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SUPPORTING INFORMATION
Amino-BODIPY as the Ratiometric Fluorescent Sensor for Monitoring Drug Release or “Power Supply” Selector for Molecular ElectronicsMartin Porubskýa, Soňa Gurskáb, Jarmila Stankováb, Marián Hajdúchb, Petr Džubákb and Jan Hlaváčb*
a Department of Organic Chemistry, Faculty of Science, Palacký University Olomouc, 17. listopadu 12, 771 46 Olomouc, Czech Republic.
b Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 5, 779 00 Olomouc, Czech Republic. E-mail: [email protected]
Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2019
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Contents
Synthesis of quinolinone derivatives 1 and 21 .........................................................................................3
Synthesis of quinolinone derivative 2 ......................................................................................................6
Synthesis of BODIPY derivatives 7 and 20 .............................................................................................7
Synthesis of conjugates 4, 18, 19 .............................................................................................................8
Synthesis of conjugates 5, 6 ...................................................................................................................111H and 13C NMR spectra of prepared compounds ..................................................................................15
Fluorescence monitoring of the conjugates 4-6 cleavage.......................................................................35
Figure S1.................................................................................................................................................35
Figure S2.................................................................................................................................................35
Figure S3.................................................................................................................................................36
Figure S4.................................................................................................................................................36
Figure S5.................................................................................................................................................37
Figure S6.................................................................................................................................................37
Figure S7.................................................................................................................................................38
Detection limit ....................................................................................................................................38
Figure S8.................................................................................................................................................39
References ..............................................................................................................................................40
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Synthesis of quinolinone derivatives 1 and 21
The derivative 1 and 21 were synthesised according to the following scheme.
O
O
OH
O
NH2
OBr
K2CO3, 2 eq.
DMF100 °C, 2 h
O
ONH2
O
O
OAcOH/H2SO4, reflux, 4h
NH
OOH
O
OH
NH
OOH
O
NHS
OO
NH
OOH
O
NHHS
OH2N
TrtTFA/DCM/ TES
20/10/0.5 ml30 min
R1R1
R1
R1R1
SH2N
O O
SHN
O O
Fmoc Trt TrtpiperidineDMF
1 R1 = 3-NO2, 4-piperidyl21 R1 = H
23a: R1 = H23b: R1 = 3-NO2, 4-Cl
25 26
27a R1 = H27b R1 = 3-NO2, 4-piperidyl
8 or 9
228: R1 = H24: R1 = 3-NO2, 4-Cl9: R1 = 3-NO2, 4-piperidyl
= Rink amide resin
i
i) piperidine, DMSO, 130°C
General procedure to methyl-1-phenacylesters of 2-aminoterephthalic acid 23.
Suspension of 2-amino-4-(methoxycarbonyl)benzoic acid 221 (359 mg, 1.84 mmol) and K2CO3 (254 mg, 1.84 mmol) in DMF (8mL) was heated up to 90°C and stirred for 1h. Then the reaction mixture was allowed to cool to room temperature and corresponding bromoacetophenone (1.84 mmol) was subsequently added portionwise. Resulting mixture was stirred overnight at room temperature. The reaction mixture was then poured on the ice and precipitate was filtered off to give corresponding phenacylester 23. Compounds were used as crude materials for the next step.
4-methyl 1-(2-oxo-2-phenylethyl) 2-aminoterephthalate (23a)
O
ONH2
O
O
O
Yield: 95 %. 1H NMR CDCl3 δ = 8.05 – 8.02 (m, 1H), 7.96 – 7.93 (m, 2H), 7.63 – 7.58 (m, 1H), 7.51 – 7.47 (m, 2H), 7.36 – 7.34 (m, J = 1.4 Hz, 1H), 7.27 – 7.24 (m, 1H), 5.54 (s, 2H), 3.89 (s, J = 5.3 Hz, 3H). 13C NMR δ = 192.38, 167.00, 166.73, 150.49, 135.38, 134.37, 134.23, 132.07, 129.15, 128.05, 118.26, 116.80, 113.30, 66.46, 52.57. MS (ESI) calcd for M+H: 314.10. Found: 314.09.
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1-(2-(4-chloro-3-nitrophenyl)-2-oxoethyl) 4-methyl 2-aminoterephthalate (23b)
O
ONH2
O
O
O
Cl
NO2
Yield: 93 %. 1H NMR DMSO-d6 δ = 8.64 (d, J = 1.9 Hz, 1H), 8.28 (dd, J = 8.4, 1.9 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 1.4 Hz, 1H), 7.09 (dd, J = 8.4, 1.5 Hz, 1H), 6.88 (s, 2H), 5.75 (s, 2H), 3.84 (d, J = 9.4 Hz, 3H). 13C NMR δ = 191.18, 166.02, 165.86, 151.32, 147.97, 134.54, 133.56, 132.58, 132.47, 131.39, 130.13, 124.83, 117.79, 114.45, 110.99, 66.83, 52.36. MS (ESI) calcd for M+H: 393.0484. Found: 393.07.
General procedure for synthesis of 3-hydroxy-2-phenylquinolin-4(1H)-one 8 and 24
Phenacylester intermediates 23a or 23b (7.65 mmol) were subjected to cyclization and subsequent hydrolysis reaction in the mixture of AcOH (60mL) and H2SO4 (15mL) under reflux for 4h. After completeness of the reaction the mixture was poured on the ice and precipitate was filtered off to afford corresponding 3-hydroxy-2-phenylquinolones 8 or 24.
3-hydroxy-4-oxo-2-phenyl-1,4-dihydroquinoline-7-carboxylic acid (8)
NH
OOH
O
OH
Yield: 75 %. 1H NMR DMSO-d6 δ= 8.45 (d, J = 1.1 Hz, 1H), 8.27 (dd, J = 8.4, 4.3 Hz, 1H), 7.86 – 7.82 (m, 2H), 7.80 (dd, J = 8.6, 1.3 Hz, 1H), 7.62 – 7.53 (m, 3H). 13C NMR δ= 168.17, 166.83, 138.71, 137.09, 134.59, 132.39, 131.83, 129.78, 129.46, 128.46, 124.86, 123.73, 122.08, 121.07. MS (ESI) calcd for M+H 282.08. Found: 282.25.
2-(4-chloro-3-nitrophenyl)-3-hydroxy-4-oxo-1,4-dihydroquinoline-7-carboxylic acid (24)
NH
OOH
O
OH
NO2
Cl
Yield: 78 %. 1H NMR DMSO-d6 δ= 8.55 (d, J = 1.9 Hz, 1H), 8.39 (s, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.18 (dd, J = 8.4, 1.9 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H) 13C NMR δ = 169.10, 166.78, 147.48, 139.34, 137.44, 134.65, 132.68, 132.20, 131.75, 130.13, 126.27, 125.86, 125.08, 123.99, 121.97, 121.02. MS (ESI) calcd for M+H: 361.02. Found: 361.04.
3-hydroxy-2-(3-nitro-4-(piperidin-1-yl)phenyl)-4-oxo-1,4-dihydroquinoline-7-carboxylic acid (9)
Solution of starting compound 24 (500 mg, 1.39 mmol) in DMSO (5mL) was treated with piperidine (344 µl, 3.48 mmol) and the resulting mixture was heated to 130°C overnight in the sealed tube. The reaction mixture was poured on the ice and resulting solid was filtered off.
