Novel triazolo-pyrrolopyridines as inhibitors of Janus kinase 1

Article abstract of DOI:10.1016/j.bmcl.2013.04.018

The identification of a novel fused triazolo-pyrrolopyridine scaffold, optimized derivatives of which display nanomolar inhibition of Janus kinase 1, is described. Prototypical example 3 demonstrated lower cell potency shift, better permeability in cells and higher oral exposure in rat than the corresponding, previously reported, imidazo-pyrrolopyridine analogue 2. Examples 6, 7 and 18 were subsequently identified from an optimization campaign and demonstrated modest selectivity over JAK2, moderate to good oral bioavailability in rat with overall pharmacokinetic profiles comparable to that reported for an approved pan-JAK inhibitor (tofacitinib).

Full text of DOI:10.1016/j.bmcl.2013.04.018

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3592–3598
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel triazolo-pyrrolopyridines as inhibitors of Janus kinase 1
g
a
g
a
Christopher A. Hurley a, , Wade S. Blair , Richard J. Bull , Christine Chang , Peter H. Crackett ,
Gauri Deshmukh d, Hazel J. Dyke a, Rina Fong f, Nico Ghilardi e, Paul Gibbons c, Peter R. Hewitt a,
Adam Johnson g, Tony Johnson a, Jane R. Kenny d, Pawan Bir Kohli g, Janusz J. Kulagowski a, Marya Liimatta g,
Patrick J. Lupardus f, Robert J. Maxey a, Rohan Mendonca c, Raman Narukulla a, Rebecca Pulk c,
Savita Ubhayakar d, Anne van Abbema g, Stuart I. Ward a, Bohdan Waszkowycz b, Mark Zak c
⇑
a Department of Medicinal Chemistry, Argenta, 8/9 Spire Green Centre, Harlow, Essex CM19 5TR, United Kingdom
b Department of Computer Aided Drug Design, Argenta, 8/9 Spire Green Centre, Harlow, Essex CM19 5TR, United Kingdom
c Department of Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States
d Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States
e Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States
f Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States
g Department of Biochemical and Cellular Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2013
Revised 5 April 2013
Accepted 6 April 2013
Available online 17 April 2013
The identification of a novel fused triazolo-pyrrolopyridine scaffold, optimized derivatives of which dis-
play nanomolar inhibition of Janus kinase 1, is described. Prototypical example 3 demonstrated lower cell
potency shift, better permeability in cells and higher oral exposure in rat than the corresponding, previ-
ously reported, imidazo-pyrrolopyridine analogue 2. Examples 6, 7 and 18 were subsequently identified
from an optimization campaign and demonstrated modest selectivity over JAK2, moderate to good oral
bioavailability in rat with overall pharmacokinetic profiles comparable to that reported for an approved
pan-JAK inhibitor (tofacitinib).
Keywords:
Janus kinase
JAK1
Ó 2013 Elsevier Ltd. All rights reserved.
JAK2
JAK1 inhibitor
Cytokine signaling pathways mediate a broad range of biologi-
cal functions, including many aspects of inflammation and immu-
nity.1 Types I and II cytokine receptors lack kinase activity and
instead transmit their signals through the receptor-associated Ja-
nus kinases (JAK1, JAK2, JAK3, and TYK2).2,3 When stimulated,
cytokine receptor complexes activate specific combinations of JAKs
in well-defined patterns,4 leading to further activation of signal
transducer and activator of transcription (STAT) proteins residing
in the cytoplasm. Upon JAK-mediated tyrosine phosphorylation,
the STATs dimerise and are translocated to the nucleus where they
regulate transcription of specific target genes.5,6 Because of the
importance of the JAK/STAT pathways in cytokine signaling, target-
ing of JAK kinases is envisioned to be useful in the treatment of a
variety of diseases including rheumatoid arthritis (RA),7 myelopro-
liferative disorders8 and cancer.9
strated efficacy in various clinical trials, likely due to its suppres-
sion of the IL-6 and cc cytokine pathways.12 Additionally, a
humanized monoclonal antibody (tocilizumab) targeting the IL-6
pathway has been approved for the treatment of moderate to se-
vere RA.13 Although IL-6 activates JAK1, JAK2, and TYK2, knockout
studies in mice have demonstrated that JAK1 plays a particularly
important role in signal transduction.14 Additionally, JAK1 has
been shown to play a critical and potentially dominant role in
the transduction of cc cytokine signaling.15 Finally, inhibition of
JAK2 is associated with anemia,16 thus limiting its suppression
may be beneficial. We, therefore, desired to develop potent JAK1
inhibitors with reduced inhibition of JAK2 to maximize anti-RA ef-
fects while limiting the potential for anemia.
