Structure–activity relationship of caffeic acid phenethyl ester analogs as new 5-lipoxygenase inhibitors

Article abstract of DOI:10.1111/cbdd.12874

Leukotrienes (LTs) are a class of lipid mediators implicated in numerous inflammatory disorders. Caffeic acid phenethyl ester (CAPE) possesses potent anti-LTs activity through the inhibition of 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of LTs. In this study, we describe the design and synthesis of CAPE analogs as radical scavengers and 5-LO inhibitors. Caffeic esters bearing propargyl and allyl linkers between the caffeoyl and aryl moieties (4a–i and 5a–i, respectively) were synthesized by Sonogashira and Heck cross-coupling reactions to probe the effects of flexibility and aryl substitution on 5-LO inhibition. Caffeoyl alcohol and ethers (6, 7a–b) as well as caffeoyl aldehyde and ketones (8a–e) were synthesized to elucidate the importance of the ester linkage for inhibitory activity. All tested compounds proved to be good radical scavengers (IC₅₀ of 10–30?μm). After preliminary anti-LTs activity screening in HEK293 cell models, 5-LO inhibition potential of selected compounds was determined in human polymorphonuclear leukocytes (PMNL). Most screened compounds outperformed CAPE 3 in concentration-dependent assays on PMNL, with ester dimers 4i and 5i along with caffeoyl ethers 7a–b being roughly eight-, seven-, and 16-fold more potent than Zileuton, with IC₅₀ values of 0.36, 0.43, and 0.18?μm, respectively.

Full text of DOI:10.1111/cbdd.12874

Received Date : 05-May-2016
Revised Date : 23-Aug-2016
Accepted Date : 18-Sep-2016
Article type : Research Article
Structure activity relationship of caffeic acid phenethyl ester analogs as new 5-
lipoxygenase inhibitors.
Jérémie A. Doirona, Luc M. Leblanca, Martin J. G. Héberta, Natalie A. Levesquea, Aurélie F. Paréa, Jacques
Jean-Françoisa, Marc Cormiera, Marc E. Surettea, Mohamed Touaibiaa*
a Département de chimie et biochimie, Université de Moncton, 18 avenue Antonine-Maillet, Moncton,
NB, Canada, E1A 3E9
*Author to whom correspondence should be addressed; E-Mail: mohamed.touaibia@umoncton.ca; Tel.:
+1-506-858-4493; Fax: +1-506-858-4541.
Keywords: CAPE, Anti-radical, Anti-leukotriene, 5-Lipoxygenase inhibitor.
Abstract
Leukotrienes (LTs) are a class of lipid mediators implicated in numerous inflammatory disorders.
Caffeic acid phenethyl ester (CAPE) possesses potent anti-LTs activity through the inhibition of 5-
lipoxygenase (5-LO), the key enzyme in the biosynthesis of LTs. In this study, we describe the design and
synthesis of CAPE analogs as radical scavengers and 5-LO inhibitors. Caffeic esters bearing propargyl and
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12874
This article is protected by copyright. All rights reserved.

allyl linkers between the caffeoyl and aryl moieties (4a-i and 5a-i, respectively) were synthesized by
Sonogashira and Heck cross coupling reactions to probe the effects of flexibility and aryl substitution on
5-LO inhibition. Caffeoyl alcohol and ethers (6, 7a-b) as well as caffeoyl aldehyde and ketones (8a-e)
were synthesized to elucidate the importance of the ester linkage for inhibitory activity. All tested
compounds proved to be good radical scavengers (IC50 of 10 – 30 µM). After preliminary anti-LTs activity
screening in HEK293 cell models, 5-LO inhibition potential of selected compounds was determined in
human polymorphonuclear leukocytes (PMNL). Most screened compounds outperformed CAPE 3 in
concentration-dependent assays on PMNL, with ester dimers 4i and 5i along with caffeoyl ethers 7a-b
being roughly 8-, 7- and 16-fold more potent than Zileuton, with IC50 values of 0.36, 0.43 and 0.18 μM,
respectively.
Introduction
5-Lipoxygenase is the key enzyme in the transformation of arachidonic acid to leukotrienes
(LTs), a class of eicosanoid inflammatory mediators which have been shown to play an important role in
numerous inflammatory disorders such as asthma, atherosclerosis, and rheumatoid arthritis (1).
Moreover, malignant cells of many cancers highly express 5-LO along with other related LTs biosynthesis
enzymes, with anti-LTs therapy showing promising anticancer activity (1-5). In response to the need for
drugs targeting the LTs biosynthesis pathway, numerous studies have been undertaken in order to
develop efficient inhibitors of 5-LO (6,7). However, the only 5-LO inhibitor currently approved for human
therapy, Zileuton (1) (fig.1), requires large and frequent dosing due to an unfavourable pharmacokinetic
profile along with metabolites possessing hepatotoxic properties (1,8-10). Amongst potential alternative
candidates for 5-LO inhibition are redox active compounds based on caffeic acid (2), notably caffeic acid
phenethyl ester 3 (CAPE).
Figure 1. Structure of known 5-LO inhibitors Zileuton (1), caffeic acid (2), and CAPE (3).
CAPE (3) is a polyphenolic ester originally found in propolis, a resinous natural mixture of
phytochemicals isolated from various plant sources by honeybees (11). Though propolis itself possesses
a great deal of pharmacological activity, its variable and complex composition, which is largely
dependent on the geographical location of collection, renders its standardisation and utilisation difficult
(12,13). CAPE (3) has been found to possess differential cytotoxicity in assays of human normal and
cancerous cell lines, preferentially targeting malignant cells. Additionally, it has shown anti-proliferative
effects in a vast range of cancerous cell lines (14-17). Not limited to anti-cancerous properties, CAPE (3)
also possesses anti-viral, immunomodulatory, anti-angiogenic, and antioxidant capacities, among others
(13). Early work by Koshihara et al. showed that caffeic acid (2) and its methyl ester both possessed
important anti-LTs activity (18,19). Based on these first studies, CAPE (3) was found to be an excellent
inhibitor of plant lipoxygenase and has also been shown to reduce LTs formation in mouse peritoneal
macrophages (20-22). Recent studies conducted in our laboratory have shown CAPE (3) to be
significantly more potent than the clinical 5-LO inhibitor Zileuton 1 for the inhibition of LTs biosynthesis
This article is protected by copyright. All rights reserved.

in human polymorphonuclear leukocytes (PMNL, IC50 = 0.52 μM), which are important producers of the
powerful chemoattractant leukotriene B4 (23). CAPE (3) was also shown to be equipotent with Zileuton
(1) in whole blood assays of 5-LO inhibition, an important consideration for the implementation of the
inhibitor in in vivo systems.
Though it has now been established that CAPE (3) and other caffeic acid-based compounds have
the potential to be potent inhibitors of 5-LO activity, little is known of the molecular basis for inhibition
with these molecules. 5-LO derives its catalytic activity from a ferric iron atom present in its substrate-
binding pocket (1). It is therefore assumed that caffeic acid-based compounds, which generally possess
radical scavenging and antioxidant properties, derive their actions from redox interactions with this
ferric iron, uncoupling the redox cycle necessary for LTs biosynthesis (6,7,24). Supporting this hypothesis
is the finding that cinnamic acid analogs of CAPE (3) or of other caffeoyl-based 5-LO inhibitors, lacking
the redox active phenolic hydroxyl groups, lose all 5-LO inhibitory activity (23,25,26). However,
antioxidant activity is not sufficient to assure efficient 5-LO inhibition in complex systems: caffeic acid (2)
and the amide analog of CAPE (3), which are roughly equipotent with CAPE (3) in radical scavenging
assays, possess far lower inhibitory activity in PMNL and whole blood assays than CAPE (3) (23).
Furthermore, assays of multivalent compounds showed that inhibitory activity of caffeates are not
proportional to the number of caffeoyl groups present in a compound (25,26). Though most of the
caffeoyl esters synthesized in these studies had excellent and similar 5-LO inhibition potential in HEK293
cell lysates, large variability in inhibition was found when performing assays on whole cells (25). Such
differences in activity amongst structurally similar compounds possessing identical pharmacophores
may indicate the important influence of structure and steric bulk on cell permeability and hydrophobic
interactions.
In order to better comprehend the specific characteristics of CAPE (3) that confer efficient 5-LO
inhibition, we hereby report the synthesis, radical scavenging capacity and 5-LO inhibition analysis of
four novel series of CAPE analogs. In this study, focus is placed on the non-redox active terminus of the
molecule, leaving the phenolic catechol group untouched (fig. 2).
Figure 2. Structures of proposed novel CAPE based 5-LO inhibitors and radical scavengers.
As can be seen in figure 2, modifications focus on the rigidification of the alkyl bridge of CAPE (3)
through the synthesis of propargyl (4a-i) and allyl (5a-i) esters of caffeic acid by using Sonogashira or
Heck cross coupling reactions with commercially available aryl iodides and the appropriate alkyne or
alkene precursors. Terminal alkene and acetylene substituent groups were chosen for their electronic
and steric properties. Additionally, as there has been very little investigation of CAPE ketone and ether
analogs as 5-LO inhibitors, molecules where the ester group of CAPE (3) was replaced with an allyl
ether/alcohol (6, 7a-b) or a conjugated aldehyde/ketone group (8a-e) were also synthesized.
This article is protected by copyright. All rights reserved.

Methods and Materials
Experimental
General
All chemicals used were purchased from Aldrich (CA). Purification of compounds was carried out by silica
gel circular chromatography (Chromatotron®, model 7924, Harrison Research) or by flash
chromatography. TLC was run on silica gel coated aluminium sheets (SiliaPlate TLC, Silicycle®) with
detection by UV light (254 nm, UVS-11, Mineralight® shortwave UV lamp). Melting points were obtained
using a MELTEMP® (model 1001D) melting point apparatus. FTIR spectra were recorded on a Nicolet®
Impact 400 spectrometer. NMR spectra were recorded on a Bruker® Avance III 400 MHz spectrometer.
High-resolution mass measurements were performed on a Bruker® Doltonics’ micrOTOF instrument in
positive or negative electrospray.
General procedure I - Sonogashira coupling
Aryl iodide (2 mmol), PdCl2(PPh3)2 (0.05 mmol, 35 mg) and CuI (0.1 mmol, 9.5 mg) are
dissolved/suspended in 7 mL 1:1 DMF/NEt3 and degassed by ultrasound for 5 minutes under a flow of
Argon. 10 (1 mmol, 302 mg) is dissolved in 1 mL DMF and added dropwise over 15 minutes to the
degassed solution with stirring at room temperature and under argon atmosphere. Progress is
monitored by TLC (AcOEt/Hex). After complete consumption of starting material (about 4 h), the
reaction mixture is diluted with 100 mL H2O and extracted with three portions of 30 mL AcOEt. The
organic fractions are washed with 2 x NH4Cl(sat), 2 x H2O, 2 x NaCl(sat), dried over MgSO4, treated with
activated charcoal, filtered and concentrated. The resulting oil is purified by flash chromatography or
silica gel circular chromatography (Chromatotron® model 7924, Harrison Research). Eluent: AcOEt/Hex.
General procedure II - Heck coupling
To a stirred solution of Pd(OAc)2 (0.05 mmol, 11.2 mg) and Ag2CO3 (0.6 mmol, 165 mg) in 7 mL benzene
is added 12 (1 mmol, 304 mg) and aryl iodide (2 mmol). The resulting mixture is brought to reflux and
left to react overnight. The benzene solution is then diluted with 60 mL AcOEt, washed with 2x NH4Cl(sat)
,
2 x H2O, 2 x NaCl(sat), dried over MgSO4, treated with activated charcoal, filtered and concentrated. The
resulting oil is purified by flash chromatography or silica gel circular chromatography (Chromatotron®
model 7924, Harrison Research). Eluent: AcOEt/Hex).
This article is protected by copyright. All rights reserved.

