Structural analysis of 2-arylidene-1-indanone derivatives by electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.6701

Rationale 2-arylidene-4-methoxy (or hydroxy)-7-methyl-1-indanone derivatives inspired from donepezil, the current drug used for the treatment of Alzheimer's disease as inhibitor of acetylcholinesterase (AChE), were studied for the first time by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). Structurally, these arylidene-indanone compounds are considered as cyclic analogues of chalcones. Methods ESI-MS and tandem mass spectra were acquired using a Q-TOF 2 instrument. Fragmentation patterns were analyzed by CID-MS^2-3 spectra acquired in a Q-TOF and in LXQ linear ion trap mass spectrometers using standard isolation and excitation procedures. RESULTS All the 2-arylidene indanones have shown a common fragmentation pathway leading to a (2¹, 1')A⁺ product ion at m/z 187 and the retro-aldol product ion [(2, 2¹)B⁺] that allow to establish the substitution in the B ring. The effect of electron-donating and -withdrawing substituents on these fragmentation pathways was noticed. The presence of the OCH₃, OH, NO₂ and Br substituents gave typical fragmentation processes that allowed their unequivocal fingerprinting. The combined loss of the ortho substituent in the B-ring plus hydrogen (H, OCH₃, Br and F) is proposed to form a stable cyclic ring product. CONCLUSIONS Arylidene indanones with different substituents on the B ring are associated with a specific fragmentation pattern. In addition, differentiation between isomers with substituents in B ring at ortho and para positions were achieved using ESI-MS/MS. These fragmentation pathways can be used to further identify and determine the fate of these molecules in all stages of drug discovery.

Full text of DOI:10.1002/rcm.6701

Research Article
Received: 21 June 2013
Revised: 29 July 2013
Accepted: 30 July 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 2461–2471
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6701
Structural analysis of 2-arylidene-1-indanone derivatives by
electrospray ionization tandem mass spectrometry†
*
*
José C. J. M. D. S. Menezes , José A. S. Cavaleiro and M. Rosário M. Domingues
Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal
RATIONALE: 2-arylidene-4-methoxy (or hydroxy)-7-methyl-1-indanone derivatives inspired from donepezil, the current
drug used for the treatment of Alzheimer’s disease as inhibitor of acetylcholinesterase (AChE), were studied for the first
time by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). Structurally,
these arylidene-indanone compounds are considered as cyclic analogues of chalcones.
METHODS: ESI-MS and tandem mass spectra were acquired using a Q-TOF 2 instrument. Fragmentation patterns were
analyzed by CID-MS2–3 spectra acquired in a Q-TOF and in LXQ linear ion trap mass spectrometers using standard
isolation and excitation procedures.
RESULTS: All the 2-arylidene indanones have shown a common fragmentation pathway leading to a (21, 1’)A+ product
ion at m/z 187 and the retro-aldol product ion [(2, 21)B+] that allow to establish the substitution in the B ring. The effect of
electron-donating and -withdrawing substituents on these fragmentation pathways was noticed. The presence of the
OCH3, OH, NO2 and Br substituents gave typical fragmentation processes that allowed their unequivocal fingerprinting.
The combined loss of the ortho substituent in the B-ring plus hydrogen (H, OCH3, Br and F) is proposed to form a stable
cyclic ring product.
CONCLUSIONS: Arylidene indanones with different substituents on the B ring are associated with a specific
fragmentation pattern. In addition, differentiation between isomers with substituents in B ring at ortho and para positions
were achieved using ESI-MS/MS. These fragmentation pathways can be used to further identify and determine the fate
of these molecules in all stages of drug discovery. Copyright © 2013 John Wiley & Sons, Ltd.
Several 2-arylidene 5,6-dimethoxy indanone derivatives
inspired by donepezil, the current drug used for inhibition of
acetylcholinesterase (AChE) in Alzheimer’s disease, have been
developed and tested to such effects.[1] Structurally, these
2-arylidene indanones can be regarded as cyclic analogues of
chalcones, a class of flavonoids with a broad range of biological
effects.[2] Similarly, 2-arylidene indanone derivatives have been
considered as potent aromatase inhibitors with potential use
for breast cancer therapy, as anticonvulsant agents and as
imaging probes for β-amyloid plaques in the brain.[3] The
synthetic utility of the 2-arylidene indanones is reviewed as
a useful template to provide important nitrogen-containing
spiroheterocyclic ring systems.[4]
Various 2-arylidene indanones have been used as molecular
tools to investigate the stereochemistry and how the electronic
structure affects the reactivity and the biological effects of
this kind of compounds.[5] The search for new biologically
important 2-arylidene indanones is still an interesting area of
research.[1,3] With the inherent anti-bacterial activity[6] of the
parent 4-hydroxy (or methoxy)-7-methyl-1-indanone (A and B)
moiety,[7] we envisaged the synthesis of new 2-arylidene
indanone moieties for biological evaluation (Scheme 1).
