Growth inhibitory activities of oxyprenylated and non-prenylated naturally occurring phenylpropanoids in cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2011.05.089

A series of 25 selected oxyprenylated natural phenylpropanoids were synthesized, and their growth inhibitory activities were evaluated in vitro together with 14 other commercially available non-alkylated compounds belonging to the same chemical series. The compounds were tested on six human cancer cell lines using MTT colorimetric assays. The data reveal that of the six chemical groups (G) studied, coumarins (G1), cinnamic and benzoic acids (G2), chalcones (G3), acetophenones (G4), anthraquinones (G5), and cinnamaldehydes and cinnamyl alcohols (G6), G2-related compounds displayed the weakest growth inhibitory activities in vitro, whereas G5-related compounds displayed the highest activities. Quantitative videomicroscopy analyses were then carried out on human U373 glioblastoma cells, which are characterized by various levels of resistance to different pro-apoptotic stimuli. These analyses revealed that compounds 20 (4,2′,4′-trihydroxychalcone), and 30 and 31 (two cinnamaldehydes) were cytostatic and able to overcome the intrinsic resistance of U373 cancer cells to pro-apoptotic stimuli.

Full text of DOI:10.1016/j.bmcl.2011.05.089

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4174–4179
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Growth inhibitory activities of oxyprenylated and non-prenylated naturally
occurring phenylpropanoids in cancer cell lines
Céline Bruyère a, , Salvatore Genovese b, , Benjamin Lallemand c, Alexandra Ionescu-Motatu a,d
,
Massimo Curini e, Robert Kiss a, Francesco Epifano b,
⇑
a Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
b Dipartimento di Scienze del Farmaco, Università ‘‘G. D’Annunzio’’ Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy
c Laboratoire de Chimie BioAnalytique, Toxicologie et Chimie Physique Appliquée, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
d Laboratoire de Chimie Organique, Faculté des Sciences, Université Libre de Bruxelles (ULB), Brussels, Belgium
e Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Università degli Studi di Perugia, Via del Liceo, 06123 Perugia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 25 selected oxyprenylated natural phenylpropanoids were synthesized, and their growth
inhibitory activities were evaluated in vitro together with 14 other commercially available non-alkylated
compounds belonging to the same chemical series. The compounds were tested on six human cancer cell
lines using MTT colorimetric assays. The data reveal that of the six chemical groups (G) studied, couma-
rins (G1), cinnamic and benzoic acids (G2), chalcones (G3), acetophenones (G4), anthraquinones (G5),
and cinnamaldehydes and cinnamyl alcohols (G6), G2-related compounds displayed the weakest growth
inhibitory activities in vitro, whereas G5-related compounds displayed the highest activities. Quantita-
tive videomicroscopy analyses were then carried out on human U373 glioblastoma cells, which are char-
acterized by various levels of resistance to different pro-apoptotic stimuli. These analyses revealed that
compounds 20 (4,20,40-trihydroxychalcone), and 30 and 31 (two cinnamaldehydes) were cytostatic and
able to overcome the intrinsic resistance of U373 cancer cells to pro-apoptotic stimuli.
Received 24 March 2011
Revised 22 May 2011
Accepted 24 May 2011
Available online 2 June 2011
Keywords:
Oxyprenylated natural phenylpropanoids
In vitro growth inhibition
Cancer
Cytotoxic versus cytostatic effects
Quantitative videomicroscopy
Ó 2011 Elsevier Ltd. All rights reserved.
Every year, approximately seven million people die from cancer,
which makes this disease responsible for at least 12% of deaths
worldwide.1 A number of important new commercialized antican-
cer drugs have been obtained from natural sources,2 including vin-
blastine, vincristine, vinorelbine, etoposide, teniposide, taxolÒ,
taxotereÒ, topotecan and irinotecan from plants.3 We contributed
to the discovery of a Catharantus roseus alkaloid, vinflunine,4 that
has been recently marketed as JavlorÒ.5 Trabectedin (YondelisÒ)
became the first marketed anticancer drug derived from a marine
source in 2007.6 In fact, as emphasized by Tan et al.7 natural prod-
ucts have been the most significant sources of drugs, accounting
for approximately 74% of anticancer drugs. Thus, as claimed both
by Coseri1 and Gordaliza,2 natural products represent the most
valuable potential source of novel anticancer agents. We have
accumulated experience in this domain over the last two decades
and have developed an original screening approach based on the
combined use of the conventional MTT colorimetric assay8–11 and
computer-assisted phase-contrast microscopy, i.e., quantitative
videomicroscopy.9–11 We have used these approaches to identify
anticancer drugs with potentially novel mechanisms of action.
