Discovery of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives as c-Src kinase inhibitors for the treatment of acute ischemic stroke

Article abstract of DOI:10.1248/cpb.55.881

We synthesized a series of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives and evaluated their ability to inhibit c-Src kinase; 7-(2-amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxyphenylamino) pyrazolo-[1,5-a]pyrimidine-3-carboxamide 7o and

Full text of DOI:10.1248/cpb.55.881

June 2007
Chem. Pharm. Bull. 55(6) 881—889 (2007)
881
Discovery of Novel 2-Anilinopyrazolo[1,5-a]pyrimidine Derivatives as
c-Src Kinase Inhibitors for the Treatment of Acute Ischemic Stroke
Harunobu MUKAIYAMA,* Toshihiro NISHIMURA, Hiroaki SHIOHARA, Satoko KOBAYASHI,
Yoshimitsu KOMATSU, Shinji KIKUCHI, Eiichi TSUJI, Noboru KAMADA,
Hideki OHNOTA, and Hiroshi KUSAMA
Central Research Laboratory, Kissei Pharmaceutical Company Ltd.; 4365–1 Azumino, Nagano 399–8304, Japan.
Received January 30, 2007; accepted April 2, 2007; published online April 9, 2007
We synthesized a series of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives and evaluated their ability
to inhibit c-Src kinase; 7-(2-amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxyphenylamino)pyrazolo-
[1,5-a]pyrimidine-3-carboxamide 7o and 7-(2-amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide 7f showed potent inhibitory activity. Compound 7f inhibited
c-Src selectively and exhibited satisfactory central nervous system (CNS) penetration. Furthermore, 7f·HCl
reduced the infarct volume in vivo in a rat middle cerebral artery (MCA) occlusion model when administrated
intraperitoneally.
Key words c-Src kinase; pyrazolo[1,5-a]pyrimidine; acute ischemic stroke; central nervous system penetration
Stroke is a leading cause of death and disability world- inhibitor as an agent for treating acute stroke. Screening of
wide, and the development of an effective therapeutic strat- our compound library led to the discovery of compound 1 as
egy for stroke has been a priority of neuroscientists for a lead compound as an inhibitor of c-Src kinase. Compound
decades. Considerable effort has been devoted to developing 1 showed a high degree of selectivity for c-Src inhibition
neuroprotective agents to save neurons from the biochemical over other kinases (data not shown), and had a structurally
and metabolic consequences of ischemia brain injury. Al- novel pyrazolo[1,5-a]pyrimidine skeleton for a c-Src kinase
though a variety of agents have demonstrated efficacy in re- inhibitor. Therefore, we sought to increase the inhibitory ac-
ducing stroke injury in preclinical studies, only tissue plas- tivity by varying this structure. In this paper, we describe the
minogen activator (t-PA) has been approved internationally synthesis and structure–activity relationship (SAR) of 2-
as a drug for stroke patients. However, its narrow therapeutic anilinopyrazolo[1,5-a]pyrimidine derivatives as c-Src kinase
window (3 h after stroke onset) and associated risks, includ- inhibitors and the neuroprotective effect of the resulting com-
ing hemorrhage, have resulted in limited therapeutic use.1) pounds in a rat model.
Therefore, new and more useful agents are needed for treat-
ing ischemia/reperfusion injury.
Chemistry The general approach to the synthesis of 5,7-
disubstituted 2-anilinopyrazolo[1,5-a]pyrimidine derivatives
Tyrosine kinase c-Src2) regulates the activity of the N- 7 and 8 is outlined in Chart 1. The intermediates 3 were
methyl-D-aspartate (NMDA) receptor, which induces a prepared by substituting the corresponding anilines on
marked and prolonged Ca2ꢀ influx into the neurons after [bis(methylthio)methylene]propanedinitrile 2. Cyclization of
cerebral ischemia3,4) and ultimately leads to neuronal dam- 3 with hydrazine monohydrate produced 3-amino-5-anilino-
age. c-Src regulates the release of cytokine and superoxide pyrazoles 4. Treatment of 4 with b-keto ester derivatives
production in neutrophils,5,6) and mediates the signaling ac- under acidic conditions gave the 2-anilinopyrazolo[1,5-
tivity in response to vascular endothelial growth factor a]pyrimidine-7-one analogs 5.13) The 7-chloro intermediates
(VEGF),7) which modulates vascular permeability and con- were obtained by converting carbonyl to chlorine with phos-
tributes to the formation of cerebral edema.8,9)
phorous oxychloride in N,N-dimethylaniline. Substitution of
Mice lacking pp60c-src show a reduction in both infarct vol- the 7-chlorine with alkylamines afforded 7-aminopyrazolo-
ume and VEGF-mediated vascular permeability after brain [1,5-a]pyrimidines 6. The amide derivatives 7 were prepared
ischemia.10) Moreover, treatment of wild-type mice with PP1, by hydrolyzing the corresponding nitriles 6 with basic hydro-
a selective Src inhibitor, reduces infarct size and decreases gen peroxide in dimethyl sulfoxide.14) Intermediates 7j—l
edema, as shown in Fig. 1.10—12) Consequently, a selectively were deprotected with HCl/EtOAc to give the amide deriva-
active inhibitor of tyrosine kinase c-Src would hold promise tives 8j—l.
as an effective new medication for stroke.
Therefore, we started a program to discover a novel c-Src Result and Discussion
We evaluated the ability of the compounds to inhibit tyro-
sine phosphorylation by c-Src kinase. These data are shown
in Tables 1 and 2. The pyrazolo[1,5-a]pyrimidine derivative 1
had potent c-Src inhibitory activity with an IC50 of 0.64 mM.
First, we focused on the substituents (R3) at the 7-position to
improve the inhibitory activity (Table 1). Modification of the
terminal primary amine to a secondary amine (7a) reduced
the c-Src inhibitory activity compared to 1, and the tertiary
amine (7b) was less potent than 7a. Replacing the amino
Fig. 1. Structure of the Src-Family Kinase Inhibitor PP1
∗ To whom correspondence should be addressed. e-mail: harunobu_mukaiyama@pharm.kissei.co.jp
© 2007 Pharmaceutical Society of Japan

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Vol. 55, No. 6
ethyl group (1) with a carbamoyl methyl (7c) or cyclopropyl tivity, and that the 7-ethylenediamino group mimics the
methyl (7d, e) group resulted in a decrease in c-Src in- triphosphate group of adenosine triphosphate (ATP). These
hibitory activity. The chain length of the diamino moiety at substituent effects were similar to reported effects for the
the 7-position has a significant effect on the c-Src inhibitory Syk family of kinase inhibitors.15,16)
activity. The inhibitory activity of the three-carbon chain de-
Next, we estimated the substituent effect of methoxy
rivative 8j was inferior to that of the two-carbon chain deriv- groups on the aniline phenyl group toward the inhibition of
ative 1. The introduction of a dimethyl group adjacent to the c-Src kinase. Previously, we reported that electron-donating
terminal nitrogen (7f) enhanced the inhibitory activity. In ad- substituents on the phenyl ring, especially the methoxy
dition, the (R)-2ꢁ-aminopropyl analog 8k showed superior ac- group, enhanced the c-Src inhibitory activity.17) A single
tivity to the (S )-2ꢁ-aminopropyl analog 8l. These results sug- methoxy group at either the 3ꢁ- (7g) or 4ꢁ-position (7h) re-
gest that both the terminal primary amine and the carbon duced the inhibitory activity against c-Src kinase compared
chain length are important factors in the c-Src inhibitory ac- to the dimethoxy derivative 7f. Removing the methoxy group
on the aniline phenyl ring (7i) resulted in activity similar to
that of monomethoxy derivatives 7g and 7h. Conversely, 2ꢁ-
Table 1. Inhibitory Activities of Pyrazolo[1,5-a]pyrimidine Derivatives
against c-Src Kinase
c-Src
Compd.
R1
R2
R3
IC50 (mM)
1
3ꢁ-MeO
3ꢁ-MeO
3ꢁ-MeO
3ꢁ-MeO
H
2ꢁ-MeO
3ꢁ-MeO
3ꢁ-MeO
4ꢁ-MeO
H
5ꢁ-MeO
5ꢁ-MeO
5ꢁ-MeO
5ꢁ-MeO
H
–CH2CH2NH2
–CH2CH2NHMe
–CH2CH2NMe2
–CH2CONH2
–CH2cPr
–CH2cPr
–CH2C(CH3)2NH2
–CH2C(CH3)2NH2
–CH2C(CH3)2NH2
–CH2C(CH3)2NH2
–CH2CH2CH2NH2
0.64
2.73
27.80
33.70
2.48
1000
0.18
1.00
1.22
1.71
8.16
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
5ꢁ-MeO
H
H
H
8j
3ꢁ-MeO
5ꢁ-MeO
Reagents: (a) Substituted or unsubstituted aniline, MeOH, reflux; (b) hydrazine
monohydrate, N,N-dimethylformamide (DMF), 100 °C; (c) R3(CO)CH2CO2Et, AcOH,
reflux; (d) POCl3, N,N-dimethylaniline, 60 °C; (e) R4NH2, NEt3, 1-methy-2-pyrrolidi-
none (NMP), r.t.; (f) NaOH, H2O2, 60 °C; (g) 4 M HCl, EtOAc, r.t.
8k
8l
3ꢁ-MeO
3ꢁ-MeO
5ꢁ-MeO
5ꢁ-MeO
0.41
2.99
Chart 1
Table 2. Inhibitory Activities against c-Src Kinase and Selectivity Profiles of Pyrazolo[1,5-a]pyrimidine Derivatives
IC50(mM)
Compd.
R4
c-Src
Lck
ZAP
Syk
PKCb
EGFR
KDR
7f
Me
Ph
Et
cPr
iPr
0.175
0.018
0.010
0.004
0.021
0.028
0.651
0.962
0.019
0.288
0.010
0.099
0.350
1.970
44.6
NTa)
27.1
124
46.6
91.6
NTa)
2.62
NTa)
3.89
3.72
12.9
11.9
NTa)
1.07
0.04
5.97
1.70
1.84
0.82
8.65
5.95
0.10
1.80
0.01
0.42
0.14
6.99
1.00
NTa)
NTa)
NTa)
NTa)
NTa)
NTa)
7m
7n
7o
7p
7q
7r
nPr
CH2COOH
a) NTꢂnot tested.

June 2007
883
methoxy derivative 7e showed no activity compared to the
cyclopropylamino derivative 7d. The complete disappearance
of the inhibitory activity of compound 7e may have been at-
tributable to the steric clash between the 2ꢁ-methoxy group
and the proximal amino acids of the binding site. These re-
sults indicate that a 3ꢁ,5ꢁ-dimethoxy group is a favorable sub-
stituent on the aniline phenyl ring in this series.
