2-Alkenylthieno[2,3-b]pyridine-5-carbonitriles: Potent and selective inhibitors of PKCθ

Article abstract of DOI:10.1016/j.bmcl.2008.06.040

A series of 2-alkenyl thieno[2,3-b]pyridine inhibitors of PKCθ were synthesized as potential inflammatory modulators. This series led to the discovery of 2-alkenyl amides, which are exceptionally potent and selective inhibitors of PKCθ. Compound 8 has an IC₅₀ of 3.8 nM against PKCθ and shows excellent selectivity over a variety of PKC isoforms.

Full text of DOI:10.1016/j.bmcl.2008.06.040

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4420–4423
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Alkenylthieno[2,3-b]pyridine-5-carbonitriles: Potent and selective inhibitors
of PKCh
a
b
b
L. Nathan Tumey a, , Diane H. Boschelli , Julie Lee , Divya Chaudhary
*
a Wyeth Research, Chemical and Screening Sciences, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Wyeth Research, Inflammation, 200 Cambridge Park Drive, Cambridge, MA 12140, 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 2-alkenyl thieno[2,3-b]pyridine inhibitors of PKCh were synthesized as potential inflammatory
modulators. This series led to the discovery of 2-alkenyl amides, which are exceptionally potent and
selective inhibitors of PKCh. Compound 8 has an IC50 of 3.8 nM against PKCh and shows excellent selec-
tivity over a variety of PKC isoforms.
Received 8 May 2008
Revised 12 June 2008
Accepted 12 June 2008
Available online 18 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
PKC
PKCh
PKCd
Indole
Inflammation
T cell
Thienopyridine
Cyano
Nitrile
T cell receptor (TCR) activation is an essential step in the
mounting of an effective immune response. The binding of an anti-
gen/major histocompatibility complex (MHC) to the TCR leads to
the formation of a supramolecular activation cluster (SMAC)
which, in conjunction with stimulation of CD28, begins a phos-
phorylation cascade that ultimately results in transcriptional acti-
vation of interleukin-2 (IL-2).1 Increased levels of IL-2, in turn,
promote T cell proliferation and differentiation, contributing to
an inflammatory response.2
Protein kinase C theta (PKCh) is a serine/threonine kinase ex-
pressed primarily in lymphocytes and mast cells. PKCh knockout
mice have been reported to have significant defects in their re-
sponse to T cell stimulation. PKCh plays an essential role in the
TCR-mediated activation of transcription factors which, in turn,
upregulate IL-2 gene expression.3 Specifically, PKCh knockout mice
have shown diminished responses in various T cell mediated dis-
ease models including the type II collagen-induced arthritis (CIA)
model,4 the experimental autoimmune encephalomyelitis (EAE)
model of multiple sclerosis,5,6 and the ovalbumin challenge
(OVA) model of asthma.7,8 Interestingly, in spite of their defective
T cell activation pathway, PKCh knockout mice have been shown
to have normal Th1 cell response in the lung and normal viral
clearance.7,9 For these reasons, the inhibition of PKCh has recently
become an attractive target for treatment of autoimmune and
inflammatory diseases.10–13
We recently reported a series of 2-phenylthieno[2,3-b]pyridine-
5-carbonitrile inhibitors of PKCh.13 The previous work13,14 sug-
gested that the presence of a solubilizing amine tail and/or a
hydrogen-bond acceptor on the phenyl ring significantly improved
the inhibitory activity against PKCh. We reasoned that the phenyl
group may serve primarily as a ‘linker’ connecting the thieno[2,3-
b]pyridine core to the important hydrogen-bond accepting residue.
With this in mind, we studied variations of this linker. The present
work focuses on attachment of various alkenyl amides that connect
the core to the requisite polar side chain.
The initial synthesis of this class of compounds is illustrated in
Scheme 1. The previously reported thieno[2,3-b]pyridine 115 was
deprotonated with LDA at ꢀ78 °C and subsequently quenched with
DMF to give aldehyde 2. Wittig coupling with the appropriate
phosphorus ylid followed by introduction of the aminoindole
headpiece by nucleophilic aromatic substitution gave ester 3.
Saponification followed by amidation gave the desired pyrrolidine
amide 4.
Compound 4 proved to be a potent inhibitor of PKCh with an
IC50 of 130 nM, but more importantly showed approximately
140-fold selectivity over PKCd. Selectivity for PKCh is desirable con-
sidering reports that PKCd deficiency in mice results in B cell
* Corresponding author. Tel.: +1 845 602 1876; fax: +1 845 602 5561.
E-mail address: nathantumey@yahoo.com (L. Nathan Tumey).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.040

