Tandem mass spectrometry based investigation of cinnamylideneacetophenone derivatives: Valuable tool for the differentiation of positional isomers

Article abstract of DOI:10.1002/rcm.5207

Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transformations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give information on th

Full text of DOI:10.1002/rcm.5207

Research Article
Received: 7 June 2011
Revised: 27 July 2011
Accepted: 29 July 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2011, 25, 3185–3195
(wileyonlinelibrary.com) DOI: 10.1002/rcm.5207
Tandem mass spectrometry based investigation of
cinnamylideneacetophenone derivatives: valuable tool for the
differentiation of positional isomers
†
,†
*
Diana I. S. P. Resende , Eduarda M. P. Silva* , Cristina Barros, M. Rosário M. Domingues ,
Artur M. S. Silva and José A. S. Cavaleiro
Department of Chemistry, QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal
Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transfor-
mations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give
information on the chemistry, reactivity and also the potential biological activity of this type of compounds. Thus, 24
derivatives were systematically studied by tandem mass spectrometry (MS2) with electrospray ionization (ESI), in posi-
tive ion mode. The protonated molecules, [M + H]+, formed under ESI conditions were induced to dissociate and the
fragmentation patterns were studied. The information collected provided important structural information on the type
of substituents present and constitute an important database concerning this family of compounds. Overall, it was
found that the substitution pattern of the cinnamylideneacetophenone derivatives changes the ESI-MS2 fragmentation
considerably. These results indicate that ESI-MS2 is a useful technique for distinguishing positional isomers of these
cinnamylideneacetophenone derivatives. Copyright © 2011 John Wiley & Sons, Ltd.
(E,E)-1,5-Diarylpenta-2,4-dien-1-ones (cinnamylideneacetophe-
nones) are a well-known and important group of unsaturated
ketones that have been widely used in a variety of synthetic
transformations.[1] One important use of these derivatives is
their exploitation as very simple starting materials to construct
cyclohexane derivatives with five new stereocenters. This trans-
formation is achieved using a simple, efficient and environmen-
tal friendly domino multicomponent reaction with complete
diastereoselectivity in high yields.[2]
Most of the biological studies for compounds with similar
structure, such as antiviral, antibacterial and antioxidant evalua-
tions, have involved the analogous chalcones.[3] These studies
allowed the biological activity of the chalcones to be attributed
to the interaction between the A and B rings via the propenone
chain. The conjugated system allows, according to several stu-
dies, the contribution of each A and B fragment to the biological
activity to be modulated by being transferred through the elec-
tronic effects of each ring. A comparison of the antistaphylococal
biological activity of the cinnamylideneacetophenones with that
of the corresponding chalcones was published in 1988.[4] This
study revealed that lengthening of the conjugated system
enhances the antibacterial activity and that the introduction of
a 2’-hydroxyl group into fragment A slightly increases the
activity against spore-forming bacteria (70–80 mg/mL).[4]
The antimicrobial activity of several ortho-, meta- and para-NO2,
OCH3 and CH3 ring A-substituted cinnamylideneacetophenones
was investigated more recently.[5] All these compounds showed
antimicrobial activity against Gram-positive and Gram-negative
bacteria, but no antifungal activity was observed against yeast-
like fungi. Of this series, the ortho-nitro-substituted compound
showed the most promising results (namely 60 mg/mL minimum
inhibitory concentration against S. aureus, B. cereus and E. faecalis).[5]
Cinnamylideneacetophenones bearing ortho-OH and para-
OCH3 substituents on ring A were also screened for their
potential use as hypolipidemic agents.[6] However, this
study was soon abandoned, since these compounds showed
less promising results in reducing cholesterol and triglyceride
levels than some of the previously studied chalcones.
There have been no studies involving the characterization of
cinnamylideneacetophenones by tandem mass spectrometry
and, as far as we are aware, this is the first report dealing with
the mass spectrometric behaviour of these useful compounds.
