3DFF8F43D0Casey, 18990248D2CE-31F3-50CD-B46F-6C0C9F6C9A1CIdentification reference: Gordon (1985)16D20865-5784-5F97-993D-F50F9265BFFFScymnus (Pullus), of which there are a number of species known from this region.Identification of this genus requires examination of male terminalia. Our single putatively female specimen was only identified to the subgenus F9F98C8D-0787-58BD-B0C2-81A880FEBC89Van Dyke, 19361FEF5503-BA54-50E3-B531-2B97AF1F7F2DIdentification reference: Pelsue and Sleeper (1972)C6F9AB97-6A4F-581A-AC1E-BC365BB12828This diverse genus is difficult to identify without genitalic dissections and we were unabe to identify our specimen to species.5A16EF68-9108-549D-AEAE-DBEE8E6CE87BIdentification reference: Morrone and Cuevas (2002)A45D0113-EBDF-56E4-925B-5DFE05895964Identification reference: Clark (1978)659D2D8E-B677-544A-AD7E-14B66887F80EIdentification reference: Clark (1978)075D76CA-278D-5C51-B062-62A2031D8C3EThis speciose genus is difficult to identify and we were unable to identify our single specimen beyond genus.DBE5116C-4EF8-59B5-AF04-99A67F441892Horn, 18949B7B87BE-6067-5DEE-9BA5-33E3749F27BEIdentification reference: Johnston and Gimmel (2020)06BB1F55-5478-5100-89F3-8400BD41E36CLeConte, 1854746DEC1C-94E7-5972-9535-447486950F20Identification reference: Beal (1998)41388122-8EB8-5F0D-A13F-93E267686A0557833888-4F01-5023-8EF9-FF61BD0DC2B5Identification reference: Miller (2002)A8D46359-8E4D-5070-810A-B4383F62DEE3DB3DD9A7-ECE8-540E-BD30-D3C728D49D64Identification reference: Arnett (1952)LeConte, 186651382046-AAD4-535D-B70F-75ED0D7D6326Identification reference: Van Dyke (1932)Horn, 1884E014B0D5-DED6-5184-ABF4-E5365FD133E6Identification reference: Horn (1884)Van Dyke, 193370B3D25E-09CF-53E7-BD53-45B7ABB371FBIdentification reference: Wells (2000)LeConte, 186335B52708-FE26-5B32-A082-44E8A8653F29Identification reference: Wells (2000)7F8F22D6-54AD-5643-A912-CDB9208A2F17Identification reference: B. Mathison unpublished data.672FCEB9-E0E6-5FFB-ABC7-83F09801D5BB26D8F485-4A6D-5940-B20A-D5986D61F022Identification reference: W.B. Warner unpublished data.E59B4134-D477-5102-ADC6-4F3DC2B64B2CIdentification reference: W.B. Warner unpublished data.F494A362-59ED-56B6-BB6E-25513AC714013BCC05C8-3458-5AEA-B1E6-5D6434A643A6Identification reference: Werner et al. (1966)Werner, 194903759E26-447F-5239-8295-0B5BC36F8EFDIdentification reference: Werner et al. (1966)4B845E05-5AF1-500A-A7D9-F0CA4F757E4FIdentification reference: Werner et al. (1966)2994E3C5-B23F-5B0D-AA67-7EFE4F5719B9Identification reference: Werner et al. (1966)Horn, 1894B2D571A7-36D4-52EB-A7A2-F41AEE4A79EEIdentification reference: Werner et al. (1966)7986BF5F-7F50-52F3-900D-8259B4C4436DFall, 19174CEF76F9-F368-5BB0-A816-0E6DFA443D24Identification reference: Marshall (1951)A3809ABC-8830-513D-AE31-A8C1C08FCFD4Identification reference: M.L. Gimmel unpublished data.Casey, 18952D3C39C8-0E0B-50AC-A351-F7AEC2161BE6Identification reference: M.L. Gimmel unpublished data.460467DE-C8CD-5A27-AFCF-ACCBF2BB50149D76D9E6-D421-578A-BBAB-4042DB4DCE84Mordellinatestacea  - a species only reported from the eastern United States.This genus has limited identification resources available. Our two specimens resemble 60732CED-843C-5110-BF65-01DF6C758CA362EECD02-029A-5D4C-B66A-8CFF6D031B81Identification reference: Arnett et al. (2002)98889A98-C8D5-5381-8E14-B87F1D921B1FC9D22D30-3385-51CF-9A6F-C5E18ACC9F1EIdentification reference: Arnett (1951)Horn, 1896019B0571-13AF-538B-805E-F20F6E125DD0Identification reference: Arnett (1951)4769596F-149E-5347-9A8F-DCE483853E9DIdentification reference: Arnett (1951)7F996EDB-A72E-5FA4-B9AA-56D8F62EED8FOxycopis species likely represent an undescribed species which we were unable to associate with any currently known from the western United States.A moderate series of this BBA07FCB-13C8-5C07-A0E6-6243F66884EB3D1F2C5A-A762-50F8-9D44-C9FF78C77B23Identification reference: Zaragoza Caballero (1986)7A2C428A-2E5F-5636-922F-14717066F3CDLeConte, 18591827E3D0-B40D-5E85-BB6F-DA74E7373B3AIdentification reference: Aalbu and Andrews (1992)Pic, 1904F9C85703-5510-56BC-AA04-037EA4318257Identification reference: Papp (1962)0049847F-36B7-5DB1-B044-585FFD254E66Tricorynuslentus .This speciose genus is in need of a modern revision. Our single specimen has elytra that lack discernible striae and may be near F850DD1B-5D15-547A-94D5-AA86DEE2F780Van Dyke, 19393012C7AC-E344-5E45-BCF7-05407807A0AEIdentification reference: Van Dyke (1939)D6F87308-AA02-5F49-83E3-26F9B8F1C844LeConte, 1858D02ACFDD-A798-59C9-A918-56465900A32DIdentification reference: Aalbu et al. (2005)43465072-5D42-5F34-B5D5-1E0024FBE770Brown, 19299E3E930E-FE41-519F-BAB3-7173D59B30C5Identification reference: W.B. Warner unpublished data.Horn, 18712606E611-4459-57A1-B56B-47302106810AIdentification reference: W.B. Warner unpublished data.Horn, 1871F1FD620C-BB9B-5011-99CE-7B3DEF27D33BIdentification reference: W.B. Warner unpublished data.Horn, 1871ADA0E144-76B2-5E37-A3DA-304FFA1F6984Identification reference: W.B. Warner unpublished data.Fall, 1909E5C6193E-B3A5-5A70-8D76-66D6C90B153CIdentification reference: W.B. Warner unpublished data.LeConte, 1856058EE653-F3BD-5996-A2AF-346375D480B8Identification reference: W.B. Warner unpublished data.Fall, 190920728724-F8BF-5049-B123-47D01BAFCD50Identification reference: W.B. Warner unpublished data.Fall, 19094092E1F7-1867-51AA-8814-B1B415CEE252Identification reference: W.B. Warner unpublished data.97CFCF65-B7A0-5946-8637-74E0C7260D70Identification reference: W.B. Warner unpublished data.AA94F8BE-0DA8-5925-B0A4-5DA1807CA488Identification reference: W.B. Warner unpublished data.D32801FA-FA35-52C2-9E84-00EAE1D62B92Identification reference: W.B. Warner unpublished data.7C0DFA9B-5636-5F55-8A39-1ED3E6E334E134D22EAB-F896-5736-990E-5C881DB7D8B5This genus lacks a comprehensive key to species, but genitalic dissections indicate that our series of specimens likely represent an undescribed species.Liljeblad, 191816CAF229-1C6F-56EC-8982-105BD06CBC3EIdentification reference: Liljeblad (1945)46409E3D-4069-556D-8DEC-7CF76B84E86BIdentification reference: Liljeblad (1945)33AA5FD2-EAA1-541C-A935-0BDF5DEE3700D4159739-69D6-5494-87DE-9BD54C900656Ahasverusrectus , but differ in several characters from the holotype of that species. We have seen conspecific specimens to ours labelled as "Ahasverus n.sp." in collections and think it is likely that it is, indeed, an undescribed species.Our specimens somewhat resemble 6C5ABF1D-5C48-511F-8223-49D7BCB5F33830C154DB-2220-50F2-87B8-FDA50CFAEF85We were unable to identify our single specimen of this species beyond the level of genus in this speciose group.2F91223E-3060-51C1-A034-86E43910BF0FCasey, 1891C14DF586-636B-5346-B9C5-5BBD84F55E2CIdentification reference: M.A. Johnston unpublished data.LeConte, 18515B5C7FB8-0683-5600-8A16-D1BB66CF25B0Identification reference: Doyen (1987)Horn, 18908E907738-172D-5015-97DF-15919066181EIdentification reference: Papp (1981)Horn, 1870C3B93157-CF3E-5FCE-89AF-FB5CABDB22B7Identification reference: Papp (1981)384F5C44-B87A-54B7-A359-0588EACBAB70Identification reference: Berry (1980)B2E50530-D8F9-5065-A52B-308959080B5CIdentification reference: Aalbu (2005)1EFE660A-59FD-5833-8480-38FB6052A935Identification reference: Doyen (1987)5F324B4A-851D-5276-B762-F69889691C48Identification reference: MacLachlan and Olson (1990)LeConte, 18661280A39C-60A2-57DA-8A87-CFFD1E69C9A3Identification reference: M.A. Johnston unpublished data.LeConte, 18517F8F9A08-D6BA-50EF-B94C-D1CA19460E00Identification reference: Arnett et al. (2002)CDDB6901-CD28-532D-BBE7-0D0A98AA6A7DIdentification reference: Aalbu (2005)613D250E-BED6-53D7-B1A3-4EF903944C7BIdentification reference: Aalbu (2005)LeConte, 1851AEE14280-EE76-5D35-878D-C3C8BC26C507Identification reference: Doyen (1968)LeConte, 1851682CC6C1-647A-5F95-ACD8-EC2D6D507510Identification