The observed biological activities of arylidene indanones
are associated with their three-dimensional shape and non-
covalent interaction with cellular macromolecules shown by
structure–activity studies.[8] While gas chromatography coupled
with mass spectrometry (MS) and 1H NMR analysis was
also used to study the in-solution and on-plate light-catalyzed
E/Z isomerization of 2-benzylidene-1-indanones,[9] studies
using electrospray ionization (ESI)-MS have not been
performed so far. Several E-2-benzylideneindanones, tetralones
and -benzosuberones with OCH3, NO2 and F substituents in
ortho, meta or para position have also been investigated by IR
and EI-mass spectrometry.[10]
Mass spectrometry (MS) plays an important role in the
structural characterization of new compounds formed during
a synthetic procedure[11] or after drug metabolism.[12] Moreover,
electrospray ionization coupled to mass spectrometry (ESI-MS)
is relatively softer and is routinely applied to investigation
related to biological samples to determine the fate of drug
molecules and in all stages of drug discovery.[13] Considering
the biological applications of the 2-arylidene indanones, it was
pertinent to develop a mass spectrometric procedure to analyze
these compounds. In this paper we describe the mass spectral
characterization of 12 new 2-arylidene-4-methoxy (or hydroxy)-
7-methyl-1-indanone derivatives (1–12) using electrospray
ionization tandem mass spectrometry (ESI-MS/MS) (Table 1).
* Correspondence to: J. C. J. M. D. S. Menezes and M. R. M.
Domingues, QOPNA, Department of Chemistry, University
of Aveiro, 3810–193 Aveiro, Portugal.
E-mail: josemenezes@ua.pt; mrd@ua.pt
†
Dedicated to Dr. Shashikumar K. Paknikar on the occasion
of his 78th birthday.
Rapid Commun. Mass Spectrom. 2013, 27, 2461–2471
Copyright © 2013 John Wiley & Sons, Ltd.

J. C. J. M. D. S. Menezes, J. A. S. Cavaleiro and M. R. M. Domingues
EXPERIMENTAL
Synthesis
The 2-arylidene-4-methoxy (or hydroxy)-7-methyl-1-indanone
derivatives were prepared by reaction of 4-hydroxy (or
methoxy)-7-methyl-1-indanone with benzaldehyde derivatives
using the aldol reaction with KOH as base and methanol
as solvent (Scheme 1).[3] The E-geometry and purity of all
synthesized compounds was assessed by 1D and 2D NMR,
and elemental analysis. The experimental details and spectral
data of the 2-arylidene indanones used in this study are
described in the Supporting Information.
Mass spectrometry
ESI mass spectra and tandem mass spectra in positive mode
were acquired using a Q-TOF 2 instrument (Micromass,
Scheme 1. Synthesis of 2-arylidene-4-methoxy (or hydroxy)-
Manchester, UK). The samples for ESI analyses were
prepared by diluting 1 μL of the arylidene indanone solutions
in chloroform (~10–5 M) in 200 μL of methanol. Nitrogen was
used as the nebulizer gas and argon as the collision gas.
Samples were introduced into the mass spectrometer at a
flow rate of 10 μL min–1, the needle voltage was set at
3000 V and the ion source temperature was 80 °C. The cone
voltage was 35 V. Collision-induced decomposition (CID)
tandem mass spectra were acquired by selecting the desired
ion with the quadrupole section of the mass spectrometer
7-methyl-1-indanone derivatives 1–12.
The substituents were chosen to study the effects of electron-
donating and -withdrawing groups in the arylidene ring,
on the fragmentation patterns and for further derivatization.
To our knowledge this constitutes the first detailed ESI-MS
analysis with new fragmentation pathways of these biologically
important 2-arylidene indanone derivatives.
Table 1. Structures and m/z values of the [M + H]+ ions of 2-arylidene-4-methoxy (or hydroxy)-7-methyl-1-indanone
derivatives 1–12
Compound
R
R1
OCH3
H
R2
R3
R4
H
R5
H
H
H
H
H
H
H
H
H
F
[M + H]+ m/z
295
1
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
2
H
OCH3
OCH3
OCH3
OH
CN
NO2
H
H
295
3
H
OCH3
OCH3
H
H
325
4
H
OCH3
H
355
5
H
281
6
H
H
H
290
7
H
H
H
310
8
Br
H
H
H
343
9
H
Br
OCH3
H
343
10
11
F
F
F
367
255
12
H
H
H
H
H
317
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Analysis of 2-arylidene-1-indanone derivatives by ESI-MS/MS
Rapid Commun. Mass Spectrom. 2013, 27, 2461–2471
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J. C. J. M. D. S. Menezes, J. A. S. Cavaleiro and M. R. M. Domingues
and using collision energy of 30–50 eV. The product ions were
well as hydroxy indanone derivatives 11 and 12, while the
inherent differences in fragmentation patterns resulting from
certain structural motifs will also be considered.
analyzed with the time-of-flight (TOF) analyzer. In MS and
MS/MS experiments, the TOF resolution was set to ~9000.
