Inhibitors and promoters of tubulin polymerization: Synthesis and biological evaluation of chalcones and related dienones as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2011.03.005

A series of dihalogenated chalcones and structurally related dienones were synthesized and evaluated for their antiproliferative activity in 10 different cancer cell lines and for their effect on microtubule assembly. All compounds showed cytotoxic activi

Full text of DOI:10.1016/j.bmc.2011.03.005

Bioorganic & Medicinal Chemistry 19 (2011) 2659–2665
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitors and promoters of tubulin polymerization: Synthesis
and biological evaluation of chalcones and related dienones
as potential anticancer agents
Christine Dyrager a, Malin Wickström b, Maria Fridén-Saxin a, Annika Friberg a, Kristian Dahlén a,
Erik A. A. Wallén a,c, Joachim Gullbo b, Morten Grøtli a, Kristina Luthman a,
⇑
a Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-412 96 Göteborg, Sweden
b Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University Hospital, SE-751 85 Uppsala, Sweden
c Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, FI-00014 University of Helsinki, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihalogenated chalcones and structurally related dienones were synthesized and evaluated for
their antiproliferative activity in 10 different cancer cell lines and for their effect on microtubule assem-
bly. All compounds showed cytotoxic activity, with IC50 values in the 5–280 lM range depending on the
chalcone structure and the cell line. Five of the compounds were found to be tubulin polymerization
inhibitors. In contrast, one of the compounds was found to stabilize tubulin to the same extent as the
anticancer drug docetaxel. Molecular modeling suggested that the tubulin inhibitors bind to the
colchicine binding site of b-tubulin while the novel tubulin stabilization agent seems to interact with
the paclitaxel binding site.
Received 30 November 2010
Revised 27 February 2011
Accepted 3 March 2011
Available online 10 March 2011
Keywords:
Chalcones
Cytotoxicity
Tubulin polymerization
Molecular modeling
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
O
Chalcones (Fig. 1) are naturally occurring compounds acting as
intermediates in the biosynthesis of flavonoids, which are of inter-
est due to their wide range of biological activities. Chalcones are
also pharmacologically active and have been shown to act as
antimitotic,1 antiproliferative,2 anti-inflammatory,3 and antiviral
agents.4,5 Due to their anticancer activities, considerable efforts
have been dedicated to identify new potential chalcone-based drug
candidates within the oncology field.6 Their cytotoxic activity is of-
ten associated with tubulin inhibition and the ability of interfering
with microtubule formation, which is essential in cellular pro-
cesses such as mitosis, cytoplasmic organelle movement, cell rep-
lication, and for regulation of cell shape.7
In general, antimitotic agents act by targeting three different
sites on the tubulin heterodimer: the colchicine, the vinca alkaloid
and the paclitaxel binding sites (Fig. 2). Agents that bind to the col-
chicine binding site (e.g., colchicine and podophyllotoxin) or to the
vinca alkaloid domain (e.g., vincristine) are defined as inhibitors of
tubulin assembly, that is, microtubule destabilizing agents. On the
contrary, agents that target the paclitaxel binding site (e.g., paclit-
Figure 1. The general structure of chalcones.
axel or docetaxel) are known to act as tubulin promoters, that is,
microtubule-stabilizing agents (Fig. 3).8
Several chalcones have been reported to act as cytotoxic agents
or as microtubule destabilizing agents, targeting the colchicine
binding site.1,7,9,10 The majority of these are naturally occurring
compounds substituted with electron donating hydroxy and/or
methoxy groups at various positions.5,7,11–13 To establish more ad-
vanced structure–activity relationships around chalcones, the syn-
thesis of compounds with more diverse substitutions patterns is of
great importance. In our research aiming at the development of no-
vel scaffold-based peptidomimetics, various dihalogenated chal-
cones have been of interest as intermediates in the synthesis of
flavones and chromones.14,15 Such halogenated chalcones have
been reported to exhibit high cytotoxic activities.16
Herein, we report the synthesis of a series of chalcones and re-
lated dienones synthesized from two structurally similar electron-
poor dihalogenated 20-hydroxyacetophenones and aryl aldehydes
with electron withdrawing or donating substituents in the para-
position. The synthesized compounds were evaluated for their
⇑
Corresponding author. Tel.: +46 31 7869031; fax: +46 31 7723840.
