Identification of 4-aminopyrazolylpyrimidines as potent inhibitors of Trk kinases

Article abstract of DOI:10.1021/jm800343j

The design, synthesis and biological evaluation of a series of 4-aminopyrazolylpyrimidines as potent Trk kinase inhibitors is reported. High-throughput screening identified a promising hit in the 4- aminopyrazolylpyrimidine chemotype. Initial optimization of the series led to more potent Trk inhibitors. Further optimization using two strategies resulted in significant improvement of physical properties and led to the discovery of 10z (AZ-23), a potent, orally bioavailable Trk A/B inhibitor. The compound offers the potential to test the hypothesis that modulation of Trk activity will be of benefit in the treatment of cancer and other indications in vivo.

Full text of DOI:10.1021/jm800343j

4672
J. Med. Chem. 2008, 51, 4672–4684
Identification of 4-Aminopyrazolylpyrimidines as Potent Inhibitors of Trk Kinases
Tao Wang,*,† Michelle L. Lamb,† David A. Scott,† Haixia Wang,† Michael H. Block,† Paul D. Lyne,† John W. Lee,†
Audrey M. Davies,† Hai-Jun Zhang,† Yanyi Zhu,† Fei Gu,† Yongxin Han,| Bin Wang,| Peter J. Mohr,| Robert J. Kaus,|
John A. Josey,| Ethan Hoffmann,§ Ken Thress,‡ Terry MacIntyre,‡ Haiyun Wang,‡ Charles A. Omer,‡ and Dingwei Yu*,†
Departments of Cancer Chemistry, Cancer Bioscience, Drug Metabolism and Pharmacokinetics, AstraZeneca R&D Boston,
35 Gatehouse DriVe, Waltham, Massachusetts 02451, Array BioPharma, 3200 Walnut Street, Boulder, Colorado 80301
ReceiVed March 26, 2008
The design, synthesis and biological evaluation of a series of 4-aminopyrazolylpyrimidines as potent Trk
kinase inhibitors is reported. High-throughput screening identified a promising hit in the 4-aminopyra-
zolylpyrimidine chemotype. Initial optimization of the series led to more potent Trk inhibitors. Further
optimization using two strategies resulted in significant improvement of physical properties and led to the
discovery of 10z (AZ-23), a potent, orally bioavailable Trk A/B inhibitor. The compound offers the potential
to test the hypothesis that modulation of Trk activity will be of benefit in the treatment of cancer and other
indications in vivo.
Introduction
Receptor tyrosine kinases (RTKs)a are a subfamily of protein
kinases that play a critical role in cell signaling and are involved
in a variety of normal and cancer related processes including
cell proliferation, survival, angiogenesis, and metastasis.1 Cur-
rently, up to 100 different RTKs including tropomyosin-related
kinases (Trks) have been identified. Trks are the high affinity
receptors activated by a group of soluble growth factors called
neurotrophins (NT). The Trk receptor family has three highly
homologous isoforms: TrkA, TrkB, and TrkC. Among the
neurotrophins, there are (i) nerve growth factor (NGF), which
activates TrkA, (ii) brain-derived neurotrophic factor (BDNF)
and NT-4/5, which activate TrkB, and (iii) NT-3, which activates
TrkC. Each Trk receptor contains an extracellular domain (NT
ligand binding), a transmembrane region, and an intracellular
domain (including kinase domain). Upon binding of the ligand,
Figure 1. Trk inhibitors in literature.
the kinase catalyzes its own phosphorylation and triggers
downstream signal transduction pathways.2
overexpression, activation, amplification, and/or mutation of
Trks are associated with many cancers including neuroblastoma,6–9
Trks are widely expressed in neuronal tissue during develop-
ment where they are critical for the maintenance and survival
ovarian,10 and colorectal cancer.11
of these cells. A postembryonic role for the Trk/neurotrophin
axis, however, remains in question. There are reports showing
that Trks play an important role in both the development and
function of the nervous system as well as in pain sensation.3,4
In the past decade, a considerable number of reports linking
Trk signaling with cancer have been published. For example,
while Trks are expressed at low levels outside the nervous
system in the adult, Trk expression is increased in late stage
prostate cancers.5 Furthermore, recent literature also shows that
Among the earliest inhibitors of Trk kinases was 1 (CEP-
701, Figure 1) and its later analogues, which are potent inhibitors
of Trks along with other kinases.12 Other inhibitors that hit Trks
along with their main targets include 1,4,5,6-tetrahydropyr-
rolo[3,4-c]pyrazoles (Aurora kinases)13 and keto-pyrrolopyri-
midines (Tie-2 kinase).14 In addition, there have been several
reports of selective Trk tyrosine kinase inhibitors in the literature.
Oxindole 2, isothiazole 3, and thiazole 4 (Figure 1) were
reported as potent Trk A inhibitors.15–17 In patent applications,
aminopyrimidines,18 pyrazolyl ureas,19 and pyrazolyl condensed
cyclic compounds20 were described as Trk inhibitors to treat
cancer or pain.
In this paper, we report the identification and optimization
of a series of 4-aminopyrazolylpyrimidines as potent Trk
inhibitors that exhibit excellent physical properties and good
oral pharmacokinetics.
High throughput screening of the AstraZeneca compound
collection against the TrkB enzyme resulted in the identification
of a 4-aminopyrazolylpyrimidine derivative 5 (Figure 2) as a
hit. The compound was found to have an IC50 against TrkB of
1.1 µM and was also potent against TrkA (IC50 ) 0.27 µM).
The series was attractive because 2,4-disubstituted pyrimidines
* To whom correspondence should be addressed. For T.W.: phone, (781)
839-4734; fax: (781) 839-4230; E-mail: tao.wang@astrazeneca.com. For
D.Y.: phone, (781) 839-4606; fax, (781)839)-4230; E-mail:
dingwei.yu@astrazeneca.com.
†
Department of Cancer Chemistry, AstraZeneca R&D Boston.
Department of Cancer Bioscience, AstraZeneca R&D Boston.
Department of Drug Metabolism and Pharmacokinetics, AstraZeneca
‡
§
R&D Boston.
|
Array BioPharma, 3200 Walnut Street, Boulder, CO 80301
a Abbreviations: RTK, receptor tyrosine kinase; Trk, tropomyosin-related
kinase; NT, neurotrophin; NGF, nerve growth factor; BDNF, brain-derived
neurotrophic factor; HTS, high throughput screening; DIAD, diisopropy-
lazodicarboxylate; CDK2, cyclin-dependent kinase 2; DMA, dimethylac-
etamide; PEG, polyethylene glycol; HPMC, hydroxypropylmethylcellulose;
D5W, dextrose 5% in water; DPPF, 1,1′-bis(diphenylphosphino)ferrocene.
10.1021/jm800343j CCC: $40.75
2008 American Chemical Society
Published on Web 07/23/2008

4-Aminopyrazolylpyrimidines as Trk Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4673
namine was initiated from compound 19 (Scheme 4). Treatment
of 19 with Zn(CN)2 in the presence of catalyst generated nitrile
20 in 72% yield. Grignard addition to 20 followed by acetylation
gave the vinyl acetamide 21. Catalytic asymmetric hydrogena-
tion23 provided the desired acetamide 22 in 88% yield and 95%
enantiomeric excess. Protection of the amide N-H with Boc
anhydride, followed by treatment with LiOH, resulted in the
Boc protected compound 23, which was then deprotected with
HCl/1,4-dioxane to give the desired amine (S)-9a.
The synthetic route of the corresponding pyrimidine ring
system (S)-9b (Scheme 5) began with 2-chloro-5-fluoropyri-
midine 24 and followed an analogous approach to that shown
in Scheme 4.
Figure 2. HTS hit.
Scheme 1. Synthesis of 10a-z, 11a
Results and Discussion
There are two potential binding modes for compound 5 to
the hinge of Trk enzymes: either through the pyrimidine or
pyrazole moieties. Because compound 5 is moderately potent
against CDK2 enzyme (IC50: 0.11 µM), a surrogate crystal
structure of 5 with CDK2 (data not shown), which has 68%
homology to TrkB in the active site, was obtained. This structure
suggested that the series binds to the ATP-binding site hinge
between N- and C-terminal domains through the pyrazole, and
that the cyclopropyl ring would stack against the phenylalanine
gatekeeper residue, which is common to both Trk and CDK2
kinases. This region of the inhibitor was then held constant and
the effect of substitutions at the pyrimidine 2-position in
compound 5 was evaluated. The isoxazole was replaced with a
phenyl group, as benzylamines are synthetically more accessible
than isoxazolylmethylamines. The benzylamine analogue 10a
exhibited an IC50 of 0.75 µM against TrkA (for simplicity,
potencies against TrkA are reported in this paper because
comparable potencies against TrkA and TrkB were observed
throughout this series of compounds). Next, a small library was
designed to probe substitutions of the newly installed phenyl
ring as exemplified in Table 1. Substitution at the 3-position of
this ring with a methoxy group (10b) did not provide a potency
improvement when compared with 10a. At the 2-position, the
methoxy group (10d) is preferred over chloro (10c). Finally,
substitution with halogen groups (10e and 10f) at the 4-position
consistently improved potency compared to 10a. Incorporation
of a halogen atom on the aromatic ring was especially attractive
because the halogen atom might reduce the potential for
metabolism at this position of the molecule. In a human
microsomal stability assay, compound 10e (Clint: 28 µL/min/
mg) was indeed more stable when compared with 10a (Clint:
66 µL/min/mg).
The presence of a methylene moiety between the 2-position
of the pyrimidine and the phenyl ring prompted an exploration
of the SAR around this linker region. It was reasoned that this
benzylic position might be prone to metabolism, and substitution
at R3 might result in compounds with better metabolic stability.
Initial evaluation of racemic methyl-substituted compounds
indicated improved potency over unsubstituted analogues (data
not shown), and molecular docking of each isomer in the TrkB
homology model (see experimental details, homology modeling)
indicated a preference for the (S)-methyl enantiomer, as il-
lustrated in Figure 3 with example 10k. It was subsequently
determined that one enantiomer is indeed more potent than the
other. For instance, small groups (e.g., methyl and hydroxym-
ethylene) (10g and 10j) show improvements in enzyme potency
(around 5-fold) over compound 10a for only one of the
enantiomers. The other enantiomers are less active (10h and
10i). It should be noted that the preferred configuration remains
could be readily synthesized in two steps from a 2,4-dichloro-
pyrimidine core and a convergent, modular synthetic approach
allowed the rapid elucidation of SAR.
Chemistry
The general synthetic sequence for the 2,4-disubstituted
pyrimidines is shown in Scheme 1. 2,4-Dichloropyrimidines 6
were reacted with aminopyrazoles 7 in the presence of base
(e.g., NEt3) to give 4-substituted-2-chloropyrimidines 8. For
inactivated pyrimidines (e.g., 5-CH3/H substitutions), the reac-
tion mixture was heated to 70 °C. Nucleophilic aromatic
substitution of intermediates 8 with suitable amines 9 was
achieved by heating under basic conditions to provide target
compounds 10a-z and 11a.
For 2,4,6-trisubstituted pyrimidines, trichloropyrimidine cores
12 were first reacted with aminopyrazoles 7 in the presence of
base to give 4-substituted-2,6-dichloropyrimidines 13a-d (Scheme
2). Nucleophilic aromatic substitutions of intermediates 13a-d
with amines 9 provided intermediates 14a-f together with their
regioisomers (6-adducts, separable by silica gel column chro-
matography). Treatment of 14a-f with either alkoxide or an
amine generated the target compounds 15a-i.
As Scheme 3 illustrates, aminopyrazoles with alkyl substitu-
tion at the 5-position can be readily synthesized from ester 16.21
For alkoxyl-substituted aminopyrazoles, this method is not
feasible and therefore a one-step synthesis was developed using
Mitsunobu conditions. Hydroxyaminopyrazole 17 was treated
with a suitable alcohol in the presence of triphenylphosphine
and diisopropylazodicarboxylate (DIAD) to give the desired
products 7a-b. Thiomethyl analogue 7c was synthesized from
3,3-bis(methylthio)acrylonitrile 1822 in the presence of hydrazine.
Most R-methyl benzylamines and R-hydroxymethyl-benzy-
lamines are commercially available in both enantiopure forms.
The synthesis of enantiopure (S)-1-(5-fluoropyridin-2-yl)etha-

