Synthesis, anti-breast cancer activity, and molecular modeling of some benzothiazole and benzoxazole derivatives

Article abstract of DOI:10.1002/ardp.201300044

A new series of benzothiazoles and benzoxazoles was synthesized using 4-benzothiazol-2-yl-phenylamine and 4-benzoxazol-2-yl-phenylamine as starting materials. All the prepared compounds were evaluated for their antitumor activities against human breast ca

Full text of DOI:10.1002/ardp.201300044

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
1
Full Paper
Synthesis, Anti-Breast Cancer Activity, and Molecular Modeling
of Some Benzothiazole and Benzoxazole Derivatives
Mohamed A. Abdelgawad1, Amany Belal2, Hany A. Omar3, Lamees Hegazy5, and Mostafa E. Rateb4
1
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef,
Egypt
2
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
Department of Chemistry, University of Florida, Gainesville, FL, USA
3
4
5
A new series of benzothiazoles and benzoxazoles was synthesized using 4-benzothiazol-2-yl-
phenylamine and 4-benzoxazol-2-yl-phenylamine as starting materials. All the prepared compounds
were evaluated for their antitumor activities against human breast cancer cell lines, MCF-7 and MDA-
231, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability analysis.
Almost all the tested compounds revealed potent antitumor activity, especially the N-methyl
piperazinyl substituted derivatives 6f and 6c, which displayed the most potent inhibitory activity with
IC50 values ranging from 8 to 17 nM. Docking the synthesized compounds into the epidermal growth
factor receptor (EGFR), which is highly expressed in breast cancer, was employed to explore the possible
interactions of these compounds with the EGFR. The activity of the reported compounds supports its
clinical promise as a component of therapeutic strategies for cancer, for which high concentrations of
chemotherapeutic agents are always a major limitation.
Keywords: Antitumor / Benzothiazoles / Benzoxazoles / MCF-7 / MDA-231 / Molecular modeling / Tyrosine kinase
Received: February 7, 2013; Revised: March 30, 2013; Accepted: April 12, 2013
DOI 10.1002/ardp.201300044
Additional supporting information may be found in the online version of this article at the publisher’s web-site
:
Introduction
developed countries with fewer chances for treatment [1–5].
Currently, a lot of anticancer drugs have been clinically used
successfully for the treatment of several malignancies.
However, solid tumors, such as breast cancer, resist most of
the clinically-available anticancer agents probably due to gene
mutations or epigenetic modifications during the course of
therapy that affect the uptake, metabolism or efflux of drugs
from cancer cells [6]. Since the response of solid tumors to
available anticancer chemotherapeutic agents is limited,
searching for novel easily accessible drugs with low cost and
superior efficacy is desired.
Receptor protein tyrosine kinases (RPTKs) have a crucial role
in the development and progression of many types of cancer.
Over-activation of these receptors is usually accompanied by
carcinogenesis [7]. Of the 30 RPTKs that are currently known
to be implicated in human cancers, the epidermal growth
factor receptor (EGFR) is frequently expressed at high levels
in certain carcinomas, especially breast, colon, and bladder
Cancer is a complex, prevalent and fatal disease that notably
affects almost every tissue in the human body. It is one of the
leading causes of death worldwide. Lung, stomach, liver,
colon, and breast cancers are the most common causes of
cancer death every year. Breast cancer comprises 23% of all
cancers (excluding non-melanoma skin cancers) in women,
and it is considered as the main and the most frequent cause
of cancer death among women all over the world. In 2008,
breast cancer caused about half million deaths in women in
USA. Awfully, the incidence and mortality rates of breast
cancer in developing countries are much higher than those in
Correspondence: Dr. Mostafa E. Rateb, Department of Pharmacognosy,
Faculty of Pharmacy, Beni-Suef University, Beni-Suef 66211, Egypt.
E-mail: ratebnplab@gmail.com
Fax: þ20 822319758
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2
M. A. Abdelgawad et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
OMe
O
O
N
R
O
HO
Cl
S
N
S
N
O
NH2
NH
N
Figure 1. Structures of some previously
reported benzothiazoles and benzoxazoles
as potent antitumor agents.
