Discovery of 1-methyl-1 H-imidazole derivatives as potent Jak2 inhibitors

Article abstract of DOI:10.1021/jm401546n

Structure based design, synthesis, and biological evaluation of a novel series of 1-methyl-1H-imidazole, as potent Jak2 inhibitors to modulate the Jak/STAT pathway, are described. Using the C-ring fragment from our first clinical candidate AZD1480 (24), optimization of the series led to the discovery of compound 19a, a potent, orally bioavailable Jak2 inhibitor. Compound 19a displayed a high level of cellular activity in hematopoietic cell lines harboring the V617F mutation and in murine BaF3 TEL-Jak2 cells. Compound 19a demonstrated significant tumor growth inhibition in a UKE-1 xenograft model within a well-tolerated dose range.

Full text of DOI:10.1021/jm401546n

Article
pubs.acs.org/jmc
Discovery of 1‑Methyl‑1H‑imidazole Derivatives as Potent Jak2
Inhibitors
Qibin Su,*,† Stephanos Ioannidis,† Claudio Chuaqui,† Lynsie Almeida,† Marat Alimzhanov,†
Geraldine Bebernitz,† Kirsten Bell,† Michael Block,† Tina Howard,‡ Shan Huang,† Dennis Huszar,†
Jon A. Read,‡ Caroline Rivard Costa,† Jie Shi,† Mei Su,† Minwei Ye,† and Michael Zinda†
†AstraZeneca, Oncology Innovative Medicines, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
‡Discovery Sciences Innovative Medicines, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom
ABSTRACT: Structure based design, synthesis, and biological
evaluation of a novel series of 1-methyl-1H-imidazole, as potent
Jak2 inhibitors to modulate the Jak/STAT pathway, are
described. Using the C-ring fragment from our first clinical
candidate AZD1480 (24), optimization of the series led to the
discovery of compound 19a, a potent, orally bioavailable Jak2
inhibitor. Compound 19a displayed a high level of cellular
activity in hematopoietic cell lines harboring the V617F
mutation and in murine BaF3 TEL-Jak2 cells. Compound 19a
demonstrated significant tumor growth inhibition in a UKE-1
xenograft model within a well-tolerated dose range.
2,4-diamine, which was progressed into clinical trials.17 We
have also explored the role of Jak kinases in solid tumors where
INTRODUCTION
■
Jak2, together with Jak1, Jak3, and Tyk2, belongs to the Jak
activation of the Jak/STAT pathway is not typically associated
(Janus-associated kinase) family of nonreceptor tyrosine
with activating mutations in Jak2.18 Herein we describe our
kinases.1 They play important roles in cytokine and growth
factor mediated signal transduction. Activated by cytokine or
growth factor engagement, autophosphorylated Jaks stimulate
the recruitment and phosphorylation of the signal transducers
structure based design efforts toward the discovery of novel
potent ATP competitive Jak2 small molecule inhibitors and
detail the structure−activity relationship within this series.
and activators of transcription (STATs).2 This in turn leads to
Detailed results of preclinical biological and pharmacokinetic
studies of the lead compounds are also discussed below.
the dimerization and translocation of phosphorylated STATs
into the nucleus, hence increasing cellular proliferation and
resistance to apoptosis.3 Discovery of a single activating somatic
CHEMISTRY
■
mutation (V617F) in the pseudokinase JH2 (Janus Homology
The nitro-imidazole 8 was regioselectively methylated using
2) domain of Jak2 stimulated significant interest in developing
methyl iodide at elevated temperature to yield 9. Subsequent
selective Jak2 inhibitors for the treatment of myeloproliferative
palladium mediated hydrogenation of 9 afforded amino
neoplasms (MPNs).4 Indeed, there is growing evidence to
imidazole 10 in excellent yields (Scheme 1).19
suggest that deregulation of constitutively activated Jak/STAT
Coupling reaction of 1-methyl-1H-imidazol-4-amine 10 with
signaling is an important event in all patients with MPNs,
the commercially or readily available dichloride intermediates
comprising essential thrombocythemia (ET), polycythemia vera
11a−g proceeded smoothly, and regioselectively, at 70 °C in
the presence of DIPEA to afford intermediates 12a−g.
However, the subsequent displacement of the second chloride
by 14 required microwave irradiation at 150 °C in a basic
alcoholic solution to yield intermediates 13a−g.20 Unfortu-
nately, these forcing reaction conditions led to a decrease in the
enantiomeric excess of the coupling products, and therefore the
final compounds required chiral purification to afford the
desired 13a−g (Scheme 2).
(PV), and myelofibrosis (MF).5 MF is associated with the
lowest survival rate among MPNs and represents a significant
unmet medical need.6 Thus, selective inhibitors capable of
targeting the Jak/STAT pathway are highly desirable.7 Several
Jak inhibitors have been evaluated in human clinical trials,
notably (Figure 1) Ruxolitinib 1 (formerly INCB018424),
which has received approval for clinical use in MF in the United
States and Europe,8 SAR302503 2 (formerly TG101348),9
Lestaurtinib 3 (CEP701),10 CYT-387 4,11 Pacritinib 5,12
LY2784544 6,13 BMS-911543 7,14 and others.15
The synthesis of pyrrolo-pyrimidine analogues started with
the capping of the NH of commercially available 15a. Removal
Our lab has pursued the discovery and development of Jak2
selective inhibitors as anticancer drugs,16 culminating in the
discovery of drug candidate (S)-5-chloro-N2-(1-(5-fluoropyr-
imidin-2-yl)ethyl)-N4-(5-methyl-1H-pyrazol-3-yl)pyrimidine-
Received: October 3, 2013
Published: December 10, 2013
© 2013 American Chemical Society
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Figure 1. Selected Jak inhibitors in human clinic trials.
Scheme 1. Preparation of 1-Methyl-1H-imidazol-4-amine
Hydrochloride Salt 10
after final deprotection of the sulfonyl group under aqueous
basic conditions (aq NaOH). Unfortunately, this reaction
sequence resulted in the partial racemization of the final
compounds, thus chiral purification by HPLC was required (see
the Experimental Section for the chiral separation conditions).
Pyrazolo-pyrimidine analogues 19d and 19e were synthesized
using the sequence depicted in Scheme 3, starting from
commercially available 15b and 17e, respectively.
23a and 23b were synthesized in a similar manner starting
from the corresponding commercially available dichloride
starting materials, 20a and 20b, in a moderate overall yield
(Scheme 4).
a
a
Reagents and conditions: (a) MeI, K2CO3, 65 °C, CH3CN, 75%; (b)
H2, Pd/C then HCl, 100%.
of the active NH functionality proved necessary for the
subsequent coupling reactions. Hence, 15a was treated with
base (NaOH or NaH) and then quenched via the addition of
either tosyl chloride or methyl iodide to afford 17a or 17b,
respectively (Scheme 3). Copper(II) acetate promoted
coupling reaction between cyclopropyl boronic acid, and 15a
proceeded smoothly to yield 17c. With these intermediates in
hand, sequential attachment of 1-methyl-1H-imidazol-4-amine
10 and (S)-1-(5-fluoropyrimidin-2-yl)ethanamine 1421 fur-
nished the desired final compounds 19b−c. 19a was obtained
RESULTS AND DISCUSSION
■
Our lab has a long-standing interest in the discovery of isosteric
replacements of the pyrazol-3-yl amine, which acts as a hinge
binder in a number of Jak2 inhibitors such as compound
25a.16b Previously, we have demonstrated that thiazol-2-yl
amine may operate as an effective replacement for the amino
pyrazole, leading to potent Jak2 inhibitors, e.g., 25b (Figure
2).16b
a
Scheme 2. Preparation of 13a−g
a
Reagents and conditions: (a) 10, DIPEA, CH3CN, 70 °C; (b) 14, DIPEA, n-BuOH, 150 °C microwave then chiral separation.
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a
Scheme 3. Preparation of 19a−e
a
Reagents and conditions: (a) For 17a, 15a, NaOH, tetra-butylammonium hydrogen sulfate, TsCl; for 17b, 15a, NaH, MeI; for 17c, cyclopropyl
boronic acid, Cu(OAc)2; for 17d, 15b, TsOH, DHP; (b) 10, DIPEA, CH3CN, 100 °C microwave; (c) for 19a, 14, DIPEA, n-BuOH, 150 °C
microwave, then NaOH (aq), 55 °C, then chiral separation; for 19b−e, 14, DIPEA, n-BuOH, 150 °C microwave; then chiral separation.
a
Scheme 4. Preparation of 23a and 23b
a
Reagents and conditions: (a) NaOH, tetra-butylammonium hydrogen sulfate, TsCl; (b) 10, DIPEA, CH3CN, 100 °C microwave; (c) 14, DIPEA, n-
BuOH, 150 °C microwave; then chiral separation; (d) NaOH (aq), 55 °C.
Figure 2. Bioisosteric replacement of amino-pyrazole with amino-
thiazole led to potent Jak2 inhibitor 25b.
