Coumarin-Based Bioactive Compounds: Facile Synthesis and Biological Evaluation of Coumarin-Fused 1,4-Thiazepines

Article abstract of DOI:10.1111/j.1747-0285.2011.01175.x

As part of our ongoing studies on the synthesis of bioactive coumarin compounds, we synthesized a series of new coumarin-fused 1,4-thiazepines using a simple method. The biological activity of target compounds along with 3-(2-hydroxybenzylidene)chroman-2,4-dione intermediates was screened by evaluation of their antioxidant and cytotoxic activities. The antioxidant activity was assessed using two methods, namely, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method and ferric reducing antioxidant power (FRAP) assay. 4-Methoxyphenolic compound 5d in thiazepine series showed the most potent scavenging activity, while the 4-bromophenolic derivative 5b was the most efficient compound in FRAP assay. Also, the result of cytotoxic evaluation using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay demonstrated that compound 5b is at least twofold more potent than etoposide against MCF-7, SK-N-MC, and MDA-MB 231 cell lines. A series of new coumarin-fused 1,4-thiazepines were synthesized using a simple method. The biological activities of target compounds were screened by evaluation of their antioxidant and cytotoxic activities.

Full text of DOI:10.1111/j.1747-0285.2011.01175.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01175.x
Chem Biol Drug Des 2011; 78: 580–586
Research Article
Coumarin-Based Bioactive Compounds: Facile
Synthesis and Biological Evaluation of
Coumarin-Fused 1,4-Thiazepines
Mehdi Khoobi1,2, Alireza Foroumadi2,3
,
Received 2 January 2011, revised 8 June 2011 and accepted for publi-
cation 2 July 2011
Saeed Emami4, Maliheh Safavi3,5
Gholamreza Dehghan6, Babak H.
,
Alizadeh7, Ali Ramazani1, Sussan K.
Ardestani5 and Abbas Shafiee3,*
Currently, cancer is a serious clinical problem that poses significant
socioeconomical effects on the human healthcare and is the second
leading cause of death in most countries (1). Apart from the use of
surgery and radiotherapy, chemotherapy has still been an important
treatment modality for cancers. Enormous progress has been made
in the chemotherapy of cancer with the development of novel anti-
cancer agents including paclitaxel, docetaxel, imatinib, and sorafe-
nib (2). However, owing to toxicity and drug-resistance problems
with current chemotherapeutic agents, it remains a great challenge
to discover and develop more effective anticancer drugs (1).
1Chemistry Department, Zanjan University, P.O. Box 45195-313,
Zanjan, Iran
2Drug Design & Development Research Center, Tehran University of
Medical Sciences, Tehran 14176, Iran
3Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran 14176, Iran
4Department of Medicinal Chemistry and Pharmaceutical Sciences
Research Center, Faculty of Pharmacy, Mazandaran University of
Medical Sciences, Sari, Iran
Among potential chemotherapeutic agents, heterocyclic compounds
represent an outstanding type of anticancer drug candidate (3). Over
the last two decades, synthesis of N and S containing heterocyclic
compounds especially thiazepines retained the interest of research-
ers because of the unique structural properties and broad spectrum
of biological activities of these compounds. The aryl- and heteroa-
ryl-fused 1,4-thiazepines are a privileged structure with fascinating
pharmacological properties, such as antiarrhythmic, antispasmodic,
angiogenic, and CNS activities, and therefore represent promising
synthetic targets (4). On the other hand, coumarin rings has been
found as a key structural unit in many bioactive natural products
and pharmaceuticals (5). However, to the best of our knowledge,
the coumarin-fused 1,4-thiazepine that combines coumarin and thia-
zepine fragments in one molecule has not been reported previously.