In this work, electrospray ionization tandem mass spectrome-
try (ESI-MS2) was used to investigate the mass spectral
fragmentation behaviour of cinnamylideneacetophenones 1,
1a–k, 2 and 2a–k (Scheme 1). The aim of the present work
was to obtain, by using ESI-MS2, detailed structural informa-
tion and to use it for the differentiation of isomeric molecules.
EXPERIMENTAL
(E,E)-Cinnamylideneacetophenones 1 and 1a–k were prepared
according to published methods through a base-catalyzed aldol
condensation of cinnamaldehydes substituted with the
appropriate acetophenones.[2,7] Compounds 2 and 2a–k were
prepared through an organocatalytic 1,4-Michael addition of
nitromethane to substituted (E,E)-cinnamylideneacetophe-
nones catalyzed by 9-thiourea-9-(deoxy)-epi-hydroquinine.[8]
The purity of all synthesized compounds was assessed by 1D
and 2D NMR, high-resolution mass spectrometry (HRMS)
and elemental analysis.
* Correspondence to: E. M. P. Silva or M. R. M. Domingues,
Department of Chemistry, QOPNA, University of Aveiro,
3810–193 Aveiro, Portugal.
E-mail: eduarda.silva@ua.pt; mrd@ua.pt
†
These authors contributed equally to this work.
Rapid Commun. Mass Spectrom. 2011, 25, 3185–3195
Copyright © 2011 John Wiley & Sons, Ltd.

D. I. S. P. Resende et al.
NO2
R
O
R
O
MeNO2
catalyst
R1
R2
R1
R2
1
R = R1 = R2 = H
2
R = R1 = R2 = H
1a R = OH, R1 = H, R2 = H
2a R = OH, R1 = H, R2 = H
2b R = NH2, R1 = H, R2 = H
2c
1b R = NH2, R1 = H, R2 = H
1c R = H, R1 = H, R2 = OCH3
1d R = H, R1 = H, R2 = NO2
1e R = H, R1 = CH3, R2 = H
1f R = H, R1 = OCH3, R2 = H
1g R = H, R1 = Cl, R2 = H
1h R = H, R1 = Br, R2 = H
1i R = H, R1 = F, R2 = H
1j R = H, R1 = CN, R2 = H
1k R = H, R1 = NO2, R2 = H
R = H, R1 = H, R2 = OCH3
R = H, R1 = H, R2 = NO2
2d
2e R = H, R1 = CH3, R2 = H
2f R = H, R1 = OCH3, R2 = H
2g R = H, R1 = Cl, R2 = H
2h R = H, R1 = Br, R2 = H
2i R = H, R1 = F, R2 = H
2j R = H, R1 = CN, R2 = H
2k R = H, R1 = NO2, R2 = H
Scheme 1. Structures of the studied compounds 1 and 1a–k, 2 and 2a–k.
Electrospray product ion mass spectra were obtained for
the [M+H]+ ions of all the studied compounds under the
same experimental conditions using helium as the collision
gas. The fragmentation patterns were studied in order to
identify typical ions for fingerprinting each specific structural
feature of the studied compounds.
For a clearer discussion, the results obtained will be exam-
ined separately. The (E,E)-cinnamylideneacetophenones 1 and
1a–k (Scheme 1), which show simpler MS2 spectra, are
discussed first. The ESI-MS spectra of all the studied cinnamy-
lideneacetophenones show an abundant [M+H]+ ion (Table 1).
The nomenclature adopted for the product ions containing
intact A and B rings was adapted from that proposed by Ma
et al.[9] The descriptions i,jA+ and i,jB+, respectively, represent
product ions containing the intact A and B rings of the
(E,E)-cinnamylideneacetophenone skeleton, where super-
scripts i and j indicate the bonds that have been broken.
