Identification of 3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives as highly potent and selective mesenchymal-epithelial transition factor (c-Met) inhibitors via metabolite profiling-based structural optimization

Article abstract of DOI:10.1016/j.ejmech.2017.03.085

c-Met/HGF signaling pathway plays an important role in cancer progression, and it was considered to be related to poor prognosis and drug resistance. Based on metabolite profiling of (S)-7-fluoro-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyrazin

Full text of DOI:10.1016/j.ejmech.2017.03.085

Accepted Manuscript
Identification of 3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline
derivatives as highly potent and selective mesenchymal-epithelial transition factor (c-
Met) inhibitors via metabolite profiling-based structural optimization
Fei Zhao, Le-Duo Zhang, Yu Hao, Na Chen, Rui Bai, Yu-Ji Wang, Chun-Chun Zhang,
Gong-Sheng Li, Li-Jun Hao, Chen Shi, Jing Zhang, Yu Mao, Yi Fan, Guang-Xin Xia,
Jian-Xin Yu, Yan-Jun Liu
PII:
S0223-5234(17)30253-2
10.1016/j.ejmech.2017.03.085
EJMECH 9345
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 January 2017
Revised Date: 28 March 2017
Accepted Date: 31 March 2017
Please cite this article as: F. Zhao, L.-D. Zhang, Y. Hao, N. Chen, R. Bai, Y.-J. Wang, C.-C. Zhang,
G.-S. Li, L.-J. Hao, C. Shi, J. Zhang, Y. Mao, Y. Fan, G.-X. Xia, J.-X. Yu, Y.-J. Liu, Identification of
3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives as highly potent
and selective mesenchymal-epithelial transition factor (c-Met) inhibitors via metabolite profiling-
based structural optimization, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.085.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Identification of
3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline
derivatives as highly potent and selective mesenchymal-epithelial transition
factor (c-Met) inhibitors via metabolite profiling-based structural optimization
Fei Zhao, Le-Duo Zhang, Yu Hao, Na Chen, Rui Bai, Yu-Ji Wang, Chun-Chun Zhang,
Gong-Sheng Li, Li-Jun Hao, Chen Shi, Jing Zhang, Yu Mao, Yi Fan, Guang-Xin Xia,
*
*
Jian-Xin Yu , Yan-Jun Liu
Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd., Building 5, No. 898
Halei Road, Shanghai 201203, P. R. China
ABSTRACT:
c-Met/HGF signaling pathway plays an important role in cancer progression, and it was
considered to be related to poor prognosis and drug resistance. Based on metabolite profiling of
(
S)-7-fluoro-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyrazin-1-yl)ethyl)quinoline
1), series of 2-substituted
-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives
(
a
or
3
was
rationally designed and evaluated. Most of the 3-substituted derivatives not only exhibited potent
activities in both enzymatic and cellular assays, but also were stable in liver microsomes among
different species (human, rat and monkey). SAR investigation revealed that introducing of
*
Corresponding authors. Tel.: +86 21 61871700; Fax: +86 21 50128885.
E-mail addresses: yujx@sphchina.com (J.-X. Yu), liuyj@sphchina.com (Y.-J. Liu).
1

ACCEPTED MANUSCRIPT
N-methyl-1H-pyrazol-4-yl group at the 3-position of quinoline moiety is beneficial to improve the
inhibitory potency, especially in the cellular assays. The influence of fluorine atom at 7-position or
5
, 7-position of quinoline moiety and substituents at the 6-position of triazolo[4,5-b]pyrazine core
on overall activity is not very significant. Racemate 14, an extremely potent and exquisitely
selective c-Met inhibitor, demonstrated favorable pharmacokinetic properties in rats, no
significant AO metabolism, and effective tumor growth inhibition in c-Met overexpressed NSCLC
(H1993 cell line) and gastric cancer (SNU-5 cell line) xenograft models. Docking analysis
indicated that besides the typical interactions of most selective c-Met inhibitors, the intramolecular
halogen bond and additional hydrogen bond interactions with kinase are beneficial to the binding.
These results may provide deep insight into potential structural modifications for developing
potent c-Met inhibitors.
Keywords: c-Met; 3-Substituted quinolines; Triazolopyrazine; Metabolism; SAR; Docking study
1
. Introduction
The tyrosine kinase mesenchymal-epithelial transition factor (c-Met), encoded by MET
proto-oncogene, belongs to a structurally distinct subfamily of receptor tyrosine kinase (RTK) and
its endogenous ligand is hepatocyte growth factor (HGF, also known as scatter factor (SF)) [1].
c-Met/HGF signaling pathway plays an important role in a diverse array of biological activities
which are essential in the development of mammalian cells, such as cell proliferation, survival,
motality and wound healing, etc. However, this pathway is also implicated in tumor angiogenesis,
invasion and metastasis, etc [2-5]. Abnormal c-Met/HGF signaling has been found in a number of
major human malignancies and was related to poor prognosis and high tumors grade [6-10].
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Furthermore, aberrant c-Met signaling was demonstrated to be associated with the resistance of
tumor cells during chemotherapy, radiotherapy and other RTK based target therapy such as
epidermal growth factor receptor (EGFR) inhibitors [11, 12].
Given its critical role in tumor cell biology, c-Met is emerging as an attractive oncology target.
Until now, two generations of ATP-competitive small molecule c-Met inhibitors with different
selectivity profiles have been developed. Among them, Crizotinib (PF-02341066) [13] and
Cabozantinib (XL184) [14] have been approved for the treatment of non-small cell lung
carcinoma (NSCLC) and medullary thyroid cancer (MTC) in 2011 and 2012, respectively.
However, both of them acted on other kinases besides c-Met, so far none of selective c-Met
inhibitor has been approved. During the past decade, a great deal of effort has been made to the
discovery of potent and exquisitely selective c-Met inhibitors [15-19]. These have enjoyed some
successes. Dozens of drug candidates progressed to human clinical studies and several of them
achieved clinical benefits in a variety of cancers, such as INCB28060 [20],Volitinib [21] and
AMG 337 [22], etc (Fig. 1).
Fig. 1. Reference compounds.
3