4674 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Scheme 2. Synthesis of 15a-i
Wang et al.
Scheme 3. Synthesis of Aminopyrazolesa
Scheme 5. Synthesis of (S)-9ba
a Reagents and conditions: (a)CH3CN, NaH, dioxane; (b)H2NNH2; (c)
R9OH, DIAD, PPH3, CH2Cl2, heating.
a Reagents and conditions: (a) Zn(CN)2, Pd2(dba)3, DPPF, Zn, DMA,
100°C; (b) MeMgBr, THF; (c) Ac2O, THF/DCM; (d) (S,S)Rh-Et-DUPHOS-
OTf, MeOH, H2 (150 psi); (e) Boc2O, DMAP, THF; (f) LiOH; (g)
HCl/1,4-dioxane.
Scheme 4. Synthesis of (S)-9aa
benzylic position would improve metabolic stability. When
substitution at the benzylic site was combined with a 4-fluo-
rophenyl ring, extremely potent compounds such as 10k and
10l were identified. Slightly larger linear groups are also
tolerated at the R3 position (10m and 10n).
As part of the evaluation of the SAR at C-5 of the pyrimidine
(Table 1), replacement of 5-Br with 5-Cl retained enzyme
potency while reducing the molecular weight of the compound.
Alternative substitutions at the pyrimidine 5-position were also
explored (10o-q in Table 2). Because enzyme potency for many
compounds in this series had reached the low end of the enzyme
assay detection limit (∼0.003 µM), a modification of a published
assay24 in which phospho-TrkA inhibition in a cell line
expressing a constitutive form of TrkA (MCF-10A TrkA-
deletion) was evaluated.25 For example, compound 10k exhib-
ited an IC50 of 0.006 µM in the more sensitive TrkA cell assay,
comparable to its enzyme assay result (0.006 µM). Based on
the cellular assay, bromine and chlorine remain among the best
moieties for potency at this position.
a Reagents and conditions: (a) Zn(CN)2, Pd2(dba)3, DPPF, Zn, DMA,
95 °C; (b) MeMgBr, THF; (c) Ac2O; (d) (S,S)Rh-Et-DUPHOS-OTf, MeOH,
H2 (150 psi); (e) Boc2O; (f) LiOH; (g) HCl/1,4-dioxane.
consistent between R-methyl and R-hydroxymethyl, but the
designation switches from (S)-configuration for the R-methyl
to (R)-configuration for the R-hydroxymethyl moiety. (S)-Methyl
was equipotent when compared to (R)-hydroxymethyl based on
the IC50’s of compounds 10g and 10j. In a human microsomal
stability assay, compound 10g (Clint: 29 µL/min/mg) was also
more stable when compared with 10a (Clint: 66 µL/min/mg).
This tended to support the original hypothesis that blocking the
To determine whether the cyclopropyl group was optimal,
other pyrazole substituents were also explored (Table 2). It was
found that TrkA in vitro cell potency improved from no
substitution (10v) to methyl (10t) and cyclopropyl (10k and

4-Aminopyrazolylpyrimidines as Trk Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4675
Table 1. SAR at R2 and R3 Positions
the solubility of the resulting compounds. Indeed the original
HTS hit compound 5 contained a heterocycle (isoxazole), this
area of chemistry was further explored, with a focus on pyridyl
and pyrimidyl moieties as a replacement for the phenyl group.
As Table 5 shows, introduction of nitrogens at the 2 and/or
6 position of the phenyl ring was tolerated in terms of cellular
potency and compound solubilities all demonstrated significant
improvement to greater than 100 µM, with the trend of phenyl
< 2-pyridyl < 2-pyrimidyl (the solubility for 10r is 4 µM, as
compared to 100 µM for 10z (AZ-23) and 440 µM for 11a). In
a human plasma protein-binding assay, 10z was found to have
6.9% free drug in plasma. A combination of the heterocycle
replacement and C-6 soluble side chain approach further
increased the solubility (compounds 15h and 15i).
On the basis of the SAR obtained in this series, several lead
compounds were selected for in vivo PK profiling. As Table 6
shows, the clearance of 10y, 10z was low to moderate across
two species (rat and dog). Half-lives (iv dosing) are somewhat
short in rat but reasonable in dog. Compound 15i also showed
a reasonable profile in rat (iv and po) and dog PK (iv) studies,
indicating that the potential metabolism of the solubilizing side
chains was not problematic in these species. The oral bioavail-
abilities for all three compounds ranged from 24% to 60% in
rat. On the basis of further evaluation in a pharmacodynamic
model and selectivity,27 compound 10z was chosen for progres-
sion into in vivo disease models.
In summary, a series of 4-aminopyrazolylpyrimidines has
been discovered as highly potent TrkA/B inhibitors. SAR
exploration started from an HTS hit, compound 5, and in vitro
potency was quickly improved to the single digit nM level (e.g.,
compounds 10k and 10l). Subsequently, two strategies were
utilized to improve the physical properties resulting in com-
pounds like 10z that has excellent overall properties in PK,
physical properties, and also retains the target potency. Being
a potent, orally bioavailable inhibitor, 10z offers the potential
to test the hypothesis that modulation of Trk activity will be of
benefit in the treatment of cancer and other indications in vivo.
The evaluation of such compounds (including 10z) will be
reported in due course.
compd
X
R2
R3
TrkA IC50 (µM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
Cl
H
3-OMe
2-Cl
2-OMe
4-F
4-Cl
H
H
H
H
F
F
F
H
H
H
H
H
H
0.75
0.72
1.17
0.12
0.10
0.36
0.10
0.65
1.90
0.12
0.006
0.010
0.013
0.015
(S)-Me
(R)-Me
(S)-CH2OH
(R)-CH2OH
(S)-Me
(R)-CH2OH
(S)-CH2CONMe2
(S)-CH2CONHMe
F
10l) and that the position can tolerate OiPr (10r) and even SCH3
groups (10s). However, further increase in linear length (10u)
or bulk (10w) resulted in 45-fold and >90-fold reduction in
cell potency respectively from compound 10k. Again com-
pounds 10k, 10l, and 10r are among the most potent Trk
compounds in this series.
Having achieved single-digit nanomolar in vitro cell potency,
pharmacokinetic properties of the lead compounds were inves-
tigated. As shown in Table 3, compound 10k showed promise
in rodent studies having reasonable in vivo clearance, moderate
half-life, and an oral bioavailability of 32%. However, the
compound displayed aqueous solubility of <3 µM and over 99%
plasma protein binding. Compound 10l had a larger free-fraction
in plasma, but in vivo clearance in rat was high and the aqueous
solubility was 1.7 µM.
Experimental Section
There are several known strategies to improve the physical
properties of compounds with low solubility.26 One strategy is
to attach side chains to the parent molecule to improve solubility
in a position where SAR is not very sensitive. For kinase
inhibitors, solubilizing groups are typically introduced in the
area of the molecule pointing to the kinase solvent channel,
which in this case is the pyrimidine C-6 position (Figure 3).
As Table 4 demonstrates, a variety of functional groups are
tolerated in the TrkA kinase solvent channel including terminal
basic rings (such as 15a) and neutral linear (15c) and branched
hydroxyl side chains (15b, 15d-g). Cellular potencies were all
comparable to that of 10l (except for methylpyrazole 15f, EC50:
0.06 µM). However, the solubilities of 15a-g were generally
10-fold higher than 10l (except for 15c). These data demonstrate
that attachment of solubilizing side chains is a reasonable way
of improving physical properties. Two potential issues associated
with this approach are that the newly added side chains might
be accessible for metabolism and the modified compounds have
higher molecular weights.
1
General. H NMR spectra were recorded on Bruker 300 MHz
or 400 MHz NMR spectrometers. Chemical shifts are expressed in
part per million (ppm, δ units). Coupling constants are in units of
hertz (Hz). Splitting patterns describe apparent multiplicities and
are designated as s (singlet), d (doublet), dd (doublet-doublet), t
(triplet), q (quartet), m (multiplet), br s (broad singlet). LC-MS
analyses were performed with an Agilent 1100 equipped with
Waters columns eluted with a gradient mixture of H2O-acetonitrile
with formic acid and ammonium acetate. All compounds analyzed
were g95% pure. HRMS were recorded on a Waters LCT TOF
mass spectrometer. Reverse phase chromatography was performed
with Gilson systems using a YMC-AQC18 reverse phase HPLC
column with dimensions 20 mm/100 and 50 mm/250 in water/
MeCN with 0.1% TFA as mobile phase. HPLC retention times (Rt)
are reported in minutes. Elemental analyses (C, H, N) were
performed by QTI, P.O. Box 470, 291 Route 22 East, Salem
Industrial Park, Building 5, Whitehouse, NJ 08888. Most of the
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 ISCO
Combi-flash systems (4700 Superior Street, Lincoln, NE) unless
otherwise mentioned using silica gel cartridges.
The second strategy used to improve physical properties was
to modify the “core template” so that the resulting compounds
were more soluble without the additional solvent channel side
chains. As heterocycles typically have lower logD compared
to phenyl rings, we reasoned that this change would improve
TrkB Enzyme Assay. TrkB kinase activity was measured against
its ability to phosphorylate synthetic tyrosine residues within a
generic polypeptide substrate using homogeneous time-resolved