We previously reported the identification and preparation of
novel fused imidazo-pyrrolopyridine tricyclic JAK inhibitors, such
as compound 2 (Fig. 1), that exhibited modest selectivity for
JAK1 over the JAK2 isoform.17 However, some members of this
structural class were compromised by low in vitro permeability
in MDCK cells and inadequate oral exposure in rat pharmacokinetic
studies. In an effort to circumvent the problems faced with com-
pounds such as 2, we undertook an exploration to find suitable
imidazo-pyrrolopyridine scaffold replacements. One restriction
Current evidence suggests that immuno-relevant cytokines
(such as IL-6 and the cc cytokines) play a pivotal role in RA disease
pathogenesis.10,11 The approved pan-JAK inhibitor tofacitinib (1,
Fig. 1) has undergone extensive evaluation for RA and has demon-
⇑
Corresponding author. Tel.: +44 1279 645 645; fax: +44 1279 645 646.
E-mail address: Chris.Hurley@glpg.com (C.A. Hurley).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.018

C. A. Hurley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3592–3598
3593
N
N
N
N
N
O
N
N
O
N
N
N
N
N
N
O
N
N
H
N
N
N
N
H
H
2
1, Tofacitinib
3
Figure 1. Structures of pan-JAK inhibitor tofacitinib 1, imidazo-pyrrolopyridine 2
and triazolo-pyrrolopyridine 3.
Figure 2. Comparison of the X-ray crystal structures of imidazo-pyrrolopyridine 2
(magenta: PDB code 4E6D) in complex with a JAK1-like JAK2 triple mutant17 and
triazolo-pyrrolopyridine 3 (green: PDB code 4I5C) in complex with JAK1. Hydrogen
bonds to the ligand are depicted with black dashed lines. Backbone hinge atoms
contacting 3 are highlighted (E957 and L959). Notable crystallographic waters are
denoted as spheres. The resolutions of the X-rays are 2.3 and 2.1 Å, respectively.
we applied was the need to readily transfer SAR to any new series
identified to expedite progression of the new scaffolds. Therefore
we limited our search to scaffolds which maintained the topology
of the existing core (compound 2). This search resulted in the iden-
tification of an alternative series of fused triazolo-pyrrolopyridine
tricycles (e.g., 3). When investigated in the JAK1 biochemical assay,
compound 3 exhibited similar, albeit slightly weaker, potency
compared to 2 (Table 1). Since the measured pKa of 3 was lower
than the pKa of compound 2, we were concerned that this reduced
basicity would be detrimental to the hydrogen bonding interac-
tions with the hinge residues (JAK1 residues E957 and L959). Con-
sequently, we obtained an X-ray co-crystal structure of 3 bound to
JAK1 and compared it to an X-ray co-crystal structure of 2 bound to
a JAK1-like JAK2 triple mutant (Fig. 2).17 We were pleased to find
that both the topology and the binding mode remained consistent
between the two scaffolds, indicating that major SAR differences
between the series would be unlikely.
Compound 3 displayed dramatically improved apical to basolat-
eral MDCK permeability compared to 2. This improved permeabil-
ity likely contributes to the observed reduction in the cell potency
shift and superior cell potency. Consistent with the imidazo-pyr-
rolopyridine scaffold, the triazole 3 demonstrated intrinsic but
modest selectivity over JAK2 in both the biochemical and cell-
based assays. Having shown a dramatic in vitro cell permeability
advantage, we decided to explore the SAR of this new chemotype
with the aim of improving selectivity against the JAK2 isoform
while ensuring favorable JAK1 potency in cells (Table 2). Synthetic
intermediate 4 exhibited modest biochemical potency and this
prompted us to prepare the less stereo-chemically complex 4-
piperidine isomer 5. Pleasingly, this compound demonstrated sin-
gle digit nanomolar JAK1 potency, suggesting a preference for the
4-piperidine ring. Compound 5 also displayed comparable potency
in the cell-based assay to 3. However, analogues 4 and 5 suffered
from poor metabolic stability in human liver microsomes. We be-
lieved that the sub-optimal metabolic profile was due to these
weakly basic compounds possessing relatively high lipophilicity
(c LogD). Therefore, in an attempt to improve microsomal stability,
we prepared compounds 6, 7 and 8 that contained substituents on
the piperidine nitrogen designed to reduce lipophilicity (LogD).
The inclusion of a cyanoethyl moiety (6) led to elevated metabolic
stability along with enhanced biochemical potency and better JAK1
isoform selectivity in cells. Compound 7 had similar selectivity and
human microsomal stability profile to compound 6, but was
slightly less potent. The introduction of the pyridine (8) was disfa-
vored and we made no further attempts to include heterocyclic
systems within this scaffold.
We were keen to identify additional structural features to im-
prove selectivity against JAK2. Previous investigations within our
laboratories demonstrated that substitution on the piperidine
nitrogen with suitable
a-amino amide groups afforded enhanced
selectivity against JAK2.17 Accordingly, the proline analogue 9
was prepared and exhibited 5.9-fold selectivity over the JAK2 en-
zyme. However, the relatively weak biochemical JAK1 potency
(121 nM) was insufficient to warrant further characterization. An
alternative approach we had previously discovered involved the
introduction of a 4-methyl substituent on the piperidine ring.17
When applied to this system, compounds 10 and 11 demonstrated
improved selectivity over the JAK2 isoform in the biochemical as-
say. Biochemically, the cyanoethyl moiety was strongly disfavored
(compare 6 with 10) highlighting that this modification was
incompatible with the previously established SAR. Conversely,
compound 11 demonstrated adequate potency and selectivity in
the cell-based assay accompanied by robust microsomal stability
to justify further progression.
