Benzene Derivatives Bearing Electron-Withdrawing Substituents

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is © The Royal Society of Chemistry 2016

Highly efficient anion transport mediated by 1,3-bis(benzimidazol-2- yl)benzene derivatives bearing electron-withdrawing substituents

Chen-Chen Peng, Meng-Jia Zhang, Xiao-Xiao Sun, Xiong-Jie Cai, Yun Chen and Wen-Hua

Chen*

Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences,

Southern Medical University, Guangzhou 510515, P. R. China

Supporting Information

Experimental

Generals. 1H NMR spectra were recorded using a Bruker Avance AV 400 spectrometer and

the solvents as internal standards. LR and HR-ESI MS spectra were measured on Waters

UPLC/Quattro Premier XE and Agilent 6210 LC/MSD TOF mass spectrometers,

respectively. Silica gel 60 Å (reagent pure, Qingdao Haiyang Chemical Co. Ltd) was used for

column chromatography. Analytical thin-layer chromatography (TLC) was performed on

silica gel plates 60 GF254. Detection on TLC was made by use of iodine and UV (254 or 365

nm). UV-Vis and Fluorescence spectra were measured on a Perkin Elmer Lambda 25

spectrophotometer and LS55 spectrofluorimeter, respectively.

EYPC, pyranine and lucigenin were purchased from Sigma Chemical Co. (St Louis, USA),

whereas calcein was from J&K Chemical Co. (Beijing, China). All the other chemicals and

reagents were obtained from commercial sources and used without further purification. Buffer

solutions were prepared in triply distilled deionized water.

S1 Chemistry

4 N (ii) NH N N 1 N N 4 2 (i) 1 3 R (3-, R1 = H) 2 H COOH + NH N N HOOC 3 R1 NH 2-, R1 = H (o-Bimbe) 1 2 2 1 Me bimbe R1 4-, R = H (p-Bimbe) 2 3-, R1 = Me (Me-Bimbe) 1 3-, R = F (F-Bimbe) N N 1 (iv) 3-, R = H (m-Bimbe) NH HN COOH NH2 (i) N Imbe + O N NO -Bimbe NO NH N 2 2 2 2 H

R1 = Cl (Cl-Bimbe) NH2 (iii) N N 1 + NH HN R = Br (Br-Bimbe) OHC CHO R1 NH 1 2 R = CF3 (CF3-Bimbe) R1 R1

Scheme S1. Synthesis of 1,3-Bis(benzimidazol-2-yl)benzene and related derivatives used in this study. Reagents and conditions: (i) concentrated H3PO4, heat; (ii) MeI, NaH, TMU; (iii)

o MeOH, room temperature; (iv) HNO3/H2SO4, 0 C then room temperature. m-Bimbe. This compound was prepared according to reported procedures. 1, 2 Specifically, a solution of isophthalic acid (200 mg, 1.72 mmol) and o-phenylenediamine (410 mg, 3.79 mmol) in concentrated phosphoric acid (2.4 mL) was heated at 220 ºC for 4 h. Then water (20 mL) was added and the resulting mixture was stirred vigorously and filtered. The obtained residue was suspended in hot aqueous sodium carbonate solution (10 %, 10 mL), stirred vigorously and filtered. The obtained residue was dissolved in methanol (10 mL) and filtered.

The filtrate was concentrated under reduced pressure. Purification was achieved by chromatography on a silica-gel column, eluted with a mixture of CHCl3/CH3OH (100/1, v/v)

1 to give m-Bimbe (196 mg, 37%) having H NMR (DMSO-d6, 400 MHz) δ 13.11 (s, 2H), 9.06

(t, J = 1.6 Hz, 1H), 8.27 (dd, J = 8.0 Hz and 2 Hz, 2H), 7.76-7.71 (m, 3H), 7.57 (d, J = 8.0 Hz,

– 2H) 7.28-7.21 (m, 4H); negative ESI-MS: m/z 310.03 ([M–H] ) and HRMS for C20H14N4

([M+H]+) Calcd: 311.1291; Found: 311.1319. The 1H NMR data were in agreement with the reported ones.1

