Design, synthesis and evaluation of seleno-dihydropyrimidinones as potential multi-targeted therapeutics for Alzheimer's disease

Article abstract of DOI:10.1039/c4ob00598h

In this paper we report the design, synthesis and evaluation of a series of seleno-dihydropyrimidinones as potential multi-targeted therapeutics for Alzheimer's disease. The compounds show excellent results as acetylcholinesterase inhibitors, being as active as the standard drug. All these compounds also show very good antioxidant activity through different mechanisms of action. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00598h

Organic &
Biomolecular Chemistry
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PAPER
Design, synthesis and evaluation of seleno-
dihydropyrimidinones as potential multi-targeted
therapeutics for Alzheimer’s disease†
Cite this: Org. Biomol. Chem., 2014,
12, 3470
Rômulo F. S. Canto,a Flavio A. R. Barbosa,a Vanessa Nascimento,a
Aldo S. de Oliveira,b Inês M. C. Brighenteb and Antonio Luiz Braga*a
Received 19th March 2014,
Accepted 23rd March 2014
In this paper we report the design, synthesis and evaluation of a series of seleno-dihydropyrimidinones as
potential multi-targeted therapeutics for Alzheimer’s disease. The compounds show excellent results as
acetylcholinesterase inhibitors, being as active as the standard drug. All these compounds also show very
good antioxidant activity through different mechanisms of action.
DOI: 10.1039/c4ob00598h
www.rsc.org/obc
high oxygen consumption rate, high lipid content, and rela-
tively limited antioxidant capacity compared to other organs,
Introduction
Alzheimer’s disease (AD), the most prevalent of the neuro- the brain is particularly susceptible to oxidative damage. Senile
degenerative diseases, affects approximately 15 million people plaques release free radicals that are extremely toxic.10 The
worldwide and nearly 50% of adults over the age of 85.1
accumulation of reactive oxygen species (ROS) results in damage
Because of the complex pathophysiology of AD, which to major cell components, such as DNA, membranes and cyto-
involves many pathways, the development of a satisfactory plasmic proteins.11 Oxidative stress is therefore included in all
therapy is problematic. The main therapy targets are reduced the pathophysiological hypotheses for AD, and studies have
levels of the neurotransmitter acetylcholine (ACh), the diffuse shown the efficacy of several antioxidant compounds.12
loss of neurons, neurofibrillary tangles and formation of
In addition to these two hypotheses, a third, known as the
β-amyloid (Aβ) plaques.2 Based on the cholinergic hypothesis, metal hypothesis, considers that metals (Fe and Cu) also play a
the mainstays of the current pharmacotherapy for AD are role in the pathogenesis of AD.13 During the disease pro-
drugs aimed at increasing the levels of ACh through the inhi- gression, metals progressively accumulate in the cerebrum.14
bition of cholinesterases (ChEs).3 ChE inhibitors have been The abnormal accumulation of metals is closely associated
approved as an efficacious treatment to reduce the symptoms with the formation of Aβ plaques and neurofibrillary tangles.15
of the early stage of AD. Several anticholinesterase agents, In addition, abnormally high levels of Cu and Fe in the brain
such as tacrine,4 ensaculin,5 donepezil6 and galanthamine,7 catalyze the production of ROS, which further elicit oxidative
have been shown to induce modest improvements in relation stress contributing to the AD pathogenesis.16 Thus, lowering
to memory and cognitive functions. Recent evidence suggests the concentration of metals in the brain through chelation rep-
that acetylcholinesterase (AChE) also plays a non-cholinergic resents another rational therapeutic approach to treating AD.
role in the development of AD,8 as it works as a chaperone,
accelerating the Aβ peptide deposition and the aggregation of advanced to the clinical stage, but they only provide relief of
Aβ into insoluble fibrils.9
the symptoms and do not tackle the underlying causes of the
Drug candidates that target single processes have been
Another current therapeutic strategy to treat AD is to reduce disease, because of the multifactorial pathogenesis of neuro-
the oxidative stress involved in cellular death. Because of its degenerative diseases. Modulation of multiple targets along
the same biological pathway could potentially lead to disease
modification rather than just control of the symptoms17 and
the development of new drugs for AD is being focused on multi-
potent molecules acting in a complementary manner, which
could be more efficacious to AD patients.18 We are therefore
interested in developing novel AChE inhibitors with antioxi-
dant function for the treatment of AD by combining, in the
same structure, biologically active dihydropyrimidinones
(DHPMs) and organoselenium compounds (Fig. 1). This strat-
aLaboratorio de Sintese de Substancias de Selenio Bioativas, Centro de Ciencias
Fisicas e Matematicas, Departamento de Quimica, Universidade Federal de Santa
Catarina, 88040-900 Florianopolis, SC, Brazil. E-mail: braga.antonio@ufsc.br;
Tel: +55 (48) 3721-6427F
bCFM, Departamento de Quimica, Universidade Federal de Santa Catarina,
88040-900 Florianopolis, SC, Brazil
†Electronic supplementary information (ESI) available: Proton NMR, carbon
NMR and HRMS spectra available. See DOI: 10.1039/c4ob00598h
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thesized and evaluated for their ability to inhibit AChE and to
act as antioxidants through the glutathione peroxidase (GPx)
mechanism, iron chelating activity, reducing power and total
antioxidant capacity. Moreover, based on the molecule struc-
ture, the number of descriptors necessary to estimate the phar-
macokinetics (PK) of the compounds was determined. As
considerable effort went into the optimization of the PK of
each substance at a very early stage of the research into its
potential as a drug; the Lipinski and other drug-likeness filters
were used to predict the PK of the compounds obtained.33
Herein, we report the design, synthesis, evaluation and pre-
dicted PK of a series of seleno-DHPM hybrids as potential
multi-targeted therapeutics for AD.
Results and discussion
Synthetic procedures
Fig. 1 Seleno-dihydropyrimidinones as multi-targeted therapeutics for
Alzheimer’s disease.
The synthesis scheme involved a two-step pathway leading to
the formation of a variety of seleno-dihydropyrimidinones
1a–g in moderate yields (Scheme 1). A three-component Biginelli
reaction of aromatic aldehydes 2a–f, urea 3a or N-methyl-urea
3b and ethyl 4-chloroacetoacetate 4 at 100 °C, under neat con-
ditions using HCl as the catalyst, produced the corresponding
6-chloromethyl-DHPMs 5a–g.34 These compounds were
obtained in 68–99% yield after precipitation from water. The
6-chloromethyl-DHPMs obtained in this way were treated with
potassium selenocyanide in MeOH at room temperature to
afford the target hybrid selenocyanides 1a–g in 22–43% yield
after column chromatography. The structures of the com-
pounds obtained are shown in Table 1. Their analytical and
spectroscopic data are in agreement with the predicted struc-
tures. The seleno-DHPMs were found to be stable solids at
room temperature, even when exposed to the atmosphere.
egy of merging two or more bioactive moieties in the same
structure has been successfully employed in the development
of new drug candidates.19
In this context, it is known that selenium (Se) might play
different roles in the progression of AD.20 In animal models
of AD, Se has been shown to prevent oxidative damage
and modulate the cholinergic system.21 Organoselenium com-
pounds, like diphenyl diselenide (Ph2Se2), can enhance the
cognitive performance of rodents without inducing neurotoxi-
city,22 while its p-methoxy analog (p-MeOPh2Se2) improves the
memory of mice, protects against Aβ-induced neurotoxicity
and inhibits the activity of AChE in the model of sporadic Alz-
heimer-type dementia, which can be explained by its antioxi-
dant properties.23 Moreover, organoselenium compounds have
the ability to act as mimetics of the enzyme glutathione peroxi-
dase (GPx),24 which is known to have an important role in
modulating oxidative stress in the brain.25 Selenocyanides are
metabolized to selenols,26 being less toxic forms of organosele-
nium compounds,27 and have been employed successfully in
the development of new bioactive compounds.28
1
In the H NMR spectra the resonance signals of the N–H’s
are usually registered as singlets in the ranges of ca.
DHPMs, readily obtained through the Biginelli reaction, are
reportedly good antioxidants, acting as radical scavengers and
being effective against lipid peroxidation,29 with reports of
some analogs being better radical scavengers than resvera-
trol.30 The compounds in this class are potent AChE inhibi-
tors,31 with recent studies showing that the potency of some
DHPM-curcuminoid hybrids is comparable to that of the stan-
dard drug galanthamine.32
Despite the advantages presented by DHPMs and organo-
selenium scaffolds in AD drug design, no studies have focused
on designing multi-targeted hybrids of these structures within
a single molecule. In this context, the aim of this study was to
design a series of novel seleno-dihydropyrimidinones as poten-
tial multi-targeted therapeutics for AD, acting as antioxidants
and AChE inhibitors. All compounds designed were syn-
Scheme 1 Reaction conditions. (i) HClconc., neat, 100 °C, 2 h, 68–99%;
(ii) KSeCN (1.2 equiv.), MeOH, r.t., 24 h.
Table 1 Structures and yields of the seleno-DHPMs
Entry
Ar
R
Compound
Yield (%)
1
2
3
4
5
6
7
C6H5–
H
H
H
H
H
H
Me
1a
1b
1c
1d
1e
1f
40
42
43
42
39
22
32
2-Me–C6H4–
4-Me–C6H4–
2-MeO–C6H4–
4-MeO–C6H4–
3-NO2–C6H4–
3-NO2–C6H4–
1g
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the treatment of Alzheimer’s disease. The result obtained with
the standard is in agreement with literature data.36 No simple
structure–activity relationship was observed for the com-
pounds tested on comparing the electron donating or with-
drawing substituents at different positions of the aromatic
ring. Compound 1a, bearing the simple phenyl substituent, is
the most active of the series.
Table 2 Inhibitory activity of the seleno-DHPMs towards AChE
AChE inhibition
IC50 (ppm)
AChE inhibition IC50
Compound
(µmol L−1
)
1a
1b
1c
1d
1e
1f
1g
7.87
2.44
6.21
6.11
2.40
7.72
3.79
4.91
2.16
8.58
16.40
15.49
6.09
18.87
8.96
17.05
Antioxidant activity
Galantamine
There are several methods available for evaluating the in vitro
antioxidant activity of biologically active substances, ranging
from chemical assays with lipid substrates to more complex
assays using many different instrumental techniques.37 Due to
the different types of free radicals and their different forms of
action in living organisms, it is difficult to obtain a simple and
universal method through which the antioxidant activity can
be measured accurately and quantitatively. Thus, the search
for more rapid and efficient tests has generated a great
number of methods for the evaluation of the activity of antioxi-
dants, through the use of a large variety of free radical generat-
ing systems.38 Due to the wide divergence in the results of
antioxidant tests, many protocols and guidelines have been
established aimed at bringing order and agreement to this
important field.39
6.07–8.14 ppm and 9.42–9.67 ppm. The aromatic protons
appear between 6.85 and 8.05 ppm, and the chiral center
appears between 5.18 and 5.70 ppm as a doublet. The two
protons attached to the selenide atom appear as two distinct
doublets at around 3.96 to 4.34 ppm. A characteristic quartet
and triplet, due to the ethyl ester moiety, can be found at
around 4.05 and 1.05 ppm, respectively. The 13C NMR spectra
show the characteristic signal of the quaternary-CN carbon
between 100 and 103 ppm.
The IR spectra show a band in the region of approximately
2139–2154 cm−1, corresponding to ν(CuN). In the mass
spectra (APPI), the molecular ion peaks M+ presented the
characteristic isotopic pattern of the organoselenium
compounds.
In this study, the antioxidant activity of the seleno-DHPMs
(Table 3) was investigated applying the following tests: redu-
cing power, iron chelating activity, total antioxidant capacity
and glutathione peroxidase assay (GPx). The results obtained
Acetylcholinesterase inhibitory activity
All compounds considered were assessed as AChE inhibitors. are presented as mean SD.
The enzymatic activity was measured using an adaptation of
We explored the potential antioxidant activity of all seleno-
the method described by Mata et al. (2007).35 The concen- DHPMs 1a–g using a GPx enzyme model based on the Tomoda
trations of the test compounds that inhibited the hydrolysis of method.40 The reduction of H2O2 was monitored through the
substrates by 50% (IC50 – Table 2) were determined by plotting formation of diphenyl sulfide and the increase in UV absorp-
the inhibition against the sample solution concentrations.
tion at 305 nm. We measured the activity by considering the
All compounds showed a high percentage inhibition of the time required to reduce the thiol concentration by 50% (T50)
enzyme acetylcholinesterase. The results demonstrate that all and used Ph2Se2 under the same conditions to compare the
of the compounds were as active as the standard alkaloid antioxidant activity.41 The results obtained are summarized in
galantamine, the active drug in Reminyl®, which is used in Table 3. Encouragingly, all compounds showed activity levels
Table 3 Antioxidant activity of seleno-DHPMs 1a–g
Iron chelating activityd
Reducing powere
(mg AAE g−1
Total antioxidant capacitye
(mg AAE g−1
Compd.
GPx T50 a,b,c (h)
(EC50 μg mL−1
)
)
)
1a
1b
1c
1d
1e
1f
1g
Ph2Se2
Rutin
BHT
EDTA
4.92 0.11
5.55 0.22
5.04 0.46
6.70 0.32
5.07 0.20
6.04 0.08
6.15 0.47
5.95 0.47
—
23.79 0.14
12.45 0.03
29.30 0.26
34.96 0.17
29.80 0.11
45.41 0.33
32.69 0.22
—
2258.68 23.54
2604.36 12.37
2054.97 11.75
1875.97 3.26
2053.20 13.44
1552.92 8.65
1937.69 11.00
—
521.81 2.86
638.17 1.15
461.74 2.22
391.42 4.21
457.61 1.88
287.88 1.22
416.36 1.09
—
41.35 0.16
20.65 0.29
4.65 0.02
1622.10 11.76
1641.89 14.23
—
310.43 1.87
548.24 1.23
—
—
—
a Under these conditions addition of H2O2 in the absence of the organoselenium compound did not produce any significant oxidation of PhSH.
c
b MeOH (1 mL); organoselenium catalyst (0.4 mM); PhSH (5 mM); H2O2 (10 mM). T50 is the time required, in hours, to reduce the thiol
concentration by 50% after the addition of H2O2. d The EC50 value for 50% chelation of iron(II) ions. e Results, in mg, for ascorbic acid per g of
the compound are calculated for sample concentrations of 100 μg mL−1. Each value is expressed as mean SD.
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comparable to that of PhSeSePh as GPx mimics. Compounds power and iron chelation showed a strong correlation (adj.
1a–c and 1e provided the best results, being approximately 1.2 R-square = 0.99851; y = −32.23369x + 3007.89974) indicating
times more active than Ph2Se2. On the other hand, the hybrids consistency in the values obtained applying to two tests.
1d and 1f–g were less efficient as GPx mimics when compared
Total antioxidant capacity was evaluated by the phospho-
to the standard. Although the seleno-DHPMs were assembled molybdenum method. This assay is based on the reduction of
with substituents which have different electronic demands, Mo(VI) to Mo(V) by the antioxidant compounds and the sub-
their catalytic activity did not follow a clear trend and the sequent formation of a green phosphate/Mo(V) complex at
effect (if any) of the substituent on their performance was acidic pH with a maximum absorption at 695 nm.46 The
negligible.
results obtained, expressed as ascorbic acid equivalents (AAE),
The reducing power is generally associated with the pres- are presented in Table 3. All samples investigated were active
ence of reductones, which exert antioxidant action by breaking in a concentration-dependent manner and their potency
the free radical chain. In addition, reductones can reduce the values were high. Of the compounds tested 1b showed the
oxidized intermediates of lipid peroxidation processes and highest total antioxidant capacity with a value of 638.17 mg
thus act as primary and secondary antioxidants. The reducing AAE per gram of the compound. The antioxidant activity,
power assay measures the electron donating ability of antioxi- assessed using this method, followed the same order observed
dants using the potassium ferricyanide reduction method. in the previous tests, with strong correlations between the
Antioxidants cause the reduction of the Fe3+/ferricyanide reducing power and the total antioxidant activity (adj. R-square
complex to the ferrous form and the activity is measured as 0.99897 and y = 0.33387x − 230.75251) and between the iron
the increase in the absorbance at 700 nm. In this assay, the chelating activity and the total antioxidant capacity (adj.
yellow color of the test solution changes to various shades of R-square 0.99886 and y = 0.33444x − 231.55161). All compounds
green and blue depending on the reducing power of the anti- assayed presented excellent results for the antioxidant poten-
oxidant samples.42 The compounds analyzed showed excellent tial on applying a variety of methods, with very good corre-
results for the reducing power, probably due to the presence of lations among the results obtained. In combination, these
the redox active selenocyanide moiety. All values were above findings indicate that the compounds have high potential to
those associated with the standards considered (rutin and BHT). act as antioxidants for the treatment of AD.
The antioxidant activity of compounds is also attributed to
their ability to chelate transition metal ions, such as those of
iron and copper, which have been proposed as catalysts for the
Preliminary pharmacokinetics
initial formation of reactive oxygen species. Chelating agents
may stabilize pro-oxidative metal ions in living systems
through complexation.43 Iron(II) ions are known to be potent
inducers of lipid peroxidation. Ferrozine can quantitatively
form complexes with Fe2+. In the presence of chelating agents,
the complex formation is disrupted resulting in a decrease in
the red color of the complex. Measurement of the reduction in
color intensity at the 562 nm wavelength allows an estimation
of the metal chelating activity of the coexisting chelator.44
All compounds demonstrated the ability to chelate Fe2+. Of
the compounds analyzed, 1b proved to be the most potent che-
lator, being more active than two standards and less potent
than the most active of the standards (EDTA) by only a factor
of 3. Of the seven compounds tested, six were more active than
rutin. The values found for the standards are in agreement
with literature data.45 Importantly, the results for the reducing
To predict some aspects of the pharmacokinetics of the com-
pounds obtained, their physicochemical and topological pro-
perties were calculated. Table 4 presents the octanol–water
partition coefficients expressed as Clog P and Mlog P (Morigu-
chi log P), the number of H-bond donors (HBD), H-bond
acceptors (HBA) and rotatable bonds (NRB), and the topologi-
cal polar surface area (tPSA).47
The descriptors obtained in silico were compared with the
filters for the prediction of the solubility and permeability of
drug candidates after oral administration described by
Lipinski,48 Oprea49 and Veber.50 The results show that the
compounds under consideration obey the Lipinski rule of 5:
molecular weight (MW) ≤ 500, HBD ≤ 5, HBA ≤ 10 and log P ≤
5.0 (Table 3). According to Lipinski, a compound whose data
do not adhere to the rule will likely to be poorly bioavailable
because of poor absorption or permeation.48 The values for
Table 4 Molecular descriptorsa in silico of seleno-DHPMs
Compd.
Clog P
Mlog P
Mol. weight
tPSA (Å2)
HBA
HBD
Vol. (Å3)
NRB
1a
1b
1c
1d
1e
1f
0.86
1.17
1.17
0.75
0.75
0.59
0.87
2.41
2.81
2.86
2.42
2.47
2.34
3.14
364.26
378.29
378.29
394.29
394.29
409.26
423.29
91.2
91.2
91.2
100.5
100.5
137.1
128.3
6
6
6
7
7
9
9
2
2
2
2
2
2
1
279.72
296.28
296.28
305.26
305.26
303.05
319.99
6
6
6
7
7
7
7
1g
a tPSA, topological polar surface area; HBA, H-bond acceptors; HBD, H-bond donors; NRB, the number of rotatable bonds.
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the compounds were MW 364.3–423.3 Da, HBD 1–2, HBA 6–9 nide (3.6 mmol). The resulting solution was stirred at room
and Clog P 0.59–1.17. temperature. The progress of the reaction was monitored by
The seleno-DHPMs also satisfied Oprea’s criteria, which TLC. After completion of the reaction, the mixture was
additionally include: the number of rings ≤5 and Mlog P in extracted with ethyl acetate, water and brine respectively and
the range of −2.0 and 4.5. The compounds possessed 2 rings dried with MgSO4. The organic phase was evaporated using a
and their Mlog P values were in the range of 2.41–3.14. Veber rotary evaporator under reduced pressure to give the crude
proposed a filter of two properties: the number of HBD and product. The crude product was purified by column chromato-
HBA ≤ 12 (tPSA ≤ 140 Å2), and the number of rotatable bonds graphy over silica gel to furnish the pure product. The product
(NRB) ≤ 10. All derivatives meet these criteria. It is postulated was characterized by the corresponding spectroscopic data
that limited molecular flexibility, expressed as the NRB, and (IR, 1H and 13CNMR, HRMS).
low polar surface area (tPSA) are important predictors of oral
bioavailability, independent of the molecular weight.
2-Oxo-4-phenyl-6-selenocyanatomethyl-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylic acid ethyl ester (1a). White solid,
The results reported herein show that the compounds syn- m.p. = 162 °C; IR (ν, cm−1): 3396, 3219, 2149, 1703, 1675;
thesized would have favorable pharmacokinetics on appli- 1H NMR (CDCl3, 200 MHz): δ (ppm) 1.10 (t, J = 7.09 Hz, 3H),
cation, that is, solubility and permeability after the oral 4.01–4.11 (m, 4H), 5.18 (d, J = 2.69 Hz, 1H), 7.26–7.34 (m, 5H),
administration of drug candidates. They possess drug-likeness 7.91 (s, 1H), 9.42 (s, 1H); 13C NMR (DMSO-d6, 50 MHz):
independent of the criterion used.
δ (ppm) 13.8, 25.8, 53.9, 60.0, 100.4, 103.8, 126.4, 127.6, 128.5,
144.0, 148.3, 151.7, 165.2; HRMS (APPI-QTOF) m/z calcd for
C15H15N3O3Se [M + H]: 366.0352; found 366.0354.
2-Oxo-6-selenocyanatomethyl-4-o-tolyl-1,2,3,4-tetrahydro-pyri-
midine-5-carboxylic acid ethyl ester (1b). Slightly yellow solid,
m.p. = 178 °C; IR (ν, cm−1): 3343, 3219, 2150, 1706, 1670;
1H NMR (CDCl3, 400 MHz): δ (ppm) 1.02 (t, J = 7.03 Hz, 3H),
2.39 (s, 3H), 3.96–4.11 (m, 4H), 5.61 (s, 1H), 6.57 (s, 1H),
7.10–7.21 (m, 3H), 7.29 (d, J = 7.42 Hz, 1H); 9.52 (s, 1H);
13C NMR (CDCl3, 100 MHz): δ (ppm) 13.7, 18.8, 26.7, 51.5,
60.6, 101.2, 103.7, 126.8, 127.3, 127.9, 130.5, 134.7,
140.8, 147.3, 152.8, 165.6; HRMS (APPI-QTOF) calcd for
C16H17N3O3Se [M + H]: 380.0509; found 380.0512.
Experimental
General experimental procedures
All reactions were carried out under an air atmosphere and
monitored by TLC using Merck 60 F254 pre-coated silica gel
plates (0.25 mm thickness) and the products were visualized
by UV detection, I2 or Vanillin staining. Flash chromatography
was carried out with silica gel (200–300 mesh). FT-IR spectra
were recorded on a Varian 3100 FT-IR spectrometer. 1H and
13C NMR spectra were recorded in CDCl3 or DMSO-d6 on a
1
Bruker Avance 200 or Varian AS-400 spectrometer. Data for H
2-Oxo-6-selenocyanatomethyl-4-p-tolyl-1,2,3,4-tetrahydro-pyri-
midine-5-carboxylic acid ethyl ester (1c). Slightly yellow solid,
NMR are reported as chemical shifts (δ ppm), multiplicity (s =
singlet, d = doublet, q = quartet, m = multiplet), coupling con-
stant J (Hz), integration, and assignment; data for 13C NMR are
reported as a chemical shift (δ ppm). High resolution mass
spectral analyses (HRMS) were carried out by APPI-Q-TOFMS
measurements and were performed with a micrOTOF Q-II
(Bruker Daltonics) mass spectrometer equipped with an auto-
matic syringe pump (KD Scientific) for sample injection. The
APPI-QTOF mass spectrometer was run at 4.5 kV with a desol-
vation temperature of 180 °C. The mass spectrometer was
operated in the positive ion mode. The standard atmospheric
pressure photoionization (APPI) source was used to generate
the ions. The sample was injected using a constant flow (3 μL
min−1). The solvent was an acetonitrile–methanol mixture.
The APPI-Q-TOF MS 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 Data
Analysis software version 4.0. All the starting materials and
catalysts were either purchased from commercial sources or
synthesized by literature known procedures.34 All the solvents
were used without special treatment.
1
m.p. = 152 °C; IR (ν, cm−1): 3342, 2153, 1730, 1675; H NMR
(CDCl3, 200 MHz): δ (ppm) 1.14 (t, J = 7.07 Hz, 3H), 2.97 (s,
3H), 3.97–4.12 (m, 4H), 5.32 (d, J = 3.03 Hz, 1H), 6.67 (s, 1H),
7.10 (d, J = 8.59 Hz, 2H), 7.20 (d, J = 8.59 Hz, 2H), 9.55 (s, 1H);
13C NMR (DMSO-d6, 50 MHz): δ (ppm) 13.8, 20.9, 26.6, 54.7,
60.6, 101.8, 103.5, 126.5, 129.25, 137.7, 139.9, 146.6, 153.5,
165.5; HRMS (APPI-QTOF) calcd for C16H17N3O3Se [M + H]:
380.0509; found 380.0510.
4-(2-Methoxy-phenyl)-2-oxo-6-selenocyanatomethyl-1,2,3,4-
tetrahydro-pyrimidine-5-carboxylic acid ethyl ester (1d).
Slightly yellow solid, m.p. = 140 °C; IR (ν, cm−1): 3359, 3225,
1
2154, 1714, 1677; H NMR (CDCl3, 200 MHz): δ (ppm) 1.07 (t,
J = 7.34 Hz, 3H), 3.86 (s, 3H), 3.99–4.08 (m, 3H), 4.22 (d, J =
12.23 Hz, 1H), 5.70 (d, J = 2.45 Hz, 1H), 6.07 (s, 1H), 6.87–6.93
(m, 2H), 7.10 (d, J = 5.87 Hz, 1H), 7.27 (t, J = 7.83 Hz, 1H),
9.58 (s, 1H); 13C NMR (CDCl3, 50 MHz): δ (ppm) 13.9, 26.8,
50.5, 55.3, 60.6, 99.3, 103.2, 110.7, 120.5, 127.1, 129.2, 129.4,
148.3, 153.6, 156.9, 165.8; HRMS (APPI-QTOF) calcd for
C16H17N3O4Se [M + H]: 396.0458; found 396.0461.
4-(4-Methoxy-phenyl)-2-oxo-6-selenocyanatomethyl-1,2,3,4-
tetrahydro-pyrimidine-5-carboxylic acid ethyl ester (1e).
Slightly yellow solid, m.p. = 155 °C; IR (ν, cm−1): 3353, 2153,
1728, 1672; 1H NMR (CDCl3, 400 MHz): δ (ppm) 1.15 (t, J =
General procedure for the synthesis of seleno-DHPMs 1a–g
To a stirred solution of 6-chloromethyl-dihydropyrimidinone 7.03 Hz, 3H), 3.78 (s, 3H), 4.03–4.09 (m, 4H), 5.32 (s, 1H), 6.26
(3.0 mmol) in MeOH (10 mL) was added potassium selenocya- (s, 1H), 6.85 (d, J = 8.60 Hz, 2H), 7.23 (d, J = 8.60 Hz, 2H), 9.55
3474 | Org. Biomol. Chem., 2014, 12, 3470–3477
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(s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 13.9, 25.9, and the subsequent formation of a green phosphate/Mo(V)
53.3, 55.1, 60.0, 100.6, 103.9, 113.8, 127.6, 136.3, 148.1, 151.8, complex at acidic pH with a maximal absorption at 695 nm.46
158.7, 165.3; HRMS (APPI-QTOF) calcd for C16H17N3O4Se
Reduction power
[M + H]: 396.0458; found 396.0457.
4-(3-Nitro-phenyl)-2-oxo-6-selenocyanatomethyl-1,2,3,4-tetra-
hydro-pyrimidine-5-carboxylic acid ethyl ester (1f). Slightly
yellow solid, m.p. = 166 °C; IR (ν, cm−1): 3427, 3320, 2151,
Fe(III) reduction is often used as an indicator of the electron-
donating activity, which is an important mechanism of pheno-
lic antioxidant action. The reducing power of compounds was
determined according to the method of Yen and Chen (1995).
Different amounts of each compound (25–250 µg mL−1) in
methanol were mixed with phosphate buffer (2.5 mL, 0.2 M,
pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%).
The mixture was incubated at 50 °C for 20 min. A portion
(2.5 mL) of trichloroacetic acid (10%) was added to the
mixture to stop the reaction, which was then centrifuged at
3000 rpm for 10 min. The upper layer of the solution (2.5 mL)
was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL,
0.1%), and the absorbance was measured at 700 nm. The
increased absorbance of the reaction mixture indicated the
increased reducing power.
1
1722, 1684; H NMR (DMSO-d6, 200 MHz): δ (ppm) 1.17(t, J =
7.07 HZ, 3H), 3.96–4.07 (m, 3H), 4.23 (d, J = 12.13 Hz, 1H),
5.38 (d, J = 3.03 Hz, 1H), 7.42–7.51 (m, 2H), 7.65 (m, 1H),
8.05–8.14 (m, 2H), 8.14 (s, 1H), 9.67 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz): δ (ppm) 13.6, 30.4, 53.4, 60.0, 99.3, 102.8, 121.2,
122.2, 129.7, 132.7, 146.0, 147.7, 149.1, 151.4, 164.7; HRMS
(APPI-QTOF) calcd for C15H14N4O5Se [M + H]: 411.0203; found
411.0208.
1-Methyl-4-(3-nitro-phenyl)-2-oxo-6-selenocyanatomethyl-
1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid ethyl ester
(1g). Slightly yellow solid, m.p. = 173 °C; IR (ν, cm−1): 3432,
3263, 2139, 1699, 1673; 1H NMR (DMSO-d6, 200 MHz): δ (ppm)
1.14 (t, J = 7.09 Hz, 3H), 3.30 (s, 3H), 4.07 (q, J = 7.09 Hz, 2H),
4.33 (s, 2H), 5.39 (d, J = 3.91 Hz, 1H), 7.42–7.61 (m, 2H), 7.95
(d, J = 3.67 Hz, 1H), 8.03–8.13 (m, 2H); 13C NMR (DMSO-d6,
100 MHz): δ (ppm) 13.7, 24.6, 29.5, 51.6, 60.4, 103.2, 103.8,
121.2, 122.3, 129.7, 132.5, 145.2, 147.7, 150.1, 152.6, 164.9;
HRMS (APPI-QTOF) calcd for C16H16N4O5Se [M + H]: 425.0359;
found 425.0364.
Iron chelating activity
Solutions of compounds (1 mL) in different concentrations
were evenly mixed with 0.05 mL FeCl2 (2 mM), and added to
0.2 mL ferrozine solution (5 mM). The mixtures were shaken
and left standing at room temperature for 20 min; the absor-
bance values (Asample) of the mixtures were measured at
562 nm. Methanol was used instead of the sample solution as
a blank control (Ablank) and Na2EDTA was used as a positive
control. Fe2+ chelating rate (%) = 100 × [(Ablank − Asample)/
Ablank].51
Acetylcholinesterase activity
The enzymatic activity was measured using an adaptation of
the method described by Mata et al. (2007). Briefly, 300 μL of
50 mmol L−1 Tris-HCl buffer, pH 8.0, 100 μL of a buffer solu-
tion containing the sample at five different concentrations dis-
solved in MeOH and 50 μL of an AChE solution containing
0.28 U mL−1 (50 mmol L−1 Tris-HCl, pH 8.0 buffer, 0.1% BSA)
were incubated for 15 min. Then, 75 μL of an acetylthiocholine
iodide solution (0.023 mg mL−1 in water) and 475 μL DTNB
(3 mmol L−1 in Tris-HCl, pH 8.0 buffer, 0.1 mol L−1 NaCl,
0.02 mol L−1 MgCl2) were added, and the final mixture was
incubated for another 30 min at room temperature. The absor-
bance of the mixture was measured at 405 nm. A control
mixture containing methanol instead of the sample was con-
sidered to have 100% AChE activity. The inhibition (%) was cal-
culated as follows: I(%) = 100 − (Asample/Acontrol) × 100 in which
Asample is the absorbance of the sample and Acontrol is the
absorbance without the sample. The tests were performed in
triplicate, and a blank containing Tris-HCl buffer was used
instead of the enzyme solution. The sample concentration
with 50% inhibition (IC50) was determined by plotting the
inhibition against the sample solution concentrations. Galan-
tamine was used as the positive control.
Determination of GPx-like activity
The experiments were carried out according to the Tomoda
method.40 The catalytic GPx model reaction was initiated by
the addition of H2O2 (final concentration: 10 mM) to a metha-
nol solution (final volume: 1 mL) of thiophenol (PhSH) (final
concentration: 5 mM) containing the selenium catalyst (final
concentration: 0.4 mM) at 25 ( 3) °C. The formation of PhSSPh
was monitored by UV spectrophotometry, at 305 nm. Absor-
bance versus time data were stored directly on a microcom-
puter. The reaction was followed for 6 minutes and three times
under the same conditions.
Conclusions
In summary, we designed, synthesized and evaluated a series
of novel seleno-DHPMs as potential multi-target therapeutics
for Alzheimer’s disease. The compounds showed excellent
activity as inhibitors of AChE, all being more active than the
standard drug. All compounds were assayed and showed very
good antioxidant activity through different mechanisms of
action. Their GPx mimetic activity, iron chelating activity, redu-
Total antioxidant capacity
Total antioxidant capacities of compounds were evaluated by cing power and total antioxidant activity were demonstrated.
the phosphomolybdenum method. This assay is based on the Moreover, some pharmacokinetic parameters were calculated
reduction of Mo(VI) to Mo(V) by the antioxidant compounds and all of the seleno-DHPMs investigated would have favour-
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