For example, these methods enabled us to identify a hemisynthetic
derivative of a cardiotonic steroid (19-hydroxy-20-oxovoruscharin)
as a novel anticancer agent and enter it into Phase I clinical trials
for oncology.12,13 The parent compound was identified in the Afri-
can plant Calotropis procera (Aiton) W.T. Aiton (Asclepiadaceae).8 It
targets the sodium-potassium pump (Na+/K+-ATPase)
a1 subunit,
which is over-expressed in renal cell carcinomas,14 non-small-cell
lung cancers (NSCLCs),9 gliomas10 and melanomas.11 It also targets
the 3 subunit,9,10 which is over-expressed in colon cancers.15 As
detailed below, we adopted a similar strategy of research in this
current work to investigate the potential of oxyprenylated natural
compounds as anticancer agents.
Oxyprenylated natural products, such as isopentenyloxy-(C5),
geranyloxy-(C10), and the farnesyloxy-(C15) related compounds,
represent a family of secondary metabolites that were considered
for years to be merely biosynthetic intermediates of the more
widespread C-prenylated derivatives. These secondary metabolites
have been recognized in the last two decades as interesting and
valuable biologically active phytochemicals. Approximately 300
compounds have been isolated and structurally characterized from
plants, primarily from the families Rutaceae and Compositae,
which are comprised of several edible vegetables and fruits. The
phytochemistry and pharmacology of prenyloxyphenylpropanoids
was recently reviewed.16
⇑
Corresponding author.
E-mail address: fepifano@unich.it (F. Epifano).
The first two authors contributed equally to the work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.089

C. Bruyère et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4174–4179
4175
R1
R2
O
R1O
O
O
R2O
OH
21 R1= H, R2 = isopentenyl
22 R1= H, R2 = geranyl
23 R1= OH, R2 = geranyl
24 R1= OH, R2 = farnesyl
25 R1= R2 = H
1 R1= geranyl, R2 = H
2 R1= isopentenyl, R2 = H
3 R1= farnesyl, R2 = H
4 R1= isopentenyl, R2 = OH
5 R1= isopentenyl, R2 = OCH3
6 R1= R2 = H
26 R1 = OH, R2 = H
R1
OH
O
OH
R3O
R2
7 R1= CH=CH-COOH, R2 = OCH3, R3 = isopentenyl
8 R1= CH=CH-COOH, R2 = OCH3, R3 = geranyl
9 R1= CH=CH-COOH, R2 = H, R3 = isopentenyl
10 R1= CH=CH-COOH, R2 = H, R3 = geranyl
11 R1= COOH, R2 = H, R3 = isopentenyl
12 R1= COOH, R2 = OCH3, R3 = isopentenyl
13 R1= CH=CH-COOH, R2 = OCH3, R3 = H
14 R1= CH=CH-COOH, R2 = R3 = H
OR1
O
27 R1 = geranyl
28 R1 = isopentenyl
29 R1 = H
R1
CH3O
R3O
15 R1= COOH, R2 = R3 = H
16 R1= COOH, R2 = OCH3, R3 = H
R2
30 R1= CHO, R2 = H, R3 = H
O
31 R1= CHO, R2 = OCH3, R3 = H
32 R1= CHO, R2 = OCH3, R3 = isopentenyl
33 R1= CHO, R2 = OCH3, R3 = geranyl
34 R1= CHO, R2 = H, R3 = geranyl
35 R1= CH2OH, R2 = OCH3, R3 = isopentenyl
36 R1= CH2OH, R2 = OCH3, R3 = geranyl
37 R1= CH2OH, R2 = H, R3 = geranyl
38 R1= CH2OH, R2 = OCH3, R3 = H
39 R1= CH2OH, R2 = R3 = H
R1O
R2
OH
17 R1= isopentenyl, R2 = H
18 R1= isopentenyl, R2 = OH
19 R1= R2 = H
20 R1= H, R2 = OH
Figure 1. Illustration of the chemical structures studied. The 39 compounds that we studied belong to six chemical groups: coumarins (compounds 1–6; Table 1), cinnamic
and benzoic acids (compounds 7–16; Table 1), chalcones (compounds 17–20; Table 1), acetophenones (compounds 21–26; Table 1), anthraquinones (compounds 27–29;
Table 1) and cinnamaldehydes and cinnamyl alcohols (compounds 30–39; Table 1).
