O-prenylated carbostyrils as a novel class of 15-lipoxygenase inhibitors: Synthesis, characterization, and inhibitory assessment

Article abstract of DOI:10.1111/cbdd.13944

Catalyzed peroxidation of unsaturated lipid in animals and plants intimately is linked to the activity of 15-Lipoxygenase enzymes. Lipoxygenases (LOXs) are well known to play an important role in many acute and chronic syndromes such as inflammation, asthma, cancer, and allergy. In this study, a series of mono prenyloxycarbostyrils were synthesized and evaluated as potential inhibitors of soybean 15-Lipoxygenase (SLO) and their inhibitory potencies were compared to mono prenyloxycoumarins which had been reported in the previous works. The synthetic compounds inhibit lipoxygenase enzyme by competitive mechanism like the prenyloxy coumarins. The results showed that position and length of the prenyl moiety play the important role in lipoxygenase inhibitory activity. Among all of the synthetic compounds (coumarin and carbostyril derivatives), 5-farnesyloxycoumarin and 8-farnesyloxycarbostyril demonstrated the best inhibitory activity by IC₅₀?values of 1.1?μM and 0.53?μM, respectively.

Full text of DOI:10.1111/cbdd.13944

DR. HAMID SADEGHIAN (Orcid ID : 0000-0003-1571-5458)
Article type
: Research Article
O-prenylated carbostyrils as a novel class of 15-lipoxygenase inhibitors:
synthesis, characterization and inhibitory assessment
Seyed Jamal Alavi a*, Amir Zebarjadi a, Mahdi Hosseni Bafghi a, Hossein Orafai b, Hamid
Sadeghian a,c*
* Corresponding author Sadeghianh@mums.ac.ir
* Postal Address: Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad
University of Medical Sciences, Mashhad 91857-63788, Iran
a Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of
Medical Sciences, Mashhad, Iran
b Department of Pharmaceutics, Faculty of Pharmacy, University of Al-Zahraa for Women,
Karbala, Iraq
c Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13944
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Abstract
Catalyzed peroxidation of unsaturated lipid in animals and plants intimately is linked to the
activity of 15-Lipoxygenase enzymes. Lipoxygenases (LOXs) are well known to play an
important role in many acute and chronic syndromes such as inflammation, asthma, cancer, allergy
etc. In this study, a series of mono prenyloxycarbostyrils were synthesized and evaluated as
potential inhibitors of soybean 15-Lipoxygenase (SLO) and their inhibitory potencies were
compared to mono prenyloxycoumarins which had been reported in the previous works. The
synthetic compounds inhibit lipoxygenase enzyme by competitive mechanism like the prenyloxy
coumarins. The results showed that position and length of the prenyl moiety play the important
role in lipoxygenase inhibitory activity. Amongst all of the synthetic compounds (carbostyril and
coumarin derivatives), 6-farnesyloxycoumarin and 8-farnesyloxycarbostyril demonstrated the best
inhibitory activity by IC50 values of 2.1 µM and 0.53 µM respectively.
Keywords: 15-LOX, Carbostyril, Coumarin, Farnesyl, Geranyl.
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Introduction
Lipoxygenases (LOX) are one of the main types of enzyme involved in regio- and stereospecific
oxidation pathways of polyunsaturated fatty acids [1]. Lipoxygenase is the common name of iron-
containing enzymes that play a key role in catalyzing polyunsaturated fatty acids conversion to
hydroyperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs) and
subsequently leukotrienes (LTs), which are the well-known mediators of hypersensitivity and
inflammatory reactions [2, 3]
.
Linoleic acid (LA) is the most common substrate for plant LOXs while arachidonic acid (AA) is
considered as the main substrate of mammalian LOXs. Moreover, peroxidation of LA, as a
preferred substrate, by some of mammalian LOXs has been identified.
There are five different types of mammalian LOXs based on the insertion site of molecular oxygen
into AA: 5-LOX (EC 1.13.11.34), 8-LOX (EC 1.13.11.40), 11-LOX, (EC 1.13.11.45), 12-LOX
(EC 1.13.11.31), and 15-LOX (EC 1.13.11.33) [1].
Up to now, two kinds of mammalian 15-lipoxygenases have been identified: 15-LOX-1 and 15-
LOX-2. LA is the main substrate of 15-LOX-1 that is metabolized to 13(S)-hydroxy-9Z,11E-
octadecadienoic acid (13-S-HODE) while the preferred substrate of 15-LOX-2 is AA oxidized to
15(S)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-S-HETE) [4].
Lipoxygenases by formation of biologically active compounds such as leukotrienes, lipoxins, and
eoxins can interfere in signaling pathways of lipids and finally regulate cell metabolism and
apoptosis [5].
It has been found that overproduction of the lipoxygenases metabolites causes many acute and
chronic diseases such as asthma, allergy, cancer, stroke, ischemia and/or post-ischemic
inflammatory response and arthritis [5, 6]. Due to lipoxygenase importance as therapeutic targets,
there is much interest for designing and discovering of novel and potent lipoxygenase inhibitors.
