Simple di- and trivanillates exhibit cytostatic properties toward cancer cells resistant to pro-apoptotic stimuli

Article abstract of DOI:10.1016/j.bmc.2010.04.047

A series of 33 novel divanillates and trivanillates were synthesized and found to possess promising cytostatic rather than cytotoxic properties. Several compounds under study decreased by >50% the activity of Aurora A, B, and C, and WEE1 kinase activity at concentrations <10% of their IC₅₀ growth inhibitory ones, accounting, at least partly, for their cytostatic effects in cancer cells and to a lesser extent in normal cells. Compounds 6b and 13c represent interesting starting points for the development of cytostatic agents to combat cancers, which are naturally resistant to pro-apoptotic stimuli, including metastatic malignancies.

Full text of DOI:10.1016/j.bmc.2010.04.047

Bioorganic & Medicinal Chemistry 18 (2010) 3823–3833
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Simple di- and trivanillates exhibit cytostatic properties toward cancer
cells resistant to pro-apoptotic stimuli
Delphine Lamoral-Theys a, Laurent Pottier b, Frédéric Kerff e, François Dufrasne c, Fabien Proutière b,
Nathalie Wauthoz d, Philippe Neven f, Laurent Ingrassia b, Pierre Van Antwerpen c, Florence Lefranc b,
Michel Gelbcke c, Bernard Pirotte f, Jean-Louis Kraus g, Jean Nève c, Alexander Kornienko h, Robert Kiss b,
Jacques Dubois a
*
,
a Laboratoire de Chimie Analytique, Toxicologie et Chimie Physique Appliquée, Institut de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
b Laboratoire de Toxicologie, Institut de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
c Laboratoire de Chimie Pharmaceutique Organique, Institut de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
d Laboratoire de Pharmacie Galénique et Biopharmacie, Institut de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
e Centre d’Ingéniérie des Protéines, Institut de Chimie B6, Université de Liège, Liège, Belgium
f Laboratoire de Chimie Pharmaceutique, Centre Interfacultaire de Recherche du Médicament, Faculté de Médecine et de Pharmacie, Université de Liège, Liège, Belgium
g Laboratoire de Chimie Biomoléculaire, CNRS, IBDML-UMR-6216, Campus de Luminy Case 907 13288, Marseille cedex 09, France
h Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 December 2009
Revised 8 April 2010
Accepted 16 April 2010
Available online 21 April 2010
A series of 33 novel divanillates and trivanillates were synthesized and found to possess promising cyto-
static rather than cytotoxic properties. Several compounds under study decreased by >50% the activity of
Aurora A, B, and C, and WEE1 kinase activity at concentrations <10% of their IC50 growth inhibitory ones,
accounting, at least partly, for their cytostatic effects in cancer cells and to a lesser extent in normal cells.
Compounds 6b and 13c represent interesting starting points for the development of cytostatic agents to
combat cancers, which are naturally resistant to pro-apoptotic stimuli, including metastatic
malignancies.
Keywords:
Polyphenol
Kinase
Ó 2010 Elsevier Ltd. All rights reserved.
Cancer
Apoptosis resistance
Cytostatic activity
1. Introduction
thereby survive after detachment from their primary site when
they travel through the lymphatic and circulatory systems.1 Fur-
Cancer remains a devastating disease and the number of cancer-
related deaths is increasing. More than 90% of cancer patients die
from tumor metastases.1 Metastatic cancer cells are intrinsically
resistant to apoptosis and, therefore, unresponsive to a large
majority of anticancer drugs available today, because the latter
generally work through apoptosis induction.2–5 As a barrier to
metastasis, cells normally undergo apoptosis after they lose con-
tact with extracellular matrix or neighboring cells.1 This cell death
process has been termed ‘anoikis’.1 Tumor cells that acquire malig-
nant potential have developed mechanisms to resist anoikis and
thermore, many cancers are naturally resistant to apoptosis and,
thus, associated with dismal prognoses. These include glioblas-
toma (GBM), the height of malignancy in the glioma group (the
most common primary brain cancer),6 melanomas,7 esophageal
cancers,8 and non-small-cell-lung cancers (NSCLC),9 among others.
Thus, drug resistance, either acquired or intrinsic, is often related
to inability of tumor cells to undergo apoptosis, resulting in cancer
cell survival and treatment failure.2,3 One solution to apoptosis
resistance entails the complementation of cytotoxic therapeutic
regimens with cytostatic agents, such as drugs targeting specific
protein tyrosine kinases or membrane receptors.4–6,10,11
Natural polyphenols, including for example the green tea poly-
phenol (À)-epigallocatechin-3-gallate (EGCG),12 genistein,13 and
curcumin,14 are intensely investigated as potential anticancer
agents, because they are able to inhibit multiple enzyme activities
and signal transduction pathways, resulting in suppression of cell
proliferation (e.g., inducing cytostatic effects) and enhancement
of apoptosis (e.g., the cytostatic effects that end with cytotoxic
Abbreviations: ATCC, American type culture collection; DSMZ, Deutsche Samm-
lung von Mikroorganismen and Zellkulturen; ECACC, European collection of cell
culture; GBM, glioblastoma; MTT, 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide; NSCLC, non-small-cell-lung carcinoma; QVM, quantitative
videomicroscopy; RA, residual activity.
* Corresponding author. Tel.: +32 477 62 20 83; fax: +322 332 53 35.