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In our previous studies, a series of imidazo[4,5-b]pyrazine derivatives as potent and selective
inhibitor of c-Met kinase was discovered. Medicinal chemistry lead optimization produced
(
S)-7-fluoro-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyrazin-1-yl)ethyl)quinoline
compound 1) as a promising c-Met inhibitor [23]. Unfortunately, a further evaluation of this
(
compound incubated with monkey liver microsomes (MLM) demonstrated it was metabolized
rapidly via both NADPH-dependent and NADPH-independent enzymes, which posed a concern
on the druggability of this scaffold. A metabolite profiling of compound 1 was then conducted to
investigate the major metabolites. As shown in Fig. 2, there are two main products generated in
MLM (incubated with or without NADPH): imidazole-oxide M1 and quinoline N-oxide M2. In
order to improve ADME properties of this scaffold, the strategy we employed is to block
metabolism by means of modifying the metabolically labile sites. Our earlier SAR investigation
revealed that the introduction of a methyl group or hydroxyl group at the 2-position of imidazole
ring resulted in the loss of c-Met inhibitory potency [23]. Considering the important π-π stacking
interaction between this fused ring and electron-rich Tyr-1230 of the kinase, a much more
electron-deficient and more metabolically stable [1,2,3]triazolo[4,5-b]pyrazine ring, by means of
bioisosteric replacement of imidazo[4,5-b]pyrazine, was selected in our follow-up study. On the
other hand, although great efforts have been made to replace the conserved quinoline moiety of
the second generation c-Met inhibitors due to the reported aldehyde oxidase (AO, a
NADPH-independent enzyme) mediated toxicity of SGX523 which was discontinued in clinical
development [21, 24], our experimental data indicated that the major AO-mediated metabolite of
compound 1 is imidazo-oxidized product M1 and none of any quinoline-oxides was detected.
4

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Additionally, the nitrogen atom of quinoline hinge motif was confirmed by several literatures to
participate in a hydrogen bond with the backbone NH of Met-1160, which is crucial for selective
binding to c-Met kinase [15, 21, 25, 26]. Taken together, it is reasonable to maintain this
ubiquitous quinoline group in our next study. Here, with the hope of reducing the formation of
N-oxide by increasing steric hindrance of biological oxidation, large substituents were attached to
the 2-position or 3-position of quinoline moiety.
Fig. 2. Major metabolites of compound 1 in monkey liver microsomes incubated with or without NADPH.
2
. Results and discussion
2
.1. Chemistry
Outlined in Scheme 1 is the synthesis of the title compounds 2-26. A neocleophilic substitution
reaction of commercially available 2-amino-3,5-dibromopyrazine (a) with corresponding
substituted quinolin-6-ylethan-1-amine (b1-b7) in the presence of N, N-diisopropyl ethylamine
2
(
DIPEA) afforded corresponding 6-bromo-N -(1-(quinolin-6-yl)ethyl)pyrazine-2,3-diamine
5

ACCEPTED MANUSCRIPT
intermediates (c1-c7). Subsequently c1-c7 were reacted with isopentyl nitrite to give
6
-(1-(6-bromo-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline
intermediates
(d1-d7),
followed by Suzuki coupling with corresponding boric acid or boric acid ester to produce title
compounds 2-26.
R2
R3
H2N
R1
N
R4
N
N
NH2
N
N
N
N
N
N
N
N
b1-b7
N
N
N
N
NH2
Br
R2
Br
NH R2
Br
R
R2
i
ii
iii
R3
R4
R3
R4
R3
R4
Br
R1
N
R1
N
R1
N
a
c1-c7
d1-d7
2-26
R , R =H or F; R , R =N-methylpyrazol-4-yl or 4-fluorophenyl
1
2
3
4
Scheme 1. Synthetic procedures of title compounds 2-26. Reagents and conditions: (i) DIPEA, NMP, 180ºC,
overnight; (ii) isopentyl nitrite, DMF, 80ºC, 2h; (iii) corresponding boric acid or boric acid ester,
Pd(dppf) Cl ·DCM complex, K CO , 1,4-dioxane/H O, 110ºC, 3h or overnight.
2
2
2
3
2
2
.2 Biological evaluation
Firstly, seven compounds (2-8) were synthesized and enzymatic assay was used as the primary
screening. As Table 1 shows, 3-substituted derivatives (5-8) displayed very potent inhibitory
activities toward c-Met. Quite unexpectedly, the introduction of a group at the 2-position of
quinoline moiety resulted in a dramatic decrease in potency, as illustrated with derivatives 2-4.
3
-Substituted compounds were further evaluated in cellular assays, including c-Met overexpressed
human NSCLC H1993 cell line and gastric cancer SNU-5 cell line. Much to our delight, compared
with compound 1 all these compounds exhibited a significant increase of potency in both cellular
6

ACCEPTED MANUSCRIPT
assays with IC values at or even below a single-digit nanomolar level, which is equivalent to the
5
0
positive control INCB28060.
Table 1
SAR of 2-substituted and 3-substituted quinoline derivatives 2-8.
a
Compd.
R1
R2
R
IC50 (nM)
Enzyme
H1993
24.7
SNU-5
11.8
1
[23]
2
1.5
>100
66.7
b
b
-A
-A
-H
-H
-
-
b
b
3
-
-
b
b
4
5
6
7
-A
-H
-H
-H
-H
-A
-B
-B
>100
0.5
-
-
b
-
0.5
2.2
3.7
1.3
1.2
4.2
3.5
8
-H
-B
2.6
0.4
8.7
2.3
7.0
2.7
INCB28060
a
IC50 are based on triple runs of experiments.
b
The – indicates not tested.
7

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Besides the inhibitory activities in vitro, what we concerned most is the metabolic stability of
this series of compounds, especially in MLM. As shown in Table 2, all compounds were stable in
liver microsomes among different species (human, rat and monkey). More significantly, the
metabolic stability in MLM was improved remarkably, which fully supported our previous
molecular
design.
3-Substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline
scaffold was therefore identified as core structure and the potential of this series was further
explored.
Table 2
Metabolic stability data of compounds in liver microsomes among different species.
a
b
Compd. CLint (µl/min/mg protein) / Parent drug recovery (%)
c
d
e
HLM
RLM
MLM
f
1
5
6
7
8
8 / 92
49 / 79
22 / 115
28 / 97
57 / 97
56 / 91
- / 1
12 / 123
16 / 105
15 / 104
14 / 105
42 / 79
112 / 109
119 / 93
65 / 97
a
b
c
CLint was determined with the cofactor NADPH; recovery was determined without the cofactor NADPH; human
d
e
f
liver microsomes; rat liver microsomes; monkey liver microsomes; The-indicates the data was not obtained
because of rapid metabolism of compound.
Previous detailed SAR study on imidazo[4,5-b]pyrazine series pointed out the introduction of
8