Positive-ion ESI-MS and ESI-MS2 spectra were acquired using
a LXQ linear ion trap mass spectrometer (ThermoFinnigan,
San Jose, CA, USA). Samples for ESI analysis were prepared
by diluting 10 mL of chloroform solutions of the cinnamylide-
neacetophenone derivatives with 200 mL of methanol/formic
acid (0.1%). Methanol-d4 (CD3OD) (Riedel-de-Haën, Seelze,
Germany) was used in the solution preparation to obtain
the deuterated molecular species, [M+D]+. Samples were
introduced into the mass spectrometer at a flow rate of 8 mL
min–1. The conditions used to obtain ESI-MS and ESI-MS2
spectra were: nitrogen sheath gas flow rate 8.00 arbitrary
units, spray voltage 5 kV, heated capillary temperature 275
ꢀC, capillary voltage 10.99 V, and tube lens voltage 75.01 V.
CID-MS2 experiments were performed on mass-selected
precursor ions using standard isolation and excitation proce-
dures (activation q value of 0.25, activation time of 30 ms).
The collision energy used was between 20 and 24 (arbitrary
units). Data acquisition was carried out with an Xcalibur data
system (ThermoFinnigan).
ESI-MS2 fragmentation pathways of the cinnamylidene-
acetophenones 1 and 1a–k
The studied cinnamylideneacetophenones have a common
core but they bear distinct functionalities. Thus, the fragmen-
tation pathways were analyzed in order to detect each speci-
fic feature and to identify the common product ions. The
main product ions observed in each MS2 spectrum are
summarized in Table 2. The tandem mass spectra (ESI-MS2)
of the [M+H]+ ions of compounds 1/1e, 1f/1c and 1k/1d
are shown in Fig. 1 as examples. Compound 1 is the non-
substituted cinnamylideneacetophenone and compound 1e
is the ring A para-methyl-substituted derivative. Compounds
1f/1c and 1k/1d are structural isomers substituted with an
electron-donating group or electron-withdrawing group in
the para-position of ring A or ring B, respectively.
The main product ion formed for most of the compounds
results from the cleavage of the C–C bond between C-1 and
C-a, forming the 0,aA+ ions (3, 3a–k) (Scheme 2, Table 2), thus
allowing us to identify this ion as characteristic of this type of
molecule. For compounds 1d (R2 = NO2) and 1f (R1 = OCH3),
this product ion has a rather smaller relative abundance
(9 and 38%, respectively) than for the other compounds. A
similar fragmentation pathway involves cleavage of the C–C
bond between C-1 and C-1’ resulting in a product ion with
the positive charge on the ring B side (4, 4a–k) (product
ion 0,1’B+, Scheme 2). This product ion has, however, very low
relative abundance for all the compounds and is not even
observed for compound 1k (R1 = NO2).
RESULTS AND DISCUSSION
This work was focused on the study of two distinct families
of a,b,g,d-unsaturated carbonylic compounds: the substi-
tuted (E,E)-cinnamylideneacetophenones 1 and 1a–k which
are the substrate used in the 1,4-Michael addition, with
nitromethane affording the nitro derivatives 2 and 2a–k
(Scheme 1).
Also common to all the studied compounds is the forma-
tion of the product ion g,dB+ (15) at m/z 91 (R2 = H) [m/z 121
and 136 for compounds 1c (R2 = OCH3) and 1d (R2 = NO2),
respectively]. The mechanism proposed for the formation of
this ion (Scheme 3) is based on an intramolecular cyclization
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Rapid Commun. Mass Spectrom. 2011, 25, 3185–3195

MS/MS investigation of cinnamylideneacetophenone derivatives
Table 1. Identification of the [M + H]+ ions observed in the ESI-MS of compounds 1, 1a–k and 2, 2a–k
[M + H]+
[M + H]+
Predicted
formula
Observed
mass (m/z) Comp.
Predicted
formula
Observed
mass (m/z)
Comp.
