Discovery of Narrow Spectrum Kinase Inhibitors: New Therapeutic Agents for the Treatment of COPD and Steroid-Resistant Asthma

Article

pubs.acs.org/jmc

Discovery of Narrow Spectrum Kinase Inhibitors: New Therapeutic

Agents for the Treatment of COPD and Steroid-Resistant Asthma

Stuart T. Onions,*^,†Kazuhiro Ito,^‡,#Catherine E. Charron,*^,‡Richard J. Brown,^†Marie Colucci,^†

Fritz Frickel,^‡George Hardy,^‡Kevin Joly,^†John King-Underwood,^§Yasuo Kizawa,^∥Ian Knowles,^⊥

P. John Murray,^‡,#Andrew Novak,^†Anjna Rani,^†Garth Rapeport,^‡,#Alun Smith,^†Peter Strong,^‡,#

David M. Taddei,^†and Jonathan G. Williams^†

^†Sygnature Discovery Limited, Biocity, Nottingham NG1 1GF, United Kingdom

^‡RespiVert Limited, 2 Royal College Street, The London Bioscience Innovation Centre, London NW1 0NH, United Kingdom

^§CompChem Resource, Old Cottage Hospital, Homend, Ledbury, Herefordshire HR8 1ED, United Kingdom

^∥Department of Physiology and Anatomy, Nihon University School of Pharmacy, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan

^⊥Pneumolabs UK Limited, Harrow, Middlesex HA1 3UJ, United Kingdom

ABSTRACT: The discovery of a novel series of therapeutic agents that has

been designed and optimized for treating chronic obstructive pulmonary disease

is reported. The pharmacological strategy was based on the identiﬁcation of

compounds that inhibit a deﬁned subset of kinase enzymes modulating

inﬂammatory processes that would be eﬀective against steroid refractory disease

and exhibit a sustained duration of action after inhaled delivery.

chemokines, reactive oxygenspecies, and electrolytic enzymes.^11,12

Macrophages are markedly elevated in number in the lung

INTRODUCTION

■

Corticosteroids are widely used in the clinic for the treatment of

pulmonary inﬂammatory disorders, including asthma and chronic

obstructive pulmonary disease (COPD). However, their

therapeutic beneﬁt is frequently limited by a steroid-refractory

and alveolar spaces of patients with COPD and are localized to

sites of alveolar destruction.¹²The higher number of macrophages

observed may result from increased recruitment of blood

monocytes as well as prolonged survival in the lung.¹³To a lesser

phenotype.^1,2This resistance to treatment can be an inherent

extent, the rise in macrophage levels may derive from increased

feature of the condition or may be induced by external factors that

provoke disease exacerbation, such as a viral infection,³or by long-

proliferation in response to oxidative stress, such as exposure

to tobacco smoke.¹⁴It is relevant to the disease symptomology

term inhalation of noxious gases and particles, such as cigarette

smoke or kitchen smoke.⁴Marked steroid resistance impedes

that alveolar macrophages obtained from COPD patients are

resistant to the anti-inﬂammatory eﬀects of corticosteroids.¹⁵

the eﬀective management of these maladies, and increasing the

These diﬀerentiated cells recruit neutrophils from the bloodstream

drug load in order to compensate for reduced eﬃcacy is often

to the site of inﬂammation by the production of cytokines such as

associated with potentially severe side-eﬀects, especially on

chronic treatment.^5,6The objective of the present study was to

identify eﬀective agents for the treatment of corticosteroid-

resistant symptoms of COPD and asthma that would be suitable

CXCL8 and TNFα.

Oral therapies targeting the p38 mitogen activated protein

kinases (MAPK) have been proposed as an alternative treatment

to corticosteroids owing to their ability to inhibit the production

for chronic use.

of proinﬂammatory cytokines.¹⁶The p38 MAP kinase family

COPD is a major and increasing global health problem with

an enormous expenditure of indirect/direct health care costs.⁷

comprises four distinct protein isoforms, α, β, γ, and δ, each of

which displays diﬀerent patterns of tissue expression.¹⁷The

COPD now aﬀects over 10% of the world population over the

age of 40,⁸and the burden of the disease is particularly high

in developing countries. COPD is a complex disease combining

inﬂammatory processes and structural changes to the aﬀected

p38 MAPK α and β isoforms are distributed widely in the body,

being present in many cell types. While the α isoform is well-

characterized as a mediator of inﬂammation, p38 MAPK δ and γ

tissues,⁹and long-acting bronchodilators (LAMA and LABA), not

are less well studied. There is evidence to suggest that inhibition

of p38 MAPK γ is the mechanism by which the β2-agonist

anti-inﬂammatory drugs, are currently the mainstay of COPD

formoterol reverses steroid resistance in asthma.¹⁸In addition,

pharmacological treatment.¹⁰While the underlying inﬂammatory

mechanisms are complex, alveolar macrophages are known

to play a critical role in the pathophysiology of the disease by

secreting proinﬂammatory proteins, including certain cytokines,

Received: July 1, 2015

A

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Figure 1. p38 MAPK inhibitors proﬁled in the clinic.

p38 MAPK γ expression is reported to be upregulated in

peripheral blood mononuclear cells obtained from severe

asthmatics.¹⁸This body of evidence has impelled numerous

eﬀorts to discover small molecule inhibitors of the p38 MAPK

family, in particular the α isoform.

rheumatoid arthritis and could be explained by activation of both

redundant and/or alternative kinase signaling pathways.³¹

For lung diseases, topical administration of p38 MAPK

inhibitors is expected to signiﬁcantly reduce their resulting

systemic exposure, especially to the liver, and thereby minimize

the likelihood of side eﬀects.³²This method of delivery is ideally

suited to treating conditions like COPD in which the principal

drug targets are epithelial cells and/or resident inﬂammatory

cells such as macrophages. It remains a concern that for selective

p38 MAPK agents redundancy in cell signaling cascades will

result in disease resistance.³³In addition, Smith and colleagues

reported while p38 MAPK α inhibitors proved to be eﬀective on

LPS-stimulated IL-6 and GM-CSF release in monocytes they

displayed limited maximal inhibition in macrophages and also no

beneﬁcial eﬀects on LPS-stimulated CXCL8 release in macro-

phages.³⁴

In addition to the p38 MAPK family, tyrosine kinases have

been implicated in inﬂammatory disease. Of particular interest

is hematopoietic kinase (HCK), which has been shown to

modulate adhesion, granulocytosis, and cytokine production in

neutrophils.³⁵HCK is also reported to possess a preeminent role

in LPS/toll like-receptor-4-induced TNF and IL-6 production in

human primary macrophages and is upregulated in PBMCs from

COPD patients.³⁶This kinase belongs to the Src family, and its

expression is limited to the hematopoietic cells, predominantly

in neutrophils.³⁷It was postulated that additional inhibition of

relevant kinases, such as HCK, could enhance the eﬃcacy of

p38 inhibitors. This approach of inhibiting a subset of kinases

is increasingly used in the oncology ﬁeld and has provided

compounds showing greater eﬃcacy when compared to those

that act on a single kinase target.^38,39

A recent example of a p38 MAPK α inhibitor entering the

clinic is 1 (Pamapimod, RO4402257; Figure 1). It was previously

reported to be eﬃcacious in several in vivo models of

inﬂammatory arthritis.¹⁹However, in a 12 week clinical study

in patients with rheumatoid arthritis, the compound did not

demonstrate signiﬁcant eﬃcacy either as a monotherapy or in

combination with methotrexate.²⁰Moreover, typical side eﬀects

associated with orally delivered p38 MAPK inhibitors were

observed, including skin rash and elevated activity levels of liver

enzymes.²¹Dose-limiting toxicology is the norm for p38 MAPK

inhibitors, and it has been suggested that ﬁrst-generation

compounds, such as 1, are much less selective agents than was

ﬁrst considered.²²The latest generation of inhibitors, typiﬁed by

2 (PH-797804), is reported to be more selective and demon-

strate little oﬀ-target kinase activity.²³In a 6 week trial involving

230 patients with COPD, the oral administration of 2 was

well-tolerated and demonstrated a statistical improvement in

FEV1, while the usual skin rash was observed in only 7% of

patients.²⁴Another oral p38 MAPK inhibitor, 3 (Losmapimod,

GW856553), was tested in a 12 week trial involving 302 patients

with COPD. This compound was also well-tolerated and demon-

strated a statistical reduction of plasma ﬁbrinogen, although there

was no signiﬁcant improvement on lung function or sputum

neutrophils.²⁵However, the eﬀect was not sustained in a 6 month

study.²⁶Selective p38 MAPK α inhibitors may not provide an

eﬀective therapeutic intervention in a chronic setting.

In contrast to 1−3, urea 4 (Doramapimod, BIRB-796) from

Boehringer Ingelheim is a pan active inhibitor of the four

p38 MAPK isoforms as well as other non-MAP kinases.²⁷This

compound has undergone clinical assessment in healthy patients

injected with LPS and was found to reduce the levels of induced

TNFα and IL-1β release, along with other cytokines.²⁸However,

in patients with Crohn’s disease, little eﬃcacy was demonstrated

and only a transient reduction in the inﬂammation marker,

C-reactive protein (CRP), was observed.²⁹These transient

eﬀects and disease resistance have been observed with other p38

MAP kinase inhibitors, such as VX702³⁰and 1 Pamapimod,²⁰in

The two main objectives for the project were to design

molecules that, ﬁrst, had p38α/γ activity and, second, had a

longer duration of action than BIRB-796 when delivered

topically to the lung. To the ﬁrst end, we were assisted by the

availability of crystallography data for our target enzymes with an

array of ligands in the Protein Data Bank (PDB).⁴⁰The design

paradigm was to identify commonalities between the p38 isoforms

that would also promote the DFG-out protein conformation

observed for BIRB-796 (1kv2.pdb). A second design parameter

was to focus on lower solubility derivatives that were anticipated

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Scheme 1. General Synthetic Strategy

to promote slow dissolution in the airways, leading to persistent

target occupancy that is not dependent on systemic PK.

This article describes the discovery of novel, small molecule

agents that inhibit a selected set of therapeutically relevant

kinases for the treatment of steroid-refractory pulmonary

disorders such as COPD. In addition, the compounds possess

pharmacokinetic properties that enable sustained activity in vivo.

either by an S_NAr displacement on 1-ﬂuoro-4-nitronaphthalene

or by a Mitsonobu coupling reaction on 4-nitronaphthalen-1-ol.

Subsequent reduction of the nitro groups, with iron in acetic acid

or by catalytic hydrogenation, gave the desired naphthylamines

47a−c. The unsymmetrical ureas 9/10 and 38 were produced

by prior activation of aminopyrazole 34 with either CDI or a

phosgene equivalent followed by treatment with the requisite

naphthylamine 47a−c and, where necessary, deprotection of the

product. In the case of 2-aminopyridine 9, the Boc group was

removed from intermediate 48 by exposure to TFA, whereas

for pyridine carboxylic acid 38, the acid was revealed by

saponiﬁcation of ester 49. These advanced intermediates were

transformed into ﬁnal test substances by an N-acylation reaction

with an acid chloride or isocyanate or amidation under standard

peptide bond forming conditions.

CHEMISTRY

■

The compound examples described herein were prepared by

one of three alternative processes as the ﬁnal or penultimate

step. Formation of the unsymmetrical central urea (Scheme 1,

Route 1) was performed via activation of aminopyrazole 34⁴¹by

its transformation into the corresponding isocyanate or

equivalent, followed by addition of the desired naphthylamine

35. However, many analogues diﬀered only in the nature of the

substituent appended to the pyridine nucleus. In some instances,

this structural divergence was readily incorporated by a

displacement reaction on chloroacetamide intermediate 36

(Scheme 1, Route 2) with an appropriate nucleophile. In most

cases, the compounds were obtained from common pyridine

amines 9, 10, 37, 40, and 41 or carboxylic acid building blocks 38

and 39 by acylation or an amide coupling reaction, respectively

(Scheme 1, Route 3).

Imidazolyl analogue 17 was readily accessible via an amination

reaction on the known alkyl iodide 50⁴⁵with 2-nitroimidazole

under basic conditions (Scheme 3). The resulting nitroarene 51

was converted into the test compound by reduction of the nitro

group followed by acylation with acetyl chloride in the presence

of DIPEA.

Most of the lower homologues (Scheme 1, L, −CH₂−) were

prepared using essentially the same synthetic strategies outlined

above, starting from the appropriately substituted pyridinemeth-

yl alcohols 52 and 53, which were available from commercial

sources (Scheme 4). The corresponding methoxy linked biaryls

54a−b were obtained by a Mitsunobu coupling. N-Methyl

methoxyacetamide 55b was prepared by alkylation of acylated

intermediate 55a with methyl iodide in the presence of

sodium hydride. The resulting nitronaphthylenes were converted

into naphthylamines 35 and 56a−b either by a dissolving

metal reduction or by catalytic hydrogenation, and the products

were subsequently exposed to a preactivated solution of

5-aminopyrazole 34. In the case of tertiary amide 35, this provided

N-methylated analogue 31 directly. The protected intermediate

obtained from the urea coupling step, ester 57, was transformed

into carboxylic acid 39 by basic hydrolysis. Final drug substances

Intermediates containing an ethylene linker (Scheme 1,

L, −CH₂CH₂−) were obtained from the corresponding ethyl

alcohol precursors (Scheme 2, 42, 44, and 45). Boc-protected

2-aminopyridine 42 was derived from ethyl 2-(2-chloropyridin-

4-yl)acetate by a Buchwald coupling⁴²with tert-butylcarbamate

followed by reduction of the ester to the alcohol, under standard

conditions. The corresponding methyl ester 44 was accessed

via hydrolysis of a protected picolinonitrile,⁴³43, which, in turn,

was obtained from commercial 2-(pyridin-4-yl)ethanol by silyl

ether protection, followed by N-oxide formation and cyanide

insertion. Treatment of 4-methyl-3-nitropyridine with base and

para-formaldehyde provided aminopyridine ethanol 45 after

nitro reduction.⁴⁴Ether intermediates 46a−c were generated

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a

Scheme 2. Synthetic Route To Access the Ethylene Linker Intermediates

a

Reagents: (a) t-BuOCONH₂, Pd₂(dba)₃, Cs₂CO₃, Xantphos, THF, 65 °C; (b) DIBAL, −78 °C, THF; (c) TBDMSCl, imidazole, DCM, rt; (d)

H₂O₂, MeReO₃, DCM, rt; (e) TMS-CN, Me₂NC(O)Cl, DCM, rt; (f) Na, MeOH, then aq. HCl, rt; (g) Triton B, paraformaldehyde, DMSO, rt; (h)

H₂, Pd/C, MeOH; (j) NaH, 1-ﬂuoro-4-nitronaphthalene, THF, 0 °C; (k) 4-nitronaphthalen-1-ol, DIAD, PPh₃, THF, −78 °C; (m) Fe, AcOH, 50 °C

(46a) or H₂, Pt/C, rt (46b and 46c); (n) CDI, DCM (47a and 47b) or diphosgene, aq. NaHCO₃(47c); (p) TFA, DCM; (q) aq. NaOH; (r)

RCOCl, base, THF/DMF, rt; (s) RNH₂, HATU, DMF, rt.

a

Scheme 3. Synthesis of the Imidazolyl Analogue

a

Reagents: (a) 2-nitro-1H-imidazole, K₂CO₃, DMF, 50 °C; (b) Fe, AcOH, 50 °C; (c) MeCOCl, DIPEA, DCM, rt.

were generated from amine precursors 37 by acylation and

sulfonylation and from acid 39 by amide couplings.

