Ophiura sarsii has previously been shown to produce a chlorin -14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) (1) with potent phototoxic activities, making it applicable to photodynamic therapy. Using extensive LC-MS metabolite profiling, molecular network analysis, and targeted isolation with de novo NMR structure elucidation, we herein identify five additional chlorin compounds from O. sarsii and its deep-sea relative O. ooplax: 10S-Hydroxypheophorbide a (2), Pheophorbide a (3), Pyropheophorbide a (4), -14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (5), and -14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (6). Chlorins 5 and 6 have not been previously reported in natural sources. Interestingly, low amounts of chlorins 1–4 and 6 could also be identified in a distant species, the basket star Gorgonocephalus cf. eucnemis, demonstrating that chlorins are produced by a wide spectrum of marine invertebrates of the class Ophiuroidea. Following the purification of these major Ophiura chlorin metabolites, we discovered the significant singlet oxygen quantum yield upon their photoinduction and the resulting phototoxicity against triple-negative breast cancer BT-20 cells. These studies identify an arsenal of brittle star chlorins as natural photosensitizers with potential photodynamic therapy applications.A Pacific brittle star  Photodynamic therapy (PDT) is a powerful alternative to other treatment options for cancer , dermatoBonellia viridis, controls the sex determination of this polychete. Larvae of B. viridis develop into males when settled in the physical proximity of an adult female; the photodynamic action of bonellin is believed to play the key role in this process 25D +300 . UV (MeCN) λmax (log ε) 269 (4.94), 320 (4.16), 375 (4.54) (sh), 393 (4.64) (sh), 408 (4.69), 504 (3.73), 535 (3.73), 604 (3.69), 661 (4.37) nm. ECD  λmax ([θ]) 405 , 660  nm. 1H NMR  δ 0.33 , 1.64 , 1.77 , 2.10 , 2.32 , 2.55 , 2.61 , 3.24 , 3.38 , 3.62 , 3.73 , 4.32 , 4.58 , 5.12 , 5.22 , 5.74 , 8.83 , 9.56 , 9.73 ; 13C NMR  δ 10.8 (CH3-1a), 11.0 (CH3-3a), 11.7 (CH3-5a), 17.5 (CH3-4b), 18.7 (CH2-4a), 22.9 (CH3-8a), 29.5 (CH2-7a), 30.9 (CH2-7b), 47.5 (CH2-10), 49.3 (CH-8), 51.1 (CH-7), 54.4 (CH2-2a), 93.6 (CH-δ), 97.0 (CH-α), 104.3 (CH-β), 106.0 (C-γ), 127.8 (C-5), 130.0 (C-6), 133.4 (C-1), 135.9 (C-3), 136.2 (C-12), 137.0 (C-15), 138.6 (C-2), 141.0 (C-11), 145.0 (C-4), 148.0 (C-16), 150.0 (C-14), 154.4 (C-13), 161.2 (C-17), 172.5 (C-18), 174.3 (C-7c), 195.3 (C-9). HR-ESI/MS analysis: m/z 539.2657 [M+H]+, . MS/MS spectrum: CCMSLIB00010128700. SMILES: OC(CC[C@H]1[C@@H](C2=N/C1=C(CC3=O)\C4=C3C(C)=C(N4)/C=C5N=C(C(C)=C\5CC)/C=C(N/6)/C(CO)=C(C6=C\2)C)C)=O. 1H, COSY, 13C-DEPTQ, HSQC, HMBC, and ROESY NMR spectra of compound 1 are shown in (2): -9-Ethenyl-14-ethyl-21-hydroxy-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (S)-Hydroxypheophorbide a(10) ) 406 , 662  nm. 1H NMR  δ 1.55 , 1.66 , 3.28 , 3.47 , 3.58 , 3.71 , 3.78 , 4.11 , 4.62 , 6.25 , 6.44 , 7.74 , 8.28 , 9.01 , 9.60 , 9.92 ; 13C NMR  δ 11.0 (CH3-3a), 11.7 (CH3-5a), 12.0 (CH3-1a), 17.5 (CH3-4b), 18.7 (CH2-4a), 22.6 (CH3-8a), 49.3 (CH-8), 51.4 (CH-7), 52.6 (CH3-10b), 94.3 (CH-δ), 97.5 (CH-α), 104.9 (CH-β), 109.5 (C-γ), 123.5 (CH2-2b), 126.8 (C-6), 128.9 (CH-2a), 129.2 (C-5), 132.3 (C-1), 135.8 (C-2), 136.7 (C-3), 137.1 (C-15), 141.5 (C-11), 145.3 (C-4), 150.6 (C-14), 154.6 (C-13), 171.3 (C-10a), 173.3 (C-18), 189.2 (C-9). HR-ESI/MS analysis: m/z 609.2718 [M+H]+, . MS/MS spectrum: CCMSLIB00010128701. SMILES: OC(CC[C@H]1[C@@H](C2=N/C1=C([C@](C(OC)=O)(O)C3=O)\C4=C3C(C)=C(N4)/C=C5N=C(C(C)=C\5CC)/C=C(N/6)/C(C=C)=C(C6=C\2)C)C)=O. 1H, COSY, HSQC, HMBC, and ROESY NMR spectra of compound 2 are shown in (rbide a) ,26: [α]23): -9-Ethenyl-14-ethyl-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (Pheophorbide a) ) 400 , 662  nm. 1H NMR  δ 0.41 , 1.62 , 1.76 , 2.15 , 2.24 , 2.44 , 2.58 , 3.21 , 3.43 , 3.64 , 3.70 , 3.83 , 4.05 , 4.58 , 6.22 , 6.40 , 6.42 , 8.21 , 8.91 , 9.44 , 9.77 , 12.05 ; 13C NMR  δ 10.9 (CH3-3a), 11.7 (CH3-5a), 12.0 (CH3-1a), 17.4 (CH3-4b), 18.6 (CH2-4a), 22.9 (CH3-8a), 29.0 (CH2-7a), 30.7 (CH2-7b), 49.2 (CH-8), 50.8 (CH-7), 52.7 (CH3-10b), 64.2 (CH-10), 94.0 (CH-δ), 97.0 (CH-α), 105.0 (CH-β), 105.4 (C-γ), 123.3 (CH2-2b), 128.5 (C-6), 128.9 (C-5), 128.9 (CH-2a), 132.3 (C-1), 135.6 (C-2), 135.9 (C-12), 136.4 (C-3), 137.2 (C-15), 141.5 (C-11), 145.3 (C-4), 148.8 (C-16), 150.4 (C-14), 154.9 (C-13), 161.8 (C-17), 169.3 (C-10a), 173.2 (C-18), 174.0 (C-7c), 189.2 (C-9). Confirmed by comparison with the standard compound. HR-ESI/MS analysis: m/z 593.2745 [M+H]+, . MS/MS spectrum: CCMSLIB00010128702. SMILES: OC(CC[C@H]1[C@@H](C2=N/C1=C([C@@H](C(OC)=O)C3=O)\C4=C3C(C)=C(N4)/C=C5N=C(C(C)=C\5CC)/C=C(N/6)/C(C=C)=C(C6=C\2)C)C)=O. 1H, COSY, 13C-DEPTQ, HSQC, HMBC, and ROESY NMR spectra of compound 3 are shown in (rbide a) : [α]25D 4): -9-Ethenyl-14-ethyl-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (Pyropheophorbide a) : [α]25D +250 . UV (MeCN) λmax (log ε) 275 (4.17), 323 (4.22), 374 (4.48) (sh), 396 (4.62) (sh), 412 (4.68), 507 (3.73), 539 (3.70), 606 (3.64), 667 (4.20) nm. ECD  λmax ([θ]) 412 , 668  nm. 1H NMR  δ 0.29 , 1.64 , 1.78 , 2.12 , 2.33 , 2.56 , 2.63 , 3.25 , 3.45 , 3.64 , 3.73 , 4.33 , 4.60 , 5.14 , 5.24 , 6.23 , 6.41 , 8.25 , 8.91 , 9.48 , 9.77 ; 13C NMR  δ 11.0 (CH3-3a), 11.7 (CH3-5a), 12.0 (CH3-1a), 17.4 (CH3-4b), 18.7 (CH2-4a), 22.9 (CH3-8a), 29.4 (CH2-7a), 30.8 (CH2-7b), 47.5 (CH2-10), 49.3 (CH-8), 51.0 (CH-7), 106.2 (C-11), 123.1 (CH2-2b), 128.1 (C-5), 129.1 (CH-2a), 130.1 (C-6), 131.9 (C-1), 135.1 (C-2), 136.2 (C-3), 137.3 (C-15), 140.7 (C-11), 145.0 (C-4), 148.0 (C-9), 150.1 (C-14), 154.2 (C-13), 172.4 (C-18), 174.2 (C-7c), 195.3 (C-9). HR-ESI/MS analysis: m/z 535.2698 [M+H]+, . MS/MS spectrum: CCMSLIB00010128703. SMILES: OC(CC[C@H]1[C@@H](C2=N/C1=C(CC3=O)\C4=C3C(C)=C(N4)/C=C5N=C(C(C)=C\5CC)/C=C(N/6)/C(C=C)=C(C6=C\2)C)C)=O. 1H, COSY, HSQC, HMBC, and ROESY NMR spectra of compound 4 are shown in (5): -14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (2-Hydroxymethyl-2-devinyl pheophorbide-a) ) 399 , 661  nm. 1H NMR  δ 0.47 , 1.64 , 1.74 , 2.11 , 2.17 , 2.20 , 2.44 , 3.25 , 3.38 , 3.65 , 3.74 , 3.82 , 4.03 , 4.57 , 5.74 , 5.84 , 6.41 , 8.84 , 9.58 , 9.79 ; 13C NMR  δ 10.8 (CH3-1a), 11.0 (CH3-3a), 11.7 (CH3-5a), 17.5 (CH3-4b), 18.7 (CH2-4a), 22.9 (CH3-8a), 49.3 (CH-8), 50.7 (CH-7), 52.6 (CH3-10b), 64.2 (CH-10), 93.8 (CH-δ), 97.5 (CH-α), 104.8 (CH-β), 128.1 (C-6), 128.6 (C-5), 134.0 (C-1), 136.2 (C-3), 136.9 (C-12), 137.1 (C-15), 139.1 (C-2), 141.6 (C-11), 145.4 (C-4), 148.8 (C-16), 150.4 (C-14), 155.1 (C-13), 169.2 (C-10a), 173.5 (C-18), 174.2 (C-7c), 189.2 (C-9). HR-ESI/MS analysis: m/z 597.2703 [M+H]+, . MS/MS spectrum: CCMSLIB00010128704. SMILES: OC(CC[C@H]1[C@@H](C2=N/C1=C([C@@H](C(OC)=O)C3=O)\C4=C3C(C)=C(N4)/C=C5N=C(C(C)=C\5CC)/C=C(N/6)/C(CO)=C(C6=C\2)C)C)=O. 1H, COSY, HSQC, HMBC, and ROESY NMR spectra of compound 5 are shown in (rbide-a) : [α]25D 6): -14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (R)-hydroxy-ETPA(10): [α]25D +200 . UV (MeOH) λmax (log ε) 283 (4.03), 312 (4.05), 360 (4.38) (sh), 398 (4.75), 494 (3.71), 500 (3.68), 525 (3.49), 606 (3.47), 665 (4.21) nm. ECD  λmax ([θ]) 400 (+8080), 667  nm. 1H NMR  δ 1.69 , 1.95 , 2.17 , 2.97 , 3.30 , 3.45 , 3.82 , 3.88 , 4.39 , 4.61 , 4.94 , 5.80 , 8.99 , 9.79 , 9.90 ; 1H NMR  δ 10.8 (CH3-1a), 11.0 (CH3-3a), 12.0 (CH3-5a), 17.7 (CH3-4b), 18.9 (CH2-4a), 23.6 (CH3-8a), 29.3 (CH2-7a), 33.8 (CH2-7b), 51.4 (CH-8), 53.9 (CH-7), 54.6 (CH2-2a), 75.1 (CH-10), 93.4 (CH-δ), 99.2 (CH-α), 102.7 (CH-β), 112.8 (C-6), 130.6 (C-5), 132.8 (C-1), 135.5 (C-12), 136.2 (C-3), 137.3 (C-15), 138.5 (C-2), 145.0 (C-4), 154.2 (C-13), 171.7 (C-18), 174.6 (C-7c). HR-ESI/MS analysis: m/z 555.2600 [M+H]+, . MS/MS spectrum: CCMSLIB00010128699. SMILES: OC(CC[C@H]1[C@@H](C2=N/C1=C([C@@H](O)C3=O)\C4=C3C(C)=C(N4)/C=C5N=C(C(C)=C\5CC)/C=C(N/6)/C(CO)=C(C6=C\2)C)C)=O. 1H, COSY, HSQC, HMBC, and ROESY NMR spectra of compound 6 are shown in  in MeOH in the wavelength range from 200 to 800 nm. Fluorescence spectra were obtained from 50 μL of 500 μM stocks of compounds in anhydrous MeOH in black 384-well plates in Tecan M200Pro reader. Excitation wavelength was 350 nm (9 nm bandpass), and emission range was from 400 to 850 nm (20 nm bandpass) with the 2 nm step.To measure the relative singlet oxygen quantum yield via its phosphorescence intensity, a 637.5 nm laser diode beam was focused through one of the ports of a cuvette holder (Thorlabs CVH100/M) to a 2-mm cuvette by an A-coated lens L1 with 5 cm focal distance (Thorlabs LBF254-050-A). The cuvette was filled with a sample and fixed in the cuvette holder. Another C-coated lens L2 with 2 cm focal distance (Thorlabs LA1074-C) was mounted in the orthogonal port of the cuvette holder to collect and collimate the singlet oxygen phosphorescence. A bandpass filter IFB with a central wavelength of 1277 nm and a bandwidth of 20 nm (Oceanoptics) was installed between this lens and the cuvette. Another C-coated lens with 3 mm focal distance (Thorlabs) focused the collected phosphorescence to an optical fiber connected to a free-running multimode single-photon detector .dR, caused by singlet oxygen relaxation is as follows:2] at the wavelength of laser excitation, 6 (0.36 μM).The number of detected phosphorescence photons −1M−1]. The concentration is given, however 2 [We start by finding 0−17 cm2 ,31 as a We averaged over 10 s measuring The resulting relative quantum yields of singlet oxygen for the new compounds are given as 2. For all experiments, the minimum cell viability considered for plating was 95%, using the trypan blue test.Triple-negative human breast cancer cells of the BT-20 cell line were cultivated in DMEM medium + GlutaMAX supplement, with FBS 10%  without antibiotics at 37 °C and 5% CO2 incubator at 37 °C. The day after removal of the medium, 50 μL DMEM was added with serial dilutions of chlorins starting at 50 μg/mL. Cells with the compounds were incubated for 72 h. Cells in DMEM without added compounds served as positive controls; DMEM wells without cells served