Design, synthesis, in silico and in vitro studies of novel 4-methylthiazole-5-carboxylic acid derivatives as potent anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2014.07.058

Since inhibitors of mucin onco proteins are potential targets for breast cancer therapy, a series of novel 4-methylthiazole-5-carboxylic acid (1) derivatives 3a-k were synthesized by the reaction of 1 with SOCl₂followed by different bases/alcohols in the presence of triethylamine. Once synthesized and characterized, their binding modes with MUC1 were studied by molecular docking analysis using Aruglab 4.0.1 and QSAR properties were determined using HyperChem. All synthesized compounds were screened for in vitro anti-breast cancer activity against MDA-MB-231 breast adenocarcinoma cell lines by Trypan-blue cell viability assay and MTT methods. Compounds 1, 3b, 3d, 3e, 3i and 3f showed good anti-breast cancer activity. Since 1 and 3d exhibited high potent activity against MDA-MB-231 cell lines, they show could be effective mucin onco protein inhibitors.

Full text of DOI:10.1016/j.bmcl.2014.07.058

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, in silico and in vitro studies of novel
4-methylthiazole-5-carboxylic acid derivatives as potent
anti-cancer agents
Ravendra Babu Kilaru a,d, Koteswara Rao Valasani a,e, Nanda Kumar Yellapu b, Hari Prasad Osuru c,
Chandra Sekhar Kuruva a, Bhaskar Matcha b, Naga Raju Chamarthi a,
⇑
a Department of Chemistry, Sri Venkateswara University, Tirupati 517502, India
b Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati 517502, India
c Department of Pathology, University of Virginia, Charlottesville, VA, USA
d Mylan Laboratories Ltd, CRD, Anrich Industrial Estate, Bollaram, Hyderabad 502325, India
e Department of Pharmacology & Toxicology and Higuchi Bioscience Center, School of Pharmacy, University of Kansas, Lawrence, KS 66047, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Since inhibitors of mucin onco proteins are potential targets for breast cancer therapy, a series of novel
4-methylthiazole-5-carboxylic acid (1) derivatives 3a–k were synthesized by the reaction of 1 with SOCl2
followed by different bases/alcohols in the presence of triethylamine. Once synthesized and character-
ized, their binding modes with MUC1 were studied by molecular docking analysis using Aruglab 4.0.1
and QSAR properties were determined using HyperChem. All synthesized compounds were screened
for in vitro anti-breast cancer activity against MDA-MB-231 breast adenocarcinoma cell lines by
Trypan-blue cell viability assay and MTT methods. Compounds 1, 3b, 3d, 3e, 3i and 3f showed good
anti-breast cancer activity. Since 1 and 3d exhibited high potent activity against MDA-MB-231 cell lines,
they show could be effective mucin onco protein inhibitors.
Received 9 March 2014
Revised 7 July 2014
Accepted 21 July 2014
Available online xxxx
Keywords:
Breast cancer
Mucin
Molecular docking
QSAR
Ó 2014 Elsevier Ltd. All rights reserved.
4-methylthiazole-5-carboxylic acid
derivatives
It is almost counter-intuitive to believe that the complexity of a
disease can be made even more complicated by the results of scien-
tific research. Breast cancer is the most common malignancy in
women and it accounts for nearly fifty per cent of cancer deaths
in women. Like all chronic diseases breast cancer also poses a series
of threats and difficulties, which may further lead to the develop-
ment of mental problems in the patients. The incidence of breast
cancer has been increasing steadily from one out of twenty in
1960 to one out of eight in women now.1–4 Mammography is the
most effective tool for screening and early detection of breast
cancer in women.5 Estrogens play crucial roles in breast cancer
development and growth, and estrogen-stimulated growth in
tumor cells requires estrogen receptors (ERs).5–8 About two-thirds
of human breast tumors reveal higher levels of ERs than normal
breast tissues.9 Inhibitions of proliferative pathways were consid-
ered an effective strategy to fight cancer and nowadays much atten-
tion has been paid to the discovery and development of new and
more selective anti-cancer drugs.9,10 A number of drug probables
are preceded to clinical trials and few of them are in clinical use.
