Identification of orally available naphthyridine protein kinase D inhibitors

Article abstract of DOI:10.1021/jm100075z

A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D (PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic mechanism with application as a heart failure therapy. As PKC was previously identified as the immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral administration. Their effects on heart morphology and function are discussed herein.

Full text of DOI:10.1021/jm100075z

5400 J. Med. Chem. 2010, 53, 5400–5421
DOI: 10.1021/jm100075z
Identification of Orally Available Naphthyridine Protein Kinase D Inhibitors
Erik L. Meredith,† Ophelia Ardayfio,† Kimberly Beattie,† Markus R. Dobler,† Istvan Enyedy,† Christoph Gaul,‡
Vinayak Hosagrahara,† Charles Jewell,† Keith Koch,§ Wendy Lee,† HansJoerg Lehmann,‡ Timothy A. McKinsey,§
Karl Miranda,† Nikos Pagratis,§ Margaret Pancost,† Anup Patnaik,† Dillon Phan,§ Craig Plato,§ Ming Qian,†
Vasumathy Rajaraman,† Chang Rao,† Olga Rozhitskaya,† Thomas Ruppen,‡ Jie Shi,† Sarah J. Siska,† Clayton Springer,†
Maurice van Eis,‡ Richard B. Vega,† Anette von Matt,‡ Lihua Yang,† Taeyoung Yoon,† Ji-Hu Zhang,† Na Zhu,† and
Lauren G. Monovich*,†
†Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, ‡Novartis Institutes for BioMedical
Research, Basel, Switzerland, and Gilead Colorado, Inc., 3333 Walnut Street, Boulder, Colorado 80301
§
Received January 18, 2010
A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D
(PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic
mechanism with application as a heart failure therapy. As PKC was previously identified as the
immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of
PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the
modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs
PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral
administration. Their effects on heart morphology and function are discussed herein.
Introduction
disruption of the MEF2 repressor HDACs, including
HDAC5, sensitizes mice to pathological cardiac growth.9
Cellular localization of class IIa HDACs is controlled by
phosphorylation state; phosphorylation of its nuclear locali-
zation sequence (NLS) targets HDAC5 for nuclear export to
the cytosol, thereby relieving repression of the transcription
factor MEF2.10 In support of this hypothesis, cardiac myo-
cytes expressing phosphorylation-resistant (e.g., Ser f Ala
mutant) HDAC5 resist stress-induced hypertrophy.
The subsequent search for the HDAC5 kinase identified
the serine/threonine kinase, protein kinase D1 (PKD1),11 one
of three PKD isoforms (including PKDs 2 and 3) from the
calmodulin kinase family. The role of PKD1 in pathologic
signaling is supported by both gain and loss of function
studies in isolated cardiac myoctes and in vivo. For example,
in isolated cardiac myocytes, knockdown of PKD1 prevents
agonist-induced hypertrophy. In vivo, loss of PKD1 pro-
tects mice from pressure overload-induced hypertrophy re-
sulting from surgical banding of the thoracic aorta (TAB).
Conversely, transgenic mice overexpressing constitutively
active PKD1 develop cardiac dysfunction. By preventing
phosphorylation and nuclear export of the MEF2 repressor
HDAC5, PKD1 inhibitors are anticipated to control activity
of the transcription factor MEF2 and the consequent patho-
logical cardiac hypertrophy and dysfunction.
Heart failure,1 a leading cause of death in industrialized
nations, is characterized by increased cardiac size and loss of
cardiovascular function. In patients with cardiac dysfunction, it
is unknown whether increased cardiac size or cardiac hyper-
trophy is causative or compensatory for cardiac dysfunction,2
but the enlarged heart, specifically hypertrophy of the left
ventricle, is an independent risk factor for adverse cardiac
events.3 Further, reduction of existing cardiac hypertrophy
decreases mortality.4 Cardiac hypertrophy does not reflect
hyperplasia of the cardiac muscle cells, or cardiac myocytes,
but rather hypertrophy of the individual myocytes.5 Thus,
aberrant hypertrophic signaling pathways in the cardiac myo-
cyte present attractive targets to intercept pathological cardiac
hypertrophy and to explore the effect on cardiac function.6
Within the normal adult cardiac myocyte, the transcription
factor myocyte enhancer factor 2 (MEF2a) dependent gene
expression is repressed by association with class IIa histone
deacetylases (HDACs 4, 5, 7, and 9).7 In failing hearts,
derepression of MEF2 is believed to be a key player in adverse
cardiac remodeling, including hypertrophy. In accordance
with this hypothesis, MEF2D-/- mice are resistant to cardiac
hypertrophy induced by stresses such as chronic β-adrenergic
stimulation and pressure overload.8 Conversely, targeted
At the inception of the present study, pan PKC inhibitors,
such as Go-6976 112 (Figure 1), had been described as a dual
*To whom correspondence should be addressed. Phone: 617-871-
7606. Fax: 617-871-7045. E-mail: lauren.monovich@novartis.com.
a Abbreviations: PKD, protein kinase D; PKC, protein kinase C;
TAB, thoracic aortic banded; DSS, Dahl salt-sensitive; HDAC, histone
deacetylase;MEF2, myocyteenhancer factor 2; GFP, green fluorescence
proteint; LV, left ventricle; TL, tibia length; IVRT, isovolumic relaxa-
tion time; MAP, mean arterial pressure; PMA, phorbol 12-myristate
13-acetate; PE, phenylephrine; PGF2R, prostaglandin F2R; ET-1, en-
dothelin-1; LPA, lipopolysaccharide A; PMBCs, peripheral blood
mononuclear cells; HTS, high throughput screen.
€
PKC/PKD inhibitors. Subsequently, additional PKD inhibi-
tors13 and an allosteric series exemplified by CID75567314
have been disclosed. PKC isoforms are described as immedi-
ate upstream activator kinases of PKD,15-17 so PKD vs PKC
selectivity, unknown at the inception of the present study,
served as an important determinant for a proof of con-
cept PKD inhibitor. Encouragingly, the PKC family, which
r
pubs.acs.org/jmc
Published on Web 07/14/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5401
belongs to the AGC group of kinases, is not closely related to
the PKDs, which belong to the calmodulin (CaMK) group of
kinases.18 Conversely, an ATP-competetive inhibitor must
distinguish between close sequence homology near the hing-
binding regions of the PKCs and PKDs, as in the sequences
VMEFLNG and VMEKLHG for PKCδ and PKD1, respec-
tively. The methionine (M) gatekeeper residue is the most
common among both the AGC and CaMK kinase groups.19
Similar to the dual PKC and PKDs inhibitors described in the
literature, an HTS from the Novartis archive identified novel
2,6-naphthyridine 2 as a dual inhibitor of PKC and PKD
(Figure 1), with 10-fold higher potency vs novel PKC iso-
forms, such as PKCδ, than PKD (Table 1). Herein, we
describe the optimization of 2 to prototype PKD inhibitors
with oral availability and 1000-fold selectivity vs PKC iso-
forms.
and trapping with MeI. Deprotonation of compound 5 with
n-BuLi and treatment with ester 7a or ester 7b afforded
ketone 8a or ketone 8b, respectively. Ring closure with
loss of tert-butylamine and dehydration to the pyranone
occurred with heating in DMF, followed by heating in AcOH.
Treatment of the pyrido-pyranones with 25% NH3 in EtOH
afforded the 2,6-naphthyridones, which were successfully
halogenated by treatment with POBr3 to give 1-bromo-2,6-
naphthyridine 9a or with POCl3 to give the 1-chloro-2,6-
naphthyridines 9b and 9c.
Displacement of the halogen of 9a or 9b with nitrogen
nucleophiles, such as BOC-protected ethylenediamine, ami-
nopropanol, or BOC-piperazine, afforded 1-amino-2,6-naph-
thyridines 10a-c. Acidic deprotection of the BOC amines
10a and 10c afforded PKD inhibitors 2 and 11, respectively.
Likewise, chloride 9c was treated with nitrogen nucleophiles
to afford 12a-c. Buchwald amination of 2-chloropyridines
12a-c with methylamine, cyclohexylamine, or 4-aminotetra-
hydropyran gave inhibitors 13. Compounds 13 were further
elaborated as indicated, for example, by acidic deprotection of
13b or 13e to give 13c and 13f, respectively. Borch reduction of
secondary amines 13c and 13f gave tertiary amines 13d and
13g, respectively.
A similar reaction sequence was used to vary the placement
of nitrogen within the core naphthyridine (Scheme 2). N-tert-
Butyl-2-methylbenzamide 14a or aza analogues 14b-d (where
A, B, or E equals nitrogen) were lithiated and trapped with
2-chloroisonicotinic acid methyl ester 7b as above. The crude
ketones 15a-d were directly treated with hot AcOH to effect
cyclocondensation to yield pyrido-pyranones, which were
then converted to naphthyridones 16a-d by treatment with
ammonia in MeOH. Tosynthesize the quinazoline-containing
analogue, treatment of nitrile 17 with HCl in methanol gave
imidate 18, which was condensed with anthranilic acid 19 to
afford the quinazolinone 20. Compounds 16a-d and 20 were
chlorinated with POCl3 and treated with BOC-piperazine
Chemistry
The synthesis of 2 and analogues began with pyridine
oxide 3, which underwent methylation followed by attack of
cyanide to afford compound 4 (Scheme 1). Nitrile 4 was
efficiently converted to the tert-butylamide 5 by treatment
with t-BuOH in concentrated H2SO4. Alternatively, com-
pound 5 was accessed from isonicotinic acid 6 by formation
of the tert-butylamide followed by amide-directed lithiation
Figure 1. PKD inhibitor structures.
Table 1. PKD, PKC, and HDAC Nuclear Export Activity

5402 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Scheme 1. Synthesis of the 2,6-Naphthyridinesa
Meredith et al.
a Reagents and conditions: (i) (MeO)2SO2, EtOH, H2O, 60 ꢀC, 18 h; (ii) KCN, EtOH, H2O, 18 h; (iii) t-BuOH, conc H2SO4, 70 ꢀC, 3 h; (iv) ethyl
chloroformate, Et3N, CH2Cl2, then t-BuNH2; (v) n-BuLi, then MeI; (vi) n-BuLi, THF, -70 ꢀC; (vii) DMF, reflux; (viii) AcOH, 100 ꢀC; (ix) 25% NH3,
EtOH; (x) POBr3 or POCl3, Me4NCl, 110 ꢀC; (xi) HNRR1, Et3N, EtOH, reflux; (xii) TFA, CH2Cl2; (xiii) H2NR2, Pd(OAc)2, t-BuOK, BINAP, PhCH3,
85 ꢀC; (xiv) isobutyraldehyde, NaBH(OAc)3, Cl(CH2)2Cl.
to afford 21a-e. As above, elaboration of chloropyridines
21a-e was accomplished by Buchwald amination with cyclo-
hexlyamine. Removal of the BOC-protecting group gave
inhibitors 22a-e.
the amine using PyBOP or converted directly by treatment of
the amine in the presence of Me3Al. Compounds 13ac and
13ae were generated by acidic deprotection of 13ab and 13ad,
respectively.
Exploration of additional diversity at the 1-position of the
naphthyridine was enabled as described in Schemes 3 and 4.
Selective displacement of the 1-chloronaphthyridine 9c with
methoxide afforded 23, which was reacted with cyclohexyla-
mine utilizing palladium catalysis, demethylated with wet t-
BuOK, and chlorinated with POCl3 to afford 24 (Scheme 3).
Nucleophilic substitution of chloride 24 with a series of cyclic
amines afforded inhibitors 13h-j. By analogy to Scheme 1,
direct treatment of dichloride 9c with amines gave intermediates
12d-i, which were converted to targets 13k-q by methods des-
cribed above. In addition, peptide coupling of piperazine 13f to
BOC-glycine and subsequent BOC removal afforded target 13r.
Reaction of 9c with piperidine-4-carboxylic acid derivatives
yielded intermediates 12j-l (Scheme 4). Amides 12j and 12k
were subjected to Pd-catalyzed coupling to cyclohexylamine or
4-aminotetrahydropyran to give compounds 13s-u (Scheme 4).
Alternatively, ester 12l was converted to a series of target
amides 13v-ae. The ester was either saponifed and coupled to
Compound Evaluation
In vitro, compounds were evaluated for their ability to
inhibit the target PKD1, PKD isoforms 2 and 3, and repre-
sentative PKC isoforms PKCR and PKCδ. All compounds
in the present study were found to competitively inhibit
the entire PKD family18 (isoforms 1, 2, and 3) with similar
potency, consistent with the high sequence identity among
PKDs 1, 2, and 3. The sequences of the PKD2 and PKD3
kinase domains are 91% and 94% identical, respectively, to
that of PKD1.20 Only the PKD1 activity is reported for
clarity, but reference throughout the text to PKD inhibition
will represent pan-PKD inhibition. The HDAC nuclear ex-
port assay, a cellular readout of PKD activity, monitored the
ability of the inhibitors to prevent PGF2R-stimulated nuclear
export of GFP-HDAC5 in the cardiac myocyte.
In vivo, selected compounds were evaluated for their ability
to block cardiac hypertrophy with daily administration for

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5403
Scheme 2. Synthesis of Alternative Coresa
a Reagents and conditions: (i) n-BuLi, 7b, THF, -70 ꢀC; (ii) AcOH, 100 ꢀC; (iii) 25% NH3, EtOH; (iv) HCl, MeOH, dioxane; (v) Na, MeOH;
(vi) POCl3, Me4NCl, 110 ꢀC; (vii) BOC piperazine, Et3N, EtOH, reflux; (viii) cyclohexylamine, Pd(OAc)2, t-BuOK, BINAP, PhCH3, 85 ꢀC; (ix) TFA,
CH2Cl2.
Scheme 3. Installation of Groups on the 1-Naphthyridinea
a Reagents and conditions: (i) NaOMe, MeOH; (ii) cyclohexylamine, Pd(OAc)2, t-BuOK, BINAP, PhCH3, 85 ꢀC; (iii) t-BuOK, t-BuOH, THF;
(iv) POCl3, Me4NCl, 110 ꢀC; (v) amine, Et3N, EtOH, reflux; (vi) TFA, CH2Cl2; (vii) N-BOC-glycine, HBTU, Et3N; (viii) formic acid.

