Synthesis, Cytotoxic Evaluation, and In Silico Studies of 4-Substituted Coumarins

Article abstract of DOI:10.1002/jhet.2458

Two series of coumarins possessing the aniline- and heterocyclic ring at 4^thposition have been synthesized and evaluated for their in vitro cytotoxic activity against MCF-7 cancer cell line in MTT assay. Structure activity relationship (SAR) studies reveal that the electron donor group at position-8 of coumarin played an important role in cytotoxic activity. Compound VIId showed the potent cytotoxic activity followed by compound Xa with IC₅₀= 6.25 and 6.50 μM, respectively. A docking study has also been carried out for the most potent compound to get an insight into molecular interactions with p50 subunit of NF-κB protein.

Full text of DOI:10.1002/jhet.2458

Month 2015
Synthesis, Cytotoxic Evaluation, and In Silico Studies of
4-Substituted Coumarins
Prabhjot Kaur,a Rupinder Kaur Gill,a,b Gagandip Singh,a and Jitender Bariwala,c
*
aDepartment of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga, Punjab 142001, India
bResearch Scholar, Punjab Technical University, Kapurthala, Punjab 144 601, India
cSatiate Research and Anatech Pvt. Ltd., Panchkula, Haryana 134102, India
*
E-mail: jitender.bariwal@gmail.com
Received February 11, 2015
DOI 10.1002/jhet.2458
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
Two series of coumarins possessing the aniline- and heterocyclic ring at 4th position have been synthe-
sized and evaluated for their in vitro cytotoxic activity against MCF-7 cancer cell line in MTT assay. Struc-
ture activity relationship (SAR) studies reveal that the electron donor group at position-8 of coumarin played
an important role in cytotoxic activity. Compound VIId showed the potent cytotoxic activity followed by
compound Xa with IC50 = 6.25 and 6.50 μM, respectively. A docking study has also been carried out for
the most potent compound to get an insight into molecular interactions with p50 subunit of NF-κB protein.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
human health, coumarins are used for diverse range of bio-
logical activities such as anti-cancer [7,8], anti-HIV [9,10],
anticoagulant [11], antimicrobial [12,13], antioxidant [14],
dyslipidemic [15], and anti-inflammatory [16]. Significant
anti-cancer effect has been shown by coumarin and its
metabolite, 7-hydroxycoumarin 6 [17,18], along with
scopoletin (6-methoxy-7-hydroxycoumarin 7) and esculetin
(6,7-dihydroxycoumarin 8), which also exhibited potent
anti-proliferative effects in leukemic cells by inducing
apoptosis [19,20]. Lee et al. [21–23] have reported a
coumarin-containing compound, neo-tanshinlactone 9,
which exhibited more potent and selective inhibition then
tamoxifen, on ER+ human breast cancer cell lines (Fig. 2).
4-Substituted coumarins are well known for interesting
anticancer activity especially 4-hydroxy-substituted couma-
rin [24]. Some of the 4-substituted coumarins known for
anti-cancer activity (Fig. 1) include di-coumarinpolysulfides
(compound 10–12) that accumulate in the G2/M phase of the
cell cycle and induce apoptosis in HCT116 colon cancer
cells [25]. Various coumarins substituted with imidazole 13
and benzothiazole ring 14, 15 have been reported as potent
anticancer agents [26,27] (Fig. 3).
Cancer is a renegade system of growth that originates
within living biosystem having one hallmark characteristic
of uncontrolled growth that invades to other tissues [1]. It
is one of the most vital health problems of the current era
and is a leading cause of death [2]. Although, many efforts
have been made to discover new agents endowed with cyto-
toxic actions, still these agents are suffering from serious
limitations such as lack of selectivity and life-threatening
side effects [3]. Hence, there is an urgent need for discovery
of potent, safe, and selective cytotoxic agents. Natural
products and their derivatives have historically served as
invaluable source of drugs and also exist as mainstay for
cancer chemotherapy. Naturally occurring compounds have
an additional ability to interact with more than one target as
exemplified by paclitaxel 1, vincristine 2, vinorelbine 3,
teniposide 4, and various water-soluble analogs of
camptothecin (e.g., topotecan 5) [4] (Fig. 1).
Coumarin is a naturally occurring potent constituent having
widespread distribution in nature and accompanied with low
toxicity profile [5,6]. Because of their beneficial effects on
© 2015 HeteroCorporation

P. Kaur, R. K. Gill, G. Singh, and J. Bariwal
Vol 000
Figure 1. Naturally occurring cytotoxic compounds.
Figure 2. Coumarins as anticancer agents.
Figure 3. 4-Substituted coumarins as anticancer agents.
