Discovery of 6-(difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline as a highly potent and selective c-Met inhibitor

Article abstract of DOI:10.1016/j.ejmech.2016.03.076

c-Met/HGF overexpression has been detected in many human malignancies including tumors which are resistant to anticancer therapy. Disrupting the aberrant c-Met/HGF axis has enjoyed significant progress in both preclinical and clinical antitumor campaign. To eliminate the OCH₂-related metabolic deficiency of our previously reported triazolotriazine 2, we synthesized a series of CH₂-/CF₂-linked triazolotriazines and assessed their c-Met activities, leading to the highly potent compound 23 with IC₅₀ values of 0.24 nM of enzymatic activity in c-Met and 0.85 nM of cellular activity in EBC-1 cancer cell line, as well as with complete tumor regression in EBC-1 xenograft mice model at dose of 25 mg/kg via oral administration. Based on its potent anti-proliferative activities and favorable pharmacokinetic properties, 23 has been selected as a drug candidate for preclinical investigation.

Full text of DOI:10.1016/j.ejmech.2016.03.076

Accepted Manuscript
Discovery of 6-(difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-
yl)methyl)quinoline as a highly potent and selective c-met inhibitor
Zhengsheng Zhan, Xia Peng, Qiufeng Liu, Fang Chen, Yinchun Ji, Shanyan Yao,
Yong Xi, Yipeng Lin, Tiantian Chen, Yechun Xu, Jing Ai, Meiyu Geng, Wenhu Duan
PII:
S0223-5234(16)30259-8
10.1016/j.ejmech.2016.03.076
EJMECH 8501
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 January 2016
Revised Date: 22 March 2016
Accepted Date: 25 March 2016
Please cite this article as: Z. Zhan, X. Peng, Q. Liu, F. Chen, Y. Ji, S. Yao, Y. Xi, Y. Lin, T. Chen, Y. Xu,
J. Ai, M. Geng, W. Duan, Discovery of 6-(difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-
yl)methyl)quinoline as a highly potent and selective c-met inhibitor, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.076.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Discovery of
6-(Difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,
2,4]triazin-3-yl)methyl)quinoline as a Highly Potent
and Selective c-Met Inhibitor
Zhengsheng Zhana,§, Xia Pengb,§, Qiufeng Liuc,§, Fang Chena,§, Yinchun Jib, Shanyan Yaoa, Yong
Xib, Yipeng Lina, Tiantian Chenc, Yechun Xuc, *, Jing Aib, *, Meiyu Gengb, *, Wenhu Duana, *
aDepartment of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences (CAS), 555 Zu Chong Zhi Road, Shanghai 201203, China
bDivision of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences (CAS), 555 Zu Chong Zhi Road, Shanghai
201203, China
cCAS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555 Zu Chong Zhi Road,
Shanghai 201203, China
*(W.D.) E-mail: whduan@simm.ac.cn. Phone: +86-21-50806032.
*(M.G.) E-mail: mygeng@simm.ac.cn.
*(J.A.) E-mail: jai@simm.ac.cn.
*(Y.X.) E-mail: ycxu@simm.ac.cn.
§(Z.Z., X.P., Q.L., and F.C.) These authors contributed equally to this work.

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ABSTRACT
c-Met/HGF overexpression has been detected in many human malignancies including tumors
which are resistant to anticancer therapy. Disrupting the aberrant c-Met/HGF axis has enjoyed
significant progress in both preclinical and clinical antitumor campaign. To eliminate the
OCH2-related metabolic deficiency of our previously reported triazolotriazine 2, we synthesized a
series of CH2-/CF2-linked triazolotriazines and assessed their c-Met activities, leading to the
highly potent compound 23 with IC50 values of 0.24 nM of enzymatic activity in c-Met and 0.85
nM of cellular activity in EBC-1 cancer cell line, as well as with complete tumor regression in
EBC-1 xenograft mice model at dose of 25 mg/kg via oral administration. Based on its potent
anti-proliferative activities and favorable pharmacokinetic properties, 23 has been selected as a
drug candidate for preclinical investigation.
1. INTRODUCTION
The receptor tyrosine kinase c-Met, also known as hepatocyte growth factor receptor (HGFR),
is a disulfide-linked transmembrane heterodimer composed of a short extracellular α chain and a
membrane spanning β chain.[1, 2] Physiologically, HGF/c-Met axis mediates a diverse array of
biological processes such as branching morphogenesis, migration, motility, proliferation, survival,
wound healing, and angiogenesis, during embryonic development and tissue repair.[3-5] Aberrant
c-Met signaling activation due to gene amplification, rearrangement, point mutation, as well as
autocrine or paracrine HGF stimulation has been involved in the progression of many types of
human malignancies.[6-12] Importantly, overexpression of HGF and/or c-Met has been associated
with poor prognosis or metastatic progression. In addition, HGF/c-Met over-activation engaged in
mediating intrinsic or acquired resistance to targeted therapies. For example, MET amplification
correlates with approximately 20% of NSCLC patients developing acquired resistance against
EGFR inhibitors.[13, 14] Thus, c-Met axis has emerged as a promising target for therapeutic
medication of cancer.
Recently, HGF/c-Met biological antagonists, antibodies against c-Met or HGF, and
small-molecule c-Met inhibitors have been employed as the strategies to inhibit the abnormal
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c-Met/HGF axis for clinical anticancer campaign.[15-25] Small-molecule c-Met inhibitors can be
roughly divided into ATP-competitive and non-ATP-competitive inhibitors.[26] Most of the c-Met
inhibitors in clinical development are ATP-competitive. ATP-competitive c-Met inhibitors of the
first generation usually target other kinases, for example, BMS-777607 is simultaneously active
against c-Met and other receptor tyrosine kinases (AXL and Ron),[27] and XL184 inhibits
VEGFR-2 besides c-Met.[28, 29] While the inhibitors of the second generation all share a
relatively conserved structure which bind to the ATP binding pocket in a “U” shape, displaying
high selectivity,[30, 31] representatively, JNJ38877605,[32] AMG337,[33] and INCB28060[34]
are selective c-Met inhibitors (Figure 1). During the development of selective c-Met inhibitors, the
OCH2-linked molecule 1 (Figure 2) suffered O-dealkylation metabolism.[33] Similar problem
emerged in our previously reported O-linked triazolotriazine 2,[35] this compound possessed
potent in vitro c-Met activity, but showed poor in vivo antitumor activities in the c-Met-driven
xenograft models through oral administration (data not shown). The previous approach to
eliminate such a liability was to replace the OCH2 linker with NHCH2 (1a) or CHCH3 (1b),
leading to a significant improvement in the metabolic properties (Figure 2).[33, 36] Here, we
introduced a more robust carbon linkage, such as CH2 or CF2 to eliminate the metabolic deficiency
(Figure 2), resulting in the identification of the highly potent and selective c-Met inhibitor
6-(difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline as an
antitumor agent for preclinical study.
<Figure 1>
<Figure 2>
2. RESULTS AND DISCUSSION
2.1. CH2- /CF2-Linked Triazolotriazines
To improve the metabolic liability of compound 2, we designed and synthesized a series of
CH2-/CF2-linked triazolotriazines. The enzymatic and cellular activities of the CH2-linked
triazolotriazines were summarized in Table 1. Compared with the lead compound 2, compounds
3−21 exhibited decreased c-Met enzymatic potency. Among them, analogues 3, 9, 10, and 14
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outperformed their counterparts with c-Met IC50 values ranging from 0.42 nM to 0.59 nM; the
indole derivatives 19, 20, and 21 displayed a significant loss in both enzymatic and cellular
activities, suggesting that the quinoline segment was essential to maintaining good c-Met potency.