reference: Johnston et al. (2015)Thomas, 2005B68460A5-FD34-57E5-B684-7E6277F244F9Identification reference: Johnston (2016)149F3F34-C10A-5A53-8C78-00AC3D675775Identification reference: Spilman (1959)Eschscholtz, 18319C41B1C7-DFBA-5498-8F26-010391111D24Identification reference: M.A. Johnston unpublished data.LeConte, 18513D3A992F-3ADB-5C4C-82FC-6D83591EE858Identification reference: Doyen (1984)Fall, 1931A6FF3988-BFEB-57A1-A761-A5323E65F897Identification reference: Fall (1931)LeConte, 186671D63690-DBEE-59EA-84DB-335C642CC72AIdentification reference: Fall (1931)Horn, 1894BAA53322-0722-5F1A-AB73-37CB7C65666BIdentification reference: Fall (1931)Waterhouse, 1880AFDF615F-7F43-50C1-B94A-8917F38B0D67Identification reference: Arnett et al. (2002)Casey, 1895F27E356E-D4B8-5362-9B2F-BE90638AAB5FIdentification reference: M.A. Johnston unpublished data.A721C9D2-A5FE-53DB-AE9B-EE8A41033C43Identification reference: M.A. Johnston unpublished data.Schaeffer, 1905CE60184A-9B50-52AA-8425-AE8217617630Identification reference: Parsons (1975)Horn, 18882CBB4229-416C-5A42-B432-1856447B7856Identification reference: Parsons (1975)8DAAA19C-9C0F-57C2-BC7C-F6614E9DA41CIdentification reference: M.A. Johnston unpublished data.A813A5C8-CA37-58DB-8EB4-0812C89DA5C9Identification reference: M.A. Johnston unpublished data.LeConte, 1851D1262B1E-EE42-5756-8C7D-AB15107A0CB5Identification reference: M.A. Johnston unpublished data.BFE4EC01-8C02-595A-BB98-67F6E39E5693Identification reference: M.A. Johnston unpublished data.4E73616A-9186-55DD-9069-EEBC996D0639Loomis, 1922D76BA4BB-4887-50E3-9ED0-ED3287DA4F9FIdentification reference: W.B. Warner unpublished data.9F2F5549-9F14-5AD0-A1FA-167FDD2BE4A5BAB8BD68-4224-5628-9EF4-931BAEE9AE1DIdentification reference: Barron (1971)94C38543-DFB4-51AC-A451-882022EBAD80Horn, 18723C544C6A-BE65-5672-A42F-30DF0F85FB4EIdentification reference: Freude (1955)Horn, 1867AD592433-0514-5662-B0ED-4145289DD199Identification reference: Stephan (1989)The checklist provided herein significantly raises the entomological knowledge of this mountain range. Our collecting efforts unfortunately were comprehensively limited as they did not include sampling during the peak flowering season that typically occurs between late February and April or in the winter which has a distinct insect fauna that often does not overlap with the taxa found during the warmer times of year. We also were unable to access a number of distinct habitats, including the relictual chaparral plant communities, that likely would have greatly increased our taxon count.Diclidiagreeni, Horistonotuslutzi, Mulsanteusarizonensis, Niptusventriculus, Oxycopismariae and Ptinuspaulonotatus). Many more represent the second digitised record or the first preserved specimen, as opposed to a human observation, from the State. These are notable in that they demonstrate specific examples of how digitised records both fall short of representing the full knowledge of the State's fauna, as well as the limited distributional information available for many species. It is also notable that three collecting events produced likely three undescribed species . Our specimens of Asbolusmexicanusangularis are the first reported from Maricopa County in Arizona and represent a roughly 50 mile (ca. 75 km) north-east range extension of that species. Many other species we report may represent additional notable range extensions, though the limited knowledge and digitised specimens from the region hinder more in-depth analyses.Many of the species reported from this study occur throughout the Lower Colorado River Basin subregion of the Sonoran Desert, but are often poorly represented in natural history collections or in digitised occurrence records. Six species recorded by us had no prior digitised records from the State of Arizona even though they are known in literature  richness failed to plateau and provided very similar results that only 70% of the full estimated species richness were observed. As all the analyses across the three scales explored here gave these similar proportional results, it seems clear that the estimates are strongly affected by incomplete sampling. We again urge readers to be cautious with the absolute numbers presented here. However, given our knowledge of the Sand Tank Mountains The analyses presented here clearly demonstrate that, according to available data, insect collecting has not been done evenly throughout the State. The underlying factors that drive the biases seen in the data are likely numerous and difficult to fully ascertain. We hypothesise that two of the primary drivers are habitat accessibility and social interactions.Habitat access for insect collectors is very important and has many facets. Proximity to roads and populations centres is clearly important, but not the only limiting factor and not all cities and roads are the same. For example, the Chiricahua Mountains have roads accessible to passenger cars that go to the highest elevations. The Mountains are almost entirely on public lands and there are nearby towns with accommodation and stores, as well as a popular research station. In contrast, all sites visited in the Sand Tank Mountains involved rugged back-country roads requiring high clearance and four-wheel drive vehicles in areas where it is unlikely to encounter other people in the event of an emergency. Habitat access is not equal at the large scale of ecoregions either. The majority of the Madrean Archipelago is covered by public lands (U.S. Forest Service and Bureau of Land Management) while large swathes of the Sonoran Desert and especially the Arizona/New Mexico Plateaus regions are Native American Reservations. Different sovereign tribal lands, private property and various public land management agencies all have unique regulations and permitting processes which affect collecting insects. The Mohave Basin and Range ecoregion is an interesting example of how these factors interact where most of the lands are public, but the terrain is very rugged and roads and population centres are limited which is likely why there are so few insect records from the region even though there are no major permitting restrictions.The Patagonia Picnic Table Effect , named aThe full scale of insect diversity has been under-documented for the State of Arizona, its constituent ecoregions or even its most popular mountain ranges, at least in available online data. It is important that the entomological community continues to survey for and collect insects everywhere in the State. Continuing and increasing efforts to mobilise specimen data from natural history collections also remains a high priority and will likely help to account for many species which are not currently represented in online data. It is estimated that not more than 5% of specimens in insect collections of the United States have been fully digitised . It is pWe urge collectors to make a concerted effort to go to new places and consider targeting specifically undercollected regions and mountain ranges. Small and targeted studies can exponentially improve our understanding of Arizona insect fauna and are likely the best way to increase knowledge of species distributions and may be crucial to understanding the entire State fauna. Our example of the Sand Tank Mountains beetles highlights how a modest collecting effort can still provide new occurrence records for species from the State and report on new localities for taxa that are otherwise considered rare in collections. We do not recommend that collectors avoid the classic and popular sites; indeed, we still need to sample those, but we would advocate that entomologists consider dividing their time in the field and only spend part of their efforts in the well-known habitats and spend the next day somewhere new.The