As a part of development in the field of coumarins as po-
RESULTS AND DISCUSSION
Synthesis of the target compounds was
accomplished through slight modification of previously
reported methods (Scheme 1) [28–30]. For this, treating
of (un)substituted phenol with malonic acid in the
presence of POCl3 and anhy ZnCl2 at 70°C for 8–10h
yielded 4-hydroxy coumarin (III) [28]. Subsequent
tent anti-cancer agents, we have decided to further explore
coumarin as a core scaffold by synthesized two series of 4-
substituted coumarins by replacing the 4-hydroxy group
with various substituted amines as in Series-I and heterocy-
clic amines as in Series-II (Fig. 4). The synthesized com-
pounds were further evaluated for cytotoxicity against
breast cancer cell line.
Chemistry.
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4-Substituted Coumarins as Effective Cytotoxic Agents
number of living cells. Biological evaluation was carried
out against MCF-7 breast cancer cell lines by using
docetaxel as a positive control. The IC50 values were
illustrated in Table 1.
Among both the series, compounds (VIIa–g and Xa–c)
comprising methyl group at the 8th position of coumarin
were found to be more potent than unsubstituted couma-
rins (VIa–g and IXa–c). Compound VIId (R = CH3,
R1 =4-OC2H5) was found to be the most potent com-
pound followed by Xa (R = CH3, X = NH) with an IC50
value of 6.25 and 6.5 μM, respectively.
From C-8-substituted coumarins, except compound
VIId (4-ethoxy), compounds comprising methoxy group
at the 3rd position of phenyl ring VIIg (IC50 = 8.5 μM) also
possess excellent cytotoxic activity. Whereas, 4-Cl deri-
vative VIIb (IC50 = 11.5 μM), unsubstituted VIIa, and
4-methoxy-substituted phenyl ring as in compound VIIf
(IC50 = 12.5μM) possess moderate cytotoxic activity.
However, few compounds from this series such as com-
pound VIIc (4-F) and VIIe (4-CH3) were found to be
relatively less cytotoxic with IC50 =20.5 and 21.0 μM,
respectively, as compared to the standard drug, Docetaxel
(IC50 = 42.5μM). All the heterocyclic amine-containing
candidates with C-8 methyl-substituted coumarins Xa-c
possess significant cytotoxic activity (Xa, IC50 = 6.5 μM
> Xb, IC50 = 9.5 μM > Xc, IC50 = 11.0μM).
Figure 4. Proposed coumarin derivatives. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
chlorination at 4-position with POCl3 under refluxing
conditions afforded the 4-chloro product IV [29]. The
targeted compounds VI(a–g) and VII(a–g) were assessed
by refluxing 4-chloro derivative IV with substituted
anilines V in the presence of organic base such as
triethylamine in ethanol [30]. Similarly, tertiary amines
IX(a–c) and X(a–c) were obtained by reaction of
4-chloro derivative IV with secondary amines VIII in the
presence of triethylamine. All the synthesized compounds
were characterized by IR and 1H NMR. The reaction
time, melting point, and percentage yield of various
synthesized compounds are mentioned in Table 1.
In vitro cytotoxic activity. All the synthesized compounds
have been evaluated for cytotoxic activity against MCF-7
cancer cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay [31]. Growth of the
breast cancer cells was quantitated by the ability of living
cells to reduce water soluble MTT to purple formazan in
the presence of mitochondrial dehydrogenase. The amount
of formazan product formed is directly proportional to the
From these results, it seems that the electron-donating
substitution at the 8th position of the coumarin plays an
important role in the cytotoxic activity as all the active
compounds have methyl group at the 8th position of the
coumarin skeleton. Although, substitutions of heterocyclic
Scheme 1. Synthesis of 4-substituted coumarin derivatives VI, VII, IX, and X.
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Table 1
Physico-chemical characterization and IC50 value of 4-substituted coumarins (VIa–g, VIIa–g, IXa–c, and Xa–c) against MCF-7 cancer cell line.
S. no.
Code
VIa
VIb
VIc
VId
VIe
VIf
VIg
VIIa
VIIb
VIIc
VIId
VIIe
VIIf
VIIg
IXa
IXb
IXc
Xa
Xb
R
R1 or X
% yield
Reaction time (h)
M.P. (°C)
IC50 MCF-7 (μM)
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
CH3
CH3
CH3
–
H
4-Cl
4-F
4-OC2H5
4-CH3
4-OCH3
3-OCH3
H
52
58
45
71
65
69
47
69
63
59
81
58
67
52
75
58
62
70
49
47
–
6
7.5
7
6
6.5
7
6.5
6
8
6.5
6
6.5
7.5
6
273–276
261–265
272–275
218–220
288–292
239–243
221–225
262–268
245–250
247–253
233–235
284–286
268–272
213–218
142–144
153–156
131–135
108–112
126–130
92–94
17.5
14.0
9.5
Inactive
11.5
8.5
Inactive
12.5
11.5
20.5
6.25
21.0
12.5
8.5
21.0
37.5
Inactive
6.5
9
4-Cl
4-F
10
11
12
13
14
15
16
17
18
19
20
21
4-OC2H5
4-CH3
4-OCH3
3-OCH3
NH
CH2
O
NH
CH2
7
6
7.5
6.5
6
9.5
11.0
42.5
Xc
Docetaxel
O
–
7
–
–
ring system at the 4th position enhance the cytotoxic activity
as in compounds Xa, Xb, and Xc. By comparing com-
pounds, VIIb (IC50 =11.5μM) and VIIc (IC50 =20.5μM),
it seems that substitution of more electronegative group at
para position of aniline part leads to a decrease in potency.
