Discovery of (S)-1-(1-(Imidazo[1,2- a ]pyridin-6-yl)ethyl)-6-(1-methyl-1 H -pyrazol-4-yl)-1 H -[1,2,3]triazolo[4,5- b ]pyrazine (Volitinib) as a Highly Potent and Selective Mesenchymal-Epithelial Transition Factor (c-Met) Inhibitor in Clinical Development for Treatment of Cancer

Article abstract of DOI:10.1021/jm500510f

HGF/c-Met signaling has been implicated in human cancers. Herein we describe the invention of a series of novel triazolopyrazine c-Met inhibitors. The structure-activity relationship of these compounds was investigated, leading to the identification of compound 28, which demonstrated favorable pharmacokinetic properties in mice and good antitumor activities in the human glioma xenograft model in athymic nude mice.

Full text of DOI:10.1021/jm500510f

Article
pubs.acs.org/jmc
Discovery of (S)‑1-(1-(Imidazo[1,2‑a]pyridin-6-yl)ethyl)-6-(1-
methyl‑1H‑pyrazol-4-yl)‑1H‑[1,2,3]triazolo[4,5‑b]pyrazine (Volitinib)
as a Highly Potent and Selective Mesenchymal−Epithelial Transition
Factor (c-Met) Inhibitor in Clinical Development for Treatment of
Cancer
Hong Jia,† Guangxiu Dai,† Jianyang Weng, Zhulin Zhang, Qing Wang, Feng Zhou, Longxian Jiao,
Yumin Cui, Yongxin Ren, Shiming Fan, Jinghong Zhou, Weiguo Qing, Yi Gu, Jian Wang, Yang Sai,
and Weiguo Su*,†
Hutchison MediPharma Limited, Building 4, 720 Cai Lun Road, Zhangjiang Hi-Tech Park, 201203, Shanghai, China
S
* Supporting Information
ABSTRACT: HGF/c-Met signaling has been implicated in
human cancers. Herein we describe the invention of a series of
novel triazolopyrazine c-Met inhibitors. The structure−activity
relationship of these compounds was investigated, leading to
the identification of compound 28, which demonstrated
favorable pharmacokinetic properties in mice and good
antitumor activities in the human glioma xenograft model in
athymic nude mice.
relatively conserved structure. In particular, the compounds all
contain a quinoline moiety, which appears to be critical for
selective binding to the enzyme. For example, PF-04217903
showed potent and selective c-Met inhibition.9 JNJ-38877605,
another selective c-Met inhibitor, was terminated in phase I
clinical study due to an increase in serum creatinine levels,
suggestive of renal toxicity and minimal pharmacodynamic
(PD) activity.10,11 Similarly, SGX523 was discontinued in
clinical development because of unexpected renal failure. This
toxicity has been hypothesized to arise as a result of insoluble
metabolites generated by aldehyde oxidase (AO) precipitating
in the kidney and causing mechanical damage12,13 (Figure 1).
Therefore, while the conserved structure of the second
generation c-Met inhibitors delivers selectivity, it also has
toxicity liabilities. Here, we wish to report the discovery of a
novel ATP-competitive kinase inhibitor which is structurally
differentiated from the known inhibitors (in particular lacking
the hitherto ubiquitous quinoline group) but surprisingly
maintains potent activity and exquisite kinase selectivity for c-
Met. It is hoped that this compound may avoid the reported
toxicity issues seen in the earlier c-Met therapies, particularly
since the compound is fairly stable to AO metabolism, and both
the parent compound and its AO metabolite are relatively
soluble in aqueous medium. This should ensure that any
products by AO metabolism should remain dissolved in the
urine, minimizing any risk of the nephropathy seen in earlier
therapeutic candidates.
INTRODUCTION
■
Mesenchymal−epithelial transition factor (c-Met), a member of
a structurally distinct family of receptor tyrosine kinases (RTK),
is a proto-oncogene encoding the high affinity receptor for
hepatocyte growth factor (HGF).1,2 The Met protein is
expressed mainly by epithelial cells, and its endogenous ligand
(HGF) is restricted to cells of mesenchymal origin.3,4 HGF/c-
Met signaling mediates a diverse array of biological activities,
including proliferation, survival, motality, migration, branching
morphogenesis, wound healing and angiogenesis. These
activities are not only essential during embryogenesis and
tissue repair, but they also contribute to cancer progression by
enabling tumor cell proliferation, survival, invasion, metastasis,
and angiogenesis. c-Met-dependent cellular proliferation can
occur in response to activating mutations, gene amplification, or
via overexpression.5,6
As a result of its central role in tumor cell biology, c-Met has
been targeted by several drug discovery programs. These have
enjoyed limited success. In particular, while drug candidates
acting on c-Met have entered clinical trials, so far none of them
has been approved. c-Met ATP-competitive inhibitors of the
first generation have broad kinase activities, usually targeting
other kinases besides c-Met. For example, XL880 is an inhibitor
of c-Met and vascular endothelial growth factor receptor 2
(VEGFR2).7 BMS-777607 is a c-Met inhibitor with additional
activities against related kinases such as AXL receptor tyrosine
kinase (Axl) and Recepteur d’Origine Nantais Kinase (Ron),
respectively.8 c-Met ATP-competitive inhibitors of the second
generation are relatively selective. Chemically, these second
generation c-Met inhibitors in clinical development all share a
Received: March 31, 2014
Published: August 22, 2014
© 2014 American Chemical Society
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Figure 1. Reference compounds.
Figure 2. Co-crystal structure of 1 with c-Met.14 Amgen reported the co-crystal structure of 1 and c-Met. PDB code: 3CCN.
interaction between the triazolopyridazine core and Tyr1230.14
Although no other binding information was reported with
selective inhibitors except 1, 6 years ago, considering the high
similarity of their structures and corresponding enzyme
selectivity, it is reasonable to assume that c-Met ATP-
competitive inhibitors of the second generation also adopt a
U-shaped conformation and are bound to the activation loop of
c-Met kinase the same way, which was confirmed by several
subsequent publications.10,15,16
RESULTS AND DISCUSSION
■
Amgen previously reported 1 as a c-Met inhibitor with an IC50
of 0.120 μM in an enzyme assay. Its co-crystal structure
revealed a unique binding mode (Figure 2): (1) a bent “U-
shaped” conformation with the inhibitor wrapped around
Met1211; (2) a hydrogen bond between the backbone NH of
Met1160 and the oxygen of the phenol, the hinge binder; (3) a
hydrogen-bonding interaction between N1 of the inhibitor and
the backbone NH of Asp1222; and (4) a π−π stacking
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To better understand the influence of the core structure on
the U-shaped binding mode between the inhibitor and the
kinase, we first investigated structure types having an N
possible for binding with Asp1222 of the c-Met enzyme and a
quinoline moiety to retain the hydrogen bond with the
backbone NH of Met1160, but with variable central heterocylic
groups.
Table 2. SAR of the Hinge Pieces
An enzymatic assay to measure the ability of a compound to
inhibit the c-Met kinase and a cellular assay to assess the
inhibition effect on phosphorylation of c-Met kinase were used
as the primary screening assays. As shown in Table 1, both
Table 1. SAR of the Core
a
b
IC50S are based on single runs of experiments. The -- indicates not
tested.
compounds 2 and 5 demonstrated high biochemical potency
toward c-Met with an IC50 of 0.005 and 0.012 μM, respectively.
However, compound 5 exhibited only moderate inhibitory
activity on phosphorylation in the cellular assay. By removing
N2 in compounds 2 and 5, compounds 3 and 6 displayed lower
c-Met kinase activities. When the N7 of compound 2 was
replaced by a C7, the corresponding analog 4 was not active
against c-Met with IC50 > 1 μM. Moving N4 of compound 2 to
N5, the resulting compound 7 also led to the loss of c-Met
activity. Compound 8 with a bridge nitrogen showed similar
potency in both enzymatic and celluar assays as compound 5.
Clearly the electronic property of the cores has a great effect on
the π-stacking interaction with Tyr1230. The most potent core,
[1,2,3]triazolo[4,5-b]pyrazine in 2, was selected for further
structure−activity relationship (SAR) exploration.
To introduce further structural differentiation from c-Met
inhibitors with known toxicity liabilities, we now began to
identify a chemical moiety to replace the quinoline hinge motif.
