N-benzoylated phenoxazines and phenothiazines: Synthesis, antiproliferative activity, and inhibition of tubulin polymerization

Article abstract of DOI:10.1021/jm200436t

A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell l

Full text of DOI:10.1021/jm200436t

ARTICLE
pubs.acs.org/jmc
N-Benzoylated Phenoxazines and Phenothiazines: Synthesis,
Antiproliferative Activity, and Inhibition of Tubulin Polymerization
Helge Prinz,*,† Behfar Chamasmani,† Kirsten Vogel,† Konrad J. B€ohm,‡ Babette Aicher,§ Matthias Gerlach,§
Eckhard G. G€unther,§ Peter Amon,|| Igor Ivanov,|| and Klaus M€uller†
†Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University, Hittorfstrasse 58-62, D-48149 M€unster,
Germany
‡Leibniz Institute for Age ResearchꢀFritz Lipmann Institute (FLI), Beutenbergstrasse 11, D-07745 Jena, Germany
§Aeterna Zentaris GmbH, Weism€ullerstrasse 50, D-60314 Frankfurt, Germany
Oncolead GmbH & Co. KG, Fraunhoferstrasse 20, D-82152 Planegg, Germany
S Supporting Information
b
ABSTRACT: A total of 53 N-benzoylated phenoxazines and phenothiazines,
including their S-oxidized analogues, were synthesized and evaluated for anti-
proliferative activity, interaction with tubulin, and cell cycle effects. Potent
inhibitors of multiple cancer cell lines emerged with the 10-(4-meth-
oxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC50 values in the range of
2ꢀ15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly
inhibited tubulin polymerization with activities higher than or comparable to
those of the reference compounds such as colchicine. Concentration-dependent
flow cytometric studies revealed that inhibition of K562 cell growth was
associated with an arrest in the G2/M phases of the cell cycle, indicative of
mitotic blockade. Structureꢀactivity relationship studies showed that best
potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in
the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable
pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.
’ INTRODUCTION
including sulfonamide E-701015 (4), 4-anilinoquinazoline16 (5),
anthracenone-derived17 6, 2-methoxyestradiol18 (7), or propene-
nitrile CC-507919 (8) (Chart 1) are known to exert their
cytotoxic activities through binding to tubulin. The promising
usage of microtubule-binding drugs for anticancer therapy stimu-
lated intense efforts aimed at the development of further micro-
tubule-targeting drugs. Therefore, great efforts have recently
been made to develop novel small-molecular tubulin binders
derived not only from natural sources but also by screening
compound libraries in combination with traditional medicinal
chemistry.3,5,20
Microtubules play a key role in organizing the spatial distribu-
tion of organelles throughout interphase and of chromsomes
during cell division. The mitotic spindle, constituted of micro-
tubules generated by noncovalent polymerization of Rβ-tubulin
heterodimers, has become an attractive and successful target to
treat many types of malignancies.1,2 Drugs acting on microtu-
bules are of wide structural heterogeneity. Many of these agents
work by inhibiting the polymerization of tubulin into micro-
tubules,3ꢀ6 thereby arresting cells in the G2/M phase of the cell
cycle, inhibiting cell division and leading to cell death.
We have earlier described a series of anthracenone-derived
tubulin polymerization inhibitors with potent in vitro antitumor
activity.17,21ꢀ25 Continuing our search strategy for novel, potent
tubulin-targeting compounds, we extended our studies to tricy-
clic heterocycles, which are important scaffolds in medicinal chem-
istry and a part of numerous important therapeutic agents.26
Phenothiazine (9, Chart 2) and its derivatives have shown
diverse biological activities including psychotropic, anticancer,27
antihelminthic, and other pharmacological properties.28 10-
Substituted phenothiazines are well-known tranquilizers and
One of the best characterized, highly effective inhibitors of
tubulin polymerization is colchicine,7 which is a toxic natural
product obtained from Colchicum autumnale (2). However,
obviously due to its narrow therapeutic window, colchicine has
not found broad application in anticancer therapy.
So far, the most successful drugs in the clinic have been the
Catharanthus bis-indole alkaloids vinblastine (1, Chart 1) and
vincristine.8 In addition, drugs stimulating tubulin polymeriza-
tion and stabilizing microtubules such as the taxanes9 paclitaxel
and docetaxel were suitable for the treatment of several cancers,
such as ovarian cancer,10 nonsmall cell lung cancer,11 and Kaposís
sarcoma.12 Many other natural products, such as combretastatin
A-413 (3) or the epothilones14 as well as some synthetic molecules
Received: April 12, 2011
Published: May 12, 2011
r
2011 American Chemical Society
4247
dx.doi.org/10.1021/jm200436t J. Med. Chem. 2011, 54, 4247–4263
|

Journal of Medicinal Chemistry
ARTICLE
Chart 1. Examples of Tubulin Interacting Agents
antineoplastic actinomycin polypeptide antibiotics. Actinomycin
D (13), bearing a phenoxazine chromophore, represents one of
the older chemotherapy drugs and is primarily used as an
investigative tool in cell biology to inhibit transcription.34 Mean-
while, the anticancer activities of several phenoxazinones on
human leukemia cell lines have also been investigated.35ꢀ37 In
addition, N-substituted phenoxazines have recently been identi-
fied as modulators of MDR38,39 and as potent specific inhibitors
of Akt signaling.40
Chart 2. Phenothiazine (9), Phenoxazine (10), and Related
Structures 11, 12, and 13
This article reports the synthesis and biological evaluation of
N-benzoylated phenoxazines and phenothiazines. Some of them
demonstrated the ability to strongly inhibit the growth of various
tumor cell lines, to act in a cell-cycle-dependent manner, and to
be remarkably potent inhibitors of tubulin polymerization. The
antitubulin activity of the most active compounds was compar-
able or superior to those of the reference compounds, such as
nocodazole, podophyllotoxin, and colchicine.
’ CHEMISTRY
Phenothiazines 9, 14, 15, and 17 (Scheme 1) were available
from commercial sources. 2-Methoxy-substituted phenothiazine
16 was obtained from diphenylamine according to a literature
protocol.41 Oxidation resulting in ring sulfoxide metabolites is a
common biotransformation pathway for all phenothiazines.42
Numerous examples of phenothiazines in which the sulfur atom
is present in higher oxidation states (sulfoxide and sulfone) can
be found in the chemical literature. As these metabolites might
contribute to the biological effects of the parent compounds, we
were also interested in the biological activities related to these
phenothiazine derivatives. 10H-Phenothiazine-derived sulfox-
ides 18aꢀ18d and 18fꢀ18h were obtained by S-oxidation using
mCPBA,43 whereas sulfone derivatives 19aꢀ19d and 19fꢀ19i
have been synthesized from the corresponding phenothiazines
by oxidation with hydrogen peroxide in glacial acetic acid.44 The
antipsychotic drugs. Chloropromazine (11), prepared in 1950, is
the prototypical phenothiazine antipsychotic drug and perhaps
the best known of the phenothiazine drugs. Phenothiazine dye
methylene blue (12) is employed as a fast-acting antidote for the
treatment of methemoglobinemia.29 It is also a photosensitizer in
the photodynamic therapy of resistant plaque psoriasis.30 Phe-
nothiazine derivatives have further been demonstrated to induce
apoptosis in cultured leukemic cells31 and to cause mitotic arrest by
the inhibition of Eg5 ATPase.32 Moreover, aryl amide derivatives of
phenothiazine are able to inhibit butyrylcholinesterase.33
Antitumor activity among phenoxazine (10) and its derivatives was
not generally known before the discovery of the Streptomyces-derived
4248
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a) benzoyl chloride, toluene, reflux; (b) mCPBA, rt, 12 h; (c) AlCl3, CH2Cl2, 0 °C; (d) HOAc, H2O2 30%, reflux, 2h.
synthesis of carboxamides 9aꢀ9d, 14aꢀ14e, 15a, 16a, and 17a
was accomplished by nucleophilic reaction of the respective
phenothiazines with appropriately substituted benzoyl chlorides
in toluene or benzene, as outlined in Scheme 1. For the synthesis
of the isovanillic acid-derived amides 9e, 18e, and 19e, benzyl-
protected acid chlorides were reacted with appropriate phe-
nothiazines and their S-oxidized analogues in refluxing toluene,
followed by the removal of benzyl protection groups using AlCl3
in dichloromethane.21
prepared using a 5-step route according to literature proce-
dures.38 This classical 2-amino-20-halodiaryl ether approach
was also applied to the synthesis of the 2,8-dichloro-10H-
phenoxazine (29), whereas the 10H-phenoxazine carbonitriles
32, 33, and 34 were prepared by a cyano-activated fluoro
displacement reaction based on a protocol by Eastmond et al.45
(Scheme 3). Similar to the above-described phenothiazine
derivatives, N-benzoylation was achieved by reacting the phe-
noxazines with the respective benzoyl chlorides in refluxing
toluene (Scheme 4). Analogues 28e and 28g were obtained by
debenzylation of 28d and 28f, respectively. N-Benzoylated 33c
was successfully synthesized from 33 without isolating the
Unsubstituted phenoxazine (10) was commercially available.
The synthesis of chloro-substituted phenoxazines is depicted
in Scheme 2. The starting 2-chloro-10H-phenoxazine (28) was
4249
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
a Reagents and conditions: (a) KOH, H2O, 120 °C, 5h; (b) H2O, Fe°, CH3COOH, 90 °C, 3h; (c) HCOOH, 140ꢀ160 °C, 4h; (d) K2CO3, CuCO3,
p-xylene, reflux; (e) 2N NaOH, toluene, reflux.
