A novel class of human 15-LOX-1 inhibitors based on 3-hydroxycoumarin

Article abstract of DOI:10.1111/cbdd.13174

Inflammations, sensitivities, and some cancers in mammals are intimately linked to the activity of lipo-oxygenase enzymes. Owing to the importance of these enzymes, mechanistic studies, product analysis, and synthesis of inhibitors have expanded. In this study, a series of hydroxycoumarins, methoxy-3-hydroxy coumarins, and 7-alkoxy-3-hydroxy coumarins were synthesized and evaluated as potential inhibitors of human 15-LOX-1. Among the synthetic coumarins, 7-methoxy-3-hydroxycoumarin derivative demonstrated potent inhibitory activity and the compound, 5f, showed the best result. Radical scavenging assessment, IC₅₀, HNMR, and DPPH bleaching results indicate that the electronic properties are the major factors for the lipo-oxygenase inhibition potency of the synthetic coumarins. Based on the theoretical studies, it was suggested that the mesomeric effect of the substituent at the seventh position of the benzene ring is one of the major factors in the stability of the oxy-radical intermediate.

Full text of DOI:10.1111/cbdd.13174

DR SEYED MOHAMAD SEYEDI (Orcid ID : 0000-0001-5542-5880)
Article type : Research Article
A novel class of human 15-LOX-1 inhibitors based on 3-hydroxycoumarin
Seyed Jamal Alavi a, Hamid Sadeghian b,c*, Seyed Mohammad Seyedi a*, Alireza Salimi a, Hossein
Eshghi a
* Corresponding author smseyedi@ferdowsi.um.ac.ir, sadeghianh@mums.ac.ir
* Postal Address: Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad,
91775-1436 Mashhad, Iran
a Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
b Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad,
Iran
c Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of
Medical Sciences, Mashhad, Iran
Abstract:
Inflammations, sensitivities, and some cancers in mammals are intimately linked to the activity of
lipoxygenase enzymes. Owing to the importance of these enzymes, mechanistic studies, product
analysis, and synthesis of inhibitors have expanded. In this study, a series of hydroxycoumarins,
methoxy-3-hydroxy coumarins and 7-alkoxy-3-hydroxy coumarins were synthesized and evaluated
as potential inhibitors of human 15-LOX-1. Among the synthetic coumarins, 7-methoxy-3-
hydroxycoumarin (7-M3HC) derivative demonstrated potent inhibitory activity and the compound,
5f, showed the best result. Radical scavenging assessment, IC50, HNMR, and DPPH bleaching results
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doi: 10.1111/cbdd.13174
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indicate that the electronic properties are the major factors for the lipoxygenase inhibition potency
of the synthetic coumarins. Based on the theoretical studies, it was suggested that the mesomeric
effect of the substituent at the 7th position of the benzene ring is one of the major factors in the
stability of the oxy radical intermediate.
Keywords: DPPH, MBTH, DMAB, inhibitory mechanism, bond energy dissociation.
Introduction:
At the past decades, the study on synthesis and evaluation of anticancer and anti-inflammation
agents has been developed.1,2 One of the causes of the mentioned disease is the lipoxygenases
enzymes.
Lipoxygenases are a kind of iron-containing enzymes that can catalyse hydroperoxidation of
polyunsaturated fatty acids derivatives, containing a cis, cis-1, and 4-pentadiene moiety (for
example: arachidonic and linoleic acids). 3,4 These enzymes are well-known for their role in many
acute and chronic diseases such as inflammation, cancer, asthma, allergy, and stroke.5 Due to their
importance as therapeutic targets, there is much interest among scientists for the discovery and
synthesis of novel and potent lipoxygenase inhibitors.
Three well-known strategies have been established to inhibit the lipoxygenase activity. They are: (i)
redox inhibitors or antioxidants that interfere with the redox cycle of hydroperoxidation, (ii) iron-
chelator agents that directly form a complex with iron core of the enzyme, and (iii) non-redox
competitive inhibitors that only compete with the substrate for occupying the active pocket of the
enzyme.6 It has been proven that most of the redox inhibitors play their inhibitory role using their
radical scavenging property. They can deactivate the enzyme by interfering with the free radical
form of the substrate after the hydrogen abstraction step of the hydroperoxidation cycle.7
One of the well-known groups of the heterocyclic compounds, which have been introduced as
lipoxygenase inhibitors, are coumarins. Coumarins consist of fused benzene and a lactone rings
(pyrone), and conform a very large class of phenolic derivatives in natural sources. More than 1,300
derivatives of coumarins, possessing anti-inflammatory and anticancer properties8, have been
isolated from green plants, fungi, and bacteria.9 Several natural and synthetic derivatives of
hydroxycoumarins have been reported as inhibitors of the lipoxygenase pathways.10-12 Most of them
inhibit the lipoxygenase activity using redox mechanism with their hydroxyl groups.13 In this study,
lipoxygenase inhibitory potency of all the mono-hydroxycoumarins against human 15-LOX-1 and
their radical scavenging activity apart from their inhibition mechanism has been comparatively
observed.
