Synthesis of honokiol analogues and evaluation of their modulating action on VEGF protein secretion and telomerase-related gene expressions

Article abstract of DOI:10.1111/cbdd.12880

A group of 36 biphenyl derivatives structurally related to honokiol were synthesized by means of Suzuki coupling reactions. Their cytotoxicities were evaluated and compared to that of honokiol. Some of the compounds were then evaluated for their ability to downregulate the secretion of the VEGF protein and the expression of the VEGF, hTERT, and c-Myc genes; the two latter involved in the activation of telomerase in tumoral cells. Some of the synthetized derivatives showed promising pharmacological features as they exhibited IC₅₀ values in low micromolar range, good therapeutic margins, and a multiple mode of action on tumor cells based on the inhibition of VEGF and, at the same time, of the expression of genes related to the activation of telomerase.

Full text of DOI:10.1111/cbdd.12880

Received Date : 14-Apr-2016
Revised Date : 27-Jul-2016
Accepted Date : 23-Sep-2016
Article type : Research Article
Synthesis of Honokiol Analogues and Evaluation of
their Modulating Action on VEGF Protein Secretion
and Telomerase-Related Gene Expressions
María Sánchez-Peris,1 Juan Murga,*1 Eva Falomir,*1 Miguel Carda,1 and J. Alberto
Marco2
1
Department of Inorganic and Organic Chemistry, University Jaume I, E-12071 Castellón,
Spain.
2
Department of Organic Chemistry, University of Valencia, Dr. Moliner, 50, E-46100
Burjassot, Spain.
* Corresponding authors: E-Mails: jmurga@uji.es (J.M.); efalomir@uji.es (E.F.); Tel.: +34-
964728243, (J.M.); Fax: +34-964728214 (J.M.).
Abstract: A group of 36 biphenyl derivatives structurally related to honokiol
were synthesised by means of Suzuki coupling reactions. Their cytotoxicities
were evaluated and compared to that of honokiol. Some of the compounds were
then evaluated for their ability to downregulate the secretion of the VEGF protein
and the expression of the VEGF, hTERT and c-Myc genes, the two latter involved
in the activation of telomerase in tumoral cells. Some of the synthetized
derivatives showed promising pharmacological features as they exhibited IC50
values in low micromolar range, good therapeutic margins and a multiple mode
of action on tumor cells based on the inhibition of VEGF and, at the same time, of
the expression of genes related to the activation of telomerase.
Keywords: honokiol analogues, angiogenesis, gene regulation, VEGF,
telomerase, hTERT, c-Myc
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12880
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Molecules containing biaryl moieties are relatively common within natural products. For
their preparation, Nature has developed an ample array of biosynthetic strategies (1). A
number of these biaryl natural products belong to the biogenetic class of lignans (2).
Honokiol (Figure 1), has aroused a great degree of interest because of their various
pharmacological properties. This product and many derivatives thereof, both of natural and
synthetic origin, have been reported to display anti-oxidative, anti-inflammatory, anti-tumor,
anti-diabetic, anti-microbial, anti-neurodegenerative, anti-depressant, pain control,
gastrointestinal, cardiovascular and liver protective properties, among others (3,4).
We have been investigating a range of analogues of natural products (5) for their
cytotoxicity and potential value in anticancer therapy (6). The latter feature may be related to
the ability of the compounds to disrupt microtubule dynamics (7), to inhibit the angiogenesis
process (8) or to inhibit the expression of genes related to telomerase activation (9) among
other alternative mechanisms.
Thanks to the intense studies on genes that mediate cancer progression and therapeutic
resistance, many gene targets that regulate apoptosis, proliferation and cell signaling have
been identified. Multiple cumulative genetic and/or epigenetic changes are needed to cause
cancer. Molecules that can inhibit expression of such genes are powerful tools in cancer
research (10). In this sense, methodologies focused on sequence-specific gene suppression
strategies involving antisense oligonucleotides and ribozymes or else involving gene
silencing using RNA interference (RNAi) have been developed (11). However, their
adaptation as broadly applicable functional genomic and therapeutic tools has proven difficult
because of problems regarding stability and poor efficiency of delivery.
