Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.05.043

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.

Full text of DOI:10.1016/j.bmcl.2017.05.043

Accepted Manuscript
Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibi-
tors
John Hynes Jr., Hong Wu, James Kempson, James J.-W. Duan, Zhonghui Lu,
Bin Jiang, Sylwia Stachura, John S. Tokarski, John S. Sack, Javed A. Khan,
Jonathan S. Lippy, Rosemary F. Zhang, Sidney Pitt, Guoxiang Shen, Kate
Gillooly, Kim McIntyre, Percy H. Carter, Joel C. Barrish, Steven G. Nadler,
Luisa M. Salter-Cid, Aberra Fura, Gary L. Schieven, William J. Pitts, Stephen
T. Wrobleski
PII:
S0960-894X(17)30524-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.043
BMCL 24988
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 March 2017
8 May 2017
13 May 2017
Please cite this article as: Hynes, J. Jr., Wu, H., Kempson, J., Duan, J.J., Lu, Z., Jiang, B., Stachura, S., Tokarski,
J.S., Sack, J.S., Khan, J.A., Lippy, J.S., Zhang, R.F., Pitt, S., Shen, G., Gillooly, K., McIntyre, K., Carter, P.H.,
Barrish, J.C., Nadler, S.G., Salter-Cid, L.M., Fura, A., Schieven, G.L., Pitts, W.J., Wrobleski, S.T., Discovery of
potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibitors, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.043
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Discovery of potent and efficacious pyrrolopyridazines
as dual JAK1/3 inhibitors.
John Hynes, Jr.∗, Hong Wu, James Kempson, James J.-W. Duan, Zhonghui Lu, Bin Jiang, Sylwia Stachura,
John S. Tokarski, John S. Sack, Javed A. Khan, Jonathan S. Lippy, Rosemary F. Zhang, Sidney Pitt, Guoxiang
Shen, Kate Gillooly, Kim McIntyre, Percy H. Carter, Joel C. Barrish, Steven G. Nadler, Luisa M. Salter-Cid,
Aberra Fura, Gary L. Schieven, William J. Pitts, and Stephen T. Wrobleski

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3
inhibitors.
John Hynes, Jr.∗, Hong Wu, James Kempson, James J.-W. Duan, Zhonghui Lu, Bin Jiang, Sylwia Stachura,
John S. Tokarski, John S. Sack, Javed A. Khan, Jonathan S. Lippy, Rosemary F. Zhang, Sidney Pitt,
Guoxiang Shen, Kate Gillooly, Kim McIntyre, Percy H. Carter, Joel C. Barrish, Steven G. Nadler, Luisa M.
Salter-Cid, Aberra Fura, Gary L. Schieven, William J. Pitts, and Stephen T. Wrobleski
Research and Development, Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective
JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups
provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good
selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine
model of chronic inflammation. X-ray co-crystal structure determination confirmed the
presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will
be described.
Revised
Accepted
Available online
Keywords:
JAK inhibitor
Pyrrolopyridazine
CIA efficacy
JAK1/3
2009 Elsevier Ltd. All rights reserved
.
———
∗ Corresponding author. Tel.: +1-609-252-4431; e-mail: john.hynes@bms.com

The therapeutic blockade of the Janus kinase (JAK) family of
non-receptor tyrosine kinases has shown promise for a variety of
human diseases. These kinases (JAK1, JAK2, JAK3, and Tyk2)
are phosphorylated in response to cytokine receptor activation,
leading to gene transcription and signal propagation.1 In the
context of inflammatory diseases, JAK1, JAK3, and Tyk2 have
been implicated as targets for therapeutic intervention and a
variety of JAK inhibitors have completed or are currently being
studied in clinical trials.2 Tofacitinib (pan-JAK1/JAK2/JAK3
inhibitor) was approved in 2012 for rheumatoid arthritis (RA)
and represents the first small molecule kinase inhibitor to reach
The dual JAK1/3 inhibition profile of 2 was particularly
attractive. JAK3 was initially targeted by many in the field but
emerging data implicates JAK1 in the pathogenesis of
inflammatory diseases such as RA.13
Key inflammatory
cytokines that signal through a JAK1 dependent process include
IL-1, IL-6 and IFN-alpha and gamma. Compounds such as 2
would provide a unique opportunity to evaluate the benefit of
dual JAK1/3 blockade in animal models of inflammatory
diseases.
