Chemical Research in Toxicology
Article
characterized as 2-(3-(4-(1-hydroxyethyl)benzoyl)phenyl)propanoic
acid. 1H NMR (CDCl3, 400 MHz): δ 7.81−7.75 (3H, m), 7.68
(1H, d, J = 7.7 Hz), 7.56 (1H, d, J = 7.7 Hz), 7.50−7.43 (3H, m), 4.99
(1H, q, J = 6.6 Hz), 3.83 (1H, q, J = 7.3 Hz), 1.58−1.51 (6H, m). 13C
NMR (CDCl3, 400 MHz) δ: 196.1, 178.6, 150.5, 140.1, 138.0, 136.5,
131.6, 130.5, 129.3, 129.2, 128.6, 125.3, 70.0, 45.0, 25.3, 18.2. ESI-MS
m/z (%): 321.0 [MNa]+ (62), 299.0 [MH]+ (95), 281.0 [MH −
H2O]+ (100).
Photolysis of Amino Acid Analogues. Illumination of a mixture
of the amino acid analogues benzylamine, 4-methylimidazole, 3-
methylindole, 4-propylphenol, and 1-octanethiol was performed for 30
min, and samples were taken after 0, 5, 10, 15, and 30 min.
Photolysis of Ketoprofen (1) in the Presence of Amino Acid
Analogues. Compound 1 and a mixture of the amino acid analogues
benzylamine, 4-methylimidazole, 3-methylindole, 4-propylphenol, and
1-octanethiol were illuminated for 10 min. Samples were taken after 0,
1, 2, 3, 4, 5, and 10 min.
a white solid. The precipitate was filtered and dried under vacuum to
yield N-acetyl-L-Trp ethyl ester (1.19 g, 54%) as a white solid. H
1
NMR (CDCl3, 400 MHz): δ 8.15 (bs, 1H), 7.53 (d, 1H, J = 8.0 Hz),
7.36 (d, 1H, J = 8.0 Hz), 7.19 (dd, 1H, J = 8.0, 7.1 Hz), 7.11 (dd, 1H, J
= 8.0, 7.1 Hz), 6.97 (s, 1H), 6.02 (d, 1H, J = 7.9 Hz), 4.96−4.91 (m,
1H), 4.21−4.07 (m, 2H), 3.38−3.26 (m, 2H), 1.96 (s, 3H), 1.23 (t,
3H, J = 7.2 Hz) 13C NMR (CDCl3, 400 MHz): δ 172.1, 169.9, 136.2,
127.9, 122.7, 122.4, 119.8, 118.7, 111.3, 110.3, 61.6, 53.2, 27.7, 23.4,
14.2. ESI-MS m/z (%): 297.0 [MNa]+ (28), 275.1 [MH]+ (100),
229.0 [M − OCHH2CH3]+ (50), 201.0 [M − COOCH2CH3]+ (60).
Photolysis of Ketoprofen (1) in the Presence of Trp and Lys.
Illumination of 1, N-acetyl-L-Trp ethyl ester, L-Lys ethyl ester
dihydrochloride, and KOH (20 mM) was performed for 15 min,
and samples were taken after 0, 1, 2, 3, 4, 5, 10, and 15 min.
Compound 17 was identified as ethyl 2-amino-6-(4-oxoquinazolin-
3(4H)-yl)hexanoate by spiking with a synthetically prepared reference;
see below for the synthetic procedure. The spiked chromatograms can
be found in the Supporting Information.
Synthesis of Compound 17 (Ethyl 2-amino-6-(4-oxoquina-
zolin-3(4H)-yl)hexanoate). A slurry of anthranilic acid (1.30 g, 9.5
mmol) in formic acid (1 mL) was heated at 80 °C for 15 min, after
which a solid product was formed. The solid still contained 15%
anthranilic acid (quantified by NMR). Formic acid (8 mL) was added,
and the obtained slurry was stirred for another 90 min at 80 °C. The
slurry was concentrated under reduced pressure at 60 °C to a
crystalline solid, which was dissolved in a mixture of acetone (100 mL)
and acetonitrile (50 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure to a white crystalline solid
(1.54 g) containing 2-formamidobenzoic acid (95%) and anthranilic
acid (5%). The compound was used for the next synthetic procedure
without any further purification.
Photolysis of Ketoprofen (1) in the presence of Benzylamine
and 3-Methylindole. Illumination of 1, benzylamine, and 3-
methylindole was performed for 4 min, and samples were taken
every minute.
Photolysis of Benzylamine and 3-Methylindole Followed by
Fractionation of the Mixture. Illumination of benzylamine and 3-
methylindole was performed for 30 min, and samples were taken after
0, 5, 10, 15, and 30 min. After 30 min, the mixture was concentrated
under reduced pressure and fractionated by flash chromatography on
silica gel. Two pure fractions (compounds 10 and 11) were obtained
after elution with ethyl acetate/hexanes (1:4), and one additional
fraction, containing a mixture of compounds 5 and 12, was obtained
after elution with ethyl acetate/hexanes (1:1). Compound 10 was
1
characterized as N-(2-acetylphenyl) formamide, and the obtained H
A solution of DCC (138 mg, 0.67 mmol) in anhydrous DMF (0.8
mL) was added dropwise to a solution of 2-formamidobenzoic acid
(100 mg, 0.61 mmol) and 1-hydroxybenzotriazole hydrate (89 mg,
0.66 mmol) in DMF (0.4 mL) under argon at 0 °C. The mixture was
stirred for 10 min at 0 °C and 20 min at room temperature. A solution
of K2CO3 (502 mg, 3.6 mmol) and L-Lys ethyl ester dihydrochloride
(167 mg, 0.68 mmol) in H2O (6.7 mL) was added dropwise, and the
resulting mixture was diluted with additional H2O (4.3 mL). The
reaction was stirred for 20 h and then extracted with ethyl acetate (4 ×
20 mL). The combined extracts were washed with brine (20 mL),
dried over Na2SO4, filtered, and concentrated to a white solid. DCM
(2 mL) was added to the solid, and the formed slurry was filtered to
remove dicyclohexylurea. The filter cake was washed with additional
DCM (3 mL), and the obtained filtrate was purified by flash
chromatography (DCM/methanol 95:5 to 9:1) to afford ethyl 2-
amino-6-(4-oxoquinazolin-3(4H)-yl)hexanoate (18 mg, 10% from
anthranilic acid) as an uncolored oil. Rf = 0.2 (DCM/methanol 9:1).
