Synthesis, inhibitory activity and in silico docking of dual COX/5-LOX inhibitors with quinone and resorcinol core

Article abstract of DOI:10.1016/j.ejmech.2020.112620

Based on the significant anti-inflammatory activity of natural quinone primin (5a), series of 1,4-benzoquinones, hydroquinones, and related resorcinols were designed, synthesized, characterized and tested for their ability to inhibit the activity of cyclooxygenase (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) enzymes. Structural modifications resulted in the identification of two compounds 5b (2-methoxy-6-undecyl-1,4-benzoquinone) and 6b (2-methoxy-6-undecyl-1,4-hydroquinone) as potent dual COX/5-LOX inhibitors. The IC₅₀ values evaluated in vitro using enzymatic assay were for compound 5b IC₅₀ = 1.07, 0.57, and 0.34 μM and for compound 6b IC₅₀ = 1.07, 0.55, and 0.28 μM for COX-1, COX-2, and 5-LOX enzyme, respectively. In addition, compound 6d was identified as the most potent 5-LOX inhibitor (IC₅₀ = 0.14 μM; reference inhibitor zileuton IC₅₀ = 0.66 μM) from the tested compounds while its inhibitory potential against COX enzymes (IC₅₀ = 2.65 and 2.71 μM for COX-1 and COX-2, respectively) was comparable with the reference inhibitor ibuprofen (IC₅₀ = 4.50 and 2.46 μM, respectively). The most important structural modification leading to increased inhibitory activity towards both COXs and 5-LOX was the elongation of alkyl chain in position 6 from 5 to 11 carbons. Moreover, the monoacetylation in ortho position of bromo-hydroquinone 13 led to the discovery of potent (IC₅₀ = 0.17 μM) 5-LOX inhibitor 17 (2-bromo-6-methoxy-1,4-benzoquinone) while bromination stabilized the hydroquinone form. Docking analysis revealed the interaction of compounds with Tyr355 and Arg120 in the catalytic site of COX enzymes, while the hydrophobic parts of the molecules filled the hydrophobic substrate channel leading up to Tyr385. In the allosteric catalytic site of 5-LOX, compounds bound to Tyr142 and formed aromatic interactions with Arg138. Taken together, we identified optimal alkyl chain length for dual COX/5-LOX inhibition and investigated other structural modifications influencing COX and 5-LOX inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2020.112620

Journal Pre-proof
Synthesis, inhibitory activity and in silico docking of dual COX/5-LOX inhibitors with
quinone and resorcinol core
Miroslav Sisa, Marcela Dvorakova, Veronika Temml, Veronika Jarosova, Tomas
Vanek, Premysl Landa
PII:
S0223-5234(20)30592-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112620
EJMECH 112620
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 April 2020
Revised Date: 22 June 2020
Accepted Date: 22 June 2020
Please cite this article as: M. Sisa, M. Dvorakova, V. Temml, V. Jarosova, T. Vanek, P.
Landa, Synthesis, inhibitory activity and in silico docking of dual COX/5-LOX inhibitors with
quinone and resorcinol core, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112620.
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Synthesis, inhibitory activity and in silico docking of dual COX/5-LOX
inhibitors with quinone and resorcinol core
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Miroslav Sisaa, Marcela Dvorakovaa, Veronika Temmlb, Veronika Jarosovaa,c, Tomas Vaneka,
Premysl Landaa,*
a
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Laboratory of Plant Biotechnologies, Institute of Experimental Botany of the Czech Academy of Sciences,
Rozvojova 263, 165 02 Prague 6 - Lysolaje, Czech Republic
b
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Department of Pharmacy/Pharmacognosy and Center of Molecular Biosciences (CMBI), University of
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Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria.
c Department of Food Science, Faculty of Agrobiology, Food and Natural Resources, The Czech University of Life
Sciences
Prague,
Kamycka
129,
165
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Prague
6
-
Suchdol,
Czech
Republic
* Corresponding author. Laboratory of Plant Biotechnologies, Institute of Experimental Botany of the Czech
Academy of Sciences, Rozvojova 263, 165 02 Prague 6 - Lysolaje, Czech Republic
E-mail address: landa@ueb.cas.cz (P. Landa)
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Abstract
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Based on the significant anti-inflammatory activity of natural quinone primin (5a), series of 1,4-
benzoquinones, hydroquinones, and related resorcinols were designed, synthesized, characterized
and tested for their ability to inhibit the activity of cyclooxygenase (COX-1 and COX-2) and 5-
lipoxygenase (5-LOX) enzymes. Structural modifications resulted in the identification of two
compounds 5b (2-methoxy-6-undecyl-1,4-benzoquinone) and 6b (2-methoxy-6-undecyl-1,4-
hydroquinone) as potent dual COX/5-LOX inhibitors. The IC50 values evaluated in vitro using
enzymatic assay were for compound 5b IC50 = 1.07, 0.57, and 0.34 µM and for compound 6b IC50
=
1.07, 0.55, and 0.28 µM for COX-1, COX-2, and 5-LOX enzyme, respectively. In addition, compound
6d was identified as the most potent 5-LOX inhibitor (IC50 = 0.14 µM; reference inhibitor zileuton IC50
= 0.66 µM) from the tested compounds while its inhibitory potential against COX enzymes (IC50
=
2.65 and 2.71 µM for COX-1 and COX-2, respectively) was comparable with the reference inhibitor
ibuprofen (IC50 = 4.50 and 2.46 µM, respectively). The most important structural modification
leading to increased inhibitory activity towards both COXs and 5-LOX was the elongation of alkyl
chain in position 6 from 5 to 11 carbons. Moreover, the monoacetylation in ortho position of bromo-
hydroquinone 13 led to the discovery of potent (IC50 = 0.17 µM) 5-LOX inhibitor 17 (2-bromo-6-
methoxy-1,4-benzoquinone) while bromination stabilized the hydroquinone form. Docking analysis
revealed the interaction of compounds with Tyr355 and Arg120 in the catalytic site of COX enzymes,
while the hydrophobic parts of the molecules filled the hydrophobic substrate channel leading up to
Tyr385. In the allosteric catalytic site of 5-LOX, compounds bound to Tyr142 and formed aromatic
interactions with Arg138. Taken together, we identified optimal alkyl chain length for dual COX/5-
LOX inhibition and investigated other structural modifications influencing COX and 5-LOX inhibitory
activity.
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Keywords: quinones; resorcinols; anti-inflammatory activity; cyclooxygenase; 5-lipoxygenase
1. Introduction
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Non-steroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of common pain as
well as for chronic inflammatory diseases such as rheumatoid arthritis. Some of them are over-the-
counter drugs and belong to the most widely used pharmaceuticals worldwide. NSAIDs reduce the
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production of prostaglandins via the inhibition of cyclooxygenase (COX) enzymes. However, chronic
intake of non-selective COX inhibitors could result in severe side effects such as gastrointestinal
bleeding [1,2]. Besides the inhibition of inducible COX-2 isoform responsible for inflammatory
processes, non-selective inhibitors also suppress the activity of COX-1 that plays cytoprotective role
in gastrointestinal system. Actually, the role of COX-1 (generally considered as homeostatic) and
COX-2 (generally considered as inducible) is complex and depends on many factors such as organ
where it actions, developmental stage, stage of inflammatory process etc. The application of
selective COX-2 inhibitors (called Coxibs) avoided gastrointestinal complications but their usage
resulted in the increased risk of cardiovascular events [3]. One of the reasons is that selective COX-2
inhibitors change homeostasis in the production of vasoactive prostanoids. Coxibs do not affect the
production of thromboxane X2 (TXA2) promoting vasoconstriction and platelet aggregation but
reduce biosynthesis of prostaglandin I2 (PGI2) that is vasodilator and potent inhibitor of platelet
aggregation. Moreover, COX-2 mediates cardioprotective effects of the late phase of ischemic
preconditioning and may also play role in angiogenesis [4]. Enzyme 5-lipoxygenase (5-LOX) catalyzes
biosynthesis of leukotrienes (LTs) and lipoxins. Leukotriene B4 (LTB4) is important chemotactic and
chemokinetic mediator regulating the immune response and inflammatory processes such as
arthritis, asthma, and atherosclerosis. The cysteinyl leukotrienes are involved in asthma, allergic
rhinitis, and chronic inflammation [5]. Until now, zileuton is only one approved 5-LOX inhibitor for
asthma treatment. Moreover, zileuton is associated with adverse effects such as hepatotoxicity and
adverse pharmacokinetic profile derived from a short half-life. The inhibition of COXs leads to the
increased production of LTs, which could increase asthma. The reason is that both COXs and 5-LOX
use as a substrate arachidonic acid (AA) and when the activity of COXs is inhibited AA availability is
increased for 5-LOX pathway. Another reason is that the production of vasodilators PGI2 and PGE2 is
reduced. Therefore, dual inhibition of COXs and 5-LOX is suggested as a promising approach that
should diminish negative effects connected with the inhibition of COXs. Moreover, dual COX/5-LOX
inhibition may exhibit better anti-inflammatory activity than NSAIDs because of the influence on
inflammatory mediators not affected by COX inhibitors [1]. Previously, we found that some natural
benzo- and naphthoquinones are able to inhibit COX as well as 5-LOX catalytic activity in vitro. As the
most promising compound was identified benzoquinone primin [6,7]. Benzoquinones were found as
potent 5-LOX inhibitors also in other studies. Hydroquinone miconidin acetate isolated from Eugenia
hiemalis inhibited human recombinant 5-LOX in vitro [8]. Natural benzoquinone embelin was
revealed as potent 5-LOX and microsomal prostaglandin E2 synthase-1 inhibitor [9]. Tests of series of
embelin derivatives revealed compounds such as 4,5-dimethoxy-3-dodecyl-1,2-benzoquinone and 5-
[(2-naphthyl)methyl]-2-hydroxy-2,5-cyclohexadiene-1,4-dione with significantly higher 5-LOX
inhibitory activity than that of the parent compound [10,11]. Further, Peduto et al. optimized alkyl
chain pattern of quinones and corresponding hydroquinones that provided the identification of
highly potent 5-LOX inhibitors with in vivo anti-inflammatory effect [12]. In our study, we used
different synthetic approach and prepared series of derivatives of natural quinone, primin. Obtained
compounds were assayed for their ability to inhibit COX-1, COX-2 and 5-LOX catalytic activity as
potential dual inhibitors. In addition, in silico docking experiments were employed to elucidate the
binding of compounds into catalytic sites of COX as well as 5-LOX enzymes.