Three well-known strategies have been established to inhibit the lipoxygenase activity: (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 [7]. It has been shown that the radical scavenging property of the redox inhibitors is the
main factor for lipoxygenase inhibition. They can react with the free radical form of the
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polyunsaturated fatty acid after the hydrogen abstraction step of the hydroperoxidation cycle, and
cause enzyme deactivation [8].
Coumarin is a heterocyclic molecule containing lactone fused benzene skeleton and some of its
derivatives have been introduced as lipoxygenase inhibitors [9-11]
.
Several natural and synthetic derivatives of hydroxycoumarins have been reported as inhibitors of
the lipoxygenase pathways [12- 14]. Most of them inhibit the lipoxygenase activity using redox
mechanism via their hydroxyl groups.
Recently, soybean 15-lipoxygenase (Soybean 13-S-Lipoxygenase-1) and human 15-lipoxygenase-
1 (15-hLOX-1) inhibitory activities of mono O-prenyloxycoumarins (isopentenyloxy, -geranyloxy
and -farnesyloxy derivatives) was reported. The structure activity relationship (SAR) studies
showed that both intermolecular interactions of coumarin ring (hydrogen bond with Fe-OH core
and π-π*/π-δ* between phenyl moiety and active site amino acids) and prenyl substitution site play
important roles in lipoxygenase inhibitory potency [9]. It was also found that prenyl moiety with
more than 10 carbons (farnesyl and geranyl) had significantly higher lipoxygenase inhibitory
potency.
Herein, we tried to study lipoxygenase inhibitory activity of mono O-prenyloxycarbostyril.
Carbostyrils (2-quinolone), which consist of fused benzene and 2-pyridone rings, are another
important group of heterocyclic compounds that are isoelectronic and homolog with coumarins.
Several natural and synthetic derivatives of carbostyril have been introduced as antimicrobial,
anti-inflammatory, anticancer, antiviral, antithrombotic and antiplatelet agents [15-17]
.
In the following of our previous studies on lipoxygenase inhibitors, a series of O-
prenyloxycarbostyril were synthesized and their lipoxygenase inhibitory activity were determined
and compared to O-prenyloxycoumarins.
Results
The desired prenyloxycarbostyril derivatives were synthesized by condensation of
hydroxycarbostyril with the desired pernyl bromide. Starting reagents 6, 7 and 8-
hydroxycarbostyril (10-12) were synthesized from the related hydroxyquinolines according to
previous literatures (Figure 1) [18]. They were produced via the following synthetic steps: (1) N-
oxidation of hydroxyl quinoline derivatives in present of hydrogen peroxide and acetic acid by
under reflux condition; (2) Thermal rearrangement (75 oC) of prepared N-oxides 4-6 to the related
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acetoxycarbostyrils 7-9; (3) Hydrolysing of O-acetyl moiety of compound 7-9 and formation of
products 10-12 (Figure 1) [18]
.
Figure 1
o
5-hydroxycarbostyril (14) was prepared by alkali fusion of 5-hydroxyqunoline at 300 C (Figure
1) [19]. O-prenylation of hydroxycarbostyril with the desired pernyl bromide in the presence of 1,8-
diazabicyclo[5,4,0]undec-7-ene (DBU) afforded the corresponded products: 5-8g and 5-8f (Figure
2) [20]
.
Figure 2
The O-prenyloxycoumarins were synthesized by reaction of the desired hydroxycoumarins with
geranyl and farnesyl bromide in the mixture of potassium carbonate and acetone (Figure 3) [9].
Figure 3
The lipoxygenase inhibitory activities of the O-ptrenylated carbostyrils and coumarins was
assessed against Soybean 13-S-Lipoxygenase-1 (SLO; type I-B; EC 1,13,11,12) using the
modified oxidative reaction of 3-methyl-2-benzothiazolinone (MBTH) with 3-(dimethylamino)
benzoic acid (DMAB), which had been reported by Iranshahi et al. [9]. All the evaluations were
compared to 4-MMPB (4-methyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine), as a
well-known 15-lipoxygenase inhibitor.
Like prenyloxycoumarins, farnesyloxy analogs of carbostyril were more potent than geranyloxy
analogs in the lipoxygenase inhibition. Among the carbostyril derivatives, 8f was the most potent
inhibitor by IC50 value of 0.53 µM. By changing the position of farnesyloxy group on carbostyril
scaffold, decreasing in lipoxygenase inhibition potency was observed for the other analoges as
follow: 6f > 7f > 5f by IC50 values of 2.13, 4.20 and 7.15 µM respectively (Table 1). Such a
variation in lipoxygenase inhibition was also seen for geranyloxycarbostyrils: 8g > 6g > 7g > 5g
by IC50 values of 7.31, 11.92, 15.34 and 17.73 µM respectively (Table 1).