R1
R1
O
R1
R1
O
CHO
CHO
R2-Br
R2-Br
R2O
K2CO3
DMF
DBU
Acetone
HO
R2O
HO
OH
OH
OCH3
OCH3
R1 = H, OH
R
1 = H, OCH3
R2 = geranyl, farnesyl
R2 = isopentenyl, geranyl
Scheme 1.
Scheme 3.
R1
R1
R2O
OH
O
OH
OH
O
OH
CHO
NaBH4
OH
R1-Br
R2O
H2O/EtOH
DBU
OH Acetone
OR1
OCH3
OCH3
O
O
R
1 = H, OCH3
R2 = H, isopentenyl, geranyl
R
1 = isopentenyl, geranyl
Scheme 4.
Scheme 2.
agent.8–11 The in vitro IC50 growth inhibitory concentrations were
determined for each compound in six human cancer cell lines.
Then, we used computer-assisted phase-contrast microscopy, that
is, quantitative videomicroscopy,9–11 to determine whether the
growth inhibitory activity of a given compound was due to cyto-
toxic or cytostatic effects.
In the current work, we characterized the in vitro growth inhib-
itory activity of 25 selected oxyprenylated phenylpropanoids com-
pared to 14 non-prenylated compounds. We adopted the same
pharmacological approach as the one described above for the novel
cardiotonic steroid that we identified as a potential anticancer

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C. Bruyère et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4174–4179
Table 1
oxyacetophenone (21), and 2-hydroxy-4-geranyloxyacetophenone
(22) have been described previously.16
Physicochemical characteristics of compounds 1–39
The compounds 20,60-dihydroxy-40-geranyloxyacetophenone
(23) and 20,60-dihydroxy-40-farnesyloxyacetophenone (24) have
been previously extracted from the fruits of Evodia merrilli Kaneh-
ira & Sasaki,30 from the aerial parts of Boronia ramosa Benth.,31
from the fruits and aerial parts of Melicope obscura (Coode) T.G.
Hartley, M. obtusifolia sp. obtusifolia var. arborea (Coode) T.G. Hart-
ley32 and M. semecarpifolia (Merr.) T.G. Hartley,33 all belonging to
the Rutaceae family. The anthranoids 30-geranyloxyemodin (27)
and madagascin (28) have been isolated from several species
belonging to the Vismia and Psorospermum genera (Fam. Clusia-
ceae),34,35 whereas (E)-3-(4-(3-methylbut-2-enyloxy)-3,5-dime-
thoxyphenyl)acrylaldehyde (32), nelumal A (33), and nelumol A
(36) have been isolated from Ligularia nelumbifolia Hand. Mazz.
(Fam. Asteraceae).36 Boropinol C (35) has been obtained from the
aerial parts of Boronia pinnata Sm. (Fam. Rutaceae).37 (2E)-3-(4-
((E)3,7-Dimethylocta-2,6-dienyloxy)-3-methoxyphenyl)acrylalde-
hyde (34) and (2E)-3-(4-((E)-3,7-dimethylocta-2,6-dienyloxy)-3-
methoxyphenyl)prop-2-en-1ol (37) have been extracted from the
bark of Fagara rhetza (Roxb.) DC (Fam. Rutaceae).38
Pa
Sa
1
2
3
4
5
6
7
8
99
99
99
98
98
99
99
99
99
99
99
99
99
99
99
99
98
98
98
99
99
99
98
98
99
99
98
98
99
99
99
99
99
99
99
99
99
99
99
100
94
43
—
—
—
—
—
—
—
—
—
—
—
—
—
100
—
—
—
—
—
—
—
—
—
—
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
To investigate the effects of O-prenylation of the phenylpropa-
noid core on the growth inhibition of cancer cells, we also investi-
gated the effects of the parent compounds with free hydroxyl
groups.
