2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes

Article abstract of DOI:10.1016/j.bmcl.2011.10.025

The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyridine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes δ, η, θ (in particular PKC/η, and display a 10-100-fold sele

Full text of DOI:10.1016/j.bmcl.2011.10.025

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7367–7372
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,6-Naphthyridines as potent and selective inhibitors of the novel protein
kinase C isozymes
Maurice J. van Eis a, , Jean-Pierre Evenou a, Philipp Floersheim a, Christoph Gaul a,
⇑
Sandra W. Cowan-Jacob a, Lauren Monovich b, Gabriele Rummel a, Walter Schuler a, Wilhelm Stark a,
Andre Strauss a, Anette von Matt a, Eric Vangrevelinghe a, Juergen Wagner a, Nicolas Soldermann a
a Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, CH-4056 Basel, Switzerland
b Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2011
Revised 5 October 2011
Accepted 7 October 2011
Available online 21 October 2011
The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyri-
dine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes d,
e, g, h (in
particular PKC , and display a 10–100-fold selectivity over the classical PKC isotypes. The prototype
e/g
compound 11 was found to inhibit PKCh-dependent pathways in vitro and in vivo. In vitro, a-CD3/a-
CD28-induced lymphocyte proliferation could be effectively blocked in 10% rat whole blood. In mice,
11 dose-dependently inhibited Staphylococcus aureus enterotoxin B-triggered IL-2 serum levels after oral
dosing.
Keywords:
Protein Kinase C
Inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Isozyme specific
2,6-Naphthyridines
T cell inhibition
Protein kinase C (PKC) comprises a family of 11 serine/threo-
nine kinases which can be grouped into three subfamilies based
on the different co-factors required for optimal catalytic activity.
progress was reported towards selective ATP-competitive PKCh
inhibitors based on the thieno[2,3-b]pyridine-5-carbonitrile4a–g
and 2,4-diamino-5-nitropyrimidine scaffolds.4h
The classical isotypes, PKC
diacylglycerol (DAG) and Ca2+ for their activation, whereas the no-
vel isotypes PKCd/ /h only require DAG. In contrast, the atypical
isotypes PKC
/k/n do not require Ca2+ or DAG to be able to exert
their full catalytic activity. Multiple members of the PKC family
play a central role in the signaling cascades of the immune system,
a
/b/
c
require the second messengers
Surprisingly, inhibitors displaying selectivity for the novel PKC
isotype family (degh) over the classical isotypes (de) have not been
reported, despite the close relationship between these isotypes in
the phylogenetic tree. In this communication we report the first
examples of low molecular weight compounds displaying such a
selectivity profile. These compounds showed promising cellular
activity and have attractive biopharmaceutical properties.
e/g
i
the key players being the classical PKC isotypes de and the novel
PKC isotype h.1 In particular for PKCh a crucial role in T cell receptor
(TCR)–mediated T cell activation and proliferation has been estab-
lished.2 The different PKC isotypes and especially PKCh, are there-
fore considered to be attractive therapeutic targets for the
treatment of autoimmune diseases and transplantation. However,
to date only Sotrastaurin (AEB071), a potent nanomolar ATP-com-
petitive inhibitor of all classical and novel PKC isotypes3a,b has pro-
gressed to clinical trials as a potential treatment of psoriasis3c and
for the prevention of allograft rejection following renal transplan-
tation.3d,e Given the crucial role of PKCh in T cell activation/prolif-
eration, many groups have initiated drug discovery programs
aiming at the development of PKCh selective compounds. Recently
In a search for PKCh inhibiting chemotypes, a high-throughput
docking (HTD) screen was performed. Docking5 of the Novartis
compound archive into the ATP binding site of PKCh using the X-
ray crystal structure of PKCh (PDB code 1XJD)6 and subsequent pro-
filing of the 641 highest ranked hits against a panel consisting of
six PKC isotypes led to the identification of compound 1 as a potent
inhibitor of PKCh. In addition to inhibition of PKCh, 1 also potently
inhibits the novel PKC isotypes d
selectivity over the classical PKC isotypes d
e
,
g
whilst displaying an excellent
(see Table 1).
e
In the binding mode identified by HTD, 1 engages in a single
canonical H-bond to Leu 461 located in the hinge region of the
ATP binding site of PKCh with the 4-pyridyl moiety serving as a
H-bond acceptor. In addition, the nitrogen atom in the 6-position
of the naphthyridine core is predicted to make a H-bond to the
catalytic lysine residue Lys 409. An alternative binding mode in
which the pyridyl ring and the naphthyridine ring switch positions
⇑
Corresponding author. Tel.: +41 61 3248293; fax: +41 61 3246735.