Scheme 3a
a (a) DMF, K2CO3, toluene, N2, 130 °C; (b) DMSO, K2CO3, N2, rt; (c) DMF, 170 °C, N2, reflux.
benzyl-protected intermediate (Scheme 4). Synthesis of 2-(2-
chloro-phenoxazin-10-yl)-1-(4-methoxyphenyl)-ethanone (35)
was performed under phase transfer catalysis conditions using
2-bromo-1-(4-methoxyphenyl)ethanone and tetra-n-butylam-
monium bromide (TBAB, Scheme 5).
unsubstituted in the phenothiazine and in the terminal phenyl
ring, were largely devoid of K562 cell growth inhibitory proper-
ties (IC50 > 30 μM). Notably, when a methoxy group was
introduced at the C-4 position of the terminal phenyl ring, a
compound with potency in the submicromolar range (9b, IC50
0.33 μM) was obtained. Regarding the series of 2-chloro-
substituted phenothiazines, 14a and its S-oxidized analogues
18f and 19f were inactive, whereas p-methoxy analogue 14b
displayed submicromolar antiproliferative activity. However,
compared with the unsubstituted phenothiazine 9b, 2-chloro
analogue 14b was considerably less effective (14b, IC50 0.84 μM
vs 9b, IC50 0.33 μM). Moreover, compound 16a, being 2-meth-
oxy-substituted in the phenothiazine, presented a cell growth
inhibitory activity very similar to that of 14b. Compared with 9b,
slightly improved potencies were observed for the isovanillic
analogues 9e, 18e, and 19e. 10-(3-Hydroxy-4-methoxybenzoyl)-
10H-phenothiazine (9e) was the most active phenothiazine
derivative, which inhibited K562 cell growth with an IC50 of
0.21 μM. Interestingly, introduction of a 3,4,5-trimethoxyphenyl
group as exemplified in 9c strongly decreased growth inhibitory
properties in comparison with that of 4-methoxyphenyl 9b and
’ BIOLOGICAL RESULTS AND DISCUSSION
In Vitro Cell Growth Inhibition Assay. In initial screens with
human chronic myelogenous leukemia K562 cells,46 the com-
pounds were assessed for their ability to inhibit cell proliferation,
measured directly by counting the cells with a hemocytometer
after 48 h of treatment. Tables 1 and 2 summarize the data for the
inhibition of K562 cell growth, compared with the inhibition of
tubulin polymerization (see below). Colchicine, nocodazole,
podophyllotoxin, vinblastine, and adriamycin served as reference
compounds. Our data revealed several interesting aspects in-
cluding a broad range of potencies. Regarding the phenothiazines
and their S-oxidized analogues, nine compounds (9b, 9e, 14b,
16a, 18b, 18e, 19b, 19e, and 19g) showed IC50 values in the
submicromolar range. The compounds 9a, 18a, and 19a, being
4250
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Scheme 4a
a Reagents and conditions: (a) benzoyl chloride, acetyl chloride in the case of 28o, toluene, reflux; (b) AlCl3, 1,2-DCE, 0 °C; (c) (i) 3-benzyloxy-4-
methoxybenzoic acid, (COCl)2, 0 °C; (ii) pyridine, reflux; (iii) AlCl3, CH2Cl2, 0 °C, 4h.
Scheme 5a
methylene spacer between the amide group and the terminal
phenyl ring did not cause an activity enhancement but even
resulted in a nearly 10-fold reduction of antiproliferative potency
(14b, IC50 0.84 μM vs 14d, IC50 8 μM).
The more or less similar antiproliferative potencies of phe-
nothiazines and their corresponding sulfoxides and sulfones (9b
vs 18b, 19b; 9c vs 18c, 19c; 9e vs 18e, 19e; 14b vs 18g, 19g; and
16a vs 18i, 19i) allow one to conclude that the sulfur oxidation
state has little influence on antiproliferative effects. However, as
mentioned above, the formation of sulfoxides and sulfones from
phenothiazines is a common metabolizing pathway which cannot
be excluded under the conditions of the cellular assay, hence
explaining the similar results from the antiproliferative assay.
Similar to the phenothiazines 9b and 9e, the sulfoxides 18b (IC50
0.42 μM) and 18e (IC50 0.30 μM), respectively, were the most
active. As expected, trimethoxyphenyl-substituted 18c displayed
substantially less activity than the isovanillic 18e, though both
were unsubstituted in the phenothiazine ring. Moreover, the
2-chloro-substituted sulfoxide 18g was nearly 3-fold less active
than 18b and 18e, again indicating that introduction of a chloro
atom into the 2-position of the tricyclic nucleus did not improve
a Reagents and conditions: (a) TBAB, NaOH 50%/THF, 2-bromo-1-(4-
methoxyphenyl)ethanone, rt, 1h.
isovanillic 9e. Likewise, this finding was reaffirmed by a distinc-
tive loss of activity for 2-chloro-substituted analogue 14c (IC50
11 μM) vs 14b. In general, introduction of 2-substituents into the
phenothiazine ring, such as chloro, trifluoromethyl (15a, IC50
K562 4.60 μM), methoxy (16a), or thiomethyl (17a) did not
lead to improved activities compared with those of 9b or 9e, both
being unsubstituted in phenothiazine. Noteworthy, inserting a
4251
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Table 1. Antiproliferative Activity of Phenothiazine Derivatives 9aꢀ9e, 14aꢀ14d, 15a, 16a, 17a, 18aꢀ18i, and 19aꢀ19i against
K562 Cells and Anti-Tubulin Activities
cmpd
n
X
R1
R2
R3
R4
K562 IC50a [μM]
ITP IC50b [μM]
9
>100
>100
>30
0.33
1.86
>30
0.21
>30
0.84
11
ND
ND
ND
2.23
4.96
ND
1.40
ND
0.87
>10
ND
>10
3.23
1.43
ND
9.39
>10
ND
8.40
ND
2.79
>10
23.31
ND
2.53
2.46
ND
3.0
14
9a
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
S
S
S
S
S
S
S
S
S
S
S
S
H
H
H
H
H
H
H
H
H
H
9b
OCH3
OCH3
OCH3
OCH3
H
9c
OCH3
OBn
OH
H
OCH3
H
9d
9e
H
14a
14b
14c
14d
15a
16a
17a
18a
18b
18c
18d
18e
18f
18g
18h
18i
19a
19b
19c
19d
19e
19f
19g
19h
19i
Cl
H
Cl
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
Cl
OCH3
H
OCH3
H
Cl
8
CF3
OCH3
SCH3
H
H
H
4.60
0.97
3.62
>30
0.42
8.03
ND
0.30
>30
1.40
35
H
H
H
H
SO
H
H
SO
H
H
OCH3
OCH3
OCH3
OCH3
H
H
SO
H
OCH3
OBn
OH
H
OCH3
H
SO
H
SO
H
H
SO
Cl
H
SO
Cl
H
OCH3
OCH3
OCH3
H
H
SO
Cl
OCH3
H
OCH3
H
SO
OCH3
H
1.65
>30
0.28
2.60
ND
0.18
>30
0.80
4.2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
OCH3
OBn
OH
H
OCH3
H
H
H
H
Cl
H
ND
1.05
>10
4.69
1.4
Cl
H
OCH3
OCH3
OCH3
H
Cl
OCH3
H
OCH3
H
OCH3
1.58
0.02
ND
ND
0.001
0.01
colchicine
nocodazole
0.76
0.35
0.13
ND
podophyllotoxin
vinblastine sulfate
adriamycin
a IC50, concentration of compound required for 50% inhibition of cell growth (K562). Cells were treated with drugs for 48 h. IC50 values are the means of at least
three independent determinations (SD < 10%). b ITP = inhibition of tubulin polymerization; IC50 values represent the compound concentration at which the
maximum of tubulin assembly level amounts 50% of the level of the control without the compound (determined after 45 min polymerization of tubulin at 37 °C).
the antiproliferative action. Replacement of 2-chloro by methoxy
resulted in comparable activities (18i, IC50 1.65 μM vs 18g,
IC50 1.40 μM). As already observed with the nonoxidized 14c,
the presence of trimethoxy in sulfoxide 18h induced a dramatic
loss of antiproliferative potency (IC50 35 μM) compared with
that of 18g. In good agreement with the findings from the
sulfoxide series, sulfones 19b and 19e were the most active
analogues.
4252
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Table 2. Antiproliferative Activity of Phenoxazine Derivatives 10aꢀ10b, 28aꢀ28n, 28o, 29a, 32a, 33aꢀ33d, 34a, and 35 against
K562 Cells and Anti-Tubulin Activities
cmpd
n
R1
R2
R3
R4
R5
R6
R7
K562 IC50a [μM]
ITP IC50a [μM]
10
>30
>30
>10
0.033
4.81
0.14
1.38
0.55
ND
0.08
ND
4.63
1.12
24
>10
>10
>10
1.10
>10
0.57
0.65
1.10
ND
0.73
ND
0.68
1.13
ND
ND
2.42
ND
0.88
5.03
ND
0.70
2.70
1.2
28
33
10a
10b
28a
28b
28c
28d
28e
28f
28g
28h
28i
28j
28k
28l
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OCH3
H
OCH3
OCH3
OCH3
OBn
OBn
OH
OCH3
OCH3
OCH3
OCH3
H
OH
OCH2O
OCH3
H
OCH3
CF3
OCH3
H
>75
20
H
Cl
H
H
Br
H
>25
1.21
>80
>30
0.49
0.16
0.23
0.014
0.011
0.49
0.12
>20
28m
28n
28o
29a
32a
33a
33b
33c
33d
34a
35
H
N(CH3)2
NO2
H
H
H
0
0
0
0
0
0
0
H
Cl
CN
H
H
Cl
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
CN
CN
CN
CN
H
H
H
H
H
H
OCH3
OCH3
Cl
0.73
0.52
2.39
0.96
H
H
OH
H
H
H
H
CN
H
OCH3
a For details, see the footnotes in Table 1.
Regarding the phenoxazines, the 10-(4-methoxybenzoyl)-
10H-phenoxazine (10a) showed strong antiproliferative potency
with an IC50 of 33 nM, thus being 10-fold more active than
phenothiazine 9b (IC50 0.33 μM). Compound 10a had no
substitution on the phenoxazine ring. Inserting a spacer as exem-
plified in 10b caused a dramatic loss of potency. 2-Chloro-[10-(3-
hydroxy-4-methoxybenzoyl)]-10H-phenoxazin (28e), which in-
corporates the isovanillic partial structures also present in
combretastatin A-4 (3), also displayed strong antiproliferative
activity (28e, IC50 0.08 μM). In addition, compound 28a was
also found to be active (IC50 0.14 μM). A marked loss in potency
was observed for the isomeric 28b, confirming that a methoxy
group placed at the para-position of the terminal phenyl is
essential, while shifting it to the meta-position is detrimental
to potency (28a, IC50 0.14 μM vs 28b, IC50 1.38 μM). Also, the
activity of the dichloro analogue 29a (IC50 0.49 μM) was
diminished compared to the monochloro 28a, indicating that
introduction of a second chloro atom in phenoxazine (29a) did
not offer any advantage in terms of potency. Again, insertion of a
spacer as seen in 2-chloro-[10-(4-methoxyphenylacetyl)]-10H-
phenoxazine (28c) resulted in about a 4-fold reduction in potency as
compared with that of 28a (28c, IC50 0.55 μM vs. 28a, IC50 0.14
μM). Thus, it seems that for the N-benzoylated phenothiazines
(14b vs 14d; see above) and the phenoxazines (10a vs 10b and
28a vs 28c), the spatial disposition of the terminal phenyl ring
and the heterocycle is an influential factor in determining
antiproliferative activity. The comparison of the IC50 values
obtained for 28a with those obtained for N-unsubstituted 28
and 10-acetyl-2-chloro-10H-phenoxazine (28o, Scheme 4) clearly
demonstrated that the terminal phenyl ring is essential. The mere
4253
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Table 3. Antiproliferative Activity of Phenoxazines 10a, 28a, 28e, 33b and 33c against Different Tumor Cell Lines
IC50a [μM]
cmpd
10a
NCIH460
SKOV3
BT549
451LU
SW480
COLO-205
DLD-1
0.27
0.21
0.12
0.06
0.24
0.24
0.25
28a
0.68
0.35
0.33
0.09
0.53
0.43
0.54
28e
0.39
0.17
0.32
0.027
0.002
0.006
0.006
0.072
0.017
0.24
2.68
0.23
33b
0.015
0.04
0.006
0.024
0.006
0.08
0.008
0.016
0.006
0.069
0.023
0.008
0.029
0.011
0.12
0.006
ND
0.009
0.016
0.05
33c
paclitaxel
nocodazole
colchicine
0.004
0.068
0.027
0.005
0.088
0.021
0.19
0.029
0.030
0.089
a IC50 values were determined from Alamar Blue proliferation assays after incubation with the test compound for 48 h. All experiments were performed
at least in two replicates (n = 2), and IC50 data were calculated from doseꢀresponse curves by nonlinear regression analysis.