We hypothesized that other heterocycles, with a similar array
of hydrogen bond interactions in a cis-donor/acceptor/donor
pattern, could provide analogous binding in the hinge region of
the ATP binding site. We therefore designed the 1-methyl-1H-
imidazol-4-amine analogue 26 (Figure 3), reasoning that the
required hydrogen bond interactions could be achieved with
the C1-NH, 2-N, and C3-H of the amino imidazole. Moreover,
the imidazole C5-H is available to form an intramolecular
hydrogen bond with the nitrogen of the pyrimidine (B ring),
a
Figure 3. Bioisosteric replacement of the A ring. Measured at high
thereby favoring a coplanar conformation with reduced
intramolecular ligand strain while making the requisite
interactions with the hinge. We envisioned that a small
hydrophobic substituent (N4-Me) could engage the methio-
nine (M929) gatekeeper in the Jak2 kinase and provide the
critical hydrophobic interaction. To test the hypothesis,
compound 26 was synthesized and evaluated in the Jak2
b
ATP concentration (5 mM); for experimental details, see ref 17. All
values are an average of at least three independent dose−response
curves. Biochemical and cellular activity of 24 was reported in ref 17.
c
biochemical and cellular assays. Compound 26 was shown to be
less potent against Jak2 (IC50 = 0.12 μM), compared to 24
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(IC50 = 0.058 μM), but maintained activity in our cellular assay
(TEL-Jak2 GI50 = 0.27 μM, Figure 3), albeit at a
correspondingly reduced level, suggesting that amino imidazole
could be an effective replacement for amino pyrazole.
Our hypothesis that amino imidazole could be a viable
replacement of amino pyrazole was strengthened when we
obtained cocrystal structures of 2716d and 2822 (Figure 4)
bound into the ATP binding site of Jak2. The binding mode
observed for 28 was consistent with that observed with 24
(Figure 3)17 and its close pyrazole analogues, 27.16d The
imidazole ring interacts with the hinge via an acceptor−donor
hydrogen-bonding motif to the backbone of leucine (L932) and
glutamate (E930), while the donor N−H hydrogen bond to the
hinge in 27 is replaced by a C−H in 28. The imidazole ring in
28 shows a small shift away from the hinge, which results in a
movement of ∼0.6 Å of the N-1 methyl group toward the
M929 gatekeeper residue.
The tolerance of a polar C−H interacting with the backbone
carbonyl at this position of the hinge is not uncommon.23 For
example, it is observed in the well characterized bis-amino-
pyrimidine24 and 4-anilinoquinazoline class of kinase inhib-
itors.25
With these encouraging results in hand, we decided to hold
the 1-methyl-1H-imidazol-4-amine constant as the hinge-
binding motif and explore the SAR of the other region (B
ring, Figure 3) of the molecule with the hope of further
improving Jak2 potency. We hypothesized that fused bicyclic B
rings would fill the hydrophobic pocket around the hinge area,
thus leading to a significant boost of Jak2 potency. To prioritize
the syntheses of bicyclic B-rings, docking studies of the newly
designed compounds into the structure of 24 bound to Jak2
(PDB code 2XA4) were carried out using Glide,26 followed by
post processing of docked poses using a MM-GBSA workflow27
that approximates ΔG of binding and ligand strain using a
continuum solvent approximation for desolvation.28 Top
scoring poses were then inspected visually to ensure that
binding modes were consistent with the known X-ray structures
of this class of Jak2 inhibitors.16d,17 From these docking studies
for [6−6]-bicyclic B-rings, several interaction preferences were
observed that helped prioritize the design and synthesis of
analogues. In particular, hydrogen bonding complementarity
between the B ring and the hinge backbone carbonyl of leucine
(L932) was key to achieving a high ΔG of binding, reduced
ligand strain, and a consistent binding mode with observed X-
ray structures (Figure 5a). B rings with a hydrogen bond donor
(N−H or polar C−H) at the C5 position were predicted to be
superior to sp2 N or O-containing rings. The predicted SAR
trends are shown in Figure 5b.
Figure 4. X-ray crystal structure overlay of Jak2 in complex with
pyrazole hinge binding compound 27 (yellow carbon atoms, PDB
code 3zmm)16d and Jak2 in complex with imidazole hinge binding
compound 28 (blue carbon atoms, PDB code 4c62). Protein backbone
for the complex of Jak2 with compound 28 is shown as a cartoon.
Selected residues are shown as sticks. Hydrogen bonds are shown as
dotted lines in the same color as the carbon atoms.
Figure 5. (a) Predicted binding mode of [6−6]-bicyclic B-rings (the structure of 24 bound to Jak2 was used for all docking studies (PDB code
2XA4)).17 (b) Predicted SAR from docking studies where the surface coloring scheme denotes hydrogen bonding preferences (blue, acceptor; red,
donor).
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Table 1. Biochemical and Cellular Activity of [6,6]-Fused B-Ring Analogues
a
b
Measured at high ATP concentration (5 mM); for experimental details, see ref 17. All values are an average of at least three independent dose−
c
d
response curves. Denotes not tested. Compound is a mixture of enantiomers; enantiomeric excess (ee) was not determined.
Table 2. Biochemical and Cellular Data of [5,6]-Fused B-Ring Analogues
a
b
Measured at high ATP concentration (5 mM); for experimental details, see ref 17. All values are an average of at least three independent dose−
c
response curves. Compound is a mixture of enantiomers; enantiomeric excess (ee) was not determined.
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To optimize Jak2 potency of 26 based on the predicted
binding mode, analogues with a [6,6]-fused pyrimidine B ring
were synthesized and evaluated (Table 1). We were pleased to
find that analogue 13a, which contained a quinazoline B ring,
was potent (IC50 = 0.003 μM, Table 1) in both the Jak2
biochemical assay at high ATP concentration and in the cellular
assay (TEL-Jak2 GI50 = 0.034 μM), which was comparable to
24. With this encouraging result, we elected to explore the SAR
around this specific scaffold. Compound 13c with a 7-OMe
substituent exhibited potent Jak2 enzyme activity (0.024 μM),
while substitution on the 6-position was detrimental to Jak2
Figure 6. (a) Predicted binding mode of [5,6]-bicyclic B-rings (The
structure of 24 bound to Jak2 was used for all docking studies (PDB
code 2XA4)).17 (b) X-ray crystal structure of compound 23b bound to
JAK2 (PDB code 4c61). Carbon atoms of the complex are colored in
green. 2FoFc electron density contoured at 1.0σ and shown as a wire
mesh. Hydrogen bonds are shown as yellow dotted lines.
potency as 13b was less potent in Jak2 enzyme assay (IC50
=
0.67 μM) and inactive in the cellular assay (3 μM), implying a
steric clash between the 6-OMe group in the Jak2 protein.
Compounds 13d and 13h displayed potent Jak2 activity (Jak2
IC50 = 0.033 μM and IC50 = 0.012 μM, respectively), while
incorporation of a sp2 nitrogen at the C5 position (13g) greatly
decreased Jak2 potency (IC50 = 0.80 μM), suggesting that there
may be electronic repulsion between this sp2 nitrogen and the
backbone carbonyl of leucine (L932). This is consistent with
our predicted binding mode depicted in Figure 5. Diminishing
Jak2 activity (IC50 = 0.49 μM) of 13e confirmed the limited
space available for substitution and hydrogen bonding
preferences shown in Figure 5b.
Being cognizant of the analogues with [6,6]-fused B rings,
which were potent against Jak2, we set out to explore the SAR
of [5,6]-fused B ring analogues in an attempt to diversify the
SAR while maintaining Jak2 activity. We elected to synthesize
the analogues 19a−e and 23a−b (Table 2), which contain
different fused 5-membered ring heterocycles. As shown in
Table 2, the fused B-ring analogue 19a retained the Jak2
enzymatic potency (IC50 < 0.003 μM). 19a exhibited excellent
cellular activity in the BaF3 TEL-Jak2 cell line (GI50 = 0.012
μM). Unsurprisingly, the alkylation of the 7-position of
pyrrolopyrimidine (19b,c) was tolerated and maintained Jak2
potency because this position is solvent accessible and points
toward the solvent channel. Pyrazolo-analogues 19d and 19e
were synthesized to lower the hydrophobicity and, to our
delight, 19e demonstrated a combination of favorable
the sulfur interacts favorably with the carbonyl oxygen via a
back-bonding mechanism.29
PK Profiling of Leads. Having achieved highly potent Jak2
inhibitors (such as the analogues in Table 3), the
a
Table 3. Rat PK Properties of Selected Potent Leads
compd
F%
CLobs (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
b
13c
13d
19a
19e
23b
NA
NA
110
100
36
6
0.98
0.92
1.6
b
2.5
2.2
1.8
1.2
64
90
25
1.3
b
NA
44
0.64
a
Han Wistar rat male; 10 mg/kg po (0.1% HPMC); 3 mg/kg iv (40%
b
DMA/40%PEG/20% saline). Denotes not tested.
pharmacokinetic (PK) properties of these leads were evaluated
and shown to display different metabolic stabilities in vivo
depending on the nature of the B-ring. While the compounds
with fused [6,6] B-rings (13c and 13d) were highly cleared in
vivo in the rat, analogues with [5,6]-fused B-rings, e.g., 19a and
19e, showed low in vivo clearance with a moderate half-life and
excellent oral bioavailability (Table 3). Because of their
favorable rat PK properties and excellent cellular potencies,
compounds 19a and 19e were progressed into further in vivo
pharmacokinetic studies in dog. We were pleased to find that
both compounds displayed excellent metabolic stability with
prolonged half-life, as well as excellent oral bioavailability in
dogs (Table 4).
enzymatic (IC50 = 0.015 μM) and cellular activity (GI50
=
0.026 μM). Interestingly, analogue 23a with a hydrogen bond
donor toward the hinge region exhibited excellent Jak2 enzyme
potency (IC50 < 0.003 μM), implying a productive interaction
between the NH and protein, thus further supporting our
binding mode hypothesis. To avoid the immunosuppression
associated with Jak3 deficiency, we have been focusing on the
development of Jak2 inhibitors with high level of selectivity
against Jak3.17 Gratifyingly, a significant level of selectivity
against Jak3 was observed in this series, however to a smaller
extent against Jak1 (Tables 1 and 2).
Interestingly, sulfur containing bicycle 23b was predicted by
MM-GBSA to be preferred and was subsequently shown to be a
potent Jak2 inhibitor (IC50 = 0.004 μM and GI50 = 0.037 μM).