Substituent
Substituent
1
1a
R = R1 = R2 = H
R = OH,
C17H15O+
235.0
251.0
2
2a
R = R1 = R2 = H
R = OH,
C18H18NO+3
C18H18NO+4
296.0
312.2
C17H15O+2
R1 = R2 = H
R = NH2,
R1 = R2 = H
R = NH2,
1b
1c
1d
1e
1f
C17H16NO+
C18H17O+2
250.0
265.0
280.2
249.0
265.2
269.2
313.0
253.0
260.0
280.0
2b
2c
2d
2e
2f
C18H19N2O+3
C19H20NO+4
C18H17N2O+5
C19H20NO+3
C19H20NO+4
C18H17ClNO+3
C18H17BrNO+3
C18H17FNO+3
C19H17N2O+3
C18H17N2O+5
311.2
326.0
341.0
310.0
326.0
330.0
374.0
314.0
321.0
341.0
R1 = R2 = H
R2 = OCH3,
R = R1 = H
R2 = NO2,
R = R1 = H
R1 = CH3,
R = R2 = H
R1 = OCH3,
R = R2 = H
R1 = Cl,
R = R2 = H
R1 = Br,
R = R2 = H
R1 = F,
R1 = R2 = H
R2 = OCH3,
R = R1 = H
R2 = NO2,
R = R1 = H
R1 = CH3,
R = R2 = H
R1 = OCH3,
R = R2 = H
R1 = Cl,
R = R2 = H
R1 = Br,
R = R2 = H
R1 = F,
C17H14NO+3
C18H17O+
C18H17O+2
1g
1h
1i
C17H14ClO+
C17H14BrO+
C17H14FO+
C18H14NO+
C17H14NO+3
2g
2h
2i
R = R2 = H
R1 = CN,
R = R2 = H
R1 = NO2,
R = R2 = H
R = R2 = H
R1 = CN,
R = R2 = H
R1 = NO2,
R = R2 = H
1j
2j
1k
2k
Table 2. Main product ions observed (m/z) in the ESI-MS2 of protonated compounds 1 and 1a–k with indication of their
relative abundances (RA %)
À18 Da À28 Da À42 Da
À46 Da
À68 Da À79 Da
À97 Da
[M + H]+ (ÀH2O) (ÀCO) (ÀC2H2O) (ÀNO2 ) (ÀC4H4O) (ÀBr) (ÀBr - H2O)
0,aA+
m/z
0,1’B+
m/z
•
m/z
m/z
m/z
m/z
m/z
m/z
m/z
m/z
g,dB+
1
235
251
250
265
280
249
265
269
313
253
260
280
217 (69) 207 (2)
193 (1)
-
-
-
-
167 (5)
183 (28)
-
-
-
105 (100)
121 (100)
120 (100)
105 (100)
105 (9)
119 (100)
135 (38)
139 (100)
183 (100)
123 (100)
130 (100)
150 (100)
157 (9)
157 (6)
157 (1)
187 (4) 121 (54)
202 (16) 136 (4)
157 (13) 91 (10)
157 (7)
157 (3)
157 (3)
157 (3)
157 (1)
-
91 (9)
91 (6)
-
1a
1b
1c
1d
1e
1f
1g
1h
1i
233 (70) 223 (14) 209 (29)
-
-
232 (1)
-
-
-
-
247 (37) 237 (6)
262 (100) 252 (6)
231 (99) 221 (5)
247 (78) 237 (5)
251 (40) 241 (2)
295 (23) 285 (1)
235 (50) 225 (2)
242 (25) 232 (2)
262 (17) 252 (6)
-
238 (3)
207 (4)
-
197 (19)
-
-
234 (10) 212 (6)
-
-
-
181 (43)
197 (100)
201 (11)
245 (9)
185 (13)
192 (1)
-
-
-
-
-
-
91 (6)
91 (4)
91 (1)
91 (6)
91 (6)
91 (2)
227 (1)
-
211 (1)
218 (1)
-
-
-
-
-
234 (21)
216 (4)
-
-
-
-
-
-
-
1j
1k
-
234 (5)
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D. I. S. P. Resende et al.
