Nuclear Magnetic Resonance (NMR), Infrared (IR) and Mass Spectrometry (MS) study of keto-enol tautomerism of isobenzofuran-1(3H)-one derivatives

Article abstract of DOI:10.1016/j.molstruc.2016.02.015

The keto-enol tautomerism of 3-(2-hydroxy-4,4-dimethyl-6-oxo-cyclohexen-1-yl)isobenzofuran-1(3H-one (1), 3-(2-hydroxy-6-oxocyclohex-1-enyl)isobenzofuran-1(3H)-one (2), 3-(2-hydroxy-4-methyl-6-oxocyclohex-1-enyl)isobenzofuran-1(3H)-one (3), 3-(2-hydroxy-5-

Full text of DOI:10.1016/j.molstruc.2016.02.015

Journal of Molecular Structure 1113 (2016) 146e152
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Nuclear Magnetic Resonance (NMR), Infrared (IR) and Mass
Spectrometry (MS) study of keto-enol tautomerism of
isobenzofuran-1(3H)-one derivatives
,
b
c
c, *
Diego Arantes Teixeira Pires a , Wagner Luiz Pereira , Robson Ricardo Teixeira
,
ꢀ
d
b, e, **
ꢀ
Jose Daniel Figueroa-Villar , Claudia Jorge do Nascimento
a
^
Department of Chemistry, Federal Institute of Education, Science and Technology of Goias, Luziania, Brazil
b Institute of Chemistry, University of Brasília, Brasília, Brazil
c Department of Chemistry, Universidade Federal de Viçosa, Viçosa, Brazil
d Department of Chemistry, Military Institute of Engineering, Rio de Janeiro, Brazil
e Institute of Biosciences, Federal University of Rio de Janeiro State, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The keto-enol tautomerism of 3-(2-hydroxy-4,4-dimethyl-6-oxo-cyclohexen-1-yl)isobenzofuran-
1(3H-one (1), 3-(2-hydroxy-6-oxocyclohex-1-enyl)isobenzofuran-1(3H)-one (2), 3-(2-hydroxy-
4-methyl-6-oxocyclohex-1-enyl)isobenzofuran-1(3H)-one (3), 3-(2-hydroxy-5-oxocyclopent-1-enyl)
isobenzofuran-1(3H)-one (4) and 2-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-1H-indene-1,3(2H)-dione
(5) were investigated. We noticed that for compounds 1 to 4 only the enol form is observed in solid, in
solution or in the gas phase. Their tautomeric equilibria are not affected by the solvent, temperature or
physical state. Compound 5 was observed in its keto form in solution (NMR) and solid state (IR). The
enol species of 5 was also observed upon Mass Spectrometry analysis. These findings were supported
by NMR, IR, MS/MS and molecular modeling analyses.
Received 3 September 2015
Received in revised form
3 February 2016
Accepted 3 February 2016
Available online 9 February 2016
Keywords:
Keto-enol tautomerism
Isobenzofuran-1(3H)-ones
Phtalides
© 2016 Elsevier B.V. All rights reserved.
Solvent effects
Physical state influence
Spectroscopy
1. Introduction
nucleus and presenting relevant biological activities have been
isolated from plants, fungi, bacterias and liverworts [7]. In partic-
The keto-enol tautomerism is important in many fields of
chemistry and biochemistry. The investigation of this phenomenon
allows us to learn about the physicochemical properties and bio-
logical activities of the substances in which it occurs. Several fac-
tors, such as intra and intermolecular interactions, the hydrogen
bond formation, solvents effects, temperature, physical state, and
others, can influence the keto-enol equilibrium, favoring the keto
or the enol species [1e6].
ular, the C-3 functionalized phtalides display several significant
medicinal properties [8e13]. It stands out the success story of
3-butylisobenzofuran-1(3H)-one (also known as n-butylphthalide),
a compound which nowadays has been clinically used as anti-
platelet drug for ischemia-cerebralapoplexy [14,15]. It also deserves
comments the importance of isobenzofuran-1(3H)-ones as building
blocks in organic synthesis [16,17].
Our own interest in this class of compounds led us to synthesize
a series of twelve C-3 functionalized isobenzofuran-1(3H)-ones
possessing alicyclic and aromatic groups. These compounds were
evaluated in vitro for their antiproliferative activity against leuke-
mia cell lines [18]. The obtained biologically data revealed that the
antiproliferative activity of two of the evaluated compounds was
superior than Etoposide (VP-16), an anticancer drug used in several
chemotheraphy regime, including leukemia. In another investiga-
tion, we also assessed the effect of the aforementioned series of
isobenzofuranones on the photosynthetic machinery. It was found
Isobenzofuran-1(3H)-ones (phtalides) are heterocyclic com-
pounds characterized by the presence of a benzene ring fused to a
g
-lactone one. Secondary metabolites containing the phtalide
* Corresponding author.
** Corresponding author. Institute of Biosciences e Federal University of Rio de
Janeiro State (UNIRIO), Brazil. Tel.: þ55 21999222021.
E-mail addresses: robsonr.teixeira@ufv.br (R.R. Teixeira), claudia.j.nascimento@
gmail.com (C.J. Nascimento).
http://dx.doi.org/10.1016/j.molstruc.2016.02.015
0022-2860/© 2016 Elsevier B.V. All rights reserved.

