Discovery of 6-phenylimidazo[2,1-b]thiazole derivatives as a new type of FLT3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.08.068

In this investigation, a series of 6-phenylimidazo[2,1-b]thiazole derivatives were synthesized. Structure-activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a number of compounds that showed potent act

Full text of DOI:10.1016/j.bmcl.2015.08.068

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 6-phenylimidazo[2,1-b]thiazole derivatives as a new
type of FLT3 inhibitors
Xing-Dong Lin a,y, Hui-Wen Yang a,y, Shuang Ma a, Wei-Wei Li a, Chun-Hui Zhang a, Wen-Jing Wang a,
Rong Xiang c, Lin-Li Li b, , Sheng-Yong Yang a,
⇑
⇑
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Sichuan 610041, China
b West China School of Pharmacy, Sichuan University, Sichuan 610041, China
c Department of Clinical Medicine, School of Medicine, Nankai University, Tianjin, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this investigation, a series of 6-phenylimidazo[2,1-b]thiazole derivatives were synthesized. Structure–
activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a
number of compounds that showed potent activity against FLT3-dependent human acute myeloid leuke-
mia (AML) cell line MV4-11, but very weak or no activity against FLT3-independent human cervical can-
cer cell line Hela. FLT3 kinase inhibition assays were then performed on the three most active
compounds. Among these compounds, 6-(4-(3-(5-(tert-butyl)isoxazol- 3-yl)ureido)phenyl)-N-(3-
(dimethylamino)propyl)imidazo[2,1-b]thiazole-3-carboxamide (19) exhibited the highest potency in
Received 12 April 2015
Revised 23 August 2015
Accepted 26 August 2015
Available online xxxx
Keywords:
6-Phenylimidazo[2,1-b]thiazole
Acute myeloid leukemia (AML)
FLT3
FLT3-ITD
MV4-11
Anti-viability
both cellular (MV4-11, IC50: 0.002
in vitro anti-AML activity and mechanism of action studies were carried out on compound 19.
Ó 2015 Published by Elsevier Ltd.
lM) and enzymatic (FLT3, IC50: 0.022 lM) assays. Further in-depth
Acute myeloid leukemia (AML) is a malignant disease of the
bone marrow and blood, characterized by the accumulation of
rapidly growing abnormal white blood cells in the bone marrow
that interferes with the production of normal blood cells.1,2 Che-
motherapies with cytotoxic agents such as cytarabine, daunoru-
bicin, and mitoxantrone are still the main treatment strategy for
AML.3,4 Nevertheless, the toxicities of these conventional cytotoxic
agents together with drug resistance and relapse force us to
develop more efficient targeted drugs.5,6 A number of studies have
revealed that mutations and/or aberrant expression of specific pro-
tein tyrosine kinases (PTKs) are often responsible for the initiation
and development of AML.7,8 Of special note is the FMS-like tyrosine
kinase 3 (FLT3). Activating mutations in FLT3 kinase are found in
up to one-third of AML cases and the most prevalent activating
mutation is ‘internal tandem duplications’ (ITDs) in the juxtamem-
brane domain that lead to constitutive, ligand-independent
activation of the kinase.9–11 Numerous studies have demonstrated
that FLT3-ITD mutations represent a driving mutation for the
development of AML and are associated with a poor prognosis
for overall survival.12–14 Therefore, FLT3 has been considered as a
potential molecular target in the treatment of AML.
Discovery of FLT3 inhibitors has attracted much attention in
recent years due to their potential therapeutic values in AML. A
number of FLT3 inhibitors have been developed, and several of
them have entered into clinical trials.15–19 However the clinical
efficacy of most of these FLT3 inhibitors in patients with AML
seems unimpressive, mainly because of their potency and/or
adverse events. Therefore, it is still necessary to discover more
novel FLT3 inhibitors with high potency and low toxicity at
present.
Currently, the ‘enzyme-cell-animal’ drug discovery mode faces
some challenges. Firstly, the screening process is increasingly
expensive, which restricts more academic institutes with limited
fund support, like us, to participate in the drug screening cam-
paign. Secondly, the screening mode sometimes does not work.
