(Z)-1-Aryl-3-arylamino-2-propen-1-ones, highly active stimulators of tubulin polymerization: Synthesis, structure-activity relationship (SAR), tubulin polymerization, and cell growth inhibition studies

Article abstract of DOI:10.1021/jm300176j

Tubulin, the major structural component of microtubules, is a target for the development of anticancer agents. A series of (Z)-1-aryl-3-arylamino-2- propen-1-one (10) were synthesized and evaluated for antiproliferative activity in cell-based assay. The most active compound (Z)-1-(2-bromo-3,4,5- trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)prop-2-en-1-one (10ae) was tested in 20 tumor cell lines including multidrug resistant phenotype and was found to induce apoptosis in all these cell lines with similar GI₅₀ values. Flow cytometry studies showed that 10ae arrested the cells in G2/M phase of cell cycle. In addition to G2/M block, these compounds caused microtubule stabilization like paclitaxel and induced apoptosis via activation of the caspase family. The observations made in this investigation demonstrate that (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) represents a new class of microtubule-stabilizing agents.

Full text of DOI:10.1021/jm300176j

Article
pubs.acs.org/jmc
(Z)-1-Aryl-3-arylamino-2-propen-1-ones, Highly Active Stimulators of
Tubulin Polymerization: Synthesis, Structure−Activity Relationship
(SAR), Tubulin Polymerization, and Cell Growth Inhibition Studies
M. V. Ramana Reddy,*,† Balaiah Akula,‡,§ Stephen C. Cosenza,†,§ Clement M. Lee,†
Muralidhar R. Mallireddigari,‡ Venkat R. Pallela,‡ D. R. C. Venkata Subbaiah,† Andrew Udofa,†
and E. Premkumar Reddy*,†
†Department of Oncological Sciences, Mount Sinai School of Medicine, Icahn Medical Institute, 1425 Madison Avenue, New York,
New York 10029-6514, United States
‡Department of Medicinal Chemistry, Onconova Therapeutics Inc., 375 Pheasant Run, Newtown, Pennsylvania 18940, United States
ABSTRACT: Tubulin, the major structural component of micro-
tubules, is a target for the development of anticancer agents. A series
of (Z)-1-aryl-3-arylamino-2-propen-1-one (10) were synthesized and
evaluated for antiproliferative activity in cell-based assay. The most
active compound (Z)-1-(2-bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)prop-2-en-1-one (10ae) was
tested in 20 tumor cell lines including multidrug resistant phenotype and was found to induce apoptosis in all these cell lines with
similar GI50 values. Flow cytometry studies showed that 10ae arrested the cells in G2/M phase of cell cycle. In addition to G2/M
block, these compounds caused microtubule stabilization like paclitaxel and induced apoptosis via activation of the caspase family.
The observations made in this investigation demonstrate that (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) represents a new class
of microtubule-stabilizing agents.
The encouraging antivascular activity of compound E has
stimulated significant interest in exploring new antitublin
agents. A variety of synthetic small molecules have been
reported as inhibitors of polymerization, which compete with
the colchicine-binding site of tubulin.5 Structurally, they involve
various heteroaromatic cores, for instance including the
benzothiophene,6 benzofuran,7 imidazole,8 thiozole,9 and
oxadiazoline10 moieties. A number of indole-based compounds,
for example, 2-aroylindoles,11 3-aroylindoles,12 3-aroyl-2-
phenylindoles,7 3-arylthioindoles-2-carboxylate,13 and indolyl-
3-glyoxamides,14 have shown strong antiproliferative and
tubulin depolymerizing activity.
Compounds like paclitaxel promote tubulin polymerization
into microtubules and block microtubule dynamics, leading to
abnormal mitosis and subsequent apoptosis.15,16 Paclitaxel was
the first microtubule-stabilizing agents (MSA) discovered.17 It
promotes the in vitro polymerization of tublin in the presence
of guanosine triphosphate (GTP), which is normally required
for microtubules assembly, and in cells, paclitaxel induces
microtubule stabilization, leading to the formation of character-
istic microtubule bundles.18
INTRODUCTION
■
Tubulin-containing structures, such as microtubules are
important for the formation of the mitotic spindle during the
process of mitosis. They play a critical role in cell growth,
division, and cytoskeletal organization of normal and tumor
cells as well as being implicated in motility, shape, and
intracellular transport.1 Microtubules are hollow tubes of α- and
β-tubulin heterodimers that polymerize parallel to a cylindrical
axis. Tubulin binding molecules interfere with the dynamic
instability of microtubules and thereby disrupt microtubules,
inducing cell cycle arrest in the M-phase, forming abnormal
mitotic spindles resulting in the apoptotic cell death.2
A
number of natural compounds (see Chart 1), such as paclitaxel
(A), epothilone (B), vinblastine (C), combretastatin A-4
(CA4) (D), dolastatin 10 (G), and colchicine (H), attack
microtubules by interfering with the dynamics of tubulin
polymerization and depolymerization, resulting in mitotic
arrest.3 However, many clinically useful chemotherapy drugs
face substantial limitations such as drug resistance, high
systemic toxicity, complex syntheses, and isolation procedures.
This has encouraged scientists to develop new antimitotic
agents. Recent studies have reported compounds targeting the
colchicine-binding domain of β-tubulins that can act as
vascular-disrupting agents, rapidly depolymerizing microtubules
of newly formed vasculature to block the blood supply to
tumors,4 while leaving blood supply to healthy cells intact.
These compounds include drug candidates combretastatin A-
4P (CA4P) (E), Oxi4503, R, R1 = OPO3Na2 (F), ZD6126 (I),
which are shown in Chart 1.
Three nontaxoid compounds have also been shown to
stabilize microtubules. The macrolides epothilone A and
epothilone B (B) from the bacterium Sorangium cellulosum,19
and discodermolide (J) from the marine sponge Discodermia
dissolute,20 share with paclitaxel the ability to arrest cells in
mitosis, cause formation of bundles of intracellular micro-
Received: February 8, 2012
© XXXX American Chemical Society
A
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Chart 1. Examples of Tubulin Interacting Agents
tubules in nonmitotic cells, and induce the formation of
hyperstable tubulin polymers.
tion of 8 with aryl amines (9) in ethanol at room temperature
resulted in the formation of (Z)-1-aryl-3-arylamino-2-propen-1-
one (10) in good yields.
A large number of small molecules have been described in
the literature which inhibit tubulin polymerization in vitro and
in vivo, resulting in the arrest of the cells in mitosis, ultimately
leading to apoptosis. So far only two reports have described
small synthetic molecules that stimulate tubulin polymerization
and microtubule stabilization.21,22 In this paper, we describe the
synthesis, structure−activity relationship (SAR), and mode of
action of a new series of (Z)-1-aryl-3-arylamino-2-propen-1-one
(10) that stimulate tubulin polymerization and stabilizes
microtubules.
Chemistry. The synthesis of target compounds (Z)-1-aryl-
3-arylamino-2-propen-1-one (10) is illustrated in Scheme 1.
Arylaldehydes 1, 2, 3, and 6 were treated with ethynylmagne-
sium bromide in dry tetrahydrofuran (THF) at 0 °C to room
temperature, producing 1-aryl-2-propyn-1-ol (7). Oxidation of
7 with 2-iodoxybenzoic acid (IBX) in the presence of dimethyl
sulfoxide (DMSO) gave 1-aryl-2-propyn-1-one (8). Condensa-
Some of the substituted arylaldehydes (2, 3, 6), which are
not commercially available, were synthesized as shown in the
Schemes 2, 3, and 4. Chlorination of 3,4,5-trimethoxybenzalde-
hyde (1c) with sulfuryl chloride (SO2Cl2) in the presence of
dichloromethane (CH2Cl2) afforded 2-chloro-3,4,5-trimethox-
ybenzaldehyde, 2 (Scheme 2). Similarly, the corresponding
bromo compound 3 was obtained according to methods
reported in literature.23 Bromination of 3,4,5-trimethoxyben-
zaldehyde (1c), with N-bromosuccinimide (NBS) in chloro-
form at reflux temperature for 3 h, afforded 2-bromo-3,4,5-
trimethoxybenzaldehyde, 3 (Scheme 3), whereas the 2-nitro-
3,4,5-trimethoxybenzaldehyde, 6, resulted by the oxidation of
(3,4,5-trimethoxy-2-nitrophenyl)methanol (5) with manganese
dioxide (MnO2) in the presence of chloroform (CHCl3) at
room temperature for 48 h. 5, in turn, was obtained by
reduction of methyl 3,4,5-trimethoxy-2-nitrobenzoate (4) with
B
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assessed using two different human tumor cell lines derived
from human prostate (DU145) and leukemic (K562) cancers.
The results of the study are presented in Table 1. These studies
Scheme 1. Synthesis of (Z)-1-Aryl-3-arylamino-2-propen-1-
one from 1-Aryl-2-propyn-1-one
a
Table 1. In Vitro Cytotoxicity of (Z)-1-Aryl-3-arylamino-2-
a
propen-1-one 10
IC50 (μM)
compd
X
Y
DU145
K562
10a
10b
10c
10d
10e
10f
4-OCH3
4-OCH3
4-OCH3-C6H4
3,4,5-(OCH3)3-C6H2
4-OCH3-C6H4
2-OH-C6H4
75
100
75
50
2,4,6-(OCH3)3
2,4,6-(OCH3)3
2,4,6-(OCH3)3
2,4,6-(OCH3)3
2,4,6-(OCH3)3
2,4,6-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
75
75
25
75
4−Cl-C6H4
25
25
2,4-(OCH3)2-C6H3
2,4,6-(OCH3)3-C6H2
3,4,5-(OCH3)3-C6H2
2-OH-C6H4
75
75
10g
10h
10i
75
50
a
Reagents and conditions: (a) ethynylmagnesium bromide, THF, 0
35
75
°C−rt, 8−9 h, 60−70%; (b) 2-iodoxybenzoic acid, DMSO, rt, 5 h, 70−
72%; (c) ethanol, rt, 4 h, 60−82%.
0.25
0.75
2.5
0.5
0.5
3
0.5
0.5
2.5
0.5
0.25
20
10j
3-OH-C6H4
10k
10l
2-OCH3-C6H4
4-OCH3-C6H4
4-Cl-C6H4
Scheme 2. Synthesis of 2-Chloro-3,4,5-
a
trimethoxybenzaldehyde
10m
10n
10o
10p
10q
10r
10s
10t
4-OCF3-C6H4
4-CF3-C6H4
0.5
0.1
0.1
0.25
0.2
0.75
75
0.5
0.1
0.25
0.25
0.25
2.5
50
3-OH,4-OCH3-C6H3
3-NH2,4-OCH3-C6H3
3-F,4-OCH3-C6H3
3-Cl,4-OCH3-C6H3
2-Cl,5-OH-C6H3
2,4,6-(OCH3)3-C6H2
3,4,5-(OCH3)3-C6H2
5-indolyl
a
Reagents and conditions: (a) sulfuryl chloride, dichloromethane, rt, 2
h, 60%.
