Janus kinase 2 inhibitors. Synthesis and characterization of a novel polycyclic azaindole

Article abstract of DOI:10.1021/jm901383u

The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.

Full text of DOI:10.1021/jm901383u

7938 J. Med. Chem. 2009, 52, 7938–7941
DOI: 10.1021/jm901383u
Janus Kinase 2 Inhibitors. Synthesis and
Characterization of a Novel Polycyclic
Azaindole†
Tiansheng Wang,‡ John P. Duffy,‡ Jian Wang,‡
Summer Halas,‡ Francesco G. Salituro,‡,^ Albert C. Pierce,‡
Harmon J. Zuccola,‡ James R. Black,‡ James K. Hogan,‡
Scott Jepson,‡ Dina Shlyakter,‡ Sudipta Mahajan,‡
Yong Gu,‡ Thomas Hoock,‡ Mark Wood,‡ Brinley
F. Furey,‡ J. Daniel Frantz,‡ Lisa M. Dauffenbach,§
Ursula A. Germann,‡ Bin Fan,‡,|| Mark Namchuk,‡
Youssef L. Bennani,‡ and Mark W. Ledeboer*,‡
Figure 1. Overlay of putative binding orientations in JAK2.
Scheme 1a
‡Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge,
Massachusetts 02139-4242, and §Mosaic Laboratories, LLC, 12
Spectrum Pointe Drive, Lake Forest, California 92630. ^ Current
address: Agios Pharmaceuticals, 38 Sidney Street, Cambridge, MA
02139. || Current address: Nektar Therapeutics, 201 Industrial Road,
San Carlos, CA 94070
Received September 17, 2009
Abstract: The synthesis and characterization of a novel polycyclic
azaindole based derivative is disclosed, and its binding to JAK2 is
described. The compound is further evaluated for its ability to block
the EPO/JAK2 signaling cascade in vitro and in vivo.
The Janus kinases (JAKa) are members of a family of
intracellular nonreceptor tyrosine kinases that play a crucial
rolein the cytokine-mediated JAK-STAT signalingpathway.1
There are four known mamalian kinases in the JAK family:
JAK1, JAK2, JAK3, and TYK2. Of particular interest is the
emergence of JAK2 as a potential new therapeutic target for
myeloproliferative disorders (MPDs).2 The MPDs include
several hematological neoplasms that include polycythemia
vera (PV), essential thombocythemia (ET), idiopathic myelo-
fibrosis (IMF), and chronic myeloid leukemia (CML). Re-
cently a common gain-of-function mutation in JAK2 (V617F)
was discovered among patients diagnosed with PV, ET, or
IMF but not CML. This has resulted in intense efforts to
understand the exact mechanism by which the proprolifera-
tive effects, resulting from the activation of the JAK STAT
pathway by valine to phenylalanine mutation at position 617,
lead to these MPDs.3,4
a (a) Pd(PPh3)4, K2CO3, DME; (b) conc HCl, reflux; (c) HCl-diox-
ane, MeOH; (d) H2, Pd-C, HCl, MeOH; (e) 6 M HCl (aq), reflux;
(f) HATU, HOAt DIEA, DMF.
containing an azaindole (1) or deazapurine (2) hinge binding
motif similar to CP-690,550.7 Overlaying the putative ATP
binding site orientations of 1 and 2, we were intrigued by the
possibility of creating novel chemotypes with improved po-
tency for JAK2 by cyclizing the 3- and 4-positions (Figure 1).
Here, we detail the synthesis, characterization, and activity
profile of 9, which contains an eight-membered central lactam
ring and exhibits in vitro potency against JAK2 mediated end
points and in vivo efficacy in a JAK2 dependent mouse model
of disease (vide infra).
