The discovery of tricyclic pyridone JAK2 inhibitors. Part 1: Hit to lead

Article abstract of DOI:10.1016/j.bmcl.2010.10.031

This paper describes the discovery and design of a novel class of JAK2 inhibitors. Furthermore, we detail the optimization of a screening hit using ligand binding efficiency and log D. These efforts led to the identification of compound 41, which demonstrates in vivo activity in our study.

Full text of DOI:10.1016/j.bmcl.2010.10.031

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7421–7425
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of tricyclic pyridone JAK2 inhibitors. Part 1: Hit to lead
Tony Siu a, , Ekaterina S. Kozina a, Joon Jung a, Craig Rosenstein b, Anjili Mathur c, Michael D. Altman a,
⇑
Grace Chan b, Lin Xu d, Eric Bachman c, Jan-Rung Mo c, Melaney Bouthillette e, Thomas Rush a,
Christopher J. Dinsmore a, C. Gary Marshall f, Jonathan R. Young a
a Department of Chemistry, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
b Department of In Vitro Sciences, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
c Department of Pharmacology, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
d Department of Drug Metabolism, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
e Department of Basic Pharmaceutical Sciences, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
f Department of Oncology, Merck & Co., 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:
This paper describes the discovery and design of a novel class of JAK2 inhibitors. Furthermore, we detail
the optimization of a screening hit using ligand binding efficiency and log D. These efforts led to the iden-
tification of compound 41, which demonstrates in vivo activity in our study.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 August 2010
Revised 4 October 2010
Accepted 6 October 2010
Available online 12 October 2010
Keywords:
JAK2
Kinase
Ligand binding efficiency
Structure based design
The JAK/STAT pathway, a common signaling pathway of cyto-
kines, regulates numerous functions of hematopoiesis and immune
response including the differentiation and proliferation of cells.1
Janus kinase 2 (JAK2) is an intracellular non receptor tyrosine ki-
nase that belongs to the JAK family of kinases (JAK1, JAK2, JAK3,
and TYK2). The presence of a catalytically inactive pseudokinase
regulatory domain distinguishes the JAKs from other tyrosine ki-
nases. Activation of JAK2 through cytokine signaling leads to phos-
phorylation of cytokine tyrosine residues, which serve as docking
sites for signal transducers and activators of transcription (STATS).
Upon binding to the receptor, the STATS are phosphorylated, then
dissociated from the receptor, dimerized, and finally translocated
to the nucleus for gene transcription.
regulating the JAK/STAT pathway has encouraged us to evaluate
JAK2 inhibitors for various therapeutic areas including MPD. In this
letter, we report the strategy used to develop structure activity
relationships (SAR) to optimize a low micromolar screening hit
into a nanomolar lead with in vivo activity.
Our strategy focused on using a virtual screening approach to
rapidly identify hits and optimizing these hits utilizing rational
drug design coupled with library synthesis. From the thousands
of leads generated from a virtual screen,5 compound 1 (Fig. 1)
was identified as an attractive starting point to initiate chemistry.
Although compound 1 has moderate activity in the in vitro JAK2
biochemical assay6 with an IC50 = 1400 nM and a ligand binding
Recently, the identification of the somatic mutation JAK2V617F
has demonstrated that the deregulation of JAK2 activity leads to
pathogenesis of myeloproliferative disorders (MPD) including
polycythemia vera (PV), essential thrombocythemia (ET), and pri-
mary myelofibrosis (PMF).2 This clear link between gain of function
mutation and pathogenesis has prompted the development of
small molecule JAK2 inhibitors in the clinic as targeted therapies
for myeloproliferative disorders,3 and numerous companies have
recently reported several classes of JAK inhibitors.4 Our focus in
HN
O
N
N
H
N
1
JAK2 IC50 = 1400 nM
LBE = 0.32
⇑
Corresponding author.
E-mail address: tony_siu@merck.com (T. Siu).