4676 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Wang et al.
Figure 3. (A) Predicted binding through the pyrazole motif for compound 10k docked into a homology model of TrkB (only polar hydrogen atoms
shown). The cyclopropyl moiety stacks against the phenylalanine gatekeeper residue. The pyrimidine C6-position is solvent-accessible. (B) View
from solvent channel into the ATP-binding site. The (S)-methyl configuration of 10k is preferred over the (R)-methyl analogue.
Table 2. SAR at X and R1 Positions
BSA, 10 mM MnCl2, 60 µM ATP, and 50nM biotinylated Poly
EAY) was incubated for 50 min at room temperature, and the kinase
reaction stopped by the addition of 60 mM ethylenediaminetet-
raacetic acid (EDTA). Reaction products are detected with the
addition of strepavidin-linked and phosphotyrosine-specific antibod-
ies using the Tecan Ultra Evolution microplate fluorometer after
additional 3 h incubation at room temperature.
TrkA Enzyme Assay. TrkA kinase activity was determined by
measuring the kinase’s ability to phosphorylate synthetic tyrosine
residues within a generic polypeptide substrate using amplified
luminescent proximity assay (Alphascreen) technology (Perkin-
Elmer, 940 Winter Street, Waltham, MA).
The intracellular domain of human TrkA kinase was HIS tagged,
compd
X
R10
R1
Trk A EC50 (µM)
expressed in SF9 insect cells, and purified using standard nickel
column chromatography. The kinase assay (0.838 ng/mL Trk A, 9
mM HEPES, 45 µg/mL BSA, 10 mM MnCl2, 5 nM Poly EY, 0.01%
Triton X-100, 70 µM ATP) was incubated for 20 min at room
temperature and the reaction was stopped by the addition of 30
mM ethylenediaminetetraacetic acid (EDTA). The reaction products
were detected with the addition of streptavidin coated donor beads
and phosphotyrosine-specific antibodies coated acceptor beads.
Plates were read using the EnVision Multilabel plate reader after
an overnight incubation at room temperature.
TrkA Mechanism of Action (MOA) Cell Assay. This is a cell-
based assay for evaluating inhibitors of TrkA phosphorylation in
MCF10A overexpressing TrkA deletion mutant cell line made in
house.25 A cDNA encoded a TrkA protein that contained a 75-aa
deletion in the extracellular domain of the receptor and was named
∆TrkA. The deletion resulted in activation of the tyrosine kinase
domain, leading to constitutive tyrosine phosphorylation of TrkA.
MCF10A cells were infected with retrovirus made from an in-house
retroviral vector that has the ∆TrkA cDNA insertion.
10k
10o
10p
10q
10l
10r
10s
10t
Br
H
F
H
H
H
H
OH
H
OH
H
OH
OH
H
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
OiPr
0.006
0.02
0.017
0.01
0.004
0.006
0.036
0.081
0.28
1.5
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
SMe
Me
OPr
10u
10v
10w
H
tBu
3
Table 3. Rat Pharmacokinetics Dataa
clearance volume of half-
(iv, mL/ distribution life bioavailability solubility plasma
min/kg)
oral
human
(L/kg)
(iv, h)
(%)
(µM)
free (%)
10k
10l
9.5
87
0.84
6.9
1.5
1.9
32
71
<3
1.7
<1
4.4
a Han Wistar Rat male; 10 mg/kg po (0.1% HPMC); 3 mg/kg iv (DMA/
PEG/saline ) 40/40/20).
After cells have been treated with different doses of TrkA
inhibitor for 75 min, the TrkA receptor was captured from cell
lysates in microtiter plates coated with an antitrk antibody (C-14)
from Santa Cruz. The amount of TrkA phosphorylation was then
measured using europium-labeled antiphosphotyrosine antibody (P-
Tyr-100 from Perkin-Elmer) with fluorometric detection. (This assay
was modified based on a TRF-based TrkA intact cell assay.24
fluorescence (HTRF) technology. The intracellular domain of human
TrkB kinase was HIS tagged, expressed in SF9 insect cells, and
purified using standard nickel column chromatography. The kinase
assay (400 ng/mL of recombinant TrkB, 10 mM HEPES, 0.005%

4-Aminopyrazolylpyrimidines as Trk Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4677
Table 4. SAR at R7 Positions
Homology Modeling. The program Modeler as implemented in
the Accelrys Insight II package28 was used to generate the TrkB
homology model. Considering the flexibility of some of the
structural elements in the ATP binding site, five template kinases
were chosen as described below. As a result, the homology model
is less likely biased by any single kinase structure. The overall
reliability of the model is very good, especially at places where
the structures are best conserved among all kinases such as residues
involved in ATP binding.
A BLAST search using the TrkB sequence against all kinases
with crystal structure information at that time provided the top
kinases with the most sequence similarity. Insulin receptor kinase
(1IR3),29 fibroblast growth-factor receptor kinase (1FGK),30 LCK2
(1QPC),31 and c-Src kinase (2SRC)32 were each among the top 10
most similar kinases to TrkB and all were crystallized with ATP
analogues. A structure of CDK2 (1H08)33 crystallized with a
bisanilinopyrimidine inhibitor 29 was included as the fifth template
due to the abundance of structural information for its inhibitors.
The five template kinases were superimposed and their sequences
aligned based on their structures. The TrkB sequence was then
aligned to the group, and Modeler generated three homology
models. The backbone of the three models varied only at the two
termini and in one extended loop region away from the ATP binding
site. There were variations at the side chain level but not so much
as to affect ATP binding. Therefore, one of the models was chosen
for further refinement.
The protein/inhibitor complex then went through limited, binding
site-focused minimization with CHARMm28 to optimize the inhibi-
tor/protein interactions.
Docking. Docking studies were carried out with Glide.34 The
TrkB homology model complexed with 30 was prepared for docking
by first modifying the atom and bond types of 30 to be compatible
with Schro¨dinger software and adding hydrogen atoms. The
Schro¨dinger protein preparation utilities pprep and impref were run
to select appropriate side-chain protonation states, refine, and
minimize the structure. Within Glide, docking grids were generated
using a 12 Å search box and 30 Å grid (defaults), with one
constraint defined such that docked ligands were each required to
form a hydrogen bond with the backbone amide NH of the hinge
methionine residue.
Three-dimensional stereoisomeric structures (with hydrogens) for
ligandssuchas10kweregeneratedfromSMILESusingCORINA,35,36
and these conformations were then minimized using the MMFF
force field in the Schro¨dinger environment using the utility scripts
sdconVert and premin.
The GlideXP docking protocol employed docked each ligand
flexibly and incorporated a van der Waals radius scaling factor of
0.8 for atoms with partial charge less than 0.15 to soften the
potential. A maximum of 100 poses were stored for each ligand,
and poses were discarded as duplicates if the rmsd was less than
1.5 Å or the maximum atomic displacement was less than 1.3 Å.
Poses were then ranked by their computed GlideXP score.
Synthesis of Aminopyrazoles 7. 5-Propan-2-yloxy-1H-pyrazol-
3-amine (7a). A mixture of triphenylphosphine (1.6 g, 6.1 mmol),
diisopropylazodicarboxylate (1.2 mL, 6.1 mmol) in DCM (20 mL)
An HTS hit (30, TrkB IC50 ) 0.646 µM) of very similar structure
to 29 (Figure 4) was modeled into the ATP pocket of TrkB based
on the overlay of the CDK2-29 complex and the TrkB model.

4678 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Table 5. SAR at Y/Y′ Positions
Wang et al.
Table 6. Pharmacokinetics of Lead Compoundsa
5-Methylsulfanyl-1H-pyrazol-3-amine (7c). 3,3-Bis(methylthio)-
acrylonitrile (compound 18; 300 mg, 2 mmol) in H2NNH2H2O (1
mL) was heated at 120 °C for 1 h. Hydrazine was removed under
vacuum to give a pale-green oil (267 mg). The crude product was
used without purification.
General Synthetic Procedure for Compounds 8a-k. A solution
of pyrimidine 6 (1.0 equiv), aminopyrazole 7 (1.0 equiv), and
triethylamine (1.5 equiv) in EtOH was stirred at room temperature
for 10 h (or at 60 °C for inactivated pyrimidines). Solvent was
removed, and EtOAc was added. The solution was washed with
water, dried over anhydrous sodium sulfate, and concentrated. Flash
column chromatography (30-100% EtOAc in hexanes) gave the
desired compound as a solid.
compd
species
iv clearance (mL/min/kg)
T1/2 (h)
F %
10y
10y
10z
10z
15i
rat
dog
rat
dog
rat
dog
7.2
8.5
17
9
23
1.3
10.3
1.4
7.5
1.6
60
NT
31
60
24
15i
9.8
8.3
NT
a Han Wistar rat male; 10 mg/kg po (0.1% HPMC); 3 mg/kg iv (DMA/
PEG/saline ) 40/40/20). Beagle dog male; 10 mg/kg po (0.1% HPMC); 3
mg/kg iv (50% PEG/D5W).
5-Bromo-2-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidin-
4-amine (8a). 1H NMR (300 MHz, DMSO-d6) δ ppm 12.34 (br s,
1H), 8.41 (m, 1H), 6.15 (m, 1H), 1.89 (m, 1H), 0.91 (m, 2H), 0.67
(m, 2H).
2,5-Dichloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)pyrimidin-4-
amine (8b). 1H NMR (300 MHz, DMSO-d6) δ ppm 12.60 (s, 1H),
9.90 (s, 1H), 8.60 (s, 1H), 6.40 (s, 1H), 2.18 (m, 1H), 1.20 (m,
2H), 0.92 (m, 2H).
Figure 4. Compound 29 from CDK2 structure, 30 from TrkB HTS
hit.
2-Chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)pyrimidin-4-
amine (8c). The reaction is similar to that for 8a except that the
reaction was performed at 70 °C. 1H NMR (300 MHz, DMSO-d6)
δ ppm 12.14 (s, 1H), 10.22 (s, 1H), 8.14 (m, 1H), 5.90 (m, 1H),
1.86 (m, 1H), 0.92 (m, 2H), 0.66 (m, 2H).
was stirred at room temperature for 20 min. The mixture was then
cooled to 0 °C and to the mixture was added 5-amino-2H-pyrazole-
3-ol (compound 17, 0.55 g, 5.6 mmol) and 2-propanol (0.34 g, 5.6
mmol) and the mixture was stirred at room temperature for 2 days.
Solid was filtered off, and the filtrate was concentrated. Flash
column chromatography on silica gel gave the desired product
5-propan-2-yloxy-1H-pyrazol-3-amine as a white solid (197 mg,
2-Chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-5-fluoropyrimidin-
4-amine (8d). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.28 (br s,
1H), 10.38 (s, 1H), 8.23 (s, 1H), 6.27 (s, 1H), 1.92 (m, 1H), 0.94
(m, 2H), 0.70 (m, 2H).
1
25%). H NMR (400 MHz, DMSO-d6) δ ppm 10.25 (br s, 1H),
7.67 (m, 1H), 4.84 (m, 1H), 4.64 (s, 1H), 4.54 (m, 1H), 1.19 (d, J
) 6 Hz, 6H).
2-Chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-5-methylpyrimidin-
4-amine (8e). The reaction is similar to that for 8a except that the
reaction was performed at 70 °C. 1H NMR (300 MHz, DMSO-d6)
δ ppm 12.18 (br, s, 1H), 9.29 (s, 1H), 7.97 (s, 1H), 6.27 (s, 1H),
2.11 (s, 3H), 1.91 (m, 1H), 0.93 (m, 2H), 0.70 (m, 2H).
5-Propoxy-1H-pyrazol-3-amine (7b). Following a similar pro-
cedure to that for 7a, compound 7c was synthesized from 5-amino-
2H-pyrazol-3-ol and 1-propanol. MS: calcd, 141; found, [M + H]+
142.

