Identification, synthesis, and biological evaluation of 6-[(6 R)-2-(4-fluorophenyl)-6-(hydroxymethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a ]pyrimidin-3-yl]-2-(2-methylphenyl)pyridazin-3(2 H)-one (AS1940477), a potent p38 MAP kinase inhibitor

Article abstract of DOI:10.1021/jm3008008

Several p38 MAPK inhibitors have been shown to effectively block the production of cytokines such as IL-1β, TNFα, and IL-6. Inhibitors of p38 MAP kinase therefore have significant therapeutic potential for the treatment of autoimmune disease. Compound 2a was identified as a potent TNFα production inhibitor in vitro but suffered from poor oral bioavailability. Structural modification of 2a led to the discovery of tetrahydropyrazolopyrimidine derivatives, exemplified by compound 3, with an improved pharmacokinetic profile. We found that blocking metabolism at the methyl group of the amine and constructing the tetrahydropyrimidine core were important to obtaining compounds with good biological profiles and oral bioavailability. Pursuing the structure-activity relationships of this series led to the discovery of AS1940477 (3f), with excellent cellular activity and a favorable pharmacokinetic profile. This compound represents a highly potent inhibitor of p38 MAP kinase with regard to in vivo activity in an adjuvant-induced arthritis model.

Full text of DOI:10.1021/jm3008008

Article
pubs.acs.org/jmc
Identification, Synthesis, and Biological Evaluation of
6‑[(6R)‑2-(4-Fluorophenyl)-6-(hydroxymethyl)-4,5,6,
7-tetrahydropyrazolo[1,5‑a]pyrimidin-3-yl]-2-
(2-methylphenyl)pyridazin-3(2H)‑one (AS1940477), a Potent p38
MAP Kinase Inhibitor
Toru Asano,*,† Hitoshi Yamazaki,† Chiyoshi Kasahara,† Hirokazu Kubota,† Toru Kontani,†
Yu Harayama,† Kazuki Ohno,† Hidekazu Mizuhara,† Masaharu Yokomoto,‡ Keiji Misumi,‡
Tomohiko Kinoshita,‡ Mitsuaki Ohta,† and Makoto Takeuchi.†
†Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
‡Drug Discovery Laboratory, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda-cho, Akitakata-shi Hiroshima 739-1195,
Japan
S
* Supporting Information
ABSTRACT: Several p38 MAPK inhibitors have been shown
to effectively block the production of cytokines such as IL-1β,
TNFα, and IL-6. Inhibitors of p38 MAP kinase therefore have
significant therapeutic potential for the treatment of auto-
immune disease. Compound 2a was identified as a potent
TNFα production inhibitor in vitro but suffered from poor
oral bioavailability. Structural modification of 2a led to the
discovery of tetrahydropyrazolopyrimidine derivatives, exem-
plified by compound 3, with an improved pharmacokinetic
profile. We found that blocking metabolism at the methyl group of the amine and constructing the tetrahydropyrimidine core
were important to obtaining compounds with good biological profiles and oral bioavailability. Pursuing the structure−activity
relationships of this series led to the discovery of AS1940477 (3f), with excellent cellular activity and a favorable pharmacokinetic
profile. This compound represents a highly potent inhibitor of p38 MAP kinase with regard to in vivo activity in an adjuvant-
induced arthritis model.
Mitogen-activated protein kinase (MAPK) p38 is a serine/
threonine kinase originally isolated from lipopolysaccharide
(LPS) stimulated monocytes. p38 MAPK positively regulates a
variety of genes involved in inflammation, such as TNFα, IL-1,
IL-6, and IL-8. Because of the broad proinflammatory role of
p38 MAPK in several in vitro systems, inhibition of this
pathway has been advocated as novel therapeutic strategy for
inflammatory disease. On the basis of this idea, many groups
have investigated whether p38 MAPK inhibitors effectively
block the production of proinflammatory cytokines and have
demonstrated prominent efficacy in vitro and in animal models
of acute inflammation and arthritis.9−18 To date, many p38
MAPK inhibitors such as BIRB-796 or VX-745 have advanced
to clinical studies for RA, but their development has been dis-
continued because of undesirable safety profiles.19−21 Two of
these compounds exhibited low efficacy, requiring high dos-
ing, which may have led to adverse side effects,13,14 and their
reported side effects may have been related to their effective dose.
INTRODUCTION
■
Tumor necrosis factor α (TNFα) is a proinflammatory cyto-
kine that plays important roles both in normal immune func-
tion and in disturbances leading to autoimmune diseases.1−3
Overexpression of TNFα can lead to the progression of inflamma-
tory diseases such as rheumatoid arthritis (RA), multiple sclerosis,
chronic obstructive pulmonary disease (COPD), psoriasis, and
inflammatory bowel disease (IBD). To date, many research pro-
grams have focused on the inhibition of TNFα production,
antagonism of TNFα, or deletion of TNFα from the cell surface.
TNFα has therefore attracted considerable attention as a molec-
ular target for the treatment of these conditions.
The recent clinical success of anti-cytokine biological thera-
peutics such as etanercept,4 infliximab,5−7 and adalimumab8 pro-
vides a strong rational for the targeting of TNFα for inflam-
matory diseases such as RA, COPD, and IBD. However, biologics
are proteins and have the general disadvantages associated with
protein drugs: poor stability, poor oral absorption and sub-
cutaneous or intravenous administration, and high costs of manu-
facturing. These characteristics point to the urgent need for orally
active small molecules that are safe, efficacious, and inexpensive.
Received: June 7, 2012
© XXXX American Chemical Society
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Figure 1. Structure of representative p38 MAP kinase inhibitors.
Figure 2. Leads 1 and 2 and derived target analogue 3.
a
Scheme 1. Synthesis of α-Bromoketone 8
a
(a) LiHMDS, 4-fluorophenyl benzoate, THF, 15−23 °C, 83%; (b) NaOAc, HOAc, 110 °C, 83%; (c) ethylene glycol, TsOH, toluene, reflux, 100%;
(d) 2-methylphenylboronic acid, Cu(OAc)2, pyridine, DMF, air, 23 °C, 56%; (e) HCl, AcOH, 23 °C, 87%; (f) PyBr3, AcOH, 23 °C, 99%.
Furthermore, Adam et al. reported that a p38 MAPK inhibitor
containing a pyridyl or pyrimidinyl moiety led to potent
inhibition of hepatic cytochrome p450 isozymes in vitro.17,22
These results indicate that the development of p38 MAPK
inhibitors for the treatment of chronic disease depends on
both highly potent TNFα inhibition and the avoidance of
CYP inhibition.
Prototypical p38 MAPK inhibitors, such as SB-203580, SD06,
RWJ67657, and TAK-715 (Figure 1),11,13 have been reported to
be effective antiarthritic drugs in rodents. Unfortunately, SD06 and
TAK-71517,23 were unsuccessful in clinical use because of a lack of
efficacy or poor pharmacokinetics. Also, as investigations have
proceeded, compounds structurally different from SB-203580 have
been reported as p38 inhibitors, including BIRB-796 and VX-745,
which represent other p38 inhibitors and which are reported to
have a potent TNFα inhibitory activity.
In our research program for TNFα production inhibitors, we
identified pyrazolopyrimidine 1 (Figure 2) with suitable
properties as optimizing leads. During the optimization process,
a series of terahydropyrazolopyrimidine derivatives 3 were
synthesized as potential inflammatory modulators that were
demonstrated to have inhibitory activity against LPS-induced
TNFα production both in vitro and in vivo. Several compounds
of this series exhibited potent anti-inflammatory effects in an
adjuvant-induced arthritis (AIA) model with ED30 values of less
than 1 mg/kg on oral administration. We report the synthesis
and structure−activity relationships of tetrahydropyrazolopyr-
imidine derivatives, leading to the discovery of the clinical
B
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a
Scheme 2. Synthesis of Aminopyrazoles 2a, 2b, and Tetrahydropirimidinopyrazole 3b
a
(g) 8, AcOH, 60 °C, 78−85%; (h) DEAD, PPh3, rt, 62%.
a
Scheme 3. Synthesis of Tetrahydropirimidinopyrazoles 3a, 3c−g
a
(i) 8, AcOH, 60 °C, 54−78%; (j) MsCl, DIPEA, MeCN, 80 °C, 10−88%; (k) Pd(OH)2, EtOH, 50 °C, 33%; (l) LiBH4, THF, rt, 82%.
a
Scheme 4. Synthesis of Tetrahydropirimidinopyrazole 3h
a
(m) PPh3, I2, THF, 0 °C, 79%; (n) methylpiperazine, 60 °C, 22%.
candidate AS1940477 (3f),24 a p38 MAPK inhibitor with novel
structure. Compound 3f showed highly potent in vitro activity
and a significant anti-inflammatory effect in the AIA model.
Scheme 2 describes the synthesis to obtain aminopyrazoles
2a and 2b and tetrahydropyrazolopyrimidine 3b. Thiosemi-
carbazides 9a and 9b were reacted with α-bromoketone 8
under acidic conditions to afford the aminopyrazoles 2a and
2b,26 followed by cyclization under Mitsunobu reaction condi-
tion to give tetrahydropyrazolopyrimidine 3b.
The synthesis route from 8 to tetrahydropyrazolopyrimidines
3a−g is shown in Scheme 3. The 5-substituted pyrazoles 10a
and 10c−f were prepared from thiosemicarbazides 9c−f) as
described above for Scheme 2. Intramolecular cyclization with
methanesulfonyl chloride and Hunig’s base afforded the
6-substituted tetrahydropyrazolopyrimidines 3a, 3c, 3d, 11e, 11f.
Compound 3e was obtained by hydrogenolysis of compound
11e with catalytic palladium hydroxide under hydrogen gas.
Reduction of ester 11f with LiBH4 afforded the racemic alcohol
in high yield. The mixture of enantiomers was separated using
chiral HPLC to afford hydroxymethyl derivatives 3f and 3g.
Synthesis of the methylpiperazine analogue 3h is shown in
Scheme 4. Iodide 12 was provided by iodination of alcohols 3f
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of the 6-substituted tetrahydro-
pyrazolopyrimidine analogues was conducted through cycliza-
tion of the α-bromoketone 8 with appropriately substituted
thiosemicarbazides (Scheme 2). The requisite α-bromoketone
8 was synthesized by the method reported by Colletti et al.16 as
shown in Scheme 1. Synthesis of α-bromoketone 8 began with
treatment of commercially available methylpyridazine 4 with
4-fluorophenyl benzoate under basic conditions, followed by
hydrolysis. Acetal protection of 5 in toluene at reflux gave 6,
which was converted into 7 by oxidative Chan−Evans−Lam
coupling25 with Cu(OAc)2 and pyridine in air and deprotection
under acidic conditions with hydrochloric acid. Pyridinium
hydrobromide perbromide in acetic acid gave α-bromoketone 8
at room temperature in high yields.
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a
Scheme 5. Synthesis of Tetrahydropyrimidinopyrazoles 3i and 3j
a
(o) 8, AcOH, 60 °C, 77−79%; (p) MsCl, DIPEA, MeCN, 80 °C, 60−76%; (q) TBAF, THF, rt, 91−93%.
a
Scheme 6. Synthesis of Pyrimidinopyrazoles 3k
a
(r) NaOMe, MeOH, reflux, 92%; (s) OsO4, NMM, H2O, acetone, MeCN, rt, 24%.
Table 1. Data Comparison of Lead Compounds 1, 2, and 3
and 3g as a racemic mixture. Methylpiperazine analogue 3h was
obtained in low yields through nucleophilic displacement of
iodide with methylpiperazine under neat conditions.
THP-1
a
b
c
LPS/TNFα, IC50 TNFα production
MLM stability, CLint
compd
(nM)
inhibition (%)
(mL min−1 kg−1
)
The 5-substituted tetrahydropyrazolopyrimidine analogues 3i
and 3j were prepared from chiral aspartic acids as shown in
Scheme 5.27 Thiosemicarbazide 9i or 9j was readily synthesized
from phenyl chlorothioformate and the corresponding amine
by a similar method reported by Arora et al.28 Cyclization of the
thiosemicarbazide 9i or 9j with α-bromoketone 8 afforded the
aminopyrazole 13i or 13j. The formation of the tetrahydropyr-
imidine rings to give the 5-substituted analogues (3i, 3j)
proceeded in quantitative yields, followed by desilylation with
tetrabutylammonium fluoride.
d
1
14
6
43
194.8
212.9
2a
3b
86
93
1
94.0
a
b
Effect on LPS-induced TNFα production from THP-1 cells. Effect
on LPS-induced TNFα production at 1 mg/kg po in rats. Percent
c
inhibition was calculated relative to the vehicle group. In vitro
metabolism with rat liver microsomes in the presence of the NADPH-
d
generating system (n = 2). Effect on LPS-induced TNFα production
at 3.2 mg/kg po in rats.