5404 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Scheme 4. Synthesis of 1-Naphthyridines with Pendent Amides and Amide Isosteresa
a Reagents and conditions: (i) amine, Et3N, EtOH, reflux; (ii) amine, Pd(OAc)2, t-BuOK, BINAP, PhCH3, 85 ꢀC; (iii) amine, Me3Al; (iv) LiOH, THF,
water; (v) amine, PyBop, HOBt; (vi) TFA, CH2Cl2; (vii) MsCl.
14 days in a rat model of disease. The two rat models utilized
in the present study were the TAB rat, a surgical model
of pressure-overload-induced cardiac hypertrophy, recalling
that PKD1knockout was shown toblunt cardiachypertrophy
in the TAB mouse. A second model, the Dahl salt-sensitive
(DSS) rat, is a genetic model of high salt-induced hyperten-
sion and cardiac hypertrophy. In all rat studies, the effect of
compound treatment on blood pressure was monitored to
control for the anticipated benefit of antihypertensives on
cardiac hypertrophy in both models.
present study. Encouragingly, the cellular activity of 13c
(IC50=32 nM) was more potent than could be expected for
micromolar inhibition across PKC isoforms. With simple
substitution changes, naphthyridine 13c reflects a ∼500-fold
increase in target activity and a ∼15,000-fold increase in PKD
vs PKCδ selectivity relative to the hit compound 2.
In Figure 2, a possible binding mode for 13c in the ATP-
binding site of PKD1 is shown, wherein the alkylaminopyr-
idine forms two hydrogen-bonding contacts with the Leu662
hinge residue, the naphthyridine 6-nitrogen contacts the cata-
lytic Lys612, and the piperazine nitrogen participates in a salt
bridge with a pendent Glu710 of the sugar pocket. However,
the pseudosymmetry of the series renders the 6-naphthyridine
nitrogen of 13c a viable alternative for the hinge contact and
the pyridine a viable alternative contact for the catalytic Lys.
Ifindeedthe binding mode depictedinFigure2 isaccurateand
a similar orientation is realized for PKC, the intolerance of
PKCs for the 2-pyridyl substituent may come from steric clash
with the residues adjacent to the hinge, which are uniformly
larger (Phe or Tyr) for the PKCs, where the PKDs contain the
more flexible Lys. In the absence of X-ray crystallographic
data for a representative naphthyridine inhibitor in PKD and
PKC, a specific structural basis for the observed selectivity
impact with substitution of the pyridine is unknown.
To better understand the contribution of the core 2,6-
naphthyridine to the PKD activity, the team explored mod-
ification of the 6-naphthyridine nitrogen, while keeping the
“2”-naphthyridine nitrogen constant (Table 2). Of the com-
pounds in Table 2, only the isoquinoline 22a, 2,5-naphthyr-
idine 22d, and quinazoline 22e achieved IC50s less than 50 nM.
In no case where the 6-nitrogen was deleted or relocated did
the PKD activity match that of the parent 13c. These results
are consistent with the function of the 6-nitrogen as a hydro-
gen-bond acceptor to the enzyme.
Results
The reported reference PKC/PKD dual inhibitor 1 showed
activity vs PKD (IC50=27 nM) and PKC isoforms (Table 1),
as wellas the cellular readout of HDAC nuclear export (IC50=
62 nM). Screening hit, compound 2, showed more modest
inhibition of PKD (IC50=321 nM) and HDAC nuclear export
(IC50=5420 nM). Like 1, compound 2 did not achieve the
desired selectivity vs a subset of the PKC isoforms. Rather, 2
exhibited 10-fold inverted selectivity vs PKCδ (IC50=20 nM).
Further, owing to the role of PKC in PKD activation, the
cellular activities of 1 and 2 cannot be firmly attributed to
PKD inhibition. That is, HDAC nuclear export activity of 1
and 2 cannot rule out the contribution of direct and exclusive
PKC inhibition.
Compound10bhighlights thesensitivityofthe1-naphthyr-
idine substituent for PKD, PKCR, and PKCδ activity. Com-
pound 13a hinted that introduction of a 2-pyridine sub-
stituent benefited PKD activity without enhancing PKC
inhibition. The piperazine 11 gained 5-fold PKD activity vs
the parent ethylenediamine 2. Combining the piperazine with
the 2-pyridine substituent, as with 12c (IC50=50 nM) or 13c
(IC50=0.6 nM), afforded potent PKD inhibitors with use-
ful (20- to 1400-fold) selectivity vs PKCs. Similarly weak
PKC activity was observed for all subsequent compounds
bearing 2-pyridyl substituents described throughout the
In certain cases, modulating the bulkiness, basicity, and/or
polarity of amine-containing compounds can improve drug-
like qualities. The compounds indicated in Table 3 were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5405
Figure 2. Proposed key contacts for PKD inhibitor 13c in PKD1.
Table 2. Naphthyridine Core Modifications
solubility at pH 6.8 of both 13c (734 μM) and 13f (887 μM),
the flexibility to introduce pyridine 2-substituents with greater
polarity, like the aminoTHP, can provide a potentially use-
ful mechanism to modify the physical properties (e.g., aqu-
eous solubility) or the off-target profile of naphthyridine
analogues.
A second study of the 1-naphthyridine substituent carried
either the cyclohexylamino or 4-tetrahydropyranamino find-
ings of the above (Table 4). As with the entries of Table 1,
untethered alcohols, such as 13n, offered modest PKD inhibi-
tion, while introdution of a ring enhanced activity (13j, IC50=
15 nM). Building from the piperazine, the first amides 13o and
13m were disappointing, especially with respect to cellular
activity. Reinserting a basic nitrogen, as with 13r, enhanced
the PKD and cellular activity relative to 13m. Thereafter, the
team was pleased to identify the piperidine carboxamide
series, including 13s and 13t, which offered good PKD
inhibition and cellular activity in the absence of a basic
nitrogen. Bulkier analogues, such as 13y and 13z, exhibited
low nanomolar IC50s vs PKD but 10-fold weaker HDAC
export activity compared to the most potent examples.
Again, in cases such as 13ae and 13v, where a pendent base
was (re)installed, the cellular activity (IC50 values of 44 and
25 nM, respectively) matched that of piperazine 13c (IC50=
32 nM).
IC50 (nM)
compd
A
B
D
E
G
PKD1
HDAC exptl
22a
22b
22c
22d
22e
CH
N
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
N
12
695
191
15
356
>10000
1558
CH
CH
CH
CH
CH
262
1521
CH
43
prepared to explore the effects of amine branching and
basicity on PKD potency. This series highlights the flat
SAR with respect to PKD and HDAC export, with only the
weakest base 13l falling 10-fold outside the IC50 of the parent
13c. Replacing the cyclohexyl with an alternative but neutral
branched aliphatic group with greater polarity (e.g., THP)
gave compounds 13f and 13g. The calculated log P values for
13c and 13f are significantly different (4.12 for 13c and 1.72 for
13f), yet compounds 13f and 13g showed equal potency
relative to their cyclohexyl comparators 13c and 13d.
Althoughthepiperazine likely contributestothe highaqueous
Further comparison of prototype PKD inhibitors amine
13c and amide 13y in vitro, in cells, and in vivo follows. In
vitro (Figure 3), compounds 13c and 13y dose-dependently
inhibited autophosphorylation of recombinant human PKD1

5406 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Table 3. 1-Naphthyridine Modifications: Piperazines and Aminopiperidine
(Ser916) and PKD2 (Ser867). At concentrations as low as
10 nM, 13c partially blunted PKD1 autophosphorylation and
nearly completely blunted PKD2 autophosphorylation in the
absence of the HDAC peptide, a 25-mer substrate peptide
designed to model one of the HDAC serine residues (Ser498)
that is phosphorylated by PKD.21
For compound 13y, partial control of PKD1 autopho-
sphorylation was observed at 10 nM, with near complete
inhibition at 100 nM. However, 100 nM of 13y was needed
to block PKD2 autophosphorylation. These experiments
compared well with the relative and absolute biochemical
assay results for 13c and 13y (PKD1 data from Tables 1 and 3,
PKD2 IC50 values of 2 and 16 nM, respectively) and con-
firmed the pan-PKD inhibitory activity of the 2,6-naphthyr-
idine series with an alternative substrate. Lastly, inhibition of
both PKDs by 13c and 13y occurred in the presence of an
HDAC5 peptide, which served as a surrogate for the native,
full length HDAC5 present in cells.
GPCR agonists, such as prostaglandin F2R (PGF2R)
utilized in the HDAC export assay or phenylephrine (PE)
and endothelin-1 (ET-1) utilized for Figure 4 below, initiate
cell surface-signaling to PKC and consequent activation of
PKD to drive GFP-HDAC5 export to the cytosol of rat
cardiac myocytes (top row, Figure 4). In addition, direct
stimulation of PKC with the phorbol ester PMA causes
nuclear export of GFP-HDAC5. With all stimuli shown to
cause export of GFP-HDAC5, pretreatment of the cells with
1 μM 13c controlled GFP-HDAC5 nuclear export, consistent
with the hypothesis that PKD integrates multiple signals
originating at the cell surface.
In PE-stimulated rat cardiac myocytes (Figure 5), 13c and
13y dose-dependently block PKD activity. PKD activity,
which is gauged by both autophosphorylation (Ser916) and
phosphorylation of the substrate HDAC5 (Ser498), occurred
without loss of PKD activation (PKC-mediated phosphory-
lation at Ser744/748 not shown). Consistent with the HDAC
export assay that tracks PGF2R-driven nuclear export of
superphysiologic GFP-HDAC, both 13c and 13y blocked
phosphorylation of GFP-HDAC5 (Ser498) at 1000 nM. Ad-
ditionally, the relative potency between 13c (IC50=32 nM) and
13y (IC50=391 nM) in the HDAC export assay is reflected in
the ability of 100 nM 13c, but not 13y, tonormalizepHDAC to
baseline. As well, both compounds dose-dependently inhibited
native PKD autophosphorylation, albeit at concentrations
higher than those required to block pHDAC. For example,
at a dose 10-fold greater than that which successfully con-
trolled pHDAC, residual pPKD was detected in the presence
of 1000 nM 13c by comparison to pPKD in unstimulated cells.
This phenomenon was matched throughout the present study.
That is, we routinely observed inhibition of pHDAC in iso-
lated cardiac myocytes at lower concentrations than those
required to blunt autophosphorylation of native PKD.
Further profiling of 13c and 13y supported their use as
prototypePKD inhibitorsfor in vivostudy. Inmale Sprague-
Dawley rats, the pharmacokinetic parameters of 13c and 13y
were evaluated following administration as solutions (Table 5).

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5407
Table 4. 1-Naphthyridine Modifications: Alcohols and Amides

5408 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Figure 3. PKD inhibitors 13c and 13y block in vitro autophosphorylation of PKD1 (Ser916) and PKD2 (Ser867) in the absence or presence of
an HDAC5 peptide. The concentrations at which PKD autophosphorylation is inhibited are consistent with the IC50 values in the PKD
biochemical assays: (a) (-) 0 μM, (/) 0.01 μM, (//) 0.1 μM, (///) 1 μM compound concentration.
Figure 4. In neonatal rat ventricular myocytes (NRVMs), 1 μM 13c blocks GFP-HDAC5 nuclear export in response to a variety of stimuli,
including 10 μM phenylephrine (PE), endothelin-1 (ET-1), prostaglandin F2R (PGF2R), lysophosphatidic acid (LPA), or phorbol 12-myristate
13-acetate (PMA).
Table 5. Pharmacokinetic Profile of 13c and 13ya
PK parameter
13c
13y
dose iv/po (mg/kg)
po Cmax (nM)
po Tmax (h)
2/5
2/5
466 ( 535
0.5
128 ( 29
0.25 ( 0.1
193 ( 175
1923 ( 460
32 ( 12
12 ( 1
po AUC0-8h (nM h)
3
iv AUC0-8h (nM h)
633 ( 647
1107 ( 313
61 ( 19
7.0 ( 7.8
5.4 ( 4.4
23
3
CL ((mL/min)/kg)
Vss (L/kg)
Thalf (h)
5.2 ( 1.9
4
bioavailability (%)
a AUC0-8h reported here, not AUC¥.
Figure 5. In NRVMs, compound 13c and 13y block PE-stimulated
PKD activity. In accordance with their relative and absolute
potencies in the HDAC export assay, compounds 13c (at 0.1 and
1 μM) and 13y (at 1 μM) block phosphorylation of the PKD
substrate HDAC (pSer498). At 1 μM, 13c effectively blocks PKD
autophosphorylation (pSer916) while 13y produces partial reduc-
tion: (a) (-) 0 μM, (/) 0.1 μM, (//) 1 μM compound concentration.
To accommodate the low aqueous solubility of 13y for iv
administration, an NMP and Cremophor EL containing
vehicle was used for the determination of PK properties. For
consistency, the vehicle used for 13c to determine PK para-
meters was matched to that used for 13y. For the subsequent
high dose exposure and efficacy studies in rats, both 13c and
13y were administered orally as suspensions in methylcellulose
or subcutaneously in acidified captisol. Following an iv dose,
13c was cleared rapidly from the plasma and showed a large
volume of distribution (12 L/kg) that was significantly greater
than the total body water in rats. When administered as a
solution via oral gavage, 13c was rapidly absorbed with a low
absolute oral bioavailability of 4%. A subset of the amides,
including 13y, showed measurable plasma exposure following
oral administration, with Cmax, AUC0-8h, and bioavailability
exceeding those of the piperazine 13c (Table 5). A 50 mg/kg po
Figure 6. DSS rat plasma exposure following 50 mg/kg oral admin-
istration of 13c and 13y as a suspension in 0.5% methylcellulose and
0.5% Tween-80.
dose of compounds 13c and 13y as suspensions to the DSS rat
produced higher plasma exposures and similar plasma curve
shapes relative to the low-dose po studies above (Figure 6).
Compound 13c for its potency and consistent plasma expo-
sure and high cardiac exposure (not shown) and the amide
13y for its different plasma PK profile, improved kinase

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5409
Figure 7. Effect of compound 13c and 13y given 50 (mg/kg)/day po as a suspension in methylcellulose for 14 days in the DSS rat on (a) mean
blood pressure and (b) cardiac hypertrophy (left ventricular mass/tibia length) on day 14. Neither compound attenuated the increase in mean
blood pressure observed with high (8%) salt diet. Compound 13c, but not 13y, blunted high-salt-induced cardiac hypertrophy (LV/TL): (/) p <
0.05 vs 0.05% NaCl plus vehicle; (†) p < 0.05 vs 8% NaCl plus vehicle.
Figure 8. Biomarker readout in the DSS rat in a surrogate cell type on day 14. In peripheral blood mononuclear cells (PBMCs), PMA-
stimulated, (a) autophosphorylation of PKD at Ser916 is blunted by compound 13c and (b) phosphorylation of the PKD substrate HDAC at
Ser498 is blunted by both 13c and 13y relative to the vehicle control: (/) p < 0.05 vs vehicle.
Table 6. Percent Activity Remaining in the Presence of 1 μM 13c or 13y
vs Set of PKD Family Member Kinases and IC50 Values versus
Adrenergic Receptors
activity (%)
kinase
PKD1
PKD2
13c
13y
2
2
3
7
GSK3β
CaMKIδ
CaMKIIβ
CaMKIIγ
CaMKIIδ
CaMKIV
MARK1
SIK1
3
38
67
13
26
45
100
28
86
100
78
72
100
73
96
91
100
80
99
GRK5
PKCδ
PKCε
100
100
80
Figure 9. Effect of compound 13c given 5, 10, and 50 (mg/kg)/day
po as a suspension in methylcellulose on day 14 of 14 days in the
DSS rat on cardiac hypertrophy (left ventricular mass/tibia length).
Only the 50 mg/kg po dose attenuates high-salt-diet-induced cardiac
hypertrophy (LV/TL): (/) p < 0.05 vs 0.05% NaCl plus vehicle; (†)
p < 0.05 vs 8% NaCl plus vehicle.
IC50 (nM)
receptor
13c
13y
R1AR
β1AR
7202
2351
>10000
>10000
Compound 13c or 13y were adminstered at 50 (mg/kg)/day
po for 14 days to DSS rats concomitantly with a switch to a
high-salt (8%) diet. Control animals remaining on normal-
salt diet (0.5%) or on high-salt (8%) diet were administered
selectivity, and spotless receptor binding profile (Table 6) were
investigated in the in vivo hypertrophy models.

5410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Figure 10. Biomarker readout in the DSS rat in a surrogate cell type on day 14. In peripheral blood mononuclear cells (PBMCs), PMA-
stimulated, (a) autophosphorylation of PKD at Ser916 is blunted by the highest dose of compound 13c and (b) phosphorylation of the PKD
substrate HDAC at Ser498 is reduced by all compound doses: (/) p < 0.05 vs vehicle; (#) p < 0.05 vs vehicle plus PMA.
Figure 11. Effect of compound 13c given 15 (mg/kg)/day sc in acidified captisol in the thoracic aortic banded (TAB) rat on (a) cardiac
hypertrophy (left ventricular mass/tibia length) and (b) a marker of diastolic function isovolumic relaxation time (IVRT) on day 14. Neither
parameter is affected by compound treatment: (/) p < 0.05 vs vehicle.
vehicle daily. As anticipated, vehicle-treated animals on high-
salt diet exhibited hypertension, or elevated mean arterial
pressure (MAP), and hypertension-driven cardiac hypertro-
phy relative to the vehicle-treated rats on normal-salt diet
(Figure 7). The mass of the left ventricle (LV) normalized to
the animal size by the tibia length (TL) gives the index of
cardiac hypertrophy. After 14 days, rats given a high-salt diet
exhibited a consistently elevated mean blood pressure
(Figure 7a) and a corresponding ∼20% increase in the LV/TL
relative to the normal-salt rats (Figure 7b). While neither
compound 13c nor 13y blocked high-salt induced hyperten-
sion (Figure 7a), the compounds were differentiated with
respect to cardiac hypertrophy. Compound 13c, but not
13y, gave a statistically significant reduction in the LV/TL
(Figure 7b). In the absence of changes to MAP, the antihy-
pertrophic behavior of 13c at 50 (mg/kg)/day po cannot be
attributed to simple relief of hypertension.
To circumvent technical difficulties with monitoring
phosphorylation state changes to the modestly abundant
HDAC5 in intact rat hearts, the team devised an ex vivo assay
to monitor PKD inhibition in a surrogate cell type, the
peripheral blood mononuclear cells (PBMCs). As with the
cardiac myocytes of Figure 4, PKD was activated in PBMCs
by treatment with phorbol ester (PMA). In PMA-stimulated
PBMCs taken from the DSS study above (Figure 8), both
compounds 13c and 13y reduced the substrate HDAC
phosphorylation, but only the efficacious 13c also reduced
PKD autophosphorylation. A dose-response study conduc-
ted with 13c given 50, 15, or 5 (mg/kg)/day in the DSS rat
(Figure 9) confirmed the antihypertrophic response at the
50 (mg/kg)/day dose as above (Figure 7b). However, while
the lower doses of 15 and 5 (mg/kg)/day achieved 6700 and
1000 nM cardiac exposures, respectively, neither compound
attenuated high salt-induced cardiac hypertrophy. From the
PKD activity biomarkers observed in PBMCs (Figure 10), all
three doses of 13c blunt pHDAC, but only the highest 50 mg/kg
dose achieves a statistically significant reduction of PKD
autophosphorylation.
To follow up on the observation that PKD1-/- limits the
pressure-overload-induced cardiac hypertrophy observed in
the TAB rat, compound 13c was evaluated in a rat TAB
model. In this case, surgical banding of the thoracic aorta was
completed prior to compound administration. Compound
was administered parenterally (15 (mg/kg)/day sc) 1 day
postsurgery to limit the postsurgical stress observed with oral
gavage. On day 14, 2 h after administration of the final oral
dose, plasma and cardiac exposures (∼60000 nM) closely
matched those observed at the same time in the DSS rat study

