Design, synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

Article abstract of DOI:10.1016/j.bmc.2018.02.032

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.

Full text of DOI:10.1016/j.bmc.2018.02.032

Accepted Manuscript
Design, synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane de-
rivatives as GPR119 agonists
Daisuke Matsuda, Madoka Kawamura, Yohei Kobashi, Fumiyasu Shiozawa,
Youichirou Suga, Keiko Fusegi, Shinichi Nishimoto, Kayo Kimura, Masako
Miyoshi, Noriko Takayama, Hiroyuki Kakinuma, Norikazu Ohtake
PII:
S0968-0896(17)32185-5
https://doi.org/10.1016/j.bmc.2018.02.032
BMC 14218
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 November 2017
16 February 2018
18 February 2018
Please cite this article as: Matsuda, D., Kawamura, M., Kobashi, Y., Shiozawa, F., Suga, Y., Fusegi, K., Nishimoto,
S., Kimura, K., Miyoshi, M., Takayama, N., Kakinuma, H., Ohtake, N., Design, synthesis and biological evaluation
of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.02.032
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Design, synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as
GPR119 agonists
Daisuke Matsudaa,*, Madoka Kawamuraa, Yohei Kobashi a, Fumiyasu Shiozawa b, Youichirou Suga b,
a
c
d
c
c
Keiko Fusegi , Shinichi Nishimoto , Kayo Kimura , Masako Miyoshi , Noriko Takayama ,
Hiroyuki Kakinuma a, Norikazu Ohtake a
a Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd.; 1-403 Yoshino-cho, Kita-ku, Saitama
331-9530, Japan.
b Pharmaceutical Science Laboratories, Taisho Pharmaceutical Co., Ltd.; 1-403 Yoshino-cho,
Kita-ku, Saitama 331-9530, Japan.
c Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd.; 1-403 Yoshino-cho, Kita-ku,
Saitama 331-9530, Japan.
d Research Support, Taisho Pharmaceutical Co., Ltd.; 1-403 Yoshino-cho, Kita-ku, Saitama
331-9530, Japan.
Abstract
The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described.
In this series, optimization of the right piperidine N-capping group (R2) and the left aryl group (R3)
led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a
desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic
rats.
1. Introduction
Diabetes mellitus is a chronic disease characterized by high blood glucose levels (hyperglycemia).
In 2015, it was estimated that the number of people with diabetes worldwide was more than 400
million, and was expected to rise to 642 million by 2040.1 Type 2 diabetes mellitus (T2DM) is the
most common form of diabetes, characterized by hyperglycemia resulting from impaired insulin
secretion and insulin resistance. Long-term hyperglycemia can cause serious complications such as
blindness, renal failure, diabetic foot disorders, heart attacks and strokes. Although multiple oral
antidiabetic agents, such as sulfonylureas, meglitinides, biguanides, thiazolidinediones,
α-glucosidase inhibitors and dipeptidyl-peptidase-4 (DPP-4) inhibitors, have been used for the
treatment of T2DM, many patients fail to achieve the desired glycemic control.2,3 Recently, a
sodium-dependent glucose co-transporter 2 (SGLT2) inhibitor has been used clinically; this inhibitor
exerts a glucose-lowering effect without causing hypoglycemia or weight gain. However, a
significant need for the development of new antidiabetic agents with greater safety and efficacy

continues to exist.
GPR119 is a G-protein coupled receptor (GPCR) that is predominantly expressed in pancreatic
β-cells and gastrointestinal L-cells. Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA)
have been identified as endogenous agonists for the GPR119 receptor.4,5 The activation of the
GPR119 receptor increases the cellular cAMP levels, leading to glucose-dependent insulin secretion
from pancreatic β-cells.6 In addition, the activation of the GPR119 receptor in the gut results in the
release of incretins, such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic
polypeptide (GIP), from enteroendocrine cells.7 GLP-1 and GIP stimulate insulin secretion from
β-cells in
a
glucose-dependent manner and protect β-cells against apoptosis.8,9 This
glucose-dependent dual mechanism of action suggests that GPR119 agonists can improve glycemic
control without inducing hypoglycemia. Furthermore, this mechanism might produce beneficial
anti-obesity effects such as suppressed food intake and body weights.4
To date, many research groups have investigated small-molecule GPR119 agonists,10,11 which have
led to the development of clinical compounds such as APD668,12 GSK1292263,13 and MBX-298214
(Figure 1).
Figure 1. Representative GPR119 receptor agonists

Figure 2. GPR119 agonists with carbon chain spacer
We
previously
reported
a
series
of
1H-pyrazolo[3,4-c]pyridine
and
3H-[1,2,3]triazolo[4,5-c]pyridine derivatives (4) as a new class of GPR119 agonists15, 16 (Figure 1).
However, these derivatives suffered from a poor aqueous solubility and a low bioavailabilitybecause
of a strong molecular interaction associated with their molecular planarity. To overcome these issues,
we explored an alternative structure class with new central spacers between the left pharmacophore
(substituted phenyl group) and the right N-capped piperidine with greater three-dimensionality.17
Using Prosidion’s (now Astellas) GPR119 agonists, which have a simple carbon chain as a central
spacer (represented by compound 5; Figure 2), as a starting point, we attempted to introduce
conformational rigidity into the carbon chain of 5 to improve its agonist activity. Merck researchers
have already identified cyclopropane derivatives, such as compound 6,18,19 which are considered the
representative of the successful realization of conformational rigidity. Our strategy was to install
spiro structures as central spacers (Figure 2) to achieve both conformational rigidity and
three-dimensionality.
Here, we describe the synthesis and evaluation of spiro-piperidine derivatives and the discovery of
novel 7-azaspiro[3.5]nonane GPR119 agonists, including their structure-activity relationships
(SARs) and an in vivo efficacy study in diabetic rats.
2. Chemistry
The synthesis of 6-azaspiro[2.5]octane derivatives 13 and 17 is shown in Schemes 1 and 2.
Compound 13 was derived from a commercially available starting material, 6-tert-butyl 1-ethyl
6-azaspiro[2.5]octane-1,6-dicarboxylate (7), which was reduced with diisobutylaluminum hydride
(DIBAL); the resulting alcohol 8 was oxidized with Dess-Martin periodinane to yield aldehyde 9.
The Wittig reaction of 9 with MeOCH2P+Ph3Cl- followed by hydrolysis with trifluoroacetic acid

(TFA) in aqueous MeCN yielded 11. The subsequent reduction of aldehyde 11 with NaBH4 yielded
12. Finally, the Mitsunobu reaction of 12 with 4-(methanesulfonyl)phenol using
N,N,N’,N’-tetramethylazadicarboxamide
6-azaspiro[2.5]octane derivative 13.
(TMAD)
and
tributylphosphine
yielded
the
Scheme 1. (a) DIBAL, THF, 0 ˚C; (b) Dess-Martin periodinane, CHCl3, rt; (c) MeOCH2P+Ph3Cl-, KO-t-Bu, toluene,
70 ˚C; (d) TFA, H2O, CH3CN, rt; (e) NaBH4, MeOH, rt, (f) 4-(methanesulfonyl)phenol, TMAD, PBu3, THF, 60 ˚C.
Compound 17 was synthesized from alcohol 8. The alcohol group was iodinated with I2 to produce
compound 14. The substitution reaction of 14 with lithium enolate of tert-butyl acetate in the
presence of N,N’-dimethylpropyleneurea (DMPU) yielded 15. The reduction of compound 15 with
LiBH4 at 60 ˚C followed by the Mitsunobu reaction under conditions similar to those described for
compound 13 provided the desired product 17.
Scheme 2. (a) I2, PPh3, imidazole, CHCl3, rt; (b) t-BuOAc, LDA, DMPU, THF, -78 ˚C; (c) LiBH4, THF,
60 ˚C; (d) 4-(methanesulfonyl)phenol, TMAD, PBu3, THF, 60 ˚C.
The synthesis of 7-azaspiro[3.5]nonane derivative 26 and 3-azaspiro[5.5]undecane derivative 27 is
shown in Scheme 3. The Horner-Wadsworth-Emmons reaction [(EtO)2POCH2CO2Et, NaH] of
commercially available starting materials 18 and 19 provided 20 and 21, respectively. Hydrogenation
of the double bonds of 20 and 21 in the presence of a catalytic amount of Pd(OH)2 and the
subsequent reduction of the ester moieties yielded alcohols 24 and 25. Finally, the Mitsunobu
reaction of the alcohols provided the desired products 26 and 27.

