Hybrid compounds as new Bcr/Abl inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.029

A series of 2,4-disubstituted thiazole derivatives were designed and synthesized as new Bcr/Abl inhibitors by hybriding the structural moieties from FDA approved imatinib, nilotinib and dasatinib. The new inhibitors strongly suppressed the activity of Bcr/Abl kinase and potently inhibited the proliferation of K562 and KU812 leukemia cancer cells. Compound 4i displayed comparable potency with that of nilotinib in both biochemical kinase assay and cancer cell growth inhibition assay. These inhibitors might serve as lead compounds for further developing new anticancer drugs.

Full text of DOI:10.1016/j.bmcl.2011.02.029

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1965–1968
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hybrid compounds as new Bcr/Abl inhibitors
Deping Wang a,b, Zhang Zhang a, Xiaoyun Lu a, Yubing Feng a, Kun Luo a, Jirong Gan a, Liu Yingxue a,
Junting Wan a, Xiang Li a, Fengxiang Zhang a, Zhengchao Tu a, Qian Cai a, Xiaomei Ren a, Ke Ding a,
⇑
a Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No.190,
Kaiyuan Avenue, Science Park, Guangzhou 510530, China
b Graduate School of Chinese Academy of Sciences, # 19 Yuquan Road, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,4-disubstituted thiazole derivatives were designed and synthesized as new Bcr/Abl inhibi-
tors by hybriding the structural moieties from FDA approved imatinib, nilotinib and dasatinib. The
new inhibitors strongly suppressed the activity of Bcr/Abl kinase and potently inhibited the proliferation
of K562 and KU812 leukemia cancer cells. Compound 4i displayed comparable potency with that of nil-
otinib in both biochemical kinase assay and cancer cell growth inhibition assay. These inhibitors might
serve as lead compounds for further developing new anticancer drugs.
Received 22 December 2010
Revised 8 February 2011
Accepted 9 February 2011
Available online 13 February 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
2,4-Disubstituted thiazole
Hybride compound
Bcr/Abl kinase
Chronic myelogenous leukemia
Anticancer
Chronic myelogenous leukemia (CML) is a hematological malig-
nancy representing about 20% of adult leukemia and being charac-
terized by the occurrence of the Philadelphia (Ph) chromosome.
The Philadelphia (Ph) chromosome is a truncated version of chro-
mosome 22 resulting from the reciprocal translocation between
chromosome 9 and 22. The chimeric Bcr/Abl gene generated by
the translocation encodes a fusion protein with constitutively acti-
vated kinase activity.1–3 Bcr/Abl kinase is a well validated target for
development of small molecular inhibitors to treat CML. The first
generation Bcr/Abl inhibitor imatinib (STI571 or Gleevec) has
achieved tremendous clinical success and become the first-line
drug in conventional treatment of CML.4,5 However, emerging ac-
quired resistance to imatinib is becoming a major challenge.
Three–four percent resistance rates were reported in newly diag-
nosed chronic phase CML patients. For imatinib treated CML pa-
tients in accelerated or blastic phases, the acquired resistance
percentages were up to 40–50% or over 80%, respectively.6 The
point mutations in the kinase domain of Bcr/Abl are the primary
mechanism for the imatinib resistance, and about 100 point muta-
tions have been identified to date.7
to imatinib. However, neither nilotinib nor dasatinib could sup-
press the proliferation of leukemia cells harboring the Bcr/Abl
T315I mutant.12–14 Only recently, a few small molecules were re-
ported to show good efficacy against the Bcr/Abl T315I mutation.15
One molecule AP24534 was advanced into phase II clinical trial in
US15g
.
In this Letter, we report the design, synthesis and biological
evaluation of a new kind of Bcr/Abl inhibitors by hybriding the
structural moieties from FDA approved imatinib, nilotinib and
dasatinib (Fig. 1).
The synthesis of compounds 4a–m was outlined in Scheme 1.
Briefly, substituted methyl 3-aminobenzoate 1 was slowly added
to a solution of acetyl chloride and NH4SCN in acetone to produce
the methyl 3-(3-acetylthioureido)benzoate 2. 3-(4-(Pyridin-
3-yl)thiazol-2-ylamino)-benzoate methyl ester 3 was synthesized
by reaction of compounds
2 with 2-bromo-1-(pyridin-3-yl)-
ethanone. Finally, the designed compounds 4a–4m were readily
prepared by direct aminolysis of intermediate 3 (for 4a–4f,
4j–4m) or condensation of the hydrolyzed 3 (acids. for 4g–4i) with
different anilines.