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2. Results and discussion
2.1 Chemistry
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For the first set of compounds – benzoquinones, hydroquinones and resorcinols – we have
employed synthetic strategy (Scheme 1) developed in our group [13] using Sonogashira cross-
coupling as the key step for the aromatic alkylation, thus affording excellent yields of desired
compounds 2a-d (60-97%). Different length of side chain was proposed with the aim to investigate
its role in inhibitory activity. Hydrogenation on palladium gave quantitative yields of products 3a-d,
which subsequently underwent either demethylation with boron tribromide giving resorcinols 4a, c,
d (82 to 92%), or chromium oxide (VI) oxidation to obtain benzoquinones 5a-d (77 to 90%).
Subsequent reduction of 5a-d with sodium dithionite yielded hydroquinones 6a-d (71 to 81%).
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Scheme 1. Synthesis of resorcinols 4a, c, d, benzoquinones 5a-d and hydroquinones 6a-d
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In order to study the influence of deoxygenation of hydroquinones in position 1 or 4 on the
inhibitory activity, compounds 10 and 11 were also synthesized (Scheme 2). Ortho-bromination of
guaiacol with bromine in the presence of tert-butyl amine gave product 8 in 93% yield. The hydroxyl
group was then protected as methoxymethylether giving compound 9, which was next submitted to
alkylation giving 2-methoxy-6-pentylphenol 10 in 87% yield. In this case, for the alkylation step, was
employed the shorter synthetic method using Suzuki cross-coupling with [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) as the catalyst and pentyl boronate as the
source of the alkyl chain. The deoxygenated hydroquinone 11, 3-methoxy-5-pentylphenol, was
obtained by partial demethylation of compound 3a with boron tribromide in 88% yield.
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Scheme 2. Synthesis of deoxygenated derivatives 10 and 11
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In an effort to replace the aliphatic lipophilic chain with hydrophilic function to improve the
solubility, we prepared bromo derivatives of previously synthesized benzoquinones 5 and
hydroquinones 6 (Scheme 3). The synthesis of bromo-hydroquinone 13 started from 5-bromovanillin
12, which was submitted to Dakin oxidation yielding the product in 78% yield. Next, iron trichloride
oxidation afforded bromo-benzoquinone 14 in 66% yield. An alternative synthetic route to obtain
compound 6a (miconidin) was employed using Suzuki cross-coupling reaction of protected
intermediate 15.
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Scheme 3. Synthesis of bromo derivatives 13 and 14, and an alternative synthesis of miconidin 6a
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In order to increase stability of quinone derivatives and also potentially increase their 5-LOX
inhibitory activity based on previously reported results [8], acetylated hydroquinones (16a, c) were
designed and prepared (Scheme 4). Standard acetylation protocol using acetic anhydride in pyridine
was used. When these conditions were employed for the acetylation of bromo derivative 13 only
monoacetate 17 was obtained. This regioselectivity might be due to strong effect of bromine
activating for acetylation ortho-hydroxyl group and deactivating meta-hydroxyl group.
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Scheme 4. Synthesis of acetylated derivatives 16a, 16c and 17
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Finally, imino derivative of primin, compound 23, was synthesized (Scheme 5). The structure 23
was inspired by docking experiments. Iodination of carbamate 18 gave iodide 19 that underwent
Sonogashira cross-coupling resulting in compound 20. Direct hydrogenation of this intermediate
failed, thus, Boc-deprotection had to be carried out first. Reduction of triple bond was at this stage
successful and afforded aniline 22 quantitatively. The favored cyclization of amine with triple bond in
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intermediate 21 to indol structure was eliminated using this intermediate immediately in the next
step without purification. The endeavor to obtain first the hydroquinone form by the oxidation of
aniline 22 with potassium persulfate was not successful even under various reaction conditions.
Therefore, direct oxidation using Co(Salen) as a catalyst in the presence of tert-butyl hydroperoxide
was employed instead leading to quantitative conversion to the desired imine 23.
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Scheme 5. Synthesis of imino derivative 23
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2.2 In vitro COX and 5-LOX assay
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From all the synthesized compounds, the best dual inhibitory activity towards COX-2 and 5-LOX
enzymes possessed compound 5b (IC50 for COX-2 = 0.57 µM, IC50 for 5-LOX = 0.34 µM) and
compound 6b (IC50 for COX-2 = 0.55 µM, IC50 for 5-LOX = 0.28 µM) (Table 1). Both these compounds
showed better activity than reference inhibitors (S)-(+)-ibuprofen (IC50 for COX-2 = 2.46 µM) and
zileuton (IC50 for 5-LOX = 0.66 µM). Both compounds also exerted mild preference towards COX-2
enzyme, with selectivity index (COX-2/COX-1) of 0.53 for compound 5b and 0.51 for compound 6b.
On the other hand, compound 6d exhibited the best inhibitory activity towards 5-LOX enzyme (IC50 =
0.14 µM) and inhibited COX enzymes to the same extent as ibuprofen. Compound 5c was strong
inhibitor of COX-1 and COX-2 while its IC50 for 5-LOX (1.05 µM) was higher than that of zileuton.
Furthermore, compounds 5a (primin), 6c, 10, 13, and 14 also showed promising activity in dual
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COXs/5-LOX inhibition. Several compounds effectively inhibited catalytic activity of 5-LOX while their
inhibitory activity towards COXs enzymes was low (5d and 23) or none (IC50 > 20 µM; compounds 6a
[miconidin], 16a, 16c, 17). In case of compound 17 the inhibitory activity towards 5-LOX was very
significant (IC50 = 0.17 µM) when compared to zileuton. Our results together with the data reported
by other authors [9-12] demonstrate that specific benzoquinones are potent COX and/or 5-LOX
inhibitors. To investigate whether prepared compounds compete with substrate in binding to the
active site of enzyme, 5b was selected as a representative compound and tested in COX-1 and 5-LOX
assays with different concentrations of AA. Inhibitory potential of compound 5b towards COX-1 was
reduced when the concentration of AA increased in the reaction. IC50 values increased from 0.80 µM
in the presence of 50 nM AA, through 2.77 µM in the presence of 250 nM AA to more than 20 µM in
the presence of 1250 nM AA. It indicates that compound 5b competed with AA in binding to COX-1.
Ibuprofen, which is known competitive COX-1 and COX-2 inhibitor [14], showed similar tendency as
its IC50 values increased from 4.96 µM when using 50 nM AA to 16.15 µM in the presence of 250 nM
AA and more than 20 µM when using 1250 nM AA. In case of 5-LOX the effect of increasing AA
concentration (final concentrations in the reaction were 5, 20, and 80 µM) on the activity of 5b was
not as evident because IC50 were in the range of 0.71 - 1.69 µM. Similarly, Schaible et al. observed
that inhibitory activity of embelin (benzoquinone with the same aliphatic 11 carbon chain as
compound 5b) towards 5-LOX was also only minutely influenced with increasing AA level in the
reaction [9]. Since inhibitory activity towards 5-LOX is often connected with antioxidant activity of
compounds [5], radical scavenging activity was investigated using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) assay. Compound 5b was not able to reduce DPPH radical at 400 µM concentration while
EC50 of Trolox (water-soluble analog of vitamin E) was 32.48 ± 8.49 µM and EC50 of L-ascorbic acid
was 31.14 ± 2.47 µM. It demonstrates that antioxidant potential of compound 5b was weak. On the
other hand, compound 17 (potent 5-LOX inhibitor with one hydroxyl group) showed only slightly
weaker radical scavenging activity (EC50 = 45.61 ± 1.00 µM) than both the reference compounds.