Data acquisitions were carried out with a Micromass Mass
Lynx data system. MS3 spectra were acquired in a LXQ linear
ion trap mass spectrometer (ThermoFinnigan, San Jose, CA,
USA) with an ESI source. Samples were introduced into the
mass spectrometer using a flow rate of 8 μL min–1. ESI
conditions used to obtain ESI-MS and ESI-MSn spectra were:
spray voltage 5 kV, nitrogen 8.00 sheath gas flow rate (arb),
heated capillary temperature 275 °C, capillary voltage 10.99 V
and tube lens voltage 75.01 V. CID-MS2–3 experiments were
performed on mass-selected precursor ions using standard
isolation and excitation procedures (activation q value of 0.25,
activation time of 30 ms). The collision energy used was
between 20–24 (arbitrary units). Data acquisition was carried
out with an Xcalibur data system (ThermoFinnigan, San Jose,
CA, USA).
The common (21, 1’)A+ cleavage
Analyzing all ESI-MS/MS spectra we were able to see a
common product ion at m/z 187 observed for 2-arylidene
indanones (1–7; R = mono-OMe, di-OMe, tri-OMe, OH, CN,
NO2), (9; R = p-Br), involving the (21, 1’)A+ cleavage (Fig. 1,
Scheme 2). The arylidene indanones with a –OH in the A ring
instead of the –OCH3 also showed the same fragmentation
pathways leading to the ion at m/z 173 (11 and 12; R = N-
methylimidazole and phenylimidazole). Although these
product ions showed distinct relative abundances (RA)
depending on the substituents, they were observed as the
base peak in most compounds with the electron-donating
methoxy groups, and with varying RA in the cases where a
hydroxyl or a CN group is linked to the B ring (Table 2). This
product ion was seen with low RA (5%) in 7 and 9 and was
absent in 8 and 10. The product ion at m/z 187 (or m/z 173
for hydroxyindanone derivatives) undergoes further losses
of 28 Da (CO) in compounds 1–6, 11, 12 and 30 Da (HCHO)
in compounds 1–5 and 6, 9. These product ions (m/z 187
and 173) and the subsequent fragmentation (loss of CO
and HCHO) clearly indicates the presence of the common
4-methoxy (or 4-hydroxy) 7-methyl indanone core in all the
studied compounds (see Fig. 2 for compounds 1 and 2;
Supplementary Figs. SF1, SF2, SF3, SF4 and SF5 for 3, 4, 5, 6
and 7; Fig. 3 for 8 and 9; Supplementary Figs. SF6, SF7 and
SF8 for 10, 11 and 12).
RESULTS
The studied 2-arylidene indanones (1–12) are shown in
Scheme 1 and Table 1. The ESI-MS spectra of the 2-arylidene
indanones (1–12) showed the [M + H]+ ion. The fragmentation
of the [M + H]+ ions were induced by collision with a gas
under tandem mass spectrometry conditions (ESI-MS/MS).
The pathways for the formation of major product ions in the
studied 2-arylidene-4-methoxy (or hydroxy)-7-methyl-1-
indanone derivatives were confirmed by MS3 experiments
obtained in a linear ion trap coupled with ESI. The main
product ions observed in the ESI-MS/MS of the [M + H]+ ion
of 2-arylidene indanones (1–12) with their most probable
identification and the corresponding relative abundance
(RA) are summarized in Table 2.
For simplicity and in analogy with chalcones and other
flavonoid derivatives the aromatic ring of the indanone moiety
was assigned as the A ring, the aryl moiety as the B ring and the
five-membered indanone ring is denoted as the C ring. The ion
nomenclature, numbering for the product ions of 2-arylidene
indanones (1–12) has been adapted and modified as proposed
in the literature (Fig. 1).[14] The (i,j) A and (i,j) B labels are used
to designate product ions containing intact A and B rings, and
the letters i and j refer to the bonds of the C ring and those
connecting the B ring that have been broken.
The retro-aldol-type fragmentation
The retro-aldol-type fragment [(2, 21)B+] (Fig. 1, Scheme 2)
was the other main fragmentation pathway observed in the
MS/MS spectra of compounds 1–6, 8–10 and 12, although
with varying RA (Table 2). This can be justified by the
resonance effects which primarily affect the electron density
at the C-2 and C-1’ carbons of similar compounds.[5]
Generally, organic compounds synthesized using well-known
reactions undergo the retro reactions in the gas phase and
contribute to the major fragmentation patterns observed.[15]
This fragmentation pathway led to the most abundant
product ion in the case of compound 10, while it was
observed in 80% RA in compound 4 (see Supplementary Figs.
SF6 and SF2, Supporting Information). It was surprisingly
noted that the p-Br compound 9 gave the retro-aldol product
ion in 46% RA while it was observed with low RA (5%) in the
o-Br compound 8. Interestingly this retro-aldol-type product
ion was absent in compounds 7 and 11. The higher degree
of methoxylation in the aromatic B ring favors the further loss
of 15 Da to give product ions 3b and 4b observed in case of
compounds 3 and 4 (Scheme 2).[16]
The fragmentation pathways of the studied 2-arylidene
indanones (1–12) showed several common features and
interesting differences. The results and discussion will be
focused on the major fragmentation pathway observed for
compounds 1–10 containing the methoxy indanone core, as
Other fragmentations
The (1,2)(3,9)B+ fragmentation (Fig. 1; 1c–6c, Scheme 3) was
observed in the MS/MS spectra of compounds 1–6 in 4–20%
RA (Table 2). An interesting benzopyrylium product ion[10]
(5d and 6d) at m/z 145 was seen in the case of compounds 5
and 6 by (1,8)(3,9)B+ fragmentation with loss of respective
Figure 1. Ion nomenclature used for the fragmentation of 2-
arylidene indanones (1–12).
substituents (H2O in
5 and HCN in 6; 8% RA; see
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Analysis of 2-arylidene-1-indanone derivatives by ESI-MS/MS
Scheme 2. Proposed fragmentation pathways for formation of (21, 1’)A+ and
(2, 21)B+ ions and subsequent fragments for compounds 1–6 [structure for (2, 21)B+
2a exemplified for compound 2].
