Study of reactions of two Mannich bases derived of 4′-hydroxychalcones with glutathione by RP-TLC, RP-HPLC and RP-HPLC-ESI-MS analysis

Article abstract of DOI:10.21577/0103-5053.20160260

4′-Hydroxychalcones have been reported to possess several beneficial biological effects. Several lines of evidence accumulated to demonstrate increased biological activities of the Mannich base derivatives of the parent 4′-hydroxychalcones. Bioactivities

Full text of DOI:10.21577/0103-5053.20160260

http://dx.doi.org/10.21577/0103-5053.20160260
J. Braz. Chem. Soc., Vol. 28, No. 6, 1048-1062, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
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Article
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones with
Glutathione by RP-TLC, RP-HPLC and RP-HPLC-ESI-MS Analysis
a,b
a
c
a
Aline Bernardes, Caridad N. Pérez, Mátyás Mayer, Cameron C. da Silva,
a
,b
Felipe T. Martins and Pál Perjési*
a
Instituto de Química, Universidade Federal de Goiás, 74690-900 Goiânia-GO, Brazil
b
c
Institute of Pharmaceutical Chemistry and Department of Forensic Medicine, University of Pécs,
H-7624 Pécs, Hungary
4
’-Hydroxychalcones have been reported to possess several beneficial biological effects.
Several lines of evidence accumulated to demonstrate increased biological activities of the
Mannich base derivatives of the parent 4’-hydroxychalcones. Bioactivities of chalcones and related
α,β-unsaturated ketones are frequently associated with their reactivity with cellular thiols, such
as GSH. For comparison of GSH reactivity, two bis Mannich bases of two 4’-hydroxychalcones
were synthesized and reacted with GSH under non-cellular conditions. Reversed-phase thin
layer chromatography (RP-TLC) and reversed-phase high performance liquid chromatography
(
RP-HPLC) analysis showed formation of two polar products which structures were confirmed by
RP-HPLC-ESI-MS (RP-HPLC-electrospray ionization mass spectrometry) as 1:1 chalcone-GSH
adducts in each case. At pH values below 8.0, the two bis Mannich bases showed higher GSH
reactivity than two 4’-hydroxychalcones. Influence of the nature of the amino groups, the ring-B
substituents and pH of the medium on reactivity was also investigated. The findings could serve
as useful structure-activity information for subsequent molecular modification of thiol-reactive
4
’-hydroxychalcones.
Keywords: hydroxychalcones, Mannich bases, glutathione, RP-TLC, RP-HPLC-ESI-MS
Introduction
mechanisms: (i) enzymatic conjugation, (ii) increased
oxidation to glutathione disulfide (GSSG), (iii) inhibition
of its synthesis and (iv) non-enzymatic reactions with
Hydroxychalcones are intermediary compounds of the
biosynthetic pathway of the natural flavonoids. The wide
1
12
electrophilic species. Electrophilic compounds such
range of biological activities of both naturally occurring and
as α,β-unsaturated ketones can be converted to GSH
conjugates by nucleophilic attack of sulfhydryl (-SH)
group, which pKa in aqueous solution is about 9.0 and the
low-energy d orbitals of the cysteinyl sulfur atom favors
2,3
synthetic analogues, among other cytotoxic, antitumor,
4
,5
5,6
anti-inflammatory and chemopreventive (antioxidant)
properties are well documented in the literature.
7
-9
13
Chalcones are α,β-unsaturated ketones and representatives
of this class of compounds have demonstrated a preferential
reactivity towards thiols in contrast to amino and hydroxyl
the spontaneous Michael reaction. Reduced GSH levels
are related to cytotoxic effects resulted from a wide range
of signaling pathways as activation of apoptosis through
mitochondrial pathways or by rendering sensitized cells
7
groups. Thus interactions with nucleic acids may be absent
14
which could eliminate the important genotoxic side effect
particularly vulnerable to oxidative damage.
10
which have been associated with certain anticancer drugs.
Aminoalkylation reactions can change lipophilicity
of a drug leading to an improved delivery of a drug to the
GSH is a water-soluble tripeptide and the prevalent
non-protein thiol present in mammalian systems at
15
active sites in the human body. Solubility of the Mannich
bases in water could be further increased as a result of
-1 11
concentrations as high as 10 mmol L . Besides that, the
reduced form of GSH plays central roles in the antioxidant
defense mechanism and it may be depleted by four primary
1
6
their protonation to form the respective ammonium salts.
They can also act as prodrugs forming reactive derivatives
17
via deaminomethylation or deamination processes. It is
therefore no surprise that Mannich reaction has become a
*
e-mail: pal.perjesi@aok.pte.hu

