In vitro study of isoflavones and isoflavans as potent inhibitors of human 12- and 15-lipoxygenases

Article abstract of DOI:10.1111/cbdd.12157

In this study, we have investigated 16 isoflavone and isoflavan derivatives as potential inhibitors of human lipoxygenase (platelet 12-lipoxygenase, reticulocyte 15-lipoxygenase-1, and epithelial 15-lipoxygenase-2). The flavonoid baicalein, a known lipoxygenase inhibitor, was used as positive control. Four compounds, 6,7-dihydroxy-3′-chloroisoflavone (1c), 7-hydroxy-8-methyl-4′-chloroisoflavan (5a), 7,8-dihydroxy-4′-methylisoflavan (5b), and 7,8-dihydroxy-3′-methylisoflavan (5c), were effective inhibitors of 12-lipoxygenases and 15-lipoxygenase-1 with IC₅₀'s <10 μm, while 6,7-dihydroxy-4′-nitroisoflavone (1b) was a selective inhibitor of 12-lipoxygenases. Docking studies, antioxidant assays, and kinetic measurements were carried out for the three best inhibitors (1b, 5b, 5c). The results showed that a catechol group in ring A is critical for the antioxidant properties of these compounds, and probably essential for their inhibitory activity. Kinetic assays showed that compounds 1b, 5b, and 5c are competitive inhibitors with K_i values in the range of 0.3-3 μm.

Full text of DOI:10.1111/cbdd.12157

Received Date : 07-Dec-2012
Revised Date : 10-Apr-2013
Accepted Date : 23-Apr-2013
Article type : Research Article
In-vitro study of isoflavones and isoflavans as potent inhibitors of human
12- and 15-lipoxygenases
Carolina Mascayanoa*, Victoria Espinosab, Silvia Sepúlveda-Bozab, Eric
K. Hooblerc and Steve Perryc
a Departamento de Ciencias del Ambiente, Facultad de Química y Biología, b Laboratorio de Investigación Científica Emory Black, Escuela de
Medicina, Facultad de Ciencias Médicas, Universidad de Santiago, Chile, Casilla 442, Correo 2 Santiago-Chile. c Department of Chemistry and
Biochemistry, University of California, Santa Cruz, California, 95064
*Author to whom correspondence should be addressed.
Tel: +56-02-27181036, email: carolina.mascayano@usach.cl
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
'Accepted Article', doi: 10.1111/cbdd.12157
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Abstract
In this study, we have investigated sixteen isoflavone and isoflavan derivatives as potential inhibitors of
human lipoxygenases (platelet 12-LOX, reticulocyte 15-LOX-1, and epithelial 15-LOX-2). The flavonoid
baicalein, a known lipoxygenase inhibitor, was used as positive control. Four compounds, 6,7-dihydroxy-
3’-chloroisoflavone (1c), 7-hydroxy-8-methyl-4'-chloroisoflavan (5a), 7,8-dihydroxy-4’-methylisoflavan
(5b) and 7,8-dihydroxy-3’-methylisoflavan (5c), were effective inhibitors of 12-LOX and 15-LOX-1 with
IC50’s less than 10 µM, while 6,7-dihydroxy-4’-nitroisoflavone (1b) was a selective inhibitor of 12-LOX.
Docking studies, antioxidant assays and kinetic measurements were done for the three best inhibitors (1b,
5b, 5c). The results showed that a catechol group in ring A is critical for the antioxidant properties of
these compounds, and probably essential for their inhibitory activity. Kinetic assays showed that
compounds 1b, 5b, 5c are competitive inhibitors with Ki values in the range of 0.3 to 3 μM.
Keywords: Human lipoxygenase assays, isoflavonoids, docking and scavenger properties
1. Introduction
Lipoxygenases (LOX's) are a family of non-heme iron-containing dioxygenases which catalyze the
stereospecific insertion of molecular oxygen into arachidonic acid [1]; the products formed are the either
(S)- or (R)-enantiomers of hydroperoxy fatty acids, which are further metabolized to the hydroxy
derivatives as end products [2]. Mammalian LOX’s are classified with respect to the positional specificity
with which they oxygenate arachidonic acid and are referred to as 5-, 8-, 12- and 15-LOX [2, 3].
The biological properties of LOXs have been widely studied because they are involved in the biosynthesis
of leukotrienes (LT's) and lipoxins (LP's), which participate in different human pathologies. For example,
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reticulocyte 15-lipoxygenase (15–LOX-1) has been implicated in colorectal [5] and prostate cancer [6],
whereas platelet 12-lipoxygenase (12–LOX) has been implicated in pancreatic [7], breast [8, 9], and
prostate cancers [10, 11]. In contrast, epithelial 15-lipoxygenase-2 (15-LOX-2) appears to be anti-
tumorigenic in prostate cancer [12]. Inhibition studies of 12-LOX and 15-LOX-1 are directly aimed at the
reduction of fatty acid metabolite production associated with inflammatory processes, cell proliferation
and carcinogenesis.
