Discovery of novel and potent orally active calcium-sensing receptor antagonists that stimulate pulselike parathyroid hormone secretion: Synthesis and structure-activity relationships of tetrahydropyrazolopyrimidine derivatives

Article abstract of DOI:10.1021/jm101452x

As part of our research for novel calcium-sensing receptor (CaSR) antagonists that can function as oral bone anabolic agents, we recently reported the discovery of a tetrahydropyrazolopyrimidine derivative featuring adamantyl group 1b with potent CaSR ant

Full text of DOI:10.1021/jm101452x

ARTICLE
pubs.acs.org/jmc
Discovery of Novel and Potent Orally Active Calcium-Sensing
Receptor Antagonists that Stimulate Pulselike Parathyroid
Hormone Secretion: Synthesis and Structure-Activity Relationships
of Tetrahydropyrazolopyrimidine Derivatives†
Masato Yoshida,* Akira Mori, Etsuo Kotani, Masahiro Oka, Haruhiko Makino, Hisashi Fujita, Junko Ban,
Yukihiro Ikeda, Tomohiro Kawamoto, Mika Goto, Hiroyuki Kimura, Atsuo Baba, and Tsuneo Yasuma
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi-2-chome, Yodogawa-ku,
Osaka 532-8686, Japan
S Supporting Information
b
ABSTRACT: As part of our research for novel calcium-sensing
receptor (CaSR) antagonists that can function as oral bone
anabolic agents, we recently reported the discovery of a tetrahy-
dropyrazolopyrimidine derivative featuring adamantyl group 1b
with potent CaSR antagonistic activity. To explore the potentialof
this calcilytic congener, we introduced the gem-dialkyl benzyl
group at the 3-position of the tetrahydropyrazolopyrimidine ring, forming a bioisostere of the adamantyl group by mimicking the
adamantyl group’s lipophilicity and bulkiness. Optimization directed toward the improvement of solubility and metabolic stability
led to the discovery of compound 9e, which stimulated transient PTH secretion when orally administered to normal rats. Further,
compound 9e proved to be fully effective in an osteopenic ovariectomized rat model.
’ INTRODUCTION
coupled receptor (GPCR) highly expressed on parathyroid cells.
CaSR detects and responds to small changes in circulating
[Ca2þ] at the surface of parathyroid cells, leading to regulation
of PTH. The receptor is negatively coupled with PTH secretion,
such that increasing the concentration of the extracellular ligand
[Ca2þ] inhibits PTH secretion. It has been proposed that an
antagonist of CaSR (calcilytic)14-17 can mimic hypocalcemia
and stimulate PTH secretion.15
Targeting this regulatory mechanism by the antagonism of
CaSR, leading to the transient and rapid secretion of endoge-
neous PTH, should promote the formation of new bone analogo-
usly to the results found for parenterally administered PTH.7-9
Therefore, we attempted to identify short-acting CaSR antago-
nists, and we recently reported the discovery of a tetrahydropyr-
azolopyrimidine derivative featuring an adamantyl group 1b with
potent CaSR antagonistic activity.18 However, after the CaSR
antagonist was orally administered to normal rats, compound 1b
did not lead to significant PTH secretion, a finding that is con-
sistent with its poor pharmacokinetic (PK) profile. Therefore, we
continued to explore the potential of such calcilytic congeners.
To modify compound 1, a diverse amide library was prepared
by high throughput organic synthesis (HTOS), and 4-trifluoro-
methyl benzyl amide 2a (IC50 = 2900 nM) and benzhydryl amide
2b (IC50 = 380 nM) showed moderate activity. Therefore, we
postulated that the bulkiness and lipophilicity of the adamantyl
Osteoporosis is characterized by a decrease in bone density,
resulting in fragile bones.1 This disorder of the skeleton weakens
bones and increases the risk for fractures. In the United States,
direct health care costs from osteoporotic fractures amount to
billions of dollars.2,3 The pathogenesis of osteoporosis involves
an imbalanced turnover and a net increase in bone resorption,
leading to reduced bone mass and increased risk for fractures.
Currently available therapies for osteoporosis include the inhibi-
tion of bone resorption through the use of antiresorptive agents
(bisphosphonates,4,5 estradiol, calcitonin, raloxifene,6 etc.) and
promotion of bone formation with anabolic agents7-9 (recom-
binant full-length human parathyroid hormone (PTH) 1-84
(Nycomed), and teriparatide, the recombinant N-terminal PTH
1-34 amino acid fragment (Lilly).
PTH is an 84-amino acid peptide, produced by the parathyr-
oid glands, that regulates calcium homeostasis through actions
on the kidney and bone. Bone-forming effects and increased bone
strength following transient exposure to PTH through intermittent,
subcutaneous administration of PTH 1-84 or PTH 1-34 have
been well documented in animal models and in healthy human
volunteers, as well as in patients with osteoporosis. However, it has
become clear that elevated levels of PTH only result in higher bone
mass if the increases are transient; continuous infusion of PTH leads
to increased bone turnover without net formation, resulting in
overall bone loss.10-12
The secretion of PTH by parathyroid glands is closely
Received: November 11, 2010
Published: February 09, 2011
regulated by the calcium-sensing receptor (CaSR),13 a G-protein
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
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carbon monoxide atmosphere. Ethyl ester 10 was reduced to
benzyl alcohol 11 using lithium aluminum hydride, and succes-
sive treatment with acetyl chloride in the presence of potassium
carbonate (K2CO3) afforded acetate 12. Phenyl ester analogue
14a was synthesized by acylation of phenol 13, which was
prepared from 8j by removal of the benzyl group, using 10%
Pd-C under a hydrogen atmosphere. Phenoxyacetate 14b was
also prepared from phenol 13 by alkylation, using ethyl bromoa-
cetate in the presence of K2CO3.
Among the compounds prepared, compound 9e stimulated
pulse-like PTH secretion in rats, and therefore, enantiomers of 9e
were prepared, as shown in Scheme 4. Racemic ester 6 was
separated by preparative high-performance liquid chromatogra-
phy (HPLC) using a chiral column [CHIRALPAK OD, hexane/
ethanol = 95:5] to afford both enantiomers, 15a and 15b with
high enantiomeric purity (>99% ee). Enantiomers 15a and 15b
were then converted to amide derivatives (-)-17a and (þ)-17b,
respectively, by alkaline hydrolysis with KOH and a subsequent
coupling reaction with 3-(4-methylphenyl)pentan-3-amine.
Their absolute configuration was confirmed by X-ray analysis
of 4-trifluoromethyl analogue 17c,19 which was derived from
ester 15b. The X-ray crystal structure of 17c is shown in Figure 2.
This result showed that the absolute configurations of (-)-17a
and (þ)-17b were (R)- and (S)-forms, respectively.
Figure 1. Introduction of gem-dialkyl benzyl amide on the 3-position.
’ RESULTS AND DISCUSSION
group are important for maintaining potent activity and intro-
duced the gem-dialkyl benzyl amide on the 3-position of the
tetrahydropyrazolopyrimidine ring as a bioisostere of the ada-
mantyl group (Figure 1). As a result of this modification, we
discovered compounds 3a-c, which showed more potent antag-
onistic activity than compound 1b. Compound 3b showed
favorable oral bioavailability (24.4%) in normal rats and stimu-
lated significant increases in PTH concentrations, but it stimu-
lated a sustained PTH pattern and the PK behavior was not
desirable for this program.
For endogenous transient PTH secretion by oral administra-
tion of a CaSR antagonist, we assumed that a CaSR antagonist
should possess high solubility and moderate microsome stability
as desirable characteristics because high solubility accelerates
absorption and rapid metabolism results in high clearance and a
pulse-like PK profile. In this report, we describe the discovery of a
short-acting CaSR antagonist and its bone-forming effect in ani-
mal osteoporosis models.
The compounds synthesized were evaluated for CaSR antag-
onistic activity using a GTP-binding assay, for which membrane
fractions were prepared from CaSR-expressing CHO cells. The
results are summarized in Tables 1 and 3.