S2 o-Bimbe. This compound was prepared according to reported procedures. 3, 4 Specifically, a solution of phthalic acid (200 mg, 1.20 mmol) and o-phenylenediamine (260 mg, 2.40 mmol) in concentrated phosphoric acid (10 mL) was heated at 200 oC for 6 h. Then water (20 mL) was added. The resulting mixture was stirred vigorously and filtered. The obtained residue was suspended in hot aqueous sodium carbonate solution (10 %, 20 mL), stirred vigorously and filtered. The obtained residue was dried and recrystallized from DMF to give o-Bimbe

1 (152 mg, 41%) having H NMR (DMSO-d6, 400 MHz) δ 13.28 (s, 2H), 8.10 (dd, J = 3.6 Hz and 3.2 Hz, 2H), 7.72 (dd, J = 3.2 Hz and 3.6 Hz, 2H), 7.68 (br, 2H), 7.55 (br, 2H), 7.22-7.21

+ + (m, 4H); ESI-MS: m/z 311.6 ([M+H] ) and HRMS for C20H14N4 ([M+H] ) Calcd: 311.1291;

Found: 311.1311. The 1H NMR data were in agreement with the reported ones.3 p-Bimbe. This compound was prepared according to reported procedures. 2 Specifically, a solution of terephthalic acid (200 mg, 1.20 mmol) and o-phenylenediamine (260 mg, 2.40 mmol) in concentrated phosphoric acid (10 mL) was heated at 200 oC for 6 h. Then water (20 mL) was added. The resulting mixture was stirred vigorously and filtered. The obtained residue was suspended in hot aqueous sodium carbonate solution (10 %, 20 mL), stirred vigorously and filtered. The obtained residue was dried and recrystallized from DMF to give

1 p-Bimbe (74 mg, 20%) having H NMR (DMSO-d6, 400 MHz) δ 13.04 (s, 2H), 8.36 (s, 4H),

7.71 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.28-7.21 (m, 4H); ESI-MS: m/z 311.6

+ + ([M+H] ) and HRMS for C20H14N4 ([M+H] ) Calcd: 311.1291; Found: 311.1324.

Imbe. This compound was prepared according to reported procedures. 5 Specifically, a solution of benzoic acid (200 mg, 1.64 mmol) and o-phenylenediamine (177 mg, 1.64 mmol) in concentrated phosphoric acid (10 mL) was heated at 150 oC for 16 h. Then water (20 mL) was added. The resulting mixture was stirred vigorously and filtered. The obtained residue

1 was washed thoroughly with water to give Imbe (82 mg, 26%) having H NMR (DMSO-d6,

400 MHz) δ 8.19 (d, J = 7.6 Hz, 2H), 7.61 (dd, J = 5.6 Hz and 3.2 Hz, 2H), 7.57 (d, J = 7.6

S3 Hz, 2H), 7.51 (d, J = 7.2 Hz, 1H), 7.22 (dd, J = 3.2 Hz and 6.0 Hz, 2H) and ESI-MS: m/z

195.3 ([M+H]+). The 1H NMR data were in agreement with the reported ones. 5

6 Me2bimbe. Similar procedures as described in literature were taken. Specifically, NaH (oil dispersion 60%, 14 mg, 0.35 mmol) was washed with petroleum ether (3 mL×3) and then suspended in dry tetramethylurea (TMU, 0.4 mL). This suspension was added to a solution of m-Bimbe (50 mg, 0.16 mmol) in TMU (0.4 mL) at 0 ºC. The resulting mixture was stirred for

30 min and then warmed to room temperature and stirred for another 1h. After the mixture was cooled to 0 ºC, MeI (22 μL) was added. The resulting mixture was stirred for 10 min and heated at 40 ºC overnight. Water (5 mL) was added to quench the reaction and the resulting mixture was concentrated under reduced pressure. The obtained residue was purified by chromatography on a silica-gel column, eluted with a mixture of CHCl3/MeOH (160/1, v/v) to

1 give Me2bimbe (29 mg, 53%) having H NMR (CDCl3, 400 MHz) δ 8.15 (s, 1H), 7.93 (dd, J

