Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.07.011

In this work the synthesis, structure-activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that

Full text of DOI:10.1016/j.ejmech.2015.07.011

European Journal of Medicinal Chemistry 101 (2015) 573e583
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and evaluation of semi-synthetic triazole-containing
caffeic acid analogues as 5-lipoxygenase inhibitors
,
b
b
b
b
Daniela De Lucia a , Oscar Mendez Lucio , Biagia Musio , Andreas Bender ,
ꢀ
Monika Listing c, Sophie Dennhardt c, Andreas Koeberle c, Ulrike Garscha c,
Roberta Rizzo d, Stefano Manfredini a ***, Oliver Werz c **, Steven V. Ley b
,
,
, *
a Department of Life Sciences and Biotechnology, University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy
b Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
c Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, 07743 Jena, Germany
d Department of Medical Sciences, University of Ferrara, Via Fossato di Mortara 74, 44100, Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work the synthesis, structureeactivity relationship (SAR) and biological evaluation of a novel
series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided
drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of
Received 13 April 2015
Received in revised form
24 June 2015
Accepted 6 July 2015
Available online 15 July 2015
0.2 and 3.2
mM in cell-based and cell-free assays, respectively. Optimization of binding and functional
potencies resulted in the identification of compound 13d, which showed an enhanced activity compared
to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15
and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their
interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests
were performed to exclude a relationship between cytotoxicity and the increased activity observed after
structure optimization.
Keywords:
5-Lipoxygenase inhibitor
Inflammation
Triazole
© 2015 Published by Elsevier Masson SAS.
Polyphenol
Caffeic acid
Zileuton
1. Introduction
implications in different diseases like asthma, allergic rhinitis as
well as cardiovascular diseases and certain types of cancer [2].
5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic
acid (AA) cascade, involved in the first two catalytic reactions in the
biosynthesis of leukotrienes (LTs) [1]. The role of LTs as inflamma-
tory mediators is well established, with pathophysiological
Some new 5-LO inhibitors of plant origin are currently under
clinical development for the treatment of osteoarthritis, such as a
combination of baicalein 1 and catechin 2, named Flavocoxid
(Limbrel) [3]. Nevertheless, the only 5-LO inhibitor to have reached
the market so far is zileuton 3, which has some major drawbacks
such as liver toxicity, low potency, and a short half-life due to rapid
metabolic breakdown (Fig. 1) [4].
With the rising number of indications for anti-LT therapy, 5-LO
inhibitor drug development becomes increasingly important. A
variety of polyphenols have been reported to be capable of pre-
venting the formation of 5-LO products, among which prominent
dietary compounds such as curcumin 4, and caffeic acid 5 are worth
mentioning (Fig. 2) [5].
Abbreviations: 5-LO, 5-Lipoxygenase; LT, Leukotriene; AA, Arachidonic Acid;
SAR, Structure-Activity Relationship; CuAAC, Copper(I)-catalysed Azide Alkyne
Cycloaddition; DCC, Dicyclohexylcarbodiimide; DCM, Dichloromethane; MTT, 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; FLAP, Five Lip-
oxygenase Activating Protein; FCC, Flash Column Chromatography; TLC, Thin Layer
Chromatography.
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: daniela.delucia@unife.it (D. De Lucia), om268@cam.ac.uk
(O.M. Lucio), bm450@cam.ac.uk (B. Musio), ab454@cam.ac.uk (A. Bender),
The current interest in polyphenols has been driven primarily by
their abundance in human diet and in plants [6]. Moreover, several
polyphenols can be isolated as products from the waste streams of
the food industry, thus providing a potentially sustainable source of
these compounds. However, the health effects of polyphenols are
Monika.Listing@uni-jena.de
(M.
Listing),
Sophie.Dennhardt@uni-jena.de
(S. Dennhardt), Andreas.Koeberle@uni-jena.de (A. Koeberle), Ulrike.Garscha@uni-
jena.de (U. Garscha), roberta.rizzo@unife.it (R. Rizzo), stefano.manfredini@unife.it
(S. Manfredini), Oliver.Werz@uni-jena.de (O. Werz), svl1000@cam.ac.uk (S.V. Ley).
http://dx.doi.org/10.1016/j.ejmech.2015.07.011
0223-5234/© 2015 Published by Elsevier Masson SAS.

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D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
Fig. 1. Zileuton and 5-LO inhibitors of plant origin.
Fig. 2. Naturally occurring bioactive polyphenols.
negatively influenced by the bioavailability and solubility issues
often associated with these phytochemicals, which largely reduce
their use in pharmaceuticals [7]. Recent attempts to overcome
these issues include the pegylation of curcumin 4 [8] and the
glycosylation of resveratrol 6 and curcumin 4 [9].
A new approach is explored in the present work with the
incorporation of a triazole-containing linker into polyphenol mol-
ecules. Triazoles are, in fact, not only passive linkers capable of
giving metabolic stability and increasing water solubility through
hydrogen-bond formation, but are also important pharmacophores
able to interact with different targets. They have been proven to be
determinant for the activities in antifungal, antibacterial, and
anticancer agents, HIV protease inhibitors [10] and anti-
inflammatory compounds [11].
Fig. 3. Structural modifications on the triazolyl caffeic acid.
formation of the triazole cycle, by means of a copper(I) catalysed
azide-alkyne cycloaddition (CuAAC, Fig. 3) [12]. Carboxylic acids 8a
and 8b were easily converted into 10ae10f and 12 after refluxing in
benzene in presence of DMF and an excess of thionyl chloride.
Propargyl alcohols 9ae9d, propargylamine 9e or the 3-
azidopropan-1-ol 11 were then added and reacted at room tem-
perature to furnish 10ae10f and 12 in good yields (Scheme 1).
Propargyl derivatives 10ae10f were subjected to the classical
CuAAC conditions, by reacting with the azidoalcohol 11 in presence
of catalytic amounts of Cu(I)iodide (Scheme 2).
2. Results and discussion
2.1. Synthesis of analogues
In this study a new class of semi-synthetic polyphenols related
to caffeic acid has been designed. Structural modifications have
been made in order to evaluate the structureeactivity relationship
(SAR) on 5-LO. Particularly, the linker length (A), the linker nature
(B), the substitution on the aromatic portion (C) and the function-
alization of the hydroxyl group (D) have been considered (Fig. 3).
The retrosynthetic analysis of these compounds suggested an
initial esterification or amidation of the caffeic acid followed by the
The molecular diversity achieved in this library of compounds
could be further increased by preparing analogue 14, which rep-
resents a regioisomer of 13a, in order to evaluate a possible distinct
activity against 5-LO. Compound 14 was smoothly obtained by

D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
575
Scheme 1. Synthetic procedures for esters (10ae10f, 12). Reagents and conditions: thionyl chloride (25 mmol), 8ae8b (1 mmol), DMF (0.1% mol), dry benzene (1 mL), reflux, then
concentrate to dryness, addition of dry benzene (4 mL), pyridine (0.1% mol) dropwise and 9ae9e (1.2 mmol) at r.t.
Scheme 2. Synthetic procedures for the preparation of triazoles (13a-13f). Reagents and conditions: t-BuOHeH2O (5 mL, ratio 1:1), 10a-10f (1.2 mmol), 11 (1 mmol), Cu(I)iodide
(0.1 mol), 125 ꢀC, 1 h.
reaction of 12 with propargyl alcohol 9a in the CuAAC conditions
applied above (Scheme 3).
ionophore A23187 together with exogenous AA (20 mM) were used
[14]. This dual strategy allows the identification of direct 5-LO in-
hibitors and provides insights into their potency in a physiologi-
cally relevant intact cell system, which generally constitutes a cut
out for actives [15]. 5-LO products formed (LTB4, its two trans-
isomers, and 5-H(P)ETE) were analysed by RP-HPLC and the 5-LO
The orthogonal derivatives 15 and 16 were prepared to further
enhance the molecular diversity within this family of compounds,
and to widen the SAR study. Compound 15 was prepared from 10a
and 12, whereas compound 16 was prepared from 10a and 3-
azidopropyl-6-acetoxy-2,5,7,8-tetramethylchromane-2-
carboxylate, 17 (Fig. 4).
inhibitor zileuton (IC50 ¼ 0.5e1
mM) served as reference com-
pound [16].