To estimate the in vivo PTH secretion stimulated by these
compounds, plasma PTH levels were assayed using a rat enzyme-
linked immunosorbent assay (ELISA) kit (Rat Bioactive Intact
PTH ELISA kit, Immutopics, Inc.) after oral administration of
the compounds to rats at a dose of 10 mg/kg. The bone-forming
effect of these compounds in osteopenic ovariectomized (OVX)
rats was evaluated after they were orally administered daily for 13
weeks.9 Increases in the plasma level of the bone formation
marker osteocalcin and bone mineral density (BMD) of the distal
femur were measured.
To develop a short-acting CaSR antagonist, we expected that
improving solubility by salt formation and optimizing metabolic
stability would impart the necessary characteristics to the CaSR
antagonist so that it could stimulate transient PTH secretion. In
the structure-activity relationship (SAR) study described in our
previous report,18 introducing a side chain containing an amide
linker at the 3-position was effective for conferring antagonistic
activity. Therefore, we expanded our SAR study of substituents
on the phenyl ring at the 3-position. As shown in Table 1,
introduction of a chlorine atom or a fluorine atom at the 2- or
3-position of the phenyl ring (8c, 8e, and 8f) resulted in slightly
decreased antagonistic activity compared with that of 4-halophe-
nyl compounds 8b and 8d. Therefore, we focused on the
substitution at the 4-position of the phenyl ring, and several
groups such as alkyl, alkoxy, and ester were introduced to adjust
metabolic stability. The methyl analogue, 8g, and alkoxy analo-
gues 8h and 8j exhibited potent antagonistic activity similar to
compound 3b. In the case of ester analogues, ethyl benzoate 10
was tolerated, but benzyl acetate 12, phenyl acetate 14a, and
phenyl propanoate 14b had reduced activities. Substitution at the
4-position of the phenyl ring provided compounds with a diverse
’ CHEMISTRY
The synthesis of 7,7-dimethyl tetrahydropyrazolopyrimidine
derivatives 3a-f and 8a-j is shown in Scheme 1. Ethyl 5-amino-
1H-pyrazole-4-carboxylate 4 was treated with 3-methyl-1-
phenylbut-2-en-1-one to afford dihydropyrazolopyrimidine 5.
Reduction of the pyrimidine ring with NaBH4 afforded tetra-
hydropyrazolopyrimidine 6, and subsequent hydrolysis with potas-
sium hydroxide (KOH) gave carboxylic acid 7. Finally, condensation
reactions were performed using 2-(1H-7-azabenzotriazole-1-yl)-
1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU) as a
coupling reagent to provide amide derivatives 3a-f and 8a-j.
Among them, the 4-chloro analogue 8b and 4-methyl analogue 8g
were converted to the corresponding salts 9a-f with various acids.
Furthermore, some ester analogues were prepared as shown in
Schemes 2 and 3. In the case of benzoate 10, the ester group was
introduced to the iodo analogue 8i using 1,10-bis(diphenyl-
phosphino)ferrocene (dppf) and palladium(II) acetate under a
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ARTICLE
Scheme 1. Synthesis of Tetrahydropyrazolopyrimidine 3, 8, and 9a
a Reagents and conditions: (a) Me2CdCHCOPh, NaH, DMF; (b) NaBH4, EtOH; (c) KOH, EtOH, H2O; (d) amine, HATU, iPr2EtN, DMF; (e) acid.
Scheme 2. Synthesis of Esters 10 and 12a
a Reagents and conditions: (a) dppf, Pd (OAc)2, Et3N, CO, EtOH; (b) LAH, THF; (c) AcCl, Et3N, K2CO3, THF.
Scheme 3. Synthesis of Ester 14a
a Reagents and conditions: (a) H2, 10% Pd-C; (b) AcCl, Et3N, THF; (c) BrCH2CO2Et, K2CO3, DMF.
range of in vitro metabolic stabilities, as shown in Table 1. All the
highly potent analogues (8a, 8b, 8d, 8g-j, and 10) showed more
rapid metabolizing profiles in rat liver microsomes than the lead
compound, 3b.
Next, to confirm the importance of solubility for rapid absorp-
tion, we selected compound 8b, which has high solubility, and
prepared salts 9a-d with various acids. The solubility of these
salts was measured under a pH 6.8 buffer with bile acid. As shown
in Table 2, all the salts (9a-d) showed increased solubility
compared to the free form, 8b. Compound 8b and its acetate salt
9d, which has highest solubility among salts, were tested in an in
vivo assay. When 9d was orally administered, the peak of PTH
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Scheme 4. Synthesis of Chiral Isomer 17a
a Reagents and condition: (a) CHIRALCEL OD; (b) KOH, EtOH, H2O; (c) SOCl2, DMF, toluene, then amine, Et3N, toluene; (d) 4 M HCl in EtOAc.
liver microsomes, a transient PTH increase is also expected in
humans after oral administration of 9e.
Membrane permeability (Caco-2) data for 3b, 8b, and 9e are
summarized in Table 2. Compounds 3b, 8b, and 9e had almost
the same antagonistic activity and membrane permeability. These
findings support our speculation that solubility and metabolic sta-
bility significantly influence rapid plasma PTH release and decrease,
respectively.
We turned our attention to the activities of enantiomers of 9e,
(-)-form 17a and (þ)-form 17b, in order to investigate the
stereochemical requirement for antagonistic activity. As expec-
ted, a significant difference in antagonistic activity was observed
between the (þ) and (-) enantiomers, and (-)-form 17a (IC50 =
2.3 nM) exhibited approximately 10-fold more potent activity
than (þ)-form 17b (IC50 = 31 nM), as shown in Table 3.
On the basis of the clinical data for teriparatide (PTH 1-34),
the PTH pattern achieved with compound 9e (magnitude and
duration) indicated that this compound is effective as a bone
anabolic agent for bone formation. Therefore, we tested com-
pound 9e in OVX rats.20 In the OVX rat model, female rats
experienced accelerated bone loss, similar to the estrogen-
deficiency bone loss in postmenopausal women. For 3 months,
OVX rats were orally administered 10 mg/kg compound 9e.
Their BMD and plasma osteocalcin (bone metabolic marker)
concentration after a 3 month treatment with compound 9e were
compared to those of vehicle control and sham-operated rats
(Table 4). Vehicle control OVX rats showed a significant
decrease in BMD compared to sham-operated rats, and treat-
ment with compound 9e significantly increased the BMD of
OVX rats. Furthermore, 9e increased the plasma osteocalcin
concentration, consistent with an elevation in bone formation
activity. Thus, 9e proved to be fully effective as a bone anabolic
agent in the OVX rat model.
Figure 2. X-ray Crystal Structure of 17c.
secretion in plasma was higher and earlier than that with the free
form, 8b; however, the plasma PTH secretion pattern was
sustained (Figure 3). This result suggests that the rat metabolic
stability (57 μL/min/mg) of 9d is still too high to achieve a
transient PTH increase in plasma. Second, we prepared salts of
the methyl analogue 8g, which showed more rapid metabolism
(164 μL/min/mg) in rat liver microsomes than compound 8b.
The HCl salt 9e and MsOH salt 9f also displayed excellent
solubility compared with the free form 8g. Figure 3 shows the
effects of PTH secretion after normal rats were orally adminis-
tered 10 mg/kg 9e. The plasma PTH level after oral administra-
tion of 9e increased to 120 pg/mL after 30 min and dropped to
normal levels after 2 h. The PK profile of compound 9e was
evaluated after it was orally administered to female SD rats. As
shown in Figure 4, compound 9e displayed good oral absorption
and the correlation between the PTH pattern and plasma con-
centration of compound 9e was observed. Because the metabolic
stability of 9e in human liver microsomes is similar to that in rat
’ CONCLUSION
An orally active tetrahydropyrazolopyrimidine derivative that
stimulated transient PTH secretion has been identified. We
found that good solubility and adequate metabolic stability are
critical factors for the rapid and transient secretion of PTH.
Given the efficacy of compound 9e in an OVX rat model of bone
loss, the magnitude and short action of this compound on plasma
PTH levels are expected to stimulate new bone formation even in
humans.
