Indian Journal of Chemistry

Vol. 50B, November 2011, pp. 1619-1629

Synthesis and cytotoxity of novel benzopyran derivatives

Anil K Tripathi*a, Debaraj Mukherjeea, Surrinder Koula, Subhash C Tanejaa, Satyam K Agrawalb,
Parduman R Sharmab & Ajit K Saxenab
a

Bio-Organic Chemistry Section and bPharmacology Division, Indian Institute of Integrative Medicines (CSIR),
Canal Road, Jammu 180 001, India
E-mail: tripathitripathi@rediffmail.com
Received 10 January 2011; accepted (revised) 26 August 2011

In-vitro cytotoxic activity of benzopyrones and benzopyran derivatives has been evaluated against a panel of several
human cancer cell lines. Out of 16 compounds prepared and screened, four compounds have shown significant cytotoxicity
against human breast, liver, colon and prostrate cancer cell lines. Apoptosis rather than necrosis has been established as the
mode of mechanism for the cytotoxicity of the studied molecules using Light Microscopic and Scanning Electron
Microscopic techniques.

Keywords: Acetophenones, chromanone, benzopyrans, cytotoxic, anti-hypertensive

Chromane, chromanones and chromenones associated

with interesting chemistry, form the core ring
structure of a number of natural products like
flavanoids, isoflavanoids, coumarins, homo-, iso- and
neo-flavonoids1,2. Compounds representing these
types of classes manifest a variety of remarkable
biological activities such as anti-allergic, tyrosine
kinase inhibitory, estrogen receptor agonist or
antagonist or inhibitor activity of steroidegenic
enzymes3,4. Chroman/chromanones or in simple terms
benzopyrans/benzopyranones can be potential
intermediates for the synthesis of heterocyclic analogs
of steroids and/or important building blocks for the
preparation of pterocarpans and isoflavones with
strong fungicidal activity5. Another important activity
associated with chromans having substitution e.g. at
2- and 4-positions has been that of its smooth muscle
relaxant activity and these compounds have been used
for the treatment of disorders such as asthma and
hypertension with chromakalim 7 being the best
representative example of these anti-hypertensives6.

7
Chromakalim

As a part of drug discovery programme at IIIM,

Jammu, the search is on for molecules that are

associated with strong growth inhibitory activity

against a panel of human cancer cells capable of
inhibiting, retarding or reversing the process of
carcinogenesis. The molecules as such in the first
place may not show high multistage potency and low
toxicity, but exhibit moderate anticancer activity.
Fine-tuning of these promising candidates can be
manoeuvred through structure activity relationship
(SAR) studies or by using molecular modeling studies
involving ligand target docking for the development
of molecules bearing minimal toxicity and high
anticancer activity. With this objective in mind, it was
envisaged to prepare chromanones and their
substituted derivatives and evaluate them for possible
cytotoxic activity and study the mechanism of action
of the active molecules. Barring 2-stryl substituted
chromanones7,8, literature survey showed scanty
references for the said class of compounds bearing
cytotoxic activity. In this communication, is described
the synthesis of novel chroman-derivatives and
thereafter their cytotoxic activity studies including
mode of action.
Results and Discussion
Chemistry
2-Substituted-7-hydroxy
chromanones
were
prepared using the procedure reported by the present9
and other groups in the literature10-12 which involves
the base catalysed condensation of substituted
acetophenones with keto compounds to give 2,2-

INDIAN J. CHEM., SEC B, NOVEMBER 2011

1620

disubstituted-7-hydroxy-benzopyran-4-one 3a-d in
high yields (80-82%). Methylation of phenolic group
in compounds 3a-d afforded 7-methoxy derivatives
4a-d. In order to introduce pyridyl group at C-4
position of chroman using reductive condensation
reaction, the key step of the reaction sequence, a
recently reported protocol was followed13. The
reaction was carried out by condensation of
benzopyran-4-one with pyridine in the presence of
aluminum-amalgam [Al/HgCl2] to afford the desired
4-pyridyl substituted benzopyrans 5a-d. Dehydration
of the hydroxyl function of the compound 5a-d
afforded 4-(2-pyridyl)-chroman derivatives 6a-d
(Scheme I). The chemical structures of the
synthesized molecules have been established by
spectroscopic analysis.

results are reported as percent growth inhibition (at

denoted concentrations) against the cell line used
(Table I). Out of sixteen compound bioevaluated (at
100 M concentration), seven compounds showed
>50% inhibition, compound 6a and 5d being the
effective molecules that showed inhibitory activity (IA)
against seven out of eight cell lines used (59-77% IA
range observed for 5d and 54-92% for 6a), followed by
6c that exhibited inhibitory activity against five cell
lines (IA 50-75%) and 5b (IA 61-70%) that proved to
be cytotoxic against four cell lines. Compound 6d
showed inhibitory activity against two cell lines and 4b
and 5a against one cell line each.
By comparing the cytotoxic potency of the
compounds 3a-d to 6a-d, a few inferences could be
drawn such as: (i) the presence of carbonyl group at
4-position does not contribute towards cytotoxicity as
can be seen in compounds 3a-d and 4a-d which
showed poor growth inhibition against all the cancer
cell lines used; (ii) the presence of pyridinyl
substituent at C-4 position contributes significantly
towards the enhancement of inhibitory activity and
(iii) presence of hydroxyl group at C-4 position or its
removal (unsaturation between C3-C4) seem to be
insignificant in terms of activity as no major step
jump in activity in positive or negative mode could be
observed except in case of 5a vs 6a.

