Synthesis and evaluation of dihydropyrimidinone-derived selenoesters as multi-targeted directed compounds against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2016.09.031

This paper describes the synthesis and evaluation of new dihydropyrimidinone (DHPM)-derived selenoesters as potential multi-targeted agents for the treatment of Alzheimer's disease. A series of DHPM-derived selenoesters were obtained with high structural

Full text of DOI:10.1016/j.bmc.2016.09.031

Accepted Manuscript
Synthesis and evaluation of dihydropyrimidinone-derived selenoesters as multi-
targeted directed compounds against Alzheimer’s disease
Flavio A.R. Barbosa, Rômulo F.S. Canto, Sumbal Saba, Jamal Rafique, Antonio
L. Braga
PII:
S0968-0896(16)30718-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.031
BMC 13284
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 July 2016
9 September 2016
12 September 2016
Please cite this article as: Barbosa, F.A.R., Canto, R.F.S., Saba, S., Rafique, J., Braga, A.L., Synthesis and evaluation
of dihydropyrimidinone-derived selenoesters as multi-targeted directed compounds against Alzheimer’s disease,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.031
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Synthesis and evaluation of dihydropyrimidinone-derived selenoesters as
multi-targeted directed compounds against Alzheimer’s disease
Flavio A. R. Barbosa,a Rômulo F. S. Canto,a Sumbal Saba,a Jamal Rafique a and Antonio L.
Braga a, 
aLaboratório de Síntese de Substâncias Bioativas de Selênio, Centro de Ciências Físicas e Matemáticas,
Departamento de Química,Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC-Brazil.
http://www.labselen.ufsc.br
* Corresponding author. Tel: +55 (48) 3721-6427;
e-mail address: braga.antonio@ufsc.br
Abbreviations: AD – Alzheimer’s disease; ROS – reactive oxygen species; AChE –
acethylcholinesterase; DHPMs – dihydropyrimidinones; GPx – glutathione peroxidase;
TBARS - thiobarbituric acid reactive substances; NMR - nuclear magnetic resonance; IR –
infrared spectroscopy; HMRS - high resolution mass spectrometry; APPI – atmosphere
pressure photoionization; DPDS – diphenyl diselenide; BHT – buthylated hydroxitoluene;
MDA – malondialdehyde; M.P. – melting point.
Abstract
This
paper
describes
the
synthesis
and
evaluation
of
new
dihydropyrimidinone(DHPM)-derived selenoesters as potential multi-targeted agents for
the treatment of Alzheimer´s disease. A series of DHPM-derived selenoesters were
obtained with high structural diversity through a short and modular synthetic route. The
antioxidant activity was evaluated by TBARS and iron chelation assays. These compounds
were also evaluated as acetylcholinesterase inhibitors (AChEi). The compounds
demonstrated good antioxidant activity, since they presented excellent lipid peroxidation

inhibition and good iron chelation activity. In addition, they showed acetylcholinesterase
inhibition activity and some of them presented activity superior to that of the standard drug
galantamine. The in silico predictions showed that the compound 1h may present a good
pharmacokinetic profile. Therefore, the series of DHPM-derived selenoesters described
herein displayed good potential for the development of antioxidant and anticholinesterasic
agents in the search for new multi-targeted therapeutics for the treatment of Alzheimer’s
disease.
Keywords: dihydropyrimidinones, selenoesters, antioxidant, Alzheimer’s disease, selenium
1. Introduction
Alzheimer’s disease (AD) is the most common neurodegenerative disease, affecting
millions of people worldwide, and the prognosis is that this number will increase with
population aging.1 The disease is characterized by progressive memory loss and cognitive
impairment, and it presents a complex pathophysiology, being described as a multifactorial
disease.2 Diverse factors such as amyloid-β deposits,3 decreased levels of acetylcholine,4 τ-
protein aggregation5 and oxidative stress6 play significant roles in the progression of the
disease.
Oxidative stress plays a pivotal role in the pathophysiology of AD, being one of the
main causes of neuronal death.7 Reactive oxygen species (ROS) are overproduced in the
-
brain of AD patients due to abnormal mitochondrial function that produces more O2 , which
increases the concentration of H2O2 in the cytoplasm.8 The decreased concentrations of
ferritin and increased free iron concentrations contribute to free radical generation through
Fenton reactions.9 ROS damage membranes, proteins and DNA, as do lipid peroxidation

products, e.g. reactive aldehydes, which present much longer half-lives in the cell than the
radicals, thus reacting with cell constituents leading to damage.10
Until now, the strategy of using single-targeted drugs has failed and the multi-
targeted drug strategy is becoming a focus of research for the development of new drugs for
the treatment of AD. The concept of multi-targeting is fully applicable to AD because of its
multifactorial pathogenic mechanisms.11 Nowadays, most of the palliative treatments
available are drugs that inhibit acetylcholinesterase (AChE), based on the deficiency of
acetylcholine in the central nervous system, e.g. tacrine,12 galantamine,13 donepezil14 and
rivastigmine,15 but their clinical usefulness is limited.16
Dihydropyrimidinones (DHPMs) are easily obtained via Biginelli multi-component
reaction, and are reported as good antioxidants acting against lipid peroxidation and being
effective as radical scavengers.17–19 Some analogs are better radical scavengers than
resveratrol.20 Also, this class of compounds presents AChE inhibitory activity,21,22 potent
examples being reported with activities comparable to the standard drug galantamine.23
Recently, we reported the synthesis and biological evaluation of a series of DHPMs
functionalized with selenocyanides as potential multi-targeted therapeutics against AD.24
Organoselenium compounds are very useful in synthetic transformations25–27 and
also for biological purposes, since such compounds are well known antioxidants and
glutathione peroxidase (GPx) mimetics,26,28–34 which is one of the most important natural
antioxidant enzymes in the brain.35 Very recently, Kumar and Engman showed that
ebselenols are very good antioxidants, being more efficient than α-tocopherol for
quenching peroxyl radicals, and better GPx mimetics than ebselen.36 Selenium might
contribute in several ways against the progression of AD.37 This element has been shown to
38
modulate the cholinergic system and prevent oxidative damage in animal models of AD.