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Table 1. SAR Summary of Tetrahydropyrazolopyrimidine Analogues as Determined via a GTP-Binding Assay
metabolic stability (μL/min/mg)
CaSR antagonistic activity
compd
R
IC50 (nM)a
rat
13
human
solubility (μg/mL)b
3b
8a
8b
8c
4-CF3
H
7.6 (5.0-12)
3.4 (1.6-7.1)
7.4 (3.7-15)
17 (6.5-44)
7.3 (4.6-12)
25 (9.6-63)
12 (8.1-16)
5.5 (2.1-14)
6.6 (4.2-10)
8.5 (3.3-22)
5.2 (2.4-11)
14 (7.8-23)
31 (12-83)
12 (5.3-27)
14
209
18
4.6
22.3
54
215
57
4-Cl
3-Cl
4-F
8d
8e
8f
129
89
8.5
3-F
2-F
8g
8h
8j
4-Me
164
158
24
122
67
15.6
59.6
24.3
5.4
4-MeO
4-BnO
48
91c
10
12
14a
14b
4-CO2Et
138c
4-CH2OCOMe
4-OCOMe
4-OCOEt
a 95% confidence intervals are shown in parentheses. b Buffer (pH6.8)þbile acid. c Data of HCl salt.
’ EXPERIMENTAL SECTION
stirring at room temperature for 20 min, inorganic product was filtered
off, and the solvent was concentrated in vacuo. Crystallization from
EtOAc-hexane afforded 6 (39.2 g, 54%) as white prisms. H NMR
1
Chemistry. Melting points were determined on a Yanagimoto
::
1
micromelting point apparatus or BUCHI B-545 and uncorrected. H
NMR spectra of deuteriochloroform (CDCl3) or dimethyl sulfoxide
(DMSO-d6) solution (internal standard tetramethylsilan (TMS), δ 0)
were recorded on a Varian Gemini-200, Mercury-300 or Bruker
AVANCE-300. Reaction were followed by TLC on Silica Gel 60 F
254 precoated TLC plates (E. Merck) or NH TLC plates (Fuji Silysia
Chemical Ltd.). Column chromatography was performed with WAKO
Gel 300 using the indicated eluents. We carried out elemental analysis
(C, H, N) to determine purity of test compounds by the Analytical
Department of Takeda Chemical Industries, and the results were within
0.4% of theoretical values. Purity of compounds (>95%) was established
by elemental analysis. The data of elemental analysis is in the Supporting
Information.
(CDCl3): δ 1.31 (3H, t, J = 7.0 Hz), 1.57 (3H, s), 1.64 (3H, s),
2.11-2.15 (2H, m), 4.23 (2H, q, J = 7.0 Hz), 4.64 (1H, dd, J = 9.4, 5.2
Hz), 6.03 (1H, s), 7.31-7.45 (5H, m), 7.64 (1H, s).
7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-
3-carboxylic acid (7). A mixture of 6 (13.5 g, 45.1 mmol) and KOH
(12.65 g, 225 mmol) in EtOH-H2O (100 þ 100 mL) was stirred at
80 °C for 14 h and concentrated in vacuo. The residue was acidified with
citric acid solution and extracted with EtOAc. The extract was washed
with water and brine, dried over MgSO4, and then concentrated to afford
a solid, which was recrystallized from EtOAc-hexane to give 7 (8.1 g,
66%) as colorless prisms; mp 156-157 °C. 1H NMR (CDCl3): δ 1.57
(3H, s), 1.63 (3H, s), 2.04-2.18 (2H, m), 4.63 (1H, dd, J = 10.2, 5.1
Hz), 5.99 (1H, s), 7.32-7.42 (5H, m), 7.68(1H, s).
Ethyl 7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyri-
midine-3-carboxylate (6). To a solution of 4 (54.3 g, 0.35 mol) in DMF
(400 mL) was added sodium hydride (14.0 g, 60% in oil, 0.35 mol) at
room temperature. After stirring at the same temperature for 30 min, a
solution of 3-methyl-1-phenyl-2-buten-1-one (40.0 g, 0.25 mol) in DMF
(20 mL) was added to the reaction mixture. After stirring at room
temperature for 2 h, the reaction mixture was quenched with EtOH (400
mL) and stirring for 5 min. After that, NaBH4 (37.8 g, 1.0 mol) was
added to the reaction mixture at 0 °C. After stirring at room temperature
for 2 h, the solvent was concentrated in vacuo. The residue was diluted
with EtOAc and washed with water. The aqueous layer separated was
extracted with EtOAc. The organic layer combined was successively
washed with 1 M HCl, water and brine, dried over MgSO4, and concen-
trated in vacuo. The residue was diluted with EtOAc-hexane (1:1,
200 mL), and silicagel (50 g, WAKO Gel 300) was added thereto. After
N-(1-Ethyl-1-phenylpropyl)-7,7-dimethyl-5-phenyl-4,5,6,7-tetrahy-
dropyrazolo[1,5-a]-pyrimidine-3-carboxamide (8a). A mixture of 7
(0.50 g, 1.84 mmol), HATU (0.84 g, 2.21 mmol), and iPr2NEt (0.71 g,
5.52 mmol) in DMF (3 mL) was stirred at room temperature for 1 h,
followed by an addition of 3-phenylpentan-3-amine hydrochloride (0.44 g,
2.21 mmol). The whole was stirred at 80 °C overnight and concentra-
ted in vacuo. The residue was diluted with EtOAc, washed with aqueous
NaHCO3 solution and brine, dried over MgSO4, and concentrated in
vacuo. The residue was chromatographed on SiO2 with EtOAc-hexane
(1:1) to give 8a as crystals (0.36 g, 47%), mp 139-140 °C (EtOAc-
hexane). 1H NMR (CDCl3): δ 0.72-0.83 (6H, m), 1.58 (3H, s), 1.66
(3H, s), 1.96-2.26 (6H, m), 4.55 (1H, dd, J = 10.6, 3.2 Hz), 5.63 (1H,
s), 6.49 (1H, s), 7.20-7.39 (10H, m), 7.58 (1H, s). Anal. (C26H32N4O)
C, H, N.
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Table 2. Solubility and Membrane Permeability (Caco-2)
Data for 3b, 8b, 8g, and 9a-f
Caco-2
compd
R
salt
free
solubility (μg/mL)a
A to B
B to A
3b
8b
9a
9b
9c
9d
8g
9e
9f
CF3
Cl
4.6
54
32.7
52.6
24.2
36.8
free
Cl
HCl
126
164
202
202
15.6
140
118
Cl
MeSO3H
H2SO4
AcOH
free
Cl
Cl
Figure 4. Pharmacokinetic Parameters for 9e after Oral Administration
in Rat.
Me
Me
Me
HCl
58.9
42.7
MeSO3H
Table 3. CaSR Antagonist Activity of 4-Methyl Analogues
a Buffer (pH6.8) þ bile acid.
compd
form
IC50 (nM)a
9e
racemic
(-)
4.9 (2.9-8.2)
2.3 (0.86-6.4)
31.0 (11-86)
17a
17b
(þ)
a 95% confidence intervals are shown in parentheses.
Table 4. Bone-Forming Effect of Compound 9e in OVX Rat
Models
OVX
sham
OVX
9e
Figure 3. Effects of CaSR Antagonists on PTH Secretion in Normal
Rats (n = 4).
osteocalcin (ng/mL)a
distal femur BMD (mg/cm3)b
proximal tibia BMD (mg/cm3)b
16.7 ( 1.1 21.1 ( 2.4 28.4 ( 2.3*c
737 ( 11
722 ( 12
622 ( 7
649 ( 8
660 ( 6**c
700 ( 5**c
The following compounds 3a-f, 8b-j were prepared by a manner
similar to that used for 8a.
a After 2 months. b Measured by animal CT. c *, P < 0.01; **, P < 0.05 vs
OVX (Student’s t-test).
7,7-Dimethyl-N-{1-methyl-1-[4-(trifluoromethyl)phenyl]ethyl}-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
(3a). White prisms (Yield 44%), mp 182-183 °C (EtOAc-hexane).
1H NMR (CDCl3): δ 1.56 (3H, s), 1.64 (3H, s), 1.70 (3H, s), 1.76 (3H,
s), 2.02-2.23 (2H, m), 4.54 (1H, dd, J = 11.4, 3.4 Hz), 5.79 (1H, s), 6.42
(1H, s), 7.30-7.31 (5H, m), 7.50 (1H, s), 7.54-7.59 (4 h, s). Anal.