Initial in life studies in a murine pharmacodynamic model of
JAK1/3 signaling (IL-15 induced phospho STAT5 inhibition)
provided an EC50 of 125 30 nM for 2.14 This level of functional
potency in combination with the sustained serum drug levels
observed in the PK studies prompted further evaluation in a
disease model of chronic inflammation. Analog 2 was studied in
a pseudo-established mouse collagen-induced arthritis (mouse
CIA) model at 2, 10, and 50 mg/kg, once daily oral dosing.
Complete suppression of the clinical signs of the disease was
achieved at 10 mg/kg (C24 390 nM), Figure 1. The 2 mg/kg dose
was not efficacious, a result consistent with minimal coverage of
the mouse EC50 over the course of the study (C24 60 nM) while
the 50 mg/kg dose was not tolerated in the study. Within the
same study, tofacitinib at 20 mg/kg was efficacious albeit with
BID dosing. Histological scores (inflammation, bone resorption)
were consistent with the visual disease activity scores.
the market in this autoimmune disease.3
Ruxolitinib
(JAK1/JAK2 inhibitor) has been approved for the treatment of
myelofibrosis targeting a gain of function mutation (V617F) in
JAK2.4 Selective JAK1 inhibition with filgotinib5 and ABT-4946
have been reported to be efficacious in RA patients and are
advancing into Phase 3 clinical trials. An NDA for the JAK1/2
dual inhibitor, baricitinib, has recently been filed with the FDA
for the treatment of RA.7
Previously, we have described pyrrolo[1,2-b]pyridazine-3-
carboxamides as JAK inhibitors.8 The focus of that effort was to
optimize JAK3 inhibition while sparing JAK2-mediated side
effects such as anemia.9 Compound 1 (Table 2) was identified as
a potent JAK3 inhibitor (IC50 5 nM) with moderate JAK family
enzyme selectivity.10 In cell-based functional assays measuring
EPO-induced JAK2 signaling vs. IL-2-induced JAK1/3 signaling,
1 was 130-fold selective over JAK2. In human whole blood, 1
had an IC50 of 210 nM (JAK1/3 dependent IL-2 induced IFN-γ
production).11 Moderate oral exposure in rodents was observed
for 1. In addition, 1 was weakly bound to serum proteins (52%
free).
Table 1. Lead Compound JAK profiles.
NH2
NH2
HN
HN
N
O
O
4
5
7
H2N
H2N
6
N
N
N
1
2
hWB
JAK3 JAK1 TYK2 JAK2 EPO/IL2
IFNγ
IC50,
nM
Ex
IC50,
nM
IC50,
nM
IC50,
nM
IC50,
nM
IC50
ratio
PKa
Figure 1. Mouse CIA efficacy – clinical scores. Values are reported
as mean SEM, n=11–15 mice per group. *p<0.05 vs. vehicle,
Mann-Whitney U Test. Vehicle, PEG 300.
Rat F% 24
Cl >100%b
5
19
97
62
130
210
1
2
Although this compound was efficacious in this arthritis
disease model, safety concerns emerged from the in vitro
profiling assays. Cardiac hERG channel inhibition (flux IC50 3.8
µM, patch clamp IC50 ~0.3 µM), and to a lesser extent Ca2+ and
Na+ channel inhibition, were viewed as key liabilities for
advancement of this compound.15 Moderate Cyp3A4 inhibition
(IC50 3 µM) was also observed. A small internal kinase panel (16
kinases) indicated good selectivity, however, a broader kinome
screen revealed potential issues (30 kinases <30% of control).16
The focus then turned towards optimizing the cardiac channel
safety profile and improving the kinase selectivity while
maintaining the whole blood potency.