Diphenyl diselenide and its analogs, have also been studied in various animal models.
These compounds can enhance the cognitive performance without inducing
neurotoxicity,39,40 inhibit AChE activity, protect against β-amyloid induced neurotoxicity
and improve the memory of mice,41–44 due to their antioxidant properties.
In this context, recent reports demonstrate the effective strategy of merging
organoselenium compounds with known AChE inhibitors in the design of potential multi-
targeted therapeutics for AD. Ebselen and donepezil were merged together in the same
structure in order to develop potent human AChE inhibitors as well as good GPx mimics
with the ability to penetrate the central nervous system with no acute toxicity.45,46 Also,
some hybrids formed with tacrine and ebselen were also designed and they presented potent
inhibitory activity against AChE and butyrylcholinesterase as well as being effective
against hydrogen peroxide and peroxynitrite oxidants.47 Recently, Wang and co-workers
reported the synthesis and evaluation of clioquinol seleno-derivatives which demonstrated
excellent antioxidant activities, inhibition of Cu(II)-induced amyloid-β aggregation and also
good blood-brain barrier penetration in vitro.48
As part of our wider research program aim at designing and developing biologically
active new organoselenium compounds as well as eco-friendly processes,24,49–53 herein we
report the design, synthesis and biological evaluation of a series of novel DHPM-derived
selenoesters. The compounds were evaluated as antioxidants through the inhibition of lipid
peroxidation in the thiobarbituric acid reactive species (TBARS) assay and also as iron
chelating agents. The most active compounds were screened as inhibitors of the enzyme
AChE, turning them into potential multi-targeted compounds for the treatment of
Alzheimer’s disease.

2. Results and Discussion
2.1. Chemistry and Biological evaluation
The synthesis of the DHPM-derived selenoesters 1a-h was performed in a two-step
pathway, affording the final compounds in moderate yields (Scheme 1). The intermediates
6-chloromethyl-DHPMs (2a-h) were synthesized in good yields from the three-component
Biginelli reaction, using aromatic aldehydes (3a-g), ethyl 4-chloroacetoacetate (4) and urea
(5a) or N-Me-urea (5b) at 100oC, under solvent-free conditions, catalyzed by HCl.24 These
6-chloromethyl-DHPMs were reacted with the selenocarboxylate (6), generated in situ from
the reaction of NaSeH with p-toluoyl chloride, at room temperature for 1 h, affording the
target seleno-DHPM 1a-h in 30-58% isolated yields.
Scheme 1. Synthesis of DHPM-derived selenoesters
A series of selenoesters functionalized with electron donating or withdrawing
groups on the DHPM aromatic portion, as well as bicyclic aromatic structures were
synthesized and the structures of the desired DHPM-derived selenoesters are shown in
Figure 1. All compounds are stable at room temperature, even when exposed to light and an
air atmosphere for prolonged times, and their analytical and spectroscopic data are in
agreement with the expected structures.

Figure 1. Structures and yields of DHPM-derived selenoesters

1
In H NMR spectra, the N-H resonance signals are usually registered as singlets in
the ranges of ca. 5.51-8.14 ppm and 7.34-9.10 ppm. The aromatic protons of the DHPM
nucleus appear at between 6.60 and 8.18 ppm and the two doublets of the aromatic moiety
attached to the selenoester at 7.18 and 7.83 ppm. The proton of the carbon near to the NH
appears as a doublet between 5.06 and 6.15 ppm. The two protons of the methylene that
links the selenium to the DHPM core appear as an AB quartet at around 4.24 and 4.55
ppm. The characteristic quartet and triplet of the ethyl ester moiety appear at around 1.10
and 4.10 ppm, respectively. The 13C NMR spectra show the characteristic carbonylic
carbon of the selenoester moiety at around 195 ppm. In the HRMS (APPI), the molecular
ion peaks M+ presented the characteristic isotopic pattern of monoselenated compounds.
2.1.1 Inhibition of Thiobarbituric Acid Reactive Substances (TBARS) Production in
Brain Homogenates
The synthesized seleno-DHPM 1a-h were evaluated as antioxidants in the TBARS
assay for lipid peroxidation. For the TBARS assay, the Ohkawa method was employed54
and the iron-induced TBARS production in phospholipids was measured. These
compounds were compared with the standard organoselenium antioxidants, ebselen and
diphenyl diselenide (DPDS). The results for the comparative screening of compounds 1a-h
at a concentration of 100µM are shown in Figure 2 (Table S1 in the supporting
information).

Figure 2. Effects of compounds 1a-h on Fe2+ (100 μmol/L)-induced TBARS production in
phospholipids extracted from egg yolk at 100µM (final concentration). Data are expressed as mean
± S.E.M of n= 3 independent experiments performed in duplicate. Data were analyzed by one-way
analysis of variance (ANOVA), followed by Bonferroni’s Multiple Comparison Test. ++p < 0.0001
#
a
b
vs control, p < 0.0001 vs iron, p < 0.0001 vs ebselen, p < 0.001 vs ebselen, cp < 0.05 vs ebselen,
****p < 0.0001 vs DPDS, ***p < 0.001 vs DPDS, **p < 0.01 vs DPDS.
As can be seen in Figure 2, the DHPM-derived selenoesters 1a-h inhibited iron-
induced TBARS formation at 100μM as compared to the experiment without any of the
compounds tested. Four of the tested compounds exhibited better activity than the standards
ebselen and DPDS (1d, 1e, 1g and 1h), whereas compounds 1a and 1f were only more
active than ebselen. Compounds 1b and 1c did not show a significant difference when
compared to the standards; however, both displayed good TBARS formation inhibition.
The observed effects of different substituents in the aromatic ring on the activity do not
adhere to a logical electronic effect, with a slightly more pronounced activity in the case of
electron donating substituents. The presence of a methyl group in the place of hydrogen in
the N-1 of the heterocycle seems to slightly reduce the activity, as can be seen on

comparing 1a and 1b. The most active compounds (1d, 1e, 1g and 1h) were used in the
concentration screening, which was carried out at concentrations of 100, 50, 25, 10, 5, and
1 μM. The compounds displayed very good inhibition activity in a dose-dependent manner,
being active even in low concentrations. For instance, 1h, the most active compound,
showed very good inhibition even at 10 μM (graphs are available in SI).
In order to check the effect of the selenoester substitution pattern on the activity, we
synthesize two new derivatives of the most active compound (1h) (Scheme 2). The acetyl
substituted DHPM-derived selenoester 7 was synthesized from the reaction of 6-
chlorometyl-DHPM 2h with the selenocarboxylate 8 in 44% yield, and the dymeric
selenoester 9 was prepared from the reaction of 2h with bis-selenocarboxylate 10 in fair
yield under the same conditions. We also synthesized the sulfur analog 11 in 46% yield,
using the thiocarboxylate 14, which was prepared using NaSH instead of NaSeH.
Scheme 2. Synthesis of 1h analogues (7, 9 and 11).