Vol. 28, No. 6, 2017
Bernardes et al.
1049
tool to improve the biological activity of many drugs as
tetracyclines and doxorubicin.
spectrometer (Bruker Optik GmbH, Leipzig, Germany).
18,19
1
13
H and C NMR spectra were recorded on a BrukerAvance
1
13
Recent literature has shown phenolic Mannich bases
derived from chalcones analogues to have remarkable
III 500 (500.15/125.77 MHz for H/ C) spectrometer
(Bruker Optik GmbH, Ettlingen, Germany). The melting
points were measured on a Barnstead Electrothermal 9100
melting point apparatus (San Francisco, USA).
20-24
cytotoxic potencies towards cancer cell lines.
However,
the exact mechanism by which chalcone compounds
and derivatives exert their cytotoxic effects in cancer
cells remains unclear. In order to investigate how
GSH-reactivity is affected by Mannich-type modification
of 4’-hydroxychalcones, bis Mannich derivatives (3 and 4)
of 4’-hydroxychalcones 1 and 2 have been synthesized.
Previous investigations showed 1 as a potent inhibitor of
Synthesis of compounds
4’-Hydroxychalcones 1 and 2 as well as their bis
Mannich derivatives 3 and 4 (Figure 1) were synthesized in
Department of Chemistry, Universidade Federal de Goiás,
Goiânia-GO, Brazil.
25
MDA-MB-231 cells (with IC of 3.8 µM). Furthermore,
50
chalcone 2 have been reported as a relatively potent
Hydroxychalcones 1 and 2 were synthesized by
Claisen-Schmidt condensation as previously described
17
inhibitor (with IC of 17.9 µM) of PC-3 cell. In the same
50
2
6
study the authors could not determine effective inhibitory
by Kumar et al. with slight modifications. Equimolar
portions of 4-hydroxyacetophenone (10 mmol) and
4-chlorobenzaldehyde(10mmol)or3-methoxybenzaldehyde
(10 mmol) were added to 15 mL of 40% NaOH methanolic
solution. The reaction mixtures were stirred at room
temperature for 24 h, when TLC indicated the end of the
reactions. Then, cold water (5 mL) was added to the media
and acidified with 10% hydrocloric acid until pH 3. The
resulting yellow solids were crystallized from methanol to
obtain the expected hydroxychalcones 1 and 2 in 87 and
63% yields, respectively. The structures of synthesized
chalcones were confirmed based on melting point
measurement and spectroscopic methods by comparison
1
3
action of compound 1. Since the C nuclear magnetic
resonance (NMR) shifts, reflecting electron density, of the
β-carbon atoms of the two compounds are rather similar
(Dd = 1.5 ppm), the compounds are expected to show similar
reactivity towards GSH. Whether or not GSH-reactivity of
the compounds can play an important role in determining
the related cytotoxic activities, comparative cell-free GSH
reactivity of the two compounds was planned to perform.
Furthermore, to the best of our knowledge, there are no
studies reporting on non-enzymatic GSH-adducts formation
of Mannich bases of 4’-hydroxychalcones. In order to
investigate the nature of such Michael addition reactions,
the present study was carried out with the aim of detecting
and identifying GSH conjugates of the bis Mannich bases
26-28
with literature data.
3
and 4 in cell free incubations carried out at different pH
(2E)-3-(4-Chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-
1-one (1)
and also comparing them to GSH-reactivity of the parent
hydroxychalcones 1 and 2.
In order to separate and monitor formation of
the expected GSH-adducts, a reversed-phase thin
layer chromatographic (RP-TLC) method with double
-1
Yield: 87%; mp 186-188 °C; IR (KBr) n / cm 3436,
1
1647, 1564; H NMR (500 MHz, DMSO-d ) d 8.09 (d, 2H,
6
J 8.80 Hz, H2’, H6’), 7.96 (d, 1H, J 15.59 Hz, Hβ), 7.92
(d, 2H, J 8.50 Hz, H6, H2), 7.69 (d, 1H, J 15.59 Hz, Hα),
7.53 (d, 2H, J 8.50 Hz, H3, H5), 6.92 (d, 2H, J 8.80 Hz,
(fluorescence and ninhydrin) visualization and a reversed-
13
phase high performance liquid chromatographic (RP-
HPLC) method with ultraviolet-visible (UV-Vis) detection
has been developed and applied. The structure of the
expected chalcone-GSH conjugates was confirmed by
HPLC-ESI-MS (HPLC-electrospray ionization mass
spectrometric) analysis.
H3’, H5’); C NMR (126 MHz, DMSO-d ) d 187.5 (C=O),
6
162.8 (C4’), 141.7 (Cβ), 135.2 (C4), 134.4 (C1), 131.7
(C2’, C6’) 130.9 (C2, C6), 129.5 (C1’), 129.4 (C3, C5),
123.4 (Cα), 115.9 (C3’, C5’).
(2E)-1-(4-Hydroxyphenyl)-3-(3-methoxyphenyl)prop-2-en-
1
-one (2)
-1
Experimental
Yield: 63%; mp 160-162 °C; IR (KBr) n / cm 3299,
1
1653, 1610, 1279; H NMR (500 MHz, DMSO-d ) d 10.43
6
Material and methods
(s, 1H, OH), 8.08 (d, 2H, J 8.69 Hz, H2’, H6’), 7.91 (d, 1H,
J 15.58 Hz, Hβ), 7.66 (d, 1H, J 15.58 Hz, Hα), 7.44-7.01
(m, 4H, H2, H4, H5, H6), 7.40 (ddd, 1H, J 8.17, 7.30,
1.75 Hz, H4’), 7.35 (d, 2H, J 8.17 Hz, H3, H5), 7.01 (m, 2H,
H3’, H5’), 6.90 (d, 2H, J 8.69 Hz, H3’, H5’) 3.82 (s, 3H,
All commercially available reagents were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Infrared
(
IR) spectra were performed on a Bruker IFS-55 FT