= 1.6 Hz and 7.6 Hz, 2H), 7.85-7.83 (m, 2H), 7.71 (t, J = 7.6 Hz, 1H), 7.42 (dd, J = 5.6 Hz and 2.4 Hz, 2H) 7.37-7.31 (m, 4H) 3.91 (s, 6H); ESI-MS: m/z 339.37 ([M+H]+) and HRMS

+ 1 for C22H18N4 ([M+H] ) Calcd: 339.1604; Found: 339.1636. The H NMR data were in agreement with the reported ones. 7

Me-Bimbe. 8 A solution of isophthalic acid (150 mg, 0.90 mmol) and 4-methyl-o-phenylene diamine (221 mg, 1.81 mmol) in concentrated phosphoric acid (12 mL) was heated at 180 ºC for 6 h. The reaction mixture was poured into iced water (20 mL) and stirred vigorously. The resulting precipitates were collected by filtration, suspended in hot aqueous sodium carbonate solution (10 %, 20 mL), stirred vigorously and filtered. The obtained residue was purified by chromatography on a silica-gel column, eluted with a mixture of CHCl3/CH3OH (100/1, v/v)

1 to afford Me-Bimbe (220 mg, 72%) having H NMR (CD3OD, 400 MHz) δ 8.70 (s, 1H), 8.18

(d, J = 7.6 Hz, 2H), 7.73 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.45 (s, 2H), 7.15 (d, J

S4 + + = 8.0 Hz, 2H), 2.51 (s, 6H); ESI-MS: m/z 339.6 ([M+H] ) and HRMS for C22H18N4 ([M+H] )

Calcd: 339.1604; Found: 339.1639.

F-Bimbe. Similar procedures as described in literature were taken. 2 Specifically, a solution of isophthalic acid (100 mg, 0.60 mmol) and 4-fluoro-o-phenylenediamine (152 mg, 1.21 mmol) in concentrated phosphoric acid (10 mL) was heated at 200 ºC for 6 h. The reaction mixture was poured into iced water (20 mL) and stirred vigorously. The resulting precipitates were collected by filtration, suspended in hot aqueous sodium carbonate solution (10 %, 20 mL), stirred vigorously and filtered. The obtained residue was purified by chromatography on a silica-gel column, eluted with a mixture of CHCl3/CH3OH (70/1, v/v) to give F-Bimbe (13

1 mg, 6%) having H NMR (DMSO-d6, 400 MHz) δ 13.28 (s, 2H), 9.04 (s, 1H), 8.27 (d, J = 7.2

Hz, 2H), 7.78-7.72 (m, 1H), 7.72 (br, 1H), 7.57 (br, 1H), 7.51 (br, 1H), 7.37 (br, 1H), 7.11 (br,

13 2H); C NMR (DMSO-d6, 100 MHz) δ 131.2, 130.2, 128.1, 125.2; ESI-MS: m/z 347.6

+ + + ([M+H] ), 369.5 ([M+Na] ) and HRMS for C20H12F2N4 ([M+H] ) Calcd: 347.11028; Found:

347.11003.

Cl-Bimbe. 8 Similar procedures as described in literature were taken. 9 Specifically, a solution of benzene-1,3-dicarbaldehyde (100 mg, 0.75 mmol) and 4-chloro-o-phenylenediamine (213 mg, 1.49 mmol) in methanol (10 mL) was stirred at room temperature for 2 d. The reaction mixture was evaporated under reduced pressure. The obtained residue was purified by chromatography on a silica-gel column, eluted with a mixture of CHCl3/CH3OH (110/1, v/v)

1 to give Cl-Bimbe (26 mg, 9%) having H NMR (CD3OD, 400 MHz) δ 8.76 (s, 1H), 8.20 (d, J

= 8.0 Hz, 2H), 7.76 (t, J = 8.0 Hz, 1H), 7.64 (br, 4H), 7.30 (d, J = 8.8 Hz, 2H); ESI-MS: m/z

+ + 379.5 ([M+H] ) and HRMS for C20H12Cl2N4 ([M+H] ) Calcd: 379.0512; Found: 379.0512.

