Bakuchiol derivatives as novel and potent cytotoxic agents: A report

Article abstract of DOI:10.1016/j.ejmech.2011.12.018

A library of 28 compounds comprising of acyl, amino, halo, nitro, styryl and cyclized derivatives of bakuchiol have been evaluated against a panel of eight human cancer cell lines. Bioevaluation studies have resulted in the identification of potent cytoto

Full text of DOI:10.1016/j.ejmech.2011.12.018

European Journal of Medicinal Chemistry 49 (2012) 55e67
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Bakuchiol derivatives as novel and potent cytotoxic agents: A report
,
,
b,
**
Rabiya Majeed a b, Mallepally V. Reddya, Praveen K. Chinthakindi a, Payare L. Sangwan a , Abid Hamid
,
*
Gousia Chashoo b, Ajit K. Saxena b, Surrinder Koul a
,
*
a Bioorganic Chemistry Section, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
b Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu-180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of 28 compounds comprising of acyl, amino, halo, nitro, styryl and cyclized derivatives of
bakuchiol have been evaluated against a panel of eight human cancer cell lines. Bioevaluation studies
have resulted in the identification of potent cytotoxic molecules exhibiting concentration dependent
growth inhibition against leukemia cancer cells with best results observed for compounds 17 and 22
exhibiting IC50 1.8 and 2.0 mM respectively. As evident from various biological end-points, inhibition of
cell proliferation by inducing G2/M cell cycle arrest, mitochondrial membrane disruption followed by
DNA fragmentation and apoptosis is demonstrated.
Received 23 August 2011
Received in revised form
7 December 2011
Accepted 11 December 2011
Available online 20 December 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Bakuchiol analogs
Mercurationedemercuration
Heck coupling
Apoptosis
Cell cycle analysis
1. Introduction
[4]. Chemically, bakuchiol is made up of styryl moiety in conjunc-
tion with a monoterpene (together known as meroterpene).
Natural products/molecules derived from plants have been
a reliable source of therapeutic agents for the use in humans and
the quest to swell these numbers goes unabated. Apart from the use
of plant derived molecules directly as drugs such as vincristine,
vinblastine, reserpine, etoposide, artemisinin; many plant derived
scaffolds, through tailoring by the chemists, have resulted in the
development of some of the most effective drugs e.g. khellin to
sodium chromoglycate an anti-asthmatic drug, papaverine to
verapamil for hypertension, galegine to metformin for diabetes.
Therefore, the exploration of plants for their possible medicinal use
needs to be continued. Psoralea corylifolia from the leguminosae
family [1] is one of the medicinally important plants (in Ayurveda
and Chinese System of Medicine) known for its inhibitory effect
against dental caries [2], DNA polymerase and Topoisomerase II [3].
Recently, we carried out a study on bakuchiol (1) the major
chemical constituent of P. corylifolia, wherein its antibacterial
activity was enhanced through chemical modification to get MIC
reduction up to eightfold against Gram þve and Gram ꢀve bacteria
Compounds having the presence of styryl unit in conjunction with
other moieties such as pyrone, chromone, quinazoline, in general,
exhibit various biological activities such as anti HIV, anti-cancer etc.
[5e8]. The fact that bakuchiol is reported to exhibit cytotoxicity
against few human cancer cell lines [9], in particular against breast
cancer [10], prompted us to carry out investigation on bakuchiol in
a bid to have molecules with more potency, and possible clinical
utility and application. In the present paper, the preparation of
bakuchiol derivatives and their cytotoxicity study is described.
2. Results and discussion
Bakuchiol (1) is reported as cytotoxic toward breast cancer with
IC50 8.29 ꢁ 10ꢀ3 mol/L (MDA-MB-231) and 2.89 ꢁ 10ꢀ5 mol/L (T-
47D) [11]. However, a detailed investigation of this molecule and its
derivatives is still awaited. The bakuchiol derivatives (Fig. 1)
prepared by modulation of phenolic group (2e10) or substitution in
the aryl part (11e13, 28), in the isopropylidene group (15e16), in
the monoterpene part (13e27) and in the ethenyl group (18e27)
[4], were tested at 50 mM concentration for their ability to induce
* Corresponding authors. Tel.: þ91 191 2569021, 191 2569001e11x232/210;
fax: þ91 191 2569333.
cytotoxicity in human cancer cell lines encompassing lung (A-549),
breast (MCF-7), prostate (PC-3), cervical (HeLa), leukemia (THP-1),
CNS/neuroblastoma (IMR-32), and ovarian (OVCAR-5) cell lines
(Table 1), taking mitomycin/adriamycin/5-FU as the gold standard.
** Corresponding author. Tel.: þ91 191 2569001e11x352; fax: þ91 191 2569333.
E-mail addresses: plsangwan@iiim.ac.in (P.L. Sangwan), ahdar@iiim.ac.in
(A. Hamid), skoul@iiim.ac.in (S. Koul).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.018

56
R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
R
R
HO
HO
RO
HO
CHO
OH
1-10
15
11 R= NO2
12 R= NH2
13 R= I
1
2
3
4
5
6
7
8
9
R= H
14 R= H
R= -COCH3
R= -COCH2CH3
R= -CO(CH2)2CH3
R= -CO(CH2)3CH3
R= -CO(CH2)4CH3
R= -CO(CH2)6CH3
R= -CO(CH2)8CH3
R= -CO(CH2)14CH3
HO
HO
O
17
16
10 R= -CH3
R
R
H3CO
R1
HO
HO
HO
19 R= OCH3
21 R= OH
23 R= F
20 R= OCH3
R1= CHO
18
OR
25 R= OCH2CH2CH3 R1= Cl
26 R= OCH3
R1= CH2OH
24 R= NH2
Br
H3CO
HO
Br
22 R= H
28
27 R= CH3
Fig. 1. Structures of bakuchiol 1 and its analogs 2e28.
Table 1
Cytotoxic activitya (%age growth inhibition) of bakuchiol and its derivatives at 50
mM concentration against various human cancer cell lines.
Compounds
Breast MCF-7
Lung A-549
Prostate PC-3
Ovary OVCAR-5
CNS IMR-32
Cervical HeLa
Leukemia THP-1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
63 ꢂ 1
75 ꢂ 2
23 ꢂ 3
20 ꢂ 2
25 ꢂ 3
18 ꢂ 3
12 ꢂ 2
10 ꢂ 1
24 ꢂ 2
18 ꢂ 3
49 ꢂ 2
17 ꢂ 4
40 ꢂ 3
46 ꢂ 2
12 ꢂ 1
33 ꢂ 3
81 ꢂ 2
42 ꢂ 3
55 ꢂ 2
36 ꢂ 3
31 ꢂ 2
79 ꢂ 2
28 ꢂ 2
50 ꢂ 2
47 ꢂ 2
42 ꢂ 2
13 ꢂ 3
42 ꢂ 1
e
63 ꢂ 2
75 ꢂ 3
42 ꢂ 1
50 ꢂ 3
38 ꢂ 2
58 ꢂ 2
15 ꢂ 3
48 ꢂ 2
61 ꢂ 3
12 ꢂ 3
52 ꢂ 1
41 ꢂ 2
12 ꢂ 2
49 ꢂ 2
35 ꢂ 3
44 ꢂ 2
76 ꢂ 2
10 ꢂ 3
40 ꢂ 2
32 ꢂ 1
33 ꢂ 1
70 ꢂ 2
31 ꢂ 1
57 ꢂ 3
5 ꢂ 3
58 ꢂ 1
67 ꢂ 2
54 ꢂ 1
38 ꢂ 3
42 ꢂ 3
28 ꢂ 2
22 ꢂ 2
50 ꢂ 3
62 ꢂ 3
44 ꢂ 2
52 ꢂ 3
28 ꢂ 1
08 ꢂ 3
40 ꢂ 2
51 ꢂ 1
46 ꢂ 3
86 ꢂ 2
46 ꢂ 1
50 ꢂ 2
34 ꢂ 3
32 ꢂ 1
93 ꢂ 1
33 ꢂ 3
59 ꢂ 2
7 ꢂ 3
66 ꢂ 1
72 ꢂ 1
52 ꢂ 1
41 ꢂ 2
3 ꢂ 1
58 ꢂ 3
74 ꢂ 3
63 ꢂ 3
54 ꢂ 3
21 ꢂ 1
19 ꢂ 1
51 ꢂ 3
52 ꢂ 2
54 ꢂ 1
30 ꢂ 2
50 ꢂ 3
39 ꢂ 3
21 ꢂ 2
53 ꢂ 2
53 ꢂ 2
59 ꢂ 3
88 ꢂ 3
68 ꢂ 1
6 ꢂ 2
55 ꢂ 1
63 ꢂ 1
48 ꢂ 3
36 ꢂ 2
23 ꢂ 2
50 ꢂ 2
53 ꢂ 3
50 ꢂ 3
51 ꢂ 2
43 ꢂ 2
49 ꢂ 1
48 ꢂ 3
43 ꢂ 3
42 ꢂ 2
40 ꢂ 3
32 ꢂ 2
68 ꢂ 1
50 ꢂ 2
20 ꢂ 3
31 ꢂ 3
32 ꢂ 3
78 ꢂ 2
43 ꢂ 1
28 ꢂ 2
4 ꢂ 3
74 ꢂ 2
82 ꢂ 1
60 ꢂ 2
54 ꢂ 2
15 ꢂ 1
54 ꢂ 2
62 ꢂ 1
52 ꢂ 1
62 ꢂ 2
45 ꢂ 3
55 ꢂ 3
48 ꢂ 3
58 ꢂ 2
58 ꢂ 2
54 ꢂ 3
52 ꢂ 3
93 ꢂ 2
62 ꢂ 2
52 ꢂ 3
45 ꢂ 3
66 ꢂ 2
88 ꢂ 2
60 ꢂ 1
54 ꢂ 3
20 ꢂ 3
58 ꢂ 2
62 ꢂ 3
51 ꢂ 3
88 ꢂ 3
e
13 ꢂ 3
42 ꢂ 3
42 ꢂ 1
64 ꢂ 2
37 ꢂ 3
55 ꢂ 2
33 ꢂ 2
11 ꢂ 3
43 ꢂ 2
22 ꢂ 3
12 ꢂ 2
75 ꢂ 1
38 ꢂ 3
20 ꢂ 2
33 ꢂ 3
33 ꢂ 2
68 ꢂ 1
29 ꢂ 2
16 ꢂ 2
12 ꢂ 1
38 ꢂ 2
25 ꢂ 2
08 ꢂ 1
75 ꢂ 3
e
35 ꢂ 3
30 ꢂ 2
69 ꢂ 2
37 ꢂ 3
28 ꢂ 2
8 ꢂ 3
25
26
27
41 ꢂ 1
15 ꢂ 1
40 ꢂ 1
78 ꢂ 2
e
39 ꢂ 2
29 ꢂ 1
50 ꢂ 2
76 ꢂ 1
70 ꢂ 1
e
34 ꢂ 2
27 ꢂ 2
21 ꢂ 2
79 ꢂ 2
e
36 ꢂ 3
11 ꢂ 1
42 ꢂ 3
71 ꢂ 3
e
28
5-Fub
Mitomycinb
Adriamycinb
e
88 ꢂ 3
76 ꢂ 1
e
e
e
e
The bold values are shown for those compounds which have proved to be active and those in normal font represent least significant.
a
Results are mean ꢂ SD of three separate experiments, conducted in triplicate at the concentration of 50
mM.
b
Concentration of 5-FU ¼ 20
m
M, Mitomycin ¼ 1
m
M, Adriamycin ¼ 1
mM.

