Olive secoiridoids and semisynthetic bioisostere analogues for the control of metastatic breast cancer

Article abstract of DOI:10.1016/j.bmc.2012.12.050

(-)-Oleocanthal (1) and ligstroside aglycone (2) are common bioactive olive oil secoiridoids. Secoiridoid 1 has been previously reported as a c-MET inhibitor. Chemically, (-)-oleocanthal is the elenolic acid ester of the common olive phenolic alcohol tyrosol. Therefore, several analogues (4-13) were synthesized by esterification and carbamoylation of tyrosol using diverse phenolic naturally occurring in olive and heterocyclic acids as elenolic acid bioisosteres to assess the effect of replacing the acid moiety of (-)-oleocanthal. Their c-MET inhibitory activity as well as their antiproliferative, antimigratory, and anti-invasive activities against the highly metastatic human breast cancer cell line MDA-MB231 has been assessed. Ligstroside aglycone (2) showed the best antimigratory activity. Generally, tyrosol esters showed better activities versus carbamate analogues. Tyrosol sinapate (5) showed the best c-MET phosphorylation inhibitory activity in Z′-LYTE kinase assay. Both 1 and 5 competitively inhibited the ATP binding into its pocket in the c-MET catalytic domain. Compound 5 showed selective activities against tumor cells without toxicity to the non-tumorigenic human breast MCF10A epithelial cell line. Tyrosol esters with a phenolic acid containing hydrogen bond donor and/or acceptor groups at the para-position have better anticancer and c-MET inhibitory activities. Olive oil secoiridoids are excellent scaffolds for the design of novel c-MET inhibitors.

Full text of DOI:10.1016/j.bmc.2012.12.050

Accepted Manuscript
Olive Secoiridoids and Semisynthetic Bioisostere Analogues for the Control of
Metastatic Breast Cancer
Belnaser A. Busnena, Ahmed I. Foudah, Tina Melancon, Khalid A. El Sayed
PII:
S0968-0896(13)00027-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.12.050
BMC 10518
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
18 October 2012
19 December 2012
28 December 2012
Please cite this article as: Busnena, B.A., Foudah, A.I., Melancon, T., El Sayed, K.A., Olive Secoiridoids and
Semisynthetic Bioisostere Analogues for the Control of Metastatic Breast Cancer, Bioorganic & Medicinal
Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2012.12.050
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Olive Secoiridoids and Semisynthetic Bioisostere Analogues for the Control of
Metastatic Breast Cancer
Belnaser A. Busnena, Ahmed I. Foudah, Tina Melancon, and Khalid A. El Sayed*
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at
Monroe, Monroe, LA 71201.
* To whom Correspondence should be addressed. Telephone 318-342-1725. Fax: 318-342-1737.
E-mail: elsayed@ulm.edu

ABSTRACT
(-)-Oleocanthal (1) and ligstroside aglycone (2) are common bioactive olive oil
secoiridoids. Secoiridoid 1 has been previously reported as a c-MET inhibitor. Chemically, (-)-
oleocanthal is the elenolic acid ester of the common olive phenolic alcohol tyrosol. Therefore,
several analogues (4-13) were synthesized by esterification and carbamoylation of tyrosol using
diverse phenolic naturally occurring in olive and heterocyclic acids as elenolic acid bioisosteres
to assess the effect of replacing the acid moiety of (-)-oleocanthal. Their c-MET inhibitory
activity as well as their antiproliferative, antimigratory, and anti-invasive activities against the
highly metastatic human breast cancer cell line MDA-MB231 has been assessed. Ligstroside
aglycone (2) showed the best antimigratory activity. Generally, tyrosol esters showed better
activities versus carbamate analogues. Tyrosol sinapate (5) showed the best c-MET
phosphorylation inhibitory activity in Z′-LYTE™ Kinase Assay. Both 1 and 5 competitively
inhibited the ATP binding into its pocket in the c-MET catalytic domain. 5 showed selective
activities against tumor cells without toxicity to the non-tumorigenic human breast MCF10A
epithelial cell line. Tyrosol esters with a phenolic acid containing hydrogen bond donor and/or
acceptor groups at the para-position have better anticancer and c-MET inhibitory activities.
Olive oil secoiridoids are excellent scaffolds for the design of novel c-MET inhibitors.
Keywords Antimigratory, Anti-invasive, Antiproliferative, Breast cancer, Carbamoylation, c-
MET, Esterification, Olive secoiridoids, Tyrosol sinapate, Wound-healing.

1. Introduction
c-MET is a tyrosine kinase receptor which is normally activated by its natural ligand
hepatocyte growth factor (HGF). c-MET/HGF pathway is critical for the normal cellular
development and hemostasis. Aberrant activation of this pathway involved in many cancer
types, which triggers diverse pathways inducing cell migration, motility, proliferation, cell
differentiation, and angiogenesis.1,2 Inhibiting c-MET kinase domain activation is among
strategies to target c-MET/HGF axis.1,2 The Mediterranean diet is associated with beneficial
health properties, including lower incidences of cardiovascular disease, age related cognitive
disease, and cancer.3 The Mediterranean countries have lower cancer incidence compared to the
rest of European countries and the United States. This includes reduced rates of the large bowel,
breast, endometrial, and prostate malignancies, which is attributed to the dietary practices; apart
from possible genetic factors.4 Olive oil is a key ingredient of the Mediterranean diet. In
addition to its unsaturated fatty acids, it is rich in other minor phenolics. These phenolics
include simple phenols, phenolic acids, flavonoids, lignans, and secoiridoids. Tyrosol (3) is the
major olive phenolic either as free phenolic alcohol or in combination as in secoiridoids
including (-)-oleocanthal (1), oleuropein aglycone, and ligstroside aglycone (2). Secoiridoid 1
recently reported to inhibit the c-MET kinase phosphorylation with IC50 value of 4.8 µM.5 It
also inhibited the proliferation, migration, and invasion of the human breast and prostate cancer
cell lines MCF7, MDA-MB231, and PC-3, respectively. On the other hand, 2 induced potent
tumoricidal effects by selectively triggering high levels of apoptotic cell death in HER2-
overexpressing breast carcinoma and showed moderate cytotoxicity against a panel of 39-
human cancer cell lines, in vitro.6,7 The alcohol moiety of both secoiridoids 1 and 2 is 3. Based
on the activity of 1 as a c-MET inhibitor, this study reports the synthesis of eight new and two