4-Aminopyrazolylpyrimidines as Trk Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4679
2,5-Dichloro-N-(5-propan-2-yloxy-1H-pyrazol-3-yl)pyrimidin-4-
amine (8f). H NMR (400 MHz, DMSO-d6) δ ppm 10.00 (br, s,
1H), 8.40 (s, 1H), 5.77 (s, 1H), 4.67 (m, 1H), 1.28 (m, 6H).
5-Bromo-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(1R)-1-phenyl-
ethyl]pyrimidine-2,4-diamine (10h). 1H NMR (300 MHz, DMSO-
d6) δ ppm 12.08 (s, 1H), 7.97 (m, 2H), 7.20-7.44 (m, 5H),
5.80-6.00 (m, 1H), 5.00 (m, 1H), 1.86 (m, 1H), 1.42 (m, 3H),
0.93 (m, 2H), 0.63 (m, 2H). HRMS (ESI) m/z calcd for C18H19BrN6
399.0933; found 399.0949 (∆ ) 4.1 ppm). Anal. (C18H19BrN6 ·
0.85H2O) C, H, N.
1
2,5-Dichloro-N-[5-(methylthio)-1H-pyrazol-3-yl]pyrimidin-4-
1
amine (8g). H NMR (300 MHz, CD3OD): δ ppm 8.24 (s, 1H),
6.72 (s, 1H), 2.49 (s, 3H).
2,5-Dichloro-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine (8h).
1H NMR (400 MHz, DMSO-d6) δ ppm 12.27 (br, s, 1H), 9.21 (s,
1H), 8.39 (s, 1H), 6.23 (s, 1H), 2.23 (s, 3H).
(2S)-2-[[5-Bromo-4-[(5-cyclopropyl-2H-pyrazol-3-yl)amino]py-
1
rimidin-2-yl]amino]-2-phenylethanol (10i). H NMR (300 MHz,
2,5-Dichloro-N-(5-propoxy-2H-pyrazol-3-yl)pyrimidin-4-
DMSO-d6) δ ppm 10.11 (m, 1H), 8.70 (s, 1H), 8.12 (m, 1H),
7.30-7.40 (m, 5H), 6.23 (m, 1H), 5.32 (m, 1H), 4.18 (m, 1H),
4.11 (m, 2H), 2.04 (m, 1H), 1.20 (m, 2H), 0.91 (m, 2H). HRMS
(ESI) m/z calcd for C18H19BrN6O 415.0882; found 415.0886 (∆ )
1.0 ppm). Anal. (C18H19BrN6O·0.3C4H8O2) C, H, N.
1
amine (8i). H NMR (300 MHz, CD3OD) δ ppm 8.28 (br s, 1H),
5.60 (br s, 1H), 4.06 (m, 2H), 1.78 (m, 2H), 1.02 (t, J ) 7.5 Hz,
3H).
2,5-Dichloro-N-(1H-pyrazol-5-yl)pyrimidin-4-amine (8j). 1H NMR
(400 MHz, DMSO-d6) δ ppm 12.64 (br s, 1H), 9.83 (br s, 1H),
8.35 (s, 1H), 7.74 (d, J ) 2 Hz, 1H), 6.51 (d, J ) 2 Hz, 1H).
N-(3-tert-Butyl-1H-pyrazol-5-yl)-2,5-dichloropyrimidin-4-
(2R)-2-[[5-Bromo-4-[(5-cyclopropyl-2H-pyrazol-3-yl)amino]py-
1
rimidin-2-yl]amino]-2-phenylethanol (10j). H NMR (300 MHz,
DMSO-d6) δ ppm 9.45 (br s, 1H), 8.40 (br s, 1H), 8.22 (s, 1H),
7.30 (m, 5H), 5.95 (s, 1H), 4.93 (m, 1H), 3.68 (m, 2H), 1.90 (m,
1H), 0.95 (m, 2H), 0.69 (m, 2H). HRMS (ESI) m/z calcd for
C18H19BrN6O 415.0882; found 415.0901 (∆ ) 4.6 ppm). Anal.
(C18H19BrN6O·1.6C2HF3O2) C, H, N.
5-Bromo-N′-(5-cyclopropyl-2H-pyrazol-3-yl)-N-[(1S)-1-(4-fluo-
rophenyl)ethyl]pyrimidine-2,4-diamine (10k). 1H NMR (300 MHz,
DMSO-d6) δ ppm 9.23 (br s, 1H), 8.92 (br s, 1H), 8.27 (s, 1H),
8.14 (s, 1H), 7.31 (m, 2H), 7.12 (m, 2H), 5.95 (s, 1H), 4.95 (m,
1H), 1.88 (m, 1H), 1.44 (m, 3H), 0.92 (m, 2H), 0.66 (s, 2H). Anal.
(C18H18BrFN6) C, H, N.
1
amine (8k). H NMR (400 MHz, DMSO-d6) δ ppm 12.37 (br s,
1H), 9.68 (br s, 1H), 8.33 (s, 1H), 6.29 (s, 1H), 1.29 (m, 9H).
General Procedure for Compounds 10. A mixture of compound
8 (1 equiv), DIPEA (1.5 equiv), and a suitable amine 9 (1.1 equiv)
in n-butanol was heated at 110 °C for 18 h (or for 4 h at 165 °C
in microwave). The solvent was removed, and EtOAc was added.
The solution was washed with water and was concentrated. Flash
column chromatography or semiprep HPLC (Gilson system)
provided product as a solid.
Compounds 10a-k were all synthesized from 5-bromo-2-chloro-
N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidin-4-amine (8a).
N-Benzyl-5-bromo-N′-(5-cyclopropyl-2H-pyrazol-3-yl)pyrimidine-
(2R)-2-[[5-Chloro-4-[(5-cyclopropyl-2H-pyrazol-3-yl)amino]py-
rimidin-2-yl]amino]-2-(4-fluorophenyl)ethanol (10l). The title com-
pound was synthesized from 2,5-dichloro-N-(3-cyclopropyl-1H-
pyrazol-5-yl)pyrimidin-4-amine (8b). 1H NMR (300 MHz, DMSO-
d6) δ ppm 12.08 (s, 1H), 8.36 (s, 1H), 7.89 (s, 1H), 7.35 (m, 2H),
7.10 (m, 2H), 6.07-6.47 (m, 1H), 5.79 (s, 1H), 4.88-4.93 (m,
2H), 3.60 (m, 2H), 1.88 (m, 1H), 0.82-0.94 (m, 2H), 0.62-0.71
(m, 2H). HRMS (ESI) m/z calcd for C18H18ClFN6O 389.1293; found
389.1302 (∆ ) 2.3 ppm). Anal. (C18H18ClFN6O·2.4H2O) C, H,
N.
1
2,4-diamine (10a). H NMR (300 MHz, DMSO-d6) δ ppm 9.53
(br s, 1H), 8.43 (br s, 1H), 8.22 (s, 1H), 7.3 (m, 5H), 6.32 (br s,
1H), 5.94 (s, 1H), 4.48 (s, 2H), 1.81 (m, 1H), 0.88 (m, 2H), 0.67
(m, 1H), 0.47 (m, 1H). HRMS (ESI) m/z calcd for C17H17BrN6
385.0776; found 385.0786 (∆
(C17H17BrN6 ·1.35C2HF3O2) C, H, N.
)
2.6 ppm). Anal.
5-Bromo-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(3-methoxyphe-
nyl)methyl]pyrimidine-2,4-diamine (10b). 1H NMR (300 MHz,
DMSO-d6) δ ppm 11.81 (s, 1H), 7.72 (m, 2H), 7.28 (m, 1H), 7.03
(m, 1H), 6.50-6.70 (m, 3H), 4.18 (m, 2H), 3.52 (s, 3H), 1.81 (m,
1H), 0.92 (m, 2H), 0.61 (m, 2H). Anal. (C18H19BrN6O) C, H, N.
5-Bromo-N-[(2-chlorophenyl)methyl]-N′-(5-cyclopropyl-1H-pyra-
zol-3-yl)pyrimidine-2,4-diamine (10c). 1H NMR (300 MHz, DMSO-
d6) δ ppm 12.08 (br s, 1H), 8.01 (m, 2H), 7.20-7.60 (m, 5H),
5.72 (m, 1H), 4.48 (m, 2H), 1.79 (m, 1H), 0.90 (m, 2H), 0.61 (m,
2H). HRMS (ESI) m/z calcd for C17H16BrClN6 419.0386; found
419.0382 (∆ ) - 0.9 ppm). HPLC analysis using a Waters Atlantis
T3 column (3 µm, 2.1 mm × 50 mm) with CH3CN/H2O as solvents
(5-95% CH3CN, flow rate: 1 mL/min, UV at 220 and 254 nm)
showed g95% purity.
(3S)-3-[[5-Chloro-4-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]py-
rimidin-2-yl]amino]-3-(4-fluorophenyl)-N,N-dimethylpropana-
mide (10m). The title compound was synthesized from (3S)-3-
amino-3-(4-fluorophenyl)-N,N-dimethylpropanamide (31) and 2,5-
dichloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)pyrimidin-4-amine (8b).
1H NMR (300 MHz, CD3OD) δ ppm 7.93 (s, 1H), 7.30 (m, 2H),
6.80 (m, 2H), 6.30 (m, 1H), 5.60 (br s, 1H), 5.07 (m, 1H), 2.95
(m, 2H), 2.60-2.80 (m, 6H), 1.80 (m, 1H), 0.90 (m, 2H), 0.60 (m,
2H). HRMS (ESI) m/z calcd for C21H23ClFN7O 444.1715; found
444.1703 (∆ ) - 2.7 ppm). HPLC analysis using a Waters Atlantis
T3 column (3 µm, 2.1 mm × 50 mm) with CH3CN/H2O as solvents
(5-95% CH3CN, flow rate: 1 mL/min, UV at 220 and 254 nm)
showed g95% purity.
5-Bromo-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(2-methoxyphe-
nyl)methyl]pyrimidine-2,4-diamine (10d). 1H NMR (300 MHz,
DMSO-d6) δ ppm 12.10 (s, 1H), 7.90-8.00 (m, 2H), 6.81-7.42
(m, 5H), 5.78 (m, 1H), 4.39 (s, 2H), 3.76 (s, 3H), 1.80 (m, 1H),
0.91 (m, 2H), 0.61 (m, 2H). Anal. (C18H19BrN6O) C, H, N.
5-Bromo-N′-(5-cyclopropyl-2H-pyrazol-3-yl)-N-[(4-fluorophenyl-
)methyl]pyrimidine-2,4-diamine (10e). 1H NMR (300 MHz, DMSO-
d6) δ ppm 12.10 (m, 1H), 8.01 (m, 2H), 7.62 (m, 1H), 7.10-7.30
(m, 4H), 6.19 (m, 1H), 4.38 (m, 2H), 1.78 (m, 1H), 0.91 (m, 2H),
0.61 (m, 2H). Anal. (C17H16BrFN6 ·0.1H2O) C, H, N.
5-Bromo-N-[(4-chlorophenyl)methyl]-N′-(5-cyclopropyl-1H-pyra-
zol-3-yl)pyrimidine-2,4-diamine (10f). 1H NMR (300 MHz, DMSO-
d6) δ ppm 12.12 (m, 1H), 8.00 (m, 2H), 7.61 (m, 1H), 7.20-7.40
(m, 4H), 5.88 (m, 1H), 4.36 (m, 2H), 1.76 (m, 1H), 0.92 (m, 2H),
0.61 (m, 2H). HRMS (ESI) m/z calcd for C17H16BrClN6 419.0386;
found 419.0384 (∆ ) - 0.5 ppm). Anal. (C17H16BrClN6) C, H, N.
5-Bromo-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(1S)-1-phenyl-
ethyl]pyrimidine-2,4-diamine (10g). 1H NMR (300 MHz, DMSO-
d6) δ ppm 12.14 (s, 1H), 8.01 (s, 1H), 7.95 (s, 1H), 7.20-7.44 (m,
5H), 5.88 (br s, 1H), 5.04 (m, 1H), 1.92 (m, 1H), 1.46 (m, 3H),
0.99 (m, 1H), 0.87 (m, 1H), 0.66 (m, 2H). Anal. (C18H19N6Br) C,
H, N.
(3S)-3-Amino-3-(4-fluorophenyl)-N,N-dimethylpropanamide (31).
A mixture of (3S)-3-[(tert-butoxycarbonyl)amino]-3-(4-fluorophe-
nyl)propanoic acid (200 mg, 0.74 mmol), EDCI (184 mg, 0.96
mmol), HOBT (160 mg, 1.2 mmol), and dimethylamine (2.0 M in
THF, 0.48 mL, 0.96 mmol) was stirred at room temperature for
48 h. To the mixture was added water and the mixture was extracted
with DCM. The combined organic layers were dried and concen-
trated. Reverse phase HPLC (Gilson) purification (monitored at 220
1
nm) gave the desired product as a white solid (506 mg, 90%). H
NMR (300 MHz, CDCl3) δ ppm 7.40 (m, 2H), 7.10 (m, 2H), 5.15
(m, 1H), 3.10 (m, 6H), 2.90 (m, 2H), 1.50 (s, 9H).
To a solution of above solid in DCM (5 mL) was added
trifluoroacetic acid (5 mL), and the reaction mixture was stirred
for 2 h. Solvent was removed and the residue was basified with
K2CO3/H2O. The mixture was extracted with CHCl3/2-propanol (3:
1). The combined organic layers were concentrated to give a yellow
1
oil (130 mg, 93%). H NMR (300 MHz, CDCl3) δ ppm 7.30 (m,
2H), 6.90 (m, 2H), 4.45 (m, 1H), 2.90 (m, 6H), 2.50 (m, 2H), 1.80
(s, 2H).

