Discovery of (E)- N1-(3-Fluorophenyl)- N3-(3-(2-(pyridin-2-yl)vinyl)-1 H-indazol-6-yl)malonamide (CHMFL-KIT-033) as a Novel c-KIT T670I Mutant Selective Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs)

Article abstract of DOI:10.1021/acs.jmedchem.9b00176

Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the gastrointestinal stromal tumors (GISTs). Despite the success of imatinib as the first-line treatment of GISTs, dozens of drug-acquired resistant mutations emerge, and c-KIT

Full text of DOI:10.1021/acs.jmedchem.9b00176

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Article
Discovery of (E)-N1-(3-fluorophenyl)-N3-(3-(2-(pyridin-2-yl)vinyl)-1H-
indazol-6-yl)malonamide (CHMFL-KIT-033) as a Novel c-KIT T670I Mutant
Selective Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs)
Xuesong Liu, Beilei Wang, Cheng Chen, Ziping Qi, Fengming Zou, Junjie Wang, Chen
Hu, Aoli Wang, Juan Ge, Qingwang Liu, Kailin Yu, Zhenquan Hu, Zongru Jiang, Wei
Wang, Li Wang, Wenchao Wang, Tao Ren, Mingfeng Bai, Qingsong Liu, and Jing Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00176 • Publication Date (Web): 02 May 2019
Downloaded from http://pubs.acs.org on May 3, 2019
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Discovery of (E)-N1-(3-fluorophenyl)-N3-(3-(2-
(pyridin-2-yl)vinyl)-1H-indazol-6-yl)malonamide
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(CHMFL-KIT-033) as a Novel c-KIT T670I Mutant
Selective Kinase Inhibitor for Gastrointestinal
Stromal Tumors (GISTs)
Xuesong Liu1,2,8, Beilei Wang1,2,8, Cheng Chen1,2,8, Ziping Qi1,3,8, Fengming Zou , Junjie
1,3
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,2
1,3
1,3,4
1,2
3,4
1,3
1,3
Wang , Chen Hu , Aoli Wang , Juan Ge , Qingwang Liu , Kailin Yu , Zhenquan Hu ,
Zongru Jiang , Wei Wang , Li Wang , Wenchao Wang1,2,3,4, Tao Ren3,4,5, Mingfeng Bai6,
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,2
1,3,4
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Qingsong Liu1,2,3,4,7*, Jing Liu1,2,3,4*
1
. High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam
Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei,
Anhui, 230031, China
2. University of Science and Technology of China, Hefei, Anhui, 230026, P. R. China
3
. Precision Medicine Research Laboratory of Anhui Province, Hefei, Anhui, 230088, P. R.
China
4. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei
Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230088, P. R.
China
5
. Precedo Pharmaceuticals Inc, Hefei, Anhui, 230088, P. R. China
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.
Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh Cancer
Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, United States
7. Institutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui,
30601, P. R. China
. These authors contribute equally
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ABSTRACT
Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the GISTs.
Despite of the success of imatinib as the first line treatment of GISTs, dozens of drug-acquired
resistant mutations emerge and c-KIT T670I is one of the most common mutants among them.
Although several kinase inhibitors are capable of overcoming the T670I mutant, none of them
can achieve the selectivity over the c-KIT wt which also plays important roles in a variety of
physiological functions such as hematopoiesis. Starting from axitinib, through fragment hybrid
type II kinase inhibitor design approach, we have discovered a novel inhibitor 24, which not only
exhibits potent activity to c-KIT T670I mutant but also achieves 12-fold selectivity over c-KIT
wt. Compound 24 displays good antiproliferative effects against c-KIT T670I mutant driven
GISTs cell lines (GIST-T1/T670I and GIST-5R), and also exhibits suitable in vivo PK profiles as
well as dose-dependent antitumor efficacy. This study provides a proof-of-concept for
developing c-KIT mutant selective inhibitor which theoretically can render better therapeutic
window.
INTRODUCTION
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasm in the
gastrointestinal tract. The overall incidence of GISTs ranges between 6.5 and 14.5 per million
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each year in Europe and America. Approximately 95% of GISTs express c-KIT, and the
majority of them harbor activating mutations of c-KIT which play a critical pathogenic role in
the development of GISTs.2,3 c-KIT kinase belongs to type III receptor tyrosine kinase family
which also includes FLT3, PDGFRs and CSF1R. Upon activation, c-KIT is involved in cell
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survival, migration and proliferation depending on the cell type . Early hematopoietic cells ,
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maturation cells such as mast cells, dendritic cells and fully differentiated cells are also
dependent on c-KIT for proliferation, survival and normal functions. In addition, c-KIT actively
triggers cell death in a variety of cancer cell lines except when engaged by its ligand stem cell
factor (SCF). c-KIT acts both as a proto-oncogene via its kinase activity and as a tumor
9
suppressor via its dependence receptor activity. Gain-of-function mutations of c-KIT drive the
pathology of several diseases especially the GITSs, therefore there have been extensive drug
discovery efforts targeting this kinase.10
Imatinib (1) was approved as the first-line therapy for advanced GISTs (Figure 1). However,
about 14% of the patients were insensitive to compound 1 initially,11,12 and more than a half of
responding patients developed drug resistance due to the acquisition of secondary mutations in
kinase domain such as T670I within 2 years treatment13-15. Sunitinib (2) and regorafenib (3)
16
17
were approved as the second and third line therapy respectively for GISTs to overcome some
imatinib-resistant mutants, including the T670I mutant (Figure 1). However, due to their very
low overall response rates and toxicities, the clinical application was limited. In addition,
1
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19
ponatinib (4) and cabozantinib (5) were also reported to be capable of overcoming the T670I
20
mutant induced resistance (Figure 1). Axitinib (6) , an approved drug for renal cell carcinoma
(
RCC), is an orally available multitarget kinase inhibitor that potently inhibits c-KIT, PDGFRα/β
and VEGFR1/2/3 (Figure 1). The kinase profiling data also revealed that it has strong binding
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affinity to c-KIT (binding Kd: 3.7 nM) and c-KIT V559D/T670I (binding Kd: 13 nM)21.