The proposed mechanism for fatty acid dioxygenation requires the oxidation of the isolated, inactive,
ferrous enzyme to the active ferric form. The ferric ion subsequently abstracts a hydrogen atom from the
substrate, with a concomitant reduction of the iron to the ferrous state. Therefore, the oxidation state of
LOX is critical to the activity of the enzyme and consequently, the search for compounds that can reduce
the ferric ion and therefore lower the activity of LOX, constitutes an important line of research [13, 14
and 15].
Lipoxygenase inhibitors have been divided into five types: reductants, iron chelators, substrate analogs,
tyrosine kinase inhibitors and lipoxygenase induction inhibitors [16]. Some examples include hydroxamic
acids [17], tropolones [18] and catechols [19]. Numerous flavonoids have been reported over the years as
human LOX inhibitors [20, 21, 22], with baicalein (5, 6, 7-trihydroxyflavone) being the most frequently
used. Baicalein is a non-specific LOX inhibitor [23], which has been shown to induce apoptosis in breast,
prostate, colon, and pancreatic cancer cell lines [23]. Other flavonoids that also inhibit LOXs are fisetin,
quercetin and luteolin [24].
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In a previous paper we reported that a catechol group in ring A, combined with a fully hydrogenated C
ring of an isoflavan backbone, are important features for the activity as lipoxygenase inhibitors [25, 26].
Now we present the evaluation as potential inhibitors of sixteen structurally related isoflavones and
isoflavans against platelet 12-LOX, reticulocyte 15-LOX-1 and prostate epithelial 15-LOX-2. Scavenging
properties and Ki values were also determined for the most prominent inhibitors (1b, 5b and 5c). Docking
studies were performed to clarify their structure-activity relationship.
2. Materials and methods
The purity of each isoflavonoid compound was confirmed by HPLC using a Merck-Hitachi Intelligent L-
6200A Pump, an L-4250 UV-Vis Detector, and a D-7000 HSM System Manager Report, with a C18
reverse phase column (Hypersil ODS-5, 250 mm x 4 mm) at a flow rate of 1 mL/min. The isoflavones
were detected at 260 nm, and the isoflavans at 295 nm. Two different solvent systems were used: system
1: (A) acetonitrile and (B) 1% acetic acid, and system 2: (A) acetonitrile and (B) a 1:1 v/v mixture of 1%
acetic acid/methanol. In both cases a gradient was used, beginning with 30% of (A), reaching 99% at 30
min, and (B) starting with 70% and ending with 1% in 30 min. All of them showed purity higher than
95%.
2.1.Synthesis of isoflavones and isoflavans
Melting points were recorded on a capillary Microthermal instrument, and were not corrected. All starting
materials were commercially available (Sigma-Aldrich), with purity higher than 98%, and used without
further purification.
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Isoflavones 1-5 were prepared by reported procedures [25, 26], from the corresponding intermediate
benzylphenylketones.
Preparation of intermediate benzylphenylketones (general procedure) [25, 26] – Dry HCl was passed into
a cooled (0 ºC), stirred mixture of the appropriate substituted phenylacetonitrile (0.34 mol) and anhydrous
zinc chloride (30 g, 0.22 mol) in dry diethyl ether (200 mL). The corresponding polyhydroxybenzene
(0.28 mol) was added portionwise with constant bubbling of gaseous HCl. The reaction mixture was then
stirred at room temperature for a few hours. The ketiminium chloride intermediate that separated as an oil
was washed with diethyl ether and hydrolyzed by refluxing in 5% HCl (1 L) for 4 to 5 hours. The ketone
that separated upon cooling was filtered and recrystallized in the appropriate solvent.
Preparation of isoflavones (general procedure) [25, 26] To a solution of the benzylketone (0.18 mol) in
dry DMF (200 mL) was added dropwise BF3.Et2O (0.88 mol). To this solution, warmed to 50 ºC, was
slowly added a solution of methanesulfonyl chloride (0.56 mol) in DMF (100 mL). The resulting mixture
was then heated to 100 ºC for 2 hours. After cooling, it was poured into water (4 L) and left overnight to
give a precipitate, which was stirred for 2 hours in cold methanol (50 mL), filtered and crystallized in the
appropriate solvent.