4680 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Wang et al.
(3S)-3-({5-Chloro-4-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]py-
rimidin-2-yl}amino)-3-(4-fluorophenyl)-N-methylpropanamide (10n).
The title compound was synthesized from (3S)-3-amino-3-(4-
fluorophenyl)-N-methylpropanamide (32) and 2,5-dichloro-N-(3-
cyclopropyl-1H-pyrazol-5-yl)pyrimidin-4-amine (8b). 1H NMR
(300 MHz, CD3OD) δ ppm 8.23 (s, 1H), 7.40 (m, 2H), 7.20 (m,
2H), 6.30 (m, 1H), 5.60 (br s, 1H), 5.07 (m, 1H), 2.95 (m, 2H),
2.80 (m, 3H), 2.10 (m, 1H), 1.25 (m, 2H), 0.90 (m, 2H). HRMS
(ESI) m/z calcd for C20H21ClFN7O 430.1558; found 430.1537 (∆
) - 4.9 ppm). Anal. (C20H21ClFN7O·1.55C2HF3O2) C, H, N.
(2R)-2-[[5-Chloro-4-[(5-propoxy-2H-pyrazol-3-yl)amino]pyrimi-
din-2-yl]amino]-2-(4-fluorophenyl)ethanol (10u). The title com-
pound was synthesized from 2,5-dichloro-N-(5-propoxy-2H-pyrazol-
3-yl)pyrimidin-4-amine (8i). 1H NMR (300 MHz, DMSO-d6) δ ppm
9.86 (br s, 1H), 8.27 (br s, 1H), 7.99 (s, 1H), 7.38 (m, 2H), 7.14
(m, 2H), 5.61 (s, 1H), 4.94 (m, 1H), 4.00 (m, 2H), 3.64 (m, 2H),
1.70 (m, 2H), 0.95 (m, 3H). HRMS (ESI) m/z calcd for
C18H20ClFN6O2 407.1398; found 407.1393 (∆ ) - 1.2 ppm). Anal.
(C18H20ClFN6O2 ·1.25C2HF3O2) C, H, N.
(2R)-2-[[5-Chloro-4-(2H-pyrazol-3-ylamino)pyrimidin-2-yl]amino]-
2-(4-fluorophenyl)ethanol (10v). The title compound was synthe-
sized from 2,5-dichloro-N-(1H-pyrazol-5-yl)pyrimidin-4-amine (8j).
1H NMR (300 MHz, DMSO-d6) δ ppm 10.14 (s, 1H), 8.18 (s, 1H),
7.73 (s, 1H), 7.28 (m, 2H), 7.13 (m, 2H), 6.23 (s, 1H), 4.88 (m,
1H), 3.65 (m, 2H). HRMS (ESI) m/z calcd for C15H14ClFN6O
349.0980; found 349.0972 (∆ ) - 2.3 ppm). Anal. (C15H14ClFN6O·
1.25C2HF3O2) C, H, N.
N′-(5-tert-Butyl-1H-pyrazol-3-yl)-5-chloro-N-[(1S)-1-(4-fluorophe-
nyl)ethyl]pyrimidine-2,4-diamine (10w). The title compound was
synthesized from N-(3-tert-butyl-1H-pyrazol-5-yl)-2,5-dichloropy-
rimidin-4-amine (8k). 1H NMR (300 MHz, DMSO-d6) δ ppm 8.07
(s, 1H), 7.32 (m, 2H), 7.11 (m, 2H), 6.22 (br s, 1H), 5.01 (m, 1H),
1.46 (m, 3H), 1.26 (s, 9H). HRMS (ESI) m/z calcd for C19H22ClFN6
389.1656; found 389.1674 (∆ ) 4.6 ppm). Anal. (C19H22ClFN6 ·
1.05C2HF3O2) C, H, N.
(3S)-3-Amino-3-(4-fluorophenyl)-N-methylpropanamide (32).
The title compound was synthesized in a similar way to compound
1
31. H NMR (300 MHz, CDCl3) δ ppm 7.40 (m, 2 H), 7.08 (m,
2H), 6.65 (br s, 1H), 4.80 (br s, 1H), 3.27 (m, 1H), 2.82 (m, 3H),
1.52 (m, 2H).
N′-(5-cyclopropyl-2H-pyrazol-3-yl)-N-[(1S)-1-(4-fluorophenyl-
)ethyl]pyrimidine-2,4-diamine (10o). The title compound was
synthesized from 2-chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)py-
rimidin-4-amine (8c). 1H NMR (300 MHz, DMSO-d6) δ ppm 12.02
(br s, 1H), 11.11 (s, 1H), 8.91 (br s, 1H), 7.83 (m, 1 H), 7.41 (m,
2H), 7.19 (m, 2H), 6.28 (m, 1H), 6.06 (s, 1H), 5.12 (m, 1H), 1.86
(m, 1H), 1.52 (m, 3H), 0.95 (m, 2H), 0.66 (m, 2H). HRMS (ESI)
m/z calcd for C18H19FN6 339.1733; found 339.1724 (∆ ) - 2.7
ppm). Anal. (C18H19FN6 ·1.55C2HF3O2) C, H, N.
N′-(5-Cyclopropyl-2H-pyrazol-3-yl)-5-fluoro-N-[(1S)-1-(4-fluo-
rophenyl)ethyl]pyrimidine-2,4-diamine (10p). The title compound
was synthesized from 2-chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-
(S)-5-Fluoro-N4-(5-cyclopropyl-1H-pyrazol-3-yl)-N2-(1-(5-fluo-
ropyridin-2-yl)ethyl)pyrimidine-2,4-diamine (10x). A mixture of
2-chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-5-fluoropyrimidin-4-
amine (compound 8d, 0.200 g, 0.788 mmol), (S)-1-(5-fluoropyridin-
2-yl)ethanamine (compound (S)-9a; 0.116 g, 0.828 mmol), and
DIPEA (0.17 mL, 0.946 mmol) in n-BuOH (2 mL) was heated in
a sealed tube at 175 °C for 40 h. The solvent was removed under
reduced pressure, and the residue was purified by flash column
chromatography (hexanes:EtOAc ) 1:1) to give the title compound
1
5-fluoropyrimidin-4-amine (8d). H NMR (300 MHz, DMSO-d6)
δ ppm 8.04 (s, 1 H), 7.38 (m, 2H), 7.15 (m, 2H), 6.07 (br s, 1H),
4.98 (m, 1H), 1.88 (m, 1H), 1.46 (m, 3 H), 0.95 (m, 2H), 0.66 (m,
2H). HRMS (ESI) m/z calcd for C18H18F2N6 357.1639; found
357.1625 (∆ ) - 3.9 ppm). Anal. (C18H18F2N6 ·0.25C4H8O2) C,
H, N.
N′-(5-Cyclopropyl-2H-pyrazol-3-yl)-N-[(1S)-1-(4-fluorophenyl-
)ethyl]-5-methylpyrimidine-2,4-diamine (10q). The title compound
was synthesized from 2-chloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-
5-methylpyrimidin-4-amine (8e). 1H NMR (300 MHz, DMSO-d6)
δ ppm 12.01 (br s, 1H), 10.04 (s, 1H), 8.65 (br s, 1H), 7.70 (s, 1
H), 7.35 (m, 2H), 7.16 (m, 2H), 6.03 (br s, 1H), 5.01 (m, 1H), 2.05
(s, 3H), 1.90 (m, 1H), 1.49 (d, 3H), 0.95 (m, 2H), 0.66 (m, 2H).
HRMS (ESI) m/z calcd for C19H21FN6 353.1890; found 353.1894
(∆ ) 1.1 ppm). Anal. (C19H21FN6 ·0.2H2O) H, C calcd, 64.10, found
63.67; N: calcd, 23.61, found, 24.13.
1
as a white solid (0.175 g, 62%). H NMR (400 MHz, DMSO-d6)
δ ppm 12.47 (s, 1H), 10.15 (s, 1H), 8.48 (s, 1H), 7.89 and 7.81 (s,
1H), 7.65 (m, 1H), 7.42 (b, 1H), 7.20 (b, 1H), 5.76 (s, 1H), 5.00
(m, 1H), 1.85 (m, 1H), 1.44 (d, J ) 6.8 Hz, 3H), 0.95 (m, 1H),
0.85 (m, 1H), 0.74 (m, 1H), 0.63 (m, 1H). HRMS (ESI) m/z calcd
for C17H17F2N7 358.1591; found 358.1577 (∆ ) - 3.8 ppm). Anal.
(C17H17F2N7 ·0.35CH4O) C, H, N.
(S)-5-Chloro-N4-(5-cyclopropyl-1H-pyrazol-3-yl)-N2-(1-(5-fluo-
ropyridin-2-yl)ethyl)pyrimidine-2,4-diamine (10y). In a similar way
described for 10x, compound 10y was synthesized from 2,5-
dichloro-4-(5-cyclopropyl-1H-pyrazole-3-ylamino)pyrimidine (8b).
1H NMR (400 MHz, DMSO-d6) δ ppmδ 12.09-12.51 (m, 1H),
9.64-9.70 (m, 1H), 8.49 (s, 1H), 8.38 (s, 1H), 7.93-8.02 (m, 1H),
7.63 (m, 1H), 7.40 (m, 1H), 5.80-5.93 (m, 1H), 5.06 (m, 1H),
1.83 (m, 1H), 1.45 (d, J ) 6.8 Hz, 3H), 0.94 (m, 1H), 0.84 (m,
1H), 0.74 (m, 1H), 0.63 (m, 1H). Anal. (C17H17ClFN7 ·0.45H2O)
C, H, N.
5-Chloro-N-[(1S)-1-(4-fluorophenyl)ethyl]-N′-(5-propan-2-yloxy-
1H-pyrazol-3-yl)pyrimidine-2,4-diamine (10r). The title compound
was synthesized from 2,5-dichloro-N-(5-propan-2-yloxy-1H-pyra-
1
zol-3-yl)pyrimidin-4-amine (8f). H NMR (400 MHz, DMSO-d6)
δ ppm 11.94 (br s, 1H), 9.66 (br s, 1H), 7.95 (s, 2H), 7.37 (d, J )
5.5 Hz, 2H), 7.13 (t, J ) 8.3 Hz, 2H), 5.52 (d, J ) 1.3 Hz, 1H),
4.88-5.05 (m, 1H), 4.52-4.74 (m, 1H), 1.42 (d, J ) 7.1 Hz, 3H),
1.27 (d, J ) 6.1 Hz, 6 H). HRMS (ESI) m/z calcd for C18H20ClFN6O
5-Chloro-N2-[(1S)-1-(5-fluoropyridin-2-yl)ethyl]-N4-(5-isopropoxy-
1H-pyrazol-3-yl)pyrimidine-2,4-diamine (10z). In a similar way
described for 10x, compound 10z was synthesized from 2,5-
dichloro-N-(5-propan-2-yloxy-1H-pyrazol-3-yl)pyrimidin-4-
amine (8f). 1H NMR (300 MHz, CDCl3) δ ppm 8.45 (s, 1H), 7.90
(s, 1H), 7,35 (m, 2H), 6.30 (br s, 1H), 5.40 (s, 1H), 5.15 (m, 1H),
4.80 (m, 1H), 1.60 (m, 3H), 1.35 (m, 6H). Anal. (C17H19ClFN7O·
0.2H2O) C, H, N.
5-Chloro-N-[(1S)-1-(5-fluoropyrimidin-2-yl)ethyl]-N′-(5-propan-
2-yloxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (11a). In a similar
way described for 10x, compound 11a was synthesized from 2,5-
dichloro-N-(5-propan-2-yloxy-1H-pyrazol-3-yl)pyrimidin-4-
amine (8f) and (S)-1-(5-fluoropyrimidin-2-yl)ethanamine (S)-9b.
1H NMR (300 MHz, CDCl3) δ ppm 8.50 (s, 2H), 7.75 (s, 1H),
5.50 (s, 1H), 5.20 (m, 1H), 4.68 (m, 1H), 1.55 (m, 3H), 1.20 (m,
6H). Anal. (C16H18ClFN8O) C, H, N.
391.1449; found 391.1445 (∆
(C18H20ClFN6O·0.25H2O) C, H, N.
)
-
1.0 ppm). Anal.
(2R)-2-[[5-Chloro-4-[(5-methylsulfanyl-2H-pyrazol-3-yl)ami-
no]pyrimidin-2-yl]amino]-2-(4-fluorophenyl)ethanol (10s). The title
compound was synthesized from 2,5-dichloro-N-[5-(methylthio)-
1H-pyrazol-3-yl]pyrimidin-4-amine (8g). 1H NMR (300 MHz,
DMSO-d6) δ ppm 9.74 (br s, 1H), 8.17 (s, 1H), 7.98 (s, 1H), 7.39
(m, 2H), 7.13 (m, 2H), 6.25 (s, 1H), 4.94 (m, 1H), 3.64 (m, 2H),
2.45 (s, 3H). HRMS (ESI) m/z calcd for C16H16ClFN6OS 395.0857;
found
395.0851
(∆
)
-
1.5
ppm).
Anal.
(C16H16ClFN6OS·1.15C2HF3O2) C, H, N.
5-Chloro-N-[(1S)-1-(4-fluorophenyl)ethyl]-N′-(5-methyl-1H-pyra-
zol-3-yl)pyrimidine-2,4-diamine (10t). The title compound was
synthesized from 2,5-dichloro-N-(5-methyl-1H-pyrazol-3-yl)pyri-
midin-4-amine (8h). 1H NMR (300 MHz, CD3OD) δ ppm 8.02 (s,
1H), 7.33 (m, 2H), 7.07 (m, 2H), 6.08 (br s, 1H), 5.05 (m, 1H),
2.34 (s, 3H), 1.57 (m, 3H). HRMS (ESI) m/z calcd for C16H16FClN6
347.1187; found 347.1199 (∆ ) 3.4 ppm). Anal. (C16H16FClN6 ·
0.8C2HF3O2) C, H, N.
General Procedure for the Synthesis of Intermediates 13.
Following a procedure similar to that of compounds 8, compounds
13a-d were synthesized.