Scheme 3. (a) (EtO)2POCH2CO2Et, NaH, DMF, rt; (b) H2, Pd(OH)2/C, EtOH, rt; (c) LiBH4, THF, reflux;
(d) 4-(methanesulfonyl)phenol, TMAD, PBu3, THF, 60 ˚C.
Scheme
4
shows the synthesis of 7-azaspiro[3.5]nonane derivatives 36a-c and
3-azaspiro[5.5]undecane derivative 37. The Wittig reaction (MeOCH2P+Ph3Cl-, KO-t-Bu) of 18 and
19 followed by acidic hydrolysis and the Horner-Wadsworth-Emmons reaction of the resulting
aldehydes yielded α,β-unsaturated esters 30 and 31. Catalytic hydrogenation of the double bonds,
the subsequent reduction, and the Mitsunobu reaction of the alcohols 34 and 35 with the
corresponding phenol provided the desired products 36a-c and 37, respectively.
Scheme 4. (a) MeOCH2P+Ph3Cl-, KO-t-Bu, toluene, 70 ˚C; (b) TFA, H2O, CH3CN, rt; (c)
(EtO)2POCH2CO2Et, NaH, DMF, THF, rt; (d) H2, Pd(OH)2/C, EtOH, rt; (e) for 34, DIBAL, THF, 0 ˚C;
for 35, LiBH4, toluene, THF, 60 ˚C; (f) ArOH, TMAD, PBu3, THF, 60 ˚C.
Compound 41 was similarly obtained from 40 (Scheme 5), which was derived via the following
four reaction steps: 1) Dess-Martin oxidation of 24, 2) Horner-Wadsworth-Emmons reaction, 3)
catalytic hydrogenation, and 4) reduction with LiBH4. The Mitsunobu reaction under conditions
similar to those described for 26 in Scheme 3 was then performed, producing 41.

Scheme 5. (a) Dess-Martin periodinane, CHCl3, rt; (b) (EtO)2POCH2CO2Et, NaH, DMF, rt; (c) H2,
Pd(OH)2/C, EtOH, rt; (d) LiBH4, THF, 60 ˚C; (d) 4-(methanesulfonyl)phenol, TMAD, PBu3, THF, 60 ˚C.
Compounds 42a, 42c, 43c, 44c, 45c, 46a-c, 47a and 49a were synthesized as shown in Scheme 6.
The removal of the tert-butoxycarbonyl group (Boc) of 36a-c followed by treatment with the
corresponding carbamating reagents yielded 42a, 42c, 43c, 44c and 45c. On the other hand,
compounds 46a-c were prepared from 36a-c via the removal of the Boc group and the subsequent
N-arylation with 2-chloro-5-ethylpyrimidine. As for 47a, N-arylation was performed using
cross-coupling reaction with 2-chloro-5-methylpyridine in the presence of Pd2(dba)3 and 2,2’-
bis(diphenylphosphino)-1,1’-binaphthyl (BINAP) as catalysts. Compound 49a was prepared via
cyanide 48a, which was reacted with N’-hydroxy-2-methylpropanimidamide and then dehydrated
under acidic conditions to produce 49a.
Scheme 6. (a) HCl, EtOAc, rt; (b) for 42a, 42c, isopropyl chloroformate, Et3N, CHCl3, rt; for 43c,
1-methylcyclopropyl 4-nitrophenyl carbonate, Et3N, CHCl3, rt; for 44c, cyclopentanol, triphosgene, Et3N,
THF, rt; for 45c, 2,2-dimethylpropan-1-ol, triphosgene, Et3N, THF, rt; (c) for 46a-c,

2-chloro-5-ethylpyrimidine, Cs2CO3, DMSO, microwave, 180 ˚C; for 47a, 2-bromo-5-methylpyridine,
Pd2(dba)3, BINAP, NaO-t-Bu, 1,2-dimethoxyethane, 60 ˚C; (d) CNBr, NaHCO3, H2O, CHCl3, rt; (e) (1)
N'-hydroxy-2-methylpropanimidamide, ZnCl2, Et2O, rt; (2) conc.HCl, EtOH, reflux.
The synthesis of amide derivatives 54a-g, 57a and 57b is shown in Scheme 7. The common
intermediate 51 was derived from 32 using the following reactions: 1) deprotection of the Boc group
2) introduction of an isopropyl carbamate group, 3) and reduction of the ester moiety with DIBAL.
The Mitsunobu reaction of 51 with ethyl 2-fluoro-4-hydroxybenzoate followed by basic hydrolysis
yielded carboxylic acid 53. Finally, the condensation of 53 with the corresponding amine under
standard conditions yielded the desired amide derivatives 54a, 54b, 54d-g. Compound 54c was
prepared from 54b through N-methylation using NaH and MeI. As for 57a and 57b, these
compounds were obtained via the Mitsunobu reaction of 51 with 5-hydroxypyridine-2-carbonitrile or
6-hydroxypyridine-3-carbonitrile, hydrolysis of the cyano groups of 55a and 55b, and the amidation
of the resulting carboxylic acid with cyclopropylamine to yield 57a and 57b.
Scheme 7. (a) (1) HCl, EtOAc, rt; (2) isopropyl chloroformate, Et3N, CHCl3, rt; (b) DIBAL, THF, rt; (c)
ArOH, TMAD, PBu3, THF, 60 ˚C; (d) NaOH, H2O, MeOH, 60 ˚C; (e) R5R6NH, EDCI·HCl, HOBt·H2O,
Et3N, DMF, rt; (f) NaH, MeI, DMF, rt; (g) NaOH, H2O, EtOH, reflux; (h) cyclopropylamine, EDCI·HCl,
HOBt·H2O, Et3N, DMF, rt.
3. Results and Discussion
The GPR119 agonist potency of the synthesized compounds was measured using a cAMP assay in
the human GPR119 cell line.

First, we evaluated the potencies of compounds with various types of spacers (including spiro
substructures) with the right pharmacophore (4-methylsulfonylphenoxy group) and the left part
(tert-butoxycarbonylpiperidine) in fixed positions (Table 1). Among the compounds, the
7-azaspiro[3.5]nonane derivative 36a containing a propylcyclobutyl spacer was the most potent
GPR119 agonist. A single carbon contraction (26) or elongation (41) of the propyl moiety of 36a led
to a 10-fold decrease in the agonist potency. Regarding the cyclobutyl spacer moiety of 36a, a ring
contraction (cyclopropyl: 17) or ring expansion (cyclohexyl: 37) resulted in a significant reduction in
potency. Furthermore, a single carbon contraction of the propyl spacer moiety of 17 or 37 led to the
identification of the potent compound 27 with an EC50 value of 55 nM. All the compounds shown in
Table 1, except 13 and 17, exhibited full intrinsic activity. As a result, we identified an alternative
chemical class of potent GPR119 agonists exemplified by 7-azaspiro[3.5]nonane derivative 36a and
3-azaspiro[5.5]undecane derivative 27. These results indicated that the rigidity of the spiro moiety
and the favorable spacer length led to the high agonist activity of 36a and 27.
Table 1
SAR of spacers
Cpd.
13
Spiro
A
n
2
3
2
3
4
2
3
hGPR119 EC50 (nM)
2388
830
183
14
17
A
26
B
36a
41
B
B
154
55
27
C
37
C
2590
Having obtained a new chemical class, we next focused on optimizing the N-capping group of the
piperidine of the more potent compound 36a (Table 2). In addition to the agonist potency, we
monitored the solubility of each compound in fasted state simulated intestinal fluid (FaSSIF) as well
as the aqueous solubility in pH6.8 phosphate buffer to compare the solubility profiles. Most of the
compounds of this series exhibited full intrinsic activity. Although 36a exhibited a low solubility:
<0.13 µg/mL (the lower limit of quantitation) in the phosphate buffer and 3.48 µg/mL in FaSSIF, the
three-dimentionallity of the compound was expected to have a positive impact on increasing not only