Several heteroaryls were investigated as new hinge pieces. As
shown in Table 2, these heteroaryls demonstrated variable c-
Met inhibition activities compared to quinoline in 2. 2-
Substituted pyrrolo[2,3-b]pyridine 9 reduced the potency
significantly, and 3-substituted pyrrolo[2,3-b]pyridine 10 was
40-fold more potent with an IC50 of 0.005 μM than compound
9 in the enzymatic assay. Pyrazolo[3,4-b]pyridine 11 with one
more N atom was also very potent against c-Met in both
a
b
IC50S are based on single runs of experiments.
Mouse liver
c
microsome stability. The -- indicates not tested.
enzymatic and cellular assays. 3-Substitued thieno[2,3-b]-
pyridine 12 and 2-substitued thieno[2,3-b]pyridine 13
exhibited much lower potency. The regioisomer thieno[3,2-
c]pyridine 14 was 17-fold more potent (IC50 is 0.011 μM in the
enzymatic assay) than 13. Thieno[3,2-c]pyrazole 15 with a
pyrazole replacing the pyridine of 13 greatly improved the
potency more than 30-fold with an IC50 of 0.006 μM in enzyme
and 0.019 μM in cell. This proved the position of N on the
hinge piece is crucial for the binding with the backbone NH of
Met 1160, and the NH of 10-11 and 15 may also contribute to
the binding as a potential hydrogen donor. Imidazo[1,2-
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a]pyridine 16 was highly potent (IC50 is 0.006 μM in enzyme
and 0.011 μM in phosphorylation), but imidazo[1,2-a]pyrazine
17, [1,2,4]triazolo[1,5-a]pyridine 18, and imidazo[1,2-b]-
pyridazine 19 with one more N all had decreased potencies
in both enzymatic and cellular assays. Similarly, pyrazolo[1,5-
a]pyridine analogue 20 was much more potent than the
corresponding aza analogues pyrazolo[1,5-a]pyrimidine 21 and
[1,2,4]triazolo[1,5-a]pyridine 22 indicating a strong electronic
effect on the binding of the hinge piece with the backbone NH
of Asp1222.
Table 3. SAR of Compounds with Selected Hinge Pieces
In summary, quinolone as a hinge binder could be replaced
by other heteroaryl moieties that retained potency against c-
Met. Although many of the compounds in Table 2
demonstrated good potencies in both enzymatic and cellular
assays with IC50 ≤ 0.1 μM, HGF-induced proliferation assay in
NCI-H441 cell line, a functional assay which has a better
correlation with the in vivo target inhibition, suggested that the
hinge binders in compounds 10, 11, 14−16, and 20 are
interesting. Further profiling study showed that 14 and 15 had
poor stabilities in mouse liver microsomes with only 23.5% and
19.4% remaining after incubation for 30 min.
To further define the SAR and improve the potency of 11,
16, and 20 series, a variety of substitutes at the 6-position of
triazolopyrazine core were explored. As shown in Table 3,
except 27B, all compounds showed very potent inhibition in
both enzymatic and cellular assays. Among them, 23C, 24A,
24C, and 26B demonstrated most potent proliferation
inhibition in NCI-H441 cell line, but 26B was unstable in
mouse liver microsomes with only 23.4% remaining after
incubation for 30 min.
Compound 10, 11, 16, 23C, 24A, and 24C were
subsequently evaluated in HGF-mediated c-Met phosphoryla-
tion in NCI-H441 in vivo in the tumor bearing mice. Figure 3
illustrates that these compounds only showed weak to
moderate inhibition at 6 h after a single oral dose of 3 mg/kg.
The selected compounds 11, 16, and 23C were further
evaluated in the preliminary pharmacokinetic studies in mice.
Injection dosing with compound 11 caused sudden death of
mice at the levels of 2.5 and 1.25 mg/kg. The reason for animal
death was not clear. The compound was therefore deprioritized.
As Table 4 shows, compound 16 was rapidly cleared in vivo.
The short T1/2 might explain its weak inhibition at 6 h in the in
vivo HGF-induced c-Met phosphorylation study. Compound
23C had desirable pharmacokinetical properties with high
exposure and bioavailability, but the moderate activity in in vivo
target inhibition assay indicates an IC50 of 0.066 μM in the
proliferation assay was not good enough to provide robust
activity in the NCI-H441 assay.
a
b
IC50S are based on single runs of experiments. Mouse liver
c
microsome stability. The -- indicates not tested.
Since the methylene attached to an aromatic ring is easily
metabolized by oxidation, the introduction of a group to block
the possible metabolism may help reduce the clearance.17 The
co-crystal of 1 with c-Met kinase revealed that methylene was
adjacent to a hydrophobic pocket with Leu-1157, Lys-1110, and
Val-1092 residues. A small hydrophobic group on the
methylene group may go into the pocket to form hydrophobic
interaction with the kinase. We therefore investigated small
modifications of the bridging methylene group. As shown in
Table 5, after introduction of a methyl, enantiomerically pure
compounds 28−31 demonstrated good inhibition of c-Met
activity. Compared with unbranched compounds 16 and 23C,
compounds 28 and 30, respectively, had equal activities against
c-Met kinase and slightly better potency in cellular assays. More
significantly, their abilities to inhibit HGF-induced proliferation
Figure 3. Inhibition on c-Met phosphorylation in NCI-H441.
Experiments were carried out in female athymic mice (n = 4); po, 3
mg/kg (0.5% carboxymethyl cellulose (CMC), pH = 2.1).
were improved. Especially compound 28 inhibited proliferation
12-fold more potently than compound 16. Compounds 28 and
30 with an (S)-configuration were found to be much more
potent than the corresponding (R)-configured compounds 29
and 31, which indicates (S)-methyl group was more suitable for
the hydrophobic pocket
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a
Table 4. Pharmacokinetic Profiles of Selected Compounds in Mice
b
b
c
c
c
c
compd
CLplasma (L/h·kg)
Vz (L/kg)
T1/2 (h)
Cmax (ng/mL)
AUC0−8h (ng·h/mL)
Foral (%)
16
4.28
1.29
5.4
2.4
1.3
3.7
1383
1920
1067
7004
46
23C
90.8
a
Experiments were carried out in male ICR mice (n = 3). Dose: iv, 2.5 mg/kg (0.25% DMSO, 10% solutol, 10% ethanol, and 79.75% saline); po,
b
c
14.1 mg/kg (0.5% CMC, pH = 2.1). Parameters obtained after iv dosing. Parameters obtained after po dosing.
Table 5. SAR of [1,2,3]Triazolo[4,5-b]pyrazine Scaffold
Figure 4. Growth inhibition of 28 in U87MG xenograft model in
mice.
kg, respectively. In addition, none of the mice in the dosing
groups exhibited body weight loss during the experiment.
As mentioned earlier, one potential hypothesis for the
toxicity seen in the known second generation c-Met inhibitors
is that it arises from the insoluble products of AO metabolism.
A metabolic study revealed that AO-mediated oxidization of
compound 28 mainly took place on the central core of
compound 28, generating a relatively polar metabolite 28-AO
(Figure 5). 28-AO′s solubility in aqueous buffers is 0.02 (pH
a
IC50S are based on single runs of experiments except for compound
28′s c-Met enzyme and p-Met (n = 3).
Given its very potent c-Met activities, compound 28 was
evaluated in vivo in a mouse pharmacokinetic study (Table 6).
It demonstrated a much more desired pharmacokinetic profile
in mice compared to the nonmethyl compound 16, suggesting
that the methyl helps to block a potential metabolic site as we
originally designed. The clearance was reduced to 0.66 L/(h·
kg) from 4.28 L/(h·kg), and the half-time was slightly enhanced
to 1.7 h from 1.3 h. Despite the moderate oral bioavailability,
the overall plasma exposure was much higher.
On the basis of the high systemic exposures, compound 28′s
inhibition of HGF-mediated c-Met phosphorylation in mice
was evaluated. 28 demonstrated 84.7% inhibition at 6 h after a
single oral dose of 3 mg/kg, much better than the 36.9%
inhibition observed with 16 at 3 mg/kg.
Encouraged by the desired PK profile and sustainable in vivo
target inhibition for at least 6 h, compound 28 was further
evaluated for its in vivo efficacy in U87MG subcutaneous
xenograft model in athymic nude mice. The compound was
administered orally once a day at various dose levels for 21
days.
Figure 5. AO metabolism of 28.
