Discovery of potent and selective pyrazolopyrimidine Janus kinase 2 inhibitors

Article abstract of DOI:10.1021/jm3012239

The discovery of somatic Jak2 mutations in patients with chronic myeloproliferative neoplasms has led to significant interest in discovering selective Jak2 inhibitors for use in treating these disorders. A high-throughput screening effort identified the p

Full text of DOI:10.1021/jm3012239

Article
pubs.acs.org/jmc
Discovery of Potent and Selective Pyrazolopyrimidine Janus Kinase 2
Inhibitors
Emily J. Hanan,† Anne van Abbema,‡,# Kathy Barrett,‡ Wade S. Blair,‡,¶ Jeff Blaney,† Christine Chang,‡
Charles Eigenbrot,§ Sean Flynn,⊗ Paul Gibbons,† Christopher A. Hurley,⊗ Jane R. Kenny,∥
Janusz Kulagowski,⊗ Leslie Lee,⊥ Steven R. Magnuson,† Claire Morris,⊗ Jeremy Murray,§
Richard M. Pastor,† Tom Rawson,† Michael Siu,† Mark Ultsch,§ Aihe Zhou,† Deepak Sampath,⊥
and Joseph P. Lyssikatos*,†
‡
§
∥
†Departments of Discovery Chemistry, Biochemical and Cellular Pharmacology, Structural Biology, Drug Metabolism and
⊥
Pharmacokinetics, Translational Oncology, Genentech, Inc. 1 DNA Way, South San Francisco, California 94080, United States
⊗Argenta, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, United Kingdom
S
* Supporting Information
ABSTRACT: The discovery of somatic Jak2 mutations in patients with chronic
myeloproliferative neoplasms has led to significant interest in discovering selective
Jak2 inhibitors for use in treating these disorders. A high-throughput screening effort
identified the pyrazolo[1,5-a]pyrimidine scaffold as a potent inhibitor of Jak2.
Optimization of lead compounds 7a−b and 8 in this chemical series for activity
against Jak2, selectivity against other Jak family kinases, and good in vivo
pharmacokinetic properties led to the discovery of 7j. In a SET2 xenograft model
that is dependent on Jak2 for growth, 7j demonstrated a time-dependent knock-
down of pSTAT5, a downstream target of Jak2.
Indeed, a number of small molecule ATP-competitive Jak2
inhibitors are currently in clinical development for the
treatment of MPDs, including TG101348,6 SB1518,7
AZD1480,8 LY2784544,9 and CYT38710 (Figure 1). The
recent FDA approval of ruxolitinib for the treatment of patients
with intermediate or high-risk MF (including apMF, post-PV
MF, and post-ET MF) has validated this target.11 As a pan-Jak
inhibitor, ruxolitinib has equivalent activity against Jak1 and
Jak2, with decreased activity against Jak3 and Tyk2.12 To
potentially increase the safety index of a Jak2 inhibitor, it would
be advantageous to identify a Jak2-selective inhibitor in light of
the anticipated chronic dosing required for MPD treatment and
the potential for immunosuppressive side effects related to
inhibition of Jak1, Jak3, or Tyk2.13,14 While the degree of
selectivity for Jak2 over Jak1, Jak3, and Tyk2 necessary for
therapeutic benefit is unclear, a 10-fold window would provide
a minimum degree of differentiation. We therefore initiated a
program to identify potent inhibitors of Jak2 with at least 10-
fold selectivity against all other Jak family members.
INTRODUCTION
■
The Janus kinase (Jak) family consists of four nonreceptor
tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) that play
important roles in hematopoiesis and immune response.1
Upon activation through cytokine signaling, Jaks subsequently
activate the downstream signal transducer and activator of
transcription (STAT) family of transcription factors, which
regulate target gene expression that govern cell proliferation
and survival.2 The identification of a single gain of function
point mutation in the JH2 pseudokinase autoinhibitory domain
of Jak2 (Jak2V617F), which renders the kinase constitutively
active, has led to interest in Jak2 as a potential therapeutic
target.3
Philadelphia (Ph)-negative myeloproliferative disorders
(MPDs) are a group of heterogeneous hematologic diseases
resulting from deregulated proliferation of differentiated
myeloid cells and include the disorders polycythemia vera
(PV), essential thrombocytemia (ET), and primary myelofib-
rosis (PMF).4 Gain of function mutations in Jak2 are frequently
observed in these disorders; the Jak2 mutation has been
identified in 77% of patients with PV, in 35% of patients with
ET, and 43% of patients with PMF.5 In preclinical cellular
models of PV, Jak2V617F expression is necessary and sufficient to
drive cytokine-independent activation of STAT1 or STAT5 and
subsequently regulates cell proliferation and survival. Therefore,
the identification of the Jak2V617F somatic mutation provides a
strong and compelling rationale to develop a targeted therapy
for Ph-negative MPDs.
The pyrazolo[1,5-a]pyrimidine scaffold was identified
through high-throughput screening to have inhibitory activity
against Jak2, and after initial medicinal chemistry exploration,
compounds 7a−b and 8 were identified as lead compounds
with good inhibitory activity against Jak2 (Table 1). These
compounds are close to the desired 10× selectivity against Jak1,
with even better selectivity against the other Jak family
Received: August 22, 2012
Published: October 12, 2012
© 2012 American Chemical Society
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the liabilities of 7a and 8 and to identify a Jak2 inhibitor with an
in vitro and in vivo profile suitable for the effective oral
treatment of MPDs. The in vitro parameters measured for
inhibitors synthesized as part of this lead-optimization
campaign are provided in Tables 2−4, with in vivo
pharmacokinetic parameters detailed in Table 5. This work
culminated in the discovery and evaluation of 7j as a potent and
selective orally active inhibitor of Jak2.
CHEMISTRY
■
The general synthetic procedure for 5-aminopyrazolo[1,5-
a]pyrimidine-3-carboxamides 7a−k is described in Scheme 1.
3-Amino-pyrazole and 1,3-dimethyluracil were heated together
with sodium ethoxide in ethanol to produce the cyclized
pyrazolo[1,5-a]pyrimidine intermediate compound 1. After
neutralization with acetic acid, iodination with N-iodosuccini-
mide provided 3. Palladium-catalyzed carbonylation and
subsequent saponification yielded intermediate 4. Alternatively,
4 could be generated in a one-step condensation of 5-amino-
1H-pyrazole-4-carboxylic acid with 1,3-dimethyluracil.18 Bis-
chlorination of 4 with POCl3 generated the key intermediate 5,
which was derivatized sequentially through amide formation
followed by an SNAr reaction with ammonia or methylamine to
yield compounds 7a−k.
5-H-Pyrazolo[1,5-a]pyrimidine-3-carboxamides 8, 10a−c,
12, 13a−g, and 14a−j were prepared in one or two steps
from pyrazolo[1,5-a]pyrimidine-3-carboxylic acid as described
in Scheme 2. Pyridine containing compounds 10a−c were
prepared as described in Schemes 2 and 3. 5-Bromo-2-
methylpyridine was oxidized with mCPBA to generate the N-
oxide 15, which was nitrated to provide compound 16.
Pyridine-N-oxide 16 was then reduced with SnCl2 to yield 5-
bromo-2-methylpyridin-4-amine 17. The acid chloride of
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was generated
using oxalyl chloride and then used to acylate each of 2-
bromo-pyridin-3-amine, 3-bromo-pyridine-4-amine, or 17 to
provide bromo-pyridines 9a−c. Suzuki coupling with 3-
chlorophenyl boronic acid yielded compounds 10a−c.19
Regioisomeric pyrazole compound 12 was prepared in two
steps. Pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was reacted
with 4-bromo-1-methyl-1H-pyrazol-3-amine under standard
HATU-mediated amide coupling conditions, followed by
coupling with 3-chlorophenyl boronic acid under Suzuki
conditions (Scheme 2).
Figure 1. Examples of Jak2 inhibitors.
Table 1. Early Lead Inhibitors
3-Aryl-4-aminopyrazole intermediates used for the synthesis
of compounds 13 and 14 were prepared as described in
Schemes 4−6. In Scheme 4, various aryl-substituted α-
bromoacetophenones were treated with potassium phthalimide
or di-tert-butyl-iminodicarboxylate to generate α-amino-ketones
18a−d. Treatment of these ketones with 1,1-dimethoxy-N,N-
dimethylmethanamine (DMFDMA) at elevated temperatures
provided intermediates 19a−d, which were cyclized with either
hydrazine or methylhydrazine to yield 1-H-4-aminopyrazoles or
1-methyl-1H-4-aminopyrazoles, respectively (20a−e).20
As an alternate preparation, as described in Scheme 5, acid
chlorides or benzoic acids activated by CDI were treated with
potassium ethyl malonate and MgCl2 to provide β-keto-esters
21a−b.21 Reaction with DMFDMA gave 22a−b. 22a was
cyclized with hydrazine to provide pyrazole 23, which was
alkylated with iodomethane to yield a regioisomeric mixture of
pyrazoles 24a and 25. Carrying forward the regioisomeric
mixture, saponification of the ester followed by Curtius
rearrangement using Shioiri’s conditions22 to the tert-
NH2
NHCH3
H
7a
7b
8
Jak2 Ki (nM)
2.5
5.1
0.43
6.5
11.9
a
LE
0.46
4.5
0.44
5.0
b
LELP
c
Jak1/2
8.6
7.8
9.4
c
Jak3/2
30.4
32.4
131
>5
31.3
24.4
259
>10
58.5
24.6
1100
4.6 (3A4)
c
Tyk2/Jak2
d
pSTAT5 IC50 (nM)
e
CYP450 inhibition IC50 (μM)
Ligand efficiency15 (LE) calculated as ΔG (in kcal/mol) divided by
a
the number of heavy atoms. LELP16 = cLogP17/LE. fold biochemical
b
c
d
selectivity for Jak2 over other Jak family kinase. pSTAT5 SET2 MSD
format. CYP isoforms tested: 1A2, 2C9, 2C19, 2D6, 3A4; IC50 is
given for most potently inhibited isoform.
e
members Jak3 and Tyk2. With these compounds as a starting
point, we undertook a lead-optimization program to address
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a
Scheme 1. General Synthetic Procedure for 5-Amino-pyrazolo[1,5-a]pyrimidine-3-carboxamides
a
Reaction conditions: (a) 21% EtONa, EtOH, 95%; (b) HOAc, H2O, 85%; (c) N-iodosuccinimide, DMF, 86%; (d) CO (1 atm), Pd(OAc)2, Et3N,
MeOH; (e) 5% aq LiOH, 68% (2 steps); (f) POCl3, DIPEA, 87%; (g) H2NR, DIPEA, DCM, 52−92%; (h) NH3, iPrOH, DIPEA, μW 120 °C, 15
min, 9−80%; (i) H2NCH3, H2O, μW 100 °C, 15 min, 62%.
a
Scheme 2. General Synthetic Procedure for Compounds 8−14
a
Reaction conditions: (a) RNH2, HATU or PyAOP, DIPEA, DMAP, 20−90%; (b) (COCl)2, DCM; (c) 2-bromopyridin-3-amine or 3-
bromopyridin-4-amine or 17, Et3N, DCM, 21−56%; (d) 3-chlorophenylboronic acid, Pd(PPh3)2Cl2, Na2CO3, CH3CN, H2O, μW, 120 °C, 42−59%;
(e) 3-chlorophenylboronic acid, Pd(PPh3)4, Na2CO3, diglyme, 100 °C, 7%; (f) R3-X, Cs2CO3, DMF, 5−61%; (g) subsituted oxirane, Cs2CO3, DMF,
46%.
a
Scheme 3. Synthesis of Aminopyridine 17
Scheme 4. 2-Bromo-1-phenylethanone-Based Synthesis of 1-
Methyl-3-phenyl-1H-pyrazol-4-amines
a
a
a
Reaction conditions: (a) potassium phthalimide, 88%; (b) HN-
Reaction conditions: (a) mCPBA, DCM, 99%; (b) HNO3, H2SO4,
(Boc)2, NaH, 34−77%; (c) DMFDMA, 38−80%; (d) methylhy-
drazine, EtOH, 38−53%; (e) hydrazine, EtOH, 80%.
90%; (c) SnCl2, HCl, 56%.
butylcarbamate and finally acid catalyzed Boc-deprotection
yielded amino-pyrazoles 26a and 27, which are easily separated
via silica gel chromatography.23 Intermediate 22b was reacted
with N-methylhydrazine to give the desired regiosiomer 24b
directly, which was carried forward in the same manner to
provide aminopyrazole 26b.
The preferred construction of the 3-aryl-4-amino-pyrazole
intermediate used for compound 14j is described in Scheme 6.
The SEM-protected 4-nitropyrazole 28 was subjected to
palladium-catalyzed direct arylation conditions24 with 2-
bromo-4-chloro-1-methoxybenzene. The biaryl product 29
was then reduced with iron in the presence of ammonium
chloride to yield SEM-protected 4-aminopyrazole 30. Inter-
mediate 30 was carried forward as described in Scheme 2, with
SEM deprotection effected with aqueous HCl in ethanol, to
provide the final compound 14j.
