Vanillin-derived antiproliferative compounds influence Plk1 activity

Article abstract of DOI:10.1016/j.bmcl.2014.09.015

We synthesized a series of vanillin-derived compounds and analyzed them in HeLa cells for their effects on the proliferation of cancer cells. The molecules are derivatives of the lead compound SBE13, a potent inhibitor of the inactive conformation of huma

Full text of DOI:10.1016/j.bmcl.2014.09.015

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5063–5069
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Vanillin-derived antiproliferative compounds influence Plk1 activity
Roberto Carrasco-Gomez a, , Sarah Keppner-Witter b, , Martina Hieke a, Lisa Lange c, Gisbert Schneider d,
c,
Manfred Schubert-Zsilavecz a, Ewgenij Proschak a,e, , Birgit Spänkuch
⇑
⇑
a Institute of Pharmaceutical Chemistry, OSF/ZAFES/TMP, Johann-Wolfgang-Goethe University of Frankfurt, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany
b Eberhard-Karls-University Tübingen, Medical School, Department of Gynecology, Molecular Oncology and Gynecology, Calwer Str. 7, 72076 Tübingen, Germany
c Friedrich-Schiller-University Jena, Institute for Biochemistry and Biophysics, Center for Molecular Biomedicine, Hans-Knöll-Straße 2, 07745 Jena, Germany
d Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Eidgenössische Technische Hochschule (ETH), Wolfgang-Pauli-Str. 10, 8093 Zürich,
Switzerland
e German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a series of vanillin-derived compounds and analyzed them in HeLa cells for their effects
on the proliferation of cancer cells. The molecules are derivatives of the lead compound SBE13, a potent
inhibitor of the inactive conformation of human polo-like kinase 1 (Plk1). Some of the new designs were
able to inhibit cancer cell proliferation to a similar extent as the lead structure. Two of the compounds
((({4-[(6-chloropyridin-3-yl)methoxy]-3-methoxyphenyl}methyl)(pyridin-4-ylmethyl)amine) and (({4-
[(4-chlorophenyl)methoxy]-3-methoxyphenyl}methyl)(pyridin-4-ylmethyl)amine)) were much stronger
in their capacity to reduce HeLa cell proliferation and turned out to potently induce apoptosis and reduce
Plk1 kinase activity in vitro.
Received 1 December 2013
Revised 2 September 2014
Accepted 3 September 2014
Available online 16 September 2014
Keywords:
Antiproliferative compounds
Polo-like kinase 1
Cell cycle
Ó 2014 Published by Elsevier Ltd.
Natural products and their derivatives are one of the major
sources for novel antiproliferative compounds, exhibiting various
modes of cytostatic action.1 Vanilloids have been described as a
valuable compound class exhibiting potential anticancer properties.
In a previous study, we identified a vanillin derivative 13 (SBE13) as
a potent Humanpolo-like kinase 1 (Plk1) inhibitor.2 It displays selec-
tivity towards the family members Plk2 and Plk3 known as tumor
suppressor genes. Plk1,3 a key regulator of mitosis, is over-expressed
in all today analyzed human tumors.4–6 Its expression level is a neg-
ative prognostic and predictive factor for cancer patients and serves
as a measure of the aggressiveness of a tumor.6,7 Thus, targeting Plk1
by small molecules might be a promising approach to cancer ther-
apy, and Plk1 inhibitors represent tools for cancer research and for
the mechanistic investigation of checkpoint control.8–10 Several
chemical entities targeting Plk1 already reached clinical trials
(Scheme 1). The dihydropteridinone-derived inhibitors BI253611,12
and BI6727 (volasertib)13 are the most advanced clinical candidates.
BI2536 and BI6727 are highly potent ATP-competitive Plk1 inhibi-
tors (BI2536 IC50 = 0.83 nM, BI6727 IC50 = 0.87 nM), which do not
discriminate between the different Plk family members. BI6727 is
currently being investigated in phase II clinical trials as mono-
therapy or combined with established chemotherapeutic agents.14
Chemically related agents TAK-96015 and NMS-P93716,17 are cur-
rently under phase I investigation. The thiophene benzimidazole
GSK46136418 has been investigated in a phase I study in patients
with advanced solid tumors.19 Two ATP-noncompetitive inhibitors,
ON0191020 and HMN-214,21 have shown efficacy in phase I trials
and are currently under advanced clinical development.
In this study, we analyzed the structure activity relationship
(SAR) of antiproliferative vanillin derivatives derived from com-
pound 13 (SBE13). We first screened analogs of compound 13,
which were commercially available from the Specs compound
library (Specs Int., The Netherlands) and featured an ortho-dimeth-
oxyphenylethylamine moiety (Table 1). We subdivided these
structures in three groups.
The first group (1–5) consists of structures with nonaromatic
amine substituents. The cyclohexane-derivative 3 was insoluble in
concentrations required for cellular assays, the introduction of ethyl
methyl ether (2, EC50 = 97
tetrahydrofuran (5, EC50 = 66
ever, the substitution with the propyl dimethylamine (1, EC50 = 5
lM), morpholine (4, EC50 > 100
lM) and
lM) resulted in loss of activity. How-
lM) restored inhibitory potency.
The second group comprises derivatives of compound 13 with
phenylethyl and benzyl substituents (6–14, 19). The para-
methoxybenzene (8) and the benzoic acid (12) were insoluble.
Removal of both methoxy groups yielded a less potent compound
6 (EC50 = 61
lM) suggesting the importance of hydrogen-bond
⇑
Corresponding authors.
acceptor functionality in the aromatic ring. Potency could not be
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.09.015
0960-894X/Ó 2014 Published by Elsevier Ltd.