General procedure III - Deacetylation of caffeoyl acetates
To a solution of the appropriate diacetylcaffeoyl derivative (0.25 mmol) in 2.5 mL anhydrous CH2Cl2
under N2 is added 2.5 mL MeOH. To the resulting stirred solution is added guanidinium hydrochloride (1
mmol, 4 equivalents) followed by triethylamine (3 mmol, 12 eq.). After consumption of the diacetylated
precursor as indicated by TLC (about 1.5 h), the reaction mixture is concentrated to remove excess NEt3
and then diluted in 45 mL AcOEt. The organic phase is then washed with 2 x H2O, 2 x NH4Cl(sat.), 2 x
NaCl(sat), treated with activated charcoal, dried over MgSO4 and concentrated to give the expected pure
caffeoyl derivative.
General Procedure IV – deprotection of tetrahydropyranyl ethers
To a stirred solution of the appropriate tetrahydropyranyl ether derivative (0.5 mmol) in 4 mL CH2Cl2
under argon is added 6 mL MeOH followed by pyridinium p-toluenesulfonate (25 mg, 0.1 mmol, 0.2 eq.).
After complete consumption of starting material as indicated by TLC (about 4 h), the reaction mixture is
concentrated and purified by flash chromatography (MeOH/CH2Cl2) to yield the appropriate catechol
derivatives as crystalline solids or powder.
Diacetylcaffeic acid (9)
Compound 9 was prepared as previously described (26). Mp 190-192 oC. 1H NMR (400 MHz, DMSO-d6,
25 °C), δ (ppm) = 7.70 (br s, 2H, Har), 7.55 (d, 1H, J = 18.6 Hz, CHCar), 7.20 (d, 1H, J = 8.6 Hz, Har), 6.5 (d,
1H, J = 18.6 Hz, CHCO), 2.3 (s, 6H, 2 x OAc). 13C NMR (101 MHz, DMSO-d6, 25 °C), δ (ppm) = 168.2, 168.1,
167.4, 143.3, 142.3, 142.2, 133.1, 126.7, 124.1, 123.0, 120.3, 20.4, 20.3. HRMS m/z calc. for C13H12O6 +
(Na+): 287.0526; found: 287.0522.
Prop-2-yn-1-yl (2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoate (10)
A mixture of 9 (1000 mg, 3.78 mmol), 3-4 drops of anhydrous DMF and 20 mL of thionyl chloride was
heated at reflux for 3 h, under Ar. The excess thionyl chloride was then evaporated at reduced pressure,
and the residue was dissolved in 5 mL of CH2Cl2. The resulting solution was added dropwise to a mixture
of propargyl alcohol (848 mg, 15.1 mmol, 4 eq.) and pyridine (1255 mg, 15.87 mmol, 4.2 eq.) in 15 mL of
o
CH2Cl2 at 0 C under argon, then left to return to ambient temperature overnight. After concentration,
the oily residue was diluted with 90 mL AcOEt and washed with 2 x 30 mL H2O, 2 x 30 mL NH4Clsat, 2 x
NaClsat, dried over MgSO4, filtered and concentrated. Compound 10 was obtained as white crystals with
84% yield (960 mg, 3.17 mmol) after silica gel circular chromatography (10-15% AcOEt/Hex); mp = 95-96
1
oC, Rf = 0.54 (40 % AcOEt/Hex). H NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 7.70 (d, 1H, J = 16.0 Hz,
=CHCar), 7.41 (d, 1H, J = 8.4 Hz, Har), 7.39 (s, 1H, Har), 7.25 (d, 1H, J = 8.4 Hz, Har), 6.43 (d, 1H, J = 16.0 Hz,
=CHCOO), 4.84 (d, 2H, J = 2.4 Hz, CH2CC), 2.54 (t, 1H, J = 2.4 Hz, CCH), 2.34 (s, 3H, OAc), 2.33 (s, 3H, OAc).
This article is protected by copyright. All rights reserved.

13C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 168.04, 167.95, 165.65, 143.97, 143.72, 142.48, 133.01,
126.50, 124.00, 122.88, 118.19, 77.64, 75.04, 52.17, 20.65, 20.61. HRMS m/z calc. for C16H14O6 + (Na+):
325.0683; found: 325.0676.
3-phenylprop-2-yn-1-yl (2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoate (11b)
Following general procedure I with iodobenzene, compound 11b was obtained with 57% yield as a white
o
solid after silica gel circular chromatography (5-15% AcOEt/Hex); mp = 87-88 C, Rf = 0.38 (30%
1
o
AcOEt/hexanes). H NMR (400 MHz, CDCl3, 25 C), δ (ppm) = 7.72 (d, 1H, J = 16.0 Hz, =CHCar), 7.51-7.24
(m, 8H, Har), 6.46 (d, 1H, J = 16.0 Hz, =CHCOO), 5.07 (s, 2H, CH2CC), 2.34 (s, 3H, OAc), 2.33 (s, 3H, OAc).
o
13C NMR (101 MHz, CDCl3, 25 C), δ (ppm) = 168.04, 167.95, 165.82, 143.80, 143.67, 142.48, 133.11,
131.93, 128.79, 128.31, 126.48, 123.99, 122.87, 122.14, 118.45, 86.65, 82.90, 53.08, 20.66, 20.61. HRMS
m/z calc. for C22H18O6 + (Na+): 401.0996; found: 401.0984
3-[4-(3-{[(2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoyl]oxy}prop-1-yn-1-yl)phenyl]prop-2-yn-1-yl (2E)-
3-[3,4-bis(acetyloxy)phenyl]prop-2-enoate (11i)
1,4-diiodobenzene (0.379 mmol, 125 mg), PdCl2(PPh3)2 (0.019 mmol, 13.3 mg, 0.05 eq) and CuI (0.019
mmol, 3.6 mg, 0.05 eq) are dissolved/suspended in 7 mL 1:1 DMF/NEt3 and degassed by ultrasound for 5
minutes under a flow of Argon. 10 (0.95 mmol, 286 mg, 2.5 eq) is dissolved in 1 mL DMF and added
dropwise over 15 minutes to the degassed solution with stirring, at room temperature and under argon
atmosphere. After 2h, TLC indicates complete consumption of starting material. The reaction mixture is
diluted with 100 mL H2O and extracted with three portions of 30 mL AcOEt. The organic fractions are
washed with 2 x NH4Cl(sat), 2 x H2O, 2 x NaCl(sat), dried over MgSO4, treated with activated charcoal,
filtered and concentrated. The resulting oil is purified by silica gel circular chromatography (0.1 – 0.35 %
MeOH/CH2Cl2). 11i is obtained in 66 % yield as a white powder. mp = 192-194 (dec.), Rf = 0.20 (40 %
AcOEt/Hex). 1H NMR (400 MHz, DMSO-d6, 25 oC), δ (ppm) = 7.75 - 7.71 (m, 6H, Har + =CHCar), 7.50 (s, 4H,
Har), 7.34 (d, 2H, J = 8.5 Hz, Har), 6.74 (d, 2H, J = 16.0 Hz, =CHCOO), 5.05 (s, 4H, CH2CC), 2.31 (s, 3H, OAc),
o
2.30 (s, 3H, OAc). 13C NMR (101 MHz, DMSO-d6, 25 C), δ (ppm) = 168.65, 168.55, 165.81, 144.30,
144.15, 142.81, 133.22, 132.33, 127.65, 124.65, 123.83, 122.45, 118.72, 86.82, 85.60, 52.97, 20.85,
20.76. HRMS m/z calc. for C38H30O12 + (Na+): 701.1629; found: 701.1648.
prop-2-yn-1-yl (2E)-3-[3,4-dihydroxyphenyl]prop-2-enoate (4a)
Following general procedure III with 10, compound 4a was obtained with 73% yield as a pale brown
o
1
o
powder, mp = 99 – 100 C, Rf = 0.46 (5% MeOH/CH2Cl). H NMR (400 MHz, DMSO-d6, 25 C), δ (ppm) =
9.40 (br s, 2H, OH), 7.52 (d, 1H, J = 15.9 Hz, =CHCar), 7.08 (s, 1H, Har), 7.04 (d, 1H, J = 8.2 Hz, Har), 6.77 (d,
13
1H, J = 8.2 Hz, Har), 6.31 (d, 1H, J = 15.9 Hz, =CHCOO), 4.79 (s, 2H, CH2CC), 3.46 (s, 1H, CCH). C NMR
o
(101 MHz, DMSO-d6, 25 C), δ (ppm) = 166.24, 149.14, 146.62, 146.04, 125.80, 122.15, 116.18, 115.40,
113.37, 79.21, 78.01, 51.93. HRMS m/z calc. for C12H10O4 + (Na+): 241.0471; found: 241.0469.
This article is protected by copyright. All rights reserved.

