Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib, and Derivatives Thereof

Article abstract of DOI:10.1021/acs.jmedchem.0c01952

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.

Full text of DOI:10.1021/acs.jmedchem.0c01952

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Article
Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib,
and Derivatives Thereof
Ryan R. Davis, Baoli Li, Sang Y. Yun, Alice Chan, Pradeep Nareddy, Steven Gunawan, Muhammad Ayaz,
̈
Harshani R. Lawrence, Gary W. Reuther, Nicholas J. Lawrence, and Ernst Schonbrunn*
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ABSTRACT: The discovery that aberrant activity of Janus kinase
2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has
led to significant efforts to develop small molecule inhibitors for
this patient population. Ruxolitinib and fedratinib have been
approved for use in MPN patients, while baricitinib, an achiral
analogue of ruxolitinib, has been approved for rheumatoid arthritis.
However, structural information on the interaction of these
therapeutics with JAK2 remains unknown. Here, we describe a
new methodology for the large-scale production of JAK2 from
mammalian cells, which enabled us to determine the first crystal
structures of JAK2 bound to these drugs and derivatives thereof.
Along with biochemical and cellular data, the results provide a
comprehensive view of the shape complementarity required for
chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as
therapeutics.
been shown to be caused by a single hyperactivating point
mutation, V617F, found in the pseudokinase domain (PKD) of
JAK2.7
INTRODUCTION
■
Protein kinases have become one of the most desired classes of
drug targets given their crucial roles in the regulation of cellular
proliferation, survival, signaling, metabolism, and homeostasis.
As a mediator of cytokine receptor activation, Janus kinase 2
(JAK2) is a cytosolic tyrosine kinase that phosphorylates signal
transducer and activator of transcription proteins (STATs)
resulting in SH2-domain mediated dimerization and STAT
activation. STATs govern many processes including cell
proliferation, differentiation, and immunological responses
vital for cell survival.1 The JAK/STAT pathway has been
listed as one of the 12 core cancer pathways demonstrating the
importance of proper JAK2 regulation to maintain a normal
cell function.1 JAK2 is a multidomain protein (Figure S1),
which undergoes trans-autophosphorylation on the activation
loop of the kinase domain (KD) involving residues Tyr1007
and Tyr1008. The purpose of this phosphorylation is not fully
known but is thought to aid in the recruitment and
phosphorylation of STATs.2 Type 1 inhibitors have been
shown to bind to the adenosine triphosphate (ATP) binding
site of the active conformation of JAK2, but inhibition
paradoxically leads to accumulation of phosphorylated residues
on the activation loop with increasing inhibitor concen-
tration.3,4 This phenomenon has been attributed to the
protection of activation loop phosphotyrosines from phospha-
tases.5,6 Higher levels of phosphorylated Tyr1007/1008, and
therefore higher levels of constitutively active JAK2, have also
Constitutively active JAK2 has been identified in several
types of cancers including breast cancer, lymphomas, and
myeloid malignancies.3,8−10 The V617F mutation is the most
commonly identified mutation found in myeloproliferative
neoplasms (MPNs), in more than 95% of polycythemia vera
patients and in more than 50% of all thrombocythemia and
primary myelofibrosis cases.8,11 The Food and Drug
Administration (FDA) approved the pyrrolopyrimidine JAK2
inhibitor ruxolitinib in 2011 and the dianilinopyrimidine
inhibitor fedratinib in 2019 for treating myelofibrosis.12−14
Baricitinib, an achiral analogue of ruxolitinib, was approved for
rheumatoid arthritis in 2018.15 JAK2 inhibitors for MPN
provide quality of life improvements but little efficacy at
antagonizing the natural course of disease. To date, no crystal
structure of ruxolitinib has been reported with any of the JAK
family proteins, making ruxolitinib the oldest FDA-approved
kinase inhibitor without a cocrystal structure with its target
Received: November 17, 2020
Published: February 11, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c01952
© 2021 American Chemical Society
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Figure 1. Ruxolitinib enhances the expression and purification of crystallization-grade JAK2 KD. (A) Expi293F cells were transiently transfected
with wild-type JAK2 KD. Cells were treated with increasing concentrations of ruxolitinib during the 24-hour transfection. Cell lysates were
subjected to immunoblotting to detect phosphorylated and unphosphorylated JAK2 KD along with the loading control GAPDH. Densitometry of
the western blot is depicted as a bar graph. (B) Same as (A) except 2 mM and 4 mM butyric acid (BA) or valproic acid (VPA) were added to cells
with 1 μM ruxolitinib. (C) Expi293F cells were transiently transfected with JAK2 KD +/− 1 μM ruxolitinib and analyzed by CETSA. Cells were
incubated at the indicated temperatures for 3 min, and lysates were probed for JAK2 KD and the loading control actin. The graph shows the
densitometry values relative to the loading control as a function of temperature. Data were fit to a four-parameter Hill equation, yielding EC50
values of 45 and 47 °C for JAK2 KD expressed in the absence and presence of ruxolitinib, respectively. (D) Cells were grown as in (C) for ITDR
against ruxolitinib at 47 °C for 3 min, and cell lysates were probed for JAK2 and GAPDH. The corresponding densitometry values were fit to a
four-parameter Hill equation yielding EC50 = 3.8 μM. (E) SDS-PAGE of typical purification of JAK2 KD from 1 L of Expi293F cell culture grown
in the presence of 1 μM ruxolitinib and 4 mM BA for 24 h. Lane 1 GenScript broad range ladder, Lane 2 soluble lysate, Lane 3 GE HisTrap flow-
through, Lane 4 GE HisTrap elution peak, and Lane 5 GE S75 elution peak. (F) Photograph of X-ray-grade crystals grown from thus purified JAK2
KD.
protein.16,17 Likewise, structural information on the interaction
of JAK2 with fedratinib and baricitinib remains unknown.
Knowledge of these structures, however, is required for the
development of more efficacious drugs to specifically target
aberrant JAK2. Here, we report the first crystal structures of
ruxolitinib, fedratinib, and baricitinib along with derivatives
RESULTS
■
Ruxolitinib Significantly Increases the Stability of
JAK2 KD Overexpressed in Expi293F Cells. Previously,
JAK2 KD had been predominantly purified from insect cells;
however, this process takes several weeks to generate and
amplify the baculovirus to produce large enough quantities of
bound to the JAK2 KD. We introduce a new methodology to
efficiently produce crystallization-grade JAK2 KD from
mammalian cells, which enabled efficient structure−activity
relationship (SAR) studies with inhibitors of different chemical
scaffolds. A total of 14 high-resolution crystal structures were
determined of JAK2 liganded with known and novel inhibitors.
The data sets detail the requirements of small molecule
inhibitors for shape complementarity with the ATP site of
JAK2 and provide a structural basis for the stereoselective
discrimination of enantiomers of ruxolitinib and derivatives
thereof.
purified protein. Upon transient expression of JAK2 KD in
Expi293F cells, the presence of ruxolitinib during the
transfection increased the levels of phosphorylated JAK2
(pY1007), as previously reported,3 as well the overall amount
of JAK2 KD in a concentration-dependent manner (Figure
1A). Protein levels of recombinant JAK2 KD from Expi293F
cells were approximately 60-fold higher with 1 μM ruxolitinib
compared to untreated cells. Additionally, by including histone
deacetylase (HDAC) inhibitors, valproic acid or butyric acid,
during expression alongside ruxolitinib, JAK2 KD levels
increased fourfold over that of ruxolitinib alone (Figure 1B).
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Figure 2. Purified JAK2 KD from Expi293 cells is suitable for SAR studies by DSF. Three series of JAK2 inhibitors were subjected to SAR studies:
(A) Ruxolitinib stereoisomers and FDA-approved derivatives, (B) piperidine−phenylamine analogues of ruxolitinib, and (C) fedratinib and derived
dual JAK2−BRD4 inhibitors. (D) DSF derivative plots of JAK2 KD in the absence (DMSO) and presence of 100 μM inhibitor. (E) Viability of
UKE-1 cells in response to increasing inhibitor concentration. (F) DSF and biochemical enzyme inhibition data correlate significantly. (G) Analysis
of DSF and UKE-1 cell growth inhibition data shows high correlation for series A, but not for series B and C. Series B likely suffers from poor cell
permeability. (H) For series C, cell inhibition data correlate significantly with the binding potential for BRD4-1, suggesting a predominant activity
through inhibition of BRD4.
Next, we investigated the thermostability of JAK2 KD upon
transient expression in the presence of ruxolitinib using a
cellular thermal shift assay (CETSA)18 and isothermal dose
response (ITDR). At 1 μM concentration, ruxolitinib elevated
the cellular thermostability of JAK2 KD by 2 °C, from 45 to 47
°C (Figure 1C). At 47 °C, the amount of expressed JAK2 KD
increased in dose-response with ruxolitinib (EC50 = 3.8 μM)
(Figure 1D). Expression in the absence of ruxolitinib yielded
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very little soluble protein and purity could not be achieved
above 50% (data not shown). Adding ruxolitinib to cells during
transfection resulted in significantly more protein, which could
be purified to homogeneity using Ni-affinity and size-exclusion
chromatography with yields of 3 mg/L culture (Figure 1E).
Purified JAK2 KD yielded X-ray grade crystals within 24 h
(Figure 1F).
SAR Studies of Diverse Small Molecule JAK2
Inhibitors. To assess the suitability of purified JAK2 KD for
SAR studies for drug development campaigns, three series of
JAK2 inhibitors (Figure 2A-C) were subjected to binding
studies by differential scanning fluorimetry (DSF). Series A
consists of ruxolitinib enantiomers and FDA-approved
derivatives baricitinib and tofacitinib, while series B consists
of piperidine−aniline analogues of ruxolitinib. Series C
comprises the FDA-approved JAK2 inhibitor fedratinib and
other diaminopyrimidines, which were further developed as
dual JAK2−BRD4 inhibitors from early lead compounds.19
The melting temperature of JAK2 in the absence of an
inhibitor was 50 °C, and thermal shifts in the presence of a 100
μM inhibitor ranged from 3.3 to 5.5 °C for series A, 6.4 to 12
°C for series B, and 2.2 to 5.2 °C for series C (Figure 2D and
Table 1).
shifts showed excellent correlation with enzymatic inhibition
values across all compounds, except for (R)-1 and rac-1 of
series B, the high inhibitory activity of which likely exceeded
the sensitivity of the radiometric assay applied (IC50 < 0.15
nM) (Figure 2F). By contrast, only series A showed significant
correlation with the cellular inhibition data (Figure 2G). Series
B probably suffered from poor cell permeability, while series C
showed a higher cell growth inhibitory activity than expected
from JAK2 inhibition alone. The increased cellular activity of
series C is likely caused by their dual activity against JAK2 and
BRD4 as UKE-1 cells are highly sensitive to the inhibition of
BRD4.19 The thermal shifts exerted by these compounds
toward BRD4 correlated significantly with the inhibitory
activity against UKE-1 cell growth (Figure 2H).