the solubility but also the dissolution rate, leading to improving the oral bioavaialbity.20 Therefore,
we started the optimization study of this series.
The replacement of the acid-labile tert-butoxycarbonyl group in 36a with an isopropyloxycarbonyl
group led to the compound 42a, which was about 5-fold less potent than 36a. However, the
installation of a fluorine atom at the 3-position of the left phenyl ring of 42a restored the potency
(42c, EC50 = 18 nM). The isopropyl group in 42c was replaced with 1-methylcyclopropyl (43c),
cyclopentyl (44c) and 2,2-dimethylpropyl (45c) to evaluate their effect on the agonist potency and
aqueous solubility. The agonist potency of the 1-methylcyclopropyl derivative (43c) was comparable
to that of 42c, while the solubility in FaSSIF was nearly half that of 42c. By contrast, the solubility
of the cyclopentyl derivative (44c) more than doubled in FaSSIF; however, its agonist potency was
not improved despite the higher lipophilicity. The 2,2-dimethylpropyl derivative (45c) also showed
no improvement in agonist potency. Moreover, replacement of the tert-butoxycarbonyl moiety of
36a with a heteroaromatic group such as pyrimidine, pyridine or oxadiazole as a carbamate isostere,
was conducted to enhance the potency.15 The pyrimidine derivative (46a) exhibited a tolerable
agonist potency (EC50: 31 nM). However, the installation of a fluorine atom into the phenyl ring of
46a significantly decreased the potency (46b and 46c). The pyridine analog (47a) showed a poor
agonist potency, while the potency of the oxadiazole analog (49a) was tolerable (EC50: 35 nM). In
this series, the carbamate groups appeared to be more favorable than the heteroaryl groups as a
piperidine N-capping group. Among these carbamate groups, we selected isopropyl carbamate (42c)
for further SAR study of the left aryl group (R3) based on its agonist potency, FaSSIF solubility, and
lipophilicity (ClogP), as shown in Table 3.
Table 2
SAR of piperidine substituents
hGPR119
EC50 (nM)
solubility (µg/mL)
Cpd.
R1
R2
ClogPa
pH6.8 buffer FaSSIF
36a
36b
36c
H
14
25
9
<0.13
NTb
3.48
NTb
NTb
4.94
4.92
5.12
2-F
3-F
NTb
42a
42c
H
78
18
0.17
7.09
2.52
4.54
4.72
3-F
<0.16

43c
44c
45c
3-F
3-F
3-F
13
23
31
<0.15
<0.14
NTb
1.27
7.65
NTb
4.76
5.36
5.74
46a
46b
46c
H
31
<0.12
NTb
0.64
NTb
NTb
4.37
4.35
4.55
2-F
3-F
225
123
NTb
47a
49a
H
H
348
35
NTb
NTb
4.60
4.34
<0.13
2.51
a The ClogP value was calculated using software from Daylight Chemical
Information Systems, Inc.
b Not tested.
In our previous study, a fluorine substituted alkylcarbamoylphenyl group as an R3 moiety was
shown to enhance potency. Therefore, the replacement of the methylsulfonyl group of 42c with a
carbamoyl (54a), N-cyclopropylcarbamoyl18 (54b), or N-cyclopropyl-N-methylcarbamoyl group
(54c) was conducted (Table 3). Among these three options, compound 54b exhibited an enhanced
potency (EC50: 8 nM) with a slightly improved solubility in the phosphate buffer (0.21 µg/mL) as
well
as
an
improved
FaSSIF
ring
solubility
of
(8.58
was
µg/mL).
The
4-(N-cyclopropylcarbamoyl)-3-fluorophenyl
54b
replaced
with
4-(N-cyclopropylcarbamoyl)-3-pyridyl (57a) or the corresponding 2-pyridyl (57b) ring, leading to a
10-fold improvement in the phosphate buffer solubility. Unfortunately, the agonist potency of 57a
and 57b was decreased by 6-fold and 8-fold, respectively. Further replacement of the
N-cyclopropylcarbamoyl moiety in 54b with a 1-pyrrolidinylcarbonyl group led to the compound
54d, which showed an improved solubility (3.85 µg/mL) and an EC50 value of 29 nM. Unfortunately,
compound 54d showed a high lipophilicity (ClogP: 5.34), often causing pharmacokinetic issues such
as the inhibition of cytochrome P450 (CYP) isozymes. To reduce the lipophilicity, further
derivatization by replacing the 1-pyrrolidinylcarbamoyl moiety of 54d with 1-azetidinylcarbonyl
(54e), 3-hydoxy-1-azetidinylcarbonyl (54f), and N-(carbamoylmethyl)carbamoyl (54g) groups was
conducted. Compound 54g exhibited reasonable lipophilicity (ClogP: 3.51) and agonist potency
(EC50: 48 nM, full intrinsic activity), while its solubility in phosphate buffer (1.77 µg/mL) and
FaSSIF (18.4 µg/mL) would require further improvement.

Table 3
SAR of R3 group
Solubility (µg/mL)
hGPR119
EC50 (nM)
CYP inhibition
Cpd.
R3
ClogPa
pH6.8
FaSSIF
buffer
IC50 < 10 µMc
42c
54a
54b
54c
57a
57b
54d
18
52
8
<0.16
NTb
2.52
NTb
8.58
NTb
NTb
NTb
92.4
4.72
4.38
5.21
5.29
5.14
5.19
5.34
NTb
NTb
NTb
NTb
NTb
NTb
0.21
NTb
67
47
63
29
2.11
2.24
3.85
1A2, 2C9,
2C19
54e
54f
54g
34
40
48
3.43
1.89
1.77
NTb
NTb
18.4
4.78
4.75
3.51
2C9, 2C19
2C9, 2C19
None
a The ClogP value was calculated using software from Daylight Chemical Information
Systems, Inc.
b Not tested.
c The inhibitory effects on CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A were
evaluated in human liver microsomes.

Given the promising profile of compound 54g (TP0459004), we conducted a pharmacokinetic
study in SD rats. As shown in Table 4, compound 54g displayed excellent oral bioavailability (F:
88.6%) despite its low solubility in the phosphate buffer. The increased three-dimensionality might
lead to increase the dissolution rate,20 resulting in the improved oral bioavailability. Subsequently,
the glucose-lowering effect of 54g was evaluated using an oral glucose tolerance test (oGTT) in
Zucker diabetic fatty rats, a model of obesity and impaired glucose tolerance. Compound 54g was
orally administered 30 min before glucose loading, and the plasma glucose concentration was then
measured over 2 h. The reductions in glucose excursion (∆AUC from 0 to 2 h) were 34.0% and
40.2% at 10 and 30 mg/kg, respectively (Figure 3). The glucose-lowering effect was not increased in
a dose-dependent manner, probably because of the low solubility or the maximum effect of 54g.
Together, these results suggest that compound 54g could potentially be useful as a therapeutic agent
in patients with T2DM.
Table 4
Pharmacokinetic parameters of 54g in SD rats
IV (3 mg/kg)a
PO (10 mg/kg)b
AUC0-t
CL
Vdss
t1/2
(h)
Compound
%F
(mL/h/kg)
(mL/kg)
(ng · h/mL)
931
1010
0.842
9510
88.6
54g
a Dosing vehicle: 20% 2-hydroxypropyl-β-cyclodextrin
b Dosing vehicle: 0.5% methylcellulose containing 0.1% Tween80
Figure 3. Effects of oral administration of 54g (10 or 30 mg/kg) on plasma glucose during oGTT in
Zucker diabetic fatty rats. Each point represents the mean SE. The numbers in parentheses indicate
the number of rats per group. ##P < 0.01 versus Lean, Welch’s t-test. **P < 0.01 versus vehicle,

