Small Library of Triazolyl Polyphenols Correlating Antioxidant Activity and Stability with Number and Position of Hydroxyl Groups

Article abstract of DOI:10.1021/acscombsci.8b00118

Polyphenolic compounds have attracted much interest because of their antioxidant properties and multiple applications, from food or cosmetic preservatives to free radical scavengers as therapeutic agents. Inspired by common OH substitutions in natural products, here we describe a small library of 1,2,3-triazoles disubstituted with polyphenol groups at 1,4-positions, in an attempt to correlate the number and position of hydroxyl groups in the aromatic rings with the antioxidant activity. Some compounds from this library exhibit strong radical scavenging activities in the oxygen radical absorbance capacity assay, similar to or even higher than resveratrol and other well-kwon flavonoids. The antioxidant activity for selected compounds was confirmed in vitro through the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) test and in vivo by a Saccharomyces cerevisiae model organism assay. The activity depends on the number and position of the hydroxyl groups, with compounds bearing a 2″5″-hydroxyl substituents on the phenyl ring at position 4 showing the best antioxidant values. The presence of two quinone-forming phenolic groups at the same molecule is behind the instability of some of these compounds in aqueous media.

Full text of DOI:10.1021/acscombsci.8b00118

Subscriber access provided by Kaohsiung Medical University
Letter
SMALL LIBRARY OF TRIAZOLYL POLYPHENOLS
CORRELATING ANTIOXIDANT ACTIVITY AND STABILITY
WITH NUMBER AND POSITION OF HYDROXYL GROUPS
M. Angeles Bonache, Silvia Moreno-Fernández, Marta
Miguel, Beatriz Sabater-Muñoz, and Rosario González-Muñiz
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00118 • Publication Date (Web): 29 Oct 2018
Downloaded from http://pubs.acs.org on October 30, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
SMALL LIBRARY OF TRIAZOLYL POLYPHENOLS CORRELATING
ANTIOXIDANT ACTIVITY AND STABILITY WITH NUMBER AND
POSITION OF HYDROXYL GROUPS
9
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
Mª Angeles Bonache†, Silvia Moreno-Fernández‡, Marta Miguel‡, Beatriz Sabater-
Muñoz§, # and Rosario González-Muñiz†*.
†Instituto de Química Médica, IQM-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain.
‡Instituto de Investigación en Ciencias de la Alimentación (CIAL-CSIC-UAM), Nicolás
Cabrera 9, 28049 Madrid, Spain.
§Instituto de Biología Molecular y Celular de Plantas (IBMCP, CSIC-UPV), Ingeniero
Fausto Elio 46022, Valencia, Spain.
#Smurfit Institute of Genetics, Trinity College of Dublin, University of Dublin, 2 College
green, Dublin2, Dublin, Ireland
Abstract
Polyphenolic compounds have attracted much interest because of their antioxidant
properties and multiple applications, from food or cosmetic preservatives to free radical
scavengers as therapeutic agents. Inspired by common OH substitutions in natural
products, here we describe a small library of 1,2,3-triazoles disubstituted with polyphenol
groups at 1,4-positions, in an attempt to correlate the number and position of hydroxyl
groups in the aromatic rings with the antioxidant activity. Some compounds from this
library exhibit strong radical scavenging activities in the oxygen radical absorbance
capacity assay, similar or even higher than resveratrol and other well-kwon flavonoids.
The antioxidant activity for selected compounds was confirmed in vitro through the 2,2’-
azinobis-(3-ethylbenzothiazoline-6-sulfonate) test and in vivo by a Saccharomyces
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 2 of 24
1
2
3
4
5
6
7
8
9
cerevisiae model organism assay. The activity depends on the number and position of the
hydroxyl groups, with compounds bearing a 2’’5’’-hydroxyl substituents on the phenyl
ring at position 4 showing the best antioxidant values. The presence of two quinone-
forming phenolic groups at the same molecule is behind the instability of some of these
compounds in aqueous media.
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
Keywords: Polyphenols, 1,4-disubstituted 1,2,3-triazoles, antioxidant capacity, ORAC
assay, ABTS assay, Saccharomyces cerevisiae, aqueous stability.
Polyphenols are a big family of secondary metabolites of plants, with more than 8,000
phenolic structures currently known. Natural polyphenols can range from simple
molecules (phenolic acids, phenylpropanoids, flavonoids) to highly polymerized
compounds (lignins, melanins, tannins). Different studies have demonstrated numerous
health benefits of polyphenols on cardiovascular, diabetes, neurodegenerative and cancer
diseases.1 In particular, some popular polyphenol antioxidants such as quercetin and
resveratrol are under clinical trials for different indications.2,3 Hydroxyl groups of phenols
are key moieties responsible for the antioxidant activity, due to its easy conversion to
phenoxy radicals through hydrogen transfer mechanisms.4 In the last two decades there is
an emerging interest in the use of antioxidant compounds in industry. In food and
cosmetics they are applied as additives, to maintain the quality of products and to extend
their half-life. In addition, as damage caused by free radicals is neutralized by
antioxidants, they are also used in cosmetics to slow skin aging. It is also known that cell
damage caused by free radicals and reactive oxygen species are behind a wide variety of
diseases, including cardiovascular problems, neurodegenerative processes and cancer. In
ACS Paragon Plus Environment

