Design, synthesis, screening, and molecular modeling study of a new series of ROS1 receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.029

A series of rationally designed ROS1 tyrosine kinase inhibitors was synthesized and screened. Compound 12b has showed good potency with IC₅₀ value of 209 nM, which is comparable with that of the reference lead compound 1. Molecular modeling studies have been performed, that is, a homology model for ROS1 was built, and the screened inhibitors were docked into its major identified binding site. The docked poses along with the activity data have revealed a group of the essential features for activity. Overall, simplification of the lead compound 1 into compound 12b has maintained the activity, while facilitated the synthetic advantages. A molecular interaction model for ROS1 kinase and inhibitors has been proposed.

Full text of DOI:10.1016/j.bmcl.2009.08.029

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5622–5626
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, screening, and molecular modeling study of a new series
of ROS1 receptor tyrosine kinase inhibitors
Ibrahim M. El-Deeb b, Byung Sun Park c, Su Jin Jung a, Kyung Ho Yoo a, Chang-Hyun Oh a, Seung Joo Cho d,e
,
Dong Keun Han a, Jae Yeol Lee c, So Ha Lee a,
*
a Life Sciences Research Division, 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 Research Institute for Basic Sciences and Department of Chemistry, College of Science, Kyung Hee University, Seoul 130-701, Republic of Korea
d Research Center for Resistant Cells, Chosun University, Gwangju 501-759, Republic of Korea
e Department of Cellular and Molecular Medicine, College of Medicine, Chosun University, 375 Seosuk-dong, Dong-gu Gwangju 501-759, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of rationally designed ROS1 tyrosine kinase inhibitors was synthesized and screened. Compound
12b has showed good potency with IC50 value of 209 nM, which is comparable with that of the reference
lead compound 1. Molecular modeling studies have been performed, that is, a homology model for ROS1
was built, and the screened inhibitors were docked into its major identified binding site. The docked
poses along with the activity data have revealed a group of the essential features for activity. Overall, sim-
plification of the lead compound 1 into compound 12b has maintained the activity, while facilitated the
synthetic advantages. A molecular interaction model for ROS1 kinase and inhibitors has been proposed.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 June 2009
Revised 28 July 2009
Accepted 7 August 2009
Available online 12 August 2009
Keywords:
Kinase inhibitors
ROS1
Glioblastoma multiforme
Homology modeling
Pyrazole
The traditional cancer treatments using classical cytotoxic
drugs cannot distinguish between normal and cancerous cells,
leading to the production of serious side effects, and limiting their
long term use in most of cases.1,2 These common drawbacks of
cytotoxic agents have encouraged scientists to search for more
selective and less toxic therapies for different types of cancers.
After the new advances in the science of genomics and the charac-
terization of the human genome, the incidence of many types of
cancers has been correlated with mutations in genes that confer
growth advantage.3–5 Among these genes are those encoding for
protein kinases, a group of important players in the process of sig-
nal transduction that controls normal cell growth and prolifera-
tion.6,7 Mutations at such genes result usually in the production
of the encoded kinases in abnormal high levels and/or constitutive
uncontrolled activity, leading to uncontrolled cell proliferation and
consequently cancer.8–10
cuses on palliative management.11–13 In a survey of 45 different
human tumor cell lines, the tyrosine kinase ROS1 was found to
be expressed in glioblastoma-derived cell lines at high levels, while
not expressed at all or expressed minimally in the remaining cell
lines.14 ROS1 kinase is a receptor tyrosine kinase that is homolo-
gous to the Drosophila Sevenless tyrosine kinase receptor.15–18 It
is encoded by ROS1-gene which is located at the chromosome 6 re-
gion 6q16 ? 6q22. This region is involved in non-random chromo-
somal arrangements in specific neoplasias.16 A microdeletion at
6q21 results in the fusion of FIG, a gene coding for a Golgi appara-
tus-associated protein, to the kinase domain of the proto-oncogene
ROS1.17,18 The fused protein product FIG-ROS is a potent oncogene,
and its transforming potential resides in its ability to interact with
and become localized at Golgi apparatus.17 The ectopic expression
of ROS1 receptor protein has been reported mainly in meningio-
mas and astrocytomas (25% of low grade and 30% of malignant gli-
oma tumors) suggesting a key role for ROS1 kinase in these CNS
malignancies.17,19 Hence the targeting of the tyrosine kinase
ROS1 could be a useful strategy for treatment of astrocytic
neoplasms.
