Synthesis, antiprolife ativeactivity and inhibition of tubulinpolymerization byanthracenone-based oxime derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.04.019

A series of anthracenone-based oxime ethers and-esters were synthesized in order to evaluate their antiproliferative activity. Several investigated compounds displayed strong antiproliferative activity against K562 leukemia cells and proved to be strong inhibitors of tubulin polymerization. In this context,anthracenone-based oxime ethers and-esters are considered to contribute to the development of novel antiproliferative drugs,based on tubulin interaction.

Full text of DOI:10.1016/j.ejmech.2010.04.019

European Journal of Medicinal Chemistry 45 (2010) 3354e3364
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative activity and inhibition of tubulin polymerization
by anthracenone-based oxime derivatives
,
Georg Surkau a, Konrad J. Böhm b, Klaus Müller a, Helge Prinz a
*
a Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University, Hittorfstrasse 58-62, D-48149 Münster, Germany
b Leibniz Institute for Age ResearcheFritz Lipmann Institute (FLI), Beutenbergstrasse 11, D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of anthracenone-based oxime ethers and -esters were synthesized in order to evaluate their
antiproliferative activity. Several investigated compounds displayed strong antiproliferative activity
against K562 leukemia cells and proved to be strong inhibitors of tubulin polymerization. In this context,
anthracenone-based oxime ethers and -esters are considered to contribute to the development of novel
antiproliferative drugs, based on tubulin interaction.
Received 9 March 2010
Received in revised form
15 April 2010
Accepted 19 April 2010
Available online 27 April 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Anthraquinone oximes
Anthracenones
10-Hydroxyimino-10H-anthracen-9-ones
Tubulin polymerization
Antiproliferative activity
1. Introduction
activities through binding to tubulin (Chart 1). Recently, much
effort has been devoted to the identification of new compounds
Microtubules remain important cellular targets for the devel-
opment of anticancer therapeutic agents [1,2]. They are composed
from both natural and synthetic sources that bind at the colchicine
site of tubulin [5,11,12].
principally of two closely related globular polypeptides,
a
- and
Regarding tubulin-binding oximes, the curacin A oxime
b
-tubulin, which exist as stable heterodimers. Microtubules play an
analogue 6 has been described to inhibit tubulin polymerization
similar to the natural product lead, with the oxime group serving as
a bioisosteric replacement of a (Z)-alkene group [13]. Furthermore,
diarylmethyloximes such as 7 containing an indole ring have
recently been shown to be strong inhibitors of tubulin polymeri-
zation [14]. Additionally, within the series of naphthylphenstatins,
E-acetyloximes as exemplified by 8 have been documented by the
same group to inhibit tubulin polymerization [15] (Chart 1).
Our group has intensively investigated tubulin polymerization
inhibitors based on anthracenone scaffolds. Among them 10-[(3-
hydroxy-4-methoxy-benzylidene)]-10H-anthracen-9-one (9), 9-[(4-
hydroxy-3,5-dimethoxy-benzylidene)]-naphtho[2,3-b]thiophen-4
(9H)-one (10) and methylaminobenzenesulfonic acid ester 11
(Chart 2) were found to have excellent in vitro antitumor activity
[16e18]. More recently, we described a series of 10-(2-oxo-2-phe-
nylethylidene)-10H-anthracen-9-ones aspotent inhibitorsof tubulin
polymerization, with 12 being the most active analogue. This
compound has an enone moiety between the anthracenone and the
terminal aromatic ring [19]. Our present strategy for the synthesis of
novel antitubulin compounds is based on the modification of the
linker between the anthracenone and the terminal phenyl ring and
important role in the formation and function of the spindle appa-
ratus, which realizes the controlled segregation of the chromo-
somes in dividing cells. Microtubules are also directly involved in
a variety of other cellular functions, such as cell movement, main-
tenance of the cell shape, as well as the transport of organelles
inside the cell. As a consequence, inhibition of microtubule function
is commonly accompanied by serious cellular dysfunctions and
might trigger cell death [1].
A still expanding family of antimitotic drugs exhibits their
anticancer properties by interfering with the dynamics of tubulin
polymerization and depolymerization, resulting in mitotic arrest
[3e6]. A structurally diverse collection of ligands, such as vinblas-
tine sulfate (1), colchicine (2), combretastatin A-4 (3) [7], the
epothilones [8] and especially the taxanes paclitaxel and docetaxel
as well as some synthetic molecules including sulfonamide E-7010
[9] (4) or triazolyl indole T115 [10] (5) are known to exert cytotoxic
* Corresponding author. Tel.: þ49 251 8332195; fax: þ49 251 8332144.
E-mail address: prinzh@uni-muenster.de (H. Prinz).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.019

G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
3355
OH
CH2CH3
N
H3CO
NHCOCH3
N
N
H
H3CO
H3CO
CH2CH3
OCOCH3
COOCH3
CH3OOC
CH3O
1
H
O
N
H3C
2
H
OCH3
OH
N
N
H3CO
H3CO
H3CO
N
H3CO
OH
O
O
S
N
H3CO
H3CO
N
OH
OCH3
H
N
CH3
HN
3
T115, 5
H3CO
4
OH
N
N
OH
OCH3
OCH3
O
N
H3C
S
6
7
OAc
OCH3
N
H3CO
OCH3
OCH3
H3CO
H3CO
8
H3CO
Chart 1. Examples of tubulin interacting agents.
incorporation of an oxime ester or ether function. Since both
1,5-dichloro- and the 1,8-dichloroanthraquinone have recently been
described as useful templates for the design of potential anticancer
agents [20e22], the corresponding anthracenones were included as
starting materials.
Herein, we describe the synthesis and characterization of
a series of anthracenone-based 10-oxyimino derivatives. Several
compounds inhibited the growth of K562 leukemia cells and were
additionally found to be potent effectors of tubulin polymerization,
whereby the inhibitory activity of most of the active compounds
was comparable to that of reference compounds, e.g. colchicine.
deprotonation, anthracenones 13e15 were reacted with isopentyl
nitrite [25] to afford the desired oximes 19e22 (Scheme 1). Anthra-
quinone oxime 18 was prepared in a two-step reaction from 16 via
bismethylated 17. In the case of 14, nitrosation led to the expected
geometrical isomers (E/Z) 20-E and 21-Z in a 3:1 ratio. Single isomers
were separated by chromatography and distinguished by NMR
spectroscopy. Synthesis of O-benzoyloxime esters 23ae23e was
performed by reaction of the oximes 19e22 with appropriately
substituted benzoyl chlorides [26] (Scheme 2). Regarding the
isomeric mixture of 20-E/21-Z as starting material, only the
E-isomeric oxime esters 23c-E and 23d-E were obtained, possibly
due to steric limitations. For the synthesis of the desired vanilline-
and isovanilline-derived esters 23f and 23g, oxime 19 was reacted
with appropriate carboxylic acids in a DCC-mediated coupling
reaction [27]. As several representatives of our recently reported
series of sulfonate derivatives, e.g. 11 proved to be highly potent
antiproliferative agents and inhibitors of tubulin polymerization, we
were also interested in the evaluation of O-benzenesulfonyl oximes,
2. Chemistry
The most commonly used method for the synthesis of oximes is
the dehydrative condensation of carbonyl compounds and hydrox-
ylamine [23]. However, oximes can also be prepared via electrophilic
nitrosation of active methylene compounds [24]. In our case, after
NHCH3
O
O
O
O
S
S
O
O
OCH3
OH
OH
O
10
9
S
OCH3
OCH3
11
12
OCH3
Chart 2. Tubulin interacting benzylidenes 9 and 10, sulfonate 11 and phenacylidene 12.

3356
G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
A
R3
O
R1
R3
R2
O
N
R1
(a)
R2
OH
13, R1 = R2 = R3 = H
19, R1 = R2 = R3 = H
14, R1 = R2 = Cl, R3 = H
15, R1 = R3 = Cl, R2 = H
20-E, 21-Z, , R1 = R2 = Cl, R3 = H
22, R1 = R3 = Cl, R2 = H
B
H3CO
O
OCH3
HO
O
OH
H3CO
O
OCH3
(b)
(a)
N
O
O
OH
18
16
17
Scheme 1. R1eR3 are defined in Table 1. Reagents and conditions: A, (a) Naꢁ, isopentyl nitrite, ethanol, rt; B, (a) (CH3O)2SO2, K2CO3, acetone, reflux; (b) NH3OH$Cl, pyridine, ethanol,
reflux.
which were synthesized according to the method of Stankevicius et
al. [28]. Again, starting with the isomeric mixture of 20-E/21-Z, we
could only isolate the E-isomeric ester 24b-E. For the synthesis of the
O-benzoyloxime ethers 25ae25k (Scheme 3), oximes 18, 19, 20-E-E
and 22 were alkylated with appropriately substituted benzylic
halides in the presence of sodium hydride [29].