1H NMR (Me2SO-d6, 400 MHz): δ 8.39 (1H, s, H2), 8.15 (1H, ddd, J
NMR and ESI-MS spectra were in agreement with those from a
previous report.21 Compound 11 was characterized as 3-methyl-2-
indolinone, and the obtained 1H and 13C NMR spectra were in
accordance with those from previous reports.22,23 During the analysis
of the mixture of compounds 5 and 12, compound 5 degraded in the
NMR solvent (CDCl3) so that, instead of a mixture of these two
compounds, a rather pure sample of compound 12 was obtained.
Compound 12 was characterized as 3-benzylquinazolin-4(3H)-one,
and the obtained 1H and 13C NMR spectra were in agreement with the
literature.24 One more compound, compound 13, was identified in the
photolysis mixture. This compound was verified as 2-acetylaniline
mixture by spiking with a reference compound. Compound 5 was
identified by chemical synthesis as 3-benzyl-4-methylene-3,4-dihydro-
quinazoline; see below for details.
Photolysis of 3-Methylindole. 3-Methylindole was illuminated
for 15 min, and samples were taken after 0, 5, 10, and 15 min. The
major photoproduct from this experiment was N-(2-acetylphenyl)
formamide 10.
= 8.0, 1.6, 0.6 Hz, H5), 7.82 (1H, ddd, J = 8.2, 7.1, 1.6 Hz, H7), 7.67
(1H, ddd, J = 8.2, 1.2, 0.6 Hz, H8), 7.54 (1H, ddd, J = 8.2, 7.1, 1.2 Hz,
H6), 4.08−4.00 (2H, m, H15), 3.97 (2H, t, J = 7.2 Hz, H9), 3.37−3.22
(1H, m, H13), 1.97 (2H, br s, NH2), 1.74−1.63 (2H, m, H10), 1.63−
1.54 (1H, m, H12), 1.53−1.42 (1H, m, H12′), 1.40−1.29 (2H, m, H11),
1.13 (3H, t, J = 7.1 Hz, H16). 13C NMR (Me2SO-d6, 400 MHz): δ
175.6 (C14), 160.1 (C4), 148.0 (C2), 147.9 (C8a), 134.2 (C7), 127.1
(C8), 127.0 (C6), 126.0 (C5), 121.5 (C5a), 59.9 (C15), 53.8 (C13), 45.8
(C9), 34.1 (C12), 28.4 (C10), 22.3 (C11), 14.1 (C16). HRMS (ES
+TOF) calculated for C16H21N3O3 [MH]+, 304.1661; found,
Synthesis of Compound 5 (3-Benzyl-4-methylene-3,4-dihy-
droquinazoline). All solvents used in the synthesis and purification
were bubbled with nitrogen before usage. N-(2-Acetylphenyl)
formamide (160 mg, 0.98 mmol) and benzylamine (105 μL, 0.96
mmol) were dissolved in EtOH (100 mL, 95%). The reaction mixture
was stirred at room temperature for 20 h under an argon atmosphere
and then refluxed for 10 h. The solvent was evaporated under reduced
pressure, and the crude product was purified by flash chromatography
(ethyl acetate/hexanes 1:4). Compound 5 was isolated and
characterized as 3-benzyl-4-methylene-3,4-dihydroquinazoline, and
1
304.1662. H NMR, 13C NMR, and 2D NMR (COSY, HSQC, and
1
the obtained H and 13C NMR spectra were in agreement with the
literature.25 Note that dihydroquinazoline 5 decomposes rapidly in the
presence of oxygen.
HMBC) spectra used for assignment can be found in the Supporting
Information.
Synthesis of N-Acetyl-L-Trp Ethyl Ester. A procedure was
adapted from the literature as follows:26 DIPEA (2.8 mL, 16 mmol)
and acetic anhydride (0.76 mL, 8.0 mmol) were added dropwise to a
stirred solution of L-Trp ethyl ester hydrochloride (2.15 g, 8.0 mmol)
in dry dichloromethane (DCM) (40 mL) under nitrogen atmosphere.
The reaction mixture was stirred at room temperature for 4 h and then
washed with H2O (2 × 15 mL), saturated NaHCO3 (2 × 15 mL), and
HCl (1 M, 15 mL). When HCl was added, the product precipitated as
RESULTS
■
Photolysis of Ketoprofen (1) in EtOH. When ketoprofen
was photolyzed in EtOH, more than 50% was consumed after
only 3 min (Figure 2). Five different peaks could be observed in
the total ion count (TIC) chromatogram from the HPLC/MS
analysis: two peaks had m/z 323 ([MNa]+), and both
C
dx.doi.org/10.1021/tx5001656 | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX