Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular,  Furthermore, this phenolic compound is characterised by proton and electron-donating capacity and, as a consequence, antioxidant properties which can occur during several important biological processesFor these reasons, nitroxoline structure has prompted the Medicinal chemists to explore the structural requirements suitable to improve antitumor effects. In previous papers, taking advantage of the reactivity of the quinoline nucleus, most authors studied the introduction of halogens and additional side chains as well as modifications of the nitro group2-C15), saturated and unsaturated alkyl chains, and substituted and unsubstituted aryl and bicyclic rings. Moreover, we inserted a carbonyl spacer between the benzylic methylene and the aryl ring to evaluate the impact of this elongation. Aliphatic ethers were also decorated with COOH, CN, and COOEt to further explore the chemical space around these positions. Benzylic ethers were characterised by the presence of electron-donating  and electron-withdrawing  substituents in ortho, meta and para positions. Polysubstitutions were also provided. Lastly, to better understand the importance of the quinoline nucleus, we aimed at simplifying the nitroxoline structure to 4-nitro(thio)phenol. On these two scaffolds, we added the same substituents that resulted to be more active in the first series giving the possibility to extrapolate robust structure-activity relationships (SARs).Starting from these premises and pursuing our efforts towards the discovery of new anti-proliferative heterocyclic agentsKRAS mutated, while BxPC-3 and AsPC-1 carry p53 mutations.All the gathered compounds have been first tested in a one-point concentration screening against three human PC cell lines to assess the best-in-class compounds for further cell-based experiments. Specifically, we selected AsPC-1, Capan-2 and BxPC-3 pancreatic cancer cell lines that display different genetic profiles: AsPC-1 and Capan-2 are 2.1–61, we followed the synthetic approaches outlined in 1–12, 14–46 , in the presence of potassium carbonate (K2CO3) and under nitrogen (N2) atmosphere. In addition, compound 12 was hydrolysed in mild conditions using lithium hydroxide (LiOH) in a mixture of water and methanol  at room temperature, to provide the carboxylic acid derivative 13. For compounds 47–61, the same reactions involving 4-nitrophenol or 4-nitrothiophenol were performed (1H/13C/19F NMR), whereas the purity of these compounds was confirmed by combustion analysis, TLC parameters, crystallographic studies (for compound 16) and melting point evaluation. In silico analysis of the most active compounds was performed by using the online free software SwissADME, a web tool that allows to appraise pharmacokinetics, as well as drug-likeness  and medicinal chemistry friendliness (PAINS) of small moleculesFor the synthesis of compounds 2, 14–46  have beeerformed . The str3.16 .Crystals of compound 16  were obt4.4.1.® melting point apparatus SMP1 and are uncorrected (temperatures are reported in °C). 1H and 13 C NMR spectra were recorded at 300 MHz and 75 MHz (Varian Mercury spectrometer), or at 400.13 MHz and 101.03 MHz on a Bruker spectrometer, using CDCl3 and DMSO-6d, as the solvents at room temperature. 19 F spectra were recorded on a Bruker AVANCE 600 spectrometer at 564.7 MHz, using CDCl3 as solvent. All the compounds were analysed with a final concentration of ∼25 mg/mL. 1H and 13 C chemical shifts are expressed as δ units (parts per millions) relative to the solvent signal , whereas 19 F chemical shifts are expressed as δ units relative to an external standard . 1H spectra are described as follows: δH : chemical shift/ppm , number of protons, assignment). 13 C spectra are described as follows: δC : chemical shift/ppm (assignment) and are fully proton decoupled. 19 F spectra are described as follows: δF : chemical shift/ppm , number of fluorine, assignment). Multiplets are abbreviated as follows: br – broad; s – singlet; d – doublet; t – triplet; q – quartette; td – triplet of doublets; m – multiplet. Coupling constants J are valued in Hertz.Unless otherwise indicated, all reactions were carried out under a positive pressure of nitrogen in washed and oven-dried glassware. All the solvents and reagents were directly used as supplied by Sigma-Aldrich  without further purification unless otherwise noted. Where mixtures of solvents are specified, the stated ratios are volume:volume. All melting points were measured on a Stuart® silica gel . All the purifications and reactions were carried out by TLC performed on 0.2 mm thick silica gel-aluminium backed plates . Spot visualisation was performed under short- and long-wavelength  ultra-violet irradiation. Where given, systematic compound names and ClogP values are those generated by ChemBioDraw Ultra 14.0 following IUPAC conventions.The processing and analyses of the NMR data were carried out with MestreNova. Elemental analyses for C, H, and N were recorded on a Perkin-Elmer 240 B microanalyzer obtaining analytical results within ± 0.4% of the theoretical values for the final compounds. Preparative chromatography was carried out employing Sigma-Aldrich4.2.4.2.1.2CO3, 1.1 equiv.) was added to a stirring solution of nitroxoline (1 equiv.) in DMF (20 ml). The yellow suspension was stirred for 30 min at room temperature; then, the proper (substituted)benzyl, diaryl or alkyl bromide or α-bromoacetophenone (1.5 equiv.) was added and the reaction stirred until disappearance of the starting reagents, as detected by thin layer chromatography (TLC). Then, the mixture was poured into ice-cold water (150 ml) and partitioned with dichloromethane . The organics were separated, reunited and added with anhydrous sodium sulphate (Na2SO4) to remove water. The salt was filtered and washed two times with 5 ml of dry DCM. The organic phase was evaporated in vacuo to afford the crude extract containing the target molecule that was recovered through column chromatography, employing silica gel (SiO2) and proper mixtures of n-hexane/ethyl acetate.Freshly ground potassium carbonate  in 30 ml of methanol. The reaction was stirred at room temperature for 24 h; then, the mixture was concentrated in vacuo to remove methanol and quenched with 3 N HCl (15 ml). The precipitate was collected by filtration and washed with n-hexane to give the title compound 13, without further purification requirements.Lithium hydroxide (1.1 equiv.) dissolved in 15 ml of water was added dropwise to a stirring solution of ethyl 2-((5-nitroquinolin-8-yl)oxy)acetate  was added to a stirring solution of 4-nitro(thio)phenol (1 equiv.) in DMF (10 ml). The yellow suspension was stirred for 30 min at room temperature; then, the proper (substituted)benzyl, diaryl and alkyl bromide or α-bromoacetophenone (1.5 equiv.) was added and the reaction stirred until disappearance of the starting reagents, as detected by thin layer chromatography. Then, the mixture was poured into ice-cold water (100 ml) and extracted with dichloromethane . The organics were reunited and added with anhydrous sodium sulphate to remove water. The salt was filtered and washed three times with 5 ml of dry DCM. The organic phase was evaporated in vacuo to afford the crude extract containing the target molecule that was recovered through column chromatography, employing silica gel and proper mixtures of n-hexane/ethyl acetate.Freshly ground potassium carbonate (K4.3.8-ethoxy-5-nitroquinoline (1). Yellow solid, 66% yield, ClogP 2.71, mp = 126–128 °C. 1H NMR : δ 1.68–1.71 , 4.44–4.49 , 7.08–7.10 , 7.69–7.72 , 8.53–8.55 , 9.07–9.08 , 9.24–9.27 ; 13 C NMR : δ 14.4 (CH3), 65.6 (CH2), 106.0 (Ar), 123.1 (Ar), 124.5 (Ar), 127.7 (Ar), 132.7 (Ar), 137.4 (Ar), 139.3 (Ar), 150.1 (Ar), 160.2 (Ar). Anal. calcd for C11H10N2O3: C, 60.55; H, 4.62; N, 12.84, found: C, 60.51; H, 4.60; N, 12.82.5-nitro-8-propoxyquinoline (2). Yellow solid, 72% yield, ClogP 3.24, mp = 110–112 °C. 1H NMR : δ 1.08–1.13 , 2.00–2.12 , 4.26–4.31 , 7.02–7.05 , 7.63–7.68 , 8.47–8.50 , 9.00–9.03 , 9.18–9.21 ; 13 C NMR : δ 10.4 (CH3), 22.0 (CH2), 71.5 (OCH2), 123.1 (Ar), 124.5 (Ar), 127.7 (Ar), 132.7 (Ar), 106.1 (Ar), 137.3 (Ar), 139.1 (Ar), 150.0 (Ar), 160.3 (Ar). Anal. calcd for C12H12N2O3: C, 62.06; H, 5.21; N, 12.06, found: C, 62.00; H, 5.19; N, 12.00.8--5-nitroquinoline (3). Brown solid, 81% yield, ClogP 2.96, mp = 102–103 °C. 1H NMR : δ 4.97–4.99 , 5.39–5.54 , 6.12–6.25 , 7.07–7.10 , 7.68–7.72 , 8.49–8.52 , 9.05–9.07 , 9.22–9.26 ; 13 C NMR : δ 70.6 (CH2), 106.9 (Ar), 119.7 (CH2=), 123.1 (Ar), 124.6 (Ar), 127.5 (Ar), 131.5 (Ar), 133.0 (CH=), 150.0 (Ar), 159.6 (Ar). Anal. calcd for C12H10N2O3: C, 62.61; H, 4.38; N, 12.17, found: C, 62.66; H, 4.40; N, 12.21.5-nitro-8-(prop-2-yn-1-yloxy)quinoline (4). Brown solid, 68% yield, CLogP 2.60, mp = 157–158 °C. 1H NMR : δ 2.62–2.63 , 5.14–5.15 , 7.31–7.34 , 7.72–7.77 , 8.55–8.58 , 9.07–9.09 , 9.26–9.29 ; 13 C NMR : δ 57.2 (OCH2), 76.6 , 77.8 (C≡), 107.9 (Ar), 123.1 (Ar), 124.7 (Ar), 127.5 (Ar), 134.0 (Ar), 138.2 (Ar), 149.7 (Ar), 157.7 (Ar). Anal. calcd for C12H8N2O3: C, 63.16; H, 3.53; N, 12.28, found: C, 63.06; H, 3.50; N, 12.20.2-((5-nitroquinolin-8-yl)oxy)acetonitrile (5). Orange solid, 65% yield, ClogP 1.22, mp = 198–200 °C [Lit. mp = 194–197 °C]. Characterisation data are in agreement with those reported in the literature8-butoxy-5-nitroquinoline (6). Brown solid, 78% yield, ClogP 3.77, mp = 104–106 °C. 1H NMR : δ 1.00–1.05 , 1.52–1.65 , 2.00–2.10 , 4.34–4.39 , 7.07–7.10 , 7.68–7.72 , 8.52–8.55 , 9.06–9.08 , 9.25–9.28 ; 13 C NMR : δ 13.8 (CH3), 19.2 (CH2), 30.6 (CH2), 70.0 (OCH2), 106.4 (Ar), 123.2 (Ar), 124.5 (Ar), 128.0 (Ar), 133.5 (Ar), 137.2 (Ar), 149.7 (Ar), 160.0 (Ar). Anal. calcd for C13H14N2O3: C, 63.40; H, 5.73; N, 11.38, found: C, 63.20; H, 5.63; N, 11.12.