Biology
Compounds 3a-d, 4a-d, 5a-d and 6a-d were bioevaluated for in vitro cytotoxicity activity using
sulforhodamine B14 and tested against a panel of eight
human cancer cell lines that contained MCF-7 (breast),
DU-145, PC-3 (prostate), HEP-2 (liver), A-549 (lung),
HT-29 and 502713 (colon) and Molt-4 cell line
(leukemia) respectively. Anticancer compounds
Paclitaxel, Mitomycin-c, Adriamycin and 5Fluorouracil were taken as positive control and the
O

(i)
HO

OH

(ii)
HO

OH

HO

R1
R2

3a-d
(iii)

N
OH

(v)
H3CO

6a-d

R1
R2

H3CO

5a-d

R1
R2

(iv)
H3CO

R1
R2

4a-d

(a) = R1, R2 =
(b) = R1, R2 =
(c) = R1, R2 =
(d) = R1, R2 =
Scheme I Synthesis of 4-(2-pyridyl)-2H-(1)-benzopyrans and 4-(2-pyridyl)-4-hydroxy-3,4-dihydro benzopyran analogues. Reagents
and conditions: (i) ZnCl2, CH3COOH, heat; (ii) Pyrrolidine, keto substrate, benzene, reflux; (iii) CH3I, acetone, K2CO3, reflux; (iv)
Pyridine, HgCl2-Al, reflux; (v) p-TSA, benzene, reflux.

TRIPATHI et al.: NOVEL BENZOPYRAN DERIVATIVES

1621

Table I Inhibition activity of unsubstituted and substituted benzopyrans against human cancer cell lines

Entry
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
Paclitaxel
Mitomycin-C
Adriamycin
5FU
*NT-Not Tested

Conc.

Breast
MCF-7

Prostate
DU-145

Prostate
PC-3

Liver
HEP-2

Lung
A-549

Colon
HT-29

Colon
502713

Leukemia
Molt-4

100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
100M
1M
10M
10M
100M

NT*
NT
NT
NT
NT
NT
NT
NT
37
61
39
73
75
37
75
70
72
84
80
48

20
18
17
14
16
18
25
25
30
29
5
42
17
31
50
48
41
52
55
50

22
22
18
15
0
0
1
21
21
22
6
63
54
25
50
27
NT
43
68
23

NT
NT
NT
NT
NT
NT
NT
NT
51
70
38
59
58
53
35
37
64
59
58
49

31
33
38
39
49
56
22
23
35
68
12
77
70
47
75
69
70
92
83
61

NT
NT
NT
NT
NT
NT
NT
NT
19
42
9
60
63
25
14
15
66
NT
57
46

NT
NT
NT
NT
NT
NT
NT
NT
18
27
25
60
62
15
37
22
68
16
NT
55

0
0
0
7
31
0
8
23
31
62
55
65
92
0
68
37
NT
NT
49
70

Since many of the synthesized compounds

displayed cytotoxicity against several human cancer
cell lines, compounds 5b, 5d, 6a, and 6c that showed
>50% growth inhibition against four or more cancer
cell lines were chosen to ascertain whether these
molecules kill cancer cells by apoptosis or necrosis.
The activation of apoptosis is often a mechanism of
cancer cell killing by anticancer drugs15. Apoptosis is
a highly organized cell death characterized by ultra
structural modification (cytoskeletal disruption, cell
shrinkage and membrane blebbing), nuclear alteration
(internucleosomal DNA cleavage and chromatin
condensation) and biochemical changes. The
morphological analysis of cancer cells has been
considered a milestone of research in the field of
apoptosis16-19. The apoptosis was studied by light
microscopy and scanning electron microscopy (SEM).
Light microscopic studies on MOLT-4 cells
revealed that untreated cells are spherical in shape and
the nucleus is large as compared to the cell size. The
nucleoli are clearly visible in the nucleus of untreated
cells (Figure 1, A; as indicated by
). After 12 hr of

treatment with 5b, 5d, 6a and 6c (110-4 M)

shrinkage in most of the cells, condensation and
fragmentation of the nucleus, loss of nucleoli and
formation of apoptotic bodies, characteristic to
apoptosis was observed (shown by arrows and arrow
heads in the Figure 1, plate B-E). The apoptosis was
more prominent in compounds 6a and 6c than 5b and
5d hence morphological analysis of 6a and 6c was
further studied by SEM.
Surface ultra structural investigations revealed the
characteristics of control and treated (12 hr, 110-4 M)
MOLT-4 cells. The untreated MOLT-4 cells depicted
numerous microvilli over the entire cell surface
(Figure 2-A, asterisk). The microvilli on the surface
of malignant cells could contribute to movement,
attachment and invasion by the malignant cells20.
Reduction of microvilli might potentially inhibit cell
proliferation. The treated MOLT-4 cells showed
smooth surface, shrinkage and formation of apoptotic
bodies (Figure 2, B-G). The treatment of 6a and 6c
caused smoothening of cell surface (S) and formation
of numerous apoptotic bodies (Figure 2, arrows) in

1622

INDIAN J. CHEM., SEC B, NOVEMBER 2011

Figure 1 Light microscopy of MOLT-4 cells after 12 hr incubation with 100 M, 5b, 5d, 6a and 6c. The untreated cells
showing spherical shape, nucleus (as shown by
) is large and has 1 or 2 nucleoli and the cytoplasm is restricted to the
periphery of the cells (A). The MOLT-4 cells treated with 5b(B), 5d(C), 6a(D) and 6c(E). Note the condensation and
fragmentation of the nucleus (as shown by ) and formation of apoptotic bodies (as shown by
).

some cells and in others, surface pits (P) were

observed due to detachment of apoptotic bodies
(Figure 2, D). The scanning electron micrographs
have shown that MOLT-4 cells following the
treatments with 6a or 6c, progressively express the
typical morphological characteristics of apoptosis.
The cells dying by apoptosis lost the organization of

superficial structures, acquiring a smooth surface.

Blebs were extruded from the cell surface and
detached from the cells (arrowheads) to form
apoptotic bodies and pits of different sizes. The above
studies unambiguously established that the mode of
action of these molecules takes place by apoptosis
rather than by necrosis.