R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
57
Among the acyl derivatives (2e9), only analog 2 showed better
growth inhibition (63e82%, bakuchiol 55e74%). Compound 17,
exhibition of different levels of activity and toxicity associated with
enantiomers is well known phenomena [14].
a
1,2,3-trisubstituted cyclopentane derivative obtained as an
Further, the molecules that exhibited >50% inhibition (Table 1)
abnormal oxymercurationedemercuration reaction product of
bakuchiol (characterized by spectral analysis), displayed excellent
activity profile against leukemia and significant cytotoxicity against
all other cell lines with 68e93% growth inhibition. Among bisstyryl
derivatives (18e27), only compounds 18, 19, 22, and 25 showed
significant but different levels of cytotoxicity which were related to
the nature and position of the substituent in the two aryl moieties.
It is worth to mention here that a limited information is available
related to the cytotoxicity of bisstyryl derivatives [11e13]. Inter-
esting results were observed for two enantiomeric compounds 19
and 22 with S-isomer displaying far better activity than the R-
isomer. While compound 19 (R-isomer) showed 50e55% growth
inhibition only, its antipode (22) displayed 68e93% inhibitory effect
against all the cancer cell lines across the board (Table 1), the
were screened at lower concentration (30 mM) which resulted in
the identification of 17, 22, and 2 as the most potent molecules
(Table 2) and these were further tested for their inhibitory potential
at lower concentrations (20, 10 and 5
showed 57% growth inhibition at 20 M against colon cancer cell
line, 17 and 22 showed inhibition at 20 M against all the cancer
cell lines. Compound 17 displayed inhibition against four cancer cell
lines and 22 against three cell lines at 10 M. Both the compounds
at 5 M concentration, showed inhibition only against THP-1 cell
line (Table 3).
mM). While compound 2
m
m
m
m
The IC50 value for 2, 17 and 22 was calculated by non-linear
regression analysis using Graph Pad Software (2236 Avenida de la
Playa La Jolla, CA 92037, USA). The derivatives displayed low IC50
values (<25e2.0 mM) against different cancer cell lines (Table 4).
Table 2
Cytotoxic activitya (%age growth inhibition) of bakuchiol and its selected derivatives at two different concentration against various human cancer cell lines.
Compounds
Conc. (
mM)
Breast MCF-7
Liver Hep-2
Lung A-549
Prostate PC-3
Ovary OVCAR-5
CNS IMR-32
Cervical HeLa
Leukemia THP-1
1
50
30
50
30
50
30
50
30
50
30
50
30
50
30
20
1
64 ꢂ 1
33 ꢂ 1
77 ꢂ 2
56 ꢂ 3
26 ꢂ 2
12 ꢂ 1
51 ꢂ 2
37 ꢂ 3
79 ꢂ 2
58 ꢂ 2
77 ꢂ 2
52 ꢂ 2
48 ꢂ 2
28 ꢂ 3
e
53 ꢂ 1
20 ꢂ 1
52 ꢂ 3
51 ꢂ 2
30 ꢂ 1
16 ꢂ 1
40 ꢂ 1
22 ꢂ 1
65 ꢂ 1
53 ꢂ 1
62 ꢂ 1
45 ꢂ 1
46 ꢂ 1
32 ꢂ 1
e
65 ꢂ 2
30 ꢂ 2
72 ꢂ 3
60 ꢂ 2
60 ꢂ 1
33 ꢂ 1
53 ꢂ 1
34 ꢂ 2
78 ꢂ 2
61 ꢂ 2
72 ꢂ 2
55 ꢂ 3
60 ꢂ 3
33 ꢂ 2
76 ꢂ 2
e
59 ꢂ 1
21 ꢂ 3
69 ꢂ 2
60 ꢂ 3
60 ꢂ 2
27 ꢂ 1
55 ꢂ 3
31 ꢂ 1
88 ꢂ 2
62 ꢂ 2
94 ꢂ 1
50 ꢂ 2
61 ꢂ 2
15 ꢂ 1
76 ꢂ 1
e
67 ꢂ 1
25 ꢂ 3
71 ꢂ 2
62 ꢂ 2
62 ꢂ 2
29 ꢂ 2
57 ꢂ 2
29 ꢂ 2
76 ꢂ 1
55 ꢂ 1
67 ꢂ 1
50 ꢂ 1
18 ꢂ 2
8 ꢂ 2
61 ꢂ 3
31 ꢂ 4
71 ꢂ 3
59 ꢂ 3
53 ꢂ 1
22 ꢂ 2
53 ꢂ 2
23 ꢂ 2
85 ꢂ 3
57 ꢂ 2
71 ꢂ 2
51 ꢂ 2
30 ꢂ 2
14 ꢂ 2
81 ꢂ 2
e
56 ꢂ 1
20 ꢂ 1
65 ꢂ 1
45 ꢂ 1
53 ꢂ 2
24 ꢂ 2
50 ꢂ 1
28 ꢂ 1
69 ꢂ 1
52 ꢂ 1
80 ꢂ 2
56 ꢂ 2
27 ꢂ 2
5 ꢂ 2
76 ꢂ 2
44 ꢂ 1
81 ꢂ 1
63 ꢂ 1
64 ꢂ 2
39 ꢂ 2
58 ꢂ 3
29 ꢂ 2
95 ꢂ 2
68 ꢂ 1
86 ꢂ 2
60 ꢂ 1
51 ꢂ 2
32 ꢂ 1
91 ꢂ 3
e
2
8
10
17
22
24
5-FUb
78 ꢂ 3
e
74 ꢂ 3
e
Mitomycinb
Adriamycinb
e
81 ꢂ 3
e
1
85 ꢂ 3
e
e
e
e
e
e
The bold values are shown for those compounds which have proved to be active and those in normal font represent least significant.
a
Results are mean ꢂ SD of three separate experiments, conducted in triplicate at the concentration of 50
mM.
b
Concentration of 5-FU ¼ 20
m
M, Mitomycin ¼ 1
m
M, Adriamycin ¼ 1
mM.
Table 3
Cytotoxic activitya (%age growth inhibition) of bakuchiol derivatives at 20
m
M, 10 mM and 5 mM concentration against human cancer cell lines.
Compounds
Conc. (
m
M)
Breast MCF-7
Liver HEP-2
Lung A-549
Prostate DU-145
Leukemia THP-1
Prostrate PC-3
CNS IMR-32
Colon HCT-15
2
20
10
5
20
10
5
20
10
5
20
1
44 ꢂ 2
31 ꢂ 2
15 ꢂ 2
50 ꢂ 2
32 ꢂ 1
27 ꢂ 2
50 ꢂ 3
37 ꢂ 1
24 ꢂ 1
e
46 ꢂ 2
30 ꢂ 1
14 ꢂ 3
50 ꢂ 1
34 ꢂ 3
17 ꢂ 2
58 ꢂ 4
46 ꢂ 1
23 ꢂ 1
e
1 ꢂ 2
22 ꢂ 1
13 ꢂ 2
53 ꢂ 3
40 ꢂ 2
32 ꢂ 3
69 ꢂ 1
47 ꢂ 1
24 ꢂ 1
78 ꢂ 2
e
42 ꢂ 2
29 ꢂ 2
19 ꢂ 1
54 ꢂ 2
50 ꢂ 1
32 ꢂ 3
73 ꢂ 2
57 ꢂ 1
20 ꢂ 4
75 ꢂ 1
e
50 ꢂ 2
44 ꢂ 2
34 ꢂ 1
76 ꢂ 2
65 ꢂ 3
54 ꢂ 2
78 ꢂ 4
62 ꢂ 1
53 ꢂ 2
85 ꢂ 3
e
45 ꢂ 3
32 ꢂ 1
21 ꢂ 2
57 ꢂ 2
35 ꢂ 2
24 ꢂ 4
58 ꢂ 2
41 ꢂ 1
23 ꢂ 2
75 ꢂ 3
e
48 ꢂ 3
34 ꢂ 2
27 ꢂ 2
62 ꢂ 2
50 ꢂ 1
46 ꢂ 1
40 ꢂ 3
32 ꢂ 1
13 ꢂ 1
83 ꢂ 2
e
57 ꢂ 2
46 ꢂ 1
31 ꢂ 2
63 ꢂ 3
55 ꢂ 3
29 ꢂ 3
61 ꢂ 3
50 ꢂ 2
28 ꢂ 3
63 ꢂ 1
e
17
22
5-Fub
Mitomycinb
Adriyamycinb
e
78 ꢂ 3
e
1
91 ꢂ 1
e
e
e
e
e
e
The bold values are shown for those compounds which have proved to be active and those in normal font represent least significant.
a
Results are mean ꢂ SD of three separate experiments, conducted in triplicate at the concentration of 50
mM.
b
Concentration of 5-FU ¼ 20
m
M, Mitomycin ¼ 1
m
M, Adriamycin ¼ 1
mM.
Table 4
IC50 values of selected compounds 2, 17, 22 against various human cancer cell lines.
Tissue type
Cell lines
Leukemia
THP-1
Prostate
DU-145
Liver
Breast
MCF-7
Lung
CNS
Colon
HL-60
PC-3
HEP-2
A-549
SF-295
HCT-15
Compounds
IC50 in mM
17
22
2
3.5 ꢂ 1.1
4.2 ꢂ 1.3
18 ꢂ 2
1.8 ꢂ 1.2
2 ꢂ 1.1
ND
14 ꢂ 2
11 ꢂ 3
26 ꢂ 3
19 ꢂ 1
15 ꢂ 3
23 ꢂ 2
19 ꢂ 2
14 ꢂ 2
20 ꢂ 1
22 ꢂ 3
23 ꢂ 3
25 ꢂ 2
17 ꢂ 3
12 ꢂ 1
40 ꢂ 3
23 ꢂ 2
22 ꢂ 3
8 ꢂ 1
13 ꢂ 1
13 ꢂ 1
11 ꢂ 2
ND ¼ not determined.