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a
Scheme 5. Benzoic Acid Based Synthesis of 3-(5-Chloro-2-methylphenyl)-1-methyl-1H-pyrazol-4-amine
a
Reaction conditions: (a) potassium ethyl malonate, CDI, MgCl2, THF, 50 °C; (b) potassium ethyl malonate, Et3N, MgCl2, CH3CN; (c)
DMFDMA, 34−70% (2 steps); (d) hydrazine, EtOH, 70 °C, quant; (e) CH3I, Cs2CO3, DMF, 40 °C, 81%; (f) methylhydrazine, AcOH, 45%; (g)
NaOH, EtOH/H2O; (h) (PhO)2P(O)N3, Et3N, 1,4-dioxane, tBuOH; (i) HCl, 1,4-dioxane, 37−46% (3 steps).
a
a
Scheme 6. 4-Nitropyrazole CH-arylation Synthesis of 30
Scheme 7. Synthesis of Aminoisoxazole 35
a
Reaction conditions: (a) SEM-Cl, NaH, THF, 99%; (b) 2-bromo-4-
chloro-1-methoxybenzene, Pd(OAc)2, (Ad)2BuP, K2CO3, tBuCO2H,
DMF, 120 °C, 89%; (c) Fe, NH4Cl, EtOH, H2O, 70 °C, quant.
a
The regioisomeric aminoisoxazole intermediates used for the
preparation of compounds 7e and 7f were synthesized as
described in Schemes 7 and 8. Acylation of 1-(3-chlorophenyl)-
ethanone provided β-keto-ester 31, which was reacted with
DMFDMA and then cyclized to the isoxazole 32 using
hydroxylamine. Hydrolysis of the ester followed by Curtius
rearrangement yielded Boc-protected amine 34, which was
deprotected with HCl to give 5-(3-chlorophenyl)isoxazol-4-
amine 35. Compound 35 was carried forward as described in
Scheme 1 to yield compound 7e. As shown in Scheme 8, 3-
chlorobenzaldehyde was condensed with hydroxylamine to give
oxime 36. Chlorination of 36 with N-chlorosuccinimide
provided α-chlorobenzaldoxime 37. After treatment with
triethylamine, 37 underwent [3 + 2] dipolar cycloaddition
with 3-pyrrolidin-1-yl-acrylic acid ethyl ester 38 via the in situ
generated benzonitrile oxide to afford isoxazole 39. Acid
catalyzed hydrolysis of the ester followed by Curtius rearrange-
ment to the tert-butylcarbamate 41 and subsequent Boc-
deprotection yielded 3-(3-chlorophenyl)isoxazol-4-amine 42,
which was carried forward as described in Scheme 1 to provide
compound 7f.
Reaction conditions: (a) diethyl carbonate, NaH, toluene, rt to reflux,
51%; (b) DMFDMA, DMF, reflux; (c) NH2OH-HCl, MeOH, reflux,
79% (2 steps); (d) HCl, AcOH, reflux; (e) SOCl2, reflux, NaN3,
acetone, 0 °C to rt, tBuOH, 85 °C, 55% (2 steps); (f) 4 M HCl in 1,4-
dioxane, 40 °C, quant.
yielded aminothiazole 43. Compound 43 was carried forward as
described in Scheme 1 to provide compound 7g.
RESULTS AND DISCUSSION
■
An HTS campaign identified the pyrazolo[1,5-a]pyrimidine 7a
as a compelling lead molecule for our selective Jak2 inhibitor
program. Compound 7a is a low nanomolar inhibitor of Jak2
(Ki = 2.5 nM) with high ligand efficiency15 (LE = 0.46), low
ligand-efficiency-dependent lipophilicity16 (LELP = 4.5), and
has potent inhibitory activity (IC50 = 131 nM) in a Jak2-driven
SET2 cell-based assay as measured by the inhibition of
pSTAT5, a downstream target of Jak2 (Table 1). Compound
7a has low potential for reversible inhibition of the 5 major
human CYP450 isoforms and good in vitro permeability.
Compound 7a has moderate selectivity (∼9−30×) against the
other Jak family members and excellent selectivity across a
panel of 177 kinases.25 Some of the liabilities of compound 7a
include low microsomal stability across five species and poor
thermodynamic aqueous solubility (Table 5). The pharmaco-
The amino-thiazole intermediate used for compound 7g was
prepared as described in Scheme 9. Heating commercially
available α-amino-(3-chlorophenyl)acetonitrile hydrochloride
in the presence of elemental sulfur and acetaldehyde directly
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a
Scheme 8. Synthesis of Aminoisoxazole 42
a
Reaction conditions: (a) NH2OH-HCl, pyridine, EtOH, quant; (b) NCS, DMF, quant; (c) ethyl propiolate, toluene, 94%; (d) Et3N, Et2O, 57%;
(e) HCl, AcOH, reflux, 99%; (f) (PhO)2P(O)N3, Et3N, tBuOH, 85 °C, 77%; (g) 4 M HCl in 1,4-dioxane, 97%.
a
However, compound 7b, in which the 2-NH2 group is replaced
with a 2-NHCH3 group, was only 2-fold less potent in the Jak2
enzyme assay (Ki = 5.1 nM, Table 1). Compound 7b
unfortunately has high clearance in rats, which is consistent
with the ∼4× higher free fraction in rat plasma (1.7% vs 0.4%)
and higher predicted clearance in rat microsomes (48 vs 39
mL/min/kg) as compared to compound 7a. Interestingly,
compound 7b has a significantly higher oral bioavailability
compared to compound 7a (Foral (%) = 30 vs 1, Table 5)
despite the higher plasma clearance. The improved oral
exposure may be a result of the greater kinetic aqueous
solubility of compound 7b.
Scheme 9. Synthesis of Aminothiazole 43
a
Reaction conditions: (a) powdered sulfur, EtOH, 0 °C; (b)
acetaldehyde, Et3N, EtOH, 60 °C, 73% (2 steps).
kinetic profile of compound 7a was determined for rats and
mice26 in order to determine how these apparent liabilities
manifested in vivo (Table 5).
Compound 7a has lower than expected plasma clearances in
both mice and rats (3 and 6.8 mL/min/kg, respectively)
because for both species microsomes predicted moderate/high
clearance (Clpred = 60 and 39 mL/min/kg, respectively). The
very low Vd in both species (Vdss = 0.27 and 0.19 L/kg,
respectively) and lower than predicted plasma clearances are
consistent with high plasma protein binding in both species
(98.7% and 99.6%, respectively). Unfortunately, compound 7a
has poor oral bioavailability in mice and rats (Foral (%) = 8.6
and 1, respectively). The limited oral exposure may be a result
of poor aqueous solubility because the compound has good
permeability. Moreover, the first pass effect should be minimal
because the plasma clearance is low (Table 5).
An X-ray structure of 8 with Jak2 was solved (Figure 2),
revealing a binding mode consistent with observed SAR. The
We surmised that the poor solubility of 7a was due to high
crystal packing forces as a consequence of the multiple aromatic
rings27 (four) and multiple potential hydrogen bond donors
(HBDs) and acceptors (HBAs). Poor aqueous solubility is also
often associated with high lipophilicity; however, because the
cLogP17 of compound 7a is in a reasonable range (2.1),
lipophilicity may not be the key driver for the poor aqueous
solubility. The rat pharmacokinetic profile of the 2-des-amino
compound 8 and the 2-methylamino compound 7b were
obtained to probe the effect of removing HBDs on oral
bioavailability (Table 5). The rat plasma clearance and Vd of
compound 8 are low as was observed with compound 7a;
however, a much-improved oral exposure was observed (Foral
(%) = 44 vs 1). The improved oral exposure may be a result of
the greater kinetic aqueous solubility of compound 8 vs 7a
because their thermodynamic solubilities, are comparable and
both compounds have similar permeability in MDCK cells.
Unfortunately, compound 8 is ∼10-fold less potent than
compound 7a against Jak2 in both the enzyme and cell assays.
Figure 2. Co-crystal structure (2.3 Å) of compound 8 in the active site
of the Jak2 kinase domain with the P-loop removed to allow a better
view of the key active site interactions. Dashed lines indicate close
contacts between ligand and protein with distances labeled in Å.
N1-nitrogen of the pyrazolopyrimidine core makes a hydrogen
bond with the backbone NH of Leu932. A possible weak
nonclassical hydrogen-bond between the C−H moiety at C-7
of the pyrazolopyrimidine core and the backbone of carbonyl
Glu930 is interesting; however, the distance of this putative
hydrogen bond is a bit long (3.4 Å).28 Both the amide carbonyl
and the pyrazole N2-nitrogen of 8 form hydrogen bonds to
waters, and the terminal phenyl ring of 8 occupies the
hydrophobic sugar region of the ATP binding site.
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Figure 3. Potential reactive metabolites of the 3-methyl-N-aryl-methylpyrazole scaffold.
While a high degree of sequence homology exists between
Jak1 and Jak2, several differences are observed in amino acids at
the periphery of the binding site. The residue Asp939 in Jak2,
which is in close proximity to the pyrazole methyl substituent,
is Glu966 in Jak1. Other residue differences with the potential
to interact with bound ATP-competitive ligands are Jak2
Tyr931→Jak1 Phe958 and Jak2 Gln853→Jak1 Arg879.29 As
these amino acids are somewhat removed from the space
occupied by 8 and other compounds in this series, improve-
ment of selectivity for Jak2 over Jak1 remained a challenging
task, with the best potential for Jak1/Jak2 differentiation offered
by positive interactions with Asp939.
In addition to the poor solubility and poor metabolic stability
in human liver microsomes, we were also concerned about two
potential reactive metabolites30 of the 3-methyl-N-arylpyrazole
core of the lead series as shown in Figure 3. The N-linked
pyrazole moiety could potentially be oxidized to the para-
quinoneimine (pathway A). Furthermore, the electron-rich
nature of the aryl ring of the N-arylpyrazole moiety may
contribute to the poor microsomal stability. The other potential
reactive metabolite is envisioned to form through a two-step
process in which the methyl group is oxidized to the alcohol,
which subsequently eliminates to give the potentially highly
reactive pyrazoleiminium species (Figure 3, pathway B and C).
Therefore, in parallel with the efforts exploring the SAR of
compound 7a, a significant effort was expended to explore
replacements for the N-arylpyrazole group.
nitrogen of 13a can form an H-bond to a water molecule
consistently observed within the binding site, whereas
regioisomer 12 is lacking a HBA at this position. Interestingly,
13a is >2× more potent in both the Jak2 kinase and cell assays
than 8 while maintaining moderate selectivity within the Jak
family. The increase in potency may be due to an increase in
the polarization of the C−H bonds of the N-methyl moiety of
compound 13a relative to the C-methyl group in compound 8.
As shown in Figure 2, a cocrystal structure of compound 8 with
the Jak2 kinase domain indicated a potential charge-partial
charge interaction (3.3 Å) of the carbonyl of Asp939 and the
pyrazole methyl hydrogens. Presumably, this weak interaction
could be enhanced with the increased polarization of the
methyl protons in a pyrazole N-methyl group as compared to a
C-linked methyl group. Although the methyl group of
compound 8 is more acidic (calculated pKa32 40 vs 43) due
to resonance stabilization of the resulting anion into the
pyrazole ring, the partial positive charge on the methyl protons
are greater in compound 13a due to greater polarization of the
N-methyl bond. Compound 13a has a similar rat plasma
clearance to its regioisomer 8 (12.5 vs 13.3 mL/min/kg; Table
5). Interestingly, 13a has high oral bioavailability despite poor
kinetic and thermodynamic solubilities (both ∼1 μM). A
possible explanation for the observed equivalence in kinetic and
thermodynamic solubilities is that compound 13a precipitated
as a crystalline form during the kinetic solubility study.
To probe the putative pyrazole N-methyl effect further, 7c
was prepared in which the 5-H of the pyrazolopyrimidine ring
was replaced with 5-NH2 in an effort to improve Jak2 potency
relative to 13a. Gratifyingly, 7c also has a >2-fold improvement
in potencies in both in the Jak2 enzyme and cell assays as
compared to its regioisomer 7a (Table 2). Moreover, the
selectivity against Jak1 is slightly increased. Unlike its
regioisomer 7a, the rat in vivo clearance of 7c was well
predicted by rat liver microsomes (Table 5). Although an
improvement in oral bioavailability was observed with 7c
relative to 7a, the oral bioavailability is still poor (7%).
Assuming the observed plasma clearance is mainly hepatic-
driven, the expected oral bioavailability is ∼60% (based on a
hepatic extraction ratio of 0.40 and rat liver blood flow of 57
mL/min/kg) for a compound that is completely absorbed
intact from the GI tract. As was observed with compound 7a,
compound 7c has poor kinetic and thermodynamic aqueous
solubility, which could explain the significantly lower than
expected oral exposure (Table 5).
The pyridine and regioisomeric pyrazole core replacements
that were prepared are shown in Table 2. 4-Pyridyl 10b is
marginally more active than 3-pyridyl 10a. Not surprisingly, 4-
pyridyl 10b exhibited potent CYP3A4 inhibition (IC50 = 0.2
μM), which is likely related to the heme-binding ability of a
sterically unhindered pyridine nitrogen. Indeed, introducing a
methyl substituent ortho to the pyridine nitrogen (compound
10c) decreased CYP3A4 inhibition.31 4-Pyridyl compounds,
10b and 10c, have Jak family enzyme potencies similar to
compound 8.