Previously, SAR studies on related compounds established
that compounds containing a 3,4-fused seven-membered cen-
tral ring were indeed potent inhibitors of JAK2 and that the
related phenolic OH plays a dual H-bond acceptor and H-
bond donor role which is required to achieve maximum
potency. Compounds without the phenolic OH or with alter-
native H donor or acceptor groups at the 4-position, such as
halogen, methoxy, carboxylic acid, aniline groups, were all
less potent on the enzyme (data not shown).8
In our program directed at identifying agents that can
attenuate the EPO/JAK/STAT pathway we sought to block
the kinase activity of JAK2 by targeting its ATP binding site.5,6
Screening of our corporate compound collection identified
several low molecular weight hits with respectable potency
Compound 9 with an eight-membered central ring was
prepared following the methods described in Scheme 1. Thus,
Suzuki coupling of 4-bromoazaindole 3 with the requisite
boronic acid gave 4-aryl azaindole 4. Deprotection of the
acetamide was achieved by heating 4 in 6 N aqueous HCl to
give the cyclization precursor 5. Pictet-Spengeler like con-
densation of 5 with the appropriate ketoester provided
cyclized product 6. Reductive ring-opening under hydrogena-
tion conditions gave ester 7, which was hydrolyzed under
acidic conditions (6 N HCl) to provide the requisite precursor
†PDB ID: The atomic coordinates and structure factors have been
deposited in the Protein Data Bank for compound 9 and the kinase
domain of JAK2 (PDB code 3JY9).
*To whom correspondence should be addressed. Phone: 617-444-
6100. Fax: 617-444-7827. E-mail: mark_ledeboer@vrtx.com.
a Abbreviations: JAK, Janus kinase; TYK, tyrosine kinase 2; STAT,
signal transducer and activator of transcription; MPD, myeloprolifera-
tive disorder; PV, polycythemia vera; ET, essential thombocythemia;
IMF, idiopathic myelofibrosis; CML, chronic myeloid leukemia; EPO,
erythropoeitin; EEC, EPO-independent endogenous erythroid colonies;
CFU-E, erythroid colony forming unit; GM-CSF, granulocyte-macro-
phage colony stimulating factor.
r
pubs.acs.org/jmc
Published on Web 11/18/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7939
Table 1. JAK2 and JAK3 Mediated Enzyme and Cell Activities for
Atropisomers 9A and 9B
KI, μM
IC50, μM
compd
JAK2
JAK3
TF1-GMCSF HT2- IL2
9A
9B
0.0010 ( 0.00016 0.0055 ( 0.0014 0.27 ( 0.16 1.53 ( 1.5
0.001 0.006
Figure 3. X-ray cocrystal structure depiction of the cocomplex of 9
and the kinase domain of JAK2. Hydrogen bonds (red) are shown
between the ligand and (from left to right) Leu-932, Glu-930, Asp-
994, Phe-995, and Glu-898.
calculating the transition state energy for a ring-flip such as
the one that interconverts the two atropisomers of 9. How-
ever, the flat, trans-amide conformation (depicted in pink)
serves as a reasonable approximation of the flat conformation
that would be a transition between the amide-up (green) and
amide-down (orange) conformations of 9. This trans-amide
conformation is approximately 25 kcal/mol above the energy
of the amide-up conformation, so 25 kcal/mol makes a good
lower-bound estimate of the transition energy. Although this
is only a rough estimate, a barrier of this magnitude would
certainly lead to slow interconversion rates such as those
described above.
In order to determine preferred stereochemistry and amide
conformation of 9 for binding to JAK2, we determined the
crystal structure cocomplex between JAK2 and 9.5,7 The
structure revealed several key hydrogen bonds to backbone
residues (Figure 3).
Figure 2. Conformational isomers of 9. The conformations with
the cis-amide are shown in green and orange. The conformation
with the trans-amide is shown in pink.
