An efficient one-pot synthesis of polyphenolic amino acids and evaluation of their radical-scavenging activity

Article abstract of DOI:10.1016/j.bioorg.2019.102983

A simple and efficient procedure for the synthesis of N-acyl 4-hydroxy, 4-hydroxy-3-methoxy and 3,4-dihydroxy phenylglycine amides by a strategy based on the multicomponent Ugi reaction is proposed. Hydroxybenzaldehyde derivatives were reacted with 4-meth

Full text of DOI:10.1016/j.bioorg.2019.102983

BioorganicChemistry89(2019)102983
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
An efficient one-pot synthesis of polyphenolic amino acids and evaluation of
their radical-scavenging activity
⁎
Luís S. Monteiroa, , Fátima Paiva-Martinsb, Sandra Oliveiraa,b, Inês Machadoa, Marlene Costab
a Chemistry Centre, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b REQUIMTE/LAQV, Department of Chemistry and Biochemistry, Faculty of Science, University of Porto, Rua do Campo Alegre 687, Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Keywords:
A simple and efficient procedure for the synthesis of N-acyl 4-hydroxy, 4-hydroxy-3-methoxy and 3,4-dihydroxy
Polyphenolic amino acids
Hydroxyphenylglycines
Cell-penetrating peptides
Ugi reaction
phenylglycine amides by
a
strategy based on the multicomponent Ugi reaction is proposed.
Hydroxybenzaldehyde derivatives were reacted with 4-methoxybenzylamine, cyclohexyl isocyanide and benzoic
acid or 2-naphthylacetic acid to give Ugi adducts that were treated with trifluoroacetic acid yielding N-acyl
hydroxyphenylglycine amides in good yields. The same procedure using as acid component protocatechuic acid
or hydrocaffeic acid gave N-catechoyl 3,4-dihydroxyphenylglycine amides. The use of N-benzylox-
ycarbonylglycine as acid component allowed the preparation of a 3,4-dihydroxyphenylglycyl dipeptide deri-
vative. Radical-scavenging activity studies of the polyphenolic amino acid derivatives showed a sharp increase in
activity with the increase in number of hydroxyl or catechol groups present. Cyclic voltammetry experiments
established a correlation between oxidation peak potentials and the radical-scavenging activity.
diHydroxyphenylglycyl peptides
Radical-scavenging activity
Oxidation potential
1. Introduction
junctions including the blood-brain barrier [7,10]. The antioxidant
action of SS peptides in scavenging radical oxygen species and in-
Phenolic amino acids represent a wide class of compounds endowed
with interesting biological activities. The natural phenolic amino acid,
tyrosine displays antioxidant properties [1], performing vital anti-
oxidant functions inside lipid bilayers and protecting cells from oxi-
dative destruction [2]. Non-proteinogenic phenolic amino acids are
crucial components of certain peptidic natural products such as the
vancomycin group of antibiotics [3] (4-hydroxyphenylglycine) and of
cell-penetrating peptides (CPPs) [4]. CPPs are short, nontoxic peptides
containing cationic and non-coded hydrophobic amino acids that can
carry small molecules. They are able to cross, not only the cellular
membrane, but also to target inside specific cellular organelles such as
nucleus and mitochondria. Of the CPPs tested as mitochondria targeted
antioxidants [5]. the Szeto-Schiller (SS) peptides are the most pro-
mising [6]. There are a number of surprising features about these
peptides. Despite being water soluble, with a net charge of 3+, they are
readily taken up by all cells via passive diffusion [7–9]. The presence of
a D-amino acid and non-coded amino acids at the tips of the peptide
renders them resistant to aminopeptidase activity. The mechanism be-
hind their cell permeability is unclear, but the aromatic rings may serve
as electron cages to shield the cationic charges via cation-π interaction.
These SS peptides may be viewed as “cloaked” or “stealth” as they can
evade cellular membranes, even penetrating cell barriers with tight
hibiting low density lipoprotein oxidation, is attributed to the 2,5-di-
methyl-L-tyrosine residue.
The ortho-dihydroxyaryl function present in catechols gives com-
pounds with a broad scope of properties that confer important bio-
chemical functions [11]. Such properties result from; the ability to es-
tablish reversible equilibria at moderate redox potentials and pHs;
irreversible cross-linking through complex oxidation mechanisms; ex-
cellent chelating properties; interaction of the vicinal hydroxyl groups
with surfaces of different nature. Despite these properties, little atten-
tion has been given to amino acids with the ortho-dihydroxyaryl func-
tion, such as 3,4-dihydroxyphenylglycine. This non-natural amino acid
has been studied as copper ligand [12] and as a substrate for tyrosinase,
being converted to 3,4-dihydroxybenzaldehyde via spontaneous dec-
arboxylation of the enzymatically generated ortho-quinone [13].
Amino acids coupled with phenolic or catecholic groups are bioac-
tive substances involved in suppression of deleterious effects of oxida-
tive stress [14–17] and have a wide range of biological activities such as
anti-atherogenic [18], anticancer [19] and antimicrobial [20–23].
Studies have confirmed that conjugation between different types of
compounds such as amino acids and phenolic acids is useful, not only to
investigate structure activity relationships, but can also constitute a
strategy to improve antioxidant efficiency and bioactivity [24,25].
⁎ Corresponding author.
E-mail address: monteiro@quimica.uminho.pt (L.S. Monteiro).
https://doi.org/10.1016/j.bioorg.2019.102983
Received 26 February 2019; Received in revised form 11 April 2019; Accepted 10 May 2019
Availableonline11May2019
0045-2068/©2019PublishedbyElsevierInc.

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
Recently, we established an innovative synthetic strategy that allowed
the preparation of a library of N-phenolic and N-catecholic dehy-
droamino acid derivatives [26].
dialkylglycines by cleavage of Ugi adducts with neat TFA [38]. Treat-
ment in these conditions of N-benzoyl-N,α,α-trialkylglycine amides
formed by reaction of 4-methoxybenzylamine, benzoic acid, cyclohexyl
isocyanide and the more bulky dibenzylketone, resulted in removal of
the methoxybenzyl group without amide cleavage. The authors re-
ported that in neat TFA the N-alkyl group of the bulkier N-benzoyl, N-
(4-methoxybenzyl) α,α-dibenzylglycine derivatives cleaves faster than
their amide bond. They concluded that for acidolytic cleavage of the C-
terminal amide to occur, the presence of an alkyl/aryl substituent at the
amino acid nitrogen atom is required. Thus, the phenyl group directly
linked to the α-carbon in adduct 1a′ seems to exert a steric effect
comparable to that reported for the α,α-dibenzyl moiety in N-acyl-
N,α,α-trialkylglycine amides.
The development of synthetic strategies that allow the synthesis of
new amino acids with phenolic or catecholic side chains and conjugates
of these amino acid with phenolic or cathecolic groups would give rise
to a new repertoire of interesting synthetic building blocks. These may
have a wide application in the preparation, not only of specific cellular
organelle targeted antioxidant peptides [5], but can also have other
intrinsic biological activities or broader applications, such as: cross-
linkers with proteins or carbohydrates [27]; transition metal ligands
[28]; or used in the design of new peptide hydrogels that mimic mussel
adhesive proteins [29–32].