Structural Basis of JAK2 Inhibition by Ruxolitinib and
Baricitinib. Structural information on the JAK2−ruxolitinib
complex was previously limited to molecular dynamics
simulations16 or predictions based on the known structures
of c-SRC with ruxolitinib21 and of JAK2 with tofacitinib.22
Crystals of JAK2 KD grown in the absence of an added
inhibitor (Figure 1F) showed that ruxolitinib was still bound to
protein despite ∼100,000-fold dilution throughout the
purification process, reflecting the high binding affinity of
ruxolitinib to JAK2 KD. However, bound ruxolitinib could be
readily displaced by other inhibitors through in-diffusion of
crystals with 1 mM inhibitor for 72 h prior to data collection.
Ruxolitinib was housed deep inside the ATP site, anchored
through H-bonding interactions between the pyrrolopyrimi-
dine moiety and main chain atoms of Glu930 and Leu932 of
the hinge region (Figure 3A-D).
The inhibitor was held in place through several van der
Waals (vdW) hydrophobic interactions with surrounding
residues, including the P-loop (Leu855 and Gly856) and the
DFG motif (Asp994). The binding pose of ruxolitinib in JAK2
agrees with molecular docking predictions16 but significantly
differs from that observed in the crystal structure of c-SRC
(Figure 3E).21 In SRC, ruxolitinib is rotated ∼180° relative to
the hinge region, likely caused by repulsion and/or steric
hindrance with the gatekeeper residue Thr338. In JAK2, the
hydrophobic and flexible gatekeeper residue Met929 accom-
modates ruxolitinib through multiple vdW interactions.
Accordingly, ruxolitinib inhibitory activity is three orders of
magnitudes higher for JAK2 over SRC.21 To confirm the
pyrrolopyrimidine binding mode of ruxolitinib in JAK2, a
cocrystal structure was determined with baricitinib showing
almost identical positioning of the two inhibitors in the ATP
site (Figure 3F).
Stereoselective Discrimination of Ruxolitinib and
Aniline Derivatives by JAK2. The high diffraction power
of the JAK2 KD crystals prompted us to evaluate the SAR of
enantiomers of ruxolitinib and analogues thereof. The (S)-
isomer of ruxolitinib is ∼10-fold less active against JAK2 than
the (R)-isomer (Table 1). The cocrystal structure revealed that
(S)-ruxolitinib adopts a binding pose similar to (R)-ruxolitinib
(Figure 4A) and achieves shape complementarity with the
ATP site through ∼180° rotation about the chiral center
(Figure 4B). Using the racemic mixture of ruxolitinib, (rac)-
ruxolitinib, the resulting cocrystal structure exclusively revealed
the (R)-isomer bound (Figure 4C). Likewise, the (R)- and (S)-
enantiomers of aniline derivatives of ruxolitinib, (R)-1 and (S)-
1, assumed the same orientation for the cyclopentyl and
propionitrile moieties in the ATP site (Figure 4D-F).
Furthermore, the racemic mixture (rac-1) showed only the
Table 1. Binding and Inhibitory Potential of Investigated
JAK2 Inhibitors
compound
ID
binding
biochemical
b
UKE-1 cell growth
a
c
series
A
potential
inhibition
inhibition
ΔTm (°C)
5.5 0.06
3.3 0.04
IC50 (nM)
0.40 0.005
5.0 0.1
IC50 (μM)
0.1 0.02
1.04 0.09
Ruxolitinib
(S)-
Ruxolitinib
(rac)-
Ruxolitinib
4.8 0.05
0.92 0.06
0.38 0.08
Baricitinib
Tofacitinib
(R)-1
(S)-1
5.2 0.08
3.5 0.08
12 0.02
8.3 0.08
11.8 0.05
0.29 0.13
2.9 0.39
0.15 0.03
0.099 0.02
< 0.05
0.19 0.03
0.91 0.1
3.2 0.76
6.3 5.4
B
(rac)-1
2.2 1.9
2
3
7
0.03
0.13 0.048
0.26 0.04
0.75 0.39
6.5 3.5
8.2 5.7
11.4 0.87
3.6 + 1.1
4.5 5.9
6.4 0.04
5.2 0.07
3.1 0.09
3.2 0.04
2.2 0.06
4.3 0.05
0.62 0.04
0.66 0.06
0.2 0.02
0.15 0.01
0.32 0.02
0.21 0.02
C
Fedratinib
4
5
6
7
a
Standard deviation (SD) from two DSF data sets, each performed in
b
quadruplicate. SD from two data sets of radiometric assay by
Reaction Biology. Standard error of the mean (SEM) for data from
c
three experiments, each performed in hexaplicate.
To evaluate if the thermal shifts reflect the inhibitory activity
against JAK2 in cells, compounds were characterized for
growth inhibitory activity of UKE-1 cells, which are driven by
V617F mutant JAK2 (Figure 2E).20 The resulting IC50 values
ranged from 0.1 to 1.0 μM for series A, 0.6 to 6.3 μM for series
B, and 0.15 to 0.66 μM for series C. Additionally, compounds
were characterized for inhibition of the enzymatic JAK2
activity using a radiometric assay by Reaction Biology Corp.,
with IC50 values ranging from 0.29 to 5.0 nM for series A, 0.05
to 0.26 nM for series B, and 0.75 to 11.4 nM for series C
(Table 1). Statistical significance of the data sets was evaluated
using Pearson’s correlation analyses (Figure 2F-H). Thermal
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Figure 3. Structural basis of ruxolitinib interaction with JAK2. Cocrystal structure of JAK2 KD liganded with ruxolitinib determined at 1.9 Å
resolution (PDB 6VGL). (A) Electrostatic surface potential of the JAK2−ruxolitinib complex. The inhibitor is shown as yellow spheres. (B)
Positioning of ruxolitinib in the ATP site. The hinge region is shown in orange, gatekeeper residue in red, DFG motif in green, P-loop in cyan, C-
helix in magenta, and other residues in grey. (C) Binding interactions of ruxolitinib in the ATP site. Potential H-bonding and hydrophobic vdW
interactions are indicated as black and green dotted lines, respectively. (D) 2Fo−Fc electron density map (1σ) of bound ruxolitinib at 1.9 Å
resolution. (E) Binding pose of ruxolitinib (beige) in c-SRC (PDB 4U5J) and upon superposition with ruxolitinib in JAK2 (yellow). (F) 1.9 Å
resolution cocrystal structure of JAK2 with baricitinib (magenta, PDB 6VN8) and superposition with ruxolitinib (yellow).
(R)-isomer bound (Figure 4G). The data demonstrate that
JAK2 discriminates between the (R)- and (S)-stereoisomers of
ruxolitinib and derivatives by preferentially interacting with the
(R)-isomer. Ruxolitinib derivatives devoid of a chiral center
and the cyclopentyl group (2 and 3) exhibited the same
preference of the respective propionitrile and butenyl moieties
for positioning in the subpocket that accommodates the
propionitrile of ruxolitinib (Figure 4H,I). Notably, the aniline
derivatives interacted in the ATP site through an additional H-
bond with the hinge region (Figure 4D-I), which explains the
substantial increase in the JAK2 inhibitory activity of series B
over ruxolitinib and series A (Table 1).
Structural Basis of JAK2 Inhibition by Diaminopyr-
imidines. To further probe the ATP site, cocrystal structures
were determined with dianilinopyrimidine-containing inhib-
itors of series C. All inhibitors interacted with the hinge region
similarly to the aniline derivatives of ruxolitinib (Figure 5).
However, the lack of a pyrrolo moiety allows only for the
establishment of H-bonds with the main chain atoms of
Leu932, but not with Glu930. This is reflected by the 10-fold
reduced biochemical potency of inhibitors of series C against
JAK2 (Table 1). Reversal of the sulfonamide moiety and
addition of fluorine (4), which are beneficial for binding to the
acetyl-lysine binding site of BRD4,19 slightly tilted the aniline
ring such that the tert-butyl moiety moves away from Val863
and positions close to the carboxyl group of Asp994 of the
DFG motif (Figure 5B). Although the reversed sulfonamide
forms potential H-bonds with the side chain of Asn981, the
unfavorably close distance between the hydrophobic tert-butyl
and Asp994 may contribute to the observed slight reduction in
inhibitory activity of 4 and related compounds 5 and 6 (Figure
5C,D). Constraining the sulfonamide moiety using an indoline
moiety (7) was well tolerated (Figure 5E) and showed the
highest inhibitory activity against JAK2 across series C (Table
1).
DISCUSSION AND CONCLUSIONS
■
JAK2 presents challenges for structural studies as it is not
stable upon recombinant overexpression, and typical protein
purification requires over 10 L of insect cultures from
bioreactors to obtain a few milligrams of crystallization-grade
protein.23−26 Recombinant overexpression of JAK2 KD using
mammalian Expi293F cells requires only 24 h of growth post-
transfection, and 1 L of culture is sufficient to purify milligram
quantities of crystallization-grade protein for biochemical and
structural studies. This was achieved using ruxolitinib during
expression, which significantly enhanced protein stability and
purification yields and enabled the first crystal structures of
JAK2 to be determined from protein expressed in mammalian
cells. The crystal structure data suggest that ruxolitinib
improves the thermostability of JAK2 by decreasing flexible
regions and forming a more rigid structure with well-defined
activation and P-loops. Notably, ruxolitinib did not affect the
proliferation of Expi293F or HEK293 cells even at 10 μM after
72 h.19 This strategy provided faster, more efficient, and cost-
effective production of recombinant JAK2 and may be
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Figure 4. JAK2 discriminates between the (R)- and (S)-stereoisomers of ruxolitinib and derivatives thereof. Distinct stereoisomers and the
enantiomeric mixture of ruxolitinib and derivatives were subjected to crystallographic studies with JAK2 KD. (A) Cocrystal structure of (S)-
ruxolitinib (PDB 6VSN). (B) Superposition of the (R)- and (S)-isomers of ruxolitinib (yellow and cyan, respectively) reveals that (S)-ruxolitinib
adopts shape complementarity with the ATP site through ∼180° rotation about the stereocenter. (C) Cocrystal structure obtained with the racemic
mixture of ruxolitinib, (rac)-ruxolitinib (PDB 6VNK) reveals only the (R)-isomer bound (green). The inset shows the superposition of (rac)-
ruxolitinib with ruxolitinib. (D) Cocrystal structure showing the H-bonding interactions of (R)-1 (yellow, PDB 6VNC) with the hinge region. (E)
Same as (D) for the (S)-1 (cyan, PDB 6VNB). (F) Superposition of (R)-1 and (S)-1 reveals the same adaptation as for ruxolitinib (B). (G)
Cocrystal structure obtained with (rac)-1 (green, PDB 6VS3) shows only the (R)-isomer bound. The inset shows the superposition of (rac)-1 with
(R)-1 (yellow). (H) Cocrystal structure with derivative 2 devoid of a stereocenter (PDB 6VNJ) reveals the same binding pose and inhibitor
conformation as (R)-1. (I) Same as (H) for derivative 3 (PDB 6VNM). The blue mesh is the 2Fo−Fc electron density of ligand contoured at 1σ.
The electron density maps upon refinement omitting the ligand are shown in Figure S2.
applicable to other difficult to overexpress kinases provided
that the inhibitor is specific, potent, and nonlethal over the
timeframe of expression.
tion of the preferred (R)-isomer through rotation about the
chiral center, but the resulting binding pose is less compatible
with the ATP site. Notably, the achiral analogue baricitinib
adopts a conformation almost identical to that of ruxolitinib,
which is reflected in the similar values obtained for binding,
enzymatic, and cellular activities of these FDA-approved
inhibitors.