known tyrosol ester and carbamate analogues (4-13). Design of these analogues was based on
replacing the 1’s elenolic acid moiety with different bioisosteres, including natural olive-derived
phenolic acids, synthetic heteroaromatic, and diverse aromatic isocyanates. The c-MET
phosphorylation inhibitory activity was evaluated using Z′-LYTE™ Kinase Assay-Tyr6 Peptide
kit. Both 1 and 5 proved ATP-competitive c-MET phosphorylation inhibitors using the Omnia®
Kinase assay. The binding mode of 5 at the ATP binding site of three different crystal structures
of c-MET kinase domain (PDB code: 3I5N, 3CD8, 2WD1) was studied in-silico. The
antimigratory, antiproliferative, and anti-invasive activities of all compounds were evaluated
against the highly metastatic human mammary epithelial breast cancer cell line MDA-MB231.
Toxicity of all analogues was also evaluated against the non-tumorigenic human breast
epithelial cell line MCF10A to assess their selectivity.
2. Results and discussion
2.1 Chemistry
Five new (4, 6-8, and 10) and two known ester analogues (5 and 9) were semisynthesized
via the highly chemoselective Mitsunobu esterification of 3 with different olive-derived phenolic
acids and nalidixic acid to generate the corresponding esters in 60-80% yields (Scheme 1).
Tyrosol sinapate (5) was synthesized in 2007 to assess its antimicrobial activity and ability to
protect the cultured cells from H2O2-induced oxidative damage and consequent apoptosis.8
Tyrosol caffeate (9) was synthesized in 2005 to evaluate its effect on stimulus-induced reactive
oxygen species production in human neutrophils.9 The activity of 5 and 9 on the c-MET and its
related anticancer activities have never been reported. In addition, three new carbamate
analogues 11-13 were synthesized via the reaction of 3 with benzyl and p-tosyl isocyanates to

generate their corresponding carbamates in 60-70% yields (Scheme 2). Their identity was
confirmed by analyses of their 1H and 13C NMR (Tables 1-4) and HREIMS data.
The HREIMS of 4 showed an [M+H]+ peak at m/z 318.1103, suggesting the molecular
formula C17H18O6 and possible ester analogue of 3. The 1H NMR data of 4 (Table 1) showed the
downfield shifting of the methylene H2-1' (4.41, +0.74), compared to that of 3, suggesting
13
possible esterification of the primary alcohol C-1'. Esterification was further confirmed by C
NMR analysis, showing the downfield ester carbonyl C-1 (166.7, Table 2). NMR data of 4 also
showed typical aromatic and methoxy signals for the syringic acid moiety. The methylene triplet
H2-1' showed a 3J-HMBC correlation with the ester carbonyl C-1, confirming the identity of 4 as
tyrosol syringate. The structure identity of each of esters 5-10 was similarly confirmed via
extensive analyses of their spectral data (Tables 1-4and Supporting Information).
Reaction of 3 with benzyl isocyanate afforded 11 and 13. The HREIMS of 11 suggested
the molecular formula C16H17NO3 and possible monocarbamoyl analogue of 3. The downfield
shifting of the methylene H2-1' (3.81, +0.14), compared to that of 3, suggested possible
carbamoylation of the primary alcohol C-1'. Carbamoylation was further confirmed in 13C-
PENDANT spectrum through the downfield carbamate carbonyl C-1 (C156.2, Table 4). The 1H
13
and C NMR data of 11 (Tables 3 and 4) also showed typical monobenzyl carbamoyl signals.
The methylene triplet H2-1' and H2-2 (4.23) showed 3J-HMBC correlation with the carbamate
carbonyl C-1, confirming the identity of 11 as tyrosol monobenzyl carbamate.
The HREIMS of 13 suggested the molecular formula C24H24N2O4 and possible dibenzyl
carbamoyl analogue of tyrosol. The NMR data of 13 was similar to 11 with additional benzyl
carbamoyl moiety. The downfiled shift of the aromatic protons H-4'/H8' and H-5'/H-7', in

addition to the upfield shifting of C-6' (C 149.6) suggested the location of the second benzyl
3
carbamoyl moiety on C-6'. The benzylic methylene doublet (H 4.34) showed a J-HMBC
correlation with the carbamate carbonyl C-1'' (C 155.2), confirming the identity of 13 as tyrosol
dibenzylcarbamate. Analysis of the HREIMS and NMR data of 12 (Tables 3 and 4) proved its
identity as tyrosol p-tosyl carbamate (Supporting Information).
2.2.Biological Activity
The antimigratory, antiproliferative, cytotoxic, and anti-invasive activities of 2-13 were
evaluated using wound-healing (WHA), MTT, and Cultrex BME assays, respectively, against the
highly metastatic human breast cancer MDA-MB231 cell line. The MTT cytotoxicity assay was
also evaluated using the non-tumorigenic human breast epithelial cell line MCF10A. The Z′-
LYTE™ Kinase Assay kit was used to evaluate the inhibitory activity of 2-13 against c-MET
phosphorylation and compare their activity versus 1. The kinetics of the c-MET phosphorylation
inhibition of 1 and the most active bioisosteric analogue 5 has been assessed by Omnia® kinase
assay.
2.2.1. Wound healing assay (WHA)5
The wound-healing assay is a simple method for the in vitro study of the directional cell
migration. Generally, all tyrosol analogues showed better antimigratory activities than their
parent alcohol 3, but less activity than the natural secoiridoids 1 and 2. Figure 1 shows the effect
of the natural secoiridoid 2 and the phenolic alcohol 3 on the cell migration across the wound
inflicted in the MDA-MB231 cell monolayer compared to the vehicle control and a 10 µM dose
of 1 as a positive control.5 Tyrosol (3) showed no activity even at 50 M dose unlike 2, which
showed about 60% inhibition of the cell migration at 25 M dose. The calculated IC50 value of 2
was 13.8 µM. This clearly highlights the importance of the acylating acid and ester

