Design of NIR Chromenylium-Cyanine Fluorophore Library for Switch-ON and Ratiometric Detection of Bio-Active Species in Vivo

Transcription

1 Supporting information for Design of NIR Chromenylium-Cyanine Fluorophore Library for Switch-ON and Ratiometric Detection of Bio-Active Species in Vivo Yanfen Wei, Dan Cheng, Tianbing Ren, Yinhui Li, Zebing Zeng, Lin Yuan* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 4182 (PR China) * To whom correspondence should be addressed: lyuan@hnu.edu.cn S1

2 Table of content 1. Supplemental Tables S1-3 S Supplemental Figures S9-23 S Synthesis and Characterization S Supplemental NMR Spectra S26-36 S2

3 DAD1 A, Sig=254,16 Ref=off (YL\BSR III 5 OH.D) DAD1 B, Sig=35,16 Ref=off (YL\BSR III 5 OH.D) DAD1 C, Sig=5,16 Ref=off (YL\BSR III 5 OH.D) DAD1 D, Sig=55,16 Ref=off (YL\BSR III 5 OH.D) DAD1 B, Sig=35,16 Ref=off (YL\NIR33.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR33.D) DAD1 D, Sig=55,16 Ref=off (YL\NIR33.D) DAD1 E, Sig=65,16 Ref=off (YL\NIR33.D) 1. Supplemental Tables Table S1. Representative HPLC-MS dates of CC1-36. Compd Structure HPLC MS CC1 :\CHEM32\1\DATA\SNAPSHOT.D ES-API, Pos, Scan Max: CC m/z S3

4 DAD1 A, Sig=254,16 Ref=off (YL\NIR 29.D) DAD1 B, Sig=35,16 Ref=off (YL\NIR 29.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR 29.D) DAD1 D, Sig=55,16 Ref=off (YL\NIR 29.D) DAD1 E, Sig=65,16 Ref=off (YL\NIR 29.D) CC14 HO O COOH N *MSD2 SPC, time=5.728 of D:\CHEM32\ CC15 N O COOH N *MSD2 SPC, time=6.343 of D:\CHEM32\1\DATA\SNAPSHOT.D ES-API, Pos, Scan S4

5 DAD1 A, Sig=254,16 Ref=off (YL\XIAOJI-H2O2-2.D) DAD1 B, Sig=35,16 Ref=off (YL\XIAOJI-H2O2-2.D) DAD1 C, Sig=5,16 Ref=off (YL\XIAOJI-H2O2-2.D) DAD1 D, Sig=55,16 Ref=off (YL\XIAOJI-H2O2-2.D) CC17 *MSD2 SPC, time=5.834 of D:\CHEM32\1\DATA CC m/z S5

6 DAD1 A, Sig=254,16 Ref=off (YL\NIR14.D) DAD1 B, Sig=35,16 Ref=off (YL\NIR14.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR14.D) DAD1 D, Sig=55,16 Ref=off (YL\NIR14.D) DAD1 E, Sig=76,16 Ref=off (YL\NIR14.D) DAD1 A, Sig=254,16 Ref=off (YL\NIR15-PTLC.D) DAD1 B, Sig=35,16 Ref=off (YL\NIR15-PTLC.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR15-PTLC.D) DAD1 D, Sig=55,16 Ref=off (YL\NIR15-PTLC.D) CC *MSD2 SPC, time=5.728 of D:\CHEM32\1\DATA\YL\NIR 29.D ES-API, Pos, Scan, Frag: CC *MSD2 SPC, time=6.47 of D:\CHEM32\1\DATA\YL\NIR15-PTLC.D ES-API, Pos, Scan, Frag S6

7 DAD1 B, Sig=35,16 Ref=off (YL\NIR34.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR34.D) DAD1 D, Sig=55,16 Ref=off (YL\NIR34.D) DAD1 E, Sig=65,16 Ref=off (YL\NIR34.D) DAD1 A, Sig=254,16 Ref=off (YL\CREV3-DTT-21.D) DAD1 B, Sig=35,16 Ref=off (YL\CREV3-DTT-21.D) DAD1 C, Sig=5,16 Ref=off (YL\CREV3-DTT-21.D) DAD1 D, Sig=55,16 Ref=off (YL\CREV3-DTT-21.D) DAD1 E, Sig=65,16 Ref=off (YL\CREV3-DTT-21.D) CC *MSD2 SPC, time=5.568 of D:\CHEM32\1\DATA CC35 *MSD2 SPC, time=8.644 of D:\CHEM32\1\DATA\YL\CREV3-DTT-2.D ES-API, Pos, Scan S7

8 1 DAD1 A, Sig=254,16 Ref=off (YL\NIR-S-XIA-HG.D) DAD1 B, Sig=35,16 Ref=off (YL\NIR-S-XIA-HG.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR-S-XIA-HG.D) DAD1 D, Sig=55,16 Ref=off (YL\NIR-S-XIA-HG.D) DAD1 E, Sig=65,16 Ref=off (YL\NIR-S-XIA-HG.D) CC *MSD2 SPC, time=5.969 of D:\CHEM32\1\D Table S2. Photo-physical properties of CC19-29 in EtOH. Compd n λ abs λ em Stokes Φ shift CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC S8

