Synthesis and biological evaluation of new pyrimidine-4-yl-ethanol derivatives as ROS1 kinase inhibitors

Article abstract of DOI:10.3109/14756366.2014.920838

As a part of trials to target ROS1 kinase with potential inhibitors, a novel series of pyrimidin-4-yl-ethanol and ethanone derivatives (4a-f, 5a-f, 6a-f and 7a-f) have been designed based on previously discovered lead compounds KIST301072 and KIST301080, and synthesized on 4-5 steps according to compounds. The structures of the newly synthesized compounds have been confirmed on ¹H-NMR, ¹³C-NMR and IR. Most of the tested compounds showed ROS1 kinase inhibitory activity in micromolar range.

Full text of DOI:10.3109/14756366.2014.920838

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.920838
!
ORIGINAL ARTICLE
Synthesis and biological evaluation of new pyrimidine-4-yl-ethanol
derivatives as ROS1 kinase inhibitors
Ahmed Z. Abdelazem1,2 and So Ha Lee2
1Department of Biological Chemistry, Korea University of Science and Technology, Gwahangno, Yuseong-gu, Daejeon, Republic of Korea and
2Chemical Kinomics Research Center, Korea Institute of Science and Technology, Hwarangno, Seongbuk-gu, Seoul, Republic of Korea
Abstract
Keywords
As a part of trials to target ROS1 kinase with potential inhibitors, a novel series of pyrimidin-4-yl-
ethanol and ethanone derivatives (4a–f, 5a–f, 6a–f and 7a–f) have been designed based on
previously discovered lead compounds KIST301072 and KIST301080, and synthesized on 4–5
steps according to compounds. The structures of the newly synthesized compounds have been
confirmed on 1H-NMR, 13C-NMR and IR. Most of the tested compounds showed ROS1 kinase
inhibitory activity in micromolar range.
Drug, non-small cell lung cancer, receptor
tyrosine kinase, ROS1
History
Received 6 March 2014
Revised 7 April 2014
Accepted 14 April 2014
Published online 17 June 2014
Introduction
the tyrosine kinase ROS1 could be a useful strategy for treatment
of such tumors.
Protein kinases are defined by their ability to catalyze the transfer
of the terminal phosphate of ATP to substrates that usually
contain a serine, threonine or tyrosine residue1. All protein
kinases, thus far characterized with regard to substrate specificity,
fall within one of three classes, tyrosine kinases, serine/threonine
kinases and dual specificity protein kinases2. Tyrosine kinases are
further divided into two subgroups; receptor tyrosine kinases
(RTKs) and non-receptor tyrosine kinases3. The protein kinase
family plays an important role in regulating various cellular
processes such as cell proliferation, cell death, cell cycle
progression, differentiation and cell survival. Therefore, it is not
surprising that the deregulation of many kinases is usually directly
linked to cancer development4. Hence, protein kinase has become
the most intensively pursued anticancer drug target5,6. It is also
worthy to mention that, since protein kinases are quite selective in
their actions, they represent a wide spectrum of specific and
exploitable targets for drugs7. Currently, more than 14 small
molecule inhibitors have become available on the market for
cancer treatments since the first kinase inhibitor (imatinib) was
approved by FDA in 2001 for chronic myeloid leukaemia8.
There are 58 RTKs in humans that belong to 20 distinct
families, and ROS1 kinase is one of them9,10. The ectopic
expression, as well as the production of variable mutant forms of
ROS1 kinase, has been reported in a number of cancers, such as
glioblastoma multiforme, cholangiocarcinoma, gastric adenocar-
cinoma and non-small cell lung cancer, suggesting a role for
ROS1 kinase in deriving such tumors11. Hence, the targeting of
In aspiration of interest in developing novel ROS1 inhibitors,
we recently detailed the discovery of KIST301072 as the first
selective inhibitor for ROS1 RTK on 45 kinases with an IC50 of
199 nM12, followed by the development of more simplified and
equipotent derivative KIST301080 with an IC50 value of
209 nM13. These two compounds represented new promising
leads for the development of more potent and more selective
ROS1 kinase inhibitors, and following this discovery, it was
important to explore the new chemical scaffold in order to have a
clear picture about the structure–activity relationship (SAR) of
the new group of ROS1 kinase inhibitors (Figure 1).
Therefore, as a continuation of our study, a further SAR study
was conducted to explore the effect of changing the central
pyrazole ring into an open form. Furthermore, considering the
importance of polar side chains, which act as water-solubilizing
groups to interact with water molecules within the kinase
enzymes14,15, efforts to improve the potency of this scaffold
was also made by exploring more water-solubilizing groups.
Experimental
Chemistry
General
NMR spectra were recorded with a Bruker NMR spectrometer
(Billerica, MA), operating at 400 MHz for 1H NMR and 100 MHz
for 13C NMR. The multiplicities were abbreviated as s: singlet, d:
doublet, t: triplet, m: multiplet and q: quartet. The coupling
constants J are recorded in hertz (Hz), and the relative shift values
of peak are recorded by ppm unit using tetramethylsilane as
standard material. Melting points were determined on a SRS
OPTIMELT (Sunnyvale, CA). FT-IR spectra were obtained on
Perkin Elmer 16E PC FT-IR Spectrometer (Waltham, MA).
Address for correspondence: So Ha Lee, PhD, Chemical Kinomics
Research Center, Korea Institute of Science and Technology, Hwarangno
14-gil 5, Seongbuk-gu, Seoul 136-791, Korea. Tel: 82-2-958-6834. Fax:
82-2-958-5189. E-mail: LSH6211@kist.re.kr

2
A. Z. Abdelazem & S. H. Lee
J Enzyme Inhib Med Chem, Early Online: 1–9
OH
OH
IR (KBr) 3274, 2918, 2843, 1636, 1590, 1461, 1224, 1193, 851
1
and 689 cmꢃ1. H NMR (CDCl3) ꢀ 1.90 (m, 2H), 2.40 (s, 3H),
3.59–3.65 (m, 4H), 3.87 (s, 3H), 5.86 (s, 1H), 6.33 (s, 1H), 6.83 (s,
1H), 7.16 (s, 1H), 7.23 (s, 1H) and 15.33 (s, 1H). 13C NMR
(CDCl3) ꢀ 21.46, 21.59, 32.60, 38.07, 55.36, 55.47, 58.85, 93.38,
105.64, 108.33, 110.10, 110.15, 117.33, 119.26, 120.94, 122.21,
136.36, 139.60, 140.03, 159.17, 159.75, 159.89, 160.40, 165.79
and 194.62.
CN
CN
N
N
OH
N
H
N
N
N
N
N
N
N
2-(2-Chloro-6-(2-morpholinoethylamino)pyrimidin-4-yl)-1-(3-
methoxy-5-methylphenyl)ethanone (3b)
KIST301072
KIST301080
Flash column chromatography was carried out using ethyl acetate-
hexane (2:1, v/v) to give the title compound as a mixture of keto/
enol tautomers (1:2.3). Yield: 630 mg (48%) and mp 114–116 ꢁC.
Figure 1. Structure of the lead compounds KIST301072 and
KIST301080.
IR (KBr) 3318, 2956, 1681, 1588, 1209, 1067, 854 and 683 cmꢃ1
.
1H NMR (CDCl3) ꢀ 2.40 (s, 3H), 2.54 (m, 4H), 2.68 (m, 2H), 3.54
(m, 2H), 3.76 (m, 4H), 3.89 (s, 3H), 5.87 (s, 1H), 6.33 (s, 1H),
6.83 (s, 1H), 7.17 (s, 1H), 7.23 (s, 1H) and 15.03 (s, 1H). 13C
NMR (CDCl3) ꢀ 21.48, 21.58, 37.57, 37.69, 47.58, 53.24, 53.28,
55.34, 55.44, 66.69, 66.78, 93.51, 105.58, 108.22, 109.84, 110.33,
117.30, 119.19, 120.63, 122.23, 136.34, 137.58, 139.58, 139.89,
158.99, 159.78, 159.85, 160.67, 165.85, 166.19, 168.18 and
195.23.
