Download PDF
Synthesis 2014; 46(22): 3103-3109
DOI: 10.1055/s-0034-1378893
paper
© Georg Thieme Verlag Stuttgart · New York

Synthesis of Novel Fluorescent Stilbenenitrones via a Mild, Ligand-Free Heck-Type Reaction of (E)-[4-(1,3-Dioxolan-2-yl)styryl]trimethylsilane with Benzene Diazonium Tetrafluoroborate Derivatives

André Karbach
Department of Chemistry, Technical University Kaiserslautern, Gottlieb-Daimler-Straße 47, P.O.Box 3049, 67653 Kaiserslautern, Germany   Fax: +49(631)2053419   Email: karbach@chemie.uni-kl.de
,
Tobias Stemler
Department of Chemistry, Technical University Kaiserslautern, Gottlieb-Daimler-Straße 47, P.O.Box 3049, 67653 Kaiserslautern, Germany   Fax: +49(631)2053419   Email: karbach@chemie.uni-kl.de
,
Christian Kopp
Department of Chemistry, Technical University Kaiserslautern, Gottlieb-Daimler-Straße 47, P.O.Box 3049, 67653 Kaiserslautern, Germany   Fax: +49(631)2053419   Email: karbach@chemie.uni-kl.de
,
Wolfgang E. Trommer*
Department of Chemistry, Technical University Kaiserslautern, Gottlieb-Daimler-Straße 47, P.O.Box 3049, 67653 Kaiserslautern, Germany   Fax: +49(631)2053419   Email: karbach@chemie.uni-kl.de
› Author Affiliations
Further Information

Publication History

Received: 04 June 2014

Accepted after revision: 07 July 2014

Publication Date:
06 August 2014 (online)

 
 PDF Download Permissions and Reprints All articles of this category


Abstract

Nitrones are important intermediates in organic syntheses. As spin traps they allow for trapping of short-lived radicals, mainly reactive oxygen species (ROS), under formation of relatively stable nitroxides. Fluorescent nitrones also offer the possibility to follow the formation of ROS with subcellular resolution. To this end, particularly stilbenenitrones have been employed. Here, we describe the synthesis of a series of stilbenenitrones from aldehyde precursors prepared by an optimized, mild Heck-type reaction. Rather unstable benzene diazonium salts required special conditions for the trans-selective reaction with styrylsilanes. Moreover, a new method was developed to produce stilbenenitrones quantitatively in most cases. Ab initio methods were used to clarify an anomaly with respect to absorption behavior.


#

Key words

ROS - spin traps - diazonium salts - nitrones - Heck reaction

The nitrone functional group is the N-oxide of an imine. Such groups are usually prepared by condensation reaction of carbonyls with N-monosubstituted hydroxylamines,[1] however, they can also be generated by oxidation of cyclic or aliphatic secondary amines and hydroxylamines in the presence of metal oxides, pertungstate ions, or oxaziridine salts.[2]

Nitrones are intermediates in a considerable number of organic syntheses. For example, they are used for the stereo­selective synthesis of isoxazolidines,[3a] for the synthesis of beta-lactams[3b] and also serve as substrates in radical polymerization.[3c]

Nitrones have also found wide application in the life sciences, for example in the trapping of short-lived radicals, mainly reactive oxygen species (ROS) as species such as the hydroxyl or the superoxide anion radical under formation of relatively stable nitroxides (Scheme [1]).[4] Whereas most ROS have very short half-lives, in the microsecond or even nanosecond range, the generated nitroxide radicals are stable for up to half an hour in spite of their α-hydrogen atoms and, thus, the original radicals can be characterized and identified by EPR spectroscopic analysis.[5] ROS are formed during normal metabolic reactions but can be the cause of many diseases and also play a role in aging.[6]

Zoom Image
Scheme 1 Example for the reaction of a fluorescent spin trap (fluorophore-tert-butyl nitrone) with a hydroxyl radical to give the spin-adduct (nitroxide radical)

In combination with fluorophores, nitrones can also be employed for the detection of ROS formation within cells with even subcellular resolution by means of confocal laser scanning microscopy. The fluorescence of a fluorophore covalently attached to the nitrone will be quenched by the generated nitroxide (Scheme [1]).[7] The opposite approach has also been tried by starting from fluorophores coupled to nitroxides, which are therefore non-fluorescent, but upon biological reduction of the nitroxide within a cell to an hydroxylamine, fluorescence is restored.[8]

The first successful application of this principle was described by us several years ago. tert-Butylnitrone was coupled to p-nitrostilbene, yielding 1d (Figure [1]). Nitrone 1d accumulated in mitochondria and allowed ROS formation to be followed in the respiratory chain depending upon the addition of different specific inhibitors of complexes I and III.[9a] [10] The absorption maximum of compound 1d is in the UV range (378 nm), which is a wavelength that may cause damage to cell components. Moreover, confocal laser microscopes are seldom equipped with lasers operating around 400 nm and below. Hence, we have synthesized a series of analogues with different substituents in the stilbene moiety with the expectation that these derivatives would absorb at a different frequency and may target different cell compartments.