However, although compounds 2-6 could overcome the c-KIT T670I mutant, none of them could
achieve the selectivity between the c-KIT wt and T670I mutant. Given the fact that c-KIT wt
plays important roles in normal physiological functions, inhibitors that are selectively potent to
the c-KIT mutant over c-KIT wt would provide potential better therapeutic window. This
concept has been successfully demonstrated by the third generation EGFR inhibitor osimertinib,
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which selectively inhibits the EGFR T790M mutant over the EGFR wt. Based on the binding
conformation of kinases, kinase inhibitors can be categorized as type I binding mode which
features a DFG-in kinase active conformation and type II binding mode which characterizes a
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DFG-out kinase inactive conformation. Considering the potential chemical spaces for SAR
exploration, the c-KIT wt/T670I dual inhibitor 6 was chosen as a starting point for further
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medicinal chemistry study. Through a fragment hybrid type II kinase inhibitor design approach ,
we have discovered a novel type II c-KIT kinase inhibitor compound 24 (CHMFL-KIT-033),
which not only overcomes the c-KIT T670I mutant, but also achieved 12-fold selectivity over the
c-KIT wt (Figure 2).
N
O
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F
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CF3
Cl
H
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HN
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HN
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N
F
H
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H
1
(Imatinib)
2 (Sunitinib)
3 (Regorafenib)
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N
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H
O
O
N
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H
N
4
(Ponatinib)
5 (Cabozantinib)
6 (Axitinib)
Figure 1. Chemical structures of representative c-KIT kinase inhibitors.
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Glu671
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Glu671
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H
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Fragment hybrid approach
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Compd. 6
Axitinib)
Head
Compd. 24
(CHMFL-KIT-033)
N
N
(
R1
Figure 2. Schematic illustration of discovery of compound 24.
RESULTS AND DISCUSSION
Design rationale and structure–activity relationship (SAR) exploration.
Currently there is no available X-ray crystal structure of compound 6 in complex with c-KIT
kinase. Based on the published X-ray crystal structure of compound 6 in complex with the ABL
2
5
kinase T315I mutant (PDB ID: 4TWP) , it might adopt type I binding mode with the c-KIT
T670I mutant. However, based on the X-ray structure of compound 6 in complex with VEGFR2
2
6
kinase (PDB ID: 4AG8) , it might adopt type II binding mode. Therefore, in order to better
understand the binding mechanism of compound 6 to c-KIT T670I, we first docked it to the
DFG-in kinase active confirmation of c-KIT T670I (generated from PDB ID: 1PKG) using the
Schrodinger induced fit docking protocol. The results showed that it could fit well in the DFG-in
conformation and the indazole moiety formed two hydrogen bonds with Cys673 and Glu671 in
the hinge binding area (Figure 3A). In addition, in the DFG-in conformation, the carbonyl group
of benzamide formed a hydrogen bond with residue Asp810 in the DFG motif. Interestingly, we
also found that compound 6 could fit well in the DFG-out kinase inactive conformation of c-KIT
T670I (generated from PDB ID: 1T46, Figure 3B). In the DFG-out conformation, the NH of
benzamide formed a hydrogen bond with Glu640 located in the αC-Helix. Considering that both
c-KIT kinase and VEGFR2 kinase belong to type III receptor tyrosine kinase family and share
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better structural similarity, we were prone to think that compound 6 adopted type II binding
mode with c-KIT kinase. In addition, by comparing these two binding modes, we envisioned that
there was limited space for affinity and selectivity improvement from type I binding mode.
Given the bulky size of Ile670 residue, the inhibitor might need a more flexible linkage moiety.
