Identification of novel SAR properties of the Jak2 small molecule inhibitor G6: Significance of the para-hydroxyl orientation

Article abstract of DOI:10.1016/j.bmcl.2011.12.042

In this study, we analyzed the structure-activity relationship properties of the small molecule Jak2 inhibitor G6. We synthesized a set of derivatives containing the native para-hydroxyl structure or an alternative meta-hydroxyl structure and examined their Jak2 inhibitory properties. We found that the para-hydroxyl derivative known as NB15 had excellent Jak2 inhibitory properties in silico, in vitro, and ex vivo when compared with meta-hydroxyl derivatives. These results indicate that NB15 is a potent derivative of the Jak2 inhibitor G6, and that maintaining the para-hydroxyl orientation of G6 is critical for its Jak2 inhibitory potential.

Full text of DOI:10.1016/j.bmcl.2011.12.042

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1402–1407
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel SAR properties of the Jak2 small molecule inhibitor G6:
Significance of the para-hydroxyl orientation
a
b
a
c
d
}
Rebekah Baskin , Meghanath Gali , Sung O. Park , Zhizhuang Joe Zhao , György M. Keseru ,
Kirpal S. Bisht b, Peter P. Sayeski a,
⇑
a Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL, USA
b Department of Chemistry, University of Florida, South Florida, Tampa, FL, USA
c Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
d Department of General and Analytical Chemistry, Budapest University of Technology and Economics Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we analyzed the structure–activity relationship properties of the small molecule Jak2 inhib-
itor G6. We synthesized a set of derivatives containing the native para-hydroxyl structure or an alternative
meta-hydroxyl structure and examined their Jak2 inhibitory properties. We found that the para-hydroxyl
derivative known as NB15 had excellent Jak2 inhibitory properties in silico, in vitro, and ex vivo when
compared with meta-hydroxyl derivatives. These results indicate that NB15 is a potent derivative of the
Jak2 inhibitor G6, and that maintaining the para-hydroxyl orientation of G6 is critical for its Jak2 inhibitory
potential.
Received 6 October 2011
Revised 5 December 2011
Accepted 8 December 2011
Available online 21 December 2011
Keywords:
Jak2 tyrosine kinase
G6
Ó 2011 Elsevier Ltd. All rights reserved.
Small molecule inhibitor
Stilbenoid
SAR
Janus kinase 2 (Jak2) is a member of the Janus family of non-
receptor tyrosine kinases and plays a key role in numerous signaling
pathways regulating cell survival, proliferation, and differentiation.
Jak2 forms associations with growth factor and cytokine receptors,
and upon activation, it phosphorylates substrates such as the Signal
Transducers and Activators of Transcription (STATs).1–4 Activated
STAT proteins thentranslocate tothe nucleus tomodulate genetran-
scription. Mutations in Jak2 can lead to constitutive activation of the
kinase and thus cause aberrant downstream Jak-STAT signaling.
Specifically, the Jak2-V617F mutation occurs within the pseudo-ki-
nase domain of Jak2 and relieves the auto-inhibitory function of this
domain, thus allowing the kinase domain to be constitutively active
in the absence of ligand.5,6
In 2005, the Jak2-V617F mutation was discovered in a large
number of myeloproliferative neoplasm (MPN) patients.7–11 These
patients suffer from an overproduction of blood cells of the mye-
loid lineage. This heterogeneous group of diseases includes polycy-
themia vera (PV), essential thrombocythemia (ET) and primary
myelofibrosis (PMF). The Jak2-V617F mutation occurs in over
90% of PV patients and a large subset of ET and PMF patients. This
mutation has also been shown to cause MPN phenotypes in bone
marrow transplant and transgenic mouse models.12,13 Currently,
there are no effective therapies for these diseases beyond palliative
treatments such as hydroxyurea and phlebotomy. Therefore, Jak2
has been investigated in recent years as a potential therapeutic tar-
get, and a number of compounds are in pre-clinical and clinical
development.14
Our lab recently used an in silico drug discovery approach to de-
velop a Jak2 small molecule inhibitor known as G6.15 We have
shown that G6 inhibits Jak2-mediated pathologic cell growth using
in vitro cell culture models, in vivo models, and ex vivo patient
samples.16 We also demonstrated that the stilbene core structure
of G6 is essential for its activity as a Jak2 inhibitor, in vitro and
ex vivo.17 Here, we further examined the structure of G6 to deter-
mine if structural modifications of the compound could enhance its
Jak2 inhibitory potential. We hypothesized that the structure of G6
could be minimized in order to enhance its Jak2 inhibitory poten-
tial and that maintaining the para-hydroxyl orientation is required
for Jak2 inhibition. To test this, we synthesized a set of G6 deriva-
tives with different structural features and examined their in silico
Jak2 binding properties and their effects on Jak2-mediated patho-
logic cell growth in vitro and ex vivo.
The structure of G6 consists of a stilbene core with ethyl groups
attached to each carbon of the core, para-hydroxyl groups on each
benzene ring, and tertiary amine moieties at the meta position of
each ring (Supplementary data Fig. 1). In order to examine the struc-
ture–function correlations of this compound, we synthesized four
structurally minimized G6 derivatives containing the native para-
hydroxyl structure or an alternative meta-hydroxyl structure
⇑
Corresponding author.
E-mail address: psayeski@ufl.edu (P.P. Sayeski).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.042