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5411
Figure 12. Biomarker readout in the TAB rat in a surrogate cell type on day 14. In PBMCs, PMA-stimulated, (a) autophosphorylation of PKD
at Ser916 with compound 13c treatment is not significantly different from vehicle control and (b) phosphorylation of the PKD substrate HDAC
at Ser498 is normalized to baseline levels: (/) p <0.05 vs vehicle; (#) p < 0.05 vs vehicle plus PMA.
Table 7. 13c and 13y Exposures in DSS Rat Plasma and Left Ventricle
2 h after Final Dose on Day 14
cardiac profile of a PKD inhibitor. The lack of efficacy in the
TAB rat with 13c represents a point of difference with the
rat dose
compd model ((mg/kg)/day) route
in plasma
(nM)
in left
ventricle (nM)
observed protection afforded by PKD1 knockout in the TAB
mouse.
Because of the high cardiac exposure together with the off-
target profile, the efficacy of 13c in the DSS rat at 50 mg/kg
cannot be firmly ascribed to PKD inhibition. Inhibition of an
alternative kinase or antagonism of an upstream receptor is also
consistent with the observed result. For example, either R-22 or
β-adrenergic23 receptor blockade (13c β1AR IC50=2351 nM,
antagonist) controls high salt-induced cardiac hypertrophy in
the DSS rat at subantihypertensive doses. Further, lower doses
of 13c (15 or 5 (mg/kg)/day) maintained 6700 and 1000 nM
cardiac exposures in the DSS rat, respectively, well in excess of
the cellular HDAC export IC50 in isolated rat cardiac myocytes.
Therefore, even the lowest observed cardiac exposure of 13c
exceeds the effective concentration in the myocytes by more
than an order of magnitude. While these exposures are likely to
produce pharmacologic inhibition of the PKDs, the strongest
evidence of PKD inhibition comes from the corroborating
biomarker evidence.
13c
DSS
DSS
DSS
TAB
DSS
50
15
5
po
po
po
sc
541 ( 99
305 ( 155
87 ( 42
288 ( 111
1653 ( 1046 2021 ( 1139
59681 ( 16330
6687 ( 3084
1001 ( 521
15
50
60992 ( 14860
13y
po
(Table 7). Unfortunately, compound 13c in the TAB rat had
no effect on cardiac hypertrophy (LV/TL) in the TAB rat
(Figure 11), in direct contrast to the efficacy observed in the
DSS rat and the reported results with PKD1 knockout in the
mouse TAB model. Neither did 13c affect cardiac function, as
gauged by the accepted marker of diastolic dysfunction,
isovolumic relaxation time (IVRT). In this case, the biomar-
ker readout in the PBMCs indicated significant blunting of
pHDAC without statistically significant control of PKD
autophosphorylation (Figure 12).
Conclusions
In addition to the availability of the compounds in the
target organ, biomarker evidence generated from PBMCs
isolated from the in vivo model affords a direct measure of
pharmacologic PKD inhibition. Most importantly, all doses
of 13c blunted PMA-stimulated phosphorylation of native
HDAC in the PBMCs. The coupling of the micromolar
concentrations in cardiac tissue to the biomarker PBMC data
is consistent with pharmacological inhibition of the target
in the heart at all three doses of 13c. Likewise, a second PKD
inhibitor, 13y, which exhibits limited receptor affinity and
higher kinase selectivity vs a set of putative HDAC kinases,
reached micromolar concentrations in cardiac tissue and
blunted PMA-stimulated HDAC phosphorylation in the
PBMCs, again in the absence of an effect on cardiac hyper-
trophy in the DSS rat. Therefore, in contrast to the target
hypothesis, no direct correlation between pHDAC levels
and cardiac hypertrophy was observed. That is, the reduced
HDAC phosphorylation observed in the PBMCs with both
test compounds at all test doses was not linked to control
of high salt-induced cardiac hypertrophy in the DSS rat.
If (1) the cardiac exposure of 13c and 13y and the pHDAC
reduction observed in the PBMCs accurately model reduced
cardiac pHDAC levels and (2) the DSS and TAB rat models
accurately predict clinical success for PKD inhibitors as
Starting from a dual PKC/PKD inhibitor identified by
HTS, potent prototype pan-PKD inhibitors were identified
with useful selectivity vs representative upstream PKD-acti-
vating PKC isoforms (>1000-fold). Further, compounds 13c
and 13y were well-tolerated by daily po or sc administration
up to 2 weeks in rat models of heart failure with micromolar
cardiac tissue exposures above the cellular IC50.
In vivo, the most potent but overall less selective (vs a broad
kinome and receptor panel) inhibitor 13c (PKD1 IC50 =
0.6 nM, HDAC export IC50=32 nM) demonstrated efficacy
at the 50 mg/kg po dose in the DSS rat model but not at the
15 mg/kg sc dose in the TAB rat model. Although the doses
and routes of administration were different (necessitated for
technical reasons), the plasma (288-541 nM) and cardiac
(∼60000 nM) exposures of 13c were matched between the two
studies. In both animal models, inhibitor 13c blunted PMA-
stimulated phosphorylation of the PKD substrate HDAC in a
surrogate cell type (PBMCs). Therefore, the same exposure of
13c and consequent control of pHDAC produced model-
dependent effects on cardiac hypertrophy. Further, the TAB
rat, which was anticipated to be the best model of PKD
inhibition based on the PKD1 knockout studies in the TAB
mouse, provideda second disconnect withthe expected in vivo

5412 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
antihypertrophic agents for the treatment of heart failure,
then the in vivo responses of compounds 13c and 13y separate
control of cardiac pHDAC with therapeutic potential in heart
failure.
Envision 2100 reader using a LANCE Eu/APC dual protocol.
As described above, a (665 nm)/(615 nm) ratio was determined
to measure substrate phosphorylation and enzyme activity.
Compounds are typically tested in an 11-point dose response
fashion in triplicate for each concentration used. IC50 values are
calculated using an activity base (IDBS) software program.
PKD2 Assay. The assay to measure protein kinase D2
(PKD2) activity was a time-resolved fluorescence resonance
energy transfer (TR-FRET) assay using PerkinElmer’s LANCE
technology. In this case, a biotinylated syntide-2 peptide was
used as the substrate in this reaction. Phosphorylation of the
syntide-2 substrate was detected by a specific antibody that
recognizes the phosphorylated peptide. A second fluorophore,
APC, was conjugated to streptavidin that binds the biotinylated
syntide-2 peptide. For detection, the europium fluorophore can
be excited by 340 nm light which then emits at 615 nm. There-
fore, when the europium labeled secondary antibody binds on
the phosphorylated peptide, it was brought into close contact
with the APC and excites this fluorophore. The APC emission
was at 665 nm, and the (665 nm)/(615 nm) ratio was a readout of
PKD2 activity.
The assay was performed with full length wild-type enzyme
purchase from Invitrogen. The reaction buffer consists of
35 mM Tris-HCl, pH7.5, 5 mM MgCl2, 0.02% Tween-20,
20 μM ATP, 1 mM DTT, and 0.2 μg/mL PKD2 enzyme. The
enzyme reaction was initiated by the addition of 2 μM syntide-2
peptide substrate and the reaction carried out for 50 min at room
temperature. The reaction was stopped by a stop/detection
buffer consisting of 50 mM EDTA, 0.18 mg/mL rabbit poly-
clonal anti-phospho syntide-2 antibody, 0.5 nM europium
labeled anti-rabbit IgG, and 10 nM streptavidin conjugated
APC. After a 1 h incubation with the stop/detection buffer,
the reaction was read on an Envision 2100 reader using a
LANCE Eu/APC dual protocol. As described above, a (665
nm)/(615 nm) ratio was determined to measure substrate phos-
phorylation and enzyme activity. Compounds are typically
tested in an 11-point dose response fashion in triplicate for each
concentration used. IC50 values are calculated using an activity
base (IDBS) software program.
In the rodent cardiac hypertrophy model studies, PKD
inhibitionmay not befunctionally equivalentto loss of PKD1.
A possible rationale lies in the protocol differences between
the relevant rat and mouse TAB experiments. That is, loss of
PKD1 is complete and consistent over the course of the
experiment where PKD inhibition is initiated postsurgery
and potentially variable. Alternatively, HDAC may represent
only one of multiple relevant PKD substrates. For example, in
the DSS rat model, the only case in which blockade of PKD
autophosphorylation was statistically significant, in vivo anti-
hypertrophic activity was also observed (50 (mg/kg)/day 13c
in the DSS rat). While inconsistent with the target hypothesis
of pHDAC/MEF2 signaling, failure to completely block
phosphorylation of multiple PKD substrates (e.g., HDAC
and PKD itself) could account for the disconnect between
PKD inhibition and PKD1 knockout in banded rodents.
On the basis of potency, tissue exposure, and biomarker
evidence, the aggregate data support that the prototype PKD
inhibitors 13c and 13y were abletoblock phosphorylationand
nuclear export of the MEF2 repressor HDAC in the target
tissue in vivo. Still, the moderate kinase selectivity of the
naphthyridine PKD inhibitors (especially 13c) complicates
interpretationofthe hypertrophic response(orlack thereof) in
both models, since pleiotropic effects (e.g., inhibition of an
antihypertrophic kinase such as GSK3β24) cannot be ruled
out. To shed further light on this question, the identification
and in vivo profiling of exquisitely potent pan-PKD inhibitors
with enhanced selectivity vs a broad kinase panel are reported
in the following paper.25 Taken in combination with the
present study, in vivo results from the second chemotype were
utilized to investigate further the therapeutic potential of
PKD inhibition as an antihypertrophic target.
PKD3 Assay. The assay to measure protein kinase D3
(PKD3) activity was a time-resolved fluorescence resonance
energy transfer (TR-FRET) assay using PerkinElmer’s LANCE
technology. In this case, a biotinylated syntide-2 peptide was
used as the substrate in this reaction. Phosphorylation of the
syntide-2 substrate was detected by a specific antibody that
recognizes the phosphorylated peptide. A second fluorophore,
APC, was conjugated to streptavidin that binds the biotinylated
syntide-2 peptide. For detection, the europium fluorophore can
be excited by 340 nM light which then emits at 615 nm. There-
fore, when the europium labeled secondary antibody binds on
the phosphorylated peptide, it was brought into close contact
with the APC and excites this fluorophore. The APC emission
was at 665 nm, and the (665 nm)/(615 nm) ratio was a readout of
PKD3 activity.
PKC Assays. The compounds of the invention are tested for
their activity on different PKC isotypes according to the follow-
ing method. Assay was performed in a white with clear bottom
384-well microtiter plate with nonbinding surface. The reaction
mixture (25 μL) contains 1.5 μM tridecapeptide acceptor sub-
strate that mimics the pseudo substrate sequence of PKCR with
the Ala f Ser replacement, 10 μM 33P-ATP, 10 mM Mg(NO3)2,
0.2 mM CaCl2, PKC at a protein concentration varying from
25 to 400 ng/mL (depending on the isotype used), lipid vesicles
(containing 30 mol % phosphatidylserine, 5 mol % DAG, and
65 mol % phosphatidylcholine) at a final lipid concentration of
0.5 mM, in 20 mM Tris-HCl buffer, pH 7.4, and 0.1% BSA.
Incubation was performed for 60 min at room temperature.
Reaction was stopped by adding 50 μL of stop mix (100 mM
EDTA, 200 μM ATP, 0.1% Triton X-100, 0.375 mg/well
streptavidin-coated SPA beads in phosphate buffered saline
Experimental Section
PKD1 Assay. The assay to measure protein kinase D1
(PKD1) activity was a time-resolved fluorescence resonance
energy transfer (TR-FRET) assay using PerkinElmer’s LANCE
technology. In this case, a biotinylated syntide-2 peptide (with
sequence PLARTLSVAGLPGKK) was used as the substrate
in this reaction. Phosphorylation of the syntide-2 substrate (at
the serine residue) was detected by a specific antibody that
recognizes the phosphorylated peptide and a europium labeled
secondary antibody. A second fluorophore, allophycocyanin
(APC), was conjugated to streptavidin that binds the biotiny-
lated syntide-2 peptide. For detection, the europium fluoro-
phore can be excited by 340 nm light which then emits at 615 nm.
Therefore, when the europium labeled secondary antibody
binds on the phosphorylated peptide, it is brought into close
contact with the APC and excites this fluorophore. The APC
emission is at 665 nm, and the (665 nm)/(615 nm) ratio is a
readout of PKD1 activity. This assay was performed with full
length wild-type enzyme that is expressed and purified from Sf9
insect cells. The reaction buffer consists of 35 mM Tris-HCl,
pH7.5, 5 mM MgCl2, 0.02% Tween-20, 20 μM ATP, 1 mM
DTT, and 0.2 μg/mL PKD1 enzyme. The enzyme reaction was
initiated by the addition of 2 μM syntide-2 peptide substrate and
the reaction carried out for 50 min at room temperature. The
reaction was stopped by a stop/detection buffer consisting of
50 mM EDTA, 0.18 mg/mL rabbit polyclonal anti-phospho
syntide-2 antibody, 0.5 nM europium labeled anti-rabbit IgG,
and 10 nM streptavidin conjugated APC. After a 1 h incubation
with the stop/detection buffer, the reaction was read on an