4-Aminopyrazolylpyrimidines as Trk Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4681
2,5,6-Trichloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidin-4-
amine (13a). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.40 (s, 1H),
9.98 (s, 1H), 6.17 (d, J ) 2.0 Hz, 1H), 1.92 (m, 1H), 0.88-0.99
(m, 2H), 0.70 (dd, J ) 4.9, 2.1 Hz, 2H).
2,5,6-Trichloro-N-(5-propan-2-yloxy-1H-pyrazol-3-yl)pyrimidin-
4-amine (13b). 1H NMR (400 MHz, DMSO-d6) δ 12.25 and 11.46
(s, 1H), 10.23 and 9.94 (s, 1H), 5.85 and 5.75 (s, 1H), 4.69 and
4.45 (m, 1H), 1.30 (d, J ) 6.0 Hz, 3H), 1.27 (d, J ) 6.0 Hz, 3H).
MS: calcd 321; found [M + H]+ 322.
2,6-Dichloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidin-4-
amine (13c). 1H NMR (300 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.66
(s, 1H), 7.76 and 6.80 (br s, 1H), 6.30 and 5.66 (br s, 1H), 1.88
(m, 1H), 0.92 (m, 2H), 0.68 (m, 2H).
2,6-Dichloro-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine
(13d). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.20 (s, 1H), 10.67
(s, 1H), 9.56 and 7.73 (m, 1 H), 6.75 and 5.81 (m, 1 H), 2.22 (s,
3 H).
General Procedure for the Synthesis of Compounds 14a-f.
Following a procedure similar to that of compounds 10, compounds
14a-f were synthesized.
5,6-Dichloro-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(1S)-1-(4-
fluorophenyl)ethyl]pyrimidine-2,4-diamine (14a). The title com-
pound was synthesized from 2,5,6-trichloro-N-(5-cyclopropyl-1H-
pyrazol-3-yl)pyrimidin-4-amine (13a). 1H NMR (300 MHz, DMSO-
d6) δ ppm 9.19 (br s, 1H), 8.83 (br s, 1H), 7.95 (m, 1H), 7.33 (m,
2H), 7.10 (m, 2H), 5.93-6.21 (m, 1H), 4.92 (m, 1H), 1.87 (m,
1H), 1.39 (m, 3H), 0.92 (m, 2H), 0.67 (m, 2H).
5,6-Dichloro-N-[(1S)-1-(4-fluorophenyl)ethyl]-N′-(5-propan-2-
yloxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (14b). The title com-
pound was synthesized from 2,5,6-trichloro-N-(5-propan-2-yloxy-
1H-pyrazol-3-yl)pyrimidin-4-amine (13b). 1H NMR (300 MHz,
DMSO-d6) δ ppm 11.89 (br s, 1H), 9.86 (br s, 1H), 8.18 (m, 1H),
7.40 (m, 2H), 7.14 (m, 2H), 5.55 (m, 1H), 4.95 (m, 1H), 4.66 (m,
1H), 1.41 (m, 1H), 1.26 (m, 6H).
s, 1H), 7.95 (br s, 1H), 7.38 (m, 2H), 7.14 (m, 2H), 5.93 (s, 1H),
4.89 (m, 1H), 3.98 (m, 2H), 3.86 (m, 2H), 3.63 (m, 4H), 3.46 (m,
2H), 3.22 (m, 2H), 3.01 (m, 2H), 1.86 (m, 1H), 0.92 (m, 2H), 0.67
(m, 2H). HRMS (ESI) m/z calcd for C24H30ClFN8O2 517.2242;
found 517.2224 (∆ ) - 3.4 ppm). Anal. (C24H30ClFN8O2 ·
2C2HF3O2 ·1.2H2O) C, H, N.
(2R)-3-[[5-Chloro-6-[(5-cyclopropyl-2H-pyrazol-3-yl)amino]-2-
[[(1S)-1-(4-fluorophenyl)ethyl]amino]pyrimidin-4-yl]amino]propane-
1,2-diol (15b). The title compound was synthesized from 5,6-
dichloro-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(1S)-1-(4-
fluorophenyl)ethyl]pyrimidine-2,4-diamine (14a). 1H NMR (300
MHz, DMSO-d6) δ ppm 9.81 (br s, 1H), 8.67 (br s, 1H), 7.42 (m,
2H), 7.14 (m, 2H), 5.86 (s, 1H), 5.02 (m, 1H), 3.30-3.54 (m, 5H),
1.93 (m, 1H), 1.45 (m, 3H), 0.98 (m, 2H), 0.69 (m, 2H). HRMS
(ESI) m/z calcd for C21H25ClFN7O2 462.1820; found 462.1833 (∆
) 2.8 ppm). HPLC analysis using a Waters Atlantis T3 column (3
µm, 2.1 mm × 50 mm) with CH3CN/H2O as solvents (5-95%
CH3CN, flow rate: 1 mL/min, UV at 220 and 254 nm) showed
g95% purity.
2-[[5-Chloro-6-[(5-cyclopropyl-2H-pyrazol-3-yl)amino]-2-[[(1S)-
1-(4-fluorophenyl)ethyl]amino]pyrimidin-4-yl]amino]ethanol (15c).
The title compound was synthesized from 5,6-dichloro-N′-(5-
cyclopropyl-1H-pyrazol-3-yl)-N-[(1S)-1-(4-fluorophenyl)ethyl]py-
rimidine-2,4-diamine (14a). 1H NMR (300 MHz, DMSO-d6) δ ppm
9.72 (br s, 1H), 8.59 (br s, 1H), 7.47 (br s, 1H), 7.39 (m, 2H), 7.14
(m, 2H), 5.85 (s, 1H), 4.97 (m, 1H), 3.37-3.54 (m, 4H), 1.90 (m,
1H), 1.45 (m, 3H), 0.96 (m, 2H), 0.68 (m, 2H). HRMS (ESI) m/z
calcd for C20H23ClFN7O 432.1715; found 462.1710 (∆ ) - 1.2
ppm). Anal. (C20H23ClFN7O·1.35C2HF3O2) C, H, N.
2-[[5-Chloro-6-[(5-cyclopropyl-2H-pyrazol-3-yl)amino]-2-[[(1S)-
1-(4-fluorophenyl)ethyl]amino]pyrimidin-4-yl]amino]propane-1,3-
diol (15d). The title compound was synthesized from 5,6-dichloro-
N′ -(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(1S)-1-(4-
fluorophenyl)ethyl]pyrimidine-2,4-diamine (14a). 1H NMR (300
MHz, DMSO-d6) δ ppm 9.73 (br s, 1H), 8.54 (br s, 1H), 7.42 (m,
2H), 7.14 (m, 2H), 6.68 (br s, 1H), 5.86 (s, 1 H), 4.98 (m, 1H),
4.04 (m, 1H), 3.33-3.56 (m, 4H), 1.91 (m, 1H), 1.45 (m, 3H),
0.96 (m, 2H), 0.68 (m, 2H). HRMS (ESI) m/z calcd for
C21H25ClFN7O2 462.1820; found 462.1805 (∆ ) - 3.2 ppm). Anal.
(C21H25ClFN7O2 ·2HCl) C, H, N.
6-Chloro-N′-(5-cyclopropyl-1H-pyrazol-3-yl)-N-[(1S)-1-(4-fluo-
rophenyl)ethyl]pyrimidine-2,4-diamine (14c). The title compound
was synthesized from 2,6-dichloro-N-(5-cyclopropyl-1H-pyrazol-
1
3-yl)pyrimidin-4-amine (13c). H NMR (300 MHz, DMSO-d6) δ
ppm 9.63 (br s, 1H), 7.78 (br s, 1H), 7.38 (m, 2H), 7.12 (m, 2H),
5.93-6.17 (m, 1H), 5.03 (m, 1H), 1.84 (m, 1H), 1.40 (m, 3H),
0.92 (m, 2H), 0.66 (m, 2H).
(2R)-3-[[6-[(5-Cyclopropyl-1H-pyrazol-3-yl)amino]-2-[[(1S)-1-(4-
fluorophenyl)ethyl]amino]pyrimidin-4-yl]amino]propane-1,2-di-
ol (15e). The title compound was synthesized from 6-chloro-N′-(5-
c y c l o p r o p y l - 1 H - p y r a z o l - 3 - y l ) - N - [ ( 1 S ) - 1 - ( 4 -
fluorophenyl)ethyl]pyrimidine-2,4-diamine (14c). 1H NMR (300
MHz, DMSO-d6) δ ppm 11.45 (br s, 1H), 10.20 (br s, 1H), 8.70
(s, 1H), 8.10 (s, 1H), 7.35 (m, 2H), 7.10 (m, 2H), 5.45 (m, 1H),
5.35 (s, 1H), 4.95 (br s, 1H), 3.10-3.50 (m, 5H), 1.80 (m, 1H),
1.45 (m, 3H), 0.90 (m, 2H), 0.65 (m, 2H). HRMS (ESI) m/z calcd
for C21H26FN7O2 428.2210; found 428.2195 (∆ ) - 3.5 ppm).
Anal. (C21H26FN7O2 ·2C2HF3O2) C, H, N.
6-Chloro-N-[(1S)-1-(4-fluorophenyl)ethyl]-N′-(5-methyl-1H-pyra-
zol-3-yl)pyrimidine-2,4-diamine (14d). The title compound was
synthesized from 2,6-dichloro-N-(5-methyl-1H-pyrazol-3-yl)pyri-
midin-4-amine (13d). 1H NMR (300 MHz, DMSO-d6) δ ppm 8.60
(m, 1H), 7.80 (m, 1H), 7.40 (m, 2H), 7.10 (m, 2H), 5.85-6.10 (m,
2H), 5.10 (m, 1H), 2.20 (m, 3H), 1.45 (s, 3H).
(2R)-2-[[4,5-Dichloro-6-[(5-cyclopropyl-1H-pyrazol-3-yl)ami-
no]pyrimidin-2-yl]amino]-2-(4-fluorophenyl)ethanol (14e). The title
compound was synthesized from 2,5,6-trichloro-N-(5-cyclopropyl-
1H-pyrazol-3-yl)pyrimidin-4-amine (13a). 1H NMR (300 MHz,
CD3OD) δ ppm 7.37 (m, 2H), 7.04 (m, 2H), 6.05 (br s, 1H), 5.00
(m, 1H), 3.76 (m, 2H), 1.90 (m, 1H), 0.97 (m, 2H), 0.73 (m, 2H).
5,6-Dichloro-N-[(1S)-1-(5-fluoropyridin-2-yl)ethyl]-N′-(5-propan-
2-yloxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (14f). The title
compound was synthesized from 2,5,6-trichloro-N-(5-propan-2-
yloxy-1H-pyrazol-3-yl)pyrimidin-4-amine (13b). 1H NMR (400
MHz, DMSO-d6) δ ppm 11.96 (s, 1H), 9.86 (s, 1H), 8.53 (bs, 1H),
8.31 (d, J ) 8.0 Hz, 1H), 7.68 (m, 1H), 7.46 (m, 1H), 5.56 (s, 1H),
5.05 (m, 1H), 4.68 (m, 1H), 1.46 (d, J ) 6.8 Hz, 3H), 1.28 (d, J )
6.0 Hz, 6H). MS: calcd 425; found [M + H]+ 426.
(2R)-2-({5-Chloro-4-[(3-cyclopropyl-1H-pyrazol-5-yl)amino]-6-
[(2-morpholin-4-ylethyl)amino] pyrimidin-2-yl}amino)-2-(4-fluo-
rophenyl)ethanol (15a). A mixture of (2-morpholin-4-ylethyl)amine
(44 µL, 0.34 mmol), (2R)-2-[[4,5-dichloro-6-[(5-cyclopropyl-1H-
pyrazol-3-yl)amino]pyrimidin-2-yl]amino]-2-(4-fluorophenyl)etha-
nol (14e, 70 mg, 0.16 mmol) in n-butanol (1.0 mL) was heated at
120 °C for 18 h. The solvent was removed and EtOAc was added.
The solution was washed with water and was concentrated.
Semiprep HPLC (Gilson system) provided product as a solid (92
mg). 1H NMR (300 MHz, CDCl3) δ ppm 9.54 (br s, 1H), 9.15 (br
(2R)-3-[[2-[[(1S)-1-(4-Fluorophenyl)ethyl]amino]-6-[(5-methyl-
1H-pyrazol-3-yl)amino]pyrimidin-4-yl]amino]propane-1,2-diol (15f).
The title compound was synthesized from 6-chloro-N-[(1S)-1-(4-
fluorophenyl)ethyl]-N′-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-
1
diamine (14d). H NMR (300 MHz, DMSO-d6) δ ppm 11.55 (br
s, 1H), 10.20 (br s, 1H), 8.78 (s, 1 H), 8.10 (s, 1H), 7.35 (m, 2H),
7.10 (m, 2H), 5.65 (m, 1H), 5.35 (s, 1H), 4.95 (br s, 1H), 3.10-3.90
(m, 5H), 2.15 (s, 3H), 1.40 (m, 3H). HRMS (ESI) m/z calcd for
C19H24FN7O2 402.2054; found 402.2043 (∆ ) - 2.7 ppm). Anal.
(C19H24FN7O2 ·0.75CH2Cl2) C, H, N.
2-[[5-Chloro-2-[[(1S)-1-(4-fluorophenyl)ethyl]amino]-6-[(5-pro-
pan-2-yloxy-1H-pyrazol-3-yl)amino]pyrimidin-4-yl]amino]propane-
1,3-diol (15g). The title compound was synthesized from 5,6-
dichloro-N-[(1S)-1-(4-fluorophenyl)ethyl]-N′-(5-propan-2-yloxy-1H-
1
pyrazol-3-yl)pyrimidine-2,4-diamine (14b). H NMR (300 MHz,
DMSO-d6) δ ppm 9.97 (br s, 1H), 8.81 (br s, 1H), 7.40 (m, 2H),
7.13 (m, 2H), 6.70 (br s, 1H), 5.63 (s, 1H), 4.98 (m, 1H), 4.55 (m,
1H), 4.04 (m, 1H), 3.33-3.55 (m, 4H), 1.46 (m, 3H), 1.30 (m,
6H). Anal. (C21H27ClFN7O3) C, H, N.