Finally, an X-ray crystal structure of compound 23b bound into
Jak2 was solved and was shown to be in good agreement with
the predicted binding mode of this series (Figure 6). In both
the predicted binding modes (Figure 6a) and the solved X-ray
cocrystal structure (Figure 6b) of [5,6]-bicyclic B-rings, the key
hydrogen bonding interactions to the hinge are consistent with
the pyrimidine B ring analogues shown in Figure 4. The fused
B-rings make an additional interaction with the backbone
carbonyl of leucine (L932) where a hydrogen bond donor or
polar C−H is preferred. In the case of 23b, we postulate that
a
Table 4. Dog PK Properties of 19a and 19e
CLobs
Vdss
(L/kg)
t1/2
(h)
compd route F% AUC (μM·h) (mL/min/kg)
19a
iv
2.07
4.56
1.9
2.1
12.4
7.6
b
b
po
iv
64
45
49
19e
6.2
po
139
7.7
a
Beagle dog male; 10 mg/kg po (0.1% HPMC, pH 2); 2.5 mg/kg iv
b
(20% TEG/D5W). AUC0−12h
.
Kinase Selectivity of Compound 19a. Compound 19a
was evaluated against a panel of 82 kinases using Upstate
Biotechnologies Kinase Profiler Service (Millipore Corporation,
Charlottesville, VA) at three concentrations (0.01, 0.10, and 1.0
μM) at or near the Km for ATP using a radiometric filter-
binding assay. The set of kinases tested were selected based on
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kinase binding site similarity and similarity in the gatekeeper
residue, thus representing the diversity of the kinome. The
selectivity profile of 19a (Jak2 IC50 < 3 nM) in this
representative kinase panel is shown in Table 5.30 More than
inhibition of phosphorylation of STAT5 in both cell lines upon
treatment with 19a (Figures 8 and 9).
19a Demonstrated a Dose Dependent Pharmacody-
namic (PD) Effect in a Mouse TEL-Jak2 Model. To evaluate
further the inhibitory effect of 19a in vivo, we measured
pSTAT5 levels in the spleen of mice implanted with BaF3 TEL-
JAK2 cells as an in vivo biomarker of Jak2 kinase inhibition as
described previously.17 A single dose of 19a was given to
tumor-bearing animals orally at 3, 5, and 10 mg/kg, and mice
were sacrificed at different time points postdose. A single 10
mg/kg dose of 19a achieved almost complete inhibition of
pSTAT5 levels at 6 h and about 50% inhibition at 12 h post
dose. With a 3 mg/kg dose of 19a about 80% pSTAT5
inhibition was seen at 6 h post dose, while only 30% inhibition
was detected at 8 h (Figure 10). This demonstrated a clear
dose/time-dependent modulation of pSTAT5 levels in spleen
lysates of tumor-bearing mice was achieved by compound 19a.
19a Showed Tumor Growth Inhibition in a UKE-1
Xenograft Model. Having established PD activity of 19a in
vivo, we proceeded with efficacy testing in the UKE-1 xenograft
Table 5. Kinase Profiling of Lead Compound 19a
compd 19a
a
b
target
IC50 (μM) (enzyme fold selectivity)
JAK2(h)
CK2α2(h)
KDR(h)
Abl(h)
<0.002
0.0027 (1)
0.022 (11)
0.025 (12)
0.033 (16)
0.035 (18)
0.035 (18)
0.040 (20)
0.045 (23)
0.046 (23)
0.050 (25)
0.050 (25)
0.058 (29)
0.063 (32)
0.19 (99)
Fgr(h)
JAK3(h)
TrkA(h)
Flt4(h)
Flt3(h)
Ret(h)
ALK(h)
FGFR1(h)
Aurora-A(h)
LIMK1(h)
SIK(h)
̈
mouse model. In a multidose tolerability study in naıve NCr
nude female mice,34 19a was tolerated at doses up to 10 mg/kg
given orally twice daily (20 mg/kg/day) for 10 days.35 UKE-1
xenograft tumors were established in SCID female mice36 by
subcutaneous implantation of UKE-1 cells mixed with matrigel.
On day 15, animals were randomized into groups at an average
tumor volume of ∼150 mm3. Mice were treated twice daily by
oral gavage with either 3 or 10 mg/kg of 19a and vehicle for 14
days. We observed a significant dose-dependent tumor growth
inhibition with 19a-treated animals (Figure 11). At the end of
the dosing phase, a maximum tumor growth inhibition of 77%
was achieved with a 10 mg/kg dose level twice daily. No
significant body weight loss was observed in any of the study
groups (Figure 11).
PKCζ(h)
0.20 (100)
a
b
Measured at Km ATP concentration. Jak2 inhibitory activity (IC50)
was measured to be <0.002 μM. The numbers in the table represent
the minimum possible fold selectivity.
10-fold selectivity was observed across the kinase panel, with
the exception of CK2α2(h), which was potently inhibited by
19a. We investigated the CK2 activity further and found 19a
was inactive (IC50 > 30 μM) in our in-house assay.31
Cellular Potency and Selectivity of the Lead Com-
pound 19a. Testing in BaF3 TEL-Jak2 cell line in an MTS
tetrazolium colorimetric assay18a 19a displayed a potent growth
inhibition of the cell line with a GI50 of 0.026 μM. This
compound exhibited cellular selectivity against Jak1, Jak3, and
Tyk2, as demonstrated by the reduced growth inhibitory effect
(Table 6) in BaF3 cells transfected with the kinase domain of
CONCLUSION
■
In conclusion, we have described a novel series of 1-methyl-1H-
imidazole derivatives as selective and potent Jak2 inhibitors,
which modulate signaling through the Jak/STAT pathway.
Compound 19a demonstrated excellent potency in a Jak2
biochemical assay, which translated into cellular activity in the
nanomolar range in hematopoietic cell lines harboring the
V617F mutation as well as in BaF3 TEL-Jak2 cells. 19a
displayed dose-dependent pharmacodynamic effects in a mouse
TEL-Jak2 model and demonstrated significant tumor growth
inhibition in a UKE-1 efficacy study within a well-tolerated dose
range. These findings warrant further investigation of 19a in
more advanced preclinical studies to fully understand its
potential as an effective anticancer therapeutic.
Table 6. Inhibition of Cellular Proliferation in Engineered
BaF3 Cell Lines and Cell Lines Bearing the V617F Mutation
by Compound 19a
cell line
Gl50 (μM)
BaF3 TEL-Jak2
BaF3 TEL-Jak3
BaF3 TEL-Jak1
BaF3 TEL-Tyk2
SET-2 (V617F)
UKE-1 (V617F)
0.026
1.8
0.22
0.76
0.014
0.055
EXPERIMENTAL SECTION
Chemistry. H NMR spectra were recorded on either Bruker 300
■
1
or 400 MHz NMR spectrometers using deuterated DMSO (DMSO-
d6) unless otherwise stated. Temperatures are given in degrees Celsius
(°C); operations are carried out at room temperature or ambient
temperature: 18−25 °C. Chemical shifts are expressed in parts per
million (ppm, δ units). Splitting patterns describe apparent multi-
plicities and are designated as s (singlet), d (doublet), dd (doublet−
doublet), t (triplet), q (quartet), m (multiplet), and br s (broad
singlet). Mass spectroscopy analyses were performed with an Agilent
1100 equipped with Waters columns (Atlantis T3, 2.1 mm × 50 mm, 3
μmor Atlantis dC18, 2.1 mm × 50 mm, 5 μm) eluted with a gradient
mixture of H2O−acetonitrile with formic acid or ammonium acetate.
The purity determination of all reported compounds was performed
other Jak family members (Jak1, Jak3 and Tyk2). These
activities were shown to tightly correlate with the inhibition of
pSTAT5 (IC50) in these cell lines (Figure 7).32
19a was tested in clinically relevant SET-2 (a human
megakaryoblastic cell line derived from an ET patient
heterozygous for V617) and UKE-1 (homozygous for
V617F) cell lines.33 Compound 19a exhibited potent growth
inhibition in both cell lines (Table 6). Western blot analysis
demonstrated that the growth inhibition correlated with the
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Figure 7. Dose-dependent inhibition of pSTAT5 (phosphorylated Stat5) by compound 19a in BaF3 TEL-Jak1, 2, 3, and Tyk2 cells.
Figure 8. Dose-dependent inhibition of pSTAT5 (phosphorylated Stat5) by 19a in UKE-1 cells.32
Figure 9. Dose-dependent inhibition of pSTAT5 (phosphorylated Stat5) by 19a in SET-2 cells.32
reverse-phase HPLC column with dimension 20 mm/100 and 50 mm/
250 in H2O/MeCN with 0.1% TFA as mobile phase. Reactions were
monitored by thin-layer chromatography on 0.25 mm E. Merck silica
gel plates (60F-254), visualized with UV light or LC/MS. Flash
column chromatography was performed on a ISCOMPLC Combi-
flash system (ISCO) (4700 Superior Street, Lincoln, NE, USA), unless
otherwise stated, using silica gel cartridges. SFC (super critical fluid
chromatography) refers to analytical SFC (ASC-1000 analytical SFC
System with diode array detector) and/or preparative SFC (APS-1000
AutoPrep Preparative SFC), obtained from SFC Mettler Toledo
AutoChem, Inc. (7075 Samuel Morse Drive Columbia, MD 21046,
USA) and used according to the manufacturer’s instruction.
