Identification of potent and selective amidobipyridyl inhibitors of protein kinase D

Article abstract of DOI:10.1021/jm100076w

The synthesis and biological evaluation of potent and selective PKD inhibitors are described herein. The compounds described in the present study selectively inhibit PKD among other putative HDAC kinases. The PKD inhibitors of the present study blunt phosphorylation and subsequent nuclear export of HDAC4/5 in response to diverse agonists. These compounds further establish the central role of PKD as an HDAC4/5 kinase and enhance the current understanding of cardiac myocyte signal transduction. The in vivo efficacy of a representative example compound on heart morphology is reported herein.

Full text of DOI:10.1021/jm100076w

5422 J. Med. Chem. 2010, 53, 5422–5438
DOI: 10.1021/jm100076w
Identification of Potent and Selective Amidobipyridyl Inhibitors of Protein Kinase D
Erik L. Meredith,*,† Kimberly Beattie,† Robin Burgis,† Michael Capparelli,† Joseph Chapo,‡ Lucian DiPietro,†
Gabriel Gamber,† Istvan Enyedy,† David B. Hood,‡ Vinayak Hosagrahara,† Charles Jewell,† Keith A. Koch,‡ Wendy Lee,†
Douglas D. Lemon,‡ Timothy A. McKinsey,‡ Karl Miranda,† Nikos Pagratis,‡ Dillon Phan,‡ Craig Plato,‡ Chang Rao,†
Olga Rozhitskaya,† Nicolas Soldermann,§ Clayton Springer,† Maurice van Eis,§ Richard B. Vega,† Wanlin Yan,†
Qingming Zhu,† and Lauren G. Monovich†
†Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, ‡Gilead Colorado, Inc., 3333 Walnut
Street, Boulder, Colorado 80301, and §Novartis Institutes for BioMedical Research, Basel, Switzerland
Received January 18, 2010
The synthesis and biological evaluation of potent and selective PKD inhibitors are described herein. The
compounds described in the present study selectively inhibit PKD among other putative HDAC kinases.
The PKD inhibitors of the present study blunt phosphorylation and subsequent nuclear export of
HDAC4/5 in response to diverse agonists. These compounds further establish the central role of PKD as
an HDAC4/5 kinase and enhance the current understanding of cardiac myocyte signal transduction.
The in vivo efficacy of a representative example compound on heart morphology is reported herein.
Introduction
and as valuable tools to further dissect of the role of PKD in
the disease. In addition to its role in heart failure, PKD has also
been found to be a key mediator of signals controlling DNA
synthesis, cell growth, and proliferation.7,8 Pharmacological in-
hibition of PKD has also been shown to inhibit prostate cancer
cell growth and migration.8 Published reports of PKD inhibi-
tors8,9 range from allosteric modulators to partially selective
type I kinase inhibitors. In most cases, the potential to under-
stand PKD-mediated pathways is limited by a lack of selectivity
for PKD.10
High throughput screening (HTS) and subsequent optimiza-
tion efforts identified 2,6-naphthyridine 111 as a potent PKD
inhibitor (Figure 1). Compound 1also povided potent inhibition
of HDAC5 nuclear export (PKD1 IC50=0.6 nM, HDACexp
IC50=32 nM) in cardiac myocytes and is 1000-fold selective for
PKD versus the upstream activator kinases (PKCs). However, 1
lacked selectivity against other putative HDAC kinases to clearly
define the role of PKD in cardiac myocyte stress response. For
example, 1 partially inhibits a set of putative HDAC kinases.
Thus, more selective PKD inhibitors were sought.
Attempts to produce an X-ray crystal structure of com-
pound 1 bound to PKD1 were unsuccessful; however, it is
known that 1 is an ATP competitive type I inhibitor of PKD1,
and thus, it is possible to propose a binding mode for 1 as
depicted in Figure 1a. Structure-activity relationship (SAR)
analysis suggests that the key interaction of 1 with PKD1 is a
hydrogen bond pair of the alkylaminopyridine to hinge region
residue Leu662. Additional interactions of note are a hydro-
gen bond of the A-ring nitrogen with the catalytic Lys612 and
a salt bridge of the distal piperazine nitrogen with Glu710.
Early SAR showed that replacement of the A-ring nitrogen in
1 by a carbon atom afforded compounds with 10- to 20-fold
loss in potency.11 This finding is in accord with proposed
binding mode of 1 wherein the A-ring nitrogen makes a
hydrogen bond contact with the catalytic Lys612. Given this
understanding of the binding mode of 1, analogues that mimic
Acute and/or chronic stresses placed on the heart can result in
compensations such as pathological cardiac hypertrophy.1
Pathological cardiac hypertrophy is not, at present, delineated
as either an adaptive or maladaptive stress response in the
cardiac myocytes. However, pathologic cardiac hypertrophy is
linked to increased morbidity and mortality.2 Specifically, left
ventricular hypertrophy is an independent risk factor for cardiac
disease.3 Given that heart failure4 continues to be a leading cause
of death, a greater understanding of the underlying mechanisms
of cardiac hypertrophy and the identification of suitable agents
to inhibit such processes is of significant interest.
The three isoforms of the protein kinase D (PKDa) family,
PKD1, PKD2, and PKD3, have been shown to play a role in
growth factor signaling and in stress-induced signaling.5 PKD1
phosphorylates histone deacetylase 5 (HDAC5) in cardiac myo-
cytes, induces the binding of 14-3-3 protein to the phospho-
serine motif of HDAC5, and leads to nuclear export through a
CRM1-dependent mechanism. The result of HDAC5 export is
increased transcriptional activity of prohypertrophic genes, such
as myocyte enhancer factor 2 (MEF2). MEF2 gene transcrip-
tion in the myocyte, in turn, alters myocyte growth, contraction,
calcium handling, and metabolism.5,6 Thus, inhibitors of PKD
may prove beneficial in slowing or altering the progressing of
pathological hypertrophy.
Growing interest in the development of small molecule
inhibitors of PKD stems from their potential in disease therapy
*To whom correspondence should be addressed. Phone: 617-871-
7586. Fax: 617-871-7045. E-mail: erik.meredith@novartis.com.
a Abbreviations: PKD, protein kinase D; PKC, protein kinase C; TAB,
thoracic aortic banded; DSS, Dahl salt-sensitive; HDAC, histone deacety-
lase; MEF2, myocyte enhancer factor 2; GFP, green fluorescence protein;
LV, left ventricle; TL, tibia length; IVRT, isovolumic relaxation time; MAP,
mean arterial pressure; PMA, phorbol 12-myristate 13-acetate; PE, pheny-
lephrine; PGF2R, prostaglandin F 2R; ET-1, endothelin-1; LPA, lipopoly-
saccharide A; PMBCs, peripheral blood mononuclear cells; TFA,
trifluoroacetic acid.
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 5423
Figure 1. (a) Proposed binding mode of 2,6-naphthyridine 1 to the PKD1 active site. (b) Truncation of A ring of 2,6-naphthyridine PKD
inhibitors (1) to provide bipyridyl compounds of type 2.
Scheme 1a
the tert-butylamide, proved to be more robust, avoided the
byproduct formation and allowed for greater diversity at R2.
To enhance diversity at R1, primary amides 12a,b,e were con-
verted to nitriles 12i-k by treatment with trifluoroacetic anhy-
dride (Scheme 2). For additional diversity at R1, intermediates
with R1 = -OTf and -Br were generated (Scheme 3). By SNAr
of 2,6-dibromo-4-nitropyridine 13 with BOC-piperazine, fol-
lowed by Suzuki coupling with 4a or 4b, compound 14a or 14b
were accessed by analogy to Schemes 1 and 2 above. Subsequent
a Reagents and conditions: (i) 4a, Pd(Ph3P)4, aqNa2CO3, CH3CN, 90 °C;
(ii) cHexNH2, Pd(t-Bu3P)2,t-BuONa, 1,4-dioxane, 130 °C; (iii) TFA, DCM,
room temp.
direct or palladium catalyzed coupling of an amine, such as
cyclohexylamine, with 14a or 14b, respectively, afforded 2-ami-
nopyridines 15a-c, which were protected with a BOC group to
give intermediates 16a-c. It is noteworthy to highlight the
conversion of 16a-c to 17a-c by treatment with potassium
hydroxide in which the nitro group served as a masked hydroxyl
the binding to the Lys612 while maintaining the hinge inter-
action were sought to improve upon 1. One such series of
compounds arose from truncation of the A-ring of 1 to
provide bipyridyls such as 2 that afford the opportunity to
introduce alternative hydrogen bond acceptor groups at R1.
Herein, we describe the design and optimization of this
demonstrated but typically with less functionalized com-
pounds.14 Conversion of the resulting 4-hydroxypyridine to tri-
bipyridyl series of PKD inhibitors.
group (Scheme 3). Utilization of a nitro group as a leaving group
in SNAr reactions is known but is less commonly utilized than
halogens. In addition, hydrolyses of 4-nitropyridines have been
flate 18b and cwas accomplished upon treatment with 2-(N,N-bis-
(trifluoromethylsulfonyl)amino)pyridine and triethylamine.
Chemistry
Alternatively, bromide 18a was prepared by reaction with
phosphorus oxytribromide. Under these reaction conditions,
the BOC groups were cleaved, thus requiring reintroduction of
the BOC group by treatment with BOC anhydride and triethy-
lamine. The bromide 18a and triflates 18b and 18c underwent
Suzuki coupling to provide pyrazoles 19a-d.
Compounds with R2 = cyclohexyl showed good in vitro
properties and profiles, so additionalexplorationof R1 and R3
was carried out leaving the R2 group constant. To that end,
stannane 21 (Scheme 4) was prepared by selective displace-
ment of the fluoride of 4-iodo-2-fluoropyridine 20 with cyclo-
hexylamine, followed by palladium mediated stannylation
with hexamethylditin. With stannane 21 in hand, Stille cou-
pling with 2-chloropyridine 22 afforded bipyridyl 23, which
was converted to targets by facile heating in dioxane with the
appropriate amine (HNR4R5). Installation of the amide to
give compounds 24a-i followed as outlined above. Likewise,
2,6-dichloro-4-substituted pyridine 25a (R1 = CF3) or 25b
(R1=CHF2) were treated with amine 9 to give intermediates
26a and 26b. Coupling of 26a and 26b by sequences outlined
above provided analogues 24j and 24k.
Compounds wherein R1 = H were prepared by a three-step
process (Scheme 1). Suzuki coupling of commercially avail-
able chloropyridine 3 to boronic acid 4a provided bipyridyl 5.
Subsequent palladium catalyzed amination using Pd(t-Bu3P)2
provided truncated analogue 6.
12
To generate analogues with R1 ¼ H (Scheme 2), treatment
of citrazinic acid 7 with phosphorus oxybromide followed by
quenching the intermediate acid bromide with methanol
provided the requisite 2,4,6-trisubstituted pyridine 8. Nucleo-
philic substitution of bromide 8 by amine 9 followed by Suzuki
coupling with pyridyl-4-boronic acids 4a and 4b provided
bipyridyls 10a and 10b, respectively. Conversion of the R1 ester
of 10a and 10b to an amide was accomplished by either of two
methods: (1) treatment with tert-butylamine under Weinreb
amidation13 conditions to generate the corresponding amide
11a; (2) treatment with methanolic ammonia to give the pri-
mary amide 11b directly. Chloropyridine 11a underwent
palladium catalyzed amination by analogy to Scheme 1.
Removal of the BOC and tert-butyl groups upon treatment
with TFA at elevated temperature provided the desired pro-
ducts 12b,c,d,f,g,h. Alternatively, suitable amines, such as
cyclohexylamine, displaced the fluoride of 11b, which, follow-
ing deprotection, provided target compounds 12a and 12g.
One of the limitations in the conversion of 11b to 12a was
competing transamidation at R1 to give an undesired cyclo-
hexylamide. The route to compounds 12 from 11a, employing
Given the promising in vitro data for amides like 12a and the
potential for in vivo hydrolysis of the primary amide, it was rea-
sonable to explore the impact of cyclizing such compounds back
onto the pyridine core as in 30 and 31 (Scheme 5). Intermediate
2715 underwent light-mediated benzylic bromination followed

5424 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Scheme 2a
a Reagents and conditions: (i) POBr3, 130 °C, then MeOH; (ii) 9, Et3N, 1,4-dioxane, 110 °C; (iii) 4a or 4b, Pd(dppf)Cl2 CH2Cl2, aq Na2CO3, DME,
3
80 °C; (iv) AlMe3, t-BuNH2, PhMe, 110 °C; (v) 7 M NH3/MeOH, 90 °C; (vi) 11b, c-hexylamine, 130 °C or NaHMDS, R2NH2, THF, 80 °C; (vii) 11a,
R2NH2, Pd(tBu3P)2, t-BuONa, 1,4-dioxane, 110 °C; (viii) TFA/DCM room temp; or TFA 120 °C, microwave for R1 = CONH-t-Bu; (ix) TFAA, Et3N,
DCM, room temp; (x) NaBH4, MeOH, room temp.
Scheme 3a
a Reagents and conditions: (i) 9, Et3N, 1,4-dioxane, 110 °C; (ii) 4a or 4b, Pd(dppf)Cl2 CH2Cl2, aq Na2CO3, DME, 90 °C; (iii) R2NH2, 110 °C;
3
(iv) R2NH2, Pd(t-Bu3P)2, t-BuONa, 110 °C, 1,4-dioxane; (v) BOC2O, DMAP, CH3CN/DCM reflux; (vi) KOH, DMSO, room temp; (vii) POBr3, 130 °C;
(viii) BOC2O, Et3N, DCM, room temp; (ix) 2-(N,N-bis(trifluoromethylsulfonyl)amino)pyridine, Et3N, DCM 0 °C to room temp; (x) R1B(OH)2,
Pd(dppf)Cl2 CH2Cl2, aq Na2CO3, DME, 130 °C, microwave; (xi) TFA/DCM, room temp.
3
by aminolysis, affording the desired lactam in one pot. Sub-
sequent displacement of onechlorinegave a separable mixture
of regioisomers 28 and 29. Stille coupling of 22 with either 28
or 29, followed by deprotection of the piperazine with TFA,
provided target compounds 30 and 31.
shown to blunt cardiac hypertrophy in the TAB mouse. A second
model, the Dahl salt-sensitive (DSS) rat, is a genetic model of
high-salt-induced hypertension and cardiac hypertrophy. In all
rat studies, the effect of compound treatment on blood pressure
was monitored to control for the anticipated benefit of antihy-
pertensives to cardiac hypertrophy in both models.
