Chemical Research in Toxicology
Article
3
7.45 (m, 3H, 2 x meta-ArH and para-ArH), 7.47 (d, 1H, JHH = 15.9
2-(13C)Cinnamyl Alcohol 1b. The title compound was prepared
according to the general procedure described above starting from 2-
(13C)cinnamaldehyde 2b (157 mg, 1.18 mmol) to give 2-(13C)-
cinnamyl alcohol 1b (135 mg, 1.00 mmol, 85% yield, E/Z > 96%) as a
3
Hz, H3), 7.53−7.59 (m, 2H, ortho-ArH), 9.70 (d, 1H, JHH = 7.7 Hz,
H1); 13C NMR (75 MHz, CDCl3) δ 128.5 (2C, meta-ArCH), 128.6
(C2), 129.2 (2C, ortho-ArCH), 131.4 (para-ArCH), 134.1 (ipso-ArC),
152.9 (C3), 193.7 (C1).
1
white solid: H NMR (500 MHz, CDCl3) δ 2.02 (br., 1H, − OH),
1
3
1-(13C)Cinnamaldehyde 2a. The title compound was prepared
according to the general procedure described above starting from 1-
(13C)acetonitrile 4a (1.26 mL, 24 mmol, 2 equiv) and benzaldehyde 5
(1.22 mL, 12 mmol, 1 equiv) to give 2a (0.732 g, 5.50 mmol, 46%
4.32 (m, 2H, H1), 6.37 (ddt, 1H, JHC = 151.9 Hz, JHH = 16.1 Hz,
3JHH = 5.5 Hz, H2), 6.62 (d, 1H, JHH = 16.1 Hz, H3), 7.24−7.40 (m,
3
5H, ArH); 13C NMR (125 MHz, CDCl3) δ 63.7 (d, JCC = 27.9 Hz,
1
3
C1), 126.6 (d, 2C, JCC = 2.7 Hz, ortho-ArCH), 127.8 (para-ArCH),
128.6 (13C2), 128.7 (2C, 2 x meta-ArCH), 131.2 (d, JCC = 43.6 Hz,
1
1
yield) as a yellow oil (E/Z > 96%): H NMR (300 MHz, CDCl3) δ
6.70 (ddd, 1H, JHH = 16.0 Hz, JHH = 7.7 Hz, JHC = 1.3 Hz, H2),
3
3
2
C3), 136.8 (ipso-ArC).
3-(13C)Cinnamyl Alcohol 1c. The title compound was prepared
according to the general procedure described above starting from 3-
(13C)cinnamaldehyde 2c (375 mg, 2.82 mmol) to give 1-(13C)
cinnamyl alcohol 1c (330 mg, 2.48 mmol, 88% yield, E/Z > 96%) as a
3
7.40−7.47 (m, 3H, 2 x meta-ArH and para-ArH), 7.48 (d, 1H, JHH
=
=
1
16.0 Hz, H3), 7.52−7.58 (m, 2H, ortho-ArH), 9.67 (dd, 1H, JHC
172.8 Hz, JHH = 7.7 Hz, H1); 13C NMR (75 MHz, CDCl3) δ 128.5
3
1
(2C, meta-ArCH), 128.6 (d, JCC = 55.4 Hz, C2), 129.2 (2C, ortho-
1
ArCH), 131.4 (para-ArCH), 134.0 (d, 3JCC = 7.6 Hz, ipso-ArC), 152.8
white solid: H NMR (300 MHz, CDCl3) δ 1.49 (br., 1H, − OH),
1
2
(d, JCC = 4.3 Hz, C3), 193.6 (13C1).
4.32−4.35 (m, 2H, H1), 6.32−6.42 (m, 1H, H2), 6.62 (ddt, 1H, JHC
3
4
2-(13C)Cinnamaldehyde 2b. The title compound was prepared
according to the general procedure described above starting from 2-
(13C)acetonitrile 4b (1.36 mL, 26 mmol, 2 equiv) and benzaldehyde 5
(1.32 mL, 13 mmol, 1 equiv) to give 2-(13C)cinnamaldehyde 2b
(0.727 g, 5.46 mmol, 42% yield) as a yellow oil (E/Z > 96%): H
NMR (500 MHz, CDCl3) δ 6.71 (ddd, 1H, JHC = 160.2 Hz, JHH
= 153.0 Hz, JHH = 15.6 Hz, JHH = 1.7 Hz, H3), 7.22−7.41 (m, 5H,
ArH); 13C NMR (75 MHz, CDCl3) δ 63.8 (C1), 126.6 (d, 2C, 2JCC
1.9 Hz, ortho-ArCH), 127.8 (para-ArCH), 128.5 (d, JCC = 72.5 Hz,
C2), 128.7 (d, 2C, 3JCC = 4.4 Hz, meta-ArCH), 131.2 (13C3), 136.8 (d,
1JCC = 55.5 Hz, ipso-ArC).