In this way the following isoflavones were obtained:
6,7-Dihydroxy-4’-chloro-isoflavone (1a). (77%). 1H NMR (DMSO-d6): δH 9.50 (1H, s, 7-OH), 9.06 (1H,
s , 6-OH ), 8.16 (1H, s, 2-H), 7.48 (2H, d, J = 8.0 Hz, H-3’, H-5’), 7.37 (1H, s, H-5), 7.29 (2H, d, J = 8.0
13
Hz, H-2’, H-6’), 6.43 (1H, s, H-8). C NMR (DMSO-d6): δC 174.7, 152.4, 151.9, 151.2, 144.7, 133.9,
129.7, 128.6, 123.5, 127.1,116.9, 108.4, 102.8. M.p. 299 °C, lit. [26] M.p. 300 °C;
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6,7-Dihydroxy-4'-nitroisoflavone (1b). (81%). 1H NMR (DMSO-d6): δH 10.11 (1H, s, 7-OH), 8.54 (1H, s,
2-H), 8.26 (2H, d, J = 8.8 Hz, H-30, H-5’), 7.89 (2H, d, J = 8.8 Hz, H-2’, H-6’), 7.40 (1H, s, H-5), 6.93
(1H, s,H-8), [signal for 8-OH not seen]. 13C NMR (DMSO-d6): δC 174.3, 155.5, 153.3, 151.7, 147.2,
145.8, 140.4, 130.5, 123.9, 121.6, 117.1, 108.9, 103.7. M.p. 168 °C, lit. [26] M.p. 166 oC.
1
6,7-Dihydroxy-3’-chloro-isoflavone (1c). (69 %). H NMR (DMSO-d6): δH 10.20 (2H, s, 6-,7-OH), 8.44
(1H, s, H-2), 7.44-7.69 (4H, m, H-2’,4’,5’,6’), 7.40 (1H, s, H-5), 6.96 (1H, s, H-8). 13C NMR (DMSO-d6):
δC 174.9, 153.3, 151.9, 151.2, 144.7, 134.1, 130.7, 129.4, 129.1, 128.3, 123.9, 117.1, 108.4, 102.8. M.p.
308 °C, lit. [27] M.p. 306 oC;
1
7,4’-Dihydroxy-8-methyl-isoflavone (2a). (66 %). H NMR (DMSO-d6): δH 10.61 (1H, s, 7-OH), 9.86
(1H, s, 4’-OH), 8.13 (1H, s, H-2), 7.97 (1H, d, J = 8.0 Hz, H-5), 7.24 (2H, m, H-2’,6’), 7.0 (1H, d, J = 8.0
13
, H-6), 6.84 (2H, m, H-3’,5’), 2.25 (3H, s, 8-CH3). C NMR (DMSO-d6): δC 175.0, 161.5, 157.5, 155.4,
153.5, 130.0, 124.3, 123.8, 117.2, 115.3, 113.9, 110.9, 8.01. M.p. 192 °C , lit. [28] M.p. 193 oC.
1
7-Hydroxy-8,4’-dimethyl-isoflavone (2b). (71%). H NMR (DMSO-d6): δH 10.55 (1H, s, 7-OH), 8.17
(1H, s, H-2), 7.98 (1H, d, J = 8.0 Hz ,H-5), 7.29 (2H, m, H-2’,H-6’), 7.19 (2H, m, H-3’,5’), 7 (1H, d, J =
13
8.0 Hz, H-6 ), 2.45 (3H, s, 4’-CH3 ), 2.25 (3H, s, 8-CH3). C NMR (DMSO-d6): δC 173.5, 158.3, 155.4,
153.5, 138.2, 129.1, 128.8, 126.1, 124.2, 123.8, 116.6, 113.9, 110.9, 21.4, 7.9. M.p. 264 °C, lit. [27] M.p.
266 oC.