58
R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
Since leukemic cell lines were found the most sensitive cells toward
the cytotoxic potential of these compounds, the IC50 value was
further calculated at two time points (48 h and 24 h) on HL-60 cells
by MTT assay. Both compounds 17 and 22 showed concentration
and time dependent inhibition of cell proliferation displaying the
Effect of 22 on proliferation of HL-60 cells at 24 h
and 48 h.
2.2
2
1.8
1.6
1.4
1.2
1
48 h IC50=2 µM
24 h IC50=16µM
IC50 values 1.8 mM and 18 mM for 17 while 2.0 mM and 16 mM for 22
after 48 h and 24 h time incubation respectively (Figs. 2 and 3).
Further experiments were carried out to verify whether the
cancer cell death induced by the 17 and 22 was apoptotic, as it
became increasingly evident that although the primary intra-
cellular targets and the pharmacological mechanisms of action of
the anti-cancer drugs vary vastly, the drug induced cell killing is
generally mediated by apoptosis [15]. Compounds 17 and 22
were observed to be potent apoptosis inducers, as evidenced
from the measurement of two important biological end-points of
the apoptosis viz., DNA fragmentation and increase in sub-G0
DNA fraction. The apoptotic potential of 17 and 22 was
confirmed through induction of DNA fragmentation in HL-60
cells, which is known as the hallmark of apoptosis. Compound
17 induced the laddering pattern of apoptosis at a concentration
0.8
0.6
0.4
0.2
0
-0.2
0
3
6
9
12 15 18 21 24 27 30 33
Concentration(µM)
Fig. 3. Effect of 22 on cell proliferation of human leukemia cell line (HL-60). Expo-
nentially growing HL-60 cells (15 ꢁ103) were seeded in 96-well plate. DMSO solution
of 22 was added to the cells at different conc. whereas the untreated control received
the vehicle only. MTT dye was added after 44 h and the plates were incubated for the
next 4 h at 37 ꢃC and the OD was measured as described in experimental section are
mean ꢂ SD (n ¼ 6 wells) and representative of two similar experiments.
of 20
dependent. The minimal concentration inducing DNA fragmen-
tation of 20 M, which on extension to 40 M showed no smear
mM, and in 22, the laddering pattern was concentration
m
m
formation which is a representative of post apoptotic necrosis
(Figs. 4 and 5).
mitochondrial membrane potential and 22 showed 55.4%, the
Further extending our study, DNA cell cycle analysis was per-
formed using HL-60 cells. Most of the differentiated cells are
arrested in the G1 phase but in case of cancer cells, this control is
lost and they go on dividing. Thus, 17 and 22 were subjected to
hypo-diploid sub-G0 DNA fraction (<2nDNA) analysis as
a measure of apoptosis. HL-60 cells treated with 17 and 22 at
untreated control showed only 6% and cells treated with camp-
tothecin showed 55.5% (at 5 mM) suggesting the central role of
mitochondria toward the apoptotic potential of both of these
molecules (Figs. 8 and 9).
30 mM concentration showed a considerable increase in the hypo-
diploid sub-G0 DNA fraction (<2nDNA), i.e. 68% in case of 17 and
66% in case of 22 at 24 h treatment, indicating DNA damage
(Figs. 6 and 7).
To rule out whether the compounds have any effect on the
mitochondrial functioning, mitochondrial membrane depolar-
ization assay was performed, both 17 and 22 caused the
disruption of mitochondrial membrane and subsequent loss of
mitochondrial membrane potential in a concentration dependent
manner. Compound 17 showed almost 54% of dissipation of
Effect of 17 on proliferation of HL-60 cells at 24 h
and 48 h.
1.6
1.4
1.2
48 h IC50=1.8 µM
24 h IC50 = 18 µM
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
Concentration(µM)
Fig. 2. Effect of 17 on cell proliferation of human leukemia cell line (HL-60). Expo-
nentially growing HL-60 cells (15 ꢁ103) were seeded in 96-well plate. DMSO solution
of 17 was added to the cells at different conc. whereas the untreated control received
the vehicle only. MTT dye was added after 44 h and the plates were incubated for the
next 4 h at 37 ꢃC and the OD was measured as described in the experimental section.
Data are mean ꢂ SD (n ¼ 6 wells) and representative of two similar experiments.
Fig. 4. Compound 17 induced DNA fragmentation in HL-60 cells. Cells 2 ꢁ 106/mL/well
were treated with indicated conc. of 17 for 24 h. Genomic DNA was isolated and put to
electrophoresis as described in experimental section to assess the fragmentation at
different concentrations.

R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
59
GCeMS/HPLC and found in the range 93e98%. HPLC/GCMS chro-
matograms of some of the analogs are provided in Supporting
information.
4.1.1. Isolation of bakuchiol
Bakuchiol was isolated in bulk quantity from the seeds of P.
corylifolia (duly authenticated by the Taxonomist of our institute).
Bakuchiol used in the present study was of >98% purity achieved by
repeated column chromatography over silica gel and the natural
product was well characterized by spectroscopic study (found in
agreement with the literature data) [16].
4.1.2. General procedure for preparation of acyl derivatives of
bakuchiol (2e9)
Compounds 2e9 were synthesized by treating bakuchiol 1 with
appropriate alkanoic anhydride in presence of catalytic amount of
N,N-dimethyl amino pyridine and the progress of reaction moni-
tored by TLC. On completion of the reaction, the contents poured in
ice water and the oily material separated and dissolved in DCM. The
organic layer washed with water, dried over anhydrous calcium
chloride and concentrated under reduced pressure. The acyl
derivatives thus obtained were purified by column chromatog-
raphy (CC) over silica gel 60e120 mesh, using hexane:Ethyl acetate
mixture (49:1) as eluent to give compounds 2e9 in >90% overall
yield. The spectral data of all the derivatives of 1 are given below.
4.1.2.1. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
acetate (2). The title compound was prepared and purified by the
procedure as described above to give 2 in 98% yield. 1H NMR
Fig. 5. Compound 22 induced DNA fragmentation in HL-60 cells. Cells 2 ꢁ 106/mL/well
were treated with indicated conc. of 22 for 24 h. Genomic DNA was isolated and put to
electrophoresis as described in the experimental section to assess the fragmentation at
different concentrations.
(200 MHz, CDCl3):
d 1.20 (3H, s, eC(CH]CH2)CH3), 1.48e1.50 (2H,
m, eC(CH3)CH2e), 1.57 and 1.67 (3H each, s, ]C(CH3)2), 1.89e2.04
(2H, m, eCH2CH2CH]), 2.28 (3H, s, eOCOCH3), 5.05 (3H, m,
eCH2CH] and eC(CH]CH2)), 5.87 (1H, dd, J ¼ 17.2 and 10.87 Hz,
eC(CH]CH2)), 6.14 (1H, d, J ¼ 16.2 Hz, AreCH]CHe), 6.30 (1H, d,
J ¼ 16.2 Hz, AreCH]), 7.01 (2H, d, J ¼ 8.58 Hz, 2ꢁ AreH), 7.35 (2H,
3. Conclusion
d, J ¼ 8.58 Hz,2ꢁ AreH). 13C NMR (125 MHz):
d 17.68, 21.14, 23.26,
We demonstrated the preparation of diversified analogs such as
acyl, nitro, amino, halo, alkyloxy, epoxy, formyl, and styryl deriva-
tives, of the natural product bakuchiol and their ability to inhibit
23.34, 25.73, 41.26, 42.68, 112.17, 121.59, 124.75, 126.33, 127.01,
131.38, 135.76, 138.24, 145.69, 149.62, 169.52. IR: 668, 757, 1019,
1084, 1160, 1216, 1403, 1457, 1513, 1541, 1635, 1765, 2853, 2924,
3421 cmꢀ1. MS m/z 321 (Mþ þ Na).
several cancer cell lines (IC50 1.8 to <25 mM). Our data revealed that
compounds 17 and 22 provide considerable evidence for their
apoptotic mode of action against various cancer cell lines especially
HL-60. Moreover, the induction of apoptosis by these molecules
was associated with perturbance in mitochondrial membrane
potential and cell cycle arrest that ultimately resulted in appre-
ciable DNA fragmentation. However, kinetic and in vivo studies in
the preclinical models need to be explored in order to ascertain
their therapeutic potency.
4.1.2.2. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
propanoate (3). The title compound was prepared and purified by
the procedure as described under Section 4.1.2 to afford 3 in 97%
yield. 1H NMR (200 MHz, CDCl3):
d 1.20, (3H, s, eC(CH]CH2)CH3),
1.25 (3H, t, J ¼ 7.3 Hz, eCH2CH3), 1.49 (2H, m, eC(CH3)CH2e), 1.58
and 1.68, (3H each, s, ]C(CH3)2), 1.94 (2H, m, eCH2CH2CH]), 2.54
(2H, q, J ¼ 15.1 and 7.27 Hz, eCOCH2e), 4.97e5.01 (3H, m, CH2CH]
and eC(CH]CH2)), 5.88 (1H, dd, J ¼ 17.24, 10.90 Hz, eC(CH]CH2)),
6.15 (1H d, J ¼ 16.27 Hz, AreCH]CHe), 6.31 (1H, d, J ¼ 16.28 Hz,
AreCH]), 7.00 (2H, d, J ¼ 8.57 Hz, 2ꢁ AreH), 7.36 (2H, d,
4. Experimental
4.1. Chemistry
J ¼ 8.58 Hz, 2ꢁ AreH). 13C NMR (125 MHz):
d 10.18, 17.63, 22.83,
23.36, 25.73, 27.76, 41.37, 42.75, 112.15, 121.73, 124.86, 126.43,
127.02, 131.23, 135.66, 138.16, 145.73, 149.87, 172.24. IR: 665, 754,
1021, 1088, 1161, 1215, 1401, 1457, 1515, 1635, 1762, 2853, 2924,
3421 cmꢀ1. MS m/z 335 (Mþ þ Na).
All reagents used for chemical synthesis were purchased from
SigmaeAldrich. Solvents were distilled before use. All the reactions
were monitored by TLC on silica gel F254 plates (E. Merck) using 2%
ceric ammonium sulfate solution for detection of the spots. Flash
chromatography was carried out for purification of the products. All
NMR spectra were recorded on Bruker DPX 200, DPX 400 and DPX
500 instruments using CDCl3 as the solvent with TMS as internal
standard. The chemical shifts are expressed in delta whereas
coupling constants in Hertz. Mass spectra were recorded on ESI-
esquire 3000 Bruker Daltonics instrument. IR recorded on FT
Bruker (270-30) spectrophotometer. The purity of all the
compounds (bakuchiol and its analogs) was determined by
4.1.2.3. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
butyrate (4). The title compound was prepared and purified by the
procedure as described under Section 4.1.2 to afford 4 in 97% yield.
1H NMR (200 MHz, CDCl3):
d
0.96 (3H, t, J ¼ 7.38 Hz, eCH2CH3), 1.19
(3H, s, eC(CH]CH2)CH3), 1.46e1.53, (2H, m, eC(CH3)CH2e),
1.57 and 1.67 (3H each, s, ]C(CH3)2), 1.74e1.98, (4H, m,
eCH2CH2CH], eCH2CH3), 2.53 (2H, t, J ¼ 7.35 Hz, eCOCH2e),
4.97e5.01 (3H, m, eCH2CH] and eC(CH]CH2)), 5.88 (1H, dd,

