Structure-based optimization of aminopyridines as PKCθ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.05.114

The identification of a novel series of PKCθ inhibitors and subsequent optimization using docking based on a crystal structure of PKCθ is described. SAR was rapidly generated around an amino pyridine-ketone hit; (6-aminopyridin-2-yl)(2-aminopyridin-3-yl)methanone 2 leading to compound 21 which significantly inhibits production of IL-2 in a mouse SEB-IL2 model.

Full text of DOI:10.1016/j.bmcl.2012.05.114

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4645–4649
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based optimization of aminopyridines as PKCh inhibitors
Juan-Miguel Jimenez a, , Christopher Davis a, , Dean Boyall a, Damien Fraysse a, Ronald Knegtel a,
Luca Settimo a, Stephen Young a, Claire Bolton b, Peter Chiu b, Adam Curnock b, Richele Rasmussen b, , Adam
Tanner b, Ian Ager b
⇑
⇑
a Department of Chemistry at Vertex Pharmaceuticals (Europe) Ltd, 88 Milton Park, Abingdon OX14 4RY, United Kingdom
b Department of Biology at Vertex Pharmaceuticals (Europe) Ltd, 88 Milton Park, Abingdon OX14 4RY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification of a novel series of PKCh inhibitors and subsequent optimization using docking based
on a crystal structure of PKCh is described. SAR was rapidly generated around an amino pyridine-ketone
hit; (6-aminopyridin-2-yl)(2-aminopyridin-3-yl)methanone 2 leading to compound 21 which signifi-
cantly inhibits production of IL-2 in a mouse SEB-IL2 model.
Received 25 April 2012
Revised 22 May 2012
Accepted 24 May 2012
Available online 6 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
PKCh
Kinase
SEB-IL2
Aminopyridine
T cell receptor
Protein kinase C theta (PKCh) is a member of a large family of
serine/threonine kinases that are involved in a diverse range of cel-
lular functions. There are 11 PKC isoforms that can be sub-divided
colitis and psoriasis.5 AEB071 is a potent inhibitor of classical
and novel PKC isoforms and is a potent inhibitor of the isoforms
involved in B cell and T cell function, PKCa, PKCb and PKCh.
into DAG- and calcium-dependent, classical isoforms (PKCs
a, bI,
bII and ) DAG-dependent, novel isoforms (PKCs d, and h)
c
e, g
and DAG- and calcium-independent atypical isoforms (PKCs k,
i
and f).1
PKCh plays a central role in the activation of T cells and integrates
signals from both the T cell receptor (TCR) and the co-stimulatory
CD28. During T cell activation, PKCh is recruited to the center of
the immune synapse (IS) and is responsible for the activation of
key transcription factors, AP-1, NFjB and NFAT that drive transcrip-
tion of the IL-2 gene, an essential step in T cell activation.2 Impor-
tantly, studies in PKCh knockout mice have demonstrated that
whilst antiviral responses are PKCh independent, T-cell responses
associated with autoimmune diseases are PKCh dependent.3,4
Consequently, it is anticipated that a selective PKCh inhibitor will
provide the desired balance of efficacy and safety for the treatment
of autoimmune diseases.
There has been significant interest in the development of PKCh
selective inhibitors for the treatment of autoimmune diseases and
there are several companies with small molecule PKCh inhibitor
programs.6–18 Whilst some have reported promising pre-clinical
data, to date, there are no selective PKCh inhibitors confirmed to
be in the clinic.
The aim of our program was to identify potent and selective PKCh
inhibitors to provide safer options to treat autoimmune diseases.
Compound 2, (2-aminopyridine-3-yl)(6-(1,4-diazepan-1-yl)pyri-
dine-2-yl)methanone was one of the most promising hits during
Novartis’ pan-PKC inhibitor, AEB071 (Sotrastaurin) 1, is cur-
rently in phase II clinical studies for transplantation, ulcerative
⇑
Corresponding authors. Tel./fax: +44 1235 438800.
E-mail addresses: juan-miguel_jimenez@vrtx.com (J.-M. Jimenez), chris_davis@
vrtx.com (C. Davis).
Present address: National Health and Medical Research Council, GPO Box 1421,
Canberra, ACT 2601, Australia.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.114