Figure 1. ESI-MS2 spectra of protonated compounds 1/1e, 1f/1c and 1k/1d.
Scheme 2. Fragmentation pathways for formation of 0,aA+ and 0,1B+.
involving the Cg–Cd double bond and ring A, resulting in the
formation of a seven-membered ring fused with the A ring.
This intermediate has several possible resonance structures
[5–7 and 5–7(a–k), as depicted in Scheme 3]. The formation of
product ion g,dB+ can be explained through hydrogen migration
and cleavage of structure 7 leading to the formation of the
benzylic carbocation 15 (which must be in a tropylium-like
structure, Scheme 3). This type of product ion is observed for
all compounds in a very low relative abundance except for 1b
(R = NH2) where it is not observed. For compound 1c, however,
this ion is observed with high relative abundance (54%) due to
the stabilization of the positive charge at the benzylic position
through the donating effect of the para-methoxyl substituent
on the B ring. The resonance structure 7 can also explain the loss
of 28 Da (CO) and 68 Da (C4H4O) leading to the formation of
the ions 12 and 14 (Scheme 3).
xanthones.[10] Evaluation of these fragmentation pathways is
quite important since they provide crucial structural informa-
tion about the substituents linked to each aromatic ring.
Loss of water (18 Da) is also common to all the studied com-
pounds. Tandem mass spectra of deuterated species, [M+D]+,
were obtained in order to provide some clarification on the
mechanism of this fragmentation and to understand the
influence of protons on it. It was found that, after deuteration,
the ion formed by ESI was [M+D]+. The product ion spectrum
of this ion revealed losses of 18 and 19 Da due to the loss of
H2O and DHO, respectively. The formation of the resonance
structure 6, 6a–k, can explain the loss of 19 Da (DHO) leading
to the observed product ion 8, 8a–k. However, the loss of
18 Da (H2O) occurs through the formation of the product ion
10, 10a–k, involving a C-a deuteration by a keto-enol equili-
brium, as depicted in Scheme 3. This is the main fragmentation
pathway for compound 1d (R2 = NO2) and it also leads to the
formation of an important product ion in the spectrum of
compound 1e (R1 = CH3) (Table 2).
The formation of A- and B-type fragments retaining the
intact rings A and B, respectively, has been also observed for
compounds such as flavonoids, chalcones, chromones and
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MS/MS investigation of cinnamylideneacetophenone derivatives
Scheme 3. Fragmentation pattern for protonated compounds 1, 1a–k.
The loss of 28 Da corresponding to the carbonyl group
(CO) is another fragmentation pathway common to the
MS2 spectra of all the studied compounds. These ions are
probably formed through cleavage of the resonance struc-
tures 5, 5a–k (Scheme 3) and migration of hydrogen with for-
mation of the carbocation 12, 12c–k and of the oxonium
cation 12a. This loss is not easy to rationalize considering
the open structures of compounds 1, 1a and 1c–k. These pro-
duct ions have a low relative abundance (1–14%) except
for 1b (R = NH2) where the ion is not observed. The higher
relative abundance of product ion 12a (14%) is due to the
presence of the hydroxyl group where the location of the
charge favours cleavage of the carbonyl group with concur-
rent formation of a stable six-membered ring fused to ring
A (Scheme 3).
The formation of the cyclic ion 5, 5a–k can also explain the
neutral losses of 42 and 68 Da (Table 2). The loss of 42 Da
(C2H2O) leads to the formation of a benzylic and allylic stable
carbocation (13, 13d,e,g,i,j, Scheme 3) although this is only
observed for some of the compounds with very low relative
abundance (1–4%, Table 2). In the case of compound 1a, how-
ever, this ion is observed with a relative abundance of 29%.
Once more, this is due to the presence of the hydroxyl group
where the location of the charge favours the loss of ketene,
COCH2, leading to the formation of a stable five-membered
ring fused to ring A (13a, Scheme 3). The loss of a 68 Da
neutral fragment (C4H4O) results in the formation of an
equally stable benzylic carbocation (14, 14a,c–j, Scheme 3).