I (R= H, I, CH3, Br & Cl)
II
UK-1 III
cancers [8]. Thus, targeting EGFR represents a potential Results and discussion
approach for the design of novel antiproliferative drugs [9].
Benzothiazoles and their related heterocycles benzoxazoles
are considered unique and versatile scaffolds used for the
design of several analogs of pharmacological interest [10].
A lot of benzothiazole derivatives exhibit a number of
interesting biological activities including anticancer activity
against several tumors [11–14]. Benzoxazole derivatives
also show diverse pharmacological activities as analgesic
[15], anti-inflammatory [16], antitubercular [17], anthelmentic
[18], antifungal [19], antimalarial [20], antidiabetic [21],
anticonvalsant [22], and antitumor [23]. Bradshaw et al. [24,
25] reported the synthesis of a series of 30-substituted-2-(40-
aminophenyl)-benzothiazoles I (Fig. 1), that showed a unique
profile of growth inhibition when tested against MCF-7
and MDA-468 cell lines. Moreover, the 2-(4-acylaminophenyl)
benzothiazole II (Fig. 1) was found to be very active against
the MCF-7 cell line [26]. The bis(benzoxazole) Streptomyces-
derived natural product UK-1 III (Fig. 1) and some of its
synthesized analogues displayed potent activity against a wide
spectrum of human cancer cell lines [27]. Benzoxazole and
benzothiazole scaffolds were designed and synthesized as Raf
kinase inhibitors [28]. It was also reported that some poly-
hydroxylated phenyl benzothiazoles have ATP antagonistic
effects and act as tyrosine kinase inhibitors by binding to the
ATP binding site of protein kinases [29, 30]. This obvious role for
benzothiazole derivatives and its related heterocyclic benzox-
azoles in the treatment of human cancer inspired us to design
some novel derivatives with similar scaffolds having the
general formula A and B (Fig. 2) and aiming to have potentially
active compounds against the solid tumors such as breast
cancer. Docking studies of these compounds into the ATP
binding site of EGFR are also presented to explore the structure-
activity relationships between the inhibitors and EGFR.
Chemistry
The synthetic pathways adopted for the preparation of the
new compounds are illustrated in Schemes 1 and 2. The
starting materials 3a,b and 5a,b were synthesized according
to the previously reported methods [26, 31–35]. Compounds
3a,b were then subjected to the reaction with 4-methox-
ybenzaldehyde in absolute ethanol to afford compounds 4a,b
(Scheme 1). The structures of 4a,b were confirmed on the basis
of their elemental and spectral data. The 1H NMR spectra
indicated the presence of a 1,2-disubstituted benzene ring
together with two 1,4-disubstituted benzene rings, a methoxy
group at d 3.85 in addition to a singlet at d 8.63 corresponding
–
to N CH proton. Moreover, the prepared structures were
–
further confirmed by 13C NMR spectra. The mass spectrum
NH2
X
N
XH
+
NH2
PPA
NH2
3a,b
COOH
2
3a X=O
1
3b
X=S
CHO
OCH3
H3CO
X
O
N
R
OMe
N
X
N
X
N
NH
N
4a,b
4a X=O
4b
A
B
X=S
Scheme 1. Synthesis of compounds 4a,b.
Figure 2. General formula of the synthesized benzothiazole and
benzoxazole derivatives.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Antitumor Activity of Benzothiazoles and Benzoxazole Derivatives
3
O
S
X
CH2Cl
X
N
NH2
ClCH2COCl
NH2
NH
N
N
5a,b
3a,b
IV
5a
5b
3a
3b
X=O
X=S
X=O
X=S
Figure 3. 4-Benzothiazol-2-yl-phenylamine IV.
Secondary
amines
O
R
X
N
NH
Table 1. IC50 of the synthesized compounds against human
breast cancer cell lines.
