New phenolic cinnamic acid derivatives as selective COX-2 inhibitors. Design, synthesis, biological activity and structure-activity relationships

Article abstract of DOI:10.1016/j.bioorg.2019.103179

Selective inhibition of cyclooxygenase (COX)-2 enzyme is an important achievement when looking for potent anti-inflammatory agents, with fewer gastrointestinal side effects. In this work, a new series of cinnamic acid derivatives, namely hexylamides, have

Full text of DOI:10.1016/j.bioorg.2019.103179

Journal Pre-Proof
New phenolic cinnamic acid derivatives as selective COX-2 inhibitors. Design,
synthesis, biological activity and structure-activity relationships
Daniela Ribeiro, Carina Poença, Carla Varela, João Janela, Elisiário J. Tavares
da Silva, Eduarda Fernandes, Fernanda M.F. Roleira
PII:
S0045-2068(19)30066-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103179
YBIOO 103179
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 January 2019
22 July 2019
1 August 2019
Please cite this article as: D. Ribeiro, C. Poença, C. Varela, J. Janela, E.J. Tavares da Silva, E. Fernandes, F.M.F.
Roleira, New phenolic cinnamic acid derivatives as selective COX-2 inhibitors. Design, synthesis, biological activity
and structure-activity relationships, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2
019.103179
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NEW PHENOLIC CINNAMIC ACID DERIVATIVES AS SELECTIVE COX-2 INHIBITORS.
DESIGN, SYNTHESIS, BIOLOGICAL ACTIVITY AND STRUCTURE-ACTIVITY RELATIONSHIPS
a
a
b,c
b
b,c
Daniela Ribeiro , Carina Poença , Carla Varela , João Janela , Elisiário J. Tavares da Silva ,
a
b,c
Eduarda Fernandes *, Fernanda M. F. Roleira *
a
LAQV, REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty
of Pharmacy, University of Porto, Porto, Portugal
b
Pharmaceutical Chemistry Laboratory, Faculty of Pharmacy, University of Coimbra, 3000-548
Coimbra, Portugal
c
CIEPQPF Centre for Chemical Processes Engineering and Forest Products, University of
Coimbra, 3030-790 Coimbra, Portugal
_____________________________
*Corresponding authors addresses: CIEPQPF, Pharmaceutical Chemistry Laboratory, Faculty of
Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; Telephone: +351 239 488 400;
Fax: +351 239 488 503; E-mail address: froleira@ff.uc.pt (Fernanda M. F. Roleira). LAQV,
REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of
Pharmacy, University of Porto, Porto, Portugal; Telephone: +351 220 428 500; Fax: +351 220 428
5
00; E-mail address: egracas@ff.up.pt (Eduarda Fernandes)
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ABSTRACT
Selective inhibition of cyclooxygenase (COX)-2 enzyme is an important achievement when
looking for potent anti-inflammatory agents, with fewer gastrointestinal side effects. In this
work, a new series of cinnamic acid derivatives, namely hexylamides, have been designed,
synthesized and evaluated in human blood for their inhibitory activity of COX-1 and COX-2
enzymes. From this, new structure-activity relationships were built, showing that phenolic
hydroxyl groups are essential for both COX-1 and COX-2 inhibition. Furthermore, the presence
of bulky hydrophobic di-tert-butyl groups in the phenyl ring strongly contributes for selective
COX-2 inhibition. In addition, a correlation with the theoretical log P has been carried out,
showing that lipophilicity is particularly important for COX-2 inhibition. Further, a plasma protein
binding (PPB) prediction has been performed revealing that PPB seems to have no influence in
the activity of the studied compounds. From the whole study, effective selective inhibitors of
COX-2 were found, namely compound 9 (IC = 3.0±0.3 μM), 10 (IC = 2.4±0.6 μM) and 23 (IC
5
0
50
50
=
1.09±0.09 μM). Those can be considered starting point hit compounds for further optimization
as potential non-steroidal anti-inflammatory drugs.
Keywords: Phenolic cinnamic acids; phenolic cinnamic amides; synthesis; COX-1 inhibition;
selective COX-2 inhibition; structure-activity relationships
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INTRODUCTION
Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are two isoenzymes involved in the
eicosanoids, prostaglandin (PG) and thromboxane, biosynthesis [1]. COX-1 is a constitutive
enzyme and COX-2 expression is primarily induced during the inflammatory process [2]. Recent
works also describe COX-2 as constitutively expressed in many important areas of the body,
being responsible for very important functions, namely in the cardiovascular system [3-5]. COX
inhibitors (CIs) diminish the biosynthesis of the referred eicosanoids and offer a therapeutic
approach for the reduction of inflammatory symptoms and pain. CIs also show antipyretic,
antithrombotic and analgesic effects. Some CIs in clinical use appear to be extremely potent and
highly specific inhibitors of COXs. However, they still show undesirable side effects, being the
most frequent, gastrointestinal complications such as the irritation of the gastric mucosa, as PGs
produced by COX-1 in the gastrointestinal tract, normally have a protective role of the referred
mucosa. Some examples of these inhibitors are indomethacin, diclofenac, ibuprofen and
naproxen, which are depicted in Figure 1A. In addition, other important side effects derived from
COX inhibition are allergic reactions, renal and cardiovascular adverse events [5]. Concerning
the gastrointestinal complications, these have been partially surpassed by COX-2 selective
inhibitors such as celecoxib, rofecoxib, etoricoxib (Figure 1B), and other members of this drug
class showing that the referred selectivity constitutes an important achievement when studying
CIs. In fact, COX-2 is primarily expressed in inflamed tissue [6], unlike COX-1 that is mainly
expressed in the gastrointestinal tract, and, for this reason, there is much less gastric irritation
related with COX-2 inhibitors and a reduced risk of gastric ulceration. However, some recent
studies postulate that inhibition of both COX-1 and COX-2 is required for non-steroidal anti-
inflammatory drugs (NSAID) induced gastric injury [7-9], since selective COX-1 inhibitors seem
to not cause gastric damage in animal studies [10, 11]. Though, the theory that inhibition of COX-
1
motivates the gastrointestinal toxicity of NSAIDs in man is generally accepted and the demand
for new selective COX-2 inhibitors remains a major goal to achieve. Several studies have also
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demonstrated that COX-2 selective inhibitors may prevent colorectal cancer [12]. Nonetheless,
other important adverse effects are associated with COX-2 inhibitors such as increased risk of
renal failure and also heart attack, thrombosis, and stroke [13, 14], through an increase of
thromboxane accumulation. Because of this, it is essential to discover new drugs to exceed the
limitations of the COX-2 selective inhibitors currently used in the clinic practice. Nowadays, there
are no really safe anti-inflammatory drugs to be used as a suitable therapy with negligible
gastrointestinal damage and cardiovascular toxicity. Regardless, COX-2 remains a very important
therapeutic target to explore in order to find efficient treatments for the inflammation related
diseases, such as the debilitating rheumatoid arthritis and osteoarthritis, and also for the
prevention of colon cancer.