It is a challenging target for synthetic chemists because of the com-
plex structures of drug problems and handling of toxic starting
materials and reagents. Under these circumstances, we aimed at
the structural modification of 4-methylthiazole-5-carboxylic acid
(1) to synthesize novel, safe and effective derivatives that can rep-
resent a promising pathway in search of new anti-cancer agents.
The present study is to predict and evaluate the efficacy of mol-
ecules against MUC1 onco protein, a member of mucin family.
Mucins are the predominantly glycoproteins and they act as phys-
ical barriers and protect the apical borders of epithelial cells in
adverse conditions. Mucins are largely unrecognized as effectors
of carcinogenesis and intimately involved in breast malignancy.
It also has been reported that mucins are predominantly over
expressed in various human malignancies in addition to breast
malignancy and their role in signaling cell growth and survival.
All of the mucin family proteins contain the tandem repeats of
proline, threonine and serine residues which are called PTS
domains and these domains are involved in the glycosylation pro-
cess. MUC1 is the heavily glycosylated high molecular weight mem-
brane protein comprising more than 50% of carbohydrate moiety
⇑
Corresponding author. Tel.: +91 9703193375.
E-mail address: rajuchamarthi10@gmail.com (N.R. Chamarthi).
http://dx.doi.org/10.1016/j.bmcl.2014.07.058
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kilaru, R. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.058

2
R. B. Kilaru et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
and can be expressed by various epithelial cells. It is overexpressed
in the entire cell membranes of carcinoma cells and allows them to
116.9–117.9 ppm is assigned to CN. 13C chemical shift in the region
169.5–172.9 ppm is attributed to C@O of the amide group (see
Schemes 1 and 2, Table 1).
interact with restricted receptors.11 They contain large
a
sub unit
and smaller b unit. The N-terminal region of
a
sub unit is fully
The models were built for ligand molecules and molecular
dynamics simulations were observed for a period of 10 ps. The
total energy graphs of dynamics simulations showed that
the confirmations were stabilized in the 10 ps dynamics run. The
stabilized conformations of the ligands were saved and a QSAR
study was carried out and their molecular descriptors were studied
in HyperChem software tools.23–25 All the ligand molecules were
filtered with Lipinski filters. The drug likeness of the molecules
was predicted from their molecular descriptors and Lipinski
data.23–25 Among all the molecules 3g is showing a molecular
weight of 518 Daltans which should be below 500 Daltans for
any molecule to behave as a drug (Table 2). Hence 3g may be anti-
genic to the host system. The remaining molecules are showing
their descriptors in optimal range indicating their potential to
behave as drugs.
The CASTp predicted binding site of MUC1 contain the amino
acid residues Pro 1061, Tyr 1066, Gln 1070, Arg 1071, Ser 1074,
Leu 1089, Ser 1090, Asn 1091 and Ile 1092. The molecular docking
between the binding domain of mucin and stabilized conforma-
tions of the molecules showed that the lowest docking energy of
ꢀ8.882 Kcal/mol was found with 3d with the formation of two
hydrogen bonds and highest docking energy of ꢀ4.387 Kcal/mol
was found with 3j with no hydrogen bond formation. Compounds
3c & 3g are not showing any docking energy, but forming single
hydrogen bond which indicates the existence of weak interaction
(Table 3). These docking results could explain that (except 3c & 3g)
exposed to cell surface and contains variable number of repeats
containing PTS domains. Glycosylation of these repeats is altered
in human carcinomas which in turn play a role in the immunosur-
veillance of cancer.12,13 This N-terminal region is anchored to cell
membrane through C-terminal region and blocks the cell–cell and
cell–extracellular matrix interactions and when it is released the
C-terminal region acts as a putative receptor which is engaged in
signaling path ways related to tumor progression.14
Hence, mucins are approved as therapeutic targets and adverse
prognosis markers. In addition, Food and Drug Administration
(FDA) approved MUC1 as a serum biomarker for breast cancer
and targeting MUC1 is the ideal choice of controlling breast can-
cer.15 Inhibitors of mucins function can become the promising
agents to control and manage the breast cancerous condition.14
The derivatives (3a–h) of 4-methylthiazole-5-carboxylic acid
(1) were prepared by the reaction of 4-methylthiazole-5-carbox-
ylic acid (1) with thionyl chloride in the presence of catalytic
amount of N,N-dimethyl formamide to form corresponding acid
chloride 2 and its further reaction with substituted benzyl amines,
aniline, 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole and bioactive
amines like cytosine, 5-fluoro-cytosine and alcohols in the pres-
ence of triehyl amine in toluene afforded the corresponding deriv-
atives 3a, 3b, 3c, 3d, 3f, 3g, 3i, 3j, and 3e, 3h (Scheme 1 & Table 1).
