Design, synthesis, in vitro antiproliferative evaluation, and kinase inhibitory effects of a new series of imidazo[2,1- b ]thiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.03.065

Design and synthesis of a new series of 5,6-diarylimidazo[2,1-b]thiazole derivatives possessing terminal aryl sulfonamide moiety are described. Their in vitro antiproliferative activities against a panel of 57 human cancer cell lines of nine different cancer types were tested at the NCI. Compounds 8a, 8b, 8n, 8q, 8t, and 8u showed the highest mean % inhibition values over the 57 cell line panel at 10 1/4M, and they were further tested in 5-dose testing mode to determine their IC₅₀ values. Among the six compounds, compound 8u possessing terminal para-hydroxybenzenesulfonamido moiety and ethylene linker showed the highest potency. It demonstrated superior potency than Sorafenib against eight different cell lines, and was equipotent to Sorafenib against COLO 205 colon cancer cell line. Its IC₅₀ values over NCI-H460 non-small cell lung cancer cell line and MCF7 breast cancer cell line were 0.845 1/4M and 0.476 1/4M, respectively. Compounds 8a, 8b, 8q, 8t, and 8u showed high selectivity indices towards cancer cells over L132 normal lung cell line. Compound 8u showed potential inhibitory effects over the components of ERK pathway. Its IC₅₀ value over V600E-B-RAF and C-RAF kinases were 39.9 nM and 19.0 nM, respectively.

Full text of DOI:10.1016/j.ejmech.2015.03.065

European Journal of Medicinal Chemistry 95 (2015) 453e463
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, in vitro antiproliferative evaluation, and kinase
inhibitory effects of a new series of imidazo[2,1-b]thiazole derivatives
a
,
b
,
c
,
1
, Mi-Ryeong Kim a , Mohammed I. El-Gamal ,
,
d
,
1
e
Mohammed S. Abdel-Maksoud
a,
b,
c
d
f
g, h
Mahmoud M. Gamal El-Din
, Jinsung Tae , Hong Seok Choi , Kyung-Tae Lee
,
i
a, b, *
Kyung Ho Yoo , Chang-Hyun Oh
Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Department of Biomolecular Science, Korea University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki-Giza 12622, Egypt
Department of Chemistry, Yonsei University, Seoul, Republic of Korea
a
b
c
d
e
f
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
Department of Pharmaceutical Biochemistry, College of Pharmacy, Chosun University, Gwangju, Republic of Korea
Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701,
g
h
Republic of Korea
i
Chemical Kinomics Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 January 2015
Received in revised form
Design and synthesis of a new series of 5,6-diarylimidazo[2,1-b]thiazole derivatives possessing terminal
aryl sulfonamide moiety are described. Their in vitro antiproliferative activities against a panel of 57
human cancer cell lines of nine different cancer types were tested at the NCI. Compounds 8a, 8b, 8n, 8q,
26 March 2015
8t, and 8u showed the highest mean % inhibition values over the 57 cell line panel at 10 mM, and they
Accepted 28 March 2015
Available online 30 March 2015
were further tested in 5-dose testing mode to determine their IC50 values. Among the six compounds,
compound 8u possessing terminal para-hydroxybenzenesulfonamido moiety and ethylene linker
showed the highest potency. It demonstrated superior potency than Sorafenib against eight different cell
lines, and was equipotent to Sorafenib against COLO 205 colon cancer cell line. Its IC50 values over NCI-
Keywords:
Antiproliferative activity
Arylsulfonamido
ERK pathway
Imidazo[2,1-b]thiazole
V600E-B-RAF
H460 non-small cell lung cancer cell line and MCF7 breast cancer cell line were 0.845 mM and 0.476 mM,
respectively. Compounds 8a, 8b, 8q, 8t, and 8u showed high selectivity indices towards cancer cells over
L132 normal lung cell line. Compound 8u showed potential inhibitory effects over the components of
ERK pathway. Its IC50 value over V600E-B-RAF and C-RAF kinases were 39.9 nM and 19.0 nM,
respectively.
C-RAF
©
2015 Elsevier Masson SAS. All rights reserved.
®
1. Introduction
Sorafenib (Nexavar , Fig. 1), a diarylurea derivative, is an example
of kinase inhibitors targeting ERK pathway. Dysregulated signaling
through RAF kinase isoforms has been detected in ~30% of human
cancers [14]. Constitutive B-RAF activity can be caused by activating
oncogenic mutations, such as B-RAF V600E mutation, which is
associated with a variety of cancer types including melanoma, non-
Hodgkin's lymphoma, colorectal adenocarcinoma, thyroid carci-
noma, non-small cell lung carcinoma, renal cell carcinoma, hepa-
tocellular carcinoma, ovarian cancer, gastrointestinal stromal
tumors, and hairy cell leukemia [15]. Wild-type RAF1 (C-RAF) has
been reported to prolong cell survival, independent of MAPK
signaling, by direct interaction with anti-apoptotic and apoptotic
regulatory proteins [14,16]. C-RAF kinase is over-expressed in cases
Imidazo[2,1-b]thiazole analogs exhibited potential anti-
proliferative activities against a variety of human cancer cell lines
1e11]. Some pyrimidinyl substituted imidazo[2,1-b]thiazole de-
rivatives were reported as RAF kinases inhibitors (Fig. 1) [12].
The RAS-RAF-MEK-ERK signaling pathway (ERK pathway) plays
an important role in tumorigenesis and cancer progression [13].
[
*
Corresponding author. Center for Biomaterials, Korea Institute of Science and
Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea.
E-mail address: choh@kist.re.kr (C.-H. Oh).
Both authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2015.03.065
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

454
M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
Fig. 1. Structures of Sorafenib, previously reported pyrimidinyl imidazo[2,1-b]thiazole derivatives [12], and the target compounds 8aev.
of renal cell carcinoma [17], hepatocellular carcinoma [18], and is
associated with poor prognosis in ovarian [19] and androgen-
insensitive prostate cancer [20]. There is also a relationship be-
tween C-RAF and disease progression and cell proliferation in
melanomas [21].
Our target compounds in the present investigation were
designed as open chain analogs to the previously reported RAF
kinase inhibitors imidazo[2,1-b]thiazole derivatives (Fig. 1) [12].
Homologation of the spacer, ethylene or propylene, was carried out
in order to study the effect of linker length on the activity. The
target compounds were tested for in vitro antiproliferative activities
against 57 human cancer cell lines of nine different cancer types,
and B-RAF/V600E-B-RAF/C-RAF kinase inhibitory effect of the most
potent compound was also examined in order to test the mecha-
nism of action at molecular level.
diisopropylethylamine led to displacement of the mesyl group of
compound 7 by amino group and formation of the target com-
pounds 8aet. This was confirmed by disappearance of the mesyl
1
signal at 3.38 ppm in H NMR analysis, and appearance of the
aliphatic and aromatic signals related to the ethylene/propylene
1
linker and terminal aromatic ring, respectively, in both H NMR and
13
C NMR analyses. The methoxy compounds 8d,n were demethy-
lated using boron tribromide to obtain the corresponding hydroxyl
compounds 8u,v. The disappearance of methoxy signals at 3.76 and
1
3.79 ppm in H NMR analysis confirms demethylation reaction. LC-
MS analysis was another tool to confirm identity of the analyzed
compounds. Table 1 illustrates the target compound structures and
their yield percentages.
2
2
.2. Biological evaluation
.2.1. In vitro antiproliferative activity over NCI-57 cancer cell line
2
. Results and discussion
panel
2
.1. Chemistry
2.2.1.1. One-dose results. Structures of the target compounds were
submitted to National Cancer Institute (NCI), Bethesda, Maryland,
USA [25], and the fifteen derivatives shown in Fig. 2 were selected
on the basis of degree of structural variation and computer
modeling techniques for testing their antiproliferative activity. The
selected compounds were subjected to in vitro anticancer assay
against tumor cells in a full panel of 57 cell lines taken from nine
different tissues (blood, lung, colon, CNS, skin, ovary, kidney,
prostate, and breast). The compounds were tested at a single-dose
The target compounds 8aev were synthesized through the
pathway illustrated in Scheme 1. It was important to synthesize the
key mesyl intermediate compound 7 and the amino tail reagents
3
treated with the appropriate arylsulfonyl chloride derivatives 2aej
in the presence of triethylamine as a base to afford the amino tail
reagents 3aet [22,23]. Refluxing 2-aminothiazole (4) with
aet. 1,2-Ethylenediamine (1a) or 1,3-propylenediamine (1b) were
a-
bromo-4-fluoroacetophenone in ethanol led to cyclization to 6-(4-
fluorophenyl)imidazo[2,1-b]thiazole (5) [24]. Coupling compound
concentration of 10 mM, and the percentages of growth inhibition
over the 57 tested cell lines were determined. The mean inhibition
percentages for each of the tested analogs over the full panel of cell
lines are illustrated in Fig. 3.