Figure 2. ESI-MS/MS spectra of positional isomers 1 and 2.
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J. C. J. M. D. S. Menezes, J. A. S. Cavaleiro and M. R. M. Domingues
Figure 3. ESI-MS/MS spectra of positional isomers 8 and 9.
Scheme 3. Proposed fragmentation pathways for formation of (1,2)(3,9)B+
in compounds 1–6 and m/z 145 from a combined loss from (1,8)(3,9)B+
with their corresponding substituents (compounds 5 and 6).
Supplementary Figs. SF3 and SF4, Supporting Information).
substituents (-OH, -NO2, -Br, -F, -OCH3, imidazole) were
seen in the case of compounds 5, 7, 8, 9, 10 and 12 along
with combined losses with other radicals or neutral
molecules. Other neutral losses of 30 Da (HCHO), 32 Da
(CH3OH) and combined losses of 33 Da (H2O + •CH3),
43 Da (CO + •CH3), 46 Da (CH3O• + •CH3), 47 Da (CH3OH +
•CH3), 48 Da (CH3OH + CH4), 58 Da (CO + HCHO) and
62 Da (CH3OH + HCHO) were seen for the compounds
having methoxy substituents in the B ring (Table 2). It is
worthwhile to note that a combined loss of 58 Da (CO+ HCHO)
gave the product ions (2e and 6e; Scheme 4) corresponding
to the base peak at m/z 237 and m/z 232 in compounds 2
and 6 and was also observed at m/z 267 and m/z 223 in 3
and 5 (Table 2).
The 2-arylidene indanones (1–12) also showed loss of neutral
molecules and/or radicals from the [M+ H]+ ion distinct for
each other (Table 2), depending on the substituents. The
common loss of 15 Da (•CH3) was seen in compounds 1–6,
10, 11 and 12 that have a methoxy and methyl group linked
to the indanone moiety. The loss of 16 Da (•CH3 and •H) was
observed for compounds 2, 3, 5, 6, 11 and 12. The methoxy-
substituted compounds (1–4) showed combined losses with
other radicals or neutral molecules similar to those observed
in flavonoids.[16] The loss of 18 Da (H2O) was observed for
the hydroxyl-substituted compound 5, and in compounds 11
and 12, which most probably occurs from the 4-hydroxy
indanone core. Fragmentation processes characterizing the
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Analysis of 2-arylidene-1-indanone derivatives by ESI-MS/MS
Scheme 4. Proposed fragmentation for the formation of product ions at m/z
204, 205, 221 and 193.
DISCUSSION
Loss of CO and HCHO from the (21, 1’)A+ product ion
Formation of the (21, 1’)A+ product ion
The loss of 28 Da (CO) from the product ions at m/z 187 and
173 (in both cases of methoxy and hydroxy indanone
moieties) can be justified by the retro-Paterno-Buchi (PB)-type
reaction to give the indene-type product ion (m/z 159)
wherein the charge would be localized on the benzylic carbon
(C-1) and would be stabilized via resonance (Scheme 2). This
type of retro-PB reaction was also observed by femtosecond
(fs)-resolved mass spectrometry,[18] photodissociation and
TOF spectra of oxetane and related cyclic ethers,[19] while the
presence of a methoxy group in the product ion (m/z 187)
gives rise to the loss of 30 Da (m/z 157; Table 2) which was
observed in the case of compounds 1–3 and 5, 6 (see Fig. 2
for 1 and 2; Supplementary Figs. SF1 for 3; SF3 and SF4 for 5
and 6, Supporting Information).
To provide additional experimental indication for the
fragmentations discussed above, we carried out representative
MS3 experiments on the product ions at m/z 187 or 173 from
compounds 1 and 11. To our delight, the fragmentation of the
ion at m/z 187 gave product ions at m/z 159 (ÀCO) and 157
(ÀHCHO) with 98% and 100% RA, while the product ion at
m/z 173 gave product ions at m/z 145 (ÀCO) and 117 (À2CO)
with 100% and 21% RA, fully suggesting the proposed
pathways discussed earlier (see Scheme 2 and Table 2
for details).