10u
10v
10w
10x
10y
10z
10aa
40
25
Scheme 3. Synthesis of 2-Bromo-3,4,5-
0.2
0.1
2.5
0.75
0.5
0.6
0.25
0.2
0.06
0.15
0.2
0.6
0.5
0.2
2.5
2.5
0.75
0.75
0.2
0.25
0.1
0.2
0.25
0.6
a
trimethoxybenzaldehyde
6-indolyl
7-indolyl
4-indazole
3-quinoline
10ab 3,4,5-(OCH3)3
10ac 2-Br,3,4,5-(OCH3)3
10ad 2-Br,3,4,5-(OCH3)3
2-CH3,5-indolyl
2-OH-C6H4
4-OCH3-C6H4
3-OH,4-OCH3-C6H3
5-indolyl
10ae
10af
10ag
2-Br,3,4,5-(OCH3)3
2-Br,3,4,5-(OCH3)3
2-Cl,3,4,5-(OCH3)3
a
Reagents and conditions: (a) NBS, chloroform, reflux, 3 h, 97%.
3-OH,4-OCH3-C6H3
10ah 2-NO2,3,4,5-(OCH3)3 4-OCH3-C6H4
diisobutylaluminum hydride (DIBAH) in the presence of
toluene at −50 °C (Scheme 4).
Structure−Activity Relationships (SAR). After the syn-
thesis of these compounds, their in vitro cytotoxicity was
a
Standard deviation for IC50 values were all within 0.05 μM.
reveal that the cytotoxicity of the 1-aryl-3-arylamino-2-propen-
1-ones (10) is totally dependent on the nature and position of
the substituents present on the two aromatic rings. For the
purpose of structure−activity relationship, we have selected a
few compounds from a library of enaminones synthesized in
our laboratory. The cytotoxicity data (Table 1 and data not
shown) clearly shows that the molecules are inactive when both
aromatic rings are mono substituted (10a). A moderate
cytoxicity was observed when benzoyl aromatic ring was tri
substituted at third, fourth, and fifth positions with methoxy
groups and anilino ring with a methoxy at second position
(10k). A 5-fold increase in the activity was observed in 10k
when the 2-methoxy group on the anilino ring was relocated to
fourth position (10l). When the pattern of substitution is
Scheme 4. Synthesis of 2-Nitro-3,4,5-
trimethoxybenzaldehyde
a
a
Reagents and conditions: (a) anhydrous toluene, diisobutylaluminum
hydride, −50 °C, 30 min, 55%; (b) chloroform, manganese dioxide, rt,
48 h, 63%.
C
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reversed by keeping 3,4,5-trimethoxy groups on the anilino ring
and 4-methoxy group on the benzoyl ring (10b), the tumor
cellular toxicity is reduced by more than 100-fold. These results
show that the molecules with three methoxy groups on the
benzoyl ring are more cytotoxic toward tumor cells than the
compounds with three methoxy groups on the anilino ring.
Once the benzoyl ring was identified as a correct moiety for tri
substitution, we then looked at the position of the methoxy
groups on the ring in modulating the cytotoxicity of the
molecule. To analyze the role of the position of the methoxy
groups, we have made a number of compounds with 2,4,6-
trimethoxy substitutions on the benzoyl ring (10c, 10d, 10e,
10f, 10g, and 10h). All the compounds with 2,4,6-trimethoxy
benzoyl substituted lost the cytotoxicity effect on the tumor
cells compared to the corresponding 3,4,5-trisubstituted
benzoyl compounds. It clearly shows that 10l, 10m, and 10o,
which are 3,4,5 trimethoxy benzoyl, are 50-fold more active
than 10c and 10e, which are 2,4,6-trimethoxy benzoyl
enaminones. This confirms that the benzoyl ring of the
enaminones presented in Table 1 require three methoxy groups
at third, fourth, and fifth positions to attain maximum
cytotoxicity toward cancer cells. To analyze the effect of
additional substituents on the aniline aromatic ring, we have
synthesized a number of analogues containing 3-hydroxy-4-
methoxy (10p), 3-amino-4-methoxy (10q), 3-fluoro-4-methoxy
(10r), 3-chloro-4-methoxy (10s), and 2-chloro-5-hydroxy (10t)
groups; the cytotoxicity analyses of these analogues showed
that the compounds with hydroxy and amino substituents at
third position (10p, 10q) exhibited the best activity in the
series compared to the halo substituents (10r, 10s, and 10t).
These compounds 10p and 10q are 5-fold more active than
10l. In an attempt to further enhance the activity of the
molecule, we have replaced aryl anilines with heteroaryl
anilines. The replacement of heteroaryl anilines did not
improve the cytotoxicity of the molecules (10w, 10x, 10y,
10z, 10aa, and 10ab). After assigning third, fourth, and fifth
positions to methoxy groups on benzoyl ring for optimum
activity, we looked for the effect of a substituent at the ortho
position of the benzoyl ring. It is clear from the cytotoxicity
data of 10ac, 10ad, 10ae, 10af, 10ag, and 10ah that the
inclusion of a halo or a nitro group at the ortho position
moderately improved the activity of the molecules compared to
the corresponding ortho-unsubstituted compounds. The SAR
analysis of these compounds also shows that replacement of a
methoxy (10k and 10l), hydroxy (10i and 10j) or chloro
(10m) or trifluoro (10o) atoms or groups in these compounds
with trimethoxy groups at 3,4,5 (10v) or 2,4,6 (10u) positions
of anilino ring lead to the total loss of activity.
Table 2. Evaluation of 10ae against a Panel of Human
Tumor Cell Lines
a
cell line
tumor type
breast (ER−)
breast (ER+)
breast (ER−)
Breast (ER+)
prostate (AR−)
pancreatic
10ae (μM)
BT20
0.75
0.4
MCF-7
SK-BR-3
BT474
DU145
MIA-Paca2
A549
0.3
0.1
0.06
0.8
NSCLC
0.9
HeLa
cervical
0.2
COLO-320
Panc-1
colorectal
0.15
0.2
pancreatic
K562
CML
0.1
SK-MEL-28
HTB-126
Caco-2
Raji
melanoma
0.2
breast carcinoma
colorectal
0.1
0.25
0.2
Burkitt’s lymphoma (B-cell)
gastric carcinoma
gastric carcinoma
colorectal
SNU-5
N87
0.25
0.3
HCT-15
MES-SA
0.04
0.25
0.50
sarcoma
MES-SA/DX5
resistant sarcoma
a
Standard deviation for IC50 values were all within 0.025 μM.
therapy and is the primary cause of treatment failure. Many
microtubulin poisons such as paclitaxel, vincristine, and
vinblastine are substrates for the multidrug resistant family
members. To further investigate the activity of these
compounds against MDR positive tumor types, we determined
the IC50 value of 10ae using a classical MDR-positive cell line.
The results shown in Table 2 show a 96 h dose response of the
uterine sarcoma cell line MES-SA and the multidrug resistant
subline MES-SA/DX524 treated with 10ae. This cell line has
been shown to express high levels of P-glycoprotein and is
resistant to a number of drugs including doxorubicin, paclitaxel,
vincristine, vinblastine, etoposide, mitoxantrone, dactinomycin,
and daunorubucin. The activity of our compound was then
compared to the activity of paclitaxel (MDR sensitive drug).
Our results show that the parental cell line was very sensitive to
paclitaxel (IC50 4 nM), but the MDR positive subline was
greater than 100-fold resistant (IC50 750 nM). When the two
cell lines were treated with 10ae, both the parental and the
MDR positive cell lines were almost equally sensitive to the cell
killing activity of the compound.
Effects of 10ae on Cell Cycle Progression of Tumor
Cells. Treatment of cancer cells with 10ae results in a time-
dependent rounding of the cells indicative of mitotic arrest.25
To study the cell cycle effects of 10ae treatment, we ran a series
of experiments by fluorescence-activated cell sorting (FACS)
and signal transduction studies using human prostate cancer
cells. DU145 cells, which are androgen-negative prostate cancer
cells, were treated with increasing concentrations of 10ae for 24
h. Because these cells were one of the more sensitive cell lines
tested, and because DU145 cells have a very predictable cell
cycle time making them suitable for cell cycle analysis, these
cells were used for the following experiment.26−28 Cell cycle
analysis performed by FACS analysis revealed that the cells
accumulated in G2/M phase of the cell cycle in a
concentration-dependent manner. Results presented in Figure
1 show that treatment with 0.25 μM of 10ae resulted in the
accumulation of tumor cells in the G2/M phase of the cell cycle
BIOLOGICAL RESULTS AND DISCUSSION
■
In Vitro Antitumor Effects of 10ae Compound. We
next tested the activity of the most active compound (10ae)
listed in Table 1 against different human tumor cell lines and,
surprisingly, they were found to induce apoptosis of all of these
cell lines with similar GI50 values (selected data shown in Table
2). The wide range of cell killing across multiple tumor types
suggests that this compound is inducing tumor cell killing by
inhibiting an intrinsically important process of tumor cell
division.
10ae Compound Is Also Active against Drug Resistant
Tumor Cell Line. Development of resistance to classical
chemotherapeutic agents is widely observed in patients who
have not responded or have relapsed after a first round of
D
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Figure 1. 10ae treatment of cancer cells results in G2/M arrest. DU145, human prostate cancer cells, were treated for 24 h with increasing
concentrations of 10ae, fixed, and stained with propidium iodide and analyzed for their DNA content by flow cytometry. G1 (2N), S-Phase (>2N <
4N), and G2/M (4N) cells are indicated solid blue, striped, and solid red, respectively. 10ae treated cells become arrested in G2/M in a dose-
responsive manner. DU145 cells were plated at a cell density of 1.0 × 106 cells per 100 mm dish. N = 3.
with a majority of cells showing G2/M arrest when treated with
0.5 μM concentration of the compound. The FACS analysis
determined that the treated cells had greater than 2N amount
of DNA but did not differentiate between G2 and mitotic
phases of the cell cycle. On the basis of these results, we next
studied the effects of 10ae on the phosphorylation status of
proteins known to be important markers of mitotic arrest and
spindle checkpoint activation. HeLa cells derived from a human
cervical tumor which have been extensively studied as a model
cell line for mitotic inhibitors29 were treated with 1 μM
concentration of 10ae for various time points and the pattern of
expression of histone H3 and its phosphorylated mitotic
(ser10) form were analyzed. Phosphorylation of histone H3
(ser10) is a widely accepted indicator of mitotic arrest as well as
a diagnostic tool for the mitotic index of tumors.30−33 As shown
in Figure 2, increased levels of phospho-histone H3 were
observed within 6 h after treatment with maximal accumulation
at 24 h (121-maximum-fold increase). Phosphorylation of BCL-
2 has also been known to be a marker for mitosis,34 and this
protein became phosphorylated at the same time as histone H3
(88 fold). Concurrently to the accumulation of phospho-
histone H3, the protein levels of both cyclin B1 (2.5 fold) and
PLK-1(2.5 fold) increased after 6 h of treatment. The
expression of these proteins is known to be closely linked to
progression through mitosis.35−37 Phosphorylation of BubR1 is
a useful marker of mitotic arrest due to the activation of the
spindle checkpoint,38,39 so we next analyzed the phosphor-
ylation status of this protein. Similar to histone H3 and Bcl-2,
BubR1 (2.95-fold) became phosphorylated at 6 h following
treatment of cancer cells with 10ae. Taken together, these data
strongly suggest that 10ae treatment induces a mitotic arrest,
with the activation of spindle checkpoint within 6 h of
treatment.