D.A.T. Pires et al. / Journal of Molecular Structure 1113 (2016) 146e152
147
Table 1
The keto and the enol structures of the investigated compounds.
Compound
Enol form
Keto form
1
2
3
4
5
Fig. 1. 1H-NMR spectra of compound 1 in methanol-d4 at different temperatures. The H-3 signal is observed near 6.70 ppm.

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D.A.T. Pires et al. / Journal of Molecular Structure 1113 (2016) 146e152
Fig. 2. APT spectra of compound 1 in methanol-d4 at different temperatures. The C-10 signal is observed at 110.1 ppm.
Fig. 3. 1H-NMR spectra of compound 1 in methanol-d4 at different temperatures. The H-30 and H-50 signals are observed around 2.34 ppm.
Fig. 4. 1H-NMR spectra of compound 5 in DMSO-d6 at different temperatures. The H-3 signal is observed at 6.25 ppm and the H-10 signal at 4.48 ppm.

D.A.T. Pires et al. / Journal of Molecular Structure 1113 (2016) 146e152
149
Fig. 5. APT spectra of compound 5 in DMSO-d6 at different temperatures. The C-10 signal is observed near 55.1 ppm and C-3 signal is near 77.5 ppm.
that the compounds are capable of interfering with electron
transport chain in spinach chloroplast via two different mecha-
nisms [19].
Spectrometry analyses of the species involved in the tautomerism
equilibria.
This paper is concerned about the investigation of keto-enol
tautomerism of isobenzofuran-1(3H)-ones containing different
appendages at C-3 position of isobenzofuranone nucleus (see
Table 1 for numbering). The structures of the investigated com-
pounds are presented in Table 1. The results herein described were
supported by the NMR and IR spectroscopy as well as Mass
2. Experimental
2.1. Study of the keto-enol tautomerism
The NMR studies were performed in a 600 MHz spectrometer
(Premium Compact, Varian) with
a 5 mm probe and with
Fig. 6. MS/MS spectrum of the compound 5, showing the fragmentation of 18 Da that indicates the presence of an OH group.

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D.A.T. Pires et al. / Journal of Molecular Structure 1113 (2016) 146e152
Fig. 7. IR spectrum (ATR) of the compound 1 (a broad band in the range 3200e2400 cmꢀ1 was associated with OeH stretching).
isobenzofuranone solutions that presented the concentration of
16.67 mg mLꢀ1. 1H-NMR (32 scans) experiments were acquired in
acetone-d6 (273e318K), methanol-d4 (288e328 K) and DMSO-d6
(288e348 K). APT (3000 scans) and 13C-NMR (2000 scans) spectra
were acquired in acetone-d6 (288e318K), methanol-d4
(288e328 K) and DMSO-d6 (288e348 K).
The IR analyses were conducted in solid state in a Varian 660-IR
with the GladiATR accessory.
The molecular modeling calculation procedures for the com-
pounds 1 to 5 were done using the DFT/B3LYP method and 6-31G*
using the Spartan'06 program [20]. The keto-enol and the diketo
conformations of all compounds were calculated with the Energy
Profile method in vacuum and in the presence of solvents (acetone,
DMSO and methanol).
The Mass spectra were acquired in a micrOTOF e QII (Bruker)
spectrometer in the positive mode of acquisition. The samples were
solubilized in acetonitrile (1% of formic acid), with a 4.5 kV voltage
at the capillary and a flow rate of 2.0 m
L minꢀ1. MS and MS/MS
experiments were performed.
Fig. 8. IR spectrum (ATR) of compound 5 (signals in 1769, 1740 and 1702 cmꢀ1: C¼O stretching).