Glioblastoma multiforme is the most advanced astrocytic neo-
plasm, and is one of the most aggressive human cancers with a
median survival of less than one year. Despite decades of therapeu-
tic research, effective chemotherapeutic treatment for high grade
astrocytomas is not yet available, and patient care ultimately fo-
In a previous work reported by our group20, a new potent and
highly selective ROS1 kinase inhibitor 1 was synthesized and eval-
uated (Fig. 1). The synthesized compound 1 was screened over 45
different kinases, and showed a high selectivity towards ROS1
* Corresponding author. Tel.: +82 2 958 6834; fax: +82 2 958 5189.
E-mail address: LSH6211@kist.re.kr (S.H. Lee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.029

I. M. El-Deeb et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5622–5626
5623
dure21, through the condensation of diethyl oxalate with acetone
in the presence of sodium ethoxide in absolute ethanol. The re-
sulted salt 2 was then cyclized into Claisen furan derivative 3 by
heating in 50% acetic acid followed by acidification with sulfuric
acid.22 The resulted Claisen compound underwent rearrangement
and aromatization into 3-hydroxy-5-methylbenzoic acid (4) within
less than one hour by heating with magnesium oxide in boiling
water, followed by acidification with hydrochloric acid to precipi-
tate the free acid.22
Methyl esterification and O-methylation of the resulted pheno-
lic acid 4 were achieved in a single step and in a high yield (94%) to
give compound 5 through a little modification of the literature pro-
cedure,23 where the acid 4 was refluxed with excess potassium car-
bonate and iodomethane in acetone, and in the presence of a
catalytic amount of DMAP.
OH
CN
N
H3C
N
H
N
N
N
N
HO
1
Figure 1. Structure of the lead compound 1.
kinase, associated with good potency (IC50 = 199 nM). In this Let-
ter, a series of rationally designed compounds have been synthe-
sized and screened over ROS1 kinase in order to study the
structure–activity relationship for this new class of inhibitors. A
molecular modeling study was also made in order to get a better
molecular insight on the mode of enzyme inhibition of these inhib-
itors, where a homology model for the enzyme ROS1 was built, and
the tested compounds were docked into the potential binding site
of the built homolog.
In Scheme 2, the benzoate ester 5 underwent a nucleophilic at-
tack at its carboxylic carbon by the activated methylene group of 2-
chloro-4-methyl-pyrimidine. The activation of this methyl group
into an active methylene was achieved by dropwise addition of
lithium bis(trimethylsilyl)amide (LHMDS) in dry THF at room tem-
perature. The resulted tautomeric
a,b-unsaturated ketone 6 was
then converted to the required pyrazole derivative 7 through two
successive steps. In the first step, compound 6 was heated with ex-
cess N,N-dimethylformamide dimethylacetal for 12 h, and the re-
sulted product was taken to the next step without further
purification, where it was cyclized with hydrazine monohydrate
in absolute ethanol into the pyrazole derivative 7. The reaction of
the resulted pyrazole 7 with iodoacetonitrile in the presence of ex-
cess potassium carbonate produced two different regioisomers; a
major isomer 8 with Rf value of 0.74 (EtOAc–hexane 1:1 v/v), and
a minor isomer 9 with Rf value of 0.84 (EtOAc–hexane 1:1 v/v). A
mixture of these two isomers was taken to the next step without
separation, where it underwent a Suzuki coupling with a series
of arylboronic acids, in the presence of dichloro-bis(triphenylphos-
phine) Pd(II) and potassium carbonate, in a mixed solvent of THF
and water in a (4:1) ratio. These series of Suzuki coupling reactions
produced two isomers in every reaction 10a–j and 11a–j, which
were separated in a pure form by preparative TLC. In all of these
reactions, the 1H-pyrazole isomer (isomer 10) was the product
with the lower Rf value while the 2H-pyrazle isomer (isomer 11)
was the product with the higher Rf. This was proved by the 2D-
NOESY NMR spectrum of compounds 10h and 11h as a model for
The synthesis work started with the preparation of the key es-
ter, methyl 3-methoxy-5-methylbenzoate (5) as illustrated in
Scheme 1. In the first step, the sodium salt of ethyl 2-hydroxy-4-
oxopent-2-enoate (2) was prepared according to literature proce-
O
O
O
OEt
OEt
O
i
ii
O
+
H3C
CH3
ONa O
2
O
OH
OCH3
O
iv
iii
O
OCH3
O
O
H3C
COOH
H3C
O
O
O
3
5
4
Scheme 1. Reaction conditions and reagents: (i) NaOEt, abs EtOH, rt, 4 h, 87%; (ii)
acetic acid: H2O (1:1), rt, 2 h, 50%; (iii) MgO, H2O, reflux, 45 min, 42%; (iv) K2CO3,
CH3I, DMAP, acetone, 65 °C, 12 h, 94%.