Compound Evaluation
Results
In vitro, compounds were evaluated for their ability to inhibit
the target PKD1, as well as PKD isoforms 2 and 3 and repre-
sentative PKC isoforms PKCR and PKCδ. All compounds in
the present study were found to inhibit the entire PKD family (1,
2, and 3) with similar potency. The HDAC nuclear export assay,
a cellular readout of PKD activity, monitored the ability of the
inhibitors to prevent prostaglandin F2R-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 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 PKD1 knockout was
The simple truncated analogue 6 proved to be a potent PKD
inhibitor (PKD1 IC50 =43 nM) with cellular activity (HDA-
Cexp IC50 =516 nM), albeit to a lesser extent than 1 (PKD1
IC50=1 nM, HDACexp IC50=32 nM). However, the biochemi-
cal and cellular efficacy of 6 was encouraging for further
exploration. Indeed, as hypothesized above, introducing a moi-
ety at R1 capable of making a hydrogen bond contact with
Lys612 such as in 12a (Figure 2) afforded a compound of equal
potency (PKD1 IC50=1 nM, HDACexp IC50=77 nM) to the
naphthyridine comparator 1. Therefore, it was demonstrated
that a primary R1 amide is capable of serving as an effective
replacement for the A-ring of 1.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5425
Scheme 4a
a Reagents and conditions: (i) cHexNH2, 120 °C; (ii) Me3SnSnMe3, Pd(Ph3P)4, PhMe, 100 °C; (iii) 22, CsF, Pd(t-Bu3P)2, 1,4-dioxane, 100 °C;
(iv) amine, Et3N, 1,4-dioxane, 90 °C; (v) 7 M NH3/MeOH, 90 °C; (vi) TFA/DCM, room temp.
Scheme 5a
a Reagents and conditions: (i) NBS, benzoylperoxide, heat lamp, CCl4,
60 °C; (ii) NH4OH, THF, room temp; (iii) 9, Et3N, 1,4-dioxane, 120 °C;
(iv) 21, CsF, Pd(tBu3P)2, 1,4-dioxane, 100 °C; (v) TFA/DCM, room temp.
Exploration of the R2 position led to the general observation
that, while a variety of substituents provided potent inhibition of
PKD1, simple branched alkyls provided optimal cellular acti-
vity. For example, R2=cyclohexyl (12a; PKD1 IC50=1 nM,
Figure 2. Proposed binding mode of bipyridyl 12a to the PKDl
active site.
HDACexp IC50=77 nM), isopropyl (12d; PKD1 IC50=1 nM,
HDACexp IC50 = 438 nM), and cyclopentyl (12g; PKD1
IC50=6 nM, HDACexp IC50 = 202 nM) offered comparable
biochemical activity but superior cellular activity to R2 = ethyl
(12h; PKD1 IC50 =4 nM, HDACexp IC50 >1000 nM). The
difference is most significant in the cellular HDAC5 export
assay. One surprising finding is that while 12c is a potent
inhibitor of PKD1 in the enzymatic assay, it failed to inhibit
nuclear export of HDAC5 below 1000 nM. Similar potency to
12a was attained with aryl and heteroaryl groups such as 12b
(PKD1 IC50 =1 nM, HDACexp IC50 =24 nM), 12e (PKD1
IC50=1 nM, HDACexp IC50=674 nM), and 12f (PKD1 IC50=
1 nM, HDACexp IC50 = 478 nM), yet only the simple phenyl
analogue 12b provided analogous cellular activity to that seen
when R2 = cyclohexyl.
assay. Intriguingly, 12i (PKD1 IC50=18 nM, HDACexp IC50=
66 nM) provided similar efficacy in the HDAC5 export assay to
that of 12a even with ∼20-fold lower potency in the PKD1 assay
(Table 1). This could be due to inherent differences in the cellular
permeability of the nitrile (Caco-2 cells, P(B-A)/P(A-B)=0.44)
compared to the amide (Caco-2 cells, P(B-A)/P(A-B)=7.89). A
similar trend in activity was seen with each of the nitriles and the
corresponding parent amides.
A variety of heterocycles were explored as potential hydrogen
bond donor/acceptor replacements for the primary amide in 12a.
Pyrazole isomers 19a-d provided the most promising examples
(Table 1). It is instructive to note the ability of pyrazole 19a to
closely mimic 12a. Pyrazoleisomer19a was more potent than the
alternative isomer 19b, which may indicate a preference for
The R1 nitrile analogues (12i-k; PKD1 IC50 =5-18 nM)
proved to be less potent than the parent amides in the enzymatic

5426 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Table 1. PKD Inhibition (IC50, nM) and HDAC5 Export (EC50, nM) Activity for Compoundsa

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5427
Table 1. Continued
a Values are the mean of at least two experiments.
Table 2. Pharmacokinetic Parameters Selected PKD Inhibitorsa
parameter
12a
BQLc
12ab
1
12j
24j
30
AUC0-8h, po (μM h)
CL ((mL/min)/kg)
Vdss (L/kg)
25.8 ( 3.7
0.19 ( 0.18
32 ( 12
12 ( 1
5.2 ( 1.9
0.12 ( 0.03
4
1.1 ( 0.2
31
7.8
0.73 ( 0.07
0.1 ( 0.05
86
35
3
97 ( 26
22 ( 6
3.8 ( 1.7
BQLc
t
1/2 (h)
max (nM)
F (%)
3.1
231 ( 49
18 ( 3
6.2
31 ( 25
5 ( 3
C
8568 ( 375
251 ( 50
31 ( 3
NCd
a Sprague-Dawley rat PK. Animals were dosed either iv (2 mg/kg, n = 2) or po (5 mg/kg, n = 3) with compound in 10% NMP, 10% Cremophor,
80% pH 4.63 buffer vehicle. PK parameters are derived from iv dosing and are reported as the mean ( SD. b Dahl-S rat: subcutaneous; 50 mg/kg; 10% 1
N HCl, 90% Captisol in pH buffer (10%); AUC is 0-24 h. c Below quantitation limits. d Not calculated, since oral exposure was BQL.
orientation of the H-bond donor/acceptor relative to both 1
and 12a. Likewise, pyrazole 19d was among the most effica-
cious PKD inhibitors evaluated to date in the HDAC5 export
cellular assay, while its comparator amide analogue 12c
provided no measurable efficacy below one micromolar.
Alteration of R3 beyond the parent piperazine proved to be
less fruitful. In general, with basic amine-containing R3
groups, such as in 24a-h, moderate levels of PKD inhibition
(IC50 < 100 nM) were achieved. However, in most cases, loss
of potency in the cell accompanied these subtle structural
changes. Again, this may be a consequence of changes in
permeability in the cellular assay. Compounds 12a with 24a,
which have similar activity in the enzymatic assays, showed
wide differences in cellular activities (EC50 for 24a is >1000
nM, while for 12a it is 77 nM). Removal of the basic amine,
such as 24i, produced compounds typically lacking activity in
the enzymatic assay.
Following low-dose iv and po administration of compound
12a to rats, a set of pharmacokinetic parameters were deter-
mined. Compound 12a had high clearance and no oral
bioavailability in rat PK (Table 2). The low bioavailability is
likely the result of poor permeability, as discussed above, since
the compound has an aqueous solubility of 860 μM (at pH
6.8). Many of the modifications described above were aimed
at replacing the amide moiety of 12a with groups to improve
the in vivo PK-ADME profile. Many of the compounds
analyzed, such as 12b,c,e showed similar behavior to 12a in
that they had high clearance and limited or no oral bioavail-
ability. The most promising changes were conversion of the
amide to a nitrile as exemplified by 12j, which provided 18%
oral bioavailability and attenuated clearance. Additionally, it
was found that when R1 = trifluoromethyl, as in 24j, reason-
able exposure was attained when dosed orally. Compounds 30
and 31 were designed specifically to ask whether or not the
lactam would provide improved oral exposure relative to the
primary amide 12a. Indeed, 30, provided some oral availabil-
ity, albeit still at only 5%. In the exploration of alternative
dosing, it was found, however, that 12a could be dose
subcutaneously (sc) (Table 2, Figure 4) to provide suitable
exposures for evaluation of the compounds in vivo in several
models of cardiac hypertrophy.
Key to further establishing the central role of PKD in
cardiac myocyte growth signaling was the identification of
an inhibitor that was more selective than 1. To that end, it was
fortuitous that 12a demonstrated improved selectivity, rela-
tive to 1, for PKD among other putative HDAC 4/5 kinases,
most notably CaMKII, MARK1, and MARK2 (Table 3).16a
In a broader kinase panel, 12a was also found to be more
selective than 1.16b If the proposed binding modes depicted in
Figures 1a and 2 accurately predict respective binding of the
inhibitors to PKD1, then it is clear from the similarity in
structure that the increased selectivity of 12a over 1 must be a
result of unfavorable interactions of the R1 amide of 12a with
the kinases in question. At present, in the absence of X-ray
crystallographic information this remains a working hypoth-
esis. By comparison of the proposed binding modes for 1 and
12a, it would be reasonable to hypothesize that substitution at
the C-5 of the naphthyridine should improve the kinase
selectivity of 1. In addition to improved kinase selectivity,
12a also shows minimal activity against a panel of receptors

5428 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Figure 3. Compound 12a (1 μM) blunts autophosphorylation of
PKD at Ser916 and phosphorylation of the PKD substrate HDAC
at Ser498 while not reducing PKC mediated phosphorylation of
PKD at Ser744/748 in ex vivo stimulation of peripheral blood
mononuclear cells (PBMCs) stimulated with PMA.
Figure 4. Dahl salt-sensitive rat plasma exposure following 50 mg/
kg sc and 100 mg/kg (solution and suspension) oral administration
of 12a: ([) 50 mg/kg sc solution; (2) 100 mg/kg po suspension; (9)
100 mg/kg po solution.
dose that could be used daily without an effect on blood
pressure was 5 (mg/kg)/day.
Table 3. PKD Inhibitor 12a Is Selective against Other Kinases
% inhibition at 1 μM a
It is known that DSS rats given a high-salt diet exhibit cardiac
hypertrophy and thus offer a model of disease. Blood pressure
and cardiac morphology results were obtained following dosing
of 12a (0.5, 1.5, or 5 (mg/kg)/day sc) in the DSS rat for 14 days.
No effect on mean blood pressure is observed at the highest
dose, 2 h after the final dose (Figure 5a). Additionally, the
compound did not affect cardiac hypertrophy (as measured by
the ratio of left ventricle mass to tibia length) at any of the three
doses (Figure 5b). Ex vivo analysis of PBMCs isolated from the
DSS rat study revealed that PKD autophosphorylation (Figure
6a) was also unaffected. By contrast, 12a at the highest dose (5
(mg/kg)/day sc) significantly reduced HDAC phosphorylation
(Figure 6b), indicating that blockade of pathway was achieved.
The concentration of 12a (LV 12 ( 2 μM, plasma 0.23 ( 0.8
μM) on day 14 (5 mg/kg sc) in the DSS rat was well above the
level required to inhibit PKD1 and HDAC5 nuclear export in
vitro (PKD1 IC50=1 nM, HDACexp IC50=77 nM, Table 1).
In the second model of cardiac hypertrophy, the thoracic
aortic bandedrat(TAB),12a (5 (mg/kg)/day sc) likewise did not
affect cardiac hypertrophy as measured by LV/TL (Figure 7).
The PBMC biomarker readouts from the TAB rat study with
12a mimicked the findings from the DSS rat. Therefore, while
no effect on PKD autophosphorylation (Figure 8a) was ob-
served, 12a significantly reduced HDAC phosphorylation
(Figure 8b) in PBMCs isolated from the TAB rat.
entry
kinase
12a
1
1
2
hCaMKIδ
rCaMKIIR
hCaMKIIβ
hCaMKIIδ
hCaMKIV
hMARK1
hMARK2
hGRK5
6
0
11
36
36
80
1
3
3
4
26
1
5
6
24
27
2
82
83
5
7
8
9
10
hPKCδ
hPKCε
22
51
34b
-3
a Mean of n = 2 measurements. Inhibition assays conducted by
Invitrogen Selectscreen. b Mean of n g 2 experiments; a more complete
comparison is provided in the Supporting Information.
and importantly is selective for PKD overR1AR(IC50 = 3000
nM) and β1AR (IC50 = 8300 nM).
Compound 12a, reported elsewhere as BPKDi,17 was shown
to inhibit endogenous PKD1 signaling. Specifically, treatment of
neonatal rat ventricular myocytes (NRVMs) with 12a (1 μM)
blunted autophosphorylation of PKD1 (Ser916) but not PKC
mediated phosphorylation of Ser744/748 in response to PMA,
ET-1, PGF2R, and PE. It was also reported that 12a (1 μM)
blocks agonist-dependent phosphorylation of both GFP tagged
and endogenous HDAC4 and HDAC5 in neonatal rat ventri-
cular myocytes (NRVMs). Treatment of NRVMs (infected
with GFP-tagged HDAC5) with 1 μM12a also blocked agonist-
driven nuclear export of HDAC5.
Conclusions
In the preceding paper (DOI: 10.1021/jm100075z),11 naph-
thyridine 1 demonstrated reduction of cardiac hypertrophy in
the DSS rat model. However, interpretation of the data is com-
plicated by suboptimal kinase and receptor selectivity. The aim
of the present study, to identify a compound with improved
kinase and receptor selectivity and sufficient exposure to study
PKD inhibition in vivo, was realized with compound 12a.
Amidobipyridyl inhibitor 12a has the desired properties to com-
plement the findings of comparator naphthyridine 1. Like 1,
potent PKD inhibitor 12a clearly demonstrated the ability to
control PKD autophosphorylation and HDAC5 phosphoryla-
tion in cells. In addition, 12a controls nuclear export without
disrupting upstream PKC signaling.16
In an ex vivo assay, 12a was also shown to blunt autopho-
sphorylation of PKD (Ser916) and downstream PKD mediated
phosphorylation of HDAC (Ser498) in PBMCs in response to
increasing concentrations of PMA (Figure 3). Importantly, this
blockade of signaling was accomplished without effecting the
phosphorylation of the PKC sites (Ser744/748) on PKD. How-
ever, it is important to note that 12a is more effective in blocking
the phosphorylation of HDAC than the phosphorylation of
PKD1 at Ser916 in response to PMA stimulation.