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H
N
N
H
N
N
H
N
N
H
N
O
N
O
N
O
N
O
O
N
O
N
N
BI 2536
BI 6727
H
N
N
H
N
N
N
N
N
F
O
CF3
N
F
F
N
H
N
N
N
N
O
O
HO
O
O
N
NMS-P937
TAK-960
N
F3C
O
O
N
N
S
COCH3
S
N
S
O
O
O
O
O
O
N
O
N
HN
COOH
NH2
O
O
ONO1910
HMN-214
GSK461364
O
O
N
H
O
O
Cl
N
SBE13
Scheme 1.
Table 1
Variation of the amine residue of the SBE13 lead structure (13)
N
H
O
O
Cl
N
Compound no.
R
EC50
5
(l
M)
Compound no.
R
EC50
18
(lM)
O
O
N
1
13
O
2
3
97
14
15
60
24
O
O
N
H
N
N
Insoluble
>100
O
H
N
N
O
N
4
16
2.7
Cl
N
O
S
N
N
5
6
66
61
17
18
86
8
N
NH
O
O
7
8
90
19
20
71
69
O
N
NH
Insoluble
N

R. Carrasco-Gomez et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5063–5069
5065
M)
Table 1 (continued)
Compound no.
R
EC50
16
(l
M)
Compound no.
R
EC50
96
(l
F
9
21
S
10
18
12
22
23
24
5
0.4
55
N
F
O
S
O
NH2
N
11
12
N
O
Insoluble
OH
Figure 1. Cell proliferation and apoptosis. Effect of compound 23 (A) and 40 (B) on cell proliferation and apoptosis in HeLa cells. Cell proliferation of HeLa cells was analyzed
24–72 h after treatment. Control cells were incubated with culture medium alone. Percentage of surviving cells is given as percentage of the number of control cells 72 h after
incubation with the inhibitors. All experiments: n = 3. Western blot analysis of Parp cleavage after treatment of HeLa cells with compound 23 (C) and compound 40 (D). To
determine the full-length Parp protein and the cleavage product in apoptotic cells, Western blot analyses targeting Parp were performed in HeLa cells 48 h after incubation.
Caspase 3/7 activation was monitored 48 h after treatment with compound 23 (E) and compound 40 (F), respectively. Luminescence is given as relative RLU levels (n = 3,
mean SD). Control cells were incubated with normal culture medium.
restored by the introduction of the carbonyl group as present in
compound 14 (EC50 = 60 M). This result is in line with the potency
yielded by compounds 9 (EC50 = 16 M) and 11 (EC50 = 12 M),
with this functionality restored by the introduction of fluorine or
a sulphonamide moiety, respectively. Surprisingly, activity was
also restored by the introduction of fluorine in ortho-position (10,
pound 7, EC50 = 90 lM) almost fully removed anti-proliferative
activity.
We examined the tolerability of the third group consisting of
diverse heterocyclic amino substituents that were available from
the commercial supplier. A thiophene analog of 7, compound 21,
l
l
l
(EC50 = 96
lM) was almost inactive, as well as the N-methyl tetra-
EC50 = 18
lM). Shortening the ethyl linker by one carbon (com-
zole- (17, EC50 = 86
l
M), 1,3-benzodioxole- (19, EC50 = 71 M),
l

