A1–A10) and trifluoromethoxy (B1–B10) substituents. The compounds were characterized by spectroscopic techniques and evaluated for their antimicrobial activity against four pathogenic Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Bacillus subtilis) bacterial and fungal  strains. In this study, the compounds with trifluoromethoxy group were more effective than those with trifluoromethyl group. Among the 20 fluorinated chalcones, compound A3/B3 bearing an indole ring attached to the olefinic carbon have been proved to possess the most antimicrobial activity compared to the standard drugs without showing cytotoxicity on human normal liver cell line (L02). Further, the minimum inhibitory concentration (MIC) for A3/B3 was determined by serial tube dilution method and showed potential activity. These results would provide promising access to future study about the development of novel agents against bacterial and fungal infections.Despite the availability of many drugs to treat infectious diseases, the problems like narrow antimicrobial spectrum, drug resistance, hypersensitivities and systemic toxicities are hampering their clinical utility. Based on the above facts, in the present study, we designed, synthesized and evaluated the antibacterial and antifungal activity of novel fluorinated compounds comprising of chalcones bearing trifluoromethyl ( In the 19F-NMR spectrum, A3 showed signal at 63.27 ppm corresponding to -CF3. The above spectral data confirmed the compound A3 as (E)-3-(1″H-indol-3″-yl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one.The FT-IR spectrum of B3 illustrated diagnostic intense carbonyl (C=O) and strong vinyl (CH=CH) absorption bands of chalcone linkage at wave numbers 1651 cm−1 and 1595 cm−1, respectively. Additionally, the other absorption bands are seen at 3681 (-NH in indole), 1206 (-OCF3), and 1248 (C-O-C). The 1H NMR spectrum showed characteristic doublet signals at 7.53 ppm and 8.05 ppm with the coupling constant value (J) around 16 Hz. The larger coupling constant value represents the trans geometry of the chalcones. The other peaks seen are three singlets corresponding to the indole amine proton at chemical shift 8.78 and two multiplets around 7.38–8.02 and 7.05–7.66 corresponding to nine aromatic protons. The 13C NMR spectra of compound B3 showed three peaks corresponding to the three carbons of the propenone moiety at 189.97, 125.33, and 137.37, respectively. The peaks of other carbon atoms are seen at the following chemical shift values: 130.23 (C-1′), 130.71 (C-2′ and 6′), 121.88 (C-3′ and 5′), 139.61 (C-4′), 121.88 , 112.08 (C-1″), 123.63 (C-2″), 137.35 (C-4″), 114.41 (C-5″), 120.47 (C-6″), 122.69 (C-7″), 117.81 (C-8″), 123.63 (C-9″). The mass spectrum of B3 recorded in positive mode showed [M + H]+ peak at m/z 332.20. In the 19F-NMR spectrum, A3 showed signal at 63.27 ppm corresponding to -CF3. The 19F-NMR spectrum of B3 showed signal at 58.64 ppm corresponding to -OCF3. Based on the above spectral data, the compound B3 was confirmed as (E)-3-(1″H-indol-3″-yl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one (The FT-IR spectrum of en-1-one .A1–A10 and B1–B10) were evaluated for their antibacterial and antifungal activities on selected pathogenic Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Proteus vulgaris) bacterial  strains (A2(B2), A4(B4), A5(B5), A8(B8), and A9(B9) represents chalcones bearing a monosubstituted aryl ring, A1(B1) denotes compounds with a disubstituted aryl ring, and A3(B3), A6(B6), A7(B7), and A10(B10) designate the compounds containing an unsubstituted heteroaromatic ring.All the 20 target compounds  of 25 and 26 mm, respectively. The activity of B3 (ZOI = 29 mm) was more than benzyl penicillin (ZOI = 27 mm) against Bacillus subtilis but the activity of A3 (ZOI = 26 mm) was less than the standard. A3 and B3 also exhibited more activity than benzyl penicillin against Escherichia coli (ZOI = 20 and 22 mm) and Proteus vulgaris with a ZOI of 23 and 21 mm, respectively. A3 and B3 showed a ZOI of 20 and 22 mm against Candida albicans and a ZOI of 25 and 26 mm, respectively, against Aspergillus niger. These ZOI values were more compared to the ZOI values obtained with standard fluconazole (19.05 and 24.41 mm).Among all the compounds, B3, all the other compounds displayed intermediate-to-low activity against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Aspergillus niger compared to the standard drugs. When the activities of other compounds against Escherichia coli were compared with the standard drugs, the compounds A1(B1), A4(B4), A5(B5), A6(B6), A7(B7), A8, and A9 were found to be more active. Further, among the above 12 compounds, A1 and A7 containing 2,3-dichlorophenyl and 2-furfuryl rings were next in potency to A3 in series-A and B1, B4, and B5 containing 2,3-dichlorophenyl, 2-nitrophenyl, and 3-nitrophenyl scaffolds after B3 in series-B. The chalcones A1(B1), B2, A4(B4), and A5(B5) were more active than benzyl penicillin against Proteus vulgaris, and the compounds A1 , A4 (2-nitrophenyl), A5 (3-nitrophenyl), B2 (3-chlorophenyl), B4 (2-nitrophenyl), and B5 (3-nitrophenyl) showed equal activity with a ZOI value of 20 mm, whereas the compound B1 containing 2,3-dichlrophenyl moiety showed activity similar to B3 (ZOI = 21 mm). These results suggest that presence of an electron-withdrawing group like chlorine or nitro at 2nd and 3rd positions of aryl ring are essential for the activity. The compounds A2 and A7 bearing 3-chlorophenyl and 2-furfuryl rings showed equal activity as benzyl penicillin (ZOI = 19 mm) against Proteus vulgaris, and B1 exhibited equal potency as fluconazole against Candida albicans with a ZOI of 19 mm. All the other compounds including A8(B8) and A9(B9) containing 4-chlorophenyl and 4-nitrophenyl rings as well as A6(B6) and A10(B10) containing heteroaromatic 2-thienyl and 2-pyrrolyl rings, respectively, showed intermediate-to-poor activity relative to the standard drugs. Based on these results, we can summarize that either in series-A or B, the conditions of the ring B-portion of chalcones being either an aromatic ring with electron-withdrawing groups in the ortho and meta positions or a bicyclic heteroaromatic scaffold are essential to the promising antimicrobial activity of the fluorinated chalcones.Except A3 and B3 were further evaluated against all the six microbial strains by serial tube dilution to assess their minimum inhibitory concentration by serial tube dilution method (A3 showed less activity against Bacillus subtilis (MIC = 101 µM) than benzyl penicillin (MIC = 95 µM). Both A3 and B3 exhibited nearly equal activity to that of fluconazole against the fungal strains and superior activity compared to benzyl penicillin. Compound A3 (MIC = 51 µM) and B3 (MIC = 48 µM) were 1.86- and 1.97-fold more active than benzyl penicillin (MIC = 95 µM) against Staphylococcus aureus and B3 (MIC = 24 µM) was 3.95 times more active than benzyl penicillin against Bacillus subtilis. Against Escherichia coli and Proteus vulgaris, A3 was 7.64 times more active than benzyl penicillin whereas B3 was 7.95 and 3.97 times more active. These results show that the activity of A3 containing a trifluoromethyl group favored the Gram-negative bacteria and B3 with a trifluoromethoxy group favored Gram-positive bacteria. The obtained results were interesting and called for a synthesis and evaluation of other analogues to improve the potency.Minimum inhibitory concentration (MIC) is the lowest concentration of the antimicrobial agent that inhibits the visible growth of a microorganism after an overnight incubation. MICs are used to determine the resistance by the diagnostic labs mainly to confirm the resistance. However, frequently MIC is a research tool to determine the in vitro activity of novel natural and synthetic compounds. The two most potent compounds that emerged out of this study, i.e., n method . Both thA3 and B3 were evaluated for their cytotoxicity study on normal human liver calls and were found to have an IC50 value greater than 50 µg/mL suggesting that the compounds were non-toxic against the tested normal human liver cell lines  and the spots were visualized under a UV lamp. 4′-trifluoromethylacetophenone and 4′-trifluoromethoxyacetophenone were obtained from Thermo Fisher Scientific-Alfa Aesar . Aldehydes were procured from Avra synthesis Pvt. Ltd. . The melting points were determined in open capillaries, using a digital melting point apparatus  (expressed in °C) and are uncorrected. FT-IR spectra were scanned using Bruker OPUS 8.0  and the 1H- and 13C-NMR spectra of the compound were recorded on a Bruker 400 Avance NMR spectrophotometer using Tetramethylsilane (TMS) as an internal standard . Mass spectra were recorded on SHIMADZU Lab Solution (ESI-MS) spectrometer at Indian Institute of Chemical Technology, Hyderabad, India -3--1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 95%; m.p. 76 °C; Rf = 0.81 (30% Ethyl acetate in Hexane); FT-IR : 1660 (C=O), 1602 (C=C Ar), 1502 (CH=CH), 1255 (-CF3), 3011 (Ar C-H stretching), 832 (C-Cl); 1H NMR , δ: 7.76 , 8.11 , 8.12–8.22 , 6.97–7.74 ; ESI-MS: 346.13 [M + H]+.A1: Synthesis of (E)-3-(3″-chlorophenyl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 90%; m.p. 68 °C; Rf = 0.8 (30% Ethyl acetate in Hexane); FT-IR : 1651 (C=O), 1611 (C=C Ar), 1507 (CH=CH), 1252 (-CF3), 3010 (Ar C-H stretching), 846 (C-Cl); 1H NMR , δ: 7.77 , 8.11 , 8.12–8.22 , 7.09–7.74 ; ESI-MS: 311.13 [M + H]+.A2: Synthesis of (E)-3-(1″H-indol-3″-yl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 50%; m.p. 62 °C; Rf = 0.9 (30% Ethyl acetate in Hexane); FT-IR : 1695 (C=O), 1611 (C=C Ar), 1573 (CH=CH), 1263 (-CF3), 3010 (Ar C-H stretching), 3230 (-NH); 1H NMR : δ: 7.77 , 8.13 , 8.78 , 7.94-8.14 , 7.05-7.61 ; 13C NMR : 189.93 (C-1), 128.60 (C-2), 142.02 (C-3), 140.11 (C-1′), 128.60 (C-2′ and 6′), 125.59 (C-3′ and 5′), 137.30 (C-4′), 123.77 , 129.52 (C-6′), 112.06 (C-1″), 125.30 (C-2″), 130.30 (C-4″), 114.06 (C-5″), 120.71 (C-6″), 122.02 (C-7″), 117.44 (C-8″), 125.56 (C-9″); ESI-MS: 316.30 [M + H]+; 19F NMR : δ: 63.27 .A3: Synthesis of (E)-3-(2″-nitrophenyl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 60%; m.p. 70 °C; Rf = 0.7 (30% Ethyl acetate in Hexane); FT-IR : 1646 (C=O), 1602 (C=C Ar), 1514 (CH=CH), 1249 (-CF3), 3010 (Ar C-H stretching), 1461 (NO2); 1H NMR : δ: 7.77 , 8.11 , 8.12–8.51 , 7.70–8.01 ; ESI-MS: 322.11 [M + H]+.A4: Synthesis of (E)-3-(3″-nitrophenyl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 60%; m.p. 85 °C; Rf = 0.5 (30% Ethyl acetate in Hexane); FT-IR : 1659 (C=O), 1623 (C=C Ar), 1536 (CH=CH), 1250 (-CF3), 3010 (Ar C-H stretching), 1446 (NO2); 1H NMR : 7.92 , 8.15 , 7.82–8.65 , 7.68–7.89 ; ESI-MS: 322.25 [M + H]+.A5: Synthesis of (E)-3-(thiophen-2″-yl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 80%; m.p. 86 °C; Rf = 0.76 (30% Ethyl acetate in Hexane); FT-IR : 1658 (C=O), 1504 (CH=CH), 1614 (C=C Ar), 3121 (Ar C-H stretching), 1250 (-CF3), 621 (C-S); 1H