Piperidine−aniline derivatives of both (R)- and (S)-
ruxolitinib showed greatly enhanced binding and inhibitory
potential toward JAK2; the picomolar activities could not be
resolved by the steady-state assays employed here. This
substantial increase in inhibitory activity is attributed to an
additional H-bond established with the hinge region. Although
these compounds suffer from poor cell penetration as indicated
by a significant loss of the cell inhibitory activity, solubilizing
groups other than piperidine may alleviate this drawback.
Diaminopyrimidine inhibitors of JAK2, including fedratinib,
mimic the binding pose of aniline derivatives of ruxolitinib.
However, they lack the potential to establish an H-bond with
Robust crystallization conditions and ease of in-diffusion of
inhibitors resulted in 14 novel cocrystal structures of JAK2
liganded with various inhibitors of three chemical scaffolds.
Along with binding and inhibition data, this information
provides a comprehensive view of the ATP site and the shape
complementarity required for small molecules to achieve
highest inhibitory activity. The (R)- and (S)-stereoisomers of
ruxolitinib and aniline derivatives thereof were thoroughly
characterized for their binding potential and inhibitory activity
against cancer cells driven by constitutively active V617F
JAK2. While (S)-ruxolitinib is a formidable inhibitor of JAK2
(IC50 = 5 nM), the (R)-isomer exerted a > 10-fold higher
activity with respect to binding and cell kill potential. Cocrystal
structures obtained with the isolated isomers along with
racemic mixtures demonstrate that JAK2 discriminates against
the (S)-configuration. The (S)-isomer mimics the conforma-
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Figure 5. Diaminopyrimidine inhibitors mimic the binding pose of ruxolitinib−aniline derivatives. (A) Cocrystal structure of JAK2 with fedratinib
(PDB 6VNE). The inset shows the superposition of fedratinib (yellow) with (R)-1 (magenta). (B) Cocrystal structure with 4 (PDB 6VNG). The
inset shows the superposition of 4 (yellow) with fedratinib (green). (C) Cocrystal structure with 5 (PDB 6VNH). (D) Cocrystal structure with 6
(PDB 6VNL). (E) Cocrystal structure with 7 (PDB 6VNF).
General Synthetic Methods. All reagents were purchased from
commercial suppliers and used without further purification. 1H
nuclear magnetic resonance (NMR) spectra were recorded using a
Bruker 500 MHz spectrometer with CDCl3, CD3OD, or DMSO-d6 as
the solvent. 13C NMR spectra were recorded at 125 MHz. All
coupling constants were measured in Hertz (Hz), and the chemical
shifts (δH and δC) were quoted in parts per million (ppm) relative to
TMS (δ 0), which was used as the internal standard. High-resolution
mass spectroscopy was carried out using an Agilent 6210 LC/MS
(ESI-TOF). High-performance liquid chromatography−mass spec-
trometry (HPLC-MS) analysis was performed using an Agilent 6120
single-quadrupole 1220 LC/MS equipped with a Zorbax SB-C18
column (4.6 × 50 mm, 1.8 μm). The purity of final compounds that
underwent biological assessment was >95% as measured by HPLC-
MS. Thin-layer chromatography (TLC) was performed using silica gel
60 F254 plates (Fisher), with observation under UV when necessary.
Anhydrous solvents (acetonitrile, dimethylformamide, ethanol,
isopropanol, methanol, and tetrahydrofuran) were used as purchased
from Aldrich. Burdick and Jackson HPLC-grade solvents (methanol,
acetonitrile, and water) were purchased from VWR for HPLC and
high-resolution mass analysis. HPLC-grade trifluoroacetic acid (TFA)
was purchased from Fisher. Compound synthesis and characterization
are detailed in the Supporting Information.
Glu930 of the hinge region, which is reflected by the reduced
but still formidable inhibitory activities (IC50 between 0.75 and
11.4 nM). These compounds also inhibit BET bromodomains
to varying degrees and appear to exert a strong cell growth
inhibitory activity by predominantly targeting BRD4. Pre-
viously, structural modeling, binding, and enzymatic assays
using full-length JAK2 expressed in insect cells showed high
affinity of fedratinib for the substrate-binding site.3 The
cocrystal structure of JAK2 KD with fedratinib (or any other
compound studied herein) showed that the inhibitor bound
exclusively to the ATP site. It is conceivable that fedratinib
interaction with the substrate-binding site occurs in the full-
length enzyme, but not in the isolated KD. Combined, our
findings may help the iterative drug design process for
developing JAK2 inhibitors with increased efficacy to combat
cancers caused by this enzyme.
EXPERIMENTAL SECTION
■
Compounds and Reagents. Reagents for biochemical and
crystallographic experiments were purchased from Fisher Scientific
and Hampton Research unless otherwise indicated. Ruxolitinib
(phosphate) was purchased from LC Laboratories (R-6688, >99%),
tofacitinib (citrate) from MedChemExpress (HY-40354A, 99.1%),
baricitinib (free base) from Combi-Blocks (QJ-1094, 98%), and
fedratinib from MedChemExpress (HY-10409, 99.9%). The following
antibodies were used for immunoblotting: His-HRP (ProteinTech,
HRP-66005, 1:5000), pJAK2 (Y1007/1008) (Cell Signaling, 3771,
1:1000), actin (Sigma, A5441, 1:1000), GAPDH-HRP (ProteinTech,
HRP-60004, 1:10,000), and anti-mouse-HRP IgG (Jackson Immu-
noResearch, 115-035-003, 1:2000).
Compound Synthesis and Characterization. Pyrrolopyrimi-
dine Inhibitors of JAK2. (S)-Ruxolitinib was prepared by a novel
resolution of pyrazole-containing carboxylic acid 9 (from conjugate
addition of 4-bromo-1H-pyrazole and ester hydrolysis of 827) as
shown in Scheme S1. Reaction of 9 with the Evans oxazolidinone
provided the diastereoisomeric oxazolidinones (4R,3R)-10 and
(4R,3S)-10, which were easily separated to homogeneity by flash
chromatography on a silica gel. The faster-running fraction was
identified as (4R,3S)-10 by correlation to the enantiomer of clinically
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used (R)-ruxolitinib using the synthetic route described in a reported
patent.27 Treatment of (4R,3S)-10 with ammonium hydroxide28 gave
the primary amide 11, which upon dehydration with phosphorus
pentoxide gave the nitrile 12. Formation of the boronate ester 13
followed by a Suzuki reaction with 4-bromo-7H-pyrrolo[2,3-d]-
pyrimidine provided (S)-ruxolitinib. The enantiomeric excess of (S)-
ruxolitinib was determined to be >99.99% by chiral HPLC (Figure
S3). A sample of (rac)-ruxolitinib was prepared from rac-13 in the
same way. The synthesis of the ruxolitinib analogues (R)- and (S)-1 is
shown in Schemes S1,S2. 2-Chloro-ruxolitinib (14) was prepared by
Suzuki coupling of 13 with 2,4-dichloropyrrolopyrimidine (Scheme
S1). Substitution of the chlorine atom with the anilinopyrimidine
substituent required protection of the pyrrole NH group. Thereby,
Buchwald−Hartwig amination of the boc-protected pyrrolopyrimi-
dine 15 gave 16 bearing anilinopiperidine at the 2-position of the
pyrimidine ring. Finally, removal of the protecting groups gave the
desired (S)-1 bearing the required piperidine group oriented toward
the solvent-accessible region. Again, a chiral HPLC analysis indicated
that the enantiomeric excess of (S)-1 was >99.99% (as determined by
the e.e. of its bis-boc protected precursor (S)-16, Figure S4). The
enantiomer (R)-1 was prepared in the same way using the
diastereoisomer (4R,3R)-10 (Scheme S2). In this case, the
enantiomeric excess of (R)-1 was 98.6% (as determined by the e.e.
of its bis-boc protected precursor (R)-22. Ruxolitinib derivatives
lacking the cyclopentyl group have been shown to retain potent JAK2
inhibitory activity.29,30 Thus, piperidine 2, incorporating the
propionitrile-substituted pyrazole, was prepared in a similar way by
Suzuki reaction of addition of 2,4-dichloropyrrolopyrimidine and the
boronic ester 27 (from acrylonitrile and 4-pyrazoleboronic
pinacolate31) to give the nitrile 28 (Scheme S3). A similar sequence
of Buchwald−Hartwig amination of the boc-protected 2-chloropyrro-
lopyrimidine 29 followed by deprotection of 30 provided piperidine
2. The piperidine 3, bearing a butenyl-substituted pyrazole group, was
prepared using the same methods used to prepare piperidine 2
(Scheme S4).
(4R)-4-Benzyl-3-((3R)-3-(4-Bromo-1H-Pyrazol-1-yl)-3-
Cyclopentylpropanoyl)Oxazolidin-2-One (4R,3R)-10. To a solution
of 9 (5.73 g, 20.0 mol, 1.0 eq) in tetrahydrofuran (172 mL), pivaloyl
chloride (4.93 mL, 40.0 mol, 2.0 eq) and triethylamine (8.34 mL, 60.0
mol, 3.0 eq) were added at 0 °C. The reaction mixture was stirred for
1 h at room temperature. To the reaction mixture, lithium chloride
(1.70 g, 40.0 mol, 2.0 eq) and oxazolidinone (3.58 g, 20.0 mol, 1.0 eq)
were added, and then the mixture was stirred overnight at room
temperature. The reaction mixture was diluted with water (100 mL),
evaporated under a reduced pressure to remove tetrahydrofuran, and
extracted with EtOAc (3 × 150 mL). The organic layer was
evaporated under a reduced pressure and the residue was purified by
SiO2 chromatography using EtOAc/hexane (20%) as an eluent to give
the title compound as a white solid (3.9 g, 42%, TLC: Rf = 0.51
developed using EtOAc/hexane (2:3)). HPLC 97% (tR = 13.40 min,
CH3OH in 0.1% TFA water 5−95% in 20 min); 1H NMR (500 MHz,
DMSO-d6) δ 8.10 (d, J = 0.7 Hz, 1H), 7.61 (s, 1H), 7.27−7.21 (m,
3H), 6.94−6.91 (m, 2H), 4.63−4.52 (m, 3H), 4.32 (t, J = 8.7 Hz,
1H), 4.17 (dd, J = 9.0, 3.1 Hz, 1H), 3.79 (dd, J = 17.6, 10.6 Hz, 1H),
3.20 (dd, J = 17.6, 2.9 Hz, 1H), 2.83−2.74 (m, 2H), 2.37−2.30 (m,
1H), 1.79−1.74 (m, 1H), 1.60−1.50 (m, 3H), 1.47−1.39 (m, 1H),
1.29−1.12 (m, 3H); HPLC−MS (ESI+): m/z 468.2 (M + Na)+.