(E)-8-(but-2-en-1-yloxy)-5-nitroquinoline (7). This compound exists in a mixture of E/Z isomers with the following ratio (4.4/1) as extrapolated by their OCH2 signal integration in the 1H NMR spectrum (peaks listed only for the major isomer). Brown solid; 79% yield, ClogP 3.49, mp = 87–89 °C; 1H NMR : δ 1.70–1.72 , 4.79–4.81 , 5.76–5.96 , 6.99–7.02 , 7.57–7.61 , 8.40–8.43 , 8.94–8.96 , 9.10–9.13 ; 13 C NMR : δ 17.9 (CH3), 70.5 (OCH2), 106.5 (Ar), 123.0 (Ar), 124.1 (Ar), 124.4 (CH=), 127.5 (Ar), 129.9 (CH=), 132.5 (Ar), 137.3 (Ar), 139.3 (Ar), 150.1 (Ar), 159.9 (Ar). Anal. calcd for C13H12N2O3: C, 63.93; H, 4.95; N, 11.47, found: C, 63.69; H, 4.80; N, 11.22.8-((3-methylbut-2-en-1-yl)oxy)-5-nitroquinoline (8). Yellow solid, 82% yield, ClogP 3.88, mp = 98–99 °C. 1H NMR : δ 1.81 , 4.94–4.96 , 5.60–5.64 , 7.05–7.08 , 7.67–7.13 , 8.51–8.54 , 9.05–9.07 , 9.24–9.27 ; 13 C NMR : δ 18.4 (CH3), 25.9 (CH3), 66.9 (OCH2), 106.6 (Ar), 118.3 (CH=), 123.1 (Ar), 124.5 (Ar), 127.8 (Ar), 133.1 (Ar), 139.5 (C), 149.9 (Ar), 160.0 (Ar). Anal. calcd for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85, found: C, 65.33; H, 5.60; N, 11.00.5-nitro-8-(pentyloxy)quinoline (9). Brown sticky oil, 83% yield, ClogP 4.30.1H NMR : δ 0.90–0-94 , 1.39–1.53 , 2.01–2.06 , 4.30–4.34 , 7.02–7.05 , 7.63–7.67 , 8.47–8.50 , 9.01–9.02 , 9.18–9.21 ; 13 C NMR : δ 14.0 (CH3), 22.4 (CH2), 28.0 (CH2), 28.4 (CH2), 70.1 (OCH2), 106.1 (Ar), 123.1 (Ar), 124.5 (Ar), 127.7 (Ar), 132.7 (Ar), 137.2 (Ar), 139.2 (Ar), 150.1 (Ar), 160.4 (Ar). Anal. calcd for C14H16N2O3: C, 64.60; H, 6.20; N, 10.76, found: C, 64.39; H, 6.09; N, 10.52.(E)-8-oxy)-5-nitroquinoline (10). Brown sticky oil, 91% yield, ClogP 5.92. 1H NMR : δ 1.54 , 1.60 , 1.78 , 2.07 , 4.94–5.02 , 5.55–5.59 , 7.00–7.03 , 7.61–7.66 , 8.45–8.48 , 8.99–9.00 , 9.17–9.20 ; 13 C NMR : δ 16.9 (CH3), 17.7 (CH3), 25.6 (CH3), 26.1 (CH2), 39.5 (CH2), 67.0 (OCH2), 106.5 (Ar), 118.2 (Ar), 123.1 (CH=), 123.5 (Ar), 124.4 (Ar), 127.6 (Ar), 131.9 (CH=), 132.7 (C), 137.2 (Ar), 139.3 (C), 142.3 (Ar), 150.0 (Ar), 160.1 (Ar). Anal. calcd for C19H22N2O3: C, 69.92; H, 6.79; N, 8.58, found: C, 69.45; H, 6.58; N, 8.22.5-nitro-8-oxy)quinoline (11). The compound exists as a mixture of E/Z isomers (peaks listed only for the major isomer). Dark brown sticky oil, 77% yield, ClogP 7.95. 1H NMR : δ 1.50 , 1.52 , 1.59 , 1.77 , 1.87–2.07 , 4.91–5.02 , 5.56 , 6.97–7.00 , 7.57–7.60 , 8.41–8.44 , 8.95 , 9.12–9.15 ; 13 C NMR : δ 16.0 (CH3), 16.8 (CH3), 17.6 (CH3), 25.4 (CH3), 26.0 (CH2), 26.6 (CH2), 39.2 (CH2), 39.6 (CH2), 66.7 (OCH2), 106.3 (Ar), 118.0 (CH=), 123.0 (Ar), 123.4 (CH=), 124.0 (Ar), 124.3 (CH=), 127.4 (C), 131.2 (Ar), 132.4 (C), 135.5 (C), 137.2 (Ar), 139.4 (C), 142.3 (Ar), 150.0 (Ar), 160.1 (Ar). Anal. calcd for C24H30N2O3: C, 73.07; H, 7.67; N, 7.10, found: C, 72.80; H, 7.49; N, 6.88.ethyl 2-((5-nitroquinolin-8-yl)oxy)acetate (12). Ochre solid, 88% yield, ClogP 2.33, mp = 178–179 °C [Lit. mp = 174–175 °C]. Characterisation data are in agreement with those reported in the literature2-((5-nitroquinolin-8-yl)oxy)acetic acid (13). Yellow solid; 81% yield, ClogP 1.47, mp = 205–207 °C [Lit. mp = 205–210 °C]. Characterisation data are in agreement with those reported in the literature8-(benzyloxy)-5-nitroquinoline (14). Dark red sticky solid, 82% yield, CloP 3.95. 1H NMR : δ 5.48 , 6.97–7.01 , 7.26–7.37 , 7.45–7.48 , 7.60–7.64 , 8.32–8.37 , 8.99–9.01 , 9.09–9.15 ; 13 C NMR : δ 71.4 (OCH2), 107.2 (Ar), 123.0 (Ar), 124.5 (Ar), 127.1 (Ar), 127.3 (Ar), 128.4 (Ar), 128.9 (Ar), 132.4 (Ar), 135.3 (Ar), 137.6 (Ar), 139.5 (Ar), 150.2 (Ar), 159.7 (Ar). Anal. calcd for C16H12N2O3: C, 68.56; H, 4.32; N, 9.99, found: C, 68.82; H, 4.41; N, 10.16.8-((2-methylbenzyl)oxy)-5-nitroquinoline (15). Yellow solid, 93% yield, ClogP 4.40, mp = 126–128 °C. 1H NMR : δ 2.45 , 5.50 , 7.06–7.10 , 7.19–7.26 , 7.44–7.46 , 7.68–7.72 , 8.46–8.49 , 9.05–9.06 , 9.22–9.25 ; 13 C NMR : δ 19.1 (CH3), 70.2 (OCH2), 107.2 (Ar), 123.2 (Ar), 124.6 (Ar), 126.2 (Ar), 127.4 (Ar), 128.2 (Ar), 128.6 (Ar), 130.7 (Ar), 132.8 (Ar), 133.0 (Ar), 136.4 (Ar), 137.8 (Ar), 139.4 (Ar), 150.2 (Ar), 159.8 (Ar). Anal. calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52, found: C, 69.01; H, 4.59; N, 9.27.5-nitro-8-((2-(trifluoromethyl)benzyl)oxy)quinoline (16). Yellow solid, 82% yield, ClogP 4.83, mp = 145–147 °C. 1H NMR : δ 5.66 , 6.93–6.96 , 7.36–7.41 , 7.47–7.52 , 7.63–7.69 , 7.75–7.77 , 8.35–8.38 , 9.02–9.04 , 9.12–9.16 ; 13 C NMR : δ 67.3 (OCH2), 107.2 (Ar), 123.0 , 124.3 , 124.6 (Ar), 126.0 (Ar), 126.1 (Ar), 127.3 (Ar), 127.9 (Ar), 128.2 (Ar), 132.6 , 132.8 (Ar), 133.9 (Ar), 138.0 (Ar), 139.3 (Ar), 150.3 (Ar), 159.0 (Ar); 19 F NMR (564.7 MHz CDCl3): δ − 58.93 . Anal. calcd for C17H11F3N2O3: C, 58.63; H, 3.18; N, 8.04, found: C, 58.40; H, 3.02; N, 7.90.2-(((5-nitroquinolin-8-yl)oxy)methyl)benzonitrile (17). Yellowish solid, 82% yield, ClogP 3.52, mp = 201–203 °C. 1H NMR : δ 5.68 , 7.09–7.13 , 7.44–7.49 , 7.61–7.81 , 8.44–8.48 , 9.05–9.07 , 9.18–9.22 ; 13 C NMR : δ 68.8 (OCH2), 107.2 (Ar), 111.1 (Ar), 116.9 (C≡N), 123.1 (Ar), 124.7 (Ar), 127.2 (Ar), 128.5 (Ar), 129.0 (Ar), 132.7 (Ar), 133.0 (Ar), 133.5 (Ar), 138.4 (Ar), 138.8 (Ar), 139.3 (Ar), 150.4 (Ar), 158.9 (Ar). Anal. calcd for C17H11N3O3: C, 66.88; H, 3.63; N, 13.76, found: C, 66.40; H, 3.50; N, 13.61.5-nitro-8-((2-nitrobenzyl)oxy)quinoline (18). Yellow solid, 82% yield, ClogP 3.61, mp = 208–210 °C. 1H NMR : δ 5.94 , 7.07–7.10 , 7.55–7.57 , 7.72–7.77 , 8.02–8.04 , 8.25 8.28 , 8.46–8.49 , 9.10 , 9.23–9.26 ; 13 C NMR : δ 68.3 (OCH2), 107.2 (Ar), 124.7 (Ar), 125.4 (Ar), 128.4 (Ar), 128.9 (Ar), 132.8 (Ar), 134.6 (Ar), 127.2 (Ar), 150.5 (Ar). Anal. calcd for C16H11N3O5: C, 59.08; H, 3.41; N, 12.92, found: C, 59.29; H, 3.57; N, 13.20.8-((2-bromobenzyl)oxy)-5-nitroquinoline (19). Yellow solid, 74% yield, ClogP 4.81, mp = 144–146 °C. 1H NMR : δ 5.61 , 7.00–7.03 , 7.20–7.31 , 7.57–7.64 , 7.71–7.75 , 8.45–8.48 , 9.10–9.11 , 9.23–9.26 ; 13 C NMR : δ 70.7 (OCH2), 107.3 (Ar), 122.0 (Ar), 123.1 (Ar), 124.7 (Ar), 127.4 (Ar), 127.9 (Ar), 128.6 (Ar), 129.8 (Ar), 132.7 (Ar), 132.9 (Ar), 134.5 (Ar), 138.0 (Ar), 139.5 (Ar), 150.4 (Ar), 159.3 (Ar). Anal. calcd for C16H11BrN2O3: C, 53.50; H, 3.09; N, 7.80, found: C, 53.12; H, 2.91; N, 7.55.8-((3-methylbenzyl)oxy)-5-nitroquinoline (20). Yellow solid, 78% yield, ClogP 4.45, mp = 94–96 °C. 1H NMR : δ 2.35 , 5.51 , 7.04–7.07 , 7.14–7.15 , 7.26–7.31 , 7.68–7.73 , 8.42–8.45 , 9.07–9.08 , 9.22–9.24 ; 13 C NMR : δ 21.4 (CH3), 71.6 (OCH2), 107.4 (Ar), 123.1 (Ar), 124.2 (Ar), 124.6 (Ar), 127.5 (Ar), 127.8 (Ar), 128.8 (Ar), 129.3 (Ar), 132.9 (Ar), 135.2 (Ar), 138.7 (Ar), 150.1 (Ar). Anal. calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52, found: C, 69.00; H, 4.52; N, 9.29.8-((3-methoxybenzyl)oxy)-5-nitroquinoline (21). White solid, 85% yield, ClogP 3.87, mp = 100–101 °C. 1H NMR : δ 3.80 , 5.55 , 6.88–6.90 , 7.07–7.10 , 7.28–7.34 , 8.43–8.45 , 9.09 , 9.22–9.24 ; 13 C NMR : δ 55.3 (OCH3), 71.4 (OCH2), 107.4 (Ar), 112.7 (Ar), 113.8 (Ar), 119.2 (Ar), 123.1 (Ar), 124.6 (Ar), 127.3 (Ar), 130.0 (Ar), 132.6 (Ar), 137.0 (Ar), 137.8 (Ar), 139.7 (Ar), 150.3 (Ar), 159.8 (Ar), 160.1 (Ar). Anal. calcd for C17H14N2O4: C, 65.80; H, 4.55; N, 9.03, found: C, 65.99; H, 4.60; N, 9.19.8-((3-fluorobenzyl)oxy)-5-nitroquinoline (22). Yellow sticky oil, 65% yield, ClogP 4.09. 1H NMR : δ 5.55 , 7.03–7.08 , 7.24–7.31 , 7.36–7.40 , 7.71–7.75 , 8.45–8.47 , 9.09–9.11 , 9.23–9.25 ; 13 C NMR : δ 70.7 (OCH2), 107.2 (Ar), 114.1 , 115.5 , 122.6 , 123.1 (Ar), 124.7 (Ar), 127.2 (Ar), 130.6 , 132.6 (Ar), 137.9 (Ar), 139.6 (Ar), 146.0 (Ar), 150.4 (Ar), 159.5 (Ar), 164.1 (Ar); 19 F NMR : δ − 110.63 . Anal. calcd for C16H11FN2O3: C, 64.43; H, 3.72; N, 9.39, found: C, 64.69; H, 3.79; N, 9.48.8-((3-chlorobenzyl)oxy)-5-nitroquinoline (23). Yellow solid, 82% yield, ClogP 4.66, mp = 126–128 °C. 1H NMR : δ 5.50 , 7.01–7.04 , 7.31–7.33 , 7.36–7.39 , 7.50 , 7.69–7.73 , 8.42–8.45 , 9.06–9.08 , 9.20–9.23 ; 13 C NMR : δ 70.6 (OCH2), 107.2 (Ar), 123.1 (Ar), 124.7 (Ar), 125.2 (Ar), 127.2 (Ar), 128.7 (Ar), 130.3 (Ar), 132.7 (Ar), 134.9 (Ar), 137.3 (Ar), 139.5 (Ar), 150.4 (Ar), 159.4 (Ar). Anal. calcd for C16H11ClN2O3: C, 61.06; H, 3.52; N, 8.90, found: C, 61.48; H, 3.59; N, 8.99.8-((3-bromobenzyl)oxy)-5-nitroquinoline (24). Beige solid, 87% yield, ClogP 4.81, mp = 141–143 °C. 1H NMR : δ 5.50 , 7.04 , 7.24–7.29 , 7.43–7.49 , 7.66 , 7.70–7.74 , 8.44–8.47 , 9.08–9.09 , 9.22–9.25 ; 13 C NMR : δ 70.6 (OCH2), 107.2 (Ar), 123.0 (Ar), 123.1 (Ar), 124.7 (Ar), 125.7 (Ar), 127.2 (Ar), 130.1 (Ar), 130.5 (Ar), 131.6 (Ar), 132.7 (Ar), 137.6 (Ar), 138.0 (Ar), 139.5 (Ar), 150.4 (Ar), 151.3 (Ar), 159.4 (Ar). Anal. calcd for C16H11BrN2O3: C, 53.50; H, 3.09; N, 7.80, found: C, 53.19; H, 3.00; N, 7.67.5-nitro-8-((3-nitrobenzyl)oxy)quinoline (25). Yellow sticky oil, 78% yield, ClogP 3.69. 1H NMR : δ 5.60 , 7.06–7.09 , 7.59–7.64 , 7.72–7.76 , 7.88–7.91 , 8.21–8.25 , 8.41 , 8.45–4.48 , 9.08–9.10 , 9.21–9.25 ; 13 C NMR : δ 70.2 (OCH2), 107.2 (Ar), 122.3 (Ar), 123.6 (Ar), 124.8 (Ar), 127.1 (Ar), 130.1 (Ar), 132.8 (Ar), 133.2 (Ar), 137.4 (Ar), 150.5 (Ar). Anal. calcd for C16H11N3O5: C, 59.08; H, 3.41; N, 12.92, found: C, 59.49; H, 3.61; N, 13.07.5-nitro-8-((3-(trifluoromethyl)benzyl)oxy)quinoline (26). Pink solid, 76% yield, ClogP 4.83, mp = 106–109 °C. 1H NMR : δ 5.56 , 7.04–7.07 , 7.51–7.56 , 7.60–7.62 , 7.70–7.78 , 8.44–8.47 , 9.08–9.09 , 9.22–9.25 ; 13 C NMR : δ 70.8 (OCH2), 107.3 (Ar), 123.15 (Ar), 124.0 , 124.7 (Ar), 125.4 (Ar), 125.5 (Ar), 127.2 (Ar), 129.5 (Ar), 130.6 (Ar), 132.9 (Ar), 136.3 (Ar), 150.3 (Ar), 159.2 (Ar); 19 F NMR (564.7 MHz CDCl3): δ − 60.92 . Anal. calcd for C17H11F3N2O3: C, 58.63; H, 3.18; N, 8.04, found: C, 58.96; H, 3.26; N, 8.15.8-((4-methylbenzyl)oxy)-5-nitroquinoline (27). Yellow solid, 66% yield, ClogP 4.45, mp = 95–97 °C. 1H NMR : δ 2.20 , 5.36 , 6.91–6.94 , 7.05–7.07 , 7.27–7.30 , 7.49–7.52 , 8.23–8.26 , 8.88 , 8.99–9.02 ; 13 C NMR : δ 21.1 (CH3), 71.3 (OCH2), 107.2 (Ar), 122.8 (Ar), 124.4 (Ar), 127.3 (Ar), 129.4 (Ar), 132.2 (Ar), 137.3 (Ar), 138.1 (Ar), 139.3 (Ar), 150.0 (Ar), 159.7 (Ar). Anal. calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52, found: C, 69.59; H, 4.87; N, 9.69.8-((4-(methylthio)benzyl)oxy)-5-nitroquinoline (28). Brown solid, 71% yield, ClogP 4.51, mp = 123–128 °C. 1H NMR : δ 2.43 , 5.46 , 7.02–7.05 , 7.20–7.23 , 7.38–7.41 , 7.66–7.70 , 8.39–8.42 , 9.03 , 9.18–9.21 ; 13 C NMR : δ 22.0 (SCH3), 77.6 (OCH2), 114.0 (Ar), 129.6 (Ar), 131.0 (Ar), 133.0 (Ar), 134.1 (Ar), 134.5 (Ar), 138.2 (Ar), 139.0 (Ar), 140.2 (Ar), 144.1 (Ar), 145.8 (Ar), 155.8 (Ar), 156.3 (Ar), 157.8 (Ar), 