L. Nathan Tumey et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4420–4423
4421
Table 1
Cl
N
Cl
N
PKCh and PKCd activities of various alkenyl amide substituted thieno[2,3-b]pyridines
CN
CN
b,c
a
OHC
NH
S
S
NH
1
2
NH
HN
O
X
CN
HN
N
d,e
HN
O
O
CN
S
N
CN
N
EtO
S
a
a
S
Compound
X
PKCh IC50 (nM)
PKCd IC50 (nM)
N
3
(PKCd/PKCh)
4
8
9
Pyrrolidine
( ) 3-Hydroxy pyrrolidine
3.8
41
1300 (340ꢁ)
450 (11ꢁ)
Scheme 1. Reagents: (a) LDA, ꢀ78 °C followed by DMF; (b) EtO2CCH@PPh3, THF; (c)
5-aminoindole, EtOH, reflux; (d) NaOH, EtOH, 80 °C; (e) pyrrolidine, EDCI, DMF.
N
10
180
430 (2ꢁ)
hyperproliferation.16,17 Due to the very high active-site sequence
identity with PKCh, selectivity over PKCd has been difficult to
achieve in the related 2-phenylthieno[2,3-b]pyridine series.13
Due to its potent activity and good selectivity, we desired a
more versatile synthesis of alkenyl amides related to compound
4. Therefore, a new synthesis was devised (Scheme 2) that short-
ened the prior sequence by one step and avoided the need for cryo-
genic lithiation. Based on our previous studies with the related 2-
phenylthieno[2,3-b]pyridine series,13 we decided to incorporate
the more active 4-methyl-5-aminoindole headpiece in place of
the 5-aminoindole headpiece in 4. The new synthetic sequence uti-
lized phosphite promoted Heck coupling18 of 513 with tert-butyl
acrylate to give compound 6. Acidic hydrolysis followed by carbo-
diimide coupling gave the various acrylamides shown in Table 1.
In agreement with our previous findings, incorporation of a 4-
Me group on the indole significantly improved inhibitory activity
against PKCh. We found that compound 8 was approximately 30-
fold more active against PKCh than compound 4 (3.8 nM vs
130 nM, respectively). More importantly, compound 8 retains
and even slightly improves upon the PKCd/PKCh ratio that was ob-
served for compound 4 (340-fold vs 140-fold, respectively).
Addition of a hydroxyl or tertiary amine solubilizing tail to the
pyrrolidine ring (9–11) resulted in a loss of over one log unit of
activity against PKCh (shown in Table 1). Furthermore, expansion
of the five-membered ring to a six-membered ring (12–15) also re-
sulted in a 10- to 15-fold loss in PKCh activity. Interestingly, most
of these examples proved to be more potent against PKCd than the
parent compound (8). This resulted in dramatic losses of selectivity
for most of these analogs. On the other hand, small dialkyl amides
such as 17 and 18 retained both the activity and selectivity of the
parent compound. Monosubstituted amides (19–21), however,
were approximately 4- to 15-fold less potent against PKCh than
the parent compound.
N
N
11
170
340 (2ꢁ)
N
12
13
14
15
16
17
18
19
20
21
22
23
Piperidine
48
53
88
43
450 (9ꢁ)
Morpholine
Piperazine
N–Me–piperazine
H2N–
Me2N–
Et2N–
EtNH–
PhNH–
MeO–(CH2)2–NH–
Me2N–CH2–CH2–N(Me)–
Me2N–NH–
4100 (78ꢁ)
200 (2ꢁ)
73 (2ꢁ)
12
67 (6ꢁ)
1.6
2.0
15
110
48
2100 (1300ꢁ)
250 (120ꢁ)
500 (34ꢁ)
500 (4ꢁ)
1800 (38ꢁ)
790 (5ꢁ)
150
100
420 (4ꢁ)
24
370
330 (1ꢁ)
N
HN
a
Values are means of two or more experiments.
In spite of the excellent activity and selectivity observed for
compounds 8, 17, and 18, these compounds suffered from very
poor aqueous solubility (data not shown). However, the incorpora-
tion of amine solubilizing groups (10–11, 14–15, and 22) resulted
in a significant loss of PKCh inhibitory activity and/or selectivity
against PKCd. Movement of the basic moiety closer to the carbonyl
via acyl hydrazines (23, 24) also resulted in compounds with poor
PKCh inhibition.
Having extensively explored the amino moiety (Table 1), we
next made variations of the carbonyl and alkene portion of amide
8 (Scheme 3). Alkene 8 could be catalytically reduced with hydro-
gen to give propanamide 25. Suzuki coupling of 5 with 3-(pyrroli-
dine-1-carbonyl)phenylboronic acid gave 26. Likewise, Suzuki
coupling of 5 with (E)-3-chloroprop-1-enylboronic acid in the pres-
ence of pyrrolidine gave allyl amine 27. Interestingly, this Suzuki
reaction requires the presence of the secondary amine. In the ab-
sence of an amine nucleophile, 5 was recovered unchanged. It is
believed that nucleophilic displacement of the allyl chloride frees
NH
NH
HN
N
HN
N
a
O
O
b
CN
CN
the palladium catalyst from the p-allyl complex that it forms with
t-Bu
I
S
(E)-3-chloroprop-1-enylboronic acid, thereby allowing the Suzuki
reaction to take place to give 27. This reaction has proven to be
rather general in scope and further examples will be reported else-
where (unpublished observations).
S
6
5
NH
NH
In addition to exploring the a,b-unsaturated carbonyl linker, we
HN
HN
c
O
O
N
were also interested in the effect of a methyl group on the thio-
phene core. The 3-methylthieno[2,3-b]pyridine skeleton was made
as illustrated in Scheme 4. The 2-aminothiophene 28 was formylat-
ed with DMF–DMA and treated with tert-butylcyanoacetate to give
enamine 29. Upon heating to 250 °C in diphenyl ether, compound
29 underwent a thermal elimination of isobutylene followed by
CN
CN
HO
R'
S
S
R
N
N
7
8-24
Scheme 2. Reagents: (a) t-Bu acrylate, P(OMe)3, Pd(OAc)2, DMF, Et3N, 80 °C, 2 h; (b)
5% TFA/DCM; (c) RR0NH, EDCI, DMF.