These compounds (7, 9, 11) and 1h were compared in the TBARS assay with the
ebselen and DPDS at 100 μM (Figure 3, Table S2 in the supporting information). These
compounds inhibited TBARS formation when compared to the reaction with iron alone;
however, only compound 1h was more active than the standards. The effect of a
modification in the selenoester structure was detrimental to the activity, as can be noted by
comparing 7 and 9 to 1h, discouraging further structural modifications in this position. The
importance of the selenium atom with regard to the activity was also demonstrated through
comparison of the activities of the selenoester 1h and its less active sulfur analog 11.
Figure 3. Effects of compounds 7, 9 , 11 and 1h on Fe2+ (100 μmol/L)-induced TBARS
production in phospholipids extracted from egg yolk at 100 µM (final concentration). Data
are expressed as mean ± S.E.M of n= 3 independent experiments performed in duplicate.
Data were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni’s
#
a
Multiple Comparison Test. ++p < 0.0001 vs control, p < 0.0001 vs iron , p < 0.0001 vs
ebselen, **p < 0.01 vs DPDS.

In order to investigate the effect of merging the selenoester moiety and the DHPM
core in the same structure, we synthesized separate fragments of the hybrid, the selenoester
12 and the DHPM 13, using the same reactions described above (see experimental section).
The TBARS assay of these compounds showed that both separate fragments possess
antioxidant activity, but the selenoester fragment 12 was more active. Nonetheless both
fragments are less active than the hybrid of the two structures 1h, indicating that both parts
of the structure contribute to its overall antioxidant activity, and there may be a synergistic
effect between the functional groups, enhancing the activity (Figure 4, (Table S3 in the
supporting information).
Figure 4. (a) Design of the fragments 12 and 13 from 1h. (b) Effects of compounds on Fe2+
(100 μmol/L)-induced TBARS production in phospholipids extracted from egg yolk at 100
µM (final concentration). Data are expressed as mean ± S.E.M of n= 3 independent
experiments performed in duplicate. Data were analyzed by one-way analysis of variance
(ANOVA), followed by Bonferroni’s Multiple Comparison Test. ++p < 0.0001 vs control,
#p < 0.001 vs iron , ap < 0.001 vs ebselen, bp < 0.01 vs ebselen, **p < 0.01 vs DPDS.

2.1.2 Iron chelating activity
The synthesized compounds (1a-h, 7, 9, 11, 12, 13) were evaluated as iron (II)
chelators in the range of 5-100 µM applying the Minotti method55 using BHT as the
standard and the results are shown in Table 1. Of the DHPM-derived selenoesters 1a-h,
three presented EC50 values comparable to the standard BHT (compounds 1a, 1e, 1g) and
two presented values lower than the standard (1d and 1h), 1d being the most active
compound, probably due to the substitution pattern (3-methoxy-4-hydroxy) in the aromatic
ring, which is a well-known pattern in the antioxidant and chelation activity of phenolic
compounds such as Apocynin, Vanillic acid and related compounds.56,57 The acyl
selenoester (7) and dimer (9) analogs of compound 1h presented lower iron chelation
capacity than 1h, indicating a detrimental effect on this activity with a variation in the
selenoester side chain as well as in the TBARS assay. The bioisosteric replacement of
selenium (1h) to sulfur (11) leads to a small decrease in activity, but compound 11 is still as
active as the standard BHT. Both separate fragments of the hybrid 1h (i.e., the selenoester
12 and the DHPM 13) show activity comparable to that of BHT, but the EC50 values for 12
and 13 are higher than that for the hybrid 1h, showing again that the hybridization
contributes to greater activity.

Table 1. The EC50 value of iron (II) chelation activity of compounds.
Compounds Fe2+chelating activitya
(EC50 μmol L−1)±SEM
24.50±0.18
32.83.±6.75
29.6±1.74
1a
1b
1c
1d
1e
1f
1g
1h
7
8.10±0.34*
22.45±1.65
35.50±1.46
21.15±1.37
10.45±1.55
41.16±2.60
29.85±5.09
20.2±3.81
9
11
12
13
BHT
22.35±0.81
15.85±2.05
23.30±0.36
aThe compounds were tested in the range of 5-100 µM, Fe2+ 120 µM (final concentration). Results
are expressed as Mean ± SEM of n = 3 independent experiments performed in duplicate. Data were
analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni's Multiple
Comparison Test. *p< 0.05 vs BHT.
2.1.3 Acetylcholinesterase inhibitory activity
The most active antioxidants and iron chelators (1d and 1h) together with the 1h
analogues (7, 9 and 11) and the fragments 12 and 13 were screened as inhibitors of AChE
applying Ellmann´s method,58 using galanthamine as the standard, and the IC50 results are
shown in Table 2. The DHPM-derived selenoesters (1d, 1h, 7 and 9) showed good
inhibitory activity against AChE, with compounds 1d, 7 and 9 presenting activity
comparable to the standard and compound 1h being once again the most active compound.
As in the case of the results for the TBARS assay and iron chelation activity, the inhibition
of AChE was sensitive to modification of the selenoester side chain, leading to lower
inhibition values (see compounds 7 and 9). The bioisosteric replacement of selenium with
sulfur also leads to a decrease in the activity and, although both fragments 12 and 13

showed good inhibition values, they were still less active than the hybrid 1h. These results
indicate that the DHPM core contributes more to the interaction with the enzyme, but the
hybridization of both fragments in the same structure produces a more active compound.
Table 2. The 50% inhibitory concentration (IC50) of AChE of compounds.
Acetylcholinesterase
Compounds
Inhibitiona
IC50 (µM)±SEM
13.57±0.48
7.21±0.48*
14.2±0.32
1d
1h
7
10.62±1.61
11.70±1.97
12.87±0.32
9.23±0.55
9
11
12
13
Galantamine
12.73±0.87
aThe compounds were tested in the range of 5-50 µM. Results are expressed as mean ± SEM of n =
3 independent experiments performed in duplicate. Data were analyzed by one-way analysis of
*
variance (ANOVA), followed by Tukey's multiple comparisons test. Represents significant
difference from galantamine at p< 0.05.
2.1.4 In silico prediction of pharmacokinetics properties
Furthermore, we performed an in silico prediction of the pharmacokinetics
properties for compound 1h. The open-source software Molinspiration59 and OSIRIS
Property Explorer60 were used for calculations. Lipinski’s rule of 561 predicts that a
potentially drug candidate will present a good pharmacokinetic profile if it meet some
requirements, such as MW(Molecular Weight) ≤ 500, HBD(Hydrogen Bond Donors) ≤ 5,
HBA(Hydrogen Bond Acceptors) ≤ 10 and Clog P ≤ 5.0 or MLogP ≤ 4.15. Veber and co-
workers62 introduced other two important criteria for this type of analysis that is, to have 10
or fewer rotatable bonds and TPSA (Topological Polar Surface Area) ≤ 140 Å2.