N
O
N
N
N
_
6a
f
6a X=O,R=piperidin
6b
R =
,
,
Compound
IC50 (nM)a)
X=O,R=morpholino
6c X=O,R=methylpiperazin
6d X=S,R=piperidin
6e
MDA-231
MCF-7
X=S,R=morpholin
6f X=S,R=methylpiperazin
4a
4b
6a
6b
6c
6d
6e
6f
>50
>50
42
37
17
34
16
8
13
>50
35
30
31
12
29
20
10
18
Scheme 2. Synthesis of compounds 6a–f.
also revealed molecular ion peaks [MþH]þ at 329.1283 and
345.1052 indicating 4a and 4b, respectively.
Furthermore, the substituted benzothiazoles and benzox-
azoles 6a–f were obtained by the reaction of compounds 5a,b
with different secondary amines in absolute ethanol in
the presence of triethylamine (Scheme 2). The structures of
compounds 6a–f were elucidated using 1H NMR spectra,
which showed a 1,2-disubstituted benzene ring together with
a 1,4-disubstituted benzene ring, and a singlet signal at d 9.99–
IV
a)
The values given are means of three experiments.
MCF-7 cell line was obtained by compound 4-methylpiperazin-
1-yl-acetamide derivative of benzothiazole 6f with IC50 value
of 10 nM, followed by 6c which showed IC50 value of 12 nM
whilst compound 4a exhibited the least cytotoxic activity.
From the results of the antitumor screening against MCF-7
and MDA-231 cell lines, some structure activity relationship
could be suggested. As a general scaffold, the benzothiazole
1
10.14 corresponding to NH proton. In addition, the H NMR
spectra showed the characteristic signals for different
secondary amine protons as morpholino, piperidino and
methyl piperazyl protons at d 2.40–3.66. The 13C NMR spectra
also showed peaks at aliphatic region (d 23–66) corresponding
to the aliphatic carbons in addition to the aromatic carbons.
The accurate mass spectral analysis also confirmed the
structures of 6a–f indicating the exact molecular ion peaks for
these compounds.
In vitro anticancer screening
All the synthesized benzothiazole and benzoxazole deriva-
tives were evaluated in vitro for their cytotoxic activity against
human breast cancer cell lines, MCF-7 and MDA-231, using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) assay [36]. 4-Benzothiazol-2-yl-phenylamine IV
(Fig. 3) was also tested for its activity against both cell lines; to
investigate the effect of structural changes on the activity, the
half maximal inhibitory concentration (IC50) was calculated.
The IC50 of the tested compounds as well as of compound IV
are shown in Table 1 and the most potent compounds are
represented graphically in Fig. 4. The obtained data revealed
that most of the newly synthesized compounds showed
potent antitumor activity in the nanomolar range. Among the
tested compounds, the most potent cytotoxic effect against
Figure 4. Viability percent at different concentrations of the most
potent compounds on MCF-7.
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M. A. Abdelgawad et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
system 4b,6d–f was more potent than the benzoxazole one
4a,6a–c for the same substituents. Acetamide substituted
benzoxazoles and bezothiazoles 6a–f (general formula A)
showed more potential cytotoxic activity than the 4-methoxy-
benzylidine derivatives 4a,b (general formula B). The methyl
piperazine substituted benzoxazole and benzothiazole deriv-
atives 6c,f were found to be more potent than morpholine
substituted derivatives 6b,e which were more active than
piperidine substituted analogues 6a,d.
predicted binding poses of 4-methoxy-benzylidine derivatives
4a and 4b make hydrophobic contacts with the hydrophobic
amino acids in the active site. However, the predicted binding
poses of the acetamide substituted benzoxazole and benzo-
thiazole derivatives 6a–6f (Figs. 5–8, S30–S37, SI) make a
hydrogen bonding interaction in addition to hydrophobic
contacts except in only one case where compound 6b was
predicted to form only hydrophobic contacts. The amide
hydrogen of compounds 6c and 6d makes hydrogen-bonding
interaction with the backbone of Pro-770. The carbonyl
oxygens of compounds 6a, 6e, and 6f make hydrogen bonding
interaction with the backbone of Cys-773.