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Materials and methods
Instruments
1H NMR (300 MHz) spectra were obtained by using a Bruker Avance DRX-300 Fourier transformer
spectrometer. Chemical shifts are reported in parts per million (δ) downfield from tetramethylsilane
(TMS). The melting points were recorded on an Electrothermal type 9100 melting point apparatus.
All measurements of DPPH bleaching and lipoxygenase activities were carried out using Spekol 1500
spectrophotometer and BioTek ELX800 plate reader. The 2-Hydroxy-3-methoxybenzaldehyde, 2-
Hydroxy-4-methoxybenzaldehyde and 2-Hydroxy-6-methoxybenzaldehyde were obtained from Alfa
Aesar and other chemicals were purchased from Sigma, Aldrich and Merck Co.
Enzyme preparation
Enzyme preparation was down according to the pervious method which had been reported by
Jabbari et al.12
Lipoxygenase Inhibitory Assessment
Linoleic acid and two assay solutions (A and B) were prepared in advance.
Solution A was 50 mM DMAB in an l00 mM phosphate buffer (pH 7.0). Solution B was a mixture of l0
mM MBTH (3 mL), hemoglobin (5 mg/mL, 3 mL) in 50 mM phosphate buffer at pH 5.0 (25 ml). A
linoleic acid solution was prepared by mixing 5.6 mg of linoleic acid (Sigma Aldrich, L1376) with 0.5
mL methanol and then diluted with KOH 100 mM to a final volume of 5 mL (4 mM).
In the standard assay, the sample in DMSO (12.5 μL), 15-LOX-1 (104 units/mL; 30 μL) and phosphate
buffer, pH 7.0 (50 mM; 435 μL) were mixed in 48 well plate and perincubation was carried out for 10
min at 30°C. A control test was done with the same volume of ethanol. After the preincubation,
linoleic acid solution (25 μL) was added to start the peroxidation reaction at 30°C, and, 10 min later,
solution A (135 μL) and then solution B (65 μL) was added to start the color formation. Further, 3
min later, 100 μL of a 2% SDS solution was added to terminate the reaction. The absorbance at 598
nm was compared with the control test. These experiments were performed in triplicate. The data
analysis was performed using GraphPad Prism 5.01.
Determination of DPPH bleaching
25 µM solution of DPPH in absolute ethanol was prepared. This solution was added to an equal
volume of the solution of the test compounds (dissolved in ethanol) to obtain a desired
concentration. Ethanol was used as control solution. After 30 min at room temperature, the
absorbance was recorded at 517 nm and compared to NDGA (nordihydroguaiaretic acid).
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General procedure for the preparation of NAC and 2:
Desired benzaldehyde (20.0 mmol), N-acetylglycine (2.34 g, 20.0 mmol) and anhydrous sodium
acetate (4.92 g, 60.0 mmol) were refluxed in Ac2O (40 mL) for 6 h. After cooling, water added to
reaction mixture and solid was filtered. The solid was recrystallized from ethanol to yield the pure
final products.
General procedure for the preparation 3-amino-2H-chromen-2-one (3AC):
2-hydroxybenzaldehyde (20.0 mmol), N-acetylglycine (2.34 g, 20.0 mmol) and anhydrous sodium
acetate (4.92 g, 60.0 mmol) were refluxed in Ac2O (40 mL) for 6 h. After cooling, water added to
reaction mixture and solid was filtered.
The previous solid was refluxed in a solution of concentrated HCl and ethanol (2:1) for 2 h, then the
solution was poured into ice water (100 mL), 30% NaOH aqueous solution was added to make pH 5–
6, the crude product was precipitated and collected. Then it was recrystallized from EtOH to obtain
pure product 3AC.
General procedure for the preparation 3-hydroxy-n-methoxy-2H-chromen-
2-one (M3HC):
The3-hydroxy-n-methoxy-2H-chromen-2-one (M3HC) was obtained by dissolving 5.14 mmol of NAC
in MeOH (36 mL) and 3N HCl (200 mL) and refluxed for 10 h. after cooling, the precipitated solid was
filtered off, washed with water and crystallization from ethanol to obtain the pure product.