166.0 (Ar). Anal. calcd for C17H14N2O3S: C, 62.56; H, 4.32; N, 8.58, found: C, 62.82; H, 4.41; N, 8.69.8-((4-fluorobenzyl)oxy)-5-nitroquinoline (29). Yellow solid, 69% yield, ClogP 4.09, mp = 177–179 °C. 1H NMR : δ 5.48 , 7.04–7.11 , 7.47–7.52 , 7.68–7.72 , 8.43–8.46 , 9.05–9.06 , 9.20–9.22 ; 13 C NMR : δ 70.9 (OCH2), 107.5 (Ar), 115.9 , 123.1 (Ar), 124.7 (Ar), 127.5 (Ar), 129.3 , 130.9 (Ar), 133.4 (Ar), 137.8 (Ar), 150.0 (Ar), 159.2 (Ar), 161.1 (Ar-F), 164.4 (Ar); 19 F NMR : δ −111.83 . Anal. calcd for C16H11FN2O3: C, 64.43; H, 3.72; N, 9.39, found: C, 64.02; H, 3.62; N, 9.25.8-((4-chlorobenzyl)oxy)-5-nitroquinoline (30). Yellow solid, 81% yield, ClogP 4.66, mp = 152–153 °C. 1H NMR : δ 5.46 , 7.42–7.60 , 7.81–7.86 , 8.54–8.57 , 9.00–9.03 ; 13 C NMR : δ 70.3 (OCH2), 108.3 (Ar), 125.3 (Ar), 128.1 (Ar), 129.1 (Ar), 130.3 (Ar), 132.2 (Ar), 135.5 (Ar), 150.8 (Ar). Anal. calcd for C16H11ClN2O3: C, 61.06; H, 3.52; N, 8.90, found: C, 61.38; H, 3.65; N, 9.12.8-((4-bromobenzyl)oxy)-5-nitroquinoline (31). Beige solid, 85% yield, ClogP 4.81, mp = 165–167 °C. 1H NMR : δ 5.48 , 7.00–7.04 , 7.37–7.40 , 7.51–7.54 , 7.69–7.74 , 8.42–8.46 , 9.07–9.08 , 9.21–9.25 ; 13 C NMR : δ 70.7 (OCH2), 107.2 (Ar), 122.4 (Ar), 123.1 (Ar), 124.6 (Ar), 127.2 (Ar), 128.9 (Ar), 132.1 (Ar), 132.6 (Ar), 134.3 (Ar), 137.9 (Ar), 139.5 (Ar), 150.3 (Ar), 159.4 (Ar). Anal. calcd for C16H11BrN2O3: C, 53.50; H, 3.09; N, 7.80, found: C, 53.19; H, 3.00; N, 7.69.8-((4-iodobenzyl)oxy)-5-nitroquinoline (32). Brown solid, 78% yield, ClogP 5.07, mp = 195–197 °C. 1H NMR : δ 5.44 , 7.37–7.44 , 7.81–7.86 , 8.55–8.57 , 9.02–9.04 ; 13 C NMR : δ 70.5 (OCH2), 108.2 (Ar), 122.6 (Ar), 125.3 (Ar), 128.1 (Ar), 129.2 (Ar), 130.6 (Ar), 132.2 (Ar), 136.2 (Ar), 137.2 (Ar), 137.7 (Ar), 137.8 (Ar), 139.2 (Ar), 150.8 (Ar), 159.8 (Ar). Anal. calcd for C16H11IN2O3: C, 47.31; H, 2.73; N, 6.90, found: C, 47.58; H, 2.80; N, 6.99.4-(((5-nitroquinolin-8-yl)oxy)methyl)benzonitrile (33). Brown solid, 94% yield, ClogP 3.38, mp = 208–212 °C. 1H NMR : δ 5.57 , 7.39–7.43 , 7.73–7.76 , 7.81–7.86 , 7.90–7.93 , 8.52–8.55 , 8.99–9.03 ; 13 C NMR : δ 70.1 (OCH2), 108.3 (Ar), 111.3 (Ar), 119.2 (CN), 122.6 (Ar), 125.3 (Ar), 128.0 (Ar), 128.7 (Ar), 132.2 (Ar), 133.0 (Ar), 138.0 (Ar), 139.2 (Ar), 142.2 (Ar), 150.9 (Ar), 159.6 (Ar). Anal. calcd for C17H11N3O3: C, 66.88; H, 3.63; N, 13.76, found: C, 67.00; H, 3.70; N, 13.82.5-nitro-8-((4-(trifluoromethyl)benzyl)oxy)quinoline (34). Brown solid, 81% yield, ClogP 4.83, mp = 128–130 °C. 1H NMR : δ 5.57 , 6.99–7.02 , 7.64–7.71 , 8.40–8.43 , 9.05–9.06 , 9.17–9.20 ; 13 C NMR : δ 70.6 (OCH2), 107.3 (Ar), 123.1 (Ar), 123.9 , 124.7 (Ar), 125.9 , 126.6 (Ar), 127.2 , 130.6 , 132.9 (Ar), 138.1 (Ar), 139.2 (Ar), 139.3 (Ar), 150.3 (Ar), 159.1 (Ar). 19 F NMR : δ − 61.32 . Anal. calcd for C17H11F3N2O3: C, 58.63; H, 3.18; N, 8.04, found: C, 58.49; H, 3.15; N, 7.96.5-nitro-8-((4-nitrobenzyl)oxy)quinoline (35). Yellow sticky oil, 98% yield, ClogP 3.69. 1H NMR : δ 5.52 , 7.35–7.38 , 8.05–8.08 ; 13 C NMR : δ 70.1 (OCH2), 108.4 (Ar), 111.4 (Ar), 122.5 (Ar), 125.4 (Ar), 128.7 (Ar), 133.1 (Ar), 138.2 (Ar), 139.0 (Ar), 142.4 (Ar), 150.7 (Ar), 159.9 (Ar). Anal. calcd for C16H11N3O5: C, 59.08; H, 3.41; N, 12.92, found: C, 59.28; H, 3.49; N, 12.99.8-oxy)-5-nitroquinoline (36). Yellow solid, 88% yield, ClogP 4.24, mp = 193–195 °C. 1H NMR : δ 5.52 , 6.94–6.99 , 7.26–7.29 , 7.32–7.40 , 7.65–7.69 , 8.52–8.55 , 9.03–9.05 , 9.20 ; 13 C NMR  δ 59.7 (OCH2), 106.6 (Ar), 111.6 (Ar), 111.8 , 123.1 (Ar), 124.5 (Ar), 127.3 (Ar), 131.4 (Ar), 131.6 , 132.6 (Ar), 139.8 (Ar), 143.4 (Ar), 150.3 (Ar), 159.7 (Ar), 160.4 (ArC-F x 2). 19 F NMR : δ − 111.74 . Anal. calcd for C16H10F2N2O3: C, 60.76; H, 3.19; N, 8.86, found: C, 60.89; H, 3.22; N, 8.95.8-oxy)-5-nitroquinoline (37). Brownish sticky oil, 81% yield, ClogP 4.95. 1H NMR : δ 2.32 , 5.49 , 6.98 , 7.07–7.09 , 7.12 , 7.69–7.73 , 8.44–8.46 , 9.08–9.09 , 9.22–9.25 ; 13 C NMR : δ 21.3 (2 × CH3), 71.7 (OCH2), 107.3 (Ar), 123.1 (Ar), 124.5 (Ar), 124.9 (Ar), 127.5 (Ar), 130.1 (Ar), 132.6 (Ar), 135.2 (Ar), 137.7 (Ar), 138.6 (Ar), 139.6 (Ar), 150.3 (Ar), 160.0 (Ar). Anal. calcd for C18H16N2O3: C, 70.12; H, 5.23; N, 9.09, found: C, 70.29; H, 5.27; N, 9.18.8-oxy)-5-nitroquinoline (38). Yellow solid, 71% yield, ClogP 4.24, mp = 135–136 °C. 1H NMR : δ 5.51 , 6.78–6.83 , 7.02–7.09 , 7.72–7.75 , 8.45–8.47 , 9.09–9.11 , 9.22–9.24 ; 13 C NMR : δ 70.1 (OCH2), 103.9 , 107.1 (Ar), 109.8 , 123.1 (Ar), 124.7 (Ar), 127.0 (Ar), 132.6 (Ar), 138.3 (Ar), 139.4 , 139.6 (Ar), 150.5 (Ar), 159.1 (Ar), 163.3 , 163.4 . Anal. calcd for C16H10F2N2O3: C, 60.76; H, 3.19; N, 8.86, found: C, 60.50; H, 3.13; N, 8.77.8-((3-chloro-5-methoxybenzyl)oxy)-5-nitroquinoline (39). Yellow solid, 72% yield, ClogP 4.72, mp = 154–155 °C. 1H NMR : δ 3.82 , 5.42 , 7.01–7.03 , 7.15–7.16 , 7.50–7.52 , 7.65–7.67 , 7.82–7.85 , 8.56–8.59 , 8.98–9.04 ; 13 C NMR : δ 56.2 (OCH3), 68.8 (OCH2), 108.0 (Ar), 113.9 (Ar), 115.3 (Ar), 122.6 (Ar), 125.3 (Ar), 125.6 (Ar), 128.2 (Ar), 132.2 (Ar), 132.6 (Ar), 134.7 (Ar), 137.8 (Ar), 139.2 (Ar), 150.7 (Ar), 160.0 (Ar), 160.8 (Ar). Anal. calcd for C17H13ClN2O4: C, 59.23; H, 3.80; N, 8.13, found: C, 59.39; H, 3.86; N, 8.20.8-oxy)-5-nitroquinoline (40). Red solid, 78% yield, ClogP 5.26, mp = 122–132 °C. 1H NMR : δ 5.44 , 6.99–7.02 , 7.32–7.34 , 7.43–7.46 , 7.59 , 7.67–7.71 , 8.40–8.43 , 9.03–9.04 , 9.16–9.19 ; 13 C NMR : δ 70.0 (OCH2), 107.1 (Ar), 123.1 (Ar), 124.7 (Ar), 126.5 (Ar), 127.2 (Ar), 129.1 (Ar), 130.9 (Ar), 132.6 (Ar), 133.1 (Ar), 135.5 (Ar), 138.1 (Ar), 139.4 (Ar), 150.5 (Ar), 159.1 (Ar). Anal. calcd for C16H10Cl2N2O3: C, 55.04; H, 2.89; N, 8.02, found: C, 55.29; H, 2.98; N, 8.11.8--5-nitroquinoline (41). Red solid, 81% yield, ClogP 5.12, mp = 177.0–178.0 °C. 1H NMR : δ 5.87 , 7.52–7.57 , 7.61–7.64 , 7.74–7.81 , 7.95–7.99 , 8.11–8.14 , 8.55–8.58 , 8.89–8.90 , 8.97–9.00 ; 13 C NMR : δ 69.8 (OCH2), 108.3 (Ar), 122.6 (Ar), 124.4 (Ar), 125.3 (Ar), 125.9 (Ar), 126.6 (Ar), 127.0 (Ar), 127.6 (Ar), 128.2 (Ar), 129.0 (Ar), 129.5 (Ar), 131.7 (Ar), 131.9 (Ar), 132.1 (Ar), 133.8 (Ar), 137.7 (Ar), 139.2 (Ar), 150.6 (Ar), 160.1 (Ar). Anal. calcd for C20H14N2O3: C, 72.72; H, 4.27; N, 8.48, found: C, 72.89; H, 4.31; N, 8.59.2-(((5-nitroquinolin-8-yl)oxy)methyl)isoindoline-1,3-dione (42). Yellow solid, 77% yield, ClogP 3.07, mp = 210 °C (dec.); 1H NMR : δ 5.92 , 7.64–7.67 , 7.80–7.84 , 7.91–8.00 , 8.53–8.56 , 8.94–8.99 ; 13 C NMR : δ 67.0 (OCH2), 110.5 (Ar), 122.5 (Ar), 124.3 (Ar), 127.5 (Ar), 131.7 (Ar), 132.3 (Ar), 135.7 (Ar), 139.1 (Ar), 151.0 (Ar), 157.8 (Ar), 167.3 (2 x N-C = O). Anal. calcd for C18H11N3O5: C, 61.89; H, 3.17; N, 12.03, found: C, 61.70; H, 3.13; N, 11.96.2-((5-nitroquinolin-8-yl)oxy)-1-phenylethan-1-one (43). Yellow-brow solid, 81% yield, ClogP 3.14, mp = 196–198 °C. 1H NMR : δ 5.80 , 6.90–6.92 , 7.51–7.56 , 7.63–7.66 , 7.69–7.74 , 8.03–8.06 , 8.42–8.45 , 9.06–9.08 , 9.22–9.25 ; 13 C NMR : δ 71.8 (OCH2), 108.5 (Ar), 122.6 (Ar), 123.3 (Ar), 125.3 (Ar), 127.9 (Ar), 128.5 (Ar), 129.4 (Ar), 132.2 (Ar), 134.5 (Ar), 137.8 (Ar), 138.9 (Ar), 150.7 (Ar), 159.8 (Ar), 193.7 (C = O). Anal. calcd for C17H12N2O4: C, 66.23; H, 3.92; N, 9.09, found: C, 66.36; H, 3.99; N, 9.19.1–(3-nitrophenyl)-2-((5-nitroquinolin-8-yl)oxy)ethan-1-one (44). Dark brown solid, 65% yield, ClogP 3.05, mp = 203–205 °C. 1H NMR : δ 5.82 , 7.07–7.10 , 7.76–7.85 , 8.48–8.53 , 8.92 , 9.15 , 9.37–9.39 . Anal. calcd for C17H11N3O6: C, 57.80; H, 3.14; N, 11.89, found: C, 57.96; H, 3.18; N, 11.99.1–(4-nitrophenyl)-2-((5-nitroquinolin-8-yl)oxy)ethan-1-one (45). Brown solid, 73% yield, ClogP 3.05, mp = 190 °C (dec.). 1H NMR : δ 6.11 , 7.38–7.41 , 7.83–7.87 , 8.27–8.30 , 8.39–8.42 , 8.46–8.49 , 9.01–9.03 . Anal. calcd for C17H11N3O6: C, 57.80; H, 3.14; N, 11.89, found: C, 57.62; H, 3.10; N, 11.78.2-((5-nitroquinolin-8-yl)oxy)-1-(thiophen-3-yl)ethan-1-one (46). Brown solid, 86% yield, ClogP 2.92, mp = 128–130 °C. 1H NMR : δ 5.89 , 7.29–7.31 , 7.63–7.64 , 7.72–74 , 7.85–7.89 , 8.50–8.52 , 8.79–8.80 , 9.03–7.06 ; 13 C NMR : δ 72.0 (OCH2), 108.4 (Ar), 122.6 (Ar), 125.3 (Ar), 127.0 (thiophene), 127.9 (thiophene), 128.2 (Ar), 132.2 (thiophene), 135.0 (Ar), 137.9 (Ar), 139.0 (Ar), 139.1 (thiophene), 150.7 (Ar), 159.7 (Ar), 188.4 (C = O). Anal. calcd for C15H10N2O4S: C, 57.32; H, 3.21; N, 8.91, found: C, 57.50; H, 3.26; N, 8.99.1-bromo-2-((4-nitrophenoxy)methyl)benzene (47). White solid, 89% yield, ClogP 4.73, mp = 122–123 °C. Characterisation data are in agreement with those reported in the literature1-nitro-2-((4-nitrophenoxy)methyl)benzene (48). Pinkish solid, 97% yield, ClogP 3.53, mp = 130–132 °C. 1H NMR : δ 5.62 , 7.08–7.12 , 7.55–7.59 , 7.73–7.77 , 7.85–7.87 , 8.22–8.27 ; 13 C NMR : δ 67.4 (OCH2), 115.0 (Ar), 125.3 (Ar), 126.1 (Ar), 129.0 (Ar), 132.2 (Ar), 134.2 (Ar), 142:2 (Ar), 147.0 (Ar), 163.0 (Ar). Anal. calcd for C13H10N2O5: C, 56.94; H, 3.68; N, 10.22, found: C, 56.74; H, 3.61; N, 10.14.1-bromo-3-((4-nitrophenoxy)methyl)benzene (49). White solid, 90% yield, ClogP 4.73, mp = 91–92 °C. Characterisation data are in agreement with those reported in the literature1-nitro-3-((4-nitrophenoxy)methyl)benzene (50). Yellow solid, 60% yield, ClogP 3.61, mp = 125–128 °C. 1H NMR : δ 5.28 , 7.07–7.11 , 7.64–7.66 , 7.79–7.81 , 8.25–8.27 , 8.35 ; 13 C NMR : δ 69.2 (OCH2), 114.9 (Ar), 122.3 (Ar), 123.5 (Ar), 126.1 (Ar), 129.9 (Ar), 133.1 (Ar), 137.7 (Ar), 142.2 (Ar), 148.6 (Ar), 163.0 (Ar). Anal. calcd for C13H10N2O5: C, 56.94; H, 3.68; N, 10.22, found: C, 56.74; H, 3.60; N, 10.16.1-((4-nitrophenoxy)methyl)-3-(trifluoromethyl)benzene (51). White solid, 72% yield, ClogP 4.75, mp = 70–74 °C. 1H NMR : δ 5.23 , 7.05–7.09 , 7.57–7.73 , 8.23–8.27 ;19F NMR : δ − 66.64  Anal. calcd for C14H10F3NO3: C, 56.57; H, 3.39; N, 4.71, found: C, 56.64; H, 3.41; N, 4.73.(3-nitrobenzyl)(4-nitrophenyl)sulfane (52). Yellow sticky oil, 70% yield, ClogP 3.87. 1H NMR : δ 4.26 , 7.26–7.30 , 7.43–7.47 , 7.63–7.65 , 8.02–8.08 , 8.19–8.20 ; 13 C NMR : δ 36.5 (SCH2), 122.9 (Ar), 123.6 (Ar), 124.1 (Ar), 127.3 (Ar), 129.9 (Ar), 134.7 (Ar), 138.0 (Ar), 145.5 (Ar), 145.7 (Ar), 148.5 (Ar). Anal. calcd for C13H10N2O4S: C, 53.79; H, 3.47; N, 9.65, found: C, 53.27; H, 3.43; N, 9.60.1-fluoro-4-((4-nitrophenoxy)methyl)benzene (53). White solid, 92% yield, ClogP 4.01, mp = 123–124 °C. [Lit. mp = 122.4 °C]. 