Compound 1h satisfied both Lipinski and Veber criteria (Table 3) and the results
showed that the compound may present good absorption and permeation after oral
administration, turning it into a good drug candidate.
Table 3. In silico pharmacokinetics properties predictions for compound 1h.
Property
MLogP
CLogP
Value
4.56
2.77
457.39
84.50
6
Molecular Weight
tPSAa (Å2)
HBAb
HBDc
2
NRBd
8
a tPSA, Topological polar surface area; b HBA, H-bond acceptors; HBD, H-bond donors; d NRB,
c
Number of rotatable bonds.
3. Conclusions
In conclusion, we have synthesized a new series of DHPM-derived selenoesters and
investigated their antioxidant activity in the TBARS assay, their capacity to chelate iron
ions and also their inhibitory activity towards AChE. These hybrid compounds containing
DHPM core and selenoester moiety presented superior activity profiles, as a result of
synergistic effect from both fragments. The in silico predictions showed that the compound
1h may present a good pharmacokinetic profile. Moreover, 1h was the most promising
compound of the series, emerging as a new lead for the development of multi-targeted
compounds directed against alzheimer’s disease.
4. Experimental Protocols
4.1. General Methods and Materials

NMR spectra (1H NMR and 13C NMR) were recorded on a Varian AS-400 or
Bruker Avance 200 spectrometer. Chemical shifts (δ) are reported (in ppm) relative to the
TMS (1H NMR) and the solvent (13C NMR). APPI-micrOTOF-Q II measurements were
performed with a micrOTOF Q-II (Bruker Daltonics) mass spectrometer equipped with an
automatic syringe pump (KD Scientific) for sample injection. The mass spectrometer was
operated in the positive ion mode. The sample was injected using a constant flow
(3µL/min). The solvent was a chloroform/methanol mixture. The APPI-micrOTOF-Q II
instrument was calibrated in the mass range of 50–3000 m/z using an internal calibration
standard (low concentration tuning mix solution) supplied by Agilent Technologies. Data
were processed employing Bruker Compass Data Analysis software (version 4.0). Column
chromatography was carried using Merck Silica Gel (230-400 mesh). Thin layer
chromatography (TLC) was conducted using Merck Silica Gel GF254 (0.25 mm thickness).
For visualization, the TLC plates were either placed under ultraviolet light or stained with
iodine vapor or acidic vanillin. The melting points were determined using a microscopy
coverslip on a Micro Chemical MQA PF digital apparatus and are uncorrected. All
common reagents and solvents were used as purchased unless otherwise noted.
4.2 Synthetic procedures
4.2.1. General procedure for the synthesis of compounds 2a-h and 13
The literature procedure was followed.24 To a two-necked round-bottom flask, the
appropriated aldehyde (10 mmol), ethyl 4-chloroacetoacetate or ethylacetoacetate (10
mmol), urea (1.2 g, 20 mmol) and 5 drops of concentrated HCl were added. The reaction
mixture was stirred at 100 °C for the time required for the consumption of the starting
materials, which was verified by TLC. After this time, the reaction mixture was poured into

crushed ice and water. The precipitate was filtered off and dried. The compounds were used
without further purification.
Ethyl 6-(chloromethyl)-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (2a):
1
Yellow solid, M.P. 160 – 163 °C (Lit.63 162-164 °C), 80% Yield; H NMR (200MHz,
DMSO-d6): δ = 1.11 (t, J=7.3Hz, 3H); 4.04 (q, J=6.8Hz, 2H); 4.62 (AB quartet, d,
J=10.7Hz, 1H); 4.78 (AB quartet, d, J=10.7Hz, 1H); 5.37 (d, J=3.4Hz, 1H); 7.62 – 7.73 (m,
2H); 8.02 (s, 1H); 8.12 – 8.16 (m, 2H); 9.69 (s, 1H); 13C NMR (50MHz, DMSO – d6): δ =
14.2; 53.8; 60.6; 101.14; 121.5; 123.0; 130.8; 133.4; 146.5; 147.5; 148.3; 152.1; 164.4.
HRMS (APPI) m/z calculated for C14H14ClN3O5 [M+H] 340.06947; found 340.06950.
Ethyl 6-(chloromethyl)-1-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (2b):
Light brown solid, M.P. 183 – 186°C, 82% Yield; 1H NMR (200MHz, DMSO-d6): δ (ppm)
1.16 (t, J=7.0Hz, 3H); 3.24 (s, 3H); 4.12 (q, J=7.0Hz, 2H); 5.03 (AB quartet, d, J=11.6Hz,
1H); 5.20 (AB quartet, d, J=12.1Hz, 1H); 5.37 (d, J=4.0Hz, 1H); 7.68 – 7.74 (m, 2H); 8.12
– 8.18 (m, 2H); 8.31 (d, J=3.5Hz, 1H); 13C NMR (100MHz, DMSO-d6): δ = 18.6; 29.6;
51.9; 56.1; 65.3; 95.7; 121.5; 122.9; 130.3; 133.7; 144.4; 147.9; 151.1; 161.9; 169.95.
HRMS (APPI) m/z calculated for C14H16ClN3O5 [M+H] 354.0851; found 354.0857.
Ethyl 6-(chloromethyl)-4-(4-(dimethylamino)phenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (2c):

1
Yellow solid, M.P. 161 – 164°C, 30% Yield; H NMR (200MHz, DMSO-d6): δ = 1.13 (t,
J=7.1Hz, 3H); 2.85 (s, 6H); 4.03 (q, J=7.1Hz, 2H); 4.60 (AB quartet, d, J=10.5Hz, 1H);
4.75 (AB quartet, d, J=10.5Hz, 1H); 5.08 (d, J=3.2Hz, 1H); 6.66 (d, J=8.8Hz, 2H); 7.06 (d,
J=8.7Hz, 2H); 7.70 (bs, 1H); 9.38 (bs, 1H); 13C NMR (50MHz, DMSO-d6): δ = 13.9; 39.3;
40.2; 53.3; 59.8; 102.4; 112.3; 126.9; 145.2; 149.8; 152.2; 164.3. IR (KBr) (ν, cm− 1): 1615;
1638; 1689; 2811; 2929; 2978; 3127; 3233; 3363. HRMS (APPI) m/z calculated for
C16H20ClN3O3 [M+H] 338.1266; found 338.1269.
Ethyl 6-(chloromethyl)-4-(4-hydroxy-3-methoxyphenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2d):
1
Yellow solid, M.P. 163 – 165°C, 78% Yield; H NMR (200MHz, DMSO-d6): δ = 1.12 (t,
J=7.1Hz, 3H); 3.72 (s, 3H); 4.04 (q, J=7.2Hz, 2H); 4.65 (AB quartet, d, J=10.6Hz, 1H);
4.72 (AB quartet, d, J=10.6Hz, 1H); 5.10 (d, J=3.3Hz, 1H); 6.61 – 6.81 (m, 4H); 7. 75 (s,
1H); 9.42 (s, 1H); 13C NMR (50MHz, DMSO-d6): δ = 14.0; 53.7; 55.6; 60.0; 102.2; 110.8;
115.5; 118.6; 135.1; 145.7; 146.2; 147.5; 152.3; 164.4. IR (KBr) (ν, cm− 1): 1650; 1683;
2835; 2974; 3106; 3245; 3367; 3567. HRMS (APPI) m/z calculated for C15H17ClN2O5
[M+H] 341.0899; found 341.0898.
Ethyl 6-(chloromethyl)-4-(4-methoxyphenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (2e):
1
Yellow solid, M.P. 183 – 185°C (Lit.64 185- 186°C), 98% Yield; H NMR (200MHz,
DMSO-d6): δ = 1.12 (t, J=6.8Hz, 3H); 3.72 (s, 3H); 4.04 (q, J=7.3Hz, 2H); 4.60 (AB
quartet, d, J=10.3Hz, 1H); 4.77 (AB quartet, d, J=10.8Hz, 1H); 5.14 (d, J=2.9Hz, 1H); 6.89
13
(d, J=8.8Hz, 2H); 7.17 (d, J=8.3Hz, 2H); 7.78 (s, 1H); 9.45 (s, 1H); C NMR (50MHz,