ACCEPTED MANUSCRIPT
fluorine atom at 7-position or 5, 7-position of quinoline moiety could improve inhibitory activities
to some degree in both enzymatic and cellular assays [23]. Therefore, the contribution of fluorine
atom to overall activity was also evaluated in the following SAR exploration of
3
-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives. As listed in
Table 3, all compounds showed high potency toward c-Met, especially for compounds substituted
with N-methylpyrazol-4-yl group at the 3-position of quinoline moiety (9-22, IC ≤1 nM). Most
5
0
compounds displayed much more potent inhibitory activities than INCB28060 in H1993 cell line,
except for compounds 17 and 21 (not tested). Analogues 23-26 with a 4-fluorophenyl group at the
3
-position exhibited relatively weak inhibitory activities in both enzymatic and H1993 cell line
assays. With the exception of compounds 24 and 26, all the synthesized compounds displayed
much more potent activities than INCB28060 in SNU-5 cell line assay. The kinds of substituents
at 6-position of triazolo[4,5-b]pyrazine core could influence the inhibitory potency, albeit not very
significant. Different from SAR of imidazo[4,5-b]pyrazine series, it seems the introduction of
fluorine atom on the quinoline moiety has little effect on the overall activity of this series.
Table 3
SAR about the 3-substituted quinoline derivatives.
a
Compd.
R1 R2 R3
R
IC50 (nM)
Enzyme H1993 SNU-5
9

ACCEPTED MANUSCRIPT
9
-H -H -A
-H -H -A
0.5
0.7
0.2
0.9
1.8
0.7
1
0
1
1
-H -H -A
0.5
0.5
0.6
1
1
1
1
2
3
4
5
-H -H -A
-F -H -A
-F -H -A
-F -H -A
0.3
0.7
0.6
0.6
0.9
0.9
1.1
0.9
1.2
1.7
2.0
0.6
1
6
-F -H -A
1.0
1.1
0.6
1
1
1
2
7
8
9
0
-F -H -A
-F -F -A
-F -F -A
-F -F -A
0.7
0.4
0.4
0.3
3.2
0.8
0.8
0.4
2.5
1.3
1.3
0.4
b
2
1
-F -F -A
0.9
-
0.9
2
2
2
2
2
2
3
4
5
6
-F -F -A
-F -H -B
-F -H -B
-F -F -B
-F -F -B
0.5
1.5
1.7
3.8
4.3
0.4
0.2
4.1
3.6
2.6
3.5
2.3
1.2
2.0
6.8
2.2
7.2
2.7
INCB28060
1
0

ACCEPTED MANUSCRIPT
a
IC50 are based on triple runs of experiments.
b
The - indicates not tested.
Given decreased potencies of derivatives with 4-fluorophenyl group, this type was discontinued
in our next study. Compounds 9-22 were subsequently evaluated for metabolic stability in liver
microsomes and inhibition effect on 5CYPs in HLM (1A2, 2C9, 2C19, 2D6 and 3A4),
respectively (Table 4). Except for compounds 21 and 22, the vast majority of these compounds
were stable in the tested liver microsomes with acceptable clearance (CLint < 100 µl/min/mg
protein). After incubation for 30 min in liver microsomes among different species (without
NADPH), most compounds demonstrated satisfactory recovery with the exception of compounds
1
3, 16 and 18 (recovery < 80% in MLM). Many compounds displayed a certain degree of direct
inhibition (DI) or time-dependent inhibition (TDI) effect on 5CYPs in HLM, but compounds 14,
5 and 16 were exceptions.
1
Table 4
Leadlikeness assessment in vitro of compounds 9-22.
a
b
Compd. CLint (µl/min/mg protein) / Parent drug recovery (%)
DI
TDI
HLM
0 / 105
3 / 120
1 / 115
22 / 104
5 / 101
5 / 95
MLM
16 / 108
30 / 98
16 / 97
78 / 90
11 / 65
50 / 107
6 / 103
24 / 69
49 / 103
RLM
6 / 104
10 / 128
5 / 114
61 / 94
34 / 103
5 / 101
1 / 115
12 / 111
55 / 106
c
9
2C9,2C19
NI
c
1
0
NI
3A4,2D6,2C9,1A2
c
1
1
2
3
4
5
6
7
3A4
NI
c
1
1
1
1
1
1
3A4
NI
c
NI
3A4
c
c
NI
NI
c
c
2 / 120
5 / 116
4 / 113
NI
NI
c
c
NI
NI
c
3A4,2C9,2C19
NI
1
1

ACCEPTED MANUSCRIPT
1
1
2
2
2
8
9
0
1
2
17 / 91
8 / 98
86 / 75
43 / 102
50 / 99
22 / 94
20 / 97
12 / 94
48 / 95
26 / 98
2C9,2C19
2C9, 2C19
3A4
c
NI
c
11 / 97
5 / 92
NI
3A4
c
187 / 95
106 / 99
2C9, 2C19
NI
c
9 / 101
3A4,2D6,2C9,2C19
NI
a
direct inhibition on 5CYPs in HLM, referred to reversible inhibition, is assessed without a preincubation step.
b
time-dependent inhibition on 5CYPs in HLM, referred to irreversible inhibition, is assessed with a preincubation
c
step. The NI indicates inhibition ratio < 50%.
Compounds 14 and 15 were selected for further evaluation of pharmacokinetic properties in
vivo (Table 5). These two compounds displayed similar pharmacokinetic profiles including
moderate clearance, equivalent apparent volume of distribution, and acceptable half-life. The
bioavailability of compound 14 was a little higher than 15.
Table 5
a
In vivo pharmacokinetic profiles of compounds 14 and 15.
b
b
b
c
c
c
Compd. CLplasma (L/h/kg) Vss (L/kg) T1/2 (h) Cmax (ng/ml) AUC0-24h (ng·h/ml) F (%)
1
1
4
5
0.58
0.56
2.1
2.0
3.7
3.2
134.6
98.9
1254
879
12.1
8.2
a
Experiments were carried out with male Sprague-Dawley rats (n=2-3). Dose: iv, 1mg/kg; po, 6 mg/kg.
b
c
Parameters obtained after iv dosing. Parameters obtained after po dosing.
A panel of 18 human tyrosine kinases was used to investigate the selectivity profile of
compound 14, including highly homologous kinase AXL (tyrosine-protein kinase receptor UFO)
and c-Met family member RON (macrophage-stimulating protein receptor). As Table 6 shows,
1
2