TRIPATHI et al.: NOVEL BENZOPYRAN DERIVATIVES

1623

Figure 2 SEM of MOLT-4 cells treated with 100 M of 6a and 6c. (Figure A) Control MOLT-4 cell. Note numerous
surface projections over the entire cell surface (*). (Figure B, C and D) The MOLT-4 cells treated with 6a compared with
the untreated cell, surface projections disappeared from the cell surface after treatment, cell surface appears smooth (S,
Figure C, D), cells decrease in size and apoptotic bodies (as shown by
, Figure B, C) are evident due to apoptosis. In
some cells apoptotic bodies appear to detach from the cell surface (Figure D). (as shown by
, E, F and G). The MOLT-4
cells treated with 6c, showing smooth surface (S, Figure F, G) and apoptotic bodies in the process of formation (as shown
by , G) and about to detach from the main cell body (as shown by
, Figure E, F and G). (Magnification A, C, D and
E, 4800X; B and F, 3600X; G, 6000X).

1624

INDIAN J. CHEM., SEC B, NOVEMBER 2011

Experimental Section
Chemistry
Solvents and other chemicals of reagent grade were
used without further purification. Laboratory grade
solvents were purified and dried by reported methods.
All melting points were determined by capillary
method on a Buchi technical apparatus (Buchi-510)
and are uncorrected. NMR spectra (1H and 13C NMR)
were obtained on Bruker Supercon 200 MHz and 500
MHz instruments and are expressed in values down
field from tetramethylsilane (TMS) as the internal
standard. Mass spectra were recorded with a Jeol MSD 300 mass spectrometer, IR (KBr pellet or neat
sample) were recorded on Perkin-Elmer-377 and
Shimadzu IR-435 spectrophotometers. Column
chromatography was performed over silica gel (100200 mesh) and TLC was performed on Silica gel 60
F254 (Merck) plates. For the visualization of spots
either UV or iodine vapor, or 10% aqueous sulfuric
acid containing 2% ceric ammonium sulfate or 2,4dinitrophenyl hydrazine (5% ethanol solution) was
used.
Preparation of 2,2-dimethyl-7-methoxy-4-hydroxy4-(2-pyridyl)-3,4-dihydro-2H-1-benzopyran 6a
Preparation of 2,2-dimethyl-7-hydroxy-3,4-dihydro-2H-1-benzopyran-4-one 3a. Pyrrolidine (3.0
mL, 50 mmol) was added to a solution of 2,4dihydroxy acetophenone (7.6 g, 50 mmol) and
acetone (2.5 mL) in dry benzene (40 mL). The
mixture was refluxed on water bath for 6 hr, the
contents cooled and poured into ice water and
acidified with 5% hydrochloric acid, extracted with
ethyl acetate (450 mL), the organic layer washed
with water, dried over anhydrous sodium sulfate and
concentrated under reduced pressure on a rotary
evaporator to give a crude product which was purified
by column chromatography over silica gel using
petroleum ether:ethyl acetate (80:20) as eluent to
afford 3a (7.9 g, 82%) as yellowish crystalline
compound, m.p. 166C. IR: 3125, 1685, 1600, 1580,
1490, 1400, 1380, 1360, 1340, 1270, 1175, 1130,
1080, 1050, 1020, 990, 860, 820, 780, 760 cm-1;
1
H NMR (200 MHz, CDCl3): 1.52(6H, s, 2CH3),
2.83(2H, s, CH2), 6.40 (1H, d, J= 2.5 Hz, 8-H), 6.56
(1H, dd, J= 8.5 and 2.5 Hz, 6-H), 7.83(1H, d, J= 8.5
Hz,5-H); 13C NMR (50 MHz, CDCl3): 26.49, 48.74,
79.40, 118.18, 120.87, 126.36, 128.48, 136.00,
160.02, 192.12; MS: m/z (%) 192(11), 190(66),
176(90), 151(10), 136(100), 135(60), 107(53), 79(21),
68(17), 63(14).

Preparation of 2,2-dimethyl-3,4-dihydro-7-methoxy-2H-1-benzopyran-4-one 4a. Methyl iodide

(1.3 mL, 20 mmol) was added to a solution of 2,2dimethyl-7-hydroxy-3,4-dihydro-2H-1-benzopyran-4one 3a (1.95 g, 10 mmol) in acetone (20 mL)
containing anhydrous potassium carbonate (2.5 g) and
the contents refluxed. After completion of the reaction
as monitored by TLC, the contents were cooled,
filtered through sintered funnel and the filtrate
concentrated on a rotary evaporator under reduced
pressure to give crude product which on column
chromatography over silica gel and petroleum
ether/ethyl acetate (96:4) as the eluant afforded, as a
pale yellow semisolid 4a (1.86 g, yield 89%). IR:
2900, 1670, 1620, 1610, 1595, 1440, 1380, 1360,
1320, 1270, 1120, 1060, 1020, 960, 980, 840, 820,
760 cm-1; 1H NMR (200 MHz, CDCl3): 1.47(6H, s,
2CH3), 2.69(2H, s, 3-H), 3.88(3H, s, OCH3),
6.43(1H, d, J=2.5Hz, 8-H), 6.60 (1H, dd, J=8.5 and
2.5Hz, 6-H), 7.84(1H, d, J=8.5Hz, 5-H); 13C NMR
(50 MHz, CDCl3): 26.68, 48.58, 58.52, 79.55,
101.20, 109.25, 114.14, 128.21, 161.99, 166.26,
190.98; MS: m/z (%) 206 (86), 192(19), 191(80),
151(100), 150(69), 122(58), 107(64), 94(21), 91(25),
82(24), 79(53), 69(21), 63(43).
Preparation of 2,2-dimethyl-4-hydroxy-7-methoxy-3,4-dihydro-4-(2-pyridyl)-2H-1-benzopyran
5a. A mixture of mercuric chloride (30 mg) and
freshly prepared aluminum powder was heated at
120C for 20 min under nitrogen until formation of
uniform gel. To that gel 4a (1.67 g, 8 mmol) in
pyridine (1.20 mL) was added with vigorous stirring
and further refluxed for 1 hr. The contents were
cooled, poured into 6N NaOH solution (20 mL) with
vigorous stirring; the aqueous layer was extracted
with ethyl acetate and washed with water, and finally
distilled to give an oily residue. The crude product
was purified by column chromatography over silica
gel (60-120 mesh) with petroleum ether:ethyl acetate
(80:20) as eluent to give 5a as a pale yellow semisolid
(0.95 g, 42%). Anal. C17H19O3N requires: C, 71.50; H,
6.71; N, 4.91. Found: C, 70.21; H, 6.63; N, 4.70%.
IR: 3350, 2900, 1620, 1585, 1500, 1440, 1385, 1370,
1340, 1260, 1240, 1200, 1140, 1110, 1080, 1030, 980,
940, 860, 840, 785, 760, 740 cm-1; 1H NMR (200
MHz,CDCl3): 1.46 (6H, s, 2CH3), 2.49 (2H, s,
CH2), 3.75 (3H, s, OCH3), 6.43 (2H, m, 6-H and 8-H),
6.67 (1H, d, J= 8.5 Hz, 5-H), 6.99 (1H, dd, J=2.2 and
8.2 Hz, 3`-H), 7.25 (1H, m , 5`-H), 7.60 (1H, m, 4`H), 8.53 (1H, d, J=4.42, 6`-H); 13C NMR (50 MHz,
CDCl3): 25.7, 25.8, 51.0, 55.5, 71.0, 75.5, 102.0,