D.A.T. Pires et al. / Journal of Molecular Structure 1113 (2016) 146e152
151
Table 2
Energy (minima) difference of the keto and the enol forms for each compound calculated by DFT (B3LYP)/6-31Ga in vacuum and in the presence of solvents (acetone, DMSO and
methanol).
Compound
1
D
Evacuum (kJ/mol)
D
Eacetone (kJ/mol)
D
EDMSO (kJ/mol)
DEmethanol (kJ/mol)
Enola
Keto
Enola
Keto
Enola
Keto
Enola
Keto
Enol
14.43
18.13
18.75
22.56
23.37
6.12
2.86
5.19
2.53
7.09
4.58
2
3
4
5
3.71
3.22
5.16
24.61
23.29
25.02
22.78
24.87
25.10
Ketoa
a
The most stable form.
3. Results and discussion
in DMSO (Fig. 4), showed the signal for the H-3 as compounds 1 to
4. However, it was also observed one signal related to H-10 (char-
acteristic of keto form). The same behavior was observed for sub-
stance 5 in acetone (see Supplementary Material). The temperature
did not influence the tautomeric equilibrium of 5.
As previously reported [18,19], the synthesis of compounds 1 to
5 (Table 1) were achieved via DBU-promoted condensation be-
tween phtalaldehydic acid and appropriated 1,3-diketones. For all
synthesized compounds, their tautomeric equilibrium was inves-
tigated by NMR, IR spectroscopy, Mass spectrometry, as well as
Molecular Modeling.
The APT spectra of 5 in DMSO showed a signal at 55.1 ppm,
related to C-10 (Fig. 5), indicating the presence of a eCH group. The
quaternary C-10 was not observed and, consequently, confirmed the
keto form for this compound. Similarly to the proton spectrum,
temperature did not affect the APT spectrum. The same behavior is
observed for compound 5 in acetone (see Supplementary Material).
It is worth to mention that 13C-NMR showed two different peaks
for ketone carbonyl groups (Supplementary Material). This obser-
vation is in agreement with the keto form of 5. The same result was
observed in acetone. The carbon spectra for compounds 1 to 4
showed only the presence of one ketone carbonyl signal.
By comparing the NMR data of compounds 4 and 5, it can be
inferred that the presence of an aromatic ring fused to the 1,3-
diketone five membered ring changes the tautomeric equilibrium
so that the keto form is observable in the case of compound 5.
Mass spectrometry experiments can provide insight about the
species involved in tautomeric equilibria. Fragmentation of 18 Da
can be related to the dehydration of the OH group in the form of
H2Oþ, and fragmentation of 28 Da can be related to the C]O group
in the form of COþ [21].
The study of the keto-enol equilibria by 1H-NMR was performed
observing the multiplicity of the H-3 signal (singlet for the enol
form and duplet for the keto form). It was also observed the pres-
ence (for the keto form) or the absence (for the enol form) of a
signal due to H-10 (see Table 1 for numbering). Furthermore, the C-
10 signal in APT spectra was also used to study the tautomeric
equilibria, observing that it is a quaternary carbon in the enol form
and a eCH in the keto form. The complete assignments of com-
pounds 1 to 5 in acetone, DMSO and methanol can be seen in the
Supplementary material.
The expansion of the aromatic region of the 1H-NMR spectrum
for compound 1 in methanol, at different temperatures, is shown in
Fig. 1. The H-3 signal is observed as a singlet, and the integration
refers to a single proton. Additionally, both the multiplicity and the
integration of the signals do not vary with the temperature
increase.
The APT spectra of compound 1 in methanol at different tem-
peratures show the signal concerning to the C-10 being a quaternary
carbon at all temperatures (Fig. 2). A similar trend is also observed
for the spectra acquired in acetone and DMSO solvents (see
Supplementary Material). Therefore, the enol form of compound 1
is favored regardless the solvent, even at higher temperatures.
It is interesting to observe the variation in the multiplicity of H-
30 and H-50 (Fig. 3) with increasing temperature. This variation can
be explained by the faster rotation around C10-C3 bond, which
leads to the coalescence of the signals at higher temperatures. This
coalescence was also observed for compound 1 in acetone and
DMSO (see Supplementary Material). This faster rotation also af-
fects the signals for C-30 and C-50 (Supplementary Material)
Compounds 2, 3 and 4 showed similar behavior to that
described for compound 1. For all of them, the enol form is favored
in different solvents (methanol, acetone and DMSO) as well as at
different temperatures (see Supplementary Material). Therefore,
for these compounds, solvent and temperature did not interfere in
the keto-enol equilibria. The presence of methyl groups (one or
two) attached to alicyclic portion also did not affect the equilib-
rium. Moreover, the replacement of the six-membered ring by a
five-membered one did not favor the keto form (see
Supplementary Material).
The MS/MS analysis of compound 5 revealed the presence of the
enol form as the sole species in the equilibrium. This statement is
corroborated by the observation of a fragmentation of 18 Da of the
OH group in the form of H2Oþ as shown in Fig. 6 for compound 5
[21,22].
The MS/MS spectra of compounds 1e4 (see Supplementary
Material) also showed that fragmentation (18 Da), indicating that
the enol form of these compounds is favored in the equilibria in the
gas phase.
The Mass spectrometry, as well as the NMR experiments for
compounds 1 to 4, revealed only the presence of enol species in the
tautomeric equilibria. On the contrary, while the NMR experiments
showed the presence of keto form in the equilibrium for compound
5, the mass experiments results showed exclusively the presence of
the enol form of compound 5. Thereby, the physical state of the
compound, gas phase (analyzed by Mass spectrometry) and solu-
tion (analyzed by NMR) can affect the keto-enol equilibrium of the
investigated compound.
We also sought information about the tautomeric equilibrium of
substances 1 to 5 analyzing them in solid phase by IR spectroscopy.
Regarding compounds 1 to 4, the IR spectra present a broad band
which can be associated to the OeH group of the enol form, as it is
shown in Fig. 7 for compound 1 (IR spectra for compounds 2 to 4 are
shown in the Supplementary Material). Thus, the enol form is
Compound 5 was insoluble in methanol, which precluded NMR
analyses using this solvent. The 1H-NMR spectrum of compound 5