19 F NMR : δ − 110.05 . Characterisation data are in agreement with those reported in the literature1-chloro-4-((4-nitrophenoxy)methyl)benzene (54). White solid, 98% yield, ClogP 4.58, mp = 113–115 °C. [Lit. mp = 114–115 °C]. Characterisation data are in agreement with those reported in the literature1-bromo-4-((4-nitrophenoxy)methyl)benzene (55). White solid, 89% yield, ClogP 4.73, mp = 120–121 °C. 1H NMR : δ 5.13 , 7.03–7.05 , 7.32–7.34 , 7.56–7.58 , 8.22–8.25 . Anal. calcd for C13H10BrNO3: C, 50.67; H, 3.27; N, 4.55, found: C, 50.69; H, 3.29; N, 4.58.4-((4-nitrophenoxy)methyl)benzonitrile (56). White solid, 94% yield, ClogP 3.30, mp = 157–158 °C. 1H NMR : δ 5.25 , 7.03–7.07 , 7.57–7.59 , 7.72–7.74 , 8.22–8.26 ; 13 C NMR : δ 69.5 (OCH2), 112.4 (Ar), 114.8 (Ar), 118.4 (C≡N), 126.0 (Ar), 127.7 (Ar), 132.6 (Ar), 140.9 (Ar), 142.1 (Ar), 163.0 (Ar). Anal. calcd for C14H10N2O3: C, 66.14; H, 3.96; N, 11.02, found: C, 66.26; H, 3.99; N, 11.11.(4-bromobenzyl)(4-nitrophenyl)sulfane (57). Yellow solid, 87% yield, ClogP 4.99, mp = 133–134° C. 1H NMR : δ 4.21 , 7.27–7.29 , 7.33–7.7.35 , 7.47–7.49 , 8.11–8.13 ; 13 C NMR : δ 36.5 (SCH2), 121.8 (Ar), 124.0 (Ar), 126.9 (Ar), 130.4 (Ar), 132.0 (Ar), 134.6 (Ar), 145.5 (Ar), 146.5 (Ar). Anal. calcd for C13H10BrNO2S: C, 48.16; H, 3.11; N, 4.32, found: C, 48.29; H, 3.14; N, 4.39.1,3-difluoro-2-((4-nitrophenoxy)methyl)benzene (58). White solid, 95% yield, ClogP 4.15, mp = 125–127 °C. 1H NMR : δ 5.29 , 7.18–7.23 , 7.26–7.29 , 7.53–7.61 , 8.23–8.25 ; 13 C NMR : δ 59.0 (OCH2), 111.9 , 112.4 , 115.7 (2 x Ar), 126.4 (2 x Ar), 132.6 , 141.8 (Ar), 161.6 , 161.7 , 163.7 (Ar); 19 F NMR : δ − 118.37 . Anal. calcd for C13H9F2NO3: C, 58.87; H, 3.42; N, 5.28, found: C, 58.99; H, 3.46; N, 5.32.1,2-dichloro-4-((4-nitrophenoxy)methyl)benzene (59). White solid, 89% yield, ClogP 5.17, 146–147 °C. 1H NMR : δ 5.13 , 7.03–7.05 , 7.29 , 7.50–7.56 , 8.23–8.25 ; 13 C NMR : δ 69.1 (OCH2), 114.8 (Ar), 124.8 (Ar), 126.0 (Ar), 126.6 (Ar), 129.3 (Ar), 130.8 (Ar), 132.6 (Ar), 133.1 (Ar), 135.7 (Ar), 142.0 (Ar), 163.1 (Ar). Anal. calcd for C13H9Cl2NO3: C, 52.38; H, 3.04; N, 4.70, found: C, 52.42; H, 3.06; N, 4.75.2-((4-nitrophenoxy)methyl)isoindoline-1,3-dione (60). Yellow solid, 75% yield, ClogP 2.99, mp = 152–154 °C. [Lit. mp = 160–161 °C]. Characterisation data are in agreement with those reported in the literature2–(4-nitrophenoxy)-1–(3-nitrophenyl)ethan-1-one (61). beige solid, 72% yield, ClogP 2.96, mp = 144–147 °C. 1H NMR : δ 5.46 , 7.03–7.05 , 7.77–7.81 , 8.22–8.25 , 8.35–8.37 , 8.50–8.52 , 8.85 ; 13 C NMR : δ 70.9 (OCH2), 114.8 (Ar), 123.1 (Ar), 126.1 (Ar), 128.5 (Ar), 130.4 (Ar), 133.8 (Ar), 135.2 (Ar), 142.5 (Ar), 148.6 (Ar), 162.4 (Ar), 191.4 (C = O). Anal. calcd for C14H10N2O6: C, 55.64; H, 3.34; N, 9.27, found: C, 55.78; H, 3.39; N, 9.33.4.4.Human PC cell lines  were cultured as previously described8-Hydroxy-5-nitroquinoline (nitroxoline) was purchased from Sigma . All compounds were dissolved in DMSO (stock solutions of 40 mM) and then in culture media for the final working concentrations. The final concentration of DMSO in the experiments was at most 0.27% and showed no cell toxicity.4.5.Cell viability was evaluated by MTT assay , essentially as previously described4.6.3 cells/well, while AsPC-1 were seeded in 6-well plates at 0.5 × 103 cells/well. After cell attachment, PC cell lines were treated for 48 h with compounds 33, 40, or with nitroxoline as indicated. Colonies were fixed when cells in the control vehicle formed colonies consisting of at least 30 cellsClonogenic assay was performed essentially as previously described (5). Capan-2 and BxPC-3 were seeded in 6-well plates at 104.7.50 values were calculated by CompuSyn softwarep values ≤0.05 was estimated as statistically significant.IC5.5.1.,,Starting from the results published on the anti-cancer potential of nitroxoline, our approach was oriented towards a wide exploration of the chemical spare around the phenolic moiety in the nitroxoline skeleton in order to attain newly synthesised derivatives with improved inhibitory activity on the viability of PC cell lines and with different chemical-physical characteristics to finely tune the predicted pharmacokinetic properties. Recent developments have started to address this issue5, we explored the effects of these novel compounds on cell viability in the same PC cells. To identify the most active compounds in the series, preliminary MTT experiments were performed by incubating the three PC cell lines with the 61 derivatives and with the lead compound nitroxoline, for 48 h at one-point screening concentration (40 μM)  (40 μM) . Severalroxoline . A similroxoline . Notably-1 cells , althoug1–13) led to a reduced inhibitory activity against the three cell lines with respect to nitroxoline disregarding the presence of double/triple bonds, functional groups, branched and long chains. Conversely, the insertion of a benzyl group (11–40) provided compounds of similar potency with respect to nitroxoline on AsPC-1  and Capan-2  cells. Preferred positions and substituents on the aryl ring were 3-Br, 4-CN, 3,5-diCH3 and 3,4-diCl. On BxPC-3 cells, these compounds displayed a more interesting scenario being some compounds  equipotent with respect to nitroxoline and compounds 18, 19, 24, 33, 36, 40 even more potent at the same tested concentration. More in detail, only electron-withdrawing substituents  on specific positions at the benzyl ring favoured the inhibitory activity. The presence of a carbonyl spacer between the OCH2 and the phenyl ring (43–46) furnished nitroxoline analogues with discrete effect on the three cell lines, whereas the molecular simplification to 4-nitro(thio)phenol derivatives (47–61) was detrimental for the inhibitory activity against AsPC-1 and Capan-2 cell lines. On BxPC-3 cells, compounds 48, 50, 55, 57 and 59 were endowed with promising activity, despite the substitution of the oxygen with the sulphur led to a loss of biological activity (compare the couples 50/52 and 55/57). Collectively, the deletion of the pyridine nucleus of the parent compound gave worse results. Thus, we selected the best-in-class compounds  for further characterisation of their chemical-physical properties and antiproliferative effects.Structure-activity relationships (SARs) within this library suggested that the functionalization of the OH with aliphatic chains  . Selected compounds affected PC cell viability in a concentration-dependent manner . IC50 values were calculated by CompuSyn software  and selectivity index (SI) values comparable or superior to nitroxoline in the three PC cell lines . In particular, compound 40 has similar selectivity as nitroxoline in Capan-2, but is much more selective in AsPC-1 and BxPC-3, suggesting that compound 40 may be more effective and safer than nitroxoline.We next tested the effects of the most active compound on viability of normal HFF-1 fibroblast cells. Notably, compound 5.2.23 are reported, aiming to describe some properties of the tested compounds that are fundamental for a proper drug development. On the basis of the well-known Lipinski’s rule of five is possible to evaluate the drug-likeness of one compound, i.e., the probability that the molecule will be effective as an oral drug. As one can see, all the evaluated compounds fully comply with the limits of the Lipinski’s rule, supporting their feasible oral use , similarly to the lead compound nitroxoline.In in silico studies rule out the possibility that these compounds could be substrate of P-gp, reinforcing their role as antitumor drugs. Finally, the presence of PAINS (Pan Assay Interference Compounds) that are substructures able to elicit promiscuous pharmacological behaviour, was also evaluated showing as all the compounds are devoid of this attribute.The capability of these compounds to be passively adsorbed in the gastrointestinal tract is an important information influencing their bioavailability after oral administration; even in this case all the examined compounds exhibited promising results. One of the Achilles’ heels of some antitumor drugs is the propensity to be substrate of the permeability glycoprotein (P-gp). This protein promotes the energy-dependent efflux of cytotoxic drugs out of the cytosol and its overexpression in tumour cells leads to multi-drug resistance (MDS), which reduces the efficacy of antitumor treatments. The outcomes extrapolated by 40), we also reported (40 is contained in the yellow area (even if it is to the limit) and is pictured with a red dot, signifying that it is likely able to cross the BBB, it is passively absorbed at the GI level and is not effluxed by P-gp.For the most active analogue  required to be bioavailable after oral administration. For the analogue 40. In 40 we did not obtain a main preference for one protein; thus, further and deep studies could shed light on its putative targets.With the aim to identify the putative target/s of our compounds we also exploited the web-tool SwissTargetPrediction6.in silico drug-likeness and cytotoxicity on normal human fibroblasts. Collectively, our data supported the exploration of this chemical scaffold to propose new compounds with alternative mechanisms of action for the treatment of pancreatic cancer.Our study explored the impact of the chemical modifications of the core nucleus of a well-known antibiotic, nitroxoline, that recently emerged as a promising candidate for its antiproliferative effects on PC cell lines. The functionalization of the phenolic OH with substituted benzyl rings led to an improvement of the biological activity with respect to the parent compound and erlotinib, whereas the introduction of alkyl groups, the deletion of the pyridine ring or the bioisosteric change from oxygen to sulphur were detrimental for the reported activity. Moreover, the most promising compounds were further evaluated in terms of clonogenicity reduction, Click here for additional data file.