Figure 1. Structure of honokiol
Many experiments have shown that targeting a single gene can inhibit the growth and
proliferation of tumor cells (12). However, interference targeting a single gene has limitations
in the prevention and treatment of cancer as it is known that tumorigenesis results in many
cases from multiple gene mutation. Therefore, therapies targeting multiple genes may have
better effects on malignant tumors.
In most solid tumors, angiogenesis is an important process for tumor growth and
metastasis (13). Many different mediators are involved in this process, including VEGF,
which has been shown to play a critical role in pathological angiogenesis (14). VEGF levels
in serum are tightly associated to a more aggressive disease state and may serve as a marker
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to evaluate diagnosis. Blocking VEGF expression can inhibit tumor growth and prevent
metastasis (15).
Most cancer cells also exhibit telomerase activity. The latter mantains the length of the
telomeres, thus preserving genomic stability (16). Telomerase is a ribonucleoprotein
composed of two main subunits which, in the case of human beings, are called human
telomerase RNA (hTR) and human telomerase protein (hTERT). Many studies have
demonstrated that interference in the expression of the hTERT gene can efficiently inhibit the
growth and tumorigenicity of cancer cells (17), as the hTERT gene is a rate-limiting factor in
telomerase synthesis and activity. Equally important is the c-Myc gene, which has been found
to be amplified in various types of human cancers such as, for example, lung carcinoma (18),
breast carcinoma (19) and colon carcinoma (20). The result of the expression of this gene, the
c-Myc protein, is a transcriptional factor with an important role in cell proliferation,
differentiation, invasion and adhesion of tumor cells (21). It is also involved in the activation
of hTERT gene transcription (17).
Since on one hand tumoral cell secretion of VEGF is an important factor in metastasis and,
on the other hand, telomerase is responsible for the immortality of tumoral cells, we consider
that the potential multiple ability (22) of some compounds to perturb microtubule dynamics
and, at the same time, to inhibit VEGF secretion by tumoral cells and the expression of the
VEGF, hTERT and c-Myc genes is a goal worth pursuing.
As the natural biphenyl derivative honokiol displays valuable anticancer activity, we
decided to prepare a number of synthetic biphenyl derivatives, such as 1-36 (Figure 2), and to
investigate their behavior in each of the aforementioned three types of biological activities. In
fact, many of these compounds can be viewed as analogues of honokiol that differ in the
relative positions of the C-allyl and OH groups. In some cases, hydroxyl functions have been
replaced by OMe groups. The C-allyl groups were introduced by means of Claisen
rearrangement (23) in precursors having O-allyl groups. The latter have also been used in the
biological evaluations. It is worth noting that the biphenyl moiety present in all compounds
can be considered a privileged structure (24) that may prove useful in the development of
lead compounds. Indeed, small molecules have always aroused interest in cancer therapy (25).
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Figure 2. Structure of biphenyl derivatives investigated in this study.
Chemistry
The starting materials for the synthesis of compounds 1-36 were hydroxylated biphenyl
derivatives prepared by means of palladium-catalyzed Suzuki coupling (26). The phenolic
functions were then O-allylated and the resulting O-allyl derivatives subjected to Claisen
rearrangement (Scheme 1) under Lewis acid catalysis (23). Compounds with O-allyl groups
ortho or para to the other benzene ring (1, 2, 5, 8-11, 14-17) gave single compounds in the
Claisen rearrangements. Those having O-allyl groups meta to the other benzene ring (3, 4, 6,
7, 12, 13, 18 and 36) gave mixtures of regioisomeric rearrangement products. In the case of
compounds 6, 12 and 18, the mixtures could be chromatographically resolved into their
individual components. These were then subjected to biological evaluation. However,
compounds 3, 4, 7, 13 and 36 gave mixtures which could not be separated into pure
components. No biological evaluation of such mixtures was carried out.