Zoom Image
Figure 1 Structural formula of the N-tert-butyl-p-nitrostilbenenitrone (1d)

By following our published procedure for the synthesis of 1d,[9] i.e., reaction of (E)-[4-(1,3-dioxolan-2-yl)styryl]tri­methylsilane (3) with higher functionalized benzene diazonium salts 2, only traces of the aldehyde precursors 4 for the corresponding nitrones 1 were obtained. The use of trimethylsilane derivatives was preferred over the unsubstituted vinyl group because otherwise extensive decomposition of the diazonium salts can occur.[11] Moreover, this bulky residue favors formation of the trans-stilbene and is also a good leaving group.[12] Decomposition of the diazonium salts 2, however, was still too fast at room temperature. Lowering the temperature resulted in considerably longer reaction times.

Most unstable under the previous conditions was 2-meth­oxy-4-nitro-diazonium tetrafluoroborate (2a), with gas formation and a color change starting immediately after dissolution in methanol. Hence, 2a was employed in a screening of yield and stereochemistry of the product, E- versus Z-configuration of the stilbene, depending on the molar ratio of the reactants, mol% of the catalyst, the temperature, and the reaction time.

Expecting thermal instability of the diazonium salt 2a due to the high functionalization, a range of temperatures was explored. Table [1], entry 2 shows that the yield of 4a was significantly increased to 62% by cooling to below –10 °C during the first hour. Cooling over the entire reaction time resulted in a lower turnover, but the reaction was completely trans-selective (entry 3).

Table 1 Screening of a Mild Heck-Type Reaction with 2a

Entry

Ratio 2a/3

Catalyst (mol%)

Time (h)a

Time (h)b

Yield of 4a (%)c

Yield of 5a (%)c

Yield of 6 (%)c

1

1:1

 2

0

 6

 1

 0

99

2

1:1

 2

1

 5

62

 8

30

3

1:1

 2

6

 0

32

 0

68

4

1:1

 4

1

 5

71

 9

20

5

2:1

 2

1

 5

53

25

22

6

1:1

 2

3

15

82

 4

14

7

1:1

 2

3

15d

21

 0

79

8

1:1

20

1

 5

82

 9

 9

a Initial reaction time at a temperature below –10 °C.

b Subsequent reaction time at r.t.

c Crude product ratio determined by integration of the 1H NMR spectra (400 MHz).

d Subsequent reaction time at 4 °C.

It was necessary to balance the conditions that favored rapid formation of 4a instead of deprotection of 3 leading to 6 and/or cis-isomerization. This was achieved by increasing the amount of catalyst (Table [1], entries 4 and 8). However, for economic reasons, an alternative method was adopted that involved extending the reaction time at a temperature below –10 °C as well as increasing the subsequent reaction time at room temperature, resulting in the preferred conditions, which gave 4a in 82% yield (entry 6).

Table [2] summarizes the scope of this mild Heck-type reaction under the optimized conditions (Table [1], entry 6). Educts 2 were chosen to represent a wide range of benzene diazonium salts, with auxochrome, antiauxochrome substituents, and some of their combinations. Comparing the results presented in Table [2] (entries 1, 3, 4, 5 and 7) reveals rather similar relative yields. This does not hold for 2b, which is a regioisomer of 2a (Table [2], entries 1 and 2); in the case of 2b, the trans-selectivity of the reaction was reduced substantially, which may be due to steric hindrance. Compound 2i failed to react at all (entry 9), which could be expected because the hydroxyl group is likely to interact with the Pd(II) species. However, the reason for the pronounced cis-selectivity in case of 2h is not yet clear (entry 8). The lower yield with the sulfonate 2f is probably due to its poor solubility in methanol.

For the synthesis of the corresponding stilbenenitrones 1, our published procedure using 4d was modified to include repeated addition of the hydroxylamine every two days for a total of 10 days under slightly alkaline conditions and removal of the formed water by the addition of anhydrous magnesium sulfate (Table [3]). Whereas some reactions were quantitative (entries 1, 2, 5, and 6), the yields in other cases varied between 61 and 65% (entries 3, 4, 7, and 8).

Table 2 Scope of the Reaction with Substituted Benzene Diazonium Salts 2 with 3 under the Optimized Conditions (Table [1, ]Entry 6)

Entry

2

R1

R2

Yield of 4 (%)a

Yield of 5 (%)a

Yield of 6 (%)a

Isolated yield of 4 (%)b

1

2a

2-OMe

4-NO2

82

 4

 14

69

2

2b

2-NO2

5-OMe

66

28

  6

52

3

2c

H

H

79

13

  8

72

4

2d

4-NO2

H

87

 9

  4

81

5

2e

4-OMe

H

84

 8

  8

76

6

2f

4-SO3H

H

38

 8

 54

25c

7

2g

4-Cl

H

80

11

  9

60

8

2h

4-NMe2

H

48

39

 13

36

9

2i

2-OH

4-NO2

 0

 0

100

 0

a Relative yields of the crude reaction products based on 1H NMR spectroscopic analysis (400 MHz).

b Purification by MPLC.

c Crystallization during processing.

