Sinapic acid phenethyl ester as a potent selective 5-lipoxygenase inhibitor: Synthesis and structure–activity relationship

Article abstract of DOI:10.1111/cbdd.13360

Given the hepatotoxicity and an unfavorable pharmacokinetic profile of zileuton (Zyflo), currently the only approved and clinically used 5-Lipoxygenase (5-LO) inhibitor, the search for potent and safe 5-LO inhibitors is highly demanded. The action of several phenolic acid phenethyl esters as potential 5-Lipoxygenase (5-LO) inhibitors has been investigated. For this purpose, a series of 14 phenethyl esters was synthesized and their impact on 5-LO inhibition was evaluated. The effects of position and number of hydroxyl and methoxy groups on the phenolic acid were investigated. The shortening of the linker between the carbonyl and the catechol moiety as well as the presence of the α,β-unsaturated carbonyl group was also explored. The sinapic acid phenethyl ester (10), which can be named SAPE (10) by analogy to caffeic acid phenethyl ester (CAPE), inhibited 5-LO in a concentration-dependent manner and outperformed both zileuton (1) and CAPE (2). With an IC₅₀ of 0.3?μm, SAPE (10) was threefold more potent than CAPE (2) and 10-fold more potent than zileuton (1), the only 5-LO inhibitor approved for clinical use. Unlike CAPE (2), SAPE (10) had no effect on 12-lipoxygenase (12-LO) and less effect on cyclooxygenase 1 (COX-1) which makes it a more selective 5-LO inhibitor.

Full text of DOI:10.1111/cbdd.13360

PROFESSOR MOHAMED TOUAIBIA (Orcid ID : 0000-0003-3744-8012)
Article type : Research Article
Sinapic acid phenethyl ester as a potent selective 5-lipoxygenase inhibitor:
synthesis and structure activity relationship
Mohamed Touaibia*, Martin J.G. Hébert, Natalie A. Levesque, Jérémie A. Doiron, Marco S.
Doucet, Jacques Jean-François, Marc Cormier, Luc H. Boudreau, Marc E. Surette.
Department of chemistry and biochemistry, Université de Moncton, Moncton, NB, Canada
*Author to whom correspondence should be addressed;
E-Mail: mohamed.touaibia@umoncton.ca;
Abstract
Given the hepatotoxicity and an unfavourable pharmacokinetic profile of Zileuton (Zyflo®),
currently the only approved and clinically used 5-Lipoxygenase (5-LO) inhibitor, the search
for potent and safe 5-LO inhibitors is highly demanded. The action of several phenolic acid
phenethyl esters as potential 5-Lipoxygenase (5-LO) inhibitors has been investigated. For this
purpose, a series of fourteen phenethyl esters was synthesized and their impact on 5-LO
inhibition was evaluated. The effects of position and number of hydroxyl and methoxy
groups on the phenolic acid were investigated. The shortening of the linker between the
carbonyl and the catechol moiety as well as the presence of the α,β-unsaturated carbonyl
group was also explored. The sinapic acid phenethyl ester (10), which can be named SAPE
(10) by analogy to caffeic acid phenethyl ester (CAPE), inhibited 5-LO in a concentration-
dependent manner and outperformed both zileuton (1) and CAPE (2). With an IC50 of 0.3
µM, SAPE (10) was 3-fold more potent than CAPE (2) and 10-fold more potent than zileuton
(1), the only 5-LO inhibitor approved for clinical use. Unlike CAPE (2), SAPE (10) had no
effect on 12-lipoxygenase (12-LO) and less effect on cyclooxygenase COX-1 which makes it
a more selective 5-LO inhibitor.
Keywords: Sinapic acid, Caffeic acid, 5-LO inhibitors, anti-leukotriene therapy.
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doi: 10.1111/cbdd.13360
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1. INTRODUCTION
5-Lipoxygenase (5-LO), a key enzyme for arachidonic acid (AA) metabolism, catalyzes the
biosynthesis of leukotriene A4 (LTA4).[1] The hydrolysis of LTA4 by leukotriene A4 hydrolase
produces the proinflammatory leukotriene B4. On the other hand, hydrolysis of LTA4 by
leukotriene C4 synthase produces the cysteinyl leukotrienes C4, D4 and E4[2]. Leukotrienes
(LTs) are a class of eicosanoid inflammatory mediators which have been shown to play a
crucial role in several inflammatory disorders including asthma, atherosclerosis and
arthritis[3]. Additionally, malignant cells highly express 5-LO and related LT biosynthesis
enzymes, consequently anti-LT therapy can not only be a promising anti-inflammatory but an
anticancer strategy as well[3–9]
.
5-LO is a validated target for inflammation and related disorders.[10] Several 5-LO inhibitors
have been reported, including iron ligand, redox, and nonredox inhibitors[11]. Given the weak
potency, hepatotoxicity and an unfavourable pharmacokinetic profile of zileuton (Zyflo®) (1)
(Figure 1), currently the only approved and clinically used 5-LO inhibitor[3,12–15], the search
for potent and safe 5-LO inhibitors is highly demanded.
Recent studies from our group have shown that CAPE (2) is significantly more potent than
Zileuton (1) for the inhibition of LTs biosynthesis in human polymorphonuclear leukocytes
(PMNL, IC50 = 0.52 µM)[16]. Our research has shown that the amide as well as the cinnamic
acid analogs are less active, which highlights the importance of the polyphenol and ester
moieties for efficient inhibition of LTs biosynthesis. On the other hand, the corresponding
ketone and ether, which may be less sensitive to hydrolysis in vivo, were more active than
CAPE (2)[11,17–19]
.
CAPE (2), like other polyphenols, undergoes both sulfatation and glucuronidation[20–26]
during metabolism, which generates several molecular entities which are rapidly eliminated
and contribute to the poor pharmacokinetic profile and bioavailability of the molecule. By
having an α,β-unsaturated carbonyl moiety, CAPE (2) and its analogues react as Michael's
acceptor with nucleophiles such as SH cation of cysteines leading to a covalent bond with the
β carbon.[27]. Therefore, derivatives of CAPE that reach their targets may be structurally quite
different from the originally administered molecule, thus complicating the discovery of new
molecules as well as the understanding of their mode of action. However, the α,β-unsaturated
carbonyl moiety can act as radical scavenger and antioxidant[28]
.
