Design, synthesis, and structure-activity relationships of novel pyrazolo[5,1-b]thiazole derivatives as potent and orally active corticotropin-releasing factor 1 receptor antagonists

Article abstract of DOI:10.1021/jm300864p

This paper describes the design, synthesis, and structure-activity relationships of a novel series of 7-dialkylamino-3-phenyl-6-methoxy pyrazolo[5,1-b]thiazole derivatives for use as selective antagonists of the corticotropin-releasing factor 1 (CRF₁) receptor. The most promising compound, N-butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-methoxy-N- (tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]thiazole-7-amine (6t), showed high affinity (IC₅₀ = 70 nM) and functional antagonism (IC₅₀ = 7.1 nM) for the human CRF₁ receptor as well as dose-dependent inhibition of the CRF-induced increase in the plasma adrenocorticotropic hormone (ACTH) concentration at a dose of 30 mg/kg (po). Further, in the light/dark test in mice, the compound 6t showed anxiolytic activity at a dose of 30 mg/kg (po).

Full text of DOI:10.1021/jm300864p

Article
pubs.acs.org/jmc
Design, Synthesis, and Structure−Activity Relationships of Novel
Pyrazolo[5,1‑b]thiazole Derivatives as Potent and Orally Active
Corticotropin-Releasing Factor 1 Receptor Antagonists
Yoshinori Takahashi,*,† Minako Hashizume,† Kogyoku Shin,† Taro Terauchi,† Kunitoshi Takeda,†
Shigeki Hibi,⊥ Kaoru Murata-Tai,‡ Masae Fujisawa,§ Kodo Shikata,∥ Ryota Taguchi,∥ Mitsuhiro Ino,∥
Hisashi Shibata,∥ and Masahiro Yonaga†
† Medicinal Chemistry, ∥ Biopharmacology, ‡ Physical Chemistry, and § Drug Metabolism and Pharmacokinetics, Tsukuba Research
Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
⊥
Project Management, Head Office, Eisai Co., Ltd., 4-20-22 Koishikawa, Bunkyo-ku, Tokyo 112-8088, Japan
ABSTRACT: This paper describes the design, synthesis, and structure−activity relationships of a novel
series of 7-dialkylamino-3-phenyl-6-methoxy pyrazolo[5,1-b]thiazole derivatives for use as selective
antagonists of the corticotropin-releasing factor 1 (CRF1) receptor. The most promising compound, N-
butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo-
[5,1-b][1,3]thiazole-7-amine (6t), showed high affinity (IC50 = 70 nM) and functional antagonism
(IC50 = 7.1 nM) for the human CRF1 receptor as well as dose-dependent inhibition of the CRF-
induced increase in the plasma adrenocorticotropic hormone (ACTH) concentration at a dose of 30
mg/kg (po). Further, in the light/dark test in mice, the compound 6t showed anxiolytic activity at a
dose of 30 mg/kg (po).
INTRODUCTION
■
Corticotropin-releasing factor (CRF), a 41-amino-acid neuro-
peptide, is a key regulator of the hypothalamus−pituitary−
adrenal axis, which coordinates the endocrine response to stress
by regulating the release of adrenocorticotropic hormone
(ACTH) from the pituitary.1,2 Two receptor subtypes (CRF1
and CRF2) belonging to the class B subfamily of G protein-
coupled receptors (GPCRs) have been identified.3−6 They are
widely distributed throughout the central and peripheral
nervous systems. Several preclinical and clinical studies suggest
the therapeutic potential of CRF1 receptor antagonists in stress-
related diseases such as depression, anxiety, and possibly,
irritable bowel syndrome;7−11 the role of the CRF2 receptor,
however, as a target for stress-related disorders has not been
fully elucidated. Subsequent to the discovery of the nonpeptide
small molecule CRF1 receptor antagonist 1 (CP-154,526),12
many research groups have reported preclinical studies that
demonstrate the efficacy of their own CRF1 receptor
antagonists in animal models of anxiety and depression.13−20
However, the clinical effectiveness of CRF1 receptor antagonists
remains to be confirmed. Evidence of antidepressant/anxiolytic
Figure 1. Reported CRF1 receptor antagonists and designed
activity of 2 (R121919) in patients with depression has been
compounds 5 and 6a.
shown in a small open-label phase IIa study,21,22 while 3 (CP-
316,311) failed to show efficacy in a double-blind, placebo-
controlled study23 (Figure 1). It is tempting to speculate that
appropriate drug-like characteristics may function as a robust,
novel antidepressant in clinical practice.
the main reason for these equivocal results lies with the
compound itself (e.g., suboptimal drug-like properties such as
high lipophilicity or low water solubility and inadequate target
engagement) and not with the mechanism of action. Therefore,
we hypothesized that a CRF1 receptor antagonist with
Received: June 20, 2012
Published: September 12, 2012
© 2012 American Chemical Society
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Figure 2. Comparison of dihedral angles in the most stable conformer of 2 (left) and the designed compound 5 (right).
a
Scheme 1
a
Reagents and conditions: (a) CS2, 2-bromo-1,1-diethoxyethane, Cs2CO3, cat. NaI, DMF, 60 °C; (b) H2NNH2·H2O, EtOH, rt; (c) 5 M HCl, 1,4-
dioxane, 60 °C, 32%, 3 steps; (d) MeI, Cs2CO3, DMF, rt, 70%; (e) (i) 5 M NaOH, EtOH, 80 °C, (ii) conc HCl, 1,4-dioxane, 60 °C, 75%, 2 steps; (f)
(i) NaNO2, 5 M HCl, H2O, rt, (ii) Pd/C, EtOH, THF, rt; (g) Boc2O, Et3N, CH2Cl2, rt, 59%, 3 steps; (h) BrF2CCF2Br, n-BuLi/hexane, THF, −78
°C−rt, 67%; (i) Ar−B(OH)2, Pd(OAc)2, PPh3, K2CO3, DME, H2O, reflux, or Ar−B(OH)2, Pd(PPh3)4, Na2CO3, toluene, EtOH, H2O, reflux, 52−
95%.
a
Scheme 2
a
Reagents and conditions: (a) (i) O-mesitylene sulfonylhydroxylamine, CH2Cl2, rt, (ii) AcONa, Ac2O, reflux, 51%, 2 steps; (b) (i) NaNO2, 5 M
HCl, H2O, rt (ii) Zn, 2 M HCl, rt, (iii) Boc2O, Et3N, CH2Cl2, rt, 56%, 3 steps; (c) BrF2CCF2Br, n-BuLi/hexane, THF, −78 °C−rt, 88%; (d) Ar−
B(OH)2, Pd(PPh3)4, Na2CO3, toluene, EtOH, H2O, reflux, 99%.
As described in our previous article,24 we focused on
modifying the central core, particularly the 5,6-fused bicyclic
heteroaromatic template, with the aim of conferring appropriate
drug-like characteristics, and identified the potent CRF1
receptor antagonist 4 (Figure 1). As part of our efforts to
identify CRF1 receptor antagonists, we investigated 5,5-fused
bicyclic systems, which have not yet been explored for their
potential as CRF1 receptor antagonists. In this report, we
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a
describe the investigation of pyrazolo[5,1-b]thiazole as a novel
core structure. The dihedral angles are related to a key
pharmacophore feature of CRF1 receptor antagonists.25 For a
known CRF1 receptor antagonist 2 and some 5,5-fused
derivatives, the dihedral angles between the bicyclic core and
the pendant aryl rings in the most stable conformation were
calculated.26 The results indicated that pyrazolo[5,1-b]thiazole
5 exhibits a conformation similar to 2 (59.2° for 5 and 59.0° for
2) (Figure 2). Then, we established efficient, synthetic routes
for investigating pyrazolo[5,1-b]thiazole derivatives.
Scheme 4
a
Reagents and conditions: (a) BrF2CCF2Br, n-BuLi/hexane, THF,
−78 °C−rt; (b) Me2Zn/hexane, Pd[P(t-Bu)3]2, 1,4-dioxane, 65 °C,
49%, 2 steps.
RESULTS AND DISCUSSION
■
Chemistry. Key intermediate 16, which is amenable to
versatile modifications at the amine moiety in the 6-methoxy
pyrazolo[5,1-b]thiazole core structure, was prepared according
to Scheme 1. The one-pot reaction of diethyl malonate 7 with
carbon disulfide and bromoacetaldehyde diethyl acetal in the
presence of cesium carbonate and catalytic sodium iodide
yielded 8, which was treated with hydrazine hydrate to yield
pyrazolone 9. Intramolecular cyclization of 9 by treatment with
hydrochloric acid followed by methylation of the resulting
bicyclic compound 10 yielded 6-methoxy pyrazolo[5,1-b]-
thiazole core 11. Hydrolysis of 11 followed by decarboxylation
under acidic conditions led to the formation of 12. Subsequent
nitrosation and reduction yielded aniline 13, which was
protected to give tert-butyl carbamate 14a. Bromination with
the desired regioselectivity by using n-butyllithium followed by
Suzuki−Miyaura coupling of 15a with various arylboronic acids
produced key intermediate 16.
Pharmacology. The affinity for human CRF1 receptors was
determined on the basis of competition with 125I-CRF by using
cell membranes prepared from human CRF1 receptors
expressed in HEK293 cells.5
Initial investigation of the pyrazolo[5,1-b]thiazole core is
summarized in Table 1. The 5,5-fused compound 5, which was
Table 1. Initial Investigation of the Binding Affinity of
Pyrazolo[5,1-b]thiazole Derivatives
Scheme 2 shows the synthesis of a 6-methyl analogue. N-
Amination of 17 with O-mesitylene sulfonylhydroxylamine,
followed by cyclization in the presence of sodium acetate in
acetic anhydride, produced a 6-methyl pyrazolo[5,1-b]thiazole
core 18.27 One-pot deacetylation and nitrosation of 18,
followed by reduction and subsequent conversion to tert-butyl
carbamate, yielded 14b. Similar to the 6-methoxy intermediate
16 described in Scheme 1, 14b was converted to 19 via
formation of 15b.
a
compd
A
X
Y
binding IC50 (nM)
clogP
4
HCCH
Et
H
50
120
46
4.7
4.2
4.2
3.6
4.4
24
5
HCCH
Me
H
S
S
S
Me
H
6a
23
OMe
Me
H
69
Me
76
a
All values are the averages of two measurements.
The final compounds 6 and 5 were prepared according to
Scheme 3. Alkylation of 16 or 19 with alkyl halides or alkyl
first designed, showed affinity comparable to those of the
representative compounds 4 and 24 from our previous
compound series.24 Replacement of the 6-methyl group in 5
with the methoxy group resulted in retention of potent binding
affinity while showing a lower clogP28 value than that shown by
5. 2-Methyl compound 23 also showed binding affinity
comparable to that of 6a. Among these pyrazolo[5,1-b]thiazole
compounds, 6a, which had a lower clogP value, was considered
to be a promising starting point for further optimization.
The 6-methoxy pyrazolo[5,1-b]thiazole compound 6a
showed relatively high human intrinsic clearance in vitro
(hCLint; 0.46 mL/min/mg). Therefore, 6a was modified
mainly with a focus on improving its metabolic stability.
Because it was presumed from our previous study24 that N-
dealkylation of the 7-dialkylamine moiety was the main
metabolic pathway, modification of the dialkylamine moiety
in 6a was first investigated (Table 2). Introduction of a polar
OH group in the 4-THP moiety to reduce lipophilicity resulted
in a decrease in the activity (6b). Introduction of the F atom to
the same carbon on the THP ring to block a potential
metabolically fragile side chain maintained the binding affinity
but increased its hCLint slightly (6c). Moreover, replacement
of the 4-THP ring with the regioisomer of the THP ring (6d
and 6e), 3- or 2-THF compounds (6f and 6g), and acyclic
a
Scheme 3
a
Reagents and conditions: (a) R2-Br or R2-I, NaH, DMF, rt, or R2-
OMs, NaOH powder, DMSO, rt−70 °C; (b) (i) TFA, CH2Cl2, rt, (ii)
aldehyde or ketone, NaBH(OAc)3, THF, rt, or NaBH(OAc)3, THF,
AcOH, rt, or α-picoline-borane, MeOH, AcOH, rt, 7−88%, 3 steps,
(iii) for 6c; DAST, CH2Cl2, 0 °C, 14%.
mesylates under basic conditions followed by removal of the
Boc group under acidic conditions and subsequent reductive
amination yielded the desired products 6 and 5.