Table 1
Complementary studies on inhibitory mechanism showed that the synthetic inhibitors reduced
lipoxygenase activity by competitive mechanism (Figure 4). None of the compounds showed
DPPH (2,2-diphenyl-1-picrylhydrazyl) bleaching activity at 100 µM.
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Figure 4
Discussion
As seen in Table 1, for both carbostyril and coumarin scaffolds’, increasing of the prenyl length in
a same substitution site has led to increase of the enzyme inhibition ability.
Replacement of carbostyril to coumarin led to significant effect on lipoxygenase inhibition for 5-
and 8- substitutions. Compared to the prenyloxycoumarines, insertion of geranyloxy at position 5
of carbostyril, decreased the lipoxygenase inhibitory potency by 4 folds and it extended to 7 folds
for farnesyloxy at the same position. About position 8, no considerable differences in the
inhibition potency was seen for geranyloxy substituent while it raised around 16 folds for 8-
farnesyloxy carbostyril (8f) in comparison with its homolog 8f' (IC50 8f = 0.53; IC50 8f' = 8.13
µM).
It is interesting that, replacing of lactone portion by lactam, has led to changing of the inhibitory
potencies variation order. For example for farnesyloxy derivatives the inhibitory potencies order
is: 8f > 6f > 7f > 5f compare to 5'f > 6'f > 7'f > 8'f. As seen in Table 1, 8-prenyloxy derivatives of
carbostyril are the best inhibitors while 5-prenyloxy coumarins are the best inhibitors among their
isomers.
None of the compounds bleached the DPPH solution. It implies that they don't have radical
scavenging activity. By considering the competitive inhibition mechanism of the
prenyloxycarbostyrils, it concluded that these compounds belong to the third group of the
lipoxygenase inhibitors (non-redox competitive inhibitors).
Conclusion
In summary, mono prenyloxy derivatives of both carbostyril and coumarin were synthesized and
their inhibitory potency against soybean 15-LOX was evaluated. The results showed that prenyl
moiety length and prenylation position play the important role in lipoxygenase inhibitory activity.
It is notable that the synthetic pernyloxycarbostyril like pernyloxycoumarin inhibits lipoxygenase
enzyme by non-redox competitive mechanism. Amongst all of the synthetic compounds (coumarin
and carbostyril derivatives), 8-farnesyloxycarbostyril and 5-farnesyloxycumarin showed the best
lipoxygenase inhibition activity, respectively.
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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 mass spectra were scanned on a Varian Mat CH-7 instrument at 70
eV. Elemental analysis was obtained on a Thermo Finnigan Flash EA microanalyzer. 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. All chemicals were purchased from Sigma,
Aldrich and Merck Co.
Lipoxygenase Inhibitory Assessment [11]
Two assay solutions (A and B), enzyme and linoleic acid were firstly provided as follow:
Solution A: DMAB (3-dimethylaminobenzoic acid) 50 mM in phosphate buffer (100 mM, PH
7.0).
Solution B: A mixture of MBTH (3-methyl-2-benzothiazolonhydrazone) (10 mM, 3mL) and
hemoglobin (5 mg/mL, 3 mL) in 25 mL phosphate buffer (50 mM, PH 5.0).
The enzyme solution was prepared by dissolving 1 mg lyophilized SLO (L1; Type I-B; EC 1, 13,
11, 12; Sigma Co.) in 5 ml phosphate buffer (50 mM, PH 7.5) and then its activity was checked by
diene formation method at 234 nm. Finally, its activity was decreased to desired quantity by
dilution with the phosphate buffer.
Linoleic acid (LA) solution: A mixture of LA (5.6 mg), ethanol (0.5 mL) and KOH (100 mM) to a
final volume of 5 ml.
Assay procedure: the sample solution in ethanol (25 µL), enzyme solution (4000 units/ml: based
on Abs increase at 234 nm per min; 25 µL) and phosphate buffer (PH 7.5, 50 mM; 900 µL), were
mixed in test-plate and pre-incubated for 5 min at room temperature. Control test was done by 25
µL of pure ethanol. After pre-incubation, LA solution (50 µL) was added (for starting of the
enzymetic reaction) and after 10 min, solution A (270 µL) and subsequently solution B (130 µL)
were added for color formation. 3 min later, 200 µL of sodium dodecyl sulfate solution (2% W/V)
was added to terminate the reaction. The absorbance was recorded at 598 nm. Each experiment
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was performed in triplicate. Calculation of IC50 values and the significant analysis between them
(one-way ANOVA multiple comparisons) was done in GraphPad Prism 6.0.
Inhibitory mechanism
25 µL of the enzyme solution (4000 unit/ml) was added to mixture of phosphate buffer (pH 7.5;
900 µL) and inhibitor (25 µL in ethanol) and incubated for 10 min at room temperature.