For example, a compound that shows potent activity in enzyme
may be not very effective in cell and animal. Cheap and reliable
screening strategies are thus needed to overcome the shortcom-
ings. In this investigation, a pure cell-based screening strategy
was adopted to identify new FLT3 inhibitors, in which two cell
lines MV4-11 and Hela were used.20–24 MV4-11 is a typical FLT3-
dependent human AML cell line, and Hela is an FLT3-independent
human cervical carcinoma cell line. The use of Hela is for ruling out
⇑
Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060 (S.-Y.Y.).
E-mail addresses: ysylilinli@sina.com.cn (L.-L. Li), yangsy@scu.edu.cn
(S.-Y. Yang).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.08.068
0960-894X/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Lin, X.-D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.068

2
X.-D. Lin et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3-position of imidazo[2,1-b]thiazole as its original ethyl ester. A
total of 13 compounds were synthesized. The synthetic routes for
these compounds are depicted in Scheme 1. Treatment of ethyl
2-aminothiazole-4-carboxylate 20 with 2-bromo-1-phenylethan-
1-one 21a or 21b provided intermediate nitrobenzene 22a or
22b, respectively,25 followed by reduction of the nitro group to
give the corresponding aniline 23a or 23b. Then a series of amide
compounds, 1–10, were obtained by condensation of 23a/b with
a variety of acyl chlorides in a basic condition. Urea series of
compounds 11–13 were synthesized from 23b by condensation
with diverse aromatic amines and triphosgene. Bioactivities of
these compounds are shown in Table 1. Obviously, compounds
with a para-substituent on the phenyl ring exhibited a higher
anti-viability activity against MV-4-11 cells than corresponding
ones with a meta-substituent. The most potent compound corre-
sponds to 13, which contains the substituent 1-(5-(tert-butyl)isox-
azol-3-yl)urea at the para-position of phenyl ring. Compound 13 as
well as others displayed a very weak or no activity against Hela,
indicating that the anti-viability activities of these compounds
against the MV4-11 cells were not due to cellular toxicity.
We next fixed the para-substituent of phenyl ring as the opti-
mal 1-(5-(tert-butyl)isoxazol-3-yl)urea and varied the 3-position
of imidazo[2,1-b]thiazole. A total of 6 compounds (14–19) were
synthesized via the synthetic method outlined in Scheme 2. Com-
pound 13 synthesized above was hydrolyzed to corresponding car-
boxylic acid 24 by sodium hydroxide. The amidation/esterification
reaction of carboxylic acid 24 with diverse moderate size amines or
alcohols was carried out to provide the desired compounds 14–19.
Table 2 summarizes the anti-viability activities of this set of com-
pounds. Replacement of the ester group of compound 13 by a vari-
ety of amides led to a significantly increased bioactivity (14, 16–19
vs 13) against MV4-11 cells, while the replacement of the ester
group of compound 13 with another ester group showed a similar
anti-viability activity (15 vs 13). These results imply that the
existence of amide groups is critical for retaining potent cellular
activity.
Figure 1. (a) The structure of compound 1. (b) Schematic showing subgroups that
are the focus of structural modifications.
activity due to non-FLT3 mediated effects (‘off target’ or cellular
toxic effects). Compounds are likely better FLT3 inhibitors if they
have high potency against MV4-11 cells but low or no activity
against Hela. The enzymatic assays were also performed but just
on compounds with higher potency at the cellular level.
With the cell-based screening strategy, we first performed a
screening against an in-house chemical database containing about
200 compounds, which led to the discovery of a number of active
compounds. Among these compounds, ethyl 6-(4-(2-chloroac-
etamido)phenyl)imidazo[2,1-b]thiazole-3-carboxylate (1, Fig. 1a)
attracted our attention since it contains a new scaffold, namely
6-phenylimidazo[2,1-b]thiazole; 6-phenylimidazo[2,1-b]thiazole
derivatives have not been reported as FLT3 inhibitors. Compound
1 displayed an IC50 (half maximal inhibitory concentration) value
of 9.07 lM against MV4-11 cells and almost no activity against
Hela cells. Obviously, the potency is not good. The purpose here
is to further optimize the potency with the cell-based strategy. A
series of 6-phenylimidazo[2,1-b]thiazole derivatives will be syn-
thesized, and the structure–activity relationship (SAR) of these
compounds will also be discussed.