Figure 1. Virtual screening hit.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.031

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T. Siu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7421–7425
N
N
MeO
N
MeO
N
O
N
O
a
OTf
b
OH
O
Cl
N
H
F
F
2
3
4
N
HN
MeO
N
O
N
NR1R2
NR1R2
c
d
F
F
5
6
Scheme 1. Synthesis of the napthyridinone core. Reagents and conditions: (a) NaOMe (5 equiv), methyl(4-fluorophenyl)acetate, MeOH, 100 °C; (b) Cs2CO3, N-phenyl-
bis(trifluoromethanesulfonimide), DMF, 0 °C; (c) NR1R2, dioxane, 130 °C microwave; (d) 6 M HCl/THF 80 °C.
efficiency (LBE)7 of 0.32, it contains a novel core from which fur-
ther modifications can be generated.
HN
HN
HN
O
N
O
N
Cl
F
O
N
F
Novel pyridone analogs of 1 were synthesized according to the
synthetic procedure in Scheme 1. Starting with known aldehyde 2,8
base induced cyclization with methyl(4-fluorophenyl)acetate
afforded bicyclic intermediate 3. Triflation of 3 provided 4 as an
intermediate for quick analoging. Heating intermediate 4 with a
variety of amines induced smooth displacement of the triflate. Sub-
sequent hydrolysis of the methyl ether 5 under forcing acidic
conditions yielded the desired pyridine compounds 6.
N
N
N
F
H
H
Cl
F
F
F
JAK2 IC50 = 29000 nM
JAK2 IC50 = 3940 nM
JAK2 IC50 = 11800 nM
7
8
9
Several analogs of the napthyridinone 1 were prepared accord-
ing to Scheme 1. The decreased potencies of analogs 7, 8, and 9
(Fig. 2) as compared to 1, suggested that further analogs made
in this region of the scaffold would not significantly improve
the potency. Thus, efforts were turned toward improving ligand
binding efficiency by modifying the scaffold. Closer examination
of structure 1 reveals that substitution of the bi-aryl portion in-
duces a 60.5° dihedral angle conformation9 between the bi-aryl
rings. This conformation might not be optimally accommodated
in the narrow ATP binding pocket of the JAK2 enzyme.10 With this
in mind, we rationalized that if the bi-aryl rings were co-planar,
the proposed structure would fit better in the ATP pocket and
increase potency. To test this hypothesis, two strategies were pro-
posed to restrain conformation as shown in Scheme 2. Strategy A
incorporated an intramolecular hydrogen bond as demonstrated
by 10 to achieve co-planarity of 7.11 In principle, the intramolec-
ular hydrogen bond between heteroatom X and the NH of the
amino pyridine would induce a co-planar relationship between
the ring systems. In contrast, strategy B involves achieving co-
planarity and conformationally restricts 7 by fusing the rings
together as depicted in 11.
Towards this end, the approach in strategy A was adopted first
due to synthetic accessibility. Using the same chemistry as shown
in Scheme 1, several heteroatom variations as represented by 12,
13, and 14 (Fig. 3) were incorporated to induce co-planarity
through intra-molecular hydrogen bonding. Unfortunately,
improvements in potency using this strategy were unsuccessful.
Compound 12 showed improved JAK2 activity compared to 7.
However, the ligand binding efficiency was still not in a desirable
range to initiate a rapid library synthesis follow on strategy.
Figure 2. Activity of napthyridinone analogs.
HN
HN
HN
O
N
X
O
N
O
N
Strategy A
Strategy B
N
H
N
X
X
N
H
H
B
X
A
X
n
F
10
X = O, N
11
7
Scheme 2. Approaches to induce co-planarity of the bi-aryl rings.
HN
HN
HN
O
N
O
O
N
N
O
N
O
N
H
N
H
N
H
N
HN
N
N
JAK2 IC50 = 630 nM
LBE = 0.35
JAK2 IC50 = 11500 nM
LBE = 0.26
JAK2 IC50 = 8350 nM
LBE = 0.28
12
13
14
Figure 3. Intramolecular hydrogen bonding strategy.

T. Siu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7421–7425
7423
N
X
HO
X
OH
O
O
O
B
NH2
O
c
a
b
O
N
Cl
N
X
X
N
N
O
NH2
I
16
15
X = N, C-F
17
19
18
HN
N
X
N
X
N
g
N
O
f
N
e
N
d
N
Cl
Cl
O
R5
R5
N
N
R6
Cl
OH
R6
X
X
23
22
21
20
Scheme 3. Synthesis of fused napthyridinones. Reagents and conditions: (a) diisopropyl amine, triethyl amine, CH2Cl2; (b) sec-BuLi, TMEDA, triisopropylborate, THF, À78 °C;
(c) 18, Pd(PPh3)4, 2.0 M Na2CO3, 100 °C; (d) NaHMDS, THF, 0 °C; (e) POCl3, MeCN, 100 °C; (f) amine, microwave, dioxane, 90–150 °C or LiHMDS THF 85 °C; (g) 6 N HCl dioxane,
85 °C.
Our second approach to improve intrinsic potency using strat-
HN
HN
egy B required a new synthetic route that allowed for maximum
diversity at a late stage in the synthesis. The construction of the tri-
cyclic core 23 was achieved based on modification of known inter-
mediates.12 Acid chloride 15 was converted to the diisopropyl
amide under basic conditions. Ortho lithiation of amide 16 with
sec-butyl lithium followed by trapping with triisopropyl borate
afforded the boronic acid 17. Suzuki cross coupling of 17 with 2-
butoxy-3-iodo-4-aminopyridine 1812 afforded bi-aryl intermediate
19. Strong base induced ring closure with NaHMDS gave the tricy-
clic core 20. Heating tricyclic 20 in POCl3 resulted in the double
chlorination to provide intermediate 21. A variety of amines added
to the core either via thermally or microwave induced reactions at
high temperature with acid or base. Interestingly, the subtle differ-
ence in reactivity of the two chlorines allows for selective substitu-
tion. In the majority of cases, the amine added to 21 to provide the
desired regioisomer as shown in 22.13 Finally, selective hydrolysis
of intermediate 22 provided pyridone 23.
With the synthesis to form fused scaffold 11 in hand, compound
24 was prepared first to test our original hypothesis of co-planar-
ity. To our delight, compound 24 (Scheme 4) improved the intrinsic
potency 1000-fold (JAK2 IC50 = 26 nM) compared to compound 7
(JAK2 IC50 = 29000 nM), confirming the feasibility of our approach.
Furthermore, significant gains were made in ligand binding effi-
ciency for 24 (LBE = 0.45) compared to 7 (LBE = 0.26).
O
N
O
N
Cyclize
N
H
N
H
F
F
JAK2 IC50 = 29000 nM
LBE = 0.26
JAK2 IC50 = 26 nM
LBE = 0.45
7
24
Scheme 4. Strategy B for improving potency.
as 33 are active, suggesting size tolerability in this area of the ki-
nase pocket.
Substitution with smaller hydrophobic amines branched at the
alpha positions such as 34, 35, and 36 trends toward improve-
ments in potency when compared to larger hydrophobic amines.
Most notable is 38 with a JAK2 IC50 = 1 nM. Remarkably, 38 is
27-fold more potent than its methyl analog 36, underscoring the
crucial contribution that the trifluoromethyl substituent makes
with the JAK2 enzyme. The enantiomer 39 is significantly less ac-
tive with an IC50 = 55 nM. Due to its excellent potency and efficient
binding (LBE = 0.49), 38 was advanced for further investigation.
To better understand how our inhibitors bind to the enzyme,
compound 38 was modeled into the JAK2 crystal structure.14 As
shown in Figure 4, compound 38 is proposed to bind in the ATP
binding pocket of JAK2 with the pyridone interacting with the
hinge region of the kinase. The pyridone makes critical H-bonds
to the backbone carbonyl of Glu930 and backbone NH of Leu932.
The planar structure nicely fits into the narrow ATP binding
pocket along the hinge, which may account for a significant pro-
portion of the potency. Furthermore, the amine linkage is proposed
to occupy the ribose pocket of the enzyme. More specifically, the
CF3 moiety is positioned into a hydrophobic pocket formed by
Leu983 and Gly993. The increased lipophilicity of the CF3 group
interacting with the hydrophobic pocket seems to explain the in-
creased potency compared to the methyl analog (36). The fluorine
substituent on the aromatic ring, however, does not interact with
the protein and is projected out into solvent.