Molecular docking studies
Carcinogenesis is usually accompanied by overactivation of
receptor tyrosine kinase (RTK) signaling pathways, so inhibitors
which block these receptors have a significant therapeutic
potential in cancer treatment [9]. On this basis, RTK was selected
as the target receptor for docking studies of the synthesized
compounds. EGFR kinase domain in complex with 4-anilino-
quinazoline inhibitor (4AQ) (PDB ID: IM17) [37] was used.
Docking calculations were carried out using DOCK6.5. The
inhibitor 4-anilinoquinazoline was extracted from the X-ray
crystal structure, then re-docked in the active site of EGFR and
the docking parameters were adjusted accordingly. DOCK6.5
[38] predicted a docking pose similar to the experimental
binding pose in the original X-ray crystal structure thereby
validating the docking approach (Fig. 25 in Supporting
Information). The protein was prepared using Dock Prep tool
in UCSF Chimera [39]. Each ligand was docked into the active
site using the flexible ligand sampling algorithm of DOCK6.5.
The surface of the EGFR binding pocket is mainly
hydrophobic (Fig. S26, SI) and the known inhibitor 4AQ forms
mostly hydrophobic contacts and only one hydrogen bond
with the backbone of Met-769. The docked inhibitors make
hydrophobic interactions with the side chains of hydrophobic
residues embedded in the active site which is consistent with
the hydrophobic interactions made by the known inhibitor
4AQ [37]. The hydrogen bonding interaction to either Pro-770
or Cys-773 made by the acetamide substituted benzoxazole
and benzothiazole derivatives 6a–6f may have increased their
binding specificity and hence increased their activity over the
4-methoxy-benzylidine derivatives 4a and 4b. The predicted
binding pose of compound IV forms a hydrogen bonding
interaction with the carboxyl group of Glu-738 (Fig. S37, SI)
which may also increased binding specificity in the active site.
Solvent was deleted from the original structure except for one Conclusion
water molecule in the active site. Each inhibitor molecule was
oriented, minimized using Amber force filed and screened for
Van der Waals and electrostatic interactions complementarity
with the EGFR active site generating the best energy scored
conformation of each docked molecule. Hydrophobic inter-
actions are principle constituents in the predicted binding
modes (Figs. S27–S35, Supporting Information, SI). The
A series of new benzothiazole and benzoxazole derivatives
have been synthesized and evaluated for their potential as
antitumor lead compounds, depending on the previously
reported antitumor activity for numerous compounds
having benzothiazole and benzoxazole scaffolds. The newly
synthesized compounds were tested in vitro on human breast
Figure 5. 2D predicted binding pose of
compound 6c in the active site of EGFR.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Antitumor Activity of Benzothiazoles and Benzoxazole Derivatives
5
Figure 6. Putative interactions in the EGFR
active site with 6c. Hydrogen bond with the
backbone of Pro-770 is shown as dashed line.
Hydrophobic contacts are demonstrated as
solid lines.
Figure 7. 2D predicted binding poses of
compound 6f in the active site of EGFR.
cancer cell lines, MCF-7 and MDA-231. Most of the tested
compounds showed potent antitumor activity in the nano-
molar level. The antitumor screening revealed that benzo-
thiazole derivatives were more potent than benzoxazole ones.
In addition, the acetamide substituted derivatives of both
benzoxazole and benzothiazole 6a–f were more potent than
the 4-methoxy-benzylidine analogues 4a,b. Moreover, the 4-
methylpiperazin-1-yl substituted acetamides of both benzox-
azole and benzothiazole 6c,f are found to be the most potent
cytotoxic derivatives. Molecular docking studies for the
Figure 8. Putative interactions in the EGFR
active site with 6f. Hydrogen bond with the
backbone of Cys-773 is shown as dashed line.
Hydrophobic contacts are demonstrated as
solid lines.