With reference to the poor 5-LO inhibitor caffeic acid 5, acety-
lation of the two hydroxyl moieties yielding compound 8a failed to
improve potency (Table 1, entry 3). However, esterification of 8a
with 9aed yielding 10aed led to highly potent derivatives in intact
2.2. Biological evaluation
In order to study the ability of the new compounds to directly
inhibit 5-LO, a cell-free assay using purified human recombinant 5-
neutrophils with IC50 values 0.18e1.8 mM (Table 1, entries 4e7). Note
LO enzyme and arachidonic acid (20
applied[13]. To study the inhibitory potency on 5-LO product for-
mation in intact cells, human neutrophils stimulated with the Ca2þ
m
M
) as the substrate was
that the compounds showed low inhibition of 5-LO in the cell-free
assay, implying that suppression of 5-LO product synthesis requires
the cellular environment and could be related to interference with
-

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D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
Compound 13d showed the highest potency for inhibiting 5-LO
within this series (IC50 values 0.2 and 3.2 in cell based and
mM
mM
cell-free assays respectively). The chain length of the bridge be-
tween the polyphenol and the triazole core was apparently critical
for the activity profile. In fact, the gain in potency by linker elon-
gation from 13a to 13d suggests the relevance of the hydrophobic
interactions in the 5-LO binding site. Based on the fact that some
compounds were much more potent against 5-LO in intact cells
than in the isolated 5-LO, it appeared possible that these com-
pounds may suppress cellular 5-LO product synthesis by interfer-
ence with 5-lipoxygenase activating protein (FLAP). Thus, in
addition to the 5-LO activity assay in neutrophils and on the iso-
lated enzyme, compound 10a, 15 and 16 were tested in a HEK cell
system that stably expresses 5-LO with or without FLAP (Fig. 5) [18].
All three tested compounds inhibited 5-LO product formation at
Scheme 3. CuAAC towards the synthesis of compound 14.
1 mM in cells expressing only 5-LO. Of significance, co-expression of
FLAP even reduced the potencies against inhibition of 5-LO. In
particular, compounds 15 and 16, that failed to reduce 5-LO activity
of the isolated enzyme, were highly potent and blocked 5-LO ac-
other enzymes within LT biosynthesis. The corresponding amide
10e of bioactive 10a was less potent, favouring the ester over amide
function (Table 1, entry 8). Replacement of the acetoxy residues at
the benzene of 10a by fluorine yielding 10f abolished 5-LO inhibi-
tion (Table 1, entry 9). Exchange of the alkyne of 10a for the azide in
12 was tolerated (Table 1, entry 10).
The triazole caffeic acid esters 13aed suppressed the 5-LO ac-
tivity in the cell-free and cell-based assays and progressive elon-
gation of the chain length from 13a to 13d increased 5-LO
inhibitory potency. Compound 13d turned out as most potent lead
tivity at a concentration of 1
mM completely. These results exclude
FLAP as target of the compounds prepared herein, but clearly
supports the inhibitory effect of the compounds on the 5-LO
product formation in intact cells.
The logP of compounds herein presented was calculated through
Marvin Sketch software 5.4.1.1. [19]. Elongating the linker length
from compound 10a (logP 1.99) to 10d (logP 4.80) and from com-
pound 13a (logP 1.01) to 13d (logP 3.65) leads to an increase in the
value of logP due to the increase in lipophilicity. Turning the linker
from an ester to an amide leads to a decrease in the logP value
confirming an enhanced hydrophilic character of compounds 10e
(logP 1.08) and 13e (logP 0.10) compared to their ester analogues
10a (logP 1.99) and 13a (logP 1.01). Substituting the groups on the
aromatic ring with fluorine atoms instead, leads to an increase in
logP values 10f (logP 3.07), 13f (logP 2.09) compared to their ho-
mologues 10a and 13a respectively. The orthogonal derivatives 15
and 16 which possess similar inhibitory activity against neutrophils
compound with IC50 values of 0.2 and 3.2 mM, in cell-based and cell-
free assays, respectively (Table 1, entry 14). The corresponding
amide 13e of the ester 13a was inactive (cell-based and cell-free
assays, Table 1, entry 15) and exchange of the acetoxy residues in
13a by fluorine in 13f led to an inactive derivative (Table 1, entry
16). Moreover, the isomeric triazole derivative 14 did not affect the
5-LO inhibitory potency in cell-free assays but resulted in loss of
activity in neutrophils (Table 1, entry 17).
For the orthogonal derivatives 15 and 16, which actually failed to
significantly suppress the activity of isolated 5-LO, potent inhibition
of 5-LO in intact cells was evident with IC50 values of 0.6 and
(0.6 and 0.16 mM respectively) have shown logP values of 3.04 and
5.70 respectively, making theoretically compound 15 a better choice
for a drug candidate than compound 16. Compound 16 possess a
logP value slightly higher than the acceptable range defined by
Lipinski (5.70 vs 5.00). All the compounds herein presented have
acceptable logP values (see supporting information for details).
0.16
mM, respectively (Table 1, entries 18 and 19). Together, com-
pounds 10d,15 and 16 are potent inhibitors of cellular 5-LO product
formation but 15 and 16 are less efficient against isolated 5-LO.
Fig. 4. Orthogonal compounds derived from caffeic acid and Trolox.

D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
577
Table 1
Effects of new semi-synthetic compounds on the activity of 5-LO in cell-based (neutrophils) and cell-free (isolated 5-LO) assays.
a
a
Entry
Cmpd
Substituents
5-LO in neutrophils IC50
(
m
M
)
Isolated 5-LO IC50
(m
M
)
1
2
3
4
5
6
7
8
3
5
8a
1.3 0.3
0.54 0.11 [17]
8.5 0.6
R1 ¼ OH
>10 (104 4.0)
>10 (84.7 3.5)
1.8 0.2
0.92 0.34
0.47 0.15
0.18 0.01
8.23 0.15
>10 (88.2 3.9)
1.3 0.4
R1 ¼ OAc
>10 (90.3 1.6)
>10 (50.2 3.7)
>10 (84.3 1.8)
>10 (70.7 4.7)
>10 (71.8 4.6)
>10 (53.5 3.1)
>10 (52.6 3.0)
>10 (73.9 8.2)
7.4 0.5
7.6 0.3
6.7 0.3
3.2 0.2
>10 (53.6 14.3)
>10 (90.3 9.7)
7.6 3.0
>10 (81.3 10.4)
>10 (60.5 8.2)
>10 (86.3 4.8)
10a
10b
10c
10d
10e
10f
12
13a
13b
13c
13d
13e
13f
14
R1 ¼ OAc; R2 ¼ CCH; X ¼ O; n ¼ 2
R1 ¼ OAc; R2 ¼ CCH; X ¼ O; n ¼ 4
R1 ¼ OAc; R2 ¼ CCH; X ¼ O; n ¼ 5
R1 ¼ OAc; R2 ¼ CCH, X ¼ O; n ¼ 8
R1 ¼ OAc; R2 ¼ CCH; X ¼ NH; n ¼ 2
R1 ¼ F; R2 ¼ CCH; X ¼ O; n ¼ 2
R1 ¼ OAc; R2 ¼ N3; n ¼ 2
9
10
11
12
13
14
15
16
17
18
19
20
R1 ¼ OAc; R3 ¼ H; X ¼ O; n ¼ 2
R1 ¼ OAc; R3 ¼ H; X ¼ O; n ¼ 4
R1 ¼ OAc; R3 ¼ H; X ¼ O; n ¼ 5
R1 ¼ OAc; R3 ¼ H; X ¼ O; n ¼ 8
R1 ¼ OAc; R3 ¼ H; X ¼ NH; n ¼ 2
R1 ¼ F; R3 ¼ H; X ¼ O; n ¼ 2
9.9
1
6.1 0.5
3.1 0.6
0.2 0.03
>10 (103.7 4.3)
>10 (101.4 6.6)
>10 (75.1 4.3)
0.6 0.09
0.16 0.01
2.6 0.1
15
16
17
R1 ¼ OAc; R3 ¼ 5; X ¼ NH; n ¼ 2
R1 ¼ OAc; R3 ¼ Troloxb; X ¼ NH; n ¼ 2
a
Remaining activity in % at 10
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
m
m.
b
presence of representative compounds, cell viability was evaluated
and expressed as EC50, which is the concentration required to
inhibit MTT-reducing mitochrondrial dehydrogenase activity by
50% (Table 2).