(a)
Thus, we investigated the influence of the substituent at
the 5-position of the pyrazolo[1,5-a]pyrimidine core on the
c-Src kinase inhibitory activity. The results are listed in Table
2. Aryl and heteroaryl groups are involved in a hydrophobic
interaction with the hydrophobic pocket adjacent to the ATP-
binding site, enhancing the tyrosine kinase inhibitory activ-
ity. For example, Chen et al. reported that replacing the
aliphatic amines with aryl or heteroaryl amines at the 4-posi-
tion of the 1,5-imidazoquinoxaline core improved the affinity
with the hydrophobic pocket of Lck kinase.18) Therefore,
substituting the phenyl group at the 5-position (7m) in-
creased the activity in comparison to 7f. Interestingly, re-
placement with a sterically bulky alkyl group, even an ethyl
group (7n), enhanced the activity more than that of the
methyl analog 7f. The introduction of bulkier substituents led
to more active compounds 7p, q. In this series, the 5-cyclo-
propyl derivative 7o had the most potent activity (IC50ꢂ
0.004 mM). Conversely, the additional introduction of a hy-
drophilic substituent (7r) led to loss of the inhibitory activity.
To gain insight into the structural basis of the inhibitory
activity of our compounds, we conducted an X-ray analysis
of the crystal structures of 7f and 7o co-crystallized with pu-
rified human Lck protein, a closely related member of the
Src family, as shown in Fig. 2. The 3-carboxamide group and
N–H of the 2-aminophenyl ring formed a pseudo-six-mem-
bered ring, and compounds 7f and 7o each interacted with
several amino acid residues to enhance their binding affinity
with the ATP binding site, as shown in Fig. 2a—c. For exam-
ple, the N–H of the 3-carboxamide group formed a hydrogen
bond with the carbonyl group of Glu 317 (Glu 341 in c-Src)
and the carbonyl group of 7f interacted with the N–H of Met
319 (Met 343 in c-Src). The N-4 of the pyrazolo[1,5-a]-
pyrimidine formed a hydrogen bond with the hydroxyl group
of Thr 316 (Thr 340 in c-Src). The terminal primary amino
group of the 7-position was hydrogen bonded to the water
bridging Asn 319.
(c)
Interestingly, five water molecules occupied the hydropho-
bic pocket (red balls; Fig. 2a), whereas the cyclopropyl group
of 7o extruded these water molecules to interact with the
pocket (Fig. 2c). The cyclopropyl moiety was approximately
90° to the plane of the pyrazolo[1,5-a]pyrimidine nucleus.
The results of the X-ray analysis explain the inhibitory activ-
ities obtained for the 5-substituted groups. Replacing the
cluster of water molecules in the hydrophobic pocket with a
hydrocarbon would stabilize the energy, as Fersht described
previously.19) The reasonable bulkiness and perpendicular
Fig. 2. Interactions of Compounds 7f and 7o with c-Src Kinases as Deter-
mined by X-Ray Crystallographic Studies
(a) The structure of the c-Src complex with 7f is shown using a stick representation.
Compound 7f is shown as a ball and stick. The hydrophobic pocket is shown as a sur-
face representation. Water molecules are represented as red balls. (b) Schematic repre-
sentation of the binding mode of compound 7f in c-Src kinase. (c) The structure of the
c-Src complex with 7o is shown using a stick representation. The hydrophobic pocket is
shown as a surface representation.
conformation of the cyclopropyl group to the plane of the c-Src kinase compared to the other kinases, such as Syk,
pyrazolo[1,5-a]pyrimidine nucleus seems to play an impor- Zap, PKCb2, EGFR, and KDR kinases (see Table 2). Fur-
tant role in binding through the stable hydrophobic interac- thermore, these compounds exhibited greater selectivity for
tion with the hydrophobic pocket.
the inhibition of c-Src relative to Lck kinase, which is a fam-
Since selectivity against other kinase families is important ily member with high sequence homology. The 5-cyclopropyl
for circumventing undesirable side effects, we evaluated the derivative 7o had the best selectivity compared to Syk and
effects of compounds 7f and 7m—r on various kinases. Zap, but displayed significant activity against EGFR.
These compounds showed excellent selectivity for inhibiting
Based on the selective inhibition of c-Src kinase, com-

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Vol. 55, No. 6
Table 3. Intracellular c-Src Kinase Inhibitory Activity of 7f·HCl and Its
middle cerebral artery (MCA) occlusion model reported by
Umemura et al.,20) and assessed the neuroprotective effect
using the infarct volume (see Fig. 3). Compound 7f·HCl fa-
cilitated neuroprotection with a dose-dependent reduction in
the infarct volume when administered intraperitoneally at
doses of 1 or 3 mg/kg 15 min after MCA occlusion. In partic-
ular, 7f·HCl at the highest dose (3 mg/kg) significantly re-
duced the infarct volume by 32% compared to the control.
Pharmacokinetic Parametersa)
c-Src inhibition CNS penetration (3 mg/kg/h, i.v. infusion)b)
Compd.
ELISA
Plasma
Brain
Ratio
IC50 (mM)
C3h (ng/ml)
C3h (ng/g) (brain/plasma)
7f
0.9
121.5ꢄ14.3 444.0ꢄ63.3 3.7ꢄ0.6
a) The results are the meansꢄS.D. of three animals in each group. b) Compound
7f was administered as a solution of EtOH : dimethyl isosorbide : propylene glycol
400 : 5% glucose (15 : 10 : 35 : 40).
Conclusion
We discovered a series of novel pyrazolo[1,5-a]pyrimidine
derivatives possessing potent in vitro inhibitory activity
against c-Src kinase. Various substituents at the 5- and 7-po-
sitions of the pyrazolo[1,5-a]pyrimidine played an important
role in modulating the kinase inhibitory activity. Of these
compounds, 7f·HCl exhibited potent intracellular kinase in-
hibition and good CNS penetration. This compound also had
a significant neuroprotective effect in rats, and could be a
useful novel therapeutic agent for acute ischemic stroke.
Table 4. Pharmacokinetic Properties of Compound 7f·HCl in Ratsa)
Pharmacokinetic parameter
i.v.b)
i.p.b)
Dose (mg/kg)
AUC0—inf. (ng·h/ml)
AUC0—4 h (ng·h/ml)
Co max (ng/ml)
Cmax (ng/ml)
Tmax (h)
T1/2 (h)
MRT (h)
Cl (ml/min/kg)
Vss (l/kg)
1.0
292.0ꢄ50.4
—
190.3ꢄ41.1
—
3.0
—
382.0ꢄ124.6
—
120.3ꢄ43.0
1.5ꢄ0.9
—
—
Experimental
2.8ꢄ1.0
3.9ꢄ1.3
58.3ꢄ9.2
13.4ꢄ4.4
Biological Procedures. (a) Kinase Assays A coupled spectrophotomet-
ric assay was used wherein ADP generated by Src kinase was converted to
ATP by pyruvate kinase (PK), with concomitant production of pyruvate
from phosphoenolpyruvate (PEP). LDH reduces pyruvate to lactate by oxi-
dizing NADH. NADH depletion was monitored at 340 nm using a mi-
croplate reader (Spectra Max 250, Molecular Device) at 30 °C for 20 min.
Reactions were performed at 30 °C in 100 mM HEPES buffer (pH 7.6), con-
taining 20 mM MgCl2, and 10% glycerol, initiated by adding ATP. PK
(100 mg/ml), LDH (50 mg/ml), PEP (2 mM), and NADH (140 mM) was also
added. Kinase activity was measured by adding 100 mM Src optimal peptide
substrate (peptide sequence: AEEEIYGEFEAKKKK, Sawady, Tokyo).
(b) Intracellular Kinase Inhibition Assay The kinase domain of
human Src kinase (NM_005417, base# 790-1650) was cloned by PCR am-
plification, using the linker-containing primers 5ꢁ-AAACTTAAGCTTCA-
TATGTCCAAGCCGCAGAC-3ꢁ and 5ꢁ-CTGCAGATATCCCTAGAAGTA-
GTCCTCCAGGAA-3ꢁ. The gene for the Src kinase domain was integrated
between the HindIII and EcoRV restriction sites within the multiple cloning
site of the pcDNA3.1(ꢀ) expression vector. The vector was transfected into
COS7 cells according to the calcium phosphate method.21) Subsequently,
8.8 mg DNA was mixed with 220 ml 1 mM Tris–HCl, 0.1 mM EDTA buffer
(pH 8.0), 250 ml 2ꢃHepes Buffered Saline, and 31 ml 2 M CaCl2. The solu-
tion was incubated at room temperature for 15 min and then used to resus-
pend a pellet of 106 COS7 cells. The cell suspension was incubated at room
temperature for 15 min; then, 4.5 ml Dulbecco’s Modified Eagle’s Medium
supplemented with 10% fetal bovine serum and antibiotics was added, and
the solution was pre-warmed to 37 °C. Cells were seeded into 96-well multi-
well plates with 2ꢃ104 cells/well/100 ml. A day after transfection, the culture
media was replaced with media containing fixed concentrations of test com-
pounds. On the third day, the cellular phosphotyrosine contents were deter-
mined using a commercially available phosphotyrosine ELISA kit (Upstate
Biotechnology, NY, U.S.A. Cellular Phosphotyrosine ELISA KitTM, Catalog
#17-182). Any basal phosphotyrosine content unrelated to c-Src activity was
screened for using vector-transfected cells. Intracellular c-Src inhibition was
expressed as the percentage of specific phosphotyrosine levels in drug-
treated cells compared to the levels in control cells without drug. The toxic-
ity of each chemical was determined with another replica plate using a col-
orimetric MTT assay kit (Chemicon International, CA, U.S.A. Cat. #CT02).
(c) Photochemically Induced Middle Cerebral Artery Occlusion in
Rats Male Sprague-Dawley rats weighing 260—320 g were anesthetized
with enflurane. The animal’s body temperature was maintained at 37.5 °C
with a heating pad. We performed middle cerebral artery (MCA) thrombosis
as previously described.20) A catheter was inserted into the femoral vein to
administer the drug and rose bengal dye. The scalp and temporalis muscle
were folded over. A subtemporal craniotomy was performed using a dental
drill under an operating microscope to open a 3-mm-diameter oval bony
window. The window was irradiated with green light (540 nm wavelength)
using a xenon lamp (L4887, Hamamatsu Photonics, Hamamatsu, Japan),
with both heat-absorbing and green filters. The irradiation was directed by a
—
—
—
a) The results are the meanꢄS.D. of three animals in each group. b) Compound
7f·HCl was administered as a solution of 5% glucose.
Fig. 3. Neuroprotective Effects of 7f·HCl in a Middle Cerebral Artery
(MCA) Thrombosis Model
∗ pꢅ0.05 vs. vehicle (Dunnett’s test; nꢂ6—12).
pound 7f was selected for an in vivo assay. Compound 7f ex-
hibited significant intracellular c-Src inhibition in the cellular
assay, in an enzyme-linked immunosorbent assay (ELISA)
using COS7 cells (Table 3).
To treat stroke effectively, it is important that compound 7f
penetrate the brain at pharmacologically active levels. We
measured the concentration of 7f in the brain and plasma 3 h
after an intravenous infusion (Table 3). Compound 7f had a
significant brain/plasma ratio (3.7) and sufficient levels in the
brain. In a pharmacokinetic study, 7f·HCl was characterized
as having a high volume of distribution (Vss) and a favorable
half-life (T1/2) and mean residence time (MRT) after intra-
venous administration (Table 4). In conjunction with the rat
data 4 h after intraperitoneal administration, we concluded
that 7f·HCl had an appropriate pharmacokinetic profile for
conducting in vivo efficacy studies.