Elimination of iodide 12 led to the exomethylene analogue
14 in high yield as shown in Scheme 6. Oxidative treatment of
exomethylene 14 with OsO4 allowed for facile aromatization to
give pyrazolopyrimidine 3k at ambient temperature.
a series of tetrahydropyrazolopyrimidine derivatives based on 3
significantly inhibited TNFα production in vivo.
Lead compounds 2a and 2b exhibited promising in vitro
activity profiles but displayed low oral bioavailability. This poor
oral bioavailability of 2a and 2b might be attributable to low
metabolic stability, as indicated by the high total clearance. We
therefore investigated strategies for decreasing metabolic oxida-
tion of the substituent on the pyrazole scaffold. In general,
compared to linear alkyl functions, the corresponding cyclic
groups are more stable.30 To probe this concept, we syn-
thesized several cyclic compounds fused to a pyrazole. Com-
pounds were evaluated in the inhibition of LPS-induced TNFα
production in THP-1 cells and in vivo at 1 mg/kg po in rat.
The results shown in Table 2 are based on the average of three
independent experiments. Although the five-membered ring
analogue 3a fused to the pyrazole was substantially less potent
than 2a in the cellular assay, the six-membered ring analogue 3b
exhibited impressive inhibitory activity against TNFα produc-
tion both in vitro and in vivo. The corresponding alkoxypyra-
zole 3c showed attenuated inhibition in THP-1 cells. Next, to
investigate whether introduction of polar substituents to the
tetrahydropyrazolopyrimidine backbone would improve in vivo
activity, hydroxyl or amino groups were attached at several
positions about a fused pyrimidine core of the scaffold. Both
compounds 3d and 3e with a hydroxyl group showed superior
Structure−Activity Relationships. Pyrazolopyridine 1,29
identified from our chemical library, showed 14 nM potency
against LPS-induced TNFα production in vitro. Although
encouraged by the in vitro potency of this hit, compound 1 was
also shown to have low solubility and poor metabolic stability,
which may have contributed to its limited in vivo efficacy.
Preliminary SAR and scaffold modifications revealed that the
aminopyrazole structures typified by 2a might have been
suitable for the development of novel TNFα production inhib-
itors. Although compound 2a29 showed more potent in vivo
activity than 1, these 1-methyl-5-(methylamino)pyrazole ana-
logues displayed high in vitro clearance in rat liver homogenates.
The low metabolic stability of compound 2a and its analogues
resulted in short half-lives (0.66 h for compound 2a). To
improve stability, we turned our attention to investigating alter-
native scaffolds without metabolically labile groups. In pre-
liminary studies, demethylation of 2a was observed as a major
metabolic pathway in rats in vitro. As can be seen from the lower
rate of intrinsic clearance in Table 1, the metabolic stabil-
ity of 2a was improved by converting the methyl(methylamino)-
pyrazole to tetrahydropyrazolopyrimidine structure 3. Furthermore,
D
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Table 2. TNFα Inhibitory Activity of Aminopyrazole Derivatives
a
b
c
d
THP-1 LPS/TNFα, IC50
inhibition of TNFα release
PAMPA (pH 6.5 ),
compd
X
n
R1
R2
(nM)
(%)
×10−6 cm/s
ClogP
2a
2b
3a
3b
3c
3d
3e
3f
−Me
−CH2CH2CH2OH
H
−Me
−H
6
86
87
30
17
3.11
3.22
3.00
3.15
3.32
2.45
2.45
2.09
2.09
1.69
2.30
2.30
2.29
NH
NH
NH
O
1
0
1
1
1
1
1
1
1
1
1
1.1
106
1
e
e
NT
NT
H
93
33
94
89
92
92
88
98
98
11
30
e
H
10
NT
NH
NH
NH
NH
NH
NH
NH
−OH (R)
−OH (S)
−CH2OH (R)
−CH2OH (S)
−CH2-methylpiperazine
0.85
1.8
0.6
0.95
1.1
0.46
0.37
23
16
17
20
e
3g
3h
3i
NT
21
23
22
3j
e
3k
NT
a
b
Effect on LPS-induced TNFα production from THP-1 cells. IC50 values are shown as the mean of triplicate experiments. Effect on LPS-induced
c
TNFα production at 1 mg/kg po in rats (n = 3−4). Percent inhibition was calculated relative to the vehicle group. pION membrane lipid was used.
d
e
pH of donor buffer. NT = not tested.
potency against TNF release. In particular, alcohol 3d showed
an IC50 of 0.85 nM in vitro. These data suggest that introduc-
tion of a polar group such as a hydroxyl group or amino group
provided stronger inhibitory activity against TNFα release in
vivo due to improved permeability. Furthermore, analogues 3f
and 3g with a methanol group at the C-6 position were more
potent than 3b in the cellular assay. Analogue 3h with a methyl-
piperazyl group at the C-6 position showed a slight decrease in
potency in vitro and in vivo due to increased hydrophilicity (3h,
ClogP = 1.69) relative to that of 3b. Changing the hydro-
xymethyl substituent from the C-6 to the C-5 position for
alcohols 3i and 3j, which possessed a hydroxymethyl group at
the C-5 position, strongly inhibited TNFα production in vitro
with IC50 values of 0.46 and 0.37 nM, respectively. It was
therefore unsurprising that both compounds inhibited LPS-
induced TNF release by 98%. Finally, aromatization of the
tetrahydropyrazolopyrimidine afforded pyrazolopyrimidine 3k,
which exhibited a 38-fold decrease in cell potency with respect
to 3f.
On the basis of the results of these studies, we selected
compounds 3d, 3e, 3f, 3g, and 3j for further in vivo evaluation.
Selected compounds were evaluated in PK assay (po and iv)
in rats, and their pharmacokinetic parameters are shown in
Table 3. The results indicated that tetrahydropyrimidinopyr-
azole derivatives displayed better oral bioavailability and had a
longer plasma half-life in rats than acyclic compounds such as
2a. Cyclization of the methylpyrazole had a significant impact
on total clearance and bioavailability of the analogues (2a vs 3d
or 3e). Extension of the hydroxyl group on the tetrahydropyr-
azolopyrimidine ring increased systemic plasma exposure (3d
or 3e vs 3f or 3j). Shifts from the C-6 position to the C-5 posi-
tion on the tetrahydropyrazolopyrimidine ring showed similar
pharmacokinetics (3f vs 3j). Compounds 3d, 3e, 3f, 3g, and 3j
Table 3. Pharmacokinetic Parameters of Selected TNFα
Release Inhibitors in Rat
a
b
b
iv (1.0 mg/kg)
po (1.0 mg/kg)
AUC
CLtot
Vss
(L/kg)
t1/2
(h)
(mL min−1 kg−1
)
(ng·h/mL)
F (%)
2a
3d
3e
3f
20.64
4.80
9.53
2.17
4.56
3.80
0.65
1.23
4.21
0.77
1.11
0.98
0.66
3.89
9.01
5.15
3.82
5.95
235
763
28.6
67.7
457
81.0
1518
1217
4502
61.8
3g
3j
103.6
100.5
a
Pharmacokinetics were determined at a dose of 1 mg/kg iv and po in
b
SD rats. Results expressed as the mean SD of n = 3.
were found to exhibit exceptionally high in vitro and in vivo
potency with a relatively long duration of action.
These tetrahydropyrazolopyrimidine derivatives (2a, 3d, 3e,
3f, 3g, and 3j) were evaluated in adjuvant-induced arthritis
(AIA) rat models. As can be seen in Table 4, 3d, 3f, and 3j
provided a significant reduction in the measured outcome of
disease progression relative to the vehicle group when dosed
orally at 1 mg/kg once a day for a period of 15−24 days.
Although these derivatives all displayed good pharmacokinetics
properties, 3f displayed the most potent inhibition in the AIA
model.
Compound 3f was further investigated to confirm its in vivo
efficacy against inflammation. As shown in Table 5, 3f and
representative compounds that are reported to be p38 MAP
kinase inhibitors were compared in vivo in a rat LPS-induced
TNFα model as an acute model and in the AIA model as a
chronic disease model. 3f displayed excellent in vivo efficacy in
both models and represents one of most potent p38 inhibitor
reported to date.31
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Table 4. Compound Efficacy in the Adjuvant-Induced
Arthritis Model after po Dosing of 1 mg/kg
a
compd
AIA, % suppression
2a
3d
3e
3f
37.0**
61.4**
51.0**
71.3**
59.7**
62.3**
3g
3j
Figure 3. Docking model of compound 3f binding to p38 MAP
kinase: (a) front view; (b) side view. Light blue and gray indicate
carbon atoms of compound 3f and p38 MAP kinase, respectively. Red,
blue, and green indicate oxygen, nitrogen, and fluorine atoms,
respectively. Arrows indicate a hydrogen bond.
a
Edema volume of foot pads in rats with the development of AIA.
Edema of the hind paw was calculated as the mean percentage of the
control value (n = 6). Double asterisks indicate statical significance at
P < 0.01 vs 0.5% MC control by Dunnett’s multiple comparison test.
Table 5. Comparative Studies of in Vivo Efficacy Assays
atom of the tetrahydropyrazolopyrimidine ring form hydrogen
bonds with Asp 168 and Lys 53, respectively. Because the
hydrogen bond between Asp 168 and the hydroxyl group is
half-exposed to solvent, this hydrogen bond was predicted to
have no substantial effect on binding affinity. In fact, there is
little difference in TNFα inhibitory activity between the
presence (compound 3f) and absence of the hydroxymethyl
group (compound 3b). On the other hand, the hydroxyl group
is thought to contribute to improving the physical properties of
compound 3f.
a
a
compd
3f
rat LPS-induced TNF ED50 (mg/kg)
AIA ED30 (mg/kg)
0.05
2.60
39.9
0.15
5.80
92
BIRB-796
VX-745
a
The results are reported as the average of at least four separate
determinations.
Further biological evaluation demonstrated that compound
3f has strong inhibition of p38α/β and high selectivity
against JNK2,32 as shown in Table 6. Compound 3f was also
CONCLUSION
■
In summary, we explored several series of tetrahydropyrazolo-
pyrimidine scaffolds as prototypical inhibitors of TNFα pro-
duction. Among these series, analogues that contained a
hydroxyl group showed improved metabolic stability compared
to 2a and displayed excellent potency in cellular and in vivo
TNFα assays. After extensive SAR studies, compounds 3d, 3e,
3f, 3g, and 3j were identified as potent inhibitors of TNFα
production and were shown to potently inhibit the production
of TNFα in vitro and in vivo. Evaluation of this series led to 3f,
which is orally bioavailable with a long duration of action and
efficacious in an AIA model. On the basis of the overall profile,
compound 3f was selected as a clinical candidate. These
findings suggested that 3f represents an attractive therapeutic
candidate for the treatment of inflammatory diseases.
Table 6. Kinase Profiling of 3f
a
kinase assay IC50 (nM)
compd
p38α
p38β
JNK2
3f
11
36
>10000
a
Assayed according to ref 30. See Experimental Section for assay
details.
completely clean with regard to CYP enzyme inhibitory activities,
indicating that it has very low potential for drug−drug interac-
tions, as shown in Table 7.
Table 7. In Vitro Profile of Compound 3f
CYP inhibition, IC50 (μM)
EXPERIMENTAL SECTION
Chemistry. H NMR spectra were measured with a JEOL EX400
b
■
PAMPA, Pe solubility
1
(10−6cm/s)
at pH 6.5
(μg/mL)
a
(400 MHz) or GX500 (500 MHz) spectrometer. Chemical shifts are
expressed in δ units using tetramethylsilane as the standard (NMR
peak description: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad peak). Mass spectra were recorded with a Hitachi
M-80 or a JEOL JMS-DX300 spectrometer. Organic solutions were
dried over anhydrous MgSO4 during workup. Column chromatog-
raphy was carried out on silica gel (Kieselgel 60). Unless otherwise
noted, all commercial reagents and solvents were used without further
purification. The purity of all compounds screened in biological assays
was >95% pure as judged by HPLC and/or combustion analysis.
HPLC analysis was obtained on an Hitachi LaChrom Elite system,
using a TOSOH TSK-gel ODS column (150 mm × 4.6 mm, 5 μm) at
40 °C with a 1.5 mL/min flow rate using acetonitrile and 0.05 M
KH2PO4 solution as the eluent over 30 min.