9 Table S3. Representative NIR fluorescent probes for Hg 2+ fluorophore platforms for fluorescence imaging. Probe λ ab /λ em (nm) R1 63/69 5 B2 37/65 SiR-H g IR / /69 Em: 78/83 CC35 67/7 2 Medium Tris HCl/CH 3 CN (1 mm, ph 7.4, 1 : 1, v/v) CH 3 CN HEPE S buffer solution (1 mm, ph 7.4, 1 : 1, v/v) HEPES buffer solution at ph 7.4 MeCN H 2 O (4:1, v/v; ph = 7.2) Methanol/water (8:2) PBS buffer (5 mm, ph 7.4, containing 2% CH 3 OH) Ratiometri c 3-fold fluorescen ce enhancem ent Fluorescen ce enhancem ent Detection limit 28 nm (S/N = 3) 15 nm (S/N = 3) Imaging application based on organic Ref. HeLa cells Analyst, 213, HeLa cells -- SH-SY5Y cells Quenching 5 µm SMMC-772 Ratiometri c (I 78 nm/i 83 nm ) 15-fold fluorescen ce enhancem ent --( nanomol ar level) 1 cells 1 nm Mouse 138, J. Mater. Chem., 212, 22, Chem. Commun., 212, 48, Chem. Lett. 215, doi:1.1246/cl Chem. Eur. J. model 21, 16, This Work S9

10 2. Supplemental Figures Figure S1. (A) Absorption spectra of compound 1 in water (ph 1) for -12 hours. (B) HPLC chromatograms of dye 1 in water (ph 1) for (a), 2 hours (b) and 12 hours (c). The samples were analyzed by HPLC with linear gradient elution (eluent A (H 2 O)/B(CH 3 CN)) 5/5, 1, /1; flow rate 1. ml/). The monitored wavelength was 55 nm. (C) The proposed fade mechanism of dye 1 in water and the mass spectra of 1-OH adduct. (D) Absorption spectra of compound CC17 in water (ph 1) for and 12 hours. (E) HPLC chromatograms of dye CC17 in water (ph 1) for (top), and 12 hours (bottom). S1

11 Normalized absorbance CC2 CC1 a) Normalized intensity CC2 CC1 b) Figure S2. Normalized absorption (a) and emission (b) spectra of CC1 ( ) and CC2 ( ). 12 a).2 b) 8 4 Absorbance Figure S3. Emission (a) and absorption (b) spectra of CC2 in ph 4 to 1 in water (5% DMF as co-solvent). Figure S4. DFT optimized structure and molecular orbital plots (LUMO and HOMO) of CC 2. S11

12 CC2 CC Figure S5. Emission spectra of CC3 ( ) and CC2 ( ). a) Normalized absorbance CC16 CC17 CC1 CC18 b) Normalized intensity CC16 CC17 CC1 CC Figure S6. Normalized absorption (a) and emission (b) spectra of CC16-18 and CC1. Figure S7. DFT optimized structure (including dihedral angle between chromenylium and indole moieties), molecular orbital plots (LUMO and HOMO) and HOMO/ LUMO energy gaps of CC19-21 and CC1. S12

13 COOH HO O CC14 N ph Figure S8. Fluorescence emission spectra of CC14 in buffer solution with ph changing from 1. to 9.. Inset: ph plot of normalized emission intensities at 655 nm for CC14. Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent Figure S9. Fluorescence emission spectra of CC3 in buffer solution with ph changing from 1. to 1.. Inset: ph plot of normalized emission intensities at 65 nm for CC3. Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent. S13

14 a) ph b) ph Figure S1. Fluorescence emission spectra of CC31 in buffer solution with ph changing from 1. to 5. (a) and 4. to 1. (b). Inset: ph plot of normalized emission intensities at 645 nm (a) or 656 nm (b). Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent a) ph b) Figure S11. Fluorescence emission spectra of CC32 in buffer solution with ph changing from 1. to 5. (a) and 4. to 1. (b). Inset: ph plot of normalized emission intensities at 637 nm (a) or 648 nm (b). Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent. S14

15 COOH COO COO HO O -H H + HO O -H H + O O N N N Figure S12. Estimated chemical equilibrium of CC31 in different ph. COOH HO O CC17 N.5 a) 25 b).4 2 Absorbance Figure S13. Absorption (a) and fluorescence emission (b) spectra of CC17 (λ ex = 67 nm) in buffer solution with ph changing from 4. to 9.. Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent..36 a) 4 b) Absorbance Figure S14. Absorption (a) and fluorescence emission (b) spectra of CC33 (λ ex = 68 nm) in buffer solution with ph changing from 4. to 9.. Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent. S15

