Structure activity optimization of 6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazines as Jak1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.09.020

Previous work investigating tricyclic pyrrolopyrazines as kinase cores led to the discovery that 1-cyclohexyl-6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (12) had Jak inhibitory activity. Herein we describe our initial efforts to develop orally bioavailable analogs of 12 with improved selectivity of Jak1 over Jak2.

Full text of DOI:10.1016/j.bmcl.2015.09.020

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure activity optimization of 6H-pyrrolo[2,3-e][1,2,4]triazolo
[4,3-a]pyrazines as Jak1 kinase inhibitors
b
b
a
a
Michael Friedman a, , Kristine E. Frank , Ana Aguirre , Maria A. Argiriadi , Heather Davis ,
⇑
Jeremy J. Edmunds a, Dawn M. George a, Jonathan S. George a, Eric Goedken a, Bryan Fiamengo c,y
,
Deborah Hyland a, Bin Li a, Anwar Murtaza d,y, Michael Morytko a, Gagandeep Somal a, Kent Stewart b,
Edit Tarcsa a, Stacy Van Epps a, Jeffrey Voss a, Lu Wang a, Kevin Woller b, Neil Wishart a
a Abbvie Bioresearch Center, 381 Plantation St., Worcester, MA 01605, USA
b Abbvie, 1 North Waukegan Rd., North Chicago, IL 60064, USA
c Materia Inc, 60 N San Gabriel Blvd., Pasadena, CA 91107, USA
d Merck Research Laboratories, 33 Avenue Louis, Pasteur Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous work investigating tricyclic pyrrolopyrazines as kinase cores led to the discovery that 1-cyclo-
hexyl-6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (12) had Jak inhibitory activity. Herein we describe
our initial efforts to develop orally bioavailable analogs of 12 with improved selectivity of Jak1 over Jak2.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 29 July 2015
Revised 4 September 2015
Accepted 7 September 2015
Available online xxxx
Keywords:
Tricyclic pyrrolopyrazines
6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]
pyrazines
Jak inhibitors
Jak selectivity
The Janus protein kinase family members (Jak1, Jak2, Jak3, and
Tyk2) are cytoplasmic tyrosine kinases often associated with mem-
brane cytokine receptors.1 Cytokine binding to these receptors ini-
tiates their Jak dependent phosphorylation and in turn results in
the formation of binding sites for Src homology 2 (SH2) containing
proteins such as signal transduction activators of transcription
(STAT) factors. The binding and subsequent activation of the STATs
play a critical role in facilitating STAT nuclear translocation, ulti-
mately resulting in cytokine production and cellular trafficking.
This process aids in a number of biological processes including reg-
ulation of immune and inflammatory responses.2,3
Clinically, Jak kinase inhibition was shown to be highly effective
in managing the signs and symptoms of rheumatoid arthritis (in
many cases achieving remission) with the first generation broad
Jak family inhibitors Tofacitinib 14–6 and Baricitinib 2 (Fig. 1).4,7
Despite encouraging efficacy, these agents have failed to reach
their full potential due to dose-limiting tolerability issues.8–10 For
example, the incidence of severe anemia was reported to be a
dose-limiting factor during Phase II development of Tofacitinib
for RA.9 It is reasonable to hypothesize that this adverse effect
could arise due to inhibition of erythropoietin signaling mediated
via Jak2.11 Based on this observation we set out to explore the
hypothesis that compounds with higher Jak1 selectivity over Jak2
could result in therapies that provide improved efficacy while lim-
iting the side effects associated with the first generation Jak inhibi-
tors, in particular, anemia. More recently, additional publications
on Jak1 selective inhibitors have appeared in the literature.12,13
In order to determine the best approach to identify selective
Jak1 inhibitors, we examined our Tyk2 crystal structures in com-
plex with a number of ligands as reported previously.14 A model
of Tofacitinib 1 in Jak1 subsequently was created (Fig. 2). Through
the examination of this model versus a published structure of
Jak215 (2B7A), we hypothesized that induced fit of the glycine-rich
loop was critical for Jak isoform selectivity. A comparison of the
primary structure of Jak1 and Jak2 revealed that the end of the gly-
cine-rich loop contains a His885 (Jak1)/Asn859 (Jak2) residue dif-
ference (seen in Fig. 2). We postulated that a histidine at this
residue position (His885) either directly or indirectly (via solvent)
interacts with the conserved catalytic base Asp1003 thereby creat-
ing a more closed glycine-rich loop conformation in Jak1. This led
⇑
Corresponding author. Tel.: +1 508 688 8013.