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In the present work, we investigated the influence of the presence of the two hydroxyls as
well as the α, β-unsaturated carbonyl group present in CAPE (2). Phenethyl esters of various
phenolic acids were synthesized and tested for 5-LO inhibition.
The development of dual or even multiple inhibitors of oxygenases is sought after, however,
the discovery of specific inhibitors to a single enzyme such as 5-LO remains relevant.
Therefore, the selectivity of the synthesized compounds was also explored. The best 5-LO
inhibitors were also tested for the inhibition of 12-lipoxygenase (12-LO) and cyclooxygenase
(COX-1). 12-LO catalyzes the stereo-specific peroxidation of polyunsaturated fatty acids
generating the bioactive lipid mediator (12S)-HPETE which regulates several biological
processes including platelet activation and angiogenesis.[29,30] COX-1 catalyzes the
biosynthesis of prostaglandins which have several important physiological roles including the
control of gastric secretions and nociception[31]
.
2. MATERIALS AND METHODS
2.1. Chemistry
All chemicals used were purchased from Aldrich (CA) and used without further purification.
Purification of compounds was carried out by flash chromatography (Isco, Inc.
CombiFlashTM Sg100c). 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 using TMS as an internal standard.
High-resolution mass measurements were performed on a Bruker® Doltonics’ micrOTOF
instrument in positive or negative electrospray.
2.1.1. General procedure for the esterification of phenolic acids
To a vigorously stirred solution of Na2CO3 (1.2 eq) in 6 mL of HMPA, 500 mg of the
appropriate phenolic acid (1 eq) is added after which the reaction vessel is put under argon
atmosphere. After 30 minutes of stirring, the appropriate bromide (1.1 eq) is dissolved in 1
mL of HMPA and added dropwise to the reaction mixture over a period of 30 minutes. A
small amount of KI is added and the reaction vessel is thoroughly flushed with argon gas and
sealed under balloon pressure. The reaction mixture is stirred in an ice bath for 72 hours
under argon atmosphere. The resulting solution is quenched with 30 mL of ice water and
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stirred for 30 minutes. The aqueous phase is extracted three times with AcOEt (30mL). The
organic fractions are then combined, washed with brine, dried over MgSO4 and concentrated.
The resulting oil is purified by flash chromatography.
2-Phenylethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (4)
Following our general procedure for the esterification of phenolic acids with para-coumaric
acid (3a) (500 mg, 3.05 mmol, 1 eq), Na2CO3 (384 mg, 3.62 mmol, 1.2 eq) and (2-
bromoethyl)benzene (0.47 mL, 3.45 mmol, 1.1 eq), compound 4 was obtained as a white
solid after flash chromatography (0 - 20 % AcOEt/Hex), yield = 24 %, Mp = 96 - 97 °C, Rf =
1
0.43 (30 % AcOEt/Hex). H-NMR (DMSO-d6, 25 °C); δ (ppm): 10.09 (s large, 1H, OH),
7.55 - 7.51 (d, J = 15.00 Hz, 1H, CAr-CH=CH), 7.55 - 7.51 (m, 2H, HAr), 7.33 - 7.27 (m, 4H,
HAr), 7.25 - 7.20 (m, 1H, HAr), 6.79 (d, J = 8.56 Hz, 2H, HAr), 6.35 (d, J = 15.97 Hz, 1H,
13
CH=CH-C(=O)), 4.33 (t, J = 6.84 Hz, 2H, CH2O), 2.95 (t, J = 6.84 Hz, 2H, CH2Ph); C-
NMR (DMSO-d6, 25 °C); δ (ppm): 167.03, 160.32, 145.29, 138.55, 130.82, 129.33, 128.84,
126.84, 125.47, 116.25, 114.48, 64.80, 34.92; HRMS m/z calc. for C17H16O3 + (H+):
269.1172; found: 269.1183.
2-Phenylethyl (2E)-3-(3-hydroxyphenyl)prop-2-enoate (5)
Following our general procedure for the esterification of phenolic acids with meta-coumaric
acid (3b) (500 mg, 3.04 mmol, 1 eq), Na2CO3 (384 mg, 3.62 mmol, 1.2 eq) and (2-
bromoethyl)benzene (0.47 mL, 3.45 mmol, 1.1 eq), compound 5 was obtained as a white
solid after flash chromatography (5 - 12 % AcOEt/Hex), yield = 13 %, Mp = 98 - 99 °C, Rf =
0.75 (50 % AcOEt/Hex). 1H-NMR (DMSO-d6, 25 °C); δ (ppm): 9.62 (s, 1H, OH), 7.53 (d, J
= 16.01 Hz, 1H, CAr-CH=CH), 7.34 - 7.29 (m, 4H, HAr), 7.25 - 7.20 (m, 2H, HAr), 7.05 - 7.01
(m, J = 1.72 Hz, 1H, HAr), 6.85 (dd, J = 8.0 0Hz, 2.24 Hz, 1H, HAr), 6.48 (d, J = 16.01 Hz,
1H, CH=CH-C(=O)), 4.55 (t, J = 6.84 Hz, 2H, CH2O), 2.95 (t, J = 6.84 Hz, 2H, CH2Ph); 13C-
NMR (DMSO-d6, 25 °C); δ (ppm): 166.57, 158.17, 145.28, 138.48, 135.65, 130.41, 129.36,
128.84, 126.86, 119.73, 118.16, 115.18, 65.04, 34.89; HRMS m/z calc. for C17H16O3 + (H+):
269.1172; found: 269.1170.
2-Phenylethyl (2E)-3-(2-hydroxyphenyl)prop-2-enoate (6)
Following our general procedure for the esterification of phenolic acids with ortho-coumaric
acid (3c) (500 mg, 3.05 mmol, 1 eq), Na2CO3 (384 mg, 3.62 mmol, 1.2 eq) and (2-
bromoethyl)benzene (0.47 mL, 3.45 mmol, 1.1 eq), compound 6 was obtained as white
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crystals after flash chromatography (0 - 10 % AcOEt/Hex), yield = 36 %, Mp = 98 - 99 °C,
Rf = 0.52 (30 % AcOEt/Hex). 1H-NMR (DMSO-d6, 25 °C); δ (ppm): 10.25 (s, 1H, OH), 7.86
(d, J = 16.17, 1H, CAr-CH=CH), 7.60 (d, J = 7.76 Hz, 1H, HAr), 7.32 - 7.22 (m, 6H, HAr),
6.91 (d, J = 8.12 Hz, 1H, HAr), 6.82 (t, J = 7.52 Hz, 1H, HAr), 6.58 (d, J = 16.13 Hz, 1H,
13
CH=CH-C(=O)), 4.35 (t, J = 6.92 Hz, 2H, CH2O), 2.97 (t, J = 6.84 Hz, 2H, CH2Ph); C-
NMR (DMSO-d6, 25 °C); δ (ppm): 157.25, 140.68, 138.54, 132.22, 129.35, 128.84, 126.83,
121.11, 119.87, 117.43, 116.62, 64.86, 34.94; HRMS m/z calc. for C17H16O3 + (H+):
269.1172; found: 269.1175.