1
050
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones
J. Braz. Chem. Soc.
Figure 1. Synthetic route of Mannich bases (3) and (4) and their precursors, 4’-hydroxychalcones (1) and (2). Reagents and conditions: (i) yield 87%,
+
-
4
-ClC H CHO/NaOH/MeOH, 24 h (reflux); (ii) yield 63%, 3-OCH C H CHO/NaOH/MeOH, 24 h (reflux); (iii) yield 78%, CH =N (CH )Cl /ACN, 78 h
6
4
3
6
4
2
3
(
reflux), HCl(g)/diethyl ether; (iv) yield 52%, C H NO/formalin/HCl/EtOH, 96 h (reflux), HCl(g)/diethyl ether.
4 5
1
3
OCH ); C NMR (126 MHz, DMSO-d ) d 187.6 (C=O),
4 in 52% yield. The yellow solid was crystallized from a
3
6
1
62.7 (C4’), 160.1 (C3), 143.2 (Cβ), 136.8 (C1), 131.7
mixture of water and methanol.
(C2’, C6’), 130.4 (C5), 129.6 (C1’), 122.9 (Cα), 122.0 (C6),
1
16.9 (C2), 115.9 (C3’, C5’), 113.7 (C4), 55.8 (OCH3).
(2E)-1-[3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl]-
3
-(4-chlorophenyl)prop-2-en-1-one hydrochloride (3)
-1
The bis Mannich chalcone 3 was synthesized
Yield: 78%; mp 110-113 °C; IR (KBr) n / cm 3418,
1
from the corresponding hydroxychalcone 1 using the
Mannich reagent N,N-dimethylmethyleneiminium
chloride, also known as Eschenmoser’s salt, previously
2703, 1657, 1568; H NMR (500 MHz, DMSO-d ) d 8.12
6
(s, 2H, H2’, H6’), 7.99 (d, 1H, J 15.41 Hz, Hβ), 7.92 (d,
2H, H2, H6), 7.65 (d, 1H, J 15.41 Hz, Hα), 7.52 (d, 2H,
J 8.43 Hz, H3, H5), 4.14 (s, 4H, H7’, H8’), 2.64 (s, 12H,
2
prepared by a literature procedure. The Mannich reagent
13
N,N-dimethylmethyleneiminium chloride (2.0 mmol) was
dissolved in a solution of hydroxychalcone 1 (1.0 mmol)
in acetonitrile (10 mL) and the mixture was heated under
reflux for 78 h. The solution was concentrated followed
by the addition of a hydrogen chloride solution in diethyl
ether to form the corresponding hydrochloride salt 3 in 78%
yield. The yellow solid was crystallized from a mixture of
water and ethyl acetate.
H9’, H10’, H11’, H12’); C NMR (126 MHz, DMSO-d )
6
d 195.3 (C=O), 172.0 (C4’), 143.4 (Cβ), 136.0 (C4), 135.5
(C2, C6), 135.5 (C1’), 130.0 (C3, C5), 130.0 (C1), 129.0
(C2’, C6’), 121.5 (C3’, C5’), 119.1 (Cα), 58.0 (C9’, C10’,
C11’, C12’), 42.4 (C7’, C8’).
(2E)-1-[4-Hydroxy-3,5-bis[(morpholin-4-ylmethyl)phenyl]-
3-(3-methoxyphenyl)prop-2-en-1-one dihydrochloride (4)
-1
In order to obtain compound 2 as a Mannich base
containing morpholino group as the amine reactants,
synthesisofthecompound4followedtheclassicalconditions
Yield: 52%; mp 195-199 °C; IR (KBr) n / cm 3140,
1
2449, 1663, 1577, 1271; H NMR (500 MHz, DMSO-d )
6
d 8.49 (s, 2H, H2’, H6’), 8.16 (d, 1H, J 15.41 Hz, Hβ), 7.73
(d, 1H, J 15.41 Hz, Hα), 7.57-7.03 (m, 4H, H2, H4, H5,
29
of the Mannich reaction for phenolic compounds. First,
morpholine (2.4 mmol) in ethanol (15 mL) was heated
under reflux with formaldehyde (3.0 mmol) for 2 h and
then, hydroxychalcone 2 (1.2 mmol) and few drops of 37%
hydrochloric acid (0.15 mL) were added to the reaction
medium and stirred for 96 h. The solution was concentrated
followed by addition of a hydrogen chloride solution in dry
diethyl ether to form the corresponding hydrochloride salt
H6), 4.46 (s, 4H, H7’, H12’), 3.87 (s, 3H, OCH ), 3.79 (t,
3
4H, H9’, H10’, H14’, H15’), 3.08 (t, 4H, H8’, H11’, H13’,
13
H16’); C NMR (126 MHz, DMSO-d ) d 198.1 (C=O),
6
162.8 (C4’), 159.3 (C3), 145.5 (Cβ), 136.4 (C2’, C6’) 136.0
(C1), 130.4 (C5), 128.8 (C1’), 122.1 (Cα), 117.9 (C3’, C5’),
117.1 (C2), 113.6 (C4), 63.7 (C8’, C11’, C13’, C16’), 55.7
(OCH ), 51.6 (C9’, C10’, C14’, C15’), 43.3 (C7’, C12’).
3