Subsequently, 50 µL of LA stock solution was added to the mixture (final concentration: 10, 20,
40, 80 and 160 µM) to start the reaction. After 10 min, solution A (270 µL) and then solution B
(130 µL) were added to initiate chromogenic reaction (preparation of solutions A and B described
in the previous section). 3 min later, 200 µL of a 2% SDS solution was added to terminate the
reaction. The absorbance was recorded at 598 nm compared to the control. These experiments
were performed in triplicate. Using the reaction rates (Abs.min-1), both inhibitor and linoleic acid
concentrations (µM), Michaelis-Menten and Lineweaver-Burk plots were graphed in GraphPad
Prism 6.0.
Determination of DPPH bleaching activity
An Ethyl alcohol solution of DPPH (50 µM) was prepared. The prepared solution was equally
added to 1 mL of the test compounds solutions (solvent: ethyl alcohol). Equal mixture of pure
o
ethyl alcohol and DPPH solution was used as control solution. After 30 min (25 C), the
absorbance was recorded at 517 nm.
Preparation of 5-Hydroxycarbostyril (14)
5-hydroxycarbostyril was prepared by alkali fusion technique as previously reported [17]. Briefly,
5-hydroxyquinoline (3.0 g) and potassium hydroxide (20 g) were placed in a stainless-steel beaker
with a capacity of 50 ml, and then heated to 300 °C. The resulting mixture (frothing black) was
heated for 3 h, when a clear black solution was obtained. After the mixture was cooled to room
temperature, water (150 ml) and excess concentrated hydrochloric acid was added to mixture,
respectively.
The solid formed (tan-color) was filtered and washed with cold water. The crud product was
recrystallized from methanol-water to give a pure product as straw-colored needles melting at 338-
340 oC.
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General procedure for preparation of carbostyril N-oxide (4-6)
To a solution of desired hydroxyquinoline (1-3) (70 mmol) in acetic acid (100 mL) was added.
Hydrogen peroxide (30%, 15 mL) and the resulting mixture were stirred at 75oC for 36 h. After
evaporated the solvent under vacuum, the mixture of water and crushed-ice was added and the
residue was basified with aqueous ammonia. The crud solid was collected and recrystallized with
proper solvent to obtain the pure crystal.
General procedure for preparation of acetoxycarbostyril (7-9)
A mixture of quinolone N-oxide (30 mmol) in Ac2O (30 mL) was refluxed for 18 h (monitored by
TLC). After cooling, the mixture was poured into ice water (100 mL) and neutralized with
aqueous ammonia. Then, reaction mixture was extracted with dichloromethane (3×60 mL). The
organic extracts were combined and washed with water, dried, and then evaporated to give a
brown solid which was crystallized from ethyl acetate [19]
.
6-Acetoxy-2-hydroxyquinoline (7)
Yellow powder, mp 200-203 oC (Lit. mp 199-200 oC) [22]
7-Acetoxy-2-hydroxyquinoline (8)
Brown powder, mp 247-250 oC (Lit. mp 247-2248 oC) [21]
8-Acetoxy-2-hydroxyquinoline (9)
mp 248-250 oC (Lit. 252-254 oC) [18]
General procedure for preparation of hydroxycarbostyril (10-12)
A stirred suspension of acetoxycarbostyril (25 mmol) in hydrochloric acid (37%, 50 mL) was
refluxed for 4 h. After completion the reaction (monitored by TLC), the mixture was poured into
ice water (50 mL). The resulting precipitate was filtered off to hydroxyl carbostyril as a brown
solid. The crud precipitate crystallized from methanol to give the pure solid [21]
.
6-Hydroxycarbostyril (10)
White powder, mp 300-305 oC (Lit. White powder, mp 299-300 oC) [22]
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7-Hydroxycarbostyril (11)
Brown powder, mp 250-252 oC (Lit. Brown powder, mp 251-252 oC) [21]
8-Hydroxycarbostyril (12)
Brown powder, mp 292-295 oC (Lit. Brown powder, mp 297-299 oC) [18]
General procedure for preparation of O-prenyloxycarbostyril (5-8 (g, f))
To a mixture of hydroxycarbostyrils 10-12 (1.5 mmol), DBU (2.3 mmol) in iso-propanol (15 mL),
prenyl bromide (155 mmol) was added and the mixture refluxed for 6. After cooling, the mixture
was diluted with water (10 mL) and then extracted with chloroform (2×15 mL).
The combined extracts were washed with HCl 1 N (2×10 mL), sodium bicarbonate solution (5%,
2×10 mL) and water (2×10 mL), respectively. The solvent was removed under reduced pressure
and the crud products were purified by crystallization from hexane or by column chromatography
(n-hexan: EtOAc).