paucity of insect data from so many mountain ranges in the State strongly limit our ability to adequately protect and conserve insect biodiversity. Entomologists and insect collections should partner with local, State and federal land management agencies to increase insect sampling throughout the State. Increasing partnerships and professional connections with tribal nations within the State are also strongly recommended. Opportunites for occurrence-data driven estimates for species diversity are in their infancy, even for a biodiversity hotspot that is accessible and popular. Nevertheless, the growing availability of occurrence data is an important resource to continue to develop to understand the diversity and distributions of insects.0A29E9FB-A9B6-550B-8E0F-58C1F5748A7610.3897/BDJ.11.e101960.suppl1Supplementary material 1Arizona insect occurrence data annotated by which ecoregion or mountain range they occur withinData typeoccurrencesBrief descriptionZip archive of two occurrence recordsets for Arizona insects. The first file contains occurrence records annotated by which ecoregion they fall within and the second file contains annotated records by which mountain range they fall within.File: oo_777372.ziphttps://binary.pensoft.net/file/777372M.A. Johnston67767A50-A46D-5AD6-8037-12364431B6E710.3897/BDJ.11.e101960.suppl2Supplementary material 2Arizona ecoregion dataData typeGeographical and biodiversity metrics of ecoregionsBrief descriptionThis table includes data for all Arizona ecoregions including their geographic size and the number of insect records, collecting events and taxa found within them.File: oo_809256.csvhttps://binary.pensoft.net/file/809256M.A. JohnstonC849E347-8C8F-5F75-B79E-C297E73DBD1D10.3897/BDJ.11.e101960.suppl3Supplementary material 3Arizona mountain range dataData typeGeographic and biodiversity metrics for mountain rangesBrief descriptionThis table includes information about the tallest mountain ranges in Arizona. A polygon in WKT format, the geographic area, prominence and height of each mountain range is given. Totals for insect occurrence records, collection events and taxa are also given.File: oo_777374.csvhttps://binary.pensoft.net/file/777374M.A. JohnstonB1AD592F-633E-52FC-BC3E-65E235D27A6610.3897/BDJ.11.e101960.suppl4Supplementary material 4Coleoptera specimen recordsSand Tank Data typeoccurrencesBrief descriptionDarwin Core Archive of new recordsFile: oo_711180.ziphttps://binary.pensoft.net/file/711180M.A. Johnston20F9779E-8E23-5849-ACA1-2935A775DFCA10.3897/BDJ.11.e101960.suppl5Supplementary material 5Data analysis and figure generation scriptData typeR script used for data analysis and for generating all the statistical figures used in this paper.File: oo_806970.Rhttps://binary.pensoft.net/file/806970M. Andrew JohnstonDDC6B965-787C-58CB-987F-A1365D78AF9410.3897/BDJ.11.e101960.suppl68098911Supplementary material 6Additional analyses for normality and log-transformed dataData typestatistical graphsBrief descriptionThis file contains graphical analyses to test the Arizona insect occurrence dataset for normality across mountain ranges and Ecoregions. Additional graphs of log-transformed data are included for untransformed analyses presented in the main text.File: oo_846719.pdfhttps://binary.pensoft.net/file/846719M. Andrew Johnston


Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum, and two pathogenic yeasts: Candida albicans and Cryptococcus neoformans var. grubii. A number of S-alkyl substituted thioglycolurils exhibited high activity against Venturia inaequalis and Rhizoctonia solani (85–100% mycelium growth inhibition), and moderate activity against other phytopathogens. S-Ethyl substituted thioglycolurils possessed a high activity against Candida albicans. Additionally, the hemolytic and cytotoxic properties of promising derivatives were determined using human red blood cells and human embryonic kidney cells, respectively. Two S-ethyl derivatives possessed both low cytotoxicity against normal human cells and high fungicidal activity against Candida albicans.A series of S-alkyl substituted thioglycolurils was prepared through the alkylation of corresponding thioglycolurils with halogenoalkanes and tested for their fungicidal activity against six phytopathogenic fungi from different taxonomic classes:  Aspergillus spp., Candida spp., and Cryptococcus spp. -5-thioxohexahydroimidazoimidazol-2(1H)-one (4-[(E)-((E)-3-phenylallylidene)amino]thioglycoluril) and evaluated their fungicidal activity. S-allyl thioglycolurils were synthesized to reveal the influence of the unsaturated fragment on the activity.Recently , we haveiana see . It was 4a–f, 5a–f, and 6a–h is outlined in 7a,b were synthesized via the cyclisation of 4,5-dihydroxyimidazolidin-2-ones 8a,b with thiosemicarbazide -5-propylthio-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (4a).1,3-Dimethyl-4-+: 358.1696; found: 358.1695.E,2E)-3-phenylallylidene)amino]-5-propylthio-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (4b).1,3-Diethyl-4+: 386.2009; found: 386.2006.E,2E)-3-(2-nitrophenyl)allylidene]amino}-5-propylthio-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (4c).1,3-Dimethyl-4-{+: 403.1547; found: 403.1541.E,2E)-3-(2-nitrophenyl)allylidene]amino}-5-propylthio-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (4d).1,3-Diethyl-4-{+: 431.1860; found: 431.1855.E,2E)-3-(2-Methoxyphenyl)allylidene]amino}-1,3-dimethyl-5-propylthio-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (4e).4-{+: 388.1802; found: 388.1811.E,2E)-3-(2-methoxyphenyl)allylidene]amino}-5-propylthio-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (4f).1,3-Diethyl-4-{+.E,2E)-3-phenylallylidene)amino]-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (5a).5-Allylthio-1,3-dimethyl-4-+: 356.1540; found: 356.1546.E,2E)-3-phenylallylidene)amino]-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (5b).5-Allylthio-1,3-diethyl-4-+: 384.1853; found: 384.1847.E,2E)-3-(2-nitrophenyl)allylidene]amino}-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (5c).5-Allylthio-1,3-dimethyl-4-{+: 401.1390; found: 401.1388.E,2E)-3-(2-nitrophenyl)allylidene]amino}-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (5d).5-Allylthio-1,3-diethyl-4-{+: 429.1703; found: 429.1695.E,2E)-3-(2-methoxyphenyl)allylidene]amino}-1,3-dimethyl-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (5e).5-Allylthio-4-{+: 386.1645; found: 386.1640.E,2E)-3-(2-methoxyphenyl)allylidene]amino}-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (5f).5-Allylthio-1,3-diethyl-4-{+: 414.1958; found: 414.1951.E,2E)-3-phenylallylidene)amino]-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6a).5-Butylthio-1,3-dimethyl-4-+: 372.1853; found: 372.1852.E,2E)-3-phenylallylidene)amino]-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6b).5-Butylthio-1,3-diethyl-4-+: 400.2166; found: 400.2164.E,2E)-3-(2-nitrophenyl)allylidene]amino}-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6c).5-Butylthio-1,3-dimethyl-4-{+: 417.1703; found: 417.1701.E,2E)-3-(2-nitrophenyl)allylidene]amino}-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6d).5-Butylthio-1,3-diethyl-4-{+: 445.2016; found: 445.2013.E,2E)-3-(2-methoxyphenyl)allylidene]amino}-1,3-dimethyl-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6e).5-Butylthio-4-{+: 402.1958; found: 402.1954.E,2E)-3-(2-methoxyphenyl)allylidene]amino}-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one . Yield 408 mg (95%); white solid; mp: 108–110 °C. IR (KBr): ν 3072, 3044 (Ar), 2960, 2932 (Alk), 2872 (C-S), 2022, 1899, 1824 (Ar), 1699, 1562  cm–1. 1H NMR : δ 0.90 , 1.01 , 1.15 , 1.30–1.50 , 1.55–1.75 , 2.90–3.10 , 3.15–3.30 , 3.40–3.55 , 3.87 , 5.67 , 5.95 , 6.90–7.01 , 7.07 , 7.20–7.40 , 7.67 , 7.85 . 