Highly selective Janus kinase 3 inhibitors based on a pyrrolo[2,3-d] pyrimidine scaffold: Evaluation of WO2013085802

Article abstract of DOI:10.1517/13543776.2014.851670

A series of pyrrolo[2,3-d]pyrimidine derivatives have previously been claimed as Janus kinase (JAK) 1 selective inhibitors. Introduction of a 4-aryl substituent onto this bicyclic scaffold modulates the JAK selectivity profile, thus providing JAK3 selective inhibitors. This patent application claims such compounds as JAK3 inhibitors. Many of the compounds exemplified show > 100,000-fold selectivity for JAK3 over JAK2. The inhibitors are claimed to be useful in the treatment of respiratory diseases, arthritis and cancer.

Full text of DOI:10.1517/13543776.2014.851670

Patent Evaluation
Highly selective Janus kinase 3
inhibitors based on a pyrrolo
[2,3-d]pyrimidine scaffold:
evaluation of WO2013085802
1. Introduction
2. Chemistry
3. Biology
Peter Norman
Norman Consulting, Bucks, UK
4. Expert opinion
A series of pyrrolo[2,3-d]pyrimidine derivatives have previously been claimed
as Janus kinase (JAK) 1 selective inhibitors. Introduction of a 4-aryl substituent
onto this bicyclic scaffold modulates the JAK selectivity profile, thus providing
JAK3 selective inhibitors. This patent application claims such compounds as
JAK3 inhibitors. Many of the compounds exemplified show > 100,000-fold
selectivity for JAK3 over JAK2. The inhibitors are claimed to be useful in the
treatment of respiratory diseases, arthritis and cancer.
Keywords: asthma, autoimmune disease, JAK3 inhibitor, rheumatoid arthritis
Expert Opin. Ther. Patents (2014) 24(1):121-125
1. Introduction
The Janus kinase (JAK) family of non-receptor tyrosine kinases comprises four
members, namely, JAK1, JAK2, JAK3 and Tyk2, with their names originally
derived from their identification as just another kinase and tyrosine kinase 2. How-
ever, the presence of two phosphate-transferring domains prompted the use of the
name Janus, in recognition of the two-faced Roman god. These kinases play a key
role in mediating cytokine receptor signalling, through their interaction with signal
transducer and activator of transcription (STAT) proteins -- the JAK-STAT path-
way [1]. Inhibition of JAK inhibitors thus provides a means for modulating
cytokine-mediated effects, with the JAK inhibitors seen as useful in the treatment
of both inflammatory diseases and some types of cancer [2].
Considerable effort has been devoted to the development of JAK inhibitors for both
indications. Incyte and its partner Novartis developed the JAK1/JAK2 inhibitor ruxo-
litinib for the treatment of myeloproliferative disorders, with United States and Euro-
pean approval gained in November 2011 and August 2012, respectively. Pfizer’s
tofacitinib, which shows little selectivity for JAK1 and JAK3 over JAK2, was approved
for the treatment of rheumatoid arthritis in the United States in November 2012 and
was approved in Switzerland in July 2013, but European approval (for tofacitinib)
was refused in May 2013 and again in July 2013.
It has been suggested that inhibition of JAK1 and/or JAK3 is desirable for the treat-
ment of inflammatory diseases, such as rheumatoid arthritis, while inhibition of
JAK2 is desirable for the treatment of myeloproliferative disorders but can lead to
dose-limiting side effects in the treatment of arthritis. Inhibition of Tyk2 has attracted
much less interest to date. The JAK1/JAK2 inhibitor baricitinib, being developed by
Incyte in partnership with Eli Lilly, is in Phase III development for the treatment of
rheumatoid arthritis and is in Phase II development for treatment of psoriasis, while
Galapagos is pursuing the development of selective JAK1 inhibitors for the same
indications. GLPG-0634, in partnership with AbbVie is in Phase II studies for the
treatment of rheumatoid arthritis, while GSK-2586184, in partnership with
GlaxoSmithKline is in Phase II studies for treatment of psoriasis and lupus.
10.1517/13543776.2014.851670 © 2014 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674
All rights reserved: reproduction in whole or in part not permitted
121

