Fluoxetine scaffold to design tandem molecular antioxidants and green catalysts

Article abstract of DOI:10.1039/d0ra03509b

Fluoxetine finds application in the treatment of depression and mood disorders. This selective serotonin-reuptake inhibitor (SSRI) also contrasts oxidative stress by direct ROS scavenging, modulation of the endogenous antioxidant defense system, and/or enhancement of the serotonin antioxidant capacity. We synthesised some fluoxetine analogues incorporating a selenium nucleus, thus expanding its antioxidant potential by enabling a hydroperoxides-inactivating, glutathione peroxidase (GPx)-like activity. Radical scavenging and peroxidatic activity were combined in a water-soluble, drug-like, tandem antioxidant molecule. Selenofluoxetine derivatives were reacted with H₂O₂in water, and the mechanistic details of the reaction were unravelled combining nuclear magnetic resonance (NMR), electrospray ionisation-mass spectrometry (ESI-MS) and quantum chemistry calculations. The observed oxidation-elimination process led to the formation of seleninic acid and cinnamylamine in atrans-selective manner. This mechanism is likely to be extended to other substrates for the preparation of unsaturated cinnamylamines.

Full text of DOI:10.1039/d0ra03509b

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Fluoxetine scaffold to design tandem molecular
antioxidants and green catalysts†
Cite this: RSC Adv., 2020, 10, 18583
a
b
a
c
Giovanni Ribaudo,
Marco Bortoli,
Alberto Ongaro,
Erika Oselladore,
a
c
b
*
Alessandra Gianoncelli,
Giuseppe Zagotto
and Laura Orian
Fluoxetine finds application in the treatment of depression and mood disorders. This selective serotonin-
reuptake inhibitor (SSRI) also contrasts oxidative stress by direct ROS scavenging, modulation of the
endogenous antioxidant defense system, and/or enhancement of the serotonin antioxidant capacity. We
synthesised some fluoxetine analogues incorporating a selenium nucleus, thus expanding its antioxidant
potential by enabling a hydroperoxides-inactivating, glutathione peroxidase (GPx)-like activity. Radical
scavenging and peroxidatic activity were combined in a water-soluble, drug-like, tandem antioxidant
molecule. Selenofluoxetine derivatives were reacted with H2O2 in water, and the mechanistic details of
the reaction were unravelled combining nuclear magnetic resonance (NMR), electrospray ionisation-
mass spectrometry (ESI-MS) and quantum chemistry calculations. The observed oxidation–elimination
process led to the formation of seleninic acid and cinnamylamine in a trans-selective manner. This
mechanism is likely to be extended to other substrates for the preparation of unsaturated cinnamylamines.
Received 19th April 2020
Accepted 7th May 2020
DOI: 10.1039/d0ra03509b
rsc.li/rsc-advances
evidence suggests that oxidative stress and an abnormally
increased generation of reactive oxygen species (ROS) may be
Introduction
implicated in the pathogenesis of many psychiatric and
degenerative disorders.6–10 Considering the mechanistic
aspects, the insurgence of such diseases may be the result of
ROS-related damage, i.e. lipid peroxidation, DNA or protein
oxidation, and mitochondrial damage. From a biochemical and
clinical point of view, the antioxidant effects of antidepressant
agents were observed in animal models by measuring variations
of GSH, malondialdehyde, nitric oxide and isoprostanes
concentrations.11–13
At molecular level, it has been highlighted that uoxetine
exerts its antioxidant effects through a combination of mecha-
nisms, involving direct ROS scavenging, modulation of the
expression and functioning of enzymatic and non-enzymatic
components of the endogenous antioxidant defence system,
and/or enhancement of the serotonin antioxidant capacity.14,15
Concerning the rst putative mechanism, the activity of uox-
etine against ROS has been thoroughly studied from
a biochemical and computational point of view. Although the
compound demonstrated an antioxidant role, its direct contri-
bution to ROS scavenging is of a minor entity with respect to
that of its metabolites.16,17 In addition, uoxetine contrasts
oxidative stress also by increasing extracellular concentration of
serotonin, which is a known strong antioxidant.17–19 This
neurotransmitter, together with its N-acetyl metabolite, exerts
neuroprotection by modulating oxidative burst mechanism
and/or the production of superoxide anion radical.19 Fluoxetine
is also thought to act indirectly on oxidative stress by modu-
lating the expression of enzymes such as superoxide dismutase
Fluoxetine
hydrochloride
(N-methyl-3-phenyl-3-[4-
(triuoromethyl)phenoxy]propan-1-amine HCl) is a selective
serotonin-reuptake inhibitor (SSRI), which was approved by the
FDA in 1987 for the treatment of depression.1,2 This drug,
marketed as Prozac by Eli Lilly, enhances the serotoninergic
tone by increasing the concentration of the neurotransmitter in
the synaptic cle by inhibiting the serotonin transporter.3 From
a structural point of view, it is constituted by a racemic mixture
of R(ꢀ)-uoxetine and S(+)-uoxetine, which show a moderate
difference in terms of inhibitory activity that becomes more
evident in the corresponding metabolites.4 Fluoxetine has been
approved worldwide for the treatment of major depression, but
its activity on a wide spectrum of mood disorders has been
reported.5
Further studies unveiled that uoxetine may protect against
the adverse effects of different types of immune system
stressors and contrast, through a combination of mechanisms,
oxidative damage. This feature is thought to play a primary role
in neuroprotection, since brain is very susceptible to oxidative
stress due to its high energetic requirement. Moreover, growing
a
`
Dipartimento di Medicina Molecolare e Traslazionale, Universita degli Studi di
Brescia, Viale Europa 11, 25123 Brescia, Italy
b
`
Dipartimento di Scienze Chimiche, Universita degli Studi di Padova, Via Marzolo 1,
35131 Padova, Italy. E-mail: laura.orian@unipd.it
c
`
Dipartimento di Scienze del Farmaco, Universita degli Studi di Padova, Via Marzolo 5,
35131 Padova, Italy
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra03509b
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(SOD), catalase (CAT) and glutathione peroxidase (GPx).14 investigation of their elementary, but biologically as well as
Finally, recent ndings suggest that uoxetine may enhance chemically signicant, reaction with H2O2. As will be discussed
cellular antioxidant capacity by tuning mitochondrial redox in the following, the oxidation–elimination process observed for
parameters and through the upregulation of thioredoxin (Trx), the studied compounds leads to the production of a cinnamyl-
a dithiol-disulde oxidoreductase that can facilitate H2O2 amine. Such scaffold is found in many drugs like unarizine
scavenging.20 Nevertheless, the indirect antioxidant role that (calcium antagonist) and naine (antifungal). It must be noted
uoxetine plays in neuroprotection is still debated. Particularly, that other strategies for the synthesis of allylamines were
Dalmizrak et al. reported that long-term use of uoxetine may previously reported in the literature, such as the preparation
be connected with glutathione reductase (GR) deciency,21 from alcohols and amines through alkoxyphosphonium salts,46
while Byeon et al. pointed out that SSRIs might mediate the reduction of secondary amides followed by N-methylation47
oxidative stress in aquatic invertebrates.22
or the reduction of propargylamines.48 Nevertheless, the cited
In the above described scenario, we have synthesised methods require hazardous and expensive reactants, harsh
chimeric derivatives of uoxetine that have been modied to experimental conditions (temperature and pressure) and do not
incorporate a selenium nucleus, thus providing an additional guarantee stereoselectivity. By contrast, the approach we
mechanism to enhance its molecular antioxidant performance propose efficiently proceeds in aqueous medium, it is trans-
(Fig. 1).