as negative controls. The resulting concentration of DMSO in the wells was maintained at 0.5%. Each experiment was conducted in four replicates.For the MTT cell survival assay, BT-20 cells were seeded in DMEM medium supplemented with 10% FBS and 1% gentamicin in a 384-well flat-bottomed transparent plate . Cells were seeded at a density of 3000 cells/well in a final medium volume of 30 μL of medium and cultured overnight in a CO2 using a Newport Optical Power Meter 842-PE, MKS Instruments, Norwood, MA, USA. For phototoxicity studies, BT-20 cells were seeded in 384-well plates in DMEM medium supplemented with 10% FBS at a density of 3000 cells/well in a final medium volume of 30 μL and cultured overnight in a CO2 incubator at 37 °C. The next day, the chlorins were dissolved in DMSO and added to the cells in DMEM medium in series dilutions starting at 25 μg/mL for each. The cells were incubated for 2 h with the chlorin compounds to allow the cellular uptake of the chlorins, after which DMEM medium was replaced with 20 μL of transparent DPBS, and the plate was irradiated with a red LED lamp for 30 min. Next, 80 μL of the culture medium was added to each well for further cultivation. Wells with no cells were used as positive controls; wells without cells were used as negative controls. The resulting concentration of DMSO in the wells was maintained at 0.5%. After 72 h, the wells were emptied, and 30 µL of MTT reagent dissolved in PBS at 0.5 mg/mL, and after 3.5 h, the formazan crystals formed were solubilized with 50 µL of DMSO. In 5 min, optical density of the solution was measured at the wavelength of 590 nm by the Infinite M Plex multifunctional plate reader .The phototoxic analysis was made using an LED lamp with a maximum wavelength of 730–870 nm and a power of 50W. Fluence was measured as 25 J/cmS,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA), was isolated from a Pacific brittle star Ophiura sarsii [O. sarsii [In our previous work, a chlorin compound,  as nodes. Two nodes are connected by an edge if their spectral similarity (based on a cosine score) is above a defined level. This visualization allows the organization of a large amount of MS2 data and can facilitate the search for analogs.This motivated a new sample collection to search for natural analogs of ETPA in May 2021. The samples were homogenized and extracted with solvents of increasing polarity . The MeOH extract was further treated by liquid–liquid extraction (butanol–water) to remove most of the salts and polar primary metabolites. In the extraction process, the hexane and water extracts were not considered for analysis because they are known to contain mainly lipids and saccharides, respectively. The EtOAc and MeOH (butanolic fraction) extracts were profiled by untargeted UHPLC-PDA-CAD-HRMS/MS in the data-dependent mode to highlight the presence of chlorin analogs. The obtained data were processed on the MZmine software and uploaded to the Global Natural Products Social (GNPS) platform [+) associated with the previously isolated compound ETPA was highlighted  in the FBMN by a diamond -14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) (1) that we previously isolated from this brittle star [S)-Hydroxypheophorbide a (2) previously isolated from the microalgae Chlorella regulans [Clerodendrum calamitosum [3) previously isolated from the plant Artemisia capillaris [4) previously isolated from diverse species such as the plant Atalantia monophyla [or the sea mussel Musculus senhousei [S,4S,21R)-14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid  previously not reported from natural sources but cited in a synthesis patent [Targeted isolation of ETPA-related compounds was performed from both MeOH and EtOAc extracts. Chromatographic conditions were first optimized on an HPLC-PDA-ELSD instrument on a CLC scale  and injeLC scale  -Hregulans  and the amitosum ; Pheophopillaris ; Pyropheonophyla  or the senhousei ; and . The 1H NMR spectrum of compound 6 showed close similarities to that of compound 1: three deshielded aromatic protons at δH 8.99 , 9.79 , and 9.90 , oxygenated methylene at δH 5.80 , three deshielded methyl singlets at δH 3.30 , 3.45 , and 3.88 , the typical ethylene group at δH 3.82 , and 1.69 , as well as a methyl doublet at δH 1.95 , and the two methine proton at δH 4.39 , and 4.94 . When compared to compound 1, the two doublets from the methylene protons in C-10  were missing and replaced by a singlet at δH/δC 4.61/75.1 indicating a hydroxyl in C-10. The ROESY correlations from H2-7a to H-8 and H-10 indicate that the three methines, H-8, H-7, and H-10, were trans-oriented. The ROESY from H-7 to the methyl H3-8a and the deshielding of H-7 (δH 4.94) compared to Pheophorbide a (δH 4.05) confirmed the trans configuration of H-7 and H-8 and that the hydroxyl in C-10 was in the same side than H-7. The positive Cotton effect at 400 nm and negative at 667 nm confirmed that, like other chlorins isolated, the configuration at C-8 and C-7 of 1 was S, S. Compound 6 was thus identified as -14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid ((10R)-hydroxy-ETPA), that has never been reported. Furthermore, to our knowledge, this is the first report of a chlorin with hydrogen and a hydroxyl group in C-10 isolated from natural sources.Compound 1 to 6 from O. sarsii are shown in O. sarsii of 40 g. It can be seen that all six isolated chlorins are major metabolites, comparable in content to the ETPA we initially isolated from this brittle star.The elucidated structures of the chlorin compounds The isolated natural chlorin compounds show typical absorption and fluorescence spectra of chlorins such as the commercially available Pheophorbide a or the ETPA previously characterized by us ,15 . Additionally, we previously used two different methods to calculate the singlet oxygen quantum yield of ETPA as 0.8 [O. sarsii possesses significant singlet oxygen quantum yield properties.The results of this analysis are depicted in A as 0.8 , which wO. sarsii, against the triple-negative breast cancer cell line BT-20. In the dark, when the chlorin compounds are not activated, their toxicities were low, with the IC50s in the range of 40–80 μM, corresponding to our previous findings on ETPA [We next assessed the phototoxicity of the chlorin compounds isolated from  on ETPA ,15 —the characteristic value of photosensitizers used in PDT. We find the PI values to vary from 12 to 41, being highest for ETPA among the tested compounds, in agreement with our previous findings [Photoactivation of the chlorins was achieved with an LED lamp, with the emission maximum partially covering the absorbance maximum of ca. 680 nm of chlorins B. Upon ir values . The ratfindings .O. sarsii [O. sarsii [O. sarsii is mostly a shallow-waters brittle star that we collected in the Sea of Japan next to Russky Island  aboard the vessel R/V Akademik M. Lavrentyev (Cruise No. 94) to the seamounts of the Emperor Chain (Pacific Ocean) in July–September 2021. During this expedition, we collected invertebrate samples from depths of 750–1000 m near the Koko guyot  has previously been identified at the coasts of Japan, Australia, and New Zealand, at depths ranging from 100 to 1170 m [Ophiura genus as O. sarsii. In contrast, Gorgonocephalus cf. eucnemis [o 1170 m ,37,38 aneucnemis  . EtOAc and MeOH extracts were profiled with UHPLC-PDA-CAD-HRMS/MS. The obtained data were again processed with MZmine with the EtOAc and MeOH extracts of O. sarsii . The generated aligned feature table was exported, and the intensities (MS peak area) for each of the six isolated compounds in each extract were plotted , but these are very minor compounds in the extracts. It should be noted, however, that the analysis we performed is only semi-quantitative, as the concentrations of the extracts were not standardized before the analysis. These data suggest that chlorins are not exclusive to the species O. sarsii, nor even to the genus Ophiura, but are likely present in different invertebrates of the class Ophiuroidea, albeit in different amounts and chemical representatives.Samples of  plotted . Most ofOphiura sarsii and Ophiura ooplax, and five of the same chlorins can be found as minor metabolites in the basket star Gorgonocephalus cf. eucnemis. The class Ophiuroidea consists of >2000 known species (of which 400 are in the North Pacific), falling into 16 families [Our findings characterize the marine invertebrates of the class Ophiuroidea as a rich source of natural photosensitizers. Six chlorins with photosensitizer properties emerge as major metabolites of the shallow- and deep-water brittle stars We provide a detailed physical and biological characterization of the six Ophiuroidea chlorins, two of which have never been found in natural sources before. All chlorins emerge as photosensitizers with significant photodynamic properties against cancer cells, being well-tolerable without photoactivation by the red-spectrum light. These features make these natural chlorins interesting candidates for development in prospective PDT applications. The most powerful of the six, the chlorin ETPA, has been applied by us in a proof-of-concept in vivo PDT treatment of glioblastoma —the mostO. sarsii represents an abundant shallow-waters brittle star in the North Pacific that is easily harvested, e.g., in the vicinity of the Russky Island, mariculture of this species appears as the best solution in case mass production becomes needed [Such developments in natural chlorins will raise the need to solve the supply chain task—the common theme for marine-derived drugs . Althougs needed . Chemicas needed ,45.In conclusion, we discover an arsenal of photosensitizers in three species of North Pacific Ophiuroidea, shallow- and deep-water inhabitants. These natural chlorins possess attractive biophysical and anticancer properties. Our discoveries pave the way for further interdisciplinary investigations of the taxonomic chemodiversity of marine invertebrates and translational developments of their metabolites for diverse PDT applications.