Vol. 28, No. 6, 2017
Bernardes et al.
1051
Single crystal X-ray diffraction
were mixed with a freshly prepared methanol solution of
.01 mmol (1 equiv.) of compounds 1or 2in a final volume of
1 mL. The final concentrations of GSH and tested compounds
0
Single crystals of compounds 3 and 4 were selected for
the X-ray diffraction experiment. The data were collected
on a Bruker-AXS Kappa Duo diffractometer with anAPEX
II CCD detector using graphite monochromated Mo Kα
radiation. After data acquisition, the crystallographic
softwares were used as follows: Bruker programs SAINT and
-1
were 30 and 10 mmol L , respectively.
All reactions were incubated at 37 °C in a water bath
and the progress of reactions were followed by HPLC at the
0, 30, 60, 120, 180 minutes and 7-day time points.
30
SADABS were used for cell refinement and data reduction.
RP-TLC and HPLC-UV-Vis investigations of the incubates
Multi-scan absorption correction was performed with
the ration between the minimum and maximum apparent
transmission of 0.36 and 0.91 for compounds 3 and 4,
respectively. The structures were solved and refined using
RP-TLC was performed on Merck TLC glass plates
precoated with RP-18 F254 using methanol-0.05%
trifluoroacetic acid:water-0.05% trifluoroacetic acid
(i) 60:40 v/v% and (ii) 50:50 v/v% mixture as eluent for
investigations of the above incubations of (i) compounds 1
and 2, and (ii) for compounds 3 and 4, respectively. For
visualization, UV illumination at 254 nm and ninhydrin
staining were used.
HPLC analysis were performed with an Agilent 1100
(Agilent Technologies, Waldbronn, Germany) integrated
HPLC system equipped with a quaternary pump, a degasser,
an autosampler, and a variable wavelength detector
(UV-Vis/VWD). Data were recorded and evaluated by using
Agilent Chem Station (Rev. B.03.01) software.
31
the programs SHELXS-97 and SHELXL-97, respectively.
Non-hydrogen and hydrogen atoms were refined by
2
full-matrix least-squares method on F , adopting free
anisotropic and constrained isotropic atomic displacement
parameters, respectively. Water and NH hydrogens were
firstly located on the difference Fourier map and then fixed
with that coordinates set. All other CH hydrogens were
added to the parent carbon following a riding model. The
Uiso values for hydrogen atoms bonded to either carbon
(
except methyl groups) or nitrogen were set to 1.2U . In
iso
the case of hydrogens bonded to either oxygen or methyl
carbon atoms their isotropic thermal parameters were set
For monitoring each incubation, the components were
separated on a Zorbax Eclipse XDB-C8 (150 × 4.6 mm,
particle size 5 µm) column (Agilent Technologies,
Waldbronn, Germany). Chromatography was performed
at (i) 20 °C (1 and 2) or (ii) 25 °C (3 and 4). The flow rate
to 1.5U . The structural data of compounds 3 and 4 are
iso
available at CCDC numbers 1479638, 1479615, respectively.
At last, MERCURY and ORTEP-3 were used for structure
32,33
analysis and graphical representations.
Crystal data and
3
-1
refinement parameters of the Mannich bases 3 and 4 are
given in Table S1 (Supplementary Information).
was (i) 1.2 or (ii) 1.0 cm min for chalcones (1 and 2) and
Mannich derivatives (3 and 4), respectively. Detection was
UV (λ = 254 nm). The injection volume was 20 µL.A binary
gradient consisting of mobile phasesA and B (A: water-0.1%
trifluoroacetic acid; B: methanol-0.1% trifluoroacetic
acid) was applied for the chromatographic separation. For
separation of compounds of the hydroxychalcone-GSH
reactions, the following gradient profile was used: 62%A for
17 min followed by a decrease to 47% in 11 min and a further
decrease to 20% for 8 min. The column was equilibrated to
the initial conditions with a 5.5-min linear gradient to 38%
A and an isocratic period of 5 min. The compounds from the
Mannich-chalcone-GSH reactions were separated with the
following gradient profile: 65% A for 5 min followed by a
decrease to 35% in 6 min. The column was equilibrated to
the initial conditions with a 1-min linear gradient to 65% A
and an isocratic period of 7 min.
Reaction of compounds 1-4 with GSH
To evaluate reactivity of the Mannich bases 3 and 4
with GSH at four different pH, a freshly prepared solution
-1
of potassium phosphate buffers (i) 0.01 mol L , pH 7.4,
-1
and (ii) 0.2 mol L , pH 6.1, 7.4 and 8.0, containing GSH
0.03 mmol, 3 equiv.) were mixed with compounds 3 and
in a final volume of 1 mL. The final concentrations of
(
4
-
1
GSH and the Mannich bases were 30 and 10 mmol L ,
respectively. The pH of the GSH solution in the 0.01 mol L
-1
(
pH 7.4) buffer was pH 3.2. The pH of the more concentrated
-1
(0.2 mol L ) buffers (pH 6.1, 7.4 and 8.0) did not change
on dissolving GSH.
To evaluate reactivity of chalcones 1 and 2 with GSH,
34
reactions were performed according to Perjési et al. with
slight modifications. The pH of a solution of 0.03 mmol
HPLC-ESI-MS investigation of the incubates
(3 equiv.) GSH in 250 µL of distilled water was set at pH
(
i) 3.2, (ii) 6.1, (iii) 7.4 and (iv) 8.0 by addition of appropriate
HPLC analyses were performed with a system
controller (LC-NetII/ADC), a pump (PU-980), a manual
-1
volume of 1 mol L NaOH solution. The aqueous solutions