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R. Carrasco-Gomez et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5063–5069
Table 2
Table 3
Variation of the ether residue of the lead structure 13
Optimization of compound 23
N
H
Compound no.
Structure
EC50
18
(lM)
N
R1
O
R3
R2
Compound no.
R1
R2
R3
EC50
0.44
(lM)
25
33
–H
26
27
87
85
34
35
36
37
–H
–H
–H
–H
6.4
4.8
5.1
2.4
28
29
>100
38
–H
5.0
18
22
39
40
41
42
43
–H
–H
5.0
0.89
0.44
4.0
30
31
–H
–H
6.1
–H
–H
–H
2.5
32
13
44
45
46
47
3.9
4.0
1,2,3-triazole- (20, EC50 = 69
oxadiazole amide moiety (15, EC50 = 24
decreased inhibition compared to 13. Activity was restored by
the introduction of the tryptamine (18, EC50 = 8 M) as well as
the more complex 4-amino-7-chloroquinoline (16, EC50 = 2.7 M).
Replacement of benzene (7) by 3-pyridine (22, EC50 = 5 M) com-
pletely restored the potency, and the 4-pyridine substituent led
to 45-fold improvement (23, EC50 = 0.4 M, Fig. 1A), thereby yield-
ing a novel lead structure for further optimization. The extension of
the linker length by one methylene resulted in a loss of potency
(24, EC50 = 55 lM).
We further examined the impact of the ether substituent by
screening a set of commercially available compounds with either
3,4-dimethoxyphenylethylamine or 2-aminomethyl-pyridine in
the eastern part of compound 13 (Table 2). Thiophene successfully
replaced the 2-chloropyridine, yielding compound 25 (EC50 = 18
l
M) bearing structures. The 1,2,5-
–H
l
M) led to a slightly
l
–H
–H
3.3
0.22
l
l
l
–H
48
0.40
compound 23 to evaluate the SAR of the central aromatic ring and
the western substituent (Table 3). The synthesis was carried out
according to Scheme 2. We exchanged the 2-chloropyridine by
para-chlorobenzene (40, EC50 = 0.89 lM), which led to slightly
worse inhibition. Interestingly, the hydrochloride salt of compound
40, which was obtained from the commercial supplier Specs yielded
a slightly altered EC50 = 0.45 lM (Fig. 1B), which might be associ-
l
M). The introduction of more polar amide substituents 26–28
led to the complete loss of potency. However, the different chloro
substitution patterns (29–32) were well tolerated suggesting that
optimization of the hydrophobic substitution pattern in the wes-
tern part might yield highly potent antiproliferative compounds.
Prescreening led to the identification of structure 23 (EC50 = 0.4
lM) as a promising inhibitor. Preliminary SAR of this lead structure
ated to different solubility characteristics of oxalates and hydro-
was explored. Our next step was the synthesis of new derivatives of
chlorides. Then, we analyzed different substituents on the benzyl