Dunnett’s test.
4. Conclusion
In our medicinal chemistry effort toward the identification of an orally active small molecule
GPR119 agonist, we designed and synthesized spiro-piperidine derivetives with the aim of
increasing solubility and bioavailability by introducing three-dimensionality into the molecule,
which resulted in the discovery of a series of novel 7-azaspiro[3.5]nonane derivatives. An
optimization study for the right piperidine N-capping group (R1) as well as the left aryl group (R3)
led to the identification of compound 54g, which possesses good PK properties (including oral
bioavailability) in SD rats and exhibits a glucose-lowering effect at 10 mg/kg (po) in Zucker diabetic
fatty rats, suggesting its potential for the treatment of T2DM. Further optimization of 54g aimed at
improving its solubility is ongoing, and will be reported in due course.
5. Experimental section
5.1. Human GPR119 agonist activity
GPR119 agonists were evaluated in Flp-In-T-Rex-HEK293 cells overexpressing human GPR119.
The cells were treated with tetracycline for 24 h and plated on to 96-well plates at 5000 cells/well in
assay buffer (D-MEM, 1 mM 3-isobutyl-1-methylxanthine, 0.01% bovine serum albumin), then
incubated with the test compound for 30 min at 37 ˚C. Changes in the cellular cAMP levels were
measured using a cAMP HiRange assay kit (Cisbio), according to the manufacturer’s protocol.
Responses were determined by subtracting the basal cAMP levels from the agonist-stimulated cAMP
levels. The EC50 values were determined as the concentration of the test compound required to
achieve 50% of the maximal response. Data were calculated from the dose-response curves using
XLfit software (IDBS).
5.2. Solubility in pH6.8 phosphate buffer
An excess amount of each compound was added to pH6.8 phosphate buffer and shaken on a shaker
(model SR-2DS; TAITEC) at 25 °C for 24 h. The suspensions were centrifuged at 3000 and 11000
rpm for 10 min, and the resulting supernatant was diluted with 50% aqueous acetonitrile solution.
The concentrations were measured using HPLC. The HPLC analysis was performed using a
Shimadzu HPLC system composed of a LC-20AD, SPD-20A and SIL-20AC. The conditions for
HPLC were as follows: mobile phase, 0.1% phosphoric acid aqueous solution/acetonitrile; flow rate,
0.8 mL/min; column, reversed- phase (Shimpack XR-ODS, 2.2 µm, 3.0 x 75 mm; Shimadzu) at
40 °C; and detection wavelength, 210 nm.

5.3. Solubility in fasted state simulated intestinal fluid (FaSSIF)
An excess amount of each compound was added to FaSSIF (pH6.5)21 and shaken on a shaker
(model SR-2s; Yamato Kagaku) at 25 °C for 2 h and then kept at 37 °C for 22 h in a water bath
(model LT-10s; Yamato Kagaku). The suspensions were centrifuged at 3000 and 11000 rpm for 10
min, and the supernatant was diluted with 50% aqueous acetonitrile solution or an acetonitrile and
methanol mixture (1:1). The concentrations were measured using HPLC. The HPLC analysis was
performed using a Shimadzu HPLC system composed of a LC-20AD, SPD-20A and SIL-20AC. The
conditions for HPLC were as follows: mobile phase, 0.1% phosphoric acid aqueous
solution/acetonitrile; flow rate, 0.8 mL/min; column, reversed- phase (Shimpack XR-ODS, 2.2 µm,
2.0 x 75 mm; Shimadzu) at 40 °C; and detection wavelength, 210 nm.
5.4. Pharmacokinetic evaluation
The pharmacokinetic profile of the test article was investigated in fasted male Sprague-Dawley
(SD) rats. After a single intravenous or oral administration of the test article, blood was obtained
from the tail vein at each sampling time point and centrifuged to prepare the plasma samples. The
quantitative analysis of the target analyte in the plasma samples was performed using liquid
chromatography-tandem mass spectrometry. The pharmacokinetic parameters were calculated using
a non-compartmental analysis with Phoenix WinNonlin (pharmacokinetic analysis software).
5.5. Oral glucose tolerance test (oGTT) in Zucker diabetic fatty rats
Male Zucker diabetic fatty and lean rats (9 weeks of age) were fasted overnight. Compound 54g
was dissolved in 20 w/v% HP-β-CD and administered orally. After 30 min, glucose solution was
orally administered at 2 g/kg body weight. Blood samples were collected from the tail vein, and the
plasma glucose levels were determined using the Glucose CII test (Wako Pure Chemical Industries).
5.6. Chemistry
All the solvents and reagents were obtained from commercial suppliers and were used without
further purification or were prepared according to published procedures. The melting points were
1
taken with a Yanaco micro-melting point apparatus MP-500D and were uncorrected. The H NMR
and 13C NMR spectra were recorded using a JOEL JNM-ECA600, Varian Inova300, or Gemini2000,
and all the chemical shifts were reported in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard. Mass spectra were recorded using a Shimadzu LCMS 2010 EV
spectrometer. High resolution mass spectral data were acquired using a Shimadzu LCMS-IT-TOF
equipped with an ESI/APCI dual ion source. Purity was determined by LC-MS (Agilent 1290
Infinity) via the following conditions. Column: Waters ACQUITY UPLC CSH C18, 2.1 x 50 mm,

1.7 µm. Flow: 0.8 ml/min. Gradient: 20 to 99% MeCN/water (0.1% formic acid) linear gradient in
1.2 min, held at 99% for 0.2 min. Detector: UV at 254 nm. All final compounds were obtained with
≥95% purity.
tert-Butyl 1-(hydroxymethyl)-6-azaspiro[2.5]octane-6-carboxylate (8)
To a solution of 6-tert-butyl 1-ethyl 6-azaspiro[2.5]octane-1,6-dicarboxylate (2.0 g, 7.1 mmol) in
tetrahydrofuran (THF) (70 mL) was added diisobutylaluminum hydride (DIBAL) (1.0 M in toluene,
20 mL, 20 mmol) under ice cooling. After stirring under ice cooling for 1 h, the reaction was
quenched with water (1.4 mL). To the mixture were added 2.5 M NaOH aqueous solution (1.4 mL)
and water (4.3 mL), and the mixture was stirred at room temperature overnight. The mixture was
filtered through a pad of Celite®, and the filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (30-60% EtOAc in hexanes) to afford 8 as
a colorless oil (1.8 g, quantitative yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.25 (t, J = 4.7 Hz, 1H), 0.58 (dd, J = 8.6, 4.7 Hz, 1H), 0.92-1.05
(m, 1H), 1.12-1.73 (m, 13H), 3.21-3.37 (m, 2H), 3.49-3.80 (m, 4H); MS ESI/APCI Dual m/z 264
[M+Na]+.
tert-Butyl 1-formyl-6-azaspiro[2.5]octane-6-carboxylate (9)
To a solution of 8 (40 mg, 0.17 mmol) in CHCl3 (1.7 mL) was added Dess-Martin periodinane (91
mg, 0.22 mmol). After stirring at room temperature for 2 h, the reaction mixture was quenched with
saturated aqueous Na2SO3 and NaHCO3 solution and extracted with CHCl3. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (20-50% EtOAc in hexanes) to afford 9 as a colorless oil (37 mg, 93%
yield).
1H NMR (600 MHz, CDCl3) δ ppm 1.13 (dd, J = 7.8, 4.5 Hz, 1H), 1.42-1.51 (m, 12H), 1.64-1.75 (m,
2H), 1.80-1.85 (m, 1H), 3.24-3.31 (m, 1H), 3.41-3.55 (m, 3H), 7.26 (s, 1H); MS ESI/APCI Dual m/z
262 [M+Na]+.
tert-Butyl 1-[2-methoxyethenyl]-6-azaspiro[2.5]octane-6-carboxylate (10)
To a solution of MeOCH2P+Ph3Cl- (689 mg, 2.01 mmol) in toluene (5.15 mL) was added t-BuOK
(226 mg, 2.01 mmol) and the mixture was stirred at room temperature for 1 h. To the mixture was
added a solution of 9 (370 mg, 1.55 mmol) in toluene (3.09 mL) under ice cooling, and the mixture
was stirred at room temperature for 2 h. The reaction was quenched with saturated aqueous NH4Cl
solution under ice cooling and the resulting mixture was extracted with EtOAc. The organic layer
was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (10-20% EtOAc in hexanes) to afford 10 as a colorless oil (257