To probe deeper and understand the formation of this
product ion [(21, 1’)A+] mentioned earlier, we synthesized
2-methylene-substituted indanone 13 by reaction of
methoxyindanone with formaldehyde (Scheme 2). ESI-MS of
this compound (m/z 189; Supplementary Fig. SF9, Supporting
Information) under similar conditions gave the product ions
at m/z 187 (2 Da; 9% RA), 174 (15 Da; 30%), 173 (16 Da; 7%),
161 (28 Da; 11%) 159 (30 Da; 11%) and 157 (32 Da; 6%)
indicating that such losses observed in the studied compounds
(1–10) originate from the indanone core. It is worthwhile to
stress that the loss of 2 Da (loss of 2H) seen in this intermediate
may give rise to a four-membered ring providing justification
The formation of the product ion at m/z 187 (A; m/z 173 in case
of 4-hydroxy derivative 11) as base peak or with higher RA in
compounds (1–5, 11; see Table 2) is proposed to be formed as
a result of a 1,5-H shift or by a homolytic cleavage of the H on
carbonyl oxygen (C-1) and the (21, 1’) bond and subsequent
neutral loss of the B ring (Scheme 2). The electron-donating
groups (methoxy in the case of 1–4, OH in 5) and the
N-methylimidazole moiety as the B ring, suggest that a
weakening and facile homolytic cleavage of the (21, 1’) bond
may be facilitated by the strong resonance effects. These
resonance transmission effects of the substituents of the aryl ring
are observed due to the planar arrangement with the enone
moiety. Similar effects of electron-donating groups causes
lowering of the IR (C = O) frequencies in 2-arylidene
indanones,[5] while recently a density functional theory (DFT)
study of ortho-substituted phenyl cations with groups like -
CHO, -CH2OH showed they undergo an unexpected ring
closure reaction to give four-membered rings in the gas phase.[17]
It was observed that such a cyclization reaction proceeds via the
nearby atom of the substituent which appears in relative vicinity
(up to ~3 Å) to the cationic center. Surprisingly, this product ion
[(21, 1’)A+] was also formed in case of compounds 6 (CN) and 12
(phenyl imidazole) with 17 and 66% RA, respectively. At this
point a comparison of the two positional isomers 1 and 2
showed that compound 1 gives the (21, 1’)A+ product ion at
m/z 187 by loss of 108 Da as the base peak of the MS/MS spectra
while, in compound 2, this peak was formed in 85% RA (Fig. 2)
indicating that they can be easily identified and differentiated
by ESI-MS/MS. Besides, the differentiation between 1 and 2
can also be achieved since their MS/MS spectra showed a
distinct fragmentation pattern, with 2 showing many more
fragment ions. In fact, the ions at m/z 129, 209, 219, 221, 237
and 265 showed a higher abundance for compound 2 and are
almost absent in compound 1 (Fig. 2).
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J. C. J. M. D. S. Menezes, J. A. S. Cavaleiro and M. R. M. Domingues
for the proposed structure (A in Scheme 2) arising at m/z 187 in
Benzopyrylium ring formation
the studied compounds. Further combined losses afforded
product ions at m/z 146 (43 Da; 100% RA), 145 (44 Da; 48%)
and 131 (58 Da; 25% RA).
An interesting loss of 2 Da was observed in compounds 6
(6i, m/z 288) and 12 (12i, m/z 315) which can be attributed to a
stable cyclic ring formation (Scheme 5; see Supplementary Figs.
SF4 and SF8, Supporting Information). The justification for the
loss of 2 Da can be obtained from the fact that these compounds
easily isomerize in solution or upon exposure to diffused
daylight to the Z geometry,[9] wherein rotation of the (21, 1’) bond
brings the molecule in the right conformation for this loss to
occur. This fact was confirmed in the present cases, wherein the
1H NMR spectra of compounds 6 and 12 showed formation of
new peaks and the chemical shift of the olefinic proton at C-21
moved upfield from ~ δ 7.50 to ~ δ 6.9. On closer observation in
the MS2 spectra of compounds 1, 8 and 10 similar loss of CH3OH,
HBr and HF was noted (product ions 1i, 8i and 10i; 7, 100 and
18% RA, respectively). Further, in the case of compound 10, the
B ring is devoid of aromatic protons; hence the justification
that this may be the only pathway for loss of 20 Da (HF) in
this molecule is fortified. Similar loss of the ortho substituent
to form a stable cyclic ring was proposed to occur for
benzylidenecyclanones under EI-MS conditions.[10] This type of
loss to afford a stable six-membered benzopyrylium ion has been
observed in analogous 2-benzylidenecyclohexanones and 2,6-
bis-benzylidenecyclohexanones containing a halogen atom as
ortho substituent in the aromatic ring under EI-MS conditions.[20]
This benzopyrylium ion was also described using EI-MS in
chalcones, where a M-1 ion arises exclusively (~85%) by loss of
the hydrogen atom from the phenyl ring of the styryl group
confirmed by deuterium labeling.[21] While the same pathway
operates for the formation of the stable benzopyrylium ion by
loss of M–H or M–Cl from ortho positions in styryl ketones.[22]
To test and confirm our hypothesis of a similar six-
membered ring formation, we carried out MS2 of compounds
1, 8 and 10 in deuterated methanol, since in these cases loss of
DR1 (R1 = OCH3, Br or F) can be identified. The ESI-MS
spectra of the deuterated experiments of compounds 1, 8
and 10 presented the [M + D]+ ions at m/z 296, 344 and 368
and the corresponding MS/MS spectra showed the ion at
m/z 187, 264 and 194, respectively, as the base peak of the
spectra (Figs. 4 and 5). The formation of m/z 264 and 263 in 1
indicates loss of CH3OH and CH3OD, respectively (7% and
6% RA). The base peak at m/z 264 in 8 indicated the major
pathway to be via loss of HBr, but a small peak at m/z 263
(15% RA; -DBr) was observed which fully supports our
hypothesis (Fig. 4), while, in 10, formation of m/z 347 (22% RA)
provides justification for loss of DF as proposed (Fig. 5). In
Fragmentations from electron-withdrawing substituents
Although the -CN group is a moderately withdrawing one,
compound 6 presented several ions similar to those appearing
for strongly donating methoxy substituents (Table 2). An
interesting fragmentation resulting via combined loss of
CO + HCHO with subsequent loss of HCN and •H gives a
product ion at m/z 204 (6f, Scheme 4; 57% RA;
Supplementary Fig. SF4, Supporting Information). A closer
examination of the MS2 spectra of compounds 2 (R3 = OCH3)
and 5 (R3 = OH) presented a product ion (2f and 5f) at m/z 205
(8% and 35% RA, respectively) which is undoubtedly forming
via the same pathway (CO + HCHO) with the subsequent loss
of CH3OH and H2O, respectively (Scheme 4).