Substituted benzyl amines, 6-fluoro-3-(piperidin-4-yl)benzo[d]
isoxazole16 and aniline directly reacted with 2 in the presence of
triethylamine as a base in toluene. But 2 did not react with cytosine
and 5-fluoro-cytosine in the presence of triehylamine; hence they
were prepared through silylated cytosine17 and 5-fluoro-cytosine17
in the presence of hexamethyldisilazane in toluene using catalytic
amount of methane sulphonic acid and triethyl amine, to get 3i and
3j. Reaction of 4 with isobutyl bromide in the presence of K2CO3
and N-methyl-2-pyrrolidone produced 5 and it is further hydro-
lyzed to afford 3k (Scheme 2).18
O
O
S
d
O
S
HO
O
O
N
N
4
5
e
Characteristic IR stretching absorptions were observed in the
regions 1245–1262 (O@CAN), 1530–1558 (NAHaliph), 1685–1692
(C@O) for aliphatic and 1648–1655 for aromatic and 1661–1671
for piperazines, 2228–2232 (CN) cmꢀ1, respectively.19,20 In the 1H
NMR spectra of compounds 3a–h, NH proton chemical shift
appeared in the region of 8.91–8.99 ppm21 but this signal is not
observed for 3e, 3g, 3h, 3i and 3j indicating the amide group for-
mation. In 13C NMR spectra of compounds 3a–k, chemical shifts
were observed in the expected regions.22 The chemical shift at
O
S
N
OH
O
3k
Scheme 2. Synthetic route for the preparation of 2-(4-isobutoxyphenyl)-4-meth-
ylthiazole-5-carboxylic acid. Reagents and conditions: (d) iso butyl bromide, K2CO3,
N-methyl-2-pyrrolidone, (e) 5% NaOH, acetone, 5% HCl.
H2N
R
O
O
N
S
N
OH
S
N
c
a
Cl
O
N
O
O
S
O
NC
NC
O
2
N
1
NC
3i-j
a, b
O
S
N
R
O
NC
3a-h
Scheme 1. Protocol for the synthesis of 4-methylthiazole-5-carboxylic acid derivatives. Reagents and conditions: (a) SOCl2, toluene, DMF, 90–95 °C. (b) R-H, TEA, toluene, 70–
75 °C. (c) TEA, N-(trimethylsilyl)-2-((trimethylsilyl)oxy)-1,2-dihydropyrimidin-4-amine, 55–60 °C, H2O.