TRIPATHI et al.: NOVEL BENZOPYRAN DERIVATIVES

108.8, 119.5, 121.6, 122.5, 130.9, 137.5, 146.9, 155.5,

160.8, 165.7; MS: m/z (%) 285(2), 284(27), 268(22),
253(25), 232(35), 211(100), 196(27), 191(6), 189(11),
160(76), 143(13), 116(25), 89(65), 63(26).
Preparation of 2,2-dimethyl-7-methoxy-4-(2pyridyl)-2H-1-benzopyran 6a. A mixture of 5a (2.85
g, 10 mmol) and p-toluenesulfonic acid (0.2 g) in
benzene (50 mL) was heated at reflux temperature for
2 hr and after the completion of the reaction, the
contents concentrated and diluted with ice cold water.
The contents was extracted with ethyl acetate (350
mL), organic layer washed with brine solution (50
mL), dried over anhydrous sodium sulfate,
concentrated on a rotary evaporator under reduced
pressure to give a brick red gummy residue which on
chromatography over silica gel column using
petroleum ether-ethyl acetate (80:20) as eluent
yielded (2.15 g, yield 78%) 6a as a gummy mass.
Anal. C17H17NO2: requires: C, 77.38; H, 6.41; N, 5.24.
Found: C, 77.81; H, 6.39; N, 5.08%. IR: 3350, 2900,
1610,1600, 1500, 1460, 1439, 1380, 1360, 1290,
1280, 1200, 1140, 1140, 1120, 1040, 985, 895, 840,
800, 780, 740 cm-1;1H NMR (200 MHz, CDCl3):
1.49(6H, s, 2CH3), 3.77 (3H, s, -OCH3), 5.80 (1H, s,
3-H), 6.47 (1H, d, J=8.5 and 2.0 Hz, 6-H and 8-H),
7.14 (1H, dd, J=2.2 and 8.2 Hz, 3`-H), 7.18 (1H, m,
5`-H), 7.60 (1H, d, J=8.5 Hz, 5-H), 7.62 (1H, m, 4`H), 8.65 (1H, d, J=4.4 Hz, 6`-H); 13C NMR (50 MHz,
CDCl3): 27.7, 55.5, 76.5, 102.9, 107.0, 114.8, 123.1,
123.6, 126.6, 129.0, 134.1, 136.7, 146.9, 155, 161.1,
165.7; MS: m/z (%) 268(46), 254(100), 239(11),
225(11), 213(20),199(19), 186(9), 163(16), 89(18).
Preparation of 2-ethyl-2-methyl-7-methoxy-4-hydroxy-4-(2-pyridyl)-3,4-dihydro-2H-1-benzopyran 6b
Preparation of 2-ethyl-2-methyl-7-hydroxy-3,4dihydro-2H-1-benzopyran-4-one 3b. The title
compound was prepared from a mixture of
resacetophenone (7.6 g, 50 mmol), ethyl methyl
ketone (2.9 mL, 50 mmol) and pyrrolidine (3.0 mL,
50 mmol) dissolved in benzene (150 mL) by the
procedure as described for the compound 3a to afford
crude product which on purification by column
chromatography over silica gel using petroleum
ether:ethyl acetate (4:1) as eluent afforded 2-ethyl-2methyl-7-hydroxy-3,4-dihydro-2H-1-benzopyran-4one 3b a pale yellow solid (7.4 g, yield 72%) m.p.
98C. IR: 3140, 2950, 1635, 1580, 1500, 1380, 1360,
1320, 1260, 1170, 1110, 1000 840, 790 cm-1;
1
H NMR (200 MHz, CDCl3): 0.93 (3H, t, J= 6.5 Hz,
CH3), 1.40 (3H, s, CH3), 1.73 (2H, q, J= 6.0 Hz, CH2),

