Novel and potent calcium-sensing receptor antagonists: Discovery of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6, 7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate (TAK-075) as an orally active bone anabolic agent

Bioorganic & Medicinal Chemistry 19 (2011) 1881–1894

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Novel and potent calcium-sensing receptor antagonists: Discovery

of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-

tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate

(TAK-075) as an orally active bone anabolic agent

Masato Yoshida, Akira Mori, Shinji Morimoto, Etsuo Kotani, Masahiro Oka, Kohei Notoya, Haruhiko Makino,

Midori Ono, Mikio Shirasaki, Norio Tada, Hisashi Fujita, Junko Ban, Yukihiro Ikeda, Tomohiro Kawamoto,

⇑

Mika Goto, Hiroyuki Kimura, Atsuo Baba, Tsuneo Yasuma

Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 2-17-85, Jusohomachi, Yodogawa-ku, Osaka 532-8686, Japan

a r t i c l e i n f o

a b s t r a c t

Article history:

The calcium-sensing receptor antagonist (CaSR) has been recognized as a promising target of anabolic

agents for treating osteoporosis. In the course of developing a new drug candidate for osteoporosis, we

found tetrahydropyrazolopyrimidine derivative 1 to be an orally active CaSR antagonist that stimulated

transient PTH secretion in rats. However, compound 1 showed poor physical and chemical stability. In

order to work out this compound’s chemical stability and further understand its in vivo efﬁcacy, we

focused on modifying the 2-position of the tetrahydropyrazolopyrimidine. As a result of chemical

modiﬁcation, we discovered (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-

tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate 10m (TAK-075), which showed

improved solubility, chemical stability, and in vivo efﬁcacy. Furthermore, we describe that evaluating

the active metabolite is important during repeated treatment with short-acting CaSR antagonists.

Received 6 January 2011

Revised 31 January 2011

Accepted 1 February 2011

Available online 24 February 2011

Keywords:

Orally active

Calcium-sensing receptor antagonists

CaSR

Bone anabolic agents

PTH

TAK-075

1. Introduction

intermittent administration of PTH 1–84 or PTH 1–34, continuous

exposure to PTH results in increases in bone turnover with subse-

The maintenance of bone mass depends on the balance between

bone resorption and formation during bone remodeling.¹Age-

related imbalances between increased bone resorption and

decreased formation results in bone loss and osteoporosis. Osteo-

porosis is a common metabolic bone disease in which bones

become fragile, increasing the risk for fractures. Furthermore,

osteoporosis affects around 200 million people worldwide and

thus represents a substantial ﬁnancial burden.^2,3Currently avail-

able osteoporosis treatments either aim to inhibit bone resorption

through the use of antiresorptive agents (bisphosphonates,^4,5

estradiol, calcitonin, raloxifene,⁶etc.) or promote bone formation

with anabolic agents^7–9(recombinant full-length human parathy-

roid hormone (PTH) 1–84 (Nycomed) or teriparatide; the recombi-

nant N-terminal PTH 1-34 amino acid fragment (Lilly); etc.).

PTH is a hormone released by the parathyroid glands. It is the

most important endocrine regulator of human calcium and phos-

phorus levels. In contrast to the anabolic effects observed after

quent losses in bone mass.^10,11The secretion of PTH is strictly

controlled by the calcium-sensing receptor (CaSR),¹²which is a

G-protein coupled receptor (GPCR) expressed on the surface of

parathyroid cells. CaSR senses extracellular levels of the calcium

ion and controls homeostasis by releasing PTH.

Recently, it was reported that antagonists of CaSR (calcilyt-

ics)^13–16can increase the endogenous levels of circulating PTH in

humans. Therefore, orally active short-acting calcilytics became

attractive and promising targets to replace PTH therapies. As part

of our CaSR antagonists program,^17,18we have previously reported

that tetrahydropyrazolopyrimidine derivative 1¹⁸has potent CaSR

antagonistic activity with an IC₅₀of 2.3 nM (Fig. 1). During our

discovery of compound 1, we also learned that solubility and

metabolic stability are key factors for rapid and transient PTH

release.¹⁸We demonstrated that salt formation improves the solu-

bility of the tetrahydropyrazolopyrimidine series, resulting in

excellent efﬁcacy.

However, our investigation of chemical stability revealed that

compound 1 is degraded under the conditions shown in Figure 1.

It was assumed that the degradation was derived from the

⇑

Corresponding author. Tel.: +81 6 6300 6117; fax: +81 6 6300 6306.

E-mail address: Yasuma_Tsuneo@takeda.co.jp (T. Yasuma).

doi:10.1016/j.bmc.2011.02.001

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M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

Figure 1. Calcilytic compound reported in previous literature.

Scheme 1. Reagents and conditions: (a) Me₂C@CHCOPh, CF₃CO₂H, 2-methoxyethanol, or Me₂C@CHCOPh, NaH, DMF; (b) NaBH₄, EtOH or NaBH₄, THF–MeOH or 10% Pd–C, H₂,

THF–MeOH; (c) KOH, H₂O–EtOH; (d) 3-(4-methylphenyl)pentan-3-amine hydrochloride, HATU, iPr₂NEt, DMF or (COCl)₂, DMF, toluene then 3-(4-methylphenyl)pentan-3-

amine hydrochloride, Et₃N, toluene; (e) 4 N HCl–EtOAc.

Scheme 2. Reagents and conditions: (a) CHIRALCEL OD; (b) KOH, H₂O, EtOH; (c) SOCl₂, DMF, toluene, then amine, Et₃N, toluene; (d) 4 N HCl–EtOAc.

liberated hydrochloride from compound 1 due to the weak basicity

of tetrahydropyrazolopyrimidine. Since the basicity of tetrahydro-

pyrazolopyrimidine depends on the electron density of the nitro-

gen atom at 1-position, we considered that introducing an

electron-donating substituent at the 2-position of the tetrahydro-

pyrazolopyrimidine ring enhances basicity to form a stable salt.

In this report, we describe further efforts to improve chemical

stability and in vivo efﬁcacy. Furthermore, the pharmacokinetic

proﬁle of active metabolites is also a signiﬁcant factor for sufﬁcient

efﬁcacy during repeated administrations.

2. Chemistry

The synthesis of 2-substituted 7,7-dimethyltetrahydropyrazol-

opyrimidine derivatives 6a–g is shown in Scheme 1. Amino

pyrazoles¹⁹2a–g were treated with 3-methyl-1-phenylbut-2-en-

1-one to afford dihydropyrazolopyrimidines 3a–g. In this ring

formation step, both the acidic condition (triﬂuoroacetic acid)

and basic condition (sodium hydride) were effective. Reduction

of the pyrimidine ring with 10% palladium on carbon under a

hydrogen atmosphere or sodium tetrahydridoborate (NaBH₄)

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

1883

afforded tetrahydropyrazolopyrimidines 4a–g, and subsequent

hydrolysis afforded carboxylic acids 5a–g. Finally, condensation

reactions were performed using 2-(1H-7-azabenzotriazole-1-yl)-

coupling reagent or through the preparation of the acid chloride

of compound 5 to synthesize amide derivatives 6a–g.

The synthesis of each enantiomer of compound 6a was accom-

plished as outlined in Scheme 2. Racemic ethyl ester 4a, prepared

from 2a, was optically resolved by preparative high performance li-

quid chromatography (HPLC) using a chiral column [CHIRALPAK

OD, hexane/ethanol = 95:5] to afford both enantiomers, 7a and

7b, with high enantiomeric purity (>99% ee). These enantiomers

were converted to carboxylic acids 8a and 8b, respectively, by

alkaline hydrolysis with potassium hydroxide (KOH). Finally, car-

boxylic acids 8a and 8b were treated with 3-(4-methylphenyl)pen-

tan-3-amine to afford amide derivatives and were subsequently

transformed to hydrochloric acid salts 9a and 9b, respectively.

The absolute conﬁguration of 9a was conﬁrmed with X-ray analy-

sis, and its X-ray crystal structure is shown in Figure 2. The abso-

lute conﬁgurations of 7a and 7b were conﬁrmed to be (R)- and

(S)-forms, respectively.²⁰

1,1,3,3-tetramethyl uronium hexaﬂuorophospate (HATU) as

a

Furthermore, some optically active alkyl, alkoxy, and thioalkoxy

substituted analogues 10a–k were prepared as shown in Scheme 3.

Carboxylic acid 8a, prepared in Scheme 2, was treated with appro-

priate amines under the same conditions as described for the syn-

thesis of 6 in Scheme 1. Additionally, in the case of ethyl analogue

10b, benzenesulfuric acid salt 10l and p-toluenesulfuric acid salt

10m were prepared to ﬁnd suitable salt forms.

Active metabolites 15–17 of compound 10m were synthesized

as shown in Scheme 4. Ethyl 4-iodobenzoate 11 was treated with

ethyl magnesium bromide to afford 3-(4-iodophenyl)pentan-3-ol.

This alcohol, without puriﬁcation, could undergo a Ritter reaction²¹

Figure 2. X-ray crystal structure of (R)-9a.

Scheme 3. Reagents and conditions: (a) amine, HATU, iPr₂NEt, DMF or SOCl₂DMF, toluene then amine, Et₃N, toluene; (b) acid.

Scheme 4. Reagents and conditions: (a) EtMgBr, Et₂O; (b) TMSCN, concd H₂SO₄; (c) 6 N HCl and then 4 N HCl–EtOAc; (d) 8a, SOCl₂DMF, Et₃N, toluene; (e) dppf, Pd (OAc)₂,

Et₃N, CO, EtOH; (f) 1 N NaOH, EtOH; (g) MeONHMe hydrochloride, WSC, HOBt, Et₃N, DMF; (h) MeMgBr, THF; (i) NaBH₄, EtOH; (j) 4 N HCl–EtOAc.

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M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

Table 1

CaSR antagonistic activity, metabolic stability, and solubility data of 2-substituted tetrahydropyrazolopyrimidine

a

Compd

R

Additive

Form

IC₅₀(nM)

Metabolic stability^b

Solubility^c

pH 6.8

Rat

Human

pH 1.2

pH 6.8+bile acid

6a

6b

6c

6d

6e

6f

6g

9a

9b

1

Me

Et

nPr

nBu

Ph

HC1

—

HC1

Racemic

R-

4.1 (2.0–8.4)

6.7 (3.3–14)

6.0 (2.9–13)

11 (4.7–25)

81 (43–151)

45 (14–145)

4.7 (1.4–16)

2.4 (1.1–5.2)

31 (9.0–106)

2.3 (0.86–6.4)

213

191

159

136

ND

118

153

157

140

ND

89

124

ND

99

24

0.19

<0.11

<0.12

ND

68

7.8

3.1

0.45

ND

<0.11

30

>94

>98

>97

ND

>99

72

Bn

ND

MeS

Me

H

154

216

ND

<0.11

0.38

ND

S-

R-

ND

5.4

ND

44

144

0.78

ND: no data.

a

95% conﬁdence intervals are shown in parentheses.

b

c

(l

l

L/min/mg).

g/mL).

(

Table 2

in the presence of trimethylsilyl cyanide (TMSCN) and sulfuric acid

(H₂SO₄) to afford N-[1-ethyl-1-(4-methylphenyl)propyl]formam-

ide. The formamide obtained was hydrolyzed with 6 N hydrochlo-

ric acid under reﬂux conditions to provide amine 12. Carboxylic

acid 8a was treated with thionyl chloride and DMF (cat.) in toluene

to form an acyl chloride, which was coupled with amine 12 to af-

ford the corresponding amide 13. An ester moiety was introduced

to the iodo analogue 13 using 1,1⁰-bis(diphenylphosphino)ferro-

cene (dppf) and palladium (II) acetate under a carbon monoxide

atmosphere. Carboxylic acid 15, which was the primary metabolite

of compound 9a, was prepared by hydrolysis of 14, using sodium

hydroxide in ethanol. On the other hand, acetyl analogue 16, which

was a metabolite of 10m, was prepared from carboxylic acid 15 via

the Weinreb amide. Finally, hydroxyl analogue 17 was prepared by

the reduction of 16 with NaBH₄.