The SAR analysis will focus on two regions: the 30/40-position
substituent of phenyl ring (ring A) and the 3-position substituent
of imidazo[2,1-b]thiazole (Fig. 1b). Firstly, we changed the
substituent of phenyl ring with different groups and fixed the
Scheme 1. Reagents and conditions: (a) dioxane, reflux, 10–12 h, 90%; (b) (i) SnCl2ꢀ2H2O, EtOH, reflux; (ii) cond. HCl, reflux, 8 h, 60–65%; (c) acyl chloride, CH2Cl2, DIEA, 0 °C–
rt, 1 h, 60–95%; (d) (i) aromatic amine, triphosgene, CH2Cl2, Et3N, 0 °C–rt; (ii) toluene, reflux, 8–10 h, 50–85%.
Please cite this article in press as: Lin, X.-D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.068

X.-D. Lin et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
Anti-viability activities of 6-phenylimidazo[2,1-b]thiazole derivatives with different meta- and para-position substituents against MV4-11 cells and HeLa cells
R1
A
EtOOC
N
N
S
Compd
Attaching position to A
3
R1
MV4-11a (IC50
17.731 1.88
,
lM)
Helaa (IC50
, lM)
H
N
Cl
Cl
1
ꢁ20
O
O
O
O
O
O
H
N
2
3
4
5
6
4
3
4
3
4
9.070 1.04
>20
>20
>20
>20
>20
>20
H
N
H
N
9.523 1.44
>20
H
N
H
N
2.929 0.27
O
O
H
N
7
3
>20
>20
O
H
N
8
4
4
4
9.917 1.63
>20
>20
>20
>20
O
H
N
O
9
S
O
H
O
S
N
10
>20
O
H
N
H
N
N
11
12
4
4
2.444 0.23
>20
>20
>20
O
Br
H
N
H
N
O
O
H
N
H
N
N
O
13
4
0.2741 0.027
>20
a
Each compound was tested in triplicate; the data are presented as the mean SD.
Collectively, the above SAR analysis led to the discovery of a
However, considering that compound 19 is the most active one
in cellular assays, further studies including kinase inhibition pro-
file, cancer cell selectivity, and signaling inhibition in intact cells
were performed just on compound 19.
number of compounds that exhibited high potency against FLT3-
dependant MV4-11 cells. Among these compounds, 16, 17 and 19
are the three most active ones, which all showed IC50 values less
than 0.01
l
M. Kinase inhibitory potencies against FLT3 were then
Kinase inhibition profiles of compound 19 against a panel of
selected recombinant human protein kinases are shown in Table 3.
Compound 19 potently inhibited FLT3 and Fms kinases
measured for the three compounds. The results showed that the
IC50 values of compounds 16, 17 and 19 against FLT3 were
22 nM, 95 nM and 22 nM, respectively. Obviously, compound 16
and 19 showed the same highest potency in enzymatic assays.
(IC50 = 0.022
lM for FLT3 and 0.043 lM for Fms). Compound 19
also exhibited considerable potency against several other kinases,
Please cite this article in press as: Lin, X.-D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.068

4
X.-D. Lin et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Scheme 2. Reagents and conditions: (a) NaOH, EtOH, reflux, overnight; (b) amines, HOBT, EDCI, DIEA, CH2Cl2, reflux, 10–12 h, 40–80%; or alcohols, DMAP, DCC, CH2Cl2, rt, 8 h,
38%.
Table 2
Table 3
6-Phenylimidazo[2,1-b]thiazole derivatives with different substituents R2, together
with their anti-viability activities against MV4-11 cells and HeLa cells
Kinase inhibition profiles of compound 19 against FLT3 and a panel of selected protein
kinases
Kinase
IC50
(l
M)
Kinase
IC50 (lM)
O
N
Flt3
cKit
Fms
PDGFR
PDGFRb
Flt1
Flt4
KDR
Pim-1
Ret
Abl
Arg
0.022
0.294
0.043
0.37
1.982
2.826
1.188
2.458
4.037
0.986
>10
>10
>10
>10
>10
>10
>10
>10
>10
EphB2
ErbB2
ErbB4
Fes
FGFR2
Fyn
GSK3b
IGF-1R
IRE1
JAK3
JNK3
Lck
Lyn
MEK1
Met
MLK1
PAK1
PAK2
PAK4
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
O
R2
NH
a
N
NH
N
S
Compd.