R. Baskin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1402–1407
1403
(Scheme 1, Supplementary data Fig. 1). These are known as NB4,
NB6, NB13, and NB15. Compounds NB-4, -6, -13, and -15 were syn-
thesized from stilbenoids 1–4 (Scheme 1). The chemical structures
of the stilbenoids obtained were confirmed by 1H and 13C NMR anal-
ysis. Experimental details are provided in the Supplementary data.
The Jak2 ATP-binding pocket contains several critical regions in-
volved in catalysis, including the hinge region, the glycine loop, the
catalytic loop and the activation loop. The glycine loop is important
for substrate and nucleotide binding. The hinge region contains
Glu930 and Leu932, which interact with the adenine group of
ATP. The activation loop contains the tyrosine 1007/1008 residues
that, once phosphorylated, allow this region to undergo a confor-
mational change to accommodate substrate binding. The catalytic
loop contains residue Arg980 which is involved in the coordination
of magnesium ions.18 Each of the G6 derivatives was docked into
the Jak2 ATP-binding pocket using AutoDock 4.2 (Scripps).19,20
Fig. 1 shows the most favorable docking orientation for each com-
pound. NB13 and NB15 showed interactions very similar to those
of the parent compound, G6.17 These include hydrogen bonds with
residues in the hinge region, the activation loop, and the catalytic
loop. NB4 and NB6 showed hydrogen bond interactions with sev-
eral residues, but lacked any interactions with the critical hinge re-
gion residues, Glu930 or Leu932. These results indicate that the
para-hydroxyl derivatives (NB13 and NB15) most closely mimic
the in silico Jak2 binding properties of G6.
activity, Jak2 in vitro kinase assays were carried out in the presence
of a known Jak2 substrate, STAT1. Experimental details are pre-
sented in the Supplementarydata. We found that NB15 significantly
reduced Jak2 autophosphorylation (Table 1, Supplementary data
Fig. 2). Both NB15 and NB13 significantly reduced the ability of
Jak2 to phosphorylate the STAT1 substrate whereas the two meta-
hydroxyl compounds had no significant effect on Jak2 autophos-
phorylation or STAT1 phosphorylation (Table 1, Supplementary
data Fig. 2).
Jak2 directly activates the transcription factor STAT5 by phos-
phorylation at Tyr 694. STAT5 activation plays an important role
in HEL cell proliferation, as STAT5 is known to regulate several
pro-survival genes.21,22 We have previously shown that G6 reduces
STAT5 phosphorylation in HEL cells.17 Therefore, we wanted to
determine whether specific structural features in G6 correlate with
reduced STAT5 phosphorylation in these cells. Fig. 3A shows a
representative phospho-STAT5 blot and Fig. 3B shows the densito-
metric quantification of all experiments. We found that only the
para-hydroxyl derivative NB15 was able to significantly reduce
STAT5 phosphorylation when compared to vehicle treated control
cells (Fig. 3).
Constitutively active Jak-STAT signaling leads to increased cell
proliferation and inhibition of apoptosis. We have previously
shown that the mechanism by which G6 decreases HEL cell viabil-
ity is induction of cell cycle arrest and apoptosis.16 Therefore, we
wanted to determine if specific changes in compound structure
correlate with changes in cell cycle properties and/or apoptosis.
We found that NB15 and NB4 caused a significant increase in the
number of cells in G2 with a corresponding decrease in G1, indicat-
ing a G2/M phase arrest (Fig. 4A). In order to further confirm the
G2/M arrest, we examined the levels of several cell cycle regulatory
proteins via Western blot analysis. We found that treatment with
NB15 and NB4 caused a reduction in cyclin A expression, but had
no effect on cyclin B1 or CDK1 expression (Fig. 4B). The other com-
pounds had no effect on cyclin A, cyclin B or CDK1 expression
(Fig. 4B). During cell cycle progression, CDK1 forms associations
with both cyclin A and cyclin B1 in order to promote mitotic en-
try.23,24 Therefore, it appears that NB15 and NB4 may cause G2 ar-
rest due to decreased cyclin A expression and a subsequent block
of mitotic entry.
G6 is known to inhibit the proliferation of human erythroleuke-
mia (HEL) cells in vitro and in vivo.16 The HEL cell line is homozygous
for the Jak2-V617F mutation and is highly proliferative due to con-
stitutive Jak2 kinase activity. Therefore, we examined the ability of
the four G6 derivatives to inhibit HEL cell growth. We found that
NB15 had the most potent effect on HEL cell growth in both a dose-
and time-dependent manner (Fig. 2A and B, respectively). This com-
pound demonstrated 50% cell death at a concentration of ꢀ0.85
lM
and completely blocked cell growth at all time points measured
(Fig. 2B). The other derivatives had varying levels of cell growth inhi-
bition, with NB6 consistently showing the lowest level of inhibition.
In order to determine the effects of these compounds on an-
other Jak2-V617F-positive cell line, we examined their impact on
cell growth in the Ba/F3-EpoR-Jak2-V617F cell line. This murine
pro B cell line has been retrovirally transduced with the Jak2-
V617F cDNA and demonstrates cytokine independent growth.
We found that again NB15 was highly effective at inhibiting cell
growth in both a dose- and time-dependent manner (Fig. 2C and
D, respectively). Also, NB6 consistently had little impact on cell
growth, except in this case at the highest concentration (Fig. 2C).
In order to determine if the reduced cell growth inhibitory po-
tential observed in Fig. 2 correlated with reduced Jak2 kinase
We also wanted to examine the effects of each of the derivative
compounds on apoptosis. We found that NB15, NB13, and NB4 in-
duced apoptosis in HEL cells whereas NB6 was without effect
(Fig. 4C). We further confirmed the induction of apoptosis by
examining expression of apoptotic markers via Western blot. We
examined cleavage of poly (ADP-ribose) polymerase, a hallmark
of apoptosis. Additionally, we examined the cleavage of Bid, Bim
R2
R3
R2
R1
Stilbene
R
R
R
3
H
H
OH
OH
% Yield
76
84
65
71
1
2
R3
R1
1
2
3
4
Me OH
H
Me
H
i
R1
O
R3
OH
H
H
R2
ii
N
R1
HO
N
R1
or
OH
N
N
HO
R1
NB13 : R = Me
OH
R1
NB4 : R = Me
1
1
1
NB6 : R = H
NB15 : R = H
1
Scheme 1. Reagents and conditions: (i) TiCl4, Zn, Dry. THF, reflux; (ii) Paraformaldehyde, dimethyl amine, MeOH, reflux.