Compound 1a could also be obtained in a one-pot procedure con-
sisting of the condensation reaction, followed by evaporation of the
solvent at reduced pressure and treatment of the residue with TFA
(Scheme 1). tert-Butyl (4-formyl-2-methoxyphenyl) carbonate and di-
tert-butyl (4-formyl-1,2-phenylene) dicarbonate (Scheme 1, compounds
b and c, respectively) were reacted in this one-pot procedure to give N-
benzoyl, 4-hydroxy-3-methoxy and N-benzoyl, 3,4-dihydroxy phe-
nylglycine cyclohexylamides in 76% and 66% yields, respectively
(compounds 1b and 1c). An attempt to carry out the condensation re-
action using the highly sterically hindered di-tert-butyl (3-formyl-1,2-
phenylene) dicarbonate (compound d) and benzoic acid gave only trace
amounts of the corresponding Ugi adduct (compound 1d′).
2. Results and discussion
A simple approach to obtain new hydroxyphenylglycines and/or
conjugates of hydroxyphenylglycines with phenolic or catecholic acids
would be a strategy where simple natural phenols are joined together in
a one-step economical way. One such multicomponent reaction is the
Ugi reaction, first reported by Ivar Ugi [33,34]. It is classified as an
isocyanide-based multicomponent reaction, consisting of the simulta-
neous joining of 4 different classes of compounds: an aldehyde; a car-
boxylic acid; an amine and an isocyanide.
Lambruschini et al. [35] using the Ugi reaction, were able to pre-
pare a series of complex polyphenols containing two to four hydroxy-
substituted aryl groups. This methodology, using hydroxyl substituted
benzoic acids and benzaldehydes, gave hydroxylated bis-amides and
their effect on quenching radicals and DNA oxidation was estimated
[36]. However, derivatives with the ortho-catechol moiety as the amino
acid side chain have not been report.
The reaction was carried out substituting 2-naphthylacetic acid for
benzoic acid and using di-tert-butyl (4-formyl-1,2-phenylene) dicarbo-
nate to give the N-(2-naphthyl)acetyl, 3,4-dihydroxyphenylglycine
amide in 78% yield (compound 2c, Scheme 1).
The results obtained led us to explore the possibility of using cate-
cholic acids as acid component in the Ugi reaction, namely, proto-
catechuic and hydrocaffeic acid. These cathecolic acids were tested
without hydroxyl protection since, when the acid component is added,
all 4-methoxybenzylamine has been consumed. Thus, tert-butyl (4-for-
mylphenyl) carbonate was reacted in the same conditions, substituting
protocatechuic acid for benzoic acid to give the Ugi adduct 3a′ in
quantitative yield (Scheme 1). Treatment of compound 3a′ with TFA
gave the N-protocatechoyl 4-hydroxyphenylglycine derivative in 72%
yield (compound 3a). By reacting tert-butyl (4-formyl-2-methox-
yphenyl) carbonate or di-tert-butyl (4-formyl-1,2-phenylene) dicarbo-
nate with protocatechuic acid, the corresponding N-protocatechoyl,
hydroxyphenylglycine amides were obtained in good yields (com-
pounds 3b and 3c, respectively). The reaction using hydrocaffeic acid
and di-tert-butyl (4-formyl-1,2-phenylene) dicarbonate gave compound
4c in 74% yield. Thus, the use of di-tert-butyl (4-formyl-1,2-phenylene)
dicarbonate and protocatechuic or hydrocaffeic acid, allows the
synthesis of amino acid derivatives baring two catecholic groups.
Synthesis of compound 4c shows that the steric effect of the phenyl
group linked to the α-carbon induces removal of the methoxybenzyl
group without amide cleavage, even with the less sterically hindered
hydrocaffeoyl substituent as N-acyl group. This was further confirmed
by reacting tert-butyl (4-formylphenyl) carbonate with acetic acid as
carboxylic component. Again, treatment of the Ugi adduct with neat
TFA led to removal of the methoxybenzyl group without amide clea-
vage (compound 5a).
A strategy based on the modified Ugi reaction, using as amine
component 4-methoxybenzylamine and as isocyanide the commercially
available and relatively stable cyclohexyl isocyanide, followed by
treatment of the adducts with trifluoroacetic acid (TFA) has previously
been explored [37,38]. This procedure using as aldehyde component
phenolic or catecholic benzyladehydes and as acid component cate-
cholic acids, could be an efficient approach for the synthesis of novel N-
(catechoyl)-hydroxy and 3,4-dihydroxy phenylglycines. Thus,
a
strategy based on the modified Ugi reaction using phenolic and/or
catecholic benzaldehydes and carboxylic acid components was ex-
plored.
A severe limitation of the application of ortho-catechol moieties in
synthesis is their high instability in basic environment. Thus, due to the
basic nature of the amine component, protection of the hydroxyl
function of the benzaldehyde would be required. O-tert-
Butyloxycarbonylated benzaldehydes can be easily obtained by reaction
of hydroxybenzaldehydes with tert-butyldicarbonate in the presence of
dimethylaminopyridine as catalyst [26]. Furthermore, this temporary
protection can be removed in the subsequent treatment with TFA, a
reagent generally used in peptide synthesis to remove acid-labile pro-
tecting groups [39,40].
A preliminary test was carried out using as reactants tert-butyl (4-
formylphenyl) carbonate, 4-methoxybenzylamine, benzoic acid and
cyclohexyl isocyanide in a 1/1 solution of ethanol and trifluorethanol
(TFE) to give compound 1a′ in high yield (Scheme 1). The proton NMR
spectrum of the compound gave the expected peaks with the diaster-
eotopic CH2 protons of the benzylamine group appearing as two signals
at 4.41 ppm and 4.63 ppm. The Ugi adduct was treated with TFA at
80 °C for 10 min. Removal of TFA gave an oily residue that precipitated
on addition of diethyl ether to give N-benzoyl, 4-hydroxyphenylglycine
cyclohexylamide in good yield (compound 1a, Scheme 1).
The procedure was also carried out using as acid component a N-
protected amino acid, namely, N-benzyloxycarbonylglycine. A Ugi ad-
duct was obtained (compound 6c′, Scheme 2) which, after treatment
with TFA gave a 3,4-dihydroxyphenylglycyl dipeptide derivative in
74% yield (compound 6c, Scheme 2). Thus, the methodology developed
also allows the preparation of hydroxyphenylglycyl dipeptide deriva-
tives.
Treatment of Ugi adduct 1a′ with TFA allowed the simultaneous
removal of the methoxybenzyl and the tert-butyloxycarbonyl groups,
without affecting the cyclohexylamide. This is in agreement with pre-
vious results by Maia et al. in the synthesis of N-acyl-α,α-
In order to establish the antioxidant capacity of the N-acyl hydro-
xyphenylglycines prepared, the radical-scavenging activity of selected
compounds was determined using 1,1-diphenyl-2-picrylhydrazyl
%
(DPPH ) as
a stable radical [41]. The EC50 values (relative
2

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
Scheme 1. Synthesis of Ugi adducts and treatment with trifluoroacetic acid to give N-acyl hydroxyphenylglycine cyclohexylamides.
activity. Thus, the introduction of the catecholic moiety in an amino
acid structure induces a significant increase in its intrinsic radical
scavenging activity.
Correlation between antioxidant profile and redox properties of low
molecular weight antioxidants is well established [42]. Cyclic voltam-
metry has been used as an important tool for the evaluation of anti-
oxidant capacity of this type of antioxidants. Compounds with lower
oxidation potentials are more readily oxidized and usually show better
antioxidant activities. Thus, oxidation peak potentials for the hydro-
xyphenylglycine derivatives were determined by cyclic voltammetry
using a glassy carbon working electrode and measured vs a Ag/AgCl
electrode (Table 1). Experiments were run using as supporting elec-
trolyte a phosphate buffer at the physiological pH of 7.30.