NMR : δ: 7.79 , 8.14 , 7.80–8.12 , 7.17–7.77 ; ESI-MS: 283.36 [M + H]+.A6: Synthesis of (E)-3-(furan-2″-yl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 80%; m.p. 72 °C; Rf = 0.71 (30% Ethyl acetate in Hexane); FT-IR : 1658 (C=O), 1514 (CH=CH), 1631 (C=C Ar), 3111 (Ar C-H stretching), 1254 (-CF3), 1700 (C-O); 1H NMR , δ: 7.75 , 8.14 , 7.59–8.12 , 6.52–7.53 ; ESI-MS: 227.68 [M + H]+.A7: Synthesis of (E)-3-(4″-chlorophenyl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 90%; m.p. 68 °C; Rf = 0.72 (30% Ethyl acetate in Hexane); FT-IR : 1656 (C=O), 1609 (C=C Ar), 1505 (CH=CH), 1247 (-CF3), 3001 (Ar C-H stretching), 841 (C-Cl); 1H NMR : δ: 7.78 , 8.14 , 7.77–8.13 , 7.43–7.61 ; ESI-MS: 311.62 [M + H]+.A8: Synthesis of (E)-3-(4″-nitrophenyl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 61%; m.p. 82 °C; Rf = 0.7 (30% Ethyl acetate in Hexane); FT-IR : 1654 (C=O), 1621 (C=C Ar), 1512 (CH=CH), 1252 (-CF3), 3010 (Ar C-H stretching), 1430 (NO2); 1H NMR : δ: 7.85 , 8.11 , 7.68–7.82 , 8.12–8.32 ; ESI-MS: 322.11 [M + H]+.A9: Synthesis of (E)-3-(1″H-pyrrol-2-yl)-1-[4′-(trifluoromethyl)phenyl]prop-2-en-1-one: Yield: 80%; m.p. 75 °C; Rf = 0.68 (30% Ethyl acetate in Hexane); FT-IR : 1650 (C=O), 1501 (CH=CH), 1618 (C=C Ar), 1242 (-CF3), 1370 (C-N), 3115 (Ar C-H stretching); 1H NMR , δ: 7.77 , 8.11 , 7.74–8.23 , 6.08–6.96 , 9.93 ; ESI-MS: 266.23 [M + H]+.A10: Synthesis of (E)-3--1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 86%; m.p. 70 °C; Rf = 0.8 (30% Ethyl acetate in Hexane); FT-IR : 1664 (C=O), 1624 (Ar C=C), 1520 (CH=CH), 1242 (-CF3), 3023 (Ar C-H stretching), 1166 (-C-O-) 838 (C-Cl); 1H NMR , δ: 7.58 , 8.03 , 7.64–8.22 , 6.97–7.62 ; ESI-MS: 362.12 [M + H]+.B1: Synthesis of (E)-3-(3″-chlorophenyl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 84%; m.p. 65 °C; Rf = 0.81 (30% Ethyl acetate in Hexane); FT-IR : 1648 (C=O), 1607 (Ar C=C), 1512 (CH=CH), 1232 (-CF3), 3015 (Ar C-H stretching), 1156 (-C-O-), 840 (C-Cl); 1H NMR , δ: 7.37 , 8.02 , 7.70–8.22 , 7.05–7.68 ; ESI-MS: 343.58 [M + H]+.B2: Synthesis of (E)-3-(1″H-indol-3″-yl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 45%; m.p. 67 °C; Rf = 0.74 (30% Ethyl acetate in Hexane); FT-IR : 1651 (C=O), 1602 (Ar C=C), 1595 (CH=CH), 1521 (C-N), 3681 (-NH), 1206 (-OCF3), 3362 (Ar C-H stretching), 1248 (-C-O-); 1H NMR : δ 7.53 , 8.05 , 8.78 , 7.38–8.02 , 7.05–7.66 ;B3: Synthesis of : 189.97 (C-1), 125.33 (C-2), 137.37 (C-3), 130.23 (C-1′), 130.71 (C-2′ and 6′), 121.88 (C-3′ and 5′), 139.61 (C-4′), 121.88 , 112.08 (C-1″), 123.63 (C-2″), 137.35 (C-4″),114.41 (C-5″), 120.47 (C-6″), 122.69 (C-7″), 117.81 (C-8″), 123.63 (C-9″); ESI-MS: 332.20 [M + H]+; 19F NMR : δ, 58.64 .E)-3-(2″-nitrophenyl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 56%; m.p. 69 °C; Rf = 0.87 (30% Ethyl acetate in Hexane); FT-IR : 1640 (C=O), 1632 (Ar C=C), 1504 (CH=CH),1232 (-CF3), 3012 (Ar C-H stretching), 1126 (-C-O-), 1462 (NO2); 1H NMR : δ: 7.85 , 8.28 , 8.27–8.51 , 7.05–8.25 ; ESI-MS: 338.08 [M + H]+.B4: Synthesis of (E)-3-(3″-nitrophenyl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 55%; m.p. 71 °C; Rf = 0.72 (30% Ethyl acetate in Hexane); FT-IR : 1659 (C=O), 1626 (Ar C=C), 1532 (CH=CH), 1248 (-CF3), 3019 (Ar C-H stretching), 1127 (-C-O-), 1448 (NO2); 1H NMR : δ: 7.06 , 8.05 , 7.76–8.03 , 7.08–7.79 ; ESI-MS: 338.35 [M + H]+.B5: Synthesis of (E)-3-(thiophen-2″-yl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 76%; m.p. 82 °C; Rf = 0.68 (30% Ethyl acetate in Hexane); FT-IR : 1656 (C=O), 1512 (CH=CH), 1610 (Ar C=C), 3126 (Ar C-H stretching), 1262 (-CF3), 1156 (-C-O-), 620 (C-S); 1H NMR : δ: ), 7.05 , 8.05 , 7.20–8.02 , 7.17–7.79 ; ESI-MS: 299.16 [M + H]+.B6: Synthesis of (E)-3-(furan-2″-yl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 78%; m.p. 68 °C; Rf = 0.66 (30% Ethyl acetate in Hexane); FT-IR : 1651 (C=O), 1524 (CH=CH), 1635 (Ar C=C), 3111 (Ar C-H stretching), 1250 (-CF3), 1159 (-C-O-); 1H NMR , δ: 7.07 , 8.05 , 7.47–8.05 , 6.52–7.59 ; ESI-MS: 283.47 [M + H]+.B7: Synthesis of (E)-3-(4″-chlorophenyl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 84%; m.p. 75 °C; Rf = 0.64 (30% Ethyl acetate in Hexane); FT-IR : 1645 (C=O), 1629 (Ar C=C), 1512 (CH=CH), 1237 (-CF3), 3011 (Ar C-H stretching), 1155 (-C-O-), 837 (C-Cl); 1H NMR : δ: 8.33 , 7.05 , 7.92–8.65 , 7.06–7.89 ; ESI-MS: 343.19 [M + H]+.B8: Synthesis of (E)-3-(4″-nitrophenyl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 57%; m.p. 68 °C; Rf = 0.68 (30% Ethyl acetate in Hexane); FT-IR : 1652 (C=O), 1614 (Ar C=C), 1510 (CH=CH), 1242 (-CF3), 3016 (Ar C-H stretching), 1122 (-C-O-), 1436 (NO2); 1H NMR : δ: 7.06 , 8.03 , 7.05–8.04 , 7.82–8.32 ; ESI-MS [M + H]+: 338.72 and 339.72.B9: Synthesis of (E)-3-(1″H-pyrrol-2-yl)-1-[4′-(trifluoromethoxy)phenyl]prop-2-en-1-one: Yield: 67%; m.p. 59 °C; Rf = 0.70 (30% Ethyl acetate in Hexane); FT-IR : 1646 (C=O), 1516 (CH=CH), 1621 (Ar-C=C-), 1232 (-CF3), 1336 (C-N), 3121 (Ar C-H stretching), 1166 (-C-O-); 1H NMR , δ: 7.07 , 8.03 , 7.08–8.23 , 6.09–7.06 , 9.93 ; ESI-MS: 282.02 [M + H]+.B10: Synthesis of (Gram-positive bacteria Bacillus subtilis (NCIM-2079), Staphylococcus aureus (NCIM-2079)Gram-negative bacteriaEscherichia coli (NCIM-2065), Proteus vulgaris (NCIM-2027)FungiCandida albicans (MDCC-227), Aspergillus niger (MTCC 5889)The antimicrobial activity was performed against six different antimicrobial strains. The organisms selected are listed below:Glassware was cleaned and kept in a hot air oven at 160 °C for 2 h. The media were sterilized and the solutions of standard drugs (Benzyl penicillin and fluconazole) and A and B series of compounds were kept ready. In the meantime, nutrient agar medium was prepared . The weighed quantities of peptone, meat extract, and sodium chloride were dissolved in 1000 mL of distilled water and the pH of the medium was adjusted to 7.2. After the dissolution of agar, the medium was distributed into conical flask each containing 25 mL. The media and sterile water were sterilized by autoclaving at 121 °C temperature and 15 lbs/sq. inch pressure for 20 min. Petri plates, test tubes, pipettes, and borer required for experiment were sterilized by dry heat sterilization using hot air oven. Cultures of respective organisms (18 h old) were taken and suspension of these microorganisms was made using sterile water. Later, 0.5 mL of this suspension was used as inoculum and pour plate technique was used for estimation of bacterial load in each sample. The inoculated agar medium was poured into sterile 10 cm-diameter petri dishes and the medium in the plates was allowed to solidify. The solutions of the test compounds in concentrations of 0.1 µg/mL were prepared in DMSO. The cups of 5 mm diameter were prepared using a borer in the corresponding medium. In each plate, 5 wells were prepared. Three wells were for test compounds, one for standard compound and another one was used as control. In each well, samples were poured and then plates were left for 45 min in a refrigerator for diffusion. After incubation for 18 h at 37 °C, the plates were examined for inhibition zones. The experiments were done in triplicate on the same day with the same conditions in order to minimize the experimental errors. The zone of inhibition values was calculated using vernier caliper and represented as a mean of three values and standard deviation was applied [A3 and B3 employing the protocol prescribed in our previously published papers [MIC has become the current standard test for antibiotic sensitivity testing because it produces more pertinent information on minimal dosages. Hence, we determined the MIC of selected compounds, i.e., d papers .A3 and B3 out of the 20 compounds were tested in vitro for their cytotoxic activity on L02  by employing MTT assay according to Mosmann’s method as described in our previous paper [−1, 100 µL) into a blue–purple formazan product, mainly by mitochondrial reductase activity inside living cells . The cells used in cytotoxicity assay were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin at 37 °C and humidified at 5% CO2. Briefly, cells were placed on 96-well plates at 100 µL total volume with a density of 1–2.5 × 104 cells per mL and were allowed to adhere for 24 h before treatment with tested drugs in DMSO solution . Triplicate wells were treated with media and agents. Cell viability was assayed after 96 h of continuous drug exposure with a tetrazolium compound. The supernatant medium was removed, and 150 µL of DMSO solution was added to each well. The plates were gently agitated using mechanical plate mixer until the color reaction was uniform and OD570 was determined using micro plate reader. The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the untreated wells by 50% of the vehicle in the MTT assay. Assays were performed in triplicate on three independent experiments. The results showed good reproducibility between replicate wells with standard errors below 10%.The most potent compounds us paper . The MTTA3 and B3, showed potential activities and were also non-toxic on the normal human liver cell lines (L02). Additionally, compounds bearing electron-withdrawing nitro or the chloro substituents at the ortho or the meta position showed valuable antimicrobial activity. Hence, these compounds are novel lead compounds identified through our study for the development of novel agents against bacterial and fungal infections. Although the present study gave us some lead molecules, future investigation needs to be done by synthesizing analogues of A3 and B3 by replacing the indole scaffold of A3 and B3 with benzofuran and benzothiophene moieties as well as by substituting more lipophilic -SCF3 for -OCF3 in B3. Further, a plausible mode of action for the proposed activities needs to be investigated.In this paper, we described the design, synthesis, characterization, and antimicrobial screening of 20 new fluorinated chalcones. Most of the compounds displayed promising antibacterial and antifungal activities and two compounds bearing indolyl scaffold, i.e.,