prop-2-en-1-yl (2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoate (12)
A mixture of 9 (1036.0 mg, 3.92 mmol), 3-4 drops of anhydrous DMF and 20 mL of thionyl chloride was
heated at reflux for 3 h, under Ar. The excess thionyl chloride was then evaporated, and the residue was
dissolved in 5-10 mL of CH2Cl2. The solution was added dropwise to a mixture of allyl alcohol (905.2 mg,
o
15.59 mmol, 4 eq.), pyridine (1302.3 mg, 16.46 mmol, 4.2 eq.) and 5 mL of CH2Cl2 stirred at 0 C under
Ar, then left to return to ambient temperature overnight. After concentration, the oily residue was
diluted with 90 mL AcOEt and washed with 2 x 30 mL H2O, 2 x 30 mL NH4Clsat, 2 x NaClsat, dried over
MgSO4, filtered and concentrated. Compound 12 obtained with 74 % yield (880.8 mg, 2.89 mmol) after
o
silica gel circular chromatography (5-15% AcOEt in hexanes); white solid, mp = 78-80 C, Rf = 0.29 (30%
1
o
AcOEt/Hex). H NMR (400 MHz, CDCl3, 25 C) δ (ppm): 7.67 (d, J = 16.0 Hz, 1H, =CHCar), 7.43 (d, J = 8.40
Hz, 1H, Har), 7.38 (s, 1H, Har), 7.24 (d, J = 8.3 Hz, 1H, Har), 6.43 (d, J = 16.0 Hz, 1H, =CHCO), 6.01 (m, 1H,
CH2CH=CH2), 5.39 (d, J = 17.25 Hz, 1H, CH2CH=CHH), 5.30 (d, J = 10.44 Hz, 1H, CH2CH=CHH), 4.73 (d, J =
5.40 Hz, 2H, CH2CH=CH2), 2.34 (s, 3H, OAc), 2.33 (s, 3H, OAc). 13C NMR (101 MHz, CDCl3, 25 oC) (ppm):
168.09, 168.01, 166.21, 143.52, 143.10, 142.44, 133.25, 132.14, 126.43, 123.96, 122.76, 119.06, 118.39,
65.35, 20.68,20.63. HRMS m/z calc. for C16H16O6 + (Na+): 327.0839; found: 327.0824.
(2E)-3-phenylprop-2-en-1-yl (2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoate (13b)
Following general procedure II with iodobenzene, compound 13b was obtained with 65% yield as a
o
yellow solid after silica gel circular chromatography (5-10% AcOEt/Hex); mp 76-77 C, Rf = 0.26 (30%
1
AcOEt/hexanes). H NMR (400 MHz, CDCl3, 25 oC) δ (ppm): 7.69 (d, 1H, J = 16.0 Hz, C=CHCar), 7.45–7.24
(m, 8H, Har), 6.73 (d, 1H, J = 15.9 Hz, CH2CH=CH), 6.45 (d, 1H, J = 16.0 Hz, =CHCO), 6.38 (dt, 1H, J = 15.9
13
Hz, 6.3 Hz, CH2CH=CH), 4.89 (d, 2H, J = 6.3 Hz, CH2CH=CH), 2.34 (s, 3H, OAc), 2.33 (s, 3H, OAc). C NMR
(101 MHz, CDCl3, 25 oC) δ (ppm): 168.10, 168.02, 166.35, 143.54, 143.16,142.45, 136.20, 134.35, 133.27,
128.63, 128.12, 126.66, 126.45, 123.97, 123.12, 122.78, 119.10, 65.30, 20.68, 20.64. HRMS m/z calc. for
C22H20O6 + (Na+): 403.1152; found: 403.1144.
(2E)-4-{4-[(1E)-3-{[(2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoyl]oxy}prop-1-en-1-yl]phenyl}but-2-en-
1-yl (2E)-3-[3,4-bis(acetyloxy)phenyl]prop-2-enoate (13i)
To a stirred solution of Pd(OAc)2 (0.075 mmol, 16.8 mg, 0.05 eq) and Ag2CO3 (0.9 mmol, 248 mg, 0.6 eq)
in 7 mL benzene is added 12 (1.5 mmol, 456 mg, 1 eq) and 1,4-diiodobenzene (0.66 mmol, 217 mg, 0.44
eq). The resulting mixture is brought to reflux and left to react overnight. The benzene solution is then
diluted with 60 mL AcOEt, washed with 2x NH4Cl(sat), 2 x H2O, 2 x NaCl(sat), dried over MgSO4, treated with
activated charcoal, filtered and concentrated. The resulting oil is purified by silica gel circular
chromatography (0 – 0.5 % MeOH/CH2Cl2). 13i was obtained with 57% yield as a yellow solid. mp = 142-
144 oC, Rf = 0.59 (2% MeOH/CH2Cl2). 1H NMR (400 MHz, CDCl3, 25 oC), δ (ppm) = 7.68 (d, 2H, J = 16.0 Hz,
=CHCar), 7.43 (dd, 2H, J = 8.4 Hz, 2.1 Hz, Har), 7.40 (s, 4H, Har), 7.39 (d, 2H, J = 2.1 Hz, Har), 7.24 (d, 2H, J =
8.4 Hz, Har), 6.70 (d, 2H, J = 15.9 Hz, CH2CH=CH), 6.44 (d, 2H, J = 16.0 Hz, =CHCOO), 6.39 (dt, 2H, J = 15.9
This article is protected by copyright. All rights reserved.

13
Hz, 6.4 Hz, CH2CH=CH), 4.89 (d, 4H, J = 6.4 Hz, CH2CH=CH), 2.33 (s, 3H, OAc), 2.32 (s, 3H, OAc). C NMR
o
(101 MHz, CDCl3, 25 C), δ (ppm) = 168.07, 167.99, 166.31, 143.54, 143.18, 142.45, 136.00, 133.80,
133.26, 126.93, 126.43, 123.96, 123.32, 122.78, 119.07, 65.25, 20.67, 20.63. HRMS m/z calc. for
C38H34O12 + (Na+): 705.1942; found: 705.1946.
(E)-methyl 3-(3,4-dihydroxyphenyl)acrylate (14)
To a stirred solution of 2 (5400 mg, 30 mmol) dissolved in 100 mL MeOH is added 1 mL conc. H2SO4 and
the mixture is heated to reflux for 4h. After complete consumption of starting material, the reaction
mixture is concentrated to 30 mL and diluted with 350 mL AcOEt. The resulting solution is washed with 2
x NaHCO3(sat.), 1 x water, 1 x brine, dried over MgSO4, filtered and concentrated. Compound 14 was
obtained with 89 % yield (5543 mg, 26.6 mmol) as a light yellow crystals; mp = 153 – 154 oC, Rf = 0.60 (7
% MeOH/CH2Cl2). 1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 9.37 (br s, 2H, OH), 7.49 (d, 1H, J = 15.9 Hz,
=CHCar), 7.06 (d, 1H, J = 1.8 Hz, Har), 7.00 (dd, 1H, J = 8.2 Hz, 1.8 Hz, Har), 6.77 (d, 1H, J = 8.2 Hz, Har), 6.28
(d, 1H, J = 15.9 Hz, =CHCO), 3.69 (s, 1H, CH3). 13C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 167.48, 148.89,
146.03, 145.63, 125.94, 121.89, 116.18, 115.22, 114.14, 51.68. HRMS m/z calc. for C10H10O4 + (H+):
195.0652; found: 195.0643.
(E)-methyl 3-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)acrylate (15)
To a stirred solution of 14 (4164 mg, 20 mmol) dissolved in 50 mL dihydropyran is added pyridinium p-
o
toluenesulfonate (251 mg, 1mmol, 0.05 eq.) and the mixture is heated to 60 C on a water bath for 90
minutes. After complete consumption of starting material, excess dihydropyran is removed under
reduced pressure and the resulting oil is dissolved in 200 mL CH2Cl2. The organic solution is washed with
2 x NaHCO3(sat.), 1 x water, 1 x brine, treated with activated charcoal, filtered and concentrated.
Compound 15, a mixture of diastereoisomers, is obtained as a translucent oil with 88 % yield (6624 mg,
17.6 mmol) after flash chromatography (6 % AcOEt/Hex); Rf = 0.52 (30 % AcOEt/Hex). 1H NMR (400 MHz,
CDCl3, 25 °C), δ (ppm) = 7.63 (d, 1H, J = 15.9 Hz, =CHCar), 7.35 (br s, 1H, Har), 7.16 (br s, 2H, Har), 6.32 (d,
1H, J = 15.9 Hz, =CHCO), 5.51 (m, 1H, OCHO), 5.46 (m, 1H, OCHO), 4.05 – 3.92 (m, 2H, CH2O), 3.81 (s, 1H,
13
CH3), 3.68 – 3.62 (m, 2H, CH2O), 2.07 – 1.86 (m, 6H, THP), 1.78 – 1.56 (m, 6H, THP). C NMR (101 MHz,
CDCl3, 25 °C), δ (ppm) = 167.68, 149.70, 149.46, 147.52, 147.30, 144.74, 144.71, 128.82, 128.69, 123.62,
123.47, 117.98, 117.78, 117.60, 117.50, 116.04, 115.99, 97.91, 97.43, 97.22, 96.82, 62.01, 61.93, 61.86,
61.75, 51.59, 30.36, 30.32, 30.23, 30.20, 25.27, 25.20, 18.62, 18.52, 18.37. HRMS m/z calc. for C20H26O6 +
(Na+): 385.1622; found: 385.1604.
This article is protected by copyright. All rights reserved.

(E)-3-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-ol (16)
To a stirred solution of 15 (2636 mg, 7 mmol) in 40 mL anhydrous toluene under argon at -78 oC is added
dropwise diisobutylaluminium hydride (15.3 ml as a 1M solution in toluene, 15.3 mmol, 2.2 eq.) over the
o
course of 30 minutes. The reaction is stirred at -78 C for 90 minutes after which 10 mL of MeOH is
slowly added to destroy excess DIBAL-H. After having returned to ambient temperature, 50 mL of
saturated potassium sodium tartrate solution is added to the organic solution and vigorously stirred for
4h. The organic and aqueous phases are then separated and the aqueous phase is extracted with 3 x 30
mL AcOEt. The pooled organic fractions are washed with 2 x NH4Cl(sat.), dried over MgSO4, filtered and
concentrated to yield compound 16, a mixture of diastereoisomers, in quantitative yields as a
translucent oil; Rf = 0.21 (30 % AcOEt/Hex). 1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 7.21 (m, 1H, Har),
7.09 (d, 1H, J = 8.34, Har), 6.99 (d, 1H, J = 8.34, Har), 6.53 (d, 1H, J = 15.8 Hz, =CHCar), 6.25 (dt, 1H, J = 15.8
Hz, 5.9 Hz, =CHCH2O), 5.47 – 5.44 (m, 2H, OCHO), 4.29 (d, 2H, CH2O), 4.06 – 3.97 (m, 2H, CH2O), 3.67 –
13
3.61 (m, 2H, CH2O), 2.07 – 1.84 (m, 6H, THP), 1.78 – 1.53 (m, 7H, THP + OH). C NMR (101 MHz, CDCl3,
25 °C), δ (ppm) = 147.59, 147.36, 147.14, 131.54, 131.38, 130.99, 130.96, 127.18, 121.23, 121.09,
118.13, 116.64, 116.31, 97.81, 97.64, 97.37, 97.20, 63.82, 61.97, 61.93, 61.80, 61.75, 30.42, 30.37,
30.34, 30.30, 25.32, 25.29, 18.68, 18.64, 18.55, 18.50. HRMS m/z calc. for C19H26O5 + (Na+): 357.1672;
found: 157.1650.
(E)-2,2'-((4-(3-(benzyloxy)prop-1-en-1-yl)-1,2-phenylene)bis(oxy))bis(tetrahydro-2H-pyran) (17a)
To a suspension of NaH (46 mg of a 60 % NaH dispersion in mineral oil, 1.15 mmol, 2.1 eq.) in anhydrous
DMF at 0 oC is added dropwise 16 (191 mg, 0.55 mmol) dissolved in 1 mL DMF and the mixture is left to
react for 30 minutes. Benzyl bromide (196 mg, 1.15 mmol, 2.1 eq) dissolved in 1 mL DMF is then added
dropwise to the alcoxide solution while maintaining the temperature of the mixture below 5 oC, and the
solution is stirred for an additional 2h and then quenched by slow addition of 0.1 mL MeOH. The
resulting solution is diluted to 75 mL with H2O and then extracted 5 x 15 mL AcOEt. The organic phase is
washed with 2 x H2O, 1 x brine, dried over MgSO4 and concentrated. Compound 17a, a mixture of
diastereoisomers, is obtained as a thick translucent oil with 70 % yield (167 mg, 0.38 mmol) after silica
1
gel circular chromatography (6 % AcOEt/Hex); Rf = 0.63 (20 % AcOEt/Hex). H NMR (400 MHz, CDCl3,
25 °C), δ (ppm) = 7.40 – 7.36 (m, 3H, Har), 7.32 – 7.28 (m, 2H, Har), 7.23 (t, 1H, J = 1.9 Hz, Har), 7.10 (dd,
1H, J = 8.3 Hz, 2.2 Hz), 7.00 (dt, 1H, J = 8.3 Hz, 1.9 Hz, Har), 6.56 (d, 1H, J = 15.9 Hz, =CHCar), 6.22 (dt, 1H, J
= 15.9 Hz, 5.9 Hz, =CHCH2O), 5.47 – 5.45 (m, 2H, OCHO), 4.58 (s, 2H, OCH2Ph), 4.19 (d, 2H, J = 5.9 Hz,
=CHCH2O), 4.07 – 3.97 (m, 2H, CH2O), 3.68 – 3.45 (m, 2H, CH2O), 2.13 – 1.83 (m, 6H, THP), 1.79 – 1.58
(m, 6H, THP). 13C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 147.57, 147.36, 147.15, 138.35, 132.39,
132.36, 131.59, 128.41, 128.36, 127.82, 124.66, 124.60, 121.27, 121.13, 118.42, 118.10, 116.68, 116.35,
97.80, 97.63, 97.36, 97.21, 72.03, 70.87, 61.96, 61.93, 61.80, 61.76, 30.43, 30.38, 30.35, 30.31, 25.33,
25.30, 18.69, 18.65, 18.56, 18.51. HRMS m/z calc. for C26H32O5 + (Na+): 447.2142; found: 447.2114.
This article is protected by copyright. All rights reserved.