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M. A. Abdelgawad et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
synthesized compounds were performed and molecular
interactions were explored. The hydrophobic contacts with
the hydrophobic amino acids embedded in the active site of
EGFR is the main constituent, consistent with the hydropho-
bic interactions of the known inhibitor 4-anilinoquinazoline
(4AQ) within the active site of EGFR. The amide group of the
acetamide substituted benzoxazole and benzothiazole deriv-
atives was predicted to make extra electrostatic interactions
which may increase their binding specificity and activity over
the 4-methoxy-benzylidine analogues. The methyl piperazinyl
substituted derivatives of both benzothiazole 6f and benzox-
azole 6c could be screened for their selective toxicity against a
panel of cancer cell lines as well as their toxicity against
normal human cell lines which could be of value for these
compounds to be used as anticancer drugs. These results
also support the clinical promise of these compounds as a
component of therapeutic strategies for cancer, for which
high concentrations of chemotherapeutic agents are always a
major limitation. Moreover, the uncomplicated methodology
used for the preparation of these potent compounds allows
for obtaining sufficient amounts for more in-depth clinical
studies. The simplicity of the prepared compounds with their
potent anti-breast cancer activity highlights the potential of
smart organic synthesis for the discovery of new drug leads.
Receptor spheres were placed on the receptor surface using the
sphgen program within the DOCK package. Receptor electrostatic
and VDW grid points were calculated by the grid program within
the DOCK package. The inhibitors coordinate files were prepared
using Maestro v9.0 software package (Schrödinger LLC) and UCSF
Chimera [38] was used further to add charges using AM1-BCC
method. All 2D interaction pictures diagrams were generated
using Maestro v9.0 software package (Schrödinger LLC). All 3D
interaction models and surfaces were rendered using UCSF
Chimera [38].
General procedure for the preparation of 4a,b
A mixture of compound 3a or 3b (0.005 mol), 4-methoxybenzal-
dehyde (0.7 g, 0.0055 mol) in absolute ethanol (25 mL) and glacial
acetic acid (0.5 mL) was heated under reflux for 3 h. The solid
formed on hot was filtered, dried and re-crystallized from
ethanol/acetone mixture to give compounds 4a and b.
(4-Benzoxazo-2-yl-phenyl)(4-methoxy-benzylidine)amine
4a
Yellow crystals, 70% yield, mp 144–146°C. IR: ymax./cmꢀ1 3050
1
–
–
(CH aromatic), 2950 (CH aliph), 1593 (C N), 1581 (C C). H NMR
–
–
(DMSO-d6, 400 MHz at 298 K):
d 3.85 (s, 3H, OCH3), 7.10
(d, J ¼ 8.7 Hz, 2H, Ar–H), 7.41–7.45 (m, 4H, Ar–H), 7.79
(t, J ¼ 8.6 Hz, 2H, Ar–H), 7.93 (d, J ¼ 8.5 Hz, 2H, Ar–H), 8.23
(d, J ¼ 7.8 Hz, 2H, Ar–H), 8.63 (s, 1H, CH N); 13C NMR (DMSO-d6):
–
–
d 55.5, 110.8, 114.4 (2C), 119.7, 121.9 (2C), 123.3, 124.8, 125.3,
128.4 (2C), 128.6, 130.9 (2C), 141.6, 150.2, 154.8, 161.4, 162.2,
162.3. HRESIMS m/z 329.1283 [MþH]þ (calcd. for C21H17N2O2
þ
Experimental
329.1285); Calcd. C, 76.81; H, 4.91; N, 8.53. Found: C, 76.70;
H, 4.80; N, 8.50.
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) on silica gel sheets pre-coated with UV fluorescent silica
(MERCK 60 F 254) and spots were developed using I2 vapor/UV
light as visualizing agents. Solvent system was chloroform/
methanol (in different ratios). 1H and 13C NMR spectra were
determined in DMSO-d6 solvent with a Varian Inova 400 MHZ
spectrometer, Department of Chemistry, University of Aberdeen.
Peak positions were given in parts per million (d). All NMR data
were processed using MestReNova 7.1.0. All reported products
showed NMR spectra in agreement with the assigned structures.