3-hydroxy-5-methoxy-2H-chromen-2-one (5-M3HC):
Brown powder, mp 158-160 oC
3-hydroxy-6-methoxy-2H-chromen-2-one (6-M3HC):
Yellow powder, mp 202-204 oC (lit. 208 oC) 21, 22
3-hydroxy-7-methoxy-2H-chromen-2-one (7-M3HC):
Yellow powder, mp 177-179 oC (lit. 175 oC) 23
3-hydroxy-8-methoxy-2H-chromen-2-one (8-M3HC):
Brown powder, mp 168-170oC, (lit. 178 oC) 24
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General procedure for the preparation N-(7-hydroxy-2-oxo-2H-chromen-3-
yl)acetamide (3):
2,4-Dihydroxy-benzaldehyde (2.76 g, 20.0 mmol), N-acetylglycine (2.34 g, 20.0 mmol) and anhydrous
sodium acetate (4.92 g, 60.0 mmol) were refluxed in Ac2O (40 mL) for 6 h. After cooling, water added
to reaction mixture and solid was filtered. The solid was recrystallized from ethanol to yield the 3-
acetamido-2-oxo-2H-chromen-7-ylacetate (2) as a yellow solid. mp 260-263°C (lit. 233–234°C).
3-Acetamide-2-oxo-2 H-chromen-7-yl acetate (1.31 g, 5.0 mmol), K2CO3 (1.38 g, 20.0 mmol) were
refluxed in MeOH (25 mL) for 1.5 h The reaction mixture was evaporated and solved in water (25
mL). After acidification with 2 N HCl to pH 3, the resulting precipitate was filtered off to give N-(7-
hydroxy-2-oxo-2H-chromen-3-yl)acetamide (3) as a light brown solid: mp 315 °C (Lit. 303–304 °C)
General procedure for the preparation of 4a-g:
To a mixture of 4.5 mmol of N-(7-hydroxy-2-oxo-2H-chromen-3-yl)acetamide (3) in 20 mL of dry
acetone was added 10 mmol of anhydrous potassium carbonate and 6.5 mmol of alkyl bromide, and
the mixture was refluxed for 6 h. After the mixture was cooled to room temperature, filtered the
solid and washed the solid with cold acetone. The solvent was removed under reduced pressure,
and the crud product was crystallized from ethanol.
N-(7-(propyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4a):
yellowish green powder (mp 188-190 oC), 1H NMR (301 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.61 (s, 1H),
7.67 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.5, 2.5 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H),
2.20 (s, 3H), 1.81 (h, J = 7.1 Hz, 2H), 1.05 (t, J = 7.4 Hz, 3H); 13C NMR (76 MHz, DMSO-d6) δ 170.26 ,
160.66 , 158.25 , 151.77 , 129.25 , 125.54, 114.78 , 113.57 , 113.01 , 102.46 , 70.11 , 24.30 , 22.33 ,
10.82 .
N-(7-butoxy-2-oxo-2H-chromen-3-yl)acetamide (4b):
Yellow powder(mp 170-172 oC), 1H NMR (301 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.61 (s, 1H), 7.67 (d, J =
8.6 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.6, 2.5 Hz, 1H), 4.12 (t, J = 6.5 Hz, 2H), 2.20 (s, 3H),
1.78 (p, J = 8.4, 6.5 Hz, 2H), 1.51 (h, 2H), 1.00 (t, J = 7.4 Hz, 3H); 13C NMR (76 MHz, DMSO-d6) δ
170.34 , 160.68 , 158.25 , 151.77 , 129.25 , 125.54 , 122.24 , 113.58 , 113.01 , 101.39 , 68.36 , 31.00 ,
24.30 , 19.16 , 14.14.
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N-(7-isobutoxy-2-oxo-2H-chromen-3-yl)acetamide (4c):
Yellow powder (mp 198-200 oC), 1H NMR (301 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.50 (s, 1H), 7.66 (d, J =
8.6 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 8.6, 2.5 Hz, 1H), 3.86 (t, J = 13.2 Hz, 2H), 2.18 (s, 3H),
2.08 (m, 1H), 1.04 (d, J = 6.6 Hz, 6H); 13C NMR (76 MHz, DMSO-d6) δ 170.33 , 160.65 , 158.24 , 151.76
, 129.23 , 125.51 , 122.24 , 113.59, 113.01 , 102.49 , 67.14 , 28.08 , 25.03 , 19.46.