(E)-2,2'-((4-(3-(3-phenylpropoxy)prop-1-en-1-yl)-1,2-phenylene)bis(oxy))bis(tetrahydro-2H-pyran)
(17b)
To a suspension of NaH (63 mg of a 60 % NaH dispersion in mineral oil, 1.58 mmol, 2.1 eq.) in anhydrous
DMF at 0 oC is added dropwise 16 dissolved in 1 mL DMF and the mixture is left to react for 30 minutes.
(3-bromopropyl)benzene (314 mg, 1.58 mmol, 2.1 eq) dissolved in 1 mL DMF is then added dropwise to
o
the alcoxide solution while maintaining the temperature of the mixture below 5 C, and the solution is
stirred for an additional 2h and then quenched by slow addition of 0.1 mL MeOH. The resulting solution
is diluted to 75 mL with H2O and then extracted 5 x 15 mL AcOEt. The organic phase is washed with 2 x
H2O, 1 x brine, dried over MgSO4 and concentrated. Compound 17b, a mixture of diastereoisomers, is
obtained as a thick translucent oil with 70 % yield (167 mg, 0.38 mmol) after silica gel circular
chromatography (5 % AcOEt/Hex); Rf = 0.65 (20 % AcOEt/Hex). 1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm)
= 7.32 – 7. 30 (m, 2H, Har), 7.23 – 7.18 (m, 4H, Har), 7.10 (dd, 1H, J = 8.4 Hz, 2.3 Hz), 7.00 (dt, 1H, J = 8.4
Hz, 2.0 Hz, Har), 6.53 (d, 1H, J = 15.9 Hz, =CHCar), 6.19 (dt, 1H, J = 15.9 Hz, 6.1 Hz, =CHCH2O), 5.48 – 5.44
(m, 2H, OCHO), 4.12 (d, 2H, J = 6.1 Hz, =CHCH2O), 4.07 – 3.98 (m, 2H, CH2O), 3.67 – 3.60 (m, 2H, CH2O),
3.50 (t, 2H, J = 6.4 Hz, OCH2CH2CH2Ph), 2.74 (t, 2H, J = 6.4 Hz, OCH2CH2CH2Ph), 2.13 – 1.84 (m, 8H, THP +
13
OCH2CH2CH2Ph), 1.80 – 1.68 (m, 6H, THP). C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 147.57, 147.35,
147.32, 147.10, 142.02, 132.05, 132.02, 131.65, 131.49, 128.50, 125.75, 124.92, 124.86, 121.25, 121.10,
118.43, 118.10, 116.67, 116.33, 97.79, 97.64, 97.35, 97.21, 71.55, 69.40, 61.95, 61.93, 61.79, 61.76,
32.40, 31.38, 30.43, 30.39, 30.35, 30.31, 25.33, 25.30, 18.68, 18.66, 18.56, 18.52. HRMS m/z calc. for
C28H36O5 + (Na+): 475.2455; found: 475.2433.
(E)-4-(3-hydroxyprop-1-en-1-yl)benzene-1,2-diol, caffeoyl alcohol (6)
Following general procedure IV with 16, compound 6 was obtained with 70 % yield as white crystals
after silica gel circular chromatography (3 - 4 % MeOH/CH2Cl2); mp = 0.45 (7.5 % MeOH/CH2Cl2), Rf =
0.45 (7.5 % MeOH/CH2Cl2). 1H NMR (400 MHz, DMSO-d6, 25 °C), δ (ppm) = 8.90 (br s, 2H, OH), 6.81 (br s,
1H, Har), 6.67 (br s, 2H, Har), 6.34 (d, 1H, J = 15.9 Hz, =CHCar), 6.04 (dt, 1H, J = 15.9 Hz, 5.4 Hz, =CHCH2OH),
13
4.73 (br s, 1H, OH), 4.05 (d, 1H, J = 5.4 Hz, CH2OH). C NMR (101 MHz, DMSO-d6, 25 °C), δ (ppm) =
145.74, 145.47, 129.52, 128.92, 127.46, 118.38, 116.06, 113.49, 62.18. HRMS m/z calc. for C9H10O3 +
(H+): 149.0597; found: 149.0586.
(E)-3-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-N-methoxy-N-methylacrylamide (18)
Compound 15 (1130 mg, 3 mmol) and N,O-dimethylhydroxylamine hydrochloride (454 mg, 4.65 mmol,
o
1.55 eq.) are dissolved/suspended in 15 mL anhydrous THF under argon and placed at -20 C with
agitation. Isopropyl magnesium chloride (4.5 mL as a 2M solution in THF, 9 mmol, 3 eq.) is then added
dropwise over 10 minutes while keeping the reaction temperature below –10 oC. After 25 minutes, the
reaction is quenched by slow addition of 5 mL of 5M NH4Cl and agitation is continued for another 10
minutes. The resulting mixture is diluted with 50 mL 5M NH4Cl and extracted with 3 x 30 mL AcOEt. The
This article is protected by copyright. All rights reserved.

organic phase is then dried over MgSO4, filtered and concentrated. Compound 18, a mixture of
diastereoisomers, is obtained as a translucent oil with 87 % yield (1056 mg, 2.6 mmol) after silica gel
1
circular chromatography (20 - 30 % AcOEt/Hex); Rf = 0.16 (30 % AcOEt/Hex). H NMR (400 MHz, CDCl3,
25 °C), δ (ppm) = 7.67 (d, 1H, J = 15.9 Hz, =CHCar), 7.37 (br s, 1H, Har), 7.21 (m, 1H, Har), 7.15 (m, 1H, Har),
6.90 (d, 1H, J = 15.9 Hz, =CHCO), 5.52 – 5.45 (m, 2H, OCHO), 4.06 – 3.93 (m, 2H, CH2O), 3.77 (s, 1H,
OCH3), 3.65 – 3.61 (m, 2H, CH2O), 3.31 (s, 3H, NCH3), 2.11 – 1.88 (m, 6H, THP), 1.77 – 1.63 (m, 6H, THP).
13C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 167.24, 149.35, 149.12, 147.39, 147.18, 143.35, 143.31,
129.68, 129.55, 123.45, 123.29, 118.38, 117.99, 117.81, 117.57, 114.12, 114.06, 98.06, 97.57, 97.26,
96.87, 62.15, 61.98, 61.93, 61.84, 61.75, 32.54, 30.40, 30.35, 30.25, 30.22, 25.28, 25.22, 18.76, 18.64,
18.55, 18.40. HRMS m/z calc. for C21H29NO6 + (H+): 392.2068; found: 392.2039.
(E)-3-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)acrylaldehyde, diacetylcaffealdehyde (19a)
To a vigorously stirred solution of compound 16 (118 mg, 0.34 mmol) in 8 mL CH2Cl2 under argon is
added MnO2 (300 mg, 3.4 mmol, 10 eq.) and the mixture is left to react overnight. After 16h the mixture
is filtered on a pad of celite and concentrated. Compound 19a, a mixture of diastereoisomers, is
obtained as a translucent oil with 53 % yield (62 mg, 0.18 mmol) after silica gel circular chromatography
(7.5 % AcOEt/Hex); Rf = 0.24 (20 % AcOEt/Hex). 1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 9.67 (d, 1H, J
= 7.8 Hz, CHO), 7.41 (d, 1H, J = 15.8 Hz, =CHCar), 7.39 (s, 1H, Har), 7.23 - 7.18 (m, 2H, Har), 6.63 (dd, 1H, J =
15.8 Hz, 7.8 Hz, =CHCHO), 5.55 (m, 1H, OCHO), 5.47 (m, 1H, OCHO), 4.05 - 3.91 (m, 2H, CH2O), 3.67 -
3.63 (m, 2H, CH2O), 2.10 - 1.88 (m, 6H, THP), 1.79 - 1.62 (m, 6H, THP). 13C NMR (101 MHz, CDCl3, 25 °C), δ
(ppm) = 193.68, 152.90, 152.85, 150.55, 150.30, 147.60, 147.38, 128.35, 128.22, 127.20, 127.16, 124.20,
124.03, 118.35, 117.96, 117.63, 117.37, 97.96, 97.47, 97.13, 96.74, 62.05, 61.96, 61.91, 61.78, 30.35,
30.30, 30.18, 30.16, 25.24, 25.16, 18.60, 18.50, 18.46, 18.32. HRMS m/z calc. for C19H24O5 + (Na+):
355.1516; found: 355.1498.
(E)-3-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1-phenylprop-2-en-1-one (19b)
To a stirred solution of 18 (285 mg, 0.70 mmol) dissolved in 7 mL anhydrous THF at 0 oC and under argon
is slowly added phenyl magnesium bromide (1.76 mL as a 1M solution in THF, 1.76 mmol, 2.5 eq.) over
o
10 minutes while keeping the solution at 0 C. After 45 minutes, the reaction is quenched by slow
addition of 7 mL of 5M NH4Cl and agitation is continued for another 10 minutes. The resulting mixture is
extracted with 3 x 30 mL CH2Cl2. The organic phase is then dried over MgSO4, filtered and concentrated.
Compound 19b, a mixture of diastereoisomers, is obtained as a thick yellow oil with 70 % yield (205 mg,
1
0.49 mmol) after silica gel circular chromatography (12 % AcOEt/Hex); Rf = 0.37 (20 % AcOEt/Hex). H
NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 8.02 (d, 2H, J = 7.3 Hz, Har), 7.45 (d, 1H, J = 15.7 Hz, =CHCar), 7.60
(t, 1H, J = 7.3 Hz, Har), 7.51 (t, 2H, J = 7.3 Hz, Har), 7.42 (br s, 1H, Har), 7.40 (d, 1H, J = 15.7 Hz, =CHCO),
7.30 (m, 1H, Har), 7.19 (m, 1H, Har), 5.56 (m, 2H, OCHO), 4.08 – 3.94 (m, 2H, CH2O), 3.69 – 3.63 (m, 2H,
CH2O), 2.10 (m, 6H, THP), 1.80 – 1.59 (m, 6H, THP). 13C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 190.73,
150.03, 149.79, 147.53, 147.31, 144.99, 144.94, 138.49, 132.55, 129.29, 129.15, 128.56, 128.47, 124.25,
This article is protected by copyright. All rights reserved.