5-((E)-3,7-dimethylocta-2,6-dienyloxy)quinolin-2(1H)-one (5g)
1
White solid, , mp 95-97 oC; H NMR (301 MHz, Chloroform-d) δ 12.25 (s, 1H), 7.64 (d, J = 9.5
Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 2.6 Hz, 1H), 6.72 (dd, J = 8.6, 2.6 Hz, 1H), 6.55 (d,
J = 9.4 Hz, 1H), 5.40 (t, J = 6.6 Hz, 1H), 5.05- 4.99 (m, 1H), 4.52 (d, J = 6.6 Hz, 2H), 2.04 (m,
4H), 1.70 (s, 3H), 1.61 (s, 3H), 1.52 (s, 3H). 13C NMR (76 MHz, Chloroform-d) δ 164.32, 154.52,
141.68, 140.62, 134.02, 131.90, 123.70, 121.60, 121.05, 120.68, 119.10, 117.52, 110.01, 65.21,
39.75, 26.30, 25.80, 17.03, 16.41. MS (EI, 70 eV): m/z= 297 [M+]; Anal. calcd for C19H23NO2: C,
78.73; H, 7.80; N, 4.71. Found: C, 78.70; H, 7.79; N, 4.70.
5-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)quinolin-2(1H)-one (5f)
1
Yellowish solid, H NMR (301 MHz, Chloroform-d) δ 12.35 (s, 1H), 7.66 (d, J = 9.4 Hz, 1H),
7.40 (d, J = 8.7 Hz, 1H), 6.81 (s, 1H), 6.70 (dd, J = 8.7, 2.2 Hz, 1H), 6.48 (d, J = 9.4 Hz, 1H), 5.40
(t, J = 6.3 Hz, 1H), 5.12 – 5.01 (m, 2H), 4.55 (d, J = 6.6 Hz, 2H), 2.12 – 1.94 (m, 8H), 1.70 (s,
3H), 1.64 (s, 3H), 1.55 (s, 6H). 13C NMR (76 MHz, Chloroform-d) δ 165.11, 160.95, 141.98,
141.01, 140.45, 135.65, 131.41, 129.05, 124.34, 123.63, 118.52, 118.129, 114.46, 113.06, 98.83,
65.24, 39.81, 27.04, 26.43, 25.72, 17.81, 16.82, 16.16. MS (EI, 70 eV): m/z= 365 [M+]; Anal.
calcd for C24H31NO2: C, 78.86; H, 8.55; N, 3.83. Found: C, 78.78; H, 8.52; N, 3.80.
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6-((E)-3,7-dimethylocta-2,6-dienyloxy)quinolin-2(1H)-one (6g)
Yellowish solid, mp 50-52 oC; 1H NMR (301 MHz, Chloroform-d) δ 12.45 (s, 1H), 7.68 (d, J = 9.5
Hz, 1H), 7.31 (d, J = 8.9 Hz, 1H), 7.10 (dd, J = 8.9, 2.6 Hz, 1H), 6.93 (d, J = 2.6 Hz, 1H), 6.65 (d,
J = 9.5 Hz, 1H), 5.43 (t, J = 6.1 Hz, 1H), 5.09 – 4.97 (m, 1H), 4.50 (d, J = 6.5 Hz, 2H), 2.04 (m,
4H), 1.69 (s, 3H), 1.60 (s, 3H), 1.53 (s, 3H). 13C NMR (76 MHz, Chloroform-d) δ 164.12, 154.48,
141.64, 140.59, 133.06, 131.89, 123.73, 121.63, 120.90, 120.48, 119.19, 117.43, 109.98, 65.44,
39.56, 26.31, 25.69, 17.73, 16.74. MS (EI, 70 eV): m/z= 297 [M+]; Anal. calcd for C19H23NO2: C,
78.73; H, 7.80; N, 4.71. Found: C, 78.78; H, 7.80; N, 4.72.
6-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)quinolin-2(1H)-one (6f)
1
Yellowish solid, mp 75-77 oC; H NMR (301 MHz, DMSO-d6) δ 11.65 (s, 1H), 7.83 (d, J = 9.6
Hz, 1H), 7.26 (d, J = 2.5 Hz, 1H), 7.21 (d, J = 2.6 Hz, 1H), 7.14 (dd, J = 8.9, 2.7 Hz, 1H), 6.50 (d,
J = 9.5 Hz, 1H), 5.45 (t, J = 6.0 Hz, 1H), 5.13 – 5.00 (m, 2H), 4.57 (d, J = 6.4 Hz, 2H), 2.12 – 1.89
13
(m, 8H), 1.72 (s, 3H), 1.63 (s, 3H), 1.56 (s, 3H), 1.55 (s, 3H). C NMR (76 MHz, DMSO-d6) δ
161.97, 153.66, 140.62, 140.21, 135.12, 133.77, 131.07, 124.54, 124.01, 122.71, 120.46, 120.20,
120.10, 116.75, 110.81, 65.21, 26.64, 26.13, 25.93, 17.98, 16.83, 16.26. MS (EI, 70 eV): m/z= 365
[M+]; Anal. calcd for C24H31NO2: C, 78.86; H, 8.55; N, 3.83. Found: C, 78.85; H, 8.54; N, 3.82.