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5413
without Ca, Mg. After 10 min of incubation at room tempera-
ture, the suspension was spun down for 10 min at 300g. Incor-
porated radioactivity was measured in a Trilux counter for
1 min. IC50 measurement was performed on a routine basis by
incubating a serial dilution of inhibitor at concentrations ran-
ging between 1 and 1000 nM. IC50 values are calculated from the
graph by curve fitting with XL fit software. Human recombi-
nant PKCR was obtained from Oxford Biomedical Research
and was used under the assay conditions as described above.
Human recombinant PKCδ was obtained from Oxford Biome-
dical Research and was used under the assay conditions as
described above.
HDAC Export Assay. Compounds are evaluated in the
HDAC5 nuclear exposrt assay, a 384-well plate-based assay
that enables HTS to identify small molecules that block agonist-
dependent nuclear export of HDAC5. This assay employs the
Cellomics High Content Imaging platform (Giuliano and
Taylor, 1998) and adenovirus encoding green fluorescent pro-
tein (GFP) tagged HDAC5. Neonatal rat ventricular myocytes
(NRVMs) are infected with GFP-HDAC5 encoding virus and
plated on gelatin-coated 384-well dishes. Cells are exposed to
compound and stimulated with a prostaglandin (PGF2R),
which is a potent stimulus for HDAC5 nuclear export. Follow-
ing 2 h of stimulation, cells are fixed and GFP-HDAC5 localiza-
tion is quantified using the Cellomics system, which provides a
readout of relative fluorescence intensity in the cytoplasmic
versus nuclear compartment.
Determination of Compound Exposure. Adult male Spra-
gue-Dawley rats, with in-dwelling jugular vein cannulae, were
obtained from Harlan (Indianapolis, IN). The animals were
fasted overnight prior to dosing and were fed 4 h after admin-
istration of dose. The rats were administered either a single
intravenous (iv) dose (2 mg/kg, n=3) or via oral (po) gavage
(5 mg/kg, n=3). Compound 13c was administered as a solution
in 10% NMP, 10% Cremophor EL, and 80% D5W, whereas
13y was administered in solution formulation consisting of
10% NMP, 10% Cremophor EL, 7.8% HCl (1 N), and 70%
phosphate buffer (pH 7.4), with pH adjustment made by adding
1 N NaOH. Blood samples were collected at 0.08, 0.25, 0.5, 1, 2,
4, 6, and 8 h after the iv dose and at 0.25, 0.5, 1, 2, 4, 6, and 8 h
after the po dose into tubes containing EDTA. Plasma was
prepared from the blood samples and analyzed for drug con-
centrations by LC-MS/MS.
DSS Rat Model. The 6-7 week-old male Dahl salt-sensitive
(DSS) rats (n=50) from Harlan Labs were fed base diet (0.49%
NaCl) and allowed to acclimate for 1 week prior being separated
into five weight-matched groups. Rats were maintained on the
grain based diet (0.49% NaCl) or switched to grain diet contain-
ing 8.0% NaCl for 2 consecutive weeks. Coincident with diet
switch rats were administered (po, 5 mL/kg) vehicle (0.5%
methylcellulose, 0.5% Tween-80) or test compound 13c or 13y
(5, 15, or 50 mg/kg) suspended in vehicle. At the completion of
study, steady-state hemodynamics were determined. The rats
were then sacrificed and tissues were collected for morphologi-
cal and biochemical analysis.
The following abbreviations are used to denote signal patterns:
s = singlet, d = doublet, t = triplet, m = multiplet, and br =
broad. Flash chromatography was conducted using grade 60
230-400 mesh silica gel from Fisher Chemical (S825-1) or by
utilizing the CombiFlash Companion from Teledyne Isco, Inc.
and RediSep Rf disposable normal phase silica gel columns
(4-120 g). Thin layer chromatography was performed using
2.5 cm ꢀ 7.5 cm glass-backed TLC silica gel 60 F254 plates from
EMD Chemicals, Inc. (15341-1) and visualized by UV light.
HPLC purifications were performed on a Gilson preparative
HPLC system controlled by Unipoint software using X-Bridge
phenyl, C8, C18, or RP18 30 mm ꢀ 50 mm columns with 5 μm
particle size. The purity of all compounds was g95% unless
otherwise noted. Low-resolution mass spectra were recorded
using an Agilent 1100 series LC-MS spectrometer. N-tert-
Butyl-2-methylnicotinamide 14d was purchased from Astatech
(catalog no. 30048).
3-Methylisonicotinonitrile (4). To 3-methylpyridine 1-oxide 3
(15.90 g, 150 mmol) was added at 0 ꢀC during 30 min dimethyl
sulfate (15.60 mL). The resulting reaction mixture was stirred
overnight at 40 ꢀC. A solution of KCN (10.75 g, 165 mmol) in a
mixture of EtOH/water 1:1 (120 mL) was added, and the
reaction mixture was stirred overnight at 40 ꢀC. The reaction
mixture was concentrated in vacuo, and the residue was parti-
tioned between EtOAc and water. The aqueous phase was
extracted with EtOAc, and the combined organic layers were
dried over Na2SO4, filtered, and concentrated at reduced pres-
sure. Purification by flash column chromatography (silica gel,
cyclohexane/EtOAc 85:15) afforded the title compound as
orange crystals (6.00 g, 50.80 mmol, 34%): 1H NMR (400
MHz, DMSO-d6) δ ppm 8.76 (s, 1H), 8.64 (d, J=4.9 Hz, 1H),
7.80 (d, J=4.9 Hz, 1H).
N-tert-Butyl-3-methylisonicotinamide (5). To the solution of 4
(18.90 g, 159.92 mmol) in CH2Cl2 (50 mL) was added t-BuOAc
(72.63 mL, 538.84 mmol), followed by concentrated H2SO4
(12.32 mL, 874.46 mmol). The mixture was stirred for 8 h at
room temperature, then diluted with a solution of saturated
aqueous NaHCO3 and CH2Cl2 (50 mL). The organic layer was
washed with H2O, brine, dried over anhydrous Na2SO4, and
then evaporated under reduced pressure to provide the title
compound as a white solid (29.30 g, 152.44 mmol, 95%).
Alternatively, the title compound was prepared from isoni-
cotinic acid 6. Acid 6 (10 g, 80.4 mmol) was added to a 750 mL
five-necked flask equipped with an overhead stirrer, internal
thermometer, and nitrogen supply. Dichloromethane (300 mL)
was added, and the suspension was cooled to 0 ꢀC. Triethyla-
mine (17.6 mL, 121 mmol) was added maintaining a tempera-
ture under 0 ꢀC, at which time the starting material dissolved. To
the clear solution, ethyl chloroformate (9.5 mL, 98.1 mmol) was
added dropwise over 25 min maintaining a temperature under
0 ꢀC. The reaction mixture was stirred at 0 ꢀC for 30 min. tert-
Butylamine (10.4 mL, 96.5 mmol) was added slowly to the
reaction mixture at 0 ꢀC, and the solution was allowed to warm
to room temperature and stirred for 3.5 h. The reaction mixture
was diluted with water (100 mL), and the dichloromethane layer
was separated. The organic phase was washed with 1 M HCl
(100 mL) and the aqueous phase, containing the product,
neutralized to pH 9 with NaOH solution. The aqueous phase
was washed twice with ethyl acetate (2 ꢀ 100 mL) and the
combined organic phases were dried over Na2SO4, filtered, and
concentrated in vacuo to give N-tert-butylisonicotinamide as a
pale-yellow solid (9.8 g, 68.4%). MS (ESI) m/z 179 (M þ 1); 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.66 (s, 2 H), 8.02 (br s, 1 H),
7.70 (s, 2 H), 1.37 (s, 9 H).
TAB Rat Model. The 7-8 week-old male Sprague-Dawley
rats (n=30) from Charles River Labs were allowed normal chow
and water ad libitum and allowed to acclimate for 1 week. Rats
were then instrumented with an aortic occlusion cuff or under-
went sham surgery. Coincident with the surgical procedure, rats
were administered (sc, 1.0 mL/kg) vehicle (acidified captisol) or
13c (15 mg/kg/day sc) solved in vehicle for two weeks. On the
final day of study, end-point cardiac performance and steady-
state hemodynamics were determined by ultrasound and direct
cardiac catheterization, respectively. Following completion of
the measurement protocol, the rats were sacrificed, and tissues
were collected for morphological and biochemical analysis.
Chemistry. General. NMR spectra were recorded on a Bru-
ker Avance II 400 MHz spectrometer. All chemical shifts are
reported in parts per million (δ) relative to tetramethylsilane.
N-tert-butylisonicotinamide (9 g, 50.5 mmol) was added to a
750 mL five-necked flask equipped with an overhead stirrer,
internal thermometer, and nitrogen supply. Tetrahydrofuran
(225 mL) was added, and the clear solution was cooled to
-75 ꢀC. A solution of n-butyllithium 1.6 M in hexane (69 mL,
110 mmol) was added dropwise over 40 min while maintaining

5414 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
the temperature under -70 ꢀC. The reaction mixture was
stirred at -70 ꢀC for 1 h. Methyl iodide (3.5 mL, 55 mmol)
was added while maintaining the temperature under -70 ꢀC.
The solution was stirred at -75 ꢀC for 30 min and then allowed
to warm to room temperature and stirred overnight. The reac-
tion mixture was cooled to 0 ꢀC, and a saturated aqueous
solution of ammonium chloride (50 mL) was added. The reac-
tion mixture was diluted with water (150 mL) and ethyl acetate
(150 mL) and the organic layer separated. The aqueous phase
was extracted with fresh ethyl acetate (150 mL). The combined
organic phases were washed with brine (100 mL), dried over
Na2SO4, filtered, and concentrated in vacuo to give a pale-
yellow solid. The yellow solid was first triturated with hexane
(30 mL) and then recrystallized with tert-butyl methyl ether
(20 mL) to give 5 as a pale-yellow solid (5.1 g, 53.1%): MS (ESI)
m/z 193 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 8.45 (dd,
J=8.2, 4.1 Hz, 2 H), 7.17 (d, J=8.2 Hz, 1 H), 5.60 (br s, 1 H), 2.39
(s, 3 H), 1.46 (s, 9 H).
1-Chloro-3-pyridin-4-yl[2,6]naphthyridine (9b). A solution of
8a (6.50 g, 20.8 mmol) in DMF (12 mL) was heated to 220 ꢀC for
5 min in a microwave oven. The product, 3-pyridin-4-ylpyrano-
[4,3-c]pyridin-1-one, precipitated from the reaction mixture and
was isolated by filtration. The filtrate was heated two more times
to 220 ꢀC for 5 min in a microwave oven to give two other crops
of the product. The precipitates were combined to yield 3-pyr-
idin-4-ylpyrano[4,3-c]pyridin-1-one (4.05 g, 17.2 mmol, 83%) as
a white solid: MS (ESI) m/z 225.1 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.09 (s, 1H), 8.83 (d, 1H), 8.74 (d, 2H), 8.04
(d, 1H), 7.85 (m, 3H).
A suspension of 3-pyridin-4-ylpyrano[4,3-c]pyridin-1-one
(6.70 g, 28.4 mmol) in NH3 (7 M in MeOH) was stirred for 2 h at
room temperature. The reaction mixture was evaporated to
dryness to yield 3-hydroxy-3-pyridin-4-yl-3,4-dihydro-2H-[2,6]-
naphthyridin-1-one (7.12 g, 28.0 mmol, 99%) as a white solid:
MS (ESI) m/z 242.3 (M þ 1); 1H NMR (400 MHz, DMSO-d6)
δ ppm 9.11 (s, NH), 8.65 (d, 1H), 8.60 (m, 3H), 7.79 (d, 1H), 7.58
(d, 2H), 6.72 (s, OH), 3.36 (m, 1H), 3.14 (m, 1H).
N-tert-Butyl-3-(2-(pyridin-4-yl)-2-oxoethyl)isonicotinamide (8a).
To 5 (10.4 g, 51.4 mmol) in THF (220 mL) was added n-BuLi
(69.0 mL, 110 mmol, 1.6 M in hexanes) at -45 ꢀC under an
inert atmosphere. The reaction mixture was stirred for 60 min
at -45 ꢀC to obtain a bright-red suspension. Then isonicotinic
acid methyl ester (6.54 mL, 54.8 mmol) was added in one portion.
The cooling bath was removed, and stirring was continued for
2 h at room temperature. The reaction mixture was diluted
with EtOAc and washed with saturated aqueous NH4Cl solution.
The organic layer was separated, and the aqueous layer was
extracted with EtOAc (3ꢀ). The combined organic layers were
dried over MgSO4, filtered, and evaporated to dryness. The
residue was purified by column chromatography (SiO2, gradient
elution, EtOAc/MeOH 100:0 f 90:10) to yield the title com-
pound (11.6 g, 37.1 mmol, 72%) as a pale-yellow solid: MS (ESI)
m/z 298.2 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 8.83
(d, 2 H), 8.56 (d, 1 H), 8.51 (s, 1 H), 8.11 (s, NH), 7.86 (d, 2 H),
7.38 (d, 1 H), 4.63 (d, 2 H), 1.21 (s, 9 H).
A suspension of 3-hydroxy-3-pyridin-4-yl-3,4-dihydro-2H-[2,6]-
naphthyridin-1-one (3.00 g, 11.8 mmol) in POCl3 (50 mL) was
heated to 80 ꢀC for 24 h. The reaction mixture was concentrated
under reduced pressure to remove excess POCl3. The residual
oil was treated with ice-cold H2O, and the suspension thus
obtained was basified to pH 14 with 10 N NaOH while keeping
the temperature below room temperature. The mixture was
filtered and the aqueous filtrate was extracted with CH2Cl2
(2ꢀ). The combined organic layers were dried over MgSO4,
filtered, and evaporated to dryness to yield a first crop of the
crude title compound. The gluey precipitate obtained from the
filtration was extracted with CH2Cl2 (stirring for 10 min at room
temperature, 2ꢀ) to yield a second crop of the crude title
compound. The combined crude products were purified by
column chromatography (SiO2, gradient elution, CH2Cl2/MeOH
100:0 f 94:6) to yield the title compound (932 mg, 3.78 mmol,
1
32%) as a white solid: MS (ESI) m/z 242.2 (M þ 1); H NMR
N-tert-Butyl-3-(2-(2-chloropyridin-4-yl)-2-oxoethyl)isonicoti-
namide (8b). The title compound was prepared from 5 above and
2-chloroisonicotinic acid methyl ester by the method described
(400 MHz, DMSO-d6) δ ppm 9.56 (s, 1H), 8.89 (m, 2H), 8.76 (m,
2H), 8.11 (m, 3H).
1-Chloro-3-(2-chloropyridin-4-yl)-2,6-naphthyridine (9c). Com-
pound 8b above (6.83 g, 20.46 mmol) was suspended in acetic
acid and heated at 100 ꢀC overnight. The mixture was cooled
to room temperature and evaporated under reduced pressure.
To the residue was added H2O, and the product was extracted
with EtOAc. The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and then concentrated. The
solid was triturated with Et2O to give 3-(2-chloropyridin-4-yl)-
1H-pyrano[4,3-c]pyridin-1-one as an off-white solid (60%): MS
(ESI) m/z 259.7 (M þ 1); 1H NMR(400 MHz, CDCl3) δ ppm9.12
(s, 1 H), 8.86 (d, J=5.5 Hz, 1 H), 8.56 (d, J=5.5 Hz, 1 H), 8.18
(d, J=5.1 Hz, 1 H), 8.03 (s, 1 H), 7.93 (d, J=5.1 Hz, 1 H), 7.77
(s, 1 H).
3-(2-Chloropyridin-4-yl)-1H-pyrano[4,3-c]pyridin-1-one (1.66 g,
6.41 mmol) was suspended in EtOH (35 mL). To the suspension
was added 28.5% NH4OH (26 mL) at room temperature, and
the mixture was stirred overnight. The solvent was then evapo-
rated, and the residue was dried under reduced pressure at 45 ꢀC
for 30 min to provide a white solid. The crude product was
suspended in EtOH (35 mL), treated with 4 N HCl (8.7 mL), and
stirred overnight at room temperature. The mixture was filtered,
and the solid obtained was dried under high vacuum (1.50 g,
91%): MS (ESI) m/z 257.7 (M þ 1); 1H NMR (400 MHz, CDCl3)
δ ppm 9.51 (s, 1 H), 8.84 (d, J=6.1 Hz, 1 H), 8.72 (d, J=6.1 Hz,
1 H), 8.62 (d, J=5.0 Hz, 1 H), 7.95 (s, 1 H), 7.80 (d, J=5.0 Hz, 1 H),
7.38 (s, 1 H).
1
above for the synthesis of 8a: MS (ESI) m/z 332.8 (M þ 1); H
NMR (400 MHz, CDCl3) δ ppm 8.63 (m, 2 H), 8.48 (s, 1 H), 7.84
(s, 1 H), 7.74 (dd, J=5.1, 1.5 Hz, 1 H), 7.33 (d, J=5.1 Hz, 1 H),
5.90 (br s, 1 H), 4.57 (s, 2 H).
1-Bromo-3-pyridin-4-yl[2,6]naphthyridine (9a). A solution of
8a (6.50 g, 20.8 mmol) in DMF (12 mL) was heated to 220 ꢀC for
5 min in a microwave oven. The product, 3-pyridin-4-ylpyrano-
[4,3-c]pyridin-1-one, precipitated from the reaction mixture and
was isolated by filtration. The filtrate was heated two more times
to 220 ꢀC for 5 min in a microwave oven to give two other crops
of the product. The precipitates were combined to yield 3-pyr-
idin-4-ylpyrano[4,3-c]pyridin-1-one (4.05 g, 17.2 mmol, 83%) as
a white solid: MS (ESI) m/z 225.1 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.09 (s, 1H), 8.83 (d, 1H), 8.74 (d, 2H), 8.04
(d, 1H), 7.85 (m, 3H).
A suspension of 3-pyridin-4-ylpyrano[4,3-c]pyridin-1-one
(6.70 g, 28.4 mmol) in NH3 (7 M in MeOH) was stirred for 2 h at
room temperature. The reaction mixture was evaporated to dryness
to yield 3-hydroxy-3-pyridin-4-yl-3,4-dihydro-2H-[2,6]naphthyridin-
1-one (7.12 g, 28.0 mmol, 99%) as a white solid: MS (ESI) m/z
1
242.3 (M þ 1); H NMR (400 MHz, DMSO-d6) δ ppm 9.11
(s, NH), 8.65 (d, 1H), 8.60 (m, 3H), 7.79 (d, 1H), 7.58(d, 2H), 6.72
(s, OH), 3.36 (m, 1H), 3.14 (m, 1H).
3-Hydroxy-3-pyridin-4-yl-3,4-dihydro-2H-[2,6]naphthyridin-
1-one (1.20 g, 5.38 mmol) and POBr3 (3.95 g, 13.7 mmol) were
mixed under argon and heated to 130 ꢀC for 6 h. After the
mixture was cooled, ice was added and the pH adjusted to 8 with
2 N Na2CO3. The precipitate was filtered, washed with water,
and dried to yield the title compound (1.20 g, 4.19 mmol, 78%) as
brown powder: MS (ESI) m/z 287 (M þ 1). The crude product
was used without further purification.
A mixture of 3-(2-chloropyridin-4-yl)-2,6-naphthyridin-1-ol
(1.29 g, 5.02 mmol), POCl3 (35 mL), and tetramethylammonium
chloride (2.6 g, 23.72 mmol) was refluxed at 110 ꢀC for 36 h. The
POCl3 was removed by distillation, and ice cold 10% K2CO3
was added carefully. The mixture was extracted with EtOAc,
washed with water and then brine, dried over anhydrous