=
1
1
1
3
Reconstructed Human Epidermis. For this study, the large 4
cm2 SkinEthic RHE model (SkinEthic, Lyon, France, http://www.
composed of keratinocytes cultured on a polycarbonate filter at the
air−liquid interface for 17 days. SkinEthic RHE were received on day
18, aseptically removed from the transport medium, and preincubated
for 2 h in a growth culture medium (SkinEthic, Lyon, France) at 37
°C, 5% CO2, and under humidified atmosphere, according to
SkinEthic’s protocol.
=
3
15.9 Hz, JHH = 7.7 Hz, H2), 7.40−7.44 (m, 3H, 2 x meta-ArH and
3
2
para-ArH), 7.47 (dd, 1H, JHH = 15.9 Hz, JHC = 1.5 Hz, H3), 7.55−
7.57 (m, 2H, ortho-ArH), 9.70 (dd, 1H, 2JHC = 25.7 Hz, 3JHH = 7.7 Hz,
H1); 13C NMR (125 MHz, CDCl3) δ 128.6 (13C2), 128.6 (2C, meta-
ArCH), 129.1 (2C, ortho-ArCH), 131.4 (para-ArCH), 134.0 (ipso-
1
1
ArC), 152.9 (d, JCC = 69.0 Hz, C3), 193.8 (d, JCC = 54.8 Hz, C1).
3-(13C)Cinnamaldehyde 2c. The title compound was prepared
according to the general procedure described above starting from
acetonitrile 4 (1.46 mL, 28 mmol, 2 equiv) and 1-(13C)benzaldehyde
5c (1.42 mL, 14 mmol, 1 equiv) to give 3-(13C)cinnamaldehyde 2c
Treatment of RHE with 1-, 2-, or 3-(13C)Cinnamaldehyde and
1-, 2-, or 3-(13C)Cinnamyl Alcohol. SkinEthic RHE were topically
and separately treated with 1-, 2- or 3-(13C)cinnamaldehyde and 1-, 2-,
or 3-(13C)cinnamyl alcohol in acetone (0.4 M, 100 μL) and incubated
for 1, 8, and 24 h, respectively. RHE negative controls were either
untreated or treated with acetone (100 μL) and incubated for 24 h.
After incubation, the RHE were rinsed with deionized water, separated
from the polycarbonate filter using a treatment with Dispase II
(neutral protease, grade II, Roche, Mannheim) in HEPES (Lancaster
Synthesis, Pelham, United States) buffer solution, washed with
deionized water, and stored at −80 °C pending NMR sample
preparation.
1
(0.751 g, 5.64 mmol, 40%) as a yellow oil (E/Z > 96%): H NMR
3
3
(300 MHz, CDCl3) δ 6.74 (dd, 1H, JHH = 16.0 Hz, JHH = 7.7 Hz,
H2), 7.40−7.44 (m, 3H, 2 x meta-ArH and para-ArH), 7.46 (dd, 1H,
1JHC = 153.4 Hz, 3JHH = 16.0 Hz, H3), 7.51−7.59 (m, 2H, ortho-ArH),
9.67 (dd, 1H, 3JHH = 7.7 Hz, 3JHC = 1.0 Hz, H1); 13C NMR (75 MHz,
3
1
CDCl3) δ 128.4 (d, 2C, JCC = 2.1 Hz, meta-ArCH), 128.5 (d, JCC
=
68.5 Hz, C2), 129.2 (d, 2C, 2JCC = 4.5 Hz, ortho-ArCH), 131.4 (para-
ArCH), 134.2 (d, JCC = 55.8 Hz, ipso-ArC), 152.9 (13C3), 193.8 (d,
1
2JCC = 4.7 Hz, C1).