2-Phenylethyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (7)
Following our general procedure for the esterification of phenolic acids with 3,4-
dimethoxycinnamic acid (3d) (500 mg, 2.40 mmol, 1 eq), Na2CO3 (320 mg, 3.02 mmol, 1.3
eq) and (2-bromoethyl)benzene (0.37 mL, 2.72 mmol, 1.1 eq), compound 7 was obtained as a
beige powder after flash chromatography (0 - 10 % AcOEt/Hex), yield = 72 %, Mp = 97 -
98°C, Rf = 0.40 (30 % AcOEt/Hex). 1H-NMR (CDCl3, 25 °C); δ (ppm): 7.64 (d, J = 15.93
Hz, 1H, CAr-CH=CH), 7.37 - 7.30 (m, 2H, HAr), 7.29 - 7.25 (m, 3H, HAr), 7.12 (dd, J = 8.28
Hz, 1.56 Hz, 1H, HAr), 7.07 (d, J = 1.44 Hz, 1H, HAr), 6.89 (d, J = 8.28 Hz, 1H, HAr), 6.32 (d,
J = 15.89 Hz, 1H, CH=CH-C(=O)), 4.45 (t, J = 7.08 Hz, 2H, CH2O), 3.94 (s, 6H, OCH3),
3.05 (t, J = 7.04 Hz, 2H, CH2Ph); 13C-NMR (CDCl3, 25 °C); δ (ppm): 167.14, 151.15,
149.23, 144.78, 137.95, 128.93, 128.52, 127.41, 126.56, 122.67, 115.74, 111.03, 109.61,
64.88, 55.98, 55.91, 35.25; HRMS m/z calc. for C19H20O4 + (H+): 313.1434; found: 313.1437.
2-Phenylethyl (2E)-3-(3,4-dichlorophenyl)prop-2-enoate (8)
Following our general procedure for the esterification of phenolic acids with 3,4-
dichlorocinnamic acid (3e) (500 mg, 2.30 mmol, 1 eq), Na2CO3 (320 mg, 3.02 mmol, 1.3 eq)
and (2-bromoethyl)benzene (0.36 mL, 2.60 mmol, 1.1 eq), compound 8 was obtained as a
white powder after flash chromatography (0 - 10 % AcOEt/Hex), yield = 69 %, Mp = 68 - 69
1
°C, Rf = 0.28 (5 % AcOEt/Hex). H-NMR (CDCl3, 25 °C); δ (ppm): 7.62 (d, J = 1.89 Hz,
1H, HAr), 7.57 (d, J = 16.05 Hz, 1H, CAr-CH=CH), 7.48 (d, J = 8.32 Hz, 1H, HAr), 7.37 - 7.33
(m, 3H, HAr), 7.29 - 7.26 (m, 3H, HAr), 6.42 (d, J = 16.01 Hz, 1H, CH=CH-C(=O)), 4.46 (t, J
= 7.04 Hz, 2H, CH2O), 3.04 (t, J = 7.00 Hz, 2H, CH2Ph); 13C-NMR (CDCl3, 25 °C); δ (ppm):
166.27, 142.08, 137.74, 134.44, 134.22, 133.26, 130.90, 129.62, 128.92, 128.56, 127.04,
126.65, 119.95, 65.29, 35.15; HRMS m/z calc. for C17H14Cl2O2 + (H+): 321.0444; found:
321.0441.
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2-Phenylethyl (2E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate (9)
Following our general procedure for the esterification of phenolic acids with ferulic acid (3f)
(501 mg, 2.57 mmol, 1 eq), Na2CO3 (325 mg, 3.06 mmol, 1.2 eq) and (2-bromoethyl)benzene
(0.40 mL, 2.94 mmol, 1.1 eq), compound 9 was obtained as a pale yellow oil after flash
1
chromatography (0 - 10 % AcOEt/Hex), yield = 24 %, Rf = 0.43 (30 % AcOEt/Hex). H-
NMR (DMSO-d6, 25 °C); δ (ppm): 9.61 (s large, 1H, OH), 7.53 (d, J = 15.89 Hz, 1H, CAr-
CH=CH), 7.34 - 7.29 (m, 5H, HAr), 7.25 - 7.21 (m, 1H, HAr), 7.10 (dd, J = 8.20 Hz, 1.84 Hz,
1H, HAr), 6.79 (d, J = 8.08 Hz, 1H, HAr), 6.45 (d, J = 15.93 Hz, 1H, CH=CH-C(=O)), 4.35 (t,
13
J = 6.84 Hz, 2H, CH2O), 3.82 (s, 3H, OCH3), 2.97 (t, J = 6.84 Hz, 2H, CH2Ph); C-NMR
(DMSO-d6, 25 °C); δ (ppm): 167.06, 149.86, 148.64, 145.64, 138.56, 129.32, 128.85,
126.83, 125.99, 123.73, 115.94, 114.77, 111.63, 64.74, 56.17, 34.95; HRMS m/z calc. for
C18H18O4 + (H+): 299.1278; found: 299.1280.
2-Phenylethyl (2E)-3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-enoate (SAPE, 10)
Following our general procedure for the esterification of phenolic acids with sinapic acid (3g)
(500 mg, 2.23 mmol, 1 eq), Na2CO3 (385 mg, 3.63 mmol, 1.6 eq) and (2-bromoethyl)benzene
(0.35 mL, 2.57 mmol, 1.2 eq), compound SAPE (10) was obtained as a pale yellow solid after
flash chromatography (0 - 20 % AcOEt/Hex), yield = 48 %, Mp = 95 - 96 °C, Rf = 0.26 (30
% AcOEt/Hex). 1H-NMR (DMSO-d6, 25 °C); δ (ppm): 8.98 (s large, 1H, OH), 7.54 (d, J =
15.85 Hz, 1H, CAr-CH=CH), 7.34 - 7.29 (m, 4H, HAr), 7.25-7.21 (m, 1H, HAr), 7.02 (s, 2H,
HAr), 6.50 (d, J = 15.85 Hz, 1H, CH=CH-C(=O)), 4.35 (t, J = 6.84 Hz, 2H, CH2O), 3.80 (s,
6H, OCH3), 2.97 (t, J = 6.84 Hz, 2H, CH2Ph); 13C-NMR (DMSO-d6, 25 °C); δ (ppm):
167.06, 148.49, 145.99, 138.85, 138.55, 129.30, 128.86, 126.84, 124.78, 115.18, 106.75,
64.75, 56.56, 34.94; HRMS m/z calc. for C19H20O5 + (H+): 329.1384; found: 329.1381.
