Identification, characterization and initial hit-to-lead optimization of a series of 4-arylamino-3-pyridinecarbonitrile as protein kinase C theta (PKCθ) inhibitors

Article abstract of DOI:10.1021/jm800214a

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCθ) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCθ enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCθ high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5- (3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low μM ATP competitive PKCθ inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3- pyridinecarbonitrile 4p, a 70 nM PKCθ inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCβ, and PKCζ, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Full text of DOI:10.1021/jm800214a

5958
J. Med. Chem. 2008, 51, 5958–5963
Identification, Characterization and Initial Hit-to-Lead Optimization of a Series of
4-Arylamino-3-Pyridinecarbonitrile as Protein Kinase C theta (PKCθ) Inhibitors
Derek C. Cole,*,† Magda Asselin,† Agnes Brennan,‡ Robert Czerwinski,§ John W. Ellingboe,† Lori Fitz,‡ Rita Greco,‡
,§
Xinyi Huang,| Diane Joseph-McCarthy, Michael F. Kelly,† Matthew Kirisits,† Julie Lee,‡ Yuanhong Li,§ Paul Morgan,‡
Joseph R. Stock,† De´sire´e H. H. Tsao,§ Allan Wissner,† Xiaoke Yang,‡ and Divya Chaudhary‡
Chemical and Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl RiVer, New York 10965, Inflammation and Chemical
and Screening Sciences, Wyeth Research, 200 Cambridge Park DriVe, Cambridge, Massachusetts 02140, Chemical and Screening Sciences,
Wyeth Research, 500 Arcola Road, CollegeVille, PennsylVania 19426
ReceiVed February 28, 2008
The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular
processes. The PKC theta (PKCθ) isoform is involved in TCR signal transduction and T cell activation and
regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus
inhibition of PKCθ enzyme activity by a small molecule represents an attractive strategy for the treatment
of asthma. A PKCθ high-throughput screening (HTS) campaign led to the identification of 4-(3-
bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low µM ATP competitive PKCθ
inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-
(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCθ inhibitor. Compound 4p was selective for
inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases,
in addition to the conventional and atypical PKCs, PKCꢀ, and PKCꢁ, respectively. Compound 4p also
inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.
Introduction
activation.5,6 PKCθ signaling regulates lung inflammation and
airway hyperresponsiveness in mouse models of antigen induced
asthma.7,8 In models of experimental autoimmune encephalo-
myelitis (EAE), PKCθ deficient mice have reduced T cell and
macrophage infiltration and demyelination in the brain compared
with wild-type control mice in response to antigen challenge.9,10
In addition PKCθ deficient mice also have a significantly
diminished response in the type II collagen-induced arthritis
(CIA) model, however mount normal protective Th1 immune
responses to clear virus infection.11,12
Therefore PKCθ is a critical enzyme that regulates selective
T cell function and represents an attractive drug target for
asthma8 and other T cell-mediated diseases,13 such as multiple
sclerosis9,10 and arthritis.12 The sequential ordered catalytic
mechanism14 and the structure of phosphorylated human
recombinant PKCθ kinase domain (KD) cocrystallized with
staurosporine15 have been reported, and these findings may aid
inhibitor optimization. Of importance is selectivity over the
closely related PKCδ since this kinase negatively regulates B
cell proliferation in mice.2,16
Several classes of inhibitors that bind to the C1 domains of
PKCs have been described, including the diterpenes such as
phorbol esters,17-19 macrocyclic lactones such as bryostatins,19,20
polyacetates such as aplysiatoxin, indole alkaloids such as
teleocidin,21 and diacylglycerol analogues.22,23 Several bisin-
dolemaleimide or staurosporine analogues including N-benzoyl-
staurosporine(midostaurin),24 7-hydroxystaurosporine(UCN01)25,26
and (9S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,
18H-5,21:12,17-dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]-
oxadiazacyclohexadecine-18,20(19H)-dione (ruboxistaurin, LY-
333531)27,28 with varying degrees of selectivity for cPKCs have
entered clinical trials for oncology and diabetes related com-
plications.29 Recently 2,4-diaminopyrimidines have been re-
ported as PKCθ inhibitors.30
The protein kinase C (PKC)a family of serine/threonine
kinases consists of 11 isoforms that share sequence and structural
homology but vary in their activation, tissue expression, and
cellular regulation.1 PKCs have been implicated in a wide
variety of cellular processes, including growth, differentiation,
secretion, apoptosis, and tumor development.2 These kinases
are classified based on their cofactor requirements: conventional
(cPKCs: R, ꢀ, γ) require both Ca2+ and diacylglycerol (DAG)
as cofactors, novel (nPKCs: δ, ε, θ, η) require DAG but are
Ca2+ independent and atypical (aPKCs: ι, λ, ꢁ) do not require
Ca2+ or DAG.3
The nPKC, PKC theta (PKCθ) was first characterized in 1993
and displays the highest homology PKCδ. PKCθ transcripts are
expressed in several cell types, with predominant expression in
T cells and thymocytes.4 PKCθ transduces the T cell receptor
(TCR) stimulation signal to the activation of transcription
factors, inducing interleukin-2 (IL-2) expression and T cell
* To whom correspondence should be addressed. Phone: 845 602-4619.
Fax: 845 602-5561. E-mail: coledc@wyeth.com.
†
Chemical and Screening Sciences, Wyeth Research, Pearl River, New
York.
‡
Inflammation Sciences, Wyeth Research, Cambridge, Massachusetts.
Chemical and Screening Sciences, Wyeth Research, Cambridge
§
Massachusetts.
|
Chemical and Screening Sciences, Wyeth Research, Collegeville,
Pennsylvania.