For compound 1f (R1 = OCH3) this ion is the base peak of
the spectrum due to the electron-donating effect of the substi-
tuent which stabilizes this carbocation.
A typical fragmentation pathway of protonated nitro
aromatic compounds[11] is the loss of NO2 (46 Da). For com-
•
pounds 1d (R2 = NO2) and 1k (R1 = NO2), this loss is observed
but with uncharacteristic very low relative abundance (10 and
5%, respectively). The characteristic loss of 79 Da typical of
compounds containing bromine atoms is also observed in the
MS2 spectrum of 1h with 21% relative abundance. The loss
•
of Br is also observed combined with the loss of H2O but in
very low relative abundance (4%). Interestingly, for the other
halogen-substituted compounds 1g (R1 = Cl) and 1i (R1 = F)
•
•
the losses of 35 Da (Cl ) and 19 Da (F ) are not observed.
These results indicate that ESI-MS2 is a useful technique
for distinguishing even positional isomers such as 1c/1f
and 1d/1k. Clearly, the difference in the relative abundances
of the common ions for the two isomers can be used to differ-
entiate them (Table 2 and Fig. S1, see Supporting Information).
ESI-MS2 fragmentation of compounds 2 and 2a–k
In contrast with the precursors 1 and 1a–k, the MS2 spectra of
the nitro derivatives 2 and 2a–k show a higher number of
product ions (Table 3). This different fragmentation pattern
is probably due to a second possible protonation position in
the nitro group. The tandem mass spectra of protonated com-
pounds 2/2e, 2f/2c and 2k/2d are shown in Fig. 2 as examples.
The loss of H2O and the formation of the product ion 0,aA+
are the only fragmentation pathways that are common to 1
and 1a–k and to all the nitro derivatives 2 and 2a–k. The
product ion 0,1’B+, also observed for all the precursors except
for 1k, is only formed for compounds 2a,b,d–f (Scheme 2).
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MS/MS investigation of cinnamylideneacetophenone derivatives
Figure 2. ESI-MS2 spectra of protonated compounds 2/2e, 2f/2c and 2k/2d.
Scheme 4. Fragmentation pathways for formation of (0,a)(1,2)B+, a,bB+ and (b,g)(1,2)A+.
Scheme 5. Fragmentation pathway for formation of (a,b)(1,2)B+.
The 0,aA+ and 0,1’B+ ions are formed for the nitro derivatives
according to the previously described mechanism (Scheme 2).
The (b,g)(1,2)A+, a,bB+ and (0,a)(1,2)B+ product ions are
observed in all the nitro derivatives 2 and 2a–k; MS2 spectra
and their structures are shown in Scheme 4. The concurrent
cleavage of the C1–C2 bond and C0–Ca, and a one proton
transfer, lead to the formation of a primary carbocation, but
a [1,2]-hydrogen shift allows the formation of a more stable
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D. I. S. P. Resende et al.
tertiary carbocation (0,a)(1,2)B+ (16, Scheme 4) at m/z at 145
(m/z 175 and 190 for 2c and 2d, respectively) observed in all
the MS2 spectra. The cleavage of the C1–C2 bond also occurs
simultaneously with the cleavage of Cb–Cg leading to the for-
mation of the product ion (b,g)(1,2)A+ (17, Scheme 4), common
to all the nitro compounds. The a,bB+ product ion (18,
Scheme 4) at m/z 176 (m/z 206 and 221 for 2c and 2d, respec-
tively) is formed due to cleavage of the Ca–Cb bond. This is
The product ion (a,b)(1,2)B+ is only observed for compounds
2c and 2d, and it is formed through cleavage of the [M+H]+
ion when protonation occurs in the nitro group (20c,d ions,
Scheme 5). The formation of the 19c (a,b)(1,2)B+ ion occurs
through stabilization of the radical cation by the electron-
donating methoxyl group. When the electron-withdrawing
nitro group is present, we suggest that the (a,b)(1,2)B+ ion corre-
sponds to a distonic ion 20d, with the positive charge located
on the nitro group and the radical stabilized by the dienic sys-
tem. For compound 2c (R2 = OCH3) this ion represents one of
the most important fragmentation pathways (m/z 160, 92%
RA); however, for the 2f isomer (R1 = OCH3), the formation
of this ion is not observed (Fig. 2). For compound 2d (R2 =
NO2) this product ion is also observed with a high relative
abundance (m/z 175; 69% RA). However, again for the 2k
isomer (R1 = NO2) this product ion is not observed (Fig. 2).