Thin-layer chromatography was performed using precoated plates
(0.25 mm, Merck) of silica gel 60 F254 (230 ꢀ 400 mesh) for
monitoring all reactions and under ultraviolet irradiation
(254 nm). Column chromatography separations are performed
using silica gel (230 ꢀ 400 mesh, Merck). All the commercially
available reagent chemicals were obtained from Aldrich, TCI,
Wako Pure Chemical, Acros and Dae-Jung Chemicals, and
generally used without further purification.
2-(2-Chloro-6-((2-(piperidin-1-yl)ethyl)amino)pyrimidin-4-yl)-1-
(3-methoxy-5-methylphenyl)ethanone (3c)
2-(2,6-Dichloropyrimidin-4-yl)-1-(3-methoxy-5-methylphenyl)
ethanone (2)
Flash column chromatography was carried out using ethyl acetate-
hexane (4:1, v/v) to give the title compound as a mixture of keto/
enol tautomers (1:2.7). Yield: 250 mg (13 %) and mp 90–92 ꢁC.
To a solution of methyl 3-methoxy-5-methylbenzoate (1) (5.0 g,
27.7 mmol)
and
2,4-dichloro-6-methylpyrimidine
(5.4 g,
33.1 mmol) in 75 mL of dry THF was added a 1.0 M solution of
lithium bis(trimethylsilyl)amide (LHMDS) in THF (55.6 mL,
55.6 mmol) at 0 ꢁC dropwise using dropping funnel, and reaction
mixture was stirred at room temperature for 12 h. The reaction
was quenched with 1 N aqueous HCl solution for neutralization
and then extracted with ethyl acetate (2 ꢂ 150 mL). The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
column chromatography using the mixed solvent of hexane and
dichloromethane (4:1, v/v) to give the title compound as a mixture
of keto/enol tautomers (1:1.5). Yield: 7 g (81%) and mp 121–
122 ꢁC. IR (KBr) 3061, 2920, 1687, 1627, 1589, 1220, 1061, 832
and 678 cmꢃ1. 1H NMR (CDCl3) ꢀ 2.42 (s, 3H), 3.86 (s, 3H), 6.01
(s, 1H), 6.88 (s, 1H), 7.01 (s, 1H), 7.21 (s, 1H), 7.26 (s, 1H) and
13.54 (s, 1H). 13C NMR (CDCl3) ꢀ 21.47, 21.60, 46.81, 55.39,
55.49, 93.09, 108.70, 110.03, 114.72, 118.12, 119.52, 120.81,
121.28, 122.00, 135.24, 136.99, 139.83, 140.23, 157.82, 159.84,
160.00, 160.48, 161.10, 162.55, 167.65, 168.11, 169.46 and
193.85.
IR (KBr) 3265, 2934, 1637, 1592, 1228, 1064, 820 and 691 cmꢃ1
.
1H NMR (CDCl3) ꢀ 1.49 (m, 2H), 1.63 (m, 4H), 2.40 (s, 3H), 2.47
(m, 4H), 2.62 (m, 2H), 3.51 (m, 2H), 3.86 (s, 3H), 5.87 (s, 1H),
6.01 (s, 1H), 6.31 (s, 1H), 6.83 (s, 1H), 7.18 (s, 1H), 7.23 (s, 1H)
and 15.17 (s, 1H). 13C NMR (CDCl3) ꢀ 21.47, 21.57, 24.22,
24.31, 25.65, 25.77, 38.01, 38.10, 54.21, 54.28, 55.34, 55.43,
57.06, 93.43, 105.28, 109.56, 110.26, 117.29, 119.21, 120.69,
122.31, 136.51, 139.54, 159.76, 160.59, 165.69, 166.12, 168.41
and 193.46.
2-(2-Chloro-6-((2-(diethylamino)ethyl)amino)pyrimidin-4-yl)-1-
(3-methoxy-5-methylphenyl)ethanone (3d)
Flash column chromatography was carried out using ethyl acetate
to give the title compound as a mixture of keto/enol tautomers
(1:2.2). Yield: 350 mg (19%) and mp 65–67 ꢁC. IR (KBr) 3275,
1
2964, 1637, 1225, 1063, 821 and 691 cmꢃ1. H NMR (CDCl3) ꢀ
1.07 (t, J ¼ 7.2, 6H), 2.41 (s, 3H), 2.60–2.63 (m, 6H), 3.45 (m,
2H), 3.87 (s, 3H), 5.87 (s, 1H), 6.01 (s, 1H), 6.32 (s, 1H), 6.83 (s,
1H), 7.18 (s, 1H), 7.24 (s, 1H) and 15.22 (s, 1H). 13C NMR
(CDCl3) ꢀ 11.31, 11.43, 21.47, 21.58, 38.86, 46.39, 46.68, 51.26,
55.35, 55.44, 93.68, 108.15, 109.63, 117.31, 119.22, 122.31,
136.52, 139.55, 139.87, 159.76, 159.83, 160.61, 165.68, 166.12,
168.45 and 195.79.
General procedure for the synthesis of compounds 3a–f
A
mixture of 2-(2,6-dichloropyrimidin-4-yl)-1-(3-methoxy-5-
methylphenyl)ethanone (2) (1.0 g, 3.23 mmol) and the appropriate
amine in THF was heated to 80 ꢁC in an oil bath for three hours.
The reaction mixture was left to cool at room temperature and
then concentrated in vacuo. The residue was partitioned between
water and ethyl acetate. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concen-
trated in vacuo. The residue was purified by column chromatog-
raphy using the proper mobile phase.
2-(2-Chloro-6-(4-ethylpiperazin-1-yl)pyrimidin-4-yl)-1-
(3-methoxy-5-methylphenyl)ethanone (3e)
Flash column chromatography was carried out using ethyl acetate
to give the title compound as a mixture of keto/enol tautomers
(1:1.6). Yield: 512 mg (28%); sticky solid. IR (KBr) 2928, 2812,
1
1637, 1591, 1231, 1061, 821 and 691 cmꢃ1. H NMR (CDCl3) ꢀ
2-(2-Chloro-6-(3-hydroxypropylamino)pyrimidin-4-yl)-1-
(3-methoxy-5-methylphenyl)ethanone (3a)
1.14 (t, J ¼ 7.2, 3H), 2.39 (s, 3H), 2.44–2.58 (m, 6H), 3.84–3.86
(m, 7H), 5.32 (s, 1H), 5.89 (s, 1H), 6.28 (s, 1H), 6.82 (s, 1H), 7.17
(s, 1H), 7.23 (s, 1H) and 15.06 (s, 1H). 13C NMR (CDCl3) ꢀ
11.89, 11.95, 21.45, 21.56, 43.69, 44.23, 47.93, 52.32, 52.40,
52.51, 53.42, 55.33, 55.41, 93.59, 105.11, 108.11, 108.79, 110.31,
Flash column chromatography was carried out using ethyl acetate-
hexane (2:1, v/v) to give the title compound as a mixture of keto/
enol tautomers (1:1.6). Yield: 550 mg (49%) and mp 105–106 ꢁC.

DOI: 10.3109/14756366.2014.920838
Synthesis and biological evaluation of ROS1 kinase inhibitors
3
117.33, 119.16, 120.59, 122.43, 136.34, 137.63, 139.55, 139.76, 139.82, 148.44, 148.57, 151.06, 151.14, 159.77, 162.73, 165.47
158.48, 159.77, 159.81, 160.66, 161.18, 161.39, 165.57, 165.75, and 196.09.
168.02 and 195.41.
