Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(Quinolin-6-ylmethyl)-1 H -[1,2,3]triazolo[4,5- b ]pyrazin-6-yl)-1 H -pyrazol-1-yl)ethanol (PF-04217903) for the treatment of cancer

Article

pubs.acs.org/jmc

Discovery of a Novel Class of Exquisitely Selective Mesenchymal-

Epithelial Transition Factor (c-MET) Protein Kinase Inhibitors and

Identiﬁcation of the Clinical Candidate 2‑(4-(1-(Quinolin-6-

ylmethyl)‑1H‑[1,2,3]triazolo[4,5‑b]pyrazin-6-yl)‑1H‑pyrazol-1-

yl)ethanol (PF-04217903) for the Treatment of Cancer

J. Jean Cui,* Michele McTigue, Mitchell Nambu, Michelle Tran-Dube, Mason Pairish, Hong Shen, Lei Jia,

́

Hengmiao Cheng, Jacqui Hoﬀman, Phuong Le, Mehran Jalaie, Gilles H. Goetz,^§Kevin Ryan,

Neil Grodsky, Ya-li Deng, Max Parker, Sergei Timofeevski, Brion W. Murray, Shinji Yamazaki,

Shirley Aguirre, Qiuhua Li, Helen Zou, and James Christensen

La Jolla Laboratories, Pﬁzer Worldwide Research and Development, 10770 Science Center Drive, San Diego, California 92121,

United States

S

* Supporting Information

ABSTRACT: The c-MET receptor tyrosine kinase is an

attractive oncology target because of its critical role in human

oncogenesis and tumor progression. An oxindole hydrazide hit

6 was identiﬁed during a c-MET HTS campaign and

subsequently demonstrated to have an unusual degree of

selectivity against a broad array of other kinases. The cocrystal

structure of the related oxindole hydrazide c-MET inhibitor 10

with a nonphosphorylated c-MET kinase domain revealed a

unique binding mode associated with the exquisite selectivity

proﬁle. The chemically labile oxindole hydrazide scaﬀold was

replaced with a chemically and metabolically stable triazolopyrazine scaﬀold using structure based drug design. Medicinal

chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)-

ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated eﬀective tumor

growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety proﬁle in preclinical

studies. 2 progressed to clinical evaluation in a Phase I oncology setting.

reasons, c-MET and its ligand HGF are emerging as attractive

targets for targeted cancer therapies.

INTRODUCTION

■

c-MET, also called hepatocyte growth factor receptor (HGFR),

belongs to a structurally unique subfamily of receptor tyrosine

kinase (RTK), along with RON protein kinase. The natural

ligand for c-MET is hepatocyte growth factor (HGF), also

known as scatter factor. The c-MET/HGF signaling pathway

plays important roles during normal development, organo-

genesis, and homeostasis. After activation by HGF, c-MET

induces an invasive program consisting of cell proliferation,

migration, invasion, and survival that is essential during normal

processes, such as morphogenesis and wound healing.¹

Aberrant c-MET/HGF signaling through constitutive activa-

tion, gene ampliﬁcation, mutations, and activation of an

autocrine loop occurs in virtually all types of solid tumors,

and as such is implicated in multiple tumor oncogenic

processes, such as mitogenesis, survival, angiogenesis, and

invasive growth, and especially in the metastatic process.²

Furthermore, overexpression of c-MET and HGF was

demonstrated to correlate with poor prognosis or metastatic

progression in a number of major human cancers.³For these

The strategies to regulate the c-MET/HGF pathway include

HGF antagonists (NK2 and NK4),⁴anti-HGF antibodies,⁵anti-

c-MET monoclonal antibodies,⁶and c-MET small molecule

inhibitors.⁷The anti-HGF antibody AMG 102 (rilotumumab),

when combined with the anti-EGFR antibody panitumumab,

showed a higher overall response rate for colorectal cancer

patients than patients treated with panitumumab plus placebo

in a randomized phase II study.⁸A durable, complete response

of metastatic gastric cancer with high c-MET gene polysomy

and evidence for an autocrine production of hepatocyte growth

factor was achieved with c-MET monoclonal antibody

MetMab.⁹Overexpression of c-MET in non-small-cell lung

cancer (NSCLC) represents more than half of the population

and is associated with a worse outcome. MetMab, in

combination with erlotinib, signiﬁcantly improved PFS and

Received: July 5, 2012

Published: August 27, 2012

8091

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

OS, resulting in a near 3-fold reduction in the risk of death.¹⁰

The ﬁrst crystal structures of c-MET were published in 2002^11a

and 2003,^11bwhich facilitate small molecule c-MET inhibitor

development. Several classes of small molecule c-MET

inhibitors with diﬀerent binding modes and selectivity proﬁles

progressed to clinical trials (Chart1). Foretinib, a type II

binding site. When given with the EGFR inhibitor erlotinib in

patients with NSCLC who have had prior chemotherapy but

are EGFR TKI naive, tivantinib may prolong PFS, extend OS,

and delay metastases compared with treatment with erlotinib

alone.²²Strong interest in targets that inhibit the HGF/c-MET

pathway is fueled by the continually emerging evidence of

clinical beneﬁt in a variety of cancers.

̈

2-(4-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903) is a

potent and exquisitely selective ATP competitive c-MET

inhibitor.²³2 demonstrated low nanomolar potency against c-

MET in both in vitro cell assays and in vivo target modulation

studies, and it showed eﬀective tumor growth inhibition with

good oral PK properties. 2 progressed to human clinical studies

for oncology. Herein, we report the discovery of a unique c-

MET autoinhibitory conformation and the medicinal chemistry

eﬀort which led to the discovery of the potent and exquisitely

selective c-MET inhibitor 2.

Chart 1. Representative c-MET Inhibitors in Clinical

Development

RESULTS AND DISCUSSION

■

Structural Elucidation of an HTS Hit. High throughput

screening (HTS) is a powerful method for the identiﬁcation of

novel chemical series for a new target. The screening results for

novel and eﬃcient hits are highly dependent on the quality and

diversiﬁcation of the compound collection. Hundreds of

thousands of compounds with diversiﬁed structural features

were screened using an enzymatic assay that assessed c-MET

inhibition. Compound 3 from the HTS campaign was an

attractive hit with a K_iof 101 nM as an ATP competitive c-

MET inhibitor. The structure of 3 was drawn according to the

original compound database record (Chart 2). As a standard

procedure for an HTS triage, attractive hits were subjected to

analytical tests including LC-MS and NMR spectroscopy

methods to conﬁrm the compound identity. The active samples

were also subjected to a process referred to as LC-MS-BA

(liquid chromatography−mass spectrometry−bioassay), which

simultaneously allowed for the assessments of sample purity

and structure conﬁrmation through generation of UV and mass

spectra. The fraction from the chromatographic step in this

process was collected for bioassay, and the comparisons

between the chromatographic and bioassay proﬁles were

made to determine whether the activity corresponded to the

desired chromatographic peak. Using the LC-MS-BA method, it

was discovered that the molecular weight of the active

component of 3 (>90%) was 309.32, which was consistent

inhibitor bound into the deep hydrophobic back pocket, is a

multitargeted c-MET/VEGFR2 inhibitor¹²and demonstrated

partial regression or stable disease in patients with papillary

renal carcinoma¹³and poorly diﬀerentiated gastric cancer.¹⁴

MK-2461 (1) is a potent c-MET inhibitor that is selective for

the inhibition of the phosphorylated c-MET enzyme.¹⁵1 is

currently in phase I/II clinic trials for cancer treatments.

Crizotinib is a potent c-MET/ALK dual inhibitor¹⁶which

demonstrated marked eﬃcacy for a subset of NSCLC patients

with an EML4-ALK fusion gene, leading to a fast track FDA

approval.¹⁷In addition, crizotinib demonstrated a rapid and

durable response for a NSCLC patient with de novo MET

ampliﬁcation, but without ALK rearrangement.¹⁸Patients with

MET-ampliﬁed esophagogastric adenocarcinoma and glioblas-

toma achieved dramatic clinical improvement and radiographic

regression upon treatment with crizotinib.^19,20Tivantinib

(ARQ-197) is reported as a non-ATP-competitive small

molecule c-MET inhibitor;²¹albeit, it is bound at the ATP

1

with the structure of 4. H NMR of the active component

Chart 2. Structural Elucidation of an HTS Hit

8092

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Chart 3. Structures of Oxindole-Based Kinase Inhibitors

indicated the absence of the azepan-1-ylmethylene group,

which further supported the proposal of the structure of 4. A

small molecule X-ray structure revealed that the real structure

of 3 was actually the structure of 5, a starting material used for

the preparation of compound 3 with the same molecular

formula and weight. As a result, the real structure of the active

component was deduced to be the structure of 6. 6 was

1

synthesized and conﬁrmed to have the same H NMR and

biological activity against c-MET as the active ingredient from

the HTS. Thus, the highly eﬃcient c-MET hit of 6 was

successfully identiﬁed through careful structural elucidation of

an HTS hit.

Discovery of an Exquisitely Selective c-MET Binding

Mode. 6 is an attractive hit with high biochemical ligand

eﬃciency (LE = −RT log K_i/the number of heavy atoms =

0.42) and lipophilic eﬃciency (lipE = −log K_i− cLogD = 4.80)

against c-MET. In addition, 6 was highly selective for c-MET,

showing an IC₅₀of >20 μM against a panel of other tyrosine

and serine/threonine kinases (VEGFR2, EGFR, SRC, DDR,

PDGFR, ZC1, PAK4, LCK, and LYN). Although the chemical

instability of the hydrazide linkage has a potential safety liability

associated with a possible release of the toxic hydrazine, the

intriguing selectivity proﬁle prompted further exploration of

this hit. Oxindole-based chemical scaﬀolds have been broadly

used as protein kinase inhibitors, with the oxindole generally

making hydrogen bonds to the backbone of the kinase hinge

segment in the active site of the kinase catalytic domain.

Examples include sunitinib (7), a potent split kinase inhibitor,²⁴

and 3-hydrazono-1,3-dihydro-indol-2-one 8 as CDK2 inhib-

itor²⁵(Chart3). Based on the cocrystal structure of 8 with

CDK2 (Figure 1a), it was rationalized that 6 should bind to c-

MET with the oxindole hydrogen bonding to the kinase hinge,

as occurs for most studied oxindole kinase inhibitors, and with

the hydrazide extending out to the protein solvent area (Figure

1b).

Even though 7 is a potent inhibitor against split kinases

including VEGFRs, PDGFR, and c-KIT, it is a relatively weak

inhibitor against c-MET with an enzymatic IC₅₀of 5 μM. A

dramatic increase in c-MET inhibitory potency was achieved

with the introduction of a 5-benzylsulfonyl group on the 3-[1-

(1H-pyrrol-2-yl)meth-(Z)-ylidene]-1,3-dihydroindol-2-one

scaﬀold, as illustrated with PHA-665752 (9), a potent and

selective c-MET inhibitor with a K_iof 9 nM.²⁶The cocrystal

structure of 9 with c-MET revealed a strong interaction of the

2,6-dichlorophenyl group with Tyr-1230,^16,27which is critical

for c-MET inhibition. A structural analysis was used to generate

hypotheses for the molecular underpinnings of the inhibitory

potency and kinase selectivity of 6. The solvent exposure area is

not expected to provide a high degree of kinase selectivity due

Figure 1. (a) Compound 8 bound in CDK2 (PDB ID 1FVT).²⁵(b)

Overlay of 8 with the proposed binding orientation of 6.

to the ﬂexibility and common structural features in this area.

Moreover, the traditional oxindole binding mode would hardly

rationalize the high ligand eﬃciency and selectivity of 6 against

c-MET (Figure 1b). It was therefore predicated that 6 have a

unique binding mode to c-MET for a proﬁle with high ligand

eﬃciency and selectivity. Chemistry optimization of 6 was

carried out to gain further insight on the potential of the series.

Demethylation of 6 improved potency dramatically, and the

optimization of the substituents on the oxindole ring aﬀorded

analogs with improved enzymatic and cellular potencies, as

illustrated with compound 10 having an enzymatic K_iof 1.3 nM

and an IC₅₀of 7 nM for the inhibition of c-MET

autophosphorylation in the A549 cell line.²⁸Inhibition of

phosphorylated c-MET KD in puriﬁed enzyme biochemical

assays conﬁrmed that the inhibitors can inhibit the active c-

MET, and the cell-based activity of the inhibitors prevented

RTK autophosphorylation, consistent with inhibitory inter-

action with the nonphosphorylated form in cells. The minor

8093

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

divergence between the enzyme and cell activities of 10 implied

the inhibition of both active and unactive c-MET.

A kinase domain (KD) construct of c-MET, in its

nonphosphorylated form, was prepared for utilization in

cocrystallization experiments. Indeed, the cocrystal structure

of 10 with nonphosphorylated c-MET protein demonstrated a

unique binding mode (Figure 2). Surprisingly, the oxindole ring

Figure 3. Overlay of c-MET protein structures from cocrystal

structures with 10 (gray) and 9 (green).

common kinase inhibitor binding areas, the chemical series of 6

represents a unique scaﬀold for the c-MET target with high

ligand eﬃciency and selectivity.

Replacement of the Chemically Unstable Oxindole

Hydrazide Scaﬀold. Although the oxindole hydrazide series

of 6 is a highly eﬃcient and selective scaﬀold for c-MET, it was

necessary to replace the chemically labile hydrazide linkage with

a stable structure. Based on the novel c-MET cocrystal

structures with oxindole hydrazide compounds, Vojkovsky et

al. fused the hydrazide with oxindole together to form a

tetracyclic aromatic scaﬀold as represented with 11 (Chart4).²⁹

The electron deﬁcient tetracyclic fused ring retained the strong

π−π stacking interaction with electron rich Tyr-1230 and could

form hydrogen bonds with NH of Asp-1226 and CO of

Arg-1208. As expected, 11 demonstrated equal potency against

c-MET. Although the tetracyclic scaﬀold solved the chemical

stability issue associated with the oxindole hydrazide series, the

druggability of the tetracyclic scaﬀold posed a concern. Zhang

et al. chopped the tetracyclic scaﬀold to the bicyclic

triazolotriazine scaﬀold as represented with 12 (Chart 4).³⁰

Surprisingly, 12 revealed a better ligand eﬃciency against c-

MET than the tetracyclic scaﬀold even though with the loss of

the hydrogen bond with CO of Arg-1208. The bicyclic

triazolopyrazine scaﬀold prepared by Cui et al. was slightly less

potent than the triazolotriazine scaﬀold, as demonstrated with

compound 13 (Chart4).³¹

Using insight from the cocrystal structure of an oxindole

hydrazide compound with c-MET, the chemically unstable

oxindole hydrazide scaﬀold was successfully replaced with

chemically stable triazolotriazine and triazolopyrazine scaﬀolds

that maintained ligand eﬃcacy (Chart4). Kinase selectivity

screening of 12 against a total of 98 protein kinases revealed an

exquisite selectivity for c-MET (Supporting Information

available). The unique binding mode results in unprecedented

selectivity; albeit, 12 is bound at the ATP binding site. The

cocrystal structure of 12 bound with nonphosphorylated c-

MET conﬁrmed the same binding mode as 10, however, with a

signiﬁcant movement of A-loop toward the inhibitor for a

better π−π stacking interaction between electron rich Tyr-1230

and the electron deﬁcient triazolotriazine ring (Figure 4). In

comparison of the cocrystal structure of 12 with 10, there is a

signiﬁcant change in the relative position of phenols. In the

cocrystal structure of 10 with c-MET, the carbonyl group from

Figure 2. Cocrystal structure of 10 with the nonphosphorylated c-

MET.

is not interacting with the hinge as for 8 bound with CDK2.

