Optimization of bipyridinyl pyrazole scaffolds via design, synthesis and screening of a new series of ROS1 kinase-modulating compounds

Article abstract of DOI:10.1002/bkcs.10077

A series of rationally designed ROS1 tyrosine kinase inhibitors 6a-9b with bipyridinyl pyrazole scaffold was synthesized and screened. The scaffold itself has showed an exclusive selectivity profile over ROS1 closely related kinases,ALKand c-Met. The aim of this study was to further explore the structure-activity relationships (SAR) of the bipyridinyl pyrazole core structure, and to improve its ROS1 inhibitory potency. The rational of this study was to explore the nature of the proposed binding site for the pyrazoleNHsubstituents. Careful selections of pyrazoleNHsubstituent groups along with their regioisomers were considered. The compounds exhibited high degree of potency, IC₅₀ values of 21-159 nM. A detailed SAR of bipyridinyl pyrazole scaffold has been finally well established and the virtual screening strategy, through molecular docking, has been performed for this type of ROS1 kinase inhibitors and the docked poses along with the activity data have gone in consistent with SAR specifications.

Full text of DOI:10.1002/bkcs.10077

Article
DOI: 10.1002/bkcs.10077
BULLETIN OF THE
M. M. Al-Sanea et al.
KOREAN CHEMICAL SOCIETY
Optimization of Bipyridinyl Pyrazole Scaffolds via Design, Synthesis and
Screening of a New Series of ROS1 Kinase-modulating Compounds
†
,‡,k
§,k
†,‡
¶
Mohammad M. Al-Sanea,
Byung Sun Park, Ahmed Z. Abdelazem, Khalid B. Selim,
‡ ‡
§
‡,
*
Kyung Ho Yoo, Taebo Sim, Jin Sung Tae, and So Ha Lee
†
Department of Biological Chemistry, Korea University of Science and Technology, Daejeon 305-333,
Republic of Korea
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul 136-791,
‡
Republic of Korea. *E-mail: LSH6211@kist.re.kr
Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea
§
¶
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt
Received September 5, 2014, Accepted October 21, 2014, Published online January 5, 2015
A series of rationally designed ROS1 tyrosine kinase inhibitors 6a–9b with bipyridinyl pyrazole scaffold was
synthesized and screened. The scaffold itself has showed an exclusive selectivity profile over ROS1 closely
related kinases, ALKandc-Met. Theaimofthisstudywastofurther explorethestructure–activityrelationships
(SAR) of the bipyridinyl pyrazole core structure, and to improve its ROS1 inhibitory potency. The rational of
this study was to explore the nature of the proposed binding site for the pyrazole NH substituents. Careful selec-
tions of pyrazole NH substituent groups along with their regioisomers were considered. The compounds exhib-
ited high degree of potency, IC50 values of 21–159 nM. A detailed SAR of bipyridinyl pyrazole scaffold has
been finallywell established and thevirtualscreening strategy, throughmolecular docking, hasbeenperformed
for this type of ROS1 kinase inhibitorsand the docked poses along with the activity data havegone inconsistent
with SAR specifications.
Keywords: ROS1 kinase inhibitor, Bipyridinyl pyrazole, Cancer, Chromosomal alteration
Introduction
showed in-vitroactivity against HCC78 celllines and inhibition
of signaling downstream of ROS1 inducing apoptosis in BaF3/
1
4
Proto-oncogene tyrosine-protein kinase ROS (ROS1) is a
receptor tyrosine kinase (RTK). RTKs are transmembrane
glycoproteins that mediate signal transduction and cell-to-cell
communication. They act as cell surface receptors for a num-
FIG-ROS positive cells (IC = 10 nM). Crizotinib, first ALK
50
inhibitor approved by FDA, is also able to inhibit ROS1 with an
IC50 value of 1.7 nM in an in-vitro assay. The 2% cases
appointedbyNSCLCharborROS1rearrangementsweretotally
1–4
18
ber of important growth factors and hormones. However,
protein kinase activation by somatic mutationorchromosomal
alteration is a key role in the initiation and progression of a
recovered in 12 weeks after crizotinib treatment.
The phenyl bipyridinyl scaffold, reported by our group,
has unleashed several ROS1 kinase inhibitors with IC50
range of 13.6–209 nM in enzyme-based screening. Com-
pound KIST301072 showed high degree of selectivity among
5–8
number of cancers.
ROS1 has become one of the most extensively studied
to date, and the ectopic expression as well as the production
of variable mutant forms of ROS1 kinase have been reported
in a number of cancers, such as glioblastoma multiforme,
cholangiocarcinoma, and nonsmall cell lung cancer
19,20
45 kinases.
Herein, the aim of the study is to further explore the struc-
ture–activity relationships (SAR) of the bipyridinyl pyrazole
core structure, and to improve its ROS1 inhibitory potency.