Please cite this article in press as: Kilaru, R. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.058

R. B. Kilaru et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Table 3
Protocol for the synthesis of 4-methylthiazole-5-carboxylic acid derivatives
Docking energies and interacting atoms of the lead molecules with amino acid
residues of binding domain of mucin protein
Entry
R
Time
2 h
Yield (%)
85
Mp (°C)
Molecule
Docking
Energy
No. of
Hydrogen bond information Length(Ao)
Hydrogen Atom of molecule & Residue involved in
3a
142–149
N
H
(Kcal/mol)
bonds
5
H- bond formation
-8.307
2.966 Ao (O &Arg 1071)
1
3b
3c
2.5 h
3 h
84
80
167–169
172–174
N
2.659 Ao (N &Gln 1070)
2.636 Ao (N & Tyr 1066)
2.904 Ao (O & Tyr 1066)
2.473 Ao (N & Ile 1092)
2.361 Ao (O &Arg 1071)
2.164 Ao (O &Gln 1070)
2.746 Ao (O & Tyr 1066)
2.757 Ao (N &Ser 1074)
2.466 Ao (N &Leu 1089)
2.058 Ao (N &Leu 1089)
2.313 Ao (N &Leu 1089)
2.766 Ao (O & Tyr 1066)
2.691 Ao (N &Gln 1070)
2.618 Ao (N & Ile 1092)
2.899 Ao (O &Gln 1070)
2.193 Ao (O &Gln 1070)
2.426 Ao (N &Leu 1089)
2.908 Ao (O & Tyr 1066)
2.942 Ao (O &Arg 1071)
2.886 Ao (O &Gln 1070)
2.953 Ao (O & Tyr 1066)
2.999 Ao (O &Arg 1071)
2.888 Ao (O &Arg 1071)
2.504 Ao (O &Arg 1071)
2.883 Ao (N &Leu 1089)
--------
H
F
N
H
F
N
H
3d
3e
1.5 h
88
95
139–141
148–150
0
4
3a
F
20 min
O
NH
3f
4 h
92
85
297–299
164–166
-7.190
0
1
1
2
3b
3c
3d
N
O
-8.082
N
3g
3.5 h
F
3h
3i
3j
O–Me
H
F
10 min
5 h
5 h
98
89
91
160–162
238–240
282–284
-7.525
-6.850
2
3
3e
3f
all other molecules are having inhibitory activity against mucin
protein. Hence, they may be considered as anti-inflammatory
agents against mucin protein. The molecular interaction of all mol-
ecules with CASTp predicted domain of MUC1 protein is shown in
Figure 1.
The anti-proliferative activity of newly synthesized 4-methylthi-
azole-5-carboxylic acid derivatives (3a–k) against MDA-MB-231
breast cancer cells was examined by Trypan blue and MTT assays.
Compounds 1, 3b, 3d, 3e, 3f and 3i exhibited more potent inhibition
against MDA-MB-231 cell lines when compared to others. The anti-
proliferative activities of test compounds against MDA-MB-231
cells are shown in Figures 2 and 3 and compared the data with the
standard Celecoxib.
0
1
3
3g
3h
-5.336
-7.704
4
3i
-4.387
-5.826
0
2
3j
2.752 Ao (N &Ser 1074)
2.901 Ao (O &Ser 1074)
3k
Table 2
Molecular descriptors of test molecules (1 and 3a–k)
Property
1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
Molecular Weight 316.00 382.00 423.00 423.00 423.00 372.00 391.00 518.00 330.00 470.00 427.00 319.00
(Daltans)
Hydrogen Bond
Donors
1
3
0
2
1
4
1
4
1
4
0
4
3
6
0
5
0
4
3
6
2
7
0
4
Hydrogen Bond
Acceptors
LogP
1.35
2.2
2.90
2.86
2.86
0.00
4.569
66.61
2.70
1.63
1.59
0.94
3.08
Molar Refractivity 90.01 115.87 117.737 117.737 117.737 66.93
(Ao3
Surface area (Ao2
Volume (Ao3
146.99 89.689 121.819 114.27 88.155
)
)
503.53 385.68 631.82 653.36 656.77 689.11 723.26 705.31 553.12 640.14 557.51 572.09
997.85 1102.02 1308.30 1316.24 1313.43 1152.38 1317.57 1535.25 1051.09 1388.47 1239.47 1037.02
)
Hydration energy -12.15
-4.34
-3.99
-4.59
-6.02
-6.96
-18.90
-7.80
-7.97
-15.26
-9.93
-3.06
(K.cal/mol)
Polarizability (Ao3
Gradient energy
(K.cal/molAo)
Total energy
)
36.20
0.096
46.66
0.087
48.89
0.099
48.89
0.097
48.89
0.099
40.47
0.093
45.99
0.099
58.51
0.099
38.03
0.099
50.55
0.099
46.73
0.099
36.68
0.089
97.779 217.771 119.001 134.196 125.194 85.192 114.316 166.611 101.002 167.109 123.368 99.653
(K.cal/mol)
Please cite this article in press as: Kilaru, R. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.058

4
R. B. Kilaru et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Image showing the molecular interaction of compounds 1, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j and 3k with mucin. Compounds are in CPK colors and protein in indigo
color. Red colored lines indicate the hydrogen bonds between the drug molecules and mucin protein.