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5415
Na2SO4, and concentrated under reduced pressure. The crude
residue was washed with cold methanol (5 mL) to give the title
compound as a greenish brown solid (1.05 g, 3.80 mmol, 76%):
(0.7 mL) and CH2Cl2 (20 mL) for 10 min at 0 ꢀC and at room
temperature for 4 h. The reaction mixture was concentrated.
Twice CH2Cl2 was added and the mixture concentrated. The
residue was crystallized from diethyl ether to yield the title
compound (94 mg, 0.17 mmol, 78%) as yellow crystals (TFA
salt): MS (ESI) m/z 266.1 (M þ 1); 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.32 (s, 1 H), 8.88 (d, J=6.0 Hz, 2 H), 8.74 (d, J=6.6
Hz, 1 H), 8.49 (d, J=6.0 Hz, 2 H), 8.17 (m, 3 H), 7.94 (m, 2 H),
3.91 (m, 2 H), 3.25 (m, 2 H).
1
MS (ESI) m/z 277.1 (M þ 1); H NMR (400 MHz, CDCl3) δ
ppm 9.55 (s, 1 H), 8.88 (d, J=6.1 Hz, 1 H), 8.75 (s, 1 H), 8.55 (d,
J=5.6 Hz, 1 H), 8.27 (s, 1 H), 8.25 (d, J=6.1 Hz, 1 H), 8.18 (d,
J=5.5 Hz, 1 H).
3-(2-Chloropyridin-4-yl)-1-methoxy[2,6]naphthyridine (23). To
a solutionof9c (0.91 g, 3.3 mmol) in methanol (20mL) was added
sodium methoxide (0.27 g, 5 mmol). The mixture was refluxed for
3 h and cooled to room temperature. Volatiles were removed by
rotary evaporation, and the residue was partitioned between
water and CH2Cl2. The separated aqueous phase was extracted
twice with CH2Cl2, and the combined organics were washed with
water and brine, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was chromatographed on silica gel to give a
light-yellow solid (0.80 g, 89%): MS (ESI) m/z 272.1 (M þ 1); 1H
NMR (400 MHz, CDCl3) δ ppm 9.3 (s, 1 H), 8.75 (d, J=6.3 Hz, 1
H), 8.5 (s, 1 H), 8.08 (s, 1 H), 8.02 (d, J=5.6 Hz, 1 H), 7.93 (dd,
J=5.1, 1.4 Hz, 1 H), 7.86 (s, 1 H), 4.28 (s, 3 H).
[4-(1-Chloro[2,6]naphthyridin-3-yl)pyridin-2-yl]cyclohexylamine
(24). To a solution of methoxynaphthyridine 23 (0.43 g, 1.29
mmol) in wet tert-butyl alcohol (10 mL) is added potassium tert-
butoxide (1 M in THF, 6.4 mL, 6.4 mmol), and the mixture was
heated at 100 ꢀC overnight. After the volatiles were removed
under reduced pressure, the residue was suspended in water. The
pH was adjusted to 7, and the resulting solid was filtered and air-
dried. The crude solid (406 mg, 94%) was pure by LCMS and
used directly in the next step without further purification: MS
(ESI) m/z 321.3 (M þ 1).
1-Piperazin-1-yl-3-pyridin-4-yl[2,6]naphthyridine (11). To a
solution of chloride 9b (50 mg, 0.197 mmol) in DMF (0.5 mL)
was added piperazine-1-carboxylic acid tert-butyl ester (77 mg,
0.393 mmol) and K2CO3 (55 mg, 0.393 mmol) at room tem-
perature. The reaction mixture was heated to 90 ꢀC and
stirred for 5 h. The reaction mixture was cooled to room
temperature, diluted with EtOAc, and washed with H2O (1ꢀ).
The organic layer was separated, and the aqueous layer was
extracted with EtOAc (3ꢀ). The combined organic layers were
dried over MgSO4, filtered, and evaporated to dryness. The
residue was purified by silica gel chromatography (4 g of SiO2,
gradient hexane/EtOAc 80:20 f 0:100) to afford 4-(3-pyridin-4-
yl[2,6]naphthyridin-1-yl)piperazine-1-carboxylic acid tert-butyl
ester (10c) as a pale-yellow solid. Compound 10c was dissolved
in TFA (0.5 mL) and CH2Cl2 (0.5 mL) and was stirred for 1 h at
room temperature. The reaction mixture was concentrated
under reduced pressure. The residue was purified by preparative
reverse phase HPLC (Waters) to yield the title compound as a
yellow solid (25 mg, 0.048 mmol, 25% over two steps, 2TFA
salt): MS (ESI) m/z 292.3 (M þ 1); 1H NMR (600 MHz, DMSO-
d6) δ ppm 9.46 (s, 1 H), 9.06 (br s, 2 H), 8.88 (d, J=6.3 Hz, 2 H),
8.74 (d, J=5.7 Hz, 1 H), 8.57 (s, 1 H), 8.42 (d, J=6.1 Hz, 2 H),
8.06 (d, J=5.9 Hz, 1 H), 3.75 (br s, 4 H), 3.43 (br s, 4 H).
4-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperazine-1-
carboxylic Acid tert-Butyl Ester (12b). To a suspension of 9c
(2.00 g, 7.20 mmol) in anhydrous ethanol (24 mL) in a dried
pressure vessel was added triethylamine (3.20 mL, 23 mmol)
followed by piperazine-1-carboxylic acid tert-butyl ester (1.70 g,
9.10 mmol). The vessel was flushed with nitrogen and then
sealed. The suspension was heated in a 100 ꢀC oil bath for
20 h. The dark-brown, nearly homogeneous reaction mixture
was cooled to room temperature, then concentrated in vacuo.
The residue was diluted with dichloromethane and water. The
layers were agitated and separated, and the aqueous layer was
extracted twice with CH2Cl2. The combined organic layers were
dried over sodium sulfate, filtered, and concentrated. The ma-
terial was purified by silica gel chromatography (120 g SiO2,
gradient 0% f 2.5% methanol/dichloromethane) to afford a
brownish yellow solid, which was refluxed in 60 mL of diethyl
ether. The mixture was cooled to room temperature and filtered
to provide the title compound as a white solid (2.2 g, 71%): MS
(ESI) m/z 426.2 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm
9.39 (d, J=0.8 Hz, 1 H), 8.70 (d, J=5.8 Hz, 1 H), 8.55 (dd, J=5.3,
0.5 Hz, 1 H), 8.24 (dd, J=1.5, 0.8 Hz, 1 H), 8.18 (dd, J=5.3, 1.5
Hz, 1 H), 7.96 (d, J=5.8 Hz, 1 H), 3.64 (br m, 4 H), 3.52 (m, 4 H),
1.45 (s, 9 H).
A mixture of the naphthyridinone above (360 mg, 1.1 mmol)
and tetramethylammonium chloride (100 mg) in POCl3 (15 mL)
was heated at 110 ꢀC overnight. Volatiles were removed by
evaporation, and the residue was treated with ice, basified with
1 N NaOH, and extracted with CH2Cl2 (3 ꢀ 20 mL). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated. The residue was chromatographed on silica gel to
give the product (260 mg, 68%): MS (ESI) m/z 339.2 (M þ 1); 1H
NMR (400 MHz, CDCl3) δ ppm 9.26 (s, 1 H), 8.61 (d, J=5.4 Hz,
1 H), 8.18 (d, J=5.2 Hz, 1 H), 7.79 (d, J=6.0 Hz, 1 H), 7.75 (s, 1
H), 7.21 (s, 1 H), 7.20 (d, J=5.4 Hz, 1 H), 4.64 (d, J=8.0 Hz, 1
H), 3.69 (br 1 H), 2.12 (m, 2 H), 1.79 (m, 2 H), 1.67 (m, 1 H),
1.6-1.4 (m, 5 H).
[2-(3-Pyridin-4-yl[2,6]naphthyridin-1-ylamino)ethyl]carbamic
Acid tert-Butyl Ester (10a). Bromide 9a (200 mg, 0.70 mmol)
and (2-aminoethyl)carbamic acid tert-butyl ester (172 mg, 1.05
mmol) in dioxane (30 mL) was heated with 5 drops of 40%
NaOH to 110 ꢀC for 3 days. After cooling, the reaction mixture
was diluted with water and extracted with ethyl acetate. The
combined organic layers were dried over sodium sulfate, filtered,
and concentrated. The residue was purified by chromatography
(ethyl acetate/ethanol 9:12), yielding the title compound (110 mg,
0.30 mmol, 42%) as a light-brown powder: MS (ESI) m/z 366.2
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 9.23 (s, 1 H),
8.69 (d, J=6.0 Hz, 2 H), 8.63 (d, J=6.6 Hz, 1 H), 8.18 (d, J=6.0
Hz, 2 H), 8.12 (d, J=6.6 Hz, 1 H), 7.96 (md, 1 H), 7.88 (s, 1 H),
7.05 (m, 1 H), 3.68 (m, 2 H), 3.33 (m, 2 H), 1.39 (s, 9 H).
3-(3-Pyridin-4-yl[2,6]naphthyridin-1-ylamino)propan-1-ol (10b).
Bromide 9a (250 mg, 0.87 mmol) and 1-hydroxypropylamine
(657 mg, 8.74 mmol) in THF (10 mL) were heated to 45 ꢀC for
2 h. After cooling, the reaction mixture was concentrated under
reduced pressure and the residue purified by chromatography
(ethyl acetate/ethanol 8:2), yielding the title compound (50 mg,
0.18 mmol, 20%) as a yellow powder: MS (ESI) m/z 281.3 (M þ
1); 1H NMR (400 MHz, DMSO-d6) δ ppm 9.24 (s, 1 H), 8.73 (d,
J=6.0 Hz, 2 H), 8.64 (d, J=6.6 Hz, 1 H), 8.17 (d, J=6.6 Hz, 1 H),
8.15 (d, J=6.0 Hz, 2 H), 7.91 (m, 1 H), 7.87 (s, 1 H), 4.54 (m, 1 H),
3.70 (m, 2 H), 3.58 (m, 2 H), 1.92 (m, 2 H).
3-(2-Chloropyridin-4-yl)-1-piperazin-1-yl[2,6]naphthyridine (12c).
A solution of compound 9c above (700 mg, 2.53 mmol) and
piperazine (262 mg, 3.04 mmol) in NMP (12 mL) was heated to
70 ꢀC overnight. After the mixture was cooled, a yellow solid was
collected by filtration and washed with MeOH (5 mL) to afford the
title compound: MS (ESI) m/z 326.2, 328.2 (M þ 1); 1H NMR (400
MHz, MeOD) δ ppm 10.00 (s, 1 H), 9.28 (d, J=5.6 Hz, 1 H), 9.07
(d, J=5.1 Hz, 1 H), 8.79 (m, 4 H), 4.43 (m, 4 H), 4.11 (m, 4 H).
3-(2-Chloropyridin-4-yl)-1-(4-cyclopropylmethylpiperazin-1-yl)-
[2,6]naphthyridine (12d). The title compound was prepared from
9c (200 mg, 0.72 mmol) and N-cyclopropylmethylpiperazine
(140 μL, 0.94 mmol) by heating in Et3N (6 μL, 4.3 mmol) and
EtOH (5 mL) in a sealed tube at 100 ꢀC overnight. The volatiles
were removed in vacuo and the residue was purified by flash
chromatography, eluting with 0-10% MeOH/CH2Cl2 to afford
N*1*-(3-Pyridin-4-yl[2,6]naphthyridin-1-yl)ethane-1,2-diamine
(2). Compound 10a (80 mg, 0.22 mmol) was stirred in TFA