presence of an amide functional group in 28o of the phenoxazine
did not necessarily induce antiproliferative potency (IC50 > 30 μM,
Table 2). Furthermore, abolishing the amide in the case of 35
(Scheme 5) by inserting a spacer led to a loss of activity as
compared with 10a and 28a, indicating the importance of the
intact amide for potency (35, IC50 > 20 μM, Table 2). Notably,
structureꢀactivity studies showed that antiproliferative potencies
of the phenoxazines were ordinarily superior to those of the
phenothiazine congeners (e.g., see 10a vs 9b; 28a vs 14b). A
hydroxy group being flanked by methoxy groups in the case of
28g showed reduced potency (IC50 4.6 μM). Similar to the
aforementioned phenothiazine 9c, 28i bearing three methoxy
moieties showed a dramatic drop in potency (28a, IC50 0.14 μM
vs 28i, IC50 24 μM). This is in agreement with similar observa-
tions from our previous reports.17,24,25 The 4,5-methylenedioxy
bridge compound 28h displayed moderate activity comparable
to that of the dimethylamino compound 28m, whereas the halo-
substituted analogues 28k and 28l were considered to be more or
less inactive (IC50 values g20 μM). Also, the compounds 28j
and 28n with an electron-withdrawing trifluoromethyl and nitro
group, respectively, revealed a dramatically decreased inhibitory
effect on cell growth (IC50 > 70 μM) and were assessed to be not
active.
Excellent antiproliferative potencies in the phenoxazine series
could be attained by introducing a cyano group into the 3-posi-
tion of the phenoxazine (33b, K562 IC50 14 nM; 33c IC50
11 nM), leading to the most active compounds of all homologues
tested. Shifting the cyano group of 33b to the 1- or 2-position
of the phenoxazine, 34a and 32a, respectively, resulted in still
potent analogues but with decreased potency (32a, IC50 0.16 μM;
34a IC50 0.12 μM). However, presently, mechanistic details
underlying the impact of the CN group for potency within this
series of compounds, such as the importance for molecular
recognition or effects on electron densities have not been
elucidated yet and are speculative. In this context, several aspects
concerning the biological function of the nitrile group reviewed
by Fleming et al.47 might be of interest.
Table 4. Most Sensitive and Resistant Cell Lines after
Treatment of Solid Tumor Cell Lines with 33c
cmpd
33c
number of tested cell lines
83
maximum concentration [μM]
mimimum concentration [μM]
10
0.0001
activity
GI50 [μM]
maximum
minimum
mean
8.4137
0.0018
0.0111
0.0072
median
most sensitive cell lines (8 shown)
GI50 [μM]
z-score
SKNSH (brain)
MG63 (bone)
0.0018
0.0018
0.0018
0.0019
0.0020
0.0020
0.0026
0.0027
ꢀ1.0677
ꢀ1.0601
ꢀ1.0467
ꢀ1.0200
ꢀ1.0042
ꢀ0.9853
ꢀ0.8390
ꢀ0.8269
SKNAS (brain)
5637 (bladder)
RD (muscle)
SKMEL28 (skin)
HT1080 (connective tissue)
A-375 (skin)
most resistant cell lines (8 shown)
GI50 [μM]
z-score
MIAPACA2 (pancreas)
HT29 (colon)
8.4137
5.6032
4.2556
2.2494
1.1309
0.0951
0.0633
0.0623
3.8507
3.6145
3.4546
3.0841
2.6845
1.2459
1.0089
1.0000
COLO205 (colon)
BXPC3 (pancreas)
MDAMB436 (breast)
MT3 (breast)
IGROV1 (ovary)
COLO678 (colon)
Effect on Growth of Different Tumour Cell Lines. Next, to
further investigate the antiproliferative potential of tumor cells,
the effect of the highly active 3-cyano analogues 33b and 33c as
well as other potent analogues against a panel of seven cell lines
derived from solid tumors was investigated. The antiproliferative
activities of the compounds were quantified by the measurement
of the cellular metabolic reduction by conversion of the dye
Resazurin, yielding a fluorescence signal at 590 nm as indicator of
cellular viability.48 Of the tested compounds, again 33b displayed
outstanding overall antiproliferative activities with IC50 values
ranging from 2 to 15 nM (Table 3), slightly improved compared
to those of colchicine. This outstanding activity of the 3-cyano
analogue 33b is further supported by the colchicine-like activity
of the 3-cyano analogue 33c. Compound 28e was also effective,
though, as expected, its potency was substantially lower than that
of 33b and 33c.
4254
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Figure 1. (A) Plot of the most sensitive and resistant cell lines (8 are shown each) from the panel screening, visualized by using the z-score. Activities
against ASPC1 and resting PBMC were >10 μM. The zero value represents the mean concentration required to inhibit 50% of the growth for all cell lines
(GI50). The relative difference in the GI50 value expressed as the z-score is represented by the horizontal bars. A positive z-score means that it is less
sensitive to 33c treatment; a negative z-score means that it is more sensitive to 33c treatment. (B) Box-and-whisker diagram.
Cell Panel Screen. Cellular screening in combination with
biochemical data can provide the rationale for optimization
cycles and can be essential for understanding the specificity of
compound action. Therefore, we explored the potencies of 28e,
33b, and 33c against a broad range (83) of solid tumor cell
lines.49 The concept of the mean graph introduced by NCI
permits visualization of a cell activity parameter for a given
anticancer drug in all cells.50 Our study included more cell lines
(83 vs 60) and a broader spectrum of therapeutic indications
(17 vs 7) than in NCI. We additionally prolonged compound
treatment to 72 h to ensure the detection of effects even in slowly
proliferating cell lines. In addition, resting peripheral blood
mononuclear cells51 (PBMC) were included into the screening.
After treatment with test compounds, cell proliferation was
determined by means of sulforhodamine B (SRB) staining.
The results obtained with this panel of cell lines further
confirmed the high potency of 28e (18ꢀ35 nM; see Supporting
Information for details), 33b (6ꢀ10 nM; see Supporting Infor-
mation for details), and 33c (1.8ꢀ2.7 nM, Table 4 and Figure 1
A,B), with all values referring to the eight most sensitive cell lines.
The most sensitive and resistant cell lines were visualized either
by using a box-plot graph (Figure 1B) or by selecting the 8 most
and least sensitive cell lines using the z-score for each agent. The
values are plotted as horizontal bars from the zero value, which
represents the mean concentration required to inhibit 50% of the
growth for all cell lines (GI50). Each bar, therefore, represents the
relative activity of the compound in the given cell lines deviating
from the mean in all cell lines.
In spite of differences in absolute activities, the activity profiles
of 28e and 33b were very similar (Pearson coefficient similarity
of r = 0.91). Comparison of these activity profiles to the internal
database identified similarities to a set of different tubulin inhibi-
tors, such as 10-[(3-hydroxy-4-methoxy-benzylidene)]-9(10H)-
anthracenone17 (6, r = 0.91), combretastatin A-413 (r = 0.86),
and 2-methoxyestradiol18 (7, r = 0.82). In contrast, the similarity
of 33c to 33b and 28e, as well as to the tubulin inhibitors, was less
significant (<0.76). Interestingly, for 33c, no significant correla-
tion could be observed to the “classical” tubulin inihibitors col-
chicine or vinblastine. Another difference was observed in activity
distribution of these agents. Figure 1 demonstrates activities for
4255
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Figure 2. (A) Inhibition of in vitro polymerization of tubulin (in total
1.2 mg/mL protein; ∼85% tubulin plus ∼15% microtubule-associated
proteins) at 37 °C by various concentrations of 33c; turbidity was
recorded at 360 nm. The steady-state tubulin assembly level in the absence
of inhibitor was set at 100%. (B) IC50 values were determined by sigmoidal
fitting of the plot of the steady state levels of tubulin assembly (taken after
45 min) against drug concentration and represent the concentration for
50% inhibition of the maximum tubulin polymerization level.
Figure 3. Effect of 28e on cellular microtubules. Immunofluorescence
images of PtK2 cells labeled with Cy3-antitubulin antibody. Top, cells
incubated for 1 h in 2% DMSO; bottom, cells incubated for 1 h in 5 μM
28e/2% DMSO.
33c within the range of 2 nM for the most sensitive cell lines to
8 μM for resistant cell lines (Table 4 and Figure 1). It is worth
mentioning that the activity of 28e in the most resistant cell lines
(see Supporting Information) was in the same range as that for
33c, whereas its potency in the most sensitive cell line was an
order of magnitude less than that for 33c.
Compounds 33b and 33c showed high potencies with cancer
cell lines originating from the skin and brain showing above-
average sensitivity (negative z-scores, see Supporting Informa-
tion for details).
The human osteosarcoma cell line MG-63, human neuroblas-
toma SKNAS and SK-N-SH cells, and human bladder carcinoma
cell line 5637 were found to be among the most responsive cell
lines against 28e, 33b, and 33c. No activity was shown in resting
PBMC, suggesting that these agents may act preferably on proli-
ferating cells.