Current address: AstraZeneca, Waltham, Massachusetts 02451.
a Abbreviations: PKC, protein kinase C; PKCR, protein kinase C alpha;
PKCꢀ, protein kinase C beta; PKCγ, protein kinase C gamma; PKCδ,
protein kinase C delta; PKCε, protein kinase C epsilon; PKCθ, protein kinase
C theta; PKCη, protein kinase C eta; PKCλ, protein kinase C iota; PKCλ,
protein kinase C lambda; PKCꢁ, protein kinase C zeta; HTS, high-throughput
screen; DAG, diacylglycerol; TCR, T cell receptor; EAE, experimental
autoimmune encephalomyelitis; CIA, collagen-induced arthritis; KD, kinase
domain; IL-2, interleukin-2; FITC, fluorescein isothiocyanate; BIM-I,
bisindolemaleimide I; FP, fluorescent polarization; FITC-BIM-I, fluorescein
isothiocyanate-BIM-I; STD, saturation transfer difference; IMAP, kinase
assay from Molecular Devices.
Here we report the identification of a novel series of
4-arylamino-3-pyridinecarbonitriles as selective nPKC, and in
10.1021/jm800214a CCC: $40.75
2008 American Chemical Society
Published on Web 09/11/2008

4-Arylamino-3-Pyridinecarbonitrile as PKCθ Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5959
Figure 2. (A and B) 1H NMR NMR spectra of 4a (80 µM) and PKCθ
(3 µM). (A) 1H NMR spectrum; (B) saturation transfer difference (STD)
spectrum, showing that 4a binds to PKCθ. (C and D) 1H NMR spectrum
of 4a and PKCθ with addition of BIM-I (5 µM). (C) 1H NMR spectrum;
(D) STD spectrum, showing reduction of 4a STD peaks due to
competition by BIM-I.
Figure 1. Lineweaver-Burk plot for inhibition of PKCθ by 4a.
Inhibitor concentration: 0.0 (b), 3.75 (O), 7.5 (1), 15.0 (3) and 30.0
µM (9). Ki ) 4.9 ( 0.3.
Scheme 1a
particular PKCθ inhibitors. Biophysical characterization experi-
ments were used to confirm an ATP competitive mode of
inhibition of the active PKCθ catalytic kinase domain (KD).
Hit-to-lead optimization led to the identification of 4p as an
attractive small molecule lead (MW ) 370, clogP ) 4.4) with
low nM PKCθ inhibitory activity, selectivity over several other
kinases, and good cellular activity in the inhibition of T cell
IL-2 production.
Results and Discussion
High-throughput screening (HTS) of Wyeth’s corporate
compound collection using a Caliper based assay31 with purified
recombinant active PKCθ (KD) (amino acids 362-706)14 led
to the identification of a small molecule inhibitor 4-(3-bromo-
phenylamino)-5-(3,4-dimethoxy-phenyl)-3-pyridinecarboni-
trile 4a with low µM enzyme inhibition activity. Binding
experiments show 4a binds competitively with ATP with a Ki
of 4.9 µM (Figure 1).
a (a) ACN, n-BuLi, -78°C, THF. (b) (i) DMF-DMA, 122°C, 16 h, DMF;
(ii) NH4OAc, EtOH 85°C, 1 h. (c) POCl3 125°C, 1.5 h. (d) ArNH2, Pyr·HCl,
135°C, 8 h, EtOEtOH.
Fluorescence polarization (FP) competition displacement
experiments32 were carried out using a fluorescent probe (FITC-
BIM-I: IC50 ) 150 nM) prepared by coupling fluorescein
isothiocyanate (FITC) and an analogue of bisindolemaleimide
I (BIM-I)33 a known potent ATP competitive PKCθ inhibitor.
Preliminary results show the HTS hit 4a partially displaced the
fluorescent probe binding to PKCθ (data not shown), consistent
with an ATP site interaction. The FP competition displacement
experiment was complicated by interactions observed between
the probe and 4a in the absence of PKCθ.
Hit characterization using NMR binding studies performed
by saturation transfer difference (STD)34 experiment showed a
strong STD signal for 4a, with PKCθ indicating binding of 4a
to the protein (Figure 2b). The interaction signal is almost
completely eliminated by the addition of BIM-I (Figure 2d),
indicating displacement by the more potent BIM-I, further
suggesting that the two inhibitors compete for the ATP binding
pocket.
Figure 3. Model of 4a (carbon atoms in gray) and 4p (carbon atoms
in green) bound to the ATP site of PKCθ.
5-(3,4-dimethoxyphenyl)-4-hydroxy-3-pyridinecarbonitrile 3 in
69% yield. Reaction in refluxing phosphorus oxychloride gave
the corresponding 4-chloro-5-(3,4-dimethoxy-phenyl)-3-pyridi-
necarbonitrile in 91% yield and displacement of the chloride
by a set of anilines by reaction in refluxing 2-ethoxyethanol in
the presence of pyridine hydrochloride gave the target com-
pounds 4a-r, which were assayed for PKCθ inhibitory activity
in a fluorescence polarization kinase assay.35
A computational model of 4a bound to the PKCθ ATP
site based on the staurosporine bound PKCθ cocrystal
structure15 indicates the molecule is locked in place by a
hydrogen bonding interaction between the pyridine nitrogen
and the backbone N-H of the hinge region residue Leu461
(Figure 3). The nitrile functionality forms an additional polar
interaction with the side chain of the gatekeeper residue,
Thr442, and the 3-bromophenyl moiety extends deep into the
An initial array of compounds was prepared to explore the
SAR of the headpiece aryl group following the route in Scheme
1. 3,4-Dimethoxyphenyl acetic acid methyl ester 1 was reacted
with the lithium anion of acetonitrile to give 4-(3,4-dimethox-
yphenyl)-3-oxo-butyro-nitrile 2 in 75% overall yield. Reaction
with DMF-DMA in DMF at 122 °C overnight followed by
reaction with ammonium acetate in refluxing ethanol gave the

5960 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Table 1. PKCθ IC50 Values for 4a-r
Cole et al.
