New imidazo[2,1-b]thiazole derivatives: Synthesis, in vitro anticancer evaluation, and in silico studies

Article abstract of DOI:10.1016/j.ejmech.2011.08.024

A series of 18 new imidazo[2,1-b]thiazole derivatives was synthesized. Their in vitro antiproliferative activities against A375P human melanoma cell line and NCI-60 cell line panel were tested. Compounds 15, 16, 18, 22, 26-28, and 31 showed superior poten

Full text of DOI:10.1016/j.ejmech.2011.08.024

European Journal of Medicinal Chemistry 46 (2011) 5769e5777
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Original article
New imidazo[2,1-b]thiazole derivatives: Synthesis, in vitro anticancer evaluation,
and in silico studies
,
,
b
,
,b,
Jin-Hun Park a c, Mohammed I. El-Gamal a d, Yong Sup Lee c, Chang-Hyun Oh a
*
a Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
b Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
c Department of Pharmaceutical Science, College of Pharmacy & Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong Dongdaemoon-ku,
Seoul 130-701, Republic of Korea
d Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 18 new imidazo[2,1-b]thiazole derivatives was synthesized. Their in vitro antiproliferative
activities against A375P human melanoma cell line and NCI-60 cell line panel were tested. Compounds
15, 16, 18, 22, 26e28, and 31 showed superior potency against A375P to sorafenib. In addition,
compounds 26 and 27 showed selectivity toward melanoma cell lines than for other cancer types. Both
compounds exerted sub-micromolar IC50 values over 7 (including A375P) and 6 melanoma cell lines,
respectively. In silico studies are also reported. ADME profiling, in silico toxicity, drug-likeness, and drug-
score data of compounds 26 and 27 are promising.
Received 11 July 2011
Received in revised form
11 August 2011
Accepted 16 August 2011
Available online 23 August 2011
Keywords:
Anticancer
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antiproliferative activity
Imidazo[2,1-b]thiazole
A375P
Melanoma
1. Introduction
developed countries, there is an urgent need to develop more
effective drugs [12e14].
Much attention has been paid to the chemistry and cytotoxicity
of imidazo[2,1-b]thiazole derivatives. Imidazo[2,1-b]thiazole
derivatives exhibited good antiproliferative activities against
a variety of human cancer cell lines [1e6].
In the present investigation, we report the synthesis, in vitro
antiproliferative activities against 61 cell lines, and in silico studies
of a new series of pyrimidinyl-imidazo[2,1-b]thiazole derivatives.
Recently, some pyrimidinyl substituted imidazo[2,1-b]thiazole
derivatives were reported as RAF kinases inhibitors [7]. It is well
known that inhibition of RAF kinases has good impact against
different cancer types including melanoma [8]. Melanoma is the
most aggressive form of skin cancer and is the fastest growing
cancer in the United States [9,10]. Early stage melanoma can be
cured surgically. However, melanoma metastasizing to major
organs (stage IV) is virtually incurable [10]. Patients with advanced
melanoma have a median survival time of less than one year, and
the estimated 5-year survival rate is less than 15% [9,11]. With the
incidence of melanoma rapidly rising in the United States and other
2. Results and discussion
2.1. Chemistry
For preparation of the target compounds, it was important at
the beginning to synthesize the key methylsulfonyl intermediate
compound 4. It was successfully synthesized as illustrated in
Scheme 1. Upon refluxing 2-aminothiazole (1) with
a-bromo-3-
methoxyacetophenone, cyclization to 6-(3-methoxyphenyl)imi-
dazo[2,1-b]thiazole (2) occurred [15]. Heating 2 with 4-iodo-2-
(methylthio)pyrimidine in the presence of palladium acetate,
cesium carbonate, and triphenylphosphine afforded the corre-
sponding methylthiopyrimidinyl compound 3. Oxidation of the
sulfide moiety of 3 using oxone gave the corresponding sulfonyl
compound 4.
* Corresponding author. Center for Biomaterials, Korea Institute of Science and
Technology, 39-1 awolgok-dong, PO Box 131, Cheongryang, Seoul 130-650, Republic
of Korea. Tel.: þ82 2 958 5160; fax: þ82 2 958 5189.
Besides the key intermediate compound 4, we had to prepare
the reagents required for introduction of the side chain of the target
E-mail address: choh@kist.re.kr (C.-H. Oh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.024

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J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
O
O
N
N
S
S
O
NH2
N
S
N
N
N
a
c
b
S
N
N
N
N
N
O
S
S
O
1
2
3
4
Scheme 1. Reagents and conditions: a)
a
-bromo-3-methoxyacetophenone, EtOH, reflux, 16 h; b) 4-iodo-2-(methylthio)pyrimidine, Pd(OAc)2, Cs2CO3, PPh3, DMF, 80 ꢀC, 12 h; c)
oxone, MeOH, H2O, rt, 16 h.
a
b
H2N
c
d
Cbz
N3
Cbz
OH
Cbz
OMs
OH
N
H
N
H
N
H
5
7
8
6
H
N
H
H
N
H
N
g
g
N
Cbz
H2N
R
R
N
H
R
e
O
O
10a-c
11a-c
Cbz
N
NH2
f
H
H
N
9
H
N
H2N
Cbz
R
N
O
H
O
13a-f
12a-f
Scheme 2. Reagents and conditions: a) benzyloxycarbonyl chloride, TEA, CH2Cl2, 0 ꢀC; b) methanesulfonyl chloride, TEA, CH2Cl2, 0 ꢀC; c) NaN3, DMSO, 70 ꢀC, 2 h; d) PPh3, MeOH,
H2O, reflux, 2 h; e) appropriate isocyanate, THF, rt, 2 h; f) appropriate carboxylic acid, HOBt, EDCI, TEA, DMF, 80 ꢀC, 8 h; g) H2/PdeC, MeOH, rt, 1 h.
compounds. These reagents were prepared according to the
sequences of reactions illustrated in Scheme 2. Interaction of 2-
aminoethanol (5) with benzyl chloroformate in the presence of
triethylamine produced the N-Cbz protected compound 6 [16].
Treatment of 6 with mesyl chloride in the presence of triethylamine
afforded the corresponding methylsulfonyl compound 7 [17].