In Vitro Tubulin Polymerization Assays. To explore whether
the growth inhibitory effect of our compounds is correlated with
an interaction with the tubulin system, we measured their effect
on the polymerization of tubulin in a cell-free system. Their
activity was compared with that of the reference anti-tubulin
compounds colchicine, podophyllotoxin, nocodazole, and vin-
blastine (Table 1). In the presence of GTP and Mg2þ, Rβ-
tubulin is known to be able to self-assemble (polymerize) in vitro
into microtubules at physiological temperature (37 °C), accompanied
by changes of light scattering (turbidity). The turbidity curves,
usually obtained at 340ꢀ360 nm, reveal a sigmoid behavior with
a plateau level reached after 20ꢀ30 min at the conditions realized
in this study. Inhibition of tubulin polymerization is reflected by a
decreased level of steady state turbidity in a dose-dependent
manner as exemplified for 33c (Figure 2A).
The phenothiazine derivative 14b (IC50 ITP 0.87 μM)
inhibited tubulin polymerization at submicromolar concentra-
tion, whereas 9e and 17a displayed inhibitory activities compar-
able to that of colchicine (Table 1). The phenothiazines 9b, 16a,
18g, 19b, 19c, and 19i were moderate inhibitors of tubulin
polymerization with IC50 values in the range of 2ꢀ3 μM,
somewhat less active than colchicine (ITP IC50 1.4 μM). It is
striking to note that potencies of the phenothiazines and the
corresponding sulfones were comparable, whereas the sulfoxides
18b,18c, 18e, 18h, and 18i were more or less ineffective tubulin
polymerization inhibitors (IC50 g 10 μM). These low potencies,
particularly in the case of 18b, 18e, and 18h were not consistent
with our findings that these compounds were good inhibitors of
K562 tumor cell growth. On the one hand, these compounds
might exert their antiproliferative action by an alternative me-
chanism. On the other hand, as already pointed out, formation
of the sulfones under the conditions of the cellular assay cannot
be excluded.
4256
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Figure 4. Induction of cell cycle arrest by 33b. Cell cycle distribution of K562 cells showing the effect of 24 h-treatment with 33b (A) and colchicine (B)
(24 h assay). K562 cells were untreated, treated with different concentrations of 33b (A) or colchicine (B). After treatment, the cells were collected and
cell cycle distribution was measured by flow cytometry with FACSCalibur (Becton Dickinson), Software CellQuest Pro, version 5.2.
In the phenoxazine series, inhibition of tubulin polymerization
generally correlated well with drug-induced growth inhibition.
The compounds with the highest antiproliferative activity (10a,
28e, 33b, and 33c) were found also among the strongest
inhibitors of tubulin polymerization. Eight phenoxazines (28a,
28b, 28e, 28g, 28m, 29a, 33b, and 33c) proved to be exception-
ally strong inhibitors of tubulin polymerization (IC50 e 1 μM).
Paralleling the results from the antiproliferative studies, the 4-
methoxy as well as 3-hydroxy-4-methoxy phenyl were crucial
moieties for the inhibition of tubulin polymerization. Four
compounds (10a, 28c, 28h, and 33a) displayed nearly the same
activity and were as active as colchicine with IC50 values in the
range of 1 μM. Notably, the phenoxazines 28g, 28k, and even
28n, which was found to be nearly inactive in the K562 assay,
showed good to moderate tubulin inhibiting activity. This is
probably due to the fact that the tubulin polymerization assay is
not a cell-based assay, thus differing in a number of important
issues, such as tubulin concentration present within the cells or
possible effects of regulatory proteins expressed in the cell but
being absent in the tubulin assay. Other important aspects could
be differences in cell permeability of the various compounds or
reduced stability of the compounds in the cell supernatant
compared to the cell-free assay.
To study the effect on the microtubule system on the cellular
level, PtK2 cells were exposed for 1 h to 5 μM of phenothiazine
28e, added to RPMI medium. Immunofluorescence microscopy
revealed that the complex microtubule network has been
destroyed, whereas in the control preparation, treated with the
solvent (DMSO) only, the intracellular network of microtubules
was seen to be organized in large bundles irradiating from the cell
center (Figure 3).
Only a few single short microtubules remained after 28e action
(Figure 3). The effect of 28e on the PtK2 microtubule system
was qualitatively and quantitatively comparable to that of 5 μM
colchicine (data not shown).
Effect on Cell-Cycle Progression. By targeting mitotic spin-
dle formation, microtubule-binding agents should alter cell cycle
parameters with preferential G2/M blockade. As a consequence,
mitosis would be blocked at the transition from metaphase
to anaphase, thereby affecting chromosome segregation.52,53
4257
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
Cell cycle arrest is a typical feature for the majority of antimitotic
agents. Using a K562 cell-based assay, we investigated the highly
active compounds 33b and 33c for effects on the cell cycle. The
cells were exposed to test compounds in different concentra-
tions, and the percentage of cells in G2/M phase after 24 h was
monitored. Phenoxazine-derived amides 33b and 33c blocked
mitosis through an arrest of cells in the G2/M phase, as illustra-
ted in typical histograms (Figure 4; see Supporting Information
for 33c).
Both compounds effectively arrested cell cycle progression at
concentrations down to 30 nM (Table 4), i.e., they are nearly as
active as colchicine. This result indicates that from the series of
compounds represented in this article at least the phenoxazine-
derived amides 33b and 33c can be classified as antimitotic drugs
with high activity.
electron beam ionization (EI) and electrospray ionization (ESI). The
atmospheric pressure chemical ionization (APCI) method was per-
formed with a microTOF-QII apparatus (Bruker). The purity of all
target compounds was determined either by elemental analyses or by
reversed phase HPLC at 254 nm. Compound purity for all target
compounds is g95%. Elemental analyses were performed at M€unster
Microanalysis Laboratory, using a Vario EL elemental analyzer from
Elementar Analysensysteme GmbH Hanau, and all values were within
(0.4% of the calculated composition. The HPLC system applied a C18
phase (Nucleosil, 3 μm, 4.0 ꢁ 125 mm, CS-Chromatographie Service
GmbH, Langerwehe, Germany) eluting the compounds with an aceto-
nitrile/H2O gradient at a flow rate of 0.40 mL/min. All organic solvents
were appropriately dried or purified prior to use. Acid chlorides were
obtained from commercial sources or prepared to literature protocols.
Purification by chromatography refers to column chromatography on
silica gel (Macherey-Nagel, 70ꢀ230 mesh). In most cases, the concen-
trated pure fractions obtained by chromatography using the indicated
eluants were treated with a small amount of hexane to induce precipita-
tion. All new compounds displayed 1H NMR and MS spectra consistent
with the assigned structure. Yields have not been optimized. Analytical
TLC was done on Merck silica 60 F254 alumina coated plates (E. Merck,
Darmstadt). Chromatography refers to column chromatography using
Acros 60ꢀ200 mesh silica gel.
’ CONCLUSIONS
Synthesis and structureꢀactivity relationship studies for a new
class of synthetic inhibitors of tubulin polymerization, based on a
phenothiazine and phenoxazine molecular skeleton, were carried
out. These compounds (e.g., 9e, 10a, 33b, and 33c) represent
novel and highly potent inhibitors of cancer cell growth and
tubulin polymerization. Replacement of sulfur in the phenothia-
zine ring with oxygen in general increased the antiproliferative
activity. Furthermore, the SAR information revealed that
the introduction of a cyano group into the 3-position of the
phenoxazine moiety substantially improved the antiproliferative
potency compared with that of the unsubstituted analogues.
The most promising phenoxazines 33b and 33c inhibited tubulin
polymerization more efficiently than the reference compound
colchicine and inhibited the growth of multiple cancer cell lines
with IC50 values in the range of 2ꢀ15 nM.
10-Benzoyl-10H-phenothiazine (9a).33,54. See Supporting
Information for details.
10-4-Methoxybenzoyl)-10H-phenothiazine (9b).33,54
A
solution containing 9 (4.04 g, 20.3 mmol) and 4-methoxybenzoyl
chloride (4.14 g, 24.35 mmol) in toluene (80 mL) was refluxed with
stirring until all of 9 was consumed (TLC control). Then, the reaction
mixture was cooled and washed successively with aqueous sodium
bicarbonate 20% (3 ꢁ 15 mL), hydrochloric acid 6% (3 ꢁ 50 mL),
and finally with water (2 ꢁ 50 mL). The solution was dried (Na2SO4)
and filtered, and the solvent was evaporated. The crude product was
purified by chromatography (CH2Cl2/hexane, 9:1) to give 9b as a white
powder (5.88 g, 87%). mp 170ꢀ175 °C (lit.33 173ꢀ174 °C). FTIR
1654 cmꢀ1; 1H NMR (DMSO, 400 MHz, 300K) δ 7.92 (dd, J = 6.9, J =
2.2 Hz, 2H), 7.75 (dd, J = 7.8, J = 1.4 Hz, 2H), 7.55ꢀ7.42 (m, 6H), 6.84
(d, J = 8.9 Hz, 2H), 3.72 (s, 3H); MS (ESI) calcd for C20H15NO2S [M þ
Na]þ 356.07; found, 356.07; Anal. C20H15NO2S.
A p-methoxy substitution pattern or a isovanillic partial
structure in the terminal phenyl ring was proved to be essential
for both potent inhibition of tumor cell proliferation and inhibi-
tion of tubulin polymerization. The observed antiproliferative
effects are most probably due to the interaction with tubulin as
those compounds with the greatest inhibitory effects on cell
growth strongly inhibited tubulin polymerization. Potent induc-
tion of G2/M arrest, similar to that of colchicine, was demon-
strated for the most active phenoxazines 33b and 33c. Given the
relative ease of synthesis and due to their high in vitro antitumor
activities, we envision that compounds of this structural class are
attractive for further structural modifications. Moreover, our
findings are expected to provide useful information for the design
of novel antitumor agents.
10-(3,4,5-Trimethoxybenzoyl)-10H-phenothiazine (9c).55
See Supporting Information for details.
10-(3-(Benzyloxy)-4-methoxybenzoyl)-10H-phenothiazine
(9d). See Supporting Information for details.
10-(3-Hydroxy-4-methoxybenzoyl)-10H-phenothiazine
(9e). AlCl3 (0.36 g, 2.7 mmol) was added at room temperature to a
solution of 9d (0.42 g, 0.96 mmol) in CH2Cl2 (40 mL). After complete
conversion, as indicated by TLC, the solution was poured into water and
extracted with CH2Cl2 (3 ꢁ 50 mL). The combined organic phases were
dried over Na2SO4. Evaporation of the solvent under reduced pressure
provided a crude solid, which was purified by silica gel chromatography
(ethyl acetate/CH2Cl2 1:4) to afford 9e as a white powder (0.30 g, 90%).