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NH
OOH
O
OHN
NO2
Yield: 78%. 1H NMR DMSO-d6 δ= 13.14 (bs, 1H), 11.74 (bs, 1H), 8.40 (s, 1H), 8.31 (d, J= 2.0 Hz, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.74 (dd, J = 8.5, 1.3 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 3.13 – 3.07 (m, 4H), 1.66 – 1.57 (m, 6H). 13C NMR δ = 170.04, 167.39, 146.76, 140.79, 139.51, 137.84, 134.82, 132.76, 130.89, 127.20, 125.53, 124.49, 123.48, 121.94, 121.24, 120.88, 52.27, 25.90, 23.92. MS (ESI) calcd for M+H: 410.1347. Found: 410.37.
General procedure for synthesis of N-(1-amino-3-mercapto-1-oxopropan-2-yl)-3-hydroxy-2-phenyl-4(1H)-quinolinone-7-carboxamides 1 and 21
NH
OOH
O
NHHS
OH2N
R1
1 R1 = 3-NO2, 4-piperidyl21 R1 = H
Resin 25
The Rink resin (1.0g, loading 0.6 mmol) was treated with solution of piperidine/DMF for 30 min, washed with DMF and DCM three times and then treated with solution of Fmoc-Cys(Trt)-OH (1.05 g, 1.8 mmol), HOBt (243 mg, 1.8 mmol) and DIC (279 ul, 1.8 mmol) in mixture of DCM/DMF (6 mL, 1:1). After 3 hours resin was washed with DMF and DCM three times to give resin 25 directly used for the next step.
Resin 26
Resin 25 was treated with piperidine/DMF for 30 minutes and washed with DMF and DCM three times. The resin was used directly for the next step.
Resin 27a or 27b
Resin 26 was treated with the solution of 8 or 9 (1.2 mmol), HOBt (135 mg, 1.2 mmol) and DIC (155ul, 1.2 mmol) in DMF/Pyridine (6 mL, 1:1) for 3 hours and washed 3 times with DMF and DCM.
Derivatives 1 and 21
Resin 25 was treated in mixture of TFA/DCM/TES (20:10:0.5) for 1h. The solvents were evaporated and the product was precipitated with Et2O to afford pure desired products 1 or 21.
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N-(1-amino-3-mercapto-1-oxopropan-2-yl)-3-hydroxy-2-(3-nitro-4-(piperidin-1-yl)phenyl)-4-oxo-1,4-dihydroquinoline-7-carboxamide (1)
NH
OOH
O
NHHS
OH2N
N
NO2
Yield: 94 %. 1H NMR DMSO-d6 δ= 11.74 (bs, 1H), 8.61 (d, J= 7.9 Hz, 1H), 8.30 (s,1H), 8.25 (s, 1H), 8.21 (d, J= 8.5 Hz, 1H), 8.03 (d, J= 8.5 Hz, 1H), 7.75 (d, J= 8.5 Hz, 1H), 7.54 (s, 1H), 7.43 (d, J= 8.8 Hz, 1H), 7.22 (s, 1H), 4.54 (dd, J= 8.3, 12.7 Hz, 1H), 3.10 (s, 4H), 2.95 – 3.03 (m, 1H), 2.83 – 2.92 (m, 1H), 2.39 (t, J= 8.4 Hz, 1H), 1.60 - 1.64 (s, 6H). 13C NMR δ= 172.20, 170.13, 166.58, 145.75, 140.80, 139.22, 137.95, 136.50, 134.83, 130.69, 127.19, 125.15, 123.67, 123.56, 120.88, 120.75, 119.28, 56.51, 52.29, 26.52, 25.91, 23.93. HRMS (ESI) m/z calcd for [C24H25N5O6S]+H: 512.1598; found: 512.1608.
N-(1-amino-3-mercapto-1-oxopropan-2-yl)-3-hydroxy-4-oxo-2-phenyl-1,4-dihydroquinoline-7-carboxamide (21)
NH
OOH
O
NHHS
OH2N
Yield: 95 %. 1H NMR DMSO-d6 δ = 11.82 (bs, 1H), 8.60 (d, J= 7.9 Hz, 1H), 8.26 (s, 1H), 8.22 (d, J= 8.50 Hz, 1H), 7.82 (d, J= 7.2 Hz, 2H), 7.76 (dd, J= 1.2, 8.5 Hz, 1H), 7.50 – 7.60 (m, 4H), 7.22 (s, 1H), 4.50 - 4.58 (m, 1H), 2.94 – 3.03 (m, 1H), 2.82 – 2.93 (m, 1H), 2.39 (t, J= 8.4 Hz, 1H) 13C NMR δ = 171.69, 169.48, 166.09, 138.55, 137.38, 135.93, 132.64, 132.09,129.03, 129.28, 128.31, 124.59, 123.09, 120.26, 118.90, 55.97, 26.00. HRMS (ESI) m/z calcd for C19H17N3O4S: 383.0940; found: 383.0944.
Synthesis of quinolinone derivative 2
The derivative 2 was synthesised according to the following scheme
NH
OOH
O
OOO
HONH
OOH OH
O
OO
HO
HO H2SO4 (cat.)
THF, reflux, on28
2-(2-(2-hydroxyethoxy)ethoxy)ethyl 3-hydroxy-4-oxo-2-phenyl-1,4-dihydroquinoline-7-carboxylate (2)
The suspension of starting acid 82 (300 mg, 1.07 mmol) in THF was mixed with triglycol (1.0 ml, 10.7 mmol) and catalytic amount of H2SO4 (3 drops). The reaction mixture was heated to reflux overnight.
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After the full consumption of the starting material, reaction was stopped and ethylacetate (100ml) was added. Resulting solution was washed with 10% aq. NaHCO3, 3 times with water and with brine. Organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain 285 mg (65 %) of pure product 2.
NH
OOH
O
OO
OHO
Yield: 65 %. 1H NMR DMSO-d6 δ= 11.87 (s, 1H), 8.46 (s, 1H), 8.29 – 8.25 (m, 1H), 7.84 (d, J = 7.2 Hz, 2H), 7.76 (dd, J = 8.6, 1.5 Hz, 1H), 7.60 – 7.53 (m, 3H), 4.47 – 4.44 (m, 2H), 3.81 – 3.78 (m, 2H), 3.62 – 3.60 (m, 2H), 3.56 – 3.53 (m, J = 2.3 Hz, 2H), 3.48 – 3.45 (m, J = 5.1 Hz, 2H), 3.43 – 3.40 (m, J = 4.4 Hz, 2H). 13C NMR δ= 169.58, 165.37, 139.10, 137.23, 132.69, 132.04, 130.91, 129.49, 129.29, 128.34, 125.29, 121.06, 120.93, 72.38, 69.92, 69.77, 68.36, 64.54, 60.22. HRMS (ESI) m/z calcd for [C22H22NO7]+H: 414.1547. Found: 414.1545.
Synthesis of BODIPY derivatives 7 and 20
The derivative 7 and 20 were synthesized according to the following scheme:
N NB
F F Cl
N NB
F F NH2
NH3
MeOH / DCM70oC, on
10 7TEA / MeOH
rt, on
NH
HON N
BF F N
20OH
1,3-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-5-amino-s-indacene (7)
N NB
F F NH2
The solution of 103 (200mg, 0.79 mmol) in dry dichloromethane (5mL) was treated with 7.0 M NH3 in methanol (2.2 mL). Resulting mixture was stirred at 70°C overnight. After full consumption of starting material, reaction mixture was cooled down to room temperature and concentrated under vacuum. Crude product was purified by column chromatography (EtOAc/n-hexane 1:2) to give dark solid compound. Yield 131 mg (71%).
