Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK)

ARTICLE

pubs.acs.org/jmc

Structure Based Drug Design of Crizotinib (PF-02341066), a Potent

and Selective Dual Inhibitor of MesenchymalꢀEpithelial Transition

Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK)^†

J. Jean Cui,* Michelle Tran-Dubꢀe, Hong Shen, Mitchell Nambu, Pei-Pei Kung, Mason Pairish, Lei Jia,

Jerry Meng, Lee Funk, Iriny Botrous, Michele McTigue, Neil Grodsky, Kevin Ryan, Ellen Padrique,

Gordon Alton, Sergei Timofeevski, Shinji Yamazaki, Qiuhua Li, Helen Zou, James Christensen,

Barbara Mroczkowski, Steve Bender, Robert S. Kania, and Martin P. Edwards

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

ABSTRACT: Because of the critical roles of aberrant signaling

in cancer, both c-MET and ALK receptor tyrosine kinases are

attractive oncology targets for therapeutic intervention. The

cocrystal structure of 3 (PHA-665752), bound to c-MET kinase

domain, revealed a novel ATP site environment, which served

as the target to guide parallel, multiattribute drug design. A

novel 2-amino-5-aryl-3-benzyloxypyridine series was created to

more eﬀectively make the key interactions achieved with 3. In

the novel series, the 2-aminopyridine core allowed a 3-benzyl-

oxy group to reach into the same pocket as the 2,6-dichloro-

phenyl group of 3 via a more direct vector and thus with a better

ligand eﬃciency (LE). Further optimization of the lead series generated the clinical candidate crizotinib (PF-02341066), which

demonstrated potent in vitro and in vivo c-MET kinase and ALK inhibition, eﬀective tumor growth inhibition, and good

pharmaceutical properties.

’ INTRODUCTION

c-MET induces an invasive program consisting of cell prolifera-

tion, migration, invasion, survival, and branching morphogenesis.

Aberrant c-MET signaling through constitutive activation, gene

ampliﬁcation, and mutations occurs in virtually all types of solid

tumors and is implicated in dysregulation of multiple tumor

oncogenic processes such as mitogenesis, survival, angiogenesis,

invasive growth, and especially the metastatic process.²Further-

more, the 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 reasons c-MET and

its ligand HGF have become leading candidates for molecular

targeted cancer therapies.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase,

grouped together with leukocyte tyrosine kinase (LTK) to a

subfamily within the insulin receptor (IR) superfamily. ALK was

ﬁrst discovered as a fusion protein, NPM (nucleophosmin)ꢀALK

in anaplastic large cell lymphoma cell lines in 1994.⁴The

transforming ability of NPMꢀALK was demonstrated, and

the oncogenesis requires the activation of ALK kinase function

as a result of oligomerization mediated by the NPM segment.⁵

ALK with other chromosomal rearrangements have been de-

tected in anaplastic large cell lymphoma (50ꢀ60%), inﬂamma-

tory myoﬁbroblastic tumors (27%), and non-small-cell lung

Receptor tyrosine kinases (RTKs) play fundamental roles in

cellular processes, including cell proliferation, migration, meta-

bolism, diﬀerentiation, and survival. RTK activity is tightly

controlled in normal cells. The constitutively enhanced RTK

activities from point mutation, ampliﬁcation, and rearrangement

of the corresponding genes have been implicated in the devel-

opment and progression of many types of cancer.¹Successful

approval of small molecule tyrosine kinase inhibitors has clini-

cally validated this mode of therapeutic intervention, along with

several pathogenic tyrosine kinases as eﬀective molecular targets

for cancer therapy. Recent examples include imatinib in gastro-

intestinal stromal tumors with mutant c-KIT kinase or chronic

myelogenous leukemia with BCR-ABL gene translocations,

erlotinib in non-small-cell lung cancer (NSCLC) with mutant

EGFR, and sunitinib targeting the VHL-dependent VEGF path-

way in renal cell carcinoma.

Signaling from the receptor tyrosine kinase c-MET, also

known as hepatocyte growth factor receptor (HGFR), and its

natural ligand hepatocyte growth factor (HGF), also known as

scatter factor, plays important roles during normal development,

organogenesis, and homeostasis. After activation by HGF,

^†PDB codes are the following: 2wkm for the PHA-665752/c-METcomplex;

2wgj for the PF-02341066/c-METcomplex; 2xp2 for the PF-02341066/ALK

complex.

Received: June 13, 2011

Published: August 03, 2011

r

2011 American Chemical Society

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Figure 1. Structures of kinase inhibitors.

cancer (4ꢀ7%).⁶The ALK fusion proteins have a common

structural feature with the amino terminal region of the fusion

protein containing an oligomerization domain to cause the

oligomerization of the fusion protein and ALK kinase-mediated

autophosphorylation. EML4ꢀALK fusion gene, comprising por-

tions of the echinoderm microtubule associated protein-like 4

(EML4) gene and the ALK gene, was ﬁrst discovered in non-

small-cell lung cancer archived clinical specimens and also cell

lines.⁷EML4ꢀALK fusion variants were demonstrated to trans-

form NIH-3T3 ﬁbroblasts and cause lung adenocarcinoma when

expressed in transgenic mice, which conﬁrm the potent onco-

genic activity of the fusion kinase.⁸Oncogenic mutations of ALK

in both familial and sporadic cases of neuroblastoma have also

been reported.⁹Therefore, ALK is an attractive molecular target

for cancer therapeutic intervention.

Herein, we detail the drug design campaign that led to

crizotinib (PF-02341066, 63), a potent and selective c-MET/

ALK dual inhibitor.¹⁰Consistent with its mechanism of action,

crizotinib demonstrates dose-dependent inhibition of phosphory-

lation of c-MET, NPMꢀALK, and selected variants, as well as

targets dependent functions in tumor cells both in vitro and

in vivo.¹⁰Crizotinib showed antitumor eﬃcacy, including marked

cytoreductive antitumor activity, in multiple tumor models im-

planted in athymic mice that expressed activated c-MET or ALK

fusion proteins.¹⁰In additional published studies, crizotinib in-

hibits tumor cell growth of cell lines harboring fusion variants and

activating mutations of ALK including Karpas299 (NPMꢀALK),

SU-DHL-1 (NPMꢀALK), Kelly neuroblastoma (active ALK

mutation), and NCI-H3122 (EML4ꢀALK variant 1) with IC₅₀

values ranging from 74 to 544 nM.¹¹On the basis of an exceptional

attribute proﬁle, crizotinib was advanced into human clinical

studies for the treatment of cancer.

sunitinib, is now approved for GIST and RCC treatment.¹²The

selectivity of indolin-2-ones for particular kinases is mediated by

substituents built onto the indolin-2-one core. Both 4-substituted

indolin-2-ones and 5-substituted indolin-2-ones were evaluated

for c-MET inhibition and attractive lead matter was found.¹³

From this starting point, key cocrystal structures guided both

prioritization decisions and drug design strategy.

To monitor the progress of optimization, lipophilic eﬃciency

(LipE = pK_i(or pIC₅₀) ꢀ cLogD) was used as a numerical index

of binding eﬀectiveness.¹⁴Drug design aimed at improvement of

LipE results in parallel optimization for desirable ADME and

greater likelihood of safe drug proﬁles, since ligand interactions

with most proteins are substantially inﬂuenced by lipophilicity.¹⁵

This convenient numeric index that reﬂects compound target

potency relative to lipophilicity is presented consistently through-

out, demonstrating the eﬀectiveness of integrated SBDD and

property based drug design.

c-MET Leads from the 3-Substituted Indolin-2-one Series.

Compound 2 (SU11274), shown in Figure 1, was identified as a

c-MET inhibitor withisolatedenzymeIC₅₀of 10 nM.¹⁶2 inhibited

HGF-induced c-MET autophosphorylation in a dose dependent

manner with complete inhibition at 1 μM in A549 c-MET

cellular assay.¹⁶Optimization of 2 led to 3 (PHA-665752)

(Figure 4), a c-MET RTK inhibitor with a significantly improved

cellular potency (IC₅₀= 9 nM in GTL-16 cell line) and selectivity

(>50-fold for c-MET compared with a panel of diverse tyrosine

and serineꢀthreonine kinases).^13c,173 was broadly used in

preclinical studies to build confidence in c-MET as a target for

cancer therapy and to identify potential patient populations.¹⁸In

a variety of tumor cells, 3 potently inhibited HGF stimulated and

constitutive c-MET phosphorylation, downstream signal trans-

duction of c-MET, and HGF/c-MET driven phenotypes, e.g.,

cell growth, cell motility, invasion, and morphology. In vivo, 3

inhibited c-MET phosphorylation in tumor xenografts, and

tumor growth in a dose-dependent manner.^17,18However, the

poor pharmaceutical properties of 3 limited its further develop-

ment as a clinic candidate.¹⁹

A cocrystal structure of 3 bound to the unphosphorylated

c-MET KD revealed the key binding interactions as presented in

Figure 2 (PDB code 2wkm). In the complex of 3, the c-MET KD

adopts the unique autoinhibitory conformation observed pre-

viously in crystal structures of the apo-enzyme and a complex

with the staurosporine analogue K252a.²⁰In these c-MET crystal

structures, the beginning of the kinase activation loop (residues

1222ꢀ1227) forms a turn that wedges between the β-sheet and

the RC-helix. Consequently, the activation loop signiﬁcantly

displaces the RC-helix from a catalytically competent position

and the downstream activation loop residues (1228ꢀ1245) to a

position that interferes with ATP and substrate binding. This

unusual kinase activation loop conformation creates a unique

’ RESULTS AND DISCUSSION

Foundations of Structure Based Design. To enable a

structure based drug design (SBDD) program, a kinase domain

(KD) construct of c-MET, in its nonphosphorylated form, was

prepared for utilization in cocrystallization experiments. Fortu-

nately, cell-based activity of the inhibitors prevented RTK autop-

hosphorylation, consistent with inhibitory interaction with the

nonphosphorylated form in cells. Inhibition of phosphorylated

c-MET KD in purified enzyme biochemical assays confirmed that

the inhibitors can inhibit both unactivated and activated c-MET.

Overall, there was little divergence between enzyme and cell data,

apart from that which could be readily understood by perme-

ability/efflux considerations. Where there was minor divergence,

the structural biology was illuminating, as will be discussed below.

The search for leads started in a potent class of kinase inhibitors,

the 3-substituted indolin-2-ones, where one representative,

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Figure 2. Cocrystal structure of 3 bound to the kinase domain of

c-MET. The backbone trace of the activation loop is highlighted in cyan,

and hydrogen bonds are indicated as dashed lines.

Figure 4. Design of 5-aryl-3-benzyloxy-2-aminopyridine scaﬀold for

c-MET inhibition.

bond between the oxygen of the sulfonyl group and the backbone

NꢀH of Asp-1222 is also stabilizing this orientation.

For clarity, it is useful to establish that all the inhibitors

discussed herein are believed to bind the above unique A-loop

conformation of c-MET and that the phosphorylation state of

c-MET protein in diﬀerent contexts is as follows: (1) c-MET

remains nonphosphorylated in cells when inhibited; (2) in crys-

tal structures of nonphosphorylated c-MET the protein adopts a

unique conformation to which the inhibitors bind. It is also

recognized that phosphorylation of the activation loop is part of

the regulatory mechanism that destabilizes the autoinhibited

activation loop conformation that is serving as the drug design

target. Impacts of this become apparent in small potency trend

diﬀerences between enzyme and cell assays for certain inhibitor

structural types. The diﬀerences arise, predictably, from structural

features optimized to bind adjacent to the activation loop.

Design of Novel 5-Aryl-3-benzyloxy-2-aminopyridine

c-MET Inhibitors. Although 3 demonstrated potent and selec-

tive inhibition of c-MET autophosphorylation and related bio-

logical functions in both in vitro and in vivo studies, the poor

pharmaceutical properties (low solubility, high metabolic clear-

ance, and poor permeability) limited further development for

human clinical studies. With improved pharmaceutical properties

as the goal, a key strategy was to design smaller and less lipophilic

inhibitors (3, MW = 641.62, measured log D = 3.20 at pH 7.4,

c-MET cell IC₅₀= 9 nM, LE = 0.25,²²LipE = 4.85) with good

kinase selectivity.

The cocrystal structure of 3 with c-MET KD was analyzed

to elucidate opportunities to make key interactions with a more

eﬃcient use of chemical structure. Starting with fundamental

scaﬀold optimization, one such opportunity was recognized by

analyzing the indolin-2-one pyrrole core, which extends the full

length of the adenine pocket. In addition to occupying a low tilt

position in the adenine pocket (vide supra) the core size requires

that the sulfone methylene linker make a U-turn to position the

2,6-dichlorophenyl group for a πꢀπ stacking interaction with

Tyr-1230. Consequently, a signiﬁcant portion of the indolinone

ring along with the sulfone linker was identiﬁed as ineﬃcient

scaﬀolding for the 2,6-dichlorophenyl group. Compounds were

designed with a smaller hinge binder, from which could be

positioned an aryl ring to interact with Tyr-1230 in a more direct

manner.

Figure 3. Structure overlay of 3/c-MET (green/gray) and 1/FGFR1K

(cyan).

inhibitor binding pocket which presents an opportunity for the

design of selective inhibitors.

As in a previously published cocrystal structure of 1 (SU5402)

with ﬁbroblast growth factor receptor 1 kinase (FGFR1K),²¹the

oxindole ring NꢀH and carbonyl oxygen of 3 form two hydrogen

bonds to the protein backbone of the kinase hinge region. Also,

similar to the 1/FGFR1K complex, the amide substituent oﬀ

the pyrrole ring extends into the solvent surrounding the kinase

hinge segment. For both inhibitors, the oxindole and pyrrole

rings are in a coplanar conformation, stabilized by resonance and

an intramolecular hydrogen bond between the pyrrole NꢀH and

the oxindole OdC. This ﬂat scaﬀold provides strong interactions

to the adenine binding cleft while ﬁtting its ﬂat dimensions well.

Unlike the 1/FGFR1K structure, the plane of the oxindoleꢀ

pyrrole system in 3 is tilted away from the c-MET glycine-rich

loop signiﬁcantly (down in Figure 3), a diﬀerence arising from

both protein and inhibitor structural diﬀerences. This lower

inhibitor position in c-MET is permitted by the more ﬂexible

(and compressed) Met-1211 and is stabilized by the critical πꢀπ

stacking interaction between the benzyl group and Tyr-1230 of

the A-loop in its unique conformation (Figure 2). A hydrogen

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3-position is the structural feature that binds to the hydrophobic

pocket and interacts with Tyr-1230, a priority area to study struc-

tural modifications. Various aryl analogues intended to interact

with Tyr-1230 were targeted, with representative results and

LipE analysis results summarized in Table 1 and Figure 6. The

convenience of the graphical plot allows for easy recognition of

overall optimization against constant LipE zones, facilitating the

mining of structural features that impart movement orthogonal

to the LipE lines (e.g., 14c, 14i, and 14j are similarly efficient

across broad potency/cLogD space).

Unsubstituted 11b serves as a benchmark for c-MET kinase

inhibition (K_i= 7.92 μM, LipE = 3.66), establishing a baseline to

analyze LipE contributions from diﬀerent phenyl ring substitu-

tion patterns. The relatively conservative phenyl ring derivatives

displayed in Table 1 have enzymatic LipE values ranging from 3

to 5. The 2-position, monosubstituted phenyl ring compounds

14aꢀd had measurable increases in potency against c-MET, with

the exception of nitrile 14c. Although with potency similar to that

of 11b, this nitrile compound had the highest biochemical LipE

of 4.31. However, 14c was not active in the c-MET cellular assay

at 10 μM tested. Compound 14e, a 4-position derivative bearing

the highly lipophilic tert-butylphenyl group, had improved enzy-

matic potency against c-MET (K_i= 0.98 μM). However, the

lower LipE (3.15), which was even worse when using cell-based

numbers, indicated that the potency gain was not attractive

relative to lipophilicity cost. The 2,4-dichloro substituted com-

pound 14f showed slightly improved enzymatic LipE due to a

lower lipophilicity. This translated to greater cell potency with an

IC₅₀of 0.41 μM and one of the three big jumps in cell LipE

observed within this table. Although a number of variables could

be contributing, the cell vs biochemical potency of 14f was an

early indication that SAR between cell and biochemical measures

may diverge because of the diﬀerent binding aﬃnity of the inhib-

itor to nonphosphorylated c-MET vs phosphorylated c-MET.

Importantly, cell/enzyme potency divergence was associated

with the benzylic structural feature, which interacts with the

unique tyrosine and protein conformation of nonphosphorylated

c-MET discussed earlier. Previous work with RTKs reinforced

the practice of targeting nonphosphorylated kinase and prioritiz-

ing the cell-based data, which is consistent with conformations

and interactions seen crystallographically.²⁵The potency con-

tribution of the 3-ﬂuoro substituent on the phenyl ring was

demonstrated by 14h compared to 14g. The addition of the

3-ﬂuoro imparted a 5-fold increase in enzymatic potency (K_i=

Figure 5. First iteration 2-aminopyridines show c-MET inhibition.

