Synthesis and PKCθ inhibitory activity of a series of 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitriles

Article abstract of DOI:10.1016/j.bmcl.2008.03.077

The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity against PKCθ. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC₅₀ value of 7.5 nM for the inhibition of PKCθ.

Full text of DOI:10.1016/j.bmcl.2008.03.077

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2850–2853
Synthesis and PKCh inhibitory activity of a series
of 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitriles
Diane H. Boschelli,a,* Biqi Wu,a Ana Carolina Barrios Sosa,a, Joan Chen,a
Magda Asselin,a Derek C. Cole,a Julie Lee,b Xiaoke Yangb and Divya Chaudharyb
aWyeth Research, Chemical and Screening Sciences, 401 N. Middletown Road, Pearl River, NY 10965, USA
bWyeth Research, Inflammation, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
Received 28 February 2008; revised 24 March 2008; accepted 31 March 2008
Available online 8 April 2008
Abstract—The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity
against PKCh. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC50 value of 7.5 nM for the inhibition of
PKCh.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The protein kinase Cs (PKCs) are a family of serine
NH
NH
threonine kinases that share sequence and structural
homology and vary in their activation requirements
and tissue expression.1 Biochemical regulation of the
classical PKC isoforms, a, b, and c, requires the second
messengers, calcium and diacylglycerol. The novel iso-
forms, d, e, g, and h, do not require calcium and the
atypical isoforms, f and k, do not require either calcium
or diacylglycerol. The three PKC inhibitors currently in
late stage clinical trials, midostaurin,2 enzastaurin,3 and
ruboxistaurin,4 all target the classical PKCs.
O
O
HN
N
HN
N
CN
CN
S
1
2
Cl
Cl
PKCh, a novel isoform, was first characterized in 1993
and plays a key role in the activation and survival of
T-cells.5,6 Studies with mice that have the PKCh gene
deleted or knocked out (KO) showed these animals to
be resistant to the development of several T-cell medi-
ated diseases including multiple sclerosis,7,8 arthritis,9
and asthma.10,11 Therefore, the inhibition of PKCh
could be of therapeutic benefit in a variety of disease
states. Interestingly, studies with PKCd KO mice re-
vealed that the inhibition of this kinase results in the in-
creased proliferation of B-cells making these animals
susceptible to autoimmune disease.12,13
CN
CN
a
I
S
N
4
S
c
N
3
b
HN
NH
HN
N
2: 5-indole
5: 4-indole
6: 6-indole
HN
N
CN
CN
S
S
7
Scheme 1. Reagents: (a) phenylboronic acid, (Ph3P)4Pd, DME, aq
NaHCO3; (b) for 2: 5-aminoindole, EtOCH2CH2OH; for 5: 4-
aminoindole, Pd2(dba)3, K3PO4, DME; for 6: 6-aminoindole, EtOH;
(c) (1) 7-aminoindole, EtOH (2) phenylboronic acid, (Ph3P)4Pd, DME,
aq NaHCO3.
Keywords: PKCh; Thieno[2,3-b]pyridine-5-carbonitrile.
*
Corresponding author. Tel.: +1 845 602 3567; fax: +1 845 602
5561; e-mail: bosched@wyeth.com
Present address: Roche Carolina, Florence, SC, USA.
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.077