Treatment of 7 with sodium azide led to formation of azido
compound 8. Reduction of the azido group of 8 using PPh3/H2O
afforded the corresponding amino compound 9, which was
subsequently treated with the appropriate isocyanates to produce
the corresponding urea derivatives 10aec. The amide derivatives
12aef were obtained by condensation of 9 with the appropriate
S
S
S
N
N
N
O
O
HO
N
N
N
a
c
N
N
N
H
N
H
N
H
N
H
N
O
S
N
N
N
H
R
N
N
H
R
O
O
O
4
17-19
14-16
b
S
S
N
N
HO
O
N
N
N
c
N
H
H
N
N
R
R
N
N
H
N
N
H
O
O
20-25
26-31
Scheme 3. Reagents and conditions: a) 11aec, DIPEA, DMSO, 80 ꢀC, 8 h; b) 13aef, DIPEA, DMSO, 80 ꢀC, 8 h; c) BBr3, CH2Cl2, ꢁ78 ꢀC, 30 min then rt, 1 h.

J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
5771
Table 1
Antiproliferative activity of urea derivatives 14e19 against A375P cell line.
Structure
Compound No.
R1
R2
IC50 (m
M)a
S
R1O
N
14
15
16
17
18
19
Sorafenib
CH3
CH3
CH3
H
H
H
H
3-CF3
3,5-bis(CF3)
H
3-CF3
>10
5.3
4.3
>10
4.2
>10
5.6
N
N
H
N
H
N
N
N
H
R2
3,5-bis(CF3)
O
a
IC50 values are expressed as means of triplicate experiments.
Table 2
Antiproliferative activity of amide derivatives 20e31 against A375P cell line.
Structure
Compound No.
R1
R2
IC50 (m
M)a
20
21
22
23
24
25
26
CH3
CH3
CH3
CH3
CH3
CH3
H
H
2-OH
3-CF3
>10
7.5
4.8
>10
>10
>10
S
3,5-bis(CF3)
R1O
N
3-CF3, 4-morpholino
3-CF3, 5-morpholino
H
N
0.5
N
27
28
29
30
31
Sorafenib
H
H
H
H
2-OH
3-CF3
3,5-bis(CF3)
3-CF3, 4-morpholino
3-CF3, 5-morpholino
3.2
2.1
>10
>10
4.4
5.6
R2
H
N
N
N
H
O
H
a
IC50 values are expressed as means of triplicate experiments.
benzoic acid derivatives using HOBt/EDCI/TEA. Deprotection of
compounds 10aec and 12aef using H2/PdeC afforded the corre-
sponding amino compounds 11aec and 13aef, respectively [6].
Heating compound 4 with the previously prepared reagents
11aec and 13aef in the presence of DIPEA produced the target
methoxy compounds 14e16 and 20e25, respectively. Demethyla-
tion of the methoxy group of 14e16 and 20e25 using boron tri-
bromide afforded the corresponding hydroxyl target compounds
17e19 and 26e31, respectively (Scheme 3).
corresponding urea compounds 15, 17, and 18. These results indi-
cate the effect of the linker on the activity.
Upon investigating the effect of substituents on the terminal
phenyl ring on activity, it was found that m-(trifluoromethyl)
phenyl-containing derivatives 15,18, and 22 were more potent than
the corresponding derivatives without m-trifluoromethyl group
(14, 17, and 20). In addition, m-CF3-possessing compounds 22 and
28 demonstrated higher potency than o-hydroxy compounds 21
and 27. This may be due to the steric and/or electronic effects of
trifluoromethyl group compared with hydroxyl group. While the
2.2. Biological evaluation
Table 3
Mean inhibition percentages observed with the final compounds in
2.2.1. Antiproliferative activity against A375P human melanoma
cell line
single dose (10
m
M) 60-cancer cell line screening.
Compound No.
Mean % inhibitiona
The antiproliferative activity of the newly synthesized
compounds against A375P human melanoma cell line was exam-
ined. The ability of target imidazo[2,1-b]thiazole compounds to
inhibit the growth of A375P cell line is summarized in Tables 1 and
2. Sorafenib was utilized as the reference standard.
As shown in Tables 1 and 2, many compounds showed moderate
potency against A375P cell line. Compounds 15, 16, 18, 22, 26e28,
and 31 showed higher potency, compared with sorafenib. The
highest potency was found in case of compounds 26 and 28
14
15
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
4.51
44.00
16.88
43.35
30.00
22.58
19.53
34.16
15.36
22.99
17.01
35.67
49.21
25.79
4.76
(IC50 ¼ 0.5 and 2.1
mM, respectively).
Compounds 18, 26e28, and 31 with m-hydroxyphenyl ring at
position 6 on the imidazothiazole nucleus showed higher potency
than the corresponding methoxy derivatives 15, 20e22, and 25.
This may be attributed to additional hydrogen bond formation by
hydroxyl group at the receptor site.
34.33
18.27
Upon comparing the potencies of derivatives with urea
and amide moieties at the side chain as a linker, it was found that
amide derivatives 22, 26, and 28 were more potent than the
a
Mean % inhibition represents the mean inhibition percentages
over the 60 cell lines. The inhibition percentages are calculated by
subtracting the growth percentages from 100.

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J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
Table 4
evaluation of their antineoplastic activity. The selected compounds
Mean IC50 values (
m
M) of the tested compounds over in vitro subpanel cancer cell
were subjected to in vitro anticancer assay against tumor cells in
a full panel of 60 cell lines taken from nine different tissues (blood,
lung, colon, CNS, skin, ovary, kidney, prostate and breast). The
lines.a
Compound No.
compounds were tested at a single dose concentration of 10 mM,
15
18
26
8.14
27
and the percentages of growth inhibition over the 60 tested cell
lines were determined. The mean inhibition percentages of all of
the tested compounds over the full panel of cell lines are illustrated
in Table 3. The figures showing the multiple inhibitions exerted by
the tested compounds are illustrated in the Supplementary data.
By comparing the structures with mean % inhibitions summa-
rized in Table 3, we find that the derivatives with m-hydroxyphenyl
ring at position 6 on the imidazo[2,1-b]thiazole nucleus 17, 26, 27,
and 30 demonstrated higher inhibition than the corresponding
methoxy compounds 14, 20, 21, and 24. The amide derivatives 20
and 26 showed higher mean % inhibition than the corresponding
urea derivatives 14 and 17. On the other hand, urea compounds 15,
18, and 19 were more active than the corresponding amides 22, 28,
and 29. 3-CF3 substituent on the terminal phenyl ring significantly
enhanced the activity of compounds 15, 18, and 22 compared with
the corresponding derivatives lacking this group 14, 17, and 20. The
increased bulkiness induced by additional group, CF3 or morpho-
lino, together with the 3-CF3 on the terminal phenyl ring reduced
the activity of 19 compared with 18, 23e25 compared with 22, in
addition to 29 and 31 compared with 28.