Compound 10ae Induces Apoptosis in Tumor Cells.
Tumor cells treated with 10ae accumulated in the G2/M phase
of the cell cycle in a dose-dependent manner and appeared to
be unable to exit from this phase, leading to the activation of
apoptotic pathways as judged by poly(ADP-ribose) polymer-
ase-1 (PARP) cleavage,40 which is a marker for caspase
activation (Figure 3).
Compound 10ae Kills Tumor Cells by Stabilizing
Microtubules. The polymerization of microtubules from
purified tubulin can be monitored in vitro by measuring an
increase in light scattering. This in vitro experiment removes
complicating factors, such as microtubule-associated proteins
(MAPs), which might be part of a putative target that leads to
disruption of microtubules as observed with microscopy. To
test our hypothesis that the target of (Z)-1-aryl-3-arylamino-2-
propen-1-one (10) is tubulin, and not a MAP, we monitored
the polymerization of tubulin after treatment with 10ag, 10ah,
and 10ae (Figure 4). In this experiment, paclitaxel, a
microtubule stabilizer, enhanced the rate of tubulin polymer-
ization, while vincristine, a microtubule destabilizer, prevented
the polymerization of tubulin. Similar to paclitaxel, 10ag, 10ah,
and 10ae stabilized tubulin polymerization at 1 μM. Compound
10ae was most potent in cytotoxicity assay compared to the
other analogues, and also from Figure 4, 10ae is superior to
paclitaxel in that it causes more microtubule assembly at the
E
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cells acquired multiple tubulin bundles (multipolar) without
chromosomes aligned at the metaphase plate with more
dispersion of the chromosomes. We compared the mitotic
spindle phenotype to cells treated with paclitaxel, a tubulin
stabilizer, and to cells treated with vincristine, a tubulin
depolymerizer. As shown in Figure 5, 10ae treatment is unique
in its effect on mitotic spindle formation, in that although the
mitotic cells are multipolar which is similar to paclitaxel, the
intensity of the tubulin staining is lower and the chromosome
spreads are different. At lower concentrations, 10ae was similar
to paclitaxel in that unbound chromosomes were observed
outside the metaphase plate while higher concentrations
resulted in a more dispersed chromosome spread with three
or more tubulin bundles. When one compares 10ae to
vincristine treated cells, it is clear that 10ae is not destabilizing
tubulin because we do not observe complete loss of tubulin
staining.
The effects of 10ae treatment on tubulin stability can also be
examined by analysis of the tubulin organization of nonmitotic
interphase cells. Figure 6 shows that treatment with high
concentrations of 10ae did not result in an overall disruption of
cytoplasmic microtubule organization as evident in cells treated
with vincristine. The microtubule organization appeared to be
somewhat shortened throughout the cytoplasm, but the overall
protofilament structure was still intact. Paclitaxel treatment
resulted in bundling of the microtubule fibers near the nucleus;
this was not observed with 10ae treatment. This data suggests
that 10ae treatment does not depolymerize tubulin but
interferes with microtubule spindle development during mitosis
without altering cytoplasmic microtubules, resulting in a mitotic
block and cell death.
Figure 2. 10ae treatment induces mitotic arrest. HeLa cells were
treated with 1 μM 10ae, and cells were harvested for Western blotting
at the indicted times. The blot was sequentially hybridized with
antibodies specific for phospho-histone H3, histone H3, cyclin B1,
PLK-1, Bub-R1, and GAPDH was used as a loading control.
Phosphorylation of histone H3, BubR1, and Bcl-2 and the
accumulation of mitotic specific proteins (cyclin B1 and PLK-1)
indicates that 10ae treatment induces a mitotic arrest, with cells
starting to accumulate in mitosis by 6 h following treatment. Primary
antibody dilutions were 1:1000 for all westerns, and 1:10000 for
secondary antibodies. N = 3. The numbers below the panel represents
the fold increase, calculated as described in the Experimental Section,
of the respective protein or phosphoprotein.
CONCLUSION
■
In this communication, we describe the synthesis of a series of
(Z)-1-aryl-3-arylamino-2-propen-1-ones (10) molecules, which
induce apoptotic death of a wide variety of human tumor cell
lines at nanomolar concentrations by promoting tubulin
polymerization and stabilizing microtubules. This is a first
report that shows a group of small synthetic molecules (10)
behave like paclitaxel and epothilones in tumor cell killing in
the nanomolar range by promoting tubulin polymerization and
microtubule stabilization in in vitro. Our studies show that the
cytotoxic activity of the (Z)-1-aryl-3-arylamino-2-propen-1-
ones (10) is completely dependent on the nature and position
of the substituents on the two aromatic rings. These structure
function studies show that a molecule with an aniline ring
having 3-hydroxy, 4-methoxy groups and a benzoyl ring with a
bromine atom at ortho and methoxy groups at 3-, 4-, and 5-
positions (10ae) showed optimum biological activity. Biological
evaluation of the activity of 10ae shows that this compound is
highly active against a wide variety of human tumor cell lines
including those that are resistant to the activity of many of the
currently used chemotherapeutic agents.
Figure 3. 10ae treatment activates caspase-3 and induces apoptosis.
HeLa cells were treated with 1.0 μM 10ae for the indicated times, and
protein lysates were resolved by SDS-PAGE and analyzed for caspase-
3 activation by Western blotting using anti-PARP specific antibodies.
PARP cleavage was observed starting at 24 h. This data indicates that
the apoptosis pathway has been activated. Primary antibody dilutions
were 1:1000 for all westerns and 1:10000 for secondary antibodies. N
= 3.
same concentration (1 μM). Thus, the enaminone-based
compounds stabilize the formation of microtubules in vitro.
Compound 10ae Treatment Induces Abnormal
Mitotic Spindle Development. Because 10ae was found to
alter tubulin in vitro polymerization kinetics, we next examined
the mitotic spindle apparatus in cancer cells treated with 10ae.
HeLa cells were treated with 10ae for 18 h and fixed and
stained for α-tubulin. Figure 5 shows that HeLa cells became
arrested in mitosis and the mitotic cells had abnormal mitotic
spindles. When cells were treated with the 0.25 μM of 10ae,
near the GI50 value, the mitotic cells displayed an atypical
bipolar metaphase plate in which the tubulin appeared bundled
at the poles and some chromosomes that were not attached
aligned at the metaphase plate (see arrows). When the cells
were treated with higher concentrations of 10ae, the mitotic
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All reagents and solvents were
obtained from commercial suppliers and used without further
purification unless otherwise stated. Solvents were dried using
standard procedures, and reactions requiring anhydrous conditions
were performed under N2 atm. Reactions were monitored by thin
layer chromatography (TLC) on preloaded silica gel F254 plates
(Sigma-Aldrich) with a UV indicator. Column chromatography was
performed with Merck 70-230 mesh silica gel 60 Å. Yields were of
F
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Figure 4. MAP-rich tubulin from bovine brain had been preincubated with various drugs (1 μM final) or vehicle dimethyl sulfoxide at room
temperature for 5 min before guanosine triphosphate (GTP) was added to start the tubulin polymerization reactions. The reaction was monitored at
various time points at OD350 nm at 37 °C. Tubulin polymerization enhancer (taxol) and inhibitor (vincristine) were included as controls.
Figure 5. 10ae treatment results in abnormal microtubule spindle development. HeLa cells were treated with 0.25 or 0.5 μM of 10ae, 0.005 μM
paclitaxel, 0.005 μM vincristine, or DMSO for 18 h and fixed and stained with anti-α-tubulin-FITC specific antibodies (green) followed by DAPI to
visualize chromosomes (blue). 10ae and paclitaxel treatment resulted in abnormal mitotic cells with chromosomes not aligned at the metaphase plate
(see arrows) without complete loss of tubulin polymers as observed in cells treated with vincristine. Cell density was 1.0 × 106 cells per 100 mm dish
containing eight coverslips each.
purified product and were not optimized. Melting points were
were measured in hertz (Hz). All LC/MS data were gathered on an
Agilent 1200 LC with Agilent 6410 triple quadrupole mass
spectrometer detectors. The compound solution was infused into
the electrospray ionization source operating positive and negative
modes in methanol/water/TFA (50:50:0.1% v/v) at 0.4 mL/min. The
sample cone (declustering) voltage was set at 100 V. The instrument
was externally calibrated for the mass range m/z 100 to m/z 1000. The
determined using an Electro Thermal Mel-Temp 3.0 micromelting
1
point apparatus and are uncorrected. H NMR spectra were obtained
with a Bruker AM 300 and 400 MHz spectrometer. The chemical
shifts are reported in parts per million (δ) downfield using
tetramethylsilane (SiMe4) as internal standard. Spin multiplicities are
given as s (singlet), d (doublet), dd (double doublet), brs (broad
singlet), m (multiplet), and q (quartet). Coupling constants (J values)
G
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Figure 6. 10ae treatment does not alter cytoplasmic microtubules. High concentrations of 10ae (0.5 μM) did not result in bundling of the
cytoplasmic microtubules as seen with paclitaxel (0.005 μM) treatment (arrow) nor did 10ae treatment depolymerize cytoplasmic microtubules as
observed in cells treated with vincristine (0.005 μM). Cell density was 1.0 × 106 cells per 100 mm dish containing eight coverslips each.
purity of the final compounds was determined by HPLC and is 95% or
higher unless specified otherwise.
Step 2: Synthesis of 3,4,5-Trimethoxy-2-nitrobenzaldehyde (6).
The compound 5 (1.0 g, 4.1 mmol) was dissolved in 50 mL of
chloroform to which MnO2 (2.14 g, 24.6 mmol) was added and stirred
for overnight, and an additional portion of MnO2 (0.53 g, 6.15 mmol)
was added and stirred for 12 h. The solids were removed by filtration
through a Celite pad and washed with chloroform. The chloroform
was evaporated in vacuo to get pure compound 6 as brown solid with
63% yield; mp 64−65 °C. 1H NMR (CDCl3, 300 MHz): δ 3.91 (s, 3H,
OCH3), 3.93 (s, 6H, OCH3), 7.15 (s, 1H, Ar−H), 9.80 (s, 1H, H−
CO). MS found (M + H)+ (m/z): 242.20. Calcd for C10H11NO6 m/
z: 241.06.
General Procedure for the Synthesis of 1-Aryl-2-propyn-1-ol (7).
A solution of aldehyde 1−6 (5 mmol) in dry tetrahydrofuran (THF)
was added to a stirred solution of ethynylmagnesium bromide in THF
(0.5 M solution, 7.5 mmol) at 0 °C. The solution was stirred at 0 °C
for 2 h and then warmed to room temperature and stirred for another
6−7 h. Saturated aqueous ammonium chloride solution 5 mL was
added, and the mixture was evaporated in vacuo and partitioned
between ethyl acetate and saturated ammonium chloride solution. The
organic layer was washed with brine, dried over anhydrous Na2SO4,
and evaporated in vacuo to get pure compounds and were used for
next step without further purification.