7-((E)-3,7-dimethylocta-2,6-dienyloxy)quinolin-2(1H)-one (7g)
White solid, mp 113-116 oC; 1H NMR (301 MHz, Chloroform-d) δ 12.51 (s, 1H), 7.65 (d, J = 9.4
Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 2.2 Hz, 1H), 6.75 (dd, J = 8.7, 2.3 Hz, 1H), 6.47 (d,
J = 9.4 Hz, 1H), 5.42 (t, J = 6.8 Hz, 1H), 5.03 (q, J = 7.7, 6.5 Hz, 1H), 4.56 (d, J = 6.7 Hz, 2H),
2.05 (d, J = 5.3 Hz, 4H), 1.71 (s, 3H), 1.59 (s, 3H), 1.53 (s, 3H). 13C NMR (76 MHz, Chloroform-
d) δ 165.11, 161.24, 142.20, 140.82, 140.43, 131.86, 128.93, 123.75, 118.74, 117.86, 114.17,
113.01, 99.18, 65.29, 39.60, 26.30, 25.68, 17.73, 16.80. MS (EI, 70 eV): m/z= 297 [M+]; Anal.
calcd for C19H23NO2: C, 78.73; H, 7.80; N, 4.71. Found: C, 78.73; H, 7.85; N, 4.72.
7-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)quinolin-2(1H)-one (7f)
White solid, mp 88-90 oC; 1H NMR (301 MHz, Chloroform-d) δ 12.34 (s, 1H), 7.65 (d, J = 9.4 Hz,
1H), 7.36 (d, J = 8.7 Hz, 1H), 6.79 (s, 1H), 6.75 (dd, J = 8.7, 2.2 Hz, 1H), 6.47 (d, J = 9.4 Hz, 1H),
5.42 (t, J = 6.3 Hz, 1H), 5.08 – 4.96 (m, 2H), 4.56 (d, J = 6.6 Hz, 2H), 2.10 – 1.88 (m, 8H), 1.72
13
(s, 3H), 1.60 (s, 3H), 1.53 (s, 6H). C NMR (76 MHz, Chloroform-d) δ 165.00, 161.25, 142.22,
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140.81, 140.39, 135.50, 131.32, 128.95, 124.34, 123.63, 118.74, 117.89, 114.16, 112.96, 99.15,
65.29, 39.69, 39.61, 26.73, 26.23, 25.72, 17.71, 16.82, 16.06. MS (EI, 70 eV): m/z= 365 [M+];
Anal. calcd for C24H31NO2: C, 78.86; H, 8.55; N, 3.83. Found: C, 78.85; H, 8.54; N, 3.82.
8-((E)-3,7-dimethylocta-2,6-dienyloxy)quinolin-2(1H)-one (8g)
Yellowish solid, mp 72-74 oC; 1H NMR (301 MHz, Chloroform-d) δ 9.16 (s, 1H), 7.65 (d, J = 9.6
Hz, 1H), 7.05 (m, 2H), 6.91 (dd, J = 7.0, 2.1 Hz, 1H), 6.59 (d, J = 9.6 Hz, 1H), 5.45 (d, J = 6.5 Hz,
1H), 5.07 – 4.97 (m, 1H), 4.59 (d, J = 6.7 Hz, 2H), 2.06 (d, J = 5.2 Hz, 4H), 1.68 (s, 3H), 1.61 (s,
13
3H), 1.56 (s, 3H). C NMR (76 MHz, Chloroform-d) δ 164.32, 159.12, 141.21, 140.74, 139.41,
130.86, 129.03, 123.55, 119.74, 117.95, 114.25, 113.22, 99.18, 65.29, 39.65, 26.30, 25.65, 17.70,
16.75. MS (EI, 70 eV): m/z= 297 [M+]; Anal. calcd for C19H23NO2: C, 78.73; H, 7.80; N, 4.71.
Found: C, 78.69; H, 7.80; N, 4.70.
8-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)quinolin-2(1H)-one (8f)
1
colorless liquid, mp <30 oC; H NMR (301 MHz, Chloroform-d) δ 9.26 (s, 1H), 7.75 (d, J = 9.6
Hz, 1H), 7.19 – 7.11 (m, 2H), 7.00 (dd, J = 7.0, 2.0 Hz, 1H), 6.68 (d, J = 9.5 Hz, 1H), 5.54 (t, J =
6.4 Hz, 1H), 5.19 – 5.08 (m, 2H), 4.69 (d, J = 6.7 Hz, 2H), 2.19 – 1.98 (m, 8H), 1.78 (s, 3H), 1.70
13
(s, 3H), 1.65 (s, 3H), 1.62 (s, 3H). C NMR (76 MHz, Chloroform-d) δ 164.10, 161.20, 142.22,
140.81, 140.21, 135.52, 131.42, 129.05, 124.45, 123.60, 118.80, 117.89, 114.20, 112.90, 99.10,
65.25, 39.71, 39.63, 26.70, 26.20, 25.82, 17.75, 16.82, 16.16. MS (EI, 70 eV): m/z = 365 [M+];
Anal. calcd for C24H31NO2: C, 78.86; H, 8.55; N, 3.83. Found: C, 78.85; H, 8.50; N, 3.85.