Given that CH2 linker might also suffer from metabolic instability[37], we further replaced
the CH2 linker with a CF2 linker. To this end, the CF2 linkage version of four CH2-linked
compounds (3, 12, 14, and 16) with excellent enzymatic and cellular potency were prepared. As
shown in Table 2, the CF2-linked analogues 24, 25, and 27 maintained the enzymatic potency
compared with their CH2-linked analogues 12, 14, and 16, respectively. The 6-phenyl CF2-linked
compound 22 displayed a six-fold drop in potency compared to its counterpart 3; to our delight,
when replacement of this phenyl with 4-fluorophenyl in the structure of 22, the resulting analogue
23 displayed a nearly twelve-fold increase in enzymatic potency compared to 22 and was
equipotent to the lead compound 2. To explore the isosteric effect, furan analogue 26 was
synthesized, and it was three-fold more potent than the thiophene analogue 24 in c-Met enzyme.
Given these enzymatic data, the CF2-linked analogues with excellent enzymatic activity (23, 25,
26, and 27) were assayed in c-Met-addicted EBC-1 cell line, and they displayed sub-nanomolar to
nanomolar IC50 values. Therefore, compounds 23, 25, 26, and 27 were chosen for further
evaluation.
<Table 1>
<Table 2>
2.2. Selection of Drug Candidate
The highly potent compounds 23, 25, 26, and 27 were assessed for their pharmacokinetic (PK)
profiles. As shown in Table 3, the PK data of 23, 25, 26, and 27 were obtained in rats after single
iv (2 mg/kg), and po (10 mg/kg) administration (iv dose of 25 was increased to 10 mg/kg).
Different from their similarity of in vitro cellular activity, compound 25 showed low
bioavailability (9.4%) and AUC0−∞. To identify the candidate for further profiling, compounds 23,
26, and 27 with both potent target inhibition and optimal PK properties were evaluated in the
EBC-1 xenograft model. Mice bearing EBC-1 cancer cells were administrated the selected
compounds orally twice daily for 21 days at the indicated doses. Body weight of the mice and
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volume of the tumor were measured twice a week. As summarized in Table 4, 23 and 27 showed
significant inhibition on tumor growth, and tumor regression was observed at the dose of 25
mg/kg, while 26 showed much weaker tumor growth inhibition (TGI = 65.8% @ 25 mg/kg). Of
note, a body weight increase was observed at both doses of 23-treated groups whereas weight loss
emerged in 27-treated (25 mg/kg) group, indicating low toxicity of compound 23 (data not shown).
Based on the above results, compound 23 stood out as a new orally-available c-Met inhibitor with
high in vitro and in vivo activities. Therefore, compound 23 was elected for further evaluation.
<Table 3>
<Table 4>
2.3. X-ray Co-Crystal Structure of Compound 23 with c-Met kinase domain
In order to elucidate the structure based activity of 23 to c-Met, we determined the co-crystal
structure of the c-Met kinase domain in complex with 23. In general, 23 bound into the ATP
binding pocket of c-Met with a “U shape” and the kinase domain adopted a “DFG-in” inactive
conformation (Figure 3). The detailed structure analysis revealed that the quinoline moiety of the
compound occupied the adenosine binding site by forming a canonical hydrogen bond (H-bond)
with the main-chain NH of Met1160 at the hinge region (Figure 3B). Replacement of the
quinoline segment with an indole core would disrupt such a H-bonding interaction, which may
explain the significant potency loss of 19, 20, 21 which all contain the indole core instead of the
quinolone in 23 (Table 1). In addition, the triazolotriazine ring, the other essential part of 23,
formed the π-π stacking interactions with Tyr1230 at the activation loop and meanwhile a H-bond
was formed between N1 of triazole and the main-chain NH of Asp1222 at the DFG motif. The
substituted p-fluoryl phenyl group on the triazolotriazine core of 23 further stabilized the π-π
stacking interactions (Figure 3B), contributing to the 10-fold biochemical potency increment
against c-Met compared to 22 (Table 2). Therefore, the complex structure revealed the binding
mode of 23 with the c-Met kinase domain and helped to explore the SARs of triazolotriazines as
inhibitors of c-Met.
<Figure 3>
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2.4. Compound 23 was a Potent and Highly Selective c-Met Inhibitor
Compound 23 was distinguished for its remarkable potency against c-Met kinase in vitro and
in vivo. To determinate the kinases selectivity, we assayed its enzymatic activity in 23 additional
kinases. As summarized in Table 5, compound 23 barely inhibited a panel of kinases, including
c-Met family member Ron and highly homologous kinase Axl, Tyro3, c-Mer (IC50 >1 ꢀM),
indicating that 23 was a selective c-Met inhibitor.
<Table 5>
2.5. Compound 23 Inhibited c-Met Phosphorylation and Its Downstream Signaling Pathways
To further assess the kinase activity of 23 in cellular level, we measured its effect on the
phosphorylation of c-Met and its downstream signaling molecules in the representative cancer cell
or model cells (EBC-1, MKN45, BaF3/ TPR-Met, and U87MG cells) that cover the frequently
occurring oncogenic forms of c-Met, including MET amplification, MET chromosomal
rearrangement (TPR-MET), and HGF stimulation.[38, 39] Among these cells, EBC-1 and MKN45
cells harbor an amplified MET gene, and BaF3/TPR-Met cells stably express a constitutively
active c-Met resulting from a chromosomal rearrangement, U87MG cells respond well to HGF
stimulation. As shown in Figure 4, 23 significantly inhibited the phosphorylation of c-Met and its
downstream signaling molecules (Akt and Erk)[38, 39] with a complete abolishment at 10 nM in
each of these cell lines. These results suggested that 23 effectively suppressed c-Met activation
and its signaling, regardless of the mechanistic complexity in c-Met activation across different
cellular contexts.
<Figure 4>
2.6. Compound 23 Significantly Inhibited c-Met-addicted Proliferation of Human Cancer Cells
Activated c-Met is known to trigger cancer cell proliferation.[5] Therefore, we assessed the
effect of 23 on cell proliferation in a broad panel of human cancer cell lines harboring different
backgrounds of c-Met expression/activation. Compound 23 significantly inhibited the proliferation
of the c-Met-constitutively activated EBC-1, and MKN-45 cells, with IC50 values of 0.85 nM and
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0.46 nM, respectively (Table 6). Moreover, 23 effectively inhibited the proliferation of the
genetically engineered BaF3/TPR-Met cells, which featured c-Met-dependent cell growth. In
contrast, 23 showed over 10000-fold less potency in cells with low c-Met expression or activation
(Figure 5). These data indicated that 23 specifically inhibited c-Met-dependent cancer cell
proliferation.
<Figure 5>
<Table 6>
2.7. Compound 23 Inhibited c-Met-addicted Cell Migration, Invasion, and Scattering
In addition to the modulation of cell proliferation, activation of the HGF/c-Met axis promotes
cell migration and invasion, which contributes to the metastatic characteristics of malignant
cells.[40] Thus the impact of 23 in this regard was evaluated by Transwell-based migration and
invasion assays of HGF-treated NCI-H441 cells. The results showed that 23 strongly suppressed
HGF-induced cell migration and invasion in a dose-dependent manner (Figure 6A, B).
Activated HGF/c-Met signaling, also known to promote the cell scattering that stimulates
cells to abandon their original environment, is a hallmark of cancer invasiveness and
metastasis.[41] Since HGF stimulation could result in disruption and scattering of the MDCK cell
colonies, we assayed the effect of 23 on cell scattering behavior using HGF-stimulated MDCK
cells. As shown in Figure 6C, compound 23 inhibited the HGF-induced cell scattering of MDCK
cells in a dose-dependent manner, completely blocking cell spreading at the dose of 10 nM.