6-[2-(4-Fluorophenyl)-2-oxoethyl]pyridazin-3(2H)-one (5).
To a solution of 3-chloro-6-methylpyridazine (50 g, 389 mmol) and
ethyl 4-fluorobenzoate (65 g, 389 mmol) in tetrahydrofuran cooled to
0 °C under a nitrogen atmosphere was added 1 M tetrahydrofuran
solution of lithium 1,1,3,3,3-hexamethyldisilazane-2-ide (779 mL, 779
mmol), and the mixture was stirred at the same temperature for 3 h.
The reaction mixture was poured into aqueous 1 M hydrochloric acid
compd 1A2 2C19 2C9 2D6
3f >50 >50 19 >50
3A4
>100
at pH 6.8
19.7
42
a
b
Midazolam was the substrate in CYP3A4 inhibition assay. pION
membrane lipid was used as a donor buffer, pH 6.5 (n = 2).
Compound 3f showed an acceptable off-target profile in both
CYP inhibition (IC50) and hERG inhibition (Rb efflux assay,
IC50 > 100 μM) in addition to desirable physicochemical prop-
erties (PAMPA,33 solubility) and mutagenic profile (negative in
the in vivo micronucleus test).
Finally, to clarify the role of the hydroxymethyl group, we
have docked compound 3f into the crystal structure of p38α
MAP kinase using the docking program GOLD, version 3.2
(Figure 3). The pyridazinone ring forms three hydrogen bonds
with hinge residues His 104, Met 106, and Gly 107. The
methylphenyl ring and fluorophenyl ring occupy the hydro-
phobic front region and the hydrophobic back pocket, re-
spectively. The hydroxymehyl group and aromatic nitrogen
F
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(1200 mL), and the resulting mixture was stirred at room temperature
for 1 h. The resulting precipitates were collected by filtration, air-dried
for 16 h, and triturated with hexane (1000 mL). The precipitates
were collected by filtration and air-dried for 24 h to give 2-(6-
chloropyridazin-3-yl)-1-(4-fluorophenyl)ethanone (74 g, 76%). 1H
NMR (400 MHz, DMSO-d6): δ 4.84 (2H, s), 7.38−7.44 (2H, m),
7.77−7.80 (1H, m), 7.91−7.96 (1H, m), 8.14−8.19 (2H, m). MS
(API-ES) m/z = 273 (M + Na).
was stirred for 1 h in an ice-cooled bath. The resulting precipitate was
filtered with the funnel and washed with water and diisopropyl ether.
The obtained solid was dried under a hood (23 g, 92%). 1H NMR (400
MHz, DMSO-d6): δ 1.86 (3H, s), 7.08 (1H, s), 7.13−7.22 (2H, m),
7.26−7.58 (6H, m), 7.79 (1H, d, J = 9.76 Hz), 8.11−8.15 (2H, m).
MS (API-ES) m/z = 424 (M + Na).
N,1-Dimethylhydrazinecarbothioamide (9a). To a solution of
methylhyrdrazine (14 mL, 274 mmol) in EtOH (55 mL) was added
dropwise a solution of methyl isothiocyanate (20 g, 274 mmol) in
EtOH (27 mL) at room temperature. A precipitate formed. After being
stirred for 6 h, the suspension was stirred at 0 °C, collected, and
washed with cold diisopropyl ether. The white solid was dried in vacuo
at 45 °C (29 g, 89%). 1H NMR (400 MHz, DMSO-d6): δ 2.86 (3H, d,
J = 4.5 Hz), 3.42 (3H, s), 4.80 (2H, s), 8.05−8.20 (1H, m).
6-[3-(4-Fluorophenyl)-1-methyl-5-(methylamino)-1H-pyra-
zol-4-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (2a). A mixture
of 8 (12 g, 30 mmol) and N,1-dimethylhydrazinecarbothioamide 9a
(3.9 g, 33 mmol) in AcOH (96 mL) was stirred for 5 h at 120 °C.
After concentration, the mixture was partitioned between EtOAc
(200 mL) and water (100 mL). The organic layer was washed with
saturated NaHCO3 (60 mL) and brine, dried, and evaporated in vacuo.
The residue was purified by column chromatography on silica gel
(methanol/chloroform = 1/9) to give 2a (9.9 g, 85%). 1H NMR
(400 MHz, DMSO-d6): δ 2.05 (3H, s), 2.66 (3H, d, J = 5.4 Hz), 3.66
(3H, s), 5.53 (1H, q, J = 5.4 Hz), 7.00 (1H, d, J = 9.6 Hz), 7.13−7.49
(9H, m). MS (ESI) m/z = 390 (M + 1).
2-(6-Chloropyridazin-3-yl)-1-(4-fluorophenyl)ethanone (110 g, 439
mmol) and sodium acetate (45 g, 549 mmol) were dissolved in acetic
acid (550 mL), and the mixture was stirred at 120 °C for 3 h. An
additional 10 g of sodium acetate was added, and stirring was con-
tinued for another 1 h. After the reaction mixture was cooled to 40 °C,
water (1300 mL) was added and the mixture was stirred with ice−
water bath cooling for 1 h. The resulting precipitates were collected by
filtration and then washed with water (580 mL). The isolated pre-
cipitates were air-dried at ambient temperature for 1 day. The
precipitates were triturated with diisopropyl ether (500 mL) for 1 h
1
and collected by filtration to give the product (83 g, 81%). H NMR
(400 MHz, DMSO-d6): δ 4.42 (2H, s), 6.86 (1H, d, J = 9.72 Hz),
7.35−7.42 (3H, m), 8.08−8.13 (2H, m), 12.91 (1H, s). MS (API-ES)
m/z = 255 (M + Na).
6-{[2-(4-Fluorophenyl)-1,3-dioxolan-2-yl]methyl}pyridazin-
3(2H)-one (6). A mixture of 6-[2-(4-fluorophenyl)-2-oxoethyl]-
pyridazin-3(2H)-one (49 g, 202 mmol), ethylene glycol (60 mL,
1073 mmol), TsOH·H2O (3.8 g, 20 mmol), and toluene (450 mL)
was refluxed for 6 h with azeotropic removal of water. The reaction
mixture was cooled to 50 °C, and 1 M NaOH (21 mL) and water
(300 mL) were added. The whole mixture was stirred on an ice bath
for 1 h, and the resulting precipitate was collected by filtration. The
obtained solid was washed with water (400 mL) and then toluene
(200 mL) to give 6 (56 g, 100%) as a gray powder. 1H NMR
(400 MHz, DMSO-d6): δ3.10 (2H, s), 3.67−3.74 (2H, m), 3.89−3.97
(2H, m), 6.76 (1H, d, J = 9.8 Hz), 7.11−7.20 (2H, m), 7.28 (1H, d, J =
9.8 Hz), 7.33−7.40 (2H, m), 12.76 (1H, s). MS (API-ES) m/z = 299
(M + Na).
6-[2-(4-Fluorophenyl)-2-oxoethyl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (7). To a mixture of 6 (32 g, 114 mmol),
2-methylphenylboronic acid (34 g, 251 mmol), and pyridine (37 mL,
457 mmol) in DMF (189 mL) was added Cu(OAc)2 (3 g, 17 mmol)
at room temperature with stirring. The mixture was stirred for 24 h
and partitioned between EtOAc and 5% citric acid. The separated
organic layer was filtered through a Celite pad. The aqueous layer was
extracted with EtOAc. The combined organic layer were washed with
5% citric acid, 10% K2CO3, and brine, dried, and concentrated in
vacuo to give an oil. The crude product was purified by column
chromatography (n-hexane/ethyl acetate = 1/1 to 1/2) to give 6-{[2-
(4-fluorophenyl)-1,3-dioxolan-2-yl]methyl}-2-(2-methylphenyl)-
pyridazin-3(2H)-one (26 g, 62%) as a white solid. 1HNMR (400 MHz,
DMSO-d6): δ1.83 (3H, s), 3.17 (2H, s), 3.71−3.81 (2H, m),
3.95−4.04 (2H, m), 6.95−7.50 (6H, m). MS (API-ES) m/z = 389
(M + Na).
N-(3-Hydroxypropyl)hydrazinecarbothioamide (9b). To a
solution of 3-amino-1-propanol (1.7 mL, 22 mmol) in CH2Cl2
(43 mL) were added phenyl chlorothioformate (2 mL, 15 mmol), water
(43 mL), and NaHCO3 (3.1 g, 37 mmol) on an ice bath, followed by
stirring at room temperature for 24 h. The reaction mixture was
extracted with AcOEt, washed with brine, and dried over MgSO4. The
crude product was purified by column chromatography to give O-
phenyl (3-hydroxypropyl)carbamothioate as a colorless oil (2.4 g,
1
77%). H NMR (400 MHz, CDCl3): δ 1.90 (2H, m), 3.70 (1H, m),
3.83 (4H, m), 7.09 (2H, m), 7.27 (1H, m), 7.39 (2H, m). MS (API-
ES) m/z = 234 (M + Na).
To a solution of O-phenyl(3-hydroxypropyl)carbamothioate (2.6 g,
12 mmol) in EtOH (20 mL) was added hydrazine hydrate (1.2 mL,
24 mmol) at room temperature, and the mixture was heated at 40 °C.
After being stirred for 4 h, the mixture was diluted with water and
extracted with diisopropyl ether. The aqueous phase was evaporated to
1
give the desired product (1.8 g, 98%). H NMR (400 MHz, DMSO-
d6): δ 1.63 (2H, m), 3.34 (1H, brs), 3.43 (2H, t, J = 5.6 Hz), 3.50 (2H,
q, J = 6.4, 6.8, 6.6 Hz), 4.49 (1H, brs), 7.89 (1H, brs), 8.57 (1H, brs).
MS (API-ES) m/z = 172 (M + Na).
6-{3-(4-Fluorophenyl)-5-[(3-hydroxypropyl)amino]-1H-pyra-
zol-4-yl}-2-(2-methylphenyl)pyridazin-3(2H)-one (2b). A solu-
tion of 6-[1-bromo-2-(4-fluorophenyl)-2-oxoethyl]-2-(2-methyl-
phenyl)pyridazin-3(2H)-one (1.0 g, 2.5 mmol) and N-(3-
hydroxypropyl)hydrazinecarbothioamide (409 mg, 2.74 mmol) in
acetic acid (100 mL) was heated at 100 °C. After heating for 1 h, the
mixture was diluted with water, extracted with EtOAc, washed with
brine, and dried over MgSO4. After removal of solvent, the residue was
purified by column chromatography (chloroform/methanol = 10/0 to
To a solution of 6-{[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]methyl}-
2-(2-methylphenyl)pyridazin-3(2H)-one(30 g, 82 mmol) in AcOH
(120 mL) was added 1 N HCl (12 mL) at room temperature. After
the mixture was stirred for 14 h, diisopropyl ether/n-hexane (1/1,
240 mL) and water (360 mL) were added to the mixture. The
resulting suspension was aged for 30 min at 0 °C. The separated solid
was collected and washed with n-hexane and water to give 7 (22 g,
1
7/3) to give 2b (812 mg, 78%). H NMR(400 MHz, CDCl3): δ 1.73
(2H, quint), 2.22 (3H, s), 3.47 (2H, t), 3.61 (2H, t), 6.81 (1H, d),
6.91 (1H, d), 7.13 (2H, t), 7.28−7.46 (6H, m). MS (API-ES) m/z =
442 (M + Na).
1
84%). H NMR (400 MHz, DMSO-d6): δ 2.01 (3H, s), 4.51 (2H, s),
6-[2-(4-Fluorophenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidin-3-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (3b). A
mixture of 2b (117 mg, 0.28 mmol), triphenylphosphine (110 mg,
0.42 mmol), and diethyl azodicarboxylate (66 μL, 0.42 mmol) in
tetrahydrofuran (5 mL) was stirred at room temperature for 2 h. To
the mixture was added water (20 mL). The mixture was extracted with
ethyl acetate (30 mL). The extract was concentrated under reduced
pressure. To the residue was added 10% hydrochloric acid (25 mL).
The mixture was washed with diethyl ether (30 mL × 4). The aqueous
layer was neutralized with sodium hydrogen carbonate and extracted
with ethyl acetate (30 mL). The extract was concentrated under
7.08 (1H, d, J = 9.6 Hz), 7.20−7.47 (4H, m), 7.53 (1H, d, J = 9.6 Hz),
8.05−8.18 (2H, m). MS (API-ES) m/z = 345 (M + Na).