During the course of our investigations, routine screening of
late stage intermediates meant that compound 12 was assessed
for activity and exhibited preferential potency for JAK1 over
JAK2. We have previously reported that hydrogen bonding ele-
ments within this region of the imidazole series can lead to en-
hanced selectivity profiles.17 Therefore, we examined a number
of additional analogues incorporating H-bond donor groups to
probe this region for beneficial selectivity enhancements (com-
pounds 13–24). Fluorinated piperidines compounds 14 and 15
were designed to reduce the basicity of the piperidine nitrogen
(ACD cpKa: 12 = 9.7; 15 = 7.7)18 and the trans-analogue (15) was
Table 1
Physicochemical and in vitro data for compounds 2 and 3
Ex JAK1 Kia,b
JAK2 Kia,b
(nM)
JAK1 cell EC50
(nM)
JAK1 cell
JAK1 cell potency
shifte
MDCK A:B Papp (Â10À6 cm/
LogDg pKa
(measured)
a,c
f
(nM)
selectivityd
s)
2
3
0.4
1.5
1.2
3.8
180
114
4.8
6.8
450
76
0.3
3.7
0.39
0.60
4.34
1.96
a
b
c
d
e
f
Arithmetic mean of at least three separate determinations.
Biochemical potency.
pSTAT3-IL6 JAK1 driven TF-1 cell-based assay.
Cell-based selectivity for JAK1 over JAK2 (pSTAT5-EPO EC50/pSTAT3-IL6 EC50).
Fold reduction in cell potency relative to biochemical potency (pSTAT3-IL6 EC50/JAK1 Ki).
Apparent permeability in MDCK transwell culture (arithmetic mean of at least two separate determinations), A:B apical to basolateral.
Measured at pH 7.2.
g

3594
C. A. Hurley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3592–3598
Table 2
Biological data and physicochemical properties for compounds 4–24
R
N
N
N
N
H
N
a,d
f
Ex
R
JAK1 Kia,b
(nM)
JAK2 Kia,b
(nM)
JAK1 enzyme
selectivityc
JAK1 cell EC50
(nM)
JAK1 cell
HLM T1/2
(h)
LogDg (c
LogD)
selectivitye
4
5
25h
8.5
40
15
1.6Â
2.0Â
ND
ND
0.2
0.5
ND (2.5)
ND (2.3)
N
N
~
~
121
3.4Â
CN
N
6
2.1
3.7
1.8Â
138
4.6Â
1.9
1.12
~
O
N
O
S
7
8
6.2
16
12
28
1.9Â
1.7Â
249
414
4.8Â
2.7Â
1.7
0.7
1.05
2.03
~
~
N
N
N
O
N
N
9
121
721
5.9Â
ND
ND
1.1
ND (À1.2)
~
~
CN
10
11
156
7.9
487
53
3.1Â
6.7Â
ND
ND
4.8
32
1.92
1.50
O
N
O
S
220
15Â
~
NH
12
13
210
745
669
3.2Â
1.7Â
ND
ND
ND
ND
ND
1.3
À0.35
~
~
1232
ND (À1.6)
NH
F
NH
14i
15i
198
21
446
57
2.3Â
2.7Â
ND
ND
ND
ND
ND
15
0.27
0.77
~
F
(+/-)
(+/-)
NH
~
NH2
16
17
21
12
121
32
5.8Â
2.7Â
729
558
7.9Â
4.2Â
1.1
1.2
À0.24
~
~
OH
1.76
H
N
CN
18
19
20
5.9
21
3.6Â
ND
165
ND
ND
6.7Â
ND
2.3
5.8
1.0
1.05
1.36
3.58
~
~
O
CN
>3763
120
ND
401
H
N
O
3.3Â
ND
O
~

C. A. Hurley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3592–3598
3595
Table 2 (continued)
a,d
f
Ex
21
22
R
JAK1 Kia,b
(nM)
JAK2 Kia,b
(nM)
JAK1 enzyme
selectivityc
JAK1 cell EC50
(nM)
JAK1 cell
HLM T1/2
(h)
LogDg (c
LogD)
selectivitye
H
N
O
12
27
17
2.3Â
3.6Â
433
350
4.0Â
5.0Â
4.8
3.7
2.08
2.7
O
O
~
~
H
N
4.7
O
23j
2.4
12
10
33
4.2Â
2.8Â
127
395
4.3Â
6.8Â
0.9
4.3
3.53
1.98
O
N
H
~
~
O
O
24i
N
H
(+/-)
ND = not determined.
a
Arithmetic mean of at least three separate determinations.