The syntheses of auraptene (1), 7-isopentenyloxycoumarin (2),
umbelliprenin (3), 8-hydroxy-7-isopentenyloxycoumarin (4), lac-
inartin (5), boropinic acid (7), 40-geranyloxyferulic acid (8), p-iso-
pentenyloxycoumaric acid (9), p-geranyloxycoumaric acid (10),
valencic acid (11), isopentenyloxy vanillic acid (12), cordoin (17),
4-hydroxycordoin (18), 20,40-dihydroxychalcone (19), 4,20,40-tri-
hydroxychalcone (20), 2-hydroxy-4-isopentenyloxyacetophenone
(21), and 2-hydroxy-4-geranyloxyacetophenone (22) were accom-
plished according to the procedures described previously.39–42 The
compounds 20,60-dihydroxy-40-geranyloxyacetophenone (23) and
20,60-dihydroxy-40-farnesyloxyacetophenone (24) were prenylated
selectively at the 40 position with geranyl and farnesyl bromide,
respectively, in the presence of DBU as the base in acetone at room
temperature for 2 h. The yields of the desired adducts were 55%
and 62%, respectively (Scheme 1).
—
95
98
81
88
85
—
—
—
—
—
—
a
P means purity expressed as %, which was assessed after selective precipitation
and/or crystallization. The stability (S), expressed as % of the products was mea-
sured by HPLC analyses following incubation in a physiological solution at 37 °C
over 3 days. Results are expressed as the % of the incubated compound recovered.
As reported previously,40 the use of a sterically hindered base
like DBU results in the selective alkylation of the more accessible
OH moiety at the 40 position. A very similar methodology, except
that the reaction time was prolonged up to 24 h, was employed
for the synthesis of 30-geranyloxyemodin (27) and madagascin
(28), which were obtained with 54% and 59% yields, respectively
(Scheme 2).
Cinnamaldehydes (30 and 31) were prenylated using either
isopentenyl bromide or geranyl bromide in DMF at 80 °C in the
presence of K2CO3 as the base, resulting in (E)-3-(4-(3-methyl-
but-2-enyloxy)-3,5-dimethoxyphenyl)acrylaldehyde (32), nelumal
One of the challenges faced by researchers when setting up new
strategies to combat cancer is that several types of cancer, includ-
ing glioblastoma (GBM),17 non-small-cell lung cancer (NSCLC),18,19
esophageal cancer,20,21 pancreatic cancer,22 melanoma11,23 and
metastatic cancers,24 display significant levels of resistance to
pro-apoptotic stimuli. This resistance indicates that cytotoxic
pro-apoptotic agents are inefficient at curing cancers associated
with intrinsic resistance to pro-apoptotic stimuli.25,26 Compounds
that induce non-apoptotic cell death or that display sustained cyto-
static effects are, therefore, needed to combat ‘apoptosis-resistant’
cancer cells. The goal of this current work was to investigate
whether certain oxyprenylated phenylpropanoids could induce
sustained cytostatic effects in cancer cells that have significant lev-
els of resistance to pro-apoptotic stimuli. We used the human
U373 GBM cell line, which indeed exhibits various levels of resis-
tance to pro-apoptotic stimuli.10,27–29
A
(33), and (2E)-3-(4-((E)3,7-dimethylocta-2,6-dienyloxy)-3-
methoxyphenyl)acrylaldehyde (34) with yields of 89%, 65%, and
60%, respectively (Scheme 3).
It is important to emphasize that the use of a ‘classic’ solvent,
such as acetone, instead of a polar aprotic solvent, such as DMF,
to perform this type of phenolic etherification failed to produce
the desired prenyloxy adduct. All of the synthesized oxyprenylated
cinnamaldehydes were also reduced to the corresponding cin-
namyl alcohol by treatment with NaBH4 in a hydroalcoholic solu-
tion at room temperature for 30 min (Scheme 4). Boropinol C
(35), nelumol A (36), and (2E)-3-(4-((E)-3,7-dimethylocta-2,6-die-
nyloxy)-3-methoxyphenyl)prop-2-en-1ol (37) were obtained with
64 %, 56 %, and 67 % yields, respectively. A similar procedure was
employed for the synthesis of unprenylated 4-[(1E)-3-hydroxy-
prop-1-enyl]-2,6-dimethoxyphenol (38) and 4-[(1E)-3-hydroxy-
The chemical structures of the compounds that we studied are
illustrated in Fig. 1.