2,6-Dimethyl-substituted pyrazolo[5,1-b]thiazole analogue
23 was prepared by bromination of 5, followed by palladium-
catalyzed alkylation of 22 with dimethylzinc (Scheme 4).
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Table 2. Effects of Dialkylamino Side Chain at 7-Position of Pyrazolo[5,1-b]thiazole Derivatives
a
b
All values are the averages of two measurements. . Rate of human intrinsic clearance in vitro. Each value represents the mean SEM of six
c
d
measurements. Kinetic solubility in phosphate buffer saline using 10 mM DMSO stock solution. NT = not tested.
methoxyethyl ether (6h) did not improve hCLint. Because the
replacement of the 4-THP ring in 6a with the above cyclic or
acyclic ethers did not result in improvement of hCLint, we
speculate that the methylene moiety with the attached nitrogen
might be susceptible to metabolism regardless of the size of the
ether ring or the position of the oxygen atom. With the aim of
blocking the metabolically fragile carbon by steric hindrance,
the directly linked THP compound (6i) was synthesized,
resulting in a significant improvement in metabolic stability.
Reduction of lipophilicity could also improve metabolic
stability. Although the compound showed a slight decrease in
binding affinity, the excellent drug-like property of 6i prompted
further optimization.
Table 3. Effects of the 7-Dialkylamino Side Chain of
Pyrazolo[5,1-b]thiazole Derivatives
binding IC50
hCLint
(mL/min/mg)
solubility
c
a
b
compd
R
(nM)
(μM)
clogP
Modification of another substituent at 7-position of 6i was
examined with the aim of enhancing binding affinity (Table 3).
Ethyl 6j and the i-propyl compound 6n showed an almost 1.5-
fold decrease in the binding affinity against 6i, and n-propyl 6k
and i-butyl compound 6o showed binding affinity comparable
to that of 6i. Meanwhile, cyclopropylethyl 6l and n-butyl
compound 6m improved activity while maintaining preferable
hCLint and solubility,29 although their lipophilicity increased.
The compounds with branched alkyl groups (6n, 6o) showed
relatively high hCLint, contrary to our expectations that N-
dealkylation might be prevented by steric bulkiness. It is
speculated that blocking dealkylation of the aniline moiety by
6i
cPrCH2
Et
101
147
98
0.18 0.01
0.20 0.01
0.22 0.01
0.18 0.03
0.20 0.01
0.67 0.03
0.36 0.02
30
38
25
12
13
53
0
3.0
2.5
3.1
3.5
3.6
2.9
3.5
6j
6k
61
6m
6n
6o
n-Pr
cPrCH2CH2
n-Bu
83
52
i-Pr
136
91
i-Bu
a
b
All values are the averages of two measurements. Rate of human
intrinsic clearance in vitro. Each value represents the mean SEM of
six measurements. Kinetic solubility in phosphate buffer saline using
10 mM DMSO stock solution.
c
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Table 4. Optimization of Pyrazolo[5,1-b]thiazole Derivatives
a
b
c
compd
R1
R2
R3
binding IC50 (nM)
hCLint (mL/min/mg)
solubility (μM)
clogP
6k
6p
6q
6r
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Bu
n-Bu
n-Bu
MeO
MeO
MeO
Cl
MeOCH2
EtOCH2
CN
98
65
0.22 0.01
0.34 0.02
0.15 0.02
0.37 0.02
25
3.1
3.5
3.0
4.1
3.6
3.6
4.0
3.5
21
126
63
16
MeOCH2
MeOCH2
MeOCH2
EtOCH2
6.3
7.9
13
d
6s
H
254
52
NT
6m
6t
MeO
MeO
MeO
0.20 0.01
0.22 0.01
0.15 0.01
70
7.1
3.9
6u
CN
46
a
b
All values are the averages of two measurements. Rate of human intrinsic clearance in vitro. Each value represents the mean SEM of six
c
d
measurements. Kinetic solubility in phosphate buffer saline using 10 mM DMSO stock solution. NT = not tested.
introducing a branched alkyl group might induce a new
metabolically fragile site on the branched alkyl substituent itself.
Finally, the substituent effect on the 3-phenyl ring was
examined using 6k and 6m (Table 4). Replacement of para
methoxymethyl on the 3-phenyl ring in 6k with an
ethoxymethyl moiety improved the binding affinity while
maintaining solubility (6p). Formation of the nitrile compound
6q, which was aimed at avoiding demethylation of the para
methoxymethyl moiety in 6k, ameliorated hCLint as expected
but decreased the binding affinity. Similar to 6p, ortho chloro
compound 6r increased the in vitro activity but decreased
hCLint. The decrease in the binding affinity on 6s indicates that
an appropriate conformation induced by the ortho disubstituent
might be important for high affinity in this series. Subsequently,
the preferable aryl group was introduced in 6m, similar to the
modification used for 6k. The compounds 6t and 6u showed
comparable binding affinity to 6m with preferable hCLint
values.
conscious rats. Compounds (10 mg/kg, po) were orally
administered to rats 60 min before IV injection of CRF (10
μg/kg). Stool weight was measured for 4 h after CRF injection.
The compounds 6r and 6t significantly inhibited CRF-induced
defecation.
These two compounds were subjected to various in vitro
assays in order to evaluate drug-likeness such as CYP inhibition,
CYP induction, P-gp substratability, and hERG inhibition.
There were few differences between these compounds, and
they did not have major problems (data not shown). On the
basis of the results, especially those related to hCLint,
compound 6t was chosen as the most promising candidate.
The functional antagonism of 6t was confirmed in a cAMP
assay by using the human neuroblastoma cell line IMR-32
expressing human CRF1 receptors. The IC50 value of 6t was 7.1
nM.32 Compound 2, which was used as a positive control, had
an IC50 value of 4.0 nM in the assay. Compound 6t exhibited
selectivity over CRF2 receptors (CRF2 IC50, >10 μM).33
The effects of 6t on the CRF-induced elevation of plasma
ACTH concentration were evaluated in F344 rats34 to confirm
oral antagonism for the CRF1 receptor (Figure 3).35 Plasma
concentration of ACTH was significantly increased on
The compounds selected through the above-mentioned
process were evaluated in a rat defecation model,30 which is a
possible IBS model, in order to confirm the in vivo antagonism
of the CRF1 receptor31 (Table 5). It is well-known that
exogenously administered CRF increases fecal pellet output in
Table 5. Effects on IV Injected CRF-Induced Fecal Pellet
Output in Ratsa
stool output (g)
inhibition of
stool output (%)
b
compd
CRF control
compound-treated
6m
6p
6r
1.48 0.30
0.95 0.19
1.65 0.20
1.15 0.20
1.15 0.20
0.91 0.24
0.44 0.22
39
54
58
65
36
c
0.70 0.31*
0.40 0.15*
0.73 0.20
c
6t
6u
a
Compounds (10 mg/kg) were orally administered 60 min before
Figure 3. Effects of 6t on the CRF-induced increase of plasma ACTH
concentration in rats with 2 as a positive control. Each value represents
the mean SEM of 8 rats. *p < 0.05 vs vehicle (CRF control) (one-
way analysis of variance, followed by Dunnett’s multiple comparison
test).
b
intravenous injection of CRF (10 |ag/kg). Inhibition ratio (%) of
stool output (g) of compounds treated groups compared to output (g)
of CRF control groups. Each value represents the mean SEM of 6
c
rats. *P < 0.05 vs CRF control groups (unpaired t test).
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subcutaneous injection of CRF (10 μg/kg). Further, 6t (30
mg/kg, po) significantly inhibited the CRF-induced increase in
plasma ACTH concentration at 30 min after CRF injection,
suggesting that 6t has an antagonistic effect on the CRF1
receptor in vivo.
The anxiolytic efficacy of 6t was investigated by performing a
light/dark test in male BALB/c mice, which is commonly used
to evaluate anxiolytics (Figure 4).36 The compound increased
CONCLUSIONS
■
Investigation of a CRF1 receptor antagonist with a 5,5-fused
ring core structure resulted in identification of novel pyrazolo-
[5,1-b]thiazole compounds as potent CRF1 receptor antago-
nists with physicochemical characteristics preferable for PO
administration; further, we established a synthetic route that
was adequate for derivatization and enabled efficient
optimization. The most promising compound, 6t, demon-
strated high affinity (IC50 = 70 nM) and functional antagonism
(IC50 = 7.1 nM) for the human CRF1 receptor; it significantly
reduced CRF-induced elevation of ACTH levels at 30 mg/kg
(po) and exhibited anxiolytic activity in the light/dark test in
mice at 30 mg/kg (po). These results not only exhibited a
promising profile of pyrazolo[5,1-b]thiazole derivatives as CRF1
receptor antagonists but could also expand the possibility of
discovering structurally diverse antagonists. The phosphate of
6t (E2009) was selected as a candidate for further investigation
to prove the clinical usefulness of CRF1 receptor antagonists in
the treatment of stress-related disorders such as depression and
anxiety.
EXPERIMENTAL SECTION
Figure 4. Anxiolytic effects of 6t in mice light/dark test with 2 as a
positive control. Each value represents the mean SEM of 12 mice.
*p < 0.05 vs vehicle (Dunnett’s multiple comparison test).
■
1
Chemistry. H NMR spectra were recorded on a Bruker Avance
spectrometer (600 MHz) or Varian Mercury 400 spectrometer (400
MHz). 13C NMR spectra were recorded on a Bruker Avance
spectrometer (150 MHz) or JEOL JNM α400 spectrometer (100
MHz). Chemical shifts were calculated in ppm (δ) from the residual
CHCl3 signal at (δH) 7.26 ppm and (δC) 77.0 ppm in CDCl3 or the
residual C5HD4N signal at (δH) 8.71 ppm and (δC) 123.5 ppm in
C5D5N, or the residual CD3SOCD2H signal at (δH) 2.49 ppm and
(δC) 40.0 ppm in CD3SOCD3. High resolution mass spectra (HRMS)
were recorded on a ThermoFisherScientific LTQ-Orbitrap XL
spectrometer (using electrospray ionization). Compounds were
purified by column chromatography on silica gel using the solvent
systems indicated below, or by preparative HPLC separations by
Waters system equipped with a Shiseido CAPCELL PAK C18 ACR
(20 mm × 50 mm, 5 μm), eluting with a linear gradient of 10−90%
MeOH in water containing 0.1% TFA at a flow rate of 30 mL/min
over 10 min and monitored using a photodiodo array detector.
The purity of the biological tested compounds was determined by
an analytical HPLC method and was found to be greater than or equal
to 95% for all compounds. The parameters of the HPLC method were
as follows: Accucore RP-MS column (2.1 mm × 50 mm, 2.6 μm);
mobile phase: A = H2O with 0.1% HCO2H, B = acetonitrile with 0.1%
HCO2H, 0−1 min, 0% B; 1−4 min, 0% B → 100% B; 4−8 min, 100%
B; 8−11 min, 0% B; flow rate =0.4 mL/min; detector: UV 254 nm;
run time = 11 min.
the time spent in the light box in a dose-dependent manner,
and a statistically significant effect was observed at 30 mg/kg
po, suggesting that 6t may have the potential to alleviate anxiety
in patients.