(4R)-4-Benzyl-3-((3S)-3-(4-Bromo-1H-Pyrazol-1-yl)-3-
Cyclopentylpropanoyl)Oxazolidin-2-One (4R,3S-10). (4R,3S)-10
was purified using the procedure described for (4R,3R)-10 to obtain
the title product as a white solid (4.0 g, 43%, TLC: Rf = 0.62
developed using EtOAc/hexane (2:3)). HPLC 98% (tR = 13.60 min,
CH3OH in 0.1% TFA water 5−95% in 20 min); 1H NMR (500 MHz,
DMSO-d6) δ 8.10 (d, J = 0.8 Hz, 1H), 7.52 (d, J = 0.6 Hz, 1H), 7.34−
7.30 (m, 2H), 7.28−7.24 (m, 1H), 7.22−7.19 (m, 2H), 4.61−4.49
(m, 2H), 4.28 (t, J = 8.5 Hz, 1H), 4.15 (dd, J = 8.8, 2.8 Hz, 1H), 3.53
(dd, J = 17.1, 10.4 Hz, 1H), 3.37 (dd, J = 17.1, 3.0 Hz, 1H), 2.97 (dd,
J = 13.6, 3.2 Hz, 1H), 2.84 (dd, J = 13.5, 8.3 Hz, 1H), 2.34−3.31 (m,
1H), 1.82−1.73 (m, 1H), 1.64−1.50 (m, 3H), 1.47−1.41 (m, 1H),
1.36−1.12 (m, 1H); HPLC−MS (ESI+): m/z 446.2 (M + H)+.
(S)-3-(4-Bromo-1H-Pyrazol-1-yl)-3-Cyclopentylpropanamide
(11). The amide 11 was synthesized using the same procedure
described for 17 from (4R,3S)-10 (7.6 mmol, 3.4 g) as a white solid
(1.9 g, 87%). HPLC 97.2% (tR = 11.4 min, CH3OH in 0.1% TFA
water 5−95% in 20 min); 1H NMR (500 MHz, DMSO-d6) δ 7.92 (s,
1H), 7.51 (s, 1H), 7.30 (s, 1H), 6.76 (s, 1H), 4.41 (td, J = 9.7, 4.1 Hz,
1H), 2.76 (dd, J = 15.3, 10.0 Hz, 1H), 2.57 (dd, J = 15.3, 4.0 Hz, 1H),
2.24 (h, J = 8.6 Hz, 1H), 1.79−1.70 (m, 1H), 1.59−1.35 (m, 4H),
1.24−1.06 (m, 3H); HPLC−MS (ESI+): m/z 286.2 (M + H)+.
(S)-3-(4-Bromo-1H-Pyrazol-1-yl)-3-Cyclopentylpropanenitrile
(12). The nitrile 12 was synthesized using the same procedure
described for 18 from amide 11 (6.6 mmol, 1.9 g) as a white solid
(1.6 g, 94%). HPLC 97.2% (tR = 12.5 min, CH3OH in 0.1% TFA
water 5−95% in 20 min); 1H NMR (500 MHz, DMSO-d6) δ 8.13 (d,
J = 0.7 Hz, 1H), 7.63 (s, 1H), 4.38 (td, J = 9.4, 4.7 Hz, 1H), 3.15−
3.05 (m, 2H), 2.36−2.26 (m, 1H), 1.80−1.69 (m, 2H), 1.62−1.37
(m, 4H), 1.30−1.19 (m, 2H), 1.08 (dq, J = 12.7, 8.3 Hz, 1H);
HPLC−MS (ESI+): m/z 268.0 (M + H)+.
Methyl 3-(4-Bromo-1H-Pyrazol-1-yl)-3-Cyclopentylpropanoate
(8). This compound was prepared according to the reported
method.27 To a solution of 3-cyclopentyl methacrylate (4.56 g,
29.58 mol, 1.0 eq) in anhydrous acetonitrile (100 mL), 4-
bromopyrazole (4.77 g, 32.49 mol, 1.1 eq) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (6.63 mL, 44.37 mol, 1.5
eq) were added. The reaction mixture was stirred overnight at room
temperature. The mixture was concentrated under a reduced pressure
and the residue was dissolved in ethyl acetate (EtOAc, 500 mL), 1 N
HCl was added to adjust the pH value to 3 or 4, and then washed with
water (100 mL) and brine (100 mL). The organic layer was dried
(Na2SO4), filtered, and concentrated under a reduced pressure to
obtain the title compound as a colorless oil (7.02 g, 80%). This
compound was used in the next step without purification. HPLC 98%
1
(tR = 13.1 min, CH3OH in 0.1% TFA water 5−95% in 20 min); H
NMR (500 MHz, DMSO-d6) δ 8.04 (s, 1H), 7.51 (s, 1H), 4.39 (m,
1H), 3.50 (s, 1H), 2.97 (m, 2H), 2.24 (m, 1H), 1.74 (m, 1H), 1.59−
1.34 (m, 4H), 1.26−1.05 (m, 3H); HPLC−MS (ESI+): m/z 301.1
(M + H)+.
(S)-3-Cyclopentyl-3-(4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-
2-yl)-1H-Pyrazol-1-yl)Propanenitrile (13). The boronic ester 13 was
synthesized using the same procedure described for 13 from (S)-
bromopyrazole 19 (1.1 mmol, 0.3 g) as a white solid (165 mg, 47%).
1H NMR (500 MHz, DMSO-d6) δ 8.08 (s, 1H), 7.95 (s, 1H), 4.43
(td, J = 9.7, 4.3 Hz, 1H), 3.15−3.09 (m, 2H), 2.37−2.27 (m, 1H),
1.80−1.71 (m, 1H), 1.58−1.37 (m, 4H), 1.25 (s, 12H), 1.20−1.15
(m, 3H).
(S)-3-(4-(7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-1-yl)-3-Cy-
clopentylpropanenitrile [(S)-Ruxolitinib]. (S)-Ruxolitinib was syn-
thesized using the same procedure described for 14 from (S)-13 (0.15
mmol, 49 mg) as a white solid (35 mg, 74%). HPLC 99.17% (tR =
12.57 min, CH3OH 75% in 0.1% TFA water, 20 min); 1H NMR (500
MHz, DMSO) δ 12.10 (s, 1H), 8.79 (s, 1H), 8.67 (s, 1H), 8.37 (s,
1H), 7.59 (dd, J = 3.5, 2.4 Hz, 1H), 6.98 (dd, J = 3.6, 1.7 Hz, 1H),
4.54 (td, J = 9.8, 4.0 Hz, 1H), 3.26 (dd, J = 17.2, 9.8 Hz, 2H), 2.42
(dd, J = 17.1, 8.3 Hz, 1H), 1.86−1.78 (m, 1H), 1.65−1.40 (m, 4H),
3-(4-Bromo-1H-Pyrazol-1-yl)-3-Cyclopentylpropanoic Acid (9).
This compound was prepared according to the reported method.27
To a solution of 8 (7.02 g, 23.40 mol, 1.0 eq), in dioxane (140 mL),
was added sodium hydroxide (2.81 g, 70.20 mol, 3.0 eq) in water (70
mL). The reaction mixture was stirred overnight at room temperature.
The mixture was concentrated under a reduced pressure and water
(100 mL) was added followed by 1 N HCl to adjust the pH to 3 or 4.
The aqueous layer was extracted with EtOAc (3 × 150 mL), dried
(Na2SO4) and filtered, and concentrated under a reduced pressure to
obtain the title compound as a white solid (6.20 g, 95%). This
compound was used in the next step without further purification.
HPLC 99% (tR = 12.3 min, CH3OH in 0.1% TFA water 5−95% in 20
min); 1H NMR (500 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.02 (s, 1H),
7.51 (s, 1H), 4.37 (m, 1H), 2.86 (m, 2H), 2.23 (m, 1H), 1.75 (m,
1H), 1.60−1.35 (m, 4H), 1.26−1.06 (m, 3H); HPLC−MS (ESI+):
m/z 287.2 (M + H)+.
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1
1.35−1.17 (m, 3H); HPLC−MS (ESI+): m/z 307.2 (M+H)+. HRMS
(ESI+) m/z calculated for C17H19N6 (M + H)+ 307.1666, found
307.1667.
7.76 min, CH3OH 50% and water 50% in 20 min); H NMR (500
MHz, DMSO-d6) δ 11.49 (s, 1H), 9.04 (s, 1H), 8.69 (s, 1H), 8.30 (s,
1H), 7.82−7.78 (m, 2H), 7.22 (dd, J = 3.7, 2.1 Hz, 1H), 7.15−7.11
(m, 2H), 6.79 (dd, J = 3.6, 1.6 Hz, 1H), 4.57 (td, J = 9.7, 4.2 Hz, 1H),
3.26−3.20 (m, 2H), 3.08 (d, J = 11.9 Hz, 2H), 2.68−2.61 (m, 2H),
1.87−1.80 (m, 1H), 1.73 (d, J = 12.6 Hz, 2H), 1.65−1.42 (m, 7H),
1.38−1.20 (m, 6H); HPLC−MS (ESI+): m/z 481.5 (M+H)+. HRMS
(ESI+) m/z calculated for C28H33N8 (M + H)+ 481.2823, found
481.2830.
(3RS)-3-(4-(7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-1-yl)-3-
Cyclopentylpropanenitrile (rac-Ruxolitinib). To a solution of 4-
bromo-7H-pyrrolo[2,3-d]pyrimidine (198 mg, 1.0 mmol, 1.0 eq), 3-
cyclopentyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazol-1-yl)propanenitrile (378 mg, 1.2 mmol, 1.2 eq)[prepared
from commercially available 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-1H-pyrazole and 3-cyclopentylprop-2-enenitrile according to
a reported procedure],32 and Na2CO3 (340 mg, 3.2 mmol, 3.2 eq) in
dioxane (4 mL) and H2O (1 mL) was added Pd(PPh3)4 (40 mg,
0.033 mmol, 0.033 eq). The pressure tube was purged with argon for
15 min, sealed, and then placed in a preheated oil bath at 120 °C. The
mixture was stirred at this temperature for 12 h and then cooled to
room temperature and partitioned between saturated NH4Cl (20 mL)
and EtOAc (10 mL). The layers were separated, and the aqueous
layer was extracted twice more with EtOAc (10 mL). The combined
organic extracts were washed with brine, dried (MgSO4), and
concentrated under a reduced pressure to give a yellow oil.
Purification by flash chromatography (SiO2, 75% EtOAc in hexane)
provided the title compound rac-ruxolitinib as a brown powder (0.224
g, 73%). 1H NMR (500 MHz, DMSO-d6) δ: 12.11 (s, 1H), 8.80 (d, J
= 0.7 Hz, 1H), 8.68 (s, 1H), 8.37 (s, 1H), 7.60 (dd, J = 3.6, 2.4 Hz,
1H), 6.99 (dd, J = 3.6, 1.8 Hz, 1H), 4.54 (td, J = 9.8, 4.1 Hz, 1H),
3.20 (m, 3H), 2.43 (m, 1H), 1.82 (m, 1H), 1.61 (m, 3H), 1.46 (m,
1H), 1.39 (m, 2H), 1.18 (m, 1H). HPLC−MS (ESI+): m/z 307.4
[100% (M+H)+], HRMS (ESI+) m/z calculated for C17H19N6 (M +
H)+ 307.1666, found 307.1662.
(S)-3-(4-(2-Chloro-7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-
1-yl)-3-Cyclopentylpropanenitrile (14). The pyrrolopyrimidine 14
was synthesized using the same procedure described for 20 from (S)-
boronic ester 13 (1.1 mmol, 0.3 g) as a white oil (151 mg, 41%).
