Discovery and biological evaluation of potent and orally active human 11β-hydroxysteroid dehydrogenase type 1 inhibitors for the treatment of type 2 diabetes mellitus

Article abstract of DOI:10.1248/cpb.c19-00211

We synthesized and evaluated novel 5-[2-(thiophen-2-yl)propan-2-yl]-4H-1,2,4-triazole derivatives as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. Optimization of the thiophene ring and the substituents on the 1,2,4-triazole ring produced

Full text of DOI:10.1248/cpb.c19-00211

824
Chem. Pharm. Bull. 67, 824–838 (2019)
Vol. 67, No. 8
Regular Article
Highlighted Paper selected by Editor-in-Chief
Discovery and Biological Evaluation of Potent and Orally Active Human
11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors for the Treatment
of Type 2 Diabetes Mellitus
Takanori Koike,* Ryota Shiraki, Daisuke Sasuga, Mitsuru Hosaka, Tomoaki Kawano,
Hiroki Fukudome, Kazuo Kurosawa, Ayako Moritomo, Shinya Mimasu, Hirofumi Ishii, and
Seiji Yoshimura
Drug Discovery Research, Astellas Pharma Inc.; 21 Miyukigaoka, Tsukuba, Ibaraki 305–8585, Japan.
Received March 4, 2019; accepted May 21, 2019
We synthesized and evaluated novel 5-[2-(thiophen-2-yl)propan-2-yl]-4H-1,2,4-triazole derivatives as
11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. Optimization of the thiophene ring and the
substituents on the 1,2,4-triazole ring produced 3,4-dicyclopropyl-5-{2-[3-fluoro-5-(trifluoromethyl)thiophen-
2-yl]propan-2-yl}-4H-1,2,4-triazole monohydrochloride (9a), which showed potent and selective inhibitory ac-
tivity against human 11β-HSD1. Compound 9a was also metabolically stable against human and mouse liver
microsomes. Oral administration of 9a to diabetic ob/ob mice lowered corticosterone levels in adipose tissue,
and thereby reduced plasma glucose and insulin levels in a dose-dependent manner.
Key words type II diabetes mellitus; 11β-hydroxysteroid dehydrogenase type 1; liver microsomal stability
normal activation of glucocorticoids in adipose tissue and dia-
Introduction
Diabetes mellitus is a metabolic disease in which hypergly- betes.4) Reports have also demonstrated increased 11β-HSD1
cemia persists chronically as a result of deficient insulin action activity in the adipose tissue of obese patients,5) and a correla-
due to failed insulin secretion in the pancreas or insulin resis- tion between 11β-HSD1 activity and body mass index (BMI),
tance in peripheral tissues.1) Diabetes mellitus is divided into homeostasis model assessment of insulin resistance (HOMA-
two types according to the cause2): type 1 diabetes is caused by IR) and fasting blood glucose level.6) In addition, a transgenic
insulin deficiency due to destruction of pancreatic β cells; and mouse selectively overexpressing 11β-HSD1 in adipose tissue
type 2 diabetes is caused by environmental factors such as ge- had raise glucocorticoids levels in adipose tissue and insulin
netic factors, obesity, binge eating, lack of exercise and a high resistance and hyperlipidemia.7,8) Therefore, an 11β-HSD1
fat diet. Numerous therapeutic agents for type 2 diabetes have selective inhibitor is expected to suppress glucocorticoid ac-
been studied,3) and insulin secretagogues and insulin sensitizers tion by inhibiting the conversion of inactive glucocorticoids to
have been developed. However, owing to limited pharmacologi- active glucocorticoids, and improve metabolic abnormalities
cal effects and harmful side effects, glycemic control in many caused by glucocorticoids such as hyperglycemia, insulin re-
type 2 diabetic patients remains insufficient. Therefore, devel- sistance and hyperlipidemia. However, inhibition of 11β-HSD2
opment of drugs with new mechanisms of action is warranted.
in the kidney is also known to induce hypertension,9) mak-
11β-Hydroxysteroid dehydrogenase is an enzyme that cata- ing selectivity for 11β-HSD1 over 11β-HSD2 of paramount
lyzes the conversion of glucocorticoids. Two isozymes have importance. Various 11β-HSD1 inhibitors have been reported
been identified (Fig. 1): 11β-hydroxysteroid dehydrogenase to date,10) and clinical trials of several compounds11–16) have
type 1 (11β-HSD1) converts inactive glucocorticoids into ac- been conducted (Fig. 2), but none of the compounds have been
tive glucocorticoids in the liver and adipose tissue, while launched yet. Therefore, we conceptualized that the discovery
11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) con- of a new type of 11β-HSD1 inhibitor is necessary to overcome
verts active glucocorticoids into inactive glucocorticoids in the current stagnation in clinical trials and initiated research
the kidney. Reports have identified a relationship between ab- from ground up.
Fig. 1. Interconversion of Glucocorticoids in Humans and Mice
*To whom correspondence should be addressed. e-mail: takanori.koike@astellas.com
© 2019 The Pharmaceutical Society of Japan

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
825
Fig. 2. Structure of 11β-HSD1 Inhibitors
10c, 10d and 10o. Compound 9d was also brominated with N-
bromosuccinimide to yield 11, which was treated with n-butyl
lithium and N,N-dimethylformamide (DMF) to give aldehyde
12. Compound 12 was converted to 13 using Wolff–Kishner
reduction. Iodization of 9d and subsequent cyanation with
copper(I) cyanide gave nitrile 15.
Synthesis of 19 is depicted in Chart 3. Cyclization of 16
and 8a using methyl trifluoromethanesulfonate followed by
removal of the tert-butoxycarbonyl (Boc) group from 17 with
hydrochloric acid yielded amine 18. Treatment of 18 with
2,5-dimethoxytetrahydrofuran gave pyrrole 19.
Fig. 3. Structure of 1 and 9d
Preparation of 21 is shown in Chart 4. Treatment of 20 with
We have been focusing on developing novel 11β-HSD1 thionyl chloride and subsequent cyclization with formyl hydra-
inhibitors as a new class of drugs for type 2 diabetes. High zine yielded 21.
throughput screening (HTS) of the Astellas compound library
Compounds 25a–e were synthesized as shown in Chart
identified compound 1 as a novel 11β-HSD1 inhibitor. In our 5. Compound 4d was condensed with cyclopropanecarbonyl
initial study, to find patentable compounds, we converted the chloride to give 22, which was cyclized with trifluorometh-
linker moiety, chlorobenzene moiety and the substituents at anesulfonic anhydride to give oxadiazole 23. Compound 24
the 3 and 4 positions on the triazole ring of compound 1 to was prepared by bromination of 23, and 24 was reacted with
produce lead compound 9d (unpublished data; Fig. 3). Com- several amines by microwave irradiation to give 25a–e.
pound 9d had no inhibitory activity against human 11β-HSD2,
Chart 6 shows the synthesis of 29a and 29b. Condensation
but showed moderate inhibitory activity against human of 26a and 26b with cyclopropanecarbonyl chloride followed
11β-HSD1 (IC50 =23nM) and insufficient metabolic stability by cyclization of 27a and 27b by treatment with phosphorus
in mice (CLint =1122 mL/min/kg).
oxychloride gave 28a and 28b. Reaction of 28a and 28b with
Therefore, we aimed to further improve the inhibitory ac- cyclopropylamine by microwave irradiation yielded 29a and
tivity of compound 9d against human 11β-HSD1 and its meta- 29b.
bolic stability in mice for testing in a diabetes mouse model.
Biological Evaluation The compounds’ enzymatic inhibi-
Here, we describe further optimization of 9d as a novel tory activity against human 11β-HSD1, 11β-HSD2 and mouse
11β-HSD1 inhibitor, including structure–activity relationship 11β-HSD1 was determined by using a homogeneous time-re-
(SAR) studies and evaluation of its in vivo efficacy.
solved fluorescence method (HTRF®). Truncated recombinant
human and mouse 11β-HSD1 proteins (residues 24–292 with
His-tag at N-terminal) were produced from Escherichia coli
Results and Discussion
Chemistry Synthesis of compounds 9a–p is shown in (E. coli), and full length recombinant human 11β-HSD2 pro-
Chart 1. Esterification of carboxylic acid 2 followed by heating tein was produced from HEK-293. Selected compounds were
of 3a and 3b with hydrazine monohydrate in ethanol (EtOH) measured for metabolic stability against mouse and human he-
yielded acylhydrazine 4a and 4b. Compound 5 was converted patic CYPs. CLogP values were calculated using ACD LogP
to 6, which was hydrolyzed to 7. Condensation of 7 and hydra- prediction software (ACD/Percepta).17)
zine monohydrate, followed by cyclization of acylhydrazine and
The effect of the conversion of the thienyl moiety is shown
N-cyclopropylcyclopropanecarboxamide (8a) using methyl tri- in Table 1, along with the corresponding data for 9d for
fluoromethanesulfonate yielded triazole 9c. Compounds 9a, 9b, comparison. Pyridine derivatives 29a and 29b showed lower
and 9d–p were synthesized in a similar manner to 9c.
inhibitory activity against human 11β-HSD1 than 9d, and
Chart 2 shows the synthesis of 10c, 10d, 10o, 11, 13 and 15. pyrazine derivative 9p showed a dramatic loss in activity.
Chlorination of 9c, 9d and 9o with N-chlorosuccinimide gave Replacement with pyrrole (19) resulted in a moderate decrease

826
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
Reagents and conditions: (a) iodomethane, K2CO3, DMF (N,N-dimethylformamide), room temperature (r.t.), 15h; (b) hydrazine monohydrate, EtOH, 80°C, 24h; (c) io-
domethane, NaH, DMF, r.t., 20min; (d) KOH, ethylene glycol, 190°C, 2.5h; (e) hydrazine monohydrate, WSCD (water-soluble carbodiimide)·HCl, HOBt·H2O (1-hydroxy-
benzotriazole hydrate), CH2Cl2, r.t., overnight; (f) 8a, methyl trifluoromethanesulfonate, triethylamine (Et3N), toluene, 60°C, 2d, 100°C, 2h; (g) methyl trifluoromethane-
sulfonate, Et3N, toluene, 60 to 110°C.
Chart 1. Synthesis of 9a–p
in inhibitory activity against human 11β-HSD1. These results 9e–j showed decreased MLM stability, possibly due to the
suggest that nitrogen-containing heteroaromatics may be un- high lipophilicity compared to 9d. In contrast, neither group
favorable at the terminal thiophene ring position. Meanwhile, of compounds showed inhibitory activity against human
compound 19 showed improved mouse liver microsomal 11β-HSD2. These results indicate that the cyclopropyl group
(MLM) stability. The difference in CLogP values between 9d was the most suitable substituent at the 3-position of the tri-
and 19 indicated that the improved MLM stability was due to azole ring.
the decrease in lipophilicity. On the basis of these results, we
concluded that the thiophene ring was the most suitable sub- we studied the effects of substituent groups such as electron
stituent at the R position.
withdrawing groups on the thiophene ring of compound 9d
To suppress oxidative metabolism of the thiophene ring,
Table 2 summarizes the effects of substituents at the 3-posi- (Table 3). Substitution with a halogen atom, such as fluorine
tion of the triazole ring. Removal of the 3-position substitu- (9m), chlorine (10d) and bromine (11), at the 5-position of the
ent (21) resulted in loss of inhibitory activity against human thiophene ring increased inhibitory activity against human
11β-HSD1, suggesting that substituents are necessary at the 11β-HSD1, but was ineffective in improving MLM stability.
3-position of the triazole ring. Substitution of the cyclopropyl A 5-methyl derivative (13) showed comparable activity to 9d,
group with an n-propyl (9e) or cyclopropylmethyl (9f) group but had 2-fold lower MLM stability. Meanwhile, substitution
decreased inhibitory activity against human 11β-HSD1 by with a 5-cyano group (15) resulted in about a 4-fold reduc-
about 2-fold. tert-Butyl (9g), cyclobutyl (9h), cyclopentyl (9i) tion in inhibitory activity, but 3-fold increase in MLM stabil-
and cyclohexyl (9j) derivatives showed a slight improvement ity compared to 9d. This improvement in MLM stability was
in inhibitory activity, indicating that bulky substituents are probably due to a decrease in lipophilicity. Substitution with
preferable at the 3-position of the triazole ring. Compounds a 5-trifluoromethyl group (9b) resulted in activity equivalent

