Optimization of 5-phenyl-3-pyridinecarbonitriles as PKCθ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.126

The key intermediate, 4-chloro-5-iodo-3-pyridinecarbonitrile, allowed for ready optimization of the PKCθ inhibitory activity of a series of 3-pyridinecarbonitriles. Analog 13b with a 4-methylindol-5-ylamino group at C-4 and a 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl group at C-5 had an IC₅₀ value of 7.4 nM for the inhibition of PKCθ.

Full text of DOI:10.1016/j.bmcl.2009.04.126

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3623–3626
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of 5-phenyl-3-pyridinecarbonitriles as PKCh inhibitors
a
a
a
a
a
Diane H. Boschelli a, , Daniel Wang , Amar S. Prashad , Joan Subrath , Biqi Wu , Chuan Niu ,
*
Julie Lee b, Xiaoke Yang b, Agnes Brennan b, Divya Chaudhary b
a Wyeth Research, Chemical Sciences, 401 N. Middletown Road, Pearl River, NY 10965, United States
b Wyeth Research, Inflammation, 200 Cambridge Park Drive, Cambridge, MA 02140, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The key intermediate, 4-chloro-5-iodo-3-pyridinecarbonitrile, allowed for ready optimization of the PKCh
inhibitory activity of a series of 3-pyridinecarbonitriles. Analog 13b with a 4-methylindol-5-ylamino
group at C-4 and a 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl group at C-5 had an IC50 value of
7.4 nM for the inhibition of PKCh.
Received 14 April 2009
Revised 24 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
PKCh
3-Pyridinecarbonitrile
The protein kinase Cs (PKCs) are a family of serine threonine ki-
nases that are subdivided into three classes based on their activa-
of 37 nM. Selectivity for PKCh over PKCd is thought to be desirable
since PKCd KO mice exhibit an increased proliferation of B cells
making them susceptible to autoimmune disease.24,25
tion requirements.1 The classical, or conventional, PKC isoforms,
a,
bIbII and
erol (DAG). The novel isoforms, d,
c
, require the second messengers calcium and diacylglyc-
The SAR studies for the thieno[2,3-b]pyridine-5-carbonitriles,
exemplified by 3, were greatly facilitated by intermediate 4 which
allowedforready variationof both the2 and 4 positionsof thecore.20
While our initial route to the 3-pyridinecarbonitriles allowed for
ready variation of the group at C-4, it required that the group at C-
5 be established early in the synthesis. Therefore, a 3-pyridinecarbo-
nitrile intermediate analogous to 4 was highly desired.
e, g
, and h, require DAG but not
calcium. The atypical isoforms, f and k, do not require either cal-
cium or DAG. The PKC inhibitors currently in clinical trials are all
structurally related to staurosporine. Midostaurin,2 enzastaurin,3
and ruboxistaurin,4 target the classical isoforms, while sotrastau-
rin5,6 targets both the classical and novel isoforms.
PKCh, a novel isoform, is crucial for the activation and survival
of T cells.7–9 Proof-of-concept studies with mice where the PKCh
gene was deleted or ‘knocked out’ (KO) validated that the inhibi-
tion of this kinase could have therapeutic utility in diseases such
as multiple sclerosis,10,11 arthritis,12 asthma,13,14 inflammatory bo-
wel disease,15 and transplant rejection.16,17 Surprisingly, there are
only limited reports in the literature of small molecules designed
to be selective inhibitors of PKCh. Boehringer Ingelheim has dis-
closed a series of 2,4-diaminopyrimidine inhibitors,18,19 and we
previously reported that a thieno[2,3-b]pyridine-5-carbonitrile20,21
and a 3-pyridinecarbonitrile22,23 ring system can be used as tem-
plates for inhibitors of this kinase. Regarding the 3-pyridinecarbo-
nitriles, the 4-indolyl isomer of 1a, namely 1b, was a more potent
inhibitor of PKCh, while the 6-indolylamino isomer, 1c, was much
less active.23 Addition of a water solubilizing group to the meta po-
sition of the C-5 phenyl ring of 1b, led to 2, which inhibited PKCh
activity with an IC50 value of 18 nM.23 PKCd was used as the pri-
mary counter assay, and 2 inhibited this kinase with an IC50 value
NH
NH
4
5
O
O
N
HN
N
6
HN
N
CN
CN
O
N
2
1a: 5-indolyl
1b: 4-indolyl
1c: 6-indolyl
NH
HN
O
Cl
CN
CN
I
S
N
S
N
3
N
4
The route used to prepare this key intermediate is shown in
Scheme 1. 3-Amino-2-butenenitrile was treated with aqueous
hydrochloric acid to give acetoacetonitrile 5. Reaction of 5 with
* Corresponding author. Tel.: +1 845 602 3567; fax: +1 845 602 5561.