E-mail address: rkiss@ulb.ac.be (R. Kiss).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.047

3824
D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
ones), as well as inhibition of cell invasion, angiogenesis, and
metastases.12–19
chain. In contrast, alkylation reactions with dibromoalkanes al-
lowed us to obtain directly in one step the desired diesters with
n P 4 (4a, 4b, 5b, 6a, 6b, 6c, 6d, 7a, 7b, 7c, 7d, 8b, 9b, 9d, 10b,
and 11b) in 7–72% yields. In addition to compounds containing
the vanillic moiety, we obtained analogues incorporating dimeth-
oxybenzoic (6a and 7a), isovanillic (6c and 7c), and dihydroxyben-
zoic ester residues (6d, 7d, and 9d). Unfortunately, the
corresponding alkylations with dibromoethane and dibromopro-
pane (n = 2 and 3) only led to the formation of mono-esters, prob-
ably as a result of bromide hydrolysis by way of anchimeric
assistance in medium-sized mono-ester intermediates. Therefore,
the short-chain products 2b and 3b were synthesized using a
three-step procedure starting from ethylene and propylene glycols
and involving the protection of the phenolic function as benzyl
ether (Scheme 2). Thus, vanillic acid was reacted with benzyl chlo-
ride to afford 1bBn in 63% yield. Subsequent esterification with n-
alkyl diols (n = 2 and 3) and hydrogenolysis then gave the desired
divanillates 2b and 3b in 21% and 25% overall yields, respectively.
Using various commercially available alkylating agents, we ex-
panded the diversity of our polyphenols by preparing compounds
incorporating unsaturated (4f and 4g) and aromatic (12a and
12b) linkers as well as trisubstituted analogues (13a–13c, Scheme
3). Because the alkylation yields decrease with the increase in the
number of free phenol functions in the products (only 7% for tetra-
phenol 9d), to obtain highly hydroxylated compounds, such as
digallates 6e and 7e, we chose again the three-step route involving
the protection of the phenolic functionality (Scheme 4). Com-
pounds 6e and 7e were obtained in overall yields of 9% and 21%,
respectively, by way of the benzylation, alkylation and deprotec-
tion sequence (Scheme 4).
Further, to increase the linker rigidity, we synthesized cyclohex-
anediol-based divanillates. Cyclohexane diols 14a–14c and 15,
were esterified with the benzyl-protected vanillic acid (1bBn)
and the benzyl group was removed by catalytic hydrogenation
using palladium on charcoal to give 17a–17c and 18 in 12–24%
overall yields (Scheme 5). When the same procedure was applied
to aminotriol 16 (chosen for the possibility of a positive charge
at the core of the molecule), only the disubstituted analogue 19
was obtained in 3% overall yield over three steps.
Finally, as esters can be cleaved by esterases in biological media,
we designed a more biologically stable, bioisosteric bis-tetrazole
analogue 22a (Scheme 6). A two-step procedure was envisaged:
formation of the tetrazole 21 from the corresponding nitrile 2030
followed by alkylation under the same conditions used for benzoic
acid derivatives. In the event, the major product was the less steri-
cally hindered 2,5-2,5 regioisomer 22a, but we also were able to
isolate a small amount of the more hindered 1,5-2,5 regioisomer
22b (Scheme 6). The structures of both regioisomers were con-
firmed by an NOE analysis (see the Supplementary data). The over-
all yields for 22a and 22b were 40% and 4%, respectively. The
synthesis of bis-tetrazoles with free phenol functions is currently
under investigation.
In this paper we describe a novel series of di- and trivanillate-
based polyphenols with cytostatic anticancer activity in vitro, as
evidenced by computer-assisted phase-contrast microscopy
(quantitative videomicroscopy).20,21 We chose scaffolds incorpo-
rating two and three vanillic ester moieties because vanillin dis-
plays anticancer activity of its own, including anti-metastatic
effects.22,23 Some of the divanillates synthesized in the current
work display structural similarities with curcumin, which there-
fore was used as an internal control.
Thirty-three compounds were synthesized and their IC50
in vitro growth inhibitory values were determined by means of
the MTT colorimetric assay20,21 in nine human and one mouse can-
cer cell lines. Because the MTT colorimetric assay can be problem-
atic when reducing compounds such as polyphenols are assayed,24
we also employed quantitative videomicroscopy20,21 to validate
the obtained data. The panel of 10 cancer cell lines under study
included four cell lines for which we accumulated experimental
evidence indicating that they display high levels of resistance to
pro-apoptotic stimuli. These four cell lines include the human
U37320,25 and T98G25 glioblastoma, the human A549 non-small-
cell-lung carcinoma (NSCLC)9,26 and the human OE21 esophageal
cancer.27 In contrast, we also obtained experimental evidence for
the sensitivity to pro-apoptotic stimuli of human Hs683 oligoden-
droglioma,25 human MCF-7 breast cancer,28 human PC-3 prostate
cancer28 and mouse B16F10 melanoma29 cell lines. Compound-in-
duced modifications in cell morphology and cell proliferation were
monitored by means of quantitative videomicroscopy.20,21 Sixteen
of the 33 compounds under study were further evaluated with
MTT colorimetric assay and computer-assisted phase-contrast
microscopy in two human normal (fibroblast) cell lines in order
to determine their bioselectivity. The ability of most of the com-
pounds under study to inhibit Aurora A, B, and C, and WEE1 kinase
activity have then been determined after having characterized the
effects of divanillate 6b on a panel of 256 kinases in order to char-
acterize its multi-kinase inhibitor profiles.