mg, 62% yield, a mixture of cis/trans isomers).
1H NMR (300 MHz, CDCl3) δ ppm 0.28-0.36 (m, 1H), 0.59-0.68 (m, 0.5H), 0.71-0.78 (m, 0.5H),
1.15-1.62 (m, 14H), 3.31-3.48 (m, 4H), 3.50 (s, 1.5H), 3.62 (s, 1.5H), 4.03-4.11 (m, 0.5H), 4.58-4.68
(m, 0.5H), 5.95-6.00 (m, 0.5H), 6.31-6.39 (m, 0.5H); MS ESI/APCI Dual m/z 290 [M+Na]+.
tert-Butyl 1-(2-oxoethyl)-6-azaspiro[2.5]octane-6-carboxylate (11)
To a solution of 10 (200 mg, 0.748 mmol) in CH3CN (7.48 mL) were added water (1.87 mL) and
trifluoroacetic acid (TFA) (0.256 g, 2.24 mmol) and the mixture was stirred at room temperature for
7 h. The reaction was quenched with saturated aqueous NaHCO3 solution under ice cooling and
organic solvent was removed in vacuo. The aqueous residue was extracted with CHCl3. The organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (20-50% EtOAc in hexanes) to afford 11 as a colorless
oil (110 mg, 58% yield).
1H NMR (600 MHz, CDCl3) δ ppm 0.16 (t, J = 5.0 Hz, 1H), 0.65-0.69 (m, 1H), 0.91-0.97 (m, 1H),
1.16-1.22 (m, 1H), 1.25-1.32 (m, 1H), 1.46 (s, 9H), 1.49-1.57 (m, 2H), 2.36-2.43 (m, 1H), 2.47-2.54
(m, 1H), 3.21-3.29 (m, 2H), 3.54-3.70 (m, 2H), 9.82 (br s, 1H); MS ESI/APCI Dual m/z 276
[M+Na]+.
tert-Butyl 1-(2-hydroxyethyl)-6-azaspiro[2.5]octane-6-carboxylate (12)
To a solution of 11 (100 mg, 0.395 mmol) in MeOH (1.97 mL) was added NaBH4 (17.9 mg, 0.474
mmol). After stirring at room temperature for 1 h, the mixture was concentrated in vacuo. The
residue was purified by silica gel column chromatography (40-60% EtOAc in hexanes) to afford 12
as a colorless oil (74.0 mg, 73% yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.04-0.12 (m, 1H), 0.47-0.56 (m, 1H), 0.57-0.71 (m, 1H),
1.06-1.18 (m, 1H), 1.20-1.66 (m, 13H), 1.70-1.87 (m, 1H), 3.16-3.30 (m, 2H), 3.51-3.79 (m, 4H);
MS ESI/APCI Dual m/z 278 [M+Na]+.
tert-Butyl 1-{2-[4-(methanesulfonyl)phenoxy]ethyl}-6-azaspiro[2.5]octane-6-carboxylate (13)
To a solution of 12 (74 mg, 0.29 mmol) in THF (2.9 mL) were added 4-(methanesulfonyl)phenol
(75 mg, 0.43 mmol), N,N,N’,N’-tetramethylazodicarboxamide (TMAD) (75 mg, 0.43 mmol) and
tributylphosphine (88 mg, 0.43 mmol). After stirring at 60 ˚C for 2 h, the reaction was quenched
with water and the resulting mixture was extracted with EtOAc. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (30-50% EtOAc in hexanes) to afford 13 as a colorless solid (95 mg, 80%
yield).
1H NMR (600 MHz, CDCl3) δ ppm 0.12 (t, J = 5.0 Hz, 1H), 0.56 (dd, J = 8.7, 4.5 Hz, 1H), 0.72-0.79

(m, 1H), 1.09-1.16 (m, 1H), 1.30-1.37 (m, 1H), 1.47 (s, 9H), 1.49-1.56 (m, 1H), 1.59-1.73 (m, 2H),
1.96-2.05 (m, 1H), 3.01-3.05 (m, 3H), 3.19-3.26 (m, 2H), 3.59-3.74 (m, 2H), 4.06-4.13 (m, 2H),
7.00-7.04 (m, 2H), 7.84-7.89 (m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm 17.1, 20.2, 21.6, 28.5,
30.4, 36.8, 43.5 (br), 44.9, 68.7, 79.4, 115.0, 129.6, 132.2, 155.0, 163.2; HRMS ESI/APCI Dual m/z
calcd for C21H31NO5S 410.1996 [M+H]+ , found 410.1981.
tert-Butyl 1-(iodomethyl)-6-azaspiro[2.5]octane-6-carboxylate (14)
Under argon atmosphere, to a solution of PPh3 (936 mg, 3.57 mmol) in CHCl3 (27.0 mL) were
added imidazole (423 mg, 6.21 mmol) and I2 (867 mg, 3.41 mmol) under ice cooling. After the
mixture being stirred at 0 ˚C for 20 min, a solution of 8 (749 g, 3.10 mmol) in CHCl3 (9.00 mL) was
added to the mixture. The mixture was allowed to warm to room temperature and was stirred for 3 h.
The reaction mixture was diluted with CHCl3 and washed with saturated aqueous Na2S2O3 solution
and then washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column chromatography (5-10%
EtOAc in hexanes) to afford 14 as a colorless oil (950 mg, 87% yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.23 (t, J = 4.8 Hz, 1H), 0.74 (dd, J = 8.4, 4.8 Hz, 1H), 1.12-1.41
(m, 3H), 1.43-1.59 (m, 10H), 1.64-1.79 (m, 1H), 3.07-3.43 (m, 4H), 3.51-3.81 (m, 2H); MS
ESI/APCI Dual m/z 374 [M+Na]+.
tert-Butyl 1-(3-tert-butoxy-3-oxopropyl)-6-azaspiro[2.5]octane-6-carboxylate (15)
Under argon atmosphere, to a solution of diisopropylamine (1.5 mL, 11 mmol) in THF (27 mL)
was added n-BuLi (2.76 M in hexane, 3.9 mL, 11 mmol) under ice cooling. After being stirred at 0
˚C for 30 min, the mixture was cooled to -78 ˚C and t-BuOAc (1.5 mL, 11 mmol) was added to the
mixture. After the mixture being stirred at -78 ˚C for 30 min, a solution of 14 (0.95 g, 2.7 mmol) in
THF (14 mL) and N,N’-dimethylpropyleneurea (DMPU) (1.3 mL) were added to the mixture and the
mixture was stirred at -78 ˚C for 1 h. The reaction was quenched with saturated aqueous NH4Cl
solution and the resulting mixture was extracted with EtOAc. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (4-10% EtOAc in hexanes) to afford 15 (0.77 g, 84% yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.01-0.07 (m, 1H), 0.44-0.51 (m, 1H), 0.53-0.67 (m, 1H),
1.05-1.85 (m, 24H), 2.23-2.37 (m, 2H), 3.18-3.30 (m, 2H), 3.49-3.70 (m, 2H); MS ESI/APCI Dual
m/z 340 [M+H]+, 362 [M+Na]+.
tert-Butyl 1-(3-hydroxypropyl)-6-azaspiro[2.5]octane-6-carboxylate (16)
To a solution of 15 (770 mg, 2.27 mmol) in THF (23.0 mL) was added LiBH4 (137 mg, 5.67 mmol)
and the mixture was stirred at 60 ˚C overnight. The reaction was quenched with saturated aqueous