Additional 2-aminopyridine N-acetyl analogues were pre-

preparing this compound class (Scheme 5). Reaction of the

parent amine 37 with chloroacetyl chloride provided chloro-

acetamide intermediate 36. This reactive alkylating agent

was isolated and then subjected to displacement reactions with

pared by a process that is highly convergent and expedient for

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a

Scheme 4. Synthetic Route To Access the Methylene Linker Intermediates

a

Reagents: (a) 4-nitronaphthalen-1-ol, DIAD, PPh₃, −78 °C, THF; (b) MeOCH₂COCl, DIPEA, THF, rt; (c) Fe, AcOH, 50 °C or H₂, Pt/C, rt; (d)

NaH, MeI, THF, 0 °C; (e) CDI, 34, DCM; or diphosgene, NaHCO₃, DCM, 0 °C; (f) LiOH, THF/H₂O, rt; (g) RCOCl, DIPEA, THF/DMF, rt; or

ROCOCl, DIPEA, THF/DMF, rt; or RSO₂Cl, DIPEA, THF/DMF, rt; or RNCO, DCM/DMF, rt; (h) HATU, RNH₂, DMF, rt.

a

Scheme 5. Synthesis of N-Acetyl Analogues

a

Reagents: (a) ClCH₂COCl, DCM, DIPEA, rt; (b) RYH, DIPEA, DCM/DMF, 40 °C (Y = N, S).

nucleophiles to provide, inter alia, aminoacetamide derivatives

26 and 27 and thioether 28.

Analogues 32 and 33 were accessed with similar chemistry as

that for compounds 18−25; however, the side chain substitution

(R²= H, Me) was introduced early in the synthesis. Treatment of

commercial methyl 2-aminoisonicotinate with methyl lithium

furnished both ketone 58 and tertiary alcohol 59a. Standard

reduction of 58 with NaBH₄gave secondary alcohol 59b.

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a

Scheme 6. Synthetic Route To Access the Side-Chain-Substituted Analogues

a

Reagents: (a) MeLi, THF, −78 °C; (b) NaBH₄, MeOH, 0 °C; (c) NaH, 1-ﬂuoro-4-nitronaphthalene, DMF, 0 °C; (d) H₂, Pt/C, rt; (e) diphosgene,

aq. NaHCO₃, 0 °C to rt; (f) MeOCH₂COCl, DIPEA, THF/DCM, 0 °C.

Elaboration of 59a−b under standard chemistry gave 32 and 33

proinﬂammatory cytokines including TNFα and CXCL8. The

potency of test compounds in this system was evaluated by

the determination of both IC₅₀values as well as their maximum

inhibitory eﬀect (E_max). This latter property was considered to

be an important criterion that resulted from the inhibition of

multiple targets and was therefore used to select compounds that

displayed the kinase proﬁle described above.

Upon evaluation of potential alternative hinge binders in

the pyrazole urea series in comparison to BIRB-796 (4), 5−8

(Table 1)²⁷demonstrated that p38 inhibition could be

maintained. 4-Pyridyl analogue 8 was superior to its regioiso-

meric analogues 6 and 7 and to imidazole 5, as the former

maintained signiﬁcant potency toward the γ isozyme and

introduced reasonable potency against HCK. Additionally,

compound 8 demonstrated moderate activity in d-U937 cells.

We next focused on further modiﬁcation of compound 8; with

the aim of promoting interactions with the peptide backbone of

the kinase hinge, a number of heteroaryl amine, amide, and urea

analogues were prepared (Table 2).

From public X-ray structures of BIRB-796 with p38 α

(1KV2.pdb) and atrial natriuretic peptide (ANP) with p38 γ

(1CM8.pdb), we identiﬁed a diﬀerence in the presentation of

hydrogen-bonding residues to the ligand from the hinge region

of the protein: BIRB-796 being monodentate in p38 α and ANP

being bidentate in p38 γ. Inspection of other p38 α structures

(e.g., 3FLN.pdb) showed that, when it is presented with a ligand

with bidentate hydrogen-bonding capacity, p38 α is able to

(Scheme 6).

RESULTS AND DISCUSSION

■

Highly potent drugs are usually required for inhaled admin-

istration owing to the small doses that can be delivered

conveniently, typically no more than a few hundred micrograms

as a single dose. A long duration of action is also highly desirable

to maintain therapeutic beneﬁt, for example, during the night and

to allow once daily dosing. In addition to these characteristics,

low oral bioavailability (<5%) is an important advantageous

property to minimize systemic availability and side eﬀects.

Slow dissolution and absorption through the lungs have also

been implicated as desirable properties, and these attributes

have recently been referred to as forming part of an “inhalation by

design” strategy.⁴⁶In view of the modest metabolizing potential

of lung epithelium and the route administration, functional groups

and molecular properties that might be considered detrimental

for the development of an oral medicine were not necessarily

excluded from consideration.^47,48Indeed, increasing the molecular

complexity by the introduction of additional polar groups and

hydrogen-bond donors was anticipated to modulate both

solubility and permeability positively for our inhaled program.

For this purpose, U937 cells were treated with phorbol

myristate acetate (PMA) to evoke their diﬀerentiation into

macrophages-like cells. Stimulation of the resulting cells (d-U937

cells) with lipopolysaccharide (LPS) induces the production of

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Table 1

undergo a rotation of the hinge region to accommodate the extra

hydrogen bond. This suggested that a bidentate hinge binder

would be tolerated by p38 α and would yield stronger binding

to p38 γ. It was also postulated that the ﬁrmer anchor of the

bidentate hinge binding in p38 γ may help to promote the trapping

of a DFG-out protein conformation, which was suspected to

be required for a longer duration of action. An unintended

consequence of this change to a bidentate ligand was to promote

theinhibition of other kinasessuch asHCK, whichwas found to be

beneﬁcial to the overall cellular proﬁle of the compounds (greater

E_maxfor the inhibition of TNFα in U937 cells).

Simple amines 9 and 10 maintained moderate activity toward

the kinases of interest and displayed a promising increase in

cellular potency. In addition, it was encouraging to see that the

pyridine nucleus could be functionalized with such a synthetically

useful and adaptable amino group. The corresponding acetamide

derivatives 11 and 12 demonstrated similar p38 MAPK proﬁles

as those of the amine precursors as well as a modest increase in

activity at HCK. Of particular interest at this stage was the ability

of ortho-substituted analogue 11 to inhibit the stimulated release

of TNFα from d-U937 cells with an IC₅₀value of <20 nM and

with a high maximal activity.

A consistent SAR trend in favor of the ortho-isomer was

evident for the two methoxyacetamido-substituted pyridines, 13

and 14. Although activity against the γ isoform of p38 MAPK

remained weak, ortho-analogue 14 displayed an impressive

cellular proﬁle, especially against TNFα release, for which the

IC₅₀value was single-digit nanomolar. This compound was also

the ﬁrst from this chemotype to potently inhibit the release of

CXCL8 from the diﬀerentiated, macrophage-like U937 cells.

In contrast, “reverse” amides 15 and 16 retained activity

against p38 MAPK α, failing to demonstrate any activity at the γ

isoform or at HCK together with little activity at the cellular level.

This data suggested that the disposition of the amidic carbonyl is

of critical importance in determining the pharmacology of this

compound series. Imidazole analogue 17 was also largely sel-

ective for the α isoform of p38 MAPK. Its cellular proﬁle was

comparable with that of 2-aminopyridine 9, and consequently

this chemotype was discounted from further study.

The in vitro biology of the compounds, summarized in Table 2,

together with that of additional analogues (data not shown)

revealed that 2-acylaminopyridine was a favored fragment that

promoted inhibitory activity against the triad of isolated enzymes

as well as potency in cellular systems. The data also indicated

that p38 MAPK inhibitors with additional HCK kinase activity

demonstrate an anti-inﬂammatory biological phenotype in a

disease-relevant cell line.

To assess the degree of CYP inhibition inherent in this series,

4-pyridyl derivative 8 was compared, against a standard isoform

panel, with the corresponding 2- and 3-N-acylamino derivatives,

11 and 13, respectively (Table 3). The former compound

exhibited signiﬁcant inhibitory activity, particularly against the

enzyme CYP 3A4. This is most likely to arise from a direct

interaction with the pyridyl nitrogen as a similar proﬁle is evident

for meta-substituted analogue 13. In contrast, 2-acetamido

derivative 11 displayed hardly any activity at either the CYP 2D6

or 3A4 isoform. The superior proﬁle shown by 11 likely derives

from the presence of the ortho substituent in the pyridine

nucleus, which presumably disrupts the undesired interaction

with the basic nitrogen.

Despite the encouraging eﬃcacy results for acylaminopyridine

11, a signiﬁcant increase in potency in d-U937 cells was deemed

to be necessary for such compounds to meet their intended

product proﬁle as inhaled medicines. We next studied shortening

of the three-atom spacer between the naphthyl and pyridine

groups (Table 4). Consistent with the early ﬁndings for these

chemotypes, pyridine carboxamides 18 and 19 remained

selective potent inhibitors of the p38 MAPK α isozyme while

being, at best, weak inhibitors of p38 MAPK γ and HCK, and

they did not reach maximum eﬃcacy in cells. In contrast, the

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Table 2

methoxy-linked compounds can possess good to excellent

activity against the triad of isolated enzymes examined in this

study and potently inhibit the inﬂammatory mediators TNFα

and CXCL8 in diﬀerentiated macrophage-type cells. Equally

gratifying was the observation that the maximal inhibitory

values reached in these latter assay systems were ≥90%, which

is a highly desirable property, postulated to be the result of

simultaneous inhibition of the α and γ protein isoforms of

p38 MAPK.

Table 3

Inhibition (%) of CYP 450 enzyme isoforms at 30 μM

Compound

2C19

2D6

3A4

2C9

BIRB-796

35

38

25

11

44

3

−23

67

6

13

31

23

37

8

11

13

73

64

The markedly superior pharmacological proﬁle of N-sulfonyl

and N-acyl 2-aminopyridines 21−23 provided the incentive to

explore this particular compound class in more detail (Table 5).

Results obtained for benzamide 24 and phenylacetamide 25 were

both surprising and disappointing. Both compounds displayed

in vitro proﬁles comparable to those of earlier structures, namely,

inhibitory activity strongly in favor of the p38 MAPK α enzyme.

While high potencies are preserved in the cellular system, the

proﬁles of a series of functionalized 2-aminopyridines was

markedly enhanced by the reduction in the chain length to a two-

atom linker. For example, carbamate derivative 20 is a potent,

dual p38 MAPK α/HCK inhibitor, and this is reﬂected by its

superior potency in cells. Sulfonamide 21 has more modest but

comparable activity at the γ isoform and HCK. Notable is the

direct comparison of the in vitro proﬁle of acetamide 22 and its

higher homologue, compound 11 (Table 2). This reveals that the

H

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Table 4

corresponding E_maxvalues are less than or equal to 80% for these

two compounds.

steric constraints from monomethyl 32 (as a racemate) to gem-

dimethyl analogue 33 gave a general decrease in isolated enzyme

and cellular proﬁle (Table 6). Further reduction to an ether or

methane linker lead to a signiﬁcant change in pharmacological

proﬁle, which is beyond the scope of this article.⁴⁹

Additional compound analogues revealed that a diversity

of functional modiﬁcation was well-tolerated and could be

exploited to reﬁne the biological phenotype. Tertiary amines

26 and 27 exhibited characteristics similar to those of earlier,

favored analogues (21−23, Table 4) and oﬀer the opportunity

to modulate global physicochemical parameters, including the

possibility of salt formation and selection. Incorporation of

a thioether, 28, or a sulfone, 29, was also tolerated as both

exhibited the desired narrow spectrum kinase inhibitor (NSKI)

activity and showed excellent potencies and eﬃcacies in cells.

These ﬁndings conﬁrm that a broad range of functionality can

be introduced at the β carbon of the acetamido group while

maintaining the desired biological proﬁle. Methoxyacetamide

analogue 30 was found to be a highly active NSKI and to potently

inhibit the release of inﬂammatory mediators in d-U937 cells.

The poor activity of N-methylated analogue 31 supports the

hypothesis that the N-acyl 2-aminopyridine pharmacophore

operates in a donor/acceptor binding mode. Perturbation of this

critical interaction has a negative impact on p38 MAPK γ and

HCK kinase inhibition. This disruption often leads to a loss of the

NSKI phenotypic proﬁle and reduction in cellular eﬃcacy.

As reducing the linker length had such a profound enhancing

eﬀect on the pharmacological proﬁle of N-acyl 2-aminopyridine

series, additional modiﬁcations were investigated that would

either perturb its conformation or shorten it further. Increasing

In view of its promising in vitro biology, methoxyacetamide

30 was selected for further proﬁling in an in vivo time course

experiment to assess the compound’s duration of action against

BIRB-796 (Figure 2). The test compound was dosed (at

0.2 mg mL⁻¹) by intratracheal (it) administration to mice

(N = 8). Following this event and after increasing intervals of time

(the preincubation time) starting with a delay of 2 h, which was

incrementally increased up to 18 hours, animals were challenged

with an endotoxin (LPS) to evoke an inﬂammatory response

in the lungs. An additional 8 h after the insult, each animal was

sacriﬁced and bronchoalveolar lavage ﬂuid (BALF) was collected.