1625

2.66 (2H, s, CH2), 6.36 (1H, d, J=2.5 Hz, 8-H), 6.53

(1H, dd, J= 8.5 and 2.5Hz, 6-H), 7.83 (1H, d, J=8.5
Hz, 5-H); MS: m/z (%) 206(37), 191(13), 177(51),
152(68), 137(100), 108(34), 81(55), 69(39), 53(47).
Preparation of 2-ethyl-2-methyl-7-methoxy-3,4dihydro-2H-1-benzopyran-4-one 4b. The title
compound prepared from a mixture of 2-ethyl-2methyl-7-hydroxy-3,4-dihydro-2H-1-benzopyran-4one (2.06 g, 10 mmol), methyl iodide (1.41 g, 10
mmol), acetone (50 mL) and potassium carbonate (1.5
g) by the procedure as described for 4a to give crude
product which was purified by column chromatography over silica gel using petroleum ether:ethyl
acetate 95:5 as eluent to yield 4b as a brick red
gummy product (2.04 g, yield 93%). IR: 2950, 1650,
1620, 1580, 1500, 1445, 1380, 1360, 1310, 1270,
1210, 1160, 1120, 1070, 1030, 840 cm-1; 1H NMR
(200 MHz, CDCl3): 0.97 (3H, t, J= 6.0 Hz, CH3),
1.40 (3H, s, CH3), 1.61 (2H, q, J=6.0 Hz, -CH2 CH3),
2.66 (2H, s, -CH2), 3.85 (3H, s, -OCH3), 6.40 (1H, d,
J=2.5 Hz, 8-H), 6.60 (1H, dd, J=8.5 Hz and 2.5 Hz, 6H), 7.83 (1H, d, J=8.5 Hz, 5-H); MS: m/z (%) 220
(94),219 (100), 204(75), 192(7), 176(20), 147(23),
135(20), 123(29), 119(28), 108(60), 106(94), 92(42),
77(38), 63(97).
Preparation of 2-ethyl-2-methyl-7-methoxy-3, 4dihydro-4-hydroxy-4-(2-pyridyl)-2H-1-benzopyran
5b. The title compound was prepared from a mixture of
4b (1.8 g, 8 mmol) and pyridine (1.20 mL) by the
procedure as described for compound 5a and the crude
product was purified by column chromatography over
silica gel and petroleum ether:ethyl acetate (80:20) as
the eluent to give 5b as a semisolid (0.94 g, yield 40%).
Anal. C18H21NO3 requires: C, 72.20; H, 7.07; N, 4.68.
Found: C, 71.78; H, 6.91; N, 4.57%. IR: 3300, 2900,
2360, 1590, 1500, 1440, 1380, 1340, 1260, 1200, 1160,
1140, 1110, 1080, 1030, 980, 935, 880, 840, 795, 745
cm-1; 1H NMR (200 MHz, CDCl3): 0.93(3H, t, J= 6.5
Hz, CH2CH3), 1.30 (3H, s, CH3), 1.90 (2H, q, J= 6.5
Hz, CH3CH2), 2.23 (2H, s, C-CH2-C), 3.76 (3H, s,
OCH3), 6.33 (1H, d, J= 2.5 Hz, 8-H), 6.41 (1H, dd, J=
8.5 Hz and 2.5 Hz, 6-H), 6.67 (1H, d, J= 8.5 Hz, 5-H),
6.98-7.4 (2H, m, 3, 5-H), 7.48-6.93 (1H , m, 4H) 8.58
(1H, d, J= 4.5 Hz, 6`-H); 13C NMR (50 MHz, CDCl3):
22.5, 29.4, 48.8), 55.5, 70.89, 78.14, 102.0, 108.7,
119.7, 121.7, 122.4, 130.8, 137.4, 146.8, 155.6, 156.7,
166.0, 165; MS: m/z (%) 299(5), 281(13), 253(19),
252(49), 229(56), 221(97), 214(26), 214(8), 191(18),
175(11), 151(100), 124(6), 122(11), 106(43), 95(16),
79(45), 63(7).

1626

INDIAN J. CHEM., SEC B, NOVEMBER 2011

Preparation of 2-ethyl-2-methyl-7-methoxy-4-(2pyridyl)-2H-1-benzopyran 6b. p-Toluenesulfonic

acid (0.1 g) was added to benzene solution of 5b (2.99
g, 10 mmol, 150 mL) and the reaction mixture
refluxed on a water bath for 3 hr. After completion of
the reaction (monitored by TLC), the contents were
cooled and basified with 5% sodium carbonate
solution, washed with water, dried over anhydrous
sodium sulfate and concentrated on a rotary
evaporator under reduced pressure. The crude product
thus obtained was purified over silica gel column
using petroleum ether:ethyl acetate (80:20) as eluent
to afford 6b as a yellowish semisolid (2.25 g, yield
80%). Anal. C18H19O2N requires: C, 76.83; H, 6.81;
N, 4.98. Found: C, 76.33; H, 6.61; N, 4.90%.
IR(KBr): 2900, 1615, 1595, 1575, 1500, 1460, 1440,
1360, 1320, 1280, 1200, 1160, 1135, 1040, 1000, 880,
840, 800, 780, 740 cm-1; 1H NMR (200 MHz, CDCl3):
1.03 (3H, t, J= 6 Hz, -CH2CH3), 1.46 (3H, s, CH3),
1.83 (2H, q, J= 6 Hz, CH2CH3), 3.76 (3H, s, -OCH3),
5.80 (1H, s, 3-H), 6.38 (1H, d, J=2.3 Hz, 6-H), 6.56
(1H, dd, J= 8.5 Hz and 2.3 Hz, 8-H), 7.23 (1H, d, J=
8.5 Hz, 5-H), 7.24 (1H, m, 5`-H), 7.38 (1H, m, 3`-H),
7.75 (1H, m, 4-H), 8.70 (1H, d, J= 4.5 Hz, 6`-H);
13
C NMR (50 MHz, CDCl3): 24.80, 33.08, 54.84,
78.4, 102.1, 106.0, 114.1, 122.1, 122.9, 125.9, 127.4,
134.0, 136.1, 149.0, 154.7, 156.7, 160.5; MS: m/z (%)
281(17), 266(25), 252(100), 237(8), 222(13), 203(9),
194(14), 180(16), 166(8), 155(9), 126(8), 89(6),
78(12).
Preparation of 2,2-spiro-cyclopentyl-7-methoxy-4hydroxy-4-(2-pyridyl)-3,4-dihydro-2H-1-benzopyran 6c
Preparation of 2,2-spiro-cyclopentyl-7-hydroxy3,4-dihydro-2H-1-benzopyran-4-one 3c. A mixture
of resacetophenone (7.6 g, 50 mmol), cyclopentanone
(4.0 mL, 50 mmol) and pyrrolidine (3.0 mL, 50
mmol) dissolved in benzene (100 mL) was refluxed
on water bath for 12 hr. The reaction mixture was
cooled and diluted with water, acidified with 2N HCl
and the contents extracted with ethyl acetate
(330 mL). The organic layer were washed with
brine, dried over anhydrous sodium sulfate and
concentrated under reduced pressure to give crude
product which on chromatography over silica gel and
elution with petroleum ether:ethyl acetate (80:20)
gave 3c a gummy mass (8.75 g, yield 76%) m.p.
175C (Lit.21 178C). IR: 3160, 1640, 1600, 1488,
1330, 1310, 1160, 1120, 995, 850, 700 cm-1; 1H NMR
(200 MHz, CDCl3): 1.51-2.26 (8H, m, 4CH2), 2.83