Subpanel
Leukemia
3.13 3.62
4.19
7.41
cancer cell lines Non-small cell 3.97 5.03 13.37
lung cancer
Colon cancer
CNS cancer
Melanoma
3.83 3.42 11.19
4.39 5.54 15.05
3.60 2.50
4.79
6.73
1.40 (4.25)b 0.79 (4.14)b
Ovarian cancer 4.07 9.96 15.67
Renal cancer 3.06 5.72 7.59
Prostate cancer 3.80 4.16 15.80
Breast cancer 3.23 3.77 5.95
7.06
6.46
5.19
3.27
a
Mean IC50 values were calculated by dividing the summation of IC50 values of
the compound over cell lines of the same cancer type by the number of cell lines in
the subpanel.
b
Selectivity ratios are shown in parentheses.
insertion of another group together with CF3, such as another CF3
group or morpholino, decreased the potency of 19 compared with
18, 23e25 compared with 22, and 29e31 compared with 28. This
effect may be due to increased bulkiness which may sterically
hinder the appropriate drugereceptor interaction.
After this initial single dose screening of the 17 selected
compounds, compounds 15, 18, 26, and 27 which showed the
highest activity at the single dose were further tested in a five-dose
testing mode, in order to determine their IC50 values over the 60
tumor cell lines. The mean IC50 values of these four compounds over
the nine cancer types are shown in Table 4. The figures and tables
showing 5-dose results in details are illustrated in the
Supplementary data.
As shown in Table 4, compounds 15 and 18 exhibited good
potency (in micromolar scale) over all the cancer types. Interest-
ingly, compounds 26 and 27 demonstrated high selectivity against
melanoma, compared with other cancer types. The selectivity ratios
of 26 and 27 were 4.25 and 4.14, respectively, as compared with the
second most susceptible cancer type, breast cancer. The mean IC50
of compound 27 over melanoma subpanel was in sub-micromolar
2.2.2. Antiproliferative activities against 60 cell line panel at the NCI
Structures of the target compounds were submitted to National
Cancer Institute (NCI) [18], Bethesda, Maryland, USA, and the 17
compounds shown in Table 3 were selected on the basis of degree
of structural variation and computer modeling techniques for
Table 5
IC50 values (mM) of compounds 26 and 27 over the NCI nine melanoma cell line
panel.
Melanoma cell line
Compound No.
26
27
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
3.22
2.65
0.205
0.434
0.578
2.94
0.336
4.15
0.152
0.335
0.433
NAa
0.683
1.22
range, 0.79 mM.
The IC50 values of compounds 26 and 27 over nine melanoma
cell lines tested at the NCI are shown in Table 5. The IC50 of both
0.489
0.211
0.651
0.161
compounds was in sub-micromolar scale over
6 cell lines.
Compound 27 was generally more potent than 26. The o-hydroxyl
a
Not tested.
Table 6
Molar refractometry, solubility, and calculated Lipinski’s rule of five for target compounds with the highest antiproliferative activities.
Compd. No.
MRa
Log Sb
Parameter
c log Pc
TPSAd
MWe
nONf
nOHNHg
nviolations
15
16
18
22
26
27
28
31
142.18
148.68
136.74
139.30
127.36
129.18
133.87
158.10
ꢁ5.49
ꢁ6.27
ꢁ5.18
ꢁ4.97
ꢁ3.88
ꢁ3.59
ꢁ4.66
ꢁ4.76
3.73
4.49
3.53
3.84
2.88
2.58
3.64
3.03
105.48
105.48
116.47
93.45
104.44
124.67
104.44
116.92
553.56
621.56
539.53
538.54
456.52
472.52
524.52
609.62
9
9
9
8
8
9
8
10
3
3
4
2
3
4
3
3
1
2
1
1
0
0
1
1
a
Molar refractometry (cm3/mol).
Solubility parameter.
b
c
d
e
f
Calculated lipophilicity.
Total polar surface area (Ǻ2).
Molecular weight.
Number of hydrogen bond acceptors.
Number of hydrogen bond donors.
g

J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
5773
Toxicity effects
Compd.
No.
15
Drug-Likeness
M
T
I
R
Low Low Low High
Low Low Low High
Low Low Low Low
Low Low Low High
Low Low Low Low
Low Low Low Low
Low Low Low Low
Low Low Low Low
-3.88
-19.7
-3.55
-7.52
3.09
Drug-score
16
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
18
22
26
27
3.14
15
16
18
22
26
27
28
31
Sorafenib
28
-7.19
-13.9
-4.20
31
Sorafenib Low Low Low Low
Fig. 1. In silico toxicity risks and drug-likeness (left panel), and drug-score (right panel) of Sorafenib and the potent antiproliferative imidazo[2,1-b]thiazole derivatives (M,
mutagenic; T, tumorigenic; I, irritant; R, reproductive).
group on the terminal phenyl ring of 27 may form additional
hydrogen bond(s) at the receptor site, which strengthen the
drugereceptor interaction and hence the potency.
presence of some fragments generally responsible for the muta-
genic, tumorigenic, irritant, or reproductive effects in these
molecules. Interestingly, compounds 18, 26e28, and 31 presented
a low in silico toxicity risk profile, similar to sorafenib (Fig. 1).
These theoretical data reinforced the cytotoxicity experimental
data described in this work pointing these compounds as lead
compounds with low toxicity risk profile. On the other hand,
compounds 15, 16, and 22 have high in silico reproductive toxicity
risk due to the methoxyphenyl ring. So it can be concluded that
compounds with hydroxyphenyl ring at position 6 of imidazo[2,1-
b]thiazole nucleus are safer than the corresponding methoxy
derivatives.