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R. Baskin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1402–1407
Gly861
A. NB4
B. NB6
Asn 859
Gly993
Asp939
Asp994
Arg980
C. NB13
Glu930
D. NB15
Glu930
Leu932
Leu932
Asp994
Arg980
Asp994
Asn981
Figure 1. Molecular docking of the NB compounds. Each compound was computationally docked into the ATP-binding pocket of the Jak2 kinase domain (PDB code 3E64). The
protein structure is represented as a ribbon, with the compounds shown as sticks. Colored regions of the protein are the glycine loop (blue), the hinge region (cyan), the
catalytic loop (magenta) and the activation loop (red). Amino acid residues that participate in hydrogen bond interactions are shown as sticks and are labeled.
Figure 2. Effects of the G6 derivatives on HEL and Ba/F3-EpoR-Jak2-V617F cell proliferation. A, HEL cells were treated with the indicated concentrations of each compound for
72 h and cell viability was determined by trypan blue exclusion. B, HEL cells were treated with a 10 lM concentration of each drug for the indicated periods of time and
viability was determined by trypan blue exclusion. C, Ba/F3-EpoR-Jak2-V617F cells were treated with the indicated concentrations for 72 h and cell viability was determined
by MTS assay. D, Ba/F3-EpoR-Jak2-V617F cells were treated with a 10
from two independent experiments, each performed in triplicate.
lM concentration for the indicated times and viability was determined by MTS assay. Shown are results
and caspase-3. Bid and Bim are pro-apoptotic factors that have
been shown to be cleaved into active forms during apoptosis via
the intrinsic pathway, and caspase-3 is an effector caspase that is
also cleaved into its active form during apoptosis.25–27 We found
that NB15, NB13, and NB4 induced cleavage of PARP while NB6
did not (Fig. 4D). NB4 also caused cleavage of caspase-3 (Fig. 4D).
However, only NB15 induced cleavage of PARP and caspase-3,
along with cleavage of Bid and Bim (Fig. 4D).
It has been shown that Jak2-V617F transgenic mice develop a
mixed MPN phenotype, with pathologically high numbers of eryth-
rocytes, white cells and platelets.13 We have shown that G6 has
efficacy in this transgenic model, as it is able to normalize the cell
counts and cause a reversal of other disease symptoms.28 There-
fore, we wanted to determine if specific structural changes to G6
correlate with the ability to reduce the clonogenic growth potential
of bone marrow cells obtained from the Jak2-V617F transgenic