Scheme 2. Synthesis of a 3,4-dihydroxyphenylglycyl dipeptide derivative.
Compound 1a, which showed very weak radical-scavenging ac-
tivity, presented the highest oxidation peak potential (0.703 V). A de-
crease in over 0.2 V in potential occurs when a methoxy group is added
in ortho position to the hydroxyl group (compound 1b) in agreement
with a significant increase in radical-scavenging activity against the
DPPH radical. The substitution of the methoxy group by a second hy-
droxyl group (compound 1c) causes a further significant decrease in the
oxidation potential (0.228 V), which also agrees with the ten-fold in-
crease observed in radical-scavenging activity. Compounds with a
single catecholic group (compounds 2c, 3a and 3b) gave anodic peaks
with similar potentials ca. 0.270 V, that corresponded to similar radical-
scavenging activities. These potentials were lower than the peak po-
tential for the catecholic reference protocatechuic acid (0.324 V).
Compound 3c baring two similar catecholic groups gave only one
anodic peak at a similar potential to the other catecholic compounds
(0.272 V). Thus, the increase in radical-scavenging activity of com-
pound 3c, when compared to the activities of the monocatecholic
compounds, must result from an additive effect of the two catechol
groups.
%
concentration of antioxidant required to lower the initial DPPH con-
centration by 50%) were determined after several reaction times and
compared to the values obtained for protocatechuic acid (Table 1). As
expected, the radical-scavenging activity against the DPPH radical of
the monophenol derivative 1a was very weak. However, a significant
increase in activity occurs when a methoxy group is added in ortho
position to the hydroxyl group (compound 1b) (a six-fold increase for
EC50 determined after 5 min). The substitution of this methoxy group
by another hydroxyl group (compound 1c), increases more than ten
times the radical-scavenging activity. A slightly higher activity is ob-
served when (2-naphthyl)acetyl is substituted for benzoyl as N-acyl
group (compound 2c). The N-protocatechoyl 4-hydroxylphenylglycine
derivatives (compound 3a) showed radical-scavenging activity com-
parable to that of compound 1c, which also has an ortho-dihydroxyaryl
function as amino acid side chain. Introduction of a methoxy group in
ortho position to the hydroxyl group (compound 3b) causes a 25% in-
creases in activity as determined after 5 min. Substitution of the
methoxy group with a second hydroxyl group (compound 3c) causes a
further 30% increase in activity. The N-hydrocaffeoyl 3,4-dihydrox-
yphenylglycine derivative (compound 4c) showed similar activity to
the N-protocatechoyl derivative 3c. Thus, the radical-scavenging ac-
tivities of compounds 3c and 4c were almost double the activities of
derivatives with a single catecholic group (compounds 1c or 3a).
These results show a direct correlation between radical-scavenging
capacity against the DPPH radical and the number of hydroxyl or ca-
techol groups present in the phenylglycine derivatives. A high increase
in activity with the increase in number of hydroxyl or catechol groups is
observed with the monocatecholic derivatives (compounds 1c and 3a)
having twice the radical-scavenging activity of protocatechuic acid,
while the N-catechoyl derivatives 3c and 4c have almost four times this
On the other hand, two anodic peaks were observed for compound
4c, one at 0.145 V and another at 0.233 V. The first peak probably re-
sults from oxidation of the N-hydrocaffeoyl group, since the potential of
this peak was similar to that determined by us for octyl hydrocaffeate
(0.145 V, data not shown). The potential of the second peak, although
slightly lower, is in line with the peak potentials presented by the other
glycine derivatives with an ortho-dihydroxyaryl side chain (compounds
1c, 2c and 3c). Despite the lower oxidation potential of the first peak,
compound 4c did not present higher radical-scavenging activity against
the DPPH radical than compound 3c, which also has catecholic groups
both as N-acyl and as side chain moiety.
These cyclic voltammetry experiments indicate
a significant
3

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
Table 1
%
DPPH Radical-scavenging capacity, EC50a, oxidation peak potentials and Log Pb of N-acyl 4-hydroxy, 4-hydroxy-3-methoxy and 3,4-dihydroxy phenylglycine
cyclohexylamides and of protocatechuic acid and tyrosine.
Compound
R1
R2
EC50
5 min
24.5
3.76
Ep,a (V vs Ag/AgCl)
Log P
60 min
11.0
1a
1b
13.8
0.20
11.9
0.60
0.703
0.500
0.016
0.006
3.25
3.06
2.08
1c
0.32
0.26
0.37
0.28
0.20
0.19
0.69
26.5
0.01
0.01
0.01
0.01
0.01
0.01
0.03
11.70
0.22
0.22
0.23
0.19
0.13
0.16
0.31
nd
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.272
0.266
0.274
0.280
0.272
0.006
0.005
0.003
0.006
0.015
2.76
4.03
2.28
2.10
1.79
2.40
0.88
−2.20
2c
3a
3b
3c
4c
0.145
0.233
0.324
0.008
0.010
0.005
Protocatechuic acid
Tyrosine
0,715
0.018
a
%
The antiradical activity was defined as the relative concentration of antioxidant required to lower the initial DPPH concentration by 50% [EC50 (M phenolic
compound per unit DPPH concentration)] obtained at different reaction times.
b
Partition coefficient between water and n-octanol. Estimated using Molinspiration Cheminformatics software (Molinspiration, Slovensky Grob, Slovak Republic,
2017, http://www.molinspiration.com).
decrease in oxidation potential with the increase in number of hydroxyl
groups. Thus, a correlation between oxidation potentials and radical-
scavenging activity can be established, with lower oxidation potentials
corresponding to higher radical-scavenging activities.
are uncorrected. 1H and 13C NMR spectra were recorded on a Bruker
Avance II+ instrument. Spectra were taken at room temperature in
CDCl3, CD3COCD3 and CD3OD at 400 MHz (1H) and 100.6 MHz (13C) by
using TMS as an internal standard (1H NMR in CDCl3: 0.000 ppm).
1H-1H spin-spin decoupling, DEPT θ45°, HMQC and HMBC were used to
attribute some signals. Chemical shifts are given in ppm and coupling
constants in Hz. HRMS data were recorded by the Laboratory for
Structural Elucidation of the Materials Centre of the University of Porto
on an LTQ OrbitrapTM XL hybrid mass spectrometer (Thermo Fischer
Scientific, Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and
Xcalibur 2.1.0. Elemental analysis was performed on a LECO CHNS 932
elemental analyzer. The reactions were monitored by thin layer chro-
matography (TLC). Column chromatography was performed on
Macherey-Nagel silica gel 230–400 mesh. Petroleum ether refers to the
boiling range 40–60 °C. Solvents were used without purification except
for acetonitrile which was dried using standard procedures.
Despite the increase in the number of hydroxyl groups in these non-
coded amino acid, their log P is significantly higher (Table 1) than that
for tyrosine. This probably confers these new non-coded amino acids
with better cell penetrating capacities.
3. Conclusions
A simple and efficient one-pot procedure for the synthesis of N-acyl
hydroxyphenylglycine amides has been established. These novel hy-
droxyphenylglycine derivatives, in particular the polyphenolic amino
acids
N-protocatechoyl
and
N-hydrocaffeoyl
3,4-dihydrox-
yphenylglycine amides exhibited high radical-scavenging activity.
The
synthesis
of
the
N-benzyloxycarbonyl-3,4-dihydrox-
%
yphenylglycyl dipeptide derivative shows that the methodology de-
veloped gives rise to a substrate that, after N-acyl deprotection, can be
coupled with other amino acid residues to give, among others, cell-
penetrating type peptides that can be tested as mitochondria targeted
antioxidants.