1
052
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones
J. Braz. Chem. Soc.
injector (Rheodyne 7725i) with a 5 µL loop and a diode
array detector (DAD) system (MD-2010) (ABL&E-Jasco
Magyarország Kft., Budapest, Hungary). Data were
evaluated with ChromNAV software (version 1.21) using
Lack of interference of NEM-treated GSH and GSSG
measurement was checked using the highest concentration
(0.4 mmol L ) GSH standard.
-1
HPLC analysis of GSH and GSSG after their
derivatization with OPA to form a fluorescent derivative was
accomplished with anAgilent 1100 (Agilent Technologies,
Waldbronn, Germany) integrated HPLC system equipped
with a quaternary pump, a degasser, an autosampler and
a fluorescent detector. The derivatives were quantified
using isocratic elution on a Zorbax Eclipse XDB-C8
(150 × 4.6 mm, particle size 5 µm) column (Agilent
Technologies, Waldbronn, Germany) at 37 °C. The mobile
TM
TM
an analytical Thermo Scientific Accucore RP-MS
150 × 2.1 mm, particle size 2.6 µm) column. The binary
gradient applied consisted of mobile phases A and B
A: 15% methanol-85% water-0.05% formic acid; B: 90%
methanol-10% water-0.05% formic acid). Chromatography
(
(
-1
was performed at 40 °C and flow rate was 0.250 mL min .
The detection was performed at 260 nm.
A Waters TQ-S triple-quadrupole mass spectrometer
with electrospray ionization in the positive ion mode
-
1
phase consisted of 15% (v/v) methanol in 25 mmol L
sodium hydrogen phosphate pH 6.0. The flow rate was
(
Manchester, United Kingdom), specifically with a
3
-1
capillary voltage of 3.00 kV at 150 °C was used for mass
detection. The following electrospray parameters were
1.0 cm min . The injection volume was 10 µL. The
response of fluorescence was observed with an excitation
and emission wavelengths of 350 and 420 nm, respectively.
Data were recorded by the use of Agilent Chem Station
(Rev.A.10.02) software. The GSH and GSSG levels were
quantified from the standard calibration curve using the
corresponding peak areas. Each value is the average of
two independent experiments. The data are presented as
mean values.
kept constant during the analysis: cone gas (N ) flow rate
2
-1
-1
of 150 L h and desolvation gas of 500 L h ; desolvation
temperature of 500 °C. The scan was monitoring m/z values
ranging from 100-1500. Data acquisition was carried out
with MassLynx V4.1 software.
Determination of GSH and GSSG levels
Measurement of GSH and GSSG content of the
samples was performed by a slight modification of the
Results and Discussion
35
fluorimetric assay of Rozmer et al. which was based on the
quantification of glutathione levels by RP-HPLC method
after derivatization with ortho phtalic aldehyde (OPA).
In this assay, the investigated samples were prepared
Mannich bases 3 and 4 were prepared from the
appropriate 4’-hydroxylchalcones (1 and 2) using
Eschenmoser’s salt (for 3) or formaline and morpholine
(for 4). The obtained compounds were isolated as
hydrochloride hydrate salts, as shown in Figure 2.
Compound 3 was solved in the orthorhombic space group
-1
by diluting 10 µL of the incubated samples at 0.2 mol L
phosphate buffer pH 7.4 or 3.2 to a final volume of 1 mL.
For GSH determination, to 100 µL of the sample,
C222 with one base unit, three water molecules and
1
-1
1
.0 mL of 0.1% EDTA in 0.1 mol L sodium hydrogen
one chloride anion per asymmetry unit. The structure of
compound 4 crystallized in the triclinic space group P-1
containing one molecule of Mannich base prepared from the
corresponding hydroxychalcone, one water molecule and
two chloride anions in the asymmetric unit. In the structure
of 3, one of the dimethylamino moiety is protonated, while
the other one is neutral. On the other hand, in 4, both amino
groups as morpholino groups are protonated.
The hydrogen bonding pattern in the three dimension-
network of 3 and 4 plays an important role in the assembly
of the crystal structures reported in this study. The analysis
of the hydrogen bonding pattern in 3 revealed that there is
an intramolecular interaction by means of N2’-H2’1···O1
contact between the hydrogen H2’1 of the protonated
phosphate, pH 8.0, was added. To 20 µL portion of this
-
1
mixture, 300 µL of 0.1% EDTA in 0.1 mol L sodium
hydrogen phosphate and 20 µL of 0.1% OPA in methanol,
was added. The reaction mixture was incubated at 25 °C
in dark for 15 minutes.
For GSSG determination, the thiol group of GSH had to
be modified by N-ethylmaleimide (NEM) reaction to form
non-OPA-reactive product. Then, 100 µL of the sample was
-1
incubated at 25 °C with 100 µL of 40 mmol L of NEM
-1
for 25 min in dark. To this mixture, 375 µL of 0.1 mol L
of NaOH was added. To 20 µL portion of this mixture,
-1
3
00 µL of 0.1 mol L of NaOH and 20 µL of 0.1% OPA in
methanol, was added. The reaction mixture was incubated
at 25 °C in dark for 15 minutes. Standard solutions of GSH
dimethylamino group and the oxygen O1 of the hydroxyl
moiety of the phenol ring forming a S (6) motif. Also, the
hydroxyl group of the phenol ring donates one hydrogen
bond to another unit of Mannich base 3 by means of
-
1
-1
1
1
(
0.4-0.025 mmol L ) and GSSG (0.3-0.00015 mmol L )
-1
freshly prepared dissolved in 0.1 mol L phosphate buffer
pH 7.4 or 3.2 were used for determining the calibration curves.

Vol. 28, No. 6, 2017
Bernardes et al.
1053
Figure 2. Asymmetric units of compounds 3 and 4. Only non-hydrogen atoms are labeled and their ellipsoids are at 50% probability level. The hydrogen
atoms are depicted as spheres of arbitrary radius.
O1-H1OH···N1’. In this case, the water O1W and the
chloride anion also assist in the connection of these two
base units. The water O1W is bridging the dimethylamino
group of one base to the chloride anion Cl1 by means of
O1W-H11W···N1’, while this same anion is connecting
the two base units by being an acceptor of hydrogen bond
through the contact N2’-H2’1···Cl1. Thus, this complex
hydrogen bonding network gives rise to a ribbon made up
of two base units connected to each other where the water
and chloride hydrogen bonding functionalities help in the
stabilization of the crystal structure (Figure 3a). In addition,
another characteristic of this crystal structure is that,
there is formation of channels filled with water molecules
and chloride anions between the ribbons (Figure 3b). At
last, the water molecules O2W and O3W, which are not
involved in the ribbon assembly, are engaged in hydrogen
bonding interactions with each other and with O1W in the
channels. The metrics of hydrogen bonds found in 3 is
shown in Table 1.
In the case of compound 4, due to the fact that both amino
functions are protonated, there is no classical intramolecular
hydrogen bond. Only intermolecular interactions of this type
Figure 3. (a) A ribbon made up of two base units connected through water and chloride species in compound 3. The arrows indicate the intramolecular
contact exhibited by 3; (b) channels filled with water molecules and chloride anions between two ribbons stacked on top of each other; (c) chain motif
connecting the base units in compound 4 where the chloride anions and the water molecule act as a bridge between the base units; (d) layers made up of
chain motifs involving both morpholine rings in compound 4. Only the hydrogen atoms involved in hydrogen bonding interactions are shown.