124.11, 120.60, 120.55, 118.27, 117.85, 117.74, 117.47, 97.94, 97.43, 97.21, 96.81, 62.08, 61.96, 61.92,
61.78, 30.40, 30.35, 30.23, 30.21, 25.29, 25.21, 25.20, 18.65, 18.55, 18.52, 18.38. HRMS m/z calc. for
C25H28O5 + (Na+): 431.1829; found: 431.1817.
(E)-4-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1-phenylbut-3-en-2-one (19c)
To a stirred solution of 18 (405 mg, 1 mmol) dissolved in 8 mL anhydrous THF at 0 oC and under argon is
slowly added benzyl magnesium chloride (2.5 mL as a 1M solution in THF, 2.5 mmol, 2.5 eq.) over 10
minutes while keeping the solution at 0 oC. After 45 minutes, the reaction is quenched by slow addition
of 7 mL of 5M NH4Cl and agitation is continued for another 10 minutes. The resulting mixture is
extracted with 3 x 30 mL CH2Cl2. The organic phase is then dried over MgSO4, filtered and concentrated.
Compound 19c, a mixture of diastereoisomers, is obtained as a thick translucent oil with 41 % yield (174
mg, 0.41 mmol) after silica gel circular chromatography (12 % AcOEt/Hex); Rf = 0.42 (25 % AcOEt/Hex).
1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 7.57 (d, 1H, J = 16.0 Hz, =CHCar), 7.40 – 7.31 (m, 5H, Har), 7.29
– 7.26 (m, 3H, Har), 7.18 – 7.13 (m, 2H, Har), 6.67 (d, 1H, J = 16.0 Hz, =CHCO), 5.51 (m, 1H, OCHO), 5.46
(m, 1H, OCHO), 4.52 – 3.91 (m, 4H, CH2O + PhCH2), 3.70 – 3.61 (m, 2H, CH2O), 2.07 – 1.89 (m, 6H, THP),
13
1.75 – 1.61 (m, 6H, THP). C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 197.37, 149.99, 149.75, 147.49,
147.27, 143.48, 143.43, 134.68, 129.48, 128.77, 128.72, 128.63, 128.58, 127.68, 126.99, 126.90, 124.04,
123.92, 123.88, 118.26, 117.85, 117.70, 117.43, 97.92, 97.42, 97.18, 96.77, 62.06, 61.94, 61.91, 61.76,
48.06, 30.36, 30.31, 30.21, 30.18, 25.27, 25.19, 18.64, 18.53, 18.49, 18.35. HRMS m/z calc. for C26H30O5 +
(Na+): 445.1985; found: 445.1963.
(E)-1-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-5-phenylpent-1-en-3-one (19d)
To a stirred solution of 18 (285 mg, 0.70 mmol) dissolved in 7 mL anhydrous THF at 0 oC and under argon
is slowly added phenylethyl magnesium chloride (2.5 mL as a 1M solution in THF, 2.5 mmol, 2.5 eq.) over
o
10 minutes while keeping the solution at 0 C. After 45 minutes, the reaction is quenched by slow
addition of 7 mL of 5M NH4Cl and agitation is continued for another 10 minutes. The resulting mixture is
extracted with 3 x 30 mL CH2Cl2. The organic phase is then dried over MgSO4, filtered and concentrated.
Compound 19d, a mixture of diastereoisomers, is obtained as a thick translucent oil with 51 % yield (212
mg, 0.51 mmol) after silica gel circular chromatography (12 % AcOEt/Hex); Rf = 0.68 (30 % AcOEt/Hex).
1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm) = 7.49 (d, 1H, J = 16.1 Hz, =CHCar), 7.37 – 7.20 (m, 6H, Har), 7.16
(m, 2H, Har), 6.63 (d, 16.1 Hz, =CHCO), 5.52 (m, 1H, OCHO), 5.47 (m, 1H, OCHO), 4.06 – 3.92 (m, 2H,
CH2O), 3.68 – 3.63 (m, 2H, CH2O), 3.04 – 3.00 (m, 4H, CH2CH2), 2.07 – 1.85 (m, 6H, THP), 1.78 – 1.59 (m,
6H, THP). 13C NMR (101 MHz, CDCl3, 25 °C), δ (ppm) = 199.37, 149.90, 149.66, 147.55, 147.33, 142.77,
142.73, 141.33, 128.82, 128.68, 128.50, 128.47, 128.40, 128.34, 126.08, 125.84, 124.74, 124.69, 123.95,
123.80, 118.04, 117.76, 117.64, 117.48, 97.88, 97.40, 97.21, 96.80, 62.02, 61.95, 61.87, 61.77, 42.24,
30.42, 30.37, 30.32, 30.26, 30.22, 30.19, 25.27, 25.20, 25.19, 18.60, 18.50, 18.37. HRMS m/z calc. for
C27H32O5 + (K+): 475.1881; found: 475.1874.
This article is protected by copyright. All rights reserved.

(E)-1-(3,4-bis((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-6-phenylhex-1-en-3-one (19e)
To 3 mL anhydrous THF is added acid washed magnesium shavings (80 mg, 3.3 mmol, 1.1 eq./bromide)
followed by a small crystal of iodine. A small portion (about 20 % of total mass) of (3-
bromopropyl)benzene (597 mg, 3 mmol, 3 eq.) is added dropwise to the magnesium suspension in order
to initiate formation of the organomagnesium reagent. Once Grignard reagent formation begins (as
indicated by light boiling) the remaining (3-bromopropyl)benzene is added slowly in order to maintain a
gentle reflux. Once the Grignard reagent formation is complete, the resulting solution of (3-
bromomagnesiumpropyl)benzene in THF is added dropwise to a stirred solution of 18 (405 mg, 1 mmol)
dissolved in 7 mL anhydrous THF at 0 oC and under argon over 10 minutes while keeping the solution at
o
0 C. After 45 minutes, the reaction is quenched by slow addition of 7 mL of 5M NH4Cl and agitation is
continued for another 10 minutes. The resulting mixture is extracted with 3 x 30 mL CH2Cl2. The organic
phase is then dried over MgSO4, filtered and concentrated. Compound 19e, a mixture of
diastereoisomers, is obtained as a thick yellow oil with 22 % yield (104 mg, 0.22 mmol) after silica gel
1
circular chromatography (12 % AcOEt/Hex); Rf = 0.71 (30 % AcOEt/Hex). H NMR (400 MHz, CDCl3,
25 °C), δ (ppm) = 7.45 (d, 1H, J = 16.1 Hz, =CHCar), 7.36 – 7.29 (m, 3H, Har), 7.24 – 7.20 (m, 3H, Har), 7.17
(m, 2H, Har), 6.61 (d, 1H, J = 16.1 Hz, =CHCO), 5.52 (m, 1H, OCHO), 5.48 (m, 1H, OCHO), 4.03 – 3.96 (m,
2H, CH2O), 3.69 – 3.61 (m, 2H, CH2O), 2.73 – 2.66 (m, 4H, COCH2CH2CH2Ph), 2.11 – 1.85 (m, 8H,
13
COCH2CH2CH2Ph + THP), 1.81 – 1.60 (m, 6H, THP). C NMR (101 MHz, CDCl3, 25 °C),
(ppm) = 200.25,
149.84, 149.61, 147.56, 147.33, 142.50. 142.46, 141.75, 128.89, 128.75, 128.55, 128.39, 125.93, 124.81,
124.76, 123.92, 123.76, 118.00, 117.79, 117.59, 117.50, 97.85, 97.37, 97.22, 96.81, 62.01, 61.95, 61.86,
61.77, 39.76, 35.20, 30.38, 30.33, 30.23, 30.20, 25.87, 25.29, 25.21, 18.60, 18.51, 18.38. HRMS m/z calc.
for C28H34O5 + (Na+): 473.2298; found: 473.2284.
Antiradical activity assay
The radical scavenging activity of test compounds was measured as previously described using 2,2-
diphenyl-1-picrylhydrazyl (DPPH) as a stable radical (20) with slight modifications. Particular care was
taken in the preparation of the control (DPPH reagent + ethanol as a diluent without test compounds).
Controls with O.D. of 0.350-0.360 at 520nm were deemed acceptable to avoid variations in IC50
calculations. 1 ml of DPPH in ethanol (60 mM) was mixed with 1 mL of the test compounds at the
indicated concentrations or their diluent (ethanol). Each mixture was then shaken vigorously and kept in
the dark for 30 min at room temperature. The absorbance of DPPH at 520 nm was then measured. The
radical scavenging activity was expressed in terms of % inhibition of DPPH absorbance:
% Inhibition = [(A control - A test)/A control)] x100
where “A control” is the absorbance of the control (DPPH solution without test compound) and “A test”
is the absorbance of the test sample (DPPH solution plus compound). Data are expressed as means ± SD
of 3 independent experiments, each performed in triplicate (table 1). IC50 values were calculated from a
This article is protected by copyright. All rights reserved.