6-[1-Bromo-2-(4-fluorophenyl)-2-oxoethyl]-2-(2-
methylphenyl)pyridazin-3(2H)-one (8). To a solution of 7 (20 g,
63 mmol) in AcOH (120 mL) was added pyridine hydrobromide
perbromide (26 g, 72 mmol). After being stirred for 1.5 h, the mixture
was added to a solution of diisopropyl ether and water. The resulting
precipitate was filtered with a funnel, and the filtrate was extracted
with EtOAc. The precipitate and the organic phase were combined to
dissolve in 5% Na2S2O4 (31.5 mL) and water (348 mL). The mixture
G
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1
reduced pressure. The residue was purified by flash column chro-
matography (gradient elution, ethyl acetate/hexane = 1/4 to 0/10) to
give 3b as yellow amorphous solid (70 mg, 62%). 1H NMR (CDCl3):
δ 2.15−2.20 (2H, m), 2.23 (3H, s), 3.36 (2H, brs), 4.15 (2H, t, J = 6.1
Hz), 5.81 (1H, brs), 6.80 (1H, d, J = 9.9 Hz), 7.02 (1H, d, J = 9.9 Hz),
7.15 (2H, dd, J = 8.7, 8.7 Hz), 7.33−7.38 (4H, m), 7.50 (2H, dd, J =
5.4, 8.7 Hz). MS (API-ES) = 402 (H + M).
6-{3-(4-Fluorophenyl)-5-[(2-hydroxyethyl)amino]-1H-pyra-
zol-4-yl}-2-(2-methylphenyl)pyridazin-3(2H)-one (10a). A sol-
ution of 6-[1-bromo-2-(4-fluorophenyl)-2-oxoethyl]-2-(2-
methylphenyl)pyridazin-3(2H)-one (400 mg, 1.0 mmol) and N-(2-
hydroxyethyl)hydrazinecarbothioamide34 (168 mg, 1.24 mmol) in
acetic acid (1.3 mL) and EtOH (2.7 mL) was heated at 50 °C. After
being heated for 30 min, the mixture was diluted with water, extracted
with EtOAc, washed with water, saturated NaHCO3, and brine, and
dried over Na2SO4. After removal of solvent, the crude product was
purified by column chromatography on silica gel (chloroform/
methanol = 10/0 to 7/3) to give the product (401 mg, 99%).
1H NMR (400 MHz, CDCl3): δ 2.20 (3H, s), 3.48 (2H, t, J = 5.2, 5.0 Hz),
3.75 (2H, t, J = 5.2, 5.0 Hz), 6.85 (1H, m), 7.10 (2H, m), 7.36
(7H, m). MS (API-ES) m/z = 428(M + Na).
6-[6-(4-Fluorophenyl)-2,3-dihydro-1H-imidazo[1,2-b]-
pyrazol-7-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (3a). To a
solution of 10a (420 mg, 1 mmol) in MeCN were added triethylamine
(347 μL, 2.5 mmol) and methanesulfonyl chloride (112 μL, 1.5 mmol),
followed by heating at 80 °C. To the mixture was added further
triethylamine (347 μL, 2.5 mmol) and methanesulfonyl chloride
(112 μL, 1.5 mmol). After being heated for 12 h, the mixture was
extracted with EtOAc, washed with 5% citric acid, aqueous NaHCO3,
and brine, and dried over MgSO4. After removal of the solvent, the
residue was purified by column chromatography (chloroform/
methanol = 10/0 to 10/1) to give the desired product (42 mg,
10%). 1H NMR (400 MHz, CDCl3): δ 2.21 (3H, s), 4.28−4.61 (2H, m),
6.94 (1H, d), 7.00−7.13 (3H, m), 7.29−7.38 (4H, m), 7.40−7.49
(2H, m). MS (API-ES) m/z = 386 (M − H). HRMS (M + H)+ found,
388.1576; calcd for C22H19N5OF, 388.1574.
4%). H NMR (CDCl3): δ 2.05 (3H, s), 2.14 (2H, m), 3.64 (2H, m),
4.47 (2H, m), 4.51 (2H, s), 6.90−7.10 (5H, m), 7.22−7.37 (7H, m),
7.48 (3H, m). MS (API-ES) m/z = 533(M + Na).
A mixture of 6-{5-[3-(benzyloxy)propoxy]-3-(4-fluorophenyl)-1H-
pyrazol-4-yl}-2-(2-methylphenyl)pyridazin-3(2H)-one (336 mg, 0.66
mmol), Pd−C (101 mg), and EtOH (3.4 mL) was stirred at room
temperature for 5 h under H2 gas. The mixture was filtered with a
funnel and the filtrate was evaporated to give 10c (247 mg, 89%).
1H NMR (400 MHz, DMSO-d6): δ 1.89 (2H, m), 1.97 (3H, s), 3.55
(2H, m), 4.31 (2H, m), 4.56 (1H, brs), 7.04−7.37 (8H, m), 7.53 (2H, m).
MS (API-ES) m/z = 443(M + Na). Anal. (C23H21FN4O3·0.2H2O) C,
H, N.
6-[2-(4-Fluorophenyl)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]-
oxazin-3-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (3c). To a
solution of 10c (247 mg, 0.59 mmol) in MeCN (2.47 mL) were added
triethylamine (197 μL, 1.4 mmol) and methanesulfonyl chloride
(55 μL, 0.71 mmol) at 0 °C, and then the mixture was stirred at the
same temperature for 30 min. After heating at 80 °C for 3 h, the
mixture was extracted with EtOAc, washed with aqueous NaHCO3
and brine, and dried over MgSO4. After removal of the solvent, the
residue was purified by column chromatography (chloroform/
methanol = 10/0 to 8/2) to give the desired product (52 mg, 22%).
1H NMR (CDCl3): δ 2.13 (3H, s), 2.36 (2H, m), 4.26 (2H, t), 4.42
(2H, t), 6.99 (3H, m), 7.30 (5H, m), 7.52 (2H, m). MS (API-ES)
m/z = 425 (M + Na). HRMS (M + H)+ found, 403.1587; calcd for
C23H20N4O2F, 403.1570.
N-[(2R)-2,3-Dihydroxypropyl]hydrazinecarbothioamide
(9d). (2R)-3-Aminopropane-1,2-diol (2.2 g, 24.0 mmol) was dissolved
in 2-propanol (84 mL) at 50 °C with stirring. After being stirred for
1 h, the solution was cooled to ambient temperature. O-Phenyl
hydrazinecarbothioate (3.37 g, 20.0 mmol) and dichloromethane were
added to the solution, and the mixture was stirred at ambient tem-
perature for 19 h. The reaction mixture was decanted to separate the
solution from undissolved materials attached to the reaction vessel
wall. The separated solution was evaporated in vacuo and the residue
was washed with diisopropyl ether (50 mL) to give 9d (4.0 g, 100%).
1H NMR (400 MHz, DMSO-d6): δ 6.753 (3H, m), 7.15 (2H, m), 9.29
O-[3-(Benzyloxy)propyl] Hydrazinecarbothioate (9c). To a
solution of NaH (606 mg, 15 mmol) in THF (2 mL) was slowly added
3-(benzyloxy)propan-1-ol (2.0 mL, 12.6 mmol) in THF (34 mL) at
0 °C, followed by stirring at room temperature for 1 h. To the mixture
was then added methanedithione (1.5 mL, 24.2 mmol) at 0 °C, with
stirring at room temperature for 4 h, followed by the addition of
iodomethane (1.2 mL, 18.9 mmol) at 0 °C. After being stirred at room
temperature for 2 h, the mixture was extracted with EtOAc, washed
with aqueous NH4Cl and brine, and dried over MgSO4. After removal
of the solvent, the crude product was purified by column
chromatography (hexane/EtOAc = 5/1) to give O-[3-(benzyloxy)-
propyl] S-methylcarbonodithioate (2.27 g, 70%). 1H NMR (CDCl3): δ
2.11 (2H, m), 2.53 (3H, s), 3.59 (2H, t, J = 5.6, 6.2 Hz), 4.52 (2H, s,
J = 5.6, 6.2 Hz), 4.73 (2H, t). MS (API-ES) m/z = 279 (M + Na).
A mixture of O-[3-(benzyloxy)propyl] S-methylcarbonodithioate
(2.27 g, 8.85 mmol), hydrazine hydrate (4.3 mL, 88.5 mmol), and
EtOH (23 mL) was stirred at 0 °C. After being stirred for 1 h, the
mixture was diluted with water, extracted with CHCl3/diisopropyl
ether (=4/1), washed with brine, and dried over MgSO4. After removal
of solvent, the crude product was purified by column chromatography
(1H, brs). MS (API-ES) m/z = 188 (M + Na).
6-[5-{[(2R)-2,3-Dihydroxypropyl]amino}-3-(4-fluorophenyl)-
1H-pyrazol-4-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one
(10d). To a solution of 9d (4.0 g, 24 mmol) in acetic acid (35 mL)
was added 8 (5.3 g, 13 mmol), and the mixture was stirred at 75 °C for
3 h. After being cooled to ambient temperature, the reaction mixture
was diluted with ethyl acetate (100 mL) and water (50 mL). Undis-
solved materials in the resultant mixture were removed by filtration.
To the filtrate were added 50 g of ice and 50 mL of 15 M aqueous
sodium hydroxide solution with stirring. The organic phase was
separated, and the aqueous phase was extracted with ethyl acetate
(50 mL). The organic phase and extracts were combined, washed with
water (100 mL) and brine (50 mL), dried over MgSO4, filtered, and
evaporated in vacuo to give 7.51 g of crude product. The obtained
materials were dissolved in methanol (80 mL) and to the solution was
added 3 M aqueous sodium hydroxide (80 mL) for 50 min at ambient
temperature. The mixture was concentrated under reduced pressure,
and water (100 mL) was added to the residue. The resulting mixture
was extracted with ethyl acetate (60 mL), and the combined extracts
were successively washed with 1 M aqueous sodium hydroxide
(60 mL), water (100 mL), and brine (50 mL), then dried over MgSO4,
filtered, and evaporated in vacuo. The residue (6.57 g) was dissolved in
ethyl acetate (15 mL), and the mixture was stirred at ambient tem-
perature for 16 h. Precipitates produced were collected by filtration,
washed with cooled ethyl acetate (5 mL), and dried under reduced
pressure at 40 °C for 1 h to give 3.78 g of 10d. By use of preparative
thin layer chromatography, 44 mg of the product was eluted with 10%
methanol in dichloromethane to provide 42 mg of 10d (4.6 g, 80%).
1H NMR (400 MHz, DMSO-d6): δ 2.11 (3H, s), 3.05 (1H, br s),
1
(hexane/EtOAc = 1/1) to give 9c as an oil (1.97 g, 93%). H NMR
(400 MHz, DMSO-d6): δ 1.91 (2H, m), 3.51 (2H, m), 4.45 (4H, m),
7.32 (5H, m). MS (API-ES) m/z = 263(M + Na).
6-[3-(4-Fluorophenyl)-5-(3-hydroxypropoxy)-1H-pyrazol-4-
yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (10c). A mixture of 6-
{5-[3-(benzyloxy)propoxy]-3-(4-fluorophenyl)-1H-pyrazol-4-yl}-2-(2-
methylphenyl)pyridazin-3(2H)-one (2.2 g, 5.5 mmol), 9c (1.9 g, 7.9
mmol), EtOH (37 mL), and acetic acid (18 mL) was heated at 100 °C.
After being stirred for 4 h, the mixture was extracted with EtOAc,
washed with aqueous Na2CO3 and brine, and dried over MgSO4. After
removal of the solvent, the crude was purified by column chro-
matography (chloroform/methanol = 10/0 to 9/1 and hexane/EtOAc =
2/1 to 0/1) to give 6-{5-[3-(benzyloxy)propoxy]-3-(4-fluorophenyl)-
1H-pyrazol-4-yl}-2-(2-methylphenyl)pyridazin-3(2H)-one (114 mg,
3.22−3.32 (2H, m), 3.54 (1H, br s), 4.57−4.67 (2H, m), 5.43 (1H,
brs), 6.05 (0.3H, brs), 6.92 (1H, d, J = 9.56 Hz), 7.02 (1H, brs), 7.35−
7.36 (6H, m), 7.50−7.53 (2H, m), 12.31 (1H, brs). MS (API-ES)
m/z = 458 (M + Na).
H
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Journal of Medicinal Chemistry
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6-[(6R)-2-(4-Fluorophenyl) -6-hydroxy-4, 5, 6, 7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (3d). To a suspension of 10d (435 mg, 0.99
mmol) and pyridine (808 μL) in acetonitrile was added the first
portion of methanesulfonyl chloride (93 μL, 1.2 mmol) at 0 °C. The
mixture was stirred at 0 °C for 30 min, then at ambient temperature
for 3 h, followed by stirring at 35 °C for 45 min. During the stirring at
ambient temperature, the reaction mixture became a clear solution.