IC50 ¼ 0.11
mM). This result correlates well with the data obtained in
the tubulin polymerization assay (see below). Unsubstituted 25a
(R1eR6 ¼ H) was only weakly potent (25a, IC50 K562 7.0
mM).
Interestingly, introduction of a 3,4,5-trimethoxyphenyl group in
25c, a typical pharmacophore for the inhibition of tubulin poly-
merization found in colchicine (2), combretastatin A-4 (3) and
podophyllotoxin, strongly decreased growth inhibitory properties
as compared with 25b and 25d. This is in agreement with similar
observations described in our previous reports [16e18]. Further
investigations were carried out with dichloro-substituted oxime
ethers 25ge25i and dimethoxy-substituted oxime ethers 25j and
25k. In sharp contrast to 25b and 25d, the pertinent dichloro
analogues 25ge25i had no significant antiproliferative activities
3. Results and discussion
3.1. In vitro cell growth inhibition assay
We screened our compounds for antiproliferative activity
against the human erythroid leukemia cell line K562 [30]. Cell
proliferation was determined after 48 h of treatment by cell
counting using a hemocytometer. Table 1 lists the effects of the
anthraquinone-derived oximes 18, 19, 20-E, 21-Z and 22 on the
growth of K562 cells in comparison with data obtained for several
reference compounds. The results presented in Table 1 show that
the nature and the location of the substituents at the anthracenone
core play a critical role for antiproliferative potency. Overall, the
antiproliferative potency of the starting oximes was not very
pronounced. Only dimethoxy-substituted oxime 18 showed an IC50
(IC50 values in the range of >12 mM). Additionally, replacement of
the chloro groups in the anthracenone core by methoxy as seen
with oxime ethers 25j and 25k also lowered potencies as compared
with 25b and 25d (Table 3). Typically, the isovanilline-derived 25k,
for example, was 6.5- and 22-fold less potent than 25b and 25d,
respectively, being unsubstituted in the anthracenone core. More-
over, the activity of 25j was remarkably lowered compared to 25b
(25j, IC50 K562 3.40 mM versus 25d, IC50 K562 0.37 mM). In
summary, our findings indicate that substituents in the peri-posi-
tion to the anthracenone carbonyl are obviously not favorable for
attaining strong antiproliferative activity within the series of
analogues reported herein.
value in the submicromolar (ꢀ1
mM) range. With respect to the 1,5-
dichloro series, geometrical isomers 20-E and 21-Z were separated
and assayed as individual compounds (indicated by E or Z following
the number). Both isomers had similar growth inhibitory activity.
Oxime esters 23ae23g were in general weak inhibitors of K562 cell
growth (Table 2), with 23a, 23f and 23g being active in the lower
micromolar range. Compound 23f, bearing the 3-hydroxy-4-
methoxy fragment, was the most potent displaying moderate
3.2. In vitro tubulin polymerization assays
To investigate whether the antiproliferative activities of the
compounds were related tothe interactionwith tubulin, we selected
several compounds to measure their effect on tubulin polymeriza-
tion, resulting in microtubule formation in a cell-free system. Their
activity was compared with that of the reference anti-tubulin
compounds colchicine, podophyllotoxin, nocodazole, and vinblas-
tine (Table 1). Microtubule suspensions are known to have an
elevated turbidity signal compared to tubulin solutions. On this
antiproliferative potency with an IC50 value of 2.0 mM. Replacement
of the isovanilline part of 23f by the vanilline fragment in 23g led to
a comparable potency. While the dichloro-substituted benzene-
sulfonyl oxime ester 24b-E was practically inactive (Table 2), the
benzyloxime ethers 25b and 25d showed comparatively strong
antiproliferative activities (K562, 25b, IC50 ¼ 0.37
mM; 25d,

G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
3357
A
R3
R2
O
R1
R3
O
N
R1
COCl
R2
(a)
R3
R1
N
O
R2
O
OH
R4
R5
19, R1 = R2 = R3 = H
R1 = R3 = H, R2 = OCH3
20-E, R1 = R2 = Cl, R3 = H R1 = R2 = R3 = OCH3
22, R1 = R3 = Cl, R2 = H
R6
23a, R1 = R2 = R3 = R4 = R6 = H, R5 = OCH3
23b, R1 = R2 = R3 = H, R4 = R5 = R6 = OCH3
23c-E, R1 = R2 = Cl, R3 = R4 = R6 = H, R5 = OCH3
23d-E, R1 = R2 = Cl, R3 = H, R4 = R5 = R6 = OCH3
23e, R1 = R3 = Cl, R2 = R4 = R6 = H, R5 = OCH3
R3
R2
R1
O
COOH
(b)
19
R1
N
O
R2
O
R4
R5
R1 = OH, R2 = OCH3
or
R1 = OCH3, R2 = OH
23f, R1 – R3 = H, R4 = OH, R5 = OCH3
23g, R1 – R3 = H, R4 = OCH3, R5 = OH
B
R3
O
R1
R3
R2
O
N
R1
SO2Cl
OCH3
(a)
R2
N
OH
O
O S O
19, R1 = R2 = R3 = H
20-E, 21-Z, R1 = R2 = Cl, R3 = H
OCH3
24a, R1 = R2 = R3 = H
24b-E, R1 = R2 = Cl, R3 = H
Scheme 2. R1eR6 are defined in Table 2. Reagents and conditions: A, (a) NaH, THF, rt; (b) DCC, DMAP, CH2Cl2, 0 ꢁC to rt; B, (a) NaOH, acetone, H2O, 0 ꢁC to rt.
basis, tubulin polymerization can be recorded by time-dependent
measurements of turbidity. The resulting turbidity curves, usually
obtained at 340e360 nm, reveal a sigmoid behaviour with a plateau
level reached after 20e30 min at the conditions realized in this
study (Fig. 1). Inhibition of tubulin polymerization is reflected by
decreasing the steady state turbidity level in a dose-dependent
manner, as exemplified for 25b (Fig.1). The results obtained with the
test compounds are summarized in Table 1. In general, inhibition of
tubulin polymerization correlated well with the drug-induced
growth inhibition. Compounds 25b and 25d, the most potent
compounds in the K562 cellular based assay, proved to be strong
phenyl ring are essential features for activity [31,32]. Both are
preferential partial structures seen with many tubulin polymeriza-
tion inhibitors [31]. Interestingly, as documented byourearlier work
and as confirmed herein, the isovanillinyl partial structure could not
be successfully replaced by trimethoxy (25c, 25h) e a prominent
pharmacophore present in many colchicine site inhibitors e with
retention of high activity. Moreover, analogues 25g, 25j and 25k
were examined for comparison with O-benzyloxime ethers 25b and
25d. These showed IC50 values in the range of ꢂ10
mM and were
regarded to have no significant antitubulin activity.
inhibitors of tubulin polymerization (IC50 ITP 25b 1.1
mM; 25d
4. Conclusion
1.4 M), with both being nearly as active as colchicine. These find-
m
ings indicate that the 3-hydroxy-4-methoxyphenyl as well as the
p-methoxyphenyl are crucial moieties for inhibition of tubulin
polymerization. The ehydroxy/emethoxy groups and the terminal
In conclusion, several oxime ethers were efficacious in inhibit-
ing tumor cell proliferation with submicromolar IC50 values, which
were superior to those of the oxime precursors. We found the

3358
G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
R3
R2
O
N
R1
R3
R2
O
N
R1
OH
18, R1 = R2 = OCH3, R3 = H
19, R1 = R2 = R3 = H
(a)
O
R4
R5
20-E, R1 = R2 = Cl, R3 = H
22, R1 = R3 = Cl, R2 = H
R6
X
25a, R1 – R6 = H
25b, R1 – R4 = H, R5 = OCH3, R6 = H
R6
R4
25c, R1 = R2 = R3 = H, R4 = R5 = R6 = OCH3
25d, R1 – R3 = H, R4 = OH, R5 = OCH3, R6 = H
25e, R1 – R4 = H, R5 = Cl, R6 = H
R5
R4 = OH, R5 = OCH3, R6 = H, X = Br
R4 = R5 = R6 = OCH3, X = Br
R4 = R5 = R6 = H, X = Br
25f, R1 – R4 = H, R5 = NO2, R6 = H
25g, R1 = R2 = Cl, R3 = R4 = R6 = H, R5 = OCH3
25h, R1 = R2 = Cl, R3 = H, R4 = R5 = R6 = OCH3
25i, R1 = R3 = Cl, R2 = R4 = R5 = H, R5 = OCH3
25j, R1 = R3 = OCH3, R2 = R4 = R6 = H, R5 = OCH3
25k, R1 = R3 = OCH3, R2 = R6 = H, R4 = OH, R5 = OCH3
R4 = R6 = H, R5 = OCH3, X = Cl
R4 = R6 = H, R5 = Cl, X = Cl
R4 = R6 = H, R5 = NO2, X = Cl
Scheme 3. R1eR6 are defined in Table 3. Reagents and conditions: (a) NaH, THF, DMF, rt.
biological activities were dependent on the substitution pattern in
the terminal phenyl ring. The best results for inhibition of cell
growth were obtained with the p-methoxy analogue 25b and the 3-
hydroxy-4-methoxy analogue 25d. The observed antiproliferative
effects are most probably due to interaction with tubulin as those
compounds with the greatest inhibitory effects on cell growth
strongly inhibited tubulin polymerization. Compounds 25b and
25d displayed IC50 values around 1 mM for inhibition of tubulin
polymerization which ranked them among the active, but not the
most active, representatives within our anthracenone-derived
tubulin polymerization inhibitors. Further structureeactivity
studies in the series of anthracenone-based tubulin polymerization
Table 2
Antiproliferative activity of oxime esters 23ae23g and 24a, 24b-E against K562 cells
Table 1
and antitubulin activities.