Hydroquinones were found in most cases more potent 5-LOX inhibitors than their quinone
counterparts in our study which could arise from their redox potential.
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2.3 Structure activity relationship (SAR)
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We decided to investigate several modifications of parent molecule primin (5a) to elucidate the
effect of structural motifs on the inhibitory activity towards COXs and 5-LOX. The elongation of
aliphatic chain was based on the known 5-LOX inhibitory activity of embelin [9], quinone compound
with long side chain. Moreover, such compounds with a long chain mimic the structure of
arachidonic acid. Preparation of imine derivative was suggested by in silico docking simulations as a
potential way to improve the inhibitory activity. Bromo derivatives were prepared with the aim to
improve solubility and stability of compounds. Reduction to hydroquinone form as well as
acetylation was based on the previous experience with promising 5-LOX inhibitor miconidin
monoacetate [8]. Finally, resorcinol derivatives were investigated to reveal the role of hydroxyl
groups on the inhibitory activity.
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Firstly, the elongation of primin (5a) aliphatic side chain was carried out. The selection of the
chain length was inspired by the activity of quinone embelin, a potent 5-LOX inhibitor, with n-
undecanyl chain [9]. Therefore, the elongation from original 5 to 11 carbons was chosen as most
promising for the bioactivity improvement. In addition, derivatives with 13 and 15 carbon chains
were prepared to test the effect of the chain length on the inhibitory activity. Bioactivity tests
demonstrated that especially the chain elongation to 11 carbons (5b) had positive effect on activity
and thus the inhibitory activity towards all three enzymes increased. Similarly, when the aliphatic
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side chain of miconidin 6a (reduced form of primin) with no activity towards COXs, was elongated to
11 carbons (6b), its ability to inhibit COX enzymes was restored. The length of aliphatic chain seemed
optimal with 11 carbons because further elongation to 13 (compounds 5c and 6c) or 15 carbons
(compounds 5d and 6d) resulted in lower inhibitory activity against COXs and 5-LOX. The only
exception was compound 6d which exerted superb inhibitory activity towards 5-LOX.
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While the elongation of aliphatic chain led to the higher activity, the primin´s imino derivative 23,
inspired by in silico modelling, gave weaker bioactivity results than primin (5a). Moreover, the
compound was unstable and underwent hydrolysis to primin.
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Bromo derivatives 13 and 14 were prepared in order to improve solubility of tested compounds.
Their advantage is simple preparation and the stabilization of the hydroquinone form by halide in
comparison with derivatives with aliphatic chain that undergo oxidization to its benzoquinone form
when exposed to the air. However, these bromo derivatives exhibited only moderate inhibitory
activity towards COXs and 5-LOX.
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The acetylation of both hydroxyl groups of hydroquinones 6a (miconidin) and 6c resulting in
diacetates 16a and 16c did not improved the inhibitory activity towards 5-LOX. However, acetylation
of one hydroxyl group of compound 13 at position 1 on the ring led to increased activity of resulting
compound 17 towards 5-LOX (IC50 decreased from 1.14 to 0.17 µM). Acetylation always led to a loss
of COX inhibitory activity.
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Prepared resorcinols (4a, c, d) showed weaker inhibitory activity towards COXs than their quinone
counterparts. This activity was again much weaker for compound 4a with 5 carbon aliphatic chain,
while compounds 4c and 4d (natural compounds grevillol and cardol, respectively) with longer
aliphatic chain inhibited COXs to similar extent as ibuprofen. It demonstrates the positive role of
aliphatic chain elongation on the bioactivity. When compared to benzoquinone or hydroquinone
structures, all resorcinols lost the ability to inhibit 5-LOX enzyme. Therefore, the presence of two
hydroxyl groups in opposite positions seems indispensable for 5-LOX inhibition.
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Table 1. IC50 values for COX-1, COX-2 and 5-LOX.
Selectivity
IC50 COX-1 ± SD IC50 COX-2 ± SD
index (COX- IC50 5-LOX ± SD
Compound
(µM)
13.63±4.09
(µM)
1/2)
0.79
0.87
2.38
9.96
1.88
0.28
(µM)
not active*
4a
17.26±0.08
2.74±0.80
2.36±1.16
1.90±0.93
0.57±0.49
0.88±0.51
8.97±3.37
not active
0.55±0.19
1.55±0.21
2.71±1.34
4.65±1.15
15.92±0.87
4c (Grevillol)
2.38±1.11
5.61±2.89
18.92±4.66
1.07±0.74
0.25±0.15
not active
not active
1.07±0.48
3.38±1.09
2.65±0.84
5.42±2.53
not active
not active
not active
1.37±0.63
0.34±0.13
1.05±0.48
0.79±0.28
0.94±0.65
0.28±0.20
1.49±0.64
0.14±0.02
3.72±1.67
not active
4d (Cardol)
5a (Primin)
5b
5c
5d
6a (Miconidin)
6b
6c
6d
10
11
1.95
2.18
0.98
1.17
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13
6.11±2.67
15.55±7.02
not active
not active
not active
15.78±0.61
4.50±1.75
not tested
4.81±2.11
4.48±1.19
not active
not active
not active
5.82±1.96
2.46±0.97
not tested
1.27
3.47
1.14±0.87
1.67±0.58
1.09±0.47
1.02±0.40
0.17±0.03
1.01±0.64
not tested
0.66±0.48
14
16a
16c
17
23
2.71
1.83
Ibuprofen
Zileuton
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*not active: compound with lower than 50% inhibition at 20 µM concentration for COX-1 and COX-2 or at 5 µM concentration for 5-LOX
2.4 Docking analysis
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The substrate of COX, arachidonic acid, is typically bound to the enzyme via ionic and hydrogen
bonds of the carboxyl group with Tyr355 and Arg120. The electron transfer of the catalyzed reaction
is mediated via Tyr385 [15]. The COX active compounds from this study also formed hydrogen bonds
with Tyr355 and Arg120, while the hydrophobic parts of the molecules filled the hydrophobic
substrate channel leading up to Tyr385. In COX-2 all the compounds docked slightly removed from
the acid binding site and especially the smaller molecules only formed interactions with Tyr355.
Some of the larger molecules (e.g. hydroquinone 5c) were turned around and bound to the catalytic
site in the opposite orientation while interacting with Tyr385.
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Long, flexible hydrophobic chains enhanced the activity, however, the optimal chain length
seemed to comprise of 11 to 13 carbon atoms, since the activity decreased in the chain with 15
carbon atoms while those molecules also had to bend more to stay inside the limits of the
hydrophobic binding pocket (see Figures 1 and 2).
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Simulation presented in Figure 3 demonstrates the comparison of the binding mode of compound
5c and flurbiprofen (member of NSAIDs family). The quinone system acts as a hydrogen bonding
partner instead of forming ionic interaction with an acidic moiety, as many known COX inhibitors
including flurbiprofen do. The alkyl chain occupies the hydrophobic channel leading up to this
binding site.
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Figure 1. Active compound 6b (green) compared to inactive compound 6a (grey) in the catalytic
site of COX-2. The longer chain pushed the ring more closely to the acid binding site.
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Figure 2. Compounds 5b (yellow) and 5d (green) in the catalytic site of COX-1. While they are both
active, 5d had to bend more strongly to stay within the hydrophobic channel and slightly loses
activity.
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Figure 3. Compound 5c (green) compared to co-crystallized inhibitor flurbiprofen (grey) in the
catalytic site of COX-1. The quinone ring forms hydrogen bonds with the typical acid binding site
formed by Arg120 and Tyr355 . The hydrophobic chain of the compound 5c fills the hydrophobic
substrate channel. Flurbiprofen in comparison forms ionic bond with Arg120 with its acidic moiety
and a hydrogen bond with Tyr355.
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The docking to the allosteric catalytic site of 5-LOX revealed a binding mode where primin (5a)
derivatives bound to Tyr142 and formed aromatic interactions with Arg138. The hydrophobic parts
of the molecule bound to a lipophilic grove between the catalytic domain and the membrane
binding domain of the enzyme. The most striking difference between the active and inactive
compounds was that a hydrogen bond donor seemed to be required in ortho position to the
hydrophobic chain. All the compounds possessing only meta substituents lost their activity. In the
docking experiments, ortho substituent was responsible for the interaction with Arg138 (Figure 4). If
this group was missing, the ring was turned, which was reflected negatively in the docking score and
it seemed to be detrimental to the experimental activity as well.
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Figure 4. 5-LOX active compound 6d (green) and inactive compound 4b (grey).