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J.-M. Jimenez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4645–4649
our HTS campaign. It is a potent inhibitor of PKCh (Ki = 164 nM) with
good selectivity over other kinases outside the PKC family.
In addition to the favorable inhibition profile, compound 2 shows
desirable features, such as low molecular weight (297.16), low cal-
culated polar surface area (84), calculated LogP of 2, good perme-
ability (Caco-2 A–B 9 Â 10À6 cm/s, B–A 11 Â 10À6 cm/s) and high
ligand and lipophilic efficiency (LE and LLE)19 (0.3 and 4.8). Com-
pound 2 was considered an excellent starting point for our optimi-
zation efforts. Our first objective was to improve the PKCh potency
of compound 2.
Figure 1. Docking and minimization of compound 2 in the active site of PKCh,
showing the two regions to be investigated.
Using docking and minimization of 2 in PKCh,20–22 the com-
pound is predicted to bind to the hinge region of PKCh via the ami-
nopyridine with the ketone pointing towards the gatekeeper
region and potentially making an interaction with Thr442. The
NH of the 1,4-diazepane is predicted to make an interaction with
the main chain carbonyl oxygen atom of Asp508 (Fig. 1). In addi-
tion, docking and minimization of 2 in PKCh also suggests that
there are two main areas of the PKCh active site where lipophilic
interactions could provide the desired increase in potency; region
A targeting the C-terminus region and region B targeting the ‘back
pocket’ near the gatekeeper (Fig. 1).
Table 1
Effect of amine ring size and positioning of amine
Compounds
R1
PKCh Kia
(
lM)
cLogP
LLE
Compounds
R1
PKCh Kia
(lM)
cLogP
LLE
LE
LE
2.0
4.8
0.3
1.7
5.4
0.3
2
3
4
0.164b
0.375
7
8
9
0.076
1.975
1.39
2.4
4.0
0.3
2.2
3.5
0.3
2.0
5.5
0.4
2.2
3.7
0.3
0.032b
1.7
4.2
0.3
2.2
ND
ND
5
6
1.52
10
11
>2.8
1.01
1.8
4.4
0.3
1.6
4.4
0.3
0.745
a
Mean from n values of 2 independent experiments.
Mean from n = 6.
b

J.-M. Jimenez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4645–4649
4647
ing a significant increase (fivefold) in PKCh activity. Methylation of
the 1,4-diazepane NH present in compound 2, Ki = 164 nM, causes
an approximately twofold loss in PKCh activity, as shown by com-
pound 3, Ki = 375 nM. From this amine set, piperazine, 4, was
chosen for further optimization due to its greater PKCh activity
and ligand efficiency.
To improve further the PKCh activity of compound 4 we postu-
lated that a lipophilic interaction could be made between the C-3
position of the piperazine and region A of the PKCh active site. To
explore this further, a set of substituted piperazines were prepared
with a diverse range of lipophilic substitutents as shown in Table 2.
Addition of an (S-)-methyl group, 12, showed little change in
activity with a PKCh Ki of 67 nM. Extending the lipophilicity to an
isopropyl group 15 or to isobutyl group 18 showed increasing
activity whilst maintaining a good level of lipophilic efficiency
with PKCh Ki’s of 4 nM and 3 nM respectively. The PKCh activity
is very sensitive to the stereochemistry at this C-3 position, with
the S-enantiomer being over 35-fold more potent than the R-enan-
tiomer, as shown by the enantiomeric pairs 19 & 20 and 15 & 16.
Disubstitution at the C-3 position of the piperazine ring, compound
Scheme 1. Reagents and conditions: (a) (i) 2,6-Dibromopyridine, <À30 °C, (ii) BuLi
2.5 M in hexanes, <30 °C; (b) amine, K2CO3, DMF, 110 °C.
In order to allow rapid optimization of these interactions, a key
intermediate A was synthesised from the commercially available
2-aminonicotinonitrile as shown in Scheme 1. This intermediate
was prepared by a ‘one pot’ anion formation and nucleophilic addi-
tion reaction giving Int-A, (2-aminopyridin-3-yl)(6-bromo pyridin-
2-yl) methanone in 50% yield.
An initial set of amines was then synthesized via displacement
of 6-bromopyridine intermediate Int-A and are shown in Table 1.
The SAR generated shows that the original 1,4-diazepane was
actually fairly close to optimum with only the piperazine 4 show-
Table 2
Effect of piperazine substitution on PKCh activity
Compounds
R1
PKCh Kia
(l
M)
cLogP
LLE
Compounds
R1
PKCh Kia
(l
M)
cLogP
LLE
LE
LE
2.0
3.3
4
0.032
5.5
0.4
16
0.275
3.3
0.3
2.4
3.7
12
13
0.182
0.067
4.8
0.3
17
18
0.038
0.003
3.8
0.3
2.4
4.4
0.3
3.7
4.9
0.3
3.8
2.7
4.4
0.3
14
15
0.085
0.004
19
20
0.238
0.006
2.9
0.2
3.3
5.3
0.4
3.8
4.5
0.3
a
Mean from n P2 independent experiments.