E-mail address: luthman@chem.gu.se (K. Luthman).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.005

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C. Dyrager et al. / Bioorg. Med. Chem. 19 (2011) 2659–2665
O
O
R5'
O
R5'
R
a) or b)
H
+
R
OH
OH
R3'
R3'
1: R5' = Cl, R3' = Br, R = 4-NEt2-Ph
2: R5' = Br, R3' = Br, R = 4-NEt2-Ph
3: R5' = Cl, R3' = Br, R = 4-CF3-Ph
5' = Cl, R3' = Br, R = 4-MeO-Ph
5' = Cl, R3' = Br, R = phenyl
5' = Cl, R3' = Br, R = 2-thienyl
4:
5:
6:
R
R
R
O
O
Cl
O
Cl
R
b)
H
+
R
OH
OH
Br
Br
R = phenyl
8: R = 1-benzyl-1H-indol-3-yl
7:
Scheme 1. Synthesis of chalcones 1–6 and dienones 7–8. Reagents and conditions:
(a) NaOH (aq) (60%), MeOH, room temperature, overnight; (b) KOH, EtOH,
50 °C ? room temperature, overnight.
Figure 2. Illustration of the tubulin a,b-heterodimer with three major binding sites
on b-tubulin; the colchicine binding site (A), the paclitaxel binding site (B), and the
vinca alkaloid domain (C).
O
O
O
Cl
Cl
a)
b)
O
OH
N
N
OH
O
O
Br
Br
N
H
O
H
CO2Me
H
O
O
HN
N
O
O
O
HO
O
HO
O
O
H
O
Cl
O
O
Colchicine
Vincristine
OH
Br
OH
O
O HO
O
O
O
O
OH
OH
O
O
O
O
O
O
Cl
N
H
O
O
O
OH
OH
O
O
O
O
9
Br
Podophyllotoxin
Paclitaxel (Taxol)
Scheme 2. Synthesis of compound 9. Reagents and conditions: (a) trans-Cinnamoyl
chloride, pyridine, room temperature, 48 h; (b) K2CO3, 2-butanone, reflux, 3.5 h.
Figure 3. Chemical structures of known tubulin inhibitors (colchicine, vincristine,
and podophyllotoxin) and paclitaxel, a promoter of microtubule assembly.
and 4-diethylaminobenzaldehyde using aqueous NaOH (60%) in
MeOH (Scheme 1).15,17 Less alkaline conditions, KOH in EtOH, were
used for the synthesis of chalcones 3–6 and dienones 7–9 from 30-
bromo-50-chloro-20-hydroxyacetophenone and the appropriate
aldehyde (Scheme 1).14 Compound 9 was prepared in two steps
from 30-bromo-50-chloro-20-hydroxyacetophenone via esterifica-
tion with benzoyl chloride followed by an intramolecular Baker–
Venkataraman rearrangement (Scheme 2).18 Interestingly, NMR
spectroscopy revealed that 9 exists as an enol tautomer.
cytotoxic activity towards 10 different cancer cell lines. They were
also investigated for their activity in a microtubular polymeriza-
tion assay. All compounds showed cytotoxic activities, most of
them were also found to act as either tubulin inhibitors or promot-
ers. In addition, computer-based docking studies have been per-
formed in an attempt to rationalize the biological results.
2. Results and discussion
2.2. Biological characterization
2.1. Synthesis
2.2.1. Cytotoxicity studies
Chalcones 1–2 were synthesized in high yields via an aldol
condensation of
Compounds 1–9 were tested for their cytotoxic effect against 10
different human cancer cell lines (Table 1) using a fluorometric
a
30,50-dihalogenated-20-hydroxyacetophenone

C. Dyrager et al. / Bioorg. Med. Chem. 19 (2011) 2659–2665
2661
Table 1
Cytotoxicity of chalcones 1–6 and dienones 7–9 against 10 cancer cell lines determined using the FMCA method.
Compd
Cytotoxicity, IC50 (lM)
CCRF-CEM
CEM/VM1
ACHN
U937-GTB
U973/Vcr
RPMI 8226
8226/Dox40
8226/LR5
NCI-H69
H69AR
1
2
3
4
5
6
7
8
9
14.8
20.1
15.4
111a
37.6
152a
129a
280a
34.1
14.5
13.1
31.1
45.8
22.3
48.9
48.6
89a
164a
9.3
8.2
8.8
9.6
29.4
26.1
20.8
80a
9.7
7.7
9.4
9.9
5.2
7.1
24.5
38.8
33.2
42.6
26.4
56.1
N.D.b
48.5
25.5
69a
49.4
49.4
48.9
44.2
93a
14.9
45.0
20.0
42.5
36.6
49.0
18.1
24.4
22.5
16.9
12.4
26.5
14.2
42.3
19.1
22.3
46.0
13.2
23.3
23.4
70a
11.5
16.4
10.3
20.0
19.9
11.1
9.4
77a
24.2
32.6
31.7
54a
18.7
79a
48.8
119a
27.9
17.0
37.8
24.1
23.9
33.5
a
Extrapolated values (highest tested concentration of 50
Not determined (no cytotoxic effect was observed).
lM).
b
microculture cytotoxicity assay (FMCA).19,20 The results, summa-
rized in Table 1, showed that all compounds exhibit cytotoxic
activities with observable variations due to structural differences.