Upon investigating the effect of linker length on anti-
proliferative activity, it was found that compounds 8a, 8b, 8e, and
8u with ethylene linker were more active the corresponding pro-
pylene derivatives 8k, 8l, 8o, and 8v. On the other hand, some
propylene analogs such as 8s and 8t showed higher activity than
5
with 4-iodo-2-(methylthio)pyrimidine in presence of palladium
acetate, cesium carbonate, and triphenylphosphine produced
compound 6. Oxidation of the methylsulfide moiety of compound 6
using oxone produced the corresponding methylsulfonyl com-
pound 7 [7]. This was confirmed through deshielding of the methyl
1
signal in H NMR from 2.64 ppm to 3.38 ppm. Heating compound 7
with the amino reagents 3aet in the presence of

M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
455
ꢀ
Scheme 1. Reagents and conditions: a) triethylamine, CH
2
Cl
2
, 0 C; rt, overnight, 50e70%; b)
a
-bromo-4-fluoroacetophenone, EtOH, reflux, 16 h, 86%; c) 4-iodo-2-(methylthio)
ꢀ
ꢀ
ꢀ
pyrimidine, Pd(OAc)
overnight.
2
, Cs
2
CO
3
, PPh
3
, DMF, 80 C, 12 h, 26%; d) oxone, MeOH, H
2
O, rt, 16 h, 81%; e) 3aet, diisopropylethylamine, DMSO, 80 C, 16 h; f) BBr
3
, CH
2
Cl
2
, ꢁ78 C, 1 h; rt,
the corresponding ethylene compounds 8i and 8j.
Sorafenib as a reference standard compound. The results of Sor-
afenib were obtained from NCI datawarehouse index [26], and are
inserted in Table 2. The six tested compounds exerted high potency
with one-digit micromolar IC50 values against most of the cell lines.
Interestingly, compound 8u demonstrated submicromolar IC50
values against COLO 205 colon cancer cell line and MCF7 breast
The effect of terminal aryl substituents on activity was also
studied. Compound 8q with para-fluorophenyl ring was more
active than compounds 8k, 8l, 8n, 8o, 8s, and 8v possessing phenyl,
para-tosyl, para-methoxyphenyl, 3,4-dimethoxyphenyl, para-(tri-
fluoromethyl)phenyl, and para-hydroxyphenyl, respectively. So in
case of propylene derivatives, para-fluorobenzenesulfonamido
moiety was optimum for activity. Among ethylene analogs, com-
pound 8u possessing para-hydroxyphenyl terminal ring showed
superior activity compared with analogs 8a, 8b, 8e, 8h, and 8i
containing phenyl, para-tosyl, 3,4-dimethoxyphenyl, para-iodo-
phenyl, and para-(trifluoromethyl)phenyl, respectively.
cancer cell line (0.845 and 0.476
mM, respectively). Compound 8a
also showed submicromolar IC50 value of 0.722
m
M against MCF7
breast cancer cell line. Compound 8a with phenyl terminal ring was
generally more potent than compound 8b possessing para-tosyl
ring. Similarly, compound 8n with para-methoxyphenyl was more
potent than compound 8q containing para-fluorophenyl terminal
ring against ten different cell lines. This may be rationalized that
hydrogen bond-forming group on the terminal ring could be
favorable for affinity at the receptor site(s), and hence potency. This
assumption can be reinforced by the superior potencies expressed
by compound 8u with para-hydroxyphenyl terminal ring,
compared with the other five compounds tested in 5-dose testing
mode. Compound 8u showed superior potencies than Sorafenib
also against eight cell lines. And it was equipotent to Sorafenib over
COLO 205 colon cancer cell lines.
In order to study the selectivity of the most potent compounds
towards cancer cells over normal cells, the cytotoxicity of com-
pounds 8a, 8b, 8q, 8t, and 8u were tested against L132 human lung
normal cell line (Table 2). The five compounds showed high IC50
values over L132 cell line, which indicates high selectivity towards
cancer cells than normal cells. The high selectivity indices of those
potent compounds could indicate high safety and diminished
toxicity.
Among all the tested compounds, compounds 8q, 8t, and 8u
showed the highest mean inhibitions. The %inhibitions of these
three compounds over each cell line of the 57 cancer cell line panel
are illustrated in Fig. 3. At 10 mM concentration, the three com-
pounds showed strong antiproliferative activities (>80% inhibition)
over eight, seven, and six cell lines, respectively. Compounds 8q, 8t,
and 8u demonstrated broad-spectrum cytotoxicities over all the
nine tested cancer types. Of special interest, compound 8q with
propylene linker and para-fluorophenyl terminal ring showed le-
thal effect over T-47D breast cancer cell line with 102.52% inhibi-
tion. It also inhibited the growth of HT29 colon cancer cell line and
UO-31 renal cancer cell line by 96.72% and 96.86%, respectively.
Compound 8t exerted lethal effect against HT29 colon cancer cell
line (107.72% inhibition). It also showed high inhibitory effect
against K-562 leukemia cell line, COLO 205, and HCC-2998 colon
cancer cell lines (96.25%, 89.90%, and 92.78%, respectively).
2
.2.1.2. Five-dose results. Compounds 8a, 8b, 8n, 8q, 8t, and 8u
The mean IC50, TGI, and LC50 for compounds 8a, 8b, 8n, 8q, 8t,
and 8u over the tested NCI-57 cancer cell line panel have been
calculated and summarized in Table 3. Compound 8u with ethylene
spacer and p-hydroxyphenyl terminal ring showed the highest
potency and efficacy, compared with the other five compounds.
Compound 8n possessing p-methoxyphenyl terminal ring was
with promising results in single-dose testing were further tested in
a five-dose testing mode in order to determine their IC50 values
over the 57 cancer cell lines. The IC50 values of those five com-
pounds over the most sensitive cell line(s) of each subpanel are
summarized in Table 2. The results were compared with those of

4
56
M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
Table 1 (continued)
Table 1
Structures of the target compounds and their yield percentages.
Compound no.
Ar
n
2
Yield%
79%
8o
8p
2
65%
8
8
q
r
2
2
2
2
63%
70%
71%
74%
Compound no.
Ar
n
1
Yield%
58%
8a
8b
8c
1
1
73%
71%
8s
8t
8
8
u
1
2
59%
62%
8
8
d
e
1
1
67%
75%
v
more potent than compound 8q with p-fluorophenyl terminal
moiety.
8f
1
63%
Some physicochemical parameters such as total polar surface
area (TPSA), cLogP, and CMR were calculated for compounds 8a, 8b,
8n, 8q, 8t, and 8u using ChemOffice 2012 software, and are sum-
8
8
8
8
g
h
i
1
1
1
1
70%
75%
72%
72%
marized in Table 4. The results showed that polar group such as
hydroxyl is required on the terminal arylsulfonamido moiety, and
this is well correlated with the results obtained by compound 8u.
Increased bulkiness and lipophilicity of that terminal moiety is
unfavorable for antiproliferative activity of this series of com-
pounds. This is obvious upon comparing the cLogP and CMR values
of compound 8u with those of the other five compounds.