E-mail address: bosched@wyeth.com (D.H. Boschelli).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.126

3624
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3623–3626
O
R
X
CN
NH
CN
b
a
CN
O
O
O
Cl
NH2
N
O
H
CN
CN
O
a
5
6
N
10a-g
N
9
c
R
O
Cl
R
NH
I
CN
NH
I
CN
d
O
O
Cl
HN
HN
N
H
7
I
CN
N
I
CN
c
CN
b
8
N
N
N
11a-d
8
10h-k
Scheme 1. Reagents: (a) aq HCl; (b) (1) t-butoxy bis(dimethylamino)methane,
DMF–DMA, (2) NH4OAc; (c) I2, aq NaOH; (d) POCl3.
Scheme 2. Reagents: (a) for 10a: 7-aminoindole, EtOH; for 10b: 5-methylamino-
indole, EtOCH2CH2OH; for 10c: 5-hydroxyindole, K2CO3, CH3CN; for 10d: 5-
indolylboronic acid, (Ph3P)4Pd, DME, aq NaHCO3; for 10e: 5-amino-4-methylindole,
EtOCH2CH2OH; for 10f: 5-amino-4-ethylindole, EtOCH2CH2OH; for 10g: 5-amino-7-
methylindole, EtOCH2CH2OH; (b) for 11a: 5-amino-6-methylindole, EtOH, Et3N; for
11b: 5-amino-4-methoxyindole, EtOH, Et3N; for 11c: 5-amino-4-fluoroindole,
EtOH, Et3N; for 11d: 5-amino-1,4-dimethylindole, EtOH; (c) 3,4-dimethoxyphenyl-
boronic acid, (Ph3P)4Pd, DME, aq NaHCO3.
t-butoxy bis(dimethylamino)methane and the dimethylacetal of
dimethylformamide followed by the addition of ammonium ace-
tate provided the pyridone 6. Iodination with iodine in aqueous so-
dium hydroxide gave 7, with chlorination resulting in the desired
intermediate 8.
The analogs in Table 1 were prepared as shown in Scheme 2,
starting from either 8, or the earlier intermediate 9. Using 1a as
the reference compound, the 7-aminoindolyl isomer 10a was pre-
pared from 9 and 7-aminoindole. This isomer was less potent in
inhibiting PKCh than 1a, having a IC50 value of only 740 nM. Next
the NH group at C-4 of 1a was replaced with NMe, O and a bond,
to provide 10b–d all of which had reduced potency compared to
1a. This SAR is consistent with what was seen in the thieno[2,3-
b]pyridine-5-carbonitriles, where these changes also resulted in
reduced inhibition of PKCh activity.20 Addition of a Me group at
C-4 of the indole ring of 1a resulted in increased activity with
10e having an IC50 value of 6.3 nM for the inhibition of PKCh. This
increase was accompanied by enhanced selectivity over PKCd
(IC50 = 81 nM). This finding, and that the 4-Et analog, 10f, had re-
duced activity, was also observed in the thieno[2,3-b]pyridine-5-
carbonitrile series. Additional substituents on the indol-5-yl group
were then studied.26 While the 7-Me analog 10g had an IC50 value
of only 120 nM, increased potency was seen with the 6-Me analog
10h. Increased selectivity over PKCd was also observed, with 10h
having IC50 values of 10.5 and 280 nM for the inhibition of PKCh
and PKCd, respectively. Good selectivity was also seen with the
4-OMe analog 10i and good PKCh potency was seen with the 4-F
analog 10j. The 1,4-diMe analog, 10k, had greatly reduced activity
illustrating the importance of the proton at N-1 of the indole.