The pharmacokinetic properties of 6t were evaluated in male
SD rats after iv and oral (po) administration at doses of 3 mg/
kg iv and 10 mg/kg po (n = 3), respectively (Table 6). The
Table 6. Pharmacokinetic Parameters for 6t (iv and po) in
a
Male Rats
iv (3 mg/kg)
CL (L/h/kg)
po (10 mg/kg)
1.93 0.16
6.32 1.23
1.58 0.15
5.2 1.3
Cmax (μg/mL)
0.54 0.11
0.5−1.0
Vdss (L/kg)
AUC (μg/mL·h)
T1/2 (h)
Tmax (h)
AUC (μg/mL·h)
BA (%)
1.24 0.36
23
a
Each value represents the mean SEM of three animals.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methyl-N-(tetrahydro-2H-pyran-4-
ylmethyl)pyrazolo[5,1-b][1,3]thiazol-7-amine (5). Compound 5
was prepared according to the procedure described for the synthesis of
6a using 19. The product was purified by column chromatography on
silica gel (n-heptane:EtOAc = 1:4), followed by column chromatog-
raphy on NH silica gel (n-heptane:EtOAc = 1:1) to afford 5 (57%, 3
steps) as a white solid. 1H NMR (400 MHz, CDCl3) δ 0.01−0.05 (m,
2H), 0.38−0.44 (m, 2H), 0.81−0.92 (m, 1H), 1.22−1.34 (m, 2H),
1.49−1.61 (m, 1H), 1.71−1.78 (m, 2H), 2.28 (s, 3H), 2.74 (d, J = 6.8
Hz, 2H), 2.88 (d, J = 7.2 Hz, 2H), 3.36 (br dd, J = 10.4, 11.6 Hz, 2H),
3.45 (s, 3H), 3.76 (s, 6H), 3.93 (br dd, J = 2.8, 11.2 Hz, 2H), 6.52 (s,
1H), 6.64 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 159.5, 150.0,
142.4, 131.9, 128.3, 123.4, 108.8, 105.6, 103.3, 74.7, 67.9, 61.9, 60.8,
58.4, 56.1, 33.8, 31.5, 12.4, 9.7, 3.6. HRMS calcd for (C26H36N3O4S)
[M + H]+ 486.2421; found 486.2422. HPLC purity: > 99%.
results indicated a half-life of 5.2 h, plasma clearance of 1.9 L/
h/kg, and an oral bioavailability of 23%. The hepatic clearances
calculated from in vitro intrinsic clearance measured by
performing a liver microsome assay were almost comparable
to the in vivo total clearances in preclinical studies in other
animals, including rat, mouse, dog, and monkey. These results
indicated that the main elimination route of 6t in animals is
probably hepatic metabolism. Therefore, hepatic metabolism
was considered the main elimination route in humans.
Screening for salt and crystal forms of 6t by using various
acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,
methanesulfonic acid, or phosphoric acid revealed that
diphosphate 24 was the most suitable crystalline form. The
apparent solubility of 24 in Fasted State Simulated Intestinal
Fluid was 50 times higher than that of the free base, suggesting
that the salt form is better for human absorption.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-[(tetrahydro-2H-
pyran-4-yl)methyl]pyrazolo[5,1-b][1,3]thiazol-7-amine (6a). To
a solution of 16a (100 mg, 0.22 mmol) in DMF (2.5 mL) was added
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NaH (60% dispersion in oil: 12 mg, 0.30 mmol) while cooling on a
water bath. After stirring for 10 min, cyclopropylmethyl bromide (26
μL, 0.27 mmol) was added, and the mixture was stirred for 1 h. Water
was added to the reaction mixture while cooling on an ice bath, and
the residue was extracted with EtOAc. The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo to afford a crude
product, which was used in next step without purification.
161.3, 159.5, 142.0, 134.2, 128.3, 111.6, 105.9, 105.6, 103.4, 94.6 (d, J
= 172.8 Hz), 74.8, 63.7 (d, J = 1.3 Hz), 62.5 (d, J = 23.0 Hz), 61.9,
58.5, 56.2, 56.1, 33.6 (d, J = 21.3 Hz), 10.1, 3.4. HRMS calcd for
(C26H35FN3O5S) [M + H]+ 520.2276; found 520.2272. HPLC purity:
> 99%.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-[(tetrahydro-2H-
pyran-3-yl)methyl)pyrazolo[5,1-b][1,3]thiazol-7-amine (6d).
Compound 6d was prepared according to the procedure described
for the synthesis of 6b using 16a. The product was purified by HPLC
to give 6d (7.5 mg, 7%, 3 steps) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 0.00−0.06 (m, 2H), 0.35−0.45 (m, 2H), 0.83−0.95 (m,
1H), 1.48−1.68 (m, 2H), 1.68−1.81 (m, 2H), 1.81−1.92 (m, 1H),
2.76 (dd, J = 6.4, 13.6 Hz, 1H), 2.81 (m, 2H), 2.93 (dd, J = 6.4, 12.4
Hz, 1H), 3.17 (dd, J = 9.6, 11.6 Hz, 1H), 3.35−3.43 (m, 1H), 3.47 (s,
3H), 3.79 (s, 6H), 3.80−3.86 (m, 1H), 3.86 (s, 3H), 4.01−4.07 (m,
1H), 4.50 (s, 2H), 6.39 (s, 1H), 6.64 (s, 2H). 13C NMR (150 MHz,
CDCl3) δ 161.7, 159.5, 142.0, 133.5, 128.2, 110.0, 105.9, 105.8, 103.4,
74.8, 72.5, 68.7, 61.3, 58.5, 56.7, 56.4, 56.1, 34.8, 28.0, 25.5, 9.5, 3.5,
3.5. HRMS calcd for (C26H35N3O5S) [M + H]+ 502.2370; found
502.2372. HPLC purity: 96.0%.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-[(tetrahydro-2H-
pyran-2-yl)methyl)pyrazolo[5,1-b][1,3]thiazol-7-amine (6e). To
a solution of 16a (90 mg, 0.20 mmol) and (tetrahydro-2H-pyran-2-
yl)methyl methanesulfonate (51 mg, 0.26 mmol) in DMSO (3 mL)
was added NaOH (10 mg, 0.26 mmol) at room temperature, and the
mixture was stirred at 70 °C for 2 h. Water was added to the reaction
mixture under ice cooling, and the residue was extracted with EtOAc.
The organic layer was dried over MgSO4, filtered, and concentrated in
vacuo to afford a crude product, which was used in next step without
purification.
To the obtained crude product was added CH2Cl2 (3 mL), followed
by TFA (1 mL), and the mixture was stirred at room temperature for 1
h. The reaction mixute was concentrated under reduced pressure.
To a solution of the obtained residue in THF (2 mL) were added
tetrahydro-2H-pyran-4-carbaldehyde (51 mg, 0.45 mmol), followed by
NaBH(OAc)3 (94 mg, 0.45 mmol), and the mixture was stirred at
room temperature for 1 h. A saturated aqueous solution of NaHCO3
was added to the reaction mixture, and the residue was extracted with
EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (n-heptane:EtOAc = 3:2) to afford 6a
(90 mg, 81%, 3 steps) as a white solid. 1H NMR (600 MHz, CDCl3) δ
0.33−0.48 (m, 2H), 0.85−0.98 (m, 1H), 1.22−1.38 (m, 2H), 1.55−
1.69 (m, 1H), 1.72−1.83 (m, 2H), 2.78−2.85 (m, 2H), 2.87−2.94 (m,
2H), 3.30−3.39 (m, 2H), 3.48 (s, 3H), 3.80 (s, 6H), 3.88 (s, 3H),
3.91−3.98 (m, 2H), 4.52 (s, 2H), 6.42 (s, 1H), 6.66 (s, 2H). 13C NMR
(150 MHz, CDCl3) δ 161.7, 159.4, 142.0, 133.4, 128.2, 110.3, 105.9,
105.7, 103.4, 74.8, 68.0, 61.4, 60.5, 58.5, 56.3, 56.1, 33.9, 31.5, 9.6, 3.4.
HRMS calcd for (C26H35N3O5S) [M + H]+ 502.2370; found 502.2379.
HPLC purity: 98.5%.
4 - { [ ( C y c l o p r o p y l m e t h y l ) { 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxypyrazolo[5,1-b][1,3]-
thiazol-7-yl}amino]methyl}tetrahydro-2H-pyran-4-ol (6b). To a
solution of 16a (100 mg, 0.22 mmol) in DMF (6 mL) was added NaH
(60% dispersion in oil: 11.6 mg, 0.29 mmol) at room temperature.
After stirring for 30 min, cyclopropylmethyl bromide (28 μL, 0.29
mmol) was added, and the mixture was stirred for 1 h. Water was
added to the reaction mixture while cooling on ice, and the residue was
extracted with EtOAc. The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo to afford a crude product, which
was used in next step without purification.
To the obtained crude product was added CH2Cl2 (2 mL), followed
by TFA (1 mL), and the mixture was stirred at room temperature for 1
h. The reaction mixute was concentrated under reduced pressure.
To a solution of the obtained residue in THF (10 mL) and AcOH
(1 mL) was added cyclopropanecarboxaldehyde (30 μL, 0.40 mmol),
followed by NaBH(OAc)3 (85 mg, 0.40 mmol), and the mixture was
stirred at room temperature for 2 h. A saturated aqueous solution of
NaHCO3 was added to the reaction mixture, and the residue was
extracted with EtOAc. The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (n-heptane:EtOAc = 3:2) to
To the obtained crude product was added CH2Cl2 (5 mL), followed
by TFA (1 mL), and the mixture was stirred at room temperature
overnight. The reaction mixute was concentrated under reduced
pressure.
To a solution of the obtained residue in MeOH (8 mL) and AcOH
(1 mL) were added 4-hydroxytetrahydro-2H-pyran-4-carbaldehyde
(289 mg, 2.2 mmol), followed by α-picolineborane (238 mg, 2.2
mmol), and the mixture was stirred at room temperature for 14 h. An
aqueous 5 M NaOH was added and the mixture was extracted with
EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (n-heptane:EtOAc = 1:2) to afford 6b
(70 mg, 61%, 3 steps) as a white solid. 1H NMR (400 MHz, CDCl3) δ
0.02−0.10 (m, 2H), 0.38−0.48 (m, 1H), 0.84−0.98 (m, 1H), 1.38−
1.62 (m, 4H), 2.84 (d, J = 6.8 Hz, 2H), 3.02 (s, 2H), 3.47 (s, 3H), 3.77
(s, 6H), 3.64−3.83 (m, 4H), 3.88 (s, 3H), 4.50 (s, 2H), 6.44 (s, 1H),
6.64 (s, 2H). HRMS calcd for (C26H36N3O6S) [M + H]+ 518.2319;
found 518.2314. HPLC purity: > 99%.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-N-[(4-fluorotetrahydro-2H-pyran-4-
yl)methyl]-6-methoxypyrazolo[5,1-b][1,3]thiazol-7-amine (6c).
To a solution of 6b (65 mg, 0.12 mmol) in CH2Cl2 (4 mL) was added
diethylaminosulfur trifluoride (25 μL, 0.19 mmol) at 0 °C. After
stirring for 5 min, a saturated aqueous solution of NaHCO3 was added
to the reaction mixture, and the residue was extracted with EtOAc.