2. Results and discussion
2.1. Chemistry
Using the above-mentioned report on vanillin,22,23 we opted to
proceed with simple linear and branched diols and triols. To this
end, esterification reactions of diversely substituted benzoic acids
1a–1d with linear alkane diols and dibromides of varying chain
length were investigated (Scheme 1). In the presence of the free
phenol functionality in 1a–1d, esterification using dicyclohexylcar-
bodiimide (DCC) and dimethylaminopyridine (DMAP) did not fur-
nish the desired products regardless of the length of the alkyl
2.2. Pharmacological evaluation
2.2.1. Determination of the IC50 in vitro growth inhibitory
values
Thirty-three compounds were synthesized in the current study
and we utilized the MTT colorimetric assay to determine their IC50
in vitro growth inhibitory values in nine human and one mouse can-
cercelllines. Thehistologicaloriginofeachcancercelllineisdetailed
in the legend to Table 1. As already mentioned in Section 1, we pre-
viously demonstrated high levels of resistance to pro-apoptotic
stimuli for the human U37320,25 and T98G25 GBM, the A549
NSCLC,9,26 and the OE21 esophageal cancer27 cell lines. In the same
manner, we demonstrated the sensitivityto apoptosis for the human
Scheme 1.

D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
3825
Scheme 2.
We could not attain the 95% purity for 4 out of the 33 com-
pounds under study, namely 8b, 12a, 18, and 19 (Table 1). These
compounds were withdrawn from further biological analyses. We
used quantitative videomicroscopy (QVM) in order to potentially
identify false positive compounds from the MTT assay data
(Fig. 2).24 All compounds that were characterized as active against
a given cancer cell line (IC50 <100 lM) by means of the MTT color-
imetric assay also appeared to be active against these cancer cells
when assayed by means of QVM (data not shown). The same obser-
vation was made with respect to the inactive compounds (IC50
>100 lM). Thus, neither false positive nor false negative IC50 values
exist in Table 1. However, while performing the QVM quality con-
trol experiments, we identified 6 of the 33 compounds, namely 4f,
6a, 6c, 7a, 22a, and 22b, which were not completely soluble in the
biological media at their IC50 in vitro growth inhibitory concentra-
tion, as illustrated in Figure 2. We took this observation into ac-
count during the interpretation of the kinase assay-related data
(vide infra).
The mean growth inhibitory IC50 values across the nine human
cancer cell lines for the 24 out of 33 compounds for which the solu-
bility and purity levels were reached are given in Figure 3. As is seen
from these data, compounds 9b, 10b, and 11b (divanillates incorpo-
rating two phenolic functions and the longest linker, n P 9), 7d
(divanillate incorporating four phenolic functions and an intermedi-
ate linker, n = 7), 13a, 13c (trivanillates) displayed the most potent
in vitro antitumor activity. The IC50 in vitro growth inhibitory values
obtainedwiththese sixcompoundsare on aparwith thosedisplayed
by the most active natural polyphenols analyzed heretofore.15 The
anticancer activitydid not seem significantlyaffected by the number
of phenolic functions. Indeed, the potencies were similar (p >0.05)
for the compounds incorporating 2 (6b, 7b), 4 (6d, 7d) or 6 (6e, 7e)
phenolic hydroxyls. In contrast, the C-chain length seemed to signif-
Scheme 3.
Hs683 oligodendroglioma,25 MCF-7 breast cancer,28 PC-3 prostate
cancer,28 and mouse B16F10 melanoma29 cell lines. We observed
no statistically significant (p >0.05) differences in the IC50 values
for any compound under study between cancer cell lines that are
sensitive or resistant to pro-apoptotic stimuli (Table 1). In the same
manner, no differences in sensitivity were observed between the hu-
man and the mouse cancer cell lines (Table 1). This indicates that
mouse cancer models can be used in the future to determine
in vivo antitumor activity for the leads that will be derived from
the presently investigated polyphenols.
Scheme 4.

3826
D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
Scheme 5.
Fig. 3).Figure 4 morphologically illustrates the levels of bioselectiv-
ity exhibited by 6b and 13c.
The data in Table 1 indicate that a number of our divanillates
display growth inhibitory potencies similar to that of curcumin
(23, Fig. 1), used as a reference for the divanillate-based polyphe-
nols. However, Figure 4 reveals that curcumin displays cytotoxic
effects at its IC50 in vitro growth inhibitory concentration, a feature
that was not observed with compound 6b (Fig. 4) sharing struc-
tural similarities with curcumin. These observations strongly sug-
gest that curcumin exerts its antitumor effects through modes of
action distinct from those associated with the divanillates under
study, as also strongly suggested by the data we report below with
respect to kinase activity measurements. Figure 4 also clearly
shows that curcumin is considerably more toxic to normal Wi38
fibroblasts than 6b or 13c.
2.2.3. The compounds under study are cytostatic rather than
cytotoxic
The QVM analyses clearly indicated that all compounds under
study are cytostatic, not cytotoxic, as illustrated in Figure 4 for
compounds 6b and 13c. The cells have not been washed and the
medium together with the dead cells have not been removed be-
fore the pictures were taken. In fact, the quantitative videomicros-
copy approach recorded all the events occurring on the bottom of
the flask during a 72-h period of observation and less than 15%
dead cells appeared during this period of observation, the reason
why we claim that the compounds under study induce cytostatic
rather than cytotoxic effects. In contrast, curcumin (23) clearly ap-
pears to be cytotoxic (Fig. 4), while displaying in vitro anticancer
activity not higher than those displayed by the most active com-
pounds under study (Fig. 3). Thus, the in vitro anticancer activity
of our polyphenols is related to cell proliferation delay or blockade,
rather than cell killing (Fig. 4). The sustained delay or blockade in
cancer cell proliferation then in turn leads to cell death several
days after the cells have been challenged with the compounds
(data not shown). Flow cytometry analyses performed at random
for various compounds under study revealed that none of them in-
duced apoptosis during the three-day period that was used to
determine the MTT colorimetric assay-related IC50 values of each
compound (data not shown). The fact that compounds 6b and
13c induce cytostatic rather than cytotoxic anticancer effects
prompted us to assay the inhibition activity of 6b on a panel of
256 kinases.