In order to evaluate the effect of 12a in a model of cardiac
hypertrophy, it was necessary to dose the compound sc, given
its limited oral bioavailability. A dramatic change in plasma
exposure with 12a is realized on changing from po to sc dosing
(Figure 4). While sc dosing at 50 mg/kg in the DSS rat
While 12a did not demonstrate suitable PK to enable oral
dosing, the compound was evaluated in models of cardiac
hypertrophy by employing sc dosing. The maximal dose of
provides good exposure (AUC0-8h = 25.8 μM h), the highest
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5429
Figure 5. Effect of compound 12a at 0.5, 1.5, 5 (mg/kg)/day sc for 14 days in the DSS rat on (a) mean blood pressure and (b) cardiac
hypertrophy (left ventricular mass/tibia length) on day 14. Compound 12a did not increase the mean blood pressure observed with high (8%)
salt diet. Compound 12a did not reduce cardiac hypertrophy (LV/TL) relative to controls: (/) p < 0.05 vs 0.05% NaCl plus vehicle.
Figure 6. 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 not blunted by compound 12a and (b) phosphorylation of the PKD substrate HDAC
at Ser498 is blunted by 12a given 5 mg/kg sc: (/) p <0.05 vs. vehicle; (#) p < 0.05 vs vehicle plus PMA.
the PBMC biomarker data, the exposures (LV 12 ( 2 μM,
plasma 0.23 ( 0.8 μM) are consistent with the inhibition of
HDAC5 phosphorylation in the heart. By analogy to naph-
thyridine 1, discussed in the preceding paper (DOI: 10.1021/
jm100075z), bipyridyl 12a produced a marked reduction
in HDAC phosphorylation in the PBMCs. Compounds 1
(PKD1 IC50 = 0.6 nM; HDACexp IC50 = 32 nM) and 12a
(PKD1 IC50=1 nM; HDACexp IC50 = 77 nM) are equivalent
with respect to in vitro potencies, and both achieve cardiac
exposure 100- to 1000-fold in excess of the cellular IC50 values.
Yet only the highest dose of 1 (50 (mg/kg)/day po, cardiac
exposure 60 μM) controls both PKD autophosphorylation and
cardiac hypertrophy. The higher cardiac exposure and lower
kinase selectivity of 1 versus 12a may be contributing factors to
the reduction of PKD autophosphorylation (Ser916) and cardi-
ac hypertrophy. Nonetheless, both 1 and 12a blunt the target
Figure 7. Effect of compound 12a at 5 (mg/kg)/day sc in the
thoracic aortic banded (TAB) rat on cardiac hypertrophy (left
ventricular mass/tibia length): (/) p <0.05 vs vehicle; (#) p < 0.05
phosphorylation event, which is the phosphorylation of the
PKD substrate HDAC5.
A major finding in this study is that the PKD selective
vs vehicle plus TAB.
inhibitor 12a failed to reduce cardiac hypertrophy in two
12a that could be employed, in the absence of effects on blood
pressure, was 5 (mg/kg)/day sc. In both models, 12a given daily
at 5 mg/kg sc produced no significant reduction of cardiac hyper-
trophy relative to vehicle controls. In the DSS rat, both plasma
and cardiac (LV) exposures were significantly greater than the
PKD1 and HDACexp IC50 values. Taken in combination with
distinct animal models (DSS and aortic banded rats). This is
seemingly in contradiction to previously published work
demonstrating that genetic loss of PKD1 protects from
pressure-overload-induced cardiac hypertrophy. There are
several possibilities to explain these results. First, it is possible
that inhibition of PKD1 kinase activity is not equivalent to

5430 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
Figure 8. Ex vivo. In peripheral blood mononuclear cells (PBMCs), PMA-stimulated, (a) autophosphorylation of PKD at Ser916 is not
blunted by compound 12a and (b) phosphorylation of the PKD substrate HDAC at Ser498 is reduced: (/) p <0.05 vs vehicle; (#) p <0.05 vs
vehicle plus PMA.
total loss of the protein as in the PKD1 knockout mouse. The
PKD1 protein may have other functions such as to serve as a
scaffold or to possess a signaling function that contributes to
its prohypertrophic activity. In addition, because of technical
limitations, we were unable to detect pHDAC5 in the heart.
Although 12a was able to reduce pHDAC5 in PBMCs, we
cannot be certain that the same is true in the heart in spite of
the high cardiac exposure of the compound. Finally, it is
possible that there is a difference in the animal models tested.
The models used here are both in the rat, while the PKD1
knockout is obviously in the mouse. There may be a funda-
mental difference in the role of PKD1 in the rat versus the
mouse in the control of cardiac cell growth. Nonetheless, 12a
represents a potent and selective PKD inhibitor that has
proven to be a valuable tool to further define the role of
PKD1 in cardiac hypertrophy and failure.
substrate phosphorylation and enzyme activity. Compounds
were typically tested in an 11-point dose response fashion in
triplicate for each concentration used. IC50 values were calcu-
lated using an activity base (IDBS) software program.
PKD2 Assay. The assay to measure protein kinase D2
(PKD2) activity was a time-resolved fluorescence resonance
transfer (TR-FRET) assay using PerkinElmer’s LANCE technol-
ogy. In this case, a biotinylated syntide-2 peptide was used as the
substrate in this reaction. Phosphorylation of the syntide-2 sub-
strate was detected by a specific antibody that recognizes the
phosphorylated peptide. A second fluorophore, APC, was con-
jugated to streptavidin that binds the biotinylated syntide-2 pep-
tide. For detection, the europium fluorophore can be excited by
340 nM light which then emits at 615 nM. Therefore, when the
europium labeled secondary antibody binds on the phosphory-
lated 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.
PKD3 Assay. 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 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 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 were typi-
cally tested in an 11-point dose response fashion in triplicate for
each concentration used. IC50 values were calculated using an
activity base (IDBS) software program.
Experimental Section
PKD1 Assay. The assay to measure protein kinase D1
(PKD1) activity was a time-resolved fluorescence resonance
transfer (TR-FRET) assay using PerkinElmer’s LANCE tech-
nology. 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 con-
jugated 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. Therefore, when
the europium labeled secondary antibody binds on the phos-
phorylated 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 PKD1
activity. This assay was performed with full length wild-type
enzyme that was 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 strepta-
vidin 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
The assay to measure protein kinase D3 (PKD3) activity was
a time-resolved fluorescence resonance 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 de-
tected by a specific antibody that recognizes the phosphorylated
peptide. A second fluorophore, APC, was conjugated to strep-
tavidin that binds the biotinylated syntide-2 peptide. For detec-
tion, the europium fluorophore can be excited by 340 nM light
which then emits at 615 nM. Therefore, when the europium
labeled secondary antibody binds on the phosphorylated pep-
tide, it was brought into close contact with the APC and excites

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5431
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 were 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 of a tridecapeptide acceptor
substrate 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
without Ca, Mg. After 10 min of incubation at room tempera-
ture, the suspension was spun down for 10 min at 300g.
Incorporated 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
ranging between 1 and 1000 nM. IC50 values were calculated
from the graph by curve fitting with XL fit software. Human
recombinant PKCR was obtained from Oxford Biomedical
Research and was used under the assay conditions as described
above. Human recombinant PKCδ was obtained from Oxford
Biomedical Research and was used under the assay conditions
as described above.
HDAC Export Assay. Compounds were 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 protein (GFP)
tagged HDAC5. Neonatal rat ventricular myocytes (NRVMs)
were infected with GFP-HDAC5 encoding virus and plated on
gelatin-coated 384-well dishes. Cells were exposed to compound
and stimulated with a prostaglandin (PGF2R), which is a potent
stimulus for HDAC5 nuclear export. Following 2 h of stimula-
tion, cells were fixed and GFP-HDAC5 localization was quan-
tified using the Cellomics system, which provides a readout of
relative fluorescence intensity in the cytoplasmic versus nuclear
compartment.
DSS Rat Model. Six to seven 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 to
being separated into five weight-matched groups. Rats were
maintained on the grain based diet (0.49% NaCl) or switched to
grain diet containing 8.0% NaCl for 2 consecutive weeks.
Coincident with diet switch rats were administered (sc, 1.0
mL/kg) vehicle (10% 1 N HCl/90% 10% captisol in pH 4.6
buffer) or test compound 12a (0.5, 1.5, or 5 mg/kg) in vehicle. At
the completion of study, steady-state hemodynamics were de-
termined. The rats were then sacrificed, and tissues were col-
lected for morphological and biochemical analysis.
TAB Rat Model. Seven to eight 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 underwent sham surgery. Coincident with the surgical
procedure, rats were administered (sc, 1.0 mL/kg) vehicle
(acidified captisol) or 12a (5 (mg/kg)/day sc) dissolved in vehicle
for 2 weeks. On the final day of study, end-point cardiac per-
formance 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
Bruker Avance II 400 MHz spectrometer. All chemical shifts
are reported in parts per million (δ) relative to tetramethylsilane.
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
(4-300 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 LCMS spectrometer.
4-(20-Chloro[2,40]bipyridinyl-6-yl)piperazine-1-carboxylic Acid
tert-Butyl Ester (5). A mixture of 4-(6-bromopyridin-2-yl)-
piperazine-1-carboxylic acid tert-butyl ester (1.98 g, 5.78 mmol),
2-chloropyridine-4-boronic acid (1.0 g, 6.35 mmol), Pd(Ph3P)4
(0.330 g, 0.289 mmol), aqueous solution of Na2CO3 (5.7 mL, 2.0
M), and CH3CN (10 mL) was sparged with argon for 10 min.
The vessel was then sealed, and the contents were heated to 90 °C
for 4 h. The mixture was then allowed to cool and concentrated
under reduced pressure. The residue was taken up in CH2Cl2
and washed with H2O. The aqueous layer was further extracted
with CH2Cl2 (2 ꢀ 50 mL). The combined organic layers were
then dried (Na2SO4), filtered, and concentrated. The residue was
purified via flash chromatography (SiO2, 20-30% EtOAc/
hexanes gradient) to give the title compound 4-(20-chloro[2,40]-
bipyridinyl-6-yl)piperazine-1-carboxylic acid tert-butyl ester:
MS (ESI) m/z 375.0, 376.9 (M þ 1); 1H NMR (400 MHz,
CDCl3) δ ppm 8.44 (d, J = 5.3 Hz, 1 H), 7.93 (s, 1 H), 7.78 (dd,
J = 5.1, 1.5 Hz, 1 H), 7.61 (dd, J = 8.5, 7.5 Hz, 1 H), 7.16 (d, J =
7.3 Hz, 1 H), 6.73 (d, J = 8.6 Hz, 1 H), 3.55-3.69 (m, 8 H), 1.50
(s, 9 H).
Cyclohexyl-(6-piperazin-1-yl[2,40]bipyridinyl-20-yl)amine (6).
A mixture of 4-(20-chloro[2,40]bipyridinyl-6-yl)piperazine-1-car-
boxylic acid tert-butyl ester (0.300 g, 0.801 mmol), Pd(t-Bu3P)2
(0.041 g, 0.080 mmol), NaO-t-Bu (0.115 g, 1.20 mmol), cyclo-
hexylamine (0.18 mL, 1.60 mmol), and 1,4-dioxane (4 mL) was
sparged with argon for 10 min. The vessel was then sealed, and
the contents were heated to 130 °C for 8 h. The mixture was then
allowed to cool followed by concentration. The residue was then
separated via flash chromatography (SiO2, EtOAc/hexanes
gradient) to give 4-(20-cyclohexylamino[2,40]bipyridinyl-6-yl)-
piperazine-1-carboxylic acid tert-butyl ester: MS (ESI) m/z
438.0 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 7.99 (d,
J = 5.3 Hz, 1 H), 7.61-7.69 (m, 1 H), 7.19 (d, J = 7.6 Hz, 1 H),
7.15 (s, 1 H), 7.01 (d, J = 5.6 Hz, 1 H), 6.88 (d, J = 8.6 Hz, 1 H),
6.32-6.61 (m, 1 H), 3.68-3.78 (m, 1 H), 3.59 (d, J = 10.4 Hz, 4
H), 3.42-3.50 (m, 4 H), 1.94 (d, J = 15.9 Hz, 2 H), 1.73 (d, J =
19.5 Hz, 2 H), 1.60 (d, J = 20.5 Hz, 1 H), 1.43 (s, 9 H), 1.26-1.40
(m, 2 H), 1.11-1.26 (m, 3 H).
To a solution of 4-(20-cyclohexylamino[2,40]bipyridinyl-6-yl)-
piperazine-1-carboxylic acid tert-butyl ester (0.220 g, 0.503
mmol) and CH2Cl2 (7 mL) was added TFA (5 mL). After being
stirred for 1 h, the solution was concentrated. The residue was
taken up in CH2Cl2 (50 mL) and washed with a saturated
aqueous solution of Na2CO3. The aqueous layer was further
extracted with CH2Cl2 (2 ꢀ 50 mL). The combined organic
layers were then dried (Na2SO4), filtered, and concentrated. The
residue was then purified via semipreperative HPLC (10-90%
CH3CN/H2O gradient with 0.1% NH4OH) to give the title
compound cyclohexyl-(6-piperazin-1-yl[2,40]bipyridinyl-20-yl)-
amine: MS (ESI) m/z 338.2 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.99 (d, J = 5.3 Hz, 1 H), 7.61 (t, J = 8.0 Hz,
1 H), 7.12 (s, 1 H), 7.10-7.17 (m, 1 H), 6.98 (d, J = 5.6 Hz, 1 H),

5432 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
6.82 (d, J=8.6 Hz, 1 H), 6.43 (d, J=7.8 Hz, 1 H), 3.66-3.80 (m, 1
H), 3.44-3.55 (m, 4 H), 3.31 (s, 1 H), 2.77-2.86 (m, 4 H),
1.87-1.98 (m, 2 H), 1.67-1.78 (m, 2 H), 1.55-1.65 (m, 1 H),
1.26-1.39 (m, 2 H), 1.12-1.25 (m, 3 H).
50%): (ESI) m/z 481.4 (M þ 1); 1H NMR (400 MHz, CD2Cl2) δ
ppm 8.01 (d, J = 5.6 Hz, 1 H), 7.53 (s, 1 H), 7.18 (s, 1 H), 7.02 (dd,
J = 5.3, 1.3 Hz, 1 H), 6.97 (s, 1 H), 4.60 (br s, 1 H), 3.55-3.65 (m, 4
H), 3.44-3.51 (m, 4 H), 1.91-2.05 (m, 2 H), 1.63-1.76 (m, 2 H),
1.53-1.63 (m, 2 H), 1.39 (s, 9 H), 1.10-1.25 (m, 4 H).
2,6-Dibromoisonicotinic Acid Methyl Ester (8). A mixture of
citrazinic acid 7 (5.0 g, 32.2 mmol) and POBr3 (27.5 g, 96.8
mmol) was heated at 130 °C. Upon completion of the reaction,
the thick slurry was cooled to 0 °C and carefully quenched with
MeOH (250 mL). The reaction mixture was concentrated in
vacuo and then partitioned between dichloromethane and a
saturated aqueous solution of NaHCO3. The organic layer was
dried over anhydrous Na2SO4 and concentrated in vacuo to give
a tan solid that was clean enough by NMR/LCMS for further
use (7.5 g, 79%): (ESI) m/z 295.8 (M þ 1); 1H NMR (400 MHz,
CD2Cl2) δ ppm 8.10 (s, 2 H), 4.05 (s, 3 H).