High resolution mass spectral data was obtained from a Thermo
Instruments MS system (LTQ XL/LTQ Orbitrap Discovery) coupled
to a Thermo Instruments HPLC system (Accela PDA detector,
Accela PDA autosampler and Accela pump), Department of
Chemistry, University of Aberdeen. The following conditions were
used: capillary voltage 45 V, capillary temperature 320°C,
auxiliary gas flow rate 10–20 arbitrary units, sheath gas flow
rate 40–50 arbitrary units, spray voltage 4.5 kV, mass range 100–
2000 amu (maximum resolution 30,000). Melting points were
determined on a Griffin instrument and are uncorrected. IR
spectra were recorded on a Shimadzu 435 spectrometer, using
KBr discs and values were represented in cmꢀ1. Elemental
analyses were performed at the Micro-Analytical Center, Cairo
University, Egypt. Compounds 3a,b and 5a,b were prepared
according to the previously reported procedures [26, 31–35].
Docking studies were performed using DOCK 6.5 [38]. Crystal
structure of protein tyrosine kinase domain (PDB codes: 1M17) is
obtained from protein data bank [37]. As for protein preparation
for docking studies and docking, procedures were performed
following the standard protocol implemented in DOCK 6.5. The
protein and ligand 4AQ are prepared in UCSF Chimera [38].
(4-Benzothiazol-2-yl-phenyl)(4-methoxy-benzylidine)-
amine 4b
Greenish crystals, 60% yield, m.p. 155–157°C. IR: ymax./cmꢀ1 3061,
–
–
–
3050 (CH aromatic), 2949 (CH aliph.), 1596 (C N), 1556 (C C);
–
1H NMR (DMSO-d6, 400 MHz at 298 K): d 3.85 (s, 3H, OCH3), 7.10
(d, J ¼ 8.6 Hz, 2H, Ar–H), 7.41 (d, J ¼ 8.2 Hz, 2H, Ar–H), 7.46
(t, J ¼ 7.8 Hz, 1H, Ar–H), 7.54 (t, J ¼ 7.9 Hz, 1H, Ar–H), 7.93
(d, J ¼ 8.6 Hz, 2H, Ar–H), 8.06 (d, J ¼ 8.1 Hz, 1H, Ar–H), 8.12–8.16
(m, 3H, Ar–H), 8.63 (s, 1H, CH N). 13C NMR (DMSO-d6): d 55.5, 114.4
–
–
(2C), 121.9 (2C), 122.3, 122.7, 125.4, 126.6, 128.3 (2C), 128.7, 130.0,
130.8 (2C), 134.4, 153.7, 154.3, 161.1, 162.2, 166.9; HRESIMS
m/z 345.1052 [MþH]þ (calcd. for C21H17N2OSþ 345.1056); Calcd. C,
73.23; H, 4.68; N, 8.13; Found: C, 73.30; H, 4.70; N, 8.20.
General procedure for the preparation of 6a–f
A well-stirred mixture of compound 5a or 5b (0.01 mol),
appropriate secondary amine (0.01 mol) and (2–3 drops) triethyl-
amine in absolute ethanol (100 mL), was heated under reflux for
24 h. The mixture was filtered while hot and the solvent was
removed by distillation under vacuum. The obtained residue
was washed with water, filtered, dried and re-crystallized from
DMF.
N-(4-Benzoxazol-2-ylphenyl)-2-piperidin-1-yl-acetamide 6a
Yellow crystals, 70% yield, m.p. 185–187°C. IR: ymax./cmꢀ1 3314
–
(NH), 3051 (CH aromatic), 2931 (CH aliph), 1687 (C O), 1608
–
1
–
–
(C N), 1595 (C C); H NMR (DMSO-d , 400 MHz at 298 K): d 1.41
–
–
6
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Antitumor Activity of Benzothiazoles and Benzoxazole Derivatives
7
(m, 2H, CH2), 1.58 (m, 4H, 2 ꢁ CH2), 2.47 (m, 4H, 2 ꢁ CH2), 3.12
m/z 354.1268 [MþH]þ (calcd. for C19H20N3O2Sþ 354.1271): Calcd.