Table 3 Synthesis of Stilbenenitrone Derivatives 1

Entry

4

R1

R2

Yield (%)a

Absorption (nm)b

Fluorescence (nm)b

1

4a

2-OMe

4-NO2

100

395

516

2

4b

2-NO2

5-OMe

100

326

444

3

4c

H

H

 61

347

392

4

4d

4-NO2

H

 65

378

510

5

4e

4-OMe

H

100

356

396

6

4f

4-SO3H

H

100

319

363

7

4g

4-Cl

H

 64

351

392

8

4h

4-NMe2

H

 65

371

474

a Isolated yield of 1 after purification by MPLC.

b Measured in EtOAc.

Although already shown for compound 1d,[9a] [10] nitrone 1a was used to explore the anticipated spin-trapping of hydroxyl radicals generated by the Fenton reaction. Figure [2] shows the EPR spectrum of the generated nitroxide radical.

Zoom Image
Figure 2 EPR spectrum of the generated nitroxide radical by reaction of 1a with Fenton’s reagent

The radical-dependent fluorescence decrease of 1a after incubation with Fenton’s reagent is shown in Figure [3]. After the total addition of 500 μL of a 100 mM solution, the fluorescence was almost completely quenched.

Zoom Image
Figure 3 Fluorescence quenching of 1a after incubation with ­Fenton’s reagent

The anticipated bathochromic shift of the absorption maximum relative to p-nitrostilbenenitrone 1d was only weakly achieved with 1a. Because of the large difference in the absorption maxima of 1a and 1b, the latter was initially believed to be the cis-derivative. Moreover, TDDFT calculations gave very similar values for 1a (391 nm) and 1b (383 nm). However, NMR data including NOESY clearly support the trans-configuration.

In summary, we have found conditions for a mild Heck-type coupling of highly functionalized aromatic diazonium salts with styrylsilanes and their subsequent conversion into the corresponding nitrones.

Commercially available starting materials were purchased from Sigma–Aldrich, Santa Cruz Biotechnology and Acros Organics, and used without further purification. Solvents were dried by standard methods, stored over 4 Å molecular sieves and degassed by a minimum of three freeze–pump–thaw cycles before use.[13] Syntheses of aldehyde and nitrone compounds were carried out under an inert atmosphere using dried glassware. Melting points were determined with a SRS DigiMelt MPA161 and are uncorrected. IR spectra were recorded with a Perkin Elmer FT-IR Spectrum 100 with a universal ATR accessory. NMR spectra were recorded with Bruker Avance DPX 200 and 400 spectrometers with CDCl3 as solvent. Chemical shifts are reported in ppm with reference to TMS at δ = 0.0 ppm and coupling constants (J) in units of Hz. The multiplicities are defined as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd doublet of doublets; td, triplet of doublets. High-resolution mass spectra (HRMS) were acquired with a GC-MS-TOF spectrometer using EI (Waters GCT premier micromass). EPR spectra were recorded in a dielectric cavity (ER4118 X-MD-5) at ambient temperature with a Bruker BioSpin Elexsys E 580 spectrometer operating at X-band in cw-mode with 100 kHz modulation. Fluorescence measurements were carried out at r.t. with a Perkin Elmer luminescence spectrometer LS 50B. Thin-layer chromatography (TLC) was performed on Macherey-Nagel Polygram Sil G/UV254 with 0.2 mm silica gel. Flash column chromatography was conducted using silica gel (40–63 μm) with different mixtures of EtOAc, petroleum ether (PE; boiling range 40–60 °C) and triethylamine as solvents. Benzenediazonium salts (2) were prepared from the corresponding aniline derivatives by using a reported procedure.[14]


#

2-(4-Bromobenzene)-[1,3]-dioxolane (8)[9] [15]

To a solution of 4-bromobenzaldehyde (20.0 g, 107 mmol) in toluene (160 mL) were added ethylene glycol (39.8 g, 35.8 mL, 642 mmol) and a catalytic amount of p-toluenesulfonic acid (204 mg, 1.07 mmol). The round-bottomed flask was fitted with a Dean–Stark trap and the mixture was heated at reflux for 22 h. The toluene solution was then poured into H2O (150 mL) and the organic phase was separated. The aqueous phase was extracted with Et2O (3 × 100 mL) and the combined organic layers were dried (MgSO4), filtered, and the solvent was evaporated under reduced pressure. The yellow oily residue was purified by vacuum distillation (3.1 × 10–1 mbar, 131–137 °C) to afford 8.

Yield: 22.7 g (93%); colorless oil.

IR (ATR): 2951, 2883, 1075, 1011, 814 cm–1.

1H NMR (400 MHz, CDCl3): δ = 3.99–4.15 (m, 4 H), 5.79 (s, 1 H), 7.37 (d, J = 8.5 Hz, 2 H), 7.54 (d, J = 8.5 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 65.3, 103.0, 123.2, 128.1, 131.4, 137.0.

HRMS (EI): m/z [M]+ calcd for C9H9BrO2: 227.9786 and 229.9766; found: 227.9784 and 229.9772.