pharmacophore in olive-derived secoiridoids for the antimigratory activity. The concentration of
1-13 that results in 50% inhibition of the cell migration, IC50, is reported in Table 6. The most
active semisynthetic analogue, 4, showed better activity at 25 M dose with 60% inhibition of
the cell migration compared to the other analogues (Figure 2). Its IC50 value was found to be
19.4 µM. Esters 5 and 6 were slightly more active than 7 at 25 M dose, suggesting the
importance of the hydrogen bond donor binding role of the hydroxyl groups of the acylating
acid’s aromatic ring. The activity of 5 was comparable to its c-MET phosphorylation inhibitory
activity. Meanwhile, although 2 and 4 were the most active in the wound-healing assay, they
showed weak c-MET activities, suggesting different molecular target(s) for their antimigratory
effect.
2.2.2. Invasion assay23
The anti-invasive activity of compounds 1-10, 12, and 13 were evaluated in the Cultrex®
BME cell invasion assay against the highly metastatic MDA-MB231 human breast cancer cell
line (Figure 3). The natural product 1 was used as apositive drug control.5 In a preliminary
screening at two different doses, 10 and 20 µM, tyrosol nalidixate (10) have shown the most
potent activity, allowing only 60 and 36 % invasion, respectively. In addition, 2, 5, and 9 have
shown more than 50 % inhibition of invasion at 20 µM. At this dose, analogues with carbonyl
group directly attached to the heterocyclic ring as in 10 inhibited the cell invasion better than
analogues with one carbon separating the carbonyl ester and the heterocyclic ring as in 2.
Removing or replacing the heterocyclic with aromatic ring decreased the activity as represented
by 1, 4-9, and 12-13. Additional carbons between the ester carbonyl and aromatic ring, in
addition to a para-hydroxyl group substitution at the aromatic ring can be alternative structural
feature to replace the heterocyclic ring and maintain the activity as represented by 5 and 9. The

activity level of 5 was parallel to its c-MET activity. Meanwhile, although the natural product 2
and the semisynthetic esters 9 and 10 were the most active in invasion assay, their weak c-MET
activity suggests molecular target(s) different from c-MET for their anti-invasive activity.
2.2.3. MTT assay10,11
The MTT assay allows the measurement of cell viability and proliferation of cell
populations in a quantitative colorimetric fashion by utilizing cellular ability to reduce the MTT
reagent to insoluble purple formazan dye. In this assay, each analogue was tested at four
concentrations; 25, 50, 75, and 100 µM. The concentration of each analogue that resulted in 50%
cell growth inhibition (IC50) was measured (Table 6).The natural olive secoiridoid 2 and the c-
MET inhibitor 5 were the most active antiproliferative analogues with IC50 values of 80.4 and
73.7 µM, respectively (Figure 4). The cytotoxic effect of all compounds 2-13 was tested against
the human mammary epithelial breast cancer cell line MDA-MB231. This experiment enabled
the confirmation of the fact that the antimigratory activity was not due to the cytotoxic effects of
tested compounds on MDA-MB231. All compounds were non-toxic at 50 µM except 10, which
was cytotoxic at this concentration. In addition, the toxicity of the most active analogues in both
WHA and MTT proliferation assays was assessed using MTT assay against the non-tumorigenic
human breast cell line MCF10A (Figure 5). All active analogues were non-toxic at
concentrations equal to their IC50 in both WHA and MTT proliferation assay, suggesting their
good selectivity toward the malignant cells.
2.2.4. Z′-LYTE™ c-MET kinase assay12
The Z′-LYTE™ Kinase Assay-Tyr6 Peptide kit (Invitrogen) was used to assess the in
vitro ability of 2, 3, and the semisynthetic analogues 4-13 to inhibit the c-MET phosphorylation.
Meanwhile, 1 was used as a positive standard control for activity comparison.5 Table 5 shows the

percentage of phosphorylation inhibition at a single 25 µM dose concentration for 1-13 except 1,
which was used at a 10 µM dose.5 Ester 5 was the most potent c-MET phosphorylation inhibitor,
with an IC50 value of 13.7 µM (Figure 6). The remarkable activity difference between 3 and 5 in
the c-MET inhibitory activity indicated the importance of the phenolic functionality in the acidic
part for binding. The importance of C-7 free phenolic hydroxy group for the c-MET activity was
highlighted by the complete activity loss of 7, which is the C-7 methyl ether of 5. This clearly
indicated the HBD role for the c-MET activity of 5. The reduced activity of carbamates suggests
the preference of the ester phramcophore for better c-MET activity. (-)-Oleocanthal (1) remained
the most potent c-MET inhibitor, compared to 2 and tyrosol esters or carbamates, indicating the
preference of elenolic acid over the rest of olive-derived phenolic acids. The calculated IC50 of 1
in this assay was 5.5 µM, which was consistent with the reported value (4.8 µM),5 validating this
study’s results.
2.2.5. Omnia® kinase assay13
Omnia® kinase Assay-Tyr12 peptide kit (Invitrogen) was used to assess the kinetic
modality of analogue 5 as c-MET inhibitor. We Studied the Michaelis-Menten parameters Km
and Vmax of an enzymatic reaction in presence of 5 (20, 10, and 5 µM) to determine the mode of
action of this c-MET inhibitor. The maximum observed velocity at enzyme saturating substrate
concentration is designated as Vmax. Michaelis-Menten constant, Km is the substrate
concentration that produces half-maximal velocity. Tyrosol sinapate (5) nearly did not affect the
Vmax at the end of experiment while the Km was increased (Figure 7). This pattern is well known
for the competitive inhibitors. To validate this assay, the known c-MET competitive inhibitor
SU11274 has been used as a positive control at 100 and 25 nM concentrations and showed
typical ATP competitive inhibitor pattern (Figure 7) .14 Similarly, (-)-oleocanthal (1) showed

