Letter to the Editor
Received: 8 November 2012
Revised: 29 May 2013
Accepted: 4 June 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 2033–2038
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6647
Dear Editor,
exhibited a molecular ion at m/z 364.2166 corresponding to
the molecular formula C23H28N2O2. The 1H-NMR spectrum
(CDCl3, 200 MHz) exhibited δ (ppm): 7.59 (d J = 15.3 Hz, 1H,
H3); 7.51–7.23 (m 10H, aromatic H); 6.06 (d J = 15.3 Hz, 1H,
H2); 1.41 (s 6H, H9 & 10); 1.42 (s 9H, H11, 12 & 13); 5.82 (s NH).
The 13C-NMR spectrum (CDCl3, 50.25 MHz) exhibited δ
(ppm): 165.8 (s C1); 119.8 (d C2); 141.5 (d C3); 63.1 (s C5); 173.8
(s C6); 50.9 (s C8); 25.5 (q C9, 10); 28.6 (q C11–13); 135.1 (s C1´);
139.5 (s C1´´); 130.6, 129.4, 129.2, 128.5, 127.7 (aromatic carbons).
The mass spectral data by GC/EI-LRMS: m/z values (% relative
abundance): [M]+.364 (0.4), 292 (1.49), 291 (1.96), 266 (1.5), 265
(11.8), 264 (29.9), 188 (1.3), 146 (1.9), 135 (9.1), 134 (100.0), 132
(5.0), 131 (49.9), 130 (2.3), 118 (7.6), 115 (1.6), 106 (2.3), 104
(6.1), 103 (31.1), 102 (2.7), 94 (1.3), 93 (2.0).
Mass spectral studies of diamide compounds obtained by
the Ugi reaction
The Ugi multicomponent reaction[1] involves the combination of
an amine, an acid, a carbonyl compound and an isocyanide to
produce
a diamide. Several diamides obtained by this
methodology have shown cytotoxic effects.[2,3] In order to
evaluate their cellular metabolism in in vitro assays it is necessary
to develop techniques that allow the unambiguous identification
of these compounds and their metabolites. Most classical
methods lack the minimal required sensitivity since the
expected concentrations in both culture media and intracellular
environments are very low (around 10–6 M). The present work
reports the synthesis of the two cinnamic acid-derived
diamides N-(1-(tert-butylamino)-2-methyl-1-oxopropan-2-yl)-
N-phenethylcinnamamide (1) and N-(1-(tert-butylamino)-2-
methyl-1-oxopropan-2-yl)-N-phenylcinnamamide (2) by a
"one-step-one-pot" procedure (Fig. 1), as well as the study
of their fragmentation pathways by mass spectrometry. The
characterization of certain fragment ions could be a tool for
the detection of these compounds and their derived
metabolites in complex biological matrices.[4–6]
The EI-HRMS measurements were performed on
a
Micromass VG AutoSpec (Manchester, UK), at the Instituto
Universitario de Bioorgánica (Universidad de La Laguna), at
a resolution of 5000 (5% valley definition), by 70 eV electron
ionization, at an accelerating voltage of 8 kV. EI-LRMS was
performed at 70 eV using an ion trap (GCQ Plus) with MSn
(Finnigan, Thermo-Quest, Austin, TX, USA), operated at a
fundamental rf-drive of 1.03 MHz. Helium was used as the
damping gas at an uncorrected gauge reading of 6 × 10–5 Torr.
For the analysis of tandem mass spectrometric (MS/MS)
product ions, the precursor ion was selected using a MS/
MS standard function, with a peak width of 0.5–1.0 m/z units,
and dynamically programmed scans. The supplementary
voltage was in the range 0.5–1.0 V, as described previously.[7]
Although peptides usually yield protonated molecules
[M + H]+ when studied by LC/MS using positive ion
electrospray ionization (ESI) or atmospheric pressure
ionization (API) methods, the diamines considered here are
not really peptidic and their high thermal stability facilitate
their study by GC/MS and GC/MSn. Thus, the compounds
have been studied by electron ionization low-resolution mass
spectrometry (LR-EIMS), EI-MSn (n = 1–5) using an ion trap
mass spectrometer, and electron ionization high-resolution
mass spectrometry (HR-EIMS). The combination of these
techniques allows us to establish the genesis[7] and the
possible structures of the fragment ions. Figures 2(a) and
2(b) show the nomenclature[8–10] used in the present work
to describe the different bond types and the possible fragment
ions that could arise from fragmentation of the molecular ions
of compounds 1 and 2.