–
(s, 2H, O C–CH ), 7.39–7.43 (m, 2H, Ar–H), 7.75–7.78 (m, 2H, Ar–
C, 64.57; H, 5.42; N, 11.89. Found: C, 64.60; H, 5.60; N, 11.90.
–
2
H), 7.90 (d, J ¼ 8.4 Hz, 2H, Ar–H), 8.15 (d, J ¼ 8.4 Hz, 2H, Ar–H),
10.03 (s, 1H, NH). 13C NMR (DMSO-d6): d 23.5, 25.4 (2C), 54.0 (2C),
62.7, 110.8, 119.5 (3C), 121.0, 124.8, 125.2. 128.1 (2C), 141.6, 141.9,
150.1, 162.2, 169.2; HRESIMS m/z 336.1703 [MþH]þ (calcd. for
C20H22N3O2þ 336.1707); Calcd. C, 71.62; H, 6.31; N, 12.53. Found:
C, 71.60; H, 6.40; N, 12.50.
N-(4-Benzothiazol-2-ylphenyl)-2-(4-methylpiperazin-1-yl)-
acetamide 6f
Yellowish green crystals, 65% yield, m.p. 198–200°C. IR:
ymax./cmꢀ1 3304 (NH), 3052 (CH aromatic), 2933 (CH aliph),
1688 (C O), 1590 (C C). 1H NMR (DMSO-d6, 400 MHz at 298 K):
–
–
–
–
d 2.19 (s, 3H, N–CH3), 2.41–2.54 (m, 8H, aliphatic protons), 3.17
N-(4-Benzoxazol-2-ylphenyl)-2-morpholin-4-yl-acetamide 6b
–
(s, 2H, O C–CH ), 7.41–7.54 (m, 2H, Ar–H), 7.85 (d, J ¼ 8.6 Hz, 2H,
–
2
Yellow crystals, 65% yield, m.p. 200–202°C. IR: ymax./cmꢀ1 3313
Ar–H), 8.01–8.07 (m, 3H, Ar–H), 8.11 (d, J ¼ 8.1 Hz, 1H, Ar–H),
10.03 (s, 1H, NH). 13C NMR (DMSO-d6): d 45.6, 52.5 (2C), 54.4 (2C),
61.7, 119.6 (2C), 122.2, 122.6, 125.2, 126.5, 127.7, 127.9 (2C), 134.3,
141.4, 153.6, 166.9, 168.7. HRESIMS m/z 367.1581 [MþH]þ (calcd.
for C20H23N4OSþ 367.1587): Calcd. C, 65.55; H, 6.05; N, 15.29.
Found: C, 65.50; H, 6.10; N, 15.20.
–
(NH), 3050 (CH aromatic), 2965 (CH aliph), 1687 (C O), 1608
–
(C N), 1596(C C). 1H NMR (DMSO-d6, 400 MHz at 298 K): d 2.50
–
–
–
–
–
(t, J ¼ 4.4 Hz, 4H, 2 ꢁ CH2), 3.18 (s, 2H, O C–CH ), 3.65
–
2
(t, J ¼ 4.5 Hz, 4H, 2 ꢁ CH2), 7.37–7.43 (m, 2H, Ar–H), 7.76
(m, 2H, Ar–H), 7.90 (d, J ¼ 8.7 Hz, 2H, Ar–H), 8.15 (d, J ¼ 8.7 Hz,
2H, Ar–H), 10.11 (s, 1H, NH). 13C NMR (DMSO-d6): d 53.1 (2C), 62.1,
66.1 (2C), 110.8, 119.5 (2C), 119.6, 121.0, 124.8, 125.2, 128.1 (2C),
141.6, 141.9, 150.1, 162.2, 168.7; HRESIMS m/z 338.1497 [MþH]þ
Pharmacological studies
Cell culture
þ
(calcd. for C19H20N3O3 338.1499): Calcd. C, 67.64; H, 5.68;
Human breast cancer cells, MCF-7 and MDA-231, were obtained
from the American Type Culture Collection (Manassas, VA) and
cultured in Dulbecco’s modified Eagle’s medium/F12 medium
(DMEM/F-12, Gibco, Grand Island, NY) supplemented with 10%
fetal bovine serum (FBS, Gibco). All cells were cultured at 37°C
in a humidified incubator containing 5% CO2.