Table 2. Enzyme Inhibition and Binding Data for 4p against PKC
Isoforms
Ar
PKCθ IC50 (µM)a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
3-bromophenyl
phenyl
4.6 ( 1.0
1.6 ( 0.2
3.9 ( 1.9
1.4 ( 0.5
>30
3-chlorophenyl
3-fluorophenyl
3-phenoxyphenyl
3-benzyloxyphenyl
4-chlorophenyl
4-methylphenyl
4-methoxyphenyl
4-phenoxyphenyl
2,4-dichlorophenyl
2,4-dimethylphenyl
2,5-dichlorophenyl
3,4-dichlorophenyl
3,4-dimethoxyphenyl
5-indolyl
PKC type
isoform
IC50 (µM)a
Ki (µM)b
conventional
novel
ꢀ
δ
ꢀ
θ
η
ꢁ
>50
0.35 ( 0.12
2.33 ( 1.19
0.07 ( 0.03
16.35 ( 1.48
>50
1.6 ( 0.2
>30
2.8 ( 0.7
0.9 ( 0.1
0.4 ( 0.0
8.4 ( 3.5
0.08 ( 0.01
0.16 ( 0.01
0.47 ( 0.10
2.4 ( 0.1
2.2 ( 0.4
0.07 ( 0.03
2.2 ( 0.4
2.0 ( 0.5
0.079 ( 0.004
atypical
a IC50 values are the average of three determinations from fluorescence
polarization assay (Molecular Devices). b Ki values are the average of three
independent determinations.
the activity of 4p is likely predominantly due to this additional
H-bonding interaction and not merely filling space in the protein
surface.
6-indolyl
5-indanyl
a IC50 values are the average of three determinations in the PKCθ
fluorescence polarization kinase assay (Molecular Devices).
Compound 4p was found to have good selectivity for PKCθ
inhibition when assayed in a panel of PKC isozyme kinase
assays (Table 2). No inhibition was observed for either the
conventional PKC isoform PKCꢀ or the atypical isoform PKCꢁ
at the highest concentration tested (50 µM). In the novel family,
4p had good selectivity against PKCε and PKCη, 33- and >200-
fold, respectively, and was 5-fold selective for inhibition of
PKCθ versus the very closely related PKCδ. Binding experi-
ments showed 4p binds approximately 20-fold more tightly to
PKCθ than PKCδ.
selectivity pocket. The model suggests the bromine atom may
be slightly too large, forcing the molecule to shift and
somewhat weaken the pyridine nitrogen to hinge the N-H
hydrogen bond. This is consistent with initial analogues
prepared that show the unsubstituted, 3-chloro, and 3-fluoro
phenyl analogues (4b-4d) have up to 3-fold higher inhibitory
activity (Table 1). Other larger substitutions such as phenoxy
(4e) and benzyloxy (4f) are also not tolerated at the
3-position. Moving the halo substitution to the 4-position as
in 4g had little effect on the inhibitory activity. The 4-methyl
analogue 4h was about 3-fold more potent, while the
4-methoxy analogue 4i was about 6-fold more active (PKCθ
IC50 ) 406 nM). Once again the larger phenoxy group (4j)
was not tolerated at the 4-position.
Additionally, compound 4p was selective for the PKC family
with an IC50 of greater than 10 µM against a wide variety of
kinases including serine/threonine kinases (MK2, Plk, Akt, PKA,
Raf, Tpl2, CDK4, p70 S6, IKK, PDK1), tyrosine kinases (Lyn,
Src, Lck, BTK, EGFR, IGFR, KDR), and phosphoinositol-3-
kinase (PI3KR).
Compound 4p was found to display kinetics consistent with
direct competition for ATP binding to PKCθ. The ATP
concentration was varied in a series of inhibition experiments
of PKCθ kinase assay with 4p. A Lineweaver-Burk double-
reciprocal dose-response plot (Figure 4) of reaction rates against
ATP concentration from the inhibition experiments was con-
sistent with 4p acting as a direct competitive inhibitor with ATP
for binding to PKCθ with an apparent Ki ) 0.079 µM, consistent
with the observed IC50 value (PKCθ IC50 ) 0.07 µM).
A small lipophilic pocket adjacent to the ortho-position of
the head-piece aryl group was exploited and gave a significant
increase in inhibitory activity as shown by the 2,4-dichloro and
2,4-dimethyl analogues (4k, 4l). Similar to the weaker meta-
substituted analogues (4a, 4c, and 4d) bis-substituted analogues
that included a meta-group, e.g., the 2,5-dichloro (4m) and the
3,4-disubstituted analogues (4n and 4o) were not well-tolerated
and led to weaker inhibitory activity.
The initial model of 4a suggested that the addition of a
hydrogen bond donating group at the 4-position of the headgroup
phenyl ring could form an additional hydrogen bonding interac-
tion with the side chain of Asp522 (the phenyl group 4-position
is 3.9 Å from the Asp522 OD1). The presence of this N-H bond
acceptor in the region adjacent to the 4-position of the headpiece
was targeted by the 5-aminoindolyl analogue 4p. The model of
4p bound to PKCθ (Figure 3, carbons in green) indicates that
the indole headpiece N-H moiety interacts with Asp522 and
allows the molecule to rotate slightly, potentially increasing the
strength of the pyridine nitrogen to hinge N-H hydrogen bond.