All the compounds tested caused only weak cytotoxic effects at
similar concentrations as caffeic acid 5. Compound 13f, which was
one of the less active 5-LO inhibitors among the compounds tested,
most potently suppressed cell viability with an EC50 value of 5
mM
(Table 2, entry 5). These data suggest that inhibition of cellular 5-LO
product synthesis is unrelated to potentially cytotoxic effects of the
compounds in the present library.
Fig. 5. Effect of compounds 10a, 15 and 16 on 5-LO product formation in HEK 293 cells
stably transfected with 5-LO with and without FLAP.
Table 2
Effects on cell viability of representative compounds on U937.
Entry
Cmpd
EC50 (mM)
1
2
3
4
5
6
7
5
9.9 0.5
9.8 1.0
10.2 0.4
10.3 1.0
5.1 0.3
9.6 0.5
10.1 0.6
2.3. Cytotoxicity assays
10a
13d
13e
13f
15
A decrease in cellular 5-LO product formation due to cytotoxic
effects of the compounds in the library has been ruled out using the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bro-
16
mide (MTT) assay. After incubation of U937 cells for 24 h in the

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D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
2.4. Molecular docking studies
A docking protocol was used to predict, at a molecular level, the
possible interactions between 5-LO and the ligands reported in this
work. All the compounds were docked into 5-LO (PDB ID: 3O8Y)
and those with the lowest predicted binding energies were
considered the most active compounds (Fig. 6). In general, com-
pounds 13aee presented the lowest predicted binding energy. As
reference, the calculated binding energy of caffeic acid is ꢁ6.5 kcal/
mol. It is worth noting that in general compounds with an
IC50 < 10
exception of caffeic acid 5).
m
M
presented a predicted binding energy ꢂe9.2 (with the
Caffeic acid 5 and zileuton 3 were docked as references. Caffeic
acid 5 is stabilized in the 5-LO binding pocket by the formation of
hydrogen bonds with Ala424, blocking the binding site of arach-
idonic acid in 5-LO (Figs. 7-A). This binding site has been reported
before for ketoconazole and ketaminazole in 5-LO. [20]. In a similar
way, the binding site of zileuton 3 is located in the inner part of the
binding pocket and is stabilized by the formation of a network of
hydrogen bonds with the main chains of Leu420 and Ala424 and
the side chain of Asn425 (Figs. 7-B).
Interestingly, the phenyl ring of compound 13d, the most active
inhibitor in both isolated 5-LO and intact neutrophils, is in close
proximity to Leu420, Ala424 and Asn425, similarly as for caffeic
acid 5, whereas the rest of the molecule extends until fulfilling the
binding site of arachidonic acid (Figs. 8-A). Compound 13f does not
form any hydrogen bond with the 5-LO pocket compared to 13d,
which is interacting with Asn425. Although the fluorines in com-
pound 13f could participate in hydrogen bonding, they are not
oriented properly for this interaction. Moreover, the hydrogen bond
between the triazole group and the catalytic Fe2þ likely could
prevent the compound 13f to get deeper in the binding site (Figs. 8-
B). It has been observed before that the interaction with the cata-
lytic Fe2þ contributes favourably to model the ligand binding, as the
reduction of the ferric atom by the ligand is a very effective mode of
action for 5-LO inhibition [20].
Fig. 7. Suggested binding conformations of caffeic acid 5 (A), zileuton 3 (B).
5-LO. Compounds lacking of the triazole group (10aed) showed
noteworthy activity in the cell based assays but not against the
isolated enzyme, suggesting the triazole core as crucial element to
specifically direct the selectivity of these compounds on 5-LO. The
orthogonal derivatives 15 and 16, although failing to inhibit 5-LO
directly, proved to be powerful inhibitors of cellular 5-LO product
formation. Experiments conducted to evidence FLAP as possible
alternative target for compounds 10a, 15 and 16 indicated that their
behaviour is not connected with inhibitory activity towards FLAP.
Possibly, the compounds act as non-redox type 5-LO inhibitors that
are up to 100-fold more potent in intact cells as compared to cell-
free assays [21]. Further studies are currently undergoing to get
more insights on the molecular mode of action of this class of
compounds.
3. Conclusions
A new series of caffeic acid analogues bearing a triazole motif
has been developed as potential 5-LO inhibitors. A clear profile in
the structureeactivity relationship emerged in dependence on the
triazole containing linker. Lead compound, 13d, was identified as a
direct 5-LO inhibitor, with a demonstrated excellent and higher
potency than the clinically approved zileuton 3 (IC50 ¼ 0.5e1
mM) in
neutrophils and slightly higher against isolated 5-LO. The linker
length proved to be a crucial factor in determining potency against
4. Experimental section
4.1. Instrumentation and reagents
Flash column chromatography (FCC) was performed using
Breckland Scientific silica gel 60, particle size 40e63 nm under air
pressure. Analytical thin layer chromatography (TLC) was per-
formed using silica gel 60 F254 pre-coated glass backed plates and
visualized by ultraviolet radiation (254 nm) and/or potassium
permanganate or ammonium molybdate as appropriate. Isolated
yields are reported to 0 decimal places and “quant.” signifies a yield
of 99.5% or higher. 1H NMR spectra were recorded on Bruker DRX-
400 (400 MHz) or DRX-600 (600 MHz) spectrometer. Chemical
Fig. 6. Predicted binding energy of the compounds in the library on 5-LO (kcal/mol).

D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
579
evaporator.
4.1.1. General procedure for the preparation of compound 8a
To a mixture of caffeic acid (1.0 eq., 1.0 mmol) and pyridine (1.0
eq., 1.0 mmol) was added acetic anhydride (5.0 eq., 5.0 mmol) and
stirred at room temperature until the reaction was completed. The
reaction mixture was then diluted with DCM (5 mL) and washed
with 3
M
HCl (3 ꢃ 5 mL) and with brine (3 ꢃ 5 mL). The organic layer
was then dried over MgSO4 and concentrated to dryness to afford
the pure product.
4.1.1.1. (E)-3-(3,4-Diacetyl)caffeic acid 8a. White solid; yield 95%;
mp 125 ꢀC. 1H NMR (600 MHz, DMSO-d6)
d
7.66 (d, J ¼ 2.0 Hz, 1H),
7.63 (dd, J ¼ 8.4 Hz, 1H), 7.57 (d, J ¼ 16.0 Hz, 1H), 7.31 (d, J ¼ 8.35 Hz,
1H), 6.53 (d, J ¼ 16.0 Hz, 1H), 3.32 (s, br, 1H), 2.29 (s, br, 3H) 2.28 (s,
br, 3H). 13C NMR (150 MHz, DMSO-d6)
d 168.07, 168.98, 167.27,
143.2, 142.22, 142.05, 130.0, 126.6, 124.0, 122.9, 120.2, 20.3, 20.2. FT-
IR (Neat, nmax cmꢁ1) 1755, 1676, 1630. HRMS m/z calculated for
C
13H13O6 [MþH]þ 265.0712, found 265.0707.
4.1.2. General procedure for the preparation of compounds 10ae10f
and 12
A mixture of diacetylcaffeic acid 8a or 3,4-difluoro cinnamic acid
8b (1.0 eq., 1.0 mmol), thionyl chloride (25.0 eq., 25.0 mmol), DMF
(0.001 eq., 0.001 mmol) and dry benzene (1.0 mL) was refluxed for
4 h. The excess of thionyl chloride was removed under vacuum. The
residue was dissolved in dry benzene (4 mL) and pyridine (0.001
eq., 0.001 mmol) was added dropwise. The appropriate reagent
9aee or 11 was added (1.2 eq., 1.2 mmol) and the mixture was
stirred overnight at room temperature under argon atmosphere.