60
R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
Fig. 6. HL-60 cells (1 ꢁ106/mL) in culture were treated with indicated conc. of 17 for 24 h. Cells were stained with PI to determine DNA fluorescence and cell cycle phase distribution
by flow cytometry. Fraction of cells for hypo-diploid (sub-G0, ꢄ2n DNA) population indicative of DNA damage was analyzed and shown as (%). Data are representative of one of two
similar experiments.
J ¼ 17.29, 10.86 Hz, eC(CH]CH2)), 6.15 (1H, d, J ¼ 16.25 Hz,
AreCH]CHe), 6.31 (1H, d, J ¼ 16.24 Hz, AreCH]), 7.01 (2H, d,
J ¼ 8.43 Hz, 2ꢁ AreH), 7.35 (2H, d, J ¼ 8.44 Hz, 2ꢁ AreH). 13C NMR
yield. 1H NMR (200 MHz, CDCl3):
d 0.91 (3H, br s, eCH2CH3), 1.20 (s,
3H, eC(CH]CH2)CH3), 1.26e1.53 (6H, m, eC(CH3)CH2e,
e(CH2)2CH2CH3), 1.58 and 1.67 (3H each, s, ]C(CH3)2), 1.75e1.97,
(4H m, eCH2CH3, eCH2CH2CH]), 2.53 (2H, t, J ¼ 7.27 Hz,
eCOCH2e), 4.97e5.01 (3H, m, C(CH]CH2), eCH2CH]), 5.87 (1H,
dd, J ¼ 17.22, 10.90 Hz, eC(CH]CH2)), 6.15 (1H, d, J ¼ 16.24 Hz,
AreCH]CHe), 6.31 (1H, d, J ¼ 16.24 Hz, AreCH]), 7.00 (2H, d,
J ¼ 8.42 Hz, 2ꢁ AreH), 7.35 (2H, d, J ¼ 8.40 Hz, 2ꢁ AreH). 13C NMR
(125 MHz):
d 13.65,.17.64, 18.46, 23.21, 23.26, 25.70, 36.23, 41.20,
42.64, 112.11, 121.57, 124.69, 126.30, 126.94, 131.38, 135.58, 138.10,
145.66, 149.62, 172.20. IR: 750, 1025, 1083, 1160, 1214, 1403, 1458,
1512, 1630, 1760, 2850, 2921, 3422 cmꢀ1. MS at m/z 349 (Mþ þ Na).
4.1.2.4. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
pentanoate (5). The title compound was prepared and purified by
the procedure as described under Section 4.1.2 to afford 5 in 97%
(125 MHz): d 14.15, 17.65, 22.72, 23.57, 24.64, 25.69, 29.39, 31.29,
34.36, 41.25, 42.75, 112.12, 121.56, 124.75, 126.36, 126.93, 131.26,
135.58, 138.08, 145.66, 149.72, 172.23. IR: 748, 1021, 1084, 1156,
1202, 1405, 1445, 1513, 1622, 1762, 2852, 2921, 3423 cmꢀ1. MS at m/
z 377 (Mþ þ Na).
yield. 1H NMR (500 MHz, CDCl3):
d
0.88 (3H, t, J ¼ 7.3 Hz, eCH2CH3),
1.12 (3H, s, eC(CH]CH2)CH3), 1.26e1.53 (4H, m, eC(CH3)CH2e,
eCH2CH2CH3), 1.58 and 1.67 (3H each, s, ]C(CH3)2), 1.76 (2H, m,
eCH2CH3), 1.95 (2H, m, eCH2CH2CH]), 2.53 (1H, t, J ¼ 7.57 Hz,
eCOCH2e), 4.96 (2H, dd, J ¼ 18.99, 11.33 Hz, eC(CH]CH2)), 5.10
(1H, t, J ¼ 7.02 Hz, eCH2CH]), 5.87 (1H, dd, J ¼ 17.43, 10.70 Hz,
eC(CH]CH2)), 6.15 (1H, d, J ¼ 16.25 Hz, AreCH]CHe), 6.30 (1H, d,
J ¼ 16.23 Hz, AreCH]), 7.00 (2H, d, J ¼ 8.48 Hz, 2ꢁ AreH), 7.35 (2H,
4.1.2.6. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
octanoate (7). The title compound was prepared and purified by
the procedure as described under Section 4.1.2 to afford 7 in 95%
yield. 1H NMR (200 MHz, CDCl3):
d 0.89 (3H, br s, eCH2CH3), 1.20
(3H, s, eC(CH]CH2)CH3), 1.30 (4H, br s, 2ꢁ CH2), 1.45 (6H, m, 3ꢁ
CH2), 1.58 and 1.67 (3H each, s, ]C(CH3)2), 1.79e1.97 (4H, m,
eCH2CH3, eCH2CH2CH]), 2.53 (2H, t, J ¼ 7.39 Hz, eCOCH2e),
4.97e5.10 (3H, m, C(CH]CH2), eCH2CH]), 5.88 (1H, dd, J ¼ 10.89,
17.24 Hz, eC(CH]CH2)), 6.15 (1H, d, J ¼ 16.27 Hz, AreCH]CHe),
6.31 (1H, d, J ¼ 16.28 Hz, AreCH]), 6.96 (2H, d, J ¼ 8.23 Hz, 2ꢁ
AreH), 7.36 (2H, d, J ¼ 8.34 Hz, 2ꢁ AreH). 13C NMR (125 MHz):
d, J ¼ 8.49 Hz, 2ꢁ AreH). 13C NMR (125 MHz):
d 13.69, 17.59, 22.22,
23.21, 23.27, 25.65, 27.19, 34.07, 41.21, 42.61, 112.09, 121.53, 124.71,
126.32, 126.91, 131.26, 135.56, 138.07, 145.63, 149.65, 172.31. IR: 751,
1024, 1085, 1158, 1212, 1401, 1448, 1510, 1628, 1761, 2851, 2919,
3425 cmꢀ1. MS at m/z 363 (Mþ þ Na).
4.1.2.5. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
hexanoate (6). The title compound was prepared and purified by
the procedure as described under Section 4.1.2 to afford 6 in 95%
d 14.08, 17.68, 22.62, 23.28, 24.94, 25.70, 28.95, 29.09, 29.36, 31.95,
34.36, 41.23, 42.63, 112.12, 121.55, 124.74, 126.35, 127.90, 131.23,
135.33, 138.02, 145.61, 149.69, 172.20. IR: 750, 1020, 1080, 1155,

R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
61
Fig. 7. HL-60 cells (1 ꢁ106/mL) in culture were treated with indicated conc. of 22 for 24 h time period. Cells were stained with PI to determine DNA fluorescence and cell cycle phase
distribution by flow cytometry. Fraction of cells for hypo-diploid (sub-G0, ꢄ2n DNA) population indicative of DNA damage was analyzed and shown as (%). Data are representative of
one of two similar experiments.
1215, 1402, 1449, 1512, 1625, 1763, 2853, 2918, 3426 cmꢀ1. MS at m/
z 405 (Mþ þ Na).
eC(CH]CH2)CH3), 1.26 (20H, br s, 10ꢁ CH2), 1.46e1.53 (6H, 3ꢁ
CH2), 1.57 and 1.67 (2H each, s, ]C(CH3)2), 1.73e1.98 (4H, m,
eCH2CH3, eCH2CH2CH]), 2.53 (2H, t, J ¼ 7.28 Hz, eCOCH2e),
4.97e5.01 (3H, eC(CH]CH2), eCH2CH]), 5.88 (1H, dd, J ¼ 17.13,
10.92 Hz, eC(CH]CH2)), 6.15 (1H, d, J ¼ 16.25 Hz, AreCH]CHe),
6.31 (1H, d, J ¼ 16.24 Hz, AreCH]), 7.00 (2H, d, J ¼ 8.46 Hz, 2ꢁ
AreH), 7.35 (2H, d, J ¼ 8.47 Hz, 2ꢁ AreH). 13C NMR (125 MHz):
4.1.2.7. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
decanoate (8). The title compound was prepared and purified by
the procedure as described under Section 4.1.2 to afford 8 in 93%
yield. 1H NMR (200 MHz, CDCl3):
d 0.83 (3H, br s, eCH2CH3), 1.20
(3H, s, eC(CH]CH2)CH3), 1.25 (8H, br s, 4ꢁ CH2), 1.46 (6H, m, 3ꢁ
CH2), 1.58 and 1.67 (3H each, s, ]C(CH3)2), 1.97 (4H, m, eCH2CH3,
eCH2CH2CH]), 2.53 (2H, t, J ¼ 7.39 Hz, eCOCH2e), 4.93e5.10 (3H,
m, eC(CH]CH2), eCH2CH]), 5.87 (1H, dd, J ¼ 10.93, 17.28 Hz,
eC(CH]CH2)), 6.07 (1H, d, J ¼ 15.97 Hz, AreCH]CHe), 6.23 (1H, d,
J ¼ 15.97 Hz, AreCH]), 7.01 (2H, d, J ¼ 8.3 Hz, 2ꢁ AreH), 7.35 (2H,
d 14.14, 17.63, 22.72, 23.23, 23.28, 24.96, 25.70, 29.13, 29.28, 29.40,
29.49, 29.62, 29.72, 31.95, 34.40, 41.23, 42.64, 112.12, 121.55, 124.72,
126.34,126.93,131.29,135.56,138.06, 145.63,149.67,172.32. IR: 750,
1020, 1080, 1155, 1215, 1402, 1449, 1512, 1625, 1763, 2853, 2918,
3426 cmꢀ1. MS at m/z 517 (Mþ þ Na).
d, J ¼ 8.38 Hz, 2ꢁ AreH). 13C NMR (125 MHz):
d
14.18, 17.63, 22.83,
4.1.2.9. Synthesis of 1-methoxy-4-(3,7-dimethyl-3-vinylocta-1,6-
dienyl)benzene (10). Bakuchiol (2 mmol) was dissolved in acetone
(5 mL) and anhydrous K2CO3 (3 mmol) was added followed by
2 mmol of methyl iodide. The mixture was allowed to reflux for 8 h.
The reaction mixture was cooled, filtered, and evaporated. The
product was purified by flash chromatography using hexane:EtOAc
(19:1) as the eluent to afford 10 in 93% yield. 1H NMR (500 MHz,
23.30, 25.05, 25.73, 29.25, 29.50, 29.59, 29.73, 29.88, 32.0, 34.43,
41.37, 42.74, 112.15, 121.70, 124.86, 126.43, 127.01, 131.23, 135.66,
138.16, 145.73, 149.87, 172.24. IR: 753, 1022, 1081, 1153, 1214, 1405,
1447, 1513, 1628, 1760, 2855, 2919, 3423 cmꢀ1. MS at m/z 433
(Mþ þ Na).
4.1.2.8. Synthesis of 4-(3,7-dimethyl-3-vinylocta-1,6-dienyl) phenyl
palmitate (9). The title compound was prepared and purified by the
procedure as described under Section 4.1.2 to afford 9 in 92% yield.
CDCl3): d 1.21 (3H, s, eC(CH]CH2)CH3), 1.51 (2H, m, eC(CH3)
CH2CH2e), 1.59 and 1.69 (3H each, s, ]C(CH3)2), 1.96 (2H, m,
eCH2CH]), 3.80 (3H, s, eOCH3), 5.03 (2H, m, eC(CH]CH2)CH3),
5.12 (1H, t, J ¼ 7.28 Hz, eCH]C), 5.89 (1H, dd, J ¼ 10.95, 17.18
1H NMR (200 MHz, CDCl3):
d 0.90 (3H, br s, eCH2CH3), 1.20 (3H,s,