2-Phenylethyl (2E)-3-(3,5-dimethoxyphenyl)prop-2-enoate (11)
Following our general procedure for the esterification of phenolic acids with 3,5-
dimethoxycinnamic acid (3h) (500 mg, 2.40 mmol, 1 eq), Na2CO3 (320 mg, 3.02 mmol, 1.3
eq) and (2-bromoethyl)benzene (0.37 mL, 2.72 mmol, 1.1 eq), compound 11 was obtained as
a white solid after flash chromatography (0 - 10 % AcOEt/Hex), yield = 72 %, Mp = 55 -
57°C, Rf = 0.25 (30 % AcOEt/Hex). 1H NMR (CDCl3, 25°C), δ (ppm)= 7.63-7.59 (d, 1H,
J=15.97 Hz, ArCH=CH-), 7.37-7.27 (m, 5H, Har), 6.69-6.68 (sd, 2H, J= 2.20 Hz, Har), 6.53-
6.52 (st, 1H, J= 2.20 Hz, Har), 6.44-6.40 (d, 1H, J= 15.97 Hz, ArCH=CH-), 4.47-4.44 (t, 2H,
J= 7.04 Hz, -COOCH2CH2Ar), 3.84 (s, 6H, 2 ArOCH3), 3.07-3.03 (t, 2H, J= 7.08 Hz, -
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COOCH2CH2Ar); 13C NMR (CDCl3, 25°C), δ (ppm)= 166.79161.03, 144.86, 137.87, 136.29,
128.93, 128.54, 126.59, 118.57, 105.99, 102.58, 65.04, 55.44, 35.21; HRMS m/z calc. for
C19H20O4 + (H+): 313.1434; found: 313.1427.
2-Phenylethyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate (12)
Following our general procedure for the esterification of phenolic acids with 3,4,5-
trimethoxycinnamic acid (3i) (500 mg, 2.40 mmol, 1 eq), Na2CO3 (320 mg, 3.02 mmol, 1.3
eq) and (2-bromoethyl)benzene (0.37 mL, 2.72 mmol, 1.1 eq), compound 12 was obtained as
a white solid after flash chromatography (0 - 10 % AcOEt/Hex), yield = 59 %, Mp = 98 -
100°C, Rf = 0.20 (30 % AcOEt/Hex). 1H-NMR (CDCl3, 25°C), δ (ppm)= 7.63-7.59 (d, 1H,
J= 15.93 Hz, ArCH=CH-), 7.37-7.25 (m, 5H, Har), 6.77 (s, 2H, Har), 6.38-6.34 (d, 1H, J=
15.89 Hz, ArCH=CH-), 4.48-4.44 (t, 2H, J= 7.08 Hz, -COOCH2CH2Ar), 3.91 (s, 6H,
ArOCH3 meta), 3.90 (s, 3H, ArOCH3 para), 3.07-3.03 (t, 2h, J= 7.04 Hz, -COOCH2CH2Ar);
13C-NMR (CDCl3, 25°C), δ (ppm)= 166.87, 153.45, 144.85, 140.16, 137.87, 129.90, 128.91,
128.54, 126.59, 117.28, 105.26, 64.98, 60.98, 56.18, 35.21. HRMS m/z calc. for C20H22O5 +
(H+): 343.1540; found: 343.1540.
2-Phenylethyl (2E)-3-(1,3-benzodioxol-5-yl)prop-2-enoate (14)
Following our general procedure for the esterification of phenolic acids with 3,4-
(methylenedioxy)cinnamic acid (13a) (500 mg, 2.60 mmol, 1 eq), Na2CO3 (338 mg, 3.19
mmol, 1.2 eq) and (2-bromoethyl)benzene (0.40 mL, 2.94 mmol, 1.1 eq), compound 14 was
obtained as a white powder after flash chromatography (0 - 10 % AcOEt/Hex), yield = 46 %,
Mp = 63 - 64 °C, Rf = 0.43 (30 % AcOEt/Hex). 1H-NMR (CDCl3, 25 °C); δ (ppm): 7.60 (d, J
= 15.93 Hz, 1H, CAr-CH=CH), 7.36 - 7.32 (m, 2H, HAr), 7.29 - 7.25 (m, 3H, HAr), 7.05 (d, J
= 1.6 Hz, 1H, HAr), 7.02 (dd, J = 8.00 Hz, 1.60 Hz, 1H, HAr), 6.83 (d, J = 8.00Hz, 1H, HAr),
6.27 (d, J = 15.89 Hz, 1H, CH=CH-C(=O)), 6.03 (s, 2H, OCH2O), 4.44 (t, J = 7.04 Hz, 2H,
13
OCH2CH2), 3.04 (t, J = 7.08 Hz, 2H, CH2Ph); C-NMR (CDCl3, 25 °C); δ (ppm): 167.09,
149.62, 148.35, 144.56, 137.94, 128.95, 128.85, 128.52, 126.57, 124.46, 115.99, 108.55,
106.25, 101.57, 64.94, 35.24; HRMS m/z calc. for C18H16O4 + (H+): 297.1121; found:
297.1118.