Therefore, a series of rationally selected derivatives of bipyr-
idinyl pyrazole scaffold were designed by carefully selecting
different substituents on pyrazole NH in order to explore their
binding role and hence discover the nature of their comple-
mentary region of interaction.
9–14
(NSCLC).
ROS1 forms fusions and defines another clin-
ically actionable oncogenic driver mutation in NSCLC.
Recently, it has been reported that around 1.4% of NSCLCs
1
5,16
harbor ROS1 rearrangements.
Becauseofthehighhomologybetweenthekinasedomainsof
ROS1 and ALK, several ALK inhibitors were assayed against
ROS1-driven cells and tumors, and they have demonstrated in-
14,15,17
Materials and Methods
vitro inhibitory activity against ROS1.
NVP-TAE684
General Remarks. Commercially available reagents and
k These authors contributed equally to this work.
anhydrous solvents were used without further purification
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Optimization of Bipyridinyl Pyrazole Scaffolds
KOREAN CHEMICAL SOCIETY
unless otherwisespecified. Thin-layer chromatography(TLC)
analyses were performed with precoated (silica gel 60 F254
prepared by E. Merck, Darmstadt, Germany). NMR spectra
were recorded with a Bruker 300 MHz spectrometeratambient
temperature with the residual solvent peaks as internal stan-
dards. The line positions of multiplets were given in ppm (δ)
and the coupling constants (J) were given in hertz. Flash chro-
matography was performed on columns of Merck silica gel
2-(4-(6-Chloropyridin-2-yl)-3-(3-methoxy-5-methyl
phenyl)-1H-pyrazol-1-yl)acetamide (4d): To a mixture of 3
(1 g, 3.34 mmol) and K CO (0.92 g, 6.67 mmol) in DMF
solution (10 mL) was added 2-iodoacetamide (1.24 g, 6.67
mmol). The reaction mixture was stirred at room temperature
for 3 h and the solvent was evaporated under vacuum. The res-
idue was partitioned between brine and ethyl acetate. The
combined organic layers were dried over anhydrous MgSO4
2
3
(
230–400 mesh, 40–63 μm) with ethylacetate/hexane or meth-
anol/dichloromethane as eluents.
-Chloro-6-(3-(3-methoxy-5-methylphenyl)-1-methyl-
H-pyrazol-4-yl)pyridine (4a) and 2-chloro-6-(5-(3-meth-
oxy-5-methylphenyl)-1-methyl-1H-pyrazol-4-yl)pyridine
and evaporated under vacuum to yield the pure product 4d
1
(1.1 g, 92%); H NMR (300 MHz, CD OD) δ 3.08 (s, 3H),
3
2
4.50 (s, 3H), 5.66 (s, 2H), 7.70–7.48 (m, 3H), 8.03 (d, J =
5.1 Hz, 1H), 8.14 (d, J = 13.2 Hz, 1H), 9.10–8.96 (m, 2H).
4-(3-(3-Methoxy-5-methylphenyl)-1-methyl-1H-pyra-
1
0
(
5a): To a mixture of 3 (1 g, 3.34 mmol) and K CO (0.92 g,
zol-4-yl)-2,3 -bipyridine (6a):Amixture of4a(172 mg, 0.55
2
3
6
.67 mmol) in dimethylformamide (DMF; 10 mL) was added
mmol), 3-pyridineboronic acid (81 mg, 0.66 mmol),
dichlorobis(triphenylphosphine)Pd(II) (12 mg, 0.017 mmol)
and K CO (76 mg, 0.55 mmol) was placed in a mixed solvent
dropwise iodomethane (0.42 mL, 6.67 mmol). The reaction
mixture was stirred at room temperature for 3 h and the solvent
was evaporated under vacuum. The residue was partitioned
between brine and ethyl acetate. The combined organic layers
2
3
of acetonitrile and water (4:1, 5 mL). N gas was bubbled into
2
this mixture for 15 min, and then the mixture was refluxed
were dried over anhydrous MgSO and evaporated under vac-
while stirring under N atmosphere for 1 h. The reaction mix-
4
2
uum. The crude product was purified by column chromatog-
raphy (silica gel, ethyl acetate–hexane) to yield 4a (0.33 g,
ture was cooled to room temperature, and then poured into ice
water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over anhydrous MgSO4,
andevaporated undervacuum. The crude product waspurified
by column chromatography (silica gel, ethyl acetate) to yield
3
2%, lower spot in TLC) and 5a (0.22 g, 21%, upper spot in
1
TLC). 4a: H NMR (300 MHz, CDCl ) δ 2.22 (s, 3H), 3.63
3
(s, 3H), 3.87 (s, 3H), 6.66 (d, J = 5.4 Hz, 2H), 6.80 (s, 1H),
1
6
.99 (d, J = 4.2 Hz, 1H), 7.17 (s, 1H), 7.53 (s, 1H), 8.12 (d,
the pure product 6a (133 mg, 68%); H NMR (300 MHz,
1
J = 4.8 Hz, 1H); 5a: H NMR (300 MHz, CDCl ) δ 2.29 (s,
CDCl ) δ 2.24 (s, 3H), 3.64 (s, 3H), 3.93 (s, 3H), 6.68 (s,
3
3
3
6
1
H), 3.68 (d, J = 10. 8 Hz, 6H), 6.54 (s, 1H), 6.61 (s, 1H),
.77 (s, 1H), 6.84 (d, J = 4.5 Hz, 1H), 7.08 (s, 1H), 7.73 (s,
H), 8.03 (d, J = 5.1 Hz, 1H).