Pharmacokinetic parameters for 9a and 15 in cynomolgus monkey plasma after oral

administration of compound 9a

(a) Pharmacokinetic parameters^a,b

9a

15

C_max(ng/mL)

T_max(h)

20.1

1.33

162.9

5.17

266.8

4.33

3680.9

10.82

AUC_{0–24 h}(ng h/mL)

MRT^c(h)

(b) Pharmacokinetic parameters^d,b

9a

7th

1st

14th

24.1

C_{l h}(ng/mL)

32.5

28.2

a

b

c

Dose: 2.5 mg/kg, po.

Mean (n = 3).

Mean residence time.

Dose: 5 mg/kg, po.

d

Table 3

3. Results and discussion

CaSR antagonistic activity and metabolic stability data of 2-methyl tetrahydro-

pyrazolopyrimidine

The compounds synthesized were evaluated for antagonist

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.

The results obtained for 2-substituted derivatives with regard

to in vitro CaSR antagonistic activity, metabolic stability, and solu-

bility are listed in Table 1. Introduction of small alkyl substituents

such as methyl 6a, ethyl 6b, and n-propyl 6c were tolerated,

although compounds with bulky substituents, for example, phenyl

group 6e and benzyl group 6f, had weak activity. Furthermore,

methylthio analogue 6g also exhibited high potency with an IC₅₀

value of 4.4 nM. However, methyl analogue 6a and ethyl analogue

6b had more rapid metabolizing proﬁles compared with compound

1, while the compounds with other alkyl substituents (6c and 6d)

and a methylthio group (6g) exhibited similar metabolic stability.

Interestingly, the solubility of compound 6a, with a methyl group

at the 2-position, was dramatically improved, especially under

a

Compd

R

IC₅₀(nM)

Metabolic stability^b

Rat

Human

10a

10b

10c

10d

10e

10f

10g

10h

10i

H

4-Et

4-iPr

4-MeO

4-EtO

3,4-Di-MeO

4-BnO

3-MeO-4-Cl

3-MeO-4-F

3-Cl-4-MeO

3-F-4-MeO

2.3 (1.0–5.3)

4.2 (1.2–15)

1.7 (0.94–3.1)

2.0 (0.88–4.6)

3.0 (1.7–5.2)

11 (5.3–22)

2.2 (1.3–3.6)

6.1 (4.0–9.1)

11 (6.3–19)

1.7 (0.55–5.5)

4.6 (1.9–11)

231

170

96

183

90

96

239

132

180

48

90

84

105

90

189

218

136

210

153

170

38

the pH 1.2 condition (6a: 24 lg/mL, 1: 5.4 lg/mL).

10j

10k

In our program, improved solubility was reﬂected as rapid PTH

secretion in the in vivo test; therefore, we focused our attention on

the activities of the two enantiomers of 6a with improved solubil-

ity, (R)-form 9a and (S)-form 9b, in order to investigate the stereo-

82

a

95% conﬁdence intervals are shown in parentheses.

b

(lL/min/mg).

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

1885

chemical requirement for inhibitory activities. As expected, a sig-

niﬁcant difference was observed between the enantiomers, and

(R)-form 9a (IC₅₀= 2.4 nM) exhibited approximately 10-fold more

potent activity than (S)-form 9b (IC₅₀= 31 nM), as shown in Table

1. To evaluate the in vivo potency of compound 9a as a CaSR

antagonist, compound 9a, which had identical in vitro activity,

more rapid and transient metabolic proﬁle, and superior solubility

than compound 1, was orally administrated to normal rats.

Figure 3 shows the effects of PTH secretion after oral administra-

tion of 5 mg/kg 1 and oral administration of 2.5 mg/kg 9a in nor-

mal rats. The plasma PTH level increased to 120 pg/mL 30 min

after compound 9a was administered and expeditiously dropped

to normal levels after 2 h. The PTH secretion pattern stimulated

by compound 9a was sharp, similar to that stimulated by com-

pound 1. Judging from the clinical data of teriparatide (PTH 1–

34)⁹and our previous data with an OVX rat model,¹⁸the PTH secre-

tion pattern stimulated by compound 9a (magnitude and duration)

suggests that compound 9a is effective as a bone anabolic agent for

bone formation.

Next, to investigate the effects of our calcilytic on other species,

we attempted to evaluate PTH secretion in cynomolgus monkeys

after they were orally administered compound 9a. As illustrated

in Figure 4, repeated administration of compound 9a (5 mg/kg)

for 1 and 2 weeks dramatically reduced PTH secretion, although

initial administration of compound 9a stimulated an ideal PTH

secretion pattern, as observed in rats. We speculated that this

disappearance of PTH secretion in cynomolgus monkeys was due

to an active metabolite of compound 9a. After identifying the

metabolite by comparison with the synthesized molecule, carbox-

ylic acid 15, with an IC₅₀value of 320 nM, was identiﬁed as the pri-

mary metabolite of compound 9a ( Fig. 5). Pharmacokinetic

parameters for 9a and 15 in cynomolgus plasma after oral admin-

istration of 2.5 mg/kg 9a are shown in Table 2(a). The C_max(266 ng/

mL) and AUC_0–24(3680.9 ng th/mL) of compound 15 were much

higher than those of compound 9a. Furthermore, the MRT of

carboxylic acid 15 was 10.82 h, which is greater than that of com-

pound 9a (MRT: 5.17 h). We also evaluated the plasma concentra-

tion of 9a after the 1st, 7th, and 14th repeated treatment in

cynomolgus (Table 2(b)). In this study, signiﬁcant reduction of ser-

um concentration of compound 9a was not observed in contrast to

the huge reduction of PTH. These ﬁndings indicated the increase

levels of carboxylic acid 15 after repeated administration lead to

continuous antagonism of CaSR, which might cause downregula-

tion of the receptor and diminish the PTH response.

Figure 3. Effects of CaSR antagonists on PTH secretion in normal rats (n = 4).

These ﬁndings indicated that further structural modiﬁcations

are required to avoid the production of carboxylic acid metabolite

15, and hence, compounds with alkyl and alkoxy substituents,

10a–k, were prepared. The CaSR antagonistic activity and

Figure 4. Effects of compound 9a on PTH secretion in cynomolgus monkeys (n = 3,

5 mg/kg, po, qd).

Figure 5. Primary metabolite of compound 9a and 10m in rats and monkeys.

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M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

metabolic stability of 10a–k are shown in Table 3. In the case of al-

kyl derivatives, 4-i-propyl analogue 10c showed high potency, but

its metabolic stability in rats and humans was better than that of

9a. On the other hand, among the alkoxy derivatives, 3,4-dime-

thoxy analogue 10f and 3-methoxy-4-ﬂuoro analogue 10i showed

decreased activity and benzyloxy analogue 10g (IC₅₀= 2.2 nM)

exhibited a stable metabolic proﬁle, contrary to the aim of tran-

sient PTH secretion.

Therefore, we tested several compounds with potent CaSR

antagonistic activity and adequate metabolic stability for their

efﬁcacies with regard to in vivo PTH secretion in rats. As shown

in Table 4, ethyl analogue 10b (2.5 mg/kg, po) stimulated an

increase in the plasma PTH level to 94.5 pg/mL after 0.5 h, and this

level dropped to normal levels after 4 h—a pattern similar to that

observed with compound 1 after it was orally administered at a

dose of 5 mg/kg.

Therefore, we selected 10b as the compound for further evalu-

ation and prepared several salt analogues of 10b. The chemical sta-

bility of related analogues is listed in Table 5. Chemical stabilities

of all 2-methyl substituted analogues (9a, 10b, 10l, and 10m) were

improved compared with compound 1. This result indicated that

introduction of a methyl group at the 2-position of the tetrahydro-

pyrazolopyrimidine ring increased the basicity and improved the

chemical stability of the hydrochloric salt. Among the ethyl ana-

logues 10b, 10l, and 10m, p-toluenesulfonic acid salt 10m showed

the highest stability. Improvement of stability for sulfonic acid

derivative 10l and 10m was assumed that hydrochloric acid is

more volatile and liberated than sulfonic acid.

The results of the in vivo PTH secretion induced by compound

10m in cynomolgus monkeys are shown in Table 6. To conﬁrm that

a reduced PTH response was completely avoided, we evaluated the

effects of oral administration of 10 mg/kg compound 10m. As ex-

pected, after the 28th dose of compound 10m, the PTH response

was maintained. The pharmacokinetic parameters for 10m and pri-

mary active metabolites 16 and 17 in rats and cynomolgus mon-

keys after oral administration are summarized in Table 7. The

MRT values of compound 10m (rat, 3.44 and monkey, 5.99 h), 16

(rat, 5.47 and monkey, 6.36 h), and 17 (rat, 4.82 and monkey,

6.16 h) were shorter than that of carboxylic acid metabolite 15

(rat, 10.82). It was assumed that the absence of carboxylic acid

type metabolite 15 allowed repeated treatment in cynomolgus

monkeys.

Table 4

Effects of CaSR antagonists on PTH secretion in normal rats

Compd

Plasma PTH concentration^a,b,c(pg/mL)

0.5 h

l h

2 h

4 h

10b

10e

10h

10k

94.5 32.2

59.3 4.6

19.1 2.4

21.6 6.0

85.7 30.2

57.0 5.4

19.9 2.0

23.4 2.0

42.0 9.8

24.4 5.8

13.7 0.5

16.8 3.4

26.8 2.1

33.6 0.9

13.3 0.4

12.1 1.1

a

b

c

Mean SD (n = 4).

Dose: 2.5 mg/kg, po.

SD rat, $, 12 weeks.

Table 5

Chemical stability of tetrahydropyrazolopyrimidine

Compd

R¹

R²

Additive

Residual ratio^a(%)

1

9a

10b

10l

10m

H

Me

Et

HC1

52.1

101.7

85.5

Me

4. Conclusion

Et

PhSO₃H

TsOH

98.9

99.1

A novel structural class of tetrahydropyrazolopyrimidines was

optimized as CaSR antagonists and orally active calcilytic agents.

For this series of compounds, it was shown that solubility and

adequate metabolic stability effectively achieved transient PTH

secretion. The introduction of a methyl group at the 2-position of

compound 1 improved solubility, chemical stability, and in vivo

efﬁcacy. In cynomolgus monkeys with repeated treatment, a

signiﬁcant reduction of PTH secretion which might be caused accu-

mulation of active metabolite was observed. To overcome the

accumulation of active metabolite, the phenyl ring at the 3-posi-

tion was subjected to further chemical modiﬁcation. Our results

revealed that compound 10m, which possesses a 4-ethyl substitu-

ent on the phenyl ring, stimulates PTH secretion in cynomolgus

monkeys without signiﬁcant changes with repeated treatment.

Compounds 1 and 9a: exposed to air for 1 week.

Compounds 10b, 10l, and 10m: exposed to air for 2 weeks.

a

Residual ratio was measured by HPLC. Condition: 60 °C, 75% RH, air open.

Table 6

The effects of 10m (TAK-075) on PTH secretion in cynomolgus monkeys

Serum intact PTH (l-84) (pg/mL)^a,b

0 h

0.5 h

8 h

1st

28th

81.2 33.5

42.2 3.4

260.5 112.4

236.6 181.8

51.6 25.6

36.2 10.4

a

Mean SD (n = 3).

Dose: 10 mg/kg, po, qd.

b

Table 7

Pharmacokinetic parameters^afor compound 10m, 16, and 17 in rat and cynomolgus monkey plasma after oral administration of 10m

Pharmacokinetic parameters

Rat^b(dose^d: 2.5 mg/kg)

Cynomolgus^c(dose^d: 5.0 mg/kg)

10m

85.7 17.7

0.50 0.00

247.3 63.0

3.44 0.26

16

17

10m

30.8 17.0

1.00 0.00

352.3 174.4

5.99 0.47

16

17

20.4 7.0

3.33 1.15

241.2 96.0

6.16 0.54

C_max(ng/mL)

T_max(h)

128.2 3.9

1.33 0.58

878.8 41.0

5.47 0.43

116.6 32.9

0.67 0.29

805.0 217.4

4.82 0.22

20.3 5.2

2.33 1.53

228.5 74.1

6.36 0.35

AUC_{0–24 h}(ng h/mL)

MRT^e(h)

a

b

c

Mean SD (n = 3).