The characteristic loss of the NO2 radical and HNO2,[10]
along with the combined losses of 61 (•NO2 + •CH3; 27%
RA), 62 (HNO2 + •CH3; 22% RA) and 63 Da (HNO2 + CH4;
12% RA) were observed for compound 7 (R3 = NO2; see
Supplementary Fig. SF5, Supporting Information). Similarly
the principal loss of HBr appearing as base peak and the
•Br radical (5% RA) gave product ions at m/z 263 and 264 in
8 and 9 (R1 and R3 = Br). Combined loss (•Br + •CH3; 18%
RA) was seen only for 9 while loss of (HBr + •CH3) was seen
in 4% and 28% RA for 8 and 9, respectively. The loss of HF,
(HF + •CH3) and (HF + HCHO) was observed in the case of
10 at m/z 347, 332 and 317 in 18, 12 and 5% RA, respectively
(Supplementary Fig. SF6, Supporting Information).
The low number of product ions observed in the case of
o-Br in comparison with p-Br indicates that the isomers can be
easily differentiated using ESI-MS/MS. Moreover, the retro-
aldol product ion was observed in 46% RA in 9 and low RA
(5%) in the o-Br compound 8. This product ion can serve as
reference peak to easily distinguish/differentiate between the
two positional isomers (Fig. 3). The probable reason for the high
number of fragments in the p-Br isomer may be due to the facile
loss of HBr and inductive effect of the halogen.
In the case of compounds 7 and 9 (p-NO2 and p-Br,
respectively), a common product ion at m/z 221 (7 g and 9 g;
Scheme 4) was formed in 45% and 25% RA, respectively.
Further, the product ion at m/z 221 losses 28 Da to give a
product ion at m/z 193 (7 h and 9 h) with 13 and 4% RA,
respectively (Scheme 4).
Scheme 5. Proposed structures for loss of 2 Da and R1X from the
ortho position in 2-arylidene indanone derivatives.
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Rapid Commun. Mass Spectrom. 2013, 27, 2461–2471

Analysis of 2-arylidene-1-indanone derivatives by ESI-MS/MS
Figure 4. ESI-MS/MS spectra of the [M + D]+ ion of compounds 1 and 8.
Figure 5. ESI-MS/MS spectrum of the [M + D]+ ion of compound 10.
fact, observation of m/z 263 and 347 in both cases (8 and 10)
ion at m/z 187 although with varying relative abundance
(RA). The electron-donating groups favoured the formation
of this product ion, while it was absent or observed in low
%RA in electron-withdrawing substituents. A retro-aldol
product ion [(2, 21)B+] was also observed as a characteristic
fragmentation pathway for this type of compounds while
the degree of methoxylation in the aromatic ring further led
to loss of 15 Da. The (1,2)(3,9)B+ fragmentation is proposed
to occur via cyclization to stable indene derivatives, while a
(1,8)(3,9)B+ fragmentation gives benzopyrylium product ions
in some cases. Specific loss of the substituents (OCH3, OH,
NO2, Br, F) and combined losses were able to add in the
assignment of the substituent pattern of these compounds.
Loss of the ortho substituent in the B ring (H, CH3O, Br and F)
combined with H via intramolecular cyclization was also
confirmed that the protonation or deuteration may also occur
at the carbonyl subsequently to undergo aromatic substitution
at the ortho position as reported earlier in styryl ketones.[22]
Moreover, the formation of the major ion at m/z 187 in 1 and
at m/z 194 in 10 indicates the deuteration at the carbonyl
oxygen and 1,5-D shift or transfer during retro-aldol
fragmentation, respectively (Figs. 4 and 5).