1-(3-Methoxy-5-methylphenyl)-2-(6-((2-(piperidin-1-yl)ethyl)
amino)-2-(pyridin-3-yl)pyrimidin-4-yl)ethanone (4c)
2-(2-Chloro-6-(4-(dimethylamino)piperidin-1-yl)pyrimidin-4-yl)-
1-(3-methoxy-5-methylphenyl) ethanone (3f)
Flash column chromatography was carried out using ethyl acetate-
Flash column chromatography was carried out using ethyl acetate- methanol (2:1, v/v) to give the title compound as a mixture of
methanol (4:1, v/v) to give the title compound as a mixture of keto/enol tautomers (1:2.6). Yield: 100 mg (40%) and mp 57–
keto/enol tautomers (1:1.8). Yield: 345 mg (18%) and mp 73– 58 ꢁC. IR (KBr) 3293, 2932, 1719, 1633, 1588, 1216, 1063, 840
1
75 ꢁC. IR (KBr) 3388, 2927, 1682, 1637, 1570, 1231, 1061, 837 and 705 cmꢃ1. H NMR (CDCl3) ꢀ 1.50 (m, 2H), 1.64–1.68 (m,
and 691 cmꢃ1. 1H NMR (CDCl3) ꢀ 1.42–1.57 (m, 2H), 1.87–1.91 4H), 2.39 (s, 3H), 2.52 (m, 4H), 2.64–2.70 (m, 2H), 3.64 (m, 2H),
(m, 2H), 2.26 (s, 6H), 2.31 (s, 3H), 2.39 (m, 1H), 2.92–2.97 (m, 3.87 (s, 3H), 6.02 (s, 1H), 6.72 (s, 1H), 6.82 (s, 1H), 6.96 (s, 1H),
2H), 3.78 (s, 3H), 4.10 (s, 1H), 4.47–4.49 (m, 2H), 5.77 (s, 1H), 7.22 (s, 1H), 7.29 (s, 1H), 7.37–7.50 (m, 1H), 8.30–8.34 (m, 1H),
6.15 (s, 1H), 6.43 (s, 1H), 6.73 (s, 1H), 7.10 (s, 1H), 7.16 (s, 1H) 8.70–8.71 (m, 1H), 9.23 (s, 1H) and 15.47 (s, 1H). 13C NMR
and 15.02 (s, 1H).
(CDCl3) ꢀ 21.47, 21.58, 24.11, 24.28, 25.49, 25.71, 29.69, 38.07,
38.13, 48.31, 54.35, 54.42, 55.34, 55.43, 57.09, 57.47, 93.59,
102.75, 106.32, 108.13, 110.33, 117.16, 11.05, 117.16, 119.26,
12.63, 122.38, 122.85, 123.46, 133.14, 133.34, 134.38, 134.50,
137.31, 137.72, 139.45, 139.84, 148.46, 148.58, 151.02, 151.11,
159.76, 159.82, 162.65, 164.47, 165.57 and 195.93.
General procedure for the synthesis of compounds 4a–f
To a glass vial containing a magnetic stir bar was added a mixture
of the starting compound 3a–f (1 mmol), 3-pyridineboronic acid
(1.5 mmol), bis(triphenylphosphine)palladium(II) dichloride
(0.05 mmol) and potassium carbonate (2 mmol) and the vial was
purged with nitrogen gas for 10 min. Then, a solvent mixture of
tetrahydrofuran/acetonitrile/water (8 mL/2:1:1) was injected, and
2-(6-((2-(Diethylamino)ethyl)amino)-2-(pyridin-3-yl)pyrimidin-4-
yl)-1-(3-methoxy-5-methylphenyl) ethanone (4d)
the vial was once again bubbled with nitrogen for 10 min and then Flash column chromatography was carried out using ethyl acetate-
capped. The reaction mixture was stirred at 90 ꢁC under nitrogen. methanol (4:1, v/v) to give the title compound as a mixture of
After three hours, the reaction mixture was left to cool to ambient keto/enol tautomers (1:2.6). Yield: 130 mg (33%); sticky solid. IR
temperature. The reaction mixture was filtered through Celite. (KBr) 3248, 2967, 1751, 1633, 1583, 1215, 1063, 838 and
1
Then the filtrate was concentrated in vacuo, and the residue was 705 cmꢃ1. H NMR (CDCl3) ꢀ 1.07 (m, 6H), 2.40 (s, 3H), 2.59–
purified by column chromatography using the proper mobile 2.63 (m, 4H), 2.75–2.78 (m, 2H), 3.58–3.60 (m, 2H), 3.85 (s, 3H),
phase.
5.90 (s, 1H), 6.02 (s, 1H), 6.72 (s, 1H), 6.82 (s, 1H), 7.23 (s, 1H),
7.40 (m, 1H), 8.33 (m, 1H), 8.69 (m, 1H), 9.23 (s, 1H) and 15.94
(s, 1H). 13C NMR (CDCl3) ꢀ 11.41, 21.47, 21.58, 38.94, 46.52,
46.78, 51.19, 51.59, 55.35, 55.43, 102.74, 106.24, 108.10, 110.30,
117.19, 119.28, 120.66, 122.41, 123.44, 134.39, 134.49, 139.46,
148.49, 148.61, 151.04, 151.11, 159.77 and 164.49.
2-(6-((3-Hydroxypropyl)amino)-2-(pyridin-3-yl)pyrimidin-4-yl)-
1-(3-methoxy-5-methylphenyl)ethanone (4a)
Flash column chromatography was carried out using ethyl
acetate–methanol (30:1, v/v) to give the title compound as a
mixture of keto/enol tautomers (1:1.7). Yield: 137 mg (35%) and
mp 99–100 ꢁC.IR (KBr) 3302, 2929, 1673, 1587, 1216, 1064, 929
2-(6-(4-Ethylpiperazin-1-yl)-2-(pyridin-3-yl)pyrimidin-4-yl)-1-(3-
methoxy-5-methylphenyl)ethanone (4e)
and 706 cmꢃ1. H NMR (CDCl3) ꢀ 1.88–1.92 (m, 2H), 2.38 (s,
1
3H), 3.64–3.71 (m, 4H), 3.82–3.85 (m, 2H), 3.87 (s, 3H), 5.97 (s, Flash column chromatography was carried out using ethyl acetate-
1H), 6.04 (t, J ¼ 6.4 Hz, 1H), 6.66 (s, 1H), 6.81 (s, 1H), 7.19 (s, methanol (4:1, v/v) to give the title compound as a mixture of
1H), 7.25 (s, 1H), 7.38–7.42 (m, 1H), 8.27 (dd, J ¼ 1.6, 8.0 Hz, keto/enol tautomers (1:1.6). Yield: 450 mg (90%) and mp 81–
1H), 8.67 (dd, J ¼ 1.6, 5.2 Hz, 1H), 9.19 (d, J ¼ 1.6 Hz, 1H) and 82 ꢁC. IR (KBr) 2926, 2852, 1737, 1634, 1575, 1217, 1062, 841
1
15.39 (s, 1H). 13C NMR (CDCl3) ꢀ 21.46, 21.60, 32.20, 32.93, and 705 cmꢃ1. H NMR (CDCl3) ꢀ 1.17 (t, J ¼ 7.2, 3H), 2.28 (s,
37.81, 47.94, 55.37, 55.46, 59.07, 59.90, 93.67, 103.10, 106.77, 3H), 2.35–2.43 (m, 6H), 3.74 (s, 3H), 3.84–3.86 (m, 4H), 5.91 (s,
108.39, 110.25, 117.26, 119.35, 120.79, 122.31, 123.57, 123.63, 1H), 6.58 (s, 1H), 6.69 (s, 1H), 7.11 (s, 1H), 7.17 (s, 1H), 7.25–
132.89, 134.41, 134.55, 137.12, 137.61, 139.54, 139.96, 148.32, 7.27 (m, 1H), 8.18 (d, J ¼ 8.0 Hz, 1H), 8.59 (s, 1H), 9.16 (s, 1H)
148.54, 151.12, 151.27, 158.59, 159.79, 159.89, 161.03, 163.14, and 15.41 (s, 1H). 13C NMR (CDCl3) ꢀ 11.89, 11.96, 21.46,
164.70 and 195.62.
21.57, 43.61, 44.17, 48.70, 52.38, 52.46, 52.53, 52.66, 53.44,
55.31, 55.38, 93.90, 102.31, 105.41, 108.03, 110.36, 117.18,
2-(6-(2-Morpholinoethylamino)-1-(3-methoxy-5-methylphenyl)-2- 119.19, 120.54, 122.58, 123.37, 123.41, 133.24, 133.29, 134.31,
(pyridin-3-yl)pyrimidin-4-yl)ethanone (4b)
134.40, 137.04, 137.71, 139.47, 139.70, 148.52, 148.63, 151.08,
158.27, 159.76, 161.38, 161.83, 162.27, 164.63, 165.29, 169.12
and 196.06.