13C NMR : δ 13.44 (2Me), 13.90 (Me), 21.37 (Me), 29.58, 30.87 (SCH2), 36.27 (NCH2), 37.64 (NCH2), 55.51 (OMe), 70.34 (CH), 78.59 (CH), 111.51, 120.73, 124.27, 125.75, 127.06, 129.99, 132.16 , 141.33 (HC=N), 156.67 (ArC-OMe), 157.08 (C=O), 165.22 (C=N). HRMS (ESI): Calculated for C22H31N5O2S [M + H]+: 430.2271; found: 430.2279.E,2E)-3-(furan-2-yl)allylidene]amino}-1,3-dimethyl-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6g).5-Butylthio-4-{+: 362.1645; found: 362.1647.E,2E)-3-(furan-2-yl)allylidene]amino})-3,3a,4,6a-tetrahydroimidazoimidazol-2(1H)-one (6h).5-Butylthio-1,3-diethyl-4-{+: 390.1958; found: 390.1945.Venturia inaequalis (V. i.), Rhizoctonia solani (R. s.), Fusarium oxysporum (F. o.), Fusarium moniliforme (F. m.), Bipolaris sorokiniana (B. s.), and Ssclerotinia sclerotiorum (S. s.). The effect of the chemicals on mycelial radial growth was determined by dissolving a concentration of 3 mg mL−1 in acetone and suspending aliquots in potato-saccharose agar at 50 °C to give a concentration of 30 µg mL−1. The final acetone concentration of both fungicide-containing and control samples was 10 mL L−1. Petri dishes containing 15 mL of the agar medium were inoculated by placing 2 mm mycelial agar discs on the agar surface. Plates were incubated at 25 °C and radial growth was measured after 72 h. The mixed medium without a sample was used as the blank control. Three replicates of each test were carried out. The mycelium elongation diameter (mm) of fungi settlements was measured after 72 h of culture. The growth inhibition rates were calculated with the following equation: I = [(DC − DT)/DC] × 100%. Here, I is the growth inhibition rates (%), DC is the control settlement diameter (mm), and DT is the treatment group fungi settlement diameter (mm). The results are summarized in The fungicidal activities were tested according to the conventional procedure ,24,25,26530), while the growth inhibition of C. neoformans was determined by measuring the difference in absorbance between 600 and 570 nm (OD600–570), after the addition of resazurin  and incubation at 35 °C for an additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The minimum inhibitory concentration (MIC) was determined following the CLSI guidelines, identifying the lowest concentration at which the full inhibition of the fungi was detected. Full inhibition of growth was defined at <=20% growth (or >80% inhibition), and concentrations were only selected if the next highest concentration displayed full inhibition  as well (eliminating ‘singlet’ active concentration). In addition, the maximal percentage of growth inhibition was reported as DMax, indicating any compounds with marginal activity. Compounds were plated as a 2-fold dose response from 32 to 0.25 μg/mL, with a maximum of 0.5% DMSO in the final assay concentration. Samples with MIC <= 16 μg/mL were declared as a hit.Preliminary antifungal screening and MIC determination assays were carried out at the University of Queensland following reported methods ,30,31. F2. Growth inhibition of HEK293 cells was determined by measuring fluorescence at ex: 530/10 nm and em: 590/10 nm (F560/590), after the addition of resazurin  and incubation at 37 °C for an additional 3 h in 5% CO2. The fluorescence was measured using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (cell culture without inhibitors) on the same plate as references.Cytotoxicity assays were carried out at the University of Queensland following reported methods ,30,31. H50 (concentration at 50% cytotoxicity) was calculated by curve fitting the inhibition values vs. log (concentration) using the sigmoidal dose–response function, with variable values for bottom, top, and slope. The curve fitting was implemented using Pipeline Pilot’s dose–response component (giving similar results to similar tools such as GraphPad’s Prism and IDBS’s XlFit). Any value with >indicates a sample with no activity  or samples with CC50 values above the maximum tested concentration .CC50 ≤ 32 µg mL−1 in either replicate (n = 2 in different plates). Cytotoxic samples were classified using CC10 (Concentration at 10% haemolytic activity) and HC50 (concentration at 50% haemolytic activity) were calculated by curve fitting the inhibition values versus log (concentration) using a sigmoidal dose–response function with variable fitting values for top, bottom, and slope. The curve fitting was implemented using Pipeline Pilot’s dose–response component (giving similar results to similar tools such as GraphPad’s Prism and IDBS’s XlFit). Hemolytic activity assays were carried out at the University of Queensland using human red blood cells (RBC) following reported methods ,30,31. H–5 mol L–1) -3,3a,6,6a-tetrahydroimidazoimidazol-2(1H)-one 3a and 5-ethylthio-6-{(E)-[(E)-3-(furan-2- yl)allylidene]amino}-1,3-dimethyl-3,3a,6,6a-tetrahydroimidazoimidazol-2(1H)-one 3g exhibited low toxicity to the normal human cell lines, while still showing high fungicidal activity. Further research should focus on in vivo assessment of the fungicidal activity of S-alkyl thioglycolurils and clarification of the mechanism of their action.The fungicidal activity of a series of S-alkyl substituted N-aminothioglycolurils was studied, and structure–activity relationships were established. Compounds exceeding or comparable to the well-known fungicidal agent triadimefon were identified. The S-alkyl-N-aminothioglycolurils with an unsubstituted phenyl or furyl ring were the most effective at inhibiting the growth of phytopathogenic fungi. Inhibitory activity increased with an increase in the length of the alkyl chain at the S atom. Compounds containing an ethyl substituent at the S atom demonstrated elevated activity against yeast


Staphylococcus epidermidis (S. epidermidis) strains. The Minimum Inhibitory Concentration (MIC) studies showed that the complex of CIP-Ad and 2,3-O-dimethyl-β-CD exhibited a similar antimicrobial activity to CIP-Ad alone, while the interaction with 2,3,6-O-trimethyl-β-CD increased MIC values. Antimicrobial assays on S. epidermidis biofilms demonstrated that the synergistic effect observed with the DspB/CIP association was partly maintained with the 2,3-O-dimethyl-β-CDs/CIP-Ad complex. To obtain this “all-in-one” drug delivery system, able to destroy the biofilm matrix and release the antibiotic simultaneously, we covalently grafted DspB on three carboxylic permethylated CD derivatives with different-length spacer arms. The strategy was validated by demonstrating that a DspB-permethylated-β-CD/ciprofloxacin-Ad system exhibited efficient antibiofilm activity.To address the high tolerance of biofilms to antibiotics, it is urgent to develop new strategies to fight against these bacterial consortia. An innovative antibiofilm nanovector drug delivery system, consisting of Dispersin B-permethylated-β-cyclodextrin/ciprofloxacin adamantyl (DspB-β-CD/CIP-Ad), is described here. For this purpose, complexation assays between CIP-Ad and (i) unmodified β-CD and (ii) different derivatives of β-CD, which are 2,3-O-dimethyl-β-CD, 2,6-O-dimethyl-β-CD, and 2,3,6-O-trimethyl-β-CD, were tested. A stoichiometry of 1/1 was obtained for the β-CD/CIP-Ad complex by NMR analysis. Isothermal Titration Calorimetry (ITC) experiments were carried out to determine Ka, ΔH, and ΔS thermodynamic parameters of the complex between β-CD and its different derivatives in the presence of CIP-Ad. A stoichiometry of 1/1 for β-CD/CIP-Ad complexes was confirmed with variable affinity according to the type of methylation. A phase solubility study showed increased CIP-Ad solubility with CD concentration, pointing out complex formation. The evaluation of the antibacterial activity of CIP-Ad and the 2,3-O-dimethyl-β-CD/CIP-Ad or 2,3,6-O-trimethyl-β-CD/CIP-Ad complexes was performed on  S. epidermidis is a major infective agent in compromised patients, such