Through re-engineering of the central core rings (three itera-

tions of thinking are depicted in Figure 4), the novel 5-aryl-3-

benzyloxy-2-aminopyridine scaﬀold was designed. This required

a recentering and truncating of the pyrroleꢀoxindole platform

down to a small, adenine-pocket core. Accordingly, the 2-amino-

pyridine NH and ring nitrogen were expected to make H-bonds to

the hinge protein residues Pro-1158 and Met-1160, similar to the

oxindoleringof3. The moreboldrequirementof this design is that

the 3-benzyloxy group must take an eﬃcient path, along a new

vector, to stack withTyr-1230. For the shortenedlinkage to stretch

back to the hinge, binding must be accompanied by a scaﬀold tilt

back to the higher adenine pocket position seen with 1. Last, the

aryl group at the 5-position of this new scaﬀold is easily anticipated

to point toward solvent in the same way oxindoleꢀpyrrole sub-

stituents do, providing a handle to modulate lipophilicity.

The design concept was tested with a small set of compounds

shown in Figure 5. Compound 7 displayed moderate inhibition

against c-MET with an enzymatic K_iof 3.83 μM (LE= 0.29,

LipE = 0.35). The introduction of a 2-morpholinoethoxyethyl

group in 8 showed a similar potency against c-MET as 7. Although

this is less eﬃcient, retained inhibition is consistent with the

5-phenyl group pointing toward solvent and the 2-aminopyridine

interacting at the hinge. Compound 11a (LE = 0.24, LipE = 3.70),

with the same amide group as 3, demonstrated improved absolute

potency against c-MET and also LipE value, again consistent with

a similar binding mode for this novel series. Additionally, given

the diﬀerent vector of the new series, optimal substituents for the

novel 2-aminopyridines were not expected to mirror those of the

oxindole core. A cocrystal structure of the 2-aminopyridine core

making H-bonds to the hinge conﬁrmed the adenine binding

orientation.²³For compounds 7, 8, and 11a, the structural features

thatare necessary to bind the hinge backbone wereincorporatedin

the monocyclic 2-aminopyridine whereas the O-methylene-2,6-

dichlorophenyl was intended to fully and eﬃciently interact with

Tyr-1230 of the A-loop. The trajectory of the solvent exposed

group and the electronic eﬀects already observed between 8 and

11a gave reason to expect that optimization from this point would

be productive. From this novel series, with greater promise of

eﬃciency, optimization was focused on improving c-MET potency

and physical properties in parallel.

0.88 μM) and a 14.5-fold increase of cell potency (IC₅₀

=

0.56 μM) relative to the 2-chloro-6-ﬂuorophenyl ring of 14g

(K_i= 5.11 μM and cell IC₅₀= 8.12 μM). The improvement of

c-MET cell LipE, from 3.45 to 4.38 for 14h, reinforced the

importance of this single atom change and is the second jump in

LipE. Overall, the phenyl group substitution optimization pro-

vided moderate improvement to c-MET potency, with notable

impacts for 2-chloro and 3-ﬂuoro substitution.

Further optimization came from modiﬁcation to the linkage

between the 2-aminopyridine core and the phenyl ring just discussed.

A small lipophilic pocket, visible to the upper left of Figure 3, is

adjacent to the putative bound location of the 3-benzyloxy linker.

Adding small lipophilic bulk to this structural feature led to enhanced

eﬃciency from an R-methyl group. As demonstrated with com-

pounds 14iꢀk, inclusion of the R-methyl group boosted both

enzymatic and cell potencies signiﬁcantly compared with the

desmethyl analogues, justifying the added lipophilicity. By com-

parison of the R-methyl 14j with the desmethyl analogue 11a, the

Optimization of the 3-Benzyloxy Group. Further optimiza-

tion of the 2-aminopyridine series was supported by the promis-

ing inhibition exhibited by 11a, which also served as a convenient

scaffold for initial analogues. According to the binding mode

of the original design hypothesis, the benzyloxy group at the

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Table 1. StructureꢀActivity Relationship of Substitutes on 3-Benzyloxy Group

c

compd

R₁

R₂

cLogD ^a

c-MET K_i(μM)^b

LipE(K_i) ^c

c-MET cell IC₅₀(μM)^b

LipE(IC₅₀)

11a

11b

14a

14b

14c

14d

14e

14f

H

2,6-di-Cl

H

2.64

1.44

1.49

2.03

0.88

2.01

3.13

2.64

1.64

1.87

2.22

2.99

3.10

0.46

7.92

2.82

1.71

6.50

2.59

0.98

1.03

5.11

0.88

0.26

0.068

0.012

3.70

3.66

4.08

3.79

4.31

3.58

3.15

3.35

4.16

4.18

4.37

4.20

4.82

1.79

>10.0

6.62

5.85

>10.0

7.11

8.53

0.41

8.12

0.56

0.39

0.14

0.020

3.11

<3.56

3.69

3.20

<4.12

3.14

1.94

3.75

3.45

4.38

4.19

3.86

4.60

H

2-F

H

2-Cl

H

2-CN

H

2-CF₃

H

4-tert-butyl

2,4-di-Cl

2-Cl-6-F

2-Cl-3,6-di-F

2,6-di-Cl

2,6-di-Cl-3-F

H

14g

14h

14i

H

CH₃

14j

14k

^aCalculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat, version 9.3. ^bInhibition constants (K_i)²⁴and cell

IC₅₀^10awere determined as described in Experimental Methods. The coeﬃcients of variance were typically less than 20% (n = 2). A549 human lung

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. ^cLipE = pK_i(or pIC₅₀) ꢀ cLogD.

on 5-position derivation to address remaining drug design

goals.

Optimization at the 5-Position of the 2-Aminopyridine

Series to Crizotinib. With the potent c-MET inhibitor 14k in

hand, design strategies focused on ensuring that potency is

maintained while more aggressively tuning physical properties

to achieve the ADME profile goals needed for a clinical candi-

date. Across series, both inhibition data and solved cocrystal

structures suggested that substituents extending through the

narrow hydrophobic cleft formed by Tyr-1159, Ile-1084, and

Gly-1163 into solvent were efficient contributors to potency,

provided the necessary planar relationship to the core is main-

tained. Modeling of 2-aminopyridines suggested that the 5-posi-

tion vector was the best opportunity to deliver polar groups to

Figure 6. LipE plot highlights 14k, 14i, and 14c as lead compounds and

where close analogues reside.

the solvent, allowing physical property modulation while opti-

mizing potency. To rapidly test this approach, truncated 2-ami-

nopyridine cores and extended basic amine derivatives from

enzyme based LipE improves from 3.70 to 4.02 and the cell-

based LipE improves from 3.11 to 3.86. Importantly, bringing

together the 2,6-dichloro and 3-ﬂuoro and R-methyl into a single

compound, 14k, resulted in the most potent inhibition against

c-MET and the highest LipE values observed within this speciﬁc

series (enzymatic LipE = 4.80 and cell LipE = 4.60) as illustrated

in Figure 6. Of note, these are racemates, and the relative poten-

cies of enantiomers will be discussed later. Accordingly, the

phenyl substitution pattern of 14k was the focus for further lead

optimization of the 2-aminopyridine series, which next focused

readily available materials were evaluated (Table 2). Expectedly,

only modest inhibition was observed for 21, which has no

5-position substituent. Additionally, 22 and 26 with 5-bromo

and 5-cyano groups had similarly weak potency as did the 5-N-

amide 25, which introduces polarity too close to the core.

Additionally, the 5-phenyl derivative 27 also resulted in micro-

molar potency. However, greater potency and LipE were

achieved with 14k and 36ꢀ38. A number of factors are poten-

tially contributing to this increase. For these latter compounds,

the amide moiety is positioned away from the adenine pocket

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Table 2. Structure and Activity Relationship of 5-Substitutes on Racemic 3-(1-(2,6-Dichloro-3-ﬂuorophenyl)ethoxy)pyridin-2-

amine

^aCalculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat, version 9.3. ^bInhibition constants (K_i)²⁴and cell

IC₅₀^10awere determined as described in Experimental Methods. The coeﬃcients of variance were typically less than 20% (n = 2). A549 human lung

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. ^cLipE = pK_i(or pIC₅₀) ꢀ cLogD.

and into solvent, eliminating the desolvation penalty seen for

25. Furthermore, the amide is electron withdrawing, perhaps

contributing to greater planarity arising from conjugation to the

amino group of the core and possibly modifying the H-bond

donating potential as a result. Last, the solvent exposed basic

groups, which lower log D, result in enzymatic and cell LipE

values that are at attractive levels, as exemplified with 14k.

In order to maximize chances of good ADME properties and

improved potencies by attenuating torsion strain associated with

planar binding, a set of smaller, less lipophilic 5-heteroaryl ana-

logues were prepared as summarized in Table 3. The preference

of a ﬁve-member heteroaryl group at the 5-position of 2-amino-

pyridine is consistent with coplanarity of the 5-aryl group with

the pyridine scaﬀold upon binding. The ﬁve-member heteroaryl

groups also lowered cLogD by about 2 units in general from the

phenyl containing 27. Consequently, compounds 24 and 29ꢀ35

showed much improved LipE in the kinase assay, with the weakest

improvements coming from compounds 24 and 34. The perfor-

mance of 34 is expected because of the methyl groups which

destabilize a planar arrangement. To help understand 24, crystal

structures of potent analogues indicate that backbone c-MET

protein carbonyl oxygen atoms of residues Met-1160 and Ile-1084

are oriented toward the “ortho” positions of these ﬁve-membered

heterocycles. Additional residues at the ligandꢀprotein interface

disfavor access of bulk water to these positions, requiring the

breaking of solvent H-bonds to the nitrogen lone pair of 24 upon

binding. The structural biology, therefore, is consistent with a

desolvation penalty and unfavorable electrostatics for com-

pounds such as 24 that present “ortho” electronegative heteroa-

toms, with capacity as H-bond acceptors only. In contrast, the

5-pyrazol-4-yl group of 33 improved enzymatic and cell potency

by 10- to 20-fold, resulting in a cell LipE of 2.83, more than a 3

unit improvement over compound 27. In this case, the heteroa-

toms are oriented toward solvent and therefore pay no desolva-

tion penalty. As a result, the pyrazol-4-yl group provided the most

potent inhibition against c-MET and the highest LipE value in

comparison with other heteroaryl and phenyl groups. Addition-

ally, N-substitution on the pyrazol-4-yl group was well tolerated,

as demonstrated by enzymatic and cell potency and an improved

cell LipE value of 2.83 for 35, indicating an attractive design path

forward.

For earlier subseries (e.g., 36ꢀ38), extending polar basic

groups into solvent was a strategy that consistently improved

potency and eﬃciency. With knowledge that the pyrazole linker

provided an extended conformation and vector that resulted in

highly eﬃcient cores, a focused lead optimization eﬀort sought to

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Table 3. Structure, Activity, and Lipophilicity Relationships of Racemic 5-Aryl Derivatives

^aCalculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat, version 9.3. ^bInhibition constants (K_i)²⁴and cell

IC₅₀^10awere determined as described in Experimental Methods. The coeﬃcients of variance were typically less than 20% (n = 2). A549 human lung

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. ^cLipE = pK_i(or pIC₅₀) ꢀ cLogD.

combine these structural features to achieve both the potency

and the pharmaceutical properties being sought. For polar

N-substituents on the pyrazole-4-yl ring, the smallest groups

were targeted to provide desirable properties. A set of represen-

tative examples are summarized in Table 4.

lipophilicity of the key 3-(1-(2,6-dichloro-3-ﬂuorophenyl)ethoxy

group on this novel 2-aminopyridine scaﬀold. Navigating the

results across series, using plots such as Figure 7, facilitates

drug design for improved LipE, where metabolic stability

improvements are achieved in parallel as summarized in

Table 4. Compounds with moderate to stable human meta-

bolic stability were achieved as exempliﬁed by 48 and 61. With

overall good pharmaceutical properties, 61 was chosen for

further evaluation.

Compounds in the tables are racemates. The two pure enan-

tiomeric forms of 61 were prepared to evaluate the relative

c-MET activity (Figure 8). The R enantiomer 63 is signiﬁcantly

more potent, consistent with speciﬁc binding as later revealed in a

cocrystal structure of crizotinib bound to c-MET (PDB code

2wgj).

The cocrystal structure of crizotinib bound to the nonpho-

sphorylated state of the c-MET kinase domain shows that, as with

3, the compound binds to an autoinhibitory kinase conformation

in which a portion of the kinase activation loop makes direct

interactions with the inhibitor (Figure 9). The potency of

crizotinib in cells, as was discussed for predecessors 14f and

14k, results from key interactions between the R-methylbenzy-

loxy unit and the unique c-MET A-loop conformation. The

halogenated phenyl group of crizotinib takes a direct and

favorable path to form a πꢀπ interaction with Tyr-1230, also

Overall, N-substituents on the pyrazol-4-yl group maintained

or enhanced c-MET inhibition potency while lowering cLogD by

almost 3 full units in some cases. Consequently, the introduced

structural features improved LipE along with targeted pharma-

ceutical properties dramatically. The c-MET cell potency vs

cLogD data from this and earlier subseries, all 5-aryl derivatives

of the 3-(1-(2,6-dichloro-3-ﬂuorophenyl)ethoxy)pyridin-2-amine

core, are plotted in Figure 7 to graphically illustrate diﬀerent

eﬃciency zones with constant LipE values. The data points

colored blue represent the 5-pyrazol-4-yl subseries which gen-

erally occupied higher LipE space over 5-phenyl subseries (red

color). Movement up and to the left, crossing LipE lines, was

analyzed for structural features that crossed zones and served as

the design goal during optimization. Table 4 indicates that 61, a

racemic version of crizotinib, is one of the most eﬃcient inhi-

bitors, with a c-MET cell IC₅₀of 0.018 μM and a LipE of 5.62.

This is also clear from the plot of all 5-aryl derivatives shown in

Figure 7.

It is important to note that the prototype 5-aryl subseries

suﬀered high metabolic clearance due, in large part, to the high

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Table 4. Structure, Activity, and Property Relationships of N-Substituents on the 5-Pyrazol-4-yl Ring

^aCalculated logarithm of the octanol/water distribution coeﬃcient at pH 7.4 using ACD pchbat, version 9.3. ^bInhibition constants (K_i)²⁴and cell

IC₅₀^10awere determined as described in Experimental Methods. The coeﬃcients of variance were typically less than 20% (n = 2). A549 human lung

carcinoma cell line was used for the evaluation of the inhibition of autophosphorylation of c-MET. ^cLipE = pK_i(or pIC₅₀) ꢀcLogD. ^dHuman liver

microsome percent remaining was determined as described in Experimental Methods. ND = not determined.

achieving better distances and aligned geometry between the aryl

groups for stacking compared to the case of 3. Another con-

sequence of the more compact linker of crizotinib is noted when

comparing cocrystal structures (Figure 10). The plane of the

novel 2-aminopyridine core in crizotinib binds to the hinge in

a position that is more consistent with the oxindole in the

1/FGFR1K complex and diﬀerent from the oxindole in 3/c-MET

complex, which was noted earlier to be tilted at approximately a 15°

angle. This observation is consistent with less conformational strain

associated with binding the more compact 3-benzyloxy-2-amino-

pyridine compared to 3. Met-1211 has closer interactions with the

phenyl group and 2-aminopyridine core via hydrophobic interac-

tions. Both the 2-chloro and 3-ﬂuoro elements on the 3-benzyloxy

group in crizotinib point toward the NꢀH of Asp-1222, indicating

that there may be beneﬁcial electrostatic interactions that replace the

sulfonyl oxygen H-bond in 3. According to the SAR, the R-methyl

and 2,6-dichloro moieties on the 3-benzyloxy group in crizotinib are

critical for establishing the low nanomolar cell potency against

c-MET. The R-methyl group not only rigidiﬁes the benzyl group

but also makes favorable hydrophobic interactions in the pocket

surrounded by side chains of residues Val-1092, Leu-1157, Lys-

1110, and Ala-1108. As the data and cocrystal structure demon-

strate, only the R-conﬁguration of the R-methyl group provides

the right ﬁt in this lipophilic pocket.

On the other side, the 5-pyrazol-4-yl group is bound through

the narrow lipophilic tunnel surrounded by Ile-1084 and Tyr-

1159. The terminal piperidine ring, attached to the N1 position

of the pyrazol-4-yl, reaches out into the solvent. The cocrystal

structure of crizotinib with c-MET conﬁrmed the original design

hypotheses, sharing a similar binding mode to 3. However,

crizotinib binds the c-MET kinase domain more eﬃciently than

3, resulting in much improved cell-based ligand eﬃcacy (LE) and

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Figure 7. c-MET cell p(IC₅₀) vs cLogD (blue for 5-pyrazol-4-yl, red for

5-phenyl, and yellow for other).