The mixture was cooled to 0 °C, and to it was added the second
portion of methanesulfonyl chloride (7.7 μL, 0.1 mmol). The resulting
mixture was stirred at 0 °C for 45 min, at ambient temperature for 1 h,
and at 70 °C for 18 h. After being cooled to ambient temperature, the
reaction mixture was partitioned between ethyl acetate (15 mL) and
water (22 mL). The aqueous phase was extracted with ethyl acetate
(15 mL), and the organic phases were combined and successively
washed with brine and then 20% aqueous citric acid (10 mL). The
combined citric acid solution was extracted with ethyl acetate (10 mL),
and the extract was combined with the organic phase, which was
successively washed with 5% aqueous citric acid, brine, 1 M aqueous
sodium hydroxide, and brine, then dried over MgSO4, filtered, and
evaporated in vacuo. The residue was crystallized from ethyl acetate to
give 3d (124 mg, 30%). 1H NMR (400 MHz, DMSO-d6): δ 2.08 (3H,
s), 3.13−3.18 (1H, m), 3.24−3.27 (1H, m), 3.89 (1H, dd, J = 12.3 and
2.95 Hz), 4.15 (1H, dd, J = 15.9 and 3.57 Hz), 4.18 (1H, s), 5.34 (1H,
d, J = 2.24 Hz), 5.93 (1H, s), 6.92 (1H, d, J = 9.76 Hz), 7.08 (1H, d,
J = 9.76 Hz), 7.21−7.25 (2H, m), 7.31−7.37 (4H, m), 7.46−7.49 (2H,
m). MS (API-ES) m/z = 440 (M + Na). [α]D −14.7 (c 0.0044 g/mL,
23.8 °C).
(2S)-3-amino-2-(benzyloxy)propan-1-ol as brown crystals (330 mg,
76%). H NMR (CDCl3): δ 2.95−3.16 (brm, 2H), 3.47−3.50 (m, 1H),
1
3.77 (dd, 1H, J = 4.5, 11.5 Hz), 3.86 (dd, 1H, J = 4.5, 11.5 Hz), 4.60
(d, 1H, J = 12 Hz), 4.67 (d, 1H, J = 12 Hz), 7.28−7.37 (m, 5H).
To a solution of (2S)-3-amino-2-(benzyloxy)propan-1-ol (730 mg,
4.03 mmol) in dichloromethane (6 mL) was added a solution of
sodium carbonate (448 mg, 4.23 mmol) in water (6 mL) and O-
phenyl chlorothionoformate (585 μL, 4.23 mmol), and the mixture
was stirred at room temperature for 90 min. To the mixture was added
diethyl ether, and the mixture was successively washed with 5% aque-
ous citric acid solution, saturated saline, 5% aqueous sodium carbonate
solution, and saturated saline. The organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
O-phenyl (2S)-2-(benzyloxy)-3-hydroxypropylthiocarbamate as brown
1
oil (1.35 g, 100%). H NMR (CDCl3): δ 2.31 (dd, 1H, J = 5, 8 Hz),
3.69−3.96 (m, 5H), 4.65 (d, 1H, J = 12 Hz), 4.69 (d, 1H, J = 12 Hz),
7.05−7.08 (m, 3H), 7.27 (dd, 2H, J = 7, 7 Hz), 7.33−7.41 (m, 6H).
To a solution of O-phenyl (2S)-2-(benzyloxy)-3-hydroxypropylth-
iocarbamate (1.35 g, 4.03 mmol) in 2-propanol (4 mL) was added
hydrazine monohydrate (1.95 mL, 40.3 mmol), and the mixture was
stirred at room temperature for 16 h. To the mixture was added
saturated saline, and the mixture was extracted with dichloromethane.
The extract was washed with saturated saline, dried over anhydrous
MgSO4, and concentrated under reduced pressure. To the oily residue
was added a mixed solvent of diethyl ether and diisopropyl ether. The
supernatant was removed by decanting twice to give the title com-
1
pound 9e as a red-brown oil (760 mg, 74%). H NMR (CDCl3): δ
3.06 (brs, 1H), 3.64 (d, 2H, J = 6 Hz), 3.70−3.80 (m, 4H), 4.12−4.17
(m, 1H), 4.63 (d, 1H, J = 11 Hz), 4.66 (d, 1H, J = 11 Hz), 7.13 (brs,
1H), 7.29−7.36 (m, 5H), 7.71 (brs, 1H).
(2S)-4-[2-(Benzyloxy)-3-hydroxypropyl]thiosemicarbazide
(9e). To a solution of (2S)-3-aminopropane-1,2-diol (4.00 g, 43.9
mmol) in N,N-dimethylformamide (65 mL) were added triethylamine
(12.8 mL, 92.2 mmol) and triphenylchloromethane (25.7 g, 92.2
mmol), and the mixture was stirred at room temperature for 2 days.
To the mixture were added triethylamine (1.22 mL, 8.78 mmol) and
triphenylchloromethane (2.45 g, 8.78 mmol), and the mixture was
stirred at room temperature for 1 day. To the mixture was added
water, and the resulting precipitate was extracted with diisopropyl
ether. The extracts were combined and washed successively with
water, 5% aqueous citric acid solution, saturated aqueous sodium
hydrogen carbonate solution, water, and saturated saline, then dried
over anhydrous MgSO4 and concentrated under reduced pressure. To
the oily residue was added a mixed solvent of diisopropyl ether and
hexane, and crystallization was induced by scratching the flask. The
crystal was filtered off to give (2S)-1-(tritylamino)-3-(trityloxy)-
(2S)-6-(5-{[2-(Benzyloxy)-3-hydroxypropyl]amino}-3-(4-fluo-
rophenyl)-1H-pyrazol-4-yl)-2-(2-methylphenyl)-3(2H)-pyridazi-
none (10e). A mixture of (2S)-4-[2-(benzyloxy)-3-hydroxypropyl]-
thiosemicarbazide 9e (251 mg, 0.98 mmol) and 8 (329 mg, 0.82 mmol)
in glacial acetic acid (2.5 mL) was stirred at 60−65 °C for 110 min. To
the mixture was added ethyl acetate (30 mL), and the resulting insoluble
materials were removed by filtration. To the mixture was added water,
and the mixture was alkalinized with sodium carbonate. The organic
layer was separated and washed successively with 5% aqueous citric acid
solution, water, saturated aqueous sodium bicarbonate solution, and
saturated saline, dried over anhydrous MgSO4, and concentrated under
reduced pressure. The residue was purified by flash column chromato-
graphy (SiO2, stepwise gradient from 0% to 10% methanol in chloroform)
to give the title compound as a pale yellow amorphous solid (315 mg,
yield 73.1%). 1H NMR (CDCl3): δ 2.20 (s, 3H), 3.44−3.61 (m, 5H), 4.50
(s, 2H), 6.01 (brs, 1H), 6.82 (d, 1H, J = 10 Hz), 6.98 (d, 1H, J = 10 Hz),
7.05−7.27 (m, 4H), 7.29−7.36 (m, 7H), 7.45−7.48 (m, 2H).
1
propan-2-ol as colorless crystals (15.0 g, 59%). H NMR (400 MHz,
CDCl3): δ 2.19 (dd, 2H, J = 6, 12 Hz), 2.45 (dd, 1H, J = 5, 12 Hz),
3.02−3.07 (m, 2H), 3.26−3.28 (m, 1H), 3.88 (brs, 1H), 7.17−7.44
(m, 30H).
(2S)-6-[6-(Benzyloxy)-2-(4-fluorophenyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2- methylphenyl)-
3(2H)-pyridazinone (11e). To a solution of 10e (240 mg, 0.46
mmol) in dichloromethane (3 mL) were added triethylamine (95 μL,
0.69 mmol) and methanesulfonyl chloride (46 μL, 0.59 mmol), and
the mixture was stirred at room temperature for 55 min. To the mix-
ture were added triethylamine (95 μL, 0.69 mmol) and methane-
sulfonyl chloride (46 μL, 0.59 mmol), and the mixture was stirred at
room temperature for 45 min and refluxed for 3 days. To the mixture
was added water, and the mixture was extracted with ethyl acetate. The
extracts were combined and successively washed with a 3% aqueous
citric acid solution, 5% aqueous sodium carbonate solution, and
saturated saline, then dried over anhydrous MgSO4 and concentrated
under reduced pressure. The residue was purified by flash column
chromatography (SiO2, stepwise gradient from 0% to 2% methanol in
chloroform) to give the title compound as a yellow amorphous solid
To a suspension of sodium hydride (55% dispersion in mineral oil,
545 mg, 12.5 mmol) in dimethylsulfoxide (50 mL), from which the
mineral oil was removed by washing with petroleum ether, was added
(2S)-1-(tritylamino)-3-(trityloxy)propan-2-ol (5.76 g, 10.0 mmol).
The mixture was stirred at room temperature for 30 min. To the
mixture was added benzyl bromide (1.37 mL, 11.5 mmol), and the
mixture was stirred at room temperature for 17 h. To the mixture was
added water, and the resulting precipitate was filtered off and washed
with hexane to give N-[(2S)-2-benzyloxy-3-(trityloxy)propyl]-N-trityl-
amine as a colorless powder (4.79 g, 72%). 1H NMR (CDCl3): δ 0.97
(brs, 1H), 2.41 (brt, 2H, J = 9 Hz), 3.26−3.30 (m, 2H), 3.71−3.75
(m, 1H), 4.42 (d, 1H, J = 12 Hz), 4.50 (d, 1H, J = 12 Hz), 7.15−7.42
(m, 35H).
To a solution of N-[(2S)-2-benzyloxy-3-(trityloxy)propyl]-N-trityl-
amine (1.60 g, 2.40 mmol) in dichloromethane (4 mL) was added
trifluoroacetic acid (2 mL), and the mixture was stirred at room
temperature for 1 day. To the mixture was added water, and the
aqueous solution was washed with diisopropyl ether. The aqueous
layer was basified with sodium hydroxide. To the mixture was added
salt, and the mixture was extracted with dichloromethane. The extr-
acts were combined and washed with saturated saline, dried over
anhydrous MgSO4, and concentrated under reduced pressure to give
1
(205 mg, yield 88%). H NMR (CDCl3): δ 2.22 (3H, s), 3.42 (2H,
brs), 4.07 (1H, m), 4.23 (2H, m), 4.67 (2H, m), 5.79 (1H, br s), 6.79
(1H, d, J = 8.4 Hz), 7.01 (1H, d, J = 7.6 Hz), 7.14 (2H, m), 7.29−7.38
(9H, m), 7.50 (2H, m). MS (API-ES) m/z = 508 (M + Na).
6-[(6S)-2-(4-Fluorophe nyl)-6-hydroxy-4, 5, 6, 7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (3e). A mixture of 11e (430 mg) and palladium
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hydroxide (250 mg, 20% wt on carbon) in EtOH (20 mL) was stirred
under a hydrogen atmosphere at 45−50 °C for 6 h. To the reaction
mixture was added further palladium hydroxide (50 mg, 20% wt on
carbon), and the mixture was stirred under a hydrogen atmosphere at
50 °C for 1 h. After the catalyst was filtered off, the filtrate was
concentrated in vacuo. The residue was purified by flash silica gel
column chromatography (gradient elution, AcOEt/hexane = 1/2 to
1/1) followed by crystallization from a mixed solvent of diethyl ether
and dichloromethane to give the title compound as pale yellow crystals
Ethyl 3-({3-(4-Fluorophenyl)-4-[1-(2-methylphenyl)-6-oxo-
1,6-dihydropyridazin-3-yl]-1H-pyrazol-5-yl}amino)-2-
(hydroxymethyl)propanoate (10f). A mixture of 8 (2.3 g, 5.73
mmol) and 9f (2.12 g, 6.31 mmol) in EtOH (12 mL) and AcOH
(12 mL) was stirred for 14 h at 60 °C. The mixture was diluted with
EtOAc, washed with saturated NaHCO3 solution and brine, dried over
Na2SO4, filtered, and concentrated. The residue was triturated with
EtOAc to give the title compound as a yellow solid (1.51 g, 54%).
1H NMR (DMSO-d6): δ 1.04 (3H, t, J = 7.2 Hz), 2.10 (3H, s), 3.37
(2H, m), 3.54 (2H, m), 3.89 (2H, q, J = 6.8 Hz), 4.82 (1H, m),
5.40 (1H, brs), 6.93 (1H, m), 7.05 (1H, m), 7.26−7.44 (6H, m), 7.52
(2H, m). MS (API-ES) m/z = 514 (M + Na).