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D.A.T. Pires et al. / Journal of Molecular Structure 1113 (2016) 146e152
favored in solid phase for compounds 1 to 4.
the financial support.
The inspection of IR spectrum of the compound 5 (Fig. 8)
showed only bands related to the C]O stretching; it was not
observed any band related to the OeH stretching, characterizing
this compound in the keto form in the solid phase.
The IR experiments (solid state) for compounds 1 to 4 showed
only the enol form as observed with the NMR (liquid state) and
Mass spectrometry (gas state) experiments. Solvent, temperature
and physical state did not change their equilibrium. However, for
compound 5, the keto form was observed when it was analyzed by
IR (solid state) and NMR (in solution), and enol form was noticed by
Mass spectrometry (gas state). The physical state could be the
responsible for the changes in the keto-enol equilibrium for the
compound 5.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2016.02.015.
References
[1] E. Ferrari, M. Saladini, F. Pignedoli, F. Spagnolo, R. Benassi, New J. Chem. 35
(2011) 2840e2847.
[2] H. Golchoubian, E. Rezaee, D. Farmanzadeh, Struct. Chem. 24 (2013) 481e489.
[3] V.L. Junior, M.G. Constantino, G.V.J. Silva, A.C. Neto, C.F. Tormena, J. Mol. Struct.
828 (2007) 54e58.
[4] A. Martinez-Richa, G. Mendonza-Diaz, P. Joseph-Nathan, App Spectrosc. 50
(1996) 1408e1412.
The keto and the enol forms of all compounds were submitted to
the geometry and energy optimization by molecular modeling and
had their energy calculated for each form (Table 2).
[5] F. Payton, P. Sandusky, W.L. Alworth, J. Nat. Prod. 70 (2007) 143e146.
[6] W. Schilf, B. Kamienski, K. Uzarevic, J. Mol. Struct. 1031 (2013) 211e215.
[7] G. Lin, S.S.K. Chan, H.S. Chung, S.L. Li, Chemistry and Biological Activities of
Naturally Occurring Phthalides. Studies in Natural Products Chemistry, first
ed.,, Elseveir, Amsterdam, 2005.
[8] G.A.R. Johnston, Br. J. Pharmacol. 169 (2013) 328e336.
[9] F. Ma, Y. Gao, H. Qiao, X. Hu, J. Chang, J. Thromb. Thrombolysis 33 (2012)
64e73.
[10] K. Yoganathan, C. Rossant, S. Ng, Y. Huang, M.S. Butler, A.D. Buss, J. Nat. Prod.
66 (2003) 1116e1117.
[11] A. Zamilpa, M.H. Ruiz, E. del Olmo, J.L.L. Perez, J. Tortoriello, A.S. Feliciano,
Bioorg. Med. Chem. Lett. 15 (2005) 3483e3486.
[12] H.M. Ge, Y. Shen, C.H. Zhu, S.H. Tan, H. Ding, Y.C. Song, R.X. Tan, Phyto-
chemistry 69 (2008) 571e576.
According to the molecular modeling data, it was observed that
solvent didn't interfere in the predominant form of the compounds
in the keno-enol equilibrium when compared to the vacuum cal-
culations. For compounds 1 to 4, the molecular modeling shows the
enol form as the most stable for all the calculations, giving support
to the NMR, MS and IR findings previously described. For com-
pound 5, the molecular modeling showed the keto form as the most
stable, which is in total agreement with the IR and NMR experi-