Figure 2. Cytotoxicity of test compounds against MDA-MB-231 cells by Trypan blue assay. Cytotoxicity of test compounds against MDA-MB-231 cells by Trypan blue assay at
10
lg/mL (Blue) and 100 lg/mL (Red) concentrations, respectively. C = control (DMSO), S = standard (Celecoxib), results mentioned as mean standard deviation for % of cell
viability. P-Value <0.001.
In conclusion, the molecular descriptors from QSAR study,
docking energies and in vitro assays on MDA-MB-231 adeno carci-
noma breast cancer cell lines indicate that the molecules 1, 3a, 3b,
3c, 3d, 3e, 3j and 3k can be used as anti-cancer agents against
mucin onco protein. The effectiveness from in vitro assays can form
the basis to proceed with the molecules to the next phase of
clinical trials to test their in vivo efficacy. The compounds 1, 3b,
3d, 3e, 3f and 3i exhibited higher anti-proliferative activity when
Please cite this article in press as: Kilaru, R. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.058

R. B. Kilaru et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Figure 3. Anti-proliferative activity of test compounds against MDA-MB-231 cells by MTT Assay. Anti-proliferative activity of test compounds against MDA-MB-231 cells by
MTT Assay. C = control (DMSO), S = standard (Celecoxib), results mentioned as mean standard deviation for OD values at 570 nm, P-Value <0.001.
compared with other derivatives. These results pave the way for
future design and development of adeno carcinoma breast cancer
compounds and also more potent drugs for cancer. The compounds
1 and 3d can be considered as lead molecules which are to be
screened in vivo to assess their efficacy.
and volume of numerous binding cavities from high resolution
structures where small molecules can be accommodated.
Docking analysis: Individual dockings were carried out between
the binding site of mucin and all the molecules using Arguslab
4.0.1 free software developed by Mark A. Thompson, Planaria
Software LLC, Seattle, WA (http://www.arguslab.com/down-
loads.htm).32,33 After docking process, the docking poses with best
energies were saved and analyzed for the reactivity of all ligand
molecules with binding site of mucin.
Pharmacology: Cell lines and culturing. The MDA-MB-231 human
breast adenocarcinoma cells were routinely maintained as mono-
layer in DMEM medium supplemented with 2 mM/L glutamine
(Himedia), 10% fetal bovine serum (FBS, Himedia) and 10 lg/mL
ciprofloxacin in humidified incubator (Binder) containing 5% CO2,
General procedure for the synthesis of title compounds: A solution
of 2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic
acid (1) (1.5 mmol), thionyl chloride (1.5 mmol) and catalytic
amount of dimethyl formamide in toluene (10 mL) was stirred at
90–95 °C for 3 h until clear solution was formed and formation of
acid chloride was determined by TLC. The reaction mixture was
cooled to 70 °C then slowly added phenylmethanamine (1.5 mmol)
was added slowly in the presence of Et3N and stirred for 2 h at
75 °C. Progress of reaction was monitored by TLC (methylene
dichloride/methanol, 9:1 v/v). The solid separated was filtered
off, washed with toluene (10 mL) followed by washed with ice-cold
water (10 mL) and then recrystallized from 2-propanol to afford
pure product. This general synthetic protocol was followed for
the preparation of 3a–h by reacting 4-methylthiazole-5-carboxylic
acid chloride (2) with various amines and alcohols in the presence
of Et3N in toluene.
Molecular dynamics and QSAR study of ligand molecules: The
models were constructed in HyperChem software tool and optimi-
zation was done.26,27 A molecular dynamics study was carried out
in AMBER 99 force field for a period of 10 pico seconds (ps) at 300 K
and the stabilized confirmations and their energy values were
saved. QSAR study was carried out to know the molecular descrip-
tors of the molecules and their reactivity against mucin protein.
Preparation of MUC1 protein: The X-ray crystallographic struc-
ture of MUC1 (PDB ID: 2ACM) was retrieved from Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do).28,29 Except A-chain all
other chains and water molecules were removed, hydrogen atoms
were added and energy minimization was done to get the stable
confirmation of the protein.
95% air at 37 °C.