1
(115 mg, 33%). H NMR (400 MHz, DMSO-d6): δ 2.08 (3H, s),
3.14−3.18 (1H, m), 3.24−3.28 (1H, m), 3.89 (1H, dd, J = 11.84 and
2.88 Hz), 4.12−4.18 (2H, m), 5.34 (1H, s), 5.93 (1H, s), 6.92 (1H, d,
J = 9.80 Hz), 7.08 (1H, d, J = 9.80 Hz), 7.21 (1H, d, J = 1.88 Hz), 7.24
(1H, d, J = 8.88 Hz), 7.31−7.36 (4H, m), 7.46−7.49 (2H, m). MS
(ESI) m/z = 418 (M + 1).
Ethyl 3-{[tert-Butyl(dimethyl)silyl]oxy}-2-{[(hydrazino-
carbothioyl)amino]methyl}propanoate (9f). To a solution of
ethyl 2-(hydroxymethyl)acrylate (5 g, 38.4 mmol) and imidazole
(2.6 g, 38.4 mmol) in CH2Cl2 (40 mL) was added dropwise a solu-
tion of tert-butyldimethylsilyl chloride (5.8 g, 38.4 mmol) in CH2Cl2
(40 mL) with cooling on ice. After being stirred for 2 h at ambient
temperature, the mixture was washed with H2O and brine. The organic
layer was dried over Na2SO4, filtered, and concentrated to give ethyl 2-
({[tert-butyl(dimethyl)silyl]oxy}methyl)acrylate as an oil (9.32 g,
99%). 1H NMR (DMSO-d6): δ 0.06 (6H, s), 0.88 (9H, s), 1.23 (3H, t,
J = 7.2 Hz), 4.15 (q, 2H, J = 6.4 Hz), 4.31 (2H, m), 5.83 (1H, m), 6.14
(1H, m). MS (ESI) m/z = 267 (M + Na).
A mixture of ethyl 2-({[tert-butyl(dimethyl)silyl]oxy}methyl)acry-
late (9.3 g, 38.1 mmol) and 1-phenylmethanamine (4.2 mL, 38.1
mmol) in EtOH (100 mL) was stirred for 14 h at 50 °C. After removal
of solvent, the crude product was purified by column chromatography
on silica gel (hexane/EtOAc = 8/1) to give ethyl 3-(benzylamino)-2-
({[tert-butyl(dimethyl)silyl]oxy}methyl)propanoate as an oil (5.12 g,
38%). 1H NMR (DMSO-d6) δ −0.01 (6H, s), 0.81 (9H, s), 1.16 (3H,
t, J = 7.0 Hz), 2.16 (1H, brs), 2.53−2.72 (3H, m), 3.65 (2H, s), 3.70−
3.78 (2H, m), 3.99−4.09 (2H, m), 7.17−7.33 (5H, m). MS (API-ES)
m/z = 352 (M + H).
Ethyl 2-(4-Fluorophenyl)-3-[1-(2-methylphenyl)-6-oxo-1,6-
dihydropyridazin-3-yl]-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate (11f). To a suspension of ethyl 3-({3-(4-
fluorophenyl)-4-[1-(2-methylphenyl)-6-oxo-1,6-dihydropyridazin-3-
yl]-1H-pyrazol-5-yl}amino)-2-(hydroxymethyl)propanoate 10f (1.45
g, 2.95 mmol) in MeCN (30 mL) were added Et3N (2.1 μL, 14.8
mmol) and methanesulfonyl chloride (343 μL, 4.42 mmol) at room
temperature followed by heating at 100 °C for 6 h. The mixture was
cooled to room temperature and evaporated in vacuo. To the residue
was added a 10% K2CO3 solution. Extraction was with CHCl3, and the
sample was washed with water and brine and dried over MgSO4. After
removal of solvent, the residue was purified by column chromatog-
raphy on silica gel (hexane/EtOAc = 4/1) to give ethyl 2-(4-fluoro-
phenyl)-3-[1-(2-methylphenyl)-6-oxo-1,6-dihydropyridazin-3-yl]-
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-6-carboxylate 11f as an
1
oil (1.1 g, 79%). H NMR (DMSO-d6): δ 1.19 (3H, t, J = 7.2 Hz),
2.08 (3H, s), 3.36−3.56 (2H, m), 4.12 (2H, m), 4.25 (2H, m),
6.10 (1H, brs), 6.93 (1H, d, J = 9.6 Hz), 7.09 (1H, d, J = 9.6 Hz), 7.23
(2H, m), 7.28−7.41 (4H, m), 7.48 (2H, m). MS (API-ES) m/z = 496
(M + Na).
6-[2-(4-Fluorophenyl)-6-(hydroxymethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (3f and 3g). A mixture of ethyl 2-(4-
fluorophenyl)-3-[1-(2-methylphenyl)-6-oxo-1,6-dihydropyridazin-3-
yl]-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-6-carboxylate 11f
(10 mg, 0.02 mmol), lithium tetrahydroborate (1 mg, 0.05 mmol),
and THF (1 mL) was stirred at room temperature for 2 h. The mixture
was quenched with water, extracted with EtOAc, dried over MgSO4,
and evaporated in vacuo. The residue was purified by preparative thin
layer chromatography to give the title product (7.5 mg, 82%).
The enantiomers of the racemic products were separated by chiral
HPLC. HPLC results were obtained on a Hitachi LaChrom Elite
system, using a DAICEL Chiralcel OJ-H column (25 cm × 0.46 cm) at
40 °C with a 1.0 mL/min flow rate using acetonitrile and n-hexane/
EtOH/MeOH/isopropyl alcohol (60:30:10:0.1) solution as the eluent
A mixture of ethyl 3-(benzylamino)-2-({[tert-butyl(dimethyl)silyl]-
oxy}methyl)propanoate (5.12 g, 14.6 mmol) in EtOH (50 mL) was
hydrogenized for 2 h under pressure with H2 gas (3 atm) at 40 °C.
After removal of catalyst, the filtrate was concentrated to give ethyl
3-amino-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)propanoate as a col-
1
orless oil (3.93 g, 100%). H NMR (DMSO-d6): δ 0.01 (3H, s), 0.02
(3H, s), 0.83 (9H, s), 1.18 (3H, t, J = 7.0 Hz), 1.46 (1H, brs), 2.47−
2.55 (1H, m), 2.60−2.77 (2H, m), 3.31 (1H, brs), 3.69−3.80 (2H, m),
4.00−4.11 (2H, m). MS (API-ES) m/z = 262 (M + H).
To a mixture of O-phenyl carbonochloridothioate (2.0 mL, 14.9
mmol) in toluene (15.6 mL) and saturated NaHCO3 solution (16 mL)
was added a solution of 3-amino-2-({[tert-butyl(dimethyl)silyl]oxy}-
methyl)propanoate (3.9 g, 14.9 mmol) in toluene (5 mL) with cooling
on ice. After being stirred for 1 h, the organic layer was separated,
washed with saturated NaHCO3 solution and brine, and dried over
Na2SO4. After removal of solvent, the residue was purified by column
chromatography on silica gel (hexane/EtOAc = 8/1) to give ethyl
3-{[tert-butyl(dimethyl)silyl]oxy}-2-{[(phenoxycarbothioyl)amino]-
over 30 min. Enantiomer 3f ([α]26 +51.3 (c 0.45, CDCl3)) was
D
collected at 11.076 min, and 3g ([α]26 −50.4 (c 0.98, CDCl3)) was
D
collected at 14.766 min.
6-[(6R)-2-(4-Fluorophenyl)-6-(hydroxymethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
1
pyridazin-3(2H)-one (3f). H NMR (DMSO-d6): δ 2.08 (3H, s),
2.23 (1H, brs), 3.04 (1H, m), 3.46 (2H, m), 3.82 (1H, m), 4.12 (1H,
m), 4.85 (1H, m), 5.98 (1H, brs), 6.92 (1H, d, J = 10 Hz), 7.08 (1H, d,
J = 10 Hz), 7.23 (2H, m), 7.30−7.40 (4H, m), 7.48 (2H, m). MS
(API-ES) m/z = 432 (M + H). [α]26 +51.3 (c 0.45, CHCl3).
6-[(6S)-2-(4-Fluorophenyl)-6D-(hydroxymethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
1
methyl}propanoate as an oil (3.76 g, 63%). H NMR (DMSO-d6): δ
0.04 (6H, s), 0.85 (9H, s), 1.21 (3H, t, J = 7.1 Hz), 2.97−3.04 (1H,
m), 3.79−3.90 (4H, m), 4.12 (2H, q, J = 7.1 Hz), 6.73−6.78 (3H, m),
7.12−7.18 (2H, m), 9.31 (1H, s). MS (API-ES) m/z = 398 (M + H).
To a solution of ethyl 3-{[tert-butyl(dimethyl)silyl]oxy}-2-
{[(phenoxycarbothioyl)amino]methyl}propanoate (3.7 g, 9.31
mmol) in EtOH (50 mL) was added hydrazine hydrate (722 mL,
14.9 mmol) at ambient temperature. The mixture was stirred for 2 h
and then for 6 h at 40 °C. After removal of solvent, the residue was
purified by column chromatography on silica gel (CHCl3/MeOH =
95/5) to give the title compound 9f as an oil (2.33 g, 75%). 1H NMR
(DMSO-d6): δ 0.021 (6H, s), 0.84 (9H, s), 1.19 (3H, t, J = 7.0 Hz),
2.88−2.97 (1H, m), 3.57−3.65 (2H, m), 3.66−3.82 (2H, m), 4.03−
4.12 (2H, m), 4.46 (2H, s), 7.87 (1H, brs), 8.73 (1H, s). MS (API-ES)
m/z = 336 (M + H).
1
pyridazin-3(2H)-one (3g). H NMR (DMSO-d6): δ 2.08 (3H, s),
2.22 (1H, brs), 3.03 (1H, m), 3.45 (2H, m), 3.81 (1H, m), 4.12 (1H,
m), 4.84 (1H, m), 5.99 (1H, brs), 6.93 (1H, d, J = 10 Hz), 7.09 (1H, d,
J = 10 Hz), 7.23 (2H, m), 7.30−7.40 (4H, m), 7.48 (2H, m). MS
(API-ES) m/z = 432 (M + H). [α]26 −50.4 (c 0.98, CHCl3).
D
6-{2-(4-Fluorophenyl)-6-[(4-methylpiperazin-1-yl)methyl]-
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl}-2-(2-
methylphenyl)pyridazin-3(2H)-one Dihydrochloride (3h). A
mixture of 6-[2-(4-fluorophenyl)-6-(iodomethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (200 mg, 0.37 mmol) and 1-methylpiperazine
(800 μL, 7.27 mmol) was stirred at 60 °C for 12 h. The reaction
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mixture was quenched with H2O, and the products were extracted with
AcOEt. The extract was dried over MgSO4, filtered, and evaporated.
The residue was purified by column chromatography (CHCl3/MeOH =
95/5 to 90/10), and the fractions were combined and evaporated.
The residue was dissolved in AcOEt, and to the mixture was added
4 M HCl−AcOEt. The precipitate was filtered and crystallized from
EtOH−AcOEt to give the title compound as a yellow powder (47 mg,
22%). 1H NMR (DMSO-d6): δ 2.09 (3H, s), 2.60−2.71 (1H, m), 2.81
(3H, s), 3.04−4.39 (14H, m), 6.95 (1H, d, J = 9.8 Hz), 7.10 (1H, d,
J = 9.8 Hz), 7.21−7.28 (2H, m), 7.32−7.39 (4H, m), 7.46−7.52 (2H,
m). MS (API-ES) m/z = 514 (M + H).
by column chromatography on silica gel (CHCl31/MeOH = 99/1 to 95/5)
to give the title compound 9i (630 mg, 51%). H NMR (DMSO-d6): δ
1.01 (9H, s), 1.73−1.90 (2H, m), 2.07 (3H, s), 3.44−3.49 (2H, m),
3.63 (1H, dd, J = 9.5, 5.5 Hz), 3.74 (1H, dd, J = 10, 3.5 Hz), 4.45−4.49
(4H, m), 7.42−7.47 (6H, m), 7.60−7.65 (4H, m), 7.86 (1H, brs), 8.73
(1H, brs). MS (ESI) m/z = 440 (M + Na).