OCH3
OCH3
OCH3
OCH3
OCH3
CN
OCH3
N
O
OH
CN
N
N
N
H3C
H3C
H3C
H3C
H3C
ii , iii
iv
NH
N
Cl
i
N
+
OCH3
H3C
N
N
N
N
N
O
Cl
Cl
Cl
Cl
Cl
6
5
9
7
8
OH
OCH3
OH
OCH3
CN
CN
CN
N
N
N
CN
H3C
N
H3C
H3C
v
H3C
vi
N
N
N
N
+
+
N
N
N
N
R
R
R
R
X
X
11a-j
X
X
12a-j
13a-j
10a-j
Scheme 2. Reaction conditions and yields: (i) LHMDS, THF, N2, rt, 18 h, 72%; (ii) DMF–DMA, reflux, 12 h; (iii) hydrazine hydrate, abs EtOH, rt, 2 h, 81%; (iv) K2CO3,
iodoacetonitrile, acetone, reflux, 4 h, 92%; (v) arylboronic acid, Pd(PPh3)2Cl2, Na2CO3, N2, THF/H2O (4:1), 70 °C, 12 h; (vi) BF3ꢀS(CH3)2, dichloromethane, N2, rt, 24 h.

5624
I. M. El-Deeb et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5622–5626
100
80
60
40
20
0
the whole series. The absence of any cross peaks between the ace-
tonitrile —CH2 and any of the aromatic protons of 3-methoxy-5-
ꢁ
methylphenyl group in the 2D-NOESY NMR spectrum of compound
10h while the presence of such peaks in the 2D-NOESY NMR spec-
trum of compound 11h has confirmed the assigned structures. The
final hydroxyl derivatives 12a–j and 13a–j were obtained by
demethylation of the methoxy group of the corresponding meth-
oxy compounds using 10 equiv of borontrifluoride–dimethylsul-
fide complex in dichloromethane.
In Scheme 3, typical procedures to those used in Scheme 2 have
been applied. The only difference was in the starting amine, where
in Scheme 3 and 2-chloro-5-methylpyridine was used instead of 2-
chloro-4-methyl-pyridine (Table 1).
-20
120
100
80
60
40
20
0
Kinase assays were performed at Reaction Biology Corporation
using the ‘HotSpot’ assay platform.24 In the initial screening step;
the 14 selected compounds showed in Figure 2 were tested over
IC50 = 209 nM
ROS1 kinase at a single dose of 20
lM, and the reaction was carried
out at 10 M ATP concentration. At this concentration, compound
l
12b showed a significant inhibition of 96% for the activity of ROS1
kinase, while the inhibition exerted by all of the other tested com-
pounds was below 30%. Accordingly, compound 12b was further
tested in a 10-dose IC50 mode with threefold serial dilutions start-
ing at 20
10-dose IC50 mode with fivefold serial dilutions starting at 20
and the reaction was carried out at 10 M ATP concentration. The
l
M. Staurosporine25 was used as a control compound in a
lM,
l
new compound 12b has showed an IC50 value of 209 nM which is
comparable with that of the reference compound 1 (Fig. 2).
In order to apply a molecular modeling study for the tested
derivatives and correlate between their activities and their binding
mode to the enzyme, a homology model for ROS1 kinase was built
using MOE software (2008.10 version). The last 1347 amino acids of
the total 2347 residues of ROS1 which are predicted to contain the
kinase domain of the enzyme26–28 were used to build the homol-
ogy model over the 3D crystal structure of the insulin-like growth
factor-1 receptor kinase (PDB code 1P4O).
Concentration (nM)
Figure 2. % of ROS1 enzyme inhibition exerted by single dose concentration of
20 M (upper panel), dose–activity curve for compound 12b on ROS1 kinase (lower
l
panel).
A good homology between the two proteins was predicted with
E value of 1.4e-57. The sequences of the two proteins were aligned
and the built homolog was relaxed applying force filed Amber99
acetonitrile nitrogen and leucine 1105 and alanine 1106, one
hydrogen bond between the phenolic OH and leucine 1122, and
a fourth hydrogen bond between the terminal pyridyl nitrogen
and lysine 1111 (Fig. 4).29
calculations.27 The site identifier has identified a major binding site
0
made up of 28 residues and having a size of 172 ÅA. The docking re-
sults for the reference compound 1 and compound 12b have re-
vealed that both of the two compounds have the same pose that
occupies the same space inside the binding pocket (Fig. 3).