1H NMR CDCl3 δ= 6.91 (d, 1H, J = 4.6 Hz), 6.72 (s, 1H), 5.89 – 5.93 (m, 2H), 5.50 (s, 2H), 2.45 (s, 3H), 2.16 (s, 3H). 13C NMR δ= 160.61, 159.39, 147.59, 134.19, 133.34, 132.12, 116.91, 115.93, 109.74, 14.12, 11.11. HRMS (ESI) m/z calcd for [C11H12BF2N3]-H: 234.1009; found: 234.1009.
N-(4-hydroxypiperidine)-1,3-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-5-amino-s-indacene (20)
N NB
F F N
OH
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Starting compound 103 (500 mg, 1.97 mmol) was dissolved in MeOH (10ml) and TEA (412 µl, 2.95 mmol), then 4-hydroxypiperidine (219 mg, 2.17 mmol) was added to the solution. Resulting mixture was stirred overnight at room temperature, extracted with EtOAc and washed three times with water. Drying over Na2SO4 was followed by evaporation to dryness. Yield 604 mg (96 %) of pure product.
1H NMR CDCl3 δ= 6.94 (d, 1H, J= 4.8 Hz), 6.65 (s, 1H), 6.13 (d, 1H, J= 4.8 Hz), 5.92 (s, 1H), 4.11 – 4.21 (m, 2H), 3.97 – 4.05 (m, 1H), 3.56 – 3.65 (m, 2H), 2.46 (s, 3H), 2.17 (s, 3H), 2.02 – 2.10 (m, 2H), 1.69 – 1.78 (m, 2H), 1.63 (bs, 1H). 13C NMR δ= 161.13, 146.52, 134.35, 133.42, 131.80, 129.92, 115.85, 115.54, 110.90, 66.73, 47.51, 34.41, 14.27, 11.03. HRMS (ESI) m/z calcd for [C16H20BF2N3O]+H: 320.1740; found: 320.1741.
Synthesis of conjugates 4, 18, 19
The derivative 4, 18 and 19 were synthesised according to the following scheme
N NB
F F HNO
O SS
NN NB
F F NH2
O
O SS NO
NN
N
DCMrt, on
7
11
12
N NB
F F N
20OH
O
O SS NO
NN
N
DCMrt, on
11
N NB
F F N
OO
O SS N
28
N NB
F F HNO
OS
HN
O
OH
O
NHS
OH2N
N NB
F F HN O
O SS N
NH
OOH
O
NHHS
OH2N
DMF60°C, on
+
4112
N NB
F F N
O
OS
SN
28
+NH
OOH
O
NHHS
OH2N21O
N NB
F F N
O
OSO
NH
OOH
O
NHS
OH2N
18
N NB
F F HN O
O SS N
O
NHHS
OH2N+
12
N NB
F F HNO
OS
O
NHS
OH2N
19
N
NO2
N
NO2
31
DMF60°C, on
DMF60°C, on
DMAP, TEA
DMAP, TEA
1,3-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-5-(carbamoyl-ethylpyridinyldisulfide)-s-indacene (12)
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N NBF F HN
O
OS
S
N
To a stirred solution of 7 (100 mg, 0.425 mmol) in dry DCM (10mL) DMAP (68 mg), TEA (178 µl) and activated disulfide linker 114 (193 mg) were added portionwise. Resulting mixture was stirred at room temperature for 3 h. After the reaction was complete solvent was evaporated and crude mixture was directly purified by column chromatography (EtOAc/n-hexane 1:2) to afford 182 mg (95 %) of pure product 12.
Yield: 95 %. 1H NMR CDCl3 δ= 8.46 (d, 1H, J= 4.3 Hz), 8.00 (s, 1H, NH), 7.59 – 7.70 (m, 2H), 7.05 – 7.10 (m, 2H), 6.99 (s, 1H), 6.97 (d, 1H, J= 4.3 Hz), 6.83 (d, 1H, J= 3.9 Hz), 6.03 (s, 1H), 4.45 (t, 2H, J= 6.4 Hz), 3.08 (t, 2H, 6.4 Hz), 2.50 (s, 3H), 2.21 (s, 3H). 13C NMR δ= 159.54, 155.70, 151.43, 149.87, 149.58, 141.25, 137.20, 133.61, 130.87, 129.74, 122.08, 121.06, 120.12, 118.98, 109.26, 63.90, 37.21, 14.64, 11.34. HRMS (ESI) m/z calcd for [C19H19BF2N4O2S2]+H: 449.1083; found: 449.1089.
1-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)piperidin-4-yl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate (28)
N NB
F F N
O
O SS
NO
To a stirred solution of 20 (40 mg, 0.13 mmol) in dry DCM (5mL) DMAP (20 mg, 0.17 mmol), TEA (53 µl, 0.17 mmol) and activated disulfide linker 114 (66 mg, 0.20 mmol) was added portionwise. Resulting mixture was stirred at room temperature overnight. After the reaction was complete solvent was evaporated and crude mixture was directly purified by column chromatography (EtOAc/n-hexane 1:2) to afford 66 mg (99 %) of pure product 28.
1H NMR CDCl3 δ = 8.49 (dd, J = 4.5, 1.3 Hz, 1H), 7.72 – 7.64 (m, 3H), 7.15 – 7.11 (m, 1H), 6.95 (d, J = 4.7 Hz, 1H), 6.68 (s, 1H), 6.11 (d, J = 4.7 Hz, 1H), 5.93 (s, 1H), 4.95 – 4.89 (m, 1H), 4.42 (t, J = 6.49 Hz, 2H), 4.05 – 3.99 (m, 2H), 3.79 – 3.73 (m, 2H), 3.09 (t, J = 6.5 Hz, 2H), 2.46 (s, 3H), 2.18 (s, 3H), 2.15 – 2.05 (m, 2H), 1.96 – 1.87 (m, 2H). 13C NMR δ = 160.98, 159.42, 154.17, 149.86, 147.06, 137.20, 134.13, 133.37, 132.42, 130.07, 121.07, 120.02, 119.28, 116.06, 110.60, 73.00, 65.48, 47.11, 37.02, 30.76, 14.31, 11.07. HRMS (ESI) m/z calcd for C24H27BF2N4O3S2: 532.1586; found: 532.1588
General procedure for synthesis of the conjugates 4,18,19.
Starting compound 12 or 28 (0.06 mmol) and 2-acetamido-3-mercaptopropanamide 31 or corresponding quinolinone 1 or 21 (1,13) (0.08 mmol) were dissolved in dry DMF (3mL) and heated to 60°C. Resulting reaction mixture was stirred overnight. After complete consumption of starting materials the reaction mixture was cooled down and diluted with mixture ethylacetate-methanol (5:1) followed by washing with water repetaed 3 times. Organic layer was dried over Na2SO4 and
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concentrated. Obtained crude product was purified by HPLC (AcCN/ammonium acetate (10mM) buffer 30:70 to 60:40 gradient) affording pure compounds 4, 18 or 19.