2.2.3. Molar refractometry and Lipinski’s rule of five
In this study, we evaluated the influence of steric factors on the
activity. Molar refractometry (MR, steric parameter) values were
determined for the compounds with high activity and are pre-
sented in Table 6. Compounds 26e28 with the lowest MR values
showed the highest potency over melanoma cell lines. The MR
value of 18 was lower than that of 15 and the potency of 18 against
A375P melanoma cell line was higher. Compound 31, whose MR
value is higher than those of compounds 26e28, was less potent
Currently, there are many approaches to assess a compound
drug-likeness based on topological descriptors, fingerprints of
molecular drug-likeness structure keys or other properties such as
c log P and molecular weight [22]. In this work, Osiris program [21]
was also used for calculating the fragment-based drug-likeness of
the most active compounds and comparing them with sorafenib.
Compounds 15 and 18 showed a little improvement of drug-
likeness values than sorafenib. Compounds 26 and 27 demon-
strated a significantly higher drug-likeness values, compared with
sorafenib. The drug-scores of the potent compounds have also been
determined in the present study. The results showed that
compounds 18, 26e28, and 31 have higher values than sorafenib. Of
special interest, the drug-score values of compounds 26 and 27
(0.66 and 0.67, respectively) were significantly more than that of
sorafenib (0.20). The drug-likeness and drug-score results are
illustrated in Fig. 1. The in silico toxicity profiles, drug-likeness, and
drug-score data of compounds 26 and 27 make these two
compounds promising leads for future development of safe and
efficient antiproliferative agents.
against melanoma and its mean % inhibition at 10 mM over the
NCI-60 cell line panel was less than those of 26e28. From these
results, we can conclude that MR and bulkiness are inversely
proportional to the antiproliferative activity of this series of
compounds.
The bioavailability of compounds 15, 16, 18, 22, 26e28, and 31
with the highest activities against both A375P and NCI-60 cell lines
was assessed using ADME (absorption, distribution, metabolism,
and excretion) prediction methods. In particular, we calculated the
compliance of compounds to the Lipinski’s rule of five [19]. This
approach has been widely used as a filter for substances that would
likely be further developed in drug design programs. In addition,
we calculated the total polar surface area (TPSA) since it is another
key property that has been linked to drug bioavailability. Thus,
passively absorbed molecules with a TPSA > 140 are thought to
have low oral bioavailability [20]. Molecules violating more than
one of these rules may have problems with bioavailability. Predic-
tions of ADME properties for the studied compounds are given in
Table 6. The results showed that all the tested compounds, except
16 which violated two rules, comply with these rules. Theoretically,
compounds 15, 18, 22, 26e28, and 31 should present good passive
oral absorption and differences in their bioactivity cannot be
attributed to this property.
3. Conclusion
A new series of pyrimidinyl-imidazo[2,1-b]thiazole deriva-
tives was synthesized based on our previous literature studies.
Among all of these derivatives, compounds 15, 16, 18, 22, 26e28,
and 31 demonstrated higher potencies against A375P human
melanoma cell line than that of the reference compound, sor-
afenib. Seventeen derivatives were also tested at a single dose of
2.2.4. In silico toxicities, drug-likeness, and drug-score profiles
Osiris program [21] was used for prediction of the overall
toxicity of the most active derivatives as it may point to the

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J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
10
m
M at the NCI over 60 cell line panel, and compounds 15, 18,
4.4. 6-(3-Methoxyphenyl)-5-(2-(methylsulfonyl)pyrimidin-4-yl)
26, and 27 were subsequently tested in 5-dose testing mode.
Compounds 26 and 27 were interestingly more selective for
melanoma cell lines than for other cancer types. At the NCI 9
melanoma cell line subpanel, both compounds showed sub-
micromolar IC50 values over 6 cell lines. The in silico ADME
profiling, toxicity, drug-likeness, and drug-score results together
with in vitro antiproliferative activities make compounds 26 and
27 promising leads for development of selective and potent
agents for treatment of melanoma. Further modifications of
these compounds in order to improve their potency are currently
in progress.
imidazo[2,1-b]thiazole (4)
To a solution of compound 3 (2.05 g, 6 mmol) in methanol
(250 mL), a solution of oxone (12.3 g, 18 mmol) in water (50 mL)
was added. The mixture was stirred at room temperature for 16 h.
The organic solvent was evaporated under reduced pressure and
the remaining aqueous solution was extracted with CH2Cl2 (50 mL)
and the organic layer was separated. The aqueous layer was
extracted with CH2Cl2 (3 ꢂ 25 mL) and the combined organic layer
extracts were washed with brine, dried over anhydrous MgSO4, and
filtered. The organic solvent was evaporated under reduced pres-
sure and the residue was purified by flash column chromatography
(silica gel, ethyl acetate:hexane 1:3 v/v then switching to ethyl
acetate:hexane 1:1 v/v). Compound 4 was obtained (2.12 g, 95%). 1H
4. Experimental
NMR (CDCl3, 300 MHz)
d
8.80 (d, 1H, J ¼ 4.5 Hz), 8.50 (d, 1H,
4.1. General
J ¼ 5.6 Hz), 7.40e7.32 (m, 2H), 7.18 (dd, 2H, J ¼ 0.9 and 1.5 Hz),
7.08e7.02 (m, 2H), 3.85 (s, 3H), 3.37 (s, 3H); 13C NMR (CDCl3,
All the reagents and solvents were purchased from Sigma
Aldrich Chemical Co. and used without purification. The purity of
all final compounds was over 95% on the basis of LCeMS analysis.
The purity was determined by the following Waters LCeMS
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
75 MHz)
d 165.7, 162.3, 160.1, 157.5, 156.8, 153.2, 135.4, 130.2, 123.3,
121.3, 119.6, 117.7, 115.6, 114.1, 113.9, 55.5, 39.3.
4.5. Benzyl 2-hydroxyethylcarbamate (6) [16]
To a stirred solution of 2-aminoethanol (5, 4.94 mL, 81.86 mmol)
in CH2Cl2 (50 mL) at 0 ꢀC, triethylamine (22.2 mL, 159.6 mmol) was
added dropwise. Benzyloxycarbonyl chloride (15.2 mL,106.42 mmol)
was added dropwise over 30 min. After completion of the addition,
the mixture was stirred at 0 ꢀC for 1 h. The mixture was quenched
with water (50 mL) and extracted with CH2Cl2 (3 ꢂ 50 mL). The
combined organic layer extracts were washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash column chromatography
(silica gel, ethyl acetate:hexane 1:1 v/v). The desired product was
obtained as white solid (9.5 g, 59%). Mp 73e75 ꢀC; 1H NMR (CDCl3,
manager, SunfireÔ C18 column (4.6 ꢂ 50 mm, 5
mm particle size);
solvent gradient ¼ 95% @ at 0 min, 1% @ at 5 min; solvent A: 0.035%
trifluoroacetic acid (TFA) in water; solvent B: 0.035% TFA in MeOH;
flow rate ¼ 3.0 mL/min. The AUC was calculated using Waters
MassLynx 4.1 software. Melting points were obtained on a Walden
Precision Apparatus Electrothermal 9300 apparatus and are
uncorrected. 1H NMR spectra were obtained using a Bruker
300 MHz FT-NMR and a Bruker 400 MHz FT-NMR spectrometers.
Mass spectra (MS) were taken in ESI mode on a Waters 3100 Mass
Detector (Waters, Milford, MA, USA).