4.1.1. DPPH radical essay
%
DPPH radical-scavenging activity was assessed as described pre-
%
viously [41,43]. Methanolic DPPH stock solution (1.93 mM) was di-
%
luted to give a 0.10 mM working solution. The reaction between DPPH
and each antioxidant was monitored at 515 nm by using a Powerwave
XS Microplate Reader (Bio-Tek Instruments, Inc) thermostated at
In addition, these non-coded amino acid derivatives can have other
intrinsic biological activities or be applied as: cross-linkers; transition
metal ligands; or used in the design of new peptide hydrogels.
T = 25.0
0.1 °C. The wells of a 96-well microplate contained a me-
%
thanolic solution of the antioxidant (3–200 µM) and 80 µM DPPH . The
absorbance of each well was recorded at 1 min intervals for a 60 min.
period. The absorbance of each solution was subtracted from the blank
4. Experimental section
%
(80 µM DPPH without antioxidant). The antiradical activity was de-
4.1. General methods
fined as the relative concentration of antioxidant required to lower the
%
initial DPPH concentration by 50% [EC50 (mol/L phenolic compound
Melting points (°C) were determined in a Gallenkamp apparatus and
per unit DPPH concentration)].
4

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
4.1.2. Cyclic voltammetry
149.99 (C]O), 187.27 (CH = O) ppm.
The cyclic voltammetry experiments were carried out using a Hi-Tek
potentiostat, type DT 2101, and a Hi-Tek wave generator type PPRl,
connected to a Philips recorder, type PM 8043, and to a three electrode,
home-built glass cell. The working electrode was a vitreous carbon disc
(diameter: 3 mm), the counter electrode a platinum spiral and the re-
ference electrode a mercury pool. The supporting electrolyte was a
phosphate buffer (0.10 M) with pH adjusted to 7.30. Stock solutions of
each compound (0.01 M) were prepared by dissolving an appropriate
amount in ethanol. The voltammetric working solutions were prepared
in the electrochemical cell by diluting 0.10 mL of the stock solution in
10 mL of supporting electrolyte in order to obtain a final concentration
of 0.0001 M. At the end of each experiment the potential of the mercury
pool was measured vs a Ag/AgCl electrode. Between experiments the
working electrode was repolished with alumina powder (∼0.05 μm).
HRMS (ESI): m/z M+ calcd for C17H22O7: 338.1366; found:
338.1597.
Procedure B: Two step synthesis of N-acyl, hydro-
xyphenylglycine cyclohexylamides. To a solution of the O-tert-buty-
loxycarbonylated benzaldehyde (1.00 mmol) in ethanol/2,2,2-tri-
fluoroethanol (1/1) (0.17 M) under a stream of nitrogen, 1.10 equiv of
4-methoxybenzylamine was added. After 4 h, 1.10 equiv of the car-
boxylic acid and 1.10 equiv of cyclohexyl isocyanide were added and
left to react for 2 days. The reaction mixture was then evaporated at
reduced pressure and the residue was dissolved in ethyl acetate
(100 mL) and washed with KHSO4 (1 M), NaHCO3 (1 M) and brine (3
times 25 mL each). The organic layer was dried with MgSO4 and the
solvent evaporated at reduced pressure to give the corresponding Ugi
adduct. Trifluoroacetic acid was added to the adduct (0.25 M) and the
solution refluxed at 80 °C for 10 min. TFA was then evaporated at re-
duced pressure. The product obtained was then recrystallized or subject
to column chromatography.
4.1.3. Statistical analysis
%
All the DPPH radical scavenging assays and cyclic voltammetry
experiments were run at least in quadruplicate. SPSS 21.0 software was
used for statistical analysis by one-way analysis of variance (ANOVA,
with Tukey′s HSD multiple comparison) with the level of significance
N-Benzoyl, (4-hydroxyphenyl)glycine cyclohexylamide (1a).
Procedure B using tert-butyl (4-formylphenyl) carbonate (0.222 g,
1.000 mmol) and benzoic acid was followed to give 1a′.
Yield: 0.537 g (94%); white solid (from diethyl ether).
Rf = 0.68 (petroleum ether/diethyl ether 1:5).
set at P < 0.05. Data are presented as means
standard deviation.
4.2. Synthesis
M.p. 65–66 °C.
1H NMR (400 MHz, CDCl3): δ = 0.86–1.93 (m, 10H, CH2 cyclo-
hexyl), 1.57 [s, 9H, C(CH3)3], 3.76 (s, 3H, OCH3), 3.79–3.82 (m, 1H, CH
cyclohexyl), 4.41 (br. s, 1H, C6H4CH2), 4.63 (br. s, 1H, C6H4CH2), 5.40
(br. s, 1H, αCH), 5.74 (br. s, 1H, NH), 6.74 (d, J = 8.4 Hz, 2H, ArH),
7.00 (br. d, J = 4.8 Hz, 2H, ArH), 7.13 (d, J = 8.8 Hz, 2H, ArH),
7.35–7.37 (m, 4H, ArH), 7.46–7.49 (m, 3H, ArH) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 24.70 (CH2), 24.78 (CH2), 25.44
(CH2), 27.66 [C(CH3)3], 32.69 (2CH2), 48.64 (CH cyclohexyl), 55.20
(OCH3), 64.08 (αCH), 65.82 (C6H4CH2), 83.80 [C(CH3)3], 113.87
(3CH), 121.61 (3CH), 126.68 (CH), 128.38 (CH), 128.49 (CH), 129.84
(CH), 130.07 (CH), 130.70 (CH), 132.65 (2C), 133.35 (CH), 136.08 (C),
151.08 (C), 151.54 (C), 158.76 (C]O), 168.04 (C]O), 173.07 (C]O)
ppm.
Procedure A: Synthesis of O-tert-butyloxycarbonylated benzal-
dehydes. To a solution of the hydroxybenzaldehyde (5.00 mmol) in dry
acetonitrile (0.20 M), 0.10 equiv of dimethylaminopyridine was added.
This was followed by addition under rapid stirring at room temperature
of 1.10 equiv of tert-butyldicarbonate for the monohydroxylated ben-
zaldehydes and 2.20 equiv for the dihydroxylated benzaldehyde. When
all the reactant had been fully tert-butyloxycarbonylated the solvent
was evaporated at reduced pressure and the residue was dissolved in
ethyl acetate (100 mL) and washed with KHSO4 (1 M) and brine (3
times 25 mL each). The organic layer was dried with MgSO4 and the
solvent evaporated at reduced pressure. The product obtained was then
recrystallized or subject to column chromatography.
tert-Butyl (4-formylphenyl) carbonate (a) [44]. Procedure A
using 4-hydroxybenzaldehyde (0.611 g, 5.000 mmol) was followed to
give a.
Anal. calcd. for C34H40N2O6 (572.69): C 71.31, H 7.04, N 4.89;
found C 71.39, H 7.32, H 4.51.
Trifluoroacetic acid was added to 1a′ (0.307 g, 0.537 mmol)
(0.25 M) and the solution refluxed at 80 °C for 10 min. TFA was then
evaporated at reduced pressure to give 1a.
Yield: 1.066 g (96%); white solid (from diethyl ether/n-hexane).
The spectroscopic properties were in agreement with those previously
reported [44].
Yield: 0.118 g (63%); white solid (from diethyl ether).
Rf = 0.48 (petroleum ether/diethyl ether 1:5).
tert-Butyl (4-formyl-2-methoxyphenyl) carbonate (b) [45]. Pro-
cedure
A
using 4-hydroxy-3-methoxybenzaldehyde (0.761 g,
M.p. 141–142 °C.