SD, $, 12 weeks, non-fasted.

#, fed.

Dose: as free base.

Mean residence time.

d

e

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

1887

Compound 10m also exhibited in vivo efﬁcacy in the OVX rat mod-

el of bone loss (manuscript in preparation). On the basis of its

excellent in vitro and in vivo efﬁcacy and favorable pharmacoki-

netic proﬁle, compound 10m (TAK-075)²²was selected as a candi-

date for clinical investigation.

7.33–7.43 (5H, m). Anal. Calcd for C₁₇H₂₁N₃O₂: C, 68.20; H, 7.07;

N, 14.04. Found: C, 68.08; H, 7.14; N, 14.07.

5.3. Ethyl 7,7-dimethyl-5-phenyl-2-propyl-4,7-dihydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylate (3c)

A solution of 2c (2.62 g, 13.3 mmol) in DMF (10 mL) was added

dropwise to a suspension of NaH (1.06 g, 60% in oil, 26.5 mmol) in

DMF (17.5 mL) on an ice-bath, and the mixture was stirred at 0 °C

for 30 min. 3-Methyl-1-phenylbut-2-en-1-one (2.55 g, 15.9 mmol)

was added dropwise to the mixture at 0 °C, and the mixture was

stirred at room temperature for 16 h. The mixture was diluted with

EtOAc, washed successively with saturated citric acid aqueous

solution, H₂O and brine, dried over anhydrous Na₂SO₄and concen-

5. Experimental

Melting points were determined on a Yanagimoto micromelting

point apparatus or BÜCHI B-545 and uncorrected. ¹H NMR spectra

of deuteriochloroform (CDCl₃) or dimethyl sulfoxide (DMSO-d₆)

solution (internal standard tetramethylsilane (TMS), d 0) were

recorded on

a Varian Gemini-200, Mercury-300 or Bruker

AVANCE-300. Reactions 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. Elemental analysis (C,

H, N) were carried out by the Analytical Department of Takeda

Chemical Industries.

trated in vacuo. The residue was puriﬁed by Purif-Pack (SI 60 lm,

SIZE 60) to give 3c as an oil (1.89 g, 42%). ¹H NMR (CDCl₃): d 0.98

(3H, t, J = 7.2 Hz), 1.37 (3H, t, J = 7.2 Hz), 1.62–1.78 (2H, m), 1.70

(6H, s), 2.76 (2H, t, J = 7.5 Hz), 4.29 (2H, q, J = 7.2 Hz), 4.91 (1H, d,

J = 2.1 Hz), 7.36–7.45 (3H, m), 7.46–7.54 (2H, m), 7.64 (1H, br s).

5.4. Ethyl 7,7-dimethyl-5-phenyl-2-propyl-4,5,6,7-tetrahydro-

5.1. 2,7,7-Trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrazolo[1,5-a]pyrimidine-3-carboxylate (4c)

pyrimidine-3-carboxylic acid (5a)

NaBH₄(468 mg, 12.4 mmol) was added to a solution of 3c

(1.89 g, 5.57 mmol) in EtOH (47 mL) in ice-bath, and the mixture

was stirred at room temperature for 19 h, and concentrated in va-

cuo. The residue was diluted with EtOAc, washed successively with

saturated citric acid aqueous solution, H₂O and brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo. The residue was

puriﬁed by Purif-Pack (SI 60 um, SIZE 60) to give 4c as an oil

(1.57 g, 83%). ¹H NMR (CDCl₃): d 0.97 (3H, t, J = 7 .0 Hz), 1.31 (3H,

t, J = 7.0 Hz), 1.58 (6H, d, J = 11.0 Hz), 1.50–1.80 (2H, m), 2.00–

2.14 (2H, m), 2.72 (2H, t, J = 5.8 Hz), 4.23 (2H, q, J = 7.2 Hz), 4.61

(1H, dd, J = 9.6, 5.2 Hz), 6.15 (1H, br s), 7.30–7.45 (5H, m).

To a solution of 2a (7.5 g, 44.3 mmol) in DMF (70 mL) was

added NaH (1.77 g 60% in oil, 44.3 mmol) at room temperature.

After stirring at the same temperature for 30 min, 3-methyl-1-phe-

nylbut-2-en-1-one (5.9 g, 36.8 mmol) was added to the reaction

mixture. After stirring at the same temperature for 3 h, the reaction

mixture was diluted with EtOH (70 mL) and NaBH₄(6.7 g,

177.1 mol) was added thereto at 0 °C. After stirring at room tem-

perature overnight, the solvent was removed in vacuo. The residue

was diluted with EtOAc, washed with brine, dried over MgSO₄, and

concentrated in vacuo. The residue was chromatographed on SiO₂

with EtOAc–hexane (1:4) to give crystals. A mixture of the crystals

and KOH (9.9 g, 176.4 mmol) in EtOH–H₂O (1:1, 300 mL) was stir-

red at 90 °C overnight, and the solvent was removed in vacuo. The

residue was diluted with EtOAc and acidiﬁed with 1 N HCl. The or-

ganic layer was washed with brine, dried over MgSO₄, and concen-

trated in vacuo to give 5a as crystals (4.33 g, 41%), mp 171–172 °C;

¹H NMR (CDCl₃): d 1.55 (3H, s), 1.61 (3H, s), 2.05–2.12 (2H, m), 2.37

(3H, s), 4.61 (1H, dd, J = 10.2, 4.5 Hz), 6.09 (1H, s), 7.34–7.41 (5H,

m). Anal. Calcd for C₁₆H₁₉N₃O₂: C, 67.35; H, 6.71; N, 14.73. Found:

C, 67.19; H, 6.74; N, 14.84.

5.5. 7,7-Dimethyl-5-phenyl-2-propyl-4,5,6,7-tetrahydropyra-

zolo[1,5-a]pyrimidine-3-carboxylic acid (5c)

A

mixture of 4c (1.57 g, 4.60 mmol) and KOH (911 mg,

16.2 mmol) in EtOH–H₂O (1:1, 32 mL) was stirred at 80 °C for

23 h. and then at 100 °C for 3 days. After cooling, the mixture

was diluted with saturated citric acid aqueous solution and ex-

tracted with EtOAc. The extract was washed successively with

H₂O and brine, dried over anhydrous Na₂SO₄and concentrated in

vacuo. The residue was puriﬁed by Purif-Pack (SI 60 lm, SIZE 60)

5.2. 2-Ethyl-7,7-dimethyl-5-phenyl-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (5b)

to give 5c as colorless crystals (849 mg, 59%). Recrystallization

from AcOEt–Et₂O gave colorless crystals, mp 156–158 °C. ¹H

NMR (CDCl₃): d 0.96 (3H, t, J = 7.2 Hz), 1.58 (6H, d, J = 12.0 Hz),

1.50–1.80 (2H, m), 1.95–2.22 (2H, m), 2.73 (2H, dt, J = 7.4,

2.8 Hz), 4.60 (1H, dd, J = 10.6, 4.8 Hz), 6.13 (1H, br s), 7.30–7.48

(5H, m). Anal. Calcd for C₁₈H₂₅N₃O₂: C, 68.98; H, 7.40; N, 13.41.

Found: C, 68.94; H, 7.46; N, 13.21.

A mixture of 2b (8.0 g, 43.7 mmol), 3-methyl-1-phenylbut-2-

en-1-one (7.0 g, 43.7 mmol) and TFA (9.97 g, 87.4 mmol) in

2-methoxyethanol (100 mL) was reﬂuxed overnight. The solvent

was concentrated in vacuo. The residue was diluted with EtOAc,

washed with aqueous NaHCO₃and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was chromatographed on

SiO₂with EtOAc–hexane (1:3) to give oil (6.9 g). A mixture of the

oil obtained and 10% Pd–C (2.0 g) in THF–MeOH (1:1, 200 mL)

was stirred under H₂atmosphere for 4 h. The inorganic material

was ﬁltered off, and the solvent was removed in vacuo to give

oil. A mixture of the oil and sodium hydroxide (16.0 g, 0.4 mol)

in EtOH–H₂O (1:1, 200 mL) was stirred at 90 °C overnight. The sol-

vent was removed in vacuo. The residue was diluted with EtOAc

and acidiﬁed with 1 N HCl. The organic layer was washed with

brine, dried over MgSO₄, and concentrated in vacuo to give 5b as

crystals (3.42 g, 26%), mp 173–174 °C; ¹H NMR (CDCl₃): d 1.23

(3H, t, J = 7.5 Hz), 1.55 (3H, s), 1.61 (3H, s), 2.04–2.11 (2H, m),

2.77 (2H, q, J = 7.5 Hz), 4.60 (1H, dd, J = 10.5, 4.8 Hz), 6.11 (1H, s),

5.6. Ethyl 2-butyl-7,7-dimethyl-5-phenyl-4,7-dihydropyrazolo-

[1,5-a]pyrimidine-3-carboxylate (3d)

A solution of 2d (3.83 g, 18.1 mmol) in DMF (15 mL) was added

dropwise to a suspension of NaH (1.45 g, 60% in oil, 36.3 mmol) in

DMF (24.5 mL) in ice-bath, and the mixture was stirred at 0 °C for

30 min. 3-Methyl-1-phenylbut-2-en-1-one (3.59 g, 22.4 mmol)

was added dropwise to the mixture at 0 °C, and the mixture was

stirred at room temperature for 16 h. The mixture was diluted with

EtOAc, washed successively with saturated citric acid aqueous

solution, H₂O and brine, dried over anhydrous Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by Purif-Pack (SI

60 l

m, SIZE 60) to give 3d as an oil (3.26 g, 49%). ¹H NMR (CDCl₃):

1888

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

d 0.94 (3H, t, J = 6.9 Hz), 1.36 (3H, t, J = 6.9 Hz), 1.32–1.48 (2H, m),

1.60–1.68 (2H, m), 1.70 (6H, s), 2.78 (2H, t, J = 8.1 Hz), 4.29 (2H, q,

J = 7.2 Hz), 4.91 (1H, d, J = 2.1 Hz), 7.36–7.45 (3H, m), 7.47–7.54

(2H, m), 7.65 (1H, br s).

crystals (374 mg, 68%). Recrystallization from EtOAc–iPr₂O gave

colorless crystals, mp 119–121 °C; ¹H NMR (CDCl₃): d 1.16 (3H, t,

J = 7.2 Hz), 1.65 (6H, d, J = 16.2 Hz), 2.08–2.24 (2H, m), 4.15 (2H,

dq, J = 7.2, 0.9 Hz), 4.69 (1H, dd, J = 10.5, 4.5 Hz), 6.36 (1H, br s),

7.30–7.48 (8H, m), 7.68 (2H, dd, J = 8.1, 1.5 Hz).

5.7. Ethyl 2-butyl-7,7-dimethyl-5-phenyl-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylate (4d)

5.11. 7,7-Dimethyl-2,5-diphenyl-4,5,6,7-tetrahydropyra-

zolo[1,5-a]pyrimidine-3-carboxylic acid (5e)

NaBH₄(775 mg, 20.5 mmol) was added to a solution of 3d

(3.26 g, 9.22 mmol) in EtOH (78 mL) in ice-bath. The mixture was

stirred at room temperature for 22 h. and concentrated in vacuo.

The residue was diluted with EtOAc, washed successively with sat-

urated citric acid aqueous solution, H₂O and brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo. The residue was

A mixture of 4e (350 mg, 0.93 mmol) and KOH (164 mg,

2.92 mmol) in EtOH–H₂O (1:1, 7 mL) was stirred at 80 °C for

23 h. After cooling, the mixture was diluted with saturated citric

acid aqueous solution and extracted with EtOAc. The extract was

washed successively with H₂O and brine, dried over anhydrous

Na₂SO₄and concentrated in vacuo. The residue was puriﬁed by

puriﬁed by Purif-Pack (SI 60 lm, SIZE 60) to give 4d as an oil

(2.41 g, 74%). ¹H NMR (CDCl₃): d 0.94 (3H, t, J = 7.2 Hz), 1.31 (3H,

t, J = 6.9 Hz), 1.36–1.48 (2H, m), 1.58 (6H, d, J = 16.8 Hz), 1.58–

1.70 (2H, m), 2.02–2.18 (2H, m), 2.74 (2H, dt, J = 8.4, 3.0 Hz), 4.23

(2H, q, J = 6.3 Hz), 4.61 (1H, dd, J = 10.5, 4.5 Hz), 6.15 (1H, br s),

7.28–7.46 (5H, m).