ATP-competitive c-MET inhibitory pattern, validating the previously reported in-silico
conclusion.5
3. Molecular docking study
About 20 c-MET crystal structures were published with and without ligands, revealing
two distinct binding modes for ATP-competitive inhibitors. Type I ligands assume U-shape
geometry through interactions with hinge region Met1160 and activation loop residue Tyr1230,
unlike type II ligands, which adopt extended orientation.15-17 c-MET ATP binding site includes:
1) Hinge region: Met1160 and Pro1158. Interaction at this site is highly characteristic for all
compounds targeting the ATP binding site.5,15 2) Central hydrophobic region. 3) Two
hydrophobic subpockets. 4) c-MET activation loop (Asp1222-Lys1245).15-17
(-)-Oleocanthal (1) was identified via a computer-assisted study as a c-MET activation
inhibitor hit, targeting its ATP binding site, and stabilizing the kinase domain in its inactive
conformation.5 Docking of 1 into the ATP binding site of c-MET crystal structure PDB: 3I5N
demonstrated the formation of a hydrogen bond (HB) between 1’s phenolic hydroxyl group and
both of Pro1158 and Met1160.5 Two additional HBs observed between 1’s C-1 aldehyde and
Tyr1230 and Arg1086. 1 Filled the space between the hinge region and activation loop
(Pro1158/Asn1167-Tyr1230).5 The ester moiety was hypothesized not to contribute any binding
role yet it provided the proper molecular size and confirmation for adequate binding. A slightly
similar binding mode of 1 was noted with another c-MET crystal structure (PDB: 1R0P).5 Both
binding modes suggested that 1 can fully fit at the ATP binding site of the c-MET, which was
experimentally proved in this study.
In this study, three highly resolved c-MET kinase domain crystal structures (PDB: 3I5N,
3CD8, 2WD1) with a resolution of 2Aº were used to assess the binding mode of 5, the most

active tyrosol analogue, at the c-MET’s ATP binding pocket. Good binding affinity for any c-
MET active hit is hypothesized to be through interactions with at least one of Asp1222, Phe1223,
or Tyr1230 at the activation loop and either of Pro1158 or Met1160 at the hinge region.5,18
To validate the docking results, each of the c-MET’s crystal structure 3I5N, 3CD8, and
2WD1 ligand (14-16, respectively) was docked into its ATP binding pocket by using the same
parameters which have been used for docking analogue 5. Each ligand was almost overlaid the
original co-crystallized structure (Figure 8 and Supporting Information Figures 1.1 and 1.3).19-21
Tyrosol’s C-6' phenolic hydroxyl of 5 exhibited two hydrogen bonding interactions (HB)
with the hinge region's essential amino acids Met1160 and Pro1158 of the three c-MET crystal
structures (Figure 8 and Supporting Information Figures 1.2 and 1.4). The MOLCAD
visualization of the docked pose of 5 emphasized its complete fitting at the c-METʼ s ATP
pocket.22 Another HB interaction was observed between the aromatic C-7 phenolic hydroxyl
group of sinapic acid with ASN1167 in both crystal structures 3I5N and 3CD8 (Figure 8A and
Supporting Information Figure 1.2). Furthermore, a - interaction has also been observed
within 5Aº distance between the Tyr1230’s aromatic ring at the activation loop and the sinapic
acid’s aromatic ring in crystal structure 2WD1 (Figure 8 and Supporting Information Figure 4).
4. Conclusion
The natural secoiridoid 2 and the semisynthetic ester 4 were the most active in the wound-
healing migration assay without any cytotoxicity to normal cells. They showed minimal c-MET
inhibitory activity, which may indicate that their antimigratory activity could be via molecular
target(s) other than the c-MET. Tyrosol sinapate (5) is a new ATP-competitive c-MET kinase
phosphorylation inhibitory scaffold with antimigratory, antiproliferative, and anti-invasive

activities. It lacks cytotoxicity to the normal epithelial breast cells. Olive secoiridoids can be
good scaffolds for the control of metastatic breast cancer and design of novel c-MET inhibitors.
5. Material and methods
5.1. General experimental procedures
The 1H and 13C NMR spectra were recorded in CDCl3 and using TMS as an internal standard
1
13
on a JEOL Eclipse-400 NMR spectrometer, operating at 400 MHz for H and 100 MHz for C
NMR. The HREIMS experiments were conducted at Louisiana State University on a 6200-TOF
LC–MS (Agilent), equipped with a multimode source (mixed source that can ionize the
compounds alternatively by ESI and APCI). Thin layer chromatography (TLC) analysis was
carried on precoated Si gel 60 F254 500 µm TLC plates (EMD Chemicals), using n-hexane-ethyl
acetate (5:5) as a mobile phase. 1% Vanillin in concentrated H2SO4 was used as a visualizing
reagent. For column chromatography, Si gel 60 (Natland, 63–200 m) and Sephadex LH-20
were used. A Dionex Summit III high performance liquid chromatography (HPLC) system with
a variable wave length UV detector set to 230 nm was used. Separation was performed on a
Phenomenex Cosmosil 5C18-AR-II column (250 mm x 4.6 mm, 5 µm; Phenomenex Inc.,
Torrance, CA) at 25 °C. Isocratic elution was performed using H2O-CH3CN (6:4) as a mobile
phase. A flow rate of 1mL/min, 25 L injection volume and 1 mg/mL sample concentration were
used. Generally, 1:100 ratios of mixtures to be chromatographed versus used stationary phase
were used in all liquid chromatographic purifications.
5.2. Compounds
(-)-Oleocanthal, tyrosol, and ligstroside aglycone were isolated from extra-virgin olive oil as
described below. HPLC, was prepared and used as previously described.23,24 SU11274 was

purchased from Sigma-Aldrich. Sinapic, syringic, caffeic, homovanillic, 3,4,5-trimethoxycinnamic,
nalidixic, and 2,3-dihydroxylphenylacetic acids as well as p-toluenesulfonyl and benzyl
isocyanates were purchased from VWR (Suwanee, GA).
5.3. Extraction and isolation of olive phenolics23,24
About 2L methanol and 3L extra-virgin olive oil (Memberʼs Mark, Batch No. VF2
US011410, Italy) were mixed, and then n-hexane (2L) was added to the methanolic fraction and
shaken twice in separating funnels. Dried methanolic layer was subjected to repeated medium
pressure liquid chromatography (MPLC) in a 50 × 3 cm column on lipophilic Sephadex LH20
(Sigma Aldrich, bead size 25–100 µ) using CH2Cl2, then CH2Cl2-MeOH, gradient elution,
followed by HPLC analysis to separate 1-3 (Supporting Information Figure 2). Identification and
purity of these compounds were based on HPLC analysis on a preparative Cosmosil AR-II
column (Waters, Milford MA, 250 mm x 20 mm) and comparison of their 1H and 13C NMR data
with the literature.23,24
5.4. Chemical reactions
5.4.1. Chemoselective esterification of tyrosol using Mitsunobu reaction25
To a cooled (0 ºC) solution of freshly prepared tyrosol (138 mg, 1 mM) and phenolic
acids (1 mM equivalent) in dry THF (3.5 mL) were added triphenylphosphine (280 mg, 1 mM)
and diisopropylazodicarboxylate (208 µL, 1 mM). After stirring for 48 h at rt, the reaction was
worked up by removal of the solvent, and the residue was partitioned between ethyl acetate and
saturated NaHCO3. The organic phase was washed with brine, dried over anhydrous Na2SO4, and
evaporated. The residue was purified on Sephadex LH-20 (35 mL after CH2Cl2 swelling) using
CH2Cl2 to remove the Mitsunobu byproducts, followed by normal phase chromatography to
isolate the esters 4-10.