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OH
O
S´
S´
O-allylation
S
S
K2CO3, allylBr,
acetone, reflux,
5 h
1-18, 36
starting biphenyl derivative
OH
S´
Claisen
(S, S´ = different ring
substitution patterns)
rearrangement
S
Et2AlCl, hexane,
r. t., 2 h
19-35
Scheme 1. Synthesis and structures of biphenyl derivatives.
Methods and Materials
Chemistry
Conditions for O-allylations (27)
A mixture of the appropriate phenol (10 mmol) and K2CO3 (2.07 g, 15 mmol) was
dissolved in acetone (15 mL). Allyl bromide (1.1 mL, ca. 12.5 mmol) was then added
dropwise and the reaction mixture was stirred at reflux for 5 h. The mixture was then cooled,
and the volatiles removed under reduced pressure. Addition of a 10% aqueous NaOH solution
(10 mL) was followed by extraction with Et2O (3 x 15 mL). The organic layers were then
washed with brine and dried on anhydrous MgSO4, followed by removal of all volatiles under
reduced pressure. This afforded an oily material which was subjected to column
chromatography on silica gel (hexane-EtOAc mixtures) to yield the desired O-allylated
derivative. Yields were in the range between 50 and 85%.
Conditions for Claisen rearrangements (28)
A mixture of the appropriate O-allylated derivative (4 mmol) in dry hexane (40 mL) was
treated dropwise under N2 with a 1M solution of Et2AlCl in dry hexane (8 mL for compounds
having one O-allyl group and 16 mL for compounds having two O-allyl groups). The mixture
was then stirred at room temperature until consumption of the starting materials (ca. 2 h, TLC
monitoring). The mixture was then cooled in an ice bath, followed by slow dropwise addition
of a 2M aqueous HCl solution (40 mL). The mixture was poured into a separation funnel, and
the organic phase was separated from the aqueous layer, which then was additionally
extracted with EtOAc (4 x 15 mL). The combined organic layers were washed with brine,
desiccated over anhydrous Na2SO4, followed by removal of all volatiles under reduced
pressure. This afforded an oily material which was subjected to column chromatography on
silica gel (hexane-EtOAc mixtures) to yield the desired C-allyl derivative. Yields were in the
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range between 30 and 60%. Crystallization of the solid products purified by means of column
chromatography was performed using methanol.
Physical data of individual compounds are given in the Supporting Information section.
Biology
Cell culture
Cell culture media were purchased from Gibco (Grand Island, NY, USA). Fetal bovine
serum (FBS) was a product of Harlan-Seralab (Belton, U.K.). Supplements and other
chemicals not listed in this section were obtained from Sigma Chemicals Co. (St. Louis, Mo.,
USA). Plastics for cell culture were supplied by Thermo ScientificTM BioLite. All tested
compounds were dissolved in DMSO at a concentration of 10 mg/mL and stored at –20ºC
until use.
Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing
glucose (1 g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 µg/mL) and
amphotericin B (1.25 µg/mL), supplemented with 10% FBS.
Cytotoxicity assays
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma
Chemical Co., St. Louis, MO) dye reduction assay in 96-well microplates was used, as
previously described (29). Some 5 x 103 cells of HT-29, MCF-7 or HEK-293 cells in a total
volume of 100 µL of their respective growth media were incubated with serial dilutions of the
tested compounds. After 3 days of incubation (37 °C, 5% CO2 in a humid atmosphere), 10 µl
of MTT (5 mg/ml in PBS) were added to each well and the plate was incubated for further 4 h
(37 °C). The resulting formazan was dissolved in 150 µL of 0.04 N HCl/2-propanol and read
at 550 nm. All determinations were carried out in triplicate.
ELISA analysis
HT-29 cells at 70–80% confluence were collected after serum starvation for 24 h. Cells
were incubated at the concentrations showed in Table 2 of the corresponding drugs in DMSO
for 72 h (Fig. 3). Culture supernatants were collected and VEGF secreted by HT-29 cells was
determined using Invitrogen Human Vascular Endothelial Growth Factor ELISA Kit
according to the manufacturer’s instructions.