1-Methyl-4-nitro-1H-imidazole (9). 4-Nitro-1H-imidazole (2 g,
17.69 mmol) was dissolved in acetonitrile (20 mL). Potassium
carbonate (3.67 g, 26.53 mmol) and iodomethane (1.327 mL, 21.22
mmol) were added. The reaction mixture was then heated at 65 °C
overnight. The reaction mixture was filtered, and the filtrate was
concentrated in vacuo, leaving a reddish-orange solid (3.2 g). This
material was purified by ISCO (0−10% MeOH/DCM). Concen-
tration of the fractions in vacuo provided 1-methyl-4-nitro-1H-
imidazole (9) as a yellow solid (2.1 g). The product was recrystallized
Figure 10. Dose/time-dependent inhibition of pSTAT5 and PK-PD
correlation for compound 19a in the mouse BaF3 TEL-Jak2 model.
Phospho-STAT5 and total-STAT5 protein epitopes were detected in
spleen lysates by Western blot analysis.17
with an Agilent 1100 equipped with Waters columns (Atlantis T3, 2.1
mm × 50 mm, 3 μm; or Atlantis dC18, 2.1 mm × 50 mm, 5 μm)
eluted for >10 min with a gradient mixture of H2O−acetonitrile with
formic or trifluoroacetic acid at wavelengths of 220, 254, and 280 nm.
All compounds analyzed were >95% pure. Reverse-phase chromatog-
raphy was performed with Gilson systems using a YMC-AQC18
1
from 2-propanol to provide an off-white solid (1.6 g, 75%). H NMR
(400 MHz, CDCl3) δ ppm 7.77 (d, J = 1.26 Hz, 1 H), 7.43 (s, 1 H),
3.84 (s, 3 H). m/z: 128 [M + H]+.
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Figure 11. 19a showed dose-dependent tumor growth inhibition in a UKE-1 xenograft model.
1-Methyl-1H-imidazol-4-amine Hydrochloride Salt (10). 1-
Methyl-4-nitro-1H-imidazole (9, 27 g) was dissolved in EtOH (800
mL), and Pd(OH)2 (2.5 g) was added. The mixture was subjected to
an atmosphere of hydrogen for 3 h at room temperature. The mixture
was filtered and the organic layer was concentrated to give 1-methyl-
1H-imidazol-4-amine. This amine was dissolved in EtOH (800 mL)
and stirred at room temperature. A saturated solution of EtOH with
HCl (g) (750 mL) was added. The mixture was stirred for 30 min, and
the solution was concentrated under reduced pressure to 100 mL. The
white solid formed. The solid was collected via filtration and washed
with ether to give 1-methyl-1H-imidazol-4-amine hydrochloride salt
(10, 28.4 g, 100%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.44 (1H,
d), 6.59 (1H, d), 3.70 (3H, s). m/z: 98 [M + H]+.
2,4-Dichloro-6-methoxyquinazoline (11b). A solution of
commercially available 6-methoxyquinazoline-2,4-diol (1.91 g, 9.94
mmol) and N,N-dimethylaniline (1.26 mL, 9.94 mmol) in POCl3
(13.90 mL, 149.09 mmol) was heated at reflux for 4 h. The reaction
mixture was cooled to room temperature and concentrated under
reduced pressure to give 2,4-dichloro-6-methoxyquinazoline (11b),
which was used directly in the next step without any further
purification. m/z: 230 [M + H]+.
yquinazoline (11c, 1.62 g, 7.1 mmol) were reacted using the same
procedure as the one described for the synthesis of 12b, providing 12c
(0.71 g, 35%), m/z 290 [M + H]+.
2-Chloro-N-(1-methyl-1H-imidazol-4-yl)pyrido[3,4-d]-
pyrimidin-4-amine (12d). 1-Methyl-1H-imidazol-4-amine hydro-
chloride salt (10, 167 mg, 1.72 mmol) and 2,4-dichloropyrido[3,4-
d]pyrimidine (11d, 500 mg, 2.50 mmol) were reacted using the same
procedure as the one described for the synthesis of 12b, providing 12d
1
(421 mg, 64%). H NMR (300 MHz, DMSO-d6) δ ppm 11.33 (s, 1
H), 9.16 (d, 1 H), 9.01 (s, 1 H), 7.54−7.72(m, 3H), 3.75 (s, 3 H). m/z
261 [M + H]+.
2-Chloro-6-fluoro-N-(1-methyl-1H-imidazol-4-yl)pyrido[2,3-
d]pyrimidin-4-amine (12e). 1-Methyl-1H-imidazol-4-amine hydro-
chloride salt (10, 0.770 g, 6.05 mmol) and 2,4-dichloro-6-fluoropyrido-
[2,3-d]pyrimidine (11e, 1.1 g, 5.1 mmol) were reacted using the same
procedure as the one described for the synthesis of 12b, providing 12e
(1.01 g, 72%), m/z 279 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ
ppm 9.06−9.39 (m, 2 H), 7.49−7.81 (m, 2 H), 3.82 (s, 3 H).
2,7-Dichloro-N-(1-methyl-1H-imidazol-4-yl)pyrido[2,3-d]-
pyrimidin-4-amine (12f). 1-Methyl-1H-imidazol-4-amine hydro-
chloride salt (10, 261 mg, 2.06 mmol) and 2,4,7-trichloropyrido[2,3-
d]pyrimidine (402 mg, 1.71 mmol) were reacted using the same
procedure as the one described for the synthesis of 12b, providing 12f
(365 mg, 72.1%), m/z 295 [M + H]+.
2-Chloro-N-(1-methyl-1H-imidazol-4-yl)pteridin-4-amine
(12g). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 400 mg,
3.15 mmol) and 2,4-dichloropteridine (11g, 633 mg, 3.15 mmol) were
reacted using the same procedure as the one described for the
synthesis of 12b, providing 12g (135 mg, 16%), m/z 262 [M + H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imida-
zol-4-yl)quinazoline-2,4-diamine, Trifluoroacetic Acid Salt
(13a). 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)quinazolin-4-amine
(12a, 460 mg, 1.77 mmol), DIPEA (5 mmol), and (1S)-1-(5-
fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 378 mg, 2.13
mmol)21 in n-BuOH (4 mL) were heated at 150 °C under microwave
irradiation for 6 h. The mixture was cooled at room temperature, and
the volatiles were evaporated in vacuo. Residue was purified by
reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA in
H2O 5% → 50%) to give N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-
methyl-1H-imidazol-4-yl)quinazoline-2,4-diamine, trifluoroacetic acid
salt (13a, 527 mg, 60%) as a mixture of enantiomers.
2-Chloro-6-methoxy-N-(1-methyl-1H-imidazol-4-yl)-quina-
zolin-4-amine (12b). A solution of 1-methyl-1H-imidazol-4-amine
hydrochloride salt (10, 1.9 g, 14.3 mmol) was added to a solution of
2,4-dichloro-6-methoxyquinazoline (11b, 2.3 g, 9.94 mmol) (12.2 mL)
and DIPEA (8.7 mL, 49.70 mmol) in MeCN. The resulting mixture
was stirred at 70 °C overnight. The reaction mixture was diluted with
water and extracted with DCM/MeOH (3 × 100 mL, 10%). The
organic layer was concentrated to a volume of 20 mL. White solid
formed. 2-Chloro-6-methoxy-N-(1-methyl-1H-imidazol-4-yl)-quinazo-
lin-4-amine 12b (710 mg, 26%) was collected after filtration as white
1
fluffy solid. H NMR (400 MHz, DMSO-d6) δ ppm 10.89 (1H, s),
8.16 (1H, d), 7.64 (1H, m), 7.55 (1H, s), 7.47 (1H, m), 3.93 (3H, s),
3.73 (3H, s). m/z: 291 [M + H]+.
The following compounds were synthesized in a similar fashion to
12b.
2-Chloro-N-(1-methyl-1H-imidazol-4-yl)quinazolin-4-amine
(12a). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 429 mg,
3.38 mmol) and 2,4-dichloroquinazoline (11a, 560 mg, 2.81 mmol)
were reacted using the same procedure as the one described for the
synthesis of 12b, providing 12a (530 mg, 72%), m/z 260 [M + H]+.