The organic layer was dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by HPLC to afford 6c (8.9 mg, 14%)
as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.01−0.10 (m, 2H),
0.36−0.44 (m, 2H), 0.83−0.98 (m, 1H), 2.16−2.25 (m, 2H), 2.80 (d, J
= 6.8 Hz, 2H), 3.47 (s, 3H), 3.59 (s, 2H), 3.74 (t, J = 5.6 Hz, 2H), 3.77
(s, 6H), 3.87 (s, 3H), 4.04−4.10 (m, 2H), 4.50 (s, 2H), 5.58−5.64
(m,1H), 6.40 (s, 1H), 6.64 (s, 2H). 13C NMR (150 MHz, CDCl3) δ
1
afford 6e (41 mg, 41%, 3 steps) as a pale-yellow solid. H NMR (400
MHz, CDCl3) δ 0.02−0.08 (m, 2H), 0.34−0.42 (m, 2H), 0.86−0.94
(m, 1H), 1.38−1.49 (m, 1H), 1.49−1.61 (m, 4H), 1.70−1.83 (m, 1H),
2.81−2.93 (m, 2H), 2.95 (dd, J = 5.6, 12.8 Hz, 1H), 3.17 (dd, J = 6.4,
12.8 Hz, 1H), 3.29−3.37 (m, 1H), 3.38 (dt, J = 2.4, 11.6 Hz, 1H), 3.47
(s, 3H), 3.79 (s, 3H), 3.87 (s, 6H), 3.92−4.00 (m, 1H), 4.50 (s, 2H),
6.41 (s, 1H), 6.65(s, 2H). 13C NMR (150 MHz, CDCl3) δ 161.6,
159.5, 142.0, 133.6, 128.1, 110.3, 106.0, 105.8, 103.4, 76.4, 74.8, 68.4,
61.1, 59.9, 58.5, 56.3, 56.1, 30.2, 26.3, 23.4, 9.7, 3.6, 3.2. HRMS calcd
for (C26H35N3O5S) [M + H]+ 502.2370; found 502.2372. HPLC
purity: 98.3%.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-[(tetrahydrofuran-3-
yl)methyl]pyrazolo[5,1-b][1,3]thiazol-7-amine (6f). Compound
6f was prepared according to the procedure described for the synthesis
of 6b using 16a. The product was purified by column chromatography
on silica gel (n-heptane:EtOAc = 1:1) to give 6f (83%, 3 steps) as a
white solid. 1H NMR (400 MHz, CDCl3) δ 0.02−0.10 (m, 2H), 0.36−
0.46 (m, 2H), 0.84−0.98 (m, 1H), 1.56−1.71 (m, 1H), 1.90−2.02 (m,
1H), 2.26−2.42 (m, 1H), 2.81 (d, J = 6.4 Hz, 2H), 2.95 (dd, J = 8.4,
12.0 Hz, 1H), 3.06 (dd, J = 6.8, 12.0 Hz, 1H), 3.47 (s, 3H), 3.56 (dd, J
= 6.0, 8.4 Hz, 1H), 3.64−3.74 (m, 1H), 3.78 (s, 6H), 3.75−3.90 (m,
2H), 3.87 (s, 3H), 4.50 (s, 2H), 6.41 (s, 1H), 6.64 (s, 2H). 13C NMR
(150 MHz, CDCl3) δ 161.9, 159.4, 142.0, 133.9, 128.3, 109.6, 105.8,
105.7, 103.4, 74.8, 72.4, 67.7, 61.2, 58.5, 57.5, 56.3, 56.1, 38.4, 30.6,
9.7, 3.5, 3.5. HRMS calcd for (C25H33N3O5S) [M + H]+ 488.2214;
found 488.2223. HPLC purity: > 99%.
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N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-[(tetrahydrofuran-2-
yl)methyl]pyrazolo[5,1-b][1,3]thiazol-7-amine (6g). Compound
6g was prepared according to the procedure described for the
synthesis of 6a using 16a. The product was purified by column
chromatography on silica gel (n-heptane:EtOAc = 3:2) to give 6g
13C NMR (150 MHz, CDCl3) δ 163.1, 159.5, 142.1, 134.8, 128.3,
106.1, 105.8, 105.8, 103.3, 74.8, 67.5, 60.4, 58.5, 56.3, 56.1, 44.9, 31.1,
13.6. HRMS calcd for (C23H31N3O5S) [M + H]+ 462.2057; found
462.2058. HPLC purity: > 99%.
3-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-6-methoxy-N-
propyl-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazole-7-amine (6k). Compound 6k was prepared according to the
procedure described for the synthesis of 6b using 16a. The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:1) to give 6k (68%, 3 steps) as a white solid.
1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 7.2 Hz, 3H), 1.32−1.45
(m, 2H), 1.53−1.66 (m, 2H), 1.78−1.88 (m, 2H), 2.90−2.99 (m, 2H),
3.00−3.12 (m, 1H), 3.38 (td, J = 2.0, 12.0 Hz, 2H), 3.47 (s, 3H), 3.79
(s, 6H), 3.86 (s, 3H), 3.94−4.03 (m, 2H), 4.50 (s, 2H), 6.42 (s, 1H),
6.65 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 162.9, 159.5, 142.0,
135.0, 128.2, 106.6, 105.8, 105.7, 103.3, 74.8, 67.5, 60.7, 58.5, 56.3,
56.1, 52.8, 31.1, 21.8, 11.7. HRMS calcd for (C24H33N3O5S) [M + H]+
476.2214; found 476.2209. HPLC purity: > 99%.
1
(64%, 3 steps) as a pale-brown solid. H NMR (400 MHz, CDCl3) δ
0.02−0.10 (m, 2H), 0.33−0.45 (m, 2H), 0.85−0.94 (m, 1H), 1.56−
1.68 (m, 1H), 1.74−1.92 (m, 2H), 1.89−2.01 (m, 1H), 2.85 (dd, J =
6.8, 13.3 Hz, 1H), 2.92 (dd, J = 6.4, 13.3 Hz, 1H), 3.00 (dd, J = 6.8,
12.8 Hz, 1H), 3.25 (dd, J = 6.0, 12.8 Hz, 1H), 3.46 (s, 3H), 3.68−3.74
(m, 1H), 3.78 (s, 6H), 3.81−3.87 (m, 1H), 3.87 (s, 3H), 3.89−3.97
(m, 1H), 4.50 (s, 2H), 6.40 (s, 1H), 6.64 (s, 2H). 13C NMR (150
MHz, CDCl3) δ 161.6, 159.5, 142.0, 133.6, 128.2, 110.1, 105.9, 105.8,
103.4, 77.8, 74.8, 67.9, 61.1, 58.7, 58.5, 56.3, 56.1, 30.0, 25.5, 9.6, 3.6,
3.2. HRMS calcd for (C25H33N3O5S) [M + H]+ 488.2214; found
488.2210. HPLC purity: > 99%.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-(2-methoxyethyl)-
pyrazolo[5,1-b][1,3]thiazol-7-amine (6h). To a solution of 16a
(100 mg, 0.22 mmol) and 2-bromoethyl methyl ether (27 μL, 0.29
mmol) in DMF (2 mL) was added NaH (60% dispersion in oil: 14
mg, 0.29 mmol) at room temperature, and then the mixture was stirred
for 4.5 h. Water was added to the reaction mixture under ice cooling,
and the residue was extracted with Et2O. The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo to afford a crude
product, which was used in next step without purification.
To the obtained crude product was added CH2Cl2 (5 mL), followed
by TFA (2 mL), and the mixture was stirred at room temperature for 1
h. The reaction mixute was concentrated under reduced pressure.
To a solution of the obtained residue in THF (10 mL) and AcOH
(1 mL) were added cyclopropanecarbaldehyde (33 μL, 0.44 mmol),
followed by NaBH(OAc)3 (94 mg, 0.44 mmol), and the mixture was
stirred at room temperature for 1 h. A saturated aqueous solution of
NaHCO3 was added to the reaction mixture, and the residue was
extracted with EtOAc. The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (n-heptane:EtOAc = 3:2) to
afford 6h (77 mg, 75%, 3 steps) as a yellow solid. 1H NMR (400 MHz,
CDCl3) δ 0.03−0.10 (m, 2H), 0.36−0.45 (m, 2H), 0.89−0.98 (m,
1H), 2.87 (d, J = 6.8 Hz, 2H), 3.25 (t, J = 6.4 Hz, 2H), 3.33 (s, 3H),
3.41−3.50 (m, 5H), 3.78 (s, 6H), 3.87 (s, 3H), 4.50 (s, 2H), 6.41 (s,
1H), 6.65 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 161.6, 159.5,
142.0, 133.3, 128.2, 109.7, 105.9, 105.8, 103.4, 74.8, 71.3, 60.9, 58.9,
58.5, 56.3, 56.1, 53.6, 9.5, 3.4. HRMS calcd for (C23H31N3O5S) [M +
H]+ 462.2057; found 462.2065. HPLC purity: 96.3%.
N-(2-Cyclopropylethyl)-3-[4-(ethoxymethyl)-2,6-dimethoxy-
phenyl]-6-methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-
b][1,3]thiazole-7-amine (6l). Compound 6l was prepared according
to the procedure described for the synthesis of 6e using 16a. The
product was purified by column chromatography on silica gel (n-
1
heptane:EtOAc = 3:2) to give 6l (83%, 3 steps) as a colorless oil. H
NMR (400 MHz, CDCl3) δ −0.03−0.04 (m, 2H), 0.34−0.43 (m, 2H),
0.63−0.74 (m, 1H), 1.20−1.37 (m, 2H), 1.54−1.69 (m, 2H), 1.86 (d, J
= 12.0 Hz, 2H), 3.02−3.15 (m, 3H), 3.40 (t, J = 12.0 Hz, 2H), 3.49 (s,
3H), 3.81 (s, 6H), 3.87 (s, 3H), 3.96−4.04 (m, 2H), 4.52 (s, 2H), 6.44
(s, 1H), 6.67 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 162.9, 159.5,
142.0, 134.9, 128.2, 106.6, 105.8, 105.8, 103.3, 74.8, 67.6, 61.0, 58.5,
56.3, 56.1, 50.8, 33.9, 31.0, 8.9, 4.2. HRMS calcd for (C26H35N3O5S)
[M + H]+ 502.2370; found 502.2373. HPLC purity: 98.6%.
N-Butyl-3-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-6-me-
thoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazole-7-amine (6m). Compound 6m was prepared according to
the procedure described for the synthesis of 6b using 16a. The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 2:3) to give 6m (80%, 3 steps) as a white solid.
1H NMR (400 MHz, CDCl3) δ 0.86 (t, J = 7.2 Hz, 3H), 1.22−1.42
(m, 4H), 1.56−1.68 (m, 2H), 1.77−1.88 (m, 2H), 2.97 (t, J = 7.2 Hz,
2H), 3.00−3.11 (m, 1H), 3.33 (td, J = 1.6, 11.6 Hz, 2H), 3.47 (s, 3H),
3.79 (s, 6H), 3.86 (s, 3H), 3.92−4.03 (m, 2H), 4.50(s, 2H), 6.42 (s,
1H), 6.65(s, 2H). 13C NMR (150 MHz, CDCl3) δ 163.0, 159.5, 142.0,
135.0, 128.2, 106.7, 105.8, 105.8, 103.3, 74.8, 67.5, 60.7, 58.5, 56.3,
56.1, 50.6, 31.1, 30.7, 20.4, 14.0. HRMS calcd for (C25H35N3O5S) [M
+ H]+ 490.2370; found 490.2363. HPLC purity: > 99%.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-6-methoxy-N-(tetrahydro-2H-pyran-
4-yl)pyrazolo[5,1-b][1,3]thiazole-7-amine (6i). Compound 6i was
prepared according to the procedure described for the synthesis of 6b
using 16a. The product was purified by column chromatography on
silica gel (n-heptane:EtOAc = 1:2) to give 6i (77%, 3 steps) as a white
solid. 1H NMR (400 MHz, CDCl3) δ −0.02−0.06 (m, 2H), 0.29−0.40
(m, 2H), 0.78−0.92 (m, 1H), 1.50−1.66 (m, 2H), 1.78−1.88 (m, 2H),
2.88 (d, J = 6.8 Hz, 2H), 3.10−3.22 (m, 1H), 3.39 (td, J = 1.6, 11.6 Hz,
2H), 3.47 (s, 3H), 3.79 (s, 6H), 3.87 (s, 3H), 3.92−4.03 (m, 2H), 4.50
(s, 2H), 6.41 (s, 1H), 6.65 (s, 2H). 13C NMR (150 MHz, CDCl3) δ
162.9, 159.5, 142.0, 135.5, 128.3, 107.3, 105.9, 105.6, 103.4, 74.8, 67.5,
59.6, 58.5, 56.3, 56.1, 55.8, 31.3, 10.2, 3.4. HRMS calcd for
(C25H33N3O5S) [M + H]+ 488.2214; found 488.2216. HPLC purity:
98.3%.