ACCEPTED MANUSCRIPT
this compound is highly selective for c-Met, showing an IC50 of >700 nM against other tested
tyrosine kinases. Furthermore, in the light of the reported AO-mediated toxicity of SGX523, a
metabolite profilling of compound 14 in monkey cytosol was also investigated. After being
coincubated with and without the AO inhibitor (Menadione) at 37ºC for 30 min, the relative
amounts of 14 are 99.6% and 99.4%, respectively. It revealed that the overall majority is parent
and the amount of AO-mediated metabolite is very small. Therefore, we think the AO metabolism
will not be significant for this compound and will not lead to a safety concern in humans.
Table 6
a
Kinase-selectivity profile of compound 14.
kinase
AxI
IC50 (nM)
1075
kinase
EGFR
IC50 (nM)
>30000
>30000
>30000
17056
RON
731
EGFR[T790]
ErbB4
ALK
>30000
>30000
18364
5396
Fit-1
c-Src
VEGFR2
c-Kit
ABL
>30000
>30000
>30000
EPH-A2
EPH-B2
PDGFRa
2357
1
3

ACCEPTED MANUSCRIPT
PDGFRb
RET
>30000
>30000
IGF1R
>30000
FGFR1
>30000
a
Compound 14 against 18 kinases in vitro were assayed with ATP concentration at Km.
Our previous study on imidazo[4,5-b]pyrazine series indicated that compounds with an
(S)-configuration displayed more excellent activities than their corresponding (R)-configured
compounds in both enzymatic and cellular assays [23]. Accordingly, the two enantiomers of
compound 14 were obtained by chiral chromatography and further evaluated. Unexpectedly, these
two enantiomerically pure compounds displayed similar activities in vitro and equivalent
bioavailability (see Supplementary Material). On the basis of these results, racemate 14 was used
for evaluation of in vivo antitumor efficacy in H1993 and SNU-5 xenograft models, respectively.
As shown in Fig. 3, this compound demonstrated significant tumor growth inhibition in the treated
groups of both subcutaneous xenograft models. The percent tumor growth inhibition (%TGI) at
3
-week in the H1993 xenograft model was 55.5%, 78.7%, and 90.8% at doses of 0.1, 1, and 10
mg/kg, respectively. In the SNU-5 xenograft model, the percent tumor regression value (%PTR) at
-week was 34.0%, 79.2%, and 87.9% at doses of 0.3, 1, and 3 mg/kg, respectively. During the
experiment, none of mice in all the dosing groups displayed body weight loss or apparent toxicity.
2
1
4

ACCEPTED MANUSCRIPT
(A)
(B)
Fig. 3. (A) Growth inhibition of 14 in H1993 xenograft model in mice. Mice bearing H1993 tumors were orally
administered 14 once daily at the indicated dose levels or vehicle alone for 3 weeks. (B) Growth inhibition of 14 in
SNU-5 xenograft model in mice. Mice bearing SNU-5 tumors were orally administered 14 once daily at the
indicated dose levels or vehicle alone for 2 weeks. Data represents the mean ± SEM. for each group over the
treatment period (n=6-7).
2
.3. Molecular docking study
In this study, compound 14 was docked into the active site of c-Met kinase to obtain detailed
information. This compound adopts a bent “U-shaped” conformation binding mode and is bound
to the activation loop of c-Met kinase in the same way as most second generations of c-Met
1
5

ACCEPTED MANUSCRIPT
inhibitors. As illustrated in Fig. 4, all the important interactions with the kinase which were
reported by several publications were retained [15, 17, 21, 25, 27]: (1) a hydrogen bond between
Met-1160 and nitrogen atom of quinoline moiety (hinge binder); (2) a π-π stacking interaction
between Tyr-1159 and the quinoline ring; (3) a hydrogen bond interaction between the N-H of
Asp-1222 residue and N-3 nitrogen of the fused ring; (4) a π-π stacking interaction between
Tyr-1230 and the large coplanar structure of triazolopyrazine core with the side chain at 6-position.
In addition to these classical interactions, the N-hydroxyethyl-pyrazol-4-yl group was directed
toward the solvent-accessible region of c-Met, forming hydrogen bond network with Tyr-1230 and
Arg-1086 via a water molecule; and the hydroxyl group forms hydrogen-bonding interaction with
Asp-1231. It indicated that there is a halogen bond interaction between 7-F and nitrogen atom at
the 2-position of core ring (displayed as blue dashed line) which is beneficial to the stability of
binding conformation. Besides, we think the scaffold of triazolopyrazine which showed more
electron deficiency than imidazopyrazine may lead to a stronger π-π stacking interaction with
Tyr-1230. All above interactions contribute to the better ligand efficiency against c-Met.
1
6

ACCEPTED MANUSCRIPT
Fig. 4. Compound 14 was docked into the active site of c-Met.
3
. Conclusions
In summary, what described here is the rationally guided optimization of a series of
-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline derivatives through
3
metabolite profiling-based structural modification of lead compound 1 in order to improve the
ADME properties. By means of bioisosteric replacement, metabolically stable
[1,2,3]triazolo[4,5-b]pyrazine ring was selected as the core ring. Large substituents were attached
to the 2-position or 3-position of quinoline moiety with the hope of increasing metabolic stability.
Among them, 3-substituted compounds not only exhibited potent inhibitory activities in vitro, but
also were stable in liver microsomes among different species. Further SAR investigation revealed
that the introduction of N-methyl-1H-pyrazol-4-yl group at the 3-position of quinoline moiety is
favorable for achieving a better potency in both enzymatic and cellular assays. Different from our
17

ACCEPTED MANUSCRIPT
earlier SAR, the effect of fluorine atom at 7-position or 5, 7-position of quinoline moiety and
substituents at 6-position of triazolo[4,5-b]pyrazine core on the overall activity is not very
significant. Racemate 14 demonstrated favorable pharmacokinetic properties in vivo, no
significant AO metabolism, and excellent antitumor efficacy in c-Met overexpressed H1993 and
SNU-5 xenograft models. Molecular docking analysis of 14 indicated this molecule assumed a
“
U-shaped” conformation binding mode, which is the typical character of selective c-Met inhibitor.
Besides the well-known important interactions, the hydrogen bond network with Tyr-1230 and
Arg-1086, the hydrogen bond interactions with Asp-1231 and the intramolecular halogen bond
may be strong supplements to the binding with kinase. These docking results may provide
additional insights into the protein-ligand interactions and potential structural modifications for
further activity improvement. 14 was selected as a candidate for further development and this
work is in progress.
4
4
4
. Experimental
.1. Chemistry
.1.1. General
All reagents and solvents employed were purchased from commercial sources and used without
further purification. Reactions were followed by thin-layer chromatography (TLC) using HSGF
54 silica-gel plates (0.15-0.2 mm thickness) and visualized with UV light (254 nm). Silica-gel
chromatography was done using the appropriate size Biotage prepacked silica filled cartridges.
2
1
8