Pharmacological results showed antiprotozoal activity with IC50 values in the sub and μM range. In addition, the in vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The substituted diphenylphenanthroline 1l was identified as the most potent antimalarial derivative with a ratio of cytotoxic to antiparasitic activities of 505.7 against the P. falciparum CQ-resistant strain W2. Against the promastigote forms of L. donovani, the phenanthrolines 1h, 1j, 1n and 1o were the most active with IC50 from 2.52 to 4.50 μM. The phenanthroline derivative 1o was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 91 on T. brucei brucei strain. FRET melting and native mass spectrometry experiments evidenced that the nitrogen heterocyclic derivatives bind the telomeric G-quadruplexes of P. falciparum and Trypanosoma. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma could be considered to be possible targets of this kind of nitrogen heterocyclic derivatives, their potential ability to stabilize the parasitic telomeric G-quadruplexes have been determined through the FRET melting assay and by native mass spectrometry.A series of novel 2,9- P. falciparum malaria in children living in regions with moderate to high transmission   , and N-biodide G  20,21].,21.Plasmreported .Leishmania species described to be infective to humans, and trypanosomiases including human African trypanosomiasis (or sleeping sickness) caused by Trypanosoma brucei and South American trypanosomiasis (or Chagas disease) caused by Trypanosoma cruzi. Classified as neglected tropical diseases according to the WHO, which wants to eliminate them as a public health problem by 2030 -1,10-phenanthroline derivatives ]-4,7-phenyl-1,10-phenanthroline derivatives salts 1 ]-4,7-phenyl-1,10-phenanthroline derivatives 1 was assessed in human HepG2 cells, and a selectivity index, the ratio of cytotoxic to antiparasitic activity, was determined for each derivative. The telomeres of the different protozoa could constitute attractive drug targets + Calculed for C34H41N6: 533.3393, Found: 533.3382.Orange oil (71%). 1H NMR (CDCl3) δ: 7.76 , 7.66 , 7.49–7.43 , 4.32 , 3.67 , 2.83 , 2.36 , 2.19 2), 1.78 . 13C NMR (CDCl3): 161.3 (C-2 and C-9), 150.3 (C-4 and C-7), 147.4 (C-1a and C-10a), 139.5 (C-1′), 131.5 (C-3′ and C-5′), 129.9 , 127.0 (C-4a and C-6a), 124.8 (C-5 and C-6), 124.0 (C-3 and C-8), 59.3 (2 NCH2), 57.6 (2 NCH2), 49.7 (2 NCH2), 46.9 (2 N(CH3)2), 29.4 (2 CH2). ESI-MS m/z [M+H]+ Calculed for C36H45N6: 561.3706, Found: 561.3705.Orange oil (83%). 1H NMR (CDCl3) δ: 7.86 , 7.72 , 7.53–7.47 , 4.35 , 2.83 , 2.29 , 2.21 2), 1.67–1.53 . 13C NMR (CDCl3): 161.5 (C-2 and C-9), 150.3 (C-4 and C-7), 147.4 (C-1a and C-10a), 139.6 (C-1′), 131.1 (C-3′ and C-5′), 129.9 (C2′ and C6′), 129.8 (C-4′), 127.0 (C-4a and C-6a), 124.8 (C-5 and C-6), 123.8 (C-3 and C-8), 61.1 (2 NCH2), 57.7 (2 NCH2), 51.4 (2 NCH2), 46.8 (2 N(CH3)2), 29.5 (2 CH2), 27.0 (2 CH2). ESI-MS m/z [M+H]+ Calculed for C38H49N6: 589.4019, Found: 589.4007.Orange oil (98%). 1H NMR (CDCl3) δ: 7.81 , 7.72 , 7.56–7.51 , 4.36 , 2.83 , 2.43 , 2.27 , 2.22 2), 1.65 , 1.51–1.38 . 13C NMR (CDCl3): 161.3 (C-2 and C-9), 150.3 (C-4 and C-7), 147.4 (C-1a and C-10a), 139.5 (C-1′), 131.1 (C-3′ and C-5′), 129.9 , 127.0 (C-4a and C-6a), 124.8 (C-5 and C-6), 124.1 (C-3 and C-8), 61.1 (NCH2), 57.6 (NCH2), 51.4 (NCH2), 46.8 (N(CH3)2), 31.5 (CH2), 29.0 (CH2), 29.6 (CH2). ESI-MS m/z [M+H]+ Calculed for C40H54N6: 618.4410, Found: 618.4393.Yellow-orange oil (65%). 3) δ: 7.81 , 7.77 , 7.56–7.50 , 4.41 , 2.91 , 2.76 , 2.61 , 2.57–2.36 , 2.27 . 13C NMR (CDCl3): 161.9 (C-2 and C-9), 150.3 (C-4 and C-7), 146.9 (C-1a and C-10a), 139.6 (C-1′), 131.1 (C-3′ and C-5′), 129.9 (C2′and C6′), 129.8 (C-4′), 127.0 (C-4a and C-6a), 124.8 (C-5 and C-6), 123.7 (C-3 and C-8), 59.4 (NCH2), 57.8 (NCH2), 56.5 (NCH2pip), 54.7 (NCH2pip), 47.9 (NCH2), 47.4 (NCH3). ESI-MS m/z [M+H]+ Calculed for C40H51N8: 643.4236, Found: 643.4229.Yellow-orange oil, (68%). 1H NMR (CDCl1H NMR (CDCl3) δ: 7.79 , 7.69 , 7.54–7.48 , 4.34 , 3.02 , 2.86 , 2.49–2.41 , 2.24 , 1.82 . 13C NMR (CDCl3): 161.2 (C-2 and C-9), 150.4 (C-4 and C-7), 147.3 (C-1a and C-10a), 139.5 (C-1′), 131.0 (C-3′ and C-5′), 130.3 (C-4′), 129.9 (C-2′ and C-6′), 127.0 (C-4a and C-6a), 124.8 (C-5 and C-6), 124.0 (C-3 and C-8), 58.2 (NCH2), 57.4 (NCH2), 56.4 (NCH2pip), 54.5 (NCH2pip), 50.0 (NCH2), 47.3 (NCH3), 28.4 (CH2). ESI-MS m/z [M+H]+ Calculed for C42H56N8: 672.4628, Found: 672.4622.Yellow oil (88%). 1H NMR (CDCl3) δ: 7.76 , 7.69 , 7.48–7.42 , 4.32 , 2.96 , 2.80 , 2.42–2.30 , 2.23 , 1.61–1.56 . 13C NMR (CDCl3): 161.6 (C-2 and C-9), 150.2 (C-4 and C-7), 147.3 (C-1a and C-10a), 139.6 (C-1′), 131.0 (C-3′ and C-5′), 129.9 (C-2′ and C-6′), 129.8 (C-4′), 126.9 (C-4a and C-6a), 124.8 (C-5 and C-6), 123.9 (C-3 and C-8), 59.9 (NCH2), 57.6 (NCH2), 56.5 (NCH2pip), 54.5 (NCH2pip), 51.3 (NCH2), 47.4 (NCH3), 29.6 (CH2), 26.1 (CH2). ESI-MS m/z [M+H]+ Calculed for C44H59N8: 699.4862, Found: 699.4839.Yellow oil (88%). 1H NMR (CDCl3) δ: 7.81 , 7.74 , 7.58–7.44 , 4.40 , 3.68 , 3.24 , 2.91 , 2.60 , 2.47 . 13C NMR (CDCl3): 161.6 (C-2 and C-9), 150.4 (C-4 and C-7), 147.4 (C-1a and C-10a), 139.6 (C-1′), 131.0 (C-3′ and C-5′), 129.9 , 127.0 (C-4a and C-6a), 124.8 (C-5 and C-6), 123.8 (C-3 and C-8), 68.3 (4 OCH2), 59.8 (2 NCH2), 57.7 (2 NCH2), 55.2 (4 NCH2morph), 47.4 (2 NCH2). ESI-MS m/z [M+H]+ Calculed for C38H45N6O4: 617.3604, Found: 617.3596.Yellow oil, (73%). 3) δ: 7.79 , 7.67 , 7.53–7.48 , 4.34 , 3.66 , 3.51 , 2.87 , 2.48–2.42 , 1.82 . 13C NMR (CDCl3): 161.0 (C-2 and C-9), 150.5 (C-4 and C-7), 147.3 (C-1a and C-10a), 139.4 (C-1′), 131.0 (C-3′ and C-5′), 130.0 (C2′and C6′), 129.9 (C-4′), 127.0 (C-4a and C-6a), 124.9 (C-5 and C-6), 124.1 (C-3 and C-8), 68.3 (2 OCH2), 58.6 (2 NCH2), 57.4 (2 NCH2), 55.1 (4 NCH2morph), 49.8 (2 NCH2), 28.0 (2 CH2). ESI-MS m/z [M+H]+ Calculed for C40H53N6O2: 645.3917, Found: 645.3912.Yellow oil, (98%). 1H NMR (CDCl1H NMR (CDCl3) δ: 7.79 , 7.74 , 7.53–7.50 , 4.39 , 3.35 , 2.90 , 2.56 , 2.43–2.39 , 1.58–1.51 , 1.43–1.38 . 13C NMR (CDCl3): 161.8 (C-2 and C-9), 150.3 (C-4 and C-7), 147.4 (C-1a and C-10a), 139.6 (C-1′), 131.1 (C-3′ and C-5′), 129.9 , 126.9 (C-4a and C-6a), 124.8 (C-5 and C-6), 123.8 (C-3 and C-8), 60.2 (NCH2), 57.7 (NCH2), 56.2 (NCH2pip), 48.1 (NCH2), 27.3 (CH2pip), 25.8 (CH2pip). ESI-MS m/z [M+H]+ Calculed for C40H49N6: 613.4019, Found: 613.4020.Orange oil, (73%). 1H NMR (CDCl3) δ: 7.76 , 7.71 , 7.50–7.44 , 4.34 , 2.82 , 2.41–2.30 , 1.78 , 1.50 , 1.40–1.36 . 13C NMR (CDCl3): 161.8 (C-2 and C-9), 150.2 (C-4 and C-7), 147.3 (C-1a and C-10a), 139.6 (C-1′), 131.0 (C-3′ and C-5′), 129.8 (C2′and C6′), 129.7 (C-4′), 126.9 (C-4a and C-6a), 124.7 (C-5 and C-6), 123.7 (C-3 and C-8), 59.1 (NCH2), 57.8 (NCH2), 56.0 (NCH2pip), 50.2 (NCH2), 28.7 (CH2), 27.3 (CH2pip), 25.8 (CH2pip). ESI-MS m/z [M+H]+ Calculed for C42H53N6: 641.4331, Found: 641.4322.Yellow-orange oil, (97%). 1H NMR (CDCl3) δ: 7.78 , 7.71 , 7.52–7.45 , 4.34 , 2.91 , 2.86 , 2.55 , 2.48–2.45 , 1.82 , 1.76–1.71 . 13C NMR (CDCl3): 161.7 (C-2 and C-9), 150.2 (C-4 and C-7), 147.5 (C-1a and C-10a), 139.6 (C-1′), 131.0 (C-3′ and C-5′), 129.9 (C2′and C6′), 129.8 (C-4′), 126.9 (C-4a and C-6a), 124.8 (C-5 and C-6), 123.8 (C-3 and C-8), 57.8 (2 NCH2), 56.1 (2 NCH2), 55.6 (4 NCH2pyrr), 50.0 (2 NCH2), 30.9 (2 CH2pyrr), 24.8 (4 CH2pyrr). ESI-MS m/z [M+H]+ Calculed for C42H49N6: 613.4019, Found: 613.4020.Yellow oil, (73%). 1H NMR (CDCl3) δ: 8.47 , 7.78 , 7.69 , 7.54 , 7.51–7.46 , 7.21 , 7.05 , 4.40 , 3.22 , 3.09 . 13C NMR (CDCl3): 161.7 , 150.6 (C-6pyr), 150.2 (C-4 and C-7), 147.3 (C-1a and C-10a), 137.7 (C-4pyr), 131.1 (C-3′ and C-5′), 129.8 , 129.7 (C-4′), 126.9 (C-4a and C-6a), 124.7 , 123.7 (C-3 and C-8), 122.6 (C-5pyr), 57.6 (NCH2), 50.8 (NCH2), 40.0 (CH2pyr). ESI-MS m/z [M+H]+ Calculed for C40H37N6: 601.3079, Found: 601.3070.Orange oil, (97%). 1H NMR (CDCl3) δ: 8.51 , 8.41 , 7.80 , 7.63 , 7.56 , 7.51–7.47 , 7.17 , 4.38 , 3.30 , 3.08 , 2.93 . 13C NMR (CDCl3): 160.9 (C-2 and C-9), 151.6 (C-2pyr), 150.5 (C-4 and C-7), 149.0 (C-6pyr), 147.3 (C-1a and C-10a), 139.4 (C-1′), 137.6 (C-4pyr), 136.8 (C-3pyr), 131.0 (C-3′ and C-5′), 129.9 , 127.1 (C-4a and C-6a), 124.9 (C-5 and C-6), 124.7 (C-5pyr), 124.0 (C-3 and C-8), 57.3 (NCH2), 52.2 (NCH2), 35.0 (CH2pyr). ESI-MS m/z [M+H]+ Calculed for C40H37N6: 601.3079, Found: 601.3069.Orange oil, (80%). 1H NMR (CDCl3) δ: 8.41 , 7.77 , 7.62 , 7.47–7.43 , 7.13 , 4.33 , 3.05 , 2.86 . 13C NMR (CDCl3): 161.2 (C-2 and C-9), 151.1 (C-2pyr and C-6pyr), 150.5 (C-4pyr), 150.4 (C-4 and C-7), 147.3 (C-1a and C-10a), 139.4 (C-1′), 131.0 (C-3′ and C-5′), 129.9 , 127.0 (C-4a and C-6a), 125.6 (C-3pyr and C-5pyr), 124.9 (C-5 and C-6), 123.8 (C-3 and C-8), 57.4 (NCH2), 51.4 (NCH2), 37.3 (CH2pyr). ESI-MS m/z [M+H]+ Calculed for C40H37N6: 601.3079, Found: 601.3073.Orange oil, (98%). 1H NMR (CDCl3) δ: 8.42 , 7.81 , 7.67 , 7.53–7.49 , 7.09 , 4.27 , 3.32 , 2.76 , 2.66 , 1.88 . 13C NMR (CDCl3): 160.9 (C-2 and C-9), 152.5 (C-4pyr), 150.9 (C-2pyr and C-6pyr), 150.5 (C-4 and C-7), 147.3 (C-1a and C-10a), 139.3 (C-1′), 131.0 (C-3′ and C-5′), 130.0 (C2′ and C6′), 129.9 (C-4′), 127.0 (C-4a and C-6a), 125.3 (C-3pyr and C-5pyr), 124.9 (C-5 and C-6), 124.2 (C-3 and C-8), 57.3 (2 NCH2), 50.4 (2 NCH2), 34.2 (CH2pyr), 31.9 (2 CH2). ESI-MS m/z [M+H]+ Calculed for C42H41N6: 629.3392, Found: 629.3382.Yellow oil, (94%). 