3-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N-isopropyl-
6-methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazol-7-amine (6n). Compound 6n was prepared according to the
procedure described for the synthesis of 6a using 16a. The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 50:1) to give 6n (59%, 3 steps) as a pale-yellow
solid. 1H NMR (400 MHz, CDCl3) δ 1.30 (d, J = 6.0 Hz, 6H), 1.80−
1.98 (m, 4H), 3.39−3.47 (m, 2H), 3.48 (s, 3H), 3.78 (s, 6H), 3.90 (s,
3H), 3.97−4.03 (m, 1H), 3.99−4.07 (m, 2H), 4.11−4.21 (m, 1H),
4.51 (s, 2H), 6.58 (s, 1H), 6.65 (s, 2H). HRMS calcd for
(C24H33N3O5S) [M + H]+ 476.2214; found 476.2213. HPLC purity:
> 98.0%.
3-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N-isobutyl-6-
methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b]thiazol-
7-amine (6o). Compound 6o was prepared according to the
procedure described for the synthesis of 6b using 16a. The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:1) to give 6o (88%, 3 steps) as a beige solid.
1H NMR (400 MHz, CDCl3) δ 0.88 (d, J = 6.8 Hz, 3H), 1.44−1.67
3-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N-ethyl-6-me-
thoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazol-7-amine (6j). Compound 6j was prepared according to the
procedure described for the synthesis of 6b using 16a. The product
was purified by column chromatography on silica gel (n-
1
(m, 3H), 1.75−1.86 (m, 2H), 2.74 (d, J = 7.2 Hz, 2H), 2.90−3.06 (m,
1H), 3.36 (td, J = 2.0, 12.0 Hz, 2H), 3.47 (s, 3H), 3.79 (s, 6H), 3.85 (s,
3H), 3.92−4.02 (m, 2H), 4.50 (s, 2H), 6.41 (s, 1H), 6.64 (s, 2H). 13C
NMR (150 MHz, CDCl3) δ 162.9, 159.5, 142.0, 135.6, 128.2, 107.1,
105.9, 105.7, 103.3, 74.8, 67.7, 61.2, 58.8, 58.5, 56.3, 56.1, 31.2, 27.3,
heptane:EtOAc = 1:1) to give 6j (59%, 3 steps) as a white solid. H
NMR (400 MHz, CDCl3) δ 0.99 (t, J = 7.2 Hz, 3H), 1.53−1.67 (m,
2H), 1.78−1.88 (m, 2H), 3.00−3.14 (m, 3H, involving a quartet at
3.05, J = 7.2 Hz), 3.32−3.45 (m, 2H), 3.47 (s, 3H), 3.79 (s, 6H), 3.86
(s, 3H), 3.94−4.03 (m, 2H), 4.51 (s, 2H), 6.42 (s, 1H), 6.65 (s, 2H).
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20.6. HRMS calcd for (C25H35N3O5S) [M + H]+ 490.2370; found
490.2369. HPLC purity: > 99%.
(m, 2H), 4.55 (s, 2H), 6.41 (s, 1H), 6.66 (s, 2H). 13C NMR (150
MHz, CDCl3) δ 163.0, 159.5, 142.4, 135.0, 128.3, 106.7, 105.8, 105.7,
103.4, 72.9, 67.5, 66.1, 60.7, 56.3, 56.1, 50.6, 31.1, 30.7, 20.4, 15.3,
14.0. HRMS calcd for (C26H37N3O5S) [M + H]+ 504.2527; found
504.2519. HPLC purity: > 99%.
4-{7-[Butyl(tetrahydro-2H-pyran-4-yl)amino]-6-
methoxypyrazolo[5,1-b]thiazol-3-yl}-3,5-dimethoxybenzoni-
trile (6u). To a solution of 16c (90 mg, 0.21 mmol) and 1-iodobutane
(36 μL, 0.32 mmol) in DMSO (0.8 mL) was added NaOH (17 mg,
0.42 mmol) at room temperature, and the mixture was stirred for 1 h.
A saturated aqueous solution of NH4Cl was added, and the residue
was extracted with EtOAc. The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo to afford a crude product, which
was used in next step without purification.
3-[4-(Ethoxymethyl)-2,6-dimethoxyphenyl]-6-methoxy-N-
propyl-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazol-7-amine (6p). Compound 6p was prepared according to the
procedure described for the synthesis of 6h using 16b. The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:1) to give 6p (82%, 3 steps) as a white solid.
1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 7.6 Hz, 3H), 1.30 (t, J =
6.8 Hz, 3H), 1.30−1.44 (m, 2H), 1.52−1.66 (m, 2H), 1.78−1.88 (m,
2H), 2.94 (dd, J = 7.2, 7.6 Hz, 2H), 3.00−3.11 (m, 1H), 3.38 (td, J =
2.0, 12.0 Hz, 2H), 3.62 (q, J = 7.2 Hz, 2H), 3.79 (s, 6H), 3.86 (s, 3H),
3.93−4.03 (m, 2H), 4.54 (s, 2H), 6.40 (s, 1H), 6.66 (s, 2H). 13C NMR
(150 MHz, CDCl3) δ 162.9, 159.5, 142.4, 135.0, 128.3, 106.6, 105.8,
105.7, 103.4, 72.9, 67.5, 66.1, 60.7, 56.3, 56.1, 52.8, 31.1, 21.8, 15.3,
11.7. HRMS calcd for (C25H36N3O5S) [M + H]+ 490.2370; found
490.2367. HPLC purity: > 99%.
3,5-Dimethoxy-4-{6-methoxy-7-[propyl(tetrahydro-2H-
pyran-4-yl)amino]pyrazolo[5,1-b][1,3]thiazol-3-yl}benzonitrile
(6q). Compound 6q was prepared according to the procedure
described for the synthesis of 6h using 16c. The product was purified
by column chromatography on silica gel (n-heptane:EtOAc = 2:1) to
give 6q (54%, 3 steps) as a yellow solid. 1H NMR (400 MHz, CDCl3)
δ 0.88 (t, J = 7.2 Hz, 3H), 1.30−1.44 (m, 2H), 1.50−1.66 (m, 2H),
1.78−1.88 (m, 2H), 2.94 (t, J = 7.2 Hz, 2H), 3.00−3.12 (m, 1H), 3.38
(t, J = 12.0 Hz, 2H), 3.82 (s, 6H), 3.85 (s, 3H), 3.94−4.03 (m, 2H),
6.49 (s, 1H), 6.93 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 163.2,
159.6, 135.2, 126.3, 118.6, 114.4, 111.8, 108.0, 107.0, 106.9, 67.5, 60.7,
56.4, 56.3, 52.8, 31.1, 21.8, 11.7. HRMS calcd for (C23H29N4O4S) [M
+ H]+ 457.1904; found 457.1898. HPLC purity: 96.9%.
To the obtained crude product was added CH2Cl2 (0.6 mL),
followed by TFA (0.2 mL), and the mixture was stirred at room
temperature for 2 h. The reaction mixute was concentrated under
reduced pressure.
To a solution of the obtained residue in THF (1 mL) were added
tetrahydro-4H-pyran-4-one (36 mg, 0.32 mmol), followed by
NaBH(OAc)3 (67 mg, 0.32 mmol), and the mixture was stirred at
room temperature for 2 h. A saturated aqueous solution of NaHCO3
was added to the reaction mixture, and the residue was extracted with
EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (n-heptane:EtOAc = 3:2) to afford 6u
1
(81 mg, 82%, 3 steps) as a pale-yellow solid. H NMR (400 MHz,
CDCl3) δ 0.82−0.92 (m, 3H), 1.22−1.38 (m, 4H), 1.50−1.64 (m,
2H), 1.78−1.86 (m, 2H), 2.94−3.01 (m, 2H), 3.06 (tt, J = 4.0, 11.2
Hz, 1H), 3.38 (dt, J = 2.0, 12.0 Hz, 2H), 3.82 (s, 6H), 3.85 (s, 3H),
3.95−4.02 (m, 2H), 6.49 (s,1H), 6.93 (s,2H). 13C NMR (150 MHz,
CDCl3) δ 163.2, 159.6, 135.2, 126.3, 118.6, 114.4, 111.8, 108.0, 107.1,
106.9, 67.5, 60.8, 56.4, 56.3, 50.6, 31.1, 30.7, 20.4, 14.0. HRMS calcd
for (C24H30N4O4S) [M + H]+ 471.2061; found 471.2058. HPLC
purity: > 99%.
3-[2-Chloro-6-methoxy-4-(methoxymethyl)phenyl]-6-me-
thoxy-N-propyl-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b]-
[1,3]thiazole-7-amine (6r). Compound 6r was prepared according
to the procedure described for the synthesis of 6a using 16d. The
product was purified by column chromatography on NH silica gel (n-
1
heptane:EtOAc = 3:1) to give 6r (61%, 3 steps) as a white solid. H
Ethyl 6-Oxo-5,6-dihydropyrazolo[5,1-b][1,3]thiazole-7-car-
boxylate (10). To a solution of diethyl malonate 7 (100 g, 624
mmol), cesium carbonate (488 g, 1.5 mol), and carbon disulfide (45.3
mL, 749 mmol) in DMF (900 mL) was added dropwise
bromoacetaldehyde diethylacetal (290 mL, 1.87 mol), followed by
sodium iodide (9.34 g, 62.4 mmol) at room temperature. The reaction
mixture was heated to 60 °C for 8 h and then cooled to ambient
temperature. The reaction mixture was filtered, and the filtrate was
partitioned between water and Et2O. The organic layer was separated,
and the aqueous layer was extracted with Et2O. The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo to afford a
crude product, which was used in the next step without further
purification.
To a solution of the crude product from the previous step in EtOH
(900 mL) was added hydrazine hydrate (60.7 mL, 1.25 mol) while
stirring on a water bath, and the mixture was stirred for 13 h at room
temperature. The reaction mixture was filtered, and the filtrate was
concentrated in vacuo to give crude compound, which was used in the
next step without further purification.
NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.2 Hz, 3H), 1.32−1.44 (m,
2H), 1.52−1.66 (m, 2H), 1.79−1.86 (m, 2H), 2.92−2.97 (m, 2H),
3.02−3.11 (m, 1H), 3.34−3.41 (m, 2H), 3.47 (s, 3H), 3.80 (s, 3H),
3.85 (s, 3H), 3.96−4.02 (m, 2H), 4.49 (s, 2H), 6.46 (s, 1H), 6.93 (br
s, 1H), 7.09 (br s, 1H). 13C NMR (150 MHz, CDCl3) δ 163.3, 159.9,
142.3, 136.1, 135.1, 128.8, 120.6, 116.5, 108.6, 107.0, 106.4, 73.9, 67.5,
67.5, 60.6, 58.6, 56.4, 56.3, 52.7, 31.2, 31.1, 21.8, 11.7. HRMS calcd for
(C23H31ClN3O4S) [M + H]+ 480.1718; found 480.1715. HPLC purity:
> 99%.
6-Methoxy-3-[2-methoxy-4-(methoxymethyl)phenyl]-N-
propyl-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazol-7-amine (6s). Compound 6s was prepared according to the
procedure described for the synthesis of 6b using 16e. The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 3:1), followed by column chromatography on NH
silica gel (n-heptane:EtOAc = 2:1) to give 6s (66%, 3 steps) as a pale-
yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.88−0.92 (m, 3H), 1.29−
1.43 (m, 2H), 1.49−1.66 (m, 2H), 1.77−1.88 (m, 2H), 2.89−3.13 (m,
3H), 3.30−3.42 (m, 2H), 3.44 (s, 3H), 3.90−4.04 (m, 8H, involving a
singlet at 3.93 and a singlet at 3.97), 4.52 (s, 2H), 6.99 (s, 1H), 7.04 (s,
2H), 7.05 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H). 13C NMR (150
MHz, CDCl3) δ 162.8, 157.2, 140.4, 136.0, 130.3, 129.5, 119.5, 116.9,
110.2, 106.7, 106.6, 74.4, 67.4, 60.6, 58.3, 56.3, 55.7, 52.6, 31.2, 21.7,
11.7. HRMS calcd for (C23H32N3O4S) [M + H]+ 446.2108; found
446.2101. HPLC purity: > 99%.
To the obtained crude product were added 1,4-dioxane (1 L) and
aqueous 5 M HCl (200 mL) in that order, and the mixture was stirred
at 60 °C for 4 h. The mixture was cooled to room temperature, and
the solvent was distilled off under reduced pressure. Water was added
to the resulting residue, and the resulting solid was filtered, washed
with water, and dried under reduced pressure to afford 10 (42.5 g,
1
32%, 3 steps) as a pale-yellow solid. H NMR (400 MHz, CDCl3) δ
N-Butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-me-
thoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazole-7-amine (6t). Compound 6t was prepared according to the
procedure described for the synthesis of 6h using 16b. The product
was purified by column chromatography on silica gel (n-
1.41 (t, J = 7.0 Hz, 3H), 4.40 (q, J = 7.0 Hz, 2H), 6.89 (d, J = 4.0 Hz,
1H), 7.69 (d, J = 4.4 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 167.0,
164.6, 141.8, 123.0, 111.2, 89.9, 60.7, 14.5. HRMS calcd for
(C8H8N2O3S) [M + H]+ 213.0328; found 213.0332.