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J.-M. Jimenez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4645–4649
Table 3
Effect of pyridine substitution of 3- or 4- position on PKCh activity and kinase selectivity
b
Compounds
R1
R2
PKCh Kia
(lM)
PKCd Kia
(lM)
PKCa
Kia
(l
M)
IL-2 Cell IC50
(
lM)
Kinases-fold selectivityb
18
21
22
23
24
25
26
27
28
29
H
CF3
Cl
Cyclopropyl
F2HCO
CF3CH2O
Propyl
Ph
H
H
H
H
H
H
H
H
H
Me
0.003
0.003
0.002
0.028
0.007
0.29
0.02
0.0006
>0.7
0.020
0.037
0.022
0.255
0.087
1.3
0.03
<0.01
>2
0.054
0.076
0.049
2.65
0.459
>1.25
1.23
0.01
>1.25
0.459
0.165
0.052
0.058
2.6
0.229
ND
0.41
0.005
ND
Src >1400 PKA 60 Gsk3b 143
Src >1300 PKA 47 Gsk3b 400
Src >4000 PKA 38 Gsk3b 255
Src >140 PKA 93 Gsk3b >140
Src >570 PKA 84 Gsk3b >570
Src >13 PKA >13 Gsk3b >13
Src >200 PKA 24 Gsk3b >200
Src 10 PKA 3 Gsk3b 30
Benzyl
H
Src ND PKA ND Gsk3b ND
Src >167 PKA 7 Gsk3b 18
0.024
0.116
0.41
a
Mean from n P2 independent experiments.
Data from n = 1.
b
Table 4
Extended selectivity profile of compound 21
a
Enzymes in TCR pathway IC50
Other kinases Kia
M)
(l
M)
Lck >2
Itk >2
Jak2 >4
PKC 0.017
PIk >4
Fyn >2
Lyn >2
Zap70 >2
c-Raf >2
Flt3 0.580
PI3 K >4
Erk-1 >2
PI3 k >4
CdK2 >4
Syk >4
PKCb 0.260
Kdr >4
Rock1 >4
Met >4
Jnk3 >4
c
(l
e
a
a
Data from n = 1 independent experiments.
also wanted to fill region B near the gatekeeper residue in order
to gain further PKCh activity. Our predicted binding mode sug-
gested that we could target this area by substitution from the C3
or C4 position of the central pyridine ring. A set of compounds
was made to explore this region of the active site as shown in
Table 3.
Table 5
Mouse PK for compound 21 (IV-bolus dose 3 mg/kg and PO dose of 10 mg/kg). Data
reported as an average of three animals.
Cl (mL/min/kg)
203
T1/2 (h)
Vz (L/kg)
19.7
VSS (L/kg)
15.8
MRT (h)
1.4
%F
45
1.3
Addition of a phenyl ring, as in compound 27, showed exquisite
PKCh potency with a Ki of 0.6 nM but also proved to be less selec-
tive over other kinases than most other compounds in the series.
Unfortunately, compound 27 showed unacceptable levels of cyto-
toxicity (data not shown). Saturated alkyl groups in this region
were detrimental to PKCh activity, such as propyl 26 and cyclopro-
pyl 23 with Ki’s of 20 and 28 nM respectively. Addition of a CF3
group (21) (LE = 21, LLE = 4.0) or Cl (22) (LE = 23, LLE = 4.5) showed
no effect on PKCh activity but at a loss of ligand and lipophilic effi-
ciency as compared with H (18) (LE = 25, LLE = 4.9), while main-
taining a good level of cellular potency and kinase selectivity
(see Table 3). However, they did improve the PK characteristics;
providing greater oral exposure and longer t1/2 in rat. Conse-
quently, compound 21 was selected for in vivo assessment on
the basis of its overall profile. Good PKCh potency (Ki = 0.003
lM)
translated to good cell activity (IL-2 IC50 0.052 M) and excellent
l
overall kinase selectivity (see Table 4). Although mouse PK of 21
was far from ideal (Table 5), it was deemed suitable to test in
our mouse SEB-IL2 model (see Fig. 2).23
Figure 2. Mouse SEB-IL2 dose response to PO dosing of compound 21.
14, showed no gain in PKCh potency as compared with mono
substituted, 13, or unsubstituted, 4 but is less efficient in terms
of LE and LLE.
Compound 18, containing the (S-)-isobutyl piperazine, was se-
lected for further optimisation due to its high level of PKCh inhib-
itory activity. Based on our models of compound 18 in PKCh, we
Compound 21 was dosed once orally at 25, 50 and 100 mg/kg in
our mouse SEB-IL2. These data showed that 21 significantly inhib-
ited the production of IL-2 in a dose dependent manner (Fig. 2). The
level of efficacy observed in vivo is in line with the free concentra-
tion achieved at the doses tested (e.g., at 100 mg/kg C-max free
concentration is 150 nM). The mechanism for this IL-2 inhibition