Compounds 1 and 2 showed similar activity profiles, with the high-
est activity against the NCI-H69 cell line (IC50 5.2 and 7.1
respectively). The difference in cytotoxicity between 1 and 2 in
the renal adenocarcinoma cell line (ACHN) (IC50 164 and 69 M,
lM,
l
respectively) suggests that ACHN is sensitive to size changes in
the 50-position of these chalcone derivatives. When comparing 3
and 4 (4-CF3-phenyl vs 4-MeO-phenyl) the electron donating 4-
methoxy group was unfavorable in three of the tested cell lines:
CCRF-CEM, U973/Vcr, and RPMI 8226. This result is more likely
due to the difference in size between the trifluoromethyl and the
methoxy group than the difference in electronic properties since
the cytotoxic activity of 5 (no substituent on the phenyl group) is
similar to that of 3. Furthermore, the 2-thienyl derivative 6 showed
decreased activity in several of the cell lines when compared with
the slightly larger bioisosteric phenyl derivative 5, most noticeable
Figure 4. Tubulin polymerization activity in the presence of compound 1–9
(25 M, mean values from duplicates of each experiment are presented) with
vincristine and docetaxel (3 M) as reference compounds. Stacks below the
horizontal (pink) line indicate tubulin inhibition, stacks above indicate tubulin
stabilization and stacks close to the horizontal line are considered to represent
inactive compounds. The y-axis demonstrates tubulin polymerization activity
measured at 15 min (arbitrary units) in the growth phase of the tubulin polymer-
ization curve.
l
l
in CCRF-CEM (IC50 152 vs 37.6 lM). Elongation of the carbon chain
between the aromatic moieties in 5 to obtain 7 also resulted in de-
creased activities. However, the introduction of a dienone fragment
as in 9 improved the activity with IC50 values corresponding to that
of 5. Furthermore, introducing an additional aromatic moiety, such
as in 8, gave low activity in several of the tested cell lines, for
2.3. Molecular modeling
example, CCRF-CEM (IC50 280
served toward the small-cell lung cancer cell line (NCI-H69) (IC50
11.1 M). In addition, 8 showed the highest activity among the
l
M), while a higher activity was ob-
Molecular modeling was performed in order to explore possible
binding modes for the compounds that showed activity toward
tubulin polymerization (Fig. 4). The microtubule destabilizing com-
pounds 1, 2, 5, 6, and 7 were docked into the colchicine binding site
of tubulin using the podophyllotoxin-tubulin complex (PDB 1SA1)
as template.24,25 Podophyllotoxin binds into a hydrophobic pocket
on b-tubulin that contains two hydrophobic centers; one sur-
rounded by Metb259, Alab316, and Lysb352 (occupied by the ben-
zodioxole fragment in podophyllotoxin), and one surrounded by
Leub242, Alab250, and Leub255 (occupied by the trimethoxy-
phenyl moiety) (Fig. 5A).26 In this study we observed that chal-
cones 5 ( Fig. 5B) and 6 bind to the colchicine binding site by
directing the phenyl or 2-thienyl moieties, respectively, into the
benzodioxole binding pocket. Furthermore, the 3-bromo-5-
chloro-2-hydroxyphenyl moiety could fit into the other hydropho-
bic pocket. However, compounds 1, 2, and 7 are more elongated
and are not able to adopt a conformation that utilizes both these
hydrophobic sites. Instead, the styryl group in 7 is directed out
from the binding site and positioned on the surface of the binding
pocket. However, tubulin activity is not lost since the 3-bromo-5-
chloro-2-hydroxyphenyl moiety is located in the same hydropho-
bic area as the corresponding moiety in chalcone 5 (Fig. 5B) and
it is actually buried deeper inside the pocket and thereby utilizes
l
tested compounds toward the ACHN cell line (IC50 17.0
lM),
known for its aggressive and high resistance profile.21
One potential mechanism of cytotoxicity could be that the chal-
cones act as electrophiles in Michael reactions.22 To investigate this
hypothesis we tested the reactivity of the chalcones using glutathi-
one as a nucleophile. No glutathione conjugate was formed (data
not shown) indicating that that the observed activity of the chal-
cones is not due to the reactivity of the
moiety.