Purif-Pack (SI 60

(231 mg, 71%). Recrystallization from EtOAc–Et₂O gave colorless

crystals, mp 172–174 °C; ¹H NMR (CDCl₃):

1.64 (6H, d,

lm, SIZE 60) to give 5e as colorless crystals

d

J = 16.5 Hz), 2.10–2.24 (2H, m), 4.67 (1H, dd, J = 10.2, 4.2 Hz), 6.36

(1H, br s), 7.30–7.50 (8H, m), 7.68 (2H, dd, J = 8.1, 1.8 Hz). Anal.

Calcd for C₂₁H₂₁N₃O₂ꢀ1.0C₄H₈O₂: C, 68.95; H, 6.70; N, 9.65. Found:

C, 69.01; H, 6.75; N, 9.68.

5.8. 2-Butyl-7,7-dimethyl-5-phenyl-4,5,6,7-tetrahydropyra-

zolo[1,5-a]pyrimidine-3-carboxylic acid (5d)

5.12. Ethyl 2-benzyl-7,7-dimethyl-5-phenyl-4,7-dihydro-

A

mixture of 4d (2.41 g, 6.78 mmol) and KOH (1.34 g,

pyrazolo[1,5-a]pyrimidine-3-carboxylate (3f)

23.9 mmol) in EtOH–H₂O (1:1, 48 mL) was stirred at 80 °C for

17 h. and then at 100 °C for 3 days. After cooling, the mixture

was diluted with saturated citric acid aqueous solution and ex-

tracted with EtOAc. The extract was washed successively with

H₂O and brine, dried over anhydrous Na₂SO₄and concentrated in

A solution of 2f (4.9 g, 20.0 mmol) in DMF (10 mL) was added

dropwise to a suspension of NaH (1.6 g, 60% in oil, 40.0 mmol) in

DMF (40 mL) in ice-bath, and the mixture was stirred at 0 °C for

10 min. 3-Methyl-1-phenylbut-2-en-1-one (3.52 g, 22.0 mmol)

was added dropwise to the mixture at 0 °C, and the mixture was

stirred at room temperature for 21 h. The mixture was diluted with

EtOAc, washed successively with saturated citric acid aqueous

solution, H₂O and brine, dried over anhydrous Na₂SO₄and concen-

vacuo. The residue was puriﬁed by Purif-Pack (SI 60 lm, SIZE 60)

to give 5d as colorless crystals (611 mg, 28%). Recrystallization

from AcOEt–Et₂O gave colorless crystals, mp 160–162 °C. ¹H

NMR (CDCl₃): d 0.912 (3H, t, J = 7.2 Hz), 1.32–1.45 (2H, m), 1.58

(6H, d, J = 18.0 Hz), 1.58–1.70 (2H, m), 2.00–2.18 (2H, m), 2.75

(2H, dt, J = 8.1, 3.9 Hz), 4.61 (1H, dd, J = 10.8, 4.2 Hz), 6.14 (1H, br

s), 7.30–7.46 (5H, m). Anal. Calcd for C₁₉H₂₅N₃O₂: C, 69.70; H,

7.70; N, 12.83. Found: C, 69.69; H, 7.67; N, 12.84.

trated in vacuo. The residue was puriﬁed by Purif-Pack (SI 60 lm,

SIZE 60) to give 3f as an oil (1.93 g, 25%). ¹H NMR (CDCl₃): d 1.25

(3H, t, J = 7.2 Hz), 1.74 (6H, s), 4.16 (2H, s), 4.20 (2H, q,

J = 7.2 Hz), 4.92 (1H, d, J = 2.4 Hz), 7.12–7.34 (5H, m), 7.36–7.44

(3H, m), 7.46–7.52 (2H, m), 7.64 (1H, br s).

5.9. Ethyl 7,7-dimethyl-2,5-diphenyl-4,7-dihydropyrazolo[1,5-

a]pyrimidine-3-carboxylate (3e)

5.13. Ethyl 2-benzyl-7,7-dimethyl-5-phenyl-4,7-tetrahydro-

pyrazolo [1,5-a]pyrimidine-3-carboxylate (4f)

A solution of 2e (2.27 g, 9.82 mmol) in DMF (10 mL) was added

dropwise to a suspension of NaH (785 mg, 60% in oil, 19.6 mmol) in

DMF (20 mL) in ice-bath, and the mixture was stirred at 0 °C for

10 min. 3-Methyl-1-phenylbut-2-en-1-one (1.73 g, 10.8 mmol)

was added dropwise to the mixture at 0 °C, and the mixture was

stirred at room temperature for 21 h. The mixture was diluted with

EtOAc, washed successively with saturated citric acid aqueous

solution, H₂O and brine, dried over anhydrous Na₂SO₄and concen-

NaBH₄(419 mg, 11.1 mmol) was added to a solution of 3f

(1.95 g, 5.03 mmol) in EtOH (42 mL) in ice-bath. The mixture was

stirred at room temperature for 23 h, and concentrated in vacuo.

The residue was diluted with EtOAc, washed successively with sat-

urated citric acid aqueous solution, H₂O and brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo. The residue was

puriﬁed by Purif-Pack (SI 60

l

m, SIZE 60) to give 4f as an amor-

trated in vacuo. The residue was puriﬁed by Purif-Pack (SI 60

l

m,

phous solid (1.47 g, 76%). ¹H NMR (CDCl₃):

d

1.18 (3H, t,

SIZE 60) to give 3e as an oil (550 mg, 15%). ¹H NMR (CDCl₃): d 1.24

(3H, t, J = 7.2 Hz), 1.77 (6H, s), 4.23 (2H, q, J = 7.2 Hz), 4.98 (1H, d,

J = 2.1 Hz), 7.26–7.48 (5H, m), 7.50–7.60 (3H, m), 7.68–7.74 (2H,

m), 7.86 (1H, br s).

J = 6.9 Hz), 1.62 (6H, d, J = 18.0 Hz), 2.02–2.20 (2H, m), 4.12 (2H,

s), 4.13 (2H, q, J = 9.6 Hz), 4.61 (1H, dd, J = 10.2, 4.2 Hz), 6.14 (1H,

br s), 7.10–7.44 (10H, m).

5.14. 2-Benzyl-7,7-dimethyl-5-phenyl-4,5,6,7-tetrahydro-

5.10. Ethyl 7,7-dimethyl-2,5-diphenyl-4,5,6,7-tetrahydropyra-

pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (5f)

zolo[1,5-a]pyrimidine-3-carboxylate (4e)

A

mixture of 4f (1.18 g, 3.03 mmol) and KOH (729 mg,

NaBH₄(124 mg, 3.28 mmol) was added to a solution of 3e

(550 mg, 8.78 mmol) in EtOH (12 mL) in ice-bath. The mixture

was stirred at room temperature for 24 h, and concentrated in va-

cuo. The residue was diluted with EtOAc, washed successively with

saturated citric acid aqueous solution, H₂O and brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo. The residue was

13.0 mmol) in EtOH–H₂O (1:1, 60 mL) was stirred at 80 °C for

23 h. After cooling, the mixture was diluted with saturated citric

acid aqueous solution and extracted with EtOAc. The extract was

washed successively with H₂O and brine, dried over anhydrous

Na₂SO₄and concentrated in vacuo. The residue was puriﬁed by

Purif-Pack (SI 60

lm, SIZE 60) to give 5f as colorless crystals

puriﬁed by Purif-Pack (SI 60 lm, SIZE 60) to give 4e as colorless

(657 mg, 60%). Recrystallization from EtOAc–Et₂O gave colorless

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

1889

crystals, mp 147–149 °C; ¹H NMR (CDCl₃):

d

1.60 (6H, d,

(6H, m), 1.55 (3H, t, J = 7.5 Hz), 1.81 (3H, s), 1.96 (3H, s), 2.00–

2.26 (6H, m), 2.31 (3H, s), 3.14–3.32 (2H, m), 4.55 (1H, t,

J = 7.5 Hz), 5.72 (1H, s), 7.13–7.20 (4H, m), 7.29–7.37 (6H, m). Anal.

Calcd for C₂₉H₃₉ClN₄O: C, 70.35; H, 7.94; N, 11.32. Found: C, 69.95;

H, 7.99; N, 11.21.

J = 18.3 Hz), 2.00–2.18 (2H, m), 4.13 (2H, dd, J = 22.5, 14.4 Hz),

4.60 (1H, dd, J = 10.5, 4.5 Hz), 6.08 (1H, br s), 7.11–7.45 (10H, m).

Anal. Calcd for C₂₂H₂₃N₃O₂: C, 73.11; H, 6.41; N, 11.63. Found: C,

73.19; H, 6.52; N, 11.50.

5.15. 7,7-Dimethyl-2-(methylthio)-5-phenyl-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (5g)

5.18. N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-5-

phenyl-2-propyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-

3-carboxamide hydrochloride (6c)

A solution of 2g (11.0 g, 54.7 mmol), 3-methyl-1-phenylbut-2-

en-1-one (9.4 g, 58.7 mmol) and TFA (13.4 g, 117 mmol) in

2-methoxyethanol (100 mL) was reﬂuxed overnight, and the sol-

vent was concentrated in vacuo. The residue was diluted with

EtOAc, washed with aqueous NaHCO₃and brine, dried over MgSO₄,

and concentrated in vacuo. The residue was chromatographed on

SiO₂with EtOAc–hexane (1:6) to give oil. NaBH₄(2.55 g,

67.4 mmol) was added to a solution of the oil obtained in

MeOH–THF (1:1, 200 mL) at 0 °C. After stirring at room tempera-

ture overnight, the solvent was concentrated in vacuo. The residue

was diluted with EtOAc, washed with brine, dried over MgSO₄, and

concentrated in vacuo to give oil. A mixture of the oil and KOH

(16.0 g, 285 mmol) in EtOH–H₂O (1:1, 400 mL) was stirred at

90 °C overnight, and the solvent was concentrated in vacuo. The

residue was diluted with EtOAc and acidiﬁed with 1 N HCl. The or-

ganic layer was washed brine, dried over MgSO₄, and concentrated

in vacuo to give 5g as crystals (4.95 g, 27%), mp 139–140 °C; ¹H

NMR (CDCl₃): d 1.55 (3H, s), 1.61 (3H, s), 2.07–2.15 (2H, m), 2.50

(3H, s), 4.57–4.64 (1H, m), 5.67 (1H, s), 6.07 (1H, s), 7.39 (5H, s).

Anal. Calcd for C₁₆H₁₉N₃O₂S: C, 60.54; H, 6.03; N, 13.24. Found:

C, 60.68; H, 6.17; N, 13.11.