Moreover, compounds VIId and VIIf have different substi-
tutions at para position of substituted anilines such as ethoxy
and methoxy, respectively, and VIIg comprising of
methoxy group at meta position of substituted anilines
showed higher activity, probably because of the presence
of two electron-donating groups.
substituted anilines, respectively, were found to be inactive
and in Series-II, compounds IXa and IXb exhibited mo-
derate cytotoxic activity with IC50 = 21.0, 37.5μM respec-
tively; this indicates that the methyl group at the 8th
position of coumarin is necessary for the potent cyto-
toxicity of these compounds. However, compound IXc
having piperidine ring at the 4th position of coumarin was
found to be inactive.
DOCKING STUDIES
From unsubstituted coumarins, compound VIf was
found to be most potent in the series with IC50 = 8.5 μM
may be because of the presence of electron-donating
methoxy group at the para position of the substituted
aniline part. Compounds VIa, VIb, and VIc have shown
moderate activity with IC50 = 17.5, 14.0, and 9.5 μM,
respectively; this explains that potency increases as electro-
negativity increases at the para position of substituted
anilines. Compounds VId and VIg having ethoxy group
at para position and ethoxy group at meta position of
To rationalize the activity profile of 4-substituted cou-
marins, docking of most potent compound was performed.
A flexible docking study of VIId was carried out at the
binding site of p50 subunit of NF-κB protein (PDB ID:
1NFK) using GOLD software [32]. The highest scoring
conformation was selected to study interaction.
The compound VIId was docked at the binding site of
p50 subunit of NF-κB protein, and the best conformation
was selected on the basis of score and visual inspection.
Figure 5 shows the binding conformation of the PB1 at
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Month 2015
4-Substituted Coumarins as Effective Cytotoxic Agents
Figure 5. Binding conformation of PB1 at binding site 1NFK. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.
com.]
the binding site of p50 subunit of NF-κB protein. The VIId
occupies the cavity of p50 subunit of NF-κB protein. The hy-
drogen bonding with binding residues helps in the stabiliza-
tion of VIId in the cavity. Two important hydrogen bonding
were observed between the carbonyl and ring oxygen of cou-
marin with oxygen and nitrogen of Ser63 and Arg56, respec-
tively. Further the VIId was found to be involved in van der
Waal interaction with residues at receptor site.
melting point apparatus. Proton (1H-NMR) and Carbon-13
nuclear magnetic resonance (13C-NMR) spectra were
recorded in CDCl3 or DMSO-d6 as solvent on Bruker
Avance II 400 NMR spectrometer at 400MHz and
100MHz, respectively. Chemical shifts are reported as δ
values in parts per million (ppm) relative to
tetramethylsilane (TMS) for all recorded NMR spectra. IR
spectra were recorded on Shimadzu 8400 S FT-IR
spectrophotometer with KBr pellets. Mass spectra were
recorded on a Q-Tof micro Mass Spectrometer. The
progress of the reaction was monitored on precoated silica-
gel plates using hexane:ethyl acetate as solvent and
visualizing with ultraviolet light. Starting materials and
reagents used in reactions were obtained commercially
from Acros, Sigma Aldrich, SD fine chemicals and were
used without purification, unless otherwise indicated.
CONCLUSION
Two series of 4-substituted coumarins were prepared
and screened for cytotoxic activity. In both series, com-
pounds comprising electron-donating group (methyl) at
the 8th position of coumarin VIIa–g and Xa–c showed ex-
cellent activity than unsubstituted compounds VIa–g and
IXa–c. Although, substitution of anilino group at the
position-4 possesses comparatively less potency than that
of the heterocyclic ring-substituted compounds. The
benchmark-active compounds are VIId, followed by Xa
with IC50 values of 6.25 and 6.5 μM, respectively, which
opens the new avenue for the novel 4-substituted coumarin
derivatives as promising anti-cancer lead candidates. The
docking study revealed the crucial interactions which make
VIId a potential inhibitor and rationalize the cytotoxic ac-
tivity of 4-substituted coumarins. Further, mechanistic study
is underway and will be communicated in the near future.
General procedure for the synthesis of compound III
[28].
(Un)substituted phenol (0.1 mole) and malonic
acid (0.1 mole) were added to a mixture of phosphorous
oxychloride (40 g) and freshly fused zinc chloride (40 g).