We confirmed the 5-aminoindolyl group potentially facilitating
this H-bonding interaction by observing a significant increase
in potency. The corresponding 6-indolyl and 5-indanyl analogue
4q and 4r were approximately 20-fold less potent, indicating
In contrast to the relatively weaker HTS hit 4a (PKCθ IC50
) 4.6 µM) the more potent inhibitor 4p (PKCθ IC50 ) 0.07
µM) completely displaced the fluorescent probe, consistent with
the tighter binding of the inhibitor to the ATP binding site of
PKCθ (Figure 5). The IC50 from FP displacement (IC50 ) 0.15
µM) was also consistent with the Ki and enzyme inhibition IC50.
Having established selectivity in a panel of kinase enzyme
assays, 4p was next evaluated for inhibition of PKCθ in T cell
activation by stimulating splenocyte derived T cells. Compound
4p was shown to inhibit IL-2 production in anti-CD3 and anti-
CD28 activated T cells from normal wild-type and PKCθ
knockout mice with IC50 values of 0.058 and 0.231 µM,
respectively (Figure 6), The approximately 4-fold selectivity in
comparing T cells deficient in PKCθ, suggests on-target

4-Arylamino-3-Pyridinecarbonitrile as PKCθ Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5961
Figure 6. Inhibition of IL-2 production by 4p. IL-2 levels from anti-
CD3 and anti-CD28 activated T cell-enriched splenocytes from wild-
type (b) and PKCθ knockout (0) mice. Results and error bars represents
an average of three independent determinations, each in triplicate.
nomenex Gemini 100 mm × 21.2 mm or Waters Xterra Prep 5
µM, 100 mm × 19 mm C18 columns and in a 10-90% acetonitrile
in water solvent gradient with 0.02% TFA buffer.
Figure 4. Lineweaver-Burk plot for inhibition of PKCθ by 4p.
Inhibitor concentration: 0.0 (b), 0.5 (O), 1.0 (1), and 2.0 (3). Ki )
0.079 ( 0.004 µM.
4-(3,4-Dimethoxyphenyl)-3-oxo-butyronitrile (2). To a 1.0 L
three-necked round-bottomed flask was added 50 mL of THF and
the reaction mixture was cooled to -78 °C. Butyl lithium (1.6 M,
14.4 mL, 23 mmol) was added dropwise keeping the temperature
below -70 °C. Acetonitrile (1.3 mL, 25 mmol) in 30 mL of THF
was added dropwise to the flask with stirring and cooling. After
2 h of stirring, 3,4-dimethoxyphenyl)acetic acid methyl ester (2.3
g, 11 mmol) was added to the resulting white colloidal mixture in
the flask. The reaction mixture was stirred for a further two hours,
followed by the addition of saturated ammonium chloride solution
(75 mL) at -78 °C. The organic layer was separated, dried with
sodium sulfate, filtered to remove the drying agent, and evaporated
to dryness to give the crude product. This crude product was purified
by flash chromatography on silica gel eluting with 30-70% ethyl
acetate in hexanes to yield a solidifying amber oil, 1.8 g (75%);
Figure 5. Fluorescent polarization (FP) competition displacement of
FITC-BIM-I (IC50 ) 150 nM) by 4p (2). In the control reaction, 4p
and the FP probe are incubated in the absence of PKCθ (9).
1
mp: 78-80 °C. H NMR (400 MHz, DMSO-d6) δ 6.89 (d, J )
8.0 Hz, 1H), 6.79 (d, J ) 2.4 Hz, 1H), 6.70 (dd, J ) 8.0, 2.4 Hz,
1H), 4.07 (s, 2H), 3.75 (s, 2H), 3.73 (s, 3H), 3.72 (s, 3H). HRMS
m/z 242.07821, calcd [M + Na]+ 242.07876, rel Int 100%, error
-0.55 mAmu. Anal. Calcd for C12H13NO3: C, 65.74; H, 5.98; N,
6.39. Found: C, 65.77; H, 6.25; N, 6.36.
inhibitory activity relative to inhibition of kinases other than
PKCθ which contribute to IL-2 production in the T cell signaling
pathway.
5-(3,4-Dimethoxyphenyl)-4-hydroxy-3-pyridinecarbonitrile (3).
To a solution of 4-(3,4-dimethoxyphenyl)-3-oxo-butyronitrile (5.0
g, 23 mmol) in DMF (12 mL) was added DMF-DMA (13.5 mL,
101 mmol) and the solution heated at 122 °C overnight. Concentra-
tion on a rotovap under high vacuum gave an orange-red solid.
This solid was dissolved in EtOH (100 mL), and excess ammonium
acetate was added and the reaction mixture was heated at 85 °C
for 1 h. The reaction mixture was allowed to cool to r.t. for 1 h,
then the solids were collected by filtration and washed with cold
EtOH to give a brown solid (4.1 g, 69%); mp: >260 °C. 1H NMR
(400 MHz, DMSO-d6) δ 12.43 (b, 1H), 8.45 (d, J ) 1.6 Hz, 1H),
7.96 (d, J ) 1.9 Hz, 1H), 7.28 (d, J ) 2.0 Hz, 1H), 7.21 (dd, J )
8.2, 2.1 Hz, 1H), 6.97 (d, J ) 8.2 Hz, 1H), 3.77 (s, 3H), 3.76 (s,
3H). HRMS m/z 257.09152, calcd [M + H]+ 257.06207, rel int
100%, error -0.55 mAmu.
4-Chloro-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile. A
solution of 5-(3,4-dimethoxyphenyl)-4-hydroxy-3-pyridinecarbonitrile
(4 g, 15.7 mmol) in POCl3 (25 mL) was heated at 125 °C for 90 min,
cooled to r.t. and poured into a mixture of ice, 3 N sodium hydroxide,
and ethyl acetate. The resulting mixture was stirred and the layers
separated. The organic layer was dried over MgSO4, filtered, and
concentrated to give 3.9 g (91% yield) of a brown solid, which was
used directly in the next step; mp: 160-162 °C. 1H NMR (400 MHz,
DMSO-d6) δ 9.07 (s, 1H), 8.87 (s, 1H), 7.15-7.09 (m, 3H), 3.82 (s,
3H), 3.79 (s, 3H). HRMS m/z 275.05871, calcd [M + H]+ 275.05819,
rel int 4%, error 0.52 mAmu. Anal. Calcd for C14H11ClN2O2: C, 61.21;
H, 4.04; N, 10.20. Found: C, 61.00; H, 4.34; N, 10.11.