16 .15 a) 6 b) Absorbance Figure S15. Absorption (a) and fluorescence emission (b) spectra of CC34 (λ ex = 67 nm) in buffer solution with ph changing from 4. to 9.. Measurements were made in 5 mm sodium phosphate buffer at various ph values in the presence of 5% DMF as a cosolvent..4.3 Absorbance Figure S16. Absorption spectra of compound CC33 (5µM) in presence of intracellular nucleophilic reagent H 2 S (5 equiv.) in water (ph 1) for to 3 hours. Figure S17. Representative fluorescence images of mice injected with CC33 in vivo. (a-b) PBS (ph 5) was injected in the peritoneal cavity of mouse, followed by injection of CC33 (6 µm). (c-d) PBS (ph 8) was injected into the peritoneal cavity of the mouse, followed by injection of CC33 (6 µm). The mice were imaged with two emission channels of Channel 1 (65 nm, a, c) and Channel 2 (72 nm, b, d). S16

17 .12 Absorbance Figure S18. Absorption spectra of NIR fluorescent probe CC35 in the presence of various concentrations of Hg 2+ ( 1 µm) in PBS buffer (5 mm, ph 7.4, containing 2% DMF). a) 1 DAD1 A, Sig=254,16 Ref=off (YL\CREV3-DTT-21.D) DAD1 B, Sig=35,16 Ref=off (YL\CREV3-DTT-21.D) 1 DAD1 C, Sig=5,16 Ref=off (YL\CREV3-DTT-21.D) 25 DAD1 D, Sig=55,16 Ref=off (YL\CREV3-DTT-21.D) 8.4 b) DAD1 E, Sig=65,16 Ref=off (YL\CREV3-DTT-21.D) 5 MSD1 TIC, MS File (D:\CHEM32\1\DATA\YL\CREV3-DTT-21.D) ES-API, Neg, Scan, Frag: 7 DAD1 A, Sig=254,16 Ref=off (YL\NIR-S-XIA-HG.D) MSD2 TIC, MS File (D:\CHEM32\1\DATA\YL\CREV3-DTT-21.D) ES-API, Pos, Scan, Frag: 7 DAD1 B, Sig=35,16 Ref=off (YL\NIR-S-XIA-HG.D) DAD1 C, Sig=5,16 Ref=off (YL\NIR-S-XIA-HG.D) 5.9 DAD1 D, Sig=55,16 Ref=off (YL\NIR-S-XIA-HG.D) DAD1 E, Sig=65,16 Ref=off (YL\NIR-S-XIA-HG.D) Figure S19. Reversed-phase HPLC with absorption (254, 35, 5, 55 and 65 nm) detection. a) Reversed-phase HPLC of 5 µm CC35. b) Reversed-phase HPLC of 5 µm CC35 in the presence of 1 µm Hg 2+ for 1. The ring-opened fluorescent dye CC36 was confirmed by Liquid chromatography mass spectrometry (LC-MS) (Figure S2). S17

18 *MSD2 SPC, time=5.969 of D:\CHEM32\1\DATA\YL\NIR-S-XIA-HG.D ES-API, Pos, Scan, Frag: Max: e Figure S2. The mass spectrometry of 5 µm CC35 in the presence of 1 µm Hg 2+ for 1. m/z Y = * X r = [Hg 2+ ], µμ Figure S21. The fluorescence response of CC35 (5 µm) to Hg 2+ (.1 µm - 4 µm) in PBS buffer (5 mm, ph 7.4, containing 2% DMF) with excitation at 67 nm. S18

19 [Hg 2+ ], nm Figure S22. The fluorescence response of CC35 (1 µm) to Hg 2+ (5 nm - 95 nm) in PBS buffer (5 mm, ph 7.4, containing 2% DMF) with excitation at 67 nm. 1 Cell Viability (%) a b c d e f g Concentration, µm Figure S23. Cytotoxic effect of CC35. MCF-7 cells were incubated with each concentration (a: ; b: 1 µm; c: 3 µm; d: 5 µm; e: 7 µm; f: 1 µm; g: 15 µm) of probes for 24 h. Cell viability was assayed with MTT test. Results are expressed as mean ± standard deviation of three independent experiments. 3. Synthesis and Characterization Synthesis of CC1-34: CC8, CC1, CC12, and CC18 were synthesized according to reported procedure (J. Am. Chem. Soc. 212, 134, 12; Biomaterials 215, 56, 1-9). Other compounds (CC1-34) were synthesized according to following procedure: A mixture of 4-(2-carboxyphenyl)-chromenylium derivatives (.22 mmol) and aldehyde (.24 mmol) was dissolved in ethanoyl ethanoate and then heated to 5 o C for 3 to 5 hours until starting materials disappearing. After cooling, water (2 S19