300 MHz)
d 7.34 (s, 5H), 5.19 (brs, 1H), 5.12 (s, 2H), 3.74 (t, 2H,
J ¼ 5.0 Hz), 3.37 (t, 2H, J ¼ 5.0 Hz), 2.31 (brs, 1H).
4.2. 6-(3-Methoxyphenyl)imidazo[2,1-b]thiazole (2) [15]
4.6. 2-(Benzyloxycarbonylamino)ethyl methanesulfonate (7) [17]
A solution of 2-aminothiazole (2.37 g, 23 mmol) and a-bromo-
3-methoxyacetophenone (5.0 g, 23 mmol) in ethanol (60 mL) was
heated under reflux for 16 h. The mixture was concentrated to
30 mL under reduced pressure. Ice water (40 mL) was added to the
remaining solution, then 30% ammonium hydroxide solution was
added. The buff colored solid formed was filtered, washed with
water, and dried overnight under vacuum at 50 ꢀC to get the title
compound (4.9 g, 90%). It was used in the next step without further
purification.
To a stirred solution of compound 6 (29.70 g, 152 mmol) in
CH2Cl2 (300 mL) at 0 ꢀC, triethylamine (31.58 mL, 227 mmol) was
added dropwise. Methanesulfonyl chloride (14.10 mL, 182 mmol)
was then added dropwise to the reaction mixture over 30 min.
After completion of the addition, the mixture was stirred at 0 ꢀC for
1 h. The mixture was quenched with water (300 mL) and extracted
with CH2Cl2 (3 ꢂ 300 mL). The combined organic layer extracts
were washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, ethyl acetate:hexane 1:2
v/v). The title product was obtained (22.0 g, 53%). Mp 70 ꢀC; 1H
4.3. 6-(3-Methoxyphenyl)-5-(2-(methylthio)pyrimidin-4-yl)
imidazo[2,1-b]thiazole (3)
NMR (CDCl3, 300 MHz) d 7.35 (s, 5H), 5.24 (brs,1H), 5.11 (s, 2H), 4.28
(t, 2H, J ¼ 5.0 Hz), 3.53 (q, 2H, J ¼ 5.3 Hz), 2.98 (s, 3H).
A mixture of compound 2 (6.0 g, 27.6 mmol), 4-iodo-2-(meth-
ylthio)pyrimidine (10.4 g, 41.3 mmol), cesium carbonate (13.4 g,
41.3 mmol), palladium acetate (1.22 g, 5.5 mmol), and triphenyl-
phosphine (2.896 g, 11.04 mmol) in anhydrous DMF (60 mL) was
stirred at 80 ꢀC for 12 h. The mixture was cooled to room temper-
ature and separated between ethyl acetate (150 mL) and water
(200 mL). The organic layer was separated and the aqueous layer
was extracted with ethyl acetate (3 ꢂ100 mL). The combined
organic layer extracts were washed with brine, dried over anhy-
drous Na2SO4, and filtered. The organic solvent was evaporated
under reduced pressure and the residue was purified by column
chromatography (silica gel, ethyl acetate:hexane 1:5 v/v). The
product was obtained (1.85 g, 20%). ESIeMS: 354.9 [M þ 1]þ.
4.7. Synthesis of compounds 8e10aec and 12aef
These compounds were synthesized according to the proce-
dures described in our previous article [6].
4.8. General procedure for synthesis of compounds 11aec and
13aef [6]
A mixture of compound 10aec or 12aef (0.52 mmol) and Pd/C
(10%) in MeOH (10 mL) was stirred under hydrogen atmosphere at
room temperature for 1 h. Pd/C was removed by celite filter, and the

J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
5775
filtrate was evaporated under reduced pressure. The product was
4.9.6. N-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
obtained and used in the next step without further purification.
pyrimidin-2-ylamino)ethyl)-3-(trifluoromethyl)benzamide (22)
Yield: 64.7%; mp 141 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 8.88 (brs,
1H), 8.18e8.09 (m, 3H), 7.56 (d, 1H, J ¼ 7.5 Hz), 7.70 (t, 1H,
J ¼ 7.7 Hz), 7.45 (d, 1H, J ¼ 4.4 Hz), 7.37 (t, 1H, J ¼ 8.1 Hz), 7.13 (d, 2H,
J ¼ 7.1 Hz), 7.10 (d, 2H, J ¼ 7.3 Hz), 6.37 (dd, 1H, J ¼ 1.0 and 1.0 Hz),
5.75 (s, 1H), 3.77 (s, 3H), 3.53 (brs, 4H); 13C NMR (DMSO-d6,
4.9. General procedure for synthesis of compounds 14e16 and
20e25
A mixture of compound 4 (0.34 g, 0.92 mmol), compound 11aec
or 13aef (2.48 mmol), and DIPEA (0.57 mL, 3.3 mmol) in DMSO
(10 mL) was stirred at 80 ꢀC for 8 h. The mixture 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 anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, ethyl acetate:hexane 1:2
v/v) to yield the desired compounds.
75 MHz) d 165.5, 162.7, 159.7, 158.1, 156.4, 151.6, 136.6, 135.8, 131.8,
130.1, 130.0, 129.8, 128.1, 126.2, 124.3, 121.8, 121.0, 114.7, 106.1, 55.6;
ESIeMS: 539.0 [M þ 1]þ.
