Ionic liquid-assisted synthesis of dihydropyrimidin(thi)one Biginelli adducts and investigation of their mechanism of urease inhibition

Article abstract of DOI:10.1039/c9nj03556g

Twenty-six Biginelli adducts were synthesized through an ionic liquid-assisted synthesis with up to 92% yield. Sixteen of these Biginelli adducts were then assayed to determine their antiureolytic activity against purified urease from jack beans. The substances BA7-S, BA9-S and BA11-S were shown to be as efficient inhibitors as hydroxyurea, a positive control used in in vitro screening assay against urease. Fluorescence studies revealed that BA7-S, BA9-S, BA11-S and BA5-S possessed high binding constant values of 5.95, 6.72, 4.55, and 4.28 M^-1, respectively, while BAS12-S, without substituents, showed a low value of log K_b = 2.16 M^-1. In addition, in the most thermodynamically favorable BA5-S and BA7-S urease complexes, the corresponding Biginelli adducts were capable of interacting with the active site of urease through non-ionic interactions, such as hydrophobic interactions, or hydrogen and van der Waals interactions, respectively. In silico studies also supported that the BAs interact with the active site, confirming the fluorescence and kinetic assay studies, which clearly indicate that BA5-S and BA7-S are competitive inhibitors (K_i = 0.96 and 0.57 mM, respectively). In silico studies also showed that the substituents in the aromatic ring interact with Ni atoms to form a stable complex.

Full text of DOI:10.1039/c9nj03556g

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de Aquino and Â. de Fátima, New J. Chem., 2019, DOI: 10.1039/C9NJ03556G.
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Ionic Liquid-assisted Synthesis of Dihydropyrimidin(thi)ones
Biginelli Adducts and Investigation of their Mechanism of Urease
Inhibition
Taniris Cafiero Braga,a Thamara Ferreira Silva,b Thamilla Maria Silva Maciel,c Edjan Carlos Dantas da
Silva,d Edeildo Ferreira da Silva-Júnior,d Luzia Valentina Modolo,b Isis Martins Figueiredo,c Josué
Carinhanha Caldas Santos,c Thiago Mendonça de Aquino,d Ângelo de Fátima,a*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Twenty-six Biginelli adducts were synthesized through an ionic liquid-assisted synthesis with up to 92% of yield. Sixteen of
these Biginelli adducts were then assayed to determine their antiureolytic activity against purified urease from jack bean.
The substances BA7-S, BA9-S and BA11-S showed to be as efficient inhibitors as hydroxyurea, a positive control used in vitro
screening assay against urease. The fluorescent studies revealed that BA7-S, BA9-S, BA11-S and BA5-S possessed high
binding constant values of logKb = 5.95, 6.72, 4.55, 4.28 M-1, respectively, while the BAS12-S, without substituents, showed
low value of logKb = 2.16 M-1. In addition, in the most thermodynamically favorable BA5-S and BA7-S ureases complexes,
the corresponding Biginelli adducts were capable of interact with the active sites of urease through non-ionic interactions,
such as hydrophobic interactions, or hydrogen and Van der Walls interactions, respectively. In silico studies also supported
that BAs display interaction in active site, confirming the fluorescence and for kinect studies which clear indicate that BA5-
S and BA7-S are competitive inhibitors (Ki = 0.96 and 0.57 mM, respectively). In silico studies also showed that, the
substituents in the aromatic ring are able to interact with Ni atoms to form a stable complex.
provides CO2 and ammonia.3,5–7 In addition, several human
pathogens, such as Mycobacterium tuberculosis, Yersinia
enterocolitica and Cryptococcus neoformans, possess ureases as
an essential virulence factor, leading to infections with urinary
stones, pyelonephritis, ammonia encephalopathies, hepatic
coma, and gastritis.6,8–10 Moreover, approximately 100
megatons of nitrogen (N) fertilizer, which is one of the most
used fertilizers worldwide, is mainly in the form of urea and
leads to the urease-driven hydrolysis of urea on the soil surface
yielding up to 70% N loss to the environment.11,12
Considering that the inhibition of ureases could
control/mitigate many problems associated with ureases, such
as human illness, agricultural production and environmental
issues, several new urease inhibitors have been developed, and
the understanding of how these new substances negatively
affect ureases has been pursued.4,13–18
Introduction
In 1926, James B. Summer reported a historical accomplishment
in biochemistry: the X-ray data for urease from jack beans - the
first enzyme ever to be examined this way.1,2 Since this pivotal
finding, other ureases have been discovered and isolated from
fungi, bacteria and other plants.1,3 Depending on the source,
altered subunits have been observed in different ureases.
However, the structures of the active sites near the nickel (II)
atoms are similar, and all ureases act by the same mechanism
for the catalytic hydrolysis of urea. Due to these similarities, an
inhibitor of one urease may also inhibit others.4
Urease catalyses urea hydrolysis, providing ammonium and
carbamate 1014 times faster than the uncatalyzed reactions.2,5
This enzyme is recognized as contributing to environmental
problems, such as acid rain, since carbamate spontaneously
Urease inhibitors include different classes of substances such as
flavonoids, quinolones, benzimidazoles, oxazoles, thiazolidines,
a. Grupo de Estudos em Química Orgânica e Biológica (GEQOB), Departamento de
Química, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
organophosphorus
compounds
and
dihydropyrimidinones/thiones (DHPMs, such as Biginelli
adducts).4,17–22 DHPMs are one of the most attractive
substances for developing new urease inhibitors since they can
be easily prepared through a multicomponent reaction (MCR).
The first Biginelli adducts were described by Pietro Biginelli in
1891, in which different 1,3-dicarbonyl compounds were
reacted with aldehydes and urea under the catalysis of HCl.
DHPMs, especially those derived from phenolic aldehydes
(Figure ), have shown good activities with IC50 values ranging
b.
Grupo de Estudos em Bioquímica de Plantas (GEBioPlan), Departamento de
Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,
Belo Horizonte, MG, Brazil.
c.
Laboratório de Instrumentação e Desenvolvimento em Química Analítica
(LINQA), Instituto de Química e Biotecnologia, Universidade Federal de Alagoas,
Maceió, AL, Brazil.
d. Núcleo de Análises e Pesquisa em Ressonância Magnética Nuclear (NAPRMN),
Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Maceió,
AL, Brazil.
23
*Corresponding author: Prof. Dr. Ângelo de Fátima: Tel.: +55-31-3409-6373. E-
mail: adefatima@qui.ufmg.br
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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from 5.4 to 34.7 µM in different studies.20,24,25 In addition to
expressive antiureolytic activities of DHPMs, there have been
few studies dedicated to elucidating the mechanism of action of
ureases for this class of substances.24,25
Herein, we describe the synthesis of 26 Biginelli adducts, the in
vitro screening for urease inhibitors of the synthesized
substances, the systematic studies of enzyme/substrate
interactions for these substances and the in silico studies for the
most potent urease inhibitors.
0
0
10 20 30 40
Catalyst / mol%
60
90
120
140
5
10
20
30
Temperature / °C
Reaction time / min
[BIMBS][Cl] (7) started with the promotion of the substitution
nucleophilic bimolecular reaction (SN2) between n-
bromobutane (1) and imidazole (2), in basic media, which
furnished the 1-butyl-1H-imidazole (3) in 69% yield.
Subsequently, 3 was treated with 1,4-butane sultone (5) to
provide the zwitterionic substance 6 in 98% yield. Finally,
treatment of 6 with HCl led to the ionic liquid [BIMBS][Cl] (7) in
68% overall yield (3 steps). The chemical characterization data
for the ionic liquid 7 are in accordance with those published
elsewhere. 32,33
Having the ionic liquid 7 in hand, our efforts then focused on the
use of this ionic liquid as a catalyst in the Biginelli reaction under
microwave irradiation and solvent-free conditions. The Biginelli
reaction of ethyl acetoacetate (8), 4-hydroxybenzaldehyde (9)
and urea (10-O) (molar ratio of 1.5:1.0:1.5, respectively), under
microwave irradiation (MW) and solvent-free conditions, was
used as a model reaction (Figure ). For the model reaction, it
was determined that 20 mol% of 7, at 90°C for 10 min under
microwave irradiation were the best conditions to provide the
compound BA4-O (71% yield) (Figure ). Notably, only traces of
the desirable products were formed when using conventional
heating or room temperature.
Results and discussion
Synthesis of the Biginelli adducts
Biginelli adducts can be obtained under the catalysis of different
Lewis or Brönsted acids, in the presence or absence of a protic
or an aprotic solvent, under conventional or microwave
heating.25–28 Examples of Lewis acids that have been used
include InCl3, SnCl3, FeCl3 and AlCl3,29 and some examples of
Brönsted acids include piperidine hydrochloride, sulfurous acid
(H2SO3), hydrochloric acid (HCl) and p-toluenesulfonic acid
(PTSA).30 Among the Brönsted acid catalyst, ionic liquids (ILs)
derived from imidazole have been recognized as efficient
catalysts for Biginelli reactions.
ILs are salts in the liquid state, which have melting point values
below an arbitrary temperature, such as 100 °C. ILs have a low
vapor pressure, which in turn makes this class of substances
interesting catalysts since their uses avoids the use of volatile
solvents. In addition, these salts are generally miscible in the
reaction media, and they are easily separated from the product,
for instance, by adding water to the reaction mixture.31 Based
on these advantages of ILs, we prepared the ionic liquid
[BIMBS][Cl] (7) (Figure ) and used it as a catalyst to prepare a
series of Biginelli adducts (BAs).The synthesis of the ionic liquid
After establishing the best reaction conditions for using 7 as the
catalyst in the Biginelli reaction, we tested different aldehydes
bearing
electron-withdrawing
and
electron-donating
substituents as well as one aliphatic aldehyde. Urea and
thiourea were also used, and 26 Biginelli adducts were
prepared; 13 BAs derived from urea (BA1-O to BA13-O) and 13
derived from thiourea (BA1-S to BA13-S) (Table 1). There was
no clear evidence that the presence of electron-withdrawing
and electron-donating substituents on the aromatic aldehydes
positively or negatively influenced the yields values for the
Biginelli reaction catalysed by 7. However, the average yields for
the aromatic aldehydes bearing electron-withdrawing
substituents (58% for urea derivatives and 62% for thiourea
derivatives) were slightly higher than those observed for the
aromatic aldehydes bearing electron-donating substituents
(55% for urea derivatives and 58% for thiourea derivatives)
(Table 1).
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Table 1. Scope of Biginelli reaction under the catalysis of ionic liquid [BIMBS][Cl] (7)a.
Effects of Biginelli adducts on urease activity
View Article Online
The criteria for choosing the Biginelli aDdOduI:c1t0s.10th39a/tC9wNoJu03ld55b6Ge
tested for their potential to inhibition of Canavalia ensiformis
(jack bean) urease relied on the selection of a representative
group of compounds bearing electron donating or withdrawing
substituents that were also soluble in ethanol.
Yield
(%)
Yield
(%)
BA
Structure
BA
Structure
BA1-O
26
52
39
BA1-S
47
46
25
The activity of jack bean urease is not affected by 10% ethanol,
26
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while DMSO and other organic solvents that are required to
BA2-O
BA3-O
BA2-S
BA3-S
dissolve substances with limited water solubility considerably
inhibit the ureolytic activity of jack bean type III urease (data not
shown).
The synthesized Biginelli adducts that contain a thiourea moiety
were usually more soluble in ethanol than their corresponding
urea derivatives. The in vitro screening revealed that the tested
Biginelli adducts were classified into four categories according
to their potential to inhibit urease (Figure ; Scott Knott test; P <
0.05). Therefore, the BA7-S, BA9-S and BA11-S were included in
group A because they were as efficient as the reference
inhibitor hydroxyurea (48.5% inhibition on average). BA1-S,
BA5-S, BA6-S and BA8-S were placed into group B and
effectively inhibited jack bean urease by 36% on average. Group
C included BA1-O, BA3-S, BA10-S and BA12-S (average
inhibition of 22.5%) while group D was constituted of
compounds that poorly inhibited the enzyme (BA2-O, BA9-O –
BA12-O; 14.3% inhibition on average). These results agree with
those reported elsewhere, in which Biginelli adducts containing
a thiourea core were much better urease inhibitors than those
bearing urea core.20,24 Additionally, the presence of electron
donating or electron withdrawing substituents at the para
position led to good antiureolytic activity of the Biginelli
adducts.
BA4-O
71
BA4-S
52
BA5-O
BA6-O
73
55
BA5-S
BA6-S
81
92
BA7-O
61
BA7-S
60
BA8-O
BA9-O
28
69
BA8-S
BA9-S
30
67
Inhibitors BA7-S, BA9-S and BA11-S (from group A) were further
used to study the interactions with jack bean urease. BA5-S and
BA12-S were also chosen for this such study since the former is
a representative of group B, while the latter is an example of a
low potency urease inhibitor from a thiourea series that lacks a
substituent on the phenyl ring (Table 1, Figure ).
BA10-O
BA11-O
74
70
BA10-S
BA11-S
81
78
80
a
60
a
a
b
a
b
b
40
20
0
b
c
c
d
c
c
BA12-O
BA13-O
68
43
BA12-S
BA13-S
85
4
d
d
d
d
S
S
S
S
S
S
BA9-
S
O
O
O
-S
-S
-S
-O
-O
-O
HU
BA1-
BA2-
BA3-BA5-BA6-BA7-BA8-
BA9-
BA1-
BA10
BA11 BA12
BA12
BA10
BA11
aReagents and reaction conditions: Aldehyde, ethyl acetoacetate, urea or thiourea
(1:1.5:1.5, respectively) and IL (20 mol%) under microwave irradiation [MW (power max
250 W, 90°C, holding time: 2 min and run time: 10 min)] in a DISCOVER CEM®.
This journal is © The Royal Society of Chemistry 20xx
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Urease (U,
1 M)
Interaction between Biginelli adducts and jack bean urease
investigated by molecular fluorescence
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DOI: 10.1039/C9NJ03556G
U
U
U
U
U
U
U
U
+
+
+
+
+
+
+
+
BA7-S 2.5 M
BA7-S 5.0 M
BA7-S 10 M
BA7-S 15 M
BA7-S 20 M
BA7-S 30 M
BA7-S 40 M
BA7-S 50 M
A
B
450
300
150
0
0.4
Interaction mechanism and thermodynamic parameters
BA7-S (free) 50 M
-0.0
-0.4
-0.8
-1.3
-1.6
The interaction studies between compounds BA5-S, BA7-S,
BA9-S, BA11-S and, BA12-S and urease were carried out to
understand the mechanism of enzyme inhibition exhibited by
selected Biginelli adducts. Urease from C. ensiformis (jack bean)
contains four Trp residues at positions 495, 648, 708, and 728;
BA5-S
BA7-S
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-5.8 -5.5 -5.2 -4.9 -4.6 -4.3 -4.0
295 315 335 355 375 395 415
which at physiological pH are the main fluorophores (
 = 0.20)
log[BA5-S] or log[BA7-S] / M
/ nm
in the enzyme, compared with Tyr ( = 0.14) and Phe (

 =
8
6
4
2
0
3.0
2.5
2.0
1.5
1.0
D
0.04),27 in native proteins, Tyr emission is often quenched,
presumably by its interaction with the peptide chain or by
energy transfer to Trp.28 Therefore, urease intrinsic
fluorescence is mainly a result of Trp residue contribution,
which can them be used as a fluorophore for the studies of
protein-ligand interactions and ligand-induced conformational
changes around a binding site.29
C
BA5-S
BA7-S
The interaction process was evaluated using steady-state
spectrofluorimetric titration based on the intrinsic urease
fluorescence. The fluorescence spectra of urease in the absence
and presence of increasing amounts of BA7-S are shown in
Figure A. Similar profiles were obtained for other compounds.
Upon excitation at 280 nm, urease presented a wide, intense
band at 335 nm (Figure ). After the addition of BA7-S to the
system, there was a decrease in the fluorescence intensity and
variation of the maximum emission wavelength from 335 to 345
nm. The signal intensity reduction and the redshift of the
maximum wavelength was attributed to conformational
changes in the structure of the enzyme.30 To evaluate the
interaction strength between urease and the Biginelli adduct
selected, the binding constant (Kb) was calculated:
0
5
10 15 20 25 30
S
S
S
9-
-S
11
-S
12
BA
5-
7-
BA
BA
BA
[BA5-S] or [BA7-S] / M
BA
Compounds
Figure 5. Interactions between jack bean urease and BA5-S or BA7-S. Values (B – D) are the
means (n = 3) ± standard deviation. (A) Emission spectral profile of urease (1.0 μM) in the
presence of 2.5 ‒ 50 M BA7-S at pH 7 and 30 °C; (B) Double logarithmic curve to determine
the binding constant (Kb) for BA5-S and BA7-S; (C) Binding constants of all tested Biginelli
adducts in relation to jack bean urease; (D) Stern-Volmer quenching plot for BA5-S and
BA7-S as a function of BAs concentration.
at 30°C) and Cu(II) (3.89×105 M-1 at 37°C when [Cu(II)] < 16
32
μM) 33, and higher than pentachlorophenol (3.85×103 M-1 at
32°C) 27 and Cr(VI) (1.96×104 M-1 at 29°C). However, the authors
of these studies did not evaluate the potential of
pentachlorophenol, Cr(VI) (K2Cr2O7) and Cu(II) as urease
inhibitors. According to equation 1, n corresponds to the
number of occupied sites by the ligand in the urease structure.
The values of n varied from 1.07 to 1.38, indicating that the
interactions among these ligands and urease occurred at a ratio
of 1:1.
(F - F)






0
log
=logKb +nlog[Q]
equation (1)
F
where F0 and F are the respective fluorescence intensities in the
absence and presence of the ligand, Kb is the binding constant
and n is the number of binding sites in the enzyme.31 Figure B
shows the respective linearization of equation 1 to calculate the
binding parameters related to Kb and n, using the compounds
BA5-S and BA7-S.
To evaluate the affinity of the selected Biginelli adducts against
urease, the respective binding constant values were compared.
Based on Figure C, compounds BA5-S and BA7-S show the
highest Kb values, hence, these compounds were employed as
models for the mechanistic enzyme inhibition studies. The
results of this study are presented in Table 2 for the
temperatures 23, 30, and 37 °C. In addition, BA20-S presented
the lowest enzyme affinity, corroborating the results of the
urease inhibition experiments (Figure ).
The binding constant (Kb) expresses the strength of the
interaction between a ligand and a protein and for the Biginelli
adducts, it was found that the interaction between BA7-S and
urease was stronger than that of BA5-S and urease (Table 2).
The Kb values for these Biginelli adducts toward the enzyme
were in the same range as those reported for the interaction
between jack bean urease and (R)-(+)-usnic acid (3.55×106 M-1
To evaluate the interaction mechanism between urease and the
Biginelli adducts, the Stern-Volmer equation was applied:
F
0
1Kq0[Q] 1KSV[Q]
equation (2)
F
where Kq is the diffusional bimolecular velocity quenching
constant, τ0 is the average life time (10-8 s), [Q] is the
concentration of the ligand (quencher), and KSV is the Stern-
Volmer quenching constant.34 Due to the elevation of the
diffusion coefficient and a greater number of collisions between
the fluorophores (protein) and the quenchers, a dynamic
quenching process would be observed if the KSV values increases
with temperature. An opposing profile was observed in the
static quenching process, since at higher temperatures, the
stability of the protein-ligand complex is affected. According to
these results (Table 2), the values of the quenching constant
(KSV) decreased with increasing temperature for both ligands.
Thus, the interaction process between BA5-S and BA7-S and
urease occurred preferentially through a static quenching
mechanism with the formation of a supramolecular enzyme-
ligand complex.35 Additionally, all Kq values were calculated to
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1012 M-1 s-1 , confirming that static deduction of the kind of forces involved in the enzyme-inhibitor
1
2
3
4
be greater than 2.0
quenching occurred.36
View Article Online
interaction (Table 2). The Gibbs free enerDgyOI(: 10.1039/C9NJ03556G
G) was calculated
The binding of small molecules to proteins usually occurs according to equation 4.
through noncovalent interactions, thus, the thermodynamic
parameters were obtained using the linearized Van’t Hoff
equation (equation 3) from interaction assays performed at
different temperatures. These parameters permit the
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Table 2. Binding and thermodynamics parameters for urease interaction with Biginelli’s adduct BA5-S and BA7-S.
Stern-Volmer constant
r
Binding constant parameters
Thermodynamic parameters
Compound
BA5-S
T (°C)
KSV
(104 M-1)
Kq
Kb
G
(kJ mol-1)
H
(kJ mol-1)
S
n
r
(1012 M-1 s-1)
(106 M-1)
(J mol-1 K-1)
22
30
38
22
30
38
4.96 ± 0.14
4.70 ± 0.10
4.46 ± 0.06
9.11 ± 0.26
8.99 ± 0.15
8.30 ± 0.09
0.9872
0.9853
0.9887
0.9861
0.9779
0.9891
4.96
4.70
4.46
9.11
8.99
8.30
0.66 ± 0.03
0.91 ± 0.10
1.20 ± 0.15
8.49 ± 0.46
5.27 ± 0.52
3.29 ± 0.37
1.36 ± 0.04
1.22 ± 0.05
1.07 ±0.03
1.29 ± 0.02
1.23 ± 0.06
1.38 ± 0.02
0.9862
0.9944
0.9873
0.9971
0.9873
0.9962
-32.88
-34.54
-36.20
+28.37
-45.19
+207.6
-20.50
-39.14
-38.98
BA7-S
-38.91
emission energy is transferred to the receiver). This critical
distance (R0) is mathematically described as:
ΔH
R
1
ΔS
R
 
logKb = -

+
equation (3)
 
T
 
R60 = 8.79×10-25K2N-4J
equation (6)
ΔG = ΔH - TΔS
equation (4)
where K2 is the dipole orientation of the donor and the receptor,
N is the mean refractive index,
 is the quantum yield of the
where T is the temperature in Kelvin (K) and R is the ideal gas
constant. Figure S1 (Supporting Information) shows a graphical
representation of the linearized Van’t Hoff equation.
Considering the negative values of ∆G obtained (Table 2) the
evaluated interaction process was thermodynamically
spontaneous. The main relationship between the forces
involved in the interaction process, and the enthalpy and
protein fluorescence, and J represents the overlapping area of
the donor's normalized emission spectrum in relation to the
receptor absorption spectrum. In this case, K2 = 2/3, N = 1.336
and
by the equation:

= 1.50 for urease.33 The overlap area (J) can be calculated
0
F()()4d
entropy variation values are as follows:
(hydrophobic forces); H < 0 and S > 0 (electrostatic forces),
S < 0 (hydrogen bonds and Van der Waals
H > 0 and S > 0
J =
equation (7)
0


F()d
and

H < 0 and
37

forces). The predominant binding forces (nonexclusive) for
Biginelli adduct BA5-S were hydrophobic interactions, and for
BA7-S the predominant forces were hydrogen bonds and Van
der Waals forces.
In this equation, F(λ) is the fluorescence of the donor and ε(λ) is
the molar absorption coefficient of the receptor, which can be
obtained through Beer’s law.38 Initially, the overlap area
between the fluorescence spectra of free urease and the
molecular absorption spectra of BA5-S and BA7-S were
evaluated (Fig. S2, Supporting Information). The calculated
parameters for the FRET process are shown in Table 3.
Förster resonance energy transfer (FRET)
FRET studies were employed to determine the distance
between the urease tryptophan residues (donor) and Biginelli
adducts (receptors) in the process of energy transfer due to
interaction. The distance (r0) between the donor and the
receptor can be calculated using equation 5:
Table 3. FRET parameters for urease interaction with Biginelli adducts BA5-S and BA7-S.
Enzyme and Biginelli adducts at 5 M.
Compound
BA5-S
J (10-15 cm3 M-1)
E (%)
34
R0 (nm)
2.16
r0 (nm)
2.42
4.0
7.5
R06
BA7-S
44
2.40
2.50
equation (5)
F
E =1-
=
F
R60 + r6
0
0
According to the results (Table 3), the energy transferred (E)
was less than 50%, implying that R0 < r0; and the values of the
distances of r0 for BA5-7 and BA7-S are similar. The critical
distances r0 between the Biginelli adducts and Trp residues (in
urease) were less than 8 nm, indicating that the energy transfer
where F0 and F are the fluorescence intensity of the donor in
the absence and presence of the ligand; respectively, E is the
fraction of energy transferred from the donor to the receptor,
and R0 is the critical distance (when 50% of the donor's
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Figure 6. Tridimensional fluorescence spectra of urease (A), BA5-S-urease complex (B) e BA7-S-urease complex (C) at pH 7. Enzyme and ligands were used at 1.0 and 40 µM,
respectively.
occurred after the interaction process 39. Once the evaluated Synchronous fluorescence studies were performed to observe
compounds interacted effectively with the urease, we assessed the preferential interactions and changes in the urease
whether the interaction process led to changes in the fluorophore (Tyr and Trp) microenvironment. Differences
conformation of the enzyme.
between the excitation and emission wavelengths were used as
parameters to evaluate polarity changes in the amino acid
residues, since a ∆λ = 15 nm (Figure S3A and C, Supporting
Information) provides information about the Tyr residue, while
a ∆λ = 60 nm (Figure S3B and D, Supporting Information) shows
changes in the Trp residue.42 The synchronous fluorescence
binding parameters for BA5-S and BA7-S towards urease are
shown in Table 4.
The maximum wavelength emission shifts for Biginelli adducts
BA5-S and BA7-S towards urease were higher for Trp than for
Tyr (Table 4). This is based on the fact that Tyr residues are less
sensitive to medium polarity changes when compared to Trp.
An increase in the microenvironment polarity is related to a
positive variation in max, while polarity reduction is associated
Evaluation of the conformational changes of urease
The three-dimensional fluorescence spectrum allows more
detailed information about the typical conformational changes
in the secondary structure of a protein to be obtained 40. The
three-dimensional fluorescence spectra for urease by itself or
complexed with BA5-S and BA7-S are presented in Figure . The
three-dimensional fluorescence spectra of free urease and
complexed urease show peak 1, which corresponds to Rayleigh
scattering, which is characterized by irradiation re-emission (ex
= em) from water (solvent). Peak 2 corresponds to the emission
of the Trp and Tyr residues while the emission of the
polypeptide backbone in the urease structure is represented by
peak 3.31,40 The fluorescence intensities of peaks 2 and 3 were
reduced by 53 and 60%, respectively, after the addition of BA5-
S (Table S1, Supporting Information). A similar spectral profile
was recorded for the systems containing BA7-S; a 72 and 79%
reduction in the fluorescence intensity was observed for peaks
2 and 3, respectively. The main parameters of tridimensional
fluorescence are listed in Table S1.
with a negative variation in max. These phenomena allow us to
describe changes in the structure of native proteins
Table 4. Synchronous fluorescence binding parameters for Biginelli’s adducts BA5-S and
BA7-S towards urease.
Stern-Volmer
parameters
KSV (104 M-1)
max

(nm)
Compound
emission shift
(nm)1
r
The reduction in fluorescence intensity corresponding to peak 2
indicated changes in theTrp and Tyr microenvironment, while
alterations related to peak 3 indicated modifications to the
native protein structure. The most significant variations in
fluorescence intensity were observed for peak 3, indicating that
alterations in the polypeptide chain and enzyme folding.41 In
addition, these results are supported by the variation of the
Stokes shift determined for native urease and the respective
inhibitor-enzyme complexes (Table S1).
15
60
15
60
4.24 ± 0.08
2.91 ± 0.04
10.21 ± 0.12
6.49 ± 0.09
0.9691
0.9880
0.9656
0.9705
-1
+6
+1
+4
BA5-S
BA7-S
1Free urease was used as reference. The error represents the SD for the three
determinations.
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Journal Name
to the presence of Tyr at positions 410 and 544 near the key His
residues (positions 409 and 545) responsible for coordinating to
Ni atoms in the jack bean urease active site.43 Considering that
the Trp residues (495 and 648) are far away from the urease
catalytic site, the Tyr residues had their microenvironment
changed more significantly than Trp.32 Therefore, the Biginelli
adducts BA5-S and BA7-S interact with the urease catalytic site.
ARTICLE
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2.5
0.2 mM
A
DOI: 10.1039/C9NJ03556G
C
I-free
0.1 mM
I-free
2.0
0.1 mM
0.2 mM
1.5
1.0
0.5
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-0.3
0.0
0.3
0.6
0.9
1.2
0
5
10 15 20 25 30 35 40
Competition assay
1/[Urea]
Urea (mM)
The effect of some classical inhibitors [N-butyl thiophosphoric
triamide (NBPT), thiourea, hydroxyurea and omeprazole] on the
interaction of urease with Biginelli adducts BA5-S and BA7-S
was evaluated by competition assay. The urease binding
constant ratio (Kb’/Kb), in which Kb’ and Kb refer to as the binding
constant in the presence or absence of a competitor,
respectively, was used for comparison purposes. The formation
of urease-ligand complexes is favored when Kb’/Kb > 1 while
ratios lower than 1 indicate that complex formation is
disfavored (Table 5).
5
2.5
B
0.2 mM
D
4
3
2
1
0
I-free
2.0
1.5
1.0
0.5
0.1 mM
I-free
0.1 mM
0.3 mM
-0.3
0.0
0.0
0.3
0.6
0.9
1.2
0
5
10 15 20 25 30 35 40
1/[Urea]
Urea (mM)
Figure 7. Representative Michaelis–Menten hyperbola (A and B) and Lineweaver-Burk (C
and D) plots for jack bean urease in the presence of BA5-S (A and C) or BA7-S (B and D).
Increasing concentrations of urea (1-32 mM) were incubated for 10 min with jack bean
type III urease in the absence (I-free) or presence of inhibitor (0.1 to 0.3 mM). I-free, free
of urease inhibitor.
Table 5. Urease binding constants ratio in the absence (Kb) or presence (Kb’) of urease
inhibitors.
Competitors (Kb’/Kb)a
Compound
NBPT
Omeprazole
0.40 ± 0.10
0.53 ± 0.03
Thiourea
0.66 ± 0.07
0.53 ± 0.02
Hydroxyurea
0.31 ± 0.03
0.33 ± 0.02b
BA5-S
BA7-S
0.32 ± 0.08
0.46 ± 0.02
source not found.). Additionally, the KM app for urea increased
with increasing of urease inhibitor concentrations in the
reaction medium. Moreover, both BA5-S and BA7-S can
compete with urea for the active site of jack bean urease to
form urease-BA5-S and urease-BA7-S complexes, respectively.
The Lineweaver-Burk plots (Figure - C and D) allowed for the
calculation of the equilibrium dissociation constants for such
complexes (Ki). The Ki value for the urease-BA5-S complex was
determined to be 0.96 ± 0.01 mM while this same value for the
urease-BA7-S complex was 0.57 ± 0.16 mM. These results
indicate that the Biginelli adduct BA7-S is a more potent
inhibitor of jack bean urease than BA5-S. Because the inhibitor
affinity for the enzyme is inversely proportional to Ki, the
kinetics results are in accordance with those obtained from the
molecular fluorescence experiments (Table 2).
aKb’ = binding constant in the presence of inhibitor (25 µM), urease (1 µM) varying
Biginelli adducts (2.5 - 50 µM). bHydroxyurea inhibitor was used at 38 µM. The error
represents the SD for the three determinations.
In general, the binding constant decreases in the presence of
inhibitors (Kb’/Kb < 1), indicating that Biginelli adducts BA5-S and
BA7-S interact with the urease active site. This result is in
agreement with the results of the synchronous fluorescence
assays (Table 4). Thiourea, hydroxyurea and NBPT are
competitive inhibitors,44 while omeprazole is classified as a
noncompetitive inhibitor by reacting with the free Cys592
residue of jack bean urease, which causes blockage of the
access of urea to the enzyme catalytic site.45 Additionally, the
affinity of BA5-S and BA7-S to the urease catalytic site was
confirmed by UV-vis spectroscopic tests, which showed
interaction between these Biginelli adducts and Ni(II) ions in
solution (Figure S4). Finally, the results presented herein show
that BA5-S and BA7-S interact with the urease active site,
however, competition assays are limited to differentiate
between competitive and mixed inhibitors. Thus, to
unequivocally assess the type of inhibitor, we applied a classic
enzymatic kinetics assay.
Molecular Dynamics and Docking simulation
For structural validation and stereochemical quality of the jack
bean urease, the refined model was confirmed by
Ramachandran plot analysis. It revealed that 89.2% of the
amino acid residues are located in the most favored region,
9.1% are located in the allowed region, and only 1.7% is located
in the outlier region (Figure Error! Reference source not
found.). Additionally, the RMSD graphic was generated for Cα
atoms, showing that the system reaches the equilibrium phase
after 20 ns (Fig. S5, Supporting Information). Thus, the
generated model of jack bean urease was geometric and
stereochemically acceptable and the stabilized structure was
used as the starting point for molecular docking studies.
Kinetics assay
BA5-S, which possesses an electron donor substituent at the
meta position, and BA7-S, which bears an electron withdrawing
group at the para position, were chosen to determine the
mechanism by which they inhibit urease activity. Both
compounds were found to be typical competitive inhibitors
because the urease Vmax was not affected by the presence of
increasing concentrations of BA5-S or BA7-S (Error! Reference
For the docking protocol validation, a self-docking experiment
was performed, where the co-crystallized acetohydroxamic acid
(HAE) was removed from the ligand-binding domain of the jack
bean urease, and then a flexibly re-docked was performed. The
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to the formation of hydrogen bonding interactions between
View Article Online
His492, Gly550, and Arg609 with carbonyl oDr OthI:i1o0c.1a0r3b9o/Cny9Nl gJ0r3o5u5p6sG.
In addition, the aryl ring is involved in π-π stacking and anion-π
interaction with His519 and Asp633
, respectively (Error!
Reference source not found.B). Binding modes of compound
BA5-S is shown in Figure 3A, where hydrogen bonding
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Figure 10. Interactions of BA5-S (A) and BA7-S (B) with Ni metals and residues of
jack bean urease. C - Comparison between the docked conformations of the most
active compound BA7-S and weak urease inhibitor BA10-S in the catalytic cavity
of the enzyme. Compound BA7-S (blue). Compound BA10-S (pink). The binding
conformation of the Biginelli adducts are shown in stick representation and the
two Ni metals are represented by green spheres. Coordination with Ni metals,
hydrogen bonding, π-π stacking, anion-π, and sulfur-π interactions are shown with
dotted lines.
interactions were observed between carbonyl and NH groups
with Arg605 and Glu492 residues, respectively. Additional sulfur-
π interactions were observed between the thiourea group and
His492 and Asp494 residues. In general, BA5-S and BA7-S
derivatives display interactions in the catalytic site similarly to
the others active Biginelli adducts described in the literature
20,46. In addition, an in silico study carried out with a less active
thiourea derivative bearing p-F at the phenyl ring (BA10-S)
revealed that such Biginelli adduct also anchors to the urease
catalytic cavity. However, the lack of coordination with Ni
metals prevents the formation of a stable ligand-enzyme
complex, which appears to be responsible for its lower
inhibitory activity on urease (Error! Reference source not
re-docked HAE-enzyme complex revealed the same ligand’s
coordination with two nickel ions via hydroxyl oxygen, when
compared with the crystallographic complex (Figure A). RMSD
value obtained by comparison between the crystallized and re-
docked ligand was computed as 0.737 Å, suggesting that this
virtual protocol is effective for the analysis of binding modes of
the Biginelli adducts. In Figure B, it is shown the superposition
of co-crystallized and re-docked HAE.
The docking study performed with the active compounds BA5-
S and BA7-S revealed a similarity in the fitting-in mechanism at
the same active site when compared with HAE. For both
compounds, was observed that the orientation of aryl ring plays
an important role in the stable complexes formation, due to the
methoxy (Figure A) or nitro coordination (Figure B) with the bi-
nickel center of the enzyme. In addition, in both BA5-S and BA7-
S complexes, Ni ions are chelated with carbamylated Lysine
KCX490 residue.
20
found.C) Finally, the theoretical studies were in agreement
with the experimental data.
Conclusions
Twenty-six BAs were successfully synthesized (yields of up to
92%) by using [BIMBS][Cl] as catalyst. BA7-S, BA9-S and BA11-S
were as effective as hydroxyurea to inhibit urease activity. In
silico tests suggest that the aromatic substituents in the BA
structures form complexes with the nickel atoms present in the
urease active site. Sulfur-π interactions between the thiourea
group and the residues His492 and Asp494 of urease contribute to
the potency of such urease inhibitors. All these theoretical
studies are supported by fluorescence (competition, 3D and
synchronous) and the kinetic assays that revealed high binding
constants (> 4) towards the jack bean urease active site for BA5-
The analysis of the predicted binding conformation of the most
active compound BA7-S showed that further stability was due
8 | J. Name., 2012, 00, 1-3
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S and BA7-S with mechanisms of action typical of competitive 28.6 (CH2), 31.2 (CH2), 48.5 (CH2, CH2), 50.4 (CH2),122.4 (CH, CH),
inhibitors. The in silico studies suggest that slight modifications 136.1 (CH). All data for 6 are in accordance with those reported
on the aromatic rings of the BAs can modulate their ability to elsewhere.48
View Article Online
DOI: 10.1039/C9NJ03556G
inhibit urease.
1-Butyl-3-(4-sulfobutyl)-1H-imidazol-3-ium
chloride
{[BIMBS][Cl]} (7): 4-(1-Butyl-1H-imidazol-3-ium-3-yl)butane-1-
sulfonate (6) (3.8 mmol) was dissolved in an aqueous solution
of HCl (37%, 3.8 mmol). The solution was stirred under reflux for
6 h, and then the water was evaporated under vacuum. 1-Butyl-
3-(4-sulfobutyl)-1H-imidazol-3-ium chloride (7) was obtained as
8
9
General experimental procedures
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The reagents used were provided by commercial sources and
were of analytical grade. For reaction monitoring, thin-layer
chromatography (TLC) Polygram-UV2540, mm MACHEREY-
NAGEL (20 x 20 cm) was used. Chromatographic purification
was performed using 100-200 mesh silica gel. The melting point
was recorded by Gehaka-PF1500 apparatus, and the observed
values were not corrected. All infrared spectra were acquired
using a Spectro One Perkin Elmer or Bruker model Alpha
spectrometer at 400-4000 cm-1. Samples were prepared using
KBr plate or ATR method. NMR was recorded with a Bruker
AVANCE DPX 200 or 400 spectrometer with a BBO multinuclear
probe.
a viscous liquid in 100% yield. IR (ATR,
1162. H-NMR (200 MHz, DMSO-d6)


cm-1): 3144, 2963, 1647,
1
0.88 (3H, t, J = 7.4 Hz),
1.24 (2H, sex, J = 7.4 Hz), 1.54 (quin, 2H, J = 7.5 Hz), 1.69-1.96
(4H, m), 2.54 - 2.57 (2H, m), 4.14 - 4.23 (4H, m), 7.83 (2H, s),
9.34 (1H, s). 13C-NMR (50 MHz, DMSO-d6)
 13.3 (CH3), 18.7
(CH2), 21.6 (CH2), 28.6 (CH2) 31.2 (CH2), 48.6 (CH2), 50.5 (CH2),
122.5 (CH, CH), 136.1 (CH).
General procedure for the synthesis of Biginelli adducts with the
catalyst [BIMBS][Cl] (7)
A mixture of ethyl acetoacetate (8) (1.5 mmol), an aldehyde (1
mmol), urea (10-O) (1.5 mmol) or thiourea (10-S) (1.5 mmol)
and 7 (0.2 mmol – 20 mol%) was heated under microwave
irradiation (MW) in a DISCOVER CEM® reactor (conditions: 90°C,
200-250 W, 10 min). Subsequently, the reaction mixture was
dissolved in hot ethanol (~50°C), and then water was added
until the solution appeared muddy. The mixture was
magnetically stirred for 10 min, and the solid obtained was
filtered and dried under vacuum. Some Biginelli adducts
required purification by silica gel column chromatography.
Synthesis and characterization
Synthesis of the ionic liquid [BIMBS][Cl] (7)
1-Butyl-1H-imidazole (3): n-Bromobutane (2.4 mmol) (1),
imidazole (3.0 mmol) (2) and KOH (6.0 mmol) were added to
acetonitrile (5.0 mL). The reaction mixture was stirred and
refluxed for 4 h. After this period, the reaction mixture was
dissolved in dichloromethane (20 mL) and extracted with
distilled water (320 mL). The organic layer was collected and
dried over magnesium sulphate, and the solvent was removed
using rotatory evaporation. The obtained crude reaction
product was then purified using silica gel column
Ethyl
4-(4-hydroxy-3,5-dimethoxyphenyl)-6-methyl-2-oxo-
chromatography,
and
a
mixture
of
ethyl
1,2,3,4-tetrahydropyrimidine-5-carboxylate (BA1-O): 26% yield
(purified by precipitation). IR (KBr, cm-1): 3614, 3232, 3102,
acetate:hexane:dichloromethane (6:1:1) was used as the
eluent. 1-Butyl-1H-imidazole (3) was obtained as a yellow liquid
1
2952, 1726, 1708, 1654, 1120. M.p. = 182.3-183.4 °C. H-NMR
1
in 69% yield. IR (ATR,
MHz, CDCl3)
1.69 (2H, quin, J = 7.2 Hz), 3.86 (2H, t, J = 7.2 Hz), 6.83 (1H, s),
6.97 (1H, s), 7.43 (1H, s). 13C-NMR (50 MHz, CDCl3)
13.4 (CH3),

cm-1): 3111, 2960, 1667. H-NMR (200
(200 MHz, DMSO-d6) 1.12 (t, 3H, J = 6.5 Hz), 2.24 (s, 3H), 3.70
(s, 6H), 4.01 (q, 2H, J = 6.5 Hz), 5.08 (s, 1H), 6.48 (s, 2H), 7.65 (s,

0.86 (3H, t , J = 7.3 Hz), 1.24 (2H, sex, J = 7.3 Hz),
1H), 8.32 (s, 1H), 9.13 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)


14.2 (CH3), 17.7 (CH3), 53.8 (CH), 56.0 (CH3), 59.1 (CH2), 99.4 (C),
103.9 (CH), 135.0 (C), 147.8 (C), 152.2 (C), 165.5 (C). All data for
BA1-O are in accordance with those reported elsewhere.49
19.7 (CH2), 33.0 (CH2), 46.8 (CH2), 188.8 (CH), 128.9 (CH), 136.9
(CH). HRMS (ESI): m/z observed: 125.1026; C7H12N2 [M+H]+
calculated: 125.1079; error (ppm): 4.2. All data for 3 are in
accordance with those reported elsewhere
47
.
Ethyl 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (BA1-S): 47% yield
4-(1-Butyl-1H-imidazol-3-ium-3-yl)butane-1-sulfonate (6)
:
1-
(purified by precipitation). IR (KBr,
2946, 1668, 1648, 1186, 1112. M.p.: 2013.0-214.3°C. H-NMR

cm-1): 3476, 3330, 3198,
Butyl-1H-imidazole (3) (1.12 mmol) and 1,4-butane sultone
(1.21 mmol) (5) were added to toluene (0.5 mL). The reaction
mixture was stirred at 80°C for 24 h. Subsequently, the reaction
mixture was cooled down to room temperature, and ethyl ether
was slowly added with magnetic stirring until a white solid
precipitated. The solid material was filtrated and dried under
vacuum, providing 6 in 78% yield. IR (ATR,
1648, 1175. M.p. = 76.8-79.3 °C (Lit. M.p. = 136°C) 56. H-NMR
(200 MHz, DMSO-d6) 0.86 (3H, t, J = 7.3 Hz), 1.24 (2H, sex, J =
1
(200 MHz, DMSO-d6)
 1.13 (t, 3H, J = 7.0 Hz), 2.28 (s, 3H), 3.71
(s, 6H), 4.04 (4, 2H, J = 7.0 Hz), 5.11 (d, 1H, J = 2.9 Hz), 6.46 (s,
2H), 8.42 (s, 1H), 9.58 (s, 1H), 10.29 (s, 1H). 13C-NMR (50 MHz,
DMSO-d6)
 14.1 (CH3), 17.1 (CH3), 53.8 (CH), 56.0 (CH3), 59.6
(CH2), 101.0 (C), 104.0 (CH), 133.7 (C), 135.4 (C), 144.7 (C), 147.9
(C), 165.3 (C), 174.3 (C). All data for BA1-S are in accordance
with those reported elsewhere.49

cm-1): 3125, 2962,
1
7.3 Hz), 1.53 (2H, quin, J = 7.0 Hz), 1.69-1.92 (4H, m), 2.42-2.46
(2H, m), 4.13-4.22 (4H, m), 7.82 (2H, brs), 9.28 (1H, brs). 13C-
NMR (50 MHz, DMSO-d6)  13.3 (CH3), 18.8 (CH2), 21.8 (CH2),
Ethyl
4-(3-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (BA2-O): 52% yield
(purified by precipitation). IR (KBr,

cm-1): 3514, 3354, 3246,
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2980, 1726, 1700, 1602, 1192. M.p.: 183.7-185.0 °C (Lit. M.p.: 53.8 (CH), 59.5 (CH2), 100.1 (C), 115.4 (CH), 127.8 (CH), 135.8
View Article Online
190-192 °C).50 1H-NMR (400 MHz, DMSO-d6) 1.11 (t, 3H, J = 7.0 (C), 148.1 (C), 152.6 (C), 156.9 (C), 165.8 D(CO)I.: 1A0l.l1d03a9t/aCf9oNrJ0B3A5456-OG

Hz), 2.23 (s, 3H), 3.99 (q, 2H, J = 7.0 Hz), 5.06 (d, 1H, J = 2.7 Hz), are in accordance with those reported elsewhere.53
6.62 (d, 1H, J = 7.9 Hz), 6.66-6.67 (m, 2H), 7.08 (t, 1H, J = 7.9 Hz),
7.69 (s, 1H), 9.16 (s, 1H), 9.36 (s, 1H). 13C-NMR (100 MHz, DMSO- Ethyl
d6)
(CH), 114.2 (CH), 116.9 (CH), 129.3 (CH), 146.3 (C), 148.1 (C), (purified by precipitation). IR (KBr,
152.3 (C), 157.4 (C), 165.4 (C). All data for BA2-O are in 3022, 1716, 1662, 1198, 1084. M.p.: 202.7-205.7 °C (Lit. M.p.:
accordance with those reported elsewhere.50 212-215 °C).54 1H-NMR (200 MHz, DMSO-d6)
1.09 (t, 3H, J = 7.1
Hz), 2.27 (s, 3H), 3.99 (q, 2H, J = 7.1 Hz), 5.06 (d, 1H, J = 3.4 Hz),
4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4- 6.70 (d, 2H, J = 8.4 Hz), 7.00 (d, 2H, J = 8.4 Hz), 9.42 (s, 1H), 9.55
tetrahydropyrimidine-5-carboxylate (BA2-S): 46% yield (s, 1H), 10.24 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)
14.1 (CH3),
(purified by precipitation), IR (KBr,
cm-1): 3308, 3184, 3116, 17.1 (CH3), 53.6 (CH), 59.5 (CH2), 101.1 (C), 115.1 (CH), 127.7
2984, 1684, 1662, 1196. M.p.: 183.4-185.1 °C (Lit. M.p.: 185-187 (CH), 134.1 (C), 144.1 (C), 156.9 (C), 165.2 (C), 173.9 (C). All data
°C).51 1H-NMR (200 MHz, DMSO-d6) 1.11 (t, 3H, J = 7.1 Hz), 2.28 for (BA4-S) are in accordance with those reported elsewhere.54
4-(4-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
14.1 (CH3), 17.8 (CH3), 53.9 (CH), 59.2 (CH2), 99.4 (C), 113.1 tetrahydropyrimidine-5-carboxylate (BA4-S): 52% yield
cm-1): 3422, 3286, 3198,


10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

Ethyl



(s, 3H), 4.05 (q, 2H, J = 7.1 Hz), 5.01 (d, 1H, J = 3.6 Hz), 6.63- 6.67
(m, 3H), 7.12 (t, 1H, J = 8.1 Hz), 9.45 (s, 1H), 9.60 (brs, 1H), 10.30 Ethyl
4-(3-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-
(s, 1H). 13C-NMR (50 MHz, DMSO-d6)

14.1 (CH3), 17.2 (CH3), tetrahydropyrimidine-5-carboxylate (BA5-O): 73% yield
54.0 (CH), 59.6 (CH2), 100.8 (C), 113.3 (CH), 114.7 (CH), 117.0 (purified by precipitation). IR (KBr,
cm-1): 3242, 3104, 2934,
(CH), 129.5 (CH), 144.9 (C), 157.5 (C), 165.2 (C), 174.2 (C). All 1702, 1648, 1600, 1164. M.p.: 221.3-223.9 °C (Lit. M.p.: 219-
data for BA2-S are in accordance with those reported 220°C). 52 1H-NMR (200 MHz, DMSO-d6)
1.11 (t, 3H, J = 7.1 Hz),


elsewhere.51
2.24 (s, 3H), 3.72 (s, 3H), 3.99 (q, 2H, J = 7.1 Hz), 5.12 (d, 1H, J =
2.9 Hz), 6.78-6.83 (m, 3H), 7.24 (t, J = 3.8, 1H), 7.74 (brs, 1H),
Ethyl 4-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4- 9.20 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)

14.1 (CH3), 17.8
tetrahydropyrimidine-5-carboxylate (BA3-O): 39% yield (CH3), 53.8 (CH), 55.0 (CH3), 59.3 (CH2), 99.2 (C), 112.2 (CH),
(purified by precipitation), IR (KBr,
cm-1): 3538, 3244, 3116, 112.4 (CH), 118.3 (CH), 129.6 (CH), 146.4 (C), 148.5 (C), 152.3
2974, 1704, 1698, 1644, 1170. M.p.: 233.9-236.0 °C (Lit. M.p.: (C), 159.2 (C), 165.4 (C). All data for BA5-O are in accordance
226 - 228 °C).52 1H-NMR (200 MHz, DMSO-d6) 1.11 (t, 3H, J = with those reported elsewhere.52


7.1 Hz), 2.24 (s, 3H), 3.72 (s, 3H), 3.99 (q, 2H, J = 7.1 Hz), 5.06 (d,
1H, J = 2.9 Hz), 6.58–6.73 (m, 2H), 6.78-6.80 (m, 1H), 7.65 (s, 1H), Ethyl
4-(3-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
8.92 (s, 1H), 9.13 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)

14.2 tetrahydropyrimidine-5-carboxylate (BA5-S): 81% yield
(CH3), 17.8 (CH3), 53.6 (CH), 55.6 (CH3), 59.2 (CH2), 99.6 (C), (purified by precipitation). IR (KBr,
cm-1): 3320, 3176, 2992,
110.9 (CH), 115.3 (CH), 118.3 (CH), 136.0 (C), 145.8 (C), 147.3 1668, 1652, 1192, 1098. M.p.: 160.9-162.8 °C (Lit. M.p.: 141-143
(C), 148.0 (C), 152.3 (C), 165.5 (C). All data for BA3-O are in °C).52 1H-NMR (200 MHz, DMSO-d6)
1.11 (t, 3H, J = 7.0 Hz), 2.28


accordance with those reported elsewhere.52
(s, 3H), 4.02 (q, 2H, J = 7.0 Hz), 5.15 (d, 1H, J = 3.0 Hz), 6.72 (d,
1H, J = 2.0 Hz), 6.79 (d, 1H, J = 7.8 Hz), 6.84 (dd, 1H, J3 = 7.8 Hz,
Ethyl
1,2,3,4-tetrahydropyrimidine-5-carboxylate (BA3-S): 25% yield 13C-NMR (50 MHz, DMSO-d6)
(purified by precipitation), IR (KBr,
cm-1): 3418, 3160, 3000, 55.0 (CH3), 59.7 (CH2), 100.6 (C), 112.5 (CH), 112.5 (CH), 118.4
2952, 1682, 1652, 1196, 1112. M.p.: 224.9-225.5 °C (Lit. M.p.: (CH), 129.8 (CH), 145.0 (C), 145.1 (C), 159.3 (C), 165.2 (C), 174.4
4-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-thioxo- J4 = 2.0 Hz), 7.26 (t, 1H, J = 7.8 Hz), 9.65 (s, 1H), 10.35 (s, 1H).

14.0 (CH3), 17.2 (CH3), 53.8 (CH),

206-208 °C).52 1H-NMR (200 MHz, DMSO-d6)
 1.11 (t, 3H, J = 7.0 (C). All data for BA5-S are in accordance with those reported
Hz), 2.28 (s, 3H), 3.72 (s, 3H), 4.02 (q, 2H, J = 7.0 Hz), 5.09 (d, 1H, elsewhere.52
J = 2.8 Hz), 6.57-6.60 (m, 1H), 6.71-6.78 (m, 2H), 9.02 (s, 1H),
9.57 (s, 1H), 10.27 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)

14.1 Ethyl
(CH3), 17.2 (CH3), 53.7 (CH3), 55.6 (CH), 59.6 (CH2), 101.0 (C), tetrahydropyrimidine-5-carboxylate (BA6-O): 55% yield
111.0 (CH), 115.4 (CH), 118.6 (CH), 134.6 (C), 144.7 (C), 146.2 (purified by precipitation). IR (KBr,
cm-1): 3256, 3108, 2958,
(C), 147.4 (C), 165.3 (C), 174.1 (C). All data for BA3-S are in 1726, 1702, 1636, 1080. M.p.: 261.0-262.6 °C (Lit. M.p: 260-262
accordance with those reported elsewhere.52 °C).55 1H-NMR (200 MHz, DMSO-d6)
1.02 (t, 3H, J = 7.0 Hz), 2.27
(s, 3H), 3.79 (s, 3H), 3.91 (q, 2H, J = 7.0 Hz), 5.49 (brs, 1H), 6.83-
4-(4-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4- 7.03 (m, 3H), 7.19-7.27 (m, 2H), 9.12 (s, 1H). 13C-NMR (100 MHz,
tetrahydropyrimidine-5-carboxylate (BA4-O): 71% yield DMSO-d6) 14.0 (CH3), 17.7 (CH3), 48.9 (CH), 55.4 (CH3), 59.0
(purified by precipitation). IR (KBr,
cm-1): 3504, 3270, 3113, (CH2), 97.6 (C), 111.1 (CH), 120.2 (CH), 127.1 (CH), 128.7
2982, 1678, 1640, 1600, 1088. M.p.: 231.9-234.8 °C (Lit. M.p.: (CH),131.6 (C), 148.9 (C), 152.2 (C), 156.5 (C), 165.4 (C). All data
237-239 °C).53 1H-NMR (200 MHz, DMSO-d6) 1.09 (t, 3H, J = 6.9 for BA6-O are in accordance with those reported elsewhere.55
4-(2-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-


Ethyl



Hz), 2.23 (s, 3H), 3.97 (q, 2H, J = 6.9 Hz), 5.04 (s, 1H), 6.68 (d, 2H,
J = 8.0 Hz), 7.03 (d, 2H, J = 8.0 Hz), 7.62 (s, 1H), 9.12 (s, 1H), 9.34 Ethyl
4-(2-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
(s, 1H). 13C-NMR (50 MHz, DMSO-d6)

14.5 (CH3), 18.1 (CH3), tetrahydropyrimidine-5-carboxylate (BA6-S): 92% yield
10 | J. Name., 2012, 00, 1-3
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(purified by precipitation). IR (KBr,
1710, 1652, 1178. M.p.: 185.5-187.9 °C (Lit. M.p.: 230-231 °C).56 2H), 5.95 (d, 1H, J = 2.8 Hz), 7.50-7.57 (mD, O2HI: )1,07.1.09339/(Ct9dN, J10H3,55J36G=
1H-NMR (400 MHz, DMSO-d6)
1.37 (t, 3H, J = 7.1 Hz), 2.29 (s, 8.2 Hz, J4 = 1.1 Hz), 7.76 (dd, 1H, J3 = 7.6 Hz, J4 = 1.1 Hz), 9.56 (s,
3H), 3.37 (s, 3H), 3.94 (qd, 2H, J3 = 7.1 Hz, J4 = 1.5 Hz), 5.50 (d, 1H), 10.44 (s, 1H). 13C-NMR (100 MHz, DMSO-d6)
13.7 (CH3),
  0.94 (t, 3H, J = 7.1 Hz), 2.31 (s, 3H), 3.81-3.93 (m,
cm-1): 3260, 3270, 2998, DMSO-d6)
View Article Online


1H, J = 3.4 Hz), 6.89 (td, 1H, J3 = 7.6 Hz, J4 = 0.7 Hz), 7.00 (d, 1H, 17.1 (CH3), 49.2 (CH), 59.5 (CH2), 99.8 (C), 124.2 (CH), 129.1 (CH),
J = 8.1 Hz), 7.04 (dd, 1H, J3 = 7.6 Hz, J4 = 1.6 Hz), 7.25 (td, 1H, J3 129.4 (CH), 134.2 (CH), 136.0 (C), 145.6 (C), 147.3 (C), 164.5 (C),
= 7.6 Hz, J4 = 1.6 Hz), 9.24 (d, 1H, J = 1.6 Hz), 10.23 (s, 1H). 13C- 174.1 (C). All data for BA8-S are in accordance with those
NMR (100 MHz, DMSO-d6)

13.9 (CH3), 17.1 (CH3), 49.5 (CH), reported elsewhere.56
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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55.5 (CH3), 59.4 (CH2), 99.4 (C), 111.3 (CH), 120.2 (CH), 127.8
(CH), 129.2 (CH), 130.6 (C), 145.2 (C),156.6 (C), 165.2 (C), 174.2 Ethyl
(C). All data for BA6-S are in accordance with those reported tetrahydropyrimidine-5-carboxylate (BA9-O): 69% yield
4-(benzo[d][1,3]dioxol-4-yl)-6-methyl-2-oxo-1,2,3,4-
elsewhere.56
(purified by precipitation). IR (KBr,
1702, 1690, 1640, 1094. M.p.: 186.9-189.4 °C. 1H-NMR (200
6-methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4- MHz, DMSO-d6) 1.10 (t, 3H, J = 7.0 Hz), 2.24 (s, 3H), 3.99 (q,

cm-1): 3222, 3106, 2966,
Ethyl

tetrahydropyrimidine-5-carboxylate (BA7-O): 61% yield 2H, J = 7.0 Hz), 5.07 (brs, 1H), 5.97 (s, 2H), 6.67- 6.68 (m, 3H),
(purified by precipitation). IR (KBr, 14.1
cm-1): 3238, 3122, 2988, 7.68 (s, 1H), 9.18 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)
1782, 1700, 1646, 1522, 1096, 856. M.p.: 201.9-206.7 °C (Lit. (CH3), 17.8 (CH3), 53.7 (CH), 59.2 (CH2) 99.3 (C), 101.0 (CH2),
M.p.: 211-212 °C).50 1H-NMR (200 MHz, DMSO-d6)
1.09 (t, 3H, 106.7 (CH), 108.0 (CH), 119.3 (CH), 138.9 (C), 146.3 (C), 147.2