Synthesis of Cinnamyl Alcohol 1: General Procedure. To a
solution of cinnamaldehyde 2 (170 mg, 1.29 mmol, 1 equiv) in
methanol (5 mL) at 0 °C was added sodium borohydride (49 mg, 1.29
mmol, 1 equiv). The mixture was stirred for 15 min at 0 °C, then
warmed up at room temperature and stirred for an additional 2 h. The
reaction medium was quenched with water (10 mL), concentrated in
vacuo, and dichloromethane (10 mL) was added. The organic layer
was separated, washed with brine (10 mL), dried over magnesium
sulfate, filtered, and concentrated in vacuo to give cinnamyl alcohol 1
Rotor Preparation and Data Acquisition by HRMAS NMR.
The rotor preparation and the data acquisition by HRMAS NMR were
carried out according to the methodology already described.22 Briefly,
each sample was prepared at −20 °C by introducing 15 to 20 mg of
frozen RHE completed with D2O into a disposable 30 μL KelF inserts.
Shortly before HRMAS analysis, the inset was placed into a standard 4
mm ZrO2 rotor and closed with a cap. The HRMAS experiments were
performed at 3 °C. Upon completion of the analysis, the inset was
taken out of the rotor and stored back at −80 °C for further
complementary NMR analysis at a later stage.
1
(160 mg, 1.19 mmol, 92% yield, E/Z > 96%) as a white solid: H
NMR (500 MHz, CDCl3) δ 2.21 (br., 1H, − OH), 4.32 (dd, 2H, 3JHH
= 5.6 Hz, 4JHH = 1.4 Hz, H1), 6.36 (dt, 1H, 3JHH = 15.8 Hz, 3JHH = 5.6
Hz, H2), 6.62 (dt, 1H, 3JHH = 15.8 Hz, 4JHH = 1.4 Hz, H3), 7.23−7.41
(m, 5H, ArH); 13C NMR (75 MHz, CDCl3) δ 63.5 (C1), 126.5 (2C,
ortho-ArCH), 127.7 (para-ArCH), 128.4 (C2), 128.6 (2C, meta-
ArCH), 131.0 (C3), 136.8 (ipso-ArC).
HRMAS spectra were recorded on a Bruker Avance III 500
spectrometer (Hautepierre University Hospital, Strasbourg) operating
at a proton frequency of 500.13 MHz, equipped with a 4 mm double
resonance (1H, 13C) gradient HRMAS probe. The conditions of
1
acquisition and processing for the 1D H experiments using standard
one pulse and Carr−Purcell−Meiboom−Gill (CPMG) pulse
1-(13C)Cinnamyl Alcohol 1a. The title compound was prepared
according to the general procedure described above starting from 1-
(13C)cinnamaldehyde 2a (500 mg, 3.76 mmol) to give 1-(13C)-
cinnamyl alcohol 1a (395 mg, 2.92 mmol, 78% yield, E/Z > 96%) as a
1
sequences coupled with water presaturation and for the 2D H−13C
g-HSQC (gradient Heteronuclear Single Quantum Coherence)
experiments using echo-antiecho gradient selection were those
described in the detailed methodology. All spectra were referenced
by setting the lactate doublet chemical shift to 1.33 ppm in 1H and to
22.7 ppm in 13C.
1
white solid: H NMR (300 MHz, CDCl3) δ 1.51 (br., 1H, − OH),
1
3
4
4.33 (ddd, 2H, JHC = 142.6 Hz, JHH = 5.7 Hz, JHH = 1.5 Hz, H1),
6.32−6.42 (m, 1H, H2), 6.63 (ddt, 1H, 3JHH = 15.8 Hz, 3JHC = 7.0 Hz,
4JHH = 1.5 Hz, H3), 7.22−7.41 (m, 5H, ArH); 13C NMR (75 MHz,
CDCl3) δ 63.9 (13C1), 126.6 (2C, 2 x ortho-ArCH), 127.9 (para-
RESULTS
■
1
Synthesis of Carbon-13 Substituted Chemicals. In
order to increase the sensitivity of the method and be able to
ArCH), 128.6 (d, JCC = 45.9 Hz, C2), 128.7 (2C, 2 x meta-ArCH),
3
131.3 (C3), 136.8 (d, JCC = 5.4 Hz, ipso-ArC).
1174
Chem. Res. Toxicol. 2016, 29, 1172−1178