The initial optimization of the substituents on the C-5 phenyl
ring was carried out with a 4-methylindol-5-yl group at C-4. As de-
picted in Scheme 3, 11e was prepared by reaction of 8 with 5-ami-
no-4-methylindole. Treatment of 11e with phenylboronic acid
under Suzuki conditions provided 12a. The three isomers of meth-
oxyphenylboronic acid were then coupled to 11e to provide 12b–
d. As shown in Table 2, the unsubstituted phenyl analog 12a, had
reduced activity compared to the 3,4-dimethoxyphenyl analog
10e. Walking the OMe group around the phenyl ring showed that
the greatest inhibition of PKCh activity was observed with the para
OMe analog 12b. The para position of the phenyl ring was therefore
chosen as the site to add water solubilizing groups.
Table 1
Table 2
PKCh and PKCd inhibitory activity of C-5 3,4-dimethoxyphenyl analogs with various
groups at C-4
PKCh and PKCd inhibitory activity of C-4 4-methylindol-5-ylamino analogs with
various groups on the phenyl ring at C-5
R
NH
NH
R1
O
O
X
HN
CN
CN
R2
R3
N
N
R1
R2
R3
PKCh IC50 PKCd IC50 h/d
Ex
Indole
isomer
X
R
PKCh IC50
PKCd IC50
h/d Ratio
Ex
(nM)27
(nM)27
(nM)27
(nM)27
Ratio
1a
1b
1c
5
4
6
7
5
5
5
5
5
5
5
5
5
5
NH
NH
NH
NH
NMe
O
Bond
NH
NH
NH
NH
NH
NH
NH
H
H
H
H
H
H
H
7022
1323
230023
740
270
1700
1700
6.3
35022
6223
NT23,27
NT
2100
NT
5.0
4.8
NA27
NA
7.8
NA
NA
13
18
NA
27
33
13
10e OMe
12a
12b OMe
OMe
H
H
OMe
H
H
H
H
H
H
H
H
H
6.3
27
5.6
36
81
63
85
210
340
150
51
13
2.3
15
5.8
4.1
7.5
6.9
6.2
2.1
H
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
12c
12d
H
H
OMe 82
13a O–CH2CH2-morpholine
13b O–CH2CH2–N-Me-piperazine
13c O–CH2CH2-pyrrolidine
13d O–CH2CH2–N–(CH2CH2OH)-
piperazine
13e O–CH2CH2–4-pyrrolidin-1-yl-
piperidine
13f O–CH2CH2–NH–CH2CH2NMe2
13g O–CH2CH2CH2–N–Me-piperazine
H
H
H
H
20
7.4
4.5
14
NT
81
4-Me
4-Et
7-Me
6-Me
4-OMe
4-F
28
29
55
980
NT
280
350
69
120
10.5
10.6
5.3
H
H
11
30
2.7
H
H
H
H
5.9
19
18
110
3.1
5.8
1,4-diMe
260
1400
5.4

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3623–3626
3625
Table 3
These analogs were prepared as shown in Scheme 3 where the
corresponding boronic acids were generated in situ from either 1-
bromo-4-(2-chloroethoxy)benzene or 1-bromo-4-(3-chloroprop-
oxy)benzene. Palladium catalyzed coupling of these boronic acids
with 11e gave the intermediate alkyl chloro derivatives which
were treated with amines in DME in the presence of NaI to provide
the desired products 13a–g. For analogs 13a–13f with a 2-ethoxy
linker, there was not a large variation in the IC50 values for the
inhibition of PKCh (4.5–20 nM). There was also not a large variation
in selectivity for PKCh over PKCd with 13a–13c being 6–7.5-fold
selective and 13d–f being 2–3-fold selective. Extension of the eth-
oxy linker of 13b by a methylene group to give 13g, decreased both
the PKCh and PKCd activity 2-fold.