We evaluated the in vivo activity of 7f·HCl using the rat 3-mm-diameter optic fiber mounted on a micromanipulator. The head of the

June 2007
885
optic fiber was placed on the window in the skull base at a distance of 2 mm viation from the ideal bond length of 0.0087 Å, and a root mean square
above the vessel, providing an irradiation dose of 0.62 W/cm2. Rose bengal (rms) deviation from the ideal bond angle of 1.21°.
(20 mg/kg) was injected intravenously. Photo-irradiation was continued for
The geometry of both models was checked using the program
another 10 min. Compound 7f·HCl was intraperitoneally administered at a PROCHECK25); no residues occurred in the disallowed regions of a Ra-
dose of 1, and 3 mg/kg at 15 min after MCA occlusion. Twenty-four hours machandran plot.
after surgery, the rats were sacrificed by administering an overdose of pento-
barbital, and their brains were quickly removed. The cerebrum was separated Micro melting point apparatus and are uncorrected. Infrared spectra were
from the other parts of the brain, and cut into six 2-mm-thick slices using a measured on a Nicolet 510 FT-IR spectrophotometer and are reported in re-
Chemical Methods Melting points were taken on a Yanako MP-3S
Brain Matrix (Muromachi Kikai, Japan). Each slice was incubated in 1% ciprocal centimeters. Proton NMR spectra were recorded at 400 or 500 MHz
tetratrihydrochloride (TTC) solution at room temperature for 30 min and with a Bruker AMX 400 or DRX 500 instrument, and chemical shifts are re-
then photographed. The area of infarction was measured for each slice using
a computerized image analysis system (Mac scope, Japan), and the ratio of
ported in parts per million (d) downfield from tetramethylsilane as the inter-
nal standard. The peak patterns are shown as the following abbreviations:
infarction size was calculated by dividing the whole area by the region of brꢂbroad, dꢂdoublet, mꢂmultiplet, sꢂsinglet, tꢂtriplet, qꢂquartet. The
cerebral ischemic damage.
(d) Analysis of c-Src Inhibitors in Plasma and Brain To inhibit c-Src
mass spectra (MS) were carried out with a Thermo Quest FINNIGAN AQA
electrospray ionization mass spectrometer. Elemental analyses were per-
kinase in the brain, we first determined the brain and plasma levels of the formed on an Elementar Vario EL analyzer (C, H, N). The analytical results
test compounds. Sprague-Dawley rats (300—350 g: nꢂ3/study) were anes- obtained were within ꢄ0.4% of the theoretical values unless otherwise
thetized with 25% urethane (1.1 g/kg, s.c.), and the femoral vein was cannu-
stated. Silica gel 60F254 precoated plates on glass from Merck KGaA or
lated to deliver the test compounds. Tracheal intubation was performed for aminopropyl silica gel (APS) precoated NH plates from Fuji Silysia Chemi-
artificial respiration. The animal’s body temperature was maintained at cal Ltd. were used for thin-layer chromatography (TLC). Flash column chro-
37.5 °C with a heating-pad. Compound 7f·HCl was administered by intra- matography was performed on silica gel 60N (particle size 40—50 mm)
venous infusion at 3 mg/kg/h. Three hours later, blood samples were taken from Kanto Chemical Co., Inc. or APS Daisogel IR-60 (particle size 25—
from the aorta, and centrifuged to separate the plasma. Brain tissue was ho-
mogenized with a 3-fold volume of saline.
40 mm) from Daiso Co., Ltd. All reagents and solvents were commercially
available unless otherwise indicated.
Plasma and brain tissue homogenate (50 ml) was mixed with 200 ml 100%
acetonitrile, 100 ml 0.1% heptafluorobutyric acid, and 200 ml internal stan-
dard solution. After centrifugation at 3300 rpm for 20 min, the supernatant
2-Cyano-3-(3,5-dimethoxyphenylamino)-3-methylsulfanylacrylonitrile
(3a) A mixture of 3,3-bis(methylsulfanyl)-2-cyanoacrylonitrile (2, 40.5 g,
0.24 mol) and 3,5-dimethoxyaniline (36.4 g, 0.24 mol) in MeOH (400 ml)
was removed and transferred to the column. Samples were quantified using was stirred for 15 h at reflux. After cooling at room temperature, collection
liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC- of the resulting precipitates gave 53.4 g (82%) of 3a as a white solid. 1H-
MS/MS system consisted of an Alliance HPLC (Waters 2690) with a SYN- NMR (CDCl3) d: 2.33 (3H, s), 3.80 (6H, s), 6.40—6.45 (2H, m), 6.35—6.40
ERGI MAX-RP 80A column (4 mm, 50ꢃ4.6 mm, Phenomenex, Torrance, (1H, m).
CA, U.S.A.), and a Sciex API 365 mass spectrometer (Perkin–Elmer Sciex,
Toronto, Canada) equipped with a turbo ion spray source. The mobile phase
consisted of 100% acetonitrile and 0.1% acetic acid in water (50 : 50 v/v).
2-Cyano-3-(3-methoxyphenylamino)-3-methylsulfanylacrylonitrile
(3b) This title compound was prepared from 2 and 3-methoxyaniline ac-
cording to the procedure described for preparation of 3a, and obtained as an
The column was maintained at 50 °C with a constant flow rate of 0.4 ml/min. off-white solid (99% yield). 1H-NMR (CDCl3) d: 2.33 (3H, s), 3.83 (3H, s),
The data was processed using Mass Chrom 1.1 software. The standard 6.79 (1H, t, Jꢂ2.0 Hz), 6.80—6.90 (2H, m), 7.33 (1H, t, Jꢂ8.0 Hz), 7.58
curves plotted for each test compound demonstrated good linearity (coeffi- (1H, br s).
cient of determination ꢆ0.99). The limits of quantification of the com-
2-Cyano-3-(4-methoxyphenylamino)-3-methylsulfanylacrylonitrile
pounds in plasma and brain were 50 ng/ml. All animal studies were approved (3c) This title compound was prepared from 2 and 4-methoxyaniline ac-
by the Kissei Pharmaceutical Co., Ltd. Committee on Ethics of Animal Ex-
perimentation, and special care was taken to prevent animal suffering.
cording to the procedure described for preparation of 3a, and obtained as an
off-white solid (93% yield). 1H-NMR (CDCl3) d: 2.34 (3H, s), 3.84 (3H, s),
X-Ray Crystallographic Analysis of Human Lck Complex with 7f and 6.90—6.95 (2H, m), 7.15—7.20 (2H, m), 7.59 (1H, br s).
7o Human Lck was purified as described,22) and concentrated to 3.0 mg/ml.
2-Cyano-3-methylsulfanyl-3-phenylaminoacrylonitrile (3d) This title
Co-crystals were obtained at 277 K using the hanging-drop vapor-diffusion compound was prepared from 2 and aniline according to the procedure de-
technique, equilibrated against a reservoir solution of 0.2 M (NH4)2SO4, 0.1 M
sodium cacodylate (pH 6.5), 30% (w/v) PEG8000. Before collecting the
data, the crystals were quickly dipped in 20% glycerol containing a cryo-so- 7.40—7.50 (2H, m), 7.82 (1H, br s).
scribed for preparation of 3a, and obtained as an off-white solid (94% yield).
1H-NMR (CDCl3) d: 2.29 (3H, s), 7.20—7.30 (2H, m), 7.30—7.40 (1H, m),
lution and plunged into liquid nitrogen. X-Ray diffraction data for the co-
2-Cyano-3-(2-methoxyphenylamino)-3-methylsulfanylacrylonitrile
crystal of 7f were collected at a wavelength of 1.0 Å with a BL32B2 station (3e) This title compound was prepared from 2 and 2-methoxyaniline ac-
(SPring-8, Harima, Japan). The diffraction data of the co-crystal of 7o were
cording to the procedure described for preparation of 3a, and obtained as an
collected with an R-AXIS IV (Rigaku, Tokyo, Japan) diffractometer. The off-white solid (80% yield). 1H-NMR (DMSO-d6) d: 2.60 (3H, s), 3.85 (3H,
crystal complex with 7f diffracted to 1.8 Å resolution and belongs to the s), 6.95—7.05 (1H, m), 7.10—7.15 (1H, m), 7.20—7.25 (1H, m), 7.30—
P212121 space group with aꢂ42.2 Å, bꢂ74.0 Å, and cꢂ91.66 Å. The crystal
complex with 7o diffracted to 1.9 Å resolution and belongs to the P212121
space group with aꢂ42.3 Å, bꢂ73.8 Å, and cꢂ93.0 Å.
7.40 (1H, m), 10.14 (1H, br s).
5-Amino-3-(3,5-dimethoxyphenylamino)pyrazole-1H-4-carbonitrile
(4a) A mixture of 2-cyano-3-(3,5-dimethoxyphenylamino)-3-methylsul-
The diffraction data for the crystal complexes with 7f and 7o were inte- fanylacrylonitrile (3a, 52.5 g, 0.19 mol) and hydrazine monohydrate (11.5 g,
grated and scaled using the programs MOSFLM23) and Scala24) with an 0.23 mol) in N,N-dimethylformamide (110 ml) was stirred for 6 h at 100 °C.
Rsym(I) of 0.138 and a completeness of 98.8% to the highest resolution of
1.8 Å and with an Rsym(I) of 0.124 and a completeness of 98.0% to the
highest resolution of 1.9 Å, respectively.
The structures were refined using the program CNX (Accelrys, San
Diego, CA, U.S.A.), starting with the existing refined Human Lck model 5.90—6.00 (1H, m), 6.25 (2H, br s), 6.70—6.75 (2H, m), 8.30 (1H, br s),
After cooling at room temperature, the mixture was diluted with water
(500 ml) and stirred for 1 h. The resulting precipitate was collected by filtra-
tion and washed with water (500 ml) to give 4a (45 g, 91%) as an off white
solid. mp 249—250 °C (MeOH). 1H-NMR (DMSO-d6) d: 3.67 (6H, s),
structure (PDB code: 3lck), excluding the ligand. A rigid-body rotation–
11.16 (1H, br s). IR (KBr) cmꢇ1: 3351, 2209, 1574, 1173. Anal. Calcd for
translation refinement was initially carried out to place the model structure C12H13N5O2: C, 55.59; H, 5.05; N, 27.01. Found: C, 55.20; H, 5.14; N,
more accurately in the new unit cell. Crystallographic refinement was con- 27.05.
tinued by conjugate-gradient minimization and individual B factor refine-
ment with CNX, and model/ligand building was performed with the pro-
gram QUANTA (Accelrys).
5-Amino-3-(3-methoxyphenylamino)pyrazole-1H-4-carbonitrile (4b)
This title compound was prepared from 3b according to the procedure de-
scribed for preparation of 4a, and obtained as an off-white solid (quant.). mp
1
The final structure complex with 7f gave Rcrystꢂ0.199 and Rfreeꢂ0.239 at 182—183 °C. H-NMR (DMSO-d6) d: 3.69 (3H, s), 6.25 (2H, br s), 6.30—
1.8 Å resolution with good stereochemistry, a root mean square (rms) devia-
tion from the ideal bond length of 0.0086 Å, and a root mean square (rms)
deviation from the ideal bond angles of 1.23°.