Further structureꢀactivity studies in the series of phenothiazine-
and phenoxazine-based tubulin polymerization inhibitors are
in progress, and results from other modifications will be reported
in due course.
1
mp 208 209 °C; FTIR 1630 cmꢀ1; H NMR (DMSO-d6, 400 MHz,
300K) δ 9.19 (s, 1H), 7.55 (m, 2H), 7.44 (m, 2H), 7.27ꢀ7.21 (m, 4H),
6.80 (d, J = 2.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.68 (dd, J = 8.3, 2.0 Hz,
1H), 3.70 (s, 3H); MS (APCI) calcd for C20H15NO3S [M þ H]þ
350.08; found, 350.08. Anal. C20H15NO3S.
’ EXPERIMENTAL SECTION
Melting points were determined with a Kofler melting point appara-
1
10-(4-Methoxybenzoyl)-10H-phenoxazine (10a). See Sup-
porting Information for details.
10-(4-Methoxyphenylacetyl)-10H-phenoxazine (10b). See
Supporting Information for details.
10-Benzoyl-2-chloro-10H-phenothiazine (14a). See Suppor-
ting Information for details.
2-Chloro-[10-(4-methoxybenzoyl)]-10H-phenothiazine
(14b). See Supporting Information for details.
tus and are uncorrected. Spectra were obtained as follows: H NMR
spectra were recorded with Varian Gemini 2000 or Varian Mercury 400
plus (400 MHz) spectrometers operating at 300 and 400 MHz, respec-
tively. NMR signals were referenced to TMS (δ = 0 ppm) or solvent
signals and recalculated relative to TMS. Fourier-transform IR spectra
were recorded on a Bio-Rad laboratories Typ FTS 135 spectrometer,
and analysis was performed with WIN-IR Foundation software. Mass
spectra were obtained on Finnigan GCQ and LCQ apparatuses applying
4258
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
2-Chloro-10-(3,4,5-trimethoxybenzoyl)-10H-phenothiazine
(14c). See Supporting Information for details.
2-Chloro-[10-(4-methoxyphenylacetyl)]-10H-phenothiazine
(14d). See Supporting Information for details.
2-Methoxy-10-(4-methoxybenzoyl)-10H-phenothiazine-
5,5-dioxide (19i). See Supporting Information for details.
1-(2-Bromophenoxy)-4-chloro-2-nitro-benzene (20).38,56
The title compound was prepared according to the literature protocol.
2-Bromo-4-chloro-1-(4-chloro-2-nitrophenoxy)-benzene
(21). See Supporting Information for details.
2-(Trifluoromethyl)-10-(2-methoxybenzoyl)-10H-pheno-
thiazine (15a). See Supporting Information for details.
2-Methoxy-10H-phenothiazine (16).41 The title compound
was prepared according to the literature protocol.
2-(2-Bromophenoxy)-5-chloro-aniline (22).38 The title com-
pound was prepared according to the literature protocol.
2-Methoxy-10-(4-methoxybenzoyl)-10H-phenothiazine
(16a). See Supporting Information for details.
10-(2-Methoxybenzoyl)-2-(methylthio)-10H-phenothiazine
(17a). See Supporting Information for details.
10-Benzoyl-10H-phenothiazine-5-oxide (18a). The title
compound was prepared from 9a (0.5 g, 1.7 mmol) and mCPBA,
70ꢀ75% (0.29 g, 1.7 mmol) in CH2Cl2 (20 mL). The mixture was
stirred at room temperature for 12 h and then concentrated in vacuo.
Purification of the residue by chromatography (ethyl acetate/CH2Cl2
1:9) afforded 18a as a white powder (0.36 g, 66%). mp 176ꢀ181 °C;
FTIR 1673 cmꢀ1; 1H NMR (DMSO-d6, 400 MHz, 300 K) δ 7.96ꢀ7.86
(m, 2H), 7.71 (m, 2H), 7.55ꢀ7.44 (m, 6H), 7.39 (t, J = 7.4 Hz, 1H), 7.30
(t, J = 7.5 Hz, 2H); MS (ESI) calcd for C19H13NO2S [M þ Na]þ
342.06; found, 342.06. Anal. (C19H13NO2S) C, H, N.
10-(4-Methoxybenzoyl)-10H-phenothiazine-5-oxide (18b).
See Supporting Information for details.
10-(3,4,5-Trimethoxybenzoyl)-10H-phenothiazine-5-oxide
(18c). See Supporting Information for details.
10-(3-(Benzyloxy)-4-methoxybenzoyl)-10H-phenothiazine-
5-oxide (18d). See Supporting Information for details.
10-(3-Hydroxy-4-methoxybenzoyl)-10H-phenothiazine-5-
oxide (18e). See Supporting Information for details.
2-(2-Bromo-4-chlorophenoxy)-5-chloro-aniline (23). See
Supporting Information for details.
N-[2-(2-bromophenoxy)-5-chlorophenyl]-formamide (24).38
The title compound was prepared according to the literature protocol.
N-(2-(2-bromo-4-chlorophenoxy)-5-chlorophenyl)forma-
mide (25). See Supporting Information related to 27 for details.
2-Chloro-10H-phenoxazine-10-carboxaldehyde (26).38,56
The title compound was prepared according to the literature protocol.
2,8-Dichloro-10H-phenoxazine-10-carboxaldehyde (27).
See Supporting Information for details.
2-Chloro-10H-phenoxazine (28).38,56 The title compound was
prepared according to the literature protocol.
2-Chloro-10-(4-methoxybenzoyl)-10H-phenoxazine (28a).
4-Methoxybenzoyl chloride (4 mmol, 0.68 g) was added to a suspension
of 2-chlorophenoxazine 28 (4 mmol, 0.87 g) in toluene (50 mL). The
mixture was heated under reflux until the reaction was complete (TLC
control, SiO2, and ethyl acetate/petrol ether 1:1). Then, the solvent was
removed in vacuo, and the residue was purified by chromatography
(ethyl acetate/petrol ether 1:1) to afford 28a (0.79 g, 56%) as a white
1
powder. mp 148ꢀ149 °C; FTIR 1658 cmꢀ1; H NMR (CDCl3, 400
MHz, 300 K) δ 7.76 (d, 1H, J = 2.35 Hz), 7.41 (d, 2H, J = 8.61 Hz),
7.12ꢀ7.02 (m, 5H), 6.90ꢀ6.87 (m, 1H), 6.78 (d, 2H, J = 9.00 Hz) 3.81
(s, 3H); MS (EI, 70 eV) m/z (%) 352.94 (2.64), 351.88 (2.60), 350.91
(9.76), 135.11 (100). Anal. (C20H14ClNO3) C, H, N.
2-Chloro-10-benzoyl-10H-phenothiazine-5-oxide (18f). See
Supporting Information for details.
2-Chloro-10-(4-methoxybenzoyl)-10H-phenothiazine-5-
oxide (18g). See Supporting Information for details.
2-Chloro-10-(3-methoxybenzoyl)-10H-phenoxazine (28b).
See Supporting Information for details.
2-Chloro-10-(3,4,5-trimethoxybenzoyl)-10H-phenothiazine-
5-oxide (18h). See Supporting Information for details.
2-Chloro-[10-(4-methoxyphenylacetyl)]-10H-phenoxazine
(28c). See Supporting Information for details.
2-Methoxy-10-(4-methoxybenzoyl)-10H-phenothiazine-
5-oxide (18i). See Supporting Information for details.
2-Chloro-[10-(3-benzyloxy-4-methoxybenzoyl)]-10H-pheno-
xazine (28d). See Supporting Information for details.
10-Benzoyl-10H-phenothiazine-5,5-dioxide (19a). A solu-
tion of 9a (1.2 g, 3.95 mmol) and 30% H2O2(10 mL) in glacial acetic
acid (30 mL) was stirred and refluxed for 2 h. The solution was then
cooled, poured into water, and extracted with CH2Cl2 (3 ꢁ 100 mL).
The combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. Chromatography of the residue (ethyl acetate/
CH2Cl2 1:9) afforded 19a as a white powder (0.73 g, 55%). mp
2-Chloro-[10-(3-hydroxy-4-methoxybenzoyl)]-10H-pheno-
xazine (28e). AlCl3 (0.27 g, 2 mmol) was added to a solution of 28d
(1 mmol, 0.46 g) in 1,2-dichloroethane (20 mL) at 0 °C, and the mixture
was stirred until the reaction was completed. The solution was then
poured into water (200 mL) and extracted with CH2Cl2 (3 ꢁ 30 mL).
The combined organic layers were washed with water (2 ꢁ 50 mL) and
dried over anhydrous Na2SO4. The solvent was then removed in vacuo,
and the residue was purified by chromatography on silica gel (CH2Cl2)
to give 28e (0.11 g, 30%) as a white powder. mp 145ꢀ147 °C; FTIR
3352, 1648 cmꢀ1; 1H NMR (CDCl3, 400 MHz, 300 K) 7.74 (d, 1H, J =
2.34 Hz), 7.12ꢀ7.09 (m, 4H), 7.05 (d, 1H, 2.35 Hz), 7.03 (d, 1H, Jo =
8.61 Hz), 6.98ꢀ6.96 (m, 1H), 6.91ꢀ6.86 (m, 1H), 6.71 (d, 1H, J = 8.61
Hz), 5.61 (s, 1H), 3.89 (s, 3H). Purity (HPLC): 97.4%.
253ꢀ257 °C; FTIR 1681 cmꢀ1
;
1H NMR (DMSO-d6, 300 MHz,
300 K) δ 8.09ꢀ8.00 (m, 2H), 7.85ꢀ7.77 (m, 2H), 7.59 (m, 4H),
7.51ꢀ7.29 (m, 5H); MS (ESI) calcd for C19H13NO3S [M þ Na]þ
358.05; found, 358.05. Anal. (C19H13NO3S) C, H, N.
10-(4-Methoxybenzoyl)-10H-phenothiazine-5,5-dioxide
(19b). See Supporting Information for details.
2-Chloro-[10-(4-benzyloxy-3,5-dimethoxybenzoyl)]-10H-
phenoxazine (28f). See Supporting Information for details.
2-Chloro-[10-(3,5-dimethoxy-4-hydroxybenzoyl)]-10H-
phenoxazine (28g). See Supporting Information for details.
2-Chloro-[10-(3,4-methylenedioxybenzoyl)]-10H-pheno-
xazine (28h). See Supporting Information for details.
10-(3,4,5-Trimethoxybenzoyl)-10H-phenothiazine-5,5-di-
oxide (19c). See Supporting Information for details.
10-(3-(Benzyloxy)-4-methoxybenzoyl)-10H-phenothiazine-
5,5-dioxide (19d). See Supporting Information for details.