1H), 6.75 (s, 1H), 6.88 (s, 1H), 7.13 (d, J = 4.2 Hz, 1H), 7.3
(d, J = 4.1 Hz, 1H), 7.6 (d, J = 9.6 Hz, 2H), 8.14 (d, J = 7.8
Hz, 1H), 8.51–8.56 (m, 2H), 8.94 (s, 1H); positive ion ESI-
+
2
-Chloro-6-(1-ethyl-3-(3-methoxy-5-methylphenyl)-
MS m/z 379.1632 (M + Na) .
1
H-pyrazol-4-yl)pyridine (4b) and 2-chloro-6-(1-ethyl-5-
4-(5-(3-Methoxy-5-methylphenyl)-1-methyl-1H-pyra-
zol-4-yl)-2,3 -bipyridine (7a): A mixture of 5a (88 mg, 0.37
0
(3-methoxy-5-methylphenyl)-1H-pyrazol-4-yl)pyridine (5b):
To a mixture of 3 (1 g, 3.34 mmol) and K CO (0.92 g,
mmol), 3-pyridineboronic acid (55 mg, 0.44 mmol),
dichlorobis(triphenylphosphine)Pd(II) (8 mg, 0.011 mmol)
and K CO (51 mg, 0.37 mmol) was placed in a mixed solvent
2
3
6.67 mmol) in a DMF solution (10 mL) was added iodoethane
(1.07 mL, 6.67 mmol) dropwise. The reaction mixture
2
3
was stirred at room temperature for 3 h and the solvent was
evaporated under vacuum. The residue was partitioned
between brine and ethyl acetate. The combined organic layers
were dried over MgSO4 and evaporated under vacuum.
The mixture of 4b and 5b (0.62 g, 57%) was used in the next
step without further purification because of the difficulty
of separation.
of acetonitrile and water (4:1, 5 mL). N gas was bubbled into
this mixture for 15 min, and then the mixture was refluxed
2
while stirring under N atmosphere for 1 h. The reaction mix-
2
ture was cooled to room temperature, and then poured into ice
water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over anhydrous MgSO4,
andevaporated undervacuum. The crude product waspurified
by column chromatography (silica gel, ethyl acetate) to yield
3
-(4-(6-Chloropyridin-2-yl)-3-(3-methoxy-5-methyl-
1
phenyl)-1H-pyrazol-1-yl)propanenitrile (4c) and 3-(4-(6-
chloropyridin-2-yl)-5-(3-methoxy-5-methyl phenyl)-1H-
pyrazol-1-yl)propanenitrile (5c): To a mixture of 3 (1 g,
the pure product 7a (88 mg, 66%); H NMR (300 MHz,
CDCl ) δ 2.33 (s, 3H), 3.73 (s, 6H), 6.62 (s, 1H), 6.70 (s,
3
1H), 6.84 (s, 1H), 7.06 (d, J = 5.1 Hz, 1H), 7.3 (t, J = 6.0
Hz, 1H), 7.45 (s, 1H), 7.83 (s, 1H), 8.12 (d, J = 7.5 Hz, 1H),
8.45 (d, J = 5.1 Hz, 1H), 8.54 (d, J = 3.9 Hz, 1H), 8.80 (s,
3
.34 mmol) and K CO (0.92 g, 6.67 mmol) in DMF solution
2 3
(10 mL) was added 3-iodopropionitrile (0.6 mL, 6.67 mmol)
+
dropwise. The reaction mixture was stirred at room tempera-
ture for 3 h and the solvent was evaporated under vacuum. The
residue was partitioned between brine and ethyl acetate. The
combined organic layers were dried over anhydrous MgSO4
and evaporated under vacuum. The mixture of 4c and 5c
1H); positive ion ESI-MS m/z 379.1542 (M + Na) .