62
R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
Fig. 8. Compound 17 induced loss of mitochondrial membrane potential (DJm) HL-60. Cells (1 ꢁ106/mL/ well) incubated with 17 at different conc. in 6 well plates for 24 h. Before
1 h of the completion of the experiment cells were treated with Rodamine-123 (5
analyzed. Data are representative of one of two similar experiments.
mM). Cells were washed with PBS and centrifuged and finally dissolved in 1 mL of PBS and
Hz,eC(CH]CH2)), 6.08 (1H, d, J ¼ 16.18 Hz, AreCH]CHe), 6.28
(1H, d, J ¼ 16.18 Hz, AreCH]CHe), 6.85 (2H, d, J ¼ 8.21 Hz, 2ꢁ
AreH), 7.31 (2H, d, J ¼ 8.21 Hz, 2ꢁ AreH). 13C NMR (125 MHz):
(50 MHz): d 17.72, 23.19, 23.26, 25.76, 41.19, 42.82, 112.56, 120.05,
121.9, 124.60, 130.95, 131.58, 133.57, 135.07, 139.33, 145.23, 154.05.
IR: 669, 762, 1019, 1078, 1216, 1322, 1404, 1422, 1537, 1626, 2854,
2924, 3020, 3377 cmꢀ1. MS at m/z 300 (Mþ ꢀ 1).
d
19.09, 24.70, 24.83, 27.15, 42.77, 43.98, 56.73, 113.3, 115.3, 126.29,
128.01, 128.61, 132.16, 132.71, 137.26, 147.45, 160.21. IR: 814, 912,
970, 1037, 1174, 1248, 1462, 1510, 1608, 1632, 2854, 2915, 2965,
3384 cmꢀ1. MS at m/z 293 (Mþ þ Na).
4.1.2.11. Synthesis
of
3-amino,4-(3,7-dimethyl-3-vinylocta-1,6-
dienyl) phenol (12). A mixture of FeeZn (3:3 mmol) and 5% HCl
(1 mL) was added to the solution of 3-nitrobakuchiol in DCM
(10 mL) with heating (40 ꢃC) and stirring. The reaction mixture was
cooled and filtered. The filtrate was extracted with DCM and
washed with aqueous saturated NaHCO3 solution. The organic layer
was dried and evaporated. The product was purified by column
chromatography over silica gel using hexane:EtOAc (17:3) as the
4.1.2.10. Synthesis of 3-nitro,4-(3,7-dimethyl-3-vinylocta-1,6-dienyl)
phenol (11). To a stirred solution of the bakuchiol (1 mmol) in
acetone (15 mL) was added nickel (II) nitrate (1 mmol) followed by
a catalytic amount of p-TSA (0.012 mmol) and the reaction mixture
was refluxed until 1 was fully consumed. Acetone was removed
under vacuum and the crude mass was partitioned between DCM
and water. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated under vacuum. The crude product was
purified by CC over silica gel using hexane:EtOAc (49:1) as the
eluent to afford 12 in 70% yield. 1H NMR (500 MHz, CDCl3):
d 1.20
(3H, s, eC(CH]CH2)CH3), 1.47 (2H, m, eC(CH3)CH2CH2e), 1.58 and
1.67 (3H each, s, ]C(CH3)2), 1.93 (2H, m, eCH2CH]), 5.03 (2H, m,
eC(CH]CH2)CH3, eCH2CH]C(CH3)2), 5.09 (1H, t, J ¼ 7.2 Hz, eCH]
C), 5.87 (1H, dd, J ¼ 17.3, 10.8 Hz,eC(CH]CH2)), 5.99 (1H, d,
J ¼ 16.27 Hz, AreCH]CHe), 6.16 (1H, d, J ¼ 16.2 Hz, AreCH]CHe),
6.62 (1H, d, J ¼ 8.0 Hz, AreH), 6.65 (1H, d, J ¼ 8.0 Hz, AreH), 6.79
eluent to furnish 11 in 86% yield. 1H NMR (200 MHz, CDCl3):
d 1.22
(3H, s, eC(CH]CH2)CH3), 1.51 (2H, m, eC(CH3)CH2CH2e), 1.59 and
1.68 (3H each, s, ]C(CH3)2), 1.96 (2H, m, eCH2CH]), 5.06 (3H,
m, eC(CH]CH2)CH3, eCH2CH]C), 5.88 (1H, dd, J ¼ 10.92,
17.22 Hz, eC(CH]CH2)), 6.17 (1H, d, J ¼ 16.18 Hz, AreCH]CHe),
6.28 (1H, d, J ¼ 16.18 Hz, AreCH]CHe), 7.10 (1H, d, J ¼ 8.62 Hz,
AreH), 7.62 (1H, d, J ¼ 8.62 Hz, AreH), 8.03 (1H, s, AreH). 13C NMR
(1H, s, AreH). 13C NMR (125 MHz):
d 17.72, 23.19, 23.26, 25.76, 41.19,
42.82, 112.56, 114.4, 115.3, 117.9, 124.8, 126.8, 131.2, 131.4, 134.4,
135.5, 143.6, 146.0. IR: 757, 825, 909, 1020, 1122, 1290, 1382, 1457,
1508, 1637, 2925, 2957 cmꢀ1. MS at m/z 294 (Mþ þ Na).