Phenolic cinnamic acid derivatives have been remarkable scaffolds in drug design, displaying
many biological activities namely antioxidant [15, 16], anticancer [16-19], and anti-inflammatory
through COX-1 and/or COX-2 inhibition [20, 21]. Nevertheless, no systematic structure-activity
relationships (SAR) studies on COX-1 versus COX-2 inhibition by this type of compounds have
been performed, in human blood, a more complex and physiological cellular model, that is closer
to what happens in the human body [22]. Recently, we have identified a series of
hydroxycinnamic acid derivatives as modulators of human neutrophils’ oxidative burst, which
demonstrate to have antioxidant and anti-inflammatory properties, and also the capacity to
inhibit colon cancer cells proliferation [16]. In the present work, it is our intention to assess if
those compounds and other new phenolic cinnamic acid derivatives have also the ability to
selectively inhibit PGs production via COX-2. For this, we designed and synthesized new
compounds of the referred class, and evaluated their aptitude to inhibit PGs production via COX-
1
and COX-2 enzymes. Additionally, a correlation of the activities with the theoretical log P
values and a plasma protein binding prediction were carried out and, from the whole study, new
SAR were established.
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RESULTS AND DISCUSSION
The synthesis of the hexylamides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 (Scheme 1) was
performed using a single-step reaction starting from the respective acids 1, 3, 5, 7, 9, 11, 13, 15,
1
7, 19 and 21 and hexylamine, in dimethylformamide and triethylamine, in the presence of the
coupling agent (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
BOP) in dichloromethane, at room temperature. The same reaction was used to prepare the
(
diethylamide 23 (Scheme 1).This procedure revealed to be adequate to afford the direct
amidation of both ,-unsaturated and saturated acids, producing the desired amides in
appropriate yields (37% to 89%) (Table 1). Then, the synthesized amides and also some of the
precursor acids were tested for their capacity to inhibit PGs production via COX-1 and COX-2
enzymes (Figures 2 and 4; Table 2) and the resulting activities were correlated with the structure
of the compounds. In terms of SAR, the discussion is focused in three main structural features,
namely: carboxylic acid function versus amide group; aromatic substitution pattern; and
presence versus absence of a -double bond in the aromatic side chain. In addition, SAR are
discussed for COX-1 and COX-2 independently and also comparatively. In this way, looking at
Figure 2 and Table 2 it is possible to check that the parent carboxylic acids do not show activity
against COX-1, such as 1 and 3, or just show low activity, as observed for 5, 9 and 19 at the
highest tested concentration (100 μM). On the contrary, the respective amide derivatives 2, 4,
6
, 10 and 23 (20 is apparently an exception) show significant activity against COX-1. One possible
reason for these results may be the low lipophilicity of carboxylic acids when comparing with
the respective hexylamides, which makes difficult to cross the membranes of blood cells. Among
the carboxylic acids, 9 and 19 with superior log P(s) (Table 2) are effectively more active than 1
and 3. The carboxylic acid 5, with the lowest log P (Table 2) among the studied compounds,
showed some activity, which may be due to the fact that it can cross the blood cell membranes
through some kind of active transport, as previously described by the authors.[16] At this point,
one can state that the presence of a lipophilic amide, such as hexylamide or diethylamide, is
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better than a carboxylic acid group for COX-1 inhibition. Further, comparing the hexyamide 10
(
IC =24±5 μM) with the diethylamide 23 (IC =4.3±0.3 μM), it seems that a teriary bulky amide
5
0
50
is superior to a secondary linear amide for COX-1 inhibition. However, the higher lipophilicity of
the amides relatively to the carboxylic acids, does not explain all the observed activities for the
referred compounds. The aromatic substitution pattern also seems to be very important for the
activity. In fact, one main conclusion is that the presence of at least one phenolic group appears
to be crucial for COX-1 inhibition, since the dimethoxy derivative 12 and the methylenedioxy
derivative 16 revealed to be not active against COX-1. In addition, comparing COX-1 inhibitory
activity of the disubstituted hexylamides 2, 4 and 14, all of them with similar log P (Table 2), it is
possible to conclude that the simultaneous presence of a methoxyl group and a hydroxyl group
in the aromatic ring, as in 4 and 14, is better than the presence of two hydroxyl groups, as in 2.
In fact, 4 and 14 even show around 50 % of inhibiton at 12.5 μM while 2 only show 30 ± 8 % of
inhibition at the same concentration (Figure 2). The position of the methoxyl group, among the
meta and para positions, relatively to the amide chain, has little influence in the COX-1
inhibition, since 4 (meta-methoxyl group) and 14 (para-methoxyl group) show similar activities,
although the presence of the methoxyl group in the meta position is preferable. Considering the
trisubstituted hexylamides 6, 8 and 10, one can say that the 3,5-di-tert-butyl-4-hydroxyl
aromatic substitution pattern, present in 10, is the more convenient, since this compound shows
an IC value of 24±5 μM followed by the 3,5-dimethoxy-4-hydroxyl 8 (IC =45±4 μM), and the
5
0
50
3
,4,5-trihydroxyl 6 (IC =87±2 μM) (Table 2). Comparing the trisubstituted hexylamides 18 and
5
0
2
0, which do not present -double bond in the aromatic side chain, the 3,5-dimethoxy-4-
hydroxyl aromatic substitution pattern, present in 18 (IC =11±2 μM), is now the more adequate
5
0
for COX-1 inhibition. Regarding the presence versus absence of a -double bond in the
aromatic side chain of the hexylamide derivatives, one can assess the influence of this particular
feature in COX-1 inhibition, by comparing the activities of the pairs 2/22, 8/18, 9/19 and 10/20.