2-Chloro-7-methoxy-N-(1-methyl-1H-imidazol-4-yl)-
quinazolin-4-amine (12c). 1-Methyl-1H-imidazol-4-amine hydro-
chloride salt (10, 1.3 g, 10.6 mmol) and 2,4-dichloro-7-methox-
The racemic mixture were separated using chiral SFC using the
following analytical instrument and conditions. Chiral SFC column,
Chiralpak AD (21 mm × 250 mm, 5 μm); solvent condition, mobile
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the synthesis of 12a, providing 13b (390 mg, 63%) after purification
Table 7. Data Processing and Refinement Statistics for X-ray
by reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA
Crystallographic Data Collected for Structures of Jak2 in
1
a
in H2O 5% → 55%). H NMR (300 MHz, MeOD) δ ppm 8.77 (s, 2
Complex with Compounds 23b and 28
H), 7.98 (d, 1 H), 7.87 (s, 1 H), 7.52 (d, 2 H), 5.37−5.59 (m, 1 H),
3.97 (s, 3 H), 3.93 (s, 3 H), 1.74 (d, 3 H). m/z: 395 [M + H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-7-methoxy-N4-(1-meth-
yl-1H-imidazol-4-yl)quinazoline-2,4-diamine, Trifluoroacetic
Acid Salt (13c). 2-Chloro-7-methoxy-N-(1-methyl-1H-imidazol-4-
yl)quinazolin-4-amine (12c, 383 mg, 1.32 mmol) and (1S)-1-(5-
fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 468 mg, 2.64
mmol) were reacted using the same procedure as the one described for
the synthesis of 13a, providing 13c (160 mg, 24%) as a mixture of
enantiomers after purification utilizing reversed phase HPLC: Column,
Waters XBridge C18 100 mm × 19 mm, particle size 5 um; mobile
phase, 0.1% NH4OH in water/MeCN; gradient, 10−60% acetonitrile
in 10 min. The racemic mixture was separated using chiral SFC. Chiral
SFC column, Chiralpak AD (21 mm × 250 mm, 5 μm); solvent
condition, mobile phase A, carbon dioxide 65%; mobile phase B, 1:1
methanol:ethanol; additive, 0.4% diethylamine 35%; flow rate (mL/
min), 60; detection (nm), 254; temperature (°C), 40; outlet pressure
(bar), 100. Enantiomeric excess (ee) of 13c (retention time 4.46 min)
was >98% determined by the following analytical instrument and
conditions. Chiral SFC column: Chiralpak AD (column dimensions:
4.6 mm × 250 mm, 5 μm); mobile phase, 60:44:0.4% carbon
dioxide:methanol:dimethylethylamine; flow rate (mL/min), 2.5;
23b
28
PDB code
4c61
4c62
space group
C2
C2
cell constants: a, b, c (Å), b
(deg)
43.9, 127.1, 135.2, 97.3
44.5, 126.9,
135.9, 97.5
resolution range (Å)
134.1−2.45(2.58−2.45)
21.2−2.75(2.90−
2.75)
completeness overall (%)
96.2(96.4)
99.4(98.7)
total no. of observations
(unique reflns)
109283(25971)
315953(19353)
multiplicity
4.2(4.3)
0.07(0.30)
13.4(2.5)
18.1
4(3.7)
0.10(0.50)
11.6(2.1)
19.7
b
Rmerge overall
c
I/σI
d
Roverall value (%)
e
Rfree value (%)
21.7
24.6
non-hydrogen protein
atoms
4334
4356
non-hydrogen ligand atoms
solvent molecules
54
58
199
117
rms deviations from ideal
values
1
detection, 254 nm. H NMR (300 MHz, MeOD) δ ppm 8.71 (s, 2
bond lengths (Å)
bond angles (deg)
average B values (Å2)
0.01
1.12
0.01
1.7
H), 7.85−8.00 (m, 1 H), 7.59 (br s, 1 H), 7.44 (d, 1H), 6.80 (dq, 2
H), 5.48 (q, 1 H), 3.89 (s, 3 H), 3.82 (s, 3 H), 1.64 (d, 3H). m/z: 394
[M + H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imida-
zol-4-yl)pyrido[3,4-d]pyrimidine-2,4-diamine, Trifluoroacetic
Acid Salt (13d). 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)pyrido-
[3,4-d]pyrimidin-4-amine (12d, 404 mg, 1.55 mmol) and (1S)-1-(5-
Fluoropyrimidin-2-yl)ethanamine hydrochloride (14) were reacted
using the same procedure as the one described for the synthesis of 13a,
providing 13d (209 mg, 28%) as a mixture of enantiomers, after
purification by reversed phase HPLC (Gilson chromatography,
MeCN/0.1%TFA in water 5% → 40%). The racemic mixture were
separated using Chiral HPLC. Chiral SFC column: Chiralpak AD (20
mm × 250 mm, 10 μm); solvent condition, 1:1 methanol:ethanol;
additive, 0.1% diethylamine 35%; flow rate (mL/min), 20; detection
220 (nm). Enantiomeric excess (ee) of 13d (retention time 7.48 min)
was >98% determined by the following chiral HPLC instrument and
conditions: Chiral HPLC column, Chiralpak AD (4.6 mm × 250 mm,
10 μm); mobile phase, 1:1:1 methanol:ethanol:0.1% dimethylethyl-
protein main chain
atoms
54.4
50.5
protein all atoms
ligand
56.9
39.4
51.5
51.7
34.6
36
solvent
Φ, Ψ angle distribution for
f
residues
in most favored
regions (%)
92.4
7.2
0.4
0
89.9
9.7
0.4
0
in additional
allowed regions (%)
in generously
regions (%)
in disallowed
regions (%)
a
Numbers in parentheses represent data collection statistics for the
b
highest resolution shell unless marked otherwise. Rmerge = ∑hkl [(∑i |
1
amine; flow rate (mL/min), 1; detection, 254 nm. H NMR (300
c
Ii − ⟨I⟩|)/∑i Ii]. I/σI average is the mean I/σ for the unique
MHz, MeOD) δ ppm 8.57 (s, 1 H), 8.27 (d, 1 H), 8.08 (d, 1 H), 7.83
(d, 1 H), 7.43−7.63 (m, 2 H), 7.27 (br s, 1 H), 5.61 (q, 1 H), 3.60 (br
s, 3 H), 1.51 (d, J = 6.97 Hz, 3H). m/z: 367.0 [M + H]+.
d
reflections in the output file. Rvalue = ∑hkl||Fobs| − |Fcalc||/∑hkl|Fobs
.
e
Rfree is the cross-validation R factor computed for the test set of 5% of
f
unique reflections. Ramachandran statistics as defined by PRO-
6-Fluoro-N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-
1H-imidazol-4-yl)pyrido[2,3-d]pyrimidine-2,4-diamine, Tri-
fluoroacetic Acid Salt (13e). 2-Chloro-6-fluoro-N-(1-methyl-1H-
imidazol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (12e, 90 mg, 0.32
mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride
(14) were reacted using a procedure similar to the one described for
the synthesis of 13a, providing 13e (139 mg, 86%) as a mixture of
enantiomers, after purification by reversed phase HPLC (Gilson
chromatography, MeCN/0.1%TFA in water 5%→30%). The racemic
mixture were separated using chiral HPLC. Chiral HPLC: Chiralpak
IC (20 mm × 250 mm, 5 μm); mobile phase A, hexane 70%, mobile
phase B, 1:1 methanol:ethanol; additive, 0.1% diethylamine; flow rate
(mL/min), 20; detection (nm), 220. Enantiomeric excess (ee) of 13e
(retention time 16.02 min) was >98% determined by the following
chiral HPLC instrument and conditions. Chiral HPLC: Chiralpak IC
(4.6 mm × 250 mm, 5 μm); mobile phase, 1:1 methanol:ethanol 0.1%
CHECK.
phase A, carbon dioxide 75%; mobile phase B, 1:1 methanol:ethanol;
additive, 0.4% diethylamine 25%; flow rate (mL/min), 60; detection
(nm), 254; temperature (°C), 40; outlet pressure (bar), 100.
Enantiomeric excess (ee) of 13a (retention time 3.86 min) was
>98% determined by the following analytical instrument and
conditions. Chiral SFC column: Chiralpak AD (4.6 mm × 100 mm,
5 μm); mobile phase A, carbon dioxide 80%; mobile phase B, 1:1
methanol:ethanol; additive, 0.4% diethylamine 20%; flow rate (mL/
min), 5; detection (nm), 220; temperature (°C), 35; outlet pressure
(bar), 120. 1H NMR (300 MHz, MeOD) δ ppm 8.66 (s, 2 H), 8.21 (d,
2 H), 8.00 (br s, 1 H), 7.79 (td, 1 H), 7.47 (m, 2 H), 5.38 (q, 1 H),
3.84 (s, 3 H), 1.61 (d, 3 H). m/z: 383 [M + H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-6-methoxy-N4-(1-meth-
yl-1H-imidazol-4-yl)quinazoline-2,4-diamine, Trifluoroacetic
Acid Salt (13b). 2-Chloro-6-methoxy-N-(1-methyl-1H-imidazol-4-
yl)quinazolin-4-amine (12b, 350 mg, 1.21 mmol) and (1S)-1-(5-
fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 428 mg, 2.42
mmol) were reacted using the same procedure as the one described for
1
dimethylethylamine; flow rate (mL/min), 1; detection, 254 nm. H
NMR (300 MHz, DMSO-d6) δ ppm 10.26 (br s, 1 H), 8.79 (s, 2 H),
8.70 (d, 1 H), 8.55 (br s, 1 H), 7.59 (d, 1 H), 7.41 (s, 1 H), 5.17−5.50
(m, 1 H), 3.65 (s, 3H), 1.50 (d, 3 H). m/z: 394 [M + H]+.
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7-Chloro-N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-
1H-imidazol-4-yl)pyrido[2,3-d]pyrimidine-2,4-diamine, Tri-
fluoroacetic Acid Salt (13f). 2,7-Dichloro-N-(1-methyl-1H-imida-
zol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (12f, 385 mg, 1.30 mmol)
and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14,
323 mg, 1.30 mmol) were reacted using the same procedure as the
one described for the synthesis of 13a, providing 13f (181 mg, 27%) as
a mixture of enantiomers, after purification by reversed phase HPLC
(Gilson chromatography, MeCN/0.1%TFA in water 5% → 35%).1H
NMR (300 MHz, MeOD) δ ppm 8.67 (s, 2 H), 8.57 (d, 1 H), 7.90 (s,
1 H), 7.45−7.59 (m, 1 H), 7.39 (d, 1 H), 5.40 (q, 1 H), 3.82 (s, 3 H),
1.62 (d, 3 H). m/z: 400 [M + H]+.
(d, J = 3.77 Hz, 1 H), 6.62 (d, J = 3.77 Hz, 1 H), 3.49−3.67 (m, 1 H),
1.01−1.31 (m, 4 H). m/z: 230 [M + H]+.