Compound 13a is the more potent of the regioisomeric
pyrazoles (12 and 13a) that were prepared. The >30-fold
decrease in the potency of 12 vs Jak2 as compared to 13a may
be ascribed to an increase in energy required to adopt a
coplanar conformation between the amide and pyrazole groups
due to lone pair repulsion between the N2 nitrogen of the
pyrazole and one of the lone pairs of the amide oxygen. This is
consistent with the binding mode for this scaffold observed in
X-ray structures (e.g., Figure 2). Moreover, the pyrazole ring
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Table 2. Pyrazole Replacement SAR
a
b
c
Fold biochemical selectivity for Jak2 over other Jak family kinase. pSTAT5 SET2 MSD format. CYP isoforms tested: 1A2, 2C9, 2C19, 2D6, 3A4;
d)
IC50 is given for most potently inhibited isoform; N.D.: not determined.
Two common strategies to improve stability toward CYP-
mediated metabolism are to increase polarity and to remove
and/or block metabolic hotspots such as N-methyl groups;33
therefore, several compounds that lacked the N-methyl group
were prepared. 1-H-Pyrazoles 7d and 13b have very similar
Jak2 enzyme potencies relative to their corresponding N-
methyl-1H-pyrazole analogs while preserving selectivity versus
the other Jak family members (Table 2). Interestingly,
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Table 3. Pyrazole N-Substitutions
a
Fold biochemical selectivity for Jak2 over other Jak family kinase.
compound 7d is ∼7× less potent than N-methyl analog 7c in
the Jak2 cell assay. Unfortunately, removing the N-methyl
moiety coupled with an increase in polarity did not lead to
improved human liver microsome (HLM) stability. Because the
pyrazole core may be the key metabolic liability in this series,
two isomeric isoxazoles (7e−f) and a methylthiazole (7g) were
prepared. All three compounds have a heteroatom (O or N)
ortho to the phenyl ring with a potential to form an H-bond
with an active site water molecule. Isoxazole 7f has comparable
Jak2 kinase potency and Jak family selectivity to compounds
7c−d. Interestingly, isoxazole 7e is less potent against all Jak
family members than its regioisomer 7f. This may be a result of
the poor H-bond accepting character of oxygen atoms in
heteroaromatic ring systems, whereas sp2 nitrogens of
heteroaromatics tend to be good H-bond acceptors.34 While
the Jak2 activity of 7f compared favorably with 13a, it
unfortunately potently inhibited CYP1A2 (IC50 = 1.2 μM).
Finally, because 2-H thiazoles are generally potent CYP
inhibitors,35 we only prepared the 2-methyl-thiazole 7g to
mitigate this known risk. This particular regioisomeric thiazole
was chosen because it was presumed that an attractive
intramolecular thiocarbonyl interaction would stabilize the
requisite coplanar geometry between the amide and five-
membered heteroaryl required for potent Jak2 activity.36
Compound 7g was determined to be equipotent with
compound 7c in the Jak2 enzyme assay and slightly more
selective vs the other Jak family members. It is, however, less
potent in the Jak2 cell assay (Ki = 176 nM vs 60.4 nM).
Moreover, compound 7g is a moderate inhibitor of CYP3A4
(IC50 = 6.7 μM), whereas compound 7c is inactive against all 5
CYP isoforms tested.
As part of our efforts to improve the aqueous solubility and
metabolic stability of our lead series, various branched and
hydrophilic substituents were prepared off the pyrazole
nitrogen because this vector points toward bulk solvent
(Table 3). These substituents are also in the region of the
Jak2 Asp939→Jak1 Glu966 amino acid difference with the
potential to impact selectivity over Jak1. The isopropyl-
substituted compound 14a was designed to increase the
percentage of sp3 centers, which has been correlated in the
literature with improved solubility.37 Moreover, by replacing
the N-methyl group with an N-isopropyl, it was hoped that the
metabolic stability might improve. Gratifyingly, 14a is indeed
more soluble than 13a (44 μM vs 1 μM kinetic aqueous
solubility) and more stable in HLMs; however, 14a has poor
Jak2 potency and selectivity vs Jak1 is eroded. The traditional
method to improve aqueous solubility is to add hydrophilic
groups such as hydroxyls or groups that are charged at
physiological pH. Furthermore, increasing polarity often leads
to an increase in metabolic stability toward CYP-mediated
metabolism. The introduction of a single hydroxyl functional
group (14b, 14e, and 14f) improved kinetic solubility
significantly vs 13a although only the tert-butyl alcohol analog
14f showed a significant improvement in HLM stability (CLpred
= 7.8 vs 18 mL/min/kg). The greatest improvement in kinetic
aqueous solubility was seen with the bis-hydroxy compounds
14c and 14d, both of which have measured kinetic aqueous
solubilities >200 μM at pH 7.4. Moreover, both of these
analogs are also more stable in HLMs than 13a, although to a
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Table 4. Phenyl Bis-substitution SAR
kinetic
Jak2 Ki
(nM)
Jak
Jak
pSTAT5 IC50
HLM, RLM Clpred
(mL/min/kg)
solubility
a
a
a
b
compd
R1
R2
R3
1/2
3/2
Tyk2/Jak2
(nM)
pH 7.4 (μM)
14h
14i
13e
14j
13g
7h
H
CH3
Cl
CH3
Cl
2.1
0.4
0.6
0.3
5.2
1.6
0.3
0.1
0.1
5.0
17.0
12.6
19.4
25
12.0
14.2
7.4
64.2
42.3
34.5
22.4
242
68.6
40.4
7.4
14, 42
12, 11
17, 38
13, 25
7, 11
9
6
H
6.9
4.4
H
CH3
OCH3
Cl
1
H
Cl
1.7
5.1
82
5
H
2,6-Cl
H
10.4
6.0
2.1
39.6
14.6
24.5
43.7
20.1
NH2
NH2
NH2
NH2
CH3
CH3
Cl
3.2
11, 22
16, 36
14, 31
15, 21
7
7i
CH3
Cl
9.7
9.8
6
7j
14.8
9.2
9.7
4
7k
CH3
Cl
13.1
9.8
7
a
b
Fold biochemical selectivity for Jak2 over other Jak family kinase. pSTAT5 TF1+EPO MSD format.38
a
Table 5. Physicochemical/Pharmacokinetic Parameters
7a
7b
<2.0
7c
7j
<2.0
8
13a
solubility, pH 6.5 (μM)
kinetic solubility, pH 7.4 (μM)
%PPB (human, rat)
MDCK A-B 10E−6 cm/sec (B-A/A-B)
HLMCL, RLMCL (mL/min/kg)
CLp (iv, mL/min/kg)
Vdss (iv, L/kg)
2.7
5.4
44
5.7
<2.0
7
121
4
>200
1
99.5, 99.6
8.4 (1.3)
15, 39
6.8
98.4, 98.3
15.1 (0.9)
17, 48
51.3
98.9, 99.7
98.5, 98.5
10.5 (0.7)
14, 31
12.6
95.8, 99.3
10.5 (0.5)
18, 36
13.3
99.4, 99.7
ND
b
ND
15, 23
23.1
0.355
0.22
7
18, 36
12.5
0.185
0.38
0.843
0.24
1.08
0.293
0.36
0.349
0.23
T1/2 (iv, hr)
1.20
Foral (%)
1
30
63
44
124
a
b
Male Sprague−Dawley rats, 0.1−1 mg/kg iv (solution in 60% aqueous PEG 400); 5 mg/kg PO (suspension in 60% aqueous PEG 400). ND = low
recovery, permeability not calculated.
lesser extent than 14f. Interestingly, the morpholino-containing
compound 14g has very poor kinetic aqueous solubility (<1
μM at pH = 7.4) and is highly unstable in HLMs. The poor
solubility of 14g may be due to the possibility that the
morpholine group of 14g is only partially protonated at pH =
7.4 (calculated pKa = 7.06). Because all but one of these analogs
are significantly less potent vs Jak2 than 13a, coupled with the
erosion of selectivity vs Jak1 with several of the compounds
(14a, 14f and 14g), this subseries was not progressed further.
Only the N-ethanol analog 14b maintained good Jak2 potency
and moderate selectivity vs Jak1; however, it has poor stability
in HLMs. It is not clear as to why Jak2 activity dropped off
significantly with most of these analogs given that these
substituents seemingly point toward solvent. One possibility is
that the side chain of the solvent-exposed Asp939 is less flexible
than presumed, leading to steric clashes with groups larger than
methyl substituted off the pyrazole.
We then focused efforts on optimizing the substituents of the
phenyl ring. Parallel work (not shown) on the regioisomeric N-
arylpyrazole series exemplified by compound 7a indicated that
any substitution at the para-position of the N-aryl group
significantly decreases inhibitory potencies for all Jak family
members. This observation is consistent with the binding mode
observed for 8 (Figure 2, Supporting Information Figure S1)
with the para-position of the phenyl ring abutting the back of
the pocket. The preferred substituents in the 3-position are
methyl, chloro, and cyano. Groups larger than chloro are not
well tolerated. The X-ray cocrystal structure of 8 with Jak2
(Figure 2) suggests that there is hydrophobic space at both the
top and bottom of the sugar-binding pocket within the ATP
binding site. This led us to make directed bis-substitutions
around the phenyl ring (Table 4). ortho-Substitution was
designed to enforce a high degree of twist between the phenyl
and pyrazole rings, thereby lowering the ground-state energy of
the active conformation adopted in cocrystal structures, as well
as decreasing the flatness of the compounds, which could
improve solubility. The 2,5-dimethylphenyl compound 14h is
slightly more potent than 13a; however, the 2,5-dichlorophenyl
analog 14i is nearly 10-fold more active with a Ki of 0.4 nM.
The Ki of the 2-methyl-5-chlorophenyl analog 13e is in-
between that of 14h and 14i, which is consistent with the SAR.
The 2,6-dichlorophenyl analog 13g is nearly 10-fold less active
against Jak2 than the corresponding 2,5-dichlorophenyl analog
14i, with similar drops in activity against the other Jak family
members. Compounds 14h−j and 13e spanned a ∼3-fold range
of activity in the Jak2 cell assay, with 2-chloro, 2-methyl, or 2-
methoxy substituents resulting in similar activities. 2,5-Bis-
substitution proved to be critical for optimal activity against
Jak2 because the 2-monosubstituted analog 7h and the 2,6-
dichloro bis-substituted analog 13g are 5−10× less potent than
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Aldrich and used directly. All reactions involving air- or moisture-
sensitive reagents were performed under a nitrogen atmosphere.
Unless otherwise stated, operations were carried out at ambient
temperature (in the range 18−25 °C). Microwave experiments were
carried out using a Biotage Initiator 60, which uses a single-mode
resonator and dynamic field tuning. Silica gel chromatography was
performed using medium-pressure liquid chromatography on a
CombiFlash Companion (Teledyne Isco) with RediSep normal-
phase silica gel columns. All final compounds were purified to ≥95% as
determined by an Agilent 1100 series HPLC using an Agilent
ZORBAX SB-C18 30 mm × 2.1 mm column, eluting with a binary
solvent system A and B using a gradient elusion [A, H2O with 0.05%
trifluoroacetic acid (TFA); B, CH3CN with 0.05% TFA] with UV
detection at 254 nm. 1H NMR spectra were recorded on a Bruker 400
or 300 MHz NMR spectrometer using the deuterated solvent stated.
Chemical shifts are reported in ppm from the solvent resonance. NMR
data are reported as follows: chemical shift, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak),
coupling constants, and number of protons. Mass spectral (MS) data
was determined with a Sciex API150ex or Agilent 6140 spectrometer
and ESI source.
their respective 2,5-bis-substituted analogs 7i and 14i.
Interestingly, only analogs 7i−k, where R1 = NH2 in the 2,5-
bis-substituted series are both highly potent vs Jak2 (Ki < 1nM)
and also meet our selectivity requirements vs the other Jak
family members (>10×).
On the basis of its Jak2 activity and selectivity, compound 7j
was dosed in rat pharmacokinetic studies (Table 5).
Compound 7j has good oral bioavailability (Foral (%) = 63)
despite poor kinetic and thermodynamic aqueous solubilities.
The good oral exposure may be a result of the combination of
low plasma clearance and high permeability. The low clearance
was not unexpected given the predicted clearance based on rat
microsomes coupled with high rat plasma protein binding. No
reversible CYP inhibition was observed for the five major
isoforms and only minimal time-dependent inhibition (TDI) of
CYP3A4 (TDI IC50 = 5.8 μM with a 38% shift in AUC).39
Compound 7j is very selective against a panel of 183 kinases;
when tested at 0.01 μM (100× the Jak2 Ki), 7j inhibited only
five kinases outside of the Jak family at >50%.40
The excellent oral exposure of 7j in rats coupled with its
potent Jak2 inhibition in cells prompted us to test whether it
could knockdown Jak2-mediated phosphorylation of STAT5 in
a SCID mouse SET2 xenograft model that is dependent on
Jak2 for growth. At an oral dose of 100 mg/kg, 64% inhibition
of pSTAT5 was observed at the 1 h time point, while the
inhibition of pSTAT5 decreased as the plasma levels of 7j
decreased over time (Figure 4). A more in-depth discussion of
the in vivo profile of 7j has been published elsewhere including
efficacy studies in preclinical PV and AML models.41
Sodium Pyrazolo[1,5-a]pyrimidin-5-olate (1). A mechanically
stirred mixture of 3-aminopyrazole (9.38 g, 113 mmol), 1,3-
dimethyluracil (14.7 g, 105 mmol), and 21% sodium ethoxide in
ethanol (170 mL) was heated to reflux for one h. The reaction mixture
was cooled, filtered, and the collected solid washed with cold ethanol
and vacuum-dried to yield 13.47 g (95%) of sodium pyrazolo[1,5-
1
a]pyrimidin-5-olate. H NMR (400 MHz, DMSO-d6) δ: 8.0 (d, 1H),
7.43 (d, 1H), 5.65 (d, 1H), 5.37 (d, 1H). MS: m/z 136.0 ([M + H]+).
