Synthesis and PKCθ inhibitory activity of a series of 4-indolylamino-5-phenyl-3-pyridinecarbonitriles

Article abstract of DOI:10.1016/j.bmcl.2009.03.053

A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCθ were synthesized as potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and variations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the discovery of compound 12d, which had an IC₅₀ value of 18 nM for the inhibition of PKCθ.

Full text of DOI:10.1016/j.bmcl.2009.03.053

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2461–2463
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and PKCh inhibitory activity of a series
of 4-indolylamino-5-phenyl-3-pyridinecarbonitriles
a
a
a
a
a
Russell G. Dushin a, , Thomas Nittoli , Charles Ingalls , Diane H. Boschelli , Derek C. Cole , Allan Wissner ,
*
Julie Lee b, Xiaoke Yang b, Paul Morgan b, Agnes Brennan b, Divya Chaudhary b
a Wyeth Research, Chemical Sciences, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Wyeth Research, Inflammation, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCh were synthesized as
potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and vari-
ations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the
discovery of compound 12d, which had an IC50 value of 18 nM for the inhibition of PKCh.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 17 February 2009
Revised 11 March 2009
Accepted 12 March 2009
Available online 18 March 2009
Keywords:
PKC
4-Amino-3-pyridinecarbonitrile
Inflammation
Kinase
The protein kinase Cs (PKCs) comprise a group of serine-threo-
nine kinases that share sequence and structural homology but dif-
fer in their activation requirements and tissue distribution.1
N
H
MeO
MeO
HN
N
CN
Conventional PKCs (
glycerol (DAG), while novel isoforms (d,
a
, b, and
c
) are regulated by calcium and diacyl
, and h) are regulated
e, g
1a: 5-indolyl
1b: 6-indolyl
1c: 4-indolyl
by DAG, and atypical isoforms (f and k) are insensitive to either
calcium or DAG.
PKCh, a novel isoform, is primarily expressed in T-cells and thy-
mocytes, and plays an important role in the activation and survival
of T-cells by mediating T-cell receptor (TCR) signalling.2,3 PKCh-
deficient or knocked out (KO) mice have demonstrated signifi-
cantly reduced responses in T-cell mediated disease models of
arthritis,4 multiple sclerosis,5,6 inflammatory bowel disease,7 and
asthma,8,9 thus indicating the potential value of a PKCh inhibitor
for the treatment of these diseases.
Recently, a number of small molecules have been reported to be
ATP-competitive inhibitors of PKCh, including 2,4-diaminopyrimi-
dines,10 4-arylamino-pyridine-3-carbonitriles11 (e.g., 1a–b, Fig. 1),
and related 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitr-
iles12–14 Herein we report on the synthesis and biological activities
of an expanded series of 4-indolylamino-5-phenyl-3-pyridinecar-
bonitriles in which we have varied the alkoxy substituents on
the phenyl ring in the context of three isomeric aminoindolyl
‘headpieces’.
Figure 1. Structures of compounds 1a–c.
Compound 1c was prepared according to the procedures de-
scribed for 1a and 1b,11 however efficient syntheses of our initially
targeted molecules (7a–d) required slight modification of our
previously reported route (Scheme 1). Thus, alkylation of the free
hydroxyl groups of substituted phenylacetic acid esters 2a,b with
2-bromoethyl methyl ether provided substituted phenylacetic acid
esters 3a,b, which were uneventfully converted to the correspond-
ing phenyl-3-oxo-butyro-nitriles 4a,b by cryogenic treatment with
the lithium anion of acetonitrile. Conversions of 4a,b to the respec-
tive pyridines by stepwise treatment with DMF–DMA followed by
ammonium acetate, as performed previously,11 only resulted in
low yields of relatively impure products. This was attributed in
part to the increased solubility of the intermediates and final prod-
ucts that in prior instances were collected from the reaction med-
ium simply by filtration. As a reliable alternative to this procedure,
oxo-butyro-nitriles 4a,b were treated with DMF–DMA as before,
but then the resulting crude bis-enamides were reacted with verat-
rylamine in refluxing toluene, thus producing the N-alkylated
* Corresponding author. Tel.: +1 845 602 3430; fax: +1 845 602 5561.
E-mail address: dushinr@wyeth.com (R.G. Dushin).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.053