20 ml) was added to the mixture and the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /acetone = 1: 1) to afford the compound CC1-CC34 as a solid. CC1: Yield: 63%. 1 H NMR (4 MHz, d 6 -DMSO) δ 8.37 (s, 1H), (m, 3H), 7.85 (t, J = 7.4 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), (m, 3H), 7.25 (s, 1H), 6.99 (s, 1H), 6.78 (d, J = 9. Hz, 1H), 3.95 (s, 3H), 3.59 (q, J = 6.9 Hz, 4H), 2.83 (2H), 2.19 (2H), 1.79 (2H), 1.21 (t, J = 6.9 Hz, 6H). 13 C NMR (1 MHz, d 6 -DMSO) δ , , 15.54, 137.5, , 13.46, , , , , , 111.1, 1.12, 99.85, 96.6, 45.14, 33.85, 28.3, 25.4, 21.11, HRMS (m/z): [M] + calcd for [C 34 H 33 N 2 O 3 ] , found CC2: Yield: 57%. 1 H NMR (4 MHz, MeOD) δ 8.61 (s, 1H), 8.19 (d, J = 7.2 Hz, 1H), 8.12 (d, J = 8.5 Hz, 1H), 7.95 (s, 1H), (2H), 7.47 (d, J = 6.5 Hz, 1H), (2H), 7.23 (d, J = 4.3 Hz, 2H), 7.9 (s, 2H), 3.68 (q, J = 6.9 Hz, 4H), 2.92 (2H), 2.3 (2H), 1.92 (2H), 1.32 (t, J = 6.9 Hz, 6H). HRMS (m/z): [M] + calcd for [C 33 H 31 N 2 O 3 ] , found CC3: Yield: 53%. 1 H NMR (4 MHz, CDCl 3 ) δ 8.45 (d, J = 7.3 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.8 (d, J = 7.4 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.46 (s, 2H), (m, 2H), 7.24 (d, J = 7.7 Hz, 1H), 6.52 (d, J = 8.9 Hz, 1H), 6.47 (s, 1H), 6.38 (d, J = 8.7 Hz, 1H), 3.38 (q, J = 7. Hz, 4H), 2.69 (s, 3H), 2.9 (t, J = 6.4 Hz, 2H), (2H), 1.43 (2H), 1.19 (t, J = 7. Hz, 6H). HRMS (m/z): [M] + calcd for [C 35 H 33 N 2 O 4 ] , found CC4: Yield: 49%. 1 H NMR (4 MHz, MeOD) δ 8.38 (s, 1H), 8.26 (d, J = 7.7 Hz, 1H), 7.77 (t, J = 7.4 Hz, 1H), (m, 4H), (m, 4H), (m, 2H), 7.21 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 9.6 Hz, 1H), 7.7 (d, J = 9.5 Hz, 1H), 7.1 (s, 1H), 3.62 (q, J = 6.9 Hz, 4H), 2.63 (t, J = 5.6 Hz, 2H), 2.41 (t, J = 5.6 Hz, 2H), (2H), 1.26 (t, J = 6.9 Hz, 6H). 13 C NMR (1 MHz, MeOD) δ 163.2, , 158.3, 155.6, 142.1, 137.5, , , , , 13.83, , , , , , , , , , , 12.71, 12.26, , , , , 11.53, 95.9, 45.34, 29.35, 26.12, 22.33, 21.51, HRMS (m/z): [M] + calcd for [C 39 H 35 N 2 O 3 ] , found S2