7.4) and 20.9 mg/mL (pH 1.2), comparable to compound 28′s
0.02 and 31.8 mg/mL, respectively. Importantly, after being co-
incubated with and without NADPH in human liver S9
fractions at 37 °C water bath for 60 min, the relative amounts
of 28 are 99.08% and 99.66% estimated by the peak intensity of
ultraviolet detection at 348 nm, and 28-AO were only 0.22%
and 0.34%, respectively. We therefore think the AO metabolism
will not be significant for compound 28, and in any case since
the solubility of the resulting metabolite is comparable to the
parent, AO metabolism should not lead to a safety concern in
humans.18
As shown in Figure 4, compound 28 demonstrated dose-
dependent tumor growth inhibition in a U87MG subcutaneous
xenograft model. The percent tumor growth inhibition (%TGI)
at day 21 was 52.8%, 76.6% ,and 92.8% at 1, 2.5, and 10.0 mg/
a
Table 6. Pharmacokinetic Profile of 28
b
b
c
c
c
c
compd
CLplasma (L/h·kg)
Vz (L/kg)
T1/2 (h)
Cmax (ng/mL)
AUC0−8h (ng·h/mL)
Foral (%)
28
0.66
1.2
1.7
4947
5594
27.2
a
The experiments was carried out in male ICR mice (n = 3). Dose: iv, 2.5 mg/kg (0.25% DMSO, 10% solutol, 10% ethanol, and 79.75% saline); po,
b
c
10 mg/kg (0.5% CMC, pH = 2.1). Parameters obtained after iv dosing. Parameters obtained after po dosing.
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a
Scheme 1. Synthesis of 2 and 3
a
Reagents and conditions: (i) DIEA, 130 °C; (ii) NaNO2, acetic acid (AcOH), H2O, H2SO4, rt; (iii) Pd(dppf)Cl2, Cs2CO3, dioxane, H2O, 110 °C;
(iv) trimethoxymethane, 120 °C; (v) Pd(dppf)Cl2, NaHCO3, DME, H2O, N2, 110 °C, microwave.
When tested against a panel of diverse kinases, compound 28
exhibited exquisite selectivity for c-Met over 274 kinases.
Chemistry. Outlined in Scheme 1 is the synthesis of
compounds 2 and 3. A reaction of 3,5-dibromopyrazin-2-amine
with quinolin-6-ylmethanamine in the presence of N,N-
diisopropylethylamine (DIEA) afforded intermediate 32.
Subsequently 32 was treated with NaNO2 under acidic
conditions to give 33, followed by Suzuki coupling with 1-
methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyr-
azole to provide compound 2. When 32 reacted with
trimethoxymethane, 34 was obtained, which was subjected to
intramolecular cyclization and Suzuki−Miyaura cross-coupling
to produce compound 3.
The synthesis of 5−7 is described in Scheme 3. The chlorine
of 37 was substituted with quinolin-6-ylmethanamine to give
38, followed by the reduction of NO2 to 39. 39 was utilized to
construct 5−7 as the similar procedures of 2 and 3.
Compounds 9-27B and 43-44 were synthesized according to
the general process as depicted in Scheme 4. Treatment of 3,5-
dibromopyrazin-2-amine with the corresponding amines under
basic conditions yielded intermediates 41. Diazotization and
subsequent intramolecular cyclization of 41 afforded 42. The
palladium-catalyzed coupling of bromide 42 with the
corresponding boric esters gave the desired compounds 9-
26C and 43-44. The substitution of bromine by morpholine in
the presence of K2CO3 provided 27B.
Scheme 5 shows compounds 28 and 29 were isolated from
racemic compounds 43 by HPLC, and compounds 30 and 31
were separated from the corresponding racemic compounds 44
by HPLC.
Preparation of compound 4 was accomplished in a similar
manner to 2 using the corresponding intermediate 35, which
was prepared from the reductive amination of 5-bromopyr-
idine-2,3-diamine and quinoline-6-carbaldehyde (Scheme 2).
CONCLUSIONS
■
In conclusion, a series of novel, highly potent, and selective c-
Met tyrosine kinase inhibitors with new hinge piece moieties
were successfully designed and synthesized. These compounds
are structurally differentiated from known compounds with
toxicity liabilities. Among them, the most promising compound,
28, has exquisite kinase selectivity and excellent potency and
showed dose-dependent antitumor efficacy in vivo. Compound
28, Volitinib, is now in phase Ib/II clinical development for
treatment of cancer.19−24
a
Scheme 2. Synthesis of 4a
EXPERIMENTAL SECTION
■
All reagents and solvents employed were purchased commercially and
used without further Centigrade, and pressure is at or near
atmospheric. NMR data were recorded on a Varian 400-MR machine.
Chemical shifts are expressed as δ units using tetramethylsilane as the
external standard (in NMR description, s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; and br, broad peak). All coupling constants
(J) are reported in Hertz. MS data were checked by agilent 6120 or
a
Reagents and conditions: (i) quinoline-6-carbaldehyde, AcOH,
MeOH, then NaBH(AcO)3, rt; (ii) NaNO2, AcOH, H2O, H2SO4,
rt; (iii) Pd(dppf)Cl2, Cs2CO3, dioxane, H2O, 110 °C.
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a
Scheme 3. Synthesis of 5−7
a
Reagents and conditions: 5: (i) Na2CO3, i-PrOH, rt; (ii) Pd/C, H2, EtOAc (ethyl acetate), rt; (iii) Pd(dppf)Cl2, Cs2CO3, dioxane, H2O, 110 °C. 6:
(i) Na2CO3, i-PrOH, rt; (ii) Pd/C, H2, EtOAc, rt; (iii) trimethoxymethane, p-TsOH, 120 °C, N2; (iv) Pd(dppf)Cl2, Cs2CO3, dioxane, H2O, 100 °C.
7: (i) Na2CO3, EtOAc, rt; (ii) SnCl2.2H2O, MeOH, 1,2-dichloroethane, 60 °C; (iii) NaNO2, AcOH, H2SO4, H2O; (iv) Pd2(dba)3, P(t-Bu)HBF4,
dioxane, H2O, 100 °C, N2.
a
Scheme 4. Synthesis of 9-27B and 43-44
a
Reagents and conditions: (i) DIEA or other bases, 100−150 °C; (ii) NaNO2, AcOH, H2O, H2SO4, rt; 9-26C, 43-44: (iii) 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl2, Cs2CO3 or other bases, dioxane, or DME, H2O, 100−110 °C. 27B: (iii):
morpholine, K2CO3, N,N-dimethylformamide, rt.
Scheme 5. Preparation of 28-31
agilent 1100. Mass spectra were measured with an Agilent quadrupole
6110 spectrometer using an ESI+APCI source coupled to an Agilent
1200 HPLC system or an 1100 series LC/MSD trap spectrometer
using an ESI source coupled to an Agilent 1100 HPLC system. IC50
values were calculated using XL-Fit 2.0 software. Unless indicated
otherwise, compound purity was determined by HPLC with a
confirming purity of ≥95% for all final biological testing compounds.
HPLC method: Aglilent 1200 HPLC, Column:ymc AQ C18 150 × 4.6
cm 5u; Mobile phase: H2O (0.1% formic acid) and MeOH(0.1%
formic acid); Gradient: 5% MeOH in 0−10 min, 95% MeOH in 10−
13 min, 5% MeOH in 13−14 min; Flow rate: 1 mL/min; Detector:
254 nm.
2-amine (1160 mg, 4.6 mmol) and DIEA (669 mg, 5.2 mmol) was
stirred overnight at 130 °C in a sealed tube. The reaction mixture was
concentrated and recrystallized with EtOAc to afford 32 as a yellow
solid (1.25 g, 82%). MS (m/z): 332 (M + 1)+.
To a solution of 32 (500 mg, 1.51 mmol) in 4 mL of H2O was
added AcOH (4 mL), followed by a solution of NaNO2 (125 mg, 1.81
mmol) in water (1 mL) dropwise at 0 °C. The mixture was stirred at 0
°C for 1.5 h, then added aq. H2SO4 (50%, 0.5 mL), and stirred at rt for
another 1.5 h. The second batch of aq. H2SO4 (50%, 0.5 mL) was
added, followed by a solution of NaNO2 (30 mg, 0.43 mmol) in water
(1 mL) dropwise, and stirred at rt for 1 h. The reaction mixture was
adjusted to pH ∼ 8 with aq. NaOH (20%) and extracted with EtOAc.
The organic layers were dried over MgSO4 and concentrated to afford
33 as a yellow solid (200 mg), which was used directly for the next
step. MS (m/z): 341 (M + 1)+.