N, 12.46. Found: C, 67.60; H, 5.60; N, 12.50.
N-(4-Benzoxazol-2-ylphenyl)-2-(4-methylpiperazin-1-yl)-
acetamide 6c
Yellow crystals, 65% yield, m.p. 190–192°C. IR: ymax./cmꢀ1 3324
–
–
(NH), 3051 (CH aromatic), 2938 (CH aliph), 1689 (C O), 1610
1
–
(C N). H NMR (DMSO-d , 400 MHz at 298 K): d 2.26 (s, 3H, N–CH ),
–
6
3
Cell viability assay
–
–
2.50–2.59 (m, 8H, aliphatic protons), 3.17 (s, 2H, O C–CH ), 7.38–
2
The effect of test compounds on cell viability was assessed using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) assay in six replicates as reported before [40]. Briefly,
MCF-7 or MDA-231 cells were seeded in 96-well plates for 24 h, and
treated with test agents in 5% FBS-supplemented DMEM/F-12 for
72 h. Controls received DMSO vehicle at a concentration equal to
that in drug-treated cells. After treatment, cells were incubated
in the same medium containing 0.5 mg/mL MTT at 37°C for
2 h. Reduced MTT was solubilized in DMSO (200 mL/well) for
determination of absorbance at 570 nm using a microplate
reader.
7.42 (m, 2H, Ar–H), 7.74–7.77 (m, 2H, Ar–H), 7.89 (d, J ¼ 8.6 Hz,
2H, Ar–H), 8.15 (d, J ¼ 8.6 Hz, 2H, Ar–H), 10.14 (s, 1H, NH).
13C NMR (DMSO-d6): d 45.1, 52.1 (2C), 54.2 (2C), 61.5, 110.8, 119.5
(3C), 121.0, 124.8, 125.2, 128.1 (2C), 141.6, 141.9, 150.1, 162.2,
þ
168.8; HRESIMS m/z 351.1813 [MþH]þ (calcd. for C20H23N4O2
351.1816): Calcd. C, 68.55; H, 6.33; N, 15.99. Found: C, 68.40;
H, 6.30; N, 15.90.
N-(4-Benzothiazol-2-ylphenyl)-2-piperidin-1-yl-acetamide 6d
Yellowish green crystals, 75% yield, m.p. 190–192°C. IR:
ymax./cmꢀ1 3316 (NH), 2933 (CH aliph), 1682 (C O), 1598
–
–
(C N). 1H NMR (DMSO-d6, 400 MHz at 298 K): d 1.40 (m, 2H,
–
–
The authors would like to thank Professor Marcel Jaspars, Marine
Biodiscovery Centre, Department of Chemistry, University of Aberdeen,
Scotland, UK for kindly carrying out the NMR and accurate mass
measurements.
CH2), 1.57 (m, 4H, 2 ꢁ CH2), 2.47 (m, 4H, 2 ꢁ CH2), 3.11 (s, 2H,
–
O C–CH ), 7.42–7.55 (m, 2H, Ar–H), 7.86 (d, J ¼ 8.8 Hz, 2H, Ar–H),
–
2
8.01–8.06 (m, 3H, Ar–H), 8.12 (d, J ¼ 8.1 Hz, 1H, Ar–H), 9.99 (s, 1H,
NH). 13C NMR (DMSO-d6): d 23.5, 25.4 (2C), 54.0 (2C), 62.7, 119.6
(2C), 122.2, 122.6, 125.2, 126.6, 127.7, 127.9 (2C), 134.3, 141.4,
153.6, 166.9, 169.1. HRESIMS m/z 352.1475 [MþH]þ (calcd. for
C20H22N3O2Sþ 352.1478): Calcd. C, 68.35; H, 6.02; N, 11.96. Found:
C, 68.30; H, 6.10; N, 11.90.
The authors have declared no conflict of interest.
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