21 CC5: Yield: 51%. 1 H NMR (4 MHz, CDCl 3 ) δ 8.4 (s, 1H), 8.22 (d, J = 5.9 Hz, 1H), 7.78 (s, 1H), 7.65 (t, J = 7. Hz, 1H), 7.53 (2H), 7.11 (d, J = 8.6 Hz, 1H), 7.6 (2H), 6.93 (d, J = 8.6 Hz, 1H), 6.8 (d, J = 8.9 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 4.2 (s, 3H), 3.59 (7H), 2.78 (2H), (2H), 1.75 (2H), 1.29 (t, J = 6.9 Hz, 6H). HRMS (m/z): [M] + calcd for [C 35 H 35 N 2 O 4 ] , found CC6: Yield: 55%. 1 H NMR (4 MHz, MeOD) δ 8.42 (s, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.8 (t, J = 7.4 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.3 (d, J = 7.5 Hz, 1H), 7.15 (q, J = 7.3 Hz, 3H), (m, 1H), 7.3 (d, J = 9.5 Hz, 1H), 3.65 (q, J = 6.9 Hz, 4H), 2.82 (t, J = 5.5 Hz, 2H), 2.61 (s, 3H), 2.44 (t, J = 6.4 Hz, 2H), (m, 2H), 1.28 (t, J = 6.9 Hz, 6H). HRMS (m/z): [M] + calcd for [C 34 H 33 N 2 O 3 ] , found CC7: Yield: 48%. 1 H NMR (4 MHz, MeOD) δ 8.22 (d, J = 7.6 Hz, 1H), 8.17 (s, 1H), (2H), 7.66 (d, J = 8.9 Hz, 2H), 7.24 (d, J = 7.4 Hz, 1H), 7.14 (s, 1H), (2H), 6.84 (d, J = 8.7 Hz, 2H), 3.66 (q, J = 7. Hz, 4H), 3.1 (s, 6H), 2.99 (t, J = 6.8 Hz, 2H), 2.42(t, J = 4.4 Hz, 2H), (m, 2H), 1.29 (t, J = 7. Hz, 6H). MS (ESI): m/z = 57.3 [M]+. HRMS (m/z): [M] + calcd for [C 33 H 35 N 2 O 3 ] , found CC9: Yield: 47%. 1 H NMR (4 MHz, CDCl 3 ) δ 8.23 (d, J = 7.8 Hz, 1H), 7.99 (s, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.11 (d, J = 7.4 Hz, 1H), 7.1 (d, J = 1.9 Hz, 1H), 6.97 (s, 1H), 6.86 (d, J = 9.4 Hz, 1H), 6.75 (dd, J = 9.4, 2. Hz, 1H), 6.7 (d, J = 3.9 Hz, 1H), (m, 1H), 3.96 (s, 3H), 3.58 (q, J = 7.1 Hz, 4H), 2.78 (t, J = 4.9 Hz, 2H), 2.2 (t, J = 7.6 Hz, 2H), (m, 2H), 1.27 (t, J = 7.1 Hz, 6H). MS (ESI): m/z = [M]+. HRMS (m/z): [M] + calcd for [C 3 H 31 N 2 O 3 ] , found CC11: Yield: 5%. 1 H NMR (4 MHz, MeOD) δ 8.69 (s, 1H), 8.3 (d, J = 7.7 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.12 (s, 1H), 7.82 (t, J = 7.2 Hz, 1H), (1H), 7.47 (d, J = 7.5 Hz, 1H), (m, 9H), 7.12 (d, J = 9.4 Hz, 1H), 7.3 (d, J = 9.4 Hz, 1H), 5.56 (s, 2H), 3.7 (q, J = 13.8, 6.9 Hz, 4H), 2.94 (t, J = 5.7 Hz, 2H), 2.44 (t, J = 4.8 Hz, 2H), (m, 2H), 1.33 (t, J = 6.9 Hz, 6H). HRMS (m/z): [M] + S21

22 calcd for [C 4 H 37 N 2 O 3 ] , found CC13: Yield: 63%. 1 H NMR (4 MHz, CDCl 3 ) δ 7.97 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.72 (1H), 7.7 (d, J = 7.6 Hz, 1H), 7.62 (s, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.53 (1H), 7.37 (t, J = 7.5 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.22 (1H), 6.82 (s, 1H), 6.61 (s, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 2.85 (t, 2H), 2.68 (t, 2H), (m, 2H). HRMS (m/z): [M] + calcd for [C 31 H 26 NO 4 ] , found CC14: Yield: 32%. 1 H NMR (4 MHz, MeOD) δ 8.47 (s, 1H), 8.15 (d, J = 6.5 Hz, 1H), 7.9 (d, J = 7.1 Hz, 1H), 7.83 (s, 1H), 7.59 (d, J = 7. Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.4 (d, J = 7.6 Hz, 1H), (2H), 7.6 (2H), 6.94 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 3.87 (s, 3H), 2.81 (s, 2H), (m, 2H), 1.84 (s, 2H). HRMS (m/z): [M] + calcd for [C 3 H 24 NO 4 ] , found CC15: Yield: 5%. 1 H NMR (4 MHz, CDCl 3 ) δ 8.26 (d, J = 4.4 Hz, 1H), 8.13 (s, 1H), 7.78 (d, J = 7.1 Hz, 1H), 7.62 (s, 1H), 7.52 (2H), 7.35 (3H), 6.97 (d, J = 6.7 Hz, 1H), 6.5 (s, 1H), 3.9 (s, 3H), 3.44 (d, J = 23.5 Hz, 4H), 3.7 (2H), 2.8 (2H), 2.6 (2H), 2.13 (8H). HRMS (m/z): [M] + calcd for [C 36 H 33 N 2 O 3 ] , found CC16: Yield: 56%. 1 H NMR (4 MHz, MeOD) δ 8.72 (d, J = 14.6 Hz, 1H), 7.99 (d, J = 7.3 Hz, 1H), (m, 3H), (m, 2H), 7.3 (d, J = 7.1 Hz, 1H), 7.7 (d, J = 7. Hz, 1H), 6.99 (s, 1H), 6.81 (d, J = 8.9 Hz, 1H), 6.75 (d, J = 8.9 Hz, 1H), 6.29 (d, J = 14.7 Hz, 1H), 3.84 (s, 3H), 3.68 (s, 3H), 2.6 (t, J = 5.6 Hz, 2H), 2.34 (t, J =6.7 Hz, 2H), (8H). MS (ESI): m/z = 518.2[M] +. HRMS (m/z): [M] + calcd for [C 34 H 32 NO 4 ] , found CC17: Yield: 44%. 1 H NMR (4 MHz, d 6 -DMSO) δ (m, 2H), 1.78 (m, 6H), (m, 1H), (m, 1H), 2.66 (1H), 3.85 (s, 1H), 6.5 (d, J = 14.2 Hz, 1H), 6.1 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 8.6, 2.2 Hz, 1H), 7. (d, J = 2. Hz, 1H), 7.32 (d, J = 7.2 Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 8. Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.8 (t, J = 7.2 Hz, 1H), 8.14 (d, J = 7.6 Hz, 1H), 8.66 (d, J = 14. Hz, 1H), 1.79 (s, 1H, -OH), 12.97(s, 1H, -COOH). 13 C NMR (1 MHz, d 6 -DMSO) 2.9, 23.63, 26.83, 27.68, 32.53, 5.23, 12.16, 13.81, , , , , , , , , S22