Preparation of 6-((6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]-
triazolo[4,5-b]pyrazin-1-yl)methyl)quinoline (2). The mixture of
quinolin-6-ylmethanamine (727 mg, 4.6 mmol), 3,5-dibromopyrazin-
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To a solution of crude 33 (50 mg, 0.15 mmol) in 5 mL of dioxane
and 0.5 mL of H2O were added 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (45 mg, 0.32 mmol), pd(dppf)Cl2 (12
mg, 0.015 mmol), and Cs2CO3 (143 mg, 0.45 mmol). The reaction
was stirred at 110 °C overnight under N2. The reaction mixture was
concentrated and purified by flash column chromatography to afford 2
as a light-yellow solid (40 mg, 80%). Purity: 96.7%, RT 8.21. MS (m/
reaction mixture was concentrated and purified by chromatography to
afford 40 (Z, W = CH) (143 mg, 96.6%). MS (m/z): 295 (M + 1)+.
A mixture of 40 (Z, W = CH) (58 mg, 0.2 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (82 mg, 0.4
mmol), and Cs2CO3 (192 mg, 0.6 mmol) in dioxane (5 mL) and H2O
(0.5 mL) was heated to 100 °C, then added Pd(dppf)Cl2 (32 mg, 0.04
mmol), and the reaction was stirred at 100 °C under N2 overnight.
After cooling to rt, the reaction mixture was concentrated and purified
by flash column chromatography to afford 6 (10 mg, 6%). Purity:
1
z): 343 (M + 1)+. H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H),
8.90 (dd, J = 4.1, 1.5 Hz, 1H), 8.65 (s, 1H), 8.38 (d, J = 7.7 Hz, 1H),
8.31 (s, 1H), 8.04 (d, J = 8.7 Hz, 1H), 8.01 (s, 1H), 7.84 (dd, J = 8.6,
1.9 Hz, 1H), 7.54 (dd, J = 8.3, 4.2 Hz, 1H), 6.16 (s, 2H), 3.95 (s, 3H).
Preparation of 6-((6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo-
[4,5-b]pyrazin-1-yl)methyl)quinoline (3). A mixture of 32 (66 mg,
0.2 mmol) and trimethoxymethane (1 mL) was stirred at 120 °C for 3
h. The reaction mixture was concentrated, and the resulting residue
was dissolved in toluene and stirred at reflux for 18 h, then
concentrated, and used directly for the next step. MS (m/z): 359.5 (M
+ 1)+.
1
98.8%, RT 8.09. MS (m/z): 341 (M + 1)+. H NMR (400 MHz,
DMSO-d6) δ 8.85 (dd, J = 4.0, 1.6 Hz, 1H), 8.56 (s, 1H), 8.34 (d, J =
7.2 Hz, 1H), 8.27 (s, 1H), 8.04−7.98 (m, 4H), 7.85 (dd, J = 8.4, 2.0
Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 8.4, 4.0 Hz, 1H), 5.69
(s, 2H), 3.87 (s, 3H).
Preparation of 6-((5-(1-methyl-1H-pyrazol-4-yl)-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-3-yl)methyl)quinoline (7). Compound
7 was prepared by a procedure similar to that described for the
synthesis of compound 5 using 2,4-dichloro-5-nitropyrimidine as a
starting material instead of 2,6-dichloro-3-nitropyridine. Purity: 98.2%,
RT 8.61. MS (m/z): 343 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ
9.70 (s, 1H), 8.91 (dd, J = 4.0, 2.0 Hz, 1H), 8.53 (s, 1H), 8.39 (d, J =
6.8 Hz, 1H), 8.17 (d, J = 0.8 Hz, 1H), 8.05 (d, J = 8.4 Hz,1H), 8.00(d,
J = 1.6 Hz,1H), 7.84 (dd, J = 8.4, 2.0 Hz, 1H), 7.56 (dd, J = 8.0, 4.0
Hz, 1H), 6.13 (s, 2H), 3.94 (s, 3H).
The crude intermediate above (36 mg, 0.10 mmol), 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (22 mg,
0.11 mol), Pd(dppf)Cl2 (7 mg, 0.01 mmol), and NaHCO3 (16 mg,
0.20 mmol) were dissolved in DME (1 mL) and H2O (0.2 mL), the
resulting mixture was bubbled with N2 for a while, then stirred at 110
°C under microwave condition for 40 min. The reaction mixture was
concentrated and purified by flash column chromatography to afford 3
as a white solid (6 mg, 17%). Purity: 99.6%, RT 7.94. MS (m/z): 342
Preparation of 6-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]-
triazolo[4,3-b]pyridazin-3-yl)methyl)quinolone (8).25 The title
compound was prepared similar to the reference WO2007/138472.
+
1
(M + 1) . H NMR (400 MHz, DMSO-d6) δ 8.89−8.87 (m, 3H),
8.41 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.13 (s, 1H), 8.02 (d, J = 8.8
Hz, 1H), 7.98 (d, J = 1.6 Hz, 1H), 7.85 (dd, J = 8.8, 2.0 Hz, 1H), 7.52
(dd, J = 8.4, 4.0 Hz, 1H), 5.73 (s, 2H), 3.91 (s, 3H).
1
Purity: 98.1%, RT 7.76. MS (m/z): 342 (M + 1)+. H NMR (400
MHz, DMSO-d6) δ 8.84 (d, J = 2.8 Hz, 1H), 8.56 (s, 1H), 8.35−8.31
(m, 2H), 8.16 (s, 1H),7.98−7.96 (m, 2H), 7.82−7.80 (m, 1H), 7.68
(d, J = 9.6 Hz Hz, 1H), 7.51−7.48 (m, 1H), 4.73 (s, 2H), 3.93 (s, 3H).
Compounds 9-27B and 43-44 were prepared by a procedure similar
to that described for the synthesis of compound 2 using the
corresponding amines instead of quinolin-6-ylmethanamine.
Preparation of 6-((6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]-
triazolo[4,5-b]pyridin-1-yl)methyl)quinoline (4). To a solution
of 5-bromopyridine-2,3-diamine (188 mg, 1 mmol) and quinoline-6-
carbaldehyde (157 mg, 1 mmol) in MeOH (5 mL) was added 4 drops
of CH3COOH, the reaction was stirred at rt for 2 h, then added
NaBH(AcO)3 (414 mg, 2 mmol), and stirred at rt for 16 h. The
reaction mixture was concentrated and purified by column
chromatography to afford 35 (329 mg).
Compound 4 was prepared in a similar manner as compound 2
using 35 as a starting material instead of 32. Purity: 95.1%, RT 8.54.
MS (m/z): 342 (M + 1)+. 1H NMR (400 MHz, CD3OD) δ 9.00 (d, J
= 2.0 Hz, 1H), 8.84 (dd, J = 4.4, 1.6 Hz,, 1H), 8.57 (d, J = 1.6 Hz, 1H),
8.36 (d, J = 8.0.Hz, 1H), 8.17 (s, 1H), 8.02−7.98 (m, 3H), 7.86 (dd, J
= 8.4, 2.0 Hz, 1H), 7.56 (dd, J = 8.4, 4.4 Hz, 1H), 6.18 (s, 2H), 3.93 (s,
3H).
Preparation of 6-((5-(1-methyl-1H-pyrazol-4-yl)-3H-[1,2,3]-
triazolo[4,5-b]pyridin-3-yl)methyl)quinoline (5). To a solution
of quinolin-6-ylmethanamine (158 mg, 1 mmol) in 5 mL of i-PrOH
were added 2,6-dichloro-3-nitropyridine (193 mg, 1 mmol), and
Na2CO3 (318 mg, 3 mmol), the reaction was stirred at rt overnight.
The reaction mixture was concentrated. The resulting residue was
diluted with EtOAc (50 mL) and washed with water and conc. NH4Cl
solution (15 mL). The organic layers were dried over anhydrous
MgSO4 and concentrated to afford the crude product 38 (Z = CH) as
a yellow solid (300 mg). MS (m/z): 316 (M + 1)+.
The mixture of crude 38 (Z = CH) (250 mg, 0.8 mmol) and Pd/C
(10%, 200 mg) in EtOAc (30 mL) was stirred at rt under H2 for 4 h.
The reaction mixture was filtered, concentrated, and purified by
column chromatography to afford 39 (Z = CH) as a yellow solid (130
mg, 57%). MS (m/z): 286 (M + 1)+.
2-(4-(1-((1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)-1H-[1,2,3]-
triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (9). Purity:
1
96.8%, RT 8.09. MS (m/z): 362 (M + 1)+. H NMR (400 MHz,
DMSO-d6) δ 11.89 (s, 1H), 9.23 (s, 1H), 8.63 (s, 1H), 8.37 (br, 1H),
8.31 (s, 1H), 8.19 (d, J = 6.4 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.05−
7.02 (m, 1H), 6.44 (s, 1H), 6.09 (s, 2H), 4.26−4.23(m, 2H), 3.81−
3.77 (m, 2H).