2462
R. G. Dushin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2461–2463
R
R
O
inhibitor than 1c, compound 7d, in which the para-methoxy sub-
a
c
b
CO2CH3
O
CO2CH3
stituent in 7a was removed, was only 2.5-fold less potent than
compound 1c (IC50 values of 13, 63, and 32 nM for 1c, 7a, and
7d, respectively). Thus, it appears that there is limited tolerance
for steric bulk in the vicinity of the dimethoxy-substituents of
bound 1a–c, but that the losses in activity seen in dialkoxy-substi-
tuted compounds containing larger meta-substituents can be par-
tially offset by removal of the para-substituent. With this
observation in mind we set out to prepare analogs of 7d that con-
tained meta-aminoalkoxy substituents on the 5-phenyl moiety in
the hopes that incorporation of amine functionality might confer
improvements in the physicochemical properties of the series, par-
ticularly solubility, which was generally poor to moderate with
1a–c and 7a–d (data not shown).
Aminoalkoxy containing derivatives 12a–g were prepared as
outlined in Scheme 2 using the same general approach used to pre-
pare 7a–d. Thus, alkylation of the free phenol in 2a with 2-chloro-
ethyl-p-toluenesulfonate provided methyl-[3(2-chloroethoxy)
phenyl]acetate (8), which was converted to the substituted phe-
nyl-3-oxo-butyro-nitrile derivative 9 by cryogenic treatment with
the lithium anion of acetonitrile. Conversion to the functionalized
pyridone 10 was accomplished by treatment of 9 with DMF–
DMA, followed by reaction of the resulting crude bis-enamide with
veratrylamine in refluxing toluene. Protected pyridone 10 was then
converted to the 4-chloropyridine 11 by treatment with phospho-
rous oxychloride and lithium chloride. Application of Buchwald–
Hartwig aminations on 11 using 4-, 5-, or 6-aminoindole gave
the corresponding isomeric aminoindolyl-substituted 5-phenyl-3-
pyridinecarbonitrile intermediates containing the chloroethoxy
substituent on the phenyl ring. Lastly, amine displacements of
the chlorine on the chloroethoxy side chains of these three inter-
mediates by treatment with cyclic secondary amines in refluxing
ethanol gave the desired derivatives 12a–g.
HO
2a: R = H
3a,b
2b: R = OCH3
R
O
O
O
CN
R
O
d
O
O
CN
CN
N
O
OMe
OMe
4a,b
5a,b
R
R
R'
Cl
HN
e
O
CN
O
O
N
N
6a,b
7a: R = OCH3, R' = 4-indolyl
7b: R = OCH3, R' = 5-indolyl
7c: R = OCH3, R' = 6-indolyl
7d: R = H, R' = 4-indolyl
Scheme 1. Reagents and conditions: (a) CH3OCH2CH2Br, Cs2CO3, Bu4N+IÀ, acetone;
(b) CH3CN, n-BuLi, À78 °C, THF; (c) (1) DMF–DMA, reflux, DMF; (2) Veratrylamine,
toluene, reflux; (d) POCl3, LiCl, reflux; (e) 4-, 5-, or 6-aminoindole, Pd2(dba)3,
DAVEPHOS15, K3PO4, DME, reflux.
pyridones 5a,b, which were isolated and purified by silica gel chro-
matography. Treatment of 5a,b with phosphorous oxychloride in
the presence of lithium chloride gave the 4-chloropyridines 6a,b
directly and without the need for a separate nitrogen-deprotection
step. Subsequent couplings of 4-, 5-, or 6-aminoindole with 4-chlo-
ropyridines 6a,b were accomplished under mild conditions by
employing a palladium catalyzed aryl amination to give the tar-
geted compounds 7a–d.
PKCh inhibitory activities for compounds 1c and 7a–c revealed
that the 4-indolylamino-analogs 1c and 7a were considerably
more potent (roughly five-fold) than the corresponding 5-indolyla-
mino compounds, 1a and 7b, which were in turn more potent
(roughly 20- to 30-fold) than the corresponding 6-indolylamino-
compounds, 1b and 7c (Table 1). It is noteworthy that this general
trend was observed previously in the related 4-(indolylamino)thi-
eno[2,3-b]pyridine-5-carbonitriles.12 Additionally, within each iso-
meric indolylamino series, replacement of the meta-methoxy
substituents on the 5-phenyl moiety with meta-methoxyethoxy
substituents uniformly resulted in a three- to five-fold loss of activ-
ity (cf. IC50 values of compounds 1c and 7a, 1a and 7b, 1b and 7c).
However, while 7a was roughly five-fold less potent as a PKCh
As shown in Table 1, all four of the 4-indolylamino-substituted
analogs of 7d bearing aminoalkoxy functionality on the phenyl
substituent (compounds 12a–d) were strong inhibitors of PKCh.
Only compound 12b, with a N-piperidinyl-2-ethoxy group on the
phenyl ring, was less potent than 7d, and even then by only a factor
of two. As was observed previously, the 4-indolylamino-substi-
tuted compounds were uniformly more potent than the corre-
sponding 5-indolylamino-substituted isomers (12a vs 12e, 12c vs
a
c
b
d
CO2CH3
Cl
CO2CH3
CN
HO
O
2a
9
8
Table 1
PKCh IC50 values for compounds 1a–c, 7a–d, and 12a–g16
O
N
R3
R1
Cl
O
HN
O
CN
R2
Cl
CN
O
O
OMe
N
OMe
10
Compound
R1
R2
R3
PKC-h
IC50 (nM)
R'
Cl
N
HN
N
1a
1b
1c
7a
7b
7c
7d
12a
12b
12c
12d
12e
12f
12g
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OCH2CH2OMe
OCH2CH2OMe
OCH2CH2OMe
OCH2CH2OMe
5-Indolyl
6-Indolyl
4-Indolyl
4-Indolyl
5-Indolyl
6-Indolyl
4-Indolyl
4-Indolyl
4-Indolyl
4-Indolyl
4-Indolyl
5-Indolyl
5-Indolyl
6-Indolyl
7011
220011
13
e,f
Cl
CN
R2N
CN
O
11
63
12a: NR2 = pyrrolidine, R' = 4-indolyl
12b: NR2 = piperidine, R' = 4-indolyl
12c: NR2 = morpholine, R' = 4-indolyl
12d: NR2 = 4-Me-piperazine R' = 4-indolyl
12e: NR2 = pyrrolidine, R' = 5-indolyl
12f: NR2 = morpholine, R' = 5-indolyl
12g: NR2 = morpholine, R' = 6-indolyl
320
6300
32
29
60
17
18
190
200
9500
H
H
H
H
H
H
H
OCH2CH2–N-pyrrolidine
OCH2CH2–N-piperidine
OCH2CH2–N-morpholine
OCH2CH2–N-(4-Me-piperazine)
OCH2CH2–N-pyrrolidine
OCH2CH2–N-morpholine
OCH2CH2–N-morpholine
Scheme 2. Reagents and conditions: (a) TsOCH2CH2Cl, Cs2CO3, Bu4N+IÀ, acetone;
(b) CH3CN, n-BuLi, À78 °C, THF; (c) (1) DMF–DMA, reflux, DMF (2) Veratrylamine,
toluene, reflux; (d) POCl3, LiCl, reflux; (e) 4-, 5-, or 6-aminoindole, Pd2(dba)3,
DAVEPHOS15, K3PO4, DME, reflux; (f) R2NH, EtOH, reflux.