23 129.54, , 13.6, 13.96, , , , , , , , 16.86, , , MS (ESI) HRMS (m/z): [M] + calcd for [C 33 H 3 NO 4 ] , found CC19: Yield: 31%. 1 H NMR (4 MHz, CDCl 3 ) δ 8.35 (d, J = 13.7 Hz, 1H), 8.26 (d, J = 7.6 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.53 (t, J = 7.4 Hz, 1H), 7.33 (2H), 7.14 (t, J = 7.3 Hz, 1H), 7.8 (d, J = 7.4 Hz, 1H), 7.1 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 9.2 Hz, 1H), 6.65 (d, J = 9.2 Hz, 1H), 6.55 (s, 1H), 5.92 (d, J = 13.4 Hz, 1H), (7H), 2.72 (s, 2H), (2H), 2.4 (2H), 1.89 (2H), 1.75 (s, 6H), 1.26 (t, J = 6.6 Hz, 6H). MS (ESI): m/z = [M]+. CC2: Yield: 53%. 1 H NMR (4 MHz, MeOD) δ 8.24 (t, J = 1.2 Hz, 2H), 7.78 (t, J = 7.5 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.28 (2H), (2H), 6.78 (d, J = 9.1 Hz, 1H), 5.97 (d, J = 14. Hz, 1H), 3.48 (s, 3H), 3.43 (q, J = 7. Hz,4H), (t, J = 6.2 Hz, 2H), (2H), 1.84 (s, 3H), 1.76 (s, 3H), 1.21 (t, J = 7. Hz, 6H). 13 C NMR (1 MHz, CDCl 3 ) δ , 168.2, , , , , 148.6, , 14.88, , , , , 131.7, , , , , , , , , , 11.56, 1.6, 97.23, 49.2, 45., 29.69, 28.46, 28.11, MS (ESI): m/z = [M] +. CC21: Yield: 29%. 1 H NMR (4 MHz, MeOD) δ 8.49 (d, J = 14.1 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), (1H), 7.65 (d, J = 8.6, 1H), 7.5 (d, J = 7.2 Hz, 1H), (2H), 7.28 (2H), 6.98 (d, J = 9.1, 1H), 6.83 (, 2H), 6.31 (1H), 6.23 (d, J = 1. Hz, 1H), (7H), 1.75 (s, 6H), 1.28 (t, 6H). MS (ESI): m/z = 519.2[M] +. HRMS (m/z): [M] + calcd for [C 34 H 35 N 2 O 3 ] , found CC22: Yield: 7%. 1 H NMR (4 MHz, MeOD) δ 8.31 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.3 (d, J = 7.9 Hz, 1H), 7.92 (s, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.72 (t, J = 7.8 Hz, 2H), 7.54 (d, J = 8.3 Hz, 1H), (2H), (m, 2H), 7.8 (d, J = 9.3 Hz, 1H), 3.95 (s, 3H), 3.65 (q, J = 6.9 Hz, 4H), 3.7(t, J = 6.4 Hz, 2H), 2.88 (t, J = 6.4 Hz,2H), 1.3 (t, J = 6.9 Hz, 6H). MS (ESI): m/z = 53.2[M] +. HRMS (m/z): [M] + calcd for [C 33 H 31 N 2 O 3 ] , found CC23: Yield: 36%. 1 H NMR (4 MHz, MeOD) δ 8.44 (d, J = 15.4 Hz, 1H), 8.12 (d, S23