2.2.1.3. In vitro kinase screening. In order to investigate the mech-
anism of action of this series of compounds at molecular level, the
j
most potent compound 8u was selected as a representative
example to be tested at a single-dose concentration of 10 mM over
B-RAF (wild-type), V600E-B-RAF, and C-RAF kinases. As illustrated
in Table 5, compound 8u showed strong inhibitory effect over the
three kinases at 10 mM. It was further tested in a 10-dose testing
mode in order to determine its IC50 values over wild-type B-RAF,
V600E mutated B-RAF, and C-RAF. The IC50 values were 428 nM,
8
8
8
k
l
2
2
2
68%
71%
77%
39.9 nM, and 19 nM, respectively. So the affinity of compound 8u
was found to be 10.73 times more towards V600E-B-RAF than wild-
type B-RAF. It was also 22.53 times more selective towards C-RAF
than wild type B-RAF. It was almost equipotent to Sorafenib over
V600E-B-RAF. Compound 8u exerted high potency against cell lines
with over-expressed V600E-B-RAF such as COLO 205, HT29 colon
cancer cell lines, and SK-MEL-5 melanoma cell line. It has been also
reported that C-RAF overexpression increases the resistance of
MCF-7 breast cancer cells to standard chemotherapeutic agents
m
8
n
2
65%
[27]. So C-RAF inhibition by compound 8u could play a role in its

M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
457
Fig. 2. Mean inhibition percentages observed with the target compounds in single-dose (10
mM) 60-cancer cell line screening. Mean % inhibition represents the mean inhibition
percentages over the 60 cell lines. The inhibition percentages were calculated by subtracting the growth percentages from 100.
Fig. 3. % Inhibition expressed by compounds 8q (a), 8t (b), and 8u (c) at a single-dose concentration of 10 mM over all cell lines of the NCI cancer cell line panel of nine different
cancer types.
sensitivity towards the compound, and hence the high potency of
u against it. As explained in the introduction section, V600E-B-
RAF and C-RAF are over-expressed in a variety of cancer types. So
antiproliferative effect.
8
Moreover, compound 8u was also tested for inhibitory effect
over MEK and ERK kinases, other components of the ERK pathway,
by Western Blot Assay (Fig. 4). The MEK/ERK-containing A375P cell
lysate was treated with three different concentrations of the test
we can conclude that the inhibitory effect of compound 8u on RAF
kinases is, at least in part,
a potential mechanism of its

458
M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
Table 2
IC50 values (
m
M) of Sorafenib and the tested compounds over the most sensitive cell line(s) of each subpanel and L132 human lung normal cell line.
Compound no.
8
a
8b
8n
8q
8.26
8t
8u
1.82
0.845
2.00
2.09
1.68
1.07
1.39
1.70
2.03
Sorafenib
Cancer cell line
SRa
NCI-H460
3.46
2.71
2.23
2.31
1.61
2.56
2.45
2.99
1.69
4.00
0.722
NTj
3.69
4.33
5.58
2.84
1.56
3.28
5.49
5.59
2.73
8.74
2.74
NTj
4.01
3.74
2.22
2.40
5.22
3.67
4.17
5.37
3.42
4.36
3.71
3.16
2.51
2.00
2.00
3.16
1.58
1.58
2.51
2.51
2.00
2.51
b
c
3.50
5.62
6.23
3.52
2.16
3.69
4.63
8.85
4.61
9.54
3.24
84.35
COLO 205
NTj
HT29c
NTj
d
SNB-75
2.65
e
NTj
MDA-MB-435
e
SK-MEL-5
3.52
3.09
2.30
NTj
1.48
70.62
f
IGROV1
g
ACHN
h
PC-3
4.51
0.476
66.56
i
MCF7
j
L132 human lung normal cell line
310.10
384.14
NT
Bold figures indicate higher potencies than Sorafenib.
a
Leukemia cell line.
Non-small cell lung cancer cell line.
Colon cancer cell line.
CNS cancer cell line.
Melanoma cell line.
Ovarian cancer cell line.
Renal cancer cell line.
Prostate cancer cell line.
b
c
d
e
f
g
h
i
Breast cancer cell line.
Not tested.
j
compound 8u (1, 3, and 10
mM) and its inhibitory activities were
activity at molecular level.
compared with that of Sorafenib. The results showed that com-
pound 8u and Sorafenib significantly suppressed MEK1/2 and
ERK1/2 phosphorylation in a dose-dependent manner. Due to the
relationship between upregulation of RAF/MEK/ERK kinases and
uncontrolled cell proliferation, increased cell survival, and tumor
progression, herein it can be concluded that the inhibitory effect of
compound 8u can be its potential mechanism of antiproliferative
3. Conclusion
In this study, a new series of imidazo[2,1-b]thiazole derivatives
possessing terminal aryl sulfonamide moiety was designed and
synthesized. The target compounds were tested for in vitro anti-
proliferative activities over 57 cancer cell line panel of nine
different cancer types at the NCI. Among them, compounds 8a, 8b,
8
n, 8q, 8t, and 8u showed the most promising results at a single-
Table 3
dose concentration of 10 M. They were further tested in 5-dose
m
Mean TGI and LC50 values of compounds 8a, 8b, 8n, 8q, 8t, and 8u over the tested
NCI-57 cancer cell line panel.
testing mode in order to determine their IC50 values, and the re-
sults were compared with those of a reference anticancer drug,
Sorafenib. Compounds 8u with para-hydroxybenzenesulfonamido
terminal moiety and ethylene spacer showed higher potencies than
Compound no.
8a
8b
72.44
8n
81.28
8q
81.28
8t
8u
Mean TGI
Mean LC50
70.79
97.72
83.18
97.72
30.90
87.10
100
100
100
Table 4
Calculated physicochemical parameters of compounds 8a, 8b, 8n, 8q, 8t, and 8u.
Compound no.
TPSAa
cLogPb
CMRc
8
8
8
8
8
8
a
b
n
q
t
98.52
98.52
107.75
98.52
98.52
118.75
4.49
4.99
4.77
4.93
5.66
4.19
13.07
13.53
13.69
13.08
14.76
13.22
u
a
b
c
Total Polar Surface Area (Å2).
Partition coefficient, a measure of lipophilicity.
Molar refractivity, a measure of bulkiness or steric factor.
Table 5
Kinase inhibition results of compound 8u, Sorafenib, and GW5074.
Kinase
Compound 8u
Inhibition at 10
Sorafenib
IC50 M)
GW5074
IC50 (mM)
(
m
%
m
M
IC50
(
m
M)
B-RAF
V600E-B-RAF
C-RAF
97.12%
97.28%
97.51%
0.428
0.0399
0.019
0.025
0.038
e
e
e
0.006
Fig. 4. Inhibitory effect of compound 8u and Sorafenib on p-MEK1/2 and p-ERK1/2.

M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
459
Sorafenib. It was also the most potent among the six compounds
compounds 3aet.
testing in 5-dose testing mode. It demonstrated submicromolar
IC50 values over NCI-H460 NSCLC cell line and MCF7 breast cancer
4.2.1. N-(2-Aminoethyl)benzenesulfonamide (3a)
ꢀ
1
cell line (0.845
mM and 0.476
m
M, respectively). Compounds 8a, 8b,
White solid; yield 69%; mp 80e81 C, H NMR (CDCl
3
, 300 MHz)
8
q, 8t, and 8u were further tested for cytotoxicity towards L132
d
7.77 (d, 2H, J ¼ 9.0 Hz), 7.46e7.38 (m, 3H), 3.99 (bs, 3H,
13
normal lung cell line, and showed high selectivity indices against
cancer cells over normal cells. These results could give an indication
about high safety of those compounds. Compound 8u showed po-
tential inhibitory effects over V600E-B-RAF, wild-type B-RAF, C-
RAF, MEK, and ERK kinases. It was almost equipotent to Sorafenib
against V600E-B-RAF kinase. Compound 8u exerted 2-digit nano-
molar IC50 against both V600E-B-RAF and C-RAF (39.9 and 19.0 nM,
respectively). So it can be concluded that inhibition of ERK pathway
could be a mechanism of antiproliferative activity of this compound
at molecular level. Hydrogen bond-forming group, such as the
hydroxyl group of compound 8u, on the terminal ring seems to be
essential for activity. The physicochemical parameter calculation
reinforced this postulation that the terminal arylsulfonamido
moiety should carry a polar group and should not be bulky or much
hydrophobic. Further structural modifications of this series of
compounds in order to improve their potencies are currently in
progress.
exchangeable), 3.86 (t, 2H, J ¼ 3.0 Hz), 2.67 (t, 2H, J ¼ 3.0 Hz).