4.9.7. N-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3,5-bis(trifluoromethyl)benzamide
(23)
Yield: 66.5%; mp 166 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 8.52 (d,
1H, J ¼ 4.5 Hz), 8.25 (s, 2H), 8.05 (d, 1H, J ¼ 5.5 Hz), 7.97 (s, 1H), 7.35
(t,1H, J ¼ 3.9 Hz), 7.17e7.15 (m, 2H), 6.98 (dd,1H, J ¼ 1.6 and 1.6 Hz),
6.91 (d, 1H, J ¼ 4.5 Hz), 6.63 (d, 1H, J ¼ 4.5 Hz), 5.65 (t, 1H,
J ¼ 5.8 Hz), 3.83 (s, 1H), 3.78e3.73 (m, 4H); ESIeMS: 607.5
[M þ 1]þ.
4.9.1. 1-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-phenylurea (14)
Yield: 68.3%; mp 146 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 8.52 (d,1H,
J ¼ 4.4 Hz), 7.95 (d, 1H, J ¼ 5.2 Hz), 7.54 (q, 2H, J ¼ 4.8 Hz), 7.38 (d,
1H, J ¼ 7.0 Hz), 7.40 (s, 4H), 7.11 (t, 2H, J ¼ 8.2 Hz), 7.10e6.50 (m, 2H),
6.88 (d, 1H, J ¼ 4.4 Hz), 6.45 (d, 1H, J ¼ 5.0 Hz), 5.88 (brs, 1H), 3.80 (s,
3H), 3.54 (t, 2H, J ¼ 5.0 Hz), 3.47 (d, 2H, J ¼ 4.8 Hz); ESIeMS: 486.0
[M þ 1]þ.
4.9.8. N-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(trifluoromethyl)-4-
morpholinobenzamide (24)
Yield: 62.0%; mp 165 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 8.49 (d,1H,
J ¼ 4.4 Hz), 8.07 (d, 1H, J ¼ 5.3 Hz), 8.01 (s, 1H), 7.81 (brs, 1H), 7.33 (t,
1H, J ¼ 8.1 Hz), 7.17e7.14 (m, 3H), 6.98 (dd, 1H, J ¼ 1.0 and 1.0 Hz),
6.90 (d, 1H, J ¼ 4.5 Hz), 6.60 (d, 1H, J ¼ 5.4 Hz), 5.73 (brs, 1H),
3.82e3.74 (m, 11H), 2.90 (m, 4H); ESIeMS: 624.0 [M þ 1]þ.
4.9.2. 1-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(3-(trifluoromethyl)phenyl)urea (15)
Yield: 65.3%; mp 151e152 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d
9.34 (s, 1H), 8.12e8.07 (m, 3H), 7.53 (s, 1H), 7.45 (brs, 1H), 7.38
(t, 1H, J ¼ 7.9 Hz), 7.15e7.12 (m, 2H), 7.01 (d, 1H, J ¼ 8.1 Hz), 6.64
4.9.9. N-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(trifluoromethyl)-5-
morpholinobenzamide (25)
(brs, 1H), 6.38 (d, 1H, J ¼ 5.3 Hz), 3.77 (s, 3H), 3.47e3.31 (m, 4H);
13C NMR (DMSO-d6, 100 MHz)
d 159.7, 156.6, 156.4, 155.7, 151.6,
141.8, 136.6, 130.3, 130.1, 129.9, 123.6, 121.9, 121.8, 121.6, 117.8,
115.6, 115.0, 114.9, 114.4, 106.5, 56.1, 55.8, 41.8; ESIeMS: 553.9
[M þ 1]þ.
Yield: 59.1%; mp 165 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 8.53 (d, 1H,
J ¼ 4.5 Hz), 8.05 (d, 1H, J ¼ 5.5 Hz), 7.57 (s, 1H), 7.36e7.31 (m, 2H),
7.18e7.15 (m, 3H), 6.97 (dd, 1H, J ¼ 2.4 and 2.5 Hz), 6.90 (d, 1H,
J ¼ 4.5 Hz), 6.60 (d, 1H, J ¼ 5.5 Hz), 5.69 (t, 1H, J ¼ 5.7 Hz), 3.88e3.81
(m, 7H), 3.78e3.72 (m, 4H), 3.25e3.19 (m, 4H); ESIeMS: 624.0
[M þ 1]þ.
4.9.3. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(4-(6-(3-
methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-ylamino)
ethyl)urea (16)
Yield: 71.3%; mp 155 ꢀC; 1H NMR (CDCl3, 300 MHz)
d 8.47 (d, 1H,
J ¼ 4.5 Hz), 8.39 (brs, 1H), 7.91 (d, 1H, J ¼ 5.4 Hz), 7.75 (s, 2H), 7.40 (s,
1H), 7.29 (t, 1H, J ¼ 8.1 Hz), 7.08e7.05 (m, 2H), 6.92 (dd, 1H, J ¼ 1.9
and 2.1 Hz), 6.83 (d, 1H, J ¼ 4.4 Hz), 6.37 (d, 1H, J ¼ 5.2 Hz), 6.16 (brs,
1H), 5.60 (brs, 1H), 3.79 (s, 3H), 3.60e3.59 (m, 2H), 3.48e3.46 (m,
2H); ESIeMS: 621.9 [M þ 1]þ.
4.10. General procedure for synthesis of compounds 17e19 and
26e31
To a mixture of compound 14e16 or 20e25 (0.1 mmol) in
methylene chloride (1 mL), BBr3 (0.02 mL of a 1 M solution in MC,
0.3 mmol) was added dropwise at ꢁ78 ꢀC under N2 and the
reaction mixture was stirred at the same temperature for 30 min.
The mixture was allowed to warm to room temperature and stir-
red for 1 h. The mixture was quenched with saturated aqueous
NaHCO3. Ethyl acetate was added and the organic layer was
separated. The aqueous layer was extracted with ethyl acetate. The
combined organic layer extracts were washed with brine and
dried over anhydrous Na2SO4. After evaporation of the organic
solvent, the residue was purified by column chromatography
(silica gel, ethyl acetate:hexane 1:2 v/v) to yield the desired
compounds.