HPLC 78.0% (tR = 11.99 min, CH3OH in 0.1% TFA water 5−95% in
20 min); 1H NMR (500 MHz, DMSO-d6) δ 12.32 (s, 1H), 8.84 (d, J
= 0.7 Hz, 1H), 8.39 (s, 1H), 7.64 (dd, J = 3.6, 2.3 Hz, 1H), 7.05 (dd, J
= 3.6, 1.8 Hz, 1H), 4.56 (td, J = 9.8, 4.1 Hz, 1H), 3.29−3.21 (m, 2H),
2.43 (q, J = 8.6 Hz, 1H), 1.82 (td, J = 11.9, 7.2 Hz, 1H), 1.66−1.41
(m, 4H), 1.33−1.23 (m, 3H); HPLC−MS (ESI+): m/z 341.2 (M +
H)+.
tert-Butyl (S)-2-Chloro-4-(1-(2-Cyano-1-Cyclopentylethyl)-1H-
Pyrazol-4-yl)-7H-Pyrrolo[2,3-d]Pyrimidine-7-Carboxylate (15). The
boc-protected pyrrolopyrimidine 15 was synthesized using the same
procedure described for 21 from (S)-pyrrolopyrimidine 14 (0.27
mmol, 92 mg) as a white solid (70 mg, 68%). HPLC 95.8% (tR = 13.6
min, CH3OH in 0.1% TFA water 5−95% in 20 min); 1H NMR (500
MHz, DMSO-d6) δ 8.91 (d, J = 0.7 Hz, 1H), 8.44 (s, 1H), 7.92 (d, J =
4.1 Hz, 1H), 7.27 (d, J = 4.2 Hz, 1H), 4.56 (td, J = 9.7, 4.1 Hz, 1H),
3.29−3.21 (m, 2H), 2.43 (q, J = 8.6 Hz, 1H), 1.82 (td, J = 12.2, 7.4
Hz, 1H), 1.64 (s, 8H), 1.58−1.41 (m, 4H), 1.35−1.23 (m, 3H);
HPLC−MS (ESI+): m/z 441.2 (M + H)+.
tert-Butyl (S)-2-((4-(1-(tert-Butoxycarbonyl)Piperidin-4-yl)-
Phenyl)Amino)-4-(1-(2-Cyano-1-Cyclopentylethyl)-1H-Pyrazol-4-
yl)-7H-Pyrrolo[2,3-d]Pyrimidine-7-Carboxylate (16). The bis-boc-
protected pyrrolopyrimidine 16 was synthesized using the same
procedure described for 22 from the (S)-pyrrolopyrimidine derivative
15 (0.09 mmol, 60 mg) as a yellow solid (47 mg, 76%). HPLC 97.0%
(tR = 15.04 min, CH3OH in 0.1% TFA water 5−95% in 20 min); 1H
NMR (500 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.43 (s, 2H), 8.75 (d, J
= 0.8 Hz, 1H), 8.33 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 4.1
Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 4.2 Hz, 1H), 4.56 (td, J
= 9.6, 4.4 Hz, 2H), 3.26−3.21 (m, 2H), 2.65−2.62 (m, 1H), 2.45−
2.40 (m, 1H), 1.85−1.80 (m, 1H), 1.76 (d, J = 13.1 Hz, 2H), 1.66−
1.52 (m, 15H), 1.43 (s, 9H), 1.36−1.26 (m, 5H); HPLC−MS (ESI
+): m/z 681.4 (M + H)+.
(R)-3-(4-Bromo-1H-Pyrazol-1-yl)-3-Cyclopentylpropanamide
(17). To a solution of (4R,3R)-10 (3.0 g, 6.7 mol, 1.0 eq) in
tetrahydrofuran (180 mL) was added ammonium hydroxide (30%,
120 mL). The reaction mixture was stirred for 2 days at room
temperature. The mixture was diluted with methanol (250 mL),
concentrated under a reduced pressure, and this process was repeated
another two times. The residue obtained was purified by reverse-
phase chromatography over a C-18 silica gel using methanol/
dichloromethane (gradient elution 0 to 5%) as an eluent to give the
title compound as a white solid (1.24 g, 64%). HPLC 95% (tR = 11.44
min, CH3OH in 0.1% TFA water 5−95% in 20 min); 1H NMR (500
MHz, DMSO-d6) δ 7.92 (d, J = 0.7 Hz, 1H), 7.51 (d, J = 0.7 Hz, 1H),
7.29 (s, 1H), 6.75 (s, 1H), 4.41 (td, J = 9.7, 4.0 Hz, 1H), 2.79−2.57
(m, 1H), 2.27−2.20 (m, 1H), 1.78−1.72 (m, 1H), 1.60−1.36 (m,
4H), 1.24−1.04 (m, 3H); HPLC−MS (ESI+): m/z 286.1 (M + H)+.
(R)-3-(4-Bromo-1H-Pyrazol-1-yl)-3-Cyclopentylpropanenitrile
(18). This compound was prepared according to the reported
method.27 To a solution of 17 (1.24 g, 4.3 mol, 1.0 eq), in
tetrahydrofuran (50 mL), was added phosphorus pentoxide (1.84 g,
13.0 mol, 3 eq) under argon. The reaction mixture was stirred for 2 h
at 70 °C, diluted with EtOAc (200 mL), and quenched by adding
saturated sodium bicarbonate (200 mL). The aqueous layer was back-
extracted with EtOAc (2 × 100 mL). The organic phases were
combined, washed with water, brine, dried over anhydrous Na2SO4,
and filtered. The filtrate was concentrated under a reduced pressure
and purified by SiO2 chromatography using methanol/dichloro-
methane (5%) as an eluent to provide the title compound as a white
solid (1.13 g, 98%). HPLC 100% (tR = 12.56 min, CH3OH in 0.1%
1
TFA water 5−95% in 20 min); H NMR (500 MHz, DMSO-d6) δ
8.12 (d, J = 0.5 Hz, 1H), 7.63 (d, J = 0.5 Hz, 1H), 4.38 (td, J = 9.4,
4.7 Hz, 1H), 3.15−3.08 (m, 2H), 2.33−2.28 (m, 1H), 1.79−1.72 (m,
1H), 1.61−1.38 (m, 4H), 1.30−1.05 (m, 3H); HPLC−MS (ESI+):
m/z 268.0 (M + H)+.
(R)-3-Cyclopentyl-3-(4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-
2-yl)-1H-Pyrazol-1-yl)Propanenitrile (19). This compound was
prepared according to the reported method.27 To a 20 mL microwave
vial, 18 (0.30 g, 1.12 mmol, 1.0 eq), 4,4,5,5-tetramethyl-2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (0.30 g, 1.19
mmol, 1.1 eq), potassium acetate (329 mg, 3.36 mmol, 3.0 eq), and
1,4-dioxane (4.0 mL) were added. The resulting reaction mixture was
degassed by bubbling argon for 5 min before being treated with
tetrakis(triphenylphosphine)palladium(0) (65 mg, 0.06 mmol, 0.05
eq). The resulting reaction mixture was heated to 120 °C in a
microwave reactor for 2 h. The reaction mixture was filtered through a
celite bed, and the celite bed was washed with dichloromethane and
the organic layer was diluted with water (10 mL). The aqueous layer
was extracted with dichloromethane (2 × 10 mL). The combined
organic layers were concentrated under a reduced pressure, and the
crude product was purified using SiO2 chromatography (EtOAc 0−
30% in hexane) to yield the title compound as a yellow oil (153 mg,
1
43%). H NMR (500 MHz, DMSO-d6) δ 8.09 (d, J = 0.7 Hz, 1H),
7.66 (d, J = 0.7 Hz, 1H), 4.43 (td, J = 9.6, 4.3 Hz, 1H), 3.19−3.05 (m,
2H), 2.33−2.31 (mz, 1H), 1.80−1.73 (m, 1H), 1.61−1.39 (m, 4H),
1.27 (s, 12H), 1.20−1.15 (m, 3H).
(R)-3-(4-(2-Chloro-7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-
1-yl)-3-Cyclopentylpropanenitrile (20). To a 50 mL round-bottom
flask were added 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (87.0 mg,
0.46 mmol, 0.95 eq), 19 (153.0 mg, 0.49 mmol, 1 eq), sodium
carbonate (191 mg, 1.38 mmol, 3 eq), water (0.5 mL), and 1,4-
dioxane (2.0 mL), and the resulting reaction mixture was degassed by
bubbling argon for 5 min before adding Pd(PPh3)4 (17 mg, 0.01
(S)-3-Cyclopentyl-3-(4-(2-((4-(Piperidin-4-yl)Phenyl)Amino)-7H-
Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-1-yl)Propanenitrile [(S)-
1]. The anilinopiperidine (S)-1 was synthesized using the same
procedure described for (R)-1 from (S)-pyrrolopyrimidine 16 (0.06
mmol, 42 mg) as a yellow solid (24 mg, 83%). HPLC 97.6% (tR =
2236
https://dx.doi.org/10.1021/acs.jmedchem.0c01952
J. Med. Chem. 2021, 64, 2228−2241

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
mmol, 0.03 eq). The resulting reaction mixture was heated to 100 °C
under argon overnight. The reaction mixture was gradually cooled
down to ambient temperature and filtered through a celite bed. The
celite bed was washed with dichloromethane (10 mL) and the organic
layer was diluted with water (10 mL). The aqueous layer was
extracted with dichloromethane (2 × 10 mL). The combined organic
layers were concentrated under a reduced pressure to remove
solvents, and the crude product was purified by SiO2 chromatography
(EtOAc 0−60% in hexane) to yield the title compound as a yellow
solid (94 mg, 68%). HPLC 74.7% (tR = 11.93 min, CH3OH in 0.1%
δ 11.49 (s, 1H), 9.01 (s, 1H), 8.68 (s, 1H), 8.30 (s, 1H), 7.80−7.76
(m, 2H), 7.21 (d, J = 3.6 Hz, 1H), 7.14−7.10 (m, 2H), 6.78 (d, J =
3.6 Hz, 1H), 4.56 (td, J = 9.6, 4.1 Hz, 1H), 3.22−3.17 (m, 2H), 3.02
(d, J = 11.9 Hz, 1H), 2.44−2.42 (m, 1H), 1.81−1.85 (m, 1H), 1.68
(d, J = 13.0 Hz, 2H), 1.61−1.44 (m, 6H), 1.36−1.22 (m, 8H);
HPLC−MS (ESI+): m/z 681.5 (M+H)+. HRMS (ESI+) m/z
calculated for C28H33N8 (M+H)+ 481.2823, found 481.2830.