4 (0.3 mmol) in isopropanol (11 mL) oxalic acid  was added. The reaction mixture was heated under reflux for 30 min. The precipitate was filtered, washed with isopropanol then with diethyl ether and dried under reduced pressure to give the ammonium oxalate salts 1.To a solution of compounds Plasmodium falciparum reference strains 3D7 and W2. The former strain is susceptible to chloroquine (CQ) but displays a decreased susceptibility to mefloquine (MQ); the latter is considered to be resistant to CQ. These two strains are obtained from the collection of the National Museum of Natural History . The parasites were cultivated in RPMI medium  supplemented with 0.5% Albumax I , hypoxanthine (Sigma Aldrich), and gentamicin (Sigma Aldrich) with human erythrocytes and were incubated at 37 °C in a candle jar, as described previously  solution followed by 4 h incubation time. The enzyme reaction was stopped by addition of 100 µL of 50% isopropanol/10% sodium dodecyl sulfate  and FPf8T [FAM-5′(GGGTTCA)3GGG3′-TAMRA], the Trypanosoma 9 and 11 chromosomic sequence FTrypBT  [FAM-5′GGGCAGGGGGTGATGGGGAGGAGCCAGGG3′-TAMRA], the human telomeric sequence F21T [FAM-(GGGTTA)3-GGG3′-TAMRA] and the human duplex sequence FdxT [FAM5′-TATAGCTATA-hexaethyleneglycol-TATAGCTATA3′-TAMRA]  ,31,47. TOligonucleotides were purchased in the lyophilized form from Eurogentec (RP-cartridge purification), and dissolved at 1 mM in pure water. TMAA (1 M) was purchased from Santa Cruz Biotechnology and potassium chloride (purity > 99.99%) from Sigma Aldrich . The sample solutions contained 10 µM oligonucleotide and 10 (for CD only) or 20 µM ligand, in a 100 mM TMAA, 1mM KCl buffer. The solutions were incubated overnight at 4 °C before analysis.The sample solutions were injected at a 10 µM strand concentration on an Agilent 6560 DTIMS-Q-TOF instrument , equipped with a dual-ESI source operated in the negative ion mode in soft conditions . The folThe MS data were extracted using the IM-MS Browser . The peak area intensity of each species at the 5- charge state was extracted from their corresponding m/z range (encompassing potassium adducts), and baseline subtracted. The concentrations of all species  and corresponding dissociation constants were determined as previously described ,50.CD experiments were performed with a JASCO J-1500 spectropolarimeter using quartz cells of 2 mm path length. The scans were recorded at 22 °C, from 220 to 350 nm with the following parameters: 1.0 nm data pitch, 2 nm bandwidth, 0.5 s response, 50 nm/min scanning speed; they are the result of three accumulations. The CD data were blank-subtracted, baseline subtracted and normalized to molar dichroic absorption as previously described ,50.1a–p were synthesized starting from the commercially available 2,9-dimethyl-4,7-di-phenyl-1,10-phenanthroline, also called bathocuproine (2 was obtained by oxidation of the bathocuproine using selenium dioxide (SeO2) -4,7-diphenyl-1,10-phenanthrolines 3a–p, which were reduced into the 2,9-bis[(substituted-aminomethyl)phenyl]-4,7-diphenyl-1,10-phenanthrolines 4a–p using sodium borohydride in methanol as previously described -4,7-phenyl-1,10-phenanthroline derivatives cuproine . First, e (SeO2) . Reactioescribed ,29,31. T1a–p were evaluated for their antimalarial activity in vitro by incubation with P. falciparum CQ-resistant strain W2  and the strain 3D7, which is CQ-sensitive and which has decreased sensitivity to MQ   . As showP. falciparum CQ-resistant strain W2, compounds 1c, 1i and 1l bearing dimethylaminobutylaminomethyl, morpholinopropylaminomethyl, or pyrrolidinopropylaminomethyl side chains at positions 2 and 9 of the phenanthroline moiety were found to be the most active compounds with an IC50 of 0.03, 0.04 and 0.03 μM, respectively, in the same range as the one observed for mefloquine (IC50 = 0.016 μM). Concerning these phenanthrolines substituted by aminoethylaminomethyl side chains, derivative 1h bearing morpholinoethylaminomethyl side chains displayed better activity than its three other analogues . In comparison, when we replaced the aminoethylaminomethyl side functions by aminopropylaminomethyl side chains , the disubstituted phenanthrolines 1i and 1l bearing morpholino C3 aminomethyl or pyrrolidino C3 aminomethyl side chains showed the best antimalarial activity, i.e., IC50 = 0.04 μM for 1i and 0.03 μM for 1l versus 11.27 μM for their analog 1f bearing methylpiperazinyl C3 aminomethyl side chains). In the sub-series of the phenanthrolines 1a–d substituted by dimethylaminoalkylaminomethyl side chains, compound 1c with butyl chains exhibited a better antimalarial activity than its di-substituted ethyl, propyl, or pentyl analogues 1a-b and 1d (IC50 = 0.03 μM for 1c versus IC50 = 1.00–6.13 μM for the other ones). In the pyridinylethylaminomethyl substituted bisphenanthroline series (compounds 1m–o), derivative 1m containing (pyridin-2-yl)ethyl)aminomethyl chains was found more active (up to 36.3 or 26.7 times) against the P. falciparum W2 strain than its counterpart compounds 1n and 1o with (pyridin-3-yl)ethyl)aminomethyl or (pyridin-4-yl)ethyl)aminomethyl chains (IC50 = 0.07 μM for 1m versus IC50 = 2.54 and 1.87 μM for 1n and 1o). Moreover, the substitution by (pyridin-4-yl)propyl)aminomethyl chains side chains (compound 1p) showed a better antimalarial activity against the W2 strain than its homologue substituted at positions 2 and 9 by (pyridin-4-yl)ethyl)aminomethyl chains (compound 1o); i.e., IC50 = 0.145 μM for 1p versus 1.87 μM for 1o.In terms of structure-activities relationships, the influence of the length of the carbon chain between the two amino functions of the side chain, and also the nature of the terminal amine group, were discussed. Against the 1, compound 1h with morpholinoethylaminomethyl side chains exhibited the most potent activity against the 3D7 strain with an IC50 of 0.67 μM but slightly more active than those observed for the other bis-aminoethylaminomethyl substituted compounds 1a, 1e, and 1j (IC50 = 1.59–2.16 μM). However, this phenanthroline derivative 1h was noticed less active than the two reference drugs, CQ and MQ, which had IC50 values of 0.11 and 0.06 μM, respectively. Moreover, the influence of the length of the carbon chain in the aminoalkylaminomethyl side chains for this phenanthroline series seems also to be detrimental: a longer alkyl chain (aminopropylamine chain to aminopentylamine chain) in comparison with an aminoethylamine chain led to a decreased in the antiplasmodial activity and this whatever the terminal amine function (dimethylamine or methylpiperazine moities) of the side chain, . In the pyridinylethylaminomethyl substituted bisphenanthroline series (compounds 1m–o), whatever the position of the pyridine moiety on the pyridinylethylaminomethyl side chain, compounds 1m–o showed similar range of antiparasitic activities against the 3D7 strain (IC50 = 0.47–0.73 μM).Among the newly reported di-substituted(aminoethylaminomethyl)phenanthroline derivatives 1, some additional antiparasitic analyses were also performed. Notably, P. falciparum belongs to the coccidian protozoan parasite family. Therefore, in vitro activity against flagellate protozoan parasite L. donovani was evaluated .To better understand the pharmacological profile of our new phenanthroline heterocycles valuated . The ref1a–p were then evaluated against T. brucei brucei. In addition, pentamidine, suramine, fexinidazole, and eflornithine were used here as reference compounds. The screening data are presented in 1a–p were active against T. brucei brucei with IC50 values ranging from 0.20 to 1.42 μM; and most of them showed an antiparasitic activity around the 1 μM. In terms of structure-activities relationships, an increase of the alkyl chain in the aminoalkylaminomethyl side chains seems to show a slight decrease in the antitrypanosomal activity, i.e., IC50 = 0.45 μM for 1e versus 0.97 and 0.96 μM for 1f and 1g, respectively. In the pyridinylethylaminomethyl substituted bisphenanthroline series (compounds 1m–o), phenanthroline 1m containing (pyridin-4-yl)ethyl)aminomethyl chains exhibited a better antitrypanosomal activity (IC50 = 0.21 μM) than its homologues bearing (pyridin-2-yl)ethyl)aminomethyl or (pyridin-3-yl)ethyl)aminomethyl side chains (compounds 1m and 1n) for which IC50 were noticed at 0.63 and 0.43 μM, respectively.These novel synthesized aza heterocyclic compounds 1 were evaluated in vitro in the human cell line HepG2 (50) were determined, and selectivity indexes (SI), defined as the ratios of cytotoxic to antiparasitic activities (SI = CC50/IC50) were calculated. The results of cytotoxicity assays and the associated SI values are presented in 1 that were found active against the various parasites showed significant cytotoxicity against the HepG2 cells with CC50 values ranging from 1.64 to 15.17 µM. Concerning the W2 strain, the calculated SIs were noticed between 0.76 and 505.7. For the CQ-sensitive strain 3D7, the SIs were found from 0.28 to 26.18. Analyses of these SI values led us to identify the phenanthrolines 1l and 1c as very interesting compounds with SI of 505.7 and 347.67 for the W2 strain, respectively. In addition, the derivative 1i also showed promising SI value of 157.25 for this CQ-resistant strain W2. In addition, this SI led to the identification of compound 1o with SI of 7.1 on L. donovani. Against the T. brucei brucei strain, 2,9-bis [2-(pyridin-4-yl)ethyl)aminomethyl]-4,7-diphenyl-1,10-phenanthroline 1o had SI of 91.0. These promising SI values could indicate that these new nitrogen heterocyclic compounds warrant further investigations into their potential use as antiparasitic drugs.To assess selectivity of action, the cytotoxicities of these newly synthesized antiparasitic phenanthroline compounds ne HepG2 , which iP. falciparum and Trypanosoma could be considered to be potential targets of this kind of nitrogen heterocyclic derivatives ), Pf8 (d[(G3T2CA)3G3]) and the human telomeric sequence 24TTG (2A2T2(CG)2]) and a G-rich unfolded single-stranded sequence, 24nonG4 (d[TG3ATGCGACA(GA)2G2ACG3A]). All tested structures contain 24 nucleotides.Native mass spectrometry is the technique of choice to determine the binding affinity and stoichiometry of small molecules with oligonucleotides . Here wece 24TTG . Two nonPlasmodium telomeric sequences used in the FRET meting experiments. Their CD signature suggests the formation of hybrid conformers 3A]) is a variant of the human telomeric sequence (similar to the 21T sequence used in the FRET melting experiments) whose hybrid structure in native MS conditions is known -4,7-phenyl-1,10-phenanthroline derivatives. These new phenanthrolines were tested for their in vitro antiprotozoal activity toward the CQ-resistant W2 and CQ-sensitive 3D7 P. falciparum strains, the promastigote form of L. donovani, and also toward a T. brucei brucei strain. In addition, the in vitro cytotoxicity of these new nitrogen heterocyclic compounds was assessed on the human HepG2 cell line. Among these novel synthesized nitrogen heterocyclic molecules, a few of them were identified as interesting potential in vitro antiplasmodial leads with IC50 ranging from 0.03 to 0.73 µM on the W2 and 3D7 strains of P. falciparum. Interestingly, the 2,9-bis [3-(pyrrolidin-1-yl)propyl)aminomethyl]-4,7-diphenyl-1,10-phenanthroline 1l was identified as the most potent and promising antimalarial candidate with a ratio of cytotoxic to antiparasitic activities of 500 against the P. falciparum CQ-resistant strain W2. Four substituted phenanthrolines showed activity against the promastigote forms of