Together, the above data suggested that 23 significantly impaired the cell motility and
invasiveness mediated by the HGF/c-Met axis.
2.8. Compound 23 Suppressed c-Met-mediated Invasive Growth
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c-Met activation in epithelial cells is known to induce a series of biological responses,
including migration, matrix degradation, cell multiplication, and survival, which collectively gives
rise to a unique multistep program known as invasive growth.[42] In vitro, this morphogenetic
program was recapitulated by stimulating the suspended MDCK epithelial cells that cultured in a
three-dimensional extracellular matrix (collagen) with HGF.[43, 44] We evaluated the inhibitory
efficacy of 23 in this phenotype. As expected, MDCK cells in the absence of HGF displayed the
round cysts, whereas HGF-stimulated MDCK cells formed the multicellular-branched structures.
The branching morphogenesis of HGF-stimulated MDCK cells was obviously inhibited by
compound 23 (Figure 6D), indicating that 23 suppressed the c-Met-mediated invasive growth
phenotype.
<Figure 6>
2.9. In vivo c-Met-driven Tumor Growth Inhibition Studies of Compound 23
To further determinate the in vivo antitumor features of compound 23, we assessed it in
MKN45 and EBC-1 xenograft models. As shown in Figure 7, 23 displayed significant antitumor
activity in a dose-dependent manner in both tumor xenografts. For the MKN45 model, treatment
with 23 at 6.25, 12.5, and 25 mg/kg resulted in significant tumor growth inhibition, with an
inhibitory rate of 46.8%, 80.1%, and 96.5%, respectively (Figure 7A). Similar results were
observed in the EBC-1 xenograft (Figure 7B). Moreover, partial or complete tumor regression was
observed in the EBC-1 model at the dose of 25 mg/kg. At all doses tested, 23 was well tolerated
with no observatory weight loss (data not shown).
<Figure 7>
3. CHEMISTRY
The preparation of 3−27 is summarized in Scheme 1. Intermediates 52−68, prepared
according to the literature method,[35] were condensed with a carboxylic acid under the
participation
of
the
coupling
reagents
hydroxybenzotriazole
(HOBT)
and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) at ambient temperature to
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give 28−51. Intermediates 28−51 were cyclized to the triazolotriazines in acetic acid under reflux.
However, only 5% yield of the desired product 23 was obtained in acetic acid under reflux, instead
85% yield of byproduct 23a was formed. Therefore, the reaction condition should be optimized to
improve the yield.
Scheme 2 summarizes the reaction conditions we investigated. We first employed pivalic acid
in place of acetic acid in consideration that the steric effect of tert-butyl could inhibit the
generation of the byproduct, but this reaction gave a low yield of 23 (32%) and a medium yield of
byproduct 23b (50%). Then using methanesulfonic acid as the cyclizing agent, 23 was obtained in
45% yield accompanying with 50% yield of intermediate 66 which was the decomposed product
of 47. When polyphosphoric acid (PPA) was used, the reaction improved slightly, and the yield of
23 increased to 55%, together with 40% yield of defluorinated byproduct 23c.
Since the reaction generates one molecule of water during the formation of the
triazolotriazine, we envisioned that the water generated in situ was the culprit of formation of 23c
which would be produced from 23 in presence of water, and such assumption was corroborated by
o
preparing 23c from 23 by adding small amount of water into PPA at the temperature of 140 C
(presented in Supporting Information). Thus 20% phosphorus pentoxide (w/w) was added to the
reaction mixture to remove the generated water, and 23 was obtained in good yield (77%)
accompanying with low yield of 23c (20%).
High reaction temperature might also be the cause for defluorination of 23, however when
o
o
the reaction temperature was lowered from 140 C to 90 C, the reaction mixture became a
gummy complex and could hardly be stirred. To decrease the high viscidity of the reaction mixture,
DMF was added to the reaction mixture and the yield of 23 increased to 85%. To our delight,
o
when the temperature further reduced to 65 C, the yield of 23 increased to 90%, with only 1.5%
yield of 23c. Using the optimized condition, we prepared 10.5 kg of 23 for preclinical study.
<Scheme 1>
<Scheme 2>
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4. CONCLUSION
A series of CH2-/CF2-linked triazolotriazines were designed and synthesized to overcome the
metabolic deficiency of the OCH2-linked triazolotriazine 2. Among them, compounds 23, 26, and
27 possessed potent enzymatic and cellular c-Met activities and favorable PK profiles. Compound
23 outperformed its counterparts due to good anti-proliferative potency on EBC-1 xenograft
model and low toxicity. Further evaluation unraveled that 23 specifically inhibited c-Met kinase
activity, impairing c-Met phosphorylation and the downstream signaling across different
oncogenic forms in c-Met overactivated cancer cells and model cells. Besides, 23-treated
inhibition was observed in c-Met driven cellular phenotype. Furthermore, 23 displayed significant
antitumor activities in c-Met-driven EBC-1 and MKN45 xenografts. Given its excellent antitumor
activities and favorable pharmacokinetic properties, 23 has been selected as an anticancer drug
candidate for further development. We thereby optimized the process of preparation of 23 and
prepared a sufficient amount of 23 for a preclinical study.
5. EXPERIMENTAL SECTION
5.1. General Methods for Chemistry
All reagents and solvents were purchased from commercial sources and used as received. 1H
NMR and 13C NMR were generated in DMSO-d6, CDCl3, or CD3OD on Varian Mercury 300, 400
or 500 NMR spectrometers. EI (Low-resolution and high-resolution mass spectra) were recorded
on a Finnigan/MAT95 spectrometer. ESI (high-resolution mass spectra) were tested on a Waters
Q-Tof Ultima apparatus. All melting points were determined on a Büchi B-510 melting point
apparatus and are uncorrected. HPLC conditions were as follows: column, Agilent Eclipse Plus
C18 3.5 ꢀM, 4.6 mm×150 mm; solvent system, acetonitrile /water; flow rate 1.0 ml/min; UV
detection, 254 nm; injection volume, 5 ꢀL; temperature, 35 °C. All the assayed compounds
displayed a purity of >95% as confirmed by HPLC.
General Procedure for Preparation of Compounds 3−27
5.1.1. 6-((6-Phenyl-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (3) A mixture of
o
28 (500 mg, 1.403 mmol) and acetic acid (15 mL) was heated at 100 C for 4 h. The reaction
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mixture was then concentrated, and the residue was purified by silica gel chromatography, eluting
with CH2Cl2/MeOH (20:1) to yield 3 as a white solid (385 mg, 81%), mp 224−226 °C. 1H NMR
(300 MHz, DMSO-d6) δ 9.31 (s, 1H), 8.87 (dd, J = 1.8, 5.2 Hz, 1H), 8.33 (dd, J = 1.2, 8.7 Hz, 1H),
8.16−8.19 (m, 2H), 7.98−8.01 (m, 2H), 7.85 (dd, J = 2.4, 8.7 Hz, 1H), 7.61−7.64 (m, 3H), 7.53 (q,
J = 5.2, 8.7 Hz, 1H), 4.80 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 150.9, 148.3, 148.0, 147.9,
147.8, 147.3, 136.2, 134.4, 132.2, 132.1, 131.5, 129.8 (2C), 129.6, 128.3, 128.0 (3C), 122.2, 30.1.
HRMS (ESI) calcd [M + H]+ for C20H15N6 339.1353, found 339.1362.