a,b-unsaturated carbonyl
2.2.2. Tubulin polymerization assay
The tubulin polymerization activity of compounds 1–9 is sum-
marized in Figure 4. Compounds 3, 4, and 9 revealed no significant
influence on tubulin assembly, which suggests a different mecha-
nism for the observed cytotoxicity than tubulin inhibition (Table
1). However, five compounds (1, 2, 5, 6, and 7) showed significant
inhibition of tubulin assembly. In contrast, chalcone 8 displayed
microtubule-stabilizing activity comparable to the well-estab-
lished antimitotic chemotherapeutic drug docetaxel, which is used
in the treatment of several types of cancer.23 To the best of our
knowledge, compound 8 is the first reported chalcone with micro-
tubule-stabilizing activity.
the area more efficiently. In contrast, compounds
1 and 2
(Fig. 5C) bind in a flipped fashion; the 4-diethylaminophenyl group

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C. Dyrager et al. / Bioorg. Med. Chem. 19 (2011) 2659–2665
Figure 5. Examples from the docking study into the colchicine binding site of b-tubulin: (A) podophyllotoxin, (B) the tubulin inhibitor 5 (the carbonyl group is not visible in
the illustration as it is hidden behind the carbon skeleton), (C) the tubulin inhibitor 2, and (D) the inactive compound 3.
Figure 6. Comparison of paclitaxel (A) and compound 8 (B) docked in the paclitaxel binding site of tubulin. Panels C and D show potential hydrogen bonding interactions
(orange dashed lines) between paclitaxel or 8 and Thr276 in the active site of the tubulin heterodimer.
is instead placed into the trimethoxyphenyl pocket and the rest of
the molecule is directed outwards from the active site. The inactive
compounds, 3 (Fig. 5D), 4 and 7, were also docked into the colchi-
cine binding site of tubulin. Interestingly, 3 and 4 adopt the same
flipped binding mode as 1 and 2 but the trifluoromethyl or the
methoxy group is not able to achieve contact with the hydrophobic
pocket to the same extent as the diethylamino moiety in 1 and 2.
Furthermore, compound 9 was positioned in a similar manner as
5 ( Fig. 5B) but the 3-bromo-5-chloro-2-hydroxyphenyl moiety
was shown to adopt
a slightly different angle and could

C. Dyrager et al. / Bioorg. Med. Chem. 19 (2011) 2659–2665
2663
therefore not reach as far into the pocket as the corresponding
moiety in 5.
internal standard (CHCl3 d 7.26, CDCl3 d 77.0). The reactions were
monitored by thin-layer chromatography (TLC), on silica plated
(Silica Gel 60 F254, E. Merck), detecting spots by UV (254 and
365 nm). Melting points were measured in a Büchi Melting Point
B-540 apparatus and were uncorrected. Elemental analyses were
performed at Kolbe Mikroanalytisches Laboratorium, Mülheim
and der Ruhr, Germany.
The microtubule-stabilizing compound 8 was docked into the
paclitaxel binding site of b-tubulin using the paclitaxel-tubulin
crystal structure (PDB 1JFF).24,27 The large and complex structure
of paclitaxel binds to tubulin through multiple hydrophobic inter-
actions (Fig. 6A). The phenyl ring of the 2-benzoyl ester moiety is
located in a pocket surrounded by Leub217, Leub219, Aspb226,
Hisb229, and Leub230. Residues Valb23, Alab233, Serb236, and
Pheb272 generate a hydrophobic pocket occupied by the 30-phenyl
ring whereas the voluminous taxane ring is enclosed by the
Prob274, Leub275, Thrb276, Serb277, Argb278, Prob360, Argb369,
Glyb370, and Leub371 residues. Furthermore, the phenyl ring of
the 30-benzamido group makes contact with Valb23 and the oxe-
tane oxygen participates in hydrogen bonding with the backbone
N–H of Thrb276 (Fig. 6C). Since dienone 8 is considerably smaller
than paclitaxel, it is not able to fill the binding pocket to the same
extent. However, the results suggest that the three different aro-
matic moieties in 8 are directed towards different hydrophobic re-
gions in the paclitaxel binding site (Fig. 6B). The indole fragment is
buried inside the hydrophobic pocket that binds to the 30-phenyl
ring in paclitaxel. The N0-benzyl moiety enters the same pocket
as the 2-benzoyl phenyl group and the 3-bromo-5-chloro-2-
hydroxyphenyl moiety is directed, via the alkene linker, down into
a hydrophobic area which is surrounded by Prob274, Thrb276,
Argb278, Glnb282, Argb284, Leub286, and Leub371. Interestingly,
the modeling study also suggested that 8 could interact via two
hydrogen bonds with Thrb276 (Fig. 6D): one between the phenolic
hydrogen and the alcohol oxygen (2.31 Å, –OHꢀ ꢀ ꢀO 134°) and one
hydrogen bond between the carbonyl oxygen and N–H in the pro-
tein backbone (2.14 Å, C@Oꢀ ꢀ ꢀH–N 171°).