The final structure complexed with 7o gave Rcrystꢂ0.216 and Rfreeꢂ0.252
at 1.9 Å resolution with good stereochemistry, a root mean square (rms) de-
6.40 (1H, m), 7.00—7.10 (2H, m), 7.10—7.15 (1H, m), 8.30 (1H, s), 11.14
(1H, s). IR (KBr) cmꢇ1: 3385, 2198, 1617, 1498. Anal. Calcd for
C11H11N5O: C, 57.63; H, 4.84; N, 30.55. Found: C, 57.54; H, 4.88; N, 30.60.
5-Amino-3-(4-methoxyphenylamino)pyrazole-1H-4-carbonitrile (4c)
This title compound was prepared from 3c according to the procedure de-

886
Vol. 55, No. 6
scribed for preparation of 4a, and obtained as an off-white solid (91%). mp
2-(3,5-Dimethoxyphenylamino)-5-ethyl-7-oxo-4,7-dihydropyrazolo-
189—190 °C. 1H-NMR (DMSO-d6) d: 3.68 (3H, s), 6.20 (2H, br s), 6.77 [1,5-a]pyrimidine-3-carbonitrile (5g) This title compound was prepared
(2H, d, Jꢂ9.1 Hz), 7.41 (2H, d, Jꢂ9.1 Hz), 8.05 (1H, s), 11.00 (1H, s). IR
from 4a and ethyl 3-oxovalerate according to the procedure described for
(KBr) cmꢇ1: 3289, 2212, 1511, 1247. Anal. Calcd for C11H11N5O: C, 57.63; preparation of 5a, and obtained as an off-white solid (88%). mp 270—
H, 4.84; N, 30.55. Found: C, 57.59; H, 4.87; N, 30.52.
5-Amino-3-phenylaminopyrazole-1H-4-carbonitrile (4d) This title
compound was prepared from 3d according the procedure to described for
272 °C. 1H-NMR (DMSO-d6) d: 1.22 (3H, t, Jꢂ7.6 Hz), 2.59 (2H, q,
Jꢂ7.6 Hz), 3.72 (6H, s), 5.79 (1H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 7.00 (2H, d,
Jꢂ2.2 Hz), 9.12 (1H, s), 12.96 (1H, br s). IR (KBr) cmꢇ1: 3328, 2219, 1696,
preparation of 4a, and obtained as an off-white solid (87%). mp 204— 1647, 1616, 1559. Anal. Calcd for C17H17N5O3·1.25H2O: C, 56.42; H, 5.43;
205 °C. 1H-NMR (DMSO-d6) d: 6.24 (2H, br s), 6.70—6.80 (1H, m), 7.10— N, 19.35. Found: C, 56.65; H, 5.23; N, 19.64.
7.20 (2H, m) 7.35—7.55 (2H, m), 8.30 (1H, br s), 11,12 (1H, br s). IR (KBr)
2-(3,5-Dimethoxyphenylamino)-7-oxo-5-phenyl-4,7-dihydropyrazolo-
cmꢇ1: 3305, 2216, 1607, 1540, 1244. Anal. Calcd for C10H9N5: C, 60.29; H, [1,5-a]pyrimidine-3-carbonitrile (5h) This title compound was prepared
4.55; N, 35.16. Found: C, 60.33; H, 4.59; N, 35.44.
5-Amino-3-(2-methoxyphenylamino)pyrazole-1H-4-carbonitrile (4e)
from 4a and ethyl 3-oxo-3-phenylpropionate according to the procedure de-
scribed for preparation of 5a, and obtained as an off-white solid (76%). mp
This title compound was prepared from 3e according to the procedure de- 274—275 °C. 1H-NMR (DMSO-d6) d: 3.73 (6H, s), 6.12 (1H, t, Jꢂ2.2 Hz),
scribed for preparation of 4a, and obtained as an off-white solid (87%). mp 6.20 (1H, s), 7.05 (2H, d, Jꢂ2.2 Hz), 7.50—7.65 (3H, m), 7.75—7.85 (2H,
165—166 °C. 1H-NMR (DMSO-d6) d: 3.87 (3H, s), 6.37 (2H, br s), 6.75 m), 9.15 (1H, s), 13.33 (1H, br s). IR (KBr) cmꢇ1: 3424, 2216, 1670, 1611,
(1H, br s), 6.81 (1H, td, Jꢂ7.9, 1.6 Hz), 6.85 (1H, td, Jꢂ7.9, 1.6 Hz), 6.97
(1H, td, Jꢂ7.9, 1.6 Hz), 7.89 (1H, td, Jꢂ7.9, 1.6 Hz), 11.22 (1H, br s). IR
(KBr) cmꢇ1: 3353, 2213, 1633, 1553, 1248. Anal. Calcd for C11H11N5O: C,
57.63; H, 4.84; N, 30.55. Found: C, 57.60; H, 4.88; N, 30.74.
1560. Anal. Calcd for C21H17N5O3·0.2H2O: C, 64.51; H, 4.49; N, 17.91.
Found: C, 64.56; H, 4.42; N, 18.26.
2-(3,5-Dimethoxyphenylamino)-5-isopropyl-7-oxo-4,7-dihydropyra-
zolo[1,5-a]pyrimidine-3-carbonitrile (5i) This title compound was pre-
2-(3,5-Dimethoxyphenylamino)-5-methyl-7-oxo-4,7-dihydropyrazolo- pared from 4a and methyl 4-methyl-3-oxopentanate according to the proce-
[1,5-a]pyrimidine-3-carbonitrile (5a) mixture of 5-amino-3-(3,5- dure described for preparation of 5a, and obtained as an off-white solid
dimethoxyphenylamino)pyrazole-1H-4-carbonitrile (4a, 8.2 g, 31.7 mmol)
A
1
(87%). mp 259—260 °C. H-NMR (DMSO-d6) d: 1.24 (6H, d, Jꢂ6.9 Hz),
and ethyl acetoacetate (4.44 ml, 34.9 mmol) in acetic acid (80 ml) was stirred 2.85—2.95 (1H, m), 3.72 (6H, s), 5.79 (1H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 7.00
for 10 h at reflux. After cooling at room temperature, the mixture was diluted (2H, d, Jꢂ2.2 Hz), 9.13 (1H, s), 12.88 (1H, br s). IR (KBr) cmꢇ1: 3380,
with water (80 ml) and stirred for 1 h. The resulting precipitates were col- 2219, 1692, 1639, 1593. Anal. Calcd for C18H19N5O3·0.25H2O: C, 60.41; H,
lected by filtration and washed with water (200 ml) to give 5a (9.4 g, 91%)
as an off white solid. mp ꢆ300 °C. 1H-NMR (DMSO-d6) d: 2.29 (3H, s),
3.72 (6H, s), 5.79 (1H, br s), 6.10 (1H, t, Jꢂ2.2 Hz), 7.00 (2H, d, Jꢂ2.2 Hz),
9.15 (1H, br s), 13.04 (1H, br s). IR (KBr) cmꢇ1: 3355, 2215, 1598, 1560,
5.49; N, 19.57. Found: C, 60.01; H, 5.14; N, 19.31.
2-(3,5-Dimethoxyphenylamino)-7-oxo-5-propyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidine-3-carbonitrile (5j) This title compound was prepared
from 4a and methyl butyrylacetate according to the procedure described for
1155. Anal. Calcd for C16H15N5O3·0.2H2O: C, 58.42; H, 4.72; N, 21.29. preparation of 5a, and obtained as an off-white solid (92%). mp 261—
Found: C, 58.45; H, 4.61; N, 21.31.
262 °C. 1H-NMR (DMSO-d6) d: 0.94 (3H, t, Jꢂ7.3 Hz), 1.60—1.75 (2H,
2-(3-Methoxyphenylamino)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5- m), 2.54 (2H, t, Jꢂ7.6 Hz), 3.72 (6H, s), 5.80 (1H, s), 6.10 (1H, t,
a]pyrimidine-3-carbonitrile (5b) This title compound was prepared from Jꢂ2.2 Hz), 7.00 (2H, d, Jꢂ2.2 Hz), 9.14 (1H, s), 12.96 (1H, br s). IR (KBr)
4b according to the procedure described for preparation of 5a, and obtained
cmꢇ1
:
3317, 2223, 1696, 1614, 1560, 1474. Anal. Calcd for
as an off-white solid (86%). mp ꢆ300 °C. 1H-NMR (DMSO-d6) d: 2.29 C18H19N5O3·H2O: C, 58.21; H, 5.70; N, 18.86. Found: C, 58.13; H, 5.62; N,
(3H, s), 3.74 (3H, s), 5.79 (1H, s), 6.51 (1H, dd, Jꢂ8.2, 2.5 Hz), 7.18 (1H, t,
Jꢂ8.2 Hz), 7.32 (1H, dd, Jꢂ8.2, 1.9 Hz), 7.38 (1H, t, Jꢂ2.4 Hz), 9.14 (1H,
s), 13.00 (1H, s). IR (KBr) cmꢇ1: 3373, 3088, 2222, 1668, 1570, 1498. Anal.
19.07.
Methyl [3-Cyano-2-(3,5-dimethoxyphenylamino)-7-oxo-4,7-dihydro-
pyrazolo[1,5-a]pyrimidin-5-yl]acetate (5k) This title compound was pre-
Calcd for C15H13N5O2·0.25H2O: C, 60.09; H, 4.54; N, 23.36. Found: C, pared from 4a and dimethyl 3-oxoglutarate according to the procedure de-
59.84; H, 4.37; N, 23.22. scribed for preparation of 5a, and obtained as an off-white solid (78%). mp
1
2-(4-Methoxyphenylamino)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5- 280—281 °C. H-NMR (DMSO-d6) d: 3.70 (3H, s), 3.72 (6H, s), 3.80 (2H,
a]pyrimidine-3-carbonitrile (5c) This title compound was prepared from s), 5.94 (1H, s), 6.11 (1H t, Jꢂ2.2 Hz), 7.00 (2H, d, Jꢂ2.2 Hz), 9.22 (1H, s),
4c according to the procedure described for preparation of 5a, and obtained
13.25 (1H, br s). IR (KBr) cmꢇ1: 3393, 2215, 1630, 1617, 1565, 1483. Anal.
as an off-white solid (97%). mp ꢆ300 °C. 1H-NMR (DMSO-d6) d: 2.28 Calcd for C18H17N5O5: C, 56.39; H, 4.47; N, 18.27. Found: C, 56.45; H,
(3H, s), 3.72 (3H, s), 5.76 (1H, s), 6.88 (2H, d, Jꢂ8.8 Hz), 7.62 (2H, d, 4.59; N, 18.38.