Figure 10. Overlay of crizotinib and 3 bound to c-MET.

more accurate picture of kinase selectivity in the whole cell

contest. Compared with c-MET cell potency, crizotinib was

found to be greater than 1000-fold selective against VEGFR2

and PDGFRβ split-RTKs (cell IC₅₀> 10 μM), with other cell

potency results within the 10 μM limit summarized in Table 5.

Selectivity is greater than 200-fold for IR and LCK and approxi-

mately 40- to 60-fold for AXL, TIE2, TRKA, and TRKB. Crizotinib

selectivity is 10-fold for the c-MET subfamily member RON,

which shares 63%sequence identity in the KDand manybiological

functions with c-MET.²⁶RON receptor tyrosine kinase is over-

expressed and activated in many cancers including breast, colon,

and lung and plays important roles in tumor invasive growth and

metastasis.²⁷RON overexpression or coexpression with c-MET

correlates with a poor disease free survival in breast, bladder, and

gastresophageal cancers.²⁸RON is an attractive molecular target

for cancer therapy. Furthermore, crizotinib demonstrated a potent

cellIC₅₀of 20nMagainst anoncogenic ALKkinasefusionprotein,

NPMꢀALK, in a human lymphoma cell line even though ALK

shares only 36% kinase domain sequence identity with c-MET.

Similar to BCR-ABL in CML, available literature indicates that

ALK fusion proteins are also key disease drivers of anaplastic large

cell lymphoma, inflammatory myofibroblastic tumor, and non-

small cell lung cancer.^7,29

Figure 8. Structure and activity of pure enantiomers of 61.

In summary, 13 diﬀerent kinases were inhibited within a 100-

fold multiple of c-MET in enzymatic assays. With cellular assays,

crizotinib demonstrated potent inhibition of c-MET/ALK, with a

10-fold selectivity window for RON. The high selectivity of

crizotinib at the cellular level is related to the unusual binding

pocket created by the unique activation loop conformation of

nonphosphorylated c-MET as discussed previously. A cocrystal

structure of ALK kinase domain complexed with crizotinib (PDB

code 2xp2) reveals a conformation similar to crizotinib bound to

c-MET. The A-loop of ALK shows a notable diﬀerence, lacking a

π-stacking interaction observed between c-MET Tyr-1230 and

crizotinib. The loss of this proteinꢀcrizotinib interaction may

partially explain the weaker potency observed with ALK. The

importance of Tyr-1230ꢀcrizotinib interaction in c-MET is

further conﬁrmed with Y1230C mutation of c-MET protein,

where crizotinib is 10-fold less potent.²⁴These results are

consistent with the weaker potency for crizotinib inhibition of

RON kinase, which has a leucine residue at the position of

c-MET Tyr-1230. One common interaction among these kinases

Figure 9. Cocrystal structure of crizotinib bound to c-MET.

lipophilic eﬃciency (LipE) (LE = 0.379 and LipE = 6.14 for

crizotinib; LE = 0.264 and LipE = 4.81 for 3).

Kinase Selectivity Profile of Crizotinib. To investigate kinase

selectivity, crizotinib was evaluated against a panel of more than

120 human kinases from Upstate Inc. Of these, 13 kinases were

inhibited with enzymatic potency within a 100-fold selectivity

window of crizotinib enzymatic c-MET potency. To fully ac-

count for the unique binding mode accessed by crizotinib, cell-

based autophosphorylation assays were employed to determine a

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Table 5. Kinase Selectivity of Crizotinib

kinase

parameter

c-MET

ALK

RON

AXL

TIE2

TRKA

TRKB

ABL

IR

LCK

% inhib (1 μM)^a

enzyme IC₅₀(nM)^a

cell IC₅₀(nM)^b

97.0

<1.0

8.0

99.0

<1.0

20

97.0

NA

80

93.0

<1.0

294

97.0

5.0

99.3

<1.0

580

99.7

2.0

91.5

24

67.7

102

96.5

<1.0

2741

448

399

1159

2887

^aData were obtained from Upstate kinase selectivity screens. ^bValues are the average of at least two experiments based on the inhibition of

autophosphorylation of targets in the corresponding cell lines with <20% variance.^10a

Scheme 1^a

is that of the Met-1211 residue in c-MET. It stabilizes the

2-aminopyridine core, the 3-benzyloxy group, and anchors the

scaﬀold in an L-shape. These important hydrophobic interac-

tions should be retained for RON because of the presence of a

methionine residue at the same position. A leucine residue in

ALK kinase at the Met-1211 position of c-MET provides similar

hydrophobic interactions with crizotinib. Moreover, the high

kinase selectivity proﬁle of crizotinib is consistent with pharmacol-

ogy stemming from a limited set of kinases with potential clinic

utility and provides mechanism-based guidance for the expansion

of crizotinib to speciﬁc additional cancer patient populations.

’ CHEMISTRY

Compounds 7, 8, and 11 were synthesized according to the

procedures outlined in Scheme 1. 4 was prepared according to

the literature procedures.³⁰Selective alkylation of the 3-hydroxyl

group was achieved under basic conditions by treating with 1

equiv of benzyl bromide and 1 equiv of Cs₂CO₃in DMF at 80 °C

for4htogive6. Conventional Suzukicouplingconditions provided

^aReagents and conditions: (a) Cs₂CO₃, DMF, 80 °C, 4 h; (b)

7 and 11. Compound 8 was synthesized via alkylation of 7.

arylboronic ester, Pd(PPh₃)₂Cl₂, Na₂CO₃, DME/H₂O, 80 °C, 12 h; (c)

For rapid optimization of substituted 3-benzyloxy groups, com-

pounds were synthesized for evaluation as outlined in Scheme 2.

11b was prepared according to the procedure described in

Scheme 1. After hydrogenolysis, 12 was obtained in a quantitative

yield and used to couple with 13 to generate compounds 14aꢀk.

Compounds 22 and 23 were prepared according to the pro-

cedures in Scheme 3, in order to access a diversity of 5-sub-

stituted analogues to support the optimization eﬀorts. Mitsuno-

bu reaction of 16 with 18 provided 20 in high yield, which was

further reduced with iron chips in acetic acid and ethanol under

reﬂux to 21. Bromination and iodination of 21 produced 22 and

23. A variety of aryl groups were introduced at the 5-position via a

Suzuki coupling reaction to provide 27ꢀ35. Compounds 24 and

25 were prepared via copper-catalyzed amination reaction under

microwave conditions. The cyano group was introduced under

palladium-catalyzed condition to give 26. Compounds 36ꢀ38

were prepared with the conventional amide coupling conditions.

A variety of N-substituted pyrazole analogues were prepared as

shown in Scheme 4 via the pyrazole boronic ester 39 or 4-bromopyr-

azole 41 which was transferred to boronic ester 43 under conventional

palladium coupling conditions. Suzuki coupling reaction generated

compounds 44ꢀ50. 52 was prepared with oxosulfonium ylide which

was generated in situ from trimethylsulfoxonium iodide using NaH in

DMSO solvent and coupled with 39 to generate compound 53.³¹

An alternative route to access N-substituted pyrazole analo-

gues is outlined in Scheme 5. 2-Amino function was protected as

a bis-tert-butyl carbamate 54 for the preparation of boronic ester

56 with high yield. Acid deprotection of 56 produced 57 which

was coupled with 42 to produce compounds 58ꢀ61.

4-(2-chloroethyl)morpholine, Cs₂CO₃, DMF, 80 °C, 4 h; (d) (R)-

1-(pyrrolidin-2-ylmethyl)pyrrolidine, HOBt, EDC, DMF, 1 h; (e) Pd-

(dppf)Cl₂, Cs₂CO₃, DME/H₂O, 80 °C, 12 h.

A total synthesis of crizotinib is outlined in Scheme 6. Boronic

ester 68 was prepared via alkylation of 66 with piperidinyl derivative

65 followed by palladium coupling with 55. 69 was prepared with

the same synthetic route as the racemic analogue 22 as shown in

Scheme 3. (S)-1-(2,6-Dichloro-3-ﬂuorophenyl)ethanol was ob-

tained via a biotransformation method.³²Mitsunobu reaction of

(S)-1-(2,6-dichloro-3-ﬂuorophenyl)ethanol with 18 provided

(R)-3-(1-(2,6-dichloro-3-ﬂuorophenyl)ethoxy)-2-nitropyridine

with >99.5% ee. Suzuki coupling reaction of 69 with 68 followed

by deprotection with HCl generated crizotinib in good yield.

’ CONCLUSIONS

The cocrystal structure of 3/c-MET complex revealed a novel

binding mode of c-MET, which was used to design the novel

5-aryl-3-benzyloxy-2-aminopyridine series. First iteration 5-aryl-

3-benzyloxy-2-aminopyridines were moderate c-MET inhibitors.

The series was optimized to a highly potent and selective c-MET/

ALK dual inhibitor, with a narrower window of selectivity to

RON kinase. As shown in Figure 7, LipE and structure based

drug design led to crizotinib, which has the highest LipE value.

Crizotinib potently inhibited c-MET phosphorylation and

c-MET-dependent proliferation, migration, and invasion of

human tumor cells in vitro (IC₅₀of 5ꢀ20 nM).^10aIn addition,

crizotinib potently inhibited HGF-stimulated endothelial cell

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Scheme 2^a

^aReagents and conditions: (a) H₂balloon, 10% Pd/C, methanol, 16 h; (b) NaH, DMF, 0 °C to ambient temperature, 2 h; (c) LiAlH₄, THF, 0 °C to

ambient temperature, 3 h; (d) Ph₃PBr₂, CH₂Cl₂.

Scheme 3^a

^aReagents and conditions: (a) Ph₃P, DEAD, THF, 0 °C, 4 h; (b) Fe, AcOH/EtOH, reﬂux, 1 h; (c) NBS, ACN, 0 °C, 15 min; (d) NIS, ACN/AcOH,

0 °C, 4 h; (e) CuI, K₃PO₄, dodecane/cyclohexanediamine, DMSO, microwave at 150 °C, 2 h; (f) Pd(dppf)Cl₂, Cs₂CO₃, DME/H₂O, 80 °C, 12 h;

(g) Zn(CN)₂, Pd₂(dba)₃, DPPF, DMF, 100 °C, 3 h; (h) EDC, HOBt, DMF, 4 h.

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survival and invasion and serum-stimulated tubulogenesis in

vitro, suggesting that this agent also exhibits antiangiogenic prop-

erties.^10aCrizotinib showed eﬃcacy at well-tolerated doses, in-

cluding marked cytoreductive antitumor activity, in several tumor

models that expressed activated c-MET.^10aIn biochemical and

cellular screens, crizotinib was shown to be selective for c-MET

and ALK at pharmacologically relevant concentrations across a

panel of >120 diverse kinases. Crizotinib demonstrated tumor

cell growth inhibitory activity against cell lines harboring fusion

variants or activating mutations of ALK including Karpas 299

(NPMꢀALK), SU-DHL-1 (NPMꢀALK), Kelly neuroblastoma

(activating mutation), and NCI-H3122 (EML4ꢀALK variant 1)

with IC₅₀values ranging from 74 to 566 nM.¹¹Crizotinib potently

inhibited cell proliferation, which was associated with G₁-S phase

cell cycle arrest and induction of apoptosis in ALK-positive ALCL

cells (IC₅₀≈ 30 nM) but not ALK-negative lymphoma cells.^10b

Oral administration of crizotinib to severe combined immunode-

ﬁcient beige mice bearing Karpas 299 ALCL tumor xenografts

resulted in dose-dependent antitumor eﬃcacy with complete

regression of all tumors at the 100 (mg/kg)/day dose within

15 days of initial compound administration.^10bA strong correla-

tion was observed between antitumor response and inhibition of

NPMꢀALK phosphorylation and induction of apoptosis in tumor

tissue.^10bCrizotinib demonstrates desirable oral pharmacokinetics

in preclinical species, consistent with target modulation and

Scheme 4^a

Scheme 6. Total Synthesis of Crizotinib^a

^aReagents and conditions: (a) Cs₂CO₃, DMF, 90 °C, 12ꢀ16 h;

(b) NaH, DMF, microwave at 110 °C, 30 min; (c) Pd(dppf)Cl₂, KOAc,

DMSO, 80 °C, overnight; (d) Pd(dppf)Cl₂or Pd(Ph₃P)₂Cl₂, CsF,

DME/H₂O, microwave at 120 °C, 1 h; (e) Pd(Ph₃P)₂Cl₂, Na₂CO₃,

DME/H₂O, 85 °C, overnight; (f) trimethylsulfoxonium iodide, NaH,

DMSO, 55 °C, 6 h; (g) NaH, DMF, 90 °C, 3 h.

^aReagents and conditions: (a) MsCl, Et₃N, CH₂Cl₂; (b) NaH, DMF,

100 °C, overnight; (c) Pd(Ph₃P)₂Cl₂, KOAc, DMSO, 80 °C, 2 h; (d)

Pd(dppf)Cl₂, Cs₂CO₃, DME/H₂O, 90 °C, 3 h; (e) 4 N HCl in dioxane,

CH₂Cl₂, 0 °C, 4 h.

Scheme 5^a

^aReagents and conditions: (a) (Boc)₂O, DMAP, DMF, ambient temperature, 18 h; (b) Pd(dppf)Cl₂, KOAc, DMSO, 80 °C, overnight; (c) 4 N HCl in

dioxane, CH₂Cl₂, 40 °C, 12 h; (d) Pd(Ph₃P)₂Cl₂, Na₂CO₃, DME/H₂O, 87 °C, 16 h; (e) 4 N HCl in dioxane, MeOH, 1 h.

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eﬃcacy, with acceptable pharmacokinetics in patients while

being well tolerated.^33,34Crizotinib has advanced to human

clinical trials and demonstrated remarkable eﬃcacy for patients

with non-small-cell lung cancer, inﬂammatory myoﬁbroblastic

tumor, and large cell anaplastic lymphoma harboring fusion ALK

genes.³⁵Early clinical eﬃcacy was also observed for patients with

lung, glioblastoma, and esophagogastric adenocarcinoma with

c-MET gene ampliﬁcation.³⁶

C₁₂H₄BrCl₂N₂O: C, 41.41; H, 2.61; N, 8.05. Found: C, 41.50; H,

2.51; N, 8.05.

4-(5-(2,6-Dichlorobenzyloxy)-6-aminopyridin-3-yl)phenol

(7). A mixture of 3-(2,6-dichlorobenzyloxy)-5-bromopyridin-2-amine

(100 mg, 0.29 mmol), 4-(4,4,5,5-tetramethyl-1,3-2-dioxabordan-2-yl)-

phenol(86mg, 0.35 mmol), bis(triphenylphosphine)palladium(II) chlor-

ide (8 mg, 0.009 mmol), and sodium carbonate (91 mg, 0.87 mmol) in

ethylene glycol dimethyl ether (10 mL) and water (0.5 mL) was degassed

and charged with nitrogen three times and then heated in a 80 °C oil bath

under nitrogen for 12 h. The mixture was cooled to ambient temperature

and diluted with ethyl acetate. The mixture was washed with water, brine

and dried over Na₂SO₄. After filtration and condensation, the residue was

purified on a silica column, eluting with EtOAc/hexane (0ꢀ50%) to

afford 7 as a light pink solid (89 mg, 85% yield): LCMS m/z 361 (100%),

363 (64%) (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.39 (s,

1H), 7.80 (d, J = 1.77 Hz, 1H), 7.53ꢀ7.61 (m, 2H), 7.40ꢀ7.52 (m, 4H),

6.81 (d, J = 8.59 Hz, 2H), 5.52 (s, 2H), 5.33 (s, 2H). HPLC purity

(method A): t_R= 8.697, 100%.