The resulting mixture was concentrated and the residue dissolved
in DCM (10 mL), washed with water (3 ꢃ 10 mL), washed with brine
(3 ꢃ 10 mL), dried over MgSO4 and solvents were removed under
vacuum to afford the crude material. The purification was per-
formed as stated for each compound.
Fig. 8. Suggested binding conformations of 13d (A) and 13f (B).
4.1.2.1. (E)-4-(3-(But-3-yn-1-yloxy)-3-oxoprop-1-en-1-yl)-1,2-
phenylene diacetate 10a. White solid; yield 93%; mp 82 ꢀC. Purified
by FCC (EtOAc/petroleum ether 30:70). Recrystallized from EtOAc/
hexane (10:90). Rf 0.23 (EtOAc/petroleum ether 30:70). 1H NMR
shifts are reported in ppm with the resonance resulting from
incomplete deuteration of the solvent as the internal standard
(CDCl3: 7.26 ppm, or DMSO-d6: 2.54 ppm, q). 13C NMR spectra were
recorded on Bruker DRX-400 (100 MHz) or DRX-600 (150 MHz)
spectrometer with complete proton decoupling. Chemical shifts are
reported in ppm with the solvent resonance as the internal stan-
dard (CDCl3: 77.0 ppm, t or DMSO-d6: 30.73, ep.). Data are reported
(600 MHz, CDCl3)
d
7.65 (d, J ¼ 16.0 Hz, 1H), 7.41 (dd, J ¼ 8.4 Hz,
1.9 Hz, 1H), 7.36 (d, J ¼ 2.0 Hz, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 6.40 (d,
J ¼ 16.0 Hz, 1H), 4.32 (t, J ¼ 6.8 Hz, 2H), 2.61 (m, J ¼ 6.8 and 2.7 Hz,
2H), 2.31 (s, 3H), 2,30 (s, 3H), 2.03 (t, J ¼ 2.7 Hz, 1H). 13C NMR
as follows: chemical shift
d
/ppm, multiplicity (s
¼
singlet,
(150 MHz, CDCl3)
d 168.0, 167.9, 166.2, 143.6, 143.3, 142.4, 133.2,
d ¼ doublet, t ¼ triplet, q ¼ quartet, br ¼ broad, m ¼ multiplet or
combinations thereof. 13C signals are singlets unless otherwise
stated), coupling constants are reported in Hz, integration (1H
only). 1H NMR signals are reported to 2 decimal places and 13C
signals to 1 decimal place unless rounding would produce a value
identical to another signal. In this case, an additional decimal place
is reported for both signals concerned. 19F NMR signals are reported
to 2 decimal places and trifluorotoluene was used as internal
standard. High resolution mass spectrometry (HRMS) was per-
formed on a Waters Micromass LCT spectrometer using electro-
spray ionization, time-of-flight analysis and Micromass MS
software HRMS signals are reported to 4 decimal places and are
within 5 ppm of theoretical values. Infrared spectra were recorded
neat as thin films on a PerkineElmer Spectrum One FTIR spec-
trometer and only selected peaks are reported (br ¼ broad). Melting
points were collected using a Stanford Research Systems Optimelt
automated melting point system using a gradient of 1 ꢀC per min.
Unless stated otherwise, reagents were obtained from commercial
sources and used without purification. The removal of solvent un-
der reduced pressure was carried out on a standard rotary
126.4, 123.9, 122.8, 118.8, 80.0, 70.0, 62.3, 20.7, 20.6, 19.1. FT-IR
(Neat, nmax cmꢁ1) 3272, 1765, 1714, 1638. HRMS m/z calculated for
C
17H17O6 [MþH]þ 317.1020, found 317.1016. Elemental analysis
calculated for C17H16O6 requires C 64.55%, 5.10%, found C 64.26%, H
5.12%.
4.1.2.2. (E)-4-[3-(Hex-5-yn-1-yloxy)-3-oxoprop-1-en-1-yl]-1,2-
phenylene diacetate 10b. White solid; yield 69%; mp 48 ꢀC. Purified
by FCC (EtOAc/petroleum ether 30:70). Recrystallized from hexane.
Rf 0.35 (EtOAc/petroleum ether 30:70). 1H NMR (400 MHz, CDCl3)
d
7.58 (d, J ¼ 16.0 Hz, 1H), 7.36 (d, J ¼ 8.5 Hz, 1H), 7.33 (overlapping,
br, s, 1H), 7.18 (d, J ¼ 8.3 Hz, 1H), 6.35 (d, J ¼ 16.0 Hz, 1H), 4.19 (t,
J ¼ 6.3 Hz, 2H), 2.26 (s, 3H), 2.25 (s, 3H), 2.22 (overlapping m, 2H),
1.96 (s, 1H), 1.86e1.73 (m, 2H), 1.65e1.58 (m, 2H). 13C NMR
(100 MHz, CDCl3)
d 167.7, 167.6, 166.2, 143.2, 142.5, 142.2, 133.0,
126.1, 123.7, 122.5, 119.0, 83.7, 68.7, 64.0, 27.6, 24.8, 20.50, 20.46,
18.0. FT-IR (Neat, nmax cmꢁ1) 3290, 2933, 2864, 1762, 1707, 1638,
1507. HRMS m/z calculated for C19H21O6 [MþH]þ 345.1338, found
345.1347. Elemental analysis calculated for C19H20O6 requires C
66.27%, 5.85%, found C 66.23%, H 5.87%.

580
D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
4.1.2.3. (E)-4-[3-(Hept-6-yn-1-yloxy)-3-oxoprop-1-en-1-yl]-1,2-
phenylene diacetate 10c. White solid; yield 41%; mp 68 ꢀC. Purified
by FCC (EtOAc/petroleum ether 30:70). Recrystallized from hexane.
Rf 0.23 (EtOAc/petroleum ether 30:70). 1H NMR (400 MHz, CDCl3)
(EtOAc/petroleum ether 40:60). Rf 0.28 (EtOAc/petroleum ether
30:70). 1H NMR (400 MHz, CDCl3)
(dd, J ¼ 8.4,1.9 Hz,1H), 7.33 (d, J ¼ 1.8 Hz,1H), 7.17 (d, J ¼ 8.3 Hz,1H),
6.34 (d, J ¼ 16.0 Hz, 1H), 4.23 (t, J ¼ 6.2 Hz, 2H), 3.38 (t, J ¼ 6.7 Hz,
2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.96e1.88 (m, 2H). 13C NMR (100 MHz,
d
7.58 (d, J ¼ 16.0 Hz, 1H), 7.36
d
7.59 (d, J ¼ 16.0 Hz, 1H), 7.37 (d, J ¼ 8.4 Hz, 1H), 7.34 (br, s, 1H), 7.19
(d, J ¼ 8.3 Hz, 1H), 6.36 (d, J ¼ 16.0 Hz, 1H), 4.18 (t, J ¼ 6.6 Hz, 2H),
2.27 (s, 3H), 2.26 (s, 3H), 2.19 (td, J ¼ 6.6 and 2.3 Hz, 2H), 1.94 (t,
J ¼ 2.4 Hz, 1H), 1.76e1.64 (m, 2H), 1.62e1.43 (m, 4H). 13C NMR
CDCl3) d 168.35, 168.25, 166.7, 143.9, 143.5, 142.8, 133.4, 126.7, 124.3,
123.1, 119.2, 61.9, 48.5, 28.5, 20.93, 20.89. FT-IR (Neat, nmax cm-1)
2096, 2933, 2864, 1770, 1710, 1639. HRMS m/z calculated for
(100 MHz, CDCl3)
d
167.9, 167.8, 166.4, 143.3, 142.6, 142.3, 133.2,
C
16H18N3 O6 [MþH]þ 348.1190, found 348.1189.
126.2, 123.8, 122.6, 119.2, 84.1, 68.4, 64.4, 28.1, 27.9, 25.0, 20.48,
20.45, 18.2. FT-IR (Neat, nmax cmꢁ1) 3294, 2940, 2861, 1764, 1705,
1638. HRMS m/z calculated for C20H23O6 [MþH]þ 359.1489, found
359.1479. Elemental analysis calculated for C20H22O6 requires C
67.03%, H 6.19%, found C 66.99%, H 6.24%.