Meanwhile, the amino acid residues around the hydrophobic pocket that are generated by DFG-
out shifting (shown in Figure 3C) are not conserved among different kinases and thus can serve
as good factors to trim the selectivity for the type II inhibitors. Therefore, exploration of the type
II binding mode, which further modifies the “linker” and “tail” moieties, might achieve the
binding affinity and selectivity against the c-KIT T670I mutant. Based on this assumption, using
the fragment hybrid type II kinase inhibitor design approach, we first designed compound 7 by
replacing the thiol linkage in compound 6 with the malonamide which presumably would
provide more space to accommodate the larger isoleucine residue, and then docked it to the
DFG-out conformation of c-KIT T670I (generated from PDB ID: 1T46, Figure 3D). As expected,
the docking results demonstrated that compound 7 prefers to adopt a typical type II binding
mode which features two canonical hydrogen bonds formed between Glu640 and Asp810 with
the amide in the linker. The indazole core forms two hydrogen bonds with Cys673 and Glu671 in
the hinge binding area and the “tail” moiety 4-chloro-3-trifloromethylphenyl occupies the
hydrophobic pocket (Figure 3E). The “head” moiety vinylpyridine occupies the shallow
hydrophobic groove adjacent to the hinge binding area (Figure 3F). On the basis of these
analyses and the fragment hybrid type II kinase inhibitor design approach23,24, we started to
explore the SAR by varying the “head”, “linker” and “tail” moieties meanwhile keeping the
indazole core scaffold of compound 7.
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Figure 3. Schematic illustration of the SAR exploration rationale. (A) Type I binding mode of
compound 6 with c-KIT T670I (generated from PDB code 1PKG). (B) Type II binding mode of
compound 6 with c-KIT T670I (generated from PDB code 1T46). (C) Surface illustration of the
hydrophobic pocket created by the DFG-out flip of c-KIT T670I (generated from PDB code
1
T46). (D) Binding mode of compound 7 with c-KIT T670I (generated from PDB code 1T46).
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(
E) Surface illustration of the hydrophobic pocket created by the DFG-out flip of c-KIT T670I.
F) Surface illustration of the shallow hydrophobic groove adjacent to the hinge binding area.
(
We used the proliferation of IL-3 independent BaF3 cells expressing the c-KIT wt (BaF3-tel-c-
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KIT) and c-KIT T670I (BaF3-tel-c-KIT-T670I) as the primary readout to evaluate the
compound’s activity and selectivity. As the starting point, we first explored the SAR of the
“
linker” part (L). Replacement of the phenyl thiol ether linkage with malonamide linkage (7)
resulted in improved potency against both c-KIT wt (GI50: 0.025 M) and c-KIT T670I (GI50:
.002 M) compared to compound 6, meanwhile exhibited 12-fold selectivity between c-KIT
0
T670I and c-KIT wt (Table 1). In addition, there is also a good selectivity window over the
parental BaF3 cells (GI : 7.4 M) indicating a good on-target antiproliferative inhibition effect
5
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in the c-KIT wt and c-KIT T670I transformed BaF3 cells. This result indicated that the design
approach of type II kinase inhibitor from compound 6 was valid. However, shorter linkers such
as amide (8), ethyleneamide (9) and urea (10) all resulted in activity loss against c-KIT T670I.
Restoring the length of the linker but increasing the size by adding the cyclopropyl group to the
linker (11) also led to activity loss to both c-KIT wt and c-KIT T670I.
Table 1. SAR Exploration Focused on the Linker Moiety (L)a
H
L
H N
2
N
N
Cl
CF3
N
BaF3
GI : μM)
BaF3-tel-c-KIT BaF3-tel-c-KIT-T670I
(GI : μM) (GI : μM)
Compd.
Linker (L)
(
5
0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
N
H
O
1.71
3.79
1.9
N
H
O
O
N
H
1
0
1.6
N
H
N
H
O
O
11
>10
1
N
H
N
H
a
All GI values were obtained by triplet testing.
5
0
1
We then fixed the “linker” moiety as malonamide and explored the “head “moiety R (Table
2). Replacement of the vinyl pyridine of 7 with hydrogen (12) and methyl group (13) both led to
significant activity loss. However, 1-methyl-1H-pyrazole (14), pyridine (15) and 2-
methylpyridine (16) all resulted in good inhibitory activities against c-KIT T670I (GI : 0.023
5
0
μM, 0.045 μM and 0.059 μM, respectively) meanwhile achieved the selectivity of c-KIT T670I
over c-KIT wt ranging from 7- to 15-fold. This indicated that the aromatic moieties in the “head”
1
region played an important role for the binding with c-KIT kinase. Changing the R moiety from
the pyridine in 15 to 5-(methylcarbamoyl)pyridin (17), fluorophenyl (18) and 3-carbamoylphenyl
19) all led to activity loss against c-KIT wt and c-KIT T670I. N-methyl formamyl (20) at R1
(
also exhibited good activity against c-KIT T670I (GI : 0.057 μM) and 6-fold selectivity over c-
5
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KIT wt. Larger groups such as 4-N-methyl piperazine (21) and N-methyl piperazinyl methylene
(22) groups both resulted in much more potent activity against c-KIT T670I and c-KIT wt but
also exhibited inhibitory activity to parental BaF3 cells. All these results indicated that the vinyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
pyridine moiety at the R position (as in compound 7) was preferred because it exhibited the best
potency against c-KIT T670I and better selectivity over c-KIT wt.
Table 2. SAR Exploration Focused on the Head Moiety (R1)a
H
H
H
N
N
N
N
O
O
Cl
CF3
R1
BaF3
(GI : μM)
BaF3-tel-c-KIT BaF3-tel-c-KIT-T670I
Compd.