J.-M. Jimenez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4645–4649
4649
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is driven by PKCh inhibition of 21, as the other kinases in the TCR
signaling pathway are not inhibited, as shown in Table 4.
In summary, we have described the synthesis and biological
evaluation of a series of (6-aminopyridin-2-yl)(2-aminopyridin-3-
yl)methanone as PKCh inhibitors. The program started with the
identification of hit 2 and subsequent optimization with the aid
of docking and minimization in PKCh, which led to the discovery
of 21. Compound 21 is a potent inhibitor of PKCh, which displayed
modest selectivity over closely related members of the PKC novel
subfamily and excellent selectivity across broad kinase space.
The compound shows activity in cellular and in vivo assays that
measures the functional activity of PKCh. The efforts to improve
further on the PKC isoform selectivity and PK characteristics will
be reported in due course.
Acknowledgement
The authors thank Don Middleton for his valuable assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
114.
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References and notes
19. Ligand efficiency LE = pKi/heavy atom count. Lipophilic ligand efficiency
LLE = pKiÀcLogP.
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20. Based on the published PKCtheta crystal stucture from Novartis: Rummel, G.;
Stark, W.; Strauss, A.; Wagner, J. PCT Int. Appl. WO2004076654, 2004.
21. Novartis PKCh crystal structure available from RCSB protein data bank,
accession code: 2JED.
22. For docking methods refer to Supplementary data.
23. For full details regarding the SEB-IL2 pharmacology model, refer to
Supplementary data.
5. Wagner, J.; Matt, P. V.; Faller, B.; Cooke, N. G.; Albert, R.; Sedrani, R.; Wiegand,
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