The main natural sources of auraptene (1), 7-isopentenyloxy-
coumarin (2), umbelliprenin (3), 8-hydroxy-7-isopentenyloxy-cou-
marin (4), lacinartin (5), boropinic acid (7), 40-geranyloxyferulic
acid (8), isopentenyloxy-p-coumaric acid (9), geranyloxy-p-couma-
ric acid (10), valencic acid (11), isopentenyloxy vanillic acid (12),
cordoin (17), 40-hydroxycordoin (18), 2-hydroxy-4-isopentenyl-

C. Bruyère et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4174–4179
4177
Table 2
In vitro growth inhibitory activity induced by treatment with the 39 compounds.
MTT colorimetric assay (IC50) (
l
M)a
Mean SEM
CL1
CL2
CL3
CL4
65
CL5
CL6
1
2
3
4
5
6
7
8
82
58
82
87
66
71 4
>100
61 4
>91
>40
>33
>100
>100
>100
>100
>100
>100
>68
>100
72
>100
70
79
30
>100
71
>100
95
>100
75
>100
46
>100
66
82
50
92
40
>100
>100
33
>100
>100
>100
>100
>100
>100
68
66
71
61
39
58
49
48
>100
37
34
18
45
42
>100
10
29
29
39
29
27
34
>100
39
23
80
60
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
81
>100
75
75
>100
44
40
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
53
87
>100
68
>100
48
29
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54
66
>100
85
>100
51
34
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
71
>100
>100
99
>100
27
48
38
>100
>100
30
6
35
35
63
97
>100
>100
>100
>100
>100
>100
95
82
94
91
58
57
81
57
>100
38
35
32
97
90
14
8
27
23
33
31
23
12
>100
38
27
>100
64
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
>66
>71
>61
54 6
>57
>49
72 7
>100
40 3
36 3
29 3
>45
33
26
33
>100
>100
18
5
31
28
56
30
8
7
>100
42
25
>100
97
>100
100
32
20
28
59
>100
68
71
5
>100
76
29
>100
85
>100
>100
40
7
35
33
66
32
26
28
>100
72
31
>100
>100
>42
23 4
10
31
2
1
35+5
>33
28
39
24
>100
40
21
>100
84
37
34
16
6
9
5
>100
47
7
25
1
>80
>100
a
IC50 growth inhibitory concentrations were determined in vitro by MTT colorimetric assays. The cell lines (CL) included the human U373 (CL1; ECACC code 89081403)
glioblastoma cell line, the OE21 (CL2; ECACC code 96062201) esophageal carcinoma cell line, the A549 (CL3; DSMZ code ACC107) NSCLC cell line, the PC-3 (CL4; DSMZ code
ACC465) prostate cancer cell line, the SKMEL-28 (CL5; ATCC code HTB-72) melanoma cell line and the LoVo (CL6; DSMZ code ACC350) colon cancer cell line
prop-1-enyl]-2-methoxyphenol (39) (Scheme 4). Finally, all prod-
ucts were purified by crystallization without the need for chro-
matographic separation, and yields were always calculated on
the isolated product.
cancer cell line with low levels of resistance to pro-apoptotic stim-
uli (the PC3 prostate cancer cell line43) and cell lines with certain
levels of resistance (the SKMEL-28 cell line,11 the U373 glioblas-
toma cell line,10,27–29 and the OE21 esophageal cancer cell line21
)
Table 1 details the levels of purity obtained for each of the 39
compounds under study for which the IC50 growth inhibitory con-
centrations have been determined in vitro by means of the MTT
colorimetric assay in six human cancer cell lines. Table 1 also pro-
vides the stability levels in MEM cell culture medium for 72 h for
those compounds for which sufficient amounts were still available
after having carried out the pharmacological analyses described
below. The data thus show that most compounds analyzed, ac-
cepted 3, were stable in MEM cell culture medium when main-
tained for 72 h at 37 °C. The fact that compound 3 revealed itself
as not being stable in cell culture medium emphasizes the need
to characterize the stability of a given compound before entering
pharmacochemical modulations to improve its anticancer activity.