Page 3 of 24
ACS Combinatorial Science
1
2
3
4
5
6
fact, numerous lines of experimental evidences support that antioxidants exert beneficial
effects on these pathologies.1
7
8
9
Except for phenolic acids and other simple compounds, most polyphenols have
at least two phenyl groups, separated by different types of linkers. Common patterns of
substitution include the hydroxyl phenolic groups at 4-, 2,4-, 2,5-, 3,4-, and 3,4-5-
positions.1-8 However, there are not clear conclusions about the number, position and best
combination of hydroxyl groups within the aromatic rings and the antioxidant activity.
Therefore, the generation of a library of polyphenolic compounds combining different
natural substitution patterns of hydroxyls could be of interest to clarify this point.
The 1,2,3-triazole ring, generated through Click-chemistry approaches, could
be a suitable spacer to combine two phenyl rings in a simple molecule. On one hand, it
could be prepared by different quite easy methodologies, including the Huisgen
cycloaddition reaction,9 a highly useful and widely applicable 1,3-dipolar cycloaddition
reaction between azides and alkynes, catalyzed by copper salts, 10,11 ruthenium, iridium
and other metals. 12-14 In addition, to avoid the use of toxic organic materials as sodium
azides, some novel methods for the synthesis of 1,2,3-triazole have been reported either
using metals or without metals. Thus, the regioselective preparation of 1,4-disubstituted
1,2,3-triazoles was achieved from commercially available anilines, ketones, and N-
tosylhydrazide in the presence of molecular iodine.15,16 This one-pot procedure allow the
C-N- and N-N-bond formation under metal-free conditions, without using azide
derivatives and assuring the absence of traces of cytotoxic copper catalysts within the
product samples.
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
On the other hand, the 1,2,3-triazole ring, a privileged scaffold, is a key
structural motif in diverse drug molecules and bioactive natural products.17 This ring
system takes part in numerous pharmacological agents with antifungal,18 antibacterial,19
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 4 of 24
1
2
3
4
5
anti-HIV,20 anti-inflammatory,21 anti-tuberculosis22 and anti-tumor activity,23 among
others, having good pharmacokinetic profiles.
6
7
8
9
Considering the interest and wide application of polyphenols in life science and
pharmaceutical research, in particular as antioxidants, we report here the preparation of a
small library of 1,4-diphenyltriazoles combining 4-, 2,4-, 2,5-, and 3,4-OH groups,
following the metal-free method commented above as the key step. This mild,
operationally simple method, followed by a demethylation step, allowed us the
identification of polyphenols with high antioxidant capacity and to establish
advantageous combinations of OH groups for this purpose and for chemical stability.
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
R4
R3
R2
R6
R8
R5
1
4
R7
N
N
R1
N
R1= R2= R3= R4= R5= R6= R7= R8= H, OH
Figure 1. New library of triazolyl compound as antioxidant agents.
The corresponding methoxy 1,4-disubstituted 1,2,3-triazole derivatives 3 were
obtained following the metal-free method described,15 heating anilines 1, acetophenones
2, and tosylhydrazide in DMSO* in the presence of I2 (Scheme 1). 4-Methoxyaniline
[1{1}], 2,5-dimethoxyaniline [1{2}] or 3,4-dimethoxyaniline [1{3}] were selected as
aniline diversity reagents (Figure 2), all able to be transformed into quinone-like
derivatives upon oxidation.24 Chemset 2, composed of 2,4-dimethoxyacetophenone
[2{1}], 2,5-dimethoxyacetophenone [2{2}], and 3,4-dimethoxyacetophenone [2{3}],
*Abbreviations: ABTS, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate; DMSO, dimethylsulfoxide;
OH, hydroxyl; ORAC, oxygen radical absorbance capacity; TE: Trolox equivalents
ACS Paragon Plus Environment

Page 5 of 24
ACS Combinatorial Science
1
2
3
4
5
6
were the selected ketones to be combined with Chemset 1 to provide the 9 library
components.
7
8
9
NH2
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
NH2
NH2
OMe
MeO
OMe
OMe
OMe
1
1
1
{3}
{1}
{2}
Chemset 1 Diversity reagents {1-3}
O
O
O
MeO
MeO
MeO
OMe
OMe
OMe
2
2
2
{3}
{1}
{2}
Chemset 2 Diversity reagents {1-3}
Figure 2. Chemsets selected for the library: Metoxyanilines (Chemset 1), metoxyacetophenones
(Chemset 2).
In our hands, the yield of the first step was from moderate to low, contrary to that
described,15 probably due to decomposition of the commercial methoxy-substituted
anilines at the high temperature of the reaction (dark/black mixtures). The mild oxidant
character of the solvent DMSO could also contribute to the transformation of the activated
anilines into undesired products. Subsequent treatment of compounds 3 with BBr3 led to
the expected polyhydroxy derivatives 4 (9 examples) in good to excellent yield (77-93%)
(Scheme 1). As described by McOmie et al., the amount of BBr3 equivalents necessary
for completing the deprotection depends on the number of methyl groups to be removed
and the number of other heteroatoms in the molecule, so we have used 2 equivalents by
each OMe group and two additional equivalents by each N atom.25
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 6 of 24
1
2
3
4
5
6
7
8
9
To understand the importance of the 2,5-OH substitution pattern within some of
the most potent antioxidant components of the library (see below), compound 4{1,4} was
prepared from aniline 1{1} and acetophenone 2{4} (Scheme 2). This compound lack of
the 2-OH group present in derivative 4{1,2}, thus hampering the formation of p-quinone
upon radical trapping.
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
R
H
H2N
3''
N
H2N
S
4''
2''
O
O
OMe
OH
2'
1
3'
4
BBr3
1
I2
+
N
4'
N
N
CH2Cl2
Ar
N
N
N
DMSO, 100 ºC
OMe
OH
3
(9 examples)
4
(9 examples)
O
R'
2
OH
HO
OH
HO
OH
OH
HO
HO
HO
N
N
N
N
N
N
N
N
N
4{1,1}: 83%
4{1,2}: 78%
4{1,3}: 83%
OH
OH
OH
HO
HO
N
HO
HO
HO
OH
N
N
N
N
N
N
N
N
OH
OH
OH
4{2,1}: 80%
4{2,2}: 77%
4{2,3}: 85%
OH
OH
HO
OH
HO
OH
N
N
HO
HO
HO
N
HO
N
N
N
N
N
N
HO
HO
4{3,1}: 83%
4{3,2}: 78%
4{3,3}: 93%
Scheme 1. Synthetic route for the generation of library components
ACS Paragon Plus Environment