R. Baskin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1402–1407
1405
Table 1
Inhibition of Jak2 in vitro kinase activity
In vitro Jak2 phosphorylation (% In vitro STAT1 phosphorylation (%
of control)
of control)
NB4
NB6
98.41 2.69
94.37 30.16
103.85 20.70
93.31 9.52
NB13 89.10 13.13
89.14 2.69*
71.48 10.53*
NB15 44.24 12.01*
*
p <0.05 versus DMSO control.
mice. For this, cells were isolated and cultured ex vivo in the pres-
ence of each compound for 0, 12, or 24 h, then transferred to meth-
ylcellulose media and cultured for an additional 6 days at which
time the number of erythroid burst forming units (BFU-E) and
granulocyte/macrophage colony forming units (CFU–GM) were
counted. We found that NB4 significantly reduced the number of
BFU–E and CFU-GM after 12 and 24 h of treatment (Fig. 5A). The
only observed effect with NB6 was a significant reduction in the
numbers of CFU–GM at the 24 hour time point (Fig. 5B). NB13 sig-
nificantly reduced both the numbers of BFU-E and CFU-GM, but
only after 24 h (Fig. 5C). Lastly, we found that NB15 significantly
reduced or completely eliminated all clonogenic growth potential
(Fig. 5D).
The results obtained here are interesting for a number of rea-
sons. Firstly, our molecular docking results indicated that the
two para-hydroxyl compounds, NB13 and NB15, interact with the
Jak2 ATP-binding pocket through hydrogen bonds with the hinge
region, the activation loop, and the catalytic loop. In contrast, the
meta-hydroxyl compounds, NB4 and NB6, interacted with the acti-
vation and catalytic loops, but lacked an interaction with the hinge
region. It has previously been shown that the hinge region is an
important area to target for Jak2 inhibition as it interacts with
the adenine group of ATP.18 Our structure–function correlations
determined here support the importance of the hinge region in
Figure 3. Effects of the G6 derivatives on STAT5 phosphorylation in HEL cells. A,
HEL cells were treated with a 25 lM concentration of each compound for 24 h and
lysates were immunoprecipitated for STAT5 and Western blot was used to detect
phospho-STAT5. Shown is one of four representative results. B, Quantification of
STAT5 phosphorylation, measured as a percent of total and normalized to the DMSO
control. n = 4. ⁄, p <0.05 versus DMSO.
Jak2 kinase inhibition as the two meta-hydroxyl compounds exhib-
ited either moderate (NB4) or poor (NB6) Jak2 inhibitory potential
in vitro when compared with NB13 and NB15.
A
B
D
C
Figure 4. Induction of cell cycle arrest and apoptosis in HEL cells. A, Cells were treated with a 10
cytometry for DNA content. Shown are results from two experiments, each measured in triplicate. ⁄, p <0.05. B, Western blot was performed on cells treated with a 25
concentration for 24 h to examine cyclin A, cyclin B1, and CDK 1 expression. b-actin was used as a loading control. Shown is one of two independent results. C, Cells were
treated with a 10 M concentration of each compound for 48 h and analyzed by flow cytometry for Annexin V and propidium iodide staining. Plotted are the percentages of
cells in early apoptosis (Annexin V positive, propidium iodide negative). Shown are the results from two independent experiments, each performed in triplicate. ⁄, p <0.05. D,
lM concentration of each compound for 24 h and analyzed by flow
lM
l
Western blot analysis was performed on cells treated with a 25
is one of two representative results for each blot.
lM concentration of each compound for 24 h to examine expression of PARP, Bid, Bim, and caspase 3. Shown

