Thiazolyl N-benzyl-substituted acetamide derivatives: Synthesis, Src kinase inhibitory and anticancer activities

Article abstract of DOI:10.1016/j.ejmech.2011.07.050

KX2-391 (KX-01/Kinex Pharmaceuticals), N-benzyl-2-(5-(4-(2- morpholinoethoxy)phenyl)pyridin-2-yl)acetamide, is a highly selective Src substrate binding site inhibitor. To understand better the role of pyridine ring and N-benzylsubstitution in KX2-391 and

Full text of DOI:10.1016/j.ejmech.2011.07.050

European Journal of Medicinal Chemistry 46 (2011) 4853e4858
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Thiazolyl N-benzyl-substituted acetamide derivatives: Synthesis, Src kinase
inhibitory and anticancer activities
Asal Fallah-Tafti a, Alireza Foroumadi a, Rakesh Tiwari b, Amir Nasrolahi Shirazi b, David G. Hangauer c,
,
a,
**
Yahao Bu c, Tahmineh Akbarzadeh a, Keykavous Parang b , Abbas Shafiee
*
a Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, 41 Lower College Road, The University of Rhode Island, Kingston, RI 02881, USA
c Kinex Pharmaceuticals, Buffalo, NY 14203, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
KX2-391 (KX-01/Kinex Pharmaceuticals), N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)
acetamide, is a highly selective Src substrate binding site inhibitor. To understand better the role of
pyridine ring and N-benzylsubstitution in KX2-391 and establish the structureeactivity relationship,
a number of N-benzyl substituted (((2-morpholinoethoxy)phenyl)thiazol-4-yl)acetamide derivatives
containing thiazole instead of pyridine were synthesized and evaluated for Src kinase inhibitory activ-
ities. The unsubstituted N-benzyl derivative (8a) showed the inhibition of c-Src kinase with GI50 values of
Received 10 May 2011
Received in revised form
12 June 2011
Accepted 14 July 2011
Available online 4 August 2011
1.34
mM and 2.30 mM in NIH3T3/c-Src527F and SYF/c-Src527F cells, respectively. All the synthesized
Keywords:
Anticancer
Cell proliferation
KX-2
compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), breast
carcinoma (BT-20), and leukemia (CCRF-CEM) cells. 4-Fluorobenzylthiazolyl derivative 8b exhibited 64
e71% inhibition in the cell proliferation of BT-20 and CCRF cells at concentration of 50 mM.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Inhibitor
Src kinase
Thiazole
1. Introduction
binding of ATP are potent, but often lack selectivity in a panel of
isolated kinase assays [8e10]. In contrary, the substrate binding site
sequences of PTKs are less conserved, which results in improved
selectivity and less toxicity of designed substrate binding site
inhibitors when compared with those of ATP mimics targeting ATP
binding site.
KX2-391 (KX-01/Kinex Pharmaceuticals) (Fig. 1) is a novel class
and highly selective non-ATP Src kinase inhibitor that targets the
substrate binding site of Src, has tubulin polymerization inhibition
as a second mechanism of action, and is currently in Phase-2 testing
for solid tumors [11]. KX2-391 was found to inhibit certain
leukemia cells that are resistant to current commercially available
drugs, such as those derived from chronic leukemia cells with the
T3151 mutation. In pre-clinical animal models of cancer, orally
administered KX2-391 was shown to inhibit primary tumor growth
and to suppress metastasis. In combination with certain chemo-
therapeutic agents, KX2-391 was synergistic, thereby, offering the
potential to prescribe lower doses of some current cytotoxic agents
that have undesirable side effects.
Src is the prototype and most widely studied member of one of
the largest family of non-receptor protein tyrosine kinases (PTKs),
known as the Src family kinases (SFKs) [1], which are key regulators
of cellular proliferation, survival, motility and invasiveness [2e4].