To a solution of 4-(4-carbamoyl-20-cyclohexylamino[2,40]bipy-
ridinyl-6-yl)piperazine-1-carboxylic acid tert-butyl ester (96.0
mg, 0.20 mmol) and CH2Cl2 (5.0 mL) was added TFA (5.0 mL).
After being stirred for 2 h, the solution was concentrated. The
residue was taken up in CH2Cl2 (10 mL) and washed with a
saturated aqueous solution of NaHCO3. The aqueous layer was
extracted with fresh CH2Cl2 (2 ꢀ 10 mL). The combined organic
layers were then dried (Na2SO4), filtered, and concentrated. The
residue was purified via semipreparative HPLC (10-90%
CH3CN/H2O gradient with 0.1% NH4OH) to give the title
6-(4-tert-Butoxycarbonylpiperazin-1-yl)-20-chloro[2,40]bipyri-
dinyl-4-carboxylic Acid Methyl Ester (10a). 2,6-Dibromoisoni-
cotinic acid methyl ester (5.0 g, 17.0 mmol), piperazine-1-
carboxylic acid tert-butyl ester (3.2 g, 17.0 mmol), and Et3N
(3.5 mL, 25.5 mmol) were stirred in 1,4-dioxane (75 mL) at 110
°C in a 150 mL pressure vessel until the reaction was completed
by LCMS. The reaction vessel was cooled to room temperature
and concentrated in vacuo. The residue was taken up in acet-
onitrile/water (1:9). A tan solid precipitate was collected by
filtration and dried to give the title compound . The purity was
sufficient by NMR/LCMS to use for further transformations
(5.4 g, 80%): MS (ESI) m/z 402.0 (M þ 1); 1H NMR (400 MHz,
CDCl3) δ ppm 7.21 (s, 1 H), 7.03 (s, 1 H), 3.85 (s, 3 H), 3.50-3.55
(m, 4 H), 3.44-3.49 (m, 4 H), 1.41 (s, 9 H).
1
compound (35.0 mg, 46%): MS (ESI) m/z 381.2 (M þ 1); H
NMR (400 MHz, DMSO-d6) δ ppm 8.17 (s, 1 H), 8.02 (d, J =
5.3 Hz, 1 H), 7.60 (s, 1 H), 7.52 (s, 1 H), 7.20 (s, 1 H), 7.16 (s, 1 H),
7.03 (dd, J = 5.4, 1.4 Hz, 1 H), 6.47 (d, J = 7.8 Hz, 1 H), 3.67-
3.84 (m, 1 H), 3.48-3.63 (m, 4 H), 2.76-2.92 (m, 4 H), 1.93 (dd,
J = 11.7, 2.4 Hz, 2 H), 1.67-1.80 (m, 2 H), 1.53-1.66 (m, 1 H),
1.10-1.41 (m, 5 H); HRMS (ESI) calcd m/z 381.2403 (M þ 1),
found m/z 381.2395 (M þ 1). Anal. (C21H28N6O 1H2O) calcd,
3
C 63.29, H 7.59, N 21.09; found, C 63.24, H 7.52, N 21.08.
20-Cyclopentylamino-6-piperazin-1-yl[2,40]bipyridinyl-4-carbo-
xylic Acid Amide (12g). The title compound was prepared by a
similar method to 12a: MS (ESI) m/z 367.2 (M þ 1); 1H NMR
(400 MHz, DMSO-d6) δ ppm 8.18 (s, 1 H), 8.04 (d, J = 5.6 Hz, 1
H), 7.61 (s, 1 H), 7.54 (s, 1 H), 7.22 (s, 1 H), 7.15 (s, 1 H), 7.05 (dd,
J = 5.3, 1.5 Hz, 1 H), 6.58 (d, J = 6.8 Hz, 1 H), 4.09-4.24 (m, 1
H), 3.52-3.65 (m, 4 H), 2.80-2.98 (m, 4 H), 1.85-1.98 (m, 2 H),
1.63-1.78 (m, 2 H), 1.37-1.62 (m, 4 H).
4-(6-Bromo-4-methoxycarbonylpyridin-2-yl)piperazine-1-car-
boxylic acid tert-butyl ester (1.5 g, 3.76 mmol) and 2-chloro-4-
pyridine boronic acid (0.71 g, 4.51 mmol) were stirred in DME
(25 mL). To this was added a 2.0 M solution of Na2CO3 (6.0 mL,
20-(1-Methyl-1H-pyrazol-3-ylamino)-6-piperazin-1-yl[2,40]bipyri-
dinyl-4-carboxylic Acid Amide (12e). A solution of NaHMDS
(0.5 mL, 0.48 mmol, 1.0 M THF) was added to a solution of
3-amino-1-methylpyrazole (0.024 g, 0.24 mmol) in THF (3 mL)
at ambient temperature. Then 4-(4-carbamoyl-20-fluoro[2,40]bi-
pyridinyl-6-yl)piperazine-1-carboxylic acid tert-butyl ester (50
mg, 0.12 mmol) was added. The reaction mixture was sealed and
heated to 80 °C for 3 h. The reaction was quenched with i-PrOH
and concentrated in vacuo. The residue was purified by flash
chromatography (2-10% MeOH/DCM) to afford 4-(4-carba-
moyl-20-(2-methyl-2H-pyrazol-3-yl)piperazine-1-carboxylic acid
tert-butyl ester: MS (ESI) m/z 479.3 (M þ 1).
11.28 mmol) and Pd(dppf)Cl2 CH2Cl2 (0.31 g, 0.37 mmol). This
3
above suspension was heated to 80 °C for 4 h. The mixture was
diluted with EtOAc (25 mL) and partitioned between organic
and saturated NaHCO3 (ꢀ2). The organic layer was washed
with brine, dried over anhydrous Na2SO4, and evaporated
under reduced pressure. The crude residue was purifed via flash
chromatography (SiO2, EtOAc/heptanes gradient) to afford the
compound as a pale-yellow solid (1.40 g, 87%): MS (ESI) m/z
433.2 (M þ 1); 1HNMR (400 MHz, CD2Cl2) δ ppm 8.37 (d, J =
4.5 Hz, 1 H), 7.92 (d, J = 1.5 Hz, 1 H), 7.79 (dd, J = 5.1, 1.5 Hz,
1 H), 7.60 (s, 1 H), 7.26 (s, 1 H), 3.86 (s, 3 H), 3.57-3.68 (m, 4 H),
3.41-3.53 (m, 4 H), 1.39 (s, 9 H).
4-(4-Carbamoyl-20-(2-methyl-2H-pyrazol-3-yl)piperazine-1-
carboxylic acid tert-butyl ester (175 mg, 0.37 mmol) and TFA
(5 mL) were stirred in CH2Cl2 (5 mL) at 25 °C for 2 h. After being
stirred for 2 h, the solution was concentrated. The residue was puri-
fied via semipreparative HPLC (10-90% CH3CN/H2O gradient
with 0.1% NH4OH) to give 20-(1methyl-1H-pyrazol-3-ylamino)-6-
piperazin-1-yl[2,40]bipyridinyl-4-carboxylic acid amide: MS (ESI)
m/z 379.2 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 8.94 (br
s, 2 H), 8.20-8.37 (m, 2 H), 8.12 (s, 1 H), 7.80 (s, 1 H), 7.76 (s, 1 H),
7.71 (s, 1 H), 7.57 (d, J = 5.4 Hz, 1 H), 7.46 (s, 1 H), 6.24 (d, J = 2.1
Hz, 1 H), 3.88-3.98 (m, 4 H), 3.85 (s, 3 H), 3.28 (br s, 4 H).
6-Piperazin-1-yl-20-(tetrahydropyran-4-ylamino)[2,40]bipyridi-
nyl-4-carboxylic Acid Amide. (12c). To a solution of toluene
(60 mL) and trimethylaluminum (23.1 mL, 46.3 mmol) was added
tert-butylamine (4.9 mL, 46.3 mmol). This solution was stirred at
room temperature for 10 min before 6-(4-tert-butoxycarbonylpi-
perazin-1-yl)-20-chloro[2,40]bipyridinyl-4-carboxylic acid methyl es-
ter (2.5 g, 5.78 mmol) was added portionwise. The resulting
suspension was heated at 110 °C until LCMS indicated complete
reaction. The mixture was cooled to ambient temperature and
quenched carefully with MeOH. The gelatinous suspension was
filtered and the filter cake washed well with MeOH. The filtrate was
concentrated in vacuo and the residue purified via flash chroma-
tography (SiO2, EtOAc/heptanes gradient) to afford the 4-(4-tert-
butylcarbamoyl-20-chloro[2,40]bipyridinyl-6-yl)piperazine-1-carbo-
xylic acid tert-butyl ester as a yellow solid (2.05 g, 75%): MS (ESI)
6-(4-tert-Butoxycarbonylpiperazin-1-yl)-20-fluoro[2,40]bipyri-
dinyl-4-carboxylic Acid Methyl Ester (10b). 10b was prepared as
described for 10a: MS (ESI) m/z 417.4 (M þ 1); 1H NMR (400
MHz, CDCl3) δ ppm 8.32 (d, J = 5.3 Hz, 1 H), 7.78-7.83 (m, 1
H), 7.70 (s, 1 H), 7.60 (s, 1 H), 7.35 (s, 1 H), 4.00 (s, 3 H), 3.70-
3.76 (m, 4 H), 3.59-3.65 (m, 4 H).
20-Cyclohexylamino-6-piperazin-1-yl[2,40]bipyridinyl-4-carbo-
xylic Acid Amide (12a). 6-(4-tert-Butoxycarbonylpiperazin-1-
yl)-20-fluoro[2,40]bipyridinyl-4-carboxylic acid methyl ester (435.0
mg, 1.00 mmol) was dissolved in a 7.0 M NH3/MeOH solution (25
mL) and heated at 90 °C in a sealed pressure vessel .Upon
completion, the reaction was concentrated in vacuo and the residue
obtained was used without further purification (398.0 mg, 95%):
(ESI) m/z402.1 (M þ 1); 1HNMR(400MHz, CD3CN) δppm 8.30
(d, J = 5.3 Hz, 1 H), 7.91-7.98 (m, 1 H), 7.71 (s, 1 H), 7.58 (d, J =
1.0 Hz, 1 H), 7.21 (d, J = 1.0 Hz, 1 H), 6.98 (s, 1 H), 6.23 (s, 1 H),
3.64-3.76 (m, 4 H), 3.48-3.59 (m, 4 H), 1.48 (s, 9 H).
4-(4-Carbamoyl-20-fluoro[2,40]bipyridinyl-6-yl)piperazine-1-
carboxylic acid tert-butyl ester (160.0 mg, 0.39 mmol) was dis-
solved in neat cyclohexylamine (8 mL) and heated at 130 °C in a
sealed pressure vessul until the reaction was complete. The reaction
was concentrated in vacuo and the residue purified via semi-
preparative HPLC (5-50% CH3CN/H2O gradient with 0.1%
NH4OH) to give 4-(4-carbamoyl-20-cyclohexylamino[2,40]bipyri-
dinyl-6-yl)piperazine-1-carboxylic acid tert-butyl ester (95.0 mg,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5433
m/z 474.1 (M þ 1); 1H NMR (400 MHz, CD2Cl2) δ ppm 8.35 (d,
J = 5.1 Hz, 1 H), 7.88 (s, 1 H), 7.76 (dd, J = 5.3, 1.5 Hz, 1 H), 7.21
(s,1H),6.93(s, 1H),5.95(brs,1H),3.55-3.65 (m, 4 H), 3.44-3.51
(m, 4 H), 1.39 (s, 18 H).
layer was dried over anhydrous Na2SO4, filtered, and concen-
trated. The residue (100 mg) was dissolved in MeOH (2.0 mL) and
treated with NaBH4 (14 mg, 0.36 mmol) at 0 °C. After 2 h, the
mixture was evaporated and the residue was taken up in CH2Cl2/
TFA (2:1) and stirred for 3 h at 0 °C. The acid was neutralized by
washing with a saturated aqueous solution of NaHCO3. The
residue was purified by column chromatography (5-10% MeOH/
CH2Cl2) to give the title compound: MS (ESI) m/z 363.3 (M þ 1);
1H NMR (400 MHz, DMSO-d6) δ ppm 8.03 (d, J = 5.3 Hz, 1 H),
7.45 (s, 1 H), 7.30 (s, 1 H), 7.15 (s, 1 H), 7.03 (dd, J = 5.4, 1.4 Hz, 1
H), 6.50 (d, J = 7.7 Hz, 1 H), 3.67-3.81 (m, 1 H), 3.51-3.65 (m, 4
H), 2.73-2.88 (m, 4 H), 1.85-1.99 (m, 2 H), 1.66-1.78 (m, 2 H),
1.54-1.66 (m, 1 H), 1.09-1.41 (m, 6 H).
A mixture of 4-(4-tert-butylcarbamoyl-20-chloro[2,40]bipyri-
dinyl-6-yl)-piperazine-1-carboxylic acid tert-butyl ester (225.0
mg, 0.47 mmol), Pd(t-Bu3P)2 (24.0 mg, 0.047 mmol), NaO-t-Bu
(141.0 mg, 1.41 mmol), 4-aminotetrahydropyran (0.14 mL, 1.41
mmol), and 1,4-dioxane (5 mL) was sparged with argon for
10 min. The vessel was sealed, and the contents were heated to
130 °C for 2 h. The mixture was allowed to cool and concentrated
under reduced pressure. The residue was purified via flash chro-
matography (SiO2, EtOAc/hexanes gradient) to give 4-[4-carba-
moyl-20-(tetrahydropyran-4-ylamino)[2,40]bipyridinyl-6-yl]piper-
azine-1-carboxylic acid tertbutyl ester (150 mg, 59%): MS (ESI)
m/z 539.2 (M þ 1); 1H NMR (400 MHz, CD2Cl2) δ ppm 8.13 (d,
J = 5.3 Hz, 1 H), 7.26 (s, 1 H), 7.13 (dd, J = 5.3, 1.5 Hz, 1 H), 7.07
(s, 1 H), 7.01 (s, 1 H), 6.07 (br s, 1 H), 4.76 (br s, 1 H), 3.93-4.16
(m, 1 H), 3.65-3.76 (m, 4 H), 3.58 (dd, J = 6.3, 4.0 Hz, 4 H), 1.50
(s, 18 H), 1.29 (d, J = 6.6 Hz, 6 H).