5.1.2. 6-((6-(4-(Benzyloxy)phenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (4)
The title compound was prepared from 29 using a method analogous to the synthesis of compound
3. White solid (78%), mp 241−243 °C. 1H NMR (300MHz, CDCl3) δ 8.95 (s, 1H), 8.89 (d, J = 4.8
Hz, 1H), 8.11 (t, J = 7.8 Hz, 2H), 7.82−7.93 (m, 4H), 7.37−7.49 (m, 6H), 7.16 (d, J = 8.7 Hz, 2H),
5.18 (s, 2H), 4.80 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 161.6, 150.9, 148.1, 147.9, 147.6,
147.5, 147.3, 137.0 (2C), 136.2, 134.5, 131.4, 129.7 (2C), 129.6, 129.0 (2C), 128.5, 128.3 (2C),
128.0, 124.5, 122.2, 116.1 (2C), 70.0, 30.1. HRMS (ESI) calcd [M + H]+ for C27H21N6O 445.1771,
found 445.1783.
5.1.3. 6-((6-(3,4-Dichlorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (5)
The title compound was prepared from 30 using a method analogous to the synthesis of compound
1
3. White solid (69%), mp >250 °C. H NMR (300MHz, DMSO-d6) δ 9.39 (s, 1H), 8.87 (dd, J =
1.8, 4.2 Hz, 1H), 8.41 (d, J = 2.1 Hz, 1H), 8.33 (d, J = 7.2 Hz, 1H), 8.18 (dd, J = 2.1, 8.4 Hz, 1H),
7.97−8.00 (m, 2H), 7.93 (d, J = 8.4 Hz, 1H), 7.83 (dd, J = 1.8, 8.7 Hz, 1H), 7.53 (dd, J = 2.1, 8.4
Hz, 1H), 4.81 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 150.9, 148.0, 147.9, 147.8, 147.3, 146.2,
136.2, 134.9, 134.3, 132.8, 132.7, 132.0, 131.5, 129.9, 129.6, 128.3, 128.1, 128.0, 122.2, 30.1.
HRMS (ESI) calcd [M + Na]+ for C20H12Cl2N6Na 429.0393, found 429.0399.
5.1.4. 6-((6-(3-Nitrophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (6) The
title compound was prepared from 31 using a method analogous to the synthesis of compound 3.
White solid (88%), mp 225−227 °C. 1H NMR (300MHz, DMSO-d6) δ 9.46 (s, 1H), 8.47−8.91 (m,
2H), 8.61 (d, J = 8.1 Hz, 1H), 8.48 (dd, J = 1.5, 4.8 Hz, 1H), 8.34 (d, J = 8.1 Hz, 1H), 7.81−8.01
(m, 4H), 7.53 (dd, J = 3.9, 8.4 Hz, 1H), 4.83 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 151.0,
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149.4, 148.3, 148.0, 147.9, 146.8, 147.5, 136.2, 134.7, 134.4, 133.3, 131.4, 129.6, 128.5, 128.2,
128.1, 127.6, 123.0, 122.1, 30.1. HRMS (EI) calcd M+ for C20H13N7O2 383.1131, found 383.1138.
5.1.5. 6-((6-(4-Chlorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (7) The
title compound was prepared from 32 using a method analogous to the synthesis of compound 3.
White solid (79%), mp >250 °C. 1H NMR (300MHz, CDCl3) δ 8.94 (s, 1H), 8.88 (dd, J = 1.2, 3.9
Hz, 1H), 8.05−8.11 (m, 2H), 7.79−7.90 (m, 4H), 7.56 (d, J = 6.9 Hz, 2H), 7.41 (dd, J = 4.2, 8.4 Hz,
1H), 4.81 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 150.9, 148.1, 147.9, 147.8, 147.3, 147.2,
137.1, 136.2, 134.4, 131.5, 131.1, 129.9 (2C), 129.8 (2C), 129.6, 128.3, 128.1, 122.2, 30.1. HRMS
(ESI) calcd [M + H]+ for C20H14ClN6 373.0963, found 373.0962.
5.1.6. 6-((6-(4-Bromophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (8) The
title compound was prepared from 33 using a method analogous to the synthesis of compound 3.
Yellow solid (79%), mp >250 °C. 1H NMR (300MHz, DMSO-d6) δ 9.37 (s, 1H), 8.87 (d, J = 3.9
Hz, 1H), 8.34 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 7.2 Hz, 2H), 7.96−8.01 (m, 2H), 7.80−7.86 (m, 3H),
7.53 (q, J = 3.9, 7.2 Hz, 1H), 4.79 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 150.9, 148.0, 147.9,
147.8, 147.3, 147.2, 136.2, 134.4, 132.8 (2C), 131.5, 131.4, 130.0 (2C), 129.6, 128.3, 128.0, 126.0,
122.2, 30.1. HRMS (ESI) calcd [M + H]+ for C20H14BrN6 417.0458, found 417.0459.
5.1.7. 6-((6-(3-Methoxyphenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (9)
The title compound was prepared from 34 using a method analogous to the synthesis of compound
3. Pale yellow solid (82%), mp >250 °C. 1H NMR (300MHz, DMSO-d6) δ 9.23 (s, 1H), 8.47−8.91
(m, 2H), 8.67 (d, J = 8.1 Hz, 1H), 8.45 (dd, J = 1.5, 4.8 Hz, 1H), 8.31 (d, J = 8.1 Hz, 1H),
7.81−8.01 (m, 4H), 7.63 (dd, J = 3.9, 8.4 Hz, 1H), 4.83 (s, 2H), 3.88 (s, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 160.3, 150.9, 148.3, 148.0, 147.8 (2C), 147.3, 136.2, 134.4, 133.4, 131.4, 131.0,
129.6, 128.3, 128.0, 122.2, 120.4, 117.9, 113.1, 55.9, 30.2. HRMS (ESI) calcd [M + H]+ for
C21H17N6O 369.1458, found 369.1465.
5.1.8. 3-(3-(Quinolin-6-ylmethyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-6-yl)phenol (10) The title
compound was prepared from 35 using a method analogous to the synthesis of compound 3.
1
Yellow solid (63%), mp >250 °C. H NMR (300MHz, DMSO-d6) δ 10.55 (s, 1H), 9.24 (s, 1H),
8.47−8.91 (m, 2H), 8.56 (d, J = 8.1 Hz, 1H), 8.45 (dd, J = 1.5, 4.8 Hz, 1H), 8.21 (d, J = 8.1 Hz,
12

ACCEPTED MANUSCRIPT
1H), 7.81−8.01 (m, 4H), 7.63 (dd, J = 3.9, 8.4 Hz, 1H), 4.79 (s, 2H); 13C NMR (150 MHz,
DMSO-d6) δ 158.5, 150.9, 148.2, 148.0, 147.9, 147.7, 147.3, 136.2, 134.4, 133.3, 131.4, 130.9,
129.6, 128.3, 128.0, 122.1, 119.2, 119.0, 114.3, 30.1. HRMS (ESI) calcd [M + H]+ for C20H15N6O
355.1302, found 355.1304.
5.1.9. 6-((6-(Quinolin-3-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (11) The
title compound was prepared from 36 using a method analogous to the synthesis of compound 3.
