Synthesis and evaluation of antiproliferative activity of a novel series of hydroxychavicol analogs

Article abstract of DOI:10.1016/j.ejmech.2014.01.016

We have recently demonstrated that hydroxychavicol is a major constituent and the most active biophenolic of Piper betel leaves with significant antiproliferative activity in the micro molar range. Herein we present the design, synthesis and evaluation of fifteen novel hydroxychavicol analogs with varying antiproliferative activities in cancer cell lines from two representative tissue types, namely, the prostate and cervix that show very encouraging results compared to the parent compounds. Our long range goal is to develop a structure-activity guided relationship to gain mechanistic insights into novel molecular targets of this class of bioactive molecules for rational drug development. Cytotoxicity-guided experimentation on these novel analogs yielded the following structural factors as the key activity regulators: 1) unlike the hydroxyl substituent at position-4, the position-3 hydroxyl is vital for enhanced activity 2) acetoxyl groups are dispensable for activity as corroborated earlier by others 3) allylic double bonds at 2′C-3′C serve to positively influence antiproliferative activity 4) long saturated side chains at 1′-position negatively regulate antiproliferative activity and 5) maneuvering position-4 with a benzyl group positively impacted the biological activity profile. Most amphiphilic compounds showed moderate to good therapeutic potential as expected on the basis of medicinal chemistry principles. Intriguingly, the most active compound with ten-fold higher activity than the parent molecule was realized by sheer serendipity to employ a silica gel based rearrangement that was further explored using nuclear magnetic resonance spectroscopy and density functional theory calculations. This is the first report to describe strategies for optimal synthesis of a novel series of 15 analogs based upon hydroxychavicol, a simple phytochemical of immense anticancer potential.

Full text of DOI:10.1016/j.ejmech.2014.01.016

European Journal of Medicinal Chemistry 75 (2014) 1e10
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of antiproliferative activity of a novel series
of hydroxychavicol analogs
,
c
,
,
c
,
d
,
a,c,d,
Yogesh Yadava, Eric A. Owens a d, Vibhuti Sharma b, Ritu Aneja b
*
, Maged Henary
*
a Department of Chemistry, Georgia State University, Petit Science Center, 100 Piedmont Avenue SE, Atlanta, GA 30303, USA
b Department of Biology, Georgia State University, Petit Science Center, 100 Piedmont Avenue SE, Atlanta, GA 30303, USA
c The Center for Diagnostics and Therapeutics, Georgia State University, Petit Science Center, 100 Piedmont Avenue SE, Atlanta, GA 30303, USA
d The Center for Biotechnology and Drug Design, Georgia State University, Petit Science Center, 100 Piedmont Avenue SE, Atlanta, GA 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently demonstrated that hydroxychavicol is a major constituent and the most active
biophenolic of Piper betel leaves with significant antiproliferative activity in the micro molar range.
Herein we present the design, synthesis and evaluation of fifteen novel hydroxychavicol analogs with
varying antiproliferative activities in cancer cell lines from two representative tissue types, namely,
the prostate and cervix that show very encouraging results compared to the parent compounds. Our
long range goal is to develop a structureeactivity guided relationship to gain mechanistic insights
into novel molecular targets of this class of bioactive molecules for rational drug development.
Cytotoxicity-guided experimentation on these novel analogs yielded the following structural factors
as the key activity regulators: 1) unlike the hydroxyl substituent at position-4, the position-3 hy-
droxyl is vital for enhanced activity 2) acetoxyl groups are dispensable for activity as corroborated
earlier by others 3) allylic double bonds at 20Ce30C serve to positively influence antiproliferative
activity 4) long saturated side chains at 10-position negatively regulate antiproliferative activity and
5) maneuvering position-4 with a benzyl group positively impacted the biological activity profile.
Most amphiphilic compounds showed moderate to good therapeutic potential as expected on the
basis of medicinal chemistry principles. Intriguingly, the most active compound with ten-fold higher
activity than the parent molecule was realized by sheer serendipity to employ a silica gel based
rearrangement that was further explored using nuclear magnetic resonance spectroscopy and density
functional theory calculations. This is the first report to describe strategies for optimal synthesis of a
novel series of 15 analogs based upon hydroxychavicol, a simple phytochemical of immense anti-
cancer potential.
Received 22 July 2013
Received in revised form
8 January 2014
Accepted 9 January 2014
Available online 21 January 2014
Keywords:
Hydroxychavicol analogs
Silica gel rearrangement
Antiproliferative
Anticancer
Density functional theory
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
phytochemical has produced the most successful anticancer drugs
such as paclitaxel and vincas.
Mother Nature remains the unchallenged source for new drug
discovery throughout the span of human history. Indeed the
dynamically-evolving diversity of bioactive compounds and novel
molecular chemo types in plants and plant-derived extracts is far
more superior and bypasses any human-made chemical library.
While plants and their extracts continue to persist as common
practice in traditional medicine for cancer treatment, isolation,
characterization and identification, of naturally-occurring bioactive
Since time immemorial, the deep green heart-shaped leaves of
Piper betel, an evergreen perennial vine, continue to be consumed
in Southeast Asian countries, highest being in India and Taiwan.
These “betel leaves” are either consumed alone as a mouth fresh-
ener or more commonly as betel quid, which consists of fresh betel
leaf, areca nut, slaked lime paste with or without tobacco. In
addition, the leaves of this plant have been employed in tropical
countries for the preparation of traditional herbal remedies [1,2]. P.
betel leaves present an excellent reservoir of phenolics with anti-
mutagenic, antitumor, antibacterial and antioxidant activities [3].
In particular, P. betel leaf extract has been shown to be active against
Chronic myelogenous leukemia (CML) cells and tumor xenografts
[4,5].
* Corresponding authors. The Center for Diagnostics and Therapeutics, Georgia
State University, Petit Science Center, 100 Piedmont Avenue SE, Atlanta, GA 30303,
USA.