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1
7-Hydroxy-8-methyl-4’-nitro-isoflavone (2c). (51%). H NMR (DMSO-d6): δH 10.85 (1H, s, 7-OH), 8.38
(1H, s, H-2), 8.18 (2H, m, H-3’,5’),7.47 (2H, m, H-2’,6’), 7.97 (1H, d, J = 8.0 Hz, H-5), 7.00 (1H, d, J =
8.0 Hz, H-6), 2.25 (3H, s, 8-CH3).13C NMR (DMSO-d6): δC 178.5, 155.2, 153.5, 147.5, 132.00, 130.1,
124.3, 124.1, 123.8, 114.00, 110.9, 8.0. Mp. 345 °C, lit. [27] M.p. 344 oC;
7-Hydroxy-8-methyl-3’-trifluoromethyl-isoflavone (2d). (68%). 1H NMR (DMSO-d6): δH 2.21 (3 H, s, 8-
CH3), 7.10 (1H, d, J = 9.0 Hz, 6-H), 7.64 (1 H, t, J = 8.0 Hz, 5’ -H), 7.71 (1 H, d, J = 8.0 Hz, 6’ -H), 7.83
(1 H, d, J = 9.0 Hz, 5-H), 7.85 (1 H, d, J = 8.0 Hz, 4’ -H), 7.97 (1 H, s, 2’ -H), 8.56 (1 H, s, 2-H), 10.68 (1
H, s, 7-OH). 13C NMR (DMSO-d6): δC 174.1, 161.2, 156.6, 153.4, 135.7, 133.1, 132.7, 129.0, 127.2,
126.4, 125.9, 124.8, 124.1, 115.6, 115.5, 112.7, 10.1. Mp. 282 °C, lit. [27] Mp. 283–284 °C.
1
7,4’-Dihydroxy-isoflavone (3a). (65%). H NMR (DMSO-d6): δH 10.90 (1H, s, 7-OH), 9.59 (1H, s, 4`-
OH), 8.24 (1H, s, 2-H), 7.96 (1H, d, 5-H), 6.77 (2H, dd, J = 8.4 Hz, 2`-H, 6`-H), 6.34 (1H, s, 8-H), 6.32
(2H, dd, J = 8.4 Hz, 3`-H, 5`-H), 6.16 (1H, d, 6-H). 13C NMR (DMSO-d6): δC 173.3, 162.9, 156.9, 153.0,
130.0, 127.6, 125.0, 122.8, 116.6, 117.0, 115.0, 102. M.p. 300 °C, lit. [29] M.p. 299 oC;
7-Hydroxy-isoflavone (3b). (78%). 1H NMR (DMSO-d6): δH 10.17 (1H, s, 7–OH), 8.18 (1H, s, H-2), 7.97
(1H, d = 7.6 Hz, H-5), 7.55 (5H, m, H-2’,3’,4’,5’ and 6’), 6.98 (1H, d, J = 7.6 Hz, H-6), 6.91 (1H, s, H-8).
13C NMR (DMSO-d6): δC 173.25, 162.86, 157.96, 154.03, 133.24, 128.74, 128.69, 128.14, 127.60,
125.00, 117.00, 116.63, 102.14. M.p. 207 °C, lit. [30] M.p. 206 oC.
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7-Hydroxy-4’-nitro-isoflavone (3c). (63%). 1H NMR (DMSO-d6): δH 11.01 (1H, s, 7-OH), 8.68 (1H, s, 2-
H), 8.35 (2H, dd, J = 7.6 Hz, 3`-H, 5`-H), 8.07 (1H, d, 5-H), 7.97 (2H, dd, J = 7.6 Hz, 2`-H, 6`-H), 7.04
(1H, s, 8-H), 6.99 (1H, d, 6-H). 13C NMR (DMSO-d6): δC 173.3, 162.9, 157.9, 154.0, 148.1, 142.2, 129.3,
127.6, 125.0, 117.0, 116.6, 102.1. M.p. 189 °C, lit. [26] M.p. 188 oC.
7-Benzyl-4’-methoxy-isoflavone (3d). (72%). 1H NMR (DMSO-d6): δH 8.87 (1H, s, H-2), 7.70 (1H, d, J =
7.6 Hz, H-5), 7.33 (2H, d, J = 7.2 Hz, H-2’,-6’), 7.24 (5H, m,-AR-H), 6.68 (1H, d,J = 7.6 Hz ,H-6), 6.66
13
(2H, d, J = 7.2Hz, H-3’,5’), 6.65 (1H, s, H-8), 5.17 (2H, s, -OCH2Bz), 3.77 (3H, s, -OCH3). C NMR
(DMSO-d6): δC 173.25, 163.73, 158.89, 156.27, 154.03, 137.09, 129.57, 128.32, 128.16, 126.63, 125.00,
124.05, 120.13, 116.79, 113.94, 103.16, 70.84, 56.04. M.p. 170 °C, lit. [26] M.p. 169 oC;
5,7-Dihydroxy-4’-nitro-isoflavone (4a). (63%). 1H NMR (DMSO-d6): δH 12.66 (1H, s, 5-OH), 10.96 (1H,
s, 7-OH), 8.56 (1H, s, 2-H), 8.26 (2H, dd, J = 7,6 Hz, 3`-H, 5`-H), 7.86 (2H, dd, J = 7,6 Hz, 2`-H, 6`-H),
13
6.40 (1H, s, 8-H), 6.22 (1H, d, 6-H). C NMR (DMSO-d6): δC 179.03, 164.88, 161.41, 158.83, 154.71,
148.14, 142.23, 129.33, 125.56, 125.01, 104.92, 99.33, 93.78. M.p. 197 °C, lit. [26] M.p. 196 oC.