8. Lactam formation was most conveniently carried out with
HATU/HOAt to provide 9A in moderate yield. However, a
second cyclization product 9B was also isolated with identical
mass and whose NMR was also consistent with the cyclized
product. When the cyclization was carried out at higher
temperature (60 ꢀC), more of 9B formed. Conversely, at
ambient temperature the reaction yielded a higher proportion
of 9A albeit at a slower rate. While 9A and 9B were separable,
there was always a small amount of cross-contamination
present.9
Compound 9 adopts an open conformation similar to that
depicted in Figure 2 (shown in green) and forms two hinge
hydrogen bonds with its azaindole moiety to the protein
(Figure 1). The hydrogen bonds are to the backbone NH of
Leu-932 and the carbonyl of Glu-930. The phenyl ring forms
some hydrophobic contacts with the glycine-rich loop above
it. The eight-membered ring is puckered such that the lactam
carbonyl is in proximity and forms a H-bond with the back-
bone NH of Asp-994 at the beginning of the activation loop of
JAK2. The importance of the phenolic OH is revealed, as it
acts both as an H-donor, by forming an H-bond with carbox-
ylate of Glu-898, and as an H-acceptor, by forming a H-bond
with Phe-995.
Finally, the complex implies a preference for the S-stereo-
chemistry at the benzylic carbon. Modeling studies suggest
that the R-isomer is likely to be far less active. All of our
attempts at docking the R-enantiomer in the active site were
unsuccessful, suggesting that all the observed activity is
ascribed to the S-enantiomer and that the R-enantiomer is
likely to be inactive. As such, we did not attempt to separate
the enantiomers of 9.
We speculated that these products might be atropisomers.
To confirm this, we monitored for interconversion of the
atropisomers, and we found that each isomer, when kept in
DMSO solution at room temperature, independently equili-
brated to a 2:1 mixture of isomers. The major isomer 9A
equilibrated over a period of 1 week, while the minor isomer
9Bequilibratedmorerapidly overthe course ofapproximately
1-2 days. However, when stored in solid form, little or no
equilibrationof atropisomers was seen over a period of several
months. For subsequent studies we used samples freshly
prepared from solid 9A.
Testing of both products against the target enzyme demon-
strated that samples of 9A and 9B were equipotent inhibitors
of JAK2 with similar preference over JAK3 (Table 1).10,11 In
addition, 9Ashowed similar foldselectivity for JAK2 vsJAK3
mediated STAT 5 phosphorylation in TF1 and HT2 cells,
respectively.12
Computational analysis suggests that 9 has two conforma-
tions with similar energies but a high barrier to intercon-
version. Both conformations contain cis-amides, as the trans-
amide conformations require a relatively flat, strained
conformation of the eight-membered ring. The two cis-con-
formations (green and orange) and one trans-conformation
(pink) can be seen in Figure 2. Given the roughly 2:1 ratio of
atropisomers at equilibrium, the energy difference between
the two should be approximately 0.4 kcal/mol. This is in
reasonably good agreement with the MMFF94s calculated
energy difference of 1.6 kcal/mol, with the discrepancy likely
due to solvent effects.13 There are no established methods for
Screening of 9 against a panel of kinases revealed the
selective nature of the compound, hitting only 3 out of 36
kinases at Ki < 500 nM (ALK Ki = 0.017 μM and cKit Ki =
0.19 μM, GCK IC50 = 0.34 μM).
Because of the favorable potency and selectivity for JAK2
of 9, we set out to characterize its in vivo pharmacokinetic
parameters. The data are summarized below (see Table 2).
Compound 9 exhibited moderate clearance, had long

7940 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Wang et al.
Table 2. Pharmacokinetic Parameters of 9 Determined in Male Charles
River Sprague-Dawley Rats and Fox Chase SCID (CB-17) Micea
rat
mouse
iv dose
po dose
iv dose
po dose
dose (mg/kg)
max (μg/mL)
max (h)
1.8
0.70
10
9.0
7.3
10
1.0
2
C
1.08
3
T
AUC0-inf (μg h/mL)
T1/2 (h)
CL ((mL/min)/kg)
1.26
3.4
7.7
3.6
12.5
2.7
8.3
3.2
3
23.8
5.7
12.1
2.3
Vss (L/kg)
F (%)
100
66.1
a Mean values determined from the concentration (as determined
using a specific LC/MS/MS method).