Antiproliferative activity of oximes 18e22 against K562 cells.
Compd
R1
R2
R3
K562
IC50
ITPb
IC50
a
[
mM]
[mM]
18
19
20-E
21-Z
OCH3
H
Cl
Cl
Cl
H
H
Cl
Cl
H
OCH3
H
H
H
Cl
0.9
3
n.d.
n.d.
n.d.
n.d.
n.d.
1.4
0.76
0.35
0.13
n.d.
Compd
R1
R2
R3
R4
R5
R6
K562
IC50
ITPb
IC50
a
[m
M]
[mM]
1.6
1.6
>9
0.02
n.d.
n.d.
23a
23b
23c-E
23d-E
23e
23f
23g
24a
24b-E
H
H
Cl
Cl
Cl
H
H
H
Cl
H
H
Cl
Cl
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
OCH3
H
OCH3
H
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
OCH3
H
OCH3
H
H
3
>12
12
>14
>12
2
3
3
>12
>10
n.d.
>10
n.d.
n.d.
n.d.
n.d.
>10
n.d.
22
Colchicine (2)
Nocodazole
Podophyllotoxin
Vinblastine sulfate (1)
Adriamycine
0.001
0.01
H
n.d: not determined.
a
IC50, concentration of drug required for 50% inhibition of cell growth (K562).
Cells were treated with drugs for two days. IC50 values are the means of at least three
independent determinations (SD < 10%). IC50 data were calculated from dos-
eeresponse curves by nonlinear regression analysis.
a
IC50, concentration of drug required for 50% inhibition of cell growth (K562).
Cells were treated with drugs for two days. IC50 values are the means of at least three
independent determinations (SD < 10%).
b
b
ITP ¼ inhibition of tubulin polymerization; IC50 represent the concentration for
ITP ¼ inhibition of tubulin polymerization; IC50 values represent the concen-
50% inhibition of the maximum tubulin assembly rate.
tration for 50% inhibition of the maximum tubulin assembly rate.

G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
3359
Table 3
performed at the Münster microanalysis laboratory, using a vario EL
III CHNOS elemental analyzer (Elementar Analysensysteme GmbH).
All values obtained were within ꢃ0.4% of the theoretical values.
Mass spectra were recorded in the EI mode using a MAT GCQ Fin-
nigan instrument. For HRMS (ESI-nanospray mode) analysis, an LTQ
Orbitrap XL (Thermo Fisher Scientific, Bremen) spectrometer was
used. All organic solvents were appropriately dried or purified prior
to use. Analytical TLC was done on Merck silica 60 F254 alumina
coated plates (E. Merck, Darmstadt). Column chromatography was
performed using Acros 60e200 mesh silica gel. In most cases, the
concentrated pure fractions obtained by chromatography using the
indicated eluents were treated with a small amount of hexane to
induce precipitation. All new compounds displayed 1H NMR, 13C
NMR, and MS spectra consistent with the assigned structure. Yields
have not been optimized. Starting anthraquinones, benzyl halides,
aroyl chlorides and benzenesulfonic acid chlorides were commer-
cially available or prepared according to standard literature
protocols.
Antiproliferative activity of oxime ethers 25ae25k against K562 cells and anti-
tubulin activities.
Compd R1
R2 R3
R4
R5
R6
K562
IC50
ITPb
M] IC50 [mM]
a
[m
25a
25b
25c
25d
25e
25f
25g
25h
25i
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
7
0.37
n.d.
1.05
n.d.
1.43
n.d.
n.d.
>10
n.d.
n.d.
>10
>10
5.1. Chemistry
OCH3 OCH3 OCH3 >12
OH
H
H
H
OCH3 OCH3 OCH3 >14
H
H
OCH3
Cl
NO2
OCH3
H
H
H
H
0.11
10
>11
>12
5.1.1. 1,5-Dichloro-10H-anthracen-9-one (14)
See Ref. [33]
5.1.2. 1,8-Dichloro-10H-anthracene-9-one (15)
Cl
OCH3
OCH3
Cl
OCH3
OCH3 OH
OCH3 OCH3 >12
OCH3
OCH3
See Refs. [34,35].
25j
25k
H
H
3.40
2.40
a
5.1.3. Synthesis of 10-hydroxyimino-10H-anthracen-9-ones
5.1.3.1. 1,8-Dimethoxy-anthracen-9,10-dione (17). See Ref. [36].
IC50, concentration of drug required for 50% inhibition of cell growth (K562).
Cells were treated with drugs for two days. IC50 values are the means of at least three
independent determinations (SD < 10%).
b
ITP ¼ inhibition of tubulin polymerization; IC50 values represent the concen-
5.1.3.2. Synthesis of 1,8-dimethoxy-10-hydroxyimino-10H-anthra-
cen-9-one (18). The title compound was prepared from 17 (0.77 g,
2.9 mmol), hydroxylamine hydrochloride (0.39 g, 4.3 mmol) and
pyridine (1 mL) in ethanol (20 mL). The mixture was heated at
reflux. Progress of the reaction was monitored by TLC. Upon
completion, the mixture was cooled to room temperature. The
oxime 18 precipitated and was collected by filtration, washed with
ice-cold water and dried in vacuo. Purification by column chroma-
tography (CH2Cl2) provided 18 as a yellow solid (0.642 g, 79%); mp:
tration for 50% inhibition of the maximum tubulin assembly rate.
inhibitors are in progress and results from other modifications will
be reported in due course.
5. Experimental protocols
Melting points were determined with a Kofler melting point
apparatus and are uncorrected. Spectra were obtained as follows:
1H NMR and 13C NMR spectra were recorded with a Varian Mercury
400 plus (1H NMR 400 MHz, 13C 101 MHz) spectrometer, using
tetramethylsilane as an internal standard. Elemental analyses were
175 ꢁC. 1H NMR (CDCl3, 400 MHz)
d: 12.2 (s, 1H, Oxime-H), 8.20 (d,
3J ¼ 7.9 Hz,1H, H-5), 7.98 (d, 3J ¼ 7.7 Hz, 1H, H-4), 7.60 (d, 3J ¼ 7.3 Hz,
2H, H-2, H-7), 7.51 (t, 3J ¼ 7.8 Hz, 2H, H-3, H-6), 3.98 (s, 6H, Ar
(OCH3)2). 13C NMR (CDCl3, 101 MHz)
d: 183.9, 141.5, 134.3, 134.1,
133.4, 133.3, 130.8, 129.3, 125.8, 121.3, 118.9, 118.3, 56.3. MS (ESI) m/
Fig. 1. A, Time-dependent turbidity curves representing the inhibition of in vitro polymerization of tubulin (in total 1.2 mg/mL protein; w80e90% tubulin plus w10e20%
microtubule-associated proteins) by various concentrations of compound 25b. B, Plot of the steady state levels of tubulin polymerization (taken at 40 min) in dependence on the
compound concentration. The corresponding level in the absence of the test compound was set 100%. From this plot the IC50 value, representing the drug concentration required for
50% inhibition, was determined by sigmoidal fitting.