5-Hydroxy-7-benzyl-4’-nitro-isoflavone (4b). (72%). 1H NMR (DMSO-d6): δH 11.05 (1H, s, 5-OH), 7.85
(1H, s, H-2), 7.55 (2H, d, J = 7.2 Hz, H-2’,6’), 7.23 (5H, m, -Bz), 6.25 (1H, s, H-8), 6.17 (1H , s, H-6),
5.17 (2H , m, O-CH2-Bz), 6.17 (1H, s, H-6), 6.25 (1H, s, H-8). 13C NMR (DMSO-d6): δC 179.03, 166.46,
161.77, 157.83, 154.71, 148.14, 142.23, 137.09, 129.33, 128.32, 128.17, 128.16, 125.56, 125.01, 106.03,
99.38, 93.94, 70.84.M.p. 185 °C, lit. [27] M.p. 184 oC.
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The isoflavans were obtained from the corresponding isoflavones by catalytic hydrogenation with Pd/C
(10%) in acetic acid containing 0.1% concentrated sulfuric acid for 14 h [26].
In this way the following isoflavans were prepared:
1
7-Hydroxy-8-methyl-4'-chloroisoflavan (5a). (53%). H NMR (DMSO-d6): δH 10.61 (1H, s, 7-OH), 7.37
(2H, d, J=8.4 Hz, H-2’,6’), 7.34 (2H, d, J=8.4Hz, H-3’,5’), 6.45 (1H, d, J=7.6 Hz, H-5), 6.31(1H, d, J=7.6
13
Hz, H-6), 2.31 (3H, s, 8-CH3) C NMR (DMSO-d6): δC 157.9, 154.3, 136.6133.0, 129.9, 128.4, 127.4,
117.6, 107.9, 70.3, 38.4, 32.9, 9.2. M.p. 159°C. lit. [27] M.p.160°C.
7,8-Dihydroxy-4’-methylisoflavan (5b). (55%). 1H NMR (DMSO-d6): δH 8.54 (1H, s, OH-7), 8.09 (1H, s,
OH-8), 7.19 (2H, d, J = 8.1 Hz, H-20, H-6’), 7.12 (2H, d, J = 8.1 Hz, H-30, H-5’), 6.36 (1H, d, J = 8.2 Hz,
H-5), 6.28 (1H, d, J = 8.2 Hz, H-6), 4.23 (1H, ddd, J = 3.4 Hz, J = 10.4 Hz, J = 1.7 Hz, H-2e), 3.97 (1H,
dd, J = 10.2 Hz; J = 10.2 Hz, H-2a), 3.09 (1H, ddd, J = 4.0 Hz, J = 5.7 Hz, J = 10.0 Hz, J = 8.9 Hz, H-3),
2.89 (1H, dd, J = 10.4 Hz, J = 16.1 Hz, H-4a), 2.80 (1H, ddd, J = 5.4 Hz, J = 15.6 Hz, J = 1.2 Hz, H-4e).
13C NMR (DMSO-d6): δC 144.7, 143.6, 139.5, 136.5, 133.7, 129.7, 127.9, 119.1, 113.9, 108.7, 70.5, 38.2,
32.0, 21.3. M.p.136°C, lit. [27] M.p.137°C;
7,8-Dihydroxy-3’-methylisoflavan (5c). (55%). 1H NMR (DMSO-d6): δH δ 9.20 (1H, s, 8-OH), 8.60 (1H,
s, 7-OH), 7.28 (1H, t, J = 7.4 Hz, H-5’), 7.17 (1H, d, J = 7.4 Hz, H-6’), 6.96 (1H, d, J = 7.6 Hz, H-4’),
6.38 (1H, d, J = 7.4 Hz, H-6), 6.19 (1H, d, J = 7.4 Hz, H-5), 4.54 (1H, dd, J = 12.4, 6.7 Hz, H-2a), 4.23
(1H, dd, J = 12.3, 6.6 Hz, H-2b), 3.60–3.47 (1H, m, H-3), 3.14 (1H, ddd, J = 12.5, 8.5, 1.0 Hz,H-4a), 2.90
(1H, ddd, J = 12.3, 8.5, 1.0 Hz, H-4b), 2.27 (3H, s, 3’-CH3). 13C NMR (DMSO-d6): δC 146.9, 146.0,
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141.7, 137.6, 133.0, 129.2, 128.4, 126.4, 126.1, 123.1, 113.9, 70.3, 34.7, 32.9,21.4. M.p.268°C, lit. [27]
M.p.265°C.