R. Carrasco-Gomez et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5063–5069
5067
O
the m-flourobenzyl (35, EC50 = 4.8
EC50 = 5.1
tuent in para-position. Next we extended the para-chlorobenzyl
substituent by chlorine in ortho- (44, EC50 = 3.9 M) and in
meta-position (45, EC50 = 4 M). The addition of a second chlorine
afforded less potent compounds. Interestingly, the introduction of
nitrogen (47, EC50 = 0.22 M) improved potency.
l
M) and o-fluorobenzyl (36,
OH
Cl
a
b
l
M) exhibited potency comparable to the fluorine substi-
R3
H
N
H
R1
R1
c
R2
O
O
R2
l
R1
R1
l
l
Scheme 2. Synthesis of vanillin derivatives. (a) vanillin derivative (1 equiv), alcohol
(1.2 equiv), TPP (1.2 equiv), DEAD (1.2 equiv), THF, 0 °C, overnight. (b) Vanillin
derivative (1 equiv), halogenide (1.2 equiv), Cs2CO3 (1 equiv), DMF, 70 °C, 2 h. (c)
Aldehyde (1 equiv), amine (1 equiv), DCE, room temperature, 3 h, then NaBH(Ac)3
(1.2 equiv), room temperature, overnight.
After optimization of the western substituent we analyzed the
impact of the methoxy group in the central aromatic ring. The
removal of the methoxy group (42, EC50 = 4
exchange of the methoxy with the ethoxy (43, EC50 = 2.5
l
M) as well as the
M) moi-
l
ety resulted in weaker inhibitors. However, the transition of the
moiety. We investigated the impact of different substituents in
methoxy substituent from ortho- to meta-position (41, EC50 = 0.4
para-position. The p-fluorobenzyl (34, EC50 = 6.4
enzyl (37, EC50 = 2.4 M), p-methylbenzyl (39, EC50 = 5
methoxybenzyl (38, EC50 = 5 M) substituents were less active.
Only the p-nitrobenzyl (33, EC50 = 0.44 M) moiety demonstrated
a comparable inhibitory effect. The next step was a fluorine scan:
l
M), p-trifluorob-
l
M) restored the inhibitory potency. The same transition applied
l
l
M) and p-
to the methoxy group in the pyridine-substituted compounds (23
and 47) led to opposite results: We observed that transferring
the methoxy group from ortho- to meta-position led to a weaker
l
l
inhibition (48, EC50 = 0.4 lM; 46, EC50 = 3.3 lM).
Figure 2. Determination of Plk1, Plk2 and Plk3 kinase activity. Kinase assays using immunoprecipitated Plk1 (A), Plk2 (B) or Plk3 (C), respectively, from HeLa cells after
incubation with compound 40. The autoradiograms show representative assays measuring kinase activity after immunoprecipitation of Plks from HeLa cells after incubation
with the compound, followed by subsequent kinase assay using casein as a substrate.