Mouse F% 67
Cl 32%c,d
0.9
0.4
7.5
9.0
300
200
a.
Clearance is reported as percent hepatic blood flow, b rat total CL
145 mL/min/kg, c mouse total CL 29 mL/min/kg, d C24 230 nM
Structurally, 1 represented an excellent starting point for
further modification on the pyrrole ring (C5–C7) with the goal of
improving potency and PK. Given the established binding mode
of this inhibitor class, substitution at C7 was anticipated to
negatively impact the hinge (Leu905) binding due to sterics and
was not pursued. C5 substitution was not tolerated (JAK3 IC50
>500 nM, data not shown). However, the C6 phenyl analog (2)
improved JAK family inhibition with the most notable
improvement in biochemical potency against JAK1 (48-fold).
Cellular selectivity against JAK2 was maintained as was the
whole blood potency.12 In mice, 2 was orally bioavailable (F%
67) and provided sustained serum exposures at or above the
mouse whole blood (JAK1/3 dependent IL-15 induced phospho-
STAT5 production, IC50 270 nM) for 24 h with a single 10 mg/kg
dose.
Structurally, 2 bound to JAK3 in an identical fashion as the
previously disclosed C6 unsubstituted inhibitors (Figure 2). The
pyrrolopyridazine N1 forms the critical hinge hydrogen bond
with Leu905 while the primary carboxamide NH interacts with
the carbonyl of Glu903 which is located deeper in the binding
pocket and proximal to the Met902 gatekeeper residue. The
cyclopentyl amine engages additional hydrogen bonding

interactions with Asn954 and the backbone carbonyl of Arg953.
The newly installed C6 phenyl group projects into the extended
hinge region of the kinase.
Figure 3. Orientation of 2 bound to JAK3 highlighting the Leu828-
Gly908 lipophilic region.
Scheme 1 outlines our synthesis of the C6-substituted
pyrrolo[1,2-b]pyridazine analogs. Methyl 1-amino-4-bromo-1H-
pyrrole-2-carboxylate17
was
converted to 6-bromo-4-
hydroxy[1,2-b]pyridazine-3-nitrile (3) followed by treatment
with POCl3 to provide chloride 4. Hydrolysis of the C3 nitrile
provide
6-bromo-4-chloropyrrolo[1,2-b]pyridazine-3-
carboxamide (5) which was reacted with (1S,3R)-1,2,2-
trimethylcyclopentane-1,3-diamine18 to afford (6). Subsequent
coupling with aromatic and heteroaromatic boronic acids
provided analogs 219, 7–38.
Figure 2. X-ray co-Crystal structure of 2 bound to the catalytic
domain of JAK3 with key residues labeled (PDB ID 5VO6).
The increased JAK family biochemical potency of 2 relative
to 1 may be due to increased lipophilic interactions of the C6
phenyl with key regions of the kinase. Specifically, in JAK3, the
C6 phenyl group is between Leu828 in the P-loop and Gly908 of
Br
Cl
OH
a
b
c
NC
NC
O
Br
Br
N
N
N
O
N
N
NH2
3
4
the extended hinge region (Figure 3).
Combined, these
NH2
NH2
interactions may explain the potency shift (at least 5-fold) against
all kinases in this family since these residues are conserved. The
subtle differences in the JAK kinase family sequence make the
significant shift in biochemical potency with regards to JAK1
more difficult to rationalize. A possible explanation may be
related to Asp912 in JAK3, conserved in JAK2 and Tyk2.
However, in JAK1 this is replaced with the longer Glu (Glu966
in JAK1, Figure 2) which may form a hydrogen bond with the
adjacent Ser963 (Cys909 in JAK3). This could expose the extra
methylene of Glu966 to the face of the aryl ring providing an
additional interaction with the kinase.
Cl
HN
HN
O
O
O
d
e
H2N
H2N
H2N
Br
Br
Ar
N
N
N
N
N
N
5
6
2, 7–38
Scheme 1. Synthesis of C6 analogs.