2-((3-amino-2-(3-hydroxy-2-(3-nitro-4-(piperidin-1-yl)phenyl)-4-oxo-1,4-dihydroquinoline-7-carboxamido)-3-oxopropyl)disulfanyl)ethyl (5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)carbamate (4)
N NB
F F HNO
OS
HN
O
OH
O
NHS
OH2N
N
NO2
1H NMR DMSO-d6 δ= δ 11.23 (s, 1H), 8.09 – 7.99 (m, 3H), 7.96 (d, J = 7.1 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.65 (s, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.00 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.74 – 6.70 (m, 1H), 6.67 – 6.63 (m, 1H), 6.42 – 6.37 (m, 1H), 6.21 (s, 1H), 5.71 (s, 1H), 4.68 – 4.58 (m, 1H), 4.18 – 4.06 (m, 2H), 3.12 – 2.99 (m, 4.4 Hz, 2H), 2.96 – 2.87 (m, 2H), 2.79 (s, 4H), 2.72 – 2.67 (m, 2H), 2.12 (s, 3H), 1.87 (s, 3H), 1.40 (s, 4H), 1.32 (s, 2H). 13C NMR δ= 166.77, 166.59, 155.12, 151.11, 149.07, 146.73, 141.81, 141.18, 140.50, 137.70, 135.94, 135.69, 134.10, 133.27, 130.80, 129.38, 127.21, 126.65, 125.00, 122.72, 122.35, 122.11, 120.25, 119.83, 118.83, 118.74, 108.87, 63.99, 52.10, 40.87, 40.77, 36.46, 25.56, 23.69, 14.34, 11.09. HRMS (ESI) m/z calcd for [C38H39BF2N8O8S2]-H : 847.2310; found: 847.2307. Yield: 34 %.
2-((3-amino-2-(3-hydroxy-4-oxo-2-phenyl-1,4-dihydroquinoline-7-carboxamido)-3-oxopropyl)disulfanyl)ethyl (1-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)piperidin-4-yl) carbonate (18)
N NB
F F N
O
OS
HN
O
OH
O
NHS
OH2N
O
1H NMR DMSO-d6 δ= 11.81 (s, 1H), 8.81 (d, 1H, J = 7.7 Hz), 8.30 – 8.20 (m, 2H), 8.04 (s, 1H), 7.82 (d, 2H, J = 6.5 Hz), 7.75 (d, 1H, J = 8.2 Hz), 7.64 – 7.48 (m, 4H), 7.21 – 7.32 (m, 2H), 6.94 (s, 1H), 6.57 (d, 1H, J = 4.2 Hz), 5.90 (s, 1H), 4.84 (s, 1H), 4.72 (s, 1H), 4.33 (s, 2H), 4.05 (s, 2H), 3.75 (s, 2H), 3.20 – 3.11 (m, 2H), 2.98 – 3.09 (m, 2H), 2.30 (s, 3H), 2.12 (s, 3H), 2.03 (s, 2H), 1.71 (s, 2H). 13C NMR δ= 171.71, 169.65, 166.07, 160.82, 153.58, 143.25, 138.58, 137.41, 135.84, 134.63, 132.38, 132.09, 129.26, 128.78, 128.27, 124.61, 123.13, 121.64, 121.33, 120.09, 119.41, 118.90, 114.97, 114.62, 112.90, 72.56, 65.18, 52.61, 46.76, 40.43, 36.04, 30.49, 13.87, 13.85, 10.61. HRMS (ESI) m/z calcd for [C38H39BF2N6O7S2]-H : 803.2299; found: 803.2298. Yield: 32 %.
3-(((2-((2-acetamido-3-amino-3-oxopropyl)disulfanyl)ethoxy)carbonyl)amino)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (19)
S11
N NB
F F HNO
OS
O
NHS
OH2N
1H NMR CDCl3 δ= 8.09 (s, 1H), 8.00 (s, 1H), 7.01 (s, 1H), 6.99 (d, J = 4.4 Hz, 1H), 6.85 (d, J = 4.3 Hz, 1H), 6.77 – 6.72 (m, 2H), 6.05 (s, 1H), 5.88 (s, 1H), 4.81 – 4.77 (m, 1H), 4.50 – 4.47 (m, 2H), 3.12 (t, J = 6.5 Hz, 2H), 3.00 (t, J = 6.5 Hz, 2H), 2.50 (s, 3H), 2.21 (s, 3H), 2.02 (s, 3H). 13C NMR δ= 172.43, 170.75, 162.71, 155.87, 151.69, 149.41, 141.48, 133.70, 130.90, 129.72, 122.21, 119.10, 109.25, 77.41, 77.16, 76.91, 64.18, 52.25, 40.80, 37.05, 36.64, 31.58, 23.20, 14.71, 11.40. HRMS (ESI) m/z calcd for [C19H23BF2N5O4S2]-H : 498.1258; found: 498.1255. Yield: 38 %.
Synthesis of conjugates 5, 6
The conjugates 5 and 6 were synthesised according to the following scheme:
NH
OOH
O
OOO
O
N NBF F HN OS
SO
O
O
DCM
5
2
N NBF F HN
OSSO
O
O
ON
NN
14
rt, on
3
N
NBF
FNH OS
SO
O
O
HN
O
OH
HN
6
N NB
F F NH2
N NB
F F HN
OHS
SO
O
N NB
F F HN
OS
SO
O
OO N
NN
O
OS
SO O
ONN N
1) TEA, DMAP, DCM, rt, 3h
2) K2CO3, THF/MeOH, rt, on
1) triphosgene, TEA, DCM; 0°C; 1h
2) HOBt, TEA, DCM; rt, on
7
13
30 14
O
OS
SOH
1) triphosgene, TEA, DCM, 0°C2) HOBt, TEA, DCM 0°C to rt
29
DMAP, TEA
DMAP, TEA
rt, onDCM
2-((2-((((1H-benzo[d][1,2,3]triazol-1-yl)oxy)carbonyl)oxy)ethyl)disulfanyl)ethyl acetate (13)
2-((2-Hydroxyethyl)disulfanyl)ethyl acetate5 29 (0.5g, 2.55 mmol) was dissolved in DCM (20mL) and cooled to 0°C. Then triethylamine (391 µl, 2.81 mmol) was added followed by addition of solution of triphosgene (250 mg, 0.84 mmol) in DCM (5 mL). After stirring for 1 hour at 0°C the solution of HOBt (379 mg, 2.81 mmol) and triethylamine (391 µl, 2.81 mmol) in DCM (5mL) was added slowly to the reaction mixture. The reaction mixture was then stirred overnight at room temperature. Reaction solution was diluted with DCM (100mL) and washed with water 3x80mL and brine. Organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain the desired product. Yield 774 mg (85%)
O
OS
SO O
ONN N
S12
1H NMR CDCl3 δ = 8.23 – 8.20 (m, 1H), 8.02 – 7.99 (m, 1H), 7.79 – 7.75 (m, 1H), 7.57 – 7.53 (m, 1H), 4.80 (t, J = 6.7 Hz, 2H), 4.34 (t, J = 6.5 Hz, 2H), 3.13 (t, J = 6.7 Hz, 2H), 2.98 (t, J = 6.5 Hz, 2H), 2.07 (s, 3H). 13C NMR δ = 170.90, 147.29, 133.52, 132.98, 126.55, 120.75, 115.96, 115.27, 77.42, 77.16, 76.91, 66.64, 62.32, 37.42, 36.62, 20.97. HRMS (ESI) m/z calcd for [C13H16N3O5S2]+H: 358.0526; found: 358.0523.