RT-qPCR analysis
HT-29 cells at 70–80% confluence were collected after serum starvation for 24 h. Cells
were incubated at the concentrations showed in Table 2 of the corresponding drugs in DMSO
for 48 h. Cells were collected and the total cellular RNA from HT-29 cells was isolated using
Ambion RNA extraction Kit according to the manufacturer’s instructions. The cDNA was
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synthesized by MMLV-RT with 1-21 μg of extracted RNA and oligo(dT)15 according to the
manufacturer’s instructions.
Amplification of the genes was performed by use of a StepOnePlus™ thermalcycler. Fast
TaqMan Gene Expression Master Mix containing the appropriate buffer for the amplification
conditions, dNTPs, thermostable DNA polymerase enzyme and a passive reference probe was
used. Each of the genes were amplified using predesigned primers by Life Technologies
TaqMan® Gene Expression Assays, Hs99999903-m1 (β-actin), Hs00900055-m1 (VEGF),
Hs00972646-m1 (hTERT) and Hs00153408-m1 (c-Myc).
Statistical analysis
Data are expressed as the mean SEM. Statistical analyses were done using Microsoft
Excel and GraphPad Prism®. Differences between means were determined using one-way
ANOVA with Dunnett's Multiple Comparison Test, and considered to be statistically
significant at α≤0.05.
Results and Discussion
Cytotoxicity of the biphenyl derivatives
The cytotoxic ability of compounds 1-36 was measured by means of their IC50 values
towards the tumoral cell lines HT-29 and MCF-7 and towards the non-tumoral cell line HEK-
293 (see Table 1). We have also evaluated the therapeutic safety margin of each compound as
expressed by means of the α and β coefficients. These are obtained by dividing the IC50 value
of each compound for the nontumoral HEK-293 line by those for the HT-29 (α) and the
MCF-7 (β) tumoral cell line, respectively. The higher the value of either coefficient, the
higher the therapeutic safety margin for the corresponding compound.
The observed IC50 values are in the low to medium micromolar range. In general terms,
these biphenyl derivatives exhibit the same cytotoxicity range in the HT-29 line as in the
MCF-7 line. Compounds 3, 6 and 36 showed comparable or better IC50 values than honokiol
towards the HT-29 cell line and, in addition, exhibited a markedly higher therapeutic margin
than honokiol (α > 7). Compounds 4, 9, 15 and 16 exhibited better IC50 values than honokiol
towards the MCF-7 cell line even though only 4 and 15 exhibited a higher therapeutic margin
than honokiol (β > 3).
Thirteen compounds (3, 6, 10, 12, 13, 15, 16, 17, 20, 22, 25, 28 and 36) were selected for
further biological evaluations. The selection was made on the basis of products showing
either comparatively low IC50 values (high cytotoxicity) or else α > 1 (good safety margin).