CONCLUSIONS
The electrospray ionization tandem mass spectroscopy of
2-arylidene-4-methoxy-7-methyl-1-indanone derivatives show
an interesting (21, 1’)A+ bond cleavage to give a diagnostic
Rapid Commun. Mass Spectrom. 2013, 27, 2461–2471
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J. C. J. M. D. S. Menezes, J. A. S. Cavaleiro and M. R. M. Domingues
carbonyl stretching frequencies and 13C NMR chemical
observed. This study also enables to distinguish between the
shifts of E-2-(X-benzylidene)-1-indanones. Comparison
with the IR data of E-2-(X-benzylidene)-1-indanones, -tetralones,
and -benzosuberones. J. Mol. Struct. 2004, 697, 41.
isomers of two different mono-substituted 2-arylidene-4-
methoxy-7-methyl-1-indanone derivatives. These fragmenta-
tions depending on the substitution pattern on the B ring will
be helpful for easy identification in biological mixtures.
[6] B. Jaki, J. Heilmann, O. Sticher. New antibacterial metabolites
from the Cyanobacterium Nostoc commune (EAWAG 122b).
J. Nat. Prod. 2000, 63, 1283.
[7] S. P. Kamat, J. C. Menezes, B. M. Siddhaye, S. K. Paknikar.
Facile syntheses of 4-hydroxy-7-methyl-1-indanone,
isolated from cyanobacterium Nostoc commune. Indian J.
Chem. 2008, 47B, 1597.
[8] (a) J. R. Dimmock, N. M. Kandepu, A. J. Nazarali,
T. P. Kowalchuk, N. Motaganahalli, J. W. Quail, P. Mykytiuk,
G. F. Audette, L. Prasad, P. Perjési, T. M. Allen, C. L. Santos,
J. Szydlowski, E. de Clercq, J. Balzarini. Conformational and
quantitative structure-activity relationship study of cytotoxic
2-arylidenebenzocycloalkanones. J. Med. Chem. 1999, 42,
1358; (b) J. R. Dimmock, G. A. Zello, E. O. Oloo, J. W.
Quail, H. B. Kraatz, P. Perjési, F. Aradi, K. Takács-Novák,
T. M. Allen, C. L. Santos, J. Balzarini, E. de Clercq, J. P.
Acknowledgements
The University of Aveiro, Fundação para a Ciência e a
Tecnologia (FCT, Portugal), European Union, QREN, FEDER
and COMPETE for funding the QOPNA research unit (project
PEst-C/QUI/UI0062/2013), the Portuguese National NMR
Network and Portuguese Mass Spectrometry Network (RNEM)
(REDE/1504/REM/2005) are gratefully acknowledged. The
authors thank C. Barros for recording the mass data. JCJMDS
Menezes thanks QOPNA for a research grant and Prof. S. P.
Kamat (Goa University) for the introduction to the chemistry
of indanones.
Stables.
Correlations
between
cytotoxicity
and
topography of some 2-arylidenebenzocycloalkanones
determined by X-ray crystallography. J. Med. Chem.
2002, 45, 3103.
REFERENCES
[9] P. Perjési, M. Takács, E. Ösz, Z. Pintér, J. Vámos, K. Takács-
Novák. In-solution and on-plate light-catalyzed E/Z
isomerization of cyclic chalcone analogues. Lipophilicity of
E- and Z-2-(X-benzylidene)-1-benzosuberones. J. Chromatogr.
Sci. 2005, 43, 289.
[1] (a) L. Huang, C. Lu, Y. Sun, F. Mao, Z. Luo, T. Su, H. Jiang,
W. Shan, X. Li. Multitarget-directed benzylideneindanone
derivatives: anti-β-amyloid (Aβ) aggregation, antioxidant,
metal chelation, and monoamine oxidase
B (MAO-B)
[10] Gy. Tarczay, K. Vékey, K. Ludányi, P. Perjési, P. Sohár. E-2-
Benzylidenecyclanones. II. IR and mass spectrometric
investigations. J. Mol. Struct. 2000, 520, 97.
[11] W. Zhu, Y. Yuan, P. Zhou, L. Zeng, H. Wang, L. Tang,
B. Guo, B. Chen. The expanding role of electrospray ionization
mass spectrometry for probing reactive intermediates in
solution. Molecules 2012, 17, 11507.
[12] (a) C. S. Ho, C. W. K. Lam, M. H. M. Chan, R. C. K. Cheung,
L. K. Law, L. C. W. Lit, K. F. Ng, M. W. M. Suen, H. L. Tai.
Electrospray ionisation mass spectrometry: principles and
clinical applications. Clin. Biochem. Rev. 2003, 24, 3; (b)
S. Banerjee, S. Mazumdar. Electrospray ionization mass
spectrometry: A technique to access the information beyond
the molecular weight of the analyte. Int. J. Anal. Chem. 2012,
Article ID 282574.
inhibition properties against Alzheimer’s disease. J. Med.
Chem. 2012, 55, 8483; (b) S. Rizzo, M. Bartolini, L. Ceccarini,
L. Piazzi, S. Gobbi, A. Cavalli, M. Recanatini, V. Andrisano,
A. Rampa. Targeting Alzheimer’s disease: Novel indanone
hybrids bearing a pharmacophoric fragment of AP2238.