Conclusion
Using a HTS Caliper kinase assay we identified a small lead
like inhibitor of PKCθ. Initial hit-to-lead optimization led to
the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-
ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCθ inhibitor
with excellent selectivity over several unrelated kinases, as well
as conventional and novel PKCs and modest selectivity over
the closely related PKCδ. Future work will describe the
continued optimization and in vivo efficacy studies of this very
promising series of PKCθ inhibitors.
Experimental Methods
NMR spectra were recorded on a Bruker Avance 300 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 b ) broad. The mass spectral data were
determined on an Agilent 1100 series LC/MSD and HRMS was
carried out on a Bruker APEX II 9.4 T mass spectrometer. Manual
silica gel flash chromatrography was performed using bulk Silica
Gel 60 (230-400 mesh) from EMD Chemicals, Inc. Automated
flash chromatography was carried out on a Teledyne Isco, Inc.
CombiFlash Companion using RediSep silica gel cartridges. Reverse
phase HPLC purifications were performed on a Gilson preparative
HPLC system controlled by Unipoint software using either Phe-

5962 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Cole et al.
Table 3. HPLC Purity and HRMS Data for 4a-r
compd A r.t. (min)a A purity (%)a B r.t. (min)b B purity (%)b
formula
calcd [M + H]+ found [M + H]+ rel int error (mAmu)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
2.65
2.16
2.06
2.43
2.91
2.91
2.55
2.29
2.07
2.82
2.84
2.34
2.89
2.89
1.97
1.96
2.09
2.51
100
97.9
2.59
2.43
2.59
2.50
2.90
2.93
2.73
2.73
2.61
3.04
2.92
2.85
2.89
2.99
2.92
2.71
2.71
3.00
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
C20H16Br1N3O2
C20H17N3O2
C20H16Cl1N3O2
C20H16F1N3O2
C26H21N3O3
C27H23N3O3
C20H16Cl1N3O2
C21H19N3O2
C21H19N3O3
C26H21N3O3
C20H15Cl2N3O2
C22H21N3O2
C20H15Cl2N3O2
C20H15Cl2N3O2
C22H21N3O4
C22H18N4O2
C22H18N4O2
C23H21N3O2
410.04987
332.13936
366.10038
350.12993
424.16557
438.18122
366.10038
346.15501
362.14992
424.16557
400.06141
360.17066
400.06141
400.06141
392.16049
371.15026
371.15026
372.17066
410.04987
332.13998
366.10032
350.13021
424.16494
438.18139
366.10109
346.15515
362.15036
424.16635
400.0612
360.17096
400.06099
400.06236
392.16076
371.15104
371.15064
372.17115
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.01
0.62
-0.06
0.28
-0.63
0.17
0.71
0.14
0.44
0.78
-0.21
0.3
-0.42
0.95
0.27
0.78
0.38
100
100
100
100
100
97.1
100
100
96.3
100
100
100
100
100
98.0
100
0.49
a HPLC method A: solvent gradient ) 90% A @ 0 min, 10% A @ 3.5 min, 10% A @ 5 min; solvent A ) 0.1% formic acid in water; solvent B ) 0.1%
formic acid in ACN; flow rate 1 mL/min; column ) Phenomenex, Gemini NX C18 30 mm × 2.1 mm, 3 µm particle size; temperature 50 °C. b HPLC
method B: solvent gradient 90% A @ 0 min, 10% A @ 3.5 min, 10% A @ 5 min; solvent A ) 0.1% NH4OH in water; solvent B ) 0.1% NH4OH in ACN;
flow rate 1 mL/min; column ) Waters Xterra C18, 30 mm × 2.1 mm, 3.5 µm particle size; temperature 50 °C.
5-(3,4-Dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecar-
bonitrile (4p). A mixture of 4-chloro-5-(3,4-dimethoxyphenyl)-3-
pyridinecarbonitrile (824 mg, 3 mmol), 5-aminoindole (396 mg, 3
mmol), and pyridine hydrochloride (345 mg, 3 mmol) in 2-ethoxy-
ethanol (25 mL) was heated at reflux for 8 h, cooled to room
temperature, and concentrated. The residue was purified by flash silica
gel column chromatography eluting with 0-25% MeOH in dichlo-
romethane to give 977 mg (88% yield) as a yellow-brown oil, which
was triturated with MeOH/ethyl ether to give 525 mg (47%) of 5-(3,4-
dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile as a
yellow-brown solid; mp: 170 °C (decompose). 1H NMR (400 MHz,
DMSO-d6) δ 11.19 (b, 1H), 8.96 (b, 1H), 8.60 (s, 1H), 8.22 (s, 1H),
7.41 (d, J ) 1.5 Hz, 1H), 7.38-7.34 (m, 2H), 7.10-7.03 (m, 3H),
6.96 (dd, J ) 8.2, 2.1 Hz, 1H), 6.41 (t, J ) 2.1 Hz, 1H), 3.78 (s, 3H),
3.77 (s, 3H). See Table 3 for HPLC purity and HRMS data.
4-Aryl-5-(3,4-Dimethoxyphenyl)-nicotinonitrile (4a-r). Using
the appropriate aniline analogues, 4a-r were prepared following
the procedure described for 4p. Compounds were purified by RP-
HPLC.