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Table 1. IC50 values (µM) and selectivity coefficients for biphenyls 1-36.a
Compound
HEK-293
HT-29
25
MCF-7
α*
β**
Honokiol
1
1.5
1.4
0.7
9.7
0.2
0.3
>9
1.9
1.3
0.8
1.1
3.6
0.7
1
35
7
3
18.2 0.5
247 24
80.0 0.9
320 30
227 24
101 13
27 12
2
67
258
49
6
3
5
242
14
4
4
5
208 14
210 30
4
5
70 30
>100
109 15
110 14
230 30
230 50
6
11
3
0.4
>1.4
0.3
2.0
0.2
>1.5
1.6
0.3
0.7
0.1
1.2
0.5
0.8
1.2
0.8
0.9
0.7
0.8
1.2
1
0.5
0.6
1.2
0.5
0.1
>1
7
104 12
140 30
232 22
>100
8
9
21
110
31
5
69.5 0.7
56.0 0.7
180 10
70 40
70 30
219 13
38 12
32 10
18
4
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
9
228 13
214 10
6
>100
97
5
0.6
1.2
8.7
1.1
0.4
0.3
1.0
1.1
1.3
1.2
1.0
0.5
1.7
1.1
0.7
1.1
0.3
0.7
0.7
0.6
0.2
0.2
0.5
120 40
60 30
210
3
52.9 2.1
2.9 0.14
6.5 2.2
130 40
187 14
230 50
26
5
4.5 2.3
53 13
44
6
48
3
>100
290 40
120 30
105 11
60 30
175 18
>100
240 11
138 14
85 10
56 18
131 13
123
66
5
6
46.4 0.6
129 13
176 23
56 13
81
93
3
4
79 23
>100
189
9
182
7
0.6
1.7
0.3
0.4
0.5
0.9
0.1
0.2
0.1
320 70
121 19
23
69
3
8
350 30
107 12
88 14
57
170 40
271
3
50 10
147.7 2.1
290 30
270 30
280 40
282 20
530 40
150 40
8
260 30
270 30
>100
239
235
303
6
3
9
209
7
292 10
240 50
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130 30
19
9
190 30
7.1
0.7
36
aValues are the average ( s.d.) of three different measurements performed as
b
c
described in the Experimental Section. α = IC50 (HEK-293) / IC50 (HT-29). β =
IC50 (HEK-293) / IC50 (MCF-7). Values of α and β have been rounded off to a
decimal figure.
Table 2. Concentrations for testing selected compounds.
Concentration
Biphenyl derivatives
6, 36
10 μM
20 μM
40 μM
60 μM
90 μM
Honokiol, 3, 16, 17
10, 12, 13, 15, 22, 25
28
20
Effect of biphenyl derivatives on VEGFA protein secretion and VEGF gene inhibition into
HT-29
The influence of the synthetic biphenyl derivatives on the secretion of the VEGFA protein
and the expression of the VEGF gene was performed on the HT-29 line. The thirteen
aforementioned compounds were tested at concentrations that were close to their respective
IC50 values in the HT-29 line (see Table 2).
The amount of VEGFA protein was first determined by means of the ELISA procedure
(30), as described in the Methods and Materials Section. Figure 3 shows the percentage of
VEGFA secreted to the culture medium after 72 h of incubation in the presence of each of the
selected compounds at concentrations showed in Table 2. The values observed when cells
were treated only with DMSO were used as the control (standardized to 100%).
Most of the studied derivatives showed an ability to diminish the secretion of VEGFA
(Fig. 3) (31). Compound 16 showed an activity (59%) similar to that of honokiol at the same
concentration whereas 36 showed a 61% decrease in VEGFA secretion at half the
concentration of honokiol. The strongest effect was observed with compounds 25 and 28,
which were able to lower VEGFA protein secretion to 40% and 47% of the control value,
respectively, an effect stronger than that observed for honokiol (55%). However, it should
also be noted that 25 and 28 were acting at a concentration twice and three times, respectively,
higher than that of the natural product.
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110%
120
110
100
90
80
70
60
50
40
30
20
10
0
99%
100%
94%
70%
81%
76%
75%
61%
62%
58%
59%
55%
47%
40%
l
l
3
6
0
1
2
1
3
1
5
1
6
1
7
1
0
2
2
2
5
2
8
2
6
3
o
o
i
r
t
k
n
o
o
n
C
o
H
Figure 3. VEGFA protein secretion from HT-29 cells determined by means of the ELISA
procedure. At least three measurements were performed in each case. Bars represent mean
values of VEGFA secretion (percentage values related to control) and error bars indicate
standard errors of the mean. The statistical analysis was evaluated using one-way ANOVA
with Dunnett's Multiple Comparison Test (P < 0.001).