Br-Bimbe. Similar procedures as described in literature were taken. 9 Specifically, a solution of benzene-1,3-dicarbaldehyde (50 mg, 0.37 mmol) and 4-bromo-o-phenylenediamine (140

S5 mg, 0.75 mmol) in methanol (10 mL) was stirred at room temperature for 2 d. The reaction mixture was evaporated under reduced pressure. The obtained residue was purified by chromatography on a silica-gel column, eluted with a mixture of CHCl3/CH3OH (160/1, v/v)

1 to give Br-Bimbe (13 mg, 8%) having H NMR (DMSO-d6, 400 MHz) δ 13.37 (s, 1H), δ

13.33 (s, 1H), 9.05 (s, 1H), 8.29 (d, J = 7.6 Hz, 2H), 7.92 (s, 1H), 7.78 (t, J = 7.6 Hz, 1H),

7.74 (br, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 7.2 Hz, 2H); 13C

NMR (DMSO-d6, 100 MHz) δ 152.4, 152.1, 145.7, 143.3, 136.8, 134.7, 130.9, 130.2, 128.4,

125.9, 125.4, 125.3, 121.7, 121.1, 115.4, 114.5, 113.8.; negative ESI-MS: m/z 467.4 ([M–H]–)

+ and HRMS for C20H12Br2N4 ([M+H] ) Calcd: 468.94821; Found: 468.95052.

9,10 CF3-Bimbe. Similar procedures as described in literature were taken. Specifically, a solution of benzene-1,3-dicarbaldehyde (50 mg, 0.37 mmol), 4-trifluoromethylbenzene-1,2- diamine (132 mg, 0.75mmol) and sodium metabisulfite (143 mg, 0.75 mmol) in DMF (6 mL) was refluxed for 6 h. After cooled to room temperature, the reaction mixture was poured into cold water (10 mL). The resulting precipitates were collected and recrystallized from

1 methanol to give CF3-Bimbe (8 mg, 5%) having H NMR (CD3OD, 400 MHz) δ 8.87 (s, 1H),

8.25 (d, J = 7.6 Hz, 2H), 7.96 (s, 2H), 7.90 (d, J = 8.0 Hz, 2H), 7.78 (t, J = 8.4 Hz, 2H), 7.58

13 (d, J = 8.8 Hz, 2H); C NMR (DMSO-d6, 100 MHz) δ 153.7, 130.8, 130.3, 128.9, 126.8,

125.8, 124.1, 123.5, 123.2, 119.5; ESI-MS: m/z 447.5 ([M+H]+), 469.4 ([M+Na]+) and HRMS

+ for C22H12F6N4 ([M+H] ) Calcd: 447.10389; Found: 447.10471.

2 NO2-Bimbe. Similar procedures as described in literature were taken. Specifically, to a solution of m-Bimbe (200 mg, 0.64 mmol) in concentrated sulfuric acid (10 mL) was added fuming nitric acid (60 μL) at 0 oC. The resulting solution was warmed to room temperature and stirred for 4 h. Then, the reaction mixture was poured into iced water (20 mL). The formed precipitates were collected by filtration and washed thoroughly subsequently with aqueous sodium carbonate solution (pH 9) and water to give NO2-Bimbe (244 mg, 94%)

S6 1 having H NMR (DMSO-d6, 400 MHz) δ 13.82 (br, 2H), 9.10 (s, 1H), 8.49 (s, 2H), 8.34 (d, J

= 7.6 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.83-7.78 (m, 3H); negative ESI-MS: m/z 399.4 ([M–

– + H] ) and HRMS for C20H12N6O4 ([M+H] ) Calcd: 401.0993; Found: 401.0993.

Preparation of EYPC vesicles

Vesicles for pH discharge were prepared according to the reported protocols. 11 Specifically,

EYPC (20 mg) was dissolved in chloroform (0.5 mL) in a pyrex test tube. While rotating the tube, the organic solvent was removed under a stream of nitrogen, resulting in a thin lipid film. The last traces of solvent were then removed under reduced pressure (room temperature,

> 4 h). To the dried lipid film was added 1.0 mL of a 0.1 mM pyranine solution in 25 mM

HEPES buffer (50 mM NaCl, pH 7.0), and the mixture was vortexed for 1 min. The dispersion was then incubated at room temperature for 5 min, followed by another 1 min of vortexing and 20 min of incubation at ambient temperature. The sample was subjected to eight freeze/thaw cycles (77 K/323 K), followed by extrusion through a 100 nm Nuclepore membrane (15 times). After extrusion, the dispersion was incubated at room temperature for 1 h. The non-entrapped pyranine was removed via gel filtration (Sephadex G-25, eluted with 25 mM HEPES buffer (50 mM NaCl, pH 7.0)).