Src was first discovered in viral sarcoma and thus was pronounced
as “sarc”. Src offers a promising molecular target for anticancer
therapy, as increased Src activity upregulates a number of signaling
cascades associated with tumor development and progression
leading to increased cell growth, migration and invasion. Moreover,
Src has been shown to play a critical role in other pathologic
disorders, such as myocardial infarction [5], stroke [6], osteoporosis
[7], and neurodegeneration [1].
In the last two decades, synthesis of Src kinase inhibitors has
been based on designing ATP binding site inhibitors and substrate
binding site inhibitors. Despite of the large variety in PTKs struc-
tural organization, their ATP binding site is mostly conserved. The
ATP binding site competitive inhibitors of Src that mimic the
In addition, previous structural studies [12e14] have proven that
occurrence of heterocyclic scaffolds such as thiazole may result in
generating effective kinase inhibitors, including potent Src kinase
inhibitors. Dasatinib (Fig. 1) with amino-thiazole moiety, is one of
* Corresponding author. Tel.: þ1 401 874 4471; fax: þ1 401 874 5787.
** Corresponding author. Tel.: þ98 21 66954708; fax: þ98 21 66461178.
E-mail addresses: kparang@uri.edu (K. Parang), ashafiee@ams.ac.ir (A. Shafiee).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.050

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A. Fallah-Tafti et al. / European Journal of Medicinal Chemistry 46 (2011) 4853e4858
chloride (EDCI) in acetonitrile afforded desired thiazolyl benzyl
acetamides 8aee.
2.2. Biological activity
Fig. 1. KX2-391 (KX-01) a potent and highly selective non-ATP Src kinase inhibitor;
Dasatinib a potent pan-Src kinase inhibitors.
2.2.1. Src kinase inhibitory activity
The compounds were evaluated in engineered Src driven cell
growth assays in NIH3T3/c-Src527Fand SYF/c-Src527F cells. NIH3T3/
c-Src527F cell line has constitutively active human Src driving
growth. SYF/c-Src527F has Src, Yes, and Fyn mouse knockouts with c-
Src527F added back. The results of Src kinase inhibitory activity of
compounds 8aee are shown in Table 1 and Fig. 2. Compound 8a with
the potent pan-Src kinase inhibitors, which has been approved by
FDA for the treatment of Gleevec-resistant CML [15,16].
Since the crystal structure of substrate binding site with Src
inhibitors is not available yet, the designing strategy for discovering
selective Src substrate binding site inhibitors has been mostly
based on screening rather than rational designing [17]. Considering
these facts, and in continuation of our efforts to design small
molecules as Src kinase inhibitor or anticancer agents [18], we
herein report the synthesis a series of substrate binding site
inhibitors by substituting pyridine ring in KX2-391 molecule with
a thiazole group and introducing substitutions on the benzyl ring.
Src kinase inhibitory and anticancer activities of the compounds
were evaluated in cell-based assays.
no substitution on N-benzyl group showed GI50 values of 1.34
mM and
2.30 M in NIH3T3/c-Src527F and SYF/c-Src527F cells, respectively,
m
and was found to be the most potent compound of this series.
Introducing of a fluoro group at position 4 of benzyl group led to
a
slight decrease of inhibitory activity in compound 8b
M) versus 8a. Introducing of a methyl group at
(GI50 ¼ 1.49e2.51
m
position 4 of benzyl group in 8e led to almost 4-fold decrease of
potency in comparison with 8a. Similarly 2-chlorobenzyl and 3,4-
dichlorobenzyl substituted analogs (8c and 8d) showed signifi-
cantly less inhibitory activities when compared to other compounds
(GI50 ¼ 7.93e13.02
mM). The data suggest that incorporation of bulky
groups, such as chlorine and methyl in compounds 8cee with higher
lipophilicity (Log P), results in decreased potency.