3-(4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-2-yl)-1H-Pyrazol-
1-yl)Propanenitrile (27).33 Using the reported method,31 a 100 mL
round-bottom flask was charged with 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (5.0 g, 25.8 mmol, 1.0 eq),
acrylonitrile (1.04 g, 52.0 mmol, 2.0 eq), DBU (1.97 g, 12.9 mmol,
0.5 eq), and acetonitrile (50 mL). The mixture was stirred at room
temperature overnight. After cooling, the mixture was diluted with
EtOAc, washed with water twice and brine once, and dried over
Na2SO4. After filtration, the filtrate was concentrated under a reduced
1
TFA water 5%∼95% in 20 min); H NMR (600 MHz, DMSO-d6) δ
12.32 (s, 1H), 8.84 (d, J = 0.8 Hz, 1H), 8.39 (s, 1H), 7.64 (d, J = 3.6,
1H), 7.05 (d, J = 3.6, 1H), 4.56 (td, J = 9.8, 4.0 Hz, 1H), 3.27−3.18
(m, 2H), 2.45−2.38 (m, 1H), 1.86−1.78 (m, 1H), 1.64−1.44 (m,
4H), 1.35−1.24 (m, 3H); HPLC−MS (ESI+): m/z 341.2 (M + H)+.
tert-Butyl (R)-2-Chloro-4-(1-(2-Cyano-1-Cyclopentylethyl)-1H-
Pyrazol-4-yl)-7H-Pyrrolo[2,3-d]Pyrimidine-7-Carboxylate (21). To
a solution of pyrrolopyrimidine 20 (94.0 mg, 0.28 mmol, 1.0 eq), in
dichloromethane (2.0 mL), N,N-diisopropylethylamine (0.058 mL,
0.33 mmol, 1.2 eq) was added and then di-tert-butyl dicarbonate (90.3
mg, 0.41 mmol, 1.5 eq) and 4-dimethylaminopyridine (7.0 mg, 0.06
mmol, 0.2 eq) were added. The reaction mixture was stirred for 2 h at
room temperature. The reaction mixture was diluted with water (10
mL) and extracted with dichloromethane (3 × 10 mL). The organic
phase was evaporated under a reduced pressure and the residue was
purified by SiO2 chromatography using EtOAc/hexane (50%) as an
eluent to give the title compound as a white solid (98 mg, 80%).
HPLC 95.2% (tR = 13.63 min, CH3OH in 0.1% TFA water 5%∼95%
1
pressure to yield the nitrile 27 as a yellow oil (3.9 g, 62%). H NMR
(500 MHz, DMSO-d6) δ 8.04 (d, J = 0.6 Hz, 1H), 7.65 (d, J = 0.7 Hz,
1H), 4.41 (t, J = 6.4 Hz, 2H), 3.07 (t, J = 6.4 Hz, 2H), 1.26 (s, 9H).
3-(4-(2-Chloro-7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-1-
yl)Propanenitrile (28). To a 50 mL round-bottom flask were added
2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (3.0 g, 12.0 mmol, 1.2 eq),
3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)-
propanenitrile 27 (0.51 g, 10.0 mmol, 1.0 eq), sodium carbonate (3.4
g, 31.6 mmol, 3.16 eq), water (H2O, 13 mL), and 1,4-dioxane (53
mL), and the resulting reaction mixture was degassed by bubbling
argon for 5 min before being treated with Pd(PPh3)4 (384 mg, 0.333
mmol). The resulting reaction mixture was heated to 100 °C under
argon overnight. The reaction mixture was gradually cooled to
ambient temperature before being filtered through a celite bed. The
celite bed was washed with dichloromethane before the filtrate and
washes were combined. The two layers were separated, and the
aqueous layer was extracted with dichloromethane. The combined
organic layers were concentrated under a reduced pressure to remove
solvents, and the residue was purified by column chromatography
(EtOAc 0 to 50% in hexane) to yield the title compound 28 as a
brown solid (1.8 g, 69%). 1H NMR (500 MHz, DMSO-d6) δ 12.33 (s,
1H), 8.81 (d, J = 0.7 Hz, 1H), 8.37 (d, J = 0.7 Hz, 1H), 7.64 (dd, J =
3.6, 2.3 Hz, 1H), 7.03 (dd, J = 3.6, 1.7 Hz, 1H), 4.53 (t, J = 6.4 Hz,
2H), 3.19 (t, J = 6.4 Hz, 2H). HPLC−MS (ESI+): m/z 273.4 (M
+1)+, 567.3 (2 M+Na)+.
tert-Butyl-2-Chloro-4-(1-(2-Cyanoethyl)-1H-Pyrazol-4-yl)-7H-
Pyrrolo[2,3-d]Pyrimidine-7-Carboxylate (29). To a solution of
chloropyrrolopyrimidine 28 (850 mg, 3.12 mmol, 1.2 eq) in DCM
(15 mL) were added (Boc)2O (1.0 g, 4.68 mmol, 1.5 eq), DIPEA
(0.49 g, 3.75 mmol, 1.2 eq), and DMAP (76 mg, 0.624 mmol, 0.2 eq).
The reaction mixture was stirred at room temperature for 12 h,
quenched with water, and extracted with EtOAc (2 × 20 mL). The
organic layer was dried (Na2SO4) and concentrated in vacuo to yield
the title compound 29 as a brown solid (1.12 g, 96%). 1H NMR (500
MHz, DMSO-d6) δ 8.87 (d, J = 0.9 Hz, 1H), 8.40 (d, J = 0.7 Hz, 1H),
7.91 (d, J = 4.1 Hz, 1H), 7.25 (d, J = 4.2 Hz, 1H), 4.53 (t, J = 6.4 Hz,
2H), 3.19 (t, J = 6.4 Hz, 2H), 1.64 (s, 9H). HPLC−MS (ESI+): m/z
373.1 [60%, (M+H)+], 769.2 [100% (2M + Na)+].
1
in 20 min); H NMR (600 MHz, DMSO-d6) δ 8.90 (d, J = 0.8 Hz,
1H), 8.43 (s, 1H), 7.91 (d, J = 4.1 Hz, 1H), 7.26 (d, J = 4.2 Hz, 1H),
4.55 (td, J = 9.8, 4.0 Hz, 1H), 3.24−3.16 (m, 2H), 1.85−1.78 (m,
1H), 1.58−1.40 (m, 4H), 1.35−1.22 (m, 12H); HPLC−MS (ESI+):
m/z 441.2 (M + H)+.
tert-Butyl (R)-2-((4-(1-(tert-Butoxycarbonyl)Piperidin-4-yl)-
Phenyl)Amino)-4-(1-(2-Cyano-1-Cyclopentylethyl)-1H-Pyrazol-4-
yl)-7H-Pyrrolo[2,3-d]Pyrimidine-7-Carboxylate (22). A mixture of
boc-pyrrolopyrimidine 21 (47.0 mg, 0.11 mmol, 1.0 eq), tert-butyl 4-
(4-aminophenyl)piperidine-1-carboxylate (30.0 mg, 0.11 mmol, 1.0
eq), and potassium carbonate (30.0 mg, 2 mmol, 2.0 eq) in anhydrous
tert-butyl alcohol (2 mL) was degassed by bubbling argon. To this
mixture, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (10.0
mg, 0.021 mmol, 0.2 eq) and tris(dibenzylideneacetone)dipalladium
(10.0 mg, 0.01 mmol, 0.1 eq) were added. The reaction was refluxed
overnight and then allowed to cool to room temperature; water was
added (10 mL) and the mixture was extracted with dichloromethane
(3 × 10 mL). The combined organic extracts were washed with brine,
dried (Na2SO4), and filtered, and the solvent was removed under a
reduced pressure. The residue was purified by SiO2 chromatography
using EtOAc/hexane (50%) as an eluent to give the title compound as
a yellow solid (54 mg, 89%). HPLC 97.1% (tR = 15.13 min, CH3OH
in 0.1% TFA water 5%∼95% in 20 min); 1H NMR (500 MHz,
DMSO-d6) δ 9.43 (s, 1H), 8.75 (d, J = 0.7 Hz, 1H), 8.33 (s, 1H),
7.93−7.90 (m, 2H), 7.56 (d, J = 4.2 Hz, 1H), 7.15 (d, J = 8.6 Hz,
2H), 7.04 (d, J = 4.2 Hz, 1H), 4.56 (td, J = 9.6, 4.3 Hz, 1H), 3.27−
3.20 (m, 2H), 2.46−2.43 (m, 1H), 1.87−1.79 (m, 1H), 1.76 (d, J =
13.0 Hz, 2H), 1.66 (s, 9H), 1.57−1.45 (m, 6H), 1.43 (s, 9H), 1.37−
1.16 (m, 8H); HPLC−MS (ESI+): m/z 681.5 (M + H)+.
(R)-3-Cyclopentyl-3-(4-(2-((4-(Piperidin-4-yl)Phenyl)Amino)-7H-
Pyrrolo[2,3-d]Pyrimidin-4-yl)-1H-Pyrazol-1-yl)Propanenitrile [(R)-
1)]. The bis-boc-protected derivative 16 (54.0 mg, 0.08 mmol, 1.0
eq) was suspended in dichloromethane (2 mL) and TFA (0.12 mL,
1.59 mmol, 20.0 eq) was added. The mixture was stirred at room
temperature for 2 h and concentrated under a reduced pressure. The
residue was dissolved in chloroform and washed with 10% potassium
carbonate (aq.). The aqueous layer was extracted with chloroform (3
× 10 mL) and the combined organic phase was dried (Na2SO4) and
filtered. The filtrate was concentrated under a reduced pressure, and
the product was dried under vacuum to afford the title compound as a
white solid (47 mg, 95%). HPLC 98.7% (tR = 11.00 min, CH3OH in
0.1% TFA water 5−95% in 20 min); 1H NMR (500 MHz, DMSO-d6)
tert-Butyl-2-((4-(1-(tert-Butoxycarbonyl)Piperidin-4-yl)Phenyl)-
Amino)-4-(1-(2-Cyanoethyl)-1H-Pyrazol-4-yl)-7H-Pyrrolo[2,3-d]-
Pyrimidine-7-Carboxylate (30). A mixture of tert-butyl-2-chloro-4-(1-
(2-cyanoethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-car-
boxylate (29) (0.37 g, 1.0 mmol, 1.2 eq), tert-butyl-4-(4-
aminophenyl)piperidine-1-carboxylate (0.33 g, 1.2 mmol, 1.2 eq),
t
and K2CO3 (0.21 g, 1.5 mmol) in anhydrous BuOH (10 mL) was
degassed under argon. To this mixture were added XPhos (48 mg, 0.1
mmol) and Pd2(dba)3 (46 mg, 0.05 mmol) and the reaction mixture
was heated for 6 h and then allowed to cool. Water (40 mL) was
added and the mixture was extracted with CH2Cl2. The combined
organic extracts were washed with brine, dried, and filtered, and the
solvent was removed under a reduced pressure. The residue was
purified by flash chromatography (50% EtOAc/hexane) to yield the
1
title compound 30 as a brown solid (0.40 g, 79%). H NMR (500
MHz, DMSO-d6) δ 9.43 (s, 1H), 8.72 (d, J = 1.6 Hz, 1H), 8.31 (d, J =
2237
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Journal of Medicinal Chemistry
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Article
1.5 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 4.2 Hz, 1H), 7.14
(d, J = 8.7 Hz, 2H), 7.01 (d, J = 4.2 Hz, 1H), 4.53 (m, 2H), 4.02 (m,
2H), 3.16 (m, 2H), 2.80 (m, 2H), 2.6 (m, 1H), 1.75 (m, 2H), 1.65 (s,
9H), 1.47 (m, 2H), 1.42 (s, 9H). HPLC−MS (ESI+): m/z 613.1
[100%, (M + H)+].
(m, 2H), 4.29 (t, J = 7.5 Hz, 2H), 2.61 (m, 2H). 2.37 (s, 3H).