5416 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
the title compound in 68% yield as a yellow solid: MS (ESI) m/z
380.3 (M þ 1).
give the title compound (90 mg, 0.29 mmol, 82%) as a yellow
1
powder: MS (ESI) m/z 310.4 (M þ 1); H NMR (400 MHz,
DMSO-d6) δ ppm 9.22 (s, 1 H), 8.59 (d, J=6.6 Hz, 1 H), 8.14 (d,
J=6.6 Hz, 1 H), 8.08 (d, J=6.0 Hz, 1 H), 7.82 (t, J=4.5 Hz, 1 H),
7.64 (s, 1 H), 7.27 (s, 1 H), 7.21 (d, J=6.0 Hz, 1 H), 6.60 (d, J=3.0
Hz, 1 H), 4.53(t, J=5.2Hz, 1 H), 3.69(m, 2 H), 3.59 (m, 2 H), 2.85
(d, J=3.6 Hz, 3 H), 1.90 (m, 2 H).
3-(2-Chloropyridin-4-yl)-1-(4-cyclopropylpiperazin-1-yl)[2,6]-
naphthyridine (12e). The title compound was prepared from 9c
and N-cyclopropylpiperazine by analogy to the method out-
lined in example 12b: MS (ESI) m/z 366.3 (M þ 1).
1-{4-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperazin-
1-yl}ethanone (12f). The title compound was prepared from 9c
and N-acetylpiperazine by analogy to the method outlined in
example 12b: MS (ESI) m/z 368.1 (M þ 1).
4-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperazine-1-carboxylic Acid tert-Butyl Ester (13b). Compound
12b (2.30 g, 5.4 mmol), cyclohexylamine (1.24 mL, 10.8 mmol),
NaO-t-Bu (1.04 g, 10.8 mmol), and Pd(P-t-Bu3)2 in dioxane
(54 mL) were heated under an Ar atmosphere in a sealed tube
at 130 ꢀC overnight. After cooling, the mixture was concentrated
under reduced pressure. The residue was diluted with 20%
acetone/CH2Cl2 and filtered through Celite. The filter cake was
washed successively with EtOH, acetone, CH3CN, and MeOH.
The combined filtrate was concentrated under reduced pressure.
The residue was diluted with EtOAc (400 mL) and washed
with water (3 ꢀ 100 mL). The separated EtOAc layer was dried
(Na2SO4), filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography using 20%
acetone/CH2Cl2 to afford the title compound: MS (ESI) m/z
489.2 (M þ 1).
2-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-ylamino]ethanol
(12g). A solution of compound 9c (536 mg, 1.94 mmol) and
2-aminoethanol (1.50 mL, 25.2 mmol) in NMP (15 mL) was
heated to 100 ꢀC overnight. After the mixture was cooled, the
volatiles were removed in vacuo and the residue was purified by
RP-HPLC (3-50% CH3CN in H2O) to afford the title com-
pound as a yellow solid: MS (ESI) m/z 301.1 (M þ 1); 1H NMR
(600 MHz, DMSO-d6) δ ppm 9.55 (m, 1 H), 8.83 (m, 1 H), 8.71
(m, 1 H), 8.57 (d, J=5.3 Hz, 1 H), 8.17 (m, 1 H), 8.12 (dd,, J=5.3,
1.4 Hz, 1 H), 8.10 (s, 1 H), 3.75-3.76 (m, 4 H).
4-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperazin-2-
one (12h). The title compound was prepared from 9c and piper-
azin-2-one by analogy to the method outlined in example 12b: MS
(ESI) m/z 340.1 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm
9.78 (s, 1 H), 9.10 (d, J=5.6 Hz, 1 H), 8.96 (d, J=5.1 Hz, 1 H), 8.85
(s, 1 H), 8.64 (s, 1 H), 8.59 (m, 1 H), 8.59 (m, 1 H), 8.41 (m, 2 H),
4.54 (m, 2 H), 4.22 (m, 2 H), 3.83 (m, 2 H).
{1-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperidin-4-
ylmethyl}carbamic Acid tert-Butyl Ester (12i). The title com-
pound was prepared from 9c and piperidin-4-ylmethylcarbamic
acid tert-butyl ester by analogy to the method outlined in 12b:
MS (ESI) m/z 454.3 (M þ 1).
Cyclohexyl-[4-(1-piperazin-1-yl[2,6]naphthyridin-3-yl)pyridin-
2-yl]amine (13c). To a solution of 13b (1.34 g, 2.74 mmol) in
CH2Cl2 (33mL) was addedTFA(6.6mL). After 1handcomplete
removal of the BOC group as judged by TLC, the mixture was
diluted with CH2Cl2 (200 mL) and a 1 M soluton of NaOH
(100 mL). The separated organic phase was washed with fresh
1 M NaOH. The combined aqueous phases were extracted with
fresh CH2Cl2 (2 ꢀ 100 mL). The combined organic phasese were
dried (Na2SO4), filtered, and concentrated under reduced pres-
sure to afford the title compound: MS (ESI) m/z 389.3 (M þ 1);
1H NMR (400 MHz, CD3OD) δ ppm 9.35 (s, 1 H), 8.62 (d, J=6.1
Hz, 1 H), 8.07 (s, 1 H), 8.05 (d, J=5.5 Hz, 1 H), 8.02 (d, J=5.6 Hz,
1 H), 7.44 (s, 1 H), 7.30 (d, J=5.6 Hz, 1 H), 3.80-3.71 (m, 1 H),
3.71(s,1 H), 3.63 (t, J=4.8Hz, 4 H), 3.20 (t, J=4.8 Hz, 4H), 2.15-
2.08 (m, 2 H), 1.90-1.82 (m, 2 H), 1.78-1.71 (m, 1 H), 1.58-1.46
(m, 2 H), 1.40-1.09 (m, 3 H).
Cyclohexyl-{4-[1-(4-isobutylpiperazin-1-yl)[2,6]naphthyridin-3-
yl]pyridin-2-yl}amine (13d). To a solution of 13c (260 mg, 0.62
mmol) and 2-methylpropionaldehyde (54 mg, 0.76 mmol) in
CH2Cl2 (5 mL) was added NaBH(OAc)3 (540 mg, 2.7 mmol).
The suspension was stirred at room temperature for 24 h. A
saturated aqueous solution of NaHCO3 (5 mL) was added,
and the mixture was extracted with CH2Cl2 (3 ꢀ 10 mL). The
combined organic layers were dried (Na2SO4), filtered, and
concentrated under reduced pressure. The oily residue was
purified by RP-HPLC (30-90% CH3CN in H2O to afford the
title compound as a white solid: HRMS (ESI) m/z445.3087 (M þ 1);
1H NMR (400 MHz, CDCl3) δ ppm 9.26 (s, 1 H), 8.61 (d,
J=5.7 Hz, 1 H), 8.20-8.14 (m, 1 H), 7.81 (d, J=5.8 Hz, 1 H),
7.75 (s, 1 H), 7.24-7.18 (m, 2 H), 4.61 (d, J = 7.7 Hz, 1 H),
3.77-3.65 (m, 1 H), 3.65-3.57 (m, 4 H), 2.74-2.65 (m, 3 H), 2.22
(d, J=7.3 Hz, 2 H), 2.13 (dd, J=12.6, 3.2 Hz, 2 H), 1.98-1.74 (m,
4 H), 1.73-1.62(m, 1 H), 1.54-1.38 (m, 2 H), 1.35-1.20 (m, 3 H),
0.96 (d, J=6.6 Hz, 6 H).
1-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperidine-4-
carboxylic Acid Amide (12j). To a suspension of 9c (200 mg, 0.72
mmol) in anhydrous ethanol (2.4 mL) in a sealable vial was
added triethylamine (0.32 mL, 2.3 mmol) followed by isonipe-
cotamide (120 mg, 0.90 mmol). The vial was flushed with
nitrogen and then sealed. The mixture was heated in a 100 ꢀC
oil bath for 16 h. The heterogeneous mixture was cooled to room
temperature, then filtered. The filtrate was washed with ethanol
and dried under high vacuum to give the title compound
as a yellowish brown solid (230 mg, 87%): MS (ESI) m/z 368.1
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (s, 1 H),
8.68 (d, J=5.8 Hz, 1 H), 8.55 (d, J=5.8 Hz, 1 H), 8.38 (s, 1H),
8.23 (s, 1 H), 8.18 (dd, J=5.3, 1.5 Hz, 1 H), 7.89 (d, J=5.8 Hz, 1
H), 7.34 (br s, 1 H), 6.82 (br s, 1 H), 4.05 (d, J=12.6 Hz, 2 H), 3.09
(m, 2 H), 2.43 (m, 1 H), 1.91 (br m, 4 H).
1-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperidine-4-
carboxylic Acid Methylamide. (12k). The title compound was
prepared from 9c and N-methylisonipecotamide by analogy to
the method outlined in example 12b: MS (ESI) m/z 382.1 (M þ 1).
1-[3-(2-Chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperidine-4-
carboxylic Acid Methyl Ester (12l). A solution of 9c (1.0 g, 3.62
mmol), Et3N (1.50 mL, 10.9 mmol), methyl isonipecotate (0.74
mL, 5.43 mmol), and DMSO (4 mL) was heated to 80 ꢀC for 3 h.
At that time, the solution was allowed to cool to room tempera-
ture and was slurried with 15 mL of water. The mixture was then
poured into 200 mL of ice water. After 10 min the solid was
collected via filtration. The solid was then driedunder vacuum to
give (1.25 g) the title compound as a brown solid: MS (ESI) m/z
383.1 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 9.29 (s, 1 H),
8.67 (d, J=5.8 Hz, 1 H), 8.49 (d, J=4.8 Hz, 1 H), 8.07 (s, 1 H),
7.95 - 7.91 (m, 1 H), 7.83 (s, 1 H), 7.79 (d, J=5.8 Hz, 1 H), 4.07 -
3.96 (m, 2 H), 3.76 (s, 3 H), 3.27-3.14 (m, 2 H), 2.73-2.61 (m, 1
H), 2.23-2.01 (m, 4 H).
4-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphthy-
ridin-1-yl}piperazine-1-carboxylic Acid tert-Butyl Ester (13e). To
a dried sealable vial was added 12b (180 mg, 0.43 mmol), sodium
tert-butoxide (120 mg, 1.30 mmol), and dioxane (6.1 mL). The
dark-red solution was sparged with argon for 10 min. 4-Amino-
tetrahydropyran (0.16 mL, 1.30 mmol) was added via syringe,
followed by palladium(0)tris(tri-tert-butylphosphine) (44 mg,
0.09 mmol). The vial was flushed with argon, then sealed and
heated in a 120 ꢀC oil bath for 14 h. The dark-brown solution was
cooled to roomtemperature, thendiluted withwater and CH2Cl2.
The layers were agitated and separated. The aqueous layer was
extracted twice with CH2Cl2. The combined organic layers were
dried over sodium sulfate, filtered, and concentrated to give a
1-(3-Hydroxypropylamino-3-(2-methylaminopyridin-4-yl)[2,6]-
naphthyridine (13a). Compound 12a (120 mg, 0.36 mmol) and
methylamine (5 mL, 33% in ethanol) were heated in an autoclave
at 135 ꢀC for 4 d. The reaction mixture was cooled, concentrated,
and purified by chromatography (ethyl acetate/ethanol 8:2) to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5417
brown residue. Purification via silica gel chromatography (40 g of
SiO2, gradient 70% f 100% ethyl acetate/hexanes followed by
0% f 10% MeOH/CH2Cl2) afforded the title compound as a
brown solid (120 mg, 80% pure) as judgedby 1H NMR:MS(ESI)
m/z 491.5 (M þ 1).
Cyclohexyl-{4-[1-(4-cyclopropylmethylpiperazin-1-yl)[2,6]na-
phthyridin-3-yl]pyridin-2-yl}amine (13k). The title compound
was prepared from 12d and cyclohexylamine by analogy to the
method described for the preparation of 13e above: HRMS
(ESI) m/z 443.2924 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm
9.27 (s, 1 H), 8.61 (d, J=5.8 Hz, 1 H), 8.18 (d, J=5.3 Hz, 1 H),
7.80 (d, J=5.8 Hz, 1 H), 7.76 (s, 1 H), 7.25-7.16 (m, 2 H), 4.59
(d, J=8.1 Hz, 1 H), 3.79-3.57 (m, 5 H), 2.84 (t, J=4.6 Hz, 4 H),
2.40 (d, J=6.6 Hz, 2 H), 2.19-2.06 (m, 2 H), 1.88-1.73 (m, 2 H),
1.73-1.61 (m, 1 H), 1.55-1.37 (m, 2 H), 1.36-1.19 (m, 3 H),
1.03-0.87 (m, 1 H), 0.64-0.51 (m, 2 H), 0.18 (q, J=4.9 Hz, 2 H).
Cyclohexyl-{4-[1-(4-cyclopropylpiperazin-1-yl)[2,6]naphthyri-
din-3-yl]pyridin-2-yl}amine (13l). The title compound was pre-
pared from 12e and cyclohexylamine by analogy to the method
described for the preparation of 13e above: HRMS (ESI) m/z
429.2768 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 9.27 (s, 1
H), 8.62 (d, J=5.8 Hz, 1 H), 8.18 (d, J=5.4 Hz, 1 H), 7.81 (d, J=
5.8 Hz, 1 H), 7.76 (s, 1 H), 7.24-7.17 (m, 2 H), 4.60 (d, J=7.9 Hz,
1 H), 3.76-3.63 (m, 1 H), 3.63-3.52 (m, 3 H), 2.98-2.85 (m, 4
H), 2.19-2.07 (m, 2 H), 1.90-1.73 (m, 4 H), 1.70 (d, J=3.4 Hz, 1
H), 1.55-1.38 (m, 2 H), 1.35-1.19 (m, 3 H), 0.60-0.46 (m, 4 H).
1-{4-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperazin-1-yl}ethanone (13m). The title compound was prepared
from 12f and cyclohexylamine by analogy to the method described
for the preparation of 13e above: MS (ESI) m/z 431.4 (M þ 1); 1H
NMR (400 MHz, CD3OD) δ ppm 9.31 (d, 1 H), 8.60 (d, 1 H), 8.04
(s, 1 H), 8.00 (m, 2 H), 7.37 (d, 1 H), 7.22 (d, 1 H), 3.88 (br d, 4 H),
3.70 (m, 1 H), 3.61 (br d, 4 H), 2.19 (s, 3 H), 2.08 (br m, 2 H), 1.80
(m, 2 H), 1.69 (br d, 1 H), 1.47 (m, 2 H), 1.28 (m, 3 H).
4-(1-Piperazin-1-yl[2,6]naphthyridin-3-yl)pyridin-2-yl](tetrahy-
dropyran-4-yl)amine (13f). To a suspension of 13e (120 mg, 0.25
mmol) in 1.80 mL of CH2Cl2 at 0 ꢀC is added 0.60 mL of TFA
drop by drop via pipet. The resulting brownish orange solution is
warmed to room temperature and stirred for 1 h and then
concentrated in vacuo to afford an amber oil. The residue was
dissolved in MeOH and a small amount of water, then purified
via preparative reverse-phase HPLC (X-Bridge C18 column, flow
rate = 40 mL/min, gradient 10% f 80% acetonitrile/5 mM
aqueous ammonium hydroxide over 20 min) to give the title
compound as a pale-yellow solid (66 mg, 68%): MS (ESI) m/z
391.2 (M þ 1); 1H NMR (400 MHz, CD3OD) δ ppm 9.30 (d, J=
1.0 Hz, 1 H), 8.58(d, J=5.8Hz, 1 H), 8.03 (m, 2 H), 7.98(d, J=5.8
Hz, 1 H), 7.42 (s, 1H), 7.29(dd, J=5.7, 1.6 Hz, 1 H), 3.99 (m, 3 H),
3.59 (m, 6H), 3.14 (dd, J=5.6, 4.0 Hz, 4 H), 2.03 (dd, J=12.2, 2.8
Hz, 2 H), 1.58 (m, 2 H).
{4-[1-(4-Isobutylpiperazin-1-yl)[2,6]naphthyridin-3-yl]pyridin-
2-yl}(tetrahydropyran-4-yl)amine (13g). The title compound is
prepared by reductive amination of 13f commercially with
available 2-methylpropionaldehyde. Thus, sodium triacetoxy-
borohydride (339 mg, 1.59 mmol) is added to a solution of 13f
(148 mg, 0.38 mmol) and 2-methylpropionaldehyde (42 μL, 0.46
mmol) in methylene chloride (8 mL) and stirred for 12 h. The
mixture is concentrated on a rotary evaporator and partially
purified on a 12 g (Redisep, Isco) silica gel column with a 0-10%
methanol/methylene chloride gradient. The resulting product
is further purified by reverse phase HPLC using a 30-95%
acetonitrile/water gradient. This yields 41.2 mg (24%) after
consolidation and concentration of fractions: HRMS (ESI)
2-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-ylami-
no]ethanol (13n). The title compound was prepared from 12g
and cyclohexylamine by analogy to the method described for the
preparation of 13e above: MS (ESI) m/z 363.4 (M þ 1); 1H NMR
(400 MHz, CD3OD) δ ppm 9.21 (s, 1 H), 8.56 (d, 1 H), 8.04 (m, 2
H), 7.66 (s, 1 H), 7.22 (m, 2 H), 3.93 (m, 4 H), 3.74 (m, 2 H), 2.08
(m, 2 H), 1.84 (m, 2 H), 1.73 (m, 1 H), 1.51 (m, 2 H), 1.32 (m, 4 H).
4-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]pi-
perazin-2-one (13o). The title compound was prepared from 12h
and cyclohexylamine by analogy to the method described for the
preparation of 13e above: MS (ESI) m/z 403.3 (M þ 1).
1
m/z 447.2717 (M þ 1); H NMR (400 MHz, CDCl3) δ ppm
9.29 (s, 1 H), 8.64 (d, J=5.8 Hz, 1 H), 8.22 (d, J=5.3 Hz, 1 H),
7.84 (d, J=5.8 Hz, 1 H), 7.78 (s, 1 H), 7.30-7.28 (m, 1 H), 7.27 (s,
1 H), 4.57 (d, J=8.0 Hz, 1 H), 4.15-3.98 (m, 3 H), 3.73-3.53 (m,
6 H), 2.73 (t, J=4.6 Hz, 4 H), 2.25 (d, J=7.3 Hz, 2 H), 2.15 (dd,
J=12.6, 2.1 Hz, 2 H), 1.98-1.77 (m, 1 H), 1.70-1.54 (m, 2 H),
0.99 (d, J=6.6 Hz, 6 H).
{1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperidin-4-ylmethyl}carbamic Acid tert-Butyl Ester (13p). The
title compound was prepared from 12i and cyclohexylamine by
analogy to the method described for the preparation of 13e
above: MS (ESI) m/z 417.2 (M þ 1).
Cyclohexyl-{4-[1-((cis-3,5-dimethylpiperazin-1-yl)[2,6]naphthy-
ridin-3-yl]pyridin-2-yl}amine (13h). The title compound was pre-
pared from chloride 24 and cis-3,5-dimethylpiperazineby analogy
to the preparation of 13i below: MS (ESI) m/z 417.4 (M þ 1); 1H
NMR (400 MHz, CDCl3) δ ppm 9.3 (s, 1 H), 8.6 (d, 1 H), 8.2 (d, 1
H), 7.8 (d, 1 H), 7.7 (s, 1 H), 7.2 (m, 2 H), 4.7 (br s, 1 H), 3.9 (d, 2
H), 3.7(br s, 5 H), 3.3(br s, 2 H), 2.7(t, 2 H), 2.1(m, 2 H), 1.5(m, 8
H), 1.2 (d, 6 H).
Cyclohexyl-{4-[1-(4-methylpiperazin-1-yl)[2,6]naphthyridin-3-
yl]pyridin-2-yl}amine (13i). A solution of chloronaphthyridine 24
(34 mg, 0.1 mmol) and 1-methylpiperazine (0.033 mL, 0.3 mmol) in
2-methoxyethanol (4 mL) was heated by microwave at 200 ꢀC for
30 min. The mixture was then poured onto a solid phase extraction
(SPE) cartridge containing strong cation exchanger (SCX) as the
media (2 g). After the sample was washed with MeOH (10 mL), the
product was eluted with 20:2:1 EtOAc-MeOH-Et3N. The eluent
was concentrated in vacuo and the residue was chromatographed
on silica gel to give the product (40 mg, 99%): MS (ESI) m/z 403.4
(M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 9.3 (s, 1 H), 8.6 (d, 1
H), 8.2 (d, 1 H), 7.8 (m, 2 H), 7.2 (m, 2 H), 4.8 (br s, 1 H), 3.6 (br s, 5
H), 2.8 (br s, 4 H), 2.4 (s, 3 H), 2.1 (m, 2 H), 1.8 (m, 2 H), 1.7 (m, 1
H), 1.5 (m, 2 H), 1.3 (m, 3 H).
1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperidine-4-carboxylic Acid Piperidin-4-ylamide (13q). The title
compound was prepared from 13p by reaction with TFA/CH2Cl2
according to the method outlined for the preparation of 13c
above: MS (ESI) m/z 514.3 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.36 (s, 1 H), 8.61 (d, J=5.8 Hz, 1 H), 8.05
(d, J=5.6 Hz, 1 H), 8.03 (s, 1 H), 7.86 (d, J=5.8 Hz, 1 H), 7.80 (d,
J=7.8 Hz, 1 H), 7.28 (s, 1 H), 7.14 (dd, J=5.4, 1.5 Hz, 1 H), 6.50
(d, J=7.8 Hz, 1 H), 4.03(m, 2 H), 374 (m, 1 H), 3.63 (m, 1 H), 3.00
(m, 4 H), 2.58 (m, 2 H), 2.40 (m, 1 H), 1.89 (m, 6 H), 1.72 (m, 4 H),
1.59 (m, 1 H), 1.26 (m, 8 H).
2-Amino-1-(4-{3-[2-(tetrahydropyran-4-ylamino)pyridin-4-yl]-
[2,6]naphthyridin-1-yl}piperazin-1-yl)ethanone (13r). Compound
13f (250 mg, 0.64 mmol) and HBTU (366 mg, 0.97 mmol) were
dissolved in DMF (3 mL). Triethylamine (89 μL, 0.64 mmol) was
added, and the mixture was stirred for 12 h. The mixture was
concentrated under reduced pressure, and the residue was pur-
ifiedby flashchromatographytoafford[2-oxo-2-(4-{3-[2-(tetrah-
ydropyran-4-ylamino)pyridin-4-yl][2,6]naphthyridin-1-
1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperidin-4-ol (13j). The title compound was prepared from
chloride 24 and 4-hydroxypiperidine by analogy to the prepara-
tion of 13i above: MS (ESI) m/z 404.4 (M þ 1); 1H NMR
(400 MHz, CDCl3) δ ppm 9.3 (s, 1 H), 8.6 (d, 1 H), 8.1 (d, 1
H), 7.8(m, 2 H), 7.2(m, 2 H), 5.6(br s, 1 H), 4.0(m, 1 H), 3.9(m, 2
H), 3.6 (m, 1 H), 3.4 (m, 2 H), 2.1-1.2 (m, 14 H).
yl}piperazin-1-yl)ethyl]carbamic acid tert-butyl ester.
The BOC-protected amine above (267 mg, 0.49 mmol) was
stirred in formic acid (5 mL) at room temperature. After 20 h,
the mixture was concentrated under reduced pressure. The
residue was purified by HPLC to afford the title compound:
HRMS (ESI) m/z 448.2459 (M þ 1); 1H NMR (400 MHz,