Enzyme Kinetics Assays. Adenosine 5′-triphosphate disodium salt
hydrate (ATP), adenosine 5′-(3-trhitriphosphate) tetralithium salt
(ATPγS), synthetic polymer of sucrose (Ficoll-400), sucrose, adenosine
diphosphate sodium salt (ADP), phosphoenolpyruvate (PEP), NADH,
pyruvate kinase (PK), lactate dehydrogenase (LDH), adenosine 5′-
(ꢀ,γ-imido)triphosphate tetralithium salt (AMPPNP), acetonitrile, DL-
dithiothreitol (DTT), and the buffer HEPES were purchased from
Sigma Chemical Co. (St. Louis, MO). Peptide substrates, inhibitors,
and phosphorylated substrate peptides were from AnaSpec (San Jose,
CA), SynPep (Dublin, CA), or Open Biosystems (Huntsville, AL).
The enzymatic activity was determined at 25 °C using the coupled
PK/LDH assay, at 340 nm on a Molecular Devices plate reader. The
standard reaction, except where indicated, was carried out in a final
volume of 80 µL, in 25 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM
DTT, 0.008% Triton X-100, 100 mM NaCl, 20 units of PK, 30 units
of LDH, 0.25 mM NADH, 2 mM PEP, and the PKCθ KD
(0.156-0.312 µg/mL). Ki values calculated according to the formula
V ) Vmax/(1 + (Km/S)(1 + I/Ki)).
BIM-I) was prepared by coupling fluorescein isothiocyanate (FITC)
with 3-[1-(2-aminoethyl)-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-
2,5-dione. A control reaction mixture contained 50 nM fluorescent
probe and various concentrations of the inhibitor. The reactions were
incubated for 5-30 min at room temperature before measurement.
NMR Binding. The STD experiment34 was performed on a 600
MHz Bruker Avance equipped with a cryoprobe at 25 °C, in 20 mM
HEPES, pH ) 7.5, 5 mM MgCl2, 5 mM DL-dithiothreitol (DTT), 100
mM NaCl, and 5% DMSO prepared in D2O. A 50 ms Gaussian pulse
was used for selective excitation at the on and off resonance frequencies
of the 0.7 ppm and -4 ppm, respectively, with a 1 ms delay in between
the pulses for a 2 s excitation time. A presaturation pulse was used to
minimize the residual water signal.
PKCθ IMAP Kinase Assay. All IC50s were measured by
employing a modified IMAP protocol from Molecular Devices. The
reaction buffer (R7209), detection reagent (R7284), and binding buffer
(R7282), as well as the peptide substrate 5-FAM-RFARKGSL-
RQKNV-OH (RP7032), were purchased from Molecular Devices. The
kinase reaction was carried out in a final volume of 20 µL reaction
buffer in a Corning Costar 384 well plate (Corning Costar 3710)
containing compound, 1.0 nM full length human PKCθ (Panvera
P2996). Three mM DL-dithiothreitol (DTT), 6 µM ATP, 100 nM of
peptide, and 5% DMSO with an incubation time of 30 min. Then 50
µL detection reagents in binding buffer with dilution ratio of 1:800
was added to the reaction mixture and the fluorescence polarization
was measured on an Envision 2100 (PerkinElmer Life Sciences).
PKC Isoform IMAP Kinase Assay. The same protocol as for
PKCθ was also used in measuring compound IC50s for PKC isoforms
including, PKCꢀ, PKCδ, PKCε, PKCη, and PKCꢁ (Panvera) using
modified enzyme concentrations and incubation times.
T Cell IL2 Production Assay. T cells were enriched from spleens
of 8-12 week old female PKCθ knockout mice or wild-type C57BL/6
mice (Taconic, Germantown, NY), using T cell enrichment columns
(R&D Systems, Minneapolis, MN). The cells were resuspended in
RPMI-1640 medium (Sigma) containing 10% fetal calf serium (FCS),
penicillin/streptomycin, glutamine, nonessential amino acids, HEPES,
sodium pyruvate, and BME vitamin solution (Sigma), and plated at 2
× 106/mL in 96-well flat-bottomed plates coated with 20 mg/mL anti-
CD3 and 4 mg/mL anti-CD28 (R&D Systems Inc., Minneapolis, MN),
with a titration of the indicated compound or DMSO control. After
20-24 h incubation at 37 °C in 5% CO2, supernatants were harvested
and IL-2 concentration was determined by ELISA (Invitrogen,
Carlsbad, CA). The limit of assay sensitivity was 3.9 pg/mL.
Molecular Modeling. The X-ray structure of a compound with
the 4-arylamino-3-pyridinecarbonitrile scaffold bound to phosphoi-
nositide-dependent kinase-1 (PDK1) was obtained through soaking
experiments (unpublished data). Backbone protein atoms from this
FP Competition Displacement. FP competition displacement
experiment was measured on a Molecular Devices Analyst AD
instrument. The 20 µL reactions were carried out in a 96-well black
LJL low-volume plate in a buffer of 20 mM MOPS (Sigma), pH 7.0,
10 mM MgCl2, 0.005% Brij-35 (Sigma), 1.4 mM ꢀ-mercaptoethanol.
The competition displacement reaction mixture contained 50 nM
fluorescent probe, 125 nM PKCθ, and various concentrations of the
inhibitor. The fluorescent probe N-(3′,6′-dihydroxy-3-oxo-3H-spiro[2-
benzofuran-1,9′-xanthen]-5-yl)-N′-(2-{3-[4-(1H-indol-3-yl)-2,5-dioxo-
2,5-dihydro-1H-pyrrol-3-yl]-1H-indol-1- yl}ethyl)thiourea (FITC-

4-Arylamino-3-Pyridinecarbonitrile as PKCθ Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5963
PDK1 structure and a PKCθ-staurosporine costructure15 were super-
imposed to determine the initial placement of the pyridinecarbonitrile
scaffold in the PKCθ ATP-binding site. As such, the models of
compounds bound to PKCθ that are discussed in this manuscript were
generated based on manual building followed by gradual minimization
using the program CHARMm36 and the all-atom force field.37 Figure
3 was created in Maestro (Schrodinger, Inc., New York, NY, 2007).