Synthesis of 5,5-difluoro-3-(((2-((2-hydroxyethyl)disulfanyl)ethoxy)carbonyl)amino)-7,9-dimethyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (30)
DMAP (68 mg, 0.557 mmol), TEA (178 µl, 1.277 mmol) and disulfide linker 13 (228 mg, 0.638 mmol) was added portionwise to a stirred solution of 7 (100 mg, 0.425 mmol) in dry DCM (10mL). Resulting mixture was stirred at room temperature for overnight. After the reaction was complete solvent was evaporated, dissolved in THF/MeOH 3:1 (10 mL) and treated with K2CO3 (176 mg, 1.275mmol). After stirring overnight ethylacetate (100mL) was added and organic phase was washed 3 times with water, dried over Na2SO4 and concentrated under vacuum. Crude product was purified by column chromatography (hexane/EtOAc 2:1) to afford pure product 31. Yield 113 mg (64 %).
NNB
F F HNOH
SS
O
O
1H NMR CDCl3 δ = 8.08 (s, 1H), 7.01 (s, 1H), 7.00 (d, J = 4.4 Hz, 1H), 6.88 (d, J = 4.3 Hz, 1H), 6.05 (s, 1H), 4.49 (t, J = 6.7 Hz, 2H), 3.91 (t, J = 5.8 Hz, 1H), 3.00 (t, J = 6.7 Hz, 2H), 2.92 (t, J = 5.8 Hz, 2H), 2.51 (s, 3H), 2.23 (s, 3H). 13C NMR δ = 155.77, 151.65, 149.57, 141.35, 133.68, 130.91, 129.78, 122.15, 119.03, 109.29, 64.36, 60.40, 41.84, 37.05, 14.66, 11.36. HRMS (ESI) m/z calcd for [C16H19BF2N3O3S2]-H: 414.0934; found: 414.0930.
3-(((2-((2-((((1H-benzo[d][1,2,3]triazol-1-yl)oxy)carbonyl)oxy)ethyl)disulfanyl)ethoxy)carbonyl)amino)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (14)
NNB
F F HN OSSO
OO
ON
NN
Solution of TEA (214 uL, 1.536 mmol) and triphosgene (146 mg, 0.492 mmol) dissolved in DCM (1 mL) was slowly added to a solution of compound 30 (510 mg, 1.229 mmol) in dry DCM (6 mL) at 0°C. Resulting reaction mixture was stirred for 2h at 0°C. Subsequently the solution of TEA (214 uL, 1.536 mmol) and HOBt (183 mg, 1.352 mmol) dissolved in DCM (2 mL) was added at 0°C. Reaction was allowed to warm to room temperature and was stirred overnight. Then EtOAc (200mL) was added to reaction mixture and organic layer was washed with water and brine. Organic layer was dried over Na2SO4 and concentrated under reduced pressure to give product 14. Yield 657 mg (93 %).
S13
1H NMR CDCl3 δ = 8.18 (d, J = 8.5 Hz, 1H), 8.04 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.75 – 7.71 (m, 1H), 7.53 – 7.48 (m, 1H), 6.98 – 6.93 (m, 2H), 6.84 (d, J = 4.3 Hz, 1H), 6.02 (s, 1H), 4.80 (t, J = 6.7 Hz, 2H), 4.49 (t, J = 5.9 Hz, 2H), 3.16 (t, J = 6.7 Hz, 2H), 3.05 (t, J = 6.5 Hz, 2H), 2.47 (s, 3H), 2.21 (s, 3H). 13C NMR δ = 155.76, 151.52, 149.49, 147.23, 141.33, 133.64, 133.45, 132.87, 130.87, 129.73, 126.40, 122.10, 119.02, 115.81, 115.26, 109.22, 66.63, 64.16, 41.85, 37.46, 37.06, 36.64, 14.62, 11.36. HRMS (ESI) m/z calcd for [C23H22BF2N6O5S2]-H: 575.1160; found: 575.1149.
General procedure for synthesis conjugates 5 and 6.
Starting compound 14 (0.06 mmol) and corresponding quinolinone derivative 2 or 3 (0.06 mmol) were dissolved in dry DCM (3mL). Subsequntly TEA (0.18 mmol) and DMAP (0.18 mmol) were added to the stirred solution of starting materials. Resulting reaction mixture was stirred overnight. After complete consumption of starting materials the mixture was diluted with ethylacetate and washed three times by water. Organic layer was dried over Na2SO4 and concentrated. Obtained crude product was purified by HPLC (AcCN/ammonium acetate buffer 30:70 to 60:40 gradient) affording pure compounds 5 or 6.
1-((5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)amino)-1,10-dioxo-2,9,11,14,17-pentaoxa-5,6-dithianonadecan-19-yl 3-hydroxy-4-oxo-2-phenyl-1,4-dihydroquinoline-7-carboxylate (5)
HN
O
OH
O
O
N
NBF
FNH
OS
SO
O
O
OO
O
1H NMR DMSO-d6 δ= 11.83 (s, 1H), 8.46 (s, 1H), 8.26 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 7.3 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H), 7.55 (dt, J = 24.6, 7.0 Hz, 4H), 7.24 (d, J = 4.0 Hz, 1H), 6.78 (t, J = 5.6 Hz, 1H), 6.20 (d, J = 9.0 Hz, 1H), 4.43 (dt, J = 12.7, 5.9 Hz, 4H), 4.30 (t, J = 6.3 Hz, 2H), 4.20 – 4.13 (m, 2H), 3.81 – 3.75 (m, 2H), 3.66 – 3.59 (m, 4H), 3.56 (dd, J = 5.7, 3.2 Hz, 2H), 3.07 (t, J = 6.4 Hz, 2H), 3.02 (t, J = 6.1 Hz, 2H), 2.42 (d, J = 6.5 Hz, 3H), 2.23 (d, J = 5.6 Hz, 3H). 13C NMR δ= 169.53, 165.30, 154.86, 154.32, 151.36, 148.92, 141.48, 139.01, 137.23, 133.02, 132.57, 131.98, 131.56, 130.89, 129.42, 129.22, 128.26, 125.20, 124.09, 123.59, 120.98, 120.86, 118.88, 109.09, 69.80, 69.70, 68.32, 68.12, 66.86, 65.13, 64.42, 63.77, 54.71, 36.29, 36.18, 14.15, 10.90. HRMS (ESI) m/z calcd for [C39H40BF2N4O11S2]-H: 853.2202; found: 853.2191. Yield: 35 %.
2-((2-(((4-(3-hydroxy-4-oxo-1,4-dihydroquinolin-2-yl)phenyl)carbamoyl)oxy)ethyl)disulfanyl)ethyl (5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)carbamate (6)
N
NBF
FNH
OSSO
O
O
HN
OOH
HN
1H NMR DMSO-d6 δ= 11.66 (s, J = 13.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.68 – 7.64 (m, 1H), 7.60 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.25 (d, J = 4.4 Hz, 1H), 6.79 (d, J = 4.4
S14
Hz, 1H), 6.71 (d, J = 8.6 Hz, 2H), 6.21 (s, 1H), 5.75 (s, 1H), 4.46 – 4.38 (m, 4H), 3.11 – 3.03 (m, 4H), 2.43 (s, 3H), 2.23 (s, 3H). 13C NMR δ= 169.44, 162.26, 154.92, 152.66, 151.32, 150.94, 148.87, 143.39, 141.54, 139.07, 133.04, 131.64, 131.58, 130.78, 129.79, 129.40, 124.69, 123.67, 122.90, 118.90, 118.47, 116.86, 113.24, 109.13, 65.90, 63.82, 54.87, 36.21, 14.17, 10.91. HRMS (ESI) m/z calcd for [C32H31BF2N5O6S2]-H: 692.1626; found: 692.1624. Yield: 40 %.