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R. Carrasco-Gomez et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5063–5069
Figure 3. Cell-cycle progression. Effect of compound 40 on the cell cycle distribution of HeLa cells. FACScan analyses were done after 24, 48, and 72 h incubation with the
compound. The graphs show the absolute number of cells in the respective cell cycle phases. The left peak represents cells in G0/G1 phase, and the right peak represents cells
in G2/M phase. The sub-G0/G1 peak is detectable at higher concentrations and later time points.
Taken together, from cell proliferation analyses in combination
with the additional visual inspection of cell morphology, two
derivatives of compound 13 (compounds 23 and 40,) turned out
to be potent Plk1 inhibitors. Other derivatives (33, 41, 47 and
48), which yielded submicromolar antiproliferative potency,
exhibited atypical cell morphology for selective Plk1 inhibition.
Therefore, further analyses were performed with compounds 23
and 40.
the SAR. It consists of a chlorinated pyridinyl or phenyl moiety cou-
pled via benzylic ether to the vanillin core. The methoxy substitu-
ent at the central aromatic ring is apparently required for the
activity. However the effect of the methoxy substituent depends
on the ether substituent. The SAR of the amine substituent is
restrictive considering both, linker length and type of heterocycle.
A chain length of one carbon atom and 4-pyridine are suitable to
yield active compounds.
To investigate whether the reduced cell proliferation after treat-
ment with the two selected derivatives of compound 13 (com-
pound 23 and 40, respectively) might be due to apoptosis we
first did Western blot analyses against Parp in HeLa cells 48 h after
treatment. We were able to detect an increasing amount of cleav-
age products of 85 kDa, accompanied by a decrease of the full
length protein of 116 kDa, after treatment with compounds 23
and 40, where complete degraded full length protein was observed
Our optimization strategy yielded several compounds (23, 33, 40,
41, 47, 48) with submicromolar activity in cellular assays. Visual
inspection of thephenotype of the treated cells implied that the anti-
proliferative effect of compounds 23 and 40 is mainly based on Plk1
inhibition. Consequently we proceeded with the characterization of
23 and 40 in order to test the proposed mode of action via kinase
assay using immunoprecipitated Plk1, Plk2 and Plk3, cell cycle anal-
ysis and PARP cleavage analysis which revealed a strong selective
inhibition of Plk1 activity and induction of apoptosis. These observa-
tions suggest compound 23 and 40 as new potent Plk1 inhibitors
with nanomolar inhibitory effect on cell proliferation.
for compound 40 with 33 lM (Fig. 1C and D). To confirm the induc-
tion of apoptosis we did Caspase 3/7 assays (Fig. 1E and F). Here we
observed a concentration-dependent induction of apoptosis after
treatment with both compounds, which was more prominent with
compound 40 than with compound 23. After treatment of HeLa
cells with compound 23 the induction of apoptosis was only signif-
Acknowledgments
icant for the two highest concentrations of 10 lM and 33 lM
This research was financially supported by Deutsche Krebshilfe
(Grant No. 108651 to B.S.), Wilhelm-Sander-Stiftung (Grant no.
2009.024.1 to B.S.), Messer-Stiftung (to B.S.), FIRST (Frankfurt
International Research Graduate School for Translational Biomedi-
cine) (to S.K.), LOEWE OSF (Oncogenic Signaling Frankfurt) and
German Cancer Consortium DKTK (to E.P.). B.S., S.K. and L.L. are
grateful to Dr. D. Wallwiener and Professor Dr. T. Heinzel for pro-
viding working facilities.
(p = 0.04 and p = 0.02, respectively). Compound 40 induced apopto-
sis more strongly than compound 23, which was significant
already at the lowest concentration of 500 pM (p = 0.007).
To analyze whether compound 40, which turned out to be potent
in first cell culture studies using HeLa cells, inhibits Plk1 kinase
activity, we performed kinase assays using immunoprecipitated
Plk1 kinase from synchronized and treated HeLa cells. Compound
40 inhibited Plk1 kinase activity with an IC50 value of 3 nM
(Fig. 2A). Reduction of kinase activity was statistically significant
Supplementary data
at all tested concentrations (500 pM–1 lM, p <0.01). Next we
checked whether compound 40 also discriminates between the
Plk family members. For that reason Plk2 and Plk3 kinase activity
was analyzed (Fig. 2B and C). Compound 40 also showed selectivity
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.015.
towards Plk2 and Plk3 with IC50 values >10 lM for both kinases.
References and notes
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as the length of the amine linker. Several compounds yielded two-
fold greater potency than compound 13 in cell-based assays. A
blueprint of an effective vanillin derivative can be deduced from
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