5.4.2. 6`-Hydroxyphenethyl-5-hydroxy-4,6-dimethoxybenzoate (Tyrosol syringate, 4)
Compound 4 was prepared in 60% yield by reaction of 138 mg of 3 with 198 mg of
syringic acid. Colorless oil; IR max(CDCl3) 3940, 3053, 1707, 1264, 898 cm-1; 1H and 13C NMR,
see Tables 1 and 2; HREIMS m/z 319.1173 [M+H]+ (calcd for C17H19O6, 319.1181).
5.4.3. (E)-6`-Hydroxyphenethyl-3-(7-hydroxyl-6,8-dimethoxyphenyl)acrylate (Tyrosol
sinapate, 5)8
Compound 5 was prepared in 85% yield by reaction of 138 mg of 3 with 224 mg of
sinapic acid. Colorless amorphous powder; IR max(CDCl3) 3747, 1702, 1514, 1263, 898 cm-1;
1H and 13C NMR, see Tables 1 and 2; HREIMS m/z 345.1336 [M+H]+ (calcd for C19H21O6,
345.1338).
5.4.4. 6`-Hydroxyphenethyl-1`-(3,4-dihydroxyphenyl)acetate (6)
Compound 6 was prepared in 50% yield by the reaction of 138 mg of 3 with 168 mg of
3,4-dihydroxyphenylacetic acid. Colorless oil; IR max(CDCl3) 3882, 1725, 1517, 1257, 890
1
cm-1; H and 13C NMR, see Tables 1 and 2; HREIMS m/z 311.0885 [M+Na]+ (calcd for
C16H16O5Na, 311.0895).
5.4.5. (E)-6`-Hydroxyphenethyl-3-(6,7,8-trimethoxyphenyl)acrylate (7)
Compound 7 was prepared in 60% yield by reaction of 138 mg of 3 with 238 mg of 3,4,5-
trimethoxycinnamic acid. Yellowish white amorphous powder; IR max(CDCl3) 3941, 3054,
1
13
1705, 1268, 903 cm-1; H and C NMR, see Tables 1 and 2; HREIMS m/z 381.1347 [M+Na]+
(calcd for C20H22O6Na, 381.1314).

5.4.6. 6`-Hydroxyphenethyl-2-(6-hydroxyl-5-methoxyphenyl)acetate (Tyrosol
homovanillate, 8)
Compound 8 was prepared in 60% yield by reaction of 138 mg of 3 with 182 mg of
homovanillic acid. Colorless oil; IR max(CDCl3) 3748, 1728, 1264, 898, cm-1; 1H and 13C NMR,
see Tables 1 and 2; HREIMS m/z 325.1043 [M+Na] + (calcd for C17H18O5Na, 325.1052).
5.4.7. (E)-6`-Hydroxyphenethyl-3-(7,8-dihydroxyphenyl)acrylate(Tyrosol caffeate, 9)9
Compound 9 was prepared in 60% yield by reaction of 138 mg of 3 with 180 mg of
1
13
caffeic acid. Colorless oil; IR max(CDCl3) 3940, 1703, 1264, 898 cm-1; H and C NMR, see
Tables 3 and 4.
5.4.8. 6`-Hydroxyphenethyl-1-ethyl-7-methyl-1,4-dihydro-1,8-naphthyridine-3-carboxylate
(Tyrosol nalidixate, 10)
Compound 10 was prepared in 70% yield by reaction of 138 mg of 3 with 232 mg of
nalidixic acid. Colorless amorphous powder; IR max(CDCl3) 3747, 1722, 1264, 914 cm-1; 1H and
13C NMR, see Tables 3 and 4; HREIMS m/z 353.1303 [M+Na]+ (calcd for C20H20N2O4Na,
375.1321).
5.4.9. Carbamoylation of tyrosol26
To solutions of tyrosol in toluene (2 mL), benzyl or p-tosyl (toluenesulfonyl) isocyanates
10 µL of triethylamine (Et3N) were added. Each solution was separately stirred and refluxed for
3 h. Water (10 mL) was added and each reaction mixture was extracted with ethyl acetate (3x10
mL). Each ethyl acetate extract was dried over anhydrous Na2SO4, concentrated under vacuum.
and subjected to Sephadex LH20 and Si gel 60 normal phase column chromatography. Reaction

of 3 with benzyl isocyanate afforded compounds 11 and 13 while the same reaction with p-tosyl
isocyanate afforded 12.
5.4.10. 6`-Hydroxyphenethylbenzyl carbamate (11)
Compound 11 was prepared in 60% yield by reaction of 138 mg of 3 with 133 mg of
1
13
benzyl isocyanate. Colorless powder; IR max (CDCl3) 3940, 1727, 1264, 898 cm-1; H and C,
see Tables 3 and 4; HREIMS m/z 272.1296 [M+H] + (calcd for C16H18NO3, 272.1287).
5.4.11. 6`-Hydroxyphenethyltosyl carbamate (12)
Compound 12 was prepared in 70% yield by reaction of 138 mg of 3 with 197 mg of p-
toluenesulfonyl isocyanate. Colorless amorphous powder; IR max (CDCl3) 3826, 1747, 1160,
1
817 cm-1; H and 13C NMR, see Tables 3 and 4; HREIMS m/z 358.0737 [M+H] + (calcd for
C16H17NO5SNa, 358.0725).
5.4.12. 1`,6`-Phenethyldibenzyl carbamate (13)
Compound 13 was prepared in 65% yield by reaction of 138 mg of 3 with 133 mg of benzyl
1
isocyanate. Colorless amorphous powder; IR max (CDCl3) 3940, 1727, 1264, 898 cm-1; H and
13C NMR, see Tables 3 and 4; HREIMS m/z 406.1886 [M+2H]+ (calcd for C24H26N2O4,
406.1893).
5.5. Molecular modeling and docking
Three-dimensional structure modeling was performed using the SYBYL-X Program
Package,27 installed on DELL desktop workstations equipped with a dual 2.0 GHz Intel® Xeon®
processor running the Red Hat Enterprise Linux (version 5) operating system. Conformations of
all compounds were generated using Confort™ conformational analysis. Energy minimizations