Page 7 of 24
ACS Combinatorial Science
1
2
3
4
O
NH2
H
N
OMe
I2
DMSO, 100 ºC
5
6
7
8
MeO
N
+
+
H2N
S
N
N
O
O
OMe
OMe
3
{1,4} (44%)
1
2
{4}
{1}
9
BBr3
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
CH2Cl2
Ar
3''
4
1
OH
HO
N
4'
N
N
4
{1,4} (85%)
Scheme 2. Synthetic route to compound 4{1,4}
Several methods have been reported to evaluate the antioxidant capacity of
different compounds, but none of them can be used as an official standardized system.
The oxygen radical absorbance capacity (ORAC) method is a Hydrogen Atom Transfer
(HAT)-based assays in which antioxidant and substrate compete for thermally generated
peroxyl radicals. This reaction is a key step in the radical chain reaction; therefore, it is
more relevant to the radical chain-breaking antioxidant capacity.26,27 In addition, the
ORAC assay stands out for their sensitivity, precision and reproducibility.28 Moreover,
the peroxyl radicals are reactive species comparable to those ROS produced by the
organism. As a first assessment, the antioxidant activity of all 1,4-diphenyl-1,2,3-triazole
derivatives 4 was measured by the ORAC assay (Table 1). The antioxidant strength is
expressed as Trolox equivalents (TE), a water-soluble analogue of Vitamin E.29
Resveratrol was included in the assay as a model antioxidant compound.
Within compounds bearing a 1-p-hydroxyphenyl group, clearly derivatives 4{1,3}
and specially 4{1,2} are the most potent derivatives, while the 2,4-OH analogue 4{1,1}
displays a much lower ORAC value. It seems that the antioxidant activity follows the
order 4-Ph(2’’,5’’-OH) > 4-Ph(3’’,4’’-OH) > 4-Ph(2’’,4’’-OH), which could be related
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 8 of 24
1
2
3
4
5
6
7
8
9
to the possible formation of p- or o-quinone on the substituent at position 4 of the triazole
ring. However, this is not a general rule, since the results obtained for compounds having
2’,5’- or 3’,4’-dihydroxyphenyl groups at position 1 were substantially different. For 1-
(2’,5’-OH)Ph derivatives, 4{2,x}, the OH-pattern of substitution on the 4-Ph group has
no significant influence on the antioxidant activity, with the three compounds showing
similar values of 8-9 TE. In the case of compounds 4{3, x}, the best substituent at position
4 was the Ph(2’’,5’’-OH) derivative 4{3,2}, although less active than compound 4{1,2}.
The preferred formation of the o-quionone over p-quinazolone at the 1-Ph group could be
responsible for the decreased activity of 4{3,2} compared to 4{1,2}, as well as of the low
and similar ORAC values for derivatives with a 2’’,4’’- or 3’’,4’’-OH substitution
(compounds 4{3,1} and 4{3,3}) (Figure 3).
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
.
O
O
N
OH
O
N
N
HO
HO
N
HO
N
N
ROO.
ROOH
HO
ROO.
HO
HO
HO
OH
OH
OH
+
.
N
N
HO
HO
O
N
N
N
O
N
N
ROO.
N
N
ROOH
O2
HO
ROO-
OOH.
HO
ROOH
ROO.
HO
HO
OH
OH
N
N
HO
O
N
N
N
ROO.
N
.
O
O
ROOH
Figure 3. Possible reaction mechanisms of compound 4{3,2} as antioxidant
Although general principles cannot be established, Ph(4’-OH) and Ph(3’,4’-OH)
at position 1, and Ph(2’’,5’’-OH) at position 4 provided the best combinations of
ACS Paragon Plus Environment