C
3
NMR (CDCl , 75 MHz) d 140.0, 132.4, 129.1, 126.8, 45.2, 40.9. IR (KBr)
ꢁ1
cm : 3563, 3272, 3065, 1586, 1479, 1446, 1323, 1157.
4.2.2. N-(2-Aminoethyl)-4-methylbenzenesulfonamide (3b)
ꢀ
1
White solid; yield 77%; mp.120e121 C, H NMR (CDCl
300 MHz)
7.75 (d, 2H, J ¼ 9.0 Hz), 7.39 (d, 2H, J ¼ 9.0 Hz), 3.45 (s,
2H, exchangeable), 2.89 (t, 2H, J ¼ 6.0 Hz), 2.66 (t, 1H, J ¼ 6.0 Hz),
3
,
d
13
3
2.44 (s, 3H). C NMR (CDCl , 75 MHz) d 143.3, 137.0, 129.7, 127.0,
ꢁ1
45.5, 41.0, 21.5. IR (KBr) cm : 3585, 3360, 3290, 2585, 1926, 1311,
LC-MS: m/z 215 (Mþ1).
4.2.3. N-(2-Aminoethyl)-3,4-dimethylbenzenesulfonamide (3c)
ꢀ
1
White solid; yield 86%; mp.93e94 C, H NMR (CDCl
7.63 (s,1H), 7.59 (dd,1H, J ¼ 3.0, J ¼ 9.0 Hz), 7.27 (s,1H), 2.95 (t, 2H,
J ¼ 6.0 Hz), 2.79 (t, 2H, J ¼ 6.0 Hz), 2.32 (s, 6H).
3
, 300 MHz)
d
4
.2.4. N-(2-Aminoethyl)-4-methoxybenzenesulfonamide (3d)
ꢀ
1
4
. Experimental
White solid, yield 76%; mp.90e91 C, H NMR (CDCl
3
, 300 MHz)
d
7.78 (d, 2H, J ¼ 9.0 Hz), 6.96 (d, 2H, J ¼ 6.0 Hz), 3.85 (s, 3H), 3.10
13
4.1. General
(bs, 2H), 2.93 (t, 2H, J ¼ 6.0 Hz), 2.76 (t, 2H, J ¼ 6.0 Hz). C NMR
CDCl , 75 MHz) 163.3, 132.2, 129.6, 114.8, 56.2, 46.0, 41.6. LC-MS:
m/z 231 (Mþ1).
(
3
d
All solvents and reagents were commercially available and used
without further purification. The target compounds and in-
termediates were purified by column chromatography using silica
gel (0.040e0.063 mm, 230e400 mesh) and technical grade sol-
vents. Analytical thin layer chromatography (TLC) was adopting on
silica gel 60 F254 plates from Merck. IR spectra (KBr disks) were
recorded with a Bruker FT-IR instrument (Bruker Bioscience, Bill-
erica, MA, USA). H NMR and C NMR spectra were recorded on a
Bruker Avance 400 or 300 spectrometer using tetramethylsilane as
an internal standard and signals are described as s (singlet),
d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet), brs
4.2.5. N-(2-Aminoethyl)-3,4-dimethoxybenzenesulfonamide (3e)
ꢀ
1
White solid; yield 89%; mp.142e143 C, H NMR (CD
3
OD,
300 MHz)
d
7.47 (dd, 1H, J ¼ 3.0, 9.0 Hz), 7.38 (d, 1H, J ¼ 3.0 Hz), 7.11
(d, 1H, J ¼ 9.0 Hz), 3.91 (s, 3H), 3.90 (s, 3H), 3.34e3.31 (m, 2H,
exchangeable), 2.90 (t, 2H, J ¼ 6.0 Hz), 2.68 (t, 2H, J ¼ 6.0 Hz).
1
13
4.2.6. N-(2-Aminoethyl)-3-fluorobenzenesulfonamide (3f)
1
Viscous light brown oil; yield 71%; H NMR (CDCl
3
, 300 MHz)
d
7.65e7.44 (m, 3H), 7.24e7.19 (m, 1H), 4.29 (bs, 2H, exchangeable),
13
(
broad singlet), or dd (doublet of doublets). LC-MS analysis was
2.59 (t, 2H, J ¼ 6.0 Hz), 2.76 (t, 2H, J ¼ 6.0 Hz). C NMR (CDCl
3
,
carried out using the following system: Waters 2998 photodiode
array detector, Waters 3100 mass detector, Waters SFO system
fluidics organizer, Waters 2545 binary gradient module, Waters
reagent manager, Waters 2767 sample manager, Sunfire™ C18
75 MHz) 163.5, 161.0, 142.1, 131.1, 122.7, 114.3, 45.3, 41.0.
d
4.2.7. N-(2-Aminoethyl)-4-fluorobenzenesulfonamide (3g)
White solid; yield 76%; mp. 108e109 C, 1H NMR (CDCl
300 MHz)
J ¼ 6.0 Hz), 2.71 (t, 2H, J ¼ 6.0 Hz). C NMR (CDCl
ꢀ
,
3
column (4.6 ꢂ 50 mm, 5
at 0 min, 1% A at 5 min; solvent A: 0.035% trifluoroacetic acid (TFA)
in water; solvent B: 0.035% TFA in CH
mm particle size); Solvent gradient ¼ 95% A
d
7.84e7.79 (m, 2H), 7.11 (t, 2H, J ¼ 6.0 Hz), 2.89 (t, 2H,
13
3
, 75 MHz) d 166.7,
3
OH; flow rate ¼ 3.0 mL/min;
136.0, 129.7 (J ¼ 8.3 Hz), 116.3 (J ¼ 22.5 Hz), 45.2, 40.9; IR (KBr)
ꢁ1
the AUC was calculated using Waters MassLynx 4.1 software. Sol-
vents and liquid reagents were transferred using hypodermic sy-
ringes. Purity % of all the target compounds were determined by
HPLC and found to be >95%.
cm : 3582, 3350, 3299, 3107, 3068, 1903, 1317, 838. LC-MS: m/z
219 (Mþ1).
4.2.8. N-(2-Aminoethyl)-4-iodobenzenesulfonamide (3h)
1
Yield 84%; H NMR (CDCl
3
, 300 MHz)
d
7.88 (d, 2H, J ¼ 8.3 Hz),
4.2. General procedure for preparation of ethylenediamine and
7.59 (d, 2H, J ¼ 9.0 Hz), 2.97 (t, 2H, J ¼ 6.0 Hz), 2.81 (t, 2H, J ¼ 6.0 Hz).
propylenediamine sulfonamides 3aet
4.2.9. N-(2-Aminoethyl)-4-(trifluoromethyl)benzenesulfonamide
To a solution of proper diamine 1a,b (10 mmol) in dichloro-
(3i)
Viscous oil; yield 72%; H NMR (CDCl
J ¼ 6.0 Hz), 7.64 (d, 1H, J ¼ 9.0 Hz), 7.52 (t, 2H, J ¼ 6.0, J ¼ 9.0 Hz),
1
methane (20 mL), triethylamine (300 mg, 0.416 mL, 30 mmol) was
3
, 300 MHz) d 7.99 (d, 1H,
ꢀ
added with stirring. The mixture was cooled to 0 C, and a solution
1
3
of the appropriate sulfonyl chloride 2aej (5 mmol) in dichloro-
methane (2 mL) was added dropwise. The mixture stirred at room
temperature overnight. The reaction mixture was washed with 10%
2.91 (t, 2H, J ¼ 6.0 Hz), 2.67 (t, 2H, J ¼ 6.0 Hz); C NMR (CDCl
3
,
ꢁ1
75 MHz)
d
138.7, 132.6, 130.6, 128.2, 128.1, 44.9, 40.8; IR (KBr) cm
:
3581, 3307, 3086, 2874, 2874, 1659, 1595, 1441, 1311, 1164, 560.