L. donovani with IC50 values ranging from 2.52 to 4.50 µM. Moreover, the antiprotozoal activity spectrum of our new synthesized phenanthrolines using a T. brucei brucei strain revealed IC50 values ranging from 0.20 to 1.42 µM, which warrant further investigations. The 2,9-bis [2-(pyridin-4-yl)ethyl)aminomethyl]-4,7-diphenyl-1,10-phenanthroline 1o was also identified as the most potent trypanosomal candidate with SI of 91 on Trypanosoma brucei brucei strain. Structure-activity relationships of these new synthetic compounds are also discussed, as well as their relative ability to target P. falciparum or Trypanosoma telomeres as a hypothetical mechanism of action and a potential way to escape the efflux resistance developed by parasites toward quinoline drugs. Thus, as the telomeres of the parasites could constitute interesting targets, we also investigated the possibility of targeting Plasmodium telomeres or Trypanosoma chromosomes by stabilizing the Plasmodium or Trypanosoma G-quadruplexes sequences through FRET melting assays with our bioactive phenanthrolines. Concerning the stabilization of the parasitic G-quadruplex, it could be noticed that the best diphenylphenanthrolines 1 which exhibited an interesting stabilization profile were those substituted by the longest aminoalkylaminomethyl side chains. However, we found no correlation between the antiparasitic activity and selectivity of these derivatives and their binding to G-quadruplexes. These derivatives are therefore unlikely to be specifically cytotoxic via G-quadruplex binding. Moreover, it would be now interesting to enlarge the biological evaluation of these new diphenylphenanthrolines derivatives 1 by studying their mechanism of action through further investigations such as the inhibition of beta-hematin formation or of the apicoplast functions. Finally, the new substituted 2,9-bis[(substituted-aminomethyl)]-4,7-phenyl-1,10-phenanthrolines could open the way to new valuable medicinal chemistry scaffolding in the antiprotozoal domain.In this work, we described the design, synthesis, antiprotozoal activities, and the in vitro cytotoxicity toward human cells of a novel series of 2,9-


In order to find novel A2AAR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A2AAR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.The A Elemental analysis calcd for C16H20N6O: C 61.52, H 6.45, N 26.90; found C 61.28, H 6.69, N 26.80.2-(4-methoxyphenethyl)-9H-purine-2,6-diamine8-Bromo-9-ethyl-N (8): 7  was dissolved in 5 mL of dry DMF and a solution of NBS  in 5 mL of dry DMF was added dropwise over 30 min in which there was the total consumption of the starting material. The mixture was evaporated to dryness under vacuum, neutralized with 1N NaOH, and extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and placed under stirring with anhydrous sodium sulphate. The suspension was filtered, the filtrate was brought to dryness, and the residue purified via flash column chromatography eluting with EtOAc-cHex (60:40). 8  was obtained as white powder after crystallization with MeCN. M.p.: 155–157 °C; 1H-NMR (DMSO-6d) δ: 1.30 , 2.77 , 3.41 , 3.73 , 4.02 , 6.42 , 6.86 , 7.18 . ESI-MS: positive mode m/z: 390.9 ([M+H]+), 414.9 ([M+Na]+). Elemental analysis calcd for C16H19BrN6O: C 49.12, H 4.89, N 21.48; found C 49.46, H 4.55, N 21.32.4-(2-((6-Amino-9-ethyl-8-bromo-9H-purin-2-yl)amino)ethyl)phenol (9): 8  was suspended in 4 mL of HBr 48% and heated at 100 °C for 2 h. The reaction was quenched with ice, neutralized at 0 °C with NH4OH 33%, followed by extraction with EtOAc (3 × 20 mL). The organic phases were dried with anhydrous sodium sulphate and filtered. The filtrate was brought to dryness and the residue purified via flash column chromatography eluting with EtOAc-cHex-MeOH (60:39:1) to obtain 9 , 10 , and 11 . 9: M.p.: 221–223 °C. 1H-NMR (DMSO-6d) δ: 1.19 , 2.74 , 3.32 , 3.67 , 5.96 , 6.25 , 6.80 , 7.05 , 9.58 . ESI-MS: positive mode m/z: 378.9 ([M+H]+), 400.9 ([M+Na]+). Elemental analysis calcd for C15H17BrN6O: C 47.76, H 4.54, N 22.28; found C 47.82, H 4.3, N 22.38.10: m.p.: 204–205 °C. 1H-NMR (DMSO-6d) δ: 1.19 , 2.74 , 3.32 , 3.67 , 3.72 , 5.98 , 6.21 , 6.85 , 7.16 , 9.53 . ESI-MS: positive mode m/z: 329.0 ([M+H]+), 351.0 ([M+Na]+). Elemental analysis calcd for C16H21N6O2: C 58.52, H 6.14, N 25.59; found C 58.23, H 6.43, N 25.57.11: m.p.: 220 °C dec. 1H-NMR (DMSO-6d) δ: 1.19 , 2.69 , 3.67 , 4.04 , 5.99 , 6.19 , 6.67 , 7.02 , 9.13 , 9.53 . ESI-MS: positive mode m/z: 315.0 ([M+H]+), 336.9 ([M+Na]+). Elemental analysis calcd for C15H18N6O2: C 57.31, H 5.77, N 26.74; found C 57.10, H 5.66, N 26.95.13) and 9-ethyl-8-(furan-2-yl)-2N-(4-methoxyphenethyl)-9H-purine-2,6-diamine (12): 2-Tributylstannylfuran  and bis(triphenylphosphine)-palladium dichloride  were, in turn, added to a solution of 9  or 8  in anhydrous THF (330 µL). The mixtures were refluxed for 4 h and 6 h, respectively, after which volatiles were evaporated to dryness and the crude mixtures purified through a flash column silica gel eluting with DCM-MeOH (100:0 to 99:1) to afford 13  and with DCM-MeOH (97:3) to obtain 12  as white solids after crystallization from MeCN. General procedure for the synthesis of 4-(2-((6-Amino-9-ethyl-8-(furan-2-yl)-9H-purin-2-yl)amino)ethyl)phenol (13: m.p.: 150–151 °C. 1H-NMR (DMSO-d6) δ: 1.32 , 2.79 , 3.44 , 3.72 , 4.28 , 6.38 , 6.71 , 6.80 , 6.86 , 6.98 , 7.18 , 7.89 ; ESI-MS: positive mode m/z: 379.2 ([M+H]+), 391.2 ([M+Na]+). Elemental analysis calcd for C20H22N6O2: C 63.48, H 5.86, N 22.21; found C 63.76, H 5.58, N 22.33.12: m. p.: 166–168 °C; 1H-NMR (DMSO-d6) δ: 1.29 , 2.71 , 3.39 , 4.26 , 6.34 , 6.66 , 6.68 , 6.79 , 6.95 , 7.03 , 7.87 , 9.13 ; ESI-MS: positive mode m/z: 365.2 ([M+H]+), 387.2 ([M+Na]+); negative mode m/z 363.1 ([M−H]−), 409.1 ([M-Cl]−). Elemental analysis calcd for C19H20N6O2: C 62.62, H 5.53, N 23.06; found C 62.86, H 5.40, N 23.20).5-(Methylthio)-1H-1,2,4-triazol-3-amine (15): Dimethyl N-cyanodithioimino-carbonate  was dissolved in toluene (3 mL) and reacted with hydrazine hydrate  under stirring in an ice bath under an inert atmosphere. The mixture was left at 0 °C for 1 h and then at r. t. for 4 h; after that it was filtered, and the residue was washed with cHex. The organic layers were dried over sodium sulfate, then filtered and evaporated and the residue was purified by flash column chromatography eluting with DCM-MeOH (97.5:2.5) to obtain the desired compound 15 as a white solid . M.p.: 136–139 °C. 1H-NMR (DMSO-d6) δ: 2.38 , 5.99 , 11.91 ppm . ESI-MS: positive mode m/z: 131.0 ([M+H]+), 153.0 ([M+Na]+). Elemental analysis calcd for C3H6N4S: C 27.68, H 4.65, N 43.04; found C 27.86, H 4.55, N 43.18.5-(Methylsulfonyl)-1H-1,2,4-triazol-3-amine (16): Compound 16 was obtained by reacting the intermediate 15  with m-CPBA , both dissolved in DCM (32 mL) at 0 °C for 4 h and subsequently at r. t. for 20 h. After removal of the solvent under vacuum, compound 16 was obtained by crystallization with EtOAc, from the crude mixture, as a white solid . M.p.: 235–237 °C. 1H-NMR (DMSO-d6) δ: 3.18 , 6,55 , 12.86 ppm . ESI-MS: positive mode m/z: 185.0 ([M+Na]+), 346.9 ([2M+Na]+); negative mode m/z: 161.1 ([M−H]−), 197.1 ([M+Cl]−), 323.0 ([2M−H]−), 358.9 ([2M+Cl]−). Elemental analysis calcd for C3H6N4O2S: C 22.22, H 3.73, N 34.55; found C 22.48, H 3.47, N 34.50).2-(Methylsulfonyl)-5-(methylthio)-triazolo triazin-7-amine (17): The intermediate 16  was reacted with dimethyl N-cyanodithioiminocarbonate  in a metal container hermetically sealed and placed at 170 ° C for 6.5 h. The product becomes a sticky yellow paste which was purified by flash column chromatography eluting with DCM-MeOH (99.5:0.5) to obtain the desired product 17 as a white solid . M.p.: 250 °C (dec). 1H-NMR (DMSO-d6) δ: 2.48 , 3.40 , 9.15 ppm . ESI-MS: positive mode m/z: 282.9 ([M+Na]+), 542.6 ([2M+Na]+); negative mode m/z: 259.1 ([M−H]−). Elemental analysis calcd for C6H8N6O2S2: C 27.69, H 3.10, N 32.29; found C 27.36, H 3.23, N 32.49).2-Ethoxy-5-(methylthio)-triazolotriazin-7-amine (18) and 2,5-diethoxy-triazolotriazin-7-amine (19): 3 mL of ethanol are reacted with sodium hydride  for 2 h at room temperature under nitrogen flow. Then compound 17  was added and the reaction mixture was left at 50 °C for 28 h. Then, the reaction was neutralized with an aqueous solution of 2N HCl and brought to dryness under vacuum. Water was added, and the product was extracted with ethyl acetate (3 × 20 mL). The organic phases were dried with anhydrous sodium sulphate and filtered. The filtrate is brought to dryness and the residue purified via flash column chromatography eluting with DCM-MeOH (99.5: 0.5) to obtain the desired compound 18  and a secondary product 19 as white solid .18: m.p.: 220 °C (dec). 1H-NMR (DMSO-d6) δ: 1.34 , 2.45 , 4.36 , 8.33 , 8.75 ppm . ESI-MS: positive mode m/z: 227.1 ([M+H]+), 249.1 ([M+Na]+), 475.0 ([2M+Na]+); negative mode m/z: 225.3 ([M−H]−). elemental analysis calcd for C7H10N6OS: C 7.16, H 4.45, N 37.14; found C 7.46, H 4.24, N 37.00.19: m.p.: 180–181 °C. 1H-NMR (DMSO-d6) δ: 1.27 , 1.33 , 4.28 , 4.34 , 8.42 . ESI-MS: positive mode m/z: 225.1 ([M+H]+), 247.1 ([M+Na]+), 471.0 ([2M+Na]+); negative mode m/z: 223.2 ([M−H]−). Elemental analysis calcd for C8H12N6O2: C 42.85, H 5.39, N 37.48; found C 42.75, H 5.51, N 37.40.2-Ethoxy-triazolotriazin-7-amine (21): Compound 18  was suspended in tetrahydrofuran  and Pd/C 10%  was added. The mixture was placed at 0 °C and then triethylsilane  was added. The reaction mixture was left at 0 °C for 30 min and then at r. t. for 7.5 h. The formed suspension was filtered, the filtrate was evaporated to dryness under vacuum, and the residue was crystallized from a mixture of DCM-MeOH (9:1) to obtain the desired product . M.p.: 212–213 °C. 1H-NMR (DMSO-d6) δ: 1.35 , 4.40 , 8.21 , 8.40 , 8.85 . ESI-MS: positive mode m/z: 181.2 ([M+H]+), 203.1 ([M+Na]+), 383.0 ([2M+Na]+); negative mode m/z: 179.3 ([M−H]−), 259.2 ([2M−H]−). Elemental analysis calcd for C14H17N7O2: C 53.32, H 5.43, N 31.09; found C 53.16, H 5.59, N 31.20).4-amino)ethyl)phenol (20): To a suspension of 18  in THF, m-CPBA  was added at r. t. and left to react for 2 h. Then, to the reaction mixture, a solution of tyramine  in THF was added. The temperature was brought to 40 °C and left for 24 h. To the reaction mixture, 2N NaHCO3 was added to neutralize the pH and extracted with EtOAc (3 × 20 mL). The organic phases were dried with anhydrous sodium sulphate and filtered. The filtrate was brought to dryness and the residue purified via flash column chromatography eluting with DCM-MeOH (98:2). The compound was obtained by crystallization with MeOH-EtOAc-Petroleum ether (9:1:1) to obtain 20 as a white powder . 