’ EXPERIMENTAL METHODS

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 chro-

matography was done using the appropriate size Biotage prepacked silica

filled cartridges. NMR spectra were generated on a Bruker 300 or 400

MHz instrument and obtained as CDCl₃or DMSO-d₆solutions

(reported in ppm), using CDCl₃as the reference standard (7.27 ppm)

or DMSO-d₆(2.50 ppm). Multiplicities were given as s (singlet), b s

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

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

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

single-quadrupole mass spec detectors running with atmospheric pres-

sure chemical ionization source. The LC instrument includes a binary

pump (Agilent model G1312A) with upper pressure limit of 400 bar

attached to an autosampler (Agilent model G1313A) that 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 ChemSta-

tion, revision B.02.01. The purity measurements were done by measuring

peak area at 254 and 224 nm and total ion chromatogram. To evaluate the

purity of each peak, UVꢀvis spectrum from 190 to 700 nm at step size of

2 nm and mass spectrum scan from 150 to 850 amu with cycle time of

0.29 cycle/sec were performed. Retention times (t_R) were in minutes,

and purity was calculated as percentage of total area. Two HPLC

methods were utilized for purity. Method A consisted of the following:

Waters Acquity UPLC BEH C18 column, 1.7 μm, 2.1 mm ꢁ 100 mm,

column temperature 80 °C; solvent A consisting of water (0.1% formic

acid and 0.05% ammonium formate); solvent B consisting of methanol

(0.1% formic acid and 0.05% ammonium formate); gradient of 5ꢀ95%

B in 10 min, 95% B in 10ꢀ12 min; flow rate of 0.6 mL/min. Method B

consisted of the following: EclipsXDB C8 column, 3.5 μm, 4.6 mm ꢁ

50 mm, column temperature 40 °C; solvent A consisting of water (5%

ACN, 2 mM ammonium acetate, 0.1% acetic acid); solvent B consisting

of ACN (5% H₂O, 2 mM ammonium acetate, 0.1% acetic acid); gradient

of 20ꢀ85% B (0.0ꢀ2.5 min), 85ꢀ95% B (2.5ꢀ3.5 min), 95% B (5 min);

flow rate of 0.8 mL/min. Compound purity was determined by combus-

tion analysis (Atlantic Microlabs, Inc.) or high pressure liquid chroma-

tography (HPLC) with a confirming purity of g95% for all of the final

biological testing compounds.

3-(2,6-Dichlorobenzyloxy)-5-(4-(2-morpholinoethoxy)-

phenyl)pyridin-2-amine (8). To a solution of 7 (100 mg, 0.247

mmol) and 4-(2-chloroethyl)morpholine (37 mg, 0.247 mmol) in DMF

(5 mL) was added Cs₂CO₃(81 mg, 0.247 mmol). The reaction mixture

was heated in a 80 °C oil bath for 4 h. After cooling to ambient tem-

perature, the reaction mixture was filtered and washed with ethyl acetate.

The filtrate was condensed and purified on a reversed phase preparative

HPLC column, eluting with methanol/water containing 0.1% formic acid.

8 was obtained as a white amorphous solid (61 mg, 52%): LCMS m/z 474

1

(100%), 476 (64%) (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ

ppm 7.85 (d, J = 1.77 Hz, 1H), 7.53ꢀ7.61 (m, 4H), 7.45ꢀ7.52 (m, 2H),

7.00 (d, J = 8.84 Hz, 2H), 5.59 (s, 2H), 5.35 (s, 2H), 4.09ꢀ4.15 (m, 2H),

3.56ꢀ3.62 (m, 4H), 2.69ꢀ2.75 (m, 2H), 2.46 (m, 4H). HPLC purity

(method A): t_R= 7.004, 100%.

(R)-(2-(Pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanone (10).

To a mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid

(9, 5.00 g, 20.2 mmol) and (3-(dimethylamino)propyl)ethylcarbodiimide

hydrochloride (EDC, 4.88 g, 24.2 mmol) in DMF (100 mL) was added

1,2,3-benzotriazol-1-ol monohydrate (HOBt, 3.57 g, 22.2 mmol). A clear

solution was observed after stirring for 30 min, and (R)-1-(pyrrolidin-2-

ylmethyl)pyrrolidine (6.22 g, 40.3 mmol) was added to the reaction

solution, which was stirred at ambient temperature overnight (12 h). The

reaction solution was diluted with EtOAc (500 mL), washed with water

(100 mL ꢁ 4), saturated Na₂CO₃solution, brine, and dried over Na₂SO₄.

After filtration, evaporation, and high vacuum drying, 10 was obtained as a

colorless syrup (4.80 g, 62%): LCMS m/z 385 (M + H)⁺; ¹H NMR (400

MHz, DMSO-d₆) δ ppm 7.70 (d, J = 7.83 Hz, 2H), 7.43 (d, J = 7.83 Hz,

2H), 4.15ꢀ4.31 (m, 1H), 3.34ꢀ3.50 (m, 2H), 3.14ꢀ3.26 (m, 1H),

2.61ꢀ2.70 (m, 1H), 2.41ꢀ2.59 (m, 4H), 1.77ꢀ1.90 (m, 4H), 1.58ꢀ1.74

(m, 4H), 1.29 (s, 12H).

(R)-(4-(5-(2,6-Dichlorobenzyloxy)-6-aminopyridin-3-yl)-

phenyl)(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

(11a). To a solution of 6a (150 mg, 0.43 mmol) and 9 (248 mg, 0.65

mmol) in DME (3 mL) was added the freshly prepared aqueous solution

of Cs₂CO₃(210 mg, 0.65 mmol) in water (1 mL), followed by the

addition of 1,1⁰-bis(diphenylphosphino)ferrocene palladium dichloride

(16.1 mg, 0.022 mmol). The reaction flask was degassed and charged with

nitrogen three times and then heated in a 80 °C oil bath overnight (15 h).

The reaction solution was diluted with EtOAc, washed with water and

brine, and dried over Na₂SO₄. After filtration and concentration, the

residue was purified on a reverse phase preparative HPLC column, eluting

withmethanol/H₂O containing0.1%formic acid (10ꢀ90%), and11a was

obtained as a white amorphous solid (78 mg, 34% yield): LCMS m/z 525

(100%), 527 (64%) (M + H)⁺; ¹H NMR (300 MHz, DMSO-d₆, 80 °C) δ

ppm 7.99 (d, J = 1.88 Hz, 1H), 7.64ꢀ7.73 (m, 2H), 7.40ꢀ7.61 (m, 6H),

3-(2,6-Dichlorobenzyloxy)-5-bromopyridin-2-amine (6a).

To a solution of 2-amino-5-bromopyridin-3-ol (1.000 g, 5.29 mmol) and

2-(bromomethyl)-1,3-dichlorobenzene (1.270 g, 5.29 mmol) in DMF

(20 mL) was added Cs₂CO₃(1.720 g, 5.29 mmol). The reaction mixture

was stirred at 80 °C in an oil bath for 4 h, and LCMS showed the com-

pletion of the reaction. The mixture was cooled to ambient temperature,

diluted with EtOAc (100 mL), washed with water (30 mL ꢁ 5) and

brine, and dried over Na₂SO₄. After filtration and condensation, the

residue was purified with a silica gel column, eluting with EtOAc/heptane

(0ꢀ50%) to provide 6a as anoff-white solid (1.120 g, 60.8%): LCMSm/z

347 (62%), 349 (100%), 351 (45%) (M + H)⁺; ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 7.62 (d, J = 2.02 Hz, 1H), 7.52ꢀ7.59 (m, 2H),

7.44ꢀ7.50 (m, 2H), 5.79 (s, 2H), 5.24 (s, 2H). Anal. Calcd for

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5.52 (s, 2H), 5.42 (s, 2H), 4.16ꢀ4.27 (m, 1H), 3.43ꢀ3.52 (m, 2H),

3.16ꢀ3.26 (m, 1H), 2.54ꢀ2.66 (m, 2H), 2.24ꢀ2.47 (m, 3H), 1.73ꢀ2.08

(m, 4H), 1.58ꢀ1.71 (m, 4H). HPLC purity (method A): t_R= 7.463,

95.7%.

δ ppm 7.95 (d, J = 1.88 Hz, 1H), 7.68ꢀ7.74 (m, 1H), 7.62ꢀ7.66 (m, 2H),

7.45ꢀ7.54 (m, 4H), 7.33ꢀ7.43 (m, 2H), 5.65 (s, 2H), 5.32 (s, 2H),

4.16ꢀ4.25 (m, 1H), 3.42ꢀ3.50 (m, 2H), 3.20ꢀ3.24 (m, 1H), 2.53ꢀ2.61

(m, 2H), 2.37ꢀ2.45 (m, 3H), 1.73ꢀ2.06 (m, 4H), 1.58ꢀ1.68 (m, 4H).

HPLC purity (method A): t_R= 6.812, 98.7%.

(R)-(4-(5-Benzyloxy-6-aminopyridin-3-yl)phenyl)(2-(pyrrolidin-

1-ylmethyl)pyrrolidin-1-yl)methanone (11b). Compound 11b

was prepared by using a similar procedure described for the synthesis of

11a: LCMS m/z 457 (M + H)⁺; ¹H NMR (300 MHz, DMSO-d₆, 80 °C)

δ ppm 7.88ꢀ7.99 (m, 1H), 7.28ꢀ7.69 (m, 10H), 5.65 (s, 2H), 5.27 (s,

2H), 4.07ꢀ4.32 (m, 1H), 3.38ꢀ3.53 (m, 2H), 2.45 (m, 6H), 1.72ꢀ2.12

(m, 5H), 1.54ꢀ1.71 (m, 3H). HPLC purity (method A): t_R= 6.419, 100%.

(R)-(4-(6-Amino-5-hydroxypyridin-3-yl)phenyl)(2-(pyrrolidin-

1-ylmethyl)pyrrolidin-1-yl)methanone (12). To a solution of 11b

(2.28 g, 5.00 mmol) in methanol (25 mL) was added 10% Pd/C (100 mg).

The mixture was degassed and charged with hydrogen three times and then

stirred under hydrogen balloon for overnight. The mixture was filtered

through a Celite pad, washed with methanol, and condensed. After high vac-

uum drying, 12 was obtained as a white solid (1.74 g, 95% yield): LCMS m/z

367 (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.79 (s, 1H), 7.54

(m, 3H), 7.46 (m, 2H), 7.14 (s, 1H), 5.68 (s, 2H), 4.22 (m, 1H), 3.45 (m,

2H), 2.66(m, 1H), 2.52(m, 4H),1.96(m, 2H), 1.84(m, 3H), 1.64(m, 4H).

1-(2,6-Dichloro-3-fluorophenyl)ethanol (16). To a solution

of 1-(2,6-dichloro-3-fluorophenyl)ethanone (15.00 g, 72 mmol) in

anhydrous THF (150 mL) at 0 °C was added lithium aluminum hydride

(2.75 g, 72 mmol) slowly. The mixture was stirred at ambient tempera-

ture for 3 h and cooled in an ice bath. Water (3 mL) was dropwise added

followed by slow addition of 15% NaOH (3 mL). The mixture was

stirred at ambient temperature for 30 min. Then 15% NaOH (9 mL) and

MgSO₄were added and the mixture was filtered to remove solids. The

solids were washed with THF (50 mL) and the filtrate was concentrated

(R)-2-((2-Amino-5-(4-(2-(pyrrolidin-1-ylmethyl)pyrrolidine-

1-carbonyl)phenyl)pyridin-3-yloxy)methyl)benzonitrile (14c).

LCMS m/z 582 (M + H)⁺; ¹H NMR (300 MHz, DMSO-d₆, 80 °C) δ

ppm 7.91ꢀ8.00 (m, 2H), 7.81ꢀ7.89 (m, 1H), 7.73ꢀ7.81 (m, 1H), 7.69

(d, J = 7.91 Hz, 2H), 7.48ꢀ7.61 (m, 4H), 5.93 (s, 2H), 5.41 (s, 2H),

4.09ꢀ4.37 (m, 1H), 3.45ꢀ3.52 (m, 4H), 2.55ꢀ2.66 (m, 3H), 2.40ꢀ2.47

(m, 1H), 1.85ꢀ2.02 (m, 4H), 1.57ꢀ1.75 (m, 4H). HPLC purity (method

A): t_R= 5.573, 98.9%.

(R)-(4-(5-(2-Trifluoromethylbenzyloxy)-6-aminopyridin-3-

yl)phenyl)(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

(14d). LCMS m/z 525 (M + H)⁺; ¹H NMR (300 MHz, DMSO-d₆, 80 °C)

δ ppm 7.97 (d, J = 1.88 Hz, 1H), 7.88 (d, J = 7.54 Hz, 1H), 7.70ꢀ7.83 (m,

2H), 7.57ꢀ7.66 (m, 3H), 7.51 (d, J = 8.29 Hz, 2H), 7.44 (d, J = 1.88 Hz,

1H), 5.54ꢀ5.81 (m, 2H), 5.39 (s, 2H), 4.15ꢀ4.32 (m, 2H), 3.47 (t, J = 5.75

Hz, 2H), 2.52ꢀ2.67 (m, 3H), 2.30ꢀ2.47 (m, 2H), 1.75ꢀ2.06 (m, 4H),

1.56ꢀ1.75 (m, 4H). HPLC purity (method A): t_R= 7.400, 100%.

(R)-(4-(5-(4-tert-Butylbenzyloxy)-6-aminopyridin-3-yl)-

phenyl)(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

1

(14e). LCMS m/z 513 (M + H)⁺; H NMR (300 MHz, DMSO-d₆,

80 °C) δ ppm 7.92 (d, J = 1.88 Hz, 1H), 7.59ꢀ7.64 (m, 2H), 7.49 (d, J =

8.10 Hz, 2H), 7.39ꢀ7.46 (m, 5H), 5.62 (br s, 2H), 5.21 (s, 2H), 4.17ꢀ

4.29 (m, 1 H), 3.39ꢀ3.51 (m, 3H), 2.51ꢀ2.65 (m, 3H), 2.33ꢀ2.47 (m,

2H), 1.75ꢀ2.06 (m, 4H), 1.62ꢀ1.71 (m, 4H), 1.31 (s, 9H). HPLC purity

(method A): t_R= 8.664, 96.5%.

(R)-(4-(5-(2,4-Dichlorobenzyloxy)-6-aminopyridin-3-yl)-

phenyl)(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

(14f). LCMS m/z 525 (100%), 527 (64%) (M + H)⁺; ¹H NMR (300

MHz, DMSO-d₆, 80 °C) δ ppm 7.97 (d, J = 1.88 Hz, 1H), 7.75 (d, J = 8.48

Hz, 1H), 7.60ꢀ7.70 (m, 3H), 7.43ꢀ7.54 (m, 4H), 5.69 (s, 2H), 5.31 (s,

2H), 4.15ꢀ4.29 (m, 1H), 3.41ꢀ3.52 (m, 2H), 3.19ꢀ3.24 (m, 1H),

2.53ꢀ2.61 (m, 2H), 2.34ꢀ2.48 (m, 3H), 1.76ꢀ2.02 (m, 4H), 1.58ꢀ1.69

(m, 4H). HPLC purity (method A): t_R= 7.987, 95.95%.

1

to give 16 (14.8 g, 95% yield) as a yellow oil: H NMR (400 MHz,

DMSO-d₆) δ ppm 7.42 (m, 1H), 7.32 (m, 1H), 5.42 (m, 2H), 1.45 (d,

J = 6.4 Hz, 3H).

2-(1-Bromoethyl)-1,3-dichloro-4-fluorobenzene (17). To a

solution of 16 (10.00 g, 47.8 mmol) in anhydrous dichloromethane was

added dibromotriphenylphosphorane (22.2 g, 52.6 mmol) portionwise

at ambient temperature. The mixture was stirred for 16 h and then

concentrated and purified by a silica gel column chromatography with

10% EtOAc in heptane to provide 17 as a colorless liquid (10.14 g, 78%

yield). ¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.41 (m, 2H), 5.90ꢀ6.09

(m, 1H), 2.2 (d, J = 7.16 Hz, 3H).

(R)-(4-(5-(2-Fluorobenzyloxy)-6-aminopyridin-3-yl)phenyl)-

(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone (14a). To

a stirred solution of 12 (100 mg, 0.27 mmol) in anhydrous DMF (15 mL)

at 0 °C was added sodium hydride (60% dispersion in mineral oil, 11 mg,

0.49 mmol). The mixture was allowed to stir at 0 °C for 30 min followed by

the addition of 1-(bromomethyl)-2-fluorobenzene (51 mg, 0.27 mmol).

The reaction mixture was stirred at room temperature for 2 h, diluted with

EtOAc, and partitioned with H₂O. The aqueous layer was extracted with

EtOAc (2 ꢁ 25 mL). The organic layers were combined, washed with H₂O

(15 mL), brine (15 mL), and dried over MgSO₄. After filtration and

concentration, the residue was purified on a reverse phase preparative

HPLC column, eluting with methanol/H₂O containing 0.1% formic acid

(10ꢀ90%), and 14a was obtained as a white amorphous solid (58 mg, 45%

yield): LCMS m/z475 (M + H)⁺;¹HNMR(300MHz, DMSO-d₆, 80 °C)

δ ppm 7.93ꢀ7.99 (m, 1H), 7.48ꢀ7.68 (m, 6H), 7.37ꢀ7.44 (m, 1H),

7.15ꢀ7.28 (m, 2H), 5.64 (br s, 2H), 5.29 (s, 2H), 4.32ꢀ4.44 (m, 1H),

3.35ꢀ3.57 (m, 3H), 2.03ꢀ2.25 (m, 2H), 1.56ꢀ2.01 (m, 9H), 1.20ꢀ1.36

(m, 2H). HPLC purity (method A): t_R= 6.515, 95.5%.