R2
MV4-11a (IC50
,
lM)
Helaa (IC50
>20
, lM)
O
13
0.2741 0.027
0.01628 0.003
0.1456 0.08
O
N
AMPK
a1
O
O
O
O
Aurora-A
Aurora-B
Axl
Bmx
BTK
CaMKIV
CDK2
CDK7
CHK1
c-RAF
DMPK
EGFR
14
15
16
>20
>20
ꢁ20
N
H
N
O
O
>10
>10
>10
>10
>10
>10
PKCl
PKD2
Plk1
Rsk1
Syk
N
S
0.0097 0.002
N
H
O
O
TrkB
17
18
0.0049 0.0018
0.0156 0.003
>20
>20
ꢁ20
N
H
O
O
and negligible activity against the remaining 29 human cancer cell
lines. These results indicate that compound 19 is relatively selec-
tive for the human AML cell line MV4-11.
The ability of compound 19 to inhibit the activation of FLT3 and
downstream signaling proteins in intact cells was assessed by
Western blot analysis.26–28 As shown in Figure 2b, compound 19
inhibited FLT3 phosphorylation in a dose-dependent manner. Con-
sistent with the downregulation of the phosphorylation of FLT3,
the phosphorylation of the downstream signaling proteins STAT5
and ERK1/2 was also significantly inhibited at concentrations lar-
HO
N
H
O
19
0.002 0.001
0.007 0.0012
N
N
H
Sorafinib
5.23 0.38
a
Each compound was tested in triplicate; the data are presented as the
mean SD.
including cKit (IC50 = 0.294
l
M), PDGFR
M), Flt4 (IC50 = 1.188
M), Pim-1 (IC50 = 4.037 M), and Ret
M). Compound 19 displayed almost no inhibitory
activity against 40 other tested protein kinases (IC50 >10 M).
a
(IC50 = 0.37
l
M), PDGFRb
ger than 0.001 lM (Fig. 2b).
(IC50 = 1.982
KDR (IC50 = 2.458
(IC50 = 0.986
l
M), Flt1 (IC50 = 2.826
l
l
M),
In conclusion, SAR studies of 6-phenylimidazo[2,1-b]thiazole
derivatives based on cellular assays combined with kinase inhibi-
tion assays led to the discovery of a number of potent FLT3 inhibi-
tors. Compound 19 is the most active one in both cellular and
enzymatic levels. It also displayed very good selectivity for FLT3
against a panel of selected kinases and considerable cell selectivity
for human AML cell line MV4-11. Western blot analysis showed
that compound 19 significantly and dose-dependently down-regu-
lated the phosphorylation of FLT3 and its downstream signal pro-
teins STAT5 and ERK. Overall, compound 19 could be taken as a
good lead compound for further lead optimization for the treat-
ment of AML.
l
l
l
l
These data demonstrate that compound 19 is a potent FLT3 inhibi-
tor with good kinase selectivity.
Table 4 shows the anti-viability activities of compound 19
against various cell lines, including leukemia and solid tumor cell
lines. Except for potently inhibiting MV4-11 cells with an IC50
value of 0.002
moderate activity against several cell lines including OCI-LY19
(lymphoma, IC50 = 3.3 M), SH-SY5Y (neuroblastoma, IC50 = 3.3 M),
lM (also see Fig. 2a), compound 19 just showed
l
l
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X.-D. Lin et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 4
Anti-viability activities of compound 19 against various cancer cell lines
Cell line
Tumor type
IC50
(
lM)
Cell line
Tumor type
IC50 (lM)
MV4-11
Jurkat
Raji
OCI-LY19
SH-SY5Y
Hela
Leukemia, AML
Leukemia, ALL
Lymphoma
0.002 0.001
10
>10
3.3 0.02
3.3 0.031
>10
10
10
10
10
10
>10
10
10
MDA-MB-157
MDA-MB-231
MDA-MB-435
MDA-MB-453
MCF7
SKBr3
ZR-75-1
BT474
H1975
PC9
Calu-6
A549
Breast cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
Lung cancer
Lung cancer
Lung cancer
Lung cancer
Esophageal cancer
Liver cancer
Liver cancer
>10
10
>10
>10
>10
10
>10
10
>10
>10
10
Lymphoma
Neuroblastoma
Cervical cancer
Bladder cancer
Colon cancer
Colon cancer
Colon cancer
Colon cancer
Prostatic cancer
Melanoma
UMUC3
HT29
HCT116
DLD-1
SW480
22Rv1
A875
A375
A2058
U251
10
>10
10
KYSE150
HepG2
SK-Hep1
Melanoma
Melanoma
Spongiocytoma
10
>10
10
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Please cite this article in press as: Lin, X.-D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.068