Na
2
CO
3
(20 mL) and then with distilled water (3 ꢂ 15 mL). The
organic layer separated and dried over anhydrous Na
2
SO . The
4
4.2.10. N-(2-Aminoethyl)naphthalene-2-sulfonamide (3j)
White solid; yield 85%; mp 132e133 C; 1H NMR (CDCl
ꢀ
,
solvent was evaporated under reduced pressure, and the residue
was purified flash column chromatography (silica gel, appropriate
ratio of hexanes-ethyl acetate) to give the purified semisolid
3
300 MHz)
d
8.43 (s, 1H), 8.15 (t, 2H, J ¼ 6.0 Hz), 8.04 (d, 1H,
J ¼ 9.0 Hz), 7.82 (d, 1H, J ¼ 9.0 Hz), 7.70 (d, 2H, J ¼ 6.0 Hz), 3.32 (s,

460
M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
2
H, exchangeable), 2.75 (t, 2H, J ¼ 6.0 Hz), 2.50 (t, 2H, J ¼ 6.0 Hz);
4.2.20. N-(3-Aminopropyl)naphthalene-2-sulfonamide (3t)
13
ꢀ
1
C NMR (CDCl
3
, 75 MHz)
d
136.8, 134.7, 132.1, 129.5, 129.2, 128.7,
White solid; yield 80%; mp 114e115 C; H NMR (CDCl
400 MHz)
8.41 (d, 1H, J ¼ 9.0 Hz), 7.94e7.83 (m, 4H), 7.58 (d, 2H,
J ¼ 9.0 Hz), 3.12 (bs, 1H), 3.06 (t, 2H, J ¼ 6.0 Hz), 2.73 (t, 2H,
3
,
ꢁ
1
1
3
28.3, 127.8, 127.5, 122.3, 45.5, 41.0; IR (KBr) cm : 3576, 3364,
306, 3055, 2563, 1927, 1819, 1592, 1312; LC-MS: m/z 251 (Mþ1).
d
13
J ¼ 6.0 Hz), 1.56 (m, 2H, J ¼ 6.0 Hz); C NMR (CDCl
136.9, 134.7, 132.1, 129.4, 129.1, 128.5, 128.4, 128.1, 127.8, 127.4,
122.3, 42.5, 40.4, 31.2. LC-MS: m/z 265 (Mþ1).
3
, 100 MHz)
4.2.11. N-(3-Aminopropyl)benzenesulfonamide (3k)
d
ꢀ
1
Buff solid; yield 67%; mp. 71e72 C, H NMR (CDCl
3
, 300 MHz)
d
2
d
3
7.90 (d, 2H, J ¼ 6.0 Hz), 7.60e7.51 (m, 3H), 3.11 (t, 2H, J ¼ 6.0 Hz),
13
.82 (t, 2H, J ¼ 6.0 Hz), 1.62 (m, 2H); C NMR (CDCl
3
, 75 MHz)
4.3. Synthesis of the mesyl intermediate compound 7
ꢁ
1
139.8, 132.4, 129.1, 126.9, 41.1, 38.7, 30.2; IR (KBr) cm : 3572,
341, 3040, 2949, 1991, 1592, 1318.
It was synthesized through a 3-step pathway as previously re-
ported [7].
4
.2.12. N-(3-Aminopropyl)-4-methylbenzenesulfonamide (3l)
ꢀ
1
White solid; yield 82%; mp 119e120 C; H NMR (CDCl
00 MHz)
7.64 (d, 2H, J ¼ 7.6 Hz), 7.19 (d, 2H, J ¼ 8.0 Hz), 3.27 (bs,
H, exchangeable), 2.89 (t, 2H, J ¼ 6.0 Hz), 2.64 (t, 2H, J ¼ 6.0 Hz),
3
,
4
3
2
d
d
4.4. General procedure for preparation of the target compounds
8aet
1
3
.31 (s, 3H), 1.48 (p, 2H, J ¼ 6.0 Hz); C NMR (CDCl
3
, 75 MHz)
ꢁ
1
143.1, 137.1, 129.6, 127.0, 42.1, 40.1, 31.4, 21.4. IR (KBr) cm : 3581,
A mixture of mesyl compound 7 (0.19 g, 0.5 mmol), amino
3
360, 3300, 3047, 1917, 1599, 1494, 1317. LC-MS: m/z 229 (Mþ1).
compound 3aet (0.55 mmol), and diisopropylethylamine (0.29 mL,
ꢀ
1.7 mmol) in DMSO (5 mL) was stirred at 80 C for 16 h. The mixture
4
.2.13. N-(3-Aminopropyl)-3,4-dimethylbenzenesulfonamide (3m)
White solid; yield 84%; mp 96e97 C; H NMR (CDCl , 300 MHz)
3
7.63 (s, 1H), 7.58 (dd,1H, J ¼ 3.0, J ¼ 9.0 Hz), 7.25 (d, 1H, J ¼ 9.0 Hz),
.06 (t, 2H, J ¼ 6.0 Hz), 2.80 (t, 2H, J ¼ 6.0 Hz), 2.32 (s, 3H), 2.29 (s,
H), 1.60 (p, 2H, J ¼ 6.0 Hz).
was cooled to room temperature, quenched with water (20 mL),
then extracted with ethyl acetate (3 ꢂ 20 mL). The combined
organic layer extracts were washed with brine, dried over anhy-
ꢀ
1
d
3
3
2 4
drous Na SO , filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (silica
gel, appropriate ratio of hexanes-ethyl acetate) to obtain the pure
target compounds.
4.2.14. N-(3-Aminopropyl)-4-methoxybenzenesulfonamide (3n)
ꢀ
1
White solid; yield 88%; mp76-77 C; H NMR (CDCl
3
, 300 MHz)
d
2
7.79 (d, 2H, J ¼ 9.0 Hz), 7.08 (d, 2H, J ¼ 9.0 Hz), 3.88 (s, 3H), 2.88 (t,
4
.4.1. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
13
H, J ¼ 6.0 Hz), 2.66 (t, 2H, J ¼ 6.0 Hz), 1.60 (p, 2H, J ¼ 6.0 Hz);
, 75 MHz) 162.6, 131.6, 129.0, 114.2, 55.6, 41.6, 39.7,
1.1; IR (KBr) cm : 3600, 3355, 3302, 2042, 1908, 1592, 1487, 1487,
317, 1167.
C
pyrimidin-2-ylamino)ethyl)benzenesulfonamide (8a)
NMR (CDCl
3
3
d
1
Colorless viscous oil; H NMR (DMSO-d
6
, 300 MHz) d 8.80 (brs,
ꢁ1
1
H, exchangeable), 8.42 (brs, 1H, exchangeable), 8.06 (d, 1H,
1
4
3
J ¼ 5.3 Hz), 7.78 (d, 2H, J ¼ 7.8 Hz), 7.65e7.55 (m, 6H), 7.46 (d, 1H,
J ¼ 3.9 Hz), 7.30 (t, 2H, J ¼ 8.8 Hz), 6.27 (d,1H, J ¼ 5.1 Hz), 2.85 (t, 2H,
.2.15. N-(3-Aminopropyl)-3,4-dimethoxybenzenesulfonamide (3o)
13
J ¼ 6.0 Hz), 2.53 (t, 2H, J ¼ 6.0 Hz); C NMR (DMSO-d
6
, 75 MHz)
1
White solid; yield 87%; mp 142e143; H NMR (CD
00 MHz)
7.47 (dd, 1H, J ¼ 3.0, J ¼ 9.0 Hz), 7.37 (d, 1H, J ¼ 3.0 Hz),
.10 (d, 1H, J ¼ 9.0 Hz), 3.91 (s, 3H), 3.90 (s, 3H), 2.92 (t, 2H,
J ¼ 6.0 Hz), 2.83 (t, 2H, J ¼ 6.0 Hz), 1.72 (p, 2H, J ¼ 6.0 Hz).
3
OD,
d
1
4
164.2, 162.5, 160.9, 158.2, 156.2, 151.6, 148.0, 140.9, 132.7, 131.7,
29.6, 126.8, 121.0, 116.1, 115.8, 114.6, 105.8, 38.8, 29.6; LC-MS: m/z
d
7
95 (Mþ1).