2-(4-(1-((1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-1H-[1,2,3]-
triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (10). Pu-
rity: 99.8%, RT 8.20. MS (m/z): 362 (M + 1)+. 1H NMR (400
MHz, DMSO-d6) δ 11.77 (s, 1H), 9.18 (s, 1H), 8.68 (s, 1H), 8.35 (s,
1H), 8.20 (d, J = 4.8 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.79−7.76 (m,
1H), 7.36 (br, 1H), 7.24 (br, 1H), 7.12−7.07 (m, 2H), 6.06 (s, 2H),
5.04 (t, J = 4.8 Hz, 1H), 4.27 (t, J = 5.2 Hz, 2H), 3.84−3.79 (m, 2H).
1-((1H-pyrazolo[3,4-b]pyridin-3-yl)methyl)-6-(1-methyl-1H-
pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (11). Purity:
1
99.0%, RT 8.86. MS (m/z): 333 (M + 1)+. H NMR (400 MHz,
DMSO-d6) δ 9.17 (s, 1H), 8.61 (s, 1H), 8.52 (dd, J = 4.4, 2.0 Hz,1H),
8.44 (br, 1H), 8.33 (dd, J = 8.4, 2.0 Hz, 1H), 8.25 (s, 1H), 7.23 (dd, J
= 8.0, 4.4 Hz, 1H), 6.26 (s, 2H), 3.93 (s, 3H).
3-((6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]-
pyrazin-1-yl)methyl)thieno[2,3-b]pyridine (12). Purity: 95.2%,
RT 9.72. MS (m/z): 349 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ
9.18 (s, 1H), 8.65 (s, 1H), 8.60 (dd, J = 4.8, 1.6 Hz, 1H), 8.50 (dd, J =
8.0, 1.6 Hz, 1H), 8.31 (d, J = 0.4 Hz, 1H), 8.07 (s, 1H), 7.53 (dd, J =
8.0, 4.4 Hz, 1H), 6.21 (s, 2H), 3.96 (s, 3H).
Compound 5 was prepared in a similar manner as compound 2
using 39 (Z = CH) as a starting material instead of 32. Purity: 100%,
RT 8.76. MS (m/z): 342 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ
8.89 (s, 1H), 8.59−8.44 (m, 2H), 8.38 (d, J = 8.2 Hz, 1H), 8.19 (s,
1H), 8.07−7.97 (m, 2H), 7.88−7.74 (m, 2H), 7.53 (dd, J = 8.1, 3.9
Hz, 1H), 6.12 (s, 2H), 3.92 (s, 3H).
Preparation of 6-((5-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)methyl)quinoline (6). A mixture of 39 (Z =
CH) (142 mg, 0.5 mmol), trimethoxymethane (5 mL), and p-TsOH
(45 mg, 0.25 mmol) was stirred at 120 °C under N2 for 2 h. The
2-((6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]-
pyrazin-1-yl)methyl) thieno[2,3-b]pyridine (13). Purity: 99.0%,
RT 9.75. MS (m/z): 349 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ
9.23 (s, 1H), 8.66 (s, 1H), 8.53 (d, J = 3.2 Hz, 1H), 8.32 (s, 1H), 8.25
(d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.44 (dd, J = 8.0, 4.8 Hz, 1H), 6.30
(s, 2H), 3.97 (s, 3H).
2-((6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]-
pyrazin-1-yl)methyl) thieno[3,2-c]pyridine (14). Purity: 100%,
1
RT 6.98. MS (m/z): 348 (M)+. H NMR (400 MHz, DMSO-d6) δ
9.23 (s, 1H), 9.09 (s, 1H), 8.65 (s, 1H), 8.40 (d, J = 5.5 Hz, 1H), 8.32
7584
dx.doi.org/10.1021/jm500510f | J. Med. Chem. 2014, 57, 7577−7589

Journal of Medicinal Chemistry
Article
(s, 1H), 8.00 (d, J = 5.6 Hz, 1H), 7.73 (s, 1H), 6.32 (s, 2H), 3.96 (s,
3H).
1.6 Hz, 1H), 8.43 (s, 1H), 8.34 (dd, J = 8.4, 1.6 Hz, 1H), 7.23 (dd, J =
8.0, 4.4 Hz, 1H), 6.29 (s, 2H), 5.32−5.26 (m, 2H).
5-((6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]-
1-(imidazo[1,2-a]pyridin-6-ylmethyl)-6-(1-(2,2,2-trifluor-
oethyl)-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine
pyrazin-1-yl)methyl)-1H-thieno[3,2-c]pyrazole (15). Purity:
1
(24B). Purity: 100%, RT 6.72. MS (m/z): 400 (M + 1)+. H NMR
1
98.1%, RT 9.18. MS (m/z): 338 (M + 1)+. H NMR (400 MHz,
(400 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.82 (s, 1H), 8.76 (s, 1H), 8.50
(s, 1H), 7.98 (s, 1H), 7.58−7.55 (m, 2H), 7.34 (d, J = 9.6 Hz, 1H),
5.99 (s, 2H), 5.34−5.28 (m, 2H).
CD3OD) δ 9.06 (s, 1H), 8.47 (s, 1H), 8.26 (s, 1H), 7.71 (s, 1H), 7.31
(s, 1H), 6.15 (d, J = 0.8 Hz, 2H), 3.99 (s, 3H).
1-(imidazo[1,2-a]pyridin-6-ylmethyl)-6-(1-methyl-1H-pyra-
zol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (16). Purity: 97.6%,
1-(pyrazolo[1,5-a]pyridin-5-ylmethyl)-6-(1-(2,2,2-trifluor-
oethyl)-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine
1
RT 6.35. MS (m/z): 332 (M + 1)+. H NMR (400 MHz, DMSO-
1
(24C). Purity: 100%, RT 9.78. MS (m/z): 400 (M + 1)+. H NMR
d6) δ 9.20 (d, J = 0.8 Hz, 1H), 8.76 (d, J = 0.8 Hz, 1H), 8.65 (s, 1H),
8.32 (s, 1H), 7.99 (s, 1H), 7.58−7.55 (m, 2H), 7.33 (dd, J = 9.2, 1.2
Hz, 1H), 5.96 (s, 2H), 3.96 (s, 3H).
(400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.81 (s, 1H), 8.68 (d, J = 6.8
Hz, 1H), 8.48 (s, 1H), 7.99 (d, J = 2.0 Hz, 1H), 7.69 (s, 1H), 6.92 (d, J
= 7.2 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H), 6.02 (s, 2H), 5.35−5.28 (m,
2H).
1-(imidazo[1,2-a]pyrazin-6-ylmethyl)-6-(1-methyl-1H-pyra-
zol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (17). Purity: 98.5%,
1
RT 8.07. MS (m/z): 333(M + 1)+. H NMR (400 MHz, DMSO-
5-(1-(imidazo[1,2-a]pyridin-6-ylmethyl)-1H-[1,2,3]triazolo-
[4,5-b]pyrazin-6-yl)picolinonitrile (25B). Purity: 100%, RT 6.10.
d6) δ 9.17 (s, 1H), 8.96 (s, 1H), 8.84 (s, 1H), 8.59 (s, 1H), 8.23 (s,
1H), 8.19 (s, 1H), 7.83 (s, 1H), 6.00 (s, 2H), 3.91 (s, 3H).
1
MS (m/z): 354 (M + 1)+. H NMR (400 MHz, DMSO-d6) δ 9.67−
9.65 (m, 2H), 8.93−8.92 (m, 1H), 8.84 (s, 1H), 8.32 (d, J = 8.4 Hz,
1H), 7.98 (s, 1H), 7.58−7.53 (m, 2H), 7.37−7.35 (m, 1H), 6.10 (s,
2H).
1-([1,2,4]triazolo[1,5-a]pyridin-6-ylmethyl)-6-(1-methyl-1H-
pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (18). Purity:
1
99.9%, RT 8.67. MS (m/z): 333 (M + 1)+. H NMR (400 MHz,
5-(1-(pyrazolo[1,5-a]pyridin-5-ylmethyl)-1H-[1,2,3]triazolo-
DMSO-d6) δ 9.26 (s, 1H), 9.19 (s, 1H), 8.65 (s, 1H), 8.53 (s, 1H),
8.30 (s, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.77 (dd, J = 9.2, 1.7 Hz, 1H),
6.07 (s, 2H), 3.96 (s, 3H).