Salmonella and Escherichia coli, which can be difficult to discriminate between in a clinical setting. In addition, differentiation between enterococci and other Gram-positive cocci was also seen.In clinical culture media inoculated with patient samples, selective inhibition of commensal bacteria is essential for accurate diagnosis and effective treatment, as they can mask the presence of pathogenic bacteria. The alanine analogue, 1-aminoethyltetrazole was investigated as a potential alanine racemase inhibitor. For effective uptake and enhanced and selective antibacterial activity, a library of C-terminal 1-aminoethyltetrazole containing di- and oligopeptides were synthesized by solid phase peptide coupling techniques. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These enabled differentiation between the closely related bacteria,  In order to treat bacterial infections effectively, and to avoid promoting the evolution of bacterial resistance against antibacterial drugs, the correct agents must be used. Therefore, rapid and accurate detection and identification of pathogenic strains are essential. Chromogenic and fluorogenic culture media are used routinely in the clinical environment for this purpose . A limitl-alanine to d-alanine in bacteria. In the past, several alanine racemase inhibitors have been reported amino}propanoate (6a) carbamate (7a) : Cbz-l/d-alanine methyl ester 6a  was reacted and worked up using general method B, to give the product as a white powder . The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate, 1:1 ratio, Rf: 0.11). Mp: 131–132 °C carbamate (8a) carbamate (9a) : Cbz-l/d-alanyl nitrile  was reacted and worked up using general method D. The product was gained as a white solid . The reaction and the work up were monitored by TLC (eluent: petroleum ether (40–60 °C) - ethyl acetate 1:1, Rf: 0.09). Mp: 134–138 °C  δH 7.99 , 7.33 , 5.01 , 1.49 ; 13C-NMR  δC 158.6 (1-C), 155.7 (6-C), 136.8 (8-C), 128.31 , 127.8 , 65.7 (7-C), 41.9 (2-C), 19.3 (3-C); νmax/cm–1 3308 (N-H), 2980, 2866, 2738, 2614 (tetrazole), 1686 (C=O), 1524 (N-H), 1255 (C-O); MS(ESI) m/z 248.1 (M + H)+.9–141 °C ); 1H-NMR(1S or R)-1-ethan-1-amine (4a) amino}butanoate (6b-L) carbamate (7b-L) carbamate (8b-L) carbamate (9b-L) carbamate (10a) carbamate (10b-L): l-1-Amino-ethyltetrazole  was reacted using general method F. The crude product was purified by recrystallization in 2 generations from acetonitrile to give the pure product as white crystals . The reaction and the work up were monitored by TLC . Mp: 209–211 °C (melted and decomposed); 1H-NMR  δH 16.26 (6-H), 8.04 , 7.89 , 7.72 , 7.42 , 7.33 , 4.81 , 4.34 , 4.23 , 1.91 , 0.88 ; 13C-NMR  δC 156.4 (7-C), 144.3 (10a-C), 144.2 (10b-C), 141.2 (15-C), 128.1 (13-C), 127.5 (12-C), 125.7 (11-C), 120.6 (14-C), 66.2 (8-C), 48.1 (2-C), 47.1 (9-C), 26.6 (3-C), 10.7 (4-C); νmax/cm–1 3300 (N-H), 2978, 2876, 2717, 2617 (tetrazole), 1682 (C=O), 1533 (N-H), 1265 (C-O); MS(ESI) m/z 350.1 (M + H)+; CHN .Benzyl-N-({[(1S)-1-ethyl]carbamoyl}methyl)carbamate (13g-L): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-glycine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure. The residue washed with petroleum ether (40 – 60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 134–136 °C; 1H-NMR  δH 8.56 , 7.35 , 5.24 , 5.03 , 3.68 , 1.49 ; 13C-NMR  δC 169.3 (5-C), 158.9 (1-C), 156.9 (9-C), 137.5 (11-C), 128.8 (13-C), 128.2 (14-C), 128.1 (12-C), 65.9 (10-C), 43.8 (6-C), 40.3 (2-C), 19.8 (3-C); νmax/cm–1 3289 (N-H), 3269 (N-H), 1697 (C=O), 1661 (C=O), 1530 (N-H), 1244 (C-O); MS(ESI) m/z 305.1 (M + H)+; HRMS (Found (M + H)+ 305.1360. Calcd. for C13H17O3N6: (M + H)+ 305.1357.); (Purity test: HPLC method D).-Amino-N-[(1S)-1-ethyl]acetamide (14g-L): Cbz-glycyl-1-l-aminoethyl-tetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with 30 mL methanol, which was discarded. The solid residue was washed into a separate flask with water, which was freeze dried to give the product as a white solid . Further purification was performed by Prep HPLC method A. Mp: 180 °C (decomposed); 1H-NMR  δH 5.27 , 3.75 , 1.52 ; 13C-NMR  δC 167.3 (5-C), 164.3 (1-C), 42.3 (2-C), 41.0 (6-C), 19.3 (3-C); νmax/cm–1 3215 (N-H), 1672 (C=O) 1547 (N-H); MS(ESI) m/z 171.1 (M + H)+; HRMS (Found (M + H)+ 171.0987. Calcd. for C5H11ON6: (M + H)+ 171.0989.); (Purity test: HPLC method A).2Benzyl-N-[(1S)-1-{[(1S)-1-ethyl]carbamoyl}ethyl]carbamate (13a-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-alanine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure and washed with petroleum ether (40–60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 186–187 °C; 1H-NMR  δH 16.18 , 8.54 , 7.35 , 5.23 , 5.05 , 5.00 , 4.09 , 1.50 , 1.20 ; 13C-NMR  δC 172.1 (5-C), 158.3 (1-C), 155.6 (10-C), 137.0 (12-C), 128.3 (14-C), 127.7 (15-C), 127.7 (13-C), 65.3 (11-C), 49.8 (6-C), 40.3 (2-C), 19.2 (7-C), 17.9 (3-C); νmax/cm–1 3287 (N-H), 3264 (N-H), 1684 (C=O), 1655 (C=O), 1535 (N-H), 1230 (C-O); MS(ESI) m/z 319.2 (M + H)+; CHN .(2S)-2-amino-N-[(1S)-1-ethyl]propanamide (14a-LL) : Cbz-l-alanyl-1-l-aminoethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with methanol, which was discarded. The solid residue was washed into a separate flask with water, then freeze dried to give the product as a white solid . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 200 °C (decomposed); 1H-NMR  δH 5.25 , 3.57 , 1.52 , 1.25 ; 13C-NMR  δC 176.2 (5-C), 164.3 (1-C), 49.9 (6-C), 42.0 (2-C), 19.4 (3-C), 19.2 (7-C); νmax/cm–1 3247 (N-H), 1643 (C=O) 1561 (N-H); MS(ESI) m/z 185.1 (M + H)+; HRMS (Found (M+H)+ 185.1145. Calcd. for C6H13ON6: (M + H)+ 185.1145.); (Purity test: HPLC method A).Benzyl-N-[(1S)-1-{[(1R)-1-ethyl]carbamoyl}ethyl]carbamate (13a-LD) : Prepared using general method G. The Fmoc group of Fmoc-1-d-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-alanine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 166–168 °C; 1H-NMR  δH 8.56 , 7.35 , 5.23 , 5.04 , 5.00 , 4.10 , 1.50 , 1.23 ; 13C-NMR  δC 172.6 (5-C), 158.0 (1-C), 156.1 (10-C), 137.4 (12-C), 128.8 (14-C), 128.2 (15-C), 128.2 (13-C), 65.9 (11-C), 50.4 (6-C), 40.3 (2-C), 19.6 (3-C), 18.8 (7-C); νmax/cm–1 3289 (N-H), 2988, 2874, 2756, 2612 (tetrazole), 1682 (C=O), 1651 (C=O), 1528 (N-H), 1224 (C-O); MS(ESI) m/z 319.1 (M + H)+; CHN .(2S)-2-Amino-N-[(1R)-1-ethyl]propanamide (14a-LD) propanamide (14d-L): Cbz-β-alanyl-1-l-amino-ethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with methanol, which was discarded. The solid residue was washed into a separate flask with water, which was freeze dried to give the product as a light brown hygroscopic amorphous solid . Further purification was performed by Prep HPLC method A. 1H-NMR  δH 5.21 , 3.17 , 2.63 , 1.48 ; 13C-NMR  δC 171.6 (5-C), 164.5 (1-C), 40.0 (2-C), 35.8 (7-C), 32.5 (6-C), 19.3 (3-C); νmax/cm–1 3232 (N-H), 1641 (C=O); MS(ESI) (M+H)+ found 185.1 m/z; HRMS (Found (M + H)+ 185.1145. Calcd. for C6H13ON6: (M + H)+ 185.1145.); (Purity test: HPLC method A).Benzyl-N-[(1S)-1-{[(1S)-1-ethyl]carbamoyl}butyl]carbamate (13e-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-L-norvaline with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 175–178 °C; 1H-NMR  δH 16.22 , 8.55 , 7.35 , 5.23 , 5.02 , 4.04 , 1.59 , 1.49 , 1.27 , 0.84 ; 13C-NMR  δC 172.2 (5-C), 159.1 (1-C), 156.4 (12-C), 137.5 (14-C), 128.8 , 128.2 (17-C), 128.1 , 65.8 (13-C), 54.6 (6-C), 40.9 (2-C), 34.3 (7-C), 19.7 (3-C), 19.1 (8-C), 14.0 (9-C); νmax/cm–1 3310 (N-H), 3271 (N-H), 2959, 2877, 2752, 2609 (tetrazole), 1682 (C=O), 1651 (C=O), 1533 (N-H), 1234 (C-O); MS(ESI) m/z 369.2 (M + Na)+; HRMS (Found (M + H)+ 347.1830. Calcd. for C16H23O3N6: (M + H)+ 347.1826.); (Purity test: HPLC method D)(S)-N-((S)-1-(1H-tetrazol-5-yl)ethyl)-2-aminopentanamide (14e-LL): Cbz-l-norvalyl-1-l-amino-ethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with methanol, which was discarded. The residue was washed with water, then freeze dried to give the product as a white solid . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 251–254 °C; 1H-NMR  δH 5.28 , 3.79 , 1.70 , 1.54 , 1.21 , 0.81 ; 13C-NMR  δC 171.2 (5-C), 164.7 (1-C), 53.5 (6-C), 42.0 (2-C), 33.8 (7-C), 19.1 (3-C), 17.6 (8-C), 12.8 (9-C); νmax/cm–1 3210 (N-H), 1687 (C=O), 1551 (N-H); MS(ESI) m/z 213.1 (M + H)+; HRMS (Found (M + H)+ 213.1458. Calcd. for C8H17ON6: (M + H)+ 213.1460.); (Purity test: HPLC method A).Benzyl-N-[(1S)-3-methyl-1-{[(1S)-1-ethyl]carbamoyl}butyl]carbamate (13h-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-leucine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and the residue washed with petroleum ether (40–60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 149–150 °C; 1H-NMR  δH 16.1 , 8.55 , 7.36 , 5.22 , 5.04 , 5.00 , 4.07 , 1.60 , 1.49 , 1.42 , 0.85 , 0.84 ; 13C-NMR  δC 172.6 (5-C), 156.4 (12-C), 137.5 (14-C), 128.8 (16-C), 128.2 (17-C), 128.1 (15-C), 65.8 (13-C), 53.3 (6-C), 41.0 (2-C), 40.3 (7-C), 24.6 (8-C), 23.6 (9a-C), 21.8 (9b-C), 19.6 (3-C); νmax/cm–1 3316 (N-H), 3271 (N-H), 2961, 2876, 2743, 2615 (tetrazole), 1682 (C=O), 1653 (C=O), 1530 (N-H), 1233 (C-O); MS(ESI) m/z 360.8 (M + H)+; CHN .(2S)-2-Amino-4-methyl-N-[(1S)-1-ethyl]pentanamide (14h-LL): Cbz-l-leucyl-1-l-aminoethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, then freeze dried and washed with diethyl ether to give the product as a white solid . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 210 °C (decomposed); 1H-NMR  δH 5.28 , 3.49 , 1.53 , 1.44 , 0.82 , 0.80 ; 13C-NMR  δC 175.6 (5-C), 169.7 (1-C), 52.9 (6-C), 42.7 (7-C), 41.8 (2-C), 24.0 (8-C), 21.9 (9a-C), 21.4 (9b-C), 16.8 (3-C); νmax/cm–1 3219 (N-H), 1653 (C=O), 1562 (N-H); MS(ESI) m/z 227.2 (M + H)+; HRMS (Found (M + Na)+ 249.1436. Calcd. for C9H18ON6Na: (M + Na)+ 249.1434.); (Purity test: HPLC method A).Benzyl-N-[(1S)-3-methyl-1-{[(1R)-1-ethyl]carbamoyl}butyl]carbamate (13h-LD): Prepared using general method G. The Fmoc group of Fmoc-1-d-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by Fmoc and TNBS tests. After the coupling of Cbz-l-leucine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 143–144 °C; 1H-NMR  δH 16.2 , 8.61 , 7.35 , 5.26 , 5.04 , 5.00 , 4.11 , 1.61 , 1.45 , 1.42 , 0.88 , 0.86 ; 13C-NMR  δC 172.4 (5-C), 158.5 (1-C), 156.4 (12-C), 137.5 (14-C), 128.8 (16-C), 128.2 (17-C), 128.1 (15-C), 65.9 (13-C), 53.4 (6-C), 41.4 (2-C), 40.3 (7-C), 24.7 (8-C), 23.5 (9a-C), 21.9 (9b-C), 19.6 (3-C); νmax/cm–1 3312 (N-H), 3269 (N-H), 2959, 2876, 2756, 2621 (tetrazole) 1690 (C=O), 1649 (C=), 1528 (N-H), 1234 (C-O); MS(ESI) m/z 361.2 (M + H); HRMS (Found (M + H)+ 361.1987. Calcd. for C17H25O3N6: (M + H)+ 361.1983.); (Purity test: HPLC method D).(2S)-2-Amino-4-methyl-N-[(1R)-1-ethyl]pentanamide (14h-LD): Cbz-l-leucyl-1-d-aminoethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried and purified by column chromatography (eluent: ethyl acetate – ethanol 1:1 – 1:2 – 0:1) to give the pure product as a white solid . The reaction and the work up were monitored by TLC . Mp: 136 °C (decomposed), 161 °C (melted); 1H-NMR  δH 5.27 , 3.95 , 1.71 , 1.53 , 0.93 , 0.91 ; 13C-NMR  δC 169.8 (5-C), 164.7 (1-C), 52.2 (6-C), 42.4 (2-C), 39.9 (7-C), 24.0 (8-C), 21.6 (9a-C), 21.3 (9b-C), 19.1 (3-C); νmax/cm–1 3194 (N-H), 1667 (C=O), 1543 (N-H); MS(ESI) m/z 227.2 (M + H)+; HRMS (Found (M + H)+ 227.1625 Calcd. for C9H19ON6: (M + H)+ 227.1620.); (Purity test: HPLC method A).Benzyl-N-[-2-methyl-1-{[(1S)-1-ethyl]carbamoyl}butyl]carbamate (13i-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-L-isoleucine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 186–189 °C; 1H-NMR  δH 8.58 , 7.35 , 7.24 , 5.24 , 5.05 , 5.00 , 3.92 , 1.70 , 1.50 , 1.35 , 1.07 , 0.77 , 0.75 ; 13C-NMR  δC 171.4 (5-C), 158.3 (1-C), 156.5 (13-C), 137.5 (15-C), 128.8 (17-C), 128.2 (18-C), 128.1 (16-C), 65.9 (14-C), 59.3 (6-C), 39.9 (2-C), 36.9 (7-C), 24.7 (9-C), 19.5 (3-C), 15.7 (8-C), 11.3 (10-C); νmax/cm–1 3279 (N-H), 2963, 2878, 2693, 2608 (tetrazole), 1690 (C=O), 1651 (C=O), 1535 (N-H), 1234 (C-O); MS(ESI) m/z 361.1 (M + H)+; CHN .