Page 9 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
hydroxyls for potent antioxidant activity. Some of the newly prepared compound
exhibited strong radical scavenging activities, similar or even much higher than
resveratrol and other well-kwon flavonoids.30
9
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
On the other hand, the 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS)
assay is an Electron Transfer (ET)-based assays which measure the capacity of an
antioxidant in the reduction of an oxidant, which also acts as the probe for monitoring the
reaction and indicator of the reaction end point.31 Reducing power of a compound is also
considered an important and chemically valid parameter and it can also present useful
information. For example, some water-soluble oxidants such as peroxynitrite and
hypochlorite are shown to be reduced to harmless species by antioxidants.32 As it has
been established that several methods involving different oxidation conditions should be
performed to evaluate the antioxidant activity of any compound, in addition to ORAC
assay, we performed the ABTS antioxidant assay for selected compounds. Polyphenols
4{1,1}, 4{1,2}, 4{1,4} with low, high and medium antioxidant ORAC activity,
respectively, and good chemical stability were chosen (Table 1). Resveratrol was again
used as the model compound. In general, the values obtained in the ABTS assay were
lower than the ORAC values, but the antioxidant activity follows the same order than in
the ORAC assay (4{1,2} > 4{1,4} > 4{1,1}). Usually ORAC numbers are higher than
ABTS values but, although the results obtained in both in vitro methods were expressed
in Trolox equivalents, these values can be not compared among them, because the
methods of measurement are performed in different oxidation conditions.
It is important to note that the in vitro antioxidant activity of compounds cannot
be directly related to their capacity in in vivo situations, since their bioavailability, in vivo
reactivity, in vivo stability and storage in tissue are not taken into consideration in these
assays. As a result, an in vivo evidence is required to support the in vitro based antioxidant
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 10 of 24
1
2
3
4
5
6
7
8
9
efficiency of compounds.33 In this context, Saccharomyces cerevisiae has become a
model organism used to study many biological processes, especially to investigate
oxidative stress.27,32 In this study, we have used S. cerevisiae as a model organism to
assess the antioxidant in vivo effect of the selected compounds 4{1,1}, 4{1,2}, 4{1,4}.
Following Martorell et al (2011)34 we have firstly stressed S. cerevisiae cells using H2O2
containing media, to a point on which survival and growth rate was compromised.35 In a
second step, stressed yeast cells were challenged to the compounds 4{1,1}, 4{1,2} and
4{1,4}, along with some controls (see experimental methods). Survival of stressed S.
cerevisiae cells challenged to each compound was in average significantly higher than
the control (stressed cells without any further treatment), especially at the two lower doses
(P-value= 1.6438E-05; 0.029; 1.474E-05; 0.048) (Figure 4). Whereas significantly higher
than control, the two higher doses of compounds 4{1,1}, and 4{1,4} showed a reduced
survivorship that the lower doses. In contrast, compound 4{1,2}, with the higher in vitro
antioxidant activities, displayed a dose-dependent survivorship for the three first doses.
As seen in Fig 4, antioxidant control compounds (Resveratrol and Vitamin C) higher
doses showed also a detrimental effect to the yeast cells when compared to the lower
doses tested.
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
ACS Paragon Plus Environment

Page 11 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
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
Figure 4. Survivors (as average colony forming units (cfu’s)± SE) after oxidative stress and
recovery with antioxidant
To obtain information that would facilitate the subsequent development of these
triazolyl derivatives, we determined their stability in aqueous solutions at room
temperature for two months (Table 1). Due to the low solubility of some compounds in
pure H2O, the study was performed in an aqueous solution containing 15% acetonitrile.
As shown in Table 1, most derivatives are quite stable, with approximately 90% or higher
of intact compound remaining after completion of the study. However, compounds 4{2,3}
and 4{3,2} showed short half-life of less than a month at room temperature, and analogue
4{2,2} of 40 days (Table 1, Figure 5). All these compounds combine quinone-precursor
substituents at both 1 and 4 positions of the triazolyl ring, suggesting an easy auto-
oxidation, especially for 2,5-OH containing molecules. Derivatives 4{1,1}, 4{2,1} and
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 12 of 24
1
2
3
4
4{3,1}, containing a 2’,4’-OH phenyl substituent at position 4 of the triazole ring, are in
5
6
general highly stable in aqueous solutions.
7
8
9
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
Figure 5. Stability behavior for some representative compounds of the library.
In conclusion, we have synthesized a small library of new 1,4-methoxyphenyl
1,2,3-triazoles 3 in one pot from commercially available anilines and ketones using a
metal-free method that not required the use of azides. Subsequent treatment of compounds
3 with BBr3 led to the corresponding polyphenol derivatives 4 in good yield. After
evaluation of all new 1,2,3-triazole 1,4-polyphenols for their antioxidant activity in vitro
by the ORAC and ABTS assays and for their aqueous stability, the most balanced
compound was triazole derivative 4{1,2}. This compound, with 4’,2’’,5’’-trihydroxy
groups, showed the highest antioxidant activities in the series, 3- and 5-times higher than
that of the natural product resveratrol, and a reasonable stability in aqueous solution. The
obtained results indicated that both properties, antioxidant capacity and stability, are
influenced by the number and position of the OH groups. The presence of 2,5-OH
substituted phenyl rings at either 1 or 4 position of the triazole led to highly potent
antioxidants, but the combination of two quinone-forming phenol moieties in the same
molecule resulted in highly unstable compounds. The antioxidant in vivo effects of these
ACS Paragon Plus Environment