Instead, the phenolic moiety of 10 functions as the hinge

binder, with the phenol oxygen bound with the N−H of the

hinge Met-1160. Also, a water network links the phenol

hydroxyl and Met-1160 carbonyl group. As the molecule makes

a turn at the methylene linker, the large coplanar structure of

the hydrazide−oxindole allows for strong interactions with the

protein backbone. The oxygen of the hydrazide carbonyl group

hydrogen bonds with the N−H of Asp-1222, and the N−H of

oxindole hydrogen bonds with the carbonyl oxygen of Arg-

1208. The intramolecular hydrogen bond between hydrazide

and oxindole secures the large coplanar structure and induces

strong π−π interaction with Tyr-1230 of the A-loop. The

protein crystal structure of nonphosphorylated c-MET with 10

(Figure 2) reveals a similar conformation as the non-

phosphorylated c-MET with 9 (PDB ID 2wkm). A unique β-

turn at the beginning of the A-loop is formed, and the c-MET

protein adopts an autoinhibitory conformation with the A-loop

occupying the binding site for the triphosphate of ATP. This

unique c-MET autoinhibitory conformation is consistent with

the unphosphorlated apo-c-MET¹¹and other c-MET−inhibitor

cocrystal structures.¹⁶Although these c-MET cocrystal

structures have the same overall protein fold, the relative

position of the A-loop is varied. The A-loop and G-loop

(glycine-rich loop) of the c-MET protein in the cocrystal

structure of 10 are closer to the inhibitor, and the binding

pocket is smaller at the adenine binding site (Figure 3) in

comparison with 9. With the structural overlay of 10 with 9

illustrating the changes (Figure 3), 10 has much reduced

molecular weight at the common hinge binding area (phenol

group in 10 vs 3-[1-(1H-pyrrol-2-yl)meth-(Z)-ylidene]-1,3-

dihydroindol-2-one) in 9; however, it has increased molecular

weight at the noncommon area, enabling a stronger interaction

with the A-loop and the catalytic loop. Because of the strong

interaction with the A-loop and reduced interactions at the

8094

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Chart 4. Eﬀorts in the Replacement of the Chemically Unstable Oxindole Hydrazide Scaﬀold

were explored (Table 1). Although the interaction with the A-

loop residue Tyr-1230 is important for overall potency and

selectivity, it is necessary to use the hinge binder to anchor the

molecule into the binding pocket. When the phenol group was

removed, 14 was not active against c-MET with a K_i> 10 μM.

The phenol hydroxyl group in concert with a water molecule

formed a hydrogen bond network with hinge residue Met-1160.

The replacement of the hydroxyl group with the methoxy

group in 15 interrupted the hydrogen bound network, and the

activity dropped more than 10-fold in the c-MET cellular assay.

16 with a ﬂuoro group dropped more than 300-fold of activity

in the c-MET enzymatic assay. 17 with an ortho-chloro group

reduced the potency signiﬁcantly. 18 with the N-substituted

pyridin-4(1H)-one hinge binder showed a signiﬁcant reduced

potency (6.51 μM), likely because of desolvation penalty from

the polar carbonyl group. More exploratory approaches

including 19−22 with an extended two-atom linker were

carried out. 22 demonstrated potent inhibition against c-MET

in both enzymatic and cellular assays. 23 and 24, with the

conventional protein kinase hinge binders quinoline and

azaindole, were potent c-MET inhibitors. In summary, the

phenol hinge binder could be replaced by other kinase hinge

binders that retained potency against c-MET. Not surprisingly,

the cocrystal structure of 24 with c-MET showed good overlay

with the cocrystal structure of 12 with c-MET (Figure 5).

In parallel with the eﬀorts to replace the phenol hinge binder,

the ADME properties were optimized. In general, the

triazolotriazine chemical series had good caco-2 permeability

(data not shown). The metabolic stability in the human

microsome varied according to the overall lipophilicity and

chemical structures (Figure 6a). However, the series demon-

strated high clearance in human hepatocyte even at very low

lipophilicity (Figure 6b). The stable compounds in the human

hepatocyte assay have O- or N-substituents at the 7-position of

the triazolotriazine core, which indicated that the highly

electron deﬁcient triazolotriazine core is subject to non-p450

enzymatic oxidation. From the analyses of cLogD and human

metabolic stability (Figure 6), it was concluded that there

would be no druggable chemistry space for the triazolotriazine

chemical series, and the series was deprioritized.

Figure 4. Overlay of cocrystal structures of 10 (gray) and 12 (purple)

bound with the nonphosphorylated c-MET.

hydrazide forms a good hydrogen bond with N−H of Asp-

1226, and the carbonyl group of oxindole does not have a direct

interaction with the protein backbone; however, the oxindole

carbonyl forms an intramolecular hydrogen bond with

hydrazide to generate a coplanar structure for a π−π stacking

interaction with Tyr-1230. For compound 12, it is the N-1 of

triazolotriazine that forms a hydrogen bond with Asp-1226

from a diﬀerent angle compared to that of the carbonyl group

of the hydrazide in 10, which places the triazolotriazine ring in a

better position for the π−π stacking interaction with Tyr-1230,

and the phenol group closer to the hinge for a better

hydrophobic interaction with Met-1226 and hydrogen bond

interaction with the hinge Met-1160 in a slightly diﬀerent

position in comparison with the phenol in 10. Without the

restriction from the interaction with the CO of Arg-1208, the

5-phenyl group in 12 orients in a diﬀerent angle as compared

with the oxindole in 10. Thus, the improved ligand eﬃciency of

the triazolotriazine series is likely due to better π−π stacking

interaction from electron rich Tyr-1230 with the electron

deﬁcient triazolotriazine ring.

Optimization to the Clinical Candidate 2-(4-(1-

(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-

yl)-1H-pyrazol-1-yl)ethanol (2). Alternatively, the less

potent triazolopyrazine series of 13 was explored for potential

acceptable ADME properties. The SAR for the phenol

replacement of 13 was similar with the triazolotriazine series

of 12, as exempliﬁed with 25−27 (Table 2). The 6-substituted

quinoline hinge binder in 27 provided good potency against c-

Replacement of the Hinge Binder Phenol. Phenols are

well-known to undergo phase II glucuronidation metabolism.

Therefore, the next design was focused on the replacement of

the phenol hinge binder to optimize the chemical scaﬀold into a

more druggable chemical series. A variety of aromatic groups

with heteroatoms as potential hydrogen bond donors and

acceptors for the interaction with hinge amino acid residues

8095

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Table 1. Structure Activity Relationship of Phenol Replacements

a

b

Calculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat version 9.3. Inhibition constants (K_i) and cell IC₅₀

were determined as described under Experimental Section. The coeﬃcients of variance were typically less than 20% (n = 2). A549 human lung

c

d

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. LipE (IC₅₀) = pIC₅₀− cLogD. Human liver

microsomal intrinsic clearance (μL/min/mg). Human hepatocyte intrinsic clearance (μL/min/million). ND, not determined.

e

MET and is about 10-fold less potent in the c-MET cellular

autophosphorylation assay than the triazolotriazine analogue

23. However, the metabolic stabilities in both human liver

microsomes and the hepatocyte cell were improved even

though the lipophilicity of 27 was about 0.71 unit higher than

that of 23, which implies that the triazolopyrazine scaﬀold was

metabolically more stable than triazolotriazine. To further

evaluate the potential of the triazolopyrazine series, the

analogues 28−32 with a 6-pyrazol-4-yl substitute were

synthesized and demonstrated improved LipE and metabolic

stabilities. 28 is 57-fold more potent than 25 (0.051 μM over

2.92 μM) in the c-MET cellular assay even though the

biochemical potencies (K_i) are similar. The enzymatic assay

used the active, autophosphorylated c-MET protein with

biochemical potency (K_i) based on the ability of the inhibitor

to prevent the phosphorylation of a substrate. The cocrystal

structure of 12 with nonphosphorylated c-MET revealed an

autoinhibitory conformation of the c-MET protein with the

activation loop occupying the ATP triphosphate binding site.

12 stabilized the nonphosphorylated c-MET in an inactive

conformation and resulted in the potent inhibition of c-MET

autophosphorylation in the cell. The N-methylpyrazol-4-yl

group in 28−32 allows a better coplanar structure with the

triazolopyrazine core than the phenyl group in 25−27. The

large coplanar structure facilitates the π−π stacking interaction

with Tyr-1230 in the A-loop and led to improved cellular

potency. The N-methylpyrazolyl group lowers the LogD by

more than two units and results in a signiﬁcant improvement of

LipE, as exempliﬁed with 31, which has a LipE of 8.32 in

comparison of 4.61 for the close analogue 27. With dramatically

lowered lipophilicity, 31 is metabolically stable in both human

microsome and hepatocyte. 32 with a quinazoline hinge binder

was 7-fold less potent than 31, as expected with the reduced

hydrophobic interaction, reduced hydrogen bond strength, and

increased desolvation penalty. In summary, a new druggable

chemical series of 6-((1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)-

8096

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

substituents, in addition to the aryl and heteroaryl groups at the

6-position of the triazolopyrazine core, were investigated

(Table 4). The essential core 42 with R = H showed a c-

MET cellular potency of 253 nM with a LipE of 6.27. 42 has

the desired ADME properties (good solubility, metabolic

stability, and permeability) for further optimization. 43 with a

lipophilic methyl group improved c-MET cellular potency by 3-

fold with a similar LipE value (of 6.30) to that for 42. The

amino function in 44 enhanced c-MET cell potency (34 nM),

with a much improved LipE of 8.42, and lowered the aqueous

solubility to 70.6 μM. 45, with a lipophilic ethylamino group,

further improved c-MET cell potency (6 nM), however with a

lower LipE of 8.06, and had a low solubility of 6.57 μM. 46,

with the hydroxyethylamino group, showed a reduced

permeability. 47, with the dimethylaminoethyl amino group,

had reduced c-MET cell potency even though the solubility is

good. 51, with the dimethylaminoethyloxy group, showed

similar properties as 47, except better permeability. The cyclic

amino substituents at the 6-position showed good potency and

ADME properties, as demonstrated with 48−50. In summary, a

variety of substituents at the 6-position of the triazolopyrazine

core are tolerated, which provides a good opportunity to

optimize potency and pharmaceutical properties.

Figure 5. Overlay of cocrystal structures of 24 and 12 with

nonphosphorylated c-MET.

After comprehensive multiparameter lead optimization, a

number of compounds were selected for in vivo rat PK studies

based on potent inhibition of c-MET at both enzymatic and

cellular assays and good in vitro ADME properties (Table 5).

The basic compounds 41 and 48 showed unexpected high in

vivo rat plasma clearance. Additional compounds with a basic

substituent at the 6-position were studied in in vivo rat PK and

showed similar results to those for 41 and 48 (data not shown).

It was suspected that non-p450 related metabolic mechanisms

may be linked with the disconnection between the in vitro

clearance prediction from rat microsomal stability and in vivo

PK results. 31, 2, and 44 showed low to moderate in vivo rat

clearance, low volume distribution, and good oral bioavail-

ability. 2 showed a better half-life and volume distribution

proﬁle and was chosen for further evaluations.

The cocrystal structure of 2 with a nonphosphorylated c-

MET kinase domain showed a similar binding mode (Figure 7)

to that of the complexes of 12 and 23 with c-MET. The A-loop

adopts an autoinhibitory conformation and possesses a strong

interaction with 2 via a c-MET characteristic π−π interaction of

Tyr-1230 with a triazolopyrazine ring. The quinoline group in 2

is docked into the hinge through a hydrogen bond with Met-

1160 and hydrophobic interactions with Ile-1084, Val-1092,

Ala-1108, and Met-1211. N-3 on the triazolopyrazine ring

forms a hydrogen bond with the N−H of the DFG Asp-1222

residue. Triazolopyrazine is anchored with Tyr-1230 and Met-

1211, and the pyrazole moiety forms a hydrogen bond network

with a water molecule which interacts with CO of Tyr-1230

and N−H of Arg-1086. The hydroxyl group from the ethanolic

group on the pyrazole forms hydrogen bonds with Asp-1231

and a water molecule.

Figure 6. (a) Human liver microsomal clearance (HLM: CLint., app)

vs cLogD; (b) Human hepatocyte clearance (HHEP: CLint., app) vs

cLogD.

methyl)quinoline was successfully identiﬁed to replace the 6-

((1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)methyl)quinoline series

with good chemical and metabolic stabilities, and c-MET

cellular potency.

31 demonstrated good cellular potency against c-MET (IC₅₀

= 1 nM), high cellular lipE (8.32), and good human microsome

stability and permeability. However, the low solubility of 31

would impact the oral absorption potentially. The cocrystal

structure of 12 with c-MET suggested an open space at the 6-

pyrazol-yl binding position between the G-loop and the A-loop

with a potential interaction with Asp-1164. A variety of polar

groups were introduced on the pyrazole to improve the

aqueous solubility (Table 3). The unsubstituted pyrazole 33

showed similar potency as 31, however with even lower

solubility and worse metabolic stability. 2 with an ethanolic

group showed a comparable potency and ADME properties as

31. 35 with a carboxyl ethyl group had a similar K_i; however, it

was less active in the cell because of low permeability. The

introduction of the more lipophilic group 1,1-dimethyl-1-

hydroxyethyl in 36 reduced the potency and decreased the

metabolic stability. The basic compounds 37 and 38 showed

much improved solubility with good potency, metabolic

stability, and permeability. 39, with a 1-ethylpyrrolidin-2-one

group, showed reduced potency. 40 and 41, with secondary

basic groups, showed good potency and reduced permeability.