4.1.3. General procedure for the preparation of compounds 13aef
A catalytic amount of Cu(I)iodide (0.1 eq., 0.1 mmol) was added
to a stirred solution of 11 (1.0 eq., 1.0 mmol) and 10aef (1.2 eq.,
1.2 mmol) in a H2O/t-butanol (5 mL, ratio 1:1). The mixture was
stirred at 125 ꢀC for 1 h until the consumption of 4 as checked by
TLC (EtOAc/petroleum ether 80:20). The reaction was allowed to
warm to room temperature and then diluted with water, extracted
with DCM (3 ꢃ 10 mL), dried over MgSO4, concentrated and puri-
fied by silica column chromatography with the appropriate eluent
system to afford the pure triazole derivatives.
4.1.2.4. (E)-4-[3-(Dec-9-yn-1-yloxy)-3-oxoprop-1-en-1-yl]-1,2-
phenylene diacetate 10d. White solid; yield 44%; mp 49 ꢀC. Purified
by FCC (EtOAc/petroleum ether 30:70). Recrystallized from hexane.
Rf 0.43 (EtOAc/petroleum ether 30:70). 1H NMR (400 MHz, CDCl3)
d
7.61 (d, J ¼ 16.0 Hz, 1H), 7.40 (dd, J ¼ 8.4 and 1.9 Hz, 1H), 7.36 (d,
J ¼ 1.8 Hz, 1H), 7.22 (d, J ¼ 8.4 Hz, 1H), 6.38 (d, J ¼ 16.0 Hz, 1H), 4.19
(t, J ¼ 6.7 Hz, 2H), 2.31 (s, 3H), 2.30 (s, 3H), 2.21e2.15 (m, 3H),1.94 (t,
J ¼ 2.6 Hz, 1H), 1.74e1.64 (m, 2H), 1.59e1.46 (m, 4H), 1.38 (m, 7H).
4.1.3.1. (E)-4-(3-(2-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)
13C NMR (100 MHz, CDCl3)
d
168.34, 168.26, 167.0, 143.7, 142.9,
ethoxy)-3-oxoprop-1-en-1-yl)-1,2-phenylene
diacetate
13a.
142.7, 133.7, 126.6, 124.2, 123.0, 119.8, 85.0, 68.4, 65.1, 29.4, 29.3,
29.0, 28.9, 28.7, 26.2, 21.0, 20.9, 18.7. FT-IR (Neat, nmax cmꢁ1) 3303,
2932, 2862, 1764, 1709, 1638. HRMS m/z calculated for C23H29O6
[MþH]þ 401.1959, found 401.1956. Elemental analysis calculated for
Yellow oil; yield 74%. Purified by column chromatography (EtOAc/
petroleum ether 70:30). Rf 0.20 (EtOAc). 1H NMR (600 MHz, CDCl3)
d
7.62 (d, J ¼ 16.0 Hz, 1H), 7.45 (s, 1H), 7.40 (dd, J ¼ 8.4 and 1.8 Hz,
1H), 7.37 (d, J ¼ 1.8 Hz, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 6.38 (d,
J ¼ 16.0 Hz, 1H), 4.52e4.48 (m, 4H), 3.64 (q, J ¼ 5.6 Hz, 2H),
3.20e3.12 (m, 2H), 2.35 (s, 1H), 2.32 (s, 3H), 2.31 (s, 3H), 2.15e2.10
C
23H28O6 requires C 68.98%, H 7.05%, found C 68.87%, H 7.03%.
4.1.2.5. (E)-4-(3-(But-3-yn-1-ylamino)-3-oxoprop-1-en-1-yl)-1,2-
phenylene diacetate 10e. White solid, yield 80%; mp 143 ꢀC. Purified
by FCC (EtOAc/petroleum ether 60:40). Recrystallized from EtOAc/
hexane (90:10). Rf 0.32 (EtOAc/petroleum ether 60:40). 1H NMR
(m, 2H). 13C NMR (150 MHz, CDCl3)
d 168.3,168.1,166.5,143.7, 143.3,
142.5, 133.3, 126.6, 124.1, 122.9, 122.3, 119.1, 63.5, 58.8, 47.0, 32.7,
31.1, 25.7, 20.77, 20.76. FT-IR (Neat, nmax cmꢁ1) 2954 (br), 1771, 1710,
1638, 1505. HRMS m/z calculated for C20H24O7N3 [MþH]þ 418.1609,
found 418.1605.
(600 MHz, CDCl3)
d
7.57 (d, J ¼ 15.6 Hz, 1H), 7.37 (dd, J ¼ 8.4 and
1.7 Hz, 1H), 7.34 (d, J ¼ 1.6 Hz, 1H), 7.19 (d, J ¼ 8.3 Hz, 1H), 6.33 (d,
J ¼ 15.5 Hz, 1H), 5.97 (s, 1H), 3.54 (q, J ¼ 6.3 Hz, 2H), 2.47 (td, J ¼ 6.3
4.1.3.2. (E)-4-(3-(4-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)
and 2.5 Hz, 2H), 2.30 (s, 3H), 2.30 (s, 3H), 2.04 (t, J ¼ 2.5 Hz, 1H). 13
C
butoxy)-3-oxoprop-1-en-1-yl)-1,2-phenylene
Yellow oil; yield 40%. Purified by FCC (EtOAc/petroleum ether
70:30). Rf 0.22 (EtOAc). 1H NMR (400 MHz, CDCl3)
7.59 (d,
diacetate
13b.
NMR (150 MHz, CDCl3)
d 168.09, 168.07, 165.4, 142.9, 142.3, 139.2,
133.7, 126.2, 123.7, 122.2, 121.7, 81.5, 70.1, 38.2, 20.58, 20.55, 19.4.
HRMS m/z found 316.1191 requires 316.1185. FT-IR (Neat, nmax
cmꢁ1) 3274, 3241, 3073, 1755, 1658, 1614, 1569. HRMS m/z calcu-
lated for C17H18NO5 [MþH]þ 316.1191, found 316.1185. Elemental
analysis calculated for C17H17NO5 requires C 64.75%, H 5.43%, N
4.44%, found C 64.37%, H 5.38%; N 4.30%.
d
J ¼ 16.0 Hz, 1H), 7.38 (d, J ¼ 8.4 Hz, 1H), 7.35 (br, s, 2H), 7.20 (d,
J ¼ 8.3 Hz, 1H), 6.35 (d, J ¼ 16.0 Hz, 1H), 4.46 (t, J ¼ 6.7 Hz, 2H), 4.20
(t, J ¼ 5.4 Hz, 2H), 3.61 (t, J ¼ 5.6 Hz, 2H), 2.74 (t, J ¼ 6.6 Hz, 2H), 2.29
(s, 3H), 2.28 (s, 3H), 2.10e2.07 (m, 2H), 1.82e1.71 (m, 4H). 13C NMR
(100 MHz, CDCl3)
d 168.2, 168.1, 166.8, 147.7, 143.6, 142.9, 142.5,
133.4, 126.5, 124.0, 122.8, 121.4, 119.4, 64.5, 58.8, 46.9, 32.8, 28.3,
25.9, 25.3, 20.7, 20.7. FT-IR (Neat, nmax cm-1) 2948 (br), 1770, 1706,
1638, 1505 HRMS m/z calculated for C22H28O7N3 [MþH]þ 446.1927,
found 446.1927.
4.1.2.6. (E)-but-3-yn-1-yl-3-(3,4-difluorophenyl)acrylate
10f.
White solid; yield 58%; mp 48 ꢀC. Purified by FCC (EtOAc/petroleum
ether 10:90). Recrystallized from petroleum ether. Rf 0.4 (EtOAc/
petroleum ether 10:90). 1H NMR (400 MHz, CDCl3)
d 7.61 (d,
J ¼ 16.0 Hz, 1H), 7.40e7.30 (m, 1H), 7.28e7.23 (m, 1H), 7.18 (dt,
J ¼ 16.6 and 8.2 Hz, 1H), 6.37 (d, J ¼ 16.0 Hz, 1H), 4.32 (t, J ¼ 6.8 Hz,
2H), 2.61 (td, J ¼ 6.8 and 2.6 Hz, 2H), 2.03 (t, J ¼ 2.6 Hz,1H). 13C NMR
4.1.3.3. (E)-4-[3-(5-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)pen-
tyl)oxy)-3-oxoprop-1-en-1-yl]-1,2-phenylene
Yellow oil; yield 61%. Purified by FCC (EtOAc/petroleum ether
70:30). Rf 0.22 (EtOAc). 1H NMR (600 MHz, CDCl3)
7.56 (d,
diacetate
13c.