NH4Cl solution and the resulting mixture was extracted with EtOAc. The organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (4-65% EtOAc in hexanes) to afford 16 as a colorless
oil (203 mg, 28% yield).
1H NMR (300 MHz, CDCl3) δ ppm -0.02-0.04 (m, 1H), 0.43-0.52 (m, 1H), 0.53-0.64 (m, 1H),
1.04-1.17 (m, 1H), 1.18-1.36 (m, 3H), 1.39-1.77 (m, 13H), 3.14-3.31 (m, 2H), 3.48-3.76 (m, 4H);
MS ESI/APCI Dual m/z 292 [M+Na]+.
tert-Butyl 1-{3-[4-(methanesulfonyl)phenoxy]propyl}-6-azaspiro[2.5]octane-6-carboxylate (17)
The title compound was synthesized according to the procedure described for compound 13 from
16 (96% yield).
1
Colorless oil; H NMR (300 MHz, CDCl3) δ ppm 0.01-0.06 (m, 1H), 0.46-0.55 (m, 1H), 0.56-0.68
(m, 1H), 1.05-1.18 (m, 1H), 1.21-1.73 (m, 14H), 1.86-2.00 (m, 2H), 3.03 (s, 3H), 3.16-3.28 (m, 2H),
3.55-3.71 (m, 2H), 4.06 (t, J = 6.4 Hz, 2H), 6.96-7.04 (m, 2H), 7.83-7.89 (m, 2H); 13C NMR (151
MHz, CDCl3) δ ppm 17.4, 21.7, 23.2, 25.3, 28.6, 29.5, 30.3, 36.9, 43.5 (br), 44.9, 68.2, 79.3, 115.0,
129.6, 132.2, 155.1, 163.3; HRMS ESI/APCI Dual m/z calcd for C22H33NO5S 424.2152 [M+H]+ ,
found 424.2144.
tert-Butyl 2-(2-ethoxy-2-oxoethylidene)-7-azaspiro[3.5]nonane-7-carboxylate (20)
To a solution of (EtO)2POCH2CO2Et (0.822 mL, 4.11 mmol) in N,N-dimethylformamide (DMF)
(10.0 mL) was added NaH (60% in oil, 164 mg, 4.11 mmol) under ice cooling. After the mixture
being stirred for 30 min, a solution of tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate (18)
(655 mg, 2.74 mmol) in DMF (4.00 mL) was added to the mixture and the mixture was allowed to
warm to room temperature. After stirring at room temperature for 3 h, the reaction mixture was
quenched with saturated aqueous NH4Cl solution and extracted with EtOAc. The organic layer was
washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (9-15% EtOAc in hexanes) to afford 20 as
a colorless oil (800 mg, 94% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.27 (t, J = 7.0 Hz, 3H), 1.46 (s, 9H), 1.52-1.62 (m, 4H),
2.55-2.61 (m, 2H), 2.85-2.92 (m, 2H), 3.25-3.44 (m, 4H), 4.15 (q, J = 7.0 Hz, 2H), 5.67-5.73 (m,
1H); MS ESI/APCI Dual m/z 332 [M+Na]+.
tert-Butyl 2-(2-ethoxy-2-oxoethyl)-7-azaspiro[3.5]nonane-7-carboxylate (22)
To a solution of 20 (797 mg, 2.58 mmol) in EtOH (13.0 mL) was added Pd(OH)2 (20% on carbon,
199 mg), and the mixture was stirred under hydrogen atmosphere at room temperature overnight.
The mixture was filtered through a pad of Celite® and the filtrate was concentrated under reduced

pressure to afford 22 as a colorless oil (730 mg, 91% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.25 (t, J = 7.0 Hz, 3H), 1.37-1.68 (m, 15H), 1.98-2.09 (m, 2H),
2.41 (d, J = 7.6 Hz, 2H), 2.57-2.72 (m, 1H), 3.19-3.39 (m, 4H), 4.11 (q, J = 7.0 Hz, 2H); MS
ESI/APCI Dual m/z 334 [M+Na]+.
tert-Butyl 2-(2-hydroxyethyl)-7-azaspiro[3.5]nonane-7-carboxylate (24)
To a solution of 22 (732 mg, 2.35 mmol) in THF (12.0 mL) was added LiBH4 (174 mg, 8.88 mmol)
and the mixture was stirred under reflux for 5 h. The reaction was quenched with water under ice
cooling and the resulting mixture was extracted with EtOAc. The organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford 24 as a colorlessoil
(580 mg, 92% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.35-1.49 (m, 13H), 1.51-1.62 (m, 2H), 1.63-1.74 (m, 2H),
1.94-2.03 (m, 2H), 2.25-2.43 (m, 1H), 3.20-3.38 (m, 4H), 3.52-3.64 (m, 2H); MS ESI/APCI Dual
m/z 292 [M+Na]+.
tert-Butyl 2-{2-[4-(methanesulfonyl)phenoxy]ethyl}-7-azaspiro[3.5]nonane-7-carboxylate (26)
The title compound was synthesized according to the procedure described for compound 13 from
24 (57% yield).
1
Colorless solid; H NMR (300 MHz, CDCl3) δ ppm 1.40-1.62 (m, 15H), 1.85-2.09 (m, 4H),
2.36-2.52 (m, 1H), 3.03 (s, 3H), 3.22-3.40 (m, 4H), 3.97 (t, J = 6.5 Hz, 2H), 6.95-7.03 (m, 2H),
7.82-7.90 (m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm 25.9, 28.5, 34.5, 36.3, 36.8, 37.9, 39.7, 40.7
(br), 44.9, 66.9, 79.3, 114.9, 129.6, 132.2, 155.0, 163.2; HRMS ESI/APCI Dual m/z calcd for
C22H33NO5S 424.2152 [M+H]+ , found 424.2144.
tert-Butyl 9-(2-ethoxy-2-oxoethylidene)-3-azaspiro[5.5]undecane-3-carboxylate (21)
The title compound was synthesized according to the procedure described for compound 20 from
tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate (19) (77% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.27 (t, J = 7.1 Hz, 3H), 1.41-1.59 (m, 17H), 2.17-2.27 (m, 2H),
2.79-2.90 (m, 2H), 3.34-3.45 (m, 4H), 4.14 (q, J = 7.1 Hz, 2H), 5.63 (s, 1H); MS ESI/APCI Dual m/z
360 [M+Na]+.
tert-Butyl 9-(2-ethoxy-2-oxoethyl)-3-azaspiro[5.5]undecane-3-carboxylate (23)
The title compound was synthesized according to the procedure described for compound 22 from
21 (92% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.06-1.74 (m, 25H), 2.20 (d, J = 7.0 Hz, 2H), 3.30-3.41 (m, 4H),
4.12 (q, J = 7.0 Hz, 2H); MS ESI/APCI Dual m/z 362 [M+Na]+.

tert-Butyl 9-(2-hydroxyethyl)-3-azaspiro[5.5]undecane-3-carboxylate (25)
The title compound was synthesized according to the procedure described for compound 24 from
23 (quantitative yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.77-1.72 (m, 24H), 3.29-3.39 (m, 4H), 3.69 (t, J = 6.7 Hz, 2H);
MS ESI/APCI Dual m/z 320 [M+Na]+.
tert-Butyl 9-{2-[4-(methanesulfonyl)phenoxy]ethyl}-3-azaspiro[5.5]undecane-3-carboxylate (27)
The title compound was synthesized according to the procedure described for compound 13 from
25 (64% yield).
Colorless amorphous; 1H NMR (300 MHz, CDCl3) δ ppm 1.01-1.36 (m, 6H), 1.40-1.80 (m, 18H),
3.03 (s, 3H), 3.29-3.44 (m, 4H), 4.02-4.11 (m, 2H), 6.98-7.04 (m, 2H), 7.81-7.90 (m, 2H); 13C NMR
(151 MHz, CDCl3) δ ppm 27.9, 28.5, 31.0, 31.7 (br), 34.8, 35.6, 36.0, 40.1 (br), 44.9, 66.6, 79.2,
115.0, 129.6, 132.1, 155.1, 163.3; HRMS ESI/APCI Dual m/z calcd for C24H37NO5S 452.2465
[M+H]+ , found 452.2455.
tert-Butyl 2-(methoxymethylidene)-7-azaspiro[3.5]nonane-7-carboxylate (28)
Under argon atmosphere, to a suspension of MeOCH2P+Ph3Cl- (32.0 g, 93.4 mmol) in toluene (200
mL) was added t-BuOK (10.5 g, 93.4 mmol) and the mixture was stirred at room temperature for 20
min. To this mixture was added a solution of tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate
(18) (17.2 g, 71.9 mmol) in toluene (160 mL), and the mixture was stirred at 70 ˚C for 4 h. The
reaction was quenched with saturated aqueous NH4Cl solution under ice cooling and the resulting
mixture was extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica gel column chromatography (5-20%
EtOAc in hexanes) to afford 28 as a pale yellow oil (12.8 g, 67% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.45 (s, 9H), 1.52-1.59 (m, 4H), 2.29-2.47 (m, 4H), 3.26-3.37 (m,
4H), 3.53-3.57 (m, 3H), 5.80-5.87 (m, 1H); MS ESI/APCI Dual m/z 290 [M+Na]+.
tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-7-azaspiro[3.5]nonane-7-carboxylate (30)
To a solution of 28 (12.8 g, 47.9 mmol) in CH3CN (456 mL) were added water (114 mL) and
trifluoroacetic acid (TFA) (4.00 mL) and the mixture was stirred at room temperature for 1 h. The
reaction was quenched with saturated aqueous NaHCO3 solution under ice cooling and organic
solvent was removed in vacuo. The resulting aqueous residue was extracted with EtOAc. The
organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo to afford tert-butyl 2-formyl-7-azaspiro[3.5]nonane-7-carboxylate.
To a solution of (EtO)2POCH2CO2Et (11.0 mL, 57.5 mmol) in DMF (60.0 mL) and THF (60.0 mL)