General procedure for preparation of O-prenyloxycoumarin (5'-8' (g, f)):
[9]
O-prenyloxycoumarin derivatives were prepared according to previously reported method
.
Briefly, a mixture of hydroxycoumarins (5 mmol), prenyl bromide (6 mmol) and anhydrous
potassium carbonate (5 mmol) in dry acetone (3 mL) was refluxed for 12 h. After cooling the
mixture, 10 mL water was added and then extracted with ether (2×20 mL). The combined extracts
were washed with sodium hydroxide solution (10%, 2×10 mL) and dried with anhydrous sodium
carbonate. The solvent was removed under reduced pressure and the crud products were purified.
Conflict of Interest
The authors declare that they have no conflict of interest.
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Supporting Information Available
Copies of 1H NMR,13C NMR and Mass spectra of compounds 5g-8g, 5f-8f and significant analysis
of the IC50 values.
.
References
[1]. Butovich I. A, Lukyanova S, M. (2008). Inhibition of lipoxygenases and cyclooxygenases by
linoleyl hydroxamic acid: comparative in vitro studies. Journal of Lipid Research, 49, 1284.
[2]. A.R. Brash. (1999). Lipoxygenases: occurrence, functions, catalysis, and acquisition of
substrate. Journal of Biological Chemistry, 274, 23679.
[3]. S. Yamamoto. (1992). Mammalian lipoxygenases: molecular structures and functions.
Biochimica et Biophysica Acta, 1128,117.
[4]. I. Shureiqi, S.M. Lippman. (2001). Lipoxygenase modulation to reverse carcinogenesis.
Cancer Research, 61, 6307.
[5]. I. A. Butovich, M. Svetlana. Lukyanova. (2008). Inhibition of lipoxygenases and
cyclooxygenases by linoleyl hydroxamic acid: comparative in vitro studies. Journal of Lipid
Research, 49, 1284.
[6]. S. Feltenmark, N. Gautam, A. Brunnström, W. Griffiths, L. Backman, C. Edenius, L.
Lindbom, M. Björkholm, H. Claesson. Proc Natl Acad. Sci. USA. (2008). Eoxins are
proinflammatory arachidonic acid metabolites produced via the 15-lipoxygenase-1 pathway in
human eosinophils and mast cells. Proceedings of the National Academy of Sciences of the United
States of America, 105, 680.
[7]. C. Charlier, C. Michaux. (2003). Dual inhibition of cyclooxygenase-2 (COX-2) and 5-
lipoxygenase (5-LOX) as a new strategy to provide safer non-steroidal anti-inflammatory drugs.
European Journal of Medicinal Chemistry , 38, 645.
[8]. R. Suardiaz, P.G. Jambrina, L. Masgrau, A. González-Lafont, E. Rosta. J.M. Lluch.(2016).
Understanding the Mechanism of the Hydrogen Abstraction from Arachidonic Acid Catalyzed by
the Human Enzyme 15-Lipoxygenase-2. A Quantum Mechanics/Molecular Mechanics Free
Energy Simulation. Journal of Chemical Theory and Computation, 12, 2079.
This article is protected by copyright. All rights reserved

[9]. M. Iranshahi, A. Jabbari, A. Orafaie, R. Mehri, S. Zeraatkar, T. Ahmadi, M. Alimardani, H.
Sadeghian. (2012).Synthesis and SAR studies of mono O-prenylated coumarins as potent 15-
lipoxygenase inhibitors. European Journal of Medicinal Chemistry, 57, 134.
[10]. H. Sadeghian, A. Jabbari. Expert Opin Ther Pat (2016). 15-Lipoxygenase inhibitors: a patent
review. Expert opinion on therapeutic patents, 26, 65-88.
[11] M. Loncaric, I. Strelec, V. Pavic, D. Šubaric, V. Rastija, M. Molnar. (2020). Lipoxygenase
Inhibition Activity of Coumarin Derivatives—QSAR and Molecular Docking Study.
Pharmaceuticals , 13, 154.
[12]. S. J. Alavi, H. Sadeghian, S. M. Seyedi, A. Salimi, H. A. Eshghi. (2018). A novel class of
human 15-LOX-1 inhibitors based on 3-hydroxycoumarin. Chemical biology & drug design, 91,
1125.
[13]. J.R.S. Hoult, M. Paya.(1996). Pharmacological and biochemical actions of simple
coumarins: natural products with therapeutic potential. Gen Pharmacol, 27, 713.
[14]. C.A. Kontogiorgis, D.J. Hadjipavlou-Litina. (2005). Synthesis and antiinflammatory activity
of coumarin derivatives. Journal of Medicinal Chemistry , 48, 6400.