Page 13 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
compounds were assessed in S. cerevisiae as a model organism. However, given that
some discrepancies exist regarding lifespan studies performed in mammals and simpler
organisms, especially where oxidative stress is concerned,36 our compounds deserve
further research. The results of this study open the possibility of designing new
antioxidant compounds of potential food, dermocosmetic and pharmaceutical interest.
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
Table 1. Results of ORAC assay and stability in aqueous solution
µmol of trolox / µmol
of pure compound
µmol of trolox / µmol
of pure compound
Stability,
%
Compound
4{1,1}
Structure
(ORAC)
(ABTS)
(t1/2)
OH
3.29 ± 0.14
1.84±0.11
98
HO
HO
HO
N
N
N
HO
4{1,2}
4{1,3}
22,2 ± 0,8
15.53 ± 0.22
90
83
OH
OH
N
N
N
HO
N
10,09 ± 0,19
_
HO
HO
N
N
OH
4{2,1}
4{2,2}
4{2,3}
8,80 ± 0,27
8,3 ± 0,3
_
_
_
95
HO
N
N
N
OH
HO
0
HO
OH
N
(26 days)
N
N
OH
HO
N
OH
9,54 ± 0,26
0
HO
(22 days)
N
N
OH
OH
4{3,1}
3,56 ±0,006
_
92
HO
N
N
HO
N
HO
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 14 of 24
1
2
3
4
5
6
7
8
9
HO
4{3,2}
4{3,3}
15,9 ± 0,3
_
_
6
OH
OH
HO
HO
(40 days)
N
N
N
HO
2,90 ± 0,14
92
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
N
HO
HO
N
N
4{1,4}
9,07 ± 0,41
7,53 ± 0,21
5.12 ±0.004
2.13 ± 0.02
98
95
OH
N
HO
HO
N
N
OH
Resveratrol
OH
EXPERIMENTAL PROCEDURES
Synthesis of 1,4-phenyl-1,2,3-triazole methoxy derivatives 3. To a solution of the
corresponding acetophenone 2 (2.77 mmol) in DMSO (11 mL) was successively added
the corresponding aniline 1 (3.32 mmol), p-toluenesulphonylhydrazide (4.15 mmol, 774
mg) and finally iodine (4.15 mmol, 1.054 g). The resulting solution was heated at 100 ºC
for 4 h. Then, the reaction mixture was cooled to room temperature and the residue was
dissolved in EtOAc:H2O (1: 1) and the phases were separated. The organic phase was
washed with a 10% Na2S2O3, saturated NaCl, dried over Na2SO4 and evaporated to
dryness. The reaction crude was purified on a silica gel column using the eluent system
indicated in each case (see SI).
Synthesis of triazolyl polyphenol derivatives 4. To a solution of the corresponding
methoxy substituted product 3 (1 equivalent) in dry CH2Cl2, previously cooled to 0 ° C
and under Argon, was slowly added a 1 M solution of BBr3 in CH2Cl2 (2 equivalents per
heteroatom). The reaction mixture was kept under stirring at room temperature until
complete deprotection of the methoxy groups (tlc, HPLC-MS). Then H2O and CH2Cl2
ACS Paragon Plus Environment

Page 15 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
were added and the precipitated formed (final compound) was separated by filtration and
washed with H2O and CH2Cl2. The organic phase was washed with brine, dried over
Na2SO4 and evaporated. Precipitation with Et2O was always attempted. When the purity
is not high enough, the precipitate or the crude was purified by column chromatography
or semipreparative HPLC as indicated in each case.
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
Oxygen radical absorbance capacity (ORAC) determination
The ORAC assay was performed following a reported procedure.28 All samples and
reagents were dissolved in phosphate buffer (75 mM; pH 7.4). Briefly, the final assay
mixture (200 µl) contained fluorescein solution (70 nM) as oxidable substrate, 2,2’-azo-
bis-(2-methylpropionamidine) dihydrochloride (14.4 mM) as oxygen radical generator
and antioxidant 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (1-
8 µM) as standard or sample (at different concentrations)] were added to the wells of a
black 96-well plate (96F untreated microwell, NuncTM, Denmark). The fluorescence was
recorded at 37 °C every 55 s for 95 min using a fluorimeter (SpectraMax M2; Molecular
Devices, CA, USA), with excitation and emission wavelengths of 480 and 520 nm,
respectively. ORAC values were expressed as μmol TE/μmol of pure compound.
The Trolox equivalent antioxidant capacity (TE) assay measures the antioxidant capacity
of a given substance, as compared to the standard, Trolox.
Antioxidant activity by ABTS method. Antioxidant activity of compounds was
measured using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
spectrophotometric decolourization method described by Pellegrini et al. (2003).38 96-
well clear polystyrene microplates (Corning, Germany) were used. 30 μL of sample or
standard were added to 270 μL of ABTS solution (PBS 5 mM pH 7,4). Trolox (6-
hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) (20-250 µM) was used as a
reference standard. Appropriate solvent blanks were run in each assay. This mixture was
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 16 of 24
1
2
3
4
5
6
7
8
9
shaken for 10 s at room temperature, and the decrease in the absorbance was recorded at
734 nm every 5 minute for 20 min. All reaction mixtures were prepared in duplicate and
at least three independent runs were performed for each sample. The absorbance was
determined spectrophotometrically by using a microplate reader (Synergy HT, BioTek
Instruments, Winoosky, USA). The percentage inhibition was calculated and plotted as a
function of concentration of antioxidants and Trolox for the standard reference data. The
results were expressed as mol of Trolox equivalent/mol of compound.
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
% Inhibition = [(Absorbancecontrol – Absorbancesample) /Absorbancecontrol] x 100
Yeast strain and maintenance conditions. Saccharomyces cerevisiae strain BY4741
(MATa; his31; leu20; met150; ura30) obtained primarily from the EUROSCARF
(European Saccharomyces cerevisiae Archive for Functional Analysis) was used as in
vivo model for antioxidant analysis following Martorell and coworkers (2011).34 This
strain was maintained and routinely cultured in YPD medium (1% yeast extract
(PRONADISA), 2% bacteriological peptone (PRONADISA) and 2% dextrose (Fisher
Sci. Chemicals)) at 28C, with shaking when required.35
In vivo (S. cerevisiae) antioxidant analysis. Growth rate and survival were measured in
yeast cells, challenged to compounds 4{1,1}, 4{1,2} and 4{1,4}, using Resveratrol and
vitamin C as controls, at four different doses (2, 10, 20 and 40 μg of each pure compound)
after exposing the cells to oxidative stress (3% H2O2 containing medium; YPOxD) for
16h. Growth parameters were determined using the BioScreen C plate-reader system (OY
Growth Curves Ab Ldt., Hensinki, Finland). Similarly to Mattenberger et al (2017) 35
a
pre-culture of yeast BY4741 was established from a -80C glycerol stock by inoculating
5 ml of YPD (28C, 220 rpm), and then subjected to pre-culture in YPOxD for 16h (28C,
220 rpm). Pre-cultures were used to inoculate 200 µl of fresh media to an initial OD595 of
ACS Paragon Plus Environment