The reaction mixture was heated on water bath at
60–70°C for 8–10h until it become viscous and turned
dark black and then poured into ice water mixture. The
solid residue was filtered out and treated with solution of
saturated sodium carbonate. The filtrate was slowly
acidified with dil. HCl. Upon neutralization, the obtained
precipitates were filtered, washed with water, dried, and
recrystallized from methanol.
4-Hydroxy-2H-chromen-2-one (IIIa).
Yield 48%, m.p.
EXPERIMENTAL
208–212°C [Lit. 210–215°C]. 1H NMR (300MHz,
DMSO-d6) δ: 6.67 (s, 1H, coumarin-H), 7.42–7.56 (m,
3H, ArH), 7.69 (d, J =8.0 Hz, 1H, ArH), 15.93 (s, 1H, OH).
General. Melting points (mp) were uncorrected and
were recorded in open capillaries on the Veego VMP-PM
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P. Kaur, R. K. Gill, G. Singh, and J. Bariwal
Vol 000
4-Hydroxy-8-methyl-2H-chromen 2-one (IIIb).
Yield
51%, m.p. 215–220°C. 1H NMR (300 MHz, DMSO-d6) δ:
2.34 (s, 3H, CH3), 6.65 (s, 1H, coumarin-H), 7.36 (m, 2H,
4-(4-Fluorophenylamino)-2H-chromen-2-one (VIc).
Yield:
45%, m.p. 272–275°C. IR νmax (cmꢀ1): 3285 (NH), 1666
1
(C¼O), 1601 (C¼C), 1218 (C―F). H NMR (300MHz,
ArH), 7.58 (d, J = 7.8 Hz, 1H, ArH), 15.93 (s, 1H, OH).
General procedure for the synthesis of compound IV
[29]. (Un)substituted 4-hydroxy-2H-chromen-2-one III
DMSO-d6) δ: 5.31 (s, 1H, coumarin-H), 6.78 (d, 2H,
J=8.0Hz, ArH), 7.19 (d, 2H, J=8.0Hz, ArH), 7.38–7.40
(m, 2H, ArH), 7.70 (t, 1H, J=8.0Hz, ArH), 8.26 (d, 1H,
J=8.0Hz, ArH), 9.34 (s, 1H, NH). Anal. Calcd. for
C15H10FNO2: C, 70.58; H, 3.95; N, 5.49. Found: C, 71.25;
(0.185 mol) and 70-mL POCl3 were refluxed for 3h,
cooled, and slowly poured into crushed ice (700g) with
vigorous stirring. The solid was collected by filtration and
washed successively with ice water. Finally, azeotropic
distillation with n-hexane, hot filtration of the by-product,
followed by evaporation of solvent and final recrystallization
with methanol give the desired product.
H, 3.56; N, 5.58.
4-(4-Ethoxyphenylamino)-2H-chromen-2-one (VId). Yield:
71%, m.p. 218–220°C. IR νmax (cmꢀ1): 3298 (NH), 2983
(C―H), 1663 (C¼O), 1622 (C¼C), 1246 (C―O). 1H
NMR (300 MHz, DMSO-d6) δ: 2.01 (t, 3H, J=7.3Hz,
CH3), 4.26–4.28 (m, 2H, CH2), 5.8 (s, 1H, coumarin-H),
6.28 (d, 2H, J=7.3Hz, ArH), 6.87 (d, 2H, J=7.3Hz,
ArH), 7.38–7.42 (m, 2H, ArH), 7.70 (t, 1H, J=7.3Hz,
ArH), 8.14 (d, 1H, J=7.3Hz, ArH), 9.19 (s, 1H, NH).
Anal. Calcd. for C17H15NO3: C, 72.58; H, 5.37; N, 4.98.
4-Chloro-2H-chromen-2-one (IVa).
87–89°C [Lit.]. H NMR (300 MHz, DMSO-d6) δ: 6.80
Yield 60%, m.p.
1
(s, 1H, coumarin-H), 7.58–7.44 (m, 3H, ArH), 7.69 (d,
J =8.0 Hz, 1H, ArH).
4-Chloro-8-methyl-2H-chromen-2-one (IVb). Yield 58%,
m.p. 96–99°C. 1H NMR (300 MHz, DMSO-d6) δ: 2.34 (s,
3H, CH3), 6.82 (s, 1H, coumarin-H), 7.34–7.28 (m, 2H,
Found: C, 72.43; H, 5.01; N, 4.67.
4-(p-Tolylamino)-2H-chromen-2-one (VIe). Yield: 65%,
m.p. 288–292°C. IR νmax (cmꢀ1): 3287 (NH), 1655
ArH), 7.60 (d, J = 7.8 Hz, 1H, ArH).
1
(C¼O), 1617 (C¼C). H NMR (300MHz, DMSO-d6) δ:
SYNTHESIS OF 4-SUBSTITUTED COUMARINS
2.38 (s, 3H, CH3), 5.93 (s, 1H, coumarin-H), 7.16–7.26
(m, 4H, ArH), 7.34–7.43 (m, 2H, ArH), 7.60 (d, 1H,
J = 8.1 Hz, ArH), 7.65 (d, 1H, J = 8.1 Hz, ArH). Anal.