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
827
Reagents and conditions: (a) N-chlorosuccinimide, acetic acid (AcOH), 50–80°C, 2h–3d; (b) N-bromosuccinimide, AcOH, 80°C, 2h; (c) n-butyl lithium, N,N,N′,N′-
tetramethyl ethylenediamine, DMF, tetrahydrofuran (THF), −78°C, 45min; (d) hydrazine monohydrate, KOH, diethylene glycol, 170°C, 4h; (e) N-iodosuccinimide, AcOH,
r.t., overnight; (f) CuCN, pyridine, 115°C, 11.5h.
Chart 2. Synthesis of 10c, 10d, 10o, 11, 13 and 15
Reagents and conditions: (a) methyl trifluoromethanesulfonate, Et3N, toluene, 60 to 100°C, 3h; (b) 4M HCl/EtOAc, EtOH, 50°C, 8h; (c) 2,5-dimethoxytetrahydrofuran,
AcOH, CHCl3, 70°C, 24h.
Chart 3. Synthesis of 19
(10c) and 3,5-dichloro derivative (10o) showed improved in-
hibitory activity compared to 9d. However, they had deterio-
rated MLM stability, especially 10o, which showed a dramatic
decrease similar to 9o. Interestingly, a 3-fluoro-5-trifluoro-
methyl derivative (9a) was the most potent human 11β-HSD1
inhibitor generated in this series (IC50 =4.8nM), and had
enhanced MLM stability (mouse CLint =578mL/min/kg).
Reagents and conditions: (a) SOCl2, DMF, CHCl3, 60°C, 1h; (b) formyl hydra-
zine, toluene, 70°C, 20h.
Results from compounds 9c, 10c and 9a suggested the fluoro
group was the most suitable substituent at the 3-position of
the thiophene ring for increasing inhibitory activity against
Chart 4. Synthesis of 21
human 11β-HSD1. In addition, results from compounds 9a
to that of 9d. Interestingly, compound 9b had about 3-fold and 9b indicated that substitution of a trifluoromethyl group
higher MLM stability, despite an increased CLogP value. at the 5-position of the thiophen ring was most effective for
This suggests that the improved MLM stability may be due improving MLM stability. Neither group of compounds had
to suppression of oxidative metabolism of the thiophene ring. inhibitory activity against human 11β-HSD2. The above re-
3-Fluoro (9c), 4-bromo (9n) and 3-chloro (9o) analogues had sults suggest that the 3-fluoro-5-trifluoromethyl group was the
increased inhibitory activity against human 11β-HSD1 but most suitable substituent for the thiophene ring.
no improvement MLM stability. In particular, the 3-chloro
The effect of substituents at the 4-position of the triazole
derivative showed a significant decrease in MLM stability, ring is shown in Table 4. To improve efficiency for SAR stud-
indicating that substitution with a chloro group is unfavorable ies, we converted 5-bromothiophene derivatives, which were
at the 3-position of the thiophene ring. Next, we investigated easier to synthesize than 3-fluoro-5-trifluoromethylthiophene
3,5-disubstituted derivatives. A 3-fluoro-5-chloro derivative derivatives. The corresponding data for 11 are also shown

828
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
Reagents and conditions: (a) cyclopropanecarbonyl chloride, Et3N, CH2Cl2, r.t., overnight; (b) trifluoromethanesulfonic anhydride, pyridine, CH2Cl2, r.t., 3d; (c) N-
bromosuccinimide, AcOH, r.t.; (d) amine, AcOH, 170°C, 40–60min (microwave).
Chart 5. Synthesis of 25a–e
Reagents and conditions: (a) cyclopropanecarbonyl chloride, Et3N, CH2Cl2, 0°C to r.t., 3–14h; (b) phosphorus oxychloride, reflux, 2–8h; (c) cyclopropylamine, AcOH,
175°C, 40min (microwave).
Chart 6. Synthesis of 29a and 29b
Table 1. Conversion of the Thienyl Moiety
Table 2. Conversion of the 3-Position Substituent of 1,2,4-Triazole
Human
11β-HSD1 11β-HSD2
IC50 (nM) IC50 (nM)
Human
Mouse CL
(mL/min/kg)
Compound
R
int CLogP
9d
21
9ea)
9f
c-Pr
H
23
>3000
51
>10000
NTb)
1122
NTb)
1705
2313
2578
1460
3024
5862
2.95
2.46
3.53
3.38
4.05
3.42
3.99
4.37
n-Pr
>10000
>10000
>3000
>3000
>3000
>3000
Cyclopropyl methyl
51
9g
9h
9i
t-Bu
16
c-Bu
c-Pen
c-Hex
13
14
9j
10
a) Hydrochloride salt. b) Not tested.
rivatives 9l and 25a–c showed inhibitory activity equivalent to
11, but 25a–c had decreased MLM stability. Substitution with
benzyl (25d) and phenethyl (25e) groups resulted in a decrease
in inhibitory activity against human 11β-HSD1, indicating that
an alkyl group at the R position may be more suitable than
a benzene ring. Neither group of compounds showed inhibi-
tory activity against human 11β-HSD2. On the basis of these
in Table 4 for comparison. Replacement of the cyclopropyl results, we concluded that a cyclopropyl group was the most
group with a methyl group (9k) resulted in a slight attenuation suitable substituent at the 4-position of the triazole ring.
of inhibitory activity, indicating that a certain bulkiness was
Among the aforementioned thiophene derivatives, com-
required at the 4-position of the thiophen ring. The alkyl de- pound 9a was chosen for further evaluation. We also evalu-

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
829
ated and showed the inhibitory activity of compound 9d glucose, plasma insulin and corticosterone levels in retro-
against mouse 11β-HSD1 for comparison. Although compound peritoneal adipose tissue were measured. Compound 9a dose-
9a showed only moderate inhibitory activity against mouse dependently reduced plasma glucose and insulin levels, and
11β-HSD1, it had potent inhibitory activity against human decreased corticosterone levels in adipose tissue. Although
11β-HSD1 and was metabolically stable against human liver compound 9a showed only moderate inhibitory activity
microsomes (Table 5). As shown in Fig. 4, we further tested against mouse 11β-HSD1 in vitro, its activity was sufficient
compound 9a to determine its activity after oral administra- to lower corticosterone levels in adipose tissue, resulting in a
tion. Seven-week-old male diabetic ob/ob mice were treated decrease in plasma glucose and insulin levels in vivo.
twice daily with 0.3, 1, 3 and 10mg/kg of 9a for four weeks.
Table 4. Conversion of the 4-Position Substituent of 1,2,4-Triazole
At 12h after the final administration, plasma and retro-
peritoneal adipose tissue samples were collected, and plasma
Table 3. Conversion of Substituent on the Thiophene Ring
Human
11β-HSD1 11β-HSD2
IC50 (nM) IC50 (nM)
Human
Mouse CL
(mL/min/kg)
Compound
R
int CLogP
11
c-Pr
Me
Et
7.9
26
>3000
NTb)
1445
NTb)
1298
2771
5746
4190
NTb)
NTb)
3.54
3.30
3.62
4.12
4.07
4.03
5.16
5.39
9ka)
9la)
Human
11β-HSD1 11β-HSD2
IC50 (nM) IC50 (nM)
Human
9.6
8.6
>3000
>3000
>10000
>10000
NTb)
Mouse CLint
(mL/min/kg)
Compound
R
CLogP
25aa)
25ba)
25c
n-Pr
i-Pr
14
9d
9ma)
10d
11
H
23
11
15
>10000
>3000
>10000
>3000
>10000
>30000
>10000
>10000
>10000
>10000
>3000
1122
1461
1494
1445
1983
385
2.95
3.00
3.34
3.54
3.06
2.49
3.59
3.50
2.90
3.34
3.06
3.71
3.53
Cyclopropyl methyl 12
25da)
Bn
22
43
5-F
5-Cl
5-Br
25e
Phenethyl
>10000
7.9
a) Hydrochloride salt. b) Not tested.
13
5-Me
19
95
24
12
12
14
15
5-CN
9b
5-CF3
4-Br
423
Table 5. Profile of Compound 9a
9n
1674
1666
6791
2116
>9066
578b)
Human
Mouse
Human
9c
3-F
Human CLint Mouse CLint
(mL/min/kg) (mL/min/kg)
Compound 11β-HSD1 11β-HSD1 11β-HSD2
9o
3-Cl
IC50 (nM) IC50 (nM) IC50 (nM)
10c
10o
9aa)
3-F, 5-Cl
3,5-diCl
3-F, 5-CF3
8.7
13
4.8
>10000
>3000
9d
9aa)
23
4.8
20
>10000
>3000
NTb)
27c)
1122
578c)
189
a) Hydrochloride salt. b) Evaluated with free form.
a) Hydrochloride salt. b) Not tested. c) Evaluated with the free form.
Fig. 4. Effects of Repeated Administration of 9a in Male ob/ob Mice (0.3–10mg/kg, Twice Daily for 4 Weeks, n=7–8)
Values are presented as the mean standard error (S.E.). *p<0.05, **p<0.01 versus vehicle-treated group, using the Dunnett’s multiple comparisons test.