A mixture of 4-[4-carbamoyl-20-(tetrahydropyran-4-ylami-
no)[2,40]bipyridinyl-6-yl]piperazine-1-carboxylic acid tert-butyl
ester (115.0 mg, 0.21 mmol) and TFA (8 mL) was stirred in a
microwave at 120 °C for 2 h. After being stirred for 2 h, the
solution was concentrated. The residue was purified via semi-
preperative HPLC (10-90% CH3CN/H2O gradient with 0.1%
NH4OH) to give the title compound (95.0 mg, 75%): MS (ESI)
m/z 383.1 (M þ 1); 1H NMR (400 MHz, MeOH-d4) δ ppm 7.90
(d, J = 5.6 Hz, 1 H), 7.46 (s, 1 H), 7.16 (s, 1 H), 7.13 (s, 1 H), 7.07
(dd, J = 5.6, 1.5 Hz, 1 H), 3.80-3.95 (m, 3 H), 3.55-3.66 (m, 4
H), 3.41-3.53 (m, 2 H), 2.78-2.94 (m, 4 H), 1.82-1.98 (m, 2 H),
1.33-1.54 (m, 2 H).
20-Phenylamino-6-piperazin-1-yl[2,40]bipyridinyl-4-carbonitrile
(12j). The title compound was prepared by a similar method as
1
described for 12: MS (ESI) m/z 357.2 (M þ 1); H NMR (400
MHz, MeOH-d4) δ ppm 8.16 (d, J = 5.6 Hz, 1 H), 7.51-7.57 (m,
2 H), 7.48-7.52 (m, 1 H), 7.39 (s, 1 H), 7.24-7.35 (m, 3 H), 7.14
(s, 1 H), 6.91-7.03 (m, 1 H), 3.57-3.74 (m, 4 H), 2.86-3.00 (m,
4 H).
20-(1-Methyl-1H-pyrazol-3-ylamino)-6-piperazin-1-yl[2,40]bipyri-
dinyl-4-carbonitrile (12k). The title compound was prepared by a
similar method as described for 12i: MS (ESI) m/z 361.2 (M þ 1);
1H NMR (400 MHz, MeOH-d4) δ ppm 8.16 (d, J = 5.3 Hz, 1 H),
7.92 (s, 1 H), 7.46 (d, J = 2.0 Hz, 1 H), 7.40 (s, 1 H), 7.32 (dd, J =
5.3, 1.5 Hz, 1 H), 7.14 (s, 1 H), 6.25 (d, J = 2.3 Hz, 1 H), 3.82 (s,
3 H), 3.64-3.73 (m, 4 H), 2.89-3.01 (m, 4 H).
4-(20-Fluoro-4-nitro[2,40]bipyridinyl-6-yl)piperazine-1-carboxy-
lic Acid tert-Butyl Ester (14a). A mixture of 2,6-dibromo-4-
nitropyridine (5.0 g, 17.8 mmol), piperazine-1-carboxylic acid
tert-butyl ester (4.0 g, 21.4 mmol), triethylamine (5 mL, 35.6
mmol), and dioxane (60 mL) was heated to 110 °C for 4 h. The
mixture was then allowed to cool to room temperature, diluted
with CH2Cl2, washed with saturated NaHCO3, brine and then
dried (Na2SO4), filtered, and concentrated. The residue was
purified via flash chromatography (SiO2, 10-30% EtOAc/
heptane gradient) to give 4-(6-bromo-4-nitropyridin-2-yl)pipe-
razine-1-carboxylic acid tert-butyl ester: MS (ESI) m/z 386.9,
388.9 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 7.42 (d, J =
1.5 Hz, 1 H), 7.22 (d, J = 1.5 Hz, 1 H), 3.64-3.69 (m, 4 H),
3.55-3.60 (m, 4 H), 1.50 (s, 9 H).
20-Ethylamino-6-piperazin-1-yl[2,40]bipyridinyl-4-carboxylic Acid
Amide (12h). The title compound was prepared by a similar
method as described for 12c: MS (ESI) m/z 327.1 (M þ 1); 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.18 (s, 1 H), 8.04 (d, J =
5.3 Hz, 1 H), 7.60 (s, 1 H), 7.53 (s, 1 H), 7.20 (s, 1 H), 7.13 (s, 1 H),
7.07 (dd, J = 5.4, 1.5 Hz, 1 H), 6.56 (t, J = 5.4 Hz, 1 H),
3.49-3.59 (m, 4 H), 3.24-3.36 (obs q, 2 H), 2.73-2.87 (m, 4 H),
2.38 (br s, 1 H), 1.15 (t, J = 7.1 Hz, 3 H).
40-tert-Butylcarbamoyl-200-isopropylamino-3,4,5,6-tetrahydro-
2H-[4,20;60,400]terpyridine-1-carboxylic Acid tert-Butyl Ester
(12d). The title compound was prepared by a similar method
as described for 12c: MS (ESI) m/z 341.1 (M þ 1); 1H NMR (400
MHz, MeOH-d4) δ ppm 7.89 (d, J = 6.1 Hz, 1 H), 7.45 (d, J =
1.0 Hz, 1 H), 7.09-7.15 (m, 2 H), 7.05 (dd, J = 5.7, 1.6 Hz, 1 H),
3.82-4.01 (m, 1 H), 3.51-3.65 (m, 4 H), 2.79-2.92 (m, 4 H),
1.15 (d, J = 6.6 Hz, 6 H).
A mixture of 4-(6-bromo-4-nitropyridin-2-yl)piperazine-1-
carboxylic acid tert-butyl ester (1.9 g, 4.9 mmol), 2-fluoropyr-
idine-4-boronic acid (0.9 g, 6.37 mmol), Pd(dppf)Cl2 CH2Cl2
3
(0.2 g, 0.245 mmol), an aqueous solution of Na2CO3 (5.0 mL, 2.0
M), and DME (45 mL) was sparged with argon for 10 min and
then heated to 90 °C for 3 h under argon. The mixture was then
allowed to cool to room temperature, diluted with CH2Cl2,
washed with saturated NaHCO3 (ꢀ2), dried (Na2SO4), filtered,
and concentrated. The residue was purified via flash chroma-
tography (SiO2, 20-30% EtOAc/heptane gradient) to give the
20-(2-Chlorophenylamino)-6-piperazin-1-yl[2,40]bipyridinyl-4-
carboxylic Acid Amide (12f). The title compound was prepared
by a similar method as described for 12c: MS (ESI) m/z 409.1 (M
þ 1); 1H NMR (400 MHz, MeOH-d4) δ ppm 8.16 (d, J = 5.4 Hz,
1 H), 7.88 (dd, J = 8.1, 1.5 Hz, 1 H), 7.66 (s, 1 H), 7.59 (s, 1 H),
7.44 (d, J = 6.8 Hz, 2 H), 7.28 (dt, 1 H), 7.23 (s, 1 H), 7.05 (dt,
J = 7.7, 1.5 Hz, 1 H), 3.61-3.73 (m, 4 H), 2.89-3.01 (m, 4 H).
20-Phenylamino-6-piperazin-1-yl[2,40]bipyridinyl-4-carboxylic
Acid Methyl Amide (12b). The title compound was prepared by a
similar method as described for 12c: (ESI) m/z 375.1 (M þ 1); 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.18 (d, J = 5.4 Hz, 1 H),
7.62 (d, J = 8.0 Hz, 2 H), 7.53 (d, J = 8.5 Hz, 2 H), 7.38 (dd, J =
5.6, 1.5 Hz, 1 H), 7.31 (app t, 2 H), 7.26 (s, 1 H), 7.00 (t, 1 H),
3.66-3.83 (m, 4 H), 2.95-3.10 (m, 4 H).
1
title compound: MS (ESI) m/z 404.0 (M þ 1); H NMR (400
MHz, CDCl3) δ ppm 8.36 (d, J = 5.3 Hz, 1 H), 7.75-7.81 (m, 2
H), 7.56-7.60 (m, 1 H), 3.78 (dd, J = 6.3, 4.0 Hz, 4 H),
3.60-3.67 (m, 4 H), 1.51 (s, 9 H).
4-(20-Cyclohexylamino-4-nitro[2,40]bipyridinyl-6-yl)piperazine-
1-carboxylic Acid tert-Butyl Ester (15a). A mixture of 14a (2.5 g,
6.2 mmol) and cyclohexylamine (250 mL) was heated to 107 °C
for 62 h. The mixture was then cooled and concentrated. The
residue was purified via flash chromatography (SiO2, 30-40%
EtOAc/heptane gradient) to give the title compound: MS (ESI)
m/z 483.1 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 8.18-
8.21 (m, 1 H), 7.72 (d, J = 1.5 Hz, 1 H), 7.35 (d, J = 1.5 Hz, 1 H),
7.10 (dd, J = 5.3, 1.5 Hz, 1 H), 6.96-6.99 (m, 1 H), 4.57 (d, J =
8.3 Hz, 1 H), 3.65-3.78 (m, 5 H), 3.62 (dd, J = 6.3, 4.0 Hz, 4 H),
2.06-2.15 (m, 2 H), 1.74-1.85 (m, 2 H), 1.62-1.73 (m, 1 H),
1.51 (s, 9 H), 1.38-1.49 (m, 2 H), 1.19-1.37 (m, 3 H).
20-Cyclohexylamino-6-piperazin-1-yl[2,40]bipyridinyl-4-carbo-
nitrile (12i). 20-Cyclohexylamino-6-piperazin-1-yl[2,40]bipyridi-
nyl-4-carboxylic acid amide (1.00 g, 2.08 mmol) in CH2Cl2
(21 mL) and Et3N (1.45 mL, 10.4 mmol) was stirred at 0 °C
before adding trifluoroacetic acid anhyride (0.9 mL, 6.33 mmol).
The solution was then allowed to warm to room temperature.
After 2 h, the mixture was diluted with CH2Cl2 and extracted with
a saturated aqueous NaHCO3 solution. The separated organic
4-[20-(tert-Butoxycarbonylcyclohexylamino)-4-nitro[2,40]bipyri-
dinyl-6-yl]piperazine-1-carboxylic Acid tert-Butyl Ester (16a).
A mixture of 15a (1.2 g, 2.49 mmol), BOC anhydride (2.72 g,

5434 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
12.4 mmol), and DMAP (0.061 g, 0.498 mmol) in acetonitrile
(50 mL) and CH2Cl2 (5 mL) was heated to 85 °C for 4.5 h. The
mixture was then cooled and concentrated under reduced
pressure. The residue was taken up in CH2Cl2 and washed with
saturated NaHCO3 and brine, respectively, and then dried (Na2-
SO4), filtered, and concentrated. The residue was purified via
flash chromatography (SiO2, 0-25% EtOAc/hexanes gradient)
mixed with 2 N NH3 in MeOH and concentrated again and then
separated via semipreparative HPLC (10-55% CH3CN/H2O
gradient with 0.1% NH4OH in 17 min) to give the title
compound: MS (ESI) m/z 404.0 (M þ 1); 1H NMR (400
MHz, DMSO-d6) δ ppm 13.07 (br s, 1 H), 8.45 (br s, 1 H),
8.19 (br s, 1 H), 8.00 (d, J = 5.3 Hz, 1 H), 7.39-7.41 (m, 1 H),
7.17-7.18 (m, 1 H), 7.08 (dd, J = 5.4, 1.5 Hz, 1 H), 7.02-7.04
(m, 1 H), 6.40 (d, J = 7.7 Hz, 1 H), 3.69-3.79 (m, 1 H),
3.53-3.58 (m, 4 H), 2.80-2.85 (m, 4 H), 1.89-1.99 (m, 2 H),
1.68-1.77 (m, 2 H), 1.55-1.65 (m, 1 H), 1.26-1.39 (m, 2 H),
1.13-1.26 (m, 3 H).
1
to give the title compound: MS (ESI) m/z 583.2 (M þ 1); H
NMR (400 MHz, CDCl3) δ ppm 8.58-8.62 (m, 1 H), 7.72-7.79
(m, 3 H), 7.39 (d, J = 1.5 Hz, 1 H), 4.09-4.20 (m, 1 H), 3.77 (dd,
J = 6.3, 4.0 Hz, 4 H), 3.62 (dd, J = 6.3, 4.0 Hz, 4 H), 1.91-2.00
(m, 2 H), 1.73-1.83 (m, 2 H), 1.54-1.65 (m, 3 H), 1.48-1.54
(m, 9 H), 1.42-1.45 (m, 9 H), 1.25-1.42 (m, 2 H), 0.98-1.12
(m, 1 H).
4-[20-(tert-Butoxycarbonylcyclohexylamino)-4-hydroxy[2,40]bi-
pyridinyl-6-yl]piperazine-1-carboxylic Acid tert-Butyl Ester (17a).
A mixture of 16a (1.9 g, 3.26 mmol), KOH (1.8 g, 32.6 mmol), and
DMSO (65 mL) was stirred at room temperature for 1 h and then
diluted with CH2Cl2, washed with H2O (2ꢀ) and then brine, dried
(Na2SO4), filtered, and concentrated. The residue was purified via
flash chromatography (SiO2, 0-50% EtOAc/heptane gradient) to
give the title compound: MS (ESI) m/z 554.2 (M þ 1); 1H NMR
(400 MHz, CDCl3) δppm 8.49 (d, J = 5.3 Hz, 1 H), 7.65-7.70(m,
1 H), 7.61-7.65 (m, 1 H), 6.58-6.61 (m, 1 H), 6.01-6.05 (m, 1 H),
4.00-4.14 (m, 1 H), 3.49-3.59 (m, 8 H), 1.92-2.01 (m, 2 H),
1.70-1.80 (m, 2 H), 1.54-1.63 (m, 1 H), 1.50 (s, 9 H), 1.22-1.48
(m, 14 H), 0.91-1.09 (m, 1 H).
Cyclohexyl-[6-piperazin-1-yl-4-(2H-pyrazol-3-yl)[2,40]bipyridi-
nyl-20-yl]amine (19b). The title compound was prepared by a
similar method to 19a: MS (ESI) m/z 404.0 (M þ 1); 1H NMR
(400 MHz, DMSO-d6) δ ppm 13.09 (br s, 1 H), 8.01 (d, J = 5.3
Hz, 1 H), 7.84 (br s, 1 H), 7.58 (br s, 1 H), 7.16-7.21 (m, 2 H),
7.06 (dd, J = 5.4, 1.5 Hz, 1 H), 6.97 (d, J = 2.0 Hz, 1 H), 6.45 (d,
J = 7.6 Hz, 1 H), 3.69-3.82 (m, 1 H), 3.53-3.61 (m, 4 H), 2.82-
2.88 (m, 4 H), 1.90-1.99 (m, 2 H), 1.67-1.78 (m, 2 H), 1.54-
1.65 (m, 1 H), 1.26-1.41 (m, 2 H), 1.11-1.26 (m, 3 H).
