Synthesis of novel derivatives of chromenone bearing an N-carbamothioyl moiety as soybean 15-LOX inhibitors

Article abstract of DOI:10.3906/kim-1604-13

Novel derivatives of chromenone bearing an N-carbamothioyl moiety were synthesized and evaluated for their soybean 15-LOX inhibitory activity. Synthesis of the target compounds was started from 7-hydroxy-2H-chromen-2-one. It was reacted with 1-fluoro-2(4)

Full text of DOI:10.3906/kim-1604-13

Turk J Chem
Turkish Journal of Chemistry
(2017) 41: 335 – 344
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1604-13
Research Article
Synthesis of novel derivatives of chromenone bearing an N -carbamothioyl moiety
as soybean 15-LOX inhibitors
Robabeh KAVIANI1, Mina SAEEDI2,3, Mohammad MAHDAVI4, Hamid NADRI5,
Alireza MORADI5, Abbas SHAFIEE6, Tahmineh AKBARZADEH3,7,∗
1Department of Medicinal Chemistry, Faculty of Pharmacy, Pharmaceutical Sciences Branch,
Islamic Azad University, Tehran, Iran
2Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
3Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
4Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
5Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences,
Yazd, Iran
6Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
7Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Received: 08.04.2016
•
Accepted/Published Online: 29.10.2016
•
Final Version: 16.06.2017
Abstract:Novel derivatives of chromenone bearing an N -carbamothioyl moiety were synthesized and evaluated for their
soybean 15-LOX inhibitory activity. Synthesis of the target compounds was started from 7-hydroxy-2H -chromen-2-one.
It was reacted with 1-fluoro-2(4)-nitrobenzene to obtain the corresponding nitrophenoxy-chromenone derivative. Reduc-
tion of the nitro group was achieved in the presence of Zn/NH4 Cl and reaction of the latter compound with in situ pre-
pared benzoyl isothiocyanate led to the formation of the title compounds. All compounds were characterized and tested
against soybean 15-LOX. Among them, 4-methyl-N -((4-((2-oxo-2H -chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide
(7l) showed the best activity as potent as the reference drug, quercetin.
Key words: Chromenone, nitro compounds, N -carbamothioyl, soybean 15-LOX
1. Introduction
2H -Chromen-2-one derivatives are significant O-heterocyclic compounds and ubiquitous in a wide range of
bioactive natural and synthetic products.1 Their role in the pharmacotherapy of breast cancer2 and cardiac
diseases3 is irrefutable. Their lipoxygenase inhibitory activity has also been recently studied.4−6
Lipoxygenases (LOXs) are nonheme ferroproteins that catalyze dioxygenation of polyunsaturated fatty
acids containing a cis, cis-1,4-pentadiene unit such as arachidonic acid (AA), linoleic acid, and linolenic acid.
There are three main lipoxygenases (5-, 12-, and 15-LOXs) that are characterized by the peroxidation site of
AA and the corresponding products play important roles in various cell functions.7,8 It has been demonstrated
that the enzyme isozymes and their metabolites are involved in the pathogenesis of numerous illnesses such
as inflammatory, hyperproliferative, and neurodegenerative diseases. For example, 15-LOX contributes in
stroke-induced brain injury.9 Moreover, recent studies confirmed the presence of increased concentration of
∗Correspondence: akbarzad@tums.ac.ir
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15-LOX in human stroke.10 In this regard, 15-LOX inhibition has been introduced as an antistroke therapy
that leads to delayed organelle degradation in the reticulocyte.11,12 15-LOX is also important in the rheumatoid
arthritis inflammatory process13 and the role of LOX inhibitors has been fully understood as potential cancer
chemopreventives.14 Apart from those biological applications, they have food-related applications in bread
making and aroma production.15 Therefore, LOX inhibitors have gained lots of attention and, in this respect,
various heterocyclic and acyclic compounds have been evaluated for their inhibitory activity.
Herein, we focused on 15-LOX inhibitors such as phthalimides bearing thiadiazoles,16 2H -chromen-
2-ones,4−6 imidazole-2(3H)-thiones,17 pyrazoles,18 imidazo[2,1-b]thiazoles,19 and thioureas.20 Recently, we
studied the 15-LOX inhibitory activity of 3-aroyl-1-(4-sulfamoylphenyl)thiourea derivatives (A, Figure 1).21
Figure 1. 3-Aroyl-1-(4-sulfamoylphenyl)thiourea derivatives as 15-LOX inhibitor.
In continuation of our research program on the synthesis of bioactive compounds22−24 as well as novel
heterocycles,25−27 we profited from both 2H -chromen-2-ones and carbamothioyl moieties for the inhibition
of 15-LOX and some novel derivatives of chromenone bearing N -carbamothioyl moiety 7 (Scheme) were
synthesized as soybean 15-LOX inhibitors.