Flash column chromatography was carried out using ethyl acetate-
methanol (10:1, v/v) to give the title compound as a mixture of
keto/enol tautomers (1:2.8). Yield: 317 mg (71%) and mp 72–
74 ꢁC. IR (KBr) 3259, 2926, 1680, 1588, 1216, 1065, 854 and
2-(6-(4-(Dimethylamino)piperidin-1-yl)-2-(pyridin-3-yl)
pyrimidin-4-yl)-1-(3-methoxy-5-methylphenyl) ethanone (4f)
1
706 cmꢃ1. H NMR (CDCl3) ꢀ 2.36 (s, 3H), 2.50 (m, 4H), 2.65
(m, 2H), 3.59 (m, 2H), 3.74 (m, 4H), 3.82 (s, 3H), 5.88 (s, 1H), Flash column chromatography was carried out using ethyl acetate-
5.99 (s, 1H), 6.70 (s, 1H), 6.80 (s, 1H), 7.20 (s, 1H), 7.39–7.40 (m, methanol-triethylamine (1:1: 0.01 v/v/v) to give the title com-
1H), 8.29 (d, J ¼ 8.0 Hz, 1H), 8.67 (d, J ¼ 3.6 Hz, 1H), 9.21 (s, pound as a mixture of keto/enol tautomers (1:2.3). Yield: 300 mg
1H) and 15.38 (s, 1H). 13C NMR (CDCl3) ꢀ 21.48, 21.58, 22.96, (83%) and mp 184–185 ꢁC. IR (KBr) 3381, 2949, 1681, 1636,
1
23.83, 28.79, 30.52, 37.70, 37.74, 38.73, 48.06, 53.37, 53.40, 1581, 1215, 1061, 843 and 707 cmꢃ1. H NMR (CDCl3) ꢀ 1.47–
55.33, 55.41, 57.33, 66.75, 66.80, 66.97, 93.71, 102.98, 106.41, 1.54 (m, 2H), 1.89–1.92 (m, 2H), 2.25 (s, 6H), 2.31 (s, 3H), 2.36–
108.16, 110.51, 117.14, 119.22, 120.40, 122.30, 123.43, 123.46, 2.42 (m, 1H), 2.94–3.00 (m, 2H), 3.77 (s, 3H), 4.61–4.64 (m, 2H),
128.76, 130.34, 133.24, 134.34, 134.5, 137.23, 137.84, 139.48, 5.93 (s, 1H), 6.57 (s, 1H), 6.72 (s, 1H), 7.15 (s, 1H), 7.21 (s, 1H),

4
A. Z. Abdelazem & S. H. Lee
J Enzyme Inhib Med Chem, Early Online: 1–9
7.28–7.31 (m, 1H), 8.21 (dt, J ¼ 2.0, 8.0 Hz, 1H), 8.61 (dd, 2-(6-((2-(Diethylamino)ethyl)amino)-2-(pyridin-3-yl)pyrimidin-4-
J ¼ 1.6, 4.8 Hz, 1H), 9.17 (d, J ¼ 1.6 Hz, 1H) and 15.53 (s, 1H). yl)-1-(3-hydroxy-5-methylphenyl) ethanone (5d)
13C NMR (CDCl3) ꢀ 21.40, 21.52, 28.08, 41.52, 41.60, 43.16,
Flash column chromatography was carried out using ethyl acetate-
43.62, 48.63, 55.22, 55.29, 62.08, 62.51, 93.69, 101.80, 104.94,
methanol (4:1, v/v) to give the title compound. Yield: 37 mg
108.05, 110.38, 117.04, 119.14, 120.39, 122.52, 123.32, 133.22,
(29%) and mp 135–136 ꢁC. IR (KBr) 3428, 2956, 2924, 1723,
134.20, 134.32, 137.13, 137.71, 139.32, 139.59, 148.40, 148.51,
1247, 1099, 874 and 758 cmꢃ1 1HNMR (DMSO-d6) ꢀ 1.05
.
150.89, 157.77, 159.69, 161.12, 161.65, 162.12, 164.35, 165.25,
169.33 and 196.04.
(t, J ¼ 7.2 Hz, 6H), 2.33 (s, 3H), 2.65 (m, 4H), 2.79 (m, 2H), 3.73–
3.75 (m, 2H), 6.59 (s, 1H), 6.81 (s, 1H), 7.15 (s, 1H), 7.22 (s, 1H),
7.44 (s, 1H), 7.52–7.54 (m, 1H), 7.63 (dd, J ¼ 4.8, 8.0 Hz, 1H),
8.52 (dt, J ¼ 1.6, 8.0 Hz, 1H), 8.80 (m, 1H), 9.36 (s, 1H) and 9.57
(s, 1H). 13C NMR (DMSO-d6) ꢀ 12.16, 21.47, 47.09, 47.33, 50.80,
93.78, 102.39, 110.77, 118.37, 119.91, 124.40, 131.51, 134.80,
135.33, 139.95, 149.06, 152.90, 154.90, 157.49, 158.15 and
163.66.
General procedure for the synthesis of compounds 5a–f
To a solution of the starting compound 4a–f (0.3 mmol) in 5 mL
of dry dichloromethane was added boron trifluoride–methylsul-
fide complex (3 mmol) dropwise. The resulting suspension was
stirred at room temperature for 12 h. The mixture was concen-
trated, and the residue was partitioned between ethyl acetate and
sodium bicarbonate solution. The layers were separated, and the
aqueous layer was extracted with ethyl acetate. The combined
organic layers were dried over anhydrous sodium sulfate and
concentrated in vacuo. The residue was purified by column
chromatography using the proper mobile phase.
2-(6-(4-Ethylpiperazin-1-yl)-2-(pyridin-3-yl)pyrimidin-4-yl)-1-(3-
hydroxy-5-methylphenyl)ethanone (5e)
Flash column chromatography was carried out using ethyl
acetate–methanol (4:1, v/v) to give the title compound. Yield:
100 mg (46%) and mp 149–150 ꢁC. IR (KBr) 3419, 2955, 2924,
1723, 1567, 1246, 1099, 874 and 759 cmꢃ1. 1H NMR (DMSO-d6)
ꢀ 1.17 (m, 3H), 2.30 (s, 3H), 2.35–2.44 (m, 4H), 3.79 (m, 4H),
6.24 (s, 1H), 6.72 (s, 1H), 7.19 (s, 1H), 7.20 (s, 1H), 7.30 (s, 1H),
7.54–7.59 (m, 1H), 8.44–8.73 (m, 1H), 8.70–8.73 (m, 1H), 9.27–
9.29 (m, 1H) and 15.23 (s, 1H).
1-(3-Hydroxy-5-methylphenyl)-2-(6-((3-hydroxypropyl)amino)-2-
(pyridin-3-yl)pyrimidin-4-yl)ethanone (5a)
Flash column chromatography was carried out using ethyl
acetate–methanol (10:1, v/v) to give the title compound. Yield:
22 mg (19%) and mp 97–99 ꢁC. IR (KBr) 3336, 2923, 1738, 1585,
2-(6-(4-(Dimethylamino)piperidin-1-yl)-2-(pyridin-3-yl)pyrimi-
din-4-yl)-1-(3-hydroxy-5-methylphenyl) ethanone (5f)
.
1243, 1019 and 851 cmꢃ1 1H NMR (DMSO-d6) ꢀ 1.82
(d, J ¼ 6.4, 12.8 Hz, 2H), 2.35 (s, 3H), 3.56 (q, J ¼ 6.0 Hz, 1H),
3.73 (q, J ¼ 6.4 Hz, 1H), 4.69 (s, 1H), 6.61 (s, 1H), 6.79 (s, 1H),
7.14 (s, 1H), 7.21 (s, 1H), 7.65 (ddd, J ¼ 0.8, 4.8, 8.0 Hz, 1H),
8.54 (dt, J ¼ 1.6, 8.0 Hz, 1H), 8.80 (dd, J ¼ 1.6, 4.8, 1H), 9.36 (d,
J ¼ 1.6 Hz, 1H) and 9.71 (s, 1H). 13C NMR (DMSO-d6) ꢀ 21.51,
21.72, 31.91, 59.10, 93.79, 102.13, 110.62, 118.30, 119.84,
124.53, 131.46, 134.69, 135.38, 140.01, 149.05, 152.95, 157.37,
158.12, 163.21 and 163.49.