To overcome the drug resistance to imatinib, several classes of
second generation kinase inhibitors have been designed and syn-
thesized.8 Type-II Bcr/Abl inhibitor nilotinib9 and type-I Bcr/Abl
inhibitor dasatinib10,11 have been approved as the second-line
drugs to treat adult patients in all phases of CML with resistance
The kinase inhibitory activities of compounds 4a–4m were eval-
uated via a well established FRET-based Z’-Lyte assay.16 Imatinib
and nilotinib were utilized as the positive controls to validate the
screening assay. Under the screening conditions, Imatinib and nil-
otinib displayed IC50 values of 309 and 39.3 nM against Bcr/Abl
fused kinase, which was highly consistent to the reported data.9
As expected, the designed hybrid compound 4a also potently inhib-
⇑
Corresponding author. Tel.: +86 20 32015276; fax: +86 20 32015299.
E-mail address: ding_ke@gibh.ac.cn (K. Ding).
ited the kinase activity of Bcr/Abl with an IC50 value of 0.67 lM.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.029

1966
D. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1965–1968
CF3
N
O
H
N
H
N
H
N
N
N
N
N
N
N
H
N
N
O
N
N
Imatinib
nilotinib
R1
O
NH
O
N
N
N
H
N
N
N
R2
S
N
H
N
Cl
H
N
S
N
R3
OH
Dasatinib
hybrideinhibitors
Figure 1. Chemical structures of FDA approved Bcr/Abl inhibitors and the designed hybrid compounds.
R1
Me
R1
R1
H
N
H
N
H
N
H
N
N
H2N
N
N
d
N
a
b
S
S
O
S
N
3
5
2
COOMe
COOH
1
COOMe
COOMe
e
c
c
N
R4
N
H
N
O
N
N
N
S
S
R1
N
Me
S
Me
NH
NH
CF3
H
4d-f
O
O
HN
R2
HN
N
N
N
N
X
R3
R2
4a-c, 4j-m
Me
4g-i
Scheme 1. Reagents and conditions: (a) acetyl chloride, NH4SCN, acetone, 80–86%; (b) 2-bromo-1-(pyridin-3-yl)ethanone, K2CO3, MeOH, 78–88%; (c) aniline, t-BuOK, THF,
79–85%; (d) NaOH, MeOH, 83%; (e) diethyl cyanophosphonate, Et3N, DMF, 50–60%.
Molecular docking study was performed to investigate the binding
modes of compound 4a with Bcr/Abl (PDB code: 3CS9) using GOLD
3.01.17 It was shown that compound 4a bind to ATP pocket of Abl
in a similar fashion to that of nilotinib (Fig. 2a). Specifically, the
4-(pyridin-3-yl)thiazole core occupied the adenine pocket of the ki-
nase, the amide formed two hydrogen bonds with Glu286 and
Asp381, and the trifluoromethylphenyl-imidazole moiety bound
deeply into the DFG-out hydrophobic pocket.
A structure–activity study revealed that the 4-methyl-1H-imi-
dazol-1-yl group in 4a could be replaced by other five-member
heterocyclic substituents (4b–4d) without significantly interfering
the inhibitory activity against Bcr/Abl kinase. However, when the
4-methyl-1H-imidazol-1-yl group was removed (4f), the kinase
inhibitory activity was almost totally abolished (Table 1). This
might be due to the fact that the removal of the 4-methyl-1H-imi-
dazol-1-yl group could reduce the hydrophobic interaction with
DFG-out pocket. The molecular modeling studies also suggested
that the CF3 group in compound 4a could be replaced with a
slightly larger hydrophobic group to achieve a better fit to the
DFG-out pocket of Abl kinase ( Fig. 2b). Compounds 4h and 4i
indeed showed better bioactivity than that of 4b. Especially, com-
pound 4i possessed an IC50 of 20.1 nM against Bcr/Ab. However,
when the CF3 group was replaced by an adamantyl group, com-
pound 4j became totally inactive, which might be due to the spatial
collision with the DFG-out pocket of Abl. When the CF3 group in
compound 4a was replaced by a relatively small Cl group (4k),
the potency decreased. It was clear that the methyl group at R1 po-
sition restricted the compound’s conformation to fit into the ATP
binding site of Abl protein, it might also form additional hydropho-
bic interactions with Ala269, Val270 and Lys271. Not surprisingly,
the removal or replacing with slightly bigger ethyl group of this
group (4l and 4m) led to a dramatic activity decrease consistent
to their potent Bcr/Abl kinase inhibitory activities, the compounds
also displayed strong suppression on the growth of human chronic
myelogenous leukaemia K562 and Ku812 cells with express high
level of Bcr/Abl protein. For instance, compound 4i potently inhib-
ited the growth of K562 and Ku812 cells with IC50 values of 10 and
11 nM, respectively, which were equally potent to nilotinib. Fur-
thermore, the compounds also potently inhibited the proliferation
of imatinib-resistant CML cells (K562-R) (Table 1).

D. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1965–1968
1967
Table 1 (continued)
Compd R1 R2
R3
Bcr/Abl
Cell Growth
inhibition IC50 (nM)
Kinase
K562 KU812 K562-Rb
inhibition IC50
(nM)
4f
Me CF3
Me Me
H
>10000
3005 1235 1440
Me
Me
Me
Me
N
N
N
N
4g
774
449 871
3290
2910
640
N
N
N
N
4h
4i
Me i-Pr
Me t-Bu
Me
177
31
10
51
11
20.1
4j
>2000
1061 2805 11,850
Me
Me
Me
N
N
N
4k
4l
Me Cl
1300
3780
5510
812 518
2360
N
N
N
H
CF3
3193 2733 1720
4m
Et CF3
1603 945
2720
a
ABL activity assays were performed using the FRET-based Z’-Lyte assay
according to the manufacturer’s instructions. The anti-proliferative activities of the
compounds were evaluated by CCK-8 kit. The data were reported as the means of at
least three independent experiments.
b
Imatinib induced resistant CML K562 cells.
Figure 2. The predicted binding modes of compounds 4a and 4i with Abl kinase.
Blue: nitrogen; red: oxygen ;. Doteed lines indicate H-bond.
Table 1
Structure–activity relationship of pyridin-thiazol-2-ylamino-benzamide derivatives.a
Figure 3. Compound 4i dose-dependently inhibits the phosphorylation of Bcr/Abl
in K562 cells. Results are representative of at least three independent experiments.
R3
O
H
N
N
N
R2
Taking compound 4i as an example, the Bcr/Abl kinase inhibi-
tory activity was further validated by using western-blot analysis.
As shown in Figure 3, compound 4i strongly inhibited the auto-
phosphrylation of Bcr/Abl fused protein in a dose-dependent man-
ner after a 4 h treatment in K562 CML cells. Flow cytometric anal-
ysis revealed that compound 4i also dose-dependently induced the
G0/G1 phase arrest and apoptosis of K562 cancer cells, which
might be a consequent response of Bcr/Abl kinase inhibition
(Fig. 4)
H
N
S
R1
Compd R1 R2
R3
Bcr/Abl
Kinase
inhibition IC50
(nM)
Cell Growth
inhibition IC50 (nM)
K562 KU812 K562-Rb
Imatinib
Nilotinib
309
39.3
380 337
6.5 3.4
6.05
260
In summary, a series of 2,4-disubstituted thiazole derivatives
were designed and synthesized as new Bcr/Abl inhibitors by hyb-
riding the structural moieties from FDA approved imatinib, niloti-
nib and dasatinib. The resulting compounds strongly suppressed
the activity of Bcr/Abl kinase and potently inhibited the prolifera-
tion of K562 and KU812 leukemia cancer cells. Furthermore, the
compounds also potently inhibited the proliferation of imatinib-
resistant CML cells (K562-R). One of the most potent compound
4i displayed comparable potency with that of nilotinib in both bio-
chemical kinase assay and cancer cell growth inhibition assay. As a
consequent response of Bcr/Abl kinase inhibition, compound 4i
also dose-dependently induced the G0/G1 phase arrest and apop-
tosis of K562 cancer cells. These inhibitors might serve as lead
compounds for further developing new anticancer drugs.
Me
N
4a
4b
4c
4d
Me CF3
Me CF3
Me CF3
Me CF3
671
507
480
491
28
28
26
27
30
1830
2140
1630
5380
N
N
N
36
N
N
38
N
Me
N
27.1
N
Me
Me
N
N
4e
Me CF3
277
57
90
3510
N

1968
D. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1965–1968
ral Science Foundation (21072192) and the National High Technol-
ogy Research and Development Program (2010CB529706,
2009CB940904) for their financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.029.
References and notes
1. Nowell, P. C.; Hungerford, D. A. Science 1960, 132, 1497.
2. Rowley, J. D. Nature 1973, 243, 290.
3. Sawyers, C. L. N. Engl. J. Med. 1999, 340, 1330.
4. Druker, B. J.; Tamura, S., et al Nature Med. 1996, 2, 561.
5. Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A. Nat. Rev. Drug Dis.
2002, 1, 493.
6. Druker, B. J.; Guilhot, F.; O’Brien, S. G., et al N. Engl. J. Med. 2006, 355,
2408.
7. O’Hare, T.; Eide, C. A.; Deininger, M. W. Blood 2007, 110, 2242.
8. Weisberg, E.; Manley, P. W.; Cowan-Jacob, S. W.; Hochhaus, A.; Griffin, J. D. Nat.
Rev. Cancer 2007, 7, 345.