#

(E)-[4-(1,3-Dioxolan-2-yl)styryl]trimethylsilane (3)[9] [16]

Compound 8 (9.16 g, 40.0 mmol) was dissolved in anhydrous acetonitrile (100 mL) and tetrabutylammonium acetate (31.1 g, 100 mmol), palladium(II) acetate (269 mg, 1.20 mmol) and vinyltrimethylsilane (8.27 g, 80 mmol) were added at r.t. The reaction mixture was heated at reflux for 28 h and the resulting black solution was diluted with H2O (80 mL). The organic phase was separated, the aqueous phase was extracted with Et2O (2 × 150 mL) and the combined organic layers were dried (MgSO4), filtered, and concentrated under vacuum. The residue was purified by vacuum distillation (2.9 × 10–2 mbar, 141–146 °C) to afford silane 3.

Yield: 9.88 g (99%); colorless oil.

IR (ATR): 2954, 2887, 1247, 1081, 835 cm–1.

1H NMR (400 MHz, CDCl3): δ = 0.00 (s, 9 H), 3.78–3.97 (m, 4 H), 5.61 (s, 1 H), 6.34 (d, J = 19.1 Hz, 1 H), 6.72 (d, J = 19.1 Hz, 1 H), 7.28 (s, 4 H).

13C NMR (100 MHz, CDCl3): δ = –1.38, 65.1, 103.4, 126.2, 126.5, 130.1, 137.4, 139.1, 143.0.

HRMS (EI): m/z [M]+ calcd for C14H20O2Si: 248.1233; found: 248.1231.


#

Synthesis of Formyl-trans-stilbenes 4a–h; General Procedure

To a solution of the corresponding benzenediazonium tetrafluoro­borate salt 2 (4 mmol) in anhydrous MeOH (50 mL) were quickly added palladium(II) acetate (0.08 mmol) and 3 (4 mmol) at a temperature of –10 °C or below. The mixture was stirred at this temperature for 3 h and then allowed to warm to r.t. overnight. The reaction mixture was poured into H2O (100 mL) and the organic phase was separated. The aqueous phase was extracted with EtOAc (3 × 100 mL) and the combined organic layers were dried (MgSO4), filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (silica gel; PE–EtOAc) to give the desired aldehyde 4.


#

(E)-4-(2-Methoxy-4-nitrostyryl)benzaldehyde (4a)

Obtained from 2a (1.07 g, 4 mmol) and 3 (994 mg, 4 mmol).

Yield: 784 mg (69%); yellow solid; mp 154–156 °C; Rf = 0.30 (PE–EtOAc, 5:1).

IR (ATR): 3070, 2972, 2837, 2752, 1697, 1600, 1339, 1250 cm–1.

1H NMR (400 MHz, CDCl3): δ = 4.03 (s, 3 H), 7.31 (d, J = 16.6 Hz, 1 H), 7.62 (d, J = 16.6 Hz, 1 H), 7.70–7.76 (m, 3 H), 7.79 (d, J = 2.2 Hz, 1 H), 7.87–7.93 (m, 3 H), 10.03 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 56.2, 86.2, 106.1, 116.2, 124.9, 126.7, 127.5, 130.3, 131.7, 132.3, 135.9, 142.8, 157.1, 191.6.

HRMS (EI): m/z [M]+ calcd for C16H13NO4: 283.0845; found: 283.0846.


#

(E)-4-(5-Methoxy-2-nitrostyryl)benzaldehyde (4b)

Obtained from 2b (1.07 g, 4 mmol) and 3 (994 mg, 4 mmol).

Yield: 584 mg (52%); yellow solid; mp 155–157 °C; Rf = 0.32 (PE–EtOAc, 4:1).

IR (ATR): 3082, 3007, 2826, 2742, 1690, 1602, 1295, 1209 cm–1.

1H NMR (400 MHz, CDCl3): δ = 3.97 (s, 3 H), 6.94 (dd, J = 9.2, 2.8 Hz, 1 H), 7.05 (d, J = 16.1 Hz, 1 H), 7.16 (d, J = 2.8 Hz, 1 H), 7.71 (d, J = 8.3 Hz, 2 H), 7.90 (d, J = 16.1 Hz, 1 H), 7.92 (d, J = 8.3 Hz, 2 H), 8.15 (d, J = 9.2 Hz, 1 H), 10.04 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 31.0, 85.6, 113.5, 127.6, 127.9, 128.5, 130.3, 132.1, 135.8, 143.5, 150.3, 151.4, 163.4, 191.6.

HRMS (EI): m/z [M]+ calcd for C16H13NO4: 283.0845; found: 283.0846.


#

(E)-4-Styrylbenzaldehyde (4c)

Obtained from 2c (768 mg, 4 mmol) and 3 (994 mg, 4 mmol).

Yield: 597 mg (72%); white solid; mp 107–110 °C; Rf = 0.35 (PE–EtOAc, 4:1).

IR (ATR): 3030, 2820, 2724, 1694, 1592, 964 cm–1.