5Department of Biochemistry, Institute of Biochemistry and
Biophysics, University of Tehran, P.O. Box 13145-1365, Tehran, Iran
6Department of Biology, Faculty of Natural Science, University of
Tabriz, Tabriz, Iran
7Department of Chemistry, Iranian Research Institute of Plant
Protection (IRIPP), Tehran, Iran
*Corresponding author: Abbas Shafiee, ashafiee@ams.ac.ir
As part of our ongoing studies on the synthesis of
bioactive coumarin compounds, we synthesized a
series of new coumarin-fused 1,4-thiazepines using
a simple method. The biological activity of target
compounds along with 3-(2-hydroxybenzylid-
ene)chroman-2,4-dione intermediates was screened
by evaluation of their antioxidant and cytotoxic
activities. The antioxidant activity was assessed
using two methods, namely, 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH) radical scavenging method and
ferric reducing antioxidant power (FRAP) assay. 4-
Methoxyphenolic compound 5d in thiazepine series
showed the most potent scavenging activity, while
the 4-bromophenolic derivative 5b was the most
efficient compound in FRAP assay. Also, the result
of cytotoxic evaluation using MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide] assay demonstrated that compound 5b is at
least twofold more potent than etoposide against
MCF-7, SK-N-MC, and MDA-MB 231 cell lines.
Synthetic approaches to 1,4-thiazepine are varied and involve addi-
tion, condensation, coupling, rearrangement, and thermolysis meth-
odologies in multistep synthesis (6). As a part of our ongoing studies
on the synthesis of bioactive chromene and coumarin compounds
(7–12), herein, we wish to report the synthesis of novel 5-(2-hydroxy-
phenyl)-2,3-dihydro-1H-chromeno[4,3-e][1,4]thiazepin-6(5H)-one deriv-
atives (5a–e) using a simple method in two steps and their
screening for antioxidant and cytotoxic activities.
Experimental Section
Chemistry
All starting materials, reagents, and solvents were purchased from
Merck AG (Darmstadt, Germany). 4-Hydroxycoumarin derivatives 2a–c
were prepared according to the literature method (13). The purity of
Key words: antioxidant activity, coumarin, cytotoxic activity, pheno-
lic compounds, thiazepine
580

Coumarin-Based Bioactive Compounds
the synthesized compounds was confirmed by thin layer chromatogra-
phy (TLC) using various solvents of different polarities. Merck silica gel
60 F254 plates were applied for analytical TLC. Melting points were
determined on a Kofler hot stage apparatus and are uncorrected.
Nuclear magnetic resonance spectra were recorded using a Bruker
500 spectrometer (Bruker, Rheinstetten, Germany), and chemical shifts
are expressed as d (ppm) with tetramethylsilane as internal standard.
The IR spectra were obtained on a Nicolet 550-FTIR spectrometer
(potassium bromide disks). Elemental analyses were carried out on a
CHN-O-rapid elemental analyzer (GmbH, Hanau, Germany) for C, H,
and N, and the results are within €0.4% of the theoretical values.
118.5, 118.9, 119.1, 120.2, 124.9, 126.7, 132.5, 137.5, 144.2, 144.4,
147.3, 153.1, 157.5, 163.2, 196.2; Anal. calcd for C17H12O5: C,
68.92; H, 4.08; Found: C, 68.70; H, 4.37.
3-(2-Hydroxy-5-methoxybenzylidene)chroman-
2,4-dione (3d)
Colorless solid (0.88 g, 35%); mp 158–160 ꢀC; IR (KBr,1 ⁄ cm) mmax
3443 (OH), 1703 (C=O); 1H NMR (CDCl3) d 3.99 (s, 3H), 6.88 (t,
J = 7.5 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H),
7.20 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.53 (m, 2H), 7.94
(s, 1H), 11.72 (bs, 1H, OH); 13C NMR (125 MHz, CDCl3) d 56.3,
115.3, 118.5, 118.9, 119.1, 120.2, 124.9, 126.7, 132.5, 137.5, 144.2,
144.4, 147.3, 153.1, 157.5, 163.2, 196.2; Anal. calcd for C17H12O5:
C, 68.92; H, 4.08; Found: C, 68.73; H, 4.31.
General procedure for synthesis of 3-(2-
hydroxybenzylidene)chroman-2,4-dione
derivatives (3)
A mixture of 4-hydroxycoumarin derivative 2 (10 mmol) and appropri-
ate 2-hydroxybenzaldehyde (12 mmol) in ethanol (15 mL) was refluxed
for 0.5–1 h. The progress of reaction was followed by TLC. After com-
pletion of the reaction, the mixture was allowed to cool and crystals
of corresponding product were formed. Immediately, the precipitated
solid was filtered off and washed with ethanol. If after the cooling of
mixture, appropriate crystals or solid did not form, the solvent was
evaporated under reduced pressure, and the residue was purified by
silica gel column chromatography to give the pure product 3.