1H-NMR (DMSO-d6) δ: 1.31 , 2.66 , 3.32 , 4.29 , 6.98 , 6.65 , 7.28 , 9.15 . ESI-MS: positive mode m/z: 316.1 ([M+H]+), 337.9 ([M+Na]+), 653.0 ([2M+Na]+). Elemental analysis calcd for C6H8N6O: C 40.00, H 4.48, N 46.65; found C 40.24, H 4.44, N 46.42.17 were collected on a Bruker APEX II single-crystal diffractometer working with monochromatic Mo-Kα radiation and equipped with an area detector. The structure was solved by direct methods and refined against F2 with SHELXL-2014/7 with anisotropic thermal parameters for all non-hydrogen atoms -CCPA, to a final volume of 200 μL. The samples were left to incubate for 3 h at room temperature, filtered in a filter 96-well plate  using the FilterMate Cell Harvester (Perkin-Elmer), and washed 3 times with cold distilled water. After drying the filter plate at 40 °C for 30′, they 20 μL of scintillating liquid  was added to each well, and then the radioactivity was quantized with the MicroBeta2 Plate Counter (Perkin-Elmer). The conditions for binding experiments at A2A and A3 receptor subtypes were essentially the same as for the A1AR using [3H]-NECA (N-ethylcarboxyamideAdo) at a concentration of 10 nM and [3H]-HEMADO (2-(1-hexyne)-N6-methylAdo) at a concentration of 1 nM as radioligands, respectively. The samples were incubated with 12 micrograms of protein for 3 h at room temperature and filtered as described above.Binding assays: The dissociation constants of the radioligand (KD) were calculated through saturation assays, while the dissociation constants of the compounds under study Ado) for the A2A and A3 receptors.The non-specific binding was determined in the presence of 1 mM theophylline for the A2 in a humidified incubator and grown in DMEM supplemented with 10% (v/v) low-endotoxin fetal bovine serum , 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin . Cells were seeded at 50,000 cells/mL in 96-well tissue culture plates for the viability assay, in 6-well tissue culture plates for the Griess assay and RT-PCR assay. BV-2 cells were treated for 24 h with ANR 94 or 13 at different concentrations and then exposed to LPS  for a further 24 h.Cell Cultures and Treatments: BV-2 murine microglial cells  were maintained at 37 °C and 5% COMTT Viability Assay: MTT assay was performed to quantify the viability of BV-2 cells as previously reported . At the Measurement of NO Production: The amount of nitrite accumulation in the culture medium of BV-2 cells was measured using Griess reagent. 50 µL of culture medium from each sample was mixed with the same volume of the Griess reagent (Sigma-Aldrich-Merck). The NO concentration was evaluated by measuring the absorbance at 540 nm with a 96-well microplate spectrophotometer . Concentration of nitrites released in the medium was determined from the standard curve generated with known concentrations of sodium nitrite. Results are expressed as mean concentration of nitrites (μM) ± SEM, from three different samples.−ΔΔCT method.Real-Time Polymerase Chain Reaction (PCR): At the end of the experiments, total RNA was extracted by BV-2 cells using RNeasy Mini Kit . RNA concentraion and quality were measured on a NanoVue Spectrophotometer . iScript cDNA Synthesis Kit  was used to synthesize cDNA starting from the extracted RNA, following the supplier’s instructions. PCR was performed by adding 2.5 μL (12.5 ng) of cDNA, 5 μL SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), and 0.5 μL (500 nM) of each primer  to a PCRReceptor refinement and energy minimization tasks were carried out using MOE. Docking experiments were performed with CCDC Gold .2AAR and A1AR crystal structures refinement: The crystal structures of the human A2AAR and A1AR in complex with ZM241385 and the antagonist PSB36, respectively, were downloaded by the Protein Data Bank webpage . A multiple alignment of the AR primary sequences was built within MOE as the preliminary step. The Homology Modeling tool of MOE was employed for this task. The obtained A3AR model was then energetically minimized with the same protocol of the other receptors (see above).Homology modeling of the human AMolecular docking analysis: Docking analyses were performed by using CCDC Gold , which wIF-E 6.0 tool retrievable at the SVL exchange service . The program calculates and displays the atomic and residue interaction forces as 3D vectors. It also calculates the per-residue interaction energies, where negative and positive energy values are associated to favorable and unfavorable interactions, respectively. For each AR structure, a shell of residues contained within a 10 Å distance from the ligand were considered for this analysis.Post Docking analysis. Residue interaction analysis: The interactions between the ligands and the AR receptors binding sites were analyzed by using the 13) and its 4-methoxyphenyl analogue 12 showed high affinity (Ki in the low nM range) and a different degree of selectivity at the A2AAR. Furthermore, 13 was found to exert anti-inflammatory properties in a microglial model of neuroinflammation, since it was able to reduce pro-inflammatory mediators and to increase anti-inflammatory cytokines in microglia cells.On the base of ZM241385 and ANR 94 structures, two series of new compounds endowed with the triazolotriazine, and purine scaffolds were designed and synthesized. Radioligand binding and functional assays at human recombinant ARs showed that the 9-ethyl-8-furyl-2-(4-hydroxyphenyl)-2-ethylaminoadenine  are substituted. Taken together, the results of our experiments reveal that the novel A


H)-ones. The method uses 5-(4-chlorophenyl)furan-2(3H)-one, triethyl orthoformate, and heterocyclic amines with different ring sizes and heteroatoms under refluxing in absolute isopropyl alcohol. The obtained enamines exist in an equilibrium of E- and Z-isomers, whose configurations relative to the double exocyclic C=C bond were confirmed with a set of NMR spectroscopy data. The E-/Z-equilibrium of the synthesized compounds is affected by the configuration of the intermediate, the volume of its substituents, the site of enolate attack, the presence of intramolecular interactions of amino components, the time of the transformation, the order of mixing of the initial reagents, and the use of polar solvents in the NMR experiment. The advantages of the method are that the reaction time is short, the product yield is high, and product purification is easy.We describe a method to synthesize a new class of hetarylaminomethylidene derivatives of furan-2(3 This could be explained by an increase in the nucleophilic properties of the amino group owing to the electron-donating effect of the hydroxyl group. Of note, the transformation proceeded smoothly in other solvents, with yields of 60–70%.Under reflux conditions, the best results were achieved with the polar protic solvent anhydrous isopropyl alcohol. After pyridine amines had been introduced, the reaction yielded new 5-(4-chlorophenyl)-3-[(R-pyridin-2-ylamino)methylidene]furan-2furan-2(3H)-one 7 reacts with imine 10 to form intermediate 11, after which another EtOH molecule is eliminated to give the desired products 9a–c,f. Pathway B can serve as an alternative mechanism, in which the initial reaction is that of the 5-(4-chlorophenyl)-furan-2(3H)-one molecule 7 with triethyl orthoformate 4 to form ethoxymethylene derivative 12, which is further converted with amino components 8a–c,f through intermediate compounds 11 to desired 3-hetarylaminomethylidenefuran-2(3H)-ones 9a–c,f -one moiety acts as an acceptor group furan-2(3H)-one 9a as an example. It was also possible to assign the signals of the protons for E- and Z-isomers separately. In the 2D NOESY spectra, the cross peaks of the NH group and the vinyl proton of the furan-2(3H)-one fragment, observed at 7.10/10.85 ppm, are due to the steric closeness of the protons. The shape of the spectra and the closeness of the protons correspond to the E-configuration. The Z-isomer showed cross peaks at 6.96/8.50 ppm, related to the proton at C-4 and the proton of the exocyclic C=C bond . In the case of a favorable combination of these factors , the push–pull nature is expected to be maximal with an isomer ratio of approximately 1:1. The assignment of the integral intensities of the signals for the NH-group protons in the 1H NMR spectra makes it possible to determine the content of the corresponding isomers in equilibrium mixtures: that of the Z-isomer, from the signal at 10.36 ppm (0.13H), and that of the E-isomer, from the signal at 10.85 ppm (0.87H) for compound 9a. E-/Z-isomers of compounds 9a–f.With a 2D NOESY experiment, it was possible to determine the configuration of compounds C=C bond . EnamineZ-form, as compared to 9a. Product 9b existed in the DMSO-d6 solution in the Z-form, and the presence of the E-isomer was not detected. This fact can be explained by additional intramolecular interactions among the proton of the OH group, the proton of the NH group, and the oxygen atom of the furanone ring; these interactions make the Z-isomer more stable. The presence of an OH group in the aminopyridine moiety at the C-3 position led to a complete shift of the equilibrium toward the E-/Z-equilibrium toward the E-diastereomer and the decrease in the proportion of Z-diastereomers in the NMR spectra in DMSO-d6 is probably due to the ability of DMSO-d6 to accept hydrogen bonds furan-2(3H)-ones 11′. The rotation around the simple C–N bond in these structures can stabilize the 11′ form through an intermolecular hydrogen bond. The antiperiplanar elimination of an ethanol molecule gives rise to a Z-form of enamines 9a–f.Alternatively, the nucleophile attack from the site where the bulkier ethoxy group is located gives rise to isomeric 5-(4-chlorophenyl)-3-[ethoxy(hetaryl-2-ylamino)methyl]furan-2 and pressed into pellets by removing water and air traces under reduced pressure. 