4
.2.16. N-(3-Aminopropyl)-3-fluorobenzenesulfonamide (3p)
4.4.2. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
1
Viscous oil; yield 72%; H NMR (CDCl
3
, 300 MHz)
d
7.52 (brs, 3H),
pyrimidin-2-ylamino)ethyl)-4-methylbenzenesulfonamide (8b)
13
1
7.27 (s, 1H), 3.28 (s, 2H), 3.08 (s, 2H), 2.79 (s, 2H), 1.62 (s, 2H);
C
Semi-solid; H NMR (DMSO-d
6
, 300 MHz)
d
8.82 (brs, 1H,
NMR (CDCl
3
, 75 MHz)
d
160.7, 142.3, 130.8, 122.6, 119.5, 114.3, 42.7,
exchangeable), 8.56 (brs, 1H, exchangeable), 8.06 (d, 1H, J ¼ 6.0 Hz),
7.66 (d, 2H, J ¼ 6.0 Hz), 7.60 (d, 2H, J ¼ 6.0 Hz), 7.45 (s, 2H), 7.33 (d,
2H, J ¼ 9.0 Hz), 7.28 (d, 2H, J ¼ 9.0 Hz), 6.29 (d, 1H, J ¼ 3.0 Hz), 3.51
ꢁ
1
4
0.5, 31.0; IR (KBr) cm : 3251, 3071, 2931, 1592, 1471, 1435, 1361,
1323, 1226.
(
brs, 2H), 2.94 (brs, 2H), 2.31 (s, 3H); LC-MS: m/z 509 (Mþ1).
4.2.17. N-(3-Aminopropyl)-4-fluorobenzenesulfonamide (3q)
ꢀ
1
White solid; yield 79%; mp.104e105 C; H NMR (CD
3
OD,
4.4.3. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
3
00 MHz)
d
7.90 (dd, 2H, J ¼ 9.0 Hz, J ¼ 6.0 Hz), 7.32 (t, 2H,
pyrimidin-2-ylamino)ethyl)-3,4-dimethylbenzenesulfonamide (8c)
J ¼ 9.0 Hz), 2.92 (t, 2H, J ¼ 6.0 Hz), 2.67 (t, 2H, J ¼ 6.0 Hz),1.61 (p, 2H,
1
H NMR (DMSO-d
.51 (brs, 1H, exchangeable), 8.06 (d, 1H, J ¼ 5.3 Hz), 7.64 (s, 1H),
.61 (d, 1H, J ¼ 3.0 Hz), 7.59 (s, 1H), 7.54 (s, 1H), 7.50 (d, 1H,
6
, 300 MHz) d 8.82 (brs, 1H, exchangeable),
13
J ¼ 6.0 Hz); C NMR (CDCl
3
, 75 MHz)
d
162.9, 136.2, 129.4, 116.1,
1.1, 39.2, 31.5; IR (KBr) cm : 3366, 3306, 3254, 3047, 2696, 2048,
912, 1317.
8
7
ꢁ
1
4
1
J ¼ 3.0 Hz) 7.48 (s, 1H), 7.33 (s, 1H), 7.29 (d, 1H, J ¼ 3.0 Hz), 7.26 (d,
1
2
H, J ¼ 3.0 Hz), 6.28 (d,1H, J ¼ 5.2 Hz), 2.94 (t, 2H, J ¼ 6.0 Hz), 2.50 (t,
H, J ¼ 6.0 Hz), 2.23 (s, 3H), 2.22 (s, 3H); LC-MS: m/z 523 (Mþ1).
4.2.18. N-(3-Aminopropyl)-4-iodobenzenesulfonamide (3r)
1
Yield 79%; H NMR (CDCl
.58 (d, 2H, J ¼ 9.0 Hz), 3.10 (t, 2H, J ¼ 6.0 Hz), 2.82 (t, 2H, J ¼ 6.0 Hz),
.60 (p, 2H, J ¼ 6.0 Hz).
3
, 300 MHz)
d
7.87 (d, 2H, J ¼ 9.0 Hz),
7
1
4.4.4. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-4-methoxybenzenesulfonamide (8d)
ꢀ
1
4
(
.2.19. N-(3-Aminopropyl)-4-(trifluoromethyl)benzenesulfonamide
3s)
Yield 78%; H NMR (CDCl
.78 (d, 2H, J ¼ 9.0 Hz), 3.13 (t, 2H, J ¼ 6.0 Hz), 2.84 (t, 2H, J ¼ 6.0 Hz),
.62 (p, 2H, J ¼ 6.0 Hz).
White solid; mp 185e186 C; H NMR (DMSO-d
6
, 300 MHz)
d 8.82 (brs, 1H, exchangeable), 8.48 (brs, 1H, exchangeable), 8.06 (d,
1
3
, 300 MHz)
d
8.00 (d, 2H, J ¼ 8.2 Hz),
1H, J ¼ 3.0 Hz), 7.85 (t, 2H, J ¼ 6.0 Hz), 7.62 (t, 2H, J ¼ 6.0 Hz),
7.41e7.30 (m, 6H), 6.29 (s, 1H), 3.76 (s, 3H), 3.34 (brs, 2H), 2.92 (brs,
2H); LC-MS: m/z 525 (Mþ1).
7
1

M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
461
4
.4.5. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3,4-dimethoxybenzenesulfonamide
8e)
mp 197e199 C; H NMR (DMSO-d
exchangeable), 8.50 (brs, 1H, exchangeable), 8.05 (d, 1H, J ¼ 6.0 Hz),
.62 (t, 2H, J ¼ 7.5 Hz), 7.46 (s, 1H), 7.37 (s, 1H), 7.33 (d, 1H,
J ¼ 3.0 Hz), 7.30 (s, 2H), 7.27 (s, 1H), 7.04 (d, 1H, J ¼ 6.0 Hz), 6.28 (d,
H, J ¼ 6.0 Hz), 3.77 (s, 3H), 3.76 (s, 3H), 2.94 (t, 2H, J ¼ 6.0 Hz), 2.51
d, 2H, J ¼ 6.0 Hz). LC-MS: m/z 555 (Mþ1).
161.4, 158.1, 156.2, 151.6, 148.1, 142.9, 138.0, 131.7, 130.5, 130.1, 129.9,
127.0, 126.9, 121.0, 116.1, 116.0, 115.8, 114.4, 105.8, 39.7, 29.5, 21.3.
(
ꢀ
1
6
, 300 MHz)
d
8.85 (brs, 1H,
4.4.13. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)propyl)-3,4-dimethylbenzenesulfonamide
(8m)
7
ꢀ
1
6
mp 220e221 C; H NMR (DMSO-d , 300 MHz) d 8.89 (brs, 1H,
1
(
exchangeable), 8.57 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 6.0 Hz),
7.64 (d, 1H, J ¼ 6.0 Hz), 7.61 (d, 1H, J ¼ 6.0 Hz), 7.54 (s, 1H), 7.49 (s,
1
H), 7.45 (d, 1H, J ¼ 6.0 Hz), 7.30 (t, 4H, J ¼ 9.0 Hz), 6.28 (d, 1H,
4
5
.4.6. 3-Fluoro-N-(2-(4-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-
-yl)pyrimidin-2-ylamino)ethyl)benzenesulfonamide (8f)
J ¼ 6.0 Hz), 2.82 (t, 2H, J ¼ 6.0 Hz), 2.51 (t, 2H, J ¼ 6.0 Hz), 2.25 (s,
3H), 2.23 (s, 3H), 1.68 (p, 2H, J ¼ 6.0 Hz); LC-MS: m/z 537 (Mþ1).
ꢀ
1
mp 192e193 C; H NMR (DMSO-d
exchangeable), 8.48 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 6.0 Hz),
.94 (brs, 1H), 7.69 (s, 1H), 7.65 (t, 4H, J ¼ 6.0 Hz), 7.55e7.47 (m, 3H),
6
, 300 MHz) d 8.83 (brs, 1H,
4.4.14. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
7
pyrimidin-2-ylamino)propyl)-4-methoxybenzenesulfonamide (8n)
ꢀ
1
7
2
.30 (t, 1H, J ¼ 9.0 Hz), 6.30 (d, 1H, J ¼ 6.0 Hz), 3.41 (s, 2H), 3.02 (s,
mp 180e181 C; H NMR (DMSO-d
6
, 300 MHz) d 8.83 (brs, 1H,
H), 2.79 (s, 2H); LC-MS: m/z 513 (Mþ1).
exchangeable), 8.50 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 6.0 Hz),
7
.66 (dt, 4H, J ¼ 9.0, J ¼ 6.0 Hz), 7.46 (s, 2H), 7.30 (t, 2H, J ¼ 9.0 Hz),
4
5
.4.7. 4-Fluoro-N-(2-(4-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-
-yl)pyrimidin-2-ylamino)ethyl)benzenesulfonamide (8g)
7.05 (d, 2H, J ¼ 6.0 Hz), 6.28 (d, 1H, J ¼ 6.0 Hz), 3.79 (s, 3H), 2.81 (brs,
2H), 2.50 (brs, 2H), 1.68 (brs, 2H); LC-MS: m/z 539 (Mþ1).