To further deﬁne the SAR of the 6-((1H-[1,2,3]triazolo[4,5-

b]pyrazin-1-yl)methyl)quinoline series, a variety of diﬀerent

As expected, 2 demonstrates an exquisite c-MET kinase

selectivity (>1000 selectivity to 208 protein kinases).²³Despite

having a similar overall protein conformation in the cocrystal

structures of 2 and crizotinib (PDB ID 2wgj) with non-

phosphorylated c-MET (Figure 8), crizotinib is less selective,

being a c-MET/ALK/ROS/RON inhibitor at the cellular

level.¹⁶Stronger interactions with A-loop Tyr-1230 and also a

hydrogen bond to the amide of Asp-1222 may be the basis for

the exquisite kinase selectivity of 2. This hypothesis is

8097

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Table 2. Structure Activity Relationship of the Triazolopyrazine Series

a

b

Calculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat version 9.3. Inhibition constants (K_i) and cell IC₅₀

were determined as described in the Experimental Section. The coeﬃcients of variance were typically less than 20% (n = 2). The A549 human lung

c

d

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. LipE (IC₅₀) = pIC₅₀− cLogD. Human liver

microsomal intrinsic clearance (μL/(min mg)). Human hepatocyte intrinsic clearance (μL/(min million)). ND, not determined.

e

supported by the dramatic decrease in potency of 2 against c-

MET when Tyr-1230 is mutated. For example, 2 is almost

1000-fold less active against the c-MET activation loop mutants

Y1230C (K_i, 9,400 nM) and Y1235H (K_i, 3,900 nM).²³

However, crizotinib is only 10-fold less potent against Y1230C

c-MET.²³

with Tyr-1230 eﬃciently and served as a basis for comparison.

In addition to the π−π stacking interaction with Tyr-1230,

fragment A formed two pairs of strong hydrogen bonds with

the protein backbone, oxindole NH with the CO of Arg-

1208 and CO of hydrazide with the NH of Asp-1222, as

shown in the complex of 10 with c-MET (Figure 2). While

fragment B retained similar interactions as fragment A with c-

MET protein, fragment C showed more blue color (Figure 9),

indicating more electron deﬁciency in the aromatic system for a

stronger π−π stacking interaction with Tyr-1230. Indeed, 12

was equally potent as 11, however, with a reduced molecular

weight, which led to improved ligand eﬃciency. The stronger

interaction with Tyr-1230 of 12 compensated the weaker C

H hydrogen bond with CO of Arg-1208. D had a higher

electron density for a weaker π−π stacking interaction as

compared with C, which was responsible for a decreased

activity of 13 relative to 12 against c-MET. The substituted

phenyl fragment E in crizotinib showed a signiﬁcantly diﬀerent

electron potential as compared with fragments A−D. The

relatively high electron density in E led to a weaker interaction

with Tyr-1230, and crizotinib lost only 10-fold potency against

Y1230C mutant c-MET as compared with 2 (>1000 fold).

Crizotinib had additional hydrophobic interactions which were

common for other kinases and led to lower selectivity in

enzymatic assays. Distinguishing from crizotinib, 2 mainly

beneﬁted from the π−π stacking interaction with Tyr-1230.

The diﬀerent selectivity proﬁles of 2 and crizotinib indicate

the relatively important roles of the Tyr-1230 interaction with

the inhibitors. To further explore the nature and the

contributions of the Tyr-1230 interaction with 10−13 and

crizotinib, the corresponding interaction fragments A−E were

optimized at the B3LYP/6-31G** level of theory with PBF

solvation, and the electronic property calculations were

completed on the fragments (Figure 9). The electron density

of only the relevant portion of each fragment was revealed for

clarity. All the corresponding bond lengths (distances) and

bond angles of optimized geometries were in good agreement.

The strength of the π−π stacking interaction of the electron

rich Tyr-1230 residue should depend on the electron deﬁciency

of the interacting aromatic system in the kinase inhibitors.

Three-dimensional-average local ionization energy (ALIE)

superimposed onto a surface of constant electron density

(0.01 e/au³) showing the most positive potential region

(deepest blue color for the most electron deﬁcient area) and

the most negative potential region (deepest red color for the

most electron rich area). The large conjugated coplanar

fragment A in the initial HTS hit chemical series interacted

8098

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Table 3. Structure Activity Relationship of Substituted 6-Pyrazol-4-yl Group

a

b

Calculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat version 9.3. Inhibition constants (K_i) and cell IC₅₀

were determined as described in the Experimental Section. The coeﬃcients of variance were typically less than 20% (n = 2). A549 human lung

c

d

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. LipE (IC₅₀) = pIC₅₀− cLogD. Kinetic apparent

e

solubility at pH 6.5. Human liver microsomal intrinsic clearance (μL/(min mg)). Caco-2 permeability (10⁻⁶cm/s). ND, not determined.

The unique autoinhibitory conformation of c-MET determined

the exquisite selectivity proﬁle of 2.

2C19, 2D6, and 3A4 (IC₅₀>30 μM) in human liver

microsomes; however, it moderately inhibited CYP2C9 (IC₅₀

∼ 5 μM). 2 was well absorbed in animals with a projected

fraction absorbed of >90% in humans. The CL_bloodand V_ss

values in humans were predicted to be 4−8 mL/(min kg) and

2.2 L/kg, respectively.³³The human PK proﬁle was evaluated

in phase I clinical studies. After a single oral administration at

the dose of 30 mg to healthy volunteers, 2 was rapidly and

extensively absorbed with a mean C_maxof 0.24 μg/mL at 1.2 h

postdose. Thereafter, 2 declined multiexponentially, with an

apparent mean t_1/2of 6.6 h. The mean area under the plasma

concentration−time curve was 0.81 (μg h)/mL, which

corresponded to an oral clearance of 9 mL/(min kg).³³

2 was negative in the Biolume Ames assay in the presence or

absence of rat liver S9 metabolic activation. In the CHO cell

micronucleus assay, 2 was also negative in the absence and

presence of S9. 2 had a low risk of QT prolongation based on

the hERG patch clamp studies (IC₅₀> 30 μM). 2 also displayed

an acceptable safety proﬁle in exploratory toxicity studies.

Taken on the whole, 2 was a potent and highly selective c-

MET inhibitor in vitro and in vivo. On the basis of the

pharmacologic and pharmacokinetic properties, a good safety

margin in preclinical animal studies, and a low risk for clinically

relevant drug−drug interaction, 2 was selected for human clinic

studies for cancer patients with abnormal c-MET activation.

The antitumor eﬃcacy (TGI) and relationship with the

inhibition of c-MET autophosphorylation of 2 were evaluated

in the c-MET ampliﬁed GTL-16 xenograft tumor model

(Figure 10). Near complete inhibition of c-MET activity for 24

h is consistent with maximal inhibition of tumor growth (30

and 10 mg/kg, 100−90% TGI), and potent inhibition of c-

MET activity for only a portion of the schedule is consistent

with signiﬁcant but submaximal eﬃcacy (3 and 1 mg/kg, 74−

64% TGI). These results suggest that near-complete inhibition

of c-MET autophosphorylation (>90% inhibition) for the

duration of the administration schedule is necessary to

maximize therapeutic beneﬁt. The antitumor eﬃcacies in

other tumor models and the mechanisms of antitumor eﬃcacy

are published elsewhere.³²

2 was a weak base with a pK_avalue of 4.6, resulting in a pH

dependent aqueous solubility. The solubility of the crystal

mesylate salt of 2 at 25 °C in simulated gastric ﬂuid (SGF, pH

1.2) was >2.0 mg/mL. In sodium phosphate buﬀer (pH 6.5,

0.05 M), the solubility was signiﬁcantly lower, at 0.6 μg/mL. 2

showed high metabolic stability in human liver microsomes,

moderate human plasma protein binding (84%), and high

permeability in the Caco-2 cell assay. Biotransformation

mediated by CYP3A4 was likely to be the major clearance

mechanism for 2. 2 was not an inhibitor of CYP1A2, 2C8,

8099

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Table 4. Structure Activity Relationship of 6-Substituents on ((1H-[1,2,3]Triazolo[4,5-b]pyrazin-1-yl)methyl)quinoline

a

b

Calculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat version 9.3. Inhibition constants (K_i) and cell IC₅₀

were determined as described in the Experimental Section. The coeﬃcients of variance were typically less than 20% (n = 2). The A549 human lung

c

d

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. LipE (IC₅₀) = pIC₅₀− cLogD. Kinetic apparent

e

f

solubility at pH 6.5. Human liver microsomal intrinsic clearance (μL/(min mg)). Caco-2 permeability (10⁻⁶cm/s). ND, not determined.

Table 5. In Vivo Rat Pharmacokinetic Properties

a

b

c

compd

MW

pK_a

cLogD

eLogD

CL_plasma(mL/(min kg))

V_ss(L/kg)

T_1/2(h)

F_oral(%)

31

2

342

372

411

277

346

4.66

0.68

0.05

1.29

1.38

ND

1.23

0.86

9.7

8.2

0.42

1.6

1.5

3.7

5.0

1.6

0.9

76

71

41

44

48

10.2

−1.45

−0.95

−1.73

81.7

26.7

27

BQL

75

4.66

8.52

0.95

3.6

83.4

BQL

a

b

Calculated ionization constant of a molecule using ACD pchbat version 9.3. Calculated logarithm of the octanol/water distribution coeﬃcient at

pH 7.4 using ACD pchbat version 9.3. Experimentally measured bilayer participating coeﬃcient at pH 7.4. BQL: below quantiﬁcation level.

c

reﬂux to provide N²-substituted 6-bromo-pyrazine-2,3-diamine

54. The Sandmeyer reaction using isoamyl nitrite in

dimethylformamide (DMF) closed the ring to provided the

key intermediate 55. 13 and 25−33 were obtained with

conventional Suzuki coupling conditions.

The synthesis of 2 was summarized in Scheme 3. 58 was

prepared under conventional alkylation conditions with

CHEMISTRY

■

12 and 14−24 were prepared according to Scheme 1. 52a−b

were prepared according to the literature procedure³⁴and were

coupled with a carboxylic acid in the presence of the coupling

reagents hydroxybenzotriazole (HOBT) and 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC) in N-methylpyrro-

lidone (NMP) at ambient temperature. After the completion of

the coupling reaction, the reaction mixture was microwaved at

170 °C until the formation of the triazolotriazine core was

completed.

13 and 25−33 were prepared according the general three-

step procedure in Scheme 2. The substituted amine replaced

the 3-bromo group of 3,5-dibromopyrazin-2-amine in n-butanol

with the presence of diisopropylethylamine (DIPEA) under

Cs₂CO₃as a base in DMF solvent at 70 °C. The boronic

ester was introduced via metalation with i-PrMgCl and then

quenched with the boronic ester reagent. 2 was prepared via

conventional Suzuki coupling conditions followed by depro-

tection under hydrogen chloride acidic conditions.

The general alkylation condition was used to introduce

various substituted groups on the pyrazole ring of 33, as

illustrated in Scheme 4.

8100

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Figure 7. Cocrystal structure of 2 with nonphosphorylated c-MET.

Figure 9. Electronic property calculations of fragments A−E.

having a strong interaction with the activation loop. Based on

insight from the cocrystal structure, a tetracyclic mimic,

represented with 11, was designed, and it demonstrated

increased potency and the same selectivity proﬁle. Further

optimization of druglike properties resulted in a triazolotriazine

series, as represented with 12. The quinoline group was

designed to replace the phenol group as a hinge binder, and the

triazolopyrazine ring was used to replace the metabolically

labile triazolotriazine ring to generate a new chemically and

metabolically stable scaﬀold, as illustrated with compound 23.

Medicinal chemistry optimization of the triazolopyrazine series

led to the discovery of 2, which demonstrated potent inhibition

of c-MET in both in vitro cell assays and in vivo target

modulation studies. In preclinical studies, 2 had signiﬁcant

tumor growth inhibition, good oral PK properties, and an

acceptable safety margin. 2 is an ATP competitive inhibitor and

bound at the adenine binding pocket. However, the kinase

selectivity screens against 208 diﬀerent protein kinases

indicated an exquisite selectivity proﬁle associated with 2,

which could be explained with the unique binding topography

of the c-MET kinase domain. 2 was advanced into phase I

clinical studies for oncology indications.

The successes of imatinib for chronic myeloid leukemia

patients having BCR-ABL abnormal gene, erlotinib for

advanced nonsmall cell lung cancer patients having EGFR

mutations, and crizotinib for lung cancer patients having

EML4-ALK fusion gene illustrate the importance to identify the

disease-driven oncogene for the eﬀective treatment of cancers.

However, genetic intratumor heterogeneity presents major

challenges to personalized medicine and can lead to tumor

adaptation and resistance to treatment.³⁵Frequently, there are

Figure 8. Overlay of cocrystal structures of 2 (gray) and crizotinib

(green) with nonphosphorylated c-MET.

The preparation of 42−51 was summarized in Scheme 5.

Hydrogenation of 55d gave 42. The methyl group was

introduced from 55d under palladium catalyst to provide 43.

44 was prepared with ammonia under microwave thermal

conditions. A variety of substituted amino groups were

introduced with potassium carbonate as a base under

microwave thermal conditions to provide 45−50. The alkoxy

compound 51 was prepared with triethylamine as a base under

microwave thermal conditions.

CONCLUSIONS

■

Oxindole hydrazide 6 with an unusual kinase selectivity proﬁle

was a hit in a c-MET receptor tyrosine kinase HTS campaign.

The selectivity proﬁle could not be rationalized with the

conventional oxindole kinase inhibitor binding mode in which

the oxindole bound to the hinge region of the ATP binding site.

A cocrystal structure of the related oxindole hydrazide

compound 10 with the nonphosphorylated c-MET kinase

domain conﬁrmed a unique binding mode with a phenol group

bound to the hinge region and the oxindole hydrazide portion

8101

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Figure 10. Tumor growth inhibition of 2 in the GTL-16 xenograft tumor model.

a

Scheme 1

a

Reagents and conditions: (a) HOBT, EDC, NMP, ambient temperature, 1−2 h; (b) microwave at 170 °C, 0.5 h or longer.

a

Scheme 2 .

a

Reagents and conditions: (a) n-butanol, DIPEA, reﬂux, 48 h; (b) isoamyl nitrite, DMF, ambient temperature, overnight, 70 °C to the completion;

(c) Pd(dppf)Cl₂·CH₂Cl₂, Na₂CO₃, DME/H₂O, 80 °C, 3.5 h.

prepacked silica ﬁlled cartridges. NMR spectra were generated on a

Bruker 300 or 400 MHz instrument and obtained as CDCl₃or

DMSO-d₆or MeOH-d₄solutions (reported in ppm), using CDCl₃as

the reference standard (7.27 ppm), DMSO-d₆(2.50 ppm), and

MeOH-d₄(3.31 ppm). Multiplicities were given as s (singlet), br s

(broad singlet), d (doublet), t (triplet), dt (double of triplets), and m

(multiplet). Mass spectral data (APCI) was gathered on an Agilent

1100 LC with MSD (Agilent model G1946B upgraded to D model)

single-quadrupole mass spectrometric detectors running with an

atmospheric pressure chemical ionization source. The LC instrument

includes a binary pump (Agilent model G1312A) with an upper

pressure limit of 400 bar attached to an autosampler (Agilent model

G1313A) which uses an external tray for sample submission. The

column compartment (Agilent model G1316A) is attached to a diode

array (Agilent model G1315A). The instrument acquisition and data

handling were done with ChemStation rev B.02.01. The purity

measurements were done by measuring peak area at 254 nm, 224 nm,

and total ion chromatogram. To evaluate the purity of each peak, the

UV−vis spectrum from 190 to 700 nm at a step size of 2 nm and mass

spectrum scan from 150 to 850 amu with a cycle time of 0.29 cycle/s

more than one abnormal genes associated with a particular

cancer. The design of a kinase inhibitor targeting a speciﬁc

subset of kinases may not be practical because of the diversiﬁed

kinase structures and intrinsic kinase activities. The combina-

tion of drugs targeting speciﬁc mechanisms associated with the

disease may represent the future of cancer treatment. Highly

selective kinase inhibitors should provide good opportunities to

combine together for maximum eﬃcacy and minimum toxicity.