Scheme 6.
icantly influence in vitro anticancer activity with a progressive drop
in potency with the increase in linker length from n = 2 to n = 5, and
then the recovery of activity with longer linkers (n = 6–11). Finally,
the replacement of the vanillic moiety (6b, 7b) by the isovanillic
one (6c, 7c) brought about a partial loss of solubility for 6c (Table
1). The replacement of the esters with bioisosteric tetrazoles (22a,
22b) led to a loss of solubility (Table 1).
2.2.2. Compound bioselectivity
We define bioselectivity as the mean IC50 (normal cells lines)/
mean IC50 (tumor cell lines) ratio. Table 2 reveals varied bioselec-
tivity ratios for the compounds under study. First, increasing the
length of the C-chain linker in divanillic polyphenols is paralleled
by the drop in bioselectivity (Table 2), while the anticancer activity
increases (Fig. 3). Second, adding the fluoro (13b) or chloro (13c)
substituents to the trivanillate scaffold (13a) seems to increase
bioselectivity (Table 2), with 13c being significantly (p <0.01) more
active than 13b in terms of in vitro anticancer activity (Table 1;

Table 1
Determination of in vitro anticancer activity in nine human and one (B16F10) mouse cancer cell lines
Compounds
% Purity
% Stabil.b
# chem. synth. stepc
In vitro IC50 growth inhibitory valuesa
(
l
M; * = >100
l
M; ** = >1000
l
M)
QVM
QC
U373
T98G
HS683
LOVO
A549
MCF-7
PC-3
OE21
OE33
m
SEM
6
B16F10
1b
1bMe
2b
3b
4b
5b
6b
7b
8b
99
99
100
98
96
99
96
96
89
96
96
95
98
96
100
100
95
100
95
96
96
100
100
84
96
99
98
100
90
82
100
98
97
99
100
100
100
96
100
99
100
96
100
100
98
CAd
CA
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
1
1
1
1
3
3
3
3
3
1
1
1
**
*
41
*
**
*
59
*
91
*
56
58
45
32
30
32
*
31
*
*
24
11
31
45
38
*
72
64
46
70
*
76
72
*
28
37
25
40
55
25
**
*
82
*
*
*
**
83
10
50
54
94
34
44
94
24
28
29
30
18
82
41
44
26
34
48
43
*
**
*
69
*
65
*
**
*
45
89
80
*
**
*
57
88
67
*
**
77
56
*
*
*
**
*
**
**
*
28
45
94
*
22
10
4
+
+
+
+
+
+
+
+
+
+
+
+
À
+
À
À
+
+
+
+
+
À
+
+
+
+
+
+
+
+
+
+
+
À
À
+
*
63
64
67
*
54
>88
>80
>96
46
49
46
29
28
27
>60
31
>73
>59
53
26
37
56
43
>97
50
53
49
50
>74
47
48
>96
28
44
31
33
98
69
22
44
38
36
30
31
68
35
46
*
54
31
37
52
35
*
39
65
66
20
53
43
27
*
28
37
31
38
91
18
63
50
40
27
25
22
50
30
49
46
44
10
24
53
32
*
40
47
58
57
*
49
69
95
27
25
31
31
80
22
27
51
39
24
30
21
54
32
46
43
46
18
32
72
30
*
39
56
35
27
23
25
55
25
*
44
69
21
28
53
58
*
39
44
30
36
26
25
90
42
86
43
88
44
68
81
44
*
46
42
42
59
77
35
34
87
30
44
36
40
*
68
46
62
28
28
27
44
34
49
53
49
41
37
31
43
73
76
56
34
41
45
55
46
81
16
73
28
28
81
27
63
47
28
28
28
28
52
36
*
64
55
34
45
73
66
*
61
66
45
66
77
56
55
*
19
71
40
36
*
6
2
6
1
1
1
9b
9
6
10b
11b
98
100
97
11
68
26
*
30
10
1
***e
6c
7c
2
***
6a
88
7a***
6d
7d
9d
6e
100
100
96
97
100
98
100
99
99
97
97
96
97
95
97
97
98
98
99
6
4
4
5
4
1
27
22
57
36
24
37
49
59
38
40
36
41
31
7
7e
***
4f
4g
36
46
26
44
64
28
31
78
29
48
33
32
41
24
40
48
63
43
76
38
65
*
23
26
26
26
52
27
39
44
57
47
75
46
32
80
55
41
33
25
71
24
6
3
4
6
12a***
12b
17a
17b
17c
5
6
***
***
18
19
13a
13b
13c
22a***
22b***
23
4
5
2
2
2
2
CA
38
*
10
96
99
96
99
>75
26
33
35
2
a
The IC50 in vitro growth inhibitory concentrations were determined by means of the MTT colorimetric assay. The cell lines include the human U373 (ECACC code 89081403), T98G (ATCC code CRL1690), and Hs683 (ATCC code
HTB-138) glioblastoma, the LoVo (DSMZ code ACC350) colon cancer, the A549 (DSMZ code ACC107) NSCLC, the MCF-7 (DSMZ code ACC115) breast cancer, the PC-3 (DSMZ code ACC465) prostate cancer, the OE21 (ECACC code
96062201), and OE33 (ECACC code 96070808) esophageal cancer.
b
The stability of products was measured by HPLC analysis following incubation in a physiological solution at 37 °C over 7 days. Results are expressed as the percentage of the incubated compound recovered.
Number of chemical synthetic steps to obtain the compound.