Scheme. Synthesis of chromenone bearing N -carbamothioyl moiety 7 as soybean 15-LOX inhibitors.
2. Results and discussion
2.1. Chemistry
Synthesis of the target compounds 7 was started from 7-hydroxy-2H -chromen-2-one 1 (Scheme). It reacted
with 1-fluoro-2-nitrobenzene or 1-fluoro-4-nitrobenzene 2 in the presence of K2 CO3 in dry DMF at 80 ◦ C to
give the corresponding nitrophenoxy-chromenone derivatives 3. Compound 3 tolerated a reduction reaction
using Zn/NH4 Cl in H2 O/MeOH to afford the related aminophenoxy-chromeone derivatives 4. Reaction of the
latter compound with in situ prepared benzoyl isothiocyanates 6 in refluxing acetone led to the formation of
the title compounds 7 (Table). It should be noted that benzoyl isothiocyanate 6 was easily prepared by the
reaction of various aroyl chloride 5 and ammonium thiocyanate in refluxing acetone.
The structures of all compounds were confirmed by characterization using 1 H NMR and 13 C NMR as
well as elemental analysis.
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Table. Synthesis and evaluation of chromenone bearing N -carbamothioyl moiety 7 as soybean 15-LOX inhibitors.
Entry Ar
1
Product 7 IC50 (µM)
C6H5
7a
7b
7c
7d
45.90 ꢀ 0.51
47.70 ꢀ 0.74
71.30 ꢀ 0.31
27.21 ꢀ 0.30
45.83 ꢀ 0.84
56.00 ꢀ 0.41
55.10 ꢀ 0.30
22.27 ꢀ 0.55
37.50 ꢀ 0.61
40.70 ꢀ 0.11
25.12 ꢀ 0.48
18.23 ꢀ 0.36
71.23 ꢀ 0.87
40.64 ꢀ 0.25
94.31 ꢀ 0.34
18.72 ꢀ 0.30
2
3
4
4-FC6H4
2-ClC6H4
2-Thienyl
5
6
4-NO2C6H4 7e
4-OMeC6H4 7f
7
8
4-MeC6H4
C6H5
7g
7h
7i
7j
7k
7l
9
2-ClC6H4
4-ClC6H4
2-MeC6H4
4-MeC6H4
4-MeOC6H4 7m
2-Thienyl 7n
4-NO2C6H4 7o
10
11
12
13
14
15
16
Quercetin
2.2. Biological evaluation: soybean 15-LOX inhibitory activity
The inhibitory activity of compounds 7a–o was evaluated against soybean 15-LOX comparing with quercetin
as the reference drug (Table). Most of compounds showed moderate to good inhibitory activity. Among
the synthesized compounds, 4-methyl-N -((4-((2-oxo-2H -chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide
(7l) was the most active compound (IC50 = 18.23 µM), and was found as potent as quercetin (IC50
=
18.72 µM). N -((4-((2-Oxo-2H -chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7h), 2-methyl-N -((4-((2-
oxo-2H -chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7k), and N -((2-((2-oxo-2H -chromen-7-yl)oxy)
phenyl)carbamothioyl)thiophene-2-carboxamide (7d) showed good activity with IC50 = 22.27, 25.12, and 27.21
µM, respectively. Moreover, moderate activity was obtained by compound 7i (IC50 = 37.50 µM). Compounds
7n, 7j, 7e–g, 7a, and 7b showed inhibitory activity with IC50 = 40.64–56.00 µM. It should be noted that
compounds 7c, 7m, and 7o were not significant 15-LOX inhibitors since the calculated IC50 values were 71.30,
71.23, and 94.31 µM, respectively.
It is obvious that the position of the aminophenoxy moiety connected to the chromeone skeleton and the
electronic property of substituents connected to the carbamothioyl moiety as well as their position play important
roles in soybean 15-LOX inhibitory activity. As can be seen in the Table, according to the calculated IC50
values compound 7l having 4-methylphenyl and 4-aminophenoxy moieties showed the best activity. Changing
the position of the methyl group led to a reduction in activity in compound 7k. Furthermore, compound 7h,
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possessing no substituents on the aryl group and 4-aminophenoxy moiety, showed lower activity compared with
7j. However, its counterpart 7a in which the N -carbamothioylbenzamide moiety was connected from the 2-
position of the phenoxy moiety showed a much lower activity compound. The inhibitory activity of compounds
having a thiophene moiety was dependent on the position of the aminophenoxy moiety. Compound 7d showed
higher activity in comparison to 7n. The outcomes from compounds 7i and 7j having a 4-aminophenoxy moiety
revealed that the presence of 2-chlorophenyl or 4-chlorophenyl led to moderate activity. However, the inhibitory
activity was reduced in compound 7c possessing 2-chlorophenyl and 2-aminophenoxy moieties. In compounds
having a 4-nitrophenyl group, the activity was dependent on the position of the aminophenoxy moiety and the
activity of 7e was less than that of 7o (almost half that of 7o). The same results were obtained for compound
pairs 7d/7n and 7f/7m. It should be noted that compound 7b possessing a 4-fluorophenyl group did not
induce remarkable inhibitory activity.