3360
G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
z: 284.20 (M þ Hþ). Anal. Calcd for C16H13NO4 (in %): C-67.84, H-
5.1.4. Preparation of 10-benzoyloxyimino-10H-anthracen-9-ones
5.1.4.1. Synthesis of 10-(4-methoxybenzoyloxyimino)-10H-anthra-
cen-9-one (23a). Sodium hydride (0.82 g, 35 mmol) was suspended
in dry THF (100 mL) and 19 (1.19 g, 5 mmol) was added. After stir-
ring for 30 min, 4-methoxy benzoic chloride (2.40 g, 28 mL,
21.0 mmol) was added. Progress of the reaction was monitored by
TLC. Upon completion of the reaction, saturated sodium hydro-
gencarbonate (10 mL) was added, followed by extraction with
CH2Cl2 (3 ꢄ 25 mL). The combined organic layers were washed with
water (20 mL) and brine (20 mL), dried over Na2SO4. Thereafter, the
solvent was evaporated under reduced pressure. Purification by
silica gel column chromatography afforded 23a as white crystals
4.63, N-4.94. Found C-67.89, H-4.65, N-4.96.
5.1.3.3. Synthesis of 10-hydroxyimino-10H-anthracen-9-one (19) e
general procedure for the synthesis of 10-hydroxyimino-10H-
anthracen-9-ones. Pentyl alcohol nitrite (22.62 g, 26 mL, 0.19 mol)
was added to a stirring solution of sodium (4.40 g, 0.18 mol) in
ethanol (500 mL) under nitrogen. To the resulting slight yellow
solution, 10H-anthracen-9-one (13, 25.75 g, 0.13 mol) was added.
Upon completion of the reaction (TLC control), the solution was
poured slowly into ice-cold water (150 mL). After treating the
solution with diluted hydrochloric acid (50 mL), the solid was
collected by filtration, washed with water and dried (Dean Stark).
The residue was collected by filtration, washed with n-hexane and
dried under reduced pressure. Purification by column chromatog-
raphy (CH2Cl2) provided the title compound 19 as a pale yellow
(1.02 g, 6); mp 182 ꢁC. 1H NMR (CDCl3, 400 MHz)
d: 8.67 (dd,
3J ¼ 7.6 Hz, 4J ¼ 1.2 Hz, 1H, H-4), 8.44 (dd, 3J ¼ 7.5 Hz, 4J ¼ 1.2 Hz, 1H,
H-8), 8.38 (dd, 3J ¼ 7.5 Hz, 4J ¼ 1.4 Hz, 1H, H-1), 8.23 (dd, 3J ¼ 7.2 Hz,
4J ¼ 1.4 Hz, 1H, H-4), 8.07 (d, 3J ¼ 9.0, 2H, H-10, H-50), 7.69e7.54 (m,
4H, H-2, H-3, H-6, H-7), 6.98 (d, 3J ¼ 6.9 Hz, 2H, H-30, H-50), 3.87 (s,
solid (16.98 g, 57%). 1H NMR (DMSO-d6, 101 MHz)
d: 13.26 (s, 1H,
Oxime-H), 9.12 (d, 3J ¼ 8.1 Hz, 1H, H-5), 8.27 (d, 3J ¼ 7.8 Hz, 1H, H-8),
8.23 (d, 3J ¼ 8.1 Hz, 1H, H-1), 8.13 (d, 3J ¼ 7.8 Hz, 1H, H-4), 7.83 (t,
3J ¼ 8.1 Hz, 1H, H-2), 7.74 (t, 3J ¼ 6.3 Hz, 2H, H-7), 7.62 (t, 3J ¼ 7.7 Hz,
3H, Ar-OCH3). 13C NMR (CDCl3, 101 MHz)
d: 183.4, 164.2, 163.5,
150.9, 133.9, 133.1, 132.9, 132.3, 131.8, 131.4, 131.1, 131.0, 129.2, 128.2,
127.2, 126.1, 120.6, 114.4, 55.7. MS (ESI) m/z: 358.10 (M þ Hþ). Anal.
Calcd for C22H15NO4 (in %): C-73.94, H-4.23, N-3.92. Found C-73.83,
H-4.25, N-3.31.
1H, H-6). 13C NMR (DMSO-d6, 101 MHz)
d: 182.6, 141.6, 136.1, 133.6,
133.5, 130.7, 130.7, 130.4, 129.7, 129.2, 128.9, 126.9, 126.3, 124.2. MS
(ESI) m/z: 225.07 (M þ Hþ). Anal. Calcd for C14H9NO2 (in %): C-
75.33, H-4.06, N-6.27. Found C-75.33, H-4.08, N-6.30.
5.1.4.2. Synthesis of 10-(3,4,5-trimethoxybenzoylimino)-10H-anthra-
cen-9-one (23b). The title compound was prepared from 19 (1.12 g,
5 mmol), sodium hydride (0.25 g, 10 mmol) and 3,4,5-trimethoxy
benzoic chloride (1.27 g, 6 mmol) in a similar manner as described
for the preparation of 23a. Purification by silica gel column chro-
matography afforded 23b as a pale yellow solid (0.43 g, 20%); mp:
5.1.3.4. Synthesis of (E)-1,5-dichloro-10-hydroxyimino-10H-anthra-
cen-9-one (20-E). The title compound was prepared from 14
(4.49 g, 0.17 mol), sodium (0.82 g, 0.03 mmol) and pentyl alcohol
nitrite (2.40 g, 2.8 mL, 21 mmol) in a similar manner as described
for the preparation of 19. Purification by silica gel column chro-
matography (CH2Cl2) afforded 20-E as a pale yellow solid (2.24 g,
199 ꢁC. 1H NMR (CDCl3, 400 MHz)
d
: 8.56 (d, 3J ¼ 8.1 Hz, 1H, H-5),
8.31 (d, 3J ¼ 8.0 Hz, 1H, H-8), 8.27 (d, 3J ¼ 7.8 Hz, 1H, H-1), 8.11 (d,
3J ¼ 7.9 Hz, 1H, H-4), 7.63e7.47 (m, 4H, H-2, H-3, H-6, H-7), 7.09 (s,
2H, H-20, H-50), 3.79 (s, 3H, Ar0OCH3), 3.77 (s, 6H Ar0(OCH3)2). 13C
45%); mp 220 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d: 13.02 (s, 1,
Oxime-H), 7.97 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.1 Hz, 1H, H-8), 7.94 (dd,
3J ¼ 7.7 Hz, 4J ¼ 1.1 Hz, 2H, H-2, H-6), 7.89 (dd, 3J ¼ 7.3 Hz, 4J ¼ 1.1 Hz,
1H, H-4), 7.83 (t, 3J ¼ 7.8 Hz, 2H, H-3, H-7). 13C NMR (DMSO-d6,
NMR (CDCl3, 101 MHz) d: 186.4, 162.0, 158.5, 157.7, 145.2, 134.7,
133.4, 131.8, 130.5, 130.4, 129.8, 126.0, 123.5, 105.0, 60.7, 56.8. MS
(ESI) m/z: 418.12 (M þ Hþ). Anal. Calcd for C24H19NO6 (in %): C-
69.06, H-4.59, N-3.36. Found C-69.10, H-4.65, N-3.40.
101 MHz) d: 182.1, 143.2, 138.9, 135.8, 135.5, 134.2, 133.1, 132.1, 131.2,
130.0, 129.5, 128.2, 125.5, 123.69. MS (ESI) m/z: 292.99 (M þ Hþ).
Anal. Calcd for C14H7Cl2NO2 (in %): C-57.56, H-2.42, N-4.79. Found
C-57.57, H-2.43, N-4.80.
5.1.4.3. Synthesis of 1,5-dichloro-10-(4-methoxybenzoyloxyimino)-
10H-anthracen-9-one (23c-E). The title compound was prepared
from 20-E/21-Z (isomeric mixture, 1.17 g, 4 mmol), sodium hydride
(0.19 g, 8 mmol) and 4-methoxy benzoic chloride (0.75 g, 4 mmol)
in a similar manner as described for the preparation of 23a. Puri-
fication by silica gel column chromatography afforded 23c-E as pale
5.1.3.5. Synthesis of (Z)-1,5-dichloro-10-hydroxyimino-10H-anthra-
cen-9-one (21-Z). The product was prepared from 14 in a similar
manner as described for the preparation of 19. Purification over
silica gel using (hexane/ethyl acetate ¼ 2:1) gave 21-Z as a white
solid (1.12 g, 30%); mp 223 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d:
yellow solid (1.09 g, 63%), mp: 235 ꢁC. 1H NMR (CDCl3, 400 MHz)
d:
12.98 (s, 1H, Oxime-H), 8.27e7.83 (m, 6H, H-Ar). 13C NMR (DMSO-
d6, 101 MHz) : 182.1, 143.2, 138.9, 135.8, 135.3, 134.1, 132.9, 131.8,
8.22 (dd, 3J ¼ 7.8 Hz, 4J ¼ 1.4 Hz, 1H, H-8), 8.13 (dd, 3J ¼ 7.0 Hz,
d
4J ¼ 1.9 Hz, 1H, H-4), 8.10 (dd, 3J ¼ 7.6 Hz, 4J ¼ 1.2 Hz, 2H, H-2, H-6),
3
130.0, 129.5, 129.0, 127.5, 125.5, 123.7. MS (ESI) m/z: 292.99
(M þ Hþ). Anal. Calcd for C14H7Cl2NO2 (in %): C-57.56, H-2.42, N-
4.79. Found C-57.57, H-2.40, N-4.81.
7.95 (d, J ¼ 8.9 Hz, 2H, H-20, H-60), 7.49 (t, 3J ¼ 7.9, 2H, H-3, H-7),
3
6.91 (d, J ¼ 8.9 Hz, 2H, H-30, H-50), 3.85 (s, 3H, ArOCH3). 13C NMR
(CDCl3, 101 MHz) d: 182.5, 164.2, 151.0, 137.3, 136.9, 136.3, 135.8,
134.6,134.3,133.9,133.8,132.4,132.3,132.2,131.9,131.1,129.0,126.1,
124.9, 120.3, 114.3, 55.7. MS (ESI) m/z: 427.03 (M þ Hþ). Anal. Calcd
for C22H13Cl2NO4 (in %): C-61.99, H-3.07, N-3.29. Found: C-61.90, H-
3.05, N-3.28.