Table 2 shows the growth inhibitory activity data of the synthe-
sized compounds. Of the six chemical groups that were studied,
(Table 2). We then submitted the human U373 GBM cell line to
quantitative videomicroscopy analyses in order to determine as
whether the compounds that were studied induced cytotoxic or
cytostatic effects. While each compound has been analyzed at its
MTT assay-related IC50 growth inhibitory concentration (Table 3),
Figure 2 shows that this concentration appeared more efficient un-
der quantitative videomicroscopy than MTT assay analyses, may be
because distinct conditions of cell cultures. Three compounds as-
sayed at their respective MTT assay-related IC50 growth inhibitory
concentration (Table 3) also reduced by about 50% the growth of
the U373 GBM cell line. These compounds are 20 (4,20,40-trihydr-
oxychalcone), and 30 and 31 (two cinnamaldehydes).
Morphological analyses as illustrated in Figure 2 revealed that
the growth inhibitory effects induced by these three compounds
were cytostatic, not cytotoxic. The remaining compounds dis-
played mixed pattern of cytostatic versus cytotoxic effects (Fig. 3).
Prenylation of phenylpropanoids and alkaloids is a common
metabolic reaction in nature, most frequently occurring in bacteria,
fungi, and plants.44 Very frequently, the addition of an isoprenoid
chain renders the molecule pharmacologically more effective than
cinnamic and benzoic acids displayed weak or no (IC50 >100 lM)
inhibitory activity, whereas anthraquinones were the most active
compounds (Table 1).
We did not notice any differences between the effects of the
0active compounds (IC50 <100 lM) on the growth inhibition of a

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C. Bruyère et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4174–4179
Table 3
Quantitative videomicroscopy dataa
% of Growth inhibition as
compared to control
% Cell death after 72 h of culture
1
2
90
—
58 14
—
3
4
90
—
40
—
9
5
6
80
—
37 10
—
7
—
—
8
—
—
9
—
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
——
—
—
—
—
—
—
—
—
—
—
—
—
—
Figure 3. Illustration of the % of cell deaths induced by each compound under study
(quantitative videomicroscopy analyses) in relation to the growth inhibitory effects
induced by these compounds. Cell cultures have been monitored in triplicate for
each experimental condition for 72 h with one image digitized each four minutes.
Thus, 1,080 images have been recorded for each experimental condition. Growth
inhibitory effects have been evaluated by counting the number of cells in the
1080th image and dividing this number by the number of cells present in the 1st
image. The results have been normalized to control conditions arbitrarily normal-
ized at 100% (Table 3).
95
70
-
60
—
80
80
70
—
88 12
17
—
7
—
17
15
42 28
—
—
100
95
99
13
2
100
100
26
—
5
3
7
2
—
90
80
90
50
50
90
80
80
—
and coworkers,45 who observed that prenylated genistein and
naringenin, among the most common flavonoids extracted from
plants, exert stronger estrogenic activities than their respective
parent compounds. In this context, selected prenyloxyphenylprop-
anoids (e.g., coumarins and cinnamic acids) were also shown to ex-
ert beneficial chemotherapeutic effects in vivo on chemically
induced colon adenocarcinoma.46
In this study, we evaluated the ability of several naturally
occurring oxyprenylated phenylpropanoids to inhibit growth in vi-
tro. Table 1 reveals that in terms of structure–activity relation-
ships, only one group of compounds among the chemical classes
tested exhibited virtually no activity. This ineffective group of com-
pounds was comprised of carboxylic acids, including prenyloxycin-
namic and prenyloxybenzoic compounds. These compounds
4
1
5
1
3
80
80
—
17
41
—
3
3
80
26 15
a
‘-’ Indicates not determined.