The number of neutrophils per milliliter of ﬂuid was counted to

determine the eﬀectiveness of the potential drug.

The decrease in the number of neutrophils at t = 2 h is an

indication of an agent’s anti-inﬂammatory activity. The persistence

of the observed eﬀect as the delay (preincubation time) before

administration of the challenge is increased indicates the

compound’s duration of action. BIRB-796 and compound 30

initially show comparable anti-inﬂammatory eﬀects in murine

lung, but the therapeutic eﬀect of BIRB-796 rapidly diminishes

and has all but disappeared at 8 h post dose. By contrast, com-

pound 30 retained a signiﬁcant proportion of its initial activity

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Table 5

Table 6

out to the 12 hour time point, indicating a t_1/2in the region of 14 h

and its likely suitability for once daily dosing in the clinic.

It is interesting to compare the physiochemical properties of

BIRB-796 and compound30. While they have similar lipophilicity

(cLog P 6.87 vs 6.66), BIRB-796 has a more strongly basic

center, which would be anticipated to lead to better solubility.

Lung permeability may also be reduced by the additional

H-bond present in compound 30 and its higher polar surface

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properties of the compound were also investigated and are

suitable for dry powder inhaled formulation, the intended method

of use in the clinic. Compound 30 is currently undergoing clinical

trials.

CONCLUSIONS

■

A series of functionalized 2-aminopyridines has been derived that

potently inhibit both p38 and SRC family kinases. It has been

demonstrated that these kinase inhibitors, such as acetamide 22,

urea 23, thioether 28, and sulfonyl derivative 29, potently reduce

the stimulated release of proinﬂammatory mediators from

disease-relevant cells, with high maximal eﬃcacy. As an example

of this chemotype, methoxy acetamide 30 has been identiﬁed to

demonstrate a long and advantageous duration of action in an

in vivo pharmacodynamic model. The same compound is also

highly eﬀective at reversing cigarette smoke-induced inﬂamma-

tion in mice, showing that it is eﬀective against a model of

the disease state that is normally resistant to treatment with

corticosteroids. It has been established that a diverse range of

substituents, appended to a common pharmacophore, is com-

patible with the desired pharmacological proﬁle. This permits the

selection of compounds having physicochemical properties that

are suitable for various pharmaceutical formulations including

those used for dry powder inhalation.

Figure 2. Comparison of the eﬀects of treatment with BIRB-796 or

compound 30 on LPS-induced airway neutrophilia in mice. it =

intratracheal.

area (tPSA 78.43 vs 116.6).⁴⁶We postulate that these proper-

ties contribute to the excellent in vivo duration of action of

compound 30.

Since the above pharmacodynamic screening method is

sensitive to corticosteroid, compound 30 was next evaluated in

an animal model of steroid-refractory inﬂammation.⁵⁰Mice

exposed repeatedly, over 11 days, to cigarette smoke (CS) develop

pulmonary inﬂammation that is inert to treatment with ﬂuticasone

propionate (FP) (Figure 3). The two test substances were

administered to mice intranasally at 40 μg mL⁻¹(35 μL per mouse,

N = 6) twice daily for 3 days following their exposure to cigarette

smoke. The ineﬀectiveness of FP, when given at 50 μg mL⁻¹,

to reduce neutrophil inﬂux into the lungs of the animals attests to

the steroid insensitivity of the stimulated inﬂammatory response.

It is particularly relevant that BIRB-796 demonstrated little or

no eﬀect on neutrophil accumulation when dosed intranasally at

40 μg mL⁻¹. This result conﬁrms that the compound is ineﬀective

at controlling inﬂammatory processes that are untreatable with

corticosteroids. However, when administered at the same dose, 30

brought about a dramatic reduction in the number of neutrophils

recruited into mouse lung. These results support the hypothesis

that a NSKI such as 30 is able to inhibit corticosteroid-refractory

inﬂammation by exerting inhibitory activity on a predeﬁned panel

of therapeutically relevant kinases.

EXPERIMENTAL SECTION

■

Enzyme Inhibition Assays. The enzyme inhibitory activities of

compounds disclosed herein were determined by FRET using synthetic

peptides labeled with both donor and acceptor ﬂuorophores (Z-LYTE,

Invitrogen Ltd., Paisley, UK). Inhibitory activities against the p38

MAPK α isoform (MAPK14: Invitrogen) were evaluated indirectly by

determining the level of activation/phosphorylation of the downstream

molecule, MAPKAP-K2. The p38 MAPK α protein (80 ng mL⁻¹, 2.5 μL

in 2× kinase buﬀer provided in the kit) was mixed with the test

compound (2.5 μL of either 4.000, 0.400, 0.040, or 0.004 μg mL⁻¹in 4%

DMSO in H₂O) for 2 h at rt. The mix solution in 1× kinase buﬀer

(2.5 μL) of the p38 α inactive target MAPKAP-K2 (Invitrogen, 600 ng

mL⁻¹) and FRET peptide (8.0 μM; a phosphorylation target for

MAPKAP-K2) was then added, and the kinase reaction was initiated by

adding ATP (40 μM, 2.5 μL in 1× kinase buﬀer). The mixture was

incubated for 1 h at rt. Development reagent (protease diluted 1:100

in development buﬀer, 5.0 μL) was added 1 h prior to detection in

a ﬂuorescence microplate reader (Varioskan Flash, ThermoFisher

Scientiﬁc).

Additional attributes of the drug substance 30 enhance its

suitability as a prospective inhaled medicine. Separate studies

(data not shown) reveal that the compound has an oral bio-

availability of less than 5% and displays high plasma protein

binding of >99%. When used at therapeutically relevant doses,

these properties should ensure that systemic exposure will be

very low, reducing the risk of side eﬀects. Relevant physical

The inhibitory activities against p38 MAPK γ (MAPK12: Invitrogen)

and HCK were evaluated in a similar fashion to that described above.

The enzyme (800 ng mL⁻¹, 2.5 μL in 2× kinase buﬀer) was incubated

with the test compound (2.5 μL at either 4.000, 0.400, 0.040, or

0.004 μg mL⁻¹in 4% DMSO in H₂O) for 2 h at rt. The FRET peptides

Figure 3. Eﬀect of treatment with either FP (A), BIRB-796 (A), or 30 (B) on tobacco smoke-induced airway neutrophilia in mice. in = intranasal.

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(8.0 μM, 2.5 μL in 1× kinase buﬀer) and the appropriate ATP solution

(2.5 μL, 400 μM for p38 MAPK γ or 2.5 μL, 60 μM for HCK in

1× kinase buﬀer) were then added to the enzyme/compound mixtures

and incubated for 1 h.

macrophages and neutrophils was determined by FACS analysis (EPICS

ALTRA II, Beckman Coulter, Inc., Fullerton, CA, USA) using anti-

mouse MOMA2 antibody (macrophage) or anti-mouse 7/4 antibody

(neutrophil), and the cell number was calculated with % population and

total BAL cell number. This study was conducted at the Department

of Physiology and Anatomy, Nihon University School of Pharmacy

(Chiba, Japan).

Chemistry. All starting materials and solvents were obtained either

from commercial sources or prepared according to the literature

citation. Unless otherwise stated, all reactions were stirred magnetically.

Organic solutions were routinely dried over anhydrous magnesium

sulfate and ﬁltered. Hydrogenations were performed on a ThalesNano

H-cube ﬂow reactor under the conditions stated. In the case of aniline

products, these were analyzed by LC-MS and used directly without

isolation.

In all cases, the site-speciﬁc protease cleaves nonphosphorylated

peptide only and eliminates the FRET signal. Phosphorylation levels of

each reaction were calculated using the ratio of coumarin emission

(donor) over ﬂuorescein emission (acceptor), for which high ratios

indicate low levels of phosphorylation and low ratios indicate high levels

of phosphorylation. The percentage inhibition of each reaction was

calculated relative to noninhibited control, compounds were tested

in quadruplicate at each concentration, and the 50% inhibitory con-

centration (IC₅₀value) was then calculated from the concentration−

response curve.

LPS-Induced TNFα/CXCL8 Release in d-U937 Cells. U937

cells, a human monocytic cell line, suspended in 50 mL of 10% FBS

RPMI-1640 media were incubated with 5.0 μL of PMA stock solution

(1.0 mg mL⁻¹in neat DMSO), resulting in a ﬁnal PMA concentration of

100 ng mL⁻¹, for 48 to 72 h and diﬀerentiated into macrophage-type

cells. The resulting cells were incubated in 5% FBS RPMI-1640 media.

The test compounds were prepared as 2.0 mg mL⁻¹neat DMSO

solution. The stock solutions were serially diluted in DMSO to be

200-fold desirable ﬁnal concentrations. This allows the ﬁnal DMSO

concentration to be constant across the plate at 0.5%. The cells were

then preincubated with the test compound in triplicate prepared above

for 2 h and stimulated with 50 μL of LPS (1 mg mL⁻¹in PBS) (from

E. coli: O111:B4, Sigma) for 4 h. Thus, the ﬁnal concentration of LPS

was 0.1 μg mL⁻¹. The supernatant was collected for determination

of TNFα and CXCL8 concentrations by sandwich ELISA (DuoSet,

R&D systems). The inhibition of TNFα and CXCL8 production

was calculated at each concentration of test compound by comparison

with vehicle control. The 50% inhibitory concentration (IC₅₀) was

determined from the resultant concentration−response curve.

LPS-Induced Neutrophil Accumulation in Mice. Nonfasted

Balb/c mice were dosed by the intratracheal route with either 20 μL of

vehicle (10% DMSO in isotonic saline) or the test substance at the

indicated times (within the range 2−18 h) before stimulation of the

inﬂammatory response by application of an LPS challenge. At t = 0, mice

were placed into an exposure chamber and exposed to LPS (7.0 mL,

0.5 mg mL⁻¹solution in PBS for 30 min). After a further 8 h, the animals

were anesthetized, their tracheas were cannulated, and bronchoalveolar

lavage ﬂuid (BALF) was extracted by infusing and then withdrawing

from their lungs 1.0 mL of PBS via the tracheal catheter. Total and

diﬀerential white cell counts in the BALF samples were measured using a

Neubaur hemocytometer. Cytospin smears of the BALF samples were

prepared by centrifugation at 200 rpm for 5 min at rt and stained using

a DiﬀQuik stain system (Dade Behring). Cells were counted using oil

immersion microscopy. Data for the number of neutrophils in BAL are

shown as mean SEM (standard error of the mean). The percentage

inhibition of neutrophil accumulation was calculated for each treatment

relative to vehicle treatment. This study was conducted at Pneumolabs

UK Ltd. (London, UK).

Cigarette Smoke-Induced Inﬂammatory Accumulation in

Mice. A/J mice (males, 5 weeks old) were exposed to cigarette smoke

(4% cigarette smoke, diluted with compressed air) for 30 min per day for

11 days using a tobacco smoke inhalation experiment system for small

animals (model SIS-CS; Sibata Scientiﬁc Technology, Tokyo, Japan).

Test substances were given intranasally (35 μL of solution in 50%

DMSO in isotonic saline) and therapeutically twice daily for 3 days

after the ﬁnal cigarette smoke exposure. At 96 h after the last tobacco

smoke exposure (meaning 12 h after the last dosing), animals were

anesthetized, the trachea was cannulated, and BALF was collected as

previously reported.⁵¹The procedure for this involved infusing at one-

third of 20 mL/kg of PBS, leaving it in the airway for 10 s, and

withdrawing infused PBS gradually at room temperature. This step was

repeated three times in total. The BALF was then centrifuged (1200 rpm

for 10 min at 4 °C), the supernatant was drawn oﬀ, and the resulting

cell pellet was resuspended in 0.2% NaCl to induce hemolysis of

erythrocytes. After isotonization with the same volume of 1.6% NaCl,

the total BAL cells were counted. The percent population of alveolar

Column chromatography was performed on prepacked silica

cartridges (230−400 mesh, 40−63 μm) using the amount indicated.

SCX resin was purchased from Supelco and treated with 1 M hydro-

chloric acid prior to use. Unless stated otherwise, the reaction mixture

to be puriﬁed was ﬁrst diluted with MeOH and then made acidic with a

few drops of AcOH. The resulting solution was loaded directly onto

a column packed with the SCX resin, which was washed with MeOH.

The desired material was then eluted with 0.7 M NH₃in MeOH.

All ¹H NMR spectra were acquired on a Bruker Avance III spectrometer

at 400 MHz using residual undeuterated solvent as reference and,

unless speciﬁed otherwise, were run in DMSO-d₆. Test compounds were

1

all >95% pure, as determined by HPLC and H NMR except where

indicated otherwise.

Preparative Reverse-Phase High-Performance Liquid Chro-

matography. Agilent Scalar column C18, 5 μm (21.2 × 50 mm), ﬂow

rate 28 mL/min, eluting with a H₂O−MeCN gradient containing 0.1%

v/v formic acid over 10 min using UV detection at 215 and 254 nm.

Gradient information: 0.0−0.5 min, 95% H₂O−5% MeCN; 0.5−7.0 min,

ramped from 95% H₂O−5% MeCN to 5% H₂O−95% MeCN;

7.0−7.9 min, held at 5% H₂O−95% MeCN; 7.9−8.0 min, returned to

95% H₂O−5% MeCN; 8.0−10.0 min, held at 95% H₂O−5% MeCN.

Analytical Reverse-Phase High-Performance Liquid Chroma-

tography-Mass Spectrometry. Agilent Scalar column C18, 5 μm

(4.6 × 50 mm), or Waters XBridge C18, 5 μm (4.6 × 50 mm), ﬂow rate

2.5 mL/min, eluting with a H₂O−MeCN gradient containing 0.1% v/v

formic acid over 7 min employing UV detection at 215 and 254 nm.