Figure 5. Treatment began 1 week after implantation of 1 million
mJAK2-Ba/F3 leukemia cells (n = 12/group). Animals were treated
by oral gavage with vehicle (5% methylcellulose solution, MC) or 9
(15 mg/kg, suspension in 5% MC) twice per day until day 32 or
achieving moribundity. All 9 treated animals survived until day 32.
Two animals experienced cage deaths that were not related to
treatment or disease; thus, data from these animals were censored.
advantage, prolonging survival to 32 days (study termina-
tion). Following sacrifice on day 32, spleens were harvested
and weighed to assess the degree of leukemic burden in treated
animals versus controls. Animals treated with 9 experienced
significantly reduced splenomegaly when compared to vehicle
controls (P < 0.05): 216 ( 15 mg vs 466 ( 26 mg, respectively.
In summary, we have characterized the binding of 9, a
selective and potent new polycyclic azaindole based inhibitor,
of JAK2, which revealed key interactions responsible for its
potency against the target protein. We have demonstrated the
ability of 9 to block the JAK2/STAT pathway in vitro in an
EPO-independent erhythroid colony formation assay. With
its ability to elicit a pharmacological benefit in a mutant
(V617F) JAK2-dependent Ba/F3 murine leukemia model,
we have shown the utility of 9 as a tool compound for the
study of relevant models of disease.
Figure 4. Effect of 9 on EPO-independent erythroid colony forma-
tion and growth of a PV patient bone marrow aspirate cultured in
vitro.
half-lives, and was 100% orally bioavailable when dosed in
Charles River Sprague-Dawley rats. Compound 9 showed
66% oral bioavailability in Fox Chase SCID (CB-17) mice
and a similar half-life.
Next, we evaluated the ability of 9 to inhibit the JAK2/
STAT pathway. Formation of EPO-independent endogenous
erythroid colonies (EEC) is a proposed diagnostic criterion
for patient with polycythemia vera.14 Thus, we established an
in vitro assay using PV patient bone marrow aspirate to
evaluate the effects of 9 on EPO-independent erhythroid
colony formation and proliferation as a surrogate for
JAK2/STAT pathway inhibition. As shown in Figure 4, 9
inhibited the spontaneous growth of erythroid colonies
(colony forming unit or CFU-E) from a PV patient bone
marrow specimen with confirmed JAK2 (V617F) mutation in
a dose-dependent manner. The average IC50 from two inde-
pendent assays using different PV patient samples with con-
firmed JAK2 (V617F) mutation was 0.87 ( 0.48 μM. These
results corroborate potency against the intended target and
indicate effective inhibition of the JAK2/STAT pathway in a
disease-relevant model system.
Acknowledgment. The authors gratefully acknowledge
Drs. C. Town and S. Pazhanisamy for helpful discussions
and critical review of the manuscript, K. Bonanno for expres-
sion of the JAK2 protein, and B. Davis, G. May and N. Ewing
for collecting HRMS data.
Supporting Information Available: Synthetic procedures; 1H
NMR and LC/MS data for all compounds and HRMS data for
both atropisomers of 9; assay protocols for Ki and IC50 deter-
minations and colony forming assays; crystallographic informa-
tion; protocols for cloning, expression, and purification of
JAK2. This material is available free of charge via the Internet
at http://pubs.acs.org.
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the second group received vehicle alone. The results of this
study are summarized in a Kaplan-Meier survival plot (see
Figure 5). Animals that received vehicle became moribund
approximately 23 days after receiving the leukemic implant.
Treatment with 9 provided a significant (P < 0.05) survival
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