In human sections, β2-AR was detectable, and its localization parallels with ECM alterations. Similarly, in IVDs of WT mice, only β2-AR was expressed, and in IVDs of SM/J mice, β2AR expression was stronger accompanied by increased collagen II, collagen XII, decorin as well as decreased cartilage oligomeric matrix protein expression. In addition, norepinephrine stimulation of isolated human IVD cells induced intracellular signaling via ERK1/2 and PKA. For the first time, the existence and functionality of ARs were demonstrated in IVD tissue samples, suggesting that the sympathicus might play a role in IVDD. Further studies will address relevant cellular mechanisms and thereby help to develop novel therapeutic options for IVDD.Healthy and degenerating intervertebral discs (IVDs) are innervated by sympathetic nerves, however, adrenoceptor (AR) expression and functionality have never been investigated systematically. Therefore, AR gene expression was analyzed in both tissue and isolated cells from degenerated human IVDs. Furthermore, human IVD samples and spine sections of wildtype mice (WT) and of a mouse line that develops spontaneous IVD degeneration  were stained for ARs and extracellular matrix (ECM) components. In IVD homogenates and cells  Degenerative changes of the intervertebral disc (IVD) represent a severe health issue resulting in devastating and disabling symptoms like pain, muscle weakness, numbness, and dysesthesia of the upper and lower limbs . In partThe IVD consists of two main parts with different embryologic origins: The ligamentous annulus fibrosus (AF) and the gelatinous nucleus pulposus (NP) . It is wα1a, α1b, α1d, α2a, α2b, α2c, β1, β2, and β3), which results in activation of two major intracellular signaling pathways, ERK1/2 and PKA [α2a- and β2-AR [During the past decades, the role of peripheral sympathetic nerve fibers and their neurotransmitters have gained in importance with regard to degeneration and regeneration of joint tissues like articular cartilage . The key and PKA ,13. Studnd β2-AR . Althougnd β2-AR , only fend β2-AR , on AR end β2-AR , or on pnd β2-AR ,16. The nd β2-AR . At presTherefore, the aim of this study was to characterize the expression profile of all AR subtypes in IVD tissue, as well as in isolated IVD cells, to specify their localization, and to correlate this expression profile with the grade of degeneration. In addition, the expression of TH, the-rate limiting enzyme of NE biosynthesis was examined.α1a- (97.7% of samples), α1b- (46.5%), α2a- (90.7%), α2b- (97.7%), α2c- (97.7%), β1- (100%), and β2-AR (100%) were highly expressed . Several AR genes were expressed at any stage of IVDD, the α1a-, α1b-, α2a-, α2b-, α2c-, β1-AR, and β2-AR  and β2-AR (p < 0.001) compared to the inner annulus and also to the nucleus (α2a p < 0.001 and β2-AR p < 0.001). In addition, significantly more β2-AR-positive cells were counted in the inner annulus compared to the nucleus (p = 0.009). In contrast, no significant differences were detected between α2a-positive cells between inner annulus and nucleus (p = 0.459). We also performed a Western Blot to detect β2-AR, because this AR was expressed at the highest level. However, β2-AR could not be detected in human tissue samples . Both ARs were detectable in the outer zone of AF, however, α2a-AR staining was not as intense as β2-AR . Further samples , possiblAbbreviations: sGAG—sulfated glycosaminoglycans, oa—outer annulus, ia—inner annulus, np—nucleus pulposus, Σ—sum of positive cells in oa + ia + np.Associations of the expression level and localization of the β2-AR with potential changes in the ECM expression were analyzed immunohistochemically. Since there were many analogies between human intervertebral discs and articular joints , we analp < 0.001) compared to WT mice and also in the inner annulus (p < 0.001) and in the nucleus (p < 0.001)  .Parallel to the β2-AR expression changes in the outer AF and NP, alterations in the expression of ECM components were observed in SM/J sections. Type II collagen staining intensity did not increase, but the type II collagen-positive chondrocytic area spread in the same manner as the β2-AR expression. Moreover, type XII collagen staining increased in the interterritorial ECM of SM/J IVDs, while COMP expression slightly decreased compared to WT samples. In addition, decorin staining became stronger in the pericellular and territorial ECM of SM/J mice .α1a-, α2a-, α2b-, α2c-, β1-, and β2-AR; α1a-, α2a-, α2b-, α2c-, β1-, and β2-AR, but in cells of some patients α1b- or β3-AR was not detectable  for 5 or 15 min revealed that both the ERK1/2 and the PKA signaling pathway were activated. After 5 min, only a slight PKA phosphorylation was visible and only after treatment with 10−8 M NE. Here, ERK was not yet activated , and TH was evaluated by RT-PCR in fresh tissue as well as in isolated IVD cells. For RNA isolation, the tissue was minced with a scalpel and further processed according to manufacturers’ instructions . Isolated IVD cells did not require special pre-treatments before RNA isolation. cDNA synthesis was performed using Quantabio qScript cDNA Synthesis Kit , and about 5 ng of cDNA per optimized primer were used for PCR . All primers were obtained from Thermo Fisher Scientific . In step 1, GAPDH band intensity of each individual patient was defined as “1” and AR expression levels were calculated in relation to that value. Then, semiquantitative relative gene expression levels depending on the grade of degeneration were calculated using grade I samples as a calibrator (mean of grade I results was defined as “1”), and correlations were performed with the degree of degeneration. Genes that did not show a detectable Ct value after 40 amplification cycles were deemed to have “zero “expression.The expression of the genes for all adrenoceptor subtypes . Sections of 8 μm were then dewaxed using a standard protocol with xylene and gradually rehydrated. After antigen demasking using citrate buffer ) for 20 min at 95 °C, the endogenous peroxidase  enzyme was blocked as well as non-specific binding sites using secondary antibody-specific horse serum  for 45 min at room temperature. Sections were then incubated with the primary antibodies rabbit anti α2a-AR , rabbit anti α2c-AR , rabbit anti β2-AR , at 4 °C overnight. Specific staining was visualized using horseradish peroxidase labeled secondary antibodies and peroxidase substrate solution . Control experiments were performed with unspecific isotype antibodies. AR-positive cells were counted in all stained sections, and different IVD areas (annulus vs. nucleus), as well as the two mouse genotypes, were compared regarding the level of AR expression. Additional sections were stained with dimethyl methylene blue (DMMB) or safranin-O/fast green to visualize tissue structure and proteoglycan distribution.The protein expression and localization of 3 major adrenoceptors (α2a-AR and β2-AR) in human and murine IVDs was investigated immunohistochemicaly. Human IVD-tissue samples were fixed in 4% paraformaldehyde  in 1x PBS overnight and after rinsed 3 times for 10 min with ddH2O2 for 10 min and blocking with a blocking solution  for 5 min at room temperature, the sections were incubated with the primary antibodies mouse anti-Col II , rabbit anti-Col XII  was used to detect the specific primary antibody bindings. The brownish staining through the oxidation of the DAB was complemented with a nuclear counterstaining with Mayer’s Heamatoxylin  for 10 s at room temperature.Furthermore, the expression of ECM proteins was analyzed in human and murine IVDs . In the case of the Col II staining, the sections were digested using pepsin  for 15 min at 37 °C at first. An enzymatic digestion with hyaluronidase  for 30 min at 37 °C and Proteinase K  for 10 min at 55 °C was conducted for all the antibodies to demask the antigens. After quenching the endogenous peroxidase with 3% H, rabbit at 4 °CMurine samples were processed and stained the same way as the human tissue.https://www.abcam.com/beta-2-adrenergic-receptor-antibody-epr707n-ab182136.html as additional information).IVD tissue and cell protein samples were loaded onto 10% SDS-PAGE and electro-transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature before incubation with primary antibody for β2-AR  at 4 °C overnight. The membranes were washed with TBST  and incubated with HRP-conjugated secondary antibody  for 1 h at room temperature. The target protein expression was detected using the chemiluminescence  reagent. A cell lysate of the human squamous carcinoma cell line A431  was used as a positive control  [2 and 2% O2. After achieving confluence, cells were detached, and pellets were stored at −80 °C until PCR analysis. The used intervertebral discs were moderately degenerated (grade II).According to a method by Tang et al. , the IVDGermany)  with 1% −8 or 10−6 M, Sigma-Aldrich, St. Louis, Missouri, USA). Protein isolation was performed using NucleoSpin RNA/Protein kit .In order to examine whether isolated IVD cells responded to NE, the 2 major AR-dependent signaling pathways, the phosphorylation of PKA and ERK1/2, were investigated. Cells were treated for 5 and 15 min with NE (10IVD protein samples were loaded onto 10% SDS-Page and electro-transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature before incubation with primary antibodies for total ERK , phosphorylated ERK , total PKA , phosphorylated PKA , and GAPDH (#(MA5-15738); Thermo Fisher Scientific, Darmstadt, Hessen, Germany) at 4 °C overnight. The membranes were washed with TBST and incubated with HRP-conjugated secondary antibody  for 1 h at room temperature. The target protein expression was detected using the chemiluminescence  reagent, with GAPDH as the endogenous control.All quantitative gene expression experiments were carried out with samples of at least 5 patients. Data were presented as box plots with medians. Multiple comparisons between groups of different degrees of degeneration were carried out using ANOVA on ranks or Wilcoxon/Mann–Whitney-Test followed by Bonferroni or Dunn’s correction. To analyze associations between AR expression and grade of degeneration, Spearman rank order correlations were performed. p-values less than 0.05 were considered significant. All statistical analyses were performed using SigmaPlot 14.0 software .