selective and requires only H2O2 as oxidant agent. Nuclear
Besides the radical scavenging activity via HAT (Hydrogen magnetic resonance (NMR) and electrospray ionisation-mass
Atom Transfer) mechanism, recently reported for uoxetine,17 spectrometry (ESI-MS) combined with quantum chemistry
selenouoxetine and its derivatives have the capacity of calculations are used to characterise intermediates and prod-
reducing hydroperoxides and H2O2 as GPx mimics.23–25 The ucts and to draw energy proles based on which the mecha-
biological role of selenium is a long-debated issue since its nistic details are unravelled.
identication in GPx, an enzyme able to protect cells from
oxidative stress by inactivating hydroperoxides.26–30 As oen
Experimental
Chemistry
happens, nature inspired medicinal chemists and synthetic
selenium-based compounds were developed through the years
to mimic GPx activity as well as to exert antioxidant activity
Commercially available chemicals were purchased from Sigma-
through other mechanisms, such as metal binding.23–25,31
Aldrich and used without any further purication if not speci-
Among GPx mimics, ebselen (2-phenyl-1,2-benzisoselenazol-
ed elsewhere. NMR experiments were performed on a Bruker
3(2H)-one) is the most popular since it was employed in clinical
Avance III 400 spectrometer (frequencies: 400.13, 100.62, and
trials.32 Aryl and alkyl selenides,33,34 together with aryl dis-
elenides,35–38 have been prepared and tested in vitro and in vivo.
In addition, unsubstituted alkylphenyl selenides and diphe-
nyldiselenide have been adopted to study the reactivity towards
1
76.37 MHz for H, 13C, and 77Se nuclei, respectively) equipped
with a multinuclear inverse z-eld gradient probe head (5 mm).
For data processing, TopSpin 4.0.8 soware was used and the
spectra were calibrated using solvent signal (1H-NMR, dH
¼
peroxides as models for understanding the behaviour of orga-
noselenides in biological environments.39 Since selenides are
largely used in organic synthesis to catalyze oxidation in pres-
ence of H2O2, combined experimental and theoretical mecha-
nistic studies on model organoselenides are important also for
designing selenium based organocatalysts.40–45
The rationale that guided the design of the selenium-
containing analogues of uoxetine aims to combine the anti-
oxidant radical scavenging capacity, within the CNS, of the
parent compound with the GPx-like activity of organic selenides
in a water-soluble drug-like small molecule (tandem antioxi-
dant). Thus, selenouoxetine and its derivatives here reported
represent models for the experimental and theoretical
7.26 ppm for CDCl3, dH ¼ 2.50 ppm for DMSO-d6, dH ¼ 4.79 ppm
for D2O; 13C-NMR, dC ¼ 77.16 ppm for CDCl3, dC ¼ 39.52 ppm
for DMSO). Multiplicities are reported as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; b, broad; dd, doublet
of doublets. Mass spectra were recorded by direct infusion ESI
on a Thermo Fisher Scientic LCQ Fleet ion trap mass spec-
trometer and on a Waters Xevo G2 QTof high-resolution mass
spectrometer (HRMS). The purity prole of the compounds was
assayed by HPLC using a Varian Pro-Star system equipped with
a Biorad 1706 UV-VIS detector and an Agilent C-18 column (5
mm, 4.6 ꢁ 150 mm). An appropriate ratio of water (A) and
acetonitrile (B) was used as mobile phase with an overall ow
rate of 1 mL minꢀ1. The general method for the analyses is
Fig. 1 Fluoxetine and its derivatives studied in this work.
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reported here: 0 min (90% A–10% B), 5 min (90% A–10% B), dissolved in a small amount of diethyl ether and 5 mL of HCl
25 min (10% A–90% B), 30 min (90% A–10% B), and 32 min 2 M in ether were added in order to obtain the hydrochloride
(90% A–10% B). The purity of all compounds was $96%, unless salt. The solvent was evaporated under reduced pressure. 10 mL
otherwise stated (254 nm).