10-(3-Hydroxy-4-methoxybenzoyl)-10H-phenothiazine-
5,5-dioxide (19e). See Supporting Information for details.
10-Benzoyl-2-chloro-10H-phenothiazine-5,5-dioxide (19f).
See Supporting Information for details.
2-Chloro-[10-(3,4,5-trimethoxybenzoyl)]-10H-pheno-
xazine (28i). See Supporting Information for details.
2-Chloro-10-(4-methoxybenzoyl)-10H-phenothiazine-5,
5-dioxide (19g). See Supporting Information for details.
2-Chloro-10-(3,4,5-trimethoxybenzoyl)-10H-phenothiazine-
5,5-dioxide (19h). See Supporting Information for details.
2-Chloro-[10-(4-trifluoromethylbenzoyl)]-10H-pheno-
xazine (28j). See Supporting Information for details.
2-Chloro-[10-(4-chlorobenzoyl)]-10H-phenoxazine (28k).
See Supporting Information for details.
4259
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
2-Chloro-[10-(4-bromobenzoyl)]-10H-phenoxazine (28l).
See Supporting Information for details.
2-Chloro-[10-(4-dimethylaminobenzoyl)]-10H-pheno-
xazine (28m). See Supporting Information for details.
2-Chloro-[10-(4-nitrobenzoyl)]-10H-phenoxazine (28n).
See Supporting Information for details.
173ꢀ175 °C; FTIR 3393, 2235, 1675 cmꢀ1
;
1H NMR (CDCl3,
300 MHz, 300 K) δ 7.67 (d, 1H, J = 8.4 Hz), 7.31 (d, 1H, J = 1.8
Hz), 7.22 (dd, 1H, J = 8.4, 1.9 Hz), 7.17ꢀ7.12 (m, 1H), 7.11ꢀ7.01
(m, 3H), 6.96ꢀ6.84 (m, 2H), 6.65 (d, 1H, J = 8.4 Hz), 5.56 (s, 1H),
3.83 (s, 3H). Anal. C21H14N2O4, MS (APCI) calcd for C21H14N2O4
[M þ H]þ 359.10; found, 359.11. Anal. C21H14N2O4.
10-(4-Chlorobenzoyl)-10H-phenoxazine-3-carbonitrile
(33d). See Supporting Information for details.
2-Chloro-10-acetyl-10H-phenoxazine (28o). See Supporting
Information for details.
10H-Phenoxazine-1-carbonitrile (34). The title compound
was prepared according to a literature protocol.45 mp 186 °C (lit.45
182ꢀ184 °C).
2,8-Dichloro-10H-phenoxazine (29). See Supporting Informa-
tion for details.
2,8-Dichloro-[10-(4-methoxybenzoyl)]-10H-phenoxazine
(29a). See Supporting Information for details.
10-(4-Methoxybenzoyl)-10H-phenoxazine-1-carbonitrile
(34a). See Supporting Information for details.
10H-Phenoxazine-2-carbonitrile (32). The title compound
was prepared according to a literature protocol;45 mp 213ꢀ214 °C
(lit.45 212ꢀ214 °C).
2-(2-Chloro-phenoxazin-10-yl)-1-(4-methoxy-phenyl)-
ethanone (35). 2-Chlorphenoxazine 28 (0.65 g, 3 mmol) and TBAB
(0.1 g, 0.3 mmol) were suspended in a mixture of NaOH 50% (5 mL),
THF (2 mL) and water (2 mL). Then, a solution of 2-bromo-1-(4-
methoxyphenyl)ethanone (1.37 g, 6 mmol) in THF (5 mL) was added
in one portion. The reaction mixture was then stirred at room tempe-
rature for 1 h, poured into water (400 mL), acidified with 6 N HCl, and
extracted with CH2Cl2 (3 ꢁ 30 mL). The combined CH2Cl2 extracts
were washed, dried over Na2SO4, and then evaporated. Purification by
chromatography (CH2Cl2) gave 35 as a white solid (0.71 g, 63%). mp
179 °C; FTIR 1677 cmꢀ1; 1H NMR (CDCl3, 400 MHz, 300 K) 8.05 (d,
2H, J = 9.0 Hz), 7.03 (d, 2H, J = 9.0 Hz), 6.74ꢀ6.70 (m, 3H), 6.63ꢀ6.60
(m, 2H), 6.19ꢀ6.14 (m, 2H), 4.90 (s, 2H), 3.92 (s, 3H); MS (APCI)
calcd for C21H16ClNO3 [M þ H]þ 365.08; found, 365.08. Anal.
(C21H16ClNO3) C, H, N.
Biological Assay Methods. Cells and Culture Conditions.
Human chronic myelogenous K562 leukemia cells were obtained from
DSMZ, Braunschweig, Germany, and cultured at 37 °C under 5% CO2
in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum
(FBS, Biochrom KG), streptomycin (0.1 mg/mL), penicillin G (100
units/mL), and L-glutamine (30 mg/L). Cell line SF268 (central nerval
system carcinoma) was obtained from NCI. The human tumor cell lines
SKOV3 (ovarian adenocarcinoma), NCI-H460 (lung), BT549 (breast
carcinoma), 451LU (lung), SW480 (colon adenocarcinoma), COLO-205
(colon), and DLD-1 (colon adenocarcinoma) were obtained from ATCC.
Screening of the agents in a panel of 83 cell lines from solid tumors by
means of sulforhodamine B staining was performed by following the
protocol described by DTP NCI (http://dtp.nci.nih.gov/branches/
btb/ivclsp.html) and in more details specific to this study in Supporting
Information.
10-(4-Methoxybenzoyl)-10H-phenoxazine-2-carbonitrile
(32a). See Supporting Information for details.
10H-phenoxazine-3-carbonitrile (33).45 A mixture of 2-ami-
nophenol (1.96 g, 18.0 mmol), dissolved in DMF (30 mL), potassium
carbonate (5.0 g), and toluene (20 mL) was heated under reflux (N2,
DeanꢀStark). It was then cooled to 80 °C, and 3,4-difluorobenzonitrile
(2.50 g, 17.97 mmol) was added. The mixture was again heated under
reflux for 4 h. Thereafter, toluene was distilled off and replaced with
DMF. The mixture was then heated at 150 °C for 2 h (TLC control).
After cooling to room temperature, NaOH (2N, 100 mL) was added.
The mixture was then extracted with CH2Cl2 (3 ꢁ 50 mL). The
combined organic layers were washed with water, dried (Na2SO4),
and concentrated under reduced pressure. Purification of the residue by
chromatography (CH2Cl2) gave 33 as a yellow crystalline solid (2.77 g,
74%). mp 202ꢀ203 °C (lit.45 198ꢀ199 °C).
10-Benzoyl-10H-phenoxazine-3-carbonitrile (33a). See
Supporting Information for details.
10-(4-Methoxybenzoyl)-10H-phenoxazine-3-carbonitrile
(33b). A mixture of 33 (0.21 g, 1 mmol) and 4-methoxybenzoyl
chloride (0.17 g, 1 mmol) in toluene (15 mL) was stirred and heated
under reflux (oil bath, 130 °C). After 5 h (TLC control), the mixture was
cooled to room temperature, and the solvent was evaporated in vacuo.
Purification of the residue by chromatography (CH2Cl2) afforded 33b
as white crystals (0.10 g, 30%). mp 184ꢀ185 °C; FTIR 2228,
1
1672 cmꢀ1; H NMR (CDCl3, 300 MHz, 300 K) δ 7.70 (d, 1H, J =
8.4 Hz), 7.40ꢀ7.34 (m, 2H), 7.31 (d, 1H, J = 1.8 Hz), 7.23 (dd, 1H, J =
8.4 Hz, J = 1.9 Hz), 7.11ꢀ7.03 (m, 3H), 6.89ꢀ6.83 (m, 1H), 6.75ꢀ6.69
(m, 2H), 3,74 (s, 3H). Anal. C21H14N2O3, MS (ESI) calcd for
C21H14N2O3 [M þ Na]þ 365.09; found, 365.09. Anal. C21H14N2O3.
10-(3-Hydroxy-4-methoxybenzoyl)-10H-phenoxazine-3-
carbonitrile (33c). A mixture of 3-benzyloxy-4-methoxybenzoic
acid57 (0.26 g, 1 mmol) and oxalyl chloride (14.55 g, 115.63 mmol)
was stirred in an ice bath (0 °C) until the solution turned clear (1 h).
Then, excess oxalyl dichloride was removed under reduced pressure.
10H-Phenoxazine-3-carbonitrile (33, 0.21 g, 1 mmol) was added, and
the mixture was heated under reflux in pyridine. After the reaction was
completed (TLC control, 6 h), the mixture was poured into ice water,
acidified with HCl (100 mL, 4 N) and extracted with CH2Cl2 (3 ꢁ
50 mL). The combined organic layers were washed with water (2 ꢁ
50 mL) and dried over anhydrous Na2SO4, and the solvent was removed
in vacuo. The crude residue (0.32 g, 0.9 mmol crude product) was
suitable for use without purification and dissolved in CH2Cl2 at 0 °C.
AlCl3 (0.60 g, 4.5 mmol) was added, and the color turned from yellow to
dark red. After the reaction was completed (4 h, TLC control), water was
added, and the mixture was extracted with CH2Cl2 (3 ꢁ 50 mL). The
combined organic layers were washed with water (2 ꢁ 50 mL) and dried
over anhydrous Na2SO4, and the solvent was removed in vacuo.
Purification of the organic phase by chromatography (ethyl acetate/
petroleum ether 3:7) afforded 33c as a beige powder (0.26 g, 73%). mp
Assay of Cell Growth. K562 cells were plated at 2 ꢁ 105 cells/mL in
24 well dishes (Costar, Cambridge, MA). Untreated control wells were
assigned a value of 100%. Test compounds were made soluble in
DMSO/methanol 1:1, and control wells received equal volumes
(0.5%) of vehicle alone. We demonstrated that 0.5% DMSO (final
DMSO concentration in all samples including the controls) did not
affect K562 cell growth. To each well, 5 μL of compound was added, and
the final volume in the well was 500 μL. Cell numbers were counted with
a Neubauer counting chamber (improved, double grid) after 48-h
compound exposure. Each assay was prepared in triplicate, and the
experiments were carried out three times. IC50 values were obtained by
nonlinear regression (GraphPad Prism) and represent the concentration
at which cell growth was inhibited by 50%. The adjusted cell number was
calculated as a percentage of the control, which was the number of cells
in wells without the test compound.