2-Phenylethyl (2E)-3-(naphthalen-2-yl)prop-2-enoate (15)
Following our general procedure for the esterification of phenolic acids with 3-naphthalen-2-
yl-acrylic acid (13b) (250 mg, 1.26 mmol, 1 eq), Na2CO3 (164 mg, 1.55 mmol, 1.2 eq), 6 mL
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of HMPA and (2-bromoethyl)benzene (0.19 mL, 1.42 mmol, 1.1 eq), compound 15 was
obtained as a white powder after flash chromatography (0 - 5 % AcOEt/Hex), yield = 70 %,
1
Mp = 77 - 78 °C, Rf = 0.38 (30 % AcOEt/Hex). H-NMR (CDCl3, 25 °C); δ (ppm): 7.95 -
7.85 (m, 5H, HAr, CAr-CH=CH), 7.69 (dd, J = 8.64 Hz, 1.08 Hz, 1H, HAr), 7.57 - 7.52 (m, 2H,
HAr), 7.39 - 7.27 (m, 5H, HAr), 6.57 (d, J = 16.01 Hz, 1H, CH=CH-C(=O)), 4.92 (t, J = 7.08
Hz, 2H, CH2O), 3.08 (t, J = 7.08 Hz, 2H, CH2Ph); 13C-NMR (CDCl3, 25 °C); δ (ppm):
166.98, 144.92, 137.94, 134.25, 133.3, 131.93, 129.96, 128.97, 128.70, 128.59, 128.56,
127.80, 127.26, 126.74, 126.61, 123.54, 118.22, 65.07, 35.26; HRMS m/z calc. for C21H18O2
+ (H+): 303.138; found: 303.1387.
2-Phenylethyl 3-(3,4-dihydroxyphenyl)propanoate (17)
Following our general procedure for the esterification of phenolic acids with hydrocaffeic
acid (16a) (300 mg, 1.65 mmol, 1 eq), Na2CO3 (211 mg, 1.99 mmol, 1.2 eq) and (2-
bromoethyl)benzene (0.25 mL, 1.84 mmol, 1.1 eq), compound 17 was obtained as a pale
brown powder after flash chromatography (0 - 15 % AcOEt/Hex), yield = 44 %, Mp = 70 -
71 °C, Rf = 0.32 (25 % AcOEt/Hex). 1H-NMR (CDCl3, 25 °C); δ (ppm): 7.34 - 7.21 (m, 5H,
HAr), 6.77 (d, J = 8.00 Hz, 1H, HAr), 6.66 (s, 1H, HAr), 6.59 (d, J = 8.00 Hz, 1H, HAr), 4.31 (t,
J = 7.00 Hz, 2H, CH2O), 2.94 (t, J = 6.96 Hz, 2H, CH2Ph), 2.82 (t, J = 7.52 Hz, 2H,
13
CH2CH2C(=O)), 2.59 (t, J = 7.68 Hz, CH2CH2C(=O)); C-NMR (CDCl3, 25 °C); δ (ppm):
173.49, 143.59, 142.08, 137.78, 133.25, 128.93, 128.52, 126.60, 120.57, 115.37, 65.16,
36.15, 35.04, 30.24; HRMS m/z calc. for C17H18O4 + (Na+): 309.1097; found: 309.1083.
2-Phenylethyl (3,4-dihydroxyphenyl)acetate (18)
Following our general procedure for the esterification of phenolic acids with (3,4-
dihydroxyphenyl)acetic acid (16b) (500 mg, 2.97 mmol, 1 eq), Na2CO3 (400 mg, 3.77 mmol,
1.3 eq) and (2-bromoethyl)benzene (0.46 mL, 3.36 mmol, 1.1 eq), compound 18 was
obtained as a brown oil after flash chromatography (0 - 20 % AcOEt/Hex), yield = 58 %, Rf
= 0.20 (30 % AcOEt/Hex). 1H-NMR (MeOD, 25 °C); δ (ppm): 7.27 - 7.14 (m, 5H, HAr), 6.72
- 6.70 (m, 2H, HAr), 6.54 (dd, J = 8.04 Hz, 1.76 Hz, 1H, HAr), 4.27 (t, J = 6.80 Hz, CH2O),
13
3.44 (s, 2H, CArCH2C(=O)), 2.89 (t, J = 6.76 Hz, 2H, CH2Ph); C-NMR (MeOD, 25 °C); δ
(ppm): 172.56, 144.91, 144.06, 137.91, 128.59, 128.04, 126.05, 125.51, 120.28, 116.04,
114.92, 65.13, 40.14, 34.60; HRMS m/z calc. for C16H16O4 + (Na+): 295.0941; found:
295.0931.
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2-Phenylethyl 3,4-dihydroxybenzoate (19)
Following our general procedure for the esterification of phenolic acids with 3,4-
dihydroxybenzoic acid (16c) (500 mg, 3.24 mmol, 1 eq), Na2CO3 (430 mg, 4.06 mmol, 1.3
eq) and (2-bromoethyl)benzene (0.50 mL, 3.67 mmol, 1.1 eq), compound 19 was obtained as
an off-white powder after flash chromatography (0 - 15 % AcOEt/Hex), yield = 39 %, Mp =
1
129 - 130 °C, Rf = 0.22 (30 % AcOEt/Hex). H-NMR (MeOD, 25 °C); δ (ppm): 7.42 - 7.39
(m, 2H, HAr), 7.31 - 7.30 (m, 4H, HAr), 7.25 - 7.19 (m, 1H, HAr), 6.80 (d, J = 8.12 Hz, 1H,
13
HAr), 4.47 - 4.43 (m, 2H, CH2O), 3.07 - 3.02 (m, 2H, CH2Ph); C-NMR (MeOD, 25 °C); δ
(ppm): 166.85, 150.32, 144.76, 138.14, 128.60, 128.11, 126.12, 122.22, 121.30, 116.00,
114.42, 65.01, 34.83; HRMS m/z calc. for C15H14O4 + (Na+): 281.0784; found: 281.0774.
2.2. 5-LO product biosynthesis assays in HEK293 cells
HEK293 cells were stably co-transfected with a pcDNA3.1 vector expressing 5-LO and a
pBUDCE4.1 vector expressing 5-LO activating protein (FLAP) as previously reported[16,32]
.
The resulting stable double transfectants were propagated in culture and aliquots were frozen.
Once thawed for a series of experiments, each aliquot of cells is cultured for a maximum of 6
weeks before being discarded. 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 compound or their
o
diluent at the indicated concentration for 5 min at 37 C. Cells were then stimulated for 15
o
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 and processed for RP-HPLC analysis as described previously
[16,33]. Data are expressed as means ± SEM of 3 independent experiments, each performed in
duplicate (Fig 2 and 3).