5.000 mmol) was followed to give b.
1H NMR (400 MHz, CD3COCD3): δ = 1.14–1.36 (m, 5H, CH2 cy-
clohexyl), 1.57–2.12 (m, 5H, CH2 cyclohexyl), 3.68–3.76 (m, 1H, CH
cyclohexyl), 5.64 (s, 1H, αCH), 6.82 (d, J = 8.4 Hz, 2H, ArH), 7.37 (d,
J = 8.4 Hz, 2H, ArH), 7.46–7.50 (m, 2H, ArH), 7.53–7.57 (m, 1H, ArH),
7.93–7.95 (m, 2H, ArH) ppm.
Yield: 1.197 g (95%); white solid (from diethyl ether/n-hexane).
The spectroscopic properties were in agreement with those previously
reported [45].
di-tert-Butyl (4-formyl-1,2-phenylene) dicarbonate (c) [46].
Procedure A using 3,4-dihydroxybenzaldehyde (0.691 g, 5.000 mmol)
was followed to give c.
13C NMR (100.6 MHz, CD3COCD3): δ = 25.48 (CH2), 25.57 (CH2),
26.22 (CH2), 33.21 (CH2), 33.30 (CH2), 49.10 (CH cyclohexyl), 57.27
(αCH), 115.91 (2CH), 128.08 (2CH), 129.17 (2CH), 129.58 (2H),
131.04 (C), 132.13 (CH), 135.40 (C), 157.83 (C), 166.36 (C]O),
170.12 (C]O) ppm.
Yield: 1.575 g (93%); light brown oil. The spectroscopic properties
were in agreement with those previously reported [46].
di-tert-Butyl
(3-formyl-1,2-phenylene)
dicarbonate
(d).
Procedure A using 2,3-dihydroxybenzaldehyde (0.691 g, 5.000 mmol)
was followed to give d.
HRMS (ESI): m/z [M+H]+ calcd for C21H25N2O3: 353.1865; found:
353.1856.
Yield: 1.622 g (96%); light brown solid.
Tentative synthesis of N-benzoyl, (2,3-dihydroxyphenyl)gly-
cine cyclohexylamide (1d). Procedure B using di-tert-butyl (3-formyl-
1,2-phenylene) dicarbonate (0.338 g, 1.000 mmol) and benzoic acid
was followed. The residue obtained was chromatographed through si-
lica using as eluent ethyl acetate/petroleum ether 1:3 to give the Ugi
adduct 1d′ in low yield.
Rf = 0.43 (petroleum ether/diethyl ether 1:1).
M.p. 58–59 °C.
1H NMR (400 MHz, CDCl3): δ = 1.56 [s, 18H, 2C(CH3)3], 7.36–7.40
(m, 1H, ArH), 7.53 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H, ArH), 7.75 (dd,
J = 8.0 Hz, J = 1.6 Hz, 1H, ArH), 10.19 (s, 1H, CHO) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 27.11 [C(CH3)3], 27.14
[C(CH3)3], 83.94 [C(CH3)3], 84.37 [C(CH3)3], 126.07 (CH), 126.39
(CH), 128.53 (CH), 129.35 (C), 143.08 (C), 143.74 (C) 149.67 (C]O),
Yield: 0.116 g (17%); light yellow oil.
Rf = 0.64 (petroleum ether/diethyl ether 1:5).
1H NMR (400 MHz, CDCl3): δ = 1.15–1.96 (m, 10H, CH2
5

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
cyclohexyl), 1.46, 1.57, 1.76 [s, 18H, C(CH3)3], 3.79 (br. s, 4H,
OCH3 + CH cyclohexyl), 4.25 (d, J = 4.8 Hz, 2H, C6H4CH2), 4.80 (br. s,
1H, αCH), 5.92 (br. d, J = 7.6 Hz, 1H, NH), 6.86 (d, J = 8.8 Hz, 2H,
ArH), 6.94 (t, J = 8.0 Hz, 1H, ArH), 7.14 (dd, J = 8.0 Hz, J = 1.6 Hz,
1H, ArH), 7.18–7.22 (m, 3H, ArH), 7.47 (t, J = 8.0 Hz, 2H, ArH), 7.59
(t, J = 7.2 Hz, 1H, ArH), 8.01 (dd, J = 8.0 Hz, J = 1.2 Hz, 2H, ArH)
ppm.
1.20–1.48 (m, 3H, CH2 cyclohexyl), 1.54 [s, 9H, C(CH3)3], 1.56–1.69
(m, 2H, CH2 cyclohexyl), 1.81–1.93 (m, 2H, CH2 cyclohexyl), 2.08 (s,
3H, CH3CO), 3.68–3.78 (m, 4H, NHCH + OCH3), 4.46 (d, J = 17.2 Hz,
1H, NCH2), 4.65 (d, J = 17.2 Hz, 1H, NCH2), 5.83 (br. s, 2H,
αCH + NH), 6.74 (d, J = 8.4 Hz, 2H, ArH), 6.91 (d, J = 8.4 Hz, 2H,
ArH), 7.08 (d, J = 8.4 Hz, 2H, ArH), 7.36 (d, J = 8.4 Hz, 2H, ArH) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 22.36 (CH3), 24.65 (CH2), 24.71
(CH2), 25.36 (CH2), 27.58 [C(CH3)3], 32.61 (2CH2), 48.60 (CH cyclo-
hexyl), 50.35 (C6H4CH2), 55.13 (OCH3), 62.04 (αCH), 83.73 [C(CH3)3],
113.83 (2CH), 121.44 (2CH), 127.32 (2CH), 129.08 (C), 130.70 (2CH),
132.68 (C), 150.98 (C), 151.47 (C), 158.56 (C]O), 168.43 (C]O),
172.63 (C]O) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 24.59 (CH2), 24.69 (CH2), 25.47
(CH2), 27.57 [C(CH3)3], 28.38 [C(CH3)3], 32.91 (2CH2), 48.06 (CH
cyclohexyl), 55.26 (OCH3), 58.43 (C6H4CH2), 82.02 [2C(CH3)3],
113.96 (2CH), 119.46 (CH), 120.20 (CH), 120.56 (C), 121.56 (CH),
124.29 (CH), 128.49 (2CH), 128.79 (CH), 129.49 (2CH), 129.62 (C),
130.51 (C), 130.87 (CH), 130.94 (C), 130.97 (C), 133.18 (CH), 144.88
(C), 158.89 (2C = O), 168.82 (C]O), 172.00 (C]O) ppm.
HRMS (ESI): m/z M+ calcd for C39H48N2O9: 688.3360; found:
688.3968.
HRMS (ESI): m/z [M+H]+ calcd for C29H39N2O6: 511.2808; found:
511.2796.
Trifluoroacetic acid was added to 5a′ (0.255 g, 0.500 mmol)
(0.25 M) and the solution refluxed at 80 °C for 10 min. TFA was then
evaporated at reduced pressure to give 5a.
N-protocatechoyl, (4-hydroxyphenyl)glycine cyclohexylamide
(3a). Procedure B using tert-butyl (4-formylphenyl) carbonate (0.444 g,
2.000 mmol) and protocatechuic acid was followed to give 3a′.
Yield: 1.208 g (quantitative); colourless oil.
White solid (from acetone).
Rf = 0.78 (petroleum ether/ethyl acetate 1:4).
M.p. 241–242 °C.
Rf = 0.31 (petroleum ether/diethyl ether 1:5).