Vesicles for ion selectivity experiments were prepared in a similar fashion, except that they were formed in a 0.1 mM pyranine solution in 25 mM HEPES buffer (pH 7.0) containing 50 mM sodium salt (= NaNO3, NaCl, NaBr or NaI), or chloride salt (= LiCl, NaCl, KCl, RbCl or

CsCl), and that 25 mM HEPES buffer (pH 7.0, 50 mM of the corresponding salt) was used to elute the Sephadex G-25 column to remove the non-entrapped pyranine.

Vesicles for mobile carrier mechanism study were prepared in a similar fashion, except that

POPC and POPC-cholesterol (7/3) were used instead of EYPC.

S7 Vesicles for chloride influx experiments were prepared in a similar fashion, except that they were formed in a 1 mM lucigenin solution in 25 mM phosphate buffer (225 mM NaNO3, pH

7.0); and that 25 mM phosphate buffer (225 mM NaNO3, pH 7.0) was used to elute the

Sephadex G-25 column to remove the non-entrapped lucigenin.

Vesicles for chloride influx inhibition experiments were prepared in a similar way, except that they were formed in 1 mM lucigenin in 25 mM phosphate buffer (225 mM Na2SO4, pH 7.0); and that 25 mM phosphate buffer (225 mM NaNO3 or Na2SO4, pH 7.0) was used to elute the

Sephadex G-25 column to remove the non-entrapped lucigenin.

Vesicles for calcein leakage experiments were prepared in a similar way, except that they were formed in 50 mM calcein in 25 mM HEPES buffer (50 mM NaCl, pH 7.0); and that 25 mM HEPES buffer (50 mM NaCl, pH 7.0) was used to elute the Sephadex G-25 column to remove the non-entrapped calcein.

Experimental procedures for pH discharge experiments 12

To 1.68 mL of 25 mM HEPES buffer (50 mM NaCl, pH 8.0) was added the vesicle dispersion

(0.3 mL), followed by the addition of each of those compounds of varying concentrations in

DMSO (20 μL). The appearance of fluorescent intensity (FI) was monitored as a function of time (ex 460 nm, em 510 nm, ex/em 3.0 nm/3.0 nm). After 20 min, 5 wt% aqueous Triton X-

100 (50 μL) was added. The ion-transporting ability was estimated from the relative FI that was calculated using the following equation: relative FI (%) = (I-I0)/(Itotal-I0)×100, where I0, I and Itotal represent the fluorescence intensities of the dispersion at the initial time, after a period of time and after addition of 5 wt% aqueous Triton X-100, respectively.

The experiments for ion selectivity study were conducted in a similar fashion, except that 25 mM HEPES buffer (pH 8.0, 50 mM of the corresponding salt under study) was used.

S8 The experiments for mobile carrier mechanism study were conducted in a similar fashion, except that POPC and POPC-cholesterol (7/3)-derived vesicles were used.

Experimental procedures for chloride influx experiments 13

To a mixture that was made from the EYPC vesicle dispersion (0.3 mL) and 225 mM NaNO3 in 25 mM phosphate buffer (pH 7.0, 1.48 mL) were added a methanol or DMF solution of each of those compounds of varying concentrations (0.02 mL) and 250 mM NaCl in 25 mM phosphate buffer (225 mM NaNO3, pH 7.0, 0.2 mL). The appearance of FI was then monitored as a function of time (ex 455 nm, em 506 nm, ex/em 3 nm/3 nm). After 20 min, 50

μL of 5 wt% aqueous Triton X-100 was added. The ability of chloride transport was estimated by the relative FI that was calculated using the following formular: relative FI (%) = (Itotal–

I)/(Itotal–I0)×100, where I0, I and Itotal represent the fluorescence intensities of the dispersion at the initial time, after a period of time and after addition of 5 wt% aqueous Triton X-100, respectively.