was added NaH (60% in oil, 2.30 g, 57.5 mmol) under ice cooling. After the mixture being stirred
for 20 min, a solution of tert-butyl 2-formyl-7-azaspiro[3.5]nonane-7-carboxylate in DMF (60.0 mL)
and THF (60.0 mL) was added to the mixture and the mixture was allowed to warm to room
temperature. After stirring at room temperature for 3 h, the reaction mixture was quenched with
saturated aqueous NH4Cl solution under ice cooling and extracted with EtOAc. The organic layer
was washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography (5-20% EtOAc in hexanes) to afford
30 as a colorless oil (13.6 g, 87% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.29 (t, J = 7.1 Hz, 3H), 1.45 (s, 9H), 1.53-1.78 (m, 4H),
2.01-2.16 (m, 4H), 2.98-3.14 (m, 1H), 3.23-3.40 (m, 4H), 4.19 (q, J = 7.1 Hz, 2H), 5.74 (dd, J = 15.5,
1.6 Hz, 1H), 7.05 (dd, J = 15.5, 6.7 Hz, 1H); MS ESI/APCI Dual m/z 346 [M+Na]+.
tert-Butyl 2-(3-ethoxy-3-oxopropyl)-7-azaspiro[3.5]nonane-7-carboxylate (32)
To a solution of 30 (13.6 g, 41.9 mmol) in EtOH (420 mL) was added Pd(OH)2 (20% on carbon,
2.72 g), and the mixture was stirred under hydrogen atmosphere at room temperature overnight. The
mixture was filtered through a pad of Celite®, and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (5-30% EtOAc in hexanes)
to afford 32 as a yellow solid (13.3 g, 97% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.25 (t, J = 7.1 Hz, 3H), 1.32-1.61 (m, 15H), 1.67-1.78 (m, 2H),
1.88-2.00 (m, 2H), 2.13-2.28 (m, 3H), 3.19-3.38 (m, 4H), 4.12 (q, J = 7.1 Hz, 2H); MS ESI/APCI
Dual m/z 348 [M+Na]+.
tert-Butyl 2-(3-hydroxypropyl)-7-azaspiro[3.5]nonane-7-carboxylate (34)
To a solution of 32 (10.4 g, 31.8 mmol) in THF (160 mL) was added DIBAL (1.0 M in toluene,
80.0 mL, 80.0 mmol) under ice cooling and the mixture was stirred at the same temperature for 1 h.
The reaction was quenched with saturated aqueous citric acid solution and extracted with EtOAc.
The organic layer was washed with saturated aqueous citric acid solution, 1 M HCl aqueous solution
and brine, and then dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (40-70% EtOAc in hexanes) to afford 34 as a
colorless oil (6.64 g, 74% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.31-1.60 (m, 19H), 1.89-2.00 (m, 2H), 2.10-2.31 (m, 1H),
3.19-3.39 (m, 4H), 3.56-3.68 (m, 2H); MS ESI/APCI Dual m/z 306 [M+Na]+.
tert-Butyl 2-{3-[4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate (36a)
The title compound was synthesized according to the procedure described for compound 13 from
34 (85% yield).

Colorless solid (mp 154-155 ˚C); 1H NMR (300 MHz, CDCl3) δ ppm 1.34-1.49 (m, 13H), 1.51-1.63
(m, 5H), 1.64-1.80 (m, 2H), 1.90-2.04 (m, 2H), 2.16-2.35 (m, 1H), 3.03 (s, 3H), 3.21-3.40 (m, 4H),
4.00 (t, J = 6.5 Hz, 2H), 6.95-7.03 (m, 2H), 7.82-7.91 (m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm
26.9, 28.5, 28.6, 34.0, 36.5, 38.0, 39.7, 41.0 (br), 44.9, 68.5, 79.2, 114.9, 129.6, 132.1, 155.0, 163.3;
HRMS ESI/APCI Dual m/z calcd for C23H35NO5S 438.2309 [M+H]+ , found 438.2297.
tert-Butyl 2-{3-[2-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate
(36b)
The title compound was synthesized according to the procedure described for compound 13 from
34 and 2-fluoro-4-(methanesulfonyl)phenol (51% yield).
1
Colorless solid; H NMR (200 MHz, CDCl3) δ ppm 1.24-1.67 (m, 17H), 1.69-1.86 (m, 2H),
1.90-2.07 (m, 2H), 2.14-2.38 (m, 1H), 3.04 (s, 3H), 3.20-3.41 (m, 4H), 4.08 (t, J = 6.4 Hz, 2H),
6.98-7.13 (m, 1H), 7.57-7.74 (m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm 26.7, 28.5, 33.9, 34.0,
36.4, 37.9, 39.7, 41.0 (br), 44.7, 69.6, 79.2, 114.0, 115.6 (d, J = 21.0 Hz), 124.6 (d, J = 5.9 Hz),
132.3, 151.9 (d, J = 252.0 Hz), 151.8 (d, J = 12.0 Hz), 155.0; HRMS ESI/APCI Dual m/z calcd for
C23H34FNO5S 456.2214 [M+H]+ , found 456.2199.
tert-Butyl 2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate
(36c)
The title compound was synthesized according to the procedure described for compound 13 from
34 and 3-fluoro-4-(methanesulfonyl)phenol (86% yield).
Colorless solid; 1H NMR (200 MHz, CDCl3) δ ppm 1.26-1.64 (m, 17H), 1.64-1.84 (m, 2H),
1.90-2.05 (m, 2H), 2.12-2.38 (m, 1H), 3.18 (s, 3H), 3.21-3.40 (m, 4H), 3.99 (t, J = 6.2 Hz, 2H),
6.64-6.85 (m, 2H), 7.76-7.94 (m, 1H); 13C NMR (151 MHz, CDCl3) δ ppm 26.8, 28.5, 34.0, 34.0,
36.5, 38.0, 39.7, 40.9 (br), 44.2, 69.0, 79.3, 103.3 (d, J = 27.0 Hz), 110.6, 120.2, 131.0, 155.1, 160.8
(d, J = 252.0 Hz), 165.2; HRMS ESI/APCI Dual m/z calcd for C23H34FNO5S 456.2214 [M+H]+ ,
found 456.2203.
tert-Butyl 9-(methoxymethylidene)-3-azaspiro[5.5]undecane-3-carboxylate (29)
The title compound was synthesized according to the procedure described for compound 28 from
tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate (19) (44% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.36-1.44 (m, 8H), 1.45 (s, 9H), 1.90-1.99 (m, 2H), 2.12-2.23 (m,
2H), 3.34-3.42 (m, 4H), 3.53 (s, 3H), 5.77 (t, J = 1.2 Hz, 1H); MS ESI/APCI Dual m/z 318 [M+Na]+.
tert-Butyl 9-(-3-ethoxy-3-oxoprop-1-en-1-yl)-3-azaspiro[5.5]undecane-3-carboxylate (31)
The title compound was synthesized according to the procedure described for compound 30 from