S15
1H and 13C NMR spectra of prepared compounds
Compound 1 (DMSO-d6)
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
7.89
1.14
1.14
1.17
4.27
1.00
1.07
1.03
1.01
1.04
1.11
1.17
1.06
1.07
1.11
1.00
1.60
1.64
2.37
2.39
2.41
2.50
2.87
2.88
2.89
2.91
2.96
2.97
2.98
2.99
3.00
3.00
3.01
3.10
4.54
4.55
7.22
7.42
7.44
7.54
7.74
7.76
8.02
8.03
8.20
8.21
8.25
8.29
8.60
8.61
11.7
4
2030405060708090100110120130140150160170180f1 (ppm)
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
0.080
0.085
0.090
23.3
725
.36
25.9
8
39.0
239
.19
39.3
539
.52
39.6
939
.78
39.8
539
.94
40.0
2
51.7
4
55.9
5
118.
7112
0.33
123.
1112
4.59
126.
6313
0.14
134.
2813
5.93
137.
4113
8.66
140.
24
146.
19
166.
0316
9.58
171.
64
NH
OOH
O
NHHS
OH2N
N
NO2
S16
Compound 2 (DMSO-d6)
2.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
2.00
2.06
2.04
2.01
2.01
2.00
3.16
1.07
2.01
1.00
1.00
0.99
35404550556065707580859095105115125135145155165175f1 (ppm)
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
60.2
2
64.5
4
68.3
669
.77
69.9
272
.38
120.
9312
1.06
125.
2912
8.34
129.
2912
9.49
130.
9113
2.04
132.
69
137.
2313
9.10
165.
37
169.
58
NH
OOH
O
OO
OHO
S17
Compound 4 (CDCl3/DMSO-d6)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
2.14
4.13
3.15
2.92
4.04
2.01
3.97
2.16
1.80
2.07
1.07
1.01
0.93
0.95
1.02
1.01
1.03
1.00
0.96
1.03
1.06
1.02
2.24
0.85
1.35
1.42
1.90
2.14
2.27
2.27
2.30
2.31
2.66
2.71
2.72
2.81
2.91
2.94
2.95
3.07
3.09
3.10
4.14
4.15
4.65
4.66
5.73
6.23
6.41
6.42
6.66
6.67
6.74
6.86
6.88
7.02
7.26
7.27
7.40
7.42
7.67
7.80
7.82
7.97
7.98
8.03
8.07
8.08
11.2
5
102030405060708090100110120130140150160170f1 (ppm)
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
0.032
0.034
10.6
2
13.8
7
23.2
225
.09
28.9
539
.00
39.1
639
.33
39.5
039
.67
39.8
340
.00
40.2
840
.39
51.6
352
.20
63.5
2
76.9
077
.16
77.4
2
108.
39
118.
2711
9.35
119.
7812
1.63
122.
2412
4.52
126.
1712
8.90
130.
3213
2.79
133.
6213
5.21
137.
2413
8.38
140.
0214
0.70
146.
2514
8.59
150.
6415
4.65
166.
11
171.
71
N NB
F F HNO
OS
HN
O
OH
O
NHS
OH2N
N
NO2
S18
Compound 5 (DMSO-d6)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
3.04
3.02
2.11
2.07
2.05
3.90
2.00
2.00
2.03
4.06
1.03
1.09
1.04
4.08
1.01
1.88
1.00
1.05
0.71
2.22
2.42
3.00
3.02
3.03
3.05
3.07
3.08
3.56
3.61
3.62
3.62
4.16
4.17
4.18
4.29
4.30
4.32
4.40
4.42
4.43
4.44
4.44
4.45
5.75
6.20
6.77
6.78
7.52
7.54
7.56
7.57
7.59
7.74
7.76
7.83
7.84
8.25
8.27
8.46
102030405060708090100110120130140150160170180f1 (ppm)
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
10.9
0
14.1
5
36.1
836
.29
54.7
1
63.7
764
.42
65.1
366
.86
68.1
268
.32
69.7
069
.80
109.
0911
8.88
120.
8612
0.98
123.
5912
4.09
125.
2012
8.26
129.
2212
9.42
130.
8913
1.56
131.
9813
2.57
133.
0213
7.23
139.
01
148.
9215
1.36
154.
3215
4.86
165.
30
169.
53
HN
O
OH
O
ON
NBF
FNH
OSSO
O
O
OOO
S19
Compound 6 (DMSO-d6)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5f1 (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
3.02
3.01
0.53
0.56
4.00
4.00
1.14
0.97
1.95
0.97
0.95
1.10
1.13
0.86
1.01
1.05
0.96
0.95
2.23
2.43
2.50
2.50
2.50
2.51
2.73
2.89
3.05
3.06
3.07
3.08
3.09
3.32
4.39
4.41
4.42
4.43
4.45
5.75
6.21
6.70
6.72
6.78
6.79
7.25
7.25
7.31
7.33
7.60
7.71
8.10
8.11
11.6
6
102030405060708090100110120130140150160170180f1 (ppm)
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
0.34
0.36
0.38
10.9
1
14.1
7
36.2
1
54.8
7
63.8
265
.90
109.
1311
3.24
116.
8611
8.47
118.
9012
2.90
123.
6712
4.69
129.
4012
9.79
130.
7813
1.58
131.
64
139.
0714
1.54
143.
39
148.
8715
0.94
151.
3215
2.66
154.
92
162.
26
169.
44
N
NBF
FNH
OS
SO
O
O
HN
OOH
HN
S20
Compound 7 (CDCl3)
1.41.82.22.63.03.43.84.24.65.05.45.86.26.67.07.27.4f1 (ppm)
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
3.06
3.08
1.95
1.92
1.03
1.00
1.58
2.18
2.47
5.52
5.93
5.94
6.74
6.93
6.94
7.26
102030405060708090100110120130140150160170f1 (ppm)
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
11.1
8
14.2
0
76.9
177
.16
77.4
1
109.
81
116.
0111
6.98
131.
0213
2.20
133.
4213
4.26
147.
66
159.
45
N NB
F F NH2
S21
Compound 8 (DMSO-d6)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
3.03
1.05
1.94
1.08
1.00
2.50
2.50
2.50
2.51
7.55
7.56
7.58
7.59
7.59
7.61
7.79
7.79
7.81
7.81
7.83
7.85
7.85
8.26
8.27
8.45
8.45
404550556065707580859095100105110115120125130135140145150155160165170f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
39.1
939
.35
39.5
239
.69
39.8
5
121.
0712
2.08
123.
7312
4.86
128.
4612
9.46
129.
7813
1.83
132.
3913
4.59
137.
0913
8.71
166.
8316
8.17
NH
OOH
O
OH
S22
Compound 9 (DMSO-d6)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.07
4.27
4.04
0.99
1.00
1.01
0.96
0.98
0.94
0.85
1.59
1.60
1.64
1.64
2.08
2.50
2.50
2.50
2.54
3.09
3.10
3.11
7.42
7.44
7.73
7.73
7.75
7.75
8.02
8.04
8.21
8.22
8.31
8.31
8.40
11.7
4
2030405060708090100110120130140150160170f1 (ppm)
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
23.4
325
.41
39.0
239
.19
39.3
539
.52
39.6
939
.78
39.8
539
.94
40.0
240
.43
51.7
8
120.
3712
0.77
121.
5112
3.05
123.
9512
4.97
126.
7213
0.50
132.