4-(20-Chloro-4-nitro[2,40]bipyridinyl-6-yl)piperazine-1-carboxy-
lic Acid tert-Butyl Ester (14b). The title compound was prepared
by a similar method to 14a: MS (ESI) m/z 420.0, 422.0 (M þ 1);
1H NMR (400 MHz, CDCl3) δ ppm 8.52 (dd, J = 5.2, 0.6 Hz, 1
H), 7.95 (dd, J = 1.5, 0.6 Hz, 1 H), 7.81 (dd, J = 5.3, 1.5 Hz, 1
H), 7.77 (d, J = 1.5 Hz, 1 H), 7.42 (d, J = 1.5 Hz, 1 H), 3.75-
3.80 (m, 4 H), 3.61-3.66 (m, 4 H), 1.51 (s, 9 H).
tert-Butyl 4-[4-Bromo-20-(cyclohexylamino)-2,40-bipyridin-6-
yl]piperazine-1-carboxylate (18a). A mixture of 17a (1.2 g, 2.17
mmol) and POBr3 (3.7 g, 13 mmol) was placed in flask with HBr
receiver and heated to 130 °C for 1 h. The mixture was cooled to
0 °C, quenched with MeOH, and concentrated. The resulting
slurry was diluted with a saturated aqueous solution of NaH-
CO3, extracted with CH2Cl2 (ꢀ5). The combined organic layer
was concentrated. The residue was purified via semipreparative
HPLC (25-55% CH3CN/H2O gradient with 0.1% NH4OH in
17 min) to give (4-bromo-6-piperazin-1-yl[2,40]bipyridinyl-20-
yl)cyclohexylamine: MS (ESI) m/z 451.9, 417.9 (M þ 1); 1H
NMR (400 MHz, DMSO-d6) δ ppm 9.06 (br s, 2 H), 8.01 (d, J =
5.6 Hz, 1 H), 7.46 (s, 1 H), 7.27 (s, 1 H), 7.21 (br s, 1 H), 7.09 (br s,
1 H), 3.85-3.90 (m, 4 H), 3.70-3.81 (m, 1 H), 3.18-3.25 (m, 4
H), 1.89-1.98 (m, 2 H), 1.69-1.78 (m, 2 H), 1.57-1.66 (m, 1 H),
1.28-1.42 (m, 2 H), 1.13-1.28 (m,3 H).
4-(20-Isopropylamino-4-nitro[2,40]bipyridinyl-6-yl)piperazine-
1-carboxylic Acid tert-Butyl Ester (15b). After a solution of 14b
(0.8 g, 1.91 mmol) in dioxane (75 mL) was sparged with argon,
isopropylamine (3.25 mL, 38.14 mmol) was added followed by
Pd(t-Bu3P)2 and cesium carbonate (1.87 g, 5.73 mmol). The
vessel was sealed and heated at 110 °C for 5 h. The mixture was
then allowed to cool and then filtered and concentrated. The
residue was purified via flash chromatography (SiO2, 25-55%
EtOAc/hexanes gradient) to give the title compound: MS (ESI)
m/z 443.1 (M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 8.19 (dd,
J = 5.4, 0.6 Hz, 1 H), 7.71 (d, J = 1.6 Hz, 1 H), 7.33 (d, J = 1.5
Hz, 1 H), 7.10 (dd, J = 5.3, 1.5 Hz, 1 H), 6.93-6.98 (m, 1 H),
3.96-4.11 (m, 1 H), 3.95-4.10 (m, 1 H), 3.70-3.78 (m, 4 H),
3.61 (dd, J = 6.4, 4.0 Hz, 4 H), 1.50 (s, 9 H), 1.29 (d, J = 6.3 Hz,
6 H).
tert-Butyl 4-{20-[(tert-Butoxycarbonyl)(isopropyl)amino]-4-ni-
tro-2,40-bipyridin-6-yl}piperazine-1-carboxylate (16b). The title
compound was prepared by a similar method to compound 16a:
A mixture of (4-bromo-6-piperazin-1-yl[2,40]bipyridinyl-20-yl)-
cyclohexylamine (crude, 0.362 mmol), BOC anhydride (0.4 g, 1.81
mmol), and triethylamine (0.252 mL, 1.81 mmol) in CH2Cl2
(25 mL) was stirred at room temperature. After 0.5 h, the mixture
was diluted with CH2Cl2, washed with a saturated aqueous
solution of NaHCO3 (ꢀ2) and then brine, dried (Na2SO4),
filtered, and concentrated. The residue was purified via flash
chromatography (SiO2, 15-25% EtOAc/heptane gradient) to
1
MS (ESI) m/z 543.3 (M þ 1); H NMR (400 MHz, CDCl3) δ
ppm 8.58 (dd, J = 5.2, 0.6 Hz, 1 H), 7.76-7.80 (m, 2 H), 7.73
(dd, J = 5.2, 1.6 Hz, 1 H), 7.38 (d, J = 1.5 Hz, 1 H), 4.53-4.65
(m, 1 H), 3.74-3.79 (m, 4 H), 3.62 (dd, J = 6.3, 4.0 Hz, 4 H), 1.51
(s, 9 H), 1.46 (s, 9 H), 1.32 (d, J = 6.8 Hz, 6 H).
tert-Butyl 4-{20-[(tert-Butoxycarbonyl)(isopropyl)amino]-4-
hydroxy-2,40-bipyridin-6-yl}piperazine-1-carboxylate (17b). The
title compound was prepared by a similar method to 17a: MS
(ESI) m/z 514.3 (M þ 1).
1
give the title compound: H NMR (400 MHz, CDCl3) δ ppm
8.13-8.16(m,1H), 7.22(d, J= 1.3 Hz, 1 H), 7.02 (dd, J=5.4, 1.5
Hz, 1 H), 6.92-6.95 (m, 1 H), 6.81 (d, J = 1.3 Hz, 1 H), 4.49-4.56
(m, 1 H), 3.60-3.73 (m, 5 H), 3.55-3.60 (m, 4 H), 2.04-2.13 (m,
2 H), 1.71-1.83 (m, 2 H), 1.62-1.71 (m, 1 H), 1.50 (s, 9 H),
1.38-1.47 (m, 2 H), 1.17-1.35 (m, 3 H).
tert-Butyl 4-(20-[(tert-Butoxycarbonyl)(isopropyl)amino]-4-{[(tri-
fluoromethyl)sulfonyl]oxy}-2,40-bipyridin-6-yl)piperazine-1-carbo-
xylate (18b). To a solution of 17b (0.379 g, 0.739 mmol) and
triethylamine (0.7 mL, 3.70 mmol) in CH2Cl2 (12 mL), 2-(N,N-
bis(trifluoromethylsulfonyl)amino)pyridine (0.265 g, 0.739
mmol) was added portionwise at 0 °C. The mixture was allowed
to warm to room temperature and stirred for 5 h and concen-
trated. The residue was diluted with CH2Cl2, washed with
saturated NaHCO3 (2ꢀ) and brine, and then dried (Na2SO4),
filtered, and concentrated. The title compound was obtained
and was used without further purification: MS (ESI) m/z 646.2
(M þ 1); 1H NMR (400 MHz, CDCl3) δ ppm 8.55 (dd, J = 5.2,
0.5 Hz, 1 H), 7.71 (d, J = 0.9 Hz, 1 H), 7.65 (dd, J = 5.2, 1.6 Hz,
1 H), 7.00 (d, J = 1.6 Hz, 1 H), 6.50 (d, J = 1.8 Hz, 1 H), 4.53-
4.65 (m, 1 H), 3.66-3.71 (m, 4 H), 3.57-3.63 (m, 4 H), 1.50 (s, 9
H), 1.45 (s, 9 H), 1.31 (d, J = 6.8 Hz, 6 H).
Cyclohexyl-[6-piperazin-1-yl-4-(1H-pyrazol-4-yl)[2,40]bipyridi-
nyl-20-yl]amine (19a). A mixture of 18a (0.22 g, 0.426 mmol), 1H-
pyrazole-4-boronic acid (0.29 g, 2.4 mmol), Pd(dppf)Cl2
3
CH2Cl2 (0.007 g, 0.085 mmol), 2 M Na2CO3 (2.4 mL), and
DME (5 mL) was sparged with argon for 10 min. The vessel was
sealed and treated with microwave at 130 °C for 20 min. The
mixture was diluted with CH2Cl2, washed with saturated NaH-
CO3, brine, dried (Na2SO4), filtered, and concentrated. The
residue was purified via flash chromatography (SiO2, 70-100%
EtOAc/heptane gradient) to give an intermediate of BOC-
protected title compound [MS (ESI) m/z 504.0 (M þ 1)]. The
intermediate was then treated with 50% TFA in CH2Cl2 at room
temperatue for 1 h and concentrated. The resulting residue was

Article
Isopropyl-[6-piperazin-1-yl-4-(1H-pyrazol-4-yl)[2,40]bipyridi-
nyl-20-yl]amine (19c). The title compound was prepared from
18b by a similar method to 19a: MS (ESI) m/z 364.2 (M þ 1); 1H
NMR (400 MHz, DMSO-d6) δ ppm 13.11 (br s, 1 H), 8.41 (br s, 2
H), 8.02 (d, J = 5.3 Hz, 1 H), 7.41 (s, 1 H), 7.16 (s, 1 H), 7.10 (dd,
J = 5.3, 1.3 Hz, 1 H), 7.04 (s, 1 H), 6.37 (d, J = 7.6 Hz, 1 H),
3.98-4.13 (m, 1 H), 3.52-3.59 (m, 4 H), 2.80-2.87 (m, 4 H),
1.16 (d, J = 6.6 Hz, 6 H).
[6-Piperazin-1-yl-4-(1H-pyrazol-4-yl)[2,40]bipyridinyl-20-yl]-
(tetrahydropyran-4-yl)amine (19c). The title compound was pre-
pared by a similar method to 19c: MS (ESI) m/z 406.2 (M þ 1); 1H
NMR (400 MHz, DMSO-d6) δ ppm 13.17 (br s, 1 H), 8.99 (br s, 2
H), 8.49 (br s, 1 H), 8.21 (br s, 1 H), 8.03 (d, J = 5.6 Hz, 1 H), 7.55
(s, 1 H), 7.25 (br s, 1 H), 7.17-7.23 (m, 2 H), 3.94-4.06 (m, 1 H),
3.85-3.93 (m, 6 H), 3.39-3.47 (m, 2 H), 3.21-3.26 (m, 4 H), 1.91
(d, J = 15.3 Hz, 2 H), 1.39-1.53 (m, 2 H).
Cyclohexyl-(4-trimethylstannanylpyridin-2-yl)amine (21). 2-
Fluoro-4-iodopyridine (4.0 g, 17.9 mmol) and cyclohexylamine
(5.1 mL, 44.8 mmol) were sealed in a pressure vessel and heated
to 120 °C for 3 h. After cooling, the mixture was concentrated
under reduced pressure. The residue was purified by flash
chromatography (from 10% to 20% to 30% EtOAc/hexanes)
to yield 5.1 g of 2-cyclohexylamino-4-iodopyridine.
To a reaction vessel containing the 2-cyclohexylamino-4-iodo-
pyridine prepared above (4.9 g, 16.2 mmol) dissolved in toluene
(175 mL) was added Me3SnSnMe3 (7.93 g, 24.2 mmol). The
solution was degassed with N2 for 10 min, Pd(Ph3P)4 (1.87 g, 1.6
mmol) was added, and the mixture was heated to 100 °C on. Upon
cooling, the mixture was filtered over Celite, concentrated under
reduced pressure, and partitioned between EtOAc and a saturated
aqueous solution of KF. The separated organic phase was washed
with a saturated aqueous solution of NaCl, dried (Na2SO4), and
concentrated in vacuo. The residue was purified by flash chroma-
tography (from 10% to 25% to 30% EtOAc/hexanes) to afford
the title compound (3.8 g, 69%) as a white solid: 1H NMR (400
MHz, CDCl3) δ ppm 0.29 (s, 7.54 H), 0.29 (d, J = 55.7 Hz, 0.77
H), 0.29 (d, J = 53.3 Hz, 0.69 H), 1.14-1.31 (m, 3 H), 1.35-1.50
(m, 2 H), 1.60-1.66 (m, 1 H), 1.71-1.80 (m, 2 H), 1.99-2.09 (m, 2
H), 3.55-3.68 (m, 1 H), 4.25-4.34 (d, J = 7.7 Hz, 1 H), 6.45 (s,
0.84 H), 6.45 (d, J = 49.6 Hz, 0.16 Hz), 6.61 (d, J = 4.8 Hz, 0.84
H), 6.61 (dd, J = 39.8, 4.8 Hz, 0.16 H), 7.96-8.03 (m, 1 H).
6-Chloro-20-cyclohexylamino[2,40]bipyridinyl-4-carboxylicAcid
Methyl Ester (23). The title compound was prepared from 2,6-
dichloroisonicotinic acid methyl ester and cyclohexyl-(4-tri-
methylstannanylpyridin-2-yl)amine by analogy to the Stille
coupling method outlined above: 1H NMR (400 MHz, CDCl3)
δ ppm 1.17-1.32 (m, 2 H), 1.38-1.52 (m, 3 H), 1.60-1.70 (m, 1
H), 1.71-1.82 (m, 2 H), 2.02-2.11 (m, 2 H), 3.69-3.80 (m, 1 H),
4.00 (s, 3 H), 4.58-4.68 (m, 1 H), 7.03 (s, 1 H), 7.05-7.10 (m, 1
H), 7.87 (d, J = 1.1 Hz, 1 H), 8.16-8.21 (m, 2 H).
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5435
(ESI) m/z 395.2 (M þ 1); 1H NMR (400 MHz, MeOD) δ ppm
1.20 (d, J = 6.32 Hz, 3 H), 1.24-1.33 (m, 3 H), 1.39-1.48 (m, 2
H), 1.65-1.72 (m, 1H), 1.80 (d, J = 13.9 Hz, 2 H), 2.04 (d, J =
9.9 Hz, 2 H), 2.58-2.66 (m, 1 H), 2.85-2.98 (m, 3 H), 3.07-3.15
(m, 1 H), 3.62-3.72 (m, 1 H), 4.39 (d, J = 12.9 Hz, 2 H), 7.12
(dd, J = 5.7, 1.4 Hz, 1 H), 7.22 (d, J = 6.8 Hz, 2 H), 7.54 (s, 1 H),
7.97 (d, J = 5.6 Hz, 1 H).