J = 7.1 Hz), 2.27 (s, 3H), 3.98 (q, 2H, J = 7.1 Hz), 5.28 (d, 1H, J = (C), 148.2 (C), 152.1 (C), 165.3 (C). All data for BA9-O are in
3.1 Hz), 7.51 (d, 2H, J = 8.7 Hz), 7.90 (s, 1H), 8.22 (d, 2H, J = 8.7 accordance with those reported elsewhere.49
Hz), 9.37 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)
 14.1 (CH3), 17.9
(CH3), 53.7 (CH), 59.4 (CH2), 98.2 (C), 123.8 (CH), 127.7 (CH), Ethyl 4-(benzo[d][1,3]dioxol-4-yl)-6-methyl-2-thioxo-1,2,3,4-
146.7 (C), 149.4 (C), 151.8 (C), 152.0 (C), 165.1 (C). All data for tetrahydropyrimidine-5-carboxylate (BA9-S): 67% yield
BA7-O are in accordance with those reported elsewhere.50
(purified by precipitation). IR (KBr,
1654, 1576, 1192, 1112. M.p.: 172,6-174,3 °C. 1H-NMR (200
6-methyl-4-(4-nitrophenyl)-2-thioxo-1,2,3,4- MHz, DMSO-d6) 1.10 (t, 3H, J = 7.1 Hz), 2.29 (s, 3H), 4.01 (q,
tetrahydropyrimidine-5-carboxylate (BA7-S): 60% yield 2H, J = 7.1 Hz), 5.09 (d, 1H, J = 3.5 Hz), 5.99 (s, 2H), 6.66-6.71 (m,
(purified by precipitation), IR (KBr,
cm-1): 3298, 3170, 2988, 2H), 6.87 (d, 1H, J = 6.9 Hz), 9.59 (s, 1H), 10,31 (s, 1H). 13C-NMR
1682, 1652, 1520, 1352, 1174, 853. M.p.: 193,0-195,6 °C ( Lit. (50 MHz, DMSO-d6) 14.1 (CH3), 17.2 (CH3), 53.7 (CH), 59.6
M.p.: 193-194 °C).56 1H-NMR (200 MHz, DMSO-d6)
1.09 (t, 3H, (CH2), 100.8 (C), 101.1 (CH), 106.7 (CH2), 108.2 (CH), 119.7 (CH),

cm-1): 3316, 3180, 2980,
Ethyl




J = 7.0 Hz), 2.31 (s, 3H), 4.00 (q, 2H, J = 7.0 Hz), 5.39 (d, 1H, J = 137.5 (C), 145.0 (C), 146.7 (C), 147.4 (C), 165.1 (C), 174.1 (C).
3,1 Hz), 7.48 (d, 2H, J = 8.4 Hz), 8.23 (d, 2H, J = 8.4 Hz), 9.77 (s, Data for BA9-S are in accordance with those reported
1H), 10.50 (s, 1H). 13C-NMR (50 MHz, DMSO-d6)

14.0 (CH3), elsewhere.49
17.3 (CH3), 53.7 (CH), 59.8 (CH2), 99.7 (C), 124.0 (CH), 127.8 (CH),
146.0 (C), 146.9 (C), 150.4 (C) 164.9 (C), 174.5 (C). All data for Ethyl
4-(4-fluorophenyl)-6-methyl-2-oxo-1,2,3,4-
BA7-S are in accordance with those reported elsewhere.56
tetrahydropyrimidine-5-carboxylate (BA10-O): 74% yield
(purified by precipitation). IR (KBr,
cm-1): 3246, 3124, 2980,
6-methyl-4-(2-nitrophenyl)-2-oxo-1,2,3,4- 1728, 1714,1648, 1220, 1100. M.p.: 179.5-180.7 °C ( Lit. M.p.:
tetrahydropyrimidine-5-carboxylate (BA8-O): 28% yield 172-173 °C).52 1H-NMR (200 MHz, DMSO-d6)
1.08 (t, 3H, J = 7.1
[purified by silica gel column chromatography using ethyl Hz), 2.25 (s, 3H), 3.98 (q, 2H, J = 7.1 Hz), 5.14 (d, 1H, J = 3.2 Hz),
acetate/hexane (3:1) as the eluant]. IR (KBr,
cm-1): 3366, 3222, 7.14 (t, 2H, J = 7.3 Hz), 7.24-7.28 (m, 2H), 7.73 (s, 1H), 9.21 (s,
2988, 1698, 1646, 1608, 1524, 1370, 1094, 860. M.p.: 220.5- 1H), 13C-NMR (50 MHz, DMSO-d6)
14.1 (CH3), 17.8 (CH3), 53.4
222.5 °C (Lit. M.p.: 205-206 °C).55 1H-NMR (400 MHz, DMSO-d6) (CH), 59.2 (CH2), 99.1 (C), 115.1 (CH), 126.2 (CH), 141.1 (C),
0.92 (t, 3H, J = 7.1 Hz), 2.28 (s, 3H), 3.82-3.85 (m, 2H), 7.51- 148.5 (C), 151.9 (C), 160.1 (C), 162.5 (C), 165.3 (C). All data for
7.57 (m, 2H), 7.71-7.73 (m, 2H), 7.88-7.90 (m, 1H), 9.36 (s, 1H). BA10-O are in accordance with those reported elsewhere.52
13C-NMR (100 MHz, DMSO-d6)
13.7 (CH3), 17.7 (CH3), 49.4
(CH), 59.1 (CH2), 96.0 (C), 123.9 (CH), 128.7 (CH), 129.0 (CH), Ethyl
134.1 (CH), 139.3 (C), 147.5 (C), 149.6 (C), 151.2 (C), 164.7 (C). tetrahydropyrimidine-5-carboxylate (BA10-S): 81% yield
All data for BA8-O are in accordance with those reported (purified by precipitation). IR (KBr,
cm-1): 3328, 3274, 2986,
1682, 1682, 1656, 1284, 1196, 1118. M.p.: 190.3-192.0 °C (Lit.
M.p.: 194-195 °C).57 1H-NMR (400 MHz, DMSO-d6)
1.09 (t, 3H,
6-methyl-4-(2-nitrophenyl)-2-thioxo-1,2,3,4- J = 7.1 Hz), 2.30 (s, 3H), 4.00 (qd, 2H, J3 = 7.1 Hz, J4 = 3.4 Hz), 5.18

Ethyl





4-(4-fluorophenyl)-6-methyl-2-thioxo-1,2,3,4-

elsewhere.55
Ethyl

tetrahydropyrimidine-5-carboxylate (BA8-S): 30% yield (d, 1H, J = 3.4), 7.15-7.20 (m, 2H), 7.23-7.26 (m, 2H), 9.64 (brs,
[purified by silica gel column chromatography using ethyl 1H), 10.35 (s,1H). 13C-NMR (100 MHz, DMSO-d6)
14.0 (CH3),
acetate/hexane (3:1) as the eluant]. IR (KBr,
cm-1): 3292, 3190, 17.2 (CH3), 53.4 (CH), 59.6 (CH2), 100.6 (C), 115.3 (CH), 128.4


2982, 2982, 1722, 1657, 1524, 1354, 1196, 1094, 860. M.p.: (CH), 139.7 (C), 145.1 (C), 160.3 (C),162.7 (C), 165.0 (C), 174.2
204.5-205.1 °C (Lit. M.p.: 195-197 °C).56 1H-NMR (400 MHz,
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(C). All data for BA10-S are in accordance with those reported 1H), 3.97-4.17 (m,2H), 7.30 (s, 1H), 8.89 (s, 1H). V1ie3wC-ANrtiMcleROn(l5in0e
elsewhere.57
Ethyl
MHz, DMSO-d6) 
14.2 (CH3), 17.7 (CH3),D2O5I.:710(C.1H0329),/C295N.9J0(3C5H562G),
26.0 (CH2), 26.3 (CH2), 28.5 (CH2), 44.9 (CH), 55.0 (CH), 59.0
4-(4-bromophenyl)-6-methyl-2-oxo-1,2,3,4- (CH2), 98.0 (C), 148.4 (C), 153.2 (C), 165.8 (C). All data for BA13-
tetrahydropyrimidine-5-carboxylate (BA11-O): 70% yield O are in accordance with those reported elsewhere.58
(purified by precipitation). IR (KBr,
1722, 1704, 1650, 1088, 678. M.p.: 212.3-215.3 °C (Lit. M.p.: Ethyl
225-226 °C).50 1H-NMR (200 MHz, DMSO-d6)
1.09 (t, 3H, J = 6.9 tetrahydropyrimidine-5-carboxylate (BA13-S): 4% yield
Hz), 2.25 (s, 3H), 3.98 (q, 2H, J = 6.9 Hz), 5.13 (s, 1H), 7.52 (d, 2H, [purified by silica gel column chromatography using hexane/
J = 8.2 Hz), 7.19 (d, 2H, J = 8.2 Hz), 7.77 (s, 1H), 9.25 (s, 1H), 13C- ethyl acetate (3:1) as the eluant]. IR (KBr, cm-1): 3188, 2980,
NMR (50 MHz, DMSO-d6) 14.4 (CH3), 18.2 (CH3), 53.9 (CH2), 2926, 1708, 1648, 1182, 1092. M.p.: 210.5-212.8 °C (Lit. M.p.:
59.6 (C), 99.2 (C), 120.7 (C), 128.9 (CH), 131.7 (CH), 144.6 (C), 207-208 °C).59 1H-NMR (200 MHz, DMSO-d6)
0.78-0.90 (m, 1H),
149.1 (C), 152.3 (C), 165.6 (C). All data for BA11-O are in 1.07-1.22 (m, 7H), 1.37 (brs, 2H), 1.58-1.65 (m, 4H), 2.21 (s, 3H),
accordance with those reported elsewhere.50
3.94-3.98 (m, 1H), 4.02-4.16 (m, 2H), 9.22-9.32 (m, 1H), 10.07
(s, 1H). 13C-NMR (50 MHz, DMSO-d6)
14.1 (CH3), 17.1 (CH3),
4-(4-bromophenyl)-6-methyl-2-thioxo-1,2,3,4- 25.5 (CH2), 25.7 (CH2), 24.9 (CH2), 26.6 (CH2), 28.3 (CH2), 44.7
tetrahydropyrimidine-5-carboxylate (BA11-S): 78% yield (CH), 55.3 (CH), 59.7 (CH2), 99.5 (C), 145.3 (C), 165.6 (C), 175.2
(purified by precipitation). IR (KBr,
cm-1): 3328, 3174, 2982, (C). All data for BA13-S are in accordance with those reported
1670, 1578, 1198, 1122, 596. M.p.: 192.0-194.4 °C (Lit. M.p.: elsewhere.59
191-192 °C). 56 1H-NMR (200 MHz, DMSO-d6)
1.09 (t, 3H, J =

cm-1): 3244, 3116, 2980,
4-cyclohexyl-6-methyl-2-thioxo-1,2,3,4-

10
11
12
13
14
15
16
17
18
19
20
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22
23
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