Gradient information: 0.0−0.1 min, 95% H₂O−5% MeCN; 0.1−5.0

min, ramped from 95% H₂O−5% MeCN to 5% H₂O−95% MeCN;

5.0−5.5 min, held at 5% H₂O−95% MeCN; 5.5−5.6 min, held at 5%

H₂O−95% MeCN, ﬂow rate increased to 3.5 mL/min; 5.6−6.6 min,

held at 5% H₂O−95% MeCN, ﬂow rate 3.5 mL/min; 6.6−6.75 min,

returned to 95% H₂O−5% MeCN, ﬂow rate 3.5 mL/min; 6.75−6.9 min,

held at 95% H₂O−5% MeCN, ﬂow rate 3.5 mL/min; 6.9−7.0 min, held

at 95% H₂O−5% MeCN, ﬂow rate reduced to 2.5 mL/min.

tert-Butyl 4-(2-Hydroxyethyl)pyridin-2-ylcarbamate (42). To

a solution of ethyl 2-(2-(tert-butoxycarbonylamino)pyridin-4-yl)-

acetate⁴¹(10.0 g, 35.7 mmol) under nitrogen in THF (100 mL) at

−78 °C was added a solution of DIBAL (1 M in THF, 71 mL, 71 mmol)

over 1 h. The reaction mixture was stirred at −78 to −60 °C for 40 min

and was then warmed to −15 °C over 1 h. The solution was recooled to

−78 °C and was treated with additional DIBAL (1 M in THF, 36 mL,

36 mmol). The mixture was allowed to warm to −40 °C and was stirred

for 1 h. Water (10 mL) was added cautiously to quench the reaction

followed by MgSO₄(20 g), and the solids were removed by ﬁltration.

The ﬁltrate was concentrated to dryness in vacuo, and the residue was

puriﬁed by ﬂash column chromatography (SiO₂, 330 g, EtOAc in

isohexanes, 65%, isocratic elution) to give the title compound (42) as a

yellow solid (6.00 g, 64%). LC-MS: m/z 239 (M + H)⁺(ES⁺). ¹H NMR:

δ 1.47 (9H, s), 2.70 (2H, t), 3.62 (2H, m), 4.68 (1H, t), 6.88 (1H, dd),

7.67 (1H, m), 8.10 (1H, d), 9.61 (1H, s).

tert-Butyl 4-(2-(4-Nitronaphthalen-1-yloxy)ethyl)pyridin-2-

ylcarbamate (46a). To a solution of tert-butyl 4-(2-hydroxyethyl)-

pyridin-2-ylcarbamate (42) (6.00 g, 25.2 mmol) in THF (70 mL) was

added sodium hydride (2.52 g, 60 wt % dispersion in mineral oil,

63.0 mmol) at 0 °C. The resulting bright yellow suspension was stirred

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for 20 min at 0 °C, and then 1-ﬂuoro-4-nitronaphthalene (4.81 g,

25.2 mmol) was added in a single portion. The reaction mixture was

warmed to rt for 2 h, and then water (100 mL) and EtOAc (100 mL)

were added. An insoluble solid formed between the layers and was

collected by ﬁltration. The organic phase was separated and washed with

saturated aq. NaHCO₃(100 mL) and brine (100 mL) and then dried

and evaporated in vacuo. The residue was combined with the collected

solid and triturated with MeOH (50 mL) to aﬀord the title compound

(46a) as a yellow solid (11.9 g, 94%). LC-MS: m/z 410 (M + H)⁺(ES⁺).

¹H NMR: δ 3.20 (2H, t), 4.55 (2H, t), 7.08 (1H, d), 7.17 (1H, d), 7.63

of the volatiles in vacuo gave the title compound (57b) as a brown oil

(1.10 g, 98%). LC-MS: m/z 323 (M + H)⁺(ES⁺).

4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)ethyl)picolinic Acid (38). To a solution of

CDI (0.85 g, 5.2 mmol) in DCM (1.0 mL) was added a solution of

3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine⁴¹(34) (1.20 g, 5.21 mmol) in

DCM (2.0 mL) dropwise over 1 h. After an additional 2 h, a solution

of methyl 4-(2-((4-aminonaphthalen-1-yl)oxy)ethyl)picolinate (47b)

(1.12 g, 3.47 mmol) in DCM (3.0 mL) was added over 5 min. After an

additional 2 h, the mixture was diluted with MeOH and was evaporated

directly onto silica gel. The mixture was puriﬁed by ﬂash column

chromatography (SiO₂, 80 g, EtOAc in isohexane, 30−100%, gradient

elution) to give methyl 4-(2-((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-

pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)ethyl)picolinate (49) as a

(1H, m), 7.82 (1H, m), 7.91 (1H, bs), 8.16 (1H, dd), 8.32 (1H, m), 8.44

(1H, d), 8.58 (1H, dt), 9.68 (1H, bs).

tert-Butyl 4-(2-(4-Aminonaphthalen-1-yloxy)ethyl)pyridin-2-

ylcarbamate (47a). A suspension of tert-butyl 4-(2-(4-nitronaph-

thalen-1-yloxy)ethyl)pyridin-2-ylcarbamate (46a) (5.20 g, 12.7 mmol)

and iron mesh (4.30 g, 76.0 mmol) in a mixture of AcOH and EtOH

(1:2 v/v, 120 mL) was placed in a preheated oil bath at 60 °C and stirred

rapidly for 2 h. The mixture was cooled to rt, poured carefully onto

saturated aq. NaHCO₃(1 L), and extracted with EtOAc (2 × 500 mL).

The combined organic extracts were washed with saturated aq. NaHCO₃

(1 L), water (1 L), and brine (1 L) and then dried and evaporated in

vacuo to furnish the title compound (47a) as a yellow oil (5.00 g, 95%).

LC-MS: m/z 380 (M + H)⁺(ES⁺). ¹H NMR: δ 1.27 (9H, s), 2.92 (2H, t),

4.05 (2H, t), 4.97 (2H, bs), 6.40 (1H, d), 6.57 (1H, d), 6.86 (1H, dd),

7.18 (2H, overlapping m), 7.69 (1H, bs), 7.81 (2H, overlapping m), 7.96

(1H, d), 9.47 (1H, bs).

1

chalky solid (1.28 g, 63%). LC-MS: m/z 578 (M + H)⁺(ES⁺). H

NMR δ 1.25 (9H, s), 2.37 (3H, s), 3.30 (2H, t), 3.85 (3H, s), 4.43

(2H, t), 6.33 (1H, s), 6.97 (1H, d), 7.30−7.35 (2H, overlapping m),

7.40−7.45 (2H, overlapping m), 7.47 (1H, m), 7.55 (1H, m), 7.60

(1H, d), 7.70 (1H, dd), 7.87 (1H, d), 8.05 (1H, dd), 8.15 (1H, dd), 8.55

(1H, bs), 8.63 (1H, dd), 8.75 (1H, bs).

To a suspension of methyl ester (550 mg, 0.95 mmol), prepared

above, in THF (10 mL) was added an aqueous solution of lithium

hydroxide (1M, 1.90 mL, 1.90 mmol). After 15 min, AcOH (1.0 mL)

was added and the reaction mixture was extracted into DCM. The

combined organic extracts were washed with water and brine and then

evaporated in vacuo to give the title compound (38) as a white solid

1

(440 mg, 81%). LC-MS: m/z 564 (M + H)⁺(ES⁺). H NMR: δ 1.25

4-(2-((4-Nitronaphthalen-1-yl)oxy)ethyl)pyridin-3-amine

(46c). To a solution of 4-nitronaphthol (0.95 g, 5.0 mmol), PPh₃

(1.97 g, 7.50 mmol), and 2-(3-aminopyridin-4-yl)ethanol⁴³(45) (1.04 g,

7.50 mmol) in THF (20 mL) at −15 °C was added DIAD (590 μL,

3.75 mmol) dropwise. The mixture was warmed to rt for 1 h and was then

evaporated in vacuo. The residue was taken up in MeOH, evaporated

onto silica (20 g), and puriﬁed by ﬂash column chromatography (SiO₂,

80 g, EtOAc in isohexane, 50−100%, gradient elution, then 5% MeOH in

EtOAc, isocratic elution) to give the title compound (46c) as a yellow

solid (1.40 g, 88%). LC-MS: m/z 310 (M + H)⁺(ES⁺). ¹H NMR: δ 3.14

(2H, t), 4.50 (2H, t), 5.31 (2H, bs), 7.10 (1H, d), 7.15 (1H, d), 7.68 (1H,

m), 7.72 (1H, d), 7.81 (1H, m), 7.96 (1H, s), 8.32 (1H, dd), 4.43 (1H, d),

8.57 (1H, d).

4-(2-((4-Aminonaphthalen-1-yl)oxy)ethyl)pyridin-3-amine

(47c). A solution of 4-(2-((4-nitronaphthalen-1-yl)oxy)ethyl)pyridin-3-

amine (46c) (700 mg, 2.26 mmol) in a mixture of MeOH (50 mL),

EtOAc (25 mL), and DCM (25 mL) was passed through a ThalesNano

H-cube (10% Pt/C, 30 mm, CatCart, 1.0 mL min⁻¹, at 40 °C in full

hydrogen mode). The volatiles were evaporated in vacuo to give the title

compound (47c) as a brown solid (612 mg, 95%). LC-MS: m/z 280

(M + H)⁺(ES⁺). ¹H NMR: δ 3.02 (2H, t), 3.17 (2H, d), 4.21 (2H, t),

5.23 (2H, bs), 6.58 (1H, d), 6.75 (1H, d), 7.08 (1H, d), 7.38−7.41

(2H, overlapping m), 7.72 (1H, d), 7.95 (1H, s), 7.98−8.02 (2H,

overlapping m).

Methyl 4-(2-((4-Nitronaphthalen-1-yl)oxy)ethyl)picolinate

(46b). To a solution of methyl 4-(2-hydroxyethyl)picolinate⁴²

(44) (1.04 g, 5.74 mmol) in THF (10 mL) at 0 °C were added

4-nitronaphthalen-1-ol (0.72 g, 3.80 mmol), PPh₃(1.31 g, 4.97 mmol),

and DIAD (0.93 mL, 4.80 mmol). The reaction was warmed to rt for

18 h. The reaction mixture was diluted with EtOAc (100 mL), washed

with saturated aq. NaHCO₃(2 × 50 mL), and then dried and con-

centrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (SiO₂, 40 g, EtOAc in isohexane, 10−100%, gradient

elution) followed by trituration from MeOH to give the title compound

(46b) as a yellow solid (0.71 g, 53%). LC-MS: m/z 353 (M + H)⁺(ES⁺).

¹H NMR δ 3.38 (2H, t), 3.88 (3H, s), 4.65 (2H, t), 7.21 (1H, d),

(9H, s), 2.30 (3H, s), 3.25 (2H, t), 4.37 (2H, t), 6.32 (1H, s), 6.93 (1H,

d), 7.28−7.36 (3H, overlapping m), 7.38−7.48 (4H, overlapping m),

7.56 (1H, d), 7.87 (1H, d), 8.01 (1H, s), 8.03 (1H, d), 8.45 (1H, d),

9.17−9.23 (2H, overlapping m).

1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-(2-(2-nitro-

1H-imidazol-1-yl)ethoxy)naphthalen-1-yl)urea (51). A mixture of

1-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-(2-iodoethoxy)-

naphthalen-1-yl)urea⁴⁵(50) (200 mg, 0.35 mmol), 2-nitroimidazole

(44 mg, 0.39 mmol), and K₂CO₃(146 mg, 1.06 mmol) in DMF (2.0 mL)

was heated at 50 °C for 3 h. The resulting mixture was cooled to rt,

poured onto water, and extracted with EtOAc. The combined organic

extracts were dried and evaporated in vacuo. The residue was puriﬁed by

ﬂash column chromatography (SiO₂, 12 g, EtOAc in isohexane, 10−80%,

gradient elution) to give the title compound (51) as a yellow solid

1

(41 mg, 21%). LC-MS: m/z 554 (M + H)⁺(ES⁺). H NMR: δ 1.26

(9H, s), 2.37 (3H, s), 4.52 (2H, t), 4.96 (2H, t), 6.33 (1H, s), 6.93 (1H,

d), 7.21 (1H, d), 7.32 (2H, m), 7.42 (2H, m), 7.46−7.57 (2H,

overlapping m), 7.60 (1H, dd), 7.86−7.90 (2H, overlapping m), 8.07

(1H, dd), 8.55 (1H, bs), 8.75 (1H, bs).

Methyl 4-(((4-Nitronaphthalen-1-yl)oxy)methyl)picolinate

(54a). To a mixture of methyl 4-(hydroxymethyl)picolinate (52)

(374 mg, 2.06 mmol), 4-nitronaphthalen-1-ol (389 mg, 2.06 mmol), and

PPh₃(648 mg, 2.47 mmol) in THF (15 mL) at −50 °C was added DIAD

(490 μL, 2.47 mmol) dropwise over 10 min. The resulting mixture was

warmed to rt for 1.5 h, and then the volatiles were evaporated in vacuo.

The residue was triturated with MeOH (8.0 mL) to give the title

compound (54a) as a yellow solid (0.60 g, 82%). LC-MS: m/z 339

(M + H)⁺(ES⁺). ¹H NMR: δ 3.89 (3H, s), 5.65 (2H, s), 7.21 (1H, d),

7.75 (1H, m), 7.83−7.88 (2H, overlapping m), 8.22 (1H, d), 8.44−8.48

(2H, overlapping m), 8.59 (1H, d), 8.77 (1H, d).

Methyl 4-(((4-Aminonaphthalen-1-yl)oxy)methyl)picolinate

(56a). A solution of methyl 4-(((4-nitronaphthalen-1-yl)oxy)methyl)-

picolinate (54a) (796 mg, 2.35 mmol) in a mixture of MeOH (150 mL)

and DCM (150 mL) was passed through a ThalesNano H-cube (10%

Pt/C, 30 mm, CatCart, 1.0 mL min⁻¹, at rt in full hydrogen mode).

The volatiles were evaporated in vacuo to give the title compound

(56a) as a purple solid (710 mg, 88%, 90% purity). LC-MS: m/z 309

(M + H)⁺(ES⁺).

7.67 (1H, td), 7.71 (1H, dd), 7.83 (1H, m), 8.16 (1H, s), 8.25 (1H, d),

8.45 (1H, d), 8.58 (1H, d), 8.67 (1H, dd).