(C25H27F3N4O) C, H, N.
N-{1-Ethyl-1-[4-(trifluoromethyl)phenyl]propyl}-7,7-dimethyl-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
(3b). White prisms (yield 60%), mp 188-189 °C (EtOAc-hexane).
1H NMR (CDCl3): δ 0.73-0.89 (6H, m), 1.58 (3H, s), 1.65 (3H, s),
1.89-2.33 (6H, m), 4.54 (1H, dd, J = 11.4, 3.6 Hz), 5.57 (1H, s), 6.41
(1H, m), 7.27-7.58 (10H, s). Anal. (C27H31F3N4O) C, H, N.
7,7-Dimethyl-5-phenyl-N-{1-propyl-1-[4-(trifluoromethyl)phenyl]-
butyl}-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
(3c). White prisms (yield 21%), mp 175-176 °C (EtOAc-hexane).
1H NMR (CDCl3): δ 0.85-0.95 (6H, m), 1.00-1.30 (4H, m), 1.57
(3H, s), 1.64 (3H, s), 1.85-2.23 (6H, m), 4.55 (1H, dd, J = 11.0, 3.2
Hz), 5.56 (1H, s), 6.37 (1H, s), 7.28-7.57 (9H, m), 7.49 (1H, s). Anal.
(C29H35F3N4O) C, H, N.
N-{1-Butyl-1-[4-(trifluoromethyl)phenyl]pentyl}-7,7-dimethyl-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
(3d). White prisms (yield 10%), mp 113-114 °C (EtOAc-hexane). 1H
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NMR (CDCl3): δ 0.82-1.43 (14H, m), 1.59 (3H, s), 1.66 (3H, s),
1.88-2.24 (6H, m), 4.54-4.57 (1H, m), 5.70 (1H, s), 6.44 (1H, s),
N-[1-Ethyl-1-(4-iodophenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (8i). White prisms
(yield, 65%), mp 182-183 °C (Et2O-hexane). H NMR (CDCl3): δ
0.71-0.83 (6H, m), 1.56 (3H, s), 1.64 (3H, s), 1.84-2.28 (6H, m), 4.54
(1H, dd, J = 11.0, 3.4 Hz), 5.50 (1H, s), 6.40 (1H, s), 7.11 (2H, d, J = 8.8
Hz), 7.27-7.37 (5H, m), 7.49 (1H, s), 7.61 (2H, d, J = 8.8 Hz). Anal.
(C26H31IN4O) C, H, N.
1
7.33-7.64 (10H, m). Anal. (C31H39F3N4O 0.5H2O) C, H, N.
3
N-{1-Isopropyl-2-methyl-1-[4-(trifluoromethyl)phenyl]propyl}-7,7-
dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-
carboxamide Hydrochloride (3e). White prisms (yield, 37%), mp
1
140-142 °C (iPr2O). H NMR (CDCl3, free form): δ 0.73 (3H, dd,
J = 6.6, 3.0 Hz), 1.11 (3H, dd, J = 6.6, 3.0 Hz), 1.21 (3H, d, J = 2.0 Hz),
1.49 (3H, s), 1.57 (3H, s), 1.63 (3H, d, J = 2.0 Hz), 2.02-2.40 (3H, m),
3.41 (1H, dd, J = 8.8, 2.0 Hz), 4.62 (1H, td, J = 11.2, 3.8 Hz), 5.24 (1H,
brs), 6.47 (1H, brs), 7.24-7.58 (10H, m). Anal. (C29H36ClF3N4O)
C, H, N.
N-{1-[4-(Benzyloxy)phenyl]-1-ethylpropyl}-7,7-dimethyl-5-phenyl-
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (8j). White
prisms (yield, 83%), mp 130-131 °C (EtOAc-hexane). 1H NMR
(CDCl3): δ 0.73-0.81 (6H, m), 1.56 (3H, s), 1.64 (3H, s), 1.94-2.25
(6H, m), 4.54 (1H, dd, J = 11.4, 3.0 Hz), 5.01 (2H, s), 5.52 (1H, s), 6.44
(1H, s), 6.92 (2H, d, J = 8.7 Hz), 7.24-7.43 (12H, m), 7.47 (1H, s). Anal.
(C33H38N4O2) C, H, N.
N-[1-(4-Chlorophenyl)-1-ethylpropyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo[1,5-a]pyrimidine-3-carboxamide hydrochloride
(9a). To a solution of 8b (350 mg, 0.78 mmol) in EtOAc (5 mL)
was added 4 M HCl (0.5 mL, 2.0 mmol) in EtOAc at room temperature.
Crystallization from EtOAc-hexane and the crystals was collected by
filtration. Recrystallization from EtOAc-hexane afforded 9a as HCl salt
(270 mg, 71%), mp 160-162 °C. 1H NMR (CDCl3): δ 0.74 (3H, t, J =
7.2 Hz), 0.82 (3H, t, J = 7.2 Hz), 1.78 (3H, s), 1.94 (3H, s), 1.99-2.44
(6H, m), 4.56 (1H, t, J = 9.0 Hz), 7.23-7.38 (10H, m), 8.26 (1H, s),
9.86 (1H, s). Anal. (C26H32Cl2N4O) C, H, N.
N-{Dicyclohexyl[4-(trifluoromethyl)phenyl]methyl}-7,7-dimethyl-
5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
Hydrochloride (3f). White prisms (yield 20%), mp 146-147 °C(EtOAc-
1
hexane). H NMR (CDCl3): δ 0.71-2.48 (29H, m), 3.40 (1H, brs),
6.47 (1H, brs), 6.95 (1H, brs), 7.43-7.51 (11H, m). Anal. (C35H44-
ClF3N4O 1.0H2O) C, H, N.
3
N-[1-(4-Chlorophenyl)-1-ethylpropyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide (8b). White prisms
(yield, 56%), mp 125-126 °C (Et2O-hexane). 1H NMR (CDCl3): δ
0.71-0.85 (6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.85-2.29 (6H, m), 4.54
(1H, dd, J = 11.0, 3.4 Hz), 5.52 (1H, s), 6.14 (1H, s), 7.26-7.34 (9H,
m), 7.50 (1H, s). Anal. (C26H31ClN4O) C, H, N.
N-[1-(3-Chlorophenyl)-1-ethylpropyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo-[1,5-a]pyrimidine-3-carboxamide (8c). White prisms
(yield, 65%), mp 195-196 °C (EtOAc-hexane). H NMR (CDCl3): δ
0.72-0.83 (6H, m), 1.58 (3H, s), 1.64 (3H, s), 1.85-2.26 (6H, m), 4.54
(1H, dd, J = 11.0, 3.0 Hz), 5.52 (1H, s), 6.40 (1H, s), 7.16-7.38 (9H, m),
7.50 (1H, s). Anal. (C26H31ClN4O) C, H, N.
N-[1-(4-Chlorophenyl)-1-ethylpropyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo[1,5-a]pyrimidine-3-carboxamide CH3SO3H salt
(9b). To a solution of 8b (350 mg, 0.78 mmol) in EtOAc (5 mL)
was added CH3SO3H (82 mg, 0.85 mmol) at room temperature. The
solvent was concentrated in vacuo. Crystallization from EtOAc-hexane
and recrystallization from EtOH-Et2O afforded 9b as CH3SO3H salt
(390 mg, 92%), mp 147-149 °C. 1H NMR (CDCl3): δ 0.70-0.82 (6H,
m), 1.71 (3H, s), 1.80 (3H, s), 1.88-2.34 (6H, m), 2.94 (3H, s), 4.56
(1H, dd, J = 9.6, 5.1 Hz), 7.13-7.36 (11H, m), 8.85 (1H, s). Anal. (C27-
1
N-[1-Ethyl-1-(4-fluorophenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo-[1,5-a]pyrimidine-3-carboxamide (8d). White prisms
1
(yield, 54%), mp 155-156 °C (EtOAc-hexane). H NMR (CDCl3): δ
0.73-0.82 (6H, m), 1.61 (3H, s), 1.70 (3H, s), 1.91-2.30 (6H, m), 4.55
(1H, dd, J = 11.4, 3.3 Hz), 5.91 (1H, s), 6.59 (1H, s), 6.96-7.02 (2H, m),
7.29-7.36 (7H, m), 7.87 (1H, s). Anal. (C26H31FN4O) C, H, N.