1
054
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones
J. Braz. Chem. Soc.
occur in the crystal structure. To assemble this compound, the
hydroxyl group of the phenol ring is donating one hydrogen
bond to the water molecule O1W through the interaction
O3-H3···O1W. Besides being engaged in hydrogen bonding
as acceptor, the water molecule is also a hydrogen bond donor
to both chloride anions by means of O1W-H11W···Cl1
and O1W-H21 ···Cl2 contacts. In addition, the amino
W
nitrogen N1’ of one morpholine ring is hydrogen bonded
to the chloride anion through the N1’-H1’N···Cl1 contact
(
Figure 3c). Likewise, the nitrogen N2’ of the second
morpholine ring donates a hydrogen bond to the second
chloride anion Cl2 by means of N2’-H2’N···Cl2. In this way,
this set of hydrogen bonding interaction leads to the formation
of chains which connect the units of Mannich base 4 using
the water molecules and chloride anions as bridge between
them (Figure 3c). On the whole, this crystal structure exhibit
layers (Figure 3d) made up of chain motifs involving both
morpholine rings of this Mannich base. The metrics of
hydrogen bonds found in compound 4 is shown in Table 1.
It is also important to mention that the crystal structure of
the Mannich bases 3 and 4 is reported for the first time here,
while the crystal structure of the precursor of compound 3,
Figure 4. Superposition of the chalcone skeleton present in compounds
1, 3 and 4. Only non-hydrogen atoms are depicted.
1
with their chemical structures. In the H NMR spectra,
a coupling constant higher than 15.4 Hz for the vinyl
H-atoms confirmed the (E)-configuration of compounds.
The methylene protons appeared as singlets at d 4.14 and
13
4.46 ppm, respectively. In the C NMR spectra, besides
the appearance of four more carbons of the morpholino
and dimethylamino groups, the carbon peaks of olefinic
group were also observed at d 143.4 and 119.1 ppm (3) and
d 145.5 and 122.1 ppm (4). In the IR spectra, the presence
of the C=C functional group was confirmed by the υC=C
36
chalcone 1, has been already known. The planarity of the
chalcone skeleton (Figure 4) of compounds 3 and 4 is very
36
similar to the one reported by Jasinski et al. (the root mean
-
1
square deviation (RMSD) for their non-hydrogen atoms are
peaks appearing about 1600 cm . Conjugation of the
0
.331 and 0.259, respectively).
As previously shown, aminomethylation of phenols
C=C to the carbonyl group decreases the C=O stretching
-1
frequencies below 1710 cm . Therefore, the strong υC=O
2
1
13
-1
occurs preferably at the ortho position. H NMR, C NMR
and IR spectra of compounds 1, 2, 3 and 4 are in accordance
peaks at 1657 (3) and 1663 cm (4) in the IR spectra support
the α,β-unsaturated ketone structure of the compounds.
Table 1. Hydrogen-bond geometry in compounds 3 and 4
D-H···A
D-H / Å
H···A / Å
Compound 3
2.59
D···A / Å
D-H···A / degree
O -H1oh···N1’
0.85
0.84
0.85
0.86
0.91
0.91
0.86
0.84
3.33(6)
3.42(8)
3.20(9)
3.25(10)
2.91(8)
3.26(6)
2.69(9)
3.06(7)
147
145
128
139
124
139
163
162
1
O -H13w···O2w
2.70
3w
O -H23w···O1w
2.60
3w
O -H22w’···O1w
2.55
2w
N -H2’1···O1
2.30
2’
N -H2’1···Cl1
2.52
2’
O -H11w···N1’
1.85
1w
O -H21w···Cl1
2.25
1w
Compound 4
1.84
O -H ···O1W
0.82
0.91
0.85
0.85
0.91
2.59(2)
3.05(2)
3.06(7)
3.12(7)
3.13(2)
152
168
172
170
162
3
3
N -H1’n···Cl1
2.15
1’
O -H21w···Cl2
2.21
1w
O -H11w···Cl1
2.28
1w
N -H2’n···Cl2
2.25
2’

Vol. 28, No. 6, 2017
Bernardes et al.
1055
The O-H stretching of the phenolic groups was observed
at 3418 (3) and 3140 cm (4).
be ninhydrin-positive spots as well. This latter observation
was in accordance with formation of the expected GSH
conjugates.
-1
In the reactions, the sulfur of GSH is added to
the β-carbon, followed by proton transfer reaction to
Formation of the expected two diastereomeric adducts
on the prochiral faces of the enone moiety was confirmed by
RP-HPLC. The RP-HPLC chromatograms of the incubates
showed three major peaks attributed to two isomeric
adducts and the starting chalcones with the following
retention times: GS-1a: 26.67, GS-1b: 27.52, 1: 35.45;
GS-2a: 14.15, GS-2b: 15.31, 2: 33.35; GS-3a: 3.94,
GS-3b: 4.59, 3:13.55; GS-4a: 3.03, GS-4b: 3.35, 4: 11.63
(Figures 5 and 6).
3
7
the α-carbon generating a chiral center. RP-TLC
of the incubates supported formation of the expected
diastereomeric conjugates in each case. Visualization of
the plates by UV (254 nm) illumination indicated presence
of the chalcones (1 and 2) and the Mannich derivatives
(
3 and 4) with lower retention factors (R ): 1: 0.05, 2: 0.08,
f
3: 0.21 and 4: 0.24. Besides, there could be seen a new
spot in each case: 1: R 0.31, 2: R 0.45, 3: R 0.53 and
f
f
f
4
: R 0.56, indicating the more polar character of the latter.
The structures of the expected GSH adducts were
confirmed by HPLC-ESI-MS (positive ion mode)
f
The new spots, along with the GSH spots, were proved to
Figure 5. Characterization of GS-1 and GS-2 conjugates by HPLC-ESI-MS analysis. Chromatograms of incubations of compounds 1 (A) and 2 (B) with
-
1
GSH in 0.2 mol L phosphate buffer pH 7.4 at 37 °C at 180 min.

1
056
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones
J. Braz. Chem. Soc.
Figure 6. Characterization of GS-3 and GS-4 conjugates by HPLC-ESI-MS analysis. Chromatograms of incubations of compounds 3 (A) and 4 (B) with
-
1
GSH in 0.2 mol L phosphate buffer pH 7.4 at 37 °C at 180 min.
2+
analysis of the 180 min incubates of compounds 1, 2, 3
and 4 (Figures 5 and 6). Besides the MH peaks of GSH
GS-3 and GS-4 double charged [M + 2H] ions displayed
at m/z 340.63 and 380.74, respectively (Figure 6).
+
(
2
m/z 308.02) and the starting compounds: 1 (m/z 259.05),
(m/z 255.15), 3 (m/z 373.02), 4 (m/z 453.12), ESI-MS
The rate of reactions of the compounds with GSH was
found to be affected by the different pH values. As it is
shown on Figure 6, a rather similar hyperbolic-type profile
of decrease of the area of the HPLC peaks of each chalcone
could be observed at pH 6.1, 7.4 and 8.0. On the contrary,
a striking difference in the reactivity of chalcones 1 and 2,
and the Mannich derivatives 3 and 4 could be observed:
analysis of each incubation confirmed presence of the
respective GSH conjugates. These latter showed MH peaks
of m/z attributed to the respective molecular ion peaks of
GS-1 (m/z 566.04), GS-2 (m/z 562.08), GS-3 (m/z 680.06)
and GS-4 (m/z 760.10) adducts. In addition, in the case of
+