Ethyl


Urease activity assay
7.0 Hz), 2.29 (s, 3H), 4.00 (q, 2H, J = 7.0 Hz), 5.15 (brs, 1H), 7.17
(d, 2H, J = 8.1 Hz), 7.55 (d, 2H, J = 8.1 Hz), 9.67 (s, 1H), 10.38 (s,
Representative Biginelli adducts bearing electron donating or
withdrawing groups that were also soluble in ethanol were
screened for their potential to inhibit type III urease from C.
ensiformis (jack bean). The reaction media contained 20 mM
phosphate buffer (pH 7.0), 1 mM ethylenediaminetetraacetic
acid (EDTA), 10 mM urea, and 12.5 mU jack bean urease in the
1H). 13C-NMR (50 MHz, DMSO-d6)
 14.0 (CH3), 17.2 (CH3), 53.6
(CH2), 59.7 (CH), 100.2 (C), 120.8 (CH), 126,6 (CH) 131.5 (CH),
142.8 (C), 145.4 (C), 165.0 (C), 174.3 (C). All data for BA11-S are
in accordance with those reported elsewhere.56
Ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-
presence or absence of 500
M Biginelli adducts. Reactions
carboxylate (BA12-O): 68% yield (purified by precipitation). IR
were incubated at 25 °C, and after 10 min, they were stopped
by adding 0.5 volumes of 1% (m/v) phenol in 5 mg L-1 sodium
nitroprusside (SNP) followed by the addition of 0.7 volumes of
0.5% (m/v) NaOH solution in 0.1% NaOCl. The mixtures were
incubated at 50°C for 5 min, and the absorbance was measured
at 630 nm to determine the amount of NH4 formed.60 Urease
inhibition was determined in terms of the percent of NH4
formed in BA-containing reactions in relation to the total urease
activity in the inhibitor-free reactions. Three independent
experiments were performed with four replicates each.
(KBr,
207.3 °C (Lit. M.p.: 202-203 °C).52 1H-NMR (400 MHz, DMSO-d6)
1.09 (t, 3H, J = 7.1 Hz), 2.25 (s, 3H), 3.98 (q, 2H, J = 7.1 Hz), 5.14
(d, 1H, J = 3.2 Hz), 7.23-7.25 (m, 3H), 7.30-7.34 (m, 2H), 7.73 (s,
1H), 9.19 (s, 1H). 13C-NMR (100 MHz, DMSO-d6)
14.0 (CH3),

cm-1): 3246, 3116, 2980, 1726, 1648, 1192. M.p.: 205.3-


+
17.8 (CH3), 54.0 (CH), 59.2 (CH2), 99.3 (C), 126.2 (CH) 127.3 (CH),
128.4 (CH), 144.0 (C), 148.3 (C), 152.2 (C), 165.4 (C). All data for
BA12-O are in accordance with those reported elsewhere.52
+
Ethyl
tetrahydropyrimidine-5-carboxylate (BA12-S): 85% yield
(purified by precipitation). IR (KBr,
cm-1): 3330, 3174, 3104,
1670, 1648, 1196, 1118. M.p.: 205.5-207.3 °C (Lit. M.p.: 208-209
°C ).55 1H-NMR (400 MHz, DMSO-d6)
1.08 (t, 3H, J = 7.0 Hz),
6-methyl-4-phenyl-2-thioxo-1,2,3,4-

Interaction studies
Interaction studies by molecular fluorescence.

Stock solutions of C. ensiformis (jack bean) type III urease
(Sigma-Aldrich) at 10 μM were prepared in 20 mM phosphate
buffer with 1 mM EDTA (pH 7), while the Biginelli adducts BA5-
S and BA7-S were dissolved in ethanol to yield 1.0 mM solutions
that were further diluted in phosphate buffer to provide the
corresponding working solutions. Molecular fluorescence
titrations were performed using quartz cuvettes with a 10 mm
optical path on a Shimadzu spectrofluorometer (model 5301PC,
Japan) equipped with a xenon lamp (150 W). The urease (1.0
μM) fluorescence emission spectra in the absence or presence
of compounds BA5-S and BA7-S (2.5 to 50 μM) were recorded
from 260 to 450 nm employing an ex of 280 nm at three
temperatures (22, 30, 38ºC). The excitation and emission slits
were 5 and 10 nm, respectively. To calculate the critical distance
2.29 (s, 3H), 4.01 (q, 2H, J = 7.0 Hz), 5.18 (d, 1H, J = 3.7 Hz), 7.21-
7.36 (m, 5H), 9.63-9.64 (m, 1H), 10.32 (s, 1H). 13C-NMR (100
MHz, DMSO-d6)
 26.9 (CH3), 30.0 (CH2), 66.9 (CH3), 72.4 (CH2),
113.6 (C), 139.3 (CH), 140.6 (CH), 141.4 (CH), 156.4 (C), 157.9
(C), 178.0 (C), 187.1 (C). All data for BA12-S are in accordance
with those reported elsewhere.55
Ethyl
4-(4-hydroxybutyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (BA13-O): 43% yield
[purified by silica gel column chromatography using
hexane/chloroform/ethyl acetate (3:3:4) as the eluant], IR (KBr,

cm-1): 3238, 3120, 2922, 1728,1704, 1646, 1096. M.p.: 234.7-
238.6 °C (Lit. M.p.: 237-238 °C).58 1H-NMR (200 MHz, DMSO-d6)
0.82-1.37 (m, 10H), 1.57-1.67 (m, 4H), 2.16 (s, 3H), 3.92 (brs,

12 | J. Name., 2012, 00, 1-3
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at which the energy transfer occurs from the urease tryptophan compressibility of 4.5 × 10−5 ·bar−1. The Parrinello-Rahman
View Article Online
residues (5 μM) to the BA5-S and BA7-S (both at 5 μM), Förster velocity rescale thermostat algorDitOhmI: 10.10w39a/sC9NJa0p3p5l5ie6Gd
Resonance Energy Transfer (FRET) studies were performed. independently for protein and water molecules at 300 K.
Tridimensional fluorescence experiments were performed Periodic boundary conditions were applied in all directions.
using an excitation wavelength in the range from 220-350 nm Molecular graphics and analyses were performed with the UCSF
and a fixed emission range from 260 to 450 nm. Synchronous Chimera package.64 The MD analysis module of the UCSF
fluorescence spectra for urease in the absence or presence of Chimera® was used to obtain the most predominant
Biginelli adducts BA5-S and BA7-S were obtained by conformation of the enzyme into the system. Root mean square
simultaneous variation of the excitation and emission deviation (RMSD) values were calculated using GROMACS®, and
monochromators. The difference in the excitation wavelength all RMSD charts were generated by using Xmgrace® software.65
(Δλ) was fixed individually at Δλ = 15 nm (Tyr) and Δλ = 60 nm Finally, the Ramachandran plot was generated from the
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(Trp) to show solely the spectroscopic behaviour of the Tyr and RAMPAGE online server 66
.
Trp residues. The inhibitors N-(n-butyl) thiophosphoric triamide After MD simulations, the most stable conformation of the jack
(NBPT), thiourea, and omeprazole were used at the fixed bean urease was selected for molecular docking studies. The
concentration of 25 μM in the competitive assay, while the optimization of the HAE (a co-crystallized ligand at the active
hydroxyurea concentration was 38