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). Partial atomic
charges were calculated using the semiempirical program MOPAC 6.0 and applying the AM1.
Surflex-Dock Program version 2.0 interfaced with SYBYL-X was used to dock all compounds at
the ATP binding site of c-MET. Surflex-Dock employs an idealized active site ligand (protomol)
as a target to generate putative poses of molecules or molecular fragments.28 These putative
poses were scored using the Hammerhead scoring function.29 The 3D structures (PDB: 3I5N,
3CD8, 2WD1) were taken from the Research Collaboratory for Structural Bioinformatics Protein
Data Bank (http://www.rcsb.org/pdb).
5.6. Cell culture
Breast cancer and non-tumorigenic cell lines, MDA-MB231 and MCF10A, respectively,
were purchased from ATCC (Manassas, VA). The cell lines were grown in RPMI 1640 medium
(GIBCO-Invitrogen, NY) with 10% fetal bovine serum (FBS) and supplemented with glutamine
(2 mmol/L), penicillin G (100 µg/mL), and streptomycin (100 µg/mL) at 37 ºC under 5% CO2.
5.6.1. Preparation of various dilutions of tested analogues for cell culture assays
A stock solution was prepared by dissolving each tested analogue in DMSO at a
concentration of 50 mM for all assays. About 2 µL of each stock solution was transferred to 998
µL of serum-free medium to obtain 100 µM concentrations (0.2% DMSO). Serial dilutions were
then conducted to obtain the desired concentrations for each assay. The negative control was
prepared as follows: adding 2 µL DMSO to 998 µL serum-free media (MTT assay); adding 3 µL
DMSO to 1497 µL serum-free media (WHA).
5.7. MTT proliferation assay

The antiproliferative activity of tyrosol and its analogues was tested in culture on the human
mammary epithelial breast cancer cell line, MDA-MB231. Cell growth was measured using
MTT kit (TACS™, Trevigen® Inc.). Cells in exponential growth were plated in a 96-well plate at
a density of 8 x 103 cells per well, and allowed to attach over-night at 37 ºC under 5% CO2.
Complete growth medium was then replaced with 100 µL of 5% RPMI fresh serum media
(GIBCO-Invitrogen, NY) containing various doses (25, 50, 75, and 100 µM) of the specific test
compound and incubation resumed at 37 ºC under 5% CO2. After 48 h, the media was replaced
and the cells retreated with compounds using 5% RPMI fresh serum media for another 24 h. The
cells were refreshed with 100 µL RPMI fresh serum free media before adding MTT solution (50
µL/well) and re-incubated for 4 h. The color reaction was stopped by the addition of
solubilization/stop solution (DMSO, 100 µL/well), and incubation at 37 ºC was continued to
ensure complete dissolution of the formazan product. Absorbance of the samples was determined
at λ570 nm with an ELISA plate reader (BioTek, VT, USA).
5.8. Cultrex® BME cell invasion assay
Anti-invasive activities were measured using Cultrex® BME cell invasion assay.30 About
50 μL of basement membrane extract (BME) coat was added per well of the top chamber. After
an over-night incubation at 37 °C in a 5% CO2 atmosphere, 50,000/50 μL of MDA-MB231 cells
in 0.5% FBS RPMI medium were added per well of the top chamber. 150 μL of RPMI medium
was then added to the lower chamber. Media contained 10% FBS and penicillin/streptomycin as
well as fibronectin (1 μL/mL) and N-formyl-Met-Leu-Phe (10 nM) as chemoattractants. Tested
compounds were prepared at 6X the desired concentration and 10 μL of each of the compounds
was added per well of the top chamber. Cells were incubated at 37 °C under 5% CO2, which
allowed for cell invasion from the top to the lower chamber. After 24 h, the top and bottom

chambers were aspirated and washed with washing buffer supplied within the kit. About 100 μL
of cell dissociation solution/calcein-AM solution was added to the bottom chamber and
incubated at 37 °C under 5% CO2 for 1h. The cells internalize calcein-AM, and the intracellular
esterases cleave the acetomethyl ester (AM) moiety to generate free calcein. Fluorescence of the
samples was determined at λexcitation 485 nm and λemission 528 nm using an ELISA plate reader
(BioTek, VT, USA). The number of cells that invaded through the BME coat was calculated
using a standard curve.
5.9. MTT cytotoxicity assay
The cytotoxic effects of the tyrosol analogues were tested in culture of the human mammary
epithelial breast cancer cell line MDA-MB231 and the non-tumorigenic human breast MCF10A
cell line. Cell growth was measured using MTT kit (TACS™, Trevigen® Inc.). Cells in
exponential growth were plated in a 96-well plate at a density of 20 x103 cells per well, and
allowed to attach over-night at 37 ºC under 5% CO2. Complete growth medium was then
replaced with 100 µL of RPMI serum free medium and 0.5% RPMI serum medium (GIBCO-
Invitrogen, NY) containing various doses (25, 50, 75, and 100 µM) of the tested compounds in
case of MCF10A and MDA-MB231, respectively. After that, the incubation resumed at 37 ºC
under 5% CO2 for 24 h. The cells were then refreshed with 100 µL RPMI fresh serum free media
before adding the MTT solution (50 µL/well) and re-incubated for 4 h. The color reaction was
stopped by the addition of solubilization/stop solution (DMSO) (100 µL/well), and incubation at
37 ºC was continued to ensure complete dissolution of the formazan product. Absorbance of the
samples was determined at λ 570 nm with an ELISA plate reader (BioTek, VT, USA). The
cytotoxicity IC50 of 2 against MDA-MB231 has been reported to be 67 µM.7
5.10. Wound healing assay