2. Results and discussions
2.1. Chemistry
All the tested compounds were significantly less potent than
KX2-391 (KX-01), suggesting that introducing thiazole replacement
of pyridine has led to decreased activity. In other words, pyridine is
possibly a pharmacophore of KX2-391 that has major interactions
with Src substrate binding site. Nevertheless, none of the
compounds were active in vitro assay against Src kinase, which
confirm earlier results that the peptide binding site is not well
formed outside of cells and these compounds indeed inhibit Src in
cellular environment when the substrate binding site is deeper for
binding interactions.
Scheme 1 outlines the procedure for the synthesis of thiazolyl
benzyl acetamides 8aee. Commercially available 4-(2-chloroethyl)
morpholine hydrochloride (1) was reacted with 4-hydroxybenzo
nitrile (2) in presence of K2CO3 in refluxing DMF for 24 h to yield
4-(2-morpholinoethoxy)benzonitrile (3). Subsequent reaction of 3
with ammonium sulfide at room temperature afforded 4-(2-
morpholinoethoxy)benzothioamide 4. Treatment of 4 with ethyl
4-chloroacetoacetate resulted in the formation of thiazolyl deriva-
tive 5, which underwent basic ester hydrolysis to generate acetic
acid derivative 6. Finally, the reaction of 6 with the corresponding
benzylamines 7aee in the presence of 1-hydroxybenzotriazole
(HOBt) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro
2.2.2. Anticancer activities
The effect of the inhibitors at the concentration of 50
mM on the
cell proliferation of human colon adenocarcinoma (HT-29) cancer
Scheme 1. Synthesis of thiazolyl benzyl acetamides 8aee.

A. Fallah-Tafti et al. / European Journal of Medicinal Chemistry 46 (2011) 4853e4858
4855
Table 1
evaluated for Src kinase inhibitory and anticancer activities.
Although the biological results revealed that introducing thiazole
replacement of pyridine in KX2-391 led to decreased activity, the
compounds retained Src kinase inhibitory activities at low micro-
Src kinase inhibitory activity and Log P values of 8aee.
molar range (1.34e13.02
mM) in NIH3T3/c-Src527F and SYF/c-
Src527F cells. Structureeactivity relationship studies revealed
that the presence of a substituent at position 4 of benzyl ring
(4-fluoro-, 3,4-dicholor, or 4-methyl) is critical for maximum
anticancer activity. For example, 4-fluorosubstituted (8b) and 3,4-
dichlorosubsitited (8d) analogs inhibited the cell proliferation of
CCRF by 71% and 69%, respectively (Fig. 3). This study provides
insights and structureeactivity relationships for further optimiza-
tion of this scaffold for generating optimal Src inhibitory and
anticancer activities.
Comp.
R
NIH3T3/c-Src527F
GI50
M)a
SYF/c-Src527F
GI50 (mM)
Log Pb
(
m
8a
8b
8c
8d
8e
KX01
H
4-F
2-Cl
3,4-di-Cl
4-CH3
e
1.34
1.49
7.93
12.32
4.76
0.023
2.30
2.51
13.02
12.50
7.23
3.54
3.70
4.10
4.66
4.03
3.09
4. Experimental protocols
0.039
a
GI50 is determined by using GraphPad Prism 5 software.
Calculated partition coefficient using ChemBioDraw Ultra 11.0.
4.1. Materials and methods
b
All starting materials, reagents, and solvents were purchased
from Merck AG (Germany). The purity of the synthesized
compounds was confirmed by thin layer chromatography (TLC)
using various solvents of different polarities. Merck silica gel 60
F254 plates were applied for analytical TLC. Column chromatog-
raphy was performed on Merck silica gel (70e230 mesh) for puri-
fication of the intermediate and final compounds. Melting points
were determined on a Kofler hot stage apparatus (Vienna, Austria)
and are uncorrected. 1H NMR spectra were recorded using a Bruker
500 MHz spectrometer (Bruker, Rheinstatten, Germany), and
cells that overexpress c-Src [19], breast carcinoma (BT-20), and
leukemia (CCRF-CEM) cells was also evaluated (Fig. 3). In general,
compounds 8a and 8c showed moderate activity against CCRF and
BT-20 compared to doxorubicin (DOX) by inhibiting the cell
proliferation by 30e40%. On the other hand, compounds 8b, 8d,
and 8e inhibited the cell proliferation of CCRF and BT-20 by 61e71%,
and HT-29 by 54e56% (Fig. 3).