Calcd. for C16H13NO2: C, 76.48; H, 5.21; N, 5.57. Found:
Preparation of 4-substituted coumarins: Series-I (VIa–g and
VIIa–g) [30]. (Un)substituted 4-chloro-2H-chromen-2-one
IV (0.05mol) and (un)substituted aniline (0.05mol) were
dissolved in ethanol (30mL), and catalytic amount of
triethyl amine was added. The resulting solution was
refluxed for 1h and left at room temperature for 4–5h.
The separated precipitate were filtered off and
recrystallized from ethanol. Using this procedure, the
C, 76.34; H, 5.66; N, 5.11.
4-(4-Methoxyphenylamino)-2H-chromen-2-one (VIf). Yield:
69%, m.p. 239–243°C. IR νmax (cmꢀ1): 3292 (NH), 3071
(C―H), 1661 (C¼O), 1622 (C¼C), 1203 (C―O). 1H NMR
(300MHz, DMSO-d6) δ: 4.02 (s, 3H, OCH3), 6.0 (s, 1H,
coumarin-H), 6.24 (d, 2H, J=7.5Hz, ArH), 6.64 (d, 2H,
J=7.5Hz, ArH), 7.42–7.58 (m, 3H, ArH), 7.79 (d, 1H,
J=7.5Hz, ArH), 8.02 (s, 1H, NH). Anal. Calcd. for
C16H13NO3: C, 71.90; H, 4.90; N, 5.24. Found: C, 71.02; H,
following compounds were synthesized.
4-(Phenylamino)-2H-chromen-2-one (VIa). Yield: 52%,
m.p. 273–276°C. IR νmax (cmꢀ1): 3299 (NH), 3027
1
(C―H), 1662 (C¼O), 1609 (C¼C). H NMR (300MHz,
DMSO-d6) δ: 5.29 (s, 1H, coumarin-H), 7.29–7.38 (m,
5H, ArH), 7.47 (d, 2H, J= 6.9 Hz, ArH), 7.65 (t, 1H,
J =7.5 Hz, ArH), 8.24 (d, 1H, J = 7.5 Hz, ArH), 9.31 (s,
1H, NH). 13C NMR (75 MHz, CDCl3): δ 161.58, 150.12,
148.26, 139.50, 134.08, 129.11, 129.01, 125.68, 124.12,
123.68, 123.01, 120.11, 119.45, 108.81, 84.21. Anal.
Calcd. for C15H11NO2: C, 75.94; H, 4.67; N, 5.90.
5.08; N, 5.00.
4-(3-Methoxyphenylamino)-2H-chromen-2-one (VIg). Yield:
47%, m.p. 221–225°C. IR νmax (cmꢀ1): 3292 (NH), 2977
(C―H), 1655 (C¼O), 1602 (C¼C), 1134 (C―O). 1H NMR
(300MHz, DMSO-d6) δ: 4.02 (s, 3H, OCH3), 5.64 (s, 1H,
coumarin-H), 6.15–6.29 (m, 3H, ArH), 7.21–7.28 (m, 3H,
ArH), 7.59 (t, 1H, J=8.0Hz, ArH), 7.91 (d, 1H, J=8.0Hz,
ArH), 8.4 (s, 1H, NH). Anal. Calcd. for C16H13NO3: C,
Found: C, 75.12; H, 4.73; N, 6.63.
4-(4-Chlorophenylamino)-2H-chromen-2-one (VIb). Yield:
71.90; H, 4.90; N, 5.24. Found: C, 71.12; H, 4.26; N, 5.00.
58%, m.p. 261–265°C. IR νmax (cmꢀ1): 3279 (NH), 1663
4-(Phenylamino)-8-methyl-2H-chromen-2-one (VIIa). Yield:
1
69%, m.p. 262–268°C. IR νmax (cmꢀ1): 3307 (NH), 2975
(C¼O), 1609 (C¼C), 747 (C―Cl). H NMR (300MHz,
1
DMSO-d6) δ: 5.29 (s, 1H, coumarin-H), 6.84 (d, 2H,
J=7.8Hz, ArH), 7.31 (d, 2H, J=7.8Hz, ArH), 7.52–7.54
(m, 2H, ArH), 7.76 (t, 1H, J=7.8Hz, ArH), 8.24 (d, 1H,
J=7.5Hz, ArH), 9.31 (s, 1H, NH). 13C NMR (75 MHz,
CDCl3): δ 161.58, 155.24, 150.12, 140.23, 132.12, 130.69,
130.01, 129.56, 125.68, 124.79, 122.98, 122.01, 112.47,
110.22, 84.11. Anal. Calcd. for C15H10ClNO2: C, 66.31; H,
3.71; N, 5.16. Found: C, 67.75; H, 4.02; N, 5.01. MS:
m/z=271 [M+].