1H NMR (400 MHz, CDCl3): δ = 7.16 (d, J = 16.2 Hz, 1 H), 7.28 (d, J = 16.2 Hz, 1 H), 7.34 (m, 1 H), 7.16 (m, 2 H), 7.57 (m, 2 H), 7.67 (d, J = 8.3 Hz, 2 H), 7.89 (d, J = 8.3 Hz, 2 H), 10.01 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 126.9, 127.3, 128.5, 128.9, 130.3, 132.2, 135.3, 136.5, 143.4, 176.4, 191.7.

HRMS (EI): m/z [M]+ calcd for C15H12O: 208.0888; found: 208.0890.


#

(E)-4-(4-Nitrostyryl)benzaldehyde (4d)

Obtained from 2d (948 mg, 4 mmol) and 3 (994 mg, 4 mmol).

Yield: 822 mg (81%); yellow solid; mp 212–216 °C; Rf = 0.26 (PE–EtOAc, 6:1).

IR (ATR): 2958, 2924, 2822, 2717, 1687, 1600, 1501, 1330 cm–1.

1H NMR (400 MHz, CDCl3): δ = 7.23 (s, 1 H), 7.27 (s, 1 H), 7.66 (m, 4 H), 7.89 (d, J = 8.5 Hz, 2 H), 8.22 (d, J = 8.5 Hz, 2 H), 10.0 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 124.2, 127.3, 127.5, 129.6, 130.3, 131.8, 136.1, 142.0, 142.9, 147.3, 191.5.

HRMS (EI): m/z [M]+ calcd for C15H11NO3: 253.0739; found: 253.0738.


#

(E)-4-(4-Methoxystyryl)benzaldehyde (4e)

Obtained from 2e (888 mg, 4 mmol) and 3 (994 mg, 4 mmol).

Yield: 720 mg (76%); pale-yellow solid; mp 131–133 °C; Rf = 0.31 (PE–EtOAc, 8:1).

IR (ATR): 3024, 2935, 2837, 2749, 1681, 1596, 1508, 1246, 1175, 1022, 967, 831 cm–1.

1H NMR (400 MHz, CDCl3): δ = 3.86 (s, 3 H), 6.94 (d, J = 8.3 Hz, 2 H), 7.02 (d, J = 16.3 Hz, 1 H), 7.24 (d, J = 16.3 Hz, 1 H), 7.51 (d, J = 8.3 Hz, 2 H), 7.64 (d, J = 8.8 Hz, 2 H), 7.87 (d, J = 8.8 Hz, 2 H), 9.99 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 55.4, 114.3, 125.2, 126.3, 126.6, 128.3, 129.3, 130.3, 131.8, 135.0, 143.9, 191.7.

HRMS (EI): m/z [M]+ calcd for C16H14O2: 238.0994; found: 238.1001.


#

(E)-4-(4-Sulfostyryl)benzaldehyde (4f)

Obtained from 2f (1.63 g, 6 mmol) and 3 (1.49 g, 6 mmol).

Yield: 430 mg (25%); beige solid; mp 241–243 °C.

IR (ATR): 3399, 3044, 3018, 2896, 2860, 1687, 1606, 1190, 1126, 1035, 829 cm–1.

1H NMR (400 MHz, CDCl3): δ = 3.35 (s, 1 H), 7.40 (d, J = 16.1 Hz, 1 H), 7.49 (d, J = 16.1 Hz, 1 H), 7.24 (br s, 4 H), 7.84 (d, J = 8.3 Hz, 2 H), 7.91 (d, J = 8.3 Hz, 2 H), 9.99 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 126.0, 126.3, 127.1, 127.7, 130.0, 131.5, 135.1, 136.5, 143.1, 148.2, 192.4.


#

(E)-4-(4-Chlorostyryl)benzaldehyde (4g)

Obtained from 2g (582 mg, 2.57 mmol) and 3 (639 mg, 2.57 mmol).

Yield: 373 mg (60%); white solid; mp 134–137 °C; Rf = 0.35 (PE–EtOAc, 5:1).

IR (ATR): 3026, 2828, 2737, 1690, 1598, 1165, 971, 829, 790 cm–1.

1H NMR (400 MHz, CDCl3): δ = 7.13 (d, J = 16.2 Hz, 1 H), 7.22 (d, J = 16.1 Hz, 1 H), 7.37 (m, 2 H), 7.49 (m, 2 H), 7.66 (d, J = 8.3 Hz, 2 H), 7.89 (d, J = 8.3 Hz, 2 H), 10.0 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 127.0, 127.9, 128.1, 129.1, 130.3, 130.8, 134.2, 135.1, 135.5, 143.0, 191.6.

HRMS (EI): m/z [M]+ calcd for C15H11ClO: 242.0498; found: 242.0496.


#

(E)-4-(4-Dimethylaminostyryl)benzaldehyde (4h)

Obtained from 2h (940 mg, 4 mmol) and 3 (994 mg, 4 mmol).

Yield: 366 mg (36%); dark-yellow solid; mp 212–214 °C; Rf = 0.29 (PE–EtOAc, 8:1).