Vol. 28, No. 6, 2017
Bernardes et al.
1057
the Mannich derivatives showed much higher reactivity
than the respective parent hydroxychalcones. The rate of
the reactions was found to be not affected by the ring B
substituents (4-chloro and 3-methoxy) at any pH (Figure 7).
On the other hand, a similar increase of the peak areas
corresponding to the formed GSH-adducts could be
recorded (Figures 8-11).
As previously mentioned, bioactivities of Mannich
bases, e.g. cytotoxicity, is frequently attributed to the
2
,18,22
high reactivity with thiols.
Thus, the nature of the
amino group is an important factor since the rate of
deaminoalkylation is inversely proportional to the basicity
17
of the liberated amine. However, in the tested conditions,
neither dimethylamine (pKa 10.78) nor morpholine
(pKa 8.49) influenced reactivity of compounds 3 and 4
with GSH and no deamination-addition products (mono-
or diadducts) were detected under tested conditions.
Intramolecular and intermolecular interactions (hydrogen
bonds) between amino and hydroxyl groups should not be
1
3
The obtained data are in accordance with the C NMR
1
3
data of the compounds. It is known that the C NMR
38
shift values are sensitive probes of electron density. The
1
3
C NMR chemical shifts of the β-carbon atoms of the
parent 1 and 2 are very similar: 141.7 and 143.2 ppm,
respectively. The corresponding chemical shift values for 3
and 4 are 143.4 and 145.5 ppm, respectively. The difference
1
7
discarded to retard or prevent the deamination process.
RP-HPLC method after derivatization with OPA was
used to determine the GSH and the GSSG levels in the
13
in the C shift values between 2 and 1, and 4 and 3 is 1.5 and
.1 ppm, respectively. Introduction of the Mannich-base
35
2
incubations. Reduced GSH reacts with OPA at pH 8.0
while GSSG reacts only at pH 12. For GSSG measurement,
GSH has to be previously modified by reaction with NEM
to form OPA-unreactive derivative. Concentrations of GSH
and GSSG were measured simultaneously in incubation
moiety into the molecules increased the electrophilic
character of the β-carbon atoms of hydroxychalcones
13
practically by the same degree. The difference in the C
shift values between 3 and 1, and 4 and 2 is 1.7 and 2.3 ppm,
respectively. Such spectroscopic data are in accordance
with the similar reactivity of the two parent chalcones (1
and 2) and the two Mannich derivatives 3 and 4.
-1
samples performed in 0.2 mol L phosphate buffer pH 7.4 at
37 °C. Measurements were performed at the 0, 30, 60, 120,
180 min and 7-day time points in two parallel incubations.
Figure 7. Time-dependent profile of decrease of HPLC peak area of compounds 1 and 2 and their bis Mannich derivatives 3 and 4 in reactions with GSH
at pH 3.2, 6.1, 7.4 and 8.0.

1
058
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones
J. Braz. Chem. Soc.
Figure 8. Time-dependent profile of HPLC peak area of adducts formed in the reaction of 4’-hydroxychalcone 1 and GSH at pH 3.2, 6.1, 7.4 and 8.0.
Figure 9. Time-dependent profile of HPLC peak area of adducts formed in the reaction of 4’-hydroxychalcone 2 and GSH at pH 3.2, 6.1, 7.4 and 8.0.
Figure 10. Time-dependent profile of HPLC peak area of adducts formed in the reaction of Mannich base 3 and GSH at pH 3.2, 6.1, 7.4 and 8.0.

Vol. 28, No. 6, 2017
Bernardes et al.
1059
Figure 11. Time-dependent profile of HPLC peak area of adducts formed in the reaction of Mannich base 4 and GSH at pH 3.2, 6.1, 7.4 and 8.0.
The mean GSH and GSSG values of the reactions with
compounds 1, 2, 3 and 4 are shown in Figures 12 and
of α-hydrogen and the retro-Michael reaction would be
favored, which implies that these two factors do not support
the relative stability of presented adducts.
1
3, respectively. As shown in Figure 12, the initial GSH
-1
concentrations were between 0.3055 and 0.3314 mmol L ,
while at the 180 min time point, the GSH levels were
found to be somewhat higher in the chalcone (1 and 2)
incubations: 0.211 and 0.2009 mmol L , respectively.
The results showed a considerable decreased amount of
GSH after 7 days, possibly as consequence of an increase
of the oxidized form GSSG. Accordingly, the measured
GSSG levels substantially increased in the 7 day samples
In a recent study, the equilibrium and reaction kinetics
of Michael addition reactions between chalcone analogues
and thiol compounds, including GSH, were investigated
-1
41
using density functional theory (DFT) calculations. While
the study in gas phase resulted in high deviated Gibbs free
energies (DG) from experimental values in water, the water
solvation model rendered well correlated measurements of
DG. Then, it is reasonable to expect that solvation effects
probably account to a significant extent for the reactivity
of the Mannich bases 3 and 4 with GSH in aqueous
environment. In addition, besides the solvent factor,
electrostatic interactions with local charges also is known
(Figure 13).
The reaction rate of α,β-unsatured ketones with
GSH was reported to be slower than their modified
Mannich derivatives, possible as a result of decrease of
the electron density of the carbonyl and/or stabilization
4
2,43
to strongly modulate cysteine sulfhydryl reactivity.
17,39,40
of the intermediate/conjugate by solvation.
Electron
Previous studies established that while conjugate
addition of the thiolate to the β-carbon to form enolate
intermediate is the rate limiting step of the Michael reaction,
the rate of retro-Michael addition is proved to be limited
by formation of the enolate anions in an elimination from
withdrawing effect of the aminoalkyl groups as well as
hydrogen bond formation between the amino and carbonyl
groups can decrease electron density of the carbonyl
and increase eletrophilicity of the β-carbon, however,
the nitrogen atoms of the Mannich bases 3 and 4 are
significantly protonated at the pH values of the present
14,41,44
conjugated base mechanism (E1cB).
4
5
The extent of ionization of GSH (pKa = 8.9) also
depends on the pH values of the solution. The increased
rate of the Michael addition reactions of the investigated
bis Mannich chalcones (3 and 4) can be rationalized by
higher concentration of thiolate anions. This may be due to
hydrogen bond formation between the phenolic hydroxyl
and the tertiary amino groups which could yield basic
enough sites (e.g., phenolate, and non-hydrogen-bonded/
non-protonated nitrogen atoms) to deprotonate thiols
and/or due to electrostatic interaction with a positively
charged thiolate-stabilizing site, e.g. the protonated amino
13
experiments. Furthermore, the carbonyl C chemical shifts
of the Mannich derivatives appear at higher d values than
those of the 4’-hydroxychalcones (see above). However,
there could be seen substantial difference in GSH-reactivity
of the hydroxychalcones and their Mannich derivatives (see
Figure 7). Therefore, we suggest the decreased electron
density of the carbonyl is not an important contributing
factor for the high reactivity of 3 and 4 and stability of
the GS-3 and GS-4 conjugates. Besides, the hydrogen
.
..
bond (N-H O-C) as well as the electron withdrawing
14,46-48
effect of Mannich moieties would also decrease the pKa
moieties.