H35ClN4O4S 0.5H2O) C, H, N.
3
N-[1-(4-Chlorophenyl)-1-ethylpropyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo[1,5-a]pyrimidine-3-carboxamide H2SO4 Salt
(9c). To a solution of 8b (120 mg, 0.27 mmol) in Et2O (2 mL) was
added H2SO4 (29 mg, 0.30 mmol) at room temperature. The crystals
was collected by filtration and washed with hexane to give 9c as H2SO4
salt (110 mg, 75%), mp 137-139 °C. 1H NMR (CDCl3): δ 0.66-0.82
(6H, m), 1.69 (3H, s), 1.77 (3H, s), 1.92-2.04 (6H, m), 4.55-4.60
(1H, m), 7.20-7.37 (10H, m), 7.66 (1H, s), 8.42 (1H, brs), 9.91 (1H, s).
N-[1-Ethyl-1-(3-fluorophenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo-[1,5-a]pyrimidine-3-carboxamide (8e). White prisms
1
(yield, 51%), mp 180-181 °C (EtOAc-hexane). H NMR (CDCl3): δ
0.72-0.84 (6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.86-2.30 (6H, m), 4.54
(1H, dd, J = 11.0, 3.2 Hz), 5.54 (1H, s), 6.41 (1H, s), 6.84-6.94 (1H, m),
7.03-7.16 (2H, m), 7.28-7.39(6H, m), 7.51(1H, s).Anal. (C26H31FN4O)
C, H, N.
Anal. (C26H33ClN4OS 2.5H2O) C, H, N.
3
N-[1-(4-Chlorophenyl)-1-ethylpropyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo[1,5-a]pyrimidine-3-carboxamide CH3CO2H Salt
(9d). To a solution of 8b (120 mg, 0.27 mmol) in EtOAc (5 mL) was
added CH3CO2H (18 mg, 0.30 mmol) at room temperature. The mix-
ture was concentrated in vacuo. Crystallization from Et2O-hexane
afforded 9d as CH3CO2H salt (70 mg, 51%), mp 123-124 °C. 1H NMR
(CDCl3): δ 0.71-0.83 (6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.89-
2.29 (6H, m), 2.10 (3H, s), 4.54 (1H, dd, J = 11.0, 3.4 Hz), 5.53 (1H, s),
6.41 (1H, s), 7.26-7.33 (9H, m), 7.52 (1H, s). Anal. (C28H35ClN4O3)
C, H, N.
N-[1-Ethyl-1-(2-fluorophenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydropy-razolo-[1,5-a]pyrimidine-3-carboxamide (8f). Amorphous
solid (yield, 64%). 1H NMR (CDCl3): δ 0.74-0.83 (6H, m), 1.56 (3H, s),
1.63 (3H, s), 2.01-2.38 (6H, m), 4.54 (1H, dd, J = 12.0, 3.3 Hz), 5.65 (1H,
s), 6.38 (1H, s), 6.94-7.48 (9H, m), 7.48 (1H, s). Anal. (C26H31FN4O)
C, H, N.
N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydrop-yrazolo[1,5-a]pyrimidine-3-carboxamide (8g). White prisms
(yield, 38%), 155-157 °C (Et2O-hexane). 1H NMR (CDCl3): δ 0.72-
0.81 (6H, m), 1.59 (3H, s), 1.64 (3H, s), 1.96-2.23 (6H, m), 2.30 (3H, s),
4.54 (1H, dd, J= 11.7, 3.3 Hz), 5.55 (1H, s), 6.45 (1H, s), 7.12 (2H, d, J=7.8
Hz), 7.23-7.38 (7H, m), 7.51 81H, s), 7.49 (1H, s). Anal. (C27H34-
N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydrop-yrazolo[1,5-a]pyrimidine-3-carboxamide Hydrochloride
(9e). To a solution of 8g (150 mg, 0.35 mmol) in Et2O (3 mL) was
added 4 M HCl in EtOAc (0.3 mL, 1.2 mmol) at room temperature. The
crystals was collected by filtration, and recrystallization from EtOAc-
N4O 0.1H2O) C, H, N.
3
N-[1-Ethyl-1-(4-methoxyphenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
Tetrahydr-opyrazolo[1,5-a]pyrimidine-3-carboxamide (8h). White prisms
(yield, 40%), mp 140-141 °C (Et2O-hexane). 1H NMR (CDCl3): δ
0.71-0.89 (6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.93-2.27 (6H, m), 3.78
(3H, s), 4.55 (1H, dd, J = 11.4, 3.4 Hz), 5.53 (1H, s), 6.45 (1H, s), 6.85
(2H, d, J = 8.8 Hz), 7.23-7.40 (7H, m), 7.48 (1H, s). Anal. (C27H34-
N4O2) C, H, N.
1
hexane afforded 9e as HCl salt (400 mg, 92%), mp 158-160 °C. H
NMR (CDCl3): δ 0.70-0.86 (6H, m), 1.78 (3H, s), 1.94 (3H, s),
1.98-2.43 (6H, m), 4.55-4.60 (1H, t, J = 8.8 Hz), 7.08 (2H, d, J = 8.2
Hz), 7.28-7.35 (8H, m), 7.66 (1H, s), 8.12 (1H, s), 9.82 (1H, s). Anal.
(C27H35ClN4O) C, H, N.
1436
dx.doi.org/10.1021/jm101452x |J. Med. Chem. 2011, 54, 1430–1440

Journal of Medicinal Chemistry
ARTICLE
N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,6,7-
tetrahydrop-yrazolo[1,5-a]pyrimidine-3-carboxamide CH3SO3H Salt
(9f). To a solution of 8g (150 mg, 0.35 mmol) in EtOAc (3 mL) was
added CH3SO3H (40 mg, 0.42 mmol) at room temperature. The mix-
ture was concentrated in vacuo. Crystallization from EtOAc-hexane
afforded 6f as CH3SO3H salt (170 mg, 93%), mp 152-154 °C. 1H NMR
(CDCl3): δ 0.69-0.83 (6H, m), 1.70 (3H, s), 1.79 (3H, s), 1.91-2.20
(6H, m), 2.29 (3H, s), 2.96 (3H, s), 4.53-4.60 (1H, m), 6.69 (1H, br s),
7.10 (2H, d, J = 8.2 Hz), 7.20-7.33 (8H, m), 8.69 (1H, br s). Anal.
temperature. After stirred at the same temperature for 1 h, the reaction
mixture was poured into aqueous NaHCO3, and extracted with EtOAc.
The extract was washed with brine, dried over MgSO4, and concen-
trated in vacuo. The residue was chromatographed on SiO2 with
EtOAc-hexane (1:1) to give crystals. Recrystallization from Et2O-
hexane afforded 14a as prisms (0.28 g, 85%), mp 171-172 °C. 1H NMR
(CDCl3): δ 0.72-0.83 (6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.97-2.22
(6H, m), 2.27 (3H, s), 4.55 (1H, dd, J = 11.0, 3.4 Hz), 5.54 (1H, s), 6.43
(1H, s), 7.04 (2H, d, J = 8.8 Hz), 7.28-7.38 (7H, m), 7.49 (1H, s). Anal.
(C28H38N4O4 0.5H2O) C, H, N.
(C28H34N4O3 0.1H2O) C, H, N.
3
3
Ethyl 4-(1-{[(7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyrimidine-3-yl)carbonyl]amino}-1-ethylpropyl)benzoate
(10). A mixture of 8i (2.0 g, 3.69 mmol), dppf (0.1 g, 0.18 mmol),
Pd(OAc)2 (40 mg, 0.18 mmol), and Et3N (0.82 g, 8.12 mmol) in EtOH
(10 mL) was stirred at 80 °C under 1 atoms CO atmosphere for 3 days.