DMSO-d6): δ = 13.9; 39.3; 53.4; 55.2; 60.1; 102.3; 113.9; 127.6; 136.2; 145.7; 152.2;
158.8; 164.4. HRMS (APPI) m/z calculated for C15H17ClN2O4 [M+H] 325.09496; found
325.09485.
Ethyl 6-(chloromethyl)-4-(naphthalen-1-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (2f):
1
Yellow solid, M.P. 132 – 135°C, 85% Yield; H NMR (200MHz, DMSO-d6): δ = 0.82 (t,
J=7.1Hz, 3H); 3.77 – 3.94 (m, 2H); 4.75 (AB quartet, d, J=10.5Hz, 1H); 4.86 (AB quartet,
d, J=10.6Hz, 1H); 6.13 (d, J=3.1Hz, 1H); 7.45 – 7.64 (m, 4H); 7.84 – 7.97 (m, 3H); 8.30 –
8.34 (m, 1H); 9.58 (s, 1H); 13C NMR (50MHz, DMSO-d6): δ = 13.7; 39.4; 49.7; 59.9;
101.9; 123.7; 124.3; 125.7; 126.2; 128.3; 128.6; 130.0; 133.6; 139.7; 146.4; 151.7; 164.2.
IR (KBr),(ν, cm− 1): 1656; 1697; 2980; 3245. HRMS (APPI) m/z calculated for
C18H17ClN3O3 [M+H] 345.1000; found 345.1004.
Ethyl 6-(chloromethyl)-2-oxo-4-p-tolyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2g):
1
Yellow solid, M.P. 165 – 168°C (Lit.63 164 - 166°C), 76% Yield; H NMR (200MHz,
DMSO-d6): δ = 1.17(t, J=7.3Hz, 3H); 2.30(s, 3H); 4.09(q, J=6.6Hz, 2H); 4.68 (AB quartet,
d, J=12.4Hz, 1H); 4.78 (AB quartet, d, J=12.4Hz, 1H); 5.35 (d, J=2.93Hz, 1H); 6.47 (s,
1H); 7.09 (d, J=8.0Hz, 2H); 7.18 (d, J=8.0Hz, 2H); 8.53 (s, 1H). 13C NMR (50MHz,
DMSO-d6): δ = 13.9; 20.6; 53.6; 59.9; 102.0; 126.2; 129.0; 136.8; 141.0; 145.8; 152.1;
164.23. . HRMS (APPI) m/z calculated for C15H17ClN2O3 [M+H] 309.1000; found
309.0999.
Ethyl 6-(chloromethyl)-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2h):

1
Yellow solid, M.P. 173 – 175°C (Lit.63 174 - 176°C), 70% Yield; H NMR (200MHz,
DMSO-d6): δ = 1.11 (t, J=7.1Hz, 3H); 4.04 (q, J=7.3Hz, 2H); 4.59 (AB quartet, d,
J=10.6Hz, 1H); 4.78 (AB quartet, d, J=10.6Hz, 1H); 5.20 (d, J=3.3Hz, 1H); 7.24 – 7.37 (m,
5H); 7.85 (s, 1H); 9.49 (s, 1H); 13C NMR (50MHz, DMSO-d6): δ = 13.8; 30.6; 53.9; 59.9;
101.8; 126.3; 127.5; 128.5; 143.9; 145.9; 152.0; 164.2. HRMS (APPI) m/z calculated for
C14H15ClN2O3 [M+H] 295.0844; found 295.0847.
Ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13):
1
White solid, M.P. 211 – 214°C, 80% Yield; H NMR (400MHz, DMSO-d6): δ = 1.09 (t,
J=6.6 Hz, 3H); 2.26 (s, 3H); 3.98 (q, J=7.0Hz, 2H); 5.16 (s, 1H); 7.24 – 7.34 (m, 5H); 7.77
(s, 1H); 9.23 (s, 1H). 13C NMR (100MHz, DMSO-d6): δ = 14.13; 17.86; 54.06; 59.28;
99.35; 126.33; 127.34; 128.46; 144.92; 148.42; 152.28; 165.41.
4.2.2. General procedure for the synthesis of compounds 1a-h, 7, 9 and 12
The literature procedure was followed.65 To a two-necked round-bottom flask,
under argon atmosphere, Se0 (0.395 g, 5.0 mmol) and ethanol (20 mL) were added followed
by the portionwise addition of NaBH4 (0.378 g, 10 mmol). The mixture was left under
stirring until the solution became colorless. After this time, the appropriate acid chloride
(5.0 mmol) was added dropwise and the reaction medium color immediately turned to
yellow/orange. After 30 min, the electrophile (DHPM or benzyl bromide) (4.2 mmol) was
added. The reaction was monitored by TLC until the total consumption of the electrophile.
The reaction mixture was extracted with ethyl acetate/water and the organic phase dried
over MgSO4 and concentrated under vacuum and the crude product was purified by column
chromatography (ethyl acetate:hexane).