Head (R1)
(GI : μM)
(GI : μM)
5
0
50
50
1
2
H
>10
>10
>10
>10
4.91
>10
>10
2.67
1.88
13
Me
N
N
1
1
1
4
5
6
0.35
0.32
0.183
0.023
0.045
0.059
N
N
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
1
1
7
8
N
H
>10
>10
>10
>10
1.35
>10
4.84
0.179
0.339
0.057
0.012
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.12
1.55
F
O
19
NH2
H
N
2
2
0
1
0.338
0.025
O
N
N
2
2
N
0.911
0.019
0.012
N
a
All GI values were obtained by triplet testing.
5
0
2
We next explored the “tail” (R ) moiety by fixing the “head” as vinyl pyridine and “linker” as
2
malonamide (Table 3). Replacement of the R moiety in compound 7 with a simple phenyl group
(
23) resulted in significant activity loss against c-KIT T670I (GI : 0.117 μM) but exhibited
50
selectivity over c-KIT wt (19-fold). Encouraged by this result, we tried a series of meta-halogen
2
substituted phenyl groups at R to increase the hydrophobicity (24-26) and m-fluoro (24)
exhibited the best potency against c-KIT T670I (GI : 0.044 μM) as well as the best selectivity
5
0
over c-KIT wt (26-fold). Subsequently we tested a series of larger substituent groups such as m-
methyl (27), m-methoxyl (28), m-N,N-dimethyl (29) and m-trifluoromethyl (30). However, all of
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them exhibited decreased activity to c-KIT T670I and lower selectivity over c-KIT wt. The even
larger group m-N-methyl piperazinyl (31) resulted in significant activity loss. Since m-
2
fluorobenzene moiety at R displayed the best activity, we also tried a series of fluoro-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
containing groups such as o-fluoro (32), p-fluoro (33) and some multi-fluoro substituents (34-36).
However, all of them exhibited activity loss against c-KIT T670I compared to compound 24.
Table 3. SAR Exploration Focused on the Tail Moiety (R2)a
H
N
H
N
H
N
R2
N
O
O
N
BaF3
BaF3-tel-c-KIT BaF3-tel-c-KIT-T670I
Tail (R2)
Compd.
(
GI : μM)
(GI : μM)
(GI : μM)
50
50
50
23
6.72
3.97
3.61
6.09
＞10
2.22
1.15
0.117
0.044
0.111
0.113
0.17
2
2
2
4
5
6
F
0.923
0.943
1.43
Cl
Br
27
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8
>10
>10
>10
2.74
3.87
0.234
0.594
0.086
O
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
3
3
0
1
0.216
CF3
N
N
>10
>10
6.17
F
32
5.09
5.86
4.24
2.16
0.554
1.87
0.312
0.111
0.399
3
3
3
4
F
F
F
F
F
3
3
5
6
5.08
3.68
3.46
2.95
0.847
1.3
F
F
F
F
F
a
All GI values were obtained by triplet testing.
50
Further biochemical and selectivity characterization of compound 24
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Since compound 24 displayed the best combined potency and selectivity profiles against the c-
KIT wt and T670I mutant, we then further examined its activity against a panel of c-KIT mutants
in the c-KIT mutants transformed BaF3 cells (Table 4). Compared with compound 1, 24
exhibited similar activities against primary gain-of-function mutations in the juxtamembrane
domain such as V559D/A/G and L576P. Furthermore, 24 displayed 26-fold selectivity between
c-KIT wt and c-KIT T670I in the BaF3 screening system compared with compound 6. In
addition, 24 exhibited better potencies against compound 1 resistant secondary mutants such as
V654A and T670I in the ATP binding pocket. However, just as compound 1, 24 did not show
potent activities against activation loop mutations either. Comparably, compound 6 was potent
against the c-KIT primary gain-of-function mutations and ATP binding pocket mutations but
relatively resistant to the activation loops mutations. More importantly, there was no selectivity
between the c-KIT wt and c-KIT T670I mutation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Antiproliferative effects of compounds 1, 6 and 24 against a panel of c-KIT mutants
transformed BaF3 cells.
GI (μM)
Mutated region
wt
1
6
24
50
BaF3-tel-c-KIT
0.364
0.039
0.005
0.001
0.115
1.57
0.105
0.012
0.005
0.002
0.007
0.013
1.15
BaF3-tel-c-KIT-V559D
BaF3-tel-c-KIT-V559A
BaF3-tel-c-KIT-V559G
BaF3-tel-c-KIT-L576P
Juxtamembrane
Juxtamembrane
Juxtamembrane
Juxtamembrane
ATP binding pocket
0.008
0.031
0.026
0.157
0.265
BaF3-tel-c-KIT-
V654A/V559D
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
BaF3-tel-c-KIT-V654A
BaF3-tel-c-KIT-T670I
ATP binding pocket
ATP binding pocket
2.30
0.014
0.108
0.129
2.02
0.525
0.044
0.122
3.46
>10
>10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BaF3-tel-c-KIT-T670I/V559D ATP binding pocket
BaF3-tel-c-KIT-D816V
BaF3-tel-c-KIT-D816H
BaF3-tel-c-KIT-D820E
BaF3-tel-c-KIT-N822K
Activation loop
Activation loop
Activation loop
Activation loop
Activation loop
>10
0.823
0.267
1.29
1.82
6.84
0.156
1.72
0.868
6.06
BaF3-tel-c-KIT-A829P
0.406
0.406
6.00
a
All GI values were obtained by triplet testing.