In this binding pose, the compound is fit into the receptor site
through four hydrogen bonds. Two hydrogen bonds between the
By investigating the binding interactions of the reference com-
pound 1 (Fig. 3), it was found that the pyrimidinyl nitrogens do not
share in any hydrogen bonding in the receptor. In addition, the 1-
aminopropan-2-ol group does not support any significant binding
for the compound in the receptor site. These observations were
consistent with the screening results for the two active compounds
OCH3
OCH3
OCH3
OCH3
OCH3
CN
OCH3
N
O
OH
CN
N
N
N
H3C
Cl
H3C
Cl
H3C
Cl
H3C
Cl
H3C
Cl
Cl
ii , iii
NH
N
iv
N
+
OCH3
i
H3C
N
N
O
N
N
N
15
17
14
16
5
OCH3
OCH3
OH
OH
CN
CN
CN
CN
N
N
N
N
H3C
H3C
H3C
H3C
v
vi
N
N
N
N
+
+
N
N
N
N
R
R
R
R
X
X
X
X
19a-c
20a-c
21a-b
18a-c
Scheme 3. Reaction conditions and yields: (i) LHMDS, THF, N2, rt, 18 h, 55%; (ii) DMF–DMA, reflux, 12 h; (iii) hydrazine hydrate, abs EtOH, rt, 4 h, 62%; (iv) K2CO3,
iodoacetonitrile, acetone, reflux, 4 h, 80%; (v) arylboronic acid, Pd(PPh3)2Cl2, Na2CO3, N2, THF/H2O (4:1), 70 °C, 12 h; (vi) BF3ꢀS(CH3)2, dichloromethane, N2, rt, 24 h.

I. M. El-Deeb et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5622–5626
5625
Table 1
Synthesized derivatives from Schemes 2 and 3
Compd
X
R
Yield
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
N
H
H
H
H
H
H
H
H
H
N
H
H
H
H
H
H
H
H
H
N
H
H
H
H
H
H
H
H
H
N
H
H
H
H
H
H
H
H
N
H
H
N
H
H
N
H
H
H
H
H
H
74
80
61
79
68
2-Ac
3-Ac
4-Ac
2-NH-Ac
3-NH-Ac
4-CN
4-N(CH3)2
4-OPh
H
H
2-Ac
3-Ac
4-Ac
2-NH-Ac
3-NH-Ac
4-CN
4-N(CH3)2
4-OPh
H
H
2-Ac
3-Ac
4-Ac
2-NH-Ac
3-NH-Ac
4-CN
4-N(CH3)2
4-OPh
H
H
2-Ac
3-Ac
4-Ac
2-NH-Ac
3-NH-Ac
4-CN
4-N(CH3)2
4-OPh
H
2-Ac
3-Ac
H
2-Ac
78
75
82
79
72
62
55
82
77
75
69
63
77
50
69
70
79
43
61
44
66
72
81
75
62
51
55
47
65
42
58
55
72
39
61
78
62
81
77
58
85
61
55
71
62
69
10j
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
12a
12b
12c
12d
12e
12f
12g
12h
12i
Figure 3. Similar docking poses for the reference compound
compound 12b (cyan).
1
(yellow) and
12j
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
18a
18b
18c
19a
19b
19c
20a
20b
20c
21a
21b
3-Ac
H
2-Ac
3-Ac
2-Ac
3-Ac
1 and 12b, where they showed nearly the same IC50 values for en-
zyme inhibition, and this means that the structural differences be-
tween them (the extra pyrimidine nitrogen and the 1-
aminopropan-2-ol side chain) has no significant effect on com-
pound activity.
Figure 4. Hydrogen bonding interactions with compound 12b.
The docking results for the remaining screened compounds
have showed different binding modes to that of compound 12b,
and in the majority of them, the compounds were loosely bound
to the receptor site with only one or maximum two hydrogen
bonds. It showed also that these derivatives failed to occupy the
same binding pose of compound 12b, due to either the lack of
hydrogen bonding capability with lysine 1111, or clashing with
the receptor surface caused by the excessive bulkiness of the sub-
stituent at the phenyl ring (Fig. 5).
It became clear from these results that the terminal pyridyl
moiety in compounds 1 and 12b is an essential feature for activity
and is highly conserved, and that the replacement of this moiety
with unsubstituted or substituted phenyl group, results in a great
loss of activity. The simple deviation in the position of this terminal
pyridyl group in space caused by the shift in its position from the
meta-position (compounds in Scheme 2) to the para-position (com-
pounds in Scheme 3) relative to the central pyridine, results in a
great loss of activity too, and this means that the orientation of
the pyridyl group is also highly conserved (Fig. 6).