20-Cyclohexylamino-6-((S)-3-methylpiperazin-1-yl)[2,40]bipyri-
dinyl-4-carboxylic Acid Amide (24d). The title compound was
prepared by a method similar to that described for 24h: MS
(ESI) m/z 395.2 (M þ 1); 1H NMR (400 MHz, MeOD) δ ppm
1.19 (d, J = 6.3 Hz, 3 H), 1.23-1.33 (m, 3 H), 1.39-1.52 (m, 2
H), 1.68 (d, J = 11.9 Hz, 1 H), 1.80 (d, J = 13.1 Hz, 2 H), 2.04 (d,
J = 12.4 Hz, 2 H), 2.57-2.65 (m, 1 H), 2.84-3.00 (m, 3 H), 3.10
(d, J = 10.9 Hz, 1 H), 3.63-3.71 (m, 1 H), 4.38 (d, J = 10.9 Hz, 2
H), 7.12 (dd, J = 5.7, 1.6 Hz, 1 H), 7.22 (d, J = 3.8 Hz, 2 H), 7.53
(s, 1 H), 7.97 (d, J = 5.6 Hz, 1 H).
20-Cyclohexylamino-6-morpholin-4-yl[2,40]bipyridinyl-4-carbo-
xylic Acid Amide (24i). The title compound was prepared by a
method similar to that described for 24h: MS (ESI) m/z 382.2 (M
þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm 1.12-1.26 (m, 3
H), 1.27-1.40 (m, 2 H), 1.54-1.66 (m, 1 H), 1.68-1.78 (m, 2 H),
1.87-1.98 (m, 2 H), 3.56-3.63 (m, 4 H), 3.71-3.78 (m, 5 H),
6.47 (d, J = 7.8 Hz, 1 H), 7.04 (dd, J = 5.3, 1.5 Hz, 1 H), 7.16 (s,
1 H), 7.24 (s, 1 H), 7.58 (s, 1 H), 7.63 (s, 1 H), 8.02 (d, J = 5.3 Hz,
1 H), 8.18 (s, 1 H).
20-Cyclohexylamino-6-((R)-pyrrolidin-3-ylamino)[2,40]bipyri-
dinyl-4-carboxylic Acid Amide (24a). The title compound was
prepared by a method similar to that described for 24h: MS
(ESI) m/z 381.2 (M þ 1); 1H NMR (400 MHz, MeOD) δ ppm
1.27 (q, J = 12.9 Hz, 3 H), 1.39-1.52 (m, 2 H), 1.69 (d, J = 14.2
Hz, 1 H), 1.80 (d, J = 13.4 Hz, 2 H), 1.85-1.94 (m, 1 H), 2.05 (d,
J = 12.4 Hz, 2 H), 2.29 (dd, J = 12.5, 7.7 Hz, 1 H), 2.96-3.02
(m, 1 H), 3.04-3.12 (m, 1 H), 3.16-3.25 (m, 1 H), 3.36 (dd, J =
11.8, 6.19 Hz, 1 H), 3.63-3.71 (m, 1 H), 4.48-4.57 (m, 1 H), 6.93
(d, J = 1.0 Hz, 1 H), 7.13 (dd, J = 5.6, 1.5 Hz, 1 H), 7.18 (s, 1 H),
7.43 (d, J = 1.3 Hz, 1 H), 7.96 (d, J = 5.6 Hz, 1 H).
20-Cyclohexylamino-6-((S)-pyrrolidin-3-ylamino)[2,40]bipyri-
dinyl-4-carboxylic Acid Amide (24b). The title compound was
prepared by a method similar to that described for 24h: MS
(ESI) m/z 381.2 (M þ 1); 1H NMR (400 MHz, MeOD) δ ppm
1.21-1.33 (m, 3 H), 1.46 (d, J = 12.4 Hz, 2 H), 1.68 (d, J = 10.4
Hz, 1 H), 1.75-1.84 (m, 2 H), 1.84-1.93 (m, 1 H), 2.05 (d, J =
11.6 Hz, 2 H), 2.23-2.34 (m, 1 H), 2.97 (dd, J = 12.0, 4.4 Hz, 1
H), 3.02-3.11 (m, 1 H), 3.14-3.23 (m, 1 H), 3.36-3.39 (m, 1 H),
3.63-3.71 (m, 1 H), 4.48-4.57 (m, 1 H), 6.93 (d, J = 1.3 Hz, 1
H), 7.13 (dd, J = 5.6, 1.5 Hz, 1 H), 7.18 (d, J = 2.3 Hz, 1 H), 7.43
(d, J = 1.0 Hz, 1 H), 7.96 (dd, J = 5.7, 0.6 Hz, 1 H).
20-Cyclohexylamino-6-(4-methylpiperazin-1-yl)[2,40]bipyridi-
nyl-4-carboxylic Acid Amide (24f). The title compound was
prepared by a method similar to that described for 24h: MS
(ESI) m/z 493.1 (M þ 1); 1H NMR (400 MHz, DMSO-d6) δ ppm
8.17 (br s, 1 H), 8.01 (d, J = 5.3 Hz, 1 H), 7.60 (br s, 1 H), 7.53 (s,
1 H), 7.23 (s, 1 H), 7.15 (s, 1 H), 7.02 (dd, J = 5.4, 1.4 Hz, 1 H),
3.69-3.80 (m, 1 H), 3.60-3.66 (m, 4 H), 2.41-2.47 (m, 4 H),
2.24 (s, 3 H), 1.89-1.97 (m, 2 H), 1.67-1.78 (m, 2 H), 1.54-1.65
(m, 1 H), 1.25-1.40 (m, 2 H), 1.11-1.26 (m, 3 H).
20-Cyclohexylamino-6-(3,3-dimethylpiperazin-1-yl)[2,40]bipyri-
dinyl-4-carboxylic Acid Amide (24h). Compound 23 was con-
verted to 6-(3,3-dimethylpiperazin-1-yl)-20-cyclohexylamino-
[2,40]bipyridinyl-4-carboxylic acid methyl ester by a method
similar to that described above for 10. The resulting product
6-(3,3-dimethylpiperazin-1-yl)-20-cyclohexylamino[2,40]bipyri-
dinyl-4-carboxylic acid methyl ester (61 mg, 0.14 mmol) and a
solution of 7 M NH3 in MeOH (10 mL) were placed in a pressure
vessel and heated to 90 °C on. The mixture was cooled and
concentrated under reduced pressure. The residue was purified
by HPLC to give the title compound: MS (ESI) m/z 409.2 (M þ
Aminomethyl-200-cyclohexylamino-3,4,5,6-tetrahydro-2H-[1,20;
60,400]terpyridine-40-carboxylic Acid Amide (24g). The title com-
pound was prepared by a method similar to that described for
24h. The BOC protecting group was removed by treatment of
the intermediate with TFA/DCM at room temperature: MS
(ESI) m/z 409.2 (M þ 1); 1H NMR (400 MHz, MeOD) δ ppm
7.94-7.98 (m, 1 H), 7.47-7.50 (m, 1 H), 7.18-7.25 (m, 2 H),
7.13 (dd, J = 5.7, 1.5 Hz, 1 H), 4.54-4.65 (m, 2 H), 3.62-3.74
(m, 1 H), 2.89-3.02 (m, 2 H), 2.64 (d, J = 6.8 Hz, 2 H),
1.99-2.10 (m, 2 H), 1.84-1.92 (m, 2 H), 1.63-1.84 (m, 4 H),
1.38-1.54 (m, 2 H), 1.21-1.35 (m, 5 H).
1
1); H NMR (400 MHz, MeOD) δ ppm 1.23 -1.24 (s, 6 H),
1.25-1.32 (m, 2H), 1.37-1.53 (m, 2 H), 1.68 (d, J = 13.9 Hz,
1 H), 1.80 (d, J = 13.9 Hz, 2 H), 2.05 (d, J = 10.9 Hz, 2 H),
2.96-3.03 (m, 2 H), 3.52 (s, 2 H), 3.64-3.72 (m, 3 H), 7.12 (dd,
J = 5.7, 1.6 Hz, 1 H), 7.20 (d, J = 1.8 Hz, 2 H), 7.50 (s, 1 H), 7.97
(d, J = 5.6 Hz, 1 H).
20-Cyclohexylamino-6-((R)-3-methylpiperazin-1-yl)[2,40]bipyri-
dinyl-4-carboxylic Acid Amide (24c). The title compound was
prepared by a method similar to that described for 24h: MS
3-Amino-200-cyclohexylamino-3,4,5,6-tetrahydro-2H-[1,20;60,400]-
terpyridine-40-carboxylic Acid Amide (24e). The title compound

5436 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Meredith et al.
was prepared by a method similar to that described for 24h.
BOC group removed by treatment of intermediate with TFA/
DCM at room temperature: MS (ESI) m/z 395.1 (M þ 1); H
concentrated under reduced pressure and used immediately for
the next step: MS(ESI) m/z 313.99; 1H NMR (400 MHz,CDCl3) δ
ppm 7.72 (s, 1 H), 4.99 (s, 2 H), 4.48 (q, J = 7.16 Hz, 2 H), 1.46 (t,
J = 7.07 Hz, 3 H).
1
NMR (400 MHz, CDCl3) δ ppm 8.15 (d, J = 5.3 Hz, 1 H),
7.20-7.25 (m, 1 H), 7.05-7.12 (m, 2 H), 7.02 (br s, 1 H), 6.18 (br
s, 1 H), 5.68 (br s, 1 H), 4.56 (d, J = 7.8 Hz, 1 H), 4.31-4.38 (m, 1
H), 4.15-4.25 (m, 1 H), 3.61-3.77 (m, 1 H), 3.06-3.16 (m, 1 H),
2.82-2.99 (m, 2 H), 1.99-2.15 (m, 3 H), 1.73-1.91 (m, 3 H),
1.60-1.72 (m, 2 H), 1.32-1.48 (m, 3 H), 1.18-1.32 (m, 3 H).
4-(6-Chloro-4-difluoromethylpyridin-2-yl)piperazine-1-carbo-
xylic Acid tert-Butyl Ester (26b). A solution of 2,6-dichloro-4-
difluoromethylpyridine (0.1 g, 0.5 mmol), piperazine-1-car-
boxylic acid tert-butyl ester (0.94 g, 0.5 mmol), and Et3N (0.28
mL, 2.0 mmol) in dioxane (3 mL) was heated in a sealed tube at
90 °C for 48 h. After cooling, the mixture was concentrated in
vacuo. The residue was purified by flash chromatography
(10-30% EtOAc/heptanes) to give the title compound as a
A mixture of 3-bromomethyl-2,6-dichloroisonicotinic acid
ethyl ester (13.4 g, 42.7 mmol), tetrahydrofuran (100 mL), and
ammonium hydroxide (300 mL of 28-30% ammonia) was
stirred at room temperature for 18 h. The solvents were then
removed by rotary evaporation. The nearly dry solid was treated
with a small amount of water. The salmon-colored solid was
isolated by filtration, washed with small amounts of water and
then diethyl ether, and dried under vacuum. Filtration of the
cooled filtrate yields additional product (7.47 g, 36.8 mmol,
86%): MS(ESI) m/z 203.2 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.29 (br s, 1 H), 7.83 (s, 1 H), 4.45 (s, 2 H).
4,6-Dichloro-2,3-dihydropyrrolo[3,4-c]pyridin-1-one (5.63 g,
27.7 mmol), tert-butyl piperazine-1-carboxylate (7.75 g, 41.6
mmol), triethylamine (14.0 g, 139 mmol), and dioxane (50 mL)
were stirred at 120 °C in a 350 mL sealed tube for 16 h. To the
cooled down reaction mixture were then added more tert-
butylpiperazine 1-carboxylate (5.2 g, 27.7 mmol) and triethyla-
mine (2.83 g, 28.0 mmol). The vessel was sealed again and stirred
at 120 °C for 24 h. The reaction mixture was then cooled to
ambient temperature, and 28 was isolated by filtration (6.18 g,
17.5 mmol, 63%): MS(ESI) m/z 353.15 (M þ 1); 1H NMR (400
MHz, DMSO-d6) δ ppm 9.04 (s, 1 H), 6.89 (s, 1 H), 4.57 (s, 2 H),
3.61-3.54 (m, 4 H), 3.47-3.41 (m, 4 H), 1.42 (s, 9 H).
1
clear oil: H NMR (400 MHz, CDCl3) δ ppm 1.48 (s, 9 H),
3.49-3.63 (m, 8 H), 6.47 (t, J = 55.7 Hz, 1 H), 6.56 (s, 1 H), 6.72
(s, 1 H).
Cyclohexyl-(4-difluoromethyl-6-piperazin-1-yl[2,40]bipyridi-
nyl-20-yl)amine (24k). A solution of 4-(6-chloro-4-difluoro-
methylpyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester
(0.06 g, 0.17 mmol) and cyclohexyl-(4-trimethylstannanylpyr-
idin-2-yl)amine (0.065 g, 0.19 mmol) in dioxane (4 mL) was
degassed with N2. CsF (0.059 g, 0.39 mmol) and Pd(t-Bu3P)2
were added. The mixture was heated under N2 to 100 °C for 5 h.
The mixture was cooled to room temperature, filtered through
Celite, rinsed with fresh dioxane, and concentrated. The residue
was purified by flash chromatography (10-50% EtOAc/
heptanes) to give 6-(4-tert-butoxycarbonylpiperazin-1-yl)-20-
cyclohexylamino[2,40]bipyridinyl-4-difluoromethane as a white
foam: 1H NMR (400 MHz, CDCl3) δ ppm 1.18-1.32 (m, 2 H),
1.36-1.53 (m, 11 H), 1.59-1.71 (m, 2 H), 1.73-1.83 (m, 2 H),
2.05-2.14 (m, 2 H), 3.53-3.73 (m, 9 H), 4.50-4.58 (m, 1 H),
6.59 (t, J = 56.0 Hz, 1 H), 6.71-6.77 (m, 1 H), 6.98 (s, 1 H), 7.07
(dd, J = 5.3, 1.3 Hz, 1 H), 7.15 (s, 1 H), 8.14 (d, J = 5.3 Hz, 1 H).