Methyl 4-(2-((4-Aminonaphthalen-1-yl)oxy)ethyl)picolinate

(47b). A solution of methyl 4-(2-((4-nitronaphthalen-1-yl)oxy)ethyl)-

picolinate (46b) (1.2 g, 3.4 mmol) in a mixture of MeOH (60 mL) and

DCM (20 mL) was passed through a ThalesNano H-cube (10% Pt/C

30 mm CatCart, 1.0 mL min⁻¹, at rt, in full hydrogen mode). Evaporation

Methyl 4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl)picolinate (57). A solution

of 3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-amine⁴¹(34) (713 mg, 3.11 mmol)

in DCM (3.0 mL) was added dropwise over 1 h to a suspension of

CDI (504 mg, 3.11 mmol) in DCM (3.0 mL). The mixture was kept at rt

M

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for 2 h and was then treated dropwise with a solution of methyl

4-(((4-aminonaphthalen-1-yl)oxy)methyl)picolinate (56a) (366 mg,

1.41 mmol) in DCM (4.0 mL). After 30 min at rt, MeOH (20 mL)

was added to the reaction mixture, which gave a precipitate. The mixture

was reduced in volume to ca. 20 mL by evaporation in vacuo, and the

precipitate was collected by ﬁltration and washedwith MeOH and diethyl

ether to give the title compound (57) as a beige solid (0.92 g, 71%,

91% purity). LC-MS: m/z 564 (M + H)⁺(ES⁺). ¹H NMR: δ 1.27 (9H, s),

2.39 (3H, s), 3.89 (3H, s), 5.47 (2H, s), 6.35 (1H, s), 7.01 (1H, d), 7.35

(2H, m), 7.43 (2H, m), 7.56−7.64 (3H, overlapping m), 7.81 (1H, dd),

7.93 (1H, m), 8.20 (1H, m), 8.31 (1H, m), 8.58 (1H, bs), 8.75 (1H, d),

8.79 (1H, bs).

and saturated aq. NaHCO₃(40 mL). The organic phase was separated

and concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (SiO₂, 80 g, MeOH in DCM, 0−10%, gradient elution).

The crude product so obtained was triturated with a mixture of diethyl

ether (20 mL) and isohexane (20 mL) to aﬀord the title compound (36)

as a light purple solid (1.07 g, 42%). LC-MS: m/z 597, 599 (M + H)⁺

(ES⁺). ¹H NMR: δ 1.25 (9H, s), 2.38 (3H, s), 4.35 (2H, s), 5.38 (2H, s),

6.35 (1H, s), 7.00 (1H, d), 7.29 (1H, dd), 7.35 (2H, m), 7.43 (2H, m),

7.55−7.63 (3H, overlapping m), 7.92 (1H, m), 8.28−8.34 (2H,

overlapping m), 8.36 (1H, dd), 8.57 (1H, bs), 8.78 (1H, bs), 10.85

(1H, bs).

2-Methoxy-N-(4-(((4-nitronaphthalen-1-yl)oxy)methyl)-

pyridin-2-yl)acetamide (55a). To a suspension of 2-amino-4-((4-

nitronaphthalen-1-yloxy)methyl)pyridine (54b) (20.0 g, 68.0 mmol) in

a mixture of DCM (100 mL) and THF (150 mL) at −5 °C was added

DIPEA (17.7 mL, 102 mmol) followed by methoxyacetyl chloride

(8.1 mL, 88 mmol) dropwise over 10 min. After 10 min, the reaction

mixture was warmed to rt for an additional 30 min and was then treated

with methanolic NH₃(7 M, 30 mL). After 1 h, the volatiles were

removed by evaporation in vacuo, and the residue was triturated with

water (500 mL). The solids were collected by ﬁltration and washed with

water (300 mL) and diethyl ether (500 mL) to give the title compound

(55a) as a yellow solid (24.0 g, 95%). LC-MS: m/z 368 (M + H)⁺(ES⁺).

¹H NMR: δ 3.37 (3H, s), 4.07 (2H, s), 5.57 (2H, s), 7.21 (1H, s), 7.30

4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)methyl)picolinic Acid (39). A suspension of

methyl 4-(((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)methyl)picolinate (57) (100 mg, 0.16 mmol) in

a mixture of THF (3.0 mL) and water (2.0 mL) was treated with lithium

hydroxide (17 mg, 0.71 mmol), and the mixture was maintained at rt for

3 h. Acetic acid (3.0 mL) was added, and the mixture was puriﬁed by

SCX capture and release. The resultant solid was triturated with diethyl

ether to aﬀord the title compound (39) as a white solid (55 mg, 62%).

1

LC-MS: m/z 550 (M + H)⁺(ES⁺). H NMR: δ 1.27 (9H, s), 2.35

(3H, s), 5.40 (3H, s), 6.31 (1H, s), 6.99 (1H, d), 7.31 (2H, m), 7.45

(2H, m), 7.53−7.60 (3H, overlapping m), 7.95 (1H, m), 8.05 (1H, s),

8.27 (1H, m), 8.55 (1H, d), 9.06 (1H, bs), 9.18 (1H, bs).

(2H, dd), 7.75 (1H, m), 7.85 (1H, m), 8.32 (1H, s), 8.36 (1H, d), 8.44−

8.47 (2H, overlapping m), 8.59 (1H, d), 10.10 (1H, bs).

2-Amino-4-((4-nitronaphthalen-1-yloxy)methyl)pyridine

(54b). To a solution of 4-nitronaphthol (5.17 g, 27.3 mmol), PPh₃

(10.8 g, 41.0 mmol), and 2-aminopyridine-4-methanol (53) (5.09 g,

41.0 mmol) in THF (50 mL) at −15 °C was added DIAD (8.07 mL,

41.0 mmol) dropwise. The reaction mixture was warmed to rt for 18 h

and was then evaporated in vacuo. The residue was triturated with

EtOAc (150 mL), and the solids were collected by ﬁltration and washed

with EtOAc (100 mL). A second trituration with MeOH (100 mL) gave

the title compound (54b) (4.54 g, 56%) as a yellow solid. LC-MS: m/z

296 (M + H)⁺(ES⁺). ¹H NMR: δ 5.35 (2H, s), 6.00 (2H, bs), 6.60 (2H,

overlapping m), 7.15 (1H, d), 7.72 (1H, m), 7.85 (1H, m), 7.97 (1H,

dd), 8.42 (2H, d), 8.60 (1H, d).

2-Methoxy-N-methyl-N-(4-(((4-nitronaphthalen-1-yl)oxy)-

methyl)pyridin-2-yl)acetamide (55b). To a solution of 2-methoxy-

N-(4-(((4-nitronaphthalen-1-yl)oxy)methyl)pyridin-2-yl)acetamide

(55a) (300 mg, 0.82 mmol) in THF at 0 °C was added sodium hydride

(74 mg, 60 wt % dispersion in mineral oil, 1.20 mmol). After 30 min at rt,

the mixture was treated with iodomethane (76 μL, 1.22 mmol), kept at rt

for 1 h, and then heated at 50 °C. An additional aliquot of iodomethane

(380 μL, 6.10 mmol) was added. After 1 h at 50 °C, the reaction mixture

was cooled to rt and was poured into water. The mixture was extracted

with EtOAc. The organic layer was separated and evaporated in vacuo.

The residue was puriﬁed by ﬂash column chromatography (SiO₂, 40 g,

EtOAc in isohexane, 0−100%, gradient elution then MeOH in EtOAc,

0−10%, gradient elution) to furnish the title compound (55b) as a

2-Amino-4-((4-aminonaphthalen-1-yloxy)methyl)pyridine

(56b). A solution of 2-amino-4-((4-nitronaphthalen-1-yloxy)methyl)-

pyridine (54b) (4.50 g, 15.24 mmol) in a mixture of MeOH (200 mL)

and AcOH (200 mL) was passed through a ThalesNano H-cube

(2.0 mL min⁻¹, 40 °C, 55 mm, 10% Pt/C Cat-Cart, full hydrogen

mode). The volatiles were removed in vacuo, and the crude product was

subjected to SCX capture and release to give the title compound (56b)

as a purple solid (3.82 g, 94%). LC-MS: m/z 266 (M + H)⁺(ES⁺).

1-(4-((2-Aminopyridin-4-yl)methoxy)naphthalen-1-yl)-3-(3-

tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (37). To a solution of

CDI (4.18 g, 25.8 mmol) in DCM (15 mL) was added a solution of

3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine⁴¹(34) (5.91 g, 25.8 mmol) in

DCM (15 mL) dropwise over 40 min. The resulting solution was stirred

at rt for 1 h and was then added dropwise to a solution of 2-amino-4-((4-

aminonaphthalen-1-yloxy)methyl)pyridine (56b) (3.80 g, 12.9 mmol)

in DCM (25 mL). The mixture was maintained at rt for 18 h. The

volatiles were removed in vacuo, and the crude product was puriﬁed by

ﬂash column chromatography (SiO₂, 120 g, MeOH in EtOAc, 0−6%,

gradient elution) to give the title compound (37) as an oﬀ-white solid

1

yellow solid (155 mg, 49%). LC-MS: m/z 382 (M + H)⁺(ES⁺). H

NMR (CDCl₃) δ: 3.40 (3H, s), 3.45 (3H, s), 4.24 (2H, s), 5.40 (2H, s),

6.85 (2H, d), 7.32 (1H, dd), 7.58 (1H, bs), 7.67 (1H, m), 7.80 (1H, m),

8.37 (1H, d), 8.46 (1H, dq), 8.51 (1H, dd), 8.78 (1H, d).

N-(4-(((4-Aminonaphthalen-1-yl)oxy)methyl)pyridin-2-yl)-2-

methoxy-N-methylacetamide (35). The title compound was

obtained in a similar manner as that for 56b to provide 37 as a purple

solid (130 mg, 90%). LC-MS: m/z 352 (M + H)⁺(ES⁺).

1-(2-Aminopyridin-4-yl)ethanone (58). To a stirred solution of

methyl-2-aminopyridine-4-carboxylate (1.00 g, 6.57 mmol) in THF

(100 mL) at −78 °C under nitrogen was added methyllithium (1.6 M in

diethyl ether, 16.4 mL, 26.3 mmol) over 10 min. After a further 30 min at

−78 °C, the viscous reaction mixture was warmed to 0 °C. After a further

3 h, the reaction was quenched at 0 °C by the cautious addition of

isopropanol (8.0 mL). The mixture was warmed to rt. Brine (200 mL)

and EtOAc (150 mL) were added, and the layers were separated. The

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

organic extracts were dried, and the solvents were removed in vacuo.

The crude residue was puriﬁed by column chromatography (SiO₂, 80 g,

MeOH in EtOAc, 0−8%, gradient elution) to give 1-(2-aminopyridin-4-

yl)ethanone (58) as a yellow powder (176 mg, 20%). LC-MS: m/z 137

(M + H)⁺(ES⁺). ¹H NMR (MeOH-d₄): δ 2.54 (3H, s), 7.01 (1H, dd),

7.03 (1H, m), 8.03 (1H, dd).

2-(2-Aminopyridin-4-yl)propan-2-ol (59a). To a stirred solution

of methyl-2-aminopyridine-4-carboxylate (2.00 g, 13.0 mmol) in THF

(200 mL) at −78 °C under nitrogen was added methyllithium (1.6 M in

diethyl ether, 33 mL, 53.0 mmol) over 10 min. After 30 min, the viscous

reaction mixture was warmed to 0 °C for 3.5 h and was quenched at 0 °C

by the cautious addition of isopropanol (15 mL). The mixture was

warmed to rt, and brine (400 mL) and EtOAc (300 mL) were added.

The aqueous layer was separated and extracted with EtOAc (3 × 200 mL).

1

(4.27 g, 63%). LC-MS: m/z 521 (M + H)⁺(ES⁺). H NMR: δ 1.24

(9H, s), 2.38 (3H, s), 5.17 (2H, s), 5.95 (2H, bs), 6.35 (1H, s), 6.58−

6.63 (2H, overlapping), 6.97 (1H, d), 7.35 (2H, m), 7.43 (2H, m),

7.54−7.63 (3H, overlapping m), 7.88−7.93 (2H, overlapping m),

8.30 (1H, dd), 8.57 (1H, bs), 8.77 (1H, bs).

N-(4-((4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yloxy)methyl) pyridin-2-yl)-2-chloroacetamide

(36). To a solution of 1-(4-((2-aminopyridin-4-yl)methoxy)naphthalen-

1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (37) (2.00 g, 3.84 mmol)

in a mixture of DCM (40 mL) and DMF (8.0 mL) were added DIPEA

(1.37 mL, 7.68 mmol) and chloroacetyl chloride (0.61 mL, 7.70 mmol).

The reaction mixture was maintained at rt for 1 h and was then treated

with a second aliquot of chloracetyl chloride (100 μL, 1.25 mmol). After

1 h at rt, the resulting mixture was partitioned between DCM (40 mL)

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The combined organic extracts were evaporated in vacuo. The crude

residue was puriﬁed by ﬂash column chromatography (SiO₂, 120 g,

MeOH in EtOAc, 0−10%, gradient elution) to aﬀord the title compound

(59a) (1.27 g, 63%) as a yellow amorphous solid. ¹H NMR (MeOH-d₄):

δ 1.47 (6H, s), 6.68−6.71 (2H, overlapping m), 7.80−7.81 (1H, dd).

4-(2-(4-Nitronaphthalen-1-yloxy)propan-2-yl)pyridin-2-

amine (60a). To a stirred solution of 2-(2-aminopyridin-4-yl)propan-

2-ol (59a) (1.55 g, 10.0 mmol) in DMF (30 mL) under nitrogen at 0 °C

was added sodium hydride (60 wt %, 0.61 g, 15.0 mmol). The resulting

mixture was stirred at 0 °C for 5 min. A solution of 1-ﬂuoro-4-

nitronaphthalene (1.95 g, 10.0 mmol) in DMF (30 mL) was added

dropwise, and the resulting dark-red mixture was stirred at 0 °C for a

further 5 min and then at rt for 2 h. The reaction mixture was quenched

by the addition of a saturated aq. NH₄Cl solution (10 mL). Water

(150 mL) and EtOAc (150 mL) were added, and the aqueous layer

was separated and extracted with EtOAc (3 × 100 mL). The combined

organic extracts were washed with brine, dried, and evaporated in vacuo.

The crude residue was puriﬁed by column chromatography (SiO₂,

80 g, EtOAc in isohexane, 0−60%, gradient elution) to aﬀord the title

compound (60a) (282 mg, 8%) as a red oil. LC-MS m/z 324 (M + H)⁺

aqueous layer was separated and extracted with EtOAc (3 × 15 mL).