N-(2-oxo-7-(pentyloxy)-2H-chromen-3-yl)acetamide (4d):
Yellow powder (mp 160-162 oC) , 1H NMR (301 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.60 (s, 1H), 7.65 (d, J
= 8.6 Hz, 1H), 7.03 (d, J = 2.3 Hz, 1H), 6.98 (dd, J = 8.6, 2.4 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 2.20 (s, 3H),
1.84 – 1.72 (m, 2H), 1.43 (m, 4H), 0.95 (t, J = 7.0 Hz, 3H); 13C NMR (76 MHz, DMSO-d6) δ 170.33 ,
160.65 , 158.24 , 151.75 , 129.22 , 125.49 , 122.23 , 113.55 , 112.99 , 101.36 , 68.64 , 31.16 , 28.11 ,
24.29 , 22.33 , 14.38.
N-(7-(isopentyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4e):
Light yellow powder (mp 163-166 oC), 1H NMR (301 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.60 (s, 1H), 7.66
(d, J = 8.7 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.6, 2.4 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 2.20 (s,
3H), 1.91 – 1.76 (m, 1H), 1.69 (q, J = 6.7 Hz, 2H), 0.99 (d, J = 6.6 Hz, 6H);13C NMR (76 MHz, DMSO-d6)
δ 170.33 , 160.65 , 158.24 , 151.76 , 129.23 , 125.51 , 122.24 , 113.59 , 113.01 , 101.40 , 67.14 , 37.68
, 25.03 , 24.30 , 22.86 .
N-(7-(hexyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4f):
Yellow powder (mp 141-142 oC), 1H NMR (301 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.61 (s, 1H), 7.67 (d, J =
8.6 Hz, 1H), 7.04 (s, 1H), 7.00 (d, J = 8.6 Hz, 1H), 4.11 (t, J = 6.3 Hz, 2H), 2.20 (s, 3H), 1.78 (m, 2H),
1.48 (m, 2H), 1.42 – 1.32 (m, 4H), 0.94 (t, J = 6.3 Hz, 3H); 13C NMR (76 MHz, DMSO-d6) δ 170.26 ,
160.67 , 158.25 , 151.76 , 129.25 , 125.55 , 122.24 , 113.58 , 101.38 , 68.66 , 31.44 , 28.92 , 25.60 ,
24.26 , 22.54 , 14.40 .
N-(7-(heptyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4g):
Yellow powder (mp 138-140 oC), 1H NMR (301 MHz, Chloroform-d) δ 8.56 (s, 1H), 7.90 (s, 1H), 7.32
(d, J = 8.6 Hz, 1H), 6.80 (dd, J = 8.6, 2.3 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 3.92 (t, J = 6.5 Hz, 2H), 2.16 (s,
3H), 1.74 (p, J = 6.6 Hz, 2H), 1.45 – 1.33 (m, 2H), 1.26 (m, 6H), 0.83 (t, J = 6.6 Hz, 3H); 13C NMR (76
MHz, Chloroform-d) δ 169.18 , 160.88 , 159.11 , 151.44 , 128.58 , 124.25 , 121.42 , 113.61 , 112.84 ,
101.21 , 68.65 , 31.75 , 29.02 , 25.94 , 24.70 , 22.60 , 14.09.
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General procedure for the preparation of 5a-g:
One gr of N-(7-alkoxy-2-oxo-2H-chromen-3-yl)acetamide (4a-g) was suspended in 15 ml of mixture
of HOAC/HCl (1:1) and refluxed for 16 h. After the reaction mixture was cooled, the precipitated
solid was filtered, washed with water and crystallization from ethanol to obtain the pure product.
3-hydroxy-7-propoxy-2H-chromen-2-one (5a):
yellow powder (mp 125 oC), 1H NMR (301 MHz, Chloroform-d) δ 7.22 (d, J = 8.5 Hz, 1H), 6.95 (s, 1H),
6.80 (dd, J = 8.5, 2.4 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.01 (s, 1H), 3.88 (t, J = 6.5 Hz, 2H), 1.75 (p, J =
7.3 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); 13C NMR (76 MHz, Chloroform-d) δ 160.82 , 159.82 , 150.68 ,
138.06 , 127.28 , 115.55 , 113.55 , 113.29 , 101.47 , 70.05 , 22.41 , 10.47.
7-butoxy-3-hydroxy-2H-chromen-2-one (5b):
light brown (mp 73 oC), 1H NMR (301 MHz, DMSO-d6) δ 7.45 (d, J = 8.6 Hz, 1H), 7.11 (s, 1H), 6.91 (d, J
= 2.9 Hz, 1H), 6.87 (dd, J = 8.4, 2.5 Hz, 1H), 6.73 (s, 1H), 4.02 (t, J = 6.5 Hz, 2H), 1.77 – 1.65 (m, 2H),
1.45 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR (76 MHz, DMSO-d6) δ 160.82 , 159.83 ,
150.69 , 138.04 , 127.28 , 115.09 , 115.14 , 113.56 , 113.27, 101.46 , 68.59 , 28.75, 22.14, 14.01.