E-mail address: maurice.van_eis@novartis.com (M.J. van Eis).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.025

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M. J. van Eis et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7367–7372
Table 1
IC50 values of isoquinoline and naphthyridine based PKC inhibitors against a panel of PKC isotypes (IC50 values in nM)ab
Compound
R
a
b1
d
e
g
h
N
N
N
1
900
859
20
5
5
77
HN
HN
HN
HN
HN
NH2
NH2
NH2
NH2
NH2
NH2
N
N
2
3
4
5
6
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
157
877
929
>1000
44
743
401
>1000
54
>1000
> 1000
>1000
355
N
N
N
N
N
N
N
N
N
N
N
N
102
38
27
>1000
HN
a
Mean values of at least two independent measurements.
Scintillation Proximity Assay (SPA), see Refs. 3a,b,7a,b.
b
resulting in H-bond formation between the nitrogen atom in the 6-
position of the naphthyridine core and the hinge of the kinase
seemed plausible as well. In order to test our binding mode
hypothesis, a series of naphthyridine derivatives were synthesized
(compounds 2–6, see Table 1). The N?CH replacement in the 6-po-
sition of the naphthyridine core (compound 2) led to a 100 fold
drop in binding affinity to all PKC isotypes. Interestingly, some
modest binding affinity to PKCeg was retained suggesting that
the naphthyridine core is not involved in binding to the hinge. As
anticipated from the binding model, shifting of the nitrogen atom
into the 5-position (compound 3) did not improve inhibitory activ-
ity on the different PKC isotypes. All PKC inhibitory activity was
lost with the 7-aza-analogue 4. Removal of the nitrogen atom in
the 2-position of the naphthyridine core of 1 (compound 5) led
to a 10-fold loss of binding affinity to PKCeg and ablation of
inhibitory activity on PKCh. We speculate that for optimal binding
affinity the pyridyl ring and the naphthyridine ring need to be co-
planar. The N?CH modification may have a detrimental steric
effect on the required co-planarity of the pyridyl and isoquinoline
rings. However, introduction of an additional nitrogen atom into
the 4-position of the naphthyridine core (compound 6) with the
aim of further favouring the co-planarity of the two aromatic rings
did not have a positive impact on the binding affinity. Although
inhibitory potency on PKCeg was retained, binding affinity to PKCh
was ablated.
Compound 1 and the majority of the compounds listed in this
paper could be synthesized by adapting the synthetic protocols
previously reported by Meredith et al. for similar compounds,
using the appropriate starting materials.8
For some compounds an alternative synthesis was used (12, 23
and 24). This is exemplified in Scheme 1 for compound 12.
Starting from the commercially available 3-methyl-pyridine-N-
oxide (7) the 3-methyl-isonicotinonitrile (8) could be obtained in
gram amounts. Deprotonation of 8 with sodium hydride and sub-
sequent reaction with isonicotinic acid methyl ester afforded 9.
We accidentally discovered that 9 was unstable under column

M. J. van Eis et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7367–7372
7369
2-methyl-propane-1,2-diamine, 12 could be isolated in 47% yield.
The scope and limitations of this reaction as well the proposed
mechanism will be the subject of a separate paper.
N
O
N
O
a,b
c
d
N
N
N
N
In a second round of optimization, the SAR around the basic
amine-containing side chain was explored. A summary of the data
is given in Table 2. Replacement of the nitrogen linker by an oxy-
gen linker led to an approximately 20-fold loss in binding affinity
to the novel PKC isotypes (10). The introduction of a small lipo-
philic substituent at the d-carbon to the basic amine further im-
proved binding affinity to the novel PKC isotypes (11). The
corresponding (R)-enantiomer of 11 (structure not shown) was
around 10-fold less potent on all PKC isotypes. In line with this,
gem-dimethyl substitution (12) did not further improve potency.