Reagents and Conditions: a. (EtO)2CHCH2CN, TsOH, 120 °C then DBU,
80 °C, 37% yield; b. POCl3, 80 °C, 34% yield; c. H2SO4, 55 °C, 65%
yield; d. (1S,3R)-1,2,2-trimethylcyclopentane-1,3-diamine, DIPEA,
DMF, 95 °C, 67% yield; e. ArB(OH)2, Pd(OAc)2, X-Phos, 2M aq.
K3PO4, dioxane, 145 °C.
While the C6 aryl substituent, oriented at the surface of the
kinase, was not anticipated to significantly modulate biochemical
potency further, an evaluation of the effects of diverse aryl
substitution was initiated in an empirical attempt to further
improve whole blood potency and decrease hERG channel
inhibition. Access to a broad array of substituted aryl groups and
heterocycles provided the compounds highlighted in Table 2. In
general, electron donating (7–12) substituents on the phenyl ring
were tolerated in the JAK3 & JAK1 biochemical assays.
Functional selectivity against JAK2 (EPO/IL2 ratio), for the most
part, was maintained. Several-fold losses in whole blood potency
was observed for the methyl substituted phenyl rings, whereas
the meta-methoxy (11) and para-methoxy (12) substitutions
retained whole blood potency. Unfortunately for this set of
compounds, hERG channel inhibition was not modulated
favorably (max 3.5 fold for 12). Similar observations with
regards to potency, selectivity, and hERG channel inhibition was
observed with electron withdrawing substituents on the C6

phenyl ring. Fluoro (13–15), chloro (16–18), and cyano (19–21)
substituents on the aryl ring provided potent JAK1/3 inhibitors,
however, several compounds were weakly potent in whole blood
(14, 16, 20). The 4-cyanophenyl analog 21 provided sub-100 nM
whole blood potency and a 3-fold improvement in hERG channel
inhibition relative to 2. Heterocyclic substitutions at C6 (22, 23)
did offer the first set of compounds with good to excellent whole
blood potency and significantly attenuated hERG channel
inhibition. Pyrimidine 24 retained the hERG liability whereas
pyrazole 25 did not but suffered from reduced biochemical
selectivity against JAK2 and Tyk2.

Table 2. C6 aryl SAR
JAK3
IC50, nM
JAK1
IC50, nM
TYK2
IC50, nM
JAK2
IC50, nM
EPO/IL2
IC50 ratio
WB IFNγ hERG flux
IC50, µM
Example
R
IC50, nM
phenyl
2-Me-Phe
3-Me-Phe
4-Me-Phe
2-OMe-Phe
3-OMe-Phe
4-OMe-Phe
2-F-Phe
0.9 0.5
1.8
0.9
0.7
1.3
0.4
1.5
1.5
1.6
2.1
1.3
1.0
0.8
1.9
3.8
1.0
0.6
6.7
11
0.4 0.2
0.7
0.9
0.8
0.5
1.0
0.6
1.2
1.2
1.2
0.8
1.3
0.9
1.6
7.2
0.9
0.7
4.9
28
7.5 1.5
51
9.0 1.5
49
300
940
50
200
3.8
2
490
700
4.9
7.7
-
7
7.9
8.6
21
11
8
14
-
>2,000
680
9
10
20
562
305
46
7.5
7.2
13.8
9.6
7.6
10.2
-
12
15
200
11
9.2
15
15
170
12
17
560
-
630
13
3-F-Phe
22
22
>1,000
420
14
15
4-F-Phe
14
27
230
-
2-Cl-Phe
3- Cl -Phe
4- Cl -Phe
2-CN-Phe
3-CN-Phe
4-CN-Phe
3-pyr
47
27
>1,000
16
20
17
230
780
6.0
-
17
10
15
nta
18
34
25
240
-
500
-
19
20
55
47
>1,000
66
-
8.6
34
7.3
11
150
550
550
180
100
12
21
110
>80
>80
8.5
>80
22
4-pyr
44
80
56
23
5-pyrimidyl
3-pyrazolyl
190
14
160
21
240
24
25
1.6
1.7
81
ant = not tested
Given the improved whole blood potency, JAK family
selectivity, and hERG channel profile of the initial pyridyl
analogs 22 and 23, we initiated follow up efforts to define the
preferred substitution patterns around the C6 pyridine (Table 3).