2, distinguished from multitargeted c-MET inhibitors currently

in clinical trials, will provide opportunities to validate the role of

the c-MET target in tumor development, and it could be used

as a single agent or combined eﬀectively with other agents for

the treatment of cancers.

EXPERIMENTAL SECTION

■

General Methods for Chemistry. All reagents and solvents were

used as purchased from commercial sources. Reactions were carried

out under nitrogen atmosphere unless otherwise indicated. Silica gel

chromatography was done using the appropriate size Biotage

8102

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

a

Scheme 3.

a

Reagents and conditions: (a) Cs₂CO₃, DMF, 70 °C, 16 h; (b) i-PrMgCl, THF, 0 °C to RT, 1 h; (c) Pd(dppf)Cl₂·CH₂Cl₂, Cs₂CO₃, DME/H₂O, 80

°C, 16 h; (d) 4 N HCl/dioxane, CH₂Cl₂, RT.

a

was performed. Retention times (RT) were in minutes, and purity was

calculated as percentage of total area. Two HPLC methods were

utilized for purity. Method A: Waters Acquity UPLC BEH C18

column, 1.7 μm, 2.1 mm × 100 mm, column temperature 80 °C;

solvent A: water (0.1% formic acid and 0.05% ammonium formate);

solvent B: methanol (0.1% formic acid and 0.05% ammonium

formate); gradient: 5−95%B in 10 min, 95%B 10−12 min; ﬂow rate

0.6 mL/min. Method B: EclipsXDB C8 column, 3.5 μm, 4.6 mm × 50

mm, column temperature 40 °C; solvent A: water (5% ACN, 2 mM

ammonium acetate, 0.1% acetic acid); solvent B: ACN (5% H₂O, 2

mM ammonium acetate, 0.1% acetic acid); gradient: 20−85%B (0.0−

2.5 min), 85−95%B (2.5−3.5 min), 95%B (5 min); ﬂow rate = 0.8

mL/min. Compound purity was determined by combustion analysis

(Atlantic Microlabs, Inc.) or high pressure liquid chromatography

(HPLC) with a conﬁrming purity of ≥95% for all ﬁnal biological

testing compounds.

Scheme 4.

a

Reagents and conditions: (a) Cs₂CO₃, DMF, 80 °C, overnight.

General Procedure A for the Preparation of Triazolotriazine

Compounds. To a solution of substituted acetic acid (1 molar equiv),

a

Scheme 5.

a

Reagents and conditions: (a) H₂, Pd/C, MeOH/AcOH/EtOAc, 18 h; (b) MeB(OH)₂, Pd(PPh₃)₂Cl₂, Na₂CO₃, DME/H₂O, 80 °C, 16 h; (c)

concentrated ammonia−water solution, microwave at 100 °C, 1 h; (d) K₂CO₃, i-PrOH, microwave at 80 °C, 20 min; (e) Et₃N, n-BuOH, microwave

at 120 °C, 20 min.

8103

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

HOBT (1 molar equiv), and EDC (1.1 molar equiv) in NMP (0.25

M) in a microwave reaction vessel was added 52a or 52b (1.0 molar

equiv). The suspension was stirred at ambient temperature until a clear

solution was obtained and a complete conversion to the intermediate

53 was observed by LC-MS (it took 1−2 h). The reaction vessel was

then microwaved at 170 °C for 30 min or longer until over 90%

conversion to the ﬁnal product was observed in LC-MS. The reaction

solution was diluted with ethyl acetate, washed with water 5 times to

remove the reaction solvent, and washed with brine. The organic

solution was dried over Na₂SO₄, ﬁltered, and concentrated. The crude

product was puriﬁed on a silica gel column, eluting with EtOAc−

hexane (50% to 100% EtOAc), or puriﬁed on a reversed phase

preparative HPLC, eluting with 10−40% (30 min) acetonitrile in water

(containing 0.1% acetic acid). A highly crystalline solid product was

obtained.

3-(4-Fluorobenzyl)-6-(4-ﬂuorophenyl)[1,2,4]triazolo[4,3-b]-

[1,2,4]triazine (16). To a solution of (4-ﬂuorophenyl)acetic acid

(154.1 mg, 1.0 mmol), HOBT (1 molar equiv), and EDC (1.1 molar

equiv) in NMP (4.0 mL) in a microwave reaction vessel was added [6-

(4-ﬂuorophenyl)[1,2,4]triazin-3-yl]hydrazine (205.2 mg, 1.0 mmol).

The suspension was stirred at ambient temperature for overnight. LC-

MS showed a complete conversion to the hydrazide intermediate. The

reaction vessel was then microwaved at 170 °C for 30 min and LC-MS

showed about 30% product was formed, at 170 °C for an additional 30

min and 60% product, and at 170 °C for another 30 min and >90%

product was formed from LC-MS. The reaction solution was diluted

with ethyl acetate, washed with water for 5 times to remove the

reaction solvent, and washed with brine. The organic solution was

dried over Na₂SO₄, ﬁltered, and concentrated. The crude product was

puriﬁed on a silica gel column eluting with EtOAc−hexane (50% to

100% EtOAc) to provide a yellow crystalline solid product (110 mg,

34% yield); LC-MS m/z 324 (M + H)⁺; ¹H NMR (400 MHz, DMSO-

d₆) δ 9.35 (s, 1H), 8.25 (dd, J = 5.43, 8.97 Hz, 2H), 7.41−7.56 (m,

4H), 7.16 (t, J = 8.97 Hz, 2H), 4.57 (s, 2H); HPLC purity (method

A): RT 11.78, >99%.

(m, 2H), 8.18 (d, J = 7.58 Hz, 1H), 8.05 (d, J = 5.56 Hz, 1H), 7.67 (d,

J = 5.56 Hz, 1H), 7.49−7.55 (m, 2H), 6.16 (d, J = 7.58 Hz, 1H), 6.13

(s, 2H); HPLC purity (method B): RT 2.08, >95%.

6-(4-Fluorophenyl)-3-((1H-pyrazolo[3,4-d]pyrimidin-4-

ylthio)methyl)[1,2,4]triazolo[4,3-b][1,2,4]triazine (21). LC-MS

1

m/z 380 (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ 14.19 (br s,

1H), 9.39 (s, 1H), 8.81 (s, 1H), 8.36 (s, 1H), 8.11−8.17 (m, 2H),

7.39−7.48 (m, 2H), 5.34 (s, 2H); HPLC purity (method A): RT

11.23, >95%.

3-(((6-(4-Fluorophenyl)[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-

yl)methyl)thio)-5-(methylamino)isothiazole-4-carboxamide

(22). LC-MS m/z 417 (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ

9.39 (s, 1H), 8.19−8.27 (m, 2H), 8.02 (br s, 1H), 7.44−7.55 (m, 2H),

7.09 (br s, 2H), 4.99 (s, 2H), 2.83 (s, 3H); HPLC purity (method B):

RT 2.71, >95%.

6-((6-(4-Fluorophenyl)[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-

1

yl)methyl)quinoline (23). LC-MS m/z 357 (M + H)⁺; H NMR

(400 MHz, DMSO-d₆) δ ppm 9.37 (s, 1H), 8.93 (d, J = 4.04 Hz, 1H),

8.44 (d, J = 8.59 Hz, 1H), 8.22−8.29 (m, 2H), 8.01−8.06 (m, 2H),

7.88 (d, J = 9.09 Hz, 1H), 7.60 (dd, J = 8.21, 4.17 Hz, 1H), 7.46−7.51

(m, 2H), 4.81 (s, 2H); HPLC purity (method B): RT 2.83, >95%

4-(3-((1H-Pyrrolo[2,3-b]pyridin-3-yl)methyl)[1,2,4]triazolo-

[4,3-b][1,2,4]triazin-6-yl)benzonitrile (24). LC-MS m/z 353.0 (M

1

+ H)⁺; H NMR (400 MHz, DMSO-d₆) δ 11.51 (br s, 1H), 9.39 (s,

1H), 8.37 (d, J = 8.59 Hz, 2H), 8.20 (dd, J = 1.52, 4.55 Hz, 1H), 8.12

(d, J = 8.84 Hz, 2H), 8.04−8.08 (m, 1H), 7.49 (d, J = 2.27 Hz, 1H),

7.06 (dd, J = 4.67, 7.96 Hz, 1H), 4.69 (s, 2H); HPLC purity (method

A): RT 10.84, >95%.

General Procedure B for the Preparation of Triazolopyr-

azine Compounds 13 and 25−33. Step 1: 6-Bromo-N²-

(quinolin-6-ylmethyl)pyrazine-2,3-diamine (54d). 3,5-Dibromo-

pyrazin-2-amine (21.8 g, 0.086 mol), quinolin-6-ylmethaneamine (13.6

g, 0.086 mol), and diisopropylethylamine (16.5 mL, 0.0946 mol) were

dissolved in n-BuOH (150 mL). The reaction mixture was reﬂuxed for

2 days and evaporated. The residue was treated with CH₂Cl₂(100

mL) and water (200 mL). The obtained mixture was vigorously stirred

for 20 min. The formed precipitate was separated by ﬁltration, washed

with water, and dried to aﬀord 54d (21 g, 74%) as a colorless solid;

Compounds 12 and 14−24 were prepared according to the general

procedure A.

4-((6-(4-Fluorophenyl)[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-

1

LC-MS m/z 330 (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ 8.87

1

yl)methyl)phenol (12). LC-MS m/z 322 (M + H)⁺; H NMR (400

(dd, J = 1.52, 4.04 Hz, 1H), 8.35 (d, J = 7.83 Hz, 1H), 8.01 (d, J = 8.59

Hz, 1H), 7.90 (s, 1H), 7.75 (dd, J = 1.89, 8.72 Hz, 1H), 7.52 (dd, J =

4.17, 8.21 Hz, 1H), 7.22 (s, 1H), 7.17 (t, J = 5.43 Hz, 1H), 6.25 (s,

2H), 4.71 (d, J = 5.56 Hz, 2H).

MHz, DMSO-d₆) δ 9.34 (s, 1H), 9.30 (s, 1H), 8.25 (dd, J = 5.31, 8.84

Hz, 2H), 7.50 (t, J = 8.84 Hz, 2H), 7.19 (d, J = 8.34 Hz, 2H), 6.70 (s,

2H), 4.43 (s, 2H); HPLC purity (method A): RT 11.22, >99%.

6-(4-Fluorophenyl)-3-methyl[1,2,4]triazolo[4,3-b][1,2,4]-

1

Step 2. 6-((6-Bromo-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)-

methyl)quinoline (55d). 54d (77 g, 0.233 mol) was dissolved in

DMF (2 L). The solution was cooled to 0 °C. A solution of isoamyl

nitrite (32.76 g, 0.28 mol) in DMF (500 mL) was added dropwise for

90 min. The reaction mixture was stirred at room temperature

overnight and then heated to 70 °C until the reaction was complete.

The reaction mixture was cooled to room temperature and

concentrated in high vacuum. The residue was crystallized from

THF to aﬀord 55d (55.7 g, 70%) as brown crystals; LC-MS m/z 341

(M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (s, 1H), 8.91 (dd, J

= 1.77, 4.29 Hz, 1H), 8.35 (d, J = 7.58 Hz, 1H), 8.03 (d, J = 8.59 Hz,

1H), 7.94 (d, J = 1.26 Hz, 1H), 7.78 (dd, J = 2.02, 8.59 Hz, 1H), 7.54

(dd, J = 4.29, 8.34 Hz, 1H), 6.19 (s, 2H); Anal. Calcd for C₁₄H₉N₆Br:

C, 49.29; H, 2.66; N, 24.63. Found: C, 49.37; H, 2.79; N, 24.41.

Step 3: 6-((6-(4-Fluorophenyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-1-yl)methyl)quinoline (27). A mixture of DME (3.0 mL)

and aqueous Na₂CO₃(1.0 M, 0.88 mL) was degassed by bubbling in

argon for 10 min. The mixture was transferred via syringe to a vial

containing 55d (100 mg, 0.29 mmol), 4-ﬂuorophenylboronic acid (45

mg, 0.32 mmol), and Pd(dppf)C1₂·CH₂C1₂(6.2 mg, 0.01 mmol). The

vial was capped and heated to 80 °C for 3.5 h. The crude reaction

mixture was diluted with dichloromethane and then washed with

water. The dichloromethane solution was dried over Na₂SO₄, ﬁltered,

and concentrated. The residue was puriﬁed via column chromatog-

raphy, eluting with ethyl acetate and dichloromethane to provide 27

triazine (14). LC-MS m/z 230 (M + H)⁺; H NMR (400 MHz,

DMSO-d₆) δ 9.34 (s, 1H), 8.23−8.30 (m, 2H), 7.46−7.53 (m, 2H),

2.75 (s, 3H); HPLC purity (method A): RT 10.96, >99%.

6-(4-Fluorophenyl)-3-(4-methoxybenzyl)[1,2,4]triazolo[4,3-

1

b][1,2,4]triazine (15). LC-MS m/z 336 (M + H)⁺; H NMR (400

MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.25 (dd, J = 5.43, 8.97 Hz, 2H), 7.50

(t, J = 8.97 Hz, 2H), 7.33 (d, J = 8.84 Hz, 2H), 6.84−6.93 (m, 2H),

4.49 (s, 2H), 3.71 (s, 3H); HPLC purity (method A): RT 11.71,

>99%.

2-Chloro-4-((6-(4-ﬂuorophenyl)[1,2,4]triazolo[4,3-b][1,2,4]-

1

triazin-3-yl)methyl)phenol (17). LC-MS m/z 356 (M + H)⁺; H

NMR (400 MHz, DMSO-d₆) δ 10.07 (s, 1H), 9.35 (s, 1H), 8.18−8.31

(m, 2H), 7.44−7.53 (m, 2H), 7.41 (d, J = 2.02 Hz, 1H), 7.16 (dd, J =

2.02, 8.34 Hz, 1H), 6.90 (d, J = 8.34 Hz, 1H), 4.46 (s, 2H); HPLC

purity (method A): RT 11.45, >95%.

1-((6-(4-Fluorophenyl)[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-

1

yl)methyl)pyridin-4(1H)-one (18). LC-MS m/z 323 (M + H)⁺; H

NMR (400 MHz, DMSO-d₆) δ 9.44 (s, 1H), 8.31 (dd, J = 5.43, 8.97

Hz, 2H), 7.78−7.86 (m, 2H), 7.52 (t, J = 8.84 Hz, 2H), 6.08−6.18 (m,

2H), 5.76 (s, 2H); HPLC purity (method A): RT 9.13, >95%.