4.2. General procedure for the synthesis of chalcones 1–2
An aqueous solution of NaOH (60%, 25 mL/mmol acetophenone)
was added dropwise to an ice-cooled solution of dihalogenated
20-hydroxyacetophenone (1.0 equiv) and 4-diethylaminobenzalde-
hyde (1.3 equiv) in MeOH (25 mL/mmol acetophenone). The reac-
tion was allowed to reach room temperature and was stirred
over night. The reaction mixture was cooled to 0 °C and acidified
with concd HCl (37%). The crude precipitate was filtered off,
washed with water and recrystallized from EtOH.
4.2.1. (E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-3-(4-diethyl-
aminophenyl)prop-2-en-1-one (1)
30-Bromo-50-chloro-20-hydroxyacetophenone (1.00 g, 4.0 mmol)
gave 1 as red crystals (1.00 g, 62%). Mp: 153–154 °C; 1H NMR
(CDCl3) d 1.22 (t, J = 7.0 Hz, 6H), 3.44 (q, J = 7.0 Hz, 4H), 6.67 (d,
J = 8.4 Hz, 2H), 7.28 (d, J = 14.3 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H),
7.69 (d, J = 1.5 Hz, 1H), 7.84 (d, J = 1.5 Hz, 1H), 7.97 (d, J = 15.0 Hz,
1H). The OH-signal was not observed in the spectrum, due to sol-
vent exchange. 13C NMR (CDCl3) d 12.6, 44.6, 111.3, 111.8, 112.7,
121.1, 121.4, 123.2, 127.8, 131.9, 137.7, 148.8, 150.6, 158.7,
191.7; IR: (KBr)
m
2963 (br), 1616, 1604, 1436, 1150 cmꢁ1. Anal.
Calcd for C19H19BrClNO2: C, 55.83; H, 4.69; N, 3.43. Found: C,
55.76; H, 4.73; N, 3.40.
3. Conclusions
4.2.2. (E)-1-(3,5-Dibromo-2-hydroxyphenyl)-3-(4-diethylamino-
phenyl)prop-2-en-1-one (2)
A series of dihalogenated chalcones and structurally related die-
nones have been synthesized using efficient methods. The com-
pounds have been evaluated for their antiproliferative activity
and for the effect on microtubule assembly. Several of the com-
30,50-Dibromo-20-hydroxyacetophenone (3.00 g, 10.2 mmol)
gave 2 (3.59 g, 78%) as red crystals. Mp 137–141 °C; 1H NMR
(CDCl3) d 1.22 (t, J = 7.0 Hz, 6H), 3.44 (q, J = 7.0 Hz, 4H), 6.67 (d,
J = 9.2 Hz, 2H), 7.22–7.31 (m, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.82 (d,
J = 2.2 Hz, 1H), 7.92–8.01 (m, 2H), 12.87 (s, 1H). 13C NMR (CDCl3)
d 12.6, 44.6, 109.9, 111.3, 111.9, 113.1, 121.1, 122.1, 130.7, 131.9,
pounds showed cytotoxic activities (IC50 values in the low
lM
range) toward individual cancer cell lines. Five of the compounds
were found to be tubulin polymerization inhibitors, and interest-
ingly, one dienone derivative was found to stabilize tubulin to
the same extent as the anticancer drug docetaxel. Tubulin inhibi-
tion was most favoured by compounds containing unsubstituted
aromatic rings (i.e., phenyl or 2-thienyl as in 5 or 6), an electron
rich 4-diethylaminophenyl group (as in 1 and 2) or an elongated
styryl moiety in the 3-position of the chalcone structure (as in 7).
Stabilization of tubulin assembly was obtained in the presence of
the significantly larger dienone structure 8 containing an addi-
tional aromatic moiety. Molecular docking studies suggested that
the tubulin inhibitors bind into the colchicine binding site of b-
tubulin while the novel tubulin-stabilizing agent seems to interact
with the paclitaxel binding site. To the best of our knowledge, com-
pound 8 is the first reported chalcone with microtubule-stabilizing
activity, which could be a starting point for the development of no-
vel small molecule microtubule-stabilizing agents.
140.2, 148.8, 150.6, 159.2, 191.6; IR (KBr)
m 2966 (br), 1623,
1608, 1540, 1516, 1182, 1146 cmꢁ1. Anal. Calcd for C19H19Br2NO2:
C, 50.36; H, 4.23; N, 3.09. Found: C, 50.31; H, 4.20; N, 2.96.