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2850–2853
2851
2,4-Diaminopyrimidines14,15 and pyridine-3-carbonitr-
iles16 have been reported to be templates for PKCh
inhibitors. The pyridine-3-carbonitrile 1 shares common
structural features with the thieno[2,3-b]pyridine-5-car-
bonitriles, compounds previously studied as Src kinase
inhibitors.17 To ascertain if this bicyclic core could also
provide inhibitors of PKCh, compound 2 was prepared
as shown in Scheme 1. The reaction of 317 with phenyl-
boronic acid under Suzuki conditions provided 4. The
subsequent treatment of 4 with 5-aminoindole gave 2,
which had an IC50 value of 460 nM for the inhibition
of PKCh activity. Since this series originated from a
Src kinase program, 2 was tested for activity against
Lyn, a member of the Src family of kinases. Activity
against Lyn is undesirable due to the finding that Lyn
KO mice develop autoimmune disease as a result of a
hyperresponsive B-cell phenotype.18,19 Fortunately, 2
had weak activity against Lyn (IC50 = 33 lM). How-
ever, as shown in Table 1, 2 inhibited PKCd with an
IC50 value of 2.0 lM. Due to the less than fivefold selec-
tivity for PKCh over PKCd and the potential deleterious
effect of inhibiting this isoform, all compounds with IC50
values of less than 500 nM for PKCh were assayed for
PKCd activity.
PKCd. Of the four indolyl isomers, 2 had the best com-
bination of potency and selectivity, and was therefore
chosen for further SAR study.
To investigate the effect of variation of the linker be-
tween the 5-indoyl headpiece and the thieno[2,3-b]pyri-
dine-5-carbonitrile core, key intermediate 4 was treated
with 5-methylaminoindole,20 5-hydroxyindole, indole-
5-methanamine, and indole-5-carboxamide, to provide
8–11. As shown in Table 1, all these compounds had re-
duced PKCh inhibitory activity compared to 2.
To determine if the PKCh inhibitory activity of 2 could
be increased by the presence of a substituent on the in-
dole ring, 4 was reacted with various 5-aminoindoles
containing a methyl group at C-1, 2, 3, or 4 to provide
12–15 (Scheme 2). 5-Amino-4-methylindole was pre-
pared by the previously reported route.21 5-Amino-3-
methylindole was obtained by the hydrogenation of
3-methyl-5-nitroindole.22 As shown in Table 1, the
4-methyl isomer 15 had increased PKCh inhibitory
activity compared to 2, but also had a corresponding in-
crease in PKCd activity. Lyn inhibition also increased,
with 15 having an IC50 value of 3.4 lM against this ki-
nase. Interestingly, the 4-ethyl analog 16 was much less
active than 15 against both PKC isoforms.23
This route used to prepare 2 was used for the prepara-
tion of 5 and 6, the 4 and 6-indolyl isomers of 2 (Scheme
1). An alternate route was used to prepare 7, the 7-indo-
lyl isomer, in that 7-aminoindole was added to 3, fol-
lowed by coupling with phenylboronic acid. Of these,
5, the 4-indolyl isomer, had the best activity against
PKCh; however, it also had decreased selectivity against
Concurrent with the optimization of the headpiece,
studies were underway to optimize the tailpiece at C-2.
In order to facilitate the preparation of these analogs,
3 was converted to the 2-iodo derivative 17 as shown
in Scheme 3. The reaction of 17 with 4-formylphenylbo-
Table 1. PKCh and PKCd inhibitory activity25
R
NH
X
R'
CN
S
N
Ex
Indole isomer
X
R
R0
PKCh IC50 (nM)
PKCd IC50 (nM)
2
5
4
6
7
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
NH
NH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
460
230
2000
310
5
H
6
NH
NH
H
>5000
>5000
>5000
>5000
3700
>5000
>5000
1800
1300
52
7
H
8
NMe
O
H
9
H
10
11
12
13
14
15
16
19
20
21
24
25
26
27
29
NHCH2
NH(CO)
NH
H
H
1-Me
2-Me
3-Me
4-Me
4-Et
H
NH
NH
NH
NH
370
3300
1100
730
200
93
830
450
NH
NH
4-CH2-morpholine
4-CH2-N-Me-piperazine
4-CH2-NMe2
H
200
130
NH
NH
H
H
3-CH2-N-Me-piperazine
3-CH2-NMe2
100
75
NH
NH
H
85
H
2-CH2-N-Me-piperazine
2-CH2-NMe2
3-CH2-NMe2
730
570
NH
NH
H
4-Me
7.5
26