Pyrazolo[1,5-a]pyrimidin-5(4H)-one (2). To 1 (13.47 g, 85.74
mmol) was added 500 mL of acetic acid and 100 mL of water. The
mixture was stirred at ambient temperature for 15 min and then
evaporated to dryness under vacuum. The residue was suspended in
300 mL of 9:1 dichloromethane (DCM):methanol and vacuum
filtered through silica gel to remove sodium acetate. The silica gel pad
was washed with 700 mL of additional 9:1 DCM:methanol solution.
The combined DCM:methanol solution was concentrated to dryness.
The solid product was suspended in 1:1 hexane:DCM and filtered to
1
yield 9.9 g (85%) of pyrazolo[1,5-a]pyrimidin-5(4H)-one. H NMR
(400 MHz, DMSO-d6) δ: 12.1 (br s, 1H), 8.47 (d, J = 7.9 Hz, 1H),
7.75 (d, J = 1.9 Hz, 1H), 5.94 (d, J = 7.5 Hz, 1H), 5.81 (d, J = 1.7 Hz,
1H). MS: m/z 136.0 ([M + H]+).
3-Iodopyrazolo[1,5-a]pyrimidin-5(4H)-one (3). A mixture of 2 (1.0
g, 7.4 mmol) and N-iodosuccinimide (1.67 g, 7.42 mmol) was
combined with 20 mL of DMF and warmed slightly until a heavy
precipitate formed. The reaction mixture was stirred an additional 1 h
at ambient temperature and then cooled on an ice−water bath and
filtered. The collected solid was washed with 20 mL of DCM and air-
dried to give 1.66 g (86%) of 3-iodopyrazolo[1,5-a]pyrimidin-5(4H)-
Figure 4. PK/PD study of 7j at an oral dose of 100 mg/kg in a SET2
xenograft model.
1
one. H NMR (400 MHz, DMSO-d6) δ: 12.1 (s, 1H), 8.5 (d, J = 7.6
Hz, 1H), 7.84 (s, 1H), 6.01 (d, J = 7.1 Hz, 1H). MS: m/z 262.2 ([M +
CONCLUSIONS
H]+).
■
In conclusion, a series of highly potent and selective novel
pyrazolo[1,5-a]pyrimidine-based Jak2 inhibitors has been
discovered. Optimization of lead compounds 7a−b and 8 for
potency and pharmacokinetic properties led to the discovery of
7j, a potent Jak2 inhibitor (Ki = 0.10 nM) with high LE (0.49)
and low LELP (5.9). Compound 7j displays an adequate
combination of cell potency, oral exposure, and in vivo knock-
down of pSTAT5 making it a reasonable tool for proof of
principal Jak2-dependent efficacy studies. Preclinical efficacy
studies of 7j in PV and acute myeloid leukemia (AML) models
have been disclosed elsewhere.41
5-Oxo-4,5-dihydropyrazolo[1,5-a]-pyrimidine-3-carboxylic Acid
(4). A mixture of 3 (1.5 g, 5.8 mmol), palladium(II) acetate (0.27 g,
1.2 mmol), triethylamine (2.3 mL, 17 mmol), and 75 mL of methanol
was stirred, degassed with nitrogen twice under vacuum, and then
blanketed with carbon monoxide under a balloon. The reaction
mixture was heated to 55 °C for 5 h and then filtered through Celite
and concentrated under vacuum. The residue was recrystallized from
water and filtered to give 0.81 g (73%) of methyl 5-oxo-4,5-
dihydropyrazolo[1,5-a]-pyrimidine-3-carboxylate. 1H NMR (400
MHz, DMSO-d6) δ: 11.0 (br s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.19
(s, 1H), 6.19 (d, J = 7.9 Hz, 1H), 3.80 (s, 3H). A solution of methyl 5-
oxo-4,5-dihydropyrazolo[1,5-a]-pyrimidine-3-carboxylate (0.75 g, 3.9
mmol), lithium hydroxide (0.466 g, 19.5 mmol), and 20 mL of water
was stirred at ambient temperature for 3 h. Acetic acid (5 mL) was
added to the reaction mixture, and the precipitated solid was collected
by vacuum filtration to give 0.65 g (93%) of 5-oxo-4,5-
dihydropyrazolo[1,5-a]pyrimidine-3-carboxylic acid. 1H NMR (400
EXPERIMENTAL SECTION
General Methods. Unless otherwise noted, all solvents and
reagents were obtained from commercial suppliers and used without
further purification. Anhydrous solvents were obtained from EMD or
■
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MHz, DMSO-d6) δ: 8.54 (d, J = 9.2 Hz, 1H), 8.06 (s, 1H), 6.11 (d, J =
7.2 Hz, 1H).
under vacuum to yield 47.5 mg (80%) of 5-amino-N-(3-(3-
chlorophenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-
carboxamide. H NMR (400 MHz, DMSO-d6) δ: 9.57 (s, 1H), 8.66
(d, J = 7.6 Hz, 1H), 8.20 (s, 1H), 8.07 (s, 1H), 7.78−7.69 (m, 2H),
7.46 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 7.6 Hz,
1H), 3.90 (s, 3H). MS: m/z 368.1 ([M + H]+).
1
As an alternative preparation, to a stirred solution of 5-amino-1H-
pyrazole-4-carboxylic acid (10.0 g, 71.4 mmol) in ethanol (100 mL)
was added sodium ethoxide (17.0 g, 245 mmol) followed by 1,3-
dimethyluracil (11.0 g, 78.6 mmol). The reaction mixture was then
stirred at reflux under an argon atmosphere overnight. The mixture
was poured into ice−water and concentrated HCl added to adjust the
pH to ∼3−4. The mixture was stirred for 2 h, and the resulting
precipitate was collected via filtration and dried under vacuum to yield
10.0 g (58%) of 5-oxo-4,5-dihydropyrazolo[1,5-a]pyrimidine-3-
N-(1-(3-Chlorophenyl)-3-methyl-1H-pyrazol-5-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide (8). To pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (202 mg, 1.24 mmol) was added phosphoryl chloride
(5.35 mL, 57.4 mmol) and then dropwise DIPEA (0.647 mL, 3.72
mmol). The reaction mixture was heated at reflux (130 °C) for 2.5 h.
The solvents were then removed under reduced pressure, exchanging
the solvent with DCM 4 times. The resulting residue was dissolved in
DCM (20 mL), and 1-(3-chlorophenyl)-3-methyl-1H-pyrazol-5-amine
(385.7 mg, 1.857 mmol) and DIPEA (0.431 mL, 2.48 mmol) were
added. The reaction mixture was stirred at ambient temperature for 5
h and then partitioned between ethyl acetate and water. The organic
portion was washed with water and brine, dried over magnesium
sulfate, and concentrated. The crude product was purified by
preparative reverse-phase HPLC and lyophilized to yield 151 mg
(35%) of N-(1-(3-chlorophenyl)-3-methyl-1H-pyrazol-5-yl)pyrazolo-
1
carboxylic acid. H NMR (400 MHz, DMSO-d6) δ: 8.54 (d, J = 9.2
Hz, 1H), 8.06 (s, 1H), 6.11 (d, J = 7.2 Hz, 1H).
5-Chloropyrazolo[1,5-a]pyrimidine-3-carbonyl Chloride (5). A
mixture of 4 (0.371 g, 2.07 mmol), phosphorus oxychloride (20
mL), and DIPEA (1.2 mL, 6.9 mmol) was refluxed for 2 h. The
reaction mixture was cooled and concentrated under reduced pressure.
The residue was taken up in DCM and concentrated again 3 times.
The residue was partitioned between water and DCM, and the DCM
layer was dried with sodium sulfate, vacuum filtered through a bed of
silica gel, and concentrated to give 390 mg (87%) of 5-chloropyrazolo-
[1,5-a]pyrimidine-3-carbonyl chloride as a yellow solid which was used
immediately without further purification. 1H NMR (400 MHz,
CDCl3) δ: 8.72 (d, J = 7.2 Hz, 1H), 8.65 (s, 1H), 7.17 (d, J = 7.6
Hz, 1H).
5-Chloro-N-(1-(3-chlorophenyl)-3-methyl-1H-pyrazol-5-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide (6a). A solution of 5 (150
mg, 0.694 mmol), 1-(3-chlorophenyl)-3-methyl-1H-pyrazol-5-amine
(140 mg, 0.674 mmol), DIPEA (0.18 mL, 1.0 mmol), and 15 mL of
DCM was stirred at ambient temperature for 3 days. The reaction
mixture was partitioned between DCM and water, and the DCM
phase was dried over sodium sulfate and vacuum filtered through a pad
of silica gel. The silica gel pad was washed with 95:5 DCM:methanol.
The combined filtrates were concentrated to give 250 mg (93%, 74%
purity by HPLC) of 5-chloro-N-(1-(3-chlorophenyl)-3-methyl-1H-
pyrazol-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide which was used
without further purification. MS: m/z 387.2 ([M + H]+).
5-Amino-N-(1-(3-chlorophenyl)-3-methyl-1H-pyrazol-5-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide (7a). A mixture of 74%
pure 6a (55.0 mg, 1.05 mmol) and 3 mL of concentrated ammonium
hydroxide was sealed and heated in a microwave reactor at 105 °C for
30 min. The reaction mixture was cooled, and the resulting precipitate
was collected by filtration, rinsed with water, and dried under vacuum
to yield 25.0 mg (48%) of 5-amino-N-(1-(3-chlorophenyl)-3-methyl-
1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was
purified by reverse-phase HPLC and lyophilized. 1H NMR (400 MHz,
DMSO-d6) δ: 9.93 (s, 1H), 8.65 (d, J = 7.5 Hz, 1H), 8.19 (s, 1H),
7.69−7.64 (m, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H),
7.43 (d, J = 8.0 Hz, 1H), 6.42−6.37 (m, 2H), 2.24 (s, 3H). MS: m/z
368.3 ([M + H]+).
N-(1-(3-Chlorophenyl)-3-methyl-1H-pyrazol-5-yl)-5-
(methylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (7b). A
mixture of 6a (172 mg, 0.444 mmol) and a 40 wt % solution of
methylamine in water (3 mL, 4.44 mmol) was sealed and heated in a
microwave reactor at 100 °C for 15 min. The reaction mixture was
cooled, and the resulting precipitate was collected by filtration, rinsed
with water, and dried under vacuum to yield 105.6 mg (62%) of N-(1-
(3-chlorophenyl)-3-methyl-1H-pyrazol-5-yl)-5-(methylamino)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide. 1H NMR (400 MHz,
DMSO-d6) δ: 9.93 (s, 1H), 8.55 (d, J = 7.6 Hz, 1H), 8.22 (s, 1H),
8.02 (d, J = 4.3 Hz, 1H), 7.64 (t, J = 1.8 Hz, 1H), 7.57−7.52 (m, 1H),
7.50 (t, J = 7.8 Hz, 1H), 7.47−7.42 (m, 1H), 6.46 (s, 1H), 6.33 (d, J =
7.6 Hz, 1H), 2.25−2.19 (m, 6H). MS: m/z 382.1 ([M + H]+).
5-Amino-N-(3-(3-chlorophenyl)-1-methyl-1H-pyrazol-4-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide (7c). Ammonia gas was
bubbled through a stirring 0 °C suspension of 6b (60 mg, 0.2 mmol)
and ethanol (3 mL) for 20 min. The reaction vessel was capped and
subjected to microwave irradiation at 120 °C for 90 min. After the
reaction mixture was cooled to ambient temperature, the resulting
precipitate was collected by filtration, rinsed with ethanol, and dried
1
[1,5-a]pyrimidine-3-carboxamide. H NMR (400 MHz, DMSO-d6) δ:
10.32 (s, 1H), 9.36 (dd, J = 7.0, 1.6 Hz, 1H), 8.71 (s, 1H), 8.66 (dd, J
= 4.2, 1.6 Hz, 1H), 7.77−7.76 (m, 1H), 7.66−7.59 (m, 2H), 7.56−7.51
(m, 1H), 7.31 (dd, J = 7.0, 4.3 Hz, 1H), 6.58 (s, 1H), 2.25 (s, 3H).
MS: m/z 353.1 ([M + H]+).