(R)-(4-(5-(2-Chloro-6-fluorobenzyloxy)-6-aminopyridin-3-

yl)phenyl)(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

1

(14g). LCMS m/z 509 (100%), 510 (32%) (M + H)⁺; H NMR (300

MHz, DMSO-d₆, 80 °C) δ ppm 7.98 (d, J = 2.07 Hz, 1H), 7.62ꢀ7.71 (m,

2H), 7.38ꢀ7.63 (m, 5H), 7.24ꢀ7.36 (m, 1H), 5.53 (s, 2H), 5.34 (d, J=1.88

Hz, 2H), 4.18ꢀ4.30 (m, 1H), 3.46ꢀ3.51 (m, 2H), 3.20ꢀ3.24 (m, 1H),

2.55ꢀ2.61 (m, 2H), 2.32ꢀ2.48 (m, 3H), 1.76ꢀ2.12 (m, 4H), 1.61ꢀ1.70

(m, 4H). HPLC purity (method A): t_R= 6.909, 100%.

(R)-(4-(5-(2-Chloro-3,6-difluorobenzyloxy)-6-aminopyridin-

3-yl)phenyl)(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

(14h). LCMS m/z 527 (100%), 529 (32%) (M + H)⁺; ¹H NMR (300

MHz, DMSO-d₆, 80 °C) δ ppm 7.99 (d, J = 1.88 Hz, 1H), 7.68 (d, J = 8.48

Hz, 2H), 7.46ꢀ7.62 (m, 4H), 7.31ꢀ7.44 (m, 1H), 5.57 (s, 2H), 5.33ꢀ5.39

(m, 2H), 4.19ꢀ4.31 (m, 1H), 3.45ꢀ3.52 (m, 2H), 3.19ꢀ3.26 (m, 1H),

2.54ꢀ2.61 (m, 2H), 2.35ꢀ2.47 (m, 3H), 1.78ꢀ2.09 (m, 4H), 1.59ꢀ1.70

(m, 4H). HPLC purity (method A): t_R= 6.821, 96.3%.

(4-(6-Amino-5-(1-(2-chloro-3,6-difluorophenyl)ethoxy)-

pyridin-3-yl)phenyl)((R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-

1-yl)methanone (14i). LCMS m/z 541 (100%), 543 (32%) (M +

H)⁺; ¹H NMR (300 MHz, DMSO-d₆, 80 °C) δ ppm 7.89 (d, J = 1.88 Hz,

1H), 7.34ꢀ7.60 (m, 5H), 7.22ꢀ7.33 (m, 1H), 7.18 (d, J = 1.88 Hz, 1H),

5.89ꢀ6.08 (m, 1H), 5.61 (s, 2H), 4.01ꢀ4.29 (m, 1H), 3.28ꢀ3.52 (m,

2H), 2.28ꢀ2.48 (m, 6H), 1.67ꢀ2.13 (m, 8H), 1.51ꢀ1.69 (m, 3H).

HPLC purity (method A): t_R= 7.865, 100%.

Compounds 14bꢀl were prepared by using a similar procedure

described for the synthesis of 14a.

(R)-(4-(5-(2-Chlorobenzyloxy)-6-aminopyridin-3-yl)phenyl)-

(2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone (14b).

(4-(6-Amino-5-(1-(2,6-dichlorophenyl)ethoxy)pyridin-3-yl)-

phenyl)((R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)methanone

(14j). LCMS m/z 539 (100%), 541 (64%) (M + H)⁺. ¹H NMR (300 MHz,

1

LCMS m/z 491 (M + H)⁺; H NMR (300 MHz, DMSO-d₆, 80 °C)

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DMSO-d₆, 80 °C) δ ppm 7.88 (s, 1H), 7.39ꢀ7.51 (m, 6H), 7.24ꢀ7.39

(m, 1H), 7.05 (s, 1H), 6.06ꢀ6.22 (m, 1H), 5.67 (s, 2H), 4.14ꢀ4.30 (m,

1H), 3.39ꢀ3.50 (m, 2H), 2.30ꢀ2.48 (m, 5H), 1.73ꢀ2.10 (m, 8H),

1.60ꢀ1.71 (m, 4 H). HPLC purity (method A): t_R= 8.329, 100%.

(R)-(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)phenyl)((R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-

1-yl)methanone (14k). LCMS m/z 557 (100%), 559 (64%) (M + H)⁺.

¹H NMR (300 MHz, DMSO-d₆, 80 °C) δ ppm 7.90 (d, J = 1.51 Hz, 1H),

7.50ꢀ7.61 (m, 1H), 7.36ꢀ7.49 (m, 5H), 7.06 (d, J = 1.88 Hz, 1H),

6.11ꢀ6.23 (m, 1H), 5.70 (s, 2H), 4.13ꢀ4.26 (m, 1H), 3.46 (t, J = 6.78 Hz,

2H), 2.32ꢀ2.49 (m, 6H), 1.75ꢀ2.04 (m, 4H), 1.56ꢀ1.70 (m, 4H). HPLC

purity (method A): t_R= 8.010, 100%.

5.82ꢀ6.06 (m, 3H), 1.76 (d, J = 6.59 Hz, 3H). Anal. Calcd for

C₁₃H₁₀Cl₂FIN₂O: C, 36.56; H, 2.36; N, 6.56. Found: C, 36.85; H,

2.28; N, 6.42.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1H-pyrazol-

1-yl)pyridin-2-amine (24). To a stirred solution of 23 (100 mg,

0.23 mmol) and pyrazole (48 mg, 0.70 mmol) in DMSO (1 mL) were

added K₃PO₄(101 mg, 0.47 mmol), dodecane (0.015 mL, 0.05 mmol),

cyclohexanediamine (0.009 mL, 0.07 mmol), and copper iodide (CuI)

(14 mg, 0.07 mmol). The solution was bubbled with nitrogen for 5 min,

then irradiated with microwave at 150 °C for 2 h. The mixture was

purified by preparative reverse phase HPLC to produce 24 as an

amorphous white solid (38 mg, yield 34%): LCMS m/z 367 (100%),

1

369 (64%) (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ ppm 8.21

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-2-nitropyridine

(20). To a stirred solution of triphenylphosphine (8.2 g, 0.03 mol) and

DEAD (13.65 mL of a 40% solution in toluene) in THF (200 mL) at 0 °C

was added a solution of 1-(2,6-dichloro-3-fluorophenyl)ethanol (4.55 g,

0.021 mol) and 3-hydroxynitropyridine (3.35 g, 0.023 mol) in THF

(200 mL). The resulting bright orange solution was stirred under a

nitrogen atmosphere at ambient temperature for 4 h at which point all

starting materials had been consumed. The solvent was removed, and the

crude material was dry loaded onto a silica gel column and eluted with

ethyl acetateꢀhexanes (20:80) to yield 20 (6.21 g, 0.021 mol, 98%) as an

off-white solid: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.08 (dd, J = 4.67,

1.14 Hz, 1H), 7.68 (dd, J = 8.59, 4.55 Hz, 1H), 7.53ꢀ7.59 (m, 1H),

7.43ꢀ7.51 (m, 2H), 6.27 (q, J = 6.74 Hz, 1H), 1.74 (d, J = 6.57 Hz, 3H).

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)pyridin-2-amine

(21). To a stirred mixture of AcOH (650 mL) and EtOH (500 mL) were

suspended 20 (9.43 g, 0.028 mol) and iron chips (15.7 g, 0.28 mol). The

mixture was heatedslowlyto reflux and allowed to stirfor 1 h. The mixture

was cooled to room temperature. Then diethyl ether (500 mL) and water

(500 mL) were added. The solution was carefully neutralized by the addi-

tion of sodium carbonate. The combined organic extracts were washed

with saturated NaHCO₃(2 ꢁ 100 mL), H₂O (2 ꢁ 100 mL), and brine

(1 ꢁ 100 mL) and then dried (Na₂SO₄), filtered, and concentrated to

dryness under vacuum to yield 21 (9.04 g, 99%) as an off-white solid:

(d, J = 2.53 Hz, 1H), 7.95 (d, J = 2.02 Hz, 1H), 7.67 (d, J = 1.77 Hz, 1H),

7.57 (dd, J = 8.97, 4.93 Hz, 1H), 7.45 (t, J = 8.72 Hz, 1H), 7.29 (d, J =

1.77 Hz, 1H), 6.46ꢀ6.52 (m, 1H), 6.16 (t, J = 6.57 Hz, 1H), 1.81 (d, J =

6.57 Hz, 3H).

N-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)benzamide (25). Compound 25 was prepared by

using a similar procedure described for the synthesis of compound 24:

1

LCMS m/z 420 (100%), 422 (64%) (M + H)⁺; H NMR (400 MHz,

DMSO-d₆) δ ppm 10.31 (s, 1H), 7.93 (s, 1H), 7.83 (d, J = 7.33 Hz, 2H),

7.48ꢀ7.60 (m, 5H), 7.37 (d, J = 1.52 Hz, 1H), 6.06 (q, J = 6.57 Hz, 1H),

1.75ꢀ1.85 (m, 4H). HPLC purity (method A): t_R= 9.457, 98.0%.

6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

nicotinonitrile (26). To a solution of 22 (5.00 g, 13.15 mmol) in DMF

(73 mL) and water (1 mL) were added Zn(CN)₂(4.50 g, 26.3 mmol),

Pd₂(dba)₃(0.602 g, 0.65 mmol), and DPPF (0.86 g, 1.55 mmol). The

mixture was degassed and charged with nitrogen three times and then stirred

under nitrogen at 100 °C for 3 h. The reaction solution was partitioned

between ethyl acetate and water. The organic layer was washed with a

solution of saturated NH₄Clꢀconcentrated NH₄OHꢀwater (4:1:4),

then dried over MgSO₄. The crude product was purified on a silica gel

column, eluting with ethyl acetateꢀhexanes (1:4) to provide 26 as a white

solid (4.15 g, 97% yield): LCMS m/z 326 (100%), 328 (64%) (M + H)⁺;

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.93 (d, J = 1.77 Hz, 1H), 7.57

(dd, J = 9.09, 5.05 Hz, 1H), 7.46 (t, J = 8.72 Hz, 1H), 6.88 (br s, 2H), 6.75

(d, J = 1.77 Hz, 1H), 6.03 (q, J = 6.57 Hz, 1H), 1.76 (d, J = 6.57 Hz, 3H).

HPLC purity (method A): t_R= 9.656, 98.9%.

1

LCMS m/z 301 (100%), 303 (64%) (M + H)⁺; H NMR (400 MHz,

DMSO-d₆) δ ppm 7.51ꢀ7.58 (m, 1H), 7.40ꢀ7.50 (m, 2H), 6.62 (dd, J =

7.83, 1.01 Hz, 1H), 6.38 (dd, J = 7.83, 5.05 Hz, 1H), 5.96 (q, J = 6.65 Hz,

1H), 5.66 (s, 2H), 1.76 (d, J = 6.57 Hz, 3H). HPLC purity (method A):

t_R= 7.185, 99.3%.

Compounds 27ꢀ35 were prepared according to a similar procedure

described for the synthesis of compound 11a.

5-Bromo-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-

2-amine (22). A stirring solution of 21 (9.07 g, 0.03 mol) in acetonitrile

was cooled to 0 °C using an ice bath. To this solution was added

N-bromosuccinimide (NBS) (5.33 g, 0.03 mol) portionwise. The mixture

was stirred at 0 °C for 15 min. The mixture was concentrated to dryness

under vacuum. The resulting dark oil was purified via a silica gel column,

eluting with ethyl acetate/hexane (1:5), and 22 was obtained as a white

crystalline solid (5.8 g, 51%): LCMS m/z 379 (62%), 381 (100%), 383

(45%) (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.52ꢀ7.62 (m,

2H), 7.43ꢀ7.51 (m, 1H), 6.75 (d, J = 1.77 Hz, 1H), 5.94ꢀ6.05 (m, 3H),

1.77 (d, J = 6.82 Hz, 3H). Anal. Calcd for C₁₃H₁₀BrCl₂FN₂O: C, 41.09; H,

2.65; N, 7.37. Found: C, 41.20; H, 2.54; N, 7.34.

5-Iodo-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-

2-amine (23). To a solution of 21 (10.0 g, 33.2 mmol) in acetonitrile

(600 mL) and acetic acid (120 mL) was added N-iodosuccinimide (11.2 g,

49.8 mmol). The mixture was stirred at room temperature for 4 h, and

the reaction was quenched with Na₂S₂O₅solution. After evaporation,

the residue was partitioned between ethyl acetate and water. The organic

layer was washed with aqueous NaOH solution (2 N), brine and dried

over Na₂SO₄. The crude product was purified on a silica gel column,

eluting with ethyl acetate/hexane (1:5) to provide 23 as an off-white

solid (7.1 g, 50% yield): LCMS m/z 427 (100%), 429 (64%) (M + H)⁺;

¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.63 (d, J = 1.70 Hz, 1H),

7.51ꢀ7.60 (m, 1H), 7.37ꢀ7.51 (m, 1H), 6.84 (d, J = 1.70 Hz, 1H),

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-phenylpyridin-

2-amine (27). LCMS m/z 377 (100%), 379 (64%) (M + H)⁺; ¹H NMR

(400 MHz, DMSO-d₆) δ ppm 7.82 (d, J = 2.02 Hz, 1H), 7.57 (dd, J = 8.97,

4.93 Hz, 1H), 7.40ꢀ7.49 (m, 1H), 7.30ꢀ7.40 (m, 4H), 7.25 (ddd, J = 5.18,

3.54, 3.41 Hz, 1H), 6.94 (d, J = 2.02 Hz, 1H), 6.07ꢀ6.17 (m, 1 H), 5.88

(s, 2H), 1.81 (d, J = 6.82 Hz, 3H). HPLC purity (method A): t_R

=

10.094, 98.4%.

4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)benzoic Acid (28). LCMS m/z 421 (100%), 423

(64%) (M + H)⁺; ¹H NMR(400 MHz, DMSO-d₆) δppm 7.80ꢀ7.91 (m,

3H), 7.59(dd, J =9.09, 5.05 Hz, 1H), 7.40ꢀ7.50 (m, 1H), 7.36 (d, J =8.08

Hz, 2H), 6.99 (d, J = 1.77 Hz, 1H), 6.15 (q, J = 6.57 Hz, 1H), 5.92 (s, 2H),

1.82 (d, J = 6.57 Hz, 3H). HPLC purity (method A): t_R= 8.335, 95.5%.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-methyl-1H-

imidazol-4-yl)pyridin-2-amine (29). LCMS m/z 381 (100%), 383

(64%) (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.30ꢀ7.39 (m,

3H), 7.22 (t, J = 8.72 Hz, 1H), 6.51 (s, 1H), 6.40 (d, J = 1.77 Hz, 1H), 5.80

(q, J = 6.65 Hz, 1H), 5.73 (s, 2H), 3.18 (s, 3H), 1.55(d, J = 6.57 Hz, 3H).

Anal. Calcd for C₁₇H₁₅N₄OCl₂F 0.1H₂O: C, 53.31; H, 4.00; N, 14.63.

3

Found: C, 53.28; H, 4.08; N, 14.31.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(thiazol-2-yl)-

pyridin-2-amine (30). LCMS m/z 384 (100%), 386 (64%) (M + H)⁺;

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.10 (d, J = 1.77 Hz, 1H), 7.76

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1

(d, J = 3.28 Hz, 1H), 7.53ꢀ7.64 (m, 2H), 7.45 (t, J = 8.72 Hz, 1H), 7.12

(d, J = 1.77 Hz, 1H), 6.35 (s, 2H), 6.10 (q, J = 6.48 Hz, 1H), 1.82 (d, J =

6.57 Hz, 3H). HPLC purity (method A): t_R= 10.417, 100%.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(isoxazol-4-yl)-

pyridin-2-amine (31). LCMS m/z 368 (100%), 370 (64%) (M + H)⁺;

¹H NMR (400 MHz, MeOD) δ ppm 8.68 (s, 1H), 8.50 (s, 1H), 7.64 (s,

1H), 7.36 (dd, J= 8.97, 4.93 Hz, 1H), 7.13 (t, J= 8.72 Hz, 1H), 6.87 (s, 1H),

6.11 (q, J = 6.57 Hz, 1H), 1.78 (d, J = 6.82 Hz, 3H). HPLC purity (method

A): t_R= 3.441, 100%.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1H-pyrazol-

5-yl)pyridin-2-amine (32). LCMS m/z 367 (100%), 369 (64%)

(M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.92 (d, J = 1.52 Hz,

1H), 7.68 (s, 1H), 7.49ꢀ7.59 (m, 1H), 7.43 (t, J = 8.72 Hz, 2H), 7.12

(s, 1H), 6.41 (s, 1H), 6.06 (s, 1H), 5.90 (s, 2H), 3.32 (s, 4H), 1.78 (d, J =

6.57 Hz, 3H).

Acetic acid salt: LCMS m/z 517 (100%), 519 (64%) (M + H)⁺; H

NMR (400 MHz, DMSO-d₆) δ ppm 8.35ꢀ8.46 (m, 1H), 7.91 (d, J = 2.02

Hz, 1H), 7.78ꢀ7.88 (m, 2H), 7.57 (dd, J = 8.84, 5.05 Hz, 1H), 7.38ꢀ7.50

(m, 3H), 6.99 (d, J = 1.52 Hz, 1H), 6.07ꢀ6.20 (m, 1H), 6.00 (s, 2H),

3.35ꢀ3.41 (m, 4H), 2.57 (t, J = 6.95 Hz, 2H), 2.43ꢀ2.48 (m, 2H), 1.90(s,

3H), 1.82 (d, J = 6.57 Hz, 3H), 1.68 (t, J = 3.28 Hz, 4H). HPLC purity

(method A): t_R= 7.801, 99.4%.