7-Hydroxy-3-(2-hydroxybenzylidene)chroman-
2,4-dione (3e)
Yellow solid (0.84 g, 30%); mp 240–242 ꢀC; IR (KBr, 1 ⁄ cm) mmax 3260
(OH), 1685 (C = O); 1H NMR (DMSO-d6) d 6.32 (s, 1H), 6.37 (d,
3
3
3J = 8.8 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H),
7.63 (d, 3J = 8.8 Hz, 1H), 7.70 (t, 3J = 7.5 Hz, 1H), 7.82 (d,
3J = 7.5 Hz, 1H), 8.26 (s, 1H), 11.48 (s, 1H, OH); 13C NMR (125 MHz,
DMSO-d6) d 102.9, 109.0, 114.3, 116.7, 118.8, 125.3, 128.4, 129.9,
133.5, 135.0, 142.8, 154.1, 158.5, 163.8, 165.7, 192.4; Anal. calcd for
C16H10O5: C, 68.09; H, 3.57; Found: C, 68.23; H, 3.31.
3-(2-Hydroxybenzylidene)chroman-2,4-dione (3a)
Yellow crystal (0.79 g, 30%); mp 173–175 ꢀC; IR (KBr, 1 ⁄ cm) mmax
3431 (OH), 1721 (C = O); 1H NMR (CDCl3) d 6.89 (td, 3J = 7.5 Hz,
4J = 1.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.38 (td, 3J = 7.5 Hz,
4J = 1.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.54 (m, 2H), 7.60 (dd,
3J = 7.5 Hz, 4J = 1.0 Hz, 1H), 7.67 (td, 3J = 7.5 Hz, 4J = 1.0 Hz,
1H), 7.97 (s, 1H), 11.73 (s, 1H, OH); 13C NMR (125 MHz, CDCl3) d
117.0, 117.9, 118.6, 119.1, 125.1, 126.4, 129.0, 132.5, 133.6, 137.5,
144.2, 154.5, 158.0, 163.2, 195.8. Anal. calcd for C16H10O4: C,
72.18; H, 3.79; Found: C, 72.36; H, 3.97.
6-Fluoro-3-(2-hydroxybenzylidene)chroman-2,4-
dione (3f)
Yellow crystal (0.99 g, 35%); mp 156–158 ꢀC; IR (KBr, 1 ⁄ cm) mmax
1
3
3429 (OH), 1722 (C = O); H NMR (CDCl3) d 7.04 (dd, J = 9.0 Hz,
4J = 4.5 Hz, 1H), 7.23 (dd, 3J = 8.5 Hz, 4J = 3.0 Hz, 1H), 7.28 (dt,
3J = 8.5 Hz, 4J = 3.0 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.44 (d,
J = 7.2 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.68 (t, J = 7.2 Hz, 1H),
7.99 (s, 1H), 11.53 (s, 1H, OH); 13C NMR (125 MHz, CDCl3) d 116.9,
117.1, 117.7, 120.0, 120.1, 125.2, 125.4, 129.2, 134.0, 144.7, 147.3,
153.8, 155.7, 157.3, 159.4, 195.5; Anal. calcd for C16H9FO4: C,
67.61; H, 3.19; Found: C, 67.28; H, 3.41.
3-(5-Bromo-2-hydroxybenzylidene)chroman-2,4-
dione (3b)
Yellow crystal (1.20 g, 35%); mp 194–196 ꢀC; IR (KBr, 1 ⁄ cm) mmax
3440 (OH), 1721 (C = O); 1H NMR (CDCl3) d 6.89 (td, 3J = 7.5 Hz,
4J = 1.0 Hz, 1H), 7.06 (dd, 3J = 8.5 Hz, 4J = 1.0 Hz, 1H), 7.30 (d,
J = 8.5 Hz, 1H), 7.50 (dd, 3J = 8.0 Hz, 4J = 2.0 Hz, 1H), 7.55 (td,
6-Fluoro-3-(2-hydroxy-3-
methoxybenzylidene)chroman-2,4-dione (3g)
Yellow crystal (0.94 g, 30%); mp 193–195 ꢀC; IR (KBr, 1 ⁄ cm) mmax
4
1
3J = 7.5 Hz, J = 2.0 Hz, 1H), 7.52 (m, 2H), 7.86 (s, 1H), 11.65 (s, 1H,
3417 (OH), 1718 (C = O); H NMR (CDCl3) d 4.02 (s, 3H), 7.05 (m,
OH); 13C NMR (125 MHz, CDCl3) d 117.7, 118.7, 119.2, 119.3, 127.4,
131.1, 132.3, 136.3, 137.7, 142.6, 153.3, 157.3, 163.2, 195.6. Anal.