2.3. 5-LO products biosynthesis assays in human PMNL cells
Human PMNL prepared from peripheral blood of healthy consenting volunteers as
described[16] were suspended in HBSS containing 1.6 mM CaCl2 (107 cells/ml) and pre-
o
incubated with compounds or their diluent for 5 min at 37 C in the presence of 0.4 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)[16]. Reactions were stopped by the
addition of 0.5 volume of cold MeOH:CH3CN (1:1) and 50 ng of PGB2 as internal standard
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and samples were stored at -20oC until processing for RP-HPLC analysis as indicated above.
Data are expressed as means ± SEM of 3 independent experiments, each performed in
duplicate (Fig. 4).
2.4. 12-LO and COX-1 products biosynthesis assays
Platelets isolation was performed as previously described[34]. Isolated platelets obtained from
healthy consenting volunteers were pre-incubated (108 cells/ml) with indicated compounds or
their diluent at 37˚C for 5 min in the presence of 5 mM CaCl2. Stimulation was initiated with
the addition of 0.5 U/ml of thrombin (Sigma-Aldrich) followed by a 15 min incubation period
at 37˚C. Reactions were stopped by the addition of 3 volumes of cold MeOH:MeCN (1:1)
containing prostaglandin B2 (100ng/ml) as internal standard. Following overnight storage at -
20˚C, the samples were centrifuged, the supernatant was evaporated under gas nitrogen, and
resuspended in 30% methanol and analyzed by reverse-phased high-performance liquid
chromatography as described above.
2.5. Free radical scavenging activity assay
The free radical scavenging activity of test compounds was measured as previously described
using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a stable radical[12] 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
as 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 held 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). Ascorbic acid
was used as a positive control. Data are expressed as means ± SD of 2 independent
experiments, each performed in triplicate (table 2). IC50 values were calculated from a
sigmoidal concentration-response curve-fitting model with a variable slope on GraphPad
Prism 5 software (GraphPad Software, San Diego, California).
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2.6. Molecular Docking
Molecular docking was performed with AutoDock Vina 1.1.2 on 5-LO (PDB code: 3O8Y[35]
)
following the procedure[36] 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
[37]
Maestro 10.7
and with LigPlot+[38]. Readers who want to reproduce our docking results
can obtain the pdb file with all ligand and target atom coordinates. This file can be sent by
email on request to the corresponding author.
2.7. Statistical analysis
Statistical analyses and graph design were performed using GraphPad Prism 5 software
(GraphPad Software, San Diego, California).
3. RESULTS AND DISCUSSION
3.1. Chemistry
To prepare compounds 4-12, coumaric acids (ortho, meta and para), 3,4-dimethoxycinnamic
acid, 3,4-dichlorocinnamic acid, ferulic acid, sinapic acid, 3,5-dimethoxycinnamic acid and
3,4,5-trimethoxycinnamic acid were respectively esterified by reacting with phenethyl
bromide in the presence of sodium carbonate (Na2CO3) and potassium iodide (KI) as a
catalyst. Target compounds were thus obtained in a single step with moderate to high yields
(Scheme 1).
As outlined in Scheme 2, phenethyl esters 14 and 15 having respectively a methylenedioxy
1,3-benzodioxolyl and a naphthyl moiety in place of the catechol of CAPE (2) were prepared
by the same esterification method described in Scheme 1. The starting acids, namely 3,4-
(methylenedioxy)cinnamic acid (13a) and 3-naphthalen-2-yl-acrylic acid (13b), were
obtained by Knoevenagel condensation of malonic acid with piperonal or β-
naphthaldehyde[39]
.
As shown in Scheme 3, catechol-bearing phenethyl esters 17, 18, 19 in which one, two or
three carbons separate the carbonyl group from the catechol moiety were also synthesized.
With the same esterification method described in Scheme 1, commercially-available 3-(3,4-
dihydroxyphenyl)propanoic acid (dihydrocaffeic acid (16a)), 2-(3,4-dihydroxyphenyl)acetic
acid (16b) and 3,4-dihydroxybenzoic acid (16c) were converted to the corresponding
phenethyl esters 18, 19, 20 (Scheme 3).
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3.2. Biological activity
3.2.1. 5-LO products biosynthesis assays
HEK293 cells stably transfected with 5-LO and 5-LO activating protein (FLAP) were used to
screen compounds for 5-LO inhibition as previously described[33]. As shown in Figure 2,
para-, meta- and ortho-coumaric acid phenethylesters (4, 5, 6) had no inhibitory activity as
opposed to Zileuton (1) and CAPE (2), which highlights the importance of multiple
oxygenated positions on this aromatic ring (Figure 2).
Comparison of compound 17, a phenethyl ester of hydrocaffeic acid, with CAPE (2) shows
that the absence of the α,β-unsaturation in 17 does not seem to reduce the inhibitory activity.
Analog 17 was equivalent with CAPE (2) (Figure 2). The shortening of the linker between
the carbonyl and the catechol moiety does not appear to be crucial for 5-LO inhibition.
Analogs 18 and 19, having only one carbon or no carbon between the carbonyl and the
catechol, were almost equipotent with Zileuton (1) but less active than CAPE (2) (Figure 2).
Replacing the 3,4-hydroxyls of CAPE (2) by 3,4-methoxy (7), 3,4-dichloro (8), 3,4-
methylenedioxy (14), or phenyl (15) group was not favorable to a good inhibition of 5-LO
since these analogs have no inhibitory activity against the biosynthesis of 5-LO products.
Compound 9, bearing 3-methoxy-4-hydroxy moiety, obtained from the ferulic acid (3f), also
had no effect on 5-LO (Figure 3).
On the other hand, the sinapic acid phenethyl ester (10), which can be named SAPE by
analogy to CAPE (2), appears to be a good inhibitor of 5-LO with potency comparable to
CAPE (2). The OH in position 4 of SAPE (10) is crucial for the inhibition of 5-LO. As shown
in Figure 3, analogs 11 and 12 in which the hydroxyl at position 4 is replaced by hydrogen or
by a methoxy respectively, had no 5-LO inhibitory activity. The methoxy groups in position 3
and 5 are also crucial for inhibition of 5-LO since the analogs 9 and 4, which conserve the 4-
OH group but have either a single methoxy in position 3 or no methoxy (neither 3 nor 5) are
not active against 5-LO (Figure 2 and 3). Compounds that approached or surpassed the
inhibitory activity of CAPE (2) were selected for further screening in human PMNL for dose-
response assays to obtain IC50 values.