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3. Conclusions
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Rational improvement of the structure of natural benzoquinone primin resulted in the design of
derivatives with increased inhibitory activity. Especially, the elongation of the side chain from 5 to 11
carbons increased the inhibitory activity towards 5-LOX and COXs (dual inhibition). The acetylation of
(one or both) hydroxyls diminished activity towards COXs, but the activity towards 5-LOX either
stayed unchanged, or in the case of monoacetylation, it increased. The data from in vitro enzymatic
assays were supported by in silico docking simulations. Taken together, compounds 5b and 6b were
the most potent compounds for dual COX/5-LOX inhibition. Compound 6d was best 5-LOX inhibitor
with COX inhibitory activity comparable with ibuprofen. Compound 17 was selective 5-LOX inhibitor
with activity comparable to compound 6d.
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4. Experimental
4.1 Chemistry
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General Remarks: All reactions requiring anhydrous or inert conditions were carried out under a
positive atmosphere of argon in oven-dried glassware. Solutions or liquids were introduced in round
bottom flasks using oven dried syringes through rubber septa. All reactions were stirred magnetically
using Teflon-coated stirring bars. If needed, reactions were warmed up using electrically-heated
silicon oil bath, and the stated temperature corresponds to the temperature of the bath. Organic
solutions obtained after aqueous work-up were dried over MgSO4. The removal of solvents was
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accomplished using a rotary evaporator at water aspirator pressure. Chemicals were purchased from
Sigma-Aldrich Chemical Company. Technical grade solvents for extractions and chromatography
were purchased from Penta Chemicals s.r.o., Czech Republic. Solvents used in reactions were
distilled from appropriate drying agents and stored under argon over activated Linde 4Å molecular
sieves. Column and flash chromatography was carried out using Merck Silica (60-200 μm). Analytical
TLC was performed with Merck Silica gel 60 F254 plates. Visualization was accomplished by UV-light
(254 nm) and staining with a vanillin solution, followed b
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y heating. H, 13C and 2D (H-COSY, HMQC) NMR spectra were all recorded on Bruker Avance III™
HD. Trimethylsilane (0 ppm, 1H NMR) and CDCl3 (77 ppm, 13C NMR) were used as internal references.
The chemical shifts (δ) are reported in ppm (parts per millions) and the coupling constants (J values)
are recorded in Hz (Hertz). Mass spectra were recorded on a LTQ Orbitrap XL spectrometer. Purity of
the final products tested for bioactivity was estimated 95% or higher according to NMR spectra.
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1,3-Dimethoxy-5-(alk-1-yn-1-yl)benzene (2a-d): general method: To a flask charged with 81.0 mg
of bis(triphenylphosphine)palladium(II) chloride (0.12 mmol, 0.05 eq) and 43.9 mg of copper(I)
iodide (0.23 mmol, 0.1 eq) was added 6 mL of triethylamine. To the resulting bright yellow
suspension was added a solution of corresponding alkyne (2.30 mmol, 1.0 eq) and 500 mg of 1-
bromo-3,5-dimethoxybenezene (2.30 mmol, 1.0 eq) in 6 mL of dichloromethane in one portion. The
flask was rinsed with 1 mL of triethylamine and added to the reaction mixture. Solvents were
degassed by deep-freezing method. The reaction vessel was stirred under argon atmosphere at r.t.
overnight and then overnight at 65°C. The reaction was monitored by NMR and stopped when full
conversion occurred. The mixture was filtered through a pad of celite and silica gel using ethyl
acetate as eluent and then concentrated by rotary evaporation in vacuo to afford brown oil. The
crude product was dry loaded onto silica gel and purified by column chromatography (19:1
petrolether:ethyl acetate) yielding the products as orange oils. 2a (456 mg, 97%): 1H NMR (400 MHz,
CDCl3): δ 6.56 (d, J = 2.3 Hz, 2H), 6.40 (t, J = 2.3 Hz, 1H), 3.77 (s, 6H), 2.38 (t, J = 7.1 Hz, 2H), 1.68 –
1.58 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 160.6, 152.2, 109.5, 101.2, 90.1,
80.8, 55.5, 22.3, 21.5, 13.7. HRMS(ESI) m/z: calcd for C13H17O2 (M + H)+: 205.1229. Found: 205.1230.
2b (483 mg, 73%): 6.55 (d, J = 2.3 Hz, 2H), 6.40 (t, J = 2.3 Hz, 1H), 3.77 (s, 6H), 2.39 (t, J = 7.1 Hz, 2H),
1.65 – 1.55 (m, 2H), 1.48 – 1.39 (m, 2H), 1.35 – 1.20 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100
MHz, CDCl3): δ 160.4, 125.4, 109.3, 101.0, 90.2, 80.5, 55.4, 31.9, 29.5, 29.3, 29.2, 29.0, 28.7, 22.7,
19.4, 14.1. HRMS(ESI) m/z: calcd for C19H28O2 (M)+: 288.2089. Found: 288.2088. 2c (440 mg, 60%): 1H
NMR (400 MHz, CDCl3): δ 6.55 (d, J = 2.2 Hz, 2H), 6.40 (t, J = 2.2 Hz, 1H), 3.77 (s, 6H), 2.39 (t, J = 7.2
Hz, 2H), 1.64 – 1.55 (m, 2H), 1.48 – 1.38 (m, 2H), 1.36 – 1.17 (m, 18H), 0.87 (t, J = 6.9 Hz, 3H);
substrate: 6.66 (d, J = 2.1 Hz, 2H), 6.38 (d, J = 2.2 Hz, 2H), 3.77 (s, 6H) – overlapped with product. 13
C
NMR (100 MHz, CDCl3): δ 160.4, 125.4, 109.3, 101.0, 90.2, 80.5, 55.4, 31.9, 29.7, 29.6, 29.5, 29.4,
29.2, 29.0, 28.7, 22.7, 19.4, 14.1. HRMS(ESI) m/z: calcd for C21H33O2 (M + H)+: 317.2475. Found:
317.2475. 2d (762 mg, 96%): 1H NMR (400 MHz, CDCl3): δ 6.55 (d, J = 2.3 Hz, 2H), 6.40 (q, J = 2.3 Hz,
1H), 3.77 (s, 6H), 2.39 (t, J = 7.1 Hz, 2H), 1.64 – 1.54 (m, 2H), 1.48 – 1.39 (m, 2H), 1.34 – 1.20 (m,
24H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 160.1, 125.2, 109.0, 100.7, 89.9, 80.2,
55.0, 31.6, 29.4, 29.4, 29.3, 29.3, 29.2, 29.0, 28.9, 28.6, 28.4, 22.4, 19.1, 13.8. HRMS(ESI) m/z: calcd
for C23H37O2 (M + H)+: 235.2788. Found: 235.2788.
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1,3-Dimethoxy-5-alkylbenzene (3a-d): Compound 2, 1.9 mmol, 1.0 eq, was dissolved in 20 mL of
methanol, palladium (10% on carbon, 42.0 mg, 0.02 eq) was added and the reaction mixture was
hydrogenated under atmospheric pressure overnight. The mixture was filtered through a pad of
celite and silica gel using ethyl acetate as eluent and then concentrated by rotary evaporation in
vacuo to afford yellow oil. The crude product was dry loaded onto silica gel and purified by column
chromatography (19:1 petrolether:ethyl acetate) to afford products as transparent colorless oils. 3a
(392 mg, 99%) 1H NMR (400 MHz, CDCl3): δ 6.35 (dd, J = 2.2, 0.6 Hz, 2H), 6.30 (t, J = 2.3 Hz, 1H), 3.78
(s, 6H), 2.60 – 2.49 (m, 2H), 1.67 – 1.54 (m, 2H), 1.41 – 1.27 (m, 4H), 0.94 – 0.85 (m, 3H). 13C NMR
(100 MHz, CDCl3): δ 160.7, 145.4, 106.5, 97.5, 55.2, 36.3, 31.5, 06, 14.1. HRMS(ESI) m/z: calcd for
C13H21O2 (M + H)+: 209.1542. Found: 209.1541. 3b (542 mg, 99%) H NMR (400 MHz, CDCl3): δ 6.34
1
(d, J = 2.2 Hz, 2H), 6.29 (d, J = 2.3 Hz, 2H), 3.78 (s, 1H), 2.58 – 2.50 (m, 2H), 1.64 – 1.55 (m, 2H), 1.35 –
1.20 (m, 16H), 0.87 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 166.6, 145.45, 97.5, 55.2, 36.3,
31.9, 31.3, 29.7, 29.6, 29.6, 29.5, 29.4, 22.7, 14.1. HRMS(ESI) m/z: calcd for C19H32O2 (M)+: 292.2402.
1
Found: 292.2401. 3c (614 mg, 99%): H NMR (400 MHz, CDCl3): δ 6.35 (d, J = 2.3 Hz, 2H), 6.30 (t, J =
2.3 Hz, 1H), 3.78 (s, 3H), 2.57 – 2.51 (m, 2H), 1.65 – 1.56 (m, 2H), 1.36 – 1.19 (m, 20H), 0.88 (t, J = 6.8
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 160.6, 145.4, 106.5, 97.5, 55.2, 36.3, 31.9, 31.3, 29.7, 29.7,
29.6, 29.5, 39.4, 22.7, 14.1. HRMS(ESI) m/z: calcd for C21H37O2 (M + H)+: 321.2788. Found: 321.2788.