E-mail addresses: raneja@gsu.edu (R. Aneja), mhenary1@gsu.edu (M. Henary).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.016

2
Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
structure alterations has not been a subject of investigation. Herein,
we report the synthesis in good to excellent yields (65e91%) and
evaluation of antiproliferative activity of 15 novel HC analogs. The
selected modifications to the aromatic core altered the steric fac-
tors, hydrogen bonding capabilities and the hydrophobicity of the
parent compound HC. Their activity was tested in human prostate
cancer (PC-3) and cervical (HeLa) cancer cells and the drug con-
centration required inhibiting the proliferation of 50% cells viz., IC50
was calculated. Intriguingly, the HC analog prepared via exploiting
an interesting silica gel catalyzed acyl transfer rearrangement
showed the highest activity against PC-3 cells. Our data lays the
foundation for the development of a SAR and will be helpful in
pharmacophore determination and identification of the yet un-
known targets of this class of compounds.
Hydroxychavicol (HC, 3,4-dihydroxyallylbenzene) and eugenol
(EU, 4-allyl-3-methoxyphenol) are important phytochemicals
found in betel leaves and endow them with several health benefits
[6e9]. Recently, our group reported that anticancer attributes of P.
betel leaf extract in both in vitro and in vivo prostate cancer models.
Employing our state-of-art analytical approaches, we identified,
characterized and purified
a number of medicinally-relevant
compounds including catechin, eugenol, chavibetol and hydrox-
ychavicol [1]. Interestingly, our study established HC as a major
contributor of the anti-prostate activity. Hydroxychavicol (HC) has
also been shown to exhibit anticancer activity upon its isolation
from a number of plant sources, including the P. betel leaf (Piper-
2. Results and discussion
2.1. Synthesis of novel hydroxychavicol analogs
The synthetic route to prepare the novel hydroxychavicol
aceae) [10e12]. Reports also indicate HC as
a
potent
derivatives is depicted in Scheme
1 and begins with the
cyclooxygenase-1/cyclooxygenase-2 (COX-1/COX-2) inhibitor, ROS
scavenger and a platelet aggregation inhibitor [13].
Although some sporadic studies report the synthesis of novel HC
analogs, a systematic evaluation of biological activity in response to
selective benzylation at the C-4-hydroxyl group of 3,4-
dihydroxybenzaldehyde which yields intermediate 2 [14]. The
precursor 2 was then subjected to two alternate synthetic routes;
the first begins with the protection of the C-3 hydroxyl group with
Scheme 1.

Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
3
tert-butyldimethylsilyl chloride (TBDMSCl) [15] followed by a
subsequent Grignard reaction using vinylmagnesium bromide [16].
Upon treatment of intermediate 4 with acetic anhydride, triethyl-
amine and 4-dimethylaminopyridine (DMAP), we observed the
acyl transfer product 5 after silica gel column chromatography,
which we decided to explore further and will be discussed more in
depth. After confirming the structure, we proceeded to cleave the
TBDMS using tetra-n-butylammonium fluoride (TBAF) [15] fol-
lowed by simultaneous benzyl group removal and alkene reduction
to afford the final diol 7. The second route for developing synthetic
analogs of compound 2 involves similar chemistry without first
protecting the C-3 hydroxyl group which allows the functionali-
zation with an acetyl group in the final product instead of the free
hydroxyl. The final compounds 8e14 were prepared using the
chemistry discussed above and the acyl transfer was observed in
the conversion of compound 11 to 14. Compounds 12 and 13 were
prepared from crude compound 11 to prevent the acyl transfer
from taking place during purification. Scheme 2 depicts the syn-
thetic route for other hydroxychavicol derivatives and starts using
the commercially obtained 3-methoxy derivative of HC (Eugenol,
EU). The synthesis started by benzylation of Eugenol to afford
compound 17 in excellent yield. The corresponding substituted
propane 1,2-diols 18 and 19 were obtained by dihydroxylation us-
ing OsO4 in presence of N-methylmorpholineN-oxide (NMO) [16].
The acetylated derivatives were also synthesized through the
effective reaction conditions that were previously reported in
Scheme 1. Further modifications, including O-allylation 22 and
incorporating the O-2,3-epoxypropane group 23, were achieved
after debenzylation under standard Pd/CeH2 conditions.
Importantly, the various synthetic steps, including debenzylation,
acetylation, deacetylation and desilylation gave us different de-
rivatives of hydroxychavicol which were then used to understand
the effect of acetate, hydroxyl and benzyl group to observe the
change in activities as anticancer agents. Before evaluating the
biological activity, we characterized each compound using 1H NMR,
13C NMR and DEPT experiments and further investigated the en-
ergetics and dynamics of the acyl transfer that was observed in two
of the final compounds 5 and 14.
2.2. NMR structural elucidations
As previously mentioned, we observed, using 1H NMR (Fig 1),
acyl migration to form compounds 5 and 14 during column
chromatography on silica gel (60e200 mesh). In order to ensure
the synthesis and confirm the structure of the compounds 5 and
14, we utilize COSY, NOESYand the DEPT NMR as shown in Fig. 2 of
compounds 11 and 14 which showed a shift in the
carbon (highlighted in the dashed blue circle) from
65.4, respectively. This clearly shows that the C-30 carbon of
compound 11 is a terminal alkene carbon and after acyl rear-
rangement the carbon chemical shift value becomes 64.4 which
d
value of C-30
117.0 to
d
d
d
suggests its aliphatic nature. The rearranged structure of com-
pounds was further confirmed by heteronuclear multipleequan-
tum correlation (HMQC, data not shown in Supplementary
information), not depicted here, which indicated the correlation
of the C-30 allylic protons with the carbon C-30 carbon
(Supplementary information).
Scheme 2.

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Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
Fig. 1. The 1H NMR for the different synthetic observations during the acyl transfer rearrangement of compound 11 to compound 14. A) The 1H NMR showing the presence of a
mixture of the two compounds that was obtained B) The 1H NMR confirming the isolation of the initial product 11 that was obtained after eluting the reaction mixture on non-acidic
alumina. C) The 1H NMR showing the rearranged product 14 after running a silica gel based column more slowly.