4-(1-Ethyl-3-(3-methoxy-5-methylphenyl)-1H-pyra-
0
zol-4-yl)-2,3 -bipyridine (6b) and 4-(1-ethyl-5-(3-meth-
0
oxy-5-methylphenyl)-1H-pyrazol-4-yl)-2,3 -bipyridine
(7b): A mixture of 4b and 5b (0.62 g, 1.89 mmol), 3-
pyridineboronic acid (0.28 g, 2.27 mmol), dichlorobis (triphe-
nylphosphine)Pd(II) (40 mg, 0.057 mmol) and K CO (0.39
(0.94 g, 80%) was used in the next step without further puri-
fication because of the difficulty of separation.
2
3
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Optimization of Bipyridinyl Pyrazole Scaffolds
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g, 2.84 mmol) was placed in a mixed solvent of acetonitrile
water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over anhydrous MgSO4,
andevaporated undervacuum. The crude product waspurified
by column chromatography (silica gel, ethyl acetate) to yield
and water (4:1, 5 mL). N gas was bubbled into this mixture
2
for 15 min, and then the mixture was refluxed while stirring
under N atmosphere for 1 h. The reaction mixture was cooled
2
ꢀ
1
to room temperature, and then poured into ice water and
extracted with ethyl acetate. The combined organic layers
the pure product 6d (50 mg, 45%); mp 206–208 C; H NMR
(300 MHz, DMSO-d ) δ 2.52 (s, 3H), 3.69 (s, 3H), 4.88 (s,
6
were washed with brine, dried over anhydrous MgSO , and
2H), 6.78 (d, J = 0.9 Hz, 1H), 6.82 (d, J = 0.6 Hz, 1H), 6.91
(d, J = 0.6 Hz, 1H), 7.24 (dd, J = 1.2, 3.9 Hz, 1H), 7.39 (s,
1H), 7.54–7.50 (m, 1H), 7.66 (s, 1H), 7.94 (d, J = 0.6 Hz,
1H), 8.36–8.33 (m, 1H), 8.37 (s, 1H), 8.59 (dd, J = 0.3, 3.9
Hz, 1H), 8.64 (dd, J = 1.2, 3.6 Hz, 1H), 9.13 (q, J = 0.7 Hz,
4
evaporated under vacuum. The mixture of 6b and 7b (0.45
g, 65%) was used for the next step without further purification
because of the difficulty of separation.
0
3
-(4-([2,3 -Bipyridin]-4-yl)-3-(3-methoxy-5-methyl
13
phenyl)-1H-pyrazol-1-yl)propanenitrile (6c) and 3-(4-
1H); C NMR (75 MHz, DMSO) δ 21.56, 54.56, 55.45,
111.42, 114.87, 117.41, 119.38, 121.75, 121.90, 124.23,
133.69, 134.34, 134.67, 139.69, 142.38, 148.14, 148.90,
150.37, 150.43, 154.51, 159.68, 168.70.
0
(
[2,3 -bipyridin]-4-yl)-5-(3-methoxy-5-methylphenyl)-1H-
pyrazol-1-yl)propanenitrile (7c): A mixture of 4c and 5c
0.9 g, 2.55 mmol), 3-pyridineboronic acid (0.38 g, 3.06
mmol), dichlorobis(triphenylphosphine)Pd(II) (54 mg,
.077 mmol) and K CO (0.53 g, 3.83 mmol) was placed
(
0
3-(4-([2,3 -Bipyridin]-4-yl)-1-methyl-1H-pyrazol-3-yl)-
0
5-methylphenol (8a): To a solution of 6a (99 mg, 0.28 mmol)
in dichloromethane (5 mL) was added borontrifluoride–
dimethylsulfide complex (568 μL, 2.8 mmol) dropwise at
2
3
in a mixed solvent of acetonitrile and water (4:1, 5 mL). N2
gas was bubbled into this mixture for 15 min, and then the
mixture was refluxed while stirring under N atmosphere
room temperature under N atmosphere. The resulting suspen-
2
2
for 1 h. The reaction mixture was cooled to room temperature,
and then poured into ice waterand extracted with ethyl acetate.