Jꢂ8.8 Hz), 8.92 (1H, s), 13.00 (1H, s). IR (KBr) cmꢇ1: 3343, 2215, 1684,
2-(3,5-Dimethoxyphenylamino)-5-methyl-7-(2-methylaminoethyl-
1639, 1594, 1559. Anal. Calcd for C15H13N5O2·1.5H2O: C, 55.90; H, 5.00; amino)pyrazolo[1,5-a]pyrimidine-3-carbonitrile (6a) (a) A mixture of
N, 21.73. Found: C, 55.71; H, 4.91; N, 20.20. 2-(3,5-dimethoxyphenylamino)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-
5-Methyl-7-oxo-2-phenylamino-4,7-dihydropyrazolo[1,5-a]pyrimi- a]pyrimidine-3-carbonitrile (5a, 3.61 g, 11.1 mmol) and in phosphorus oxy-
dine-3-carbonitrile (5d) This title compound was prepared from 4d ac-
cording to the procedure described for preparation of 5a, and obtained as an
off-white solid (quant.). mp ꢆ300 °C. 1H-NMR (DMSO-d6) d: 2.29 (3H, s),
chloride (21 ml, 0.22 mol) in N,N-dimethylaniline (21 ml, 0.17 mol) was
stirred for 2 h at 60 °C. After cooling at room temperature, the mixture was
poured into ice-water. The resulting precipitates were collected by filtration
5.79 (1H, s), 6.85—7.00 (1H, m), 7.25—7.35 (2H, m), 7.70—7.75 (2H, m), and washed with water (500 ml). After drying under vacuum, 3.7 g (96%) of
9.16 (1H, s), 13.05 (1H, s). IR (KBr) cmꢇ1: 3415, 3072, 2215, 1668, 1624,
7-chloro-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-a]pyrimi-
1596, 1559. Anal. Calcd for C14H11N5O: C, 63.39; H, 4.18; N, 26.40. Found: dine-3-carbonitrile was obtained as a crude mixture. (b) To a suspension of
C, 63.13; H, 4.04; N, 26.49. 7-chloro-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-a]pyrimi-
2-(2-Methoxyphenylamino)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5- dine-3-carbonitrile (222 mg, 0.65 mmol) in 1-methyl-2-pyrrolidinone (2.0
a]pyrimidine-3-carbonitrile (5e) This title compound was prepared from ml) was added 2-(methylamino)ethylamine (0.28 ml, 3.23 mmol), and the
4e according the procedure described for preparation of 5a, and obtained as
mixture was stirred for 24 h at room temperature. The resulting mixture was
1
an off-white solid (98%). mp ꢆ300 °C. H-NMR (DMSO-d6) d: 2.29 (3H, diluted with water (3.0 ml), and the obtained precipitates were collected by
s), 3.89 (3H, s), 5.79 (1H, s), 6.90—7.10 (3H, m), 7.64 (1H, br s), 8.04 (1H, filtration and washed with water (3.0 ml) to give 6a (226 mg, 92%) as an off
dd, Jꢂ7.6, 1.9 Hz), 13.06 (1H, br s). IR (KBr) cmꢇ1: 3415, 3091, 2214, white solid. mp 283—284 °C. 1H-NMR (DMSO-d6) d: 2.30 (3H, s), 2.40
1676, 1635, 1594, 1559. Anal. Calcd for C15H13N5O2·0.2H2O: C, 60.27; H, (3H, s), 2.74 (2H, t, Jꢂ6.0 Hz), 3.43 (2H, t, Jꢂ6.0 Hz), 3.74 (6H, s), 6.08
4.52; N, 23.43. Found: C, 60.25; H, 4.38; N, 23.65.
(1H, t, Jꢂ2.2 Hz), 6.33 (1H, s), 6.98 (2H, d, Jꢂ2.2 Hz), 9.21 (1H, s). IR
5-Cyclopropyl-2-(3,5-dimethoxyphenylamino)-7-oxo-4,7-dihydropyra- (KBr) cmꢇ1: 3310, 2210, 1593, 1584. Anal. Calcd for C19H23N7O2: C, 59.83;
zolo[1,5-a]pyrimidine-3-carbonitrile (5f) This title compound was pre- H, 6.08; N, 25.70. Found: C, 59.63; H, 6.01; N, 25.66.
pared from 4a and methyl 3-cyclopropyl-3-oxopropionate according the pro-
2-(3,5-Dimethoxyphenylamino)-7-(2-dimethylaminoethylamino)-5-
cedure to described for preparation of 5a, and obtained as an off-white solid methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6b) This title com-
(73%). mp 186—187 °C. 1H-NMR (DMSO-d6) d: 0.92—1.14 (4H, m), pound was prepared from 5a according to the procedure described for prepa-
1.92—1.99 (1H, m), 3.72 (6H, s), 5.52 (1H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 7.00
ration of 6a, and obtained as an off-white solid (90%). mp ꢆ300 °C. 1H-
(2H, d, Jꢂ2.2 Hz), 9.11 (1H, s), 13.13 (1H, br s). IR (KBr) cmꢇ1: 2988, NMR (DMSO-d6) d: 2.21 (6H, s), 2.41 (3H, s), 2.50—2.60 (2H, m), 3.45
2217, 1684,1635, 1529. Anal. Calcd for C18H17N5O3: C, 61.53; H, 4.88; N, (2H, q, Jꢂ6.0 Hz), 3.74 (3H, s), 6.08 (1H, t, Jꢂ2.2 Hz), 6.33 (1H, s), 6.99
19.93. Found: C, 61.22; H, 4.85; N, 20.03.
(2H, d, Jꢂ2.2 Hz), 7.24 (1H, t, Jꢂ5.0 Hz), 9.27 (1H, s). IR (KBr) cmꢇ1
:

June 2007
887
3315, 2208, 1622, 1593, 1487. Anal. Calcd for C20H25N7O2·0.25H2O: C,
60.06; H, 6.43; N, 24.51. Found: C, 60.20; H, 6.24; N, 24.31.
zolo[1,5-a]pyrimidin-7-ylamino]-1-methylethylcarbamate (6k) This
title compound was prepared from 5a according to the procedure described
2-[3-Cyano-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-a]-
for preparation of 6a, and obtained as an off-white solid (71%). mp 199—
pyrimidin-7-ylamino]acetamide (6c) This title compound was prepared 200 °C. [a]D28 ꢇ15.4° (cꢂ1.05, DMSO). H-NMR (DMSO-d6) d: 1.09 (3H,
from 5a according to the procedure described for preparation of 6a, and ob- d, Jꢂ6.0 Hz), 1.26 (9H, s), 2.40 (3H, s), 3.35—3.45 (2H, m), 3.74 (6H, s),
1
1
tained as an off-white solid (84%). mp 296—297 °C. H-NMR (DMSO-d6) 3.75—3.85 (1H, m), 6.08 (1H, t, Jꢂ2.2 Hz), 6.39 (1H, s), 6.87 (1H, d,
d: 2.40 (3H, s), 3.74 (6H, s), 4.01 (2H, d, Jꢂ5.7 Hz), 6.09 (1H, t, Jꢂ2.2 Hz),
Jꢂ7.9 Hz), 6.95 (2H, d, Jꢂ2.2 Hz), 7.67 (1H, t, Jꢂ5.7 Hz), 9.17 (1H, s). IR
6.14 (1H, s), 7.01 (2H, d, Jꢂ2.2 Hz), 7.35 (1H, s), 7.65 (1H, s), 7.73 (1H, t, (KBr) cmꢇ1: 3362, 2212, 1684, 1616, 1594. Anal. Calcd for C24H31N7O4: C,
Jꢂ5.7 Hz), 9.25 (1H, s). IR (KBr) cmꢇ1: 3321, 3176, 2216, 1709, 1595, 59.86; H, 6.49; N, 20.36. Found: C, 59.58; H, 6.44; N, 20.33.
1490. Anal. Calcd for C18H19N7O3·1.5H2O: C, 52.93; H, 5.43; N, 24.01.
Found: C, 53.18; H, 5.35; N, 23.90.
t-Butyl (S)-2-[3-Cyano-2-(3,5-dimethoxyphenylamino)-5-methylpyra-
zolo[1,5-a]pyrimidin-7-ylamino]-1-methylethylcarbamate (6l) This title
7-Cyclopropylmethylamino-5-methyl-2-phenylaminopyrazolo[1,5-a]- compound was prepared from 5a according to the procedure described for
pyrimidine-3-carbonitrile (6d) This title compound was prepared from preparation of 6a, and obtained as an off-white solid (71%). mp 199—
5d according to the procedure described for preparation of 6a, and obtained
200 °C. [a]D28 14.7° (cꢂ1.09, DMSO). 1H-NMR (DMSO-d6) d: 1.09 (3H, d,
as an off-white solid (89%). mp 293—294 °C. 1H-NMR (DMSO-d6) d: Jꢂ6.0 Hz), 1.26 (9H, s), 2.40 (3H, s), 3.35—3.45 (2H, m), 3.74 (6H, s),
0.30—0.40 (2H, m), 0.45—0.55 (2H, m), 1.10—1.25 (1H, m), 2.40 (3H, s), 3.75—3.85 (1H, m), 6.08 (1H, t, Jꢂ2.2 Hz), 6.39 (1H, s), 6.87 (1H, d,
3.25—3.35 (2H, m), 6.37 (1H, s), 6.93 (1H, t, Jꢂ7.3 Hz), 7.25—7.35 (2H, Jꢂ7.9 Hz), 6.95 (2H, d, Jꢂ2.2 Hz), 7.67 (1H, t, Jꢂ5.7 Hz), 9.17 (1H, s). IR
m), 7.81 (2H, d, Jꢂ7.9 Hz), 7.91 (1H, t, Jꢂ6.4 Hz), 9.19 (1H, s). IR (KBr)
(KBr) cmꢇ1
:
3345, 2212, 1683, 1617, 1568. Anal. Calcd for
cmꢇ1: 3324, 2206, 1609, 1594, 1569. Anal. Calcd for C18H18N6: C, 67.90; H, C24H31N7O4·0.25H2O: C, 59.31; H, 6.53; N, 20.17. Found: C, 59.30; H,
5.70; N, 26.40. Found: C, 67.69; H, 5.60; N, 26.59.
6.40; N, 20.26.
7-Cyclopropylmethylamino-2-(2-methoxyphenylamino)-5-methyl-
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
pyrazolo[1,5-a]pyrimidine-3-carbonitrile (6e) This title compound was phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6m) This title com-
prepared from 5e according to the procedure described for preparation of 6a, pound was prepared from 5h according to the procedure described for
and obtained as an off-white solid (92%). mp 198—199 °C. 1H-NMR
preparation of 6a, and obtained as an off-white solid (81%). mp 166—
(DMSO-d6) d: 0.30—0.40 (2H, m), 0.45—0.55 (2H, m), 1.15—1.25 (1H, 167 °C. 1H-NMR (DMSO-d6) d: 1.13 (6H, s), 3.39 (2H, s), 3.75 (6H, s),
m), 2.40 (3H, s), 3.25—3.35 (2H, m), 3.91 (3H, s), 6.39 (1H, s), 6.95—7.10 6.08 (1H, t, Jꢂ2.2 Hz), 7.03 (1H, s), 7.07 (2H, d, Jꢂ2.2 Hz), 7.50—7.60
(3H, m), 7.60 (1H, br s), 8.00 (1H, t, Jꢂ6.3 Hz), 8.20—8.30 (1H, m). IR
(3H, m), 8.20—8.26 (2H, m), 9.42 (1H, s). IR (KBr) cmꢇ1: 3362, 2299,
(KBr) cmꢇ1: 3410, 3331, 2202, 1607, 1590, 1560. Anal. Calcd for 1617, 1570. Anal. Calcd for C25H27N7O2: C, 65.63; H, 5.95; N, 21.43.