6-(4-Fluorophenyl)-3-((pyridin-3-yloxy)methyl)[1,2,4]-

1

triazolo[4,3-b][1,2,4]triazine (19). LC-MS m/z 323 (M + H)⁺; H

NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 8.49 (br s, 1H), 8.31 (br s,

1H), 7.99−8.12 (m, 2H), 7.55 (dd, J = 8.46, 1.89 Hz, 1H), 7.29−7.37

(m, 3H), 5.75 (s, 2H); HPLC purity (method B): RT 2.71, >95%.

6-(4-Fluorophenyl)-3-((thieno[3,2-b]pyridin-7-yloxy)-

methyl)[1,2,4]triazolo[4,3-b][1,2,4]triazine (20). LC-MS m/z 379

(M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.41 (s, 1H), 8.23−8.35

1

(77 mg, 74%). LC-MS m/z 357 (M + H)⁺; H NMR (400 MHz,

DMSO-d₆) δ 9.19−9.24 (m, 1H), 8.98 (dd, J = 4.29, 1.52 Hz, 1H),

8.64 (s, 1H), 8.52 (d, J = 7.83 Hz, 1H), 8.30 (s, 1H), 8.04−8.10 (m,

8104

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

1

2H), 7.91 (dd, J = 8.72, 1.89 Hz, 1H), 7.64 (dd, J = 8.34, 4.29 Hz, 1H),

6.17 (s, 2H), 3.94 (s, 3H); HPLC purity (method B): RT 3.41, >95%.

Compounds 13, 25−26, and 28−33 were prepared according to

general procedure B.

to provide 58 as a yellow oil (14.78 g, 87% yield). H NMR (300

MHz, DMSO-d₆) δ 7.89 (s, 1H), 7.52 (s, 1H), 4.47−4.56 (m, 1H),

4.25−4.35 (m, 2H), 3.81−3.96 (m, 1H), 3.66−3.75 (m, 1H), 3.45−

3.57 (m, 1H), 3.32−3.40 (m, 1H), 1.34−1.71 (m, 6H).

4-((6-(4-Fluorophenyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)-

Step 2: 1-(2-(Tetrahydro-2H-pyran-2-yloxy)ethyl)-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (60). To a

solution of 57 (1.0 g, 3.1 mmol) in anhydrous THF (8 mL) was

added i-PrMgCl (2 M in THF, 3.10 mL, 6.21 mmol) at 0 °C dropwise

under nitrogen. The reaction was stirred for 1 h at 0 °C under

nitrogen. To the solution was added 2-methoxy-4,4,5,5-tetramethyl-

1,3,2-dioxaborolane (0.736 g, 4.66 mmol) at 0 °C, and the resulting

yellow solution was allowed to stir for 1 h at ambient temperature

under nitrogen. The reaction was quenched with a saturated aqueous

solution of NH₄Cl (10 mL). EtOAc (50 mL) and saturated aqueous

NH₄Cl solution (10 mL) were added. The organic layer was separated,

and the aqueous layer was extracted with EtOAc (3 × 50 mL), dried

over Na₂SO₄, and concentrated to give the crude product as a yellow

oil. The oil was puriﬁed in a silica gel column, eluting with EtOAc and

hexanes to provide 60 as clear oil (800 mg, 80% yield). ¹H NMR (300

MHz, DMSO-d₆) δ 7.91 (s, 1H), 4.48−4.54 (m, 1H), 4.26−4.33 (m,

2H), 3.86−3.90 (m, 1H), 3.66−3.76 (m, 1H), 3.45−3.57 (m, 1H),

3.33−3.39 (m, 1H), 1.33−1.70 (m, 6H), 1.24 (s, 12H).

1

methyl)phenol (13). LC-MS m/z 322 (M + H)⁺; H NMR (400

MHz, DMSO-d₆) δ 9.5−9.57 (m, 1H), 9.48 (s, 1H), 8.36−8.44 (m,

2H), 7.47 (t, J = 8.72 Hz, 2H), 7.32 (d, J = 8.34 Hz, 2H), 6.74 (d, J =

8.34 Hz, 2H), 5.88 (s, 2H); HPLC purity (method B): RT 3.24, >95%.

6-(4-Fluorophenyl)-1-(4-methoxybenzyl)-1H-[1,2,3]triazolo-

1

[4,5-b]pyrazine (25). LC-MS m/z 336 (M + H)⁺; H NMR (400

MHz, DMSO-d₆) δ 9.48 (s, 1H), 8.40 (dd, J = 5.43, 8.97 Hz, 2H),

7.39−7.51 (m, 4H), 6.93 (d, J = 8.59 Hz, 2H), 5.95 (s, 2H), 3.31 (s,

3H); HPLC purity (method A): RT 11.99, >99%.

1-(4-Fluorobenzyl)-6-(4-ﬂuorophenyl)-1H-[1,2,3]triazolo[4,5-

1

b]pyrazine (26). LC-MS m/z 324 (M + H)⁺; H NMR (400 MHz,

DMSO-d₆) δ 9.49 (s, 1H), 8.38 (dd, J = 5.43, 8.97 Hz, 2H), 7.54 (dd, J

= 5.56, 8.59 Hz, 2H), 7.46 (t, J = 8.84 Hz, 2H), 7.21 (t, J = 8.84 Hz,

2H), 6.02 (s, 2H); HPLC purity (method A): RT 11.59, >99%.

1-(4-Methoxybenzyl)-6-(1-methyl-1H-pyrazol-4-yl)-1H-

[1,2,3]triazolo[4,5-b]pyrazine (28). LC-MS m/z 322 (M + H)⁺; ¹H

NMR (400 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.65 (s, 1H), 8.31 (s,

1H), 7.42 (d, J = 8.84 Hz, 2H), 6.93 (d, J = 8.84 Hz, 2H), 5.85 (s, 2H),

3.96 (s, 3H), 3.72 (s, 3H); HPLC purity (method A): RT 11.37,

>99%.

1-((2,3-Dihydrobenzofuran-5-yl)methyl)-6-(1-methyl-1H-

pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (29). LC-MS m/z

334 (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.62−

8.69 (m, 1H), 8.33 (s, 1H), 7.33 (s, 1H), 7.25 (d, J = 8.34 Hz, 1H),

6.75 (d, J = 8.08 Hz, 1H), 5.83 (s, 2H), 4.49 (t, J = 8.72 Hz, 2H), 3.97

(s, 3H), 3.13 (t, J = 8.72 Hz, 2H); HPLC purity (method B): RT 2.94,

>95%.

Step 3: 6-((6-(1-(2-(Tetrahydro-2H-pyran-2-yloxy)ethyl)-1H-

pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)methyl)-

quinoline (61). To a solution of 55d (845 mg, 2.48 mmol) in DME

(16 mL) was added 60 (800 mg, 2.48 mmol) and Cs₂CO₃(2.42 g,

7.43 mmol) in H₂O (4 mL). The reaction mixture was degassed and

charged with nitrogen three times. The palladium catalyst Pd(dppf)-

Cl₂·CH₂Cl₂(101 mg, 0.124 mmol) was added, and the reaction

mixture was degassed and charged with nitrogen three times, and

stirred for 16 h at 80 °C under nitrogen. The reaction mixture was

then ﬁltered over a pad of Celite and washed with EtOAc (50 mL) and

water (25 mL). The ﬁltrate was extracted with EtOAc (3 × 50 mL).

The combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude product was puriﬁed via Biotage

silica gel column chromatography, eluting with hexane/EtOAc (0−

1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-6-(1-meth-

yl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine (30). LC-

1

MS m/z 350 (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ 9.17 (s,

1H), 8.63 (s, 1H), 8.29 (s, 1H), 6.96 (d, 1H), 6.89−6.92 (dd, 1H),

6.81−6.83 (d, 1H), 5.78 (s, 2H), 4.18 (s, 4H), 3.94 (s, 3H); HPLC

purity (method A): RT 11.27, >99%.

6-((6-(1-Methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-1-yl)methyl)quinoline (31). LC-MS m/z 343 (M + H)⁺;

¹H NMR (400 MHz, DMSO-d₆) δ 9.22 (s, 1H), 8.90 (dd, J = 1.77,

4.29 Hz, 1H), 8.65 (s, 1H), 8.38 (dd, J = 1.01, 8.34 Hz, 1H), 8.31 (d, J

= 0.51 Hz, 1H), 7.97−8.07 (m, 2H), 7.84 (dd, J = 2.02, 8.59 Hz, 1H),

7.54 (dd, J = 4.29, 8.34 Hz, 1H), 6.16 (s, 2H), 3.95 (s, 3H); HPLC

purity (method A): RT 10.90, >99%.

6-((6-(1-Methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-1-yl)methyl)quinazoline (32). LC-MS m/z 344 (M + H)⁺;

¹H NMR (400 MHz, DMSO-d₆) δ 9.60 (s, 1H), 9.28 (s, 1H), 9.21 (s,

1

100%) to provide 61 (910 mg, 81% yield) as a white solid. H NMR

(300 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.82−8.95 (m, 1H), 8.67 (s,

1H), 8.28−8.45 (m, 2H), 7.92−8.08 (m, 2H), 7.77−7.90 (m, 1H),

7.53 (dd, J = 8.29, 4.14 Hz, 1H), 6.15 (s, 2H), 4.49−4.62 (m, 2H),

4.30−4.47 (m, 2H), 3.91−4.00 (m, 1H), 3.67−3.87 (m, J = 5.46 Hz,

1H), 3.47−3.60 (m, 1H), 3.35−3.42 (m, 1H), 1.48−1.66 (m, 2H),

1.32−1.45 (m, 3H).

Step 4: To a solution of 61 (780 mg, 1.71 mmol) in CH₂Cl₂(20

mL) was added the anhydrous HCl/dioxane solution dropwise (4 N,

1.07 mL, 4.27 mmol). A white solid precipitated out. The reaction

mixture was stirred for 1 h, and the LCMS showed the completion of

the reaction. The reaction mixture was concentrated, and the residue

was dissolved in distilled water (15 mL). The solution was adjusted to

pH 7 with Na₂CO₃. An oﬀ-white solid crashed out, which was ﬁltered,

washed with water, and dried on a high vacuum for 1 h. The solid was

recrystallized from EtOH (50 mL) to provide 2 (400 mg, 63% yield)

as a white crystalline solid: mp 222 °C; LC-MS m/z 373 (M + H)⁺; ¹H

NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.94 (dd, J = 1.52, 4.29

Hz, 1H), 8.64 (s, 1H), 8.46 (d, J = 8.59 Hz, 1H), 8.33 (s, 1H), 8.02−

8.08 (m, 2H), 7.88 (dd, J = 1.89, 8.72 Hz, 1H), 7.59 (dd, J = 4.29, 8.34

Hz, 1H), 6.17 (s, 2H), 4.25 (t, J = 5.43 Hz, 2H), 3.79 (t, J = 5.43 Hz,

2H); HPLC purity (method A): RT 10.679, 99.6%; Anal. Calcd for

C₁₉H₁₆N₈O·1.0H₂O: C, 58.45; H, 4.65; N, 28.70. Found: C, 58.45; H,

4.57; N, 28.50.

1H), 8.62 (s, 1H), 8.29 (s, 1H), 8.08−8.15 (m, 2H), 8.00−8.06 (m,

1H), 6.19 (s, 2H), 3.93 (s, 3H); HPLC purity (method A): RT 10.58,

>95%.

6-((6-(1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)-

1

methyl)quinoline (33). LC-MS m/z 329 (M + H)⁺; H NMR (400

MHz, DMSO-d₆) δ 13.47 (br s, 1H), 9.26 (s, 1H), 8.89 (dd, J = 4.17,

1.64 Hz, 1H), 8.55 (br s, 2H), 8.38 (d, J = 8.59 Hz, 1H), 7.99−8.07

(m, 2H), 7.84 (dd, J = 8.97, 1.64 Hz, 1H), 7.53 (dd, J = 8.34, 4.29 Hz,

1H), 6.15 (s, 2H); HPLC purity (method B): RT 2.38, >95%.

2-(4-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (PF-04217903) (34). Step

1: 4-Iodo-1-(2-(tetrahydro-2H-pyran-2-yloxy)ethyl)-1H-pyra-

zole (58). In a round-bottom ﬂask was added 4-iodopyrazole (10.22

g, 52.70 mmol), Cs₂CO₃(20.6 g, 63.2 mmol), and anhydrous DMF

(100 mL). The suspension was stirred at 23 °C for 5 min. 2-(2-

Bromoethoxy)tetrahydro-2H-pyran (9.95 mL, 63.2 mmol) was added,

and the reaction mixture was stirred at 70 °C for 16 h. After the

reaction mixture was cooled down, EtOAc (100 mL) and water (100

mL) were added. The organic layer was collected, and the aqueous

layer was extracted with EtOAc (3 × 100 mL). The combined organic

layers were washed with water (3 × 100 mL), dried over Na₂SO₄, and

concentrated to aﬀord a dark brown oil. The crude product was

puriﬁed on a silica gel column, eluting with ethyl acetate and hexanes

2-Methyl-1-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo-

[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)propan-2-ol (36). To a

suspension of 33 (50 mg, 0.15 mmol) and Cs₂CO₃(50 mg, 0.15

mmol) in DMF (2 mL) was added 2,2-dimethyl-oxirane (21.6 mg,

0.30 mmol). The reaction was stirred at 80 °C for 16 h. The reaction

was then puriﬁed with a reverse-phased preparative HPLC eluting with

acetonitrile/water having 0.1% acetic acid to yield 36 as a white

amorphous solid after lyophilization (13 mg, 22% yield); LC-MS m/z

401 (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ 9.25 (s, 1H), 8.90

(dd, J = 1.64, 4.17 Hz, 1H), 8.58 (s, 1H), 8.34−8.41 (m, 1H), 8.32 (s,

1

8105

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

1H), 7.98−8.06 (m, 2H), 7.83 (dd, J = 2.02, 8.59 Hz, 1H), 7.54 (dd, J

= 4.17, 8.21 Hz, 1H), 6.16 (s, 2H), 4.81 (s, 1H), 4.12 (s, 2H), 1.11 (s,

6H); HPLC purity (method A): RT 11.10, >99%.

8.62−8.76 (m, 2H), 8.48 (d, J = 7.58 Hz, 1H), 8.37 (s, 1H), 8.01−8.10

(m, 2H), 7.88 (d, J = 8.59 Hz, 1H), 7.61 (dd, J = 4.29, 8.08 Hz, 1H),

6.17 (s, 2H), 4.61 (t, J = 10.74 Hz, 1H), 3.36−3.46 (m, J = 12.60 Hz,

2H), 3.01−3.19 (m, 2H), 2.10−2.29 (m, 4H); HPLC purity (method

A): RT 10.00, >99%.

N,N-Dimethyl-2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]-

triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanamine (37).