In order to find out whether the selected biphenyl derivatives were able to downregulate
the secretion of VEGFA protein by means of a mechanism based on interference at the
transcriptional level, we tested the ability of the compounds to inhibit the expression of the
VEGFA gene. In this case, HT-29 cells were incubated for 48 h with the selected derivatives
at the concentrations shown in Table 2, as well as with DMSO as the control test. The real
time quantitative PCR (RT-qPCR) methodology (32) was then used as described in the
Experimental Section to determine the percentage of VEGFA gene expression related to the
control value. The results are shown in Figure 4.
Seven of the thirteen studied synthetic derivatives showed an ability to diminish the
expression of VEGFA gene to a noticeable degree (< 50% of the control value, with honokiol
showing a 38% value). The most active compound was 6, which inhibited gene expression to
16% of the control value and at the lowest concentration of all of the tested compounds (half
the concentration of honokiol). At the same concentration as the natural product, biphenyl
derivatives 16 and 17 were able to decrease the expression of the VEGFA gene to less than
40% of the control value. Finally, compounds 12, 15 and 22 lowered the expression of the
VEGFA to 23, 24 % and 35%, respectively, of the control value, even though at a
concentration twice higher than for honokiol. We can observe that most of these compounds
display one allyloxy group at one at least of the two aromatic rings and the ortho position is
usually not occupied. Furthermore, the most frequent substitution patterns for the oxygen
atoms in these structures are meta-meta, meta-para and para-para.
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100%
100
75
50
25
0
87%
87%
72%
52%
55%
51%
36%
38%
42%
35%
33%
23%
24%
16%
Figure 4. Expression percentage of the VEGFA gene after 48 h of incubation of HT-29 cells
determined by means of the RT-qPCR methodology. At least three measurements were
performed in each case. Error bars indicate standard errors of the mean. The statistical
analysis was evaluated using one-way ANOVA with Dunnett's Multiple Comparison Test (P
< 0.001).
It is worth mentioning here that the results discussed above do not show a good
correlation between the VEGFA amount excreted to the medium and the degree of gene
expression. This suggests that these compounds exert the control of VEGFA production at a
phase different from that of gene transcription (33).
Effect of biphenyl derivatives on the inhibition of the hTERT and c-Myc genes
In order to determine whether the studied compounds were able to downregulate the
expression of the hTERT and c-Myc genes, we have performed an RT-qPCR analysis on HT-
29 tumoral cells. The cells were incubated for 48 h in the presence of DMSO (control) and
the selected compounds (see Table 2). Figures 5 and 6 show the results observed for the
expression of the hTERT and c-Myc genes, respectively.
110
100
90
80
70
60
50
40
30
20
10
0
102%
100%
85%
78%
76%
72%
56%
51%
45%
45%
46%
42%
34%
25%
25%
l
l
3
6
0
1
2
1
3
1
5
1
6
1
7
1
0
2
2
2
5
2
8
2
6
3
o
o
i
r
t
k
n
o
o
n
C
o
H
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Figure 5. Expression percentage of the hTERT gene after 48 h of incubation. At least three
measurements were performed in each case. Error bars indicate standard errors of the mean.
The statistical analysis was evaluated using one-way ANOVA with Dunnett's Multiple
Comparison Test (P < 0.001).
100%
100
75
63%
63%
64%
58%
49%
45%
47%
45%
50
25
0
36%
32%
26%
23%
13%
7%
l
l
3
6
0
1
2
3
1
5
1
6
1
7
1
0
2
2
2
5
2
8
2
6
3
o
o
i
1
r
t
k
n
o
o
n
C
o
H
Figure 6. Expression percentage of the c-Myc gene after 48 h of incubation. At least three
measurements were performed in each case. Error bars indicate standard errors of the mean.
The statistical analysis was evaluated using one-way ANOVA with Dunnett's Multiple
Comparison Test (P < 0.001).
Honokiol and several of the synthetic biphenyl derivatives were able to inhibiting hTERT
gene expression to levels below 50% of the control (45% for honokiol). Again, compound 6
and 36 deserve mention as they were able to lower hTERT gene expression to 34 % and 42%,
respectively, of the control and at concentration one half that of the natural product (Fig. 5).