2.3. Proposed mechanism and DFT calculations
proceeding through an SN10 allylic pathway through a carbocation
intermediate which would remain non-dissociated on silica gel.
This intermediate would proceed to form the most energetically
favorable product after eluting from the column. Recently, Ray-
mond et al. [18] reported that the acidic nature of silica gel effi-
ciently promotes the rearrangement of allylic acetates into their
most stable regioisomers under microwave irradiation. Our syn-
thetic method for preparing the acyl-transferred compounds 5 and
14 is more efficient and avoids the use of microwave irradiation as
previously reported [18].
The mode of action of silica gel to catalyze the rearrangement is
unclear. The hypothesis of this rearrangement can be explained by
the susceptibility of the oxygen atom of the carbonyl group to
attack the allylic 30-carbon, which then initiates a double bond
shift and migration of acyl transfer from 10-carbon to 30-carbon
under the acidic silica gel conditions via a six-membered transi-
tion state (Scheme 3). This mechanism was earlier proposed by
Sukh Dev et al. [17]. They reported that this rearrangement is
Fig. 2. The 13C NMR to confirm the structure of the final products A) initial compound 11 and B) rearranged product 14.

Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
5
Scheme 3.
To confirm the acyl-transfer we performed density functional
2.4. Evaluation of antiproliferative activity in cancer cell lines
theory (DFT) calculations on the starting materials, both products
and the proposed carbocation intermediate. DFT and many other
computational methods are used to determine the barrier of re-
action and also the pathway of reaction. The structure and relative
free energy values for the starting material, expected product,
rearranged product and transition state structure were calculated
by using Gaussian at the B3LYP level (Fig. 3). As expected, the ab-
solute energy of the transition state is higher than the starting
compound and the expected product though the silica gel could
effectively stabilize this structure to lower the energy of activation.
We also observed that the absolute energy of the rearranged
product was lower than the energy of the initial product. This helps
account for ability of the rearrangement to provide the most
energetically stable product.
In addition to calculating the relative energetics of the observed
acyl transfer, the physicochemical properties of all synthesized
compounds were calculated (Table 1). The lipophilicity of com-
pounds has an important effect on their biological activity. The
prediction of lipophilicity and other physicochemical properties
such as log D, molecular weight, topological polar surface area
(TPSA), hydrogen bond acceptors, hydrogen bond donors and
rotatable bonds for compound 6e23 was calculated using Marvin
Sketch and JChem (ChemAxon, Budapest, Hungary) software in an
attempt to correlate physicochemical properties of the compounds
with their anticancer activity.
We next evaluated the antiproliferative activity of these HC
analogs by performing the MTT assay on prostate cancer PC-3 cells
and cervical cancer HeLa cells. Fig. 4(A and B) displays the analogs
that were most potent and exhibited IC50 below 100
cells. HC analogs 6, 7, 10, 12, 14 and HC showed IC50 values of 3.5,
83, 42, 48, 38 and 55 M, respectively. Fig. 4B is a bar graph of the
mM in PC-3
m
IC50 values with the standard deviation calculated between
batches.
HC analogs that were active in PC-3 cells also exhibited similar
activity in HeLa cells. Fig. 4C displays IC50 value less than 100
HeLa cells. HC analogs 6, 7, 8, 10, 12, 14, 21, 22 and HC displayed an
IC50 value of 13, 22, 76, 53, 40, 42, 60, 96 and 83 M correspond-
mM in
m
ingly. Fig. 4D displays the IC50 values in a bar graph format with the
standard deviation observed between batches.
From the in vitro cell proliferation data, we can gather that the
methyl ether derivatives at position 3 of hydroxychavicol are less
active when compared to both the hydroxyl and acylated analogs
suggesting that this methyl structural modification compromises
the biological activity. Along similar lines, we have seen that the
hydroxyl group at position 3 significantly improves the activity as
shown by a comparison of compounds 6 and 14. Other molecular
modifications, including benzylation and reduction, altered the
biological response but not to the same extent. We have noticed
that the more hydrophilic compounds with lower distribution co-
efficients (logD) did not show appealing anticancer profiles using
IC50 values. The compounds’ relative efficacy cannot be entirely
correlated to log D but the most active compounds have log D
values ranging between approximately 2 and 4 with compounds
displaying lower log D having lower potency.
3. Experimental
3.1. Synthesis
The chemical reagents used in the synthesis of these compounds
were obtained from Acros Organics, Alfa Aesar and Matrix Scien-
tific. The reactions were followed using silica gel 60 F254 thin layer
chromatography plates (Merck EMD Millipore, Darmstadt, Ger-
many). Open column chromatography was utilized for the purifi-
cation of all final compounds using 60e200 u, 60 A classic column
silica gel (Dynamic Adsorbents, Norcross, GA). Melting points were
determined on a MEL-TEMP II capillary tube apparatus and were
uncorrected. The 1H NMR and 13C NMR spectra were obtained using
high quality kontes NMR tubes (Kimble Chase, Vineland, NJ) rated
to 500 MHz and were recorded on a BrukerAvance (400 MHz)
spectrometer using CDCl3, DMSO-d6 or MeOD-d4 containing tetra-
methylsilane (TMS) as an internal calibration standard. Chemical
shift data for the proton resonances were reported in parts per
million (ppm, scale) relative to internal standard TMS (0.0), and
Fig. 3. Reaction energy diagram showing the relative energetic stabilities of the initial
product and rearranged product with the proposed silica gel stabilized acyl transfer
intermediate. The energies were calculated using Gaussian 03W and show that the
rearranged product is more stable which helps facilitate the acyl transfer.

6
Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
Table 1
The physicochemical properties of the prepared compounds and the parent compounds that have been isolated and examined for their anticancer profile. These descriptors
where calculated using ChemAxon MarvinSketch and JChem plugins (Budapest, Hungary).