Flash column chromatography was carried out using ethyl
acetate–methanol–triethylamine (1:3:0.01, v/v/v) to give the title
compound. Yield: 20 mg (16%) and mp 105–106 ꢁC. IR (KBr)
3419, 2954, 2926, 1725, 1568, 1264, 813 and 756 cmꢃ1. 1H NMR
(CDCl3) ꢀ 1.54–1.61 (m, 2H), 1.98–2.01 (m, 2H), 2.33–2.35 (m,
9H), 2.49–2.57 (m, 1H), 2.97–3.03 (m, 2H), 4.68–4.72 (m, 2H),
5.95 (s, 1H), 6.23 (s, 1H), 6.75 (s, 1H), 7.15 (s, 1H), 7.17 (s, 1H),
7.34–7.43 (m, 1H), 8.28–8.32 (m, 1H), 8.65–8.68 (m, 1H), 9.21–
9.22 (m, 1H) and 15.57 (s, 1H).
1-(3-Hydroxy-5-methylphenyl)-2-(6-((2-morpholinoethyl)amino)-
2-(pyridin-3-yl)pyrimidin-4-yl) ethanone (5b)
General procedure for the synthesis of compounds 6a–f
Flash column chromatography was carried out using ethyl acetate-
methanol (10:1, v/v) to give the title compound. Yield: 22 mg
(17%) and mp 143–145 ꢁC IR (KBr) 3428, 2955, 2924, 1723,
Sodium borohydride was placed in round bottom flask, and the
flask was closed with a rubber septum and purged with nitrogen
gas for 10 min. Then 10 mL of ethanol was injected, and the
flask was once again bubbled with nitrogen and placed in an ice-
bath. Then the compound 4a–f dissolved in 10 mL ethanol was
injected. The reaction mixture was stirred at room temperature
under nitrogen for 12 h. The mixture was concentrated, and the
residue was partitioned between ethyl acetate and water. The
layers were separated, and the aqueous layer was extracted with
ethyl acetate. The combined organic layers were dried over
sodium sulfate and concentrated in vacuo. The residue was
purified by column chromatography using the proper mobile
phase.
1248, 1099, 874 and 728 cmꢃ1 1H NMR (DMSO-d6) ꢀ 2.33
.
(s, 3H), 2.50 (m, 4H), 2.65 (t, J ¼ 6.4, 2H), 3.61 (t, J ¼ 4.4, 4H),
3.75 (m, 2H), 6.61 (s, 1H), 6.80 (s, 1H), 7.14 (s, 1H), 7.22 (s, 1H),
7.48 (s, 1H), 7.56 (s, 1H), 7.65 (dd, J ¼ 4.8, 8.0 Hz, 1H), 8.52
(dt, J ¼ 2.0, 8.0 Hz, 1H), 8.79 (d, J ¼ 3.6 Hz, 1H), 9.36 (s, 1H) and
9.69 (s, 1H).
1-(3-Hydroxy-5-methylphenyl)-2-(6-((2-(piperidin-1-yl)ethyl)
amino)-2-(pyridin-3-yl)pyrimidin-4-yl)ethanone (5c)
Flash column chromatography was carried out using ethyl acetate-
methanol (4:1, v/v) to give the title compound. Yield: 34 mg
(26%) and mp 126–128 ꢁC. IR (KBr) 3424, 2955, 2924 1723,
.
1593, 1248, 1099, 875 and 728 cmꢃ1 1H NMR (DMSO-d6) ꢀ
3-((6-(2-Hydroxy-2-(3-methoxy-5-methylphenyl)ethyl)-2-(pyridin-
3-yl)pyrimidin-4-yl)amino)propan-1-ol (6a)
1.43–1.46 (m, 2H), 1.53–1.58 (m, 4H), 2.33 (s, 3H), 2.45–2.48
(m, 4H), 2.61 (t, J ¼ 6.4 Hz, 1H), 3.70–3.75 (m, 2H), 6.59 (s, 1H), Flash column chromatography was carried out using dichlor-
6.80 (s, 1H), 7.15 (s, 1H), 6.96 (s, 1H), 7.22 (s, 1H), 7.44 (s, 1H), omethane–methanol (10:1, v/v) to give the title compound. Yield:
7.53–7.54 (m, 1H), 7.63 (ddd, J ¼ 0.4, 4.4, 8.0 Hz, 1H), 8.52 23 mg (58%); sticky solid. IR (KBr) 3314, 2922, 2851, 1584,
1
(dt, J ¼ 1.6, 8.0 Hz, 1H), 8.79 (dd, J ¼ 1.6, 4.4 Hz, 1H), 9.35 1556, 1065, 846 and 704 cmꢃ1. H NMR (CD3OD) ꢀ 1.86–1.93
(d, J ¼ 1.6 Hz, 1H) and 9.56 (s, 1H). 13C NMR (DMSO-d6) ꢀ (m, 2H), 2.31 (s, 3H), 2.96–3.07 (m, 2H), 3.61 (t, 2H), 3.73
21.47, 24.47, 26.03, 54.16, 56.31, 93.78, 102.27, 110.76, 118.37, (t, 2H), 3.77 (s, 3H), 5.10 (dd, J ¼ 5.2, 8.4 Hz, 1H), 6.66 (s, 1H),
119.89, 124.41, 131.53, 134.82, 135.31, 139.95, 149.05, 152.88, 6.80 (s, 1H), 6.83 (s, 1H), 6.97 (s, 1H), 7.53–7.64 (m, 1H), 8.45
154.93, 157.44, 158.15, 163.58 and 163.65.
(d, J ¼ 8.0 Hz, 1H), 8.63 (dd, J ¼ 1.6, 4.8 Hz, 1H) and 9.16

DOI: 10.3109/14756366.2014.920838
Synthesis and biological evaluation of ROS1 kinase inhibitors
5
(d, J ¼ 1.6 Hz, 1H). 13C NMR (CD3OD) ꢀ 20.23, 32.09, 37.85, 105.95, 108.49, 113.22, 118.87, 123.86, 133.80, 135.07, 139.01,
54.18, 59.23, 72.79, 106.01, 108.23, 113.29, 118.74, 123.88, 145.63, 147.40, 149. 98, 159.79, 161.24, 161.61 and 169.43.
133.77, 135.13, 139.07, 145.53, 147.44, 150.01, 159.82, 162.67
and 169.59.
2-(6-(4-(Dimethylamino)piperidin-1-yl)-2-(pyridin-3-yl)pyrimi-
din-4-yl)-1-(3-methoxy-5-methylphenyl) ethanol (6f)
1-(3-Methoxy-5-methylphenyl)-2-(6-((2-morpholinoethyl)amino)-
2-(pyridin-3-yl)pyrimidin-4-yl)ethanol (6b)
Flash column chromatography was carried out using dichlor-
omethane–methanol (5:1, v/v) to give the title compound. Yield:
Flash column chromatography was carried out using ethyl 33 mg (44%) and mp 95–96 ꢁC. IR (KBr) 3341, 2925, 2853, 1583,
acetate–methanol (3:1, v/v) to give the title compound. Yield: 1552, 1055, 1026, 842 and 706 cmꢃ1. H NMR (CDCl3) ꢀ 1.51–
1
29 mg (58%); sticky solid. IR (KBr) 3331, 2927, 2855, 1583, 1.55 (m, 2H), 1.57–1.60 (m, 2H), 1.99–2.02 (m, 9H), 2.52–2.58
1556, 1067, 844 and 706 cmꢃ1. 1H NMR (CD3OD) ꢀ 2.31 (s, 3H), (m, 1H), 2.98–3.05 (m, 4H), 3.83 (s, 3H), 4.92 (m, 2H), 5.13 (dd,
2.58 (m, 4H), 2.65 (t, J ¼ 6.8 Hz, 2H), 2.96–3.08 (m, 2H), 3.65 (t, J ¼ 4.0, 8.4 Hz, 1H), 6.67 (s, 1H), 6.81 (s, 1H), 6.85 (s, 1H), 6.88
J ¼ 6.8 Hz, 2H), 3.74 (m, 4H), 3.77 (s, 3H), 5.13 (q, J ¼ 4.8, 1H), (s, 1H), 7.41 (dd, J ¼ 4.8, 8.0 Hz, 1H), 8.32 (dt, J ¼ 1.6, 8.0 Hz,
6.53 (s, 1H), 6.79 (s, 1H), 6.82 (s, 1H), 7.53 (dd, J ¼ 4.8, 7.6, 1H), 1H), 8.71 (d, J ¼ 4.0 Hz, 1H) and 9.23 (s, 1H). 13C NMR (CDCl3)
8.43 (d, J ¼ 8.0 Hz, 1H), 8.63 (dd, J ¼ 1.6, 4.8 Hz, 1H) and 9.21 ꢀ 21.59, 27.98, 28.02, 41.46, 43.41, 45.77, 55.22, 62.67, 72.63,
(d, J ¼ 1.6 Hz, 1H).13C NMR (CD3OD) ꢀ 20.27, 37.53, 53.43, 105.13, 108.26, 113.91, 118.96, 123.47, 133.21, 134.46, 139.49,
54.19, 57.45, 66.35, 72.77, 106.22, 108.31, 113.25, 118.74, 145.09, 148.59, 151.22, 159.79, 161.02, 162.62 and 169.51.