-2-amino-3-methyl-N-[(1S)-1-ethyl]pentanamide (14i-LL): Cbz-l-isoleucyl-1-l-aminoethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried to give the product as white solid . Due to hygroscopicity, the corresponding hydrochloride salt was made with 2M HCl in diethyl ether solution (20 mL) for characterization. Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 164 - 167 °C (melted and decomposed); 1H-NMR  δH 5.39 , 3.85 , 1.92 , 1.65 , 1.31 , 1.14 , 0.89 , 0.83 ; 13C-NMR  δC 169.1 (5-C), 158.1 (1-C), 57.7 (6-C), 40.7 (2-C), 36.3 (7-C), 23.9 (9-C), 17.6 (3-C), 14.1 (8-C), 10.4 (10-C); νmax/cm–1 3240 (N-H), 1682 (C=O), 1574 (N-H); MS(ESI) m/z 227.2 (M + H)+; HRMS (Found (M + Na)+ 249.1436. Calcd. for C9H18ON6Na: (M + Na)+ 249.1434.); (Purity test: HPLC method A).Benzyl-(5S)-2-oxo-5-{[(1S)-1-ethyl]carbamoyl}pyrrolidine-1-carboxylate (13f-LL): Prepared using general method G. The Fmoc group of the Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed. After coupling of Cbz-L-pyroglutamine with the resin, the title compound was cleaved off. The cleaving mixture  was subsequently removed by evaporation under reduced pressure. The crude product was purified by column chromatography  to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 195 °C (decomposed); 1H-NMR  δH 16.05 , 8.93 , 7.36 , 5.17 , 4.64 , 2.41 , 2.26 , 1.94 , 1.42 ; 13C-NMR  δC 174.1 (9-C), 171.0 (5-C), 158.3 (1-C), 150.9 (12-C), 136.0 (14-C), 128.8 , 128.5 (17-C), 128.0 (15-C), 67.5 (13-C), 59.3 (6-C), 40.2 (2-C), 31.3 (8-C), 22.3 (7-C), 19.6 (3-C); νmax/cm–1 3289 (N-H), 2993, 2868, 2739, 2615 (tetrazole), 1780 (C=O), 1701 (C=O), 1655 (C=O), 1545 (N-H), 1236 (C-O); MS(ESI) m/z 381.2 (M + Na)+. CHN .(S)-N-((S)-1-(1H-tetrazol-5-yl)ethyl)-5-oxopyrrolidine-2-carboxamide (14f-LL): Cbz-l-pyroglutamyl-1-l-aminoethyltetrazole  was reacted by general method E. Following filtration, the solid residue was washed with methanol and the filtrate was evaporated under reduced pressure. The residual oil was triturated with diethyl ether to give the title compound as a white solid . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 160 °C (decomposed); 1H-NMR  δH 5.35 , 4.34 , 2.52 , 2.40  2.04 , 1.61 ; 13C-NMR  δC 182.3 (9-C), 174.6 (5-C), 159.0 (1-C), 56.9 (6-C), 41.0 (2-C), 29.3 (8-C), 25.0 (7-C), 18.1 (3-C); νmax/cm–1 3262 (N-H), 1649 (C=O), 1547 (N-H); MS(ESI) m/z 225.1 (M + H)+. HRMS (Found (M + H)+ 225.1096. Calcd. for C8H13O2N6: (M + H)+ 225.1095.); (Purity test: HPLC method A).Benzyl-N-[(1S)-2-phenyl-1-{[(1S)-1-ethyl]carbamoyl}ethyl]carbamate (13j-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Cbz-l-phenylalanine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals . Further purification was performed using a CombiFlash EZ Prep system . The reaction and the work up were monitored by TLC . Mp: 191–193 °C; 1H-NMR  δH 16.21 , 7.44 , 7.33 - 7.24 , 5.27 , 4.96 , 4.92 , 4.30 , 3.03 , 2.72 , 1.52 ; 13C-NMR  δC 171.7 (5-C), 158.7 (1-C), 156.3 (14-C), 138.5 (8-C), 137.5 (16-C), 129.6, 128.7, 128.5, 128.1, 127.9, 126.7 , 65.7 (15-C), 56.5 (6-C), 40.0 (2-C), 37.8 (7-C), 19.7 (3-C); νmax/cm–1 3300 (N-H), 2984, 2874, 2741, 2615 (tetrazole), 1684 (C=O), 1651 (C=O), 1531 (N-H), 1234 (C-O); MS(ESI) m/z 417.2 (M + Na)+; CHN .(2S)-2-Amino-3-phenyl-N-[(1S)-1-ethyl]propanamide (14j-LL): Cbz-l-phenylalanyl-1-l-aminoethyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried, then purified by column chromatography  to give the pure product as a white solid . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 152–155 °C; 1H-NMR  δH 7.15 , 7.01 , 5.24 , 3.77 , 2.97 , 2.89 , 1.43 ; 13C-NMR  δC 163.5 (1-C), 150.6 (5-C), 135.5 (8-C), 129.2 (9-C), 128.6 (10-C), 127.1 (11-C), 55.5 (6-C), 41.3 (2-C), 39.3 (7-C), 19.3 (3-C); νmax/cm–1 3217 (N-H), 1651 (C=O), 1528 (N-H); MS(ESI) m/z 283.2 (M + Na)+. HRMS (Found (M + H)+ 261.1462. Calcd. for C12H17ON6: (M + H)+ 261.1458.); (Purity test: HPLC method A).Benzyl-N-[(1S)-1-{[(1S)-1-propyl]carbamoyl}ethyl]carbamate (13b-LL): Prepared using general method G. The Fmoc group of the Fmoc-1-l-aminopropyltetrazole attached to 2-chlorotrityl chloride resin  was removed. After coupling of Cbz-l-alanine with the resin, the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure. The crude product was subsequently washed with petroleum ether (40–60 °C) to obtain the product as white crystals . Mp: 174–175 °C; 1H-NMR  δH 8.44 , 7.41 , 7.34 , 5.06 , 5.02 , 4.10 , 1.88 , 1.19 , 0.87 ; 13C-NMR  δC 173.0 (6-C), 156.8 (1-C), 156.1 (11-C), 137.5 (13-C), 128.8 (15-C), 128.2 (16-C), 128.1 (14-C), 65.8 (12-C), 50.4 (7-C), 45.8 (2-C), 26.8 (3-C), 18.4 (8-C), 10.6 (4-C); νmax/cm–1 3300 (N-H), 3265 (N-H), 2976, 2876, 2735, 2617 (tetrazole), 1691 (C=O), 1651 (C=O), 1533 (N-H), 1246 (C-O); MS(ESI) m/z 333.1 (M + H)+; CHN .(2S)-2-Amino-N-[(1S)-1-propyl]propanamide (14b-LL): Cbz-l-alanyl-1-l-aminopropyltetrazole  was deprotected by general method E. Following filtration, the solid residue was washed with ethanol, which was discarded. The residue was washed with water, which was freeze dried to give the product as a white solid . Due to poor solubility, the corresponding hydrochloride salt was prepared using 2M HCl in diethyl ether solution (20 mL) for characterization (hygroscopic solid). Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . 1H-NMR  δH 9.17 , 8.34 , 5.09 , 3.93 , 1.92 , 1.38 , 0.90 ; 13C-NMR  δC 170.0 (6-C), 158.0 (1-C), 48.6 (7-C), 46.4 (2-C), 26.8 (3-C), 17.4 (8-C), 10.6 (4-C); IR; MS(ESI) m/z 199.1 (M + H)+; HRMS (Found (M + H)+ 199.1302. Calcd. for C7H15ON6: (M + H)+ 199.1302.); (Purity test: HPLC method A).N-[(1S)-1-{[(1S)-1-{[(1S)-1-ethyl]carbamoyl}ethyl]carbamoyl} ethyl]carbamate (13-LLL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin  was removed, this was monitored by the Fmoc and TNBS tests. After the coupling of Fmoc-l-alanine with the resin, the Fmoc group was removed again. Finally, Cbz-l-alanine was attached to the resin, and the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and washed with petroleum ether (40–60 °C) to obtain the product as white crystals . Further purification was performed by Prep HPLC method A. Mp: 199–201 °C (melted and decomposed); 1H-NMR  δH 16.09 (13-H), 8.48 , 7.93 , 7.42 , 7.35 , 5.21 , 5.02 , 4.29 , 4.06 , 1.49 , 1.19 ; 13C-NMR  δC 172.6 (9-C), 172.3 (5-C), 159.1 (1-C), 156.1 (14-C), 137.5 (16-C), 128.8 (18-C), 128.2 (19-C), 128.1 (17-C), 65.8 (15-C), 50.4 (10-C), 48.4 (6-C), 40.9 (2-C), 19.7 (3-C), 18.5 (7-C), 18.4 (11-C); νmax/cm–1 3298(N-H), 1694 (C=O), 1643 (C=O), 1526 (N-H), 1219 (C-O); MS(ESI) m/z 390.2 (M + H)+; HRMS (Found (M + H)+ 390.1883. Calcd. for C17H24O4N7: (M + H)+ 390.1884.); (Purity test: HPLC method C).(S)-N-((S)-1-(1H-tetrazol-5-yl)ethyl)-2-((S)-2-aminopropanamido)propanamide (14c-LLL): Cbz-l-alanyl-l-alanyl-1-l-aminoethyltetrazole  was reacted by the general method E. Following the filtration, the solid residue was washed with methanol, then the filtrate was evaporated under reduced pressure to give the crude product , further recrystallization from a methanol - ethyl acetate mixture provided the pure product as white crystals . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 135 °C (melted and decomposed); 1H-NMR  δH 5.25 , 4.30 , 3.95 , 1.52 , 1.43 , 1.33 ; 13C-NMR  δC 173.6 (5-C), 171.7 (9-C), 164.1 (1-C), 49.8 (6-C), 49.0 (10-C), 42.0 (2-C), 19.4 (3-C), 17.0 (11-C), 16.5 (7-C); νmax/cm–1 3252 (N-H), 1639 (C=O), 1539 (N-H); MS(ESI) m/z 256.1 (M + H)+; HRMS (Found (M + H)+ 256.1520. Calcd. for C9H18O2N7: (M + H)+ 256.1516.); (Purity test: HPLC method A).3-{[(1S)-1-{[(1S)-1-ethyl]carbamoyl}ethyl]carbamoyl}propanoic acid (16-LL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin  was removed. After the coupling of Fmoc-L-alanine with the resin, the Fmoc group was removed again. Finally, a succinyl group was attached to the resin using succinic anhydride, and the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and recrystallized from a water – acetone solvent mixture to obtain the product as white crystals . The reaction and the work up were monitored by TLC . Mp: 170–171 °C; 1H-NMR  δH 5.32 , 4.28 , 2.64 , 2.54 , 1.62 , 1.35 ; 13C-NMR  δC 176.9 (12-C), 175.0 (9-C), 174.9 (5-C), 158.5 (1-C), 49.7 (6-C), 40.7 (2-C), 29.9 (10-C), 29.0 (11-C), 17.8 (3-C), 16.4 (7-C); νmax/cm–1 3358 (N-H), 3254 (O-H), 2992, 2945, 2886, 2778, 2712 (tetrazole), 1713 (C=O), 1636 (C=O), 1543 (N-H), 1526 (N-H), 1231 (C-O); MS(MALDI) m/z 285.30 (M + H)+; HRMS (Found (M + H)+ 285.1310. Calcd. for C10H17O4N6: (M + H)+ 285.1306.); (Purity test: HPLC method B).3-{[(1S)-1-{[(1S)-1-{[(1S)-1-ethyl]carbamoyl}ethyl]carbamoyl}ethyl]-carbamoyl}propanoic acid (17-LLL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin  was removed. After the coupling of Fmoc-l-alanine with the resin, the Fmoc group was removed, and the free amino group was coupled with Fmoc-l-alanine again. After removing the Fmoc protection, a succinyl group was attached to the resin using succinic anhydride, and the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure, and triturated with diethyl ether to obtain the product as white crystals . Further purification was performed by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 186 °C (melted and decomposed); 1H-NMR  δH 5.32 , 4.29 , 4.21 , 2.66 , 2.55 , 1.62 , 1.35 , 1.34 ; 13C-NMR  δC 177.0 (17-C), 175.3 (14-C), 175.2 (9-C), 174.6 (5-C), 158.1 (1-C), 50.0 (10-C), 49.6 (6-C), 40.7 (2-C), 29.8 (16-C), 29.0 (15-C), 17.8 (3-C), 16.3 (7-C), 16.2 (11-C); νmax/cm–1 3343 (N-H), 3381 (O-H), 1707 (C=O), 1651 (C=O), 1634 (C=O), 1539 (N-H), 1201 (C-O); MS(MALDI) m/z 356.30 (M + H)+. HRMS (Found (M + H)+ 356.1682. Calcd. for C13H22O5N7: (M + H)+ 356.1677.) (Purity test: HPLC method A).3-{[(1S)-1-{[(1S)-1-{[(1S)-1-{[(1S)-1-ethyl]carbamoyl}ethyl]carbamoyl}ethyl]-carbamoyl}ethyl]carbamoyl}propanoic acid (18-LLLL): Prepared using general method G. The Fmoc group of Fmoc-1-l-aminoethyltetrazole attached to the 2-chlorotrityl chloride resin  was removed, and resin was coupled with Fmoc-l-alanine, the Fmoc deprotection and coupling with Fmoc-l-alanine procedure was repeated 2 more times. After removing the Fmoc protection for the final time, a succinyl group was attached to the resin using succinic anhydride, and the title compound was cleaved off. The cleaving mixture  was removed by evaporation under reduced pressure. The residue was triturated with diethyl ether to obtain the product as a white solid . Further purification was carried out by column chromatography  and by Prep HPLC method A. The reaction and the work up were monitored by TLC . Mp: 231 °C (decomposed); 1H-NMR  δH 5.32 , 4.26 , 2.64 , 2.58 , 1.63 , 1.36 ; 13C-NMR  δC 177.9 (20-C), 175.5 (17-C), 175.3, 174.9, 174.4 , 158.7 (1-C), 50.1, 49.8, 49.6 , 40.9 (2-C), 29.9 (18-H), 29.2 (19-H), 18.0 (3-C), 16.4, 16.3, 16.2 ; νmax/cm–1 3265 (N-H), 1690 (C=O), 1653 (C=O), 1531 (N-H), 1223 (C-O); MS(ESI) m/z 425.1 (M-H)-; HRMS (Found (M + H)+ 427.2047. Calcd. for C16H27O6N8: (M + H)+ 427.2048.); (Purity test: HPLC method B).8 CFU/mL) and then diluted 1 in 15. A 1 µL aliquot of each diluted suspension was then delivered onto plates with a multipoint inoculator to give a final inoculum of approximately 10,000 CFU/spot as recommended [All MICs were determined using an agar dilution method . This neommended . SelecteThis paper introduces novel antibacterial agents specifically inhibiting the growth of certain bacteria. Such compounds can potentially be exploited to increase the selectivity and effectiveness of the detection of clinically important pathogens by chromogenic or fluorogenic culture media.Antibacterial agents targeting the common bacterial enzyme alanine racemase were developed and investigated. Two 1-aminoalkyltetrazoles were synthesized as potential alanine racemase inhibitors. All molecules were tested against a collection of clinically relevant pathogenic bacteria. As the transportation of a single alanine unit into the cell is not provided by any bacteria, these molecules did not inhibit the growth of any bacteria when deployed as the simple amino acid analogue.l-1-aminoethyltetrazole were designed and synthesized by a novel solid phase peptide coupling method to enable the transportation of these molecules into specially targeted bacterial cells. Many of these peptides displayed significant antibacterial activity. Depending upon the attached amino acids, the substrates caused growth inhibition of bacteria with different levels of selectivity. The selective inhibition of enterococci while not affecting the growth of other Gram-positive cocci by l-pyroglutamyl dipeptide 14f-LL is potentially a valuable result. The most significant example is the selective inhibition of E. coli and some Enterobacterales by l-alanyl- 14a-LL, l-leucyl- 14h-LL and dialanyl- 14c-LLL substrates. These compounds should be investigated in clinical applications, as they could provide a selective antibacterial ingredient in a Salmonella selective culture medium.Selected di- and tri- and succinyl oligo-peptide analogues of E. coli and growth of Salmonella in the presence of these inhibitors, as only two representative strains of each have been tested. However, the successful application of an analogous inhibitor, alafosfalin, in culture media for Salmonella gives us some expectation that these new inhibitors could demonstrate equivalent utility [Salmonella isolates as well as large numbers of other species of Enterobacterales, using procedures that have previously been documented for analogous compounds such as alafosfalin [In this report an important limiting factor is that very few results are presented to demonstrate inhibition of  utility . Furtherfosfalin . Furtherfosfalin .R. J. Anderson, M. Gray, L. Kondacs, S. Orenga, J. Perry, WO 2019/058074 A1 2019.


Salvia cinnabarina afforded two undescribed labdane diterpenoids 1 and 2  along with two known labdane diterpenoids, trans-communic acid (3) and trans-communol (4). Additionally, seven known metabolites were also isolated; two isopimarane diterpenoids 5 and 6, two sesquiterpenoids identified as β-eudesmol (7) and cryptomeridiol (8), and three aromatic compounds identified as phthalic acid (9), a mixture of tyrosol fatty acid esters (10) and the flavone salvigenine (11). While compounds compounds 1–3 showed significant inhibition of yeast α-glucosidase, compounds 2, 3 and 7 had no anti-inflammatory activity in the edema model induced by TPA. This paper is not only the first report on a wild population of Salvia cinnabarina, but also of the presence of labdane-type diterpenoids in a Mexican Salvia sp.The aerial parts of  Salvia L. is the largest genus of Lamiaceae family of plants, with over 1000 species distributed worldwide D589 = +2.2 ; IR (CHCl3) νmax 3607, 3512, 2933, 2854, 1732, 1624 cm−1; 1H and 13C NMR, see m/z [M + H]+ 375.25347 .6β-Hydroxy-trans-communic acid (2): white powder; m.p. 170–173 °C; [α]D589 = +23.0 ; IR (CHCl3) νmax 3601, 3591, 2941, 2853, 1726, 1519, 1406, 1239, 1223 cm−1; 1H and 13C NMR, see m/z [M + H]+ 319.24747 .Trans-communic acid (3): colorless crystals; m.p. 118–120 (reported 135–137 °C); [α]D589 = +45.3  ; NMR data were essentially the same as reported D589 = +4.6  ; IR (CHCl3) νmax 3629, 2872, 2852, 1723, 1642, 1451, 1384, 1209, 1018, 908, 895 cm−1. 1H NMR  δ 6.33 , 5.40 , 5.04 , 4.88 , 4.81 , 4.46 , 3.77 , 3.41 , 2.42–2.37 , 2.13 , 1.96 , 1.77–1.86 , 1.75 , 1.52 , 1.32 , 1.28 , 1.13 , 1.01-1.95 , 0.99 , 0.70 ; 13C NMR  δ 148.2 (C-8), 141.8 (C-14), 134.1 (C-12), 133.6 (C-13), 110.0 (C-17), 107.9 (C-15), 65.3 (C-19), 57.3 (C-9), 56.4 (C-5), 39.6(C-10), 39.3 (C-1), 39.0 (C-4), 38.5 (C-7), 35.5 (C-3), 27.2 (C-18), 24.4 C-6), 23.4 (C-11), 19.2 (C-2), 15.4 (C-20), 12.0 (C-16). DARTMS m/z 289.Isopimara-7,15-dien-3-one (5): white powder; m.p. 80–82 °C (reported 91–92 °C); [α]D589 = −70   D589 = −8.0 . Conformational analysis was performed with the same software under a PM3 semiempirical force field. The resulting conformers were filtered and checked for redundancy. All conformers within 4 kcal/mol were minimized, optimized and the thermochemical properties, IR and vibrational frequencies calculated using the DFT-B3LYP/DGDZVP force field in Gaussian 09. The TD-SCF with the default solvent model was used to perform the ECD calculations of the major conformers in MeOH solution at the B3LYP/6-31G* (d) level of theory. The calculated excitation energy (nm) and rotatory strength (R) in dipole velocity (velR) form were simulated into an ECD curve using the Harada–Nakanishi equation (Equation (1)) as implemented in the SpecDis software  × 100. Where A is the absorbance at 405 nm of sample and control.α-Glucosidase inhibition was evaluated using an adapted method of Xiao-Ping et al. 2010 and Zhou et al. 2010 ,71. A so2CO3 (1 M). Acarbose and miglitol were used as a positive control and DMSO 5% as negative control. Enzyme activity was quantified by measuring the absorbance at 405 nm in a BioTek microplate reader Synergy HT . Experiments were done in triplicates. The percentage of enzyme inhibition by the sample was calculated by the following formula: % Inhibition = [(AC − AS) / AC)] × 100, where AC is the absorbance of the negative control and AS is the absorbance of the tested sample. The concentration of an inhibitor required to inhibit 50% of enzyme activity under the mentioned assay conditions is defined as the inhibition concentration 50 (IC50)Mammalian α-glucosidase was prepared following the modified method of Jo . Rat-intSalvia cinnabarina, several natural products were isolated, including two unpublished labdane-type diterpenoids named malonylcommunol (1) and 6β-hydroxy-trans-communic acid (2). Two already known labdane diterpenoids, trans-communic acid (3) and trans-communol (4) were also isolated and identified by spectroscopic means and comparison with literature date. Two isopimarane-type diterpenoids 5 and 6 were isolated together with two eudesmane-type sesquiterpenoids identified as β-eudesmol (7) and cryptomeridiol (8). Three aromatic natural products identified as phtalic acid (9), tyrosol derivatives (10) and the flavone salvigenin (11) were also isolated from this plant. Compounds 5 and 6 could be considered as biogenetic precursor of compound 12, a bioactive seco-isopimarane diterpenoid previously isolated from a cultivated population of S. cinnabarina. This work represented the first phytochemical analysis of a wild population of this plant.From the dichloromethane extract of a wild population of 1–3 as α-glucosidase inhibitors indicated high specificity towards yeast α-glucosidase. Malonylcommunol (1) was the most active compound (IC50 20.96 ± 0.58 µM), near to the reference compound quercetin.Some products were tested in the TPA induced edema, anti-inflammatory assay with no significant results. The assay of compounds


Doriprismatica stellata  nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina  led to the isolation of the structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin, with an unprecedented position of the cyclopropane ring annelated to the ring A. Unlike other scalaranes, which are most often functionalized at C-12 of ring C, it bears two acetoxy groups at C-11 and C-24 instead. The compound was present in all three samples, supporting the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. The results also indicate that D. stellata passes the scalarane metabolite on to its egg ribbons, most likely for protective purposes. The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32).Investigations on the biochemical relationship between  In habitats with intense competition and feeding pressure, such as coral reefs, sessile or slow-moving organisms commonly defend themselves with toxic or deterrent molecules –8. SpongHexabranchus sanguineus D +40.5. The molecular formula C29H42O6 was established based on 13C NMR data and HRAPCIMS measurements, yielding m/z 487.3054 [M + H]+ , and two oxygen bearing groups: C-11 (δ 68.4) and C-19 (δ 98.9)), and seven quaternary carbons, as obvious from a DEPT135 spectrum. The 1H NMR spectrum showed unusual upfield resonances, diagnostic for a cyclopropyl ring H2-22 . Furthermore, this spectrum proved the presence of the olefinic proton H-16 (δ 5.49 brs), the downfield shifted methine proton H-11 (δ 5.49 brs), and the hemiacetal hydrogen atom H-19 . The 1H NMR spectrum also featured two downfield shifted methylene systems H2-20  and H2-24 , as well as two acetoxy groups H3-11-OAc (δ 2.06 s) and H3-24-OAc (δ 2.08 s), and three methyl groups H3-21 (δ 0.94 s), H3-23 (δ 0.95 s), and H3-25 (δ 0.98 s).The planar structure of 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin was established by extensive 1D and 2D NMR experiments  and the carbon atoms C-24-OAc . The location of C-24 was apparent from HMBC cross peaks between the methylene protons H2-24  and the carbon atoms C-7 (δ 36.5) and C-14 (δ 55.0). The cyclopropyl group was assigned to the C-3 (δ 17.9) and the C-4 (δ 16.1) carbon atoms, based on a 1H,1H-COSY correlation between the methylene protons H2-22 , and the methine proton H-3 (δ 0.55 m), based on HMBC cross peaks between the protons H2-22 and the carbon atoms C-2 (δ 19.0), C-5 (δ 53.2) and C-21 (δ 23.3). The entire assignment of all NMR data is given in The analysis of the 2D NMR data and comparison to literature values  suggesteSpongia . The twoJ = 3.9, 9.2 Hz), H3-21 (δ 0.94 s), and H3-23 (δ 0.95 s), as well as between H3-23 and H-24a , H-24b  and H3-25 (δ 0.98 s), and between H3-25 and H-19 , indicated that these protons share the same orientation on the molecular plane. The chemical shifts of the angular methyl groups CH3-23 (δ 14.0) and CH3-25 (δ 16.1) suggested that all ring junctions are trans D20 +40.5 ; IR (ATR) vmax: 3416, 2922, 2861, 1732, 1234 cm−1; 1H and 13C NMR (m/z): [M + H]+ calcd. for C29H43O6, 487.3060; found, 487.3054.C 13C NMR ; HRAPCIMFile 1Spectroscopic data and other relevant information for 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin.