2,6-Dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-
d]pyrimidine (17d). To a solution of 4,6-dichloro-1H-pyrazolo[3,4-
d]pyrimidine (15b, 2 g, 10.58 mmol) and p-Ts-OH (0.201 g, 1.06
mmol) in DCM (30 mL) and THF (30.0 mL) was added 3,4-dihydro-
2H-pyran (1.335 g, 15.87 mmol). The resulting solution was stirred
overnight at ambient temperature, whereupon the volatiles were
removed under reduced pressure. The residue was dissolved in DCM,
and the organic layer was washed with saturated aqueous sodium
carbonate solution, water, brine, and dried MgSO4. Evaporation of the
volatiles under reduced pressure gave 17d (2.80g, 100%). m/z: 273 [M
+ H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imida-
zol-4-yl)pteridine-2,4-diamine (13g). 2-Chloro-N-(1-methyl-1H-
imidazol-4-yl)pteridin-4-amine (12g, 135 mg, 0.52 mmol) and (1S)-1-
(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 183 mg, 1.03
mmol) were suspended in butan-1-ol (2 mL), and DIPEA (0.360 mL,
2.06 mmol) was added. The reaction was irradiated in a microwave at
150 °C for 1 h. The reaction mixture was concentrated in vacuo,
leaving an amber oil (423 mg). This material was purified by ISCO
(2−10% MeOH/DCM). Concentration of the fractions in vacuo
provided 13g as a mixture of enantiomers as a yellow solid (72 mg,
29%). The racemic mixture were separated using chiral SFC. Chiral
SFC: Chiralpak AD (21 mm × 250 mm, 5 μm); mobile phase, 20%
methanol with 0.4% dimethylethylamine; flow rate (mL/min), 40;
detection (nm), 254; outlet pressure (bar), 100. Enantiomeric excess
(ee) of 13g (retention time 15.52 min) was >98% determined by the
following chiral SFC instrument and conditions. Chiral SFC:
Chiralpak AD (4.6 mm × 250 mm, 5 μm); mobile phase: 20%
methanol with 0.4% dimethylethylamine; flow rate (mL/min), 2.5;
detection, 254 nm; pressure (bar), 120. 1H NMR (300 MHz, MeOD)
δ ppm 8.49−8.88 (m, 3 H), 8.37 (d, 1 H), 7.88 (br s, 0.5 H), 7.56 (br
s, 0.5 H), 7.46 (d, 1 H), 5.37−5.70 (m, 1 H), 3.81 (d, 3 H), 1.67 (d, 3
H). m/z: 367 [M + H]+.
2-Chloro-N-(1-methyl-1H-imidazol-4-yl)-7-[(4-
methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(18a). 1-Methyl-4-nitro-1H-imidazole hydrochloride salt (10, 50 mg,
0.39 mmol) was added to a solution of 4-dichloro-7-[(4-
methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidine (17a, 108 mg,
0.31 mmol) and TEA (0.11 mL, 0.79 mmol) in CH3CN. The reaction
mixture was stirred at 100 °C in a microwave reactor for 2 h. After
completion of the reaction as indicated by TLC, the reaction mixture
was evaporated in vacuo to obtain a light-yellow solid which was
purified by column chromatography (3% MeOH, 0.3% NH4OH in
1
DCM) to provide 18a (90 mg, 72%) as a white solid. H NMR (400
MHz, CDCl3) δ ppm 8.92 (s, 1 H), 8.03 (d, 2 H), 7.41 (s, 1 H), 7.39
(d, 1 H), 7.25 (d, 2H), 6.48 (s, 1 H), 3.67 (s, 3 H), 2.33 (s, 3 H). m/z:
403 [M + H]+. The following compounds were synthesized in a
similar fashion to 18a.
2-Chloro-7-methyl-N-(1-methyl-1H-imidazol-4-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, Trifluoroacetic Acid Salt
(18b). 1-Methyl-4-nitro-1H-imidazole hydrochloride salt (10, 1.38
g) was added to a solution of 2,4-dichloro-7-methyl-7H-pyrrolo[2,3-
d]pyrimidine (17b, 500 mg) and DIPEA (0.92 mL, 5.32 mmol). The
resulting mixture was stirred at 90 °C for 15 h. The reaction mixture
was diluted with water and extracted with DCM/MeOH (10%).
Evaporation of the volatiles under reduced pressure gave a residue
which was purified by reverse phase HPLC (Gilson chromatography,
MeCN/0.1%TFA in water 5%→45%) to provide 18b (300 mg, 21%).
m/z: 265 [M + H]+.
2-Chloro-7-cyclopropyl-N-(1-methyl-1H-imidazol-4-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, Trifluoroacetic Acid Salt
(18c). 2,4-Dichloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine (17c,
270 mg, 1.18 mmol) and 1-methyl-1H-imidazol-4-amine hydro-
chloride (10, 604 mg, 3.55 mmol) were reacted using a procedure
similar to the one described for the synthesis of 18a, providing 18c
(200 mg, 42%) after purification by reverse phase HPLC (Gilson
chromatography, MeCN/0.1%TFA in water 0%→50%).m/z: 291 [M
+ H]+ .
2,4-Dichloro-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-
d]pyrimidine (17a). 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (15a,
1.00 g, 5.32 mmol), 4-methylbenzene-1-sulfonyl chloride (1.115 g,
5.85 mmol), and tetra-butylammonium hydrogen sulfate (0.090 g, 0.27
mmol) were dissolved in DCM (20 mL) at room temperature, and
NaOH (50% aq, 1 mL) was added. The reaction mixture stirred at
room temperature for 30 min. After completion of the reaction as
indicated by TLC, the reaction mixture was diluted with H2O and
DCM. The organic layer was evaporated in vacuo to obtain a light-
yellow solid, which was purified by column chromatography (100%
1
DCM) to provide 17a (1.76 g, 97%) as a white solid. H NMR (400
MHz, CDCl3) δ ppm 8.14 (d, 2 H), 7.78 (d, 1 H), 7.39 (d, 2 H), 6.70
(d, 1 H), 2.45 (s, 3 H). m/z: 342 [M + H]+.
6-Chloro-N-(1-methyl-1H-imidazol-4-yl)-1-(tetrahydro-2H-
pyran-2-yl)-1H pyrazolo[3,4-d]pyrimidin-4-amine (18d). To a
solution of 4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo-
[3,4-d]pyrimidine (17d, 3.17 g, 11.60 mmol) in ethanol (60 mL)
was added TEA (4.04 mL, 29.00 mmol) followed by 1-methyl-1H-
imidazol-4-amine hydrochloride (10, 1.549 g, 11.60 mmol). The
resulting mixture was heated at 60 °C for 2 h. Evaporation of the
volatiles under reduced pressure gave a residue which was purified
utilizing ISCO (EtOAc/hexanes 0→80%) to give 18d (1.56 g, 41%).
m/z: 334 [M + H]+.
2,4-Dichloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (17b).
2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (15a, 2.37 g, 12.61 mmol)
was dissolved in acetonitrile (8.32 mL), and sodium hydride (529 mg,
13.24 mmol) was added portionwise. The reaction mixture was stirred
at room temperature for 0.5 h until gas evolution ceased. Methyl
iodide (0.87 mL, 13.87 mmol) was added, and the resulting mixture
was stirred for 0.5 h. The reaction mixture was then poured into water
and extracted with DCM/MeOH. Concentration of the organic layers
under reduced pressure provided a residue, which was purified utilizing
1
ISCO (0% → 100% DCM/EtOAc) to afford 17b (2.1g, 84%). H
6-Chloro-1-methyl-N-(1-methyl-1H-imidazol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (18e). Commercially available
4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine (17e, 4.49 g,
22.12 mmol) and 1-methyl-1H-imidazol-4-amine hydrochloride (10,
2.96 g, 22.12 mmol) were suspended in ethanol (104 mL), and TEA
(6.17 mL, 44.25 mmol) was added. The reaction mixture was heated at
70 °C overnight. The reaction mixture was cooled to 0 °C. Solid
formed and was collected via filtration to provide 18e as a purple−gray
solid (2.94 g, 50%). 1H NMR (300 MHz, MeOD) δ ppm 8.69 (s, 2 H)
7.85 (br s, 1 H) 7.53 (br s, 1 H) 7.42 (s, 1 H) 5.42 (q, 1 H) 3.65−3.89
(m, 6 H) 1.61 (d, 3 H). m/z: 264 [M + H]+.
NMR (300 MHz, DMSO-d6) δ ppm 7.75 (s, 1 H), 6.71 (s, 1 H), 3.82
(s, 3 H). m/z: 204 [M + H]+.
2,4-Dichloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine
(17c). 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (15a, 1 g, 5.32
mmol), copper(II) acetate (1.45 g, 7.98 mmol), pyridine (2.15 mL,
26.59 mmol), and cyclopropylboronic acid (1.1 g, 13.30 mmol) were
heated at 90 °C under dry air for 36 h. The reaction mixture was
concentrated under reduced pressure, and the residue was partitioned
between EtOAc and water. The organic layer was collected, dried, and
concentrated under reduced pressure to provide a crude mixture,
which was purified utilizing ISCO (0% → 30% hexanes/EtOAc) to
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imida-
zol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (19a). In a
1
afford 17c (270 mg, 22%). H NMR (300 MHz, MeOD) δ ppm 7.53
154
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Journal of Medicinal Chemistry
Article
microwave tube, 2-chloro-N-(1-methyl-1H-imidazol-4-yl)-7-[(4-
methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (18a, 90
mg), (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14,
881 mg, 4.96 mmol), and DIPEA (1.084 mL, 6.21 mmol) were
dissolved in n-BuOH (5 mL). The reaction mixture was heated in a
microwave reactor at 150 °C for 3 h. After completion of the reaction
as indicated by LCMS, the reaction mixture was evaporated in vacuo
to obtain a brown residue. This residue was purified by column
chromatography (4% MeOH, 0.4% NH4OH in DCM) to provide the
N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)-
7-[(4 methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-dia-
mine (350 mg, 56%) as a mixture of enantiomers as a yellow solid.
m/z: 508 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.35 (s,
1H), 8.79 (s, 2H), 7.91−7.97 (m, 2H), 7.46 (s, 1H), 7.33 (t, 3H), 7.08
(d, 1H), 6.90 (d, 1H), 6.67 (d, 1H), 5.28−5.35 (m, 1H), 3.67 (s, 3H),
2.36 (s, 3H), 1.56 (d, 3H).