3-hydroxy-7-isobutoxy-2H-chromen-2-one (5c):
brown powder(mp 107-110 oC), 1H NMR (301 MHz, DMSO-d6) δ1H NMR (301 MHz, DMSO-d6) δ 7.50
(d, J = 8.5 Hz, 1H), 7.16 (s, 1H), 7.00 (d, J = 2.4 Hz, 1H), 6.96 (dd, J = 8.5, 2.4 Hz, 1H), 6.68 (s, 1H), 3.86
(d, J = 6.5 Hz, 2H), 2.09 (dq, J = 13.3, 6.6 Hz, 1H), 1.04 (d, J = 6.7 Hz, 6H); 13C NMR (76 MHz, DMSO-d6)
δ 159.25 , 159.05 , 150.87 , 139.66 , 127.60 , 116.23 , 113.92 , 113.19 , 101.45 , 74.67 , 28.08 , 19.46.
3-hydroxy-7-(pentyloxy)-2H-chromen-2-one (5d):
yellow powder (mp 74-76 oC), 1H NMR (301 MHz, Chloroform-d) δ 7.22 (d, J = 8.5 Hz, 1H), 6.95 (s,
1H), 6.80 (dd, J = 8.5, 2.4 Hz, 1H), 6.77 (d, J = 2.2 Hz, 1H), 5.96 (s, 1H), 3.91 (t, J = 6.5 Hz, 2H), 1.74 (p,
J = 6.7 Hz, 2H), 1.45 – 1.25 (m, 4H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR (76 MHz, Chloroform-d) δ 160.82
, 159.83 , 150.69 , 138.04 , 127.28 , 115.09 , 113.56 , 113.27 , 101.46 , 68.59 , 28.75 , 28.14 , 22.44 ,
14.01.
3-hydroxy-7-(isopentyloxy)-2H-chromen-2-one (5e):
light yellow powder (mp 100-102 oC), 1H NMR (301 MHz, Chloroform-d) δ7.22 (d, J = 8.3 Hz, 1H), 6.95
(s, 1H), 6.85 – 6.73 (m, 2H), 5.96 (s, 1H), 3.95 (t, J = 6.6 Hz, 2H), 1.78 (dp, J = 13.1, 6.6 Hz, 1H), 1.63 (q,
J = 6.7 Hz, 2H), 0.90 (d, J = 6.6 Hz, 6H); 13C NMR (76 MHz, Chloroform-d) δ 160.81 , 159.81 , 150.69 ,
138.04 , 127.28 , 115.07 , 113.56 , 113.28 , 101.47 , 66.99 , 37.72 , 25.04 , 22.56.
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7-(hexyloxy)-3-hydroxy-2H-chromen-2-one (5f):
brownish yellow color (mp 78-80 oC), 1H NMR (301 MHz, Chloroform-d) δ 7.23 (d, J = 8.4 Hz, 1H),
6.95 (s, 1H), 6.80 (dd, J = 8.4, 2.5 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 5.89 (s, 1H), 3.92 (t, 2H), 1.79 – 1.68
(m, 2H), 1.41 (p, J = 7.1, 6.5 Hz, 2H), 1.34 – 1.15 (m, 4H), 0.85 (t, J = 7.0 Hz, 3H); 13C NMR (76 MHz,
Chloroform-d) δ 160.82 , 159.83 , 150.69 , 138.14 , 127.28 , 115.09 , 113.56 , 113.27 , 101.46 , 68.59 ,
28.75 , 28.14 , 24.56 , 22.44 , 14.01.
7-(heptyloxy)-3-hydroxy-2H-chromen-2-one (5g):
light yellow powder (mp 75-76 oC), 1H NMR (301 MHz, Chloroform-d) δ 7.22 (d, J = 8.4 Hz, 1H), 6.95
(s, 1H), 6.79 (dd, J = 8.4, 2.5 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 5.94 (s, 1H), 3.91 (t, J = 6.5 Hz, 2H), 1.73
(p, J = 6.6 Hz, 2H), 1.46– 1.33 (m, 2H), 1.26 (m, 6H), 0.88– 0.77 (t, J = 7.0 Hz, 3H); 13C NMR (76 MHz,
Chloroform-d) δ 160.82 , 159.83 , 150.69 , 138.03 , 127.28 , 115.07 , 113.56 , 113.26 , 101.47 , 68.61 ,
31.76 , 29.06 , 29.03 , 25.95 , 22.61 , 14.09.