[15]. B.S. Jayashree, S. Thomas, Y. Nayak. Med. Chem. R. (2010). Design and synthesis of 2-
quinolones as antioxidants and antimicrobials: A rational approach. Medicinal Chemistry
Research, 19, 193.
[16]. B.S. Creaven, M. Devereuxm, A. Foltyn, S. McClean, G. Rosair, V.R. Thangella, M. Walsh.
(2010).Quinolin-2(1H)-one-triazole derived Schiff bases and their Cu(II) and Zn(II) complexes:
Possible new therapeutic agents. Polyhedron, 29, 813.
[17]. N.J. Thumar, M.P. Patel. (2011). Synthesis, characterization, and antimicrobial evaluation of
carbostyril derivatives of 1H-pyrazole. Saudi Pharmaceutical Journal, 19, 75.
[18]. G.R. Pettit, W.C. Fleming, Paull KD. (1968). Synthesis of the 6- and 7-hydroxy-5,8-
dioxocarbostyrils. Journal of Organic Chemistry, 33, 1089.
[19]. W.C. Fleming, G.R. Pettit. (1971).Synthesis of 2-dialkylamino-6- and 7-hydroxy-5,8-
dioxoquinolines. J Org Chem. Journal of Organic Chemistry, 36, 3490.
[20]. M. Nikpour, H. Sadeghian, M. R. Saberi, R. Shafiee Nick, S. M. Seyedi, A. Hosseini, H.
Parsaee, A. Taghian Dasht Bozorg. (2010).Design, synthesis and biological evaluation of 6-
(benzyloxy)-4-methylquinolin-2(1H)-one derivatives as PDE3 inhibitors. Bioorganic & Medicinal
Chemistry, 18, 855.
This article is protected by copyright. All rights reserved

[21]. Y. L. Chen, T. C. Wang, K. C. Fang, N. C. Chang, C. C. Tzeng. (1999). Synthesis of Certain
Quinolin-2(1H)-one α-Methylene-γ-butyrolactones as Potential Antiplatelet Agents. Heterocycles,
50, 453.
[22]. T. C. Wang, Y. L. Chen, K. H. Lee, C. C. Tzeng.(1997). Lewis Acid Catalyzed Reaction of
Cinnamanilides: Competition of Intramolecular and Intermolecular Friedel-Crafts Reaction.
Synthesis, 1, 87.
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Table 1: Lipoxygenase Inhibitory assessment and DPPH bleaching data of the O-prenyloxycarbostyrils and O-
prenyloxycoumarins in comparison with 4-MMPB (± SD). Significant analysis of the data is accessible in
supplementary section. (NE = no effect)
% DPPH
bleaching
(100 µM)
% DPPH
bleaching
(100 µM)
Compound
IC50 (μM)
Compound
IC50 (μM)
5g
17.73 ± 1.81
11.92 ± 1.62
15.34 ± 1.43
7.31 ± 0.73
7.15 ± 0.64
2.13 ± 0.21
4.20 ± 0.17
0.53 ± 0.06
18.6 ± 1.44
NE
NE
5'g
6'g
7'g
8'g
5'f
6'f
7'f
8'f
3.93 ± 0.92
13.46 ± 1.66
8.86 ± 1.21
11.35 ± 1.41
1.10 ± 0.11
2.93 ± 0.26
4.45 ± 0.71
8.15± 1.13
NE
NE
NE
NE
NE
NE
NE
NE
6g
7g
NE
8g
NE
5f
NE
6f
7f
NE
NE
8f
NE
4-MMPB
57.5 ± 3.6
Ac2O
AcOH/H2O2
75oC/36h
HCl
HO
AcO
HO
HO
75oC/18h
Reflux
O
N
O
N
N
N
H
O
H
1-3
4-6
7-9
10-12
OH
OH
KOH
300oC, 3 h
N
N
O
H
13
14
Figure 1: General procedure for the synthesis of compounds 10-14.
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DBU, RBr
HO
RO
i-PrOH, reflux
N
H
O
N
H
O
5-8 (g, f)
10-12, 14
R =
*
*
OR
5g
6g
5f
6f
N
H
O
O
O
O
RO
RO
N
H
7g
8g
7f
8f
N
H
N
H
OR
Figure 2: General procedure for the synthesis of compounds 5-8 (g, f). (DBU = 1,8-diazabicyclo[5,4,0]undec-7-
ene)
K2CO3, RBr
Acetone, reflux
HO
RO
O
O
O
O
5'- 8' (g, f)
R =
*
*
g
f
Figure 3: General procedure for the synthesis of O-prenyloxycoumarins 5'-8' (g, f).
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Figure 4: Lineweaver-Burk plot of SLO inhibition by 8f at concentrations 0, 0.3, 0.6 and 1.2 µM (LA: Linoleic
acid). Km = 22.15 ± 1.65 µM and Ki = 0.24 ± 0.018 µM.
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