1H NMR (400 MHz, DMSO): δ = 1.16–1.24 (m, 5H, CH2 cyclo-
hexyl), 1.49–1.75 (m, 5H, CH2 cyclohexyl), 1.85 (s, 3H, CH3CO),
3.40–3.45 (m, 1H, NHCH), 5.31 (d, J = 8.4 Hz, 1H, αCH), 6.66 (d,
J = 8.4 Hz, 2H, ArH), 7.15 (d, J = 8.4 Hz, 2H, ArH), 7.99 (d,
J = 8.0 Hz, 1H, NH), 8.29 (d, J = 8.4 Hz, 1H, NH), 9.34 (s, 1H, OH)
ppm.
M.p. 119–120 °C.
1H NMR (400 MHz, CDCl3): δ = 1.03–1.86 (m, 10H, CH2 cyclo-
hexyl), 1.57 [s, 9H, C(CH3)3], 3.72–3.75 (m, 4H, OCH3 + CH cyclo-
hexyl), 4.39 (br. s, 1H, C6H4CH2), 4.76 (br. s, 1H, C6H4CH2), 5.16 (br. s,
1H, αCH), 5.81 (br. s, 1H, NH), 6.72–6.79 (m, 3H, ArH), 6.89 (br. d,
J = 8.4 Hz, 1H, ArH), 7.00–7.02 (m, 2H, ArH), 7.10–7.12 (m, 3H, ArH),
7.32 (br. d, J = 8.4 Hz, 2H, ArH) ppm.
13C NMR (100.6 MHz, DMSO): δ = 22.46 (CH3), 24.37 (CH2), 24.49
(CH2), 25.17 (CH2), 32.18 (CH2), 32.30 (CH2), 47.52 (CH cyclohexyl),
55.38 (αCH), 114.88 (2CH), 128.11 (2CH), 129.62 (C), 156.59 (C),
168.69 (C]O), 169.29 (C]O) ppm.
13C NMR (100.6 MHz, CD3COCD3): δ = 24.60 (CH2), 24.66 (CH2),
25.32 (CH2), 27.67 [C(CH3)3], 32.46 (2CH2), 48.94 (CH cyclohexyl),
55.21 (OCH3), 60.40 (αCH), 83.89 [C(CH3)3], 113.96 (3CH), 114.86
(CH), 115.05 (CH), 119.67 (CH), 121.70 (3CH), 126.75 (C), 128.68
(CH), 130.03 (C), 130.60 (CH), 132.22 (C), 144.34 (C), 147.07 (C),
151.18 (C), 151.55 (C), 158.87 (C]O), 168.84 (C]O), 171.18 (C]O)
ppm.
HRMS (ESI): m/z [M+H]+ calcd for C16H23N2O3: 291.1709; found:
291.1705.
N-Benzyloxycarbonylglycyl, (3,4-dihydroxyphenyl)glycine cy-
clohexylamide (6c). Procedure B using di-tert-butyl (4-formyl-1,2-
phenylene) dicarbonate (0.338 g, 1.000 mmol) and N-benzylox-
ycarbonylglycine was followed to give 6c′.
HRMS (ESI): m/z [M+H]+ calcd for C34H41N2O8: 605.2863; found:
605.2856.
Yield: 0.731 g (94%); light pink solid (from ethyl acetate/petroleum
ether).
Trifluoroacetic acid was added to 3a′ (0.453 g, 0.750 mmol)
(0.25 M) and the solution refluxed at 80 °C for 10 min. TFA was then
evaporated at reduced pressure. The residue was dissolved in ethyl
acetate (100 mL) and washed with KHSO4 (1 M), NaHCO3 (1 M) and
brine (3 times 25 mL each). The organic layer was dried with MgSO4
and the solvent evaporated at reduced pressure to give 3a.
Yield: 0.207 g (72%); white solid (from ethyl acetate/di-
chloromethane).
Rf = 0.60 (petroleum ether/diethyl ether 1:5).
M.p. 113–114 °C.
1H NMR (400 MHz, CDCl3): δ = 1.10–1.12 (m, 3H, CH2 cyclohexyl),
1.31–1.34 (m, 3H, CH2 cyclohexyl), 1.56 [s, 18H, 2C(CH3)3], 1.65–1.70
(m, 2H, CH2 cyclohexyl), 1.84–1.88 (m, 2H, CH2 cyclohexyl), 3.74–3.80
(m, 4H, OCH3 + CH cyclohexyl), 3.93 (br. d, J = 16.8 Hz, 1H, αCH2),
4.10 (br. d, J = 16.8 Hz, 1H, αCH2), 4.43 (br. d, J = 17.2 Hz, 1H,
C6H4CH2), 4.60 (br. d, J = 17.2 Hz, 1H, C6H4CH2), 5.10 (s, 2H, CH2 Z),
5.66 (d, J = 7.6 Hz, 1H, αCH), 5.71–5.73 (m, 2H, 2NH), 6.75 (d,
J = 8.4 Hz, 2H, ArH), 6.94 (d, J = 8.4 Hz, 2H, ArH), 7.15 (s, 2H, ArH),
7.30–7.35 (m, 6H, ArH) ppm.
Rf = 0.90 (petroleum ether/ethyl acetate 1:4).
M.p. 158–159 °C.
1H NMR (400 MHz, CD3COCD3): δ = 1.11–1.39 (m, 5H, CH2 cy-
clohexyl), 1.57–1.78 (m, 4H, CH2 cyclohexyl), 1.87–1.90 (m, 1H, CH2
cyclohexyl), 3.69–3.75 (m, 1H, CH cyclohexyl), 5.60 (d, J = 7.6 Hz, 1H,
αCH), 6.80–6.82 (m, 2H, ArH), 6.89 (d, J = 8.0 Hz, 1H, ArH),
7.34–7.36 (m, 3H, ArH), 7.46 (d, J = 2.0 Hz, 1H, ArH), 7.75 (d,
J = 7.6 Hz, 1H, NH), 8.42 (br. s, 3H, OH) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 24.73 (CH2), 24.81 (CH2), 25.40
(CH2), 27.58 [2C(CH3)3], 29.63 (CH2), 31.90 (CH2), 32.65 (CH2), 43.46
(αCH2), 48.91 (CH cyclohexyl), 49.27 (C6H4CH2), 55.17 (OCH3), 62.75
(αCH), 66.85 (CH2 Z), 84.03 [2C(CH3)3], 114.17 (CH), 123.37 (CH),
125.00 (CH), 127.69 (CH), 127.95 (2CH), 128.03 (2CH), 128.46 (3CH),
133.04 (C), 136.39 (C), 142.59 (C), 142.75 (C), 150.39 (C), 150.43 (C),
156.14 (C]O), 158.91 (2C]O), 167.37 (C]O), 170.12 (C]O) ppm.
HRMS (ESI): m/z [M+H]+ calcd for C42H54N3O11: 776.3758;
found: 776.3768.
13C NMR (100.6 MHz, CD3COCD3): δ = 25.48 (CH2), 25.56 (CH2),
26.21 (CH2), 33.22 (CH2), 33.29 (CH2), 49.21 (CH cyclohexyl), 57.29
(αCH), 115.50 (CH), 115.61 (CH), 115.92 (2CH), 120.38 (CH), 127.16
(C), 129.53 (2CH), 131.30 (C), 145.60 (C), 149.20 (C), 157.79 (C),
166.23 (C]O), 170.41 (C]O) ppm.
HRMS (ESI): m/z [M+H]+ calcd for C21H25N2O5: 385.1764; found:
385.1698.