IR (ATR): 3021, 2892, 2851, 2803, 1686, 1584, 1352, 1162, 967, 820 cm–1.

1H NMR (400 MHz, CDCl3): δ = 3.02 (s, 6 H), 6.73 (m, 2 H), 6.95 (d, J = 16.1 Hz, 1 H), 7.22 (d, J = 16.1 Hz, 1 H), 7.46 (m, 2 H), 7.61 (d, J = 8.3 Hz, 2 H), 7.84 (d, J = 8.3 Hz, 2 H), 9.97 (s, 1 H).

13C NMR (100 MHz, CDCl3): δ = 40.4, 112.24, 122.7, 126.3, 128.2, 129.2, 129.6, 130.3, 131.5, 132.5, 134.5, 191.7.

HRMS (EI): m/z [M]+ calcd for C17H17NO: 251.1310; found: 251.1315.


#

Stilbene Nitrones; General Procedure

To a well-stirred solution of aldehyde 4 (0.5 mmol) in anhydrous CH2Cl2 (50 mL) was added a mixture of N-tert-butylhydroxylamine hydrochloride (502 mg, 4 mmol) and Et3N (445 mg, 612 μL, 4.4 mmol) in anhydrous CH2Cl2 (10 mL) at r.t. in one portion. During stirring for 9 d at ambient temperature, a fresh, extra solution of N-tert-butylhydroxylamine hydrochloride (502 mg, 4 mmol) and Et3N (445 mg, 612 μL, 4.4 mmol) in anhydrous CH2Cl2 (10 mL) was added every second day. After each addition, a small amount of anhydrous MgSO4 (481 mg, 4 mmol) was also added. Upon completion, the mixture was filtered, the filtrate was evaporated to dryness, and the obtained solid was washed with H2O (3 × 20 mL), dissolved in EtOAc, and dried with MgSO4. After evaporation of the solvent, the crude product was purified by column chromatography (silica gel; PE–EtOAc–Et3N), if required, to afford the fluorescent nitrone 1.


#

(Z)-N-{4-[(E)-2-methoxy-4-nitrostyryl]benzylidene}-2-methylpropan-2-amine Oxide (1a)

Obtained from 4a (200 mg, 706 μmol).

Yield: 249 mg (100%); yellow solid; mp 203–206 °C.

IR (ATR): 3083, 2981, 2921, 2853, 1510, 1340, 1248, 1089, 969, 860, 831 cm–1.

1H NMR (400 MHz, CDCl3): δ = 3.02 [s, 9 H, C(CH3)3], 4.01 (s, 3 H, H-4), 7.28 (d, J = 16.6 Hz, 1 H, H-5), 7.54 (d, J = 16.6 Hz, 1 H, H-6), 7.57 (s, 1 H, H-7), 7.62 (d, J = 8.4 Hz, 2 H, H-8,9), 7.72 (d, J = 8.5 Hz, 1 H, H-10), 7.77 (d, = 2.1 Hz, 1 H, H-11), 7.88 (dd, J = 8.5, 2.1 Hz, 1 H, H-12), 8.31 (d, J = 8.4 Hz, 2 H, H-13,14).

13C NMR (100 MHz, CDCl3): δ = 28.4 (C1,2,3), 56.1 (C4), 71.0 (C5), 106.0 (C6), 116.2 (C7), 118.4 (C8), 122.5 (C9), 126.3 (C10), 127.1 (C11,12), 129.1 (C13,14), 129.5 (C15), 131.0 (C16), 132.6 (C17), 138.4 (C18), 147.7 (C19), 156.9 (C20).

HRMS (EI): m/z [M]+ calcd for C20H22N2O4: 354.1580; found: 354.1578.

UV/Vis (EtOAc): λmax (ε) = 261 (3070), 326 (6940), 395 (13910) nm.


#

(Z)-N-{4-[(E)-5-Methoxy-2-nitrostyryl]benzylidene}-2-methylpropan-2-amine Oxide (1b)

Obtained from 4b (283 mg, 1 mmol).

Yield: 353 mg (100%); yellow solid; mp 137–139 °C.

IR (ATR): 3077, 2980, 2937, 2845, 1501, 1293, 1126, 1086, 863, 831 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.64 (s, 9 H), 3.95 (s, 3 H), 6.90 (dd, J = 9.2, 2.8 Hz, 1 H), 7.04 (d, J = 16.4 Hz, 1 H), 7.16 (d, J = 2.8 Hz, 1 H), 7.58 (s, 1 H), 7.72 (d, J = 8.5 Hz, 2 H), 7.61 (d, J = 16.4 Hz, 1 H), 7.88 (d, J = 9.2 Hz, 1 H), 8.32 (d, J = 8.4 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 56.0, 71.0, 112.8, 113.5, 125.8, 127.1, 127.7, 129.1, 129.5, 131.1, 133.0, 136.9, 138.0, 141.0, 163.2.

HRMS (EI): m/z [M]+ calcd for C20H22N2O4: 354.1580; found: 354.1575.

UV/Vis (EtOAc): λmax (ε) = 326 (6554), 363 (5637) nm.