1
White solid (82%), mp 219−221 °C. H NMR (400MHz, DMSO-d6) δ 9.54 (d, J = 2.1 Hz, 1H),
9.40 (s, 1H), 9.03 (d, J = 1.5 Hz, 1H), 8.83 (d, J = 4.2 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.21 (d, J
= 8.1 Hz, 1H), 7.88−8.11 (m, 5H), 7.79 (t, J = 7.8 Hz, 1H), 7.53 (q, J = 4.8, 8.7 Hz, 1H), 4.92 (s,
2H); 13C NMR (125 MHz, CF3COOD) δ 154.2, 148.5, 148.4, 148.2, 147.5, 144.0, 143.7, 142.6,
139.2, 138.1, 137.0, 134.9, 132.4, 130.5, 129.8, 129.5, 128.8, 123.8, 122.0, 121.0, 120.3, 115.6,
29.1. HRMS (ESI) calcd [M + H]+ for C23H16N7 390.1462, found 390.1463.
5.1.10. 6-((6-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (12)
The title compound was prepared from 37 using a method analogous to the synthesis of compound
3. White solid (85%), mp 224−226 °C. 1H NMR (300MHz, DMSO-d6) δ 10.39 (s, 1H), 9.70 (br s,
1H), 9.00 (s, 1H), 8.87 (d, J = 4.2 Hz, 1H), 8.34 (d, J = 8.1 Hz, 1H), 7.91−7.99 (m, 2H), 7.65−7.79
(m, 3H), 7.54 (dd, J = 4.5, 8.4 Hz, 1H), 7.21 (dd, J = 0.9, 5.4 Hz, 1H); 13C NMR (150 MHz,
DMSO-d6) δ 150.8, 148.4, 147.7, 147.5, 147.2, 136.2, 134.6, 134.5, 134.0, 132.1, 131.4, 129.5,
128.3, 128.0, 124.0, 122.1, 115.4, 30.1. HRMS (EI) calcd M+ for C18H12N6S 344.0844, found
344.0853.
5.1.11.
6-((6-(1-Benzyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (13)
The title compound was prepared from 38 using a method analogous to the synthesis of compound
3. White solid (83%), mp 237−239 °C. 1H NMR (300MHz, CDCl3) δ 8.87−8.85 (m, 1H), 8.68 (s,
1H), 8.11 (s, 1H), 8.07−8.02 (m, 3H), 7.81 (s, 1H), 7.78−7.74 (m, 1H), 7.40−7.28 (m, 6H), 5.40 (s,
2H), 4.73 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 150.8, 148.3, 147.8, 147.4, 147.3, 143.8,
139.1, 137.2 (2C), 136.2, 134.4, 131.7, 131.5, 129.6, 129.1 (2C), 128.4, 128.2 (2C), 128.1, 122.1,
115.9, 55.8, 30.1. HRMS (ESI) calcd [M + H]+ for C24H19N8 419.1727, found 419.1734.
13

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5.1.12.
6-((6-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline
(14) The title compound was prepared from 39 using a method analogous to the synthesis of
1
compound 3. White solid (81%), mp >250 °C. H NMR (300MHz, DMSO-d6) δ 9.11 (s, 1H),
8.85−8.86 (m, 1H), 8.65 (s, 1H), 8.35 (d, J = 6.9 Hz, 1H), 8.26 (s, 1H), 7.98−8.01 (m, 2H), 7.83 (d,
J = 8.4 Hz, 1H), 7.49−7.53 (m, 1H), 4.71 (s, 2H), 3.96 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ
150.9, 148.3, 147.9, 147.4, 147.3, 143.9, 138.7, 136.2, 134.5, 132.1, 131.5, 129.6, 128.3, 128.1,
122.2, 115.5, 39.6, 30.1. HRMS (ESI) calcd [M + H]+ for C18H15N8 343.1414, found 343.1417.
5.1.13.
6-((6-(1-Ethyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (15)
The title compound was prepared from 40 using a method analogous to the synthesis of compound
1
3. White solid (88%), mp >250 °C. H NMR (300MHz, CDCl3) δ 8.88 (dd, J = 1.5, 4.5 Hz, 1H),
8.72 (s, 1H), 8.04−8.11 (m, 4H), 7.78−7.83 (m, 2H), 7.40 (q, J = 4.2, 8.1 Hz, 1H), 4.76 (s, 2H),
4.25−4.33 (m, 2H), 1.60 (t, J = 6.9 Hz, 3H); 13C NMR (150 MHz, DMSO-d6) δ 150.9, 148.3,
147.9, 147.4, 147.3, 143.9, 138.7, 136.2, 134.5, 131.8, 131.5, 129.6, 128.3, 128.1, 122.2, 115.3,
45.3, 30.1, 15.1. HRMS (EI) calcd M+ for C19H16N8 356.1498, found 356.1501.
5.1.14.
6-((6-(1-Propyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (16)
The title compound was prepared from 41 using a method analogous to the synthesis of compound
3. White solid (66%), mp 120−121 °C. 1H NMR (300MHz, DMSO-d6) δ 10.96 (br s, 1H), 9.08 (s,
1H), 8.69 (s, 1H), 8.28 (s, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 8.4 Hz, 2H), 6.99−7.09 (m,
2H), 4.57 (s, 2H), 4.18 (t, J = 6.9 Hz, 2H), 1.28−1.89 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6) δ 150.8, 148.3, 147.8, 147.4, 147.3, 143.9, 138.6, 136.2, 134.5, 131.5,
131.4, 129.6, 128.3, 128.1, 122.1, 115.2, 53.9, 30.1, 23.5, 11.3. HRMS (ESI) calcd [M + H]+ for
C20H19N8 371.1727, found 371.1734.
5.1.15.
Tert-butyl
4-(4-(3-(quinolin-6-ylmethyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-6-yl)-1H-pyrazol-1-yl)piperi
dine-1-carboxylate (17) The title compound was prepared from 42 using a method analogous to
14

ACCEPTED MANUSCRIPT
1
the synthesis of compound 3. White solid (14%), mp 112−114 °C. H NMR (300MHz, CDCl3) δ
8.89 (d, J = 3.9 Hz, 1H), 8.72 (s, 1H), 8.05−8.12 (m, 4H), 7.79−7.83 (m, 2H), 7.42 (q, J = 3.9, 7.8
Hz, 1H), 4.77 (s, 2H), 4.32−4.37 (m, 3H), 2.89−2.97 (m, 2H), 2.12−2.21 (m, 2H), 1.94−2.04 (m,
2H), 1.49 (s, 9H); 13C NMR (150 MHz, DMSO-d6) δ 154.3, 150.9, 148.4, 147.8, 147.4, 147.3,
143.9, 138.4, 136.2, 134.5, 131.5, 129.8, 129.6, 128.3, 128.1, 122.1, 115.3, 79.4, 59.2, 32.3 (2C),
30.1 (3C), 28.6 (3C). HRMS (ESI) calcd [M + H]+ for C27H30N9O2 512.2517, found 512.2531.
5.1.16.
6-((6-(1-(Piperidin-4-yl)-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)qui
noline (18) A mixture of 17 (350 mg, 0.648 mmol), CF3COOH (0.5 mL, 6.842 mmol), and DCM
(25 mL) was stirred at room temperature for 5 h. The reaction mixture was then concentrated, and
the residue was purified by silica gel chromatography, eluting with CH2Cl2/MeOH (10:1) to yield
1
18 as a white solid (59 mg, 21%), mp 125−127 °C. H NMR (300 MHz, CDCl3) δ 9.12 (s, 1H),
8.85−8.86 (m, 1H), 8.78 (s, 1H), 8.32−8.35 (m, 1H), 8.28 (s, 1H), 7.97−7.99 (m, 2H), 7.79−7.83
(m, 1H), 7.53 (q, J = 3.9, 7.8 Hz, 1H), 4.71 (s, 2H), 4.45−4.58 (m, 2H), 3.92−3.97 (m, 1H),
3.12−3.31 (m, 3H), 2.70−2.75 (m, 1H), 2.05−2.14 (m, 1H), 1.75−1.94 (m, 2H); 13C NMR (150
MHz, DMSO-d6) δ 150.9, 148.4, 147.8, 147.4, 147.3, 143.9, 138.4, 136.2, 134.5, 131.5, 129.8,
129.6, 128.3, 128.1, 122.1, 115.3, 59.2, 44.9 (2C), 32.9, 32.1, 30.1. HRMS (ESI) calcd [M + H]+
for C22H22N9 412.1993, found 412.2002.