Likewise, the introduction of large lypophilic substituents (13)
was poorly tolerated. Alkylation of the linker nitrogen atom or
the basic amine led to a 5- and 20-fold drop in binding affinity
N
CN
CN
7
8
9
HN
12
NH2
Scheme 1. Reagents and conditions: (a) Dimethyl sulfate (1.1 equiv), 40 °C, 16 h;
(b) KCN (1.1 equiv), EtOH/H2O (1:1), 40 °C, 16 h, 34%; (c) NaH (4.0 equiv),
isonicotinic acid methyl ester (1.0 equiv), DME, 95 °C, 16 h., 100%; (d) 2-methyl-
propane-1,2-diamine (10 equiv), EtOAc/acetic acid (6:4), Silica Gel 60 (15 weight
equiv), rt, 2 h, 47%.
chromatography conditions and that cyclisation gave rise to a
naphthyridinone. When 9 was stirred in a mixture of ethyl acetate
and acetic acid in the presence of silica gel and an excess of
Table 2
Variations around the flexible side-chain of 1 (IC50 values in nM)ab
N
N
N
R
Compound
R
a
b1
d
e
g
h
10
O
>1000
>1000
378
703
120
82
948
NH2
NH2
NH2
HN
11
12
829
967
8
5
2
9
55
HN
942
44
18
141
13
818
>1000
932
403
452
>1000
HN
*
NH2
NH2
14
15
N
>1000
>1000
>1000
>1000
371
254
24
79
34
93
350
997
HN
N
H
N
16
>1000
823
31
8
6
109
N
H
N
17
18
433
771
661
865
63
7
8
4
77
N
H
N
214
13
132
*
NH2
19
20
676
779
53
13
81
10
97
HN
HN
NH2
>1000
>1000
635
188
743
OH
N
21
>1000
>1000
>1000
802
735
>1000
N
O
H
a
Mean values of at least two independent measurements.
Scintillation Proximity Assay (SPA), see Refs. 3a,b,7a,b.
b

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M. J. van Eis et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7367–7372
Table 4
Inhibition of a-CD3/aCD28 induced rat T lymphocyte proliferationa,b,c
Compound
IC50
5
12
11
1041
356
138
a
b
c
Mean values of at least two independent measurements.
See Ref. 11.
Assay run in the presence of 10% rat blood.
We were not able to obtain an X-ray crystal structure of one of
the naphthyridines bound to PKCh. However, we did obtain an X-
ray crystal structure of 12 bound to the kinase domain of PKC
(see Fig. 1).9 PKC
adopts an active conformation despite the fact
g
g
that tyrosine 513 in the activation loop is non-phosphorylated.
The X-ray crystal structure unambiguously demonstrates that 12
adopts the binding mode found by HTD in which the pyridyl moi-
ety makes a single hydrogen bond to the hinge region of the pro-
tein (Val 436). This binding mode is identical to the recently
proposed binding mode of similar compounds to Protein Kinase
D.8 No indication was found for the occurrence of an alternative
binding mode in which the key interaction to the hinge would in-
volve the naphthyridine core as it has been already hypothesized
for other naphtyridine-based inhibitors targeting SYK10a and
CK210b kinases. The nitrogen in the 6-position of the naphthyridine
core is involved in hydrogen bonding to the catalytic lysine residue
(Lys 384) whereas the basic amine forms a salt bridge to Asp 440
located in the sugar pocket. The inhibitor is tightly packed in the
ATP binding pocket and strong lipophilic interactions occur be-
tween Leu 486 and the pyridyl ring and Val 369 and the naphthyri-
dine core. In addition a strong face-to-edge interaction occurs
between the naphthyridine ring and a phenyl alanine residue
(Phe 366) situated in the P-loop.
Figure 1. X-ray crystal structure of 12 (green)
g (carbons in magenta) bound to the
ATP-binding site of PKC9 solved at 2.05 Å resolution. Hydrogen bonds are indicated
by dashed red lines and water molecules by red spheres.
(14 and 15, respectively). Binding affinity was retained when the
linker nitrogen atom or the basic amine were incorporated into a
cyclic fragment (16, 17 and 18). A chain length of two carbon
atoms proved to be ideal. A three-carbon spacer (19) was tolerated
but longer chain lengths were unfavourable. The basic amine
proved to be essential for achieving a high binding affinity to the
novel PKC isozymes which is in line with the binding hypothesis
suggesting the occurrence of a salt bridge between an aspartate
residue of the protein and the protonated amine. Substitution of
the amine by a hydroxyl group (20) strongly attenuated binding
affinity whereas an amide group ablated binding affinity (21).