5213
4.34
137.
3913
9.00
140.
28
146.
27
167.
00
NH
OOH
O
OHN
NO2
S23
Compound 12 (CDCl3)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
3.00
2.90
2.05
1.96
0.95
0.94
1.96
1.19
2.18
1.01
1.08
2.23
2.52
3.09
3.10
3.12
4.46
4.47
4.48
6.05
6.85
6.86
6.98
6.99
7.01
7.08
7.09
7.10
7.26
7.65
7.67
7.67
7.69
7.70
8.02
8.47
8.48
0102030405060708090100110120130140150160170f1 (ppm)
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
11.4
0
14.6
9
37.2
7
63.9
6
76.9
177
.16
77.4
1
109.
31
119.
0212
0.17
121.
1212
2.14
129.
7913
0.92
133.
6813
7.26
141.
30
149.
6414
9.92
151.
4815
5.73
159.
59
N NB
F F HNO
O
SS
N
S24
Compound 13 (CDCl3)
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
3.00
2.14
1.92
2.07
1.97
1.15
1.04
0.95
1.00
2.07
2.96
2.98
2.99
3.12
3.13
3.15
4.33
4.34
4.35
4.79
4.80
4.81
7.53
7.53
7.55
7.55
7.55
7.56
7.57
7.76
7.76
7.77
7.78
7.78
7.79
7.79
8.00
8.00
8.00
8.01
8.02
8.02
8.21
8.21
8.22
0102030405060708090100110120130140150160170180f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.020
.97
36.6
237
.42
62.3
2
66.6
4
76.9
177
.16
77.4
2
115.
2711
5.96
120.
75
126.
55
132.
9813
3.52
147.
29
170.
90
O
OS
SO O
ONN N
S25
Compound 14 (CDCl3)
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.89
2.97
1.98
2.00
2.07
1.97
0.94
0.94
1.94
1.02
1.00
0.97
0.97
1.00
1.23
1.25
1.26
2.03
2.21
2.21
2.47
2.50
3.04
3.05
3.07
3.15
3.16
3.17
4.10
4.12
4.49
4.80
6.02
6.83
6.84
6.96
6.96
6.98
7.00
7.26
7.50
7.52
7.52
7.72
7.73
7.96
7.97
8.04
8.17
8.19
102030405060708090100110120130140150160f1 (ppm)
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.6511.3
6
14.6
2
36.6
437
.06
37.4
6
41.8
5
64.1
666
.63
109.
22
115.
2611
5.81
119.
0212
2.10
126.
4012
9.73
130.
8713
2.87
133.
4513
3.64
141.
33
147.
2314
9.49
151.
52
155.
76
NNB
F F HN OSSO
OO
ON
NN
S26
Compound 18 (DMSO-d6)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
1.99
1.96
2.95
2.62
2.07
2.09
1.93
1.92
2.22
1.12
1.11
0.71
0.79
0.86
2.06
4.16
1.09
2.13
2.13
1.04
1.00
1.72
2.03
2.07
2.12
2.30
2.50
3.04
3.13
3.17
3.33
3.74
4.05
4.32
4.72
4.84
5.90
6.58
6.94
7.23
7.26
7.57
7.60
7.81
7.82
8.22
8.23
8.26
8.81
8.82
11.8
1
102030405060708090100110120130140150160170f1 (ppm)
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
0.032
0.034
10.6
2
13.8
8
29.0
130
.51
39.1
939
.35
39.5
239
.69
39.8
540
.02
46.7
6
52.6
0
65.1
9
72.5
7
112.
9111
4.64
114.
9811
8.91
119.
4212
0.10
123.
1412
4.62
128.
2812
8.79
129.
2713
2.10
132.
3813
4.64
135.
8413
7.41
137.
7713
8.59
143.
26
149.
63
153.
59
160.
83
166.
0816
9.66
171.
72
N NB
F F N
O
OS
HN
O
OH
O
NHS
OH2N
O
S27
Compound 19 (CDCl3)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
3.09
3.08
3.07
2.14
1.67
2.00
1.19
1.03
1.03
2.02
1.04
1.10
1.10
1.00
1.03
2.02
2.21
2.50
3.00
3.12
4.46
4.47
4.48
4.48
4.49
4.49
4.78
4.79
4.80
5.88
6.05
6.73
6.75
6.84
6.85
6.98
6.99
7.01
8.00
8.10
0102030405060708090100110120130140150160170180190f1 (ppm)
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.010
0.011
0.012
0.013
0.014
0.015
0.016
0.017
0.018
0.019
0.020
0.021
11.3
9
14.7
1
23.2
0
31.5
8
36.6
437
.05
40.8
0
52.2
5
64.1
8
109.
25
119.
1012
2.21
129.
7213
0.90
133.
70
141.
48
149.
4115
1.69
155.
87
162.
71
170.
7517
2.43
N NB
F F HNO
OS
O
NHS
OH2N
S28
Compound 20 (CDCl3)
1.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
2.13
2.05
2.94
2.85
2.05
1.03
2.07
0.89
0.98
0.94
1.00
1.70
1.70
1.71
1.72
1.73
1.74
1.75
1.76
1.76
2.04
2.05
2.06
2.06
2.07
2.18
2.46
3.58
3.59
3.60
3.61
3.62
3.63
3.64
4.00
4.01
4.02
4.02
4.03
4.14
4.15
4.15
4.16
4.17
4.17
4.18
4.19
5.93
6.13
6.14
6.65
6.94
6.95
7.26
102030405060708090100110120130140150160f1 (ppm)
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
0.08011.0
9
14.3
3
34.4
8
47.5
347
.58
47.6
2
66.8
0
76.9
177
.16
77.4
1
110.
97
115.
6011
5.90
129.
9913
1.86
133.
4813
4.41
146.
59
161.
19
N NB
F F N
OH
S29
Compound 21 (DMSO-d6)
2.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
0.94
1.11
1.07
1.08
0.97
4.28
1.05
2.13
1.12
1.07
1.01
1.00
2.40
2.50
2.50
2.50
2.87
2.88
2.89
2.90
2.91
2.96
2.97
2.98
2.99
3.00
3.00
4.52
4.53
4.53
4.54
4.55
4.56
7.23
7.56
7.58
7.82
7.83
7.83
8.22
8.27
8.27
8.60
8.62
11.8
5
2030405060708090100110120130140150160170f1 (ppm)
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
26.0
1
39.0
239
.19
39.3
539
.52
39.6
939
.78
39.8
539
.94
40.0
2
55.9
8
118.
9112
0.26
123.
1012
4.60
126.
2612
8.32
129.
0412
9.29
129.
4313
2.09
132.
6413
5.93
137.
3913
8.56
143.
79
166.
10
171.
70
NH
OOH
O
NHHS
OH2N
S30
Compound 23a (CDCl3)
4.04.55.05.56.06.57.07.58.08.5f1 (ppm)
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
2.96
1.97
0.92
0.93
2.07
1.03
2.01
1.00
3.91
5.56
7.26
7.27
7.28
7.29
7.37
7.37
7.49
7.50
7.51
7.53
7.61
7.61
7.61
7.63
7.64
7.96
7.96
7.97
7.98
7.98
8.05
8.06
5060708090100110120130140150160170180190f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.052.5
0
66.3
9
76.8
477
.16
77.4
7
113.
2211
6.73
118.
18
127.
9812
9.08
131.
9913
4.16
134.
3013
5.31
150.
42
166.
6616
6.92
192.