[4,5-b]pyrazin-6-yl)picolinonitrile (25C). Purity: 97.7%, RT
1
9.2.MS (m/z): 354 (M + 1)+. H NMR (400 MHz, DMSO-d6) δ
9.65−9.62(m, 2H), 8.81−8.88 (dd, J = 8.4, 2.0 Hz,1H), 8.65 (d, J = 7.2
Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.75 (s,
1H), 6.93 (dd, J = 7.2, 1.6 Hz, 1H), 6.59 (d, J = 1.6 Hz, 1H), 6.11 (s,
2H).
1-(imidazo[1,2-b]pyridazin-6-ylmethyl)-6-(1-methyl-1H-pyr-
azol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (19). Purity: 98.8%,
1
RT 7.42. MS (m/z): 333 (M + 1)+. H NMR (400 MHz, CD3OD) δ
9.08 (s, 1H), 8.43 (s, 1H), 8.21 (s, 1H), 8.04−8.00 (m, 2H), 7.72 (s,
1H), 7.40 (d, J = 9.2 Hz, 1H), 6.21 (s, 2H), 3.97 (s, 3H).
6-(4-fluorophenyl)-1-(imidazo[1,2-a]pyridin-6-ylmethyl)-1H-
[1,2,3]triazolo[4,5-b]pyrazine (26B). Purity: 95.2%, RT 7.26. MS
(m/z): 346 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H),
8.79 (s, 1H), 8.42−8.39 (m, 2H), 7.98 (s, 1H), 7.58−7.56 (m, 2H),
7.49−7.44 (m, 2H), 7.36 (d, J = 10.0 Hz, 1H), 6.06 (s, 2H).
6-(4-fluorophenyl)-1-(pyrazolo[1,5-a]pyridin-5-ylmethyl)-
1H-[1,2,3]triazolo[4,5-b]pyrazine (26C). Purity: 100%, RT 10.66.
MS (m/z): 346 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ 9.27 (s,
1H), 8.78 (s, 1H), 8.67 (d, J = 7.2 Hz, 1H), 8.48 (d, J = 0.8 Hz, 1H),
7.99 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 0.8 Hz, 1H), 6.92 (dd, J = 7.2, 1.6
Hz, 1H), 6.61 (dd, J = 2.4, 0.8 Hz, 1H), 6.01 (s, 2H), 5.63 (s, 2H).
4-(1-(imidazo[1,2-a]pyridin-6-ylmethyl)-1H-[1,2,3]triazolo-
[4,5-b]pyrazin-6-yl)morpholine (27B). Purity: 100%, RT 5.82. MS
(m/z): 337 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H),
8.57 (s, 1H), 7.9972 (s, 1H), 7.58 (br, 2H), 7.27 (d, J = 10.4 Hz, 1H),
5.75 (s, 2H), 3.75−7.54 (m, 8H).
6-(1-methyl-1H-pyrazol-4-yl)-1-(pyrazolo[1,5-a]pyridin-5-yl-
methyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (20). Purity: 98.1%, RT
1
9.42. MS (m/z): 332 (M + 1)+. H NMR (400 MHz, DMSO-d6) δ
9.19 (s, 1H), 8.65−8.63 (m, 2H), 8.27 (s, 1H), 7.96 (d, J = 2.4 Hz,
1H), 7.65 (s, 1H), 6.88 (dd, J = 7.2, 2.0 Hz, 1H), 6.58 (d, J = 1.6 Hz,
1H), 5.96 (s, 2H), 3.92 (s, 3H).
6-(1-methyl-1H-pyrazol-4-yl)-1-(pyrazolo[1,5-a]pyrimidin-5-
ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (21). Purity: 95.5%,
1
RT 8.66. MS (m/z): 333 (M + 1)+. H NMR (400 MHz, CD3OD) δ
9.10 (s, 1H), 8.91(dd, J = 7.2, 0.8 Hz, 1H), 8.43 (s, 1H), 8.21 (d, J =
0.4 Hz, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.08 (d, J = 7.2 Hz, 1H), 6.54
(dd, J = 2.4, 0.8 Hz, 1H), 6.18 (s, 2H), 3.96 (s, 3H).
1-([1,2,4]triazolo[1,5-a]pyridin-7-ylmethyl)-6-(1-methyl-1H-
pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (22). Purity:
1
99.5%, RT 8.55. MS (m/z): 333 (M + 1)+. H NMR (400 MHz,
Preparation of (S)-1-(1-(imidazo[1,2-a]pyridin-6-yl)ethyl)-6-
(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine
(28) and (R)-1-(1-(imidazo[1,2-a]pyridin-6-yl)ethyl)-6-(1-meth-
yl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (29). The
racemic compound 43 (prepared by a procedure similar to that
described for the synthesis of compound 2 using the corresponding 1-
(imidazo[1,2-a]pyridin-6-yl)ethanamine instead of quinolin-6-ylme-
thanamine) was resolved by an outsourced service to produce the
optically pure two enantiomers 28 and 29. Compound 43: Purity:
DMSO-d6) δ 9.20 (s, 1H), 8.94 (d, J = 7.2 Hz, 1H), 8.62 (s, 1H), 8.48
(s, 1H), 8.27 (s, 1H), 7.85 (s, 1H), 7.21 (d, J = 6.8 Hz, 1H), 6.10 (s,
2H), 3.92 (s, 3H).
2-(4-(1-((1H-pyrazolo[3,4-b]pyridin-3-yl)methyl)-1H-[1,2,3]-
triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (23A). Pu-
rity: 99.2%, RT 8.17. MS (m/z): 363 (M + 1)+. 1H NMR (400 MHz,
DMSO-d6) δ 9.20 (s, 1H), 8.61 (s, 1H), 8.53 (dd, J = 8.4, 4.4 Hz, 1H),
8.35 (dd, J = 8.0, 1.6 Hz, 1H), 8.28 (s, 1H), 7.25 (dd, J = 8.0, 4.4 Hz,
1H), 6.28(s, 2H), 4.25−4.23 (m, 2H), 3.80−3.77 (m, 2H).
2-(4-(1-(imidazo[1,2-a]pyridin-6-ylmethyl)-1H-[1,2,3]-
triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (23B). Pu-
1
100%, RT 6.78. MS (m/z): 346 (M + 1)+. H NMR (400 MHz,
DMSO-d6) δ 9.17 (s, 1H), 8.80 (s, 1H), 8.65 (s, 1H), 8.29 (s, 1H),
7.98 (s, 1H), 7.57−7.54 (m, 2H), 7.38 (dd, J = 9.4, 1.8 Hz, 1H), 6.40
(q, J = 7.2 Hz, 1H), 3.94 (s, 3H), 2.17 (d, J = 7.2 Hz, 3H). Compound
28: MS (m/z): 346 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ 9.18
(s, 1H), 8.79 (s, 1H), 8.64 (s, 1H), 8.30 (s, 1H), 7.98 (s, 1H), 7.58−
7.54 (m, 2H), 7.38 (dd, J = 9.2, 1.6 Hz, 1H), 6.42 (q, J = 7.2 Hz, 1H),
3.95 (s, 3H), 2.17 (d, J = 7.2 Hz, 3H). Purity: 99.9%, RT 15.07. ee:
100%. Compound 29: MS (m/z): 346 (M + 1)+. 1H NMR (400 MHz,
DMSO-d6) δ 9.18 (s, 1H), 8.79 (s, 1H), 8.63 (s, 1H), 8.30 (s, 1H),
7.97 (s, 1H), 7.58−7.54 (m, 2H), 7.38 (dd, J = 9.6, 2.0 Hz, 1H), 6.41
(q, J = 7.2 Hz, 1H), 3.95 (s, 3H), 2.18 (d, J = 7.2 Hz, 3H). Purity:
99.5%, RT 17.48, ee: 99%. HPLC analysis conditions: Gilson system,
Column: Chiralpak Ia 4.6 mm I.D. × 25 cm L; Mobile phase: EtOH/
DEA = 100/0.1; Flow rate: 0.5 mL/min; Detector: 254 nm.
1
rity: 100%, RT 5.74. MS (m/z): 362 (M + 1)+. H NMR (400 MHz,
DMSO-d6) δ 9.22 (s, 1H), 8.76 (s, 1H), 8.66 (s, 1H), 8.34 (s, 1H),
7.98 (s, 1H), 7.57−7.55 (m, 2H), 7.33−7.31 (dd, J = 9.2, 1.6 Hz, 1H),
5.97 (s, 2H), 5.01−4.98 (m, 1H), 4.27−4.24 (m, 2H), 3.83−3.78 (m,
2H).