The former two compounds are the only ones in this family
possessing a substituent on ring B. Interestingly, cleavage of
the Ca–Cb bond also leads to the formation of the a,bA+
product ion when protonation occurs on the carbonyl group
(21a,b,e,f, Scheme 6). This product ion is only observed for
compounds possessing electron-donating groups at either
the R or the R1 position on ring A (compounds 2a,b,e,f).
the main product ion observed for compound 2c (R2
=
OCH3) and, curiously, for the isomer 2f (R1 = OCH3) it is
only formed with 5% relative abundance, due to the charge
stabilization in the product ion caused by the presence of
an electron-donating substituent in B ring.
Scheme 6. Fragmentation pathway for formation of (a,b)A+.
Scheme 7. Dehydration of protonated nitro derivatives 2, 2a–k.
Scheme 8. Proposed mechanism of the concurrent loss of H2O and NO.
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MS/MS investigation of cinnamylideneacetophenone derivatives
The loss of 18 Da is also observed in the MS2 spectra of all the
studied compounds. However, for the nitro derivatives 2 and
2a–k a new mechanism can be envisaged (Scheme 7) adding
to that depicted earlier for compounds 1 and 1a–k where the
charge was located on the carbonyl group (Scheme 3). The
alternative mechanism, illustrated in Scheme 7, depends on
charge location at the nitro group (19). In pathway a, the elim-
ination of H2O occurs through formation of a triple bond as a
consequence of a [1,5]-hydrogen shift leading to the formation
of 23. Another mechanism can be rationalized by considering
a [1,3]-hydrogen shift of the acidic hydrogen neighbour to the
nitro group (25, pathway b). The loss of H2O constitutes the
main fragmentation pathway for compounds 2f (R1 = CH3)
and 2g (R1 = Cl).
protonated molecule. This fragmentation pathway is not
observed for compound 2b (R = NH2) (Table 3).
In the MS2 spectrum of protonated 2a, 2e and 2f we can
observe product ions resulting from the loss of 122 Da. For
compound 2d this same type of ion is observed at m/z 174
(24% RA) and results from the loss of 167 Da. The formation
of these ions can be envisaged through the loss of styrene
from structure 27 (Scheme 8) leading to the ion 31. For all
the other compounds this product ion is formed with low
relative abundance (2–9%), except for 2k and 2j where they
are not observed in the MS2 spectrum.
The typical loss of HNO2 (47 Da), common to all the nitro
derivatives, is depicted in Scheme 9 (pathway a). This frag-
mentation pathway leads to product ions with RA ranging
from 2 to 47% (Table 3). For compound 2d the product ion
32 (Scheme 9) is obtained with considerably higher relative
abundance (47%) than for the other structures. This finding
can be rationalized as resulting from the presence of a
para-NO2 substituent at the B ring that induces higher acidity
of the H-b (pathway a, Scheme 9). Interestingly, and support-
ing the effect already referred to, the presence of a NO2 substi-
tuent in ring A (compound 2k) does not lead to the same
result (m/z 294, 17% RA), since this group do not have the
same effect on the acidity of the H-b. The product ion formed
A further mechanism can explain the loss of water for these
nitro derivatives involving, in this case, the oxygen atom of the
carbonyl group. This fragmentation pathway involves the
formation of a stable six-membered ring through reaction
between the negatively charged oxygen atom of the nitro group
and the carbonyl group (Scheme 8). This intermediate 26 can
•
then lose H2O (18 Da; pathway a) or NO (30 Da; pathway b),
leading to the formation of structure 27 or 29, respectively. Sub-
•
sequently, 27 can lose NO (pathway a) and 29 (pathway b) can
lose H2O resulting in both pathways in the formation of a stable
•
dihydrofuran-type radical cation 28 (Scheme 8). The concurrent
due to the loss of NO2 , which is a typical fragmentation
•
loss of NO + H2O (48 Da), observed for all the studied com-
pathway of nitro compounds, is not observed in any of the
studied nitro-derivatives.