of thionyl chloride were added to the round-bottomed ask and
Synthesis of N,N-dimethyl-3-oxo-3-phenylpropan-1-aminium the solution was stirred under reux. The reaction was followed
chloride (2). Dimethylamine hydrochloride (2.03 g, 24.9 mmol, through TLC (DCM/MeOH/TEA 97 : 2.5 : 0.5) and was stopped
1.5 eq.) and paraformaldehyde (0.65 g, 21.6 mmol, 1.3 eq.) were aer 2 hours when the starting material spot was no longer
weighted in a 50 mL round-bottomed ask and dissolved in detected. The solvent was evaporated under reduced pressure
2.5 mL of ethanol. Acetophenone (2.00 g, 16.6 mmol, 1 eq.) was obtaining the compound as hydrochloride salt. Yield 1.88 g
added to the solution together with 40 mL of concentrated (96%), white solid; 1H NMR (400 MHz, DMSO): dH (ppm) 10.90
hydrochloric acid. The reaction mixture was stirred at reux and (br, 1H, NH), 7.52–7.49 (m, 2H, Ph-H), 7.45–7.35 (m, 3H, Ph-H),
checked through TLC (DCM/MeOH/TEA 97 : 2.5 : 0.5). Aer 2 5.31 (dd, J ¼ 9.1, 5.2 Hz, 1H, CH(Cl)), 3.23–3.18 (m, 1H, CH(Cl)
hours, the solution was le to cool to room temperature. A solid CHAHB), 3.10–3.04 (m, 1H, CH(Cl)CHAHB), 2.76 (s, 6H, N(CH3)2),
precipitate of N,N-dimethyl-3-oxo-3-phenylpropan-1-aminium 2.63–2.44 (m, 2H, CH2N); 13C-NMR (101 MHz, DMSO): dC (ppm)
chloride salt formed and the solid was ltered with a Buchner 141.0 (s, Ph-C), 129.3 (s, 2C, Ph-C), 129.2 (s, 2C, Ph-C), 127.5 (s,
funnel, washed with cold acetone (3 ꢁ 10 mL) and with hexane Ph-C), 61.1 (s, CH(Cl)), 42.7 (s, CH2N), 42.4 (s, 2C, N(CH3)2), 33.6
(1 ꢁ 10 mL). Yield 3.48 g (98%); white solid; 1H-NMR (400 MHz, (s, CH(Cl)CH2); (HRMS ESI+) m/z calcd for C11H17ClN+ [M + H]+:
DMSO): dH (ppm) 10.57 (br, 1H, NH), 8.02 (d, 2H, J ¼ 7.2 Hz, Ph- 198.1044, found: 198.1043.
H), 7.69 (t, 1H, J ¼ 7.4 Hz, Ph-H), 7.57 (t, 2H, J ¼ 7.6 Hz, Ph-H),
Synthesis of 1,2-bis(4-(triuoromethyl)phenyl)diselane (5).
3.63 (t, 2H, J ¼ 7.2 Hz, C(O)CH2), 3.40 (t, 2H, J ¼ 7.2 Hz, CH2N), Under nitrogen atmosphere, magnesium chips (108 mg,
2.80 (s, 6H, N(CH3)2); 13C-NMR (101 MHz, DMSO): dC (ppm) 4.44 mmol, 1 eq.) were added to a solution of 4-bromobenzo-
196.8 (s, C(O)), 135.9 (s, Ph-C), 133.7 (s, 2C, Ph-C), 128.8 (s, Ph- triuoride (1.00 g, 4.44 mmol, 1 eq.) in dry ether in a 50 mL
C), 128.0 (s, 2C, Ph-C), 51.8 (s, CH2N), 42.2 (s, 2C, N(CH3)2), 33.1 three-necked round-bottomed ask. The halobenzene solution
(s, C(O)CH2); (HRMS ESI+) m/z calcd for C11H16NO+ [M + H]+: was added dropwise at gentle reux and le stirring for another
178.1232; found: 178.1308.
30 minutes. Aerwards selenium powder (351 mg, 4.44 mmol, 1
Synthesis of 3-hydroxy-N,N-dimethyl-3-phenylpropan-1- eq.) was added maintaining gentle reuxing and the reaction
amine (3). Compound 2 (1.80 g, 8.39 mmol, 1 eq.) was dis- mixture was stirred for another 30 minutes. Then the mixture
solved in 5 mL of distilled water and 1.2 mL of a solution 8 M of was poured in a mixture of cracked ice and concentrated
KOH was added to the mixture. A white solid precipitate formed hydrochloric acid. The cold mixture was separated and the
and the mixture was extracted with DCM (4 ꢁ 20 mL). The water phase was extracted with ether (3 ꢁ 20 mL). The
organic phases were combined, and the solvent was evaporated combined organic layer was dried over anhydrous magnesium
under reduced pressure, yielding the free base of compound 2. sulphate, which was then removed by ltration. The solvent was
The oily compound was dissolved in 10 mL of methanol and removed under reduced pressure. Yield 1.21 g (61%); orange oil;
a couple of drops of KOH 8 M were added in order to provide an 1H-NMR (400 MHz, CDCl3): dH (ppm) 7.71 (d, J ¼ 8.0 Hz, 4H, Ph-
alkaline environment. The solution was cooled to 0 C with an H), 7.52 (d, J ¼ 8.2 Hz, 4H, Ph-H); 13C-NMR (101 MHz, CDCl3): dC
ꢂ
ice bath and sodium borohydride (0.47 g, 12.6 mmol, 3 eq.) was (ppm) 135.0 (s, 2C, Ph-C), 130.9 (s, 4C, Ph-C), 129.9 (q, J ¼
added to the solution. When all the reactants were dissolved, 28.9 Hz, 2C, Ph-C), 126.2 (q, J ¼ 3.7 Hz, 4C, Ph-C), 123.4 (q, J ¼
the ice bath was removed and the mixture was stirred at room 328.1 Hz, 2C, CF3). (ESI+) m/z calcd for C14H9F5Se2 [M–F +
+
temperature for 1.5 hours. Aer said time, concentrated H]+:431.89, found: 431.44.
hydrochloric acid was added dropwise to the solution until acid
Synthesis of N,N-dimethyl-3-phenyl-3-((4-(triuoromethyl)
pH, then the solution was again basied with KOH 8 M. The phenyl)selanyl)propan-1-amine (1-CF3). The diselenide
5
methanol was evaporated under reduced pressure and the (373 mg, 0.85 mmol, 1 eq.) was introduced in a 50 mL round-
precipitate was dissolved in 100 mL of DCM and washed with bottomed ask and dissolved in ethanol, KOH (143 mg,
alkaline water (4 ꢁ 10 mL). The organic phase was dried with 2.55 mmol, 3 eq.) was added and the solution was cooled in an
anhydrous magnesium sulphate and ltered, then the solvent ice bath. Then, sodium borohydride (193 mg, 5.10 mmol, 6 eq.)