sigmoidal concentration-response curve-fitting model with a variable slope on GraphPad Prism 5
software (GraphPad Software, San Diego, California).
5-LO product biosynthesis assays in HEK293 cells
HEK293 cells stably co-transfected with a pcDNA3.1 vector expressing 5-LO and a pBUDCE4.1 vector
expressing 5-LO activating protein (FLAP) were utilized (25,26) to screen compounds for 5-LO inhibition.
.
For cell stimulation of 5-LO products, transfected HEK293 cells were collected following trypsinization,
washed and the cell pellet was resuspended in Hank’s balanced salt solution (HBSS) (Lonza, Walkerville,
MD) containing 1.6 mM CaCl2 at a concentration of 5 x 105 cells mL-1. Cells were pre-incubated with each
o
o
test compound at 1 µM for 5 min at 37 C. Cells were then stimulated for 15 minutes at 37 C with the
addition of 10 µM calcium ionophore A23187 (Sigma–Aldrich, Oakville, ON, Canada) and 10 µM
arachidonic acid (Cayman Chemical, Ann Arbor, MI). Stimulations were stopped by the addition of 0.5
volume of cold MeOH:CH3CN (1:1) containing 50 ng of PGB2 as internal standard and samples were
stored at -20oC until processing on octadecyl (C18) columns and analysis by RP-HPLC as described
previously (25,26). Data are expressed as means ± SEM of 3 independent experiments, each performed
in duplicate (fig. 3).
Stimulation of human polymorphonuclear leukocytes (PMNL) for 5-LO products
Human PMNL prepared from peripheral blood as described (23) were suspended in HBSS containing 1.6
o
mM CaCl2 (107 cells/ml) and pre-incubated with test compounds for 5 min at 37 C in the presence of 1
U/ml of adenosine deaminase (Sigma-Aldrich, Oakville, On, Canada). Cells were then stimulated for 15
min at 37oC with 1 µM thapsigargin (Sigma-Aldrich) and 10 µM AA (Cayman Chemical, Ann Arbour, MI)
as previously described (23). Reactions were stopped, processed and analysed by RP-HPLC as indicated
above. Data are expressed as means ± SEM of 3 independent experiments, each performed in duplicate
(fig. 4). Initial screening of compounds were performed in the presence of 1 µM of test compounds
followed by concentration-response assays of selected compounds at 0.1, 0.3, 1.0 and 3.0 µM.
Molecular docking
Molecular docking was done with AutoDock Vina 1.1.2 on 5-LO (PDB code: 3O8Y) following a recently
described procedure (27) outlined in "Design, synthesis and evaluation of semi-synthetic triazole-
containing caffeic acid analogues as 5-lipoxygenase inhibitors." by Daniela De Lucia et al. For
optimization, the MMFF94 force field was used. Results were analyzed with Maestro 10.4 (27)
This article is protected by copyright. All rights reserved.

Results and discussion
Chemistry
Caffeic propargyl esters (5a-h) (scheme 1) were synthesized starting from caffeic acid 2, which was
acetylated (26) to yield diacetylcaffeic acid (9). Acetylated propargyl caffeate (10) was obtained by
condensing diacetylcaffeoyl chloride, obtained by refluxing (9) in excess thionyl chloride and catalytic
DMF, with propargyl alcohol in pyridine/CH2Cl2. Compounds (11b-h), acetylated precursors of the final
esters, were obtained in moderate to good yields through optimized Sonogashira cross coupling
conditions with propargyl ester 10 and the appropriate aryl iodide in the presence of catalytic
PdCl2(PPh3)2 and copper iodide in dry and degassed DMF/NEt3. Dimer (11i) was obtained in similar
conditions with 1,4-diiodobenzene as the aryl iodide and a slight excess of compound (10).
Deacetylation of compounds (10) and (11b-i) to afford caffeic esters (4a-i) was performed with
guanidinium hydrochloride and triethylamine in MeOH/CH2Cl2, thus generating the expected phenolic
compounds (28).
o
Scheme 1. Synthetic route to propargyl esters (4a-i). Reagents: i) AcOAc, DMAP, pyridine, 0 C to rt
overnight; ii) SOCl2, DMFcat, reflux 3h then HCCCH2OH, pyridine/CH2Cl2, rt overnight ; iii) aryl iodide,
PdCl2(PPh3)2, CuI, DMF/NEt3, rt overnight; iv) guanidinium HCl, NEt3, MeOH/CH2Cl2, rt 4h.
Caffeic allyl esters 5a-i (scheme 2) were synthesized in a similar fashion to compounds (4a-i). Acetylated
esters (13b-h) were obtained in moderate to good yields through optimized Heck cross coupling
conditions by reacting allyl ester (12) with the required aryl iodides in the presence of catalytic Pd(OAc)2
and Ag2CO3 in refluxing benzene. Dimer (13i) was obtained in similar conditions with 1,4-diiodobenzene
as the aryl iodide and a slight excess of compound (12). Deacetylation of compounds (12) and (13b-i) to
afford caffeic esters (5a-h) was performed with guanidinium hydrochloride and triethylamine in
MeOH/CH2Cl2, identical to the propargyl derivatives (28).
Scheme 2. Synthetic route to allyl esters (5a-i). Reagents: i) SOCl2, DMFcat, reflux 3h then
CH2=CHCH2OH, pyridine/CH2Cl2, rt overnight; ii) aryl iodide, Pd(OAc)2, AgCO3, PhH, reflux overnight;
iii) guanidinium HCl, NEt3, MeOH/CH2Cl2, rt 4h.
Caffeoyl alcohol (6) and allyl ethers (7a) and (7b) (scheme 3) were synthesized starting from
caffeic acid 2, which was esterified in MeOH with concentrated sulphuric acid, yielding ester
(14). Methyl caffeate (14) was then protected as the tetrahydropyranyl ester (15) through PPTS
catalyzed acetal formation in DHP. The resulting mixture of diastereoisomers was quantitatively
reduced to the tetrahydropyranylated allylic alcohol (16) by DIBAL-H in dry toluene at -78.
This article is protected by copyright. All rights reserved.

Allyl ethers (17a-b) were obtained through Williamson conditions with (16) and NaH in DMF to
generate the alkoxide, which was subsequently reacted with either benzyl bromide or (3-
bromopropyl)benzene to produce the expected ethers in good yields. Deprotection of
tetrahydropyranylated compounds (16) and (17a-b) by methanolysis in the presence of PPTS
yielded the caffeoyl alcohol (6) as well as allyl ethers (7a) and (7b) in good yields.
Scheme 3. Synthetic route to caffeoyl alcohol 6 and allylic ethers (7a-b). Reagents: i) MeOH, H2SO4,
o
reflux 4h; ii) DHP, PPTS, reflux 1.5h; iii) DIBAL-H, PhCH3, -78 C 2h; iv) NaH, PhCH2Br or PhCH-
2CH2CH2Br, DMF, 0 oC 2h; v) (6) from (16), (7a-b) from (17a-b); ppts, MeOH/CH2Cl2, rt 4h.
Tetrahydropyranylated caffealdehyde (19a) (scheme 4) was conveniently synthesized by oxidation of
allyl alcohol (16) with excess MnO2 in CH2Cl2 (29). Acetylated caffeoyl ketones (19b-e) were obtained in
two steps from the previously synthesized tetrahydropyranylated methyl ester (15). Initial conversion of
(15) to the Weinreb amide (18) with N,O-dimethylhydroxylamine hydrochloride and
isopropylmagnesium chloride (30) was followed by Grignard reaction with the required
organomagnesium reagents to yield the desired ketones in moderate yields. Deprotection of
tetrahydropyranylated compounds (19a-e) by methanolysis in the presence of PPTS yielded the
caffealdehyde (8a) as well as caffeoyl ketones (8b-e) in excellent yields.
Scheme 4. Synthetic route to caffeoyl aldehyde 8a and ketones (8b-e). Reagents: i) DIBAL-H, PhCH3, -
78 oC 2h; ii) CH3NHOCH3 - HCl, i-prMgCl, THF, -20 oC 35 mins; iii) (19a) from (16), MnO2, CH2Cl2, rt
overnight; iv) (19b-e) from (18), R1MgBr, THF, O oC 1h; v) ppts, MeOH/CH2Cl2, rt 4h.
Radical scavenging activity
Radical scavenging activities of synthesized caffeoyl propargyl esters (4a-i), allyl esters (5a-i),
alcohol/ethers (6, 7a and 7b) and aldehyde/ketones (8a-e) were assayed using 2,2-Diphenyl-1-
picrylhydrazyl (DPPH) as a stable radical (20) and are expressed as IC50 concentrations in table 1. As was
expected of catechol-containing molecules, all compounds possessed considerable radical scavenging
ability with most having IC50 values in the range of 10 – 15 μM, similar to radical scavenging activities of
caffeic acid (2, 15.3 μM) and CAPE (3, 11.9 μM). Little variation in radical scavenging activity of
monovalent esters (4a-h and 5a-h) was found, both between related propargyl or allyl esters as well as
between analogous esters of both series. This finding underlines the fact that radical scavenging activity
stems from the catechol moiety of caffeoyl derivatives and is relatively insensitive to modification that
do not directly alter this group (31-33). Logically, the radical scavenging activities of caffeoyl ester
dimers (4i and 5i), containing two catechol moieties and thus four phenolic hydroxyl groups, is roughly
twice as high as that of monovalent caffeoyl esters (4a-h) and (5a-h).
This article is protected by copyright. All rights reserved.

Caffeoyl alcohol (6) and ethers (7a-b) again had IC50 values in the 10-15 μM range, similar to their
related ester analogs. These findings were surprising as the conjugated electron withdrawing ester
groups of compounds (4a-h) and (5a-h) have long range influences on the catechol rings electron
1
density, as is made evident in HNMR analysis of compounds. For example, the phenolic hydrogens of
caffeoyl ester (4a) have significantly higher chemical shifts than those of allyl alcohol (6) (9.40 ppm and
8.90 ppm in DMSO-d6, respectively). Similarly, all (3) aromatic catechol hydrogens of ester (4a) are
significantly downshifted when compared to those of allyl alcohol (6), suggesting reduced electron
density due to the α,β-unsaturated ester. Nonetheless, no drastic change is observed in radical
scavenging activity, though the lipophilic benzyl and phenpropyl allyl ethers are somewhat more potent
radical scavengers than the more hydrophilic allyl alcohol. Caffeoyl aldehyde (8a), caffeoyl phenyl
ketone (8b) and caffeoyl benzyl ketone (8c) showed similar radical scavenging capacities with IC50 of
about 10 μM. However, elongation of the linker separating the carbonyl from the terminal aryl group, as
in compound (8d: IC50 = 19.3 μM) and (8e: IC50 = 28.2 μM), resulted in a steady decrease in potency.
Table 1. Radical scavenging activity of synthesized caffeoyl derivatives. Data are expressed as means
SD of 3 independent experiments, each performed in triplicate.
Compound
R
Radical scavenging
IC50 (μM) [SD]
11.1 1.0
H
4a
4b
4c
4d
4e
4f
Ph
12.7 0.2
4-CH3-Ph
4-CH3O-Ph
4-NO2-Ph
4-F-Ph
11.5 0.9
13.7 0.4
10.6 0.2
10.7 1.9
1-naphtyl
4-Ph-Ph
13.7 0.8
4g
4h
4i
15.0 2.2
5.6 0.33
4-(C≡CCH2OCaffeoyl)-Ph
H
5a
5b
5c
5d
12.3 0.2
Ph
12.4 0.1
4-CH3-Ph
4-CH3O-Ph
13.1 0.3
11.6 0.1
This article is protected by copyright. All rights reserved.