R. G. Dushin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2461–2463
2463
12f), and the 5-indolylamino-substituted compound bearing an N-
Acknowledgements
morpholino-2-ethoxyphenyl substituent (compound 12f) was
more potent than its 6-indolylamino isomer (12g).
We thank the Wyeth Chemical Technologies department for
compound characterizations and solubility measurements, and
Drs. John Ellingboe, Janis Upeslacis, and Tarek Mansour for their
support.
Compound 12d, an 18 nM PKCh inhibitor that displayed excel-
lent aqueous solubility (>100
further evaluation against an expanded panel of kinases from the
PKC family. This compound inhibited PKCd, PKC , and PKC , which
lg/mL at pH 7.4), was selected for
e
g
all belong to the novel PKC class, with IC50 values of 37, 39, and
References and notes
940 nM, respectively. In assays measuring inhibition of PKCb, a
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15. The IUPAC name for DAVEPHOS is 2-(2-dicyclohexylphosphanylphenyl)-N,N-
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16. See Ref. 11 for assay protocols. IC50 values represent the mean of at least two
determinations.
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18. Miyamoto, A.; Nakayama, K.; Imaki, H.; Hirose, S.; Jiang, Y.; Abe, M.;
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conventional PKC, 12d was a 2.0
value of >100 against PKCf, an atypical PKC. Thus, although 12d
was only two-fold selective against PKCd and PKC , it displayed
lM inhibitor, and it had an IC50
e
excellent selectivity against members of other classes of PKCs.
Inhibitory activity against PKCd remains a concern, however, due
to the known and undesirable role of PKCd in stimulating B-cell
hyperresponsiveness in mice.17,18
Inhibitory properties of compound 12d were also measured
against the Src family kinases Lyn and Lck as well as against Akt.
Compound 12d was found to be inactive against Lyn and Akt at
the highest concentration tested (IC50 values >100
only moderately active against Lck (IC50 value of 24
l
l
M) and was
M).
Lastly, compound 12d was evaluated in a functional cellular
assay that measures IL-2 production from T-cells derived from
wild-type (WT) and PKCh KO mice following T-cell activation by
anti-CD3 and anti-CD28.11 Compound 12d inhibited IL-2 produc-
tion from WT cells with an IC50 value of 854 nM, but had a mark-
edly reduced effect (IC50 value of >15 lM) on cells derived from
PKCh KO mice. This >20-fold difference in activity is consistent
with a mechanism that involves PKCh selective inhibition.
In summary, we have described the synthesis and biological
evaluation of a series of 4-indolylamino-3-pyridinecarbonitriles
as PKCh inhibitors that expand upon compounds discovered in
our earlier hit-to-lead studies.11 This effort has culminated in the
identification of compound 12d, a potent inhibitor of PKCh that dis-
played modest selectivity over closely related members of the PKC
family of kinases, and has shown activity in a cellular assay that
measures the functional activity of PKCh. The on-going efforts to
further improve the potency, selectivity, and physicochemical
properties of this series of compounds will be reported in due
course.