30
O
ONH2
O
O
O
S31
Compound 23b (DMSO-d6)
2.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
0.69
3.01
1.98
1.99
0.99
1.02
1.06
0.99
1.01
1.00
2.50
3.31
3.85
5.75
6.87
7.08
7.08
7.10
7.10
7.48
7.48
7.91
7.92
8.00
8.02
8.26
8.27
8.28
8.28
8.64
8.64
102030405060708090100110120130140150160170180190200f1 (ppm)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.4538
.90
39.1
039
.31
39.5
239
.73
39.9
440
.15
52.3
7
66.8
4
111.
0011
4.46
117.
80
124.
8413
0.14
131.
4013
2.48
132.
5913
3.57
134.
55
147.
98
151.
33
165.
8716
6.02
191.
19
O
ONH2
O
O
O
Cl
NO2
S32
Compound 24 (DMSO-d6)
2.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
0.99
1.02
0.94
1.04
0.97
1.00
2.50
7.77
7.78
8.00
8.02
8.17
8.17
8.18
8.19
8.24
8.26
8.39
8.54
8.55
30405060708090100110120130140150160170180f1 (ppm)
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
38.8
939
.10
39.3
139
.52
39.7
339
.94
40.1
5
121.
0312
1.98
124.
0012
5.09
125.
8712
6.28
130.
1413
1.76
132.
2113
2.69
134.
6613
7.45
139.
35
147.
49
166.
7916
9.11
172.
12
NH
OOH
O
OH
NO2
Cl
S33
Compound 28 (CDCl3)
2.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
1.94
2.81
2.81
2.00
1.94
1.91
2.03
1.00
0.82
0.87
0.95
0.96
1.23
2.20
0.96
1.93
1.94
1.94
2.10
2.10
2.11
2.12
2.14
2.18
2.46
3.08
3.09
3.11
3.75
3.76
3.77
3.77
3.78
3.78
3.79
4.00
4.01
4.02
4.39
4.40
4.41
4.42
4.43
4.90
4.91
4.91
4.92
4.93
5.93
6.11
6.11
6.68
6.95
6.96
7.13
7.13
7.13
7.14
7.26
7.64
7.64
7.67
7.67
7.68
7.69
7.69
7.71
8.49
8.49
8.50
8.50
102030405060708090100110120130140150160f1 (ppm)
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
11.0
7
14.3
1
30.7
6
37.0
2
47.1
1
65.4
8
73.0
076
.91
77.1
677
.41
110.
60
116.
0611
9.28
120.
0212
1.07
130.
0713
2.42
133.
3713
4.13
137.
20
147.
06
149.
86
154.
17
159.
4216
0.98
N NB
F F N
O
O
SS
N
O
S34
Compound 30 (CDCl3)
1.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
2.93
3.00
2.10
2.09
2.07
2.03
0.95
0.94
1.03
1.01
0.97
2.23
2.51
2.91
2.92
2.93
2.98
3.00
3.01
3.90
3.91
3.92
4.48
4.49
4.51
6.05
6.88
6.89
6.99
7.00
7.01
7.26
8.08
102030405060708090100110120130140150160f1 (ppm)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
11.3
6
14.6
6
37.0
5
41.8
4
60.4
0
64.3
6
109.
29
119.
03
122.
15
129.
7813
0.91
133.
68
141.
35
149.
5715
1.65
155.
77
N NB
F F HN
OHS
SO
O
S35
Fluorescence monitoring of the conjugates 4-6 cleavage
Figure S1. Decreasing of emission intensity of conjugate 4 (left) upon excitation by 510 nm and increasing of emission intensity upon excitation by 480 nm (right) in time during cleavage of conjugate 4 (5µM) by GSH (5mM, DMSO/HEPES 2:1, pH 7.4, 37°C).
Figure S2. Increasing of emission intensity change of conjugate 5 upon excitation by 510 nm (left) and upon excitation by 480 nm (right) in time during cleavage of conjugate 5 (5µM) by GSH (5mM, 0.1M HEPES Buffer, pH 7.4, 37°C).
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Figure S3. Decreasing of emission intensity of conjugate 5 (left) upon excitation by 510 nm and increasing of emission intensity upon excitation by 480 nm (right) in time during cleavage of conjugate 5 (5µM) by GSH (5mM, DMSO/HEPES 2:1, pH 7.4, 37°C).
Figure S4. Increasing of emission intensity change of conjugate 6 upon excitation by 510 nm (left) and upon excitation by 480 nm (right) in time during cleavage of conjugate 6 (5µM) by GSH (5mM, 0.1M HEPES Buffer, pH 7.4, 37°C).
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Figure S5. Decreasing of emission intensity of conjugate 6 (left) upon excitation by 510 nm and increasing of emission intensity upon excitation by 480 nm (right) in time during cleavage of conjugate 6 (5µM) by GSH (5mM, DMSO/HEPES 2:1, pH 7.4, 37°C).
Figure S6. Comparison of the ratio of fluorescence emission at 525nm upon excitation at 480nm and 510nm (I480/I510) for the Probe 4 (5µM) in HEPES Buffer (0.1M, pH 7.4, 37°C) and DMSO/HEPES Buffer 2:1 (0.1M, pH 7.4, 37°C) during GSH (5mM) cleavage.
S38
Figure S7. Emission intensity change of conjugate 19 (5µM) upon excitation by 510 nm (left upper) and upon excitation by 480 nm (right upper) in time during cleavage by GSH (2.5mM, 0.1M HEPES Buffer, pH 7.4, 37°C) and representation by the fluorescence ratio (I480/I510) change in time (bottom).
Detection limitDetection limit (LOD) was calculated as follows:
𝐿𝑂𝐷 = 3·𝜎
𝑠
σ – standard deviation of response
s – slope of the calibration curve
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0 20 40 60 80 100
0.49
0.56I4
85/I5
10
Concentration ()
Equation y = a + b*xAdj. R-Square 0.99999
Value Standard ErrorF Intercept 0.48021 1.02533E-4F Slope 0.00101 1.82687E-6
0 20 40 60 80 100
0.42
0.49
0.56
I485
/I510
Concentration ()
Equation y = a + b*xAdj. R-Square 0.99994
Value Standard ErrorE Intercept 0.40474 4.21248E-4E Slope 0.00168 7.50555E-6
Figure S8. Detection limit determination for compounds 5 (LOD = 305 nM) and 6 (LOD = 752 nM). (GSH concentration 0 µM to 90µM, 0.1M HEPES Buffer, 7.4 pH, 37°C, 2h).
S40
References
(1) Ursuegui, S.; Yougnia, R.; Moutin, S.; Burr, a; Fossey, C.; Cailly, T.; Laayoun, a; Laurent, a; Fabis, F. Org. Biomol. Chem. 2015, 13 (12), 3625–3632.
(2) Soural, M.; Funk, P.; Kvapil, L.; Hradil, P.; Hlaváč, J.; Bertolasi, V. Arkivoc 2010, 2010 (10).
(3) Leen, V.; Leemans, T.; Boens, N.; Dehaen, W. European J. Org. Chem. 2011, No. 23, 4386–4396.
(4) Krajčovičová, S.; Gucký, T.; Hendrychová, D.; Kryštof, V.; Soural, M. J. Org. Chem. 2017, 82 (24), 13530–13541.
(5) Azéma, L.; Lherbet, C.; Baudoin, C.; Blonski, C. Bioorganic Med. Chem. Lett. 2006, 16 (13), 3440–3443.