5
0
We then used the Z’-LYTE based biochemical activity assay with purified kinase proteins to
confirm the inhibition activities of compound 24 against the c-KIT wt and c-KIT T670I kinases.
The results showed that 24 inhibited the c-KIT T670I kinase with an IC of 45 nM and inhibited
50
the c-KIT wt kinase with an IC of 536 nM (Figure 4A). Compound 24 exhibited about 12-fold
5
0
selectivity against c-KIT T670I over c-KIT wt. In order to further confirm this selectivity in cells,
we proceeded to examine the phosphorylation of c-KIT at Y703, 719 and 823 sites in the colon
cancer cell line COLO320DM which expresses c-KIT wt kinase. As expected, 24 did not
apparently affect all of these three phosphorylation sites up to 1 μM (EC s: >1 μM) (Figure 4B).
5
0
Similarly, in the c-KIT wt transformed BaF3 cells, 24 did not affect these c-KIT phosphorylation
sites up to 1 μM (EC s >1 μM) either (Figure 4C). However, in the c-KIT T670I transformed
5
0
BaF3 cells, 24 could potently inhibit the phosphorylation of c-KIT at Y703, 719 and 823 sites
EC : 77 nM, 750 nM and 34 nM, respectively) (Figure 4D). The results demonstrated that both
(
5
0s
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in protein level and in cellular level, 24 could achieve the selectivity between c-KIT wt and c-
KIT T670I, which further confirmed the selective antiproliferative effects observed from the
transformed BaF3 cells.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. (A) Z’-LYTE biochemical kinase assay characterization of compound 24 against c-
KIT wt/T670I kinases. (B) Inhibitory activity of 24 to the phosphorylation of c-KIT wt in the
colon cancer cell line COLO320DM. (C) Inhibitory activity of 24 to the phosphorylation of c-
KIT wt in BaF3-tel-c-KIT cells. (D) Inhibitory activity of 24 to the phosphorylation of c-KIT
T670I in BaF3-tel-c-KIT-T670I cells.
To further characterize the selectivity of compound 24 among other protein kinases, we
examined its kinome-wide selectivity profile with DiscoverX’s KINOMEscan binding
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
21
technology (Figure 5). The results demonstrated that 24 bore a good selectivity (S score (1) =
0
.02) in a panel of 468 kinases and mutants at 1 μM concentration (Figure 5A). Besides high
binding affinity to c-KIT kinase, it also displayed strong binding to CSF1R, CSNK1A1, NEK,
PAK2, PDGFRβ, PIM and YSK1 kinases (percent of control value less than 1% at 1 μM. For
further details, please refer to https://www.discoverx.com/services/drug-discovery-development-
services/kinase-profiling/kinomescan/scanelect) (Figure 5 and Supplementary Table 1). In
addition, among the c-KIT mutant panel in the assay, 24 exhibited strong binding to c-KIT
L576P, V559D, V559D/T670I and V559D/V654A (%Ctrl value less than 1% at 1 μM) which
was consistent with the results observed from the transformed BaF3 cells in Table 4. Given the
fact that KINOMEscan is a binding assay, which sometimes may not reflect the real inhibitory
activity of the compound, we also used the Z’-LYTE activity-based assay to test the inhibitory
activity of 24 against these potential targets revealed by KINOMEscan assay. The data showed
that 24 could also potently inhibit CSF1R kinase (IC = 14.2 nM) besides c-KIT T670I (Figure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
5
C). This could be explained that both of c-KIT T670I and CSF1R belong to the type III receptor
tyrosine kinase family and the ATP pocket of these two kinases are highly conserved.
Interestingly, 24 did not show potent inhibitory activity against other targets from KINOMEscan
assay such as CSNK1A1, NEK1, PAK2, PDGFR, PIM3 and STK25, indicating that 24 was only
selectively potent toward c-KIT T670I mutant and CSF1R kinase.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Kinome-wide selectivity profiling of compound 24. (A) KINOMEscan profiling of 24
at a concentration of 1 μM against 468 kinases and mutants. (B) and (C) Z’-LYTE kinase assay
of 24 against the kinase targets revealed from the KINOMEscan profiling in isogenic cell lines.
The table shows the target kinases that remained activity of less than 1% control in the presence
of 1 μM of 24 and the IC values of these kinases.
5
0
In order to better understand its binding mechanism, we then docked it into the c-KIT wt (PDB
code: 1T46) and T670I mutant kinase (generated from PDB code: 1T46) by molecular modeling.
In the c-KIT wt (PDB code: 1T46) model, 24 adopts a canonical type II binding mode. The two
nitrogen atoms of indazol form two hydrogen bonds with the c-KIT residues Cys673 and Glu671
in the hinge binding area (Figure 6A). The amide in the linker moiety forms two hydrogen bonds
with Glu640 located in the c-helix and Asp810 in the DFG motif. The “tail” part occupies the
hydrophobic pocket generated by the DFG-out shifting. In the homology model of c-KIT T670I
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
mutant, 24 adopts a similar type II binding mode. The malonamide linkage is flexible which
generates enough space to accommodate the larger residue Ile670, which could explain its
potency against the T670I mutant (Figure 6B). However, the modeling study from the published
and homology structures could not explain the selectivity between the c-KIT wt and c-KIT T670I,
we reasoned that there might be some kinetic factors that associate with T670I mutant27,28 or the
drug binding might induced special conformational change either in the c-KIT wt or c-KIT
T670I mutant which makes the inhibitor 24 more favorable for c-KIT T670I. All of these
speculations would require further detailed kinetic and structural biology studies.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Schematic illustration of the binding mechanism of compound 24. (A) Binding mode
of 24 with c-KIT wt (PDB code: 1T46). (B) Binding mode of 24 with c-KIT T670I (generated
from PDB code: 1T46).