Enantiomeric excess (ee) of 19c (retention time 3.44 min) was >98%
determined by the following chiral SFC instrument and conditions.
Chiral SFC: Chiralpak AD (4.6 × 100 mm, 5 μm); mobile phase,
60:40:0.4% carbon dioxide:methanol; flow rate (mL/min), 5;
1
detection, 254 nm. H NMR (300 MHz, MeOD) δ ppm 8.70 (s, 2
H), 7.47 (d, 1 H), 7.39 (d, 1 H), 6.70 (d, 1 H), 6.33 (d, 1 H), 5.27−
5.52 (m, 1 H), 3.80 (s, 3 H), 3.17−3.29 (m, 1 H), 1.62 (d, 3 H), 0.74−
1.07 (m, 4 H). m/z: 394 [M + H]+.
N6-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imida-
zol-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (19d). 6-
Chloro-N-(1-methyl-1H-imidazol-4-yl)-1-(tetrahydro-2H-pyran-2-yl)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine (18d, 158 mg, 0.47 mmol) and
(1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 84 mg,
0.47 mmol) were dissolved in butan-1-ol (2.5 mL), followed by the
addition of triethylamine (0.165 mL, 1.18 mmol). The reaction
mixture was heated under microwave irradiation at 160 °C for 6 h.
LCMS analysis indicated that the protecting group was cleaved under
the employed conditions. The volatiles were evaporated under reduced
pressure, and the residue left was purified to give 19d (14.2 mg, 8%) as
A solution of N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-
1H-imidazol-4-yl)-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]-
pyrimidine-2,4-diamine (7.61 g, 15 mmol) and NaOH (12g, 300.00
mmol) in water (10 mL), methanol (10 mL), and 1,4-dioxane (52
mL) was heated at 55 °C overnight. The reaction mixture was acidified
with HCl to pH = 3 and washed with DCM. The aqueous layer was
neutralized with NaHCO3 to pH = 8 and extracted with DCM/MeOH
(10%). The organic layer was concentrated under reduced pressure to
give a residue. This residue was purified utilizing ISCO (0%→80%
DCM/acetone/2% NH4OH) to provide 19a (3.91 g, 74%) as a
mixture of enantiomers. The racemic mixture was separated using
chiral HPLC. Chiral HPLC: Chiralpak AD (50 mm × 250 mm, 5 μm);
mobile phase, 1:1:0.1% methanol:ethanol:diethylamine; flow rate
(mL/min), 120; detection (nm), 220. Enantiomeric excess (ee) of
19a (retention time 3.21 min) was >98% determined by the following
chiral HPLC instrument and conditions. Chiral HPLC: Chiralpak AD
(4.6 mm × 250 mm, 5 μm); mobile phase, 60:40:0.4% carbon
dioxide:methanol; flow rate (mL/min), 5; detection, 220 nm. 1H
NMR (300 MHz, MeOD) δ ppm 8.68 (s, 2 H), 7.47 (d, 1 H), 7.39 (d,
1 H), 6.74 (d, 1 H), 6.37 (d, 1 H), 5.39 (q, 1 H), 3.80 (s, 3H), 1.59 (d,
4 H). m/z: 354 [M + H]+.
1
a mixture of enantiomers. H NMR (300 MHz, MeOD) δ ppm 8.70
(s, 2 H), 7.87 (br s, 1 H), 7.55 (br s, 1 H), 7.56 (br s, 1H), 5.40 (q, 1
H), 3.81 (s, 3 H), 1.62 (d, 3 H). m/z: 355 [M + H]+.
N6-[1-(5-Fluoropyrimidin-2-yl)ethyl]-1-methyl-N4-(1-methyl-
1H-imidazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
(19e). 6-Chloro-1-methyl-N-(1-methyl-1H-imidazol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (18e, 2 g, 7.58 mmol) and (1S)-1-
(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 1.482 g, 8.34
mmol) were suspended in butan-1-ol (21.05 mL), and TEA (4.23 mL,
30.34 mmol) was added. The reaction mixture was subjected to
microwave irradiation at 150 °C for 3 h. The reaction mixture was
filtered, and the filtrate was concentrated in vacuo, leaving a brown
semisolid (3.504 g). This material was purified by ISCO (5% MeOH/
DCM, isocratic) to provide 19e as a mixture of enantiomers in the
form of a yellow solid (1.58 g, 57%). The racemic mixture were
separated using chiral HPLC. Chiral HPLC: Chiralpak AD (50 mm ×
500 mm, 20 μm); mobile phase, 100% methanol; flow rate (mL/min),
120; detection (nm), 220. Enantiomeric excess (ee) of 19e (retention
time 2.30 min) was >98% determined by the following chiral SFC
instrument and conditions. Chiral SFC: Chiralpak AD (4.6 mm × 100
mm, 5 μm); mobile phase, 40% methanol with 0.1% dimethylethyl-
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-7-methyl-N4-(1-methyl-
1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine,
Trifluoroacetic Acid Salt (19b). (1S)-1-(5-Fluoropyrimidin-2-yl)-
ethanamine hydrochloride (14, 303 mg, 1.71 mmol) and 2-chloro-7-
methyl-N-(1-methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine, trifluoroacetic acid salt (18b, 300 mg, 1.14 mmol), were reacted
using a procedure similar to the one described for the synthesis of 19a,
providing 19b (211 mg, 38%) as a mixture of enantiomers, after
purification by reverse phase HPLC (Gilson chromatography, MeCN/
0.1%TFA in water 5%→45%). The racemic mixture were separated
using chiral SFC. Chiral SFC: Chiralpak AD (21 × 250 mm, 5 μm);
mobile phase, 65:35:0.4% carbon dioxide:methanol; flow rate (mL/
min), 60; detection (nm), 254. Enantiomeric excess (ee) of 19b
(retention time 4.33 min) was >98% determined by the following
chiral HPLC instrument and conditions. Chiral HPLC: Chiralpak AD
(4.6 × 250 mm, 5 μm); mobile phase, 60:40:0.4% carbon
1
amine; flow rate (mL/min), 5; detection, 220 nm. H NMR (300
MHz, MeOD) δ ppm 8.69 (s, 2 H), 7.85 (br s, 1 H), 7.53 (br s, 1 H),
7.42 (s, 1 H), 5.42 (q, 1 H), 3.65−3.89 (m, 6 H), 1.61 (d, 3 H). m/z:
369 [M + H]+.
2,4-Dichloro-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2-
d]pyrimidine (21). Commercially available 2,4-dichloro-5H-pyrrolo-
[3,2-d]pyrimidine (20a, 500 mg, 2.66 mmol) and 4-methylbenzene-1-
sulfonyl chloride (558 mg, 2.93 mmol) were reacted using a procedure
similar to the one described for the synthesis of 17a, providing 21 (900
1
mg, 100%). H NMR (400 MHz, CDCl3) δ ppm 8.34 (d, 1 H), 7.75
(d, 2 H), 7.34 (d, 2 H), 6.87 (d, 1H), 2.44 (s, 3 H). m/z: 342 [M +
H]+.
2-Chloro-7-methyl-N-(1-methyl-1H-imidazol-4-yl)thieno[3,2-
d]pyrimidin-4-amine (22a). 1-Methyl-1H-imidazol-4-amine hydro-
chloride salt (10, 194 mg, 2 mmol) and 2,4-dichloro-7-methylthieno-
[3,2-d]pyrimidine (20a, 438 mg, 2 mmol) were reacted using a
procedure similar to the one described for the synthesis of 18a,
1
dioxide:methanol; flow rate (mL/min), 2.5; detection, 254 nm. H
NMR (300 MHz, MeOD) δ ppm 8.58 (s, 2 H), 7.36 (d, 1 H), 7.28 (d,
1 H), 6.58 (d, 1 H), 6.26 (d, 1 H), 5.30 (q, 1 H), 3.68 (s, 3 H), 3.47 (s,
3 H), 1.50 (d, 3 H). m/z: 368 [M + H]+.
1
providing 22a (294 mg, 52%). H NMR (300 MHz, MeOD) δ ppm
7-Cyclopropyl-N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-
methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-dia-
mine, Trifluoroacetic Acid Salt (19c). (1S)-1-(5-Fluoropyrimidin-
2-yl)ethanamine hydrochloride (14, 252 mg, 1.42 mmol) and 2-
chloro-7-cyclopropyl-N-(1-methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, trifluoroacetic acid salt (18c, 205 mg, 0.71
mmol), were reacted using a procedure similar to the one described for
the synthesis of 19a, providing 19c (40 mg, 14%) as a mixture of
enantiomers after purification by reverse phase HPLC (Gilson
chromatography, MeCN/0.1%TFA in water 5%→45%). The racemic
mixture were separated using chiral SFC. Chiral SFC: Chiralpak AD
(21 × 250 mm, 5 μm); mobile phase, 75:25:0.4% carbon
dioxide:methanol; flow rate (mL/min), 60; detection (nm), 254.
7.85 (s, 1H), 7.53 (s, 1H), 7.40 (s, 1 H), 3.71 (s, 3 H), 2.30 (s, 3H).
m/z: 280 [M + H]+.