The combined organic extracts were washed with brine and dried, and

the solvents were removed in vacuo. The crude material was puriﬁed

by column chromatography (SiO₂, 12 g, EtOAc in isohexane, 0−80%,

gradient elution) to give 4-(1-(4-nitronaphthalen-1-yloxy)ethyl)-

pyridin-2-amine (60b) (94.6 mg, 57%) as an orange gum. LC-MS:

m/z 310 (M + H)⁺(ES⁺). ¹H NMR: δ 1.69 (3H, d), 5.75 (1H, q), 5.93

(2H, bs), 6.45 (1H, s), 6.55 (1H, dd), 6.93 (1H, d), 7.75 (1H, m), 7.84−

7.89 (2H, overlapping m), 8.35 (1H, d), 8.50 (1H, dd), 8.58 (1H, d).

4-(1-(4-Aminonaphthalen-1-yloxy)ethyl)pyridin-2-amine

(61b). 4-(1-(4-Nitronaphthalen-1-yloxy)ethyl)pyridin-2-amine (60b)

(91 mg, 0.29 mmol) in MeOH (15 mL) and AcOH (3.0 mL) was passed

through a ThalesNano H-cube (1.0 mL min⁻¹, 30 °C, 55 mm 10% Pt/C

Cat-Cart, full hydrogen mode). The volatiles were removed in vacuo,

and the residue was subjected to SCX capture and release to give

4-(1-(4-aminonaphthalen-1-yloxy)ethyl)pyridin-2-amine (61b) (81 mg,

99%) as a purple oil. LC-MS: m/z 280 (M + H)⁺(ES⁺).

1-(4-(1-(2-Aminopyridin-4-yl)ethoxy)naphthalen-1-yl)-3-(3-

tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (41). The title com-

pound was prepared in a similar manner as that for 40 from 4-(1-(4-

aminonaphthalen-1-yloxy)ethyl)pyridin-2-amine (61b) to give 41 as

a beige powder (63 mg, 38%). LC-MS: m/z 535 (M + H)⁺(ES⁺).

¹H NMR: δ 1.24 (9H, s), 1.63 (3H, d), 2.37 (3H, s), 5.50 (1H, q),

5.80 (2H, bs), 6.31 (1H, s), 6.48 (1H, s), 6.55 (1H, dd), 6.75 (1H, d),

7.34 (2H, m), 7.42 (2H, m), 7.47 (1H, d), 7.55 (1H, m), 7.82 (1H, dd),

7.87 (1H, m), 8.32 (1H, m), 8.60 (1H, bs), 8.76 (1H, bs).

1-(4-(2-(2-Aminopyridin-4-yl)ethoxy)naphthalen-1-yl)-3-(3-

tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (9). To a suspension of

CDI (3.00 g, 18.2 mmol) in DCM (15 mL) was added a solution of

3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine⁴¹(34) (4.17 g, 18.2 mmol) in

DCM (40 mL) over 1.5 h. After 2 h at rt, a solution of tert-butyl 4-(2-(4-

aminonaphthalen-1-yloxy)ethyl)pyridin-2-ylcarbamate (48a) (3.00 g,

7.91 mmol) in DCM (15 mL) was added. After 18 h, the solution

was diluted with MeOH (10 mL), evaporated onto silica gel (30 g), and

subjected to column chromatography (SiO₂, 330 g, EtOAc in isohexane,

30−100%, gradient elution then MeOH in EtOAc, 0−6%, gradient

elution) to give tert-butyl (4-(2-((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-

pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)ethyl)pyridin-2-yl)carbamate

as a beige solid (4.2 g, 80%).

1

(ES⁺). H NMR: δ 1.52 (6H, s), 5.92 (2H, br s), 6.45 (1H, s), 6.55

(1H, dd), 6.93 (1H, d), 7.75 (1H, m), 7.84−7.90 (2H, overlapping m),

8.35 (1H, d), 8.49 (1H, dd), 8.58 (1H, d).

4-(2-(4-Aminonaphthalen-1-yloxy)propan-2-yl)pyridin-2-

amine (61a). A solution of 4-(2-(4-nitronaphthalen-1-yloxy)propan-2-

yl)pyridin-2-amine (60a) (282 mg, 0.87 mmol) in MeOH (45 mL) was

passed through a ThalesNano H-cube (1.0 mL min⁻¹, 30 °C, 55 mm

10% Pt/C Cat-Cart, full hydrogen mode). The reaction mixture was

evaporated in vacuo to aﬀord the title compound (61a) (253 mg, 89%)

as a brown foam. LC-MS m/z 294 (M + H)⁺(ES⁺).

1-(4-(2-(2-Aminopyridin-4-yl)propan-2-yloxy)naphthalen-1-

yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (40). A solution

of 3-(tert-butyl)-5-isocyanato-1-(p-tolyl)-1H-pyrazole (see compound

10) (447 mg, 1.75 mmol) in THF (15 mL) was added to a solution of

4-(2-(4-aminonaphthalen-1-yloxy)propan-2-yl)pyridin-2-amine (61a)

(253 mg, 0.86 mmol) in THF (2.0 mL). DIPEA (451 μL, 2.59 mmol)

was added, and the reaction mixture was stirred at rt for 2 h. Water

(30 mL) and EtOAc (20 mL) were added, and the aqueous layer

was separated and extracted with EtOAc (3 × 15 mL). The combined

organic layers were washed with brine (40 mL) and evaporated in vacuo.

The residue was puriﬁed by ﬂash column chromatography (SiO₂, 40 g,

MeOH in DCM, 0−10%, gradient elution) to aﬀord the title compound

(40) (249 mg, 51%) as a purple amorphous solid. LC-MS: m/z 549

(M + H)⁺(ES⁺). ¹H NMR: δ 1.27 (9H, s), 1.37 (6H, s), 2.38 (3H, s),

6.37 (1H, s), 6.97 (1H, dd), 7.36 (2H, m), 7.45 (2H, m), 7.47−7.55

(2H, overlapping m), 7.72 (2H, m), 7.92 (1H, d), 7.97 (1H, dd), 8.12

(1H, dd), 8.66−8.71 (2H, overlapping m), 8.93 (1H, bs).

1-(2-Aminopyridin-4-yl)ethanol (59b). To a mixture of 1-(2-

aminopyridin-4-yl)ethanone (58) (168 mg, 1.23 mmol) in MeOH

(10 mL) under nitrogen at 0 °C was added sodium borohydride (47 mg,

1.23 mmol). The resulting reaction mixture was stirred at rt for 2 h, and

then the solvents were removed in vacuo. The residue was taken up into

EtOAc (25 mL) and washed with a saturated aq. NaHCO₃solution

(30 mL), and the layers were separated. The aqueous layer was extracted

with EtOAc (2 × 20 mL). The combined organic extracts were washed

with brine (30 mL) and dried, and the solvents were removed in vacuo

to give 1-(2-aminopyridin-4-yl)ethanol (59b) (77 mg, 45%) as a yellow

oil. LC-MS: m/z 139 (M + H)⁺(ES⁺). ¹H NMR: δ 1.23 (3H, d), 4.50

(1H, m), 5.13 (1H, d), 5.75 (2H, br s), 6.40−6.43 (2H, overlapping m),

7.78 (1H, d).

4-(1-(4-Nitronaphthalen-1-yloxy)ethyl)pyridin-2-amine

(60b). To a stirred solution of 1-(2-aminopyridin-4-yl)ethanol (59b)

(73 mg, 0.53 mmol) in DMF (1.5 mL) under nitrogen at 0 °C was added

sodium hydride (32 mg, 0.79 mmol, 60 wt % in mineral oil). The

resulting mixture was stirred at 0 °C for 40 min before a solution of

1-ﬂuoro-4-nitronaphthalene (101 mg, 0.53 mmol) in DMF (1.5 mL)

was added dropwise. The resulting dark-red mixture was stirred at 0 °C

for a further 5 min and warmed to rt. After a further 40 min, the reaction

was quenched by the addition of a saturated aq. NH₄Cl solution

(1.0 mL). Water (20 mL) and EtOAc (20 mL) were added, and the

A portion of the above material (1.35 g, 2.20 mmol) was suspended in

DCM (10 mL) and was treated with TFA (10 mL). The reaction

mixture was kept at rt for 2 h and was then evaporated in vacuo. The

residue was taken up in EtOAc (50 mL), washed with saturated aq.

NaHCO₃(50 mL) and brine (50 mL), and then dried and evaporated

in vacuo to give the title compound (9) as a pale pink solid (1.20 g,

1

80% over two steps). LC-MS: m/z 535 (M + H)⁺(ES⁺). H NMR:

δ 1.26 (9H, s), 2.37 (3H, s), 3.00 (2H, t), 4.32 (2H, t), 5.80 (2H, s),

6.34 (1H, s), 6.45 (1H, s), 6.52 (1H, dd), 6.95 (1H, d), 7.32−7.36 (2H,

overlapping m), 7.40−7.45 (2H, overlapping m), 7.50−7.55 (2H,

overlapping m), 7.60 (1H, d), 7.81 (1H, dd), 7.87 (1H, m), 8.11 (1H,

dd), 8.54 (1H, s), 8.74 (1H, s).

1-(4-(2-(3-Aminopyridin-4-yl)ethoxy)naphthalen-1-yl)-3-(3-

(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (10). To a solution of

3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine⁴¹(34) (1.00 g, 4.36 mmol) in

DCM (90 mL) was added a saturated aq. NaHCO₃solution (60 mL).

The heterogeneous mixture was stirred vigorously at 0 °C, treated with

diphosgene (2.10 mL, 17.4 mmol) in a single portion, and then warmed

to rt for 1 h. The organic layer was separated, dried, and evaporated in

vacuo to give a brown oil, which was triturated from isohexane (5.0 mL)

and ﬁltered. The ﬁltrate was concentrated in vacuo to give 3-tert-butyl-5-

isocyanato-1-p-tolyl-1H-pyrazole as a light brown oil (1.00 g, 3.92 mmol,

90%). LC-MS: m/z 288 (in MeOH) (M + H+MeOH)⁺(ES⁺).

A solution of 3-tert-butyl-5-isocyanato-1-p-tolyl-1H-pyrazole (530 mg,

2.10 mmol) in THF (2.0 mL) was added to a solution of 4-(2-(4-

aminonaphthalen-1-yloxy)ethyl)pyridin-3-amine (47c) (580 mg, 2.10 mmol)

and DIPEA (1.1 mL, 6.20 mmol) in a mixture of THF (10 mL) and

MeCN (1.0 mL). The reaction mixture was stirred at rt for 18 h, poured

into brine (25 mL), and extracted with EtOAc (2 × 25 mL). The

combined organics were dried, and the volatiles were removed in vacuo.

The residue was taken up in MeOH, evaporated onto Hyﬂo Super Cel

O

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Journal of Medicinal Chemistry

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(10 g), and puriﬁed by reverse-phase ﬂash column chromatography (C18

reverse-phase silica gel, 40 g, MeCN in H₂O, 0−100%, gradient elution)

to aﬀord the title compound (10) as an oﬀ-white solid (410 mg, 36%).

LC-MS: m/z 535 (M + H)⁺(ES⁺). ¹H NMR: δ 1.26 (9H, s), 2.38 (3H, s),

3.06 (2H, t), 4.34 (2H, t), 5.25 (2H, bs), 6.34 (1H, s), 6.95 (1H, d), 7.07

(1H, d), 7.35 (2H, m), 7.42 (2H, m), 7.45−7.57 (2H, overlapping m),

7.60 (1H, d), 7.71 (1H, dd), 7.87 (2H, d), 7.95 (1H, s), 8.15 (1H, dd),

8.55 (1H, bs), 8.75 (1H, bs).

N-(4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)ethyl)pyridin-2-yl)acetamide (11).

The title compound was prepared using the process described to make

14 by replacing methoxyacetyl chloride with acetyl chloride to give 11

as a white solid (41 mg, 77%). LC-MS: m/z 577 (M + H)⁺(ES⁺). ¹H

NMR: δ 1.26 (9H, s), 2.08 (3H, m), 2.38 (3H, s), 3.20 (2H, t), 4.37 (2H, t),

6.34 (1H, s), 6.95 (1H, d), 7.15 (1H, dd), 7.35 (2H, m), 7.40−7.50

(3H, overlapping m), 7.55 (1H, m), 7.60 (1H, m), 7.90 (1H, d), 8.15−8.23

(3H, overlapping m), 8.57 (1H, s), 8.77 (1H, bs), 10.40 (1H, s).

N-(4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)ethyl)pyridin-3-yl)acetamide (12).

The title compound was prepared in the same manner as that for 14

from 1-(4-(2-(3-aminopyridin-4-yl)ethoxy)naphthalen-1-yl)-3-(3-(tert-

butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (10) as the amine substrate by

reaction with acetyl chloride to provide 12 as a white solid (20 mg, 46%).

LC-MS: m/z 577 (M + H)⁺(ES⁺). ¹H NMR: δ 1.24 (9H, s), 2.10 (3H,

s), 2.38 (3H, s), 3.20 (2H, t), 4.38 (2H, t), 6.34 (1H, s), 6.95 (1H, d),

7.35 (2H, m), 7.42 (2H, m), 7.45−7.50 (2H, overlapping m), 7.55

(1H, m), 7.61 (1H, d), 7.87 (1H, d), 8.07 (1H, dd), 8.31 (1H, d), 8.52−

8.56 (2H, overlapping m), 8.75 (1H, bs).

4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)ethyl)-N-(2-methoxyethyl)picolinamide

(16). The title compound was prepared by a similar procedure as that

used for 15 by substituting methylamine with 2-methoxyethanamine to

give 16 (58 mg, 53%). LC-MS: m/z 621 (M + H)⁺(ES⁺). ¹H NMR: δ

1.25 (9H, s), 2.38 (3H, s), 3.25 (3H, s), 3.30 (2H, obscured by DOH),

3.45−3.48 (4H, overlapping m), 4.42 (2H, t), 6.33 (1H, s), 6.97 (1H, d),

7.33 (2H, m), 7.42 (2H, m), 7.45 (1H, m), 7.53 (1H, m), 7.60 (1H, d),

7.65 (1H, dd), 7.86 (1H, d), 8.05 (1H, dd), 8.12 (1H, d), 8.52−8.58

(2H, overlapping m), 8.65 (1H, m), 8.75 (1H, bs).