Page 17 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
0.05-0.07, being distributed in 100-well honeycomb plates (Oy Growth Curves) with
multi-step pipette to reduce pipetting errors. Plates were then incubated at 28C, during
120h, with shaking (High force) for 3 minutes prior OD measurement (each 15 minutes)
in the Bioscreen C. Maximum growth rate (max) and lagging time (tLag) were determined
for each media using GrowthRates software version 2.1.39,40 Survival was determined as
number of colony forming units per treatment, when treatment was performed at the four
abovementioned doses, after 16h of oxidative stress followed by antioxidant exposure for
another 16h. Exposed cells were serial-diluted to determine the effective number of
colony forming units, before being plated in agar YPD plates. All treatments were
performed in triplicate. Means were compared using t-Student between dose and between
compound and control (no post-oxidative stress treatment).
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
Aqueous Stability. The corresponding polyphenol (1 mg) was placed in an HPLC vial
and dissolved in 225 L of acetonitrile and H2O (1.5 mL total volume). The vial was
placed in the automatic injector of an Agilent 1120 Compact LC apparatus, under a dark
screen, and maintained for two months at room temperature. Chromatograms were
recorded periodically by injection of 30 L of sample each time (wavelength 256 nm).
Column: Eclipse Plus C18 (4.6 × 150 mm, 5 μm). Flow: 1.5 mL/min. Eluent: CH3CN (A)
and 0.05% of TFA in H2O (B), gradient from 5 to 100% of A in 20 min.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website
at …….
General synthetic information, characterization data of all compounds, and copies of 1H
and 13C-NMR spectra.
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 18 of 24
1
2
3
4
5
6
7
8
AUTHOR INFORMATION
Corresponding Author
9
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
*E-mail: rosario.gonzalezmuniz@iqm.csic.es
ORCID Rosario González-Muñiz: 0000-0001-8833-4328
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the Spanish Ministerio de Economía y Competitividad
(MINECO) SAF2015-66275-C2-R and CSIC (201580E073, 201880E109).
REFERENCES
(1) Quiñones, M.; Miguel M.; Aleixandre, A. Beneficial effects of polyphenols on
cardiovascular disease. Pharmacol. Res. 2013, 68, 125–131.
(2) Tomé-Carneiro, J.; Larrosa, M.; González-Sarrías, A.; Tomás-Barberán, F. A.;
García-Conesa, M. T.; Espín, J. C. Resveratrol and clinical trials: the crossroad
from in vitro studies to human evidence. Curr. Pharm. Des. 2013, 19, 6064–6093.
(3) Oboh, G., Ademosun, A.O., Ogunsuyi, O.B. Quercetin and Its Role in Chronic
Diseases. Adv. Exp. Med. Biol. 2016, 929, 377–387.
(4) Rezk, B. M.; Haenen, G. R. M.M.; Van Der Vijgh, W. J. F.; Bast, A. The
antioxidant activity of phloretin: the disclosure of a new antioxidant
pharmacophore in flavonoids. Biochem. Biophys. Res. Commun. 2002, 295, 9–13.
ACS Paragon Plus Environment