2.2.Kinetics and IC50 assay
The H6-tagged proteins, 12-LOX, 15-LOX-1 and 15-LOX-2, were expressed and purified as described
previously [31, 32, and 33]. The inhibition of their activities was determined by following the formation
of the conjugated diene product at 234 nm (ε = 25,000 M-1cm-1) with a Perkin-Elmer Lambda 40 UV/Vis
spectrophotometer, relative to control rates of carrier solvent DMSO as previously published [34]. All
reactions were done in a volume of 2 mL constantly stirred using a magnetic stir bar at room temperature
(23 ºC) with approximately 2.040 Units of 12-hLOX (evaluated by iron content), 4.200 Units 15-hLOX-1
(by iron content), and 6.600 Units of 15-hLOX-2. Units of specific activity were defined as (µmol
substrate consumed/ mg enzyme x min). Reactions with 12-hLOX were carried out in 25 mM HEPES
(pH 8.0), 0.01% Triton X-100 and 10 µM arachidonic acid (AA). Reactions with 15-hLOX-1 and 15-
hLO-2 were carried out in 25 mM HEPES buffer (pH 7.5), 0.01% Triton X-100 and 10 µM AA and 30
µM AA, respectively. The concentration of AA (for 12-hLOX and 15-hLOX-2) was quantitatively
determined by allowing the enzymatic reaction to reach completion. IC50 values were obtained by
determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor
concentration, followed by a hyperbolic saturation curve fit. The data used for the saturation curves were
obtained in duplicate or triplicate, depending on their quality. Additionally the Ki and inhibitor behavior of
the three most interesting compounds (1b, 5b, and 5c) were determined against soybean 15-LOX (specific
activity 100000 U/mg, Cayman Chemical Catalog Number 60712) using 10 µM AA as substrate in
Hepes-Triton X-100 buffer, pH 7.4. The reaction velocities at different inhibitor concentration expressed
as logarithm, (0-50µM) in triplicate for three independent experiments for each compound were plotted
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and the Ki values were interpolated from the Cheng-Prusoff [35] equation, using GraphPad Prism v.5
DEMO [36]
2.3. Evaluation of radical-trapping activity using DPPH
The DPPH scavenging capacity assay was performed using 20 µL of different compound concentrations
in methanol, which were added to 1 mL of 0.1 mM DPPH in methanol. Each mixture was vortexed for a
few seconds and left with agitation in the dark for 30 min at room temperature. The absorbance (A) of
each reaction mixture was measured at 517 nm against a blank of methanol using a UV-visible
spectrometer (Genesys 5). The scavenging capacity of each sample was calculated on the basis of an
ascorbic acid standard curve. Duplicate reactions were carried out with each sample [37]. The correlation
among the IC50 values for enzyme inhibition and the scavenger properties of compounds was evaluated
using a correlation matrix including the IC50 for enzyme inhibition of 12 compounds and the percentage
of remaining DPPH radical for 3 independent experiments with each enzyme. The Pearson correlation for
the IC50 and % remaining DPPH was done using GraphPad Prism v.5 DEMO, considering an alpha value
of 0.05.
2.4.Docking studies
All structures were built with the GaussianView software [38]. The Chelpg charges were obtained at the
B3LYP/6-31G** level of theory, employing the Gaussian 03 package [38]. Docking into the active site of
human 12- and 15-lipoxygenase models and the crystal structure of soybean LOX-1 (PDB ID: 1N8Q)
[39] was done using AutoDock 4.2 [40] with the Lamarckian algorithm and assuming total flexibility of
the inhibitors and partial flexibility of the His residues coordinated to Fe+3 inside the binding site. The
grid maps were built with 60 x 60 x 60 points, with a grid-point spacing of 0.375 Å. The AutoTors option
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was used to define the ligand torsions, and the docking results were then analysed by a ranked cluster
analysis, resulting in conformations with the highest overall binding energy (most negative –ΔGbinding
value).
3. Results and Discussion
The studied isoflavones were prepared by a two-step procedure. Reduction of the isoflavones by catalytic
hydrogenation led to the corresponding isoflavans, as shown in Scheme 1.
Table 1 shows the sixteen isoflavonoids that were investigated as potential inhibitors of human platelet
12-LOX, reticulocyte 15-LOX-1 and epithelial 15-LOX-2. The compounds evaluated were thirteen
isoflavones, three isoflavans and the flavone baicalein as a positive control [23].