1-(4-Methoxyphenyl)prop-2-yn-1-ol (7a). Starting from 4-methox-
ybenzaldehyde 1a, 70% of 7a was obtained according to the method
described in general procedure 7; liquid. 1H NMR (CDCl3, 400
MHz): δ 2.65 (d, 1H, J = 2.2 Hz, ≡H), 3.79 (s, 3H, OCH3), 5.37−5.40
(m, 1H, H-C-OH), 6.87−6.90 (m, 2H, Ar−H), 7.43−7.47 (m, 2H,
Ar−H). MS found (M − H2O + H)+ (m/z): 145.20. Calcd for
C10H10O2 m/z: 162.07.
1-(2,4,6-Trimethoxyphenyl)prop-2-yn-1-ol (7b). Starting from
2,4,6-trimethoxybenzaldehyde 1b, 65% of 7b was obtained as brown
solid according to the method described in general procedure 7; mp
119−120 °C. 1H NMR (CDCl3, 400 MHz): δ 2.42 (d, 1H, J = 2.3 Hz,
≡H), 3.80 (s, 3H, OCH3), 3.86 (s, 6H, OCH3), 5.80−5.84 (m, 1H, H-
C-OH), 6.14 (s, 2H, Ar−H). MS found (M − H2O + H)+ (m/z):
205.30. Calcd for C12H14O4 m/z: 222.09.
Synthesis of 2-Chloro-3,4,5-trimethoxybenzaldehyde (2). 3,4,5-
Trimethoxybenzaldehyde 1c (1.0 g, 5 mmol) was dissolved in 25 mL
of dichloromethane, and sulfuryl chloride (0.49 mL, 6.1 mmol) was
added and stirred at 10 °C for 2 h. After reaction was completed
(checked by TLC), the solvent was concentrated under vacuum and
washed with hexane to get pure 2-chloro-3,4,5-trimethoxybenzalde-
1
hyde 2 with 60% yield; semi solid. H NMR (CDCl3, 300 MHz): δ
3.91 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.27 (s,
1H, Ar−H), 10.39 (s, 1H, H−CO). MS found (M + H)+ (m/z):
231.20. Calcd for C10H11ClO4 m/z: 230.03.
Synthesis of 2-Bromo-3,4,5-trimethoxybenzaldehyde (3). To a
solution of 3,4,5-trimethoxy benzaldehyde 1c (5.0 g, 25.4 mmol) in 50
mL chloroform (CHCl3) was added N-bromosuccinimide (5.44 g,
30.5 mmol). The solution was heated at reflux temperature for 3 h.
After the reaction was completed by TLC, the reaction mixture was
brought to room temperature, and the solution was washed with water
and extracted with diethyl ether (Et2O). The combined extracts were
dried with anhydrous Na2SO4 and concentrated. The crude
bromobenzaldehyde was recrystallized from hexanes and Et2O to get
pure white solid with 97% yield; mp 69−70 °C. 1H NMR (CDCl3, 300
MHz): δ 3.90 (s, 3H, OCH3), 3.92 (s, 6H, OCH3), 7.30 (s, 1H, Ar−
H), 10.15 (s, 1H, H−CO). MS found (M + H)+ (m/z): 275.10.
Calcd for C10H11BrO4 m/z: 273.98.
Synthesis of 3,4,5-Trimethoxy-2-nitrobenzaldehyde (6). . Step 1:
Synthesis of (3,4,5-Trimethoxy-2-nitrophenyl)methanol (5). Methyl
3,4,5-trimethoxy-2-nitrobenzoate 4 (1.5 g, 3.69 mmol) was dissolved
in 20 mL of anhydrous toluene, cooled to −50 °C, and treated with a
solution of diisobutylaluminum hydride (1.0 M solution in hexane, 4.6
mL, 4.6 mmol) over a period of 10 min. After 30 min, the reaction was
quenched with 10 mL of methanol and brought to room temperature.
The resulting reaction mixture was diluted with water and extracted
with ethyl acetate. The extract was dried over anhydrous Na2SO4 and
concentrated under vacuum to give (3,4,5-trimethoxy-2-nitrophenyl)-
methanol 5, which was purified by column chromatography (silica gel,
30% ethyl acetate in hexane, Rf = 0.33) as brown solid with 55% yield;
1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-ol (7c). Starting from
3,4,5-trimethoxybenzaldehyde 1c, 65% of 7c was obtained as brown
solid according to the method described in general procedure 7; mp
1
mp 59−60 °C. H NMR (CDCl3, 300 MHz): δ 2.34 (brs, 1H, OH),
3.90 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.85 (s,
1H, Ar−H). MS found (M + H)+ (m/z): 244.10. Calcd for
C10H13NO6 m/z: 243.07.
1
51−52 °C. H NMR (CDCl3, 400 MHz): δ 2.62 (d, 1H, J = 2.1 Hz,
≡H), 3.83 (s, 3H, OCH3), 3.86 (s, 6H, OCH3), 5.39−5.54 (m, 1H, H-
H
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C-OH), 6.77 (s, 2H, Ar−H). MS found (M − H2O + H)+ (m/z):
205.20. Calcd for C12H14O4 m/z: 222.09.
3H, OCH3), 3.98 (s, 3H, OCH3), 7.47 (s, 1H, Ar−H). MS found (M
+ H)+ (m/z): 298.90. Calcd for C12H11BrO4 m/z: 297.98.
1-(2-Chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-ol (7d). Starting
from 2-chloro-3,4,5-trimethoxybenzaldehyde 1d, 70% of 7d was
obtained as brown solid according to the method described in general
procedure 7; mp 65−66 °C. 1H NMR (CDCl3, 400 MHz): δ 2.66 (d,
1H, J = 2.4 Hz, ≡H), 3.89 (s, 3H, OCH3), 3.90 (s, 6H, OCH3), 5.79−
5.80 (m, 1H, H-C-OH), 7.14 (s, 1H, Ar−H). MS found (M − H2O +
H)+ (m/z): 239.10. Calcd for C12H13ClO4 m/z: 256.05.
1-(3,4,5-Trimethoxy-2-nitrophenyl)prop-2-yn-1-one (8f). Starting
from 1-(3,4,5-trimethoxy-2-nitrophenyl)prop-2-yn-1-ol 7f, 70% of 8f
was obtained as brown solid according to the method described in
above-mentioned general procedure 8; mp 87−88 °C. 1H NMR
(CDCl3, 400 MHz): δ 3.42 (s, 1H, ≡H), 3.88 (s, 3H, OCH3), 3.91 (s,
3H, OCH3), 3.93 (s, 3H, OCH3), 7.39 (s, 1H, Ar−H). MS found (M
+ H)+ (m/z): 266.30. Calcd for C12H11NO6 m/z: 265.06.
1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-ol (7e). Starting
from 2-bromo-3,4,5-trimethoxybenzaldehyde 1e, 70% of 7e was
obtained as brown solid according to the method described in general
procedure 7; mp 59−60 °C. 1H NMR (CDCl3, 400 MHz): δ 2.66 (d,
1H, J = 2.1 Hz, ≡H), 3.88 (s, 3H, OCH3), 3.89 (s, 6H, OCH3), 5.79−
5.80 (m, 1H, H-C-OH), 7.18 (s, 1H, Ar−H). MS found (M − H2O +
H)+ (m/z): 283.20. Calcd for C12H13BrO4 m/z: 300.00.
General Procedure for the Synthesis of (Z)-1-Aryl-3-arylamino-2-
propen-1-one (10). The ethynylketone 8 (5 mmol) was dissolved in
absolute ethanol, and arylamine (5 mmol) was added. The reaction
was stirred for 4 h at room temperature. After the completion of
reaction (checked by TLC), the reaction mixture was diluted with
water and the product filtered. The crude product was recrystallized
from methanol to get pure compound 10.
1-(3,4,5-Trimethoxy-2-nitrophenyl)prop-2-yn-1-ol (7f). Starting
from 2-nitro-3,4,5-trimethoxybenzaldehyde 1f, 60% of 7f was obtained
as brown solid according to the method described in general
(Z)-1-(4-Methoxyphenyl)-3-(4-methoxyphenylamino)prop-2-en-
1-one (10a). Starting from 1-(4-methoxyphenyl)prop-2-yn-1-one 8a
and 4-methoxyphenylamine, 76% of 10a was obtained as yellow solid
according to above-mentioned general procedure 10; Rf = 0.71 (30%
1
procedure 7; mp 75−76 °C. H NMR (CDCl3, 400 MHz): δ 2.50
1
ethyl acetate in hexane); mp 187−188 °C. H NMR (CDCl3, 400
(d, 1H, J = 2.2 Hz, ≡H), 3.72 (s, 3H, OCH3), 3.77 (s, 3H, OCH3),
3.80 (s, 3H, OCH3), 5.42−5.52 (m, 1H, H-C-OH), 6.91 (s, 1H, Ar−
H). MS found (M − H2O + H)+ (m/z): 250.20. Calcd for C12H13NO6
m/z: 267.07.
MHz): δ 3.80 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 5.94 (d, 1H, J = 7.8
Hz, COCH), 6.88−6.95 (m, 4H, Ar−H), 7.02−7.05 (m, 2H, Ar−
H), 7.37−7.42 (dd, 1H, J = 7.8, 12.3 Hz, CH-NH), 7.90−7.93 (m,
2H, Ar−H), 12.06 (d, 1H, J = 12.1 Hz, NH). MS found (M + H)+ (m/
z): 284.20. Calcd for C17H17NO3 m/z: 283.12.
General Procedure for the Synthesis of 1-Aryl-2-propyn-1-one
(8). A solution of 2-iodoxy-benzoic acid (IBX) (12.5 mmol) in
dimethyl sulfoxide (DMSO) (90 mL) was stirred for 5 min at room
temperature until homogeneous. A solution of secondary alcohol 7 (5
mmol) in DMSO (5 mL) was added, and the mixture was stirred for 5
h. Water (10 mL) was added, and the mixture was stirred at room
temperature for 10 min, cooled in ice, and partitioned between water
and ethyl acetate. The mixture was filtered through Celite, and the
aqueous layer was further extracted with ethyl acetate. The organic
extracts were combined, washed with water followed by saturated
sodium bicarbonate solutions and brine, dried over sodium sulfate, and
evaporated in vacuo to get the pure product and was used for next step
without further purification.
(Z)-1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenylamino)prop-
2-en-1-one (10b). Starting from 1-(4-methoxyphenyl)prop-2-yn-1-
one 8a and 3,4,5-trimethoxyphenylamine, 74% of 10b was obtained as
yellow solid according to above-mentioned general procedure 10; Rf =
0.43 (30% ethyl acetate in hexane); mp 100−101 °C. 1H NMR
(CDCl3, 400 MHz): δ 3.82 (s, 3H, OCH3), 3.87 (s, 9H, OCH3), 5.98
(d, 1H, J = 12.9 Hz, COCH), 6.31 (s, 2H, Ar−H), 6.94−6.99 (m,
2H, Ar−H), 7.39−7.44 (dd, 1H, J = 7.8, 12.3 Hz, CH-NH), 7.91−
7.94 (m, 2H Ar−H), 12.12 (d, 1H, J = 12.0 Hz, NH). MS found (M +
H)+ (m/z): 344.20. Calcd for C19H21NO5 m/z: 343.14.