(100 MHz, CDCl3)
d
166.2, 152.9, 151.9 (d, J ¼ 13.2 Hz), 150.3 (d,
J ¼ 12.9 Hz), 149.4 (d, J ¼ 13.2 Hz), 142.9, 124.9 (dd, J ¼ 6.6 and
3.6 Hz), 118.9, 117.9 (dd, J ¼ 17.8 and 0.7 Hz), 116.5 (dd, J ¼ 17.7 and
0.9 Hz), 80.2, 77.5, 77.2, 76.8, 70.1, 62.6, 19.3. 19F NMR (376 MHz,
d
J ¼ 16.0 Hz, 1H), 7.36 (dd, J ¼ 8.4 and 1.9 Hz, 1H), 7.33 (d, J ¼ 1.9 Hz,
1H), 7.31 (s, 1H), 7.18 (d, J ¼ 8.4 Hz, 1H), 6.34 (d, J ¼ 16.0 Hz, 1H), 4.41
(t, J ¼ 6.9 Hz, 2H), 4.15 (t, J ¼ 6.6 Hz, 2H), 3.57 (t, J ¼ 5.8 Hz, 2H), 3.12
(s, 1H), 2.69 (t, J ¼ 7.6 Hz, 2H), 2.26 (s, 3H), 2.25 (s, 3H), 2.07e2.01
(m, 2H), 1.73e1.63 (m, 4H), 1.47e1.37 (m, 2H). 13C NMR (150 MHz,
CDCl3)
d
ꢁ63.61 (d, J ¼ 3.5 Hz), -134.83 (d, J ¼ 20.9 Hz), e137.41 (d,
J ¼ 20.8 Hz). FT-IR (Neat, nmax cmꢁ1) 3296, 1710, 1640, 1612, 1602,
1513. HRMS m/z calculated for C13H11F2O2 [MþH]þ 237.0722, found
237.0710. Elemental analysis calculated for C13H10F2O2 requires C
66.10%, H 4.27%, found C 66.07%, H 4.30%.
CDCl3)
d 168.0, 167.9, 166.5, 147.7, 143.3, 142.6, 142.3, 133.1, 126.3,
123.8, 122.6, 121.1, 119.2, 64.4, 58.4, 46.7, 32.6, 28.8, 28.2, 25.4, 25.3,
20.5, 20.5. FT-IR (Neat, nmax cmꢁ1) 2939, 2860, 1770, 1707, 1639,
4.1.2.7. (E)-4-(3-(3-Azidopropoxy)-3-oxoprop-1-en-1-yl)-1,2-
phenylene diacetate 12. Yellow oil; yield 72%. Purified by FCC
1505. HRMS m/z calculated for C23H30O7N3 [MþH]þ 460.2071
,
found 460.2084.

D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
581
4.1.3.4. (E)-4-[3-((8-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)
359.1501 found 4359.1495.
,
octyl)oxy)-3-oxoprop-1-en-1-yl]-1,2-phenylene
diacetate
13d.
White solid; yield 62%; mp 73 ꢀC. Purified by FCC (EtOAc/petroleum
4.1.3.8. 4-[(E)-3-(3-(4-(2-(((E)-3-(3,4-Diacetoxyphenyl)acryloyl)oxy)
ethyl)-1H-1,2,3-triazol-1-yl)propoxy)-3-oxoprop-1-en-1-yl]-1,2-
phenylene diacetate 15. White solid; yield 40%; mp 125 ꢀC. Purified
by FCC (EtOAc). Recrystallized from EtOAc/hexane (20:80). Rf 0.27
ether 70:30). Recrystallized from hexane. Rf 0.25 (EtOAc). 1H NMR
(400 MHz, CDCl3)
d
7.60 (d, J ¼ 16.0 Hz, 1H), 7.40 (d, J ¼ 8.4 Hz, 1H),
7.36 (s, 1H), 7.29 (s, 1H), 7.21 (d, J ¼ 8.4 Hz, 1H), 6.38 (d, J ¼ 16.0 Hz,
1H), 4.47 (t, J ¼ 6.7 Hz, 2H), 4.18 (t, J ¼ 6.6 Hz, 2H), 3.63 (t, J ¼ 5.8 Hz,
2H), 2.70 (t, J ¼ 7.6 Hz, 2H), 2.30 (s, 3H), 2.29 (s, 3H), 2.13e2.07 (m,
2H), 1.70e1.65 (m, 4H), 1.40e1.30 (m, 8H). 13C NMR (100 MHz,
(EtOAc/petroleum ether 80:20). 1H NMR (400 MHz, CDCl3)
d 7.61
(dd, J ¼ 16.0 and 2.3 Hz, 2H), 7.43 (s, 1H), 7.40 (dd, J ¼ 8.4 and 2.2 Hz,
2H), 7.37 (s, 2H), 7.23 (d, J ¼ 8.4 Hz, 1H), 7.21 (d, J ¼ 8.5, 1H),
6.40e6.34 (m, 2H), 4.50e4.47 (m, 4H), 4.25 (t, J ¼ 5.9 Hz, 2H),
3.20e3.10 (m, 2H), 2.37e2.33 (m, 2H), 2.31 (s, 3H), 2.30 (s, 3H), 2.29
CDCl3)
d 168.0, 167.9, 166.6, 143.3, 142.5, 142.3, 133.3, 126.3, 123.8,
122.6, 120.9, 119.4, 77.1, 64.7, 58.8, 46.6, 32.6, 29.3, 29.1, 29.0, 29.0,
28.6, 25.8, 25.5, 20.6, 20.5. FT-IR (Neat, nmax cmꢁ1) 3460, 2920,
2853, 1760, 1731, 1702, 1635, 1504. HRMS (ESIþ) calculated for
(s, 3H), 2.29 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 168.2, 168.1, 168.1,
168.1, 166.5, 166.4, 144.4, 143.8, 143.7, 143.6, 143.3, 142.6, 142.6,
133.3, 133.2,126.6,124.1, 124.07, 122.9,122.9,121.9,119.1, 118.7, 63.5,
61.3, 47.3, 29.7, 25.7, 25.7, 20.8, 20.8, 20.7, 20.7. FT-IR (Neat, nmax
cmꢁ1) 2962, 1768, 1708, 1638, 1504. HRMS m/z calculated for
C
26H36O7N3 m/z 502.2567 found 502.2553. Elemental analysis
,
calculated for C26H35O7N3 requires C 62.11%, H 7.00%, N 8.38%,
found C 62.12%, H 6.97%, N 8.30%.
C
33H34O12N3 [MþH]þ 664.2137 found 664.2130 Elemental analysis
,
4.1.3.5. (E)-4-[3-((2-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)
ethyl)amino)-3-oxoprop-1-en-1-yl]-1,2-phenylene diacetate 13e.
White solid; yield 76%; mp 84 ꢀC. Purified by FCC (MeOH/DCM
5:95). Rf 0.25 (MeOH/DCM 10:90). Recrystallized from EtOAc/hex-
calculated for C33H33O12N3 requires C 59.73%, H 5.01%, N 6.33%,
found C 59.06%, H 4.97%, N 6.02%.