Cellular Viability Assay. The AlamarBlue reagent is based on the dye
Resazurin, which exhibits fluorescence change in the appropriate
oxidationꢀreduction range relating to cellular metabolic reduction.48
The increase of fluorescence at 590 nM is an indicator of cellular
viability/cell number. The cells were seeded in the respective growth
medium in 125 μL per 96 wells and were grown for 24 h at 37 °C/5%CO2.
4260
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
For doseꢀresponse analysis, 10 mM compound stocks in 100% DMSO
were diluted in half logarithmic steps in cell medium. The final DMSO
concentration in compound and control wells is adjusted to 0.316%.
Twenty-five microliters of the respective compound dilutions are added
per 96 wells. After 45 h of compound incubation at 37 °C/5% CO2,
15 μL of the detection reagent AlamarBlue (AbD Serotec, cat. no.
BUF012B) was added for an additional 3 h, and after a total of 48 h of
compound incubation, the cellular metabolic activity was quantified by
measurement of fluorescence at 590 nm. Nontreated cells and blank
controls without cells are set as reference values. IC50 values were
obtained by nonlinear regression (GraphPad Prism).
’ ACKNOWLEDGMENT
We wish to thank Roland Lindstedt, Katrin Zimmermann,
Angelika Zinner and Marina Wollmann for excellent technical
assistance.
’ ABBREVIATIONS
Akt, a serine-threonine kinase; APCI, atmospheric pressure chemi-
cal ionization; ITP, inhibition of tubulin polymerization; MTP,
microtubule protein; ND, not determined; SRB, sulforhodamine B
Isolation of MTP and In Vitro MT Assembly Assay by Turbidimetric
Measurement. Microtubule protein (MTP) consisting of 80 to 90%
tubulin and 10 to 20% microtubule associated proteins (MAPs) was
isolated from the porcine brain by two cycles of temperature-dependent
disassembly (0 °C)/reassembly (37 °C), according to the described
procedures.58,59 Throughout the preparation, a buffer containing 20 mM
PIPES (1,4-piperazine diethane sulfonic acid, pH 6.8), 80 mM NaCl,
0.5 mM MgCl2, 1 mM ethylene bis-(oxyethylenenitrilo) tetraacetic acid,
and 1 mM dithiothreitol was used. To start tubulin polymerization, stock
MTP solutions were diluted with the preparation buffer to 1.2 mg/mL
(corresponding to a final tubulin concentration of about 10 μmol/L) in
glass cuvettes, guanosin-50-triphosphate was added to 0.6 mM (final
concentration), and the samples were transferred into a Cary 4E spectro-
photometer (Varian Inc.), equipped with a temperature-controlled multi-
channel cuvette holder, adjusted to 37 °C. Turbidity, which increased as a
result of tubulin polymerization,60 was recorded at 360 nm over 45 min.
The test compounds were added from stock solutions made in
DMSO. The final DMSO concentration was 1%. Control measurements
were made with DMSO only. To quantify the drug activity, the turbidity
signal after 45 min (plateau level, representing the assembly/disassem-
bly 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 the drug.
’ REFERENCES
(1) Honore, S.; Pasquier, E.; Braguer, D. Understanding microtu-
bule dynamics for improved cancer therapy. Cell. Mol. Life Sci. 2005,
62, 3039–3056.
(2) Pellegrini, F.; Budman, D. R. Review: tubulin function, action of
antitubulin drugs, and new drug development. Cancer Invest. 2005, 23,
264–273.
(3) Hearn, B. R.; Shaw, S. J.; Myles, D. C. Microtubule targeting
agents. Compr. Med. Chem. II 2007, 7, 81–110.
(4) Jordan, M. A. Mechanism of action of antitumor drugs that
interact with microtubules and tubulin. Curr. Med. Chem. Anticancer
Agents 2002, 2, 1–17.
(5) Li, Q.; Sham, H. L. Discovery and development of antimitotic
agents that inhibit tubulin polymerisation for the treatment of cancer.
Expert Opin. Ther. Patents 2002, 12, 1663–1702.
(6) Mahindroo, N.; Liou, J. P.; Chang, J. Y.; Hsieh, H. P. Antitubulin
agents for the treatment of cancer: a medicinal chemistry update. Expert
Opin. Ther. Patents 2006, 16, 647–691.
(7) Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Anti-
mitotic activity of colchicine and the structural basis for its interaction
with tubulin. Med. Res. Rev. 2008, 28, 155–183.
(8) Duflos, A.; Kruczynski, A.; Barret, J. M. Novel aspects of natural
and modified Vinca alkaloids. Curr. Med. Chem. Anticancer Agents 2002,
2, 55–70.
(9) Ferlini, C.; Gallo, D.; Scambia, G. New taxanes in development.
Expert. Opin. Invest. Drugs 2008, 17, 335–347.
Flow Cytometric Analysis of Cell Cycle Status. K562 cells (2 ꢁ 105
cells/mL) were exposed to each compound at different concentrations
for 24 h at 37 °C. The control cells were treated with vehicle (DMSO/
MeOH 1:1) only. After treatment, K562 cells were washed twice with
phosphate buffered saline (PBS), centrifuged (5 min, 500g), resuspended
in 1 mL of PBS, fixed with 5 mL ice-cold 70% ethanol, and stored at 4 °C.
DNA content was then measured after staining with propidium iodide
(Sigma) solution (1 mL, 0.02 mg/mL propidium iodide, and 0.2 mg/mL
RNase) for 30 min. Finally, the cells were analyzed using a FACSCalibur
flow cytometer (Becton Dickinson), equipped with an argon-ion laser
operating at a wavelength of 488 nm. The distribution of cells in the cell
cycle was analyzed with the CellQuest Pro Software Version 5.2.
(10) Markman, M. Antineoplastic agents in the management of
ovarian cancer: current status and emerging therapeutic strategies.
Trends Pharmacol. Sci. 2008, 29, 515–519.
(11) Gridelli, C.; Aapro, M.; Ardizzoni, A.; Balducci, L.; De Marinis,
F.; Kelly, K.; Le Chevalier, T.; Manegold, C.; Perrone, F.; Rosell, R.;
Shepherd, F.; De Petris, L.; Di Maio, M.; Langer, C. Treatment of
advanced non-small-cell lung cancer in the elderly: Results of an
international expert panel. J. Clin. Oncol. 2005, 23, 3125–3137.
(12) Dhillon, T.; Stebbing, J.; Bower, M. Paclitaxel for AIDS-asso-
ciated Kaposi’s sarcoma. Expert Rev. Anticancer Ther. 2005, 5, 215–219.
(13) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Isolation and structure of the strong cell growth and
tubulin inhibitor combretastatin A-4. Experientia 1989, 45, 209–211.
(14) Goodin, S.; Kane, M. P.; Rubin, E. H. Epothilones: mechanism
of action and biologic activity. J. Clin. Oncol. 2004, 22, 2015–2025.
(15) Yoshimatsu, K.; Yamaguchi, A.; Yoshino, H.; Koyanagi, N.;
Kitoh, K. Mechanism of action of E7010, an orally active sulfonamide
antitumor agent: inhibition of mitosis by binding to the colchicine site of
tubulin. Cancer. Res. 1997, 57, 3208–3213.
(16) Sirisoma, N.; Kasibhatla, S.; Pervin, A.; Zhang, H.; Jiang, S.;
Willardsen, J. A.; Anderson, M. B.; Baichwal, V.; Mather, G. G.; Jessing,
K.; Hussain, R.; Hoang, K.; Pleiman, C. M.; Tseng, B.; Drewe, J.; Cai,
S. X. Discovery of 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-
4-amine (EP128265, MPI-0441138) as a potent inducer of apoptosis
with high in vivo activity. J. Med. Chem. 2008, 51, 4771–4779.
(17) Prinz, H.; Ishii, Y.; Hirano, T.; Stoiber, T.; Camacho Gomez,
J. A.; Schmidt, P.; D€ussmann, H.; Burger, A. M.; Prehn, J. H.;
G€unther, E. G.; Unger, E.; Umezawa, K. Novel benzylidene-9(10H)-
anthracenones as highly active antimicrotubule agents. Synthesis,
’ ASSOCIATED CONTENT
S
Supporting Information. Supporting analytical and ex-
b
perimental data for compounds 9aꢀ9d, 10a, 10b, 14aꢀ14d,
15a, 16a, 17a, 18bꢀ18i, 19bꢀ19i, 21, 23, 27, 28bꢀ28o, 32a,
and 34a; methodology of the panel screen; visualization of
z-scores for 28e, 33b, and 33c; cell cycle analysis data of K562
cells treated with 33b and colchicine; FACS histograms for 33c;
and table of elemental analysis results of all target compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ49 251-8332195. Fax: þ49 251-8332144. E-mail: prinzh@
uni-muenster.de.
4261
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
antiproliferative activity, and inhibition of tubulin polymerization.
J. Med. Chem. 2003, 46, 3382–3394.
(34) Sobell, H. M.; Actinomycin, D. N. A. Transcription. Proc. Natl.
Acad. Sci. U.S.A. 1985, 82, 5328–5331.
(18) D’Amato, R. J.; Lin, C. M.; Flynn, E.; Folkman, J.; Hamel, E.
2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits
tubulin polymerization by interacting at the colchicine site. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3964–3968.
(19) Zhang, L. H.; Wu, L.; Raymon, H. K.; Chen, R. S.; Corral, L.;
Shirley, M. A.; Narla, R. K.; Gamez, J.; Muller, G. W.; Stirling, D. I.;
Bartlett, J. B.; Schafer, P. H.; Payvandi, F. The synthetic compound CC-
5079 is a potent inhibitor of tubulin polymerization and tumor necrosis
factor-alpha production with antitumor activity. Cancer. Res. 2006,
66, 951–959.
(35) Miyano-Kurosaki, N.; Ikegami, K.; Kurosaki, K.; Endo, T.;
Aoyagi, H.; Hanami, M.; Yasumoto, J.; Tomoda, A. Anticancer effects
of phenoxazine derivatives revealed by inhibition of cell growth and
viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-
positive leukemia cells. J. Pharmacol. Sci. 2009, 110, 87–97.
(36) Motohashi, N.; Mitscher, L. A.; Meyer, R. Potential antitumor
phenoxazines. Med. Res. Rev. 1991, 11, 239–294.
(37) Shimamoto, T.; Tomoda, A.; Ishida, R.; Ohyashiki, K. Anti-
tumor effects of a novel phenoxazine derivative on human leukemia cell
lines in vitro and in vivo. Clin. Cancer Res. 2001, 7, 704–708.
(38) Eregowda, G. B.; Kalpana, H. N.; Hegde, R.; Thimmaiah, K. N.