In the case of 4-pyridines, substitution meta to the pyridine
nitrogen was favored for reducing activity in the hERG flux
assay (26 vs. 27 for methyl; 28 vs. 29 for fluoro). Methoxy
substitution ortho to the nitrogen (30) reduced whole blood
potency. In the case of 3-pyridyl groups at C6, potency and
selectivity was generally retained for methyl (31, 32) and fluoro
(33, 34) substitutions. Methoxy substitution ortho to the 3-
pyridine nitrogen (35) did offer an advantage against hERG
liabilities and maintained the desired selectivity and whole blood
potency. Interestingly, when the alkoxy group size was increased
(36, 37), whole blood potency decreased and hERG inhibition
returned suggesting that lipophilicity may play a role in this
liability trend. Cyano substitution ortho to the pyridine nitrogen
(38) was tolerated at the enzyme level but not in the whole blood
assay or hERG flux assay.

Table 3. C6 pyridine SAR
JAK3
IC50, nM
JAK1
IC50, nM
TYK2
IC50, nM
JAK2
IC50, nM
EPO/IL2
IC50 ratio
WB IFNγ hERG flux
IC50, µM
Example
R
IC50, nM
4
2.4
0.6
29
19
250
8
75
>80
26
0.6
5.7
3.5
55
30
27
28
4.6
1.6
2.8
3.6
3.3
2.3
46
59
36
70
38
36
150
35
120
>80
270
350
41
nta
29
30
86
6.1
0.9
1.3
2.0
180
29
140
22
120
80
97
84
18
8
31
32
1.9
6.6
60
44
160
220
5
33
0.4
0.9 0.5
1.1
2.2
1.9 0.6
4.0
27
25 9.7
38
19
15 6.9
30
13
130
120
2
100
100 30
800
35
>80
17
34
35
36
37
0.5
1.2
30
20
400
5.3
4.1
1.0
6.6
47
29
160
430
38
ant = not tested
Table 4. Coarse PK & Profiling data for select analogs. PO dosing at
Compounds with good whole blood potency and improved
hERG channel selectivity were advanced to mouse PK studies
(Table 4). Compounds 23, 25 and 28 did not provide adequate
exposures at Cmax (<0.25 µM). Addition of a fluoro substituent
(34) in the 3-pyridyl series provided a moderate Cmax (0.3 µM)
and reasonably sustained drug exposures over an 8 h treatment
window. Ultimately, the methoxy-3-pyridyl analog (35)
provided the best circulating exposures in the series (Cmax 1.1
µM) over a 24 h treatment window.
10 mg/kg, PEG 300 vehicle.
Cmax
Cmin
Caco2b
(nm/s)
MLMc
PPBd
(%)
Example
(µM)
(µM)
BLLQa
(%rem)
23
25
28
34
35
<0.25
<0.25
<0.25
0.3
<15
65
75
83
68
88
95
69
49
78
nt
BLLQ
BLLQ
23
0.1 (8 h)
0.3 (24 h)
<15
26
1.1
93
aBLLQ = below the level of quantitation, bCaco-2 permeability, apical to
basolateral cmouse liver microsomal stability, percent remaining after 10
minutes dmouse plasma protein binding
A more detailed profile of 35 is shown in Table 5. Moderate
permeability with minimal efflux in the Caco-2 assay was
observed. In human microsomal incubations, an extended T1/2
was observed. Minimal Cyp 3A4 inhibition was noted and
hERG channel inhibition relative to 2 was improved 10-fold as

measured in the patch clamp assays.20 Protein binding was
moderate (~10% free, human). Crystalline 35 possessed suitable
aqueous solubility for in vivo formulations and PK in rats was
consistent with the data obtained from mice (Table 6).