4.1.3.9. (E)-4-[3-(2-(1-(3-((6-acetoxy-2,5,7,8-tetramethylchromane-
2-carbonyl)oxy)propyl)-1H-1,2,3-triazol-4-yl)ethoxy)-3-oxoprop-1-
en-1-yl]-1,2-phenylene diacetate 16. White solid; yield 30%; mp
58 ꢀC. Purified by FCC (EtOAc/petroleum ether 20:80). Rf 0.44
(EtOAc/petroleum ether 30:70). Recrystallized from hexane. 1H
ane (20:80). 1H NMR (600 MHz, CDCl3)
d
7.54 (d, J ¼ 15.6 Hz, 1H),
7.45 (s, 1H), 7.37 (dd, J ¼ 8.4 and 2.0 Hz, 1H), 7.33 (d, J ¼ 2.0 Hz, 1H),
7.21 (d, J ¼ 8.2 Hz, 1H), 6.48 (m, 1H), 6.33 (d, J ¼ 15.6 Hz, 1H), 4.51 (t,
J ¼ 6.7 Hz, 2H), 3.78e3.75 (m, 2H), 3.64e3.61 (m, 2H), 2.99 (t,
J ¼ 6.3 Hz, 2H), 2.32 (s, 3H), 2.31 (s, 3H), 2.19 (s, 1H), 2.16e2.10 (m,
NMR (600 MHz, DMSO)
d 7.75 (s, 1H), 7.69e7.65 (m, 1H), 7.64 (d,
2H),1.97 (t, J ¼ 4.9 Hz,1H). 13C NMR (150 MHz, CDCl3)
d
168.2, 168.2,
J ¼ 15.5 Hz, 2H), 7.31 (d, J ¼ 8.3 Hz,1H), 6.63 (d, J ¼ 16.0 Hz,1H), 4.38
(t, J ¼ 6.2 Hz, 2H), 4.22e4.07 (m, 2H), 4.04e3.88 (m, 2H), 3.02 (t,
J ¼ 6.1 Hz, 2H), 2.67e2.33 (m, 4H), 2.28 (d, J ¼ 3.6 Hz, 8H), 2.09 (s,
3H), 2.04 (s, 2H) 1.93 (s, 3H), 1.86 (s, 3H), 1.85e1.76 (m, 2H), 1.55 (s,
165.7, 142.9, 142.3, 142.3, 139.0, 133.8, 126.2, 123.8, 122.3, 122.0,
58.6, 46.9, 38.8, 32.4, 29.7, 25.5, 20.6, 20.6. FT-IR (Neat, nmax cmꢁ1
)
3348, 3243, 3060, 2933, 2256, 1768, 1663.21, 1620, 1574, 1505.
HRMS m/z calculated for C20H25O6N4 [MþH]þ 417.1778, found
417.1774. Elemental analysis calculated for C20H24O6N4 requires C
57.69%, 5.81%, N 13.45%, found C 58.19%, H 6.04%, N 10.25%.
3H). 13C NMR (150 MHz, DMSO)
d 173.0, 169.5, 168.6, 168.5, 166.4,
149.3, 144.0, 143.6, 143.4, 142.8, 141.4, 133.3, 127.4, 127.1, 125.5,
124.6, 123.6, 123.0, 122.2, 119.4, 117.7, 77.5, 63.6, 62.2, 46.3, 30.3,
29.2, 25.5, 25.4, 20.8, 20.7, 20.7, 20.6, 13.2, 12.3, 12.1. FT-IR (Neat,
nmax cmꢁ1) 2935, 1749, 1712, 1638, 1505. HRMS m/z calculated for
4.1.3.6. (E)-2-[1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl]ethyl-3-
(3,4-difluorophenyl)acrylate 13f. White solid; yield 98%; mp 82 ꢀC.
Purified by FCC (EtOAc/petroleum ether 70:30). Recrystallized from
petroleum ether. Rf 0.11 (EtOAc/petroleum ether 80:20). 1H NMR
C
36H42O11N3 [MþH]þ 692.2814, found 692.2823. Elemental analysis
calculated for C36H41N3O11 requires C 62.51%, H 5.97%, N 6.07%
found C 61.76%, H 5.90%, N 5.92.
(400 MHz, CDCl3)
d
7.53 (d, J ¼ 16.0 Hz, 1H), 7.47 (s, 1H), 7.34e7.27
(m, 1H), 7.20 (s, 1H), 7.16e7.06 (m, 1H), 6.30 (d, J ¼ 16.0 Hz, 1H), 4.46
(dt, J ¼ 13.2 and 6.6 Hz, 4H), 3.62 (t, J ¼ 5.5 Hz, 2H), 3.10 (t, J ¼ 6.2 Hz,
4.1.4. General procedure for the preparation of compound 17
To 6-acetyl-2,5,7,8-tetramethylchromane-2-carboxylic acid (1.0
eq.) in dichloromethane was added DCC (1.0 eq.) and DMAP (1.0
eq.). The mixture was allowed to stir for 10 min at room temper-
ature before the azide or alcohol (1.0 eq.) was added. The mixture
was stirred at room temperature overnight under an atmosphere of
argon. The resulting mixture was filtered, washed with a 10% so-
lution of KHSO4 three times and dried over MgSO4 before the sol-
vent removed in vacuo to afford the crude material.
2H), 2.14e2.01 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 165.9 (s),
d
152.0 (dd, J ¼ 96.8, 13.0 Hz), 149.5 (d, J ¼ 79.7 Hz) 143.7 (s),
142.6e142.3 (m),131.2 (dd, J ¼ 6.0 and 4.1 Hz), 124.5 (dd, J ¼ 6.6 and
3.5 Hz), 121.7 (s), 118.6 (d, J ¼ 2.3 Hz), 117.5 (d, J ¼ 17.8 Hz), 116.0 (d,
J ¼ 17.7 Hz), 63.2 (s), 58.6 (s), 46.7 (s), 32.4 (s), 25.4 (s). 19F NMR
(376 MHz, CDCl3)
d
ꢁ62.75 (s), e133.92 (s), e133.98 (s), e136.52 (d,
J ¼ 20.9 Hz). FT-IR (Neat, nmax cm-1) 3328, 3124, 3055, 2962, 2885,
1712, 1638, 1600, 1516. HRMS m/z calculated for C16H18O3N3F2
[MþH]þ 338.1316, found 338.1316. Elemental analysis calculated for
4.1.4.1. 3-Azidopropyl 6-acetoxy-2,5,7,8-tetramethylchromane-2-
carboxylate 17. Colourless oil; yield 78%. Purified by FCC (EtOAc/
petroleum ether 40:60). Rf 0.24 (EtOAc/petroleum ether 60:30) 1H
C
16H17N3O3F2 requires C 56.97%, H 5.08%, N 12.46%, found C 56.97%,
H 5.19%, N 12.16%.
NMR (400 MHz, DMSO-d6)
d 4.13 (m, 1H), 3.97e4.03 (m, 1H),
4.1.3.7. (E)-4-[3-(3-(4-(2-Hydroxyethyl)-1H-1,2,3-triazol-1-yl)pro-
poxy)-3-oxoprop-1-en-1-yl]-1,2-phenylene diacetate 14. White
solid; yield 46%; mp 78 ꢀC. Purified by FCC (EtOAc). Rf 0.14 (EtOAc).
3.10e3.03 (m, 2H), 2.68e2.56 (m, 1H), 2.50e2.32 (m, 2H), 2.29 (s,
3H), 2.09 (s, 3H), 1.96 (s, 3H), 1.87 (s, 3H), 1.86e1.77 (m, 1H),
1.75e1.65 (m, 2H), 1.57 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
Recrystallized from EtOAc. 1H NMR (400 MHz, CDCl3)
d
7.60 (d,
d
172.4, 168.8, 148.7, 140.9, 126.5, 124.9, 121.7, 117.0, 77.0, 61.8,
J ¼ 16.0 Hz, 1H), 7.44 (s, 1H), 7.40 (dd, J ¼ 8.5 and 1.9 Hz, 1H), 7.37 (d,
J ¼ 1.7 Hz, 1H), 7.22 (d, J ¼ 8.3 Hz, 1H), 6.35 (d, J ¼ 16.0 Hz, 1H), 4.46
(t, J ¼ 6.9 Hz, 2H), 4.23 (t, J ¼ 5.9 Hz, 2H), 3.92 (t, J ¼ 5.8 Hz, 2H), 2.93
(t, J ¼ 5.9 Hz, 2H), 2.34e2.31 (m, overlapping s, 2H), 2.30 (s, 3H),
47.2, 29.9, 27.5, 25.1, 20.2, 20.2, 12.7, 11.8, 11.6. FT-IR (Neat, max
n
cmꢁ1
) 2934, 2097, 1731, 1752. HRMS m/z calculated for
C
19H25N3O5Na [MþNa]þ 398.1705, found 398.1692.