5.1.3.6. Synthesis of 1,8-dichloro-10-hydroxyimino-10H-anthracen-
9-one (22). The title compound was prepared from 15 (5.03 g,
20 mmol), sodium (0.68 g, 3 mmol) and pentyl alcohol nitrite
(2.40 g, 2.8 mL, 20 mmol) in a similar manner as described for the
preparation of 19. Purification by silica gel column chromatog-
raphy (CH2Cl2) afforded 22 as a white solid (2.46 g, 44%); mp
5.1.4.4. Synthesis of 1,5-dichloro-10-(3,4,5-trimethoxybenzoylimino)-
10H-anthracen-9-one (23d-E). The title compound was prepared
from 20-E/21-Z (isomeric mixture, 1.03 g, 3.5 mmol), sodium
hydride (0.19 g, 5.4 mmol) and 3,4,5-trimethoxy benzoic chloride
(1.22 g, 5 mmol) in a similar manner as described for the prepara-
tion of 23a. Purification by silica gel column chromatography
afforded 23d-E as pale yellow solid (0.43 g, 20%); mp: 200 ꢁC. 1H
220 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d: 13.20 (s, 1H, Oxime-H),
8.67 (dd, 3J ¼ 7.9 Hz, 4J ¼ 1.2 Hz, 1H, H-5), 8.36 (dd, 3J ¼ 7.9 Hz,
4J ¼ 1.2 Hz, 1H, H-7), 8.21 (d, 3J ¼ 8.2 Hz, 1H, H-2), 8.00 (d,
3J ¼ 8.1 Hz, 1H, H-4), 7.84 (t, 3J ¼ 8.0 Hz, 2H, H-3, H-6). 13C NMR
(DMSO-d6, 101 MHz) d: 181.9, 142.0, 137.1, 133.4, 133.1, 133.0, 131.8,
131.1, 130.6, 130.2, 129.3, 128.8, 124.8, 123.3. MS (ESI) m/z: 292.99
(M þ Hþ). Anal. Calcd for C14H9NO2 (in %): C-75.33, H-4.06, N-6.27.
Found C-75.10, H-4.07, N-6.27.
NMR (CDCl3, 400 MHz)
d
: 8.22 (d, 3J ¼ 7.5 Hz, 1H, H-8), 8.13 (d,
3J ¼ 7.0 Hz, 1H, H-4), 7.95 (d, 3J ¼ 7.0 Hz, 2H, H-2, H-6), 7.49 (t,
3J ¼ 7.9, 2H, H-3, H-7), 7.39 (s, 2H, H-Ar0), 3.94 (s, 3H, ArOCH3), 3.85

G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
3361
(s, 6H, Ar(OCH3)2). 13C NMR (CDCl3, 400 MHz)
d: 189.1, 162.0, 157.7,
5.1.5. Synthesis of 10-(4-methoxyphenylsulphonyloxyimino)-10H-
156.6, 145.2, 140.5, 136.4, 133.6, 133.4, 133.0, 131.4, 130.5, 128.2,
126.9, 124.3, 123.5, 105.0, 60.6, 56.8. MS (ESI) m/z: 468.30 (M þ Hþ).
Anal. Cald for C24H17Cl2NO6 (in %): C-59.28, H-3.52, N-2.88. Found:
C-59.27, H-3.49, N-2.87.
anthracen-9-ones
5.1.5.1. Synthesis of 10-(4-methoxyphenylsulphonyloxyimino)-10H-
anthracen-9-one (24a). To an ice-cold mixture of 19 (1.15 g,
5 mmol) and 4-methoxybenzenesulphonyl chloride (1.08 g,
5 mmol) in acetone (75 mL) a solution of sodium hydroxide (0.25 g)
in water (5 mL) was added. The solution immediately turned
orange and then the colour changed slowly to yellow. Progress
of the reaction was monitored by TLC. Upon completion, the solu-
tion was added to ice-cold water, the precipitate was filtered off,
washed with water and hexane. Purification by column chroma-
tography with ethyl acetate/hexane (1:2) as eluent afforded 24a as
5.1.4.5. Synthesis of 1,8-dichloro-10-(4-methoxybenzoylimino)-10H-
anthracen-9-one (23e). The title compound was prepared from 22
(0.50 g,1.7 mmol), sodium hydride (0.08 g, 3 mmol) and 4-methoxy
benzoic chloride (0.34 g, 2 mmol) in a similar manner as described
for the preparation of 23a. Purification by silica gel column chro-
matography afforded 23e as pale yellow solid (0.07 g, 10%); mp:
194 ꢁC. 1H NMR (CDCl3, 400 MHz)
d
: 8.30 (d, 3J ¼ 7.9 Hz, 1H, H-5),
a beige solid (0.65 g, 57%); mp: 200 ꢁC. 1H NMR (CDCl3, 400 MHz)
d:
8.15 (dd, 3J ¼ 7.9 Hz, 4J ¼ 1.1 Hz, 1H, H-4), 8.00 (d, 3J ¼ 9.0 Hz, 2H, H-
20, H-60), 7.68 (dd, 3J ¼ 8.1, 4J ¼ 1.1 Hz, 2H, H-2, H-7), 7.60 (t,
3J ¼ 8.0 Hz,1H, H-3), 7.53 (t, 3J ¼ 7.9 Hz, 2H, H-6), 6.96 (d, 3J ¼ 9.0 Hz,
8.67 (dd, 3J ¼ 8.0 Hz, 5J ¼ 0.8 Hz, 1H, H-4), 8.44 (dd, 3J ¼ 7.9 Hz,
5J ¼ 0.8 Hz,1H, H-8), 8.38 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.4, 1H, H-1), 8.23 (dd,
3J ¼ 7.6 Hz, 4J ¼ 1.2 Hz, 1H, H-4), 8.07 (d, 3J ¼ 9.0 Hz, 2H, H-20, H-60),
7.76 (td, 3J ¼ 7.7 Hz, 4J ¼ 1.4 Hz, 1H, H-3), 7.72e7.65 (m, 2H, H-6, H-
7), 7.62 (td, 3J ¼ 7.5 Hz, 4J ¼ 1.2 Hz, 1H, H-2), 6.98 (d, 3J ¼ 9.0 Hz, 2H,
2H, H-30, H-50), 3.87 (s, 3H, ArOCH3). 13C NMR (CDCl3, 101 MHz):
d:
182.2, 164.4, 151.4, 135.6, 134.8, 134.1, 133.9, 133.4, 133.2, 132.4,
132.3, 132.2, 131.0, 130.5, 128.7, 124.7, 120.3, 55.8. MS (ESI) m/z:
426.02 (M þ Hþ). Anal. Calcd for C22H13Cl2NO4 (in %): C-61.99, H-
3.07, N-3.29. Found: C-62.00, H-3.10, N-3.31.
H-30, H-50), 3.87 (s, 3H, ArOCH3). 13C NMR (CDCl3, 101 MHz)
d: 183.4,
164.3, 163.6, 150.9, 135.0, 133.9, 133.3, 133.1, 133.0, 132.4, 131.9,
131.5, 131.1,129.2, 128.3, 127.2, 126.1,120.7,114.4, 55.8, 31.2. MS (ESI)
m/z: 394.41 (M þ Hþ). Anal. Calcd for C21H15NO5S (in %): C-64.11, H-
3.84, N-3.56. Found: C-64.10, H-3.85, N-3.55.