Results and discussion:
Among the mono-hydroxycoumarins, 5- and 8-hydroxycoumarin that were not commercially
accessible were synthesized according to the procedure reported by Iranshahi et al.14
The lipoxygenase inhibitory potency of all of the mono-hydroxycoumarins was studied against
human 15-LOX-1 using the modified oxidative coupling of 3-methyl-2-benzothiazolinone (MBTH)
with 3-(dimethylamino) benzoic acid (DMAB), which was reported by Jabbari et al.15 Human 15-LOX-
1 was prepared from isolated human eosinophil based on the procedure reported by Jabbari et al.15
In this procedure, the peroxide concentration measurement is the basis of lipoxygenase activity
determination.
Among the mentioned coumarins, 3-hydroxy derivative was the potent lipoxygenase inhibitor by IC50
value of 9.5 µM and after that, 4-, 5-, 6-, 8- and 7-hydroxy isomers were in the next rank,
respectively. Replacement of 3-hydroxy with 3-amino group (compound 3AC)16 led to the
elimination of the lipoxygenase inhibition activity. Determination of the type of enzyme inhibition,
using the Lineweaver–Burk plot, showed that 3-hydroxycoumarin inhibits lipoxygenase activity by
non-competitive mechanism (Figure 1). The Ki can be obtained via Lineweaver–Burk equation:
1/V = D. (Km/Vmax.[S]) + D/Vmax
When V = Vmax/2
D = 1 + [I]/Ki
[I] = IC50
=>
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Figure 1
Radical scavenging activities of the mono-hydroxycoumarins were comparatively tested using the
DPPH bleaching method. It was interesting to see that the radical scavenging activity of the
coumarins were in accordance to their lipoxygenase inhibition potency.
In order to find the chemical factors that make 3-hydroxycoumarin the potent radical scavenger,
first, we studied the reaction mechanism of the mono-hydroxycoumarins with DPPH using the
HNMR spectra of their mixture in CDCl3. The NMR spectra of the hydroxycoumarins showed that the
hydrogen abstraction from hydroxyl groups of the coumarin is down by DPPH. Methylation of the
hydroxy group (compound 3MC) led to the elimination of DPPH bleaching and lipoxygenase
inhibition (Table 1).
Table 1
Observing the conformity among IC50 variations and DPPH bleaching results apart from the HNMR
data indicates that the electronic properties is the major factor for the lipoxygenase inhibition
potency of the aforementioned coumarins.
The stabilization energies related to the optimized structures of 3HC to 8HC in both natural and oxy-
radical states were computed using the B3LYP/6-311++G (d, p) level of theory.17,18 An acceptable
relationship was only found between the stabilization energy of the radical forms (Er) and the DPPH
bleaching potencies. The aforementioned relationship has been shown by making a diagram of the
DPPH bleaching IC50 versus Er difference between 3HC and the other isomers (Figure 2).
Table 2
Figure 2
In order to rationalise the electronic effect on the radical scavenging activity of the
hydroxycoumarins, the DPPH bleaching potency of methoxy derivatives of 3HC (the best radical
scavenger) were evaluated. For this purpose, 5, 6, 7, and 8-methoxy derivatives of 3HC were
prepared by coupling the related methoxyhydroxybenzaldehyde (MHB) with N-acetylglycine and
then hydrolysing the formed product NAC in acid (Figure 3).19, 20
Figure 3
Among the methoxy derivatives, 7-methoxy-3-hydroxycoumarin (7-M3HC) showed the best radical
scavenging activity, followed by 5-, 6- and 8-methoxy isomers, respectively. The observed variation
in the radical scavenging potency is in accordance to the mesomeric effect of the methoxy
substituent on the stability of the oxy radical intermediate. Acceptable relationship was found
between DPPH bleaching potencies and both of Er and bond energy dissociation (BDE) (Table 2). It is
interesting that the same variation was seen for lipoxygenase inhibition. 7-M3HC showed the best
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lipoxygenase inhibition potency, hence, it was decided to study the effect of the chain length of 7-
alkoxy moiety on the human 15-LOX-1 inhibitory activity of 3HC.
Table 3
Seven alkoxy derivatives of 3HC, compounds 5a-g, were synthesized according to the former
procedure mentioned for the preparation of methoxy-3-hydroxycoumarins. In this study, the
synthesis of compounds 5a-g was started by coupling 2, 4-dihydroxycoumarinwith N-acetylglycine
and the formation of diacetyl derivative of 3-Amino-7-hydroxycoumarin (2). Mild hydrolysis of O-
acetyl moiety of 2 produces compound 3. O-Alkylation of 3 and subsequent hydrolysing of
acetamide moiety with a slight modification the previous method led to the formation of products
5a-g (Figure 4).19 Varying the size of the alkoxy moiety led to no significant changes in lipoxygenase
inhibition (less than 2 fold variation in IC50). In contrast with 7-M3HC, an increase of the alkoxy
chain length to six carbon, only led to a twofold increase in the lipoxygenase inhibition activity (IC50 =
2.9 µM for compound 5f) while increasing the alkoxy length to seven carbon led to a 1.5-time
decrease in lipoxygenase inhibition (IC50 = 9.2 µM for compound 5g). It showed that the electronic
effect (redox potency) is dominant against the steric effects of the alkoxy moieties for compounds
5a-g (Table 3).