HPLC−MS (ESI+): m/z 428.1 (M+1)+, 877.1 (2M + Na)+.
tert-Butyl 4-(4-((4-(1-(But-3-en-1-yl)-1H-Pyrazol-4-yl)-7-Tosyl-
7H-Pyrrolo[2,3-d]Pyrimidin-2-yl)Amino)Phenyl)Piperidine-1-Car-
boxylate (34). A mixture of 4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-2-
chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine 33 (300 mg, 0.7 mmol,
1.0 eq), tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (200
mg, 0.74 mmol, 1.06 eq), and K2CO3 (200 mg, 1.4 mmol, 2.0 eq) in
3-(4-(2-((4-(Piperidin-4-yl)Phenyl)Amino)-7H-Pyrrolo[2,3-d]-
Pyrimidin-4-yl)-1H-Pyrazol-1-yl)Propanenitrile (2). To a solution of
the bis-boc-protected pyrrolopyrimidine 30 (0.306 g, 0.5 mmol) in
DCM (3 mL) was added TFA (3 mL) under an argon atmosphere,
and the reaction mixture was stirred at room temperature for 4 h.
After this time, there was no starting material present (as measured by
HPLC), and the reaction was quenched with sat. NaHCO3 solution
and extracted with DCM (2 × 10 mL). The combined organic layers
were dried over Na2SO4 and evaporated to dryness in vacuo to yield
the title compound 2 as a brown solid (0.121 g, 81%). 1H NMR (500
MHz, DMSO-d6) δ 11.49 (s, 1H), 9.06 (s, 1H), 8.67 (s, 1H), 8.29 (s,
1H), 7.80 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 3.6 Hz, 1H), 7.13 (d, J =
8.3 Hz, 2H), 6.77 (d, J = 3.6 Hz, 1H), 4.53 (t, J = 6.4 Hz, 2H), 3.13
(m, 4H), 2.69 (m, 2H), 2.60 (m, 1H), 1.77 (dd, J = 13.6, 3.4 Hz, 2H),
1.58 (m, 2H). HPLC−MS (ESI+): m/z 207.2 [100% (1/2 M+H)2+],
413.3 [40% (M + H)+]. m/z calculated for C23H25N8 (M+H)+
413.2197, found 413.2191.
1-(But-3-en-1-yl)-4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-2-
yl)-1H-Pyrazole (31). A 100 mL round-bottom flask was charged with
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.5 g,
2.58 mmol, 1.0 eq), 4-bromobutene (0.48 g, 3.6 mmol, 1.4 eq),
cesium carbonate (1.67 g, 5.16 mmol, 2 eq), and acetonitrile (10 mL).
The reaction mixture was stirred at 90 °C overnight. After cooling, the
mixture was quenched with water (30 mL) and extracted with EtOAc
(3 × 30 mL). The organic layers were combined, washed with brine
(50 mL), dried (Na2SO4), and filtered. The solvent was evaporated
under a reduced pressure to yield the title product as a yellow oil. 1H
NMR (500 MHz, chloroform-d) δ 7.78 (s, 1H), 7.67 (s, 1H), 5.78
(m, 1H), 5.09 (m, 2H), 4.20 (t, J = 7.5 Hz, 2H), 2.63 (m, 2H), 1.31
(s, 12H).
t
anhydrous BuOH (15 mL) was degassed by bubbling argon for 5
min. XPhos (33 mg, 0.07 mmol, 0.1 eq) and Pd2(dba)3 (32 mg, 0.035
mmol, 0.05 eq) were added and the mixture was refluxed overnight.
The mixture was allowed to cool to room temperature and quenched
with water (40 mL). The mixture was extracted with CH2Cl2 (3 × 30
mL). The combined organic extracts were washed with brine, dried
(Na2SO4), and filtered, and the solvent was removed under a reduced
pressure. The residue was purified by SiO2 chromatography (0 to 60%
EtOAc in hexane) to afford the title compound as a yellow solid (330
1
mg, 85%). H NMR (500 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.60 (s,
1H), 8.19 (s, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H),
7.70 (d, J = 4.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz,
2H), 5.79 (m, 1H), 5.07 (m, 2H), 4.30 (t, J = 7.5 Hz, 2H), 4.09 (br s,
2H), 2.82 (br s, 2H), 2.69−2.58 (m, 4H), 2.32 (s, 3H), 1.80 (m, 2H),
1.53 (m, 2H), 1.48 (s, 9H). HPLC−MS (ESI+): m/z 668.3 (M + 1)+.
tert-Butyl 4-(4-((4-(1-(but-3-en-1-yl)-1H-Pyrazol-4-yl)-7H-
Pyrrolo[2,3-d]Pyrimidin-2-yl)Amino)Phenyl)Piperidine-1-Carboxy-
late (35). To a solution of tert-butyl 4-(4-((4-(1-(but-3-en-1-yl)-1H-
pyrazol-4-yl)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-
phenyl)piperidine-1-carboxylate 34 (165 mg, 0.29 mmol, 1.0 eq) in
MeOH (5 mL) and H2O (3 mL) was added K2CO3 (165 mg, 1.20
mmol, 4.0 eq) and the mixture was refluxed for 3 h. The mixture was
diluted with H2O (30 mL) and extracted with EtOAc (3 × 30 mL).
The combined organic extracts were washed with brine, dried
(Na2SO4), and filtered, and the solvent was removed under a reduced
pressure to give a yellow residue. The residue was purified by SiO2
flash chromatography (0 to 70% EtOAc in hexane) to give the title
4-(1-(But-3-en-1-yl)-1H-Pyrazol-4-yl)-2-Chloro-7H-Pyrrolo[2,3-
d]Pyrimidine (32). A 50 mL round-bottom flask was charged with
2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (0.3 g, 1.6 mmol, 1.0 eq),
1-(but-3-en-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole 31 (0.51 g, 2.08 mmol, 1.5 eq), sodium carbonate (0.33 g,
3.2 mmol, 2.0 eq), water (H2O, 5 mL), and 1,4-dioxane (15 mL). The
reaction mixture was degassed by bubbling argon for 5 min before
treating with Pd(PPh3)4 (199 mg, 0.172 mmol, 0.107 eq). The
mixture was heated to 100 °C under argon overnight. The reaction
mixture was gradually cooled down to ambient temperature,
quenched with water (20 mL), and extracted with EtOAc (3 × 30
mL). The organic layers were combined, washed with brine (50 mL),
dried (Na2SO4), and filtered. The solvent was evaporated under a
reduced pressure, and the residue was triturated with Et2O and
1
compound (105 mg, 87%) as a white solid. H NMR (500 MHz,
DMSO-d6) δ 11.47 (s, 1H), 9.03 (s, 1H), 8.55 (s, 1H), 8.20 (s, 1H),
7.81 (d, J = 9.0 Hz, 2H), 7.19 (m, 1H), 7.14 (d, J = 9.0 Hz, 2H), 6.76
(m, 1H), 5.86 (m, 1H), 5.10 (m, 2H), 4.32 (t, J = 7.0 Hz, 2H), 4.10
(br s, 2H), 2.79 (br s, 2H), 2.65−2.58 (m, 3H), 1.76 (m, 2H), 1.49
(m, 2H), 1.42 (s, 9H). HPLC−MS (ESI+): m/z 514.4 (M + 1)+.
4-(1-(But-3-en-1-yl)-1H-Pyrazol-4-yl)-N-(4-(Piperidin-4-yl)-
Phenyl)-7H-Pyrrolo[2,3-d]Pyrimidin-2-Amine (3). tert-Butyl-4-(4-
((4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-
2-yl)amino)phenyl)piperidine-1-carboxylate 35 (0.22 g, 0.43 mmol,
1.0 eq) was suspended in CH2Cl2 (2 mL) and TFA (0.65 mL, 8.5
mmol, 20 eq) was added. The mixture was stirred at room
temperature for 2 h and concentrated under a reduced pressure.
The residue was dissolved in CHCl3 (20 mL) and washed with 10%
K2CO3 (aq). The aqueous layer was extracted with CHCl3 (3 × 20
mL). The organic layers were combined, dried (Na2SO4), and filtered.
The filtrate was concentrated under a reduced pressure, and the
residue was dried under vacuum to afford the title compound as an
off-white solid (0.13 g, 75%). HPLC 99.8% (tR = 8.90 min, CH3OH
45% in H2O (0.1% TFA), 1 mL/min, 20 min); 1H NMR (500 MHz,
DMSO-d6) δ 11.47 (s, 1H), 9.00 (s, 1H), 8.55 (s, 1H), 8.20 (s, 1H),
7.79 (d, J = 7.5 Hz, 2H), 7.19 (d, J = 4 Hz, 1H), 7.12 (d, J = 7.5 Hz,
2H), 6.76 (d, J = 4 Hz, 1H), 5.86 (m, 1H), 5.10 (m, 2H), 4.32 (t, J =
7.0 Hz, 2H), 3.02 (br d, J = 12.0 Hz, 2H), 2.65 (q, J = 7 Hz, 2H), 2.59
(t, J = 12.5 Hz, 2H), 1.68 (d, J = 12.0 Hz, 2H), 1.53 (m, 2H); 13C
NMR (125 MHz, DMSO-d6) δ 156.4, 154.2, 151.3, 140.0, 139.3,
139.1, 135.3, 131.3, 126.9, 123.6, 121.2, 118.7, 117.7, 107.6, 100.5,
51.3, 47.2, 42.4, 35.0, 34.3; HPLC−MS (ESI+): m/z 414.3 (M + 1)+;
HRMS (ESI+) m/z calculated for C24H28N7 (M + H)+ 414.2401,
found 414.2402.
1
hexane to give 32 as a yellow solid (0.32 g, 82%). H NMR (500
MHz, DMSO-d6) δ 12.28 (s, 1H), 8.69 (s, 1H), 8.28 (s, 1H), 7.60
(dd, J = 3.0, 1.5 Hz, 1H), 7.02 (d, J = 3.0 Hz, 1H), 5.84 (m, 1H), 5.08
(m, 2H), 4.31 (t, J = 7.5 Hz, 2H), 2.64 (m, 2H). HPLC−MS (ESI+):
m/z 274.2 (M+1)+, 569.2 (2 M + Na)+.