MDA-MB231 cells were plated onto sterile 24-well plates and allowed to form a confluent
cell monolayer per well (>95% confluence) over-night. Wounds were then inflicted in each cell
monolayer using a sterile 200 µL pipette tip. Media was removed and cells were washed twice
with PBS and once with fresh serum free media. Test compounds at the desired concentrations in
0.5% fresh serum media were added to each well. The incubation was carried out for 24 h, after
which media was removed and cells were washed, fixed and stained using Diff-Quick™ staining
(Dade Behring Diagnostics, Aguada, Puerto Rico). Cells which migrated across the inflicted
wound were counted under the microscope in three or more randomly selected fields (400X).31,32
5.11. Z′-LYTE™ kinase assay
Z′-LYTE™ Kinase Assay-Tyr 6 Peptide kit (Invitrogen) was used to assess the ability of
tyrosol analogues to inhibit c-MET phosphorylation. Briefly, 20 µL/well reactions were set up in
96-well plates containing kinase buffer, 200 µM ATP, 4 µM Z′-LYTE™ Tyr6 Peptide substrate,
2500 ng/mL c-MET kinase and compound of interest as an inhibitor. After 1 h of incubation at
rt, 10 µL development solution containing site-specific protease was added to each well.
Incubation was continued for 1 h. The reaction was then stopped, and the fluorescent signal ratio
of 445 nm (coumarin)/520 nm (fluorescin) was determined on a plate reader (BioTek-
FLx800™), which reflects the peptide substrate cleavage status and/or the kinase inhibitory
activity in the reaction.
5.12. Omnia® kinase assay
Omnia® kinase Assay-Tyr12 peptide kit (Invitrogen) was used to assess the kinetics of c-
MET inhibitors. Assay reagents prepared as outlined by the manufacturer’s manual.33 The assay
was performed in half-area 96-well plate (Perkin Elmer, Waltham, MA). The volume of reaction
in each well was 40 µL. For kinetics study, kinase (100 ng/well), SU11274 dilutions (100 and 25

nM), analogue 1 and 5 dilutions (20, 10, 5 µM) were used. ATP concentrations used were: 1000,
800, 500, 333, 250, 200, 125, 100, 50 and 20 µM. The peptide control Master Mix,
phosphopeptide generation Master Mix, and kinetic reaction Master Mix as well as the assay
steps were adopted according to the manufacturer’s manual.30 Reaction mixtures were then
incubated for 60 min at 30 °C. The relative fluorescence units (RFU) data was collected every 70
seconds by fluorescence microplate reader (FLx800, Biotek Instruments, Inc.). The ∆RFU was
then calculated by subtracting the RFU value of peptide control wells (containing no kinase)
from the RFU value of kinase reaction for each time point. The background was subtracted and
∆RFU is plotted against time and log MET concentration. Curve-fitting to Michaelis-Menten
equation was conducted using Prism 5.0 software from GraphPad Prism Software, Inc.
Acknowledgment
The Libyan Government is greatly acknowledged for the fellowship support of B.B.
References and notes
1. Satter, M.; Salgia, R. Curr. Oncol. Rep. 2007, 9, 102.
2. Liu, X.; Newton, C. R.; Scherle, A. P. Expert Opin. Invest. Drugs. 2011, 20, 1225.
3.Colomer, R.; Menendez, J. A. Clin. Transl. Oncol. 2006, 8, 15.
4. Lastra, C.; Barranco, M. D.; Motilva, V.; Herrerias, J. M. Curr.Pharm. Des. 2001, 7, 933.
5. Elnagar, A. Y.; Sylvester, P. W.; El Sayed, K. A. Planta Medica. 2011, 77, 1013.
6. Menendez, J. A.; Vazquez, A.; Garcia, R.; Carrasco, A.; Oliveras, C.; Fernandez, A.; Segura,
A. BMC Cancer. 2008, 8, 377.
7. Kikuchi, M.; Uehara, Y.; Kikuchi, M.; Mano, N.; Machida, K. J. Nat. Med. 2011, 65, 237.

8. Stevenson, E. D.; Parkar, G. S.; Zhang, J.; Stanley, A. R; Jensen, J. D.; Cooney, M. J. J.
Enzyme Microb. Techno. 2007, 40, 1078.
9. Lee, Y. T.; Don, M. J.; Liao, C. H.; Chiou, H. W.; Chen, C. F.; Ho, L. K. Clin. Chem. Acta.
2005, 352, 135.
10. TACS™ MTT Cell Proliferation Assays Protocol; www.trevigen.com, accessed on
22/05/2011.
11. Mosmann, T. J. Immunol. Methods. 1983, 65, 55.
12. Rodems, S. M.; Hamman, B. D.; Lin, C.; Zhao, J.; Shah, S.; Heidary, D.; Makings, L.; Stack,
J. H.; Pollok, B. A. Assay Drug Dev. Technol. 2002, 1, 9.
13. Shults, M. D.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 14248.
14. Berthou, S.; Aebersold, D. M.; Schmidt, L. S.; Stroka, D.; Heigl, C.; Streit, B.; Stalder, D.;
Gruber, G.; Liang, C.; Howlett, A. R.; Candinas, D.; Greiner, R. H.; Lipson, K. E.; Zimmer, Y.
Oncogene 2004, 23, 5387.
15. Schiering, N.; Knapp, S.; Marconi, M.; Flocco, M. M.; Cui, J.; Perego, R.; Rusconi, L.;
Cristiani, C. PNAS. 2003, 100, 12654.
16. Wang, W.; Marimuthu, A.; Tsai, J.; Kumar, A.; Krupka, H. I.; Zhang, C.; Powell, B.; Suzuki,
Y.; Nguyen, H.; Tabrizizad, M.; Luu, C.; West, BL. PNAS 2006, 103, 3563.
17. Christensen, J.; Burrows, J.; Salgia, R. Cancer Lett. 2005, 225, 1.
18. Peach, L. M.; Tan, N.; Choyke, J. S.; Giubellino, A.; Athauda, G.; Burke, R. T.; Jr.; Nicklaus,
C. M.; Bottaro, P. D. J. Med. Chem. 2009, 52, 943.
19. Boezio, A. A.; Berry, L.; Albrecht, K. B.; Bauer, D.; Bellon, F. S.; Bode, C.; Chen, A.;
Choquette, D.; Dussault, I.; Fang, M.; Hirai, S.; Kaplan, P.; Larrow, J. F.; Lin, J. M.; Lohman, J.;