Structureeactivity relationship studies suggest that the presence
of substituents at position 4 of benzyl ring (4-fluoro-, 3,4-dicholor, or
4-methyl) is critical for maximum anticancer activity as seen in
compounds 8b, 8c, or 8e. Compound 8b that showed high Src
inhibitory potency against Src was consistently also active against all
cancer cells. On the other hand, poor correlation was observed
between Src kinase inhibitory potency and the inhibition of cell
proliferation in cancer cells for other compounds. For example, while
compound 8a that was one of the most potent Src kinase inhibitors
in this class, showed much weaker anticancer activities versus other
compounds. Thus, some other mechanisms may be involved rather
than Src kinase inhibition in generating anticancer activity.
Compounds 8bee have slightly higher lipophilicity than 8a as
shown in Log P values. As can be seen in Fig. 3, after 72 h
compounds 8bee exhibited significantly higher cell proliferation
inhibitory activity when compared to that of 24 h in colon cancer
cells HT-29. These data suggest that other factors like lipid solu-
bility and cellular uptake may also contribute in anticancer activi-
ties of these compounds.
chemical shifts are expressed as
d (ppm) with tetramethylsilane
(TMS) as an internal standard. Proton coupling patterns are
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). The IR spectra were obtained on a Shimadzu
470 (Shimadzu, Tokyo, Japan) spectrophotometer (potassium
bromide disks). The mass spectra were run on a Finnigan TSQ-70
spectrometer (Finnigan, USA) at 70 eV. Elemental analyses were
carried out on a CHNeO-rapid elemental analyzer (Heraeus GmbH,
Hanau, Germany) for C, H, and N, and the results were within ꢀ0.4%
of the theoretical values.
4.2. Chemistry
4.2.1. 4-(2-Morpholinoethoxy)benzonitrile (3)
A mixture of 4-(2-chloroethyl)morpholine hydrochloride 1
(9.5 g, 0.05 mol), 4-hydroxybenzonitrile 2 (6 g, 0.05 mol) and K2CO3
(17.3 g, 0.13 mol) in anhydrous DMF (150 mL) was refluxed for 24 h.
After cooling the reaction mixture to room temperature, it was
poured in to cold water(100 mL) with crushed ice (100 mL) and the
resulted light brown precipitate was collected and dried to obtain
3. Conclusions
In summary, a number of N-benzyl substituted (((2-morpholi-
noethoxy)phenyl)thiazol-4-yl)acetamide were prepared and
pure 3 in 76% yield. IR (KBr):
n
¼ 2217 (CN). 1H NMR (CDCl3):
d 7.59
Fig. 2. MTT assay and inhibition of Src kinase activity by 8aee in NIH3T3/c-Src527F and SYF/c-Src527F cells.

4856
A. Fallah-Tafti et al. / European Journal of Medicinal Chemistry 46 (2011) 4853e4858
Fig. 3. Inhibition of HT-29, CCRF, and BT-20 cells by compounds 8aee (50 mM). The results are shown as the percentage of the control that has no compound (set at 100%). All the
experiments were performed in triplicate.