The catalyst was removed by filtration, and the filtrate was concentrated
in vacuo. The residue was diluted with EtOAc, washed with aqueous
NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo. The
residue was chromatographed on SiO2 with EtOAc-hexane (2:3) to
N-{1-Ethyl-1-[4-(2-oxobutoxy)phenyl]propyl}-7,7-dimethyl-5-phenyl-4,
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (14b). A
mixture of 13 (0.40 g, 0.92 mmol), K2CO3 (0.51 g, 3.68 mmol), and
ethyl bromoacetate (0.18 g, 1.10 mmol) in DMF (10 mL) was stirred at
room temperature for 2 h. The reaction mixture was poured into H2O
and extracted with EtOAc. The extract was washed with aqueous
NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo to
give crystals. Recrystallization from EtOAc-hexane afforded 14b as
1
prisms (0.42 g, 88%), mp 178-179 °C. H NMR (CDCl3): δ 0.70-
1
give crystals 10 as prisms (1.55 g, 86%), mp 181-182 °C. H NMR
0.81 (6H, m), 1.29 (3H, t, J = 7.4 Hz), 1.56 (3H, s), 1.64 (3H, s),
1.94-2.22 (6H, m), 4.26 (2H, q, J = 7.4 Hz), 4.51-4.58 (1H, m), 4.58
(2H, s), 5.51 (1H, s), 6.43 (1H, s), 6.85 (2H, d, J = 8.8 Hz), 7.25-7.38
(7H, m), 7.48 (1H, s). Anal. (C30H38N4O3) C, H, N.
(CDCl3): δ 0.72-0.84 (6H, m), 1.35 (3H, J = 6.8 Hz), 1.57 (3H, s), 1.64
(3H, s), 1.91-2.32 (6H, m), 4.34 (2H, q, J = 6.8 Hz), 4.54 (1H, dd, J =
11.0, 3.2 Hz), 5.57 (1H, s), 6.39 (1H, s), 7.26-7.35 (5H, m), 7.43 (2H,
d, J = 8.4 Hz), 7.52 (1H, s), 7.99 (2H, d, J = 8.4 Hz). Anal. (C29-
H36N4O3) C, H, N.
Ethyl (5R)-7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-
a]pyrimidine-3-carboxylate (15a) and Ethyl (5S)-7,7-Dimethyl-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxylate
(15b). Compound 6 (79.9 g, 0.27 mol) was separated by CHIRAL
column HPLC (CHIRALCEL OD 50 mm I.D. ꢀ 500 mmL, hexane-
EtOH/95:5, flow rate 60 mL/min, temperature 30 °C, detection (UV)
254 nm, 1shot approximately 800 mg, 100 times) to give 15a (39.4 g,
retention time 19.7 min: 99.8% ee) and 15b (37.8 g, retention time 14.8
min: 99.8% ee). Analytical condition of 15a and 15b was CHIRAL
column HPLC (CHIRALCEL OD 4.6 mm I.D. ꢀ 500 mmL, hexane:
EtOH/95:5, flow rate 0.5 mL/min, temperature 30 °C, detection (UV)
254 nm, injection volume 10 μL). 1H NMR (CDCl3): δ 1.31 (3H, t, J =
7.0 Hz), 1.57 (3H, s), 1.64 (3H, s), 2.11-2.15 (2H, m), 4.23 (2H, q, J =
7.0 Hz), 4.64 (1H, dd, J = 9.4, 5.2 Hz), 6.03 (1H, s), 7.30-7.45 (5H, m),
7.64 (1H, s).
(5R)-(þ)-7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidine-3-carboxylic acid (16a). A mixture of 15a (0.67 g, 2.24
mmol) and KOH (0.38 g, 6.77 mmol) in H2O-EtOH (1:1, 40 mL) was
stirred at 90 °C for 12 h and the solvent was concentrated in vacuo. The
residue was diluted with EtOAc and acidified with 1N HCl. The organic
layer was washed brine, dried over MgSO4, and concentrated in vacuo to
give 16a as crystals (0.55 g, 82%), mp 205-206 °C; [R]D20 = 86.01 (c
0.48, CHCl3). 1H NMR (CDCl3): δ 1.59 (3H, s), 1.66 (3H, s), 2.05-
2.15 (2H, m), 4.64 (1H, dd, J = 9.6, 5.4 Hz), 6.04 (1H, s), 7.30-7.41
(5H, m), 7.73 (1H, s).
(5S)-(-)-7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidine-3-carboxylic Acid (16b). A mixture of 15b (0.73 g, 2.44
mmol) and KOH (0.41 g, 7.32 mmol) in H2O-EtOH (1:1, 40 mL) was
stirred at 90 °C for 12 h, and the solvent was concentrated in vacuo. The
residue was diluted with EtOAc and acidified with 1N HCl. The organic
layer was washed brine, dried over MgSO4, and concentrated in vacuo to
give 16b as crystals (0.55 g, 83%), mp 205-206 °C; [R]D20 = -85.33 (c
0.46, CHCl3,). 1H NMR (CDCl3): δ 1.59 (3H, s), 1.66 (3H, s), 2.05-
2.15 (2H, m), 4.64 (1H, dd, J = 9.6, 5.4 Hz), 6.04 (1H, s), 7.30-7.41
(5H, m), 7.73 (1H, s).
(5R)-(-)-N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
Hydrochloride (17a). To a mixture of 16a (25.0 g, 92.1 mmol) and DMF
(1.5 mL) in toluene (250 mL) was added SOCl2 (13.75 mL, 189 mmol)
at room temperature. After stirring at the same temperature for 1 h, the
solvent was evaporated off. A mixture of 3-(4-methylphenyl)pentan-3-amine
N-{1-Ethyl-1-[4-(hydroxymethyl)phenyl]propyl}-7,7-dimethyl-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
(11). To a solution of 10 (500 mg, 1.02 mmol) in THF (50 mL) was
added LAH (117 mg, 3.0 mmol) at 0 °C. After stirred at the same
temperature for 1 h, Na2SO4 10H2O (0.96 g, 3 mmol) was added to the
3
reaction mixture at 0 °C. The whole was stirred at room temperature for
30 min and then filtered through Celite. The solvent was concentrated in
vacuo to give crystals. Recrystallization from EtOAc-hexane afforded
1
11 as prisms (0.38 g, 83%), mp 178-179 °C. H NMR (CDCl3): δ
0.72-0.83 (6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.93-2.26 (6H, m), 4.54
(1H, dd, J = 11.4, 3.6 Hz), 4.66 (2H, s), 5.56 (1H, s), 6.43 (1H, s),
7.26-7.39 (9H, m), 7.50 (1H, s). Anal. (C27H34N4O2) C, H, N.
4-(1-{[(7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-
a]pyrimidine-3-yl)carbonyl]amino}-1-ethylpropyl)benzyl Acetate
(12). To a mixture of 11 (200 mg, 0.45 mmol), Et3N (91 mg, 0.90
mmol), and K2CO3 (75 mg, 0.54 mmol) in THF (10 mL) was added
AcCl (42 mg, 0.54 mmol) at 0 °C. After being stirred at the room
temperature overnight, the reaction mixture was poured into H2O and
extracted with EtOAc. The extract was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was chromatographed
on SiO2 with EtOAc-hexane (1:1) to give 12 as crystals (0.10 g, 46%),
mp 185-186 °C. 1H NMR (CDCl3): δ 0.72-0.83 (6H, m), 1.56 (3H,
s), 1.64 (3H, s), 1.96-2.26 (6H, m), 2.08 (3H, s), 4.54 (1H, dd, J = 11.0,
3.2 Hz), 5.06 (2H, s), 5.54 (1H, s), 6.42 (1H, s), 7.25-7.38 (9H, m),
7.49 (1H, s). Anal. (C29H36N4O3) C, H, N.