ACCEPTED MANUSCRIPT
Melting points were determined using a Shenguang WRR digital apparatus, and were uncorrected.
NMR data were recorded on a Bruker 400 MHz Digital NMR Spectrometer using CDCl or
3
DMSO-d as solvents. Chemical shifts are expressed as δ units (in NMR description, s, singlet; d,
6
doublet; t, triplet; q, quartet; m, multiplet; dd, double doublet; and brs, broad singlet). All coupling
constants (J) are reported in Hertz (Hz). High resolution mass spectra were measured with a
Waters Synapt G2-S Q-Tof spectrometer using an ESI source coupled to a Waters Acquity UPLC
system.
4
.1.2. General synthetic procedure of title compounds 2-26
To a solution of d1-d7 (1 equiv., synthetic procedures see supplementary material) in the
mixture of dioxane/H O (4:1, v/v), corresponding boric acid or boric acid ester (1.5 equiv.),
2
Pd(dppf) Cl ·DCM complex (0.05 equiv.), and K CO (3 equiv.) were added. The reaction mixture
2
2
2
3
was bubbled with N for 2 min and then stirred at 110ºC for 4h. The mixture was concentrated and
2
purified by flash column chromatography to afford the corresponding title compounds 2-26.
4
2
.1.2.1.
-(1-Methyl-1H-pyrazol-4-yl)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazi
1
n-1-yl)ethyl)quinoline (2). Yellow solid. Yield: 57%. Mp: 168-170ºC. H NMR (400 MHz,
DMSO-d ) δ 9.17 (s, 1H), 8.61 (s, 1H), 8.44 (s, 1H), 8.33 (d, J=8.8 Hz, 1H), 8.27 (s, 1H), 8.13 (s,
6
1
3
1
H), 7.99 (s, 1H), 7.91 (d, J=8.8 Hz, 1H), 7.83-7.80 (m, 2H), 6.54 (q, J=7.2 Hz, 1H), 3.94 (s, 3H),
1
3
.90 (s, 3H), 2.24 (d, J=7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 152.77, 148.42, 147.72,
6
47.60, 142.43, 138.91, 138.24, 137.91, 137.50, 137.35, 132.04, 130.99, 129.32, 128.99, 126.60,
1
9

ACCEPTED MANUSCRIPT
+
1
26.11, 123.32, 119.82, 119.75, 57.95, 39.44, 39.26, 20.70. HRMS (ESI): [M+H] =437.1953,
C H N calcd for 436.1872; retention time 1.99 min, >95% pure.
2
3
20 10
4
2
4
2
.1.2.2.
-Fluoro-N-methyl-4-(1-(1-(2-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[
,5-b]pyrazin-6-yl)benzamide
-fluoro-4-(1-(1-(2-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyra
was prepared by the general procedure using methyl
-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate as the reagent. This intermediate
30 mg, 0.06 mmol) was added into methanamine solution (33% in alcohol, 10ml). The mixture
(3).
Intermediate
methyl
zin-6-yl)benzoate
2
(
was stirred at rt for 3h. Evaporated the solvent and the residue was purified by column
1
chromatography to give the title compound as yellow solid. Yield: 30%. Mp: 245-247ºC. H NMR
(
400 MHz, DMSO-d ) δ 9.56 (s, 1H), 8.45 (s, 1H), 8.40 (brs, 1H), 8.35 (d, J=8.4 Hz, 1H),
6
8
.23-8.20 (m, 2H), 8.14 (s, 1H), 8.06 (s, 1H), 7.93 (d, J=8.4 Hz, 1H), 7.87-7.81 (m, 3H), 6.71 (q,
1
3
J=7.2 Hz, 1H), 3.92 (s, 3H), 2.82 (d, J=4.4 Hz, 3H), 2.28 (d, J=7.2 Hz, 3H). C NMR (100 MHz,
DMSO-d ) δ 163.90, 161.17, 158.70, 152.82, 149.99, 148.89, 147.76, 142.90, 139.50, 139.42,
6
1
38.25, 137.78, 137.34, 137.28, 131.45, 131.42, 131.00, 129.34, 129.06, 126.60, 126.28, 124.21,
1
24.18, 123.32, 119.77, 115.90, 115.66, 58.25, 39.26, 26.76, 20.85. HRMS (ESI):
+
[
M+H] =508.2012, C H FN O calcd for 507.1931; retention time 2.11 min, >95% pure.
2
7
22
9
4
4
.1.2.3.
-(1-(1-(2-(1-Methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)
2
0

ACCEPTED MANUSCRIPT
1
aniline (4). Yellow solid. Yield: 52%. Mp: 275-277ºC. H NMR (400 MHz, DMSO-d ) δ 9.28 (s,
6
1
H), 8.45 (s, 1H), 8.34 (d, J=8.4 Hz, 1H), 8.14 (s, 1H), 8.04 (d, J=8.4 Hz, 2H), 7.99 (d, J=1.2 Hz,
H), 7.92 (d, J=8.4 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.82 (dd, J =8.8 Hz, J =2.0 Hz, 1H), 6.70 (d,
1
1
2
1
3
J=8.4 Hz, 2H), 6.58 (q, J=7.2 Hz, 1H), 5.90 (s, 2H), 3.91 (s, 3H), 2.24 (d, J=7.2 Hz, 3H).
C
NMR (100 MHz, DMSO-d ) δ 152.96, 152.74, 152.48, 147.71, 147.44, 141.89, 138.24, 137.97,
6
1
37.70, 137.32, 130.99, 129.76, 129.31, 128.98, 126.60, 126.04, 123.34, 122.30, 119.73, 114.34,
+
5
7.73, 39.26, 20.82. HRMS (ESI): [M+H] =448.1988, C H N calcd for 447.1920; retention
2
5
21
9
time 2.11 min, >95% pure.
4
3
.1.2.4.
-(1-Methyl-1H-pyrazol-4-yl)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazi
1
n-1-yl)ethyl)quinoline (5). Yellow solid. Yield: 62%. Mp: 230-232ºC. H NMR (400 MHz,
DMSO-d ) δ 9.20 (s, 1H), 9.18 (d, J=2.4 Hz, 1H), 8.63 (s, 1H), 8.49 (d, J=2.0 Hz, 1H), 8.38 (s,
6
1
H), 8.29 (s, 1H), 8.08 (s, 1H), 8.00 (d, J=8.8 Hz, 1H), 7.91 (d, J=2.0 Hz, 1H), 7.82 (dd, J =8.4
1
Hz, J =2.0 Hz, 1H), 6.59 (q, J=6.8 Hz, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 2.27 (d, J=6.8 Hz, 3H).
2
+
HRMS (ESI): [M+H] =437.1951, C H N calcd for 436.1872; retention time 2.09 min, >95%
2
3
20 10
pure.
4
3
.1.2.5.
-(4-Fluorophenyl)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)eth
1
yl)quinoline (6). Yellow solid. Yield: 25%. Mp: 196-198ºC. H NMR (400 MHz, DMSO-d ) δ 9.23
6
(d, J=2.0 Hz, 1H), 9.20 (s, 1H), 8.66 (d, J=2.4 Hz, 1H), 8.63 (s, 1H), 8.29 (s, 1H), 8.06 (d, J=8.8
2
1