2.3. Docking study
The docking study was performed using Autodock Vina (1.1.2) to clarify the binding mode of the target
compounds in the active site of 15-LOX. Then the most energetically favored binding mode was further
analyzed to clarify interactions between compound 7l and the 15-LOX enzyme. A close examination of residues
surrounding the ligand as depicted in Figure 2 reveals that the 4-methylbenzoyl moiety is oriented toward a
hydrophobic packet composed of side chains of Leu277, Ile557, Leu560, Leu565, and Leu773. This binding
mode places this moiety in the vicinity of catalytic site Fe3+ ion, forming a ?-cation interaction. The NH of
the thioamide group is also involved in hydrogen bond interaction with carbonyl of Gln514 residue. A close-up
of the chromene moiety shows that this ring is located perpendicularly to the aromatic ring of Phe576. In
this orientation, the chromene ring interacts with Phe576 by means of H–? interaction that could help the
establishment of ligand in the active site of 15-LOX.
Figure 2. The best docked pose of compound 7l in the active site of 15-LOX. The H-bond is shown as a red dashed
line and important hydrogens are colored in light green.
3. Experimental
3.1. Apparatus and chemicals
Melting points were recorded on a Kofler hot stage apparatus and are uncorrected. 1 H and 13 C NMR spectra
were recorded on Bruker FT-400 and 500 using TMS as an internal standard. The IR spectra were obtained on
a Nicolet Magna FTIR 550 spectrometer (KBr disks). Mass spectra were documented on an Agilent Technology
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(HP) mass spectrometer operating at an ionization potential of 70 eV. The elemental analysis was performed
with an Elementar Analysensysteme GmbH Vario EL in CHNS mode.
3.2. Preparation of chromenones bearing N -carbamothioyl moiety 7
A mixture of 7-hydroxy-2H -chromen-2-one 1 (1 mmol), 1-fluoro-2-nitrobenzene or 1-fluoro-4-nitrobenzene 2 (1
mmol), and K2 CO3 (1 mmol) in dry DMF (10 mL) was heated at 80 ◦ C for 2–3 h. After completion of the
reaction (checked by TLC), the reaction mixture was cooled to room temperature and poured into crushed ice.
The precipitated product was filtered, washed with cold water, and dried at 50–60 ◦ C to give pure compound 3.
Powder zinc (20 mmol) was added in small portions (within 10 min) to the mixture of aqueous solution
of NH4 Cl (6 mmol in 2 mL) and methanol solution of compound 3 (1 mmol in 12 mL) at room temperature.
It was stirred for 3 h and after completion of the reaction (checked by TLC) the solid was removed by filtration
through a bed of Celite and washed with hot methanol. Concentration of the filtrate gave a white solid, which
was used without purification.
A solution of aroyl chloride 5 (1 mmol) and ammonium thiocyanate (1 mmol) in acetone (8 mL) was
heated under reflux for 10–20 min. After completion of the reaction (checked by TLC), the reaction mixture
was cooled to room temperature and the formed precipitate (NH4 Cl) was filtered off. Compound 4 (1 mmol)
was added to the freshly prepared solution of benzoyl isothiocyanate derivative 6, and the mixture was stirred
at reflux overnight. Upon completion of the reaction (checked by TLC), the resulting precipitate was collected
by filtration and recrystallized from EtOH to give the pure product 7.