24 J = 7.3 Hz, 1H), 8.7 (d, J = 7.7 Hz, 1H), 7.98 (s, 1H), (2H), 7.62 (s, 1H), 7.53 (d, J = 8. Hz, 1H), (m, 2H), 7.32 (d, J = 7.4 Hz, 1H), 7.29 (d, J = 9.7 Hz, 1H), 7.19 (s, 1H), 7.12 (d, J = 15.7 Hz, 1H), 7.5 (s, 1H), 3.9 (s, 3H), 3.62 (q, J = 6.8 Hz, 4H), 1.29 (t, J = 6.8 Hz, 6H). HRMS (m/z): [M] + calcd for [C 31 H 29 N 2 O 3 ] , found CC24: Yield: 52%. 1 H NMR (4 MHz, MeOD) δ 8.23 (d, J = 7.6 Hz, 1H), 8.11 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), (3H), 7.36 (d, J = 7.5 Hz, 1H), 7.32 (d, J = 7.4 Hz, 1H), (3H), 7.4 (d, J = 9.5 Hz, 1H), 6.98 (s, 1H), 3.62 (q, J =7. Hz, 4H), 2.87 (t, J = 4.4 Hz, 2H), 2.79 (t, J = 4.4 Hz, 2H), 1.3 (t, J = 7. Hz, 6H). HRMS (m/z): [M] + calcd for [C 32 H 29 N 2 O 3 ] , found CC25: Yield: 4%. 1 H NMR (4 MHz, MeOD) δ 8.51 (d, J = 15.5 Hz, 1H), 8.17 (d, J = 7.2 Hz, 1H), 8.7 (d, J = 4.9 Hz, 1H), 8.2 (s, 1H), (2H), 7.48 (dd, J = 7.6, 3.6 Hz, 1H), 7.42 (d, J = 7.4 Hz, 1H), 7.3 (d, J = 4.4 Hz, 1H), 7.27 (s, 1H), 7.26 (s, 1H), 7.21 (s, 1H), 7.16 (d, J = 15.6 Hz, 1H), 7.7 (d, J = 9.2 Hz, 1H), 7.5 (s, 1H), 3.62 (q, J = 7. Hz, 4H), 1.28 (t, J = 7. Hz, 6H). HRMS (m/z): [M] + calcd for [C 3 H 27 N 2 O 3 ] , found CC26: Yield: 57%. 1 H NMR (4 MHz, MeOD) δ 8.23 (d, J = 7.7 Hz, 1H), 8.9 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 7.4 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 7.2 Hz, 2H), (3H), 7.47 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 7.7 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.17 (d, J = 9.6 Hz, 1H), 7.1 (d, J = 9.7 Hz, 1H), 7.1 (s, 1H), 3.62 (q, J = 7.1 Hz, 4H), 2.73 (t, J = 7.2 Hz, 2H), 2.6(t, J = 7.2 Hz, 2H), 1.27 (t, J = 7.1 Hz, 6H). 13 C NMR (1 MHz, MeOD) δ 17.57, , , , , , , , , , 13.98, 13.6, , , 129.2, , 129.3, , , , 123.4, , 12.42, 116.1, , , , 96.37, 45.25, 29.35, 27.89, MS (ESI): m/z = 565.2[M] +. CC27: Yield: 42%. 1 H NMR (4 MHz, MeOD) δ 8.28 (d, J = 15.4 Hz, 1H), (2H), 7.66 (s, 2H), 7.61 (5H), 7.45 (d, J = 7. Hz, 1H), 7.33 (d, J = 9.1 Hz, 2H), 7.28 (d, J = 5.2 Hz, 2H), 7.7 (3H), 6.69 (s, 1H), 3.58 (q, J = 6.9 Hz, 4H), 1.26 (t, J = 6.9 Hz, 6H). MS (ESI): m/z = 539.2[M] +. HRMS (m/z): [M] + calcd for [C 36 H 31 N 2 O 3 ] + S24

25 , found CC28: Yield: 52%. 1 H NMR (4 MHz, MeOD) δ 8.27 (d, J = 7.7 Hz, 1H), 8.1 (s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.81 (t, J = 7.3 Hz, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.35 (t, J = 7.7 Hz, 2H), 7.19 (d, J = 7.2 Hz, 1H), 7.16 (s, 1H), (m, 2H), 7.4 (d, J = 9. Hz, 1H), 3.63 (q, J = 6.9 Hz, 4H), 3.24 (t, J =9.8 Hz, 2H), 2.74 (t, J = 9.8 Hz, 2H), 2.69 (s, 3H), 1.28 (t, J = 6.9 Hz, 6H). 13 C NMR (1 MHz, MeOD) δ 171.8, , 158.8, , , , 137.1, , , , 131.9, , 129.2, , , , , 123., , 12.79, , , , , , 96.69, 45.7, 31.68, 29.35, 22.34, 13.4, MS (ESI): m/z = 53.2[M] +. CC29: Yield: 36%. 1 H NMR (4 MHz, MeOD) δ 8.6 (d, J = 14.8 Hz, 1H), 7.98 (s, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.54 (s, 2H), 7.33 (d, J = 9.1 Hz, 1H), (3H), 7.13 (d, J = 6.8 Hz, 1H), 6.92 (s, 1H), 6.87 (d, J = 9.4 Hz, 1H), 6.74 (d, J = 14.6 Hz, 1H), 6.67 (s, 1H), 3.55 (q, J = 6.3 Hz, 4H), 2.66 (s, 3H), 1.28 (t, J = 6.1 Hz, 6H). MS (ESI): m/z = [M] +. HRMS (m/z): [M] + calcd for [C 31 H 29 N 2 O 3 ] , found CC3: Yield: 28%. 1 H NMR (4 MHz, MeOD) δ 8.78 (s, 1H, NH), 8.27 (d, J = 7.5 Hz, 1H), 8.12 (s, 1H), 8.6 (d, J = 7.2 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.7 (d, J = 7.2 Hz, 1H), 7.62 (dd, J = 7.6 Hz, 1H), 7.52 (dd, J = 7.6 Hz, 1H), 7.35 (d, J = 4.8 Hz, 1H), 7.33 (s, 1H), 7.29 (d, J = 7.4 Hz, 1H), 7.6 (d, J = 9. Hz, 1H), 6.97 (d, J = 9.1 Hz, 1H), 2.93 (t, J = 3.6 Hz, 2H), 2.47 (t, J = 3.6 Hz, 2H), 2.32 (t, J = 3.6 Hz, 2H). MS (ESI): m/z = 448. [M] +. HRMS (m/z): [M] + calcd for [C 29 H 22 NO 4 ] , found CC31: Yield: 27%. 1 H NMR (4 MHz, MeOD) δ 8.3 (dd, J = 6.2, 3.6 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.87 (s, 1H), 7.67 (s, 1H), 7.58 (d, J = 3.6 Hz, 1H), 7.56 (d, J = 3.6 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.2 (dd, J = 7.6, 3.6 Hz, 1H), 7.15 (d, J = 9.3 Hz, 1H), 6.76 (d, J = 1.8 Hz, 1H), 6.68 (dd, J = 7.4, 1.6 Hz, 1H), 3.87 (s, 3H), (m, 2H), (m, 2H). HRMS (m/z): [M] + calcd for [C 29 H 22 NO 4 ] , found CC32: Yield: 21%. 1 H NMR (4 MHz, DMSO) δ 7.97 (d, J = 7.7 Hz, 1H), 7.84 (d, J S25