4-NO2-Ph
12.3 0.6
5e
5f
5g
5h
5i
4-F-Ph
12.31 0.04
13.0 0.6
1-naphtyl
4-Ph-Ph
12.03 0.01
6.1 0.2
4-(CH=CHCH2OCaffeoyl)-Ph
H
15.20 0.01
12.0 0.14
11.3 0.3
6
CH2Ph
7a
7b
CH2CH2CH2Ph
H
10.7 0.2
8.97 1.2
11.6 2.1
19.3 2.8
28.2 2.6
15.3
8a
8b
8c
8d
8e
2*
3*
Ph
CH2Ph
CH2CH2Ph
CH2CH2CH2Ph
OH
Caffeic acid
CAPE
OCH2CH2Ph
11.9
*Leblanc et al., 2012
Inhibition of 5-LO product synthesis in whole HEK293 cells.
Inhibition of leukotriene biosynthesis was assayed first in intact HEK293 cells that are stably transfected
with 5-LO and FLAP, thereby possessing all of the necessary cellular machinery for LTs biosynthesis. This
model serves as a highly reproducible model of 5-LO product biosynthesis in which compounds can be
preliminarily screened for anti-LTs activity before moving on to more complex systems (25). All
compounds were assayed at 1 μM, including known inhibitors CAPE (3) and Zileuton (1) whose inhibitory
potencies were used as reference points (figure 3). As can be seen in the upper left of figure 3, among
the propagyl caffeoyl esters of caffeic acid (4a-i), only compound (4i), a dimer type compound bearing
two caffeoyl residues, out performed known 5-LO inhibitor CAPE (3) with roughly 80% inhibition of LTs
biosynthesis in the HEK293 cell model. One could hypothesize that since compound (4i) is the most
potent radical scavenger amongst this series (Table 1), a trend may exist between radical scavenging
activity and LTs biosynthesis inhibition. However, all other inhibitors in this series (4a-h) only possess
one caffeoyl residue and have fairly similar radical scavenging activities between them, though they
possess markedly different anti-LTs activity, a finding which is supported by past studies (23, 34).
This article is protected by copyright. All rights reserved.

Figure 3. Inhibition of 5-LO products synthesis by synthetic CAPE analogs (1 μM) in whole HEK293
cells.
For example, CAPE (3) and compounds (4e) and (4f) have near identical IC50 values in the radical
scavenging assays (table 1), however CAPE (3) and compound (4e) show much better 5-LO inhibition
than compound (4f). This discrepancy between radical scavenging capacity and anti-LTs activity is
echoed in the anti-LTs activity of related caffeoyl esters (5a-e), alcohol/ethers (6, 7a-b) and
aldehyde/ketones (8a-e). Notably, ketones (8b-e), which have progressively lower radical scavenging
activity as the alkyl bridge linking their terminal phenyl group is extended, retain near identical anti-LTs
activity with CAPE (3) in this assay.
Interestingly, some analogous caffeoyl esters have markedly different 5-LO inhibition activity. Propargyl
esters (4b: R = Ph) and (4f: R = 4-F-Ph), for example, possess much lower anti-LTs activity than their
related allyl esters (5b) and (5f). These compounds differ only by the presence of a trans alkene vs linear
alkyne link between the caffeoyl ester and terminal aromatic group, highlighting the importance of
geometry in these compounds for inhibition.
Caffeoyl ethers (7a-b), a class of caffeic acid derived 5-LO inhibitors which have not previously been
tested for anti-LTs activity, outperformed caffeoyl alcohol (6), CAPE (3) and Zileuton (1) in HEK293 cell
based assays. That lipophilic ethers (7a-b) were better inhibitors than the more polar alcohol (6) may
highlight the importance of polarity in inhibitory activity, as it has previously been found that caffeic acid
itself is a very poor inhibitor of 5-LO in cell based assays (23). As mentioned above, caffeoyl ketones (7b-
e) were found to be roughly equipotent with CAPE for 5-LO inhibition in HEK293 cells.
Compounds that approached or surpassed the anti-LTs activity of CAPE (3) were selected for further
screening in human PMNL.
Inhibition of 5-LO product synthesis in thapsigargin-stimulated human PMNL.
To further probe inhibitory activity of lead compounds selected from preliminary HEK293 screenings (fig.
3), 5-LO inhibition assays were undertaken in stimulated human PMNL in the presence of 1 μM of
inhibitors. 5-LO is highly expressed in PMNL and these cells are important physiological producers of
LTB4 (23, 35).
As can be seen in figure 4, propargyl esters (4c) and (4d-e), which possessed similar potency in HEK293
cells, differ significantly in PMNL based assays. 4-Me-phenyl substituted (4c) is significantly less active
than (4d-e) (4-MeO-Phenyl and 4-NO2-Phenyl, respectively), though these compound differ in structure
only in the presence of the 4-substituent of their terminal phenyl group. These subtle structural changes
may influence availability or cell permeation of inhibitors in the more complex PMNL versus HEK293
cells. Impressively, dimer ester (4i) induced almost complete inhibition of LTs biosynthesis at 1 μM,
widely surpassing the activity of known inhibitor CAPE (3).
This article is protected by copyright. All rights reserved.

As can be seen in figure 4, inhibition results for allyl esters (5b-f, 5i) were similar their propargyl
counterparts. Specifically, compounds (5c) and (5d-e), which differ from each other in the same way as
esters (4c) and (4d-e) (R = 4-Me-Phenyl vs 4-MeO-Phenyl and 4-NO2-Phenyl, respectively), show a nearly
identical inhibition pattern to their propargyl counterparts, (5d-e) outperforming (5c). Again, dimer
compound (5i) was the best inhibitor in this class, surpassing CAPE (3), though was not as potent as its
propargyl analog (4i).
Impressively, novel caffeoyl ethers (7a-b) produced near total inhibition of LTs biosynthesis in PMNL
based assays, suggesting these lipophilic ethers may be a privileged structure for 5-LO inhibition in a
physiological setting.
Ketones (8b-e) became progressively more potent as the alkyl chain linking the caffeoyl ketone moiety
to the terminal phenyl group became longer (with 0, 1, 2 or 3 methylene units respectively separating
carbonyl and phenyl groups).
Figure 4. Inhibition of 5-LO products synthesis by selected synthetic CAPE analogs (1 μM) in
thapsigargin stimulated human PMNL.
It is interesting to note that the inverse pattern is apparent in radical scavenging activity, with
compound (8b) being most potent with diminishing activity up to compound (8e).
In order to better analyse the anti-LTs activity of these compounds, selected inhibitors with LTs
biosynthesis inhibition of at least 50% at 1 µM in PMNL (all compounds from figure 4 except 4c, 5c, 5f, 6,
8b and 8c) were screened further through concentration-response studies in stimulated PMNL (table 2).
Selected compounds were assayed between 0.1 – 3 µM in order to obtain dose response curves, from
which were calculated IC50 values. All compounds inhibited 5-LO in a concentration-dependent manner
and showed promising activities, with IC50 values ranging from 0.18-1.03 µM.
This article is protected by copyright. All rights reserved.

Table 2. IC50 values of selected inhibitors.
Compound
Human PMNL (μM) [SEM]
3.0 [0.2]
Compound
Human PMNL (μM) [SEM]
0.88 [0.05]
Zileuton (1)
5e
5i
CAPE (3)
0.99 [0.08]
0.61 [0.04]
0.83 [0.07]
0.43 [0.04]
4d
4e
4i
7a
7b
8d
8e
0.68 [0.04]
0.18 [0.01]
0.36 [0.03]
0.84 [0.05]
1.03 [0.02]
0.64 [0.05]
5b
5d
0.73 [0.04]
Most compounds outperformed CAPE 3 and all possessed IC50 values significantly lower than the clinical
5-LO inhibitor Zileuton 1 in PMNL, with propargyl ester dimer 4i, allyl dimer 5i and caffeoyl ether 7a-b
being roughly 8-, 5-, 7- and 16-fold more potent than Zileuton in this model.
Molecular Docking
Molecular docking was done on a modified 5-LO (PDB code: 3O8Y) protein with various ligands. Zileuton
was docked as reference. Its binding energy was determined with AutoDock Vina of -6.6 kcal/mol and
forms two hydrogen bonds with Leu420 (OH···O, d = 2.48 Å: NH···O, d = 1.64 Å). For molecule (7b), the
affinity energy was -8.2 kcal/mol with one hydrogen bond (OH···O, d = 2.24 Å) with Leu420. Both
molecules take a similar position as can be seen in figure 5. Ligand (7b) positions itself over most of the
length of a docked arachidonic acid molecule.
Figure 5. Results of AutoDock Vina docking displayed in Maestro of Zileuton (left) and 7b (right)
Comparing the best inhibitor, 7b, with CAPE (3) and less flexible analogues (4b and 5b) shows that the
flexibility of the ester part is essential 5-LO inhibition. Even these analogues have similar affinity as 7b,
they were clearly less active (table 2 and 3).
This article is protected by copyright. All rights reserved.

Table 3. Molecular docking data of selected inhibitors.
Compound
Affinity (kcal/mol)
Hydrogen bonding
π-π Interaction
-
(S)-Zileuton*
(R)-Zileuton*
- 6.5
- 6.6
- 8.5
- 8.2
- 8.2
- 8.2
- 8.7
-
Leu420 x 2
Phe421 x2
His367
CAPE (3)
-
-
Phe177, His367
-
4b
5b
7b
8e
Asn407
Leu420
-
His372
Phe177, His367
* Zileuton is formulated as a racemic mixture.
Hydrogen bond with Leu420 seems to be necessary for 5-LO inhibition, 7b which formed this bond with
this specific amino acid was our best 5-LO inhibitor (IC50: 0.18 µM). The same bond was also present
when Zileuton was modeled with 5-LO (table 3).
π-π Interaction with His372 was also identified as crucial for a good inhibition of 5-LO. This interaction
was only found with 7b and not with any other molecules (table 3). Hydrogen bond with Asn407 and π-
π interactions with Phe421, Phe 177, and His367 do not seem to be crucial for a strong 5-LO inhibition.
The absence of the hydrogen bond with Leu420 as well as π-π interaction with Hist372 may explain why
the ketone 8e (IC50: 0.64 µM) was slightly less active than the ether 7b (IC50: 0.18 µM) (table 3).
Further molecular modeling studies should confirm these hypotheses to better understand the mode of
action of such inhibitors and even for further structural refinement of new 5-LO inhibitors.
Conclusion
In summary, 26 caffeic acid phenethyl ester analogs, including propargyl (4a-i) and allyl (5a-i) esters of
caffeic acid as well as caffeoyl alcohol (6), ethers (7a-b), aldehyde (8a) and ketones (8b-e) were
designed, synthesized and tested for their radical scavenging and anti-LTs activity. All compounds were
This article is protected by copyright. All rights reserved.