5418 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
CDCl3) δ ppm 9.31 (s, 1 H), 8.66 (d, J=5.8 Hz, 1 H), 8.21 (d, J=
5.4 Hz, 1 H), 7.83 (s, 1 H), 7.80 (d, J=5.8 Hz, 1 H), 7.23 (dd, J=
5.4, 0.9 Hz, 1 H), 7.16 (s, 1 H), 4.55 (d, J = 7.9 Hz, 1 H),
4.11-3.98 (m, 3 H), 3.98-3.91 (m, 2 H), 3.74-3.66 (m, 2 H),
3.64-3.53 (m, 8 H), 2.11 (dd, J=12.5, 1.9 Hz, 2 H), 1.70 (br s, 2
H), 1.63-1.51 (m, 2 H).
1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperidine-4-carboxylic Acid Amide (13s). The title compound
was prepared from 12j and cyclohexylamine by analogy to the
method described for the preparation of 13e above: MS (ESI) m/z
431.2 (M þ 1);1H NMR (400 MHz, CD3OD) δppm9.22 (d, 1 H),
8.56 (d, 1H), 7.98(d, 1 H), 7.90 (br m, 2 H), 7.31 (d, 1 H), 7.18(d, 1
H), 4.01 (br d, 2 H), 3.61 (m, 1 H), 3.08 (dd, 2 H), 2.58 (m, 1 H),
2.01-1.92 (br m, 6 H), 1.71 (br d, 2 H), 1.60 (d, 1 H), 1.37 (m, 2
H), 1.19 (m, 3 H).
2.15-2.01 (m, 4 H), 1.98-1.88 (m, 2 H), 1.79-1.64 (m, 2 H), 1.14
(t, J=7.3 Hz, 3 H).
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphth-
yridin-1-yl}piperidine-4-carboxylic Acid Isopropylamide (13y).
To a solution of 12k (2.65 g, 6.93 mmol), THF (30 mL), and
H2O (10 mL) was added LiOH H2O (1.45 g, 34.6 mmol). After
3
20 min, an additional 30 mL of THF was added. After 5 h, the
mixture was complete as judged by LCMS. At that point, 1 M HCl
in Et2O (35 mL) was added. The mixture was stirred for 10 min
and then concentrated in vacuo. The residue was then azeotro-
ped with toluene (3 ꢀ 150 mL) to give 1-[3-(2-chloropyridin-4-
yl)[2,6]naphthyridin-1-yl]piperidine-4-carboxylic acid. The crude
1-[3-(2-chloropyridin-4-yl)[2,6]naphthyridin-1-yl]piperidine-4-car-
boxylic acid was taken up in DMF (60 mL) before DIEA (5.75 mL,
34.60 mmol), i-PrNH2 (2.9 mL, 34.6 mmol), PyBOP (10.80 g,
20.80 mmol), and HOBt (0.94 g, 6.93 mmol) were added in
sequence. The mixture was stirred at room temperature for 24 h,
then concentrated in vacuo. The residue was taken up in CH2Cl2
(500 mL) and H2O (500 mL). The layers were mixed and then
separated. The aqueous layer was extracted further with CH2Cl2
(2 ꢀ 500 mL), and each organic layer was washed with an aliquot
of H2O (500 mL). The combined organic layers were then dried
over Na2SO4, filtered, and concentrated. The residue was stirred
with hot EtOAc (50 mL) and then filtered. The filtrate was then
washed several times with cold EtOAc to give 1-[3-(2-chloropyr-
idin-4-yl)[2,6]naphthyridin-1-yl]piperidine-4-carboxylic acid iso-
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphthy-
ridin-1-yl}piperidine-4-carboxylic Acid Amide (13t). The title
compound was prepared from 12j and 4-aminotetrahydropyran
by analogy to the method described for the preparation of
1
13e above: MS (ESI) m/z 433.3 (M þ 1); H NMR (400 MHz,
DMSO-d6) δ ppm9.36 (s, 1 H), 8.63 (d, J=6.1 Hz, 1 H), 8.07 (m, 2
H), 7.86 (d, J=5.6 Hz, 1 H), 7.34 (s, 2 H), 7.19 (d, J=4.0 Hz, 1 H),
6.83 (s, 1 H), 6.70 (br s, 1 H), 4.00 (m, 3 H), 3.89 (br dt, J=8.0, 3.2
Hz, 2 H), 3.43 (td, J=11.0, 2.0 Hz, 2 H), 3.40 (m, 1 H), 3.06 (m, 2 H),
2.42 (m, 1 H), 1.91 (br m, 5 H), 1.47 (m, 2 H).
1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]pi-
peridine-4-carboxylic Acid Methylamide (13u). The title com-
pound was prepared from 12k and cyclohexylamine by analogy
to the method described for the preparation of 13e above: MS
(ESI) m/z 445.4 (M þ 1); 1H NMR (400 MHz, CD3OD) δ ppm
9.28 (s, 1 H), 8.57 (d, 1 H), 8.00 (m, 2 H), 7.99 (d, 1 H), 7.38 (s, 1
H), 7.26 (d, 1 H), 4.13 (br d, 2 H), 3.68 (m, 1 H), 3.14 (dd, 2 H),
2.77 (s, 3 H), 2.50 (m, 1 H), 2.07 (br m, 4 H), 1.95 (m, 2 H), 1.80,
(m, 2 H), 1.69 (br d, 1 H), 1.46 (m, 2 H), 1.29 (m, 3 H).
1
propylamide as a white solid: MS (ESI) m/z 410.2 (M þ 1); H
NMR (400 MHz, DMSO-d6) δ ppm 9.36 (s, 1 H), 8.68 (d, J=5.8
Hz, 1 H), 8.56 (d, J=5.3 Hz, 1 H), 8.38 (s, 1 H), 8.23 (s, 1 H),
8.20-8.15 (m, 1 H), 7.90 (d, J=5.8 Hz, 1 H), 7.71 (d, J=7.6 Hz,
1 H), 4.06 (d, J=13.1 Hz, 2 H), 3.94-3.79 (m, 1 H), 3.13-3.02
(m, 2 H), 2.47-2.34 (m, 1 H), 2.00-1.81 (m, 4 H), 1.07 (d, J=
6.6 Hz, 6 H).
A pressure reaction vessel was charged with 1-[3-(2-chloropyr-
idin-4-yl)[2,6]naphthyridin-1-yl]piperidine-4-carboxylic acid iso-
propylamide (0.50 g, 1.22 mmol), 4-aminotetrahydropyran
(0.25 g, 2.44 mmol), Pd(t-Bu3P)2 (0.06 g, 0.12 mmol), t-BuONa
(0.35 g, 3.66 mmol), and 1,4-dioxane. The mixture was sparged
with argon for 10 min, and the vessel is then sealed and heated to
130 ꢀC for 2.5 h. The contents of the vessel are allowed to cool to
room temperature before being diluted with CH2Cl2 (150 mL)
and brine (150 mL). The layers were mixed and then separated.
The aqueous layer was extracted further with CH2Cl2 (3 ꢀ
150 mL), and the combined organic layers were then dried over
Na2SO4, filtered, and concentrated. The residue was then
separated via RP-HPLC (5-60% CH3CN/H2O/0.1%NH4OH
gradient) to give the title compound: MS (ESI) 475.1 m/z
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (s, 1 H),
8.62 (d, J=5.6 Hz, 1 H), 8.08 (d, J=5.3 Hz, 1 H), 8.04 (s, 1 H),
7.86 (d, J=5.8 Hz, 1 H), 7.70 (d, J=7.6 Hz, 1 H), 7.31 (s, 1 H),
7.22-7.14 (m, 1 H), 6.66 (d, J=7.6 Hz, 1 H), 4.09-3.93 (m, 3 H),
3.94-3.77 (m, 3 H), 3.49-3.36 (m, 2 H), 3.05 (t, J=12.0 Hz, 2
H), 2.46-2.31 (m, 1) H, 2.01-1.78 (m, 6 H), 1.56-1.38 (m, 2 H),
1.07 (d, J=6.6 Hz, 6 H).
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphth-
yridin-1-yl}piperidine-4-carboxylic Acid Isobutylamide (13z).
The title compound was prepared from ester 12k by the method
outlined for the preparation of 13y above: MS (ESI) m/z 489.2
(M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 9.27 (s, 1 H), 8.62
(d, J=5.8 Hz, 1 H), 8.20 (d, J=5.6 Hz, 1 H), 7.79 (d, J=5.8 Hz, 1
H), 7.76 (s, 1 H), 7.26-7.22 (m, 1 H), 7.20 (s, 1 H), 5.58 (t, J=5.6
Hz, 1 H), 4.50 (d, J=8.1 Hz, 1 H), 4.14-3.98 (m, 5 H), 3.65-3.54
(m, 2 H), 3.19-3.07 (m, 4 H), 2.46-2.33 (m, 1 H), 2.22-2.01 (m,
6 H), 1.87-1.75 (m, 1 H), 0.95 (d, J=6.6 Hz, 6 H).
1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]pi-
peridine-4-carboxylic Acid (2-Pyrrolidin-1-ylethyl)amide (13v).
The title compound was prepared from ester 12l by the method
outlined for the preparation of 13y below: MS (ESI) m/z 528.4
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (s, 1 H),
8.62 (d, J=5.3 Hz, 1 H), 8.03 (m, 2 H), 7.83 (m, 2 H), 7.29 (s, 1 H),
7.14 (d, J=5.8 Hz, 1 H), 6.51 (d, J=7.6 Hz, 1 H), 4.01 (m, 2 H),
3.74 (br s, 1 H), 3.18 (m, 2 H), 3.03 (m, 2 H), 1.92 (m, 8 H),
1.79-1.56 (m, 8 H), 1.42-1.10 (m, 8 H).
{1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]pi-
peridin-4-yl}-(4-hydroxypiperidin-1-yl)methanone (13w). A mix-
ture of ester 12k (500 mg, 1.3 mmol), 4-hydroxycyclohexylamine
(400 mg, 3.9 mmol), and Me3Al (4 mL of a 2 M solution, 8 mmol)
in toluene (10 mL) was heated for 6 h at 110 ꢀC. After cooling, the
mixture was poured into MeOH (250 mL) and stirred for 10 min.
The mixture was filtered and concentrated under reduced pres-
sure. The residue was purified by flash chromatography (1-2%
MeOH/CH2Cl2) to afford the amide as a yellow solid.
The amide above was converted to the target by coupling to
cyclohexylamine using the method outlined for the preparation
of 13e above: MS (ESI) m/z 515.4 (M þ 1); 1H NMR (400 MHz,
CD3OD) δ ppm 9.27 (s, 1 H), 8.56 (d, J=5.8 Hz, 1 H), 8.02-7.90
(m, 3 H), 7.37 (s, 1 H), 7.25 (d, J=5.6 Hz, 1 H), 4.18-4.07 (m, 2
H), 4.02-3.93 (m, 1 H), 3.92-3.83 (m, 1 H), 3.75-3.63 (m, 1 H),
3.45-3.34 (m, 1 H), 3.27-3.10 (m, 3 H), 3.09-2.98 (m, 1 H),
2.17-1.96 (m, 4 H), 2.00-1.76 (m, 6 H), 1.75-1.62 (m, 1 H),
1.59-1.38 (m, 4 H), 1.36-1.21 (m, 4 H).
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphthy-
ridin-1-yl}piperidine-4-carboxylic Acid Ethylamide (13x). The title
compound was prepared from ester 12l by the method outlined
for the preparation of 13y below: MS (ESI) 461.2 m/z (M þ 1); 1H
NMR (400 MHz, MeOD) (TFA salt) δ ppm 8.68 (d, J=5.8 Hz,
1 H), 8.22 (s, 1 H), 8.02 (d, J = 6.1 Hz, 1 H), 8.00-7.96
(m, 1 H), 7.95-7.86 (m, 1 H), 7.69-7.61 (m, 1 H), 4.26-
4.16 (m, 2 H), 4.09-3.99 (m, 2 H), 3.96-3.84 (m, 1 H),
3.63-3.53 (m, 2 H), 3.29-3.14 (m, 4 H), 2.58-2.45 (m, 1 H),
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphthy-
ridin-1-yl}piperidine-4-carboxylic Acid (2-Pyrrolidin-1-ylethyl)-
amide (13aa). The title compound was prepared from ester 12k
by the method outlined for the preparation of 13y above: MS
(ESI) m/z 530.4 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm
9.29(s, 1 H), 8.55 (d, J=5.8 Hz, 1 H), 8.00 (d, J=5.6 Hz, 1 H), 7.98

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5419
(s, 1 H), 7.84-7.71 (m, 2 H), 7.25 (s, 1 H), 7.11(d, J=5.6 Hz, 1 H),
6.59 (d, J=7.6 Hz, 1 H), 3.87-4.01 (m, 3 H), 3.87-3.78 (m, 2 H),
3.41-3.30 (m, 2 H), 3.12(d, J=19.2 Hz, 2 H), 2.99 (d, J=23.2 Hz,
2 H), 1.92-1.74 (m, 8 H), 1.60 (br s, 4 H), 1.49-1.28 (m, 6 H).
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphth-
yridin-1-yl}piperidine-4-carboxylic Acid (2-tert-Butoxyethyl)-
amide (13ab). The title compound was prepared from ester 12k
by the method outlined for the preparation of 13y above: MS
(ESI) m/z 533.2 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm
9.27 (s, 1 H), 8.63 (d, J=5.8 Hz, 1 H), 8.20 (d, J=5.3 Hz, 1 H),
7.79 (d, J=5.8 Hz, 1 H), 7.76 (s, 1 H), 7.25-7.22 (m, 1 H), 7.20 (s,
1 H), 6.01 (br s, 1 H), 4.54-4.46 (m, 1 H), 4.15-3.97 (m, 5 H),
3.65-3.54 (m, 2 H), 3.49-3.44 (m, 4 H), 3.19-3.07 (m, 2 H),
2.49-2.34 (m, 1 H), 2.18-1.97 (m, 6 H), 1.21 (s, 9 H).
1-{3-[2-(Tetrahydropyran-4-ylamino)pyridin-4-yl][2,6]naphth-
yridin-1-yl}piperidine-4-carboxylic Acid (2-Hydroxyethyl)amide
(13ac). The title compound was prepared from 13ab above by
acidic deprotection (TFA in CH2Cl2) of the tert-butyl ether to
afford the title alcohol: MS (ESI) m/z 477.1 (M þ 1); 1H NMR
(400 MHz, CDCl3) δ ppm 9.29 (s, 1 H), 8.65 (d, J=5.6 Hz, 1 H),
8.22-8.15 (m, 1 H), 7.83-7.76 (m, 2 H), 7.27-7.24 (m, 2 H),
6.08-5.99 (m, 1 H), 4.17-3.98 (m, 5 H), 3.84-3.76 (m, 2 H),
3.67-3.57 (m, 2 H), 3.56-3.47 (m, 2 H), 3.20-3.09 (m, 2 H),
2.53-2.41 (m, 1 H), 2.39-2.26 (m, 1 H), 2.23-2.02 (m, 7 H),
1.70-1.60 (m, 2 H).
dropwise. After 30 min at -78 ꢀC, ethyl 2-chloroisonicotinate
(3.0 g, 17.4 mmol) in THF (10 mL) was added rapidly dropwise.
The mixture was further stirred at -78 ꢀC for 30 min before
being quenched with AcOH (10 mL). The mixture was allowed
to warm to room temperature and then concentrated in vacuo to
remove volatiles. The residue was dissolved in AcOH (50 mL),
and the mixture was heated at 80 ꢀC with stirring. After cooling
to room temperature, the resulting slurry was diluted with
MeOH (50 mL) and the precipitate was collected by filtration,
washed with MeOH, and air-dried to give 15a as a white powder
(2.8 g, 63%): MS (ESI) m/z 258.1 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.72 (d, J=5.3 Hz, 1 H), 8.38 (d, J=7.9 Hz,
1 H), 8.2-8.0 (m, 4 H), 8.0-7.8 (m, 2 H).
3-[2-(2-Chloropyridin-4-yl)-2-oxoethyl]pyridine-2-carboxylic
Acid tert-Butylamide (15b). The title compound was prepared
from 14b in a manner analogous to the method described above
for the preparation of 15a: 1H NMR (400 MHz, DMSO-d6)
δ ppm 8.83 (s, 1 H), 8.58 (d, J=5.2 Hz, 1 H), 8.35 (d, J=8.3 Hz,
1 H), 8.22 (s, 1 H), 8.20-8.15 (m, 2 H), 8.00-7.90 (m, 2 H).
N-tert-Butyl-4-[2-(2-chloropyridin-4-yl)-2-oxoethyl]nicotina-
mide (15c). The title compound was prepared from 14c in a
manner analogous to the method described above for the
preparation of 15a: MS (ESI) m/z 259.1 (M þ 1); 1H NMR
(400 MHz, DMSO-d6) δ ppm 9.68 (s, 1 H), 9.3(d, J=4.8 Hz, 1 H),
8.96(d, J=5.3 Hz, 1 H), 8.34 (s, 1 H), 8.24(m, 2 H), 8.00(d, J=5.3
Hz, 1 H).
N-tert-Butyl-2-[2-(2-chloropyridin-4-yl)-2-oxoethyl]nicotinamide
(15d). The title compound was prepared from 14d in a manner
analogous to the method described above for the preparation
of 15a: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.38 (dd, J=4.4,
1.5 Hz, 1 H), 8.90 (d, 5.2 Hz, 1 H), 8.86 (d, J=8.0 Hz, 1 H), 8.38 (s, 1
H), 8.29 (dd, J=5.2, 1.5 Hz, 1 H), 8.21 (s, 1 H), 8.0 (dd, J=8.0, 4.4
Hz, 1 H).
3-(2-Chloropyridin-4-yl)isoquinolin-1-ol (16a). To a suspen-
sion of 15a (2.8 g, 11 mmol) in MeOH (10 mL) was added a 2 M
solution of NH3 in MeOH (40 mL). The mixture was heated to
80 ꢀC in a sealed tube, and after it was cooled, the precipitate
was collected by filtration. The crude product, which LCMS
analysis indicated to be a 1:1 mixture of 16a and uncyclized
amide, was resuspended in MeOH. Concentrated HCl (0.1 mL)
was added, and the mixture was heated to 60 ꢀC for 1 h. The
resulting slurry was cooled to 0 ꢀC, filtered, washed with cold
MeOH, and air-dried to give 16a as a yellowish solid (2.6 g,
93%): MS (ESI) m/z 257.1 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 11.65 (br, 1 H), 8.52 (d, J=5.3 Hz, 1 H),
8.24 (d, J=8.0 Hz, 1 H), 8.00 (s, 1 H), 7.87 (d, J=5.5 Hz, 1 H),
7.77 (m, 2 H), 7.58 (m, 1 H), 7.3 (s, 1 H).
4-({1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperidine-4-carbonyl}amino)piperidine-1-carboxylic Acid tert-Bu-
tyl Ester (13ad). The title compound was prepared from ester
12k by the method outlined for the preparation of 13y above: MS
(ESI) m/z 614.4 (M þ 1).
1-[3-(2-Cyclohexylaminopyridin-4-yl)[2,6]naphthyridin-1-yl]-
piperidine-4-carboxylic Acid Piperidin-4-ylamide (13ae). The
title compound was prepared from 13ad above by acidic depro-
tection (TFA in CH2Cl2) to afford the title amine: MS (ESI) m/z
514.3 (M þ 1); 1HNMR(400MHz, DMSO-d6) δppm9.36(s, 1H),
8.61 (d, J=5.8 Hz, 1 H), 8.05(d, J=5.6 Hz, 1 H), 8.03 (s, 1 H), 7.86
(d, J=5.8Hz, 1 H), 7.80 (d, J=7.8Hz, 1 H), 7.28 (s, 1 H), 7.14 (dd,
J=5.4, 1.5 Hz, 1 H), 6.50 (d, J=7.8 Hz, 1 H), 4.09-3.96 (m, 2 H),
3.82-3.66 (m, 1 H), 3.69-3.57 (m, 1 H), 3.11-2.92 (m, 4 H),
2.64-2.52 (m, 2 H), 2.46-2.34 (m, 1 H), 2.00-1.79 (m, 6 H),
1.78-1.66 (m, 4 H), 1.66-1.53 (m, 1 H), 1.40-1.12 (m, 8 H).
N-tert-Butyl-2-methylbenzamide (14a). To an ice-cold solu-
tion of aqueous Na2CO3 (1 M, 30 mL) containing tert-butyla-
mine (3.2 mL, 30 mmol) was added 2-methylbenzoyl chloride
(3.9 g, 25 mmol) in dioxane (5 mL) slowly dropwise. After the
addition, the mixture was allowed to warm to room temperature
and further stirred for 4 h. The resulting slurry was diluted with
water and the precipitate was filtered and air-dried to give 14a as
a white solid (3.6 g, 75%) matching the reported 1H NMR
data.26
6-(2-Chloropyridin-4-yl)[1,7]naphthyridin-8-ol (16b). The title
compound was prepared from 15b by analogy to the method
described above for the preparation of 16a: MS (ESI) m/z 258.1
1
3-Methylpyridine-2-carboxylic Acid tert-Butylamide (14b). To
a solution of 2-cyano-3-methylpyridine (1.2 g, 12 mmol) in tert-
butyl acetate (10 mL) was added concentrated H2SO4 (1 mL),
and the mixture was stirred at room temperature overnight
before it was diluted with water. The mixture was then carefully
neutralized by adding NH4OH at 0 ꢀC and then extracted twice
with 1:1 hexane-EtOAc. The combined organics were washed
with saturated brine, dried (Na2SO4), filtered, and concentrated.
The residue was chromatographed on silica gel to give 14b as a
colorless oil (1.85 g, 95%): 1H NMR (400 MHz, CDCl3) δ ppm
8.35 (d, J=4.3 Hz, 1 H), 8.2 (br s, 1 H), 7.55 (m, 1 H), 7.27 (m,
1 H), 2.74 (s, 3 H), 1.48 (s, 9 H).
N-tert-Butyl-4-methylnicotinamide (14c). The title compound
was prepared in an analogous manner to the method described
for the preparation of 14b above: 1H NMR (400 MHz, CDCl3) δ
ppm 8.52 (s, 1 H), 8.46 (d, J=5.1 Hz, 1 H), 7.13 (d, J=5.1Hz, 1 H),
5.63 (br s, 1 H), 2.45 (s, 3 H), 1.48 (s, 9 H).
(M þ 1); H NMR (400 MHz, DMSO-d6) δ ppm 12.22 (br s,
1 H), 9.15 (d, J=4.0 Hz, 1 H), 8.87 (d, J=5.6 Hz, 1 H), 8.51 (d,
J=8.1 Hz, 1 H), 8.32 (s, 1 H), 8.18 (d, J=5.1 Hz, 1 H), 8.07 (dd,
J=8.1, 4.0 Hz, 1 H), 7.58 (s, 1 H).
3-(2-Chloropyridin-4-yl)[2,7]naphthyridin-1-ol (16c). The title
compound was prepared from 15c by analogy to the method
described above for the preparation of 16a: MS (ESI) m/z 258.1
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 12.38 (br s, 1
H), 9.75 (s, 1 H), 9.17 (d, J=4.6 Hz, 1 H), 8.95 (d, J=5.6 Hz, 1
H), 8.38 (s, 1 H), 8.24 (d, J=5.1 Hz, 1 H), 8.04 (d, J=5.6 Hz, 1
H), 7.61 (s, 1 H).
7-(2-Chloropyridin-4-yl)[1,6]naphthyridin-5-ol (16d). The title
compound was prepared from 15d by analogy to the method
described above for the preparation of 16a: MS (ESI) m/z 258.1
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 12.27 (br s, 1
H), 9.18 (d, 1 H), 8.89 (d, J=5.1 Hz, 1 H), 8.53 (d, J=8.1 Hz, 1
H), 8.34 (s, 1 H), 8.20 (d, J=5.6 Hz, 1 H), 8.09 (dd, J=8.1, 4.6
Hz, 1 H), 7.60 (s, 1 H).
3-(2-Chloropyridin-4-yl)isochromen-1-one (15a). To a solu-
tion of 14a (3.4 g, 18 mmol) in THF (60 mL) at -78 ꢀC was
added n-butyllithium (2.5 M in hexane, 16 mL, 40 mmol) slowly
2-(2-Chloropyridin-4-yl)quinazolin-4-ol (20). To a solution of
17 (6 g, 43 mmol) in dioxane (50 mL) was added MeOH (7 mL,