N-(1-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)ethyl)-1H-imidazol-2-yl)-

acetamide (17). To a suspension of 1-(3-(tert-butyl)-1-(p-tolyl)-1H-

pyrazol-5-yl)-3-(4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)naphthalen-1-

yl)urea (51) (80 mg, 0.15 mmol) in a mixture of EtOH and AcOH

(2:1v/v, 3.0 mL) was added iron mesh (48 mg, 0.87 mmol). The mixture

was heated at 60 °C for 1 h, cooled to rt, and adjusted to pH 9 by

the addition of a saturated aq. Na₂CO₃solution, which resulted in the

formation of a thick suspension. The volatiles were evaporated in vacuo,

and the residue was subjected to SCX capture and release to aﬀord a

crude sample of 1-(4-(2-(2-amino-1H-imidazol-1-yl)ethoxy)naphthalen-

1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (80 mg). A portion

of this material (53 mg, ∼0.10 mmol) in DCM (2.0 mL) was treated with

DIPEA (35 μL, 0.20 mmol) and acetyl chloride (8 μL, 0.11 mmol). After

18 h at rt, the mixture was diluted with a saturated aq. NaHCO₃solution

(1.0 mL) and the layers were separated. The organic layer was evaporated

in vacuo, and the residue was puriﬁed by ﬂash column chromatography

(SiO₂, 12 g, MeOH in EtOAc, 0−5%, gradient elution). The material so

obtained was triturated from a mixture of diethyl ether and isohexane and

was then subjected to SCX capture and release to give the title compound

(17) as pale yellow solid (7 mg, 9%, 87% purity). LC-MS: m/z 566

(M + H)⁺(ES⁺). ¹H NMR: δ 1.24 (9H, s), 2.05 (3H, s), 2.37 (3H, s), 4.25

(2H, m), 4.35 (2H, t), 6.35 (1H, s), 6.79 (1H, s), 6.90 (1H, d), 7.29−7.39

(3H, overlapping m), 7.42 (2H, m), 7.50−7.60 (2H, overlapping m),

7.61 (1H, d), 7.88 (1H, d), 8.15 (1H, d), 8.55 (1H, bs), 8.76 (1H, bs),

10.05 (1H, bs).

4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)methyl)-N-(2-methoxyethyl)picolinamide

(18). A mixture of 4-(((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-

yl)ureido)naphthalen-1-yl)oxy) ethyl)picolinic acid (39) (35 mg,

0.06 mmol), 2-methoxyethylamine (6.0 μL, 0.07 mmol), DMAP (7.8 mg,

0.06 mmol), and EDC·HCl (16 mg, 0.08 mmol) in a mixture of DCM

(1.0 mL) and DMF (1.0 mL) was kept at rt for 72 h. The neat reaction

mixture was puriﬁed by ﬂash column chromatography (SiO₂, 12 g,

MeOH in DCM, 0−20%, gradient elution). The crude product so

obtained was puriﬁed further by SCX capture and release to aﬀord the

title compound (18) as a light brown solid (7 mg, 17%). LC-MS: m/z

607 (M + H)⁺(ES⁺). ¹H NMR: δ 1.25 (9H, s), 2.37 (3H, s), 3.27 (3H, s),

3.47 (4H, overlapping m), 5.48 (2H, s), 6.35 (1H, s), 7.00 (1H, s), 7.35

(2H, m), 7.43 (2H, m), 7.57−7.63 (3H, overlapping m), 7.75 (1H, dd),

7.92 (1H, m), 8.18 (1H, d), 8.31 (1H, m), 8.58 (1H, bs), 8.67 (1H, d),

8.72 (1H, m), 8.78 (1H, bs).

N-(4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)ethyl) pyridin-3-yl)-2-methoxya-

cetamide (13). The title compound was prepared in the same manner

as that for 12 from 10 by reaction with methoxyacetyl chloride to pro-

vide 13 as a pale pink solid (45 mg, 77%). LC-MS: m/z 607 (M + H)⁺

(ES⁺). ¹H NMR: δ 1.25 (9H, s), 2.38 (3H, s), 3.16 (3H, s), 4.07 (2H, s),

4.37 (2H, t), 6.34 (1H, s), 6.95 (2H, s), 7.33 (2H, m), 7.43 (2H, m),

7.46−7.55 (3H, overlapping m), 7.60 (1H, d), 7.88 (1H, d), 8.07

(1H, dd), 8.35 (1H, d), 8.51 (1H, s), 8.55 (1H, bs), 8.75 (1H, bs),

9.65 (1H, bs).

N-(4-(2-(4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yloxy)ethyl)pyridin-2-yl)-2-methoxyacetamide

(14). To a suspension of 1-(4-(2-(2-aminopyridin-4-yl)ethoxy)-

naphthalen-1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (9)

(35 mg, 0.07 mmol) in DCM (0.5 mL) were added DIPEA (23 μL,

0.13 mmol) and methoxyacetyl chloride (7.0 μL, 0.07 mmol). The

mixture was maintained at rt for 2 h and was then diluted with saturated

aq. NaHCO₃(1.5 mL). The organic layer was collected and evaporated

in vacuo, and the residue was subjected to SCX capture and release.

The resulting crude product was puriﬁed by preparative RP HPLC to

give the title compound (14) as a white solid (5 mg, 13%). LC-MS: m/z

1

607 (M + H)⁺(ES⁺). H NMR: δ 1.26 (9H, s), 2.37 (3H, s), 3.20

(2H, t), 3.37 (3H, s), 4.06 (2H, s), 4.38 (2H, t), 6.33 (1H, s), 6.95

(1H, d), 7.19 (1H, dd), 7.33 (2H, m), 7.42−7.47 (3H, overlapping m),

7.54 (1H, m), 7.59 (1H, d), 7.87 (1H, d), 8.12 (1H, d), 8.18 (1H, bs),

8.23 (1H, d), 8.67 (1H, s), 8.84 (1H, s), 9.89 (1H, s).

4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)methyl)-N-(2-morpholinoethyl)-

picolinamide (19). The title compound was prepared in the same

manner as that described for 18 by replacing 2-methoxyethanamine with

2-morpholinoethanamine to furnish 19 as an oﬀ-white solid (10 mg,

4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)ethyl)-N-methylpicolinamide (15). To a

mixture of 4-(2-((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yl)oxy)ethyl)picolinic acid (38) (100 mg, 0.18 mmol),

HOBt (36 mg, 0.27 mmol), and EDC (51 mg, 0.27 mmol) in THF was

added methylamine (2 M in THF, 890 μL, 1.8 mmol). The reaction was

maintained at 45 °C for 2 h and then at rt for 18 h. The resulting mixture

was diluted with saturated aq. NaHCO₃and extracted with DCM. The

organic extracts were evaporated in vacuo, and the residue was puriﬁed

by ﬂash column chromatography (SiO₂, 12 g, MeOH in DCM, 0−5%,

gradient elution) to provide the title compound (16) (24 mg, 24%).

LC-MS: m/z 577 (M + H)⁺(ES⁺). ¹H NMR: δ 1.25 (9H, s), 2.37 (3H, s),

2.80 (3H, d), 3.30 (2H, obscured by DOH), 4.41 (2H, t), 6.32 (1H, s),

6.97 (1H, d), 7.32 (2H, m), 7.42 (2H, m), 7.46 (1H, m), 7.54 (1H, m),

7.60 (2H, d), 7.63 (1H, dd), 7.87 (1H, d), 8.05 (1H, dd), 8.10 (1H, d),

8.53−8.57 (2H, overlapping m), 8.70−8.76 (2H, overlapping m).

1

16%). LC-MS: m/z 662 (M + H)⁺(ES⁺). H NMR: δ 1.27 (9H, s),

2.35−2.45 (7H, overlapping s), 2.45 (2H, obscured by DOH), 3.45 (2H,

t), 3.55 (4H, t), 5.44 (2H, s), 6.35 (1H, s), 7.00 (1H, d), 7.35 (2H, m),

7.43 (2H, m), 7.58−7.62 (3H, overlapping, m), 7.75 (1H, dd), 7.93

(1H, m), 8.17 (1H, d), 8.31 (1H, m), 8.58 (1H, bs), 8.67 (1H, dd), 8.74

(1H, t), 8.79 (1H, bs).

Methyl (4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)ethyl)pyridin-2-yl)carbamate (20).

To a solution of 1-(4-((2-aminopyridin-4-yl)methoxy)naphthalen-1-yl)-

3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (37) (40 mg, 0.08 mmol)

in a mixture of DCM (0.9 mL) and DMF (0.1 mL) were added DIPEA

(15 μL, 0.09 mmol) and methyl chloroformate (6.5 μL, 0.09 mmol).

After 1 h, an additional aliquot of methyl chloroformate (6.5 μL,

0.09 mmol) was added. The mixture was maintained at rt for 1 h and was

P

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then evaporated in vacuo. The residue was triturated with methanol

(2.0 mL). The resulting solid was collected by ﬁltration and washed with

MeOH (1.0 mL) and diethyl ether (2.0 mL) to give the title compound

(20) as a beige powder (15 mg, 33%). LC-MS: m/z 579 (M + H)⁺(ES⁺).

¹H NMR: δ 1.25 (9H, s), 2.38 (3H, s), 3.67 (3H, s), 5.35 (2H, s), 6.35

(1H, s), 6.99 (1H, d), 7.19 (1H, dd), 7.35 (2H, m), 7.43 (2H, m), 7.56−

7.63 (2H, overlapping m), 7.92 (1H, m), 8.05 (1H, s), 8.26−8.32 (2H,

overlapping m), 8.58 (1H, bs), 8.79 (1H, bs), 10.25 (1H, bs).

8.25−8.29 (2H, overlapping m), 8.33 (1H, dd), 8.57 (1H, bs), 8.78

(1H, bs), 10.77 (1H, bs).

N-(4-((4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yloxy)methyl)yridin-2-yl)-2-(dimethylamino)-

acetamide (26). To a solution of N-(4-((4-(3-(3-tert-butyl-1-p-tolyl-

1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy) ethyl) pyridin-2-yl)-2-

chloroacetamide (36) (50 mg, 0.08 mmol) in a mixture of DCM

(1.0 mL), DMF (0.1 mL), and DIPEA (17 μL, 0.10 mmol) was added a

solution of dimethylamine (2.0 M in THF, 41 μL, 0.08 mmol). The

reaction mixture was heated at 40 °C for 12 h. The resulting mixture was

puriﬁed by ﬂash column chromatography (12 g SiO₂, MeOH in DCM,

0−10%, gradient elution). The product obtained was triturated with a

mixture of diethyl ether, DCM, and isohexane (2:1:2, 5.0 mL) to aﬀord

the title compound (26) as an orange solid (18 mg, 35%). LC-MS: m/z

N-(4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl)yridin-2-yl)-

methanesulfonamide (21). To a solution of 1-(4-((2-aminopyridin-

4-yl)methoxy)naphthalen-1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-

yl)urea (39) (100 mg, 0.19 mmol) in pyridine (1.0 mL) was added

methanesulfonyl chloride (59 μL, 0.76 mmol), and the resulting yellow

solution was kept at rt for 72 h. The mixture was treated with 1% NH₃

in MeOH (2.0 mL), and after 30 min, it was preabsorbed onto silica gel

and puriﬁed by ﬂash column chromatography (SiO₂, 12 g, MeOH in

DCM, 0−5%, gradient elution) to furnish the title compound (23) as

a light orange solid (49 mg, 50%). LC-MS: m/z 599 (M + H)⁺(ES⁺).

¹H NMR: δ 1.25 (9H, s), 2.39 (3H, s), 3.25 (3H, s), 5.32 (2H, s), 6.35

(1H, s), 7.00 (1H, d), 7.10 (1H, m), 7.21 (1H, m), 7.35 (2H, m), 7.43

(2H, m), 7.55−7.63 (3H, overlapping m), 7.92 (1H, dd), 8.21 (1H, m),

8.31 (1H, dd), 8.57 (1H, bs), 8.79 (1H, bs), 10.80 (1H, bs).

N-(4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl)yridin-2-yl)acetamide (22).

To a solution of 1-(4-((2-aminopyridin-4-yl)methoxy)naphthalen-1-yl)-

3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (37) (40 mg, 0.08 mmol)

and DIPEA (15 μL, 0.09 mmol) in a mixture of DCM (1.0 mL) and DMF

(0.1 mL) was added acetyl chloride (6 μL, 0.08 mmol). After 40 min at rt,

additional aliquots of acetyl chloride (4 μL, 0.05 mmol) and DIPEA

(10 μL, 0.06 mmol) were added. The resulting mixture was kept at rt for

an additional 30 min, treated with acetic acid (0.5 mL), and subjected to

SCX capture and release. The crude material so obtained was triturated

with DCM to aﬀord the title compound (22) as a pale orange solid

(30 mg, 69%). LC-MS: m/z 563 (M + H)⁺(ES⁺). ¹H NMR: δ 1.26 (9H,

s), 2.09 (3H, s), 2.35 (3H, s), 5.35 (2H, s), 6.35 (1H, s), 7.00 (1H, d),

7.22 (1H, m), 7.35 (2H, m), 7.42 (2H, m), 7.55−7.63 (3H, overlapping

m), 7.92 (1H, dd), 8.28−8.33 (2H, overlapping m), 8.57 (1H, bs), 8.78

(1H, bs), 10.54 (1H, bs).

1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((2-(3-

methylureido)pyridin-4-yl)ethoxy)naphthalen-1-yl)urea (23).

To a solution of 1-(4-((2-aminopyridin-4-yl)methoxy)naphthalen-1-yl)-

3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (37) (70 mg, 0.13 mmol)

in pyridine (1.5 mL) was added methyl isocyanate (14 μL, 0.24 mmol).

The reaction mixture was kept at rt for 72 h. The reaction mixture was

evaporated in vacuo, and the residue was triturated with DCM (3.0 mL)

to aﬀord the title compound (23) as an oﬀ-white powder (36 mg, 45%).

LC-MS: m/z 578 (M + H)⁺(ES⁺). ¹H NMR: δ 1.27 (9H, s), 2.39 (3H, s),

2.74 (3H, d), 5.30 (2H, s), 6.36 (1H, s), 6.99 (1H, d), 7.05 (1H, d), 7.35,

(2H, m), 7.44 (2H, m), 7.54−7.64 (4H, overlapping m), 7.93 (1H, d),

8.19 (1H, d), 8.23 (1H, bs), 8.35 (1H, d), 8.58 (1H, bs), 8.79 (1H, bs),

9.36 (1H, bs).