(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)phenyl)((3S,5R)-3,5-dimethylpiperazin-1-yl)-

methanone (38). LCMS m/z 516, (100%), 518 (64%) (M + H)⁺;

¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.88 (d, J = 1.77 Hz, 1H), 7.57

(dd, J = 8.97, 4.93 Hz, 1H), 7.40ꢀ7.49 (m, 3H), 7.31ꢀ7.40 (m, 2H), 7.00

(d, J = 1.77 Hz, 1H), 6.09ꢀ6.21 (m, 1H), 5.97 (s, 2H), 4.28ꢀ4.46 (m,

1H), 3.39ꢀ3.54 (m, 1H), 2.57ꢀ2.73 (m, 3H), 2.18ꢀ2.29 (m, 1H), 1.81

(d, J = 6.57 Hz, 3H), 0.78ꢀ1.05 (m, 6H). HPLC purity (method A): t_R=

7.661, 100%

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-isopropyl-

1H-pyrazol-4-yl)pyridin-2-amine (44). Step 1. To a solution of

4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (39, 1.00 g,

5.15 mmol) inDMF(25mL, 0.2M) were added2-iodopropane (0.55 mL,

5.15 mmol) and Cs₂CO₃(2.50 g, 7.73 mmol). The mixture was stirred

at 90 °C for 16 h, cooled, diluted with water, and extracted with EtOAc

(3 ꢁ 50 mL). The combined organic layers were washed withbrine, dried

over Na₂SO₄, filtered, and concentrated to give 1-isopropyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole as a white solid (1.20

g, 100% yield): ¹H NMR(400MHz, CDCl₃) δ ppm7.80 (s, 1H), 7.75 (s,

1H), 4.53 (dt, J = 13.39, 6.69 Hz, 1H), 1.51 (d, J = 6.57 Hz, 6H), 1.32 (s,

12H).

Step 2. In a microwave vessel were added 1-isopropyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (122mg, 0.515mmol),

3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-iodopyridin-2-amine (200 mg,

0.468 mmol), DME (4 mL), and a freshly made solution of CsF (234 mg,

1.545 mmol) in water (1 mL). The reaction mixture was purged with N₂

for 5 min. Pd(PPh₃)₂Cl₂(16.4 mg, 0.023 mmol) was added, and the

system was purged with N₂for 5 min. The mixture was heated in a

microwave for 40 min at 120 °C. To the mixture were added EtOAc and

water (10 mL each). The aqueous layer was extracted with EtOAc (3 ꢁ

10mL), driedoverNa₂SO₄, filtered, and concentrated. The crude material

was purified with a reversed phase C-18 preparative HPLC column,

eluting with acetonitrile/water with 0.1% acetic acid to afford 44 as a white

amorphous solid (121 mg, 63% yield): LCMS m/z 409 (100%), 411

(64%) (M + H)⁺; ¹H NMR (400 MHz, CDCl₃) δ ppm 7.53ꢀ7.56 (m,

1H), 7.48 (s, 1H), 7.44 (d, J = 1.77 Hz, 1H), 7.37 (dd, J = 8.84, 4.80 Hz,

1H), 7.10ꢀ7.17 (m, 1H), 7.00ꢀ7.06 (m, 1H), 6.17 (q, J = 6.82 Hz, 1H),

4.42ꢀ4.60 (m, 1H), 1.91ꢀ1.97 (m, 3H), 1.51ꢀ1.57 (m, 6H). HPLC

purity (method A): t_R= 9.577, 100%.

2-(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)-1H-pyrazol-1-yl)-N-(2-(dimethylamino)ethyl)-2-

methylpropanamide (45). Step 1. To a solution of 4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (5 g, 25.77 mmol)

and methyl 2-bromo-2-methylpropanoate (12.6 g, 27.06 mmol) in

DMF (85 mL) was added Cs₂CO₃(12.6 g, 38.65 mmol). The reaction

mixture was heated in a 90 °C oil bath overnight. The reaction solution

was cooled to room temperature and partitioned between water and

ethyl acetate. The combined ethyl acetate solution was washed with

water five times, dried over Na₂SO₄, and concentrated to give methyl

2-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-

1-yl)propanoate (4.776 g, 63% yield): ¹H NMR (400 MHz, CDCl₃) δ

ppm 7.89 (s, 1H), 7.84 (s, 1H), 3.71 (s, 3H), 1.85 (s, 6H), 1.32 (s, 12H).

Step 2. To a solution of 23 (6.363 g, 14.90 mmol) and methyl

2-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-

1-yl)propanoate (4.6 g, 15.64 mmol) in DME (27 mL) was added a solution

of CsF (6.79 g, 44.7 mmol) in water (9.3 mL). The reaction mixture was

degassed 3 times with N₂. 1,1⁰-Bis(diphenylphosphino)ferrocene palladium

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1H-pyrazol-

4-yl)pyridin-2-amine (33). Yield: 53.6%. LCMS m/z 367 (100%),

369 (64%) (M + H)⁺; ¹H NMR (400 MHz, CDCl₃) δ ppm 7.80 (d, J =

1.52 Hz, 1H), 7.67 (s, 2H), 7.22ꢀ7.33 (m, 1H), 6.96ꢀ7.06 (m, 1H),

6.89 (d, J = 1.77 Hz, 1H), 6.06 (q, J = 6.57 Hz, 1H), 5.07 (s, 2H), 1.84 (d,

J = 6.57 Hz, 3H), 1.60 (s, 1H). Anal. Calcd for C₁₆H₁₃N₄OCl₂F 0.5

3

CH₃OH 0.1CH₂Cl₂: C, 50.90; H, 3.91; N, 14.30. Found: C, 50.60; H,

3.94; N, 14.57.

3

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(3,5-dimethyl-

1H-pyrazol-4-yl)pyridin-2-amine (34). LCMS m/z 395 (100%),

397 (64%) (M + H)⁺; ¹H NMR (400 MHz, CDCl₃) δ ppm 7.36 (dd, J =

8.84, 4.80 Hz, 1H), 7.21 (d, J = 1.26 Hz, 1H), 7.11ꢀ7.19 (m, 1H), 6.71 (s,

1H), 6.05ꢀ6.16 (m, 1H), 2.14 (s, 6H), 1.93 (d, J = 6.57 Hz, 3H). HPLC

purity (method A): t_R= 8.335, 100%.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-methyl-1H-

pyrazol-4-yl)pyridin-2-amine (35). Yield: 68.4%. LCMS m/z 381

(100%), 383 (64%) (M + H)⁺; ¹H NMR (400 MHz, CDCl₃) δ ppm 7.74

(d, J = 1.77 Hz, 1H), 7.53 (s, 1H), 7.40 (s, 1H), 7.28 (dd, J = 8.97, 4.93 Hz,

1H), 7.03 (m, 1H), 6.84 (d, J = 1.77 Hz, 1H), 6.05 (q, J = 6.57 Hz, 1H),

4.76 (s, 2H), 3.89 (s, 3H), 1.84 (d, J = 6.82 Hz, 3H). Anal. Calcd for

C₁₇H₁₅N₄OCl₂F 0.5H₂O: C, 52.32; H, 4.13; N, 14.36. Found: C, 52.51;

3

H, 4.00; N, 14.30.

General Procedure A for Amide Formation. To a solution of

6-amino-5-(substituted benzyloxy)pyridin-3-yl]benzoic acid (1 mol equiv),

1-hydroxybenzotriazole hydrate (HOBT, 1.2 mol equiv), and 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 1.2

mol equiv) in DMF (0.2 M) was added amine (1.2 mol equiv). The

reaction solution was stirred at room temperature overnight, then diluted

with EtOAc and partitioned with H₂O. The organic layer was separated,

and the aqueous layer was extracted with EtOAc. The combined organic

layers were washed with water, saturated NaHCO₃, and brine. The

solution was dried over Na₂SO₄, filtered, and concentrated to dryness

under vacuum. The residue was purified using column chromatography

(silica gel, 99:1 to 95:5 CH₂Cl₂/MeOH). The fractions containing

product were concentrated under vacuum to yield the amide product

with a yield of ∼50%.

Compounds 36ꢀ38 were prepared according to general procedure B

for amide formation.

(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)phenyl)(4-(pyrrolidin-1-yl)piperidin-1-yl)meth-

anone (36). LCMS m/z 557 (100%), 559 (64%) (M + H)⁺; ¹H NMR

(400 MHz, DMSO-d₆) δ ppm 7.88 (d, J = 1.77 Hz, 1H), 7.57 (dd, J =

9.09, 5.05 Hz, 1H), 7.40ꢀ7.50 (m, 3H), 7.33ꢀ7.40 (m, 2H), 7.01 (d, J =

1.77 Hz, 1H), 6.15 (q, J = 6.57 Hz, 1H), 5.95 (s, 2H); 4.24 (m, 1H), 3.59

(m, 1H), 2.99ꢀ3.12 (m, 2H), 2.40ꢀ2.47 (m, 1H), 2.16ꢀ2.31 (m, 1H),

1.73ꢀ1.97 (m, 5H), 1.62ꢀ1.73 (m, 4H);, 1.27ꢀ1.45 (m, 2H). HPLC

purity (method A): t_R= 6.293, 100%.

4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (37).

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dichloride (545 mg, 0.745 mmol) was added, and thereaction mixture was

degassed 3 times with N₂and then microwaved at 120 °C for 1 h. The

reaction solution was diluted with water and extracted with EtOAc. The

combined extracts were dried over Na₂SO₄, concentrated, purified by a

silica gel column chromatography with a gradient of 25ꢀ50% EtOAc/

hexanes to provide methyl 2-(4-(6-amino-5-(1-(2,6-dichloro-3-fluoro-

phenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)-2-methylpropanoate (1.46 g,

21% yield): LCMS m/z 467 (100%), 469 (64%) (M + H)⁺; ¹H NMR

(400 MHz, CDCl₃) δ ppm 7.79 (d, J = 1.77 Hz, 1H), 7.62ꢀ7.66 (m, 2H),

7.30 (dd, J = 8.84, 4.80 Hz, 1H), 7.06 (t, J = 8.46 Hz, 1H), 6.89 (d, J = 1.52

Hz, 1H), 6.05ꢀ6.11 (m, 1H), 4.76ꢀ4.87 (m, 2H), 3.72ꢀ3.74 (m, 3H),

1.85ꢀ1.89 (m, 9H).

Step 2: tert-Butyl 4-Hydroxy-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)-1H-pyrazol-1-yl)methyl)piperidine-1-carboxylate (53).

A reaction mixture of 52 (214 mg, 1.0 mmol) and 4-(4,4,5,5-tetra-

methyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (39, 194 mg, 1.0 mmol)

with NaH of 60% dispersion in mineral oil (60 mg, 1.5 mmol) in DMF

(3 mL) was stirred at 90 °C for 3 h. The reaction mixture was partitioned

between EtOAc (200 mL) and saturated NaHCO₃solution (50 mL)

and washed with brine (50 mL). The organic layer was dried over

Na₂SO₄and concentrated to give 53 as a yellow grease (361 mg, 89%

1

yield): LCMS m/z 408 (M + H)⁺; H NMR (400 MHz, CDCl₃) δ

ppm 8.00 (s, 1H), 7.80 (s, 1H), 7.65 (s, 1H), 4.05 (s, 2H), 3.72ꢀ3.91 (m,

2H), 3.13ꢀ3.15 (m, 2H), 1.56ꢀ1.78 (m, 4H), 1.45 (s, 9H), 1.30 (s,

12H).

Step 3. To a solution of methyl 2-(4-(6-amino-5-(1-(2,6-dichloro-3-

fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)-2-methylpropano-

ate (2.92 g, 6.25 mmol) in MeOH (31 mL) was added a solution of

LiOH (450 mg, 18.76 mmol) in water (6.25 mL). The mixture was

heated at 60 °C for 45 min and cooled to ambient temperature. The pH

of the reaction solution was adjusted to ∼5 with 1 N HCl, and the prod-

uct was precipitated out. 2-(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)-

ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)-2-methylpropanoic acid was obtained

after filtration (2.825 g, 100% yield): ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 8.09 (s, 1H), 7.82 (d, J = 1.52 Hz, 1H), 7.57ꢀ7.62 (m, 2H),

7.43ꢀ7.48 (m, 1H), 6.98 (s, 1H), 5.94ꢀ6.24 (m, 3H), 1.83 (d, J = 6.57

Hz, 3H), 1.75 (s, 6H).

Step 3: 4-((4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)-1H-pyrazol-1-yl)methyl)tetrahydro-2H-pyran-4-ol (47).

To a reaction mixture of 53 (361 mg, 0.89 mmol) and 23 (378 mg,

0.89 mmol) in ethylene glycol dimethyl ether (DME) (9 mL) were

added Na₂CO₃solution (1.0 N, 3.9 mL, 3.9 mmol) and Pd(II)-

(PPh₃)₂Cl₂(32 mg, 0.05 mmol). The reaction mixture was purged with

N₂for 15 min and stirred at 85 °C under N₂overnight. The mixture was

partitioned between EtOAc (200 mL) and saturated NaHCO₃solution

(2 ꢁ 50 mL) and washed with brine (50 mL). The organic layer was

dried over Na₂SO₄, concentrated, and purified by a reversed phase C-18

preparative HPLC column, eluting with 25ꢀ95% MeCN in H₂O with

0.1% HOAc. tert-Butyl 4-((4-(6-amino-5-(1-(2,6-dichloro-3-fluorophe-

nyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)methyl)-4-hydroxypiperidine-

1-carboxylate was obtained as a white solid (147 mg, 28% yield), which

was dissolved in 5 mL of CH₂Cl₂. To the solution was added 4.0 M HCl

in dioxane (2.0 mL, 8.1 mmol). The reaction mixture was stirred at

ambient temperature for 2.0 h, concentrated, and purified with reversed

phase C-18 preparative HPLC column, eluting with 5ꢀ95% MeCN in

H₂O with 0.1% HOAc, and 47 was obtained as an off-white solid (76 mg,

63% yield): LCMS m/z 481 (100%), 483 (64%) (M + H)⁺; ¹H NMR

(400 MHz, CDCl₃) δ ppm 7.65ꢀ7.69 (m, 1H), 7.57ꢀ7.62 (m, 1H),

7.44ꢀ7.49 (m, 1H), 7.32 (dd, J = 8.84, 4.80 Hz, 1H), 7.08 (d, J = 8.08

Hz, 1H), 6.87 (d, J = 1.77 Hz, 1H), 6.04ꢀ6.14 (m, 1H), 5.44 (br s, 2H),

4.09 (s, 2H), 3.72ꢀ3.85 (m, 4H), 1.87 (d, J = 6.57 Hz, 3H), 1.59ꢀ1.69 (m,

2H), 1.32ꢀ1.40 (m, 2H). HPLC purity (method B): t_R= 0.765, 95.1%.

5-(1-(Azetidin-3-yl)-1H-pyrazol-4-yl)-3-(1-(2,6-dichloro-3-

fluorophenyl)ethoxy)pyridin-2-amine (50). Step 1. tert-Butyl

3-(Methylsulfonyloxy)azetidine-1-carboxylate. To a solution of tert-

butyl 3-hydroxyazetidine-1-carboxylate (466 mg, 2.69 mmol), Et₃N

(0.75 mL, 5.38 mmol), and 4-(dimethylamino)pyridine (33 mg, 0.269

mmol) in CH₂Cl₂(10 mL) at 0 °C was added methanesulfonyl chloride

(0.25 mL, 3.23 mmol). The resulting brown mixture was stirred at 0 °C

to ambient temperature overnight. The reaction was quenched with

NaHCO₃, and then the mixture was partitioned between CH₂Cl₂

(200 mL) and saturated NaHCO₃solution (50 mL). The organic layer

was dried over Na₂SO₄, filtered through a silica gel pad, and eluted with

hexane/EtOAc, 1:1. The filtrate was concentrated by vacuum to give

tert-butyl 3-(methylsulfonyloxy)azetidine-1-carboxylate as a yellow oil

(614 mg, 91% yield): ¹H NMR (400 MHz, CDCl₃) δ ppm 5.26ꢀ5.11

(m, 1H), 4.26 (dd, J = 10.36, 6.82 Hz, 2H), 4.08 (dd, J = 10.36, 4.29 Hz,

2H), 3.05 (s, 3H), 1.43 (s, 9H).