Page 19 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
(5) Soobrattee , M. A.; Neergheen , V. S.; Luximon-Ramma, A.; Aruoma , O. I.;
Bahorun, T. Phenolics as potential antioxidant therapeutic agents: Mechanism and
actions. Mutation Res. 2005, 200–213.
9
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
(6) Rice-Evans, C. A.; Miller, N. J.; Paganga A. G. Structure-Antioxidant Activity
Relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20,
933–956.
(7) Liu, T.; Shirai, R.; Matsui, T.; Umezawa, K.; Iwasaki, S. Synthesis and biological
activity of 5-[(2,5-dihydroxybenzyl)amino]salicylic acid analogs as inhibitors or
EGF receptor-associated protein tyrosine kinase. Bioorg. Med. Chem. Lett. 1997,
7, 365–368.
(8) Su, C.; Liu, Z.; Wang, Y.; Song, E.; Song, Y. The electrophilic character of
quinines is essential for the suppression of Bach 1. Toxicology 2007, 387, 17–26.
(9) Huisgen, R. 1,3-diopolar cycloadditions. Past and future. Angew. Chem. Int. Ed.
Engl. 1963, 2, 565–598.
(10) Kolb, H. C.; Finn, M.; Sharpless, K. B. Click chemistry: Diverse chemical
function from a few good reactions. Angew. Chem. Int. Ed. 2001, 40, 2004–2021.
(11) Rodionov, V. O.; Fokin, V.V.; Finn, M. G. Mechanism of the ligation-free CuI-
catalized azide-alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 2005, 44,
2210–2215.
(12) Zhang L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.; Sharpless, K. B.;
Fokin, V. V.; Jia, G. Ruthenium-catalyzed cycloaddition of alkynes and organic
azides. J. Am. Chem. Soc. 2005, 127, 15998–15999.
(13) Ding, S.; Jia, G.; Sun. J. Iridium-Catalyzed Intermolecular Azide–Alkyne
Cycloaddition of Internal Thioalkynes under Mild Conditions. Angew. Chem,
2014, 126, 1908–1911.
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 20 of 24
1
2
3
4
5
6
7
8
(14) Barluenga, J.; Valdés, C.; Beltrán, G.; Escribano, M.; Aznar, F. Developments
in Pd Catalysis: Synthesis of 1H-1,2,3-Triazoles from Sodium Azide and Alkenyl
Bromides. Angew. Chem, 2006, 118, 7047–7050.
9
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
(15) Chen, Z.; Yan, Q.; Liu, Z.; Zhang, Y. Metal-Free CN- and NN-Bond Formation:
Synthesis of 1,2,3- Triazoles from Ketones, N-Tosylhydrazines, and Amines in
One Pot. Chem. Eur. J. 2014, 20, 17635–17639.
(16) Cai, Z.-J.; Lu, X.-M.; Zi, Y.; Yang, C.; Shen, L.-J.; Li, J.; Wang, S.-J. I2/TBPB
Mediated Oxidative Reaction of N-Tosylhydrazones with Anilines: Practical
Construction of 1,4-Disubstituted 1,2,3-Triazoles under Metal-Free and Azide-
Free Conditions Org. Lett. 2014, 16, 5108–5111.
(17) Lau, Y. H.; Rutledge, P. J.; Watkinson, M.; Todd, M. H. Chemical sensors that
incorporate click-derived triazoles. Chem. Soc. Rev. 2011, 40, 2848–2866.
(18) Messina, F.; Botta, M.; Corelli, F.; Paladino, A. Chiral azole derivatives. Part 5:
Synthesis of enantiomerically pure 1-[α-(benzofuran-2-yl)arylmethyl]-1H-1,2,4-
triazoles, antifungal and antiaromatase agents. Tetrahedron: Asymmetry 2000,
11, 4895–4901.
(19) Genin, M. J.; Allwine, D. A.; Anderson, D. J.; Barbachyn, M. R.; Emmert, D.
E.; Garmon, S. A.; Graber, D. R.; Grega, K. C.; Hester, J. B.; Hutchinson, D. K.;
Morris, J.; Reischer, R. J.; Ford, C. W.; Zurenko, G. E.; Hamel, J. C.; Schaadt, R.
D.; Stapert, D.; Yagi, B. H. Substituent effects on the antibacterial activity of
nitrogen-carbon-linked (azolylphenyl)oxazolidinones with expanded activity
against the fastidious gram-negative organisms Haemophilus influenzae and
Moraxella catarrhalis. J. Med. Chem. 2000, 43, 953–970.
(20) Alvarez, R.; Velazquez, S.; San Felix, A.; Aquaro, S.; De Clercq, E.; Perno, C.
F.; Karlsson, A.; Balzarini, J.; Camarasa, M. J. 1,2,3-Triazole-[2',5'-bis-O-(tert-
ACS Paragon Plus Environment