(2H, s, CH2), 6.40 (1H, d, J= 2.5 Hz, 8-H), 6.56 (1H,

dd, J= 8.5 and 2.5 Hz, 6-H), 7.90 (1H, d, J= 8.5 Hz, 5H); MS: m/z: 218 (59), 189 (100), 152 (56), 137 (78),
108 (34), 95 (14), 81 (67), 69 (47), 63 (24).
Preparation of 2,2-spiro-cyclopentyl-7-methoxy3,4-dihydro-2H-1-benzopyran-4-one 4c. A solution
of 3c (2.10 g, 5 mmol) in acetone (50 mL) containing
anhydrous potassium carbonate (2 g) and methyl
iodide (3.2 mL, 10 mmol) was refluxed for 3 hr and
then cooled and filtered. The filtrate was concentrated
and the crude product purified by chromatography
over silica gel using ethyl acetate:petroleum ether
(1:19) as eluent to give 4c as a yellowish semi solid
(2.27 g, yield 98%). IR: 2950, 1680, 1620, 1580,
1500, 1440, 1275, 1220, 1160, 1140, 1120, 1025, 985,
840, 820 cm-1; 1H NMR (200 MHz, CDCl3): 1.402.16 (8H, m, 4CH2), 2.75 (2H, s, CH2), 3.80 (3H, s,
OCH3), 6.40-6.6 (2H, m, 6 and 8-H), 6.82 (1H, d, J=
8.5 Hz, 5-H), 7.13-7.47 (2H,m, 3and 5-H) 7.53-7.9
(1H,m, 4H), 8.70 (1H,d, J= 4.5 Hz, 6H; MS: m/z
(%) 232 (10), 202(83), 150(100), 122(39), 107(24),
79(37), 69(5), 63(19).
Preparation of 2,2-spirocyclopentyl-7-methoxy4-hydroxy-4-(2-pyridyl)-3,4-dihydro-2H-1-benzopyran 5c. The title compound was prepared from a
mixture of 4c (1.90 g, 8 mmol) and pyridine (1.20
mL) by the procedure as described for compound 5a
and the crude product was purified by column
chromatography over silica gel and elution with
petroleum ether:ethyl acetate (80:20) to yield 5c as a
yellowish semisolid (0.845 g, yield 45%). Anal.
C19H21NO3 requires: C, 73.29; H, 6.80; N, 4.5. Found:
C, 73.42; H, 6.67; N, 4.77%. IR: 2900, 2360, 1615,
1500, 1430, 1375, 1340, 1280, 1260, 1200, 1160,
1140, 1035, 980, 860, 840, 800 cm-1; 1H NMR (200
MHz, CDCl3): 1.40-2.16 (8H, m, 4CH2), 2.75 (2H,
s, CH2), 3.80 (3H, s, OCH3), 6.40-6.6 (2H, m, 6 and
8-H), 6.82 (1H, d, J= 8.5 Hz, 5-H), 7.13-7.47 (2H,m,
3and 5-H), 7.53-7.9 (1H,m, 4H), 8.70 (1H,d, J=
4.5 Hz,6H). 1.24-1.96 (8H, m, CH2), 2.26 (2H, s,
CH2), 3.83 (3H, s, OCH3), 6.35 (1H, d, J=2.5 Hz, 8H), 6.56 (1H, dd, J=9.0 and J=2.0 Hz, 6-H), 6.81 (1H,
d, J=8.5 Hz, 5-H), 7.20 (1H,m 5`-H), 7.40 (1H, m, 3`H), 7.73 (1H,m, 4`-H), 8.70 (1H, d, J=4.0 Hz, 6`-H);
MS: m/z (%) 311 (4), 293 (29), 264 (40), 233 (56),
215 (31), 203 (25), 151 (100), 132 (12), 106 (39), 78
(84), 69 ((14), 63 (14).
Preparation of 2,2-spirocyclopentyl-7-methoxy4-(2-pyridyl)-2H-1-benzopyran 6c. A mixture of 5c
(3.1 g, 10 mmol) and p-toluenesulphonic acid (0.1 g)
taken in benzene (50 mL) was refluxed for 3 hr on a