To a solution of 33 (50 mg, 0.152 mmol) in anhydrous DMF (1.52

mL, 0.1 M) was added cesium carbonate (100 mg, 0.304 mmol). The

1-(2-chloroethyl)pyrrolidine-HCl salt (26 mg, 0.152 mmol) was

added, and the reaction mixture was allowed to stir under nitrogen

for 3 h at 80 °C. The reaction mixture was puriﬁed directly by

preparative HPLC using acetonitrile and H₂O with 0.1% HOAc to give

6-((1H-[1,2,3]Triazolo[4,5-b]pyrazin-1-yl)methyl)quinoline

(42). A solution of 55d (200 mg, 0.586 mmol) in MeOH (10 mL)/

AcOH (1 mL)/EtOAc (1 mL) was degassed 3 times with nitrogen. To

this solution was added Pd/C (20 mg). A balloon containing hydrogen

was added via syringe, and the reaction was allowed to stir for 18 h at

room temperature. The reaction did not complete, and more Pd/C

was added (20 mg) and stirred for 18 h at room temperature. The

reaction was stopped when LCMS showed a ratio of 1:1 (product/

starting material). The reaction was ﬁltered over Celite and washed

with EtOAc (50 mL). The ﬁltered solution was concentrated and

puriﬁed by Biotage silica gel column chromatography with hexane/

EtOAc to give 42 as a white solid (30 mg, 20% yield); LC-MS m/z

1

37 (15 mg, 23% yield); LC-MS m/z 400 (M + H)⁺; H NMR (400

MHz, DMSO-d₆) δ ppm 9.51 (br s, 1H), 9.27 (s, 1H), 8.93 (dd, J =

4.17, 1.64 Hz, 1H), 8.75−8.79 (m, 1H), 8.45−8.52 (m, 1H), 8.43 (d, J

= 8.34 Hz, 1H), 8.05 (d, J = 8.59 Hz, 1H), 8.00 (d, J = 1.26 Hz, 1H),

7.85 (dd, J = 8.72, 1.89 Hz, 1H), 7.58 (dd, J = 8.21, 4.17 Hz, 1H), 6.17

(s, 2H), 4.64 (t, J = 6.19 Hz, 2H), 3.63 (br s, 2H), 2.83 (br s, 6H);

HPLC purity (method A): RT 9.64, 95.30%.

1

263.0 (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ 8.85−8.92 (m,

38 and 39 were prepared with a similar procedure as that used for

3H), 8.31−8.39 (m, 1H), 8.00 (d, J = 8.59 Hz, 1H), 7.96 (d, J = 1.52

Hz, 1H), 7.77 (dd, J = 2.15, 8.72 Hz, 1H), 7.52 (dd, J = 4.17, 8.21 Hz,

1H), 6.22 (s, 2H); HPLC purity (method A): RT 10.11, 97.2%.

6-((6-Methyl-1H-[1,2,3]triazolo[4,5-b]pyrazin-1-yl)methyl)-

quinoline (43). To a solution of 55d (100 mg, 0.29 mmol) and

methyl boronic acid (20 mg, 0.32 mmol) in DME/H₂O (4:1, 0.1 M)

was added sodium carbonate (92 mg, 0.87 mmol). The mixture was

degassed with nitrogen three times. Pd(PPh₃)₂Cl₂(10 mg, 0.015

mmol) was added, and the reaction was degassed again three times.

After it was heated to 80 °C for 16 h, the reaction mixture was ﬁltered

through a pad of Celite and washed with EtOAc. The aqueous layer

was extracted with EtOAc (3 × 10 mL), dried over Na₂SO₄, ﬁltered,

and concentrated in vacuo. The crude material was puriﬁed directly by

preparative HPLC using ACN/H₂O with 0.1% HOAc to aﬀord 43 (13

mg, 16%); LC-MS m/z 277.1 (M + H)⁺; ¹H NMR (400 MHz,

DMSO-d₆) δ 8.90 (dd, J = 1.77, 4.29 Hz, 1H), 8.79 (s, 1H), 8.36 (d, J

= 7.58 Hz, 1H), 8.01 (d, J = 8.84 Hz, 1H), 7.91 (s, 1H), 7.75 (dd, J =

2.02, 8.59 Hz, 1H), 7.53 (dd, J = 4.29, 8.34 Hz, 1H), 6.17 (s, 2H), 2.74

(s, 3H); HPLC purity (method B): RT 2.60, 95%.

1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-

amine (44). To a microwave vessel were added 55d (300 mg, 0.88

mmol) and NH₄OH (3 mL). The reaction was irradiated in a

microwave at 100 °C for 1 h. The solid was ﬁltered and washed with

water and then recrystallized in MeOH, ﬁltered, and dried to give 44

as a white crystal solid (100 mg, 41%); mp 266 °C; LC-MS m/z 278

(M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (dd, J = 1.64, 4.17

Hz, 1H), 8.35 (dd, J = 1.01, 8.34 Hz, 1H), 8.04 (s, 1H), 7.99−8.03 (m,

1H), 7.80 (d, J = 1.52 Hz, 1H), 7.68 (dd, J = 2.02, 8.84 Hz, 1H), 7.49−

7.56 (m, 3H), 5.88 (s, 2H); HPLC purity (method A): RT 9.33,

>99%.

(R)-1-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-6-yl)pyrrolidin-3-amine (48). Step 1. A mixture of 55d

(200 mg, 0.59 mmol), potassium carbonate (243 mg, 1. 76 mmol), and

(R)-tertbutylpyrrolidin-3-ylcarbamate (218 mg, 1.17 mmol) in 2-

propanol (6 mL) was heated in the microwave at 80 °C for 20 min.

The mixture was allowed to cool, and the solids were collected by

ﬁltration and then puriﬁed by ﬂash chromatography, eluting with

chloroform/ethyl acetate (25−75%) to aﬀord the N-Boc protected 48

(239 mg, 91%).

Step 2. To a solution of (R)-tert-butyl 1-(1-(quinolin-6-ylmethyl)-

1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)pyrrolidin-3-ylcarbamate (100

mg, 0.224 mmol) in dichloromethane (2.2 mL) was added

hydrochloric acid (4 N in dioxane 0.56 mL, 2.24 mmol). After stirring

at room temperature for 6 h, the reaction was diluted with

dichloromethane and quenched with saturated sodium bicarbonate

(5 mL). The organic layer was separated and concentrated to aﬀord

the desired product 48 (65 mg, 84%); LC-MS m/z 347 (M + H)⁺; ¹H

NMR (400 MHz, methanol-d₄) δ 8.85 (dd, J = 1.5, 4.3 Hz, 1H), 8.36

(d, J = 8.3 Hz, 1H), 8.15 (s, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.95 (d, J =

1.0 Hz, 1H), 7.85 (dd, J = 2.0, 8.8 Hz, 1H), 7.55 (dd, J = 4.4, 8.5 Hz,

1H), 5.95 (s, 2H), 3.88−3.78 (m, 2H), 3.75−3.66 (m, 2H), 3.48−3.40

37.

6-((6-(1-(2-(Pyrrolidin-1-yl)ethyl)-1H-pyrazol-4-yl)-1H-[1,2,3]-

triazolo[4,5-b]pyrazin-1-yl)methyl)quinoline (38). LC-MS m/z

426.1 (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.88

(dd, J = 1.77, 4.29 Hz, 1H), 8.67 (s, 1H), 8.35−8.39 (m, 1H), 8.30 (s,

1H), 7.96−8.05 (m, 2H), 7.82 (dd, J = 2.02, 8.59 Hz, 1H), 7.51 (s,

1H), 6.14 (s, 2H), 4.30 (t, J = 6.44 Hz, 2H), 3.35−3.42 (m, 4H), 2.86

(t, J = 6.44 Hz, 2H), 2.44−2.48 (m, 4H), 1.63 (td, J = 3.22, 6.69 Hz,

4H); HPLC purity (method A): RT 9.86, >99%.

1-(2-(4-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-6-yl)-1H-pyrazol-1-yl)ethyl)pyrrolidin-2-one (39). LC-

MS m/z 440.0 (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s,

1H), 8.90 (dd, J = 1.77, 4.04 Hz, 1H), 8.69 (s, 1H), 8.38 (d, J = 7.33

Hz, 1H), 8.34 (s, 1H), 7.99−8.06 (m, 2H), 7.84 (dd, J = 2.02, 8.84 Hz,

1H), 7.54 (dd, J = 4.29, 8.34 Hz, 1H), 6.16 (s, 2H), 4.34 (t, J = 5.94

Hz, 2H), 3.62 (t, J = 5.94 Hz, 2H), 3.10−3.20 (m, 2H), 2.07−2.17 (m,

2H), 1.82 (t, J = 7.45 Hz, 2H); HPLC purity (method A): RT 10.79,

>99%.

2-(4-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-6-yl)-1H-pyrazol-1-yl)acetic acid (35). The methyl ester

of 35 was prepared with a similar procedure as that used for 37. To a

solution of 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-6-yl)-1H-pyrazol-1-yl)acetic acid methyl ester (242 mg, 0.60

mmol) in MeOH (4 mL) was added a freshly prepared solution of

LiOH (72 mg, 3.02 mmol) in water (1 mL). The reaction was stirred

for 16 h at ambient temperature. The white suspension was then

neutralized to pH 7 with 1 N HC1, and a white solid precipitated out.

The solid was ﬁltered, washed with water, and dried under a high

vacuum for 16 h to provide 35 (131 mgs, 56% yield); LC-MS m/z 387

(M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.15 (s, 1H), 8.82 (d, J

= 2.78 Hz, 1H), 8.52 (s, 1H), 8.31 (d, J = 7.58 Hz, 1H), 8.19 (s, 1H),

7.90−8.00 (m, 2H), 7.78 (s, 1H), 7.42−7.50 (m, 1H), 6.09 (s, 2H),

4.54−4.69 (m, 2H); HPLC purity (method A): RT 10.21, 96.19%.

(R)-6-((6-(1-(Pyrrolidin-3-yl)-1H-pyrazol-4-yl)-1H-[1,2,3]-

triazolo[4,5-b]pyrazin-1-yl)methyl)quinoline (40). The N-Boc

analogue of 40 was prepared according to a similar procedure as

that used for 37. The Boc-protecting group was removed with 4 N

HCl in dioxane in methanol solvent; LC-MS m/z 398 (M + H)⁺; H

1

NMR (400 MHz, DMSO-d₆) δ 9.26 (s, 1H), 9.16 (br s, 2H), 8.94 (dd,

J = 2.78, 1.52 Hz, 1H), 8.82 (s, 1H), 8.44 (s, 2H), 8.00−8.10 (m, 2H),

7.86 (d, J = 8.84 Hz, 1H), 7.59 (ddd, J = 6.19, 4.17, 2.02 Hz, 1H), 6.17

(s, 2H), 5.26−5.38 (m, J = 7.14, 7.14, 4.04, 3.66 Hz, 1H), 3.65−3.74

(m, 1H), 3.57−3.65 (m, 1H), 3.44 (d, J = 16.67 Hz, 2H), 2.41−2.48

(m, 1H), 2.29−2.40 (m, 1H); HPLC purity (method A): RT 9.90,

99%.

6-((6-(1-(Piperidin-4-yl)-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo-

[4,5-b]pyrazin-1-yl)methyl)quinoline (41). The N-Boc analogue of

41 was prepared according to a similar procedure as that used for 37.

The Boc-protecting group was removed with 4 N HCl in dioxane in

methanol solvent; LC-MS m/z 412 (M + H)⁺; H NMR (400 MHz,

1

DMSO-d₆, 2 equiv HCl-salt) δ 9.27 (s, 1H), 8.84−8.98 (m, 2H),

8106

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

(m, 1H), 2.35−2.22 (m, 1H), 2.03−1.86 (m, 1H); HPLC purity

(method A): RT 8.96, >99%.

Compounds 45−47 and 49−50 were prepared with similar

procedures as that for 48.

competitive inhibition by the method of nonlinear least-squares

(GraphPad Prism, GraphPad Software, San Diego, CA).

Cellular Kinase Phosphorylation ELISA Assays.^36,32All

experiments were done under standard conditions (37 °C and 5%

CO₂). IC50 values were calculated by concentration−response curve

ﬁtting using a Microsoft Excel-based four-parameter method. Cells

were seeded in 96-well plates in media supplemented with 10% fetal

bovine serum (FBS) and transferred to serum-free media [with 0.04%

bovine serum albumin (BSA)] after 24 h. In experiments investigating

ligand-dependent RTK phosphorylation, corresponding growth factors

were added for up to 20 min. After incubation of cells with an inhibitor

for 1 h and/or appropriate ligands for the designated times, cells were

washed once with HBSS supplemented with 1 mmol/L Na₃VO₄, and

protein lysates were generated from cells. Subsequently, phosphor-

ylation of selected protein kinases was assessed by a sandwich ELISA

method using speciﬁc capture antibodies used to coat 96-well plates

and a detection antibody speciﬁc for phosphorylated tyrosine residues.

Antibody-coated plates were (a) incubated in the presence of protein

lysates at 4 °C overnight; (b) washed seven times in 1% Tween 20 in

PBS; (c) incubated in a horseradish peroxidase-conjugated anti-total-

phosphotyrosine (PY-20) antibody (1:500) for 30 min; (d) washed

seven times again; (e) incubated in 3,3,5,5-tetramethylbenzidine

peroxidase substrate (Bio-Rad) to initiate a colorimetric reaction that

was stopped by adding 0.09 N H₂SO₄; and (f) measured for

absorbance in 450 nm using a spectrophotometer. The A549 cell line

was used for the c-MET cellular kinase phosphorylation ELISA assay.

Human Microsomal Stability Studies. Compounds (1 μM)

were incubated at 37 °C for 30 min in a ﬁnal volume of 200 μL of 100

mM potassium phosphate buﬀer (pH 7.4) containing pooled human

liver microsomes (0.8 mg/mL protein) and 2 mM NADPH. Reactions

were initiated with the addition of NADPH following a 10-min

preincubation. Aliquots of incubation samples were protein

precipitated with cold methanol containing 0.1 μM buspirone (internal

standard) and centrifuged, and supernatants were analyzed by LC-

MS/MS. All incubations were performed in triplicate, and the percent

remaining of parent drug at the end of incubation was determined by

LC-MS/MS peak area ratio.

N-Ethyl-1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]-

1

pyrazin-6-amine (45). LC-MS m/z 306 (M + H)⁺; H NMR (400

MHz, DMSO-d₆) δ 8.88 (dd, J = 1.64, 4.17 Hz, 1H), 8.32−8.38 (m,

1H), 8.19 (br s, 1H), 7.96−8.04 (m, 2H), 7.89−7.94 (m, 1H), 7.74

(dd, J = 2.02, 8.84 Hz, 1H), 7.53 (dd, J = 4.29, 8.34 Hz, 1H), 5.87 (s,

2H), 3.34−3.39 (m, 2H), 1.16 (t, J = 7.20 Hz, 3H); HPLC purity

(method A): RT 11.13, >99%.