Table 5 Compound 35 ADME properties.
Caco-2 permeability
Human metabolic stability (T1/2
Cyp 3A4 IC50
26 nm/s
>90 min
)
11 µM
>17 µM
PXR EC50
hERG patch clamp IC50
Ca patch clamp
Na patch clamp
plasma protein binding (%
bound)
Aq. Solubility, pH 6.5
3 µM
16% inhib at 10 µM
71% inhib at 10 µM
90 (hum), 88 (rat), 93 (mse)
0.079 mg/mL
Table 6 Pharmacokinetic profile of 35 in mice and rats.
balb/c mice
10 (po)b
SD rats
Dose (mg/kg)
2 (iv)a
–
2 (iv)c
–
10 (po)d
0.6
1.1
4
Cmax (µM)
Tmax (h)
–
–
7.3
4.9
6.5
16.7
8.2
–
17.9
10.2
5.8
9.8
14.1
11.5
–
12.9
12.2
AUC (µm.h)
T1/2 (h)
CL (mL/min/kg)
Vss (L/kg)
F%
73
44
avehicle 30% PEG 300–70% citrate buffer; bvehicle 5% ethanol–90%
PEG 300–5%TPGS; cvehicle 10% N,N-dimethylacetamide–30%
PEG 300–60% water; dvehicle 5% ethanol–90% PEG 300–5%TPGS
Inhibitor 35 provided exceptional inhibition of JAK1/3
mediated ex vivo processes in the mouse PD model (EC50 67
13 nM) and was advanced into IV rabbit telemetry studies to
assess potential cardiovascular liabilities, specifically, QT
interval prolongation. Unexpectedly, dose escalation (>3 mg/kg)
resulted in a significant decrease in mean arterial blood pressure
with a corresponding increase in heart rate, which confounded
any QT interval assessment. Further studies in conscious,
telemetrized rats confirmed a direct effect on blood pressure
(~14% decrease) at 30 mg/kg, PO. Dose escalation in rats (200
mg/kg) resulted in a rapid decrease of MAP leading to death.
Our preliminary investigations as to the cause of this
cardiovascular effect implicated off-target inhibition of Rho
family kinases as a potential factor. Rho kinase inhibitors have
been explored as potential anti-hypertensive agents21 and 35 was
found to inhibit both Rho Kinase 1 (IC50 95 nM) and Rho Kinase
2 (IC50 43 nM). Although the overall kinome selectivity of 35
relative to 2 had been improved (14 kinases <30% of control),
ROCK activity was persistent. The advancement of this series
was halted due to the significant pre-clinical CV safety findings.
Further studies detailing our efforts to mitigate the in vivo
cardiovascular liabilities will be the focus of future reports.
Acknowledgments
We gratefully acknowledge Richard A. Rampulla and the
Department of Discovery Synthesis at Biocon Bristol-Myers
Research Center for the large scale synthesis of intermediate 5
and Georgia Cornelius for analyzing PK samples.
Supporting Information
Supplementary data associated with this article can be found,
in the online version, at XXXXXX

(m, 1H), 2.05 (m, 1H), 1.84 (m, 1H), 1.50 (s, 3H), 1.18 (s, 3H), 1.08 (s, 3H).
LRMS m/z 378.14 (M+H)+.
References and notes
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clamp assay is noteworthy for this series, albeit limited in examples
(Compound 2 and 35). The high throughput hERG flux assay was used to
triage compounds for advanced in vitro liability studies. Patch clamp
assessments were limited to key compounds.
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7. Press release, www.lilly.com January 19, 2016.
8. J.-W. Duan, J.; Lu, Z.; Jiang, B.; Yang, B. V.; Doweyko, L. M.; Nirschl, D.
S.; Haque, L. E.; Lin, S.; Brown, G.; Hynes, Jr., J.; Tokarski, J. S.; Sack, J. S.;
Khan, J.; Lippy, J. S.; Zhang, R. F.; Pitt, S.; Shen, G.; Pitts, W. J.; Carter, P.