N-(2-Bromopyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxa-
mide (9a). To a suspension of pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid (253.9 mg, 1.556 mmol) in DCM (10 mL) was added DMF (0.1
mL) followed by oxalyl chloride (2.0 M in DCM, 1.0 mL, 2.0 mmol).
The reaction mixture was stirred at ambient temperature for 30 min,
and then additional oxalyl chloride (0.8 mL, 1.6 mmol) was added.
After a further 30 min, the reaction mixture was concentrated. The
resulting residue was resuspended in DCM (10 mL), and 2-
bromopyridin-3-amine (215.4 mg, 1.245 mmol) and triethylamine
(0.8 mL, 6 mmol) were added. The reaction mixture was stirred at
ambient temperature for 1.5 h and then concentrated onto silica gel.
The crude product was purified via flash chromatography on silica gel
(gradient elution: 20−80% ethyl acetate in DCM) to yield 151.3 mg
(38%) of N-(2-bromopyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-car-
boxamide. MS: m/z 317.9 ([M + H]+).
N-(2-(3-Chlorophenyl)pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (10a). A mixture of 9a (76.2 mg, 0.240 mmol), 3-
chlorobenzeneboronic acid (100.2 mg, 0.6408 mmol), bis-
(triphenylphosphine)palladium(II) chloride (22.6 mg, 0.032 mmol),
sodium carbonate (1.0 M aqueous solution, 1.0 mL, 1.0 mmol), and
acetonitrile (3.0 mL) was subjected to microwave irradiation at 120 °C
for 30 min. The reaction mixture was partitioned between ethyl acetate
and water, and the organic layer was dried over magnesium sulfate,
filtered, and concentrated. The crude product was purified by reverse
phase HPLC and lyophilized to give 49.4 mg (59%) of N-(2-(3-
chlorophenyl)pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
1H NMR (400 MHz, DMSO-d6) δ: 9.97 (s, 1H), 9.33 (dd, J = 7.0, 1.6
Hz, 1H), 8.82 (dd, J = 8.3, 1.4 Hz, 1H), 8.69 (s, 1H), 8.42 (dd, J = 4.6,
1.4 Hz, 1H), 8.39 (dd, J = 4.2, 1.6 Hz, 1H), 7.71−7.69 (m, 1H), 7.63−
7.58 (m, 3H), 7.49 (dd, J = 8.4, 4.6 Hz, 1H), 7.27 (dd, J = 7.0, 4.2 Hz,
1H). MS: m/z 350.0 ([M + H]+).
N-(4-Bromo-1-methyl-1H-pyrazol-3-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide (11). A mixture of pyrazolo[1,5-a]-
pyrimidine-3-carboxylic acid (165.5 mg, 1.014 mmol), 4-bromo-1-
methyl-1H-pyrazol-3-amine (177.4 mg, 1.008 mmol), HATU (497.0
mg, 1.307 mmol), DIPEA (0.25 mL, 1.4 mmol), 4-dimethylaminopyr-
idine (33.8 mg, 0.277 mmol), and 5 mL of DMF was heated at 50 °C
for 4 days. The reaction mixture was partitioned between ethyl acetate
and water, and the organic portion washed with brine, dried over
magnesium sulfate, filtered, and concentrated. The crude product was
purified by flash chromatography on silica gel (gradient elution: 20−
80% ethyl acetate in 5% methanol:DCM) to yield 203.6 mg (63%) of
N-(4-bromo-1-methyl-1H-pyrazol-3-yl)pyrazolo[1,5-a]pyrimidine-3-
1
carboxamide. H NMR (400 MHz, CD3OD) δ: 9.17 (dd, J = 7.0, 1.6
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Hz, 1H), 8.85 (dd, J = 4.2, 1.6 Hz, 1H), 8.68 (s, 1H), 7.76 (s, 1H),
7.28 (dd, J = 7.0, 4.2 Hz, 1H), 3.88 (s, 3H). MS: m/z 321.1 ([M +
H]+).
= 8.0, 1.0 Hz, 1H), 7.33 (dd, J = 7.0, 4.2 Hz, 1H), 4.73 (s, 1H), 4.09 (s,
2H), 1.13 (s, 6H). MS: m/z 411.1 ([M + H]+).
5-Bromo-2-methylpyridine 1-oxide (15). A solution of 5-bromo-2-
methylpyridine (2.0 g, 12 mmol) and m-chloroperoxybenzoic acid
(70%, 2.89 g, 12.9 mmol) in 30 mL of chloroform was stirred at room
temperature for 4 h. The reaction mixture was then partitioned
between DCM and 2 M aqueous sodium carbonate. The aqueous layer
was extracted once more with DCM, and the combined organic
portions were dried over magnesium sulfate, filtered, and concentrated
to yield 2.1575 g (99%) of 5-bromo-2-methylpyridine 1-oxide. 1H
NMR (400 MHz, DMSO-d6) δ: 8.57 (d, J = 1.4 Hz, 1H), 7.51 (dd, J =
8.4, 1.6 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 2.30 (s, 3H). MS: m/z
189.0 ([M + H]+).
5-Bromo-2-methyl-4-nitropyridine 1-oxide (16). 15 (2.269 g,
12.07 mmol) was dissolved in sulfuric acid (4 mL, 80 mmol) and
cooled in an ice−water bath. Fuming nitric acid (3 mL, 60 mmol) was
added dropwise. After addition of the nitric acid was complete, the
reaction mixture was first warmed to room temperature and then
heated to 90 °C. After heating for 2 h, the reaction mixture was cooled
in an ice−water bath and slowly adjusted to pH 10 with 2 M aqueous
sodium carbonate. This mixture was extracted twice with DCM. The
combined organic extracts were dried over magnesium sulfate, filtered,
and concentrated to yield 2.54 g (90%) of 5-bromo-2-methyl-4-
nitropyridine 1-oxide. MS: m/z 233.0 ([M + H]+).
5-Bromo-2-methylpyridin-4-amine (17). To a solution of 16 (2.54
g, 10.9 mmol) in 10 mL of concentrated hydrochloric acid was added
tin chloride dihydrate (9.96 g, 43.75 mmol). The reaction mixture was
heated at 90 °C for 24 h, at which time additional tin chloride
dihydrate (3.15 g, 13.84 mmol) and 5 mL of concentrated
hydrochloric acid were added. The reaction mixture was held at 90
°C for an additional 24 h and then cooled to room temperature and
adjusted to neutral pH with 2 M aqueous sodium carbonate. The
resulting mixture was extracted three times with DCM, and the
combined organic extracts dried over magnesium sulfate, filtered, and
concentrated to yield 1.15 g (56%) of 5-bromo-2-methylpyridin-4-
amine. 1H NMR (400 MHz, DMSO-d6) δ: 8.07 (s, 1H), 6.51 (s, 1H),
6.13 (s, 2H), 2.22 (s, 3H). MS: m/z 187.2 ([M + H]+).
N-(1-Methyl-4-(3-chlorophenyl)-1H-pyrazol-3-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide (12). To a mixture of 11 (59.5 mg, 0.185
mmol) and 3-chlorobenzeneboronic acid (44.6 mg, 0.285 mmol) in
diglyme (5.0 mL) was added sodium carbonate (2 M aqueous
solution, 0.3 mL, 0.6 mmol) and tetrakis(triphenylphosphine)-
palladium(0) (19.5 mg, 0.0169 mmol). The reaction mixture was
purged with nitrogen and then stirred at 100 °C for 24 h. The reaction
mixture was partitioned between ethyl acetate and water and the
organic portion washed with brine, dried over magnesium sulfate,
filtered, and concentrated. The crude product was purified by reverse
phase HPLC and lyophilized to yield 4.3 mg (7%) of N-(1-methyl-4-
(3-chlorophenyl)-1H-pyrazol-3-yl)pyrazolo[1,5-a]pyrimidine-3-car-
boxamide. 1H NMR (400 MHz, DMSO-d6) δ: 9.70 (s, 1H), 9.38 (dd, J
= 7.0, 1.6 Hz, 1H), 8.86 (dd, J = 4.2, 1.6 Hz, 1H), 8.68 (s, 1H), 8.16 (s,
1H), 7.56 (t, J = 1.8 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.35−7.34 (m,
1H), 7.32 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 3.86 (s, 3H).
MS: m/z 353.0 ([M + H]+).
N-(3-(3-Chlorophenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide (13a). A mixture of 20a (400.0 mg, 1.926
mmol), pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (342.9 mg, 2.102
mmol), 7-azabenzotriazol-1-yloxy-tris-(pyrrolidino)phosphonium hex-
afluorophosphate (PyAOP) (1.205 g, 2.324 mmol), DIPEA (0.80 mL,
4.6 mmol), and 4-dimethylaminopyridine (43.4 mg, 0.355 mmol) in
15.0 mL DMF was stirred at 50 °C for 15 h. The reaction mixture was
partitioned between DCM and water and the aqueous layer extracted
once more with DCM. The combined organic portions were dried
over magnesium sulfate and concentrated onto silica gel. The crude
product was separated by flash chromatography on silica gel (solvent
gradient: 0−70% ethyl acetate (containing 2% methanol) in DCM).
The resulting solid material was triturated with ethyl acetate, sonicated,
and the solids were collected by filtration and dried under vacuum to
yield 0.502 g (74%) of N-(3-(3-chlorophenyl)-1-methyl-1H-pyrazol-4-
yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide. 1H NMR (400 MHz,
DMSO-d6) δ: 10.00 (s, 1H), 9.38 (dd, J = 7.0, 1.6 Hz, 1H), 8.84 (dd, J
= 4.2, 1.6 Hz, 1H), 8.70 (s, 1H), 8.33 (s, 1H), 7.84 (t, J = 1.8 Hz, 1H),
7.77 (dt, J = 7.8, 1.8 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.47 (d, J = 7.9
Hz, 1H), 7.34 (dd, J = 7.0, 4.2 Hz, 1H), 3.93 (s, 3H). MS: m/z 353.0
([M + H]+).
N-(3-(3-Chlorophenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (14a). 13b (68.9 mg, 0.203 mmol) was
dissolved in 4 mL of DMF. To this solution was added cesium
carbonate (148 mg, 0.454 mmol) and isopropyl iodide (23.0 μL, 0.230
mmol). The reaction mixture was stirred at 50 °C for 2 h. The reaction
mixture was then partitioned between ethyl acetate and water and the
organic portion washed with brine, dried over magnesium sulfate, and
concentrated. The crude product was purified by reverse phase HPLC
and lyophilized to give 47.2 mg (61%) of N-(3-(3-chlorophenyl)-1-
isopropyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
1H NMR (400 MHz, DMSO-d6) δ: 9.99 (s, 1H), 9.38 (dd, J = 7.0, 1.6
Hz, 1H), 8.84 (dd, J = 4.2, 1.6 Hz, 1H), 8.70 (s, 1H), 8.36 (s, 1H),
7.85 (t, J = 1.8 Hz, 1H), 7.78 (td, J = 7.7, 1.4 Hz, 1H), 7.57 (t, J = 7.9
Hz, 1H), 7.50−7.44 (m, 1H), 7.34 (dd, J = 7.0, 4.2 Hz, 1H), 4.66−
4.51 (m, 1H), 1.49 (d, J = 6.7 Hz, 6H). MS: m/z 381.1 ([M + H]+).
N-(3-(3-Chlorophenyl)-1-(2-hydroxy-2-methylpropyl)-1H-pyra-
zol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14f). 13b (58.9
mg, 0.174 mmol) was dissolved in 5 mL of DMF. To this solution was
added isobutylene oxide (0.5 mL, 6 mmol) and cesium carbonate
(56.4 mg, 0.173 mmol). The reaction mixture was stirred at 50 °C for
7.5 h. The reaction mixture was then partitioned between ethyl acetate
and water and the organic portion washed with brine, dried over
magnesium sulfate, and concentrated. The crude product was purified
by reverse phase HPLC and lyophilized to give 33.1 mg (46%) of N-
(3-(3-chlorophenyl)-1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide. 1H NMR (400 MHz,
DMSO-d6) δ: 10.02 (s, 1H), 9.37 (dd, J = 7.0, 1.5 Hz, 1H), 8.84
(dd, J = 4.2, 1.6 Hz, 1H), 8.69 (s, 1H), 8.35 (s, 1H), 7.84 (t, J = 1.7
Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.48 (dd, J
2-(2-(3-Chlorophenyl)-2-oxoethylisoindoline-1,3-dione (18a). A
mixture of 2-bromo-3′-chloroacetophenone (0.927 g, 3.97 mmol) and
potassium phthalimide (0.813 g, 4.39 mmol) in 15 mL of DMF was
stirred at 50 °C for 1 h. The solvent was removed under vacuum, and
the resulting solids were triturated with ethyl acetate and filtered. The
collected solids were dried under vacuum to give 2-(2-(3-
chlorophenyl)-2-oxoethylisoindoline-1,3-dione, which was carried
1
forward without further purification. H NMR (400 MHz, DMSO-
d6) δ: 8.13 (t, J = 1.9 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 8.00−7.90 (m,
4H), 7.85−7.80 (m, 1H), 7.65 (t, J = 7.9 Hz, 1H), 5.29 (s, 2H).