(C―H), 1659 (C¼O), 1609 (C¼C). H NMR (300MHz,
DMSO-d6) δ: 1.19 (s, 3H, CH3), 5.34 (s, 1H, coumarin-H),
6.27 (d, 2H, J=7.5Hz, ArH), 6.67–7.21 (m, 5H, ArH), 7.84
(d, 1H, J=7.5Hz, ArH), 9.31 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3): δ 158.85, 149.12, 148.26, 139.50,
134.08, 131.23, 128.11, 126.22, 124.13, 122.52, 122.01,
120.81, 120.08, 108.81, 84.21, 16.01. Anal. Calcd. for
C16H13NO2: C, 76.48; H, 5.21; N, 5.57. Found: C, 76.98; H,
5.26; N, 5.63.
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4-Substituted Coumarins as Effective Cytotoxic Agents
4-(4-Chlorophenylamino)-8-methyl-2H-chromen-2-one
8.52 (s, 1H, NH). Anal. Calcd. for C17H15NO3: C, 72.58;
H, 5.37; N, 4.98. Found: C, 72.22; H, 5.89; N, 4.22.
Preparation of 4-Substituted coumarins: Series-II (IXa–c
(VIIb). Yield: 63%, m.p. 245–250°C. IR νmax (cmꢀ1):
1
3291 (NH), 1659 (C¼O), 1608 (C¼C), 726 (C―Cl). H
NMR (300 MHz, DMSO-d6) δ: 1.19 (s, 3H, CH3), 5.45
(s, 1H, coumarin-H), 6.55 (d, 2H, J= 7.5 Hz, ArH), 7.19–
7.21 (m, 3H, ArH), 7.23 (t, 1H, J= 7.5 Hz, ArH), 8.20 (d,
1H, J =7.5 Hz, ArH), 9.25 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3): δ 159.56, 152.11, 150.42, 133.31,
128.39, 127.63, 127.01, 126.78, 125.23, 124.79, 122.01,
121.76, 117.34, 112.21, 84.11, 16.01. Anal. Calcd. for
C16H12ClNO2: C, 67.26; H, 4.23; N, 4.90. Found: C,
67.45; H, 4.13; N, 4.95.
and Xa–c).
The 4-chloro-2H-chromen-2-one IVa/4-
chloro-8-methyl-2H-chromen-2-one IVb (0.05mol) and
secondary amines (0.05mol) were dissolved in ethanol
(30 mL) and added catalytic amount of triethyl amine.
The resulting solution was refluxed for 1 h and left at
room temperature for 4–5h. The separated precipitate
was filtered off and recrystallized from ethanol.
4-(Piperazin-1-yl)-2H-chromen-2-one (IXa). Yield: 58%,
m.p. 153–156°C. IR νmax (cmꢀ1): 3397 (NH), 2852
(C―H), 1712 (C¼O), 1603 (C¼C), 1321 (C―N). 1H
NMR (300 MHz, DMSO-d6) δ: 2.51 (t, 4H, J=4.1Hz,
piperazin-CH2), 2.78 (t, 4H, J=4.1Hz, piperazin-CH2),
5.23 (s, 1H, ArH), 7.79 (d, 1H, J=8.0Hz, ArH), 7.45–7.54
(m, 3H, ArH). Anal. Calcd. for C13H14N2O2: C, 67.81; H,
4-(4-Fluorophenylamino)-8-methyl-2H-chromen-2-one
(VIIc). Yield: 59%, m.p. 247–253°C. IR νmax (cmꢀ1):
3295 (NH), 2976 (C―H), 1611 (C¼O), 1600 (C¼C),
1
1233 (C―F). H NMR (300 MHz, DMSO-d6) δ: 1.20 (s,
3H, CH3), 5.43 (s, 1H, coumarin-H), 6.78 (d, 2H,
J =7.8 Hz, ArH), 7.17 (d, 2H, J= 7.8 Hz, ArH), 7.23–7.25
(m, 2H, ArH), 8.21 (d, 1H, J = 7.8 Hz, ArH), 9.25 (s, 1H,
NH). Anal. Calcd. for C16H12FNO2: C, 71.37; H, 4.49;
N, 5.20. Found: C, 70.28; H, 5.20; N, 5.15.
6.13; N, 12.17. Found: C, 67.05; H, 6.18; N, 12.76.