Trypan blue assay: The MDA-MB-231 cells were seeded at
2 ꢁ 105 cells per well in 6-well tissue culture plates in DMEM
media supplemented with 10% FBS, incubated for 24 h at 37 °C in
CO2 incubator for cell viability assay by Trypan blue dye exclu-
sion.34 Later the medium was replaced with fresh medium contain-
ing various concentrations of synthesized compounds and 0.1% of
DMSO (served as solvent control), incubated for 24–48 h at 37 °C.
Celecoxib, a potent inhibitor of MDA MB 231 cell lines was used
as Standard control to evaluate the potency of the test com-
pounds.35 To assess the cell viability after incubation aliquots from
both floating and trypsinized adherent cells were mixed with a
0.4% trypan blue solution in 1:1 (v/v) and loaded on to a haemocy-
tometer. The cells were observed and counted under light micro-
scope, live cells are clear and the dead cells are blue in color. The
concentration of the compound that inhibited cell growth by 50%
(IC50) was determined from cell survival plots. Data are expressed
as mean SE (n = 3) percentage of viable cells per sample was
calculated by;
Viable cellsð%Þ ¼ ½ðTotal cells ꢀ Dead cellsÞ=Total cellsꢂ ꢁ 100%:
Prediction of binding site on MUC1: The binding site of MUC1
protein was predicted from CASTp server30,31 (Computed Atlas of
Surface Topography of proteins) which predicts the binding
regions based on the pocket algorithm of the alpha shape theory.
This algorithm facilitates the accurate computation of surface area
MTT assay: Cell proliferation was assayed by MTT (3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay
as described by Mosmann.36 The exponentially growing MDA-
MB-231 cells were collected and resuspended in fresh culture
Please cite this article in press as: Kilaru, R. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.058

6
R. B. Kilaru et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
8. Gunnarsson, C.; Ahnstrom, M.; Kirschner, K.; Olsson, B.; Nordenskjold, B.;
Rutqvist, L. E.; Skoog, L.; Stal, O. Oncogene 2003, 22, 34.
9. Hong-Goka, B. C.; Chang, F. L. Neurosci. Lett. 2004, 360, 113.
10. Rao, V. K.; Reddy, S. S.; Krishna, B. S.; Reddy, C. S.; Reddy, N. P.; Reddy, T. C. M.;
Raju, C. N.; Ghosh, S. K. Lett. Drug Des. Discov. 2011, 8, 59.
11. Raina, D.; Ahmad, R.; Joshi, M. D.; Yin, L.; Wu, Z.; Kawano, T.; Vasir, B.; Avigan,
D.; Kharbanda, S.; Kufe, D. Cancer Res. 2009, 69, 5133.
12. Finn, O. J. J. Immunol. 2008, 181, 1589.
13. Ichige, K.; Perey, L.; Vogel, C. A.; Buchegger, F.; Kufe, D. Clin. Cancer Res. 1995, 1,
565.
14. Kufe, D. W. Cancer Biol. Ther. 2008, 7, 81.
15. Albrecht, H.; Carraway, K. L., 3rd Cancer Biother. Radiopharm. 2011, 26, 261.
16. Priya, B. S.; Basappa; Swamy, S. N.; Rangappa, K. S. Bioorg. Med. Chem. 2005, 13,
2623.
17. Goodyear, M. D.; Hill, M. L.; West, J. P.; Whitehead, A. J. Tetrahedron Lett. 2005,
46, 8535.
medium with 10% FBS. Before the treatment with synthesized com-
pounds and the standard (Celecoxib), cells were washed with PBS
and fresh medium was added and incubated in 96-well plates in
the presence or absence of various concentrations of synthesized
compounds for 24 h in a final volume of 100
treatment, 20 L of MTT (5 mg/mL in PBS) was added to each well
and incubated for an additional 4 h at 37 °C. The purple-blue forma-
zan precipitate was dissolved in 100 L of DMSO and the optical
lL. At the end of the
l
l
density was measured at 570 nm on Bio-Rad, micro titer plate
reader. An increase in number of living cells results in an increase
in the total metabolic activity in the sample. This increase directly
correlates to the amount of purple formazan crystals formed. Data
are expressed as mean SD, (n = 3).