ACCEPTED MANUSCRIPT
Hz, 1H), 8.05 (s, 1H), 7.95-7.90 (m, 3H), 7.38 (t, J=8.8 Hz, 2H), 6.60 (q, J=6.8 Hz, 1H), 3.94 (s,
1
3
3
1
1
H), 2.28 (d, J=6.8 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 164.06, 161.62, 150.27, 148.47,
6
47.62, 146.73, 142.46, 139.27, 138.92, 137.98, 133.84, 133.81, 133.51, 132.67, 132.04, 129.80,
29.72, 128.86, 127.84, 126.39, 119.80, 116.66, 116.44, 57.95, 39.44, 20.73. HRMS (ESI):
+
[
M+H] =451.1831, C H FN calcd for 450.1717; retention time 2.71 min, >95% pure.
2
5
19
8
4
2
.1.2.6.
-Fluoro-4-(1-(1-(3-(4-fluorophenyl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-N
-methylbenzamide (7). This compound was prepared as a yellow solid by a procedure similar to
that described for the synthesis of compound using methyl
3
2
-fluoro-4-(1-(1-(3-(4-fluorophenyl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)be
1
nzoate as a starting material. Yield: 51%. Mp: 188-190ºC. H NMR (400 MHz, DMSO-d ) δ 9.58
6
(s, 1H), 9.23 (d, J=2.0 Hz, 1H), 8.66 (d, J=2.0 Hz, 1H), 8.39 (brs, 1H), 8.22 (s, 1H), 8.21-8.18 (m,
1
7
H), 8.12 (d, J=1.2 Hz, 1H), 8.07 (d, J=8.8 Hz, 1H), 7.96-7.91 (m, 3H), 7.82 (t, J=8.0 Hz, 1H),
1
3
.38 (t, J=8.8 Hz, 2H), 6.77 (q, J=7.2 Hz, 1H), 2.82 (d, J=4.8 Hz, 3H), 2.30 (d, J=7.2 Hz, 3H). C
NMR (100 MHz, DMSO-d ) δ 164.06, 163.88, 161.62, 161.17, 158.69, 150.31, 150.04, 150.02,
6
1
1
48.90, 146.76, 142.92, 139.48, 139.39, 139.09, 137.85, 133.83, 133.80, 133.49, 132.70, 131.46,
31.43, 129.80, 129.72, 128.92, 127.84, 126.51, 126.33, 126.18, 124.21, 124.18, 116.66, 116.44,
+
1
15.90, 115.66, 58.21, 26.76, 20.91. HRMS (ESI): [M+H] =522.1851, C H F N O calcd for
2
9
21
2
7
5
21.1776; retention time 2.78 min, >95% pure.
4
.1.2.7.
2
2

ACCEPTED MANUSCRIPT
3
-(4-Fluorophenyl)-6-(1-(6-(pyridin-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)quinoline
1
(
8). Yield: 59%. Mp: 199-201ºC. H NMR (400 MHz, DMSO-d ) δ 9.61 (s, 1H), 9.23 (d, J=2.4 Hz,
6
1
1
2
1
1
H), 8.82 (d, J=6.0 Hz, 2H), 8.66 (d, J=2.0 Hz, 1H), 8.24 (d, J=6.0 Hz, 2H), 8.11 (d, J=1.6 Hz,
H), 8.07 (d, J=8.8 Hz, 1H), 7.96-7.90 (m, 3H), 7.38 (t, J=8.8 Hz, 2H), 6.77 (q, J=7.2 Hz, 1H),
1
3
.32 (d, J=7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 164.05, 161.61, 151.16, 150.31, 149.77,
6
49.33, 146.76, 142.92, 142.75, 139.04, 137.97, 133.81, 133.78, 133.49, 132.68, 129.83, 129.79,
29.70, 128.86, 127.84, 126.48, 122.09, 116.65, 116.44, 58.40, 20.84. HRMS (ESI):
+
[
M+H] =448.1687, C H FN calcd for 447.1608; retention time 2.62 min, >95% pure.
2
6
18
7
4
2
.1.2.8.
-(4-(1-(1-(3-(1-Methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-
1
yl)-1H-pyrazol-1-yl)ethan-1-ol (9). Yellow solid. Yield: 56%. Mp: 131-133ºC. H NMR (400 MHz,
DMSO-d ) δ 9.22 (s, 1H), 9.18 (d, J=2.0 Hz, 1H), 8.64 (s, 1H), 8.49 (d, J=1.6 Hz, 1H), 8.38 (s,
6
1
H), 8.32 (s, 1H), 8.08 (s, 1H), 8.00 (d, J=8.8 Hz, 1H), 7.92 (d, J=1.6 Hz, 1H), 7.82 (dd, J =8.8
1
Hz, J =2.0 Hz, 1H), 6.60 (q, J=7.2 Hz, 1H), 4.98 (t, J=5.2 Hz, 1H), 4.25 (t, J=5.6 Hz, 2H), 3.92 (s,
2
1
3
3
1
1
H), 3.80 (q, J=5.2 Hz, 2H), 2.26 (d, J=7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 149.70,
6
48.58, 147.59, 146.07, 142.50, 139.20, 138.95, 137.98, 137.05, 131.95, 130.07, 129.86, 129.06,
28.19, 127.81, 126.87, 125.65, 119.52, 119.05, 60.23, 57.86, 55.06, 39.27, 20.79. HRMS (ESI):
+
[
M+H] =467.2069, C H N O calcd for 466.1978; retention time 1.94 min, >95% pure.
2
4
22 10
4
2
.1.2.9.
-Fluoro-4-(1-(1-(3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyra
2
3