(Z)-3-(4-Methoxyphenylamino)-1-(2,4,6-trimethoxyphenyl)prop-
2-en-1-one (10c). Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-
1-one 8b and 4-methoxyphenylamine, 77% of 10c was obtained as
yellow solid according to above-mentioned general procedure 10; Rf =
0.28 (30% ethyl acetate in hexane); mp 130−131 °C. 1H NMR
(CDCl3, 400 MHz): δ 3.80 (s, 6H, OCH3), 3.82 (s, 3H, OCH3), 3.84
(s, 3H, OCH3), 5.52 (d, 1H, J = 7.7 Hz, COCH), 6.14 (s, 2H, Ar−
H), 6.95−6.98 (m, 2H, Ar−H), 7.20−7.26 (m, 1H, CH-NH and
2H, Ar−H), 11.86 (d, 1H, J = 12.2 Hz, NH). MS found (M + H)+ (m/
z): 344.20. Calcd for C19H21NO5 m/z: 343.14.
1-(4-Methoxyphenyl)prop-2-yn-1-one (8a). Starting from 1-(4-
methoxyphenyl)prop-2-yn-1-ol 7a, 72% of 8a was obtained as pale-
yellow solid according to the method described in above-mentioned
1
general procedure 8; mp 81−82 °C. H NMR (CDCl3, 400 MHz): δ
3.36 (s, 1H, ≡H), 3.88 (s, 3H, OCH3), 6.94−6.96 (m, 2H, Ar−H),
8.11−8.13 (m, 2H, Ar−H). MS found (M + H)+ (m/z): 161.30. Calcd
for C10H8O2 m/z: 160.05.
1-(2,4,6-Trimethoxyphenyl)prop-2-yn-1-one (8b). Starting from 1-
(2,4,6-trimethoxyphenyl)prop-2-yn-1-ol 7b, 70% of 8b was obtained as
pale yellow solid according to the method described in above-
mentioned general procedure 8; mp 83−84 °C. 1H NMR (CDCl3, 400
MHz): δ 3.14 (s, 1H, ≡H), 3.76 (s, 6H, OCH3), 3.77 (s, 3H, OCH3),
6.03 (s, 2H, Ar−H). MS found (M + H)+ (m/z): 221.20. Calcd for
C12H12O4 m/z: 220.07.
(Z)-3-(2-Hydroxyphenylamino)-1-(2,4,6-trimethoxyphenyl)prop-
2-en-1-one (10d). Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-
yn-1-one 8b and 2-aminophenol, 69% of 10d was obtained as yellow
solid according to above-mentioned general procedure 10; Rf = 0.10
1
(30% ethyl acetate in hexane); mp 164−165 °C. H NMR (CDCl3,
1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one (8c). Starting from 1-
(3,4,5-trimethoxyphenyl)prop-2-yn-1-ol 7c, 70% of 8c was obtained as
pale-yellow solid according to the method described in above-
400 MHz): δ 3.47 (s, 6H, OCH3), 3.56 (s, 3H, OCH3), 5.27 (d, 1H, J
= 7.4 Hz, COCH), 5.85 (s, 2H, Ar−H), 6.43−6.54 (m, 3H, Ar−H),
6.80−6.82 (m, 1H, Ar−H), 7.08−7.13 (dd, 1H, J = 7.6, 12.8 Hz, 
CH-NH), 11.85 (d, 1H, J = 12.2 Hz, NH). MS found (M + H)+ (m/
z): 330.20. Calcd for C18H19N2O5 m/z: 329.13.
1
mentioned general procedure 8; mp 123−124 °C. H NMR (CDCl3,
400 MHz): δ 3.35 (s, 1H, ≡H), 3.86 (s, 3H, OCH3), 3.88 (s, 6H,
OCH3), 7.36 (s, 2H, Ar−H). MS found (M + H)+ (m/z): 221.20.
Calcd for C12H12O4 m/z: 220.07.
(Z)-3-(4-Chlorophenylamino)-1-(2,4,6-trimethoxyphenyl)prop-2-
en-1-one (10e). Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-
one 8b and 4-chlorophenylamine, 72% of 10e was obtained as yellow
solid according to above-mentioned general procedure 10; Rf = 0.26
1-(2-Chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (8d). Start-
ing from 1-(2-chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-ol 7d, 70%
of 8d was obtained as brown solid according to the method described
1
(30% ethyl acetate in hexane); mp 180−181 °C. H NMR (CDCl3,
1
in above-mentioned general procedure 8; mp 84−85 °C. H NMR
400 MHz): δ 3.80 (s, 6H, OCH3), 3.84 (s, 3H, OCH3), 5.54 (d, 1H, J
= 7.8 Hz, COCH), 6.13 (s, 2H, Ar−H), 6.97−7.00 (m, 2H, Ar−H),
7.23−7.28 (m, 1H, CH-NH and 2H, Ar−H), 11.85 (d, 1H, J = 12.1
Hz, NH). MS found (M + H)+ (m/z): 348.20. Calcd for
C18H18ClNO4 m/z: 347.09.
(CDCl3, 400 MHz): δ 3.50 (s, 1H, ≡H), 3.91 (s, 3H, OCH3), 3.93 (s,
3H, OCH3), 3.99 (s, 3H, OCH3), 7.44 (s, 1H, Ar−H). MS found (M
+ H)+ (m/z): 255.20. Calcd for C12H11ClO4 m/z: 254.03.
1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (8e). Start-
ing from 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-ol 7e, 70%
of 8e was obtained as brown solid according to the method described
(Z)-3-(2,4-Dimethoxyphenylamino)-1-(2,4,6-trimethoxyphenyl)-
prop-2-en-1-one (10f). Starting from 1-(2,4,6-trimethoxyphenyl)-
prop-2-yn-1-one 8b and 2,4-dimethoxyphenylamine, 70% of 10f was
obtained as yellow solid according to above-mentioned general
1
in above-mentioned general procedure 8; mp 80−81 °C. H NMR
(CDCl3, 400 MHz): δ 3.50 (s, 1H, ≡H), 3.90 (s, 3H, OCH3), 3.93 (s,
I
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procedure 10; Rf = 0.10 (30% ethyl acetate in hexane); mp 105−106
°C. 1H NMR (CDCl3, 400 MHz): δ 3.79 (s, 6H, OCH3), 3.84 (s, 6H,
OCH3), 3.89 (s, 3H, OCH3), 5.47 (d, 1H, J = 7.6 Hz, COCH), 6.12
(s, 2H, Ar−H), 6.44−6.50 (m, 2H, Ar−H), 7.03 (d, 1H, J = 8.6 Hz,
Ar−H), 7.27−7.33 (dd, 1H, J = 7.6, 12.8 Hz, CH-NH), 11.90 (d,
1H, J = 12.1 Hz, NH). MS found (M + H)+ (m/z): 374.30. Calcd for
C20H23NO6 m/z: 373.15.
1-one 8c and 4-chlorophenylamine, 72% of 10m was obtained as
yellow solid according to above-mentioned general procedure 10; Rf =
0.80 (50% ethyl acetate in hexane); mp 147−148 °C. 1H NMR
(CDCl3, 300 MHz): δ 3.92 (s, 3H, OCH3), 3.94 (s, 6H, OCH3), 6.01
(d, 1H, J = 8.1 Hz, COCH), 7.02−7.05 (m, 2H, Ar−H), 7.20 (s,
2H, Ar−H), 7.30−7.33 (m, 2H, Ar−H), 7.42−7.49 (dd, 1H, J = 7.8,
12.3 Hz, CH-NH), 12.13 (d, 1H, J = 12.3 Hz, NH). MS found (M +
H)+ (m/z): 348.10. Calcd for C18H18ClNO4 m/z: 347.09.
(Z)-3-(4-Trifluoromethoxyphenylamino)-1-(3, 4, 5-
trimethoxyphenyl)prop-2-en-1-one (10n). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 4-trifluoromethoxyphe-
nylamine, 75% of 10n was obtained as yellow solid according to
above-mentioned general procedure 10; Rf = 0.80 (50% ethyl acetate
in hexane); mp 107−108 °C. 1H NMR (CDCl3, 300 MHz): δ 3.92 (s,
3H, OCH3), 3.94 (s, 6H, OCH3), 6.02 (d, 1H, J = 7.8 Hz, COCH),
7.08−7.12 (m, 2H, Ar−H), 7.20−7.24 (m, 4H, Ar−H), 7.42−7.47
(dd, 1H, J = 8.1, 12.3 Hz, CH-NH), 12.15 (d, 1H, J = 12.0 Hz,
NH). MS found (M + H)+ (m/z): 398.10. Calcd for C19H18F3NO5 m/
z: 397.11.
( Z ) - 3 - ( 4 - T r i fl u o r o m e t h y l p h e n y l a m i n o ) - 1 - ( 3 , 4 , 5 -
trimethoxyphenyl)prop-2-en-1-one (10o). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 4-trifluoromethylphenyl-
amine, 68% of 10o was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.80 (50% ethyl acetate in
hexane); mp 92−93 °C. 1H NMR (CDCl3, 400 MHz): δ 3.86 (s, 3H,
OCH3), 3.87 (s, 6H, OCH3), 6.01 (d, 1H, J = 8.0 Hz, COCH), 7.10
(d, 2H, J = 8.4 Hz, Ar−H), 7.15 (s, 2H, Ar−H), 7.43−7.48 (dd, 1H, J
= 8.0, 12.0 Hz, CH-NH), 7.58 (d, 2H, J = 8.5 Hz, Ar−H), 12.05 (d,
1H, J = 12.3 Hz, NH). MS found (M + H)+ (m/z): 382.20. Calcd for
C19H18F3NO4 m/z: 381.12.
( Z ) - 1 - ( 2 , 4 , 6 - T r i m e t h o x y p h e n y l ) - 3 - ( 2 , 4 , 6 -
trimethoxyphenylamino)prop-2-en-1-one (10g). Starting from 1-
(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 2,4,6-trimethoxyphe-
nylamine, 80% of 10g was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.06 (30% ethyl acetate in
1
hexane); mp 129−130 °C. H NMR (CDCl3, 400 MHz): δ 3.78 (s,
6H, OCH3), 3.80 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.86 (s, 6H,
OCH3), 5.35 (d, 1H, J = 7.5 Hz, COCH), 6.12 (s, 2H, Ar−H), 6.17
(s, 2H, Ar−H), 7.85−7.90 (dd, 1H, J = 7.5, 12.7 Hz, CH-NH),
12.00 (d, 1H, J = 12.2 Hz, NH). MS found (M + H)+ (m/z): 404.20.
Calcd for C21H25NO7 m/z: 403.16.
( Z ) - 1 - ( 2 , 4 , 6 - T r i m e t h o x y p h e n y l ) - 3 - ( 3 , 4 , 5 -
trimethoxyphenylamino)prop-2-en-1-one (10h). Starting from 1-
(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 3,4,5-trimethoxyphe-
nylamine, 75% of 10h was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.08 (30% ethyl acetate in
hexane); mp 177−178 °C. 1H NMR (CDCl3, 300 MHz): δ 3.82−3.90
(m, 18H, OCH3), 5.55 (d, 1H, J = 7.6 Hz, COCH), 6.19 (s, 2H,
Ar−H), 6.40 (s, 2H, Ar−H), 7.30−7.37 (dd, 1H, J = 7.6, 12.8 Hz, 
CH-NH), 11.98 (d, 1H, J = 12.3 Hz, NH). MS found (M + H)+ (m/
z): 404.20. Calcd for C21H25NO7 m/z: 403.16.