Page 21 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"oxathiole-
2",2"-dioxide) (TSAO) analogues: synthesis and anti-HIV-1 activity. J. Med.
Chem. 1994, 37, 4185–4194.
9
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
(21) Mahmoud, A. M.; Abdel-Moty, S. G.; Salem, O. I. A.; Abdel-Alim, M.;
Aboraia, A. S. Synthesis, anti-inflammatory and molecular docking of some new
1,2,4-triazolobenzimidazol-3-yl acetone thiosemicarbazone cyclized derivatives
as PLA-2 inhibitors. J. Adv. Chem. 2015, 11, 3609–3621.
(22) Castelino, P. A.; Dasappa, J. P.; Bhat, K. G.; Joshi, S. A.; Jalalpure, S. Some
novel Schiff bases of [1,2,4]triazole bearing haloarene moiety-synthesis and
evaluation of antituberculosis properties and neutrophil function test. Med. Chem.
Research 2016, 25, 83–93.
(23) Bakunov, S. A.; Bakunova, S. M.; Wenzler, T.; Ghebru, M.; Werbovetz , K. A.;
Reto Brun, R.; Tidwell, R. R. Synthesis and antiprotozoal activity of cationic 1,4-
diphenyl-1H-1,2,3- triazoles. J. Med. Chem. 2010, 53, 254–272.
(24) Bendary, E.; Francis, R.R.; Ali, H.M.G.; Sarwat, M.I.; El Hady, S. Antioxidant
and structure-activity relationships (SARs) of some phenolic and anilines
compounds. Ann. Agricult. Sci. 2013, 58, 173–181.
(25) McOmie, J. F. W.; Watts, M. L.; West, D. E. Demethylation of aryl methyl
ethers by boron tribromide. Tetrahedron 1968, 24, 2289–2292.
(26) Lucarini, M.; Pedrielli, P.; Pedulli, G.F.; Valginigli, L.; Gigmes, D.; Tordo, P.
Bond dissociation energies of the N-H bond and rate constants for the reaction
with alkyl, alkoxyl, and peroxyl radicals of phenothiazines and related
compounds. J. Am. Chem. Soc. 1999,121, 11546–11553.
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 22 of 24
1
2
3
4
5
6
7
8
(27) McDonald-Wicks, L.K.; Wood LG, Garg ML. Methodology for the
determination of biological antioxidant capacity in vitro: a review. J. Sci. Food
Agric. 2006,86, 2046–2056.
9
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
(28) Lucas-Abellán, C.; Mercader-Ros, M.T.; Zafrilla, M.P.; Gabaldón, J.A.; Núñez-
Delicado, E. Comparative study of different methods to measure antioxidant
activity of resveratrol in the presence of cyclodextrins. Food Chem. Toxicol,
2011,49, 1255–1260.
(29) Forrest, V. J.; Kang, Y.-H.; McClain, D. E.; Robinson, D. H.; Ramakrishnan,
N. Oxidative stress-induced apoptosis prevented by Trolox. Free Radical Biol.
Med. 1994, 16, 675–684.
(30) Sueishi, Y.; Ishikawa, M.; Yoshioka, D.; Nobuyuki, E.; Shigeru, O.; Shimmei,
M.; Fujii, H.; Kotake, Y. Oxygen radical absorbance capacity measured with the
ORAC-EPR method. J. Clin. Biochem. Nutr. 2012, 50, 127–132.
(31) Zulueta, A.; Esteve, M.J.; Frigola, A. ORAC and TEAC assays comparison to
measure the antioxidant capacity of food products. Food Chem. 2009,114, 310–
316).
(32) Huang, D.; Ou, B.; Prior, R.L. The chemistry behind antioxidant capacity assays.
J. Agric. Food Chem. 2005,53, 1841–1856)
(33) Petranovic, D.; Nielsen, J. Can yeast systems biology contribute to the
understanding of human disease? Trends Biotechnol. 2008, 26, 584–590;
(34) Martorell, P.; Forment, J.V.; de Llanos, R.; Monton, F.; Llopis, S.; Gonzalez, N.;
Genoves, S.; Cienfuegos, E.; Monzo, H.; Ramon, D. Use of Saccharomyces
cerevisiae and Caenorhabditis elegans as Model Organisms To Study the Effect
of Cocoa Polyphenols in the Resistance to Oxidative Stress. J. Agric. Food
Chem. 2011, 59, 2077–2085.
ACS Paragon Plus Environment

Page 23 of 24
ACS Combinatorial Science
1
2
3
4
5
6
7
8
(35) Mattenberger, F.; Sabater-Muñoz, B.; Hallsworth, J.E.; Fares, M.A. Glycerol
stress in Saccharomyces cerevisiae: Cellular responses and evolved adaptations.
Environm. Microbiol. 2017, 19, 990–1007.
9
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
(36) Perez, V.; van Remmen, H.; Bokov, A.; Epstein, C. J.; Vijg, J. Richardson, A.
The overexpression of major antioxidant enzymes does not extend the lifespan
of mice. Aging Cell 2009, 8, 73–75
(37) Dávalos, A.; Gómez-Cordovés, C.; Bartolomé, B. Extending applicability of the
oxygen radical absorbance capacity (ORAC−fluorescein) assay. J. Agric. Food
Chem. 2004, 52, 48–54.
(38) Pellegrini, N.; Serafini, M.; Colombi, B.; Del Rio, D.; Salvatore, S.; Bianchi, M.;
Brighenti, F. Total antioxidant capacity of plant foods, beverages and oils
consumed in Italy assessed by three different in vitro assays. J. Nutr. 2003, 133,
2812–2819.
(39) Baty, F.; Delignette-Muller, M.L. Estimating the bacterial lag time: which
model, which precision? Intl. J. Food Microbiol. 2004,91, 261–277.
(40) Hall, B.G.; Acar, H.; Nandipati, A.; Barlow, M. Growth rates made easy. Mol.
Biol. Evol. 2014,31, 232–238.
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 24 of 24
1
2
3
4
5
6
7
8
9
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
150x50mm (96 x 96 DPI)
ACS Paragon Plus Environment