TRIPATHI et al.: NOVEL BENZOPYRAN DERIVATIVES

water bath and the contents cooled, basified with 5%

sodium carbonate solution, washed with water, the
organic layer dried over anhydrous sodium sulfate and
concentrated on a rotary evaporator under reduced
pressure. The residue obtained was purified by silica
gel column chromatography with petroleum
ether : ethyl acetate, (80:20) as eluent to give 6c as a
gummy mass (2.54 g, yield 87%). Anal. C19H19NO2
requires: C, 77.79; H, 6.53; N, 4.77. Found: C, 76.99;
H, 6.50; N, 4.93%. IR: 2900, 2360, 2340, 1605, 1500,
1440, 1370, 1360, 1300, 1260, 120, 1200, 1160, 1145,
1020, 985, 860, 840, 820 cm-1; 1H NMR (200 MHz,
CDCl3): 1.30-2.02 (8H, bm, CH2), 3.89 (3H, s,
OCH3), 5.90 (1H, s, 3-H), 6.43 (1H, d, J=2.0 Hz, 8H), 6.60 (1H, dd, J=8.5 and 2.5 Hz, 6-H), 6.90 (1H, d,
J=8.5 Hz, 5-H), 7.25 (1H, m, 5`-H), 7.43 (1H,m, 3`H), 7.78 (1H, m, 4`-H), 8.78 (1H, d, J=4.4 Hz, 6`-H);
MS: m/z (%) 293 (22), 258 (76), 225 (52), 195 (39),
143 (63), 124 (48), 106 (22), 71 (18).
Preparation of 2,2-spirocyclohexyl-7-methoxy-4hydroxy-4-(2-pyridyl) 3,4-dihydro-2H-1-benzopyran 6d
Preparation of 2,2-spirocyclohexyl-7-hydroxy3,4-dihydro-2H-1-benzopyran-4-one 3d. Pyrrolidine
(3.0 mL, 50 mmol) was added to a solution of 2,4dihydroxy acetophenone (7.6 g, 50 mmol) and
cyclohexanone (5.0 mL, 50 mmol) in dry benzene (40
mL) and the contents refluxed. The mixture was
heated under reflux conditions for 12 hr, the reaction
monitored by TLC. After the completion of the
reaction (12 hr) the contents were poured in 6N HCl.
The product was extracted with ethyl acetate
(325 mL), washed with water, dried over anhyd.
Na2SO4 and concentrated under reduced pressure to
give the crude product which on column
chromatography over silica gel and elution with
petroleum ether:ethyl acetate (80:20) afforded 3d as a
semisolid
2,2-spirocyclohexyl-7-hydroxy-3,4dihydro-2H-1-benzopyran-4-one (9.50 g,yield 82%).
m.p. 166C (Lit.21 m.p. 168C); IR: 3140, 1620, 1590,
1500, 1390, 1245, 1195, 1140, 1120, 995, 980, 795
cm-1; 1H NMR (200 MHz, CDCl3): 1.31-1.98 (10H,
m, 5CH2), 2.70 (2H, s, CH2), 6.46 (1H, d, J= 2.5 Hz,
8-H), 6.54 (1H, dd, J= 8.5 Hz and 2.5 Hz, 6-H), 7.70
(1H, d, J=8.5 Hz, 5-H); MS: m/z (%) 232 (8), 191 (7),
189 (15), 164 (11), 152 (38), 137 (35), 135 (11), 123
(10), 81 (100), 69 (26), 63 (14).
Preparation of 2,2-spiro cyclohexyl-7-methoxy3,4-dihydro-2H-1-benzopyran-4-one 4d. To a

1627

solution of 3d (2.32 g, 0.01 mole) in acetone (50 mL)

was added, methyl iodide (3.2 mL, 0.01 mole) and
potassium carbonate (0.2 g) and the mixture refluxed
for 6 hr on a water bath. After cooling the contents,
the reaction mixture was concentrated on a rotary
evaporator and the residue thus obtained was
chromatographed over silica gel column, using
petroleum ether:ethyl acetate, 95:5 as eluent to afford
4d as a yellowish gummy mass (2.09 g, yield 84%).
IR: 2900, 1685, 1620, 1580, 1500, 1440, 1340, 1320,
1280, 1260, 1205, 1170, 1120, 1030, 985, 840, 820,
760 cm-1; 1H NMR (200 MHz, CDCl3): 1.36-1.85
(10H, m, 5CH2), 2.63 (2H, s, CH2), 3.85 (3H, s,
OCH3), 6.40 (1H, d, J=2.5 Hz, 8-H), 6.56 (1H, dd, J=
8.5 Hz and 2.5 Hz, 6-H), 7.83 (1H, d, J=8.5 Hz, 5-H);
MS: m/z (%) 246 (6), 244 (28), 215 (7), 201 (36), 189
(7), 150 (100), 136 (9), 121 (31), 106 (40), 94 (24), 79
(15), 78 (57), 68 (10), 63 (33).
Preparation of 2,2-spirocyclohexyl-7-methoxy-4hydroxy-4-(2-pyridyl)-3,4-dihydro-2H-1-benzopyran 5d. The title compound was prepared from a
mixture of 4d (2.04 g, 8 mmol) and pyridine (1.20
mL) by the procedure as described for compound 5a
and the crude mixture purified by column
chromatography over silica gel and petroleum
ether : ethyl acetate (80:20) as eluent to give 5d as a
semisolid (1.14 g, yield 58%). Anal. C20H23O3
requires: C, 73.82; H, 7.12; N, 4.30. Found: C, 74.33;
H, 6.87; N, 4.18%. IR: 3365, 2850, 1620, 1600, 1500,
1440, 1340, 1280, 1260, 1200, 1160, 1140, 1085,
1040, 985, 840, 785, 745 cm-1; 1H NMR (200 MHz,
CDCl3): 1.45-1.96 (10H, m, 5CH2), 2.26 (2H, s, CCH2), 3.83 (3H, s, OCH3), 6.53-6.66 (2H, m, 6 and 8H), 6.83 (1H, d, J=8.5 Hz, 5-H), 7.0-7.43 (2H, m,
3,5-H), 7.73 (1H, m, 4`-H), 8.63 (1H, d, J= 4.5 Hz,
6`-H); 13C NMR (50 MHz, CDCl3): 22.4, 22.5, 26.0,
32.9, 38.9, 50.2, 55.6, 70.9, 76.6, 102.1, 108.8, 120.2,
122.2, 126.5, 132.5, 138.7, 147.8, 155.2, 160.8, 165.9;
MS: m/z (%) 325 (18), 307 (12), 264 (23), 247 (40),
230 (52), 200 (13), 166 (8), 151 (100), 149 (21), 134
(36), 106 (54), 78 (36), 69 (8), 63 (6).
Preparation of 2,2-spiro cyclohexyl-7-methoxy4-(2-pyridyl)-2H-1-benzopyran
6d.
Benzene
solution of 5d (1.65 g, 5 mmol) and p-toluenesulfonic
acid (0.1 g) were refluxed for 2.5 hr and the contents
cooled, basified with 5% sodium carbonate solution,
washed with water, the organic layer dried over
anhydrous sodium sulfate and concentrated on a
rotary evaporator under reduced pressure to give the
crude product, which on purification by column