(17) Yaney, G. C.; Fairbanks, J. M.; Deeney, J. T.; Korchak, H. M.;
Tornheim, K.; Corkey, B. E. Potentiation of insulin secretion by
phorbol esters is mediated by PKC-R and nPKC isoforms. Am. J.
Physiol. 2002, 283, E880-E888.
(18) Kotsonis, P.; Funk, L.; Prountzos, C.; Iannazzo, L.; Majewski, H.
Differential abilities of phorbol esters in inducing protein kinase C
(PKC) down-regulation in noradrenergic neurones. Br. J. Pharmacol.
2001, 132, 489–499.
(19) Lee, H.-W.; Smith, L.; Pettit, G. R.; Smith, J. B. Bryostatin 1 and
phorbol ester down-modulate protein kinase C-R and -ε via the
ubiquitin/proteasome pathway in human fibroblasts. Mol. Pharmacol.
1997, 51, 439–447.
(20) Kortmansky, J.; Schwartz, G. K. Bryostatin-1: A Novel PKC Inhibitor
in Clinical Development. Cancer InVest. 2003, 21, 924–936.
(21) Irie, K.; Ohigashi, H. Synthesis and functional analysis of the cysteine-
rich domains of protein kinase C (PKC) for development of new
medicinal leads with PKC isozyme and C1 domain selectivity. Yuki
Gosei Kagaku Kyokaishi 2002, 60, 563–572.
Acknowledgment. We thank James LaRocque and Mariya
Gazumiyan for HTS assay, Xidong Feng for HRMS data, Karl
Malakian, Laura Lin, Ron Kriz and Mark Stahl for PKCθ protein
expression and purification.
Supporting Information Available: HPLC chromatographic
data and NMR spectra for 4a-4r. This material is available free
of charge via the Internet at http://pubs.acs.org.
(22) Choi, Y.; Pu, Y.; Peach, M. L.; Kang, J. H.; Lewin, N. E.; Sigano,
D. M.; Garfield, S. H.; Blumberg, P. M.; Marquez, V. E. Conforma-
tionally constrained analogues of diacylglycerol (DAG). 28. DAG-
dioxolanones reveal a new additional interaction site in the C1b domain
of PKC delta. J. Med. Chem. 2007, 50, 3465–3481.
(23) Goekjian, P. G.; Jirousek, M. R. Protein kinase C inhibitors as novel
anticancer drugs. Expert Opin. InVestig. Drugs 2001, 10, 2117–2140.
(24) Fabbro, D.; Ruetz, S.; Bodis, S.; Pruschy, M.; Csermak, K.; Man, A.;
Campochiaro, P.; Wood, J.; O’Reilly, T.; Meyer, T. PKC412: a protein
kinase inhibitor with a broad therapeutic potential. Anti-Cancer Drug
Des. 2000, 15, 17–28.
(25) Mizuno, K.; Noda, K.; Ueda, Y.; Hanaki, H.; Saido, T. C.; Ikuta, T.;
Kuroki, T.; Tamaoki, T.; Hirai, S.; Osada, S.; et al. UCN-01, an anti-
tumor drug, is a selective inhibitor of the conventional PKC subfamily.
FEBS Lett. 1995, 359, 259–261.
(26) Mizuno, K.; Saido, T. C.; Ohno, S.; Tamaoki, T.; Suzuki, K.
Staurosporine-related compounds, K252a and UCN-01, inhibit both
cPKC and nPKC. FEBS Lett. 1993, 330, 114–6.
(27) McGill, J. B.; King, G. L.; Berg, P. H.; Price, K. L.; Kles, K. A.;
Bastyr, E. J.; Hyslop, D. L. Clinical safety of the selective PKC-beta
inhibitor, ruboxistaurin. Expert Opin. Drug Saf. 2006, 5, 835–845.
(28) Jirousek, M. R.; Gillig, J. R.; Gonzalez, C. M.; Heath, W. F.;
McDonald, J. H.; Neel, D. A.; Rito, C. J.; Singh, U.; Stramm, L. E.;
Melikian-Badalian, A.; Baevsky, M.; Ballas, L. M.; Hall, S. E.;
Winneroski, L. L.; Faul, M. M. (S)-13-[(Dimethylamino)methyl]-
10,11,14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyr-
rolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione (LY333531)
and Related Analogs: Isoenzyme Selective Inhibitors of Protein Kinase
Cꢀ. J. Med. Chem. 1996, 39, 2664–2671.
References
(1) Newton, A. C. Protein kinase C: structural and spatial regulation by
phosphorylation, cofactors, and macromolecular interactions. Chem.
ReV. 2001, 101, 2353–2364.
(2) Miyamoto, A.; Nakayama, K.; Imaki, H.; Hirose, S.; Jiang, Y.; Abe,
M.; Tsukiyama, T.; Nagahama, H.; Ohno, S.; Hatakeyama, S.;
Nakayama, K. I. Increased proliferation of B cells and auto-immunity
in mice lacking protein kinase C delta. Nature 2002, 416, 865–869.
(3) Hug, H.; Sarre, T. F. Protein kinase C isoenzymes: divergence in signal
transduction. Biochem. J. 1993, 291, 329–343.
(4) Baier, G.; Telford, D.; Giampa, L.; Coggeshall, K. M.; Baier-Bitterlich,
G.; Isakov, N.; Altman, A. Molecular cloning and characterization of
PKC theta, a novel member of the protein kinase C (PKC) gene family
expressed predominantly in hematopoietic cells. J. Biol. Chem. 1993,
268, 4997–5004.