(d, J ¼ 9 Hz, 2H, eArH), 6.96 (d, J ¼ 9 Hz, 2H, eArH), 4.16 (t,
J ¼ 11.5 Hz, 2H, eCH2), 3.74 (t, J ¼ 9 Hz, 4H, eCH2), 2.83 (t,
J ¼ 11.5 Hz, 2H, eCH2), 2.58 (t, J ¼ 9 Hz, 4H, eCH2). MS m/z (%): 232
[M]þ (45), 114 (12.5), 100 (100), 70 (25), 56 (48), 42 (36).
eArH), 7.40 (s, 1H, eCHethiazole), 7.04 (d, J ¼ 8.8 Hz, 2H, eArH),
4.15 (t, J ¼ 11.45 Hz, 4H, eCH2), 3.75 (s, 2H, eCH2), 3.58 (t, J ¼ 9 Hz,
4H, eCH2), 2.71 (t, J ¼ 11.45 Hz, 2H, eCH2), 2.50 (t, J ¼ 9 Hz, 4H,
eCH2). MS m/z (%): 348 [M]þ (28), 114 (64), 100 (100), 71 (20), 56
(28), 42 (16).
4.2.2. 4-(2-Morpholinoethoxy)benzothioamide (4)
Ammonium sulfide (23 mL, 0.068 mol) was added slowly to
a stirring mixture of 3 (8 g, 0.034 mol) in DMF (45 mL) at room
temperature. The mixture was stirred at the same temperature
overnight and then was poured in to crushed ice (150 mL). The
yellow precipitate was collected and dried to give pure 4 in 71%
4.2.5. N-Benzyl-2-(2-(4-(2-morpholinoethoxy)phenyl)thiazol-4-yl)
acetamide (8a)
A mixture of acid 6 (0.5 g, 1 mmol), EDCI (0.21 g, 1.1 mmol), and
HOBt (0.15 g, 1 mmol) in dry CH3CN (10 mL) was stirred at room
temperature for 30 min and then treated with the benzyl amine 7a
(0.11 mL, 1 mmol). The mixture was stirred at room temperature for
an additional 24 h. Then the solution was evaporated to dryness in
vacuum. The residue was dissolved in ethyl acetate (20 mL) and
washed sequentially with brine (2 ꢁ 5 mL), 10% aqueous sodium
carbonate (2 ꢁ 5 mL), 10% aqueous citric acid (2 ꢁ 5 mL), and water
(2 ꢁ 5 mL). The organic layer was dried over anhydrous sodium
sulfate. Concentration of the dried extract yielded an oily residue,
which was crystallized (CH2Cl2/n-C6H14) to give pure 8a amide in
yield. IR (KBr):
n
¼ 3370, 3155 (NH2), 1654 (C]S). 1H NMR (CDCl3):
d
7.90 (d, J ¼ 9 Hz, 2H, eArH), 7.58 (br, 1H, eNH2), 7.16 (br, 1H,
eNH2), 6.91 (d, J ¼ 9 Hz, 2H, eArH), 4.16 (t, J ¼ 11.5 Hz, 2H, eCH2),
3.74 (t, J ¼ 9 Hz, 4H, eCH2), 2.83 (t, J ¼ 11 Hz, 2H, eCH2), 2.59 (t,
J ¼ 9 Hz, 4H, eCH2). MS m/z (%): 266 [M]þ (88), 114 (12), 100 (100),
70 (16), 56 (32), 42 (20).