TMPRSS2 and ACE2 is a viable treatment strategy to prevent SARS-CoV-2 infection. In particular, inhibitors of androgen receptor (AR) or bromodomain and extraterminal domain (BET) proteins are effective against SARS-CoV-2 infection. AR inhibitors are already approved in the clinic for treatment of prostate cancer and are under investigation in COVID-19 patients; BET inhibitors are also in clinical development for other indications and could be rapidly repurposed for COVID-19.New therapeutic targets are urgently needed against SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic. Results in this study show that targeting the transcriptional regulation of host entry factors  TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2).  The COVID-19 pandemic has become one of the greatest public health challenges in modern times, leading to an unprecedented surge in research efforts to develop vaccines and treatments . The SpiTMPRSS2 transgenic knockout mice have shown that loss of TMPRSS2 can reduce coronavirus replication in lungs, elicit a weaker proinflammatory response, and result in a milder lung pathology   . Oncogen cancers , 9.TMPRSS2 host entry factor in males. This has raised the hypothesis that inhibition of AR activity and down-regulation of TMPRSS2 may prevent SARS-CoV-2 infection  proteins, known epigenetic regulators of AR transcriptional activity . On the other hand, we discovered that the BET inhibitor JQ1 and degrader ZBC260 consistently down-regulated ACE2 in Calu-3  and TMPRSS2 and ACE2 in Caco-2 cells , suggesting that BET proteins may play a role in regulating SARS-CoV-2 entry factor expression. Additionally, in other cell lines originating from the lung lineage with high endogenous expression of AR, TMPRSS2, and ACE2, we found that both androgen and AR antagonists also did not change TMPRSS2 and ACE2 messenger RNA (mRNA) levels in bulk gene expression analysis . The varying endogenous expressions of AR, TMPRSS2, and ACE2 in lung cell lines limit their use in SARS-CoV-2 research; thus, there is a need for understanding their expression patterns in the lung at the single-cell level.To determine whether androgen signaling regulates the expression of SARS-CoV-2 entry factors RS-CoV-2 , 18. DesAR, TMPRSS2, and ACE2 genes will be critical to understanding the biology of SARS-CoV-2 infection. Thus, we performed bioinformatics analysis of published single-cell RNA sequencing (scRNAseq) data of human and murine lungs  lung epithelial cells, including alveolar (AT1 and AT2) epithelial cells, with maximal expression in ciliated and secretory epithelial cells . This suggested that pulmonary TMPRSS2 and ACE2 in alveolar and bronchial cells had the potential to be regulated by AR.Given the complexity of the lungs, which comprise more than 25 distinct cell types including bronchial and alveolar cells –23, idenne lungs –23. The of human A and murAr, Ace2, and Tmprss2 as compared to scRNAseq ; however, in mice, Tmprss2 was expressed at comparable levels in AT1  and ciliated cells  of lung tissues with prostate luminal cells . ACE2 detection was sparse in both lungs and prostates from humans and mice, but, noticeably, outlier expression of this gene was higher in lung AT2 cells compared to other cells in the lung and epithelial cells in the prostate of both humans and mice. AR detection was low in both human lung and prostate, whereas it was higher in mouse prostate epithelial cells compared to mouse lung epithelial cells. This analysis indicates that TMPRSS2 and ACE2 are expressed in both lung and prostate tissues in humans and mice, with higher relative TMPRSS2 expression in human prostate epithelial cells.To compare expression levels of neages 20, 25, 26.SI Appendix, Fig. S4A). Systemic testosterone levels from mouse serum confirmed down-regulation of testosterone upon castration in male mice and significant up-regulation by testosterone treatment in both castrated male and intact female mice .To evaluate the effect of systemic androgen levels on murine lungs, we castrated adult immune-competent C57BL/6 male mice, and a group of castrated male mice were restimulated with testosterone for 5 d. To also test whether lungs from female mice are responsive to androgen stimulation, we treated intact female mice with testosterone . Similar to mRNA levels, protein expression of ACE2 was not different among groups of pooled samples . We further tested ACE2 protein expression from individual lung samples obtained from intact and castrated severe combined immunodeficiency male mice but again observed no difference . However, given the coexpression patterns of Ar, Tmprss2, and Ace2 at the single-cell level in lung and their regulation by castration  and female mice . In murine lungs, AR was detected by immunohistochemistry (IHC) in nuclei, with localization in both bronchial and nonbronchial epithelial cells . AR protein levels were calculated as an AR IHC score by factoring in IHC staining intensity and subcellular localization of AR protein. The AR IHC score was significantly lower in lungs of castrated male mice compared to intact male mice without changing subcellular localization of AR protein , suggesting that pulmonary AR expression in bronchial and nonbronchial cells is positively regulated by androgen.Levels of stration , we deve protein A and B. Tmprss2 and Ace2 were analyzed using lung sections of the same cohort. Consistent with single-cell sequencing analysis, Tmprss2 RNA visualized by Tmprss2 in situ hybridization (ISH) localized in both bronchial and nonbronchial cells  of murine lungs. Similar to AR protein, Tmprss2 decreased in male bronchial cells upon castration and reactivated with testosterone treatment . Tmprss2 RNA in nonbronchial cells was shown to have the same regulation by androgen as in bronchial cells . However, Ace2 RNA was only detectable in bronchial cells of murine lung. Bronchial expression of Ace2 was down-regulated upon castration in male mice and significantly reactivated with testosterone stimulation in castrated male and intact female mice . To investigate ACE2 protein expression in lungs, we optimized ACE2 IHC to compare with Ace2 ISH staining; lack of a suitable antibody for TMPRSS2 precluded its evaluation by IHC. Consistent with it being a known transmembrane protein, ACE2 IHC showed strong positive membrane staining in both bronchial and nonbronchial cells. This suggests that Ace2 RNA may have a short half-life, or the Ace2 ISH signal is too low to be detected in nonbronchial cells. Since almost all bronchial cells showed positive ACE2 IHC staining, quantitative analysis of ACE2 positive cells in nonbronchial cells was calculated and presented here. ACE2 protein levels significantly decreased upon castration in male lungs and increased with testosterone stimulation in lungs of both castrated male and intact female mice . Together, our data indicate that pulmonary Tmprss2 and Ace2 expression in bronchial and nonbronchial cells are positively regulated by androgen.reatment C and D. Ar and the SARS-CoV-2 entry factors. ISH analysis showed that Ar and Tmprss2 were coexpressed at the cellular level in bronchial cells, and Ace2 and Tmprss2 were also coexpressed in bronchial cells . Tmprss2 is partially coexpressed with lung epithelial marker Sftpb in both bronchial and alveolar cells . This evidence indicates that SARS-CoV-2 entry factors TMPRSS2 and ACE2 are coexpressed with AR in subsets of pulmonary epithelia, including bronchial and alveolar cells.Additionally, we performed costaining experiments with al cells G and H. TMPRSS2 was examined by ISH on the same cohort and showed bronchial and nonbronchial expression of TMPRSS2 in human lung tissues . However, quantitative analysis of TMPRSS2 ISH did not show significant differences between males and females in either bronchial or nonbronchial cells , but there was no differential expression in males and females (Tmprss2 and Ace2 (and ACE2 protein) levels, intact male and female mice had similar expression levels of these factors .We extended the in situ analysis to human lung tissues using normal lung sections from male, female, and metastatic castration-resistant prostate cancer (mCRPC) patients. AR protein had clear nuclear localization in bronchial and alveolar cells of human lungs. The intensity and quantity of AR protein, represented by AR IHC score, in lungs indicated that males had significantly higher levels of AR than females, whereas mCRPC  had the lowest AR levels A and B. al cells C. Additi females D. These AR and TMPRSS2 RNA were also coexpressed in both bronchial and nonbronchial cells , whereas similar (relatively lower) expression levels were observed in the under 30-y-old group . Smoking is a known risk factor for lung disease and a more severe COVID-19 disease course . Importantly, elderly male smokers had significantly higher AR and ACE2 protein expression than nonsmokers in lungs of males over 70 y of age . These data demonstrate that aged males have increased AR and SARS-CoV-2 host entry factor ACE2 protein expression in lungs, and their expression is enhanced by smoking. This suggests that older smoking men have a higher vulnerability to SARS-CoV-2 infection due to elevated entry factor levels.SARS-CoV-2 has been reported as having higher detrimental impacts in elderly populations, especially men with a smoking history . Here, we course ; therefoTMPRSS2 is a bona fide AR-regulated gene through direct binding of AR, FOXA1, and BRD4 (a BET protein) at the promoter region of TMPRSS2 , suggesting that ACE2 is a direct target of AR and BRD4 in the prostate. Here, our data suggest that pulmonary TMPRSS2 and ACE2 expression is also mediated by AR signaling in murine and human lungs. Thus, we examined whether blocking AR signaling could impair the infection ability of SARS-CoV-2 in the AR-positive LNCaP prostate cancer cell line, which we found to be permissive to infection with isolated SARS-CoV-2 virus. Using LNCaP cells, we screened Food and Drug Administration (FDA)-approved and experimental small-molecule AR and BET inhibitors for their effects on SARS-CoV-2 infection (50) values of 79, 768, and 90 nM, respectively. Our experimental small-molecule AR protein degrader ARD-61 ; thus, we examined whether pulmonary AR, Tmprss2, and Ace2 levels were responsive to systemic treatment with BET inhibitor JQ1 in male mice. After 5 d, individual lungs were collected from intact C57BL/6 male mice treated with either vehicle or 50 mg/kg JQ1 . Levels of Tmprss2 and Ace2 were then examined using bulk lung tissue harvested from individual mice. The results showed significant transcriptional repression of Tmprss2 and Ace2 by JQ1 in this bulk lung tissue analysis , which we did not observe using bulk lung tissue in intact and castrated male mice . RNA ISH of the same cohort confirmed that Tmprss2 and Ace2 levels were significantly lower in mice treated with JQ1 compared to vehicle in bronchial cells . Furthermore, ACE2 IHC in lungs demonstrated that ACE2 protein levels were reduced by JQ1 in nonbronchial cells . Protein levels of AR measured by AR IHC score were lower in lungs of JQ1-treated mice .Furthermore, we previously reported that androgen signaling in prostate cancer requires BET protein activity  and iden50 values of 10, 8, and 3.5 nM, respectively  and a BET protein degrader (ZBC260) . JQ1, OTectively C and D, and ACE2 E. Similaompounds F and G, ompounds H and I. TMPRSS2 expression was found to be regulated by androgens in certain cells in the lungs, and we also identified ACE2 as an AR-regulated target; these findings support an early hypothesis that differences in androgen levels may be one reason for gender disparities in COVID-19 outcomes . Thus, in cell types where AR regulation of ACE2 and TMPRSS2 does not exist, the broader transcriptional repression by BET inhibitors may block expression of these key host factors, and agents targeting BET proteins may have a different therapeutic efficacy in COVID-19 than direct AR inhibitors. BET inhibitors can also affect innate and adaptive immune responses, through processes such as repression of IFNG (IFN gamma)  , which cSeveral clinical trials are already underway to evaluate targeting of androgen signaling for treatment of COVID-19 . Some ofTMPRSS2 knockout mice exhibit reduced lung pathogenesis and decreased viral spread after infection with different strains of influenza virus, including H1N1, the virus responsible for the 2009 swine flu pandemic  were purchased from ScienCell Research Laboratories. All cell lines were tested negative for mycoplasma and authenticated by genotyping.H1437, HCC4006, Caco-2, Calu-3, and LNCaP cells were obtained from American Type Culture Collection (ATCC) and maintained under 5% COApalutamide, darolutamide, dexamethasone, enzalutamide, mifepristone, OTX015, and JQ1 were purchased from Selleckchem. R1881, dihydrotestosterone (DHT), and beta-estradiol were purchased from Sigma-Aldrich. BET protein degrader ZBC260 and AR protein degrader ARD-61 were described previously , 29.SI