ACCEPTED MANUSCRIPT
zin-6-yl)benzamide
(10).
Intermediate
methyl
2
-fluoro-4-(1-(1-(3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyra
zin-6-yl)benzoate (30 mg, 0.06 mmol) was added into ammonia solution (7 M in methanol, 10ml).
The mixture was heated to 50ºC overnight in a sealed tube. Evaporated the solvent and the residue
was purified by column chromatography to give the title compound as yellow solid. Yield: 62%.
1
Mp: 258-260ºC. H NMR (400 MHz, DMSO-d ) δ 9.58 (s, 1H), 9.18 (d, J=2.0 Hz, 1H), 8.50 (d,
6
J=2.0 Hz, 1H), 8.38 (s, 1H), 8.22-8.18 (m, 2H), 8.08 (s, 1H), 8.00 (d, J=8.8 Hz, 1H), 7.99 (s, 1H),
1
3
7
.88-7.84 (m, 3H), 7.78 (s, 1H), 6.74 (q, J=7.2 Hz, 1H), 3.92 (s, 3H), 2.29 (d, J=7.2 Hz, 3H). C
NMR (100 MHz, DMSO-d ) δ 165.12, 161.30, 158.82, 150.14, 150.12, 148.91, 144.21, 143.01,
6
1
1
41.66, 139.49, 139.41, 137.91, 137.86, 137.54, 137.40, 131.89, 131.56, 131.53, 130.13, 129.11,
28.18, 126.30, 126.15, 124.16, 124.13, 123.06, 117.11, 115.93, 115.68, 57.83, 39.45, 20.80.
+
HRMS (ESI): [M+H] =494.1848, C H FN O calcd for 493.1775; retention time 2.11 min, >95%
2
6
20
9
pure.
4
2
4
.1.2.10.
-Fluoro-N-methyl-4-(1-(1-(3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[
,5-b]pyrazin-6-yl)benzamide (11). This compound was prepared as a yellow solid by a procedure
similar to that described for the synthesis of compound
3
using methyl
2
-fluoro-4-(1-(1-(3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]pyra
1
zin-6-yl)benzoate as a starting material. Yield: 67%. Mp: 132-134ºC. H NMR (400 MHz,
DMSO-d ) δ 9.57 (s, 1H), 9.17 (d, J=1.6 Hz, 1H), 8.49 (s, 1H), 8.39 (s, 1H), 8.37 (s, 1H), 8.21 (s,
6
1
H), 8.19 (d, J=2.0 Hz, 1H), 8.07 (s, 1H), 8.00-7.98 (m, 2H), 7.86-7.80 (m, 2H), 6.74 (q, J=6.8 Hz,
2
4

ACCEPTED MANUSCRIPT
1
H), 3.91 (s, 3H), 2.82 (d, J=4.4 Hz, 3H), 2.28 (d, J=6.8 Hz, 3H). HRMS (ESI):
+
[
M+H] =508.2007, C H FN O calcd for 507.1931; retention time 2.21 min, >95% pure.
2
7
22
9
4
3
.1.2.11.
-(1-Methyl-1H-pyrazol-4-yl)-6-(1-(6-(pyridin-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)ethyl)q
1
uinoline (12). Yellow solid. Yield: 35%. Mp: 210-212ºC. H NMR (400 MHz, DMSO-d ) δ 9.60
6
(s, 1H), 9.17 (d, J=2.4 Hz, 1H), 8.82 (dd, J =4.8 Hz, J =1.6 Hz, 2H), 8.49 (d, J=2.0 Hz, 1H), 8.38
1 2
(
s, 1H), 8.24 (dd, J =4.8 Hz, J =1.6 Hz, 2H), 8.08 (s, 1H), 8.00 (d, J=8.4 Hz, 1H), 7.97 (d, J=1.6
1
2
Hz, 1H), 7.85 (dd, J =8.8 Hz, J =2.0 Hz, 1H), 6.74 (q, J=6.8 Hz, 1H), 3.91 (s, 3H), 2.30 (d, J=6.8
1
2
1
3
Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 151.15, 149.74, 149.31, 146.11, 142.91, 142.73,
6
1
38.91, 137.93, 137.03, 130.04, 129.91, 129.03, 128.19, 127.81, 126.88, 125.82, 122.08, 119.01,
+
5
8.40, 39.27, 20.85. HRMS (ESI): [M+H] =434.1835, C H N calcd for 433.1763; retention
2
4
19
9
time 1.98 min, >95% pure.
4
7
.1.2.12.
-Fluoro-3-(1-methyl-1H-pyrazol-4-yl)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5
1
-
b]pyrazin-1-yl)ethyl)quinoline (13). Yellow solid. Yield: 56%. Mp: 228-230ºC. H NMR (400
MHz, DMSO-d ) δ 9.21 (d, J=2.0 Hz, 1H), 9.20 (s, 1H), 8.58 (s, 2H), 8.36 (s, 1H), 8.23 (s, 1H),
6
8
3
4
.07 (d, J=8.0 Hz, 1H), 8.06 (s, 1H), 7.80 (d, J=12.0 Hz, 1H), 6.72 (q, J=7.2 Hz, 1H), 3.93 (s, 3H),
+
.91 (s, 3H), 2.24 (d, J=7.2 Hz, 3H). HRMS (ESI): [M+H] =455.1864, C H FN calcd for
2
3
19
10
54.1778; retention time 2.26 min, >95% pure.
2
5

ACCEPTED MANUSCRIPT
4
2
.1.2.13.
-(4-(1-(1-(7-Fluoro-3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-b]p
1
yrazin-6-yl)-1H-pyrazol-1-yl)ethan-1-ol (14). Yellow solid. Yield: 49%. Mp: 158-160ºC. H NMR
(
400 MHz, DMSO-d ) δ 9.22 (s, 1H), 9.20 (d, J=2.0 Hz, 1H), 8.60 (s, 1H), 8.58 (d, J=1.6 Hz, 1H),
6
8
.36 (s, 1H), 8.26 (s, 1H), 8.09 (d, J=8.0 Hz, 1H), 8.06 (s, 1H), 7.79 (d, J=12.0 Hz, 1H), 6.74 (q,
J=7.2 Hz, 1H), 4.96 (t, J=5.2 Hz, 1H), 4.24 (t, J=5.2 Hz, 2H), 3.92 (s, 3H), 3.78 (q, J=5.2 Hz, 2H),
1
3
2
1
1
.25 (d, J=7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 161.11, 158.63, 150.74, 148.60, 147.47,
6
46.81, 146.69, 142.52, 138.89, 137.86, 136.98, 131.92, 130.29, 128.99, 128.91, 128.73, 128.32,
28.27, 126.45, 126.42, 125.51, 119.47, 118.86, 113.33, 113.13, 60.22, 55.04, 52.19, 39.28, 20.01.
+
HRMS (ESI): [M+H] =485.1949, C H FN O calcd for 484.1884; retention time 0.09
2
4
21
10
min, >95% pure.
4
2
5
.1.2.14.
-Fluoro-4-(1-(1-(7-fluoro-3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,
-b]pyrazin-6-yl)benzamide (15). This compound was prepared as a yellow solid by a procedure
similar to that described for the synthesis of compound 10 using methyl
2
5
-fluoro-4-(1-(1-(7-fluoro-3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,
1
-b]pyrazin-6-yl)benzoate as a starting material. Yield: 74%. Mp: 243-245ºC. H NMR (400 MHz,
DMSO-d ) δ 9.57 (s, 1H), 9.22 (d, J=2.4 Hz, 1H), 8.59 (d, J=2.4 Hz, 1H), 8.37 (s, 1H), 8.19 (d,
6
J=8.4 Hz, 1H), 8.16 (d, J=8.4 Hz, 1H), 8.14 (d, J=12.0 Hz, 1H), 8.07 (s, 1H), 7.86-7.77 (m, 4H),
1
3
6
1
.88 (q, J=7.2 Hz, 1H), 3.92 (s, 3H), 2.29 (d, J=7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ
6
65.11, 162.35, 161.28, 159.82, 158.80, 150.09, 150.07, 148.79, 146.66, 143.01, 140.74, 140.60,
2
6