Di-tert-butyl 2-(2,5-Dimethylphenyl)-2-oxoethyliminodicarbon-
ate (18b). In an oven-dried flask, di-tert-butyl iminodicarboxylate
(2.566 g, 11.81 mmol) was combined with sodium hydride (60 wt %
in mineral oil, 0.59 g, 15 mmol) and 30 mL of DMF. The reaction
mixture was stirred at ambient temperature for 1.5 h, and then 2-
bromo-1-(2,5-dimethylphenyl)ethanone (2.432 g, 10.71 mmol) was
added. The reaction was stirred at ambient temperature for an
additional 1.5 h and then partitioned between ethyl acetate and water.
The organic portion was washed with water and brine, dried over
magnesium sulfate, and concentrated under vacuum. The crude
product was purified by flash chromatography on silica gel (gradient
elution: 0−40% ethyl acetate in heptanes) to obtain 3.008 g (77%) of
di-tert-butyl 2-(2,5-dimethylphenyl)-2-oxoethyl-iminodicarbonate. MS:
m/z 386.2 ([M + Na]+).
2-(3-(3-Chlorophenyl)-1-(dimethylamino)-3-oxoprop-1-en-2-yl)-
isoindoline-1,3-dione (19a). A stirred mixture of 18a (782.2 mg,
2.610 mmol) and DMFDMA (1.5 mL, 11 mmol) was heated at 100
°C for 18 h. Excess DMFDMA was removed under vacuum. The crude
product was purified by flash chromatography on silica gel (gradient
elution: 50−100% ethyl acetate in heptanes) to yield 740 mg (80%) of
2-(3-(3-chlorophenyl)-1-(dimethylamino)-3-oxoprop-1-en-2-yl)-
1
isoindoline-1,3-dione. H NMR (400 MHz, CDCl3) δ: 7.91 (dd, J =
5.4, 3.1 Hz, 2H), 7.76 (dd, J = 5.5, 3.1 Hz, 2H), 7.57 (s, 1H), 7.45 (d, J
10101
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= 7.5 Hz, 1H), 7.42−7.35 (m, 2H), 7.31 (t, J = 7.8 Hz, 1H), 3.00 (s,
6H). MS: m/z 355.2 ([M + H]+).
ethyl 5-(5-chloro-2-methylphenyl)-1-methyl-1H-pyrazole-4-carboxy-
late. MS: m/z 279.2 ([M + H]+).
3-(3-Chlorophenyl)-1-methyl-1H-pyrazol-4-amine (20a). To a
solution of 19a (2.30 g, 6.48 mmol) in 50 mL of ethanol was added
N-methylhydrazine (1.4 mL, 26 mmol). The reaction mixture was
stirred at 80 °C for 2 h and then concentrated onto silica gel. The
crude mixture of regioisomers was separated and purified by flash
chromatography on silica gel (gradient elution: 0−80% ethyl acetate in
DCM) to yield 715.0 mg (53%) of 3-(3-chlorophenyl)-1-methyl-1H-
Ethyl 3-(2,6-Dichlorophenyl)-1-methyl-1H-pyrazole-4-carboxy-
late (24b). To a stirred solution of 22b (12.6 g, 40 mmol) in acetic
acid (100 mL) was added N-methyl-hydrazine (3.84 g, 83.4 mmol).
The reaction mixture was stirred at ambient temperature for 3 days.
The mixture was then concentrated under reduced pressure, and the
residue was partitioned between ethyl acetate and water. The organic
portion was dried over magnesium sulfate and concentrated under
vacuum. The crude product was purified by flash chromatography on
silica gel to yield 5.4 g (45%) of ethyl 3-(2,6-dichlorophenyl)-1-
methyl-1H-pyrazole-4-carboxylate. MS: m/z 298.9 ([M + H]+).
3-(5-Chloro-2-methylphenyl)-1-methyl-1H-pyrazol-4-amine
(26a) and 5-(5-Chloro-2-methylphenyl)-1-methyl-1H-pyrazol-4-
amine (27). To a solution of 24a and 25 (1:1 mixture of regioisomers,
2.179 g, 7.818 mmol) in ethanol (10 mL) was added 1 M aqueous
NaOH (18 mL, 18 mmol). The reaction mixture was heated at 50 °C
for 20 h, and then the solvent was removed under reduced pressure.
The resulting residue was adjusted to pH 2 with 1 M aqueous H3PO4
and extracted with DCM (3 × 100 mL). The combined organic
extracts were dried over magnesium sulfate, filtered, and concentrated
under vacuum. The resulting material was dissolved in 1,4-dioxane (15
mL) and treated with triethylamine (2.20 mL, 15.8 mmol) and
diphenylphosphonic azide (1.90 mL, 8.82 mmol). The mixture was
stirred at ambient temperature for 1 h and then treated with tert-butyl
alcohol (15 mL) and heated at 90 °C for 1 h. After being cooled to
room temperature, the mixture was partitioned between ethyl acetate
and water. The organic portion was dried over magnesium sulfate and
concentrated. The crude product was purified by flash chromatography
on silica gel (solvent gradient: 0−50% ethyl acetate in DCM) to yield
a mixture of the title compounds as the tert-butyl-carbamates (1.637 g,
66%). To a solution of this material (1:1 mixture of regioisomers,
1.637 g, 5.088 mmol) in 10 mL of DCM was added hydrogen chloride
(10.0 mL of a 4.0 M solution in 1,4-dioxane, 40 mmol). The reaction
mixture was stirred at ambient temperature for 16 h and then
evaporated to dryness. The solid residue was partitioned between
DCM and a saturated aqueous solution of sodium bicarbonate. The
aqueous portion was extracted once more with DCM, and the
combined organic extracts were dried over magnesium sulfate, filtered,
and concentrated under vacuum. The crude product was purified and
regioisomers separated by flash chromatography on silica gel (gradient
elution: 0−100% ethyl acetate in DCM) to yield 429.7 mg (38%) of 3-
(5-chloro-2-methylphenyl)-1-methyl-1H-pyrazol-4-amine (26a). 1H
NMR (400 MHz, CDCl3) δ: 7.35 (d, J = 1.8 Hz, 1H), 7.24−7.17
(m, 2H), 7.04 (s, 1H), 3.84 (s, 3H), 2.76 (s, 2H), 2.30 (s, 3H). MS,
m/z 222.1 ([M + H]+). And 420.2 mg (37%) of 5-(5-chloro-2-
methylphenyl)-1-methyl-1H-pyrazol-4-amine (27). 1H NMR (400
MHz, CDCl3) δ: 7.33 (dd, J = 8.3, 2.1 Hz, 1H), 7.28 (s, 1H), 7.24−
7.19 (m, 2H), 3.57 (s, 3H), 2.71 (s, 2H), 2.15 (s, 3H). MS: m/z 222.1
([M + H]+).
1
pyrazol-4-amine. H NMR (400 MHz, CDCl3) δ: 7.78 (t, J = 1.8 Hz,
1H), 7.64 (dt, J = 7.7, 1.4 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.28−7.23
(m, 2H), 7.04 (s, 1H), 3.84 (s, 3H). MS: m/z 208.2 ([M + H]+). Also
isolated was 274.6 mg (20%) of 5-(3-chlorophenyl)-1-methyl-1H-
pyrazol-4-amine. 1H NMR (400 MHz, CDCl3) δ: 7.46−7.40 (m, 1H),
7.40−7.35 (m, 2H), 7.29−7.25 (m, 1H), 7.23 (s, 1H), 3.76 (s, 3H).
MS: m/z 208.2 ([M + H]+).
Ethyl 3-(5-Chloro-2-methylphenyl)-3-oxopropanoate (21a). To a
stirring solution of 5-chloro-2-methylbenzoic acid (4.8547 g, 28.458
mmol) in 30 mL of THF was added N,N-carbonyldiimidazole (4.8688
g, 30.027 mmol). Stirring was continued at ambient for 30 min to
generate the acyl-imidazole. Separately, potassium ethyl malonate
(11.612 g, 68.224 mmol) and MgCl2 (3.2689 g, 34.333 mmol) were
suspended in 50 mL of THF. To the magnesium chloride mixture was
added the acyl-imidazole solution. The resulting reaction mixture was
heated at 50 °C for 10 h. The reaction mixture was partitioned
between ethyl acetate and water, and the organic portion dried over
magnesium sulfate, filtered through a pad of Celite, and concentrated
to provide 8.07 g of ethyl 3-(5-chloro-2-methoxyphenyl)-3-oxopropa-
noate, which was used without purification. MS: m/z 241.2 ([M +
H]+).
Ethyl 3-(2,6-Dichlorophenyl)-3-oxopropanoate (21b). Potassium
ethyl malonate (63.0 g, 370 mmol) was placed in a flask under
nitrogen. Acetonitrile (1 L) was added, and the mixture was cooled to
10−15 °C. To the cooled solution was added triethylamine (55 mL,
0.39 mol) followed by MgCl2 (42.8 g, 0.449 mol), and the mixture was
then stirred at ambient temperature for 2.5 h. After cooling the
reaction mixture to 0 °C, 2,6-dichlorobenzoyl chloride (37.7 g, 180
mmol) was added slowly over 25 min, followed by addition of more
triethylamine (5 mL, 40 mmol). The reaction mixture was stirred at
ambient temperature overnight. The solvent was removed under
reduced pressure and the residue partitioned between toluene and
10% aqueous HCl. The organic layer was concentrated to provide a
quantitative yield of ethyl 3-(2,6-dichlorophenyl)-3-oxopropanoate,
which was carried forward without purification.
Ethyl 2-(5-Chloro-2-methylbenzoyl)-3-(dimethylamino)acrylate
(22a). A stirred mixture of 21a (28.458 mmol) and DMFDMA
(10.0 mL, 75.3 mmol) was heated at 90 °C for 3 h. After evaporation
of excess DMFDMA under reduced pressure, the crude product was
purified by flash chromatography on silica gel (gradient elution: 0−
80% ethyl acetate in DCM) to yield 2.87 g (34%) of ethyl 2-(5-chloro-
2-methylbenzoyl)-3-(dimethylamino)acrylate. MS: m/z 296.3 ([M +
H]+).
Ethyl 5-(5-Chloro-2-methylphenyl)-1H-pyrazole-4-carboxylate
(23). A solution of 22a (2.87 g, 9.70 mmol) and hydrazine (0.50
mL, 16.0 mmol) in 30 mL of ethanol was heated at 70 °C for 2 h. The
crude reaction mixture was concentrated under vacuum to provide
2.57 g (100%) ethyl 5-(5-chloro-2-methoxyphenyl)-1H-pyrazole-4-
carboxylate, which was used without further purification. MS: m/z
265.2 ([M + H]+).
Ethyl 3-(5-Chloro-2-methylphenyl)-1-methyl-1H-pyrazole-4-car-
boxylate (24a) and Ethyl 5-(5-Chloro-2-methylphenyl)-1-methyl-
1H-pyrazole-4-carboxylate (25). To a solution of 23 (9.70 mmol) in
35 mL of DMF was added cesium carbonate (3.831 g, 11.76 mmol)
and iodomethane (0.90 mL, 14 mmol). The reaction mixture was
stirred at 40 °C for 7 h and was then partitioned between ethyl acetate
and water. The organic portion was dried over magnesium sulfate and
concentrated. The crude product was purified by flash chromatography
on silica gel (gradient elution: 0−40% ethyl acetate in DCM) to yield
2.179 g (81%) of a 1:1 mixture of the regioisomeric products, ethyl 3-
(5-chloro-2-methylphenyl)-1-methyl-1H-pyrazole-4-carboxylate, and
4-Nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (28). In
an oven-dried flask equipped with a stir bar, 4-nitro-1H-pyrazole
(6.598 g, 58.35 mmol) was dissolved in 50 mL of THF. Sodium
hydride (60 wt % dispersion in mineral oil, 4.827 g, 120.7 mmol) was
added while cooling the reaction vessel with an ice−water bath, and
the reaction was then stirred at ambient temperature for 10 min. [β-
(Trimethylsilyl)ethoxy]methyl chloride (12.0 mL, 67.8 mmol) was
then added, and the reaction stirred at ambient temperature for 1.5 h.
The reaction mixture was quenched with 50 mL of water and extracted
into ethyl acetate. The organic extract was dried over magnesium
sulfate, filtered, and concentrated under vacuum. The resulting crude
product was purified by flash chromatography on silica gel (gradient
elution: 0−30% ethyl acetate in heptanes) to obtain 14.1368 g (99%)
of 4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole. 1H NMR
(400 MHz, CDCl3) δ: 8.30 (s, 1H), 8.10 (s, 1H), 5.45 (s, 2H), 3.73−
3.54 (m, 2H), 1.02−0.84 (m, 2H), −0.00 (s, 9H). MS: m/z 244.2 ([M
+ H]+).