calcd for C16H9BrO4: C, 55.68; H, 2.63; Found: C, 55.89; H, 2.34.
1H), 7.19 (d, J = 8.0 Hz, 1H), 7.23 (m, 2H), 7.32 (m, 2H), 7.99 (s,
3
1H), 11.49 (s, 1H, OH); 13C NMR (125 MHz, CDCl3) d 56.4, 115.5,
116.9, 117.1, 118.4, 119.9, 120.0, 120.3, 125.1, 125.2, 126.2, 144.9,
147.3, 153.8, 155.7, 157.3, 159.4, 195.5; Anal. calcd for C17H11FO5:
C, 64.97; H, 3.53; Found: C, 65.10; H, 3.39.
3-(2-Hydroxy-3-methoxybenzylidene)chroman-
2,4-dione (3c)
Colorless solid (0.88 g, 35%); mp 217–219 ꢀC; IR (KBr, 1 ⁄ cm) mmax
3428 (OH), 1723 (C = O); 1H NMR (CDCl3) d 3.99 (s, 3H), 6.88 (t,
J = 7.5 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H),
7.20 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.53 (m, 2H), 7.94
(s, 1H), 11.72 (s, 1H, OH); 13C NMR (125 MHz, CDCl3) d 56.3, 115.3,
General procedure for synthesis of 5-(2-
hydroxyphenyl)-2,3-dihydro-1H-chromeno[4,3-
e][1,4]thiazepin-6(5H)-one derivatives (5)
A mixture of 3-(2-hydroxybenzylidene)chroman-2,4-dione derivatives
3 (0.5 mmol) and 2-aminoethanethiol hydrochloride (1.5 mmol) was
Chem Biol Drug Des 2011; 78: 580–586
581

Khoobi et al.
dissolved in ethanol (2 mL). The mixture was stirred at room tem-
perature overnight. The precipitated solid was filtered off and
washed with water to give pure thiazepine derivative 5.
124.6, 132.8, 143.6, 151.8, 156.1, 156.8, 159.8; Anal. calcd for
C19H17NO4S: C, 64.21; H, 4.82; N, 3.94; Found: C, 64.56; H, 4.51; N,
3.69.
5-(2-Hydroxyphenyl)-2,3-dihydro-1H-
9-Hydroxy-5-(2-hydroxyphenyl)-2,3-dihydro-1H-
chromeno[4,3-e][1,4]thiazepin-6(5H)-one (5e)
White solid (0.13 g, 78%); mp 190–193 ꢀC; IR (KBr, 1 ⁄ cm) mmax
chromeno[4,3-e][1,4]thiazepin-6(5H)-one (5a)
White solid (0.14 g, 85%); mp 223–225 ꢀC; IR (KBr, 1 ⁄ cm) mmax
3441 (OH), 1705 (C=O); 1H NMR (DMSO-d6) d 2.60–2.73 (m, 2H),
2.79–2.83 (m, 2H), 5.36 (s, 1H), 7.34 (td, 3J = 8.0 Hz, 4J = 2.0 Hz,
1
3431 (OH), 1698 (C = O); H NMR (DMSO-d6) d 2.56–2.69 (m, 2H),
4
2.83–2.86 (m, 2H), 5.34 (s, 1H), 6.83 (d, J = 2.2 Hz, 1H), 6.93 (dd,
3
1H), 7.43–7.44 (m, 2H), 7.48–7.52 (m, 2H), 7.67 (d, J = 7.5 Hz, 1H),
3J = 8.7 Hz, 4J = 2.2 Hz, 1H), 7.33 (t, 3J = 7.5 Hz, 1H), 7.39–7.46
(m, 2H), 7.63 (d, 3J = 7.5 Hz, 1H), 7.71 (bs, 1H, NH), 7.89 (d,
3J = 8.7 Hz, 1H), 10.36 (bs, 1H, OH); 13C NMR (125 MHz, DMSO-d6)
d 27.2, 36.9, 38.8, 97.8, 102.8, 105.7, 114.0, 117.0, 121.9, 124.7,
126.4, 129.6, 130.3, 150.0, 154.4, 157.6, 160.8, 162.6; Anal. calcd
for C18H15NO4S: C, 63.33; H, 4.43; N, 4.10; Found: C, 63.10; H, 4.71;
N, 3.82.