3.2.1. N-((2-((2-Oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7a)
White solid, Yield 75%; mp 200–201 ◦ C; IR (KBr, νmax cm−1) 3286, 3032, 1722, 1676, 1620; 1 H NMR (400
MHz, DMSO-d6): δ 6.34 (d, J = 9.4 Hz, 1H, H3), 6.95 (d, J = 2.4 Hz, 1H, H8), 7.01 (dd, J = 8.5, 2.4 Hz,
1H, H6), 7.21 (dd, J = 8.0, 2.0 Hz, 1H, H6’), 7.34 (td, J = 8.0, 2.0 Hz, 1H, H4’), 7.42–7.52 (m, 3H, H5’, H3”,
H5”), 7.62 (t, J = 7.6 Hz, 1H, H4”), 7.66 (d, J = 8.5 Hz, 1H, H5), 7.86 (d,J = 7.6 Hz, 2H, H2”, H6”), 8.00
(d, J = 9.4 Hz, 1H, H4), 8.33 (dd, J = 8.0, 2.0 Hz, 1H, H3’), 11.60 (s, 1H, NH), 12.70 (s, 1H, NH); 13 C NMR
(100 MHz, DMSO-d6): δ 105.3, 114.5, 114.6, 115.0, 121.0, 125.3, 127.1, 128.3, 128.8, 129.1, 130.5, 130.6, 132.3,
133.6, 144.4, 148.2, 155.3, 160.0, 161.1, 169.0, 180.0; Anal. calcd. for C23 H16 N2 O4 S: C, 66.33; H, 3.85; N,
6.73. Found: C, 66.21; H, 3.71; N, 6.58.
3.2.2. 4-Fluoro-N-((2-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7b)
White solid, Yield 70%; mp 210–211 ◦ C; IR (KBr, νmax cm−1) 3286, 3044, 1727, 1670, 1617; 1 H NMR (400
MHz, DMSO-d6): δ 6.36 (d, J = 9.6 Hz, 1H, H3), 6.97 (d, J = 2.4 Hz, 1H, H8), 7.01 (dd, J = 8.8, 2.4 Hz,
1H, H6), 7.22 (dd, J = 7.8, 2.0 Hz, 1H, H6’), 7.30–7.40 (m, 4H, H4’, H5’, H3”, H5”), 7.71 (d, J = 8.8 Hz,
1H, H5), 7.90 (dd, J = 8.5, 2.0 Hz, 2H, H2”, H6”), 8.00 (d, J = 9.6 Hz, 1H, H4), 8.30 (dd, J = 7.8, 2.0 Hz,
1H, H3’), 11.60 (s, 1H, NH), 12.70 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 105.3, 114.5, 114.7, 115.0,
115.9 (d, JC−F = 22.0 Hz), 121.0, 125.3, 127.1, 128.4, 128.8 (d, JC−F = 2.9 Hz), 130.6, 132.1, 132.2, 144.4,
148.2, 155.3, 160.1, 160.3, 165.4 (d, JC−F = 250.0 Hz), 167.8, 180.0; Anal. calcd. for C23 H15 FN2 O4 S: C,
63.59; H, 3.46; N, 6.45. Found: C, 63.70; H, 3.25; N, 6.31.
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3.2.3. 2-Chloro-N-((2-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7c)
White solid, Yield 80%; mp 203–204 ◦ C; IR (KBr, νmax cm−1) 3427, 3188, 1722, 1685, 1616; 1 H NMR (400
MHz, DMSO-d6): δ 6.38 (d, J = 9.6 Hz, 1H, H3), 6.98–6.63 (m, 2H, H6, H8), 7.27 (dd, J = 7.4, 2.0 Hz,
1H, H6’), 7.34–7.51 (m, 6H, H4’, H5’, H3”, H4”, H5”, H6”), 7.71 (d, J = 8.4 Hz, 1H, H5), 8.04 (d, J = 9.6
Hz, 1H, H4), 8.33 (dd, J = 7.4, 2.0 Hz, 1H, H3’), 11.60 (s, 1H, NH), 12.30 (s, 1H, NH); 13 C NMR (100 MHz,
DMSO-d6): δ 105.0, 114.2, 114.9, 121.4, 125.6, 127.1, 127.5, 128.1, 128.5, 129.5, 129.9, 130.3, 130.5, 130.5,
132.6, 134.5, 144.5, 148.0, 155.3, 160.1, 160.3, 168.3, 179.4; Anal. calcd. for C23 H15 ClN2 O4 S: C, 61.27; H,
3.35; N, 6.21. Found: C, 61.40; H, 3.51; N, 6.48.
3.2.4. N-((2-((2-Oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)thiophene-2-carboxamide (7d)
Yellow solid, Yield 75%; mp 202–204 ◦ C; IR (KBr, νmax cm−1) 3547, 3196, 1712, 1650, 1620; 1 H NMR (400
MHz, DMSO-d6): δ 6.35 (d, J = 9.2 Hz, 1H, H3) 6.96 (d, J = 2.4 Hz, 1H, H8), 6.99 (dd, J = 8.5, 2.4 Hz 1H,
H6), 7.18–7.22 (m, 2H, H4’, H6’), 7.30–7.38 (m, 2H, H5’, H4”), 7.69 (d, J = 8.5 Hz, 1H, H5), 7.99 (d, J = 9.2
Hz, 1H, H4), 8.01 (dd, J = 4.7, 0.8 Hz, 1H, H5”), 8.27 (dd, J = 7.6, 2.0 Hz, 1H, H3’), 8.32 (dd, J = 4.7, 0.8
Hz, 1H, H3”), 11.70 (s, 1H, NH), 12.60 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 105.4, 114.5, 114.7,
115.0, 120.9, 125.3, 127.4, 128.4, 129.2, 130.4, 130.6, 133.4, 136.0, 136.7, 144.3, 148.4, 155.3, 1601.1, 160.3,
162.7, 179.8; Anal. calcd. for C21 H14 N2 O4 S2 : C, 59.70; H, 3.34; N, 6.63. Found: C, 59.84; H, 3.11; N, 6.84.