was evaporated under reduced pressure. Yield 1.43 g (95%); was added. Once that a colour change was observed (between 30
transparent oil; 1H-NMR (400 MHz, CDCl3): dH (ppm) 7.41–7.31 minutes and 1 hour aer the reaction began), compound 4
(m, 4H, Ph-H), 7.28–7.22 (m, 1H, Ph-H), 4.91 (dd, J ¼ 7.1, 4.7 Hz, (200 mg, 0.85 mmol, 1 eq.) was added to the solution. The
1H, CH(OH)), 2.66–2.58 (m, 1H, CH(OH)CHAHB), 2.48–2.42 (m, reaction was stirred at room temperature for 3 hours. To quench
1H, CH(OH)CHAHB), 2.28 (s, 6H, N(CH3)2), dH 1.86–1.79 (m, 2H, the unreacted NaBH4, concentrated hydrochloric acid was
CH2N); 13C-NMR (101 MHz, CDCl3): dC (ppm) 145.1 (s, Ph-C), added to the mixture until acidic pH. Aerwards KOH 8 M was
128.1 (s, 2C, Ph-C), 126.8 (s, Ph-C), 125.5 (s, 2C, Ph-C), 75.3 (s, added to basic pH. Ethanol was evaporated under reduced
CH(OH)), 58.1 (s, CH2N), 45.2 (s, 2C, N(CH3)2), 34.7 (s, CH(OH) pressure and the precipitate that formed was dissolved in DCM.
CH2); (HRMS ESI+) m/z calcd for C11H18NO+ [M + H]+: 180.1383, The solution was washed with alkaline water (3 ꢁ 20 mL), dried
found: 180.1133.
Synthesis of
over anhydrous magnesium sulphate and ltered, then the
3-chloro-N,N-dimethyl-3-phenylpropan-1- solvent was evaporated under reduced pressure. The product
aminium chloride (4). Compound 3 (1.50 g, 8.37 mmol) was was puried by column chromatography (silica gel, DCM/
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1
MeOH/TEA, 92 : 7.5 : 0.5). Yield 112 mg (34%); yellow oil; H- experiments of 16–40 scans were accumulated and processed
NMR (400 MHz, CDCl3): dH (ppm) 7.49 (d, J ¼ 8.0 Hz, 2H, Ph- with a magnitude calculation; an evolution delay of 33.3 ms was
H), 7.43 (d, J ¼ 8.4 Hz, 2H, Ph-H), 7.29–7.19 (m, 5H, Ph-H), 4.49– used for 1H–77Se long-range coupling constants; the spectral
4.46 (m, 1H, CH(Se)), 2.38–2.22 (m, 4H, CHCH2CH2N), 2.22 (s, width was 13 ppm in F2 and 1000 ppm in F1. Zero-lling in the F1
6H, N(CH3)2); 13C-NMR (101 MHz, CDCl3): dC (ppm) 141.72 (s, and F2 dimensions, multiplication with a Gaussian function (in
Ph-C), 134.9 (s, 2C, Ph-C), 134.7 (s, Ph-C), 129.7 (q, J ¼ 32.6 Hz, F2) and a squared sine function (in F1) were performed prior to 2D
Ph-C), 128.6 (s, 2C, Ph-C), 127.9 (s, 2C, Ph-C), 127.4 (s, Ph-C), Fourier transformation.
125.6 (q, J ¼ 3.7 Hz, 2C, Ph-C), 124.2 (q, J ¼ 272.19 Hz, CF3), 57.8
(s, CH(Se)), 46.3 (s, CH2N), 45.4 (s, 2C, N(CH3)2), 34.0 (s, CH(Se)
CH2); (HRMS ESI+) m/z calcd for C18H21F3NSe+ [M + H]+:
388.0786, found: 388.0866.
ESI-MS study of the reaction with H2O2
Mechanistic ESI-MS studies were performed under the same
experimental conditions (concentration and temperature) used
in the NMR analysis, except for the fact that Milli-Q water was
used instead of D2O. Solutions were diluted 1 : 1000 in meth-
anol before the analysis. Spectra were recorded by direct infu-
sion ESI on a Thermo Fisher Scientic (Waltham, MA) LCQ Fleet
ion trap mass spectrometer. ESI parameter for positive ionisa-
tion mode are here described: 4.0 kV spray voltage, 225 ꢂC
capillary temperature, 5 mL minꢀ1 ow rate. For negative ion-
isation mode: 5.0 kV spray voltage, 180 ꢂC capillary temperature,
5 mL minꢀ1 ow rate.
Synthesis
of
N,N-dimethyl-3-phenyl-3-(phenylselanyl)
propan-1-amine (1-H). The diphenyl diselenide (400 mg,
1.28 mmol, 1 eq.) was introduced in a 50 mL round-bottomed
ask and dissolved in ethanol, KOH (215 mg, 3.84 mmol, 3
eq.) was added and the solution was cooled in an ice bath. Then,
sodium borohydride (290 mg, 7.68 mmol, 6 eq.) was added.