Ethyl 6-((4-methylbenzoylselanyl)methyl)-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1a):
Slightly yellow solid, M.P. 169 – 173°C, 30% Yield; 1H NMR (200MHz, CDCl3): δ = 1.19
(t, J=7.1Hz, 3H); 2.41 (s, 3H); 4.11 (q, J=7.2Hz, 2H); 4.27 (AB quartet, d, J=12.3 Hz, 1H);
4.39 (AB quartet, d, J=12.3 Hz, 1H); 5.48 (d, J=3.2Hz, 1H); 7.01 (s, 1H); 7.26 (d, J=8.2
Hz, 2H); 7.43 (t, J=7.9Hz, 1H); 7.63 – 7.70(m, 2H); 7.79 (d, J=8.2 Hz, 2H); 8.03 – 8.09 (m,
13
1H); 8.16 (t, J=1.9 Hz, 1H); C NMR (50MHz, CDCl3): δ = 13.82; 21.53; 22.80; 54.29;
60.44; 99.90; 121.56; 122.56; 127.35; 129.41; 129.55; 132.67; 135.17; 145.34; 147.92;
149.48; 152.70; 164.62; 195.39.IR (KBr) (ν, cm− 1): 1603; 1634; 1644; 1693; 1713; 1937;
2868; 2935; 2947; 2978; 3114; 3196; 3239; 3310. HRMS (APPI) m/z calculated for
C22H21N3O6Se [M+H] 504.0670; found 504.0668.
Ethyl 1-methyl-6-((4-methylbenzoylselanyl)methyl)-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1b):
1
White solid, M.P. 63 – 65°C, 55% Yield; H NMR (CDCl3, 200MHz): δ (ppm) 1.22 (t,
J=7.1Hz, 3H); 2.41 (s, 3H); 3.29 (s, 3H); 4.16 (q, J=7.1Hz, 2H); 4.64 (bs, 2H); 5.50(d,
J=3.5Hz, 1H); 7.27 (d, J=8.3Hz, 3H); 7.46 (t, J=7.6 Hz, 1H); 7.59 (d, J=7.9 Hz, 1H); 7.80
(d, J=8.0Hz, 1H); 8.07 – 8.15 (m, 2H); 13C NMR (50MHz, CDCl3): δ (ppm) 13.95; 21.59;
22.44; 29.94; 52.39; 60.73; 104.09; 121.48; 122.57; 127.26; 129.51; 129.56; 132.03;
135.39; 144.86; 145.17; 148.11; 150.82; 154.20; 164.96; 192.81. IR (KBr) (ν, cm− 1): 1685;
2880; 2925; 2955; 2980; 3090; 3235. HRMS (APPI) m/z calculated for C23H23N3O6Se
[M+H] 518.0826; found 518.0827.

Ethyl 4-(4-(dimethylamino)phenyl)-6-((4-methylbenzoylselanyl)methyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1c):
1
Yellow solid, M.P. 179 – 182°C, 34% Yield; H NMR (200MHz, CDCl3): δ = 1.20 (t,
J=7.3Hz, 3H); 2.42 (s, 3H); 2.91 (s, 6H); 4.08 (q, J=7.1Hz, 2H); 4.27 (AB quartet, d,
J=12.2Hz, 1H); 4.36 (AB quartet, d, J=12.2Hz, 1H); 5.27 (d, J=2.7Hz, 1H); 5.51 (bs, 1H);
6.63 (d, J=8.8Hz, 2H); 7.16 (d, J=8.8Hz, 2H); 7.27 (d, J=8.1Hz, 2H); 7.34 (bs, 1H); 7.81
(d, J=8.3Hz, 2H); 13C NMR (100MHz, CDCl3): δ = 14.10; 21.79; 22.94; 40.45; 55.14;
60.26; 101.70; 112.36; 127.50; 127.66; 129.58; 131.41; 135.52; 145.45; 147.71; 150.19;
152.15; 165.44; 196.27. IR (KBr) (ν, cm− 1): 1615; 1630; 1687; 1713; 2809; 2923; 2980;
3110; 3227; 3369. HRMS (APPI) m/z calculated for C24H27N3O4Se [M+H] 502.1241;
found 502.1241.
Ethyl 4-(4-hydroxy-3-methoxyphenyl)-6-((4-methylbenzoylselanyl)methyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1d):
1
White solid, M.P. 168 – 170°C, 41% Yield; H NMR (200MHz, DMSO-d6): δ = 1.10 (t,
J=7.0Hz, 3H); 2.37 (s, 3H); 3.67 (s, 3H); 4.02 (q, J=7.0Hz, 2H); 4.27 (AB quartet, d,
J=11.2Hz, 1H); 4.37 (AB quartet, d, J=11.0Hz, 1H); 5.07 (d, J=3.2 Hz, 1H); 6.61 – 6.71
(m, 2H); 6.79 (s, 1H); 7.37 (d, J=8.2Hz, 2H); 7.78 (d, J=8.2Hz, 3H); 8.95 (s, 1H); 9.09 (s,
1H); 13C NMR (50MHz, DMSO-d6): δ = 14.06; 21.29; 23.81; 53.72; 55.52; 59.74; 100.87;
110.61; 115.32; 118.66; 127.01; 129.95; 135.37; 135.42; 145.23; 146.00; 147.42; 147.93;
152.08; 164.94; 193.35. IR (KBr) (ν, cm− 1): 1676; 1715; 2843; 2937; 2978; 3012; 3125;
3243; 3406. HRMS (APPI) m/z calculated for C23H24N2O6Se [M+H] 505.0874; found
505.0873.

Ethyl 4-(4-methoxyphenyl)-6-((4-methylbenzoylselanyl)methyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1e):
Slightly yellow solid, M.P. 169 – 170°C, 58% Yield; 1H NMR (200MHz, CDCl3): δ = 1.17
(t, J=7.8Hz, 3H); 2.41 (s, 3H); 3.75 (s, 3H); 4.08 (q, J=6.8Hz, 2H); 4.27 (AB quartet, d,
J=11.7Hz, 1H); 4.36 (AB quartet, d, J=12.7Hz, 1H); 5.31 (d, J=2.9Hz, 1H); 6.20 (s, 1H),
6.79 (d, J=8.8Hz, 2H); 7.19 – 7.28 (m, 4H); 7.54 (s, 1H); 7.80 (d, J=7.8Hz, 2H); 13C NMR
(50MHz, CDCl3): δ = 13.99; 21.68; 22.93; 54.76; 55.08; 60.21; 101.41; 113.87; 127.55;
127.75; 129.50; 135.48; 135.82; 145.34; 148.02; 152.48; 159.07; 165.25; 195.84. IR (KBr)
(ν, cm− 1): 1697; 2835; 2929; 2957; 3104; 3222; 3351. HRMS (APPI) m/z calculated for
C23H24N2O5Se [M+H] 489.0924; found 489.0918.
Ethyl 6-((4-methylbenzoylselanyl)methyl)-4-(naphthalen-1-yl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1f):
Slightly yellow solid, M.P. 174 – 177°C, 30% Yield; 1H NMR (200MHz, CDCl3): δ = 0.86
(t, J=7.1Hz, 3H); 2.43 (s, 3H); 3.84 – 4.01 (m, 2H); 4.31 (AB quartet, d, J=12.2Hz, 1H);
4.56 (AB quartet, d, J=12.2Hz, 1H); 5.57 (s, 1H); 6.26 (d, J=2.1Hz, 1H); 7.26 – 7.59 (m,
13
7H); 7.74 – 7.89 (m, 4H); 8,09(d, J=8.1Hz, 1H); C NMR (50MHz, CDCl3): δ = 13.69;
21.70; 22.89; 50.79; 60.07; 100.27; 122.16; 124.50; 125.58; 126.51; 127.59; 128.46;
128.85; 129.55; 130.14; 133.85; 135.50; 138.38; 145.39; 149.05; 151.86; 165.21; 195.82.
IR (KBr) (ν, cm− 1): 1640; 1695; 2853; 2925; 2955; 2976; 3110; 3233. HRMS (APPI) m/z
calculated for C26H24N2O4Se [M+H] 509.0976; found 509.0975.
Ethyl 6-((4-methylbenzoylselanyl)methyl)-2-oxo-4-p-tolyl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (1g):