Figure 4
Conclusion
In summary, we have successfully introduced and synthesised 3-hydroxycoumarin as a potent and
redox inhibitor of human 15-LOX-1. Radical scavenging assessment, IC50, DPPH bleaching and
stabilization energies assessment indicate that the electronic properties are the major factors for the
lipoxygenase inhibition potency of the synthetic coumarins. These result showed that the
lipoxygenase inhibition potency of compounds is related to the role of radical intermediate stability
in variation of radical scavenging potency of the mono-hydroxycoumarins. Due to the important role
of hydroxyl group at the third position of coumarin ring, development of the synthetic structures
based on 3-hydroxycoumarin was carried out and synthesized 7-alkoxy-3-hydroxycoumarin as
potent human 15-LOX-1inhibitors.
Acknowledgment:
The authors gratefully acknowledged for partially financial support of this study (Grant No. 37712)
by Research Council of Ferdowsi University of Mashhad.
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Conflict of Interest
The authors declare that they have no conflict of interest.
Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this
article. Appendix S1-S28. Nuclear magnetic resonance (NMR) spectra.
Figure Legends
Figure 1: Lineweaver-Burk plots of human 15-LOX-1 inhibition by 3HC (the error bars are
shown as ± SD).
Figure 2: Diagram of IC50 versus Er of 3HC-8HC.
Figure 3: General procedure for the synthesis of compounds n-M3HC.
Figure 4: General procedure for the synthesis of compounds 5a-g.
Table Legends
Table 1: Human 15-LOX-1 Inhibitory assessment data and DPPH bleaching potency of the
synthetic compounds 3HC- 8HC, 3AC and 3-8M3HC in comparison with 4-methyl-2-(4
methylpiperazinyl)pyrimido[4, 5-b]benzothiazine (4-MMPB). Data are shown as ± SD.
Table 2: En, Er and bond energy dissociation (BDE) of the synthetic compounds.
Table 3: Human 15-LOX-1 inhibitory assessment data of the synthetic compounds 5a-g (± SD).
Supplementary data captions
Figure S1. (a), (b): 1H NMR spectra of N-(7-(propyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4a)
dissolved in DMSO-d6.
Figure S2: 13C NMR spectra of N-(7-(propyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4a)
dissolved in DMSO-d6.
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Figure S3 (a), (b), (c): 1H NMR spectra of N-(7-butoxy-2-oxo-2H-chromen-3-yl)acetamide (4b)
dissolved in DMSO-d6.
Figure S4: 13C NMR spectra of N-(7-butoxy-2-oxo-2H-chromen-3-yl)acetamide (4b) dissolved in
DMSO-d6.
Figure S5: 1H NMR spectra of N-(7-isobutoxy-2-oxo-2H-chromen-3-yl)acetamide (4c) dissolved in
DMSO-d6.
Figure S6: 13C NMR spectra of N-(7-isobutoxy-2-oxo-2H-chromen-3-yl)acetamide (4c) dissolved in
DMSO-d6.
Figure S7 (a), (b), (c): 1H NMR spectra of N-(2-oxo-7-(pentyloxy)-2H-chromen-3-yl)acetamide (4d)
dissolved in DMSO-d6.
Figure S8: 13C NMR spectra of N-(2-oxo-7-(pentyloxy)-2H-chromen-3-yl)acetamide (4d) dissolved in
DMSO-d6.
Figure S9 (a), (b), (c): 1H NMR spectra of N-(7-(isopentyloxy)-2-oxo-2H-chromen-3-yl)acetamide
(4e) dissolved in DMSO-d6.
Figure S10: 13C NMR spectra of N-(7-(isopentyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4e)
dissolved in DMSO-d6.
Figure S11 (a), (b), (c): 1H NMR spectra of N-(7-(hexyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4f)
dissolved in DMSO-d6.
Figure S12: 13C NMR spectra of N-(7-(hexyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4f) dissolved in
DMSO-d6.
Figure S13 (a), (b), (c): 1H NMR spectra of N-(7-(heptyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4g)
dissolved in Chloroform-d.