the parent non-prenylated compound. One of the clearest exam-
ples of this phenomenon was recently reported by Kretzschmar
displayed IC50 values >100 lM. These IC50 values are higher than
those recorded for their non-prenylated counterparts (13–16), for
which marginal activity was revealed (Table 2). For the other oxy-
prenylated phytochemicals, prenylation of the hydroxyl group en-
hanced the observed anticancer effects in vitro (e.g.,
anthraquinones, acetophenones, and cinnamic aldehydes) (Table
2). Exceptions included coumarins, for which umbelliferone (6)
exhibited a level of activity comparable to compounds 1–5, and
cinnamic alcohols, for which compound 35 had a markedly lower
effect than the unprenylated parent molecules 38 and 39 (Table
2). The effect of the length of the O-side chain was strongly depen-
dent on the class of oxyprenylated secondary metabolites. Among
coumarins (compounds 1–5), no significant differences were ob-
served with respect to in vitro growth inhibitory activity (Table
2), although a farnesyloxy- or an isopentenyloxy- moiety appeared
to enhance this activity. In the case of acetophenones, the presence
of a C5 side chain was not a necessary structural requirement for
anticancer effects, at least in vitro.
Increasing the length of the side chain from C10 (geranyl) to C15
(farnesyl) resulted in a significant improvement of the observed
growth inhibitory activity (Table 2). In contrast, among the anthra-
quinones, madagascin (28), which has an isopentenyloxy chain,
was significantly more effective than 30-geranyloxyemodin (27)
(Table 2). Finally, for cinnamic aldehydes and alcohols, the
influence of side chain length on the observed activity was
similar. For these secondary metabolites, the most significant
Figure 2. Morphological illustrations (quantitative videomicroscopy analyses) of
changes induced by compounds 1 and 20 on the growth of the human U373
glioblastoma cell line. Cytostatic effects are defined as growth delay without
observing numerous cell death as illustrated for compound 20. In contrast,
cytotoxic effects induce numerous cell deaths as illustrated for compound 1. The
% of cell deaths induced by each compound under study are detailed in Table 3.

C. Bruyère et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4174–4179
4179
13. Mijatovic, T.; Van Quaquebeke, E.; Delest, B.; Debeir, O.; Darro, F.; Kiss, R. BBA
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14. Seligson, D. B.; Rajasekaran, S. A.; Yu, H.; Liu, X.; Eeva, M.; Tze, S.; Ball, W., Jr.;
Horvath, S.; de Kernion, J. B.; Rajasekaran, A. K. J. Urol. 2008, 179, 338–345.
15. Sakai, H.; Suzuki, T.; Maeda, M.; Takahashi, Y.; Horikawa, N.; Minamimura, T.;
Tsukada, K.; Takeguchi, N. FEBS Lett. 2004, 563, 151–154.
enhancement of the observed effects occurred when the length of
the O-chain increased from isopentenyl to geranyl (compounds
32–37) (Table 2). When evaluating the growth inhibitory activity
of substituents other than the prenylated ones, each class of natu-
ral products can be considered separately. Thus, for coumarins, the
effects were slightly higher for compound 5, which possesses a
methoxy group in position 8, than for compounds 1–4, which are
deprived of this moiety (Table 2). When we compared chalcones
17 and 18, the recorded growth inhibitory activity was improved
for the compounds that do not have an hydroxy group in the 40 po-
sition (Table 2). The presence of two hydroxy substituents in posi-
tions 20 and 60 (compounds 23 and 24), rather than only one
hydroxy substituent (compound 22), increased the anticancer ef-
fects of prenyloxyacetophenones. For cinnamic aldehydes and
alcohols, the structural features leading to higher growth inhibi-
tory effects were similar (Table 2). Compounds having only one
methoxy group in the 30 position performed more effectively than
those having two in positions 30 and 50 (Table 2).
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In conclusion, oxyprenylated natural phenylpropanoids display
either cytostatic or cytotoxic growth inhibitory effects in vitro.
Three compounds, that is, 20 (4,20,40-trihydroxychalcone), and 30
and 31 (two cinnamaldehydes), displayed cytostatic effects in hu-
man U373 glioblastoma cells that usually resist to pro-apoptotic
cytotoxic insults.
28. Mégalizzi, V.; Mathieu, V.; Mijatovic, T.; Gailly, P.; Debeir, O.; De Neve, N.; Van
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32. Adsersen, A.; Smitt, U. W.; Simonsen, H. T.; Christensen, S. B.; Jaroszewski, J. W.
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F. Epifano and S. Genovese wish to thank Maria Serena Colases-
sano for her friendly collaboration. R. Kiss is a director of research
with the Fonds National de la Recherche Scientifique (FNRS,
Belgium).
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