Oxalyl chloride (0.17 mL, 2.01 mmol) and a drop of DMF were

added dropwise to a solution of 5c (393 mg, 1.25 mmol) in THF

(6 mL), and the mixture was stirred at room temperature for 3 h

and concentrated in vacuo. A solution of the residue in toluene

(2 mL) was added dropwise to

a mixture of 3-(4-methyl-

phenyl)pentan-3-amine hydrochloride (268 mg, 1.26 mmol) and

Et₃N (0.53 mL, 3.80 mmol) in toluene (4 mL) at 60 °C, and the mix-

ture was stirred at 60 °C for 1 h. The mixture was diluted with

EtOAc, washed successively with H₂O and brine, dried over anhy-

drous Na₂SO₄and concentrated in vacuo. The residue was puriﬁed

by Purif-Pack (SI 60 lm, SIZE 60) to give colorless crystals (218 mg,

37%). 4 N HCl in EtOAc (0.099 ml, 0.40 mmol) was added dropwise

to a solution of the crystals obtained (170 mg, 0.36 mmol) in EtOAc

(2 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h. and concen-

trated in vacuo to give 6c (121 mg, 66%) as colorless crystals, mp

102–104 °C; ¹H NMR (CDCl₃): d 0.78 (6H, dt, J = 17.0, 7.4 Hz),

1.15 (3H, t, J = 7.3 Hz), 1.81 (3H, s) 1.95 (3H, s), 1.98–2.26 (9H,

m), 2.31 (3H, s), 3.04–3.29 (2H, m), 4.56 (1H, t, J = 7.3 Hz), 5.70

(1H, s), 7.15 (2H, d, J = 8.0 Hz), 7.18 (2H, d, J = 8.4 Hz), 7.29–7.33

(2H, m), 7.33–7.40 (3H, m). Anal. Calcd for C₃₀H₄₁ClN₄Oꢀ0.5C₄H₈O₂:

C, 69.48; H, 8.20; N, 10.13. Found: C, 69.48; H, 8.33; N, 10.25.

5.16. N-[1-Ethyl-1-(4-methylphenyl)propyl]-2,7,7-trimethyl-5-

phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carbox-

amide hydrochloride (6a)

5.19. 2-Butyl-N-[1-ethyl-1-(4-methylphenyl)propyl]-7,7-

dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (6d)

A mixture of 5a (3.0 g, 10.5 mmol), HATU (4.80 g, 12.6 mmol)

and iPr₂NEt (4.08 g, 31.6 mmol) in DMF (50 mL) was stirred at

room temperature for 1 h, and then 3-(4-methylphenyl)pentan-

3-amine hydrochloride (2.70 g, 12.7 mmol) was added. The whole

was stirred at 70 °C overnight, and concentrated in vacuo. The res-

idue was diluted with EtOAc, washed with H₂O and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was chro-

matographed on SiO₂with EtOAc–hexane (2:3) to give crystals.

The crystals were dissolved in Et₂O and 4 M HCl in EtOAc was

added. The crystals were collected by ﬁltration and washed with

Et₂O (2.58 g, 51%), mp 161–162 °C; ¹H NMR (CDCl₃): d 0.75–0.88

(6H, m), 1.80 (3H, s), 1.95 (3H, s), 2.00–2.30 (6H, m), 2.31 (3H, s),

2.85 (3H, s), 4.56 (1H, br s), 5.66 (1H, br s), 7.17–7.34 (10H, m).

Anal. Calcd for C₂₈H₃₇ClN₄O: C, 69.91; H, 7.75; N, 11.65. Found:

C, 69.84; H, 7.78; N, 11.59.

Oxalyl chloride (0.13 mL, 1.54 mmol) and a drop of DMF were

added dropwise to a solution of 5d (300 mg, 0.92 mmol) in THF

(6 mL). The mixture was stirred at room temperature for 2 h and

concentrated in vacuo. A solution of the residue in toluene

(2 mL) was added dropwise to

a mixture of 3-(4-methyl-

phenyl)pentan-3-amine hydrochloride (195 mg, 0.92 mmol) and

Et₃N (0.39 mL, 2.80 mmol) in toluene (4 mL) at 60 °C, and the mix-

ture was stirred at 60 °C for 1.5 h. The mixture was diluted with

EtOAc, washed successively with H₂O and brine, dried over anhy-

drous Na₂SO₄and concentrated in vacuo. The residue was puriﬁed

by Purif-Pack (SI 60

(148 mg, 33%). 4 N HCl in EtOAc (0.062 mL, 0.25 mmol) was added

dropwise to solution of the crystals obtained (110 mg,

lm, SIZE 60) to give as colorless crystals

a

0.23 mmol) in EtOAc (1 mL) at 0 °C, and the mixture was stirred

at 0 °C for 1 h. and concentrated in vacuo to give 6d as colorless

crystals (80 mg, 68%). Recrystallization from EtOAc–Et₂O gave col-

orless crystals, mp 103–105 °C; ¹H NMR (CDCl₃): d 0.78 (6H, dt,

J = 18.1, 7.3 Hz), 1.00 (3H, t, J = 7.3 Hz), 1.49–1.60 (2H, m), 1.78

(3H, s), 1.82–1.91 (2H, m), 1.92 (3H, s), 1.95–2.26 (7H, m), 2.31

(3H, s), 3.03–3.29 (2H, m), 4.55 (1H, t, J = 6.4 Hz), 5.70 (1H, s),

7.14 (2H, d, J = 8.0 Hz), 7.19 (2H, d, J = 8.4 Hz), 7.29–7.39 (5H, m).

Anal. Calcd for C₃₁H₄₃ClN₄Oꢀ0.9H₂O: C, 69.03; H, 8.37; N, 10.39.

Found: C, 69.11; H, 8.17; N, 10.18.

5.17. 2-Ethyl-N-[1-ethyl-1-(4-methylphenyl)propyl]-7,7-

dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (6b)

A mixture of 5b (1.0 g, 3.34 mmol), HATU (1.52 g, 4.00 mmol)

and iPr₂NEt (1.30 g, 10.1 mmol) in DMF (30 mL) was stirred at

room temperature for 1 h, and then, 3-(4-methylphenyl)pentan-

3-amine hydrochloride (1.07 g, 5.03 mmol) was added. The whole

was stirred at 70 °C overnight, and concentrated in vacuo. The res-

idue was diluted with EtOAc, washed with H₂O and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was chro-

matographed on SiO₂with EtOAc–hexane (2:3) to give crystals.

The crystals were dissolved in Et₂O and 4 M HCl in EtOAc was

added. The crystals were collected by ﬁltration and washed with

Et₂O (0.10 g, 6%), mp 182–183 °C; ¹H NMR (CDCl₃): d 0.74–0.83

5.20. N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-2,5-

diphenyl-4,5,6,7-tetrahydopyrazolo[1,5-a]pyrimidine-3-

carboxamide (6e)

Oxalyl chloride (0.079 mL, 0.93 mmol) and a drop of DMF were

added dropwise to a solution of 5e (200 mg, 0.58 mmol) in THF

1890

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

(5 mL). The mixture was stirred at room temperature for 30 min,

and concentrated in vacuo. A solution of the residue in toluene

5.23. Ethyl (5R)-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylate (7a) and Ethyl (5S)-

2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo-[1,5-a]-

pyramiddine-3-carboxylate (7b)

(2 mL) was added dropwise to

a mixture of 3-(4-methyl-

phenyl)pentan-3-amine hydrochloride (135 mg, 0.63 mmol) and

Et₃N (0.24 mL, 1.72 mmol) in toluene (3 mL) at 60 °C, and the mix-

ture was stirred at 60 °C for 1 h. The mixture was diluted with

EtOAc, washed successively with H₂O and brine, dried over anhy-

drous Na₂SO₄and concentrated in vacuo. The residue was puriﬁed

Compound 4a (43.2 g, 138 mmol) was separated by CHIRAL Col-

umn HPLC (CHIRALCEL OD 50 mm ID ꢁ 500 mml, hexane/EtOH

95:5, ﬂow rate 60 mL/min, temperature 30 °C, detection(UV)

254 nm, one shot 600 mg) to give 7a (20.7 g, retention time big:

99.9% ee) and 7b (20.6 g, retention time small: 99.9% ee). ¹H

NMR (CDCl₃): d 1.31 (3H, t, J = 7.5 Hz), 1.55 (3H, s), 1.61 (3H, s),

2.05–2.12 (2H, m), 2.37 (3H, s), 4.23 (2H, q, J = 7.5 Hz), 4.62 (1H,

dd, J = 9.9, 4.2 Hz), 6.10 (1H, s), 7.33–7.44 (5H, m).

by Purif-Pack (SI 60 lm, SIZE 60) to give 6e as colorless crystals

(207 mg, 71%). Recrystallization from AcOEt–Et₂O gave colorless

crystals, mp 204–206 °C. ¹H NMR (CDCl₃): d 0.53 (6H, dt, J = 12.0,

7.2 Hz), 1.65 (6H, d, J = 15.0 Hz), 1.68–1.98 (4H, m), 2.02–2.12

(1H, m), 2.23 (1H, t, J = 12.0 Hz), 2.26 (3H, s), 4.61 (1H, dd,

J = 11.1, 2.1 Hz), 5.44 (1H, br s), 6.74 (1H, br s), 6.93 (2H, d,

J = 7.8 Hz), 7.01 (2H, d, J = 8.4 Hz), 7.24–7.36 (3H, m). 7.40 (2H, d,

J = 6.9 Hz), 7.42–7.54 (3H, m), 7.64 (2H, d, J = 8.1 Hz). Anal. Calcd

for C₃₃H₃₈N₄O: C, 78.23; H, 7.56; N, 11.06. Found: C, 78.14; H,

7.53; N, 10.94.

5.24. (5R)-2,7,7-Trimethyl-5-phenyl-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (8a)

A mixture of 7a (11.8 g, 37.7 mmol) and KOH (20.7 g, 0.37 mol)

in H₂O–EtOH (1:1, 200 mL) was stirred at 90 °C for 16 h, and then

concentrated in vacuo. The residue was diluted with H₂O and ad-

justed at pH 4 with 1 N HCl to give 8a as crystals (17.8 g, 91%).

5.21. 2-Benzyl-N-[1-ethyl-1-(4-methylphenyl)propyl]-7,7-

dimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (6f)

½

a ²_D¹= 103.65 (c 1.17, CHCl₃). ¹H NMR (CDCl₃): d 1.55 (3H, s),

1.61 (3H, s), 2.05–2.12 (2H, m), 2.37 (3H, s), 4.61 (1H, dd,

ꢂ

Oxalyl chloride (0.095 mL, 1.12 mmol) and a drop of DMF were

added dropwise to a solution of 5f (250 mg, 0.69 mmol) in THF

(5 mL). The mixture was stirred at room temperature for 30 min,

and concentrated in vacuo. A solution of the residue in toluene

J = 10.2, 4.5 Hz), 6.09 (1H, s), 7.34–7.41 (5H, m). Anal. Calcd for

C₁₆H₁₉N₃O₂: C, 67.35; H, 6.71; N, 14.73. Found: C, 67.40; H, 6.83;

N, 14.63.

(2 mL) was added dropwise to

a mixture of 3-(4-methyl-

5.25. (5S)-2,7,7-Trimethyl-5-phenyl-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (8b)

phenyl)pentan-3-amine hydrochloride (163 mg, 0.77 mmol) and

Et₃N (0.29 mL, 2.08 mmol) in toluene (3 mL) at 60 °C, and the mix-

ture was stirred at 60 °C for 1 h. The mixture was diluted with

EtOAc, washed successively with H₂O and brine, dried over anhy-

drous Na₂SO₄and concentrated in vacuo. The residue was puriﬁed

A mixture of 7b (7.0 g, 22.3 mmol) and KOH (5.0 g, 89.1 mmol)

in H₂O–EtOH (1:1, 200 mL) was stirred at 90 °C overnight, and then

concentrated in vacuo. The residue was diluted with EtOAc and

acidiﬁed with 1 N HCl. The organic layer was washed brine, dried

over MgSO₄, and concentrated in vacuo to give 8b as crystals

by Purif-Pack (SI 60 lm, SIZE 60) to give colorless crystals (256 mg,

71%). 4 N HCl in EtOAc (0.11 mL, 0.44 mmol) was added dropwise

to a solution of the crystals obtained (200 mg, 0.38 mmol) in EtOAc

(2 mL) at 0 °C, and the mixture was stirred at 0 °C for 1 h. and con-

centrated in vacuo to give 6f as colorless crystals (114 mg, 53%).

Recrystallization from MeOH–EtOAc gave colorless crystals, mp

194–196 °C; ¹H NMR (CDCl₃): d 0.47 (6H, dt, J = 7.5, 4.8 Hz),

1.40–1.80 (4H, m), 1.92 (6H, d, J = 44.0 Hz), 2.12–2.30 (2H, m),

2.26 (3H, s), 4.55 (1H, dd, J = 10.1, 5.1 Hz), 4.65 (2H, dd, J = 86.1,

16.8 Hz), 5.49 (1H, br s), 6.91 (2H, d, J = 8.4 Hz), 7.13 (2H, d,

J = 7.8 Hz), 7.20–7.42 (10H, m). Anal. Calcd for C₃₄H₄₁ClN₄O: C,

73.29; H, 7.42; N, 10.06. Found: C, 73.04; H, 7.46; N, 10.13.