1
3d (650 mg, 99%): H NMR (400 MHz, CDCl3): δ 6.35 (d, J = 2.3 Hz, 2H), 6.30 (d, J = 2.3 Hz, 1H), 3.78
(s, 6H), 2.58 – 2.51 (m, 2H), 1.64 – 1.57 (m, 2H), 1.37 – 1.21 (m, 24H), 0.91 – 0.86 (m, 3H). 13C NMR
(100 MHz, CDCl3): δ 160.6, 145.4, 106.5, 97.5, 55.2, 36.3, 31.9, 31.3, 29.9, 29.7, 29.7, 29.6, 29.5, 29.4,
22.7, 14.1. HRMS(ESI) m/z: calcd for C23H41O2 (M + H)+: 349.3101. Found: 349.3102.
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1,3-Dihydroxy-5-alkylbenzene (4a,c,d): To the solution of 1,3-dimethoxy-5-alkylbenzenes 3a,c,d
(0.96 mmol, 1.0 eq), respectively, in 5-10 mL of dry dichloromethane at -15°C was added dropwise
BBr3 (1M solution in dichloromethane, 2.11 mmol, 2.2 eq) and the reaction was stirred under argon
atmosphere with rising temperature to r.t. for about 1.5 h. The mixture was quenched with H2O and
neutralized with NaHCO3 and extracted with dichloromethane. Organic phase was washed with
water and brine and dried over anhydrous sodium sulphate. After concentration, the crude product
was purified on silica column chromatography with petrolether:ethyl acetate (9:1) as an eluent. Pure
1
resorcinols 4a,c,d were obtained as yellow solids. 4a (160 mg, 92%): H NMR (400 MHz, CDCl3): δ
6.26 (t, J = 2.2 Hz, 1H), 6.19 (t, J = 2.2 Hz, 1H), 2.5 – 2.38 (m, 2H), 1.36 – 1.21 (m, 4H), 0.87 (q, J = 6.6
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 156.4, 108.1, 100.2, 35.8, 31.5, 30.7, 22.5, 14.0. HRMS(ESI)
1
m/z: calcd for C11H17O2 (M + H)+: 181.1184. Found: 181.1184. 4c (235 mg, 82%): H NMR (400 MHz,
MeOD): δ 6.12 (d, J = 2.2 Hz, 2H), 6.08 (t, J = 2.2 Hz, 1H), 2.43 (dd, J = 8.5, 6.8, 2H), 1.56 (t, J = 7.5 Hz,
2H), 1.35 – 1.25 (m, 20H), 0.90 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, MeOD): δ 159.3, 146.3, 107.9,
100.9, 37.0, 33.1, 32.5, 30.9, 30.8, 30.8, 30.8, 30.7, 30.6, 30.5, 30.4, 23.7, 14.5. HRMS(ESI) m/z: calcd
1
for C19H31O2 (M - H)+: 291.2330. Found: 291.2330. 4d (264 mg, 86%): H NMR (400 MHz, MeOD): δ
6.12 (d, J = 2.2 Hz, 2H), 6.08 (t, J = 2.2 Hz, 1H), 2.43 (dd, J = 8.5, 6.8 Hz, 2H), 1.56 (t, J = 7.5 Hz, 2H),
1.34 – 1.25 (m, 29H), 0.90 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, MeOD): δ 159.3, 146.3, 107.9,
100.9, 37.0, 33.1, 32.5, 30.9, 30.8, 30.8, 30.7, 30.6, 30.5, 30.4, 23.7, 14.5. HRMS(ESI) m/z: calcd for
C21H35O2 (M - H)+: 319.2643. Found: 319.2640.
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2-Methoxy-6-alkyl-1,4-benzoquinone (5a-d): Compound 3a-d (0.48 mmol, 1.0 eq), respectively,
was dissolved in 4 mL of acetic acid-water mixture (1:1) and 48.0 mg of chromium(VI) oxide (0.48
mmol, 1.0 eq) was added. The reaction mixture was stirred at 50°C for 2-3h. Then the reaction
mixture was diluted with water, extracted three times with ethyl acetate and the collected organic
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phase was washed with saturated bicarbonate solution, water and brine and dried over MgSO4.
After evaporation the crude material was loaded on silica gel column and eluted with
petrolether:ethyl acetate (7:3) to afford products as a yellow crystalline material. 5a (91.0 mg, 91%)
1H NMR (400 MHz, CDCl3): δ 6.47 (dt, J = 2.4, 1.5 Hz, 1H), 5.87 (d, J = 2.3 Hz, 1H), 3.81 (s, 3H), 2.48 –
2.36 (m, 2H), 1.56 – 1.43 (m, 2H), 1.39 – 1.26 (m, 4H), 0.93 – 0.82 (m, 3H). 13C NMR (100 MHz, CDCl3):
δ 187.7, 182.1, 158.8, 147.6, 132.9, 107.1, 56.3, 31.4, 28.7, 27.4, 22.4, 13.9. HRMS(ESI) m/z: calcd for
C12H17O3 (M + H)+: 209.1172. Found: 209.1173; calcd for (M + Na)+: 231.0992. Found: 231.0991. 5b
(119 mg, 85%) 1H NMR (400 MHz, CDCl3): δ 6.48 (dt, J = 2.7, 1.5 Hz, 1H), 5.87 (d, J = 2.4 Hz, 1H), 3.81
(s, 1H), 2.48 – 2.37 (m, 2H), 1.49 (q, J = 7.5 Hz, 1H), 1.38 – 1.20 (m, 16H), 0.88 (t, J = 8.0 Hz, 3H). 13
C
NMR (100 MHz, CDCl3): δ 187.7, 182.1, 158.9, 147.6, 132.9, 107.1, 56.3, 31.9, 29.6, 29.5, 29.3, 29.2,
28.7, 27.7, 22.7, 14.1. HRMS(ESI) m/z: calcd for C18H28O3 (M + H)+: 293.2111. Found: 293.2109. 5c
(119 mg, 77%) 1H NMR (400 MHz, CDCl3): δ 6.47 (dt, J = 2.7, 1.5 Hz, 1H), 5.87 (d, J = 2.4 Hz, 1H), 3.81
(s, 1H), 2.45 – 2.38 (m, 2H), 1.55 – 1.44 (m, 2H), 1.34 – 1.20 (m, 20H), 0.86 (t, J = 7.8 Hz, 3H). 13C NMR
(100 MHz, CDCl3): δ 187.7, 182.1, 158.8, 147.6, 132.9, 107.1, 56.3, 31.9, 29.7, 29.6, 29.6, 29.5, 29.4,
29.3, 29.2, 28.7, 22.7, 14.1. HRMS(ESI) m/z: calcd for C20H32O3 (M + Na)+: 343.2244. Found: 343.2243.
1
5d (134 mg, 80%) H NMR (400 MHz, CDCl3): δ 6.47 (dt, J = 2.7, 1.5 Hz, 1H), 5.87 (d, J = 2.3 Hz, 1H),
3.81 (s, 1H), 2.46 – 2.38 (m, 2H), 1.49 (dq, J = 11.1, 7.1 Hz, 2H), 1.35 – 1.20 (m, 24H), 0.87 (t, J = 6.8
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 187.7, 182.1, 158.8, 147.6, 132.9, 107.1, 56.3, 31.9, 29.7, 29.7,
29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 28.7, 27.7, 22.7, 14.1. HRMS(ESI) m/z: calcd for C22H36O3 (M +
H)+: 349.2737. Found: 349.2739; calcd for (M + Na)+: 371.2557. Found: 371.2557.
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2-Methoxy-6-alkyl-1,4-hydroquinone (6a-d): Compound 5a-d (0.24 mmol, 1.0 eq), respectively,
was dissolved in 2 mL of ether (5% H2O) mixture and 50.2 mg of sodium dithionite (0.29 mmol, 1.2
eq) was added. The reaction mixture was stirred at r.t. for 1-2h. Then the reaction mixture was
diluted with water, extracted three times with ethyl acetate and the collected organic phases were
washed with saturated bicarbonate solution, water and brine and dried over Na2SO4. After
evaporation the crude material was loaded on short silica gel column and eluted with
petrolether:ethyl acetate (9:1) to afford products as a white-grey crystalline material. Note: the
products are air sensitive and susceptible to oxidation back to corresponding benzoquinones 5a-d.