4.2.3. Ethyl 2-(2-(4-(2-morpholinoethoxy)phenyl)thiazol-4-yl)
acetate (5)
27% yield. M.P. 104e108 ꢂC; 1H NMR (CDCl3):
d
7.74 (d, J ¼ 8.9 Hz,
To a mixture of 4 (6 g, 0.022 mol) in ethanol (60 mL), ethyl
4-chloroacetoacetate (3.5 mL, 0.025 mol) and a few drops of
pyridine were added. The reaction mixture was then refluxed for
4 h. After cooling to ambient temperature, the solvent was
evaporated and the oily residue was purified by flash column
chromatography (CH3COOC2H5/n-C6H14) to give pure brownish oil
2H, eArH), 7.41 (br, 1H, eNH), 7.28 (m, 5H, eArH), 7.03 (s, 1H,
eCHethiazole), 6.91 (d, J ¼ 8.9 Hz, 2H, eArH), 4.48 (d, J ¼ 5.5 Hz, 2H,
eCH2), 4.24 (m, 2H, eCH2), 3.81 (m, 4H, eCH2), 3.78 (s, 2H, eCH2),
2.93 (m, 2H, eCH2), 2.71 (m, 4H, eCH2); 13C NMR (CDCl3):
d 39.09,
43.67, 53.93, 57.41, 65.58, 66.49, 114.88, 115.05, 126.42, 127.32,
127.54, 127.92, 128.64, 138.17, 150.40, 160.19, 168.52, 169.04; MS m/z
(%): 437 [M]þ (12), 114 (72), 100 (100). Anal. calcd. for C24H27N3O3S:
C, 65.88; H, 6.22; N, 9.60; found: C, 65.46; H, 5.98; N, 9.79.
of 5 in 54% yield. 1H NMR (CDCl3):
d
7.87 (d, J ¼ 9 Hz, 2H, eArH),
7.46 (s, 1H, eCHethiazole), 7.11 (d, J ¼ 9 Hz, 2H, eArH), 4.11 (m,
4H, eCH2), 3.92 (t, J ¼ 11 Hz, 4H, eCH2), 3.85 (s, 2H, eCH2), 2.82 (t,
J ¼ 11 Hz, 2H, eCH2), 2.58 (t, J ¼ 9 Hz, 4H, eCH2), 1.28 (t, J ¼ 10 Hz,
3H, eCH3). MS m/z (%): 376 [M]þ (8), 121 (100), 105 (72), 77 (36),
57 (24), 43 (28).
4.2.6. N-(4-Fluorobenzyl)-2-(2-(4-(2-morpholinoethoxy)phenyl)
thiazol-4-yl)acetamide (8b)
The same procedure described for 8a was used starting with
4-fluorobenzylamine 7b, Yield 23%. M.P. 148e151 ꢂC; 1H NMR
4.2.4. 2-(2-(4-(2-Morpholinoethoxy)phenyl)thiazol-4-yl)acetic
acid (6)
(CDCl3):
d
7.73 (d, J ¼ 8.8 Hz, 2H, eArH), 7.42 (br, 1H, -NH), 7.23 (m,
2H, eArH), 7.02 (s, 1H, eCHethiazole), 6.97 (t, J ¼ 17.4 Hz, 2H,
eArH), 6.91 (d, J ¼ 8.8 Hz, 2H, eArH), 4.43 (d, J ¼ 5.5 Hz, 2H, eCH2),
4.21 (m, 2H, eCH2), 3.77 (m, 5H, eCH2), 2.89 (m, 2H, eCH2), 2.66 (m,
Sodium hydroxide solution (30 mL, 1 N) was added to a mixture
of 5 (4 g, 0.01 mol) in ethanol (20 mL) and was refluxed overnight.
The mixture was then cooled to ambient temperature and
neutralized with HCl. The remaining solvent was evaporated and
the oily residue was purified by flash column chromatography
4H, eCH2); 13C NMR (CDCl3):
d 39.05, 42.95, 53.99, 57.44, 65.75,
66.65, 114.89, 115.06, 115.36, 115.53, 126.26, 127.85, 129.21, 129.27,
134.02, 150.28, 160.33, 161.08, 168.57, 169.04; MS m/z (%): 455 [M]þ
(32), 114 (76), 100 (100). Anal. calcd. for C24H26FN3O3S: C, 63.28; H,
5.75; N, 9.22; found: C, 63.87; H, 6.02; N, 9.53.