In general, it is possible to conclude that the presence of this feature influence COX-1 inhibition.
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However, this influence may be positive (10/20), or negative (8/18, 9/19, 2/22) depending on
the substitution pattern of the aromatic ring. Actually, there are more situations where the
absence of the double bond is beneficial. In summary, the most important SAR for COX-1
inhibition by the studied compounds are the presence of an amide group and disubstituted
aromatic rings with one hydroxyl group and one methoxyl group or two hydroxyl groups,
preferably without -double bond (Figure 3). These features are combined in 4, 14 and 22. In
addition, the absence of a -double bond along with a trisubstituted aromatic ring with two
methoxyl groups and one hydroxyl group seems to favour the activity (Figure 3). These features
are combined in compound 18. Further, the 3,5-di-tert-butyl-4-hydroxyl aromatic substitution
pattern combined with the -double bond along with the tertiary diethylamide group,
features that are present in compound 23 (IC =4.3±0.3 μM), seems to be the better
5
0
combination to achieve higher COX-1 inhibition, since 23 was the best COX-1 inhibitor, in this
work.
Concerning COX-2 inhibition, it is possible to notice that carboxylic acids 1, 3, 5 and 19 do not
show activity against COX-2 (Figure 4, Table 2). Interestingly, the carboxylic acid 9 demonstrates
to be a very potent inhibitor of COX-2 reaching an IC value of 3.0±0.3 µM. On the contrary to
5
0
the majority of carboxylic acids, their corresponding hexylamides 2, 4, 6 and 20 are effectively
active against COX-2. In the case of amides 10 and 23, they are even more potent than the
corresponding carboxylic acid 9, reaching IC values of 2.4±0.6 and 1.09±0.09 µM, respectively.
5
0
As in COX-1 inhibition, one can state that the amide group is better than a carboxylic acid group
for COX-2 inhibition and that a teriary bulky amide as in 23 is superior to a secondary linear
amide as in 10 for COX-2 inhibition. Once more, the low lipophilicity of carboxylic acids 1, 3 and
5
should be responsible for its low COX-2 inhibition. Nevertheless, other reasons than
lipophilicity will account for the great COX-2 inhibition of carboxylic acid 9, as discussed below.
By analyzing the aromatic substitution pattern one can notice that the presence of at least one
phenolic group seems to be also crucial for COX-2 inhibition, since the methylenedioxy
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derivative 16 and the dimethoxy derivative 12 are again not active against COX-2. In addition,
comparing COX-2 inhibitory activity of the disubstituted hexylamides 2, 4 and 14, it appears that,
in this case, the presence of two hydroxyl groups (cathecol) in the aromatic ring as in 2, is more
advantageous than the simultaneous presence of one hydroxyl group and one methoxyl group,
as in 4 and 14. In fact, 2 shows an IC value of 12.7±0.5 µM, against 20±1 µM for 4 and 21±2 µM
5
0
for 14 (Table 2). Again, the position of the methoxyl group, among the meta and para positions
relatively to the amide chain, does not seem to influence the COX-2 inhibitory activity, since 4
(meta-methoxyl group) and 14 (para-methoxyl group) show similar activities. Considering the
trisubstituted hexylamides 6, 8 and 10, one can say that, in this case, the aromatic substitution
pattern has great influence in COX-2 inhibition. In fact, the 3,5-di-tert-butyl-4-hydroxyl aromatic
substitution pattern, present in 10, seems to be very appropriate for COX-2 inhibition. The 3,5-
dimethoxyl-4-hydroxyl aromatic substitution pattern, present in 8, is the second best approach
and the 3,4,5-trihydroxyl, present in 6, is the less suitable aromatic substitution. These results
can be correlated with the active site of COX-2. In fact, it is known that the most significant
difference between COX-1 and COX-2 isoenzymes is the presence of an isoleucine residue, at
position 523 in COX-1, instead of a valine residue in COX-2. The smaller size valine residue
permits a better access to a hydrophobic side-pocket in COX-2, allowing molecules with
hydrophobic groups to better interact with its active site [13]. This can explain the great COX-2
inhibitory activity of the carboxylic acid 9 and even better of the hexylamide 10 and diethylamide
2
3, all of them bearing two hydrophobic tert-butyl groups in the aromatic ring. These groups
confer great lipophilicity to the referred compounds, which present log P values of 4.95, 6.61
and 5.45 (Table 2), respectively. Besides the tert-butyl groups in the aromatic ring, compound
2
3 presents a bulky hydrophobic diethylamine group that can also contribute to favor the access
and the interaction with the active site of COX-2. Based on this approach, it can be explained
why compound 8, with less hydrophobic methoxyl groups and with log P of 2.95 (Table 2), show
lower COX-2 inhibitory activity, followed by 6, with hydrophilic hydroxyl groups and with log P
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of 2.43 (Table 2). Regarding the presence versus absence of a -double bond in the aromatic
side chain of the hexylamide derivatives, one can assess the influence of this particular feature
in COX-2 inhibition, by comparing the activities of the pairs 2/22, 8/18, 9/19 and 10/20.