Toddalia asiatica. The separation and purification of ingredients from T. asiatica is an important procedure to acquire high-purity compounds for subsequent pharmacological investigation to discover leading compounds. In the present work, an offline two-dimensional high-performance liquid chromatography (HPLC) method was successfully established for the separation of high-purity glycosides from T. asiatica. Based on the separation results obtained with two different chromatographic stationary phases, a phenyl-bonded silica-based reversed-phase column was employed as the first HPLC preparation, and three fractions were obtained from the sample. Then, the fractions were isolated and purified on an octadecyl-bonded silica-based reversed-phase column to obtain high-purity compounds in the second HPLC separation. As a result, three coumarin glycosides, including two undescribed and one known, along with one known flavonoid glycoside with more than 98% purity were isolated from the sample. The structures of the isolated compounds were elucidated on the basis of extensive spectroscopic evidence derived from optical rotation, mass spectrometry, and nuclear magnetic resonance experiments. Two-dimensional HPLC with different stationary phases has the potential to be an efficient method for the separation of high-purity compounds from T. asiatica.Coumarins and flavonoids are the major constituents of  Toddalia asiatica (L.) Lam. (Rutaceae) , well known as Feilongzhangxue in Chinese and commonly known as orange climber in English, is widely distributed in the east, south, and southeast of Asia, Madagascar, Africa, and the Mascarene Islands D25 -41). The molecular formula of 1 was found to be C22H30O11 by the HR-ESIMS ion at m/z 493.1679 [M+Na]+ , which was further supported by its 1H and 13C NMR data. The 1H NMR spectrum exhibited the characteristic AB system signals of the lactone ring at δ 6.23  and 8.02  and a singlet signal at δ 6.75 , indicative of the presence of a 5,6,7-triisubstituted coumarin moiety. The characteristic signal of the hydroxylated isopentyl group was observed at δH 2.73 , 3.01 , 3.89 , 1.31 , and 1.26  and δC 26.9 (C-1′), 88.0 (C-2′), 75.0 (C-3′), 26.4 (C-4′), and 24.1 (C-5′), following the HSQC, 1H-1H COSY, and HMBC correlations (δC 119.8) of the coumarin moiety. A two-methoxy-group-substituted coumarin moiety was confirmed by the presence of the methoxy groups resonating at δH 3.92  and 3.93  in the 1H NMR spectrum and δC 56.8 and 64.1 in the 13C NMR spectrum, along with the HMBC correlations from the methoxy hydrogens resonating at δH 3.92 and 3.93 to C-5 (δC 157.8) and C-7 (δC 163.9) of the coumarin moiety, respectively. The 13C NMR spectrum displayed the existence of 22 carbons, including 9 coumarin carbons, 2 methoxy carbons, and a set of isopentyl group carbons. The remaining six oxygenated carbons were assignable to one sugar unit. By comparison of the NMR data with those reported . The aforementioned data indicated that the chemical structure of 1 resembled that of toddalolactone 3′-O-β-d-glucopyranoside (compound 3) D25 -41 , which was consistent with that of compound 3 [α]D25 -44 . On the basis of the abovementioned evidence, the structure of compound 1 was determined as 6-[(2′S)-2′-O-β-d-glucopyranosyloxy-3′-hydroxy-3′-methylbutyl]-5,7-dimethoxy-2H-1-benzopyran-2-one, and the compound was given the trivial name of (−)-toddalolactone 2′-O-β-d-glucopyranoside  , except ranoside .2 exhibited the molecular formula C22H30O11, as determined by 1D NMR data and the HRESIMS ion at m/z 471.1862 [M+H]+ . Its 1H as well as the 13C NMR data demonstrated that compound 2 possessed 22 carbons displaying coumarin glycoside features closely similar to those of (−)-toddalolactone 3′-O-β-d-glucopyranoside (3) D25 +44 , which was opposite to that of 3 [α]D25 -44 . Thus, 2 was identified as the enantiomer of 3 and given the trivial name (+)-toddalolactone-3′-O-β-d-glucopyranoside (Compound side (3) . Howeverranoside .1: colorless oil; [α]D25 -41 . 1H NMR  δ 8.02 , 6.75 , 6.23 , 4.19 , 3.93 , 3.92 , 3.89 , 3.27 , 3.21 , 3.19 , 3.06 , 3.05 , 3.01 , 2.87 , 2.73 , 1.31 , 1.26 ; 13C NMR  δ 163.9 (C-7), 163.5 (C-2), 157.8 (C-5), 156.2 (C-9), 141.3 (C-4), 119.8 (C-6), 112.4 (C-3), 108.4 (C-10), 106.0 (C-1″), 96.2 (C-8), 88.0 (C-2′), 78.0 (C-3″), 77.1 (C-5″), 75.9 (C-2″), 75.0 (C-3′), 71.6 (C-4″), 64.1 (5-OCH3), 62.8 (C-6″), 56.8 (7-OCH3), 26.9 (C-1′), 26.4 (C-4′), 24.1 (C-5′); HRESIMS m/z 493.1679 [M+Na]+ .Compound 2: colorless oil; [α]D25 +44 , 1H NMR δ 8.02 , 6.74 , 6.23 , 4.56 , 3.91 , 3.90 , 3.82 , 3.80 , 3.62 , 3.37 , 3.31 , 3.29 , 3.19 , 2.88 , 2.76 , 1.36 , 1.36 ; 13C NMR  δ 163.7 (C-7), 163.3 (C-2), 157.7 (C-5), 156.2 (C-9), 141.2 (C-4), 120.2 (C-6), 112.6 (C-3), 108.4 (C-10), 98.6 (C-1″), 96.3 (C-8), 81.9 (C-3′), 78.1 (C-3″), 77.7 (C-5″), 77.5 (C-2′), 75.2 (C-2″), 71.7 (C-4″), 63.9 (5-OCH3), 62.7 (C-6″), 56.7 (7-OCH3), 27.1 (C-1′), 23.9 (C-5′), 21.8 (C-4′); HRESIMS 471.1862 [M+H]+ .Compound 6H5-3152-N column , octadecyl-bonded silica-based reversed-phase YMC-Pack ODS-AQ column , and YMC-Pack ODS-AQ column .HPLC separation was carried out on a system consisting of a Prepstar SD-1 solvent delivery unit, a G1316A thermostatted column compartment, a G1362A refractive index detector, and an Agilent HPLC workstation . HPLC analysis was performed on an Agilent 1260 system equipped with a G1311C quaternary pump, a G1314F variable-wavelength detector coupled with an analytical workstation, a G1329B autosampler, and a G1316A thermostatted column compartment. The columns used in this study were: phenyl-bonded silica-based reversed-phase Senshu Pak C3OD resonances (1H δ 3.31 and 13C δ 49.0) as internal reference. Mass spectra were obtained using an LTQ Orbitrap XL mass spectrometer . A JASCO P-1020 automatic digital polarimeter  was used for the optical rotation experiments. Nuclear magnetic resonance spectra were measured on a Bruker AV-500 FT-NMR spectrometer  in deuterated methanol, using visual CDn-butanol, ethanol, methanol, and formic acid purchased from Fuyu Fine Chemical  were of analytical grade. Deionized water was purified using a Milli-Q water purification system . All samples and reagents prepared for chromatographic analysis and separation were filtered through 0.45 μm membranes  before use.Chromatographic-grade acetonitrile and methanol were bought from Oceanpak . Ethyl acetate, T. asiatica were purchased from Jingde Pharmaceutical Company, Bozhou, Anhui Province of China, in October 2017, and authenticated by Prof. Yingxia Li, School of Pharmacy, Qingdao University, China. A voucher specimen (accession number: TA20171001) was deposited at the School of Pharmacy, Qingdao University, China.The air-dried roots of n-butanol (2 L × 3), successively, and then concentrated to yield 596.4 g of the ethyl acetate extract and 241.2 g of the n-butanol extract. The n-butanol fraction (8 g) was chromatographed on an octadecyl-bonded silica-based reversed-phase column and eluted with methanol–water (30:70 and 60:40), successively. The fraction eluted with 60% methanol (1.2 g) was chosen for subsequent two-dimensional HPLC separation. The target sample was dissolved in 6 mL of methanol and stored at 4 °C until further separation.The raw materials (11.80 kg) were powdered and reflux-extracted with 90% ethanol for 2 h at 80 °C. After evaporating the solvent under reduced pressure, the residue (1.56 kg) was dissolved in distilled water (2 L) and partitioned with ethyl acetate (2 L × 3) and 6H5-3152-N column (150 × 8 mm). The mobile phase consisted of water and methanol  at a constant flow rate of 1.5 mL/min. The column temperature was maintained at 30 °C, and the chromatogram was recorded using a refractive index detector. The sample injection volume was 100 μL.The first HPLC isolation of the fraction eluted with 60% methanol was carried out on a phenyl-bonded silica-based reversed-phase Senshu Pak Cv/v) was adopted to separate the fractions collected during the first HPLC separation at a constant flow rate of 2.0 mL/min. The column temperature was maintained at 30 °C, and the chromatogram was recorded using a refractive index detector. The sample injection volume was 200 μL.The second HPLC preparation was performed on a YMC-Pack ODS-AQ (250 × 10 mm) column. The mobile phase of water and acetonitrile  at 25 °C. The mobile phase was the same as that of the second HPLC preparation, and the flow rate was 0.42 mL/min. The detection wavelength was 269 nm, and the sample injection volume was set at 10 μL.The HPLC analysis of the compounds purified from the three fractions was performed on a YMC-Pack ODS-AQ column (250 × 4.6 mm) at 25 °C. The mobile phase consisted of water with 0.2% formic acid (A) and acetonitrile (B). The gradient elution program was as follows: 0–50 min, 10–30% B, 50–65 min, 30% B at a constant flow rate of 1.0 mL/min. The detection wavelength was 269 nm, and the sample injection volume was set at 10 μL.T. asiatica at high-purity. The Senshu Pak C6H5-3152-N column was used to simplify the sample in the first HPLC separation. Then, the YMC-Pack ODS-AQ column was adopted to isolate and purify compounds in the second HPLC preparation. Benefiting from the optimized collection mode, four glycosides with purity higher than 98% were obtained in the second HPLC separation. Among them, (−)-toddalolactone 2′-O-β-d-glucopyranoside and (+)-toddalolactone-3′-O-β-d-glucopyranoside are new coumarin glycosides. The methodology used in this investigation based on two different chromatographic stationary phases with different separation mechanisms allowed obtaining the target compounds in a satisfactory degree of purity, which will facilitate the investigation on their pharmacological activities.An offline two-dimensional HPLC system based on two reversed-phase columns with phenyl- and octadecyl-bonded silica-based stationary phases was used for the isolation of the target compounds from