1H (400 MHz) and 13C NMR (100 MHz) spectra in DMSO-d6 were recorded with a Varian (Agilent) 400 spectrometer , and the internal standard was TMS (see δ) are reported in ppm. Elemental analysis was done on an Elementar Vario MICRO cube CHNS analyzer . Melting points were determined on a Stuart™ SMP10 melting point apparatus . The progress of the reaction and the purity of the synthesized compounds were monitored by TLC on ALUGRAM® SIL G UV254 plates , with hexane–ethyl acetate–acetone (2:2:1) as the eluent.FTIR spectra were collected on an FSM-1201 Fourier spectrometer  in the range 4000–400 cm TMS see . ChemicaH)-one (7), 9 mmol of triethyl orthoformate (4), and 1 mmol of the corresponding heterocyclic amine (8a–d) was refluxed in 7 mL of absolute isopropyl alcohol. The precipitated crystals were filtered, washed with isopropyl alcohol, recrystallized from DMF, and dried.A mixture of 1 mmol of 5-(4-chlorophenyl)furan-2-5-(4-chlorophenyl)-3-[(pyridin-2-ylamino)methylidene]furan-2(3H)-one (9a). Yellow crystals (DMF), yield 0.22 g (75%), mp 275–276 °C; FTIR (KBr), ν, cm−1: 3265–3223 (NH), 1722 (C=O), 1637 (C=C); 1H NMR : δ 6.96 , 7.06–7.06 , 7.10 , 7.49 , 7.56 , 7.75–7.79 , 8.30 , 8.35 , 8.54 , 10.35 , 10.86 , 13C NMR : δ 100.83 (4-Fu), 110.00 (3-Fu), 112.06 (Py), 119.49 (Py), 125.34, 125.70, 125.75, 128.28, 129.59, 133.27, 135.12 (6-Fu), 139.42 (Py), 146.85 (5-Fu), 148.85 (Py), 151.76 (Py), 170.16 (C=O). Anal. calcd. for C16H11ClN2O2: C: 64.33%; H: 3.71%; N: 9.38%; Cl: 11.87%; Found: C: 64.75%; H: 3.98%; N: 9.82%; Cl: 12.06%. (Z)-5-(4-Chlorophenyl)-3-{[(3-hydroxypyridin-2-yl)amino]methylidene}furan-2(3H)-one (9b). Yellow crystals (DMF), yield 0.25 g (80%), mp 258–260 °C; FTIR (KBr), ν, cm−1: 3348 (OH), 3289 (NH), 1774 (C=O), 1689 (C=C); 1H NMR : δ 6.99 , 7.02 , 7.24  7.53 , 7.60 , 7.82  8.57 , 10.39 , 10.92 . 13C NMR : δ 101.2 (4-Fu), 104.2 (3-Fu), 120.9, 121.3, 121.7, 122.2, 126.0, 128.3, 132.6, 137.4 (6-Fu), 138.7 (Py), 141.0 (C-OH), 146.2, 155.2 (Py), 170.1 (C=O). Anal. calcd. for C16H11ClN2O3: C: 61.06%; H: 3.52%; N: 8.90%; Cl: 11.26%; Found: C: 61.55%; H: 3.98%; N: 9.15%; Cl: 11.78%.-4-{[(5-[4-chlorophenyl]-2-oxofuran-3[2H]-ylidene)methyl]amino}-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazole-3-one (9c). Yellow crystals (DMF), yield 0.25 g (81%), mp 229–239 °C; FTIR (KBr), ν, cm−1: 3414 (NH), 1751 (C=O), 1685 (C=C), 1649 (C=O); 1H NMR : δ 2.31 , 3.05 , 6.86 , 7.10 , 7.33-7.56 , 7.88 , 8.06 , 9.54 , 9.69 ; 13C NMR : δ 10.79 (CH3), 36.40 (CH3-N), 101.13 (4-Fu), 111.10 (3-Fu), 124.48, 124.99, 125.23, 127.26, 128.82, 129.92, 129.49, 129.64, 129.67, 132.44, 134.96, 143.22 (6-Fu), 144.48, 146.62 (5-Fu), 160.31 (C=O), 170.03 (C=O). Anal. calcd. for C22H18ClN3O3: C: 64.79%; H: 4.45%; N: 10.30%; Cl: 8.69%; Found: C: 65.08%; H: 4.13%; N: 9.98%; Cl: 9.13%.-5-(4-chlorophenyl)-3-{[(1-amino-4-phenyl-1H-imidazol-2-yl)amino]methylene}furan-2(3H)-one (9f). Yellow crystals (DMF), yield 0.16 g (59%), mp 246–248 °C; FTIR (KBr), ν, cm−1: 3327 (NH2), 3267 (NH), 1713 (C=O), 1653 (C=C); 1H NMR : δ 6.04 , 6.14 , 7.00 , 7.20 , 7.16 , 7.18 , 8.03 , 8.24 , 10.40 , 10.69 ; 13C NMR : δ 100.98 (4-Fu), 103.63, 109.39 (3-Fu), 116.45 (Cimidazole), 124.34, 125.42, 125.66, 128.39, 128.79, 129.37, 129.54, 134.89, 138.27 (6-Fu), 145.31 (5-Fu), 156.71, 169.44 (C=O). Anal. calcd. for C20H15ClN4O2: C: 63.41%; H: 3.99%; N: 14.79%; Cl: 9.36%; Found: C: 63.86%; H: 4.13%; N: 14.58%; Cl: 9.13%.H)-one (7) and 9 mmol of triethyl orthoformate (4) was heated for 30 min in 7 mL of absolute isopropyl alcohol. Next, the corresponding heterocyclic amine  was added portionwise for 1 h. The precipitated crystals were filtered, washed with isopropyl alcohol, recrystallized from DMF, and dried. A mixture of 1 mmol of 5-(4-chlorophenyl)furan-2(3(E)-5-(4-chlorophenyl)-3-[methylidene]-3H-furan-2-one (9d). Brown crystals (DMF), yield 0.19 g (65%), mp 255–256 °C; FTIR (KBr), ν, cm−1: 3435 (NH), 3346 (NH), 1738 (C=O), 1650 (C=C); 1H NMR : δ 7.17 , 7.46-7.54 , 7.92 , 8.46 , 11.31 , 13.82 ; 13C NMR : δ 100.92 (4-Fu), 113.74 (3-Fu), 125.64, 127.78, 128.35, 129.57, 130.27, 133.15, 137.53 (6-Fu), 144.26, 146.16 (5-Fu), 159.05 (Ctriazole), 170.14 (C=O). Anal. calcd. for C13H9ClN4O2: C: 54.09%; H: 3.14%; N: 19.41%; Cl: 12.28%; Found: C: 53.92%; H: 3.24%; N: 19.61%; Cl: 11.93%.(E)-5-(4-chlorophenyl)-3-[(thiazol-2-ylamino)methylidene]furan-2(3H)-one (9e). Brown crystals (DMF), yield 0.18 g (60%), mp 256–257 °C; FTIR (KBr), ν, cm−1: 3265 (NH), 1726 (C=O), 1662 (C=C); 1H NMR : δ 7.05 , 7.24 , 7.42 , 7.51 , 7.58 , 7.90 , 11.84 ; 13C NMR : δ 100.42 (4-Fu), 103.48 (3-Fu), 113.99 (Cthiazole), 125.86, 126.00, 127.96, 129.43, 129.62, 133.68, 134.94 (6-Fu), 140.00 (Cthiazole), 147.49 (5-Fu), 162.12 (Cthiazole), 169.78 (C=O). Anal. calcd. for C14H9ClN2O2S: C: 55.18%; H: 2.98%; N: 9.19%; Cl: 11.73%; S: 10.52%; Found: C: 54.99%; H: 3.16%; N: 9.50%; Cl: 12.02%; S: 10.87%. E- and Z-forms) were fully optimized by using Becke’s three-parameter hybrid functional combined with the Lee–Yang–Parr correlation functional  was used. All structures  w,36 wE- aE-/Z-transitions, we used a formalism proposed for rotation around the partly delocalized C=C bond [E-forms were used for relaxed scans of the C(=O)–C=C–N torsion angle with an increment of 2°. The energy profiles of the rotation processes were analyzed for extrema to evaluate the barrier energy. Calculations were also done at two stages—in vacuo and in a DMSO solution (PCM approach). The structure for each of enamines 9a–f at the global maxima on the curve was optimized as a transition state (TS). The Hamiltonians of the TSs were analyzed for imaginary vibrational modes.To evaluate the magnitude of the energy of the possible C=C bond ,37. The H)-one. The synthesized compounds exist in solution as a mixture of E- and Z-isomers, the ratio between which is determined by a set of factors. One of these factors is the reaction mode. In the one-pot mode, the E-form predominates, and the sequential mixing of the starting reagents in the case of aminotriazole and aminothiazole yields only the E-form, which can also be explained by the higher energy barrier values for the E-/Z-transition, as compared with those for other amines. Stereochemically, the synthesis of enamines proceeds preferably through the initial interaction of the triethyl orthoformate and the amine reagent with the formation of an ethoxyimine intermediate. The configuration of the final product, enamine, is determined by the attack of the enolized form of furanone. The proposed mechanism is the most preferable for the formation of E-form enamines.We have shown that it is possible to synthesize enamines based on furan-2(3


In this study, six new benzo[a]phenoxazinium chlorides possessing sulfonamide groups at 5-amino-positions were synthesized and their optical and biological properties were tested. Compared with previous probes evaluated using fluorescence microscopy, using different S. cerevisiae strains, these probes, with sulfonamide groups, stained the vacuole membrane and/or the perinuclear membrane of the endoplasmic reticulum with great specificity, with some fluorochromophores capable of even staining the plasma membrane. Thus, the addition of a sulfonamide group to the benzo[a]phenoxazinium core increases their specificity and attributes for the fluorescent labeling of cell applications and fractions, highlighting them as quite valid alternatives to commercially available dyes.The demand for new fluorophores for different biological target imaging is increasing. Benzo[ The use of fluorescent probes for bioimaging has proved to be an essential and robust tool in the fields of biochemistry, medicine, and environmental sciences ,2,3. Thea]phenoxazinium chlorides are long-wavelength, NIR fluorescent probes that possess exceptional optical properties. They strongly absorb and emit fluorescence up to about 720 nm, with modest Stokes shifts (20–60 nm) and have high photostability, quantum yields, and molar absorption phenoxazine compounds have also been reported for their pharmaceutical properties. These compounds were reported for their validity as antitumor agents [a]phenoxazinium chlorides to possess different functional groups, resulting in compounds with variable behaviors.In addition to their outstanding optical properties, benzophenoxazinium chlorides possessing these functional groups. In fact, sulfonamide or sulfonyl functional groups are of great importance in medicinal chemistry as they exhibit a broad reactivity profile and are considered safe for drug development phenoxazinium chlorides depends on the substituents at the 5- and 9-positions of the heterocycle [a]phenoxazinium chlorides, functionalized with sulfonamide groups. Our main objective was to evaluate if the introduction of the sulfonamide group into the scaffolds of precursor compounds would be beneficial to the overall biological activity and/or fluorescent labeling capacity.In previous publications, we have reported that the behavior of benzophenoxazinium chlorides capable of specifically staining cell organelles, and the purpose of exploring the effects of sulfonamide groups as terminals of 5-amino substituents in combination with different substituents at the 9-amino position of the polycyclic core, a set of new benzo[a]phenoxazinium chlorides 3a–f was designed. With the aim of obtaining benzophenoxazinium chlorides 3a–f started with the synthesis of the precursors 1a–c and 2a–c. 5-(Dipropylamino)-2-nitrosophenol hydrochloride 1a, 2-nitroso-5-(propylamino)phenol hydrochloride 1b, and 5-(ethylamino)-4-methyl-2-nitrosophenol hydrochloride 1c were prepared with a reaction of 3-(dipropylamino)phenol, 3-(propylamino)phenol, and 3-(ethylamino)-4-methylphenol, respectively, with sodium nitrite in an acid solution under ice cold conditions phenoxazin-9-ylidene)propan-1-aminium chloride 3a, N-(5-((3-(4-methylphenylsulfonamido)propyl)amino)-9H-benzo[a]phenoxazin-9-ylidene)-N-propylpropan-1-aminium chloride 3b and N-(5-((3-(4-nitrophenylsulfonamido)propyl)amino)-9H-benzo[a]phenoxazin-9-ylidene)-N-propylpropan-1-aminium chloride 3c, respectively. In similar conditions, 5-amino position monoalkylated nitrosophenol 1b reacted with precursors 2a and 2b to yield N-(5-((3-(propylsulfonamido)propyl)amino)-9H-benzo[a]phenoxazin-9-ylidene)propan-1-aminium chloride 3d and N-(5-((3-(4-methylphenylsulfonamido)propyl)amino)-9H-benzo[a]phenoxazin-9-ylidene)propan-1-aminium chloride 3e, respectively. At last, nitrosophenol precursor 1c reacted with naphthylamine precursor 2a to yield N-(10-methyl-5-((3-(propylsulfonamido)propyl)amino)-9H-benzo[a]phenoxazin-9-ylidene)ethanaminium chloride 3f. Compounds 3a–f were obtained as blue solids in yields ranging from 8–48% and were fully characterized using high resolution mass spectrometry and IR and NMR (1H and 13C) spectroscopy.Benzophenoxazine