Encouraged by the potency enhancement and gains in ligand
binding efficiency, 24 was used as a frame of reference for probing
structure–activity relationships (SAR) of
a diverse subset of
amines. Table 1 highlights the in vitro data of amine analogs that
were synthesized. The focus for this amine library centered on
improving potency as well as understanding the structural envi-
ronment in this region of the enzyme. Polar substituents, especially
with basic amines such as in examples 25 and 27, are examined in
this region of the kinase, but are not well tolerated. Secondary
amines, such as 29, are tolerated, but did not improve potency
compared to 24. Interestingly, aniline 30 is only moderately active
with IC50 = 4100 nM; however, the activity is dramatically im-
proved when ortho substituents are incorporated as exemplified
by 31 (IC50 = 4 nM) and 32 (IC50 = 14 nM). This result suggests that
the strong conformational preference for the aniline ring to adopt a
perpendicular conformation to the core significantly contributes to
the binding affinity. Furthermore, larger hydrophobic groups such

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T. Siu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7421–7425
Table 1
SAR on fused napthyridinone analogs
R
Enzyme
IC50 (nM)
JAK2
R
Enzyme
IC50 (nM)
JAK2
N
H
NH2
N
H
3157
200
25
33
O
S
O
N
H
N
H
512
75
34
26
27
N
N
H
N
H
1115
139
Figure 4. Proposed binding mode of compound 38 bound to JAK2 X-ray structure.
35
Cl
N
H
N
H
predicted, the incorporation of this nitrogen reduces the experi-
53
27
mental HPLC log D from 3.34 to 2.33,18 and the HCl salt of 41 has
Cl
36
aqueous solubility up to 120 lM in a solubility assay. Furthermore,
28
the activities in both the enzymatic and cell based assays are main-
tained, validating the hypothesis for modifying physicochemical
properties without diminishing potency. In addition, as an added
benefit, incorporating the polar heteroatom decreases the hERG
binding.19 However, the lower log D diminishes the pharmacoki-
netic properties of 41 with a Cl = 68 mL/min/kg. Nevertheless, on
the basis of the excellent intrinsic and cellular potency coupled
with improved aqueous solubility, 41 was evaluated in an in vivo
model.20 Administration of 41 at doses of 30 and 90 mpk (p.o.) re-
duces pSTAT5 signals to basal levels in whole blood of erythropoi-
F
F
N
N
N
H
540
750
37
29
H
CF3
N
F
N
H
4100
1
38
30
F
etin (Epo) treated mice at
1 and 3 h (Fig. 5), with an
IC50 = 2700 nM.21
CF3
H
N
In conclusion, low micromolar focused screening hit 1 was opti-
mized by rational targeted design along with diversity oriented
synthesis using ligand binding efficiency as a guiding parameter
for optimization. We were able to identify 38 as a lead, improving
the potency by 1400-fold while enhancing the ligand binding effi-
ciency to 0.49. With the aid of molecular modeling, judicious intro-
duction of a polar functionality led to 41, which reduces hERG
binding and increases aqueous solubility. Compound 41 potently
reduces pSTAT5 levels in mice and allowed us to achieve in vivo
proof of concept. The detailed optimization of the pharmacokinetic
properties and in vivo potency of 41 will be disclosed in a future
communication.
N
H
4
55
Cl
F
39
31
Cl
H
N
H
OH
N
14
274
Cl
40
32
Having optimized the intrinsic potency, compound 38 was fur-
ther profiled to understand the liabilities associated with this com-
pound (Table 2). In addition to having excellent enzymatic
potency, 38 demonstrates good activity in our cell based assay
JAK2 Cell IC50 = 50 nM.15 Further investigation in vivo revealed
modest pharmacokinetic properties in rat with a Cl = 21 mL/min/
kg and Vd = 6 L/kg.16 However, a liability of 38 is the off-target
binding affinity to the Ikr potassium channel hERG (human Ether-
a-go-go-Related Gene) with IC50 = 3500 nM.17 Moreover, while
quite potent, 38 suffers from poor aqueous solubility as measured
in our solubility assay. Unfortunately, the reduced solubility lim-
ited in vivo studies with 38, and a strategy to improve aqueous sol-
ubility while maintaining the potency was needed.