(5.0 g, 78%). ½a ²_D¹

ꢂ

= ꢃ109.21 (c 1.25, CHCl₃). ¹H NMR (CDCl₃): d

1.55 (3H, s), 1.61 (3H, s), 2.05–2.12 (2H, m), 2.37 (3H, s), 4.61

(1H, dd, J = 10.2, 4.5 Hz), 6.09 (1H, s), 7.34–7.41 (5H, m). Anal. Calcd

for C₁₆H₁₉N₃O₂: C, 67.35; H, 6.71; N, 14.73. Found: C, 67.19; H,

6.73; N, 14.51.

5.26. (5R)-N-[1-Ethyl-1-(4-methylphenyl)propyl]-2,7,7-tri-

methyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (9a)

To a mixture of 8a (19.8 g, 69.4 mmol) and DMF (0.5 mL) in

toluene (200 mL) was added SOCl₂(12.4 g, 104 mmol) at room

temperature. After stirring at the same temperature for 1 h, the

solvent was evaporated off. A mixture of 3-(4-methylphenyl)pen-

tan-3-amine hydrochloride (17.1 g, 80.4 mmol) and Et₃N (20.2 g,

200 mmol) in toluene (200 mL) was stirred at 70 °C for 20 min,

and then acid chloride obtained in toluene was added to the reac-

tion mixture at 65 °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, 10% critic acid and

brine, dried over MgSO₄, and concentrated in vacuo. The residue

5.22. N-[1-Ethyl-1-(4-methylphenyl)propyl]-7,7-dimethyl-2-

(methylthio)-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (6g)

A mixture of 5g (1.0 g, 3.15 mmol), HATU (1.44 g, 3.79 mmol),

and iPr₂NEt (1.22 g, 9.44 mmol) in DMF (20 mL) was stirred at

room temperature for 1 h, and then, 3-(4-methylphenyl)pentan-

3-amine hydrochloride (0.80 g, 3.76 mmol) was added thereto.

The whole was stirred at 70 °C overnight, and concentrated in va-

cuo. The residue was diluted with EtOAc, washed with H₂O and

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was chromatographed on SiO₂with EtOAc–hexane (2:3) to give

crystals. The crystals were dissolved in Et₂O and 4 N HCl in EtOAc

was added thereto. The crystals were collected by ﬁltration and

recrystallization from EtOAc–hexane afforded 6g as prisms

(0.58 g, 36%), mp 106–107 °C; ¹H NMR (CDCl₃): d 0.72–0.84 (6H,

m), 1.85 (3H, s), 1.95 (3H, s), 2.00–2.22 (6H, m), 2.31 (3H, s), 3.04

(3H, s), 4.52–4.60 (1H, m), 7.10–7.36 (11H, m). Anal. Calcd for

was puriﬁed by Purif-Pack (SI 60 lm, SIZE 60) to give an oil. To

a solution of the oil obtained in EtOAc was added 4 N HCl in

EtOAc (30 mL, 0.12 mol) at 0 °C. The crystals were collected by ﬁl-

tration (26.7 g, 80%), mp 206–207 °C. ½a _D²⁵

= ꢃ8.1 (c 0.52, CHCl₃).

ꢂ

¹H NMR (CDCl₃): d 0.73–0.82 (6H, m), 1.79 (3H, s), 1.94 (3H, s),

1.98–2.25 (6H, m), 2.31 (3H, s), 2.84 (3H, s), 4.55 (1H, t,

J = 7.2 Hz), 5.65 (1H, s), 7.12–7.36 (10H, m). Anal. Calcd for

C₂₈H₃₇ClN₄O: C, 69.91; H, 7.75; N, 11.65. Found: C, 69.66; H,

C₂₈H₃₇ClN₄OS: C, 65.54; H, 7.27; N, 10.92. Found: C, 65.47; H,

7.94; N, 11.70.

7.28; N, 10.92.

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

1891

5.27. (5S)-N-[1-Ethyl-1-(4-methylphenyl)propyl]-2,7,7-tri-

methyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrim-

idine-3-carboxamide hydrochloride (9b)

2.85 (3H, s), 4.56 (1H, t, J = 7.0 Hz), 5.68 (1H, s), 7.19–7.34 (10H,

m). Anal. Calcd for C₂₈H₃₉ClN₄O: C, 70.35; H, 7.94; N, 11.32. Found:

C, 70.36; H, 7.91; N, 11.32.

To a mixture of 8b (0.5 g, 1.75 mmol) and DMF (one drop) in tol-

uene (5 mL) was added SOCl₂(0.42 g, 3.53 mmol) at room temper-

ature. After stirring at the same temperature for 1 h, the solvent

was evaporated off. A mixture of 3-(4-methylphenyl)pentan-3-

amine hydrochloride (0.49 g, 2.30 mmol) and Et₃N (0.53 g,

5.24 mmol) in toluene (5 mL) was stirred at 70 °C for 1 h, and then

acid chloride obtained in toluene was added to the reaction mix-

ture 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. The residue was puriﬁed by Purif-Pack

5.30. (5R)-N-[1-Ethyl-1-(4-isopropylphenyl)propyl]-2,7,7-tri-

methyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimi-

dine-3-carboxamide hydrochloride (10c)

Compound 10c was prepared in a manner similar to that de-

scribed for 10a as a solid. Yield: 45%. Mp: 134–136 °C (EtOH–

Et₂O); ¹H NMR (CDCl₃): d

0.72–0.84 (6H, m), 1.23 (6H, d,

J = 7.0 Hz), 1.80 (3H, s), 1.95 (3H, s), 2.00–2.28 (6H, m), 2.82–2.94

(4H, m), 4.53–4.61 (1H, m), 5.66 (1H, s), 7.20 (4H, s), 7.25–7.40

(6H, m). Anal. Calcd for C₃₀H₄₁ClN₄Oꢀ0.1H₂O: C, 70.52; H, 8.13; N,

10.97. Found: C, 70.27; H, 8.13; N, 10.95.

(SI 60 lm, SIZE 60) to give an oil. To a solution of the oil obtained in

EtOAc was added 4 N HCl in EtOAc (0.5 mL, 2.00 mmol) at 0 °C.

Crystallization from EtOAc–Et₂O afforded 9b (0.56 g, 80%), mp

206–207 °C; ¹H NMR (CDCl₃): d 0.73–0.83 (6H, m), 1.80 (3H, s),

1.95 (3H, s), 1.98–2.26 (6H, m), 2.31 (3H, s), 2.85 (3H, s), 4.55

(1H, t, J = 7.2 Hz), 5.69 (1H, s), 7.13–7.37 (10H, m). Anal. Calcd for

5.31. (5R)-N-[1-Ethyl-1-(4-methoxyphenyl)propyl]-2,7,7-tri-

methyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimi-

dine-3-carboxamide hydrochloride (10d)

Compound 10d was prepared in a manner similar to that de-

scribed for 10a as a solid. Yield: 51%. Mp: 158–160 °C (EtOAc–

Et₂O); ¹H NMR (CDCl₃): d 0.74–0.82 (6H, m), 1.80 (3H, s), 1.94

(3H, s), 1.97–2.26 (6H, m), 2.85 (3H, s), 3.78 (3H, s), 4.56 (1H, t,

J = 6.9 Hz), 5.68 (1H, s), 6.87 (2H, d, J = 8.7 Hz), 7.21–7.39 (8H, m).

Anal. Calcd for C₂₈H₃₇ClN₄O₄: C, 67.66; H, 7.50; N, 11.27. Found:

C, 67.61; H, 7.64; N, 11.39.

C

₂₈H₃₇ClN₄Oꢀ0.25H₂O: C, 69.26; H, 7.78; N, 11.54. Found: C,

69.13; H, 7.69; N, 11.25.

5.28. (5R)-N-(1-Ethyl-1-phenylpropyl)-2,7,7-trimethyl-5-

phenyl-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-

carboxamide hydrochloride (10a)

To a mixture of 8a (1.0 g, 3.50 mmol) and DMF (one drop) in tol-

uene (10 mL) was added SOCl₂(0.83 g, 7.00 mmol) at room tem-

perature. After stirring at the same temperature for 1 h, the

solvent was evaporated off. A mixture of 3-phenylpentan-3-amine

hydrochloride (0.77 g, 3.87 mmol) and Et₃N (0.97 g, 9.59 mmol) in

toluene (10 mL) was stirred at 70 °C for 1 h, and then the acid chlo-

ride 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 ex-

tract was washed with brine, dried over MgSO₄, and concentrated

in vacuo. The residue was chromatographed on SiO₂with EtOAc–

hexane (1:1) to give an amorphous solid. To a solution of the amor-

phous solid obtained in Et₂O was added 4 N HCl in EtOAc at 0 °C.

The precipitated crystals were collected by ﬁltration and washed

with Et₂O (0.85 g, 52%), mp 185–186 °C; ¹H NMR (CDCl₃): d 0.74–

0.83 (6H, m), 1.80 (3H, s), 1.95 (3H, s), 2.00–2.25 (6H, m), 2.86

(3H, s), 4.53–4.58 (1H, m), 5.68 (1H, s), 7.24–7.36 (11H, m). Anal.

Calcd for C₂₇H₃₅ClN₄O: C, 69.43; H, 7.55; N, 12.00. Found: C,

69.19; H, 7.82; N, 11.97.

5.32. (5R)-N-[1-(4-Ethoxyphenyl)-1-ethylpropyl]-2,7,7-tri-

methyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimi-

dine-3-carboxamide hydrochloride (10e)

Compound 10e was prepared in a manner similar to that de-

scribed for 10a as a solid. Yield: 42%. Mp: 127–129 °C (EtOH–

Et₂O); ¹H NMR (CDCl₃):

d

0.73–0.82 (6H, m), 1.39 (3H, t,

J = 6.9 Hz), 1.80 (3H, s), 1.95 (3H, s), 1.97–2.22 (6H, m), 2.84 (3H,

s), 4.00 (2H, q, J = 6.9 Hz), 4.56 (1H, t, J = 8.4 Hz), 5.64 (1H, s),

6.86 (2H, d, J = 8.7 Hz), 7.20 (2H, d, J = 8.7 Hz), 7.25–7.37 (6H, m).

Anal. Calcd for C₂₉H₃₉ClN₄O₂ꢀ0.5H₂O: C, 66.97; H, 7.75; N, 10.77.

Found: C, 66.82; H, 7.73; N, 10.77.

5.33. (5R)-N-[1-(3,4-Dimethoxyphenyl)-1-ethylpropyl]-2,7,7-

trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (10f)

Compound 10f was prepared in a manner similar to that de-

scribed for 10a as a solid. Yield: 53%. Mp: 183–184 °C (EtOH–

Et₂O); ¹H NMR (CDCl₃): d 0.74–0.83 (6H, m), 1.80 (3H, s), 1.94

(3H, s), 1.98–2.26 (6H, m), 2.85 (3H, s), 3.86 (6H, s), 4.56 (1H, t,

J = 6.6 Hz), 5.66 (1H, s), 6.81–6.88 (3H, m), 7.29–7.39 (6H, m). Anal.

Calcd for C₂₉H₃₉ClN₄O₃ꢀ0.1H₂O: C, 65.85; H, 7.47; N, 10.59. Found:

C, 65.58; H, 7.49; N, 10.41.