2-Phenylethanamine (2.02 mmol) or aniline and acetone
(2.43 mmol) were dissolved in methanol and stirred at
roomtemperature. After 3 h, cinnamic acid (1.35 mmol) and
tert-butyl isocyanide (1.48 mmol) were added and stirred for
24 h. Reaction progress was checked by thin-layer
chromatography (TLC) and purification was carried out by
flash chromatography using mixtures of n-hexane/ethyl
acetate as eluent to afford 306.23 mg and 282.94 mg of
compounds 1 and 2, respectively.
Compound 1: N-(1-(tert-butylamino)-2-methyl-1-oxopropan-2-
yl)-N-phenethyl-cinnamamide: 306.23 mg (0.78 mmol, yield
58%) of compound 1 was obtained as a colorless oil, which
exhibited a molecular ion at m/z 392.2462 corresponding to
1
the molecular formula C25H32N2O2. The H-NMR spectrum
(CDCl3, 200 MHz) exhibited δ (ppm): 7.68 (d J = 15.3 Hz,
1H, H3); 7.51–7.23 (m 10H, aromatic H); 6.77 (d J = 15.3 Hz,
1H, H2); 3.75 (dd J = 7.8, 8.4 Hz, 2H, H14); 3.0 (dd J = 7.8, 8.4
Hz, 2H, H15); 1.61 (s 6H, H9 & 10); 1.33 (s 9H, H11, 12 & 13);
5.65 (s NH). The 13C-NMR spectrum (CDCl3, 50.25 MHz)
exhibited δ (ppm): 167.4 (C1), 118.8 (d C2); 142.9 (d C3); 62.9
s (C5); 174.3 (s C6); 50.9 (s C8); 46.0 (t C14); 37.8 (t C15); 25.7
(q C9 & 10); 28.5 (q C11-15); 135.1 (s C1´); 138.5 (s C1´´); 129.7,
128.86, 128.82, 128.6, 127.9, 126.8 (aromatic carbons). The
gas chromatography/electron ionization low-resolution mass
spectrometry (GC/EI-LRMS) spectra showed: m/z values
(% relative abundance): [M]+.392 (0.8), 320 (1.0), 299 (1.1), 293
(6.1), 292 (29.4), 216 (1.4), 209 (1.0), 202 (4.5), 163 (12.7), 162
(100.0), 145 (10.0), 132 (2.6), 131 (21.4), 106 (1.1), 105 (22.7), 104
(1.9), 103 (29.1), 102 (1.3), 79 (2.1), 77 (9.5), 70 (1.0).
The EI-LRMS spectrum of compound 1 and the study of its
fragment ions by MSn, combined with the EI-HRMS spectra, led
to the fragmentation pathway shown in Scheme 1 (Tables 1 and 2).
Routes R1 and R2 involve the elimination of 71 and 72 Da
as neutral species from the molelcular ion at m/z 392. These
pathways can be described as removal of t-butene and a
methyl group, and the N-t-butylamino radical, respectively,
to form the fragment ions at m/z 321 ([C20H21N2O2]+) and
320 ([C21H22NO2]+).
Compound 2: N-(1-(tert-butylamino)-2-methyl-1-oxopropan-2-
yl)-N-phenyl-cinnamamide: 287.94 mg (0.79 mmol, yield 59%) of
compound 2 was obtained as a clear and colorless oil, which
Route R3 corresponds to the loss of a neutral radical of 91
Da ([C7H7].) involving the B ring, to form the fragment ion
at m/z 301 ([C18H25N2O2]+). Route R4 involves cleavage of
Rapid Commun. Mass Spectrom. 2013, 27, 2033–2038
Copyright © 2013 John Wiley & Sons, Ltd.