Appendix for scoring details.IHC was performed on 4-μm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections using anti-AR rabbit monoclonal primary antibody  and anti-ACE2 rabbit monoclonal primary antibody . IHC was carried out on the Benchmark XT automated slide staining system  using the UltraView Universal diaminobenzidine (DAB) detection kit  and Hematoxylin II  for counterstain. Dual IHC was performed consecutively, and signals were developed using the Universal DAB detection kit and the Discovery purple kit . Staining was evaluated under 100× and 200× magnification using a bright-field microscope. See PPIB. Assay background was monitored using a negative control probe against bacterial gene DapB. RNA ISH was performed as previously described (SI Appendix for probes and scoring information.RNA ISH was performed on 4-μm-thick FFPE tissue sections using RNAscope 2.5 high definition (HD) Brown kit (Advanced Cell Diagnostics) for single target and RNAscope 2.5 HD Duplex kit (322430) for dual targets. RNA quality was evaluated using a positive control probe against human escribed , 42. FFPn = 3) using an HPD300e digital compound dispenser and allowed to incubate 24 h prior to infection, with a final DMSO vehicle concentration of 0.1%. SARS-CoV-2 WA1/2020 strain  was added in BSL3 containment at a final working dilution equivalent to a multiplicity of infection of 10 and was allowed to incubate for 48 h at 37 °C. Wells were fixed with 4% paraformaldehyde, permeabilized with 0.03% Triton X-100, and blocked with antibody buffer . Following blocking, plates were sealed, surface decontaminated, and transferred to a BSL2 laboratory for staining. Cells were stained overnight with SARS-CoV-2 nucleoprotein primary antibody  and then stained with anti-mouse IgG:AlexaFluor 647 secondary  and Hoechst 33342 .LNCaP and H1437 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum and seeded at 8,000 cells per well in poly-D-lysine−coated 384-well plates , or 10,000 cells per well in 96-well plates , respectively. Cells were allowed to attach and recover for 12 h to 18 h. BET and AR antagonists were solubilized in dimethyl sulfoxide (DMSO) and dispensed  and 32 uninfected (100% effect) control wells (mean LNCaP infectivity range: 18 to 25%). Viability was assessed by comparing cell counts in treated wells to the average cell count in the 32 uninfected control wells (100% viability).Plates were imaged on a Thermo-Fisher CX5 high-content microscope with a UPlanFLN 10×/0.3NA objective. Nine fields were acquired per well for each of the two fluorescent channels . Images were analyzed using CellProfiler to quantify the percentage of infected cells at the well level . InfecteACE2/TMPRSS2/AR expression in lung tissues at the cell level, we searched for publicly available scRNAseq datasets based on the following criteria: 1) profiled healthy human and/or mouse lung tissues; 2) generated with 10× Chromium platform; and 3) libraries prepared from single cells. Four human datasets and three mouse datasets were selected (1944ACE2/TMPRSS2/AR. Log-normalized expression value by Seurat was used to generate the bubble plot. For the published prostate scRNAseq dataset (https://singlecell.broadinstitute.org/single_cell/study/SCP859. Log-normalized expression value by Seurat v3 was used to generate the violin plots. See SI Appendix for data analysis of the lung and prostate comparisons.To evaluate lected 19, 44–46.  dataset , fastq fP values were calculated by GraphPad Prism 8 using two-tailed unpaired t test, and exact P values are provided.Sample sizes are listed on the figures or in figure legends. Supplementary File


While the androgen receptor (AR) may influence the progression of clear cell renal cell carcinoma (ccRCC), its role to impact vasculogenic mimicry (VM) to alter the ccRCC progression and metastasis remains obscure. Here, we demonstrated that elevated AR expression was positively correlated with tumor-originated vasculogenesis in ccRCC patients. Consistently, in vitro research revealed AR promoted VM formation in ccRCC cell lines via modulating lncRNA-TANAR/TWIST1 signals. Mechanism dissection showed that AR could increase lncRNA-TANAR (TANAR) expression through binding to the androgen response elements (AREs) located in its promoter region. Moreover, we found that TANAR could impede nonsense-mediated mRNA decay (NMD) of TWIST1 mRNA by direct interaction with TWIST1 5′UTR. A preclinical study using in vivo mouse model with orthotopic xenografts of ccRCC cells further confirmed the in vitro data. Together, these results illustrated that AR-mediated TANAR signals might play a crucial role in ccRCC VM formation and metastasis, and targeting this newly identified AR/TANAR/TWIST1 signaling may help in the development of a novel anti-angiogenesis therapy to better suppress the ccRCC progression. Clear cell renal cell carcinoma (ccRCC), the major subtype of aggressive human malignancies, accounted for approximately 1,000,000 cases and 175,000 deaths worldwide in 2018 , 4. HencThe androgen receptor (AR) can promote progression and hematogenous metastasis in ccRCC through ASS1P3/miR-34a-5p/ASS1 and miR-185-5p/HIF-2a/VEGF signaling, respectively . This isBesides the classical tumor angiogenesis, Maniotis et al. demonstrated a de novo pattern of tumor perfusion in melanomas, named vasculogenic mimicry (VM), in which cancer cells form their own supplying channel without endothelial cells . Due to Long noncoding RNAs (lncRNAs), a class of untranslated transcripts longer than 200 nucleotides barely with protein-coding capacity, participate in diverse biological processes . RecentlIn this study, we aimed to determine the biological function of AR in ccRCC VM. Mechanism dissection revealed that the lncRNA-TANAR, which is regulated by AR, might increase the oncogene TWIST1 expression through competitive binding to TWIST1 mRNA to reduce the activity of Up-frameshift protein 1 (UPF1), the core factor required for nonsense-mediated mRNA decay .To clarify the clinical significance of VM on ccRCC, we collected 51 ccRCC human specimens along with their clinicopathological data and performed the HE & PAS/CD31 double staining to detect VM Fig. . The VM 1# and shAR2#) in RCC AR-positive SW839 cells  and 10 µM enzalutamide (Enz), an FDA approved antiandrogen, respectively. As shown in Fig. lls Fig.  and OSRClls Fig. . In contlls Fig.  led to mTaken together, results from Fig. To dissect the molecular basis for AR’s effect on ccRCC VM formation, we screened the known critical genes linked with VM via qRT-PCR assay in RCC 786O cells after adding AR-cDNA Fig. . ConsistIn order to confirm whether AR induced VM formation through TWIST1, we applied rescue experiments adding TWIST-cDNA (oeTWIST1) into SW839 cells and TWIST1-shRNA (shTWIST1) into 786O cells Fig. . The resTogether, results from Fig. p < 0.001; n = 23) , we found elevating TWIST1 levels (using 25% of samples with the highest TWIST1 expression) led to a significantly lower survival rate in ccRCC patients  Fig. , which w23) Fig. . Further23) Fig. . Additionts Fig. . In addiSince AR could elevate TWIST1 expression at both protein and mRNA levels see Fig. a, b, we P < 0.05) in Sunitinib resistance cells from the GSE69535 dataset  predicting the location of TANAR  in the 2 kb region of the TANAR promoter by using the JASPAR database Fig.  and deteTogether, results from Fig. To dissect the molecular mechanism underlying AR/TANAR’s regulation on TWIST1 expression, especially the impact of AR on TWIST1 mRNA stability see Fig. , we hypohttp://uswest.ensembl.org/) and individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) performed by Zünd [Intriguingly, based on Ensembl database . We also generated two mutant reporters with mutations in the conserved splicing sequence to eliminate the NMD initiation (pWPI-luc-mutant 1) and TANAR-interacting region in the 5′UTR (pWPI-luc-mutant 2) Fig. . As expeTogether, results from Fig. 6 786O cells mixed with matrigel were inoculated into the left kidney capsule of nude mice and tumor progressions were evaluated via the non-invasive in vivo imaging system (IVIS). After 6 weeks, we observed that increased bioluminescence signals in the whole mice . A total of 1 × 10ice Fig. , as wellice Fig. . Upon anice Fig. . Importaice Fig. .Fig. 7PrTogether, the results are shown in Fig. The epithelium-dependent angiogenesis has been long regarded as the sole blood supplement initiated by proangiogenic factors. The conception of VM, epithelium-independent angiogenesis, reinforces the tumor neovascularization theory, indicating that targeting critical factors regulating VM formation in the tumor could be a novel anti-angiogenesis therapy to stop tumor cells .Recent literature revealed that VM formation is an independent and relevant prognostic criterion for disease-free survival in patients with ccRCC , 44. SimR = 0.3947; P < 0.001; Fig. Mounting evidence has demonstrated that AR functions as an oncogene in ccRCC, promoting progression and hematogenous metastasis , 6, 22, R = 0.34; P < 0.001), which is consistent with our in vivo or in vitro data. Furthermore, we treated ccRCC cell with Harmine, a naturally occurring beta-carboline alkaloid widely used herb, as a TWIST1 inhibitor selected through the unbiased screen and validated in the lung cells [A previous study revealed that AR could upregulate TWIST1 via ETV1 in prostate cancer . Similarng cells , 34, andIncreasing evidence indicates that TWIST1 could be post-transcriptionally modulated via noncoding RNA –53. The In conclusion, we characterized a new long noncoding RNA TANAR transcriptionally regulated by AR, which influences VM formation by decreasing TWIST1 mRNA nonsense-mediated decay. Targeting the AR/lncRNA-TANAR/TWIST1 axis could be a promising strategy for the development of better treatment of ccRCC.A total of 51 histologically confirmed ccRCC tissue samples with 23 paired adjacent noncancerous tissues were collected between August 1, 2014 and February 1, 2016 from the Department of Urology, the Second Affiliated Hospital of Harbin Medical University . Patients were excluded if they had been treated previously with neoadjuvant chemotherapy or TKIs. All samples collected for use in research after patients signed the consent of the scientific ethics were fixed in 10% formalin and then embedded in paraffin. Our research was approved by the Institutional Review Board of the hospital in advance.2 incubator at 37 °C. According to ATCC’s protocol, all cell lines used in the paper have been authenticated and proven to be mycoplasma and bacteria-free and were periodically re-authenticated by PCR.786O, SW839, and HEK293T cell lines were purchased from the American Type Culture Collection . Cells were cultured in DMEM media with 1% penicillin and streptomycin, containing 10% fetal bovine serum (FBS). All cells were maintained in a humidified 5%(v/v) COCells were washed twice with cold PBS and lysed in cell lysis buffer and equal proteins (30–50 μg) were loading, mixed, boiled, and separated on 6–12% SDS/PAGE gel, then transferred onto PVDF membranes . We blocked the PVDF membranes for 1 h via 5% skim milk and incubated with the specific primary antibodies overnight. After incubated with HRP-conjugated secondary antibodies, the PVDF membranes were visualized using the ECL system . Primary antibodies used in the study for western blot are listed in Table 6 cells/mouse, into the left renal capsule of 8-week-old male athymic nude mice (n = 5 mice/group). After 6 weeks, mice were sacrificed, and tumors were excised and measured.All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and use of laboratory animals, conformed to the regulations for animal experimentation, and reviewed by the Institutional Laboratory Animal Care and Use Committee of the University of Rochester Medical Center. Male athymic BALB/c nude mice (6–8 weeks old) were obtained from the Animal Production Area of the National Cancer Institute-Fredrick Cancer Research and Development Center in Frederick, MD, USA. 786-O cells transfected with Scr-luc, oeAR-luc, shTANAR-luc, or oeAR+shTANAR, were injected at 1 × 10t-test and two-way ANOVA test by SPSS 22  or GraphPad Prism 6 . P values less than 0.05 were considered statistically significant .Experiments were repeated independently at least three times with data points completed in triplicate. Results are shown as mean ± S.D. Statistical significance was determined using the Student’s supplement figure legendsupplement figure 1supplement figure 2supplement figure 3supplement figure 4Supplemental table1Supplemental table2supplement method