1
060
Study of Reactions of Two Mannich Bases Derived of 4’-Hydroxychalcones
J. Braz. Chem. Soc.
Figure 12. GSH levels in each time point (0, 30, 60, 120, 180 min, 7 days) of incubations of compounds 1, 2, 3, 4 and GSH. Determination of GSH
3
5
-1
levels were performed according to Rozmer et al. based on RP-HPLC method after derivatization with OPA. Incubations were performed in 0.2 mol L
phosphate buffer pH 7.4 at 37 °C.
Figure 13. GSSG levels in each time point (0, 30, 60, 120, 180 min, 7 days) of incubations of compounds 1, 2, 3, 4 and GSH. Determination of GSSG
3
5
-1
levels were performed according to Rozmer et al. based on RP-HPLC method after derivatization with OPA. Incubations were performed in 0.2 mol L
phosphate buffer pH 7.4 at 37 °C.

Vol. 28, No. 6, 2017
Bernardes et al.
1061
Recent literature reveals that neighboring positive
charges were shown to induce pKa shifts of thiols
increasing its reaction with electrophiles and change
the transition state energy affecting the reaction kinetics
whereas the opposite effect was observed with nearby
of compounds 1-4] are available free of charge at
http://jbcs.sbq.org.br as PDF file.
Crystallographic data for the structures 3 and 4 were
deposited in the Cambridge Crystallographic Data Centre
as supplementary publication numbers CCDC 1479638
and 1479615 for compounds 3 and 4, respectively. Copies
of the data can be obtained, free of charge, via www.ccdc.
cam.ac.uk/conts/retrieving.html or from the Cambridge
Crystallographic Data Centre, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033. E-mail:
deposit@ccdc.cam.ac.uk.
4
2,49,50
negative charged groups.
Indeed, positive charged
amino acids proximal to a Cys residue increased the
Michael reaction rate as the protonation of the N atom in
vinyl-substituted pyridine derivatives also promoted higher
42,49
reactivity towards Cys.
Based on this findings and how
the effect of thiolate concentration overwhelms any other
factor on Michael reaction kinetics, it is suggested that
protonated amino groups in compounds 3 and 4 can be
Acknowledgments
49
determinant for their high reactivity with GSH.
Since E1cB reaction takes place by a base-induced
abstraction of an α-hydrogen unusually acidic by
resonance stabilization of the carbanion intermediate
The authors express their special thanks to Coordenadoria
deAperfeiçoamento de Pessoal de Ensino Superior (CAPES)
(Grant No. BEX 6234/15-1) for a fellowship (to A. B.) for
supporting this work. The authors express their sincere thanks
to the Faculty of Medicine (University of Pécs) Research
Fund (Grant No. AOK-KA-213/20) and the Conselho
Nacional de Desenvolvimento Científico Tecnológico
(conjugated base), a slower retro-Michael reaction would
be observed possibly if compounds 3 and 4 destabilize the
enol intermediate rendering more stable GS-3 and GS-4
4
1,51
conjugates.
(CNPq) (Grant No. 478337/2013-2) for providing financial
Conclusions
support. The authors express their thanks for Dr Monika
Kuzma (Institute of Pharmaceutical Chemistry, University
of Pécs) for her advice in the HPLC works.
Present study showed bis Mannich bases 3 and 4 derived
from 4’-hydroxychalcones (1 and 2) had a high spontaneous
reactivity towards GSH, which demonstrated not to be
significantly affected by the basicity of the morpholino
and the dimethylamino groups. Considering how easily the
compounds reacted with GSH, it would seem highly likely
that they can also conjugate with another thiols in cellular
environment. The reactions rendered stable monoadducts
estimated in 85% of GSH conjugation of the starting material
at 7-day time point. Taking into consideration the diverse
biological effects of the investigated hydroxychalcones, it
seems reasonable to suppose thiol-reactivity is not the only
molecular mechanism that is involved in development of
their biological effects. Application of HPLC analysis for
this preliminary screening of the thiolalkylation reaction
of Mannich bases proved to be an efficient and practical
method to be used in further investigations. The findings
could serve as useful structure-activity information for
subsequent molecular modification of thiol-reactive
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