Thus, the attention must be paid to all the operations and handling after the reaction is completed,
including analyses. 6a (38.0 mg, 78%) 1H NMR (400 MHz, CDCl3): δ 6.31 (d, J = 2.8 Hz, 1H), 6.21 (d, J =
2.7 Hz, 1H), 5.23 (s, 1H), 4.36 (s, 1H), 3.84 (s, 3H), 2.57 (m, J = 2.6 Hz, 2H), 1.64
– 1.54 (m, 2H), 1.38 –
1.21 (m, 4H), 0.92
–
0.86 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 148.3, 146.7, 137.4, 129.0, 107.9,
97.1, 56.0, 31.7, 29.7, 29.4, 22.6, 14.1. HRMS(ESI) m/z: calcd for C12H17O3 (M + H)+: 211.1289. Found:
211.1289. 6b (50.0 mg, 71%) 1H NMR (400 MHz, CDCl3): δ 6.31 (d, J = 2.8 Hz, 1H), 6.21 (d, J = 2.8 Hz,
1H), 5.23 (s, 1H), 3.84 (s, 3H), 2.60 – 2.53 (m, 2H), 1.62 – 1.52 (m, 2H), 1.37 – 1.15 (m, 16H), 0.87 (t, J
= 8.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 148.3, 146.7, 137.3, 129.0, 107.9, 97.1, 56.0, 31.9, 29.7,
29.7, 29.6, 29.6, 29.5, 29.4, 22.7, 14.1. HRMS(ESI) m/z: calcd for C18H30O3 (M)+: 294.2195. Found:
294.2193. 6c (55.0 mg, 72%) 1H NMR (400 MHz, CDCl3): δ 6.31 (d, J = 2.8 Hz, 1H), 6.21 (d, J = 2.8 Hz,
1H), 3.84 (s, 3H), 2.60 – 2.52 (m, 2H), 1.62 – 1.52 (m, 2H), 1.35 – 1.18 (m, 20H), 0.87 (t, J = 8.0 Hz,
3H). 13C NMR (100 MHz, CDCl3): δ 187.7, 182.1, 158.9, 147.6, 132.9, 107.1, 56.3, 31.9, 29.7, 29.7,
29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 27.7, 22.7, 14.1. HRMS(ESI) m/z: calcd for C20H34O3 (M)+: 322.2508.
Found: 322.2506. 6d (68.0 mg, 81%) 1H NMR (400 MHz, CDCl3): δ 6.30 (d, J = 2.8 Hz, 1H), 6.21 (d, J =
2.8 Hz, 1H), 5.22 (s, 1H), 3.84 (s, 3H), 2.61 – 2.53 (m, 2H), 1.36 – 1.17 (m, 22H), 0.91 – 0.85 (m, 3H).
13C NMR (100 MHz, CDCl3): δ 148.3, 146.7, 137.4, 129.0, 107.9, 97.0, 56.0, 31.9, 29.7, 29.7, 29.6,
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29.6, 29.5, 29.4, 22.7, 14.1. HRMS(ESI) m/z: calcd for C22H38O3 (M + H)+: 350.2821. Found: 350.2819.
Alternative route to 6a (Scheme 3): Compound 15 (200 mg, 651 mmol), K3PO4 (415 mg, 1.95 mmol,
3.0 eq), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (23.8 mg, 32.6 mmol, 0.05 eq),
and pentylboronic acid (113 mg, 977 mmol, 1.5 eq) were dissolved in anh. toluene and heated under
argon overnight. The mixture was filtered through a pad of celite and silica gel using ethyl acetate as
an eluent and then concentrated by rotary evaporation in vacuo to afford brown oil. The crude
product was dry loaded onto silica gel and purified by column chromatography (7:3 petrolether:ethyl
acetate) yielding the product 6a as white solid (92.0 mg, 47%).
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2-Bromo-6-methoxyphenol (8): Tert-butylamine (0.60 g, 8.06 mmol) was dissolved in 10 mL of
toluene and cooled to -30°C. Bromine (1.29 g, 8.06 mmol, 1.0 eq) was added dropwise and the final
mixture was cooled to -60°C and then 2-methoxyphenol (1.00 g, 8.06 mmol, 1.0 eq) dissolved in 1 mL
of dichloromethane (DCM) was slowly added. The reaction mixture was stirred for 5 h and then let
to warm up to r.t. The mixture was quenched with 10% solution of Na2S2O3 and the product was
extracted 3x with DCM. DCM fraction was washed with brine and dried over Na2SO4. Column
1
chromatography (8:2 petrolether:ethyl acetate) afforded product as white crystals (1.52 g, 93%). H
NMR (400 MHz, CDCl3): δ 7.09 (dd, J = 8.0, 1.5 Hz, 1H), 6.81 (dd, J = 8.1, 1.5 Hz, 1H), 6.75 (t, J = 8.1
Hz, 1H), 5.92 (s, 1H), 3.90 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 147.3, 143.1, 124.8, 120.7, 109.9,
108.4, 56.3. HRMS(ESI) m/z: calcd for C7H7BrO2 (M + H)+: 202.9707. Found: 202.9708.
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1-Bromo-3-methoxy-2-(methoxymethoxy)benzene (9): Compound 8 (286 mg, 1.41 mmol) was
dissolved in 3 mL of anh. DCM and cooled to 0°C. To this mixture was added DIPEA (273 mg, 2.11
mmol, 1.5 eq) and MOMCl (147 mg, 1.83 mmol, 1.3 eq) and the reaction mixture was stirred
overnight. Then saturated NH4Cl solution was added and the product was extracted 3x into DCM.
DCM fraction was washed with brine and dried over Na2SO4. Evaporation afforded pure product as
an transparent oil, which was used directly for the next step without further purification (345 mg,
1
99%) H NMR (400 MHz, CDCl3): δ 7.14 (dd, J = 8.0, 1.5 Hz, 1H), 6.93 (t, J = 8.1 Hz, 1H), 6.86 (dd, J =
8.3, 1.5 Hz, 1H), 5.17 (s, 2H), 3.84 (s, 3H), 3.66 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 153.6, 143.4,
125.1, 125.1, 117.9, 111.7, 98.6, 58.0, 56.1. HRMS(ESI) m/z: calcd for C9H11BrO3 (M + Na)+: 268.9784.
Found: 268.9786.
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2-Methoxy-6-pentylphenol (10): Compound 9 (345 mg, 1.40 mmol), K3PO4 (889 mg, 4.19 mmol,
3.0 eq), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (51.1 mg, 69.8 µmol, 0.05 eq),
and pentylboronic acid (243 mg, 2.09 mmol, 1.5 eq) were dissolved in anh. toluene and heated
under argon overnight. The mixture was filtered through a pad of celite and silica gel using ethyl
acetate as eluent and then concentrated by rotary evaporation in vacuo to afford brown oil. The
crude product was dry loaded onto silica gel and purified by column chromatography (9:1
1
petrolether:ethyl acetate) yielding the product as an orange oil (235 mg, 87%). H NMR (400 MHz,
CDCl3): δ 6.82 – 6.66 (m, 3H), 5.66 (s, 1H), 3.88 (s, 3H), 2.68 – 2.54 (m, 2H), 1.67 – 1.56 (m, 1H), 1.39
– 1.29 (m, 3H), 0.93 – 0.85 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 146.3, 143.4, 128.8, 122.3, 119.1,
108.1, 56.0, 31.8, 29.7, 29.5, 22.6, 14.1. HRMS(ESI) m/z: calcd for C12H18O2 (M + H)+: 195.1385.
Found: 195.1386.
460
461
462
463
3-Methoxy-5-pentylphenol (11): To a solution of compound 3a (100 mg, 0.48 mmol) in dry DCM
(4 mL) was added dropwise BBr3 (1M solution in dichloromethane, 0.53 mmol, 1.1 eq) and the
reaction was stirred under argon atmosphere with temperature rising to r.t. over approximately 1.5
h. The mixture was quenched with H2O and neutralized with NaHCO3 and extracted with DCM.
15

464
465
466
467
468
469
470
471
Organic phase was washed with water and brine and dried over anhydrous sodium sulphate. After
concentration, the crude product was purified on silica column chromatography with
petrolether:ethyl acetate (9:1) as an eluent. Pure resorcinol 11 was obtained as yellow solid. 11 (82.0
mg, 88%): 1H NMR (400 MHz, CDCl3): δ 6.33 (ddd, J = 2.1, 1.3, 0.6 Hz, 1H), 6.26 (ddd, J = 1.9, 1.3, 0.6
Hz, 1H), 6.24 (t, J = 2.3 Hz, 1H), 4.70 (s, 1H), 3.77 (s, 3H), 2.57 – 2.46 (m, 2H), 1.67 – 1.51 (m, 3H), 1.40
– 1.21 (m, 4H), 0.93 – 0.84 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 166.8, 156.5, 145.8, 107.9, 106.8,
98.7, 55.3, 36.0, 31.5, 30.9, 22.6, 14.0. HRMS(ESI) m/z: calcd for C12H18O2 (M + H)+: 195.1385. Found:
195.1386.