PKCh and PKCd inhibitory activity of C-5 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl
analogs with various groups at C-4
RAr
CN
O
N
HN
N
N
Ex
RAr
PKCh IC50 (nM)27
PKCd IC50 (nM)27
h/d Ratio
13b
14a
14b
14c
4-Me-5-indolyl
4-Indolyl
5-Indolyl
7.4
14
73
51
41
160
130
6.9
2.9
2.2
3.9
6-Me-5-indolyl
33
Keeping the group on the phenyl ring of 13b constant, analogs
were prepared varying the indole at C-4. Key intermediate 8 was
reacted with 4-aminoindole and 5-aminoindole to provide 11f
and 11g (Scheme 4). Conversion of 1-bromo-4-(2-chloroeth-
oxy)benzene to the corresponding boronic acid, Pd catalyzed cou-
pling with 11a, 11f and 11g and displacement of the alkyl chloride
with 1-methylpiperazine gave 14a–c. As shown in Table 3, these
three analogs were all weaker PKCh inhibitors than 13b and were
also less selective over PKCd. The decrease in selectivity observed
with the 6-methylindol-5-yl analog 14c was surprising since in
the C-5 3,4-dimethoxyphenyl series 10h showed 24-fold selectiv-
ity for PKCh over PKCd.
PDGFR and ROCK1, demonstrating the selectivity of 13b for inhibi-
tion of the novel PKCs.
The cellular activity of 13b was evaluated in an assay using
murine T cells stimulated with anti-CD3 and anti-CD28 to induce
IL-2 expression.27 With T cells from WT mice, 13b blocked the pro-
duction of IL-2 with an IC50 value of 160 nM, a 5-fold increase in
activity compared to that of 2 (IC50 = 850 nM23). Reduced activity
was seen in a corresponding assay with T cells isolated from PKCh
KO mice where 13b had an IC50 value of greater than 15 lM. In
pharmaceutical profiling assays, 13b had a moderate permeability
of 0.28 ꢀ 10ꢁ6 cm/s in a PAMPA format, with good solubility at pH
7.4 (67 lg/mL). However, in stability studies with liver microsomes
Analog 13b was profiled against additional PKC family mem-
bers. While 13b only weakly inhibited PKCb (IC50 = 22
l
M) a clas-
from rats, mice, dogs, monkeys and humans, 13b had half-lives of
less than 15 min across the species.
sical isoform, more potent inhibition of PKC and PKC
e
g, two novel
PKCs was observed, with 13b having IC50 values of 54 and 450 nM,
respectively. No inhibition of PKCf, an atypical isoform, was ob-
Via the versatile 3-pyridinecarbonitrile intermediate 8, we are
continuing to develop the SAR of this series, with the goals of
retaining potent activity against PKCh and increasing both the
selectivity against PKCd and the metabolic stability. These efforts
are focusing on variation of the ring at C-5 and the nature of the
water solubilizing group.
served (IC50 >100
lM). Additional kinase profiling of 13b provided
IC50 values of greater than 10
l
M for Lyn, Lck, MK2, p38, IKK,
Acknowledgments
NH
b
NH
R1
R2
We thank the Wyeth Chemical Technology department for
compound characterization and the pharmaceutical profiling re-
sults, the Wyeth Screening Sciences department for the kinase
selectivity results, the Wyeth Discovery Synthetic Chemistry and
Chemical Development departments for the preparation of multi
gram batches of both 8 and 5-amino-4-methylindole and Dr. Tarek
Mansour for his support.
Cl
HN
HN
I
CN
I
CN
CN
a
R3
N
8
N
11e
N
12a-d
c
NH
RR'N-(CH2)n-O
HN
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Scheme 3. Reagents: (a) 5-amino-4-methylindole, EtOH; (b) phenylboronic acids,
(Ph3P)4Pd, DME, aq.NaHCO3; (c) for 13a–f: (1) 1-bromo-4-(2-chloroethoxy)ben-
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N
N
N
N
14a-c
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determinations. NT: Compound was not tested; NA: Selectivity ratio is not
available.