Compounds 1a and 1b, with a catechol group on ring A and a chloro or nitro group at the para position of
B ring, respectively, had IC50 values >100 µM toward 15-LOX-1. On the other hand, 1b proved to be a
selective inhibitor of platelet 12-LOX (IC50 13 µM). In addition to the inhibitory assays, docking studies
were performed for all compounds based on human 12-LOX and 15-LOX-1 models. In 12-LOX, the nitro
group of 1b is positioned close to Arg402 and Gln406 (1.79 Å and 1.78 Å, respectively), and also near the
exit of the binding site, enabling electrostatic interactions. Ring B is parallel to Ile399 in the enzyme
cavity, allowing hydrophobic stabilization of this aromatic moiety, as has been reported for soybean LOX
[41] (Figure 1). In 15-LOX-1, the small, neutral aminoacid Gly replaces the polar, ionic Gln406, which
might be an important modification to explain the weak inhibitory effect of 1b on this enzyme.
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Compound 1c with a chloro substituent in the meta position of ring B inhibits 12-LOX and 15-LOX-1
with IC50 values of 0.28 and 0.59 µM, respectively. The analysis of its interaction in the binding cavity of
12-LOX indicates that the meta chloro group in 1c participates in a stabilizing hydrophobic interaction
with Leu172, unlike the behavior of ring B in compound 1a with a para chloro group. In addition, ring B
of 1c can rotate in the active site of 15-LOX-1 producing more favorable hydrophobic interactions,
similar to those recently reported for other inhibitors [42]. This difference in inhibitory activity towards
LOX might be due to the different shape and size of the binding cavity [44] of the enzyme isoforms. This
effect was observed with compounds 1b and 1c against 12-LOX, 15-LOX-1 and 15-LOX-2 (see Table 1).
Compounds 2a, 2b, 2c and 2d, were unable to inhibit 12-LOX and 15-LOX-1 (IC50 > 100 µM). This
could be due to the presence of the methyl group on C-8 of ring A and the availability of a single hydroxyl
group instead of a catechol group to coordinate with the iron atom in the active site. Other studies have
reported that catechol moieties as found in many lipoxygenase inhibitors are an important structural
features [41]. The evaluation of 3a, 3b, 3c and 3d, which likewise do not contain a catechol ring, also
revealed no inhibitory activity against 12-LOX and 15-LOX-1, confirming the importance of the catechol
group on ring A. Besides, the evaluation of compounds 4a and 4b exhibiting low activities against human
LOX (IC50 >100 µM) also showed that one OH group attached to C-7 on ring A is not sufficient to make a
compound a good inhibitor.
Following with our study, the biological evaluation of isoflavan derivatives showed that these compounds
were effective but not selective for the three lipoxygenases studied. The IC50 values were 4.4 (5b), 5.8
(5c) and 29 (5a) µM for 15-hLOX-1 and 6.4 (5c) 7.7 (5b) and > 40 (5a) µM for human 12-LOX. The
analysis of the inhibitory activity of these isoflavans revealed decreased potency of compound 5a versus
compounds 5b and 5c. This behavior could be explained by the presence of a methyl group at C-8 instead
of a hydroxyl group in 5a, so that the presence of a catechol moiety on ring A is prevented. Furthermore,
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isoflavans that have a fully hydrogenated ring C are able to fit more efficiently into the binding site of
human LOXs and consequently proved to be better inhibitors. Also, we found that the IC50 value observed
for 5b against 15-LOX-2 (34 μM) is eight times higher than its IC50 value against 15-LOX-1 (4.4 μM),
15-LOX-1 favoring cancer development while 15-LOX-2 had shown anti-tumor activity [12].
The antioxidant capacity of twelve compounds was estimated by reaction with DPPH [37] and the results
are summarized in table2.
The compounds without catechol moieties, which did not show LOX inhibition, were also unable to
bleach the DPPH radical. Although only 12 of 16 compounds were evaluated, a good correlation was
observed among the IC50s for enzyme inhibition and the IC50s for the reduction of DPPH (P = 0.0002 and
P = 0.0042 for 12-LOX and 15LOX-1, respectively). These results support the previous evidence that
there is a close relationship between the inhibitory capacity of lipoxygenases by catechol isoflavonoids
and the antioxidant properties of the latter [26]. Because of the great similarity among the active sites of
the different lipoxygenases, kinetic studies were performed with three inhibitors (1b, 5b and 5c) to
determine if the isoflavonoids were competitive inhibitors of soybean lipoxygenase, as a model of the
different LOXs. All compounds assayed proved to be competitive inhibitors with low Ki, especially
isoflavans 5b and 5c, with K an order of magnitude lower than 1b (3.4 , 0.53 and 0.25 μM), as shown in
Figure 2 and summarized in table 3 [43].