5-(5-Chloro-2-methoxyphenyl)-4-nitro-1-((2-(trimethylsilyl)-
ethoxy)methyl)-1H-pyrazole (29). To a solution of 28 (4.259 g, 17.50
mmol) in 40 mL DMF was added 2-bromo-4-chloroanisole (3.35 mL,
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24.6 mmol), palladium(II) acetate (197.2 mg, 0.8784 mmol), di(1-
adamantyl)-n-butylphosphine (469.5 mg, 1.309 mmol), potassium
carbonate (7.2763 g, 52.648 mmol), and trimethylacetic acid (0.4523
g, 4.428 mmol). While stirring at room temperature, nitrogen gas was
bubbled through the reaction mixture for 10 min, and the reaction was
then heated under a nitrogen balloon at 120 °C for 6 h. The reaction
mixture was then cooled to room temperature, diluted into ethyl
acetate, washed with water and brine, dried over magnesium sulfate,
filtered, and concentrated under vacuum. The crude product was
purified by flash chromatography on silica gel (gradient elution: 0−
25% ethyl acetate in heptanes) to obtain 6.719 g (89%) of 3-(5-chloro-
2-methoxyphenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
1H NMR (400 MHz, CDCl3) δ: 8.65 (s, 1H), 8.18 (t, J = 1.9 Hz, 1H),
8.04 (dt, J = 7.7, 1.4 Hz, 1H), 7.53 (ddd, J = 8.1, 2.1, 1.2 Hz, 1H),
7.49−7.44 (m, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).
5-(3-Chlorophenyl)-isoxazole-4-carboxylic Acid (33). A suspen-
sion of 32 (422 mg, 1.68 mmol) in 6 M aqueous HCl (10 mL) and
acetic acid (6 mL) was heated to reflux for 5 h. After cooling, ethyl
acetate was added to the suspension and the layers were separated.
The aqueous layer was then extracted with ethyl acetate (2×). The
organic extracts were combined, dried with anhydrous sodium sulfate,
and concentrated under reduced pressure to yield 5-(3-chlorophenyl)-
isoxazole-4-carboxylic acid as a white solid. This was used in the next
step without further purification. 1H NMR (400 MHz, CDCl3) δ: 8.70
(d, J = 2.3 Hz, 1H), 8.13 (d, J = 2.3 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H),
7.54 (m, 1H), 7.47 (td, J = 7.9, 2.3 Hz, 1H).
[5-(3-Chlorophenyl)-isoxazole-4-yl]-carbamic Acid tert-Butyl
Ester (34). A solution of crude 33 (254 mg, 1.14 mmol) in thionyl
chloride (3 mL) was heated to reflux for 3 h. After cooling, the mixture
was concentrated under reduced pressure to yield an orange residue.
The residue was dissolved in acetone (2 mL) and cooled to 0 °C (ice−
water bath). Sodium azide (133 mg, 2.04 mmol) was added, and the
resulting mixture was stirred for 1 h and then at ambient temperature
for 1 h. Ethyl acetate was added and the layers separated. The organic
extract was washed with water (2×) and brine (2×), dried with
anhydrous sodium sulfate, and concentrated under reduced pressure to
leave an orange solid. The residue was then treated with tert-butanol
(8 mL) at reflux for 16 h. After cooling, the solvent was removed
under vacuum and the residue purified by flash chromatography on
silica gel (gradient elution: 0−20% ethyl acetate in cyclohexane) to
afford 184 mg (55%) of [5-(3-chlorophenyl)-isoxazole-4-yl]-carbamic
acid tert-butyl ester as an orange solid. 1H NMR (400 MHz, CDCl3) δ:
7.70 (s, 1H), 7.59 (d, J = 6.9 Hz, 1H), 7.48−7.37 (m, 3H), 1.52 (s,
9H).
1
pyrazole. H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.47 (dd, J =
8.9, 2.6 Hz, 1H), 7.36 (d, J = 2.5 Hz, 1H), 6.96 (d, J = 8.9 Hz, 1H),
5.34−5.20 (m, 2H), 3.76 (s, 3H), 3.69−3.46 (m, 2H), 0.96−0.79 (m,
2H), −0.02 (s, 9H). MS: m/z 384.2 ([M + H]+).
5-(5-Chloro-2-methoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-pyrazol-4-amine (30). To a solution of 29 (5.97 g, 15.6
mmol) in 25 mL of ethanol was added water (50 mL), ammonium
chloride (3.367 g, 62.94 mmol), and iron powder (4.367 g, 78.2
mmol). The reaction mixture was heated at 75 °C for 1.5 h. After
being cooled to ambient temperature, the mixture was diluted with
DCM and filtered through a Celite pad, rinsing with more DCM. The
combined filtrate was added to 150 mL of saturated aqueous sodium
bicarbonate and extracted twice with DCM. The combined organic
extracts were dried over magnesium sulfate, filtered, and concentrated
under vacuum to yield 5.50 g (100%) of 3-(5-chloro-2-methox-
yphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine,
1
which was carried forward without purification. H NMR (500 MHz,
CDCl3) δ 7.44 (d, J = 2.6 Hz, 1H), 7.35 (dd, J = 8.8, 2.6 Hz, 1H), 7.28
(s, 1H), 6.93 (d, J = 8.9 Hz, 1H), 5.24 (s, 2H), 3.82 (s, 3H), 3.52 (t, J
= 8.2 Hz, 2H), 0.91−0.79 (m, 2H), −0.04 (s, 9H). MS: m/z 354.3
([M + H]+).
5-(3-Chlorophenyl)-isoxazole-4-ylamine Hydrochloride (35).
First, 4 M HCl solution in 1,4-dioxane (1.5 mL, 6.0 mmol) was
added to a stirred solution of 34 (184 mg, 0.624 mmol) in 1,4-dioxane
(2 mL). Then the resulting mixture was heated to 40 °C for 20 h. After
cooling, the mixture was concentrated under reduced pressure to give
147 mg (quant) of 5-(3-chlorophenyl)-isoxazole-4-ylamine hydro-
chloride as a yellow solid. This was used in the next step without
3-(3-Chlorophenyl)-3-oxopropoinic Acid Ethyl Ester (31). Sodium
hydride (60% in mineral oil, 1.03 g, 25.9 mmol) was added to a stirred
solution of diethyl carbonate (3.9 mL, 32 mmol) in toluene (25 mL),
under nitrogen. 3-Chloroacetophenone (0.84 mL, 6.5 mmol) in
toluene (5 mL) was then added dropwise to the suspension over 30
min. The resulting mixture was heated to reflux for 16 h. After cooling,
the reaction was carefully quenched with acetic acid (20 mL). Water
(20 mL) was added and the mixture extracted into ethyl acetate (3×).
The combined organic extracts were washed with saturated aqueous
sodium bicarbonate solution, dried with anhydrous sodium sulfate, and
concentrated under reduced pressure to yield a dark-orange oil, which
was purified by Kugelrohr bulb to bulb distillation, at 150 °C, under
vacuum, to yield 0.996 g of a colorless oil. Further purification by flash
chromatography on silica gel (gradient elution: 0−15% ethyl acetate in
cyclohexane) afforded 0.75 g (51%) of 3-(3-chlorophenyl)-3-
oxopropoinic acid ethyl ester, 2:1 mixture of keto−enol tautomers,
as a colorless oil. 1H NMR (300 MHz, CDCl3) δ: 12.56 (s, 1H), 7.93
(t, J = 1.9 Hz, 1H), 7.83 (ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 7.77 (t, J = 1.9
Hz, 1H), 7.65 (dt, J = 7.7, 1.5 Hz, 1H), 7.58 (ddd, J = 8.0, 2.1, 1.1 Hz,
1H), 7.47−7.32 (m, 3H), 5.66 (s, 1H), 4.33−4.16 (m, 4H), 3.98 (s,
2H), 1.35 (t, J = 7.1 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H).
5-(3-Chlorophenyl)-isoxazole-4-carboxylic Acid Ethyl Ester (32).
DMF-DMA (1.5 mL, 11.6 mmol) was added to a solution of 31 (0.75
g, 3.3 mmol) in DMF (25 mL), under nitrogen. The mixture was
heated to reflux for 5 h. After cooling, ethyl acetate and water were
added and the layers separated. The organic extract was dried with
anhydrous sodium sulfate and concentrated under reduced pressure to
yield 0.79 g of a thick orange oil. The residue (0.595 g, ca. 2.11 mmol)
was dissolved in methanol (5 mL) and treated with hydroxylamine
hydrochloride (0.147 g, 2.11 mmol) at reflux for 1 h. After cooling,
ethyl acetate and water were added and the layers separated. The
aqueous phase was then extracted with ethyl acetate (2×). The
combined organic extracts were dried with anhydrous sodium sulfate
and concentrated under reduced pressure to yield an orange solid.
Purification by flash chromatography on silica gel (gradient elution:
0−10% ethyl acetate in cyclohexane) afforded 422 mg (79%) of 5-(3-
chlorophenyl)-isoxazole-4-carboxylic acid ethyl ester as a white solid.
1
further purification. H NMR (400 MHz, CD3OD) δ: 8.61 (br, m,
1H), 7.85 (br, m, 1H), 7.75 (br, m, 1H), 7.62 (br, m, 2H).
3-Chlorobenzaldehyde Oxime (36). To a solution of 3-
chlorobenzaldehyde (0.81 mL, 7.1 mmol) in EtOH (25 mL) was
added hydroxylamine hydrochloride (0.54 g, 7.8 mmol), followed by
pyridine (0.57 mL, 7.1 mmol). The resulting colorless mixture was
stirred at ambient temperature for 5 h. Then 1 M aqueous HCl was
added until pH reached 2, and then the mixture was extracted into
DCM (3×). The organic layers were combined and dried with
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure to yield 1.22 g (quant) of 3-chlorobenzaldehyde oxime as a
white solid. This was used in the next step without further purification.
1H NMR (400 MHz, CDCl3) δ: 8.11 (s, 1H), 7.59 (m, 1H), 7.45 (m,
1H), 7.36 (m, 1H), 7.32 (m, 1H).
(Z)-3-Chloro-N-hydroxybenzene-1-carbonimidoyl Chloride (37).
To a solution of 36 (436 mg, 2.80 mmol) in DMF (8 mL) was added
N-chlorosuccinimide (307 mg, 2.30 mmol), and the resulting yellow
mixture was stirred at ambient temperature for 1.5 h. The reaction was
quenched with ice−water and extracted into diethyl ether (2×). The
organic layers were combined, washed with water (4×), dried with
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure to yield 480 mg (quant) of (Z)-3-chloro-N-hydroxybenzene-
1-carbonimidoyl chloride as an orange solid. This was used in the next
step without further purification. 1H NMR (300 MHz, CDCl3) δ: 7.85
(m, 1H), 7.75 (ddd, J = 7.72, 1.75, 1.25 Hz, 1 H), 7.43 (ddd; J = 8.02,
2.06, 1.25 Hz, 1H), 7.36 (m, 1H).
(E)-3-Pyrrolidin-1-ylacrylic Acid Ethyl Ester (38). To a solution of
ethyl propiolate (2.9 mL, 29 mmol) in toluene (20 mL) was added
dropwise a solution of pyrrolidine (2.4 mL, 29 mmol) in toluene (5
mL) over 10 min. The resulting orange mixture was stirred at ambient
temperature for 16 h and concentrated under reduced pressure to
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leave a thick orange oil, which was purified by Kugelrohr distillation, at
140−150 °C, under vacuum, to afford 4.56 g (94%) of (E)-3-
pyrrolidin-1-ylacrylic acid ethyl ester as pale-yellow oil. 1H NMR (400
MHz, CDCl3) δ: 7.65 (d, J = 12.9 Hz, 1H), 4.48 (d, J = 12.9 Hz, 1H),
4.13 (q, J = 7.1 Hz, 2H), 3.27 (s, 4H), 1.93 (s, 4H), 1.26 (t, J = 7.1 Hz,
3H).
(washing with DCM and eluting with 2 M NH3 in methanol solution)
to afford 0.42 g (73%) of 4-(3-chlorophenyl)-2-methylthiazol-5-
1
ylamine as a pale-orange oil, which solidified on standing. H NMR
(400 MHz, CDCl3) δ: 7.75 (ddd, J = 2.1, 1.7, 0.4 Hz, 1H), 7.61 (ddd, J
= 7.8, 1.7, 1.1 Hz, 1H), 7.33 (m, 1H), 7.23 (ddd, J = 8.0, 2.1, 1.1 Hz,
1H), 3.84 (s, 2 H), 2.58 (s, 3H). MS: m/z 224.9/226.9 ([M + H]+).
Enzyme Assays. The activity of the isolated Jak1, Jak2, Jak3, or
Tyk2 kinase domain was measured by monitoring phosphorylation of
a peptide derived from Jak3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-
Leu-Thr-Thr) fluorescently labeled on the N-terminus with 5-
carboxyfluorescein using the Caliper LabChip technology (Caliper
Life Sciences, Hopkinton, MA). To determine the inhibition constants
(Ki), compounds were diluted serially in DMSO and added to 50 μL
kinase reactions containing 1.5 nM purified Jak1 enzyme (or 0.2 nM
purified Jak2 enzyme or 5 nM purified Jak3 enzyme or 1 nM purified
Tyk2 enzyme), 100 mM Hepes pH 7.2, 0.015% Brij-35, 1.5 μM
peptide substrate, 25 μM ATP, 10 mM MgCl2, 4 mM DTT at a final
DMSO concentration of 2%. Reactions were incubated at 22 °C in
384-well polypropylene microtiter plates for 30 min and then stopped
by addition of 25 μL of an EDTA containing solution (100 mM Hepes
pH 7.2, 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA
concentration of 50 mM. After termination of the kinase reaction, the
proportion of phosphorylated product was determined as a fraction of
total peptide substrate using the Caliper LabChip 3000 according to
the manufacturer’s specifications. Ki values were then determined
using the Morrison tight binding model.42 The activity of ruxolitinib
was measured as follows, with data representing either three or four
independent experiments and error described as the standard
deviation: Jak1 Ki 0.09 nM ( 0.01 nM), Jak2 Ki 0.24 nM ( 0.04
nM), Jak3 Ki 3.22 nM ( 0.27 nM), Tyk2 Ki 0.55 nM ( 0.07 nM).