(5) Pfeifhofer, C.; Kofler, K.; Gruber, T.; Tabrizi, N. G.; Lutz, C.; Maly,
K.; Leitges, M.; Baier, G. Protein kinase C theta affects Ca+2
mobilization and NFAT cell activation in primary mouse T cells. J.
Exp. Med. 2003, 197, 1525–1535.
(6) Berg-Brown, N. N.; Gronski, M. A.; Jones, R. G.; Elford, A. R.;
Deenick, E. K.; Odermatt, B.; Littman, D. R.; Ohashi, P. S. PKCtheta
signals activation versus tolerance in vivo. J. Exp. Med. 2004, 199,
743–52.
(7) Marsland, B. J.; Soos, T. J.; Spath, G.; Littman, D. R.; Kopf, M. Protein
kinase C theta is critical for the development of in vivo T helper (Th)2
cell but not Th1 cell responses. J. Exp. Med. 2004, 200, 181–189.
(8) Salek-Ardakani, S.; So, T.; Halteman, B. S.; Altman, A.; Croft, M.
Differential regulation of Th2 and Th1 lung inflammatory responses
by protein kinase C theta. J. Immunol. 2004, 173, 6440–6447.
(9) Salek-Ardakani, S.; So, T.; Halteman, B. S.; Altman, A.; Croft, M.
Protein kinase C theta controls Th1 cells in experimental autoimmune
encephalomyelitis. J. Immunol. 2005, 175, 7635–7641.
(10) Tan, S. L.; Zhao, J.; Bi, C.; Chen, X. C.; Hepburn, D. L.; Wang, J.;
Sedgwick, J. D.; Chintalacharuvu, S. R.; Na, S. Resistance to
experimental autoimmune encephalomyelitis and impaired IL-17
production in protein kinase C theta-deficient mice. J. Immunol. 2006,
176, 2872–2879.
(11) Giannoni, F.; Lyon, A. B.; Wareing, M. D.; Dias, P. B.; Sarawar,
S. R. Protein kinase C theta is not essential for T-cell-mediated
clearance of murine gammaherpesvirus 68. J. Virol. 2005, 79, 6808–
6813.
(12) Healy, A. M.; Izmailova, E.; Fitzgerald, M.; Walker, R.; Hattersley,
M.; Silva, M.; Siebert, E.; Terkelsen, J.; Picarella, D.; Pickard, M. D.;
LeClair, B.; Chandra, S.; Jaffee, B. PKC-theta-deficient mice are
protected from Th1-dependent antigen-induced arthritis. J. Immunol.
2006, 177, 1886–1893.
(29) Mackay, H. J.; Twelves, C. J. Targeting the protein kinase C family:
are we there yet? Nat. ReV. Cancer 2007, 7, 554–562.
(30) Cywin, C. L.; Dahmann, G.; Prokopowicz, A. S.; Young, E. R. R.;
Magolda, R. L.; Cardozo, M. G.; Cogan, D. A.; 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, K. M.; White, D. M.; Woska, J. R.; Brown, M. L.
Discovery of potent and selective PKCθ inhibitors. Bioorg. Med.
Chem. Lett. 2007, 17, 225–230.
(31) Caliper Life Sciences.
(32) Huang, X. Equilibrium competition binding assay: inhibition mechanism
from a single dose response. J. Theor. Biol. 2003, 225, 369–376.
(33) Bisindolylmaleimide I. 2-[1-(3-Dimethylaminopropy)-1H-indol-3-yl]-
3-(1H-indol-3-yl)-maleimide.
(34) Mayer, M.; Meyer, B. Characterization of ligand binding by saturation
transfer difference NMR spectroscopy. Angew. Chem., Int. Ed. 1999,
38, 1784–1788.
(13) Chaudhary, D.; Kasaian, M. PKCtheta: A potential therapeutic target
for T-cell-mediated diseases. Curr. Opin. InVestig. Drugs 2006, 7, 432–
437.
(14) Czerwinski, R.; Aulabaugh, A.; Greco, R. M.; Olland, S.; Malakian,
K.; Wolfrom, S.; Lin, L.; Kriz, R.; Stahl, M.; Huang, Y.; Liu, L.;
Chaudhary, D. Characterization of protein kinase C theta activation
loop autophosphorylation and the kinase domain catalytic mechanism.
Biochemistry 2005, 44, 9563–9573.
(15) Xu, Z. B.; Chaudhary, D.; Olland, S.; Wolfrom, S.; Czerwinski, R.;
Malakian, K.; Lin, L.; Stahl, M. L.; Joseph.McCarthy, D.; Benander,
C.; Fitz, L.; Greco, R.; Somers, W. S.; Mosyak, L. Catalytic domain
crystal structure of protein kinase C-theta (PKCtheta). J. Biol. Chem.
2004, 279, 50401–50409.
(16) Mecklenbrauker, I.; Saijo, K.; Zheng, N. Y.; Leitges, M.; Tarakhovsky,
A. Protein kinase C delta controls self-antigen-induced B-cell tolerance.
Nature 2002, 416, 860–865.
(35) Molecular Devices. http://www.moleculardevices.com/pages/reagents/
imap_intro.html.
(36) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.;
Swaminathan, S.; Karplus, M. CHARMM: a program for macromo-
lecular energy, minimization, and dynamics calculations. J. Comput.
Chem. 1983, 4, 187–217.
(37) MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L.;
Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.;
Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos,
C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.,
III; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.;
Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-
Atom Empirical Potential for Molecular Modeling and Dynamics
Studies of Proteins. J. Phys. Chem. B 1998, 102, 3586–3616.
JM800214A