2-Chloro-N-(1-methyl-1H-imidazol-4-yl)-5-[(4-
methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]pyrimidin-4-amine
(22b). 2,4-Dichloro-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]-
pyrimidine (21, 240 mg, 0.70 mmol) and 1-methyl-1H-imidazol-4-
amine (10, 1.5 equiv) were reacted using a procedure analogous to
that described for the synthesis of 18a, providing the title product 22b
(90 mg, 32%). 1H NMR (400 MHz, CDCl3) δ ppm 9.92 (s, 1 H), 7.71
(d, 1 H), 7.61 (d, 2 H), 7.44 (br.s., 1 H), 7.24 (s, 1H), 7.16 (d, 2 H),
6.62 (d, 1 H), 3.68 (s, 3 H), 2.28 (s, 3 H). m/z: 403 [M + H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-7-methyl-N4-(1-methyl-
1H-imidazol-4-yl)thieno[3,2-d]pyrimidine-2,4-diamine, Tri-
155
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Journal of Medicinal Chemistry
Article
fluoroacetic Acid Salt (23a). 2-Chloro-7-methyl-N-(1-methyl-1H-
imidazol-4-yl)thieno[3,2-d]pyrimidin-4-amine (22a, 276 mg, 0.99
mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride
(14, 175 mg, 0.99 mmol) were reacted using a procedure similar to the
one described for the synthesis of 19a, providing 23a as a mixture of
Data were processed using MOSFLM, SCALA, and reduced using
CCP4 software.38 The structures were solved by molecular
replacement using coordinates of the JAK2 kinase domain (PDB
entry 2XA4) as a trial model using CCP4 software. Protein and
inhibitor were modeled into the electron density using COOT.39 The
models were refined using Refmac40 and BUSTER.41 Atomic
coordinates and structure factors for the Human JAK2 complex42,43
with compound 23b and 28 have been deposited in the Protein Data
Bank (4c61 and 4c62, respectively), together with structure factors and
detailed experimental conditions (Table 7).
1
enantiomers, in the form of a yellow solid (126 mg, 25%). H NMR
(300 MHz, MeOD) δ ppm 8.79 (s, 2 H), 7.97 (bs, 1H), 7.86 (s, 1H),
7.52 (s, 1H), 5.46 (q, 1 H), 3.91 (s, 3 H), 2.40 (s, 3H), 1.70 (d, 3 H).
m/z: 385 [M + H]+.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imida-
zol-4-yl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (23b). 2-
Chloro-N-(1-methyl-1H-imidazol-4-yl)-5-[(4-methylphenyl)sulfonyl]-
5H-pyrrolo[3,2-d]pyrimidin-4-amine (22b, 65 mg, 0.16 mmol) and
(1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 114
mg, 0.64 mmol) were reacted using a procedure similar to the one
described for the synthesis of 19a, providing N2-[1-(5-fluoropyrimidin-
2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)-5-[(4-methylphenyl)-
sulfonyl]-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (25 mg) as a
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 781 839 4683. E-mail: Qibin.su@astrazeneca.com.
■
Notes
The authors declare no competing financial interest.
1
mixture of enantiomers. H NMR (400 MHz, MeOD) δ ppm 8.60
ACKNOWLEDGMENTS
■
(s, 1 H), 8.54 (s, 2 H), 7.62 (d, 1 H), 7.54 (d, 2 H), 7.34 (s, 1 H), 7.14
(d, 2H), 6.33 (d, 1 H), 5.21 (q, 1 H), 3.69 (s, 3 H), 2.18 (s, 3 H), 1.46
(d, 3 H). m/z: 508 [M + H]+.
We thank Nancy DeGrace and Kanayochukwu Azogu for chiral
separations work. Special thanks to Melissa Vasbinder, Andrew
Mortlock, James Dowling, and Neil Grimster for valuable
discussions during the preparation of the manuscript.
N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-
yl)-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]pyrimidine-2,4-di-
amine (25 mg, 0.05 mmol) was reacted using a procedure similar to
the one described for the synthesis of 19a, providing 23b (13 mg, 23%
for 2 steps) as a mixture of enantiomers. 1H NMR (400 MHz, MeOD)
δ ppm 8.59 (s, 2 H), 7.39 (s, 1 H), 7.31 (s, 1 H), 7.21 (d, 1 H), 6.09
(d, 1 H), 5.29 (q, 1H), 3.70 (s, 3 H), 1.52 (d, 3 H). m/z: 354 [M +
H]+.
ABBREVIATIONS USED
■
DIAD, diisopropylazodicarboxylate; DHP, dihydropyran;
DIPEA, diisopropylethyl amine; D5W, 5% dextrose (w/v) in
water; ET, essential thrombocythemia; HPMC, hydroxypro-
pylmethylcellulose; Jak2, Janus kinase 2; Jak1, Janus kinase 1;
Jak3, Janus kinase 3; MF, idiopathic myelofibrosis; MM-GBSA,
molecular mechanics-generalized Born solvation approxima-
tion; mpk, mg/kg; MPNs, myeloproliferative neoplasms; PV,
polycythemia vera; STAT, signal transducers and activators of
transcription; p-Ts-OH, p-toluenesulfonic acid; pSTAT,
phosphorylated signal transducers and activators of tran-
scription; SCID, severe combined immunodeficiency; tSTAT,
total signal transducers and activators of transcription; TGI,
tumor growth inhibition; Tyk2, tyrosine kinase 2
5-Chloro-N2-(1-(5-fluoropyrimidin-2-yl)ethyl)-N4-(1-methyl-
1H-imidazol-4-yl)pyrimidine-2,4-diamine (26). A mixture of 1-
methyl-1H-imidazol-4-amine hydrochloride salt (10, 500 mg) was
added to a solution of 2,4,5-trichloro-pyrimidine (0.36 mL) in MeCN
(12.2 mL) and TEA (1.10 mL, 7.87 mmol) and the resulting mixture
stirred at room temperature overnight. 2,5-Dichloro-N-(1-methyl-1H-
imidazol-4-yl)pyrimidin-4-amine (710 mg, 39%) was isolated as a
white fluffy solid after filtration. m/z: 236 [M + H]+. In a microwave
vessel, a suspension of 2,5-dichloro-N-(1-methyl-1H-imidazol-4-yl)-
pyrimidin-4-amine (100 mg, 0.41 mmol) and (1S)-1-(5-fluoropyr-
imidin-2-yl)ethanamine hydrochloride (14, 91 mg, 0.51 mmol) in
butan-1-ol was treated with DIPEA (0.29 mL, 1.6 mmol). The mixture
was subjected to microwave irradiation at 150 °C for 5 h. The mixture
was concentrated and purified using an ISCO system (2−10%
MeOH/DCM) to provide the desired compound 26 (100 mg, 70%).
1H NMR (300 MHz, MeOD) δ ppm 8.70 (s, 2 H), 7.83 (s, 1 H), 7.40
REFERENCES
■
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(s, 1 H), 5.25 (q, 1H), 3.78 (s, 3 H), 1.58 (d, 3 H). m/z: 349 [M +
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Computational Chemistry. The structure of 24 bound to Jak2
was used for all docking studies (ref 17, PDB code 2XA4). The
structure was prepared for docking using the Schrodinger protein
̈
preparation utilities prep and impref, applying the appropriate side-
chain protonation states, refine, and structure minimization. Docking
grids were generated and defined based on the centroid of 24 in the
ATP binding site incorporating hydrogen-bonding constraints to the
hinge. Ligands were prepared using proprietary in-house software.
Ligand receptor docking was carried out using XP-Glide26 and
constrained to satisfy all three of the hydrogen bonds to hinge
illustrated in Figure 4. The top five poses generated by Glide were
retained and used to compute estimates of the ΔG of binding and
ligand strain energies via the MM-GBSA.27
Protein Expression, Purification, Crystallization, and Struc-
ture Determination. Protein expression and purification, crystal-
lization, and cryoprotection procedures have been described
previously.17 Pictures of the crystal structures of Jak2 kinase with
compounds have been generated by PYMOL.37 Diffraction data for
complexes of JAK2 with compounds 23b and 28 were collected ”in-
house” using a Rigaku MM007 X-ray generator equipped with a
MAR345 image plate X-ray detector, using Cu Kα radiation at a
wavelength of 1.5418 Å focused using Rigaku VariMax HF mirrors.
156
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Journal of Medicinal Chemistry
Article
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following figure, with hits overlaid on the kinome phylogenetic tree
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157
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(42) Crystallographic statistics for JAK2 bound to 23b are as follows.
Numbers in parenthesis characterize the higher resolution shell. Space
group C2, unit cell a, b, and c dimensions 43.9, 127.1, 135.2, 97.3 Å, β
= 97.3°, resolution 134−2.45 Å (2.58−2.45 Å), 25971 unique
reflections with an overall redundancy of 4.2 (4.3), 96.2% (96.4%)
completeness with Rmerge of 7% (30%) and mean I/σ(I) of 13.4 (2.5).
The final model containing 4334 proteins, 1199 solvents, and 54
compound atoms has an R-factor of 18.1% (Rfree using 5% of the data
21.7%). Mean temperature factors for the protein and the ligand are
43.0 and 50.1 Å2, respectively.
(43) Crystallographic statistics for the JAK2 bound to 28 are as
follows. Numbers in parenthesis characterize the higher resolution
shell. Space group C2, unit cell a, b, and c dimensions 44.5, 126.9,
135.9 Å, β = 97.5°, resolution 21−2.75 Å (2.9−2.75 Å), 19353 unique
reflections with an overall redundancy of 4.0(3.7), 99.4% (98.7%)
completeness with Rmerge of 10% (50%) and mean I/σ(I) of 11.6 (2.1).
The final model containing 4356 proteins, 117 solvents, and 58
compound atoms has an R-factor of 19.7% (Rfree using 5% of the data
24.6%). Mean temperature factors for the protein and the ligand are
50.5 and 51.7 Å2, respectively.
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