The combined organic layers were washed with brine, dried
sion was stirred at room temperature for 12 h and the reaction
mixture was concentrated under reduced pressure. The residue
was partitioned between water and ethyl acetate. The com-
over anhydrous MgSO , and evaporated under vacuum. The
bined organic layers were dried over MgSO and evaporated
4
4
crude product was purified by column chromatography (silica
under vacuum. The crude product was purified by column
gel, ethyl acetate-hexane) to yield 6c and 7c (0.37 g, 37%,
upper spot in TLC). 6c: mp 59–61 C; H NMR (300 MHz,
chromatography (silica gel, ethyl acetate) to yield the pure
ꢀ
1
1
product 8a (60 mg, 63%); H NMR (300 MHz, CDCl ) δ
3
CDCl ) δ 2.32 (s, 3H), 3.07 (t, J = 4.8 Hz, 2H), 3.72 (s,
2.15 (s, 3H), 3.85 (s, 3H), 6.5–6.81 (m, 3H), 7.07 (d, J =
4.8 Hz, 1H), 7.19–7.23 (m, 1H), 7.51 (d, J = 14.1 Hz, 2H),
8.06 (d, J = 7.8 Hz, 1H), 8.36 (dd, J = 5.1, 4.5 Hz, 2H), 8.72
(d, J = 7.5 Hz, 1H); positive ion ESI-MS m/z 343.1559 (M
3
3H), 4.48 (t, J = 5.0 Hz, 2H), 6.78 (d, J = 0.5 Hz, 1H), 6.83
(t, J = 1.2 Hz, 1H), 6.94 (d, J = 0.3 Hz, 1H), 7.21 (dd, J =
1
1
.2, 3.9 Hz, 1H), 7.39–7.36 (m, 1H), 7.66 (d, J = 0.6 Hz,
+
H), 7.84 (s, 1H), 8.21 (dt, J = 1.1, 5.5 Hz, 1H), 8.63–8.60
+ H) .
1
3
0
(m, 2H), 9.02 (d, J = 1.5 Hz, 1H); C NMR (75 MHz, CDCl )
3-(4-([2,3 -Bipyridin]-4-yl)-1-methyl-1H-pyrazol-5-yl)-
3
δ 19.42, 21.54, 47.97, 55.27, 111.12, 115.34, 116.95, 118.57,
5-methylphenol (9a): To a solution of 7a (80 mg, 0.22 mmol)
in dichloromethane (5 mL) was added borontrifluoride–
dimethylsulfide complex (455 μL, 2.2 mmol) dropwise at
1
1
1
2
3
21.75, 121.81, 123.66, 130.40, 133.36, 134.56, 134.92,
40.00, 141.65, 147.97, 149.69, 150.18, 151.08, 154.83,
ꢀ
1
59.77; 7c: mp 125–127 C; H NMR (300 MHz, CDCl ) δ
room temperature under N atmosphere. The resulting suspen-
3
2
.44 (d, J = 0.3 Hz, 3H), 3.00 (t, J = 4.9 Hz, 2H), 3.85 (s,
H), 4.32 (t, J = 5.0 Hz, 2H), 6.78 (t, J = 1.2 Hz, 1H), 6.84
sion was stirred at room temperature for 12 h and the reaction
mixture was concentrated under reduced pressure. The residue
was partitioned between water and ethyl acetate. The com-
(d, J = 0.6 Hz, 1H), 6.98 (q, J = 0.6 Hz, 1H), 7.16 (dd, J =
1
1
6
3
1
1
1
.2, 3.9 Hz, 1H), 7.29 (s, 1H), 7.43 (dd, J = 3.8, 5.8 Hz,
H), 7.56 (q, J = 0.6 Hz, 1H), 8.02 (s, 1H), 8.26 (dt, J = 1.5,
.1 Hz, 1H), 8.57 (dd, J = 0.6 and 3.9 Hz, 1H), 8.66 (d, J =
bined organic layers were dried over anhydrous MgSO and
4
evaporated under vacuum. The crude product was purified
by column chromatography (silica gel, ethyl acetate) to yield
1
3
1
.0 Hz, 1H), 8.91 (s, 1H); C NMR (75 MHz, CDCl ) δ
the pure product 9a (55 mg, 73%); H NMR (300 MHz,
3
8.81, 29.72, 44.58, 55.55, 112.86, 116.45, 116.87, 118.17,
20.53, 122.89, 124.02, 129.69, 135.51, 138.81, 141.39,
CDCl ) δ 2.28 (s, 3H), 3.75 (s, 3H), 6.62 (d, J = 9.6 Hz,
3
2H), 6.90 (s, 1H), 7.19 (s, 3H), 7.38 (s, 2H), 7.819 (s, 1H),
42.23, 146.84, 148.70, 149.95, 160.49.
8.29 (d, J = 7.8 Hz, 1H), 8.45 (d, J = 5.1 Hz, 1H); positive
0
+
2
-(4-([2,3 -Bipyridin]-4-yl)-3-(3-methoxy-5-methylphe-
ion ESI-MS m/z 365.1389 (M + Na) .