3
4
7.74 (td, J = 8.0 Hz, J = 1.5 Hz, 1H), 7.98 (bs, 2H, OH, NH), 8.06
(dd, J = 8.0 Hz, J = 1.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) d
28.8, 36.3, 38.3, 95.3, 101.1, 113.5, 116.7, 116.7, 121.3, 122.8,
124.8, 126.2, 129.3, 129.9, 133.1, 149.4, 152.0, 156.3, 159.9; Anal.
calcd for C18H15NO3S: C, 66.44; H, 4.65; N, 4.30; Found: C, 66.11;
H, 4.97; N, 4.16.
3
4
5-(5-Bromo-2-hydroxyphenyl)-2,3-dihydro-1H-
chromeno[4,3-e][1,4]thiazepin-6(5H)-one (5b)
White solid (0.18 g, 88%); mp 265–267 ꢀC; IR (KBr, 1 ⁄ cm) mmax
Biological activity
Cytotoxic assay
1
3441 (OH), 1705 (C = O); H NMR (DMSO-d6) d 2.63–2.72 (m, 2H),
Human cancer cell lines including MCF-7, SK-N-MC, and MDA-MB
231 were obtained from Pasture institute, Tehran (Iran). Cells were
maintained in RPMI 1640 with added 10% FBS, 1% L-glutamine, and
penicillin ⁄ streptomycin, and then cells were incubated at 37 ꢀC in a
5% concentration of CO2. Cells were harvested by trypsin ⁄ EDTA and
resuspended in fresh medium. Cell survival was determined by MTT
[3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colori-
metric assay (14). Exponentially growing cells (4 · 104 cells ⁄ well)
were seeded in 96-well plates in RPMI with 10% FBS and incubated
for 24 h. After treatment of cells with different concentrations of test
compounds 3a–g and 5a–e for 24 h at 37 ꢀC, the medium was
removed and phenol red-free medium with FBS was added to cells.
Then, MTT solution was added to each well (2 mg ⁄ mL), followed by
4-h incubation. The viable cell number is directly proportional to the
production of formazan, which, following solubilization with isopropyl
alcohol, can be measured spectrophotometrically at 570 nm by an
ELISA plate reader. In each plate, there were control wells (cells with-
out test compounds) and blank wells (the medium only or 0.1%
DMSO). The percentage of cell viability versus controls was assessed
by the formula: [1–(absorbance of treated cells ⁄ absorbance of control
cells)] ·100.
3
2.85–2.88 (m, 2H), 5.39 (s, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.48–7.53
3
4
3
(m, 2H), 7.62 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H), 7.73 (t, J = 8.0 Hz,
1H), 7.90 (d, 4J = 2.5 Hz, 1H), 7.91 (bs, 2H, OH, NH), 8.05 (d,
3J = 8.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) d 26.9, 35.8, 38.2,
100.9, 113.3, 116.7, 117.6, 119.0, 122.8, 123.9, 124.8, 132.0, 132.1,
133.2, 148.7, 152.0, 156.1, 159.7; MS, m ⁄ z (%) 405 (M+ + 2, 6),
403 (M+, 6), 329 (100), 327 (93), 248 (20), 220 (17), 163 (23); Anal.
calcd for C18H14BrNO3S: C, 53.48; H, 3.49; N, 3.46; Found: C, 53.69;
H, 3.78; N, 3.21.