Generally, and on the contrary to what was noticed for COX-1, for COX-2 inhibition the presence
of the double bond in the side chain is very beneficial, since the trisubstituted compounds with
the double bond, 8, 9 and 10 are more active than the corresponding compounds without the
double bond, 18, 19 and 20, respectively. For the disubstituted pair 2/22, this difference is not
evident, showing both compounds similar activities, although 2 is slightly better. In summary,
the most important SAR for COX-2 inhibition by the studied compounds are: presence of an
amide group; disubstituted aromatic rings with two hydroxyl groups, preferably with -double
bond; more important, trisubstituted aromatic rings with 3,5-di-tert-butyl-4-hydroxyl aromatic
substitution, definitely with a double bond in the side chain of either the acid or the amides
(Figure 5). In the case of disubstituted compounds, these features are combined in caffeic acid
hexylamide 2 (IC = 12.7±0.5 µM) and hydrocaffeic acid hexylamide 22 (IC = 13.4±0.5 µM). In
5
0
50
the case of trisubstituted compounds, the referred features are combined in compounds 9, 10,
and 23 which are the best COX-2 inhibitors studied in this work, reaching IC values of 3.0±0.3,
5
0
2
.4±0.6 and 1.09 ± 0.09 µM (Table 2).
More remarkable is the comparison between COX-1 and COX-2 inhibition by the studied
compounds. From this, we can assess if there is some selectivity for either COX-1 or COX-2
inhibition. In fact, the carboxylic acids 5 and 19 show a slight inhibition, but total selectivity for
COX-1, since they are not active against COX-2 (Figures 2 and 4, Table 2). Also, compounds 4,
and mainly 14, and 18 are selective COX-1 inhibitors. Moreover, the carboxylic acid 9, the
hexylamide 10, and the diethylamide 23, besides being very powerful compounds in COX-2
inhibition, are very selective for the referred enzyme with COX-1/COX-2 ratio values of >33, 10.0,
and 3.9 respectively, demonstrating their higher affinity to COX-2 (Table 2). This is in agreement
with established SAR for the COX inhibitor flurbiprofen, where chemical modifications in the
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phenyl group are capable of inducing steric constraint resulting in an increased selectivity for
COX-2 [23]. Compounds 9, 10, and 23 bearing two bulky tert-butyl groups in their phenyl rings
are capable of inducing the abovementioned steric restrictions when interacting with COX-2
resulting in very effective and selective COX-2 inhibitors.
Finally, as the present study was performed in human whole blood, a plasma protein binding
(PPB) prediction for the studied compounds was done using the PreADMET web-based
application (Table 2). The positive controls tested, indomethacin and celecoxib, presented PPB
values of 89.5 % and 91.1 %, respectively, really similar to the ones found in the literature,
around 90 % and 97 %, respectively [24, 25]. It was observed that the most active compounds
bind considerably to plasma proteins. However, the same was observed for the reference
compounds indomethacin and celecoxib, showing that apparently, the plasma protein binding
has no influence in the activity of the studied compounds. An in vitro Caco-2 cell permeability
prediction was also carried out, for the studied compounds, using the PreADMET web-based
application. All the compounds analyzed seem to have capacity to cross the membranes of Caco-
2
cells in a similar or superior way to indomethacin, being the amide derivatives more able to
do so than the parent carboxylic acids. This is in accordance with the predicted log P and justifies
once more, the superior COX-1 and COX-2 inhibition showed by the amide derivatives relatively
to the correspondent carboxylic acids. Based on all the work we can consider compounds 9, 10,
and 23 hit compounds for further optimization and a starting point for the preparation of
selective COX-2 inhibitors as potential non-steroidal anti-inflammatory drugs
MATERIAL AND METHODS
Chemistry
Reactions were controlled by thin layer chromatography (TLC) using silica gel 60 HF254 plates.
Column chromatography was performed using silica gel 60 (0.063 – 0.200 mm). Melting points
1
0

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(Mps) were determined on a Reichert Thermopan hot block apparatus, except for compounds
1
2 and 14, which were measured in a Büchi Melting Point B-540 apparatus. Mp values were not
corrected. IR spectra were recorded on a Jasco 420FT/IR spectrometer, except for compounds
2, 14, 22, and 23 which IR spectra were recorded on a Perkin Elmer Spectrum 400 FT-IR/FT-NIR
1
1
13
spectrometer. The H NMR and C NMR spectra were recorded at 400 MHz and 100 MHz,
respectively, on a Varian Unity 400, using DMSO-d as solvent. Peaks positions are given in parts
6
per million (ppm) using the residual non-deuterated solvent as the internal standard. Data are
reported as follows: chemical shift (ppm), integrated intensity, multiplicity (indicated as: s,
singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet and combination
thereof), coupling constants (J) values in Hertz (Hz) and corresponding nucleus. Caffeic acid 1,
ferulic acid 3 and 3,4-dimethoxycinnamic acid 11 were purchased from Sigma-Aldrich
(Schnelldorf, Germany); 3,4,5-trihydroxycinnamic acid 5, 4-hydroxy-3,5-dimethoxycinnamic acid
7
and 3-(4-hydroxy-3,5-dimethoxyphenyl)propionic acid 17 from Apin Chemicals Limited
(
Abingdon, Oxon, United Kingdom); 3,5-di-tert-butyl-4-hydroxycinnamic acid 9, 3,4-
methylenedioxy)cinnamic acid 15 and 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid 19,
(
from Alfa Aesar (Karlsruhe, Germany); 3-hydroxy-4-methoxycinnamic acid 13 from Carbosynth
Limited (Berkshire, United Kingdom); and 3-(3,4-dihydroxyphenyl)propionic acid 21 from
Honeywell Fluka (Göterborg, Sweden).
General procedure to obtain the hexylamides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22
Carboxylic acids 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 (Scheme 1) were dissolved in
dimethylformamide (DMF) and triethylamine (TEA). The solution was then cooled in an ice-
water bath and hexylamine or diethylamine was added, followed by a solution of (benzotriazol-
1
-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) in dichloromethane. The
o
mixture was stirred at 0 C for 30 min and then at room temperature for specific periods of time.
Dichloromethane was removed under reduced pressure and the remaining solution was diluted
1
1

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with water (100 mL). The aqueous phase was extracted with ethyl acetate (2x 100 mL) and the
organic phases were washed with 1N HCl (2x 100 mL), water (2x 100 mL), NaHCO 5% (3x 100
3
mL) and water (2x 100 mL), dried over anhydrous MgSO , filtered and concentrated. The
4
obtained residues were purified by silica gel column chromatography or crystallization yielding
the corresponding hexylamides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.