5.1.4.6. Synthesis of 10-(3-hydroxy-4-methoxybenzoyloxyimino)-
10H-anthracen-9-one (23f). To a solution of 3-hydroxy-4-methoxy
benzoic acid (0.84 g, 5 mmol) in CH2Cl2 (50 mL), 4-dimethylami-
nopyridine (DMAP, 0.11 g, 0.9 mol) and 19 (1.12 g, 5 mmol) were
added. The resulting solution was cooled to 0 ꢁC, and supplemented
with N,N0-dicyclohexylcarbodiimide (DCC, 1.23 g, 6 mmol). The
mixture was stirred for 10 min at 0 ꢁC and then warmed to room
temperature. Progress of the reaction was monitored by TLC. Upon
completion, the precipitate was filtered off and the filtrate was
concentrated in vacuo to a mass which was diluted with CH2Cl2
(15 mL) and washed with saturated NH4Cl solution (15 mL) and
NaHCO3 solution (15 mL). The organic layers were dried over
Na2SO4, and the solvent was then evaporated under reduced
pressure. Purification by column chromatography with CH2Cl2 as
eluent afforded 23f as a pale yellow solid (0.59 g, 33%), mp: 193 ꢁC.
5.1.5.2. Synthesis of (E)-1,5-dichloro-10-(4-methoxyphenylsulphonylox-
yimino)-10H-anthracen-9-one (24b-E). The title compound was
prepared according to 24a from 20-E/21-Z (isomeric mixture, 1.17 g,
4 mmol), 4-methoxybenzenesulphonyl chloride (0.84 g, 4 mmol)
and sodium hydroxide (0.22 g, 6 mmol) in acetone (75 mL). Puri-
fication by column chromatography (CH2Cl2) afforded 24b-E as
a pale orange solid (1.00 g, 54%); mp: 185 ꢁC. 1H NMR (CDCl3,
400 MHz)
d
: 8.18 (dd, 3J ¼ 7.5 Hz, 4J ¼ 1.4 Hz, 1H, H-8), 7.98 (d,
3J ¼ 9.1 Hz, 1H, H-4), 7.94 (d, J ¼ 9.0 Hz, 2H, H-20, H-60), 7.68e7.43
3
(m, 4H, H-20, H-3, H-60, H-7), 6.98 (d, 3J ¼ 9.1 Hz, 2H, H-Ar), 3.86 (s,
3H, ArOCH3). 13C NMR (CDCl3, 101 MHz)
d: 182.2, 164.7, 150.5, 136.5,
135.7, 135.0, 134.4, 133.7, 132.9, 132.0, 131.6, 131.3, 130.8, 130.2,
129.5, 128.8, 128.1, 126.0, 124.3, 114.5, 55.9. MS (ESI) m/z: 463.30
(M þ Hþ). Anal. Calcd for C21H13Cl2NO5S (in %): C-54.56, H-2.83, N-
3.03. Found: C-54.55, H-2.84, N-3.00.
1H NMR (CDCl3, 400 MHz)
d
: 8.69 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.1 Hz, 1H, H-
5), 8.45 (dd, 3J ¼ 7.8 Hz, 4J ¼ 0.9 Hz, 1H, H-8), 8.39 (dd, 3J ¼ 7.7 Hz,
4J ¼ 1.3 Hz, 1H, H-1), 8.25 (dd, 3J ¼ 7.8 Hz, 4J ¼ 1.4 Hz, 1H, H-4),
7.86e7.56 (m, 5H, H-2, H-3, H-6, H-60, H-7), 6.95 (d, 3J ¼ 8.5 Hz, 1H,
H-50), 5.73 (s, 1H, ArOH), 3.93 (s, 3H, ArOCH3). 13C NMR (CDCl3,
5.1.6. Synthesis of 10-benzyloxyimino-10H-anthracen-9-ones
5.1.6.1. Synthesis of 10-benzyloxyimino-10H-anthracen-9-one (25a).
Sodium hydride (0.19 g, 8 mmol) and 19 (0.9 g, 4 mmol) were sus-
pended in dry DMF (5 mL). After stirring for 30 min, benzyl
bromide (1.03 g, 0.7 mL, 6 mmol) in THF (10 mL) was added and
stirred again at room temperature. Progress of the reaction mixture
was monitored by TLC. Upon completion, water (20 mL) was added.
The compound was extracted with CH2Cl2 (3 ꢄ 30 mL), the organic
layer was washed with water (25 mL) and brine solution (25 mL).
After drying with Na2SO4, the solvent was removed under reduced
pressure. Purification by column chromatography (CH2Cl2) of the
resulting solid afforded 25a as a beige solid (0.63 g, 51%); mp:
101 MHz) d: 183.5,163.5,151.4,151.1,145.9,135.0,134.0,133.2,133.0,
132.0, 131.5, 131.2, 131.1, 129.2, 128.3, 127.3, 126.1, 123.8, 121.5, 116.1,
110.5, 56.38. MS (ESI) m/z: 374.09 (M þ Hþ). Anal. Calcd for
C22H15NO5 (in %): C-70.67, H-4.05, N-3.75. Found C-70.77, H-4.06,
N-3.78.
5.1.4.7. Synthesis of 10-(4-hydroxy-3-methoxybenzoyloxyimino)-
10H-anthracen-9-one (23g). The title compound was prepared
according to 23f from 19 (1.08 g, 5 mmol), 4-hydroxy-3-methoxy
benzoic acid (0.86 g, 5 mmol), DCC (1.27 g, 6 mmol) and DMAP
(0.11 g, 0.9 mmol) in CH2Cl2 (50 mL). Purification by column
chromatography (CH2Cl2) afforded 23g as a yellow-orange solid
225 ꢁC. 1H NMR (CDCl3, 400 MHz)
d
:
8.94 (dd, 3J ¼ 7.9 Hz,
4J ¼ 1.4 Hz, 1H, H-5), 8.39 (dd, 3J ¼ 7.5 Hz, 4J ¼ 1.4 Hz, 1H, H-4), 8.26
(dd, 3J ¼ 7.9 Hz, 4J ¼ 1.4 Hz, 2H, H-1, H-8), 7.70e7.33 (m, 9H, H-2, H-
3, H-6, H-7, H-20 to H-60), 5.47 (s, 2H, O-CH2-Ar). 13C NMR (CDCl3,
(0.31 g, 17%). 1H NMR (DMSO-d6, 400 MHz)
d
: 8.77 (d, 3J ¼ 8.0 Hz,
1H, H-5), 8.28 (d, 3J ¼ 7.8 Hz, 2H, H-1, H-8), 8.16 (dd, 3J ¼ 7.7 Hz,
4J ¼ 1.4 Hz, 1H, H-4), 7.98 (td, 3J ¼ 7.7 Hz, 4J ¼ 1.3 Hz, 1H, H-2),
7.91e7.82 (m, 2H, H-3, H-6), 7.77 (t, 3J ¼ 7.4 Hz, 1H, H-7), 7.62 (d,
3J ¼ 8.3 Hz, 1H, H-60), 7.55 (s, 1H, H-20), 6.99 (d, 3J ¼ 8.3 Hz, 1H, H-
50), 5.75 (s, 1H, ArOH), 3.93 (s, 3H, ArOCH3). 13C NMR (CDCl3,
400 MHz) d: 183.8, 143.3, 137.1, 136.0, 133.3, 132.0, 131.2, 130.7,
130.6, 129.5, 129.4, 128.8, 128.6, 128.5, 127.8, 127.0, 124.9. MS (ESI)
m/z: 314.10 (M þ Hþ). Anal. Calcd for C21H15NO2 (in %): C-80.49, H-
4.82, N-4.47. Found: C-80.50, H-4.85, N-4.49.
101 MHz) d: 183.5, 163.5, 151.4, 151.1, 145.9, 135.0, 134.0, 133.2,
133.0, 132.0, 131.5, 131.2, 131.1, 129.2, 128.3, 127.3, 126.1, 123.8,
121.5, 116.1, 110.5, 56.4. MS (ESI) m/z: 373.09 (M þ Hþ). Anal. Calcd
for C22H15NO5 (in %): C-70.77, H-4.15, N-3.75. Found: C-70.77, H-
4.05, N-3.75.