9. Weisberg, E.; Manley, P. W.; Breitenstein, W., et al Cancer Cell 2005, 7,
129.
10. Shah, N. P.; Tran, C.; Lee, F. Y.; Chen, P.; Norris, D.; Sawyers, C. L. Science 2004,
305, 399.
11. Lombardo, L. J.; Lee, F. Y.; Chen, P., et al J. Med. Chem. 2004, 47, 6658.
12. O’Hare, T.; Walters, D. K.; Stoffregen, E. P., et al Cancer Res. 2005, 65, 4500.
13. Deguchi, Y.; Kimura, S.; Ashihara, E.; Niwa, T.; Hodohara, K.; Fujiyama, Y.;
Maekawa, T. Leuk. Res. 2008, 32, 980.
14. Redaelli, S.; Piazza, R.; Rostagno, R., et al J. Clin. Oncol. 2009, 27, 469.
15. Selected examples see: (a) Zhou, T.; Parillon, L.; Li, F.; Wang, Y.; Keats, J.;
Lamore, S.; Xu, Q.; Shakespeare, W.; Dalgarno, D.; Zhu, X. Chem. Biol. Drug Des.
2007, 70, 171; (b) Choi, H. G.; Ren, P.; Adrian, F.; Sun, F.; Lee, H. S.; Wang, X.;
Ding, Q.; Zhang, G.; Xie, Y.; Zhang, J.; Liu, Y.; Tuntland, T.; Warmuth, M.;
Manley, P. W.; Mestan, J.; Gray, N. S.; Sim, T. J. Med. Chem. 2010, 53, 5439; (c)
Giles, F. J.; Cortes, J.; Jones, D.; Bergstrom, D.; Kantarjian, H.; Freedman, S. J.
Blood 2007, 109, 500; (d) Gontarewicz, A.; Balabanov, S.; Keller, G.; Colombo,
R.; Graziano, A.; Pesenti, E.; Benten, D.; Bokemeyer, C.; Fiedler, W.; Moll, J.;
Brummendorf, T. H. Blood 2008, 111, 4355; (e) Howard, S.; Berdini, V.;
Boulstridge, J. A.; Carr, M. G.; Cross, D. M.; Curry, J.; Devine, L. A.; Early, T. R.;
Fazal, L.; Gill, A. L.; Heathcote, M.; Maman, S.; Matthews, J. E.; McMenamin, R.
L.; Navarro, E. F.; O’Brien, M. A.; O’Reilly, M.; Rees, D. C.; Reule, M.; Tisi, D.;
Williams, G.; Vinkovic, M.; Wyatt, P. G. J. Med. Chem. 2009, 52, 379; (f) Shiotsu,
Y.; Kiyoi, H.; Ishikawa, Y.; Tanizaki, R.; Shimizu, M.; Umehara, H.; Ishii, K.; Mori,
Y.; Ozeki, K.; Minami, Y.; Abe, A.; Maeda, H.; Akiyama, T.; Kanda, Y.; Sato, Y.;
Akinaga, S.; Naoe, T. Blood 2009, 114, 1607; (g) Huang, W.; Metcalf, C.;
Sundaramoorthi, R.; Wang, Y.; Zou, D.; Thomas, R.; Zhu, X.; Cai, L.; Wen, D.; Liu,
S.; Romero, J.; Qi, J.; Chen, I.; Banda, G.; Lentini, S.; Das, S.; Xu, Q.; Keats, J.;
Wang, F.; Wardwell, S.; Ning, Y.; Snodgrass, J.; Broudy, M.; Russian, K.; Zhou, T.;
Commodore, L.; Narasimhan, N.; Mohemmad, Q.; Iuliucci, J.; Rivera, V.;
Dalgarno, D.; Sawyer, T.; Clackson, T.; Shakespeare, W. J. Med. Chem. 2010,
53, 4701; (h) Deng, X.; Lim, S. M.; Zhang, J.; Gray, N. S. Bioorg. Med. Chem. Lett.
2010, 20, 4196; (i) Crespan, E.; Radi, M.; Zanoli, S.; Schenone, S.; Botta, M.;
Maga, G. Bioorg. Med. Chem. 2010, 18, 3999.
Figure 4. A. Compound 4i dose-dependently induces G0/G1 arrest (A) and
apoptosis of K562 cancer cells. Results are representative of at least three
independent experiments.
Acknowledgments
16. The assay was performed based on the manufacturer’s instructions
(Invitrogen).
17. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
We thank the 100-Talent Program of the Chinese Academy of
Sciences (CAS), the CAS grant (KSCX2-YWR-27), the National Natu-