Slightly yellow solid, M.P. 158 – 160°C, 53% Yield; 1H NMR (200MHz, CDCl3): δ = 1.18
(t, J=7.1HZ, 3H); 2.29 (s,3H); 2.41 (s, 3H); 4.09 (q, J=7.1Hz, 2H); 4.29 (AB quartet, d,
J=12.4Hz, 1H); 4.35 (AB quartet, d, J=12.4Hz, 1H); 5.33 (d, J=2.9Hz, 1H); 5.97 (s, 1H);
7.09 (d, J=8.1Hz, 2H); 7.19 (d, J=8.1Hz, 2H); 7.27 (d, J=8.1Hz, 2H); 7.47 (s, 1H); 7.81 (d,
13
J=8.2Hz, 2H); C NMR (50MHz, CDCl3): δ = 13.99; 21.00; 21.70; 22.91; 55.06; 60.24;
101.30; 126.46; 127.57; 129.23; 129.51; 135.48; 137.47; 140.55; 145.35; 148.22; 152.50;
165.25; 195.90. IR (KBr) (ν, cm− 1): 1628; 1699; 2823; 2853; 2925; 2980; 3049; 3106;
3222; 3349. HRMS (APPI) m/z calculated for C23H24N2O4Se [M+H] 473.0975; found
473.0979.
Ethyl 6-((4-methylbenzoylselanyl)methyl)-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (1h):
Slightly yellow solid, M.P. 135 – 138°C, 40% Yield; 1H NMR (200MHz, CDCl3): δ = 1.15
(t, J=6.8Hz, 3H); 2.39 (s, 3H); 4.07 (q, J=6.8Hz, 2H); 4.29 (AB quartet, d, J=12.8Hz, 1H);
4.35 (AB quartet, d, J=12.8Hz, 1H); 5.35 (d, J=2.9Hz, 1H); 6.62 (s, 1H); 7.22 – 7.27 (m,
13
7H); 7.65 (s, 1H); 7.79 (d, J=7.8Hz, 2H); C NMR (50MHz, CDCl3): δ = 13.94; 21.68;
22.89; 55.30; 60.21; 101.07; 126.54; 127.54; 127.74; 128.54; 129.49; 135.46; 143.41;
145.32; 148.42; 152.46; 165.19; 195.80. IR (KBr) (ν, cm− 1): 1605; 1636; 1662; 1691;
1717; 1882; 1903; 1956; 1980; 2855; 2872; 2927; 2959; 2976; 3027; 3127; 3245. HRMS
(APPI) m/z calculated for C22H22N2O4Se [M+H] 459.0819; found 459.0822.
Ethyl 6-(acetylselanylmethyl)-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(7):

1
Yellow solid, M.P. 156 – 158°C, 44% Yield; H NMR (200MHz, CDCl3): δ = 1.14 (t,
J=7.1Hz, 3H); 2.47 (s, 3H); 4.05 (q, J=7.1Hz, 2H); 4.14 (s, 2H); 5.34 (d, J=2.9Hz, 1H);
13
6.28 (bs, 1H); 7.28 (s, 5H); 7.37 (bs, 1H); C NMR (100MHz, CDCl3): δ = 13.88; 23.58;
33.95; 55.09; 60.18; 101.12; 126.46; 127.71; 128.48; 143.33; 148.01; 152.63; 165.05;
199.76. IR (KBr) (ν, cm− 1): 1632; 1685; 2853; 2927; 2953; 2974; 3029; 3106; 3223; 3359.
HRMS (APPI) m/z calculated for C16H18N2O4Se [M+H] 383.0505; found 383.0507.
Phenyl-1,3-bis-ethyl4-(phenyl)-6-((selenoformate)methyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (9):
1
Yellow solid, M.P. 126 – 128°C, 20% Yield; H NMR (200MHz, CDCl3): δ = 1.18 (t,
J=7.1Hz, 3H); 4.11 (q, J=7.1Hz, 2H); 4.38 (s, 2H); 5.40 (d, J=2.0Hz, 1H); 6.50 (bs, 1H);
7.25 – 7.29 (m, 6H); 7.58 (t, J=8.0Hz, 1H); 7.85 (bs, 1H); 8.10 (d, J=6.5Hz, 1H); 8.35 (bs,
1H). 13C NMR (100MHz, CDCl3): δ = 14.00; 23.84; 55.35; 60.41; 101.58; 126.11; 126.56;
127.91; 128.64; 129.72; 132.30; 138.68; 143.25; 147.70; 152.66; 165.14; 195.39. IR (KBr)
(ν, cm− 1): 1638; 1695; 2906; 2933; 2978; 3031; 3088; 3237; 3351. HRMS (APPI) m/z
calculated for C36H34N4O8Se2 [M+H] 811.0788; found 811.0786.
Se-Benzyl 4-Methylbenzoselenoate (12):
Yellowish solid, M.P. 54 – 56ºC, 85% Yield; 1H NMR (400MHz, CDCl3): δ = 2.35 (s, 3H);
4.31 (s, 2H); 7.20 (d, J=7.8Hz, 3H); 7.27 (t, J=7.6Hz, 2H); 7.35 (d, J=7.4Hz, 2H); 7.78 (d,
J=8.2Hz, 2H). 13C NMR (100MHz, CDCl3): δ = 21.6; 28.8; 126.8; 127.3; 128.5; 128.9;
129.4; 136.2; 139.1; 144.6; 193.8.
4.2.3. General procedure for the synthesis of compound 11