Ethyl 6-Methoxypyrazolo[5,1-b][1,3]thiazole-7-carboxylate
(11). To a solution of 10 (41.3 g, 195 mmol) in DMF (624 mL)
was added cesium carbonate (127 g, 389 mmol), followed by
iodomethane (24.2 mL, 389 mmol) at room temperature. The
reaction mixture was stirred at ambient temperature for 1 h, and then
1
heptane:EtOAc = 1:1) to give 6t (75%, 3 steps) as a white solid. H
NMR (400 MHz, CDCl3) δ 0.87 (t, J = 6.8 Hz, 3H), 1.30 (t, J = 6.8
Hz, 3H), 1.24−1.40 (m, 4H), 1.52−1.67 (m, 2H), 1.78−1.87 (m, 2H),
2.97 (t, J = 6.8 Hz, 2H), 2.99−3.11 (m, 1H), 3.38 (td, J = 1.6, 11.6 Hz,
2H), 3.63 (q, J = 7.2 Hz, 2H), 3.79 (s, 6H), 3.86 (s, 3H), 3.94−4.03
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water and a mixed solvent of EtOAc/Et2O (1/1) were added. The
organic layer was separated, washed with brine, dried over MgSO4 and
filtered. The solvent was distilled off under reduced pressure. The
residue was purified by silica gel column chromatography on silica gel
(n-heptane:EtoAc = 1:2.3) to afford 11 (30.7 g, 70%) as a brown solid.
1H NMR (400 MHz, CDCl3) δ 1.39 (t, J = 7.0 Hz, 3H), 4.08 (s, 3H),
4.35 (q, J = 7.0 Hz, 2H), 6.87 (d, J = 4.4 Hz, 1H), 7.66 (d, J = 4.4 Hz,
1H). 13C NMR (150 MHz, CDCl3) δ 166.1, 161.9, 145.1, 122.6, 111.3,
92.0, 60.1, 56.8, 14.5. HRMS calcd for (C9H10N2O3S) [M + H]+
227.0485; found 227.0488.
The organic layer was washed with brine, dried over MgSO4, and
filtered, and then the solvent was distilled off under reduced pressure,
which was used in the next step without further purification.
To a solution of the obtained crude product in CH2Cl2 (20 mL)
were added triethylamine (0.83 mL, 5.9 mmol) and Boc2O (0.95 g, 4.4
mmol) at room temperature. After stirring at room temperature
overnight, water was added and the mixture was extracted with
CH2Cl2. The organic layer was dried over MgSO4, and filtered, and
then the solvent was distilled off under reduced pressure. The residue
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:1) to afford 14b (0.81 g, 56%, 3 steps) as a
pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.53 (s, 9H), 2.33 (s,
3H), 6.03 (br s, 2H), 6.71 (d, J = 4.0 Hz, 1H), 7.59 (d, J = 4.0 Hz,
1H). 13C NMR (150 MHz, CDCl3) δ 153.2, 144.6, 132.0, 121.9, 112.0,
110.6, 80.8, 28.3, 11.7. HRMS calcd for (C11H16N3O2S) [M + H]+
254.0958; found 254.0957.
6-Methoxypyrazolo[5,1-b][1,3]thiazole (12). To a solution of
11 (30.7 g, 136 mmol) in EtOH (407 mL) was added aqueous 5 M
NaOH (136 mL, 680 mmol) at room temperature. The mixture was
stirred at 80 °C for 2 h and then cooled to ambient temperature. The
reaction mixture was adjusted to neutral pH with aqueous 5 M HCl at
0 °C. The resulting mixture was concentrated to remove EtOH, and
then it was filtered. Solid cake was washed with H2O and dried under
vacuum to afford a crude product, which was used in the next step.
To the obtained crude product were added 1,4-dioxane (400 mL)
and concentrated HCl (200 mL) in that order, and the mixture was
stirred at 60 °C for 1.5 h. The reaction mixute was concentrated under
reduced pressure, and then was adjusted to weakly acidic pH with solid
NaOH under ice cooling. The residue was extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4, and filtered,
and then the solvent was distilled off under reduced pressure. The
residue was purified by column chromatography on silica gel (n-
heptane:EtOAc = 2.3:1) to afford 12 (15.8 g, 75%, 2 steps) as brown
tert-Butyl (3-Bromo-6-methoxypyrazolo[5,1-b][1,3]thiazol-
7-yl)carbamate (15a). To a solution of 14a (16.5 g, 61.4 mmol)
in THF (410 mL) was added n-butyllithium (2.77 M solution in n-
hexane: 62.1 mL, 172 mmol) at −78 °C. After stirring the mixture at
−78 °C for 40 min, 1,2-dibromotetrafluoroethane (10.2 mL, 86 mmol)
was added and then the mixture was allowed to warm to room
temperature over 2 h while stirring. To the reaction mixture were
added a saturated aqueous solution of NH4Cl and EtOAc, followed by
AcOH to adjust to a weakly acidic pH. The mixture was separated and
the organic layer was washed with brine, dried over MgSO4, and
filtered, and the solvent was distilled off under reduced pressure. The
residue was purified by column chromatography on silica gel (n-
1
oil. H NMR (400 MHz, CDCl3) δ 3.95 (s, 3H), 5.81 (d, J = 0.8 Hz,
1
1H), 6.60 (d, J = 4.0 Hz, 1H), 7.58 (dd, J = 0.8, 4.4 Hz, 1H). 13C
NMR (150 MHz, CDCl3) δ 167.7, 140.5, 122.2, 107.8, 81.7, 56.5.
HRMS calcd for (C6H6N2OS) [M + H]+ 155.0274; found 155.0276.
tert-Butyl (6-Methoxypyrazolo[5,1-b][1,3]thiazol-7-yl)-
carbamate (14a). To a solution of 12 (15.8 g, 103 mmol) in
aqueous 5 M HCl (350 mL) was added a mixture of sodium nitrite
(10.6 g, 154 mmol) and water (115 mL) under ice cooling. The
mixture was stirred at room temperature for 0.5 h and then adjusted to
neutral pH with aqueous 5 M NaOH under ice cooling. The
precipitate was collected by filtration and washed with water.
To the obtained crude product were added EtOH (200 mL), THF
(300 mL), and 10% palladium−carbon (50% wet: 16 g) in that order,
and the mixture was stirred at room temperature for 5 h at an
atmospheric pressure under a hydrogen atmosphere. The mixture was
filtered with Celite and concentrated under reduced pressure.
To a solution of the obtained crude product in CH2Cl2 (425 mL)
were added triethylamine (17.8 mL, 128 mmol) and Boc2O (24.1 g,
111 mmol) at room temperature. The mixture was stirred at room
temperature for 11 h and then concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel (n-
heptane:EtOAc = 2:1) to afford 14a (16.5 g, 59%, 3 steps) as light-
pink solid. 1H NMR (400 MHz, CDCl3) δ 1.51 (s, 9H), 3.98 (s, 3H),
6.12 (br s, 1H), 6.54 (d, J = 4.0 Hz, 1H), 7.48 (d, J = 4.4 Hz, 1H). 13C
NMR (150 MHz, CDCl3) δ 157.6, 153.1, 131.5, 122.1, 108.9, 98.9,
80.7, 56.5, 28.3. HRMS calcd for (C11H15N3O3S) [M + H]+ 270.0907;
found 270.0910.
tert-Butyl (6-Methylpyrazolo[5,1-b][1,3]thiazol-7-yl)-
carbamate (14b). To a solution of 18 (1.0 g, 5.7 mmol) in aqueous
5 M HCl (24 mL) was added a mixture of sodium nitrite (0.78 g, 11.3
mmol) and water (1.5 mL) under ice cooling. The mixture was stirred
at 0 °C for 2 h and then warmed to room temperature. After stirring
overnight, the mixture was adjusted to alkalic pH with aqueous 5 M
NaOH under ice cooling. The precipitate was collected by filtration
and washed with water, which was used in the next step without
further purification.
To the obtained crude product were added 2 M HCl (22 mL) and
Zn powder (0.37 g, 5.7 mmol) in that order, and the mixture was
stirred at room temperature for 15 min at room temperature.
Additional Zn powder (0.37 g, 5.7 mmol) was added, and then the
mixture was stirred at room temperature for 1 h at room temperature.
The mixture was filtered with Celite. The mixture was adjusted to
neutral pH with aqueous 5 M NaOH and then extracted with EtOAc.
heptane:EtOAc = 4:1) to afford 15a (14.3 g, 67%). H NMR (400
MHz, CDCl3) δ 1.51 (s, 9H), 4.04 (s, 3H), 6.16 (br s, 1H), 6.50 (s,
1H). 13C NMR (150 MHz, CDCl3) δ 157.2, 152.9, 129.6, 106.6, 103.6,
101.0, 81.0, 56.8, 28.3. HRMS calcd for (C11H14BrN3O3S) [M + H]+
348.0012; found 348.0020.
tert-Butyl (3-Bromo-6-methylpyrazolo[5,1-b][1,3]thiazol-7-
yl)carbamate (15b). Compound 15b was prepared according to
the procedure described for the synthesis of 15a using 14b (1.2 g, 4.7
mmol) in THF (40 mL), 1,2-dibromotetrafluoroethane (0.62 mL, 5.2
mmol), and n-butyllithium (1.57 M solution of n-hexane: 6.6 mL, 10.4
mmol). The product was purified by column chromatography on silica
gel (n-heptane:EtOAc = 1:2) to afford 15b (1.4 g, 88%) as a pale-
1
yellow solid. H NMR (400 MHz, CDCl3) δ 1.52 (s, 9H), 2.37 (s,
3H), 6.07 (br s, 2H), 6.68 (s,1H). 13C NMR (150 MHz, CDCl3) δ
153.0, 144.1, 130.1, 112.7, 110.0, 103.2, 81.2, 28.3, 11.8. HRMS calcd
for (C11H15BrN3O2S) [M + H]+ 332.0063; found 332.0058.
tert-Butyl {3-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-6-
methoxypyrazolo[5,1-b][1,3]thiazol-7-yl}carbamate (16a). To
a solution of 15a (998 mg, 2.87 mmol) in DME (107 mL) and water
(36 mL) were added [2,6-dimethoxy-4-(methoxymethyl)phenyl]-
boronic acid (973 mg, 4.31 mmol), K2CO3 (791 mg, 5.74 mmol),
triphenylphosphine (374 mg, 1.43 mmol), and palladium acetate (64.5
mg, 0.285 mmol). The mixture was stirred at 90 °C for 1.5 h under a
nitrogen stream. The reaction mixture was cooled, and then water was
added. The mixture was extracted with EtOAc, washed with brine,
dried over MgSO4, and filtered, and then the solvent was distilled off
under reduced pressure. The residue was purified by column
chromatography on silica gel (n-heptane:EtOAc = 1:1) to afford 16a
(1.22 g, 95%) as a beige solid. 1H NMR (400 MHz, CDCl3) δ 1.52 (s,
9H), 3.46 (s, 3H), 3.75 (s, 6H), 3.88 (s, 3H), 4.49 (s, 2H), 6.08 (br s,
1H), 6.43 (s, 1H), 6.63 (s, 2H). 13C NMR (150 MHz, CDCl3) δ
159.5, 157.1, 153.2, 142.0, 131.0, 127.9, 107.7, 106.1, 103.3, 98.3, 80.4,
74.8, 58.4, 56.8, 56.0, 28.3. HRMS calcd for (C21H27N3O6S) [M + H]+
450.1693; found 450.1703.
tert-Butyl {3-[4-(Ethoxymethyl)-2,6-dimethoxyphenyl]-6-
methoxypyrazolo[5,1-b][1,3]thiazol-7-yl}carbamate (16b).