4-(Piperidin-1-yl)-2H-chromen-2-one (IXb). Yield: 62%,
m.p. 131–135°C. IR νmax (cmꢀ1): 3063 (NH), 2940
(C―H), 1708 (C¼O), 1604 (C¼C), 1320 (C―N). 1H
NMR (300MHz, DMSO-d6) δ: 1.58 (t, 4H, J =4.1 Hz,
piperidin-CH2), 1.62 (t, 2H, J =4.1 Hz, piperidin-CH2),
3.21 (t, 4H, J = 4.1 Hz, piperidin-CH2), 5.25 (s, 1H,
coumarin-H), 7.47–7.56 (m, 3H, ArH), 7.79 (d, 1H,
J = 8.0 Hz, ArH). Anal. Calcd. for C14H15NO2: C, 73.34;
4-(4-Ethoxyphenylamino)-8-methyl-2H-chromen-2-one
(VIId). Yield: 81%, m.p. 233–235°C. IR νmax (cmꢀ1):
3306 (NH), 2974 (C―H), 1660 (C¼O), 1610 (C¼C),
1
1246 (C―O). H NMR (300 MHz, DMSO-d6) δ: 2.47 (s,
3H, CH3), 4.06 (m, 2H, CH2), 4.77 (s, 3H, ArCH3), 5.52
(s, 1H, coumarin-H), 6.92–6.95 (m, 2H, ArH), 7.17–7.23
(m, 3H, ArH), 7.41–7.48 (m, 2H, ArH). Anal. Calcd. for
C18H17NO3: C, 73.20; H, 5.80; N, 4.74. Found: C, 73.53;
H, 4.73; N, 4.10.
H, 6.59; N, 6.11. Found: C, 73.85; H, 6.24; N, 6.86.
4-Morpholino-2H-chromen-2-one (IXa). Yield: 75%, m.
p. 142–144°C. IR νmax (cmꢀ1): 3032 (NH), 2862 (C―H),
1707 (C¼O), 1600 (C¼C), 1332 (C―N), 1137 (C―O).
1H NMR (300 MHz, DMSO-d6) δ: 3.24 (t, 4H, J =3.9 Hz,
morpholino-CH2), 3.93 (t, 4H, J= 3.9 Hz, morpholino-
CH2), 5.76 (s, 1H, coumarin-H), 7.24–7.29 (m, 1H,
ArH), 7.36 (d, 1H, J= 8.4 Hz, ArH), 7.50–7.55 (m, 1H,
ArH), 7.62 (d, 1H, J = 7.8 Hz, ArH). 13C NMR (75 MHz,
CDCl3): δ 158.20, 152.87, 150.58, 126.73, 123.24,
122.28, 115.07, 113.33, 74.98, 67.46, 67.46, 48.82,
48.82. Anal. Calcd. for C13H13NO3: C, 67.52; H, 5.67;
4-(p-Tolylamino)-8-methyl-2H-chromen-2-one (VIIe). Yield:
58%, m.p. 284–286°C. IR νmax (cmꢀ1): 3300 (NH), 2973
1
(C―H), 1658 (C¼O), 1608 (C¼C). H NMR (300MHz,
DMSO-d6) δ: 2.05 (s, 3H, CH3), 2.30 (s, 3H, ArCH3), 5.61
(s, 1H, coumarin-H), 6.26 (d, 2H, J=8.0Hz, ArH), 6.82 (d,
2H, J=8.0Hz, ArH), 7.24–7.25 (m, 1H, ArH), 7.58 (d, 1H,
J=8.0Hz, ArH), 7.65 (d, 1H, J=8.0Hz, ArH). Anal. Calcd.
for C17H15NO2: C, 76.96; H, 5.70; N, 5.28. Found: C,
76.44; H, 6.01; N, 5.15.
N, 6.06. Found: C, 67.29; H, 5.20; N, 6.13.
4-(Piperazin1-yl)-8-methyl-2H-chromen-2-one (Xa). Yield:
4-(4-Methoxyphenylamino)-8-methyl-2H-chromen-2-one
(VIIf). Yield: 67%, m.p. 268–272°C. IR νmax (cmꢀ1):
49%, m.p. 126–130°C. IR νmax (cmꢀ1): 3301 (NH),
2959 (C―H), 1692 (C¼O), 1600 (C¼C), 1322 (C―N).
1H NMR (300 MHz, DMSO-d6) δ: 2.08 (s, 3H, CH3),
2.65 (t, 4H, J = 3.8 Hz, piperazin-CH2), 3.08 (t, 4H,
J = 3.8 Hz, piperazin-CH2), 5.50 (s, 1H, coumarin-H),
7.20 (t, 1H, J = 7.5 Hz, ArH), 7.28 (d, 1H, J = 7.8 Hz,
ArH), 7.75 (d, 1H, J = 7.8 Hz, ArH). Anal. Calcd. for
3301 (NH), 2952 (C―H), 1663 (C¼O), 1591 (C¼C),
1
1247 (C―O). H NMR (300 MHz, DMSO-d6) δ: 2.12 (s,
3H, CH3), 3.78 (s, 3H, OCH3), 5.24 (s, 1H, coumarin-H),
6.00 (s, 1H, ArH), 6.24 (d, 2H, J = 7.5 Hz, ArH), 6.64 (d,
2H, J = 7.5Hz, ArH), 7.23–7.26 (m, 2H, ArH), 8.00 (s,
1H, NH). Anal. Calcd. for C17H15NO3: C, 72.58; H,
5.37; N, 4.98. Found: C, 72.35; H, 5.12; N, 4.58.