26 = 7.6 Hz, 1H), 7.8 (t, J = 1. Hz, 1H), (m, 2H), 7.51 (s, 1H), 7.45 (d, J = 8. Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H), (1H), 6.89 (s, 1H), 6.63 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), (2H), (2H). MS (ESI): m/z = 434.[M] +. HRMS (m/z): [M] + calcd for [C 28 H 2 NO 4 ] , found CC33: Yield: 25%. 1 H NMR (4 MHz, MeOD/CDCl 3 ) δ 8.24 (d, J = 14.3 Hz, 1H), 8.8 (d, J = 7.5 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.54 (d, J = 7.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 8.8 Hz, 1H), 7.3 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.3 Hz, 1H), 6.96 (s, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.69 (d, J = 8.3 Hz, 1H), 6.3 (d, J = 14.3 Hz, 1H), 3.67 (s, 3H), 2.83(t, J = 5.6 Hz, 2H), 2.7(t, J = 5.6 Hz, 2H), 1.72 (s, 6H). 13 C NMR (1 MHz, MeOD/CDCl 3 ) δ , 167.8, , , , , 141.3, 14.27, , , 131.2, 13.59, 129., , , , , , 12.97, , , , 12.84, 11.78, 49.81, 31.12, 27.22, 27.12, MS (ESI): m/z = 49.[M] +. HRMS (m/z): [M] + calcd for [C 32 H 28 NO 4 ] , found CC34: Yield: 23%. 1 H NMR (4 MHz, MeOD) δ 8.55 (t, J = 13.2 Hz, 1H), 7.89 (dd, J = 6.8, 3.6 Hz, 1H), (3H), 7.47 (t, J = 7.5 Hz, 1H), 7.4 (d, J = 7.9 Hz, 1H), (2H), 7.2 (d, J = 8.8 Hz, 1H), (m, 2H), 6.78 (dd, J = 8.8, 2. Hz, 1H), 6.48 (d, J = 13.1 Hz, 1H), 6.27 (d, J = 12.6 Hz, 1H), 3.71 (s, 3H), 1.76 (s, 6H). MS (ESI): m/z = 464.3[M]+. HRMS (m/z): [M] + calcd for [C 3 H 26 NO 4 ] , found S26

27 4. Supplemental Spectra 1 H NMR of CC 1 in d 6 -DMSO. 1 C NMR of CC 1 in d 6 -DMSO. S27

28 1 H NMR of CC 4 in MeOD. 13 C NMR of CC 4 in MeOD. S28

29 1 H NMR of CC 5 in CDCl 3. 1 H NMR of CC 6 in MeOD S29

30 1 H NMR of CC 15 in CDCl 3. 1 H NMR of CC17 in d 6 -DMSO. S3

31 1 C NMR of CC17 in d 6 -DMSO. 1 H NMR of CC19 in CDCl 3. S31

32 1 H NMR of CC2 in MeOD. 13 C NMR of CC2 in CDCl 3. S32

33 1 H NMR of CC26 in MeOD. 13 C NMR of CC26 in MeOD. S33

34 1 H NMR of CC33 in MeOD. 33 C NMR of CC33 in MeOD/CD 3 Cl. S34

35 1 H NMR of CC34 in MeOD. 1 H NMR of CC35 in CDCl 3. S35

36 13 C NMR of CC35 in CDCl 3. S36