C19H20N6O·0.2H2O: C, 64.83; H, 5.84; N, 23.87. Found: C, 64.76; H, 5.68; Found: C, 65.32; H, 5.89; N, 21.34.
N, 24.01.
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5- ethylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6n) This title compound
methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6f) This title com- was prepared from 5g according to the procedure described for preparation
pound was prepared from 5a according to the procedure described for prepa- of 6a, and obtained as an off-white solid (75%). mp 250—251 °C. 1H-NMR
ration of 6a, and obtained as an off-white solid (89%). mp ꢆ300 °C. 1H- (DMSO-d6) d: 1.10 (6H, s), 1.25 (3H, t, Jꢂ7.6 Hz), 2.66 (2H, q, Jꢂ7.6 Hz),
NMR (DMSO-d6) d: 1.09 (6H, s), 2.40 (3H, s), 3.20 (2H, s), 3.74 (6H, s), 3.22 (2H, s), 3.74 (6H, s), 6.06 (1H, t, Jꢂ2.2 Hz), 6.38 (1H, s), 7.04 (2H, d,
6.06 (1H, t, Jꢂ2.2 Hz), 6.38 (1H, s), 7.04 (2H, d, Jꢂ2.2 Hz), 9.29 (1H, s).
Jꢂ2.2 Hz), 9.32 (1H, s). IR (KBr) cmꢇ1: 3336, 2203, 1596, 1534, 1486.
IR (KBr) cmꢇ1: 3321, 2205, 1623, 1594, 1487. Anal. Calcd for C20H25N7O2: Anal. Calcd for C21H27N7O2: C, 61.60; H, 6.65; N, 23.94. Found: C, 61.31;
C, 60.74; H, 6.37; N, 24.79. Found: C, 60.58; H, 6.33; N, 24.68.
H, 6.60; N, 23.55.
7-(2-Amino-2-methylpropylamino)-2-(3-methoxyphenylamino)-5-
7-(2-Amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxy-
methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6g) This title com- phenylamino)pyrazolo[1,5-a]pyrimidine-3-carbonitrile (6o) This title
pound was prepared from 5b according to the procedure described for compound was prepared from 5f according to the procedure described for
preparation of 6a, and obtained as an off-white solid (86%). mp 269— preparation of 6a, and obtained as an off-white solid (68%). mp 246—
1
270 °C. 1H-NMR (DMSO-d6) d: 1.10 (6H, s), 2.40 (3H, s), 3.22 (2H, s), 247 °C. H-NMR (DMSO-d6) d: 0.95—1.05 (4H, m), 1.10 (6H, s), 2.00—
3.76 (3H, s), 6.40 (1H, s), 6.49 (1H, ddd, Jꢂ8.2, 2.2, 0.9 Hz), 7.15 (1H, t,
Jꢂ8.2 Hz), 7.23 (1H, ddd, Jꢂ8.2, 2.2, 0.9 Hz), 7.52 (1H, t, Jꢂ2.2 Hz), 9.29
2.10 (1H, m), 3.23 (2H, s), 3.73 (6H, s), 6.05 (1H, t, Jꢂ2.2 Hz), 6.43 (1H,
s), 7.03 (2H, d, Jꢂ2.2 Hz), 9.28 (1H, s). IR (KBr) cmꢇ1: 3330, 2204, 1617,
(1H, s). IR (KBr) cmꢇ1: 3330, 2200, 1591, 1536, 1480. Anal. Calcd for 1590, 1488. Anal. Calcd for C22H27N7O2·0.25H2O: C, 62.03; H, 6.51; N,
C19H23N7O: C, 62.45; H, 6.34; N, 26.83. Found: C, 62.19; H, 6.29; N, 26.98.
23.02. Found: C, 61.93; H, 6.33; N, 22.75.
7-(2-Amino-2-methylpropylamino)-2-(4-methoxyphenylamino)-5-
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6h) This title com- isopropylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6p) This title com-
pound was prepared from 5c according to the procedure described for prepa-
pound was prepared from 5i according to the procedure described for prepa-
ration of 6a, and obtained as an off-white solid (90%). mp 225—226 °C. 1H-
ration of 6a, and obtained as an off-white solid (62%). mp 129—130 °C. 1H-
NMR (DMSO-d6) d: 1.09 (6H, s), 2.38 (3H, s), 3.23 (2H, s), 3.72 (3H, s), NMR (DMSO-d6) d: 1.10 (6H, s), 1.25 (6H, d, Jꢂ6.9 Hz), 2.85—3.00 (1H,
6.39 (1H, s), 6.86 (2H, d, J ꢂ9.1 Hz), 7.65 (2H, d, Jꢂ9.1 Hz), 9.04 (1H, s). m), 3.23 (2H, s), 3.74 (6H, s), 6.06 (1H, t, Jꢂ2.2 Hz), 6.37 (1H, s), 7.03
IR (KBr) cmꢇ1: 3333, 2204, 1595, 1568, 1512, 1235. Anal. Calcd for (2H, d, Jꢂ2.2 Hz), 9.78 (1H, s). IR (KBr) cmꢇ1: 3315, 2968, 2208, 1599,
C19H23N7O: C, 62.45; H, 6.34; N, 26.83. Found: C, 62.23; H, 6.29; N, 26.96. 1486. Anal. Calcd for C22H29N7O2: C, 62.39; H, 6.90; N, 23.15. Found: C,
7-(2-Amino-2-methylpropylamino)-5-methyl-2-phenylaminopyra- 62.08; H, 6.87; N, 22.97.
zolo[1,5-a]pyrimidine-3-carbonitrile (6i) This title compound was pre-
pared from 5d according to the procedure described for preparation of 6a,
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
propylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (6q) This title com-
and obtained as an off-white solid (89%). mp 271—272 °C. 1H-NMR pound was prepared from 5j according to the procedure described for prepa-
(DMSO-d6) d: 1.10 (6H, s), 2.39 (3H, s), 3.25 (2H, s), 6.42 (1H, s), 6.90— ration of 6a, and obtained as an off-white solid (83%). mp 133—134 °C. 1H-
7.00 (1H, m), 7.20—7.35 (2H, m), 7.70—7.80 (2H, m), 9.24 (1H, s). IR NMR (DMSO-d6) d: 0.94 (3H, t, Jꢂ6.7 Hz), 1.10 (6H, s), 1.65—1.80 (2H,
(KBr) cmꢇ1: 3336, 2197, 1590, 1566, 1411. Anal. Calcd for C18H21N7: C,
64.46; H, 6.31; N, 29.23. Found: C, 64.22; H, 6.23; N, 29.48.
m), 2.55—2.65 (2H, m), 3.22 (2H, s), 3.74 (6H, s), 6.06 (1H, t, Jꢂ2.2 Hz),
6.38 (1H, s), 7.03 (2H, d, Jꢂ2.2 Hz), 9.29 (1H, s). IR (KBr) cmꢇ1: 3327,
t-Butyl 3-[3-Cyano-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo- 2206, 1617, 1591, 1570. Anal. Calcd for C22H29N7O2: C, 62.39; H, 6.90; N,
[1,5-a]pyrimidin-7-ylamino]propylcarbamate (6j) This title compound
was prepared from 5a according to the procedure described for preparation
of 6a, and obtained as an off-white solid (94%). mp 210—211 °C. 1H-NMR
23.15. Found: C, 62.05; H, 6.82; N, 22.78.
Methyl [7-(2-Amino-2-methylpropylamino)-3-cyano-2-(3,5-dimethoxy-
phenylamino)pyrazolo[1,5-a]pyrimidin-5-yl]acetate (6r) This title com-
(DMSO-d6) d: 1.38 (9H, s), 1.60—1.80 (2H, m), 2.40 (3H, s), 2.95—3.05 pound was prepared from 5k according the procedure described for prepara-
1
(2H, m), 3.35—3.45 (2H, m), 3.73 (6H, s), 6.08 (1H, t, Jꢂ2.2 Hz), 6.31 (1H,
br s), 6.92 (1H, t, Jꢂ5.7 Hz), 6.95 (2H, d, Jꢂ2.2 Hz), 7.79 (1H, t, Jꢂ6.3 Hz),
tion of 6a, and obtained as an off-white solid (68%). mp 217—218 °C. H-
NMR (DMSO-d6) d: 1.11 (6H, s), 3.22 (2H, s), 3.66 (3H, s), 3.74 (6H, s),
9.17 (1H, s). IR (KBr) cmꢇ1: 3307, 2211, 1690, 1617, 1540. Anal. Calcd for 3.77 (2H, s), 6.07 (1H, t, Jꢂ2.2 Hz), 6.49 (1H, s), 7.03 (2H, d, Jꢂ2.2 Hz),
C24H31N7O4: C, 59.86; H, 6.49; N, 20.36. Found: C, 59.67; H, 6.49; N, 9.35 (1H, s). IR (KBr) cmꢇ1: 3332, 2205, 1617, 1592, 1490. Anal. Calcd for
20.43.
C22H27N7O4: C, 58.27; H, 6.00; N, 21.62. Found: C, 58.33; H, 6.08; N,
t-Butyl (R)-2-[3-Cyano-2-(3,5-dimethoxyphenylamino)-5-methylpyra- 21.94.

888
Vol. 55, No. 6
2-(3,5-Dimethoxyphenylamino)-5-methyl-7-(2-methylaminoethyl-
amino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (7a) To a solution of
7-(2-Amino-2-methylpropylamino)-2-(4-methoxyphenylamino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7h) This title com-
2-(3,5-dimethoxyphenylamino)-5-methyl-7-(2-methylaminoethylamino)- pound was prepared from 6h according to the procedure described for
pyrazolo[1,5-a]pyrimidine-3-carbonitrile (6a, 106 mg, 0.28 mmol) and 5 M
aqueous NaOH (0.39 ml, 1.95 mmol) in DMSO (1 ml) and EtOH (1 ml) was
added dropwise 30% aqueous hydrogen peroxide (0.22 ml, 1.95 mmol) at
60 °C, and the mixture was stirred for 3 h at same temperature. After cooling
preparation of 7a, and obtained as an off-white solid (81%). mp 235—
236 °C. 1H-NMR (DMSO-d6) d: 1.11 (6H, s), 2.41 (3H, s), 3.27 (2H, s),
3.73 (3H, s), 6.36 (1H, s), 6.89 (2H, d, Jꢂ8.8 Hz), 7.31 (1H, d, Jꢂ2.8 Hz),
7.58 (1H, d, Jꢂ2.8 Hz), 7.64 (2H, d, Jꢂ8.8 Hz), 9.40 (1H, br s). IR (KBr)
at room temperature, water (5.0 ml) was added to the mixture and the result- cmꢇ1: 3346, 1653, 1590, 1562, 1233. Anal. Calcd for C19H25N7O2: C, 59.51;
ing precipitate was collected by filtration. The obtained solid was crystal- H, 6.57; N, 25.57. Found: C, 59.13; H, 6.46; N, 25.70.