4.9.4. N-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)benzamide (20)
Yield: 58.7%; mp 146 ꢀC; 1H NMR (CDCl3, 400 MHz)
d 8.50 (d,1H,
J ¼ 4.4 Hz), 8.04 (d, 1H, J ¼ 5.4 Hz), 7.75e7.67 (m, 2H), 7.41e7.29 (m,
4H), 6.96 (d, 1H, J ¼ 8.2 Hz), 6.86 (d, 1H, J ¼ 4.3 Hz), 6.55 (d, 1H,
J ¼ 5.3 Hz), 5.94 (brs,1H), 3.81 (s, 3H), 3.73 (s, 4H); 13C NMR (DMSO-
d6, 100 MHz)
d 167.0, 162.7, 159.7, 158.3, 156.4, 151.6, 149.0, 136.6,
135.0, 131.6, 130.1, 128.7, 127.6, 123.4, 121.8, 121.0, 114.8, 106.2, 99.0,
55.6, 31.4; ESIeMS: 471.0 [M þ 1]þ.
4.9.5. N-(2-(4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-2-hydroxybenzamide (21)
4.10.1. 1-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
Yield: 63.0%; mp 169 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d
12.40
pyrimidin-2-ylamino)ethyl)-3-phenylurea (17)
(s, 1H), 8.48 (d, 1H, J ¼ 4.4 Hz), 8.10 (d, 1H, J ¼ 5.4 Hz), 7.36e7.26
(m, 2H), 7.19e7.16 (m, 2H), 6.98 (dd, 1H, J ¼ 1.6 and 4.8 Hz),
6.96e6.91 (m, 2H), 6.32 (d, 1H, J ¼ 5.4 Hz), 5.65 (t, 1H, J ¼ 5.7 Hz),
3.82 (s, 3H), 3.79e3.75 (m, 2H), 3.69e3.67 (m, 2H); ESIeMS: 486.9
[M þ 1]þ.
Yield: 80.8%; mp 161 ꢀC, 1H NMR (DMSO-d6, 300 MHz)
d 9.56 (s,
1H), 8.70 (s, 1H), 8.10 (d, 1H, J ¼ 5.2 Hz), 7.49e7.37 (m, 5H),
7.28e7.18 (m, 4H), 6.99 (s, 1H), 6.97 (s, 1H), 6.95 (d, 1H, J ¼ 7.3 Hz),
6.83 (t, 1H, J ¼ 6.7 Hz), 6.38 (d, 1H, J ¼ 5.3 Hz), 3.45e3.39 (m, 4H);
ESIeMS: 471.9 [M þ 1]þ.

5776
J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
4.10.2. 1-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(3-(trifluoromethyl)phenyl)urea (18)
Yield: 82.4%; mp 162e164 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
4.10.9. N-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(trifluoromethyl)-5-
morpholinobenzamide (31)
d
9.57 (s, 1H), 8.94 (s, 1H), 8.10 (d, J ¼ 4.8 Hz), 7.96 (s, 1H), 7.51 (d,
Yield: 69.4%; mp 168 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d 9.58 (s,
1H, J ¼ 8.2 Hz), 7.43 (t, 3H, J ¼ 7.8 Hz), 7.23 (t, 1H, J ¼ 7.3 Hz),
6.98e6.94 (m, 12H), 6.82 (d, 1H, J ¼ 7.6 Hz), 6.43e6.38 (m, 2H),
1H), 8.80 (s, 1H), 8.09 (d, 1H, J ¼ 5.2 Hz), 7.63 (s, 1H), 7.56 (s, 1H),
7.45 (d, 1H, J ¼ 4.2 Hz), 7.32 (s, 1H), 7.25 (t, 1H, J ¼ 8.0 Hz), 6.96 (s,
2H), 6.82 (d, 1H, J ¼ 7.8 Hz), 6.38 (d, 1H, J ¼ 5.3 Hz), 3.74 (s, 4H),
3.51(brs, 4H), 3.24 (s, 4H). ESIeMS: 610.0 [M þ 1]þ.
3.54e3.39 (m, 4H); 13C NMR (DMSO-d6, 100 MHz)
d 162.7, 157.8,
156.5, 155.7, 151.6, 141.8, 136.5, 130.2, 130.0, 129.7, 126.1, 123.4,
121.6, 120.9, 120.3, 117.7, 116.4, 116.0, 114.1, 106.2, 41.5, 35.6;
ESIeMS: 539.8 [M þ 1]þ.
4.11. Evaluation of the antiproliferative activity against A375P
human melanoma cell line
4.10.3. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(4-(6-(3-
hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-ylamino)
ethyl)urea (19)
A375P cells were purchased from American Type Culture
Collection (ATCC, Rockville, MD, USA) and maintained in Dul-
becco’s modified eagle medium (DMEM, Welgene, Daegu,
Republic of Korea) supplemented with 10% fetal bovine serum
(FBS, Welgene, Daegu, Republic of Korea) and 1% penicillin/
streptomycin (Welgene, Daegu, Republic of Korea) in a humidi-
fied atmosphere with 5% CO2 at 37 ꢀC. A375P cells were taken
from culture substrate with 0.05% trypsine0.02% EDTA and
plated at a density of 5 ꢂ103 cells/well in 96-well plates and then
incubated at 37 ꢀC for 24 h in a humidified atmosphere with 5%
CO2 prior to treatment with various concentrations (3-fold serial
dilution, 12 points) of the tested compounds. The cells were
incubated for 48 h after treatment with the test compounds. The
A357P cell viability was assessed by the conventional 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reduction assay. MTT assays were carried out with CellTiter 96Ò
(Promega) according to the manufacturer’s instructions. The
absorbance at 590 nm was recorded using EnVision 2103 (Perkin
Elmer; Boston, MA, USA). The IC50 values were calculated using
GraphPad Prism 4.0 software. Triplicate testing was performed
for each test compound.
Yield: 71.3%; mp 159 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d 9.53 (s,
1H), 8.97 (d, 1H, J ¼ 4.5 Hz), 8.40 (brs, 1H), 7.99 (d, 1H, J ¼ 5.2 Hz),
7.81e7.79 (m, 2H), 7.52e7.50 (s, 1H), 7.36e7.34 (m, 1H), 7.13e7.08
(m, 2H), 6.99 (dd,1H, J ¼ 1.5 and 1.3 Hz), 6.90 (d,1H, J ¼ 4.2 Hz), 6.45
(d, 1H, J ¼ 5.2 Hz), 6.20 (brs, 1H), 5.73 (brs, 1H), 3.71e3.48 (m, 4H);
ESIeMS: 607.8 [M þ 1]þ.