0
nyl)-1H-pyrazol-1-yl)acetamide (6d): A mixture of 4d (100
mg, 0.28 mmol), 3-pyridineboronic acid (41 mg, 0.34 mmol),
dichlorobis(triphenylphosphine)Pd(II) (6 mg, 0.008 mmol)
and K CO (39 mg, 0.28 mmol) was placed in a mixed solvent
3-(4-([2,3 -Bipyridin]-4-yl)-1-ethyl-1H-pyrazol-3-yl)-5-
0
methylphenol (8b) and 3-(4-([2,3 -bipyridin]-4-yl)-1-ethyl-
1H-pyrazol-5-yl)-5-methylphenol (9b): To a solution of 6b
and 7b (0.26 g, 0.7 mmol) in dichloromethane (5 mL) was
added borontrifluoride–dimethylsulfide complex (1.4 mL, 7
2
3
of acetonitrile and water (4:1, 5 mL). N gas was bubbled into
2
this mixture for 15 min, and then the mixture was refluxed
mmol) dropwise at room temperature under N atmosphere.
2
while stirring under N atmosphere for 1 h. The reaction mix-
ture was cooled to room temperature, and then poured into ice
The resulting suspension was stirred at room temperature
for 12 h and the reaction mixture was concentrated under
2
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Optimization of Bipyridinyl Pyrazole Scaffolds
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reduced pressure. The residue was partitioned between water
and ethyl acetate. The combined organic layers were dried
temperature, which in turn converts 4-methyl group into an
active methylene, and achieved the adduct with a mixture of
keto and enol tautomers 2.
over anhydrous MgSO and evaporated under vacuum. The
4
crude product was purified by preparative TLC (silica gel, tol-
uene–MeOH–AcOH–acetone, 1:1:0.25:0.25, v/v/v/v) to yield
The conversion of the resulted tautomers 2 to the required
pyrazole 3 was achieved through two successive steps. In
the first step, compound 2 was heated with excess N,N-
dimethylformamide dimethylacetal for 12 h, and the resulted
product was taken to the next step without further purification,
where it was cyclized with hydrazine monohydrate in absolute
ethanol into the pyrazole derivative 3 as reported. The reac-
tion of the resulted pyrazole 3 with 4 different alkyl iodides in
the presence of excess potassium carbonate produced two dif-
ferent regioisomers, compounds 4a–d and 5a–d.
8
b(32 mg, 13%, lower spot in TLC) and 9b (22 mg, 9%, upper
1
spot in TLC). 8b: H NMR (300 MHz, CDCl ) δ 1.48 (t, J =
7
1
3
.2 Hz, 3H), 2.17 (s, 3H), 4.16 (q, J = 7.2 Hz, 2H), 6.57 (s,
H), 6.70 (s, 1H), 6.84 (s, 1H), 7.11–7.13 (m, 1H), 7.26 (q,
21
J = 4.3 Hz, 1H), 7.52 (s, 1H), 7.60 (s, 1H), 8.11 (d, J = 7.8
Hz, 1H), 8.4 (d, J = 5.1 Hz, 2H), 8.75 (s, 1H), 9.61 (s, 1H);
1
9
2
1
2
8
b: H NMR (300 MHz, CDCl ) δ 1.44 (t, J = 7.35, 3H),
3
.39 (s, 3H), 4.15 (q, J = 7.1 Hz, 2H), 7.3 (d, J = 4.2 Hz,
H), 6.93 (s, 1H), 7.24 (d, J = 5.1 Hz, 1H), 7.48–7.57 (m,
H), 7.94 (s, 1H), 8.12 (s, 2H), 8.26 (t, J = 6.3 Hz, 1H),
.55 (d, J = 5.1 Hz), 8.74 (s, 1H).
The structures of 1H-pyrazole and its regioisomer, 2H-
pyrazole were confirmed before by the two-dimensional
Nuclear Overhauser effect spectroscopy (NOESY) NMR
19
ROS1 Kinase Assays. The compounds were screened by the
spectrum. Generally, the 1H-pyrazole isomer was the prod-
uct with the lower R at which the CH protons of the NH sub-
2
0
same method in the literature.
Molecular Modeling. The compounds were studied by the
f
2
stituent group exhibited NOE interactions with two ortho
aromatic protons at carbon 2 and 6. However, the second
2
0
same method in the literature.
2
H-pyrazle isomer was the product with the higher R and does
f
Results and Discussion
not show any cross peak between the CH protons of the NH
2
substituent group and any of the aromatic protons of 3-meth-
oxy-5-methylphenyl group. The distance factor is the sole fac-
The preparation of compounds 6–9 is outlined in Scheme 1.
A nucleophilic substitution reaction using lithium hexam-
ethyldisilazide (LHMDS) was selected for attacking a benzo-
ate ester 1 by 2-chloro-4-methylpyridine. Thenucleophile was
produced in situ by abstracting one proton of the methyl group
via dropwise addition of LHMDS in dry THF at ambient
tor for making the CH proton being accessible or not for
2
making spatial coupling with the two ortho aromatic protons.