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R. Baskin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1402–1407
Figure 5. Effects of the G6 derivatives on the ex vivo clonogenic growth potential of Jak2-V617F-positive, murine bone marrow cells. Bone marrow cells were isolated from
6 month old Jak2-V617F transgenic mice and were incubated with media containing a 25 M concentration of each drug for 0, 12, or 24 h. Cells were then washed and plated
l
in semi-solid media, and the number of BFU-E and CFU-GM were counted 6 days later. Panels A–D show the results for NB4, NB6, NB13, and NB15, respectively. Each
condition was measured in duplicate. ⁄, p <0.05 versus 0 h.
It is also notable that, although NB13 and NB15 both demon-
strated in silico Jak2 binding and in vitro Jak2 kinase inhibition,
NB13 was still much weaker than NB15 as a Jak2 inhibitor as mea-
sured by the cell-based assays. Overall, these results indicate that
the para-hydroxyl orientation on a stilbene scaffold alone is not
sufficient to confer Jak2 inhibitory activity, but appears to be a nec-
essary component.
Interestingly, NB4 was able to inhibit Jak2-mediated cell growth
and induce apoptosis and cell cycle arrest. However, it was unable
to reduce in vitro Jak2 kinase activity or reduce STAT5 phosphory-
lation in HEL cells. Furthermore, it demonstrated weak binding
interactions with Jak2 in silico. Therefore, we believe the effects
of NB4 on cell growth are via Jak2-independent mechanisms and
underscore the importance of the para-hydroxyl structure in not
only maintaining potency, but also specificity.
The results obtained with NB6 are also of interest. This meta-hy-
droxyl derivative had a minimized stilbene core. We found that it
had the poorest Jak2 binding properties in silico and was largely
without effect in the protein and cell based assays performed in vi-
tro. From this, we conclude that minimization of the stilbene core
on the meta-hydroxyl scaffold results in poor Jak2 inhibitory
potential.
The parent compound, G6, has previously been shown to interact
with Jak2 in silico via hydrogen bond interactions with the hinge re-
gion, the activation loop, and the catalytic loop.17 Additionally, we
have previously shown that G6 inhibits HEL cell proliferation and
induces apoptosis and cell cycle arrest.16 Of the four G6 derivatives
tested in the current study, NB15 was clearly the most effective
inhibitor as determined in these assays. This compound maintained
the para-hydroxyl orientation of G6, but had a minimized stilbenoid
core. Based on the results obtained here, we conclude that having a
minimized stilbenoid core on the para-hydroxyl structure provides
the highest degree of Jak2 specific inhibition.
In summary, we have identified important structural features of
the Jak2 small molecule inhibitor G6. We found that the para-hy-
droxyl structure with a reduced stilbene core conferred strong
Jak2 interactions in silico as well as greater Jak2 inhibitory poten-
tial in vitro and ex vivo as measured by reduced cell viability, re-
duced Jak2 kinase activity, reduced levels of phospho-STAT,
increased cell cycle arrest and apoptosis and reduced Jak2 clono-
genic growth potential. As such, these results may be useful in
the future development of stilbene-derived Jak2 inhibitors for the
purpose of treating Jak2-mediated pathologies.
Stilbene compounds such as resveratrol and its derivatives have
been implicated as potential therapeutic agents for hematological
malignancies. These compounds have antioxidant properties, but
also induce apoptosis and cell cycle arrest in leukemia cells.29 It
has been shown that the positioning of hydroxyl groups in resvera-
trol derivatives greatly impacts their antioxidant and cell growth
inhibitory potential. Interestingly, the overall cytotoxic effects of
para-hydroxyl resveratrol derivatives are lower than those of
ortho-hydroxyl derivatives.29 This may indicate that using a para-
hydroxyl stilbene scaffold for the development of tyrosine kinase
inhibitors may be beneficial in reducing non-specific cytotoxicity.
Also, this type of structure allows for manipulation of other groups,
such as amine moieties, in order to enhance the specificity for the
target protein.
Acknowledgments
We thank Christopher Fuhrman for assistance with the molec-
ular docking experiments. We thank Steve McClellan for assistance
with flow cytometry. We also thank Anurima Majumder for signif-
icant intellectual contribution. This work was supported by Na-
tional Institutes of Health Award R01-HL67277, a University of
Florida Opportunity Fund Award, and a University of Florida/Mof-
fitt Cancer Center Collaborative Initiative Award.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.042.

R. Baskin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1402–1407
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