Compounds 12 and 25 also diminished the expression of the gene to about 25% of the control
value although at a concentration twice higher than that of honokiol. In this case, the most
frequent substitution patterns for the oxygen atoms in the most active derivatives are meta-
meta or meta-para.
As regards the downregulation of the expression of the c-Myc gene (Fig. 6), compounds
13, 15, 16, 22, 25 and 36 were able to diminish the expression of this gene to levels below
45% (the value for honokiol) of the control value. As above, compound 36 is also noteworthy
as it caused a decrease of gene expression to 36 % of the control at half the concentration of
honokiol. Compounds 15 and 25 were also quite active, with gene expression reduced to 13%
and 7%, respectively, of the control value, but at a concentration twice than that of honokiol.
Another compound that showed a noticeable activity was 16, which lowered gene expression
to 23% of the control at the same concentration as the natural product. Thus, the most
frequent oxygen substitution patterns in this case are para-para or meta-para.
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Conclusions
Thirty-six biphenyl derivatives structurally related to honokiol have been synthesized and
biologically evaluated. All of them bear either O- or C-allyl groups.
IC50 values were first determined for all these biphenyl derivatives towards the HT-29,
MCF-7 and HEK-293 cell lines. Some of these derivatives exhibited comparable or lower
IC50 values than honokiol towards the HT-29 cell line or else higher therapeutic margin (α
values) than the natural product.
Thirteen of the derivatives (3, 6, 10, 12, 13, 15, 16, 17, 20, 22, 25, 28 and 36) were then
selected for further biological evaluation on the basis of showing either comparatively low
IC50 values or else α > 1 (good safety margin). These derivatives were specifically evaluated
for their ability to inhibit the secretion of the VEGFA protein and to inhibit the expression of
the VEGFA, hTERT and c-Myc genes. We observed that, in general, the most active
derivatives in these particular types of biological properties exhibited in many cases
substitution patterns meta-meta or meta-para in their oxygen atoms.
Table 3 summarizes the results obtained for compounds that showed the highest activity
in simultaneously inhibiting the VEGFA protein secretion and the expression of the VEGFA,
hTERT and c-Myc genes. All these derivatives exhibit two oxygen functions (OH or OR) and
also two allyl units, either of the C-allyl or the O-allyl type.
Table 3. Compounds with the highest activity in simultaneously inhibiting the VEGFA
protein secretion and the expression of the VEGFA, hTERT and c-Myc genes.
Biological
activity
IC50 (μM)
HT-29
25
11
38
79
19
1.5
55
>9
76
0.7
58
1.2
40
7.1
61
α
% VEGFA
secretion
% VEGFA
gene expr.
36
45
16
34
24
45
42
25
55
42
% hTERT
gene expr.
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% c-Myc
gene expr.
45
58
13
7
36
Compound 6 turns out to be a very promising derivative as a potential anticancer agent
because it is able to simultaneously diminish the expression of all three targeted genes at a
concentration in the low micromolar range (half the concentration used for honokiol). In
addition, 6 exhibits the highest therapeutic margin of all the tested derivatives, honokiol
included, and has no free hydroxyl groups. This feature could be advantageous because, as it
is not able to form glucuronide conjugates, it may exhibit a lower metabolization rate.
Compounds of this type therefore deserve to be the object of further pharmacological
investigation.
Acknowledgments
Financial support has been granted to M.C. by the Ministerio de Economía y
Competitividad of Spain (project CTQ2014-52949-P), by the Consellería d´Empresa,
Universitat i Ciencia de la Generalitat Valenciana (projects PROMETEO/2013/027 and
ACOMP/2014/274) and by the University Jaume I (project PI-1B2011-37). M. S.-P. thanks
the University Jaume I for a predoctoral fellowship.
Conflict of Interest
The authors declare no conflict of interest.
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Supporting Information
Physical data. Physical and spectral data of compounds 1-36.
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