ꢀ
1
mp 235e236 C; H NMR (DMSO-d
6
, 300 MHz) d 8.82 (brs, 1H,
exchangeable), 8.48 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 3.4 Hz),
4.4.15. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)propyl)-3,4-dimethoxybenzenesulfonamide
7
.85 (t, 3H, J ¼ 5.9 Hz), 7.62 (t, 2H, J ¼ 6.0 Hz), 7.45e7.26 (m, 6H),
6
.29 (d, 1H, J ¼ 3.8 Hz), 2.96 (s, 2H); LC-MS: m/z 513 (Mþ1).
(8o)
1
Colorless viscous oil; H NMR (DMSO-d
6
, 300 MHz)
d
8.84 (brs,
4
.4.8. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
1H, exchangeable), 8.55 (brs, 1H, exchangeable), 8.07 (d, 1H,
J ¼ 6.0 Hz), 7.65e7.61 (m, 3H), 7.45 (d, 1H, J ¼ 6.0 Hz), 7.34 (dd, 2H,
J ¼ 3.0, J ¼ 6.0 Hz), 7.29 (d, 1H, J ¼ 3.0 Hz), 7.28 (d, 1H, J ¼ 3.0 Hz),
7.07 (d, 1H, J ¼ 6.0 Hz), 6.28 (d, 1H, J ¼ 6.0 Hz), 3.79 (s, 3H), 3.78 (s,
3H), 2.83 (t, 2H, J ¼ 6.0 Hz), 2.51 (t, 2H, J ¼ 6.0 Hz), 1.65 (p, 2H,
J ¼ 6.0 Hz); LC-MS: m/z 569 (Mþ1).
pyrimidin-2-ylamino)ethyl)-4-iodobenzenesulfonamide (8h)
ꢀ
1
mp 213e214 C; H NMR (DMSO-d
6
, 300 MHz) d 8.80 (brs, 1H,
exchangeable), 8.47 (brs, 1H, exchangeable), 8.06 (d, 1H, J ¼ 5.2 Hz),
7
.89 (d, 2H, J ¼ 8.3 Hz), 7.63 (q, 3H, J ¼ 4.8 Hz), 7.53 (d, 2H,
J ¼ 8.4 Hz), 7.46 (s, 1H), 7.45 (d, 2H, J ¼ 8.8 Hz), 6.29 (d, 1H,
J ¼ 5.1 Hz), 2.98 (t, 2H, J ¼ 6.4 Hz), 2.50 (s, 2H). LC-MS: m/z 621
(
Mþ1).
4.4.16. 3-Fluoro-N-(3-(4-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-
5
-yl)pyrimidin-2-ylamino)propyl)benzenesulfonamide (8p)
ꢀ
1
4.4.9. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
mp 209e210 C; H NMR (DMSO-d , 300 MHz) d 8.84 (brs, 1H,
6
pyrimidin-2-ylamino)ethyl)-4-(trifluoromethyl)
benzenesulfonamide (8i)
exchangeable), 8.54 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 6.0 Hz),
7.80 (s, 1H), 7.62e7.56 (m, 5H), 7.45 (d, 2H, J ¼ 3.0 Hz), 7.30 (t, 2H,
J ¼ 9.0 Hz), 6.28 (d, 1H, J ¼ 3.0 Hz), 3.35 (brs, 2H), 2.89 (brs, 2H), 1.69
(brs, 2H).
ꢀ
1
mp 164e165 C; H NMR (DMSO-d
6
, 300 MHz) d 8.84 (brs, 1H,
exchangeable), 8.45 (brs, 1H, exchangeable), 8.03 (q, 4H, J ¼ 6.5 Hz),
7
.92 (d, 2H, J ¼ 8.3 Hz), 7.61 (t, 2H, J ¼ 7.1 Hz), 7.46 (s,1H), 7.29 (t, 2H,
J ¼ 8.8 Hz), 6.28 (d, 1H, J ¼ 4.9 Hz), 3.40 (s, 2H), 3.01 (s, 2H). LC-MS:
4.4.17. 4-Fluoro-N-(3-(4-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-
m/z 563 (Mþ1).
5-yl)pyrimidin-2-ylamino)propyl)benzenesulfonamide (8q)
1
6
H NMR (DMSO-d , 300 MHz) d 8.84 (brs, 1H, exchangeable),
4
.4.10. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
8.54 (brs, 1H, exchangeable), 8.03 (d, 1H, J ¼ 5.3 Hz), 7.65 (d, 4H,
J ¼ 7.7 Hz), 7.53 (s, 1H), 7.45 (s, 1H), 7.32 (t, 4H, J ¼ 7.0 Hz), 6.28 (d,
1H, J ¼ 4.8 Hz), 2.82 (s, 2H), 2.54 (s, 2H), 1.70e1.66 (m, 2H).
pyrimidin-2-ylamino)ethyl)naphthalene-2-sulfonamide (8j)
ꢀ
1
mp 158e159 C; H NMR (DMSO-d
6
, 300 MHz) d 8.74 (brs, 2H,
exchangeable), 8.40 (s, 1H), 8.03 (t, 4H, J ¼ 6.1 Hz), 7.91 (s, 1H), 7.81
(
3
d, 2H, J ¼ 6.9 Hz), 7.59 (s, 5H), 7.43 (s, 1H), 7.28 (s, 2H), 6.21 (s, 1H),
4.4.18. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
.02 (brs, 2H), 2.49 (brs, 2H).). LC-MS: m/z 545 (Mþ1).
pyrimidin-2-ylamino)propyl)-4-iodobenzenesulfonamide (8r)
ꢀ
1
6
mp 206e207 C; H NMR (DMSO-d , 300 MHz) d 8.84 (brs, 1H,
4
.4.11. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
exchangeable), 8.49 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 6.0 Hz),
7.91 (d, 2H, J ¼ 9.0 Hz), 7.72 (s, 1H), 7.64 (dd, 2H, J ¼ 3.0, J ¼ 6.0 Hz),
7.52 (t, 2H, J ¼ 9.0 Hz), 7.45 (s, 1H), 7.30 (t, 2H, J ¼ 9.0 Hz), 6.29 (d,
1H, J ¼ 6.0 Hz), 2.85 (brs, 2H), 2.50 (brs, 2H), 1.67 (t, 2H, J ¼ 6.0 Hz);
LC-MS: m/z 635 (Mþ1).
pyrimidin-2-ylamino)propyl)benzenesulfonamide (8k)
ꢀ
1
mp 188e189 C; H NMR (DMSO-d
exchangeable), 8.48 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 5.3 Hz),
.78 (d, 2H, J ¼ 7.8 Hz), 7.65e7.55 (m, 6H), 7.46 (d, 1H, J ¼ 3.9 Hz),
6
, 300 MHz) d 8.82 (brs, 1H,
7
7
2
.30 (t, 2H, J ¼ 8.8 Hz), 6.28 (d, 1H, J ¼ 5.1 Hz), 3.31 (s, 2H), 2.86 (t,
H, J ¼ 6.3 Hz), 1.69e1.66 (m, 2H).