Furthermore, the anticipated higher shielding of the two pro-
tons of the NH substituent CH in all of the 2H-pyrazole iso-
2
mers caused by the anisotropic effect of the nearby aromatic
O
O
O
O
O
R
N
N
O
OH b, c
N
d
N
O
NH
a
N R
O
N
N
N
N
N
O
Cl
3
Cl
4a-d
Cl
5a-d
Cl
Cl
1
2
OH
OH
O
O
R
N
N
R
=
R
a =
N
N
N
e
f
N R
N R
N
-CH3,
=
b
-CH CH ,
2
3
N
N
N
N
N
N
N
c =
=
-
CH CH CN,
CH CONH
2 2
2
2
-
d
N
8
a-d
9a-d
2
, room temperature (rt), 24 h, 72%; (b) DMF-
6
a-d
7a-d
Scheme 1. Reaction conditions and reagents: (a) LHMDS, 2-chloro-4-methylpyridine, THF, N
ꢀ
DMA, 90 C, 12 h; (c) NH
2
NH
2
ꢁH
2
O, abs. EtOH, rt, 2 h, 81%; (d) K
2
CO
3
, RI, DMF, rt, 3 h; (e) 3-pyridineboronic acid, Pd(PPh
3
)
2
2
2
3
Cl , K CO ,
N
2
, CH
3
CN/H
2
O (4/1), reflux, 1 h; (f ) BF
3
ꢁS(CH , CH Cl , N , rt, 24 h.
)
3 2
2
2
2
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Optimization of Bipyridinyl Pyrazole Scaffolds
KOREAN CHEMICAL SOCIETY
electron cloud, was proved also before and in the experimental
section as the H NMR spectra of two pairs of compounds 6c,
mode and the detailed roles of all scaffold structural features
have become well known except the pyrazole moiety and its
NH substituents. Herein, the exact binding mode of pyrazole
NH substituents has been also clarified and emphasized.
Previously, SAR data analysis has been disclosed after dis-
covering some potent ROS1 kinase inhibitors and the detailed
SAR profile has been well understood. Isosteric replacement
of distal pyridine with phenyl moiety in some reported com-
pounds harboring the same skeleton has proven the role of dis-
tal pyridine N atom as an essential complementary group of
1
7c and 8b, 9b. In the 2H-isomers, such protons appeared at
upfield δ values; however in the 1H-isomers, these protons
were more downfield shifted.
OCH3
OCH3
H
H
H
H
R
2
2
H
N
N
21
H C
H C
3
3
6
H
H
hinge residue, methionine Met2029. However, internal pyr-
idine or pyrimidine moieties does not have any binding role
with hinge-interacting residues. Furthermore, ortho-
substitution of distal pyridine will deteriorate the activity as
it sterically hinders the pyridine N from approaching hinge
residues and forming the essential hydrogen bond with
Met2029. Also, simple deviation in the position of this termi-
nal pyridyl group in space caused by shift in its position from
the meta-position to the para-position relative to the central
pyridine, resultsinagreatlossofactivitytoo, andthisconfirms
the importance of keeping the orientation of the terminal pyr-
N
6
H
N
R
1
H-pyrazole
2H-pyrazole
Then Suzuki coupling of compound 4a–d and 5a–d
with 3-pyridineboronic acid, in the presence of
dichlorobis(triphenylphosphine)Pd(II) and potassium carbo-
nate, in a mixed solvent of acetonitrile and water in a ratio
(4:1), produced compounds 6a–d and 7a–d, respectively.
19,21
The corresponding hydroxyl products 8a–d and 9a–d were
obtained by demethylation of the methoxy group of com-
pounds 6a–d and 7a–d using 10 equiv of borontrifluoride–
dimethylsulfide complex in dichloromethane.
idyl group (Figures 1 and 2).
Further SAR studies were then undertaken. The biological
screening data have revealed that the pyrazole moiety was
essential for ROS1 kinase inhibitory activity, aliphatic replace-
22
Kinase assays were performed at Reaction Biology Corpo-
mentsofthepyrazolemoiety, resultingininactivecompound.
2
0
ration using the “HotSpot” assay platform. In the initial
screening step of the synthesized compounds, the compounds
were tested over ROS1 kinase at a single dose of 20 μM, and
the reaction was carried out at 10 μM ATP concentration.
After long term of handling bipyridinyl pyrazole as a core
structure with good ROS1 inhibitory potency, the binding
Pyrazole ring has an important role in directing both phenyl
and the internal pyridine ring toward their complementary
sites of interaction at ATP-binding pocket. With regard to
the pyrazole NH substitution, distal pyrazole NH is more
favorable for substitution, as in compounds 6a–d, 8a, and
8b, than their regioisomers, compounds 7a, 7c, 9a and 9b.