From Figure 5, we can also conclude that the 2H-pyrazole iso-
mers of this series cannot fit well in the intended pose at the recep-
tor site, because of the loss of the two hydrogen bonds formed with
the acetonitrile nitrogen, in addition to the possible clash between
the acetonitrile group at this position and the receptor surface. For
a more clear comparison between the different binding modes of
all of the screened compounds, the bound complexes were aligned

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I. M. El-Deeb et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5622–5626
these two active compounds (1 and 12b) have typical binding pro-
files, which accounts for their identical inhibitory activity.
In conclusion, the molecular modeling study has showed that the
more simple structure of compound 12b contains three essential
features for activity (terminal pyridyl, 1H-acetonitrile, and phenolic
hydroxyl), and that this structure is highly conserved in most of its
parts. A possible site that is yet viable for more modifications is the
methyl group at the 3-hydroxy-5-methylphenyl ring. The extension
of structure originated from this point may produce derivatives with
extending binding features and possible enhancement in activity.
Acknowledgments
This research was supported by Korea Institute of Science and
Technology and Pioneer Research Program for converging technol-
ogy through the Korea Science and Engineering Foundation funded
by the Ministry of Education, Science and Technology
(M10711060001-08M1106-00110). We also appreciate to Dr. Sean
W. Deacon and Dr. Haiching Ma from Reaction Biology Corporation
for kinase screening.
Figure 5. Proposed clash between compound 13g and the receptor surface when
occupy the same pose of compound 12b.
References and notes
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Figure 6. Binding mode of compound 20a.
21. Wipf, P.; Mahler, S. G.; Okumura, K. Org. Lett. 2005, 7, 4483.
22. Turner, F. A.; Gearien, J. E. J. Org. Chem. 1959, 24, 1952.
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24. Kinase assays were performed at Reaction Biology Corporation using the
‘HotSpot’ assay platform. Kinase Assay Protocol: Reaction Buffer: 20 mM Hepes
(pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM
Na3VO4, 2 mM DTT, 1% DMSO. Reaction Procedure: To a freshly prepared buffer
together, and a barcode presentation mode was established to view
all the interacting residues against the docked compounds, and the
characteristic bindings with each of them. As showed in Figure 7, a
unique common binding feature in compounds 1 and 12b is the
binding with leucine 1122. This characteristic binding was not ob-
served at any of the other derivatives. This figure reveals also that
solution was added ROS1 kinase at a concentration of 20 lM. The contents
were mixed gently, then the compound under test dissolved in DMSO was
added to the reaction mixture in the appropriate concentration. 339-ATP
(specific activity 500 lCi/ll) was added to the mixture in order to initiate the
reaction, and the mixture was incubated at room temperature for 2 h. Initial
single dose screening: The compound was tested by single dose duplicate
made at
compound in a 5-dose IC50 mode with 10-fold serial dilutions starting at
20 M. Reaction was carried out at 10 M ATP concentration. Testing in IC50
mode: Compound 12b was tested in a 10-dose IC50 mode with threefold serial
dilutions starting at 20 M. Staurosporine was used as a control compound in a
10-dose IC50 mode with fivefold serial dilutions starting at 20 M. Reaction
was carried out at 10 M ATP concentration.
a concentration of 10 lM. Staurosporine was used as a control
l
l
l
l
l
25. Yang, S.; Malaviya, R.; Wilson, L. J.; Argentieri, R.; Chen, X.; Yang, C.; Wang, B.;
Cavender, D.; Murray, W. V. Bioorg. Med. Chem. Lett. 2007, 17, 326.
26. Birchmeier, C.; Birnbaum, D.; Waitches, G.; Fasano, O.; Weigler, M. Mol. Cell
Biol. 1986, 6, 3109.
27. Birchmeier, C.; O’Neill, K.; Riggs, M.; Wigler, M. Proc. Natl. Acad. Sci. U.S.A. 1990,
87, 4799.
28. Wang, J.; Cieplak, P.; Kollman, P. A. J. Comput. Chem. 2001, 21, 1049.
29. The showed residues’ numbers refer to the amino acid sequence in the built
model which is 1000 less than the actual number in the whole original protein.
Figure 7. Barcode presentation mode for the characteristic interactions of the
screened compounds at the receptor site (the order from top to bottom: 1, 12b, 12a,
12c, 12d, 12e, 12f, 13f, 13g, 12h, 12i, 12j, 20a, 20b, 20c).