5.1.6.2. Synthesis of 10-(4-methoxybenzyloxyimino)-10H-anthracen-
9-one (25b). The title compound was prepared according to 25a
from 19 (1.17 g, 5 mmol), 4-methoxybenzyl chloride (1.02 g, 0.9 mL,
7 mmol) and sodium hydride (0.25 g, 10 mmol). Purification of the

3362
G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
resulting solid afforded 25b as a white solid (0.24 g, 14%); mp:
H-30, H-50), 7.63e7.52 (m, 4H, H-2, H-3, H-6, H-7), 7.34 (d,
3J ¼ 8.8 Hz, 2H, H-20, H-60), 5.55 (s, 2H, O-CH2-Ar). 13C NMR (CDCl3,
235 ꢁC. 1H NMR (CDCl3, 400 MHz)
d
: 8.91 (d, 3J ¼ 8.0 Hz, 1H, H-4),
8.37 (d, 3J ¼ 8.0 Hz, 1H, H-8), 8.28 (d, 3J ¼ 7.6 Hz, 1H, H-1), 8.24 (d,
3J ¼ 7.6, 1H, H-4), 7.77e7.52 (m, 4H, H-2, H-3, H-6, H-7), 7.42 (d,
3J ¼ 8.7 Hz, 2H, H-20, H-60), 6.91 (d, 3J ¼ 8.7 Hz, 2H, H-30, H-50), 5.38
(s, 2H, O-CH2-Ar), 3.80 (s, 3H, Ar-OCH3). 13C NMR (CDCl3, 101 MHz)
101 MHz) d: 183.9, 147.2, 145.0, 144.8, 143.2, 134.6, 133.9, 133.8,
133.0, 131.4, 130.9, 130.1, 129.0, 127.9, 127.3, 126.8, 126.4, 124.5,
123.7, 123.1, 76.5. MS (ESI) m/z: 359.20 (M þ Hþ). Anal. Calcd for
C21H14N2O4 (in %): C-70.39, H-3.94, N-7.82. Found: C-70.40, H-3.95,
N-7.84.
d: 207.7, 184.1, 183.6, 160.0, 143.4, 136.4, 134.6, 134.0, 133.7, 132.3,
131.5, 130.9, 130.8, 130.4, 129.8, 129.45, 128.1, 127.7, 127.3, 125.2,
114.4, 31.4. MS (ESI) m/z: 344.12 (M þ Hþ). Anal. Calcd for
C22H17NO3 (in %): C-76.95, H-4.99, N-4.08. Found: C-76.95, H-4.99,
N-4.09.
5.1.6.7. Synthesis of (E)-1,5-dichloro-10-(4-methoxybenzyloxyimino)-
10H-anthracen-9-one (25g). The title compound was prepared
according to 25a from 20-E (1.17 g, 4 mmol), 4-methoxybenzyl
chloride (0.69 g, 0.6 mL, 4 mmol) and sodium hydride (0.19 g,
8.0 mmol). Purification of the resulting solid afforded 25g as
a yellow solid (0.22 g, 13%); mp: 196 ꢁC. 1H NMR (CDCl3, 400 MHz)
5.1.6.3. Synthesis of 10-(3,4,5-trimethoxybenzylimino)-10H-anthra-
cen-9-one (25c). The title compound was prepared according to
25a from 19 (1.12 g, 5 mmol), 3,4,5-trimethoxybenzyl bromide
(1.33 g, 5 mmol) and sodium hydride (0.14 g, 6.0 mmol). Purifica-
tion of the resulting solid afforded the title compound 25c as
d
: 8.29 (dd, 3J ¼ 7.6 Hz, 4J ¼ 1.4 Hz, 1H, H-8), 8.05 (d, 3J ¼ 7.7 Hz, 1H,
H-6), 7.98 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.3 Hz, 1H, H-2), 7.73 (dd, 3J ¼ 8.1 Hz,
4J ¼ 1.4 Hz, 1H, H-4), 7.65 (t, 3J ¼ 7.8 Hz, 1H, H-7), 7.53e7.31 (m, 3H),
a white solid (0.70 g, 35%); mp: 235 ꢁC. 1H NMR (CDCl3, 400 MHz)
d
:
6.89 (d, J ¼ 8.7 Hz, 2H, H-30, H-50), 5.27 (s, 2H, O-CH2-Ar), 3.80 (s,
3
8.95 (dd, 3J ¼ 8.0 Hz, 4J ¼ 1.3 Hz, 1H, H-8), 8.39 (dd, 3J ¼ 7.7 Hz,
4J ¼ 1.7 Hz, 1H, H-4), 8.27 (dd, 3J ¼ 8.0 Hz, 4J ¼ 1.2 Hz, 2H, H-1, H-8),
7.66e7.62 (m, 4H, H-2, H-3, H-6, H-7), 6.70 (s, 2H, H-20, H-60), 5.39
(s, 2H, O-CH2-Ar), 3.84 (s, 9H, Ar(OCH3)3). 13C NMR (CDCl3,
3H, ArOCH3). 13C NMR (CDCl3, 101 MHz)
d: 189.0, 159.2, 154.7, 140.6,
136.4, 133.6, 133.4, 133.0, 131.4, 130.5, 130.0, 129.4, 128.2, 126.9,
124.3, 113.9, 72.4, 56.0. MS (ESI) m/z: 412.04 (M þ Hþ). Anal. Calcd
for C22H15Cl2NO3 (in %): C-64.09, H-3.67, N-3.40. Found: C-64.10, H-
3.65, N-3.41.
101 MHz) d: 186.7, 156.5, 153.8, 138.7,134.7,133.4,133.1,131.8, 130.5,
130.4, 129.8, 126.0, 106.7, 71.7, 60.7, 56.8. MS (ESI) m/z: 404.43
(M þ Hþ). Anal. Calcd for C24H21NO5 (in %): C-71.45, H-5.25, N-3.47.
Found: C-71.46, H-5.28, N-3.51.
5.1.6.8. Synthesis of (E)-1,5-dichloro-10-(3,4,5-trimethoxybenzylox-
yimino)-10H-anthracen-9-one (25h). The title compound was
prepared according to 25a from 20-E (1.17 g, 4 mmol), 3,4,5-tri-
methoxybenzyl bromide (0.69 g, 4 mmol) and sodium hydride
(0.19 g, 8.0 mmol). Purification of the resulting solid afforded 25h as
a pale yellow solid (0.36 g, 19%); mp: 200 ꢁC. 1H NMR (CDCl3,
5.1.6.4. Synthesis of 10-(3-hydroxy-4-methoxybenzyloxyimino)-10H-
anthracen-9-one (25d). The title compound was prepared accord-
ing to 25a from 19 (0.3 g, 1.3 mmol), 3-hydroxy-4-methoxybenzyl
bromide (0.5 g, 1.3 mmol) and sodium hydride (0.05 g, 2 mmol).
Purification of the resulting solid afforded 25d as a yellow solid
400 MHz)
d
: 8.29 (dd, 3J ¼ 7.6 Hz, 4J ¼ 1.4 Hz, 1H, H-8), 8.05 (d,
3J ¼ 7.7 Hz, 1H, H-6), 7.98 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.3 Hz, 1H, H-2), 7.73
(dd, 3J ¼ 8.1 Hz, 4J ¼ 1.4 Hz, 1H, H-4), 7.65 (t, 3J ¼ 7.8 Hz, 1H, H-7),
7.53e7.31 (m, 1H, H-3), 6.89 (s, 2H, H-20, H-60), 5.27 (s, 2H, O-CH2-
(0.164 g, 34%); mp: 231 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d: 9.13 (d,
3J ¼ 8.1 Hz, 1H, H-5), 8.90 (d, 3J ¼ 8.5 Hz, 2H, H-1, H-8), 8.14 (d,
3J ¼ 7.8 Hz, 1H, H-4), 7.88e7.59 (m, 4H, H-2, H-3, H-6, H-7),
6.94e6.91 (m, 3H, H-20, H-50, H-60), 5.74 (s, 1H, Ar-OH), 5.35 (s, 2H,
Ar), 3.80 (s, 9H, Ar(OCH3)3). 13C NMR (CDCl3, 101 MHz)
d: 189.0,
154.7, 153.8, 140.6, 138.7, 136.4, 133.6, 133.4, 133.1, 133.0, 131.4,
130.5, 128.2, 126.9, 124.3, 106.7, 71.7, 60.7, 56.8. MS (ESI) m/z: 473.32
(M þ Hþ). Anal. Calcd for C24H29Cl2NO5 (in %): C-61.03, H-4.05, N-
2.97. Found: C-61.10, H-4.09, N-2.98.
O-CH2-Ar), 3.74 (s, 3H, Ar-OCH3). 13C NMR (DMSO-d6, 101 MHz)
d:
186.7, 156.5, 148.1, 147.8, 134.7, 133.4, 131.9, 131.8, 130.5, 130.4, 129.8,
126.0, 120.3, 115.0, 112.3, 72.0, 56.8. MS (ESI) m/z: 359.9 (M þ Hþ).
Anal. Calcd for C22H17NO4 (in %): C-75.53, H-4.77, N-3.90. Found: C-
75.53, H-4.78, N-3.90.