E-mail address: michael.friedman@abbvie.com (M. Friedman).
Current address.
y
http://dx.doi.org/10.1016/j.bmcl.2015.09.020
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Friedman, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.020

2
M. Friedman et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
N
TMS
N
Br
N
Br
O
S
N
N
Br
b
a
N
N
NH2
N
N
NH2
N
O
NH2
N
N
N
3
4
5
N
N
N
Boc
Boc
NH2
HN
NH
Br
O
N
N
N
N
N
c
d
e
N
N
N
N
H
N
N
Ts
N
Ts
N
8
H
Ts
6
7
1
2
O
R
O
X
X
Figure 1. First generation Jak inhibitors: Tofacitinib 1 and Baricitinib 2.
S
O
N
N
N
N
X
NH
g,h
f
N
i,j
N
HN
N
us to hypothesize that Jak1 inhibitors with improved selectivity
over Jak2 could be obtained by optimization of the substituent pro-
jecting under the glycine-rich loop. This ‘closed glycine-rich loop’
structural hypothesis was later confirmed by the crystal structure
of 1 in Jak1 (3EYG)16 where a solvent-mediated interaction
between His885 and Asp1003 was observed. Under this premise,
we postulated that in addition to the cyanoacetamide moiety in
Tofacitinib 1, other capping groups could be considered to provide
an improved van der Waals interaction with the glycine-rich loop
of Jak1 thus improving the Jak1/2 selectivity ratio.
N
Ts
N
H
N
N
11
N
Ts
N
9
10
Scheme 1. Reagents and conditions: (a) NBS, Acetontrile/DMF, 0–5 °C, 3 h, 76%; (b)
TMS-acetylene, PdCl2(PPh3)2, CuI, TEA, THF, ꢀ5 to 0 °C, 1.5 h, 88%; (c) NaH, TsCl,
DMF, 0 °C to rt, 1 h, 54%; (d) di-tert-butyl hydrazodicarboxylate, Pd(OAc)2,
Xantphos, K2CO3, t-AmOH/dioxane, 95 °C, 2 h, 91%; (e) H3PO4, THF/heptane, 70 °C,
2 h, then K2CO3, H2O, rt, 1 h, 94%; (f) X-COOH, EDC-HCl, DCM, rt, 15 h, 43–85%; (g)
thionyl chloride, TEA, dioxane, 80 °C, 1 h, 40–95%; (h) 4 M HCl in dioxane, dioxane,
60 °C, 4 h, 75–92%; (i) RSO2Cl, TEA, DMF, rt, 75 min, 62–91%; (j) 1 M aq NaOH,
dioxane, 60 °C, 2 h, 30–95%.
To identify leads for Jak1 selective inhibitors, we utilized work
previously described17 to afford 6H-pyrrolo[2,3-e][1,2,4]triazolo
basic conditions and subsequent removal of the tosyl protecting
group provided sulfonamide 11h. In some cases in which hydra-
zides 9 contained a Boc-protected amine, partial deprotection
occurred during cyclization. In such examples, the resulting mix-
ture was subjected to anhydrous acidic conditions (such as trifluo-
roacetic acid) to effect complete amine deprotection then treated
as above.