CA: commercially available.
c
d
e
The compounds labeled ‘***’ have been withdrawn from kinase assay analyses because either not pure enough (purity <95%), not stable enough (stability at 37 °C for 7 days <85%) or precipitating at the IC50 concentration as
revealed by quantitative videomicroscopy-related quality control (see the QVM QC column and Fig. 2).

3828
D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
Figure 1. Structures of vanillic acid, methyl vanillate and curcumin.
Figure 3. IC50 in vitro growth inhibitory concentrations for 25 out of 33 compounds
for which the solubility and purity levels were reached, determined by means of the
MTT colorimetric assay. The data are presented as means SEM calculated on nine
values, each value representing in turn the mean value calculated on sextuplicates.
The nine values correspond to the nine human cancer cell lines given in Table 1.
2.2.4. Divanillate 6b targets multiple kinases
Compound 6b, which displaysa bioselectivityindex>2.2(Table 2),
was assayed at 20
concentration represents ca. 50% of its IC50 in vitro growth inhibitory
concentration(Table1).At20 M6bdecreasedby>50%theactivityof
lM on a panel of 256 human kinases. This 20 lM
When Table 1 reports a mean value >100
lM for a given cell line, we used the
l
‘105 M’ value in calculation of the mean SEM IC50 numbers. Thus, the loss of
l
eight kinases, that is, Aurora A, Aurora B, Aurora C, CDK7/CycH, FER,
SAK, WEE1, and WNK2 (data not shown). Aurora,31,32 CDK7,33,34
Fer,35,36 SAK,37,38 WEE1,39 and WNK2,40,41 that is, the kinases targeted
by 6b, are over expressed in various types of cancers and display ma-
jor roles in cancer cell biology.
Cancer cells exhibit deregulation in multiple cellular signaling
pathways and treatments using specific agents that target only
one pathway usually fail in cancer therapy, especially for those can-
cer types that display natural resistance to pro-apoptotic stim-
uli.32,42–48 As recently emphasized by Sarkar and Li,49
combination treatments using chemotherapeutic agents with dis-
tinct molecular mechanisms are considered more promising and
some natural products, including polyphenols, have growth inhibi-
tory effects on human apoptosis-resistant cancer cells mediated by
targeting multiple cellular signaling pathways without causing un-
wanted toxicity in normal cells. Well-known modes of action by
antitumor activity for the less active compounds is underestimated, while the
potencies of the most active analogues are reflected accurately.
which polyphenolic compounds exert their anticancer activity in-
clude for example redox-dependent and redox-independent mech-
anisms underlying cytotoxic/cytostatic effects toward tumor cells
and cytoprotection of normal cells,16 phototoxicity against tumor
cells and photoprotection of non-tumor cells,16 and inhibition of
specific proteases17 in addition to well-documented effects on cell
cycle and/or the inhibition of the NF-j
B pathway.19 In contrast,
the polyphenol-induced anticancer effects mediated through ki-
nase targeting have not been studied adequately as we indicated
in our recent review of this topic.15 The polyphenol-related multi-
kinase inhibition usually occurs in the double digit micromolar con-
centration range,15 unlike that of synthetic small molecules de-
signed to selectively inhibit the activity of a given kinase, which
Figure 2. Quantitative videomicroscopy analyses of in vitro anticancer activity displayed by compounds 7d and 22a in the human U373 glioblastoma cell line. Compounds 7d
and 22a were assayed at their IC50 in vitro growth inhibitory concentrations, that is, 32 and 35 lM, respectively (Table 1). Compound 22a appeared non soluble in the
biological media as illustrated by marked precipitation processes.

D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
3829
Table 2
Comparison of in vitro global cell population growth development in two human normal versus nine human cancer (see Table 1) cell lines
Compounds
In vitro IC50 growth inhibitory values (
lM)
Ratio normal/cancerb
Normal human cell lines
Human cancer cell line (meana)
WI38
NHDF
Mean
2b
3b
4b
5b
6b
7b
6d
7d
6e
7e
8b
9b
10b
13a
13b
13c
>100
>100
>100
>100
>100
74
86
89
>100
>100
61
92
>96
>100
>100
>100
>100
78
79
87
>90
>98
75
54
>88
>80
>96
46
49
53
26
56
43
46
29
28
28
44
31
>1.8
n.c.c
n.c.
n.c.
>2.2
1.6
1.5
3.3
>1.6
>2.3
1.6
>100
>100
>100
>100
81
72
84
79
95
89
32
29
30
69
37
30
46
20
74
31
38
25
72
1.1
1.3
0.9
1.6
65
51
1.6
a
The m SEM values were calculated from the values reported in Table 1.
The mean IC50 value in the normal group was divided by the mean IC50 value in the cancer group. The normal cell lines analyzed include the human WI-38 (ATCC code
b
CCL-75) and NHDF (Promo Cell c-12300) fibroblast cell lines.
c
n.c. means ‘not calculated’. The ratio was not calculated when both mean IC50 values in normal and cancer cell lines were associated with ‘>’.
often occurs in nanomolar concentrations.31,44 The current data
clearly show that several compounds under study (as detailed be-
low) display multi-anti-kinase activity in single digit micromolar
concentration range.
binds in the hydrophobic pocket adjacent to the third phosphate-
binding site, the ester being involved in two hydrogen bonds
(Fig. 6A). The linker does not appear to make significant contribu-
tions. For compound 13c, the lowest energy solution (À10.7 kcal/
mol) gives a conformation very similar to compound 6b with
two phenols in almost identical positions (Fig. 6B). The third phe-
nol covers Thr217, an active site residue not conserved in Aurora B
and C. The third ester forms a hydrogen bond with a nitrogen back-
bone of the N-terminal lobe loop delimiting the upper side of the
nucleotide-binding site.