2-(4-(1-(pyrazolo[1,5-a]pyridin-5-ylmethyl)-1H-[1,2,3]-
triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (23C). Pu-
rity: 99.2%, RT 8.85. MS (m/z): 362 (M + 1)+. 1H NMR (400 MHz,
DMSO-d6) δ 9.21 (s, 1H), 8.64 (d, J = 7.2 Hz, 1H), 8.61 (s, 1H), 8.30
(s, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.67 (s, 1H), 6.88 (dd, J = 7.2, 1.6
Hz, 1H), 6.59 (dd, J = 2.0, 0.8, 1H), 5.98 (s, 2H), 4.98 (t, J = 5.2 Hz,
1H), 4.23 (t, J = 5.6 Hz, 2H), 3.80−3.75 (m, 2H).
1-((1H-pyrazolo[3,4-b]pyridin-3-yl)methyl)-6-(1-(2,2,2-tri-
fluoroethyl)-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine
Preparation of (S)-2-(4-(1-(1-(pyrazolo[1,5-a]pyridin-5-yl)-
ethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)-
ethanol (30) and (R)-2-(4-(1-(1-(pyrazolo[1,5-a]pyridin-5-yl)-
1
(24A). Purity: 100%, RT 9.17. MS (m/z): 401 (M + 1)+. H NMR
(400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.76 (s, 1H), 8.52 (dd, J = 4.8,
7585
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Journal of Medicinal Chemistry
Article
ethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)-
ethanol (31). The racemic compound 44 (prepared by a procedure
similar to that described for the synthesis of compound 2 using the
corresponding 1-(pyrazolo[1,5-a]pyridin-5-yl)ethanamine instead of
quinolin-6-ylmethanamine) (5 mg) was resolved by chiral HPLC to
produce optically pure enantiomers 30 (1.0 mg) and 31 (1.9 mg).
HPLC resolution conditions: Gilson system, Column: Dicel IA 20 ×
250 mm; Mobile phase: n-Hexane/i-PrOH/DEA = 6/4/0.1; Flow
rate: 8 mL/min; Detector: 254 nm). Compound 44: Purity: 95.8%,
Inhibition(%) = 100 − 100 × {[OD − OD570 compd
]
450
− [OD − OD570 lysis buffer
]
}/{[OD − OD570 cell
]
450
450
− [OD − OD570 lysis buffer
]
}
450
where [OD450 − OD570
]
represents the optical density of wells
compd
with compound treatment; [OD450 − OD570
]cell represents the optical
density of wells without compounds treatment; and [OD450
−
OD]lysis buffer represents the optical density of wells containing lysis
buffer, but without cells.
1
RT 9.28. MS (m/z): 376 (M + 1)+. H NMR (400 MHz, CD3OD) δ
9.07 (s, 1H), 8.49−8.47 (m, 2H), 8.26 (s, 1H), 7.93 (d, J = 2.4 Hz,
1H), 7.78 (s, 1H), 7.01 (dd, J = 7.2, 2.0 Hz, 1H), 6.62 (d, J = 2.4 Hz,
1H), 6.47 (q, J = 6.8 Hz, 1H), 4.33 (t, J = 4.2 Hz, 2H), 3.95 (t, J = 4.2
Hz, 2H), 2.25 (d, J = 6.8 Hz, 3H). Compound 30: MS (m/z): 376 (M
HGF-Induced Proliferation Assay. NCI-H441 cells were seeded
in 96-well plates at 8000 cells/well in RPMI 1640 medium containing
10% FBS. After incubation overnight, cells were starved in FBS-free
medium (containing 0.5% BSA) for 16−20 h, then treated with test
compounds at different concentrations for 2 h at 37 °C. Recombinant
human HGF (R&D System China Co., Ltd.) was added to the
working concentration of 50 ng/mL and incubated for additional 72 h
at 37 °C in incubator. Ten μL/well of 5 mg/mL of MTT solution was
added, and the plates were incubated at 37 °C for additional 4 h. Cell
survival was read at 492 nm on Labsystems Multiskan K3. Inhibition
rate of compound at each test point was calculated according to
following equation and IC50 was calculated using XLFit2.0 software.
1
+ 1)+. H NMR (400 MHz, CD3OD) δ 9.08 (s, 1H), 8.50 (s,1 H),
8.50 (d, J = 7.2 Hz, 1H), 8.27 (s, 1H), 7.94 (d, J = 2.4 Hz, 1H), 7.79
(s, 1H), 7.01(dd, J = 7.2, 2.0 Hz, 1H), 6.62 (d, J = 1.6 Hz, 1H), 6.48
(q, J = 6.8 Hz, 1H), 4.33 (t, J = 4.2 Hz, 2H), 3.95(t, J = 4.2 Hz, 2H),
2.26 (d, J = 6.8 Hz, 3H). Purity: 98.1%, RT 18.44, ee: 96%.
Compound 31: MS (m/z): 376 (M + 1)+. 1H NMR (400 MHz,
CD3OD) δ 9.08 (s, 1H), 8.51 (s, 1H), 8.49 (d, J = 7.6 Hz, 1H), 8.27
(s, 1H), 7.94 (d, J = 2.4 Hz, 1H), 7.79 (s, 1H), 7.01 (dd, J = 7.2, 2.0
Hz,1H), 6.62 (d, J = 2.0 Hz, 1H), 6.48 (q, J = 6.8 Hz, 1H), 4.33 (t, J =
4.2 Hz, 2H), 3.95 (t, J = 4.2 Hz, 2H), 2.26 (d, J = 6.8 Hz, 3H). Purity:
90.7%, RT 24.22, ee: 81%. HPLC analysis conditions: Gilson system,
Column: Chiralpak Ia 4.6 mm I.D. × 25 cm L; Mobile phase: n-
Hexane/i-PrOH/DEA = 6/4/0.1; Flow rate: 1 mL/min; Detector: 254
nm.
Inhibition(%) = 100 − 100 × {[OD
]
− [OD ] }
492 cell
492 compd
/{[OD
]
− [OD ] }
492 cell
492 HGF
where [OD492
test compound in the presence of HGF; [OD492
]
compd represents the optical density of cells treated with
represents the
]
cell
optical density of cells without test compound and HGF; and [OD492
]
In Vitro Enzymatic Assay. Inhibition of test compound on c-Met
kinase activity was measured by using of Transcreener FP ADP Assay
(Bellbrook Lab) that was developed to detect ADP generated in the
enzyme reaction. The kinase reaction buffer was composed of 67 mM
of HEPES/pH = 7.4, 0.013% of Triton X-100, 27 mM of MgCl2, 0.67
mM of MnCl2, 1.25 mM of DTT. Test compound was tested in a 8-
point series dilution. The enzymatic reaction contains 0.2 μg/mL of
recombinant c-Met catalytic domain (Invitrogen Co.), 25 μg/mL of
Poly E4Y substrate (Sigma-Aldrich), 5 μL of tested compound diluted
in 20% DMSO, and 10 μM of ATP. All components were diluted in
assay buffer. The final concentration of DMSO in the reaction was 4%.
ATP was the last component to be added, for initiating the reaction.
The reaction mixture was incubated at 25 °C for 45 min. Then ADP
detection mixture was added and incubated at 25 °C for additional 1 h.
Florescence polarization is measured on TECAN F500 at excitation of
610 nm and emission of 670 nm. Produced ADP concentration was
calculated using standard curve obtained in the same study based on
the recommendation of manufacturer.
Cell-Based Activity Assay. IC50 of test compounds on c-Met
autophosphorylation was determined in NCI-H441 cells (American
Type Culture Collection, ATCC, Rockville, MD) using human
phospho-HGF R/c-MET ELISA kit (R&D System China Co., Ltd.).
The assay was carried out according the manufacturer’s instruction.
Briefly, NCI-H441 cells were plated at a density of 15,000 cells/well in
RPMI-1640 medium with 10% FBS in 96-well plates. After incubation
overnight, cells were then treated with serially diluted test compounds
at 37 °C for 1 h. Then the medium was removed, and cells were lysed
in 100 μL/well lysis buffer (1% NP-40, 20 mM Tris/pH = 8.0, 137
mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium
orthovanadate, 10 mg/mL Aprotinin, 10 mg/mL Leupeptin). The
plates containing cell lysate were kept at −80 °C overnight. The next
day, the plates were thawed on ice, mixed gently. Twenty-five μL/well
of lysates were added into the assay plates precoated with anti-p-Met
antibody to detect p-c-Met signal. p-c-Met level was determined at 450
and 570 nm using Labsystems Multiskan K3. The inhibition rate was
calculated according to the following equation, and IC50 was
determined using XLFit2.0 software.
represents the optical density of cells stimulated with HGF, but
HGF
without test compound.
In Vivo Inhibition on c-Met Phosphorylation in NCI-H441.