as drug abusers or immunocompromised patients . The ability to adhere and subsequently form biofilms on indwelling devices is among the potential virulence factors associated with S. epidermidis species t) in the aqueous medium (Equation (1)):0 is the intrinsic solubility of CIP-Ad  and K1/1 is the apparent stability constant of the drug/CD complex. The plot of St versus [CD]t gives a straight line with a slope K1/1 S0/(1 +K1/1 S0)) less than unity and an intercept (Sint) equal to S0. Next, K1/1 can be determined from the slope and S0 according to Equation (2):CIP-Ad fication , the solEquation (2) according to Loftsson et al. .0 for all CDs and a negative value was obtained for host CD IV. This negative deviation from linearity at low CD concentration , reported as the A-L-type phase-solubility profile in the literature, is currently observed for poorly soluble drugs (S0 < 1.10−4 M) -, M = 1541.75 g/mol−1.; IR ν(cm−1): ring vibration 946 cm−1, (C-C/C-O) 1032–1079,(C-H/O-H) ν = 1297–1449,(CO-NH) 1731,(CO-OH) 1774, (CH2) 2931, (N-H) 3405. TLC: Rf 0.052 (DCM/MeOH:95/5); mp (°C): 138; RMN 1H : δ (ppm) 1.60–1.61 2-CH2-COOH), 2.15–2.25 , 3.13 , 3.31 , 3.34 , 3.43 , 3.54 , 3.56 , 3.73–3.75 ; RMN 13C : δ (ppm) 24.5–25.2 (-CH2-(CH2)2-COOH), 33.3 (-CH2-COOH), 36.1 (-NH-CO-CH2-COOH), 39.9 (-CH2-NH-CO-), 58.5 (-OCH3), 58.9 (-CH2-OCH3), 61.8 (-CH-OCH3), 70.8 (-CH-O-CH), 71.3 (-CH2-OCH3), 80.3 (-CH-CH-OCH3), 81.5 (-CH-CH-OCH3), 81.7 (-CH-CH-OCH3), 98.5–98.9 (-CH-O-); ESI-MS: calcd for C68H119NO37 1540,75. Found 1540.73 [M-H]-; HR-MS: TOF-MS-ES- This compound was obtained as the same procedure described above. The compound 4  was added to the compound b . The desired product CD3 was obtained as white solid .m/z = 1625, 84 [M-H]-, mass found m/z = 1625.0 [M-H]-; IR ν(cm−1): ring vibration 946 cm−1, (C-C/C-O) 1032–1079, (C-H/O-H) 1243–1436, (CO-NH) 1574, (-CO-OH) 1628; (CH2) 2849–2928, (N-H) 3329; HR-MS: TOF-MS-ES- m/z = 1625.83 [M-H]-, M = 1626.83 g/mol−1.TLC: Rf = 0.064 DCM/MeOH (95:5); RMN 1H : δ (ppm) 1.05–1.09 3-(CH2)3-COOH), 1.21–2.25 2-(CH2)2-COOH), 1.55 , 2.12–2.23 , 3.13 , 3.32 , 3.36 , 3.43–3.44 , 3.46 , 3.54 , 3.57–3.58 , 3.70–3.75 , 5.04–5.10 ; RMN 13C : δ (ppm) 23.2–25.8 (-(CH2)3-(CH2)3-COOH), 24.8 -(CH2-CH2-COOH), 29.1–29.5 (-(CH2)2-(CH2)2-COOH), 34.2 (-CH2-COOH), 36.7 -(CH2-CO-NH), 39.9 (-CONH-CH2-OCH3), 58.3–58.6 (-OCH3), 58.8-59.9 (-OCH3), 61.2–61.5 (-OCH3), 71.0 (-CH-OCH3), 71.2–71.3 (-CH2-OCH3), 79.9–80.6 (-CH-OCH3), 81.3–82.2 (-CH-CH-OCH3), 98.8–99.0 (-CH-O-CH-), 173.5 (-CONH-), 177.4 (-COOH); ESI-MS calcd N-Boc protection of ciprofloxacin 2.The mixture containing ciprofloxacin , Boc2O  and 16.9 mL du NaOH aqueous solution in THF was stirred at room temperature overnight. The product precipitated from the reaction mixture and was collected by filtration as white solid, 100% yield (3.5 g).TLC: Rf 0.55, DCM/MeH 90:10; 1H NMR : δ 1.19–1.20 , 1.36–1.43 , 1.49 , 3.27–3.30 , 3.65–3.68 , 7.34–7.36 , 7.97–8.20 , 8.74 , 14.94 . 13C NMR : δ 8.2 (CH2-cyclopropyl), 28.4 (C-Boc), 35.3 (CH-cyclopropyl), 49.9 (CH2-piperazin), 104.7 (CH-Ar), 107.9 (C-Ar), 111.7 (CH-Ar), 119.9 (C-Ar), 138.8 (C-Ar), 146.4 (C-Ar), 152.1 (C-Ar), 155.2 (C-F), 167.2 (-COOH), 177.3 (Ar-C=O), 177.3 (Ar-C=O), 177.3 (-N-C=O); ESI-MS: calcd for (C28H33FN4O4+Na)+: 431.19. Found: 454.0 [M+Na]+.7-)-adamantane-1-carbonyl) piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4 dihydroquinoline-3-carboxylic (tert-butyl carbonic) anhydride 2To a solution of the compound 2  in DMF (16.7 mL), HBTU  was added followed by HOBt , DIPEA , and 1-amino adamantane . The mixture was stirred at room temperature under N2 for 30 min, and further DIPEA  was added. The reaction mixture was stirred at room temperature for 1 h, poured into water, and extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated. The crude residue was purified by column chromatography on silica gel using DCM:MeOH 5:95 as eluant to provide the compound 3 (90% yield) as yellow solid.m/z = 564.31 found: 587 [M+Na]+; 19F NMR : δ (ppm) −123.33 (F-Ar), −113.36 (F-Ar); IR ν (cm−1): (C=C), 1656, (C=O) 1698, (CH2) 2231, 2906, (CH2), (N-H) 3439; Anal. Calcd/Found: calcd for (C27H33FN4O2): C, 69.80; H, 7.16; N, 12.06; found 62.80, 6.65, 9.77; TLC: Rf 0.34 (DCM/MeOH 95:5); mp (°C): 232; 1H NMR : δ (ppm) 1.14–1.16 , 1.28–1.31 , 1.48 , 1.65–1.71 , 2.09 , 2.15 , 3.20–3.24 , 3.40–3.46 , 3.63–3.66 , 7.29–7.32 , 8.01–8.05 , 8.80 , 9.86 ; 13C NMR : δ (ppm) 8.0 (CH2-cyclopropyl), 28.4 ((CH3)3-C-BOC), 29.5 (Ad-CH), 34.6 (cyclopropyl-CH), 36.5 (Ad-CH2), 41.7 (Ad-CH2), 42.5 (CH2-piperazin), 49.9 (CH2-piperazin), 104.8 (CH-Ar), 112.3 (CH-Ar), 112.6 (CH-Ar), 146.4 (C-Ar). 155.2 (C-F), 154 (Boc-C=O), 163.6 (NH-C=O), 175.7 (Ar-C=O); ESI: calcd calcd for C32H41FN4O4 m/z = 565.31 [M+H]+, M = 565.31 g/mol−1.HR-MS: TOF-MS-ES- 7--adamantane-1-carbonyl) piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4 dihydroquinoline-3-carboxylic acid 4.Cleavage of Boc group was achieved by treatment of conjugate 3  with 10% TFA solution in dichloromethane (19.7 mL). Complete reaction was confirmed by TLC. The compound was extracted with 25 mL of AcOEt and organic phase was then collected and evaporated under vacuum. The resulting residue was washed three times with 200 mL of hexane, dissolved in minimum volume of methanol, precipitate by addition of cold diethyl ether and kept at −20 °C overnight. The solid precipitate was obtained by decantation after solvent removing by high vacuum. The residue was recovered in 200 mL of water and the pH was then adjusted to 9 by dropwise adding of aqueous 1 M NaOH solution. The desired product was precipitated and extracted by DCM to eliminate the trifluoroacetic acid sodium salt. The organic layer was washed with brine, dried over anhydrous Na2SO4 and evaporated to dryness.TLC: Rf 0.27 (DCM/MeOH 95:5); mp (°C): 232; 1H NMR : δ 1.1–1.14 , 1.24–1.28 , 1.67 , 2.06 , 2.11 , 3.06–3.09 , 3.22–3.26 , 3.40–3.45 , 7.26–7.28 , 7.92–7.96 , 8.74 , 9.86 ; 13C NMR : δ 8.0 (CH2), 28.5 (Ad-CH), 33.7 (cyclopropyl-CH), 35.5 (Ad-CH2), 40.7 (Ad-CH2), 44.7.m/z= 464.26 [M+H]+, M = 465.26 g/mol−1; IR ν (cm−1): (C=C), 1625, (C=O) 1297–1449, (CH2) 2349, 2844, 2908, (CH2) 2931, (N-H) 3446.; Anal. Calcd/Found: calcd for (C27H33FN4O2): C, 69.80; H, 7.16; N, 12.06; found 62.80, 6.65, 9.77.(piperazin-CH2), 49.8 (piperazin-CH2), 103.58–103.62 (CH-Ar), 112.2 (CH-Ar), 112.5 (CH-Ar), 121.8 (C-Ar), 138.2 (C-Ar), 145.1 (CH-Ar), 151.4 (C-Ar), 155.2 (C-F), 163.6 (NH-C=O), 175.7 (Ar-C=O), 19F : δ 123.36 ; ESI-MS: calcd for (C27H33FN4O2+H)+: 465.30; found: 465.40 [M+H]+; HR-MS: TOF-MS-ES- ®, France) before use. Experiments were realized at atmospheric pressure and at 310 K. For all experiments, corrections have be made to the heat data to account for heat effects associated with titrant dilution and any temperature difference between titrant and titrate solutions. These corrections were made by performing a blank titration experiment and subtracting the blank heat data from the experimental thermogram. The blank titration was carried out by introducing βCD or derivatives (4 mM) in the sample cell containing buffer alone (without CIP-Ad), according the same experimental conditions . All experiments were reproduced at least two times. The binding isotherms (without the first data point) were analyzed using an independent binding model of NanoAnalyse software (which models an interaction of “n” ligands with a macromolecule that has one binding site) to estimate the thermodynamic parameters of interaction such as the apparent binding stoichiometry (n), association constant (Ka) and enthalpy of binding (ΔH). Then, since temperature is held constant throughout, the free Gibbs energy (ΔG) of the binding reaction was determined by ΔG = −RTlnKa. Consequently, the entropy change (ΔS) was determined by ΔS = (ΔH − ΔG)/T.Calorimetric studies were performed using the Nano ITC 2G Isothermal Titration Microcalorimeter from TA instruments . This power compensation differential instrument has a reference cell and a sample cell of approximately 1.040 mL. Each experiment involved 25 injections of 10 μL of β-CD or derivatives into the thermostated sample cell containing solution of CIP-Ad. The reference cell was filled with degassed MilliQ-water. The time interval between two consecutive injections was fixed to 300 s and the agitation speed (via the paddle stirrer at the end of the syringe) at 250 rpm. Solutions of βCD or derivatives (4mM) and CIP-Ad (0.3 mM) were prepared in chloroacetate buffer  and filtered through 0.2 μm cellulose acetate membrane  to eliminate the insol-uble supernatant. The filtrate was analyzed by NMR. A solution of 2,3-O-dimethyl-β-CD at 4 mM in D2O was prepared.Preparation of Complex between 2,3-di-O-methyl-β-CD and (CIP–Ad).