Once that a colour change was observed (between 30 minutes
and 1 hour aer the reaction began), compound 4 (300 mg,
1.28 mmol, 1 eq.) was added to the solution. The reaction was
stirred at room temperature for 3 hours. To quench the
unreacted NaBH4, concentrated hydrochloric acid was added to
the mixture until acidic pH. Aerwards KOH 8 M was added to
Computational methodology
basic pH. The solvent was evaporated under reduced pressure For hydrogen atom transfer (HAT) reactions, geometry optimi-
and the precipitate that formed was dissolved in DCM. The sations of the reactants and products were performed in the gas
solution was washed with alkaline water (3 ꢁ 20 mL), dried over phase without any constraint, using the M06-2X functional49
anhydrous magnesium sulphate and ltered, then the solvent combined with the 6-31G(d) basis set, as implemented in
was evaporated under reduced pressure. The product was Gaussian 16.50 Spin contamination was checked for the doublet
puried by column chromatography (silica gel, DCM/MeOH/ ground state species to assess the reliability of the wave-
TEA, 92 : 7.5 : 0.5). Yield 155 mg (38%); yellow oil; 1H-NMR function. To conrm the nature of the stationary points and to
(400 MHz, CDCl3): dH (ppm) 7.43–7.40 (m, 2H, Ph-H), 7.28– obtain the thermodynamic corrections at 1 atm and 298 K,
7.18 (m, 8H, Ph-H), 4.38–4.33 (m, 1H, CH(Se)), 2.31–2.19 (m, 4H, frequency calculations at the M06-2X/6-31G(d) level of theory
CH(Se)CH2CH2N), 2.17 (s, 6H, N(CH3)2); 13C-NMR (101 MHz, were run to ascertain that only positive frequencies were
MeOD): dC (ppm) 140.6 (s, Ph-C), 135.5 (s, Ph-C), 128.8 (s, 2C, present. In order to obtain more accurate energy values, single-
Ph-C), 128.4 (s, Ph-C), 128.3 (s, 2C, Ph-C), 128.0 (s, 2C, Ph-C), point energy calculations were performed at M06-2X/6-
127.3 (s, Ph-C), 127.2 (s, 2C, Ph-C), 56.4 (s, CH(Se)), 43.7 (s. 311+G(d,p) in the gas phase, and subsequently, at the same level
CH2N), 42.1 (c, 2C, N(CH3)2), 30.5 (s, CH(Se)CH2); (HRMS ESI+) of theory, in benzene and water using the continuum Solvation
m/z calcd for C17H22NSe+ [M + H]+: 320.0912, found: 320.1072. Model based on Density (SMD).51 This level of theory is denoted
in the text (SMD)-M06-2X/6-311+G(d,p)//M06-2X/6-31G(d,p).
The choice fell on benzene and water because the model an
apolar and a polar environment, respectively.52 Energy barriers
NMR study of the reaction with H2O2
In order to study the oxidation reaction of compounds 1-CF3 and were calculated for the most reactive sites (identied on the
1-H, their reaction with H2O2 was investigated by 1H-NMR in an basis of DGHꢂ AT), at the (SMD)-M06-2X/6-311+G(d,p)//M06-2X/6-
aqueous environment at room temperature (22 ꢂC). The 31G(d,p) level of theory. Analysis of the single imaginary
compounds were used as hydrochloride salts (28 mM: 1-CF3 ¼ frequency of transition states conrmed that the normal mode
4.9 mg, 11.6 mmol and 1-H ¼ 5.6 mg, 17.6 mmol) and dissolved in involved was correct for the HAT process. Energy barriers were
D2O, t0 spectra were registered with 8 scans, 1 dummy scan and calculated, referring to the free reactants in the gas phase as
a delay time d1 of 2 s. Aerwards, H2O2 (1.1 eq., H2O2 3% w/w well as in the solvent.
solution: 1-CF3 ¼ 15 mL, 1-H ¼ 18 mL) was added and the
The quantum chemistry calculations for the Se oxidation
spectra were registered every 2 or 5 minutes until the reaction was mechanism were performed using the Amsterdam Density
complete (47 min for 1-CF3 and 104 min for 1-H) (8 scans, 1 Functional (ADF).53–55 The energy proles were obtained from
dummy scan, d1 of 2 s). The reaction was studied plotting the geometries and energies computed by using the OLYP func-
results of the integration of the signals corresponding to the tional,56,57 which is known to perform well for reactivity studies
protons bound to the carbon adjacent to the selenium atom for on organic compounds, and it has been recently benchmarked58
the starting material, and the two diastereoisomers and the allylic and applied59 to organic dichalcogenides. OLYP was combined
hydrogens for the cinnamylamine. 1H–77Se HMBC NMR spectra with the TZ2P basis set for all the atoms.60 The TZ2P basis set is
were acquired using a repetition delay of 1 s; a total of 300 of triple-z quality and has been augmented with two sets of
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Scheme 1 (a) dimethylamine HCl/HCOH/EtOH; (b) NaBH4/KOH/MeOH; (c) SOCl2.
polarisation functions. Core shells of the atoms (1s for C, F, N procedures and were used for the experimental reactivity
and O and up to 3p for Se) were treated by using the frozen-core studies. Compounds 1-CF3 and 1-H, were obtained through
approximation. Scalar relativistic effects were treated using the a multi-step synthesis. Acetophenone was initially subjected to
Zeroth Order Regular Approximation (ZORA).61–63 The numer- a Mannich reaction with dimethylamine hydrochloride and
ical integration was performed by using the fuzzy-cell integra- formaldehyde giving compound 2.69 The Mannich base was then
tion scheme developed by Becke.64,65 Energy minima and reduced with sodium borohydride providing compound 3 as
transition states have been veried through vibrational anal- two enantiomers,70 and subsequently chlorinated with thionyl
ysis. All minima were found to have zero imaginary frequencies chloride obtaining the intermediate synthon 4 (Scheme 1).71
and all transition states have one that correspond to the mode
The chloride was then displaced by the appropriate selenide
of the reaction under consideration. For single point calcula- nucleophile, formed in situ by the reduction of the corre-
tions in water the conductor-like screening Model was sponding diphenyl diselenide with sodium borohydride,
employed (COSMO), as implemented in ADF.66–68 Water was providing 1-CF3 and 1-H (Scheme 2; please refer to ESI† for
parameterised using a dielectric constant of 78.39 and a solvent detailed experimental procedures and analytical data). Partic-
˚
radius of 1.93 A.
ularly, 1,2-bis(4-(triuoromethyl)phenyl)diselane (compound 5)
was obtained through the reaction with elemental selenium of
the Grignard reagent formed from p-triuoromethyl bromo-
benzene (see Fig. S1–S22† for 1H, 13C and mass spectra).72,73 The
unsubstituted diphenyl diselenide is commercially available.