830
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
Fig. 6. (a) Molecular Modeling of the Interaction between Compound
9a (Ball and Stick Diagram: Blue Is Nitrogen, Yellow Is Sulfur, Green Is
Fluorine and Pale Pink Is Carbon) and Human 11β-HSD1
The depicted residues are those that differ between human and mouse 11β-HSD1.
For example, Val180 in human, Ile in mouse. (b) Simplified view of (a) focusing
on the difference between human Val180 and mouse Ile180. Atoms relevant for
the discrepancy in interactions are shown in Space-filling spheres. Mouse Ile180
is colored cyan.
docking study suggested that 9a was positioned in the steroid
substrate binding site and that the nitrogen atom at the 1-po-
sition of the triazole ring interacted with Tyr183, while the
nitrogen atom at the 2-position of the triazole ring interacted
with Ser170. In addition, this model indicated that the thio-
phene moiety of 9a fit into the hydrophobic pocket of human
11β-HSD1. These findings may form the basis for the potent
inhibitory activity of 9a against human 11β-HSD1. To analyze
the species difference in the in vitro activity of 9a against
human and mouse 11β-HSD1, we also examined the sequence
homology of 11β-HSD1 in humans and mice. We found differ-
ences in several amino acid residues around the ligand bind-
ing site of 11β-HSD1 between humans and mice, as shown
Fig. 6a. Docking analysis of the interaction between 9a and
human 11β-HSD1 suggests that the trifluoromethyl moiety of
compound 9a fit into the hydrophobic pocket, which contained
Val180. The corresponding residue to Val180 in mice was
isoleucine (Ile), which is more sterically bulky than Val as
shown in Fig. 6b. Therefore the trifluoromethyl moiety of 9a
cannot exist at this position in mouse 11β-HSD1 due to steric
hindrance, and this may be an explanation for the attenuated
inhibitory activity of 9a against mouse 11β-HSD1 compared to
Fig. 5. (a) Molecular Modeling of the Interaction between 9a (as a Free
Base) and Human 11β-HSD1
Best docking solution (lowest binding energy) was calculated using GOLD ver-
sion 5.5.119–21) for compound 9a (ball and stick diagram: blue is nitrogen, yellow
is sulfur, green is fluorine and pale pink is carbon) when surrounded by human
11β-HSD1 active site residues. (b) Two-dimensional diagram prepared using the
ligand interactions application in MOE.22)
Finally, to explore the reason behind the very potent inhibi- human 11β-HSD1.
tory activity of 9a against human 11β-HSD1, we performed
a docking analysis of the interaction between 9a and human
11β-HSD1. The human 11β-HSD1 model was created based on
Conclusion
In our investigation of novel 11β-HSD1 inhibitors,
the crystal structure of human 11β-HSD1 in a complex with optimization of 9d resulted in the discovery of com-
a triazole inhibitor (PDB code 3D5Q).18) Compound 9a was pound 9a, 3,4-dicyclopropyl-5-{2-[3-fluoro-5-(trifluoromethyl)-
docked using GOLD version 5.5.1,19–21) as shown in Fig. 5. The thiophen-2-yl]propan-2-yl}-4H-1,2,4-triazole monohydrochlo-

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
831
ride, which showed potent and selective inhibitory activity hydrazide (4b) The title compound was prepared in a
against human 11β-HSD1, and good human and mouse liver similar manner to that described for 4a from ethyl 2-methyl-2-
microsomal stability. Oral administration of compound 9a [5-(trifluoromethyl)thiophen-2-yl]propanoate
(3b,
1.51g,
reduced corticosterone levels in adipose tissue, resulting in 5.67mmol, CAS No.; 950604-90-7) at 89% yield (1.27g,
1
reduced plasma glucose and insulin levels in diabetic ob/ob 5.03mmol). H-NMR (400MHz, DMSO-d6) δ: 1.56 (6H, s),
mice. Therefore, 9a may be a new type of insulin sensitizer 4.27 (2H, brs), 7.04–7.07 (1H, m), 7.51–7.55 (1H, m), 9.09 (1H,
with the potential to be an effective treatment for type II dia- brs). Electrospray ionization (ESI)-MS m/z: 253 [M+H]+.
betes.
2-(3-Fluorothiophen-2-yl)-2-methylpropanenitrile (6) A
mixture of iodomethane (2.14mL, 34.4mmol) and (3-
fluorothiophen-2-yl)acetonitrile (5, 1.62g, 11.5mmol, CAS
Experimental
Chemistry Starting materials and reagents are com- No.; 950604-64-5) in DMF (15mL) was slowly added to an
1
mercially available. H-NMR spectra were recorded on Var- ice-cooled mixture of sodium hydride (NaH) (60% in mineral
ian 300-MR, Varian 400-MR, Varian VNS-400 or JEOL oil; 1.19g, 29.8mmol) in DMF (25mL), and the mixture was
LAMBDA and chemical shifts were expressed in δ (ppm) stirred at r.t. for 20min. The reaction mixture was poured
values with trimethylsilane as an internal reference (s=sin- into ice water and extracted with EtOAc. The organic layer
glet, d=doublet, dd=double doublet, ddd=double double was washed with brine, dried over MgSO4, filtered and con-
doublet, t=triplet, q=quartet, m=multiplet, br=broad centrated in vacuo. The resulting residue was purified by
and brs=broad singlet). MS were recorded on JEOL JMS- column chromatography on silica gel (EtOAc–hexane) to give
LX2000, Waters ZQ2000, Thermo Electron TRACE DSQ, the title compound 6 (1.75g, 10.3mmol, 90%) as a colorless
1
Thermo Electron LCQ Advantage or Thermo Scientific Exac- oil. H-NMR (300MHz, CDCl3) δ: 1.81 (6H, s), 6.81 (1H, d,
tive Plus. Elemental analyses were performed with Elementar J=5.6Hz), 7.08–7.13 (1H, m).
Vario EL III (C, H, N) and DIONEX ICS-5000 (S, halogen)
2-(3-Fluorothiophen-2-yl)-2-methylpropanoic Acid (7)
instruments, and results were within 0.4% of theoretical Potassium hydroxide (1.74g, 31.0mmol) was added to a solu-
values. All reactions were carried out using commercially tion of 6 (1.75g, 10.3mmol) in ethylene glycol (17.5mL) and
available reagents and solvents without further purification. the mixture was stirred at 190°C for 2.5h. The reaction mix-
The following abbreviations are used: AcOH, acetic acid; ture was cooled to r.t. and water was added to the mixture.
DMF, N,N-dimethylformamide; DMSO, N,N-dimethylsulfox- The mixture was washed with Et2O and the aqueous layer was
ide; Et2O, diethyl ether; EtOAc, ethyl acetate; EtOH, ethanol; cooled in an ice bath, and conc. hydrochloric acid (ca. 30mL)
Et3N, triethylamine; HOBt, 1-hydroxybenzotriazole; MeOH, was added. The mixture was extracted with Et2O and washed
methanol; THF, tetrahydrofuran; WSCD, water-soluble car- with 1M hydrochloric acid and brine. The organic layer was
bodiimide; CAS No., Chemical Abstracts Service Registry dried over MgSO4, filtered and concentrated in vacuo, then
Number.
dried to give the title compound 7 (1.91g, 10.1mmol, 98%)
1
Methyl 2-[3-Fluoro-5-(trifluoromethyl)thiophen-2-yl]-2- as an ochre solid. H-NMR (300MHz, CDCl3) δ: 1.67 (6H, s),
methylpropanoate (3a) Dipotassium carbonate (6.20g, 6.76 (1H, d, J=5.6Hz), 7.04 (1H, dd, J=3.7, 5.6Hz).
44.9mmol) and iodomethane (2.27mL, 36.5mmol) were added
3,4-Dicyclopropyl-5-[2-(3-fluorothiophen-2-yl)propan-
to an ice-cooled solution of 2-[3-fluoro-5-(trifluoromethyl)- 2-yl]-4H-1,2,4-triazole (9c) WSCD·HCl (2.41g, 12.6mmol)
thiophen-2-yl]-2-methyl propanoic acid (2, 7.67g, 29.9mmol, was added to an ice-cooled solution of 7 (2.15g, 11.4mmol),
CAS No.; 950604-93-0) in DMF (30mL), and the mixture was hydrazine monohydrate (1.11mL, 22.9mmol) and HOBt·H2O
stirred at r.t. for 15h. The reaction mixture was diluted with (1.84g, 12.0mmol) in CH2Cl2 (21.5mL), and the mixture
water (60mL) and extracted with Et2O. The organic layer was was stirred at r.t. overnight. WSCD·HCl (240mg, 1.25mmol)
washed with water, dried over Na2SO4, filtered and concen- was added and the reaction mixture was stirred at r.t. for 2h.
trated in vacuo. The resulting residue was purified by column Saturated aqueous sodium hydrogen carbonate solution was
chromatography on silica gel (hexane–Et2O) to give the title added and the reaction mixture was extracted with CHCl3,
compound 3a (6.81g, 25.2mmol, 84%) as a pale yellow syrup. and washed with H2O and brine. The organic layer was
1H-NMR (400MHz, CDCl3) δ: 1.65 (6H, s), 3.73 (3H, s), dried over MgSO4, filtered and concentrated in vacuo, then
7.12–7.13 (1H, m).
dried to give a pale yellow solid (2.10g, 10.4mmol). A mix-
2-[3-Fluoro-5-(trifluoromethyl)thiophen-2-yl]-2-methyl- ture of N-cyclopropylcyclopropanecarboxamide (8a, 1.56g,
propanehydrazide (4a) Hydrazine monohydrate (1.0mL, 12.5mmol) and methyl trifluoromethanesulfonate (1.59mL,
21mmol) was added to a solution of 3a (322mg, 1.19mmol) in 13.5mmol) was stirred at 60°C for 30min. Toluene (26mL),
EtOH (2mL) and the mixture was stirred at 80°C for 24h. The Et3N (1.88mL, 13.5mmol) and the obtained solid described
reaction mixture was concentrated in vacuo. The resulting above (2.10g, 10.4mmol) were added and the mixture was
residue was diluted with saturated aqueous sodium hydrogen stirred at 60°C for 2d and 100°C for 2h. CHCl3 and saturated
carbonate solution and extracted with CHCl3. The organic aqueous sodium hydrogen carbonate solution were added to
layer was dried over Na2SO4, filtered and concentrated in the mixture, and the organic layer was separated. The organic
vacuo. The resulting residue was purified by column chroma- layer was washed with brine, dried and concentrated in vacuo.
tography on silica gel (CHCl3–MeOH) to give the title com- The resulting residue was purified by column chromatogra-
pound 4a (286mg, 1.06mmol, 89%) as a pale yellow syrup. phy on silica gel (EtOAc–MeOH) to give a pale yellow solid.
1H-NMR (400MHz, CDCl3) δ: 1.66 (6H, s), 2.10–3.90 (2H, CHCl3 was added to the resulting solid and the precipitated
br), 6.80–7.04 (1H, m), 7.17 (1H, s). Chemical ionization mass solid was filtered off. Diisopropyl ether was added to the re-
spectrometry (CI-MS) m/z: 271 [M+H]+.
2-Methyl-2-[5-(trifluoromethyl)thiophen-2-yl]propane- with a solvent comprising hexane, EtOAc and Et2O, followed
sulting residue and solidified. The obtained solid was washed