R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
63
4.1.2.12. Synthesis of 4-(3-ethyl-3,7-dimethyloctyl)-2-iodophenol
(13). A mixture of 0.5 mmol of 14 (hexahydrobakuchiol, prepared
by hydrogenation of bakuchiol), molecular iodine (0.5 mmol), and
CAN (10 mol%) in acetonitrile (5 mL) was stirred at 25 ꢃC and the
reaction monitored by TLC. After the completion of the reaction, the
contents were treated with aqueous Na2S2O3 solution (10 mL),
extracted with ethyl acetate (3 ꢁ 10 mL), the organic layer washed
with water (2 ꢁ 10 mL), dried over sodium sulfate and concen-
trated. The crude product purified by CC over silica gel
(60e120 mesh) using hexane:EtOAc (19:1) as the eluent to afford
127.7, 127.8, 129.8, 134.7, 146.4, 157.4. IR: 814, 912, 970, 1037, 1174,
1248, 1462, 1510, 1608, 1632, 2854, 2915, 2965, 3384. MS at m/z 271
(Mþ ꢀ 1).
4.1.2.16. Synthesis of 4-[2-{4-(2-hydroxypropan-2-yl)-1,2-dime-
thylcyclopentyl}vinyl]phenol (17). Bakuchiol (1.95 mmol) dissolved
in THF:H2O (1:1) (10 mL) at 0 ꢃC, added Hg(OAc)2 (0.625 g,
1.95 mmol), after stirring the solution for 10 min, 10% NaOH solu-
tion was added to the reaction mixture. Reaction mixture was
further stirred for half an hour, followed by addition of NaBH4,
reaction monitored through TLC. After the completion of the
reaction, the contents neutralized with dilute HCl and extracted
with EtOAc (3 ꢁ 100 mL). The combined organic layers washed with
water (2 ꢁ 50 mL), and dried over anhydrous Na2SO4. The crude
product purified by CC over silica gel using Hexane:EtOAc (9:1) as
the eluent to afford 17 in 90% yield. 1H NMR (500 MHz, CDCl3):
13 in 88% yield. 1H NMR (200 MHz, CDCl3):
d 0.81e0.93 (12H, m),
1.23e1.59 (11H, m), 2.52 (2H, m, AreCH2e), 7.08 (1H, d, J ¼ 8.5 Hz,
AreH), 7.44 (1H, dd, J ¼ 8.5, 2.01 Hz, AreH), 7.92 (1H, d, J ¼ 2.04 Hz,
AreH). 13C NMR (125 MHz):
d 8.06, 21.25, 22.74, 24.52, 27.98, 29.59,
31.47, 35.29, 39.09, 40.05, 41.10, 119.81, 123.70, 133.38, 136.11,
138.19, 153.33. IR: 668, 764, 824, 1079, 1181, 1247, 1324, 1382, 1427,
1464, 1488, 1539, 158, 1630, 2868, 2931, 2957, 3250 cmꢀ1. MS at m/z
411(Mþ þ Na).
d
0.95 (3H, d, J ¼ 6.67 Hz, eCHCH3), 0.98 (3H, s, eCeCH3), 1.24 (6H,
br s, eCe(CH3)2), 1.49 (1H, q, J ¼ 8.6 Hz, 2.2 Hz, eCH), 1.59 (2H, m,
eCH2), 1.72 (2H, m, CHeCH2eCH2) 1.87 (1H, m,eCHe(CH3)2OH),
6.03 (1H, d, J ¼ 16.2 Hz, AreCH]CHe), 6.24 (1H, d, J ¼ 16.2 Hz,
AreCH]CHe), 6.76 (2H, d, J ¼ 8.5 Hz, AreH), 7.22 (2H, d, J ¼ 8.5 Hz,
4.1.2.13. Synthesis of 4-(3-ethyl-3,7-dimethyloctyl)phenol (14). The
title compound was prepared by hydrogenation of methanolic
solution of 1 (1.0 mmol, 25 mL) at 35 psi with Pd/C to afford 14 in
AreH). 13C NMR (125 MHz):
d 16.07, 18.48, 26.12, 27.68, 28.40,
98% yield. 1H NMR (200 MHz, CDCl3):
d
0.80e0.89 (12H, m),
39.80, 44.51, 48.20, 55.62, 74.27, 115.47, 125.81, 127.23, 130.58,
137.97, 154.98. IR: 757, 816, 854, 928, 968, 1021, 1164, 1237, 1379,
1451, 1512, 1608, 2927, 2963, 3360 cmꢀ1. MS at m/z 273 (Mþ ꢀ 1).
1.12e1.61 (11H, m), 2.41 (2H, m, PheCH2e), 6.74 (2H, d, J ¼ 8.3 Hz,
2ꢁ AreH), 7.03 (2H, d, J ¼ 8.3 Hz, 2ꢁ AreH). 13C NMR (100 MHz):
d
8.27, 21.67, 24.48, 27.97, 29.47, 31.66, 35.30, 39.32, 40.20, 41.62,
115.27, 129.32, 136.1, 153.51. IR: 825, 1021, 1170, 1230, 1381, 1462,
4.1.3. General procedure for preparation of compounds 18e22 by
Heck coupling reaction
1513, 1612, 2867, 2930, 2957, 3356 cmꢀ1. MS at m/z 261 (Mþ ꢀ 1).
A mixture of substituted aromatic halide (1.2 equiv), bakuchiol
(1.0 equiv) and K2CO3 (2 equiv) in dry DMF in presence of tetrakis
(triphenylphosphine) palladium (O) complex was refluxed under
N2 current at 140 ꢃC for 7 h. The contents cooled, diluted with 5%
HCl to attain pH 5, and extracted with diethyl ether (3 ꢁ 100 mL),
the organic layer washed with water (2 ꢁ 50 mL), dried over
Na2SO4, and concentrated under reduced pressure. The crude
product purified by CC over silica (60e120) with hexane:EtOAc
mixture as the eluent to afford the required compound.
4.1.2.14. Synthesis of 6-(4-hydroxystyryl)-2,6-dimethylocta-2,7-
dienal (15). Bakuchiol (0.5 g, 1.9 mmol) was dissolved in
a mixture of acetic acid and water (9:1), SeO2 (216 mg, 1.9 mmol)
was added and the contents stirred for 4 h at 20 ꢃC. The reaction
mixture was diluted with ice-cold water and extracted with EtOAc
(2 ꢁ 100 mL). The organic layer washed with water (2 ꢁ 50 mL),
dried over anhydrous Na2SO4 and concentrated under reduced
pressure to give crude product, which was purified on silica gel
column using Pet. ether 60e80:EtOAc (9:1) as the eluent to afford
15 in 67% yield. 1H NMR (200 MHz, CDCl3):
d
1.25 (3H, s, eC(CH]
4.1.3.1. Synthesis of 4-{3-(2-methoxystyryl)-3,7-dimethylocta-1,6-
dienyl}phenol (18). Using 2-iodoanisole as the substrate, the title
compound was prepared by the procedure as described under
Section 4.1.3 and the crude product purified by CC over silica gel
using hexane:EtOAc (17:3) as the eluent to afford 18 in 52% yield. 1H
CH2)CH3), 1.64 (2H, m, eC(CH3)CH2CH2e), 1.72 (3H, s, ]C(CH3)),
2.01 (2H, m, eCH2CH]), 4.99 (2H, m, eC(CH]CH2)CH3), 5.89 (1H,
dd, J ¼ 17.3, 10.8 Hz, eC(CH]CH2)), 6.02 (1H, d, J ¼ 16.28 Hz,
AreCH]CHe), 6.28 (1H, d, J ¼ 16.28 Hz, AreCH]CHe), 6.50 (1H, t,
J ¼ 7.0 Hz, eCH]C), 6.78 (2H, d, J ¼ 8.56 Hz, 2ꢁ AreH), 7.24 (2H, d,
J ¼ 8.56 Hz, 2ꢁ AreH), 9.36 (1H, s, eCHO). 13C NMR (50 MHz):
NMR (200 MHz, CDCl3):
d 1.33 (3H, s, eCeCH3), 1.63 (2H, m,
eC(CH3)CH2CH2), 1.65 and 1.69 (3H each, s, ](CH3)2), 2.03 (2H, m,
eCH2CH]), 3.84 (3H, s, AreOCH3), 5.13 (1H, t, J ¼ 6.9 Hz,eCH]C),
6.25 (2H, m, AreCH]CHe, Ar0eCH]CHe), 6.81 (5H, m, 2ꢁ
AreCH]CH, 2ꢁ AreH, AreH), 7.23 (4H, m, 2ꢁ AreH, 2ꢁ Ar0eH),
d
18.5, 24.64, 24.73, 42.65, 43.8,113.51,115.56,127.85,129.68,131.81,
137.33, 140.42, 147.35, 152.86, 155.6, 195.1. IR: 757, 1020, 1084, 1120,
1169, 1217, 1456, 1513, 1610, 1715, 2851, 2922, 3384 cmꢀ1. MS at m/z
269 (Mþ ꢀ 1).
7.46 (1H, d, J ¼ 8.4 Hz, Ar0eH). 13C NMR (50 MHz):
d 18.11, 23.83,
24.45, 26.13, 42.16, 44.22, 55.91, 111.33, 115.78, 121.06, 122.15,
125.37, 126.75, 126.94, 127.84, 127.85, 128.40, 132.1, 132.4, 136.7,
138.9, 156.1, 158.7. IR: 757, 925, 1040, 1064, 1233, 1371, 1457, 1509,
1578, 1606, 2853, 2924, 2959, 3411 cmꢀ1. MS at m/z 361 (Mþ ꢀ 1).
4.1.2.15. Synthesis
of
12,13-epoxybakuchiol
(16). Bakuchiol
(5.0 mmol) was dissolved in 30 mL DCM at 0 ꢃC and m-CPBA
(5.5 mmol) in 60 mL DCM was added slowly. After being stirred for
3 h at 25 ꢃC, aqueous saturated NaHSO3 solution added and the
contents stirred for an additional 0.5 h. The reaction mixture was
extracted with DCM and the organic layer was washed with water,
dried, and evaporated. The product was purified by CC over silica
gel using hexane:EtOAc (9:1) as the eluent to afford 16 in 80% yield.
4.1.3.2. Synthesis of 4-{3-(4-methoxystyryl)-3,7-dimethylocta-1,6-
dienyl}phenol (19). The title compound was prepared using the
procedure as described under Section 4.1.3 by coupling Bakuchiol 1
with 4-iodoanisole and the product purified by CC over silica gel
using hexane:EtOAc (17:3) as the eluent to afford 19 in 55% yield. 1H
1H NMR (500 MHz, CDCl3):
d 1.20 (3H, s, eC(CH]CH2)CH3), 1.22
and 1.23 (3H each, s, C(CH3)2), 1.57 (4H, m, eC(CH3)CH2CH2e), 2.67
(1H, t, J ¼ 6.0 Hz,eCH2eCHe), 5.03 and 5.06 (1H each, m, eC(CH]
CH2)CH3), 5.93 (1H, m, eCH]CH2), 6.10 (1H, d, J ¼ 16.2 Hz,
AreCH]CHe), 6.31 (1H, d, J ¼ 16.2 Hz, AreCH]CHe), 6.79 (2H, d,
J ¼ 8.5 Hz, 2ꢁ AreH), 7.31 (2H, d, J ¼ 8.5 Hz, 2ꢁ AreH). 13C NMR
NMR (200 MHz, CDCl3): d 1.28 (3H, s, eCeCH3), 1.57 (2H, m, ]
CHCH2CH2), 1.59 and 1.67 (3H each, s, ]C(CH3)2), 1.98 (2H, m, ]
CHCH2e), 3.79 (3H, s, AreOCH3), 5.12 (1H, t, J ¼ 6.9 Hz, eCH2CH),
6.11 (2H, d, J ¼ 16.2 Hz, AreCH]CHe, Ar0eCH]CHe), 6.30 (2H, d,
J ¼ 16.2 Hz, AreCH]CHe, Ar0eCH]CHe), 6.76 (2H, d, J ¼ 8.5 Hz,
2ꢁ AreH), 6.85 (2H, d, J ¼ 8.7 Hz, 2ꢁ Ar0eH), 7.26 (2H, d, J ¼ 6.85 Hz,
(50 MHz):
d 18.3, 23.1, 23.2, 24.6, 37.9, 42.3, 57.8, 64.2, 112.0, 115.7,