TFA was added dropwise to a solution of 6-(4-tert-butox-
ycarbonylpiperazin-1-yl)-20-cyclohexylamino[2,40]bipyridinyl-
4-difluoromethane (110 mg, 0.23 mmol) in CH2Cl2 (5 mL) until
TLC showed complete consumption of the starting material. A 3
N solution of NH3 in MeOH was added, and the mixture was
concentrated under reduced pressure. The residue was purified
by reverse phase HPLC to yield the title compound as a white
solid: MS (ESI) m/z 388.2 (M þ 1); 1H NMR (400 MHz, MeOD)
δ ppm 1.19-1.33 (m, 3 H), 1.37-1.53 (m, 2 H), 1.68 (m, 1 H),
1.75-1.85 (m, 2 H), 1.98-2.08 (m, 2 H), 2.92-3.00 (m, 4 H),
3.62-3.73 (m, 5 H), 6.74 (t, J = 55.8 Hz, 1 H), 6.93 (s, 1 H), 7.09
(dd, J = 5.6, 1.5 Hz, 1 H), 7.20 (s, 1 H), 7.26 (s, 1 H), 7.96 (dd,
J = 5.6, 0.76 Hz, 1 H).
Intermediate 29 was obtained following concentration of the
above filtrate. The solid was treated with methanol and filtered to
1
give 29 as light-yellow solid: MS(ESI) m/z 353.30 (M þ 1). H
NMR (400 MHz, DMSO-d6) δppm 8.93 (s, 1 H), 7.01 (s, 1 H), 4.28
(s, 2 H), 3.58-3.53 (m, 4 H), 3.45-3.40 (m, 4 H), 1.42 (s, 9 H).
6-(2-Cyclohexylaminopyridin-4-yl)-4-piperazin-1-yl-2,3-dihy-
dropyrrolo[3,4-c]pyridin-1-one (30). To an argon-degassed mix-
ture of 4-(6-chloro-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-
yl)piperazine-1-carboxylic acid tert-butyl ester (0.997 g, 2.83 mmol),
cyclohexyl-(4-trimethylstannanylpyridin-2-yl)amine (1.15 g, 3.39
mmol), and cesium fluoride (0.988 g, 6.50 mmol) in dioxane
(100 mL) was added bis(tri-tert-butylphosphine)palladium(0)
(0.116 g, 0.226 mmol). The reaction mixture was stirred at
100 °C for 18 h. The room temperature reaction mixture was
then filtered through Celite and concentrated down to dryness.
Treatment of the brown residue with diethyl ether (50 mL) yields
an off-white solid which was isolated by filtration (1.37 g, 2.78
mmol, 98%). The yield was slightly inflated because the product
was less than 95% pure: MS(ESI) m/z 493.29 (M þ 1); 1H NMR
(400 MHz, DMSO-d6) δ ppm 8.97 (s, 1 H), 8.02 (d, J = 5.56 Hz,
1 H), 7.44 (s, 1 H), 7.20 (s, 1 H), 7.08 (d, J = 7.07 Hz, 1 H), 6.42
(d, J = 7.83 Hz, 1 H), 4.61 (s, 2 H), 3.85-3.70 (m, 1 H),
3.70-3.58 (m, 4 H), 3.57-3.43 (m, 4 H), 2.02-1.87 (m, 2 H),
1.81-1.66 (m, 2 H), 1.65-1.55 (m, 1 H), 1.44 (s, 9 H), 1.40-1.27
(m, 2 H), 1.27-1.13 (m, 3 H).
Cyclohexyl-(6-piperazin-1-yl-4-trifluoromethyl[2,40]bipyridi-
nyl-20-yl)amine (24j). The title compound was prepared by a
method similar to that described for 24k: MS (ESI) m/z 406.2 (M
þ 1); 1H NMR (400 MHz, MeOD) δ ppm 1.19-1.33 (m, 3 H),
1.38-1.51 (m, 2 H), 1.63-1.72 (m, 1 H), 1.74-1.84 (d, 2 H),
1.99-2.08 (m, 2 H), 2.94-2.99 (m, 4 H), 3.68-3.73 (m, 5 H),
7.02 (s, 1 H), 7.09 (dd, J = 5.6, 1.5 Hz, 1 H), 7.18-7.20 (m, 1 H),
7.31 (s, 1 H), 7.98 (dd, J = 5.6, 0.8 Hz, 1 H).
4-(6-Chloro-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yl)-
piperazine-1-carboxylic Acid tert-Butyl Ester (28) and 4-(4-Chlo-
ro-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl)piperazine-
1-carboxylic Acid tert-Butyl Ester (29). To a mixture of 2,6-
dichloro-3-methylisonicotinic acid ethyl ester (10.0 g, 42.7 mmol)
and acetic acid (2.69 g, 44.9 mmol) in carbon tetrachloride
(147 mL) were added N-bromosuccinimide (8.36 g, 47.0 mmol)
and then benzoyl peroxide (1.03 g, 4.27 mmol). The mixture
was stirred in an oil bath at 60 °C under a heat lamp for 5 h.
The mixture was then cooled to room temperature. About half of
the solvent was removed by rotary evaporation. The white
succinimide solid was removed by filtration. The filtrate was
To a suspension of 4-[6-(2-cyclohexylaminopyridin-4-yl)-
1-oxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yl]piperazine-1-
carboxylic acid tert-butyl ester (1.37 g, 2.78 mmol) in dichlor-
omethane (20 mL) was added trifluoroacetic acid (5 mL, 7.4 g,
65 mmol). The solution was stirred at ambient temperature for
2 h. The solvents were removed by rotary evaporation. The
crude residue was treated with triethylamine (5 mL) and di-
chloromethane (200 mL) and then washed with water (40 mL).
After separation of the layers, the organic layer was dried over
sodium sulfate, filtered, and concentrated down to dryness by
rotary evaporation. The tan solid was then treated with hot
2-propanol (50 mL). After cooling of the suspension to room
temperature, a light-yellow solid was isolated by filtration and
dried under reduced pressure(0.616 g, 1.57 mmol, 56%). MS(ESI)
m/z 393.24 (M þ 1). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.98
(s, 1 H), 8.02 (d, J = 5.30 Hz, 1 H), 7.45 (s, 1 H), 7.17 (s, 1 H), 7.09
(d, J = 5.56 Hz, 1 H), 6.42 (d, J = 8.08 Hz, 1 H), 4.60 (s, 2 H),
3.88-3.59 (m, 5 H), 3.15-2.94 (m, 4 H), 2.03-1.86 (m, 2 H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5437
1.80-1.67 (m, 2 H), 1.65-1.54 (m, 1 H), 1.42-1.26 (m, 2 H),
1.26-1.13 (m, 3 H).
Ohyama, Y.; Kurabayashi, M. Mechanisms that underlie diastolic heart
failure and diastolic dysfunction. J Card. Failure 2008, 14 (Suppl. 1),
S142.(c) For WHO mortality data by country or region, see http://www.
who.int/whosis/whostat2006_mortality.pdf.
4-(2-Cyclohexylaminopyridin-4-yl)-6-piperazin-1-yl-2,3-dihy-
dropyrrolo[3,4-c]pyridin-1-one (31). To a nitrogen-degassed
mixture of 4-(4-chloro-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyri-
din-6-yl)piperazine-1-carboxylic acid tert-butyl ester (0.103 g,
0.2919 mmol) and cyclohexyl-(4-trimethylstannanylpyridin-2-yl)-
amine (0.114 g, 0.336 mmol) in toluene (10 mL) was added trans-
dichlorobis(triphenylphosphine)palladium(II) (0.021 g, 0.029
mmol). The reaction mixture was stirred under nitrogen at
110 °C for 16 h. The mixture was cooled to room temperature
and the solvent was removed by rotary evaporation. The crude
was purified through two successive silica gel columns (first
solvent system, ethyl acetate and then 95:5 ethyl acetate/
methanol; second solvent system, 98:2:0.5 and then 96:4:0.9
dichloromethane/methanol/ammonium hydroxide). A third
column was used for contaminated fractions. A yellow solid
was obtained as a result (0.043 g, 0.087 mmol, 30%): MS(ESI)
(5) (a) Fielitz, J.; Kim, M.-S.; Shelton, J. M.; Qi, X.; Hill, J. A.;
Richardson, J. A.; Bassel-Duby, R.; Olson, E. N. Requirement of
protein kinase D1 for pathological cardiac remodeling. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 3059–3063. (b) Ha, C. H.; Wang, W.;
Jhun, B. S.; Wong, C.; Hausser, A.; Pfizenmaier, K.; McKinsey, T. A.;
Olson, E. N.; Jin, Z.-G. Protein kinase D-dependent phosphorylation
and nuclear export of histone deacetylase 5 mediates vascular endo-
thelial growth factor-induced gene expression and angiogenesis.
J. Biol. Chem. 2008, 283, 14590–14599. (c) Backs, J.; Backs, T.;
Bezprozvannaya, S.; McKinsey, T. A.; Olson, E. N. Histone deacetylase
5 acquires calcium/calmodulin-dependent kinase II responsiveness by
oligomerization with histone deacetylase 4. Mol. Cell. Biol. 2008, 28,
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kinetic mechanism. Arch. Biochem. Biophys. 2006, 450, 141–148. (e)
Vega, R. B.; Harrison, B. C.; Meadows, E.; Roberts, C. R.; Papst, P. J.;
Olson, E. N.; McKinsey, T. A. Protein kinases C and D mediate agonist-
dependent cardiac hypertrophy through nuclear export of histone
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1
m/z 493.33 (M þ 1); H NMR (400 MHz, DMSO-d6) δ ppm
8.94 (s, 1 H), 8.04 (d, J = 5.43 Hz, 1 H), 7.08 (s, 1 H), 6.98 (s, 1 H),
6.95 (d, J = 5.43 Hz, 1 H), 6.50 (d, J = 7.58 Hz, 1 H), 4.59 (s, 2 H),
3.77-3.69 (m, 1 H), 3.67-3.59 (m, 4 H), 3.50-3.44 (m, 4 H), 1.95
(d, J = 10.11 Hz, 2 H), 1.77-1.69 (m, 2 H), 1.64-1.56 (m, 1 H),
1.43 (s, 9 H), 1.39-1.31 (m, 2 H), 1.25-1.17 (m, 3 H).
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Qi, X.; Richardson, J. A.; Hill, J. A.; Bassel-Duby, R.; Olson, E. N. The
MEF2D transcription factor mediates stress-dependent cardiac remo-
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A solution of 4-[4-(2-cyclohexylaminopyridin-4-yl)-1-oxo-
2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-yl]piperazine-1-carboxy-
lic acid tert-butyl ester (0.042 g, 0.085 mmol) in dichloromethane
(5 mL) and trifluoroacetic acid (3.0 mL, 4.4 g, 39 mmol) was
stirred for 2 h. The solvents were removed by rotary evapora-
tion. The residue was then dissolved into dichloromethane and
washed with 2 N aqueous sodium hydroxide solution and then
brine. The aqueous layers were extracted three times with fresh
dichloromethane. The combined organic layers were dried over
sodium sulfate, filtered, concentrated by rotary evaporation,
and dried in vacuo to yield a yellow solid (0.030 g, 0.077 mmol,
90%): MS (ESI) m/z 393.24 (M þ 1); 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.94 (s, 1 H), 8.04 (d, J = 4.55 Hz, 1 H), 7.05
(s, 1 H), 6.97 (s, 1 H), 6.94 (d, J = 4.29 Hz, 1 H), 6.52 (d, J = 7.58
Hz, 1 H), 4.59 (s, 2 H), 3.73 (br s, 1 H), 3.62 (s, 4 H), 2.92 (s, 4 H),
1.94 (d, J = 9.85 Hz, 2 H), 1.72 (d, J = 10.36 Hz, 2 H), 1.59 (d,
J = 9.35 Hz, 1 H), 1.36-1.27 (m, 2 H), 1.25-1.14 (m, 3 H).
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Protein kinase D distribution in normal human epidermis, basal
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dependant c-Jun phosphorylation in cancer cells. Biochem. Biophys.
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Protein kinase D in small cell lung cancer cells: rapid activation through
protein kinase C. Cancer Res. 1999, 59, 572–577. (d) Johannes, F. J.;
Horn, J.; Link, G.; Haas, E.; Siemienski, K.; Wajant, H.; Pfitzenmaier,
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dependant protective genes. Eur. J. Biochem. 1998, 257, 47–54. (e)
Zhukova, E.; Sinnett-Smith, J.; Rozengurt, E. Protein kinase D potenti-
ates DNA synthesis and cell proliferation induced by bombesin,
vasopressin or phorbol esters in Swiss 3T3 cells. J. Biol. Chem.
2001, 276, 40298–40305.
(8) Sharlow, E. R.; Giridhar, K. V.; Lavalle, C. R.; Chen, J.; Leimgruber,
S.; Barrett, R.; Bravo-Altamirano, K.; Wipf, P.; Lazo, J. S.; Wang,
Q. J. Potent and selective disruption of protein kinase D functionality
by benzoxoloazepinolone. J. Biol. Chem. 2008, 283, 33516–33526.
(9) (a) Gschwendt, M.; Dieterich, S.; Rennecke, J.; Kittstein, W.;
Mueller, H.-J.; Johannes, F.-J. Inhibition of protein kinase C μ
by various inhibitors. Differentiation from protein kinase c iso-
enzymes. FEBS Lett. 1996, 392, 77–80.(b) Singh, R.; Li, H.; Zhao, H.;
Payan, D. G.; Kolluri, R.; Tso, K.; Ramphal, J.; Gu, S. Preparation of
Cyclic Amine Substituted Pyrimidinediamines as PKC Inhibitors. WO
2009012421, 2009. (c) Yokoyama, S. AP2 Inhibitor. WO 2008088006,
2008. (d) Raynham, T. M.; Hammonds, T. R.; Charles, M. D.; Pave,
G. A.; Foxton, C. H.; Blackaby, W. P.; Stevens, A. P.; Ekwuru, C. T.
Pyridine Benzamides and Pyrazine Benzamides as PKD Inhibitors and
Their Preparation, Pharmaceutical Compositions and Use in the Treat-
ment of Diseases. WO 2008074997, 2008. (e) Raynham, T. M.;
Hammonds, T. R.; Gilliatt, J. H.; Charles, M. D.; Pave, G. A.; Foxton,
C. H.; Carr, J. L.; Mistry, N. S. Aminoethylamino-aryl (AEAA) Compounds
as PKD Inhibitors and Their Preparation, Pharmaceutical Compositions and
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Gschwendt, M.; Kittstein, W.; Johannes, F.-J. Differential effects of suramin
on protein kinase C isoenzymes, a novel tool for discriminating protein
kinase C activities. FEBS Lett. 1998, 421, 165–168.
Acknowledgment. We thank Patrick Cronan for high re-
solution mass spectrometry data and Analytical Sciences for
LCMS and NMR support. We thank the Metabolism and
Pharmacokinetics Group for the in vivo and bioanalytical
support in the pharmacokinetic studies.
Supporting Information Available: A table of additional com-
parative kinase selectivity for compounds 1 and 12a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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