Compound 16b was prepared according to the procedure described
for the synthesis of 16a using 15a (2.0 g, 5.7 mmol) and [4-
(ethoxymethyl)-2,6-dimethoxyphenyl]boronic acid (2.1 g, 8.6 mmol).
The product was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:1) to afford 16b (2.5 g, 94%) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 1.29 (t, J = 7.2 Hz, 3H), 1.52 (s, 9H), 3.61
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(q, J = 7.2 Hz, 2H), 3.75 (s, 6H), 3.87 (s, 3H), 4.53 (s, 2H), 6.09 (br s,
1H), 6.42 (s, 1H), 6.64 (s, 2H). 13C NMR (150 MHz, CDCl3) δ
159.5, 157.2, 153.2, 142.4, 131.0, 127.9, 107.7, 106.0, 103.4, 98.3, 80.4,
72.9, 66.1, 56.8, 56.0, 28.3, 15.2. HRMS calcd for (C22H29N3O6S) [M
+ H]+ 464.1850; found 464.1851.
tert-Butyl [3-(4-Cyano-2,6-dimethoxyphenyl)-6-
methoxypyrazolo[5,1-b][1,3]thiazol-7-yl]carbamate (16c).
Compound 16c was prepared according to the procedure described
for the synthesis of 16a using 15a (461 mg, 1.3 mmol) and (4-cyano-
2,6-dimethoxyphenyl)boronic acid (412 mg, 2.0 mmol). The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 2:1) to afford 16c (296 mg, 52%) as a yellow
solid. 1H NMR (400 MHz, CDCl3) δ 1.52 (s, 9H), 3.78 (s, 6H), 3.87
(s, 3H), 6.11 (br s, 1H), 6.50 (s, 1H), 6.91 (s, 2H). 13C NMR (150
MHz, CDCl3) δ 159.6, 157.1, 153.2, 130.7, 125.9, 118.7, 114.3, 112.0,
109.0, 108.0, 98.7, 80.6, 56.8, 56.3, 28.3. HRMS calcd for
(C20H23N4O5S) [M + H]+ 431.1384; found 431.1381.
tert-Butyl {3-[2-Chloro-6-methoxy-4-(methoxymethyl)-
phenyl]-6-methoxy-pyrazolo[5,1-b][1,3]thiazol-7-yl}carbamate
(16d). To a solution of 15a (250 mg, 0.72 mmol) in toluene (5 mL)
and EtOH (2.5 mL) was added [2-chloro-6-methoxy-4-
(methoxymethyl)phenyl]boronic acid (248 mg, 1.1 mmol), Pd(PPh3)4
(83 mg, 0.072 mmol), and a 1 M aqueous solution of Na2CO3 (1.4
mL, 1.4 mmol). After refluxing for 3 h under a nitrogen stream, the
reaction mixture was cooled and then water was added. The mixture
was extracted with EtOAc, washed with brine, dried over MgSO4, and
filtered, and the solvent was distilled off under reduced pressure. The
residue was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:1) to afford 16d (281 mg, 86%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 1.52 (s, 9H), 3.46 (s, 3H), 3.76 (s,
tert-Butyl {3-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-6-
methylpyrazolo[5,1-b]thiazol-7-yl}carbamate (19). Compound
19 was prepared according to the procedure described for the
synthesis of 16d using 15b (1.8 g, 5.5 mmol) and [2,6-dimethoxy-4-
(methoxymethyl)phenyl]boronic acid (1.9 g, 8.2 mmol). The product
was purified by column chromatography on silica gel (n-
heptane:EtOAc = 1:9) to afford 19 (2.4 g, 99%) as a pale-yellow
solid. 1H NMR (400 MHz, CDCl3) δ 1.54 (s, 9H), 2.27 (s, 3H), 3.44
(s, 3H), 3.73 (s, 6H), 4.48 (s, 2H), 5.99 (br s, 1H), 6.55 (s, 1H), 6.61
(s, 2H). 13C NMR (150 MHz, CDCl3) δ 159.5, 153.4, 144.3, 142.4,
132.0, 127.9, 110.4, 110.0, 105.6, 103.2, 80.5, 74.7, 58.4, 56.0, 28.3,
11.9. HRMS calcd for (C21H27N3O5S) [M + H]+ 434.1744; found
434.1742.
N - ( C y c l o p r o p y l m e t h y l ) - 3 - [ 2 , 6 - d i m e t h o x y - 4 -
(methoxymethyl)phenyl]-2,6-dimethyl-N-(tetrahydro-2H-
pyran-4-ylmethyl)pyrazolo[5,1-b][1,3]thiazol-7-amine (23). To
a solution of 5 (120 mg, 0.25 mmol) in THF (2 mL) was added n-
butyllithium (2.69 M solution in n-hexane: 0.10 mL, 0.27 mmol) at
−78 °C. After stirring the mixture at −78 °C for 20 min, 1,2-
dibromotetrafluoroethane (0.034 mL, 0.28 mmol) was added and then
the mixture was allowed to warm to room temperature over 1 h while
stirring. A saturated aqueous solution of NH4Cl was added to the
reaction mixture, and the residue was extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4, and filtered,
and the solvent was distilled off under reduced pressure to afford a
crude product, which was used in next step without purification.
To a solution of the obtained residue in 1,4-dioxane (1.5 mL) was
added dimethylzinc (1.0 M solution in n-hexane: 0.49 mL, 0.49 mmol)
and Pd[P(t-Bu)3]2 (5 mg, 0.010 mmol), and the mixture was stirred at
65 °C for 1 h under a nitrogen stream. The reaction mixture was
cooled, and then a saturated aqueous solution of NH4Cl was added.
The mixture was extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and filtered, and the solvent was
distilled off under reduced pressure. The residue was purified by
3H), 3.87 (s, 3H), 4.48 (s, 2H), 6.13 (br s, 1H), 6.48 (s, 1H), 6.91 (s,
1H), 7.08 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 159.9, 157.2,
153.2, 142.3, 136.1, 130.6, 128.4, 120.5, 116.8, 108.6, 108.5, 98.9, 80.5,
73.9, 58.6, 56.9, 56.2, 28.3. HRMS calcd for (C20H24ClN3O5S) [M +
H]+ 454.1198; found 454.1196.
1
HPLC to afford 23 (60 mg, 49%, 2 steps) as a white solid. H NMR
tert-Butyl {6-Methoxy-3-[2-methoxy-4-(methoxymethyl)-
phenyl]pyrazolo[5,1-b][1,3]thiazol-7-yl}carbamate (16e). Com-
pound 16e was prepared according to the procedure described for the
synthesis of 16d using 15a (150 mg, 0.43 mmol) and [2-methoxy-4-
(methoxymethyl)phenyl]boronic acid (127 mg, 0.65 mmol). The
product was purified by column chromatography on NH silica gel (n-
heptane:EtOAc = 5:1) to afford 16e (150 mg, 83%) as a white solid.
1H NMR (400 MHz, CDCl3) δ 1.52 (s, 9H), 3.43 (s, 3H), 3.90 (s,
3H), 3.98 (s, 3H), 4.51 (s, 2H), 6.12 (br s, 1H), 6.96 (s, 1H), 7.02 (d,
J = 7.6 Hz, 1H), 7.03 (s, 1H), 8.25 (d, J = 7.6 Hz, 1H). 13C NMR (150
MHz, CDCl3) δ 157.3, 156.9, 153.1, 140.5, 131.5, 130.3, 129.5, 119.5,
117.3, 110.3, 108.4, 98.4, 80.5, 74.4, 58.3, 56.7, 55.7, 28.3. HRMS calcd
for (C20H25N3O5S) [M + H]+ 420.1588; found 420.1582.
1-(6-Methylpyrazolo[5,1-b][1,3]thiazol-7-yl)ethanone (18).
To a solution of 2-methylthiazole (5.8 g, 58.2 mmol) in CH2Cl2 (35
mL) were added O-mesitylene sulfonylhydroxylamine (12.5 g, 58.2
mmol, CAUTION!) dissolved with CH2Cl2 (35 mL) at 0 °C, and then
reaction mixture was stirred at room temperature for 9.5 h. The
reaction mixture was concentrated and dried under reduced pressure
overnight to afford a crude product, which was used in the next step
without further purification.
To the obtained crude product (15.6 g) were added acetic
anhydride (125 mL, 1.32 mol) and sodium acetate (6.1 g, 74.4 mmol)
in that order at room temperature, and the mixture was refluxed for 5
h. The mixture was cooled to room temperature, and the solvent was
distilled off under reduced pressure. Water was added to the resulting
residue, and then a saturated aqueous solution of K2CO3 was added at
0 °C. The mixture was extracted with EtOAc, washed with brine, dried
over MgSO4, and filtered, and then the solvent was distilled off under
reduced pressure. The resulting solid was sonicated in diisopropylether
(40 mL), and filtered to afford 18 (5.3 g, 51%, 2 steps) as a brown
solid. 1H NMR (400 MHz, CDCl3) δ 1.51 (s, 3H), 2.65 (s, 3H), 7.00
(d, J = 4.0 Hz, 1H), 7.76 (d, J = 4.0 Hz, 1H). 13C NMR (150 MHz,
CDCl3) δ 190.2, 155.3, 145.2, 122.1, 114.2, 114.0, 28.9, 15.5. HRMS
calcd for (C8H9N2OS) [M + H]+ 181.043; found 181.0428.
(400 MHz, CDCl3) δ −0.04−0.07 (m, 2H), 0.34−0.46 (m, 2H),
0.78−0.94 (m, 1H), 1.20−1.37 (m, 2H), 1.48−1.62 (m, 1H), 1.67−
1.84 (m, 2H), 2.14 (s, 3H), 2.25 (s, 3H), 2.67−2.76 (m, 2H), 2.81−
2.90 (m, 2H), 3.26−3.39 (m, 2H), 3.47 (s, 3H), 3.76 (s, 3H), 3.87−
3.99 (m, 2H), 4.49 (s, 2H), 6.64 (s, 2H). 13C NMR (150 MHz,
CDCl3) δ 159.4, 148.3, 142.3, 130.3, 124.5, 122.9, 120.6, 105.1, 103.4,
74.9, 68.0, 62.0, 60.8, 58.5, 56.1, 33.8, 31.5, 13.3, 12.3, 9.6, 3.6. HRMS
calcd for (C27H38N3O4S) [M + H]+ 500.2578; found 500.2576. HPLC
purity: 96.9%.
N-Butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-me-
thoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]-
thiazol-7-amine bis(phosphate) (24). To a solution of 6t (3.0 g,
6.0 mmol) in EtOH (7 mL) was added 85% phosphoric acid (0.84
mL, 12.3 mmol) at 60 °C, and then the mixture was cooled to room
temperature. To the mixture was added a seed crystal of 24, followed
by n-heptane (5 mL) over 10 min. An additional n-heptane (25 mL)
was added, and then the mixture was stirred at room temperature for 3
h.