Cell Lines. NCI-H441, a human nonsmall cell lung adenocarcinoma
epithelial cell line with high level of c-Met expression, was grown in
RPMI1640 medium containing 10% FBS. MRC-5, a human lung
fibroblast cell line with a capability to secrete bioactive human HGF,
was cultured in EMEM with 10% FBS. Female athymic mice (6−8
weeks old) were used for study.
Animals. Female athymic mice (6−8 weeks old) were purchased
from Shanghai SLAC laboratory animal CO. Ltd. The animals were
housed in SPF environment, 4 animals/cage under standard
conditions (12:12 h light/dark, 40−70% relative humidity at 20−25
°C), and given free access to Co60 radiated-sterile diet and sterile
water. The nude mice were quarantined for at least 3 days before
implantation.
Establishment of Subcutaneous Xenograft Model. The tumor
cells were incubated at 37 °C in a 5% CO2 incubator until reaching
approximately 80% confluence. Cells were detached by 0.05% Trypsin-
EDTA, centrifuged at 800 rpm, and the cells were suspended in serum
free-medium, and the concentration was adjusted for tumor
implantation in nude mice. NCI-H441 2 × 106 and MRC-5 1 × 106
cells were subcutaneously co-implated to the right lateral flank of nude
mice.
c-Met Phosphorylation ELISA Assay. Two weeks after cells
implantation, when average tumor volume reached 300 mm3, the
animals were randomized and divided into vehicle and compound
treated groups. At 6 h after single oral dosing with 3 mg/kg, animals
were euthanized, and tumor tissues were harvested and placed into
lysis buffer (Cell Signaling Technologies, CAT #9803) containing
phosphatase and protease inhibitors (Thermo Scientific, Cat:
1861282). Tumors were homogenized at 28000 rpm for 8 s
(Homogenizers, FA25, Fluko), lysate was spun down at 12000 rpm
for 15 min twice, and the supernatant was transferred to eppendorf
tubes and stored at −80 °C prior to ELISA assay. Protein
concentrations were determined by Bio-Rad Protein Assay (Bio-Rad
Cat# 500-0006). Total-Met (R&D Systems, DYC358) and phospho-
Met ELISA assays (R&D Systems, DYC2480) were performed
according to the manufacturer’s instructions.
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Journal of Medicinal Chemistry
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In Vivo Efficacy Study in U87MG Xenograft Model. Test
article. Compound 28 was formulated in 0.5% CMC-Na (pH = 2.1)
and stored at 4 °C. Cell line: U87MG, a human glioma cell line with
high expression of c-Met, was incubated in EMEM medium with FBS
and sodium pyruvate.
Animals. Female athymic mice (6−8 weeks old) were purchased
from Shanghai SLAC laboratory animal CO. Ltd. The animals were
housed in SPF environment, 4 animals/cage under standard
conditions (12:12 h light/dark, 40−70% relative humidity at 20−25
°C) and given free access to Co60 radiated-sterile diet and sterile water.
The nude mice were quarantined for at least 3 days before
implantation. All studies were done in accordance with Hutchison
Medipharma Limited Institutional Animal Care and Use Committee
(HMPLACUC) guidelines.
putting the incubation system into 37 °C water bath open to the air
and maintained for 0 and 60 min and stopped with the addition of an
equal volume of ice-cold acetonitrile. Blank samples were also
prepared simultaneously with same contents except of using blank
reagents to instead compound 28 and incubated for 60 min. All
terminated incubation samples were centrifuged at 3000 rpm for 10
min at 4 °C, and then 2400 μL supernatant was aspirated, transferred
to other clean tubes, and dried with a nitrogen flow at room
temperature. The residues were then reconstituted with 200 μL 50%
methanol and injected for LC-MSn/PDA analysis.
Solubility Test. pH 1.2 buffer: Dissolve 3.73g of potassium chloride
in 250 mL of purified water. Add 425 mL of 0.2 M hydrochloric acid.
Mix thoroughly, dilute with water to 1000 mL, and adjust the pH of
the buffer to 1.2 0.05. pH 7.4 buffer: Dissolve 6.81 g of monobasic
sodium phosphate in 250 mL of purified water. Add 195.5 mL of 0.2
M sodium hydroxide solution. Mix thoroughly, dilute with water to
1000 mL, and adjust the pH of the buffer to 7.4 0.05. Accurately
weigh 7.9 mg of 28-AO into a 50 mL volumetric flask, add about 40
mL of mobile phase to dissolve, and dilute to volume with mobile
phase, or weigh 10.66 mg of 28 into a 50 mL volumetric flask, add
about 40 mL of mobile phase to dissolve, and dilute to volume with
mobile phase. Place excess 28-AO in a 10 mL tube containing 5 mL of
buffer solution, or 28 in a 5 mL tube containing 2 mL of buffer
solution and shake (3 cm/200time s/min) for 48 h at 20 °C. Then
centrifuge for 10 min at 4000 rpm, transfer 1 mL of supernatant into a
clean glass container, dilute with mobile phase to a suitable
concentration, and measure by a HPLC method.
Establishment of subcutaneous xenograft model and in vivo
efficacy. U87MG tumor cells were inoculated into the flanks of
athymic nude mice (3 × 106cells/mouse). When tumor volume
reached 200 mm3, the mice were randomized into vehicle treated and
compound 28 treated groups. The animals were orally administered
with compound 28 once a day at 1.0, 2.5, and 10.0 mg/kg or treated
with vehicle 0.5% CMC-Na at pH = 2.1. Tumor size was measured 2−
3 times per week. Tumor volumes were calculated using the formula:
TV = (Length × Width2)/2. The percentage of tumor growth
inhibition (%TGI = [1 − (Vt − V0)drug treated/(Vt − V0)vehicle control] ×
100%) was used for evaluation of the antitumor efficacy. V0 represents
mean tumor volume at the first day of treatment; and Vt represents
mean tumor volume at the last day of treatment. Animal body weight
and behavior were observed during the experiment. Statistical analysis
for tumor volume was performed by the student t test for a mean
comparison.
In vivo PK study. Twelve male mice were randomly divided into
two groups (6 mice per group). One group was given an intravenous
dose of 2.5 mg/kg dissolved in saline containing 10% Solutol HS 15,
10% ethanol, and 0.25% DMSO. The other group was given an oral
dose of 10 mg/kg as a suspension in acidic 0.5% CMC-Na (acidified
by HCl to pH = 2.1 before adding compound). In each group, 3 mice
were blood-sampled at predose (0), 0.25, 1, 2, 6, and 24 h postdose,
while the remaining at 5 min, 0.5, 1.5, 4, and 8 h postdose. At the due
time, 100 μL of blood was collected. After centrifugation to get the
plasma, the concentration was determined by a LC/MS/MS method.
Mouse Liver Microsome (MLM) Stability Screening Study. The
incubations were conducted in duplicate. In general, incubation
mixtures consisted of mouse liver microsomes (0.5 mg/mL), test
compound (1 μM), MgCl2 (final 3 mM), EDTA (final 1 mM), and
NADPH regenerating system (containing 5 mM G-6-P, 1U/mL G-6-
PD, and 1 mM NADP, which was prepared in 50 mM potassium
phosphate buffer at pH 7.4 and preincubated at 37 °C water-bath for
10 min and then cooled on ice until added into the reaction system)
with a final volume of 125 μL. The stock solution of test compound
was prepared with DMSO first and then diluted to designated
concentration. The contents of organic solvents in the final incubation
system were no more than 1% (for DMSO, the controlled margin was
0.1%). The incubation was commenced by putting the incubation
system into 37 °C water bath open to the air and maintained for 0 and
30 min. The incubation was typically terminated by adding 125 μL
cold acetonitrile containing internal standard. The terminated
incubation mixtures were centrifuged at 3000 g for 10 min, and
then 10 mL of supernatant was injected for analysis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Kinase selectivity data of compound 28, body weight of
compound 28 on U87MG xenograft model in mice, and
additional synthetic procedures of the intermediates 1−16 for
compounds 9-27B and 43-44. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: weiguos@hmplglobal.com. Phone: (+86)-21-
20673002.
■
Author Contributions
†These authors contributed equally.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
AcOH, acetic acid; AO, aldehyde oxidase; Axl, AXL receptor
tyrosine kinase; c-Met, mesenchymal−epithelial transition
factor; CMC, carboxymethyl cellulose; DIEA, N,N-diisopropy-
lethylamine; EtOAc, ethyl acetate; HGF, hepatocyte growth
factor; ng, nanogram; Ron, Recepteur d’Origine Nantais
Kinase; RTK, receptor tyrosine kinases
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