Compounds were profiled in Jak1, Jak2, Tyk2, Jak3 enzyme
assays18 and Jak1, Jak2 cellular assays.19 We focused on assessing
Jak1 over Jak2 selectivity by screening in IL-6-stimulated TF1 and
EPO-stimulated UT-7 based cellular assays to benefit from full
length Jak enzymes and physiologically relevant ATP concentra-
tions. While there is some literature on IL-6 driven signaling also
involving Jak2, our data indicate that IL-6 signaling can be sepa-
rated from Jak2-dependent Epo signaling. This suggests that Jak2
is unlikely to be important for the IL-6-induced phospho-STAT3
signal we measured.
Lead tricyclic pyrrolopyrazine 12 showed reasonable potency
against Jak1 (Table 1). Initial analogs with the piperidine amide
moiety from 1 (albeit without the methyl group) as represented
by compound 13 did not result in improvement of potency or
selectivity compared to 12. We hypothesized that this could be a
result of the additional constraint posed by the tricyclic core caus-
ing the cyano group to project in a different orientation compared
to 1. Therefore we attempted to optimize both the size and trajec-
tory of group appended to the core (piperidine to pyrrolidine) and
the nature of the terminal group (amide to sulfonamide) to see if
potency and/or selectivity could be increased. Neither the piperi-
dine nor the pyrrolidine linking group attached to the tricyclic moi-
ety (compounds 13–16) produced potency levels comparable to
the pyrrolopyrimidine hinge binders, such as Tofacitinib. In fact,
these initial changes led to a loss of potency in both enzyme and
cellular assays.
[4,3-a]pyrazines.
A general synthetic route (Scheme 1) was
designed to provide access to analogs to explore the Gly-rich loop
region of the protein. Commercially available aminopyrazine 3 was
treated with NBS to give dibromide 4 which underwent a regiose-
lective Sonogashira cross-coupling reaction with TMS-acetylene.
The resulting alkyne 5 was reacted with sodium hydride in the
presence of tosyl chloride and after aqueous acidic workup tosyl
protected pyrrolopyrazine 6 was obtained. Buchwald–Hartwig
amination with bis-Boc protected hydrazine yielded compound 7
which was deprotected with acid and neutralized to give the free
hydrazine 8. Hydrazides of general structure 9 were formed using
peptide coupling reagents such as EDC with a variety of carboxylic
acids, some of which contained protected amines. The cyclization
of hydrazides of general structure 9 was accomplished with thionyl
chloride in the presence of TEA and heat to give triazolopyrazines
10. In the cases where the triazolopyrazines contained protected
amines in X, these compounds were deprotected and then further
functionalized as appropriate. For example, Boc deprotection of
tert-butyl ((1S,3R)-3-(6-tosyl-6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-
a]pyrazin-1-yl)cyclopentyl)carbamate (precursor to 11h) with
HCl followed by reaction with cyclopropylsulfonyl chloride under
Given the undesired results from compounds 13–16, additional
modeling on 5- and 6-membered rings with other terminal groups
indicated that alkylsulfonamide substituents via an exocyclic
amine should provide an alternative and improved trajectory to
interact with the Gly-rich loop. Thus we turned our attention to
compounds with sulfonamide-substituted cyclohexyl and cyclo-
pentyl groups (Table 2). Both the stereochemical configuration
and length of the linking group significantly affected Jak1 in vitro
Figure 2. Overlay of Tofacitinib 1 modeled in Jak1 (blue) and Jak2 crystal structure
2B7A13 (green).