2-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-

6-ylamino)ethanol (46). LC-MS m/z 322 (M + H)⁺; ¹H NMR (400

MHz, DMSO-d₆) δ 8.89 (dd, J = 1.6, 4.2 Hz, 1H), 8.36 (d, J = 8.3 Hz,

1H), 8.26 (t, J = 5.8 Hz, 1H), 8.10 (s, 1H), 8.00 (d, J = 8.6 Hz, 1H),

7.93 (d, J = 1.3 Hz, 1H), 7.76 (dd, J = 2.0, 8.6 Hz, 1H), 7.53 (dd, J =

4.3, 8.3 Hz, 1H), 5.87 (s, 2H), 4.82 (t, J = 5.3 Hz, 1H), 3.58 (q, J = 5.6

Hz, 2H), 3.44 (q, J = 5.7 Hz, 2H); HPLC purity (method A): RT

10.20, >99%.

N¹,N¹-Dimethyl-N²-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]-

triazolo[4,5-b]pyrazin-6-yl)ethane-1,2-diamine (47). LC-MS m/

1

z 349 (M + H)⁺; H NMR (400 MHz, methanol-d₄) δ 8.83 (dd, J =

1.52, 4.29 Hz, 1H), 8.34 (d, J = 8.08 Hz, 1H), 7.96−8.05 (m, 2H),

7.92 (s, 1H), 7.79 (dd, J = 2.02, 8.84 Hz, 1H), 7.53 (dd, J = 4.29, 8.34

Hz, 1H), 5.93 (s, 2H), 3.59 (t, J = 6.57 Hz, 2H), 2.59 (t, J = 6.57 Hz,

2H), 2.24 (s, 6H); HPLC purity (method A): RT 8.34, >99%.

1-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-

1

6-yl)pyrrolidin-3-ol (49). LC-MS m/z 348 (M + H)⁺; H NMR

(400 MHz, DMSO-d₆) δ 8.89 (dd, J = 1.77, 4.04 Hz, 1H), 8.36 (d, J =

7.58 Hz, 1H), 8.22 (s, 1H), 8.00 (d, J = 8.59 Hz, 1H), 7.90−7.96 (m,

1H), 7.76 (dd, J = 1.89, 8.72 Hz, 1H), 7.53 (dd, J = 4.17, 8.21 Hz, 1H),

5.90 (s, 2H), 5.10 (br s, 1H), 4.43 (br s, 1H), 3.56−3.80 (m, 3H),

3.48−3.55 (m, 1H), 1.84−2.08 (m, 2H); HPLC purity (method A):

RT 10.01, >99%.

6-((6-(4-Methylpiperazin-1-yl)-1H-[1,2,3]triazolo[4,5-b]-

pyrazin-1-yl)methyl)quinoline (50). LC-MS m/z 361 (M + H)⁺;

¹H NMR (400 MHz, chloroform-d) δ 8.92 (br s, 1H), 8.30 (s, 1 H),

8.12 (d, J = 8.3 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.82 (s, 1 H), 7.77

(dd, J = 1.9, 8.7 Hz, 1H), 7.41 (dd, J = 4.3, 8.3 Hz, 1H), 5.88 (s, 2H),

3.86−3.78 (m, 4H), 2.59 (br s, 4H), 2.39 (s, 3H); HPLC purity

(method A): RT 8.45, >99%.

Cocrystal Structures. c-MET cocrystals were obtained at 13 °C

by the hanging drop vapor diﬀusion method by mixing 1.2 μL of

protein solution (containing 7−13 mg/mL c-MET KD (residues

1051−1348) with a 5-fold molar excess of selected c-MET inhibitor)

with 1.2 μL of solution containing 0.05 M citrate-phosphate pH 4.6,

0−0.275 M NaCl, and 21% polyethylene glycol MW = 3350. Details of

the crystal structure determinations can be accessed from the PDB

database.

N,N-Dimethyl-2-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo-

[4,5-b]pyrazin-6-yloxy)ethanamine (51). A mixture of 55d (100

mg, 0.293 mmol), triethylamine (0.123 mL, 0.879 mmol), and N,N-

dimethylethanolamine (0.959 mL, 0.586 mmol) in n-butanol (3.0 mL)

was heated in the microwave at 120 °C for 20 min and then

concentrated. The crude product was puriﬁed by ﬂash chromatography

using a Horizon puriﬁcation system on a 25S column eluting with

chloroform/7 N ammonia in methanol (0.1−5%) to aﬀord 49 (75 mg,

74%); LC-MS m/z 350 (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ

8.89 (dd, J = 1.6, 4.2 Hz, 1H), 8.42 (s, 1H), 8.36 (d, J = 7.6 Hz, 1H),

8.01 (d, J = 8.6 Hz, 1H), 7.97 (d, J = 1.5 Hz, 1H), 7.78 (dd, J = 2.0, 8.8

Hz, 1H), 7.53 (dd, J = 4.3, 8.3 Hz, 1H), 6.06 (s, 2H), 4.50 (t, J = 5.7

Hz, 2H), 2.61 (t, J = 5.7 Hz, 2H), 2.13 (s, 6H); HPLC purity (method

A): RT 8.09, 99%.

Biochemical Kinase Assays. c-MET enzyme inhibition was

measured by a continuous coupled spectrophotometric assay as

previously described.²³The assay monitored ATP consumption

coupled to oxidation of NADH (measured at 340 nm) while

regenerating ATP in the presence of phosphoenol pyruvate (PEP)

and coupling enzymes, pyruvate kinase (PK), and lactic dehydrogenase

(LDH). Assay reactions contained 0.30 mM ATP (4K_m), 0.5 mM

Met2 peptide (Ac-ARDMYDKEYYSVHNK), 20 mM MgCl₂, 1 mM

PEP, 330 μM NADH, 2 mM DTT, 15 units/mL LDH, 15 units/mL

PK, test compound (1% DMSO ﬁnal) in 100 mM HEPES, pH 7.5, 37

°C, and the reactions were initiated by adding 50 nM c-Met N-

terminal His6-tagged recombinant human enzyme, aa residues 974−

1390 (Millipore Corp./Upstate Ltd., Billerica, MA). The inhibitors

were shown to be ATP-competitive from kinetic and crystallographic

studies, and the dose−response data were ﬁt to the equation for

ASSOCIATED CONTENT

■

S

* Supporting Information

Kinase selectivity screening data for 12 against a total of 98

protein kinases. This material is available free of charge via the

internet at http://pubs.acs.org.

Accession Codes

Coordinates of the c-MET crystal structures have been

deposited in the Protein Data Bank for compounds 2 (3zxz),

10 (3zze), 12 (4ap7), and 24 (4aoi).

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 858-638-6333. Fax: 877-481-1783. E-mail: jean.cui@

p ﬁ zer.com.

Present Address

^§Groton Laboratories, Pﬁzer Worldwide Research and

Development, Eastern Point Road, Groton, Connecticut 06340.

Notes

The authors declare no competing ﬁnancial interest.

8107

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Radinsky, R.; Kendall, R. Fully human monoclonal antibodies to

hepatocyte growth factor with therapeutic potential against hepatocyte

growth factor/c-Met-dependent human tumors. Cancer Res. 2006, 66,

1721−1729.

(6) (a) Jin, H.; Yang, R.; Zheng, Z.; Romero, M.; Ross, J.; Bou-

Reslan, H.; Carano, R. A.D.; Kasman, I.; Mai, E.; Young, J.; Zha, J.;

Zhang, Z.; Ross, S.; Schwall, R.; Colbern, G.; Merchant, M. MetMAb,

the one-armed 5D5 anti-c-Met antibody, inhibits orthotopic pancreatic

tumor growth and improves survival. Cancer Res. 2008, 68, 4360−

4368. (b) van der Horst, E. H.; Chinn, L.; Wang, M.; Velilla, T.; Tran,

H.; Madrona, Y.; Lam, A.; Ji, M.; Hoey, T. C.; Sato, A. K. Discovery of

fully human anti-MET monoclonal antibodies with antitumor activity

against colon cancer tumor models in vivo. Neoplasia 2009, 11, 355−

364.

ACKNOWLEDGMENTS

■

We are grateful to Muhammad Alimuddin for analytical

support, to the Pﬁzer PDM department for in vitro ADME

and in vivo pharmacokinetic studies, and to the Pﬁzer DSRD

department for in vitro exploratory toxicity studies.

ABBREVIATIONS

■

A-loop, activation loop; HGF, hepatocyte growth factor;

HGFR, hepatocyte growth factor receptor; KD, kinase domain;

c-MET, mesenchymal-epithelial transition factor; OS, overall

survival; PFS, progression free survival; RTK, receptor tyrosine

kinase; TKI, tyrosine kinase inhibitor

(7) Liu, X.; Newton, R. C.; Scherle, P. A. Development of c-MET

pathway inhibitors. Expert Opin. Invest. Drugs 2011, 20, 1225−1241.

(8) Tuma, R. S. Novel antibody, rilotumumab (AMG 102) shows

activity in metastatic colorectal cancer patients. Oncology Times 2011,

33, 22−23.

(9) (a) Catenacci, D. V. T.; Henderson, L.; Xiao, S.-Y.; Patel, P.;

Yauch, R. L.; Hegde, P.; Zha, J.; Pandita, A.; Peterson, A.; Salgia, R.

Durable complete response of metastatic gastric cancer with anti-Met

therapy followed by resistance at recurrence. Cancer Discovery 2011, 1,

573−579. (b) Surati, M.; Patel, P.; Peterson, A.; Salgia, R. Role of

MetMAb (OA-5D5) in c-MET active lung malignancies. Expert Opin.

Biol. Ther. 2011, 11, 1655−1662.

(10) Spigel, D. R.; Ervin, T. J.; Ramlau, R.; Daniel, D. B.;

Goldschmidt, J. H.; Blumenschein, G. R.; Krzakowski, M. J.;

Robinet, G.; Clement-Duchene, C.; Barlesi, F.; Govindan, R.; Patel,

T.; Orlov, S. V.; Wertheim, M. S.; Zha, J.; Pandita, A.; Yu, W.; Yauch,

R. L.; Patel, P. H.; Peterson, A. C. Final eﬃcacy results from

OAM4558g, a randomized phase II study evaluating MetMAb or

placebo in combination with erlotinib in advanced NSCLC. Presented

at ASCO 2011 Annual Meeting, Chicago, IL, June 3−7, 2011; Abstr.

7505.

REFERENCES

■

(1) (a) Comoglio, P. M.; Tamagnone, L.; Boccaccio, C.

Plasminogen-related growth factor and semaphorin receptors: a gene

superfamily controlling invasive growth. Exp. Cell Res. 1999, 253, 88−

99. (b) Comoglio, P. M.; Trusolino, L. Invasive growth: from

development to metastasis. J. Clin. Invest. 2002, 109, 857−862.

(2) (a) Maulik, G.; Shrikhande, A.; Kijima, T.; Ma, P. C.; Morrison,

P. T.; Salgia, R. Role of the hepatocyte growth factor receptor, c-MET,

in oncogenesis and potential for therapeutic inhibition. Cytokine

Growth Factor Rev. 2002, 13, 41−59. (b) Ma, P. C.; Maulik, G.;

Christensen, J.; Salgia, R. c-MET: structure, functions and potential for

therapeutic inhibition. Cancer Metastasis Rev. 2003, 22, 309−325.

(c) Christensen, J.; Burrows, J.; Salgia, R. c-MET as a target in human

cancer and characterization of inhibitors for therapeutic intervention.

Cancer Lett. (Amsterdam, Neth.) 2005, 225, 1−26. (d) Benvenuti, S.;

Comoglio, P. M. The met receptor tyrosine kinase in invasion and

metastasis. J. Cell. Physiol. 2007, 213, 316−325. (e) Knudsen, B. S.;

Vande Woude, G. Showering c-MET-dependent cancers with drugs.

Curr. Opin. Genet. Dev. 2008, 18, 87−96.

(3) (a) Cheng, H.-L.; Trink, B.; Tzai, T.-S.; Liu, H.-S.; Chan, S.-H.;

Ho, C.-L.; Sidransky, D.; Chow, N.-H. Overexpression of c-MET as a

prognostic indicator for transitional cell carcinoma of the urinary

bladder: a comparison with p53 nuclear accumulation. J. Clin. Oncol.

2002, 20, 1544−1550. (b) Lengyel, E.; Prechtel, D.; Resau, J. H.;

Gauger, K.; Welk, A.; Lindemann, K.; Alanti, G.; Richter, T.; Knudsen,

B.; Vande Woude, G. F.; Harneck, N. c-MET overexpression in node-

positive breast cancer identifies patients with poor clinical outcome

independent of Her2/neu. Int. J. Cancer 2005, 113, 678−682. (c) Lo

Muzio, L.; Farina, A.; Rubini, C.; Coccia, E.; Capogreco, M.; Colella,

G.; Leonardi, R.; Campisi, G.; Carinci, F. Effect of c-MET expression

on survival in head and neck squamous cell carcinoma. Tumor Biol.

2006, 27, 116−121. (d) Sawada, K.; Radjabi, A. E.; Shinomiya, N.;

Kistner, E.; Kenny, H.; Becker, A. R.; Turkyilmaz, M. A.; Salgia, R.;

Yamada, S. D.; Vande Woude, G. F.; Tretiakova, M. S.; Lengyel, E. c-

MET overexpression is a prognostic factor in ovarian cancer and an

effective target for inhibition of peritoneal dissemination and invasion.

Cancer Res. 2007, 67, 1670−1679. (e) Drebber, U.; Baldus, S. E.;

(11) (a) Mroczkowski, B.; McTigue, M. A..; Sarup, J.; Murray, B. W.;

Hickey, M.; Parge, H.; Zhu. J. Catalytic domains of the human

hepatocyte growth factor receptor kinase and material and methods for

identiﬁcation thereof. USP 277968, EP1243596A1, 2001. (b) Schiering,

N.; Knapp, S.; Marconi, M.; Flocco, M. M.; Cui, J.; Perego, R.;

Rusconi, L.; Cristiani, C. Crystal structure of the tyrosine kinase

domain of the hepatocyte growth factor receptor c-MET and its

complex with the microbial alkaloid K-252a. Proc. Natl. Acad. Sci.

U.S.A. 2003, 100, 12654−12659.

(12) Qian, F.; Engst, S.; Yamaguchi, K.; Yu, P.; Won, K.-A.; Mock, L.;

Tracy Lou, T.; Tan, J.; Li, C.; Tam, D.; Lougheed, J.; Yakes, F. M.;

Bentzien, F.; Xu, W.; Zaks, T.; Wooster, R.; Greshock, J.; Joly, A. H.

Inhibition of tumor cell growth, invasion, and metastasis by EXEL-

2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF

receptor tyrosine kinases. Cancer Res. 2009, 69, 8009−8016.

(13) Srinivasan, R.; Choueiri, T. K.; Vaishampayan, U.; Rosenberg, J.

E.; Stein, M. N.; Logan, T.; Bukowski, R. M.; Mueller, T.; Keer, H. N.;

Linehan, W. M. A phase II study of the dual MET/VEGFR2 inhibitor

XL880 in patients (pts) with papillary renal carcinoma (PRC).

Presented at ASCO 2008 Annual Meeting, Chicago, IL, May 30−June

3, 2008; Abstr. 5103.