4682 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Wang et al.
(R)-3-(5-Chloro-2-((S)-1-(5-fluoropyridin-2-yl)ethylamino)-6-(5-
isopropoxy-1H-pyrazol-3-ylamino)pyrimidin-4-ylamino)propane-
1,2-diol (15h). In a similar way described for 15a, compound 15h
was synthesized from (S)-5,6-dichloro-N2-(1-(5-fluoropyridin-2-
yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-di-
amine (14f). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.91 (s, 1H),
9.02 (s, 1H), 8.47 (s, 1H), 7.64 (m, 2H), 7.43 (m, 1H), 6.24 (t, J )
5.6 Hz, 1H), 5.37 (d, J ) 1.6 Hz, 1H), 4.99 (m, 1H), 4.72 (m, 1H),
4.65 (m, 1H), 4.55 (m, 1H), 3.43 (m, 1H), 3.37 (m, 1H), 3.26 (m,
2H), 3.10 (m, 1H), 1.43 (d, J ) 6.8 Hz, 3H), 1.26 (d, J ) 6.0 Hz,
6H). Anal. (C20H26ClFN8O3 ·0.35H2O) C, H, N.
2-[5-Chloro-2-[[(1S)-1-(5-fluoropyridin-2-yl)ethyl]amino]-6-[(5-
propan-2-yloxy-1H-pyrazol-3-yl)amino]pyrimidin-4-yl]oxypropane-
1,3-diol (15i). In a slightly different way described for 15a,
compound 15i was synthesized from 5,6-dichloro-N-[(1S)-1-(5-
fluoropyridin-2-yl)ethyl]-N′-(5-propan-2-yloxy-1H-pyrazol-3-yl)py-
rimidine-2,4-diamine (14f) and 2-phenyl-1,3-dioxan-5-ol: sodium
hydride (1.1 equiv) was added to a suspension of 2-phenyl-1,3-
dioxan-5-ol in toluene followed by addition of 14f. The mixture
was heated overnight and to the mixture was added water. Solvent
was removed and excess TFA was added. Flash column chroma-
tography on silica gel provided the desired product. 1H NMR (400
MHz, DMSO-d6) δ ppm 11.90 (s, 1H), 9.36 (s, 1H), 8.49 (s, 1H),
7.98 (d, J ) 7.2 Hz, 1H), 7.66 (m, 1H), 7.43 (m, 1H), 5.45 (s, 1H),
4.97 (m, 1H), 4.71 (m, 1H), 4.64 (m, 1H), 4.56 (m, 1H), 3.43 (m,
1H), 3.61 (m, 2H), 3.30 (m, 2H), 1.46 (d, J ) 7.2 Hz, 3H), 1.27
(d, J ) 6.0 Hz, 6H). HRMS (ESI) m/z calcd for C20H25FClN7O4
482.1719; found 482.1709 (∆ ) - 2.1 ppm). Anal. (C20H25Cl-
FN7O4 ·0.1H2O) C, H, N.
5-Fluoropicolinonitrile (20). A mixture of 2-bromo-5-fluoropy-
ridine (compound 19, 93.0 g, 528 mmol), Zn dust (8.29 g, 127
mmol), zinc cyanide (40.3 g, 343 mmol), DPPF (11.7 g, 21.1
mmol), and Pd2(dba)3 (9.68 g, 10.6 mmol) in anhydrous dimethy-
lacetamide (300 mL) was heated at 95 °C for 3 h. After cooling to
room temperature, brine (100 mL) and ether (500 mL) were added.
The solid formed was removed by filtration and washed with ether
(300 mL). The organic layer was separated, washed with brine (200
mL), and was dried over anhydrous sodium sulfate and concen-
trated. The crude product was purified by flash column chroma-
tography (hexanes:DCM ) 1:1) to give 5-fluoropicolinonitrile as
a white solid (49 g, 72%). 1H NMR (400 MHz, DMSO-d6) δ ppm
8.82 (d, J ) 2.8 Hz, 1H), 8.21 (dd, J ) 4.4 and 8.8 Hz, 1H), 8.05
(dd, J ) 2.8 and 8.8 Hz, 1H).
N-(1-(5-Fluoropyridin-2-yl)vinyl)acetamide (21). A solution of
MeMgBr (170.3 mL, 510.98 mmol) in ether was diluted with 170
mL of anhydrous THF and cooled to 0 °C. 5-Fluoropicolinontrile
(compound 20, 53.6 g, 425.82 mmol) in THF (170 mL) was added
dropwise. The reaction was stirred at 0 °C for 30 min and then
diluted with DCM (170 mL). Acetic anhydride (48.3 mL, 510.98
mmol) in DCM (100 mL) was added dropwise at 0 °C. After
addition, the reaction was warmed to room temperature and stirred
at room temperature for 8 h. Saturated sodium bicarbonate solution
(50 mL) was added and extracted with ethyl acetate (2 × 200 mL).
The combined organic was dried over sodium sulfate. After removal
of solvent, the resulting residue was purified by flash column
chromatography (hexanes:EtOAc ) 2.5:1) to give N-(1-(5-fluoro-
pyridin-2-yl)vinyl)acetamide as a white solid (26.6 g, 35%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 9.37 (s, 1H), 8.57 (d, J ) 2.8
Hz, 1H), 7.81 (m, 2H), 6.01 (s, 1H), 5.52 (s, 1H), 2.08 (s, 3H).
MS: calcd 180; found [M + H]+ 181.
DMSO-d6) δ ppm 8.49 (d, J ) 2.4 Hz, 1H), 8.32 (d, J ) 7.6 Hz,
1H), 7.66 (m, 1H), 7.39 (dd, J ) 4.4 and 8.8 Hz, 1H), 4.95 (m,
1H), 1.85 (s, 3H), 1.34 (d, J ) 7.2 Hz, 3H). MS: calcd 182; found:
[M + H]+ 183. Enantiomeric excess determined by HPLC
(Chiralpak IA 250 mm × 4.6 mm; solvent 70:30 CO2/MeOH,
isocratic; run: 2 mL/min, 10 min run; pressure: 120 bar; temperature:
45 °C; wavelength: 220 nm; retention time: 1.77 and 2.19 min
(desired enantiomer) 95.3% ee).
(S)-tert-Butyl-1-(5-fluoropyridin-2-yl)ethylcarbamate (23). A so-
lution of (S)-N-(1-(5-fluoropyridin-2-yl)ethyl)acetamide (compound
22, 11.0 g, 60.37 mmol), DMAP (1.48 g, 12.07 mmol), and Boc2O
(26.35 g, 120.7 mmol) in THF (100 mL) was stirred at 50 °C for
20 h. After cooling to room temperature, lithium hydroxide
monohydrate (5.19 g, 123.8 mmol) and water (100 mL) were added.
The reaction was stirred at room temperature for 5 h and diluted
with ether (200 mL). The organic layer was separated, washed with
brine (100 mL), and dried over sodium sulfate. After removal of
solvent, the resulting residue was purified by flash column chro-
matography (hexanes:EtOAc ) 5:1) to give (S)-tert-butyl-1-(5-
fluoropyridin-2-yl)ethylcarbamate as a pale-yellow oil (13.6 g,
94%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.46 (d, J ) 2.8 Hz,
1H), 7.69 (m, 1H), 7.35-7.41 (m, 2H), 4.67 (m, 1H), 1.37 (s, 9H),
1.32 (d, J ) 7.2 Hz, 3H). MS: calcd 240; found [M + H]+ 241.
(S)-1-(5-Fluoropyridin-2-yl)ethanamine (S)-9a. To the solution
of (S)-tert-butyl-1-(5-fluoropyridin-2-yl)ethylcarbamate (compound
23, 12.8 g, 53.3 mmol) in DCM (100 mL) was added HCl/dioxane
solution (107 mL, 4 N, 428 mmol). The reaction was stirred at
room temperature for 3 h. The solvent was removed and 50 mL of
saturated sodium bicarbonate was added. The resulting aqueous
solution was extracted with ether (6 × 400 mL), dried over sodium
sulfate, and concentrated to give (S)-1-(5-fluoropyridin-2-yl)etha-
1
namine (7.30 g, 98%) as a pale-yellow oil. H NMR (400 MHz,
DMSO-d6) δ ppm 8.44 (d, J ) 2.8 Hz, 1H), 7.66 (m, 1H), 7.53
(m, 1H), 4.01 (q, J ) 6.8 Hz, 1H), 1.94 (m, 2H), 1.26 (d, J ) 6.8
Hz, 3H). MS: calcd 140; found: [M + H]+ 141.
5-Fluoropyrimidine-2-carbonitrile (25). A 10 mL microwave vial
was charged with 2-chloro-5-fluoropyrimidine (compound 24, 2.0 g,
15.09 mmol), Pd2(dba)3 (0.549 g, 0.6 mmol), DPPF (0.67 g, 1.21
mmol), zinc cyanide (1.15 g, 9.81 mmol), and zinc dust (0.237
mg, 3.62 mmol). The flask was evacuated and backfilled with N2
and anhydrous dimethylacetamide. The vial was mounted onto a
Personal Chemistry microwave reactor and heated at 100 °C for
10 h. The reaction mixture was diluted with EtOAc and then washed
with brine three times. The organic layer was obtained and
evaporated to dryness. The dried residue was purified by silica gel
chromatography (by ISCO Combiflash with gradient EtOAc and
hexanes) to afford the title compound as an off-white solid (1.50
1
g, 80%). GC-MS: 123 (M); H NMR (300 MHz, CDCl3) δ ppm
8.80 (s, 2H).
N-(1-(5-Fluoropyrimidin-2-yl)vinyl)acetamide (26). To a solution
of 5-fluoropyrimidine-2-carbonitrile (compound 25, 1.0 g, 8.1
mmol) in THF (10 mL) was added a solution of MeMgBr (3.3
mL, 9.75 mmol) in ether dropwise at 0 °C. After addition, the
reaction was warmed to room temperature, stirred at room tem-
perature for 1 h, and then diluted with DCM (10 mL). Acetic
anhydride (1.23 mL, 13.0 mmol) was added in one portion. The
reaction was stirred at room temperature for 1 h and 40 °C for 1 h.
Saturated sodium bicarbonate solution (10 mL) was added and
extracted with ethyl acetate (2 × 20 mL). The combined organic
was dried over sodium sulfate. After removal of solvent, the
resulting residue was purified by flash column chromatography
(hexanes:EtOAc ) 2.5:1) to give N-(1-(5-fluoropyrimidin-2-yl)vi-
nyl)acetamide as a white solid (0.38 g, 26%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.34 (s, 1H), 8.95 (s, 2H), 6.25 (s, 1H), 6.03 (s,
1H), 2.11 (s, 3H). MS: calcd 181; found [M + H]+ 182.
(S)-N-(1-(5-Fluoropyrimidin-2-yl)ethyl)acetamide (27). A solu-
tion of N-(1-(5-fluoropyrimidin-2-yl)vinyl)acetamide (compound 26,
0.10 g, 0.55 mmol) in MeOH (5 mL) under N2 was added (+)-
1,2-bis((2S, 5S)-2,5-diethylphospholano)benzene (cyclooctadien-
e)rhodium(I)trifluoromethanesulfonate (0.04 g, 0.0055 mmol). The
solution was transferred to a high pressure bomb and charged 150
(S)-N-(1-(5-Fluoropyridin-2-yl)ethyl)acetamide (22). To a solu-
tion of N-(1-(5-fluoropyridin-2-yl)vinyl)acetamide (compound 21,
11.0 g, 61.1 mmol) in MeOH (120 mL) under N2 was added (+)-
1,2-bis((2S, 5S)-2,5-diethylphospholano)benzene (cyclooctadien-
e)rhodium(I) trifluoromethanesulfonate (0.441 g, 0.611 mmol). The
solution was transferred to a high pressure bomb and charged 150
psi H2. The reaction was stirred at room temperature and maintained
inside pressure between 120-150 psi for 7 h. The solvent was
removed, and the resulting residue was purified by flash column
chromatography (EtOAc) to give (S)-N-(1-(5-fluoropyridin-2-yl)-
ethyl)acetamide as a white solid (9.8 g, 88%). 1H NMR (400 MHz,