−1. The gradient duration δ/2 was adjusted to observe a near complete signal loss at 32.35 G.cm−1. The δ/2 delay was fixed at 2 ms. A 2s recycle delay was used between scans for all data shown. The number of scans was fixed to 32 (for the free molecules) and 64 scans (for the complex). The number of scans was fixed to 32 (for the free molecules) and 64 scans (for the complex).2 equivalents of 2,3-O-dimethyl-β-CD  were added to 1 equivalent of CIP–Ad  in 1 mL of D2O. The cloudy solution obtained was stirred and then filtrated through a 0.2 μm syringe filter  to eliminate the insoluble supernatant. The filtrate was analyzed by NMR. The reactants, 2,3-O-dimethyl-β-CD (in D2O) and CIP-Ad (in D2O and CDCl3), were analyzed with a Bruker AVIII300 NMR spectrometer. Experiments were carried out at 300 MHz for 1H and at 75 MHz for 13C. The complete assignment of all H and C atoms of 2,3-O-dimethyl-β-CD and CIP-Ad was achieved by conventional 2D experiments: HMQC (1H-13C) and HMBC (1H-13C). For 2D spectra, a total of 1024 (HMQC) or 2048 (HMBC) points in F2 and 128 experiments in F1 were recorded. The complex 2,3-O-dimethyl-β-CD:CIP–Ad (in D2O) was analyzed with a Bruker AVIII600 NMR spectrometer equipped with a 10 A gradient amplifier and a 5 mm CPTXI  including shielded z-gradients. 1D 1H and 2D 1H-1H NMR were recorded at 298 K. All chemical shifts were measured relative to external TMS. 2D NOESY (1H-1H): was used to evidence the interaction between 2,3-O-dimethyl-β-CD and CIP–Ad. For 2D NOESY (1H-1H) spectrum, a total of 2048 points in F2 and 256 experiments in F1 were recorded. A mixing time of 300ms was used. The formation of the complex was confirmed using the diffusion coefficients measured by 1H DOSY experiments. The diffusion 1H NMR experiments were carried out by using the AVIII600 spectrometer, for 2,3-O-dimethyl-β-CD, CIP-Ad and complex. A pulsed-gradient stimulated echo (LED-PFGSTE) sequence, using bipolar gradient was used. Sequence delays were: diffusion delay (Δ) 150 ms, recovery delay after gradient (τ) 0.2 ms, and LED recovery delay 5 ms. For each data set, 66,560 complex points were collected for each 32 experiments in which the gradient strength was linearly incremented from 0.681 to 32.35 G.cmm/z = 1625, 84 [M-H]-, mass found m/z = 1625.0 [M-H]-; IR ν(cm−1): ring vibration 946 cm−1, (C-C/C-O) 1032–1079, (C-H/O-H) 1243–1436, (CO-NH) 1574, (-CO-OH) 1628; (CH2) 2849–2928, (N-H) 3329; HR-MS: TOF-MS-ES- m/z= 1625.83 [M-H]-, M = 1626.83 g/mol−1.TLC: Rf = 0.064 DCM/MeOH (95:5); RMN 1H : δ (ppm) 1.05–1.09 3-(CH2)3-COOH), 1.21–2.25 2-(CH2)2-COOH), 1.55 , 2.12–2.23 , 3.13 , 3.32 , 3.36 , 3.43–3.44 , 3.46 , 3.54 , 3.57–3.58 , 3.70–3.75 , 5.04–5.10 ; RMN 13C : δ (ppm) 23.2–25.8 (-(CH2)3-(CH2)3-COOH), 24.8 -(CH2-CH2-COOH), 29.1–29.5 (-(CH2)2-(CH2)2-COOH), 34.2 (-CH2-COOH), 36.7 -(CH2-CO-NH), 39.9 (-CONH-CH2-OCH3), 58.3–58.6 (-OCH3), 58.8–59.9 (-OCH3), 61.2–61.5 (-OCH3), 71.0 (-CH-OCH3), 71.2–71.3 (-CH2-OCH3), 79.9–80.6 (-CH-OCH3), 81.3–82.2 (-CH-CH-OCH3), 98.8–99.0 (-CH-O-CH-), 173.5 (-CONH-), 177.4 (-COOH); ESI-MS calcd 2 = 0.99).Triplicate saturated solutions of CIP-Ad in water were prepared by adding excess amount of compound in distilled water. Flasks with concentrated solutions were capped with plastic stoppers and mechanically shaken for 24 h at 25 °C and 250 rpm. The solutions were then taken up into syringe filter, and filtered through 0.2 μm microcellulose filter . UV measurements  were performed to quantify the CIP-Ad amount in solutions (Perkin Elmer Carry 100 BIO UV-vis spectrophotometer) by using a calibration curve  using 5 mL glass flasks. 13.96–14 mg of CIP Ad was added to the aqueous CDs solution for a concentration of 14 mg/L. The tubes were recapped and mechanically shaken for 48 h at 37 °C and 250 rpm. After 48 h, the solution was extracted using a 1 mL syringe and filtered with a 0.2 μm microcellulose filter. 0.5 ML of sample was diluted with 0.5 mL of ethanol in order to dissociate the μm microcellulose filter. 0.5 ML of sample was diluted with 0.5 mL of ethanol in order to dissociate the complex. Dilution (1/500) of each sample with 50% aqueous ethanol was necessary to performed UV absorption spectrophotometry and quantified the CIP-Ad in solution. A 50% aqueous ethanol solution was used as a blank. For solubility test with β-CD, the same procedure was followed. β-CD was added to four tubes at various concentrations in a range of 2; 4; 6; 8; 12; 16 mM. The experiment was performed in triplicate (n = 3).Preparation of Solid Inclusion Complex: The inclusion complex was prepared by freeze-drying method water by adding CIP-Ad into CD solution prepared using Milli-Q water. The concentrations of each component were calculated at the highest solubility given by the solubility curves. The aqueous CIP-Ad mixture was shaken for 1 night at −4 °C at 250 rpm . The solution was filtered and placed into wide shallow dishes covered with plastic wrap and frozen to −80 °C. Samples were freeze-dried over four days . The solid sample was stored in sealed bottles at −4 °C until analysis.Preparation of Dispersin B–CD conjugates: EDAC (30 mg) and 16 mg of N-hydroxysuccinimide were added to the reaction mixtures containing 0.5 mg of Dispersin B dissolved in 0.5 mL of 80 mM borate buffer, pH 8.0, and 76 mg of each CD derivative, i.e, CD1, CD2, and CD3. The solutions were stirred for 1 h at room temperature and then for 16 h at 4 °C. Ultrafiltration of enzymatic solution was performed using centrifugal filter  for concentrating and desalting the enzyme and elimination of the entire reagent using a cut off 10 KDa. The modified Dsp B was purified by coprecipitation using a 2-D Clean-Up Kit .Minimal Inhibitory Concentration (MIC) determination: Antimicrobial activities of compounds were evaluated by MIC measurements through the micro dilution method based on ISO standard 20776-1 . Briefly, microplate wells containing TSB were enriched by a solution of CIP hydrochloride at various concentrations between 1024 and 0.25 μg/mL in 0.15 M, Ph = 7.4 phosphate buffer (PBS). Each well was inoculated by a bacterial suspension . After 24 h of incubation at 37 °C, the bacterial growth was visualized by the broth turbidity. MIC was defined as the lowest concentration of CIP-Ad that inhibited the visible bacterial growth. Negative (without bacteria) and positive controls (without antibiotic) were included in each assay. The same procedure was used for CIP-Ad and each CDs complexes, i.e., CIP-Ad:2,3-O-dimethyl-β-CDs and CIP-Ad:permethyl β-CDs. In these cases CIP-Ad was solubilized in steril dimethylsulfoxide DMSO and CDs complexes in PBS.Biofilm formation assay: Strain 5 was used. Biofilms were formed in 96-well microplates containing TSB inoculated from a preculture  as previously described . After 2v/v) and the OD measured at 590 nm . The OD590 values obtained at times zero (before DspB exposure) and after treatment were compared. For each strain the experiment was performed 3 times.Tests were adapted from the method described by O’Toole and Kolter . BiofilmAntibiotic resistance assays: Biofilms were formed in 96-well microplates as described above. After 24 h of incubation at 37 °C, the medium was removed and cells washed three times with PBS. Wells were then filled with 200 μL of TSB containing different concentrations of ciprofloxacin, i.e., 5 MIC.Biological tests.S. epidermidis clinical strains  were kindly provided by Pr. J. Kaplan from the University of Medicine and Dentistry of New Jersey (USA). Strains were stored at −80 °C in glycerol and used as required. For precultures, bacteria were grown for 24 h in Tryptic soy broth (TSB)  at 37 °C with shaking. All antibiotics, CIP or CIP-Ad was conserved at −4 °C.Bacterial strains: Bacterial growth monitoring: The growth curve was obtained by incubating the strain 5 in TSB for 26 h at 37 °C and monitoring the OD595 . 