Trifluoroacetic acid was added to 6c′ (0.583 g, 0.750 mmol)
(0.25 M) and the solution refluxed at 80 °C for 10 min. TFA was then
evaporated at reduced pressure to give 6c.
N-Acetyl, (4-hydroxyphenyl)glycine cyclohexylamide (5a).
Procedure B using tert-butyl (4-formylphenyl) carbonate (0.222 g,
1.000 mmol) and acetic acid was followed to give 5a′.
Yield: 0.510 g (quantitative); light yellow oil.
Yield: 0.252 g (74%); white solid (from ethyl acetate/diethyl ether).
Rf = 0.82 (petroleum ether/ethyl acetate 1:4).
M.p. 162–163 °C.
Rf = 0.31 (petroleum ether/diethyl ether 1:5).
1H NMR (400 MHz, CD3COCD3): δ = 1.11–1.71 (m, 10H, CH2 cy-
clohexyl), 3.63–3.67 (m, 1H, CH cyclohexyl), 3.87 (d, J = 6.0 Hz, 2H,
1H NMR (400 MHz, CDCl3): δ = 1.01–1.15 (m, 3H, CH2 cyclohexyl),
6

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
αCH2), 5.10 (s, 2H, CH2 Z), 5.32 (d, J = 7.6 Hz, 1H, αCH), 6.70 (br. d,
J = 7.6 Hz, 1H, NH), 6.73 (s, 2H, ArH), 6.91 (s, 1H, ArH), 7.26–7.39
(m, 6H, ArH + NH), 7.66 (d, J = 7.2 Hz, 1H, NH) ppm.
HRMS (ESI): m/z [M+H]+ calcd for C21H25N2O4: 369.1814; found:
369.1811.
N-(2-Naphthyl)acetyl, (3,4-dihydroxyphenyl)glycine cyclohex-
ylamide (2c). Procedure C using di-tert-butyl (4-formyl-1,2-phenylene)
dicarbonate (0.338 g, 1.000 mmol) and 2-naphthylacetic acid was fol-
lowed. The residue obtained after removal of TFA was dissolved in ethyl
acetate (100 mL) and washed with KHSO4 (1 M), NaHCO3 (1 M) and
brine (3 times 25 mL each). The organic layer was dried with MgSO4
and the solvent evaporated at reduced pressure to give 2c.
Yield: 0.338 g (78%); light yellow solid (from ethyl acetate/di-
chloromethane).
13C NMR (100.6 MHz, CD3COCD3): δ = 25.62 (CH2), 26.27 (CH2),
29.26 (CH2), 33.26 (CH2), 33.38 (CH2), 45.01 (αCH2), 49.20 (CH cy-
clohexyl), 57.16 (αCH), 66.93 (CH2 Z), 115.45 (CH), 115.90 (CH),
119.80 (CH), 128.63 (2CH), 129.24 (3CH), 131.60 (C), 138.16 (C),
145.66 (C), 145.78 (C), 157.63 (C]O), 169.13 (C]O), 170.00 (C]O)
ppm.
HRMS (ESI): m/z [M+H]+ calcd for C24H30N3O6: 456.2135; found:
456.2141.
Procedure C: One-pot synthesis of N-acyl hydro-
xyphenylglycine cyclohexylamides. To a solution of the O-tert-buty-
loxycarbonylated benzaldehyde (1.00 mmol) in ethanol/2,2,2-tri-
fluoroethanol (1/1) (0.17 M), 1.10 equiv of 4-methoxybenzylamine was
added under a stream of nitrogen. After 4 h, 1.10 equiv of the car-
boxylic acid and 1.10 equiv of cyclohexyl isocyanide were added and
left to react for 2 days. The reaction mixture was then evaporated at
reduced pressure and trifluoroacetic acid added to the residue (0.25 M).
The solution was refluxed at 80 °C for 10 min. after which TFA was
evaporated at reduced pressure. The product obtained was then re-
crystallized or subject to column chromatography
Rf = 0.75 (petroleum ether/ethyl acetate 1:4).
M.p. 220–221 °C.
1H NMR (400 MHz, CD3COCD3): δ = 1.06–1.33 (m, 5H, CH2 cy-
clohexyl), 1.63–1.83 (m, 5H, CH2 cyclohexyl), 3.59–3.66 (m, 1H, CH
cyclohexyl), 3.81 [d, J = 2.8 Hz, CH2 (2-naphthyl)acetyl], 5.38 (d,
J = 8.0 Hz, 1H, αCH), 6.75 (s, 2H, ArH), 6.95 (s, 1H, ArH), 7.17 (d,
J = 8.0 Hz, 1H, C6H3CONH), 7.46–7.53 (m, 3H, ArH), 7.70 (d,
J = 7.6 Hz, 1H, NHC6H11), 7.84–7.89 (m, 6H, ArH + 2OH) ppm.
13C NMR (100.6 MHz, CD3COCD3): δ = 25.42 (CH2), 25.52 (CH2),
26.18 (CH2), 33.19 (CH2), 33.28 (CH2), 43.57 [CH2 (2-naphthyl)
acetyl], 49.01 (CH cyclohexyl), 57.28 (αCH), 115.46 (CH), 115.84
(CH), 119.79 (CH), 126.32 (CH), 126.80 (CH), 128.40 (2CH), 128.54
(CH), 128.59 (CH), 128.65 (CH), 131.80 (C), 133.29 (C), 134.49 (C),
134.85 (C), 145.57 (C), 145.74 (C), 170.04 (C]O), 170.25 (C]O)
ppm.
N-Benzoyl, (4-hydroxyphenyl)glycine cyclohexylamide (1a).
Procedure C using tert-butyl (4-formylphenyl) carbonate (0.222 g,
1.000 mmol) and benzoic acid was followed to give 1a.
Yield: 0.221 g (63%).
N-Benzoyl, (4-hydroxy-3-methoxyphenyl)glycine cyclohex-
HRMS (ESI): m/z [M+H]+ calcd for C26H29N2O4: 433.2127; found:
433.2131.
ylamide (1b). Procedure
C using tert-butyl (4-formyl-2-methox-
yphenyl) carbonate (0.252 g, 1.000 mmol) and benzoic acid was fol-
lowed to give 1b.
N-Protocatechoyl, (4-hydroxyphenyl)glycine cyclohexylamide
(3a). Procedure C using tert-butyl (4-formylphenyl) carbonate (0.222 g,
1.000 mmol) and benzoic acid was followed. The residue obtained after
removal of TFA was dissolved in ethyl acetate (100 mL) and washed
with KHSO4 (1 M), NaHCO3 (1 M) and brine (3 times 25 mL each). The
organic layer was dried with MgSO4 and the solvent evaporated at re-
duced pressure to give 3a.
Yield: 0.291 g (76%); light yellow solid (from diethyl ether).
Rf = 0.31 (petroleum ether/diethyl ether 1:5).
M.p. 169–170 °C.
1H NMR (400 MHz, CD3COCD3): δ = 1.15–1.36 (m, 5H, CH2 cy-
clohexyl), 1.57–1.91 (m, 5H, CH2 cyclohexyl), 3.69–3.75 (m, 1H, CH
cyclohexyl), 3.83 (s, 3H, OCH3), 5.67 (s, 1H, αCH), 6.81 (d, J = 8.0 Hz,
1H, ArH), 7.01 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H, ArH), 7.19 (d,
J = 2.0 Hz, 1H, ArH), 7.46–7.57 (m, 3H, ArH), 7.94–7.96 (m, 2H, ArH),
8.04 (s, 1H, NH) ppm.
Yield: 0.279 g (72%).