1

607 (M + H)⁺(ES⁺). H NMR: δ 1.27 (9H, s), 2.31 (6H, s), 2.39

(3H, s), 3.14 (2H, s), 5.39 (2H, s), 6.35 (1H, s), 7.01 (1H, d), 7.29

(1H, d), 7.35 (2H, d), 7.44 (2H, d), 7.65−7.55 (3H, overlapping m),

7.94 (1H, m), 8.38−8.28 (3H, overlapping m), 8.59 (1H, bs), 8.79

(1H, bs), 9.93 (1H, bs).

N-(4-((4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yloxy)methyl)yridin-2-yl)-2-morpholinoaceta-

mide (27). To a solution of N-(4-((4-(3-(3-tert-butyl-1-p-tolyl-

1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)ethyl)pyridin-2-yl)-2-

chloroacetamide (36) (50 mg, 0.08 mmol) in a mixture of DCM

(1.0 mL), DMF (0.1 mL), and DIPEA (21.9 μL, 0.13 mmol) was added

morpholine (11.0 μL, 0.13 mmol). The reaction mixture was kept at rt

for 3 h, heated at 40 °C for 12 h, and then treated with a second aliquot

of morpholine (11.0 μL, 0.13 mmol). After an additional 5 h at 40 °C,

the crude reaction mixture was puriﬁed by ﬂash column chromatography

(12 g, SiO₂, MeOH in DCM, 0−10%, gradient elution). The impure

product obtained was triturated with MeOH (5.0 mL), and the solid was

collected by ﬁltration to provide the title compound (27) as a light

yellow solid (11 mg, 20%). LC-MS: m/z 648 (M + H)⁺(ES⁺). ¹H NMR:

δ 1.27 (9H, s), 2.39 (3H, s), 2.54 (4H, m), 3.20 (2H, s), 3.63 (4H, m),

5.39 (2H, s), 6.35 (1H, s), 7.01 (1H, d), 7.28 (1H, d), 7.35 (2H, d),

7.43 (2H, d), 7.56−7.62 (3H, overlapping m), 7.92 (1H, d), 8.29−8.37

(3H, overlapping m), 8.58 (1H, bs), 8.90 (1H, bs), 10.01 (1H, bs).

N-(4-((4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yloxy)methyl)yridin-2-yl)-2-(methylthio)-

acetamide (28). To a stirred suspension of sodium thiomethoxide

(35 mg, 0.50 mmol) in MeOH (5.0 mL) at rt was added portionwise

N-(4-((4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-

yloxy)methyl)pyridin-2-yl)-2-chloroacetamide (36) (100 mg, 0.17 mmol).

After 1 h at rt, the resulting mixture was evaporated in vacuo and the

residue was partitioned between brine (20 mL) and DCM (30 mL).

The organic layer was separated and concentrated in vacuo. The residue

was puriﬁed by ﬂash column chromatography (SiO₂, 12 g, EtOAc in

isohexane, 10−100%, gradient elution) to give the title compound (28)

as a light yellow solid (28 mg, 26%). LC-MS: m/z 610 (M + H)⁺(ES⁺).

¹H NMR: δ 1.27 (9H, s), 2.16 (3H, s), 2.39 (3H, s), 3.53 (2H, s),

5.37 (2H, s), 6.35 (1H, s), 7.01 (1H, d), 7.26 (1H, dd), 7.35 (2H, m),

7.44 (2H, m), 7.55−7.64 (3H, m), 7.92 (1H, m), 8.30−8.35 (3H,

overlapping m), 8.58 (1H, s), 8.78 (1H, s), 10.60 (1H, s).

N-(4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl)yridin-2-yl)benzamide

(24). The title compound was prepared by the procedure described for

22 using benzoyl chloride in place of acetyl chloride to give 24 as a beige

powder (19 mg, 39%). LC-MS: m/z 625 (M + H)⁺(ES⁺). ¹H NMR: δ

1.25 (9H, s), 2.39 (3H, s), 5.41 (2H, s), 6.35 (1H, s), 7.04 (1H, d), 7.31−

7.37 (3H, overlapping m), 7.42 (2H, m), 7.50 (2H, m), 7.56−7.64 (4H,

overlapping m), 7.93 (1H, m), 8.02 (2H, m), 8.32 (1H, m), 8.40−8.45

(2H, overlapping m), 8.58 (1H, bs), 8.79 (1H, bs), 10.90 (1H, bs).

N-(4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl)yridin-2-yl)-2-phenylace-

tamide (25). The title compound was obtained in a similar manner as

that for 22 using phenylacetyl chloride in place of acetyl chloride to give

25 as a beige powder (7 mg, 13%). LC-MS: m/z 639 (M + H)⁺(ES⁺).

¹H NMR: δ 1.25 (9H, s), 2.37 (3H, s), 4.72 (2H, s), 5.34 (2H, s), 6.34

N-(4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl)yridin-2-yl)-2-

(methylsulfonyl)acetamide (29). To a suspension of methanesulfo-

nylacetic acid (40 mg, 0.29 mmol) and oxalyl chloride (29 μL, 0.34 mmol)

in DCM (1.0 mL) was added DMF (1 drop). The reaction mixture was

stirred at rt for 1 h. The resulting solution was added dropwise to a mixture

of 1-(4-((2-aminopyridin-4-yl)methoxy)naphthalen-1-yl)-3-(3-tert-butyl-

1-p-tolyl-1H-pyrazol-5-yl)urea (37) (50 mg, 0.10 mmol) and DIPEA

(167 μL, 1.00 mmol) in DCM/DMF (10:1 v/v, 1.1 mL). After 18 h, the

reaction mixture was treated with 1% NH₃in MeOH (2.0 mL) and was

then evaporated in vacuo. The residue was subjected to SCX capture and

release to aﬀord the title compound (29) as a pale pink solid (11 mg,

18%). LC-MS: m/z 641 (M + H)⁺(ES⁺). ¹H NMR: δ 1.27 (9H, s), 2.39

(3H, s), 3.17 (3H, s), 4.44 (2H, s), 5.40 (2H, s), 6.35 (1H, s), 7.02 (1H, d),

7.33 (1H, dd), 7.36 (2H, m), 7.44 (2H, m), 7.56−7.64 (3H, overlapping

m), 7.93 (1H, m), 8.30−8.33 (2H, overlapping m), 8.39 (1H, dd),

8.59 (1H, bs), 8.79 (1H, bs), 10.98 (1H, bs).

(1H, s), 6.97 (1H, d), 7.20−7.26 (2H, overlapping m), 7.27−7.37

(6H, overlapping m), 7.53−7.62 (3H, overlapping m), 7.92 (1H, m),

Q

DOI: 10.1021/acs.jmedchem.5b01029

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-(4-((4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)-

naphthalen-1-yloxy)methyl)yridin-2-yl)-2-methoxyacetamide

(30). To a solution of 1-(4-((2-aminopyridin-4-yl)methoxy)-

naphthalen-1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (37)

(526 mg, 0.96 mmol) and DIPEA (184 μL, 1.06 mmol) in a mixture

of DCM and DMF (10:1, 11 mL) was added methoxyacetyl chloride

(92 μL, 1.01 mmol). After 1 h at rt, additional aliquots of DIPEA

(184 μL, 1.06 mmol) and methoxyacetyl chloride (92 μL, 1.01 mmol)

were added sequentially. Stirring was continued for 1 h. A solution of 1%

NH₃in MeOH (40 mL) was added, and after 15 min, the mixture was

evaporated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (SiO₂, 40 g, MeOH in DCM, 0−6%, gradient elution)

to furnish the title compound (30) as a white solid (286 mg, 49%).

LC-MS: m/z 593 (M + H)⁺(ES⁺). ¹H NMR: δ 1.27 (9H, s), 2.39 (3H,

s), 3.32 (3H, s), 4.08 (2H, s), 5.39 (2H, s), 6.36 (1H, s), 7.03 (1H, d),

7.28 (1H, dd), 7.36 (2H, m), 7.44 (2H, m), 7.56−7.64 (3H, overlapping

m), 7.93 (1H, m), 8.30−8.35 (3H, overlapping m), 8.58 (1H, bs),

8.79 (1H, bs), 10.02 (1H, bs).

DEDICATION

■

This paper is dedicated to the memory of Fritz Frickel.

ABBREVIATIONS USED

■

AcOH, glacial acetic acid; BALF, bronchoalveolar lavage ﬂuid; b,

broad; CatCart, catalytic cartridge; CDI, 1,1-carbonyl-diimida-

zole; COPD, chronic obstructive pulmonary disease; CS,

cigarette smoke; Cs₂CO₃, cesium carbonate; CXCL8, interleukin

8; CYP, cytochrome P450; d-, diﬀerentiated; DFG, aspartic

acid−phenylalanine−glycine; DIAD, diisopropylazadicarboxy-

late; DIPEA, N,N-diisopropylethylamine; EDC, 1-(3-

(dimethylamino)propyl)-3-ethylcarbodiimide; EtOAc, ethyl ac-

etate; EtOH, ethanol; (ES⁺), electrospray ionization, positive

mode; FACS, ﬂuorescence-activated cell sorting; FBS, fetal

bovine sample; FEV1, forced expiratory volume in 1 s; FP,

ﬂuticasone propionate; FRET, ﬂuorescence resonance energy

transfer; GM-CSF, granulocyte-macrophage colony-stimulating

factor; H₂O₂, hydrogen peroxide; HCl, hydrochloric acid;

HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-

nium hexaﬂuorophosphate; HCK, hematopoietic kinase;

HOBt, 1-hydroxybenzotriazole; IBD, inﬂammatory bowel

disease; K₂CO₃, potassium carbonate; IL, interleukin; IT,

intratracheal; LiOH, lithium hydroxide; MAP, mitogen activated

protein; MAPKAP-K2, mitogen activated protein kinase-

activated protein kinase 2; MeCN, acetonitrile; MeI, methyl

iodide; MeLi, methyllithium; MeOH, methanol; (M + H)⁺,

protonated molecule; min, minute(s); MOMA2, monocyte/

macrophage marker antibody; m/z, mass-to-charge ratio;

MgSO₄, magnesium sulfate; NaBH₄, sodium borohydride;

NaCl, sodium chloride; NaHCO₃, sodium bicarbonate; NaOH,

sodium hydroxide; NH₃, ammonia; NH₄Cl, ammonium

chloride; NSKI, narrow spectrum kinase inhibitor; PBMC,

peripheral blood mononucleated cell; Pd₂(dba)₃, tris-

(dibenzylideneacetone)dipalladium(0); PMA, phorbol myristate

acetate; PPh₃, triphenylphosphine; RA, rheumatoid arthritis; RP,

reverse phase; rt, room temperature; SCX, strong cation

exchange (resin); SiO₂, silica; S_NAr, nucleophilic aromatic

substitution; TBDMSCl, tert-butyldimethylsilyl chloride; TMS-

CN, trimethylsilyl cyanide; Xantphos, 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene

N-(4-(((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)methyl) yridin-2-yl)-2-methoxy-N-

methylacetamide (31). The title compound was prepared in a similar

manner as that for 37 from N-(4-(((4-aminonaphthalen-1-yl)oxy)-

methyl)pyridin-2-yl)-2-methoxy-N-methylacetamide (35) to give 31

as a light beige solid (60 mg, 27%). LC-MS: m/z 607 (M + H)⁺(ES⁺).

¹H NMR: δ 1.27 (9H, s), 2.38 (3H, s), 3.20 (3H, s), 3.29 (3H, s), 4.13

(2H, s), 5.39 (2H, s), 6.34 (1H, s), 7.02 (1H, d), 7.35 (2H, m), 7.41−

7.45 (3H, overlapping m), 7.55−7.65 (3H, overlapping m), 7.70 (1H, s),

7.92 (1H, m), 8.31 (1H, m), 8.46 (1H, d), 8.57 (1H, bs), 8.78 (1H, bs).

N-(4-(1-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)ethyl)pyridin-2-yl)-2-methoxyace-

tamide (32). The title compound (32) was prepared in a similar

manner as that for 30 from 1-(4-(1-(2-aminopyridin-4-yl)ethoxy)-

naphthalen-1-yl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea

(41) as a beige solid (24 mg, 51%). LC-MS m/z 607 (M + H)⁺(ES⁺).

¹H NMR: δ 1.26 (9H, s), 1.67 (3H, d), 2.38 (3H, s), 4.03 (2H, s), 5.75

(1H, q), 6.32 (1H, s), 6.81 (1H, d), 7.22 (1H, dd), 7.35 (2H, m), 7.42

(2H, m), 7.48 (1H, s), 7.59 (2H, m), 7.88 (1H, m), 8.24 (1H, bs), 8.27

(1H, d), 8.36 (1H, m), 8.59 (1H, bs), 8.76 (1H, bs), 9.99 (1H, bs).

N-(4-(2-((4-(3-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-

ureido)naphthalen-1-yl)oxy)propan-2-yl)pyridin-2-yl)-2-me-

thoxyacetamide (33). The title compound (33) was prepared in a

similar manner as that for 30 from 1-(4-(2-(2-aminopyridin-4-yl)propan-

2-yloxy)naphthalen-1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea

(42) as a white solid (18 mg, 24%). LC-MS m/z 621 (M + H)⁺(ES⁺).

¹H NMR: δ 1.28 (9H, s), 1.39 (6H, s), 2.39 (3H, s), 3.22 (3H, s), 3.80

(1H, d), 4.16 (1H, d), 5.27 (1H, s), 6.41 (1H, s), 7.26 (1H, dd), 7.37 (2H,

m), 7.46 (2H, m), 7.56 (1H, d), 7.60 (2H, overlapping m), 7.76 (1H, br

s), 7.95 (1H, m), 8.01 (1H, d), 8.09 (1H, m), 8.22 (1H, d), 8.94 (1H, bs),

9.28 (1H, bs).

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AUTHOR INFORMATION

Corresponding Authors

*(S.T.O.) E-mail: s.onions@sygnaturediscovery.com. Phone:

+44 (0)1159 415401.

■

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^#Kazuhiro Ito, P. John Murray, Garth Rapeport, and Peter

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The authors declare the following competing ﬁnancial

interest(s): Stuart T. Onions, Richard J. Brown, Marie Colucci,

Kevin Joly, Andrew Novak, Anjna Rani, Alun Smith, David M.

Taddei, and Jonathan G. Williams were full-time employees of

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Discovery of Narrow Spectrum Kinase Inhibitors: New Therapeutic Agents for the Treatment of COPD and Steroid-Resistant Asthma

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