5.29. (5R)-N-[1-Ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-

5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-

carboxamide hydrochloride (10b)

A mixture of 8a (0.30 g, 1.05 mmol), HATU (0.52 g, 1.37 mmol)

and iPr₂NEt (0.47 g, 1.37 mmol) in DMF (5 mL) was stirred at room

temperature for 1 h, and then, 3-(4-ethylphenyl)pentan-3-amine

hydrochloride (0.31 g, 1.37 mmol) was added thereto. The whole

was stirred at 70 °C overnight, and concentrated in vacuo. The res-

idue was diluted with EtOAc, washed with H₂O and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was chro-

matographed on SiO₂with EtOAc–hexane (1:1) to give an amor-

phous solid. The amorphous solid was dissolved in EtOAc and

4 N HCl in EtOAc was added thereto. Crystallization from EtOAc–

hexane afforded 10b as HCl salt (0.27 g, 52%), mp 163–164 °C; ¹H

NMR (CDCl₃): d 0.3–0.84 (6H, m), 1.22 (3H, t, J = 7.2 Hz), 1.80

(3H, s), 1.95 (3H, s), 2.05–2.20 (6H, m), 2.63 (2H, q, J = 7.2 Hz),

5.34. (5R)-N-{1-[4-(Benzyloxy)phenyl]-1-ethylpropyl}-2,7,7-

trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (10g)

Compound 10g was prepared in a manner similar to that de-

scribed for 10a as a solid. Yield: 32%. Mp: 167–169 °C (EtOH–

Et₂O); ¹H NMR (CDCl₃): d 0.77–0.80 (6H, m), 1.80 (3H, s), 1.94

(3H, s), 2.00–2.30 (6H, m), 2.84 (3H, s), 4.56 (1H, br s), 5.03 (2H,

s), 5.65 (1H, s), 6.95 (2H, d, J = 6.6 Hz), 7.22–7.43 (13H, m). Anal.

Calcd for C₃₄H₄₁ClN₄O₂: C, 1.25; H, 7.21; N, 9.77. Found: C, 70.97;

H, 7.21; N, 9.74.

1892

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

5.35. (5R)-N-[1-(4-Chloro-3-methoxyphenyl)-1-ethylpropyl]-

2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (10h)

5.39. (5R)-N-[1-Ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-

5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-

carboxamide benzenesulfonate (10l)

Compound 10h was prepared in a manner similar to that

Free base of compound 10b (0.90 g, 1.96 mmol) and benzene-

sulfonic acid monohydrate (0.38 g, 2.16 mmol) were dissolved in

EtOH (4 mL). Crystallization from EtOH–Et₂O and recrystallization

from EtOH–Et₂O afforded 10l as benzenesulfonate (1.15 g, 96%),

mp 165–166 °C. ¹H NMR (CDCl₃): d 0.73–0.81 (6H, m), 1.22 (3H,

t, J = 7.8 Hz), 1.72 (3H, s), 1.80 (3H, s), 2.04–2.22 (6H, m), 2.63

(2H, q, J = 7.8 Hz), 2.80 (3H, s), 4.57 (1H, t, J = 8.7 Hz), 5.68 (1H,

s), 7.18–7.34 (13H, m). Anal. Calcd for C₃₅H₄₄N₄O₄S: C, 68.15; H,

7.19; N, 9.08. Found: C, 68.07; H, 7.19; N, 9.13.

described for 10a as

a solid. Yield: 8%. Mp: 167–169 °C

(AcOEt–Et₂O); ¹H NMR (CDCl₃): d 0.802 (6H, dt, J = 11.4, 7.2 Hz),

1.84 (6H, d, J = 41.4 Hz), 1.86–2.30 (6H, m), 2.82 (3H, s), 3.88 (3H,

s), 4.56 (1H, t, J = 6.6 Hz), 5.65 (1H, br s), 6.80–6.90 (2H, m),

7.13 (1H, br s), 7.22–7.40 (6H, m). Anal. Calcd for C₂₇H₃₇Cl₂-

N₅Oꢀ0.4H₂O: C, 61.69; H, 7.25; N, 13.32. Found: C, 61.79; H, 7.28;

N, 13.04.

5.36. (5R)-N-[1-Ethyl-1-(4-ﬂuoro-3-methoxyphenyl)propyl]-

2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-

a]pyrimidine-3-carboxamide (10i)

5.40. (5R)-N-[1-Ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-

5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-

carboxamide p-toluenesulfonate (10m)

Compound 10i was prepared in a manner similar to that

Free base of compound 10b (120 mg, 0.26 mmol) and

p-toluenesulfonic acid monohydrate (50 mg, 0.29 mmol) were dis-

solved in EtOH (2 mL). Crystallization from EtOH–Et₂O and recrys-

tallization from EtOH–Et₂O afforded 10m as p-toluenesulfonate

(130 mg, 79%), mp 160–161 °C. ¹H NMR (CDCl₃): d 0.73–0.81 (6H,

m), 1.22 (3H, t, J = 7.8 Hz), 1.72 (3H, s), 1.80 (3H, s), 2.04–2.22

(6H, m), 2.37 (3H, s), 2.62 (2H, q, J = 7.8 Hz), 2.80 (3H, s), 4.56

(1H, t, J = 7.5 Hz), 5.69 (1H, s), 7.18–7.34 (12H, m), 7.85 (2H, d,

J = 8.1 Hz). Anal. Calcd for C₃₆H₄₆N₄O₄S: C, 68.54; H, 7.35; N,

8.88. Found: C, 68.54; H, 7.48; N, 8.92.

described for 10a as

a solid. Yield: 45%. Mp: 167–169 °C

(MeOH–Et₂O); ¹H NMR (CDCl₃): d 0.796 (6H, dt, J = 10.8, 7.5 Hz),

1.82 (6H, d, J = 39.6 Hz), 1.92–2.30 (6H, m), 2.78 (3H, s), 3.88 (3H,

s), 4.56 (1H, t, J = 6.6 Hz), 5.63 (1H, br s), 6.82–6.94 (2H, m), 7.03

(1H, dd, J = 11.1, 8.7 Hz), 7.09 (1H, br s), 7.28–7.40 (5H, m). Anal.

Calcd for C₂₈H₃₆ClFN₄O₂: C, 65.29; H, 7.04; N, 10.88. Found: C,

65.26; H, 7.05; N, 10.77.

5.37. (5R)-N-[1-(3-Chloro-4-methoxyphenyl)-1-ethylpropyl]-

2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide hydrochloride (10j)

5.41. 3-(4-Iodophenyl)pentan-3-amine hydrochloride (12)

To a solution of ethyl 4-iodobenzoate 11 (31.4 g, 114 mmol) in

Et₂O (200 mL) was added EtMgBr (95 mL, 3 M in Et₂O solution,

285 mmol) at 0 °C. After stirring at room temperature for 3 h, the

reaction mixture was quenched with 1 N HCl. Organic layer sepa-

rated was washed with brine, dried over MgSO₄, and concentrated

in vacuo to give as oil. To a mixture of the oil obtained and TMSCN

(20.3 g, 205 mmol) was added concd H₂SO₄(30.0 g) at ꢃ10 °C, and

the whole was stirred at room temperature for 2 day. The reaction

mixture was quenched with 28% NH₃solution and extracted with

EtOAc. The extract was washed with brine, dried over MgSO₄, and

concentrated in vacuo. The mixture of residue obtained and 6 N

HCl (100 mL) were reﬂuxed for 3 h. The reaction mixture was baci-

diﬁed with 8 N NaOH and extracted with Et₂O. The extract was

washed with brine, dried over MgSO₄, and concentrated in vacuo.

The residue was diluted with EtOAc and 4 N HCl in EtOAc was

added thereto. The precipitated crystals were collected by ﬁltration

and washed with Et₂O (8.25 g, 23%). ¹H NMR (DMSO-d₆): d 0.73

(6H, t, J = 7.5 Hz), 1.87–2.04 (4H, m), 7.24 (2H, d, J = 8.7 Hz), 7.81

(2H, d, J = 8.7 Hz), 8.58 (2H, s).

Compound 10j was prepared in a manner similar to that

described for 10a as

a solid. Yield: 34%. Mp: 178–180 °C

(i-PrOH-Et₂O); ¹H NMR (CDCl₃): d 0.75–0.83 (6H, m), 1.80 (3H, s),

1.95 (3H, s), 1.91–2.25 (6H, m), 2.86 (3H, s), 3.87 (3H, s),

4.56 (1H, t, J = 6.3 Hz), 5.69 (1H, s), 6.89 (1H, d, J = 8.7 Hz), 7.15–

7.26 (2H, m), 7.29–7.39 (6H, m). Anal. Calcd for C₂₈H₃₆Cl₂-

N₄O₂ꢀ0.25H₂O: C, 62.74; H, 6.86; N, 10.45. Found: C, 62.68; H,

6.99; N, 10.42.

5.38. (5R)-N-[1-Ethyl-1-(3-ﬂuoro-4-methoxyphenyl)propyl]-

2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-

a]pyrimidine-3-carboxamide hydrochloride (10k)

To a mixture of 8a (0.35 g, 1.23 mmol) and DMF (one drop) in

toluene (3 mL) was added SOCl₂(0.29 g, 2.44 mmol) at room

temperature. After stirring at the same temperature for 1 h, the sol-

vent was evaporated off. A mixture of 3-(3-ﬂuoro-4-methoxy-

phenyl)pentan-3-amine hydrochloride (0.40 g, 1.62 mmol) and

Et₃N (0.37 g, 3.66 mmol) in toluene (3 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 temper-

ature for 3 h, the reaction mixture was poured into H₂O and ex-

tracted with EtOAc. The extract was washed with brine, dried

over MgSO₄, and concentrated in vacuo. The residue was chro-

matographed on SiO₂with EtOAc–hexane (1:1) to give an amor-

phous solid. To a solution of the amorphous solid obtained in

EtOAc (1 mL) was added 4 N HCl in EtOAc (0.4 mL, 1.60 mmol) at

0 °C. The solvent was evaporated off. Crystallization from EtOAc–

Et₂O and recrystallization from EtOAc–Et₂O afforded 10k as HCl

salt (0.10 g, 16%), mp 180–182 °C. ¹H NMR (CDCl₃): d 0.80 (6H, s),

1.81 (3H, s), 1.95 (3H, s), 2.00–2.40 (6H, m), 2.88 (3H, s), 3.86

(3H, s), 4.57 (1H, br s), 5.65 (1H, br s), 6.80–7.40 (9H, m). Anal.

Calcd for C₂₈H₃₆ClFN₄O₂ꢀ0.25H₂O: C, 64.73; H, 7.08; N, 10.78.

Found: C, 64.55; H, 7.04; N, 10.77.

5.42. (5R)-N-[1-Ethyl-1-(4-iodophenyl)propyl]-2,7,7-trimethyl-

5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-

carboxamide (13)

To a mixture of 8a (6.0 g, 21.0 mmol) and DMF (three drops) in

toluene (60 mL) was added SOCl₂(3.75 g, 31.5 mmol) at room tem-

perature. After stirring at the same temperature for 1 h, the solvent

was evaporated off. A mixture of 12 (8.21 g, 25.2 mmol) and Et₃N

(4.67 g, 46.2 mmol) in toluene (60 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 tempera-

ture for 3 h, the reaction mixture was poured into H₂O and ex-

tracted with EtOAc. The extract was washed with brine, dried

over MgSO₄, and concentrated in vacuo. The residue was chro-

matographed on SiO₂with EtOAc–hexane (1:2) to give 13 as an

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

1893

oil (6.26 g, 54%); ¹H NMR (CDCl₃): d 0.73–0.82 (6H, m), 1.54 (3H, s),

1.61 (3H, s), 1.93–2.21 (6H, m), 2.50 (3H, s), 4.52 (1H, dd, J = 11.7,

3.0 Hz), 5.58 (1H, s), 6.55 (1H, s), 7.10 (2H, d, J = 8.4 Hz), 7.27–7.36

(5H, m), 7.61 (2H, d, J = 8.4 Hz).

C₂₉H₃₆N₄O₂: C, 73.70; H, 7.68; N, 11.85. Found: C, 73.46; H, 7.72;

N, 11.72.

5.46. (5R)-N-{1-Ethyl-1-[4-(1-hydroxyethyl)phenyl]propyl}-

2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide (17)

5.43. Ethyl 4-[1-ethyl-1-({[(5R)-2,7,7-trimethyl-5-phenyl-4,5,6,7-

tetrahydropyrazolo-[1,5-a]-pyrimidin-3-yl]-carbonyl}amino)-

propyl]benzoate (14)

To a solution of 16 (0.80 g, 1.69 mmol) in EtOH was added

NaBH₄(0.26 g, 6.77 mmol) at room temperature. After stirring at

the same temperature for 4 h, the solvent was evaporated off.