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M. Friedman et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
In vitro Jak enzyme and cellular activity
R
N
N
N
N
H
N
Compound
R
Jak1 HTRF (
l
M)
Jak2 HTRF (
l
M)
Jak3 HTRF (
lM)
Tyk2 HTRF (
l
M)
Jak1 cell (
l
M)
Jak2 cell (lM)
Tofacitinib 1
Baricitinib 2
na
na
0.003
0.006
0.004
0.006
0.003
0.18
0.14
0.036
0.045a
0.017a
1.12
0.31
12
0.03
0.09
3.23
0.53
0.29
0.29a
11.8
13
0.01
0.08
1.02
0.36
6.42
14
15
16
0.23
0.08
0.49
0.59
na
5.65
10.8
20.4
2.6
na
>20
>20
na
3.95
8.1
>1.0
na
na
a
Assays performed in 384 well format.19
Table 2
In vitro Jak enzyme and cellular activity
X
1
S
O
N
N
O
N
N
N
H
Compound
X
Jak1 HTRF (
lM)
Jak1 cell (
l
M)
Jak2 cell (lM)
Jak2/Jak1 cellular selectivity
11a
0.02
0.37
0.27
2.4
9ꢁ
racemic mixture
of cis-isomers
11b
1.9
>20
>10ꢁ
racemic mixture
of trans-isomers
11c
11d
11e
11f
0.21
0.15
0.05
0.01
>1.0
0.94
>1.0a
0.02
>20
>20
14.6
4.5
—
>20ꢁ
—
225ꢁ
11g
11h
0.06
0.02
>1.0a
0.11
8.2
1.9
—
17ꢁ
(continued on next page)
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4
M. Friedman et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2 (continued)
Compound
X
Jak1 HTRF (
0.70
lM)
Jak1 cell (
l
M)
Jak2 cell (
lM)
Jak2/Jak1 cellular selectivity
—
11i
na
>20
11j
0.70
na
na
—
a
Assays performed in 384 well format.19
Table 3
In vitro Jak enzyme and cellular activity
H
N
H
N
2HCl
f
NH2
N
CN
N
N
N
N
O
N
e
O
N
N
N
N
N
N
H
N
H
N
N
H
N
19
18
20
b,c,d
O
Boc
HN
NH2
HN
NH
HN
N
Compound
R
Jak1 HTRF
M)
Jak1 cell
M)
Jak2 cell
(lM)
a
N
N
(l
(l
N
N
N
Ts
Ts
8
17
11h
18
0.02
1.9
0.11
na
1.9
na
g,h
H
H
N
2HCl
H
N
NH2
n =0,1
19
0.70
na
>20
i,j or
k,l
N
N
N
N
O
N
N
N
o,p
N
N
N
N
N
N
CN
20
22
23
0.09
0.44
1.2
>1.0
na
>20
>20
>20
N
N
H
N
N
H
Ts
21
22 n = 0
23 n = 1
25
m,n
na
H
N
O
N
S
N
N
O
24
25
0.05
0.04
>1.0a
0.5a
1.8
N
N
H
N
>20
24
a
Assays performed in 384 well format.19
Scheme 2. Reagents and conditions: (a) (1R,3S)-3-((tert-butoxycarbonyl)amino)-
cyclopentanecarboxylic acid, EDCꢂHCl, DCM, rt, 2 h, 97%; (b) SOCl2, TEA, dioxane,
80 °C, 1.5 h; (c) 2 M aq Na2CO3, dioxane, 80 °C, 20 h, 85% over 2 steps; (d) 4 M HCl in
dioxane, dioxane, 60 °C, 2 h, 95%; (e) pyrrolidine-1-carbonyl chloride, TEA, THF,
60 °C, 16 h, 40%;(f) 2-cyanoacetic acid, EDC, HOBt, DIEA, perfluorophenyl 2-
cyanoacetate, rt, 5 h, 21%; (g) SOCl2, TEA, dioxane, 80 °C, 1.5 h, 85%; (h) 4 M HCl
in dioxane, dioxane, 60 °C, 2 h, 93%; (i) cyclopropanecarboxylic acid, EDCꢂHCl, DIEA,
DCM, rt, 20 h, 89%; (j) 2 M aq Na2CO3, dioxane/ethanol (1:1), 60 °C, 16 h, 55%; (k)
cyclopropylacetic acid, EDCꢂHCl, DIEA, DCM, rt, 20 h, 79%; (l) 2 M aq Na2CO3,
dioxane/ethanol (1:1), 60 °C, 16 h, 61%; (m) piperidine-1-sulfonyl chloride, DIEA,
DMF, rt, 8 h, 72%; (n) 1 M aq NaOH, dioxane, 60 °C, 1 h, 91%; (o) 5-chloropyrazine-2-
potency in both enzymatic and cellular assays compared to 11g.
We were encouraged by the fact that the initial set of analogs
demonstrated good in vitro permeability and solubility. The
increased chemical reactivity of the cyclopentyl amine versus the
hindered [2,2,2]-bicyclooctyl amine allowed for exploration of
more diverse chemical space, and thus 11h was selected over 11f
as a starting point for further exploration.
carbonitrile, DIEA, n-propanol,
lW, 150 °C, 30 min, 80%; (p) 1 M aq NaOH, dioxane,
We next sought to determine the optimal terminal substituent
in the context of the cyclopentyl group. A versatile synthetic route
allowed exploration of a wide range of terminal groups as outlined
in Scheme 2. Hydrazide 17 was accessed through the coupling of
hydrazine 8 and (1R,3S)-3-((tert-butoxycarbonyl)amino)cyclopen-
tane-carboxylic acid with EDC and was readily cyclized to the
desired 6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazine with thio-
nyl chloride and TEA. Basic hydrolysis of the tosyl group followed
by acidic cleavage of the Boc-protected amine afforded amine 18
as a dihydrochloride salt. Reaction of 18 with pyrrolidine-1-car-
bonyl chloride and TEA yielded urea 19. Cyclopentylamine salt
18 was also subjected to amidation using 2-cyanoacetic acid with
coupling agents EDC and HOBt; when this initial coupling failed,
addition of perfluorophenyl-2-cyanoacetate to the reaction mix-
ture resulted in conversion to 20. Alternatively, hydrazide 17 can
60 °C, 80 min, 64%.
potency. In the cyclohexyl series, cis-3-sulfonamide substituted
11a possessed superior Jak1 potency compared to the trans analog
11b and both 4-substituted analogs (11c and 11d). Interestingly,
although bridged bicycle 11e was less potent than parent 11a,
the bulkier bicycle 11f demonstrated excellent enzymatic and cel-
lular potency as well as greatly improved selectivity versus Jak2. In
the cyclopentyl series, the absolute R-stereochemistry at the point
of attachment of the group (X) to C-1 of the tricycle proved to be
critical as compounds 11g and 11h showed significantly better
potency in the Jak1 enzymatic assay compared to compounds 11i
and 11j. Absolute stereochemistry at the sulfonamide attachment
point was also important as 11h showed 3–9ꢁ improved Jak1
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M. Friedman et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 5
be cyclized with thionyl chloride and TEA, followed by Boc
PK in male Sprague–Dawley rats
deprotection under acidic conditions to give amine 21, also as a
dihydrochloride salt. Coupling of 21 with either cyclopropanecar-
boxylic acid or cyclopropylacetic acid and EDC followed by basic
tosyl group hydrolysis led to amides 22 and 23. Reaction of 21 with
piperidine-1-sulfonyl chloride and Hunig’s base followed by tosyl
group removal yielded sulfonyl urea 24. Finally, chlorine displace-
ment of 5-chloropyrazine-2-carbonitrile with 21 and subsequent
deprotection afforded aminoheterocycle 25.
The primary amine, urea, amide, sulfonyl urea, and aminohete-
rocycle analogs (18–20 and 22–25, Table 3) all led to decreased
potency in enzyme and/or cellular assays, confirming that the sul-
fonamide linker (as in 11h) was optimal for Jak1 potency. Based on
this data, we refocused our attention on optimization of the sulfon-
amide capping group in order to improve Jak1 potency and Jak1
versus Jak2 cellular selectivity.
Multiple sulfonamide capping groups were evaluated (Table 4).
Although both alkyl- (26–28) and cycloalkyl- (29–31) sulfon-
amides were tolerated, the cycloalkyl-analogs 11h, 29, and 30
appeared to be optimal from a combined potency and selectivity
standpoint. 11h was selected for further profiling based on the
highest microsomal stability. Aryl sulfonamides 32–34 were gener-
ally potent but had significantly lower microsomal stability and
were not advanced.
b
b
Compound IV CLpa
(L/h/kg)
PO AUC0-inf
gꢂh/mL)
0.28
0.05
0.41
1.1
PO Cmax
(lg/mL)
PO
F(%) @ 10 mg/kg
Rat Con A19 (%)
(l
12
na
na
3.1
2.9
2.5
0.17
0.01
0.06
0.23
0.31
na
na
13
29
32
—
0%
31%
55%
40%
11a
11f
11h
29
0.86
a
IV dose and vehicle—5 mg/kg in 10% EtOH, 65%PEG-400 qs. D5 W.
PO dose and vehicle—10 mg/kg in 0.02% Tween 80 in 0.5% HPMC.
b
observed upon dose escalation. In contrast, 11h and 29 demon-
strated acceptable oral exposure and IV half-life although plasma
clearance was relatively high, warranting further optimization. In
addition, 11h and 29 demonstrated greater than 100-fold selectiv-
ity in an in-house kinome panel of 80 kinases broadly covering the
kinome tree. Next, we evaluated these compounds in an acute
in vivo model of pro-inflammatory cytokine production as a read-
out of Jak1 inhibition. Encouragingly, both compounds demon-
strated significant levels of inhibition of Con A induced IFN
c
production in rat.21
In summary, we have described initial optimization of
6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazines as Jak1 inhibitors.
Compounds of interest were advanced into rat PK studies
(Table 5). Compound 11a had poor oral absorption as indicated
by the low Cmax and AUC. Compound 11f demonstrated moderate
Cmax and total AUC compared to 11a; however non-linear PK was
Compounds demonstrating
a promising combination of Jak1
potency, Jak1 versus Jak2 cellular selectivity and desirable PK prop-
erties were identified. Further efforts to generate optimized Jak1
inhibitors from this series with improved in vivo efficacy will be
described in due course.
Table 4
Disclosure
In vitro Jak enzyme and cellular activity
M.F., K.E.F., A.A., M.A.A., H.D., J.J.E, D.M.G., J.S.G., E.G., D.H., B.L.,
M.M., G.S., K.S., E.T., S.V.E., J.V., L.W., K.W and N.W. are employees
at AbbVie.
The design, study conduct, and financial support for the
research were provided by AbbVie. B.F. is an employee at Materia
Inc. A.M. is an employee at Merck Research Laboratories.
All authors participated in the interpretation of data, review,
and approval of the publication.
Compound
R
Jak1
cell
Jak2
cell
Jak2/Jak1
cellular
Microsomal
stability, %
remaining,
human/rat20
Acknowledgments
(
lM)
(lM) selectivity
The authors would like to thank members of the Department of
Molecular and Cellular Pharmacology for their contributions in
obtaining in vitro enzyme activity and cellular data.
11h
26
0.11
1.9
17ꢁ
34ꢁ
100/98
na
0.69a 23.7
0.6a
0.92a >20
References and notes
27
28
21.1
101ꢁ
>20ꢁ
—/100
na
1. Schindler, C.; Levy, D. E.; Decker, T. J. Biol. Chem. 2007, 282, 20059.
2. Shuai, K.; Liu, B. Nat. Rev. Immunol. 2003, 3, 900.
3. Ghoreschi, K.; Laurence, A.; O’Shea, J. J. Immunol. Rev. 2009, 228, 273.
4. Gomez-Puerta, J. A.; Mocsai, A. Curr. Top. Med. Chem. 2013, 13, 760.
5. Meyer, D. M.; Jesson, M. I.; Li, X.; Elrick, M. M.; Funckes-Shippy, C. L.; Warner, J.
D.; Gross, C. J.; Dowty, M. E.; Ramaiah, S. K.; Hirsch, J. L.; Saabye, M. J.; Barks, J.
L.; Kishore, N.; Morris, D. L. J. Inflamm. 2010, 7, 41.
6. Kremer, J. M.; Bloom, B. J.; Breedveld, F. C.; Coombs, J. H.; Fletcher, M. P.;
Gruben, D.; Krishnaswami, S.; Burgos-Vargas, R.; Wilkinson, B.; Zerbini, C. A.;
Zwillich, S. H. Arthritis Rheum. 2009, 60, 1895.
7. Fridman, J. S.; Scherle, P. A.; Collins, R.; Burn, T. C.; Li, Y.; Li, J.; Convington, M.
B.; Thomas, B.; Collier, P.; Favata, M. F.; Wen, X.; Shi, J.; Mcgee, R.; Haley, P. J.;
Shepard, S.; Rodgers, J. D.; Yeleswaram, S.; Hollis, G.; Newton, R. C. J. Immunol.
2010, 184, 5298.
29
30
31
32
0.08
0.2
18.2
20.5
227ꢁ
100ꢁ
>75ꢁ
34ꢁ
85/80
90/68
na
0.67a >50
0.69a 23.4
42/31
8. Fleischmann, R.; Cutolo, M.; Genovese, M. C.; Lee, E. B.; Kanik, K. S.; Sadis, S.;
Connell, C. A.; Gruben, D.; Krishnaswami, S.; Wallenstein, G.; Wilkinson, B. E.;
Zwillich, S. H. Arthritis Rheum. 2012, 64, 617.
9. Riese, R. J.; Krishnaswami, S.; Kremer, J. Best Pract. Res. Clin. Rheumatol. 2010,
24, 513.
10. Lee, E. B.; Fleischmann, R.; Hall, S.; Wilkinson, B.; Bradley, J. D.; Gruben, D.;
Koncz, T.; Krishnaswami, S.; Wallenstein, G.; Zang, C.; Zwillich, S. H.; van
Vollenhoven, R. F. NEJM 2014, 370, 2377.
33
34
0.69
18.2
26ꢁ
54ꢁ
35/61
8/6
0.12a 6.51
Assays performed in 384 well format.19
a
Please cite this article in press as: Friedman, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.020

6
M. Friedman et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
11. Laurence, A.; Pesu, M.; Silvennoinen, O.; O’Shea, J. Open Rheumatol. J. 2012, 6,
232.
18. In vitro Jak1 kinase activity assay used Jakl kinase domain (aa 845–1142),
biotin-TYR2 peptide (Biotin-(Ahx)-AEEEYFFLFA-amide; 2 M) and 1 M ATP in
l
l
12. Zak, M.; Hurley, C.; Ward, S.; Bergeron, P.; Barrett, K.; Balacz, M.; Blair, W.; Bull,
R.; Chakravarty, P.; Chang, C.; Crackett, P.; Deshmukh, G.; DeVoss, J.; Dragovich,
P.; Eigenbrot, C.; Ellwood, C.; Gaines, S.; Ghilardi, N.; Gibbons, P.; Gradl, S.;
Gribling, P.; Hamman, C.; Harstad, E.; Hewitt, P.; Johnson, A.; Johnson, T.;
Kenny, J.; Koehler, M.; Kohli, P. B.; Labadie, S.; Lee, W.; Liao, J.; Liimatta, M.;
Mendonca, R.; Narukulla, R.; Pulk, R.; Reeve, A.; Savage, S.; Shia, S.; Steffek, M.;
Ubhayakar, S.; Abbema, A.; Aliagas, I.; Avitabile-Woo, B.; Xiao, Y.; Yang, J.;
Kulagowski, J. J. Med. Chem. 2013, 65, 4764.
13. Menet, C.; Fletcher, S.; Van Lommen, G.; Geney, R.; Blanc, J.; Smits, K.;
Jouannigot, N.; Deprez, P.; Van Der Aar, E.; Clement-Lacroix, P.; Lepescheux, L.;
Galien, R.; Vayssiere, B.; Nelles, L.; Christophe, T.; Brys, R.; Uhring, M.;
Ciesielski, F.; Van Rompaey, L. J. Med. Chem. 2014, 66, 9323.
50 mM MOPSO pH 6.5, 10 mM MgC12, 2 mM MnC12 (2 mM), DTT (2.5 mM),
BSA (0.01% w/v), 0.1 mM Na3VO4 and ATP (0.001 mM). After 60 min, the
enzymatic reaction was quenched by addition of EDTA (to final concentration
100 mM) and developed by addition of PT66-K (europium labeled
antiphosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, MA) and
3.12 lg/mL phycolink streptavidin-allophycocyanin acceptor (cat #PJ25S,
Prozyme, San Leandro, CA). Following incubation for 60 min, 96-well plates
were read via a Rubystar detector (BMG) using a 337 nm laser for excitation
and emission wavelength of 665 nm. In vitro Jak2, Tyk2 and Jak3 enzymatic
assays were performed in a similar fashion.
19. IL-6 dependent pSTAT3 in TF-1 and Epo-dependent pSTAT5 assays in UT-7 cells
were conducted in 96 well plates unless indicated by
K in which case they
14. Argiriadi, M. A.; Goedken, E. R.; Banach, D.; Borhani, D. W.; Burchat, A.; Dixon,
R. W.; Marcotte, D.; Overmeyer, G.; Pivorunas, V.; Sadhukhan, R.; Sousa, S.;
Moore, N.; Tomlinson, M.; Voss, J.; Wang, L.; Wishart, N.; Woller, K.; Talanian,
R. BMC Struct. Biol. 2012, 12, 22.
were conducted in 384 well plates as described in the Supplemental methods
in Goedken, E. R.; Devanarayan, V.; Harris, C. M.; Dowding, L. A.; Jakway, J. P.;
Voss, J. W.; Wishart, N.; Jordan, D. C.; Talanian, R. V. J. Biomol. Screen. 2012, 17,
857.
15. Lucet, I. S.; Fantino, E. S.; Styles, M.; Barmet, R.; Patel, O.; Broughton, S. E.;
Walter, M.; Burns, C. J.; Treutlein, H.; Wilks, A. F.; Rossjohn, J. Blood 2006, 107,
176.
16. Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P. M.; Wilks, A. F.;
Fantino, E.; Rossjohn, J.; Lucet, I. S. J. Mol. Biol. 2009, 387, 219.
17. Van Epps, S.; Fiamengo, B.; Edmunds, J.; Ericsson, A.; Frank, K.; Friedman,
M.; George, D.; George, J.; Goedken, E.; Kotecki, B.; Martinez, G.; Merta, P.;
Morytko, M.; Shekhar, S.; Skinner, B.; Stewart, K.; Voss, J.; Wallace, G.;
Wang, L.; Wang, L.; Wishart, N. Bioorg. Med. Chem. Lett. 2013,
23, 693.
20. Microsomal stability assay: 25 min incubation at 37 °C, 0.25 mg microsomal
protein, 1 mM NADPH and 1 lM compound. Measured % remaining at 25 min.
For human a pool of male/female microsomes was used; for rat a pool of male
SD rats microsomes was used.
21. Concanavalin A (con A) induced IFN inhibition assay: male lewis rats were
given Con A at 10 mg/kg (0.002 mL/g body weight) iv at t = 0. Con A was
formulated in Dulbecco’s Phosphate Buffered Saline 1X. Compounds were
dosed at t = ꢀ30 min. All compounds were formulated in 0.5% HPMC/0.02%
tween80 (polyoxyethylene-sorbitan Mono-oleate) in MQ water (95%). Rats
were cardiac bled at t = 4 h. IFN-gamma and IL-2 were measured by ELISA.
Please cite this article in press as: Friedman, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.020