N-Hexyl-3-(3,4-dihydroxyphenyl)-2-propenamide (2)
Obtained as described before [15].
N-Hexyl-3-(4-hydroxy-3-methoxyphenyl)-2-propenamide (4)
Obtained as described before [15].
N-Hexyl-3-(3,4,5-trihydroxyphenyl)-2-propenamide (6)
Obtained as described before [16].
N-Hexyl-3-(4-hydroxy-3,5-dimethoxyphenyl)-2-propenamide (8)
Compound 7 (1.12 g, 5 mmol); DMF (10 mL); TEA (0.7 mL); hexylamine (0.66 mL, 5 mmol); BOP
(
2.21 g, 5 mmol); CH Cl (10 mL); reaction time: 2 h; ethyl acetate crystallization; 37 % yield.
2
2
-1
Mp(ethyl acetate) 121-123°C. IR (ATR) νmax cm : 3523 (O-H), 3279 (N-H), 1653 (C=O), 1210 (C-O), 1113
1
(
C-O). H NMR (400 MHz, DMSO-d )  : 0.85 (3H, t, J=6.9, CH ), 1.23-1.27 (6H, m, CH , (C-3’-C-
6
3
2
5
’)), 1.40-1.45 (2H, m, CH (C-2’)), 3.14 (2H, dt, J=5.4, 6.6, CH (C-1’)), 3.78 (6H, s, OCH (C-3 and
2
2
3
C-5)), 6.47 (1H, d, J=15.6, CH()), 6.83 (2H, s, CH (C-2 and C-6)), 7.31 (1H, d, J=15.6, CH()), 7.96
1
3
(
1H, t, J=5.4, NH). C NMR (100 MHz, DMSO-d )  : 14.0 (CH ), 22.2 (C-5’), 26.2 (C-4’), 29.2 (C-
6
3
3
1
’), 31.1 (C-2’), 38.8 (C-1’), 56.0 (-OCH C-3 and C-5), 105.2 (C-2 and C-6), 119.5 (C-), 125.4 (C-
3
), 137.3, 139.3 (C-), 148.1, 165.4 (C=O).
1
2

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N-Hexyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-propenamide (10)
Compound 9 (2.76 g, 10 mmol); DMF (20 mL); TEA (1.4 mL); hexylamine (1.32 mL, 10 mmol);
BOP (4.42 g, 10 mmol); CH Cl (20 mL); reaction time: 3 h; ethyl acetate crystallization; 81 %
2
2
o
-1
yield. Mp(ethyl acetate) 173-174 C. IR (ATR) ν cm : 3523 (O-H), 3280 (N-H), 1651 (C=O), 1218 (C-
max
1
O), 1113 (C-O). H NMR (400 MHz, DMSO-d )  : 0.86 (3H, t, J=6.9, CH ), 1.26-1.29 (6H, m, CH
6
3
2
(
C-3’-C-5’)), 1.36-1.44 (2H, m, CH (C-2’)), 1.39 (18H, s, (CH ) ), 3.12-3.16 (2H, m, CH (C-1’)), 6.45
2
3 3
2
(1H, d, J=15.7, CH()), 7.28 (1H, br s, OH), 7.29 (2H, s, CH (C-2 and C-6)), 7.32 (1H, d, J=15.7,
1
3
CH()), 7.92 (1H, t, J=5.5, NH). C NMR (100 MHz, DMSO-d )  : 13.8 (CH ), 21.9 (C-5’), 26.1 (C-
6
3
4
1
’), 29.0 (C-3’), 30.1 (C(CH ) ), 30.9 (C-2’), 34.4 (C(CH ) ), 38.5 (C-1’), 118.9 (C-1), 124.1 (C-2),
3
3
3 3
26.2 (C-1), 139.1 (C-3 and C-5), 139.2 (C-), 155.3 (C-4), 165.1 (C=O).
N-Hexyl-3-(3,4-dimethoxyphenyl)-2-propenamide (12)
Compound 11 (500.0 mg, 2.40 mmol); DMF (5.5 mL); TEA (0.34 mL); hexylamine (0.32 mL, 2.40
mmol); BOP (1.06 g, 2.40 mmol); CH Cl (5 mL); reaction time: 5 h; ethyl acetate crystallization;
2
2
o
-1
1
7
6% yield. Mp(ethyl acetate) 95-97 C. IR (ATR) ν cm : 3283 (N-H), 1650 (C=O), 1136 (C-O). H NMR
max
(
400 MHz, DMSO-d )  : 0.87 (3H, t, J=6.5, CH ), 1.27-1.31 (6H, m, CH (C-3’-C-5’)), 1.40-1.45 (2H,
6
3
2
m, CH (C-2’)), 3.12-3.17 (2H, m, CH (C-1’)), 3.78 (3H, s, OCH ), 3.79 (3H, s, OCH ), 6.49 (1H, d,
2
2
3
3
J=15.7, CH()), 6.97 (1H, d, J=8.3, CH (C-6)), 7.09 (1H, dd, J =8.3, J =1.5, CH (C-5)), 7.14 (1H, d,
5
,6
5,2
1
3
J =1.5, CH (C-2)), 7.33 (1H, d, J=15.7, CH()), 7.95 (1H, t, J=5.5, NH). C NMR (100 MHz, DMSO-
2
,5
d )  : 13.8 (CH ), 21.9 (C-5’), 26.1 (C-4’), 29.1 (C-3’), 30.9 (C-2’), 38.5 (C-1’), 55.3 (OCH ), 55.4
6
3
3
(
OCH ), 109.8 (C-), 111.7 (C-6), 120.0 (C-5), 121.2 (C-2), 127.7 (C-1), 138.3 (C-), 148.8 (C-2),
3
1
49.9 (C-3), 164.9 (C=O).