5.1.17. 3-((1H-indol-3-yl)methyl)-6-(4-(benzyloxy)phenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazine
(19) The title compound was prepared from 43 using a method analogous to the synthesis of
1
compound 3. White solid (69%), mp 241−243 °C. H NMR (300MHz, DMSO-d6) δ 10.95 (br s,
1H), 9.29 (s, 1H), 8.15 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.32−7.50 (m, 7H), 7.24 (d, J
= 9.0 Hz, 2H), 6.96−7.09 (m, 2H), 5.25 (s, 2H), 4.62 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ
161.5, 148.3, 147.7, 147.6, 147.3, 137.0, 136.6, 129.7 (2C), 129.0 (2C), 128.5, 128.3 (2C), 127.4,
124.6, 121.6, 119.1, 119.0, 116.1 (2C), 111.9, 108.4 (2C), 70.0, 20.7. HRMS (ESI) calcd [M + H]+
for C26H21N6O 433.1771, found 433.1773.
5.1.18. 3-((1H-indol-3-yl)methyl)-6-(4-bromophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazine (20)
The title compound was prepared from 44 using a method analogous to the synthesis of compound
15

ACCEPTED MANUSCRIPT
3. White solid (49%), mp 241−243 °C. 1H NMR (300MHz, DMSO-d6) δ 10.96 (br s, 1H), 9.32 (s,
1H), 8.14 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.32−7.34 (m,
2H), 6.97−7.10 (m, 2H), 4.64 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 148.5, 147.7, 147.6,
147.0, 136.6, 132.8 (2C), 131.5, 130.0 (2C), 127.3, 125.9, 124.6, 121.6, 119.0 (2C), 111.9, 108.3,
20.7. HRMS (ESI) calcd [M + H]+ for C19H14BrN6 405.0458, found 405.0463.
5.1.19. 3-(3-((1H-indol-3-yl)methyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-6-yl)phenol (21) The
title compound was prepared from 45 using a method analogous to the synthesis of compound 3.
1
White solid (56%), mp 214−216 °C. H NMR (300MHz, DMSO-d6) δ 10.95 (br s, 1H), 9.92 (s,
1H), 9.26 (s, 1H), 7.57−7.69 (m, 3H), 7.44 (t, J = 7.8 Hz, 1H), 7.33−7.35 (m, 2H), 6.96−7.09 (m,
3H), 4.63 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 158.5, 148.3, 147.9, 147.8, 147.7, 136.6,
133.4, 130.9, 127.3, 124.5, 121.7, 119.2, 119.1, 119.0 (2C), 114.2, 111.9, 108.4, 20.7. HRMS (ESI)
calcd [M + H]+ for C19H15N6O 343.1302, found 343.1303.
5.1.20. 6-(Difluoro(6-phenyl-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (22)
The title compound was prepared from 46 using a method analogous to the synthesis of compound
3. White solid (39%), mp 164−166 °C. 1H NMR (300MHz, CDCl3) δ 9.14 (s, 1H), 9.03 (d, J = 2.4
Hz, 1H), 8.25−8.33 (m, 3H), 8.00−8.10 (m, 3H), 7.60−7.67 (m, 3H), 7.54 (dd, J = 3.9, 8.7 Hz, 1H);
13C NMR (150 MHz, DMSO-d6) δ 153.1, 150.2, 149.3, 149.0, 148.8, 143.0 (t, J = 36.3 Hz), 137.6,
132.4, 131.6, 131.5 (t, J = 24.9 Hz), 130.4, 129.9 (2C), 128.1 (2C), 127.6, 127.4 (t, J = 5.6 Hz),
126.7, 123.0, 117.7 (t, J = 239.4 Hz). HRMS (ESI) calcd [M + H]+ for C20H13F2N6 375.1164,
found 375.1168.
5.1.21.
6-(Difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline (23)
Method A: the title compound was prepared from 47 using a method analogous to the synthesis of
1
compound 3. Off-white solid (5%), mp 208−210 °C. H NMR (300MHz, DMSO-d6) δ 9.53 (s,
1H), 9.06 (dd, J = 1.8, 3.9 Hz, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.50 (s, 1H), 8.15−8.24 (m, 3H), 8.06
(dd, J = 1.8, 8.1 Hz, 1H), 7.69 (q, J = 3.9, 8.1 Hz, 1H), 7.51 (t, J = 9.0 Hz, 2H); 13C NMR (100
MHz, DMSO-d6) δ 166.0, 163.5, 152.9, 150.1, 149.1 (d, J = 86.8 Hz), 148.5, 143.1 (t, J = 35.6
16

ACCEPTED MANUSCRIPT
Hz), 137.7, 131.6 (t, J = 24.9 Hz), 130.8 (d, J = 12.0 Hz, 2C), 130.1, 128.1 (d, J = 2.8 Hz), 127.5,
127.3 (t, J = 5.2 Hz), 127.2 (t, J = 5.1 Hz), 123.0, 118.4 (t, J = 239.5 Hz), 117.1 (d, J = 22.6 Hz,
2C). HRMS (ESI) calcd [M + H]+ for C20H12F3N6 393.1070, found 393.1074.
Method B: a mixture of 200 g polyphosphoric acid, 40 g phosphorus pentoxide, and 200 mL
o
DMF was mechanically stirred, when the temperature reduced to about 45 C, 47 (20 g, 0.0487
o
mol) was added to the mixture with stirring, the resulting complex was heated at 65 C for 72 h.
The gummy reaction complex was poured into ice with stirring until homogeneous phase formed,
then ammonia water was added dropwise to alkalize the mixture, the brown solid was harvested
by filtration. Then the brown solid was stirred in isopropanol for 3 h, after filtration and dry,
yielded 23 as off-white solid (17.1 g, 90%).
5.1.22. 6-(Difluoro(6-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline
(24) The title compound was prepared from 48 using a method analogous to the synthesis of
1
compound 3. Pale yellow solid (41%), mp 227−229 °C. H NMR (300MHz, CDCl3) δ 8.99−9.01
(m, 2H), 8.23−8.33 (m, 3H), 8.10 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 3.9 Hz, 1H), 7.72 (d, J = 5.4 Hz,
1H), 7.52 (q, J = 4.8, 8.7 Hz, 1H), 7.24−7.27 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ 153.1,
149.4, 149.0, 148.5, 146.3, 143.0 (t, J = 36.0 Hz), 137.8, 134.5, 134.0, 132.1, 131.5 (t, J = 24.6
Hz), 130.1, 127.7, 127.2 (t, J = 5.6 Hz), 126.7, 124.3, 123.1, 117.6 (t, J = 240.0 Hz). HRMS (EI)
calcd M+ for C18H10F2N6S 380.0656, found 380.0660.
5.1.23.
6-(Difluoro(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quin
oline (25) The title compound was prepared from 49 using a method analogous to the synthesis of
1
compound 3. White solid (43%), mp 238−240 °C. H NMR (300MHz, CDCl3) δ 9.02 (d, J = 1.8
Hz, 1H), 8.83 (s, 1H), 8.23−8.26 (m, 3H), 8.02−8.06 (m, 3H), 7.45−7.52 (m, 1H), 4.04 (s, 3H);
13C NMR (125 MHz, DMSO-d6) δ 153.0, 150.3, 149.1, 148.8, 145.0, 142.8 (t, J = 35.8 Hz), 138.9,
137.5, 132.4, 131.7 (t, J = 25.3 Hz), 130.3, 127.6, 127.3 (t, J = 6.3 Hz), 126.7, 123.0, 118.0 (t, J =
241.6 Hz), 115.1, 39.6. HRMS (ESI) calcd [M + H]+ for C18H13F2N8 379.1226, found 379.1230.
5.1.24.