Cell Assays. The activities of compounds in Tables 1, 2, and 4
were determined in cell-based assays that are designed to measure
Jak2-dependent signaling. The activity of ruxolitinib was measured in
both assay formats as follows, with data representing either two (SET2
format) or five (TF1+EPO format) independent experiments and
error described as the standard deviation: pSTAT5 SET2 MSD IC50
1.84 nM ( 1.70 nM), pSTAT5 SET2 MSD IC50 6.85 nM ( 3.57
nM).
pSTAT5 SET2 MSD Format. Compounds were serially diluted in
DMSO and incubated with SET2 cells (German Collection of
Microorganisms and Cell Cultures (DSMZ); Braunschweig, Ger-
many), which express the JAK2V617F constitutively active mutant
protein, in 96-well microtiter plates for 1 h at 37 °C in RPMI medium
at a final cell density of 100000 cells per well and a final DMSO
concentration of 0.57%. Compound-mediated effects on STAT5
phosphorylation were then measured in the lysates of incubated cells
using the Meso Scale Discovery (MSD) technology (Gaithersburg,
Maryland) according to the manufacturer’s protocol, and EC50 values
were determined.
pSTAT5 TF1+EPO MSD Format. Compounds were serially diluted
in DMSO and incubated with TF-1 cells (American Type Culture
Collection, ATCC), Manassas, VA) in 384-well microtiter plates in
OptiMEM medium without phenol red, 1% charcoal/dextran stripped
FBS, 0.1 mM NEAA, 1 mM sodium pyruvate (Invitrogen Corp.;
Carlsbad, CA) at a final cell density of 100000 cells per well and a final
DMSO concentration of 0.2%. Human recombinant EPO (Invitrogen
Corp., Carlsbad, CA) was then added at a final concentration of 10
units/mL to the microtiter plates containing the TF-1 cells and
compound, and the plates were incubated for 30 min at 37 °C.
Compound-mediated effects on STAT5 phosphorylation were then
measured in the lysates of cells incubated in the presence of EPO,
using the MSD technology (Gaithersburg, Maryland) according to the
manufacturer’s protocol and IC50 values were determined.
3-(3-Chlorophenyl)-isoxazole-4-carboxylic Acid Ethyl Ester (39).
A mixture of 38 (0.427 g, 2.52 mmol) and triethylamine (0.413 mL,
2.96 mmol) in diethyl ether (5 mL), under nitrogen, was cooled using
an ice bath. To this was added dropwise a solution of 37 (0.48 g, 2.5
mmol) in diethyl ether, over 25 min. A cream-colored solid precipitate
formed, and the suspension was stirred at ambient temperature for 18
h. The mixture was partitioned between water and diethyl ether, and
the aqueous layer was extracted with diethyl ether. The organic
extracts were combined and dried with anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure to give a viscous
yellow oil. Purification by flash chromatography on silica gel (gradient
elution: 0−20% ethyl acetate in cyclohexane) afforded 0.359 g (57%)
of 3-(3-chlorophenyl)-isoxazole-4-carboxylic acid ethyl ester as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ: 9.02 (s, 1H), 7.81 (ddd,
J = 2.1, 1.6, 0.5 Hz, 1H), 7.68 (ddd, J = 7.6, 1.6, 1.2 Hz, 1H), 7.48
(ddd, J = 8.1, 2.1, 1.2 Hz, 1H), 7.43−7.39 (m, 1H), 4.31 (q, J = 7.1 Hz,
2H), 1.32 (t, J = 7.1 Hz, 3H).
3-(3-Chlorophenyl)-isoxazole-4-carboxylic Acid (40). A stirred
solution of 39 (0.359 g, 1.43 mmol) in acetic acid (6 mL) was treated
with 6 M aqueous HCl (10 mL) at reflux for 5.5 h. After cooling, ethyl
acetate was added causing phase separation of the organic and aqueous
layers. The resulting aqueous layer was extracted again with ethyl
acetate. The combined organic extracts were dried with anhydrous
sodium sulfate, filtered, and concentrated under reduced pressure to
yield 0.315 g (99%) of 3-(3-chlorophenyl)-isoxazole-4-carboxylic acid
as a white solid. This was used in the next step without further
1
purification. H NMR (400 MHz, CDCl3) δ: 9.12 (m, 1H), 7.81 (m,
1H), 7.69 (d, J = 7.6 Hz, 1H), 7.49 (m, 1H), 7.43 (m, 1H).
[3-(3-Chlorophenyl)-isoxazol-4-yl]-carbamic Acid tert-Butyl Ester
(41). A mixture of 40 (0.215 g, 0.962 mmol), diphenylphosphorylazide
(0.21 mL, 0.96 mmol), and triethylamine (0.134 mL, 0.961 mmol) in
tert-butanol was heated to reflux (85 °C) for 16 h. After cooling, ethyl
acetate was added and the mixture successively washed with 1 M
aqueous HCl (2×), saturated aqueous sodium bicarbonate (2×), and
brine (2×). The organic layer was dried with anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure to yield a
cream-colored residue. Purification by flash chromatography on silica
gel (gradient elution: 0−15% ethyl acetate in cyclohexane) afforded
217 mg (77%) of [3-(3-chlorophenyl)-isoxazol-4-yl]-carbamic acid
tert-butyl ester as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 8.91
(s, 1H), 7.63 (m, 1H), 7.50 (m, 3H), 6.09 (s, 1H), 1.51 (s, 9H).
3-(3-Chlorophenyl)-isoxazol-4-ylamine Hydrochloride (42). A
solution of 41 (0.217 g, 0.736 mmol) in 1,4-dioxane (2 mL) was
treated with 4 M HCl solution in 1,4-dioxane (1.5 mL) at ambient
temperature for 20 h. The mixture was concentrated under reduced
pressure to yield 165 mg (97%) of 3-(3-chlorophenyl)-isoxazol-4-
ylamine hydrochloride as a cream-colored solid. This was used in the
1
next step without further purification. H NMR (400 MHz, CD3OD)
δ: 9.06 (s, 1H), 7.80 (td, J = 1.8, 0.5 Hz, 1H), 7.69 (dt, J = 7.2, 1.6 Hz,
1H), 7.63 (m, 1H), 7.59 (m, 1H).
4-(3-Chlorophenyl)-2-methylthiazol-5-ylamine (43). A mixture of
α-amino-(3-chlorophenyl)acetonitrile hydrochloride (0.52 g, 2.6
mmol) and powdered sulfur (80 mg, 2.6 mmol) in ethanol (3 mL)
was stirred in a sealed tube at 0 °C. Triethylamine (0.53 mL, 3.8
mmol) and a solution of acetaldehyde in ethanol (0.11 g, 2.6 mmol in
0.5 mL) were added sequentially. The resulting mixture was then
heated to 60 °C for 2.5 h. After cooling, mixture was concentrated
under reduced pressure, and the resulting residue was then dissolved in
ethanol (2 mL) and treated with 1 M aqueous HCl (2 mL) at ambient
temperature for 16 h. The pH was then adjusted to 12 (using saturated
aqueous sodium carbonate solution) and the mixture extracted into
DCM (3 × 10 mL). The combined organic extracts were dried with
anhydrous sodium sulfate and concentrated under reduced pressure.
The resulting residue was purified using an Isolute SCX-2 cartridge
In Vivo Pharmacodynamic Assay. Twenty million SET2 cells
containing 1:1 matrigel were inoculated subcutaneously in the right
flank of female, 8−12-week-old, severe combined immunodeficient
mice (SCID beige) obtained from Charles River (Hollister, CA).
Treatment was initiated when mean tumor volume reached 250−300
mm3. For pharmacodynamic studies, mice were administered a single
oral dose of compound in 0.5% methylceullulose/0.2% Tween-80
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(MCT). At 1, 4, 8, and 24 h after dosing, mice were euthanized
according to IACUC guidelines and tumor samples were harvested
and snap frozen on dry ice or fixed in 10% neutral buffered formalin.
Tris lysis buffer containing 150 mM NaCl, 20 mM Tris PH 7.5, 1 mM
EDTA, 1 mM EGTA, 1% Triton X-100 supplemented with
phosphatase and protease inhibitors (Sigma), 1 mM NaF, and 1
mM PMSF was added to frozen tumor biopsies. Tumors were
dissociated with a small pestle (Konte Glass Company, Vineland, NJ),
sonicated briefly on ice, and centrifuged at maximum RPM for 20 min
at 4 °C. Then 20 μg of protein from tumor lysates was used to
determine phosphorylation status. Samples were assayed in duplicates
per manufacturer’s protocol for pSTAT5/tSTAT5 (MSD). Plasma
samples were obtained by centrifugation, and drug concentrations
were determined using liquid chromatography with tandem mass
spectrometric detection (LC/MS).
Kinetic Solubility. Compounds were dissolved in DMSO to a
concentration of 10 mM. These solutions were diluted into PBS buffer
(pH 7.2, composed with NaCl, KCl, Na2HPO4, and KH2PO4) to a
final compound concentration of 100 μM, DMSO concentration of
2%, at pH 7.4. The samples were shaken for 24 h at room temperature,
followed by filtration. LC/CLND was used to determine compound
concentration in the filtrate, with the concentration calculated by a
caffeine calibration curve and the sample’s nitrogen content. An
internal standard compound was spiked into each sample for accurate
quantification.
ACKNOWLEDGMENTS
■
We thank Christopher Hamman, Michael Hayes, and Mengling
Wong for assistance with compound purification, Baiwei Lin for
conducting kinetic solubility experiments, Sashi Gopaul, Hoa
Le, Emile Plise, Savita Ubhayakar, and Jacob Chen for
conducting DMPK experiments, and Sarah Hymowitz and
Ivan Bosanac for collecting diffraction data. The Advanced
Light Source is supported by the Director, Office of Science,
Office of Basic Energy Sciences, of the U.S. Department of
Energy under contract no. DE-AC02-05CH11231.
ABBREVIATIONS USED
■
CDI, 1,1′-carbonyldiimidazole; Cl, clearance; DIPEA, N,N-
diisopropylethylamine; DMFDMA, 1,1-dimethoxy-N,N-dime-
thylmethanamine; ET, essential thrombocytemia; EtOH,
ethanol; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronoium hexafluorophosphate; HBA, hydrogen bond
acceptor; HBD, hydrogen bond donor; Jak, Janus kinase;
HLM, human liver microsome; HOAc, acetic acid; LE, ligand
efficiency; mCPBA, meta-chloroperoxybenzoic acid; MDCK,
Madin−Darby canine kidney cells; MF, myelofibrosis; MPD,
myeloproliferative disorder; MSD, meso scale discovery; NCS,
N-chlorosuccinimide; PK/PD, pharmacokinetic/pharmakody-
namic; PV, polycythemia vera; PyAOP, 7-azabenzotriazol-1-
yloxy-tris-(pyrrolidino)phosphonium hexafluorophosphate;
SEM, 2-(trimethylsilyl)ethoxymethyl; STAT, signal transducer
and activator of transcription; tBuOH, tert-butanol; TDI, time-
dependent inhibition; TFA, trifluoroacetic acid; THF, tetrahy-
drofuran
Thermodynamic Solubility. Compounds were dissolved in
DMSO to a concentration of 10 mM, and these solutions were used
to make serial dilutions to 2, 20, and 200 μM. A calibration curve for
each compound was generated by LC/UV (254 nM) measurement.
Solid compound (2 mg) was placed into Whatman UniPrep filter vials
with 0.45 mL PBS buffer (pH 6.5). The vials were shaken for 24 h at
room temperature and filtered. The samples were diluted 10× with
PBS buffer and analyzed by LC/UV, and the solubility calculated by
comparison with the calibration curves.
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■
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MDCK Permeability. Experiments were carried out as described.44
Crystallography. Jak2 JH1 (kinase) domain was prepared,
complexed with compound 8, crystallized, and its molecular structure
determined as described as previously described.45 See Supporting
Information Table S2.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Mouse pharmacokinetic parameters for selected compounds, X-
ray data collection and refinement details, cocrystal structure
(2.3 Å) of compound 8 in the active site of the Jak2 kinase with
protein surface displayed, and preparation and characterization
of compounds 6b−j, 7d−k, 9b−c, 10b−c, 13b−g, 14b−e,
14g−j, 18c−d, 19b−d, 20b−e, 22b, and 26b. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID for the 8−Jak2 complex: 4HGE.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 650-467-6671. E-mail: lyssikatos.joseph@gene.com.
Present Addresses
#Department of Cell Biology, Novartis, Emeryville, California.
¶Department of Infectious Disease, MedImmune, Gaithersburg,
Maryland.
Notes
The authors declare no competing financial interest.
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