4-(1-(But-3-en-1-yl)-1H-Pyrazol-4-yl)-2-Chloro-7-Tosyl-7H-
Pyrrolo[2,3-d]Pyrimidine (33). To a solution of 4-(1-(but-3-en-1-yl)-
1H-pyrazol-4-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine 32 (1.0 g,
5.32 mmol, 1.0 eq), p-toluenesulfonyl chloride (1.1 g, 5.85 mmol,
1.1 eq), and tetra-butylammonium hydrogen sulfate (0.090 g, 0.27
mmol, 0.05 eq) in dichloromethane (20 mL) was added NaOH (50%
aq, 0.2 mL). The reaction mixture was stirred at room temperature for
30 min. After completion of the reaction (as indicated by TLC), the
reaction mixture was diluted with H2O (20 mL). The organic layer
was separated, washed with brine (50 mL), dried (Na2SO4), and
filtered. The organic layer was evaporated under a reduced pressure to
obtain a light yellow solid, which was purified by SiO2
chromatography using hexane/EtOAc (5:1) as an eluent to give the
title product (1.3 g, 85%). 1H NMR (500 MHz, DMSO-d6) δ 8.75 (s,
1H), 8.29 (s, 1H), 8.04 (d, J = 4.5 Hz, 1H), 8.03 (d, J = 8.5 Hz, 2H),
7.50 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 4.5 Hz, 1H), 5.81 (m, 1H), 5.05
Dianilinopyrimidine Inhibitors of JAK2. N-(2-Fluoro-5-((2-
((3-fluoro-4-(1-methylpiperidin-4-yl)phenyl)amino)-5-methylpyrimi-
din-4-yl)amino)phenyl)-2-methylpropane-2-sulfonamide (4)
This was prepared according the method reported.34
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Article
N-(5-((2-((3,5-Difluoro-4-(1-methylpiperidin-4-yl)phenyl)amino)-
5-methylpyrimidin-4-yl)amino)-2-fluorophenyl)-2-methylpropane-2-
sulfonamide (5)
experiment. Expi293F cells were transfected with His-JAK2 KD. The
cells were centrifuged at 300 × g for 3 min at room temperature. Cells
were resuspended at a density of 4 × 107 cells/mL. Serial dilutions
were performed yielding threefold dilutions ranging from 10 nM to 20
μM inhibitor with constant amounts of DMSO. Cells (approximately
1.2 × 106 cells) were added to the compounds and were incubated for
30 min at 37 °C with shaking every 10 min. The tubes were heated at
47 °C for 3 min. The cells were then vitrified and thawed twice at 25
°C before being vortexed at 20,000 × g for 20 min at 4 °C. The
soluble supernatant was transferred to a new tube, mixed with a 5 ×
SDS-loading dye, and resolved on SDS-PAGE. The western blot
transfer and incubation are the same as described above. Data were
normalized to 0% and 100%.
Cell Viability Assays. UKE-1 cells were cultured in RPMI-1640
with 10% FBS at 37 °C with 5% CO2. Suspension cells were seeded at
20,000 cells per well. Cells were incubated with increasing
concentrations of compound ranging from 1 nM to 10 μM (as
denoted in the figure legends) in the presence of a vehicle (0.2%
DMSO) with six replicates per concentration for 72 h at 37 °C. After
drug treatment, 15 μL of CellTiter Blue (Promega) was added to each
well, mixed for 1 min using an orbital shaker, and incubated at 37 °C
for 3 h. Fluorescence was measured using a Wallac EnVision 2130
plate reader (PerkinElmer). Excitation and emission filters of 570 and
615 nm were used, respectively. The cell viability data were analyzed
using GraphPad Prism6.
Protein Crystallography. Purified His-JAK2 KD at 8.1 mg/mL was
incubated with 1 mM ruxolitinib and then added to 0.2 M NaCl, 0.1
M Bis−Tris pH 5.5, and 25% PEG 3350 in a 1:1 v/v ratio on a
hanging drop coverslip with the crystallization solution at 293 K.
Rodlike crystals were formed within 10 h and grew to a maximum size
over 5 days. Ligand exchange was performed by moving JAK2−
ruxolitinib crystals into the crystallization solution containing 1 mM
inhibitor of interest for 3 days. The crystals were preserved in a
cryoprotectant containing the crystallization solution and 30%
glycerol. All data were collected on the 23-ID beamline, GM/CA at
the Advanced Photon Source (Argonne, IL). Data were processed and
scaled with XDS. Molecular replacement (using PDB 2XA4 as the
search model) and refinements were carried out using PHENIX, and
model building was performed using Coot. Figures were prepared
This was prepared according the method reported.34
4-((4-((4-Chloro-3-((1,1-dimethylethyl)sulfonamido)phenyl)-
amino)-5-methylpyrimidin-2-yl)amino)-2-fluoro-N-(1-methylpiperi-
din-4-yl)benzamide (6)
This was prepared according the method reported.35
N4-(1-(Tert-Butylsulfonyl)indolin-6-yl)-N2-(3-fluoro-4-(1-methyl-
piperidin-4-yl)phenyl)-5-methylpyrimidine-2,4-diamine (7)
This was prepared according the method reported.34
Cloning and Expression of JAK2 KD. The DNA of human JAK2
KD (JH1), encoding amino acids 840−1132, was synthesized and
cloned into pcDNA 3.3 (GeneArt). The construct was expressed
using a CMV promoter and contained a His8 N-terminal affinity tag
followed by a TEV cleavage site. Expi293F cells (Invitrogen) were
grown, maintained, and treated in a shaking culture at 37 °C with 8%
CO2 in Expi293 medium (Invitrogen). Recombinant JAK2 was
expressed in Expi293F cells using Transporter 5 transfection reagent
as described by the manufacturer (Polysciences). One hour after
adding the plasmid/transfection complex, varying concentrations of
the JAK2 inhibitor or the transcription enhancer were added as
indicated in Figure 1. The transfection proceeded for 24 h before
harvest. Cells used for the preparation of crystallization-grade JAK2
were incubated with 1 μM ruxolitinib and 4 mM butyric acid for 23 h.
Purification of JAK2 KD. Purification of JAK2 KD was performed
at 4 °C. Expi293F cells were centrifuged at 1000 × g for 30 min at 4
°C. Cells were resuspended in lysis buffer (50 mM HEPES pH 7.5,
250 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 5 mM
MgCl2, 0.1 mM ATP, 10 mM imidazole, 1 mM PMSF, and 0.5%
Triton X-100). The cells were sonicated at 40% power for 2 min on
ice and then centrifuged at 40,000 × g for 1 h at 4 °C to clarify the
lysate. The soluble lysate was loaded onto two 5 mL HisTrap FF
columns in tandem (GE Healthcare). The columns were washed with
10 mM imidazole, 40 mM imidazole, 100 mM imidazole, and 200
mM imidazole before applying a gradient up to 600 mM imidazole.
The protein was concentrated to 10 mL using a 10,000 MWCO filter
and loaded onto an S75 gel filtration column (GE Healthcare) that
was pre-equilibrated with 40 mM bicine pH 8.6, 100 mM NaCl, and
10% glycerol. The single JAK2 peak, as determined by sodium
dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and
western blot analysis (>99% purity), was collected, concentrated to
8.1 mg/mL using a 3000 MWCO spin concentrator, flash-frozen in
liquid N2, and stored at −80 °C.
Cellular Thermal Shift Assay. CETSA was used to compare the
melting curves from ligand-based thermal stabilization of JAK2 KD.
Expi293F cells were transfected with His-JAK2 KD from pcDNA 3.3
as described above. Ruxolitinib (20 μM), or the equivalent amount of
DMSO, was added to the cells and incubated for 1 h at 37 °C. After 1
h, the cells were centrifuged at 300 × g for 3 min at room
temperature. The cells were washed with 15 mL of PBS and
centrifuged again at 300 × g for 3 min. The cells were resuspended in
1 mL of PBS and approximately 3 × 106 cells were added to PCR
tubes that were incubated at 40, 43, 46, 49, 52, 55, 58, 61, 64, or 67
°C for 3 min. The tubes were then flash-frozen in liquid N2 and
thawed twice at 25 °C. The cells were vortexed at 20,000 × g for 20
min at 4 °C. The soluble lysate was transferred to a new tube, a 5 ×
SDS-PAGE loading buffer was added, and 13 μL (equivalent of 3.3 ×
105 cells) was added to an SDS-PAGE gel. The gel was transferred
using the eBlot transfer system (GenScript), blocked with 5% BSA in
TBS-T, and blotted for anti-His-HRP, anti-actin, or anti-GAPDH-
HRP for 2 h at room temperature. The anti-actin blot was washed and
then incubated with an anti-mouse-HRP-conjugated secondary
antibody at 1:2000 for 1 h at room temperature. The blots were
incubated with SignalFire ECL (Cell Signaling, 6883) and imaged
using a GE Healthcare AmerSham Imager 600.
̈
using PyMOL (Schrodinger, LLC). All structures were validated and
deposited in the PDB with accession codes listed in Supplementary
Tables S1-S2.
PDB ID CODES
■
6VGL (JAK2/ruxolitinib), 6VSN (JAK2/S-ruxolitinib), 6VNK
(JAK2/rac-ruxolitinib), 6VN8 (JAK2/baricitinib), 6VNC
(JAK2/R-1), 6VNB (JAK2/S-1), 6VS3 (JAK2/rac-1), 6VNJ
(JAK2/2), 6VNM (JAK2/3), 6VNE (JAK2/fedratinib),
6VNG (JAK2/4), 6VNH (JAK2/5), 6VNL (JAK2/6), and
6VNF (JAK2/7). Authors will release the atomic coordinates
and structure factors upon article publication.
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01952.
■
sı
Molecular formula strings (CSV)
Supplementary figures, schematics and tables (PDF)
AUTHOR INFORMATION
Corresponding Author
■
̈
Ernst Schonbrunn − Drug Discovery Department, Moffitt
Isothermal Dose Response. ITDR measures protein stabilization
as a function of increasing inhibitor concentration. After performing
the CETSA assay described above, the data were graphed and a
temperature at the IC50 value (47 °C) was used for the ITDR
Cancer Center, Tampa, Florida 33612, United States;
orcid.org/0000-0002-3589-3510;
Email: ernst.schonbrunn@moffitt.org
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Article
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Baoli Li − Drug Discovery Department, Moffitt Cancer Center,
Tampa, Florida 33612, United States
Sang Y. Yun − Chemical Biology Core, Moffitt Cancer Center,
Tampa, Florida 33612, United States
Alice Chan − Drug Discovery Department, Moffitt Cancer
Center, Tampa, Florida 33612, United States
Pradeep Nareddy − Drug Discovery Department, Moffitt
Cancer Center, Tampa, Florida 33612, United States
Steven Gunawan − Drug Discovery Department, Moffitt
Cancer Center, Tampa, Florida 33612, United States
Muhammad Ayaz − Chemical Biology Core, Moffitt Cancer
Center, Tampa, Florida 33612, United States
Harshani R. Lawrence − Chemical Biology Core, Moffitt
Cancer Center, Tampa, Florida 33612, United States;
orcid.org/0000-0002-9263-7775
Gary W. Reuther − Molecular Oncology Department, Moffitt
Cancer Center, Tampa, Florida 33612, United States
Nicholas J. Lawrence − Drug Discovery Department, Moffitt
Cancer Center, Tampa, Florida 33612, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01952
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the GM/CA station at the Argonne National
Laboratory for assistance with synchrotron data collection
(National Cancer Institute grant Y1-CO-1020, National
Institute of General Medical Sciences grant Y1-GM-1104,
and U.S. Department of Energy contract W-31-109-ENG-38).
We thank the Moffitt Chemical Biology Core for use of the
crystallization and crystallography facility, and NMR and mass
spectrometry instruments (National Cancer Institute grant
P30-CA076292). H.R.L. acknowledges support by the Na-
tional Cancer Institute grant R50CA211447. This work was
supported by the Leukemia & Lymphoma Society (Screen-to-
lead grant 8012-18) and the Florida Department of Health
(Bankhead−Coley grant 20B10).
ABBREVIATIONS
■
JAK2, Janus kinase 2; MPN, myeloproliferative neoplasms;
KD, kinase domain; SAR, structure−activity relationship;
STATs, signal transducer and activator of transcription
proteins
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