(CH3OH/CH3COOC2H5) to give 6 in 42% yield. IR (KBr):
n
¼ 3431
(OH), 1710 (C]O). 1H NMR (DMSO-d6):
d
7.83 (d, J ¼ 8.8 Hz, 2H,

A. Fallah-Tafti et al. / European Journal of Medicinal Chemistry 46 (2011) 4853e4858
4857
4.2.7. N-(2-Chlorobenzyl)-2-(2-(4-(2-morpholinoethoxy)phenyl)
thiazol-4-yl)acetamide (8c)
(C ꢃ T0) ꢁ 100%orODvalueof thetest well exposuretotest drugꢃ OD
value at time zero/(OD of the control well without drug
treatment ꢃ OD value at time zero) ꢁ 100%. Growth inhibition curves
and GI50 were determined using GraphPad Prism 5 software.
The same procedure described for 8a was used starting with 2-
chlorobenzylamine 7c, Yield 21%. M.P. 114e116 ꢂC; 1H NMR (CDCl3):
d
7.81 (d, J ¼ 9 Hz, 2H, eArH), 7.65 (br, 1H, -NH), 7.37 (m, 1H, eArH),
7.33 (m, 1H, eArH), 7.19 (m, 2H, eArH), 7.00 (s, 1H, eCHethiazole),
6.93 (d, J ¼ 8.8 Hz, 2H, eArH), 4.56 (d, J ¼ 6 Hz, 2H, eCH2), 4.17 (t,
J ¼ 11 Hz, 2H, eCH2), 3.75 (m, 5H, eCH2), 2.84 (t, J ¼ 10 Hz, 2H,
4.3.2. Cell culture and cell proliferation assay
4.3.2.1. Cell culture. Human colon adenocarcinoma HT-29 (ATCC
no. HTB-38), breast carcinoma cells BT-20 (ATCC no. HTB-19),
leukemia CCRF-CEM (ATCC no. CCL-119) were obtained from
American Type Culture Collection. Cells were grown on 75 cm2 cell
culture flasks with RPMI-16 medium for leukemia and EMEM
medium for colon adenocarcinoma and breast carcinoma and
supplemented with 10% fetal bovine serum (FBS), and 1% pen-
icillinestreptomycin solution (10,000 units of penicillin and 10 mg
of streptomycin in 0.9% NaCl) in a humidified atmosphere of 5% CO2,
95% air at 37 ꢂC.
eCH2), 2.60 (m, 4H, eCH2); 13C NMR (CDCl3):
d 39.03, 41.47, 54.00,
57.45, 65.76, 66.70, 114.84, 114.97, 126.27, 127.00, 127.90, 128.71,
129.39, 129.83, 133.57, 135.72, 150.26, 160.31, 168.62, 169.03; MS m/
z (%): 473 [Mþ2]þ (8), 471 [M]þ (16), 114 (76),100 (100). Anal. calcd.
for C24H26ClN3O3S: C, 61.07; H, 5.55; N, 8.90; found: C, 60.87; H,
5.89; N, 8.35.