Bioorg. Med. Chem. 2010, 18, 1749; (c) R. Sheng, Y. Xu, C. Hu,
J. Zhang, X. Lin, J. Li, B. Yang, Q. He, Y. Hu. Design, synthesis
and AChE inhibitory activity of indanone and aurone
derivatives, Eur. J. Med. Chem. 2009, 44, 7; (d) Z. Omran,
T. Cailly, E. Lescot, J. Sopkova-de Oliveira Santos,
J. H. Agondanou, V. Lisowski, F. Fabis, A. M. Godard,
S. Stiebing, G. Le Flem, M. Boulouard, F. Dauphin,
P. Dallemagne, S. Rault. Synthesis and biological evaluation
as AChE inhibitors of new indanones and thiaindanones
related to donepezil. Eur. J. Med. Chem. 2005, 40, 1222.
[13] W. A. Korfmacher. Principles and applications of LC-MS in
new drug discovery. Drug Discov. Today 2005, 10, 1357.
[14] Y. L. Ma, Q. M. Li, H. Van den Heuvel, M. Claeys.
Characterization of flavone and flavonol aglycones by
collision-induced dissociation tandem mass spectrometry.
Rapid Commun. Mass Spectrom. 1997, 11, 1357.
[15] J. C. J. M. D. S. Menezes, M. G. P. M. S. Neves,
J. A. S. Cavaleiro, C. Barros, S. M. Santos, F. C. da Silva,
V. F. Ferreira, M. R. M. Domingues. Gas phase reactions of
β-substituted hetero-Diels-Alder adducts of meso-
tetraphenylporphyrin using tandem mass spectrometry. Int.
J. Mass Spectrom. 2013, 343/344, 1.
[16] J. C. J. M. D. S. Menezes, J. A. S. Cavaleiro, S. P. Kamat,
C. M. R. F. Barros, M. R. M. Domingues. Electrospray
tandem mass spectrometry analysis of methylenedioxy
chalcones, flavanones and flavones. Rapid Commun. Mass
Spectrom. 2013, 27, 1303.
[17] S. V. Bondarchuk, B. F. Minaev. Density functional study of
ortho-substituted phenyl cations in polar medium and in
the gas phase. Chem. Phys. 2011, 389, 68.
[2] (a) Z. Nowakowska. A review of anti-infective and anti-
inflammatory chalcones. Eur. J. Med. Chem. 2007, 42, 125;
(b) N. K. Sahu, S. S. Balbhadra, J. Choudhary, D. V. Kohli.
Exploring pharmacological significance of chalcone
scaffold: A review. Curr. Med. Chem. 2012, 19, 209.
[3] (a) R. Bansal, G. Narang, C. Zimmer, R. W. Hartmann.
Synthesis of some imidazolyl-substituted 2-benzylidene
indanone derivatives as potent aromatase inhibitors for breast
cancer therapy. Med. Chem. Res. 2011, 20, 661; (b) N. Siddiqui,
M. F. Arshad, S. A. Khan, W. Ahsan, R. Ali, M. S. Alam,
S. Ahmed. Synthesis of new piperidyl indanone derivatives
as anticonvulsant agents. Med. Chem. Res. 2012, 21, 726;
(c) J. P. Qiao, C. S. Gan, C. W. Wang, J. F. Ge, D. D. Nan,
J. Pan, J. N. Zhou. Novel indanone derivatives as potential
imaging probes for β-amyloid plaques in the brain.
ChemBioChem 2012, 13, 1652.
[4] (a) A. Lévai. Synthesis of exocyclic α,β-unsaturated ketones.
Arkivoc 2004, vii, 15; (b) A. Lévai, T. Patonay. Reaction of
E-2-arylidene-1-indanones, Z-aurones, Z-1-thioaurones and
Z-2-arylidene-2,3-dihydro-1H-indol-3-ones with diazomethane.
J. Heterocyclic Chem. 1999, 36, 747.
[5] P. Perjési, A. Perjéssy, E. Kolehmainen, E. Ősz, M. Samaliková,
J. Linnanto, E. Virtanen. E-2-Benzylidenebenzocycloalkanones.
III. Studies on transmission of substituent effects on IR
[18] A. A. Scala, E. W. G. Diau, Z. H. Kim, A. H. Zewail.
Femtosecond β-cleavage dynamics: Observation of the
diradical intermediate in the nonconcerted reactions of
cyclic ethers. J. Chem. Phys. 1998, 108, 7933.
wileyonlinelibrary.com/journal/rcm
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 2461–2471

Analysis of 2-arylidene-1-indanone derivatives by ESI-MS/MS
[19] S. H. Lee. The photodissociation of oxetane at 193 nm as the
reverse of the Paterno–Buchi reaction. J. Chem. Phys., 2009,
131, 224309.
[22] P. J. Smith, J. R. Dimmock, W. A. Turner. Mass spectrometry
of some nuclear substituted styryl ketones. Can. J. Chem.
1972, 50, 871.
[20] P. J. Smith, J. R. Dimmock, W. A. Turner. Mass spectrometry
of some substituted 2-benzylidenecyclohexanones and 2,6-
bis-benzylidenecyclohexanones. Can. J. Chem. 1973, 51, 1458.
[21] H. Budzikiewicz, C. Djerassi, D. H. Williams. Mass
Spectrometry of Organic Compounds. Holden-Day, San
Francisco, 1967, p. 169.
SUPPORTING INFORMATION
Additional supporting information may be found in the
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