Results and discussion
Synthesis of the selenium-based uoxetine analogues
Selenouoxetine closely resembles uoxetine, in which oxygen
was substituted with a selenium atom. N,N-dimethyl derivatives
1-CF3 and 1-H (Fig. 1) were designed to simplify synthetic
Oxidation by H2O2: NMR results
In our experimental model reaction, the process initiates upon
addition of H2O2 and evolves to an oxidation-triggered elimi-
nation, which is peculiar of selenides having protons in the b-
position with respect to the chalcogen nucleus (Scheme 3). It is
a highly trans-selective process affording olens, and it was
demonstrated that it occurs through a syn mechanism.74
The rst step of the process consists in the oxidation of the
starting selenide to the corresponding selenoxide. It has been
reported that this reaction can occur using hydrogen peroxide
(H2O2), meta-chloroperoxybenzoic acid (mCPBA) and
ozone.39,75,76 This process promotes the formation of a new
chiral centre, giving rise to two enantiomers or to two diaste-
reoisomers, depending on the initial compound. Then, an
intramolecular syn elimination takes place: a proton is trans-
ferred from the b-position to the oxygen of the selenoxide, and
the selenium–carbon bond breaks leading to the formation of
a carbon–carbon double bond (Scheme 4).77
Scheme 2 (d) Mg/Et2O; (e), Se; (f) NaBH4/KOH/EtOH; (g) compound
3/EtOH.
Scheme 3 Oxidation of 1-CF3 and 1-H by H2O2. This scheme represents the overall reaction leading to the formation of cinnamylamine and
seleninic acid, which were experimentally observed as final products.
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Scheme 4 Mechanistic details of selenoxide elimination leading to the formation of selenenic acid and olefin. Selenenic acid undergoes
disproportionation and only seleninic acid was experimentally observed.
The above described selenoxide elimination was studied material (4.42 ppm) occurred, accompanied by the appearance
considering the selenium-based analogues of uoxetine as of the signals of the two selenoxide diastereoisomers (4.48 ppm
models. As anticipated, uoxetine was modied with the aim of and 4.23 ppm) (Fig. 2). The same behaviour was observed for 1-
improving its antioxidant properties through the substitution of CF3, but the signal of one of the two selenoxide diastereoiso-
its oxygen atom with selenium, leading to compound 1-CF3. In mers unluckily overlap to the signals of the starting material
this connection, compound 1-H was designed as a further (approximately 4.55 ppm), thus complicating the mechanistic
simplied model for our investigation aiming at drawing widely investigation (Fig. S23–S25†).
general conclusions. Finally, due to synthetic feasibility and to
When a chiral centre is present in the starting compound,
improve water solubility, both analogues differ from uoxetine since in the transition state the carbon–hydrogen and carbon–
also for the presence of a tertiary amine instead of a secondary selenium bonds are co-planar, the two selenoxide diastereo-
amine. To explore the potential application of these derivatives isomer products may react with different formation and
in the eld of green chemistry and to better mimic a biologically subsequent different elimination rate (Fig. 2). This phenom-
relevant environment, the oxidation of 1-H was performed in enon will be discussed more in detail in the computational
water. Indeed, although selenium and its oxidised species were section, since even slight differences in the activation energies
reported to show toxic effect at high concentrations,78 growing for the elimination reactions that are strictly related to the
attention is recently being paid to organoselenides catalysing structure of the transition states may affect the reaction speed.
organic reactions in environmentally friendly conditions.79 In
As the reaction proceeded, it was possible to observe the
particular, this holds true when aqueous medium is consid- disappearance of the selenoxide diastereoisomers (depicted in
ered.80 In this context, good water solubility was achieved for 1- Fig. 2(a)) and the formation of the elimination products: (E)-
H and 1-CF3, since the compounds were prepared as hydro- N,N-dimethyl-cinnamyl amine 6, which was characterised by
chloride salts. Firstly, to gain insight into the reaction mecha- NMR, and the corresponding highly oxidised Se-containing
nism and to fully characterise intermediates and products, the species, i.e. seleninic acids 7 and 8. At this stage, it must be
whole process was followed by 1H-NMR spectroscopy according noted that seleninic acid itself is endowed with synthetic value
to the procedure reported in the Experimental section. in the context of organic catalysis. It is indeed generally referred
Compounds were dissolved in deuterated water and the oxida- to as a pre-catalyst,81 which can be converted to the corre-
tion was carried out at room temperature using 1 equivalent of sponding benzeneperoxyseleninic acid,
a known oxygen-
H2O2. During the reaction of 1-H, prompt disappearance of the transfer agent, in presence of an excess of peroxide.82,83 For
signal corresponding to the a position proton of the starting the studied compounds, the overall reaction demonstrated to
Fig. 2 1H-NMR mechanistic study of the reaction of 1-H with H2O2. The graph reported in (a) was obtained by integration of the a-hydrogen
1
signals for the starting material and the two diastereoisomers and the allylic-hydrogens signals for the cinnamylamine in the H-NMR spectra
acquired at different time points ( starting material, R–R (and its enantiomer), R–S (and its enantiomer) cinnamylamine 6). In (b), repre-
sentative 1H-NMR spectra are reported showing the variation over time during the oxidation-reaction of compound 3, focusing on the region
between 4.7 and 3.8 ppm (area of interest to plot the graph; see Fig. S31† for additional spectra).