3.2.5. 4-Nitro-N-((2-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7e)
Yellow solid, Yield 70%; mp 210–211 ◦ C; IR (KBr, νmax cm−1) 3485, 3180, 1710, 1650, 1620, 1570, 1375; 1 H
NMR (400 MHz, DMSO-d6): δ 6.36 (d, J = 9.6 Hz, 1H, H3), 6.96 (d, J = 2.4 Hz, 1H, H8), 7.02 (dd, J =
8.4, 2.4 Hz 1H, H6), 7.21 (dd, J = 7.6, 1.6 Hz, 1H, H6’), 7.33–7.40 (m, 2H, H4’, H5’), 7.72 (d, J = 8.4 Hz, 1H,
H5), 8.03 (d, J = 9.6 Hz, 1H, H4), 8.07 (d, J = 8.8 Hz, 2H, H2”, H6”), 8.30 (d, J = 8.8 Hz, 2H, H3”, H5”),
8.31 (dd, J = 7.6, 1.6 Hz, 1H, H3’), 12.03 (s, 1H, NH), 12.57 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ
105.3, 114.5, 114.7, 115.1, 121.0, 123.7, 125.3, 127.0, 128.4, 130.4, 130.5, 130.7, 138.2, 144.5, 148.2, 150.3, 152.3,
160.0, 160.3, 167.5, 179.7; Anal. calcd. for C23 H15 N3 O6 S: C, 59.87; H, 3.28; N, 9.11. Found: C, 59.67; H,
3.40; N, 9.28.
3.2.6. 4-Methoxy-N-((2-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7f)
White solid, Yield 75%; mp 205–207 ◦ C; IR (KBr, νmax cm−1) 3480, 3168, 1710, 1648, 1618; 1 H NMR (400
MHz, DMSO-d6): δ 3.83 (s, 3H, OMe), 6.35 (d, J = 9.6 Hz, 1H, H3), 6.96 (d, J = 2.4 Hz, 1H, H8), 7.00–7.02
(m, 3H, H6, H3”, H5”), 7.21 (dd, J = 7.8, 2.0 Hz, 1H, H6’), 7.32–7.41 (m, 2H, H4’, H5’), 7.71 (d, J = 8.4
Hz, 1H, H5), 7.91 (d, J = 8.8 Hz, 2H, H2”, H6”), 8.02 (d, J = 9.6 Hz, 1H, H4), 8.32 (dd, J = 7.8, 2.0 Hz,
1H, H3’), 12.03 (s, 1H, NH), 12.57 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 56.1, 105.3, 114.2, 114.5,
114.6, 115.0, 121.0, 124.0, 125.3, 127.2, 128.3, 130.5, 130.7, 131.4, 144.4, 148.2, 155.3, 160.1, 160.3, 163.7, 168.1,
180.2; Anal. calcd. For C24 H18 N2 O5 S: C, 64.56; H, 4.06; N, 6.27. Found: C, 64.32; H, 3.89; N, 6.41.
3.2.7. 4-Methyl-N-((2-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7g)
White solid, Yield 70%; mp 215–216 ◦ C; IR (KBr, νmax cm−1) 3475, 3160, 1715, 1650, 1625; 1 H NMR (500
MHz, DMSO-d6): δ 2.38 (s, 3H, Me), 6.40 (d, J = 9.5 Hz, 1H, H3), 6.98 (d, J = 2.5 Hz, 1H, H8), 7.05 (dd, J
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= 8.0, 2.5 Hz, 1H, H6), 7.20 (dd, J = 8.0, 2.0 Hz, 1H, H6’), 7.30–7.40 (m, 2H, H4’, H5’), 7.34 (d, J = 8.5 Hz,
2H, H3”, H5”), 7.68 (d, J = 8.0 Hz, 1H, H5), 7.87 (d, J = 8.5 Hz, 2H, H2”, H6”), 8.03 (d, J = 9.5 Hz, 1H, H4),
8.32 (dd, J = 8.0, 2.0 Hz, 1H, H3’),11.58 (s, 1H, NH), 12.72 (s, 1H, NH); 13 C NMR (125 MHz, DMSO-d6): δ
20.0, 105.1, 114.0, 114.7, 121.1, 125.0, 126.5, 127.2, 128.4, 129.1, 129.4, 129.6, 130.3, 130.6, 135.2, 143.5, 144.8,
155.6, 160.5, 160.8, 168.1, 179.6; Anal. calcd. For C24 H18 N2 O4 S: C, 66.96; H, 4.21; N, 6.51. Found: C, 66.71;
H, 4.10; N, 6.38.