The best solutions for these two compounds in Aurora B are dif-
ferent from the ones observed for Aurora A. In the lowest energy
conformation of compound 6b (À6.8 kcal/mol), the linker partially
occupies the purine position and participates in hydrophobic inter-
actions. In addition, one vanillic ester interacts with the hinge re-
gion, while the second one is in the sugar binding region making
three hydrogen bonds, including one with Glu177 which substi-
tutes Thr217 in Aurora B and C (Fig. 6C). On the other hand, in
the best solution (À9 kcal/mol), compound 13c virtually wraps
around Glu177, with one ester group interacting with the hinge re-
gion, another one with the sugar region and the last one with the
solvent accessible region (Fig. 6D).
2.2.5. Characterization of anti-Aurora and anti-WEE1 kinase
activity
Although not as potently as some of the specifically designed
reported Aurora kinase inhibitors,31,32 compound 6b targeted the
three Aurora kinases as detailed above. Figure 5A illustrates the
dose–response inhibition curves for a dozen of compounds with
respect to Aurora A kinase activity, including curcumin (23). We
performed the same types of analyses with respect to Aurora B,
Aurora C, and WEE1 kinases. However, we report in Figure 5B
the
lM concentrations inducing 50% decrease in Aurora A, B, C,
and WEE1 kinases instead of illustrating each dose–response curve
as in Figure 5A. We thus determined the IC50 inhibitory values of
various divanillates (with curcumin as a reference) and trivanil-
lates on the three Aurora kinases to compare with the WEE1 kinase
that was also targeted by 6b in our initial screening using a 20 lM
concentration (data not shown). Incorporating 4 (6d, 7d; data not
shown) or 6 (6e, 7e; Fig. 5B) phenolic hydroxyls leads to the ability
of the compounds to progressively inhibit WEE1 activity in addi-
tion of inhibiting Aurora kinase activity. The same feature was ob-
served when adding fluoro (13b) or chloro (13c) substituents on
the trivanillate scaffold (13a) (Fig. 5B).
According to AUTODOCK 4.1, the affinities of compound 6b for
Aurora A and B correspond to estimated inhibition constants (ki) of
about 5
l
M. For 13c, the estimated ki are below 50 nM. The affinity
M. The
of ATP for a kinase is usually considered to be around 10
l
Curcumin (23), which shares structural similarities with divan-
illates such as 6b and 7b, displayed similar Aurora/WEE1 kinase
inhibition profiles when compared to 6b and 7b, while exerting
less potent activity inhibition than 6b and 7b (Fig. 5B). Curcumin
did not inhibit WEE1 kinase activity, even at concentrations as high
binding energies reported are therefore compatible with a compet-
itive inhibition of the Aurora kinases.
An additional factor that needs to be taken into account while
analyzing the computer-generated solutions is flexibility of the ki-
nase active site.50,51
as 100 lM (Fig. 5B).
2.2.7. Aurora kinase inhibition is responsible, at least partly, for
the in vitro anticancer activity
2.2.6. Molecular docking of 6b and 13c to Aurora A and Aurora B
kinases
We investigated whether the in vitro anticancer activity of the
compounds under study could be related, at least partly, to Aurora
kinase inhibition. We observed a weak but nevertheless statisti-
cally significant (p <0.05) correlation between the compound-in-
duced inhibitory activities for the three Aurora kinases and their
anticancer activities. Indeed, the compounds manifesting the high-
est anti-Aurora kinase activities, also display the highest in vitro
anticancer activity. However, this analysis revealed the absence
To further validate the role of Aurora kinase targeting in anti-
cancer effects of our synthetic polyphenols, we performed molecu-
lar docking experiments. The best docking solution for compound
6b on Aurora A is characterized by a binding energy of À7.2 kcal/
mol. One of the vanillic esters binds in the purine binding pocket,
allowing for favorable hydrophobic interactions and one hydrogen
bond with the hinge region (Fig. 6A). The second vanillic ester

3830
D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
Figure 4. Illustration of modest levels of bioselectivity observed for compounds 6b and 13c, but the absence of bioselectivity for curcumin (23). The compounds were assayed
at their respective IC50 in vitro growth inhibitory concentrations, that is, 22 M for 6b (Table 1), 31 M for 13c (Table 1), and 18 M for 23 (Table 1), in the human U373
glioblastoma cell line and 50 M for the three compounds in the human Wi38 fibroblast cell line. The cell lines were challenged with the compounds for 72 h and the
l
l
l
l
morphological illustrations are shown in the figure. Bioselectivity is defined as the mean IC50 (normal cells lines)/mean IC50 (tumor cell lines) ratio. The bioselectivity indices
are provided in Table 2.
of any specificity of inhibitory activity of a given compound for a
given Aurora kinase (data not shown).
ether from Na/benzophenone, CH2Cl2 and toluene from CaCl2.
The 1H NMR and 13C NMR spectra were recorded on a BRUKER
AVANCE 300 instrument (Brucker, Wissembourg, France) in
CDCl3 or DMSO-d6 or a mixture for solubility reasons. For com-
pounds 22a, and 22b, 1D spectra and NOE difference experi-
ments were recorded on a VARIAN VNMR SYSTEM 600. The
chemical shifts d are in ppm and the coupling constants J are
in Hz. Analytical thin-layer chromatography was carried out on
Macherey–Nagel SILG/UV254 precoated silica gel plates
(0.25 mm) and visualization was performed by UV and/or by
development using a 5% solution of phosphomolybdic acid in
ethanol (heating to 110 °C for 5–20 min). Flash column chroma-
In conclusion, the current study reveals that very simple divan-
illic or trivanillic polyphenols display promising anticancer cyto-
static activity through multi-kinase inhibition (including Aurora
A, B, and C, and WEE1) and exhibit modest levels of bioselectivity.