Step 4. To a solution of 2-(4-(6-amino-5-(1-(2,6-dichloro-3-fluoro-

phenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)-2-methylpropanoic acid

(1.00 g, 2.20 mmol) in DMF (5.5 mL) were added HOBT (300 mg,

2.20 mmol), EDC (633 mg, 3.30 mmol), and N¹,N¹-dimethylethane-

1,2-diamine (225 mg, 2.20 mmol). The mixture was stirred overnight

and then purified by a reversed phase C-18 preparative HPLC column,

eluting with acetonitrile/water with 0.1% acetic acid to afford 45 as a

white solid (170 mg, 14% yield): LCMS m/z 523 (100%), 525 (64%)

(M + H)⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.65ꢀ7.72 (m, 2H),

7.64 (s, 1H), 7.32 (dd, J = 8.84, 4.80 Hz, 1H), 7.07 (t, J = 8.34 Hz, 1H),

6.90ꢀ6.96 (m, 1H), 6.87 (d, J = 1.52 Hz, 1H), 6.04ꢀ6.11 (m, 1H), 5.25

(br s, 2H), 3.43ꢀ3.51 (m, 2H), 2.80 (t, J = 5.68 Hz, 2H), 2.51 (s, 6H),

2.06 (s, 3H), 1.81ꢀ1.92 (m, 9H). HPLC purity (method B): t_R

=

3.156, 95.2%.

2-(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)-1H-pyrazol-1-yl)-N-(3-(dimethylamino)propyl)-

acetamide (46). Compound 46 was prepared with similar procedures

as compound 45: LCMS (APCI) m/z 509 (100%), 511 (64%) (M + H)⁺.

¹H NMR (400 MHz, CDCl₃) δ ppm 7.78 (d, J = 1.77 Hz, 1H),

7.70ꢀ7.77 (m, 1H), 7.67 (s, 1H), 7.50 (s, 1 H), 7.31 (dd, J = 8.84,

4.80 Hz, 1H), 7.01ꢀ7.09 (m, 1H), 6.86 (d, J = 1.77 Hz, 1H), 6.07 (q, J =

6.65 Hz, 1H), 4.74ꢀ4.86 (m, 4H), 3.30ꢀ3.41 (m, 2H), 2.22ꢀ2.34 (m,

2H), 1.94 (s, 6H), 1.87 (d, J = 6.57 Hz, 3H), 1.57 (dt, J = 11.94, 6.03 Hz,

2H). Anal. Calcd for C₂₃H₂₇N₆O₂Cl₂F 1.5H₂O: C, 51.50; H, 5.64; N,

3

15.67. Found: C, 51.47; H, 5.39; N, 15.70.

4-((4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-

pyridin-3-yl)-1H-pyrazol-1-yl)methyl)-tetrahydro-2H-pyran-

4-ol (47). Step 1: tert-Butyl 1-Oxa-6-azaspiro[2.5]octane-6-carbox-

ylate (52). To a solution of dimethylsulfoxonium methylide, which was

prepared under N₂from NaH of 60% dispersion in mineral oil (440 mg,

11.0 mmol) and trimethylsulfoxonium iodide (2.421 g, 11.0 mmol) in

5 mL of anhydrous DMSO, was added 1-Boc-4-oxo-1-piperidincarboxy-

late (50, 1.993 g, 10.0 mmol) in 5 mL of DMSO dropwise. The resulting

mixture was stirred at 55 °C for 6 h. The cooled reaction mixture was

poured into iceꢀwater and extracted with EtOAc (2 ꢁ 200 mL). The

combined organic layers were washed with H₂O (50 mL), brine (50 mL)

and then dried over Na₂SO₄. After concentration, 52 was obtained as a

yellow oil (1.479 g, 69% yield): ¹H NMR (400 MHz, CDCl₃)

δ ppm 3.62ꢀ3.78 (m, 2H), 3.35ꢀ3.49 (m, 2 H), 2.63ꢀ2.72 (m, 2 H),

1.71ꢀ1.84 (m, 2 H), 1.37ꢀ1.52 (m, 11 H).

Step 2: tert-Butyl 3-(4-Bromo-1H-pyrazol-1-yl)azetidine-1-carboxy-

late. A microwave tube (5 mL) was charged with tert-butyl 3-(methyl-

sulfonyloxy)azetidine-1-carboxylate (304 mg, 1.21 mmol), 4-bromopyr-

azole (178 mg, 1.21 mmol), NaH 60% in mineral oil (73 mg, 1.82 mmol),

and anhydrous DMF (2 mL). The resulting mixture was microwaved

at 110 °C for 30 min and then partitioned between EtOAc (200 mL)

and saturated NaHCO₃solution (2 ꢁ 50 mL) and washed with brine

(50 mL). The organic layer was dried over Na₂SO₄and concentrated by

vacuum to afford tert-butyl 3-(4-bromo-1H-pyrazol-1-yl)azetidine-1-car-

boxylate as a yellow oil (360 mg, 98%): ¹H NMR (400 MHz, DMSO-d₆)

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1

δ ppm 8.14 (s, 1H), 7.67 (s, 1H), 5.22ꢀ5.12 (m, 1H), 4.31ꢀ4.18 (m,

2H), 4.08 (s, 2H), 1.43ꢀ1.36 (m, 9H).

78%. LCMS m/z 436 (100%), 438 (64%) (M + H)⁺; H NMR (400

MHz, MeOD) δ ppm 7.69 (s, 1H), 7.57 (s, 1H), 7.48 (s, 1H), 7.35 (dd,

J = 8.84, 4.80 Hz, 1H), 7.14 (t, J = 8.59 Hz, 1H), 6.83 (s, 1H), 6.08 (d, J =

6.57 Hz, 1H), 4.29 (d, J = 6.82 Hz, 2H), 4.00 (t, J = 9.73 Hz, 2H),

3.84ꢀ3.95 (m, 2H), 3.32 (d, J = 8.08 Hz, 1H), 1.78 (d, J = 6.57 Hz, 3H).

Anal. Calcd for C₂₀H₂₀N₅OCl₂F 2.5HCl 2H₂O 2.5CH₃CO₂H: C,

Step 3: tert-Butyl 3-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-

yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate. A reaction mixture of tert-

butyl 3-(4-bromo-1H-pyrazol-1-yl)azetidine-1-carboxylate (225 mg, 0.74

mmol) and bis(pinacolate)diboron (227 mg, 0.89 mmol) with KOAc

(247 mg, 2.52 mmol) in DMSO (3 mL) was purged with N₂for 15 min.

Then PdCl₂(dppf)₂CH₂Cl₂(30 mg, 2.52 mmol) was added. The result-

ing mixture was stirred at 80 °C under N₂overnight. After it cooled to

room temperature, the mixture was filtered through a Celite pad and

washed well with EtOAc. The filtrate was washed with H₂O (2 ꢁ 50 mL)

and brine (50 mL). The organic layer was dried over Na₂SO₄and then

concentrated by vacuum. The residue was purified on a silica gel column,

eluting with hexane/EtOAc (3:2) to provide tert-butyl 3-(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidine-1-carbox-

ylate as a clear oil (250 mg, 97% yield): ¹H NMR (400 MHz, CDCl₃))

δ ppm 7.83 (s, 2H), 5.13ꢀ4.98 (m, 1H), 4.36 (t, J = 8.59 Hz, 2H),

4.33ꢀ4.22 (m, 2H), 1.49ꢀ1.41 (m, 6H), 1.34ꢀ1.28 (m, 6H), 1.27ꢀ1.18

(m, 9H).

3

42.08; H, 5.16; N, 9.81. Found: C, 42.34; H, 4.98; N, 9.41.

1-(3-((4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)-

ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)methyl)azetidin-1-yl)-

2-(dimethylamino)ethanone (49). LCMS m/z 521 (100%), 523

(64%) (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.91 (d, J =

1.52 Hz, 1H), 7.74 (d, J = 1.77 Hz, 1H), 7.52ꢀ7.61 (m, 2H), 7.40ꢀ7.47

(m, 1H), 6.88 (s, 1H), 6.08 (q, J = 6.57 Hz, 1H), 5.66 (s, 2H), 4.31 (d,

J = 7.33 Hz, 2H), 4.21 (t, J = 8.59 Hz, 1H), 3.98 (dd, J = 8.97, 5.43 Hz,

1H), 3.90 (t, J = 9.09 Hz, 1H), 3.68 (dd, J = 9.98, 5.43 Hz, 1H),

2.84ꢀ2.92 (m, 2H), 2.15 (s, 6H), 1.80 (d, J = 6.57 Hz, 3H). HPLC

purity (method A): t_R= 6.731, 100%.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-

4-yl)-1H-pyrazol-4-yl)pyridin-2-amine (61). Step 1. Toa solution

of 5-bromo-3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-2-ylamine

(22, 12.83 g, 33.76 mmol) in anhydrous DMF (100 mL) were added di-

tert-butyl dicarbonate (21.25 g, 97.35 mmol) and 4-dimethylaminopyr-

idine (0.793 g, 6.49 mmol). The mixture was stirred at ambient

temperature for 18 h. Saturated NaHCO₃solution (300 mL) was added

to the mixture and extracted with EtOAc (3 ꢁ 250 mL). The combined

extracts were washed with water (5 ꢁ 100 mL), saturated NaHCO₃, and

brine, then dried over Na₂SO₄. After filtration, evaporation, and high

vacuum drying, 54 was obtained as an off-white foam solid (19.59 g, 100%

yield): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.18 (d, J = 1.77 Hz, 1H),

7.83 (d, J = 2.02 Hz, 1H), 7.52ꢀ7.61 (m, 1H), 7.43ꢀ7.52 (m, 1H),

6.21ꢀ6.33 (m, 1H), 1.75 (d, J = 6.57 Hz, 3H), 1.39 (s, 9H), 1.16ꢀ1.27

(m, 9H).

Step 4: tert-Butyl 3-(4-(6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)-

ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate. A reac-

tion mixture of tert-butyl 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate (459 mg, 1.31 mmol) and

3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-iodopyridin-2-amine (27,

374 mg, 0.88 mmol) in 13 mL of ethylene glycol dimethyl ether (13 mL)

was purged with N₂for 15 min. Then Pd(II)(PPh₃)₂Cl₂(46 mg, 0.07

mmol) was added and purging continued with N₂for another 15 min.

Another 1.0 N Na₂CO₃solution (3.9 mL, 3.9 mmol) was added after

purging with N₂for 15 min. The resulting mixture was stirred at 85 °C

under N₂overnight. The reaction mixture was filtered through a Celite

pad and washed well with MeOH. The filtrate was concentrated by

vacuum. The residue was partitioned between EtOAc (200 mL) and

saturated NaHCO₃solution(2 ꢁ 50 mL) and washed with brine (50 mL).

The organic layer was dried over Na₂SO₄, concentrated, and purified by a

flash chromatography, eluting with 0ꢀ10% methanol in CH₂Cl₂to afford

tert-butyl 3-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-

3-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate as a brown grease (421 mg,

92% yield): LCMS m/z 522 (100%), 524 (64%) (M + H)⁺; ¹H NMR (400

MHz, CDCl₃) δ ppm 7.78ꢀ7.72 (m, 1H), 7.65ꢀ7.59 (m, 1H), 7.58ꢀ7.53

(m, 1H), 7.52ꢀ7.44 (m, 1H), 7.41ꢀ7.33 (m, 1H), 7.04 (t, J=8.46Hz, 1H),

5.02 (d, J = 7.58 Hz, 1H), 4.79 (s, 2H), 4.41ꢀ4.34 (m, 1H), 4.33ꢀ4.20 (m,

2H), 4.18ꢀ4.04 (m, 2H), 1.87ꢀ1.80 (m, 3H), 1.26ꢀ1.17 (m, 9H).

Step 5. 5-(1-Azetidin-3-yl-1H-pyrazol-4-yl)-3-[1-(2,6-dichloro-3-

fluorophenyl)ethoxy]pyridin-2-amine (50). A reaction mixture of tert-

butyl 3-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-

3-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylate (421 mg, 0.81 mmol) with

dry HCl in dioxane (4.0 M, 2.0 mL, 8.1 mmol) in CH₂Cl₂(5 mL) was

stirred at ambient temperature for 2.0 h. The reaction mixture was

concentrated by vacuum. The residue was treated with EtOAc. The pre-

cipitated solid was filtered off and washed well with EtOAc, hexane, then

dried under vacuum to give 50 as a sand color solid of HCl salt (275 mg,

81% yield), which was further purified on a reversed phase HPLC

column, eluting with acetonitrile in water with 0.1% acetic acid to provide

50 as a white amorphous solid after lyophilization: LCMS m/z 422

Step 2. To a solution of 54 (19.58 g, 33.76 mmol) in DMSO (68 mL)

was added potassium acetate (11.26 g, 114.78 mmol) and bis-

(pinacolato)diboron (10.29 g, 40.51 mmol). The mixture was degassed

and charged with nitrogen three times. Then Pd(dppf)Cl₂CH₂Cl₂(1.38 g,

3

1.69 mmol) was added. The reaction mixture was degassed and charged

with nitrogen three times and then stirred in a 80 °C oil bath for 12 h. The

reaction was cooled to ambient temperature, diluted with ethyl acetate

(100 mL), and filtered through a Celite pad which was washed with ethyl

acetate. The combined ethyl acetate solution (700 mL) was washed with

water (5 ꢁ 100 mL), brine (100 mL) and dried over Na₂SO₄. After filtra-

tion and concentration, the residue was purified on a silica gel column,

eluting with EtOAc/hexane (0ꢀ50%) to provide 56 as a foam solid

(20.59 g, 97% yield): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.18 (d, J =

1.26 Hz, 1H), 7.51ꢀ7.61 (m, 2H), 7.42ꢀ7.50 (m, 1H), 6.12ꢀ6.21 (m,

1H), 1.74 (d, J = 6.57 Hz, 3H), 1.16ꢀ1.42 (m, 30H).

Step 3. To a solution of 56 (20.34 g, 32.42 mmol) in CH₂Cl₂(80 mL)

was added a solution of dry HCl in dioxane (4 N, 40.5 mL, 162 mmol).

The reaction solution was stirred in a 40 °C oil bath for 12 h. The reac-

tion mixture was cooled to ambient temperature, diluted with EtOAc

(400 mL), then washed carefully but quickly with saturated NaHCO₃

until the water layer was basic (pH > 8). The organic layer was washed

with brine and dried over Na₂SO₄. After filtration, evaporation, and high

vacuum drying, 57 was obtained as an off-white foam solid (13.48 g, 97%

1

(100%), 424 (64%) (M + H)⁺; H NMR (400 MHz, DMSO-d₆) δ

1

ppm 9.20 (s, 1H), 8.12 (s, 1H), 7.86 (s, 1H), 7.73ꢀ7.83 (m, 1H), 7.59

(dd, J ꢀ 8.84, 5.05 Hz, 1H), 7.40ꢀ7.54 (m, 1H), 7.09 (s, 1H), 6.23 (d, J =

6.57 Hz, 2H), 5.40 (s, 1H), 4.35 (s, 4H), 3.56 (s, 1H), 1.79ꢀ1.89 (m,

3H). Anal. Calcd forC₁₉H₁₈N₅OCl₂F 2H₂O 1CH₃CO₂H: C, 48.66;H,

yield): H NMR (400 MHz, DMSO-d₆) δ ppm 7.71ꢀ7.78 (m, 1H),

7.54 (dd, J = 8.97, 4.93 Hz, 1H), 7.36ꢀ7.49 (m, 1H), 6.87 (d, J = 1.01

Hz, 1H), 6.13 (br s, 2H), 5.99 (q, J = 6.65 Hz, 1H), 1.77 (d, J = 6.82 Hz,

3H), 1.22 (s, 6 H) 1.20 (s, 6 H).

3

5.06; N, 13.51. Found: C, 48.41; H, 4.89; N, 13.14.

Compounds 48 and 49 were prepared with similar procedures as

compound 50.

Step 4. To a stirred solution of 57 (4.2711 g, 10.0 mmol) and tert-

butyl 4-(4-bromo-1H-pyrazol-1-yl)piperidine-1-carboxylate (3.9628 g,

12.0 mmol) in DME (40 mL) was added a solution of Na₂CO₃(3.1787

g, 30.0 mmol) in water (10 mL). The solution was degassed and charged

with nitrogen three times. To the solution was added Pd(PPh₃)₂Cl₂

5-(1-(Azetidin-3-ylmethyl)-1H-pyrazol-4-yl)-3-(1-(2,6-di-

chloro-3-fluorophenyl)ethoxy)pyridin-2-amine (48). Yield:

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ARTICLE

(351 mg, 0.50 mmol). The reaction solution was degassed and charged

with nitrogen again three times and then stirred in an 87 °C oil bath for 16 h.

After cooling to ambient temperature, the reaction mixture was diluted with

EtOAc (200 mL), filtered through a pad of Celite, and washed with EtOAc.