The X-ray crystal structure does not provide a satisfactory
explanation for the observed PKC isotype selectivity. Of all the res-
idues of the protein which make a direct contact with the inhibitor
Table 3
Variations around pyridyl ring of 12 (IC50 values in nM)a
R
N
N
HN
NH2
Compound
R
a
b1
d
e
g
h
N
22
>1000
>1000
>1000
>1000
>1000
>1000
>1000
N
N
23
24
378
294
147
60
496
NH2
NH
>1000
>1000
961
>1000
>1000
862
>1000
F
N
25
>1000
124
62
18
140
Cl
N
N
26
27
>1000
>1000
>1000
>1000
221
386
47
15
52
93
133
738
a
Mean values of at least two independent measurements.
Scintillation Proximity Assay (SPA), see Refs. 3a,b,7a,b.
b

M. J. van Eis et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7367–7372
7371
Table 5
Pharmacokinetic properties of 11 in rats and efficacy in a mouse SEB modela,b
Intravenous (iv) dose = 5 mg /kg
AUC (ng h mLꢀ1
Oral (po) dose = 10 mg /kg
Mouse SEB modelb inhibition [%] standard deviation
)
1770
8.1
49
AUC (ng h mLꢀ1
)
712
2.0
53
50 mg/kg
37 25
70 15
p = 1.6 ꢁ 10ꢀ2
1
1
t
(h)
Tmax (h)
Cmax (ng/mL)
F(%)
100 mg/kg
p = 3 ꢁ 10ꢀ4
½
E
Cl (mL minꢀ1 kgꢀ1
)
Vss (L/kg)
14.0
20
a
Data reported as the average of three animals (Lewis rats). Vehicle for iv: 85% water–15% PEG200. Vehicle for po: 92.5% water/HCl (pH 4) – 7.5% PEG200. AUC1: Area
under the curve extrapolated to infinity.
b
SEB model, see Refs. 16,17.
only one difference exists between the novel and classical PKC iso-
types (Leu 486 (PKCg, see Fig. 1) ? Met 470 (PKCd)). We feel that
erties. The iv pharmacokinetic profile of 11 is characterised by a
large volume of distribution which is typical for a strongly basic
compound. Clearance is medium high, resulting in an elimination
half-life of 8.1 h. The po pharmacokinetic profile is characterised
by a relatively rapid absorption which is in line with the good
aqueous solubility and medium permeability of 11. Oral bioavail-
ability is modest (20%).
Compound 11 was subsequently tested in vivo using the model
of SEB-induced IL-2 production in BALB/c mice.16,17 In this experi-
ment, 11 showed dose-dependent, statistically significant inhibi-
tion of SEB-triggered release of IL-2. At an oral dose of 100 mg/kg
70% inhibition was achieved whereas inhibition was 37% at the
lower dose of 50 mg/kg.
In summary, we have reported the first examples of a new class
of PKC inhibitors based on the 2,6-naphthyridine scaffold which
show high selectivity for the novel over the classical PKC isotypes.
Selected derivatives show activity in a PKCh dependent cellular
assay and have attractive biopharmaceutical properties. For the
prototype compound 11 this translated into promising inhibition
of IL-2 release in vivo after SEB stimulation. We believe that these
compounds may also serve as tool compounds to further dissect
the role of the different PKC isotypes in signaling pathways. Efforts
are ongoing to further improve the binding affinity of these
compounds to PKCh.
this difference is insufficient to rationalize the observed isotype
selectivity. We speculate that the origin of the observed isotype
selectivity may reside in a difference in the flexibility of the P-loop
between the different isotypes. A more flexible P-loop in the novel
PKC isotypes may allow for more favourable van der Waals con-
tacts between the protein and the inhibitor.
In another round of optimisation, the SAR around the hinge
binding pyridyl moiety was investigated (see Table 3). Changing
the connectivity of the naphthyridine core to the pyridyl fragment
(22) was not tolerated, which is in line with the observed binding
mode. Adding a hydrogen bond donor to the 2-position of the pyr-
idyl ring, with the potential to make an additional hydrogen bond
to the hinge region of the protein, reduced binding affinity (23).