4.2.8. N-(3,4-Dichlorobenzyl)-2-(2-(4-(2-morpholinoethoxy)
phenyl)thiazol-4-yl)acetamide (8d)
The same procedure described for 8a was used starting with 3,4-
4.3.2.2. Cell proliferation assay. Cell proliferation assay of
compounds was evaluated in HT-29, CCRF-CEM, and BT-20 cells,
and was compared with that of doxorubicin (DOX) according to the
previously reported procedure [21]. Cell proliferation assay was
carried out using CellTiter 96 aqueous one solution cell prolifera-
tion assay kit (Promega, USA). Briefly, upon reaching about 75e80%
confluency, 5000 cells/well were plated in 96-well microplate in
dichlorobenzylamine 7d, Yield 33%. M.P. 129e131.5 ꢂC; 1H NMR
(CDCl3):
d
7.76 (d, J ¼ 8.8 Hz, 2H, eArH), 7.52 (br, 1H, -NH), 7.34 (d,
J ¼ 2.6 Hz, 2H, eArH), 7.33 (s, 1H, eArH), 7.10 (d, J ¼ 10 Hz, 1H,
eArH), 7.03 (s, 1H, eCHethiazole), 7.94 (d, J ¼ 8.8 Hz, 2H, eArH),
4.42 (d, J ¼ 5.8 Hz, 2H, eCH2), 4.18 (m, 2H, eCH2), 3.79 (s, 2H, eCH2),
3.75 (m, 4H, eCH2), 2.84 (m, 2H, eCH2), 2.60 (m, 4H, eCH2); 13C
NMR (CDCl3):
d
39.01, 42.46, 53.92, 57.39, 65.62, 66.47, 114.98,
100
50
as the positive control. At the end of the sample exposure period
(24e72 h), 20 L CellTiter 96 aqueous solution was added. The plate
m
L media. After seeding for 24 h, the cells were treated with
115.19, 126.30, 126.88, 127.89, 129.32, 130.54, 131.25, 132.62, 138.68,
150.11, 159.72, 160.30, 168.74, 169.20; MS m/z (%): 507 [Mþ2]þ (8),
505 [M]þ (12),114 (76),100 (100). Anal. calcd. for C24H25Cl2N3O3S: C,
61.43; H, 5.68; N, 8.77; found: C, 61.98; H, 6.02; N, 8.14.
m
M compound in triplicate. Doxorubicin (DOX, 10 M) was used
m
m
was returned to the incubator for 1 h in a humidified atmosphere at
ꢂ
37 C. The absorbance of the formazan product was measured at
4.2.9. N-(4-Methylbenzyl)-2-(2-(4-(2-morpholinoethoxy)phenyl)
thiazol-4-yl)acetamide (8e)
490 nm using microplate reader. The blank control was recorded by
measuring the absorbance at 490 nm with wells containing
medium mixed with CellTiter 96 aqueous solution but no cells.
Results were expressed as the percentage of the control (without
compound set at 100%). The percentage of cell survival was calcu-
lated as OD value of cells treated with test compound ꢃ OD value of
culture medium/(OD value of control cells ꢃ OD value of culture
medium) ꢁ 100%.
The same procedure described for 8a was used starting with 4-
methylbenzylamine 7e, Yield 18%. M.P. 142e147 ꢂC; 1H NMR
(CDCl3):
d
7.74 (d, J ¼ 8.7 Hz, 2H, eArH), 7.35 (br, 1H, -NH), 7.15 (d,
J ¼ 7.9 Hz, 2H, eArH), 7.09 (d, J ¼ 7.9 Hz, 2H, eArH), 7.02 (s, 1H,
eCHethiazole), 6.90 (d, J ¼ 8.75 Hz, 2H, eArH), 4.42 (d, J ¼ 5.45 Hz,
2H, eCH2), 4.17 (t, J ¼ 11.2 Hz, 2H, eCH2), 3.77 (s, 2H, eCH2), 3.75 (t,
J ¼ 9.4 Hz, 4H, eCH2), 2.85 (t, J ¼ 11.2 Hz, 2H, eCH2), 2.61 (m, 4H,
eCH2), 2.32 (s, 1H, eCH3); 13C NMR (CDCl3):
d 21.07, 39.08, 43.40,
Acknowledgments
54.01, 57.44, 65.81, 66.72, 114.83, 114.97, 126.31, 127.53, 127.90,
128.71, 129.27, 135.13, 136.90, 150.40, 160.29, 168.47, 168.97; MS m/z
(%): 451 [M]þ (12), 114 (60), 100 (100). Anal. calcd. for C25H29N3O3S:
C, 66.49; H, 6.47; N, 9.31; found: C, 67.21; H, 6.78; N, 9.03.
This work was supported by grants from the American Cancer
Society Grant # RSG-07-290-01-CDD, National Science Foundation,
Grant Number CHE 0748555, and Research Council of Tehran
University of Medical Sciences and Iran National Science Founda-
tion (INSF).
4.3. Biological assays
4.3.1. MTT assay
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