1628

INDIAN J. CHEM., SEC B, NOVEMBER 2011

chromatography over silica gel with ethyl

acetate:pet.ether (80:20) as the eluent afforded 6d as a
gummy mass (1.35 g, yield 88%). Anal. C20H21O2N
requires: C, 78.15; H, 6.89; N, 4.56. Found: C, 77.93;
H, 6.76; N, 4.49%. IR: 2850, 1620, 1600, 1500, 1460,
1440, 1360, 1320, 1280, 1260, 1200, 1160, 1120,
1040, 1000, 985, 920, 840, 810, 795 cm-1; 1H NMR
(200 MHz, CDCl3): 1.33-2.00 (10H, m, 5CH2),
3.87 (3H, s, OCH3) 5.87 (1H, s, 3-H), 6.40 (1H, d, J=
2.5 Hz, 8-H), 6.63 (1H, dd, J= 8.5 and 2.5 Hz, 6-H),
7.20 (1H, d, J= 8.5 Hz, 5-H), 7.32 (1H, m, 5`-H), 7.45
(1H, m, 3`-H), 7.68 (H, m, 4`-H), 8.57 (1H, d, J=4.4
Hz, 6`-H); MS: m/z (%) 306 (46), 278 (18), 263 (100),
250 (35), 228 (10), 214 (19), 192 (15), 180 (16), 166
(15), 153 (13), 79 (14), 77 (18), 52 (19).
Biology
In vitro cytotoxicity against human cancer cell
lines. The human cancer cell lines procured from
National Cancer Institute, Frederick, U.S.A, were
used in the present study. Cells were grown in tissue
culture flasks in complete growth medium (RPMI1640 medium with 2 mM glutamine, 100 g/mL
streptomycin, pH 7.4, sterilized by filtration and
supplemented with 10% fetal calf serum and 100
units/mL penicillin before use) at 37C in an
atmosphere of 5% CO2 and 90% relative humidity in
a carbon dioxide incubator. The cells at subconfluent
stage were harvested from the flask by treatment with
trypsin (0.05% in PBS containing 0.02% EDTA) for
determination of cytotoxicity. Cells with viability of
more than 98%, as determined by trypan blue
exclusion, were used for assay. The cell suspension of
1 105 cells/mL was prepared in complete growth
medium for determination of cytotoxicity.
Stock solutions of 2 10-2 M of all the samples
were prepared in DMSO. The stock solutions were
serially diluted with complete growth medium
containing 50 g/mL of gentamycin to obtain
working test solutions of required concentrations.
In vitro cytotoxicity against eight human cancer
cell lines was determined13 by three different
experiments, using 96-well tissue culture plates. The
100 L of cell suspension was added to each well of
the 96-well tissue culture plate. The cells were
incubated for 24 hr. Test materials in complete growth
medium (100 L) were added after 24 hr incubation
to the wells containing cell suspension. The plates
were further incubated for 48 hr (at 37C in an
atmosphere of 5% CO2 and 90% relative humidity in

a carbon dioxide incubator) after addition of test

material and then the cell growth was stopped by
gently layering trichloroacetic acid (TCA, 50% w/v,
50 L) on top of the medium in all the wells. The
plates were incubated at 4C for 1 hr to fix the cells
attached to the bottom of the wells. The liquid of all
the wells was gently pipetted out and discarded. The
plates were washed five times with distilled water to
remove TCA, growth medium, low molecular weight
metabolites, serum proteins, etc. and air-dried. Cell
growth
was
measured
by
staining
with
Sulforhodamine B dye21,22. The adsorbed dye was
dissolved in Tris-Buffer (100 L, 0.01 M, pH 10.4)
and the plates were gently shaken for 10 min on a
mechanical shaker. The optical density (OD) was
recorded on an ELISA reader at 540 nm. Percent
growth inhibition was calculated using the following
formula
Cell viability % = 100 [OD
OD (control) - OD (blank)] 100%

(test sample)

- OD

(blank)

Light microscopy. The cells were spun onto glass

slides using a centrifuge, and fixed for 1 min in
methanol and stained for 10 min with modified
Giemsa stain. The morphology was studied under
Olympus Research Microscope (VANOX). The
photography was done using an Olympus Digital
Camera (C4000).
Scanning electron microscopic studies (SEM).
Compound 6a and 6c, 110-4 M treated MOLT-4 cells
after 12 hr were subjected to SEM studies. The cells
on a cover slip were fixed in 2.5% glutaraldehyde in
0.1 M cacodylate buffer (pH 7.2) for 2 hr at 4C, postfixed with OsO4 for 2 hr in the same buffer,
dehydrated with graded acetone and dried in a critical
point drier using CO2 (Balzers Union). The samples
were then mounted on stubs, coated first with carbon
in a Jeol-JEE4X vacuum evaporator, and then coated
with gold in a Polaron sputter coater. Finally, the
samples were analyzed by using a Jeol 100 CX II
electron microscope with ASID at 40 KV. The
photography was done using a 120 roll film.
Conclusion
Synthesis of 4-(2-pyridyl)-2H-1-benzopyrans and
4-(2-pyridyl) 4-hydroxy-3,4-dihydro benzopyran
compounds hitherto not known in the literature has
been achieved in moderate to high yields (58-88%)
through the key step of introduction of pyridyl moiety
in the benzopyran system. In vitro studies (100 g/mL

TRIPATHI et al.: NOVEL BENZOPYRAN DERIVATIVES

conc.) against eight human cancer cells have resulted

in the identification of four potent cytotoxic
molecules active against many of the cancer cells
tested. The mechanistic aspects of the active
compounds have been carried out using SEM and
apoptosis has been established as the mode of cancer
cell death. Further inputs in term of modification at C4 position by bioisoteres other than pyridyl moiety
leave scope for development of more potent cytotoxic
molecules.
Acknowledgements
Authors are thankful to CSIR, New Delhi for the
project grant.
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