ACCEPTED MANUSCRIPT
1
39.44, 139.36, 137.72, 137.30, 136.02, 131.55, 131.52, 130.66, 130.49, 129.77, 129.49, 129.44,
27.39, 127.38, 126.31, 126.26, 126.16, 124.10, 124.07, 117.46, 115.86, 115.61, 109.12, 108.88,
1
+
5
2.50, 39.42, 19.83. HRMS (ESI): [M+H] =512.1761, C H F N O calcd for 511.1681; retention
2
6
19
2
9
time 2.26 min, >95% pure.
4
2
5
.1.2.15.
-Fluoro-4-(1-(1-(7-fluoro-3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,
-b]pyrazin-6-yl)-N-methylbenzamide (16). This compound was prepared as a yellow solid by a
procedure similar to that described for the synthesis of compound 3 using methyl
2
5
-fluoro-4-(1-(1-(7-fluoro-3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,
1
-b]pyrazin-6-yl)benzoate as a starting material. Yield: 80%. Mp: 226-228ºC. H NMR (400
MHz, DMSO-d ) δ 9.58 (s, 1H), 9.23 (s, 1H), 8.60 (s, 1H), 8.38 (s, 2H), 8.20-8.13 (m, 3H), 8.08 (s,
6
1
H), 7.82-7.79 (m, 2H), 6.88 (q, J=6.8 Hz, 1H), 3.93 (s, 3H), 2.82 (d, J=4.4 Hz, 3H), 2.30 (d,
+
J=6.8 Hz, 3H). HRMS (ESI): [M+H] =526.1915, C H F N O calcd for 525.1837; retention time
2
7
21
2
9
2
.36 min, >95% pure.
4
7
.1.2.16.
-Fluoro-3-(1-methyl-1H-pyrazol-4-yl)-6-(1-(6-(pyridin-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-
1
yl)ethyl)quinoline (17). Yellow solid. Yield: 65%. Mp: 227-229ºC. H NMR (400 MHz, DMSO-d )
6
δ 9.60 (s, 1H), 9.20 (d, J=2.0 Hz, 1H), 8.80 (d, J=6.0 Hz, 2H), 8.58 (d, J=1.6 Hz, 1H), 8.36 (s, 1H),
8
.18 (d, J=6.0 Hz, 2H), 8.16 (d, J=8.4 Hz, 1H), 8.06 (s, 1H), 7.79 (d, J=12.0 Hz, 1H), 6.88 (q,
1
3
J=6.8 Hz, 1H), 3.91 (s, 3H), 2.30 (d, J=6.8 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 161.13,
6
27

ACCEPTED MANUSCRIPT
1
58.65, 151.16, 150.82, 149.73, 149.22, 146.88, 146.75, 142.95, 142.68, 137.82, 136.97, 130.27,
28.99, 128.59, 128.53, 128.49, 128.42, 126.47, 126.45, 125.53, 122.02, 118.84, 113.40, 113.19,
1
+
5
2.75, 52.72, 39.29, 19.98. HRMS (ESI): [M+H] =452.1744, C H FN calcd for 451.1669;
2
4
18
9
retention time 2.14 min, >95% pure.
4
5
.1.2.17.
,7-Difluoro-3-(1-methyl-1H-pyrazol-4-yl)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo
1
[
4,5-b]pyrazin-1-yl)ethyl)quinoline (18). Yellow solid. Yield: 95%. Mp: 172-174ºC. H NMR (400
MHz, DMSO-d ) δ 9.30 (d, J=2.0 Hz, 1H), 9.15 (s, 1H), 8.61 (d, J=1.2 Hz, 1H), 8.50 (s, 1H), 8.48
6
(s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.74 (d, J=11.6 Hz, 1H), 6.76 (q, J=7.2 Hz, 1H), 3.92 (s, 6H),
1
3
2
1
1
.38 (d, J=7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 161.08, 160.99, 158.60, 158.51, 157.55,
6
57.45, 154.97, 154.87, 151.97, 148.14, 147.46, 146.12, 146.07, 145.98, 145.93, 142.22, 138.61,
38.03, 137.30, 131.73, 129.47, 126.95, 122.86, 119.74, 118.51, 116.38, 116.22, 114.49, 114.35,
114.29, 114.14, 110.05, 110.01, 109.83, 109.79, 49.90, 39.28, 18.56. HRMS (ESI):
+
[
M+H] =473.1766, C H F N calcd for 472.1684; retention time 2.38 min, >95% pure.
23 18 2 10
4
2
.1.2.18.
-(4-(1-(1-(5,7-Difluoro-3-(1-methyl-1H-pyrazol-4-yl)quinolin-6-yl)ethyl)-1H-[1,2,3]triazolo[4,5-
1
b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethan-1-ol (19). Yellow solid. Yield: 70%. Mp: 210-212ºC. H
NMR (400 MHz, DMSO-d ) δ 9.30 (d, J=1.6 Hz, 1H), 9.17 (s, 1H), 8.59 (s, 1H), 8.51 (s, 1H),
6
8
.48 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.72 (d, J=12.0 Hz, 1H), 6.78 (q, J=7.2 Hz, 1H), 4.96 (t,
J=5.2 Hz, 1H), 4.22 (t, J=5.2 Hz, 2H), 3.92 (s, 3H), 3.77 (q, J=5.2 Hz, 2H), 2.37 (d, J=7.2 Hz, 3H).
2
8