N-[1-Ethyl-1-(4-hydroxyphenyl)propyl]-7,7-dimethyl-5-phenyl-4,5,-
6,7-tetrahydro-pyrazolo[1,5-a]pyrimidine-3-carboxamide (13). A mix-
ture of 8j (0.15 g, 0.29 mmol) and 10%Pd-C (50 mg) in MeOH (20 mL)
was stirred at room temperature under hydrogen atmosphere for 4 h. The
catalyst was removed by filtration, and the filtrate was concentrated in
vacuo to give crystals. Recrystallization from THF-hexane afforded 13 as
prisms (0.12 g, 99%), mp 152-153 °C. 1H NMR (CDCl3): δ 0.72-0.81
(6H, m), 1.57 (3H, s), 1.64 (3H, s), 1.93-2.21 (6H, m), 4.56 (1H, dd, J =
11.4, 3.8 Hz), 5.54 (1H, s), 5.69 (1H, s), 6.45 (1H, s), 6.73 (2H, d, J =
8.4 Hz), 7.18-7.35 (7H, m), 7.50 (1H, s). Anal. (C26H32N4O2) C, H, N.
4-(1-{[(7,7-Dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrimidine-3-yl)carbonyl]-amino}-1-ethylpropyl)phenyl Acetate (14a). To
a mixture of 13 (0.30 g, 0.69 mmol) and Et3N (0.21 g, 2.08 mmol)
in THF (20 mL) was added AcCl (60 mg, 0.76 mmol) at room
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hydrochloride (23.65 g, 111 mmol) and Et3N (28.0 g, 277 mmol) in
toluene (250 mL) was stirred at 70 °C for 1 h, and then acid chloride
obtained in toluene was added to the reaction mixture at 70 °C. After
stirring at the same temperature for 3 h, the reaction mixture was poured
into H2O and extracted with EtOAc. The extract was washed with brine,
dried over MgSO4, and concentrated in vacuo. The residue was
chromatographed on SiO2 with EtOAc-hexane (1:1) to give crystals.
To a solution of the crystals obtained in Et2O (150 mL) was added 4N
HCl in EtOAc (25.0 mL, 100 mmol) at 0 °C. The crystals were collected
by filtration and washed with Et2O (35.5 g, 83%), mp 142-143 °C;
Measurement of in Vivo PTH Secretion. To estimate in vivo PTH
secretion of these compounds in rat, plasma intact PTH levels were
assayed using a rat enzyme-linked immunosorbent assay (ELISA) kit
(Rat Bioactive Intact PTH ELISA kit, Immutopics, Inc.) after oral
administration.
Bone-Forming Test in OVX Rat Models. Three-month-old virgin Crj:
CD(SD)IGS rats were subjected either to a bilateral ovariectomy or to a
sham surgery under anesthesia. The OVX rats were divided into two
groups that were matched by the body weight. Sham rats and one group
of OVX rats received an oral dose of a vehicle (0.5% aqueous solution of
methylcellulose). The remaining OVX groups orally received 9e (10
mg/kg) suspended in the vehicle. From the next day of the operation,
the oral treatment continued daily for 3 months. Two months after
starting treatment, a blood sample was collected for osteocalcin mea-
surement. The plasma was obtained by centrifugation, and the super-
natants were collected and plasma osteocalcin levels were measured by
RIA (Immupopics, San Clemente, CA). One day after the last treatment,
animals were sacrificed and the right femur and tibia were removed for
the bone mineral density (BMD) evaluation. The BMD of the distal
femur and proximal tibia were quantified by X-ray-CT scanner for small
animals (LaTheta LCT-100A, ALOKA, Japan).
Metabolic Stability Assay. Metabolic stability assay hepatic micro-
somes from mice, rats, and humans were purchased from Xenotech, LLC
(Lenexa, KS). An incubation mixture with a final volume of 0.1 mL
consisted of microsomal protein in 50 mmol/L KH2PO4-K2HPO4
phosphate buffer (pH 7.4) and 1 mmol/L test compound. The con-
centration of hepatic microsomal protein was 0.2 mg/mL. An NADPH-
generating system containing 50 mmol/L MgCl2, 50 mmol/L glucose-6-
phosphate, 5 mmol/L β-NADPþ, and 15 unit/mL glucose-6-phosphate
dehydrogenase was prepared and added to the incubation mixture with a
10% volume of the reaction mixture. After the addition of the NADPH-
generating system, the mixture was incubated at 37 °C for 0 and 20 min.
The reaction was terminated by the addition of acetonitrile equivalent to
the volume of the reaction mixture. All incubations were made in dup-
licate. Test compound in the reaction mixture was measured by HPLC
system equipped with a UV detector. For metabolic stability determina-
tions, chromatograms were analyzed for parent compound disappear-
ance from the reaction mixtures.
21
1
[R]D = -27.99 (c 0.80, CHCl3). H NMR (CDCl3): δ 0.69-0.86
(6H, m), 1.78 (3H, s), 1.94 (3H, s), 1.98-2.46 (6H, m), 4.54 (1H, t, J =
8.8 Hz), 7.08 (2H, d, J = 8.2 Hz), 7.29-7.35 (8H, m), 8.15 (1H, s), 9.83
(1H, s). Anal. (C27H35ClN4O 0.25H2O) C, H, N.
3
(5S)-(þ)-N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-5-
phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
Hydrochloride (17b). To a mixture of 16b (0.10 g, 0.37 mmol) and
DMF (1 drop) in toluene (1 mL) was added SOCl2 (0.05 mL, 0.69
mmol) at room temperature. After stirring at the same temperature for
1 h, the solvent was evaporated off. A mixture of 3-(4-methylphenyl)-
pentan-3-amine hydrochloride (95 mg, 0.44 mmol) and Et3N (0.11 g,
1.11 mmol) in toluene (1 mL) was stirred at 70 °C for 1 h, and then acid
chloride obtained in toluene was added to the reaction mixture at 70 °C.
After stirring at the same temperature for 3 h, the reaction mixture was
poured into H2O and extracted with EtOAc. The extract was washed
with brine, dried over MgSO4, and concentrated in vacuo. The residue
was chromatographed on SiO2 with EtOAc-hexane (1:1) to give crys-
tals. To a solution of the crystals obtained in Et2O was added 4N HCl in
EtOAc (0.2 mL, 0.80 mmol) at 0 °C. The crystals were collected by
21
filtration and washed with Et2O (0.08 g, 47%), mp 142-143 °C; [R]D
=
31.05 (c 0.65, CHCl3). 1H NMR (CDCl3): δ 0.76-0.85 (6H, m), 1.78
(3H, s), 1.94 (3H, s), 1.98-2.44 (6H, m), 4.55-4.60 (1H, dd, J = 9.6,
5.4 Hz), 7.09 (2H, d, J = 7.8 Hz), 7.28-7.34 (8H, m), 8.04 (1H, s), 9.76
(1H, s). Anal. (C27H35ClN4O 0.25H2O) C, H, N.
3
(5S)-N-{1-Ethyl-1-[4-(trifluoromethyl)phenyl]propyl}-7,7-dimeth-
yl-5-phenyl-4,5,6-,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxa-
mide (17c). To a mixture of 16b (100 mg, 0.37 mmol) and DMF (1
drop) in toluene (2 mL) was added SOCl2 (66 mg, 0.91 mmol) at room
temperature. After stirring at the same temperature for 1 h, the solvent
was evaporated off. A mixture of 3-(4-trifluoromethylphenyl)pentan-3-
amine hydrochloride (129 mg, 0.61 mmol) and Et3N (75 mg, 0.74
mmol) in toluene (2 mL) was stirred at 70 °C for 1 h, and then the acid
chloride obtained in toluene was added to the reaction mixture at 70 °C.
After stirring at the same temperature for 1 h, the reaction mixture was
poured into H2O and extracted with EtOAc. The extract was washed
with H2O and brine, dried over MgSO4, and concentrated in vacuo to
give crystals. Recrystallization from iPrOH afforded 17c (85 mg, 47%),
mp 163-164 °C. 1H NMR (CDCl3): δ 0.71-0.84 (6H, m), 1.21 (6H,
d, J = 6.3 Hz), 1.57 (3H, s), 1.64 (3H, s), 1.90-2.31 (6H, m), 4.00-4.04
(1H, m), 4.54 (1H, dd, J = 11.4, 3.0 Hz), 5.56 (1H, s), 6.38 (1H, s),
Measurement of Solubility. The compounds were added to the
aqueous buffer solution (pH6.8 þ bile acid: 20 mmol/L sodium glyco-
chenodeoxycholate). After incubation, precipitates were separated by
filtration. The thermodynamic solubility was determined by HPLC ana-
lysis of each filtrate.