Biochemical potency.
Biochemical selectivity for JAK1 over JAK2 (JAK2 Ki/JAK1 Ki).
pSTAT3-IL6 JAK1 driven TF-1 cell-based assay.
Cell-based selectivity for JAK1 over JAK2 (pSTAT5-EPO EC50/pSTAT3-IL6 EC50).
Half-life in the presence of in vitro preparations of human liver microsomes.
Measured at pH 7.2 or calculated (c LogD) at pH 7.2 see Ref. 18 for details.
Biochemical determination N = 2.
b
c
d
e
f
g
h
i
Racemate.
Single enantiomer.
j
Table 3
In vitro and rat pharmacokinetic profiles of compounds 3, 6, 7, 11 and 18
Kinase selectivityb
(>40% inhibition)
JAK1 cell
MDCK A:B Papp
(Â10À6 cm/s)
In vivo rat PKa,f
d,e
Ex
potency shiftc,d
Fd (%)
Cmax
(lM) (po)
AUCd
(lM h) (po)
CLd,g (mL/min/kg)
d
3
6
7
11
18
0/50
ND
ND
0/50
1/50
76 (450)
66 (72)
40 (120)
28 (123)
28 (98)
3.7 (0.3)
17 (8.5)
13 (1.6)
14 (2.5)
7.2 (2.3)
41 (5)
0.4 (0.05)
4.9 (1.8)
4.6 (2.9)
0.7 (0.8)
0.9 (0.6)
1.0 (0.3)
1.8 (1.6)
18 (2.3)
1.0 (2.3)
1.4 (0.7)
110 (85)
44 (96)
16 (45)
211 (82)
88 (120)
28 (48)
>100 (36)
82 (74)
44 (28)
ND = not determined.
a
PK data are the mean profiles from three animals.
b
c
d
e
f
Number of enzymes inhibited >40% out of a panel of 50 non-JAK kinases, at a test concentration of 100Â JAK1 Ki.
Fold reduction in cell potency relative to biochemical potency (pSTAT3-IL6 EC50/JAK1 Ki).
Values in parentheses correspond to imidazo-pyrrolopyridine analogues reported in Ref. 17.
Apparent permeability in MDCK transwell culture (arithmetic mean of at least two separate determinations), A:B apical to basolateral.
PK data normalized to 1 mg/kg iv and 5 mg/kg po.
g
Plasma clearance following iv dosing.
Ph
N
Ph
N
10-fold more potent biochemically than 12. Unfortunately, the in-
creased structural complexity coupled with the poor reactivity of
the piperidine nitrogen prevented a meaningful follow-up cam-
paign around compound 15. The introduction of a methylene
spacer between the tricyclic core and the piperidine ring (13)
was strongly disfavored. Whereas when the H-bonding moiety
was relocated (16) this resulted in enhanced potency (compared
to 12) with slightly improved selectivity. Preparation of the corre-
sponding cis isomer was not undertaken as previous investigations
found this was disfavored.17 Compound 17, possessing a hydroxyl
group as a H-bond donor moiety, had similar potency to 16, how-
ever the selectivity against the JAK2 isoform degraded in both the
biochemical and cell-based assays. In an attempt to improve the
potency of compound 16, the amine moiety was capped with a
cyanoethyl group (18) to reduce the basicity of the nitrogen (ACD
cpKa: 16 = 10.3; 18 = 8.2). This transformation led to enhanced bio-
chemical and cell potency and was accompanied by greater micro-
somal stability. Furthermore, compound 18 maintained the same
a, b
HN
N
N
N
N
H2N
N
N
N
H
SO2Ph
25
4
c
N
N
NH
d
O
N
N
N
N
N
N
N
H
N
H
N
N
3
26
Scheme 1. Reagents and conditions: (a) n-Butyl nitrite, CuBr2, CH3CN, 70 °C, 2 h,
65% or sodium nitrite, AcOH, rt (ca. 95%); (b) NaOH (aq 2 M), MeOH/THF, 98%; (c)
ammonium formate, Pd(OH)2, MeOH, reflux, 91%; (d) cyanoacetic acid, HOBt, EDCI,
magnitude of cell selectivity against JAK2 as analogue 16. Appending DMAP, DCM, 92%.