P. Norman
N
N
H
N
O
N
O
N
O
NH
S
N
O
N
O
N
N
N
N
OH
N
HN
N
N
H
N
N
N
N
N
H
CF
3
O
N
O
F C
N
3
N
N
H
N
H
N
H
N
Adelatinib
1
N
Tofacitinib
Ruxolitinib
Baricitinib
CN
CN
CN
N
N
N
O
O
O
HN
O
HN
HN
N
N
O
O
N
N
O
N
N
N
N
N
H
Cl
O
N
H
N
H
N
H
2
3
4
A = aryl or heteroaryl
R1 = aryl, heteroaryl,
cycloalkyl, heterocycle,
carbalkoxy or carboxamide
R2 = H, m-NH2,
A
R
2
O
O
R
1
N
N
N
N
N
H
N
m-NHCOCH=CHR3 or various
R3 = H, alkyl or F
N
N
N
7
N
H
O
CF
3
Cl
N
N
H
N
H
N
(compounds 2 -- 4) [6-8]. Two reviews have highlighted the
range of selective JAK3 inhibitors that are claimed by various
companies in their patent applications [9,10].
5
6
The later review summarised the first patent filings from
Merck which claimed 3-amino-4-carboxamidopyrazole and
5-amino-4-carboxamidothiazole derivatives as JAK inhibitors
with the former described as showing JAK2 selectivity [11,12].
These were followed by a filing that claimed pyrrolo[2,3-b]
pyridine derivatives, for example compound (5), as potent
and selective JAK3 inhibitors [13]. More recently, a series of
applications has been published claiming pyrazole carboxa-
mide derivatives as JAK1 selective inhibitors [14-17], while a
single application claimed pyrrolo[2,3-d]pyrimidines, such
as compound (6), as JAK1 inhibitors [18]. The application
that forms the basis of this evaluation is also from Merck,
and it claims selective JAK3 inhibitors based on a 4-arylpyr-
rolo[2,3-d]pyrimidine scaffold [19].
R
2
R
2
H
N
R
1
R
1
O
O
O
R
3
N
N
N
H
N
H
N
N
8
9
A number of companies are pursuing the development of
selective JAK3 inhibitors for the treatment of rheumatoid
arthritis. Vertex’s adelatinib (VX-509) has progressed to
Phase IIb studies in rheumatoid arthritis [3,4]. A number of
other companies have described selective JAK3 inhibitors,
for example, Novartis (compound 1) [5], although none are
yet reported as having entered clinical development. Roche
has been the most prominent of these companies with a series
of publications describing highly selective JAK3 inhibitors
based on a 5H-pyrrolo[2,3-b]pyrazine-7-carboxamide scaffold
2. Chemistry
The sole generic claim of this application is very broad, encom-
passing an extensive range of 4-arylpyrrolo[2,3-d]pyrimidines.
The Markush structure (7) represents a simplified form which
122
Expert Opin. Ther. Patents (2014) 24(1)