lized from MeOH and DMSO to give 7a (100 mg, 90%) as an off white
solid. mp 256—257 °C. H-NMR (DMSO-d6) d: 2.32 (3H, s), 2.43 (3H, s),
2.77 (2H, t, Jꢂ6.0 Hz), 3.46 (2H, t, Jꢂ6.0 Hz), 3.78 (6H, s), 6.09 (1H, t,
Jꢂ2.2 Hz), 6.29 (1H, s), 6.90 (2H, d, Jꢂ2.2 Hz), 7.41 (1H, d, Jꢂ2.8 Hz), tained as an off-white solid (80%). mp 223—224 °C. H-NMR (DMSO-d6)
7.61 (1H, d, Jꢂ2.8 Hz), 9.59 (1H, s). IR (KBr) cmꢇ1: 3374, 1647, 1604, d: 1.11 (6H, s), 2.42 (3H, s), 3.28 (2H, br s), 6.39 (1H, s), 6.93 (1H, t,
1566, 1458. Anal. Calcd for C19H25N7O3·0.25H2O: C, 56.49; H, 6.36; N, Jꢂ7.3 Hz), 7.25—7.35 (2H, m), 7.37 (1H, d, Jꢂ2.8 Hz), 7.60 (1H, d,
7-(2-Amino-2-methylpropylamino)-5-methyl-2-phenylaminopyrazolo-
[1,5-a]pyrimidine-3-carboxamide (7i) This title compound was prepared
1
from 6i according to the procedure described for preparation of 7a, and ob-
1
24.27. Found: C, 56.32; H, 6.19; N, 24.20.
Jꢂ2.8 Hz), 7.71 (2H, d, Jꢂ7.9 Hz), 9.62 (1H, s). IR (KBr) cmꢇ1: 3355,
7-(2-Dimethylaminoethylamino)-2-(3,5-dimethoxyphenylamino)-5- 1648, 1590, 1564. Anal. Calcd for C18H23N7O·0.25H2O: C, 60.40; H, 6.62;
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7b) This title com- N, 27.39. Found: C, 60.33; H, 6.41; N, 27.65.
pound was prepared from 6b according to the procedure described for
t-Butyl 3-[3-Carbamoyl-2-(3,5-dimethoxyphenylamino)-5-methylpyra-
preparation of 7a, and obtained as an off-white solid (73%). mp 254— zolo[1,5-a]pyrimidin-7-ylamino]propylcarbamate (7j) This title com-
255 °C. 1H-NMR (DMSO-d6) d: 2.23 (6H, s), 2.44 (3H, s), 2.56 (2H, t, pound was prepared from 6j according to the procedure described for prepa-
Jꢂ6.3 Hz), 3.45—3.55 (2H, m), 3.78 (6H, s), 6.09 (1H, t, Jꢂ2.2 Hz), 6.29
ration of 7a, and obtained as an off-white solid (94%). mp 231—232 °C. 1H-
(1H, s), 6.90 (2H, d, Jꢂ2.2 Hz), 7.25 (1H, d, Jꢂ5.4 Hz), 7.42 (1H, d, NMR (DMSO-d6) d: 1.39 (9H, s), 1.65—1.80 (2H, m), 2.43 (3H, s), 2.95—
Jꢂ2.8 Hz), 7.60 (1H, d, Jꢂ2.8 Hz), 9.60 (1H, s). IR (KBr) cmꢇ1: 3369, 3.10 (2H, m), 3.35—3.50 (2H, m), 3.77 (6H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 6.27
1653, 1590, 1570, 1465. Anal. Calcd for C20H27N7O3·0.5H2O: C, 56.56; H, (1H, s), 6.87 (2H, d, Jꢂ2.2 Hz), 6.95 (1H, t, Jꢂ5.7 Hz), 7.39 (1H, d,
6.68; N, 23.21. Found: C, 56.85; H, 6.40; N, 23.21.
Jꢂ3.0 Hz), 7.63 (1H, d, Jꢂ3.0 Hz), 7.70 (1H, t, Jꢂ6.4 Hz), 9.58 (1H, s). IR
7-Carbamoylmethylamino-2-(3,5-dimethoxyphenylamino)-5-methyl- (KBr) cmꢇ1: 3364, 1602, 1569, 1457, 1167. Anal. Calcd for C24H33N7O5: C,
pyrazolo[1,5-a]pyrimidine-3-carboxamide (7c) This title compound was 57.70; H, 6.66; N, 19.63. Found: C, 57.58; H, 6.60; N, 19.61.
prepared from 6c according to the procedure described for preparation of 7a,
and obtained as an off-white solid (67%). mp 286—287 °C. 1H-NMR
(DMSO-d6) d: 2.43 (3H, s), 3.78 (6H, s), 4.04 (2H, d, Jꢂ6.0 Hz), 6.09 (1H,
s), 6.10 (1H, t, Jꢂ2.2 Hz), 6.91 (2H, d, Jꢂ2.2 Hz), 7.35 (1H, br s), 7.41 (1H,
d, Jꢂ2.8 Hz), 7.61 (1H, d, Jꢂ2.8 Hz), 7.67 (1H, br s), 7.74 (1H, t,
t-Butyl (R)-2-[3-Carbamoyl-2-(3,5-dimethoxyphenylamino)-5-methyl-
pyrazolo[1,5-a]pyrimidin-7-ylamino]-1-methylethylcarbamate (7k) This
title compound was prepared from 6k according to the procedure described
for preparation of 7a, and obtained as an off-white solid (89%). mp 227—
1
228 °C. [a]D28 ꢇ10.3° (cꢂ1.01, DMSO). H-NMR (DMSO-d6) d: 1.11 (3H,
Jꢂ6.0 Hz), 9.61 (1H, s). IR (KBr) cmꢇ1: 3365, 1700, 1603, 1566, 1458. d, Jꢂ6.9 Hz), 1.28 (9H, s), 2.43 (3H, s), 3.30—3.50 (2H, m), 3.75—3.90
Anal. Calcd for C18H21N7O4·1.5H2O: C, 50.70; H, 5.67; N, 22.99. Found: C, (1H, m), 3.77 (6H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 6.35 (1H, s), 6.88 (2H, d,
50.96; H, 5.54; N, 23.06.
Jꢂ2.2 Hz), 6.90—6.95 (1H, m), 7.39 (1H, d, Jꢂ2.5 Hz), 7.55—7.70 (2H,
7-Cyclopropylmethylamino-5-methyl-2-phenylaminopyrazolo[1,5-a]- m), 9.57(1H, s). IR (KBr) cmꢇ1: 3353, 1696, 1590, 1458. Anal. Calcd for
pyrimidine-3-carboxamide (7d) This title compound was prepared from C24H33N7O5·0.5H2O: C, 56.68; H, 6.74; N, 19.28. Found: C, 56.29; H, 6.61;
6d according to the procedure described for preparation of 7a, and obtained
N, 19.32.
t-Butyl (S)-2-[3-Carbamoyl-2-(3,5-dimethoxyphenylamino)-5-methyl-
as an off-white solid (85%). mp 246—247 °C. 1H-NMR (DMSO-d6) d:
0.30—0.40 (2H, m), 0.45—0.55 (2H, m), 1.15—1.30 (1H, m), 2.43 (3H, s), pyrazolo[1,5-a]pyrimidin-7-ylamino]-1-methylethylcarbamate (7l) This
3.35 (2H, t, Jꢂ6.6 Hz), 6.34 (1H, s), 6.94 (1H, t, Jꢂ7.3 Hz), 7.30—7.36 title compound was prepared from 6l according to the procedure described
(2H, m), 7.38 (1H, d, Jꢂ2.8 Hz), 7.63 (1H, d, Jꢂ2.8 Hz), 7.75 (2H, d, for preparation of 7a, and obtained as an off-white solid (81%). mp 226—
Jꢂ7.6 Hz), 8.02 (1H, t, Jꢂ6.6 Hz), 9.63 (1H, s). IR (KBr) cmꢇ1: 3369, 3151, 227 °C. [a]D28 13.7° (cꢂ1.02, DMSO). 1H-NMR (DMSO-d6) d: 1.11 (3H, d,
1654, 1590, 1564. Anal. Calcd for C18H20N6O: C, 64.27; H, 5.99; N, 24.98. Jꢂ6.9 Hz), 1.28 (9H, s), 2.43 (3H, s), 3.30—3.50 (2H, m), 3.75—3.90 (1H,
Found: C, 64.02; H, 5.90; N, 25.02.
m), 3.77 (6H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 6.35 (1H, s), 6.88 (2H, d,
7-Cyclopropylmethylamino-2-(2-methoxyphenylamino)-5-methyl- Jꢂ2.2 Hz), 6.90—6.95 (1H, m), 7.39 (1H, d, Jꢂ2.5 Hz), 7.55—7.70 (2H,
pyrazolo[1,5-a]pyrimidine-3-carboxamide (7e) This title compound was m), 9.57 (1H, s). IR (KBr) cmꢇ1: 3353, 1696, 1590, 1458. Anal. Calcd for
prepared from 6e according to the procedure described for preparation of 7a,
C24H33N7O5·0.5H2O: C, 56.68; H, 6.74; N, 19.28. Found: C, 56.72; H, 6.60;
and obtained as an off-white solid (91%). mp 267—268 °C. 1H-NMR N, 19.48.
(DMSO-d6) d: 0.30—0.40 (2H, m), 0.45—0.55 (2H, m), 1.15—1.30 (1H,
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
m), 2.43 (3H, s), 3.30—3.40 (2H, m), 3.90 (3H, s), 6.33 (1H, s), 6.85—6.95 phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7m) This title com-
(1H, m), 6.95—7.05 (2H, m), 7.22 (1H, br s), 7.61 (1H, br s), 7.95—8.05
pound was prepared from 6m according to the procedure described for
(1H, m), 8.55—8.65 (1H, m), 9.94 (1H, s). IR (KBr) cmꢇ1: 3398, 3147, preparation of 7a, and obtained as an off-white solid (80%). mp 288—
1654, 1560, 1459. Anal. Calcd for C19H22N6O2·0.1H2O: C, 61.97; H, 6.08; 289 °C. 1H-NMR (DMSO-d6) d: 1.15 (6H, s), 3.41 (2H, s), 3.79 (6H, s),
N, 22.82. Found: C, 61.95; H, 5.96; N, 22.91.
6.08 (1H, t, Jꢂ2.2 Hz), 6.95—7.00 (3H, m), 7.45—7.60 (4H, m), 7.69 (1H,
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5- d, Jꢂ2.8 Hz), 8.20—8.25 (2H, m), 9.69 (1H, s). IR (KBr) cmꢇ1: 3424, 1648,
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7f) This title com- 1592, 1571, 1457. Anal. Calcd for C25H29N7O3·0.5H2O: C, 61.97; H, 6.24;
pound was prepared from 6f according to the procedure described for prepa-
N, 20.23. Found: C, 61.79; H, 6.05; N, 20.22.