64
R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
Fig. 9. Compound 22 induced loss of mitochondrial membrane potential (DJm) HL-60. Cells (1 ꢁ106/mL/ well) incubated with 22 at different conc. in 6 well plate for 24 h. Before
1 h of the completion of the experiment cells were treated with Rodamine-123 (5
mM). Cells were washed with PBS and centrifuged and finally dissolved in 1 mL of PBS and
analyzed on flow cytometer. Data are representative of one of two similar experiments.
2ꢁ AreH), 7.32 (2H, d, J ¼ 8.7 Hz, 2ꢁ Ar0eH). 13C NMR (50 MHz):
4.1.3.4. Synthesis of 4-{3-(4-hydroxystyryl)-3,7-dimethylocta-1,6-
dienyl} phenol (21). This compound was prepared and purified by
the same procedure as described under Section 4.1.3.1 to afford 21
d
18.10, 23.81, 24.50, 26.13, 42.22, 42.49, 55.75, 114.40, 115.83,
125.31, 127.00, 127.65, 127.84, 131.18, 131.19, 131.21, 136.54, 155.1,
159.5. IR: 817, 970, 1035, 1098, 1246, 1439, 1511, 1608, 2854, 2925,
2963, 3030, 3159 cmꢀ1. MS at m/z 361 (Mþ ꢀ 1).
in 64% yield. 1H NMR (200 MHz, CDCl3):
d 1.28 (3H, s, eCeCH3), 1.57
(2H, m, ]CHCH2CH2e), 1.59 and 1.67 (3H each, s, C(CH3)2), 2.0 (2H,
m, ]CCH2CH2), 5.14 (1H, t, J ¼ 6.9 Hz, eCH]C), 6.11 (2H, d,
J ¼ 16.2 Hz, 2ꢁ AreCH]CHe), 6.29 (2H, d, J ¼ 16.2 Hz, 2ꢁ AreCH]
CHe), 6.77 (4H, d, J ¼ 8.6 Hz, 4ꢁ AreH), 7.26 (4H, d, J ¼ 8.6 Hz, 2ꢁ
4.1.3.3. Synthesis of 4-{3-(3-formyl,4-methoxystyryl)-3,7-dimethy-
locta-1,6-dienyl}phenol (20). The title compound prepared by
coupling Bakuchiol with 2-formyl, 4-iodoanisole and purified by
the procedure as described under Section 4.1.3.1 to afford 20 in 63%
AreH). 13C NMR (125 MHz):
d 17.71, 23.39, 24.03, 25.74, 41.77, 42.06,
115.45, 124.87, 126.54, 127.43, 130.81, 131.40, 136.08, 154.7. IR: 757,
816, 970, 1015, 1101, 1171, 1234, 1375, 1446, 1511, 1609, 2854, 2925,
2961, 3022, 3363 cmꢀ1. MS at m/z 347 (Mþ ꢀ 1).
yield. 1H NMR (200 MHz, CDCl3):
d 1.29 (3H, s, eCeCH3), 1.57 (2H,
m, ]CHCH2CH2e), 1.59 and 1.67 (3H each, s, C(CH3)2), 1.97 (2H, m,
]CCH2), 3.92 (3H, s, OCH3), 5.12 (1H, t, J ¼ 6.9 Hz, eCH]C), 6.21
(4H, m, 2ꢁ AreCH]CHe, 2ꢁ Ar0eCH]CHe), 6.78 (2H, d, J ¼ 8.6 Hz,
2ꢁ AreH), 6.93 (1H, d, J ¼ 8.6 Hz, Ar0eH),7.26 (4H, d, J ¼ 8.6 Hz, 2ꢁ
AreH), 7.54 (1H, d, J ¼ 8.6 Hz, Ar0eH), 7.85 (1H, s, Ar0eH), 10.45 (1H,
4.1.3.5. Synthesis of 4-{3-(4-methoxystyryl)-3,7-dimethylocta-1,6-
dienyl}phenol (22). This compound was prepared (by coupling
methyl ether Bakuchiol with 4-iodophenol) and purified by the
procedure as described under Section 4.1.3.1 to afford 22 in 58%
s, CHO). 13C NMR (125 MHz):
d 17.71, 23.39, 23.93, 25.72, 41.72,
42.19, 55.92, 111.86, 115.5, 124.61, 125.48, 126.88, 127.43, 130.89,
131.43, 132.17, 135.45, 138.0, 155.13, 160.99, 190.32. IR: 757, 816, 970,
1015, 1101, 1171, 1234, 1375, 1446, 1511, 1609, 2854, 2925, 2961,
3022, 3363 cmꢀ1. MS at m/z 389 (Mþ ꢀ 1).
yield. 1H NMR (500 MHz, CDCl3):
d 1.32 (3H, s, eCeCH3), 1.62 (2H,
m, eCH2CH2CH]), 1.64 and 1.71 (3H each, s, ]C(CH3)2), 2.04 (2H,
m, eCH2CH]), 3.83 (3H, s, AreOCH3), 5.17 (1H, t,
J ¼ 6.9 Hz,eCH2CH]), 6.17 (2H, d, J ¼ 16.2 Hz, 2ꢁ Ar0eCH]CHe),

R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
65
6.33 (2H, d, J ¼ 16.2 Hz, 2ꢁ AreCH]CH), 6.80 (2H, d, J ¼ 8.6 Hz, 2ꢁ
AreH), 6.88 (2H, d, J ¼ 8.6 Hz, 2ꢁ Ar0eH), 7.29 (2H, d, J ¼ 8.6 Hz, 2ꢁ
AreH), 7.34 (2H, d, J ¼ 8.6 Hz, 2ꢁ Ar0eH). 13C NMR (125 MHz):
(1H, t, J ¼ 6.9 Hz, eCH2CH]C), 6.15 (4H, m, 2ꢁ AreCH]CHe, 2ꢁ
Ar0eCH]CHe), 6.80 (3H, m, 1ꢁ Ar0eH, 2ꢁ AreH), 7.21 (3H, m, 2ꢁ
AreH, 1ꢁ Ar0eH), 7.41 (1H, s, 1ꢁ Ar0eH). 13C NMR (125 MHz):
d
16.73, 22.42, 23.10, 24.74, 40.83, 41.09, 54.37, 113.01, 114.46,
d 14.24, 18.7, 24.09, 24.15, 25.75, 27.8, 41.80, 42.12, 71.4, 114.06,
123.92, 125.61, 126.26, 126.44, 129.79, 129.81, 130.35, 135.08, 135.18,
153.85, 157.73. IR: 817, 970, 1034, 1102, 1173, 1246, 1302, 1374, 1441,
1511, 1608, 2853, 2925, 2964, 3030, 3305 cmꢀ1. MS at m/z 361
(Mþ ꢀ 1).
115.50, 122.50, 124.12, 126.10, 126.46, 126.74, 127.38, 127.41, 128.10,
130.81, 131.78, 132.2, 135.78, 136.66, 152.20, 155.32. IR: 762, 803,
909, 1108, 1258, 1402, 1462, 1500, 1602, 2852, 2922, 3385 cmꢀ1. MS
at m/z 423 (Mþ ꢀ 1).
4.1.4. General procedure for preparation of compounds 23e25, 27
by oxidative Heck coupling reaction
4.1.4.4. Synthesis of 4-{3-(3-(hydroxymethyl)-4-methoxystyryl)-3,7-
dimethylocta-1,6-dienyl}phenol (26). Sodium borohydride (0.5 mmol)
reduction of compound 20 (1 mmol) in methanol at 0 ꢃC afforded
title compound on purification by CC over silica gel using hex-
ane:EtOAc (9:1) as the eluent in 97% yield. 1H NMR (500 MHz,
Bakuchiol dissolved in DMF (0.2 M) stirred at room temperature
(<25 ꢃC) and to the clear solution, substituted phenyl boronic acid
(1.2 equiv) followed by a single addition of Na2CO3 and Pd(OAc)2
catalyst. The reaction flask fitted with an oxygen balloon, heated up
to 100 ꢃC with stirring for 7 h. The contents diluted with diethyl
ether (20 mL), and washed with aqueous NaCl solution (2 ꢁ 25 mL).
The organic layer dried over Na2SO4 and filtered. The filtrate
concentrated under reduced pressure and the crude products
purified by CC over silica gel (60e120 mesh) with EtOAc:Hexane
mixture as the eluent to afford the appropriate compounds.
CDCl3):
d 1.29 (3H, s, eCeCH3), 1.56 (2H, m, eCH2CH2CH]), 1.59
and 1.67 (3H each, s, ]C(CH3)2), 1.99 (2H, m, eCH2CH2CH]), 3.82
(3H, s, AreOCH3), 4.69 (2H, s, ArCH2OH), 5.11 (1H, t,
J ¼ 6.9 Hz,eCH2CH]C), 6.12 (2H, d, J ¼ 16.2 Hz, 2ꢁ AreCH]CH),
6.28 (2H, d, J ¼ 16.2 Hz, 2ꢁ AreCH]CH), 6.75 (2H, d, J ¼ 8.4 Hz, 2ꢁ
AreH), 6.81 (1H, d, J ¼ 8.6 Hz, Ar0eH), 7.24 (3H, m, 2ꢁ AreH, Ar0eH),
7.32 (1H, s, Ar0eH). 13C NMR (125 MHz):
d 17.74, 23.43, 24.09, 25.75,
41.80, 42.12, 55.49, 62.26, 110.40, 115.50, 124.92, 126.42, 126.46,
126.74, 126.97, 127.38, 127.41, 128.7, 130.81, 131.35, 135.78, 136.66,
155.20, 156.67. IR: 757, 813, 970, 1033, 1107, 1251, 1375, 1461, 1503,
1512, 1609, 2853, 2961, 3332 cmꢀ1. MS at m/z 391 (Mþ ꢀ 1).
4.1.4.1. Synthesis of 4-{3-(4-fluorostyryl)-3,7-dimethylocta-1,6-die
nyl}phenol (23). The title compound prepared by the method as
described above by coupling of 4-fluorophenyl boronic acid and
compound 1 and purified by CC over silica gel using hexane:EtOAc
(9:1) as the eluent to afford 23 in 51% yield. 1H NMR (500 MHz,
4.1.4.5. Synthesis of 1-methoxy-[4-{3-(4-methoxystyryl)-3,7-dime-
thylocta-1,6-dienyl}] benzene (27). The title compound prepared by
coupling of compound 10 with 4-iodoanisole following the proce-
dure as described under Section 4.1.4.1 and purified by CC over
silica gel using hexane:EtOAc (9:1) as the eluent to afford 27 in 66%
CDCl3): d 1.30 (3H, s, eCeCH3), 1.58 (2H, m, ]CCH2CH2e), 1.59 and
1.68 (3H each, s, ]C(CH3)2), 2.00 (2H, m,eCH2CH2CH]), 5.13 (1H, t,
J ¼ 6.9 Hz, eCH]C), 6.13 and 6.18 (1H each, d, J ¼16.2 Hz, 2ꢁ
AreCH]CHe), 6.30 and 6.33 (1H each, d, J ¼ 16.2 Hz, 2ꢁ AreCH]
CH), 6.78 (2H, d, J ¼ 8.4 Hz, 2ꢁ AreH), 6.99 (2H, d, J ¼ 8.6 Hz, 2ꢁ
AreH), 7.26 (2H, d, J ¼ 8.4 Hz, 2x AreH), 7.33 (2H, d, J ¼ 8.6 HZ, 2ꢁ
yield. 1H NMR (400 MHz, CDCl3):
d 1.26 (3H, s, eCeCH3), 1.52 (2H,
m, eCH2CH2CH]), 1.54 and 1.67 (3H each, s, ]C(CH3)2), 1.97 (2H,
m, eCH2CH]), 3.83 (6H, s, 2ꢁ AreOCH3), 5.11 (1H, t, J ¼ 6.9 Hz,
eCH2CH]C), 6.13 (2H, d, J ¼ 16.2 Hz, AreCHeCH and Ar0eCHeCH),
6.30 (2H, d, J ¼ 16.2 Hz, AreCH]CH and Ar0eCH]CH), 6.85 (4H, d,
J ¼ 8.34 Hz, 2ꢁ AreH and 2ꢁ Ar0eH), 7.30 (4H, d, J ¼ 8.34 Hz, 2ꢁ
Ar0eH). 13C NMR (125 MHz):
d 18.40, 24.07, 24.65, 26.42, 42.41,
42.85, 115.74, 116.12, 125.45, 126.79, 127.46, 128.13, 128.20, 131.41,
132.16, 134.69, 136.39, 138.68, 155.51, 163.66. IR: 757, 1018, 1123,
1157, 1216, 1403, 1456, 1508, 1602, 2850, 2920, 2960, 3405 cmꢀ1. MS
at m/z 349 (Mþ ꢀ 1).
AreH and 2ꢁ Ar0eH). 13C NMR (100 MHz):
d 17.68, 23.39, 24.10,
25.71, 41.79, 42.07, 55.21, 113.95, 124.89, 126.64, 127.24, 130.73,
131.30, 136.11, 158.82. IR: 756, 814, 970, 1014, 1100, 1173, 1234, 1376,
1444, 1511, 1609, 2852, 2924 cmꢀ1. MS at m/z 375 (Mþ ꢀ 1).
4.1.4.2. Synthesis of 4-{3-(4-aminostyryl)-3,7-dimethylocta-1,6-
dienyl}phenol (24). The title compound prepared by the method
as described under Section 4.1.4.1 by coupling 4-iodobenzamine
and compound 1 and purified by CC over silica gel using hex-
ane:EtOAc (9:1) as the eluent to afford 24 in 51.7% yield. 1H NMR
4.1.4.6. Synthesis of 2,6-dibromo-4-(3-ethyl-3,7-dimethyloctyl)phe
nol (28). Bromine (0.5 mmol) was added to CCl4 solution (10 mL)
of 14 (hexahydro bakuchiol) (0.5 mmol), the contents stirred for
2 h, and the reaction monitored by TLC. The reaction mixture on
usual work up and purification by CC over silica gel using hex-
ane:EtOAc (19:1) as the eluent to afford 28 in 96% yield. 1H NMR
(200 MHz, CDCl3):
d 1.25 (3H, s, eCeCH3), 1.57 (2H, m,
eCH2CH2CH]),1.58 and 1.67 (3H each, s, ]C(CH3)2),1.99 (2H, m, ]
CHCH2), 5.13 (1H, t, J ¼ 6.9 Hz, eCH]C), 6.07 and 6.10 (1H each, d,
J ¼ 16.2 Hz, 2ꢁ AreCH]CH), 6.25 and 6.27 (1H, d, J ¼ 16.2 Hz, 2ꢁ
AreCH]CH), 6.64 (2H, d, J ¼ 8.6 Hz, 2ꢁ AreH), 6.75 (2H, d,
J ¼ 8.4 Hz, 2ꢁ Ar0eH), 7.19 (2H, d, J ¼ 8.4 Hz, 2ꢁ AreH) 7. 24 (2H, d,
(200 MHz, CDCl3):
d
0.80 (3H, t, J ¼ 7.5 Hz, CH3eCH2), 0.83 (6H, d,
J ¼ 6.5 Hz, (CH3)2eCH), 0.85 (3H, s, CH3eC), 1.05e1.45 (11H, m),
J ¼ 8.6 Hz, 2ꢁ Ar0eH). 13C NMR (125 MHz):
d 17.68, 23.40, 24.15,
2.34e2.43 (2H, m, PheCH2e), 7.24 (2H, br s, 2ꢁ AreH). 13C NMR
25.70, 41.85, 41.99, 115.48, 124.78, 124.96, 126.45, 126.53, 126.91,
127.20, 127.38, 129.75, 131.88, 132.65, 134.76, 136.27, 146.1, 154.85.
IR: 617, 655, 911, 972, 1014, 1110, 1195, 1369, 1412, 1452, 1505, 1603,
1633, 2855, 2922, 2967, 3033 cmꢀ1. MS at m/z 346 (Mþ ꢀ 1).
(125 MHz): d 8.04, 21.21, 22.73, 24.46, 27.94, 29.05, 31.43, 35.23,
39.07, 40.03, 41.22, 109.56, 131.70, 138.39, 147.22. IR: 735, 862, 1019,
1197, 1239, 1271, 1319, 1382, 1407, 1472, 1652, 2867, 2930, 2956,
3508 cmꢀ1. MS at m/z 413 (Mþ ꢀ 1).
4.1.4.3. Synthesis of 4-{-3-(3-chloro-4-propoxystyryl)-3,7-dimethy-
locta-1,6-dienyl}phenol (25). The title compound prepared and
purified by the method as described under Section 4.1.4.1 by
coupling of 3-chloro-4-propoxylphenyl boronic acid and
compound 1 and purified by CC over silica gel using hexane:EtOAc
(9:1) as the eluent to afford 25 in 49.6% yield. 1H NMR (200 MHz,
4.2. Biology
RPMI-1640 medium (#N 3520), rhodamine-123 (Rh-123) (#R
8004), propidium iodide (PI) (#P4170), proteinase-K, 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(#M 2128), penicillin (#P 3032), streptomycin (#S 9137), campto-
thecin (#C 911), fetal bovine serum (# 7524), mitomycin (#M 4287),
sodium bicarbonate (#S 5761), phosphate buffer saline (PBS) (#P
3831), sulphorhodamine (SRB) (#1402), trypsin (#T 4799), paclitaxel
CDCl3):
d
1.06 (3H, t, J ¼ 7.3 Hz, eCH2CH3), 1.28 (3H, s, eCeCH3), 1.57
(2H, m, CH2CH2CH]), 1.59 and 1.67 (3H each, s, ]C(CH3)2), 1.92
(4H, m, eCH2CH3, eCH2CH]), 3.95 (2H, t, J ¼ 6.5 Hz,eOCH2e), 5.07