6-[5-{[(2R)-1-{[tert-Butyl(diphenyl)silyl]oxy}-4-hydroxybu-
tan-2-yl]amino}-3-(4-fluorophenyl)-1H-pyrazol-4-yl]-2-(2-
methylphenyl)pyridazin-3(2H)-one (13i). To a solution of 8 (630
mg, 1.6 mmol) in EtOH/acetic acid (1/1, 8.4 mL) was added 9i
(655.8 mg, 1.6 mmol) at room temperature, and the mixture was
stirred at 50 °C for 2 h. The mixture was quenched with NaHCO3
solution, and extraction was with ethyl acetate. The organic layer was
separated, successively washed with NaHCO3 solution and brine, dried
over MgSO4, and evaporated under reduced pressure to give the crude
product. The crude product was purified by column chromatography
on silica gel (CHCl3/MeOH = 100/0 to 90/10) to give the title
N-[(2R)-1-{[tert-Butyl(diphenyl)silyl]oxy}-4-hydroxybutan-2-yl]-
hydrazinecarbothioamide (9i). To a solution of (2R)-4-{[tert-
butyl(dimethyl)silyl]oxy}-2-(dibenzylamino)butan-1-ol (1.47 g, 3.7
mmol) in DMF (29.4 mL) were added imidazole (601 mg, 8.8
mmol) and tert-butyl(chloro)diphenylsilane (1.15 mL, 4.4 mmol) at
room temperature, and the mixture was stirred for 15 h. The mixture
was partitioned between ethyl acetate and water. The organic layer was
separated, successively washed with water and brine, dried over
MgSO4, and evaporated under reduced pressure to give the crude
product. The crude product was purified by column chromatography
on silica gel (hexane/EtOAc = 100/0 to 95/5) to give (6R)-N,N-
dibenzyl-2,2,10,10,11,11-hexamethyl-3,3-diphenyl-4,9-dioxa-3,10-disi-
1
compound (850 mg, 79%). H NMR (DMSO-d6): δ 0.80 (9H, s),
1.52−1.62 (1H, m), 1.70−1.83 (1H, m), 2.07 (3H, s), 3.46−3.86 (5H,
m), 4.46 (1H, brs), 5.53 (1H, brs), 6.92 (1H, d, J = 10 Hz), 7.03 (1H,
d, J = 10 Hz), 7.24−7.56 (18H, m), 12.27 (1H, brs). MS (API-ES)
m/z = 688 (M + H).
6-[(5R)-2-(4-Fluorophenyl)-5-(hydroxymethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (3i). To a solution of 13i (850 mg, 1.2 mmol)
in acetonitrile (17 mL) were added methanesulfonyl chloride
(143.5 μL, 1.8 mmol) and triethylamine (516.7 μL, 3.7 mmol), and
the mixture was stirred at 80 °C for 2 h. The mixture was partitioned
between ethyl acetate and water. The organic layer was separated,
successively washed with 10% citric acid solution and brine, dried over
MgSO4, and evaporated under reduced pressure to give the crude
product, which was purified by column chromatography on silica gel
(CHCl3/MeOH = 99/1 to 95/5) to give 6-[(5R)-5-({[tert-butyl-
(diphenyl)silyl]oxy}methyl)-2-(4-fluorophenyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
1
ladodecan-6-amine (1.97 g, 84%). H NMR (400 MHz, CDCl3): δ
−0.04 (6H, s), 0.82 (9H, s), 1.07 (9H, s), 1.63−1.72 (1H, m), 1.80−
1.89 (1H, m), 2.87−2.94 (1H, m), 3.50−3.58 (1H, m), 3.60−3.82
(7H, m), 7.16−7.21 (2H, m), 7.23−7.28 (4H, m), 7.31−7.44 (10H,
m), 7.64−7.69 (4H, m). MS (API-ES) m/z = 639 (M + H).
To a solution of (6R)-N,N-dibenzyl-2,2,10,10,11,11-hexamethyl-3,3-
diphenyl-4,9-dioxa-3,10-disiladodecan-6-amine (1.97 g, 3.0 mmol) in
dioxane (19.7 mL) was added 4 N HCl (3.94 mL) at room tem-
perature, and the mixture was stirred for 1 h. The mixture was parti-
tioned between ethyl acetate and water. The organic layer was
separated, successively washed with NaHCO3 solution and brine, dried
over MgSO4, and evaporated under reduced pressure to give the crude
product. The crude product was purified by column chromatography
on silica gel (hexane/EtOAc = 9/1 to 4/1) to give (3R)-4-{[tert-
butyl(diphenyl)silyl]oxy}-3-(dibenzylamino)butan-1-ol (1.53 g, 95%).
1H NMR (400 MHz, CDCl3): δ 1.10 (9H, s), 1.47−1.56 (1H, m),
1.86−1.99 (1H, m), 3.02−3.11 (1H, m), 3.56 (2H, d, J = 13 Hz),
3.69−3.80 (2H, m), 3.83−3.88 (2H, m), 3.93 (2H, d, J = 13 Hz),
7.20−7.33 (10H, m), 7.38−7.47 (6H, m), 7.66−7.71 (4H, m). MS
(API-ES) m/z = 524 (M + H).
1
pyridazin-3(2H)-one (495.6 mg, 60%). H NMR (DMSO-d6): δ 0.90
(9H, s), 1.97−2.00 (1H, m), 2.03 (3H, s), 2.15−2.23 (1H, m), 3.52−
3.68 (3H, m), 4.00−4.05 (2H, m), 6.03 (1H, brs), 6.92 (1H, d, J =
10 Hz), 7.07 (1H, d, J = 10 Hz), 7.19−7.57 (18H, m). MS (ESI)
m/z = 692 (M + Na).
To a solution of 6-[(5R)-5-({[tert-butyl(diphenyl)silyl]oxy}-
methyl)-2-(4-fluorophenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidin-3-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (450 mg,
0.67 mmol) in THF (4.5 mL) was added a solution of 1 M
tetrabutylammonium fluoride in THF (0.67 mL, 0.67 mmol) at room
temperature. After being stirred for 30 min, the mixture was parti-
tioned between EtOAc and H2O. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (CHCl3/MeOH = 98/2 to
95/5). The obtained oil was crystallized from iPrOH−Hex to give the
To a solution of (3R)-4-{[tert-butyl(diphenyl)silyl]oxy}-3-
(dibenzylamino)butan-1-ol (1.57 g, 3.0 mmol) in ethanol (24 mL)
were added ammonium formate (2.84 g, 45.0 mmol) and palladium on
carbon (628 mg, 10% wt on carbon) at room temperature, and the
mixture was refluxed for 4 h. After cooling to room temperature, the
mixture was filtered through a funnel. The filtrate was evaporated
under reduced pressure to give (3R)-3-amino-4-{[tert-butyl(diphenyl)-
1
1
title compound (269 mg, 93%). H NMR (DMSO-d6): δ 1.71−1.84
silyl]oxy}butan-1-ol (1 g, 97%). H NMR (400 MHz, CDCl3): δ 1.06
(1H, m), 2.05−2.09 (1H, m), 2.11 (3H, s), 3.26−3.33 (1H, m), 3.40
(1H, m), 3.48−3.52 (1H, m), 3.98−4.14 (2H, m), 4.89 (1H, t, 5 Hz),
6.11 (1H, brs), 6.92 (1H, d, J = 10 Hz), 7.03 (1H, d, J = 10 Hz), 7.26
(2H, t, J = 9 Hz), 7.31−7.37 (4H, m), 7.49 (2H, dd, J = 9, 5 Hz). MS
(ESI) m/z = 454 (M + Na).
N-[(2S)-1-{[tert-Butyl(diphenyl)silyl]oxy}-4-hydroxybutan-2-yl]-
hydrazinecarbothioamide (9j). (6S)-N,N-Dibenzyl-2,2,10,10,11,
11-hexamethyl-3,3-diphenyl-4,9-dioxa-3,10-disiladodecan-6-amine
was synthesized from (2S)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-
(dibenzylamino)butan-1-ol according to the procedure for the R-
isomer ((6R)-N,N-dibenzyl-2,2,10,10,11,11-hexamethyl-3,3-diphenyl-
4,9-dioxa-3,10-disiladodecan-6-amine) (99%). 1H NMR (400 MHz,
CDCl3): δ 0.03 (6H, s), 0.85 (9H, s), 1.05 (9H, s), 1.63−1.73
(1H, m), 1.81−1.90 (1H, m), 2.86−2.94 (1H, m), 3.51−3.58 (1H, m),
3.59−3.82 (7H, m), 7.18−7.23 (2H, m), 7.25−7.30 (4H, m),
7.31−7.46 (10H, m), 7.65−7.68 (4H, m). MS (API-ES) m/z = 639
(M + H).
(9H, s), 1.53−1.60 (2H, m), 3.01−3.05 (1H, m), 3.44 (1H, dd, J = 6,
10 Hz), 3.56 (1H, dd, J = 4, 10 Hz), 3.79−3.82 (2H, m), 7.34−7.46
(6H, m), 7.62−7.68 (4H, m). MS (API-ES) m/z = 344 (M + H).
To a solution of (3R)-3-amino-4-{[tert-butyl(diphenyl)silyl]oxy}-
butan-1-ol (1.0 g, 2.9 mmol) in CH2Cl2/H2O (1/1, 30 mL) were
added O-phenyl carbonochloridothioate (471 μL, 3.5 mmol) and
NaHCO3 (489.1 mg, 5.8 mmol) at 0 °C followed by stirring at room
temperature for 30 min. The mixture was partitioned between ethyl
acetate and water. The organic layer was separated, successively
washed with water and brine, dried over MgSO4, and evaporated
under reduced pressure to give 1.42 g of O-phenyl [(2R)-1-{[tert-
butyl(diphenyl)silyl]oxy}-4-hydroxybutan-2-yl]carbamothioate. MS
(ESI) m/z = 502 (M + Na). To a solution of the above product
(1.42 g, 3.0 mmol) in isopropyl alcohol (8.0 mL) was added hydrazine
hydrate (1.4 mL, 29.6 mmol) at room temperature, and the mixture
was stirred for 2 h. The mixture was partitioned between ethyl acetate
and water. The organic layer was separated, washed successively with
1 N NaOH solution and brine, dried over MgSO4, and evaporated
under reduced pressure to give the crude product, which was purified
(3S)-4-{[tert-Butyl(diphenyl)silyl]oxy}-3-(dibenzylamino)butan-1-ol
was synthesized from (6S)-N,N-dibenzyl-2,2,10,10,11,11-hexamethyl-3,
K
dx.doi.org/10.1021/jm3008008 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
3-diphenyl-4,9-dioxa-3,10-disiladodecan-6-amine according to the
procedure for the R-isomer ((3R)-4-{[tert-butyl(diphenyl)silyl]oxy}-3-
(dibenzylamino)butan-1-ol) (98%). 1H NMR (400 MHz, CDCl3):
δ 1.12 (9H, s), 1.47−1.55 (1H, m), 1.88−2.00 (1H, m), 3.01−3.12
(1H, m), 3.58 (2H, d, J = 13 Hz), 3.68−3.80 (2H, m), 3.83−3.89 (2H,
m), 3.95 (2H, d, J = 13 Hz), 7.25−7.33 (10H, m), 7.35−7.46 (6H, m),
7.66−7.73 (4H, m). MS (API-ES) m/z = 524 (M + H).
title product (1.0 g, 92%). H NMR (CDCl3): δ 2.22 (3H, s), 3.90
(2H, s), 4.81 (2H, s), 5.31 (2H, d), 5.97 (1H, brs), 6.82 (1H, d, J = 8.8
Hz), 7.02 (1H, d, J = 10 Hz), 7.17 (2H, m), 7.30−7.42 (4H, m), 7.53
(2H, m). MS (ESI) m/z = 436 (M + Na).
6-[2-(4-Fluorophenyl)-6-(hydroxymethyl)pyrazolo[1,5-a]-
pyrimidin-3-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (3k). A
mixture of 14 (150 mg, 0.36 mmol), OsO4 (46 mg, 0.18 mmol), N-
methylmorpholine N-oxide (55.3 mg, 0.47 mmol), H2O (0.6 mL),
acetone (0.6 mL), and MeCN (0.6 mL) was stirred for 3 weeks and
then filtered through a Celite pad. The filtrate was evaporated and the
residue was purified by column chromatography on silica gel (CHCl3/
MeOH = 10/0 to 98/2) to give the title compound (37.0 mg, 24%).