To further probe the inhibitory activity of lead compounds selected from preliminary
HEK293 cell screenings (Figures 2 and 3), 5-LO inhibition assays were undertaken in
stimulated human immune cells. 5-LO is highly expressed in polymorphonuclear leukocytes
(PMNL) and these cells are important physiological producers of LTB4[16]. The
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phenethylester of hydrocaffeic acid (17) was equipotent to slightly more active than Zileuton
(1) and equipotent with CAPE (2) (Figure 4). Analogs 18 and 19 did not significantly inhibit
the biosynthesis of 5-LO products in these cells (Figure 4). The number of carbons between
the carbonyl and the catechol appears to be important for the inhibition of 5-LO in PMNL
cells. As shown in Figure 4, whenever the carbon number decreases activity decreases.
Compounds 18 and 19 having one and no carbon, respectively, were less active than 17,
which had two carbons (Figure 4).
Among the selected molecules tested for 5-LO inhibition in PMNL, SAPE (10) was found to
be the best inhibitor exceeding the inhibitory capacity of both zileuton (1) and CAPE (2) in
this model. The presence of the two methoxy groups in position 3 and 5 and the OH in
position 4 seems to be an ideal combination for inhibition of 5-LO in PMNL cells. These
structural changes may influence the availability, cell permeation or even the stability of
inhibitors in PMNL versus HEK293 cells. The steric hindrance created by the presence of
both methoxy may have an effect on the stability of the SAPE (10) by sterically shielding the
phenolic hydroxyl group, possibly rendering it less sensitive to sulfatation and
glucuronidation than a catechol such as CAPE (2).
The superiority of the inhibitory activity of SAPE (10) was confirmed in dose response
experiments. Selected compounds with inhibition of at least 50% at 1 µM in PMNL were
screened further through concentration-response assays in PMNL (Table 1).
Table 1. 5-LO inhibition in human PMNL cells.
IC50 (μM) [SEM]
3.1 [0.2]
Compounds
Zileuton (1)
CAPE (2)
17
1.01 [0.03]
1.01 [0.05]
0.3 [0.01]
SAPE (10)
SAPE (10) inhibited 5-LO in a dose-dependent manner and outperformed both Zileuton (1)
and CAPE (2). With an IC50 of 0.3 µM, this phenethylester of sinnapic acid was 3-fold more
potent than CAPE (2) and 10-fold more potent than zileuton, the only approved 5-LO
inhibitor. Compound 17, hydrocaffeic acid phenethylester, was equipotent with CAPE (2)
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and was three times more active than Zileuton (1) (IC50 = 3.1 μM) (Table 1). SAPE (10),
which was previously synthesized along with a few other analogues by a different
esterification strategy by Shi et al., has almost no cytoprotective and neuritogenic activities[40]
but was never tested for its inhibition of 5-LO, 12-LO or COX-1.
3.2.2. 12-LO and COX-1 products biosynthesis assays
Human platelets were used for the evaluation of products synthesized by the 12-LO and
COX-1 pathways. As shown in Figure 5, CAPE (2) inhibits the biosynthesis of both 12-LO
and COX-1 products. Including 5-LO, these results indicate that CAPE (2) is an inhibitor of
the biosynthesis of products from all three pathways. In contrast, SAPE (10) had no effect on
12-LO and less effect on COX-1 indicating that this compound is a more selective 5-LO
inhibitor. Therefore, the presence of the 3,5-dimethoxy and the 4-hydroxy moieties makes the
phenethyl ester of sinapic acid completely inactive against 12-LO inhibition at this
concentration. It is interesting to note that CAPE (2) surpasses the inhibitory capacity of
Baicalein which is the standard commonly used as a 12-LO inhibitor. Concentration-response
assays in human platelets demonstrated that CAPE (2) (IC50 = 0.07 μM) is 20-fold more
active for 12-LO inhibition than Baicalein (IC50 = 1.52 μM).
3.3. Free radical scavenging activity assay
The free radical scavenging activity of our compounds was compared against a known
antioxidant, the hydrosoluble vitamin C or ascorbic acid. The results from the DPPH assay
show that all the tested compounds in this study have a higher free scavenging radical activity
than vitamin C (Table 2). This potentially make these compounds good candidates as lead
anti-inflammatory molecules solely from their increased free radical scavenging activity.
We previously demonstrated that the anti-radical effect of the esters of certain phenolic acid
esters is not the only factor that explains their inhibitory activity against 5-LO[16,18,33]
.
Similarly, the free radical scavenger activity of the best 5-LO inhibitors in HEK293 cells
(Figure 2 and 3) exhibit similar radical scavenging activity to CAPE (2) (Table 2). Analogs
17, 18 and 19 bear the important catechol group and only differ from CAPE (2) by the extent
of the carboxylate linker and the absence of the ethylene moiety, but only 17 shows
equivalent activity against 5-LO to that of CAPE (2). This indicates that the linker and not
differences in radical scavenging capacity is what distinguished their 5-LO inhibitory
activities.
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Table 2. Free radical scavenging activity
IC50 (μM) [SEM]
16.5 [4.0]
Compounds
CAPE (2)
17
24.6 [4.8]
18.0 [3.6]
18
20.4 [2.9]
19
SAPE (10)
Sinapic acid (3g)
Ascorbic acid
25.4 [0.7]
21.9 [2.0]
68.7 [1.6]
SAPE (10), which was the best inhibitor of 5-LO in HEK293 and PMNL cells, also had
antiradical activity that was similar to that of CAPE (2). Therefore, esterification of sinapic
acid (3g) does not seem to induce any dramatic change in radical scavenging activity. SAPE
(10) has essentially the same anti-radical activity as its corresponding acid, which is
completely inactive against 5-LO.
3.4. Molecular Docking
Molecular docking of CAPE (2) and the ether substituted compounds SAPE (10), 11 and 12
was performed using the modified 5-LO protein (PDB ID: 3O8Y). CAPE (2) and SAPE (10)
are almost superimposed, root-mean-square deviation (RMSD) = 0.4158 for heavy and
similarly placed atoms, with the substituted benzene at the end of the hydrophobic cavity near
Ala424 (Figure 6). The length of the molecules follows the cavity until the unsubstituted
benzene approaches the iron atom. For both CAPE (2) and SAPE (10), the closest atom to Fe
is at a distance of 6.6 Å. The two ether substituted compounds with no measurable 5-LO
inhibition, 11 and 12, are partially superimposed, RMSD = 1.1928. Both benzene rings have
the same "center" while the connecting chains take differing paths resulting in the ester
oxygens pointing in opposite directions. Both molecules are the closest to the iron atom with
11, 4.11 Å, being slightly further away than 12, 3.88 Å.