The chloride influx inhibition experiments were conducted in a similar fashion, except that the vesicles having internal Na2SO4 (250 mM) were used.

Experimental procedures for calcein leakage assay 14

To a mixture that was made from the EYPC vesicle dispersion (0.35 mL) and 25 mM HEPES buffer (50 mM NaCl, pH 7.0, 1.63 mL) was added a DMSO solution (20 μL) of CF3-Bimbe

-2 (0.25 mol%) or NO2-Bimbe (3.13×10 mol%). The appearance of fluorescence at 520 nm was monitored with time (ex 490 nm, ex/em 3 nm/3 nm). At the end of the experiment (20 min), aqueous Triton X-100 (5 wt%, 50 μL) was added to lyse the vesicles. The leakage of calcein was characterized by the relative FI that was calculated using the following formular: relative

FI (%) = (Itotal–I)/(Itotal–I0)×100, where I0, I and Itotal represent the fluorescence intensities of

S9 the dispersion at the initial time, after a period of time and after addition of 5 wt% aqueous

Triton X-100, respectively.

Spectrophotometric titrations 15, 16

Spectrophotometric titrations were performed by keeping the concentrations of each compound constant, while gradually increasing the concentration of tetrabutylammonum chloride. Typically, to a solution of m-Bimbe (1.5×10-5 M) in a mixture of acetonitrile and water (9/1, v/v) were added aliquots of m-Bimbe (1.5×10-5 M) and tetrabutylammonum chloride (1.0×10-3 M) in the same solvent. The mixing was achieved by stirring for 2 min.

Then, the corresponding absorption spectra were recorded. This operation was repeated until saturation in the absorbance was observed. The spectrophotometric titrations of the other compounds with tetrabutylammonum chloride were conducted in a similar way. The association constants (Ka’s) were derived from nonlinear least-squares fit of the experimental data according to a 1 : 1 binding model, A = A0 + ((A∞ –A0)/2[C]0){([N]0+[C]0+1/Ka) –

2 0.5 (([N]0+[C]0+1/Ka) – 4[N]0[C]0) }, wherein [N]0 and [C]0 are the initial analytical concentrations of tetrabutylammonum chloride and each compound, respectively; A and A0 represent the absorbance of the sample and compound alone, respectively, and A∞ is the absorbance when compound is totally bound.

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S11 1 Fig. S1. H NMR (DMSO-d6, 400 MHz) of m-Bimbe.

Fig. S2. HR MS of m-Bimbe.

S12 1 Fig. S3. H NMR (DMSO-d6, 400 MHz) of o-Bimbe.

Fig. S4. HR MS of o-Bimbe.

S13 1 Fig. S5. H NMR (DMSO-d6, 400 MHz) of p-Bimbe.

Fig. S6. HR MS of p-Bimbe.

S14 1 Fig. S7. H NMR (DMSO-d6, 400 MHz) of Imbe.

1 Fig. S8. H NMR (CDCl3, 400 MHz) of Me2bimbe.

S15 Fig. S9. HR MS of Me2bimbe.

1 Fig. S10. H NMR (CD3OD, 400 MHz) of Me-Bimbe.

S16 Fig. S11. HR MS of Me-bimbe.

1 Fig. S12. H NMR (DMSO-d6, 400 MHz) of F-Bimbe.

S17 13 Fig. S13. C NMR (DMSO-d6, 100 MHz) of F-Bimbe.

Fig. S14. HR MS of F-Bimbe.

S18 1 Fig. S15. H NMR (CD3OD, 400 MHz) of Cl-Bimbe.

Fig. S16. HR MS of Cl-bimbe.

S19 1 Fig. S17. H NMR (DMSO-d6, 400 MHz) of Br-Bimbe.

13 Fig. S18. C NMR (DMSO-d6, 100 MHz) of Br-Bimbe.

S20 Fig. S19. HR MS of Br-bimbe.

1 Fig. S20. H NMR (DMSO-d6, 400 MHz) of CF3-Bimbe.

S21 13 Fig. S21. C NMR (DMSO-d6, 100 MHz) of CF3-Bimbe.

Fig. S22. HR MS of CF3-Bimbe.