4.3. General procedure for the synthesis of chalcones 3–6 and
dienones 7–8
KOH (2.9 equiv) was added to a solution of 30-bromo-50-chloro-
20-hydroxyacetophenone (1 equiv) and the appropriate aldehyde
(1.05 equiv) in EtOH (6 mL/mmol acetophenone). The reaction
was stirred at 50 °C for 3 h and was then allowed to reach room
temperature over night. The reaction mixture was poured into
water and acidified with aqueous HCl (1 M). The crude precipitate
was filtered off.
4.3.1. (E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-3-(4-(triflu-
oromethyl) phenyl)prop-2-en-1-one (3)
4. Experimental
4.1. Chemistry
4-Trifluoromethylbenzaldehyde (2.19 g, 12.6 mmol) gave
3
(4.86 g, 98%), which could not be recrystallized from EtOH, due
to instant cyclization upon heating. Mp 160 °C (decomp.). How-
ever, NMR spectrum and elemental analysis revealed that the
crude precipitate had high purity; 1H NMR (CDCl3) d 7.62 (d,
J = 15.4 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.79–7.81 (m, 3H), 7.86
(d, J = 2.4 Hz, 1H), 7.98 (d, J = 15.4 Hz, 1H). The OH-signal was not
observed in the spectrum, due to solvent exchange. 13C NMR
All reagents and solvents were of analysis or synthesis grade. 1H
and 13C NMR-spectra were recorded on a JEOL JNM-EX 400-spec-
trometer at 400 and 100 MHz, respectively, in CDCl3. Chemical
shifts are reported in ppm with the solvent residual peak as

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C. Dyrager et al. / Bioorg. Med. Chem. 19 (2011) 2659–2665