Highly selective JAK3 inhibitors based on a pyrrolo[2,3-d]pyrimidine scaffold: evaluation of WO2013085802
R
O
2
H
N
H
N
H
N
R
S
1
O
O
O
O
O
O
O
N
N
N
N
H
N
H
N
N
H
N
N
10
11
12
assays. Examples of most of the more potent and JAK3-specific
inhibitors are shown in Table 1.
N
O
The examples shown in Table 1 indicate that the presence of a
vinyl amide, or an isostere (12), combines high potency for JAK3
(< 100 pM) with excellent selectivity for JAK3 (17,000 to
> 100,000). Only one of these examples, compound (13), does
not incorporate a 4-carboethoxy substituent. Within the exam-
ples shown, it appears that substitution of the aryl ring and vari-
ous substituents on the vinylic centre are tolerated.
OH
N
N
H
N
13
is more closely supported by various examples. The exemplified
compounds fall into two major groups. The first group are vinyl
amide-substituted compounds, better defined by the Markush
structure (8), the larger group comprises ethyl 4-arylpyrrolo
[2,3-d]pyrimidine-3-carboxylates defined by the Markush struc-
ture, where R1 may be a vinyl amide (9). Some 270 compounds
are specifically claimed, of which 30 compounds are within the
definition of compound (7), 217 are carboxylate derivatives
(10) and there is a small number of 4,5-bisaryl substituted pyr-
rolo[2,3-d]pyrimidines. Their use is claimed for the treatment
of JAK3-mediated diseases and specifically for the treatment of
arthritis, asthma and obstructive airways diseases, autoimmune
diseases and cancer.
The 4-substituted pyrrolo[2,3-d]pyrimidines were prepared
via Suzuki coupling of 4-chloro-pyrrolo[2,3-d]pyrimidine-
3-carboxylate derivatives. The acrylamide derivatives were
prepared via reduction of nitroaryl-substituted pyrrolo[2,3-d]
pyrimidines followed by acylation. Bisaryl derivatives were
prepared via sequential Suzuki couplings from 4-chloro-7H-
pyrrolo[2,3-d]pyrimidine as illustrated in Scheme 1.
4. Expert opinion
This application claims a series of compounds that appear to
be the most potent and, based on the data provided, most
selective JAK3 inhibitors described to date. In contrast, adela-
tinib and Astellas’ ASP-015K, the JAK3 selective inhibitors in
clinical development, are both < 10-fold selective for
JAK3 relative to other JAK inhibitors in isolated kinase
assays [3,20]. Adelatinib is reported to show much better selec-
tivity, and lower potency, in cellular assays. Galapagos has also
reported how the cellular selectivity of selective JAK kinase
inhibitors often differs from the selectivity observed in iso-
lated enzyme assays. However, in this instance, the pro-
nounced difference in potency between JAK3 and JAK2 is
such that considerable cellular selectivity would be anticipated
for the claimed 4-arylpyrrolo[2,3-d]pyrimidine derivatives.
These compounds, thus, appear to represent a significant
advancement in the 5H-pyrrolo[2,3-b]pyrazine derivatives
that have been recently described by Roche. In a series of
papers, compound (3) was highlighted as one of the most selec-
tive, a 1 nM potency JAK3 inhibitor with 20- and 30-fold
selectivity over JAK2 and JAK1, respectively [8]. Given the sim-
ilarity of this scaffold to the claimed 4-arylpyrrolo[2,3-d]pyri-
midines, it should be possible to more clearly delineate why
the latter are so potent and selective for JAK3. But it is possible
that the presence of a vinyl amide results in non-competitive
inhibition that is specific to JAK3. Alternatively, it may form
a slowly reversible, covalent bond with an active site cysteine.
The compounds claimed in the current application also
represent a novel variation on the pyrrolopyrimidine motif
in the kinase inhibitor field. The use of this scaffold with a
4-aryl substituent has barely been explored. However, fused
analogues, such as compound (13), were claimed by Vertex
as JAK inhibitors, and were described as either
JAK2 selective or JAK2/JAK3 inhibitors [21].
3. Biology
Compounds were tested as inhibitors of all four JAK family
kinases using purified glutathione-S-transferase (GST)-tagged
catalytic domains of each kinase, and a common peptide sub-
strate, LCB-EQEDEPEGDYFEWLW-NH2, in 384-well plate
homogeneous time resolved fluorescence (HTRF) assays. Com-
pounds were incubated with the kinases for 120 min and were
quenched with a Eu-tagged pY20 antibody. The fluorescence
signals were read 60 min later. IC50 values are stated to be lower
against JAK3 than either JAK1 or JAK2. Data are tabulated for
all exemplified compounds against JAK2 and JAK3. Some data
are just shown as IC50 values < 10, 10 -- 500 or > 500 nM, while
specific IC50 values are presented for 112 compounds. Of these
compounds, 48 have IC50 values > 1500 nM against JAK2, with
IC50 values between 13 pM and 1 nM against JAK3 with two
examples displaying JAK3 selectivity of > 100,000 in these
Expert Opin. Ther. Patents (2014) 24(1)
123

P. Norman
O N
O N
2
2
Cl
N
Br
A.
B.
C.
N
N
N
N
H
N
H
N
H
N
N
H
N
O
O
H
N
O
H N
2
H N
O
2
D.
Br
+
N
N
B
N
H
OH
N
HO
N
H
N
NH
2
H
N
E.
O
N
N
H
N
Scheme 1. The preparation of an acrylamide-substituted derivative. (A) 3-nitrophenylboronic acid, Pd(dppf)Cl2-CH2Cl2,
Na2CO3, dimethylformamide (DMF), 115ꢀC, 1 h; (B) N-bromosuccinimide, DMF, 30 min; (C) H2, 3%Pt/C, MeOH, 18 h; (D) (i)Pd
(dppf)Cl2-CH2Cl2, KH2PO4, dioxane, 110ꢀC, 17 h, (ii) SiliaMetSÒ DMT, 20 h; (E) 2-methylprop-2-enoic acid, 1-propanepho-
sphonic acid cyclic anhydride, Et3N, DMF, 70 h.
Another interesting facet of the application being evaluated
is the inclusion of claims for the use of the compounds in
Table 1. Potent and selective JAK3 inhibitors
exemplified in the application.
treating respiratory diseases, notably asthma and chronic
obstructive pulmonary disease. These claims are made sepa-
rately from the claims relating to the use of the compounds
in treating arthritis and other autoimmune diseases. It is,
thus, possible that Merck is evaluating the potential of some
of the compounds in animal models of respiratory disease,
as well as arthritis. Given the high potency, and selectivity,
of the claimed compounds, it would appear probable that
Merck is pursuing a more detailed preclinical evaluation of
selected examples. Since it has been postulated that
JAK3 regulates a number of responses in allergic asthma [22]
and that some tyrosine kinases contribute to airway inflamma-
tion in severe asthma [23], such studies would represent a log-
ical exploration of their potential. Since JAK3 expression is
principally confined to haematopoietic cells [24], the utility
of JAK3 inhibitors is likely to be confined to conditions in
which such cells are pivotally involved.
Structure R1
R2
JAK3
JAK2
IC50 (nM) IC50 (nM)
(10)
(10)
(11)
(10)
(10)
(10)
(10)
(10)
(12)
(10)
(10)
(10)*
(10)*
(10)
(10)
(10)
(10)
(10)
(10)
(10)+
Et
F
4-Cl
4-F
0.013
0.013
0.016
0.02
0.024
0.024
0.025
0.033
0.037
0.04
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
790
CH2OH
CH2F
F
CH2OMe
H
H
H
H
H
4-Me
H
Br
Et
Et
H
H
H
0.043
0.049
892
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
> 1496
5-MeO 0.049
5-F
H
Et
CH2NHSO2Me
CH2NMe2
H
Et
Et
Et
0.05
0.052
0.066
0.066
0.068
0.07
H
Declaration of interest
4-Cl
5-CN
4-F
H
The author received an honorarium by Informa for prepara-
tion of the manuscript.
0.085
*4-pyridyl ring, + 3-pyridyl ring.
124
Expert Opin. Ther. Patents (2014) 24(1)

Highly selective JAK3 inhibitors based on a pyrrolo[2,3-d]pyrimidine scaffold: evaluation of WO2013085802
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