S22 1 Fig. S23. H NMR (DMSO-d6, 400 MHz) of NO2-Bimbe.

Fig. S24. HR MS of NO2-Bimbe.

S23 (a) (b)

Fig. S25. Discharge of a pH gradient by 3.0 mol% of m-Bimbe across EYPC-based liposomal membranes. (a) Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM MCl, pH 7.0); and external vesicles: 25 mM HEPES (50 mM MCl, pH 8.0) (M = Li, Na, K, Rb and

Cs). The experiment that was conducted in NaCl media and in the absence of m-Bimbe was used as a control. (b) Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM

NaX, pH 7.0) and external vesicles: 25 mM HEPES (50 mM NaX, pH 8.0) (X = NO3, Cl, Br and I). Each profile represents the real increment in the presence of m-Bimbe.

(a) (b)

Fig. S26. Chloride influx into EYPC vesicles promoted by 5 mol% of m-Bimbe. (a)

Intravesicular conditions: 1 mM lucigenin in 25 mM phosphate buffer (225 mM NaNO3 or

Na2SO4, pH 7.0); and extravesicular conditions: 25 mM NaCl in 25 mM phosphate buffer

(225 mM NaNO3, pH 7.0). (b) Intravesicular conditions: 1 mM lucigenin in 25 mM phosphate

S24 buffer (225 mM Na2SO4, pH 7.0); and extravesicular conditions: 25 mM NaCl in 25 mM phosphate buffer (225 mM NaNO3 or Na2SO4, pH 7.0). Ex 455 nm; em 506 nm.

Fig. S27. Discharge of a pH gradient promoted by 5 mol% of m-Bimbe across POPC and

POPC-cholesterol (7/3)-based liposomal membranes, respectively. Measuring conditions for internal vesicles: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0); and for external vesicles: 25 mM HEPES (50 mM NaCl, pH 8.0). Ex 460 nm; em 510 nm.

S25 (a) (b)

(e) (f)

Fig. S28. Chloride influx into EYPC vesicles promoted by (a) Me-Bimbe, (b) F-Bimbe, (c)

Cl-Bimbe, (d) Br-Bimbe, (e) CF3-Bimbe and (f) NO2-Bimbe of varying concentrations.

Intravesicular conditions: 1 mM lucigenin in 25 mM phosphate buffer (225 mM NaNO3, pH

7.0); Extravesicular conditions: 25 mM NaCl in 25 mM phosphate buffer (225 mM NaNO3, pH 7.0). Ex 455 nm; em 506 nm.

S26 (a) (b)

Fig. S29. Discharge of a pH gradient across EYPC-based liposomal membranes in the presence of (a) Me-Bimbe, (b) F-Bimbe, (c) Cl-Bimbe and (d) Br-Bimbe of varying concentrations. Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0); Extravesicular conditions: 25 mM HEPES (50 mM NaCl, pH 8.0). Ex 460 nm; em

510 nm.

S27 (a) (b)

(e) (f)

(g)

Fig. S30. Hill plots of the initial rate constants (kin’s) versus the mol% concentrations for (a) m-Bimbe, (b) Me-Bimbe, (c) F-Bimbe, (d) Cl-Bimbe, (e) Br-Bimbe, (f) CF3-Bimbe and (g)

S28 NO2-Bimbe in EYPC liposomes. The solid lines are nonlinear least-squares fit of the data

n n n according to the Eq. kin = k0 + kmax×[compound] /([compound] + [EC50] ).

‒1 Table S1. Initial rate constants (kin’s, min ) of pH discharge across EYPC vesicles containing

a,b m-Bimbe, Me-Bimbe, F-Bimbe, Cl-Bimbe, Br-Bimbe, CF3-Bimbe and NO2-Bimbe

Compound mol% kin Compound mol% kin m-Bimbe 1 (4.79±1.22)×10-2 Me-Bimbe 1 (2.25±0.35)×10-2 2 (8.33±1.55)×10-2 2 (5.99±0.43)×10-2 3 (1.30±0.09)×10-1 3 (1.52±0.07)×10-1 4 (2.35±0.21)×10-1 4 (7.55±0.35)×10-1 5 (2.59±0.04)×10-1 5 (9.23±0.53)×10-1 F-Bimbe 0.125 (9.44±0.39)×10-2 Cl-Bimbe 0.03125 (2.18±0.36)×10-2 0.250 (1.13±0.07)×10-1 0.06250 (3.21±0.23)×10-2 0.500 (1.68±0.04)×10-1 0.12500 (7.37±0.18)×10-2 1.000 (2.52±0.05)×10-1 0.25000 (1.01±0.04)×10-1 2.000 (3.00±0.04)×10-1 0.50000 (1.46±0.04)×10-1 3.000 (3.09±0.05)×10-1 1.00000 (1.88±0.05)×10-1