2.29 (s, 3H). 13C NMR (100 MHz, CDCl3)
d
168.2, 168.1, 166.5, 143.8,
4.2. Biological assays
143.6, 142.6, 133.1, 126.6, 126.6, 124.1, 122.9, 122.1, 118.7, 61.7, 61.3,
47.3, 29.6, 28.8, 20.8, 20.7. FT-IR (Neat, nmax cmꢁ1) 2933, 2099, 1770,
1710, 1639, 1505. HRMS m/z calculated for C20H23O6N3 [MþH]þ
4.2.1. Expression and purification of human recombinant 5-LO
E. coli Bl21 (DE3) cells were transformed with pT3e5LO plasmid,

582
D. De Lucia et al. / European Journal of Medicinal Chemistry 101 (2015) 573e583
lysed in 50 m
M
triethanolamine/HCl pH 8.0 plus EDTA (5 m
g/mL), phenylmethanesulphonyl
), dithiothreitol (1 m ) and lysozyme (1 mg/mL) and
M
),
4.2.6. Statistics
soybean trypsin inhibitor (60
fluoride (1 m
m
Data obtained from biological experiments are expressed as
mean S.E.M. of single determinations performed in three or four
independent experiments at different days. IC50 values obtained
from at least four different compound concentrations were calcu-
lated by nonlinear regression using SigmaPlot 9.0 (Systat Software
Inc., San Jose, USA) one site binding competition. Statistical evalu-
ation of the data was performed by one-way ANOVA followed by a
Bonferroni or TukeyeKramer post-hoc test for multiple compari-
sons respectively. A p value <0.05 (*) was considered significant.
M
M
then sonicated (3 ꢃ 15 s). The homogenate was centrifuged at
10,000 ꢃ g for 15 min and the remaining supernatant at 40,000 ꢃ g
for 70 min at 4 ꢀC. 5-LO in the supernatant was partially purified by
affinity chromatography on an ATP-agarose column as described by
Fisher et al., in 2003 [22]. Semi-purified 5-LO was diluted in PBS
containing EDTA (1 mM) and ATP (1 mM) and immediately used for
activity assays.
4.3. Computational analysis
4.2.2. Activity assay for human recombinant 5-LO
Human recombinant 5-LO was pre-incubated with the test
4.3.1. Ligand and protein preparation for docking
compounds for 10 min at 4 ꢀC and pre-warmed for 30 s at 37 ꢀC. 5-
The structure of all compounds used in this study was optimized
using the MMFF94x force field. The torsional root and branches of
the ligands were chosen using AutoDockTools (version May_03_13),
allowing flexibility for all rotatable bonds of the ligand [26]. Subse-
quently, AutoDockTools was used to assign GasteigereMarsilli
atomic charges to all ligands. On the other hand, all water molecules
were removed from the 5-LO crystal structure (PDB ID: 3O8Y). It is
worth mentioning that this structure of 5-LO was crystallized
without any ligand. In order to obtain an active conformation of this
enzyme, the coordinates of arachidonic acid were extracted from
crystal structure 3V99 and docked in 3OY8 using a rigid docking
approach. Afterwards, the complex was submitted to a structure
optimization using the MMFF94x force field and the resulting pro-
tein structure was used for the calculations in this study. Also,
AutoDockTools was used to merge all non-polar hydrogen atoms and
to assign Gasteiger charges for each atom of the macromolecule.
LO product formation was initiated by addition of 2 m
M CaCl2 and
20
m
M
arachidonic acid. After 10 min at 37 ꢀC, the reaction was
terminated by addition of 1 mL ice-cold methanol. Formed 5-LO
metabolites (all-trans isomers of LTB4 and 5-H(P)ETE) were ana-
lysed by RP-HPLC as described by Koeberle et al., in 2009 [23].
4.2.3. Determination of 5-LO product formation in neutrophils
Freshly isolated neutrophils (1 ꢃ 107/mL) were pre-incubated
with the test compounds for 15 min at 37 ꢀC. Then, 5-LO product
formation was started by addition of 2.5
mM
Ca2þ-ionophore A23187
plus 20
mM
AA. The reaction was stopped after 10 min at 37 ꢀC with
1 mL of methanol. Major 5-LO metabolites (LTB4 and its all-trans
isomers and 5-H(P)ETE) were extracted and analysed by HPLC as
described by Werz et al. [24]. Cysteinyl-LTs C4, D4 and E4 and
oxidation products of LTB4 were not determined.
4.3.2. Molecular docking
4.2.4. Determination of 5-LO product formation in stably
transfected HEK 293 cells
Docking calculations were performed with AutoDock Vina 1.1.2
software [27]. The searching area was defined by a box of
30 ꢃ 20 ꢃ 30 Å centred at the coordinates of the catalytic Fe2þ. In
this research, the conformational search of the ligand was carried
using and exhaustiveness value of 8 to generate maximum 9
binding modes with a maximum difference in energy of 4 kcal/mol
between the best and the worst conformation. The conformation
with the lowest predicted binding energy was used for further
analysis. In order to maintain the reproducibility of this study, all
the calculations were performed using a seed equal to 1.
Two different HEK cell lines were tested (HEK_5-LO; HEK-5LO/
FLAP) as described by Pergola et al. [18]. 1 ꢃ 106 cells were sus-
pended in 1 mL PGC buffer (PBS; 0.1% glucose, 1 m
M CaCl2, pre-
incubated with the test compound for 10 min at 37 ꢀC and subse-
quently stimulated by 2.5
mM mM
Ca2þ-ionophore A23187) plus 2
arachidonic acid for 10 min at 37 ꢀC. The reaction was stopped by
1 mL methanol, and the metabolites (all-trans isomers of LTB4 and
5-H(P)ETE) were extracted and analysed by HPLC as described
above.
Funding sources
4.2.5. Cytotoxicity assays
U937 (lymphoblast from lung) a cell line derived from malig-
nant cells of a pleural effusion of 37 year old caucasian male with
diffuse histiocytic lymphoma were grown in RPMI 1640 (Gibco)
added with 10% FBS (Foetal Bovine Serum), 10% Hepes buffer, 5%
penicillin\streptomycin at 37 ꢀC in a humidified 5% CO2 atmosphere
The authors would like to thank the European Social Fund,
through “Sovvenzione Globale Spinner” Spinner Consortium,
Regione Emilia Romagna, Italy for Daniela De Lucia PhD fellow-
ships; Ministry of Education, University and Research (MIUR Italy-
PRIN, Grant 20082L3NFT_003); the EPSRC (Award Nos. EP/
K009494/1 and EP/K039520/1); Unilever and the European
for 72 h 100
m
L of cells at the density of 1 ꢃ 106/mL were seeded
into 96-well plates (Nunc) and treated with the different com-
Research Council for
a Starting Grant (ERC-2013-StG-336159
pounds at different concentrations (Caffeic acid at 1, 10, 20
mM;
MIXTURE) for financial support. CONACyT (No. 217442/312933),
Cambridge Overseas Trust and the Secretariat of Public Education
10a,13d, 13e,13f, 15, 16 at 3, 10, 20 ) and analysed by MTT assay
mM
after 24 h incubation. MTT stock solution (5 mg/mL) was added to
each culture being assayed to equal one-tenth the original culture
volume and incubated for 4 h. At the end of the incubation period,
ꢀ
(Mexico) for funding Oscar Mendez Lucio PhD fellowship.
Appendix A. Supplementary data
we added acidic isopropanol (100 mL of 0.04N HCl in absolute iso-
propanol). Absorbance of converted dye was measured at a wave-
length of 570 nm with background subtraction at 630e690 nm
using ELISA-reader (Victor, PerkineElmer). We expressed the effect
on cell viability as EC50, which is the concentration required to
inhibit MTT-reducing mitochrondrial dehydrogenase activity by
50% [25]. The absorbance of untreated cells (RPMI þ DMSO) was
taken as 100% viability to calculate cytotoxicity.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.07.011.
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