moiety (δ 94.01–164.62 ppm). It is possible to observe the differences between compound 3f and the remaining from the change in the chemical shift of C-10 from 112.02–117.55 ppm to 149.64 ppm.The 3a–c show the signals of the propyl group, namely N(CH2CH23CH)2 (δ 11.42–11.48 ppm), N(CH22CHCH3)2 (δ 21.81–21.87 ppm), and N(2CHCH2CH3)2 (δ 54.59–54.64 ppm). These values are slightly different from the values for compounds with only one propyl group as a substituent , which show the signal of the terminal CH3 carbon at δ 11.79–11.80 ppm, from the central methylene group at δ 23.03–23.05 ppm and the CH2 group directly linked to the amine at δ 46.43–46.44 ppm. The spectrum of compound 3f shows the methyl group at the 10-position at δ 17.61 ppm, and the ethyl group at the 9-position at δ 14.12 ppm for CH3 and δ 39.71 ppm for CH2. Regarding the 5-position, the methylene carbons are also highlighted; namely NHCH22CHCH2NHSO2 (δ 26.56–30.76 ppm), NH2CHCH2CH2NHSO2 (δ 42.28–43.18 ppm), and NHCH2CH22CHNHSO2 (δ 38.35–41.58 ppm). Compounds 3a, 3d, and 3f show the signals of the carbons of the propyl group linked to the sulfonyl function, namely the methyl group (δ 13.21–13.25 ppm) and the methylene carbons, NHSO2CH22CHCH3 (δ 18.39–18.41 ppm) and NHSO22CHCH2CH3 (δ 54.44–54.45 ppm). On the other hand, compounds 3b and 3e exhibit the signals of the methyl group present in the aromatic ring attached to the sulfonyl function (δ 21.40–21.45 ppm) and the signals of aromatic carbons . In the case of compound 3c, these signals are also displayed .Regarding the substituents on the 9-position amine, compounds a]phenoxazinium chlorides 3a–f were studied in different solvents to assess their photophysical properties. The solvents used were dry ethanol, water, and aqueous solutions of different biologically relevant pH values. The maximum absorption (λabs) and emission (λemi) wavelengths (ε), the relative fluorescence quantum yield (ΦF), using Oxazine 1 as a standard (ΦF = 0.11 in ethanol) at 590 nm excitation, and the Stokes shifts (Δλ) were obtained for each compound in each solvent. The results obtained are summarized in Benzophenoxazinium chlorides 3a–f was evaluated through a broth microdilution method for testing the antifungal activity against yeasts , using S. cerevisiae PYCC 4072 as a model organism phenoxazines, without the sulfonamide group phenoxazines for the fluorescent staining of biological samples as they have low toxicity.Among the six tested compounds, the di-alkylated derivatives (f 25 μM) . The cona]phenoxazinium chlorides 3a–f was assessed using fluorescence microscopy. To obtain preliminary information on the staining ability of these compounds, MIC values were used as reference concentrations. S. cerevisiae W303-1A cells were incubated with MIC concentrations for 1 h and observed under the fluorescence microscope. This incubation resulted in the accumulation of the compounds in the cells, as NIR fluorescence emission was observed for all compounds upon excitation , at theter Vba1 ) and at ter Jen1 ). We alss family .3a demonstrated the specificity of this compound to accumulate and stain the vacuolar membrane, as seen from the co-localization of NIR fluorescence and green fluorescence of Vba1-GFP  values—Overall, the compounds synthesized were found to show high resolution fluorescence staining patterns demonstrating the ability to strain relevant intracellular structures. Indeed, compounds operties . CompounIn fact, the compounds containing sulfonamide groups have proven to be the ones that exhibit the best fluorescence labeling patterns of our entire library. Moreover, the fact that these compounds have high MICs and retain their ability to stain cellular structures at concentrations well below their MICs is a positive aspect for their use as fluorescent probes, since it is desirable that a probe does not affect the viability of a given organism.1H and 100 MHz for 13C using the solvent peak as internal reference at 25 °C, Billerica, MA, USA. All chemical shifts are given in ppm using δH Me4Si = 0 ppm as reference and J values are given in Hz. Assignments were made through a comparison of chemical shifts, peak multiplicities, and J values, and were supported spin decoupling-double resonance and bidimensional heteronuclear correlation techniques. Mass spectrometry analyses were performed at the “C.A.C.T.I.—Unidad de Espectrometria de Masas”, at University of Vigo, Pontevedra, Spain. All commercial reagents and solvents were used as received.Melting points were correct and were measured on a Stuart SMP3 melting point apparatus. TLC analyses were carried out on 0.20 mm thick precoated silica plates , and spots were visualized under UV light on a CN-6 camera. Chromatography on silica gel was carried out on Acros Organics 60 (0.035–0.070 mm) . IR spectra were determined on a BOMEM MB 104 spectrophotometer, Quebec City, QC, Canada. Samples were prepared in 1% KBr pellets. UV–Vis–NIR absorption spectra (200–800 nm) were obtained using Shimadzu UV/3101PC, Kyoto, Japan, spectrophotometer and fluorescence spectra with Fluoromax-4 spectrofluorometer, Horiba, NJ, USA. NMR spectra were obtained on a Bruker Avance III 400 at an operating frequency of 400 MHz for N1-propane-1,3-diamine hydrobromide phenoxazinium chloride 3a–f was obtained.To a solution of the corresponding nitrosophenol hydrochloride 1a +: 509.2580; C28H37N4O3S requires [M+1]+: 509.2581.The reaction between 5-(dipropylamino)-2-nitrosophenol hydrochloride 1a  , concentrated hydrochloric acid (0.070 mL), and 2b  (reflux time: 14 h) gave compound 3b as a blue solid . Mp > 300 °C. Rf = 0.52 (dichloromethane/methanol 95:5). FTIR (KBr 1%): υmax 3436, 3207, 3062, 2963, 2931, 2874, 1641, 1587, 1547, 1497, 1452, 1437, 1384, 1332, 1290, 1240, 1159, 1135, 1097, 1008, 964, 918, 869, 816 cm−1. 1H NMR δH  1.08 2), 1.80 2), 2.05 , 2.38 , 3.06 , 3.60 2), 3.74 , 6.77 , 6.79 , 7.23 , 7.34 , 7.71–7.76 , 7.84 , 8.20 , 8.69  ppm. 13C NMR δC  11.48 (N(CH2CH23CH)2), 21.43 (Ph-3CH), 21.87 (N(CH22CHCH3)2), 29.56 (NHCH22CHCH2NHSO2), 41.58 (NHCH2CH22CHNHSO2), 43.18 (NH2CHCH2CH2NHSO2), 54.64 (N(2CHCH2CH3)2), 94.42 (C-6), 97.17 (C-8), 117.04 (C-10), 123.81 (C-4), 124.55 (Ar-C), 125.43 (C-1), 128.08 (2 × Ar-H m-CH3), 130.80 (2 × Ar-H o-CH3), 130.84 (C-3), 131.69 (Ar-C), 132.26 (Ar-C), 132.90 (C-2), 134.01 (C-11), 134.59 (Ar-C), 138.45 (C-SO2), 144.83 (C-CH3), 149.44 (Ar-C), 152.79 (Ar-C), 156.13 (C-9), 158.99 (C-5) ppm. HRMS: m/z (ESI): Found [M+1]+: 557.2576; C32H37N4O3S requires [M+1]+: 557.2581.The reaction between 5-(dipropylamino)-2-nitrosophenol hydrochloride 1a , concentrated hydrochloric acid (0.055 mL), and 2c  (reflux time: 23 h) gave compound 3c as a blue solid . Mp = 236.1–238.0 °C. Rf = 0.82 (dichloromethane/methanol 95:5). FTIR (KBr 1%): υmax 3410, 3190, 3055, 2962, 2935, 2876, 1640, 1589, 1547, 1529, 1495, 1453, 1436, 1383, 1331, 1293, 1239, 1161, 1136, 1100, 1059, 1011, 962, 888, 854, 820 cm−1. 1H NMR δH  1.08 2), 1.83 2), 2.06 , 3.18 , 3.62–3.67 2), 3.77 , 4.60 , 6.91 , 6.98 , 7.36 , 7.86 , 7.93–7.99 , 8.08 , 8.33 , 8.36 , 8.98  ppm. 13C NMR δC  11.42 (N(CH2CH23CH)2), 21.81 (N(CH22CHCH3)2), 30.76 (NHCH22CHCH2NHSO2), 41.52 (NHCH2CH22CHNHSO2), 42.90 (NH2CHCH2CH2NHSO2), 54.63 (N(2CHCH2CH3)2), 94.36 (C-6), 97.23 (C-8), 117.32 (C-10), 123.82 (C-4), 124.75 (Ar-C), 125.45 (2 × Ar-C o-NO2), 125.72 (C-1), 129.44 (2 × Ar-C m-NO2), 130.96 (C-3), 132.30 (Ar-C), 132.76 (Ar-C), 133.06 (C-2), 134.27 (C-11), 134.89 (Ar-C), 147.60 (Ar-C), 149.93 (Ar-C), 151.35 (Ar-C), 153.33 (Ar-C), 156.46 (C-9), 159.31 (C-5) ppm. HRMS: m/z (ESI): Found [M+1]+: 588.2269; C31H34N5O5S requires [M+1]+: 588.2275.The reaction between 5-(dipropylamino)-2-nitrosophenol hydrochloride 1b +: 467.2115; C25H31N4O3S requires [M+1]+: 467.2111.The reaction between 5-(propylamino)-2-nitrosophenol hydrochloride 1b  , concentrated hydrochloric acid (0.059 mL), and 2b  (reflux time: 16 h) gave compound 3e as a blue solid . Mp > 300 °C. Rf = 0.76 (dichloromethane/methanol 95:5). FTIR (KBr 1%): υmax 3435, 2966, 2928, 2854, 1660, 1641, 1591, 1549, 1530, 1464, 1384, 1322, 1158, 1094, 971, 803 cm−1. 1H NMR δH  1.10 , 1.79 , 2.05 , 2.36 , 3.06 , 3.38 , 3.80 , 6.81 , 6.95 , 7.14 , 7.34 , 7.74 , 7.80–7.86 , 7.94 , 8.32 , 8.91  ppm. 13C NMR δC  11.80 (NHCH2CH23CH), 21.40 (Ph3CH), 23.05 (NHCH22CHCH3), 29.53 (NHCH22CHCH2NHSO2), 41.49 (NHCH2CH22CHNHSO2), 42.98 (NH2CHCH2CH2NHSO2), 46.44 (NH2CHCH2CH3), 94.30 (C-6 and C-8), 112.02 (C-10), 123.76 (C-4), 124.80 (Ar-C), 125.56 (C-1), 128.07 (2 × Ar-C m-CH3), 130.77 (2 × Ar-C o-CH3), 130.86 (C-11), 132.48 (Ar-C), 132.66 (Ar-C), 132.89 (C-2), 134.11 (C-3), 134.42 (Ar-C), 138.50 (C-CH3), 144.81 (C-SO2), 153.09 (Ar-C), 158.41 (C-9), 159.09 (C-5) ppm. HRMS: m/z (ESI): Found [M+1]+: 515.2117; C29H31N4O3S requires [M+1]+: 515.2111.The reaction between 5-(propylamino)-2-nitrosophenol hydrochloride 1c +: 467.2109; C25H31N4O3S requires [M+1]+: 467.2111.The reaction between 5-(ethylamino)-4-methyl-2-nitrosophenol hydrochloride 1c ,61,62 , with concentrations between 10−7 and 10−5 M. These last solutions were prepared using boric acid, citric acid, and sodium phosphate buffers, or phosphate–saline buffer PBS (pH 7.4).Photophysical properties of benzophenoxazinium chlorides functionalized with different sulfonamide groups at the 5-position were synthesized. These compounds display fluorescence emission in the NIR region, with maximum emission wavelengths (λemi) between 642 and 687 nm, in the different tested solvents.A set of six new benzophenoxazinium chlorides capable of staining the plasma membrane. All compounds showed interesting fluorescent labeling patterns, but compounds 3d and 3f are remarkable for their labeling specificity. Their structural features, such as mono-alkylation at the 9-position and a propyl sulfonamide at the 5-position, are likely a key factor in this behavior. In addition, both compounds exhibit the highest fluorescence quantum yields of the set and are associated with the highest MIC values, making them promising bioimaging probes.Our results show that at a low concentration of 2.5 μM, these compounds are able to stain with high specificity the vacuolar membrane and/or the endoplasmic reticulum, and in the case of compounds a]phenoxazinium chlorides as an alternative to commercial probes, as we were able to obtain very robust compounds that can be used in studies with fluorescence microscopy.In this sense, this work contributes to the valorization of benzo[