Figure S14: 13C NMR spectra of N-(7-(heptyloxy)-2-oxo-2H-chromen-3-yl)acetamide (4g) dissolved
in Chloroform-d.
Figure S15 (a), (b): 1H NMR spectra of 3-hydroxy-7-propoxy-2H-chromen-2-one (5a) dissolved in
Chloroform-d.
Figure S16: 13C NMR spectra of 3-hydroxy-7-propoxy-2H-chromen-2-one (5a) dissolved in
Chloroform-d.
Figure S17 (a), (b), (c): 1H NMR spectra of 7-butoxy-3-hydroxy-2H-chromen-2-one (5b) dissolved in
DMSO-d6.
Figure S18: 13C NMR spectra of 7-butoxy-3-hydroxy-2H-chromen-2-one (5b) dissolved in
DMSO-d6.
Figure S19 (a), (b), (c): 1H NMR spectra of 3-hydroxy-7-isobutoxy-2H-chromen-2-one (5c)
dissolved in DMSO-d6.
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Figure S20: 13C NMR spectra of 3-hydroxy-7-isobutoxy-2H-chromen-2-one (5c) dissolved
in DMSO-d6.
Figure S21 (a), (b): 1H NMR spectra of 3-hydroxy-7-(pentyloxy)-2H-chromen-2-one (5d)
dissolved in Chloroform-d.
Figure S22: 13C NMR spectra of 3-hydroxy-7-(pentyloxy)-2H-chromen-2-one (5d) dissolved
in Chloroform-d.
Figure S23 (a), (b): 1H NMR spectra of 3-hydroxy-7-(isopentyloxy)-2H-chromen-2-one (5e)
dissolved in Chloroform-d.
Figure S24: 13C NMR spectra of 3-hydroxy-7-(isopentyloxy)-2H-chromen-2-one (5e)
dissolved in Chloroform-d.
1
Figure S25 (a), (b), (c): H NMR spectra of 7-(hexyloxy)-3-hydroxy-2H-chromen-2-one
(5f) dissolved in Chloroform-d.
Figure S26: 13C NMR spectra of 7-(hexyloxy)-3-hydroxy-2H-chromen-2-one (5f) dissolved
in Chloroform-d.
1
Figure S27 (a), (b), (c): H NMR spectra of 7-(heptyloxy)-3-hydroxy-2H-chromen-2-one
(5g) dissolved in Chloroform-d.
Figure 28: 13C NMR spectra of 7-(heptyloxy)-3-hydroxy-2H-chromen-2-one (5g) dissolved
in Chloroform-d.
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Table 1
IC50 (µM)
IC50 (µM)
(DPPH bleaching)
> 1000
IC50 (µM) LOX
inhibition
IC50 (µM) LOX
inhibition
compounds
compounds
(DPPH bleaching)
31.2 ± 2.1
3HC
4HC
5HC
6HC
7HC
8HC
3AC
9.5 ± 1.3
3-MC
>1000
153.9 ± 9.8
184.7 ± 14.5
185.1 ± 16.1
782.0 ± 23.8
>1000
199.5 ± 8.6
494.1 ± 13.1
853.4 ± 18.3
790.8 ± 19.4
> 1000
5-M3HC
6-M3HC
7-M3HC
8-M3HC
4-MMPB
7.8 ± 1.0
11.9 ± 0.9
5.7 ± 0.4
21.1 ± 1.8
19.1 ± 2.2
15.2 ± 1.6
29.6 ± 2.5
10.8 ± 1.1
48.9 ± 3.8
-
>1000
> 1000
Table 2
Compound
3HC
En
Er
BDE
-572.268940
-572.269165
-572.264404
-572.261880
-572.264924
-572.265246
-686.790123
-686.789132
-686.790432
-686.785345
-571.635297
-571.632793
-571.632362
-571.632288
-571.632173
-571.627908
-686.159073
-686.155129
-686.160100
-686.151105
0.133746
0.136475
0.129145
0.129695
0.132854
0.137441
0.131215
0.134162
0.130032
0.134000
4HC
5HC
6HC
7HC
8HC
5-M3HC
6-M3HC
7-M3HC
8-M3HC
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Table 3
OH
O
R
O
O
compound
R
IC50 (µM)
5.7 ± 1.1
5.1 ± 0.7
4.1 ± 0.3
3.1 ± 0.2
3 ± 0.1
7-M3HC
5a
Methyl
n-Propyl
n-Butyl
5b
5c
Isobutyl
n-Pantyl
Isopantyl
n-Hexyl
n-Heptyl
5d
5e
3.7 ± 0.3
2.9 ± 0.2
9.2 ± 0.7
5f
5g
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