5-(2-Hydroxy-3-methoxyphenyl)-2,3-dihydro-1H-
chromeno[4,3-e][1,4]thiazepin-6(5H)-one (5c)
White solid (0.15 g, 85%); mp 203–205 ꢀC; IR (KBr, 1 ⁄ cm) mmax
1
3444 (OH), 1727 (C = O); H NMR (DMSO-d6) d 2.56–2.69 (m, 2H),
3
2.82–2.85 (m, 2H), 3.95 (s, 3H), 5.35 (s, 1H), 7.15 (dd, J = 8.0 Hz,
3
3
4J = 1.3 Hz, 1H), 7.19 (d, J = 7.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H),
7.52–7.55 (m, 2H), 7.74 (bs, 2H, OH, NH), 7.76 (dt, 3J = 7.0 Hz,
4J = 1.6 Hz, 1H), 7.96 (dd, J = 7.0 Hz, J = 1.6 Hz, 1H); 13C NMR
(125 MHz, DMSO-d6) d 26.7, 36.4, 38.2, 56.2, 95.3, 111.7, 113.6,
116.7, 120.6, 121.9, 122.5, 124.8, 126.0, 133.0, 139.0, 147.4, 151.9,
156.1, 159.8; Anal. calcd for C19H17NO4S: C, 64.21; H, 4.82; N, 3.94;
Found: C, 64.58; H, 4.49; N, 3.71.
3
4
DPPH radical scavenging assay
Several concentrations of test compounds 3b–g and 5a–e in
DMSO were prepared. The compound solution (1.0 mL) was added
to the methanolic DPPH solution (2.0 mL, 0.1 mM), and the mix-
ture was kept in the dark for 15 min. The absorbance at 517 nm
was then measured by an UV ⁄ visible spectrophotometer. The
percent scavenging activity was calculated using the following
5-(2-Hydroxy-5-methoxyphenyl)-2,3-dihydro-1H-
chromeno[4,3-e][1,4]thiazepin-6(5H)-one (5d)
White solid (0.14 g, 80%); IR (KBr, 1 ⁄ cm) mmax 3442 (OH), 1720
1
(C = O); H NMR (DMSO-d6) d 2.40–2.49 (m, 2H), 2.56–2.59 (m, 2H),
3.80 (s, 3H), 5.25 (s, 1H), 7.00 (dd, 3J = 9.0 Hz, 4J = 3.0 Hz, 1H),
7.15 (d, 4J = 3.0 Hz, 1H), 7.36 (d, 3J = 9.0 Hz, 1H), 7.47–7.51 (m,
2H), 7.73 (dt, 3J = 7.5 Hz, 4J = 1.5 Hz, 1H), 8.03 (dd, 3J = 7.5 Hz,
4J = 1.5 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) d 31.60, 36.5,
40.5, 55.6, 100.6, 113.3, 113.5, 115.1, 116.6, 117.50, 122.4, 122.6,
formula: inhibition (%) = 100 · (Abscontrol ) Abscompound) ⁄ Abscontrol.
The DPPH radical scavenging activity of compounds was expressed
in terms of IC50 (mM), which is obtained from linear regression
plot between concentrations of test compound and percent inhibi-
tions (15).
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Chem Biol Drug Des 2011; 78: 580–586

Coumarin-Based Bioactive Compounds
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay reagent was prepared by adding 10 vol of 300 mM
acetate buffer, pH 3.6 (3.1 g sodium acetate and 16 mL glacial ace-
tic acid), 1 vol of 10 mM 2,4,6-tripyridyl-s-triazine prepared in
40 mM HCl, and 1 vol of 20 mM FeCl3. The mixture was diluted to
1 ⁄ 3 with methanol and prewarmed at 37 ꢀC. This reagent (3 mL)
was mixed with 0.1 mL diluted test compounds 3b–g and 5a–e.
The mixture was shaken and incubated at 37 ꢀC for 8 min, and the
absorbance was read at 593 nm. A blank with only 0.1 mL metha-
nol was used for calibration. The difference in absorbance between
the tested sample and the blank reading was calculated, and the
data were expressed as mM of ferric reduced to ferrous form (16).