4.10.4. N-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)benzamide (26)
Yield: 84.5%; mp 146 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d 9.58 (s,
1H), 8.63 (s, 1H), 8.11 (d, 1H, J ¼ 5.3 Hz), 7.85 (d, 1H, J ¼ 7.1 Hz),
7.57e7.45 (m, 5H), 7.25 (t, 1H, J ¼ 8.0 Hz), 6.98e6.96 (m, 2H), 6.82
(d, 1H, J ¼ 6.1 Hz), 6.39 (d, 1H, J ¼ 5.3 Hz), 3.51 (s, 4H); ESIeMS:
456.9 [M þ 1]þ.
4.10.5. N-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-2-hydroxybenzamide (27)
Yield: 77.6%; mp 169 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d 12.60
(s, 1H), 9.57 (s, 1H), 8.97 (s, 1H), 8.11 (d, 1H, J ¼ 5.3 Hz), 7.83 (d, 1H,
J ¼ 6.5 Hz), 7.62 (brs, 1H), 7.43e7.36 (m, 2H), 7.25 (t, 1H, J ¼ 7.8 Hz),
6.97 (s, 2H), 6.90e6.82 (m, 3H), 6.39 (d, 1H, J ¼ 5.3 Hz), 3.55 (s, 4H);
ESIeMS: 472.9 [M þ 1]þ.
4.12. 60-Cancer cell line screening at the NCI
Screening against a panel of 60 cancer cell lines was applied at
the National Cancer Institute (NCI), Bethesda, Maryland, USA
[18], applying the following procedure. The human cell lines are
grown in RPMI 1640 medium containing 5% fetal bovine serum
and 2 mM L-glutamine. For a typical screening experiment, cells
4.10.6. N-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(trifluoromethyl)benzamide (28)
Yield: 78.7%; mp 148 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d 9.58 (s,
1H), 8.90 (s, 1H), 8.20 (s, 1H), 8.15 (d, 1H, J ¼ 8.3 Hz), 8.10 (d, 1H,
J ¼ 5.3 Hz), 7.90 (d, 1H, J ¼ 7.7 Hz), 7.10 (t, 1H, J ¼ 7.5 Hz), 7.45 (d, 1H,
J ¼ 4.4 Hz), 7.25 (t, 1H, J ¼ 8.0 Hz), 6.97e6.96 (m, 2H), 6.82 (d, 1H,
J ¼ 8.2 Hz), 6.38 (d, 1H, J ¼ 5.3 Hz), 3.53 (s, 4H); ESIeMS: 524.9
[M þ 1]þ.
are inoculated into 96-well microtiter plates in 100 mL at plating
densities ranging from 5000 to 40,000 cells/well depending on
the doubling time of individual cell lines. After cell inoculation,
the microtiter plates are incubated at 37 ꢀC, 5% CO2, 95% air and
100% relative humidity for 24 h prior to addition of experimental
drugs. After 24 h, two plates of each cell line are fixed in situ with
TCA, to represent a measurement of the cell population for each
cell line at the time of drug addition (Tz). Experimental drugs are
solubilized in dimethyl sulfoxide at 400-fold the desired final
maximum test concentration and stored frozen prior to use. At
the time of drug addition, an aliquot of frozen concentrate is
thawed and diluted to twice the desired final maximum test
4.10.7. N-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3,5-bis(trifluoromethyl)benzamide
(29)
Yield: 66.5%; mp 166 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d 9.57 (s,
1H), 9.13 (s,1H), 8.50 (s, 2H), 8.31 (s,1H), 8.10 (d,1H, J ¼ 5.3 Hz), 7.45
(d, 1H, J ¼ 4.5 Hz), 7.25 (t, 1H, J ¼ 8.1 Hz), 6.96 (s, 2H), 6.82 (d, 1H,
J ¼ 6.6 Hz), 6.38 (d, 1H, J ¼ 5.3 Hz), 3.55 (s, 4H); ESIeMS: 593.0
[M þ 1]þ.
concentration with complete medium containing 50 mg/mL
gentamicin. Additional four, 10-fold or 1/2 log serial dilutions are
4.10.8. N-(2-(4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-ylamino)ethyl)-3-(trifluoromethyl)-4-
morpholinobenzamide (30)
made to provide a total of five drug concentrations plus control.
Aliquots of 100
the appropriate microtiter wells already containing 100
m
L of these different drug dilutions are added to
L of
m
Yield: 70.0%; mp 168 ꢀC; 1H NMR (DMSO-d6, 300 MHz)
d
9.57 (s,
medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an addi-
tional 48 h at 37 ꢀC, 5% CO2, 95% air, and 100% relative humidity.
For adherent cells, the assay is terminated by the addition of cold
1H), 8.80 (s, 1H), 8.15 (s, 1H), 8.11 (d, 2H, J ¼ 5.0 Hz), 7.55e7.43 (m,
3H), 7.25 (t, 1H, J ¼ 7.8 Hz), 6.97 (s, 2H), 6.83 (d, 1H, J ¼ 7.2 Hz), 6.39
(d, 1H, J ¼ 5.2 Hz), 3.70 (s, 4H), 3.52 (s, 4H), 2.90 (s, 4H); 13C NMR
(DMSO-d6, 100 MHz)
d
165.9, 162.1, 157.5, 156.3, 154.4, 151.7, 148.8,
TCA. Cells are fixed in situ by the gentle addition of 50 mL of cold
144.2, 136.0, 132.6, 130.2, 126.7, 123.8, 122.8, 122.3, 120.3, 116.1,
50% (w/v) TCA (final concentration, 10% TCA) and incubated for
114.3, 106.3, 66.8, 53.3, 53.0; ESIeMS: 610.0 [M þ 1]þ.
60 min at 4 ꢀC. The supernatant is discarded, and the plates are

J.-H. Park et al. / European Journal of Medicinal Chemistry 46 (2011) 5769e5777
5777
washed five times with tap water and air dried. Sulforhodamine
B (SRB) solution (100 L) at 0.4% (w/v) in 1% acetic acid is added
Appendix. Supplementary data
m
to each well, and plates are kept for 10 min at room temperature.
After staining, unbound dye is removed by washing five times
with 1% acetic acid and the plates are air dried. Bound stain is
subsequently solubilized with 10 mM trizma base, and the
absorbance is read on an automated plate reader at a wavelength
of 515 nm. For suspension cells, the methodology is the same
except that the assay is terminated by fixing settled cells at the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.08.024.
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Korea Institute of Science and Technology (KIST) for financial
support.
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