4.4.19. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)propyl)-4-(trifluoromethyl)
benzenesulfonamide (8s)
4
.4.12. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
ꢀ
1
pyrimidin-2-ylamino)propyl)-4-methylbenzenesulfonamide (8l)
mp 118e120 C; H NMR (DMSO-d , 300 MHz) d 8.82 (brs, 1H,
6
ꢀ
1
mp 210e211 C; H NMR (DMSO-d
exchangeable), 8.49 (brs, 1H, exchangeable), 8.06 (d, 1H, J ¼ 5.1 Hz),
.63 (d, 4H, J ¼ 7.3 Hz), 7.54 (s, 1H), 7.45 (s, 2H), 7.31 (t, 4H,
J ¼ 6.5 Hz), 6.28 (s, 1H), 3.30 (s, 2H), 2.82 (brs, 2H), 2.32 (brs, 3H),
.69e1.67 (m, 2H); 13C NMR (DMSO-d
, 75 MHz) 163.8, 162.5,
6
, 300 MHz)
d
8.84 (brs, 1H,
exchangeable), 8.45 (brs, 1H, exchangeable), 8.07 (d, 1H, J ¼ 5.3 Hz),
7.99 (t, 4H, J ¼ 7.6 Hz), 7.80 (brd, 1H, J ¼ 8.1 Hz), 7.62 (t, 2H,
J ¼ 7.1 Hz), 7.46 (d, 1H, J ¼ 3.8 Hz), 7.29 (t, 2H, J ¼ 8.8 Hz), 6.28 (d,1H,
J ¼ 4.3 Hz), 2.92e2.88 (t, 2H, J ¼ 6.9 Hz), 2.50 (s, 2H), 1.70 (brs, 2H).
LC-MS: m/z 577 (Mþ1).
7
1
6
d

462
M.S. Abdel-Maksoud et al. / European Journal of Medicinal Chemistry 95 (2015) 453e463
4.4.20. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
phosphoric acid and once in methanol prior to drying and scintil-
pyrimidin-2-ylamino)propyl)naphthalene-2-sulfonamide (8t)
lation counting.
ꢀ
1
mp 198e199 C; H NMR (DMSO-d
exchangeable), 8.42 (d, 1H, J ¼ 8.6 Hz), 8.06 (q, 3H, J ¼ 8.4 Hz), 7.97
d, 1H, J ¼ 7.8 Hz), 7.80 (d, 2H, J ¼ 8.6 Hz), 7.71e7.57 (m, 5H), 7.41 (s,
H), 7.30 (t, 2H, J ¼ 8.7 Hz), 6.25 (d, 1H, J ¼ 4.6 Hz), 3.29 (s, 2H), 2.91
6
, 300 MHz) d 8.79 (brs, 2H,
4.8. MEK/ERK kinase screening
(
1
4.8.1. Protein immunoblotting and immunoprecipitation
For immunoblotting, A375 melanoma cells grown to 70%e80%
confluence were harvested in RIPA lysis buffer and disrupted by
sonication and centrifuged at 12,000 rpm for 10 min. The quantity
of protein was determined with DC protein assay kit (Bio-Rad Lab.,
Hercules, CA). Protein samples were subjected to SDS-PAGE and
immunoblotting.
13
(
t, 2H, J ¼ 6.6 Hz), 1.70 (p, 2H, J ¼ 6.0 Hz). C NMR (DMSO-d
5 MHz) 163.8, 162.5, 161.4, 158.0, 156.2, 151.6, 148.1, 137.9, 134.5,
32.2, 131.8, 131.7, 129.8, 129.5, 129.1, 128.2, 127.9, 127.7, 122.7, 121.0,
16.2, 116.0, 115.8, 105.7, 41.1, 38.7, 30.0.
6
,
7
1
1
d
4.5. Preparation of N-(2-(4-(6-(4-fluorophenyl)imidazo[2,1-b]
thiazol-5-yl)pyrimidin-2-ylamino)ethyl)-4-
hydroxybenzenesulfonamide (8u) and N-(3-(4-(6-(4-fluorophenyl)
imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-ylamino)propyl)-4-
hydroxybenzenesulfonamide (8v)
4.8.2. Suppression of RAF-1/MEK/ERK signaling pathway in A375
cells
To assess the effect of compound 1u on the MEK/ERK kinases,
A375 cells were treated with compound 1u and Sorafenib (1, 3, and
A solution of the corresponding methoxy compound (8d or 8n,
10
mM) for 24 h and immunoblotted with antibodies against
0
.2 mmol) in anhydrous dichloromethane (3 mL) was cooled
phosphor-MEK1/2, phospho-ERK1/2 and b
-actin, respectively.
ꢀ
to ꢁ78 C, and BBr
3
(17% in DCM solution, 1.2 mL, 2.1 mmol) was
added dropwise thereto under nitrogen over a period of 10 min.
The reaction mixture was stirred at the same temperature for 1 h,
then the temperature was allowed to reach room temperature and
4.9. Cytotoxicity screening over L132 normal cells
4.9.1. Cell culture
kept overnight at same temperature. 10% Na
2
CO
3
solution (15 mL)
The human lung normal cell line (L132; human embryonic
pulmonary epithelial cells) were obtained from the Korean cell line
bank (KCLB, Seoul, Korea). Cells were cultured in RPMI 1640 sup-
plemented with 10% heat-inactivated FBS, penicillin (100 units/ml)
and streptomycin sulfate (100
in an atmosphere of 5% CO2.
and ethyl acetate (15 mL) were added, and the alkaline aqueous
layer was extracted with ethyl acetate (3 ꢂ 20). The combined
organic layer extracts were washed with brine, separated, and dried
ꢀ
mg/mL). Cells were cultured at 37 C
over anhydrous Na
2
SO
4
. The organic solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography to obtain the purified title compounds.
4
.9.2. Quantification of cytotoxicity
4
.5.1. N-(2-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
MTT assay was used to determine sample cytotoxicity. Cells
4
pyrimidin-2-ylamino)ethyl)-4-hydroxybenzenesulfonamide (8u)
(5 ꢂ 10 ) were seeded in each well containing 100
dium supplemented with 10% FBS in a 96-well plate. After 24 h,
various concentrations of sample were added. After 48 h, 20 L of
MTT (5 mg/mL stock solution, in phosphate buffered saline (PBS))
was added, and the plates were incubated for an additional 4 h. The
medium was discarded and the formazan blue, which was formed
in the cells, was dissolved with 200 mL DMSO. The optical density
was measured at 540 nm using microplate readers (Molecular
Devices, CA, USA).
mL of the me-
ꢀ
1
White solid; mp 194e195 C; H NMR (DMSO-d
10.34 (s, 1H), 8.85 (brs, 1H, exchangeable), 8.21 (brs, exchange-
able), 8.07 (d, 1H, J ¼ 5.2 Hz), 7.61 (d, 4H, J ¼ 8.4 Hz), 7.45 (s, 2H),
.30 (t, 2H, J ¼ 8.7 Hz), 6.87 (d, 2H, J ¼ 8.5 Hz), 6.29 (d, 1H,
J ¼ 5.0 Hz), 2.90 (t, 2H, J ¼ 6.0 Hz), 2.52 (brs, 2H). LC-MS: m/z 511
Mþ1).
6
, 300 MHz)
d
m
7
(
4.5.2. N-(3-(4-(6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)propyl)-4-hydroxybenzenesulfonamide (8v)
1
H NMR (DMSO-d
6
, 300 MHz)
d
8.08 (d, 1H, J ¼ 5.3 Hz),
Acknowledgments
7
6
2
.65e7.57 (m, 4H), 7.46 (d, 1H, J ¼ 4.5 Hz), 7.30 (t, 3H, J ¼ 8.8 Hz),
.86 (d, 2H, J ¼ 7.6 Hz), 6.29 (d, 1H, J ¼ 6 Hz), 5.75 (s, 1H), 3.17 (brs,
This work was supported by Korea Institute of Science and
Technology (KIST), Seoul, Republic of Korea, KIST Project (2E24680).
We would like to thank the National Cancer Institute (NCI),
Bethesda, Maryland, USA, for performing the in vitro anticancer
testing over the cell lines.
H), 2.79 (t, 2H, J ¼ 6.0 Hz), 1.68 (p, 2H, J ¼ 6.0 Hz).
4
.6. Cancer cell line screening at the NCI
Screening against the cancer cell lines was carried out at the
National Cancer Institute (NCI), Bethesda, Maryland, USA [25]
applying the standard protocol of the NCI [28].
Appendix A. Supplementary data
Supplementary data related to this article containing charts of
the NCI-57 cell line screening results, and NMR and mass spectra
can be found at http://dx.doi.org/10.1016/j.ejmech.2015.03.065.
4
.7. B-RAF, V600E-B-RAF, and C-RAF kinase profiling
Reaction Biology Corp. Kinase HotSpotSM service [29] was used
for screening of compound 1g. Assay protocol: In a final reaction
volume of 25 L, kinase (5e10 mU) is incubated with 25 mM Tris
pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic protein, 10 mM
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