6-(Difluoro(6-(furan-2-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline
(26) The title compound was prepared from 50 using a method analogous to the synthesis of
17

ACCEPTED MANUSCRIPT
1
compound 3. Yellow solid (39%), mp 217−219 °C. H NMR (300MHz, CDCl3) δ 9.08 (s, 1H),
9.02 (d, J = 3.9 Hz, 1H), 8.24−8.29 (m, 3H), 8.08 (d, J = 9.3 Hz, 1H), 7.78 (s, 1H), 7.48−7.52 (m,
1H), 7.39 (d, J = 3.3 Hz, 1H), 6.72 (d, J = 2.1 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 153.0,
149.5, 149.0, 148.7, 148.6, 146.5, 143.1 (t, J = 36.0 Hz), 141.4, 137.8, 131.6 (t, J = 24.8 Hz),
130.1, 127.6, 127.3 (t, J = 5.4 Hz), 126.8, 123.0, 118.0, 117.8 (t, J = 240.0 Hz), 113.8. HRMS (ESI)
calcd [M + H]+ for C18H11F2N6O 365.0957, found 365.0961.
5.1.25.
6-(Difluoro(6-(1-propyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quin
oline (27) The title compound was prepared from 51 using a method analogous to the synthesis of
compound 3. Yellow solid (45%), mp 81−83 °C. 1H NMR (300MHz, CDCl3) δ 9.05 (s, 1H), 8.84
(s, 1H), 8.26 (m, 3H), 8.01−8.11 (m, 3H), 7.51 (m, 1H), 4.21 (t, J = 6.9 Hz, 2H), 1.90−2.00 (m,
2H), 0.98 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 153.0, 150.5, 149.2, 148.7, 145.1,
142.9 (t, J = 36.0 Hz), 138.9, 137.7, 133.0, 131.9 (t, J = 25.3 Hz), 130.3, 127.8, 127.3 (t, J = 6.4
Hz), 126.1, 123.4, 118.1 (t, J = 240.3 Hz), 115.7, 53.9, 23.4, 11.3. HRMS (ESI) calcd [M + H]+ for
C20H17F2N8 407.1539, found 407.1545.
5.2. ELISA Kinase Assay
The effects of compounds on the activities of various tyrosine kinases were determined using
enzyme-linked immunosorbent assays (ELISAs) with purified recombinant proteins. Briefly, 20
ꢀg/mL poly (Glu,Tyr)4:1 (Sigma, St Louis, MO, USA) was pre-coated in 96-well plates as a
substrate. A 50-ꢀL aliquot of 10 ꢀmol/L ATP solution diluted in kinase reaction buffer (50 mmol/L
HEPES [pH 7.4], 50 mmol/L MgCl2, 0.5 mmol/L MnCl2, 0.2 mmol/L Na3VO4, and 1 mmol/L
DTT) was added to each well; 1 ꢀL of various concentrations of compounds diluted in 1% DMSO
(v/v) (Sigma, St Louis, MO, USA) were then added to each reaction well. DMSO (1%, v/v) was
used as the negative control. The kinase reaction was initiated by the addition of purified tyrosine
kinase proteins diluted in 49 ꢀL of kinase reaction buffer. After incubation for 60 min at 37 °C, the
plate was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20
(T-PBS). Anti-phosphotyrosine (PY99) antibody (100 ꢀL; 1:500, diluted in 5 mg/mL BSA T-PBS)
was then added. After a 30-min incubation at 37 °C, the plate was washed three times, and 100 ꢀL
18

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horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000, diluted in 5 mg/mL BSA T-PBS)
was added. The plate was then incubated at 37 °C for 30 min and washed 3 times. A 100-ꢀL
aliquot of a solution containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 mol/L
citrate buffer (pH 5.5) was added. The reaction was terminated by the addition of 50 ꢀL of 2 mol/L
H2SO4 as the color changed, and the plate was analyzed using a multi-well spectrophotometer
(SpectraMAX 190, from Molecular Devices, Palo Alto, CA, USA) at 490 nm. The inhibition rate
(%) was calculated using the following equation: [1 − (A490/A490 control)] × 100%. The IC50 values
were calculated from the inhibition curves in two separate experiments.
5.3. Cell Culture
The human gastric cancer cell lines SNU-1, SNU-5,SNU-16, AGS, MGC-803, BGC-823, and
BGC-7901; the human lung cancer cell lines SPC-A1, SPC-A4, Calu-3, NCI-H441, NCI-H661,
NCI-H226, NCI-H3122, NCI-H596, and NCI-H1299; the human breast cancer cell line
MDA-MB-231; the human colon cancer cell line HCT-116; the human renal cell adenocarcinoma
cell line 786-O; the human epithelioid carcinoma cell line PANC-1 and the Madin-Daby canine
kidney (MDCK) cell line were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). The hepatic carcinoma cell line SMMC-7721 and the human lung cancer
cell lines HCC827,BEL-7404, NCI-H292, NCI-H358, NCI-H460 were obtained from the Institute
of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The human
gastric cell line MKN-45 and the human NSCLC cell line EBC-1 were purchased from Joint
Conference of Restoration Branches (JCRB). The murine pre-B cell line BaF3 was obtained from
ATCC and grown in RPMI 1640 containing 10% fetal bovine serum (FBS) and 10%
WEHI-conditioned medium as a source of murine interleukin 3. The genetical architectures was
generated from transfecting the BaF3 cell line with expression vector containing the TPR-MET
cDNA (Addgene) and subsequent selecting in puromycin (Invitrogen Corporation). All the cell
lines were routinely maintained in media according to the suppliers’ recommendations. Except of
special instructions, cell culture reagents were obtained from Life Technologies, Inc. Cells were
routinely maintained according to recommendations of their suppliers.
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5.4. Western Blot Analysis
U87MG cells were serum-starved for 24 h, treated with the indicated dose of 23 for 1 h at
37 °C, stimulated with HGF (100 ng/mL, PeproTech, Rocky Hill, NJ, USA) for 15 min, and then
lysed in 1×sodium dodecyl sulfate (SDS) sample buffer. EBC-1, MKN-45, and BaF3/TPR-Met
cells were treated with the indicated dose of 23 for 1 h at 37 °C and then lysed in 1×SDS sample
buffer. The cell lysates were subsequently resolved by 10% SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were probed with the appropriate primary antibodies
[c-Met (Santa Cruz, CA, USA), phospho-c-Met, phospho-ERK1/2, phospho-AKT, and AKT (all
from Cell Signaling Technology, Beverly, MA, USA), and GAPDH (KangChen Biotech, Shanghai,
China)] and then with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG. The
immunoreactive proteins were detected using an enhanced chemiluminescence detection reagent
(Thermo Fisher Scientific, Rockford, IL, USA).
5.5. Cell Proliferation Assay
Cells were seeded in 96-well tissue culture plates. On the next day, the cells were exposed to
various concentrations of compounds and further cultured for 72 h. Cell proliferation was then
determined using sulforhodamine B (SRB, from Sigma-Aldrich, St Louis, MO, USA) or the
thiazolyl blue tetrazolium bromide (MTT, from Sigma-Aldrich, St Louis, MO, USA) assay. The
IC50 values were calculated by concentration-response curve fitting using the four-parameter
method. Each reagent and concentration was tested at least in triplicate during each experiment,
and each experiment was performed at least 2 times.
5.6. Cell Migration and Matrigel Invasion Assays
For the migration assays, 1.5 ×105 cells in serum-free media were placed into the upper
chamber of an insert (8 ꢀm pore size; Corning Inc.). For the invasion assays, 1.5 ×105 cells in
serum-free media were placed into the upper chamber of an insert coated with Matrigel (BD
Bioscience). Serum-free medium was added to the lower chamber with or without recombinant
o
human HGF (100 ng/mL). After 24 h of incubation at 37 C , the cells remaining on the upper
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membrane were removed with cotton wool, whereas the cells that had migrated or invaded
through the porous membrane were stained with 0.1% crystal violet for 15 min at room
temperature. Finally, migrated and invasive cells were imaged in 5 different fields of each filter.