10 MIC,The same protocol was used for the complex CIP-Ad:2,3-O-dimethyl-β-CD by using concentrations corresponding to 5 MIC, 10 MIC, 20 MIC, 50 MIC. The solid CIP-Ad:2,3-O-dimethyl-β-CD complex was prepared as described above and solubilized in PBS.S. epidermidis:Combined effect of Dsp B and ciprofloxacin on established biofilms of Biofilms were performed as described above. After 24 h of incubation at 37 °C, the supernatant was gently aspirated with a micropipette in each well. Wells were then washed three times with PBS under aseptic conditions to eliminate the unbound bacteria, without disturbing the adherent film. Ciprofloxacin (10 × MIC) and/or, Dsp B (5 μg/mL) were added in 200 μL PBS. Plates were incubated at 37 °C under shaking. After 2 h of incubation, the growth medium was removed and wells washed three times with PBS. Biofilm cells were recovered by scrapping and enumerated as described above.Combined effect of DspB with  complex, Dsp B-CD3 conjugate and Dsp B-CD3:CIP-Ad conjugate on biofilms.The same procedure was used to test the effect of the Dsp B CIP-Ad:2,3-O-dimethyl-β-CD complex (5 MIC) association. The complex was prepared as described above and then solubilized in PBS. The solution was sterilized by filtration on 0.2 μm filter and then diluted in PBS at 1000 mg/L.Likewise, the Dsp B-CD3 and Dsp B-CD3:CIP-Ad conjugate conjugates was tested , alone or associated with CIP-Ad. Finally, the antibiofilm activity of the Dsp B-CD3-:CIP-Ad conjugate was tested .v/v). After 30 min of reaction, the absorbance was read at 595 nm and the protein concentration was determined using a calibration curve realized with a range concentration of Bovine Serum Albumin (BSA).Bradford protein assay: Twenty μL of protein sample was added to 1 mL of a Bradford solution (BioRad Protein Assay) diluted , nosocomial-, or depressed-immune-system-related infections. Among the s aureus ,2,3,73, s aureus ,75. Howes aureus .® , a recombinant human deoxyribonuclease I (rhDNase), is inhaled to improve lung function in cystic fibrosis [To conclude, and in light of this assessment, anti-biofilm-enzyme-based approaches (as the drug delivery system presented here) constitute a promising alternative. Due to their ability to degrade the biofilm polymer matrix, they significantly increase the efficiency of some antibiotics by both modifying the environmental conditions within the biofilm  and by increasing drug diffusivity. Some of these enzymes are already marketed. Thus, the pulmozymefibrosis  and mechfibrosis . However


Streptomyces sp. BI0788, exhibiting neuroprotective activity in vitro, was selected and cultivated in large-scale. The chemical analysis of its organic extract resulted in the isolation of four new butanolides , along with two previously reported butanolides (2 and 3) and eight previously reported butenolides (7–14). Compounds 2–4 and 7–14 were evaluated for their neuroprotective effects on TrkB-expressing NIH-3T3 cells. Among them, metabolites 3, 4, 7, 10, 11, 13 and 14 exhibited significant protective activity on the aforementioned cells through the activation of TrkB, the high-affinity receptor for the Brain-Derived Neurotrophic Factor (BDNF), which is well known to play a crucial role in neuronal cell survival and maintenance.Neurodegenerative diseases are incurable and debilitating conditions, characterized by progressive loss and degeneration of vulnerable neuronal populations. Currently, there are no effective therapies available for the treatment of most neurodegenerative disorders. A panel of extracts exhibiting interesting chemical profiles among a high number of bacterial strains isolated from East Mediterranean marine sediments and macroorganisms were evaluated for their activity on TrkB-expressing cells. Among them, the actinobacterial strain  Currently there is an urgent need to identify and develop new therapeutics for the treatment of neurodegenerative diseases . NeurodeOver the last years, the marine environment has been recognized as an ecosystem hosting unique taxa of microorganisms, especially actinobacteria , which a1–14, 1 and 4–6).In search of new molecules with neuroprotective activity, several extracts exhibiting interesting chemical profiles among a high number of bacterial strains isolated from East Mediterranean marine sediments and macroorganisms were evaluated for their activity on the TrkB-stably expressing cell line NIH-3T3 (NIH-3T3-TrkB). Among them, the actinobacterial strain BI0788, exhibiting protective activity in vitro, was selected for further chemical investigation. Extraction of a large-scale culture of the bacterial strain BI0788 and fractionation of the resulting organic residue allowed for the isolation of fourteen γ-lactone-containing metabolites 1–14, , includi1 and 4–6, as well as the evaluation of the protective activity of compounds 2–4 and 7–14 on NIH-3T3-TrkB cells through selective activation of the TrkB receptor.Herein, we report the isolation and structure elucidation of the new butanolides Streptomyces sp. BI0788 was the most potent, decreasing cell death levels to half as compared to those of the control (serum free)  for their ability to reduce serum deprivation-induced apoptosis in comparison to BDNF, the native ligand of TrkB receptor. Cell death was caused by serum deprivation for 24 h and subsequently BDNF or the different bacterial extracts were added to the media for 24 h to assess (using CellTox kit) whether they were able to reduce the levels of cell death. Among them, the organic extract of um free) .Streptomyces sp., underwent several chromatographic fractionations to yield, in pure form, four new natural products, namely -2-ethyl-3-hydroxy-4-methylbutan-1,4-olide (1), -11-oxo-10-methyldodecan-1,4-olide (4), -11-hydroxy-10-methyldodecan-1,4-olide (5), and (4S)-10-hydroxy-10-methyldodecan-1,4-olide (6), and ten previously reported metabolites .Subsequently, the organic extract of strain BI0788, isolated from a marine sediment collected in the Ionian sea and tentatively identified as 1, obtained as an amorphous white powder, displayed the molecular formula C7H12O3, as deduced from high-resolution electrospray ionization mass spectrometry (HR-ESIMS) measurements where a sodium adduct ion peak [M + Na]+ at m/z 167.0679 was detected. The 1H and 13C NMR data of compound 1 (δC 11.3 and δH 1.07 (CH3-7) and one doublet at δC 18.3 and δH 1.44 (CH3-5), one methylene at δC 21.3 and δH 1.88/1.68 (CH2-6), three methines resonating at δC 49.9 and δH 2.50 (CH-2), δC 78.6 and δH 3.84 (CH-3), and δC 79.7 and δH 4.18 (CH-4), and one quaternary carbon, corresponding to a carbonyl at δC 175.6 (C-1). The planar structure of 1 was determined on the basis of the homonuclear and heteronuclear correlations observed