5420 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
172 mmol), followed by 4 M HCl in dioxane (50 mL, 200 mmol).
The mixture was stirred at room temperature overnight, and the
resulting slurry was filtered, washed with dioxane, and air-dried
to give crude imidate salt 18. To an ice-cold solution of the crude
18 obtained as above in MeOH (50 mL) was added Na metal
(2 g, 86 mmol) slowly in small pieces. After the addition, the
mixture was heated to reflux with 19 for 6 h before it was allowed
to cool to room temperature and filtered, washed with water,
and air-dried to give the quinazolone 20, which was used in the
next step without further purification (4.6 g, 42%).
1-(1-Piperazinyl)-3-(2-cyclohexylaminopyrid-4-yl)isoquinoline
(22a). A suspension of the above 16a (510 mg, 1.99 mmol) in
POCl3 was heated at 100 ꢀC with stirring for 2 h. After cooling to
room temperature, the mixture was concentrated under reduced
pressure, triturated with water, filtered, and air-dried to a yellow
solid. The solid was dissolved in CH2Cl2, washed with a satu-
rated aqueous solution of NaHCO3, dried (Na2SO4), and fil-
tered through silica gel to afford 1-chloro-3-(2-chloropyridin-4-
yl)isoquinoline (0.47 g, 86%): MS (ESI) m/z 259.1 (M þ 1);
1H NMR (400 MHz, CDCl3) δ ppm 8.83 (s, 1 H), 8.58 (d, J=5.2
Hz, 1 H), 8.35 (d, J =8.3 Hz, 1 H), 8.22 (s, 1 H), 8.20-8.15
(m, 2 H), 7.99 (dd, J = 8.3, 7.0 Hz, 1 H), 7.91 (dd, J = 8.3,
7.1 Hz, 1 H).
The 1-chloroisoquinoline above (360 mg, 1.3 mmol) and N-
BOC piperazine (300 mg, 1.6 mmol) in NMP (1 mL) were heated
to 180 ꢀC in a microwave reactor for 45 min. After cooling, the
mixture was poured into water and the resulting precipitate was
isolated by filtration. The collected tan solid was dissolved in
CH2Cl2 andpurifiedbycolumnchromatographyusing a gradient
of0-20% EtOAc/CH2Cl2. Compound21a was isolatedas a solid
285 mg (51%): MS (ESI) m/z 425.3 (M þ 1); 1H NMR (400 MHz,
CDCl3) δ ppm 8.47 (d, J=5.2 Hz, 1 H), 8.11 (d, J=8.1 Hz, 1 H),
8.08 (s, 1 H), 7.94 (dd, J=5.2, 1.5 Hz, 1 H), 7.87 (d, J=8.1 Hz, 1
H), 7.82 (s, 1 H), 7.68 (dd, J=7.5, 6.9 Hz, 1 H), 7.59 (dd, J=8.1,
7.6 Hz, 1 H), 3.74 (m, 4 H), 3.50 (m, 4 H), 1.52 (s, 9 H).
A vial containing 21a (42 mg, 0.1 mmol) in toluene (0.5 mL)
and cyclohexylamine (12 mg, 0.12 mmol) in toluene (0.6 mL)
was degassed with N2. To the vial was added Pd(OAc)2 and
BINAP (0.25 mL of a 0.01 M solution in toluene, 2.5 ꢀ 10-3
mmol) followed by t-BuOK (0.12 mL of a 1 M solution in THF,
0.12 mmol). The vial was heated at 80 ꢀC overnight. After
cooling, the mixture was partitioned between toluene and water.
The toluene layer was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by flash chromatog-
raphy and then by preparative TLC to afford cyclohexylami-
nopyridine.
The title compound was prepared from 21b by Buchwald
amination followed by acidic deprotection: MS (ESI) m/z 389.4
(M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 8.99 (d, J=4.0
Hz, 1 H), 8.45 (d, J=8.1 Hz, 1 H), 8.18 (d, J=5.1 Hz, 1 H), 7.88
(s, 1 H), 7.82 (dd, J=8.6, 3.54 Hz, 1 H), 7.41 (s, 1 H), 7.25 (d, J=
5.1 Hz, 1 H), 6.61 (d, J =7.6 Hz, 1 H), 4.22-4.10 (m, 4 H),
3.98-3.84 (m, 1 H), 3.16-3.04 (m, 4 H), 2.16-2.05 (m, 2 H),
1.95-1.83 (m, 2 H), 1.80-1.70 (m, 1 H), 1.56-1.25 (m, 5 H).
Cyclohexyl-[4-(1-piperazin-1-yl[2,7]naphthyridin-3-yl)pyridin-2-
yl]amine (22c). The title compound was prepared from 16c by
analogy to the method described for the preparation of 22b. The
intermediate 21c was isolated as a solid: MS (ESI) m/z 426.2
(M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 9.48 (s, 1 H), 8.69 (d,
J=5.7 Hz, 1 H), 8.50 (d, J=5.2 Hz, 1 H), 8.05 (d, J=1.5 Hz, 1 H),
7.91 (dd, J=5.2, 1.5 Hz, 1 H), 7.69 (s, 1 H), 7.64 (d, J=5.7 Hz,
1 H), 3.75 (m, 4 H), 3.65 (m, 4 H), 1.52 (s, 9 H).
Buchwald amination of 21c followed by deprotection under
acidic conditions afforded the title compound 22c: MS (ESI) m/z
389.4 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 9.67 (s, 1
H), 9.08 (br s, 2 H), 8.90-8.85 (m, 1 H), 8.24-8.17 (m, 2 H), 8.03
(d, J=5.56 Hz, 1 H), 7.54 (br s, 1 H), 7.37 (br s, 1 H), 3.93 (br s, 4
H), 3.59 (br s, 4 H), 3.30-3.19 (m, 1 H), 2.13-2.07 (m, 2 H),
1.94-1.85 (m, 2 H), 1.82-1.72 (m, 1 H), 1.59-1.34 (m, 5 H).
Cyclohexyl-[4-(5-piperazin-1-yl[1,6]naphthyridin-7-yl)pyridin-2-
yl]amine (22d). The title compound was prepared from 16d by
analogy to the method described for the preparation of 22a: MS
(ESI) m/z 389.3 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm
9.02 (dd, J=4.2, 1.6 Hz, 1 H), 8.50-8.39 (m, 1 H), 8.05 (d, J=5.3
Hz, 1 H), 7.90 (s, 1 H), 7.57 (dd, J=8.5, 4.2 Hz, 1 H), 7.32 (d,
J=0.8Hz, 1 H), 7.19 (dd, J=5.4, 1.5 Hz, 1 H), 6.50 (d, J=7.8 Hz, 1
H), 3.83-3.71 (m, 1 H), 3.47-3.39 (m, 4 H), 3.05-2.95 (m, 4 H),
2.02-1.90 (m, 2 H), 1.80-1.67 (m, 2 H), 1.66-1.57 (m, 1 H),
1.43-1.13 (m, 5 H).
Cyclohexyl-[4-(4-piperazin-1-ylquinazolin-2-yl)pyridin-2-yl]-
amine (22e). The title compound was prepared from 20 by
analogy to the method described for the preparation of 22a.
The intermediate 21e was isolated as a solid: 1H NMR (400 MHz,
CDCl3) δ ppm 8.52 (d, J=5.2 Hz, 1 H), 8.41 (s, 1 H), 8.31 (dd, J=
5.1, 1.3 Hz, 1 H), 8.01(d, J=8.4 Hz, 1 H), 7.93 (d, J=8.3 Hz, 1 H),
7.80 (app t, J=8.3 Hz, 1 H), 7.52 (app t, J=8.2 Hz, 1 H), 3.86 (m,
4 H), 3.71 (m, 4 H), 1.51 (s, 9 H).
Deprotection of 21e was effected under acidic conditions
to afford the title compound 22e as a solid: MS (ESI) m/z
1
389.3 (M þ 1); H NMR (400 MHz, CDCl3) δ ppm 8.19 (d,
J=5.1 Hz, 1 H), 7.98 (d, J=8.1 Hz, 1 H), 7.91 (d, J=8.6 Hz, 1 H),
7.74 (t, J=7.3 Hz, 1 H), 7.61 (d, J=6.1 Hz, 1 H), 7.52 (s, 1 H),
4.67 (br s, 1 H), 3.89-3.80 (m, 4 H), 3.80-3.68 (m, 1 H),
3.17-3.08 (m, 4 H), 2.17-2.07 (m, 2 H), 1.87-1.61 (m, 3 H),
1.33-1.19 (m, 3 H), 1.52-1.38 (m, 2 H).
The pure fractions of the above compound were dissolved in
CH2Cl2 (2 mL) and TFA (0.4 mL). After being stirred at room
temperature overnight, the mixture was concentrated in vacuo
and partitioned between CH2Cl2 and a saturated aqueous
solution of NaHCO3. The phases were separated using a Biotage
phase separator cartridge. The organic layer was concentrated
in vacuo. The residue was purified using silica gel solid phase
extraction to afford the title compound 22a: MS (ESI) m/z 388.3
(M þ H); 1H NMR (400 MHz, DMSO-d6) δ ppm 8.52 (d, J=8.1
Hz, 1 H), 8.44 (d, J=5.1 Hz, 1 H), 8.42-8.35 (m, 2 H), 8.13 (t, J=
8.1 Hz, 1 H), 8.03 (t, J=8.1 Hz, 1 H), 7.68 (s, 1 H), 7.55 (d, J=5.6
Hz, 1 H), 6.85 (d, J=7.6 Hz, 1 H), 4.22-4.08 (m, 1 H), 3.96-3.86
(m, 4 H), 3.67-3.59 (m, 4 H), 3.29-3.16 (m, 1 H), 2.40-2.31 (m,
2 H), 2.19-2.07 (m, 2 H), 2.06-1.95 (m, 1 H), 1.82-1.67 (m,
2 H), 1.68-1.53 (m, 3 H).
Acknowledgment. We thank Patrick Cronan for high re-
solution mass spectrometry data, Analytical Sciences for
LCMS and NMR support, and Angela Mackay for the
preparation of 12g. We thank the Metabolism and Pharma-
cokinetics Group for the in vivo and bioanalytical support in
the pharmacokinetic studies.
Supporting Information Available: Figures S1 and S2 showing
the PK iv and po curves for 13c and 13y; Table T1 listing percent
activity remaining for kinases. This material is available free of
charge via the Internet at http://pubs.acs.org.
Cyclohexyl-[4-(8-piperazin-1-yl[1,7]naphthyridin-6-yl)pyridin-2-
yl]amine (22b). The title compound was prepared from 16b by
analogy to the method described for the preparation of 22a. The
intermediate 21b was generated under milder conditions than 21a
above (80 ꢀC, overnight): MS (ESI) m/z 259.1 (M þ 1); 1H NMR
(400 MHz, CDCl3) δ ppm 8.87 (dd, J=4.2, 1.8 Hz, 1 H), 8.48 (d,
J=5.2 Hz, 1 H), 8.11 (dd, J=8.3, 1.9 Hz, 1 H), 8.04 (d, J=1.6 Hz, 1
H), 7.91 (dd, J=5.2, 1.6 Hz, 1 H), 7.6 (s, 1 H), 7.56 (dd, J=8.3, 4.2
Hz, 1 H), 4.12 (br, 4 H), 3.72 (m, 4 H), 1.51 (s, 9 H).
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