H.; Barrish, J. C.; Nadler, S. G.; Salter-Cid, L. M.; McKinnon, M.; Fura, A.;
Schieven, G. L.; Wrobleski, S. T. Bioorg. & Med. Chem. Letters 2014, 24,
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9. Sonbol, M. B.; Firwana, B.; Zarzour, A.; Morad, M.; Rana, V.; Tiu, R. V.
Ther. Adv. Hematol. 2013, 4, 15.
10. See reference 8 for a description of the kinase assays. Assay Variability
was measured using internal standards as controls. Individual assay
variability SD against a standard is highlighted in the table below:
JAK3 1.9 1.3 nM, n = 111
JAK1 3.5 5.7 nM, n = 128
Tyk2 82 41 nM, n = 61
JAK2 4.9 2.2 nM, n = 135
EPO STAT5 67 35 nM, n = 124
IL2 STAT3 3.1 1.4 nM, n = 134
hWB 41 24 nM, n = 214
11. Human whole blood assay protocol: Human whole blood was stimulated,
in the presence or absence of test compounds, with 1 µg/mL soluble anti-CD3
(R&D Systems), 10 ng/mL anti-CD28 (R&D systems) and 10 ng/mL IL-2
(BD Biosciences). The final volume of the assay was 200 µL with 50% whole
blood in RPMI 1640 (Gibco). The blood was incubated at 37 °C in 5% CO2 in
a humidified incubator for 18 h. The plates were centrifuged and supernatant
was removed. IFN-γ levels in the supernatant were determined using a human
IFN-γ ELISA set (BD Biosciences). Compounds were dissolved at 10 mM in
dimethylsufoxide (DMSO) and tested at 6 concentrations in triplicate. IC50
values were derived by nonlinear regression analysis.
12. Compounds 1 and 2 have a similar level of potency in the whole blood
assay, despite a significant improvement in JAK1/3 enzyme potency for 2.
This may be related to increased serum protein binding of 2 relative to 1
(97% vs. 48%).
13. Menet, C. J.; Mammoliti, O.; Lopez-Ramos, M. Future Med. Chem.
2015, 7, 203.
14. Female BALB/c mice, >10 weeks of age, were dosed orally with vehicle
or compound (PEG300, 5 mg/mL, n=8 per dose group). 4 hours later, blood
was collected for PK analysis and evaluation in an ex vivo IL-15 induced
phospho-STAT5 assay.
15. Pollard, D.E.; Gerges, N.A.; Bridgland-Taylor, M.H.; Easter, A.;
Hammond, T.; Valentin, J-P. Br. J. Pharmacol. 2010, 159, 12.
16. Ambit (now DiscoverX) KinomeScan was used to assess kinome
selectivity. Data for 2 and 35 are included in the Supporting Information.
17. Hynes, J., Jr.; Doubleday, W. W.; Dyckman, A. J.; Godfrey, J. D., Jr.;
Grosso, J. A.; Kiau, S.; Leftheris, K. J. Org. Chem., 2004, 69, 1368.
18. Jaramillo, D.; Buck, D. P.; Collins, J. G.; Fenton, R. R.; Stootman, F. H.;
Wheate, N. J.; Aldrich-Wright, J. R. Eur. J. Inor. Chem. 2006, 4, 839.
19. Wrobleski, S. T.; Brown, G.; Doweyko, L.; Duan, J.; Gou, J.; Hynes, J.;
Jiang, B.; Kempson, J.; Lin, S.; Lu, Z.; Spergel, S. H.; Tokarski, J. S.; Wu,
H.; Yang, B. V. WO 2012/125893, 2012. Compound 2: 1H NMR (400 MHz,
CD3OD) δ 8.17 (s, 1H), 7.97 (s, 1H), 7.69 (d, J=8.0 Hz, 2H), 7.40 (t, J=8.0
Hz, 2H), 7.26 (t, J=8.0 Hz, 1H), 7.20 (m, 1H), 4.71 1H), 2.49 (m, 1H), 2.14