The combined EtOAc solution was washed with brine, dried over Na₂SO₄,

and concentrated. The crude product was purified on a silica gel column,

eluting with EtOAc/hexane system (0ꢀ100% EtOAc) to afford tert-butyl

4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-

pyrazol-1-yl)piperidine-1-carboxylate as a foam solid (3.4167 g, 65% yield).

Step 5. To a solution of tert-butyl 4-(4-(6-amino-5-(1-(2,6-dichloro-

3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-car-

boxylate (566.7 mg, 1.03 mmol) in methanol (5 mL) was added 4 N HCl

in dioxane (1 mL, 4 mmol). The solution was stirred for about 1 h or

until the deprotection was complete. The solvents were evaporated and

the residue was dissolved in methanol and purified on a reversed phase

C-18 preparative HPLC column, eluting with acetonitrile/water with

0.1% acetic acid from 5% to 30% with a linear gradient. After lyophiliza-

tion, 61 was obtained as a white solid (410 mg, 78% yield): LCMS m/z

450 (100%), 452 (64%) (M + H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 7.92 (s, 1H), 7.75 (d, J = 1.77 Hz, 1H), 7.58 (dd, J = 8.97, 4.93 Hz,

1H), 7.52 (s, 1H), 7.42ꢀ7.48 (m, 1H), 6.89 (d, J = 1.77 Hz, 1H), 6.03ꢀ

6.14 (m, 1H), 5.65 (s, 2H), 4.08ꢀ4.20 (m, 1H), 2.99ꢀ3.07 (m, 2H),

2.57 (td, J = 12.38, 2.27 Hz, 2H), 1.90ꢀ1.97 (m, 2H), 1.80 (d, J = 6.82

Hz, 3H), 1.74 (dd, J = 12.00, 3.92 Hz, 2H). HPLC purity (method A):

t_R= 6.694, 100%.

NaH (16.32 g, 0.68 mol) was added portionwise to a stirred solution

of 4-iodopyrazole (66, 110.57 g, 0.57 mol) in DMF (2 L) at 4 °C. The

resulting mixture was stirred for 1 h at 4 °C, and 65 (176.00 g., 0.63 mol)

was then added. The resulting mixture was heated to 100 °C for 12 h.

The reaction was quenched with H₂O, and the aqueous portion was

extracted with EtOAc several times. The combined organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated to aﬀord an orange oil.

The residue was puriﬁed by a silica gel column, eluting with 5% EtOAc in

pentane to give tert-butyl 4-(4-iodo-1H-pyrazol-1-yl)piperidine-1-car-

boxylate 67 as a white solid (140 g, 66%): ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 7.98 (s, 1H), 7.52 (s, 1H), 4.30ꢀ4.43 (m, 1H), 3.93ꢀ4.07

(m, 2H), 2.77ꢀ2.97 (m, 2H), 1.90ꢀ2.00 (m, 2H), 1.66ꢀ1.83 (m, 2H),

1.40 (s, 9H).

Bis(pinacilato)diboron (55, 134 g, 0.52 mol) and potassium acetate

(145 g, 1.48 mol) were added sequentially to a solution of 67 (140 g, 0.37

mol) in DMSO (1.5 L). The mixture was purged with nitrogen several

times, and dichlorobis(triphenylphosphino)palladium(II) (12.9 g, 0.018

mol) was then added. The resulting mixture was heated at 80 °C for 2 h.

The reaction mixture was cooled to ambient temperature and ﬁltered

through a bed of Celite and washed with EtOAc. The ﬁltrate was washed

with brine (2 ꢁ 500 mL), dried over Na₂SO₄, ﬁltered, and concentrated.

The residue was puriﬁed by a silica gel column, eluting with 5% EtOAc in

hexanes to give 68 as a white solid (55 g, 40%): ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 7.97 (s, 1H), 7.59 (s, 1H), 4.29ꢀ4.44 (m, 1H), 4.01

(d, J = 13.14 Hz, 2H), 2.77ꢀ3.01 (m, 2H), 1.92ꢀ1.99 (m, 2H), 1.78 (dd,

J = 12.00, 4.17 Hz, 2H), 1.41 (s, 9H), 1.24 (s, 12H).

Compounds 58ꢀ60 were prepared by using similar procedures

described for the synthesis of compound 61.

A reaction solution of (R)-5-bromo-3-(1-(2,6-dichloro-3-ﬂuorophe-

nyl)ethoxy)pyridin-2-amine (69, 14.4 g, 37.9 mmol) and 68 (17.2 g,

45.5 mmol) in DME (114 mL) was purged with nitrogen. Then

Pd(dppf)Cl₂(1.24 g, 1.52 mmol) and Cs₂CO₃aqueous solution (2.0 N,

57 mL, 114 mmol) were added. The resulting mixture was purged with

nitrogen and stirred at 90 °C for 3 h. The reaction mixture was cooled to

ambient temperature and ﬁltered through a Celite pad and washed well

with MeOH. The ﬁltrate was concentrated by vacuum. The residue was

partitioned between EtOAc (800 mL) and saturated NaHCO₃solution

(2 ꢁ 150 mL) and brine (150 mL). The organic layer was dried over

Na₂SO₄and then ﬁltered through a silica gel pad and washed well with

EtOAc to remove black solid. The ﬁltrate was concentrated by vacuum.

The residue was puriﬁed by a silica gel column, eluting with EtOAc/

hexanesystemtocollectdesiredfractionandthenconcentratedbyvacuum.

The residue of oﬀ-white foam was treated with ether (100 mL) and hexane

(500 mL). The precipitated solid was ﬁltered oﬀ and washed well with

hexane, then dried under vacuum to aﬀord tert-butyl 4-(4-(6-amino-

5-((R)-1-(2,6-dichloro-3-ﬂuorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-

1-yl)piperidine-1-carboxylate as an oﬀ-white solid (19.55 g, 94% yield).

To a solution of 4-(4-(6-amino-5-((R)-1-(2,6-dichloro-3-ﬂuorophe-

nyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (19.54 g,

35.5 mmol) in CH₂Cl₂(250 mL) at 0 °C was added 4.0 N HCl in dioxane

(133 mL, 532 mmol) dropwise. The resulting mixture was stirred at 0 °C

to room temperature for 4 h. The white suspension was ﬁltered oﬀ, and

the yellowishcrude product was dissolvedin H₂O (500 mL) and extracted

with CH₂Cl₂(500 mL). The pH of the aqueous layer was adjusted to ∼10

by adding Na₂CO₃, and then the aqueous portion was extracted with

EtOAc (1000 mL). The organic layer was washed with saturated aqueous

NaHCO₃(400 mL) and brine (150 mL), then dried over Na₂SO₄, and

concentratedbyvacuum. Theprecipitatedsolidwastreated withether and

hexane and ﬁltered oﬀ, washed well with ether and hexane, then dried

under vacuum to aﬀord crizotinib (63) as a white powder (15.34 g, 96%

yield): mp 196ꢀ199 °C; LCMS m/z 450 (100%), 452 (64%) (M + H)⁺;

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.92 (s, 1H), 7.75 (d, J = 1.77 Hz,

1H), 7.58 (dd, J = 8.97, 4.93 Hz, 1H), 7.52 (s, 1H), 7.42ꢀ7.48 (m, 1H),

6.89 (d, J = 1.77 Hz, 1H), 6.03ꢀ6.14 (m, 1H), 5.65 (s, 2H), 4.08ꢀ4.20

(m, 1H), 2.99ꢀ3.07 (m, 2H), 2.57 (td, J = 12.38, 2.27 Hz, 2H),

1.90ꢀ1.97 (m, 2H), 1.80 (d, J = 6.82 Hz, 3H), 1.74 (dd, J = 12.00, 3.92

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-((S)-pyrro-

lidin-3-yl)-1H-pyrazol-4-yl)pyridin-2-amine (58). LCMS m/z

1

436 (100%), 438 (64%) (M + H)⁺; H NMR (400 MHz, CDCl₃) δ

ppm 7.65 (s, 1H), 7.56 (s, 1H), 7.46 (s, 1H), 7.39 (dd, J = 8.59, 5.05 Hz,

1H), 7.15 (t, J = 8.08 Hz, 1H), 6.99ꢀ7.03 (m, 1H), 6.13ꢀ6.22 (m, 1H),

5.06ꢀ5.13 (m, 1H), 3.74ꢀ3.84 (m, 2H), 3.57ꢀ3.72 (m, 2H), 2.55ꢀ2.70

(m, 1H), 2.33ꢀ2.47 (m, 1H), 1.95 (d, J = 6.82 Hz, 3H). HPLC purity

(method B): t_R= 2.872, 100%.

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-

3-yl)-1H-pyrazol-4-yl)pyridin-2-amine (59). LCMS m/z 450

1

(100%), 452 (64%) (M + H)⁺; H NMR (400 MHz, CDCl₃) δ ppm

7.70 (d, J = 1.52 Hz, 1H), 7.56 (s, 1H), 7.53 (s, 1H), 7.32 (dd, J = 8.84,

4.80 Hz, 1H), 7.02ꢀ7.10 (m, 1H), 6.87 (d, J = 1.77 Hz, 1H), 6.03ꢀ6.13

(m, 1H), 5.22 (br s, 2H), 4.18ꢀ4.31 (m, 1H), 3.37ꢀ3.44 (m, 1H),

3.03ꢀ3.12 (m, 2H), 2.72ꢀ2.83 (m, 1H), 1.98ꢀ2.05 (m, 3H), 1.87 (d, J =

6.57 Hz, 3H), 1.60ꢀ1.72 (m, 1H). HPLC purity (method B): t_R

=

2.892, 100%

3-(1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-(tetrahydro-

2H-pyran-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine (60). LCMS

m/z 451 (100%), 453 (64%) (M + H)⁺; 1H NMR (400 MHz, DMSO-

d₆) δ ppm 7.96 (s, 1H), 7.76 (d, J = 1.77 Hz, 1H), 7.54ꢀ7.63 (m, 2H),

7.41ꢀ7.48 (m, 1H), 6.90 (d, J = 1.52 Hz, 1H), 6.06ꢀ6.13 (m, 1H), 5.65

(s, 2H), 4.30ꢀ4.41 (m, 1H), 3.90ꢀ4.01 (m, 2H), 3.43ꢀ3.53 (m, 2H),

1.93ꢀ2.00 (m, 2H), 1.84ꢀ1.91 (m, 2H), 1.81 (d, J = 6.82 Hz, 3H). HPLC

purity (method A): t_R= 8.712, 100%.

3-((R)-1-(2,6-Dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-

4-yl)-1H-pyrazol-4-yl)pyridin-2-amine (Crizotinib). To a stirred

solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (7.94 g, 39.45 mmol)

in CH₂Cl₂(100mL), cooledto0°C, was slowly added Et₃N (5.54 mL, 39.45

mmol) followed by methane sulfonyl chloride (3.06 mL, 39.45 mmol) and

DMAP (48 mg, 0.39 mmol). The mixture was stirred at room temperature

overnight. To the mixture was added water (30 mL). Extraction with CH₂Cl₂

(3 ꢁ 30 mL) followed by drying (Na₂SO₄) and removal of the solvent in

vacuo afforded tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate

65 as a white solid (11.00 g, >99% yield): ¹H NMR (400 MHz, CDCl₃) δ

4.89 (m, 1H), 3.69 (m, 2H), 3.31 (m, 2H), 3.04 (s, 3H), 1.95 (m, 2H),

1.83 (m, 2H), 1.46 (s, 9H).

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ARTICLE

Hz, 2H). HPLC purity (method A): t_R= 6.694, 100%. Anal. Calcd for

2wkm. The crizotinib and compound 3 cocrystal structures were refined

C₂₁H₂₂N₅OCl₂F 0.5H₂O: C, 54.91; H, 5.05; N, 15.25. Found: C,

55.20; H, 4.91; N, 15.13.

to resolution limits of 2.0 and 2.2 Å, respectively.

3

Biochemical Kinase Assays. c-MET enzyme inhibition was

meaured by Omnia (Invitrogen Inc.) continuous fluorometric assay as

described previously.²⁴The reactions were conducted in 50 μL volumes

in 96-well plates at 30 °C. Mixtures contained 1 nM human recombinant

c-MET kinase domain (aa 1051ꢀ1348), 2 μM phosphoacceptor peptide

Ac-EEEEYI(cSx)-IV-NH2 (Invitrogen Inc.), test compound (11-dose

3-fold serial dilutions, 2% DMSO final) or DMSO only, 0.2 mM DTT,

and 10 mM MgCl₂in 20 mM Hepes, pH 7.5, and the reactions were

initiated by addition of ATP (100 μM final concentration) following a

20 min preincubation. The initial rates of phosphopeptide formation

were measured over 20 min using a Tecan Safire microplate reader with

wavelength settings of 360 nm for excitation and 485 nm for emission.

The inhibitors were shown to be ATP-competitive from kinetic and

crystallographic studies. The K_ivalues were calculated by fitting the data

to the equation for competitive inhibition using nonlinear regression

method (GraphPad Prism, GraphPad Software, San Diego, CA) and

experimentally measured ATP K_m= 56 μM.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: 858-638-6333. Fax: 877-481-1783. E-mail: jean.cui@

pﬁzer.com.

’ ACKNOWLEDGMENT

We are grateful to Dr. Nikolaus Schiering for generating the

ﬁrst cocrystal structure of PHA-665752 bound to c-MET, to

Muhammad Alimuddin for analytical support, to the Pﬁzer PDM

group for metabolic stability studies, to Dr. Klaus Dress for

discussions and making Figures 6 and 7, and to Dr. Beth Lunney

for c-MET/ALK sequence identity analyses.

’ ABBREVIATIONS USED

Cellular Kinase Phosphorylation ELISA Assays^10a. All experi-

ments were done under standard conditions (37 °C and 5% CO₂). IC₅₀

values were calculated by concentrationꢀresponse curve fitting using a

Microsoft Excel based four-parameter method. Cells were seeded in 96-

well plates in medium supplemented with 10% fetal bovine serum (FBS)

and transferred to serum-free medium [with 0.04% bovine serum

albumin (BSA)] after 24 h. In experiments investigating ligand-depen-

dent 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, phosphorylation 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

specificfor phosphorylatedtyrosine residues. Antibody-coatedplateswere

(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 horse-

radish peroxidase conjugated anti-total-phosphotyrosine (PY-20) anti-

body (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. Cell lines

used for individual kinases include A549 for c-MET, Karpas 299 for ALK,

3T3-RON for RON, 293-AXL for AXL, 3T3-E/TIE2 for TIE2, PAE-

TRKA for TRKA, PAE-TRKB for TRKB, BaF3-BCL-ABL for ABL, 293-

hIR for IR, and Jurkat for LCK.

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 LCMS/MS. All

incubations were performed in triplicate, and the percent remaining of

parent drug at the end of incubation was determined by LCMS/MS peak

area ratio.

c-MET, mesenchymalꢀepithelial transition factor; HGF, hepato-

cyte growth factor; HGFR, hepatocyte growth factor receptor; ALK,

anaplastic lymphoma kinase; RTK, receptor tyrosine kinase; c-KIT,

proto-oncogene tyrosine-protein kinase kit; BCR, breakpoint-

cluster region; ABL, Abelson leukemia; NSCLC, non-small-cell

lung cancer; GIST, gastrointestinal stromal tumor; RCC, renal cell

carcinoma; EGFR, epidermal growth factor receptor; VHL, Von

HippelꢀLindau; VEGF, vascular endothelial growth factor; LTK,

leukocyte tyrosine kinase; IR, insulin receptor; NPM, nucleophos-

min; EML4, echinoderm microtubule associated protein-

like 4; SBDD, structure based drug design; KD, kinase domain; A-

loop, activation loop; FGFR1K, ﬁbroblast growth factor receptor

1 kinase; PDGFR, platelet derived growth factor receptor; LCK,

lymphocyte speciﬁc kinase; AXL, tyrosine-protein kinase receptor

UFO (AXL gene); TIE2, tyrosine kinase with Ig; TRKA, tropo-

myosin receptor kinase A; TRKB, tropomyosin receptor

kinase B; RON, receptor d'origine nantais; ADME, absorption,

distribution, metabolism, and excretion; ALCL, anaplastic large

cell lymphoma

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Ahmed, S.; Nallasura, V.; Hetzel, J. T.; Jagadeeswaran, R.; Karczmar, G.;

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lipophilicity and large molecular weight, PHA-665752 demonstrated

high metabolic clearance (CL = 77 (mL/min)/kg in rat in vivo PK

study), low permeability, and a pH dependent solubility. PHA-665752

was originally positioned as a preclinic candidate with an intravenous

drug proﬁle. However, the low solubility at pH 7.4 (0.9 μg/mL)

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dx.doi.org/10.1021/jm2007613 |J. Med. Chem. 2011, 54, 6342–6363

Article Doi

Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK)

DOI: 10.1021/jm2007613

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Full text of DOI:10.1021/jm2007613

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