832
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
by washing with diisopropyl ether to give the title compound (2H, m), 1.01–1.11 (5H, m), 1.87–2.03 (2H, m), 2.00 (6H, s),
9c (878mg, 3.01mmol, 26%) as a white solid. 1H-NMR 2.95–3.02 (1H, m), 3.10–3.18 (2H, m), 6.80 (1H, dd, J=1.2,
(300MHz, CDCl3) δ: 0.79–1.05 (6H, m), 1.15–1.22 (2H, m), 3.6Hz), 6.97 (1H, dd, J=3.5, 5.3Hz), 7.23–7.28 (1H, m). FAB-
1.88–2.00 (1H, m), 1.93 (6H, s), 2.70–2.79 (1H, m), 6.71 (1H, MS m/z: 276 [M+H]+. HR-MS (ESI) m/z: 276.1524 [M+H]+
d, J=5.6Hz), 7.01–7.05 (1H, m). ESI-MS m/z: 292 [M+H]+. (Calcd for C15H22N3S: 276.1529).
High resolution (HR)-MS (ESI) m/z: 292.1276 [M+H]+
(Calcd for C15H19N3FS: 292.1278).
4-Cyclopropyl-3-(cyclopropylmethyl)-5-[2-(thiophen-
2-yl)propan-2-yl]-4H-1,2,4-triazole (9f) The title com-
3,4-Dicyclopropyl-5-{2-[3-fluoro-5-(trifluoromethyl)thio- pound was prepared in a similar manner to that described for
phen-2-yl]propan-2-yl}-4H-1,2,4-triazole Monohydrochlo- 9a, except for the salt-forming procedure, from 4d (883mg,
ride (9a) A mixture of 8a (220mg, 1.76mmol) and methyl 4.79mmol) and N,2-dicyclopropylacetamide (1.00g, 7.18mmol)
1
trifluoromethanesulfonate (200µL, 1.77mmol) was stirred at at 51% yield (709mg, 2.47mmol) as a white solid. H-NMR
60°C for 30min and diluted with toluene (4mL). A solution (400MHz, CDCl3) δ: 0.23–0.29 (2H, m), 0.48–0.60 (4H, m),
of Et3N (500µL, 3.59mmol) and 4a (279mg, 1.03mmol) in 0.80–0.87 (2H, m), 1.15–1.24 (1H, m), 1.96 (6H, s), 2.71–2.78
toluene (6mL) was added and the mixture was stirred at 60°C (3H, m), 6.73 (1H, dd, J=1.2, 3.6Hz), 6.90 (1H, dd, J=3.5,
for 24h, 90°C for 9h and 110°C for 15h. The reaction mixture 5.1Hz), 7.15 (1H, dd, J=1.1, 5.1Hz). FAB-MS m/z: 288
was concentrated in vacuo, diluted with water (20mL) and ex- [M+H]+. HR-MS (ESI) m/z: 288.1527 [M+H]+ (Calcd for
tracted with EtOAc. The organic layer was dried over Na2SO4, C16H22N3S: 288.1529).
filtered and concentrated in vacuo. The resulting residue was
3-tert-Butyl-4-cyclopropyl-5-[2-(thiophen-2-yl)propan-
purified by column chromatography on silica gel (CHCl3– 2-yl]-4H-1,2,4-triazole (9g) The title compound was pre-
MeOH) to give a colorless syrup. The syrup was diluted pared in a similar manner to that described for 9a, except for
with EtOAc (5mL), and 4M HCl solution in EtOAc (0.3mL, the salt-forming procedure, from 4d (814mg, 4.42mmol) and
1.2mmol) was added to the mixture. The reaction mixture N-cyclopropyl-2,2-dimethylpropanamide (2.50g, 17.7mmol) at
1
was stirred at r.t. for 30min and concentrated in vacuo. The 6.5% yield (83.0mg, 0.287mmol) as a white solid. H-NMR
resulting solid was washed with diisopropyl ether, collected (300MHz, DMSO-d6) δ: 0.44–0.51 (2H, m), 0.86–0.94 (2H,
by filtration and dried to give the title compound 9a (166mg, m), 1.44 (9H, s), 1.85 (6H, s), 3.09–3.19 (1H, m), 6.76 (1H, dd,
0.419mmol, 41%) as a white solid. 1H-NMR (400MHz, J=1.1, 3.5Hz), 6.95 (1H, dd, J=3.5, 5.1Hz), 7.37 (1H, dd,
DMSO-d6) δ: 0.78–0.84 (2H, m), 0.98–1.05 (2H, m), 1.14–1.20 J=1.1, 5.1Hz). ESI-MS m/z: 290 [M+H]+. HR-MS (ESI) m/z:
(4H, m), 1.91 (6H, s), 2.19–2.28 (1H, m), 3.15–3.22 (1H, 290.1690 [M+H]+ (Calcd for C16H24N3S: 290.1685).
m), 7.80 (1H, d, J=1.0Hz). FAB-MS m/z: 360 [M+H]+.
3-Cyclobutyl-4-cyclopropyl-5-[2-(thiophen-2-yl)propan-
HR-MS (ESI) m/z: 360.1151 [M+H]+ (Calcd for C16H18N3F4S: 2-yl]-4H-1,2,4-triazole (9h) The title compound was pre-
360.1152). Anal. Calcd for C16H17F4N3S.HCl: C, 48.55; H, 4.58; pared in a similar manner to that described for 9a, except for
N, 10.62; S, 8.10; Cl, 8.96; F, 19.20. Found: C, 48.95; H, 4.82; the salt-forming procedure, from 4d (1.32g, 7.18mmol) and
N, 10.60; S, 8.06; Cl, 8.85; F, 19.10.
N-cyclopropylcyclobutanecarboxamide (1.00g, 7.18mmol) at
1
3,4-Dicyclopropyl-5-{2-[5-(trifluoromethyl)thiophen-2-yl]- 29% yield (590mg, 2.05mmol) as a colorless solid. H-NMR
propan-2-yl}-4H-1,2,4-triazole (9b) The title compound (400MHz, DMSO-d6) δ: 0.27–0.33 (2H, m), 0.74–0.81 (2H,
was prepared in a similar manner to that described for m), 1.80–1.92 (1H, m), 1.85 (6H, s), 1.95–2.08 (1H, m),
9a, except for the salt-forming procedure, from 4b (1.00g, 2.25–2.41 (4H, m), 2.80–2.87 (1H, m) 3.63–3.73 (1H, m) 6.77
3.96mmol) and 8a (843mg, 6.73mmol) at 34% yield (456mg, (1H, dd, J=1.1, 3.6Hz), 6.94 (1H, dd, J=3.5, 5.1Hz), 7.37
1.34mmol). 1H-NMR (400MHz, DMSO-d6) δ: 0.48–0.53 (1H, dd, J=1.1, 5.1Hz). FAB-MS m/z: 288 [M+H]+. HR-MS
(2H, m), 0.88–1.02 (6H, m), 1.89 (6H, s), 2.00–2.07 (1H, m), (ESI) m/z: 288.1534 [M+H]+ (Calcd for C16H22N3S: 288.1529).
3.02–3.09 (1H, m), 6.92–6.95 (1H, m), 7.55–7.58 (1H, m). ESI-
MS m/z: 342 [M+H]+. HR-MS (ESI) m/z: 342.1243 [M+H]+ 2-yl]-4H-1,2,4-triazole (9i) The title compound was pre-
(Calcd for C16H19N3F3S: 342.1246). pared in a similar manner to that described for 9a, except for
3-Cyclopentyl-4-cyclopropyl-5-[2-(thiophen-2-yl)propan-
3,4-Dicyclopropyl-5-[2-(thiophen-2-yl)propan-2-yl]-4H- the salt-forming procedure, from 4d (1.00g, 5.43mmol) and
1,2,4-triazole (9d) The title compound was prepared in N-cyclopropylcyclopentanecarboxamide (1.25g, 8.14mmol)
1
a similar manner to that described for 9a, except for the at 37% yield (611mg, 2.03mmol) as a white solid. H-NMR
salt-forming procedure, from 2-methyl-2-(thiophen-2-yl)- (400MHz, CDCl3) δ: 0.51–0.57 (2H, m), 0.84–0.91 (2H, m),
propanehydrazide (4d, 1.00g, 5.43mmol, CAS No.; 1.59–1.72 (2H, m), 1.84–2.15 (6H, m), 1.96 (6H, s), 2.71–2.78
880166-34-7) and 8a (1.02g, 8.14mmol) at 29% yield (425mg, (1H, m), 3.21–3.32 (1H, m), 6.72–6.75 (1H, m), 6.91 (1H, dd,
1.55mmol) as a solid. 1H-NMR (400MHz, DMSO-d6) δ: J=3.8, 4.8Hz), 7.15–7.19 (1H, m). ESI-MS m/z: 302 [M+H]+.
0.45–0.50 (2H, m), 0.83–0.89 (2H, m), 0.92–1.03 (4H, m), 1.85 HR-MS (ESI) m/z: 302.1682 [M+H]+ (Calcd for C17H24N3S:
(6H, s), 2.01–2.08 (1H, m), 2.96–3.03 (1H, m), 6.81 (1H, dd, 302.1685).
J=1.2, 3.6Hz), 6.95 (1H, dd, J=3.5, 5.0Hz), 7.39 (1H, dd,
J=1.2, 5.2Hz). FAB-MS m/z: 274 [M+H]+. HR-MS (ESI) 2-yl]-4H-1,2,4-triazole (9j) The title compound was pre-
m/z: 274.1366 [M+H]+ (Calcd for C15H20N3S: 274.1372).
pared in a similar manner to that described for 9a, except for
3-Cyclohexyl-4-cyclopropyl-5-[2-(thiophen-2-yl)propan-
4-Cyclopropyl-3-propyl-5-[2-(thiophen-2-yl)propan- the salt-forming procedure, from 4d (1.00g, 5.43mmol) and
2-yl]-4H-1,2,4-triazole Monohydrochloride (9e) The title N-cyclopropylcyclohexanecarboxamide (1.18g, 7.06mmol) at
compound was prepared in a similar manner to that described 40% yield (687mg, 2.18mmol) as a white solid. 1H-NMR
for 9a from 4d (449mg, 2.44mmol) and N-cyclopropylbutan- (400MHz, CDCl3) δ: 0.50–0.55 (2H, m), 0.84–0.90 (2H, m),
amide (310mg, 2.44mmol) at 11% yield (80mg, 0.26mmol) 1.27–1.43 (3H, m), 1.71–2.01 (7H, m), 1.95 (6H, s), 2.68–2.76
as a white solid. 1H-NMR (400MHz, CDCl3) δ: 0.72–0.78 (1H, m), 2.82–2.92 (1H, m), 6.73 (1H, dd, J=1.2, 3.6Hz), 6.90