4-(3-Methoxyphenylamino)-8-methyl-2H-chromen-2-one
(VIIg). Yield: 52%, m.p. 213–218°C. IR νmax (cmꢀ1):
C14H16N2O2: C, 68.83; H, 6.60; N, 11.47. Found: C,
68.12; H, 6.82; N, 11.11.
4-(Piperidin-1-yl)-8-methyl-2H-chromen-2-one (Xb). Yield:
47%, m.p. 92–94°C. IR νmax (cmꢀ1): 3299 (NH), 2937
3301 (NH), 2993 (C―H), 1662 (C¼O), 1601 (C¼C),
1
1264 (C―O). H NMR (300 MHz, DMSO-d6) δ: 2.12 (s,
(C―H), 1655 (C¼O), 1603 (C¼C), 1319 (C―N).
1H NMR (300 MHz, DMSO-d6) δ: 1.60–1.48 (m, 6H,
piperidin-CH2), 2.20 (s, 3H, CH3), 2.88 (t, 4H, J=4.0Hz,
piperidin-CH2), 5.45 (s, 1H, coumarin-H), 7.13 (t, 1H,
3H, CH3), 4.10 (s, 3H, O CH3), 5.24 (s, 1H, coumarin-H),
5.98–6.15 (m, 3H, ArH), 6.75 (t, 1H, J = 8.0 Hz, ArH),
7.23–7.25 (m, 2H, ArH), 7.67 (d, 1H, J = 8.0 Hz, ArH),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

P. Kaur, R. K. Gill, G. Singh, and J. Bariwal
Vol 000
Acknowledgments. Authors are thankful to the Department of
Sciences and Technology (DST) for providing Junior Research
fellowship to Ms. Rupinder Kaur Gill through research grant to
Dr. Jitender Bariwal (Letter No. SR/FT/CS-59/2011). Authors
are also thankful to Shri Parveen Garg (Chairman), I.S.F
College of Pharmacy Moga, Punjab, for providing us supportive,
encouraging environment and excellent research facilities in
the college.
J = 7.8 Hz, ArH), 7.33 (d, 1H, J = 7.8 Hz, ArH), 7.45 (d,
1H, J= 7.8 Hz, ArH). Anal. Calcd. for C15H17NO2: C,
74.05; H, 7.04; N, 5.76. Found: C, 74.85; H, 7.82; N, 5.21.
4-Morpholino-8-methyl-2H-chromen-2-one (Xc).
Yield:
70%, m.p. 108–112°C. IR νmax (cmꢀ1): 3368 (NH), 2918
(C―H), 1710 (C¼O), 1601 (C¼C), 1318 (C―N), 1131
1
(C―O). H NMR (300MHz, DMSO-d6) δ: 2.46 (s, 3H,
CH3), 3.23 (t, 4H, J=4.5Hz, morpholine-CH2), 3.93
(t, 4H, J=4.8Hz, morpholine-CH2), 5.74 (s, 1H,
coumarin-H), 7.14 (t, 1H, J=7.5Hz, ArH), 7.35 (d, 1H,
J=7.2Hz, ArH), 7.44 (d, 1H, J=7.8Hz, ArH). 13C NMR
(75MHz, CDCl3): δ 157.92, 151.85, 149.16, 130.28,
122.10, 121.01, 114.42, 110.79, 75.26, 68.00, 68.00,
47.56, 47.56, 15.26. Anal. Calcd. for C14H15NO3: C,
68.56; H, 6.16; N, 5.71. Found: C, 68.43; H, 6.18; N, 6.93.
MS: m/z=243 [M+]
In vitro cytotoxicity assay [31]. Cytotoxic effects were
examined in MCF-7 breast cancer cell lines. The MCF-7
was at a concentration of 3 ×80cells/mL, plated onto 96
well flat bottom culture plates for 24 h, in which cell
number was kept at 4000–5000 using neubauer cell
counting chamber. After 24h of incubation, cells were
treated with various concentrations of test drugs. Then
cultures were incubated for 72 h at 37°C in a humidified
incubator. After 72h of incubation (37°C, 5% CO2 in
humidified atmosphere), 5μL of MTT (5mg/mL in DMSO)
was added to each well, and the plate was incubated for a
further 4h at 37°C in dark. The resulting formazan was
dissolved in 50-μL dimethyl sulphoxide, and absorbance of
the solution was read at 595nm using an ELISA plate
reader (Biorad, Model 680, Japan). All determinations were
carried out in triplicate. Concentrations of test drugs
showing 50% reduction in cell viability (i.e., IC50 values)
were determined by a nonlinear regression analysis.
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CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
Journal of Heterocyclic Chemistry
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