In Cell Evaluation of Compound 24.
We next investigated the antiproliferative effects of compound 24 against a panel of
established GISTs cancer cell lines (Table 5). Compound 24 was potent against GIST-T1 (c-KIT
del 560-578, GI : 0.052 M) and moderately potent against GIST882 (c-KIT K642E, GI :
5
0
50
0
.296 M) cell lines. In the compound 1-resistant cell line GIST-T1-T670I (c-KIT T670I,
generated by CRISPR-Cas9 knock-in T670I mutant to the GIST-T1 cell line) (GI >10 M), 24
5
0
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displayed a GI value of 0.288 M. In addition, in another compound 1-resistant cell line GIST-
5
0
5
R (c-KIT T670I, generated by continuous treatment of 1 and colon selection), 24 also exhibited
better antiproliferative effects than 1 (GI : 0.475 M versus 10 M). Neither of these two
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
inhibitors has effect against GIST-48B cell line which is c-KIT independent.
Table 5. Antiproliferative effects of compounds 1 and 24 against GIST cells.
Compd. 1
Compd. 24
Cell line
c-KIT status
(
GI : μM)
(GI : μM)
5
0
50
GIST-T1
∆560-578
0.026
0.058
>10
0.052
0.296
0.288
0.475
>10
GIST-882
K642E
GIST-T1-T670I
GIST-5R
∆560-578/T670I
∆560-578/T670I
c-KIT-independent
>10
GIST-48B
>10
a
All GI values were obtained by triplet testing.
5
0
We also evaluated the effect of compound 24 to the c-KIT-mediated signaling pathways in the
GISTs cancer cell lines (Figure 7). As expected, 24 potently inhibit the c-KIT
autophosphorylation sites p-Y719, p-Y703, p-Y823 and downstream signaling mediators such as
p-AKT (S473), p-ERK (T202/Y204) and p-S6 (S235/S236) starting from 0.1 M concentration
in the c-KIT growth-dependent GISTs cancer cell line GIST-T1 and two compound 1-resistant
cell lines GIST-T1-T670I and GIST-5R (Figure 7A). Interestingly, in the GIST-T1-T670I and
GIST-5R cells, compound 24 but not 1 could inhibit the phosphorylation of STAT3 dose-
dependently. While in the GIST-T1 cells, neither 24 nor 1 could affect the phosphorylation of
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2
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STAT3. In accordance with the effect of 24 on the c-KIT-mediated signaling pathways, 24 could
also induce the cell cycle arrest and apoptosis dose-dependently in these cell lines (Figure 7B, C).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Characterization of the cellular effects of compound 24. (A) Effects of 24 on the c-
KIT-mediated signaling transduction pathways in GIST-T1, GIST-T1-T670I and GIST-5R cells
after 1 h inhibitor treatment. (B) Apoptosis induction effects of 24 in GIST-T1, GIST-T1-T670I
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and GIST-5R cells after 24 h inhibitor treatment. (C) Cell cycle arresting effects of 24 in GIST-
T1, GIST-T1-T670I and GIST-5R cells after 24 h inhibitor treatment.
In vivo PK/PD Evaluation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We then examined the in vivo PK profile of compound 24 in rats and mice following
intravenous and oral administration (Table 6). The results demonstrated that in rats 24 displayed
an acceptable bioavailability (F = 27.5%) with a relatively longer half-life (T1/2 = 4.9 h) and
moderate drug exposure (AUC = 712 ng/mL·h) with 10 mg/kg of oral administration. While in
0
-t
mice 24 exhibited a bioavailability of 16.4% with good drug exposure (AUC = 1469 ng/mL·h)
0
-t
and moderate half-life (T =1.6 h). These data indicated that the oral administration of 24 was
1
/2
acceptable for the in vivo efficacy study.
Table 6. Pharmacokinetic Study of Compound 24 on Rats and Micea
Rats
Mice
Compd. 24
I.V.
P.O.
I.V.
P.O.
(1 mg/mg)
(10 mg/mg)
(1 mg/mg)
(10 mg/mg)
AUC(0-t) (ng/mL·h)
AUC(0-∞) (ng/mL·h)
Tmax (h)
258.9±30.6
307.2±27.1
0.017±0
712.5±172.5
808.9±46.8
1.7±2.1
897.9
901.4
0.03
4.9
1468.9
1478.3
0.5
T (h)
10.4±1.5
4.9±0.5
1.6
1
/2
Vz (L/kg)
49.3±10.3
3.3±0.3
86.8±11.8
12.4±0.7
97.1±27.9
5.4±1.9
5.7
19.0
6.8
CLz (L/h/kg)
Cmax (ng/mL)
MRT (0- ∞) (h)
1.1
378.5±104.4
6.4±0.3
3017.0
0.4
1124.0
2.2
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2
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2
2
2
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3
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F%
--
27.5
--
16.4
aAll data were obtained by triple testing (±SD) except mice.