66
R. Majeed et al. / European Journal of Medicinal Chemistry 49 (2012) 55e67
(#T 7402), 5 fluorouracil (5-FU) (#F 6627), doxorubicin (#D 1515),
gentamycin sulfate (#G 1264) were procured from SigmaeAldrich.
Tris buffer (#RM 262), electrophoresis reagents, bromophenol blue
(#RM 117) were procured from Himedia while Glacial acetic acid
(#21055) was purchased from Fisher Scientific. Trichloroacetic acid
was purchased from Merck Specialties Private Ltd.
for 24 h. After treatments cells were centrifuged at 1500 rpm for
10 min, and washed in PBS. The pellet was lysed in 250 L of lysis
buffer (100 mM NaCl, 5 mM EDTA, 10 mM TriseHCl, pH 8.0, 5%
Triton X-100) containing (200 g/mL) proteinase-K and incubated at
50 ꢃC for 1 h followed by 90 min incubation with 400
g/mL DNase-
free RNase. The DNA was extracted with 100 phenol:-
m
m
m
m
L
chloroform:isoamylalcohol (25:24:1) and centrifuged. DNA was
precipitated from aqueous phasewith 3 volumes of chilled alcohol and
0.3 M sodium acetate at 20 ꢃC overnight. The precipitate was centri-
fuged at 13,000 ꢁ g for 10 min. The DNA pellet was washed in 80%
4.2.1. Cell culture, growth conditions and treatment
Human promyelocytic leukemia cell line (HL-60), human
ovarian cancer cell line (OVCAR-5), human neuroblastoma cancer
cell line (IMR-32), were procured from National Centre for Cell
Sciences (NCCS), Pune, India. Human lung carcinoma cell line (A-
549) was obtained from National Cancer Institute, Frederick, USA.
Human prostate cancer cell line (PC-3) was obtained from National
Cancer Institute (NCI), Bethesda, USA. Cells were grown in RPMI-
1640/ MEM medium containing 10% FCS, 100 unit penicillin/
alcohol, dried, dissolved in 50 mL TE buffer, mixed in loading buffer and
electrophoresed in 1% agarose gel at 80 V for 1.5 h in TAE buffer [19].
4.2.5. DNA content and cell cycle phase distribution
HL-60 cells (2 ꢁ 106cells/well/2 mL) were seeded in 6 well plates
and treated for 24 h with 1 mM, 5 mM 10 mM, 20 mM and 30 mM conc.
100
m
g streptomycin per mL medium. Cells were grown in CO2
of compound 17 and compound 22. After 24 h cells were collected,
washed in PBS and fixed in 70% cold ethanol for 30 min. Cells were
incubator (Thermo Scientific, USA) at 37 ꢃC with 98% humidity and
5% CO2 gas environment. Cells were treated with different struc-
tural analogs of bakuchiol dissolved in DMSO while the untreated
control cultures received only the vehicle (DMSO, <0.2%).
again washed with PBS, subjected to RNase digestion (400
at 37 ꢃC for 45 min. Finally, cells were incubated with propidium
iodide (10 g/mL) [18]. Cells are then analyzed immediately on flow
mg/mL)
m
cytometer FACS Aria (Becton Dickinson, USA). The fluorescence
intensity of sub-G0 cell fraction represents the apoptotic cell pop-
ulation [20].
4.2.2. Sulpharhodamine B assay for % growth inhibition
The Sulpharhodamine B (SRB) assay was used to evaluate
inhibitory effect of different structural analogs of bakuchiol. The
assay relies on the ability of SRB to bind to protein components of
cells that have been fixed to tissue-culture plates by trichloroacetic
acid (TCA). SRB is a bright-pink aminoxanthene dye with two
sulfonic groups that bind to basic amino-acid residues under mild
acidic conditions, and dissociate under basic conditions. As the
binding of SRB is stoichiometric, the amount of dye extracted from
stained cells is directly proportional to the cell mass. To determine
the effect of different structural analogs of bakuchiol on cell
number over time, SRB assays were performed as described. Cells
were seeded in flat-bottomed 96-well plates. The cells were
allowed to adhere overnight, and then media containing samples
were added at different concentrations. The plates were incubated
4.2.6. Measurement of mitochondrial membrane potential for
cellular energy status
Changes in mitochondrial transmembrane potential (DJm) as
a result of mitochondrial perturbation were measured after stain-
ing with Rhodamine-123 [18,21]. HL-60 cells (1 ꢁ106/mL/well)
were grown in 6 well plates and treated with indicated concen-
tration of compounds 17 and 22. Rh-123 (5 mM) was added 1 h
before the termination of experiment. Cells were washed in PBS
and centrifuged at 1500 rpm for 5 min, suspended in PBS and
analyzed for loss in mitochondrial membrane potential on flow
cytometer.
for 48 h. The cells were fixed by adding 50
50% TCA to each well for 60 min. The plates were washed five
times in running tap water and stained with 100 L per well SRB
reagent (0.4% w/v SRB in 1% acetic acid for 30 min). The plates were
washed five times in 1% acetic acid to remove unbound SRB and
mL per well of ice-cold
Acknowledgements
m
The authors are thankful to Dr. SN Sharma (Taxonomist, IIIM,
Jammu) for providing the authentic plant material. Authors (MVR
and PKC) gratefully thank Council of Scientific and Industrial
Research (CSIR), New Delhi for the award of senior research
fellowships. Director IIIM is well acknowledged for encouragement
of the work.
allowed to dry overnight. SRB was solubilized with 100 mL per well
96-well plate 10 mM Tris-base, shaken for 5 min and the OD was
measured at 570 nm with reference wavelength of 620 nm [17].
Further the IC50 values on the cancer cells of different tissue origin
used for screening were determined by non-linear regression
analysis using Graph Pad Software (2236 Avenida de la Playa La
Jolla, CA 92037, USA).
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