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The ether substitutions of SAPE (10), 11 and 12 occupy different orientations usually
depending on their placement in the meta or para positions. For the strong 5-LO inhibitor
SAPE (10), both ethers that are in a meta position point in the same direction (towards
Tyr181 and Trp599 for the first meta ether and towards Val604 for the second meta ether).
The same is true for 12, both ethers in the meta position point in the same direction as their
counterparts in SAPE (10), however the additional ether in para points in the opposite
direction (towards Ala424). For 11 with only 2 substitutions which are the ethers in the meta
position, it positions itself so that both point in opposite directions, one is pointing towards
Tyr181 and Trp599 while the second one is pointing towards Ala424. For 11 to have both
ethers in opposite direction, it must rotate the substituted benzene differently compared to the
other molecules.
In the tested molecules, the only detected hydrogen bond using LigPlot+ is between CAPE (2)
and Leu420 (OH∙∙∙O, d = 3.09 Å) (Table 3). Leu420 helps define the active site cavity[35]
.
Maestro detects several π-π interactions (Table 3). For the tested molecules, a minimum of
one π-π interaction is present. His372 seems to be the most important, being present in four
out of the five tested molecules (including Zileuton) and in the two most stable molecules.
The residue His372 is an iron coordinating residue[35]
.
CAPE (2) and SAPE (10) are the most stable molecules tested, with their respective affinity
of - 8.8 kcal/mol and - 8.7 kcal/mol, while 12 is the least stable molecule, - 8.1 kcal/mol. It
appears that the molecules that have a hydroxyl substitution have more affinity for the active
site than the molecules with solely ether substitutions. Overall, these molecular docking
studies indicate that the molecules that are strong inhibitors of 5-LO show different
positioning and affinities for 5-LO than the poorly active ether-substituted compounds that
lack a hydroxyl substituent.
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Table 3. Molecular modeling results for modeling affinity, π-π interactions and hydrogen
bonds.
Affinity
(kcal/mol)
Compounds
CAPE (2)
Maestro π-π Interactions
His372
LigPlot+ H-Bond
-8.8
Leu420
SAPE (10)
-8.7
His372
-
-
-8.5
Phe177, His367
Phe177, His367, His372,
Phe421
11
-
-8.1
-6.6
12
(R)-Zileuton
Phe421
Leu420 x 2, Asn425
Leu420 x 2, Ala424,
Phe421
(S)-Zileuton
-6.5
-
4. CONCLUSIONS
Fourteen phenolic acid phenethyl esters were synthesized and evaluated for their 5-LO
inhibition activities. Compared to zileuton (1) and CAPE (2), sinapic acid phenethyl ester
(SAPE, 10), showed high potency in HEK293 cell and in PMNL (IC50 = 0.3 µM) assays.
Even with a lower antiradical potency than CAPE (2), SAPE (10) was found to be the best 5-
LO inhibitor. SAPE (10) is more selective to 5-LO since, unlike CAPE (2), it is inactive
against 12-LO and less active against COX-1. The presence of the two methoxy in position 3
and 5 and the OH in position 4 in SAPE (10) seems to be an ideal configuration for inhibition
of only 5-LO. Although a large number of investigations have been carried out on the study
of the biological effects of CAPE (2), one of the main constituents of honeybee propolis,
reports about the activity of SAPE (10) are scarce. Sinapic acid (3g) is found in many edible
[41]
plants and fruits
such as rapeseed where it is the predominant phenolic compound [42]. In
addition, anti-inflammatory activity of sinapic acid was reported and attributed the
suppression of inducible nitric oxide synthase, COX-2, and proinflammatory cytokines
expressions via nuclear factor-κB inactivation [43]. Overall, this first study on the anti-
inflammatory properties of SAPE (10) suggests that additional studies are warranted to better
understand the biological effects of this compound in comparison to those of CAPE (2).
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ACKNOWLEDGMENTS
M.T. 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. M.E.S. would
like to thank the NBIF Innovation Research Chairs program. L.H.B. would like to
acknowledge the Canadian Institutes of Health Research (CIHR), the NBIF and the
Université de Moncton.
ETHICAL APPROVAL
Blood was obtained from healthy consenting volunteers. This research project was approved
by the Comité d’éthique de la recherche avec les êtres humains at the Université de Moncton.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Figures Legends
Figure 1. Structure of Zileuton (1) and CAPE (2)
Scheme 1. Synthesis of compounds 4-12. Reagents and conditions: i) Na2CO3, PhCH2CH2Br, KI,
HMPA, 0oC to RT, 12h.
Scheme 2. Synthesis of compounds 14 and 15. Reagents and conditions: i) Na2CO3, PhCH2CH2Br,
KI, HMPA, 0oC to RT, 12h.
Scheme 3. Synthesis of compounds 17-19. Reagents and conditions: i) Na2CO3, PhCH2CH2Br, KI,
HMPA, 0oC to RT, 12h.
Figure 2. Inhibition of 5-LO product biosynthesis by synthetic CAPE (2) analogs (1 M) in
stimulated HEK293 cells. Data are expressed as means ± SEM of at least 3 independent experiments.
Values without one common superscript are significantly different determined by one-way ANOVA
with Tukey’s multiple comparison test (p < 0.05).
Figure 3. Inhibition of 5-LO products synthesis by synthetic CAPE (2) analogs (1 M) in stimulated
HEK293 cells. Data are expressed as means ± SEM of at least 3 independent experiments. Values
without one common superscript are significantly different determined by one-way ANOVA with
Tukey’s multiple comparison test (p < 0.05).
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Figure 4. Inhibition of 5-LO products synthesis by synthetic CAPE (2) analogs (1 M) in human
PMNL cells.
Data are expressed as means ± SEM of 3 independent experiments. Values without one common
superscript are significantly different determined by one-way ANOVA with Tukey’s multiple
comparison test (p < 0.05).
Figure 5. Inhibition of 12-LO and COX-1 product synthesis by indicated compounds (1 M) in
human platelets. Data are expressed as means ± SEM of at least 3 independent experiments. Values
without one common superscript are significantly different determined by one-way ANOVA with
Tukey’s multiple comparison test (p < 0.05).
Figure 6. Visual representation of SAPE (10) docked with 5-LO in 3D (left, Maestro) and 2D (right,
LigPlot+).
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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.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.