in the HSQC-DEPT, HMBC and COSY spectra (3-5 (δH 1.44) with C-3 (δC 78.6) and C-4 (δC 79.7), and of H2-6 (δH 1.88/1.68) with C-1, C-2, C-3, and C-7 . Comparison of the spectroscopic characteristics of compound 1 with those of the co-occurring metabolites 2 and 3, in combination with the correlations of H-2/H-4 and H-3/H3-5 observed in 1D NOE experiments, allowed for the determination of the relative configuration of the chiral centers at C-2, C-3 and C-4. Specifically, the chemical shifts of H-2, H-3, H-4 and H3-5, as well as of the corresponding carbons C-2, C-3, C-4 and C-5 of 1, were similar to those of 2 and 3, while differing significantly from those of other synthetic analogues featuring the 3-hydroxy-γ-butyrolactone system + at m/z 249.1456, indicating the molecular formula C13H22O3, which in turn suggested that 4 had three degrees of unsaturation. The planar structure of compound 4 was assigned by interpretation of its 1D and 2D NMR spectroscopic data. In particular, the 1H and 13C NMR spectroscopic data of compound 4 (δC 28.0 and δH 2.11 (CH3-12), one doublet methyl at δC 16.3 and δH 1.06 (CH3-13), seven methylenes, one oxygenated methine at δC 83.3 and δH 4.45 (CH-4), one deshielded methine at δC 47.1 and δH 2.48 (CH-10), and two quaternary carbons at δC 177.2 (C-1) and 212.8 (C-11), corresponding to one ester and one ketone carbonyl, respectively, with the latter two accounting for two of the three degrees of unsaturation, indicating the monocyclic structure of compound 4. The correlations observed in the COSY spectrum revealed the presence of a single spin system, while the HMBC correlations of H2-2, H2-3 and H-4 with C-1 supported the presence of a γ-lactone. Additionally, the HMBC correlations of H-10, H3-12 and H3-13 with C-11 connected C-10 with an acetyl group (δC 212.8 and δC 28.0/δH 2.11) and concluded the structure of 4 -11-oxo-10-methyldodecan-1,4-olide (4), described here for the first time as a natural product, are identical to those reported for a semisynthetic derivative of compound 7 c 0.05, MeOH); UV (MeOH) λmax (log ε) 200.5 (2.82) nm; IR (thin film) vmax 3450, 2980, 2920, 2880, 1750, 1460, 1380, 1180, 1050 cm−1; 1H and 13C NMR data, see m/z 167.0679 [M + Na]+ .(22): white solid; [α]c 0.10, MeOH); 1H NMR, see m/z 172 [M]+.(−)-Blastmycinolactol (3): white solid; [α]c 0.10, MeOH); 1H NMR, see m/z 186 [M]+.(−)-8-Methylblastmycinolactol -11-Oxo-10-methyldodecan-1,4-olide (4): yellow oil; [α]c 0.09, MeOH); UV (MeOH) λmax (log ε) 200.5 (3.23), 215 (3.04) nm; IR (thin film) vmax 2930, 2860, 1770, 1710, 1460, 1350, 1180 cm−1; 1H and 13C NMR data, see m/z 249.1456 [M + Na]+ .-11-Hydroxy-10-methyldodecan-1,4-olide (5): yellow oil; [α]c 0.17, MeOH); UV (MeOH) λmax (log ε) 200.5 (3.21), 221 (2.84) nm; IR (thin film) vmax 3450, 2920, 2850, 1740, 1460, 1380, 1180 cm−1; 1H and 13C NMR data, see m/z 251.1614 [M + Na]+ .(4S)-10-Hydroxy-10-methyldodecan-1,4-olide (6): yellow oil; [α]c 0.54, MeOH); UV (MeOH) λmax (log ε) 201 (3.63), 219 (3.52) nm; IR (thin film) vmax 3450, 2920, 2850, 1740, 1460, 1380, 1180 cm−1; 1H and 13C NMR data, see m/z 251.1617 [M + Na]+ .-11-Oxo-10-methyldodec-2-en-1,4-olide (7): yellow oil; [α]c 0.20, MeOH); 1H NMR, see [m/z 224 [M]+.-11-Hydroxy-10-methyldodec-2-en-1,4-olide (8): yellow oil; [α]c 0.10, MeOH); 1H NMR, see [m/z 226 [M]+.-11-Hydroxy-10-methyldodec-2-en-1,4-olide (9): yellow oil; [α]c 0.10, MeOH); 1H NMR, see [m/z 226 [M]+.-10-Hydroxy-10-methyldodec-2-en-1,4-olide (10): yellow oil; [α]c 0.13, MeOH); 1H NMR, see [m/z 226 [M]+.-10-Hydroxy-11-oxo-10-methyldodec-2-en-1,4-olide (11): yellow oil; [α]c 0.10, MeOH); 1H NMR, see [m/z 240 [M]+.-10,11-Dihydroxy-10-methyldodec-2-en-1,4-olide (12): yellow oil; [α]c 0.10, MeOH); 1H NMR, see [m/z 242 [M]+.-10-Methyldodec-2-en-1,4-olide (13): yellow oil; [α]c 0.10, MeOH); 1H NMR, see [m/z 210 [M]+.-10-Hydroxy-10-methylundec-2-en-1,4-olide (14): yellow oil; [α]c 0.10, MeOH); 1H NMR, see [m/z 212 [M]+. in EtOAc (4 mL), Pd/C (1 mg) was added and the resulting mixture was stirred under H2 atmosphere overnight. Then, it was passed through a celite pad and the filtrate was evaporated to dryness. To an ice-cold solution of the hydrogenated intermediate (8.5 mg) in MeOH (0.1 mL), a solution of NaBH4  in MeOH (0.3 mL) was added dropwise. The reaction mixture was left at RT for 1 h (monitored with TLC and ESIMS). It was quenched with 3 drops of water and evaporated to dryness, affording a mixture of the two epimeric alcohols at C-11. Consecutive purifications on chiral-phase HPLC, using a mixture of n-hexane/iso-propanol (94:6 and 97:3) as eluent, afforded 5 in pure form (3.0 mg).To a solution of 10 (6.0 mg) in EtOAc (3 mL), Pd/C (1 mg) was added and the resulting mixture was stirred under H2 atmosphere overnight. Then, it was passed through a celite pad and the filtrate was evaporated to dryness. Consecutive purifications on chiral-phase HPLC, using a mixture of n-hexane/iso-propanol (94:6 and 97:3) as eluent, afforded 6 (3.5 mg).To a solution of 2 atmosphere at 37 °C.Stably transfected NIH-3T3-TrkB-expressing cells were used to test the ability of the extracts or compounds to activate the TrkB receptor under serum deprivation conditions. Cultures were carried out in High-Glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin in a humidified 5% COhttps://imagej.nih.gov/ij/, accessed on 30 April 2023) was used.Naïve NIH-3T3 or NIH-3T3-TrkB-expressing cells plated in 96-well plates at 10,000 cells/well were serum starved for 24 h. Compound treatments were performed at 1–100 μM for 24 h, while BDNF was used as a positive control at 500 ng/mL. For dead and total cell counting, CellTox™ Green Dye  and Hoechst 33342 Solution , respectively, were used. For image capture, a ZEISS Axio Vert.A1 microscope and the ImageJ software  with a Millipore phospho-protease inhibitor cocktail by Millipore for 10 min. After adding the loading buffer (5× Laemni) to 25 μg of the protein sample, samples were incubated at 95 °C for 5 min and subjected to SDS-PAGE. Nitrocellulose membrane transfer was performed at 350 mA for 2 h, followed by blocking at room temperature with 5% bovine serum albumin (BSA) for 1 h and primary antibody incubation in blocking solutions at 4 °C overnight and detection with HRP-conjugated secondary antibodies. ECL solution was used to detect chemiluminescence. Primary antibodies used are Phospho-TrkB (Tyr816) Millipore # ABN1381 and Anti-TrkB Abcam #ab33655, β-Actin.Streptomyces sp. BI0788, an actinobacterial strain isolated from a marine sediment collected from the Ionian sea that showed an ability to significantly reduce cell death in a TrkB-dependent cell population, led to the isolation and structure elucidation of four new butanolides (1 and 4–6), along with two previously reported butanolides (2 and 3) and eight previously reported butenolides (7–14). Among compounds 2–4 and 7–14 that were isolated in sufficient amounts and evaluated for their activity on NIH-3T3-TrkB cells, compound 7 showed the greatest reduction at 1 μM, similar to the levels of the native neurotrophin ligand BDNF.The chemical investigation of the organic extract of This study demonstrates for the first time the action of natural small molecules that have the ability to activate the TrkB receptor and promote cell survival, one of the most important functions mediated by the receptor. Over the last years, many research efforts have focused on developing BDNF mimetics with suitable pharmacological properties in order to be used as new therapeutics against neurodegenerative diseases. Hence, our results suggest that these γ-lactone-containing compounds could have great potential in various neurological disorders as lead molecules for further investigations in relevant mouse models and eventually in human clinical trials.