4422
L. Nathan Tumey et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4420–4423
Table 2
NH
PKCh and PKCd activities of various pyrrolidinyl-thieno[2,3-b]pyridines
NH
a
HN
N
HN
N
O
N
O
N
CN
NH
CN
S
S
HN
25
R
8
CN
N
L
O
HN
S
N
N
CN
Compound
L
R
PKCh IC50a (nM) PKCd IC50a (nM) (PKCd/PKCh)
S
N
NH
26
O
b
8
H
3.8
250
58
1300 (340ꢁ)
1800 (7ꢁ)
180 (3ꢁ)
HN
HN
CN
c
CN
O
N
I
25
26
27
H
H
H
S
N
S
N
27
5
O
Scheme 3. Reagents: (a) H2, Pd/C, EtOH/toluene; (b) 3-(pyrrolidine-1-car-
bonyl)phenylboronic acid, Pd(PPh3)4, NaHCO3, dioxane, reflux; (c) (E)-3-chloro-
prop-1-enylboronic acid, pyrrolidine, Cs2CO3, Pd(PPh3)2Cl2, DMF, 130 °C (lW).
22
100 (5ꢁ)
decarboxylation and concomitant cyclization to form thieno[2,3-
b]pyridone 30. Iodination followed by treatment with POCl3 gave
intermediate 31. This compound was converted in 4 steps to 33
by the previously described route.
O
33
Me 3.5
380 (110ꢁ)
a
Values are means of two or more experiments.
As shown in Table 2, propanamide 25 lost nearly two log units
of inhibitory activity against PKCh as compared to the parent com-
pound 8. This suggested a significant structural role for the rigidity
provided by the alkene. Replacement of the alkene with a phenyl
(26) resulted in loss of both potency and selectivity. Replacement
of the carbonyl with a methylene (27) resulted in a loss of approx-
imately one log unit of activity suggesting that the carbonyl plays a
role in the binding to PKCh. However, the significant improvement
of PKCd inhibitory activity observed for this compound suggested
that the carbonyl is key for selectivity against this PKC isoform.
Addition of a methyl group to C-3 of the core thieno[2,3-b]pyridine
seemed to have little effect on potency and selectivity. Compound
33 retained the excellent potency of parent compound 8 (3.5 nM vs
3.8 nM) and the selectivity of the parent compound (110ꢁ vs
340ꢁ).
tion of PKCh, the compound had a significantly reduced effect on
IL-2 release upon stimulation of T cells that were isolated from
PKCh knockout mice (IC50 > 1000 nM). Compound 8 had a T1/2
greater than 30 min in rat liver microsomes and had limited inhi-
bition of various cytochrome P450 isozymes at 3
lM (<50% inhibi-
tion of 2D6 and 2C9 and ꢂ60% inhibition of 3A4).
Table 3 illustrates the kinase selectivity profile for this com-
pound. Compound 8 is exceptionally selective over other PKC iso-
forms. However, significant inhibitory activity was observed
against Src family kinases. Due to their role in T cell activation,
the inhibition of certain Src family kinases such as Lck and Fyn
may actually be beneficial in a PKCh targeting therapeutic.19 How-
ever, inhibition of other Src family kinases such as Lyn may lead to
impaired B-cell response.20 The in vitro selectivity necessary for
safe clinical use is unknown at this time.
While extensive SAR was determined for compound 8, no sig-
nificant improvements were identified in potency (Tables 1 and
2) or in pharmaceutical profile (data not shown). We therefore sub-
jected compound 8 to further biological characterization in order
to determine if it was a suitable candidate for advancement. We
found that compound 8 inhibited the anti-CD3/anti-CD28 pro-
moted release of IL-2 from wild-type murine T cells, with an IC50
of 170 nM.12 As was expected based on the rather selective inhibi-
In spite of the promising overall profile of compound 8, its poor
aqueous solubility placed great limitations on our ability to obtain
meaningful pharmacokinetic (PK) results (data not shown). Addi-
tional work will be reported at a later time regarding efforts to im-
prove upon the PK profile and Src family selectivity of this family of
compounds.
O
CO2Et
CO2Et
CN
d,e
c
a,b
CO2tBu
N
S
NH2
S
S
H
N
CN
H
29
28
30
NH
g,h,i
NH
Cl
CN
f
HN
N
HN
N
I
O
N
S
CN
N
CN
I
31
S
S
33
32
Scheme 4. (a) DMF–DMA, 100° C; (b) tert-butyl cyanoacetate, t-BuOH, rt; (c) PhOPh, reflux, 2 h; (d) (CF3CO2)2PhI, I2, CHCl3; (e) POCl3, 100 °C; (f) 5-amino-4-methylindole,
EtOH, reflux; (g) t-Bu acrylate, P(OMe)3, Pd(OAc)2, DMF, Et3N, 80 °C, 2 h; (h) 5% TFA/DCM; (i) pyrrolidine, EDCI, DMF.