No
Distribution
coefficient
(Log D at pH ¼ 7.4)
TPSA
H-bond
acceptors
H-
bond
donors
Rotatable
bonds
R1
R2
R3
EU
HC
6
3.70
1.77
55.76
3
3
1
2
7
5
7
66.76
49.69
8
3.26
3
2
5
9
1.53
1.80
60.69
78.90
3
3
3
0
2
8
10
12
13
2.54
4.30
72.83
52.60
3
3
1
0
6
9
14
16
3.61
0.08
61.83
93.06
3
4
0
2
9
7
18
19
20
21
2.27
0.40
3.16
1.28
58.92
69.92
71.06
82.06
4
4
4
4
2
3
0
1
7
4
11
8
22
23
2.16
1.04
71.06
4
5
0
0
11
12
100.66
coupling constants (J) were reported in hertz (Hz). All moisture-
and/or air-sensitive reactions were performed under a maintained
positive pressure of nitrogen.
4-(Benzyloxy)-3-hydroxybenzaldehyde (2): this compound
was synthesized according to procedure reported earlier and used
as starting material for the synthesis of desired molecules [14].
4-(Benzyloxy)-3-{(tert-butyldimethylsilyl)oxy}benzaldehyde
(3): this compound was synthesized according to procedure re-
ported earlier [15].
1-[4-(Benzyloxy)-3-{(tert-butyldimethylsilyl)oxy}phenyl]prop
-2-en-1-ol (4): an oven-dried three-necked flask was charged with
compound 3 (1.0 g) in 25 mL of dry THFand vinylmagnesium bromide
(1.0 M inTHF) was added drop wise under N2 atmosphereover30min.
The corresponding white suspension was stirred at 0 ꢀC for 1 h and
was then left to stir for an additional for 4e5 h at room temperature.
The reaction was quenched with slow addition of saturated aqueous
solution of NH4Cl at 0 ꢀC. The organic layer was collected and the
aqueous layer was extracted with EtOAc. The combined extracts were

Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
7
Fig. 4. PC-3 cancer cells were treated for 48 h with increasing concentration of HC and its analogs, to measure the percentage cell proliferation using MTT assay. A. Line graph
represents percent cell survival in PC-3 cells upon treatment with HC and its analogs 6, 7, 10, 12 and 14. B. Bar graph represents the IC50 M) values in PC-3 cells upon treatment
(
m
with HC analogs C. Line graph represents percent cell survival in HeLa cells upon treatment with HC and its analogs 6, 7, 8, 10, 12, 14, 21 and 22. D. Bar graph represents the IC50 (mM)
values of all the HC analogs in HeLa cells.
washed with water, brine, dried over MgSO4 and concentrated under
reduced pressure. The residue was subjected to purification on silica
gel (EtOAc/hexane30/70)to givethe desired product4 in 79% yield.1H
solution of TBAF in THF (3.0 mmol) slowly at 0 ꢀC under nitrogen
atmosphere. After stirring at room temperature for 2 h, the re-
action mixture was poured into ice-water and extracted with
EtOAc. The combined organic layer was washed with brine,
saturated solution NaHCO3 and dried over anhydrous Na2SO4. The
solvent was evaporated in vacuo, and the crude product was
purified by column chromatography to obtain compound 6 in 82%
yield. IR (cmꢂ1) 3218 (OeH), 1707 (C]O), 1509 (C]C), 1259 (e
NMR (400 MHz, CDCl3)
d
0.15 (s, 6H, 2ꢁ CH3),1.00 (s, 9H, 3ꢁ CH3),1.95
(brs,1H, OH), 5.07 (s, 2H, CH2), 5.12 (brs,1H, CH), 5.20 (d, Jcis ¼ 10.4 Hz,
1H, CH2), 5.33 (d, Jtrans ¼ 17.2 Hz, 1H, CH2), 6.05 (ddt, J ¼ 16.4, 4.4,
4.4 Hz, 1H, CH), 6.91e6.92 (m, 3H, AreH), 7.32e7.41 (m, 3H, AreH),
7.45e7.47 (m, 2H, AreH). 13C NMR (100 MHz)
d
ꢂ4.5, 18.3, 25.9, 70.8,
74.8, 113.9, 114.8, 119.4, 119.5, 127.7, 127.9, 128.4, 135.8, 137.0, 140.3,
145.4, 149.8.
CH2), 1000 (CeO), 744 (CeH), 1H NMR (400 MHz, CDCl3)
d 2.11 (s,
3H, CH3), 4.71 (d, J ¼ 6.4 Hz, 2H, CH2), 5.69 (brs, 1H, OH), 6.13e
6.18 (m, 1H, CH), 6.57 (d, J ¼ 16.0 Hz, 1H, AreH), 6.88 (s, 2H, Are
H), 7.06 (s, 1H, AreH), 7.38e7.43 (m, 5H, AreH) 13C NMR
(E)-3-[4-(Benzyloxy)-3-{(tert-butyldimethylsilyl)oxy}phenyl]
allyl acetate (5): triethylamine (3 mmol) was added to a stirring
solution of 4 (1 mmol) and 4-(dimethylamino)pyridine (DMAP)
(0.1 mmol) in CH2Cl2 (20 mL) at 0 ꢀC under nitrogen atmosphere
and followed by acetic anhydride (1.2 mmol). After stirring at 0 ꢀC
for 1 h and at room temperature for 4e5 h, the reaction mixture
was quenched with water. The organic layer was washed with
water, saturated solution NaHCO3 and brine and dried over anhy-
drous MgSO4. The solvent was evaporated in vacuo, and the crude
product was purified by column chromatography on silica gel (3:7
EtOAc/Hexane) to give 5 in 87% yield. 1H NMR (400 MHz, CDCl3)
(100 MHz, CDCl3)
d 21.0, 65.2, 71.2, 112.1, 112.4, 119.2, 121.6, 127.8,
128.8, 128.8, 130.3, 134.0, 136.2, 145.9, 146.0, 170.9. HRMS (ESI)
Calculated for C18H18O4 (M þ Na)þ 321.1097, found (M þ Na)þ
321.1093.