#

(Z)-2-Methyl-N-{4-[(E)-styryl]benzylidene}propan-2-amine Oxide (1c)

Obtained from 4c (104 mg, 0.5 mmol).

Yield: 85 mg (61%); white solid; mp 142–145 °C; Rf = 0.39 (PE–EtOAc–Et3N, 1:2:0.01).

IR (ATR): 3026, 2975, 2934, 2873, 1359, 1178, 1124, 966, 830, 722, 688 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.64 (s, 9 H), 7.12 (d, J = 16.2 Hz, 1 H), 7.20 (d, J = 16.2 Hz, 1 H), 7.29 (dd, J = 7.5, 7.5 Hz, 1 H), 7.38 (t, J = 7.5, 7.5 Hz, 2 H), 7.54 (m, 2 H), 7.56 (s, 1 H), 7.88 (d, J = 8.5 Hz, 2 H), 8.32 (d, J = 8.5 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 71.0, 126.5, 126.7, 127.9, 128.0, 128.8, 129.1, 129.7, 129.9, 130.2, 137.1, 139.0.

HRMS (EI): m/z [M]+ calcd for C19H21NO: 279.1623; found: 279.1617.

UV/Vis (EtOAc): λmax (ε) = 276 (7124), 332 (23278), 347 (27928), 360 (24805) nm.


#

(Z)-2-Methyl-N-{4-[(E)-4-nitrostyryl]benzylidene}propan-2-amine Oxide (1d)

Obtained from 4d (127 mg, 0.5 mmol).

Yield: 105 mg (65%); yellow solid; mp 204–207 °C; Rf = 0.35 (PE–EtOAc–Et3N, 1:2:0.01).

IR (ATR): 3102, 2983, 2925, 2854, 1522, 1338, 1107, 964, 861 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.64 (s, 9 H), 7.22 (d, J = 16.5 Hz, 1 H), 7.28 (d, J = 16.5 Hz, 1 H), 7.58 (s, 1 H), 7.61 (d, J = 8.6 Hz, 2 H), 7.66 (d, J = 8.8 Hz, 2 H), 8.24 (d, J = 8.8 Hz, 2 H), 8.33 (d, J = 8.6 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 71.1, 124.2, 127.0, 127.1, 127.3, 129.2, 129.4, 131.3, 132.5, 137.6, 143.6, 146.9.

HRMS (EI): m/z [M]+ calcd for C19H20N2O3: 324.1473; found: 324.1485.

UV/Vis (EtOAc): λmax (ε) = 262 (3923), 317 (9615), 378 (19083) nm.


#

(Z)-N-{4-[(E)-4-Methoxystyryl]benzylidene}-2-methylpropan-2-amine Oxide (1e)

Obtained from 4e (119 mg, 0.5 mmol).

Yield: 249 mg (100%); yellow solid; mp 210–215 °C.

IR (ATR): 2999, 2968, 2931, 2838, 1508, 1246, 1177, 1034, 837 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.63 (s, 9 H), 3.85 (s, 3 H), 6.92 (m, 2 H), 6.99 (d, J = 16.4 Hz, 1 H), 7.16 (d, J = 16.4 Hz, 1 H), 7.58 (m, 2 H), 7.53 (d, J = 8.5 Hz, 2 H), 7.54 (s, 1 H), 8.28 (d, J = 8.5 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 55.4, 70.7, 114.2, 125.9, 126.2, 127.9, 129.1, 129.4, 129.8, 129.9, 131.6, 139.4, 159.6.

HRMS (EI): m/z [M]+ calcd for C20H23NO2: 309.1729; found: 309.1737.

UV/Vis (EtOAc): λmax (ε) = 279 (11839), 334 (35105), 356 (36376), 369 (31674) nm.


#

(Z)-2-Methyl-N-{4-[(E)-4-sulfostyryl]benzylidene}propan-2-amine Oxide (1f)

Obtained from 4f (204 mg, 706 μmol).

Yield: 253 mg (100%); beige solid; mp 209–211 °C.

IR (ATR): 2962, 2932, 2874, 1199, 1030, 1177, 727, 704 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.62 (s, 9 H), 7.10 (d, J = 16.3 Hz, 1 H), 7.18 (d, J = 16.3 Hz, 1 H), 7.48 (d, J = 8.3 Hz, 2 H), 7.55 (s, 1 H), 7.56 (d, J = 8.5 Hz, 2 H), 7.91 (d, J = 8.3 Hz, 2 H), 8.28 (d, J = 8.5 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 59.9, 126.1, 126.5, 126.7, 128.3, 129.1, 129.5, 129.7, 130.2, 137.6, 138.9, 146.6.

HRMS (EI): m/z [M]+ calcd for C19H21NO4S: 359.1191; found: 359.1180.

UV/Vis (EtOAc): λmax (ε) = 319 (39640), 334 (31596), 368 (10788) nm.


# (Z)-N-{4-[(E)-4-Chlorostyryl]benzylidene}-2-methylpropan-2-amine Oxide (1g)

Obtained from 4g (121 mg, 0.5 mmol).