123.87, 133.70, 135.08, 139.07, 145.59, 147.44, 150.04, 159.82,
161.69, 162.44 and 169.65.
General procedure for the synthesis of compounds 7a–f
Synthesis of compounds 7a–f could be achieved through reaction
with compounds 5a–f by the same method for synthesis of
compounds 6a–f.
1-(3-Methoxy-5-methylphenyl)-2-(6-((2-(piperidin-1-yl)ethyl)
amino)-2-(pyridin-3-yl)pyrimidin-4-yl)ethanol (6c)
Flash column chromatography was carried out using dichlor-
omethane–methanol (10:1 to 4:1, v/v) to give the title compound. 3-(1-Hydroxy-2-(6-((3-hydroxypropyl)amino)-2-(pyridin-3-yl)
Yield: 16 mg (32%) and mp 123–125 ꢁC. IR (KBr) 3397, 2926, pyrimidin-4-yl)ethyl)-5-methylphenol (7a)
1
2854, 1583, 1556, 1064, 847 and 706 cmꢃ1. H NMR (CDCl3) ꢀ
Flash column chromatography was carried out using dichlor-
1.53 (m, 2H), 1.74 (m, 4H), 2.36 (s, 3H), 2.65 (m, 4H), 2.78
omethane–methanol (10:1, v/v) to give the title compound. Yield:
7 mg (35%) and mp 112–114 ꢁC. IR (KBr) 3323, 2970, 2929,
(t, J ¼ 5.6 Hz, 1H), 3.02–3.05 (m, 2H), 3.72 (m, 2H), 3.83 (s, 3H),
5.13 (dd, J ¼ 3.6, 8.4 Hz, 1H), 6.10 (s, 1H), 6.67 (s, 1H), 6.85
(s, 1H), 6.86 (s, 1H), 6.87 (s, 1H), 7.43 (ddd, J ¼ 0.8, 4.8, 8.0 Hz,
1H), 8.33 (dt, J ¼ 2.0, 8.0 Hz, 1H), 8.71 (dd, J ¼ 1.6, 4.8 Hz, 1H)
and 9.21 (s, 1H). 13C NMR (CDCl3) ꢀ 21.59, 23.83, 25.12, 37.91,
54.41, 55.23, 57.29, 72.63, 106.24, 108.30, 113.91, 118.97,
123.54, 133.03, 134.56, 139.52, 145.08, 148.49, 151.24, 159.79
and 161.74.
1
1585, 1558, 1048, 849 and 705 cmꢃ1. H NMR (CD3OD) ꢀ 1.90
(dd, J ¼ 6.4, 12.8 Hz, 2H), 2.27 (s, 3H), 2.96–3.08 (m, 2H), 3.61
(t, J ¼ 6.4, 2H), 3.72 (t, J ¼ 6.4, 2H), 5.05 (dd, J ¼ 4.8, 8.4 Hz,
1H), 6.55 (s, 1H), 6.69 (s, 1H), 6.72 (s, 1H), 6.98 (s, 1H), 7.56
(ddd, J ¼ 0.8, 4.8, 8.0 Hz, 1H), 8.46 (d, J ¼ 8.0 Hz, 1H), 8.64 (dd,
J ¼ 1.6, 4.8 Hz, 1H) and 9.18 (d, J ¼ 1.6 Hz, 1H). 13C NMR
(CD3OD) ꢀ 20.12, 32.08, 37.83, 59.22, 72.80, 106.01, 109.68,
114.58, 117.69, 123.88, 133.82, 135.16, 139.01, 145.49, 147.45,
149.99, 157.04, 162.68 and 169.63.
2-(6-((2-(Diethylamino)ethyl)amino)-2-(pyridin-3-yl)pyrimidin-4-
yl)-1-(3-methoxy-5-methylphenyl) ethanol (6d)
Flash column chromatography was carried out using dichlor-
omethane–methanol (6:1, v/v) to give the title compound. Yield:
12 mg (40%); sticky solid. IR (KBr) 3302, 2964, 2926, 1583,
1556, 846 and 705 cmꢃ1. 1H NMR (CD3OD) ꢀ 1.15 (t, J ¼ 7.2 Hz,
6H), 2.31 (s, 3H), 2.75 (q, J ¼ 7.2 Hz, 4H), 2.83 (t, J ¼ 7.2 Hz,
2H), 2.97–3.10 (m, 1H), 3.65 (t, J ¼ 7.2, 2H), 3.76 (s, 3H), 5.11
(dd, J ¼ 5.2, 8.4 Hz, 1H), 6.66 (s, 1H), 6.79 (s, 1H), 6.82 (s, 1H),
7.01 (s, 1H), 7.56 (ddd, J ¼ 0.8, 5.2, 8.4 Hz, 1H), 8.46
(d, J ¼ 8.0 Hz, 1H), 8.64 (dd, J ¼ 1.6, 4.8 Hz, 1H) and 9.19
(s, 1H). 13C NMR (CD3OD) ꢀ 10.06, 20.23, 38.04, 51.31, 54.17,
72.81, 106.35, 108.27, 113.25, 118.72, 123.87, 133.76, 135.12,
139.07, 145.57, 147.45, 150.04, 159.83, 161.87, 162.54 and
169.68.
3-(1-Hydroxy-2-(6-((2-morpholinoethyl)amino)-2-(pyridin-3-
yl)pyrimidin-4-yl)ethyl)-5-methylphenol (7b)
Flash column chromatography was carried out using dichlor-
omethane-methanol (6:1, v/ꢁv) to give the title compound. Yield:
10 mg (50%) and mp 68–69 C. IR (KBr) 3323, 2919, 2848, 1583,
1557, 1068, 844 and 704 cmꢃ1. 1H NMR (CD3OD) ꢀ 2.27 (s, 3H),
2.64 (m, 4H), 2.72 (t, J ¼ 6.8 Hz, 2H), 2.97–3.09 (m, 2H), 3.69 (t,
J ¼ 6.4 Hz, 2H), 3.76 (m, 4H), 5.08 (dd, J ¼ 4.8, 8.4 Hz, 1H), 6.54
(s, 1H), 6.67 (s, 1H), 6.71 (s, 1H), 7.01 (s, 1H), 7.57 (ddd, J ¼ 0.8,
4.8, 8.0 Hz, 1H), 8.47 (d, J ¼ 8.0 Hz, 1H), 8.64 (dd, J ¼ 1.6,
4.8 Hz, 1H) and 9.19 (d, J ¼ 1.6 Hz, 1H).
3-(1-Hydroxy-2-(6-((2-(piperidin-1-yl)ethyl)amino)-2-(pyridin-3-
2-(6-(4-Ethylpiperazin-1-yl)-2-(pyridin-3-yl)pyrimidin-4-yl)-1-(3- yl)pyrimidin-4-yl)ethyl)-5-methylphenol (7c)
methoxy-5-methylphenyl)ethanol (6e)
Flash column chromatography was carried out using dichlor-
Flash column chromatography was carried out using dichlor- omethane-methanol (6:1, vꢁ/v) to give the title compound. Yield:
omethane-methanol (20:1, v/v) to give the title compound. Yield: 8 mg (53%) and mp 72–73 C. IR (KBr) 3265, 2930, 2848, 1581,
52 mg (52%); sticky solid. IR (KBr) 3223, 2929, 2852, 1582, 1557, 847, 805 and 705 cmꢃ1. 1H NMR (CD3OD) ꢀ 1.54–1.56 (m,
1
1065, 846 and 705 cmꢃ1. H NMR (CDCl3) ꢀ 1.17 (t, J ¼ 7.2 Hz, 2H), 1.64–1.1.71 (m, 4H), 2.27 (s, 3H), 2.64 (m, 4H), 2.71
3H), 2.30 (s, 3H), 2.50 (q, J ¼ 7.2, 2H), 2.56 (t, J ¼ 5.2 Hz, 4H), (t, J ¼ 6.8 Hz, 2H), 2.96–3.09 (m, 2H), 3.69 (t, J ¼ 6.8 Hz, 2H),
2.99–3.11 (m, 2H), 3.76 (s, 3H), 3.94 (m, 4H), 5.13 (dd, J ¼ 5.2, 5.04–5.07 (dd, J ¼ 5.2, 2.8, 1H), 6.54 (s, 1H), 6.67 (s, 1H), 6.72 (s,
8.4 Hz, 1H), 6.64 (s, 1H), 6.78 (s, 1H), 6.81 (s, 1H), 6.98 (s, 1H), 1H), 7.00 (s, 1H), 7.56 (ddd, J ¼ 0.8, 4.8, 8.0 Hz, 1H), 8.47 (d,
7.51 (ddd, J ¼ 0.8, 4.8, 8.0 Hz, 1H), 8.42 (dt, J ¼ 1.6, 8.0 Hz, 1H), J ¼ 8.0 Hz, 1H), 8.64 (dd, J ¼ 1.2, 4.8 Hz, 1H) and 9.19 (s, 1H).
8.6 (dd, J ¼ 1.6, 4.8 Hz, 1H) and 9.15 (d, J ¼ 1.6 Hz, 1H). 13C 13C NMR (CD3OD) ꢀ 20.13, 23.59, 24.97, 37.60, 54.23, 57.17,
NMR (CDCl3) ꢀ 10.41, 20.30, 43.10, 52.04, 52.26, 54.20, 72.75, 72.71, 106.29, 109.72, 114.59, 115.36, 117.68, 121.39, 123.88,

6
A. Z. Abdelazem & S. H. Lee
J Enzyme Inhib Med Chem, Early Online: 1–9
133.78, 135.13, 138.99, 145.51, 147.45, 150.01, 157.05, 161.60, to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve
162.46 and 169.71.
fits were obtained using Prism (GraphPad Software). Compounds
were tested against ROS1 kinase in a 10-dose IC50 mode with
threefold serial dilutions starting at 20 mM, using staurosporine as
a positive control.
3-(2-(6-((2-(Diethylamino)ethyl)amino)-2-(pyridin-3-yl)pyrimi-
din-4-yl)-1-hydroxyethyl)-5-methylphenol (7d)
The combined organic layers were dried over sodium sulfate and
concentrated in vacuo to give 20 mg (80%) of the target product;
mp 62–63 ꢁC. IR (KBr) 3272, 2925, 2853, 1584, 1557, 1066, 845
Docking studies
Docking studies were performed using Molecular Operating
Environment (MOE version 2008.10; Chemical Computing
Group Canada). The program operated under ‘‘Windows XP’’
operating system installed on an Intel Pentium IV PC with a
2.8 MHz processor and 512 RAM. Compounds to be studied were
built using the builder interface of the MOE program and
subjected to energy minimization tool using the included MOPAC
7.0. The produced model was subjected to Systematic
Conformational Search where all items were set as default with
RMS gradient of 0.01 kcal/mol and RMS distance of 0.1 A. The
X-ray crystallographic structure of ROS1 kinase enzyme co-
crystallized with crizotinib (PDB code 3zbf) was obtained from
the Protein Data Bank17. The enzyme was prepared for docking
studies where: (i) the ligand molecule with any existing solvent
molecules were removed from the enzyme active site; (ii)
hydrogen atoms were added to the structure with their standard
geometry; and (iii) MOE Alpha Site Finder was used for the
active sites search in the enzyme structure and dummy atoms
were created from the obtained alpha spheres. The obtained
ligand–enzyme complex model was then used in calculating the
energy parameters using MMFF94x force field energy calculation
and predicting the ligand–enzyme interactions at the active site.
1
and 705 cmꢃ1. HNMR (CD3OD) ꢀ 1.16 (t, J ¼ 7.2 Hz, 6H), 2.27
(s, 3H), 2.77 (q, 4H), 2.86 (t, J ¼ 6.8 Hz, 3H), 2.95–3.09 (m, 1H),
3.66 (t, J ¼ 7.2, 3H), 5.06 (dd, J ¼ 4.8, 3.2 Hz, 1H), 5.52 (s, 1H),
6.55 (s, 1H), 6.68 (s, 1H), 6.871 (s, 1H), 7.01 (s,1H), 7.55 (dd,
J ¼ 5.2, 8.0 Hz, 1H), 8.46 (d, J ¼ 8.0 Hz, 1H), 8.64 (dd, J ¼ 1.6,
5.2 Hz, 1H) and 9.19 (s, 1H). 13C NMR (CD3OD) ꢀ 9.96, 20.14,
29.30, 37.97, 51.30, 72.82, 106.39, 109.70, 114.60, 117.67,
123.87, 133.76, 135.13, 139.00, 145.54, 147.45, 150.02, 157.07,
161.79, 162.53 and 169.73.
3-(2-(6-(4-Ethylpiperazin-1-yl)-2-(pyridin-3-yl)pyrimidin-4-yl)-1-
hydroxyethyl)-5-methylphenol (7e)
Flash column chromatography was carried out using
dichloromethane–methanol (5:1, v/v). Yield: 50 mg (83%) and
mp 92–93 ꢁC. IR (KBr) 3349, 2920, 2852, 1583, 1554, 1026, 842
and 702 cmꢃ1. 1H NMR (CD3OD) ꢀ 1.18 (t, J ¼ 7, 6 Hz, 3H), 2.26
(s, 3H), 2.48–2.64 (m, 6H), 2.99–3.09 (m, 2H), 3.95 (t, J ¼ 5.2 Hz,
4H), 5.08 (dd, J ¼ 5.2, 8 Hz, 1H), 6.54 (s, 1H), 6.78 (s, 1H), 6.68
(s, 1H), 6.71 (s, 1H), 6.98 (s, 1H), 7.52 (ddd, J ¼ 0.8, 4.8, 8.0 Hz,
1H), 8.42 (dt, J ¼ 1.6, 8.0 Hz,1H), 8.6 (dd, J ¼ 1.6, 4.8 Hz, 1H)
and 9.15 (d, J ¼ 1.6 Hz, 1H). 13C NMR (CD3OD) ꢀ 10.40, 20.18,
43.09, 52.03, 52.26, 72.80, 105.94, 109.83, 114.58, 117.82,
123.87, 133.85, 135.10, 138.95, 145.57, 147.39, 149.94, 157.03,
161.23, 161.62 and 169.45.
Results and discussion
Chemistry
3-(2-(6-(4-(Dimethylamino)piperidin-1-yl)-2-(pyridin-3-yl)
pyrimidin-4-yl)-1-hydroxyethyl)-5-methyl phenol (7f)
The target compounds were readily prepared according to
Scheme 1. Briefly, the benzoate ester 1 underwent a nucleophilic
attack at its carboxylic carbon by the activated methylene group of
2,4-dichloro-6-methylpyrimidine. The activation of this methyl
group into an active methylene was achieved by dropwise addition
of LHMDS in dry THF at room temperature. The resulted
tautomeric a,b-unsaturated ketone 2 was then subjected to a
nucleophilic substitution reaction with the amino group of the
appropriate amine by heating at 80 ꢁC in THF for 3 h to give
compounds 3a–3f. The intermediates 3a–f was reacted with 3-
pyridineboronic acid under palladium catalysis to afford the
Suzuki coupling products 4a–f. Compounds 5a–f was obtained by
demethylation of the methoxy group of the corresponding
compounds 4a–f using 10 equivalent of boron trifluoride–
dimethylsulfide complex in dichloromethane. The reduction of
4a–f and 5a–f with sodium borohydride in ethanol, afforded the
alcohol derivatives 6a–f and 7a–f, respectively (Scheme 1).