(Z)-3-(2-Hydroxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (10i). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one 8c and 2-aminophenol, 72% of 10i was obtained as yellow solid
according to above-mentioned general procedure 10; Rf = 0.22 (30%
(Z)-3-(3-Hydroxy-4-methoxyphenylamino)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (10p). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 2-methoxy-5-aminophenol,
70% of 10p was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.26 (30% ethyl acetate in
1
ethyl acetate in hexane); mp 190−191 °C. H NMR (CDCl3, 400
MHz): δ 3.89 (s, 3H, OCH3), 3.93 (s, 6H, OCH3), 6.02 (d, 1H, J = 7.7
Hz, COCH), 6.91 (s, 2H, Ar−H), 7.18−7.26 (m, 4H, Ar−H),
7.57−7.62 (dd, 1H, J = 7.7, 12.8 Hz, CH-NH), 12.35 (d, 1H, J =
12.4 Hz, NH). MS found (M + H)+ (m/z): 330.20. Calcd for
C18H19N2O5 m/z: 329.13.
1
hexane); mp 142−143 °C. H NMR (CDCl3, 400 MHz): δ 3.59 (s,
3H, OCH3), 3.62 (s, 3H, OCH3), 3.65 (s, 6H, OCH3), 5.64 (d, 1H, J
= 7.7 Hz, COCH), 6.28−6.31(m, 1H, Ar−H), 6.46 (d, 1H, J = 2.6
Hz, Ar−H), 6.53 (d, 1H, J = 8.6 Hz, Ar−H), 6.91 (s, 2H, Ar−H),
7.10−7.15 (dd, 1H, J = 7.7, 12.3 Hz, CH-NH), 11.80 (d, 1H, J =
12.1 Hz, NH). MS found (M + H)+ (m/z): 360.10. Calcd for
C19H21NO6 m/z: 359.14.
(Z)-3-(3-Hydroxyyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (10j). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one 8c and 3-aminophenol, 70% of 10j was obtained as yellow solid
according to above-mentioned general procedure 10; Rf = 0.64 (50%
1
ethyl acetate in hexane); mp 120−121 °C. H NMR (CDCl3, 400
Synthesis of (Z)-3-(3-Amino-4-methoxyphenylamino)-1-(3,4,5-
trimethoxyphenyl)-prop-2-en-1-one (10q). . Step 1: Synthesis of
(Z)-3-(4-nitro-3-methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)-
prop-2-en-1-one. The ethynylketone 8c (1.94 g, 5 mmol) was
dissolved in absolute ethanol, and 4-methoxy-3-nitrophenylamine
(0.84 g, 5 mmol) was added. The reaction was stirred for 4 h at room
temperature. After the completion of reaction by TLC, the reaction
mixture was diluted with water and filtered the product. The crude
product was recrystallized from methanol to get pure compound with
74% yield as yellow solid, Rf = 0.40 (30% ethyl acetate in hexane); mp
MHz): δ 3.74 (s, 3H, OCH3), 3.76 (s, 6H, OCH3), 5.36 (s, 1H, OH),
5.80 (d, 1H, J = 7.8 Hz, COCH), 6.38−6.51 (m, 3H, Ar−H), 7.01−
7.05 (m, 3H, Ar−H), 7.28−7.33 (dd, 1H, J = 8.0, 12.3 Hz, CH-
NH), 11.88 (d, 1H, J = 12.3 Hz, NH). MS found (M + H)+ (m/z):
330.10. Calcd for C18H19NO5 m/z: 329.13.
(Z)-3-(2-Methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (10k). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one 8c and 2-methoxyphenylamine, 81% of 10k was obtained as
yellow solid according to above-mentioned general procedure 10; Rf =
0.46 (30% ethyl acetate in hexane); mp 118−119 °C. 1H NMR
(CDCl3, 400 MHz): δ 3.84 (s, 3H, OCH3), 3.86 (s, 6H, OCH3), 3.91
(s, 3H, OCH3), 5.94 (d, 1H, J = 7.8 Hz, COCH), 6.85−6.97 (m,
4H, Ar−H), 7.16 (s, 2H, Ar−H), 7.44−7.49 (dd, 1H, J = 7.6, 12.6 Hz,
CH-NH), 12.10 (d, 1H, J = 12.1 Hz, NH). MS found (M + H)+
(m/z): 344.20. Calcd for C19H21NO5 m/z: 343.14.
1
115−116 °C. H NMR (CDCl3, 300 MHz): δ 3.93 (s, 3H, OCH3),
3.96 (s, 3H, OCH3), 3.98 (s, 6H, OCH3), 6.05 (d, 1H, J = 7.8 Hz,
COCH), 7.13 (d, 1H, J = 9 Hz, Ar−H), 7.21 (s, 2H, Ar−H), 7.28−
7.30 (m, 2H, Ar−H), 7.42−7.46 (dd, 1H, J = 7.8, 12.6 Hz, CH-
NH), 12.22 (d, 1H, J = 11.7 Hz, NH). MS found (M + H)+ (m/z):
389.20. Calcd for C19H20N2O7 m/z: 388.13.
Step 2: Synthesis of (Z)-3-(3-Amino-4-methoxyphenylamino)-1-
(3,4,5-trimethoxyphenyl)-prop-2-en-1-one (10q). The above nitro
compound (2.0 g, 5.11 mmol) obtained from step 1 was dissolved in
acetone/water (2:1 ratio) 100 mL and heated to 50 °C for 30 min.
After completion of the reaction by TLC, the contents were cooled to
room temperature and the reaction mixture was extracted with ethyl
acetate. The organic extract was washed with water and brine solution
and dried over anhydrous Na2SO4 and concentrated under vacuum to
get crude product. The pure product 10q was obtained by flash
chromatography, Rf = 0.22 (30% ethyl acetate in hexane). Yield: 69%;
(Z)-3-(4-Methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (10l). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one 8c and 4-methoxyaniline, 82% of 10l was obtained as yellow
solid according to above-mentioned general procedure 10; Rf = 0.40
1
(30% ethyl acetate in hexane); mp 107−108 °C. H NMR (CDCl3,
300 MHz): δ 3.81 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.94 (s, 6H,
OCH3), 5.93 (d, 1H, J = 7.8 Hz, COCH), 6.89−6.92 (m, 2H, Ar−
H), 7.04−7.07 (m, 2H, Ar−H), 7.20 (s, 2H, Ar−H), 7.41−7.48 (dd,
1H, J = 7.8, 12.3 Hz, CH-NH), 12.18 (d, 1H, J = 12.3 Hz, NH). MS
found (M + H)+ (m/z): 344.20. Calcd for C19H21NO5 m/z: 343.14.
(Z)-3-(4-Chlorophenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (10m). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1
yellow solid; mp 98−99 °C. H NMR (CDCl3, 400 MHz): δ 3.65 (s,
3H, OCH3), 3.72 (s, 3H, OCH3), 3.73−3.79 (m, 6H, OCH3 and 2H,
J
dx.doi.org/10.1021/jm300176j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
NH2), 5.70 (d, 1H, J = 7.7 Hz, COCH), 6.25−6.30 (m, 2H, Ar−H),
6.55 (d, 1H, J = 8.4 Hz, Ar−H), 7.00 (s, 2H, Ar−H), 7.19−7.24 (dd,
1H, J = 7.7, 12.5 Hz, CH-NH), (d, 1H, J = 12.0 Hz, NH). MS found
(M + H)+ (m/z): 359.20. Calcd for C19H22N2O5 m/z: 358.15.
(Z)-3-(3-Fluoro-4-methoxyphenylamino)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (10r). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 3-fluoro-4-methoxyaniline,
68% of 10r was obtained as yellow solid according to above-mentioned
general procedure 10; Rf = 0.40 (30% ethyl acetate in hexane); mp
(Z)-3-(1H-Indole-6-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (10x). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-
one 8c and 6-aminoindole, 69% of 10x was obtained as yellow solid
according to above-mentioned general procedure 10; Rf = 0.60 (30%
1
ethyl acetate in hexane); mp 131−132 °C. H NMR (CDCl3, 300
MHz): δ 3.86 (s, 3H, OCH3), 3.88 (s, 6H, OCH3), 6.07 (d, 1H, J = 8.1
Hz COCH), 7.16 (s, 2H, Ar−H), 7.19 (s, 1H, Ar−H), 7.48−7.60
(m, 1H, CH-NH and 1H, Ar−H), 7.68−7.71 (m, 2H, Ar−H), 8.00
(d, 1H, J = 8.4 Hz, Ar−H), 8.72 (brs, 1H, NH), 12.26 (d, 1H, J = 12.3
Hz, NH). MS found (M + H)+ (m/z): 353.20. Calcd for C20H20N2O4
m/z: 352.14.
1
110−111 °C. H NMR (CDCl3, 400 MHz): δ 4.05 (s, 3H, OCH3),
4.08 (s, 3H, OCH3), 4.10 (s, 6H, OCH3), 6.13 (d, 1H, J = 7.8 Hz,
COCH), 6.98−7.14 (m, 3H, Ar−H), 7.36 (s, 2H, Ar−H), 7.51−
7.56 (dd, 1H, J = 7.8, 12.2 Hz, CH-NH), 12.0 (d, 1H, J = 12.1 Hz,
NH). MS found (M + H)+ (m/z): 362.20. Calcd for C19H20FNO5 m/
z: 361.13.
(Z)-3-(1H-Indole-7-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (10y). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-
one 8c and 7-aminoindole, 66% of 10y was obtained as yellow solid
according to above-mentioned general procedure 10; Rf = 0.82 (50%
ethyl acetate in hexane); mp 86−87 °C. 1H NMR (CDCl3, 400 MHz):
δ 3.86 (s, 3H, OCH3), 3.87 (s, 6H, OCH3), 6.01 (d, 1H, J = 7.4 Hz,
COCH), 6.55 (s, 1H, Ar−H), 6.96−7.09 (m, 3H, Ar−H), 7.15 (s,
2H, Ar−H), 7.40 (d, 1H, J = 7.6 Hz, Ar−H), 7.57−7.62 (dd, 1H, J =
7.8, 12.3 Hz, CH-NH), 8.75 (s, 1H, NH), 12.45 (d, 1H, J = 12.3 Hz,
NH). MS found (M + H)+ (m/z): 353.30. Calcd for C20H20N2O4 m/z:
352.14.