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analysis. In analogy with the NMR protocol, the reaction of 1-H
was studied more in detail. In particular, the conversion of the
starting material into the products was followed by sampling
the reaction at the same time points used during the NMR
experiments, and the two analytical techniques showed that the
oxidation/elimination process was accomplished in a similar
time frame (approximately 70 min, see Fig. S43†). Upon addi-
tion of H2O2, the signals corresponding to the selenoxide (m/z ¼
334) and cinnamylamine 6 (m/z ¼ 162) were detected in the
mass spectra obtained in positive ionisation mode. Moreover,
MS analyses performed in negative ionisation mode highlighted
the presence of the signal corresponding to seleninic acid 8 (m/z
¼ 189, see Fig. S33–S36† for representative mass spectra), in
agreement with the data reported by Wang and colleagues.85
Finally, MS analysis was also performed on the NMR samples
used in the mechanistic study above described. Positive and
negative ionisation mode MS spectra acquired on the
Scheme 5 Redox equilibrium involving differently oxidised selenium
species.
be relatively fast. Particularly, a 90% conversion was observed
for 1-CF3 aer 36 minutes, whereas for 1-H the same conversion
was achieved aer 95 minutes (Fig. S26† and 2).
Importantly, it has to be pointed out that selenenic acid is
involved in the chemical equilibrium reported in Scheme 5,77
and according to this mechanism it disproportionates
providing seleninic acid, diphenyl diselenide and water.
On the other hand, also diphenyl diselenide can be oxidised
by H2O2 giving seleninic acid.84 The formation of these less
water soluble species was also suggested by the fact that a solid
precipitate was observed in the NMR sample aer the reaction
study. Moreover, a signal at 1172.0 ppm was detected in the 77Se
NMR spectrum acquired at the end of the reaction, likely due to
the presence of seleninic acid 8 (Fig. S32†).
Thus, to better clarify this behaviour and conrm the iden-
tity of the products, a more focused 1H–77Se HMBC NMR
experiment was carried out. A mid-reaction sample from the
oxidation study carried out on 1-CF3 was diluted 1 : 1 in MeOD
to completely dissolve the precipitate. The NMR analysis
demonstrated the presence of p-triuoromethyl seleninic acid 7
and 1,2-bis(4-(triuoromethyl)phenyl)diselane 5, respectively
testied by the 77Se signals detected at 447.9 and 1211.8 ppm
(Fig. S29†). These ndings are in agreement with previous
observations by Wang and colleagues.85 The authors studied the
selenoxide elimination reaction in a simple model system by
reacting a phenylalkylselenide with 2 equivalents of H2O2 in
CD3CN, observing the appearance of a signal at 1175.1 ppm in
the 77Se NMR spectrum, which is consistent with the presence
of seleninic acid. Our results were further conrmed by ESI-MS
experiments, as described in the following.
Investigation of the reaction mechanism by ESI-MS
Mass spectrometry, and ESI-MS in particular, can be used to
identify the species in solution and complete the information
obtained from NMR studies, even if few examples of its appli-
cation to the chemistry and biochemistry of selenium are
available.39,86,87 In this study, mass spectra analysis was used as
an independent technique for the identication and charac-
terisation of the species involved in the transformation of the
uoxetine analogues and the compound identity was conrmed
by collision-induced dissociation (CID) studies.
The monitoring of the reaction with H2O2 was carried out
under the same experimental conditions (solvent, concentra-
tion, temperature) used to investigate the mechanistic details by
NMR, working on independently prepared samples and
according to the procedure reported in the Experimental
section. Briey, the reaction was carried out in water and time
Fig. 3 DGꢂHAT ðkcal molꢀ1Þ in the gas phase (a), in benzene (b), and in
water (c) for the scavenging of HOc, HOOc and CH2]CHOOc from
non-aromatic sites of selenofluoxetine. Values for fluoxetine taken
from Muraro et al.17 are showed in a lighter shade for comparison.
point samples were diluted 1 : 1000 in methanol before ESI-MS Level of theory: (SMD)-M06-2X/6-311+G(d,p)//M06-2X/6-31G(d).
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and C17. DGꢂHAT values become more negative when going from
the gas to the condensed phase and with increasing solvent
polarity. In benzene, the same trend of gas-phase is found;
conversely, in water, HAT from the amino site is the most fav-
oured, as found for uoxetine. DG‡HAT were computed for all ve
non-aromatic sites to HOc, i.e. those from C7, C14, C15, N16 and
C17 and are reported in Table 1. In gas-phase, the lowest acti-
vation energy is computed for HAT from C7, which is also the
most exergonic process. i.e. 2.8 kcal molꢀ1. This value slightly
increases in benzene where HAT from C7 becomes also less
thermodynamically favoured process and decreases to
0.9 kcal molꢀ1 in water where DGHꢂ AT reaches the lowest negative
value, i.e. ꢀ32.0 kcal molꢀ1. Notably, in selenouoxetine, HAT
from all the ve sites to HOc is thermodynamically as well as
kinetically more favoured than in uoxetine, denoting a better
radical scavenging potential.
Table 1 DG‡HAT ðkcal molꢀ1Þ in the gas phase (a), in benzene (b), and in
water (c) for the scavenging of HOc, from non-aromatic sites of
selenofluoxetine. Level of theory: (SMD)-M06-2X/6-311+G(d,p)//
M06-2X/6-31G(d)
HOc
Site
Gas phasea
Benzenea
Watera
C7
2.8 (6.6)
8.9 (7.5)
4.6 (5.4)
3.5 (3.7)
6.1 (7.8)
3.8 (8.6)
10.2 (8.9)
5.3 (5.9)
3.8 (4.0)
4.1 (8.3)
4.1 (10.0)
11.0 (9.5)
3.0 (4.2)
0.9 (0.8)
6.4 (5.5)
C14
C15
N16
C17
a
Values referring to the analogous sites of uoxetine,17 computed at the
same level of theory, are reported in parentheses for comparison.
completely reacted sample (t ¼ 24 h) diluted in methanol
conrmed the presence of seleninic acid 8 (m/z ¼ 189), diphenyl
diselenide (m/z ¼ 316) and cinnamylamine 6 (m/z ¼ 162) as
reaction products, that were identied using CID experiments
(Fig. S37–S42†). A similar behaviour was observed for 1-CF3.
Again, MS analysis on the sample reacted with H2O2 unam-
biguously conrmed the presence in solution of 6 (m/z ¼ 162)
and 7 (m/z ¼ 255) as nal products (Fig. S44 and S45†).