3596
C. A. Hurley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3592–3598
H
N
NHBoc
NHBoc
R1
a
b
N
N
N
HN
N
N
N
N
H2N
N
N
N
N
H
N
SO2Ph
SO2Ph
20, R1 = Boc
16, R1 = H
18, R1 = -CH2CH2CN
28
27
c
e
d
R2
H
NH2
N
O
f or g, then h
N
N
N
N
N
N
N
N
N
H
N
SO2Ph
29
21, R2 = -OMe
22, R2 = -CH2CH(Me)2
Scheme 2. Reagents and conditions: (a) sodium nitrite, AcOH, 45 min, rt, 99%; (b) NaOH (aq 2 M), MeOH, THF, rt, 1 h, 87%; (c) TFA, H2O, rt, 2 h, 58%; (d) acrylonitrile, EtOH,
reflux, 90 min, 18%; (e) TFA, H2O, rt, 1 h, quantitative; (f) ClCO2Me, iPr2EtN, DCM, rt, 25 min, 90%; (g) ClC(O)R2, iPr2EtN, DMAP, DMF, rt, overnight; (h) NaOH (aq 1 M), MeOH,
THF, rt, 45 min.
the cyanoethyl group to the alcohol (19) was not tolerated. With
O
a
HO
these results, we focused on identifying alternative substitutions
to the cyanoethyl group found in 18. Synthetic intermediate 20
was screened for biochemical potency at JAK1, and exhibited activ-
ity, albeit weak. We noted that this structural change was accom-
panied by reduced stability in human liver microsomes (compared
to 18). We attributed this reduction to an increase in the lipophil-
icity of the compound and this prompted us to prepare analogues
which were more hydrophilic. The methyl carbamate 21 and amide
22 both demonstrated greater metabolic stability accompanied by
improved biochemical potency. Disappointingly, both examples
lacked sufficient potency in cells to justify further progression.
We also investigated the effect of ring size on JAK1 potency.
Accordingly, cyclopentane analogue 23 was prepared, and this dis-
played a ꢀ50-fold improvement in biochemical potency (com-
pared to 20) with favorable cell potency. Despite the marginally
reduced lipophilicity of 23, poor metabolic stability was observed.
Therefore, we deployed an analogous strategy to ease microsomal
turnover within the cyclopentane system, and truncated the tert-
butyl moiety to generate methyl carbamate 24. Although stability
in human liver microsomes improved, 24 exhibited reduced bio-
chemical potency.
Several compounds were subjected to further evaluation and
their in vitro and rat pharmacokinetic profiles are summarized
in Table 3. Compounds 3, 11, and 18 were screened in JAK3
and TYK2 biochemical assays and were found to be >10-fold less
potent against those isoforms compared to JAK1.19 To assess the
kinase selectivity of this series, compounds 3, 11 and 18 were
screened against a panel of 50 non-JAK kinases at a concentra-
tion 100-fold higher than their JAK1 Ki values. Examples 3 and
11 demonstrated excellent selectivity against all non-JAK related
enzymes in the panel; however, 18 exhibited some activity
against protein kinase D1 (inhibition = 44%). Cell permeability
determination in MDCK cells indicated that the triazole ana-
logues had moderate to good permeability that was routinely
better than the equivalent imidazoles. As a result, analogues 3,
6, 7, 11 and 18 demonstrated a lower potency shift from bio-
chemical to cell-based assays than their corresponding fused
imidazole analogues. We next examined examples 3, 6, 7, 11
and 18 in male Sprague–Dawley rat to determine whether the
NHBoc
NHBoc
O
N
H
31
NHBoc
O
30
Cl
O2N
b
N
N
SO2Ph
N
N
33
N
H2N
NHBoc
c, d, e, f
N
H
23
N
32
Scheme 3. Reagents and conditions: (a) DPPA, Et3N, toluene, then allyl alcohol,
DMAP, 72%; (b) 1,3-dimethylbarbituric acid, Pd(PPh3)4, DCM, reflux, 56%; (c) 33,
iPr2EtN, propan-2-ol, reflux, 2 h, 88%; (d) Fe, NH4Cl, MeOH, H2O, reflux, quantita-
tive; (e) sodium nitrite, AcOH, rt, 79%; (f) NaOH (aq 1 M), MeOH, THF, rt, 35%.
F
F
a
F
NBoc
NBoc
(+/-)
NBoc
R3
b
R3
+
O
N
H
N
H
(+/-)
36a, R3 = Bn
36b, R3 = H
34
35a, R3 = Bn
35b, R3 = H
b
c, d, e, f, g
c, d, e, f, g
H
N
H
N
F
F
N
N
N
N
N
N
N
H
N
H
N
N
15 (+/-)
14 (+/-)
Scheme 4. Reagents and conditions: (a) BnNH2, NaCNBH4, MeOH, AcOH, rt,
overnight, 30% (35a), 36% (36a); (b) H2, 10% Pd on C, EtOAc; (c) 33, iPr2EtN,
propan-2-ol, reflux; (d) H2, 10% Pd on C, EtOAc; (e) sodium nitrite, AcOH; (f) TFA,
DCM, 45 min; (g) NaOH (aq 1 M), EtOH, 60 °C, 2 h, 23% (15).

C. A. Hurley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3592–3598
3597
OR4
OR4
OAc
OH
a, b, c, d
e
g
N
N
N
N
N
N
N
N
N
H2N
N
N
N
N
N
H
N
SO2Ph
SO2Ph
39, R4 = H
40, R4 = -CH2CH2CN
17, R4 = H
37
38
f
19, R4 = -CH2CH2CN
Scheme 5. Reagents and conditions: (a) 33, iPr2EtN, propan-2-ol, reflux, overnight, 96%; (b) acetyl chloride, iPr2EtN, DMAP, DCM, rt, overnight; (c) Fe, NH4Cl, MeOH, H2O,
reflux; (d) sodium nitrite, AcOH, rt, 45 min, 34% over three-steps; (e) LiOH (aq 1 M), THF, rt; (f) sodium hydride, MeCN, rt, then 3-bromopropionitrile, rt, overnight; (g) NaOH
(aq 2 M), MeOH, rt, overnight, 84% (17) and 16% (19).
observed improvement in permeability would lead to improved
oral exposure compared to the imidazole series. Analogues 3,
7, 11 and 18 exhibited superior oral bioavailabilities when
benchmarked against their corresponding imidazole equivalents.
Furthermore, compounds 6, 7 and 18 also displayed lower plas-
ma clearance following intravenous administration. Finally all
compounds, except 11, had higher oral AUC and Cmax values.
The extremely high clearance observed for compound 3 follow-
ing iv dosing was reduced in compounds 6 and 7. Conversely,
compound 11 still showed exceptionally high clearance in vivo
which prohibited further development of this compound. Overall,
the rat pharmacokinetic profiles of compounds 3, 6, 7 and 18
compare favorably to that reported for the pan-JAK inhibitor
tofacitinib (1).20
ate 39 was alkylated with 3-bromopropionitrile to give 40; subse-
quent unmasking of the pyrrole furnished analogue 19.
In summary, we have identified a novel fused triazolo-pyrrolo-
pyridine tricyclic scaffold which exhibited nanomolar JAK1 en-
zyme potency with intrinsic but modest selectivity against the
JAK2 isoform and excellent selectivity against other non-JAK ki-
nases. Representative compounds 3, 7, 11 and 18 demonstrated
significantly improved in vitro permeability and better oral bio-
availability compared to corresponding imidazo-pyrrolopyridine
analogues.17 Furthermore rat PK profiles for analogues 3, 6, 7 and
18 compared favorably to the reported profile of tofacitinib (1).20
Acknowledgments
Preparation of the triazolo-pyrrolopyridine scaffold is outlined
in Scheme 1.21 The synthesis of key diamine intermediate 25 has
been disclosed previously.17 Cyclisation of 25 was achieved using
either n-butyl nitrite/copper bromide or sodium nitrite in acetic
acid. Subsequent cleavage of the sulfonyl protecting group under
basic conditions furnished analogue 4. Removal of the benzyl
group was facile under transfer hydrogenation conditions affording
amine 26 in good yield. Amine 26 was converted to analogue 3
using an EDCI-mediated coupling with cyanoacetic acid.
The syntheses of amino cyclohexane analogues 16, 18, 20, 21,
and 22 are illustrated in Scheme 2. Cyclisation of diamino interme-
diate 27,17 using sodium nitrite, generated triazole 28. Deprotec-
tion of the sulfonamide moiety furnished boc-protected amine 20
which was readily converted to amine 16 and then 18. To prepare
carbamate 21 and amide 22, compound 16 could not be utilized as
the competing di-acylation occurred. In the event, compounds 21
and 22 were best produced via boc-deprotection of intermediate
28 followed by amine derivatization and unmasking of the pyrrole
nitrogen under basic conditions.
The synthesis of chiral cyclopentyl analogue 23 is summarized in
Scheme 3. Commercially available amino acid 30 was converted to
protected amine 31 utilizing a Curtius rearrangement. Deprotection
oftheallylcarbamateprotectinggroupfurnished32, whichwassub-
sequently coupled to intermediate 33,17 and progressed to analogue
23 using standard methodology. Preparation of racemic cyclopentyl
analogue24 utilized similarprocedures as describedherein and con-
struction of racemic trans-diaminocyclopentane followed previ-
ously reported methodology.22
We wish to thank Russell Scammell, Michael Podmore and Neil
Bayliss for analytical support, Ning Liu (microsomal stability),
Emile Plise and Jonathan Cheng (MDCK permeability) for in vitro
ADME support, and Peter Dragovich for editorial assistance.
References and notes
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J. N. Cell 1998, 93, 385.
15. Haan, C.; Rolvering, C.; Raulf, F.; Kapp, M.; Druckes, P.; Thoma, G.; Behrmann, I.;
Zerwes, H.-G. Chem. Biol. 2011, 18, 314.
16. (a) 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.;
Fluorinated piperidines 14 and 15 were generated using the
route illustrated in Scheme 4. Amines 35b and 36b were prepared
according to literature procedures and were converted to triazole
analogues 14 and 15 using established conditions.23
The route to analogues 17 and 19 is summarized in Scheme 5.
Intermediate 38 was generated by reaction of 33 with 4-aminocy-
clohexanol (37), followed by protection of the alcohol moiety
(acetyl), reduction of the nitro group, and subsequent cyclisation
with sodium nitrite. Deprotection of 38 provided 39, which was
converted to analogue 17 under hydrolytic conditions. Intermedi-

3598
C. A. Hurley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3592–3598
Zwillich, S. H. Arthritis Rheum. 2012, 64, 617; (b) Frenzel, K.; Wallace, T. A.;
100 mL) at ambient temperature for 20 min. The mixture was partially
concentrated under vacuum and the aqueous residue was extracted with
ethyl acetate (3Â). The combined extracts were washed with brine, dried with
sodium sulfate and concentrated under vacuum to leave an orange residue.
Purification by column chromatography on silica gel (gradient: 0–100% ethyl
acetate in DCM) afforded 1.17 g (98%) of 1-[(3R)-1-benzylpiperidin-3-yl]-1,6-
dihydropyrrolo[2,3-b][1,2,3]triazolo[4,5-d]pyridine (4) as a yellow residue. MS
(ESI): m+H = 333.2; 1H NMR (400 MHz, DMSO-d6) d: 12.34 (s, 1H), 8.99 (s, 1H),
7.55 (t, J = 2.8 Hz, 1H), 7.34–7.17 (m, 5H), 6.78 (dd, J = 3.5, 1.8 Hz, 1H), 5.10–
5.00 (m, 1H), 3.72–3.49 (m, 2H), 3.22 (m, 1H), 2.95 (m, 1H), 2.57–2.50 (m, 1H),
2.32 (m, 1H), 2.26–2.09 (m, 2H), 1.98–1.83 (m, 2H). A solution of 4 (1.16 g,
3.49 mmol) in MeOH (100 mL) was treated with palladium hydroxide (20 wt %
on C, 244 mg) and ammonium formate (2.20 g, 34.9 mmol) at reflux for 2 h.
After cooling the mixture was filtered through Celite and concentrated. The
product was purified by column chromatography on silica gel (eluting with 5–
10% [2 M NH3 in MeOH] in DCM) to provide 768 mg (91%) of 1-[(3R)-piperidin-
3-yl]-1,6-dihydropyrrolo[2,3-b][1,2,3]triazolo[4,5-d]pyridine (26) as a white
solid. MS (ESI): m+H = 243.3; 1H NMR (400 MHz, CDCl3) d: 10.18 (s, 1H), 9.11
(s, 1H), 7.45 (m, 1H), 6.90 (m, 1H), 4.96 (m, 1H), 3.55 (m, 1H), 3.44 (dd, J = 12.4,
9.5 Hz, 1H), 3.20–3.13 (dt, J = 12.4, 3.9 Hz, 1H), 2.88 (m, 1H), 2.48–2.40 (m, 2H),
2.02–1.94 (m, 1H), 1.86–1.71 (m, 1H). To a stirred solution of 26 (72 mg,
McDoom, I.; Xiao, H. D.; Capecchi, M. R.; Bernstein, K. E.; Sayeski, P. P. Exp. Cell
Res. 2006, 312, 2735; (c) Neubauer, H.; Cumano, A.; Muller, M.; Wu, H.;
Huffstadt, U.; Pfeffer, K. Cell 1998, 93, 397.
17. Kulagowski, J. J.; Blair, W.; Bull, R. J.; Chang, C.; Deshmukh, G.; Dyke, H. J.;
Eigenbrot, C.; Ghilardi, N.; Gibbons, P.; Harrison, T. K.; Hewitt, P. R.; Liimatta,
M.; Hurley, C. A.; Johnson, A.; Johnson, T.; Kenny, J. R.; Kohli, P. B.; Maxey, R. J.;
Mendonca, R.; Mortara, K.; Murray, J.; Narukulla, R.; Shia, S.; Steffek, M.;
Ubhayakar, S.; Ultsch, M.; van Abbema, A.; Ward, S. I.; Waszkowycz, B.; Zak, M.
J. Med. Chem. 2012, 55, 5901.
18. ACD/labs 2012 release (build 1996. 1 June 2012) was used to calculate cpKa and
c LogD values.
19. Biochemical potencies for 3: JAK3 Ki = 24 nM and TYK2 Ki = 20 nM. Biochemical
potencies for 11: JAK3 Ki = 369 nM and TYK2 Ki = 234 nM. Biochemical
potencies for 18: JAK3 Ki = 122 nM and TYK2 Ki = 80 nM.
20. (a) Reported rat PK profile for 1: CL = 62 mL/min/kg; F = 27%; Cmax = 442 ng/
mL; AUC (po) (normalized to 5 mg/kg) = 1.16
lM h. AUC was calculated in
house using the reported values for CL and
%
F.; (b) Flanagan, M. E.;
Blumenkopf, T. A.; Brissette, W. H.; Brown, M. F.; Casavant, J. M.; Shang-Poa,
C.; Doty, J. L.; Elliott, E. A.; Fisher, M. B.; Hines, M.; Kent, C.; Kudlacz, E. M.;
Lillie, B. M.; Magnuson, K. S.; McCurdy, S. P.; Munchhof, M. J.; Perry, B. D.;
Sawyer, P. S.; Strelevitz, T. J.; Subramanyam, C.; Sun, J.; Whipple, D. A.;
Changelian, P. S. J. Med. Chem. 2010, 53, 8468.
297
hydroxybenzotriazole (56.0 mg, 416
(58.0 mg, 475 mol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (91.0 mg, 475 mol) were added. The mixture was then
l
mol) in DCM (15 mL) at 0 °C, cyanoacetic acid (30.0 mg, 357
lmol), N-
lmol), 4-(dimethylamino)pyridine
21. (a) Compounds 5–12 were prepared using either 4-amino-1-benzylpiperidine
or 4-amino-4-methyl-1-benzylpiperidine adapting the procedures described in
Ref. 17. (b) Preparation of 3-oxo-3-[(3R)-3-(pyrrolo[2,3-b][1,2,3]triazolo[4,5-
d]pyridin-1(6H)-yl)piperidin-1-yl]propanenitrile (3): A stirred solution of 25
(300 mg, 0.65 mmol) in acetonitrile (6 mL) was treated with copper(II)
l
l
stirred at ambient temperature for 18 h. The crude reaction mixture was
purified by column chromatography on silica gel (gradient: 0–6% methanol in
DCM) affording 82 mg (92%) of
3 as an off-white solid. LCMS (ESI):
bromide (174 mg, 0.78 mmol) and n-butyl nitrite (114
lL, 0.97 mmol) and
m+H = 310.1; 1H NMR was consistent with amide rotamers (400 MHz,
DMSO-d6) d: 12.38 (br s, 1H), 9.02 (s, 1H), 7.64–7.59 (m, 1H), 7.16–7.08 (m,
1H), 5.28–4.96 (m, 1H), 4.81–4.31 (m, 1H), 4.20–3.70 (m, 3H), 3.36–2.96 (m,
2H), 2.44–1.68 (m, 4H).
then heated to 70 °C for 2 h. After cooling, the reaction was quenched by the
addition of 1 M aqueous HCl solution (ca. 5 mL) and stirred for 5 min. The
mixture was basified with saturated sodium hydrogen carbonate solution and
extracted with ethyl acetate (3Â). The combined extracts were washed with
brine, dried with sodium sulfate and concentrated under vacuum to leave a
brown residue. Purification by column chromatography on silica gel (gradient:
0–60% ethyl acetate in cyclohexane) gave 200 mg (65%) of 1-[(3R)-1-
benzylpiperidin-3-yl]-6-(phenylsulfonyl)-1,6-dihydropyrrolo[2,3-
22. Racemic trans (3-amino-cyclopentyl)-carbamic acid tert-butyl ester was
prepared following the methods outlined in (a) Li, F.; Brogan, J. B.; Gage, J. L.;
Zhang, D.; Miller, M. J. J. Org. Chem. 2004, 69, 4538; (b) Ranganathan, S.; George,
K. S. Tetrahedron 1997, 53, 3347; (c) Gregson, M.; Ayres, B. E.; Ewan, G. B.; Ellis,
F.; Knight, J. Patent WO9417090.; (d) Ray, A. K.; Sigfridsson, E. M.; Linusson, A.
S. M.; Sandberg, P. M.; Inghardt, T.; Svensson, A. M.; Brickmann, K. Patent
WO2004004726.
23. Synthesis of intermediate 34: (a) Budzik, B.; Garzya, V.; Shi, D.; Walker, G.;
Lauchart, Y.; Lucas, A. J.; Rivero, R. A.; Langmead, C. J.; Watson, J.; Wu, Z.;
Forbes, I. T.; Jin, J. Bioorg. Med. Chem. Lett. 2010, 20, 3545; (b) Synthesis of
amines 35b and 36b: Dale, G. E.; Pierau, S.; Cappi, M.; Gray, C.; Hubschwerlen,
C.; Surivet, J.-P.; Zumbrunn, C. Patent WO2007042325.
b][1,2,3]triazolo[4,5-d]pyridine as a brown residue. MS (ESI): m+H = 473.5; 1H
NMR (400 MHz, CDCl3) d: 9.18 (s, 1H), 8.21 (m, 2H), 7.82 (d, J = 3.7 Hz, 1H), 7.57
(m, 1H), 7.53–7.46 (m, 2H), 7.30 (s, 5H), 6.75 (d, J = 3.7 Hz, 1H), 4.88 (m, 1H),
3.61 (m, 2H), 3.25 (m, 1H), 3.03 (m, 1H), 2.57 (m, 1H), 2.39–2.17 (m, 3H), 2.10–
1.87
(m,
2H).
1-[(3R)-1-Benzylpiperidin-3-yl]-6-(phenylsulfonyl)-1,6-
dihydropyrrolo[2,3-b][1,2,3]triazolo[4,5-d]pyridine (1.70 g, 3.60 mmol) was
treated with 2 M aqueous NaOH solution (10 mL) and methanol/THF (1:1,