The precipitate was collected by suction filtration, washed with n-
heptane (9 mL), and dried by heating at 40 °C under reduced pressure
to afford 24 as a white solid (3.8 g, 90%). 1H NMR (600 MHz,
DMSO-d6) δ 0.82 (t, J = 7 Hz, 3H), 1.19 (t, J = 7 Hz, 3H), 1.23 (tt, J =
7, 7 Hz, 2H), 1.29 (tq, J = 7, 7 Hz, 2H), 1.34 (dddd, J = 4, 11, 11, 11
Hz, 1H), 1.70 (br d, J = 11 Hz, 2H), 2.91 (t, J = 7 Hz, 2H), 2.98 (tt, J
= 4, 11 Hz, 1H), 3.25 (br dd, J = 11, 11 Hz, 2H), 3.56 (q, J = 7 Hz,
2H), 3.70 (s, 6H), 3.71 (s, 3H), 3.83 (br d, J = 11 Hz, 2H), 4.50 (s,
2H), 6.74 (s, 2H), 6.75 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ
162.4, 159.1, 143.1, 135.2, 127.8, 106.8, 106.4, 104.9, 103.5, 71.8, 66.5,
65.5, 59.8, 56.2, 56.1, 49.6, 31.2, 30.7, 19.8, 15.3, 14.0.
Biology. Binding Assays. HEK293 cells expressing human CRF1
receptor were cloned using essentially the same method as that
described in the literature.5 CRF1 receptor binding assay was
performed using the homogeneous technique of scintillation proximity
(SPA, Amersham Pharmacia, UK) with 96-well plates. Cell membrane
(5 μg/well), wheat germ agglutinin-coated SPA beads (1 mg/well),
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[
125I] human/rat CRF (0.1 nM), and diluted test compound solution
ACTH concentration was calculated from a standard curve that was
prepared using the standard ACTH solution.
Data are expressed as the mean SEM. The differences between
the CRF control and compound-treated groups were analyzed using
one-way analysis of variance (ANOVA), followed by Dunnett’s
multiple comparison test. A value of p < 0.05 (two-sided) was
considered statistically significant.
were suspended in 150 μL of assay buffer (137 mM NaCl, 8.1 mM
Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 10 mM MgCl2, 2 mM
EGTA, 1.5% bovine serum albumin [BSA], pH 7.0). Total binding and
nonspecific binding were measured in the absence and presence of 0.4
μM of unlabeled sauvagine, respectively. Plates were shaken gently and
incubated for more than 2 h at room temperature. After plate
centrifugation (260g, 5 min, room temperature), radioactivity was
detected using TopCount (Packard, USA, MA) with a 1 min counting
time per well. Each count was corrected by subtracting the value for
nonspecific binding and was represented as a percentage of the total
binding. The IC50 value of each compound was calculated using a
concentration−response curve. The affinity for human CRF2α
receptors was evaluated using HEK293 cell membrane expressing
human CRF2α receptors, which were cloned as described in the
literature.33 The assay was conducted in similar fashion as the above-
described CRF1 receptor binding assay, except that cell membrane (5
μg/well) was incubated with [125I] sauvagine (0.1 nM) instead of
Light/Dark Test in Mice. Male BALB/c mice (Charles River Japan
Inc., Kanagawa, Japan) weighing 18.9−25.6 g were used. Test
compounds (10 and 30 mg/kg; n = 12) were orally administered to
the mice 1 h before the test. A control group received an equivalent
volume of vehicle (0.5% methyl cellulose, 10 mL/kg; n = 12). The test
apparatus was a modified version of that described by Belzung et al.37
It comprised two acrylic boxes, one of which was a black darkened box
(dark box; 10 cm × 15 cm × 20 cm high) and the other was a white
open-top box (light box; 20 cm × 15 cm × 20 cm high). A black
acrylic tunnel (7 cm × 10 cm × 4.5 cm high) separated the dark box
from the light box. To record the behavior of the animals, the front-
and back sides of the light box (20 cm × 20 cm) were made of clear
acrylic glass. The light intensity on the floor of the light box was fixed
at 150 lx. At the beginning of the experiment, a mouse was placed in
the dark box. Its behavior was recorded on videotape over a 5 min
period, and the time spent in the light box was measured by an
observer. A mouse whose four paws were in the light box was
considered as being in the light box.
[
125I] human/rat CRF.
Functional Assay. To determine antagonistic activity, the effect of
the test compound on CRF-stimulated intracellular cAMP accumu-
lation was examined using IMR-32 cells, which is a human
neuroblastoma cell line, as described in the literature32 but with slight
modification. cAMP production was measured using an enzyme
immunoassay kit (Amersham Pharmacia, UK). IMR-32 cells (100000
cells/well) in MEM Earle’s medium containing 10% fetal bovine serum
were seeded in 96-well plates in the presence of 1 mM 3-isobutyl-1-
methylxanthine, which is a phosphodiesterase inhibitor. After
preincubation for 30 min, the diluted test compound was added and
incubated for 30 min at 37 °C. Cells were stimulated with 1 nM of
human/rat CRF for 30 min at 37 °C and collected by centrifugation
(630g, 5 min, 4 °C). After aspiration of the medium, the cells were
lysed with lysis buffer, and the amount of intracellular cAMP was
measured according to the procedures detailed in the instruction
manual of the kit. In each case, the basal amounts of cAMP (i.e., in the
absence of CRF) were subtracted from the produced cAMP and were
expressed as a percentage of the total amount produced. The IC50
value of the compound was calculated using a concentration−response
curve.
Effects on CRF-Induced Defecation in Rats. Male Fischer 344 rats
(Charles River Japan Inc. Kanagawa, Japan) weighing 192−223 g were
used. Test compounds were orally administered to the nonfasted rats
(n = 6) 1 h before the iv injection of CRF (10 μg/kg). Rats in the CRF
control group were orally administered an equivalent volume of
vehicle (dimethyl sulfoxide/cremophor/saline (5:5:90, v/v/v), 5 mL/
kg; n = 6). Then 1 h after the po administration of tested compounds
(10 mg/kg), the rats were anesthetized lightly with ether and CRF was
injected intravenously via the lateral tail vein. The rats were placed in
individual compartments of unit cages. The feces excreted by each rat
were collected and weighed 4 h after injection of CRF.
Data are expressed as the mean SEM. Differences between the
vehicle control and the compound-treated groups were evaluated using
one-way analysis of variance, followed by Dunnett’s multiple
comparison test. A value of p < 0.05 (two-sided) was considered
statistically significant.
Determination of in Vitro Hepatic Clearance. The in vitro hepatic
clearance data were obtained by measuring depletion in human liver
microsomes. Pooled human liver microsomes (n = 150) were
purchased from BD (MA, USA). A stability assay was conducted
using 0.3 μmol/L substrate and 0.1 mg/mL microsomal protein in
which the final concentration of organic solvent was 0.01% DMSO. A
microsomal matrix contained 0.1 mmol/L EDTA, 100 mmol/L
phosphate buffer (pH 7.4), an NADPH-generating system, liver
microsomes, and substrates. The NADPH-generating system was
prepared as a mixture containing 3.3 mmol/L β-NADP+, 80 mmol/L
G6P, 60 mmol/L MgCl2 and 1 unit/mL G6PDH. The incubation was
conducted at 37 °C for 0 and 15 min by adding the NADPH-
generating system. After the incubation, the microsomal matrix was
deproteinized by adding acetonitrile/methanol containing the internal
standard. After centrifugation, the resulting supernatant was analyzed
with LC/MS/MS.
Rat Pharmacokinetic Study. Pharmacokinetic parameters were
estimated in fasted male Sprague−Dawley (SD) rats (Charles River
Japan Inc., Kanagawa, Japan) after iv (3 mg/kg; n = 3) and po (10 mg/
kg; n = 3) administration. The dosing solution for iv administration
was prepared in 1% ethanol−0.1 mol/L HCl−glucose at a
concentration of 3 mg/mL. The posing solutions for PO
administration were prepared in 5% ethanol−0.1 mol/L HCl−-glucose
at concentrations of 2 mg/mL. Blood samples were collected from the
jugular vein at 0.083 (5 min; for IV), 0.25, 0.5, 1, 2, 4, 6, and 8 h after
dosing (n = 3 at each time point). Plasma was separated by
centrifugation and stored in a frozen state until analysis. Plasma was
separated by centrifugation and stored frozen until analysis. Plasma
concentrations were measured using the LC/MS/MS method. Plasma
samples (100 μL) were deproteinized by adding 250 μL of acetonitrile
containing the internal standard (imipramine). The samples were
mixed and centrifuged. After centrifugation, the resulting supernatant
(5 μL) was subjected to LC/MS/MS. Chromatography was performed
in the reverse phase mode with a CAPCELL PAK C18 MGIII (2.0
mm i.d. × 150 mm, Shiseido Co. Ltd., Tokyo, Japan). Mobile phase A
comprising distilled water containing 0.1% HCOOH and mobile phase
B comprising acetonitrile containing 0.1% HCOOH were pumped at
0.5 mL/min, by using the linear gradient program [B%, 30% (0−1
min), 30−95% (1−3 min), 95% (3−5 min)]. Ionization was initiated
using an electrospray (positive mode) along with monitoring of
Data are expressed as a percentage of the inhibition of stool output
(g/4 h) of compound-treated groups compared to stool output of
CRF control groups, using the mean standard error of the mean
(SEM). The results were analyzed using an unpaired t test. A value of
p < 0.05 (two-sided) was considered statistically significant.
Effects on the CRF-Induced Increase of Plasma ACTH
Concentration in Rats. Male Fischer 344 rats (Charles River Japan
Inc. Kanagawa, Japan) weighing 173−200 g were used. Test
compounds (10, 30 mg/kg; n = 8) were orally administered to the
rats 1 h before subcutaneous injection of CRF (10 μg/kg). The CRF
control groups received an equivalent volume of vehicle (0.5% methyl
cellulose, 5 mL/kg; n = 8). Then 30 min after the subcutaneous
injection of CRF, blood samples obtained via the decapitated animals
were stirred with 100 μL of EDTA-2Na (100 mg/mL) and kept on ice.
The blood samples were centrifuged (1000g, 4 °C, 5 min), and plasma
samples were prepared. Plasma ACTH concentrations were
determined using an immunoradiometric assay kit (ACTH Irma
Mitsubishi, Mitsubishi Chemical Medience). Radioactivity of the beads
was measured using a scintillation counter (ARC-1000M, Aloka). The
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parent−daughter peaks of 504.26 > 419.2 for 19ax and 281.1 > 86.1
for the internal standard.
mediator of anxiety or stress responses? Brain Res. Rev. 1990, 15, 71−
100.
Kinetic Solubility Assay. Water solubility was determined as
follows: Sample solutions were prepared by dilution of 2.5 μL of 10
mM DMSO stock solution with 250 μL of Dulbecco’s phosphate
buffered saline in a 96-well filter plate. The plate was shaken for 15
min at room temperature to allow the compounds to equilibrate. The
sample solutions were filtered into another 96-well plate by vacuum.
Standard solutions were prepared by dilution of 2.5 μL of 10 mM
stock DMSO solution with 250 μL of DMSO in a 96-well plate. The
filtrated sample solutions and standard solutions were analyzed by
HPLC to determine the solubility.
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AUTHOR INFORMATION
Corresponding Author
■
(13) Zorrilla, E. P.; Koob, G. F. Progress in corticotropin-releasing
factor-1 antagonist development. Drug Discovery Today 2010, 15,
371−383.
*Phone: +81 (0)29 847 7395. Fax: +81 (0)29 847 4952. E-
mail: y4-takahashi@hhc.eisai.co.jp.
Notes
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A. C.; Learned-Coughlin, S. M.; Holsboer, F.; Grigoriadis, D. E. High-
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Kazuya Nagaoka for the in silico calculations, Dr.
Takuma Minamisono for salt and crystal screening, Mami
Gomibuchi for HRMS, Eri Ena and Yumi Yokoyama for NMR
analysis, and Tomoki Nishioka, Dr. Yuichi Suzuki, Dr. Noritaka
Kitazawa, and Dr. Teiji Kimura for their assistance in the
preparation of the manuscript.
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2003, 305, 86−96.
ABBREVIATIONS USED
CRF, corticotropin-releasing factor; hCLint, intrinsic clearance
in human liver microsomes; THP, tetrahydropyranyl
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