The residue was diluted with EtOAc, washed with 1 N HCl and

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was chromatographed on SiO₂with EtOAc–hexane (1:1) to give

an oil. The oil was diluted with EtOAc, and 4 N HCl in EtOAc

(0.5 mL, 2.0 mmol) was added thereto. Crystallization from

EtOAc–Et₂O and recrystallization from EtOH–Et₂O afforded 17 as

prisms (0.61 g, 71%), mp 182–184 °C; ¹H NMR (CDCl₃): d 0.75–

0.83 (6H, m), 1.49 (3H, d, J = 6.6 Hz), 1.80 (3H, s), 1.94 (3H, s),

2.00–2.30 (6H, m), 2.85 (3H, s), 4.55 (1H, t, J = 6.0 Hz), 4.88 (1H,

q, J = 6.6 Hz), 5.67 (1H, s), 7.22–7.36 (10H, m). Anal. Calcd for

A mixture of 13 (6.2 g, 11.1 mmol), dppf (0.33 g, 0.60 mmol),

Pd(OAc)₂(0.14 g, 0.60 mmol) and Et₃N (2.25 g, 22.3 mmol) in EtOH

(120 mL) was stirred at 90 °C under 1 atom CO atmosphere for

24 h. The inorganic products were removed by ﬁltration, and ﬁl-

trate was concentrated in vacuo. The residue was diluted with

EtOAc, washed with NaHCO₃solution and brine, dried over MgSO₄,

and concentrated in vacuo. The residue was chromatographed on

SiO₂with EtOAc–hexane (1:2) to give 14 as an amorphous solid

(3.50 g, 63%). ¹H NMR (CDCl₃): d 0.74–0.83 (6H, m), 1.33 (3H,

J = 6.9 Hz), 1.54 (3H, s), 1.61 (3H, s), 1.98–2.23 (6H, m), 2.51

(3H, s), 4.33 (2H, q, J = 6.9 Hz), 4.51 (1H, dd, J = 11.4, 3.0 Hz), 5.63

(1H, s), 6.53 (1H, s), 7.24–7.34 (5H, m), 7.41 (2H, d, J = 8.1 Hz),

7.52 (1H, s), 7.98 (2H, d, J = 8.1 Hz).

C₂₉H₃₉Cl N₄O₂: C, 68.15; H, 7.69; N, 10.96. Found: C, 67.84; H,

7.70; N, 10.84.

5.44. 4-[1-Ethyl-1-({[(5R)-2,7,7-trimethyl-5-phenyl-4,5,6,7-

tetrahydropyrazolo[1,5-a]-pyrimidin-3-yl]carbonyl}amino)-

propyl]benzoic acid (15)

5.47. GTP

c

S binding assay

S binding activity was measured as follows. The

The GTP

c

CaR-expressing cell membrane was incubated with test com-

pounds for 10 min. The assays were carried out at room tempera-

ture for an hour in a reaction solution mixture containing 20 mM

A mixture of 14 (3.5 g, 6.96 mmol) and 1 N NaOH (30 mL,

30.0 mmol) in EtOH (50 mL) was stirred at 60 °C for 12 h, and the

solvent was concentrated in vacuo. The residue was diluted with

EtOAc and acidiﬁed with 1 N HCl. The organic layer was washed

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was chromatographed on SiO₂with EtOAc–hexane (4:1) to give

15 as crystals (2.70 g, 82%), mp 234–236 °C. ¹H NMR (CDCl₃): d

0.76–0.88 (6H, m), 1.56 (3H, s), 1.62 (3H, s), 1.97–2.25 (6H, m),

2.53 (3H, s), 4.53 (1H, dd, J = 11.7, 3.0 Hz), 5.67 (1H, s), 6.56 (1H,

s), 7.24–7.36 (5H, m), 7.45 (2H, d, J = 8.4 Hz), 8.01 (2H, d,

J = 8.4 Hz). Anal. Calcd for C₂₈H₃₄N₄O₂ꢀ0.25H₂O: C, 70.19; H, 7.26;

N, 11.69. Found: C, 70.25; H, 7.16; N, 11.49.

HEPES (pH.7.4), 100 mM NaCl, 1 mM MgCl₂, 167

V guanosine 5⁰-diphosphate, 0.4 nM [35S]-guanosine 5⁰-(

thio) triphosphate ([35S]-GTP S) and 6 mM CaCl₂. The mixture

was ﬁltrated through a GF/C ﬁlter. After washing fourth with

300 L of phosphate-buffered saline, radioactivity of the ﬁlter

was measured using a Top-count scintillation counter. Data from

GTP S binding assays for antagonists were analyzed with the use

lg/mL DTT,

5

l

c-

c

l

c

of the Prism program (GraphPad Software, Inc). IC₅₀values were

determined through nonlinear regression analysis performed with

Prism.

5.45. (5R)-N-[1-(4-Acetylphenyl)-1-ethylpropyl]-2,7,7-

trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-

pyrimidine-3-carboxamide (16)

5.48. 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. In case of monkey,

plasma intact PTH levels were measured using an immunoradio-

metric assay human kit (Allegro intact PTH, Kyowa Medex Co., Ltd).

A mixture of 15 (2.65 g, 5.58 mmol), WSC (1.60 g, 8.38 mmol),

HOBt (1.28 g, 8.38 mmol), MeONHMe hydrochloride (0.82 g,

8.38 mmol) and Et₃N (1.13 g, 11.2 mmol) in DMF (20 mL) was stir-

red at 60 °C for 3 h. The reaction mixture was poured into H₂O and

extracted with EtOAc. The extract was washed brine, dried over

MgSO₄, and concentrated in vacuo. The residue was chromato-

graphed on SiO₂with EtOAc–hexane (3:1) to give an oil (2.5 g,

87%). ¹H NMR (CDCl₃): d 0.75–0.88 (6H, m), 1.55 (3H, s), 1.61

(3H, s), 1.99–2.25 (6H, m), 2.51 (3H, s), 3.34 (3H, s), 3.56 (3H, s),

4.54 (1H, dd, J = 11.1, 3.0 Hz), 5.63 (1H, s), 6.55 (1H, s), 7.24–7.39

(7H, m), 7.65 (2H, d, J = 8.7 Hz). To a solution of the oil obtained

(2.5 g) in THF was added MeMgBr (50 mL, 1 M in THF solution,

50.0 mmol) at 0 °C. After stirring at room temperature for 1 h,

the reaction mixture was quenched with 1 N HCl and extracted

with EtOAc. The extract was washed brine, dried over MgSO₄,

and concentrated in vacuo. The residue was chromatographed on

SiO₂with EtOAc–hexane (1:2) to give crystals. Recrystallization

from EtOAc–hexane afforded 16 as prisms (1.21 g, 53%), mp

144–145 °C; ¹H NMR (CDCl₃): d 0.75–0.84 (6H, m), 1.55 (3H, s),

1.61 (3H, s), 1.96–2.28 (6H, m), 2.52 (3H, s), 2.56 (3H, s), 4.51

(1H, dd, J = 11.4, 3.0 Hz), 5.64 (1H, s), 6.52 (1H, s), 7.24–7.31 (5H,

m), 7.44 (2H, d, J = 8.4 Hz), 7.80 (2H, d, J = 8.4 Hz). Anal. Calcd for

5.49. Metabolic stability assay

Metabolic stability assay Hepatic microsomes from rats and hu-

mans were purchased from Xenotech, LLC (Lenexa, KS). An incuba-

tion mixture with a ﬁnal volume of 0.1 mL consisted of microsomal

protein in 50 mmol/L KH₂PO₄–K₂HPO₄phosphate buffer (pH 7.4)

and 1 lmol/L test compound. The concentration of hepatic micro-

somal protein was 0.2 mg/mL. An NADPH-generating system

containing 50 mmol/L MgCl₂, 50 mmol/L glucose-6-phosphate,

5 mmol/L beta-NADP+ and 15 unit/mL glucose-6-phosphate dehy-

drogenase 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 duplicate. Test compound in the reac-

tion mixture was measured by HPLC system equipped with a UV

1894

M. Yoshida et al. / Bioorg. Med. Chem. 19 (2011) 1881–1894

detector. For metabolic stability determinations, chromatograms

were analyzed for parent compound disappearance from the reac-

tion mixtures.

(B) acetonitrile = 8.5:1.5; gradient condition, 0–0.5 min: 15–95%

B; 0.5–3.0 min: 95% B; 3.0–3.1 min: 95–15% B; 3.1–5.0 min: 15%

B; ﬂow rate, 0.25 mL/min; column temperature, 40 °C.

5.50. Solubility determination

Acknowledgments

Small volumes of the compound DMSO solutions were added to

the aqueous buffer solution (pH’s 1.2, 6.8, and 6.8+bile acid). After

incubation, precipitates were separated by ﬁltration. The solubility

was determined by HPLC analysis of each ﬁltrate.

We thank Dr. S. Ohkawa, Dr, Y. Ikeura, Mr. H. Maezaki for their

encouragement and helpful advice. We thank Ms. K. Higashikawa

for X-ray crystallographic analysis, and Dr. T. Yamano and

Mr. S. Yamamoto for optical resolution, Dr. M. Yamaguchi for

obtaining rat and monkey pharmacokinetic study, Mr. Y. Suzuki

for measurement of chemical stability, Dr. T. Okuda for measure-

ment of metabolic stability. We thank Mr. T. Kobayashi and

Mr. N. Tsuruta for in vivo assays.

5.51. Chemical stability determination

The compounds were stored under 75% RH at 60 °C for 2 weeks.

The residual percentage of the compound was analyzed by HPLC,

after dissolved with CH₃CN.

Supplementary data

5.52. Plasma concentration in monkeys (compound 9a)

Supplementary data (X-ray crystallographic data for (R)-9a)

associated with this article can be found, in the online version, at

doi:10.1016/j.bmc.2011.02.001.

Compound 9a was administered orally to fed cynomolgus mon-

keys (male, n = 3) at a dose of 2.5 or 5.0 mg/kg in 0.5% methylcel-

lulose suspension. At 0.25, 0.5, 1, 2, 4, 8, and 24 h after oral

administration, blood samples were collected, and then immedi-

ately centrifuged to obtain the plasma fraction. The plasma sam-

ples were deproteinized with acetonitrile containing an internal

standard. After centrifugation, the supernatant obtained was di-

luted with 0.01 mol/L ammonium formate (pH 3.0) and centrifuged

again. The compound concentration in the supernatant was mea-

sured by high performance liquid chromatography/triple quadru-

pole mass spectrometry system, API3000 (Applied Biosystem, CA,

USA), equipped with a binary solvent manager (LC-10AD_vp), sam-

ple organizer/manager (SIL-HT_c) and column oven (CTO-10AC)

from SHIMADZU (Kyoto, Japan). The mass spectrometer was

equipped with a turbo ionspray source and operated in positive

ion mode. The HPLC conditions were as follows: column, Inertsil

C8–3 (2.1 ꢁ 33 mm) from GL Sciences (Tokyo, Japan); mobile

phase, (A) 0.01 mol/L ammonium formate (pH 3.0)/(B) acetoni-

trile = 8.5:1.5; gradient condition, 0–1.4 min: 15% B; 1.4–1.5 min:

15–91.5% B; 1.5–5.5 min: 91.5% B; 5.5–8.0 min: 91.5–15% B; 8.0–

9.0 min: 15% B; ﬂow rate, 0.2 mL/min; column temperature, 40 °C.

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umn, Inertsil ODS-3 (2.1 ꢁ 50 mm) from GL Sciences (Tokyo, Ja-

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22. The practical synthesis of TAK-075 will be reported in due course.

Article Doi

Novel and potent calcium-sensing receptor antagonists: Discovery of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6, 7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate (TAK-075) as an orally active bone anabolic agent

DOI: 10.1016/j.bmc.2011.02.001

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Full text of DOI:10.1016/j.bmc.2011.02.001

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