1H NMR (CDCl3): δ 2.14 (3H, s), 4.83 (2H, s), 7.04 (2H, t), 7.14
(3S)-4-{[tert-Butyl(diphenyl)silyl]oxy}-3-(dibenzylamino)butan-1-
ol was synthesized from (3S)-4-{[tert-butyl(diphenyl)silyl]oxy}-3-
(dibenzylamino)butan-1-ol according to the procedure for the R-
isomer ((3S)-4-{[tert-butyl(diphenyl)silyl]oxy}-3-(dibenzylamino)-
1
butan-1-ol) (100%). H NMR (400 MHz, CDCl3): δ 1.05 (9H, s),
1.55−1.60 (2H, m), 3.01−3.06 (1H, m), 3.44 (1H, dd, J = 6, 10 Hz),
3.56 (1H, dd, J = 4, 10 Hz), 3.78−3.83 (2H, m), 7.33−7.46 (6H, m),
7.63−7.68 (4H, m). MS (API-ES) m/z = 344 (M + H).
(3H, m), 7.30 (2H, m), 7.70 (2H, dd), 7.92 (1H, d), 8.58 (1H, s), 8.71
(1H, s). MS (API-ES) m/z = 450 (M + Na).
Biological Methods. Inhibition of TNF-α Production in THP-1
Cells. THP-1 cells, from a human monocytic cell line, were main-
tained in RPMI 1640 (Sigma R8758) supplemented with penicillin
(50 U/mL), streptomycin (50 μg/mL), and 10% fetal bovine serum
(Moregate BioTech) at 37 °C and 5% CO2 in a humidified incubator.
Initial stock solutions of test compounds were made in DMSO. All
cells, reagents, and test compounds were diluted in culture media.
THP-1 cells (1 × 105 cells/well final) and lipopolysaccharide (LPS, 10
μg/mL final, Sigma L-4005, from E. coli serotype 055:B5) were added
to 96-well polypropylene culture plates (Sumilon, MS-8196F5; sterile)
containing test compound or 0.1% DMSO vehicle. The cell mixture
was incubated for 20 h in a humidified incubator at 37 °C, 5% CO2.
The culture supernatants were harvested, and TNF-α levels from
LPS-stimulated cells in the presence of 100 nM test compound were
calculated and compared with those from control cells stimulated in
the presence of 0.1% DMSO.
N-[(2S)-1-{[tert-Butyl(diphenyl)silyl]oxy}-4-hydroxybutan-2-yl]-
hydrazinecarbothioamide (9j) was synthesized from (3S)-3-amino-4-
{[tert-butyl(diphenyl)silyl]oxy}butan-1-ol according to the procedure
1
for the R-isomer (9i). H NMR (DMSO-d6): δ 1.01 (9H, s), 1.74−
1.91 (2H, m), 2.07 (3H, s), 3.45−3.48 (2H, m), 3.64 (1H, dd, J = 9.5,
5.5 Hz), 3.74 (1H, dd, J = 10, 3.5 Hz), 4.46−4.48 (4H, m), 7.41−7.49
(6H, m), 7.61−7.66 (4H, m), 7.89 (1H, brs), 8.74 (1H, brs). MS
(ESI) m/z = 440 (M + Na).
6-[5-{[(2S)-1-{[tert-Butyl(diphenyl)silyl]oxy}-4-hydroxybutan-
2-yl]amino}-3-(4-fluorophenyl)-1H-pyrazol-4-yl]-2-(2-
methylphenyl)pyridazin-3(2H)-one (13j). 6-[5-{[(2S)-1-{[tert-
Butyl(diphenyl)silyl]oxy}-4-hydroxybutan-2-yl]amino}-3-(4-fluoro-
phenyl)-1H-pyrazol-4-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one
(13j) was synthesized from N-[(2S)-1-{[tert-butyl(diphenyl)silyl]-
oxy}-4-hydroxybutan-2-yl]hydrazinecarbothioamide (11j) according
1
to the procedure for the R-isomer (13i) (77%). HNMR (DMSO-
Inhibition of Hind Paw Swelling in Adjuvant-Induced
Arthritis in Rats. Arthritis was induced by injection of 0.5 mg of
Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI) in 50 μL
of liquid paraffin into the right hind footpad of female Lewis rats aged
7 weeks (day 0). Normal untreated rats were used as negative controls.
Animals were randomized and grouped (n ≥ 5) for drug treatment
based on an increase in left hind paw volume and body weight on day
15. Drugs were suspended in vehicle (0.5% methylcellulose) and orally
administered once a day from day 15 to day 24. The volume of the left
hind paw was measured on day 25 by a water displacement method
using a plethysmometer for rats (MK-550; Muromachi Kikai Co., Ltd.,
Tokyo, Japan).
In Vivo Assays. LPS-Induced TNFα in the Rat. Four hours prior
to LPS administration, rats were dosed orally with the compounds
suspended in 0.5% methylcellulose/0.1% Tween-80. LPS (10 μg/
animal) was administered intraperitoneally, and 1.0 or 1.5 h later, rats
were anesthetized and retroorbital blood was collected in heparin
tubes. Plasma was separated by centrifugation and diluted one-fifth
prior to assaying in a standard rat TNF-α ELISA.
Kinase Isoform Selectivity Assays. The human p38α and p38β
kinase activities of 3f were evaluated using a Z′-LyteTM kinase assay
system (Life Technologies, Madison, FL, U.S.). JNK2 kinase activity
was examined by following an in-house protocol. Preactivated
recombinant human JNK2α2/SAPK1a (40 ng/well; Upstate Bio-
technology, Lake Placid, NY, U.S.) was diluted in 1.5× assay dilution
buffer I (1× ADB-I, 20 mM MOPS, pH 7.2, 25 mM β-glycerol
phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT),
and then 20 μL of 1.5× ADB-I (containing recombinant JNK2)
was mixed with 20 μL of Mg/ATP/1× ADBI buffer (37.5 mM MgCl2,
2 μM ATP diluted with 1× ADB-I) and 10 μL of 3f at final
concentrations of 10−1000 nM in 96-well MaxiSorp Immuno Plates
(Nalge Nunc International, Rochester, NY, U.S.) precoated with
recombinant human ATF-2 (0.1 μg/well, Upstate Biotechnology).
After 30 min of incubation at 30 °C, the plate was washed three times
with phosphate buffered saline containing Tween 0.05% and then
incubated with 1:1000 diluted anti-phospho-ATF-2 (Thr71) (Cell
Signaling Technology, Danvers, MA, U.S.) antibody for 60 min at
room temperature. The plate was then washed as described above
and reacted with 1:5000 diluted anti-rabbit IgG-conjugated HRP
d6): δ 0.90 (9H, s), 1.53−1.62 (1H, m), 1.70−1.83 (1H, m), 2.07
(3H, s), 3.44−3.85 (5H, m), 4.46 (1H, brs), 5.52 (1H, brs), 6.93 (1H,
d, J = 10 Hz), 7.00 (1H, d, J = 10 Hz), 7.23−7.56 (18H, m), 12.27
(1H, brs). MS (ESI) m/z = 688 (M + H).
6-[(5S)-2-(4-Fluorophenyl)-5-(hydroxymethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (3j). 6-[(5S)-5-({[tert-Butyl(diphenyl)silyl]-
oxy}methyl)-2-(4-fluorophenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidin-3-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one was synthesized
from 12j according to the procedure for the R-isomer (6-[(5R)-5-
({[tert-butyl(diphenyl)silyl]oxy}methyl)-2-(4-fluorophenyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
1
pyridazin-3(2H)-one) (76%). HNMR (DMSO-d6): δ 0.91 (9H, s),
1.97−2.05 (1H, m), 2.03 (3H, s), 2.14−2.24 (1H, m), 3.53−3.66
(3H, m), 4.01−4.05 (2H, m), 6.04 (1H, brs), 6.92 (1H, d, J = 10 Hz),
7.09 (1H, d, J = 10 Hz), 7.20−7.57 (18H, m). MS (ESI) m/z = 692
(M + Na).
6-[(5S)-2-(4-Fluorophenyl)-5-(hydroxymethyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (3j) was synthesized from 6-[(5S)-5-({[tert-
butyl(diphenyl)silyl]oxy}methyl)-2-(4-fluorophenyl)-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one according to the procedure for the R-isomer (3i)
(91%). Chiral HPLC AS-H, 99.9% (15.82 min), Hex/EtOH/MeOH/
Et2NH = 60/30/10/0.1, flow = 0.5 mL/min, 254 nm). 1HNMR
(DMSO-d6): δ 1.73−1.86 (1H, m), 2.06−2.08 (1H, m), 2.11 (3H, s),
3.25−3.33 (1H, m), 3.42 (1H, m), 3.49−3.53 (1H, m), 4.01−4.13
(2H, m), 4.89 (1H, t, 5 Hz), 6.12 (1H, brs), 6.92 (1H, d, J = 10 Hz),
7.03 (1H, d, J = 10 Hz), 7.25 (2H, t, J = 9 Hz), 7.30−7.36 (4H, m),
7.48 (2H, dd, J = 9, 5 Hz). MS (ESI) m/z = 454 (M + Na).
6 - [ 2 - ( 4 - F l u o r o p h e n y l ) - 6 - m e t h y l e n e - 4 , 5 , 6 , 7 -
tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)-
pyridazin-3(2H)-one (14). A mixture of 6-[2-(4-fluorophenyl)-6-
(iodomethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-
methylphenyl)pyridazin-3(2H)-one 12 (1.43 g, 2.64 mmol) and 28%
NaOMe (612 mg, 3.17 mmol) in MeOH (14.3 mL) was refluxed for
12 h. The mixture was partitioned between ethyl acetate and water.
The organic layer was separated, washed with 5% citric acid and brine,
dried over MgSO4, and evaporated under reduced pressure to give the
L
dx.doi.org/10.1021/jm3008008 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(Zymed Laboratories, South San Francisco, CA, U.S.) for 30 min at
room temperature. After the plate was washed, TMB substrate (BD
Biosciences Pharmingen, San Diego, CA, U.S.) was added, and the
plate was incubated for 20 min in the dark. The reaction was stopped
by adding 50 μL of 2 N H2SO4, and sample absorbance was measured
with a microplate reader at 450 nm.
Pharmacokinetic Studies. 2a, 3d, 3e, 3f, 3g, or 3j was dissolved
in PEG400 and administered to Sprague−Dawley rats at a dose of
1 mg/kg. Blood was collected and centrifuged to obtain plasma, and
the plasma concentrations of each compound were determined by
LC/MS/MS.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We deeply acknowledge the contributions of Tatsuo Inoue, Dr.
Katsue Magari, and Masaomi Terajima for the establishment
and measurement of the TNFα assay and in vivo assays
and Fujiko Takamura and Dr. Hidetsugu Murai for the
pharmacokinetic study and valuable discussions. The authors
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G.; Hickey, E.; Kroe, R. R.; Madwed, J. R.; Moriak, M.; Nelson, R.;
Pargellis, C. A.; Swinamer, A.; Torcellini, C.; Tsang, M.; Moss, N.
Structure−activity relationships of the p38 alpha MAP kinase inhibitor
1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-
ABBREVIATIONS USED
■
TNFα, tumor necrosis factor α; MAPK, mitogen-activated
protein kinase; LPS, lipopolysaccharide; COPD, chronic
obstructive pulmonary disease; IBD, inflammatory bowel
disease; CYP, cytochrome; °C, degree Celsius; ClogP,
calculated log P; SAR, structure−activity relationship; IC50,
half-maximum inhibitory concentration; ED50, 50% effective
dose; F, % oral bioavailability; AUC, area under the curve; t1/2
,
half-life; PAMPA, parallel artificial membrane permeability
assay; po, oral administration; iv, intravenous; PK, pharmaco-
kinetics; Ac, acetyl; Bn, benzyl; Et, ethyl; Me, methyl; Ts, p-
toluenesulfonyl; Ms, methanesulfonyl; ph, phenyl; DEAD,
diethyl azodicarboxylate; DMF, dimethylformamide; TBAF,
tetrabutylammonium fluoride; HPLC, high-performance liquid
chromatography; MS, mass spectrometry; HRMS, high-
resolution mass spectrometry; NMR, nuclear magnetic
resonance; ppm, parts per million; J, coupling constant; RA,
rheumatoid arthritis; LiBH4, lithium borohydride; OsO4,
osmium tetroxide; LiHMDS, lithium bis(trimethylsilyl)amide;
PyBr3, pyridinium tribromide; NMM, N-methylmorpholine;
DIPEA, N,N-diisopropylethylamine; CLint, intrinsic clearance;
CLtot, total body clearance; Vss, volume of distribution at steady
state; AIA, adjuvant-induced arthritis; MC, methylcellulose;
ED30, 30% effective dose
M
dx.doi.org/10.1021/jm3008008 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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