-2 -2 Br-Bimbe 0.03125 (1.35±0.42)×10 CF3-Bimbe 0.00049 (1.25±0.23)×10 0.06250 (2.99±0.36)×10-2 0.00195 (1.76±0.20)×10-2 0.12500 (5.68±0.45)×10-2 0.00781 (2.66±0.30)×10-2 0.25000 (8.21±0.31)×10-2 0.01563 (4.33±0.40)×10-2 0.50000 (1.21±0.04)×10-1 0.03125 (5.78±0.46)×10-2 1.00000 (1.35±0.04)×10-1 0.06250 (8.98±0.34)×10-2 0.12500 (1.51±0.04)×10-1 0.25000 (1.75±0.06)×10-1 -3 NO2-Bimbe 0.00012 (7.93±2.54)×10 0.00098 (1.80±0.11)×10-2 0.00195 (2.11±0.14)×10-2 0.00391 (3.49±0.33)×10-2 0.00781 (5.41±0.41)×10-2 0.01563 (8.97±0.42)×10-2 0.03125 (9.58±0.15)×10-2 a See Fig. 2a, 4 and S16 for the measuring conditions. All the measurements were performed in triplicate, and the mean values were taken. b -3 ‒1 The rate constant for the background (k0) was (4.40±3.16)×10 min under the same conditions.

S29 -2 Fig. S31. Calcein leakage by CF3-Bimbe (0.25 mol%) and NO2-Bimbe (3.13×10 mol%) from unilamellar EYPC vesicles loaded with 50 mM calcein buffered to pH 7.0 with 25 mM

HEPES buffer and 50 mM NaCl. The vesicles were dispersed in 50 mM NaCl buffered to pH

7.0 with 25 mM HEPES buffer, followed by the addition of a DMSO solution of CF3-Bimbe or NO2-Bimbe. At the end of the experiment (20 min), an aqueous solution of Triton X-100 (5 wt%) was added to lyse the vesicles. DMSO was used as a control. The results are shown as

% calcein leaked from the vesicles.

S30 (a) (b)

(e) (f)

(g)

Fig. S32. Plots of the absorbance of (a) m-Bimbe, (b) Me-Bimbe, (c) F-Bimbe, (d) Cl-Bimbe,

(e) Br-Bimbe, (f) CF3-Bimbe and (g) NO2-Bimbe (15 μM) versus the concentrations of

S31 + – tetrabutylammonium chloride (Bu4N Cl ). The solid lines are the nonlinear least-squares fit of the experimental data according to a 1 : 1 binding model.

‒1 Table S2. Binding constants (Ka’s, M ) of m-Bimbe, Me-Bimbe, F-Bimbe, Cl-Bimbe, Br-

a Bimbe, CF3-Bimbe and NO2-Bimbe with tetrabutylammonium chloride

‒1 Ka (M ) Compound 1 2 3 4 mean ± SD m-Bimbe 2619 1403 3400 2489 (2.5±0.8)×103 Me-Bimbe 2696 2445 2896 / (2.7±0.2)×103 F-Bimbe 3597 4128 2316 4755 (3.7±1.0)×103 Cl-Bimbe 1468 2216 2319 2700 (2.2±0.5)×103 Br-Bimbe 4002 4293 4993 / (4.4±0.5)×103

3 CF3-Bimbe 5433 3472 4372 4809 (4.5±0.8)×10 3 NO2-Bimbe 2062 1523 3073 / (2.2±0.8)×10 a Measured by means of spectrophotometric titrations in a mixture of acetonitrile and water (9/1, v/v) at room temperature.

S32