Synthesis and analysis of structural features of phenoxazine analogues
needed to reverse vinblastine resistance in multidrug resistant (MDR)
cancer cells. Indian J. Chem. 2000, 39B, 243–259.
(39) Horton, J. K.; Thimmaiah, K. N.; Harwood, F. C.; Kuttesch,
J. F.; Houghton, P. J. Pharmacological characterization of N-substituted
phenoxazines directed toward reversing Vinca alkaloid resistance in
multidrug-resistant cancer cells. Mol. Pharmacol. 1993, 44, 552–559.
(40) Thimmaiah, K. N.; Easton, J. B.; Germain, G. S.; Morton, C. L.;
Kamath, S.; Buolamwini, J. K.; Houghton, P. J. Identification of N10-
substituted phenoxazines as potent and specific inhibitors of Akt
signaling. J. Biol. Chem. 2005, 280, 31924–31935.
(20) Hadfield, J. A.; Ducki, S.; Hirst, N.; McGown, A. T. Tubulin and
microtubules as targets for anticancer drugs. Prog. Cell Cycle Res. 2003,
5, 309–325.
(21) Nickel, H. C.; Schmidt, P.; B€ohm, K. J.; Baasner, S.; M€uller, K.;
Gerlach, M.; Unger, E.; G€unther, E. G.; Prinz, H. Synthesis, antiproli-
ferative activity and inhibition of tubulin polymerization by 1,5- and 1,8-
disubstituted 10H-anthracen-9-ones bearing a 10-benzylidene or 10-(2-
oxo-2-phenylethylidene) moiety. Eur. J. Med. Chem. 2010, 45, 3420–3438.
(22) Prinz, H.; Schmidt, P.; B€ohm, K. J.; Baasner, S.; M€uller, K.;
Unger, E.; Gerlach, M.; G€unther, E. G. 10-(2-oxo-2-Phenylethylidene)-
10H-anthracen-9-ones as highly active antimicrotubule agents: synth-
esis, antiproliferative activity, and inhibition of tubulin polymerization.
J. Med. Chem. 2009, 52, 1284–1294.
(23) Surkau, G.; B€ohm, K. J.; M€uller, K.; Prinz, H. Synthesis, anti-
proliferative activity and inhibition of tubulin polymerization by anthrace-
none-based oxime derivatives. Eur. J. Med. Chem. 2010, 45, 3354–3364.
(24) Zuse, A.; Schmidt, P.; Baasner, S.; B€ohm, K. J.; M€uller, K.;
Gerlach, M.; G€unther, E. G.; Unger, E.; Prinz, H. 9-Benzylidene-
naphtho[2,3-b]thiophen-4-ones as novel antimicrotubule agents-synth-
esis, antiproliferative activity, and inhibition of tubulin polymerization.
J. Med. Chem. 2006, 49, 7816–7825.
(41) Kadaba, P. K.; Massie, S. P. Ring derivatives of phenothiazine.
IV. Further studies on the thionation reaction, and the synthesis of
phenothiazinols. J. Org. Chem. 1959, 24, 986–987.
(42) Williams, D. A. Part I: Principles of Drug Discovery. Chapter
10: Drug Metabolism. In Foyꢀes Principles of Medicinal Chemistry, 6th ed.;
Lemke, T. L., Williams, D. A., Eds.; Lippincott Williams & Wilkins:
Philadelphia, PA, 2008; pp 253ꢀ326.
(43) Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Boger, D. L.
Heterocyclic sulfoxide and sulfone inhibitors of fatty acid amide hydro-
lase. Bioorg. Med. Chem. Lett. 2005, 15, 103–106.
(25) Zuse, A.; Schmidt, P.; Baasner, S.; B€ohm, K. J.; M€uller, K.;
Gerlach, M.; G€unther, E. G.; Unger, E.; Prinz, H. Sulfonate derivatives of
naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone as
highly active antimicrotubule agents. Synthesis, antiproliferative activity,
and inhibition of tubulin polymerization. J. Med. Chem. 2007, 50,
6059–6066.
(44) Smith, N. L. Formation and oxidation of some phenothiazine
derivatives. J. Org. Chem. 1951, 16, 415–418.
(45) Eastmond, G. C.; Gilchrist, T. L.; Paprotny, J.; Steiner, A. Cyano-
activated fluoro displacement reactions in the synthesis of cyanophenox-
azines and related compounds. New J. Chem. 2001, 25, 385–390.
(46) Lozzio, C. B.; Lozzio, B. B. Human chronic myelogenous
leukemia cell-line with positive philadelphia chromosome. Blood 1975,
45, 321–334.
(47) Fleming, F. F.; Yao, L. H.; Ravikumar, P. C.; Funk, L.; Shook,
B. C. Nitrile-containing pharmaceuticals: efficacious roles of the nitrile
pharmacophore. J. Med. Chem. 2010, 53, 7902–7917.
(26) Baldessarini, R. J. In Goodman & Gilmaꢀns The Pharmacological
Basis of Therapeutics, 11th ed.; Brunton, L. L., Lazo, J. S., Parker, K. L.,
Eds.; McGraw-Hill: New York, 2006; pp 429ꢀ459.
(27) Motohashi, N.; Gollapudi, S. R.; Emrani, J.; Bhattiprolu, K. R.
Antitumor properties of phenothiazines. Cancer Invest. 1991, 9, 305–
319.
(28) Ohlow, M. J.; Moosmann, B. Phenothiazine: the seven lives of
pharmacologꢀys first lead structure. Drug Discovery Today 2011, 16,
119–131.
(48) Nociari, M. M.; Shalev, A.; Benias, P.; Russo, C. A novel one-
step, highly sensitive fluorometric assay to evaluate cell-mediated
cytotoxicity. J. Immunol. Methods 1998, 213, 157–167.
(29) Harvey, J. W.; Keitt, A. S. Studies of the efficacy and potential
hazards of methylene blue therapy in aniline-induced methaemoglobi-
naemia. Br. J. Hamaetol. 1983, 54, 29–41.
(49) Sharma, S. V.; Haber, D. A.; Settleman, J. Cell line-based
platforms to evaluate the therapeutic efficacy of candidate anticancer
agents. Nat. Rev. Cancer 2010, 10, 241–253.
(30) Salah, M.; Samy, N.; Fadel, M. Methylene blue mediated
photodynamic therapy for resistant plaque psoriasis. J. Drugs Dermatol.
2009, 8, 42–49.
(31) Zhelev, Z.; Ohba, H.; Bakalova, R.; Hadjimitova, V.; Ishikawa,
M.; Shinohara, Y.; Baba, Y. Phenothiazines suppress proliferation and
induce apoptosis in cultured leukemic cells without any influence on the
viability of normal lymphocytes: phenothiazines and leukemia. Cancer
Chemother. Pharmacol. 2004, 53, 267–275.
(32) Okumura, H.; Nakazawa, J.; Tsuganezawa, K.; Usui, T.;
Osada, H.; Matsumoto, T.; Tanaka, A.; Yokoyama, S. Phenothiazine
and carbazole-related compounds inhibit mitotic kinesin Eg5 and
trigger apoptosis in transformed culture cells. Toxicol. Lett. 2006, 166,
44–52.
(50) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, a.; Scudiero,
D. a.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display and analysis of
patterns of differential activity of drugs against human-tumor cell-lines:
development of mean graph and compare algorithm. J. Natl. Cancer Inst.
1989, 81, 1088–1092.
(51) Bøyum, A. Isolation of mononuclear cells and granulocytes
from human blood: isolation of mononuclear cells by one centrifugation
and of granulocytes by combining centrifugation and sedimentation at 1
G. Scand. J. Clin. Lab. Invest. 1968, S 21, 77–89.
(52) Kanthou, C.; Greco, O.; Stratford, A.; Cook, I.; Knight, R.;
Benzakour, O.; Tozer, G. The tubulin-binding agent combretastatin
A-4-phosphate arrests endothelial cells in mitosis and induces mitotic
cell death. Am. J. Pathol. 2004, 165, 1401–1411.
(33) Darvesh, S.; McDonald, R. S.; Darvesh, K. V. B.; Mataija, D.;
Conrad, S.; Gomez, G.; Walsh, R.; Martin, E. Selective reversible
inhibition of human butyrylcholinesterase by aryl amide derivatives of
phenothiazine. Bioorg. Med. Chem. 2007, 15, 6367–6378.
(53) Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.; Rondanin,
R.; Piccagli, L.; Baruchello, R.; Rossi, M.; Romagnoli, R.; Invidiata, F. P.;
Grimaudo, S.; Jung, M. K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa,
V.; Di Cristina, A.; Dusonchet, L.; Meli, M.; Tolomeo, M. Heterocyclic
4262
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263

Journal of Medicinal Chemistry
ARTICLE
and phenyl double-bond-locked combretastatin analogues possessing
potent apoptosis-inducing activity in HL60 and in MDR cell lines.
J. Med. Chem. 2005, 48, 723–36.
(54) Mackie, A.; Cutler, A. A. Preparation of phenothiazine deriva-
tives as possible anthelmintics. J. Chem. Soc. 1954, 2577–2579.
(55) Turbanti, L.; DiPaco, G. F. Acyl derivatives of phenothiazine
with presumed sedative action. Boll. Chim. Farm. 1961, 100, 490–495.
(56) Olmsted, M. P.; Zirkle, C. L.; Craig, P. N.; Pavloff, a. M.;
Lafferty, J. J. Analogs of phenothiazines. II. Phenoxazine and phenose-
lenazine analogs of phenothiazine drugs. J. Org. Chem. 1961, 26,
1901–1907.
(57) Wang, M.; Gao, M.; Mock, B. H.; Miller, K. D.; Sledge, G. W.;
Hutchins, G. D.; Zheng, Q.-H. Synthesis of carbon-11 labeled fluori-
nated 2-arylbenzothiazoles as novel potential PET cancer imaging
agents. Bioorg. Med. Chem. 2006, 14, 8599–8607.
(58) Shelanski, M. L.; Gaskin, F.; Cantor, C. R. Microtubule
assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci.
U.S.A. 1973, 70, 765–768.
(59) Vater, W.; B€ohm, K. J.; Unger, E. A simple method to obtain
brain microtubule protein poor in microtubule-associated proteins. Acta
Histochem. Suppl. 1986, 33, 123–129.
(60) Gaskin, F.; Cantor, C. R.; Shelanski, M. L. Turbidimetric
studies of the in vitro assembly and disassembly of porcine neurotubules.
J. Mol. Biol. 1974, 89, 737–755.
4263
dx.doi.org/10.1021/jm200436t |J. Med. Chem. 2011, 54, 4247–4263