N-Protocatechoyl, (4-hydroxy-3-methoxyphenyl) glycine cy-
clohexylamide (3b). Procedure C using tert-butyl (4-formyl-2-meth-
oxyphenyl) carbonate (0.252 g, 1.000 mmol) and protocatechuic acid
was followed. The residue obtained after removal of TFA was dissolved
in ethyl acetate (100 mL) and washed with KHSO4 (1 M), NaHCO3 (1 M)
and brine (3 times 25 mL each). The organic layer was dried with
MgSO4 and the solvent evaporated at reduced pressure to give 3b.
Yield: 0.283 g (68%); white solid (from ethyl acetate/di-
chloromethane).
13C NMR (100.6 MHz, CD3COCD3): δ = 25.48 (CH2), 25.56 (CH2),
26.22 (CH2), 33.21 (CH2), 33.29 (CH2), 49.09 (CH cyclohexyl), 56.23
(CH3), 57.60 (αCH), 111.94 (CH), 115.53 (CH), 121.15 (CH), 128.12
(2CH), 129.16 (2CH), 131.49 (C), 132.13 (CH), 135.43 (C), 147.05 (C),
148.17 (C), 166.46 (C]O), 170.07 (C]O) ppm.
HRMS (ESI): m/z [M+H]+ calcd for C22H27N2O4: 383.1971; found:
383.1973.
Rf = 0.47 (petroleum ether/ethyl acetate 1:4).
N-Benzoyl, (3,4-dihydroxyphenyl)glycine cyclohexylamide
M.p. 129–130 °C.
(1c). Procedure
C
using di-tert-butyl (4-formyl-1,2-phenylene) di-
1H NMR (400 MHz, CD3COCD3): δ = 1.17–1.35 (m, 5H, CH2 cy-
clohexyl), 1.58–1.89 (m, 5H, CH2 cyclohexyl), 3.68–3.73 (m, 1H, CH
cyclohexyl), 3.81 (s, 3H, OCH3), 5.63 (d, J = 7.2 Hz, 1H, αCH), 6.80 (d,
J = 8.0 Hz, 1H, ArH), 6.89 (d, J = 8.4 Hz, 1H, ArH), 6.99 (dd,
J = 8.4 Hz, J = 2.0 Hz, 1H, ArH), 7.17 (d, J = 2.0 Hz, 1H, ArH), 7.36
(dd, J = 8.4 Hz, J = 2.0 Hz, 1H, ArH), 7.40 (br. s., 1H, NH), 7.48 (d,
J = 2.0 Hz, 1H, ArH), 7.81 (d, J = 7.2 Hz, 1H, NH) ppm.
13C NMR (100.6 MHz, CD3COCD3): δ = 25.48 (CH2), 25.56 (CH2),
26.21 (CH2), 33.24 (CH2), 33.30 (CH2), 49.20 (CH cyclohexyl), 56.22
(CH3), 57.66 (αCH), 111.91 (CH), 115.58 (2CH), 115.62 (CH), 120.44
(CH), 121.07 (CH), 127.15 (C), 131.70 (C), 145.63 (C), 147.05 (C),
148.16 (C), 149.25 (C), 166.41 (C]O), 170.39 (C]O) ppm.
HRMS (ESI): m/z [M+H]+ calcd for C22H27N2O6: 415.1869; found:
415.1864.
carbonate (0.338 g, 1.000 mmol) and benzoic acid was followed. The
residue obtained after removal of TFA was chromatographed through
silica using as eluents from ethyl acetate/petroleum ether 1:3 to neat
ethyl acetate to give 1c.
Yield: 0.241 g (66%); white solid.
Rf = 0.88 (petroleum ether/ethyl acetate 1:4).
M.p. 127–128 °C.
1H NMR (400 MHz, CD3COCD3): δ = 1.28–1.36 (m, 5H, CH2 cy-
clohexyl), 1.57–1.92 (m, 5H, CH2 cyclohexyl), 3.69–3.77 (m, 1H, CH
cyclohexyl), 5.59 (d, J = 7.6 Hz, 1H, αCH), 6.80 (d, J = 8.4 Hz, 1H,
ArH), 6.88 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, ArH), 7.04 (d, J = 2.0 Hz,
1H, ArH), 7.33 (d, J = 7.6 Hz, 1H, C6H5CONH), 7.46–7.55 (m, 3H,
ArH), 7.90–7.97 (m, 5H, 2ArH + 2OH + NHC6H11) ppm.
13C NMR (100.6 MHz, CD3COCD3): δ = 25.49 (CH2), 25.57 (CH2),
26.22 (CH2), 33.24 (CH2), 33.32 (CH2), 49.23 (CH cyclohexyl), 57.53
(αCH), 115.58 (CH), 115.91 (CH), 119.97 (CH), 128.07 (2CH), 129.17
(2CH), 131.73 (C), 132.12 (CH), 135.42 (C), 145.66 (C), 145.77 (C),
166.39 (C]O), 170.17 (C]O) ppm.
N-Protocatechoyl, (3,4-dihydroxyphenyl)glycine cyclohex-
ylamide (3c). Procedure C using di-tert-butyl (4-formyl-1,2-phenylene)
dicarbonate (0.338 g, 1.000 mmol) and protocatechuic acid was fol-
lowed. The residue obtained after removal of TFA was chromato-
graphed through silica using as eluents from ethyl acetate/petroleum
7

L.S. Monteiro, et al.
BioorganicChemistry89(2019)102983
ether 1:3 to neat ethyl acetate to give 3c.
This article does not contain any studies with human participants or
Yield: 0.292 g (69%); white solid (from ethyl acetate/di-
chloromethane).
animals performed by any of the authors.
Rf = 0.41 (petroleum ether/ethyl acetate 1:4).
References
M.p. 177–178 °C.
1H NMR (400 MHz, CD3COCD3): δ = 1.21–1.33 (m, 6H, CH2 cy-
clohexyl), 1.57–1.91 (m, 4H, CH2 cyclohexyl), 3.71–3.74 (m, 1H, CH
cyclohexyl), 5.55 (d, J = 7.6 Hz, 1H, αCH), 6.78 (d, J = 8.0 Hz, 1H,
ArH), 6.85 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, ArH), 6.90 (d, J = 8.0 Hz,
1H, ArH), 7.03 (d, J = 2.0 Hz, 1H, ArH), 7.35 (dd, J = 8.4 Hz,
J = 2.0 Hz, 1H, ArH), 7.48 (d, J = 2.0 Hz, 1H, ArH), 7.72 (d,
J = 7.2 Hz, 1H, C6H3CONH), 7.95 (br. s, 1H, OH), 8.08 (br. s, 1H, OH),
8.45 (br. s, 1H, OH), 8.49 (br. s, 1H, OH) ppm.
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Acknowledgements
This work received financial support from the Foundation for
Science and Technology (FCT, Portugal), through projects PTDC/QUI-
QOR/29015/2017, UID/QUI/00686/2016 (CQUM) and UID/QUI/
50006/2013–POCI-01-0145-FEDER-007265, co-financed by European
Union (FEDER under the Partnership Agreement PT2020), and from
North of Portugal Regional Operational Programme (NORTE 2020),
under the PORTUGAL 2020 Partnership Agreement, through the
European Regional Development Fund (ERDF) (project NORTE-01-
0145-FEDER-24). We also acknowledge the Doctoral grant SFRH/BD/
100889/2014.We are thankful to Laboratory for Structural Elucidation
of the Materials Centre of the University of Porto CEMUP for MS/NMR
analysis and able technical support.
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Conflict of interest
The authors declare that they have no conflict of interest.
8