pounds, it is not observed for the structurally similar nitro-
•
chalcones although the loss of NO has been observed in
The loss of the nitromethane group at the b position through a
retro-Michael addition is shown in pathway b of Scheme 9. The
formation of this product ion 1 at m/z 251 is particularly impor-
tant to compound 2a (77% RA, Table 3), where the hydroxyl
substituent is involved in a hydrogen bond with the carbonyl
group leaving it more prone to leave the hydrogen in the a
position. For all the other compounds this ion is formed with
low relative abundance (3–30%).
the tandem mass spectra of other nitro derivatives.[10c,11b,12]
This fragmentation mechanism is of particular importance
for compound 2a (R = OH) leading to the formation of a
product ion 28 at m/z 264 with a high relative abundance
•
(95%). The loss of NO is the main fragmentation pathway
for compound 2b (R = NH2, m/z 281) but it is not observed
for all the compounds that possess an electron-withdrawing
substituent at R1 (compounds 2g–k).
The main product ion observed for compound 2h (R1 = Br)
•
The formation of the base peak for compounds 2, 2d, 2e,
2i–k is due to loss of HNO (31 Da) (30, Scheme 8) and can
be explained through the formation of the ion 26, a
is formed through a loss of 62 Da [ CH2N(OH)2] that occurs
by cleavage of 33 (Scheme 10). This ion 33 results from the
formation of the N–O bond between the nitro and the
Scheme 9. Proposed mechanism for the loss of HNO2 and CH3NO2.
Scheme 10. Fragmentation pathway for the formation of 26.
Scheme 11. Proposed fragmentation pathways for the loss of 168 Da.
Rapid Commun. Mass Spectrom. 2011, 25, 3185–3195
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D. I. S. P. Resende et al.
carbonyl groups favoured by the electron-donating effect of
the bromine substituent at R1. This ion at m/z 312 is the base
peak of the MS2 spectrum of protonated 2h.
Acknowledgements
Thanks are due to the University of Aveiro, to the Portu-
guese Foundation for Science and Technology (FCT) and
FEDER for funding the Organic Chemistry Research Unit.
E. M. P. Silva (Post-Doc grant SFRH/BPD/66961/2009)
and D. I. S. P. Resende (PhD grant SFRH/BD/62696/2009)
thank FCT for their grants.
The formation of the base peak at m/z 144 of compound 2a,
due to loss of 168 Da, is depicted in Scheme 11. The formation
of the same product ion for compound 2b is also observed
although at m/z 143, with considerable lower relative
abundance (7%), proving that this ion contains the ring A
fragment. Curiously, the loss of 168 Da is also observed for
compounds 2 and 2c at m/z 128 and 158, respectively, reveal-
ing that the fragmentation pathway involves, for these two
cases, the ring B fragment. Deuterium-labelling experiments
were performed to prove the formation of product ion 35
(Scheme 11). The ESI-MS2 spectrum of the [M+D]+ ion of
compound 2 revealed the presence of a product ion at
m/z 128 which confirms the loss of 169 Da and supports the
suggested product ion formation.
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•
The product ion formed due to the loss of NO2 , which is a
typical fragmentation pathways of nitro compounds, is not
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•
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•
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
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Copyright © 2011 John Wiley & Sons, Ltd.
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