The oxidation of the uoxetine analogues by H2O2 was
investigated in silico at ZORA-OLYP/TZ2P level of theory. In
Fig. 4, the computed intermediates and transition states for
selenouoxetine are shown, while energy data are reported in
Table S2.†
As can be seen from the energy prole shown in Fig. 5(a), in
gas phase, the transition states are preceded and followed by
the formation of a reactant complex (RCox) and a product
complex (PCox), which are stabilised with respect to the free
reactants and products, respectively. Upon reaction with H2O2,
the selenide is oxidised to selenoxide. Since the attack of the
peroxide may occur from two opposite sides, two paths are
envisioned and two diastereoisomeric products form.
The oxidation occurring at selenium keeps the stereochem-
istry of C7 intact (R), therefore the two diastereoisomeric oxi-
dised product differ only in the stereochemistry of selenium
and are labelled R–R and R–S (Fig. 4). Despite sterically more
hindered, there are no large differences in the energetics along
the R–R path and R–S path, and the former is predicted to be
slightly more stabilised. From the two products, i.e. R–R-Ox and
R–S-Ox, elimination also occurs via two distinct paths with
rather similar energetics, leading to the seleninic acid 7 and the
alkene 6. The energy proles shown in Fig. 5(c) and (d) are those
of 1-CF3. No large mechanistic and energy differences are
computed between uoxetine and 1-CF3, suggesting that small
Mechanistic DFT analysis
The radical scavenging activity via HAT of uoxetine has been
recently investigated in silico by some of us.17 HAT occurs from
the non-aromatic sites C7, C14, C15, N16 and C17. We have
considered the corresponding sites of selenouoxetine (Scheme
1) and focused on the scavenging of HOc, which is the most
reactive and electrophilic oxygen centred radical, of the peroxyl
radical HOOc and of CH2]CHOOc, which mimics larger
unsaturated peroxyl radicals. DGꢂHAT values are shown in Fig. 3
and listed in Table S1.†
As reported for uoxetine, selenouoxetine is not selective
for peroxyl radicals; in fact, all HATs from all the ve sites to
HOOc and most HATs to CH2]CHOOc are endergonic in gas-
phase as well as in solvent. Focusing on HAT to HOc, in gas
phase, it is thermodynamically most favoured from C7, which is
close to Se, and more favoured from C15 and N16 than from C14
Fig. 4 Fully optimised geometries of intermediates and transition states of the oxidation of selenofluoxetine shown in Scheme 3 (R ¼ H, R0 ¼
CF3). Reactant and product complexes are omitted for clarity. Level of theory: ZORA-OLYP/TZ2P.
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Fig. 5 Energies of the stationary points of the investigated reactions (Scheme 3) for different selenofluoxetine derivatives in the gas-phase (left
column) and in water (right column). Level of theory: (COSMO)-ZORA-OLYP/TZ2P.
geometry modications do not affect signicantly the reactivity. selenium nucleus. In addition, the here reported reactions are
Calculations have been carried out also for 1-H. Considering the also endowed with synthetic value, since the basis for a novel
oxidation step, the barrier decreases when decreasing the approach for the synthesis of cinnamylamine, a pharmaco-
electron-withdrawing character of the substituent on the phenyl logically-relevant scaffold are presented. More in general, the
ring R0, i.e. DE‡(R0 ¼ CF3) > DE‡(R0 ¼ H) (Table S2†). Conversely, here described procedure provides an efficient alternative, on
in the elimination step, selenouoxetine (R0 ¼ CF3) has the the green chemistry side, for the preparation of the allylamine.
lowest activation energy. The energy proles of uoxetine and 1- The oxidation step can indeed be carried out in water, only
CF3 have been recalculated also in solvent using the COSMO requiring H2O2 and affording a quantitative conversion.
continuum approach for water. They are shown in Fig. 5(b) and Notably, the reaction is highly selective providing the trans
(d). As reported for similar reactions,88 we assist to the disap- products. Moreover, we veried that 1-CF3 and 1-H react
pearance of the reactant and product complexes, which leads to following the same mechanism to give cinnamylamine. This
a signicant decrease of the activation energy for the oxidation prompted us to further investigate the oxidation–elimination
step. Consequently, the highest barrier in water is the one reaction on other substrates in our ongoing investigation. Our
computed for the elimination.
perspective goal, supported by preliminary results, consists in
probing the potential application of this methodology to
a broader range of substrates for the production of unsatu-
rated amines.
Conclusions
The results of our multi-approach experimental and theoret-
ical investigation on the reaction of selenouoxetine deriva-
tives with H2O2 in water demonstrate that the substrates
undergo an oxidation–elimination process, providing cinna-
mylamine and seleninic acid as nal products. The model Se-
based compounds here presented are closely related, from
a structural point of view, to uoxetine. Thus, they are ex-
pected to maintain the pharmacokinetic properties, such as
Conflicts of interest
There are no conicts to declare.
Acknowledgements
`
CNS permeation, and biological activities of the original drug. This research was funded by the Universita degli Studi di
Analogues of uoxetine bearing tertiary amines were demon- Padova, thanks to the P-DiSC (BIRD2018-UNIPD) project MAD3S
strated to bind SERT with similar affinity.89 Consequently, (Modelling Antioxidant Drugs: Design and Development of
toxicological and medical research is prompted to further computer-aided molecular Systems); P. I. L. O. All the calcula-
explore this aspect by probing the antidepressant potential of tions were carried out on Galileo (CINECA: Casalecchio di Reno,
selenouoxetine and its derivatives. Moreover, the studied Italy) thanks to the ISCRA Grant REBEL2 (REdox state role in
organoselenides are novel tandem antioxidants, combining the Bio-inspired ELementary reactions 2), P. I.: L. O. L.O. contrib-
radical scavenging properties of uoxetine, which are mainly uted to this research as part of the scientic activity of the
related to C7 and N16 sites, and serotonin-mediated redox international multidisciplinary network ‘SeS Redox and
activity, with the GPx-like reactivity, due to the presence of the Catalysis’.
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´
A. Polimeno, L. Flohe and F. Ursini, Free Radical Biol.
Med., 2015, 87, 1–14.
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