Compounds 6b (a divanillate) and 13c (a trivanillate) could serve
as hits for optimization of the cytostatic activity through the
improvement of the efficacy in inhibiting kinases over expressed
in cancer as compared to normal cells, and therefore the bioselec-
tivity of this type of polyphenols.
tography was carried out using (40–63 lm) silica gel at moder-
ate pressure. HPLC analyses were performed on an Agilent
3. Experimental
1100 series HPLC system (Agilent, Diegem, Belgium). The chro-
3.1. General experimental procedures
3.1.1. Chemistry
matographic system was an ultrasphere 5
l
 4.6 mm  250 mm
C18 (Beckman Coulter (Analis, Namur, Belgium)) using the same
mobile phase for all compounds (except 13b and 13c): MeOH–
water 50:50 to 100:0 for 10 min, then 100:0 for 15 min. For
3.1.1.1. General methods.
Before their use, the solvents
were distilled and dried by standard methods, that is, THF and

D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
3831
by silica gel chromatography using an eluent specified for the Rf of
each product.
3.1.1.3. General procedure for the three-step synthesis using
benzyl ether as phenol protecting group.
Selective protec-
tion of the phenol function of vanillic acid: To a stirred solution
of a required substituted benzoic acid (1 equiv) in THF (15 mL),
a solution of NaOH (2.5 equiv) in water (37 mL) is added. This
mixture is cooled to 0 °C and benzyl chloride (4.1 mL,
34.8 mmol) is added. The reaction mixture is allowed to warm
to rt and is then heated at 70 °C for 18 h. The temperature of
the reaction is then raised to 90 °C for 4 h. After cooling to rt,
the organic solvent is evaporated and the residual aqueous phase
is acidified with 2 M HCl. The precipitate is filtered and washed
with cyclohexane to afford a white solid. Selective protection of
dihydroxybenzoic and gallic acids is performed according to Bagga-
ley et al.52 esterification: A required diol (100–200 mg), DMAP
(2.1 equiv) and the phenol-protected benzylic acid derivative
(2.5 equiv) are dissolved in toluene (10 mL per 100 mg diol).
Then, DCC (2.3 equiv) are added. The reaction mixture is stirred
at rt for 3–4 days. The solvent is evaporated to dryness and the
crude product is directly purified by silica gel chromatography
using an eluent given for the Rf of each product. Deprotection:
The benzylated compound (200–500 mg, 1 equiv) is added to
MeOH (20 mL for 100 mg of starting product) and the solution
is cooled by a water-ice bath before the addition of the 10%
Pd/C (same weight as that of the benzylated compound). This
mixture is then placed under hydrogen atmosphere and
stirred at rt overnight. The reaction mixture is filtered through
silica gel and then
necessary.
a flash chromatography is performed if
Figure 5. (A): Dose–response curves of Aurora A residual activity with compounds
6b, 7b, 6d, 7d, 6e, 7e, 9b, 11b, 13a, 13b, 13c, and 23 (curcumin). (B) Concentrations
(lM) inducing 50% decrease in Aurora A, B, C, and WEE1 kinase activities. The data
3.1.1.4. Three-step synthesis involving alkylation instead of
esterification. The protocols for protection and deprotection
are presented as means SEM values calculated in triplicates.
are the same as in Procedure 2. The alkylation step is described in
Procedure 1.
13b and 13c, the mobile phase was 70% H2O/30% MeCN + 0.1%
TFA without gradient. The detection system was an agilent Diode
Array Detector G1315B (monitoring wavelength given for each
compound) (Agilent, Diegem, Belgium). The purity given is the
purity measured by this HPLC method but we are aware of the
fact that it does not take into account the co-crystallized sol-
vents detected by NMR and elemental analysis. When there
are some, the elemental analysis gives the compound-to-solvent
molar ratio. The elemental analyses were recorded on a thermo
Flash EA1112 series elemental analyzer (Thermo Electron Corpo-
ration, Rodano, Italy) and were within 0.4% of the theoretical
values. Mass spectra (ESI, positive mode) were recorded using
a Q-TOF 6520 (Agilent, Palo Alto, CA, USA). The error between
the measured exact mass and the calculated exact mass is ex-
pressed in ppm. Below 3 ppm, the compounds were considered
to have the predicted formula. The infra-red spectra were re-
corded as KBr pellets on a Perkin Elmer 1750 FT-spectrophotom-
eter (Perkin–Elmer, Waltham, MA, USA) and the wavelength are
3.1.2. Pharmacology
3.1.2.1. Determination of in vitro anticancer activity.
The
histological types and the origin of the 10 cancer cell lines and
the two normal fibroblast cell lines are detailed in the legends to
Tables 1 and 2, respectively. The 10 cancer cell lines under study
were cultured in RPMI (Invitrogen, Merelbeke, Belgium) media
supplemented with 10% heat inactivated fetal calf serum, 4 mM
gluta mine, 100
(200 U/mL and 200
blasts were cultured in MEM medium supplemented with 10% heat
lg/mL gentamicin, and penicillin–streptomycin
l
g/mL) (Invitrogen). Wi38 and NHDF fibro-
inactivated fetal bovine serum and 100
acids.
lM non-essential amino
The overall growth level of human and mouse cancer cell lines,
and human normal cell lines, was determined using the colorimetric
MTT (3-[4,5-dimethylthiazol-2yl-diphenyl tetrazolium bromide,
Sigma, Belgium) assay.20,21 Briefly, the cell lines were incubated for
24 h in 96-microwell plates (at a concentration of 10,000–40,000
cells/mL culture medium depending on the cell type) to ensure ade-
quate plating prior to cell growth determination. The assessment of
cell population growth by means of the MTT colorimetric assay is
based on the capability of living cells to reduce the yellow product
MTT to a blue product, formazan, by a reduction reaction occurring
in the mitochondria. The number of living cells after 72 h of culture
in the presence (or absence: control) of the various compounds is di-
rectly proportional to the intensity of the blue, which is quantita-
tively measured by spectrophotometry—in our case using a Biorad
Model 680XR (Biorad, Nazareth, Belgium) at a 570 nm wavelength
(with a reference of 630 nm). Each experiment was carried out in
sextuplicate.
expressed in cmÀ1
.