Figure1. Two-dimensionalinteractionschemeofbipyridinylpyrazolescaffoldinROS1receptor. SARsareillustratedinbriefaccordingtopink-
colored squares. Hydrogen bonds are shown as dotted lines.
Bull. Korean Chem. Soc. 2015, Vol. 36, 305–311
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BULLETIN OF THE
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Optimization of Bipyridinyl Pyrazole Scaffolds
KOREAN CHEMICAL SOCIETY
Table 1. Enzymatic assay results of compounds against kinases.
ROS1
ALK
c-Met
Comp. No. R
IC50 (nM)
IC50 (μM)
IC50 (μM)
6
6
6
7
7
8
8
9
9
a
c
CH3
23.2
40
159
31.1
74
35
28.1
21
19.9
—
20<
—
a
CH
CH
2
2
CH
CONH
2
CN
d
a
c
a
b
a
b
2
—
—
CH3
17.5
—
20<
—
CH
CH
2
3
2
CH CN
16.4
20<
10.8
20<
20<
20<
15.6
20<
CH CH3
2
CH
CH
3
2
CH
3
79.5
Crizotinib
1.7 (Ref. 23) 0.024 (Ref. 24) 0.011
a
Not determined.
Figure 2. Hydrogen bonding interaction between terminal pyridine
of compound 8b and hinge Met2029.
Distal pyrazole NH substitutions can closely direct the sub-
stituent group toward the intended small hydrophobic pocket.
The effect of this regioisomerism quietly appears with ethyl
and cyanoethyl groups that show significant improvements
in potency with 2.5- and 2-folds, respectively (Table 1). The
hydrophobic nature of amino acids forming pocket around
the pyrazole ring has positive discrimination toward hydro-
phobic groups being attached to the pyrazole NH moiety.
Therefore, groups such as methyl, ethyl, and cyanoethyl
groups have revealed magnificent binding interactions as
shown in Table 1.
On contrary, pyrazole NH substituent groups with polar
hydrophilic nature does not show such kind of potent inhibi-
tory activity as shown by IC50 values for 6d. The phenolic
hydroxyl group and its methyl ether have yet undisclosed
effect in influencing the inhibitory activity on ROS1 protein.
In case of compounds 6a and 8a, the methoxy analog 8a
showed more potency than its hydroxy analog 6a. However,
the hydroxy analog 9a showed more potent activity than its
methoxy analog 7a.
With regard to the selectivity profile, six compounds have
been screened against ROS1 closely related and often crossly
inhibited kinases, ALK and c-Met. The results exhibit good
ROS1 selectivity profile over them that are considering a
new tool in the field of discovering ROS1 kinase inhibitor.
Table 1 illustrates how much the improvement in the IC50 is
going in consistent with the aforementioned points of SAR
profile plus the selectivity pattern of six compounds over c-
MET and ALK kinases
Figure 3. Docking mode of representative ROS1 inhibitors 8b bound
13
to the ATP-binding pocket of ROS1 protein. It shows key H-bond
interaction with hinge residue. The binding motif 8b shows how
much the ethyl group is closely approaching to its complementary
hydrophobicarea. The convexsurface ofthis areahinders more bulky
groups from approaching.
shows a hydrophobic area, with convex surface, which has a
steric clash possibility with any bulky group attached to pyr-
azole NH group. It has become also obvious how the IC50 data
of final compounds be in consistent with the nature of pyrazole
NH substituent groups regarding hydrophobicity and size.
Also, we can conclude that the 2H-pyrazole isomer does not
support orienting pyrazole NH substituent groups to their
intended hydrophobic area. This is because of the possible
steric clash between the substituent groups and the receptor
surface. Therefore, positive discrimination revealed by hydro-
phobic area toward pyrazole NH substituent group has
become understandable and the observations were consistent
with the screening results of all compounds.
By investigating the docking mode of compound 8b
using Molecular Operating Environment (MOE) software
(2008.10 version, Chemical Computing Group, Inc., Mont-
real, Quebec, Canada) it has become clear that the nitrogen
of the internal pyridine does not share any hydrogen bonding
in the receptor; however, the terminal pyridine binds through
hydrogen bond with hinge region residue, Met 2029. Figure 3
Bull. Korean Chem. Soc. 2015, Vol. 36, 305–311
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BULLETIN OF THE
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Optimization of Bipyridinyl Pyrazole Scaffolds
KOREAN CHEMICAL SOCIETY
Compound 6a was selected as a representative member of
9. L. Nagarajan, E. Louie, Y. Tsujimoto, P. C. Balduzzi, K.
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to its potential cardiotoxic activity.
8
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The molecular modeling study has showed the three essential
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and this research was supported by a grant (NRF-2011-
0028676) from the creative/challenging research program of
National Research Foundation of Korea. We also appreciate
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Bull. Korean Chem. Soc. 2015, Vol. 36, 305–311
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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