Phlebia tremellosa. The structures of all isolated compounds were established by extensive spectroscopic analyses, including those involving extensive two-dimensional nuclear magnetic resonance. The absolute configurations of phlebidiol, phlebioic acid, and phlebiolide were determined by comparisons of experimental and calculated electronic circular dichroism spectra. Phlebidiol and phlebioic acid have previously unreported carbon skeletons, for which we propose the skeletal names “seco-sterpurane” and “phlebiane,” respectively. Phlebiolide is also the second published example of a merulane sesquiterpene.Three new sesquiterpenes, phlebidiol, phlebioic acid, and phlebiolide, as well as the known compound tremetriol, were isolated from cultures of the basidiomycete The online version of this article (10.1007/s11418-019-01286-8) contains supplementary material, which is available to authorized users. Phlebia tremellosa (Schrad.) Nakasone & Burds. (formerly Merulius tremellosus), a species in class Basidiomycetes, is classified in the family Meruliaceae and is also known to be one of the wood-decay fungi  to C-1, C-8, and C-9 demonstrated the presence of an α-methyl enone moiety, while another methyl resonance [δH 1.16 ] exhibited four HMBC correlations to two methylene carbons at δC 40.7 (C-6) and 37.8 (C-12), a ketonic carbon (C-8), as well as an undistinguishable carbon signal assigned to a methine carbon at δC 42.8 (C-5) or a quaternary carbon at δC 42.6 (C-7). The observation of four correlations from a methyl-proton singlet indicated a linkage between C-14 and a quaternary carbon, i.e., C-7. Accordingly, the remaining carbon atoms C-6, C-8, and C-12 that correlated with H3-14 were all assumed to be bound to C-7. HMBC correlations from H2-6  to C-8, C-12, and C-14 and from H2-13 [δH 3.73 ] to C-7 also sustained the partial structure around C-7. The other methyl proton signals [δH 1.02  and δH 1.14 ] exhibited HMBC correlations with C-2, C-3, C-4, and C-11/C-10. In addition, H2-2 [δH 2.46 and 2.25 ] correlated with C-1, C-3, C-4, C-5, and C-9, which was suggestive of linkages from C-1 to C-4 and between C-1 and C-5, as well as the presence of two methyl groups at C-3. Hence, the structure of phlebidiol (1) was proposed to be a four-membered ring-opening product of a sterpurane sesquiterpene. The relative configuration of 1 was confirmed by the essential NOEs  observed in the NOESY spectrum  spectrum was calculated by quantum-chemical methods and compared with the experimental spectrum. The preliminary conformers, which were generated using the GMMX add-on module , were optimized using density functional theory (DFT) at the CAM-B3LYP/6-31+G level. ECD spectra of the DFT-optimized conformers were calculated using time-dependent DFT at the CAM-B3LYP/6-31+G level with the conductor-like polarizable continuum model (CPCM) solvation model in MeOH. The obtained ECD spectra were Boltzmann averaged on the basis of the calculated relative energies of the DFT-optimized conformers. The experimental and calculated ECD spectra of 1 were in good agreement for the  absolute configuration , + ion peak at 289.1396, indicating the quasi-molecular formula C15H22O4Na . The IR spectrum exhibited absorptions for hydroxy groups (3435 cm−1) and carbonyl groups (1699 cm−1). The 13C NMR and DEPT135 spectra (Table  sp2 carbons assigned to a tetra-substituted double bond [δC 132.8 (C-1) and 155.8 (C-10)], a carboxy group [δC 182.8 (C-14)], and a ketonic carbonyl group [δC 208.2 (C-2)]. The DQF–COSY spectrum showed two solitary C-4/C-5/C-6 and C-8/C-9 sequences  within the double bond. HMBC correlations from H3-13 [δH 1.35 ] to two methylene carbons (C-6 and C-8), a quaternary sp3 carbon (C-7), and a carboxy carbon (C-14) suggested the C-6/C-7/C-8 sequence, with both a methyl group and a carboxy group bonded to C-7, as sustained by the HMBC correlations . Furthermore, both methyl-proton signals [δH 1.00  and δH 1.11 ] correlated with a ketonic carbon (C-2), a quaternary sp3 carbon (C-3), and a methylene carbon (C-4), suggestive of the C-2/C-3/C-4 sequence with two methyl groups at C-3. The HMBC correlation from H2-6 to C-1 confirmed the linkage between C-1 and C-5. The remaining indices of hydrogen deficiency and the chemical shifts of C-1 and C-10 implied a linkage between C-1 and C-2. Hence, the planar structure of 2 was determined to be a sesquiterpene with a previously unreported skeleton. The relative structure of 2 depicted in Fig. 3-11/H-5; H-5/H3-13; H3-12/H-4]. Finally, we determined the absolute structure of 2 to be the  configuration because the experimental ECD spectrum was in good agreement with the time-dependent density functional theory (TDDFT) spectrum calculated at the CAM-B3LYP/6-31+G//CAM-B3LYP/6-311+G level , + ion at m/z 269.1133, indicating the molecular formula C15H18O3Na . The IR spectrum exhibited a strong absorption for a carbonyl group (1749 cm−1). The 1H NMR spectrum , a quaternary carbon (C-3), and a methylene (C-4), as well as mutual correlations (C-14 or C-13). The HMBC correlations  suggested a linkage between C-1 and C-5, revealing the presence of a 3,3-dimethylcyclopentene moiety. The HMBC spectrum also revealed correlations from the uncoupled methyl group [δH 1.33 ] to a methine at δC 66.0 (C-6), a nonprotonated carbon at δC 61.0 (C-7), and a methylene at δC 30.5 (C-8). Furthermore, H2-8 correlated with two sp2 quaternary carbons at δC 122.9 (C-9) and 153.8 (C-12) and a carbonyl carbon at δC 174.3 (C-10). The carbon signal of C-12 also correlated with an olefinic proton (H-2) within a 3,3-dimethylcyclopentene moiety. These correlations allowed a seven-membered ring bearing a methyl group and a carbonyl group at C-7 and C-9, respectively, to be constructed. The remaining oxygenated methylene (H2-11) was proposed to be attached to C-12 based on HMBC correlations from H2-11 to C-9 and C-12. The location of C-2 was also corroborated by an NOE between H-2 and H2-11, as observed in the NOESY spectrum. Finally, the correlation from H2-11 to C-10 confirmed the formation of a γ-lactone ring. The oxygen functional groups at C-6 and C-7 were determined to be an epoxy group based on the remaining indices of hydrogen deficiency and the chemical shifts in the 1H and 13C NMR spectra. The presence of the epoxy group was also supported by the fact that the IR spectrum showed no absorption for hydroxy groups. The relative configurations of C-5, C-6, and C-7 were deduced to be as shown in Fig. 3-13/H-4β; H-4β/H-5; H-5/H-8β; H3-14/H-4α; H-4α/H-6; H3-15/H-8α] in the NOESY spectrum. Based on the comparison of the experimental spectrum and the TDDFT ECD spectrum calculated at the CAM-B3LYP/6-31+G//APFD/6-311+G level of theory, the absolute structure of 3 was determined to have a  configuration , the first described seco-sterpurane, is possibly explained by the oxidative aliphatic C–C bond cleavage of the α-hydroxy ketones within the cyclobutane ring of an oxidized tremetriol , is potentially biosynthesized by the secondary ring-opening rearrangement of the isolactarane sesquiterpene, as shown in Fig. P. tremellosa. Furthermore, the biosynthetic pathway to phlebiolide (3) also seems to involve the secondary ring-opening rearrangement of an isolactarane sesquiterpene. Merulactone + .Colorless oil; mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 257 (4.03) nm; IR (KBr) νmax 3435, 2969, 2932, 2874, 1699, 1616, 1464, 1211, 1078 cm−1; ECD  λext (Δε) 346 (+ 2.01), 261 (+ 2.31), 227 (+ 1.42), 217 (+ 1.96), 210 (+ 2.31) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table m/z 289.1396 [M+Na]+ .Colorless oil;mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 275 (4.03) nm; IR (KBr) νmax 2957, 2926, 2864, 1749, 1651, 1647, 1456, 1224, 1159, 1041 cm−1; ECD  λext (Δε) 254 (+ 4.47), 205 (– 5.01) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table m/z 269.1133 [M+Na]+ .Colorless oil; mentclass2pt{minim1–3 were generated using the GMMX add-on module of GaussView 6 with an energy window of 10 kcal/mol. Optimization of suggested conformers followed by TDDFT calculations were performed using Gaussian 16 with various combinations of functionals  and basis sets  with the CPCM solvent model. ECD spectra were generated by the SpecDis program using a Gaussian band shape with 0.3–0.35 eV [Conformers of –0.35 eV , 25. TheSupplementary file1 (PDF 7337 kb)Below is the link to the electronic supplementary material.