472
473
474
475
476
477
478
479
480
481
2-Bromo-6-methoxy-1,4-hydroquinone
(13):
4.60
g
of
3-bromo-4-hydroxy-5-
methoxybenzaldehyde (19.9 mmol) was added to the mixture of 1M KOH (21.0 mL) and 3% H2O2
(45.0 mL) and stirred at 40°C for 1-2h. The conversion was controlled by NMR. When finished, the
reaction mixture was cooled down to r.t and acidified with 10% HCl to reach pH~5, and extracted 3x
with ethyl acetate. The organic phases were collected and washed with brine a dried over Na2SO4.
The evaporation and subsequent column chromatography (petrolether:ethyl acetate 8:2) afforded
1
the product 13 as the brown needles (3.41 g, 78%). H NMR (400 MHz, CDCl3): δ 6.58 (d, J = 2.7 Hz,
1H), 6.42 (d, J = 2.7 Hz, 1H), 5.48 (s, 1H), 3.87 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 149.1, 147.7,
137.4, 110.5, 108.0, 99.4, 56.3. HRMS(ESI) m/z: calcd for C7H7BrO2 (M + H)+: 217.9579. Found:
217.9580.
482
483
484
485
486
487
488
489
2-Bromo-6-methoxy-1,4-benzoquinone (14): 100 mg of compound 13 (457 mmol) was dissolved
in 4 mL of MeOH and FeCl3·6H2O (617 mg, 2.28 mol, 5.0 eq) in H2O (3 mL) was added. The reaction
mixture was then stirred at r.t. for 5h. The reaction was quenched with 10% KHCO3 and extracted 3x
with ethyl acetate, washed with brine a dried over Na2SO4. Column chromatography (9:1
petrolether:ethyl acetate) afforded product 14 as brown needles (65.0 mg, 66%). 1H NMR (400 MHz,
CDCl3): δ 7.20 (d, J = 2.3 Hz, 1H), 5.96 (d, J = 2.3 Hz, 1H), 3.85 (s, 3H). 13C NMR (400 MHz, CDCl3): δ
184.6, 174.5, 158.3, 138.5, 132.3, 107.6, 56.8. HRMS(ESI) m/z: calcd for C7H5BrO3 (M + H)+: 215.9422.
Found: 215.9425.
490
491
492
493
494
495
496
497
498
499
1-Bromo-3-methoxy-2,5-bis(methoxymethoxy)benzene (15): Compound 13 (1.00 g, 4.57 mmol)
was dissolved in 10 mL of anh. DCM and cooled to 0°C. To this mixture was added DIPEA (1.77 g,
13.70 mmol, 3.0 eq) and MOMCl (956 mg, 11.9 mmol, 2.6 eq) and the reaction mixture was stirred
overnight. Then saturated NH4Cl solution was added and the product was extracted 3x with DCM.
DCM phases were washed with brine and dried over Na2SO4. After evaporation the crude product
was purified on column chromatography (8:2 petrolether:ethyl acetate) yielding the product 15 as a
red oil (1.34 g, 95%). 1H NMR (400 MHz, CDCl3): δ 6.85 (d, J = 2.7 Hz, 1H), 6.57 (d, J = 2.7 Hz, 1H), 5.08
(s, 2H), 5.10 (s, 2H), 3.81 (s, 3H), 3.65 (s, 3H), 3.47 (d, J = 0.7 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ
148.9, 148.5, 133.0, 128.0, 123.4, 112.4, 106.4, 96.2, 93.4, 89.6, 52.6, 50.8, 50.7. HRMS(ESI) m/z:
calcd for C11H15BrO5 (M + Na)+: 328.9995. Found: 328.9994.
500
501
502
503
504
505
506
2-Methoxy-6-alkyl-1,4-benzoquinone diacetate (16a,c): Compound 6a,c (0.24 mmol),
respectively, was dissolved in pyridine (2mL) and acetic anhydride (54.5 mg, 542 mmol, 2.2 eq) was
added and the reaction mixture was stirred overnight. Then the mixture was acidified with 10% HCl
to reach pH~6 and extracted 3x with ethyl acetate, washed with brine and dried over Na2SO4. After
evaporation, the preparative TLC (9:1 petrolether:ethyl acetate) afforded products as yellowish
solids. 16a (45.0 mg, 76%): 1H NMR (400 MHz, CDCl3): δ 6.57 (s, 2H), 3.78 (s, 3H), 2.52 – 2.42 (m, 2H),
2.32 (s, 3H), 2.28 (s, 3H), 1.36 – 1.23 (m, 6H), 0.91 – 0.86 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 148.3,
16

507
508
509
510
511
512
146.7, 137.4, 129.0, 107.9, 97.0, 56.0, 31.9, 29.7, 29.7, 29.6, 29.4, 22.7, 14.1. HRMS(ESI) m/z: calcd
for C16H22O5 (M + Na)+: 317.1359. Found: 317.1360. 16c (79.0 mg, 81%): 1H NMR (400 MHz, CDCl3): δ
6.57 (d, J = 1.0 Hz, 2H), 3.78 (s, 3H), 2.53 – 2.42 (m, 2H), 2.32 (s, 3H), 2.28 (s, 3H), 1.33 – 1.19 (m,
22H), 0.90 – 0.85 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 168.8, 148.4, 136.6, 114.0, 103.9, 100.0,
56.1, 31.9, 30.1, 29.7, 29.7, 29.7, 29.5, 29.4, 29.4, 29.4, 22.7, 21.2, 20.5, 14.1. HRMS(ESI) m/z: calcd
for C24H38O5 (M + Na)+: 429.2612. Found: 429.2611.
513
514
515
516
517
518
2-Bromo-6-Methoxy-1,4-benzoquinone acetate (17): The reaction was carried out following the
above described acetylation procedure. For the reaction, compound 13 (50.0 mg, 0.23 mmol) was
used as the starting material. 18 (38.0 mg, 64%): 1H NMR (400 MHz, CDCl3): δ 6.87 (d, J = 2.5 Hz, 1H),
6.60 (d, J = 2.5 Hz, 1H), 5.83 (s, 1H), 3.87 (s, 3H), 2.27 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 169.6,
147.0, 143.5, 141.2, 117.5, 107.6, 104.7, 56.5, 21.0. HRMS(ESI) m/z: calcd for C9H9BrO4 (M + H)+:
259.9684. Found: 259.9685.
519
520
521
522
523
524
525
526
527
528
529
530
Tert-butyl(2-iodo-6-methoxyphenyl)carbamate (19): To tert-butyl 2-methoxyphenylcarbamate 18
(4.82 g, 21.6 mmol) in dry Et2O (20 mL) at -20 °C was added dropwise tert-butyllithium (28.0 mL, 47.4
mmol). The reaction was stirred for 3 hours at -20 °C, then cooled to -100 °C with a liquid N2/Et2O
bath. Iodine (5.48 g, 21.6 mmol) in Et2O (50 mL) was added to the solution. The reaction was slowly
warmed up to r.t. overnight. Na2S2O3 (saturated, 40 mL) was then added to the reaction mixture and
phases were separated. The aqueous phase was extracted with Et2O, and the combined organic
layers were dried over Na2SO4, filtered and concentrated. DCM (10 mL) was added, followed by
hexanes (40 mL). The solution was concentrated to remove DCM. The product crashed out, and was
collected by filtration and washed with hexanes (20 mL) to give the crude product as a transparent
1
viscous oil (4.39 g, 58%). H NMR (400 MHz, CDCl3): δ 7.44 (dd, J = 7.8, 1.4 Hz, 1H), 7.05 – 6.78 (m,
2H), 5.97 (s, 1H), 3.83 (s, 3H), 1.51 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 130.8, 129.0, 111.4, 100.3,
80.5, 56.0, 28.3. HRMS(ESI) m/z: calcd for C12H16INO3 (M + Na)+: 372.0067. Found: 372.0067.
531
532
533
534
535
536
537
Tert-butyl(2-methoxy-6-(pent-1-yn-1-yl)phenyl)carbamate (20): The reaction was carried out
following the general method for the preparation of compounds 2a-d. Iodide 19 (500 mg, 1.43
mmol, 1.0 eq) was used as halobenzene in this Sonogashira coupling. Pure product was obtained as a
1
brown oil without purification (410 mg, 99%): H NMR (400 MHz, CDCl3): δ 7.13 – 6.98 (m, 2H), 6.83
(dd, J = 8.0, 1.6 Hz, 1H), 3.83 (s, 3H), 2.41 (t, J = 7.0 Hz, 2H), 1.70 – 1.57 (m, 2H), 1.05 (t, J = 7.3 Hz,
3H). 13C NMR (100 MHz, CDCl3): δ 151.0, 147.4, 141.9, 131.4, 122.7, 112.5, 104.4, 104.2, 83.2, 55.2,
30.1, 37.7, 22.2, 14.0. HRMS(ESI) m/z: calcd for C17H23O3N (M + Na)+: 312.1570. Found: 312.1571.