Images were obtained using an Olympus BX51 microscope.
5.7. Cell-scatter Assay
MDCK cells (1.5×103 cells per well) were plated in 96-well plates and grown overnight.
Increasing concentrations of 23 and HGF (100 ng/mL) were added to the appropriate wells, and
the plates were incubated at 37 °C and 5% CO2 for 24 h. The cells were fixed with 4%
paraformaldehyde for 15 min at room temperature and then stained with 0.2% crystal violet,
washed with water, and dried. Images were obtained using an Olympus IX51 microscope.
5.8. Cell Branching Morphogenesis Assay
MDCK cells at a density of 2×104 cells/mL in DMEM medium were mixed with an equal
volume of collagen I solution and plated at 0.1 mL/well in a 96-well culture plate. After incubation
for 60 min at 37 °C and 5% CO2 to allow the collagen to gel, HGF (100 ng/mL) with or without
23 at various concentrations dissolved in 100 ꢀL of growth medium was added to each well. The
medium was replaced with fresh growth medium every 2 d. Images were obtained using an
Olympus IX51 microscope after 5d.
5.9. S.c. Xenograft Models in Athymic Mice and in vivo Antitumor Activity Studies
Nude mice were used for all in vivo studies. The mice were housed and maintained under
specific-pathogen free conditions in accordance with Institutional Animal Care and Use
Committee. Cells at density of 5−10×106 in 200 ꢀL firstly implanted s.c. into the right flank of
each nude mice and then allowing to grow to 700−800 mm3, defined as a well-developed tumor.
After that, the well-developed tumors were cut into 1.5 mm3 fragments and transplanted s.c. into
the right flank of nude mice. When the tumor volume reached 100 to 200 mm3, the mice were
randomly assigned into vehicle and treatment groups (n = 6 in treated group, n = 12 in vehicle
group). Vehicle groups were given vehicle alone, and treatment groups received 23 as indicated
doses via po administration once daily for indicated days. The sizes of the tumors were measured
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twice per week using microcaliper. The tumor volume (TV) was calculated as: TV =
(length×width2)/2 and the individual relative tumour volume (RTV) was calculated as follows:
RTV = Vt/V0, where Vt is the volume on each day, and V0 is the volume at the beginning of the
treatment. RTV was shown on indicated days as the median RTV ± SE indicated for groups of
mice. Percent (%) inhibition values (TGI) were measured on the final day of study for drug-treated
compared with vehicle-treated mice and are calculated as 100 × {1 – [(VTreated Final day – VTreated Day 0
)
/ (VVehicle Final day – VVehicle Day 0)]}. Significant differences between the treated versus the vehicle
groups (P≤0.05) were determined using Student’s t test.
5.10. Co-crystallization of 23 and c-Met
Co-crystallization of the c-Met kinase domain with the compound 23 was carried out by
mixing a solution of the protein-ligand complex with an equal volume of precipitant solution (100
mM Tris-HCl pH 7.3, 5% isopropanol, 12% MPD, 12−14% PEG5KMME). The protein-ligand
complex was prepared by adding the compound to the protein solution to a final concentration of 1
mM 23. Co-crystallization utilized the vapour-diffusion method in hanging drops. Crystals were
flash frozen in liquid nitrogen in the presence of well solution supplemented with 25% glycerol.
Details of the crystal structure can be obtained from the PDB database.
Supplementary Material. Synthetic procedures and analytical data for intermediates reported in
this article and structure determination of c-Met in complex with 23.
ACKNOWLEDGEMENT
We thank the National Natural Science Foundation of China (81273365, 81573271,
81422047, 81321092, and 81473243), National Science & Technology Major Project ‘‘Key New
Drug
Creation
and
Manufacturing
Program’’
(2014ZX09304002-008-001
and
2012ZX09301001-007), the Shanghai Science and Technology Commission (1315431901300) for
their financial support. SA-SIBS Scholarship Program is also gratefully acknowledged.
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Captions
Figure 1. The representative selective c-Met inhibitors.
Figure 2. Strategies to eliminate the OCH2-related metabolic deficiency.
Figure 3. Co-crystal structure of 23 in complex with the kinase domain of c-Met. The pdb code of
the structure is 5EOB. (A) Compound 23 bound at the ATP-binding site of c-Met with a “U” shape.
(B) The interactions of 23 with the surrounding residues, including two H-bonds between the
compound and the backbone amide of Met1160 and Asp1222 at the hinge region and the DFG
motif, respectively.
Figure 4. Compound 23 suppressed the phosphorylation of c-Met and its downstream signaling in
various cells (EBC-1 Cells (A), MKN45 cells (B), BaF3/TPR-Met cells (C), and U87MG cells (D).
Cells were treated with indicated concentrations of 23 for 1 h and analyzed by immunoblot.
Figure 5. Compound 23 specifically inhibited c-Met-dependent cancer cell proliferation. The
anti-proliferation activity of 23 against a panel of tumor cell lines originating from different tissue
types was determined by a sulforhodamine B (SRB) or an MTT assay. The IC50 values were
plotted as the mean±SD (nM) from three separate experiments or estimated values from two
separate experiments.
Figure 6. Compound 23 prevented HGF-induced migration, invasion, scattering, and invasive
growth phenotypes. Compound 23 impaired migratory (A) and invasive (B) ability of NCI-H441
cells induced by 100 ng/mL of HGF (Scale bar, 100 ꢀm); MDCK cell scattering induced by HGF
was inhibited by 23 (C) (Scale bar, 10 ꢀm); Compound 23 prevented HGF-induced branching
morphogenesis (D). MDCK cells were cultured in collagen gel with 100 ng/mL of HGF, and
tubule-like structure was photographed after 5-day treatment (Scale bar, 10 ꢀm). Representative
pictures presented in A−D were taken from three independent experiments.
Figure 7. Compound 23 potently inhibited c-Met-dependent tumor growth in vivo. Inhibitory
activity of 23 on the tumor growth in MKN45 (A) and EBC-1 (B) xenografts. The tumor-bearing
mice were administrated (po) 23 twice daily for 3 weeks. Mean relative tumor volume ± SE was
shown (n = 6 in treated group, n = 12 in vehicle group). **P<0.01, ***P<0.001 vs vehicle group,
determined using Student’s t test.
Table 1. c-Met Enzymatic and Cellular Activities of the CH2-Linked Triazolotriazines.
Table 2. c-Met Enzymatic and Cellular Activities of the CF2-Linked Triazolotriazines.
Table 3. Pharmacokinetic Profiles of 23, 25, 26, and 27 in Rats.
Table 4. Antitumor Efficacy of Compounds 23, 26, and 27 in the EBC-1 Xenograft Model.
Table 5. Kinase Selectivity Profile of 23.
Table 6. Anti-proliferative activity of 23 on c-Met-addicted cell lines.
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Scheme 1. Synthesis of Compounds 3−27. Reagents and conditions: (a) 2-(quinolin-6-yl)acetic
acid for 28−42, 2-(1H-indol-3-yl)acetic acid for 43−45, 2,2-difluoro-2-(quinolin-6-yl)acetic acid
for 46−51, HOBT, EDCI, DIPEA, DMF; (b) HOAc, 100 oC; (c) CF3COOH, DCM.
Scheme 2. Optimization of Reaction Conditions for the Preparation of 23.
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Figure 1. The representative selective c-Met inhibitors.
28