Nolden, B.; Grass, G.; Bollschweiler, E.; Dienes, H. P.; Holscher, A.

̈

H.; Monig, S. P. Overexpression of c-MET as a prognostic indicator

̈

for gastric carcinoma compared to p53 and p21 nuclear accumulation.

Oncol. Rep. 2008, 19, 1477−1483.

(14) Jhawer, M. P.; Kindler, H. L.; Wainberg, Z. A.; Hecht, J. R.;

Kerr, R. O.; Ford, J. M.; Henderson, C.; Mueller, T.; Keer, H. N.;

Shah, M. A. Preliminary activity of XL880, a dual MET/VEGFR2

inhibitor, in MET ampliﬁed poorly diﬀerentiated gastric cancer

(PDGC): Interim results of a multicenter phase II study. Presented

at ASCO 2008 Annual Meeting, Chicago, IL, May 30−June 3, 2008;

Abstr. 4572.

(15) (a) Katz, J. D.; Jewell, J. P.; Guerin, D. J.; Lim, J.; Dinsmore, C.

J.; Deshmukh, S. V.; Pan, B.-S.; Marshall, C. G.; Lu, W.; Altman, M.

D.; Dahlberg, W. K.; Davis, L.; Falcone, D.; Gabarda, A. E.; Hang, G.;

Hatch, H.; Holmes, R.; Kunii, K.; Lumb, K. J.; Lutterbach, B.;

Mathvink, R.; Nazef, N.; Patel, S. B.; Qu, X.; Reilly, J. F.; Rickert, K.

W.; Rosenstein, C.; Soisson, S. M.; Spencer, K. B.; Szewczak, A. A.;

(4) (a) Matsumoto, K.; Nakamura, T. Mechanism and significance of

bifunctional NK4 in cancer treatment. Biochem. Biophys. Res. Commun.

2005, 333, 316−327. (b) Otsuka, T.; Jakubczak, J.; Vieira, W.; Bottaro,

D. P.; Breckenridge, D.; Larochelle, W. J.; Merlino, G. Disassociation

of met-mediated biological responses in vivo: the natural hepatocyte

growth factor/scatter factor splice variant NK2 antagonizes growth but

facilitates metastasis. Mol. Cell. Biol. 2000, 20, 2055−2065.

(c) Matsumoto, K; Nakamura, T.; Sakai, K.; Nakamura, T. Hepatocyte

growth factor and Met in tumor biology and therapeutic applications

with NK4. Proteomics 2008, 8, 3360−3370.

(5) Burgess, T.; Coxon, A.; Meyer, S.; Sun, J.; Rex, K.; Tsuruda, T.;

Chen, Q.; Ho, S.-Y.; Li, L.; Kaufman, S.; McDorman, K.; Cattley, R.

C.; Sun, J.; Elliott, G.; Zhang, K.; Feng, X.; Jia, X.-C.; Green, L.;

8108

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Journal of Medicinal Chemistry

Article

Walker, D.; Wang, W.; Young, J.; Zeng, Q. Discovery of a 5H-

benzo[4,5]cyclohepta[1,2-b]pyridin-5-one (MK-2461) inhibitor of c-

Met kinase for the treatment of cancer. J. Med. Chem. 2011, 54, 4092−

4108. (b) Rickert, K. W.; Patel, S. B.; Allison, T. J.; Byrne, N. J.; Darke,

P. L.; Ford, R. E.; Guerin, D. J.; Hall, D. L.; Kornienko, M.; Lu, J.;

Munshi, S. K.; Reid, J. C.; Shipman, J. M.; Stanton, E. F.; Wilson, K. J.;

Young, J. R.; Soisson, S. M.; Lumb, K. J. Structural basis for selective

small molecule kinase inhibition of activated c-Met. J. Biol. Chem.

2011, 286, 11218−11225.

J.; Montana, V.; Rocque, W. J.; Rusnak, D.; Shewchuk, L.; Veal, J. M.;

Walker, D. H.; Kuyper, L. F. Oxindole-based inhibitors of cyclin-

dependent kinase 2 (CDK2): design, synthesis, enzymatic activities,

and x-ray crystallographic analysis. J. Med. Chem. 2001, 44, 4339−

4358.

(26) Christensen, J. G.; Schreck, R.; Burrows, J.; Kuruganti, P.; Chan,

E.; Le, P.; Chen, J.; Wang, X.; Ruslim, L.; Blake, R.; Lipson, K. E.;

Ramphal, J.; Do, S.; Cui, J. J.; Cherrington, J. M.; Mendel, D. B. A

selective small molecule inhibitor of c-MET kinase inhibits c-MET-

dependent phenotypes in vitro and exhibits cytoreductive antimutor

activity in vivo. Cancer Res. 2003, 63, 7345−7355.

(27) PDB ID 2wkm for the PHA-665752/c-MET complex.

(28) Koenig, M.; Cui, J.; Wei, C. C.; Do, S. H.; Zhang, F.-J.;

Vojkovsky, T.; Ramphal, J.; Yang, G.; Mattson, M.; Nelson, C.; Tang,

P. C. Indolinone hydrazides as c-met inhibitors. PCT Int. Appl.

WO2005005378, 2005.

(29) Vojkovsky, T.; Koenig, M.; Zhang, F.-J.; Cui, J. Tetracyclic

compounds as c-Met inhibitors. PCT Int. Appl. WO2005004808,

2005.

́

(16) Cui, J. J.; Tran-Dube, M.; Shen, H.; Nambu, M.; Kung, P. P.;

Pairish, M.; Jia, L.; Meng, J.; Funk, L.; Botrous, I.; McTigue, M.;

Grodsky, N.; Ryan, K.; Padrique, E.; Alton, G.; Timofeevski, S.;

Yamazaki, S.; Li, Q.; Zou, H.; Christensen, J.; Mroczkowski, B.;

Bender, S.; Kania, R. S.; Edwards, M. P. Structure based drug design of

crizotinib (PF-02341066), a potent and selective dual inhibitor of

mesenchymal-epithelial transition factor (c-MET) kinase and ana-

plastic lymphoma kinase (ALK). J. Med. Chem. 2011, 54, 6342−6363.

(17) Kwak, E. L.; Bang, Y.-J.; Camidge, R.; Shaw, A. T.; Solomon, B;

Maki, R. G.; Ou, S.-H. I.; Dezube, B. J.; Janne, P. A.; Costa, D. B.;

̈

(30) Zhang, F.-J.; Vojkovsky, T.; Huang, P.; Liang, C.; Do, S. H.;

Koenig, M.; Cui, J. Preparation of triazolotriazines as c-Met

modulators for treating cancer. PCT Int. Appl. WO2005010005, 2005.

(31) Cui, J.; Botrous, I. Arylmethyl triazolo and imidazopyrazines as

c-MET inhibitors. PCT Int. Appl. WO2005004607, 2005.

(32) Zou, H. Y.; Li, Q.; Lee, J. H.; Arango, M. E.; Burgess, K.; Qiu,

M.; Engstrom, L.; Yamazaki, S.; Parker, M.; Timofeevski, S.; Cui, J. J.;

McTigue, M.; Los, M. G.; Bender, S.; Smeal, T.; Christensen, J. G.

Sensitivity of Selected Human Tumor Models to PF-04217903, a

Novel Selective c-Met Kinase Inhibitor. Mol. Cancer Ther. 2012, 11,

1036−1047.

(33) Yamazaki, S.; Skaptason, J.; Romero, D.; Vekich, S.; Jones, H.

M.; Tan, W.; Wilner, K.; Koudriakova, T. Prediction of oral

pharmacokinetics of cMet kinase inhibitors in humans: physiologi-

cally-based pharmacokinetic modeling versus traditional one-compart-

ment model. Drug Metab. Dispos. 2011, 39, 383−393.

(34) Trust, R. I.; Albright, J. D.; Lovell, F. M.; N. A. Perkinson, N. A.

6- and 7-aryl-1,2,4-triazolo[4,3-b]-1,2,4-triazines. Synthesis and char-

acterization. J. Heterocycl. Chem. 1979, 16, 1393−1403.

(35) (a) Marusyk, A.; Vanessa Almendro, V.; Polyak, K. Intra-tumour

heterogeneity: a looking glass for cancer? . Nat. Rev. Cancer 2012, 12,

323−334. (b) Szerlipa, N. J.; Pedrazab, A.; Chakravartyb, D.; Azimc,

M.; McGuirec, J.; Fangd, Y.; Ozawae, T.; Hollande, E. C.; Hused, J. T.;

Jhanward, S.; Levershac, M. A.; Mikkelseni, T.; Brennan, C. W.

Intratumoral heterogeneity of receptor tyrosine kinases EGFR and

PDGFRA amplification in glioblastoma defines subpopulations with

distinct growth factor response. Proc. Natl. Acad. Sci. U.S.A 2012, 109,

3041−3046. (c) Gerlinger, M.; Rowan, A. J.; Horswell, S.; Larkin, J.;

David Endesfelder, D.; Gronroos, E.; Martinez, P.; Matthews, N.;

Stewart, A.; Tarpey, P.; Varela, I.; Phillimore, B.; Begum, S.;

McDonald, N. Q.; Butler, A.; Jones, D.; Raine, K.; Latimer, C.;

Santos, C. R.; Nohadani, M.; Eklund, A. C.; Spencer-Dene, B.; Clark,

G; Pickering, L.; Stamp, G.; Gore, M.; Szallasi, Z.; Downward, J.;

Futreal, P. A.; Swanton, C. Intratumor heterogeneity and branched

evolution revealed by multiregion sequencing. N. Engl. J. Med. 2012,

366, 883−892.

Varella-Garcia, M.; Kim, W.-H.; Lynch, T. J.; Fidias, P.; Stubbs, H.;

Engelman, J. A.; Sequist, L. V.; Tan, W.; Gandhi, L.; Mino-Kenudson,

M.; Wei, G. C.; Shreeve, S. M.; Ratain, M. J.; Settleman, J.;

Christensen, J. G.; Haber, D. A.; Wilner, K.; Salgia, R.; Shapiro, G. I.;

Clark, J. W.; Iafrate, A. J. Anaplastic lymphoma kinase inhibition in

non-small-cell-lung cancer. N. Engl. J. Med. 2010, 363, 1693−1703.

(18) Ou, S.-H. I.; Kwak, E. L.; Siwak-Tapp, C.; Dy, J.; Bergethon, K.;

Clark, J. W.; Camidge, D. R.; Solomon, B. J.; Maki, R. G.; Bang, Y.-J.;

Kim, D.-W.; Christensen, J.; Tan, W.; Wilner, K. D.; Salgia, R.; Iafrate,

A. J. Activity of crizotinib (PF02341066), a dual mesenchymal-

epithelial transition (MET) and anaplastic lymphoma kinase (ALK)

inhibitor, in a non-small cell lung cancer patient with de novo MET

amplification. J. Thorac. Oncol. 2011, 6, 942−946.

(19) Chi, A. S.; Kwak, E. L.; Clark, J. W.; Wang, D. L.; Louis, D. N.;

Iafrate, A. J.; Batchelor, T. Clinical improvement and rapid

radiographic regression induced by a MET inhibitor in a patient

with MET-ampliﬁed glioblastoma. Presented at ASCO 2011 Annual

Meeting, Chicago, IL, June 3−7, 2011; Abstr. 2072.

(20) Lennerz, J. K.; Kwak, E. L.; Michael, M.; Fox, S. B.; Ackerman,

A.; Bergethon, K.; Lauwers, G. Y.; Christensen, J. G.; Wilner, K. D.;

Haber, D. A.; Salgia, R.; Bang, Y.; Clark, J. W.; Solomon, B. J.; Iafrate,

A., J. Identiﬁcation of a small and lethal subgroup of esophagogastric

adenocarcinoma with evidence of responsiveness to crizotinib by MET

ampliﬁcation. Presented at ASCO 2011 Annual Meeting, Chicago, IL,

June 3−7, 2011; Abstr. 4130.

(21) Munshi, N.; Jeay, S.; Li, Y.; Chen, C. R.; France, D. S.; Ashwell,

M. A.; Hill, J.; Moussa, M. M.; Leggett, D. S.; Li, C. J. ARQ 197, a

novel and selective inhibitor of the human c-Met receptor tyrosine

kinase with antitumor activity. Mol. Cancer Ther. 2010, 9, 1544−1553.

(22) Sequist, L. V.; von Pawel, J.; Garmey, E. G.; Akerley, W. L.;

Brugger, W.; Ferrari, D.; Chen, Y.; Costa, D. B.; Gerber, D. E.; Orlov,

S.; Ramlau, R.; Arthur, S.; Gorbachevsky, I.; Schwartz, B.; Schiller, J. H.

Randomized phase II study of erlotinib plus tivantinib versus erlotinib

plus placebo in previously treated non-small-cell lung cancer. J. Clin.

Oncol. 2011, 29, 3307−3315.

(23) Timofeevski, S. L.; McTigue, M. A.; Ryan, K.; Cui, J.; Zou, H.

Y.; Zhu, J. X.; Chau, F.; Alton, G.; Karlicek, S.; Christensen, J. G.;

Murray, B. W. Enzymatic characterization of c-MET receptor tyrosine

kinase oncogenic mutants and kinetic studies with aminopyridine and

triazolopyrazine inhibitors. Biochemistry 2009, 48, 5339−5349.

(24) Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.;

Fukuda, J. Y.; Chu, J.-Y.; Nematalla, A.; Wang, X.; Chen, H.; Sistla, S.;

Luu, T. L.; Tang, F.; Wei, J. Cho, Tang. Discovery of 5-[5-fluoro-2-

oxo-1,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-

3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase

inhibitor targeting vascular endothelial and platelet-derived growth

factor receptor tyrosine kinase. J. Med. Chem. 2003, 46, 1116−1119.

(25) Bramson, H. N.; Corona, J.; Davis, S. T.; Dickerson, S. H.;

Edelstein, M.; Frye, S. V.; Gampe, R. T., Jr.; Harris, P. A.; Hassell, A.;

Holmes, W. D.; Hunter, R. N.; Lackey, K. E.; Lovejoy, B.; Luzzio, M.

(36) Zou, H. Y.; Li, Q.; Lee, J. H.; Arango, M. E.; McDonnell, S. R.;

Yamazaki, S.; Tatiana B. Koudriakova, T. B.; Alton, G.; Cui, J. J.; Kung,

P.-P.; Nambu, M. D.; Los, G.; Bender, B. L.; Mroczkowski, B.;

Christensen, J. G. An orally available small-molecule inhibitor of c-

Met, PF-2341066, exhibits cytoreductive antitumor efficacy through

antiproliferative and antiangiogenic mechanisms. Cancer Res. 2007, 67,

4408−4417.

8109

dx.doi.org/10.1021/jm300967g | J. Med. Chem. 2012, 55, 8091−8109

Article Doi

DOI: 10.1021/jm300967g

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm300967g

Full text of DOI:10.1021/jm300967g

Products guided by the article

R&D Labs maybe for 1399857-77-2

Relevant to this article

Hot Product