Larger substituents in the 2-position (24) fully ablated binding
affinity. Halogen atoms in the 3-position of the pyridyl ring were
tolerated (25/26) but did not have a favourable impact on the bind-
ing affinity whereas a methyl group had a detrimental effect (27).
A number of derivatives were tested for their ability to inhibit
anti-CD3/anti-CD28 induced proliferation of rat T-lymphocytes in
10% whole blood.11 This assay is expected to be PKCh-dependent
as this isoform integrates signaling pathways downstream of the
T cell receptor and the CD28 co-receptor. The role of the other no-
vel PKC isoforms in T cell activation is not well understood. Indeed
inhibition of the novel PKC isotypes translated into inhibition in
the proliferation assay. The limited data set suggests that the IC50
values for inhibition in the proliferation assay track the inhibition
of PKCh in the biochemical assay (see Table 4).
Acknowledgment
Christoph Burkhart is kindly acknowledged for performing the
mouse SEB experiments.
Compound 11 showed good activity in the proliferation assay
(IC50 = 138 nM) and was selected for further profiling against a pa-
nel of 224 human serine/threonine and tyrosine protein kinases.12
Compound 11 shows a promising kinase selectivity with only 38
kinases inhibited by more than 50% and only 16 kinases (including
the novel PKC isotypes) inhibited by more than 80% at a compound
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.025.
concentration of 1 lM. Among the kinases which were strongly
inhibited in addition to the novel PKC isotypes are closely related
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DiSalvo, D.; Ginn, J. D.; Kashem, M. A.; Wolak, J. P.; Homon, C. A.; Farrell, T. M.;
Grbic, H.; Hu, H.; Kaplita, P. V.; Liu, L. H.; Spero, D. M.; Jeanfavre, D. D.; O’Shea,
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9. Full experimental details and crystallographic data for the structure of 12
bound to PKX
g have been deposited at the RCSB Protein Databank as PDB 3txo.
10. (a) Cywin, C. L.; Zhao, B.-P.; McNeil, D. W.; Hrapchak, M.; Prokopowicz, A. S.;
Goldberg, D. R.; Morwick, T. M.; Gao, A.; Jakes, S.; Kashem, M.; Magolda, R. L.; Soll,
R. M.; Player, M. R.; Bobko, M. A.; Rinker, J.; DesJarlais, R. L.; Winters, M. P. Bioorg.
Med. Chem. Lett. 2003, 13, 1415; (b) Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-
Jain, A.; Bourbon, P.; Haddach, M.; Michaux, J.; Nagasawa, J.; Schwaebe, M. K.;
Stefan, E.; Vialettes, A.; Whitten, J. P.; Chen, T. K.; Darjania, L.; Stansfield, R.;
Anderes, K.; Bliesath, J.; Drygin, D.; Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.;
Trent, K.; Rice, W. G.; Ryckman, D. M. J. Med. Chem. 2011, 54, 635.
11. For a description of the assay, see Supplementary data.
12. Upstate KinaseProfiler™.
13. High-throughput permeability assay: Wohnsand, F.; Faller, B. J. Med. Chem.
2001, 44, 923.
14. Measurement of plasma protein binding via rapid equilibrium dialysis: Waters,
N. J.; Jones, R.; Williams, G.; Sohal, B. J. Pharm. Sci. 2008, 97, 4586.
15. Radioligand binding assay for hERG channel blockers: Finlayson, K.; Turnbull,
L.; January, C. T.; Sharkey, J.; Kelly, J. S. Eur. J. Pharmacol. 2001, 430, 147.
16. For a description of the SEB model, see Supplementary data.
17. For the use of the SEB model in mice for testing immunosuppressive
compounds, see: Kuschnaroff, L. M.; Valckx, D.; Goebels, J.; Rutgeerts, O.;
Heremans, H.; Froyent, G.; Waer, M. Scand. J. Immunol. 1997, 46, 459.
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L.; Stahl, M. L.; Joseph-McCarthy, D.; Benander, C.; Fitz, L.; Greco, R.; Somers, W.
S.; Mosyak, L. J. Biol. Chem. 2004, 48, 50401.
6. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shaw, D. E.; Shelley, M.; Perry, J. K.; Francis, P.;
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