found to possess good radical scavenging activity, with IC50 values proving relatively insensitive to
modifications not affecting the catechol ring of inhibitors. Compounds were submitted to an initial
screening for 5-LO inhibition in 5-LO/FLAP-transfected HEK293 cells before being assayed in human
polymorphonuclear leukocytes (PMNL). Interestingly, no correlation between 5-LO inhibition and radical
scavenging activity were evident in these assays. Though aromatic substitution patterns in assayed allyl
and propargyl esters influenced inhibitory activity, many proved no better than CAPE (3) in potency,
though most surpassed Zileuton (1) in PMNL. Dimer caffeoyl esters (4i) and (5i) however proved to be
excellent inhibitors, along with caffeoyl ethers (7a-b) which showed near complete inhibition of LTs
biosynthesis at assayed concentrations. Further structural investigation of these scaffolds, particularly
the caffeoyl ethers which are a novel class of 5-LO inhibitors, are currently underway along with
pharmacodynamics studies in more complex biological models.
Conflict of Interest
The authors confirm that this article content has no conflict of interest.
Supplementary material
Supporting information including synthetic details and spectroscopic analysis of compounds 4b-i, 5a-i,
7a-b, 8a-e, 11c-h and 13c-h is available online.
Acknowledgments
MT would like to acknowledge the contribution of the Natural Sciences and Engineering Research
Council of Canada (NSERC), the New Brunswick Innovation Foundation (NBIF), the Canadian Foundation
for Innovation (CFI), and Université de Moncton. JD and MH were supported by fellowships from NSERC.
MES was supported by a Canada Research Chair.
References
1. Peters-Golden, M., & Henderson, W. R. Jr. (2007). Leukotrienes. New England Journal of
Medecine, 357(18), 1841-1854.
2. Chen, X., Li, N., Wang, Su, W. N., Hong, J., Jiao, X., Krasna, M. J., Beer, D. G., & Yang C. S.
(2003). Leukotriene A4 hydrolase in rat and human esophageal adenocarcinomas and inhibitory
effects of bestatin. The Journal of the National Cancer Institute, 95(14), 1053-1061.
3. Ghosh, J., & Myers, C. E (1998). Inhibition of arachidonate 5-lipoxygenase triggers massive
apoptosis in human prostate cancer cells. Proceedings of the National Academy of Sciences of the
This article is protected by copyright. All rights reserved.

United States of America, 95(22), 13182–13187.
4. Rioux, N., & Castonguay, A. (1998). Inhibitors of lipoxygenase: a new class of cancer
chemopreventive. Carcinogenesis, 19(8), 1393–1400.
5. Sun, Z., Sood, S., Li, N., Ramji, D., Yang, P., Newman, R. A., Yang, C. S., & Chen, X. (2006).
Involvement of the 5-lipoxygenase/leukotriene A4 hydrolase pathway in 7,12-
dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch, and
inhibition of carcinogenesis by its inhibitors. Carcinogenesis, 27(9), 1902-1908.
6. Pergola, C., & Werz, O. (2010). 5-Lipoxygenase inhibitors: a review of recent developments and
patents. Expert Opinion on Therapeutic Patents, 20(3), 355-375.
7. Werz, O., & Steinhilber, D. (2005). Pharmacological intervention with 5-lipoxygenase: new
insights and novel compounds. Expert Opinion on Therapeutic Patents, 15(5), 505-519.
8. Berger, W., De Chandt, M. T., & Cairns, C. B. (2007). Zileuton: clinical implications of 5-
Lipoxygenase inhibition in severe airway disease. International Journal of Clinical Practice,
61(4), 663–676.
9. Garcia-Marcos, L., Schuster, A., & Perez-Yarza, E. G. (2003). Benefit-risk assessment of
antileukotrienes in the management of asthma. Drug Safety, 26(7), 483–518.
10. Liu, M. C., Dube, L. M., & Lancaster, J. (1996). Acute and chronic effects of a 5-lipoxygenase
inhibitor in asthma: a 6-month randomized multicenter trial. Zileuton Study Group. Journal
of Allergy and Clinical Immunology, 98(5 Pt 1), 859-871.
11. Bankova, V., Dyulgerov, Al., Popov, S., & Marekov, N. (1987). A GC/MS study of the propolis
phenolic constituents. Zeitschrift fuer Naturforschung, C: Journal of Biosciences, 42(1-2), 147-
151.
12. Sawicka, D., Car, H., Borawska, M. H., & Niklinski, J. (2012). The anticancer activity of
propolis. Folia Histochemica et Cytobiologica, 50(1), 25–37.
13. Watanabe, M. A. E., Amarante, M. K., Conti, B. J., & Sforcin, J. M. (2011). Cytotoxic
constituents of propolis inducing anticancer effects: a review. Journal of pharmacy and
pharmacology, 63(11), 1378–1386.
14. Akyol, S., Ozturk, G., Ginis, Z.; Armutcu, F.; Yigitoglu, M. R., & Akyol, O. (2013). In Vivo and
In Vitro Antıneoplastic Actions of Caffeic Acid Phenethyl Ester (CAPE): Therapeutic
Perspectives. Nutrition and Cancer, 65(4), 515–526.
15. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E. M., Efros, L., Caldwell, M., Estevez, V., &
Nakanishi, K. (1988). Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester
isolated from propolis. Experientia, 44(3), 230–232.
16. Lin, H.-P., Lin, C.-Y., Huo, C., Su, L.-C., & Chuu, C.-P. (2012). Anticancer Effect of Caffeic
Acid Phenethyl Ester. Pharmacologia, 3(1), 26-30.
17. Najafi, M. F., Vahedy, F., Seyyedin, M., Jomehzadeh, H. R., & Bozary, K. (2007). Effect of the
water extracts of propolis on stimulation and inhibition of different cells. Cytotechnology, 54(1),
49–56.
18. Koshihara, Y., Neichi, T., Murota, S.-I., Lao, A.-N., Fujimoto, Y., & Tatsuno T. (1983). Selective
inhibition of 5-lipoxygenase by natural compounds isolated from Chinese plants, Artemisia
rubripes Nakai. FEBS letters, 158(1), 41–44.
19. Koshihara, Y., Neichi, T., Murota, S.-I., Lao, A.-N., Fujimoto, Y., & Tatsuno, T. (1984). Caffeic
acid is a selective inhibitor for leukotriene biosynthesis. Biochimica et Biophysica Acta (BBA) -
Lipids and Lipid Metabolism, 792(1), 92–97.
This article is protected by copyright. All rights reserved.

20. Lin, C. F., Chang, T. C., Chiang, C. C., Tsai, H. J., & Hsu, L. Y. (2005). Synthesis of Selenium-
Containing Polyphenolic Acid Esters and Evaluation of Their Effects on Antioxidation and 5-
Lipoxygenase Inhibition. Chemical & Pharmaceutical Bulletin, 53(11), 1402-1407.
21. Mirzoeva, O. K., & Calder, P. C. (1996). The effect of propolis and its components on eicosanoid
production during the inflammatory response. Prostaglandins, Leukotrienes and Essential Fatty
Acids, 55(6), 441-449.
22. Sud'ina, G. F., Mirzoeva, O. K., Pushkareva, M. A., Korshunova, G. A., Sumbatyan, N. V., &
Varfolomeev, S. D. (1993). Caffeic acid phenethyl ester as a lipoxygenase inhibitor with
antioxidant properties. FEBS Letters, 329(1-2), 21-24.
23. Boudreau, L. H., Maillet, J., Leblanc, L. M., Jean-François, J., Touaibia, M., Flamand, N., &
Surette, M. E. (2012). Caffeic Acid Phenethyl Ester and Its Amide Analogue Are Potent
Inhibitors of Leukotriene Biosynthesis in Human Polymorphonuclear Leukocytes. PLoS ONE,
7(2), e31833.
24. Pontiki, E., & Hadjipavlou-Litina, D. (2008). Lipoxygenase inhibitors: a comparative QSAR
study review and evaluation of new QSARs. Medical Research Reviews, 28(1), 39–117.
25. Boudreau, L. H., Picot, N., Doiron, J., Villebonnet, B., Surette, M. E., Robichaud, G. A., &
Touaibia, M (2009). Caffeoyl and cinnamoyl clusters with anti-inflammatory and anti-cancer
effects. Synthesis and structure-activity relationship. New Journal of Chemistry, 33(9), 1932-
1940.
26. Doiron, J., Boudreau, L. H., Picot, N., Villebonet, B., Surette, M. E., & Touaibia, M. (2009).
Synthesis and 5-Lipoxygenase Inhibitory Activity of New Cinnamoyl and Caffeoyl Clusters.
Bioorganic and Medicinal Chemistry Letters, 19(4), 1118-1121.
27. De Lucia, D., Lucio, O. M., Musio, B., Bender, A., Listing, M., Dennhardt, S., Koeberle, A.,
Garscha, U., Rizzo, R., Manfredini, S., Werz, O., Ley, S. V. (2015). Design, synthesis and
evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase
inhibitors. European Journal of Medicinal Chemistry, 101(28), 573-583. Schrödinger Release
2015-4: Maestro, version 10.4, Schrödinger, LLC, New York, NY, 2015.
28. Kunesch, N., Meit, C., & Poisson, J. (1987). Utilisation de la guanidine comme agent
désacétylant sélectif: une method de désacétylation instantanée applicable aux sucres.
Tetrahedron Letters, 28(31), 3569-3572.
29. Chen, F., Parvathi, K., Blount, J. W., Dixon, R. A. (2001). Chemical syntheses of caffeoyl and 5-
OH coniferyl aldehydes and alcohols and determination of lignin O-methyltransferase activities
in dicot and monocot species. Phytochemistry, 58(7), 1035–1042.
30. Williams, J. M., Jobson, R. B., Yasuda, N., Marchesini, G, Dolling, U.-H., & Grabowski, E. J. J.
(1995). A new general method for reparation of N-methoxy-N-methylamides. Application in
direct conversion of an ester to a ketone. Tetrahedron Letters, 36(31), 5461-5464.
31. Chen, J. H., & Ho, C.-H. (1997). Antioxidant Activities of Caffeic Acid and Its Related
Hydroxycinnamic Acid Compounds. The Journal of Agricultural and Food Chemistry, 45(7),
2374−2378.
32. Torres de Pinedo, A., Penalver, P., Morales, J. C. (2007). Synthesis and evaluation of new
phenolic-based antioxidants: Structure–activity relationship. Food Chemistry, 103(1), 55-61.
33. LeBlanc, L. M., Paré, A. F., Jean-François, J., Hébert, M. J. G., Surette, M. E., & Touaibia, M.
(2012). Synthesis and Antiradical/Antioxidant Activities of Caffeic Acid Phenethyl Ester and Its
Related Propionic, Acetic, and Benzoic Acid Analogues. Molecules, 17(12), 14637-14650.
This article is protected by copyright. All rights reserved.

34. Doiron, J. A., Métayer, B., Richard, R. R.; Desjardins, D.; Boudreau, L.H., Lesveque, N. A.,
Jean-François, J., Poirier, S. J., Surette, M. E., Touaibia, M. (2014). Clicked cinnamic/caffeic
esters and amides as radical scavengers and 5-lipoxygenase inhibitors. International journal of
medicinal chemistry, Vol. 2014, Article ID 931756, 12 pages.
35. Walsh C, E., Waite, B. M., Thomas, M. J., DeChatelet, L. R. (1981). Release and metabolism of
arachidonic acid in human neutrophils. Journal of Biological Chemistry, 256(14), 7228–7234.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.