Based on our modeling study, the trajectory of the aryl fluoride
substituent of 38 is pointing away toward the open end of the cat-
alytic site and into solvent. Consequently, the incorporation of po-
lar functional groups in this region of the molecule to modify
physicochemical properties without affecting the potency would
be a viable strategy. Towards that end, compound 41 incorporating
a nitrogen heteroatom was synthesized according to Scheme 3. As
Table 2
Profile of compound 38 and 41
HN
HN
CF3
CF3
N
N
O
O
N
N
H
H
N
F
38
41
JAK2 (IC50
Cell (IC50
hERG (IC50
Rat CIp
VD
)
1 nM
50 nM
3500 nM
21 mL/min/kg
6 L/kg
1 nM
40 nM
1400 nM
68 mL/min/kg
8 L/kg
)
)
HPLC log D pH 7.4
Solubility @ pH 7
In vivo IC50
3.34
2.33
3
l
M
120
lM
NA
2700 nM

T. Siu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7421–7425
7425
reported as the averages of at least two independent determinations;
standard deviations are within 25–50% of IC50 values. For further details
see: WO2009035575.
7. (a) Abad-Zapatero, C. Expert Opin. Drug Disc. 2007, 2, 469; (b) Hopkins, A. L.;
Groom, C. R.; Alex, A. Drug Discov. Today 2004, 9, 430.
8. Siu, T.; Liang, J.; Arruda, J.; Li, Y.; Jones, R. E.; Defeo-Jones, D.; Barnett, S. F.;
Robinson, R. G. Bioorg. Med. Chem. Lett. 2008, 18, 4186.
9. Conformational analysis was performed with Macromodel module from
Schrodinger, LLC. Mixed torsional/low-mode sampling algorithm was used to
generate conformers with OPLS-2005 force field and implicit solvent model. To
ensure sufficient sampling, the maximum energy cutoff was set at 8 kcal/mol.
10. X-ray structure of JAK2 (PDB ID: 2B7A)
11. Kuhn, B.; Mohr, P.; Stahl, M. J. Med. Chem. 2010, 53, 2601.
12. Rodgers, J. D; Robinson D. J.; Arvanitis, A. G.; Maduskuie, T. P. Jr.; Shepard, S.;
Storace, L.; Wang, H.; Rafalski, M.; Jalluri, R. K.; Combs, A. P.; Crwely, M. L.
Preparation of tetracyclic inhibitors of Janus kinases for treating immune-
related diseases and cancer. PCT Int. Appl. WO2005105814, 2005.
13. The structure was determined by observing the nOe interaction between the
N–H and aromatic proton H1 in the 1H NMR spectrum.
Figure 5. Inhibition of pSTAT5 signal in C57Bl/6 mice (n = 6) by oral dosing of
compound 41 at 30 and 90 mpk.
N
Cl
N
Acknowledgments
R
N
H
T.S. thanks Christopher Cox, Lily Moy, and Kerrie Spencer for
careful reading of this manuscript and Bruce Adams for NMR
support.
F
H1
14. Docking studies were performed using Glide software from Schrodinger, LLC.
Based on the X-ray structure of JAK2 (PDB ID: 2B7A). Extra precision mode (XP)
with default settings were used to generate 20 docked poses which were
subsequently filtered based on visual inspection.
References and notes
1. Shuai, K.; Liu, B. Nat. Rev. Immunol. 2003, 3, 900.
15. Detection of STAT phosphorylation was performed using AlphaScreen™
SureFire™ p-STAT5 assay (Perkin–Elmer and TGR Biosciences) using both
biotinylated anti-phospho-STAT5 antibody, which is captured by Streptavidin-
coated donor beads, and anti-total STAT5 antibody, which is captured by
Protein A conjugated acceptor beads. IC50 values are reported as the averages of
at least two independent determinations; standard deviations are within
25À50% of IC50 values. For further details see: WO2008156726.
2. (a) James, C.; Ugo, V.; Le Coudeic, J. P.; Staerk, J.; Delhommeau, F.; Lacout, C.;
Garcon, L.; Raslova, H.; Berger, R.; Bennacaur-Griscelli, A.; Villeval, J. L.;
Constantinescu, S. N.; Casadevall, N.; Vainchenker, W. Nature 2005, 434, 1144;
(b) Baxter, E. J.; Scott, L. M.; Campebell, P. J.; East, C.; Fourouclas, N.; Swanton,
S.; Vassiliou, G. S.; Bench, A. J.; Boyd, E. M.; Curtin, N.; Scott, M. A.; Erber, W. N.;
Green, A. R. Lancet 2005, 365, 1054; (c) Levine, R. L.; Wadleigh, M.; Cools, J.;
Ebert, B. L.; Wernig, G.; Huntly, B. J. P.; Boggon, T. J.; Wlodarska, I.; Clark, J. J.;
Moore, S.; Adelsperger, J.; Koo, S.; Lee, J-C.; Gabriel, S.; Mercher, T.; D’Andrea,
A.; Frohling, S.; Dohner, K.; Marynen, P.; Vandenberghe, P.; Mesa, R. A.; Tefferi,
A.; Griffine, J. D.; Eck, M. J.; Sellers, W. R.; Meyerson, M.; Golub, T. R.; Lee, S. J.;
Gilliland, D. G. Cancer Cell 2005, 7, 387.