We also evaluated the pharmacodynamic profile of 24 in the GIST-5R mouse model at the 100
mg/kg/day dosage for the impact on levels of p-c-KIT and downstream markers p-ERK and p-
AKT (Figure 8 and Supplementary Figure 1). Meanwhile, we also monitored the drug
concentration in the blood plasma. Following a single dose treatment, in 2 h time course the
levels of p-c-KIT/ERK/AKT decreased to lowest and then started to recover. The drug
concentration in the plasma reached highest level in 30 min and then gradually decreased to
basal levels in 4 h, which was correlated to the levels of phosphorylation of the signaling
mediators.
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1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Pharmacodynamic effects of compound 24 on p-c-KIT, p-ErK and p-AKT in tumor
from the GIST-5R xenograft mouse model with a single dosage of 100 mg/kg/day p.o.
administration.
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Finally, the in vivo antitumor efficacy of compound 24 was evaluated in the GIST-5R cells
inoculated xenograft mouse model. Upon 28 days continuous oral gavage treatment, none of the
25, 50 and 100 mg/kg/day (QD) administration affected the mouse body weight (Figure 9A) and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
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2
3
4
5
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9
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1
2
3
4
5
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8
9
0
2
4 dose-dependently inhibited the tumor growth (Figure 9B). The 100 mg/kg/day dosage
achieved a tumor growth inhibition (TGI) of 83% (Figure 9C, D) which was much better than
compound 1.
Figure 9. In vivo antitumor efficacy of compound 24 in the GIST-5R xenograft mouse model.
Female nu/nu mice bearing established GIST-5R tumor were treated with 24 at 25, 50 and 100
mg/kg/d or vehicle control. Daily oral administration was initiated when GIST-5R tumors had
3
reached a size of 200 mm . Each group contained 4 animals. Data = mean±SEM. (A) Body
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weight change in mice for each daily dosing group of 24 and imatinib. Initial body weight was
set as 100%. (B) Tumor size measurements of GIST-5R xenograft mice after 24 and imatinib
treatment. (C) Representative photographs of tumors in each group after 25, 50, 100 mg/kg/d of
0
1
2
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4
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8
9
0
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2
3
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9
0
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2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4 or vehicle treatment. (D) Comparison of the final tumor weight in each group of 28-day
treatment period.
CHEMISTRY
The syntheses of 7, 11 and 23-36 are illustrated in Scheme 1. Methyl ester 38 was obtained by
amide coupling reaction of 37 with carboxylic acid. Then hydrolysis under basic condition
afforded carboxylic acid 39. Finally, amide coupling reaction with amine and removal of the
tetrahydropyranyl (THP) protective group furnished the target compounds. As shown in Scheme
2
, 8 and 9 were obtained from amide coupling reaction of 37 with carboxylic acid followed by
deprotection of the THP group. To obtain 10, 37 was first converted to urea and then the THP
protective group was removed. In Scheme 3, Suzuki coupling reaction of 40 with arylboronic
acid and protection of 41 with THP group afforded intermediate 42. Then the nitro group was
reduced to provide amine 43. Coupling reaction of 44 with methyl hydrogen malonate followed
by hydrolysis of methyl ester 45 offered carboxylic acid 46. The final compounds 12-22 were
obtained by amide coupling reaction of 43 with 46 and removal of the THP protective group.
Scheme 1. Synthetic Route of Compounds 7, 11 and 23-36a
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9
O
O
O
H
N
H
X
OH
NH2
N
X
O
N
N
N
b
a
N
N
N
O
O
O
O
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
38a-b
39a-b
3
3
8a-39a, X = CH2
8b-39b, X = 1,1-cyclopropane
1
7
1
, R = 4-chloro-3-(trifluoromethyl)phenyl, X = CH
1, R = 4-chloro-3-(trifluoromethyl)phenyl, X = 1,1-cyclopropane
2
1
1
23, R = phenyl, X = CH
2
1
2
4, R = 3-fluorophenyl, X = CH
2
H
H
H
1
N
X
N
25, R = 3-chlorophenyl, X = CH
N
R1
2
1
N
26, R = 3-bromophenyl, X = CH
2
c, d
O
O
1
2
7, R = m-tolyl, X = CH
2
1
28, R = 3-(trifluoromethyl)phenyl, X = CH
2
1
2
9, R = 3-methoxyphenyl, X = CH
2
1
N
30, R = 3-(dimethylamino)phenyl, X = CH
1, R = 3-(4-methylpiperazin-1-yl)phenyl, X = CH
2
1
3
3
2
7, 11, 23-36
2, R = 2-fluorophenyl, X = CH
1
2
1
33, R = 4-fluorophenyl, X = CH
2
1
3
3
3
4, R = 3,5-difluorophenyl, X = CH
2
2
1
5, R = 2,5-difluorophenyl, X = CH
1
6, R = perfluorophenyl, X = CH2
aReagents and conditions: (a) methyl hydrogen malonate or 1,1-yclopropanedicarboxylic acid
1
monomethyl ester, HATU, DIPEA, DMF, rt, 2 h; (b) LiOH, MeOH, H O, rt, 6 h; (c) R NH ,
2
2
HATU, DIPEA, DMF, rt, 2 h; (d) CF COOH, DCM, rt, 4 h.