5.1.6.9. Synthesis of 1,8-dichloro-10-(4-methoxybenzyloxyimino)-
10H-anthracen-9-one (25i). The title compound was prepared
according to 25a from 22 (1.08 g, 3.7 mmol), 4-methoxybenzyl
chloride (0.87 g, 0.8 mL, 6 mmol) and sodium hydride (0.26 g,
11.0 mmol). Purification of the resulting solid afforded 25i as
5.1.6.5. Synthesis of 10-(4-chlorobenzyloxyimino)-10H-anthracen-9-
one (25e). The title compound was prepared according to 25a from
19 (0.22 g, 1 mmol), 4-chlorobenzyl chloride (0.18 g, 1 mmol) and
sodium hydride (0.03 g, 1.2 mmol). Purification of the resulting
solid afforded 25e as a yellow solid (0.19 g, 78%); mp: 231 ꢁC. 1H
a yellow solid (0.40 g, 26%). 1H NMR (CDCl3, 400 MHz)
d: 8.45 (dd,
3J ¼ 7.9 Hz, 4J ¼ 1.1 Hz, 1H, H-5), 7.90 (dd, 3J ¼ 7.8 Hz, 4J ¼ 1.2 Hz, 1H,
H-4), 7.53 (dd, 3J ¼ 8.1 Hz, 4J ¼ 1.1 Hz, 1H, H-7), 7.50 (dd, 3J ¼ 8.0 Hz,
4J ¼ 1.2 Hz, 1H, H-2), 7.43 (t, 3J ¼ 8.0 Hz, 2H, H-3, H-7), 7.36 (d,
3J ¼ 8.7 Hz, 2H, H-20, H-60), 6.90 (d, 3J ¼ 8.7 Hz, 2H, H-30, H-50), 5.31
NMR (CDCl3, 400 MHz)
d
: 8.90 (dd, 3J ¼ 7.9 Hz, 4J ¼ 1.4 Hz, 1H, H-5),
8.39 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.6 Hz, 1H, H-8), 8.25 (dd, 3J ¼ 7.7 Hz,
4J ¼ 1.5 Hz, 1H, H-1), 8.23e8.19 (m, 1H, H-4), 7.67e7.52 (m, 4H, H-2,
3
H-3, H-6. H-7), 7.41 (d, J ¼ 8.6 Hz, 2H, H-20, H-60), 7.35 (d,
(s, 2H, O-CH2-Ar), 3.80 (s, 3H, ArOCH3). 13C NMR (CDCl3,101 MHz)
d:
3J ¼ 8.6 Hz, 2H, H-30, H-50), 5.41 (s, 2H, O-CH2-Ar). 13C NMR (CDCl3,
186.9, 159.2, 152.9, 135.1, 132.4, 131.9, 130.9, 130.3, 130.0, 129.4,
128.0, 127.5, 113. 9, 72.4, 56.0. MS (ESI) m/z: 413.10 (M þ Hþ). Anal.
Calcd for C22H15Cl2NO3 (in %): C-64.09, H-3.67, N-3.40. Found C-
64.10, H-3.71, N-3.42.
101 MHz) d: 182.3,142.5, 136.0,134.9,133.8,133.7,132.9, 131.2,131.1,
130.7, 130.3, 129.9, 129.8, 128.6, 128.4, 127.2, 126.3, 124.5, 77.1, 30.8.
MS (ESI) m/z: 348.80 (M þ Hþ). Anal. Calcd for C21H14ClNO2 (in %):
C-72.52, H-4.06, N-4.03. Found: C-72.55, H-4.05, N-4.03.
5.1.6.10. Synthesis of 1,8-dimethoxy-10-(4-methoxybenzylimino)-
10H-anthracen-9-one (25j). The title compound was prepared
according to 25a from 18 (1.08 g, 4 mmol), 4-methoxybenzyl
chloride (0.87 g, 0.8 mL, 6 mmol) and sodium hydride (0.26 g,
11.0 mmol). Purification of the resulting solid afforded 25j as
a yellow solid (0.40 g, 26%); mp: 180 ꢁC. 1H NMR (CDCl3, 400 MHz)
5.1.6.6. Synthesis of 10-(4-nitrobenzyloxyimino)-10H-anthracen-9-
one (25f). The title compound was prepared according to 25a from
19 (0.75 g, 3.4 mmol), 4-nitrobenzyl chloride (0.58 g, 4 mmol) and
sodium hydride (0.19 g, 8.0 mmol). Purification of the resulting
solid afforded 25f as a yellow solid (0.41 g, 34%); mp: 228 ꢁC. 1H
NMR (CDCl3, 400 MHz)
d
: 8.90 (dd, 3J ¼ 7.9 Hz, 4J ¼ 1.2 Hz, 1H, H-4),
d: 8.09e8.05 (m, 2H, H-4, H-5), 7.84e7.75 (m, 2H, H-2, H-7), 7.65 (t,
8.40 (dd, 3J ¼ 7.7 Hz, 4J ¼ 1.6 Hz, 1H, H-8), 8.30 (d, 3J ¼ 7.8 Hz, 1H, H-
1), 8.15 (dd, 3J ¼ 8.1 Hz, 4J ¼ 1.0 Hz, 1H, H-4), 7.79 (d, 3J ¼ 8.8 Hz, 2H,
3J ¼ 8.1 Hz, 2H, H-3, H-6), 7.34 (d, 3J ¼ 8.3 Hz, 2H, H-20, H-60), 6.86 (d,
3J ¼ 8.7 Hz, 2H, H-30, H-50), 5.31 (s, 2H, O-CH2-Ar), 3.98 (s, 6H, Ar

G. Surkau et al. / European Journal of Medicinal Chemistry 45 (2010) 3354e3364
3363
(OCH3)2), 3.80 (s, 3H, ArOCH3). 13C NMR (CDCl3, 101 MHz)
d: 185.6,
Inc.) spectrophotometer, equipped with a temperature-controlled
multi channel cuvette holder. Tubulin polymerization was initiated
by shifting the temperature to 37 ꢁC and turbidity was recorded
over 45 min at 360 nm. The tubulin effectors were added from
stock solutions in DMSO. The final DMSO concentration was 1%.
Control measurements were made with DMSO, only. To quantify
compound activity, the turbidity signal after 40 min (plateau level,
representing the assembly/disassembly steady state) was
compared with that of the control samples. The IC50 value is
defined as the drug concentration that causes a 50% inhibition in
relation to the assembly level without test compound.
159.2, 157.7, 155.4, 134.6, 134.0, 132.6, 130.0, 129.4, 125.5, 122.8,
114.0, 113.9, 72.4, 57.1, 56.0. MS (ESI) m/z: 404.14 (M þ Hþ). Anal.
Calcd for C24H21NO5 (in %): C-71.45, H-5.25, N-3.47. Found: C-71.49,
H-5.24, N-3.49.
5.1.6.10.1. Synthesis of 1,8-dimethoxy-10-(3-hydroxy-4-methox-
ybenzylimino)-10H-anthracen-9-one (25k). The title compound
was prepared according to 25a from 18 (1.08 g, 4 mmol) and 3-
hydroxy-4-methoxybenzyl alcohol e prepared in situ from 3-
hydroxy-4-methoxybenzyl alcohol (0.48 g, 3.1 mmol) and PBr3
(0.65 g, 0.2 mL, 2.4 mmol) in CH2Cl2 (15 mL) e and sodium hydride
(0.04 g, 1.6 mmol); mp: 177 ꢁC. Purification of the resulting solid
afforded 25k as a yellow solid (0.1 g, 23%). 1H NMR (CDCl3,
Acknowledgement
400 MHz)
d
: 8.45 (dd, 3J ¼ 7.9 Hz, 4J ¼ 1.1 Hz, 1H, H-5), 7.90 (dd,
3J ¼ 7.8 Hz, 4J ¼ 1.2 Hz, 1H, H-4), 7.53 (dd, 3J ¼ 8.1 Hz, 4J ¼ 1.1 Hz, 1H,
H-7), 7.50 (dd, 3J ¼ 8.0 Hz, 4J ¼ 1.2 Hz, 1H, H-2), 7.43 (t, 3J ¼ 8.0 Hz,
2H, H-3, H-6), 7.36 (d, 3J ¼ 8.7 Hz, 1H, H-60), 6.94 (s, 1H, H-20), 6.90
(d, 3J ¼ 8.7 Hz, 1H, H-50), 5.74 (s, 1H, Ar0-OH), 5.31 (s, 2H, O-CH2-Ar),
3.98 (s, 6H, Ar(OCH3)2), 3.80 (s, 3H, ArOCH3). 13C NMR (CDCl3,
We wish to thank Marina Wollmann for excellent technical
assistance.
References
101 MHz) d: 185.6, 157.7, 155.4, 148.1, 147.8, 134.6, 134.0, 132.6, 131.9,
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3364
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