3-(3,4-Dihydroxyphenyl)propyl acetate (7): compound 6 (1.0
equiv) was dissolved in methanol (5 mL) at room temperature.
Palladium on carbon 5% (0.15 equiv) was added under inert con-
dition and applies hydrogen source. The reaction mixture was
monitored using TLC. After completion the hydrogen source was
removed and the reaction mixture was filtered through celite and
concentrated. The crude material was purified by flash column
chromatography to obtain compound 7 in 87% yield. IR (cmꢂ1) 3365
(OeH), 1704 (C]O), 1253 (-CH2), 1036 (CeO), 811 (-C-H); 1H NMR
d
0.13 (s, 6H, 2ꢁ CH3), 0.99 (s, 9H, 3ꢁ CH3), 2.12 (s, 3H, CH3), 4.73 (d,
J ¼ 6.4 Hz, 2H, CH2), 5.06 (s, 1H, CH2), 6.10e6.17 (m, 1H, CH), 6.56 (d,
J ¼ 16.0 Hz, 1H, CH), 6.85e6.90 (m, 1H, AreH), 6.92e6.96 (m, 2H,
AreH), 7.28e7.41 (m, 3H, AreH), 7.44e7.46 (m, 2H, AreH), 13C NMR
(100 MHz, CDCl3)
d
ꢂ4.5, 18.3, 21.0, 25.7, 65.3, 70.7, 113.7, 119.0,
(400 MHz, CDCl3) d 1.86e1.91 (m, 2H, CH2), 2.06 (s, 3H, CH3), 2.55 (t,
120.6,121.0,127.8,127.9,128.4,129.7,134.1,136.8,145.3,150.4,170.8.
(E)-3-{4-(Benzyloxy)-3-hydroxyphenyl)allyl acetate (6): to a
stirred solution of 5 (1.0 mmol) in THF (10 mL) was added 1.0 M
J ¼ 7.6 Hz, 2H, CH2), 4.07 (t, J ¼ 6.8 Hz, 2H, CH2), 2.55 (brs, 2H, OH),
6.58 (d, J ¼ 8.0 Hz, 1H, AreH), 6.69 (s, 1H, AreH), 6.77 (d, 1H,
J ¼ 8.0 Hz, AreH) 13C NMR (100 MHz, CDCl3)
d 21.1, 30.1, 31.4, 64.2,

8
Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
115.3, 115.5, 120.6, 134.0, 141.9, 143.7, 172.2. HRMS (ESI) Calculated
for C11H13O4 (M ꢂ H)ꢂ 209.0819, found (M ꢂ H)ꢂ 209.0814.
2-(Benzyloxy)-5-(1-hydroxyallyl)phenol (8): this compound
was synthesized from compound 2 by the same procedure as
described for compound number 4. The crude product was purifi-
cation on silica gel (EtOAc/hexane 40/60) to obtained desired
product 8 in 79% yield. IR (cmꢂ1) 3538 (OeH), 3250 (OeH), 1247 (e
1-(3-Acetoxy-4-(benzyloxy)phenyl}propyl acetate (13): the
compound 13 was isolated with compound 12 in the same reaction
in 41% yield. IR (cmꢂ1) 1765 (C]O), 1731 (C]O), 1509 (C]C), 1232
(eCH2), 1011 (CeO), 735 (CeH), 1H NMR (400 MHz, CDCl3)
d 0.90 (t,
J ¼ 7.2 Hz, 3H, CH3), 1.76e1.87 (m, 1H, CH2), 1.89e1.96 (m, 1H, CH2),
2.08 (s, 3H, CH3), 2.29 (s, 3H, CH3), 5.12 (s, 2H, CH2), 5.65 (t,
J ¼ 6.8 Hz, 1H, CH), 6.98 (d, J ¼ 8.8 Hz, 1H, AreH), 7.09 (s, 1H, AreH),
7.16 (d, J ¼ 8.4 Hz, 1H, AreH), 7.34e7.41 (m, 5H, AreH), 13C NMR
CH2), 805 (eCeH) 1H NMR (400 MHz, CDCl3)
d 2.04 (s, 1H, OH), 5.09
(s, 2H, CH2), 5.17 (d, Jcis ¼ 10.0 Hz, 1H, CH2), 5.32 (d, Jtrans ¼ 17.2 Hz,
1H, CH2), 5.85 (brs, 1H, OH), 6.01 (ddt, J ¼ 16.4, 4.4, 4.4 Hz, 1H, CH),
6.84 (d, J ¼ 8.4 Hz, 1H, AreH), 6.89 (d, J ¼ 8.4 Hz, 1H, AreH), 6.97 (s,
1H, AreH), 7.36e7.41 (m, 5H, AreH), 13C NMR (100 MHz, CDCl3)
(100 MHz, CDCl3) d 9.9, 20.6, 21.2, 29.0, 113.6, 121.2, 125.3, 127.1,
127.2, 127.9, 128.5,133.5, 136.6, 140.1, 149.8, 168.9, 170.3. HRMS (ESI)
Calculated for C20H22O5 (M þ NH4)þ 360.1805, found (M þ NH4)þ
360.1800.
d
71.2, 74.9, 112.2, 113.1, 114.9, 118.0, 127.8, 128.5, 128.8, 136.3, 136.5,
3-{3-Acetoxy-4-(benzyloxy)phenyl}allyl acetate (14): the
compound 14 was the silica gel mediated rearranged product of 11
during column chromatography from compound 8. 1H NMR
140.2, 145.4, 146.0. HRMS (ESI) Calculated for C16H15O3 (M ꢂ H)ꢂ
255.1016, found (M ꢂ H)ꢂ 255.1018.
4-(1-Hydroxypropyl)benzene-1,2-diol (9): this compound was
synthesized from 8 by the same procedure as described for com-
pound number 7. The crude product was purification on silica gel
(EtOAc/hexane 40/60) to obtained desired product 9 in 88% yield. IR
(cmꢂ1) 3437 (OeH), 3065 (OeH),1275 (eCH2), 810 (eCeH) 1H NMR
(400 MHz, CDCl3) d 2.11 (s, 3H, CH3), 2.30 (s, 3H, CH3), 4.71 (d,
J ¼ 6.8 Hz, 2H, CH2), 5.11 (s, 2H, CH2), 6.12e6.20 (m, 1H, CH), 6.57 (d,
J ¼ 16.0 Hz, 1H, CH), 6.96 (d, J ¼ 8.4 Hz, 1H, AreH), 7.16e7.20 (m, 2H,
AreH), 7.39e7.40 (m, 5H, AreH), 13C NMR (100 MHz, CDCl3)
d 20.6,
21.0, 65.0, 70.6, 113.8, 120.6, 122.2, 125.4, 127.1, 128.0, 128.7, 129.8,
133.0, 136.5, 140.3, 150.1, 169.0, 170.8.
(400 MHz, DMSO-d6)
d
0.77 (t, J ¼ 6.8 Hz, 3H, CH3), 1.46e1.57 (m,
2H, CH2), 4.21e4.22 (m, 1H, CH), 4.77e4.80 (m, 1H, OH), 6.51 (d,
J ¼ 8.0 Hz, 1H, AreH), 6.63 (d, J ¼ 8.0 Hz, 1H, AreH), 6.70 (s, 1H, Are
H), 8.61e8.70 (m, 2H, OH), 13C NMR (100 MHz, DMSO-d6) 10.7, 32.4,
74.0,113.8, 115.3,117.2, 137.6, 144.2, 145.2. HRMS (ESI) Calculated for
C9H11O3 (M ꢂ H)- 167.0714, found (M ꢂ H)ꢂ 167.0714.
4-Allyl-1,2-phenylene diacetate (15): this compound was
synthesized from compound 9 by the same procedure as described
for compound number 5. The crude product was purification on
silica gel (EtOAc/hexane 10/90) to obtained desired product 15 in
89% yield. 1H NMR (400 MHz, CDCl3)
d 2.29 (s, 6H, COCH3), 3.40 (d,
4-(1-Acetoxypropyl)-1,2-phenylene diacetate (10): this com-
pound was synthesized from compound 9 by the same procedure
as described for compound number 5. The crude product was pu-
rification on silica gel (EtOAc/hexane 40/60) to obtained desired
product 10 in 91% yield. IR (cmꢂ1) 1769 (C]O),1732 (C]O),1200 (e
J ¼ 6.8 Hz, 2H, CH2), 5.10e5.14 (m, 2H, CH2), 5.90e6.00 (m, 1H, CH),
7.03 (s, 1H, AreH), 7.07e7.13 (m, 2H, AreH), 13C NMR (100 MHz,
CDCl3)
d 20.6, 39.4, 116.6, 123.1, 123.4, 126.6, 136.4, 138.9, 140.3,
141.9, 168.3, 168.4.
4-(2,3-Dihydroxypropyl)-1,2-phenylene diacetate (16): com-
pound 15 (3.24 mmol) was added to a mixture of acetone-water
(27 mL, 8:2) followed by the successive addition of NMO
(3.6 mmol) and OsO4 (8.76 mol, 4% solution in H2O). The mixture
was stirred at room temperature for 24 h. TLC indicated total
consumption of the starting material. An aqueous solution of
Na2SO3 (10 mL) was added to the reaction mixture and the mixture
was stirred for 30 min at room temperature. Then, the solvent was
evaporated in vacuo and the residue was extracted with EtOAc
(3 ꢁ 50 mL). The extract was washed with brine (3 ꢁ 25 mL). The
organic layer was dried over MgSO4 and concentration under
reduced pressure, furnished a residue which was subsequently
purified by silica gel chromatography (EtOAc/hexane 40/60) to
afford compound 16 in 77% yield. IR (cmꢂ1) 3391 (OeH), 1761 (C]
O), 1505 (C]C), 1207 (eCH2), 1012 (CeO), 824 (CeH), 1H NMR
CH2), 1011 (CeO), 897 (CeH), 1H NMR (400 MHz, CDCl3)
d 0.89 (t,
J ¼ 7.2 Hz, 3H, CH3), 1.76e1.86 (m, 1H, CH2), 1.88e1.93 (m, 1H, CH2),
2.07 (s, 3H, CH3), 2.28 (s, 6H, CH3), 5.67 (t, J ¼ 6.8 Hz, 1H, CH), 7.14e
7.22 (m, 3H, AreH), 13C NMR (100 MHz, CDCl3)
d 9.8, 20.6, 20.6, 21.2,
29.2, 76.1, 121.7, 123.3, 124.8, 139.3, 141.5, 142.0, 168.1, 168.2, 170.2.
HRMS (ESI) Calculated for C15H18O6 (M þ Na)þ 317.0996, found
(M þ Na)þ 317.0991.
(E)-3-[3-(Acetoxy)-4-(benzyloxy)phenyl]allyl acetate (11):
this compound was synthesized from compound 9 by the same
procedure as described for compound number 5. The crude product
was purification on silica gel (EtOAc/hexane 40/60) to obtained
desired product 11 in 81% yield. IR (cmꢂ1) 1763 (C]O), 1743 (C]O),
1508 (C]C), 1218 (-CH2), 1012 (CeO), 735 (CeH), 1H NMR
(400 MHz, CDCl3)
d 2.11 (s, 3H, CH3), 2.29 (s, 3H, CH3), 5.11 (s, 2H,
CH2), 5.26e5.34 (m, 2H, CH2), 5.99 (ddt, J ¼ 16.4, 4.8, 4.8 Hz,1H, CH),
(400 MHz, CDCl3) d 1.62 (brs, 2H, OH), 2.30 (s, 6H, COCH3), 2.71e
6.24 (d, J ¼ 5.2 Hz, 1H, CH), 6.99 (d, J ¼ 8.4 Hz, 1H, AreH), 7.11 (s, 2H,
2.81 (m, 2H, CH2), 3.49e3.52 (m, 1H, CH2a), 3.67e3.70 (m, 1H,
AreH), 7.19 (d, J ¼ 8.4 Hz, 1H, AreH), 7.34e7.40 (m, 5H, AreH), 13
C
CH2b), 3.91 (brs, 1H, CH), 7.08e7.13 (m, 3H, AreH), 13C NMR
NMR (100 MHz)
d
20.6, 21.2, 70.7, 75.2, 113.7, 116.9, 122.0, 125.9,
(100 MHz, CDCl3) d 20.6, 38.9, 65.7, 67.6, 72.7, 123.3, 124.3, 127.7,
127.1, 128.01, 128.5, 131.8, 135.9, 136.5, 140.1, 150.1, 168.9, 169.9.
HRMS (ESI) Calculated for C20H20O5 (M þ Na)þ 363.1203, found
(M þ Na)þ 363.1196.
137.4, 140.4, 141.7, 168.7, 168.8. HRMS (ESI) Calculated for C13H16O6
(M þ H)þ 269.1020, found (M þ H)þ 269.1016.
4-Allyl-1-(benzyloxy)-2-methoxybenzene (17): the compound
was synthesized according to procedure reported earlier and used
as starting material for the synthesis of desired molecules [16].
3-{4-(Benzyloxy)-3-methoxyphenyl}propane-1,2-diol (18):
the compound was synthesized according to procedure reported
earlier and used as starting material for the synthesis of desired
molecules [16].
3-(4-Hydroxy-3-methoxyphenyl)propane-1,2-diol (19): this
compound was synthesized from 18 by the same procedure as
described for compound number 7. The crude product was puri-
fication on silica gel (MeOH/DCM 5/95) to obtained desired
product 19 in 73% yield. IR (cmꢂ1) 3337 (OeH), 1515 (C]C), 1270
(-CH2), 1025 (CeO), 795 (CeH), 1H NMR (400 MHz, DMSO-d6)
1-(3-Acetoxy-4-hydroxyphenyl)propyl acetate (12): this
compound was synthesized from the crude compound 11 to avoid
the acyl transfer taking place during purification by the same
procedure described for compound number 7. The reaction mixture
was purification on silica gel (EtOAc/hexane30/70) to obtained
desired product 12 in 38% yield. IR (cmꢂ1) 3374 (OeH), 1738 (C]O),
1515 (C]C), 1230 (eCH2), 1111 (CeO), 818 (CeH), 1H NMR
(400 MHz, CDCl3) d 0.85e0.90 (m, 3H, CH3), 1.74e1.85 (m, 1H, CH2),
1.87e1.94 (m, 1H, CH2), 2.08 (d, J ¼ 6.8 Hz, 3H, CH3), 2.31 (s, 3H,
CH3), 5.95 (t, J ¼ 6.0 Hz, 1H, CH), 6.34 (brs, 1H, OH), 6.85e6.90 (m,
1H, AreH), 6.93 (s, 1H, AreH), 6.02e7.07 (m, 1H, AreH), 13C NMR
(100 MHz, CDCl3)
d 9.9, 20.9, 21.2, 29.1, 76.7,115.9,117.5,118.8,120.9,
122.4, 125.4, 169.5, 170.8.
d
2.40e2.50 (m, 1H, CH2), 2.61e2.66 (m, 1H, CH2), 3.27 (t,

Y. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 1e10
9
J ¼ 8.0 Hz, 2H, CH2), 3.55e3.62 (m, 1H, CH), 3.73(m, 3H, OCH3),
3.2. DFT calculations
4.48e4.53 (m, 2H, OH), 6.58 (d, J ¼ 8.0 Hz,1H, AreH), 6.65 (d,
J ¼ 8.0 Hz,1H, AreH), 6.76 (s, 1H, AreH), 8.64 (s, 1H, OH), 13C NMR
In order to perform the energy level calculations, the chemical
structures were prepared in GaussView 4.1.2 and the structures
were then minimized to the conformer of the lowest energy. En-
ergy values were calculated in Gaussian 03W Version 6.1 using the
B3LYP basis set.
(100 MHz, DMSO-d6)
d 31.1, 55.9, 65.7, 73.1, 113.9, 115.5, 121.9,
130.8, 144.9, 147.5. HRMS (ESI) Calculated for C10H14O4 (M ꢂ H)ꢂ
197.0819, found (M ꢂ H)- 197.0814.
3-{4-(Benzyloxy)-3-methoxyphenyl}propane-1,2-diyldiacetate
(20): this compound was synthesized from 18 by the same procedure
as described for compound number 5. The crude product was puri-
fication on silica gel (EtOAc/Hexane 20/80) to obtained desired
product 20 in 86% yield. IR (cmꢂ1) 1736 (C]O), 1513 (C]C), 1219 (e
3.3. Cell culture and drug treatment
RPMI and DMEM culture were supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin for PC-3
(prostate) cancer cells and MIA PaCa-2 (pancreatic) respectively.
All analogs, stocks and dilutions were dissolved in sterile 100%
DMSO.
CH2), 1034 (CeO), 730 (CeH), 1H NMR (400 MHz, CDCl3)
d 2.00 (s, 3H,
CH3), 2.04 (s, 3H, CH3), 2.76e2.88 (m, 2H, CH2), 3.84 (s, 3H, OCH3),
3.99e4.03 (m, 1H, CH2a), 4.20e4.24 (m, 1H, CH2b), 5.09 (s, 2H, CH2),
5.22e5.25 (m, 1H, CH), 6.66 (d, 1H, J ¼ 8.0 Hz, AreH), 6.74 (s, 1H, Are
H), 6.79 (d, 1H, J ¼ 8.0 Hz, AreH), 7.26e7.42 (m, 5H, AreH), 13C NMR
(100 MHz, CDCl3) d 20.7, 21.0, 36.6, 55.9,U 64.2, 71.0, 72.1, 113.0,114.2,
3.4. Cell proliferation assay
121.3, 127.3, 127.7, 128.4, 129.4, 137.2, 147.1, 149.6, 170.2, 170.5. HRMS
(ESI) Calculated for
C
21H24O6 (M
þ
NH4)þ 390.1911, found
To measure the percent cell proliferation of cells in vitro, MTT 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(M þ NH4)þ 390.1906.
3-(4-Hydroxy-3-methoxyphenyl)propane-1,2-diyl diacetate
(21): this compound was synthesized from 20 by the same proce-
dure as described for compound number 7. The crude product was
purification on silica gel (EtOAc/Hexane 25/75) to obtained desired
product 21 in 88% yield. IR (cmꢂ1) 3436 (OeH), 1732 (C]O), 1515
(C]C), 1220 (eCH2), 1032 (CeO), 796 (CeH), 1H NMR (400 MHz,
was used. A 96 well plate was seeded with 100
ml of medium
containing 2 ꢁ 103 cells in density and incubated for 24 h. The plate
was treated with increasing concentration made in medium of 0,
0.01, 0.1, 1, 10, 25, 50, 75, 100 and 250
DMSO. After 48 h incubation with the drug, medium was removed
and fresh 90 L of medium carrying 10 L (5 mg/mL in PBS) of MTT
mM of HC analogs dissolved in
m
m
CDCl3) d 2.02 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.76e2.88 (m, 2H, CH2),
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
dye was added to each well. The plates were again incubated for 4 h
followed by addition of 100 mL of 100% DMSO. The plates were then
scanned at 570 nm using a Spectra Max Plus multi-well plate reader
(Molecular Devices, USA).
3.84 (s, 3H, OCH3), 3.99-4.04 (m, 1H, CH2a), 4.21e4.25 (m, 1H, CH2b),
5.21e5.24 (m, 1H, CH), 5.83 (brs, 1H, OH), 6.66e6.69 (m, 2H, AreH),
6.80e6.82 (m, 1H, AreH), 13C NMR (100 MHz, CDCl3)
d 20.7, 21.0,
36.6, 55.8, 64.2, 72.3, 111.7, 114.4, 122.1, 128.0, 144.5, 146.6, 170.4,
170.7. HRMS (ESI) Calculated for C14H18O6 (M ꢂ H)- 281.1020, found
(M ꢂ H)- 281.1021.
4. Conclusions
3-{4-(Allyloxy)-3-methoxyphenyl}propane-1,2-diyl diacetate
(22): a solution of 21 (1.0 mmol) and NaH (1.0 mmol) in DMF
(10 mL) was stirred at ambient temp for 15 min. Then allyl bromide
(1.2 mmol) was added and the reaction mixture was stirred at 25 ꢀC
for another 4e5 h. Ethyl acetate was added to the reaction mixture
and the organic layer was washed with H2O, and saturated NaCl,
dried over MgSO4, filtered, and concentrated under reduced pres-
sure. Purification by flash column chromatography (silica gel, 30/
70% EtOAc/hexanes) yielded 71% of the desired compound as
viscous oil. IR (cmꢂ1) 1735 (C]O), 1513 (C]C), 1218 (-CH2), 1035
The hydroxychavicol analogs that were synthetically prepared
in good yield and biologically evaluated have provided valuable
directions for further structural modifications through SAR corre-
lation studies. The active compound exploited an interesting acyl
transfer mechanism on silica gel during column purification; the
structure was determined using 13C DEPT and the energetics of the
reaction showed increased stability of the final rearranged product
compared to the initially obtained product.
(CeO), 804 (CeH), 1H NMR (400 MHz, CDCl3)
d 2.00e2.08 (m, 6H,
Acknowledgments
COCH3), 2.75e2.89 (m, 2H, CH2), 3.82 (s, 3H, OCH3), 3.97e4.05 (m,
1H, CH2a), 4.19e4.25 (m, 1H, CH2b), 4.54e4.59 (m, 2H, OCH2), 5.22e
5.29 (m, 2H, CH2), 5.34e5.42 (m, 1H, CH), 6.00e6.10 (m, 1H, CH),
6.66e6.73 (m, 2H, AreH), 6.76e6.81 (m, 1H, AreH), 13C NMR
Synthetic work was supported by the Georgia Research Alliance
grant to MH. EAO was supported through the Center for Diagnostics
and Therapeutics (CDT). The biological work was supported by
grants to RA from the National Cancer Institute at the National
Institutes of Health. The authors would also like to thank the NMR
director, Dr. Zhenming Du, at Georgia State University for help
acquiring the 2D NMR data.
(100 MHz, CDCl3) d 20.7, 21.0, 36.5, 55.8, 64.2, 69.8, 72.1, 112.7, 113.5,
117.8, 121.3, 129.1, 133.3, 146.8, 149.3, 170.2, 170.6. HRMS (ESI)
Calculated for C17H22O6 (M þ Na)þ 345.1309, found (M þ Na)þ
345.1301.
3-{3-Methoxy-4-(oxiran-2-ylmethoxy)phenyl}propane-1,2-
diyl diacetate (23): this compound was synthesized from 20 by
the same procedure as described for compound number 21. The
crude product was purification on silica gel (EtOAc/Hexane 25/75)
to obtained desired product 23 in 65% yield. IR (cmꢂ1) 1733 (C]
O), 1514 (C]C), 1220 (eCH2), 1030 (CeO), 734 (CeH), 1H NMR
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.016.
(400 MHz, CDCl3) d 2.77 (s, 3H, COCH3), 2.88 (s, 3H, COCH3), 2.85e
2.88 (m, 4H, CH2), 3.78 (s, 3H, OCH3), 3.94e3.97 (m, 2H, CH2),
4.12e4.17 (m, 2H, CH2), 5.15 (brs, 1H, CH), 6.58e6.79 (m, 3H, Are
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d 20.6, 20.9, 44.7, 50.1, 55.8, 64.1,
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