(CDCl3) d 113.3, 120.7, 121.3, 123.7 (q, 1JCF = 272.3 Hz), 123.9, 126.1
(q, 3JCF = 3.7 Hz), 128.1, 129.0, 132.8 (q, 2JCF = 32.9 Hz), 137.3, 139.1,
145.4, 158.8, 192.2. Anal. Calcd for C16H9BrClF3O2: C, 47.38; H,
2.24. Found: C, 47.28; H, 2.32.
20.0 mmol) in dry pyridine (20 mL) and the reaction was stirred
at room temperature for 48 h. Ice-water was added and the pre-
cipitate was filtered off. Recrystallization from EtOH (reflux) gave
the esterified acetophenone as white crystals (6.63 g, 87%). Anhy-
drous K2CO3 (4.21 g, 30.48 mmol) was added to a solution of the
esterified acetophenone (4.63 g, 12.19 mmol) in 2-butanone
(125 mL) and the reaction was refluxed for 3.5 h. The reaction
mixture was neutralized (pH 7) with concentrated AcOH and
water was added. The precipitate was filtered off. Recrystalliza-
tion from MeOH (reflux, 1 h) gave 9 as yellow crystals (3.40 g,
73%). Mp 154–155 °C; 1H NMR (CDCl3) d 6.25 (s, 1H), 6.61 (dd,
J = 1.2, 15.8 Hz, 1H), 7.38–7.45 (m, 3H), 7.53–7.59 (m, 2H),
7.61–7.65 (m, 1H), 7.67–7.70 (m, 1H), 7.73 (s, 1H), 12.93 (s,
1H). The enolate signal was not observed in the spectrum, due
to solvent exchange. 13C NMR (CDCl3) d 96.6, 113.1, 120.1,
121.4, 123.9, 127.0, 128.2, 129.0, 130.5, 134.6, 137.9, 141.4,
157.7, 176.0, 193.7. Anal. Calcd for C17H12BrClO3: C, 53.78; H,
3.19. Found: C, 53.99; H, 3.51.
4.3.2. (E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-3-(4-metho-
xyphenyl)prop-2-en-1-one (4)
The crude precipitate was recrystallized from EtOH. p-Anisalde-
hyde (1.7 g, 12.6 mmol) gave 4 (3.9 g, 90%) as a yellow powder. Mp
167–168 °C; 1H NMR (CDCl3) d 3.88 (s, 3H), 6.97 (d, J = 8.6 Hz, 2H),
7.43 (d, J = 15.3 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 2.2 Hz,
1H), 7.86 (d, J = 2.2 Hz, 1H), 7.97 (d, J = 15.3 Hz, 1H). The OH-signal
was not observed in the spectrum, due to solvent exchange. 13C
NMR (CDCl3) d 55.5, 113.0, 114.6, 116.1, 121.0, 123.6, 126.8,
128.0, 131.0, 138.4, 147.5, 158.8, 162.6, 192.2. Anal. Calcd for
C
16H12BrClO3: C, 52.28; H, 3.29. Found: C, 52.35; H, 3.25.14
4.3.3. (E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-3-phenyl-
prop-2-en-1-one (5)
The crude precipitate was recrystallized from EtOH. Benzalde-
hyde (0.45 g, 4.2 mmol) gave compound 5 (1.3 g, 96%). Mp 128–
129 °C; 1H NMR (CDCl3) d 7.47–7.50 (m, 3H), 7.57 (d, J = 15.4,
1H), 7.70–7.72 (m, 2H), 7.78 (d, J = 2.2 Hz, 1H), 7.88 (d, J = 2.2 Hz,
1H), 8.01 (d, J = 15.4 Hz, 1H), 13.51 (s, 1H). 13C NMR (CDCl3) d
113.3, 119.0, 121.1, 124.0, 128.4, 129.2, 129.4, 131.8, 134.3,
139.0, 147.9, 159.0, 192.7.14
4.5. Biology
4.5.1. Cell lines
A panel of human cancer cell lines was used for the cytotoxicity
studies. The panel has previously been described and consists of
the parental sensitive cell lines: RPMI 8226 (myeloma), CCRF-
CEM (leukemia), U937-GTB (lymphoma) and NCI-H69 (small-cell
lung cancer) and the drug resistant sublines 8226/Dox40
(doxorubicin resistant myeloma), 8226/LR5 (melphalan resistant
myeloma), CEM/VM1 (teniposide resistant leukemia), U937/Vcr
(vincristine resistant lymphoma), H69AR (doxorubicin resistant
small-cell lung cancer), and the primary resistant ACHN (renal ade-
nocarcinoma) cell line.28 This panel has been designed to represent
different histologies as well as different mechanisms of drug
resistance.28 The cells were cultivated in complete cell medium
consisting of culture medium RPMI 1640 supplemented with 10%
4.3.4. (E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-3-(thiophen-
2-yl)prop-2-en-1-one (6)
The crude precipitate was recrystallized from EtOH. 2-Thio-
phenecarboxaldehyde (0.47 g, 4.2 mmol) gave 6 (1.35, 96%). Mp
165–166 °C; 1H NMR (CDCl3) d 7.15 (dd, J = 3.5, 5.0 Hz, 1H), 7.31
(d, J = 15.0 Hz, 1H), 7.46 (d, J = 3.5 Hz, 1H), 7.54 (d, J = 5.0 Hz, 1H),
7.76 (d. J = 2.3 Hz, 1H), 7.82 (d, J = 2.3 Hz, 1H), 8.12 (d, J = 15.0 Hz,
1H). The OH-signal was not observed in the spectrum, due to sol-
vent exchange. 13C NMR (CDCl3) d 113.0, 117.4, 120.8, 123.7,
128.0, 128.8, 130.7, 133.8, 138.6, 139.7, 139.9, 158.8, 191.9.14
heat-activated fetal calf serum, 2 mM glutamine, 100 lg/mL
streptomycin and 100 U/mL penicillin (all purchased from
Sigma–Aldrich, Stockholm, Sweden). The cells were grown in
37 °C in a humidified atmosphere containing 5% CO2 and harvested
in log-phase for experimental use.
4.3.5. (2E,4E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-5-phen-
ylpenta-2,4-dien-1-one (7)
The crude precipitate was recrystallized from EtOH. Cinnamic
aldehyde (1.59 g, 12 mmol) gave 7 (3.45 g, 95%) as a yellow pow-
der. Mp 148–149 °C; 1H NMR (CDCl3) d 7.06–7.15 (m, 3H), 7.37–
7.42 (m, 3H), 7.52–7.54 (m, 2H), 7.74–7.81 (m, 3H), 13.59 (s, 1H).
13C NMR (CDCl3) d 113.1, 120.9, 122.1, 123.7, 126.4, 127.7, 128.1,
129.1, 130.0, 135.7, 138.6, 144.6, 147.6, 158.8, 192.4.
4.5.2. Cytotoxicity studies
The cytotoxicity study was performed using the fluorometric
microculture cytotoxicity assay (FMCA) method. FMCA is a total
cell kill assay, based on the ability of cells with intact cell mem-
branes to convert non-fluorescent fluorescein diacetate (FDA;
Sigma–Aldrich) to fluorescent fluorescein.19 The details of the
method have been presented previously in the literature.20 The cell
suspensions (10,000–20,000 cells per mL) were exposed, at 37 °C,
to varying concentrations of the test substances 1–9 (0.016, 0.08,
4.3.6. (2E,4E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-5-(1-
benzyl-1H-indol-3-yl)penta-2,4-dien-1-one (8)