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
833
(1H, dd, J=3.6, 5.1Hz), 7.16 (1H, dd, J=1.1, 5.1Hz). ESI- a similar manner to that described for 9a, except for the
MS m/z: 316 [M+H]+. HR-MS (ESI) m/z: 316.1843 [M+H]+ salt-forming procedure, from 2-methyl-2-(pyrazin-2-yl)-
(Calcd for C18H26N3S: 316.1842).
propanehydrazide (800mg, 4.44mmol, CAS No.; 950604-81-6)
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopropyl- and 8a (885mg, 7.07mmol) at 43% yield (513mg, 1.90mmol)
1
4-methyl-4H-1,2,4-triazole Monohydrochloride (9k) The as a white solid. H-NMR (300MHz, DMSO-d6) δ: 0.28–0.35
title compound was prepared in a similar manner to that (2H, m), 0.61–0.69 (2H, m), 0.89–1.00 (4H, m), 1.82 (6H, s),
described for 9a from 2-(5-bromothiophen-2-yl)-2-methylpro- 1.94–2.05 (1H, m), 2.71–2.80 (1H, m), 8.55–8.57 (2H, m),
panehydrazide (690mg, 2.62mmol, CAS No.; 950604-87-2) 8.59–8.61 (1H, m). ESI-MS m/z: 270 [M+H]+. HR-MS (ESI)
and N-methylcyclopropanecarboxamide (390mg, 3.93mmol) m/z: 270.1712 [M+H]+ (Calcd for C15H20N5: 270.1713).
at 13% yield (127mg, 0.350mmol) as a solid. 1H-NMR
3-[2-(5-Chlorothiophen-2-yl)propan-2-yl]-4,5-dicyclopro-
(400MHz, DMSO-d6) δ: 1.16–1.21 (4H, m), 1.78 (6H, s), pyl-4H-1,2,4-triazole (10d) N-Chlorosuccinimide (51.3mg,
2.13–2.21 (1H, m), 3.45 (3H, s), 6.88 (1H, d, J=3.9Hz), 7.16 0.384mmol) was added to solution of 9d (100mg,
(1H, d, J=3.9Hz). ESI-MS m/z: 326 [M+H]+. HR-MS (ESI) 0.366mmol) in AcOH (3mL) and the mixture was stirred
m/z: 326.0324 [M+H]+ (Calcd for C13H17N3BrS: 326.0321).
at 80°C for 2h. The reaction mixture was concentrated in
a
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopropyl- vacuo and diluted with CHCl3. One molecule aqueous sodium
4-ethyl-4H-1,2,4-triazole Monohydrochloride (9l) The hydroxide solution was added and the reaction mixture was
title compound was prepared in a similar manner to that extracted with CHCl3. The organic layer was washed with
described for 9a from 2-(5-bromothiophen-2-yl)-2-methyl- brine, dried over MgSO4, filtered and concentrated in vacuo.
propanehydrazide (500mg, 1.90mmol) and N-ethylcyclopro- The resulting residue was purified by column chromatogra-
panecarboxamide (323mg, 2.85mmol) at 16% yield (111mg, phy on silica gel (CHCl3–MeOH). The obtained product was
1
0.295mmol) as a solid. H-NMR (400MHz, CDCl3) δ: 1.20 washed with Et2O, and dried to give the title compound 10d
(3H, t, J=7.2Hz), 1.39–1.46 (2H, m), 1.87–1.94 (3H, m), 1.89 (66.7mg, 0.217mmol, 59%) as a light pink solid. 1H-NMR
(6H, s), 4.02 (2H, q, J=7.3Hz), 6.67 (1H, d, J=3.8Hz), 6.97 (400MHz, DMSO-d6) δ: 0.56–0.61 (2H, m), 0.90–1.01 (6H,
(1H, d, J=3.8Hz). ESI-MS m/z: 340 [M+H]+. HR-MS (ESI) m), 1.83 (6H, s), 1.98–2.07 (1H, m), 3.00–3.07 (1H, m), 6.70
m/z: 340.0479 [M+H]+ (Calcd for C14H19N3BrS: 340.0478).
(1H, d, J=3.7Hz), 6.96 (1H, d, J=3.9Hz). FAB-MS m/z: 308
3,4-Dicyclopropyl-5-[2-(5-fluorothiophen-2-yl)propan- [M+H]+. HR-MS (ESI) m/z: 308.0984 [M+H]+ (Calcd for
2-yl]-4H-1,2,4-triazole Monohydrochloride (9m) The title C15H19N3ClS: 308.0983).
compound was prepared in a similar manner to that described
3-[2-(5-Chloro-3-fluorothiophen-2-yl)propan-2-yl]-4,5-
for 9a from 2-(5-fluorothiophen-2-yl)-2-methylpropanehy- dicyclopropyl-4H-1,2,4-triazole (10c) The title compound
drazide (515mg, 2.55mmol CAS No.; 950604-98-5) and 8a was prepared in a similar manner to that described for
(637mg, 5.09mmol) at 19% yield (161mg, 0.491mmol) as a 10d from 9c (150mg, 0.515mmol) at 63% yield (106mg,
1
colorless solid. H-NMR (400MHz, DMSO-d6) δ: 0.74–0.80 0.325mmol) as a white solid. 1H-NMR (300MHz, CDCl3)
(2H, m), 1.00–1.07 (2H, m), 1.19–1.31 (4H, m), 1.85 (6H, s), δ: 0.87–0.96 (2H, m), 0.97–1.07 (4H, m), 1.14–1.22 (2H, m),
2.23–2.32 (1H, m), 3.21–3.28 (1H, m), 6.60–6.63 (2H., m). 1.85–1.99 (1H, m), 1.91 (6H, s), 2.77–2.88 (1H, m), 6.61 (1H,
FAB-MS m/z: 292 [M+H]+. HR-MS (ESI) m/z: 292.1276 s). ESI-MS m/z: 326 [M+H]+. HR-MS (ESI) m/z: 326.0888
[M+H]+ (Calcd for C15H19N3FS: 292.1278).
3-[2-(4-Bromothiophen-2-yl)propan-2-yl]-4,5-dicyclopro-
[M+H]+ (Calcd for C15H18N3ClFS: 326.0889).
3,4-Dicyclopropyl-5-[2-(3,5-dichlorothiophen-2-yl)pro-
pyl-4H-1,2,4-triazole (9n) The title compound was pre- pan-2-yl]-4H-1,2,4-triazole (10o) The title compound
pared in a similar manner to that described for 9a, except was prepared in a similar manner to that described for
for the salt-forming procedure, from 2-(4-bromothiophen- 10d from 9o (100mg, 0.325mmol) at 51% yield (56.8mg,
2-yl)-2-methylpropanehydrazide (7.4g, 28mmol, CAS No.; 0.166mmol) as a white solid. 1H-NMR (300MHz, CDCl3)
950604-78-1) and 8a (4.58g, 36.6mmol) at 41% yield (4.07g, δ: 0.89–1.05 (6H, m), 1.13–1.20 (2H, m), 1.87–1.98 (1H, m),
1
11.6mmol) as a white solid. H-NMR (300MHz, DMSO-d6) δ: 1.90 (6H, s), 2.57–2.66 (1H, m), 6.71 (1H, s). ESI-MS m/z: 342
0.47–0.55 (2H, m), 0.87–1.02 (6H, m), 1.84 (6H, s), 1.98–2.08 [M+H]+. HR-MS (ESI) m/z: 342.0596 [M+H]+ (Calcd for
(1H, m), 2.98–3.08 (1H, m), 6.88 (1H, d, J=1.5Hz), 7.54 (1H, C15H18N3Cl2S: 342.0593).
d, J=1.4Hz). ESI-MS m/z: 352 [M+H]+. HR-MS (ESI) m/z:
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-4,5-dicyclopro-
pyl-4H-1,2,4-triazole (11) N-Bromosuccinimide (68mg,
352.0478 [M +H]+ (Calcd for C15H19N3BrS: 352.0478).
3-[2-(3-Chlorothiophen-2-yl)propan-2-yl]-4,5-dicyclopro- 0.38mmol) was added to
a solution of 9d (100mg,
pyl-4H-1,2,4-triazole (9o) The title compound was pre- 0.366mmol) in AcOH (3mL) and the mixture was stirred at
pared in a similar manner to that described for 9a, except 80°C for 2h. The reaction mixture was concentrated in vacuo
for the salt-forming procedure, from 2-(3-chlorothiophen- and diluted with CHCl3. One molar aqueous sodium hydroxide
2-yl)-2-methylpropanehydrazide (8.32g, 38.0mmol, CAS No.; solution was added and the reaction mixture was extracted
950604-75-8) and 8a (5.71g, 45.7mmol) at 18% yield (2.10g, with CHCl3. The organic layer was washed with brine, dried
1
6.82mmol) as a pale yellow solid. H-NMR (300MHz, CDCl3) over MgSO4, filtered and concentrated in vacuo. The resulting
δ: 0.76–0.93 (4H, m), 0.95–1.04 (2H, m), 1.12–1.19 (2H, m), residue was purified by column chromatography on silica gel
1.85–1.97 (1H, m), 1.93 (6H, s), 2.48–2.58 (1H, m), 6.85 (CHCl3–MeOH). The obtained product was washed with Et2O,
(1H, d, J=5.3Hz), 7.16 (1H, d, J=5.4Hz). ESI-MS m/z: 308 and dried to give the title compound 11 (72.8mg, 0.207mmol,
1
[M+H]+. HR-MS (ESI) m/z: 308.0983 [M+H]+ (Calcd for 57%) as a colorless solid. H-NMR (400MHz, DMSO-d6) δ:
C15H19N3ClS: 308.0983).
0.54–0.60 (2H, m), 0.89–1.01 (6H, m), 1.83 (6H, s), 1.99–2.07
2-[2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)propan- (1H, m), 3.00–3.06 (1H, m), 6.67 (1H, d, J=3.6Hz), 7.06 (1H,
2-yl]pyrazine (9p) The title compound was prepared in d, J=3.9Hz). FAB-MS m/z: 354 [M+H]+. HR-MS (ESI) m/z:

834
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
352.0477 [M +H]+ (Calcd for C15H19N3BrS: 352.0478).
(400MHz, DMSO-d6) δ: 0.48–0.54 (2H, m), 0.88–1.02 (6H,
5-[2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)propan- m), 1.89 (6H, s), 1.99–2.07 (1H, m), 3.01–3.07 (1H, m), 7.02
2-yl]thiophene-2-carbaldehyde (12) N,N,N′,N′-Tetramethyl (1H, d, J=3.9Hz), 7.84 (1H, d, J=3.9Hz). FAB-MS m/z: 299
ethylenediamine (1.64mL, 10.9mmol) was added to a solution [M+H]+. HR-MS (ESI) m/z: 299.1324 [M+H]+ (Calcd for
of 11 (1.92g, 5.45mmol) in THF (40mL) and the mixture was C16H19N4S: 299.1325).
cooled to −78°C. n-Butyl lithium (1.6M in hexane; 5.1mL,
tert-Butyl [2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)-
8.2mmol) was added and the mixture was stirred at −78°C propan-2-yl]carbamate (17) A mixture of 8a (500mg,
for 45min. A solution of DMF (1.27mL, 16.4mmol) in THF 3.99mmol) and methyl trifluoromethanesulfonate (452µL,
(10mL) was added and the mixture was stirred at −78°C for 3.99mmol) was stirred at 60°C for 30min. Tolu-
30min. The reaction mixture was poured into water and ex- ene (9mL), Et3N (557µL, 3.99mmol) and tert-butyl
tracted with CHCl3. The organic layer was washed with brine, (1-hydrazinyl-2-methyl-1-oxopropan-2-yl)carbamate
(16,
dried over MgSO4, filtered and concentrated in vacuo. The 723mg, 3.33mmol, CAS No.; 184002-61-7) were added and
resulting residue was purified by column chromatography on the reaction mixture was stirred at 60°C for 2h and 100°C for
silica gel (CHCl3–MeOH). The obtained product was washed 1h. CHCl3 and saturated aqueous sodium hydrogen carbonate
with diisopropyl ether, and dried to give the title compound solution were added to the mixture, and the organic layer was
1
12 (950mg, 3.15mmol, 58%) as a pale yellow solid. H-NMR separated and washed with brine, dried over MgSO4, filtered
(400MHz, DMSO-d6) δ: 0.47–0.53 (2H, m), 0.86–1.01 (6H, and concentrated in vacuo. The residue was purified by col-
m), 1.89 (6H, s), 1.99–2.07 (1H, m), 3.00–3.07 (1H, m), 7.03 umn chromatography on silica gel (CHCl3–MeOH) to give the
(1H, d, J=3.9Hz), 7.89 (1H, d, J=3.9Hz), 9.85 (1H, s). ESI- title compound 17 (464mg, 1.51mmol, 46%) as a white solid.
MS m/z: 302 [M+H]+.
1H-NMR (300MHz, CDCl3) δ: 0.94–1.24 (8H, m), 1.35 (9H,
3,4-Dicyclopropyl-5-[2-(5-methylthiophen-2-yl)propan- brs), 1.78 (6H, s), 1.88–2.01 (1H, m), 3.00–3.13 (1H, m), 5.28
2-yl]-4H-1,2,4-triazole (13)
mixture of 12 (300mg, (1H, brs). ESI-MS m/z: 307 [M+H]+.
0.995mmol), diethylene glycol (10mL) and hydrazine mono- 2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)propan-
A
hydrate (121µL, 2.49mmol) was stirred at 130°C for 3h. 2-amine (18) A mixture of 17 (480mg, 1.57mmol), 4M HCl
Potassium hydroxide (140mg, 2.49mmol) was added and the solution in EtOAc (1.96mL, 7.83mmol) and EtOH (10mL) was
mixture was stirred at 170°C for 4h. The reaction mixture stirred at 50°C for 8h. CHCl3, 1M aqueous sodium hydroxide
was cooled and water was added. The reaction mixture was solution and water were added to the mixture. The organic
extracted with CHCl3 and the organic layer was washed with layer was separated and washed with brine, dried over MgSO4,
brine, dried over MgSO4, filtered and concentrated in vacuo. filtered and concentrated in vacuo, then dried to give the title
The resulting residue was purified by column chromatog- compound 18 (320mg, 1.55mmol, 99%) as a pale yellow oil.
raphy on silica gel (CHCl3–MeOH). The obtained product 1H-NMR (300MHz, CDCl3) δ: 0.98–1.06 (2H, m), 1.12–1.27
was washed with diisopropyl ether and dried to give the title (4H, m), 1.33–1.41 (2H, m), 1.67 (6H, s), 1.89–2.01 (1H, m),
compound 13 (54.9mg, 0.191mmol, 19%) as a pale yellow 3.17–3.26 (1H, m). ESI-MS m/z: 207 [M+H]+.
1
solid. H-NMR (400MHz, DMSO-d6) δ: 0.52–0.57 (2H, m),
3,4-Dicyclopropyl-5-[2-(1H-pyrrol-1-yl)propan-2-yl]-4H-
0.85–1.01 (6H, m), 1.80 (6H, s), 1.98–2.06 (1H, m), 2.36 (3H, 1,2,4-triazole Monohydrochloride (19) A mixture of 18
s), 2.95–3.01 (1H, m), 6.56–6.62 (2H, m). ESI-MS m/z: 288 (500mg, 2.42mmol), 2,5-dimethoxytetrahydrofuran (373µL,
[M+H]+. HR-MS (ESI) m/z: 288.1529 [M+H]+ (Calcd for 2.91mmol), AcOH (5mL) and CHCl3 (5mL) was stirred at
C16H22N3S: 288.1529).
70°C for 24h. CHCl3, 1M aqueous sodium hydroxide solu-
3,4-Dicyclopropyl-5-[2-(5-iodothiophen-2-yl)propan- tion and water were added to the mixture. The organic layer
2-yl]-4H-1,2,4-triazole (14) N-Iodosuccinimide (286mg, was separated and washed with brine, dried over MgSO4,
1.27mmol) was added to a solution of 9d (331mg, 1.21mmol) filtered and concentrated in vacuo. The residue was purified
in AcOH (5mL) and the mixture was stirred overnight at r.t. by column chromatography on silica gel (CHCl3–MeOH).
CHCl3 and water were added, and the mixture was extracted The obtained product was dissolved in Et2O (20mL), and 4M
with CHCl3. The organic layer was washed with 1M aque- HCl solution in EtOAc (606µL, 2.42mmol) was added to the
ous sodium hydroxide solution and brine, dried over MgSO4, mixture. The reaction mixture was stirred at r.t. for 30min
filtered and concentrated in vacuo. The resulting residue was and the solid was precipitated. The precipitate was collected
purified by column chromatography on silica gel (CHCl3– by filtration and dried to give the title compound 19 (590mg,
MeOH) to give the title compound 14 (437mg, 1.09mmol, 2.02mmol, 83%) as a colorless solid. 1H-NMR (400MHz,
1
90%) as a colorless solid. H-NMR (400MHz, DMSO-d6) δ: DMSO-d6) δ: 0.38–0.43 (2H, m), 0.87–0.94 (2H, m), 1.21–1.35
0.51–0.57 (2H, m), 0.87–1.00 (6H, m), 1.82 (6H, s), 1.97–2.06 (4H, m), 1.99 (6H, s), 2.24–2.31 (1H, m), 3.07–3.13 (1H, m),
(1H, m), 2.97–3.04 (1H, m), 6.54 (1H, d, J=3.5Hz), 7.16 (1H, 6.08 (2H, t, J=2.2Hz), 6.75 (2H, t, J=2.2Hz). FAB-MS m/z:
d, J=3.6Hz). FAB-MS m/z: 400 [M+H]+.
257 [M+H]+. HR-MS (ESI) m/z: 257.1755 [M+H]+ (Calcd
5-[2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)propan- for C15H21N4: 257.1761).
2-yl]thiophene-2-carbonitrile (15) Copper cyanide (196mg,
4-Cyclopropyl-3-[2-(thiophen-2-yl)propan-2-yl]-4H-
2.19mmol) was added to a solution of 14 (437mg, 1.09mmol) 1,2,4-triazole (21) Thionyl chloride (3.49mL, 47.8mmol)
in pyridine (10mL) and the mixture was stirred at 115°C for and DMF (74µL, 0.96mmol) were added to a solution
11.5h. The reaction mixture was concentrated in vacuo. The of N-cyclopropyl-2-methyl-2-(thiophen-2-yl)propanamide (20,
residue was purified by column chromatography on silica gel 2.00g, 9.56mmol, CAS No.; 950604-59-8) in CHCl3 (30mL),
(CHCl3–MeOH) and the resulting solid was washed with Et2O and the mixture was stirred at 60°C for 1h. The reaction mix-
and recrystallized from toluene to give the title compound 15 ture was concentrated in vacuo, and toluene (40mL) and for-
1
(170mg, 0.568mmol, 52%) as a pale yellow solid. H-NMR myl hydrazine (631mg, 10.5mmol) were added to the resulting

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
835
mixture. The mixture was stirred at 70°C for 20h, and CHCl3 for 60min using a microwave reaction device. The reaction
and 1M aqueous sodium hydroxide solution were added. The mixture was cooled to r.t. Toluene was added and the mix-
organic layer was separated and washed with brine, dried over ture was concentrated in vacuo. The residue was dissolved in
MgSO4, filtered and concentrated in vacuo. The residue was EtOAc and the mixture was washed with H2O. The organic
purified by column chromatography on silica gel (CHCl3– layer was dried over MgSO4 and filtered. 4M HCl solution
MeOH). The obtained product was washed with hexane and in EtOAc (1mL, 4mmol) was added and the mixture was
dried to give the title compound 21 (565mg, 2.42mmol, concentrated in vacuo. The residue was washed with EtOAc
1
25%) as a colorless solid. H-NMR (400MHz, DMSO-d6) δ: and diisopropyl ether, then dried to give the title compound
0.74–0.80 (4H, m), 1.86 (6H, s), 2.75–2.81 (1H, m), 6.89 (1H, 25a (231mg, 0.591mmol, 37%) as a white solid. 1H-NMR
dd, J=1.1, 3.5Hz), 6.97 (1H, dd, J=3.5, 5.3Hz), 7.42 (1H, dd, (400MHz, CDCl3) δ: 0.87 (3H, t, J=7.4Hz), 1.37–1.53 (4H,
J=1.2, 5.1Hz), 8.37 (1H, s). EI-MS m/z: 233 [M]+. HR-MS m), 1.84–2.00 (3H, m), 1.90 (6H, s), 3.86–3.93 (2H, m), 6.70
(ESI) m/z: 234.1053 [M+H]+ (Calcd for C12H16N3S: 234.1059). (1H, d, J=3.9Hz), 6.98 (1H, d, J=3.8Hz). FAB-MS m/z: 356
N′-[2-Methyl-2-(thiophen-2-yl)propanoyl]cyclopropan- [M+H]+. HR-MS (ESI) m/z: 354.0634 [M+H]+ (Calcd for
ecarbohydrazide (22) Et3N (15mL, 108mmol) was added C15H21N3BrS: 354.0634).
to a solution of 4d (19.0g, 103mmol) in dichloromethane
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopropyl-
(150mL) and the mixture was cooled in an ice bath. A solu- 4-(propan-2-yl)-4H-1,2,4-triazole Monohydrochloride (25b)
tion of cyclopropanecarbonyl chloride (9.80mL, 109mmol) in The title compound was prepared in a similar manner to that
dichloromethane (50mL) was added and the mixture stirred described for 25a from 24 (500mg, 1.60mmol) and isopropyl-
overnight at r.t. Water was added, and the mixture was ex- amine (1.37mL, 16.7mmol) at 12% yield (74mg, 0.19mmol)
1
tracted with CHCl3. The organic layer was dried over MgSO4, as a white solid. H-NMR (400MHz, CDCl3) δ: 1.41–1.48 (8H,
filtered and concentrated in vacuo. The obtained solid was m), 1.87 (6H, s), 1.96–2.08 (3H, m), 4.52–4.63 (1H, m), 6.62
washed with Et2O and dried to give the title compound 22 (1H, d, J=3.9Hz), 6.97 (1H, d, J=3.9Hz). FAB-MS m/z: 356
1
(24.0g, 95.1mmol, 92%) as a white solid. H-NMR (400MHz, [M+H]+. HR-MS (ESI) m/z: 354.0635 [M+H]+ (Calcd for
DMSO-d6) δ: 0.66–0.75 (4H, m), 1.53–1.62 (1H, m), 1.56 (6H, C15H21N3BrS: 354.0634).
s), 6.94 (1H, dd, J=3.5, 5.1Hz), 7.07 (1H, dd, J=1.0, 3.5Hz),
7.37 (1H, dd, J=1.1, 5.2Hz), 9.41 (1H, brs), 9.87 (1H, brs). 4-(cyclopropylmethyl)-4H-1,2,4-triazole (25c) Cycloprop-
EI-MS m/z: 252 [M]+.
anemethylamine (568mg, 7.99mmol) was added to a solution
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopropyl-
2-Cyclopropyl-5-[2-(thiophen-2-yl)propan-2-yl]-1,3,4- of 24 (500mg, 1.60mmol) in AcOH (5mL) and reacted at
oxadiazole (23) Pyridine (16.9mL, 209mmol) was added 170°C for 40min using a microwave reaction device. The
to a solution of 22 (24.0g, 95.1mmol) in dichloromethane reaction mixture was cooled to r.t. and concentrated in vacuo.
(480mL) and the mixture was cooled in an ice bath. Trifluo- The residue was dissolved in EtOAc and extracted with 1M
romethanesulfonic anhydride (21.8mL, 133mmol) was added hydrochloric acid. The aqueous layer was neutralized with 1M
and the mixture was stirred at r.t. for 3d. Saturated aqueous aqueous sodium hydroxide solution and extracted with EtOAc.
sodium hydrogen carbonate solution was added and the mix- The organic layer was dried over MgSO4, filtered and concen-
ture was extracted with CHCl3. The organic layer was dried trated in vacuo. The obtained syrup was dissolved in EtOAc,
over MgSO4, filtered and concentrated in vacuo. The residue and 4M HCl solution in EtOAc (1mL, 4mmol) was added and
was purified by column chromatography on silica gel (hex- the mixture was concentrated in vacuo. The obtained solid
ane–EtOAc). The obtained solid was washed with hexane and was washed with diisopropyl ether to give a white solid. The
dried to give the title compound 23 (6.34g, 27.1mmol, 28%) solid was mixed with 1M aqueous sodium hydroxide solu-
as a white solid. 1H-NMR (400MHz, CDCl3) δ: 1.06–1.11 tion and extracted with EtOAc. The organic layer was dried
(4H, m), 1.86 (6H, s), 2.05–2.13 (1H, m), 6.89 (1H, dd, J=1.2, over MgSO4, filtered and concentrated in vacuo. The obtained
3.6Hz), 6.94 (1H, dd, J=3.6, 5.1Hz), 7.20 (1H, dd, J=1.2, solid was washed with diisopropyl ether, then dried to give the
5.1Hz). EI-MS m/z: 234 [M]+.
title compound 25c (92mg, 0.25mmol, 16%) as a white solid.
2-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopro- 1H-NMR (400MHz, CDCl3) δ: 0.17–0.22 (2H, m), 0.52–0.58
pyl-1,3,4-oxadiazole (24) N-Bromosuccinimide (5.28g, (2H, m), 0.77–0.87 (1H, m), 0.96–1.03 (2H, m), 1.14–1.20 (2H,
29.7mmol) was added to a solution of 23 (6.32g, 27.0mmol) m), 1.71–1.79 (1H, m), 1.86 (6H, s), 3.61 (2H, d, J=6.2Hz),
in AcOH (95mL) and the mixture was stirred at r.t. The reac- 6.55 (1H, d, J=3.7Hz), 6.88 (1H, d, J=3.9Hz). FAB-MS m/z:
tion mixture was concentrated in vacuo, diluted with EtOAc 368 [M+H]+. HR-MS (ESI) m/z: 366.0635 [M+H]+ (Calcd
and washed with 1M aqueous sodium hydroxide solution. The for C16H21N3BrS: 366.0634).
organic layer was dried over MgSO4, filtered and concentrated
4-Benzyl-3-[2-(5-bromothiophen-2-yl)propan-2-yl]-5-cy-
in vacuo. The residue was purified by column chromatography clopropyl-4H-1,2,4-triazole Monohydrochloride (25d) The
on silica gel (hexane–EtOAc). The obtained solid was washed title compound was prepared in a similar manner to that
with hexane and dried to give the title compound 24 (6.32g, described for 25a from 24 (500mg, 1.60mmol) and benzyl-
1
20.2mmol, 75%) as a white solid. H-NMR (400MHz, CDCl3) amine (1.74mL, 15.9mmol) at 33% yield (231mg, 0.526mmol)
δ: 1.07–1.13 (4H, m), 1.82 (6H, s), 2.06–2.13 (1H, m), 6.65 as a white solid. 1H-NMR (400MHz, CDCl3) δ: 1.14–1.21
(1H, d, J=3.9Hz), 6.88 (1H, d, J=3.9Hz). FAB-MS m/z: 315 (2H, m), 1.62–1.70 (1H, m), 1.77–1.85 (2H, m), 1.83 (6H, s),
[M+H]+.
5.20 (2H, s), 6.64 (1H, d, J=3.9Hz), 6.80–6.87 (2H, m), 6.86
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopropyl- (1H, d, J=3.9Hz), 7.34–7.38 (3H, m). FAB-MS m/z: 404
4-propyl-4H-1,2,4-triazole Monohydrochloride (25a) n- [M+H]+. HR-MS (ESI) m/z: 402.0634 [M+H]+ (Calcd for
Propylamine (1.37mL, 16.7mmol) was added to a solution of C19H21N3BrS: 402.0634).
24 (500mg, 1.60mmol) in AcOH (5mL) and reacted at 170°C
3-[2-(5-Bromothiophen-2-yl)propan-2-yl]-5-cyclopropyl-