(POCl3) as condensing agent (13). Then, compounds 2a–c were con-
verted to the corresponding benzylidene derivatives 3a–g using a
simple aldol condensation with appropriate 2-hydroxybenzaldehyds
refluxing in absolute ethanol (Scheme 1). The low yields (20–40%)
of the desired benzylidene derivatives 3a–g may be relatively
because of the formation of bis-coumarins 4. Reaction of 2-amino-
ethanethiol hydrochloride with benzylidene derivatives 3a–g in eth-
anol at room temperature gave the desired coumarin-fused
thiazepines 5a–e (Scheme 2). It seems that slightly acidic condi-
tions provided by the excess 2-aminoethanethiol hydrochloride were
necessary for the reaction to proceed. The structures of all com-
pounds were ascertained by their spectroscopic data (1H- and 13C-
NMR, MS) and elemental analyses. Based on 1H-NMR spectra of
the crude product, it was concluded that the compound 5 was the
only product of this reaction and formation of 6 was not detected.
An indication for this interpretation is the existence of just one sin-
glet signal in 5.25–5.39 ppm.
Results and Discussion
Chemistry
The synthetic routes for synthesis of key intermediates 3-(2-hydrox-
ybenzylidene)chroman-2,4-diones 3a–g and target compounds 5-(2-
hydroxyphenyl)-2,3-dihydro-1H-chromeno[4,3-e][1,4]thiazepin-6(5H)-one
derivatives 5a–e are depicted in Schemes 1 and 2, respectively.
Biological activity
Cytotoxic activity
First, 4-hydroxycoumarin derivatives 2a–c were prepared by the lit-
erature method using an appropriately substituted phenol and
malonic acid, ZnCl2 as Lewis acid, and phosphorus oxychloride
The cytotoxic activity of compounds 3a–g and 5a–e was evalu-
ated against three human cell lines MCF-7, SK-N-MC, and MDA-
MB 231 using MTT assay. The amount of produced purple formazan
O
OH
O
O
R
R'
H
OH
3a-g
OH
O
R
O
R'
EtOH, reflux
Malonic acid
R
ZnCl2, POCl3,
70 oC
OH
O
R
O
1a-1c
2a-c
O
O
O
OH
R= H, 6-F, 7-OH
Scheme 1: Synthesis of key
intermediates 3-(2-hydroxybenzylidene)
chroman-2,4-diones 3a–g.
R'= H, 5-Br, 3-OMe, 5-OMe
O
S
R
R'
4
HN
OH
O
O
R
R'
O
O
OH
5a-e
HS
NH3 Cl
EtOH, r.t.
O
R'
R
N
OH
3a-e
S
Scheme 2: Synthesis of 5-(2-
hydroxyphenyl)-2,3-dihydro-1H-chro-
meno[4,3-e][1,4]thiazepin-6(5H)-one
derivatives 5a–e.
R = H, 7-OH
R' = H, 5-Br, 3-OMe, 5-OMe
O
O
R
R'
6
Chem Biol Drug Des 2011; 78: 580–586
583

Khoobi et al.
Table 1: Chemical structures and cytotoxic activity (IC50, lg ⁄ mL) of compounds 3a–g and 5a–e
O
OH
OH
O
O
O
O
O
O
Br
OH
O
O
O
O
OH
O
O
O
O
OH
HO
F
O
O
O
OH
O
O
O
OH
F
O
O
O
HN
OH
S
O
O
OH
Br
HN
S
O
O
HN
OH
S
O
O
O
HN
OH
S
O
O
O
S
HN
OH
O
HO
O
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Chem Biol Drug Des 2011; 78: 580–586

Coumarin-Based Bioactive Compounds
from MTT was assayed by spectrophotometer and is proportional to
the number of viable cells. The IC50 values (lg ⁄ mL) were calculated
by linear regression analysis, expressed in mean € SD. The result
of cytotoxic evaluation in comparison with etoposide as a standard
drug was summarized in Table 1.
5e showed good scavenging activity, surprisingly benzylidene-7-hy-
droxycoumarin analog 3e exhibited very weak activity. In both ser-
ies, 4-bromo and 4-methoxyphenolic derivatives were more active
than 2-methoxyphenolic compounds.
The growth inhibitory concentrations of compounds indicate that 3-
(2-hydroxybenzylidene)chroman-2,4-diones 3a–g showed no signifi-
cant activity against tested cell lines. Among thiazepine derivatives,
4-bromophenolic compound 5b and 2-methoxyphenolic counterpart
5c exhibited good cytotoxic activity against all tested cell lines.