3
Scheme 2. Synthetic Route of Compounds 8-10a
H
H
N
O
N
N
R1
a, b
1
8
, R = 4-chloro-3-(trifluoromethyl)phenyl
9, R = 4-chloro-3-(trifluoromethyl)benzyl
1
O
N
8, 9
NH2
N
N
H
H
H
N
N
N
CF3
Cl
N
37
N
c, b
O
N
10
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aReagents and conditions: (a) 4-chloro-3-(trifluoromethyl)benzoic acid or 4-chloro-3-
(
trifluoromethyl)phenylacetic acid, HATU, DIPEA, DMF, rt, 2 h; (b) CF COOH, DCM, rt, 4 h;
3
(c) 4-chloro-3-(trifluoromethyl)phenyl isocyanate, DCM, rt, 8 h.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthetic Route of Compounds 12-22a
O
NO2
N
N
a
b
O
O
c
I
NO2
NH2
N
N
40
N
N
H
NO2
R1
R1
N
H
H
H
N
N
N
4
2a-k
43a-k
f, g
N
N
R1
O
O
Cl
41a-b
R1
CF3
H
H
N
12-22
NH2
N
O
OH
d
e
O
O
O
O
Cl
Cl
Cl
CF3
CF3
CF3
44
45
46
1
4
4
4
1a-43a, 12, R = H
1
1b-43b, 13, R = Me
1
2c-43c, 14, R = methyl-1H-pyrazol-4-yl
1
42d-43d, 15, R = pyridin-3-yl
1
4
4
4
4
2e-43e, 16, R = 6-methylpyridin-3-yl
1
2f-43f, 17, R = 5-(methylcarbamoyl)pyridin-3-yl
1
2g-43g, 18, R = 3-fluorophenyl
1
2h-43h, 19, R = 3-(3-carbamoylphenyl)
1
42i-43i, 20, R = 3-(4-(methylcarbamoyl)phenyl
1
4
4
2j-43j, 21, R = 3-(4-(4-methylpiperazin-1-yl)phenyl
1
2k-43k, 22, R = 3-(4-((4-methylpiperazin-1-yl)methyl)phenyl
a
Reagents and conditions: (a) arylboronic acid, Pd(Ph P) , K CO , 1,4-dioxane, H O, reflux, 12 h;
3
4
2
3
2
(
b) dihydropyran, CH SO H, THF, reflux, 8 h; (c) H , 10% Pd/C, MeOH, rt, 20 h; (d) methyl
3 3 2
hydrogen malonate, HATU, DIPEA, DMF, rt, 2 h; (e) LiOH, MeOH, H O, rt, 6 h. (f) HATU,
2
DIPEA, DMF, rt, 2 h; (g) CF COOH, DCM, rt, 4 h.
3
CONCLUSIONS
In summary, starting from a type I multiple kinase inhibitor 6 which displayed moderate c-KIT
wt and c-KIT T670I dual inhibitory effects, through fragment hybrid drug design approach, we
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discovered a novel indazole scaffold based c-KIT T670I mutant selective type II kinase inhibitor
2
4. In the purified protein biochemical assay, transformed BaF3 cell growth inhibition assay and
in cell on-target phosphorylation assay, 24 achieved 10-30-fold selectivity between c-KIT T670I
and c-KIT wt. In addition, it also displayed great selectivity profile over other protein kinases in
the kinome except CSF1R kinase. 24 exhibited potent antiproliferative effect against imatinib-
sensitive GIST cell line GIST-T1 and imatinib-resistant cell lines GIST-T1-T670I and GIST-5R
through potent inhibition of the c-KIT-mediated signaling pathways, arrest of the cell cycle
progression and induction of the apoptosis. Furthermore, 24 displayed a suitable PK profile and
potent in vivo antitumor efficacy. Although the selectivity mechanism of the inhibitor between
the c-KIT T670I mutant and c-KIT wt remains to be elucidated, the high selectivity, in cell and
in vivo properties of 24 might make it a useful pharmacological tool for the c-KIT wt/mutants
associated physiological and pathological studies.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemistry. All reagents and solvents were purchased from commercial sources and were used
as received unless specified otherwise, or prepared as described in the literature. All moisture
sensitive reactions were carried out using dry solvents under ultrapure argon protection.
Glassware was dried in an oven at 140 °C for at least 12 h prior to use and then assembled
quickly while hot, sealed with rubber septa, and allowed to cool under a stream of argon.
Reactions were stirred magnetically using Teflon-coated magnetic stirring bars. Commercially
available disposable syringes were used for transferring the reagents and solvents. LC/MS were
performed on an Agilent 6224 TOF using an ESI source coupled to an Agilent 1260 Infinity
HPLC system operating in reverse mode with an Agilent XDB-C18 column (4.6 × 50 mm, 1.8
μm) using a water/acetonitrile (each with 0.2% (v/v) formic acid) gradient at a flow rate at 0.5
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