836
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
4-(2-phenylethyl)-4H-1,2,4-triazole (25e) The title com- (300mg, 1.31mmol) in AcOH (3mL) at 0°C, and the mixture
pound was prepared in a similar manner to that described for was reacted at 175°C for 40min using a microwave reaction
25a, except for the salt-forming procedure, from 24 (500mg, device. The reaction mixture was neutralized with 1M aque-
1.60mmol) and 2-phenylethylamine (2.00mL, 15.8mmol) at ous sodium hydroxide solution and extracted with CHCl3.
20% yield (131mg, 0.314mmol) as a white solid. 1H-NMR The organic layer was washed with brine, dried over MgSO4,
(400MHz, CDCl3) δ: 1.03–1.09 (2H, m), 1.19–1.24 (2H, m), filtered and concentrated in vacuo. The residue was purified
1.64–1.73 (1H, m), 1.85 (6H, s), 2.63–2.70 (2H, m), 3.88–3.94 by column chromatography on silica gel (EtOAc–MeOH then
(2H, m), 6.64 (1H, d, J=3.7Hz), 6.95 (1H, d, J=3.8Hz), CHCl3–MeOH) to give 2-[2-(4,5-dicyclopropyl-4H-1,2,4-
6.96–7.00 (2H, m), 7.22–7.33 (3H, m). FAB-MS m/z: 416 triazol-3-yl)propan-2-yl]pyridine (200mg, 0.746mmol, 57%)
[M+H]+.
as a pale yellow oil. Four molar HCl solution in 1,4-di-
N′-[2-Methyl-2-(pyridin-2-yl)propanoyl]cyclopropane- oxane (169µL, 0.677mmol) was added to a solution of
carbohydrazide (27a) Et3N (3.42mL, 24.6mmol) was added 2-[2-(4,5-dicyclopropyl-4H-1,2,4-triazol-3-yl)propan-2-yl]-
to a solution of 2-methyl-2-(pyridin-2-yl)propanehydrazide pyridine (182mg, 0.677mmol) in EtOAc (2mL). The mixture
(26a, 4.00g, 22.3mmol, CAS No.; 880166-60-9) in dichloro- was stirred at r.t. for 1h and the solid was precipitated. The
methane (20mL), and then a solution of cyclopropanecarbonyl precipitate was collected by filtration, washed with EtOAc,
chloride (2.11mL, 23.4mmol) in dichloromethane (20mL) was and dried to give the title compound 29a (158mg, 0.518mmol,
added dropwise to the mixture at 0°C. The reaction mixture 76%) as a white solid. 1H-NMR (300MHz, DMSO-d6) δ:
was stirred at 3h and saturated aqueous sodium hydrogen 0.40–0.47 (2H, m), 0.74–0.82 (2H, m), 1.23–1.30 (4H, m), 1.85
carbonate solution was added. The mixture was extracted with (6H, s), 2.26–2.36 (1H, m), 3.05–3.14 (1H, m), 7.35–7.40 (1H,
CHCl3, and the organic layer was washed with brine, dried m), 7.53 (1H, d, J=8.0Hz), 7.90 (1H, ddd, J=1.8, 7.8, 7.8Hz),
over MgSO4, filtered and concentrated in vacuo. The residue 8.47–8.51 (1H, m). ESI-MS m/z: 269 [M+H]+. HR-MS (ESI)
was purified by column chromatography on silica gel (CHCl3– m/z: 269.1759 [M+H]+ (Calcd for C16H21N4: 269.1761).
MeOH) to give the title compound 27a (5.25g, 21.2mmol,
4-[2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)propan-
95%) as a white solid. 1H-NMR (400MHz, CDCl3) δ: 2-yl]pyridine Monohydrochloride (29b) The title com-
0.75–0.82 (2H, m), 0.97–1.03 (2H, m), 1.51–1.59 (1H, m), 1.68 pound was prepared in a similar manner to that described for
(6H, s), 7.23 (1H, dd, J=5.0, 7.3Hz), 7.44 (1H, d, J=8.0Hz), 29a from 28b (36.4mg, 0.159mmol) at 22% yield (10.5mg,
1
7.72 (1H, ddd, J=1.7, 7.8, 7.8Hz), 8.63 (1H, d, J=4.0Hz), 0.0344mmol) as a white solid. H-NMR (300MHz, DMSO-
8.93–9.02 (1H, m), 10.06–10.18 (1H, m).
d6) δ: 0.53–0.60 (2H, m), 0.81–0.89 (2H, m), 1.18–1.26 (4H,
N′-[2-Methyl-2-(pyridin-4-yl)propanoyl]cyclopropane- m), 1.86 (6H, s), 2.17–2.28 (1H, m), 3.03–3.12 (1H, m), 7.74
carbohydrazide (27b) The title compound was prepared in (2H, d, J=6.5Hz), 8.82 (2H, d, J=6.4Hz). ESI-MS m/z: 269
a similar manner to that described for 27a from 2-methyl-2- [M+H]+. HR-MS (ESI) m/z: 269.1759 [M+H]+ (Calcd for
(pyridin-4-yl)propanehydrazide (26b, 166mg, 0.926mmol, C16H21N4: 269.1761).
CAS No.; 950604-79-2) at 45% yield (103mg, 0.418mmol) as
11β-HSD1 and 11β-HSD2 Assay Recombinant human
a white solid. 1H-NMR (300MHz, DMSO-d6) δ: 0.68–0.76 and mouse 11β-HSD1 enzymes were prepared in accordance
(4H, m), 1.45–1.63 (1H, m), 1.47 (6H, s), 7.38–7.42 (2H, m), with a previous study.23) Test compounds were dissolved in
8.47–8.51 (2H, m), 9.47 (1H, brs), 9.87 (1H, brs). ESI-MS m/z: DMSO and diluted to the desired concentrations. The reaction
248 [M+H]+.
was initiated by adding the compound to a reaction mixture
2-[2-(5-Cyclopropyl-1,3,4-oxadiazol-2-yl)propan-2-yl]- containing 10mM phosphate buffer (pH 6.6), 200nM corti-
pyridine (28a) A mixture of 27a (10.1g, 40.8mmol) and sone, 40µM reduced nicotinamide adenine dinucleotide phos-
phosphorus oxychloride (76mL, 0.82mol) was stirred at reflux phate (NADPH) and the 11β-HSD1 enzyme, and incubating at
temperature for 8h and poured into ice water. The reaction r.t. for 1h.
mixture was neutralized with 4M aqueous sodium hydroxide
The 11β-HSD2 assay was conducted using the same method
solution and extracted with EtOAc. The organic layer was as that used for the 11β-HSD1 assay, except for the enzyme
washed with saturated aqueous sodium hydrogen carbonate reaction conditions. The human 11β-HSD2 enzyme was pro-
solution and brine, dried over MgSO4, filtered and concentrat- duced in HEK293 cells transfected with an expression vec-
ed in vacuo. The residue was purified by column chromatog- tor (pcDNA3.1, Invitrogen) encoding human 11β-HSD2. The
raphy on silica gel (CHCl3–MeOH) to give the title compound crude extract from HEK293 cell homogenates was used as the
28a (5.81g, 25.3mmol, 62%) as a colorless oil. 1H-NMR enzyme source for the human 11β-HSD2 assay. The enzyme
(300MHz, CDCl3) δ: 1.03–1.11 (4H, m), 1.83 (6H, s), 2.03–2.16 reaction was initiated by adding the compound to a reaction
(1H, m), 7.12–7.21 (2H, m), 7.60–7.68 (1H, m), 8.53–8.59 (1H, mixture containing 40mM Tris–HCl buffer (pH 8.0), 200nM
m). ESI-MS m/z: 230 [M+H]+.
4-[2-(5-Cyclopropyl-1,3,4-oxadiazol-2-yl)propan-2-yl]- and the 11β-HSD2 enzyme, and incubating at 37°C for 2h.
cortisol, 200µM nicotinamide adenine dinucleotide (NAD)
pyridine (28b) The title compound was prepared in a
After the enzymatic reaction, the enzymatic inhibitory
similar manner to that described for 28a from 27b (100mg, activity was measured by detecting cortisol using a HTRF®.
0.404mmol) at 45% yield (41.7mg, 0.182mmol) as a colorless Each of the XL-665-labeled cortisol containing 400µM car-
oil. 1H-NMR (300MHz, CDCl3) δ: 1.03–1.14 (4H, m), 1.79 benoxolone and cryptate-labeled cortisol antibody (Cisbio
(6H, s), 2.03–2.14 (1H, m), 7.15–7.19 (2H, m), 8.54–8.60 (2H, Bioassays, Codolet, France) was added and incubated at r.t.
m). ESI-MS m/z: 230 [M+H]+.
for 2h, and the fluorescence intensity was measured using a
2-[2-(4,5-Dicyclopropyl-4H-1,2,4-triazol-3-yl)propan- fluorophotometer (ARVO HTS, PerkinElmer, Inc., Waltham,
2-yl]pyridine Monohydrochloride (29a) Cyclopropyl amine MA, U.S.A.). The enzymatic inhibitory activity was calcu-
(900 µL, 13.1mmol) was slowly added to a solution of 28a lated from the fluorescence intensity ratio at two wavelengths