Although compound 5a showed no activity (IC50 > 100 lg ⁄ mL)
against MCF-7 and MDA-MB-231 cell lines but had potent inhibitory
activity against SK-N-MC cell (IC50 < 10 lg ⁄ mL). The comparison of
IC50 values of the most potent compound 5b and standard drug
etoposide demonstrated that compound 5b was at least twofold
more potent than etoposide. Also, the activity of compound 5c
against MCF-7 and SK-N-MC cells was comparable to that of eto-
poside.
FRAP ability
The FRAP assay measures the ability of a compound to reduce the
ferric 2,4,6-tripyridyl-s-triazine complex to the colored ferrous com-
plex. FRAP values are obtained by comparing the absorbance
change at 593 nm in test reaction mixtures with those containing
ferrous ions in known concentration. The FRAP values for test com-
pounds (Table 2) revealed that the fluoro analog of 3-(2-hydroxyben-
zylidene)chroman-2,4-diones (3f and 3g) and thizepine derivatives
5b, 5d, and 5e exhibited more potent reducing power in compari-
son with reference drug quercetin as a poly-phenolic flavonoid.
Thiazepine compound 5b having 4-bromophenolic moiety showed
the most antioxidant power in FRAP assay. It is noticeable that this
compound also showed respectable DPPH radical scavenging activ-
ity. Compounds 5d and 5e that showed the most potent antioxi-
dant activity in DPPH model had good reducing power but their
efficacy was half in respect to that of 5b.
DPPH radical scavenging activity
Many synthetic and natural coumarin derivatives have special ability
to scavenge free radicals. Thus, primarily, the free radical scaveng-
ing activity of coumarin-based compounds 3b–g and 5a–e was
evaluated by DPPH colorimetric method. Several dilutions of com-
pounds were made and assayed to obtain concentration of the
sample required to scavenge 50% (IC50) of DPPH-free radical apply-
ing suitable regression analysis of the mean values. The results are
given in Table 2. Among the tested compounds, thiazepine deriva-
tives 5d and 5e showed a significant DPPH radical scavenging
activity with IC50 values of 0.12 mM. However, their activity was
less than quercetin as reference drug. From the results of 3-(2-hy-
droxybenzylidene)chroman-2,4-diones 3b–g and coumarin-fused
thiazepines 5a–e, it is evident that thiazepine formation improved
radical scavenging ability of the compounds. Also, the substitution
effect on activity was different in these two series of compounds.
For example, while the 7-hydroxycoumarin-fused thiazepine analog
Conclusion
A series of new coumarin-fused 1,4-thiazepines were synthesized
with a simple method in two steps starting from 4-hydroxycouma-
rins. The biological activity of target compounds along with 3-(2-hy-
droxybenzylidene)chroman-2,4-dione intermediates was screened by
evaluation of their antioxidant and cytotoxic activities. 4-Methoxy-
phenolic compound 5d in thiazepine series showed the most potent
scavenging activity, while the 4-bromophenolic derivative 5b was
the most efficient compound in FRAP assay. Also, the result of cyto-
toxic evaluation using MTT assay demonstrated that compound 5b
is at least twofold more potent than etoposide against MCF-7, SK-
N-MC, and MDA-MB 231 cell lines. The results revealed that the
coumarin-fused 1,4-thiazepine structure could be served as a scaf-
fold for finding new bioactive compounds.
Table 2: Antioxidant activities of compounds 3b–g and 5a–e
Acknowledgments
DPPH radical
scavenging
activity (IC50, mM)
FRAP value
(mM Fe2+) (100 lg)
This work was supported by grants from the research council of
Tehran University of Medical Sciences and Iran National Science
Foundation (INSF).
Compound
3b
3c
1.56 € 0.01
3.19 € 0.02
1.47 € 0.01
17.03 € 0.06
0.68 € 0.01
0.58 € 0.01
4.52 € 0.03
0.29 € 0.01
0.97 € 0.01
0.12 € 0.01
0.12 € 0.01
7.92 € 0.3a
39.4 € 0.6
41.8 € 0.8
44.0 € 1.1
30.2 € 0.5
145.7 € 3.4
158.4 € 5.1
41.9 € 1.9
186.4 € 4.5
29.2 € 0.8
89.3 € 2.3
68.1 € 1.7
53.9 € 1.5
3d
3e
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