Flufenamic acid (FFA) is a representative of the fenamic acids, an important group of NSAIDs. In the present study, we study the effects of FFA on adipogenesis of human mesenchymal stem cells (MSCs) and we explore the potential mechanism. in vitro. The effects of FFA in vivo were evaluated using a heterotopic adipose formation assay in nude mice as well as ovariectomized (OVX) and aged mice. To explore the mechanism of FFA, Western blot was used to determine activation of the PI3K/AKT signaling pathway.  To investigate the effects of FFA on adipogenic differentiation of hMSCs, human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs), representative of hMSCs, were treated with FFA during adipogenic differentiation in vitro and in vivo. Mechanistically, FFA inhibited adipogenesis of human MSCs by inhibiting the PI3K/AKT pathway.  Our results demonstrate that, at certain concentrations, FFA inhibited adipogenesis of human MSCs both  The present study indicated that FFA could be used to inhibit adipogenesis of human MSCs in tissue engineering and diseases related to excessive adipogenic differentiation of MSCs. Tissue engineering has become a hotspot in recent years . As a coNonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for anti-inflammatory and analgesic purposes. NSAIDs inhibit cyclooxygenases (COXs) which inhibit the synthesis of prostaglandin –12. NSAIγ [The lineage commitment of MSCs is regulated by a variety of transcription and signaling factors, including the AKT signaling pathway. AKT signaling plays an important role in cellular proliferation, differentiation, and apoptosis , as wellγ , which iin vitro and in vivo effects of flufenamic acid (FFA), a representative of the fenamic acids, on the adipogenesis of human MSCs. We report a potential mechanism of action, which provides valuable insight into the potential use of FFA in tissue engineering and diseases related to excessive adipogenic differentiation of MSCs.In the present study, we investigate the in vitro studies were repeated at least three times, using hMSCs from three different donors.Primary hASCs and hBMMSCs were obtained from ScienCell Research Laboratories . All cell-based α-MEM, and 100x penicillin and streptomycin mixture were obtained from Gibco . Human ASCs and BMMSCs were cultured at 37°C with a 5% CO2 atmosphere in proliferation medium (PM), consisting of penicillin/streptomycin, 10% (v/v) FBS, and either fresh DMEM (for hASCs) or α-MEM (for hBMMSCs). The adipogenic medium (AM) was comprised of DMEM or α-MEM containing 10 μM insulin, 100 nM dexamethasone, 200 μM indomecin, 500 μM 3-isobutyl-1-methylxanthine (IBMX), 10% (v/v) FBS, and penicillin/streptomycin.FBS, DMEM, in vitro, we dissolved flufenamic acid in DMSO to obtain a concentrated FFA solution of 200 mM FFA.In order to examine the effects of FFA at different concentrations on the adipogenesis of hMSCs and to identify the optimal concentration for adipogenesis of hMSCs Cells were inoculated and cultured in PM and AM separately, and Oil red O staining was performed on the 21st day after adipogenic induction. Briefly, cells were fixed with 10% formalin for 1 hour and rinsed with 60% isopropanol, and 0.3% Oil red O working solution was applied. The cells were then washed with distilled water, observed, and photographed under a microscope. For quantitative assessment, stained cells were eluted with 100% isopropyl alcohol and quantified by spectrophotometric absorbance at 520 nm against a blank .in vitro. Afterwards, cDNA was reverse-transcribed using a PrimeScript RT Reagent Kit . Real-time quantitative PCR assays were conducted with SYBR Green Master Mix  on an ABI Prism 7500 real-time PCR System . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The primer sequences used are described in TRIzol reagent  was used to extract total RNA from hASCs and hBMMSCs cultured μg) of total protein extracts were subjected to 10% SES-PAGE and transferred to a polyvinylidene fluoride membrane . Membranes were blocked in 5% skim milk for 1 hour and then incubated with anti-PPARγ , anti-p-PI3K, anti-PI3K, anti-p-AKT, anti-AKT, or anti-GAPDH  overnight at 4°C. Subsequently, membranes were incubated with peroxidase-conjugated secondary antibody solution at room temperature. The immunoreactive protein bands were detected using an ECL kit . Image J software  was used for quantitative densitometric analysis of protein bands. All protein levels were quantified by the target gene/GAPDH densitometric ratio.Human ASCs were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phosphatase inhibitor (Roche) and 2% protease inhibitor cocktail (Roche) at 4°C for 30 min. Cell lysates were sonicated and centrifuged at 13362g at 4°C for 30 min to obtain the supernatant. Total protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific). Aliquots . All experiments were performed under the approved guidelines.ad libitum.All the mice were obtained from Vital River Corporation . Mice were maintained in a pathogen-free facility on a 12-hour light/dark cycle with water and food provided n = 24) female BALB/C homozygous nude mice were used for heterotopic adipose tissue formation. The 24 mice were randomly divided into four groups (6 mice per group): AM, AM+FFA of hASCs, and AM, AM+FFA of hBMMSCs. Human ASCs or BMMSCs were cultured in AM or AM with 25 μM FFA for 1 week prior to inoculation in nude mice. The cells were then collected and incubated with a Collagen Sponge  (8 mm × 8 mm × 2 mm) at 37°C for 2 h. After centrifugation, the complex was implanted into the dorsal subcutaneous space of the nude mice. The implants were collected after six weeks and fixed with 4% paraformaldehyde. After that, each sample was cut in half for H&E staining and Oil red O staining.6-week-old (n = 40) female C57BL6 mice underwent ovariectomy (OVX) or sham operation. The forty mice were randomly divided into two groups (20 mice per group): Sham and OVX. Pentobarbital sodium (50 mg·kg−1) was used for general anesthesia by intraperitoneal injection. OVX or sham operations were performed on each mouse following standard procedure [8-week-old : (1) Sham mice with normal saline (N.S.) and (2) Sham mice with FFA. Similarly, the 20 OVX mice were randomly divided into two groups (10 mice per group): (3) OVX mice with N.S. and (4) OVX with FFA. Four weeks later , FFA or n = 20) female C57BL6 mice were randomly divided into two groups (10 mice per group). Mice in group one (FFA) were given FFA once a day; mice in group two (N.S.) were given N.S., in an equivalent volume as the FFA solution, once a day.For aged mice, 12-month-old  . Adipocyt-tests, one-way ANOVA, and a Tukey's post hoc test were used to determine the level of significance. P values of P < 0.05 were considered statistically significant.Data are given as mean ± standard error (SE). SPSS Statistics 20.0 software (IBM) was used for statistical analyses. Independent two-tailed Student's μM FFA had an inhibitory effect on the adipogenic differentiation of hASCs cultured in AM on day 21 (Figures μM FFA significantly attenuated the expression of PPARG (CEBPA (μM did not. The inhibitory effect was attenuated with increasing FFA concentration, and 25 μM FFA showed the strongest inhibitor effect on adipogenic differentiation (Figures in vitro (Supporting Oil red O staining and quantification indicated that 12.5, 25, 50, 100, and 200  Figures . Meanwhiof PPARG  and CEBPG (CEBPA  at day 1 Figures . FurtherμM FFA had an inhibitory effect on the adipogenic differentiation of hBMMSCs cultured in AM on day 21 (Figures μM attenuated the expression of PPARG (CEBPA (μM did not. The inhibitory effect was attenuated with increasing FFA concentration. Like the hASCs, treatment of hBMMSCs with 25 μM FFA showed the strongest inhibitory effect on adipogenic differentiation (Figures in vitro (Supporting To test the effects of FFA on the adipogenesis of other types of hMSCs, we treated hBMMSCs with FFA during adipogenic induction. Similar to the results obtained in hASCs, Oil red O staining and quantification showed that 12.5, 25, 50, 100, and 200  Figures . Additioof PPARG  and CEBPG (CEBPA  at day 1 Figures . FurtherμM FFA resulted in the optimal inhibitory effect on adipogenesis of hASCs and hBMMSCs in in vitro, 25 μM FFA was the concentration selected for the in vivo experiments. Human ASCs were treated with AM or with AM+25 μM FFA for 7 days, separately loaded onto Collagen Sponge scaffolds, and implanted into nude mice. Six weeks later, the implanted complexes were collected and analyzed. H&E (Since 25 zed. H&E  and Oil zed. H&E  stainingzed. H&E .To further explore whether FFA could suppress adipogenesis of MSCs and adipose formation in animal disease models and define clinic value for our results, FFA was given to OVX and aged mice. Forty female mice were divided into four groups with ten mice in each group: SHAM+N.S., SHAM+FFA, OVX+N.S., and OVX+FFA. H&E staining  showed tIn aged mice, H&E staining indicated that FFA treatment partially attenuated adipose accumulation in the femurs . SpecifiμM FFA in both PM and AM to mRNA analysis for a set of key regulators of adipogenesis. Treatment with 25 μM FFA suppressed the expression of the target genes of AKT, FOXO1 (FOXO3 (CEBPα (μM FFA treatment, the phosphorylation of PI3K and AKT and total PI3K and AKT as well as p-PI3K/PI3K and p-AKT/AKT were downregulated in both PM and AM conditions (Figures γ, which was upregulated in the AM condition (μM FFA. Oil red O staining and quantification showed that the inhibitory effect of 25 μM FFA on adipogenesis of hASCs was attenuated by IGF-1 treatment (Figures CEBPα and Western blot showed that IGF-1 upregulated the phosphorylation of PI3K and AKT, total PI3K and AKT, and p-PI3K/PI3K and p-AKT/AKT as well as PPARγ, which were reduced by FFA treatment alone (Figures To explore the mechanism by which FFA regulates adipogenesis, we subjected cells treated with 25 T, FOXO1 , FOXO3 (1 (FOXO3 , and CEB3 (CEBPα  in hASCs Figures . Meanwhiin vitro and in vivo. The inhibitory effect was attenuated as the concentration of FFA increased, and 25 μM FFA showed the optimal inhibitory effect on adipogenesis. Mechanistically, FFA inhibited adipogenesis of hMSCs by inhibiting the AKT pathway. These data indicate the potential application of FFA in tissue engineering and diseases related to excessive adipogenic differentiation of MSCs.In the present study, we found that treatment with FFA inhibited adipogenesis of two kinds of hMSCs both in vitro experiments of this study show that FFA inhibits adipogenesis of hMSCs. According to the qRT-PCR results, the inhibitory effects of FFA on the expression of adipogenesis markers declined as the concentration of FFA increased from 25 μM to 200 μM. However, Oil red O staining showed that 200 μM FFA inhibited adipogenesis better than 100 μM FFA. This may be because 200 μM FFA also inhibited the proliferation of hMSCs, which has been documented in our previous study [in vitro and in vivo. This might be because they are both populations of postnatal tissue-specific stem cells with similar gene expression profiles and display similar characteristics [in vitro results, we showed that FFA suppressed adipogenesis of hMSCs in vivo. This evidence indicates that FFA could be a potential therapeutic option to treat diseases related to excessive adipogenic differentiation of MSCs.The eristics . We founExcessive adipogenic differentiation of MSCs can be seen in several diseases, such as obesity and osteoporosis , 29, 30.NSAIDs are often used to treat osteoarthritis and other bone-related diseases, and some studies have reported that NSAID drugs effect the biological behavior of bone and MSCs, including adhesion, proliferation, and differentiation. However, few studies have reported on the effects of NSAIDs on the adipogenesis of MSCs except that indomethacin promotes adipogenesis of MSCs . FurtherOur study indicated that FFA inhibited adipogenesis of hMSCs by inhibiting the PI3K/AKT pathway. The AKT pathway is closely associated with the lineage commitment of MSCs , 21. PreNevertheless, there are several limitations of our study. This study only examined the signaling pathways by which certain concentrations FFA inhibit adipogenic differentiation of hMSCs. Why the inhibitory effect declines as FFA concentration increases remains unclear. This might be because low concentrations of FFA and high concentrations of FFA influenced adipogenesis of hMSCs by different mechanism and we will conduct deeper exploration in future study. Furthermore, to promote the clinical transformation of our findings, further exploration is needed to determine the effective concentration for treatment in humans.in vitro and in vivo by inhibiting the PI3K/AKT pathway. These data suggest that FFA is applicable in regulating adipogenesis of MSCs and highlight FFA as a potential therapeutic option to treat diseases related to excessive adipogenic differentiation of MSCs.Overall, our results indicate that FFA can inhibit the adipogenesis of hMSCs in vitro, at an optimal inhibitory concentration of 25 μM FFA. Treatment with 25 μM FFA also inhibits adipogenesis of hMSCs in vivo. Mechanistically, FFA inhibits adipogenesis through inhibition of the PI3K/AKT signaling pathway. These data suggest that FFA might be used to inhibit adipogenesis of human MSCs in tissue engineering and diseases related to excessive adipogenic differentiation of MSCs.FFA inhibits adipogenesis of hMSCs