N-Hexyl-3-(3-hydroxy-4-methoxyphenyl)-2-propenamide (14)
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Compound 13 (500.0 mg, 2.57 mmol); DMF (6 mL); TEA (0.36 mL); hexylamine (0.34 mL, 2.57
mmol); BOP (1.14 g, 2.57 mmol); CH Cl (6 mL); reaction time: 5 h; column chromatography
2
2
o
-1
(
hexane/ethyl acetate); 43% yield. Mp(ethyl acetate) 102-103 C. IR (ATR) ν cm : 3321 (O-H and N-
max
1
H), 1642 (C=O), 1126 (C-O). H NMR (400 MHz, DMSO-d )  : 0.86 (3H, t, J=6.8, CH ), 1.27-1.29
6
3
(
6H, m, CH (C-3’-C-5’)), 1.39-1.45 (2H, m, CH (C-2’)), 3.12-3.16 (2H, m, CH (C-1’)), 3.79 (3H, s,
2
2
2
OCH ), 6.38 (1H, d, J=15.7, CH()),6.93-6.97 (2H, m, CH (C-5 and C-6)), 6.96 (1H, br s, CH (C-2)),
3
1
3
7
.25 (1H, d, J=15.7, CH()), 7.96 (1H, t, J=5.5, NH), 9.15 (1H, s, OH). C NMR (100 MHz, DMSO-
d )  : 13.8 (CH ), 21.9 (C-5’), 26.1 (C-4’), 29.1 (C-3’), 30.9 (C-2’), 38.5 (C-1’), 55.4 (OCH ), 111.9
6
3
3
(C-), 113.2 (C-5), 119.6 (C-6), 120.0 (C-2), 127.7 (C-1), 138.4 (C-), 146.6 (C-4), 148.9 (C-3), 164.9
(C=O).
N-Hexyl-3-(3,4-methylenedioxyphenyl)-2-propenamide (16)
Compound 15 (500.0 mg, 2.60 mmol); DMF (6 mL); TEA (0.37 mL); hexylamine (0.34 mL, 2.60
mmol); BOP (1.15 g, 2.60 mmol); CH Cl (6 mL); reaction time: 5 h; ethyl acetate crystallization;
2
2
o
-1
1
6
6% yield. Mp(ethyl acetate) 80-81 C. IR (ATR) ν cm : 3305 (N-H), 1647 (C=O), 1037 (C-O). H NMR
max
(
400 MHz, DMSO-d )  : 0.86 (3H, t, J=7, CH ), 1.26-1.30 (6H, m, CH (C-3’-C-5’)), 1.40-1.45 (2H,
6
3
2
m, CH (C-2’)), 3.12-3.16 (2H, m, CH (C-1’)), 6.06 (2H, s, O-CH -O), 6.45 (1H, d, J=15.7, -CH()),
2
2
2
6
.93 (1H, d, J=7.65, CH (C-6)), 7.05 (1H, dd, J =7.7, J =1.6, CH (C-5)), 7.12 (1H, d, J =1.6, -CH
5
,6
5,2
2,5
1
3
(
C-2)), 7.31 (1H, d, J=15.7, CH()), 7.95 (1H, t, J=5.5, NH). C NMR (100 MHz, DMSO-d )  : 13.8
6
(
CH ), 21.9 (C-5’), 26.1 (C-4’), 29.0 (C-3’), 30.9 (C-2’), 38.6 (C-1’), 101.3 (O-CH -O), 106.0 (C-),
3
2
1
08.4 (C-6), 120.4 (C-5), 122.9 (C-2), 129.3 (C-1), 138.0 (C-), 147.8 (C-4), 148.2 (C-3), 164.8
(C=O).
N-Hexyl-3-(4-hydroxy-3,4-dimethoxyphenyl)propanamide (18)
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Compound 17 (1.13 g, 5 mmol); DMF (10 mL); TEA (0.7 mL); hexylamine (0.66 mL, 5 mmol); BOP
(
2.21 g, 5 mmol); CH Cl (10 mL); reaction time: 1h; ethyl acetate crystallization; 56 % yield.
2
2
-1
1
Mp(ethyl acetate) 93-94 °C. IR (ATR) νmax cm : 3319 (N-H), 1643 (C=O), 1225 (C-O), 1125 (C-O). H
NMR (400 MHz, DMSO-d )  : 0.85 (3H, t, J=6.9, CH ), 1.20-1.26 (6H, m, CH (C-3’-C-5’)), 1.33-
6
3
2
1
6
.38 (2H, m, CH (C-2’)), 2.34 (2H, t, J=7.8, CH ()), 2.71 (2H, t, J=7.8, CH ()), 3.02 (2H, dt, J=5.4,
2
2
2
.0, CH (C-1’)), 3.72 (6H, s, CH (C-3 and C-5)), 6.44 (2H, s, CH (C-2 and C-6), 7.76 (1H, t, J=5.4,
2
3
1
3
NH), 8.04 (1H, s, OH). C NMR (100 MHz, DMSO-d )  : 13.9 (CH ), 22.0 (C-5’), 26.1 (C-4’), 29.1
6
3
(
C-3’), 31.0 (C-2’), 31.3, 37.4, 38.4 (C-1’), 55.8 (2 x OCH ), 105.5 (C-2 and C-6), 131.3 (C-1), 133.6,
3
1
47.8 (C-3 and C-5), 171.3 (C=O).
N-Hexyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanamide (20)
Compound 19 (2.78 g, 10 mmol); DMF (20 mL); TEA (1.4 mL); hexylamine (1.32 mL, 10 mmol);
BOP (4.42 mg, 10 mmol); CH Cl (20 mL); reaction time: 3 h; ethyl acetate crystallization; 37 %
2
2
o
-1
1
yield. Mp(ethyl acetate) 77-78 C. IR (ATR) ν cm : 3284 (N-H), 1619 (C=O), 1205 (C-O). H NMR (400
max
MHz, DMSO-d )  : 0.85 (3H, t, J=6.9, CH ), 1.23-1.26 (8H, m, CH (C-2’-C-5’)), 1.35 (18H, s, (CH ) ),
6
3
2
3 3
2
.28 (2H, t, J=7.8, CH ()), 2.68 (2H, t, J=7.8, CH ()),3.00-3.02 (2H, m, CH (C-1’)), 6.7 (1H, s,
2
2
2
1
3
OH), 6.89 (1H, s, CH (C-2 and C-6)), 7.75 (1H, t, J=5.5, NH). C NMR (100 MHz, DMSO-d )  : 13.8
6
(
CH ), 21.9 (C-5’), 25.9 (C-4’), 29.1 (C-3’), 30.3 (C(CH ) ), 30.9 (C-2’), 31.1, 34.3 (C(CH ) ), 37.4,
3
3 3
3 3
3
8.4 (C-1’), 124.0 (C-2 and C-6), 132.1 (C-1), 138.9 (C-3 and C-5), 151.7 (C-4), 171.2 (C=O).