4-Aminopyrazolylpyrimidines as Trk Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4683
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Receptor Interactions. J. Med. Chem. 2003, 46, 52775291.
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psi H2. The reaction stirred at room temperature for 4 h. The solvent
was removed and the resulting residue was purified by flash column
chromatography (EtOAc) to give (S)-N-(1-(5-fluoropyrimidin-2-
yl)ethyl)acetamide as a white solid (0.096 g, 95%). 1H NMR (400
MHz, DMSO-d6) δ 8.84 (d, J ) 0.8 Hz, 2H), 8.34 (d, J ) 7.6 Hz,
1H), 5.00 (m, 1H), 1.84 (s, 3H), 1.37 (d, J ) 6.8 Hz, 3H). MS:
calcd 183; found [M + H]+ 184. Enantiomeric excess determined
by HPLC (Chiralpak IA; 95:5 CO2/MeOH), conditions similar to
that for compound 22; >99% ee.
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Enigma. Nat. ReV. Cancer 2003, 3, 203–216.
(S)-tert-Butyl-1-(5-Fluoropyrimidin-2-yl)ethylcarbamate (28). A
mixture of (S)-N-(1-(5-fluoropyrimidin-2-yl)ethyl)acetamide (com-
pound 27, 0.20 g, 1.09 mmol), DMAP (0.027 g, 0.22 mmol), and
Boc2O (0.60 g, 2.73 mmol) in THF (10 mL) was stirred at 50 °C
for 40 h. After cooling to room temperature, lithium hydroxide
monohydrate (0.094 g, 2.24 mmol) and water (10 mL) was added.
The reaction mixture was stirred at room temperature for 9 h. Ether
(30 mL) was added and the organic layer was separated, washed
with brine (20 mL), and dried over sodium sulfate. After removal
of solvent, the resulting residue was purified by flash column
chromatography (hexanes:EtOAc ) 5:1) to give (S)-tert-butyl-1-
(5-fluoropyrimidin-2-yl)ethylcarbamate as a pale-yellow oil (0.21
g, 80%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.84 (s, 2H), 7.24
(d, J ) 7.6 Hz, 1H), 4.74 (m, 1H), 1.35 (s, 12H). MS: calcd 241;
found [M + H]+ 242.
(S)-1-(5-Fluoropyrimidin-2-yl)ethanamine HCl salt (S)-9b (HCl
Salt). To a solution of (S)-tert-butyl-1-(5-fluoropyrimidin-2-yl)eth-
ylcarbamate (compound 28, 0.21 g, 0.87 mmol) in DCM (5 mL)
was added HCl (1.3 mL, 5.2 mmol) in dioxane. The reaction
mixture was stirred at room temperature for 3 h. The solvent was
removed to give (S)-1-(5-fluoropyrimidin-2-yl)ethanamine as HCl
salt as white solid (quantitative). MS: calcd 141; found [M + H]+
142.
Microsome Stability Assay. This primary in vitro test assesses
the propensity of a compound to be metabolized by human liver
microsomal enzymes. The time-dependent disappearance of
compound (2 µM initial concentration) incubated with mi-
crosomes was measured by LC-MS/MS. Results are reported as
intrinsic clearance of compound (CLint, in µL/min/mg protein).
Aqueous Equilibrium Solubility Assay. This primary in vitro
test assesses the equilibrium solubility of a compound in pH 7.4
phosphate buffer. Solid compound was prepared by evaporation
of a DMSO solution. Dried compound was incubated for 24 h with
agitation in buffer. Undissolved compound was removed by
filtration and the concentration of test compound in the filtrate was
measured by HPLC-UV with MS confirmation. The upper and
lower limits of the assay are typically 1 mM and ∼1 µM,
respectively.
Plasma Protein Binding Assay. This primary in vitro test
assesses the extent of binding of a compound to plasma proteins
using equilibrium dialysis to separate free from bound compound.
The amount of compound in plasma (10 µM initial concentration)
and in dialysis buffer (pH 7.4 phosphate buffer) is measured by
LC-MS/MS after equilibration in a dialysis chamber. The fraction
unbound (fu) is reported.
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Acknowledgment. We thank the Analytical Chemistry
group for providing HRMS and chiral HPLC data, Zhaiwei
Lin and Jeremy Disch for experimental contributions, Ken
Mattes for HTS analysis, Andy Thomas for helpful sugges-
tions, and Julie Tucker from Global Structural Chemistry for
the CDK2-5 crystal structure.
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Supporting Information Available: Elemental analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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