2852
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2850–2853
proved inhibitory activity against PKCh, the ortho iso-
mers 26 and 27 had reduced activity.
NH
Cl
N
X
N
The optimized tailpiece and headpiece were added to the
core as shown in Scheme 4. The reaction of 3 with 5-
amino-4-methylindole resulted in 28. The treatment of
28 with 3-[(N,N-dimethylamino)methyl]phenylboronic
acid pinacol ester gave 29, which had an IC50 value of
7.5 nM for the inhibition of PKCh. Based on the earlier
SAR, it was not surprising that 29 also showed a corre-
sponding increase in the inhibition of PKCd
(IC50 = 26 nM), and of Lyn (IC50 = 520 nM).
CN
CN
a
S
S
4
R
8: X = NMe
9: X = O
NH
b
10: X = NHCH2
11: X = NH(CO)
HN
CN
S
N
Analog 29 was profiled against other PKC family mem-
bers. While 29 only weakly inhibited PKCb,
(IC50 = 1.6 lM), more potent inhibition of PKCg and
PKCe, two novel PKCs, was observed, with 29 having
IC50 values of 62 and 17 nM, respectively. Limited addi-
tional kinase profiling of 29 provided IC50 values of
28 nM for Lck and of 2.2 lM for PKA. Weaker activity
against MK2 and AKT was observed (IC50 values of
>20 lM). 29 demonstrated ATP-competitive binding
with a Ki of 9 nM for PKCh and 96 nM for PKCd.16,24
12: R = 1-Me
13: R = 2-Me
14: R = 3-Me
15: R = 4-Me
16: R = 4-Et
Scheme 2. Reagents: (a) for 8: 5-methylaminoindole, EtOH, for 9: 5-
hydroxyindole, K2CO3, acetonitrile, for 10: indole-5-methanamine,
Hunig’s base, 2-ethoxyethanol, for 11: indole-5-carboxamide, NaH,
DMF; (b) substituted 5-aminoindole, EtOH.
ronic acid provided 18, with subsequent reductive ami-
nation with morpholine, N-methylpiperazine and
dimethylamine resulting in analogs 19–21, respectively.
As shown in Table 1, the N-methylpiperazine and
dimethylamine derivatives 20 and 21 had increased
PKCh inhibitory activity compared to 2. The meta and
ortho isomers of 20 and 21 were prepared as shown in
Scheme 3. While the meta isomers 24 and 25 had im-
To assay cell activity, T-cells isolated from both PKCh
wild type (WT) and KO mice were stimulated with
anti-CD3 and anti-CD28 to produce IL-2.16 Compound
29 blocked the production of IL-2 from the WT cells
with an IC50 value of 230 nM with decreased potency
in the PKCh KO cell assay (IC50 = 1300 nM) as would
be expected for a PKCh inhibitor. It should be noted
that some of the activity in these cell assays may be
due to the inhibition of Lck by 29.
NH
In pharmaceutical profiling assays, 29 had a permeabil-
ity of 4.6 · 106 cm/s in a PAMPA assay, with low solu-
bility at neutral pH. However, as expected due to the
presence of the dimethylamine group, decreasing the
pH to 3.0 increased the solubility of 29 to >100 lg/
mL. In a stability study with rat liver microsomes, 29
had an estimated half-life of 17 min. These results indi-
cate that this new class of PKCh inhibitors should have
acceptable physical chemical properties.
Cl
N
HN
N
CN
CN
I
a
I
S
S
3
17
NH
NH
NH
d
b
HN
HN
N
CN
CN
OHC
S
S
N
NH
18: para
22: meta
23: ortho
27
N
Cl
HN
c
CN
CN
a
I
I
HN
S
S
N
N
CN
3
R'RN
28
NH
S
N
b
HN
N
19: para; NRR' = morpholine
20: para; NRR' = N-Me-piperazine
21: para; NRR' = NMe2
CN
24: meta; NRR' = N-Me-piperazine
25: meta; NRR' = NMe2
S
26: ortho; NRR' = N-Me-piperazine
N
29
Scheme 3. Reagents: (a) 5-aminoindole, EtOH; (b) 4, 3, or 2-formyl-
phenylboronic acid, (Ph3P)4Pd, DME, aq NaHCO3; (c) RR0NH,
Na(OAc)3BH, CH2Cl2, NMP; (d) 2-[(dimethylamino)methyl]-phenyl-
boronic acid, (Ph3P)4Pd, DME, aq NaHCO3.
Scheme 4. Reagents: (a) 5-amino-4-methylindole, EtOH; (b) 3-[(N,N-
dimethylamino)methyl]phenylboronic acid pinacol ester, (Ph3P)4Pd,
DME, aq NaHCO3.

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Acknowledgments
We thank the Wyeth Chemical Technologies depart-
ment for compound characterization and the pharma-
ceutical profiling results, Jerry Sun and Pingzhong
Huang for the scale-up of 3, Robert Czerwinski for
the Ki data, Paul Morgan for the PKA, AKT, and
MK2 assays, Agnes Brennan for the cell assay, Gulnaz
Khafizova for the preparation of 5-methylaminoindole,
and Drs. John Ellingboe, Janis Upeslacis, and Tarek
Mansour for their support.
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