N-Hexyl-3-(3,4-dihydroxyphenyl)propanamide (22)
Compound 21 (500.8 mg, 2.7 mmol); DMF (5.5 mL); TEA (0.38 mL); hexylamine (0.36 mL, 2.7
mmol); BOP (1.19 g, 2.7 mmol); CH Cl (5.4 mL); reaction time: 6 h; column chromatography
2
2
o
-1
(
hexane/ethyl acetate); 89% yield. Mp(ethyl acetate) 65-67 C. IR (ATR) ν cm : 3486 (O-H), 3369 (N-
max
1
H), 1606 (C=O), 1189 (C-O), 1108 (C-O) H NMR (400 MHz, DMSO-d )  : 0.86 (3H, t, J=6.9, CH ),
6
3
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JOURNAL PRE-PROOF
1
.21-1.27 (6H, m, CH (C-3’-C-5’)), 1.33-1.36 (2H, m, CH (C-2’)), 2.24 (2H, t, J=7.7, CH ()), 2.60
2
2
2
(
2H, t, J=7.7, CH ()),2.97-3.02 (2H, m, CH (C-1’)), 6.41 (1H, dd, J =7.97, J =2.0, CH (C-5)), 6.55
2
2
5,6
5,2
(
1H, d, J =2.0, CH (C-2)), 6.59 (1H, dd, J =8.0, CH (C-6)), 7.71 (1H, t, J=5.4, NH), 8.58 (1H, s, OH),
2
,5
6,5
1
3
8
3
1
.67 (1H, s, OH). C NMR (125 MHz, DMSO-d )  : 13.8 (CH ), 21.9 (C-5’), 25.9 (C-4’), 29.0 (C-3’),
6
3
0.6 (C-2’), 30.9, 37.5, 38.3 (C-1’), 115.2 (C-5), 115.6 (C-2), 118.6 (C-6), 132.1 (C-1), 143.2, 144.8,
71.2 (C=O).
N,N-Diethyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-propenamide (23)
Compound 9 (500.2 mg, 1.81 mmol); DMF (4.2 mL); TEA (0.25 mL); diethylamine (0.19 mL, 1.81
mmol); BOP (800.7 mg, 1.81 mmol); CH Cl (4.2 mL); reaction time: 6 h; ethyl acetate
2
2
o
-1
crystallization; 56 % yield. Mp(ethyl acetate) 161-164 C. IR (ATR) ν cm : 3217 (O-H), 1638 (C=O),
max
1
1
261 (C-O), 1191 (C-N). H NMR (400 MHz, DMSO-d )  : 1.07 (3H, t, J=6.3, CH ), 1.15 (3H, t,
6
3
J=5.6, CH ), 1.40 (18H, s, (CH ) ), 3.37-3.39 (2H, m, CH (C-1’)), 3.49-3.54 (2H, m, CH (C-1’)), 6.80
3
3 3
2
2
1
3
(
1H, d, J=15.3, CH()), 7.34 (2H, s, CH (C-2 and C-6)), 7.43 (1H, d, J=15.3, CH()). C NMR (100
MHz, DMSO-d )  : 13.2 (CH ), 15.0 (CH ), 30.1 (C(CH ) ), 34.4 (C(CH ) ), 40.1 (C-1’), 41.3 (C-1’),
6
3
3
3 3
3 3
1
15.1 (C-), 124.5 (C-2 and C-6), 126.4 (C-1), 139.0 (C-3 and C-5), 142.3 (C-), 155.6 (C-4), 164.9
(C=O).
Biological assays
The following reagents were purchased from Sigma-Aldrich Co. LLC (St. Louis, USA):
dimethylsulfoxide (DMSO), acetylsalicylic acid, gentamicin sulfate, cremophor® EL, Dulbecco's
phosphate-buffered saline (DPBS), lipopolysaccharides from Escherichia coli 026:B6 (LPS),
calcium ionophore (A23187). Ethanol absolute was purchased from Fisher Chemical
(
Loughborough, Leic, UK). The thromboxane synthase inhibitor (TXBSI) was synthesized as
described [26]. The “PGE2 Enzyme Immunoassay (EIA) Kit” was obtained from Enzo Life Sciences
Lausen, Switzerland).
(
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Human whole blood assay
+
Venous blood was collected by antecubital venipuncture into heparin-Li vacuum tubes from
healthy human volunteers, following informed consent. The human whole blood assays to
assess the inhibition of PGE production - via COX-1 and -2 were performed as previously
2
reported [27, 28].
COX-1 assay
Briefly, the collected blood (500 μL) was placed in microtubes and incubated in a water bath at
3
7 °C with TXBSI (1 μM, in DPBS) and the tested compounds (3.1 - 100 μM, in DMSO), for 15 min.
Then, the calcium ionophore A23187 (12.2 μg/mL, in ethanol) was added and the mixture was
incubated for another 15 min. After, the samples were placed on ice, for 5 min. The samples
were subsequently centrifuged at 1,000 g for 20 min, at 4 °C. The supernatant was then collected
and stored at -20 °C until use. The quantity of solvents used did not have inhibitory effects and
neither affected the cellular viability (data not shown).