18. Hasegawa, M. Heterocycles 1998, 47, 857.
19. Rao, V. K.; Babu, B. H.; Babu, K. R.; Srinivasulu, D.; Raju, C. N. Synth. Commun.
2012, 42, 3368.
20. Rao, V. K.; Reddy, S. S.; Krishna, B. S.; Naidu, K. R. M.; Raju, C. N.; Ghosh, S. K.
Green Chem. Lett. Rev. 2010, 3, 217.
Acknowledgments
21. Rao, V. K.; Reddy, S. S.; Peer, E. D.; Rao, A. J.; Raju, C. N. Chin. J. Chem. 2009, 27,
2379.
22. Koteswara Rao, V.; Janardhan Rao, A.; Subba Reddy, S.; Naga Raju, C.;
Visweswara Rao, P.; Ghosh, S. K. Eur. J. Med. Chem. 2010, 45, 203.
23. Valasani, K. R.; Hu, G.; Chaney, M. O.; Yan, S. S. Chem. Biol. Drug Des. 2013, 81,
238.
The authors thank the management of Mylan Laboratories
Limited, Hyderabad. The authors are also thankful to BRNS for
the financial assistance.
Supplementary data
24. Valasani, K. R.; Vangavaragu, J. R.; Day, V. W.; Yan, S. S. J. Chem. Inform. Model.
2014, 54, 902.
25. Valasani, K. R.; Chaney, M. O.; Day, V. W.; Shidu Yan, S. J. Chem. Inform. Model.
2013, 53, 2033.
26. Brzezinska, E. Acta Pol. Pharm. 2003, 60, 3.
27. Kaminskaia, N. V.; Ullmann, G. M.; Fulton, D. B.; Kostic, N. M. Inorg. Chem. 2000,
39, 5004.
28. Berman, H. M.; Bhat, T. N.; Bourne, P. E.; Feng, Z.; Gilliland, G.; Weissig, H.;
Westbrook, J. Nat. Struct. Biol. 2000, 7, 957.
29. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
30. Dundas, J.; Ouyang, Z.; Tseng, J.; Binkowski, A.; Turpaz, Y.; Liang, J. Nucleic Acids
Res. 2006, 34, W116.
31. Binkowski, T. A.; Naghibzadeh, S.; Liang, J. Nucleic Acids Res. 2003, 31, 3352.
32. Joy, S.; Nair, P. S.; Hariharan, R.; Pillai, M. R. In Silico Biol. 2006, 6, 601.
33. Naz, A.; Bano, K.; Bano, F.; Ghafoor, N. A.; Akhtar, N. Pak. J. Pharm. Sci. 2009, 22,
78.
Supplementary data (spectral data for all the title compounds
and Tables 4 and 5, and 1H and 13CNMR spectra of the title
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.058.
References and notes
1. Albrand, G.; Terret, C. Drugs Aging 2008, 25, 35.
2. Guenel, P.; Raskmark, P.; Andersen, J. B.; Lynge, E. Br. J. Ind. Med. 1993, 50, 758.
3. Sont, W. N.; Zielinski, J. M.; Ashmore, J. P.; Jiang, H.; Krewski, D.; Fair, M. E.;
Band, P. R.; Letourneau, E. G. Am. J. Epidemiol. 2001, 153, 309.
4. Weigel, S.; Batzler, W. U.; Decker, T.; Hense, H. W.; Heindel, W. RoFo 2009, 181,
1144.
34. Kumi-Diaka, J.; Nguyen, V.; Butler, A. Biol. Cell 1999, 91, 515.
35. Wang, L.; Liu, L. H.; Shan, B. E.; Zhang, C.; Sang, M. X.; Li, J. Chin. J. Cancer 2009,
28, 569.
5. Kubista, E. Wien. Med. Wochenschr. 2001, 151, 548.
6. Giretti, M. S.; Fu, X. D.; De Rosa, G.; Sarotto, I.; Baldacci, C.; Garibaldi, S.;
Mannella, P.; Biglia, N.; Sismondi, P.; Genazzani, A. R.; Simoncini, T. PloS One
2008, 3, e2238.
36. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
7. Baumann, C. K.; Castiglione-Gertsch, M. Drugs 2007, 67, 2335.
Please cite this article in press as: Kilaru, R. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.058