3.1.1.2. General procedure for one step selective alkylation of
benzoic acid derivatives bearing unprotected phenol func-
tion(s) with dibromo- or dichloroalkanes.
An alkylating
agent (100–300 mg, 1 equiv), a required substituted benzoic acid
(1.1 equiv) and NaHCO3 (1.1 equiv per halide) are added to DMF
(7 mL for 100 mg of starting material) and the reaction mixture is
heated at 105–110 °C overnight. A partition between water and
AcOEt is then performed. The aqueous phase is extracted three times
with AcOEt and organic phases are dried over Na2SO4, filtered and
concentrated in vacuo. The crude product obtained is then purified

3832
D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
Figure 6. Best solutions of the docking experiments performed with compound 6b (green) and 13c (yellow) in the active site of Aurora A (panels A and B) and B (panels C and
D). The overall structures of the proteins are shown as cartoons and the area surrounding the compounds as a transparent surface. The blue area highlights the residue
substitution between Aurora A (T217) and B (E177) close to the nucleotide-binding pocket. The different regions of the Aurora active site are highlighted in panel A: solvent
accessible (green), hinge (red), purine binding (yellow), sugar binding (magenta), phosphate binding (blue), and hydrophobic (orange) regions.
3.1.2.2. Quality controls for the determination of in vitro
anticancer activity. The direct visualization of compound-in-
duced effects on cell proliferation and cell migration was carried
out by means of computer-assisted phase-contrast microscopy, that
is, quantitative videomicroscopy as detailed elsewhere.53,54
gen atoms were added to the PDB files; Gasteiger charges were
merged to the receptor. For compounds 6b and 13c, Gasteiger
charges were assigned and non-polar hydrogen atoms were merged.
The docking energy grids were calculated with the program
AutoGrid. The grid dimensions were 78 Â 84 Â 70 points along
the x-, y-, and z-axes, with points separated by 0.375 Å. The grids
were chosen to include the entire ATP binding site as well as a re-
gion potentially involved in ligand binding, and cover the positions
of all the cofactors and inhibitors in complex with Aurora A or B
available in the Protein Data Bank. Ligand docking was performed
using the Lamarckian genetic algorithm implemented in AUTO-
DOCK 4.1.57 The energy evaluations, number of individuals in the
population and number of runs were, respectively, set to
25 Â 106, 250, and 50. Other parameters were set to default values.
The figure presenting the solution with the lowest energy was gen-
erated using Pymol.58
3.1.2.3. Kinase activity determination.
The profiling of com-
M) has
pounds 6b (carried out at 20 M) and 13c (carried out at 2
l
l
been performed at ProQinase GmbH (Freiburg, Germany) on 256 hu-
man protein kinases which are listed in Table 3 (in the Supplemen-
tary data) along with their substrates. The materials and methods
used by ProQinase to perform these assays are fully detailed in the
Supplementary data. Compounds 6b and 13c were tested twofold
in singlicate in each kinase assay. This assay was carried out in order
to identify which kinase had residual activity decreased by at least
50% with 20 lM 6b and by at least 75% with 2 lM 13c. Then, com-
pounds 1b, 2b, 3b, 4g, 6a, 6b, 6c, 6d, 6e, 7a, 7b, 7c, 7d, 7e, 9b, 11b,
12b, 13a, 13b, 13c, 17a, 17b, and 17c were tested in triplicate at each
of the eight concentrations, that is, 0 (control), 1, 5, 10, 25, 50, 75, and
Acknowledgments
100
l
M in the Aurora A, Aurora B, Aurora C, and WEE1 kinase assays.
The authors thank the Fonds National de la Recherche Scientif-
ique (FNRS, Belgium), the Fonds Yvonne Boël (Belgium) and the
National Institutes of Health (USA, CA135579) for financial support
of this work. Robert Kiss is a director of research, Florence Lefranc a
MD, Postdoctoral Fellow, and Frederic Kerff a PhD, Postdoctoral
Fellow with the FNRS. We greatly thank Amandine Djeffal for her
excellent technical assistance.
This second set of experiments was carried out to determine the IC50
concentration of the compound leading to 50% of kinase residual
activity.
3.1.2.4. Molecular docking.
The crystallographic structures of
the human Aurora A and the Xenopus laevis Aurora B with the respec-
tive PDB code 1OL555 and 2VRX56 were selected to perform the
docking experiments. The two structures were chosen for their open
active sites whose superimposition exhibits the lowest root mean
square deviation calculated for residues within 10 Å around the ac-
tive site. The structures were prepared for docking study as follows:
for both proteins, water molecules were removed from and hydro-
Supplementary data
Supplementary data (the characterization data for all com-
pounds synthesized and biologically evaluated in this study (HPLC,
1H NMR, 13C NMR, mass spectra, etc.), as well as detailed biological

D. Lamoral-Theys et al. / Bioorg. Med. Chem. 18 (2010) 3823–3833
3833
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N.; Gaussin, J. F.; Dehoux, M.; Lefranc, F.; Berger, W.; Kiss, R. J. Cell. Mol. Med.
2009, 13, 3960.
and biochemical protocols) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2010.04.047.
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