Plasma Concentration in Rats. Compound 9e was administered
orally to nonfasted Crl:CD(SD) rats (female, 12 weeks old, n = 3) at a
dose of 10 mg/kg in 0.5% methylcellulose suspension. At 0.25, 0.5, 1, 2,
4, 8, and 24 h after oral administration, blood samples were collected and
immediately centrifuged to obtain the plasma fraction. The plasma
samples were deproteinized with acetonitrile. After centrifugation, the
supernatant obtained was diluted with 0.01 mol/L ammonium acetate
and centrifuged again. The compound concentration in the supernatant
was measured by high performance LC system (SHIMADZU, Kyoto,
Japan) consisting of a binary solvent manager (LC-10ADvp), sample
organizer (SCL-10ADvp), sample manager (SIL-10Avp), and column
oven (CTO-10AC). The HPLC conditions were as follows: column,
L-column ODS (4.6 mm ꢀ 250 mm) from Chemicals Evaluation and
Research Institute (Tokyo, Japan); mobile phase, (A) 0.01 mol/L
ammonium acetate/(B) acetonitrile = 3/7; flow rate, 1.0 mL/min;
column temperature, 40 °C; wavelength, 262 nm.
7.26-7.58 (9H, m). Anal. (C27H31F3N4O 1.0i-PrOH) C, H, N.
3
Biological Method. GTPγS Binding Assay. The GTPγS binding
activity was measured as follows. The CaR-expressing cell membrane
was incubated with test compounds for 10 min. The assays were carried
out at room temperature for an hour in a reaction solution mixture
containing 20 mM HEPES (pH.7.4), 100 mM NaCl, 1 mM MgCl2, 167
μg/mL DTT, 5 μM guanosine 50-diphosphate, 0.4 nM [35S]-guanosine
50-(γ-thio) triphosphate ([35S]-GTPγS), and 6 mM CaCl2. The mix-
ture was filtered through a GF/C filter. After washing the fourth time
with 300 μL of phosphate-buffered saline, radioactivity of the filter was
measured using a Top-Count scintillation counter. Data from GTPγS
binding assays for antagonists were analyzed with the use of the Prism
program (GraphPad Software, Inc.). IC50 values were determined through
nonlinear regression analysis performed with Prism.
Permeability of Test Compounds across Caco-2 Monolayers. Caco-
2 monolayers were grown to confluence on collagen-coated, micropor-
ous, polycarbonate membranes in 12-well Costar Transwell plates. The
permeability assay buffer was Hanks’ Balanced Salt Solution containing
10 mmol/L HEPES, 15 mmol/L glucose, and 1% bovine serum albumin
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at a pH of 7.3-7.5. The test compound dosing concentrations were 10
μmol/L in the assay buffer. The cells were dosed on the apical side (A-to-
B) or basolateral side (B-to-A) and incubated at 37 °C with 5% CO2 in a
humidified chamber. At each time point, 1 and 2 h, 200 μL were taken
from the A-to-B receivers, and 50 μL were taken from the B-to-A recei-
vers. Fresh assay buffer was added to the receivers after the 1 h sampling.
Also, at 2 h, 50 μL of the donors were taken. Each determination was
performed in duplicate. The permeability through a cell-free (blank)
membrane was studied to determine nonspecific binding and free
diffusion of the test compounds through the device. The lucifer yellow
flux was also measured for each monolayer after being subjected to the
test compounds to ensure no damage was inflicted to the cell mono-
layers during the flux period. All samples were assayed by LC/MS/MS
using electrospray ionization. The apparent permeability, Papp, and
percent recovery were calculated as follows: Papp = (dCr/dt) ꢀ Vr/
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(A ꢀ C0) (eq 1). Percent recovery = 100((Vr ꢀ Crfinal) þ (Vd
ꢀ
Cdfinal))/(Vd ꢀ C0) (eq 2). Where, dCr/dt is the slope of the cumulative
concentration in the receiver compartment versus time in μmol/L s-1
.
Vr is the volume of the receiver compartment in cm3. Vd is the volume
of the donor compartment in cm3. A is the area of the cell monolayer
(1.1 cm2 for 12-well Transwell). C0 is the nominal concentration of the
dosing solution in μmol/L. Crfinal is the cumulative receiver concentra-
final
tion in μmol/L at the end of the incubation period. Cd is the con-
centration of the donor in μmol/L at the end of the incubation period.
’ ASSOCIATED CONTENT
S
Supporting Information. Methods and instrumentation
b
(8) Dobnig, H. A review of teriparatide and its clinical efficacy in the
treatment of osteoporosis. Expert Opin. Pharmacother. 2004, 5, 1153–
1162.
used to obtain the X-ray crystal structure of compound 17c;
elemental analyses data for compounds 3, 8-14, and 17. This
material is available free of charge via the Internet at http://pubs.
acs.org.
(9) Neer, R. M.; Arnaud, C. D.; Zanchetta, J. R.; Prince, R.; Gaich,
G. A.; Reginster, J. Y.; Hodsman, A. B.; Eriksen, E. F.; Ish-Shalom, S.;
Genant, H. K.; Wang, O.; Mitlak, B. H. Effect of parathyroid hormone
(1-34) on fractures and bone mineral density in postmenopausal
women with osteoporosis. N. Engl. J. Med. 2001, 344, 1434–1441.
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Augsburger, P. L.; Sato, M.; Hock, J. M. Anabolic and catabolic bone
effects of human parathyroid hormone (1-34) are predicted by
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Accession Codes
†CCDC codes: CCDC 800340.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ81-6-6308-9081. Fax: þ81-6-6300-6306. E-mail:
Yoshida_Masato@takeda.co.jp.
’ ACKNOWLEDGMENT
(12) Gerspacher, M; Altmann, E; Beerli, R; Buhl, T; Endres, R;
Gamse, R; Kameni-Tcheudji, J; Kneissel, M; Krawinkler, K. H.; Mis-
sbach, M; Schmidt, A; Seuwen, K; Weiler, S; Widler, L. Penta-sub-
stituted benzimidazoles as potent antagonists of the calcium-sensing
receptor (CaSR-antagonists). Bioorg. Med. Chem. Lett. 2010, 17, 5161–
5164.
(13) Chattopadhyay, N. Biochemistry, physiology and pathophy-
siology of the extracellular calcium-sensing receptor. Int. J. Biochem. Cell
Biol. 2000, 32, 789–804.
We thank Dr. S. Ohkawa and the late Dr. M. Kawase for their
encouragement and helpful advice. We thank K. Higashikawa for
X-ray crystallographic analysis, and Dr. T. Yamano and S.
Yamamoto for optical resolution, Dr. M. Yamaguchi for obtain-
ing rat pharmacokinetic study, and Dr. T. Okuda for measure-
ment of metabolic stability. We thank N. Tsuruta for in vivo
assays.
(14) Fox, J.; Miller, M. A.; Stroup, G. B.; Nemeth, E. F.; Miller, S. C.
Plasma levels of parathyroid hormone that induce anabolic effects in
bone of ovariectomized rats can be achieved by stimulation of endo-
genous hormone secretion. Bone 1997, 21, 163–169.
’ ABBREVIATIONS USED
CaSR, calcium sensing receptor; PTH, parathyroid hormone;
OVX rat, osteopenic ovariectomized rat; GPCR, G-protein
coupled receptor; HTOS, high throughput organic synthesis;
HATU, 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyl uro-
nium hexafluorophosphate; BMD, bone mineral density
(15) Gowen, M.; Stroup, G. B.; Dodd, R. A.; James, I. E.; Votta, B. J.;
Smith, B. R.; Bhatnagar, P. K.; Lago, A. M.; Callahan, J. F.; DelMar, E. G.;
Miller, M. A.; Nemeth, E. F.; Fox, J. Antagonizing the parathyroid
calcium receptor stimulates parathyroid hormone secretion and bone
formation in osteopenic rats. J. Clin. Invest. 2000, 105, 1595–1604.
(16) Kumar, S.; Matheny, C. J.; Hoffman, S. J.; Marquis, R. W.;
Schultz, M.; Liang, X.; Vasko, J. A.; Stroup, G. B.; Vaden, V. R.; Haley,
H.; Fox, J.; Delmar, E. G.; Nemeth, E. F.; Lago, A. M.; Callahan, J. F.;
Bhatnagar, P.; Huffman, W. F.; Gowen, M.; Yi, B.; Danoff, T. M.;
Fitzpatrick, L. A. An orally active calcium-sensing receptor antagonist
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