L. Nathan Tumey et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4420–4423
4423
Table 3
ing team for selectivity testing, and Drs. Tarek Mansour and Janis
Upeslacis for their support.
Kinase selectivity panel for compound 8
Kinase
IC50 (nM)
Kinase
IC50 (nM)
References and notes
PKCh (target)
3.8
MK2
VEGFR2
ERK2
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>20,000
330
PKCa
>50,000
>100,000
1300
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PDGFR
ITK
PKCf
RSK1
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CHK1
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SRC
FYN
HCK
LCK
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PKA
ROCK1
CK1
c1
LYN
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In summary, we have described the synthesis and SAR of a
series of 2-alkenylthieno[2,3-b]pyridines as PKCh inhibitors.
Incorporation of the alkenylamide moiety resulted in the identi-
fication of several low nM compounds with >100-fold selectivity
over the closely related PKCd isoform. One of these compounds
(8) was more fully characterized biologically and found to have
good selectivity over other PKC isoforms and a reasonable phar-
maceutical profile. Compound 8 was shown to potently inhibit
the activation of wild-type murine T cells. Comparison of this
activity with that against T cells lacking PKCh strongly suggests
that this compound is exerting its activity by blockade of the
PKCh pathway.
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Acknowledgments
We thank the Wyeth Chemical Technologies department for
spectroscopic characterization and pharmaceutical profiling, the
Discovery Synthetic Chemistry team for scaleup of 4-methyl-5-
aminoindole, Agnes Brennan for cellular testing, the kinase profil-
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