Flash column chromatography was carried out using
dichloromethane–methanol (5:1, v/v). Yield: 47 mg (94%) and
mp 153–154 ꢁC. IR (KBr) 3208, 2924, 2857, 1583, 1553, 1051,
844 and 705 cmꢃ1. 1H NMR (CDCl3) ꢀ 1.39–1.48 (m, 2H), 1.96–
1.99 (m, 2H), 2.26 (s, 3H), 2.33 (s, 6H), 2.52–2.58 (m, 1H), 2.87–
3.10 (m, 4H), 5.00 (m, 2H), 5.07 (dd, J ¼ 5.6, 8.0 Hz, 1H), 6.54 (s,
1H), 6.68 (s, 1H), 6.72 (s, 1H), 6.93 (s, 1H), 7.52 (ddd, J ¼ 0.8,
4.8, 8.0 Hz, 1H), 8.42 (dt, J ¼ 1.6, 8.0 Hz, 1H), 8.60 (dd, J ¼ 1.6,
4.8 Hz, 1H) and 9.23 (d, J ¼ 1.6 Hz, 1H). 13C NMR (CDCl3) ꢀ
20.20, 27.67, 40.30, 42.95, 48.26, 62.66, 72.81, 105.42, 109.88,
114.59, 117.82, 123.86, 133.95, 135.08, 138.95, 145.55, 147.37,
149.90, 157.03, 161.26, 161.44 and 169.43.
Enzyme inhibition assay
Kinase assays was performed at Reaction Biology Corporation
using the ‘‘HotSpot’’ assay platform16. Briefly, specific kinase/
substrate pairs along with required cofactors were prepared in
Evaluation of biological activity
reaction buffer; 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM In order to study the effect of structural modifications in the new
EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM group of compounds, all synthesized compounds were in vitro
DTT and 1% DMSO. Compounds were delivered into the screened against ROS1 kinase enzyme. By referring to the IC50’s
reaction, followed ꢀ20 min later by addition of a mixture of of the tested compounds as in Table 1, we can observe micromolar
ATP (Sigma) and 33P ATP (Perkin Elmer) to a final concentration activity of most of them. But, unfortunately, it is obvious that the
of 10 mM. Reactions were carried out at 25 ꢁC for 120 min, new compounds are much less active than the lead compound
followed by spotting of the reactions onto P81 ion exchange filter KIST301072. We think this is because of changing the central
paper (Whatman # 3698–915). Unbound phosphate was removed pyrazole ring into an open form. Keeping the rigid pyrazole ring
by extensive washing of filters in 0.1% phosphoric acid.
linking the two aromatic moieties together seems essential for
After subtraction of background derived from control reactions good ROS1 inhibitory activity. Changing the central pyrazole ring
containing inactive enzyme, kinase activity data were expressed into an open form might lead to loss of proper orientation of the
as the percent remaining kinase activity in test samples compared different groups in the active site of ROS1.

DOI: 10.3109/14756366.2014.920838
Synthesis and biological evaluation of ROS1 kinase inhibitors
7
OCH3
OCH3
OCH3
OCH3
OCH3
H3C
R
H3C
R
i
ii
OH
O
OH
H3C
Cl
O
H3C
Cl
OCH3
H3C
O
N
N
N
N
N
N
N
N
Cl
Cl
Cl
1
Cl
2
3a-f
OH
OH
OCH3
OCH3
H3C
R
H3C
R
O
H3C
OH
H3C
R
O
OH
v
iii
iv
R
N
N
N
N
N
N
N
N
N
N
N
N
7a-f
5a-f
4a-f
v
OCH3
N
O
HN
OH
HN
b:R=
a:R=
H3C
R
OH
N
N
d:R=
f:R=
c:R=
e:R=
HN
N
N
H
N
N
N
N
N
N
6a-f
Scheme 1. Synthesis of target compounds: (i) LHMDS, 2,4-dichloro-6-methylpyrimidine, THF, N2, rt, 24 h, 81%; (ii) THF, various amine, 80 ꢁC, 3 h;
(iii) 3-pyridineboronic acid, Pd(PPh3)2Cl2, K2CO3, N2, THF/CH3CN/H2O (2:1:1), 90 ꢁC, 3 h; (iv) BF3 ꢄ S(CH3)2, dichloromethane, N2, rt, 12 h; and (v)
NaBH4, ethanol, N2, 0 ! rt, 12 h.
Table 1. The IC50 values of the target compounds against ROS1 kinase.
this set of compounds. Other changes just have minor effect
on activity.
Compound no. ROS1 (IC50, mM) Compound no. ROS1 (IC50, mM)
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
420
4.6
420
420
420
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
13.7
12.6
420
420
13.5
8.1
Docking studies
˚
The 2.2 A co-crystal structure of crizotinib with ROS1-kinase
domain discloses how the inhibitor binds in the ATP-binding site.
ROS1 binds crizotinib at the ATP-binding site in the cleft between
the N-terminal and C-terminal domains of the kinase (Figure 2).
The G2032 residue sits at the solvent front in the distal end of the
kinase hinge and creates a turn, putting the G2032 a-carbon in
position to engage in a Van der Waals interaction with the
pyrazole ring of crizotinib17. The hinge interactions include a
hydrogen bond between the pyridine of crizotinib and the
backbone nitrogen of Met2029. Remarkably, this is the only
direct polar interaction observed between ROS1 kinase domain
420
No inhibition
No inhibition
15.6
9.1
420
420
420
5.8
420
17.7
420
4.9
In addition, from all tested compounds, the compounds (4d, and crizotinib.
5d, 6d and 7d) having N,N-diethyl amino ethylamine group as the In order to find an explanation for the potency of the lead
polar side chain were the least active. This group seems to be not molecules and loss of potency of the designed analogues against
good water-solubilizing group in this compounds’ set. ROS1 kinase, we applied docking simulation in the active site of
Finally, reduction of ketone group into alcohol or demethyla- ROS1 kinase using MOE.2008.10 software. Using the crystal
tion of methoxy group did not have significant effect in ROS1 structure ROS1 kinase enzyme co-crystallized with crizotinib
inhibitory activity.
(PDB code 3zbf), we found that KIST301072 exhibits similar
Therefore, we think changing the central pyrazole ring into binding behavior to that of crizotinib. The most stable docking
an open form is the major cause of decreasing ROS1 activity in pose of KIST301072 aligned with crizotinib in the binding pocket

8
A. Z. Abdelazem & S. H. Lee
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 2. Binding mode of crizotinib in ROS1 active pocket in 2D and 3D, respectively.
Figure 3. Overlay of crizotinib (green stick) and KIST301072 (element Figure 4. Overlay of KIST301072 (yellow stick) and compound 7a
colored stick) bound to ROS1 active site. (orange stick) bound to ROS1 active site.
is shown in Figure 3. KIST301072 occupies a comparable space enzyme as it was supported by docking studies. Therefore, in
in the binding pocket and overlays the bioactive conformation future studies, we plan to preserve the rigid scaffold through
of crizotinib.
replacing central pyrazole moiety with suitable isosteres.
Then, compound 7a was selected as a representative from the
synthesized analogues due to its similarity to KIST301072 except
the absence of central pyrazole ring. Aligning the most stable
docking pose of both compounds 7a and KIST301072 showed a
similar orientation of both pyridine rings and solvent exposure
moieties, but the lead compound occupied a different and more
expanded space in ROS1 kinase active site (Figure 4). Therefore,
we think that the loss of potency in the designed analogues is
due to their failure to occupy the same space and to have the same
orientation as the lead compounds.
Declaration of interest
The authors declare no conflict of interest.
The research was supported by Korea Institute of Science and
Technology.
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In conclusion, through synthesis and screening of this new set of
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