(Z)-3-(3-Chloro-4-methoxyphenylamino)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (10s). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 3-chloro-4-methoxyphenyl-
amine, 70% of 10s was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.66 (50% ethyl acetate in
1
hexane); mp 133−134 °C. H NMR (CDCl3, 300 MHz): δ 3.90 (s,
3H, OCH3), 3.91 (s, 3H, OCH3), 3.94 (s, 6H, OCH3), 5.96 (d, 1H, J
= 7.8 Hz, COCH), 6.90−6.99 (m, 2H, Ar−H), 7.17 (d, 1H, J = 2.5
Hz, Ar−H), 7.19 (s, 2H, Ar−H), 7.35−7.42 (dd, 1H, J = 7.6, 12.6 Hz,
CH-NH), 12.15 (d, 1H, J = 12.3 Hz, NH). MS found (M + H)+
(m/z): 378.20. Calcd for C19H20ClNO5 m/z: 377.10.
(Z)-3-(1H-Indazole-5-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (10z). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one 8c and 4-aminoindazole, 65% of 10z was obtained as yellow
solid according to above-mentioned general procedure 10; Rf = 0.16
1
(50% ethyl acetate in hexane); mp 160−161 °C. H NMR (CDCl3,
(Z)-3-(2-Chloro-5-hydroxyphenylamino)-1-(3, 4, 5-
trimethoxyphenyl)prop-2-en-1-one (10t). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 3-amino-4-chlorophenol,
73% of 10t was obtained as yellow solid according to above-mentioned
general procedure 10; Rf = 0.56 (50% ethyl acetate in hexane); mp
99−100 °C. 1H NMR (CDCl3, 300 MHz): δ 3.87 (s, 3H, OCH3), 3.88
(s, 6H, OCH3), 6.02 (d, 1H, J = 8.0 Hz, COCH), 6.40−6.42 (m,
1H, Ar−H), 6.67 (d, 1H, J = 2.1 Hz, Ar−H), 7.16 (s, 2H, Ar−H),
7.34−7.36 (m, 1H, CH-NH and 1H, Ar−H), 12.20 (d, 1H, J = 12.2
Hz, NH). MS found (M + H)+ (m/z): 364.10. Calcd for
C18H18ClNO5 m/z: 363.09.
300 MHz): δ 3.92 (s, 3H, OCH3), 3.95 (s, 6H, OCH3), 6.00 (d, 1H, J
= 7.8 Hz, COCH), 7.20 (s, 2H, Ar−H), 7.46−7.60 (m, 1H, CH
and 4H, Ar−H), 8.06 (s, 1H, NH), 12.31 (d, 1H, J = 12.3 Hz, NH).
MS found (M + H)+ (m/z): 354.20. Calcd for C19H19N3O4 m/z:
353.14.
(Z)-3-(Quinolin-3-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-
1-one (10aa). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-
one 8c and 3-aminoquinoline, 64% of 10aa was obtained as yellow
solid according to above-mentioned general procedure 10; Rf = 0.36
1
(50% ethyl acetate in hexane); mp 120−121 °C. H NMR (CDCl3,
300 MHz): δ 3.92 (s, 3H, OCH3), 3.95 (s, 6H, OCH3), 5.96 (d, 1H, J
= 7.8 Hz, COCH), 6.52−6.54 (m, 1H, Ar−H), 6.94−6.97 (m, 1H,
Ar−H), 7.13 (brs, 1H, Ar−H), 7.17−7.19 (m, 1H, Ar−H), 7.22 (s,
2H, Ar−H), 7.55−7.62 (m, 1H, CH-NH and 1H, Ar−H), 8.26 (brs,
1H, Ar−H), 12.35 (d, 1H, J = 12.3 Hz, NH). MS found (M + H)+ (m/
z): 365.10. Calcd for C21H20N2O4 m/z: 364.14.
( Z ) - 1 - ( 3 , 4 , 5 - T r i m e t h o x y p h e n y l ) - 3 - ( 2 , 4 , 6 -
trimethoxyphenylamino)prop-2-en-1-one (10u). Starting from 1-
(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 2,4,6-trimethoxyphe-
nylamine, 80% of 10u was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.20 (30% ethyl acetate in
1
hexane); mp 132−133 °C. H NMR (CDCl3, 400 MHz): δ 3.78 (s,
( Z ) - 3 - ( 2 - M e t h y l - 1 H - i n d o l e - 5 - y l a m i n o ) - 1 - ( 3 , 4 , 5 -
trimethoxyphenyl)prop-2-en-1-one (10ab). Starting from 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8c and 5-amino-2-methylindole,
65% of 10ab was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.56 (50% ethyl acetate in
3H, OCH3), 3.86 (s, 6H, OCH3), 3.87 (s, 3H, OCH3), 3.89 (s, 6H,
OCH3), 5.81 (d, 1H, J = 7.7 Hz, COCH), 6.16 (s, 2H, Ar−H), 7.17
(s, 2H, Ar−H), 7.89−7.94 (dd, 1H, J = 7.6, 12.7 Hz, CH-NH),
12.05 (d, 1H, J = 12.1 Hz, NH). MS found (M + H)+ (m/z): 404.10.
Calcd for C21H25NO7 m/z: 403.16.
1
hexane); mp 176−177 °C. H NMR (CDCl3, 400 MHz): δ 2.39 (s,
( Z ) - 1 - ( 3 , 4 , 5 - T r i m e t h o x y p h e n y l ) - 3 - ( 3 , 4 , 5 -
trimethoxyphenylamino)prop-2-en-1-one (10v). Starting from 1-
(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 3,4,5-trimethoxyphe-
nylamine, 78% of 10v was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.22 (30% ethyl acetate in
3H, CH3), 3.85 (s, 3H, OCH3), 3.88 (s, 6H, OCH3), 5.86 (d, 1H, J =
7.6 Hz, COCH), 6.13 (s, 1H, Ar−H), 6.83−6.85 (m, 1H, Ar−H),
7.16 (s, 2H, Ar−H), 7.17−7.20 (m, 2H, Ar−H), 7.48−7.53 (dd, 1H, J
= 7.6, 12.5 Hz, CH-NH), 7.87 (s, 1H, NH), 12.25 (d, 1H, J = 12.3
Hz, NH). MS found (M + H)+ (m/z): 367.20. Calcd for C21H22N2O4
m/z: 366.16.
1
hexane); mp 181−182 °C. H NMR (CDCl3, 400 MHz): δ 3.75 (s,
3H, OCH3), 3.80 (s, 6H, OCH3), 3.84 (s, 3H, OCH3), 3.87 (s, 6H,
OCH3), 5.90 (d, 1H, J = 7.8 Hz, COCH), 6.25 (s, 2H, Ar−H), 7.13
(s, 2H, Ar−H), 7.36−7.41 (dd, 1H, J = 7.8, 12.2 Hz, CH-NH),
12.20 (d, 1H, J = 12.2 Hz, NH). MS found (M + H)+ (m/z): 404.20.
Calcd for C21H25NO7 m/z: 403.16.
( Z ) - 1 - ( 2 - B r o m o - 3 , 4 , 5 - t r i m e t h o x y p h e n y l ) - 3 - ( 2 -
hydroxyphenylamino)prop-2-en-1-one (10ac). Starting from 1-(2-
bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 2-amino-phe-
nol, 70% of 10ac was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.66 (50% ethyl acetate in
1
(Z)-3-(1H-Indole-5-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (10w). Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one 8c and 5-aminoindole, 65% of 10w was obtained as yellow solid
according to above-mentioned general procedure 10; Rf = 0.24 (30%
hexane); mp 189−190 °C. H NMR (CDCl3, 300 MHz): δ 3.85 (s,
3H, OCH3), 3.94 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 5.77 (d, 1H, J
= 7.5 Hz, COCH), 6.78−6.80 (m, 1H, Ar−H), 6.87−6.90 (m, 2H,
Ar−H), 6.94 (s, 1H, Ar−H), 7.17−7.20 (m, 1H, Ar−H), 7.56−7.63
(dd, 1H, J = 7.8, 13.2 Hz, CH-NH), 12.33 (d, 1H, J = 13.2 Hz,
NH). MS found (M + H)+ (m/z): 408.10. Calcd for C18H18BrNO5 m/
z: 407.04.
1
ethyl acetate in hexane); mp 174−175 °C. H NMR (CDCl3, 400
MHz): δ 3.98 (s, 3H, OCH3), 4.01 (s, 6H, OCH3), 6.01 (d, 1H, J = 7.6
Hz, COCH), 6.60 (s, 1H, Ar−H), 7.07 (d, 1H, J = 6.7 Hz, Ar−H),
7.29−7.45 (m, 5H, Ar−H), 7.62−7.67 (dd, 1H, J = 7.6, 12.8 Hz, 
CH-NH), 8.29 (bs, 1H, NH), 12.38 (d, 1H, J = 12.1 Hz, NH). MS
found (M + H)+ (m/z): 353.20. Calcd for C20H20N2O4 m/z: 352.14.
( Z ) - 1 - ( 2 - B r o m o - 3 , 4 , 5 - t r i m e t h o x y p h e n y l ) - 3 - ( 4 -
methoxyphenylamino)prop-2-en-1-one (10ad). Starting from 1-(2-
bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 4-methoxy-
K
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Article
phenylamine, 68% of 10ad was obtained as yellow solid according to
above-mentioned general procedure 10; Rf = 0.74 (50% ethyl acetate
in hexane); mp 70−71 °C. H NMR (CDCl3, 300 MHz): δ 3.81 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.92 (s, 3H,
OCH3), 5.63 (d, 1H, J = 7.5 Hz, COCH), 6.86 (s, 1H, Ar−H),
6.89−6.93 (m, 2H, Ar−H), 7.05−7.09 (m, 2H, Ar−H), 7.37−7.44
(dd, 1H, J = 7.5, 12.6 Hz, CH-NH), 11.94 (d, 1H, J = 12.6 Hz,
NH). MS found (M + H)+ (m/z): 424.10. Calcd for C19H20BrNO5 m/
z: 421.05.
Flow Cytometry. Cancer cell line DU145 (human prostate tumor)
cells, were grown in DMEM (Cellgro) supplemented with 10% fetal
bovine serum and 1 unit/mL penicillin−streptomycin. The cells were
plated onto 100 mm2 dishes at a cell density of 1.0 × 106 cells/dish,
and 24 h later, they were treated with 2.5 μM of the compound. The
cells were harvested 24, 48, and 72 h after treatment. The cells were
removed from the plate by trypsin digestion and combined with the
nonattached cells found in the medium. The cell pellets were washed
in phosphate buffered saline (PBS) and fixed in ice cold 70% ethanol
for at least 24 h. The fixed cells were then washed with room
temperature PBS and stained with propidium iodide (50 mg/mL) and
RNase A (0.5 mg) for 30 min at 37 °C. The stained cells were then
analyzed on a Becton-Dickinson (BD) (FACScan) flow cytometer and
the data analyzed by cell cycle analysis software (Modfit, BD).
Western Blotting Analysis. HeLa cells were harvested at the
indicated times, and cell pellets were frozen on dry ice before lysis.
Cell pellets were lysed (25 mM HEPES, 0.1% Triton-X100, 300 mM
NaCl, 20 mM β-glycerophosphate, 0.5 mM DTT, 1.0 mM EDTA, 1.5
mM MgCl2, pH 7.5, 0.2 mM sodium orthovanadate, and protease
inhibitors). Then 100 μg of each lysate was resolved by 10%-SDS-
polyacrylamide gel electrophoresis. The resolved proteins were
transferred onto nitrocellulose filter paper and hybridized to the
following antibodies: phosphospecific histone H3 (Upstate, cat. 05−
806), histone H3 (upstate, cat. 07−690), bcl-2 (BD Biosciences, cat.