mental data. The MS results for compound 5 (which shows only the
enol form) were the only one not supported by the molecular
modeling results.
ꢀ
[13] L.P.L. Logrado, C.O. Santos, L.A.S. Romeiro, A.M. Costa, J.R.O. Ferreira,
B.C. Cavalcanti, O.M. de Moraes, L.V.C. Lotufo, C. Pessoa, M.L. dos Santos, Eur. J.
Med. Chem. 45 (2010) 3480e3489.
[14] X. Wang, Y. Li, Q. Zhao, Z. Min, C. Zhang, Y. Lai, H. Ji, S. Peng, Y. Zhang, Org.
Biomol. Chem. 9 (2011) 5670e5681.
[15] X. Diao, P. Deng, C. Xie, X. Li, D. Zhong, Y. Zhang, X. Chen, Drug Metab. Dispos.
41 (2013) 430e444.
4. Conclusion
It was showed that the keto-enol equilibrium for compounds 1
to 4 is not affected by changes in solvent, temperature and physical
state. Only the enol form was observed for these compounds by
NMR, Mass Spectroctrometry, IR and Molecular Modeling. For
compound 5, it was observed the enol form by Mass Spectroscopy.
Interestingly, the observation of only enol form of compound 5 by
Mass Spectroscopy and exclusively the keto form by IR, NMR and
Molecular Modeling, indicates that the physical state can influence
the tautomerism for this compound.
Comparing the structures of compounds 4 and 5, and looking at
the NMR, IR and Molecular Modeling results, it can be inferred that
the presence of an aromatic ring changes the tautomeric
equilibrium.
[16] R. Karmakar, P. Pahari, D. Mal, Chem. Rev. 114 (2014) 6213e6284.
[17] D. Mal, P. Pahari, Chem. Rev. 107 (2007) 1892e1918.
[18] R.R. Teixeira, G.C. Bressan, W.L. Pereira, J.G. Ferreira, F.M. de Oliveira,
D.C. Thomaz, Molecules 18 (2013) 1881e1896.
[19] R.R. Teixeira, W.L. Pereira, D.C. Tomaz, F.M. de Oliveira, S. Giberti, G. Forlani,
J. Agric. Food Chem. 61 (2013) 5540e5549.
[20] Y. Shao, L.F. Molnar, Y. Jung, J. Kussmann, C. Ochsenfeld, S.T. Brown,
A.T.B. Gilbert, L.V. Slipchenko, S.V. Levchenko, D.P. O'Neill, J.R.A. DiStasio,
R.C. Lochan, T. Wang, G.J.O. Beran, N.A. Besley, J.M. Herbert, C.Y. Lin, T. Van
Voorhis, S.H. Chien, A. Sodt, R.P. Steele, V.A. Rassolov, P.E. Maslen,
P.P. Korambath, R.D. Adamson, B. Austin, J. Baker, E.F.C. Byrd, H. Dachsel,
R.J. Doerksen, A. Dreuw, B.D. Dunietz, A.D. Dutoi, T.R. Furlani, S.R. Gwaltney,
A. Heyden, S. Hirata, C.P. Hsu, G. Kedziora, R.Z. Khalliulin, P. Klunzinger,
A.M. Lee, M.S. Lee, W.Z. Liang, I. Lotan, N. Nair, B. Peters, E.I. Proynov,
P.A. Pieniazek, Y.M. Rhee, J. Ritchie, E. Rosta, C.D. Sherrill, A.C. Simmonett,
J.E. Subotnik, H.L. Woodcock III, W. Zhang, A.T. Bell, A.K. Chakraborty,
D.M. Chipman, F.J. Keil, A. Warshel, W.J. Hehre, H.F. Schaefer, J. Kong,
A.I. Krylov, P.M.W. Gill, M. HeadGordon, Phys. Chem. Chem. Phys. 8 (2006)
3172e3191.
Acknowledgment
[21] H. Jiang, A. Somogyi, N.E. Jacobsen, B.N. Timmermann, D.R. Gang, Rapid
Commun. Mass Spectrom. 20 (2006) 1001e1012.
The authors would like to thank to CNPq, FAPEMIG and IFG for
[22] S. Kawano, Y. Inohana, Y. Hashi, J. Lin, Chin. Chem. Lett. 24 (2013) 685e687.