538
539
540
541
542
2-Methoxy-6-(pent-1-yn-1-yl)aniline (21): Compound 20 (300 mg, 1.04 mmol) was dissolved in
DCM (5 mL) and trifluoroacetic acid was added (354 mg, 3.11 mmol, 3.0 eq). The resulting mixture
was stirred at r.t. for 1h and quenched with sat. NaHCO3. Product was extracted 3x with DCM,
washed with brine and dried over Na2SO4. After evaporation, it was used directly in the next
reduction step without further purification.
543
544
545
546
547
548
549
2-Methoxy-6-pentylaniline (22): Compound 21 (150 mg, 793 mmol) was dissolved in ethyl
acetate, and 0.02 eq of Pd/C(10%) was added. The reaction mixture was stirred under hydrogen
overnight. Catalyst was filtered off through a pad of celite and evaporation afforded quantitative
yield of product as brown oil (152 mg, 99%). 1H NMR (400 MHz, CDCl3): δ 6.73 – 6.67 (m, 3H), 3.85 (s,
3H), 2.53 – 2.47 (m, 2H), 1.68 – 1.56 (m, 2H), 1.43 – 1.30 (m, 4H), 0.94 – 0.87 (m, 3H). 13C NMR (100
MHz, CDCl3): δ 147.2, 133.7, 127.3, 121.6, 117.6, 108.0, 55.6, 31.9, 31.2, 28.5, 22.6, 14.1. HRMS(ESI)
m/z: calcd for C12H19NO (M + H)+: 194.1539. Found: 194.1538.
17

550
551
552
553
554
555
556
557
558
559
560
4-Imino-3-methoxy-5-pentylcyclohexa-2,5-dienone (23): Aniline 22 (61.0 mg, 316 mmol) was
dissolved in dichloroethane (DCE), tert-butyl hydroperoxide as 5.5M solution in hexane (0.46 mL,
2.52 mmol, 8.0 eq) and N,N′-bis(salicylidene)ethylenediaminocobalt(II) (20.5 mg, 63.1 mmol, 0.2 eq)
were added. The reaction mixture was stirred under argon at r.t. for 3h. After filtration through a
pad of celite, the mixture was quenched with sodium bisulfite and the product was extracted 3x with
ethyl acetate and dried over Na2SO4. Product was isolated on preparative TLC (7:3 petrolether:ethyl
acetate) as yellow solid (32.0 mg, 49%). 1H NMR (400 MHz, CDCl3): δ 11.45 (s, 1H), 6.31 (d, J = 1.8 Hz,
1H), 5.65 (d, J = 2.0 Hz, 1H), 3.82 (s, 3H), 2.64 (t, J = 7.5 Hz, 2H), 1.66
– 1.45 (m, 4H), 1.28 – 1.20 (m,
2H), 0.93
–
0.86 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 187.7, 182.1, 158.9, 147.6, 132.9, 107.1, 56.3,
31.4, 28.7, 27.4, 22.4, 13.9. HRMS(ESI) m/z: calcd for C12H17NO2 (M + H)+: 208.1338. Found:
208.1340.
561
4.2 COX-1 and COX-2 assay
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
The inhibitory activity of compounds was tested using in vitro enzymatic assay. Ovine COX-1 (1
unit/reaction) or human recombinant COX-2 (0.5 unit/reaction; both enzymes Sigma-Aldrich, USA)
was added to 180 µL of 100 mM Tris buffer (pH 8.0), containing 5 μM porcine hematin, 18 mM L-
epinephrine, and 50 μM Na2EDTA. Ten µL of tested compound dissolved in DMSO or pure DMSO (as
a blank) were added to the reaction. (S)-(+)-ibuprofen (Sigma-Aldrich, USA) was used as a reference
inhibitor. After 5 min incubation at room temperature, reaction was initiated by 5 µL of arachidonic
acid (10 µM) and incubated 20 min at 37°C. Then the reaction was stopped by 20 µL of formic acid
(10% v/v). The concentration of prostaglandin E2 (PGE2) was measured with Prostaglandin E2 ELISA
kit (Enzo Life Sciences, USA). Reaction mixture was diluted 1:15 in assay buffer (provided in the kit)
and incubated according to the manufacturer instructions. The concentration of PGE2 produced
during the reaction was estimated according to absorbance measured at 405 nm by a Tecan Infinite
M200 microplate reader (Tecan Group, Switzerland). The inhibitory activity was calculated as
percentual inhibition of PGE2 production compared to blank. The experiments were repeated at least
three times with at least two replicates in each test. To test whether increasing substrate
concentration influences the activity of compounds, AA was used in the reaction in final 50, 250, and
1250 nM concentrations. Compound 5b was selected as a representative compound for the tests
and (S)-(+)-ibuprofen was used as a reference compound. The inhibitory activity was calculated as
percentual inhibition of PGE2 production compared to blank with respective concentration of AA.
580
4.3 5-LOX assay
581
582
583
584
585
586
587
588
589
590
591
592
593
Human recombinant 5-LOX (Cayman Chemical, MI, USA) was used for in vitro enzymatic assay.
Enzyme (1 unit/reaction) was added to 180 μL of phosphate buffer saline (pH 7.4) containing 1 mM
Na2EDTA, and 200 μM ATP. Ten µL of tested compound dissolved in DMSO or pure DMSO (as a
blank) was added to the reaction and incubated 10 min at 4°C. Zileuton (Farmak a.s., Czech Republic)
was used as a reference inhibitor. Then 5 μL of 80 mM CaCl2 was added and reaction was initiated by
5 µL of arachidonic acid (800 μM). After 10 min incubation at 37°C, the reaction was stopped by 20
µL of formic acid (10% v/v). The concentration of leukotriene B4 (LTB4) produced during the reaction
was measured with Leukotriene B4 ELISA kit (Enzo Life Sciences, USA). The reaction mixture was
diluted 1:15 in assay buffer (provided in the kit) and incubated according to the manufacturer
instructions. The calculation of inhibitory activity was identical as in the case of COX assay. The
impact of increasing concentration of substrate on the activity of compounds was assessed using
three AA concentrations. Final 5, 20, and 80 µM concentrations of AA in the 5-LOX assay were used.
Compound 5b was selected as a representative compound for the tests and zileuton was used as a
18

594
595
reference compound. The inhibitory activity was calculated as percentual inhibition of LTB4
production compared to blank with respective concentration of AA.
596
4.4 DPPH assay
597
598
599
600
601
602
603
604
Radical scavenging activity was estimated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay
previously described by Sharma and Bhat [16]. Compounds 5b and 17 diluted in methanol (final
concentrations in the reaction were 3.125 - 400 µM using two-fold serial dilution) were 30 min
incubated with stable radical DPPH (Sigma-Aldrich, USA) dissolved in methanol (final concentration
in the reaction 125 mM) in room temperature in the darkness. After the incubation, the absorbance
at 517 nm was measured using Tecan Infinite M200 microplate reader (Tecan Group, Switzerland).
Trolox and L-ascorbic acid (both Sigma-Aldrich, USA) were used as positive references. Activities
were expressed as EC50 values.
605
4.5 Docking
606
607
608
609
610
611
612
613
614
615
616
617
618
Docking simulations were conducted on a windows 10 machine, running GOLD version 5.2
(www.ccdc.cam.ac.uk), using GOLD Score Fitness as a scoring function for all simulations. For COX-1
pdb entry 2AYL [17] was used and the binding site was defined in an 8 Å radius around the A chain
co-crystallized ligand. For COX-2 pdb entry 5IKV [18] was used and the binding site was defined in an
8 Å radius around the A chain co-crystallized ligand. To validate the docking simulations, five known
COX inhibitors, ibuprofen, indomethacin, lumiracoxib (COX-2 selective), flufenamic acid, and
flurbiprofen were docked into both isoforms. The resulting poses were compared to crystal
structures of these ligands within COX (flurbiprofen [17], flufenamic acid [18], ibuprofen and
lumiracoxib [19], indomethacin [20]). With the exception of lumiracoxib, which only docked correctly
into COX-2, all compounds were oriented correctly within the binding sites and formed the same key
interactions as known from the crystal structures. For 5-LOX pdb entry 3O8Y [21] was used and the
binding site was defined in an 8 Å radius around the C2 of Ile 167. This binding site definition was
acquired in other studies on 5-LOX allosteric binding [22].
619
Acknowledgments
620
621
622
623
The authors gratefully acknowledge the financial support by the grant LT18065 from the Ministry
of Education, Youth and Sports of the Czech Republic and networking contribution by the COST
Action CM16225 “Realising the therapeutic potential of novel cardioprotective therapies”. Further,
Prague OPPK grant CZ.2.16/3.1.00/21519 is honestly acknowledged.
624
Supplementary Material
625
626
Supplementary information contains 1H and 13C NMR spectra.
627
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628
629
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Highlights
Series of novel 1,4-benzoquinones, hydroquinones, and resorcinols were synthesized.
Compounds 5b and 6b possessed effective dual inhibition of COX-1, COX-2 and 5-LOX.
Most important modification was the elongation of alkyl chain from 5 to 11 carbons.
SAR study and docking revealed other modifications important for bioactivity.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: