Design and pharmacophore modeling of biaryl methyl eugenol analogs as breast cancer invasion inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.12.019

Cell invasion and migration are required for the parent solid tumor cells to metastasize to distant organs. Microtubules form a polarized network, enabling organelle and protein movement throughout the cell. Cytoskeletal elements coordinately regulate cell's motility, adhesion, migration, exocytosis, endocytosis, and division. Thus, microtubule disruption can be a useful target to control cancer cell invasion and metastasis. The phenolic ether methyl eugenol (1), the major component of the essential oil of the leaves of Melaleuca ericifolia Sm. (Myrtaceae), was used as a starting scaffold to design eleven new and three known anti-tubulin agents 2-15 using carbon-carbon coupling reactions. A computer-assisted approach was used to design these new biaryl derivatives using colchicine-binding site of tubulin as the molecular target and colchicine as an active ligand. Several derivatives showed potent inhibitory activity against MDA-MB-231 cell migration at the 1-4 μM dose range. The Z isomers, 4 and 15 were more active as invasion inhibitors compared to their structurally related E isomers, 2 and 14. The cytotoxic activities of compounds 2-15 against two breast cancer cell lines MDA-MB-231 and MCF-7 were evaluated. Anti-invasive activity of the semisynthetic derivatives is not due to a direct cytotoxic effect on MDA-MB-231. Analogs 2-15 may promote their anti-invasive activity through the induction of changes in cell morphology. A pharmacophore model was generated involving seven essential features for activity, which was consistent with a previously generated colchicine site inhibitors model.

Full text of DOI:10.1016/j.bmc.2009.12.019

Bioorganic & Medicinal Chemistry 18 (2010) 496–507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and pharmacophore modeling of biaryl methyl eugenol analogs
as breast cancer invasion inhibitors
Fatma M. Abdel Bar ,à, Mohammad A. Khanfar , Ahmed Y. Elnagar , Farid A. Badria à,
à
à
,
*
Ahmed M. Zaghloul , Kadria F. Ahmad , Paul W. Sylvester , Khalid A. El Sayed
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, 700 University Avenue, Monroe, LA 71209, USA
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Cell invasion and migration are required for the parent solid tumor cells to metastasize to distant organs.
Microtubules form a polarized network, enabling organelle and protein movement throughout the cell.
Cytoskeletal elements coordinately regulate cell’s motility, adhesion, migration, exocytosis, endocytosis,
and division. Thus, microtubule disruption can be a useful target to control cancer cell invasion and
metastasis. The phenolic ether methyl eugenol (1), the major component of the essential oil of the leaves
of Melaleuca ericifolia Sm. (Myrtaceae), was used as a starting scaffold to design eleven new and three
known anti-tubulin agents 2–15 using carbon–carbon coupling reactions. A computer-assisted approach
was used to design these new biaryl derivatives using colchicine-binding site of tubulin as the molecular
target and colchicine as an active ligand. Several derivatives showed potent inhibitory activity against
Received 6 October 2009
Revised 3 December 2009
Accepted 5 December 2009
Available online 11 December 2009
Keywords:
Anti-invasive
Biaryl
Breast cancer
Heck and Suzuki coupling reactions
Methyl eugenol
Microtubules
MDA-MB-231 cell migration at the 1–4 lM dose range. The Z isomers, 4 and 15 were more active as inva-
sion inhibitors compared to their structurally related E isomers, 2 and 14. The cytotoxic activities of com-
pounds 2–15 against two breast cancer cell lines MDA-MB-231 and MCF-7 were evaluated. Anti-invasive
activity of the semisynthetic derivatives is not due to a direct cytotoxic effect on MDA-MB-231. Analogs
2–15 may promote their anti-invasive activity through the induction of changes in cell morphology. A
pharmacophore model was generated involving seven essential features for activity, which was consis-
tent with a previously generated colchicine site inhibitors model.
Pharmacophore modeling
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
microtubules is a dynamic process that involves the assembly of
heterodimers formed by a- and b-tubulin subunits and degradation
Breast cancer is the most common cancer representing nearly
25% of total diagnosed cancer cases and the second leading cause
of female cancer deaths in America.1 Metastasis rather than the
primary tumor kills 90% of cancer patients. Patient progression at
the metastatic stage of disease is very poor, and currently there
are few available treatment options.1 Controlling the invasion
and metastasis of primary tumor cells and preventing their spread
to distant and vital organs are major challenges in cancer therapy.
The cytoskeleton is composed of actin, microtubule, and inter-
mediate microfilaments.1,2 Cytoskeleton proteins constitute over
25% of total proteins in the cell.1–3 Cytoskeleton proteins play an
important role in invasion and metastasis of tumor cells.1,2 Micro-
tubules play a critical role in formation of the mitotic spindle, which
provides the structural framework for the physical segregation of
chromosomes during cell division (mitosis).5–7 The formation of
or disassembly of the linear polymers.3–7 The normal function of
tubulin assembly and disassembly is thus crucial for cell division,
and any interference with this process will disrupt cell division
and induce cell death by apoptosis.3–7 Microtubule dynamics are
implicated in many cellular processes including adhesion, migra-
tion, and morphology.5–7 Thus, tubulin inhibitors can impair the
invasiveness potential of cancer cells.5–7
Tubulin polymerization inhibitors, for example, vinca alkaloids,
combretastatins and colchicine and tubulin polymerization pro-
moters, for example, taxanes like paclitaxel, are examples of anti-
cancer tubulin/microtubule-targeting drugs.6,7 The importance of
microtubules as molecular targets has been amplified by the dis-
covery of potent and selective toxicity of combretastatin A4
(CA4) as a vascular-disrupting agent (VDA).2,3 Vascular-disrupting
agents (VDAs) selectively impair tumor’s vasculature which is
essential for tumor progression and metastasis. The water-soluble
prodrug of CA4, combretastatin-A4 phosphate (CA4P), is currently
in phase II clinical trials.4 Paclitaxel is unique in its mechanism,
promoting the assembly of tubulin into highly stable non-func-
tional polymers.6,7 The mechanism by which paclitaxel stabilizes
* Corresponding author. Tel.: +1 318 342 1725; fax: +1 318 342 1737.
E-mail address: elsayed@ulm.edu (K.A. El Sayed).
University of Louisiana at Monroe, USA.
à
Mansoura University, Egypt.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.019

F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
497
the microtubules is still under investigation.6 So far, three binding
domains have been identified in the crystal structure of tubulin; (i)
the cholchicine site, which is close to the /b interface, (ii) the area
2. Results and discussion
a
2.1. Molecular modeling and docking study
where vinca alkaloids bind, and (iii) the taxane binding pocket.7
Methyl eugenol (1, 4-allyl-1,2-dimethoxybenzene) is a widely
distributed naturally occurring phenolic ether in a variety of plant
species. It represents 96.8% of the content of the volatile oil of Mel-
aleuca ericifolia Sm. grown in Egypt.8 This study reports a series of
rationally designed tubulin-targeting biaryl derivatives (2–15),
which were prepared semisynthetically starting with 1 using Heck
and Suzuki coupling reactions. Their structure design was based on
molecular docking studies in SYBYL 8.1 and close structure similarity
to chalcones, the biaryl enones that showed potent toxicity to sev-
eral cancer cell lines and interact with tubulin at its colchicine-
binding site.7
Grams of methyl eugenol 1 were isolated from the volatile oil of
M. ericifolia Sm. grown in Egypt.8 To design a series of coupling
products of 1 with diverse aryl moieties, a computer-aided molec-
ular modeling study was carried out within the colchicine-binding
site of the high-resolution crystal structure (resolution = 3.80 Å) of
the tubulin–colchicine–soblidotin: stathmin-like domain complex
(PDB 3e22).9 The protein crystal structure was retrieved from the
Research Collaboratory for Structural Bioinformatics Protein Data
Bank. The structure of colchicine was used as a reference ligand
along the docking and modeling studies.
In silico docking indicated two interactions with b-tubulin (ala-
The new biaryl compounds have been tested for ability to inhi-
bit cell invasion in the MDA-MB-231 breast cancer cell line. The
cytotoxicity of 2-15 against the MDA-MB-231 and MCF-7 breast
cancer cell lines has also been studied.
nine 250, ALA 250) and asparagine 250, ASN 249) and one with a-
tubulin (asparagine 101, ASN 101) via strong H-bonding with the
3,4-dimethoxy functionality in the methyl eugenol moiety
(Fig. 1). These results were consistent with the fact that colchi-
cine-binding site is deeply buried within the b-tubulin.10,11 The
binding modes of the designed structures were found to be compa-
rable to the interaction maps of other anti-tubulin agents.12
Several biaryl carbon–carbon coupling structures of 1 with 2-
acetoxy, 2-methoxy, 4-methoxy, and 3,4,5-trimethoxyphenyl func-
tionalities showed improved binding affinities at the colchicine-
binding site as suggested by the high total scores of 2–4, 6, 9,
and 11–14, compared to colchicine (Table 1). To test whether elec-
tron-donating alkyl groups like methyl will show improved activ-
ity versus methoxy groups, compounds 14 and 15 were
proposed.13 Thus, compounds 2–15 were synthesized and tested
for their cytotoxic and anti-invasive activities.
2.2. Chemical synthesis
Compounds 2–4, 14, and 15 were prepared by using Heck’s cou-
pling reaction between the alkene group of methyl eugenol (1) and
aryl halides (Scheme 1).14 Compounds 5–13 were prepared using
oxygen-promoted Pd(II) catalysis for the coupling of aryl boronic
acids and the D2,3 alkene system of 1 (Suzuki coupling, Scheme
2).15 Both E and Z isomers have been observed as products in the
Heck reaction with the E-adduct more predominant than the Z-ad-
duct, which was a minor product (Scheme 1). Suzuki coupling was
Table 1
Virtual binding affinity of compounds 2–15 at the colchicine-binding site of tubulin
(PDB 3e22) using SYBYL 8.1 Surflex-Docka
Compound
Total
Crash
Polar
C Score
3
6
12
9
2
4
13
11
8.51
8.40
8.28
8.25
7.97
7.97
7.81
7.57
7.53
7.52
7.45
7.03
6.92
6.92
6.19
ꢀ1.64
ꢀ1.93
ꢀ1.51
ꢀ3.22
ꢀ2.79
ꢀ2.16
ꢀ2.66
ꢀ1.61
ꢀ2.92
ꢀ1.50
ꢀ1.79
ꢀ1.22
ꢀ3.74
ꢀ3.74
ꢀ1.08
2.64
2.90
1.73
2.78
2.64
1.06
0.57
0.00
2.65
1.01
1.20
0.00
1.39
1.39
0.00
4
2
2
4
2
5
2
4
2
5
4
2
4
2
2
14
Colchicine
10
7
5
8
15
a
Total score was expressed in ꢀlog (Kd) units to represent binding affinities.
Crash is the degree of inappropriate penetration by the ligand into the protein and
of interpenetration between ligand atoms that are separated by rotatable bonds.
Polar score is the contribution of the polar non-hydrogen bonding interactions to
the total score. The polar score may be useful for excluding docking results that
make no hydrogen bonds.
Figure 1. Detailed view of docked structures 4 (A), 9 (B), and 14 (C) with the
corresponding interacting amino acids of tubulin binding site.

498
F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
2
1
O
7``
2``
7``
7``
OCH3
3
OCH3
1``
8``
7``
2``
O
2``
1``
6``
1``
3``
1``
1``
4``
OCH3
OCH3
OCH3
5``
OCH3
9``
8``
8``
OCH3
7``
a
c
e
b
1
d
1
1
3
3
1
2
3
R1
1`
3`
1`
R3
2
1`
2
R2
4`
H3CO
H3CO
4`
H3CO
4`
3`
3`
8`
8`
8`
OCH3
O7C` H3
O7C` H3
7`
R1
R2
a
c
R3
c
d
2
5
8
10
a
b
c
d
3
6
9
7
11
14
d
e
O
7``
2``
8``
7``
2``
3
O
2
3
2
1``
6``
1``
1
1
1`
1`
8``
3`
3`
OCH3
OCH3
OCH3
OCH3
4`
4`
7`
7`
8`
8`
4
15
7``
OCH3
7``
OCH3
O
1
3
O
1
3
1``
1``
3``
4``
3``
4``
1`
1`
2
2
OCH3
OCH3
H3CO
H3CO
4`
4`
5``
5``
O3C` H3
O3C` H3
8``
8``
8`
8`
OCH3
OCH3
7`
7`
9``
9``
12
13
Ar
Ar
Et3N, Pd(OAc)2, PPh3
CH3CN, 85°C, N2, 3 h
Ar
OCH3
X
OCH3
OCH3
Product (E/Z)
OCH3
OCH3
OCH3
1
Scheme 1. Heck coupling reaction between methyl eugenol olefinic side chain and aryl halides used for the preparation of 2–4, 14, and 15.
Ar
Ar
Pd(OAc)2
ArB(OH)2
Na2CO3, DMF, 50°C, air, 3 h
OCH3
OCH3
OCH3
OCH3
Major product (E)
OCH3
OCH3
1
Scheme 2. Suzuki coupling reaction between methyl eugenol olefinic side chain and aryl boronic acids used for the preparation of 5–13.

F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
499
Table 2
1H NMR data of compounds 2–4, 6, and 7a
Position
dH
2
3
4
6
7
1
2
3
6.35, d (15.7)
6.09, dt (15.8, 6.6)
3.44, d (6.6)
3.60, s
—
5.07, d (1.5)
5.08, br s
3.48, d (5.5)
6.34, dt (7.7, 5.8)
6.36, d (7.7)
3.74, s
—
4.95, s
5.40, s
—
6.76, s
—
—
6.74, d (8.1)
6.74, d (8.1)
3.81, s
3.83, s
—
7.35, d (8.4)
6.80, d (8.1)
—
3.46, d (7.0)
6.15,dt (15.8, 7.0)
6.36, d (15.8)
10
—
—
—
—
20
6.88, d (1.8)
—
—
6.78, d (8.0)
6.85, dd (8.0, 1.8)
3.86, s
3.87, s
—
6.64, d (1.8)
—
—
6.75, d (8.0)
6.67, dd (8.0, 1.8)
3.80, s
3.84, s
—
6.73, d (2.2)
—
—
6.81, d (8.4)
6.74, dd (8.0, 1.8)
3.87, s
3.86, s
—
6.74, d (1.8)
—
—
6.80, d (8.1)
6.76, dd (8.1, 1.8)
3.86, s
30
40
50
60
70
80
3.86, s
—
100
200
300
400
500
600
700
800
—
—
—
7.28, d (8.8)
6.82, d (8.8)
—
6.80, d (8.8)
7.28, d (8.8)
3.80, s
7.03, dd (7.7, 1.5)
7.24, dd (7.7, 1.8)
7.19, dd (7.3, 1.5)
7.29, dd (7.3, 1.5)
—
7.01, d (8.0)
7.23, dd (2.2, 8.4)
7.11, dd (7.7, 1.1)
7.09, dd (7.7, 2.2)
—
6.99, dd (7.7, 1.1)
7.51, dd (7.7, 1.8)
7.21, dd (7.7, 1.8)
7.18, dd (7.4, 1.5)
—
6.80, d (8.1)
7.35, d (8.4)
3.78, s
2.28, s
2.26, s
2.26, s
a
In CDCl3, at 400 MHz. Coupling constants (J) are in Hz.
selective to E-geometry only. The location of the
D
2,3 system adja-
of 2 as a carbon–carbon coupling product. The presence of the acet-
oxy methyl singlet H3-800 at d 2.27 in addition to seven aromatic
protons further supported this conclusion. The proton doublet H-
cent to the aliphatic methylene carbon C-1 in 1 allowed its migra-
tion to the D1,2 position in some products during the coupling
reaction. Additionally, coupling may occasionally target the ole-
finic methine carbon C-2 instead of the olefinic methylene carbon
C-3 of 1, resulting in the formation of minor C6–C2–C6 products
with an exomethylene group as in compounds 3, 6, and 9 along
with the major C6–C3–C6 products. Compounds 5, 12, and 13 are al-
ready known in literature.16,17
3
1 showed J-HMBC correlations with C-3, C-20, and C-60 (dC 33.9,
108.5, and 119.2, respectively). The proton H-2 showed 3J-HMBC
correlations with C-10, and C-100 (dC 130.5 and 132.2, respectively).
The 3J-HMBC correlation of the methylene proton doublet H2-3 (dH
3.44) with carbons C-200 and C-600 (dC 148.5 and 127.6, respectively)
indicated the migration of D2,3 of the parent 1 to D1,2 during the
reaction. Therefore, the structure of 2 was determined to be (E)-
2-[3-(3,4-dimethoxyphenyl)allyl]phenyl acetate.
The HRESIMS data of 2 (m/z 335.1265 for [M+Na]+) suggested
the molecular formula C19H20O4. 1H NMR data of 2 (Table 2)
showed a doublet at d 6.35 (J = 15.7) assigned to H-1 and a double
triplet at d 6.09 (J = 15.8, 6.6) assigned to H-2 and indicated a pos-
Analysis of HRESIMS of 3 (m/z 335.1250 for [M+Na]+) suggested
the same molecular formula as 2 and indicated a coupling product.
The 1H NMR data of 3 (Table 2) showed a benzylic methylene singlet
at d 3.60, assigned to H2-1 and exomethylene protons H-3a and H-3b
(dH 5.07 and 5.08, respectively). This indicated a possible coupling
product at C-2 rather than the expected C-3 of the parent compound
sible E-geometry for
D
1,2 system and hence trans-coupling of the 2-
acetoxyphenyl moiety with 1. The 13C NMR data of 2 (Table 3)
showed 19 carbon signals, which further confirmed the identity
1. The 13C NMR data of 3 (Table 3) showed a
D
2,3 typical exomethyl-
ene pattern with a quaternary carbon at d 145.8 (C-2) and olefinic
exomethylene at d 116.7 (C-3). The exomethylene protons H-3a
and H-3b showed 3J-HMBC correlations of with the methylene car-
bon C-1 (dC 42.8) and the quaternary aromatic C-100 (dC 135.6). It also
showed a 2J-HMBC correlation with C-2, confirming the coupling at
C-2 of the side chain of 1. Therefore, compound 3 was determined to
be 2-[3-(3,4-dimethoxyphenyl)prop-1-en-2-yl]phenyl acetate.
The HRESIMS data of 4 (m/z 335.1257 for [M+Na]+) suggested it is
an isomerof 2 and 3. The 1HNMR data of4 (Table 2) showed twoover-
lapped protons H-2 and H-3 (dH 6.34, J = 7.7, 5.8 and 6.36, J = 7.7,
respectively). Their coupling constant values indicated the Z-geome-
try for D2,3 system and hence cis-coupling of the 2-acetoxyphenyl
moiety with 1. The methylene protons H2-1 (dH 3.48) showed
3J-HMBC correlations with C-3, C-20, and C-60 (dC 132.3, 111.9, and
120.7, respectively), in addition to 2J-HMBC correlation with the qua-
ternary carbon C-10 (dC 132.3), indicated an expected carbon–carbon
coupling pattern at C-3 of 1. Therefore, compound 4 was determined
to be (Z)-2-[3-(3,4-dimethoxyphenyl)prop-1-enyl]phenyl acetate.
Analysis of the HRESIMS data of 6 (m/z 307.1310 for [M+Na]+)
suggested the molecular formula C18H20O3 and indicated a coupling
product of 1 with 4-methoxyphenyl moiety. The 1H and 13C NMR
spectrum of 6 (Tables 2 and 3, respectively) suggested a coupling
product similar to 3. The methoxy proton singlet H3-700 (dH 3.78)
Table 3
13C NMR data of compounds 2–4, 6, and 7a
Position
dC
2
3
4
6
7
1
2
3
132.2, CH
125.8, CH
33.9, CH2
130.5, qC
108.5, CH
149.1, qC
149.0, qC
111.1, CH
119.2, CH
56.0, CH3
55.9, CH3
132.2, qC
148.5, qC
122.6, CH
130.7, CH
126.4, CH
127.6, CH
169.6, qC
21.1, CH3
42.8, CH2
145.8, qC
116.7, CH2
131.5, qC
112.4, CH
148.7, qC
147.4, qC
111.0, CH
121.3, CH
55.9, CH3
55.8, CH3
135.6, qC
147.6, qC
122.6, CH
128.3, CH
126.0, CH
130.0, CH
169.6, qC
21.2, CH3
39.3, CH2
124.3, CH
132.3, CH
132.3, qC
111.9, CH
149.0, qC
147.8, qC
111.2, CH
120.7, CH
55.9, CH3
56.0, CH3
130.1, qC
147.5, qC
122.6, CH
126.8, CH
128.1, CH
126.3, CH
169.5, qC
20.97, CH3
41.4, CH2
146.4, qC
112.9, CH2
132.3, qC
111.2, CH
148.9, qC
147.4, qC
112.2, CH
120.9, CH
55.9, CH3
55.8, CH3
133.4, qC
127.3, CH
113.7, CH
159.1, qC
113.7, CH
127.3, CH
55.3, CH3
39.0, CH2
127.4, CH
130.3, CH
133.1, qC
112.0, CH
149.0, qC
147.5, qC
111.4, CH
120.6, CH
55.9, CH3
56.0, CH3
130.6, qC
127.3, CH
114.0, CH
159.0, qC
114.0, CH
127.3, CH
55.4, CH3
10
20
30
40
50
60
70
80
100
200
300
400
500
600
700
800
a
In CDCl3, at 100 MHz. Carbon multiplicities were determined by APT experi-
ment. qC = quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.

500
F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
Table 4
1H NMR data of compounds 8–11 and 14–15a
Position
dH
8
9
10
11
14
15
1
2
3
6.34, d (15.7)
6.16, dt (15.7, 7.0)
3.46, d (7.0)
—
6.90, d (1.8)
—
3.73, s
—
5.00, s 5.41, s
—
6.78, br s
—
—
6.37, d (15.8)
6.15, dt (15.4, 7.0)
3.46, d (7.0)
—
6.92, d (1.8)
—
—
3.47,d (5.9)
6.21, dt (15.4, 6.6)
6.33, d (15.8)
—
6.75, d (1.8)
—
—
3.52, d (7.0)
5.77, dt (16.1, 7.0)
6.39, d (16.1)
—
6.81, d (2.6)
—
3.53, d (4.4)
6.13, dt (5.5, 4.4)
6.14, d (5.5)
—
6.85, d (1.8)
—
10
20
30
40
50
60
70
80
100
200
300
400
500
600
700
800
900
—
—
—
6.78, d (8.4)
6.86, dd (8.1, 1.8)
3.86, s
3.87, s
—
7.15, d (8.8)
6.85, d (8.8)
—
6.85, d (8.8)
7.15, d (8.8)
3.79, s
6.75, br d (10.6)
6.75, br d (10.6)
6.79, d (8.1)
6.89, dd (8.1, 1.8)
6.82, d (8.0)
6.79, d (8.0, 1.8)
6.81, d (2.6)
6.81, d (2.6)
3.87, s
3.87, s
—
6.76, d (8.0)
6.81, d (8.0, 1.8)
3.85, s
3.86, s
—
3.84, s
3.82, s
—
6.62, s
—
—
—
6.62, s
3.81, s
3.82, s
3.81, s
3.87, s
3.88, s
—
6.45, s
—
—
—
6.45, s
3.85, s
3.83, s
3.85, s
3.87, s
3.87, s
—
6.58, s
—
—
—
6.58, s
3.85, s
3.83, s
3.85, s
—
—
7.02, m
7.02, m
7.02, m
—
2.30, s
2.30, s
7.02, d (1.8)
7.02, d (1.8)
7.02, d (1.8)
—
2.34, s
2.34, s
a
In CDCl3, at 400 MHz. Coupling constants (J) are in Hz.
showed a 3J-HMBC correlation with the quaternary aromatic oxy-
genated carbon C-400 (dC 159.1). The aromatic proton doublet H-200
(dH 7.35) showed 3J-HMBC-correlations with the quaternary olefinic
carbons C-2 (dC 146.4) and C-400. Therefore, the structure of 6 was
determined to be 1,2-dimethoxy-4-[2-(4-methoxyphenyl)allyl]
benzene.
structure of 7 was determined to be (E)-1,2-dimethoxy-4-[3-(4-
methoxyphenyl)allyl]benzene.
The HREIMS data of 8 (m/z 307.1313 for [M+Na]+) has the same
molecular formula and was very closely similar to 7. The 1H and
13C NMR data of 8 (Tables 4 and 5, respectively) revealed a trans-
coupling product closely similar to 2, with the replacement of 2-
acetoxyphenyl with a 4-methoxyphenyl moiety. 13C NMR data of
8 show some differences in chemical shift values were clearly ob-
served than in case of 7. The symmetric aromatic carbons C-200/C-600
were downfield shifted (+2.4 ppm) while C-20 and C-60 were up-
field shifted (ꢀ3.5 and ꢀ1.4 ppm, respectively) compared to those
of 7. These chemical shift differences were mainly due to the
migration of D2,3 of 1 to D1,2 during the coupling reaction similar
to 2 and 5. Therefore, the structure of 8 was determined to be
(E)-1,2-dimethoxy-4-[3-(4-methoxyphenyl)prop-1-enyl]benzene.
The HRESIM of 9 (m/z 367.1509 for [M+Na]+) suggested the
molecular formula C20H24O5, suggesting a coupling product of 1
The HRESIMS data of 7 (m/z 307.1313 for [M+Na]+) suggested a
similar molecular formula as 6. The 1H and 13C NMR data of 7 (Ta-
bles 2 and 3, respectively) indicated an expected coupling product
at C-3 of 1. The large coupling value between H-2 (dH 6.15, J = 15.8,
7.0) and H-3 (dH 6.36, J = 15.8) indicated a possible E-geometry for
D
2,3 system and hence the trans-coupling of the 3,4,5,-trimethoxy-
phenyl moiety with C-3 of 1. The methylene H2-1 (dH 3.46) showed
3J-HMBC correlations with carbons C-3, C-20, and C-60 (dC 130.3,
112.0, and 120.6, respectively). It also showed a 2J-HMBC correla-
tion with the quaternary carbon signal at d 133.1 (C-10) indicated
a similar pattern of the double bond position to 1. Therefore, the
Table 5
13gC NMR data of compounds 8–11 and 14–15a
Position
dC
8
9
10
11
14
15
1
2
3
130.4, CH
127.9, CH
38.5, CH2
130.7, qC
108.5, CH
149.1, qC
149.0, qC
111.1, CH
119.2, CH
55.9, CH3
56.0, CH3
132.5, qC
129.7, CH
114.0, CH
157.8, qC
114.0, CH
129.7, CH
55.4, CH3
41.4, CH2
147.3, qC
114.2, CH2
132.1, qC
111.2, CH
148.5, qC
147.5, qC
112.1, CH
121.0, CH
55.9, CH3
56.0, CH3
136.9, qC
103.7, CH
152.9, qC
137.7, qC
152.9, qC
103.7, CH
56.1, CH3
61.0, CH3
56.1, CH3
130.8, qC
127.2, CH
39.8, CH2
130.5, qC
108.7, CH
149.1, qC
148.6, qC
111.2, CH
119.2, CH
56.0, CH3
55.9, CH3
136.2, qC
105.6, CH
153.3, qC
137.2, qC
153.3, qC
105.6, CH
56.2, CH3
61.0, CH3
56.2, CH3
38.9, CH2
129.1, CH
130.8, CH
132.7, qC
112.2, CH
150.8, qC
149.1, qC
111.5, CH
120.7, CH
56.0, CH3
56.1, CH3
133.3, qC
103.3, CH
153.4, qC
137.7, qC
153.4, qC
103.3, CH
56.2, CH3
61.0, CH3
56.2, CH3
39.5, CH2
134.3, CH
128.8, CH
133.1, qC
111.3, CH
149.0, qC
149.0, qC
111.9, CH
120.5, CH
55.9, CH3
56.0, CH3
137.4, qC
136.0, qC
127.7, CH
126.4, CH
127.7, qC
136.0, CH
21.1, CH3
21.1, CH3
129.8, CH
126.2, CH
32.9, CH2
130.8, qC
108.7, CH
149.1, qC
148.4, qC
111.2, CH
119.0, CH
55.9, CH3
56.0, CH3
136.4, qC
136.9, qC
128.2, CH
125.5, CH
128.2, qC
136.9, qC
20.1, CH3
20.1, CH3
10
20
30
40
5‘
60
70
80
100
200
300
400
500
600
700
800
900
a
In CDCl3, at 100 MHz. Carbon multiplicities were determined by APT experiment. qC = quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.

F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
501
with a 3,4,5-trimethoxy-1-phenyl moiety. The 1H and 13C NMR
data of 9 (Tables 4 and 5, respectively) suggested a coupling prod-
uct similar to 3. The methoxy proton singlets H3-700, H3-800, H3-900
(dH 3.81, 3.82, and 3.81, respectively) showed 3J-HMBC correlations
with the quaternary aromatic oxygenated carbons C-300, C-400, and
C-500 (dC 152.9, 137.7, and 152.9, respectively). The symmetric aro-
matic proton singlets H-200/H-600 (dH 6.62) showed 3J-HMBC-corre-
lations with the quaternary olefinic carbons C-2 (dC 147.3) and the
aromatic oxygenated carbon C-400. Therefore, compound 9 was
determined to be 5-[3-(3,4-dimethoxyphenyl)prop-1-en-2-yl]-
1,2,3-trimethoxybenzene.
Compound 10 had the same molecular formula as 9 (C20H24O5),
which indicated a coupling product with 3,4,5-trimethoxy-1-phe-
nyl moiety. The 1H and 13C NMR data of 10 (Tables 4 and 5, respec-
tively) suggested a coupling product similar to 2, 5 and 8.
Therefore, the structure of 10 was determined to be (E)-5-[3-
(3,4-dimethoxyphenyl)allyl]-1,2,3-trimethoxybenzene.
(dC 130.8 and 136.4, respectively. Therefore, compound 15 was
determined to be (Z)-2-[3-(3,4-dimethoxyphenyl)allyl]-1,3-
dimethylbenzene.
2.3. Pharmacology
Metastasis is the leading cause of cancer death. Cell migration is
a prerequisite for cancer invasion and metastasis. Therefore, cell
motility is suggested as a potential therapeutic target for cancer
treatment.18–21 Compounds 2–15 were evaluated for their poten-
tial use as anti-invasive agents against the metastatic breast cancer
MDA-MB-231 cells using a CultrexÒ Basement Membrane Extract
(BME) cell invasion assay.22–24
Basement membranes are thin continuous sheets separating
epithelial tissues from adjacent stroma (Fig. 2).23,24 The main com-
ponents of basement membranes are laminin, collagen IV, and a
heparan sulfate proteoglycan.23,24 The primary function of the
basement membrane is to anchor down the epithelium to its loose
connective tissue underneath. This is achieved by cell-matrix adhe-
sions through cell adhesion molecules (CAMs).25 The basement
membrane acts as a mechanical barrier, preventing malignant cells
from invading the deeper tissues.25 Early stages of malignancy that
are thus limited to the epithelial layer by the basement membrane
are called carcinoma in situ.25 Tumor invasion of basement mem-
branes is a vital step in the complex multistage process, which
leads to the formation of a metastasis.23,24 Tumor cells cross base-
ment membranes as they initially invade the lymphatic or blood
vessels for dissemination into the circulation, and then undergo
metastatic growth in the target organ. The penetration of the tu-
mor cells into basement membranes involves steps including (i)
attachment of the tumor cells to the basement membrane; (ii)
Secretion of enzymes by the tumor cells leading to degradation
of the adjacent basement membrane; and (iii) migration of the tu-
mor cells into the target tissue in response to specific chemotactic
stimuli.18
Compound 11 was found to have the same molecular formula as
9 and 10 (C20H24O5), which indicated a coupling product with the
3,4,5-trimethoxy-1-phenyl moiety. The 1H and 13C NMR data of 11
(Tables 4 and 5, respectively) suggested a coupling product similar
to 7. Minor differences were observed in dH values for 11 than 10,
however, major differences were observed in dC values. Carbons C-
2, C-20, and C-60 were downfield shifted as compared to those of 10
(+1.9, +3.5, and +1.5 ppm, respectively) unlike carbons C-200/600,
which were upfield shifted (ꢀ2.3 ppm). These chemical shift differ-
ences may be attributed to the double bond position (D2,3 versus
D
1,2) and the effect of ring substitution. Therefore, compound 11
was determined to be (E)-5-[3-(3,4-dimethoxyphenyl)prop-1-
enyl]-1,2,3-trimethoxybenzene.
The HRESIMS data of 14 (m/z 305.1526 for [M+Na]+) suggested
the molecular formula C19H22O2, which suggested a coupling prod-
uct with a xylyl moiety. The 1H and 13C NMR data of 14 (Tables 4
and 5, respectively) suggested a coupling product similar to 7
and 11. The methyl singlets H3-700/H3-800 (dH 2.30) showed 3J-HMBC
correlations with the quaternary aromatic carbon C-100 (dC 137.4),
which was 3J-HMBC correlated with H-2 (dH 5.77), confirming
the position of the xylyl moiety. The unexpected splitting pattern
in the protons H-50, H-60 and H-300-H-500, as they appeared as nar-
row doublets or broad singlets rather than straight doublets with
regular o-coupling may be due a dihedral angle approaching 90°
between these protons, resulting in a J value near zero. Therefore,
the structure of 14 was determined to be (E)-2-[3-(3,4-dimethoxy-
phenyl)prop-1-enyl]-1,3-dimethylbenzene.
CultrexÒ Basement Membrane Extract is a soluble basement
membrane extract of the Engelbreth–Holm–Swarm (EHS) tumor
that gels at 37 °C to form a reconstituted basement membrane. It
consists of laminin I, type IV collagen, entactin, and heparan sulfate
proteoglycan. Therefore, CultrexÒ BME cell invasion assay provides
a standardized model in vitro to quantify the degree at which inva-
sive cells penetrate a barrier consisting of basement membrane
component in response to chemoattractants and/or inhibiting
compound.23,24 It employs a simplified Boyden chamber design
in which basement membrane extract form a matrigel coating on
The HRESIMS of 15 (m/z 305.1522 for [M+Na]+) suggested the
same molecular formula as compound 14 and a coupling product
with the xylyl moiety. Analysis of 1H NMR data of 15 (Table 4)
showed two overlapped cis-coupled olefinic protons H-1 and H-2
(dH 6.14, J = 5.5 and 6.13, J = 5.5, 4.4, respectively). Proton H-1
showed 3J-HMBC correlations with the aliphatic methylene carbon
C-3, in addition to the aromatic methane carbons C-20, and C-60 (dC
32.9, 108.7, and 119.0, respectively. Proton H-2 showed 3J-HMBC
correlations with the quaternary aromatic carbons C-10 and C-100
the top of a porous filter made of 8
(PET) (Fig. 2). Cells were treated with 2–15 at different concentra-
tions 1–4 M for 24 h, and 10% FBS was used as a chemoattractant.
lm polyethylene terephthalate
l
Compounds 2–15 inhibited the invasion of MDA-MB-231 in dose-
dependent manner (Fig. 3). The anti-invasive activities for the C6–
C3–C6 semisynthesized methyl eugenol derivatives (Fig. 3A and C)
were markedly better than the C6–C2–C6 (3, 6, and 9) derivatives
(Fig. 3B). The Z isomers represented by 4 and 15 were more active
Chemicals
Cells (MDA-MB-231)
a
b
c
Upper chamber
(BME) coating
No BME coat (control)
PET membrane
Lower chamber
Chemoattractant
Figure 2. Schematic diagram of the cell invasion chamber used in invasion assays.

502
F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
120.0
100.0
80.0
60.0
40.0
20.0
0.0
A
Control
4
15
Colchicine
1
2
4
Concentration µM
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
B
Control
9
3
6
Colchicine
1
2
4
Concentraction µM
120.0
100.0
80.0
60.0
40.0
20.0
0.0
C
Control
2
5
8
10
7
11
14
Colchicine
1
2
4
Concentration µM
Figure 3. Anti-invasive activity of the biaryl methyl eugenol, (Z)-C6–C3–C6, compounds 4 and 15 (A), C6–C2–C6, compounds 3, 6, and 9 (B), and (E)-C6–C3–C6, compounds 2, 5,
7, 8, 10, 11, and 14 (C) using CultrexÒ 96 well BME cell invasion assay against the human metastatic breast cancer cell line MDA-MB-231.
as invasion inhibitors than the E isomers 2 and 14, respectively
(Fig. 3A–C). The 200-acetoxy-containing Z-isomer 4 showed potent
at the same dose level (Fig. 3A). However, the trimethoxychalcone
12 showed better activity than colchicines at 2 M dose, data not
l
activity at 4
lM (9.0% invasion, compared to vehicle control
shown (8.5% invasion vs 12.5% for colchicine). The E-trimethoxy-
(Fig. 3A). This activity was comparable to the activity of colchicine
phenyl and p-methoxyphenyl analogs 10 and 7, respectively, also

F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
503
showed potent anti-invasive activity (11.0%, 13.7% invasion,
respectively) at 4 M concentration compared to colchicine (8.9%
invasion). These results were consistent with the virtual docking
ous cycle of interdependent steps including polarization and elon-
gation of cells.12 Microtubule disruption is reported to provoke
cytoskeleton and cell adhesion changes, which end by cell round-
ing.17,23 Therefore, the methyl eugenol biaryl analogs 2–15 may
promote their anti-invasive activity through the induction of
changes in cell morphology, which alter the motility of cells in
three-dimensional matrices.
Colchicine has been tested by the National Cancer Institute’s
Developmental Therapeutics Program (NSC757). Their results
show the cytostatic effect of colchicine against the breast cancer
l
study (Table 1).
The cytotoxicity of compounds 2–15 was evaluated and com-
pared to those of colchicine. Most methyl eugenol biaryl analogs
were not cytotoxic at the 10–50
cate that the anti-invasive activity of compounds 2–15 at concen-
tration 1–4 M is not due to a direct cytotoxic effect (Fig. 4). Cell
lM dose range. These results indi-
l
migration through the extracellular matrix results from a continu-
DMSO
Colchicine
2
3
4
5
6
7
8
40000
A
35000
30000
25000
20000
15000
10000
5000
0
Concentration (µM)
B
DMSO
40000
35000
30000
25000
20000
15000
10000
5000
Colchicine
9
10
11
12
13
14
15
0
Concentration (µM)
Figure 4. Cytotoxic activity of biaryl methyl eugenol derivatives 2–8 (A) and 9–15 (B) against MDA-MB-231 cells. Colchicine was used as a positive control and 10
at concentration as a vehicle control.
lM DMSO

504
F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
cells MCF-7 but not to MDA-MB-231.21,26 They also established a
structure–activity relationship model that describes the selective
activity of colchicine toward a cell line but not to other closely re-
lated one, which was consistent with the results shown in Figure
4.21 Therefore, the cytotoxic activities of 2–15 against MCF-7 cells
were tested and compared with colchicine at four-dose levels (10–
techTM identifies features that could be elements in a pharmaco-
phore model.27,28 DISCOtechTM operates in distance space and
can perform clique detection to generate pharmacophore hypoth-
eses on up to 300 conformers per molecule.27,28
These diverse conformers are used in DISCOtech’s clique detec-
tion routine to find 3D alignments of the pharmacophore features
in different molecules. A clique is a subgraph in which every node
is connected to every other’s node. DISCOtechTM reduces the con-
formers to sets of pharmacophore features (nodes) and inter-fea-
ture distances (connections).27,28
100
4 showed markedly less cytotoxicity versus colchicine at a dose
range of 10–50 M (Fig. 5A). Interestingly, the trimethoxyphenyl
lM dose range, Fig. 5A and B). The 2-acetoxyphenyl analogs 2–
l
analog 11 showed limited cytotoxicity versus the related known
trimethoxychalcone 12 (Fig. 5B). This fact led to conclude that
the removal of C-1 ketone of 12 did not affect the anti-invasive
activity but reduced its cytotoxicity.
For assignment of the pharmacophore features, a DISCOtechTM
run was undertaken using 4 as the reference, 1.0 Å tolerances,
and requiring models to have between three and eight matched
features. DISCOtechTM produced 20 different pharmacophore mod-
els satisfying these constraints. Each of the models showed at least
five matched features. Visual inspection of the resulting structural
superpositions showed one model with a good structural overlay of
the four compounds, while the other models did not match the
biaryl system common to the four molecules.
Figure 6 shows the structural overlay corresponding to generated
model. Clearly, the model has seven essential features required for
highreceptorbindingaffinity. Thesevenfeaturesincludetwohydro-
phobic sites, two hydrogen bond acceptor atom, and three receptor
2.4. Pharmacophore modeling
A 3D pharmacophore mapping methodology based on distance
comparison technique was built for the four most active analogs (4,
7, 10, and 12) using DISCOtech™ module implemented in SYBYL
8.1.27,28 DISCOtech™ is a well established module for designing
pharmacophoric maps and frequently used in the process of virtual
screening to discover new leads.29,30 Given a set of molecules that
are related by their ability to bind to same protein receptor, DISCO-
40000
A
DMSO
Colchicine
35000
30000
25000
20000
15000
10000
5000
0
2
3
4
5
6
7
8
Concentration (µM)
35000
30000
25000
20000
15000
10000
5000
0
B
DMSO
Colchicine
9
10
11
12
13
14
15
Concentration ( µM)
Figure 5. Cytotoxic activity of biaryl methyl eugenol derivatives 2–8 (A) and 9–15 (B) against the human breast cancer cells MCF-7. Colchicine was used as a positive control
and DMSO at 10 M concentration as a vehicle control.
l

F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
505
Figure 6. Pharmacophoric features and their distance relation generated by DISCOtechTM module. AA—acceptor atom, DS—donor site, HD—hydrophobic center.
donor sites. A biaryl system, di or trimethoxyphenyl moiety, and a
constrained conformation features are common in all the 14 analogs
and consistent with the pharmacophoric maps generated by previ-
ous colchicine site inhibitors (CSIs).22
The hydrophobic and the planar group serve as the rigid portion
of the molecular scaffold that satisfies the overall geometric and
steric requirements of binding.
Docking studies showed that the hydrophobic feature (HD1)
embedded in a hydrophobic pocket consisting of the side chains of
residues of side chains of Val 179 and Met 257, while the trime-
thoxyphenyl moiety (HD2 feature) occupies a pocket bounded by
side chains of Leu 255, Ala 316, Val 318 and Ile 378. Two methoxy
oxygen atoms on the HD2 ring are located within hydrogen-bond
distances 2.8 and 3.4 Å to the backbone N–H groups of residues
Asp (DS1) 249 and Ala 250 (DS2), respectively. AA2 features H-bond-
ing with sulfur atom of Cys 239 (DS3) (AA2-DS3 distance of 3.2–
4.2 Å). The distance between the biaryl systems is 6.99 1 Å, which
is in the range of the biaryl system of the different structural classes
of CSIs (5.1–7.4 Å).22 This model has the characteristic features re-
quired for an ideal pharmacophoric query, because it possessed
the important interactions required for this series of CSIs and was
consistent with previously reported pharmacophore models.22
tions were performed using the Tripos force field with
a
distance-dependent dielectric and the Powell conjugate gradient
algorithm with a convergence criterion of 0.01 kcal/(mol A).32 Par-
tial atomic charges were calculated using the semiempirical pro-
gram MOPAC 6.0 and applying the AM1.33
Surflex-Dock program version 2.0 interfaced with SYBYL 8.1 was
used to dock the biaryl methyl eugenol coupling derivatives to
the colchicine-binding site of tubulin.34,35 Surflex-Dock employs
an idealized active site ligand (protomol) as a target to generate
putative poses of molecules or molecular fragments.36,37 These
putative poses were scored using the Hammerhead scoring func-
tion.36,37 The 3D structure (PDB 3e22) was taken from the Research
Collaboratory for Structural Bioinformatics Protein Data Bank
(http://www.rcsb.org/pdb).9
3.3. Preparation of compounds 2–4, 14, 15 by Heck reaction14
In a 50 mL 3-necked flask were placed equimolar quantities of
methyl eugenol 1 (163.8 mg, 0.92 mM) and 2-acetoxy-iodoben-
zene (241.0 mg, 0.92 mM) in case of 2–4 or bromoxylene
(170.3 mg, 0.92 mM) in case of 14 and 15. Acetonitrile (14 mL), tri-
ethylamine (4 mL), palladium acetate (4.5 mg, 2 mol %) and tri-
phenylphosphine (12.1 mg, 5 mol %) were then added and the
mixtures were stirred for few minutes. Reaction flasks were then
fitted to condensers and attached to a N2 supply and a pressure re-
lief bubbler. Each reaction mixture was stirred and heated to 85 °C
for 3 h. Reactions were monitored by TLC every 1 h. At the end of
reaction period, each reaction mixture was quenched by the addi-
tion of 5 g of ice. The mixtures were then extracted with EtOAc
(3 ꢁ 15 mL) and the organic layers were dried over anhydrous
Na2SO4 and concentrated under vacuum to give crude brown mix-
tures, which were then purified using chromatographic methods
described later. Reaction of 2-iodobenzene with Ac2O in pyridine
afforded 2-acetoxyiodobenzene.
3. Experimental section
3.1. General experimental procedures
IR spectra were recorded on a Varian 800 FT-IR spectrophotom-
eter. The 1H and 13C NMR spectra were recorded in CDCl3, using
TMS as an internal standard, on a JEOL Eclipse NMR spectrometer
operating at 400 MHz for 1H and 100 MHz for 13C. The HREIMS
experiments were conducted at the University of Michigan on a
Micromass LCT spectrometer. TLC analysis was carried out on pre-
coated Si Gel 60 F254 500 lm TLC plates, using the developing sys-
tems n-hexane–EtOAc (8:2) or (6:4). For column chromatography,
Si Gel 60 (EMD Chemicals Inc.), 70–230 mesh, or Si Gel (Natland
International Corporation), 230–400 mesh, were used. For medium
pressure column chromatography (MPLC) Bakerbond octadecyl
3.4. Preparation of compounds 5–13 by Suzuki coupling
reaction15
C18, 40 lm and MeOH–H2O system were used. Photographs of
Methyl eugenol 1 (89 mg, 0.5 mM, 1 equiv) was dissolved in
DMF (2.5 mL, 0.2 M solution), and stirred at room temperature.15
To the solution, was added 2-methoxy phenylboronic acid in case
of 5 (91.2 mg, 0.6 mM, 1.2 equiv), 4-methoxy phenylboronic acid
in case of 6–8 (91.2 mg, 0.6 mM, 1.2 equiv) or 3,4,5-trimethoxy
phenylboronic acid in case of 9–13 (127.2 mg, 0.6 mM, 1.2 equiv)
followed by addition of Na2CO3 (106 mg, 1.0 mM, 2 equiv) and
Pd(OAc)2 (11 mg, 0.05 mM, 0.1 equiv) to each reaction flask. The
reaction was carried out under air atmosphere instead of O2 and
therefore 5 mol % of Pd(OAc)2 was used as previously described be-
fore.15 The reaction mixtures were heated to 50 °C, and stirred for
3 h. The mixtures were then diluted with ethyl acetate (20 mL),
cells were captured using a NikonÒ Eclipse TE2000-U inverted
microscope (Nikon Instruments Inc.).
3.2. Molecular modeling and docking
Three-dimensional structure building and all modeling were
performed using the SYBYL Program Package, version 8.1,31 installed
on DELL desktop workstations equipped with a dual 2.0 GHz IntelÒ
XeonÒ processor running the Red Hat Enterprise Linux (version 5)
operating system. Conformations of each compound were gener-
ated using ConfortTM conformational analysis. Energy minimiza-

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F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
and washed with aqueous NaCl solution (3 ꢁ 10 mL). The organic
layers were dried over anhydrous Na2SO4 and filtered. The filtrates
were concentrated under vacuum, and purified using chromato-
graphic methods described below.
3.5.5.3.
(Z)-2-[3-(3,4-Dimethoxyphenyl)prop-1-enyl]phenyl
acetate (4). Colorless amorphous powder, IR mmax (CHCl3) 3002,
2960, 2938, 2912, 2839, 1761, 1682, 1595, 1514, 1514, 1465,
1371, 1269, 1139, 1026, 968, 914, 860 cmꢀ1 1H and 13C NMR,
;
see Tables 2 and 3. HRESIMS m/z 335.1257 [M+Na]+ (calculated
3.5. Chromatographic purification of 2-15
for C19H20O4, 335.1259).
3.5.1. Acetoxyphenyl products 3 and 4
3.5.5.4.
1,2-Dimethoxy-4-[2-(4-methoxyphenyl)allyl]benzene
The crude brown residue obtained from Heck’s reaction of 1
with 2-acetoxy-iodobenzene, was chromatographed over Si Gel
60 and eluted with (n-hexane–EtOAc, 8:2). The selected fraction
was further subjected to MPLC using RP-C18 Si Gel (MeOH–H2O,
6:4) to afford 2 (15 mg, 8.5%), 3 (3 mg, 38.5%), and 4 (9 mg, 23.1%).
(6). Colorless amorphous powder, IR mmax (CHCl3) 3002, 2958,
2938, 2912, 2839, 1607, 1513, 1465, 1262, 1140, 1029, 898,
837 cmꢀ1 1H and 13C NMR, see Tables 2 and 3. HRESIMS m/z
;
307.1310 [M+Na]+ (calculated for C18H20O3, 307.1310).
3.5.5.5. (E)-1,2-Dimethoxy-4-[3-(4-methoxyphenyl)allyl]ben-
3.5.2. Xylyl products 14 and 15
zene (7). Colorless amorphous powder, IR mmax (CHCl3) 3001,
2959, 2938, 2912, 2839, 1608, 1512, 1465, 1268, 1155, 1139,
1027, 967, 836 cmꢀ1; 1H and 13C NMR, see Tables 4 and 5. HRESIMS
m/z 307.1313 [M+Na]+ (calculated for C18H20O3, 307.1310).
The crude product of Heck’s reaction of 1 with bromoxylene
was subjected to MPLC using RP-C18 Si Gel (MeOH–H2O, gradient
elution). Elution with (MeOH–H2O, 7:3, isocratic) afforded 15
(6 mg, 23.4%) and 14, (7.5, 29.3% mg).
3.5.5.6. (E)-1,2-Dimethoxy-4-[3-(4-methoxyphenyl)prop-1-enyl]-
benzene (8). Colorless amorphous powder, IR mmax (CHCl3) 3001,
2958, 2938, 2913, 2839, 1608, 1513, 1465, 1267, 1139, 1026, 966,
3.5.3. 2-Methoxyphenyl product 5
The crude product of Suzuki coupling reaction of 1 with 2-
methoxy phenylboronic acid coupling reaction was chromato-
graphed over Si Gel column using (n-hexane–EtOAc, gradient elu-
tion). The selected fraction eluted with (n-hexane–EtOAc, 9:1)
was further subjected to medium pressure column chromatogra-
phy using RP-C18 Si Gel (MeOH–H2O, 7:3, isocratic) afforded 5
(25 mg, 80%).
860, 834 cmꢀ1 1H and 13C NMR, see Tables 4 and 5. HRESIMS m/z
;
307.1313 [M+Na]+ (calculated for C18H20O3, 307.1310).
3.5.5.7. 5-[3-(3,4-Dimethoxyphenyl)prop-1-en-2-yl]-1,2,3-tri-
methoxybenzene (9). Colorless amorphous powder, IR mmax
(CHCl3) 3001, 2939, 2839, 1586, 1511, 1464, 1412, 1338, 1266,
1131, 1027, 1002 cmꢀ1 1H and 13C NMR, see Tables 4 and 5. HRE-
;
3.5.4. 4-Methoxyphenyl products 6–8
SIMS m/z 367.1509 [M+Na]+ (calculated for C20H24O5, 367.1521).
The crude brown product from Suzuki coupling reaction of 1
with 4-methoxy phenylboronic acid was purified using Si Gel col-
umn chromatography and (n-hexane–EtOAc, 9.5:0.5). The selected
fractions were subjected to medium pressure column chromatog-
raphy using RP-C18 Si Gel (MeOH–H2O, 8:2, isocratic) to afford 6
(12 mg, 15%) and a mixture of two isomers. The isomers were iso-
3.5.5.8. (E)-5-[3-(3,4-Dimethoxyphenyl)allyl]-1,2,3-trimethoxy-
benzene (10). Colorless amorphous powder, IR mmax (CHCl3) 3000,
2960, 2940, 2911, 2840, 1592, 1510, 1464, 1331, 1267, 1130,
1026, 1002, 966 cmꢀ1 1H and 13C NMR, see Tables 4 and 5. HRE-
;
SIMS m/z 367.1519 [M+Na]+ (calculated for C20H24O5, 367.1521).
lated using HPLC using a Phenomenex Luna 5 lm PFP(2) 100A col-
umn, 250 ꢁ 10 mm, and MeOH–H2O, 7:3 isocratic system to afford
3.5.5.9. (E)-5-[3-(3,4-Dimethoxyphenyl)prop-1-enyl]-1,2,3-tri-
methoxybenzene (11). Colorless amorphous powder, IR mmax
(CHCl3) 3001, 2960, 2940, 2913, 2840, 1592, 1514, 1465, 1330,
7 (3 mg, 37%) and compound 8 (3.5 mg, 37%).
3.5.5. 3,4,5-Trimethoxyphenyl products 9–13
1266, 1130, 1027, 1001, 965 cmꢀ1 1H and 13C NMR, see Tables 4
;
The crude brown product of Suzuki coupling reaction of 1
with 3,4,5-trimethoxy phenylboronic acid was purified over Si
gel 60 (n-hexane–EtOAc, gradient elution). The selected fraction
eluted with (n-hexane–EtOAc, 8:2) was subjected to MPLC using
RP-C18 Si Gel (MeOH–H2O, 5:5) to afford 9 (4.5 mg, 11%) and a
mixture of two isomers. These isomers were further purified using
and 5. HRESIMS m/z 367.1518 [M+Na]+ (calculated for C20H24O5,
367.1521).
3.5.5.10.
(E)-2-[3-(3,4-Dimethoxyphenyl)prop-1-enyl]-1,3-
dimethylbenzene (14). Colorless amorphous powder, IR mmax
(CHCl3) 3003, 2937, 2839, 1592, 1515, 1465, 1264, 1153, 1139,
1028, 976, 857 cmꢀ1; 1H and 13C NMR, see Tables 4 and 5. HRESIMS
m/z 305.1526 [M+Na]+ (calculated for C19H22O2, 305.1517).
semipreparative HPLC using a Phenomenex Luna 5 lm PFP(2) 100A
column, 250 ꢁ 10 mm, and MeOH–H2O, 7:3 isocratic system to
afford 10 (3.5 mg, 25%) and 11 (2.8 mg, 25%). The fraction eluted
with (n-hexane–EtOAc, 7:3) was purified using RP-C18 Si Gel
MPLC using (MeOH–H2O, 7:3, isocratic elution) to give the satu-
rated chalcone 13 (10 mg, 15.1%) and the chalcone 12 (25 mg,
12.3%).
3.5.5.11.
(Z)-2-[3-(3,4-Dimethoxyphenyl)allyl]-1,3-dimethyl-
benzene (15). Colorless amorphous powder, IR mmax (CHCl3) 3002,
2958, 2939, 2913, 2839, 1585, 1512, 1466, 1267, 1139, 1026,
965, 858 cmꢀ1 1H and 13C NMR, see Tables 4 and 5. HRESIMS m/
;
z 305.1522 [M+Na]+ (calculated for C19H22O2, 305.1517).
3.5.5.1. (E)-2-[3-(3,4-Dimethoxyphenyl)allyl]phenyl acetate
(2). Colorless amorphous powder, IR mmax (CHCl3) 3002, 2960,
2938, 2913, 2840, 1753, 1597, 1514, 1465, 1370, 1269, 1157, 1139,
1025, 966, 916, 861 cmꢀ1; 1H and 13C NMR, see Tables 2 and 3. HRE-
SIMS m/z 335.1265 [M+Na]+ (calculated for C19H20O4, 335.1259).
3.6. Invasion assay
Invasion activities of the methyl eugenol derivatives were mea-
sured using Trevigen’s CultrexÒ 96 well Basement Membrane Ex-
tract (BME) cell invasion assay kit against the highly metastatic
human breast cancer cell line MDA-MB-231.23,24 This assay employs
3.5.5.2. 2-[3-(3,4-Dimethoxyphenyl)prop-1-en-2-yl]phenyl ace-
tate (3). Colorless amorphous powder, IR mmax (CHCl3) 3002, 2937,
2839, 1755, 1593, 1515, 1465, 1370, 1263, 1140, 1084, 1028, 914,
a simplified Boyden Chamber design with an 8 lm polyethylene
terephthalate (PET) membrane. Detection of cell invasion is quanti-
fied using calcein-AM. The cells internalize calcein-AM, and intracel-
lular esterases cleave the acetomethylester (AM) moiety to generate
858 cmꢀ1 1H and 13C NMR, see Tables 2 and 3. HRESIMS m/z
;
335.1250 [M+Na]+ (calculated for C19H20O4, 335.1259).

F. M. Abdel Bar et al. / Bioorg. Med. Chem. 18 (2010) 496–507
507
free calcein, which fluoresces brightly, and this fluorescence used to
quantify the number of cells that have invaded across BME. The bot-
tom plate was read at 485 nm excitation, and 520 nm emission using
BioTeckÒ microplate reader to obtain relative fluorescence unit
(RFU). The RFU values were converted into cell number using a stan-
dard curve. The % invasion was obtained by dividing the number of
cells that invaded (through the BME) by the number of cells that mi-
grated (no coating).23,24
7, 8, 10, and 11) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2009.12.019.
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was derived using DISCOtechTM 22,28–30
Assignment of the initial
.
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ring centroids as hydrophobic centers, hydrogen bond donors and
acceptors, and external site points representing receptor-associ-
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Acknowledgment
31. Tripos Associates. SYBYL Molecular Modeling Software, version 8.0; Tripos
Associates: St. Louis, MO, 2007; http://www.tripos.com.
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The Egyptian Government is acknowledged for the fellowship
support to F.M.A.B.
Supplementary data
36. Welch, W.; Ruppert, J.; Jain, A. N. Chem. Biol. 1996, 3, 449.
37. Ruppert, J.; Welch, W.; Jain, A. N. Protein Sci. 1997, 6, 524.
Supplementary data (1H and 13C NMR spectra of compounds 2–
11 and 14–15 and HPLC chromatogram of isolations of compound