The literature procedure was followed for the synthesis of NaSH.66 In a two-necked
round-bottom flask, Na2S.9H2O (0.361 g, 1.5 mmol) and NaHCO3 (0.126 g, 1.5 mmol)
were solubilized in 10 ml of a mixture of ethanol-water (1:1). After 15 min, p-toluoyl
chloride (0.198 ml, 1.5 mmol) was added. After 30 min, 2h was added (0.294 g, 1.0 mmol).
The consumption of 2h was monitored by TLC. The reaction mixture was extracted with
ethyl acetate/water and the organic phase dried over MgSO4 and concentrated under
vacuum and the crude product was purified by column chromatography (ethyl
acetate:hexane).
Ethyl
6-((4-methylbenzoylthio)methyl)-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (11):
Slightly brown solid, M.P. 119 – 121°C, 46% Yield; 1H NMR (200MHz, CDCl3): δ = 1.14
(t, J=7.2Hz, 3H); 2.39 (s, 3H); 4.07 (q, J=7.2Hz, 2H); 4.38 (s, 2H); 5.34 (d, J=2.9Hz, 1H);
13
6.74 (bs, 1H); 7.18 – 7.27 (m, 7H); 7.86 (m, 3H); C NMR (50MHz, CDCl3): δ = 13.87;
21.57; 27.88; 55.20; 60.25; 101.94; 126.47; 127.51; 127.69; 128.48; 129.22; 133.34;
143.22; 144.87; 146.48; 152.53; 165.01; 192.44. IR (KBr) (ν, cm− 1): 1640; 1664; 1699;
1717; 2931; 2976; 3104; 3125; 3241; 3386. HRMS (APPI) m/z calculated for C22H22N2O4S
[M+H] 411.1373; found 411.1371.
4.4 Pharmacological evaluation
4.4.1. Thiobarbituric acid reactive substances (TBARS) assay
Production of thiobarbituric acid reactive species (TBARS) from phospholipid
The oxidative degradation of lipids by reactive oxygen species (ROS), called lipid
peroxidation, results in the formation of highly reactive and unstable lipid peroxides.

Decomposition of lipid peroxides results in the formation of Thiobarbituric Acid Reactive
Substances (TBARS), including malondialdehyde (MDA) which react with TBA
(Thiobarbituric acid) resulting a pink coloured MDA-TBA adduct. Measuring TBARS
levels offers a convenient method of determining the relative lipid peroxide content in a
sample.
The production of TBARS from phospholipid was determined using the method of
Ohkawa et al. (1979) with some modifications.54 Thirty grams of egg yolk was mixed with
isopropanol (90 mL), followed by the addition of water (90 mL). The mixture was stirred
for 5 min and after filtration the liquid phase was centrifuged at 2000 x g to 10 min. The
remaining supernatant was discarded and the white phase of the pellet was collected and re-
suspended in 0.8 mL of water. The final concentration of triglycerides (1 mg/mL) was
determined using an assay kit (Lab Test, MG, Brazil) according to the supplier’s
specifications.
The phospholipids derived from the yolk (0.05 mL) were incubated with and
without freshly prepared iron sulfate (100 μM) and with different concentrations of the
organoselenium compounds (as show in the figures) together with potassium phosphate
buffer (0.05 M, pH 7.5) and an appropriate volume of distilled water to give a total volume
of 0.2 mL at 37oC for 1 h. The color reaction was carried out by adding 100 μL of each
acetic acid (pH 3.4) and 0.6% TBA, respectively. The reaction mixtures, including those
diluted with 0.03 mM MDA standard, were incubated at 97◦C for 1 h. The tubes were
cooled and finally 0.58 mL of n-butanol was added followed by centrifugation at 1000 × g.
The organic layer (supernatant) was collected and absorbance was measured at 532 nm
using the Cary 50 UV-Vis spectrophotometer. The TBARS unit was expressed as nmol of
MDA/mg of triglycerides.

4.4.2. Iron chelation assay
The ability of compounds to chelate Fe2+ was determined using a modified version
of the method of Minotti and Aust (1987) as described by Olabinri et al. (2010).55,67 In a
tube, 268 µL of freshly prepared 120 µM FeSO4 was added to a reaction mixture containing
336 µL of 0.1 M Tris–HCl (pH 7.4) and 200 µL of the test compounds at five different
concentrations (5-100 µM; prepared in methanol), followed by the addition of 436 µL of
saline solution (0.9% NaCl, w/v). The reaction mixture was incubated for 10 min at 37oC
before the addition of 260 µL of 1,10-phenanthroline (0.25%, w/v). The absorbance was
subsequently measured at 510 nm using the Cary 50 UV-Vis spectrophotometer. Methanol
was used instead of the sample solution as a control (A control) and BHT was used as a
standard chelating agent.
Fe2+ chelating activity (%) was calculated by using the following formula:
Fe2+ chelating activity (%) = [(Abs control – Abs sample) / Abs control)] ×100
where Abs control is the absorbance of the control without the sample and Abs sample is
the absorbance in the presence of the sample.
4.4.3. In Vitro Protocol of AChE inhibition
In the presence of the enzyme AChE, the compound ATCI (acetylthiocholine
iodide) is hydrolyzed to produce acetate and thiocholine. The thiol (R-SH) group of
thiocholine can react with the indicator compound DTNB (5-5'-dithio-bis-2-nitrobenzoic
acid) to form TNB (5-thio-2-nitrobenzoate) and this reaction resulted in the development of
a yellow color. The color intensity of the product is measured at 412 nm, and it is
proportional to the enzyme activity. If an inhibitor, tested compounds, inhibits the enzyme,

then the rate of reaction will be slower and the optical density (absorbance) of the sample
during the assay will be lower and hence no or less yellow color will be developed.
Acetylcholinesterase enzymatic activity was measured using the Ellman et al.
(1961) method, with some modifications.58 The assay medium (1 mL) consisted of
deionized water, 0.1M phosphate buffer (pH 7.4), 0.01 M DTNB, the test compounds at
five different concentrations dissolved in MeOH and an AChE (from electric eel) solution
containing 0.8U mL−1 and it was incubated for 15 min at 25°C. In the next step, 0.01 M
acetylthiocholine iodide solution was added and the activity was determined by measuring
the absorbance at 412 nm every 15 s for 5 or 10 min on a Cary 50 UV-Vis
spectrophotometer at 25 °C. A control mixture containing methanol instead of the test
samples was used. The absorbance value obtained was considered to be 100% activity. The
percentage of AChE inhibitory activity (% IA) was calculated using the following equation:
I(%) = 100 − (A sample/A control) × 100
where “A sample” is the absorbance of the sample and “A control” is the absorbance
without the sample. Plotting the inhibition percentage against the sample solution
concentrations gave estimates of the IC50 (concentration of the drug resulting in 50%
inhibition of enzyme activity). Galantamine was used as the positive control.
Acknowledgements
The authors thank C BIM , CNP , INCT-Cat lise, CAPES, TWAS and FAPESC for
financial support.
References