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
ration of 7a, and obtained as an off-white solid (22%). mp 288—289 °C. 1H-
NMR (DMSO-d6) d: 1.30 (6H, s), 2.45 (3H, s), 3.55 (2H, br s), 3.78 (6H, s), ethylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7n) This title com-
6.06 (1H, t, Jꢂ2.2 Hz), 6.34 (1H, s), 6.95 (2H, d, Jꢂ2.2 Hz), 7.42 (1H, d, pound was prepared from 6n according to the procedure described for
Jꢂ2.5 Hz), 7.59 (1H, d, Jꢂ2.5 Hz), 9.61 (1H, s). IR (KBr) cmꢇ1: 3355,
1654, 1593, 1472. Anal. Calcd for C20H27N7O3·0.25H2O: C, 57.47; H, 6.63; 246 °C. H-NMR (DMSO-d6) d: 1.12 (6H, s), 1.27 (3H, t, Jꢂ7.6 Hz), 2.70
N, 23.64. Found: C, 57.51; H, 6.49; N, 23.49. (2H, q, Jꢂ7.6 Hz), 3.24 (2H, s), 3.77 (6H, s), 6.06 (1H, t, Jꢂ2.2 Hz), 6.34
preparation of 7a, and obtained as an off-white solid (76%). mp 245—
1
7-(2-Amino-2-methylpropylamino)-2-(3-methoxyphenylamino)-5- (1H, s), 6.95 (2H, d, Jꢂ2.2 Hz), 7.42 (1H, d, Jꢂ2.8 Hz), 7.62 (1H, d,
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7g) This title com- Jꢂ2.8 Hz), 9.59 (1H, s). IR (KBr) cmꢇ1: 3349, 1654, 1593, 1564, 1464.
pound was prepared from 6g according to the procedure described for prepa- Anal. Calcd for C21H29N7O3·0.4H2O: C, 58.02; H, 6.91; N, 22.55. Found: C,
ration of 7a, and obtained as an off-white solid (91%). mp 243—244 °C. 1H-
NMR (DMSO-d6) d: 1.11 (6H, s), 2.43 (3H, s), 3.24 (2H, s), 3.81 (3H, s),
6.35 (1H, s), 6.49 (1H, dd, Jꢂ8.2, 2.2 Hz), 7.04 (1H, dd, Jꢂ8.2, 2.2 Hz),
7.18 (1H, t, Jꢂ8.2 Hz), 7.38 (1H, br s), 7.55—7.65 (2H, m), 9.62 (1H, s). IR
(KBr) cmꢇ1: 3351, 1649, 1590, 1566, 1547. Anal. Calcd for C19H25N7O2: C,
59.51; H, 6.57; N, 25.57. Found: C, 59.23; H, 6.50; N, 25.66.
58.23; H, 6.87; N, 22.18.
7-(2-Amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxy-
phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (7o) This title
compound was prepared from 6o according to the procedure described for
preparation of 7a, and obtained as an off-white solid (72%). mp 246—
1
247 °C. H-NMR (DMSO-d6) d: 1.00—1.05 (4H, m), 1.12 (6H, s), 2.00—

June 2007
889
ration of 8j, and obtained as an off-white solid (79%). mp 272—273 °C.
[a]D28 8.4° (cꢂ1.02, DMSO). 1H-NMR (DMSO-d6) d: 1.07 (3H, d,
Jꢂ6.0 Hz), 2.43 (3H, s), 3.05—3.20 (2H, m), 3.30—3.40 (1H, m), 3.77 (6H,
s), 6.08 (1H, t, Jꢂ2.2 Hz), 6.31 (1H, s), 6.91 (2H, d, Jꢂ2.2 Hz), 7.39 (1H, d,
Jꢂ2.6 Hz), 7.61 (1H, d, Jꢂ2.6 Hz), 9.59 (1H, s). IR (KBr) cmꢇ1: 3178,
1670, 1599, 1458, 1205. Anal. Calcd for C19H25N7O3·0.25H2O: C, 56.49; H,
6.36; N, 24.27. Found: C, 56.16; H, 6.22; N, 23.98.
2.10 (1H, m), 3.24 (2H, m), 3.77 (6H, s), 6.06 (1H, t, Jꢂ2.2 Hz), 6.40 (1H,
s), 6.94 (2H, d, Jꢂ2.2 Hz), 7.36 (1H, d, Jꢂ2.5 Hz), 7.43 (1H, d, Jꢂ2.5 Hz),
9.58 (1H, s). IR (KBr) cmꢇ1: 3403, 1654, 1589, 1472. Anal. Calcd for
C22H29N7O3·0.2H2O: C, 59.63; H, 6.69; N, 22.13. Found: C, 59.58; H, 6.55;
N, 22.14.
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
isopropylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7p) This title com-
pound was prepared from 6p according to the procedure described for
preparation of 7a, and obtained as an off-white solid (85%). mp 235—
236 °C. 1H-NMR (DMSO-d6) d: 1.12 (6H, s), 1.27 (6H, d, Jꢂ6.9 Hz),
2.90—3.00 (1H, m), 3.26 (2H, s), 3.77 (6H, s), 6.07 (1H, t, Jꢂ2.2 Hz), 6.33
(1H, s), 6.96 (2H, d, Jꢂ2.2 Hz), 7.42 (1H, d, Jꢂ2.8 Hz), 7.63 (1H, d,
Jꢂ2.8 Hz), 9.58 (1H, s). IR (KBr) cmꢇ1: 3323, 1654, 1590, 1563, 1459.
Anal. Calcd for C22H31N7O3·0.2H2O: C, 59.36; H, 7.11; N, 22.03. Found: C,
59.25; H, 6.90; N, 21.78.
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
propylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7q) This title com-
pound was prepared from 6q according to the procedure described for
preparation of 7a, and obtained as an off-white solid (42%). mp 252—
253 °C. 1H-NMR (DMSO-d6) d: 0.95 (3H, t, Jꢂ7.3 Hz), 1.11 (6H, s),
1.70—1.80 (2H, m), 2.65 (2H, t, Jꢂ7.6 Hz), 3.24 (2H, s), 3.77 (6H, s), 6.06
(1H, t, Jꢂ2.2 Hz), 6.33 (1H, s), 6.95 (2H, d, Jꢂ2.2 Hz), 7.41 (1H, d,
Jꢂ2.8 Hz), 7.61 (1H, d, Jꢂ2.8 Hz), 9.59 (1H, s). IR (KBr) cmꢇ1: 3371,
3153, 1653, 1593, 1458. Anal. Calcd for C22H31N7O3: C, 59.85; H, 7.08; N,
22.21. Found: C, 59.59; H, 7.01; N, 22.14.
Acknowledgments We thank Dr. Nobuyuki Tanaka and Dr. Hiromu
Harada for helpful comments while reviewing the manuscript.
References and Notes
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[7-(2-Amino-2-methylpropylamino)-3-carbamoyl-2-(3,5-dimethoxy-
phenylamino)pyrazolo[1,5-a]pyrimidin-5-yl]acetic acid (7r) This title
compound was prepared from 6r according to the procedure described for
preparation of 7a, and obtained as an off-white solid (74%). mp 282—
283 °C. 1H-NMR (DMSO-d6) d: 1.20 (6H, s), 3.48 (2H, s), 3.57 (2H, s),
3.77 (6H, s), 6.08 (1H, t, Jꢂ2.2 Hz), 6.57 (1H, s), 6.95 (2H, d, Jꢂ2.2 Hz),
7.35—7.40 (1H, m), 7.55—7.65 (1H, m), 9.59 (1H, s). IR (KBr) cmꢇ1
:
3384, 1653, 1576, 1364. Anal. Calcd for C21H27N7O5·4.25H2O: C, 47.23; H,
6.70; N, 18.36. Found: C, 47.12; H, 6.63; N, 18.50.
7-(2-Amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (7f·HCl)
To a solution of 7-(2-amino-2-methylpropylamino)-2-(3,5-dimethoxypheny-
lamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7f, 240 mg,
0.58 mmol) in MeOH (3 ml) was added 4 M HCl/EtOAc (0.22 ml, 0.87
mmol). The mixture was stirred for 1 h at room temperature, and evaporated
in vacuo. The residue was crystallized from MeOH to give 7f·HCl (196 mg,
1
75%) as a white solid. mp 291—292 °C. H-NMR (DMSO-d6) d: 1.30 (6H,
s), 2.45 (3H, s), 3.57 (2H, s), 3.78 (6H, s), 6.09 (1H, t, Jꢂ2.2 Hz), 6.56 (1H,
s), 6.93 (2H, d, Jꢂ2.2 Hz), 7.10—7.80 (3H, m), 7.42 (1H, d, Jꢂ2.5 Hz),
7.61 (1H, d, Jꢂ2.5 Hz), 9.61 (1H, s). IR (KBr) cmꢇ1: 3383, 2963, 1643,
1594, 1574, 1150. Anal. Calcd for C20H27N7O3·HCl: C, 53.39; H, 6.27; N,
21.79. Found: C, 53.62; H, 6.36; N, 21.92.
7-(3-Aminopropylamino)-2-(3,5-dimethoxyphenylamino)-5-methyl-
pyrazolo[1,5-a]pyrimidine-3-carboxamide (8j) To a solution of t-butyl
3-[3-carbamoyl-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-
a]pyrimidin-7-ylamino]propylcarbamate (7j, 95 mg, 0.19 mmol) in MeOH
(2 ml) was added 4 M HCl/EtOAc (210 ml, 0.95 mmol). The mixture was
stirred for 2 h at room temperature, and evaporated in vacuo. The residue
was triturated with the saturated aqueous sodium hydrogen carbonate solu-
tion, and the resulting precipitate was collected by filtration to give 8j
(75 mg, 90%) as a white solid. mp 125—126 °C. 1H-NMR (DMSO-d6) d:
1.65—1.80 (2H, m), 2.43 (3H, s), 2.75 (2H, t, Jꢂ6.3 Hz), 3.49 (2H, t,
Jꢂ6.3 Hz), 3.76 (6H, s), 6.10 (1H, t, Jꢂ2.2 Hz), 6.23 (1H, s), 6.90 (2H, d,
Jꢂ2.2 Hz), 7.36 (1H, d, Jꢂ2.8 Hz), 7.61 (1H, d, Jꢂ2.8 Hz), 9.54 (1H, s). IR
(KBr) cmꢇ1: 3158, 1680, 1614, 1464. Anal. Calcd for C19H25N7O3·0.25H2O:
C, 56.49; H, 6.36; N, 24.27. Found: C, 56.41; H, 6.23; N, 24.42.
7-((R)-2-Aminopropylamino)-2-(3,5-dimethoxyphenylamino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (8k) This title com-
pound was prepared from 7k according to the procedure described for
preparation of 8j, and obtained as an off-white solid (61%). mp 269—
270 °C. [a]D28 ꢇ8.7° (cꢂ1.06, DMSO). 1H-NMR (DMSO-d6) d: 1.07 (3H, d,
Jꢂ6.0 Hz), 2.43 (3H, s), 3.05—3.20 (2H, m), 3.30—3.40 (1H, m), 3.77 (6H,
s), 6.08 (1H, t, Jꢂ2.2 Hz), 6.31 (1H, s), 6.91 (2H, d, Jꢂ2.2 Hz), 7.39 (1H, d,
Jꢂ2.6 Hz), 7.61 (1H, d, Jꢂ2.6 Hz), 9.59 (1H, s). IR (KBr) cmꢇ1: 3189,
1671, 1602, 1465, 1205. Anal. Calcd for C19H25N7O3·0.25H2O: C, 56.49; H,
6.36; N, 24.27. Found: C, 56.46; H, 6.26; N, 23.92.
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7-((S )-2-Aminopropylamino)-2-(3,5-dimethoxyphenylamino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (8l) This title com-
pound was prepared from 7l according to the procedure described for prepa-