3.2.8. N-((4-((2-Oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7h)
White solid, Yield 80%; mp 200–201 ◦ C; IR (KBr, νmax cm−1) 3261, 3042, 1715, 1672, 1610; 1 H NMR (400
MHz, DMSO-d6): δ 6.39 (d, J = 9.6 Hz, 1H, H3), 6.98 (d, J = 2.4 Hz, 1H, H8) 7.00 (dd, J = 8.8, 2.4 Hz,
1H, H6), 7.20 (d, J = 8.8 Hz, 2H, H2’, H6’), 7.54 (t, J = 7.5 Hz, 2H, H3”, H5”), 7.66 (t, J = 7.5 Hz, 1H,
H4”), 7.73–7.78 (m, 3H, H5, H3’, H5’), 8.00 (d, J = 7.5 Hz, 2H, H2”, H6”), 8.04 (d, J = 9.6 Hz, 1H, H4),
11.60 (s, 1H, NH), 12.70 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 105.6, 114.5, 114.9, 115.1, 120.3,
126.8, 128.9, 129.2, 130.6, 132.6, 133.6, 135.1, 144.4, 153.3, 155.4, 160.3, 160.5, 168.7, 179.7; Anal. calcd. For
C23 H16 N2 O4 S: C, 66.33; H, 3.87; N, 6.73. Found: C, 66.51; H, 3.61; N, 6.51.
3.2.9. 2-Chloro-N-((4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7i)
White solid, Yield 80%; mp 218–219 ◦ C; IR (KBr, νmax cm−1) 3267, 3024, 1710, 1680, 1610; 1 H NMR (400
MHz, DMSO-d6): δ 6.38 (d, J = 9.4 Hz, 1H, H3), 6.97 (d, J = 2.4 Hz, 1H, H8), 7.07 (dd, J = 8.4, 2.4 Hz,
1H, H6), 7.20 (d, J = 8.8 Hz, 2H, H2’, H6’), 7.46 (td,J = 8.0, 1.6 Hz, 1H, H5”), 7.50–7.64 (m, 2H, H3”, H4”),
7.74–7.78 (m, 3H, H5, H3’, H5’), 8.01 (dd, J = 8.0, 1.6 Hz, 1H, H6”), 8.06 (d, J = 9.4 Hz, 1H, H4), 11.60
(s, 1H, NH), 12.70 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 105.6, 114.5, 114.9, 120.3, 125.2, 126.9,
127.6, 129.7, 130.0, 130.4, 130.6, 132.6, 134.7, 134.9, 144.5, 153.5, 155.5, 160.3, 160.5, 168.1, 179.2; Anal. calcd.
For C23 H15 ClN2 O4 S: C, 61.27; H, 3.35; N, 6.21. Found: C, 61.42; H, 3.50; N, 6.38.
3.2.10. 4-Chloro-N-((4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7j)
White solid, Yield 80%; mp 213–214 ◦ C; IR (KBr, νmax cm−1) 3600, 3129, 1713, 1672, 1619; 1 H NMR (400
MHz, DMSO-d6): δ 6.32 (d, J = 9.6 Hz, 1H, H3), 6.97 (d, J = 2.4 Hz, 1H, H8), 7.00 (dd, J = 8.0, 2.4 Hz,
1H, H6), 7.20 (d, J = 8.8 Hz, 2H, H2’, H6’), 7.61 (d, J = 8.8 Hz, 2H, H3’, H5’), 7.73–7.77 (m, 3H, H5, H3”,
H5”), 8.01 (d, J = 8.4 Hz, 2H, H2”, H6”), 8.04 (d, J = 9.6 Hz, 1H, H4), 11.60 (s, 1H, NH), 12.70 (s, 1H, NH);
13 C NMR (100 MHz, DMSO-d6): δ 105.6, 114.5, 114.9, 120.4, 126.9, 129.0, 130.6, 131.1, 131.4, 135.0, 138.5,
141.0, 144.5, 153.5, 155.5, 160.3, 160.5, 167.6, 179.6; Anal. calcd. For C23 H15 ClN2 O4 S: C, 61.27; H, 3.35; N,
6.21. Found: C, 61.37; H, 3.18; N, 6.11.
3.2.11. 2-Methyl-N-((4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7k)
White solid, Yield 80%; mp 214–215 ◦ C; IR (KBr, νmax cm−1) 3260, 1715, 1675, 1605; 1 H NMR (400 MHz,
DMSO-d6): δ 2.40 (s, 3H, Me), 6.36 (d, J = 9.5 Hz, 1H, H3), 6.93 (d, J = 2.4 Hz, 1H, H8), 7.00 (dd, J =
7.5, 2.4 Hz, 1H, H6), 7.17 (d, J = 8.8 Hz, 2H, H2’, H6’), 7.26–7.30 (m, 2H, H3”, H5”), 7.40–7.49 (m, 2H, H4”,
H6”), 7.74–7.77 (m, 3H, H5, H3’, H5’), 8.06 (d, J = 9.5 Hz, 1H, H4), 11.64 (s, 1H, NH), 12.47 (s, 1H, NH);
13 C NMR (100 MHz, DMSO-d6): δ 19.9, 105.5, 114.4, 114.9, 120.3, 126.0, 126.8, 127.0, 130.7, 131.1, 131.4,
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134.4, 135.0, 136.5, 143.1, 144.5, 153.3, 155.4, 160.4, 160.5, 170.9, 179.5; Anal. calcd. For C24 H18 N2 O4 S: C,
66.96; H, 4.21; N, 6.51. Found: C, 67.18; H, 4.41; N, 6.38.
3.2.12. 4-Methyl-N-((4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7l)
White solid, Yield 80%; mp 215–216 ◦ C; IR (KBr, νmax cm−1) 3258, 1717, 1678, 1602; 1 H NMR (400 MHz,
DMSO-d6): δ 2.40 (s, 3H, Me), 6.38 (d, J = 9.4 Hz, 1H, H3), 6.96 (d, J = 2.4 Hz, 1H, H8), 7.00 (dd, J =
8.0, 2.4 Hz, 1H, H6), 7.19 (d, J = 8.8 Hz, 2H, H2’, H6’), 7.34 (d, J = 8.2 Hz, 2H, H3”, H5”), 7.74–7.77 (m,
3H, H5, H3’, H5’), 7.90 (d, J = 8.2 Hz, 2H, H2”, H6”), 8.06 (d, J = 9.4 Hz, 1H, H4), 11.60 (s, 1H, NH), 12.70
(s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 20.0, 105.5, 114.5, 114.9, 120.4, 125.2, 126.8, 127.0, 129.2,
129.5, 129.7, 130.7, 135.1, 143.0, 144.5, 155.5, 160.4, 160.5, 168.4, 179.7; Anal. calcd. For C24 H18 N2 O4 S: C,
66.96; H, 4.21; N, 6.51. Found: C, 67.22; H, 4.38; N, 6.74.
3.2.13. 4-Methoxy-N-((4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7m)
White solid, Yield 75%; mp 215–217 ◦ C; IR (KBr, νmax cm−1) 3581, 3038, 1717, 1667, 1599; 1 H NMR (400
MHz, DMSO-d6): δ 3.86 (s, 3H, OMe), 6.38 (d, J = 9.2 Hz, 1H, H3), 6.96 (d, J = 2.4 Hz, 1H, H8), 6.99 (dd,
J = 8.0, 2.4 Hz, 1H, H6), 7.01 (d, J = 7.2 Hz, 2H, H3’, H5’), 7.2 (d, J = 7.2 Hz, 2H, H2’, H6’), 7.72–7.80 (m,
3H, H5, H3”, H5”), 8.02–8.06 (m, 3H, H4, H2”, H6”), 11.60 (s, 1H, NH), 12.70 (s, 1H, NH); 13 C NMR (100
MHz, DMSO-d6): δ 56.0, 105.5, 114.5, 114.2, 114.4, 114.8, 114.9, 120.4, 124.3, 126.8, 130.6, 135.1, 144.5, 153.3,
155.5, 160.3, 160.5, 163.7, 168.0, 179.8; Anal. calcd. For C24 H18 N2 O5 S: C, 64.56; H, 4.06; N, 6.27. Found: C,
64.37; H, 3.87; N, 6.40.
3.2.14. N-((4-((2-Oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)thiophene-2-carboxamide (7n)
White solid, Yield 80%; mp 215–216 ◦ C; IR (KBr, νmax cm−1) 3450, 3130, 3022, 1729, 1663, 1605; 1 H NMR
(400 MHz, DMSO-d6): δ 6.37(d, J = 9.5 Hz, 1H, H3), 6.95 (d, J = 2.4 Hz, 1H, H8), 6.99 (dd, J = 8.0, 2.4 Hz
1H, H6), 7.18 (d, J = 7.2, 2.0 Hz, 2H, H2’, H6’), 7.25 (dd, J = 4.8, 4.0 Hz, 1H, H3”), 7.76–8.73 (m, 3H, H3’,
H5’, H5”), 8.05–8.07 (m, 2H, H4, H4”), 8.39 (dd, J = 9.5 Hz, 1H, H4), 11.60 (s, 1H, NH), 12.70 (s, 1H, NH);
13 C NMR (100 MHz, DMSO-d6): δ 105.5, 114.4, 114.9, 120.4, 126.8, 129.2, 130.6, 131.1, 133.1, 135.2, 135.8,
137.2, 144.5, 155.3, 155.5, 160.4, 160.5, 162.4, 179.3; Anal. calcd. For C21 H14 N2 O4 S2 : C, 59.70; H, 3.34; N,
6.63. Found: C, 59.61; H, 3.51; N, 6.48.
3.2.15. 4-Nitro-N-((4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7o)
Yellow solid, Yield 70%; mp 214–216 ◦ C; IR (KBr, νmax cm−1) 3480, 3185, 1710, 1650, 1620, 1575, 1375; 1 H
NMR (400 MHz, DMSO-d6): δ 6.38 (d, J = 9.6 Hz, 1H, H3), 6.95 (d, J = 2.0 Hz, 1H, H8), 7.00 (dd, J =
8.4, 2.0 Hz 1H, H6), 7.19 (d, J = 8.8 Hz, 2H, H2’, H6’), 7.74–7.77 (m, 3H, H5, H3’, H5’), 8.06 (d, J = 9.6 Hz,
1H, H4), 8.18 (d, J = 8.8 Hz, 2H, H2”, H6”), 8.34 (d, J = 8.8 Hz, 2H, H3”, H5”), 11.97 (s, 1H, NH), 12.38
(s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6): δ 105.5, 114.4, 114.9, 120.4, 123.8, 125.1, 126.8, 130.6, 130.7,
135.1, 138.6, 144.5, 150.2, 153.4, 155.5, 160.4, 160.5, 167.0, 179.3; Anal. calcd. For C23 H15 N3 O6 S: C, 59.87;
H, 3.28; N, 9.11. Found: C, 59.67; H, 3.49; N, 9.28.
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3.3. Biological assay
The stock solution of tested compounds was prepared in DMSO (1 mL) and phosphate buffer (9 mL, 0.1 M,
pH 8). This stock solution was added to test solution containing enzyme (final concentration: 167 U/mL)
and phosphate buffer (pH 8) to achieve the final concentrations of 10−3 to 10−6 . After incubation of the
test solution for 4 min, linoleic acid was added to give the final concentration of 134 mM. Then changes in
absorbance were measured by UV Unico Double Beam Spectrophotometer for 60 s at 234 nm. The enzyme
solution was kept in ice and controls were measured at intervals throughout the experimental periods to ensure
that the enzyme activity was constant. All experiments were performed at 25 ◦ C in triplicate.28
3.4. Molecular docking study
Docking simulations were performed with Autodock Vina (ver. 1.1.2)29 employing the 3D structure of soybean
lipoxygenase in complex with 13(S)-hydroproxy-9(Z)-2,11(E)-octadecadienoic acid (code ID: 1IK3). First,
the aforementioned pdb file was retrieved from the Protein Data Bank (www.pdb.org). Then the co-crystallized
ligand and water molecules were removed and the protein was converted to pdbqt format using Autodock Tools
(1.5.6).30 For ligand preparation, the 2D chemical structures of ligands were sketched using Marvin Sketch 5.8.3,
2012, ChemAxon (http://www.chemaxon.com) and then converted to 3D format by Open Babel (ver 2.3.1).31
Finally, the pdbqt format of ligands was prepared using an Autodock Tools python script, prepare ligand4.py.
The docking simulation was performed using the following parameters: size x = 20; size y = 20; size z =
20; center x = 19.693; center y = 0.054; center z = 17.628. The exhaustiveness was set to 100 and the max.
number of retrieved final docked poses was set to 15 using the num modes parameter. The other docking
parameters were left as default. Finally, the best docking solutions were selected for further analysis of enzyme-
inhibitor interactions. The graphics are depicted using Chimera 1.6 software.32
4. Conclusion
Various derivatives of chromenone bearing an N -carbamothioyl moiety were synthesized and evaluated for
their soybean 15-LOX inhibitory activity. Most of the compounds showed moderate to good inhibitory activ-
ity. Among them, 4-methyl-N -((4-((2-oxo-2H -chromen-7-yl)oxy)phenyl)carbamothioyl)benzamide (7l) was as
potent as the reference drug, quercetin.
Acknowledgment
The authors gratefully acknowledge the Research Council of Tehran University of Medical Sciences with project
No. 95-01-33-30885.
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