The crude precipitate was recrystallized from EtOH. (E)-3-(1-
Benzyl-1H-indol-3-yl)acrylaldehyde (0.82 g, 3.13 mmol) gave 8
(0.39 g, 26%) as a red powder. Mp 180 °C (decomp.); 1H NMR
(CDCl3) d 5.34 (s, 2H), 6.99–7.21 (m, 4H), 7.24–7.39 (m, 7H), 7.43
(s, 1H), 7.71 (d, J = 2.3 Hz, 1H), 7.80–7.89 (m, 2H), 7.96–8.00 (m,
1H). The OH-signal was not observed in the spectrum, due to sol-
vent exchange. 13C NMR (CDCl3) d 50.5, 77.2, 110.6, 112.8, 114.5,
116.1, 118.2, 120.7, 121.7, 122.7, 123.4, 126.1, 127.0, 127.9,
128.2, 129.0, 132.0, 136.0, 137.8, 138.0, 139.0, 150.0, 158.7,
192.0; HRMS (FT-ICR-MS): [M+H]+ calcd for C26H19BrClNO2,
492.0361. Found: 492.0363.
0.04, 2, 10, and 50 lM dissolved in DMSO with maximum 1% DMSO
in cell suspensions) in 96-well microtiter plates (NUNC Brand
Products, Denmark) for 72 h. Each compound concentration was
tested in duplicate and the experiments were repeated twice.
The cells were washed with phosphate-buffered saline (PBS) fol-
lowed by the addition of FDA (dissolved in DMSO and diluted with
physiological buffer to 10 lg/mL). The fluorescence was measured
after 40 min incubation at 485/520 nm in a fluorometer (Fluorostar
Optima, BMG Technologies; Germany). The fluorescence is propor-
tional to the number of living cells, and the results were presented
as survival index (fraction of surviving cells vs un-treated control
wells). Log IC50 values (inhibitory concentration 50%) were calcu-
lated from the obtained data from log-concentration-effect curves
in Graph Pad Prism (GraphPad software Inc., CA, USA) using non-
linear regression analysis.
4.4. (1Z,4E)-1-(3-Bromo-5-chloro-2-hydroxyphenyl)-1-hydroxy-
5-phenylpenta-1,4-dien-3-one (9)
trans-Cinnamoyl chloride (4.00 g, 24.0 mmol) was added to a
solution of 30-bromo-50-chloro-20-hydroxyacetophenone (5.00 g,

C. Dyrager et al. / Bioorg. Med. Chem. 19 (2011) 2659–2665
2665
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Effects on tubulin polymerization were monitored using a com-
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utilize fluorescent reporter enhancement (the standard assay pro-
tocol was used).29,30 The fluorescence was measured using FLUO-
star OPTIMA and the test substances 1–9 (dissolved in DMSO)
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twice (mean values are presented). Docetaxel (Apoteket AB,
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and vincristine were diluted with PBS and used in the concentra-
tion of 3 lM.
4.6. Molecular modeling
Molecular modeling was performed using the Schrödinger
Package, MAESTRO interface version 9.0 (r211). The structure of tubu-
lin in complex with podophyllotoxin (PDB 1SA1) and the structure
of tubulin in complex with taxol (PDB 1JFF) was utilized for the
study.24,25,27,31 Tubulin, chains A and B, together with the ligand
were extracted and missing residues in the protein were added
by the Prime application v. 2.1 (r211). The tubulin structure and
the test compounds (1–9) were prepared and energy minimized
according to standard procedure (e.g., ligand preparation). Docking
was performed by using GLIDE v. 5.5 (r211) with extra precision (XP)
settings and standard parameters for ligand docking. Figure 2 was
made from three different X-ray structures (PDB: 1SA1, 1JFF, and
1Z2B).
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24. http://www.pdb.org.
25. Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.;
Knossow, M. Nature 2004, 428, 198.
Acknowledgments
26. Kim, D. Y.; Kim, K. H.; Kim, N. D.; Lee, K. Y.; Han, C. K.; Yoon, J. H.; Moon, S. K.;
Lee, S. S.; Seong, B. L. J. Med. Chem. 2006, 49, 5664.
27. Lowe, J.; Li, H.; Downing, K. H.; Nogales, E. J. Mol. Biol. 2001, 313, 1045.
28. Dhar, S.; Nygren, P.; Csoka, K.; Botling, J.; Nilsson, K.; Larsson, R. Br. J. Cancer
1996, 74, 888.
The authors thank the Knut and Alice Wallenberg Foundation,
the Swedish Research Council (#621-2010-4846) and the Academy
of Finland for the financial support.
29. http://www.cytoskeleton.com/products/biochem/bk011.html.
30. Bonne, D.; Heusele, C.; Simon, C.; Pantaloni, D. J. Biol. Chem. 1985, 260, 2819.
31. MAESTRO [9.0], Schrödinger, LLC, Portland, 2009; GLIDE [2.1], Schrödinger, LLC,
Portland, 2009.
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