A number of ligands (L2–L8) and the analogs of L1, with various substituents and chiral backbones, were synthesized and examined in the oxidative coupling reactions.An iron-catalyzed asymmetric oxidative homo-coupling of 2-naphthols for the synthesis of 1,1′-Bi-2-naphthol (BINOL) derivatives is reported. The coupling reaction provides enantioenriched BINOLs in good yields (up to 99%) and moderate enantioselectivities (up to 81:19 er) using an iron-complex generated in situ from Fe(ClO Axially chiral compounds (atropisomers) have aroused much attention from organic chemists due to their prevalence in natural products, bioactive molecules, functional materials, and their wide applications in asymmetric transformations . Many elIn the past few decades, enormous efforts have been devoted to the enantioselective assembly of BINOL scaffolds. Transition-metal-catalyzed asymmetric oxidative coupling of 2-naphthols have shown its power in the synthesis of BINOLs a. EfficiN,N′-dimethyl-N,N′-bis(8-quinolyl)-cyclohexanediamine (BQCN), developed by the Che group and successfully applied in iron-catalyzed asymmetric cis-dihydroxylation of alkenes -2,2′-diamine (L6), (12R)-9,10-dihydro-9,10-ethanoanthracene-11,12-diamine (L7), and -1,2-diphenylethane-1,2-diamine (L8). Ligands with a variety of electronically differentiated substituents at the C6 position of the quinoline moiety (L2–L4) and an acridine-derived ligand (L5) were also prepared to probe the electronic and steric effects of the ligands on the reaction.The Buchwald-Hartwig C–N coupling reaction ,56 was u1a) as the model substrate for optimization of reaction conditions (4)2 and L1 for 30 min before the addition of the substrate, and under atmospheric dioxygen. Various solvents  were screened first (entries 1–5) and resulted in moderate conversions and enantioselectivities, except toluene and methanol. The reaction in chlorobenzene gave a better balance between reactivity and selectivity . The addition of 4Å MS led to a slightly higher enantioselectivity with partial conversion in a much shorter reaction time (entries 5 vs. 6). We were delighted to find that increasing the temperature from 30 °C to 50 °C delivered 39% conversion with comparable enantioselectivity (entry 7). Further increase in temperature  resulted in lower enantioselectivities (entries 8 and 9). Then, the reaction with different iron salts were investigated, including Fe(ClO4)3, Fe(OAc)2, Fe(OTf)2, Fe(acac)2, and FeCl2, but failed to provide better results (entries 10–14). The ratio of iron precursor versus L1 was also examined (entries 14–19). Surprisingly, increasing Fe(ClO4)2-loading from 5 mol% to 10 mol% improves the efficiency without affecting the enantioselectivity and delivered the coupling product 2a in 84% isolated yield with 80:20 er (entries 15 and 16). Although further increasing Fe(ClO4)2 to 12.5 mol% improved the yield, a slightly diminished er was also observed (entry 17). Finally, reactions with the catalysts derived from the diamine ligand (L2–L8) were inspected. Electron-withdrawing groups-substituted ligands (L2 and L3) showed excellent reactivities but with low enantioselectivities (entries 20 and 21). In contrast, ligand L4 with an electron-donating substituent delivered the product in reduced yield, albeit with good er (entry 22). Upon further screening, the ligands (L5–L8) bearing different chiral backbones, lower yield, and er were obtained with L5, while ligands L6–L8 failed to give any product (entries 23–26).To test our hypothesis, we selected 2-naphthol  in 56–88% yields with 56:44 to 81:19 er. The substrate bearing an electron-withdrawing Br substitution at the C3-position was also converted into the corresponding product (2f) in 51% yield with 79:21 er. The electronic effects of different functionalities are clearly demonstrated by the observation that electron-donating groups delivered higher yield . However, C3-substituted substrates with carbonyl functionalities like CO2H, COPh, and CO2Bn failed to give any products (2g–2i). When C6-Br-substituted 2-naphthol was applied, the desired product (2j) was obtained in 82% yield with 79:21 er. A C6-phenyl-substituted 2-naphthol also resulted in 64% yield and 74:26 er (2k). Moreover, C7-substituted  substrates were also effectively coupled and delivered the corresponding products (2l–2o) in 65–99% yields, albeit with dramatically diminished er.Next, we investigated the scope of the iron-catalyzed asymmetric oxidative coupling reaction, and the results were summarized in 3, MeOH, and acetone were freshly distilled after drying over CaH2. 1H-NMR spectra were recorded at room temperature on a Bruker ADVANCE III 400 MHz spectrometer and were reported relative to residual Chloroform-d (δ 7.26 ppm) or DMSO-d6 (δ 2.50 ppm). 13C-NMR spectra were recorded on a Bruker ADVANCE III 400 MHz spectrometer (100 MHz) and were reported relative to Chloroform-d (δ 77.16 ppm). 19F-NMR spectra were recorded on a Bruker ADVANCE III 400 MHz spectrometer (376 MHz). Data for 1H-NMR were reported as chemical shift (δ ppm) , and integration) using standard abbreviations for multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet. Data for 13C-NMR and 19F-NMR were reported in terms of chemical shifts (δ ppm). High-resolution mass spectra (HRMS) were obtained by using a Bruker Compact TOF mass spectrometer in electrospray ionization mode (ESI). Enantiomeric ratio (er) was determined by an Agilent 1260 Series HPLC utilizing DAICEL Chiralpak  or Chiralcel (OD-H) columns (4.6 mm × 250 mmL). Optical rotations were measured with a Perkin Elmer 343 polarimeter and were reported as: [α]DT . The NMR spectra of all new compounds and HPLC spectra of oxidative coupling products were provided in the Unless otherwise noted, all reagents were purchased commercially and used without further purification. Petroleum ether (PE) (60–90 °C), ethyl acetate (EA), and dichloromethane (DCM) were used for silica gel chromatography. MeCN, toluene, DMF, and THF were purchased commercially or were dried by passage through an activated alumina column under argon . PhCl, C3 (1.0 equiv), Pd2(dba)3 (5 mol%), rac-BINAP (10 mol%), NaOtBu (3.0 equiv), and toluene under Ar atmosphere. Then 8-haloqunoline 4 (2.2 equiv) was added directly. The flask was sealed, and the reaction was stirred at 85 °C until the complete consumption of the starting material 3. The mixture was cooled to room temperature, filtered through a silica plug, and the plug was washed with EA. The combined filtrates were concentrated under reduced pressure, and the residue was purified by silica gel chromatography to give the desired product Ln.General Procedure : To an o1,N2-Di(quinolin-8-yl)cyclohexane-1,2-diamine-N (L1) +: 405.1885, found 405.1880;   ; 13C-NMR.7, 24.5 ; 19F NMRδ –110.9 ; HRMS quinolin-8-yl)cyclohexane-1,2-diamine-N (L3): Following the general procedure, the reaction was carried out with -cyclohexane-1,2-diamine 3a ; Pd2(dba)3 ; rac-BINAP ; NaOtBu ; and 8-bromo-6-trifluoromethylquinoline 4c  in 25 mL of toluene. The desired product was obtained  as a yellow green solid after purification by silica gel chromatography (PE/DCM = 10/1 to 5/1 to 1/1). 1H-NMR  δ 8.61 , 7.95 , 7.32 , 7.14–7.03 , 6.95–6.83 , 6.55 , 3.87–3.62 , 2.45–2.24 , 2.03–1.85 , 1.70–1.48   δ 148.4, 144.9, 138.8, 136.9, 129.4 , 127.5, 124.5 , 122.3, 110.6 , 100.0, 57.3, 32.3, 24.8  δ –62.8 (+) calcd for C26H23F6N4 [M + H]+: 505.1821, found 505.1817; c = 1.0, CHCl3); M. p. 120–124 °C.  ; 13C-NMR.3, 24.8 ; 19F NMR δ –62.8 ; HRMS quinolin-8-yl)cyclohexane-1,2-diamine-N (L4): Following the general procedure, the reaction was carried out with -cyclohexane-1,2-diamine 3a ; Pd2(dba)3 ; rac-BINAP ; NaOtBu ; and 8-bromo-6-(tert-butyl)quinoline 4d  in 35 mL of toluene. The desired product was obtained  as a yellow solid after purification by silica gel chromatography (PE/DCM = 10/1 to PE/EA = 5/1). 1H-NMR  δ 8.55 , 7.99 , 7.31–7.25 , 7.02–6.89 , 3.87–3.81 , 2.38 , 1.91–1.83 , 1.71–1.50 , 1.36   δ 150.6, 146.2, 143.5, 137.4, 136.0, 128.5, 121.3, 109.4, 104.3, 55.5, 35.2, 31.4, 30.7, 24.0 (+) calcd for C32H41N4 [M + H]+: 481.3326, found 481.3323; c = 1.0, CHCl3); M. p. 172–174 °C. ; 13C-NMR.7, 24.0 ; HRMS cyclohexane-1,2-diamine-N (L5): Following the general procedure, the reaction was carried out with -cyclohexane-1,2-diamine 3a ; Pd2(dba)3 ; rac-BINAP ; NaOtBu ; and 4-iodoacridine 4e  in 30 mL of toluene. The desired product was obtained  as a yellow solid after purification by silica gel chromatography (PE/DCM = 2/1 to PE/DCM = 1/1 to DCM). 1H-NMR  δ 8.78 , 8.01 , 7.91 , 7.67 , 7.49 , 7.43 , 7.18 , 6.97 , 6.75 , 3.98–3.90 , 2.37–2.30 , 1.86–1.80 , 1.62–1.55   δ 146.5, 144.2, 140.7, 135.1, 129.7, 128.9, 127.8, 127.3, 127.2, 127.0, 125.5, 113.8, 103.1, 56.6, 31.6, 24.4 (+) calcd for C32H29N4 [M + H]+: 469.2387, found 469.2371; c = 0.5, CHCl3); M. p. 198–202 °C.  ; 13C-NMR.6, 24.4 ; HRMS --2,2′-diamine(R)-N (L6) -2,2′-diamine 3b ; Pd2(dba)3 ; rac-BINAP ; NaOtBu ; and 8-bromoqunoline 4a  in 10 mL of toluene. The desired product was obtained  as a yellow solid after purification by recrystallization from EA. 1H-NMR  δ 8.42 , 7.99–7.95 , 7.94–7.86 , 7.37 , 7.30–7.19 , 6.94–6.89 .ine (L6) : Followi11,N12-Di(quinolin-8-yl)-9,10-dihydro-9,10-ethanoanthracene-11,12-diamine(12R)-N (L7) +: 275.9630, found 275.9620; M. p. 58–62 °C. Hz, 1H) ; 13C-NMR72.8 Hz) ; 19F NMR δ –62.5 ; HRMS (E8-Bromo-6-(tert-butyl)quinoline (4d)   was added dropwise by syringe. Upon the completion of the addition, the mixture was stirred at 0 °C for another 30 min. Then ZnCl2•TMEDA  was added at 0 °C, and the resultant mixture was stirred for 20 min before acridine  was added. After the reaction was warmed up to 25 °C, I2  in THF (20 mL) was added dropwise. The reaction mixture was stirred for 2 h and then quenched with saturated Na2S2O3 solution and extracted with EA (3 × 30 mL). The combined organic phase was washed with brine and dried over anhydrous Na2SO4. The desired product was obtained  as a yellow solid after purification by silica gel chromatography (PE/DCM = 20/1 to 5/1). 1H-NMR  δ 8.72 , 8.46 , 8.39 , 8.09–7.93 , 7.83 , 7.63–7.52 , 7.31–7.19   δ 149.8, 147.1, 141.0, 137.2, 130.9, 130.1, 129.4, 127.9, 127.2, 126.7, 126.63, 126.58, 104.0 (+) calcd for C13H9IN [M + H]+: 305.9774, found 305.9763; M.p. 100–104 °C.To a 100 mL Schlenk flask were added TMP  and 20 mL of THF. The solution was cooled to 0 °C, and   ; 13C-NMR8, 104.0 ; HRMS ; NaH ; and MeI  in 10 mL of DMF. The desired product was obtained  as a white solid after purification by silica gel chromatography (DCM). 1H-NMR  δ 7.78 , 7.47 , 7.36 , 7.22–7.13 , 3.94 .hthalene : Followi2-Bromo-3-methoxynaphthalene -2,2′-diol (2a) -2,2′-diol (2b) -2,2′-diol (2c) -2,2′-diol (2d) -2,2′-diol (2e) -2,2′-diol (2f) -2,2′-diol (2j) -2,2′-diol (2k) -2,2′-diol (2l) -2,2′-diol (2m) -2,2′-diol (2n): The reaction was conducted with Fe(ClO4)2  and L1 ; 7-((tert-butyldimethylsilyl)oxy)naphthalen-2-ol ; and MS 4Å (153.7 mg). The desired product was obtained  as a brown solid after purification by silica gel chromatography (PE to PE/EA = 5/1). 68:32 er : major = 6.0, minor = 8.5). 1H-NMR  δ 7.86 , 7.75 , 7.21 , 6.95 , 6.46 , 5.07 , 0.83 , −0.03 , −0.06   δ 155.3, 153.2, 134.9, 131.1, 129.9, 125.1, 119.9, 115.3, 111.7, 109.8, 25.8, 18.3, −4.5 (−), m/z calc′d for C32H41O4Si2 [M − H]−: 545.2549, found 545.2578; c = 1.0, CHCl3); M.p. 118–122 °C.  ; 13C-NMR.3, −4.5 ; HRMS (E(S)-7,7′-Dimethoxy--2,2′-diol (2o) [4)2  and L1 ; 7-methoxynaphthalen-2-ol ; and MS 4Å (156.3 mg). The desired product was obtained  as a white solid after purification by silica gel chromatography (PE to PE/EA = 5/1). 59:41 er : major = 11.1, minor = 18.8). 1H-NMR  δ 7.88 , 7.78 , 7.22 , 7.03 , 6.48 , 3.57 .iol (2o) : The rea4)2 and readily available ligand L1 and uses 1 atm oxygen as the oxidant. The atom economy of this transformation, the easily available catalyst, and operationally simple procedure provide new applications of asymmetric iron catalysis. Further studies on synthesizing a library of nitrogen ligands and extending their applications are underway in our laboratory.In summary, we have developed an iron/bisquinolyldiamine-catalyzed asymmetric oxidative coupling of 2-naphthols. This method employs in situ-formed iron complexes from Fe(ClO