M.
site from urease enzyme used for the re-docking experiment)
and Biginelli adducts studied was performed with the classic
method, using AMBER 99bsc0 force field and assigning partial
charges AM1-BCC, automatically performed by the USCF
Chimera® dock prep module. The UCSF Chimera® package was
utilized to perform all molecular docking simulations. Water
molecules and counterions previously added in MD simulations
were removed from the resulting structure. All simulations
were carried out considering the area of the active site from the
jack bean urease. In order to validate the docking protocol, the
molecular alignment of co-crystallized ligands was performed
using PyMOL® version 2.3.1 software (https://pymol.org). In
sense, the co-crystallized HAE ligand was extracted and aligned
with the HAE binding mode obtained by molecular docking by
using the urease enzyme after 50 ns dynamics simulations.
Thus, the molecular alignment value (RMSD in Ångström) was
obtained to support our virtual protocol and proceeds.
Furthermore, it is assumed that a reliable virtual protocol
involving DM simulations and molecular docking should provide
Kinetics studies
Reactions were set exactly as described for the screening
experiments except that the Biginelli adducts BA5-S and BA7-S
were used individually at concentrations that inhibit urease in
the range from 30 to 40% (0.1 to 0.3 mM) in the presence of
increasing concentrations of urea (1-32 mM). Reactions were
stopped and analyzed as described for the screening
experiments. The Michaelis-Menten hyperbolas and
Lineweaver-Burk plots were obtained using the software
OriginPro 8 (Origin Lab, Northampton, MA), and Ki values were
determined using the experimental values of KM, Vmax, KM app
Vmax app, α and α’.
,
Molecular Dynamics and Docking simulation
For the molecular modeling studies, the acetohydroxamic acid
(HAE) and Biginelli adducts (BA5-S, BA7-S and BA10-S) were
constructed using the Marvin software 61 and jack bean urease
(C. ensiformis) was obtained from the Protein Data Bank
website (PDB entry: 4H9M). In order to obtain this hydrolase in
its native state, molecular dynamics (MD) simulations were
performed prior to docking studies. Thus, OPLS solvation model
atomic alignment values up to 2.0 Å 67
.
Reference
1
2
J. B. Sumner, J. Biol. Chem., 1926, 69, 435-441.
K. Kappaun, A. R. Piovesan, C. R. Carlini and R. Ligabue-Braun,
J. Adv. Res., 2018, 13, 3–17.
62
was added to the urease structure. The simulations were
performed using the GROMACS® package 63. Water molecules
and hydrogenionic concentration (pH 7.0) were added at the
atomic level, considering a triclinic box. Geometries of the
urease were optimized with the steepest descent algorithms
followed by the conjugate gradient minimization. Two previous
stabilization steps were performed at 1000 ps with a time step
of 2 fs using NVT and, then, sets of 300 K NPT isobaric and
isothermal to relax the water molecules, and to stabilize the box
density. Then, MD simulations were carried out at 300 K for 30
ns (1 fs time step). The Lennard-Jones and the real-space part
of electrostatic interactions were cut-off at 1.0 nm. For long-
range electrostatic interactions, the particle-mesh Ewald (PME)
method was used with the reciprocal-space interactions
evaluated on a 0.12 nm grid with cubic interpolation of order
four. The P-LINCS algorithm was used to constrain all bond
lengths. Isotropic pressure coupling was applied using the
Berendsen algorithm at 1 bar with a time constant of 3.0 ps and
3
L. V. Modolo, A. X. de Souza, L. P. Horta, D. P. Araujo and Â. de
Fátima, J. Adv. Res., 2015, 6, 35–44.
4
5
P. Kafarski and M. Talma, J. Adv. Res., 2018, 13, 101–112.
B. P. Callahan, Y. Yuan and R. Wolfenden, J. Am. Chem. Soc.,
2005, 127, 10828–10829.
6
7
B. Krajewska, J. Mol. Catal. B Enzym., 2009, 59, 9–21.
L. V. Modolo, C. J. da-Silva, D. S. Brandão and I. S. Chaves, J.
Adv. Res., 2018, 13, 29–37.
8
9
M. J. Maroney and S. Ciurli, Chem. Rev., 2014, 114, 4206–
4228.
C. Follmer, J. Clin. Pathol., 2010, 63, 424–430.
10 D. Y. Graham and M. Miftahussurur, J. Adv. Res., 2018, 13, 51–
57.
11 K. X. Ruthrof, E. Steel, S. Misra, J. McComb, G. O’Hara, G. E. St.
J. Hardy and J. Howieson, Sci. Total Environ., 2018, 625, 1–7.
12 H. Cantarella, R. Otto, J. R. Soares and A. G. de B. Silva, J. Adv.
Res., 2018, 13, 19–27.
13 F. Colpaert, F. Ledoux, V. R. Belzen, US 2019 / 0039962 A1,
2019, 1–6.
This journal is © The Royal Society of Chemistry 20xx
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Page 14 of 16
ARTICLE
Journal Name
1
2
3
4
5
6
7
8
9
14 A. Hameed, M. Al-Rashida, M. Uroos, S. U. Qazi, S. Naz, M. 42 Y.-Q. Wang, G.-C. Zhang and H.-M. Zhang, J. Solut. Chem.,
View Article Online
Ishtiaq and K. M. Khan, Expert Opin. Ther. Pat., 2019, 29, 181–
189.
2011, 40, 458–469.
DOI: 10.1039/C9NJ03556G
43 B. Valeur and M. N. Berberan-Santos, Molecular fluorescence:
principles and applications, Wiley-VCH Verlag GmbH & Co.
KGaA, Weinheim, 2001, pp.273-350.
44 C. C. Santana, E. F. Silva-Júnior, J. C. N. Santos, É. E. da S.
Rodrigues, I. M. da Silva, J. X. Araújo-Júnior, T. G. do
Nascimento, L. A. O. Barbosa, C. B. Dornelas, I. M. Figueiredo,
J. C. C. Santos and L. A. M. Grillo, Bioorganic. Chem., 2019, 87,
169–180.
15 D. Tzemi and J. Breen, Ecol. Model., 2019, 394, 18–26.
16 J. Bzura and R. Koncki, Enzyme Microb. Technol., 2019, 123, 1–
7.
17 Y. F. Rego, M. P. Queiroz, T. O. Brito, P. G. Carvalho, V. T. de
Queiroz, Â. de Fátima and F. Macedo Jr., J. Adv. Res., 2018, 13,
69–100.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18 Â. de Fátima, C. de P. Pereira, C. R. S. D. G. Olímpio, B. G. de F.
Oliveira, L. L. Franco and P. H. C. da Silva, J. Adv. Res., 2018, 45 M. de M. Silva, T. S. Macedo, H. M. P. Teixeira, D. R. M.
13, 113–126.
Moreira, M. B. P. Soares, A. L. da C. Pereira, V. de L. Serafim,
F. J. B. Mendonça-Júnior, M. C. A. de Lima, R. O. de Moura, E.
F. da Silva-Júnior, J. X. de Araújo-Júnior, M. D. A. Dantas, E. de
O. O. Nascimento, T. M. S. Maciel, T. M. de Aquino, I. M.
Figueiredo and J. C. C. Santos, J. Photochem. Photobiol. B,
2018, 189, 165–175.
19 L.V. Modolo, A. de Fátima, M. Giovanni, D. L. da Silva, V. S. S.
Morais, BR 102013017356-8 A2, 2016, 1–14.
20 U. Rashid, I. Batool, A. Wadood, A. Khan, Z. ul-Haq, M. I.
Chaudhary and F. L. Ansari, J. Mol. Graph. Model., 2013, 43,
47–57.
21 F. Tamaddon and S. Ghazi, Catal. Commun., 2015, 72, 63–67. 46 P. D. Ross and S. Subramanian, Biochemistry, 1981, 20, 3096–
22 E. Artola, S. Cruchaga, I. Ariz, J. F. Moran, M. Garnica, F. 3102.
Houdusse, J. M. G. Mina, I. Irigoyen, B. Lasa and P. M. Aparicio- 47 F. Mehranfar, A. K. Bordbar, M. Keyhanfar and M. Behbahani,
Tejo, Plant. Growth. Regul., 2011, 63, 73–79. J. Lumin., 2013, 143, 687–692.
23 (a) P. Biginelli, Gazz. Chim. Ital., 1981, 21, 455–461. (b) P. 48 Y. Shu, W. Xue, X. Xu, Z. Jia, X. Yao, S. Liu and L. Liu, Food
Biginelli, Gazz. Chim. Ital., 1891, 21, 497–500. (c) P. Biginelli, Chem., 2015, 173, 31–37.
Berichte Dtsch. Chem. Ges., 1891, 24, 1317–1319. (d) P. 49 J. C. N. Santos, I. M. da Silva, T. C. Braga, Â. de Fátima, I. M.
Biginelli, Berichte Dtsch. Chem. Ges., 1891, 24, 2962–2967. (e)
P. Biginelli, Gazz. Chim. Ital., 1893, 23, 360–413.
Figueiredo and J. C. C. Santos, Int. J. Biol. Macromol., 2018,
113, 1032–1040.
24 L. Horta, Y. Mota, G. Barbosa, T. Braga, I. Marriel, Â. de Fátima 50 Q. Wang, X. Liu, M. Su, Z. Shi and H. Sun, Spectrochim. Acta.
and L. Modolo, J. Braz. Chem. Soc., 2016, 27, 1512-1519.
A. Mol. Biomol. Spectrosc., 2015, 136, 321–326.
25 A. Khan, J. Hashim, N. Arshad, I. Khan, N. Siddiqui, A. Wadood, 51 M. R. Siddiqui, Z. A. AlOthman and N. Rahman, Arab. J. Chem.,
M. Ali, F. Arshad, K. M. Khan and M. I. Choudhary, Bioorganic.
Chem., 2016, 64, 85–96.
2017, 10, S1409–S1421.
52 A. Balasubramanian and K. Ponnuraj, J. Mol. Biol., 2010, 400,
274–283.
26 C. O. Kappe, J. Org. Chem., 1997, 62, 7201–7204.
27 K. K. Pasunooti, H. Chai, C. N. Jensen, B. K. Gorityala, S. Wang 53 F. M. Oliveira, L. C. A. Barbosa, A. J. Demuner, C. R. A. Maltha,
and X.-W. Liu, Tetrahedron Lett., 2011, 52, 80–84.
28 J. Yu, F. Shi and L. Z. Gong, Acc. Chem. Res., 2011, 44, 1156–
71.
S. R. Pereira, L. P. Horta and L. V. Modolo, Med. Chem. Res.,
2014, 23, 5174–5187.
54 Z. Amtul, B. S. P. Atta-ur-Rahman, R. Siddiqui and M.
Choudhary, Curr. Med. Chem., 2002, 9, 1323–1348.
29 Q. Wang and W. Pei, J. Iran. Chem. Soc., 2010, 7, 318–321.
30 L. M. Ramos, A. Y. P. L. Tobio, M. R. dos Santos, H. C. B. de 55 F. Iftikhar, Y. Ali, F. Ahmad Kiani, S. Fahad Hassan, T. Fatima,
Oliveira, A. F. Gomes, F. C. Gozzo, A. L. de Oliveira and B. A. D.
A. Khan, B. Niaz, A. Hassan, F. Latif Ansari and U. Rashid,
Bioorganic Chem., 2017, 74, 53–65.
Neto, J. Org. Chem., 2012, 77, 10184–10193.
31 M. Terada, K. Machioka and K. Sorimachi, J. Am. Chem. Soc., 56 J.-Y. Cheng and Y.-H. Chu, Tetrahedron Lett., 2006, 47, 1575–
2007, 129, 10336–10337.
1579.
32 D. J. Adams, P. J. Dyson and S. J. Tavener, Chemistry in 57 F. M. A. Geilen, B. Engendahl, A. Harwardt, W. Marquardt, J.
Alternative Reaction Media, John Wiley & Sons, 2003, vol. 41,
pp. 75–93.
Klankermayer and W. Leitner, Angew. Chem. Int. Ed., 2010,
49, 5510–5514.
33 S. Duan, X. Jing, D. Li and H. Jing, J. Mol. Catal. Chem., 2016, 58 D. L. da Silva, F. S. Reis, D. R. Muniz, A. L. T. G. Ruiz, J. E. de
411, 34–39.
Carvalho, A. A. Sabino, L. V. Modolo and Â. de Fátima, Bioorg.
Med. Chem., 2012, 20, 2645–2650.
59 B. Zeynizadeh, K. A. Dilmaghani and M. Yari, Phosphorus Sulfur
Silicon Relat. Elem., 2009, 184, 2465–2471.
34 Y. Jeong, D.-Y. Kim, Y. Choi and J.-S. Ryu, Org. Biomol. Chem.,
2011, 9, 374–378.
35 T. O. Brito, A. X. Souza, Y. C. C. Mota, V. S. S. Morais, L. T. de
Souza, Â. de Fátima, F. Macedo and L. V. Modolo, RSC Adv., 60 D. L. da Silva, S. A. Fernandes, A. A. Sabino and Â. de Fátima,
2015, 5, 44507–44515.
36 W. Yan-Qing and Z. Hong-Mei, Spectrochim. Acta. A. Mol. 61 N. S. Nandurkar, M. J. Bhanushali, M. D. Bhor and B. M.
Biomol. Spectrosc., 2012, 96, 352–357. Bhanage, J. Mol. Catal. Chem., 2007, 271, 14–17.
Tetrahedron Lett., 2011, 52, 6328–6330.
37 A. Ghisaidoobe and S. Chung, Int. J. Mol. Sci., 2014, 15, 22518– 62 J. Zhu, M. Zhang, B. Liu and X. Li, Chem. Lett., 2009, 38, 56–57.
22538.
63 F. Shirini, K. Rad-Moghadam and S. Akbari-Dadamahaleh,
Green Solvents II: Properties and Applications of Ionic Liquids,
Springer Netherlands, Dordrecht, 2012, pp. 289–334.
38 H.-M. Zhang, G.-C. Zhang and Y.-Q. Wang, Biol. Trace Elem.
Res., 2011, 141, 53–64.
39 T. Li, Z. Cheng, L. Cao and X. Jiang, Food Chem., 2016, 194, 64 A. Mobinikhaledi, M. Zendehdel, M. H. Nasab and M. A. B.
740–748. Fard, Heterocycl. Commun., 2009, 15, 451-458.
40 M. D. de A. Dantas, H. de A. Tenório, T. I. B. Lopes, H. J. V. 65 P. Kumar, A. Kumar, S. S. Verma, N. Dwivedi, N. Singh, M. I.
Pereira, A. J. Marsaioli, I. M. Figueiredo and J. C. C. Santos, Int.
J. Biol. Macromol., 2017, 102, 505–514.
Siddiqi, R. P. Tripathi, A. Dube and N. Singh, Exp. Parasitol.,
2008, 120, 73–79.
41 T. C. A. Lage, T. M. S. Maciel, Y. C. C. Mota, F. Sisto, J. R. Sabino, 66 A. R. Gholap, K. Venkatesan, T. Daniel, R. J. Lahoti and K. V.
J. C. C. Santos, I. M. Figueiredo, C. Masia, Â. de Fátima, S. A. Srinivasan, Green Chem., 2004, 6, 147–150.
Fernandes and L. V. Modolo, New J. Chem., 2018, 42, 5356– 67 K. S. Atwal, B. C. O’Reilly, J. Z. Gougoutas and M. F. Malley,
5366.
Heterocycles, 1987, 26, 1189–1192.
68 M. W. Weatherburn, Anal. Chem., 1967, 39, 971–974.
14 | J. Name., 2012, 00, 1-3
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69 M. F. Sanner, J. Mol. Graph. Model., 1999, 17, 57–61.
ARTICLE
1
2
3
4
5
6
7
8
View Article Online
DOI: 10.1039/C9NJ03556G
70 X. Jiang, W. Li, G. Chen and L. Wang, J. Chem. Inf. Model., 2017,
57, 288–297.
71 S. Pronk, S. Páll, R. Schulz, P. Larsson, P. Bjelkmar, R.
Apostolov, M. R. Shirts, J. C. Smith, P. M. Kasson, D. van der
Spoel, B. Hess and E. Lindahl, Bioinformatics, 2013, 29, 845–
854.
72 M. M. Silva, E. O. O. Nascimento, E. F. Silva, J. X. de Araújo, C.
C. Santana, L. A. M. Grillo, R. S. de Oliveira, P. R.R.Costa, C. D.
Buarque, J. C. C. Santos and I. M. Figueiredo, Int. J. Biol.
Macromol., 2017, 96, 223–233.
9
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
25
26
27
28
29
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34
35
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
73 P. J. Turner, Xmgrace, 2005.
74 S. C. Lovell, I. W. Davis, W. B. Arendall, P. I. W. de Bakker, J. M.
Word, M. G. Prisant, J. S. Richardson and D. C. Richardson,
Proteins Struct. Funct. Bioinforma., 2003, 50, 437–450.
75 E. F. Da Silva-Júnior, P. H. B. França, F. F. Ribeiro, F. J. B.
Mendonça-Júnior, L. Scotti, M. T. Scotti, T. M. De Aquino and
J. X. De Araújo-Júnior, Curr. Comput. Aided Drug Des., 2018,
14, 68–78.
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Three out of twenty-six synthesized Biginelli adducts were identified as potent competitive
urease inhibitors.