610558), bub R1 (BD Biosciences, cat. 612503), plk-1(Santa Cruz, sc-
17783), cyclin B1(Santa Cruz, sc-594), PARP (BD Biosciences, cat.
556362), and GAPDH (Fitzgerald, cat. RDI-TRK5G4−6C5).
Following the primary hybridization, the blots were then treated
with secondary antibodies conjugated to infrared dyes (IRDye 800 or
IRDye 680) and analyzed on an infrared scanning system (Odyssey,
Li-Cor Biosciences, NE). All hybridization and washing conditions
were performed as specified by the manufacturer. Quantitation of the
bands was performed using Odyssey software. Fold increase was
determined by first normalizing each band against GAPDH and then
dividing that number by the value of the normalized control.
PARP Western. DU145 cells were plated at a density of 3.0 × 106
cells per 150 mm2 plate and treated 24 h later with either DMSO or
10ae. The cells were collected at the indicated time points, and cell
pellets were frozen. The frozen cell pellets were lysed in 1% NP40/
PBS lysis buffer containing protease inhibitors. Equal amounts of total
cellular protein was then resolved on a 10%-SDS-polyacrylamide gel.
The gels were transferred onto nitrocellulose paper (S/S), hybridized
with anti-PARP antibodies (BD), and developed using ECL (Perkin-
Elmer, MA) solution.
1
(Z)1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxy-
phenylamino)-prop-2-en-1-one (10ae). Starting from 1-(2-bromo-
3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 2-methoxy-5-amino-
phenol, 65% of 10ae was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.60 (50% ethyl acetate in
1
hexane); mp 150−151 °C. H NMR (CDCl3, 300 MHz): δ 3.81 (s,
3H, OCH3), 3.89 (s, 3H, OCH3), 3.91 (s, H, OCH3), 3.92 (3H,
OCH3) 5.64 (d, 1H, J = 7.5 Hz, COCH), 5.73 (brs, 1H, OH),
6.58−6.62 (m, 1H, Ar−H), 6.76−6.84 (m, 2H, Ar−H), 6.86 (s, 1H,
Ar−H), 7.34−7.41 (dd, 1H, J = 7.5, 12.6 Hz, CH-NH), 11.86 (d,
1H, J = 12.3 Hz, NH). MS found (M + H)+ (m/z): 438.10. Calcd for
C19H20BrNO6 m/z: 437.05.
(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-(1H-indol-5-ylamino)-
prop-2-en-1-one (10af). Starting from 1-(2-bromo-3,4,5-
trimethoxyphenyl)prop-2-yn-1-one 8e and 5-aminoindole, 64% of
10af was obtained as yellow solid according to above-mentioned
general procedure 10; Rf = 0.70 (50% ethyl acetate in hexane); mp
1
109−110 °C. H NMR (CDCl3, 300 MHz): δ 3.78 (s, 3H, OCH3),
3.83 (s, 6H, OCH3), 5.54 (d, 1H, J = 7.5 Hz, COCH), 6.42 (s, 1H,
Ar−H), 6.80 (s, 1H, Ar−H), 6.87−7.91 (m, 1H, Ar−H), 7.13−7.18
(m, 1H, Ar−H), 7.25−7.30 (m, 2H, Ar−H), 7.42−7.49 (dd, 1H, J =
7.6, 12.7 Hz, CH-NH), 8.50 (s, 1H, NH), 12.00 (d, 1H, J = 12.6 Hz,
NH). MS found (M + H)+ (m/z): 431.10. Calcd for C20H19BrN2O4
m/z: 430.05.
(Z)-1-(2-Chloro-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-
methoxyphenylamino)prop-2-en-1-one (10ag). Starting from 1-(2-
chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8d and 2-methoxy-5-
aminophenol, 65% of 10ag was obtained as yellow solid according to
above-mentioned general procedure 10; Rf = 0.60 (50% ethyl acetate
in hexane); mp 102−103 °C. 1H NMR (CDCl3, 300 MHz): δ 3.89 (s,
3H, OCH3), 3.90 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.94 (s, 3H,
OCH3) 5.73 (d, 1H, J = 7.5 Hz, COCH), 5.76 (brs, 1H, OH),
6.59−6.63 (m, 1H, Ar−H), 6.76−6.85 (m, 2H, Ar−H), 6.86 (s, 1H,
Ar−H), 7.35−7.42 (dd, 1H, J = 7.5, 12.6 Hz, CH-NH), 11.93 (d,
1H, J = 12.3 Hz, NH). MS found (M + H)+ (m/z): 394.20. Calcd for
C19H20ClNO6 m/z: 393.10.
(Z)-3-(4-Methoxyphenylamino)-1-(3,4,5-trimethoxy-2-
nitrophenyl)prop-2-en-1-one (10ah). Starting from 1-(3,4,5-trime-
thoxy-2-nitrophenyl)prop-2-yn-1-one 8f and 4-methoxyphenylamine
60% of 10ah was obtained as yellow solid according to above-
mentioned general procedure 10; Rf = 0.68 (50% ethyl acetate in
Tubulin Polymerization. MAP-rich tubulin form bovine brain
(Cytoskeleton), resuspended in PEM (80 mM PIPES, (pH 6.9), 0.5
mM EGTA, 2 mM MgCl2) was preincubated with drug or its vehicle
dimethyl sulfoxide at room temperature for 5 min. PEG containing
GTP was added to start the tubulin polymerization reaction. The
reaction was monitored by a spectrophotometer at OD350 nm at 37
°C. The final concentrations of tubulin and GTP were 1.15 mg/mL
and 4 mM, respectively.
1
hexane); mp 132−133 °C. H NMR (CDCl3, 300 MHz): δ 3.82 (s,
3H, OCH3), 3.96 (s, 6H, OCH3), 4.00 (s, 3H, OCH3), 5.63 (d, 1H, J
= 7.5 Hz, COCH), 6.89−6.92 (m, 2H, Ar−H), 6.97 (s, 1H, Ar−H),
7.04−7.07 (m, 2H, Ar−H), 7.39−7.45 (dd, 1H, J = 7.5, 12.6 Hz, 
CH-NH), 11.93 (d, 1H, J = 12.9 Hz, NH). MS found (M + H)+ (m/
z): 389.10. Calcd for C19H20N2O7 m/z: 388.13.
Biology. Tissue Culture and Reagents. Paclitaxel was purchased
from Sigma. Cell lines were purchased from ATCC. Cell lines were
routinely grown in DMEM or RPM1 (CellGro) supplemented with
10% fetal bovine serum (CellGeneration, CO) and 1 unit/mL
penicillin−streptomycin (Gibco).
Cytotoxicity Assay. We have tested a number of tumor cell lines
using a dose response end point assay system. The cells were grown in
either DMEM or RPMI supplemented with 10% fetal bovine serum
and 1unit/mL penicillin−streptomycin solution. The tumor cells were
plated into six well dishes at a cell density of 1.0 × 105 cells/mL/well,
and compounds were added 24 h later at various concentrations. Cell
counts were determined from duplicate wells after 96 h of treatment.
The total number of viable cells was determined by trypan blue
exclusion.
Tubulin Staining. HeLa cells were grown on glass coverslips and
treated with 10ae (0.25 μM, 0.5 uM), paclitaxel (0.005 μM), or
vincristine (0.005 μM) for 18 h. The coverslips were washed in
phosphate buffered saline and fixed in freshly prepared 4%
paraformaldehyde. The cells were blocked and permeabilized and
stained with 1:400 dilution of anti-α-tubulin conjugated with FITC
(Sigma) followed by staining with DAPI (0.5 ug/mL). The coverslips
were mounted onto glass slides using Prolong Gold Antifade
(Molecular Probes/InVitrogen, CA) solution and visualized using an
Olympus IX-71 inverted fluorescent microscope at 100× magnifica-
tion. The cells were photographed using the DP-71 camera system
using two filters followed by merging and representative merged
images are shown.
L
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Journal of Medicinal Chemistry
Article
5372−5376. (i) Zhang, Q; Peng, Y.; Wang, X. I.; Keenan, S. M.; Arora,
S.; Welsh, W. J. Highly potent triazole-based tubulin polymerization
inhibitors. J. Med. Chem. 2007, 50, 749−754.
(6) Pinney, K. G.; Bounds, A. D.; Dingeman, K. M.; Mocharla, V. P.;
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AUTHOR INFORMATION
Corresponding Author
*For M.V.R.R.: phone, (212) 659-6875; fax, (215) 893-6989;
E-mail, r.reddy@mssm.edu. For E.P.R.: phone, (212) 659-
5571; E-mail, ep.reddy@mssm.edu.
■
Author Contributions
§These authors contributed equally.
Notes
The authors declare the following competing financial
interest(s): Dr. E.P. Reddy is a stockholder, Board member,
grant recipient and paid consultant of Onconova Therapeutics
Inc. Dr. M.V.R. Reddy is a stockholder and paid consultant of
Onconova Inc. Dr. S. Cosenza is a paid consultant of Onconova
Therapeutics Inc.
(8) Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.;
Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y. H.; Marsh, K.;
Warner, R.; Lee, J. Y.; Zielinski-Mozng, N.; Frost, D.; Rosenberg, S.
H.; Sham, H. L. Potent, oral active heterocycle-based combretastatin
C-4 analogues: synthesis, structure−activity relationship, pharmacoki-
netics, and in vivo antitumor activity evaluation. J. Med. Chem. 2002,
45, 1697−1711.
(9) Niher, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T. Syntheses
and antitumor activity of cis-restricted combretastatins: 5-membered
heterocyclic analogues. Bioorg. Med. Chem. Lett. 1998, 8, 3153−3158.
(10) Szczepankiewicz, B. G.; Liu, G.; Jae, H. S.; Tasker, A. S.;
Gunawardana, I. W.; von Geldern, T. W.; Gwaltney, S. L., II; Wu-
Wong, J. R.; Gehrke, L.; Chiou, W. J.; Credo, R. B.; Alder, J. D.;
Nukkala, M. A.; Zielinski, N. A.; Jarvis, K.; Mollison, K. W.; Frost, D.
J.; Bauch, J. L.; Hui, Y. H.; Claiborne, A. K.; Li, Q.; Rosenberg, S. H.
New mitotic agents with activity in multidrug-resistant cell lines and in
vivo efficacy in murine tumor models. J. Med. Chem. 2001, 44, 4416−
4430.
ACKNOWLEDGMENTS
This work was supported by grants from the NIH (CA 109820)
and Onconova Therapeutics Inc.
■
ABBREVIATIONS USED
■
MSA, microtubule-stabilizing agents; GTP, guanosine triphos-
phate; FACs, fluorescence-activated cell sorting; PLK-1, Polo-
like kinase-1; Bcl-2, B-cell lymphoma-2; MAPS, microtubule-
associated proteins; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; PARP, poly(ADP-ribose)polymerase
(11) Mahboobi, S.; Pongratz, H.; Hufsky, H.; Hockemeyer, J.;
Frieser, M.; Lyssenko, A.; Paper, D. H.; Burgermeister, J.; Bohmer, F.
̈
̈
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