Potashman, H. M.; Rex, K.; Santostefano, M.; Shah, K.; Shimanovich, R.; Springer, K. S.;
Teffera, Y.; Yang, Y.; Zhang, Y.; Harmange, J. J. Bioorg. Med. Chem. Lett. 2009, 19, 6307.
20. Albrecht, K. B.; Harmange, J.; Bauer, D.; Berry, L.; Bode, C.; Boezio, A. A.; Chen, A.;
Choquette, D.; Dussault, I.; Fridrich, C.; Hirai, S.; Hoffman, D.; Larrow, J. F.; Kaplan, P.; Lin,
J.; Lohman, J.; Long, M. A.; Moriguchi, J.; O’Connor, A.; Potashman, H. M.; Reese, M.; Rex,
K.; Siegmund, A.; Shah, K.; Shimanovich, R.; Springer, K. S.; Teffera, Y.; Yang, Y.; Zhang, Y.;
Bellon, F. S. J. Med. Chem. 2008, 51, 2879.
21. Porter, J.; Lumb, S.; Franklin, J. R.; Gascon, M. G.; Calmiano, M.; Le Riche, K.; Lallemand,
B.; Keyaerts, J.; Edwards, H.; Maloney, A.; Delgado, J.; King, L.; Foley, A.; Lecomte, F.;
Reuberson, J.; Meier, C.; Batchelor, M. Bioorg. Med. Chem. Lett. 2009, 19, 2780.
22. Heiden, W.; Goetze, T.; Brickmann, J. J. Comp. Chem. 1993, 14, 246.
23. Christophoridou, S.; Dais, P.; Tseng, L. H.; Spraul, M. J. Agric. Food Chem. 2005, 53, 4667.
24. Montedoro, G.; Servili, M.; Baldioli, M.; Selvaggini, R.; Miniati, E.; Macchioni, A. J. Agric.
Food Chem. 1993, 41, 2228.
25. Appendino, G.; Minassi, A.; Daddario, N.; Bianchi, F.; Tron, G. Org. Lett. 2002, 4, 3839.
26. Hassan, M. H.; Elnagar, Y. A.; Khanfar, A. M.; Sallam, A. A.; Rabab, M.; Shaala, A. L.;
Youssef, T. D.; Hifnawy, S. M.; El Sayed, A. K. Eur. J. Med. Chem. 2011, 46, 1122.
27. Tripos Associates. SYBYL Molecular Modeling Software, version 8.0; Tripos Associates: St.
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December 1, 2012.

1'
OH
O
OCH3
H3CO
H3CO
3'
OCH3
DIAD, TPP, THF
rt / 48 hr
OH
+
7
1
O
5'
OCH3
12
7'
1'
5'
3
HO
OCH3
O
OH
Tyrosol trimethoxycinnamate (7)
Tyrosol (3)
Trimethoxycinnamic acid
Scheme 1. Synthesis of tyrosol ester analogue 7.
OH
O
H
O
1
O
N
8
S
S
5
Toluene
2
+
N
O
HO
O
O
TEA / Reflux 2 hr
O
OH
p-Toluenesulfonyl isocyanate
4- Hydroxyphenylethyltosyl carbamate (12)
3
Tyrosol ( )
Scheme 2. Synthesis of tyrosol carbamate analogue 12.

1
CHO
MeO2C
O
6
8
( )
E
HO
3`
O
7
O
(S)
5
8'
7`
OR2
1`
3'
1'
O
O
HO
10
5`
OHC
R1O
CHO
3
5'
Oleocanthal (1)
Ligstroside aglycone (2)
10
OCH3
9
OCH3
OCH3
OH
4
OCH3
5
OH
OH
OH
5
5
O
1
7
7
4
1
1
1
OCH3
12
OCH3
8
OCH3
7
3
9
9
3
7
3
O
O
O
a
b
c
d
O
9
5
4
8
OH
10
OCH3
O
4
OH
5
H
O
S
OH
5
7
1
H
N
O
7
1
1 2
N
N
9
N
2
i
1
11
7
O
2
8
7
O
12
3
9
O
3
1
O
e
f
g
h
Compound R1 R2
H
H
H
H
H
H
H
H
H
H
h
H
a
b
c
d
e
f
g
h
i
3
4
5
6
7
8
9
10
11
12
13
h
N
N
N
N
N
NH2
N
X
O
S
O
N
O
O2N
N
OCH3
X
14 NH
15 O
16

120
100
80
60
40
20
0
DMSO
1
3
2
Controls
25
50
Concentration µM
Figure 1. Antimigratory activity of 2 and 3 against the human breast cancer cells MDA-MB231.
Error bars indicate the SD of n = 3/dose. DMSO was used as a negative control while 1 was used
as a positive control at 10 µM dose.5
120
100
DMSO
1
80
3
5
4
7
6
60
40
20
0
Controls
25
50
75
100
Concentration µM
Figure 2. Antimigratory activity of 3-7 against the human breast cancer cells MDA-MB231.
Error bars indicate the SD of n = 3/dose. DMSO was used as a negative control while 1 was used
as a positive control at 10 µM dose.5

120
100
80
60
40
20
0
10 μM 20 μM
Compound
Figure 3. Anti-invasive activities of 1-10, 12, and 13 against the human breast cancer cell line
MDA-MB231 using Cultrex® BME cell invasion assay kit. Each concentration was run in
triplicate and the data are expressed as the mean±SEM. 1 was used as a positive control.5
80000
70000
60000
DMSO
50000
40000
30000
20000
10000
0
5
4
3
2
Control
25
50
75
100
Concentration µM
Figure 4. Antiproliferative activity of 2-5 against MDA-MB231 cells. Error bars indicate the SD
of n = 3/dose.

70000
60000
50000
40000
30000
20000
10000
0
DMSO
7
8
6
4
5
2
Control
25
50
Concentration µM
Figure 5. Cytotoxic activity of 2-6 against non-tumorigenic human breast MCF10A epithelial
cell line. Error bars indicate the SD of n = 3/dose.
120
100
80
60
DMSO
40
1
5
20
0
Controls
10
20
25
50
-20
Concentration µM
Figure 6. Percentage of c-MET phosphorylation by various doses of 5 using Z′-LYTE™ assay
kit. Error bars indicate the SD of n = 3 per dose. 1 was used as a positive control.5