Vol. 67, No. 8 (2019)
Chem. Pharm. Bull.
837
(665nm/620nm). The ratio when DMSO was added instead of metabolic clearance assay, elemental analysis, and spectral
the compound was regarded as 0% and the ratio when neither measurements.
11β-HSD1 nor 11β-HSD2 was added was regarded as 100%.
Effect of Repeated Administration in ob/ob Mice All
Conflict of Interest The authors declare no conflict of
experiments were performed in accordance with the regula- interest.
tion of the Animal Ethics Committee of Astellas Pharma Inc.
Male diabetic ob/ob mice were purchased from Charles
River Laboratories Japan (Kanagawa, Japan). Compound 9a
was dissolved in 6% 2-hydroxypropyl-β-cyclodextrin (Sigma-
Aldrich Co. LLC., St. Louis, MO, U.S.A.). Seven-week-old
male ob/ob mice (n=7–8) were orally administered vehicle or
0.3–10mg/kg of 9a twice daily for four weeks. At 12h after
the final administration, plasma and retroperitoneal adipose
tissue samples were collected. Plasma glucose levels were
determined using the Glucose CII test (Wako, Osaka, Japan).
Plasma insulin levels were determined using the Insulin
enzyme-linked immunosorbent assay (ELISA) kit (Shibayagi,
Gunma, Japan). Corticosterone levels in retroperitoneal adi-
pose tissue were determined using the Corticosterone ELISA
kit (Enzo Life Sciences, Farmingdale, NY, U.S.A.).
In Vitro Intrinsic Clearance with Mouse and Human
Liver Microsomes To estimate the metabolic stability of
compounds against mouse or human hepatic CYPs, the test
compound (0.2µM) was incubated with pooled male CD1
mouse or human liver microsomes (0.2mg protein/mL),
NADPH (1mM) and ethylenediaminetetraacetic acid (EDTA)
(0.1mM) in pH 7.4 Na+–K+ phosphate buffer (100mM) at
37°C. Incubations were conducted for 0, 15, 30, and 45min.
The peak area of the compound and internal standard was
measured using liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) and analyzed to calculate CLint
(mL/min/kg).24)
References
1) Taylor S. I., Accili D., Imai Y., Diabetes, 43, 735–740 (1994).
2) American Diabetes Association, Diabetes Care, 35 (Suppl. 1),
S64–S71 (2012).
3) Ross S. A., Gulve E. A., Wang M., Chem. Rev., 104, 1255–1282
(2004).
4) Montague C. T., O’Rahilly S., Diabetes, 49, 883–888 (2000).
5) Rask E., Olsson T., Söderberg S., Andrew R., Livingstone D. E.,
Johnson O., Walker B. R., J. Clin. Endocrinol. Metab., 86, 1418–
1421 (2001).
6) Lindsay R. S., Wake D. J., Nair S., Bunt J., Livingstone D. E.,
Permana P. A., Tataranni P. A., Walker B. R., J. Clin. Endocrinol.
Metab., 88, 2738–2744 (2003).
7) Masuzaki H., Paterson J., Shinyama H., Morton N. M., Mullins J. J.,
Seckl J. R., Flier J. S., Science, 294, 2166–2170 (2001).
8) Masuzaki H., Yamamoto H., Kenyon C. J., Elmquist J. K., Morton
N. M., Paterson J. M., Shinyama H., Sharp M. G., Fleming S., Mul-
lins J. J., Seckl J. R., Flier J. S., J. Clin. Invest., 112, 83–90 (2003).
9) Mune T., Rogerson F. M., Nikkilä H., Agarwal A. K., White P. C.,
Nat. Genet., 10, 394–399 (1995).
10) Goldberg F. W., Dossetter A. G., Scott J. S., Robb G. R., Boyd S.,
Groombridge S. D., Kemmitt P. D., Sjögren T., Gutierrez P. M.,
deSchoolmeester J., Swales J. G., Turnbull A. V., Wild M. J., J.
Med. Chem., 57, 970–986 (2014).
11) Abrahmsen L., Nilsson J., Opperman U., Svensson S.,
WO2005068646A1, 2005.
12) Siu M., Johnson T. O., Wang Y., Nair S. K., Taylor W. D., Cripps S.
J., Matthews J. J., Edwards M. P., Pauly T. A., Ermolieff J., Castro
A., Hosea N. A., LaPaglia A., Fanjul A. N., Vogel J. E., Bioorg.
Med. Chem. Lett., 19, 3493–3497 (2009).
13) Feig P. U., Shah S., Hermanowski-Vosatka A., Plotkin D., Springer
M. S., Donahue S., Thach C., Klein E. J., Lai E., Kaufman K. D.,
Diabetes Obes. Metab., 13, 498–504 (2011).
14) Véniant M. M., Hale C., Hungate R. W., Gahm K., Emery M. G.,
Jona J., Joseph S., Adams J., Hague A., Moniz G., Zhang J., Bart-
berger M. D., Li V., Syed R., Jordan S., Komorowski R., Chen M.
M., Cupples R., Kim K. W., St. Jean D. J. Jr., Johansson L., Hen-
riksson M. A., Williams M., Vallgårda J., Fotsch C., Wang M., J.
Med. Chem., 53, 4481–4487 (2010).
15) Zhuang L., Tice C. M., Xu Z., Zhao W., Cacatian S., Ye Y. J., Singh
S. B., Lindblom P., McKeever B. M., Krosky P. M., Zhao Y., Lala
D., Kruk B. A., Meng S., Howard L., Johnson J. A., Bukhtiyarov
Y., Panemangalore R., Guo J., Guo R., Himmelsbach F., Hamilton
B., Schuler-Metz A., Schauerte H., Gregg R., McGeehan G. M.,
Leftheris K., Claremon D. A., Bioorg. Med. Chem., 25, 3649–3657
(2017).
Molecular Modeling
Human 11β-HSD1 Model
The crystal structure of human 11β-HSD1 was obtained
from the Research Collaboratory for Structural Bioinformatics
(RCSB) Protein Data Bank (PDB code: 3D5Q).25) After delet-
ing C and D chains, hydrogen atoms were added to the protein
using the Protonate3D module with the Amber10EHT force
field in MOE.22)
Docking Study
The ligand molecule was prepared using LigPrep26) and
MacroModel27) and energy-minimized conformation was used
to input molecules. Compounds were docked to the human
11β-HSD1 model using the docking program GOLD version
5.5.1. The ligand molecule was docked 10 times. The top
scoring position, as assessed by GoldScore, was employed for
discussion.
16) Wan Z.-K., Chenail E., Li H.-Q., Kendall C., Wang Y., Gingras S.,
Xiang J., Massefski W. W., Mansour T. S., Saiah E., J. Org. Chem.,
76, 7048–7055 (2011).
17) ACD/Percepta, version 14.0.0, Advanced Chemistry Development,
Inc., Toronto, ON, Canada, ‹www.acdlabs.com›, 2015.
18) Tu H., Powers J. P., Liu J., Ursu S., Sudom A., Yan X., Xu H.,
Meininger D., Degraffenreid M., He X., Jaen J. C., Sun D., Labelle
M., Yamamoto H., Shan B., Walker N. P., Wang Z., Bioorg. Med.
Chem., 16, 8922–8931 (2008).
19) GOLD5.5.1, The Cambridge Crystallographic Data Centre, Cam-
bridge, U.K.
20) Jones G., Willett P., Glen R. C., J. Comput. Aided Mol. Des., 9,
532–549 (1995).
Sequence Homology between Human and Mouse
The crystal structure of mouse 11β-HSD1 was down-
loaded from RCSB (PDB code: 4K2628)). The homol-
ogy between human 11β-HSD1(PDB code: 3D5Q) and mouse
11β-HSD1(PDB code: 4K26) sequences was evaluated using
MOE.
Acknowledgments The authors thank Koji Aoyama
and Taisuke Nakazawa for their contribution to the biologi-
cal evaluations, Susumu Watanuki for his helpful support in
preparing this manuscript, and Yuichiro Sato and Hiroshi
Kayakiri for their support. The authors also thank the staff of
Astellas Research Technologies Co., Ltd., for conducting the
21) Jones G., Willett P., Glen R. C., Leach A. R., Taylor R., J. Mol.

838
Chem. Pharm. Bull.
Vol. 67, No. 8 (2019)
Biol., 267, 727–748 (1997).
25) Tu H., Powers J. P., Liu J., Ursu S., Sudom A., Yan X., Xu H.,
Meininger D., Degraffenreid M., He X., Jaen J. C., Sun D., Labelle
M., Yamamoto H., Shan B., Walker N. P., Wang Z., Bioorg. Med.
Chem., 16, 8922–8931 (2008).
22) Molecular Operating Environment (MOE), version MOE2016.0802,
Chemical Computing Group Inc., 1010 Sherbrooke St. West, Suite
#910, Montreal, QC, Canada, H3A 2R7, 2016.
23) Walker E. A., Clark A. M., Hewison M., Ride J. P., Stewart P. M., J.
Biol. Chem., 276, 21343–21350 (2001).
26) LigPrep (version 44011), Schrodinger, LLC, New York, NY.
27) MacroModel, Schrodinger, LLC, New York, NY.
24) Naritomi Y., Terashita S., Kimura S., Suzuki A., Kagayama A.,
Sugiyama Y., Drug Metab. Dispos., 29, 1316–1324 (2001).
28) Böhme T., Engel C. K., Farjot G., Güssregen S., Haack T., Tschank
G., Ritter K., Bioorg. Med. Chem. Lett., 23, 4685–4691 (2013).