COX-2 assay
Briefly, the collected human whole blood (800 μL) was placed in a six-wells plate and incubated
in a humidified incubator at 37 °C with TXBSI (1 μM, in DPBS-gentamicin), the acetylsalicylic acid
(10 μg/mL, in DPBS-gentamicin), and the tested compounds [0.08 - 100 μM, in
DMSO/chremophor/ethanol 1 % (1:10)], for 15 min. Then, LPS (10 μg/mL, in DPBS-gentamicin)
was added and the mixture was incubated for 5 h. After this period of time, DPBS-gentamicin
buffer (1 mL) was added to the samples and they were then placed on ice, for 10 min. The
samples were subsequently centrifuged at 1,000 g for 15 min, at 4 °C. The supernatant was then
collected and stored at -20 °C until use. The quantity of solvents used did not have inhibitory
effects and neither affected the cellular viability (data not shown).
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Determination of PGE production
2
The above mentioned commercial EIA kit was used to determine the amount of PGE in the
2
samples (thawed plasma supernatants), according to the manufacturer’s instructions, as an
indicator of COX-1 and -2 activities. COX inhibitors, indomethacin (1 μM) and celecoxib (1 μM),
were used as positive controls. Results are expressed as the percent inhibition of control PGE2
production. Each study corresponds to at least three independent experiments.
Statistical Analysis:
GraphPad Prism™ (version 6.0; GraphPad Software, San Diego, CA, USA) was used to perform
the statistical analysis. Results are expressed as mean ± standard error of the mean (SEM).
Statistical comparison between groups was estimated using the one-way analysis of variance
(ANOVA), followed by the Bonferroni’s post-hoc test. In all cases, p-values lower than 0.05 were
considered as statistically significant.
Acknowledgements
This work was supported by UID/QUI/50006/2019 with funding from Fundação para a Ciência e
Tecnologia (FCT) and Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through
national funds, and “Programa Operacional Competitividade e Internacionalização” (COMPETE)
(POCI-01-0145-FEDER-029253). Carina Proença acknowledges FCT the financial support for the
PhD grant (SFRH/BD/116005/2016), in the ambit of "QREN - POPH - Tipologia 4.1 - Formação
Avançada", co-sponsored by Fundo Social Europeu (FSE) and by national funds of Ministério da
Ciência, Tecnologia e Ensino Superior (MCTES). We gratefully acknowledge Graça Porto and the
nursing staff of the Centro Hospitalar do Porto-Hospital de Santo António blood bank for the
collaboration in the recruitment of blood donors to participate in the study.
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NMR data was collected at the UC-NMR facility which is supported in part by FEDER – European
Regional Development Fund through the COMPETE Programme (Operational Programme for
Competitiveness) and by National Funds through FCT – Fundação para a Ciência e a Tecnologia
(Portuguese Foundation for Science and Technology) through grants REEQ/481/QUI/2006,
RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, and Rede Nacional de Ressonância
Magnética Nuclear (RNRMN).
Note
The molecular structure of COX-2 used in the graphical abstract was created by using the PDB
ID: 3LN1 (Structure of celecoxib bound at the COX-2 active site; Wang, J.L., Limburg, D.,
Graneto, M.J., Springer, J., Hamper, J.R., Liao, S., Pawlitz, J.L., Kurumbail, R.G., Maziasz, T., Talley,
J.J., Kiefer, J.R., Carter, J.; (2010) Bioorg Med Chem Lett 20 7159-7163), the NGL Viewer (A.S.
Rose, A.R. Bradley, Y. Valasatava, J.D. Duarte, A. Prlić, P.W. Rose (2018) NGL viewer: web-based
molecular graphics for large complexes. Bioinformatics (doi:10.1093/bioinformatics/bty419),
and RCSB PDB (www.rcsb.org).
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Inflammation, 40 (2017) 1155-1165.
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23] C.I. Bayly, W.C. Black, S. Leger, N. Ouimet, M. Ouellet, M.D. Percival, Structure-based design
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26] K. Kato, S. Ohkawa, S. Terao, Z. Terashita, K. Nishikawa, Thromboxane synthetase inhibitors
TXSI). Design, synthesis, and evaluation of a novel series of omega-pyridylalkenoic acids, J Med
(
Chem, 28 (1985) 287-294.
2
3

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[
27] D. Ribeiro, M. Freitas, S.M. Tomé, A.M.S. Silva, S. Laufer, J.L.F.C. Lima, E. Fernandes,
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Inflammopharmacology, 16 (2008) 155-161.
A
O
N
OH
H
O
OH
O
O
HO
N
OH
O
O
Cl
Cl
O
ibuprofen
naproxen
Cl
diclofenac
indomethacin
F
B
F
F
N
N
N
O
S
O
S
O
O
O
N
O
O
S
O
rofecoxib
H N
2
Cl
celecoxib
etoricoxib
2
4

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Figure 1. A Some examples of COX-1/COX-2 inhibitors; B Some examples of selective COX-2
inhibitors.
2
5

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Figure 2. Inhibition of PGE production via COX-1, in human whole blood by the indicated studied
2
compounds determined by EIA. Each value represents mean ± SEM of at least three experiments.
2
6

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****p<0.0001, ***P<0.001, **P<0.01, *P<0.5 compared with the stimulated control (TXBSI/
calcium ionophore A23187). Indomethacin was used as positive control and presented an
inhibitory activity of 91 ± 4 %, for 1 µM.
2
7

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Disubstituted aromatic:
Preferably absence of double bond
O
R = OH
1
R = OCH
2
3
or
R = OCH
R1
R5
R4 = H
R5 = (CH2)5CH3
or
1
3
R = OH
N
2
or
R4
R4 = R5 = CH2CH3
R = R = OH
1
2
R2
R3
Trisubstituted aromatic:
R = R = OCH
Presence of hexyl or diethylamide side chain
1
3
3
R = OH
2
or
R = R = C(CH )
1
3
3 3
R = OH
2
Figure 3. Principal structural features for COX-1 inhibition.
2
8

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Figure 4. Inhibition of PGE production via COX-2, in human whole blood by the indicated studied
2
compounds determined by EIA. Each value represents mean ± SEM of at least three experiments.
*
***p<0.0001, ***P<0.001, **P<0.01, *P<0.5 compared with the stimulated control
TXBSI/LPS). Celecoxib was used as positive control and presented an inhibitory activity of 71 ±
%, for 1 µM.
(
1
2
9