The docking studies also show the most favorable interactions between the binding site of soybean LOX
and compounds 1b, 5b and 5c, as shown in Figure 3.
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Conclusion
Sixteen isoflavonoids were evaluated as potential inhibitors of lipoxygenases. We found that catechol
groups and the flexibility of ring C were important features for higher inhibitory capacity of these
compounds, as can be observed particularly in isoflavones 1b, 1c and isoflavans 5b and 5c, which proved
to be potent and selective inhibitors of the human isoforms 12- and 15-LOX-1. Additionally, the presence
of a chlorine atom at the meta instead of the para position on ring B enhances the inhibitory effect of the
isoflavonoids toward LOX. Because various studies have suggested a protective role of 15-hLOX-2
against cancers, we have included this enzyme in our study, butit was not inhibited by any of the tested
compounds. Also in this work, kinetic assays were performed for 1b, 5b and 5c, showing that all three are
competitive inhibitors. Another important feature observed was the direct correlation between the
inhibition of 12- and 15-hLOX1 and the antioxidant capacity of the inhibitors, determined by reaction
with DPPH. Finally docking studies supported the experimental evidence. Therefore, this paper confirm
the previously results [26] concerning the importance of the catechol group and reduction of the central
ring on the inhibition of 12 - and 15-lipoxygenase and alsowas observed that a chlorine atom in position
3' of the B ring of the isoflavonoid improved the capacity as lipoxygenase inhibitor as seen in 1c.
Acknowledgments
Financial support from FONDECYT (project # 1120379) is gratefully acknowledged.
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Figure and Scheme Captions
Scheme 1. General route for the preparation of isoflavones and isoflavans.
Figure 1. Principal interactions of isoflavonoid 1b and the binding site of human 12-LOX
Figure 2. Relation between Log inhibitor concentrations and reaction velocity (u.a/min) of soybean 15-
LOX and three inhibitors
Figure 3. Superimposition and most important interactions of 1b (green), 5b (light blue) and 5c
(magenta) in the binding site of soybean LOX by docking studies
Scheme 1
(i)ZnCl2/H2O/H+; (ii) DMF/MeSO2Cl in BF3OEt2; (iii) H2/Pd/C/HAc-0.1%conc. H2SO4
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Table 1. Structures of isoflavonoids and baicalein as inhibitors of human lipoxygenases and their
inhibitory activity (IC50) against platelet 12-LOX, reticulocyte 15- LOX-1, epithelial 15-LOX-2.
Compounds 12-LOX 15-LOX-1 15-LOX-2 R5
R6
R7
R8
R3’
R4’
[µM]
[µM]
[µM]
1a
1b
>100
>100
N.D
H
H
OH
OH
OH
OH
H
H
H
H
Cl
13
>100
0.59
>30
>30
NO2
1c
0.28
H
OH
OH
H
Cl
H
2a
2b
>100
>100
>100
>100
N.D
N.D
H
H
H
H
OH
OH
CH3
CH3
H
H
OH
CH3
2c
>100
>100
N.D
H
H
OH
CH3
H
NO2
2d
3a
3b
>100
>100
>100
>100
>100
>100
N.D
N.D
N.D
H
H
H
H
H
H
OH
OH
OH
CH3
H
H
H
H
CF3
OH
H
H
3c
>100
>100
>100
>100
N.D
N.D
H
H
H
H
OH
H
H
H
H
NO2
3d
OBn
OCH3
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4a
>100
>40
N.D
OH
H
OH
H
H
NO2
4b
5a
>100
>40
>100
29
N.D
N.D
OH
H
H
H
OBn
OH
H
H
H
NO2
Cl
CH3
5b
7.7
4.4
34
H
H
H
H
OH
OH
OH
H
CH3
H
5c
6.4
3.3
5.8
33
71
OH CH3
Baicalein
11.5
*N.D: Not Determined
Table 2. Radical DPPH scavenger properties of twelve compounds. Activity expressed as percentage of
remaining radical.
Compound % DPPH remaining
1b
1c
2a
2b
2c
2d
3a
3b
3c
4a
5b
5c
17.2
4.12
Without activity
Without activity
Without activity
Without activity
Without activity
Without activity
Without activity
Without activity
8.41
6.7
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Table 3. Ki for inhibition of soybean 15-LOX by compounds 1b, 5b, and 5c obtained by nonlinear
regression for one site binding fit of Cheng-Prusoff equation, considering Km = 13 µM [43] and 10 µM
substrate.
Compound
Ki (μM)
R2
Inhibition type
3.434
0.5290
0.2455
0.8683
0.9737
0.9536
Competitive
Competitive
Competitive
1b
5b
5c
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