3. (a) Fridman, J.; Nussenzveig, R.; Liu, P.; Rodgers, J.; Burn, T.; Haley, P.; Scherle,
P.; Newton, R.; Hollis, G.; Freidman, S.; Verstovsek, S.; Vaddi, K. Blood, ASH
Annual Meeting Abstracts, 2007, Abstr. 110, 3538.; (b) Verstovsek, S.;
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F.; Tefferi, A.; Kantarjian, H. M.; Bui, L. A.; Clary, D. O. Blood ASH Annual Meeting
Abstracts, 2007, Abstr. 110, 553.; (c) Geron, I.; Abrahamsson, A. E.; Barraoga, C.
F.; Kavalerchik, E.; Gotlib, J.; Hood, J. D.; Durocher, J.; Mak, C. C.; Noronha, G.;
Soll, R. M.; Tefferi, A.; Kaushansky, K.; Catriona, K.; Jamieson, C. H. M. Cancer
Cell 2008, 13, 321.
16. Dosed in Sprague–Dawley rats (n = 1) IV 1 mg/kg as 1 mg/mL solution
PEG400:Captisol (1:1).
17. The hERG IC50 values were acquired by radioligand binding competition
experiments using membrane preparations from human embryonic kidney
cells that express hERG. IC50 values are reported as the averages of at least two
independent determinations; standard deviations are within 25–50% of IC50
values. For assay detail see Bell, I. M.; Gallicchio, S. N.; Abrams, M.; Beshore, D.
C.; Buser, C. A.; Culberson, J. C.; Davide, J.; Ellis-Hutchings, M.; Fernandes, C.;
Gibbs, J. B.; Graham, S. L.; Hartman, G. D.; Heimbrook, D. C.; Homnick, C. F.;
Huff, J. R.; Kassahun, K.; Koblan, K. S.; Kohl, N. E.; Lobell, R. B.; Lynch, J. J., Jr.;
Miller, P. A.; Omer, C. A.; Rodrigues, A. D.; Walsh, E. S.; Williams, T. M. J. Med.
Chem. 2001, 44, 2933. and references therein.
18. Waring, M. J. Expert Opin. Drug Disc. 2010, 5, 235.
19. Siu, T.; Li, Y.; Nagasawa, J.; Liang, J.; Tehrani, L.; Chua, P.; Jones, R. E.; Defeo-
Jones, D.; Barnett, S. F.; Robinson, R. G. Bioorg. Med. Chem. 2008, 18, 4191.
20. Mathur, A.; Mo, J.; Kraus, M.; O’Hare, E.; Sinclair, P.; Young, J.; Zhao, S.; Wang,
Y.; Kopinja, J.; Qu, X.; Reilly, J.; Walker, D.; Xu, L.; Aleksandrowicz, D.; Marshall,
G.; Scott, M. L.; Kohl, N. E.; Bachman, E. Biochem. Pharmacol. 2009, 78, 382.
21. Compound 41 was dosed by oral gavage to C56bl/6 mice as a solution in
40%PEG400: 50%Trappsol: 10% water. Drug concentrations and pSTAT5 signal
were measured at 1, 3, and 8 h.
4. Wilson, L. J. Expert Opin. Ther. Pat. 2010, 5, 609.
5. A virtual screen was performed to identify molecules in Merck’s collection that
would be predicted to bind to the ATP binding pocket of JAK2 (PDB ID: 2B7A).
The GLIDE docking software from Schrodinger was run in parallel mode against
structures of JAK2 with and without water molecules bound to the active site.
The best scoring hits were visually inspected, and the most interesting and
novel molecules were chosen for experimental follow-up.
6. Detection of peptide phosphorylation was performed by homogenous time
resolved fluorescence (HTRF) using
a europium chelate. IC50 values are