Yield: 100 mg (64%); white solid; mp 196–198 °C, Rf = 0.36 (PE–EtOAc–Et3N, 1:2:0.01).

IR (ATR): 3019, 2977, 2938, 1362, 1131, 970, 845, 825 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.64 (s, 9 H), 7.08 (d, J = 16.1 Hz, 1 H), 7.15 (d, J = 16.1 Hz, 1 H), 7.34 (m, 2 H), 7.46 (m, 2 H), 7.56 (s, 1 H), 7.56 (d, J = 8.4 Hz, 2 H), 8.30 (d, J = 8.4 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 70.8, 126.6, 127.8, 128.5, 128.6, 128.9, 129.1, 133.5, 135.6, 137.6, 138.6, 142.4.

HRMS (EI): m/z [M]+ calcd for C19H20ClNO: 313.1234; found: 313.1232.

UV/Vis (EtOAc): λmax (ε) = 319 (14771), 334 (16621), 351 (17119) nm.

(Z)-N-(4-{(E)-4-[Dimethylamino]styryl}benzylidene)-2-methylpropan-2-amine Oxide (1h)

Obtained from 4h (200 g, 706 μmol).

Yield: 249 mg (100%); yellow solid; mp 157–159 °C, Rf = 0.40 (PE–EtOAc–Et3N, 1:2:0.01).

IR (ATR): 3093, 2972, 2929, 2801, 1521, 1359, 1190, 1106, 811 cm–1.

1H NMR (400 MHz, CDCl3): δ = 1.63 (s, 9 H), 3.00 (s, 6 H), 6.72 (m, 2 H), 6.92 (d, J = 16.2 Hz, 1 H), 7.07 (d, J = 16.2 Hz, 1 H), 7.44 (m, 2 H), 7.53 (d, J = 8.4 Hz, 1 H), 7.53 (s, 1 H), 8.26 (d, J = 8.4 Hz, 2 H).

13C NMR (100 MHz, CDCl3): δ = 28.4, 40.4, 70.4, 112.4, 123.6, 125.9, 127.8, 129.2, 129.3, 130.0, 131.4, 140.0, 141.0, 144.4.

HRMS (EI): m/z [M]+ calcd for C21H26N2O: 322.2045; found: 322.2061.

UV/Vis (EtOAc): λmax (ε) = 272 (9667), 327 (15161), 371 (18688) nm.


#

EPR Measurement of 1a after Incubation with Fenton’s Reagent­

Compound 1a (1.77 mg, 5.00 mmol) was dissolved in degassed EtOAc (1 mL). To an aliquot (200 μL) of this solution were added aq hydrogen peroxide solution (100 mM, 400 μL) and aq iron(II) sulfate solution (100 mM, 400 μL). The mixture was vortexed vigorously for 2 min, then the organic layer was separated and the EPR spectrum was acquired in a capillary with the following instrument settings: conversion time: 81 ms; microwave power: 20 mW; modulation amplitude: 1 G; receiver gain: 90 dB; spectrum width: 80 G; number of points: 2048.


#

Fluorescence Measurements of 1a after Incubation with Fenton­’s Reagent

To a solution of 1a in EtOAc (100 nM) was added aq hydrogen peroxide (100 mM, 25 μL) and aq iron(II) sulfate (100 mM, 25 μL) and the mixture was thoroughly mixed for 1 min. Excitation at 395 nm and emission at 516 nm. This procedure was repeated with additional 25, 50, and 150 μL (Fe2+/H2O2).


#

Ab Initio Calculations for the Absorption Maxima of 1a and 1b; Time-Dependent Density Functional Theory (TDDFT)

B3LYP and PBE0 functional with the basis theorems TZVP and def2-TZVP. m4 was chosen as integration grid. The geometry optimizations were performed with Gaussian 09 / TURBOMOLE program package (version 6.5). Solvent effects were simulated with COSMO.

TZVP-basis (B3LYP) transition beta-electron from orbital 93 to 95: 1a = 392.85 nm, 1b = 380.73 nm.

def2-TZVP basis (B3LYP) transition alpha-electron from orbital 93 to 95: 1a = 389.61 nm, 1b = 386.27 nm.


#
#

Acknowledgment

We are grateful to Ruth Bergsträßer for the HRMS measurements, as well as to Andreas Molberg for the TDDFT calculations.

Supporting Information

    for this article is available online at http://www.thieme-connect.com.accesdistant.sorbonne-universite.fr/products/ejournals/journal/ 10.1055/s-00000084.
  • Supporting Information


   Permissions and Reprints All articles of this category
Zoom Image
Scheme 1 Example for the reaction of a fluorescent spin trap (fluorophore-tert-butyl nitrone) with a hydroxyl radical to give the spin-adduct (nitroxide radical)
Zoom Image
Figure 1 Structural formula of the N-tert-butyl-p-nitrostilbenenitrone (1d)
Zoom Image
Figure 2 EPR spectrum of the generated nitroxide radical by reaction of 1a with Fenton’s reagent
Zoom Image
Figure 3 Fluorescence quenching of 1a after incubation with ­Fenton’s reagent