29 (82% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.06-1.80 (m, 24H), 2.06-2.23 (m, 1H), 3.28-3.44 (m, 4H), 4.19
(q, J = 7.2 Hz, 2H), 5.74-5.82 (m, 1H), 6.86-6.98 (m, 1H); MS ESI/APCI Dual m/z 374 [M+Na]+.
tert-Butyl 9-(3-ethoxy-3-oxopropyl)-3-azaspiro[5.5]undecane-3-carboxylate (33)
The title compound was synthesized according to the procedure described for compound 32 from
31 (90% yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.99-1.82 (m, 27H), 2.23-2.38 (m, 2H), 3.27-3.42 (m, 4H), 4.12
(q, J = 7.2 Hz, 2H); MS ESI/APCI Dual m/z 376 [M+Na]+.
tert-Butyl 9-(3-hydroxypropyl)-3-azaspiro[5.5]undecane-3-carboxylate (35)
To a solution of 33 (470 mg, 1.33 mmol) in THF (7.00 mL) and toluene (7.00 mL) was added
LiBH4 (87.0 mg, 3.99 mmol) and the mixture was stirred at 60 ˚C for 16 h. The reaction was
quenched with saturated aqueous NH4Cl solution and the mixture was extracted with EtOAc. The
organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (0-20% EtOAc in hexanes) to
afford 35 as a colorless oil (370 mg, 89% yield).
1H NMR (300 MHz, CDCl3) δ ppm 0.98-1.73 (m, 26H), 3.28-3.40 (m, 4H), 3.57-3.67 (m, 2H); MS
ESI/APCI Dual m/z 334 [M+Na]+.
tert-Butyl 9-{3-[4-(methanesulfonyl)phenoxy]propyl}-3-azaspiro[5.5]undecane-3-carboxylate (37)
The title compound was synthesized according to the procedure described for compound 13 from
35 (61%yield).
Colorless solid; 1H NMR (300 MHz, CDCl3) δ ppm 1.02-1.20 (m, 4H), 1.20-1.43 (m, 6H), 1.45 (s,
9H), 1.52-1.63 (m, 3H), 1.64-1.73 (m, 2H), 1.76-1.92 (m, 2H), 3.03 (s, 3H), 3.29-3.43 (m, 4H), 4.01
(t, J = 6.5 Hz, 2H), 6.97-7.03 (m, 2H), 7.82-7.89 (m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm 26.5,
28.0, 28.5, 31.1, 31.6, 33.2, 35.7, 37.7, 39.5 (br), 40.1, 44.9, 68.9, 79.2, 115.0, 129.6, 132.1, 155.1,
163.3; HRMS ESI/APCI Dual m/z calcd for C25H39NO5S 466.2622 [M+H]+ , found 466.2612.
tert-Butyl 2-[(2E)-4-ethoxy-4-oxobut-2-en-1-yl]-7-azaspiro[3.5]nonane-7-carboxylate (38)
To a solution of 24 (526 mg, 1.95 mmol) in CHCl3 (10.0 mL) was added Dess-Martin periodinane
(1.10 g, 2.54 mmol) under ice cooling and the mixture was stirred at room temperature for 2 h. The
residue was diluted with CHCl3 and washed with saturated aqueous Na2SO3 and NaHCO3 solution.
The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford
tert-butyl 2-(2-oxoethyl)-7-azaspiro[3.5]nonane-7-carboxylate.
To a solution of (EtO)2POCH2CO2Et (0.552 mL, 2.93 mmol) in DMF (10.0 mL) was added NaH

(60% in oil, 117 mg, 2.93 mmol) under ice cooling. After the mixture being stirred for 20 min, a
solution of tert-butyl 2-(2-oxoethyl)-7-azaspiro[3.5]nonane-7-carboxylate in DMF (3.00 mL) was
added to the mixture and the mixture was allowed to warm to room temperature. After stirring at
room temperature for 3 h, the reaction mixture was quenched with saturated aqueous NH4Cl solution
and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (8-15% EtOAc in hexanes) to afford 38 as a colorless oil (360 mg, 55% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.29 (t, J = 7.1 Hz, 3H), 1.36-1.49 (m, 13H), 1.50-1.60 (m, 2H),
1.93-2.06 (m, 2H), 2.23-2.46 (m, 3H), 3.21-3.37 (m, 4H), 4.18 (q, J = 7.1 Hz, 2H), 5.73-5.83 (m,
1H), 6.78-6.94 (m, 1H); MS ESI/APCI Dual m/z 360 [M+Na]+.
tert-Butyl 2-(4-ethoxy-4-oxobutyl)-7-azaspiro[3.5]nonane-7-carboxylate (39)
The title compound was synthesized according to the procedure described for compound 32 from
38 (96% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.25 (t, J = 7.0 Hz, 3H), 1.30-1.61 (m, 19H), 1.89-2.00 (m, 2H),
2.12-2.31 (m, 3H), 3.19-3.39 (m, 4H), 4.12 (q, J = 7.0 Hz, 2H); MS ESI/APCI Dual m/z 362
[M+Na]+.
tert-Butyl 2-(4-hydroxybutyl)-7-azaspiro[3.5]nonane-7-carboxylate (40)
The title compound was synthesized according to the procedure described for compound 16 from
39 (92% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.16-1.61 (m, 21H), 1.87-1.99 (m, 2H), 2.10-2.30 (m, 1H),
3.20-3.38 (m, 4H), 3.58-3.70 (m, 2H); MS ESI/APCI Dual m/z 320 [M+Na]+.
tert-Butyl 2-{4-[4-(methanesulfonyl)phenoxy]butyl}-7-azaspiro[3.5]nonane-7-carboxylate (41)
The title compound was synthesized according to the procedure described for compound 13 from
40 (92% yield).
1
Colorless solid; H NMR (300 MHz, CDCl3) δ ppm 1.28-1.60 (m, 19H), 1.71-1.85 (m, 2H),
1.89-2.01 (m, 2H), 2.16-2.29 (m, 1H), 3.03 (s, 3H), 3.22-3.38 (m, 4H), 4.01 (t, J = 6.5 Hz, 2H),
6.97-7.04 (m, 2H), 7.81-7.90 (m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm 23.7, 28.5, 28.8, 29.0,
34.0, 36.5, 37.6, 38.0, 39.8, 40.9 (br), 44.9, 68.6, 79.2, 115.0, 129.6, 132.1, 155.1, 163.3; HRMS
ESI/APCI Dual m/z calcd for C24H37NO5S 452.2465 [M+H]+ , found 452.2456.
Propan-2-yl 2-{3-[4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate (42a)
To a suspension of 36a (2.7 g, 6.1 mmol) in EtOAc (31 mL) was added 4 M HCl in EtOAc (31 mL).
After stirring at room temperature overnight, the mixture was concentrated in vacuo. To the resulting

residue were added 1 M NaOH aqueous solution and CHCl3. The organic layer was separated, dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford
2-{3-[4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane (2.0 g).
To a solution of the above crude material (60 mg, 0.18 mmol) in CHCl3 (2.0 mL) were added Et3N
(0.050 mL, 0.36 mmol) and isopropyl chloroformate (0.031 mL, 0.27 mmol) under ice cooling. After
stirring at room temperature for 6 h, the reaction mixture was quenched with water and extracted
with CHCl3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography (25-40% EtOAc in hexanes) and
solidified with EtOAc and hexanes to afford 42a as a colorless solid (47 mg, 62% yield).
Mp 130 ˚C; 1H NMR (600 MHz, CDCl3) δ ppm 1.20-1.25 (m, 6H), 1.37-1.43 (m, 2H), 1.43-1.50 (m,
2H), 1.54-1.61 (m, 4H), 1.69-1.78 (m, 2H), 1.95-2.01 (m, 2H), 2.20-2.32 (m, 1H), 3.03 (s, 3H),
3.25-3.44 (m, 4H), 4.00 (t, J = 6.6 Hz, 2H), 4.86-4.94 (m, 1H), 6.97-7.02 (m, 2H), 7.81-7.87 (m,
2H); 13C NMR (151 MHz, CDCl3) δ ppm 22.3, 26.9, 28.6, 34.0, 36.4, 37.9, 39.7, 40.6, 40.9, 44.9,
68.3, 68.4, 114.9, 129.6, 132.1, 155.4, 163.3; HRMS ESI/APCI Dual m/z calcd for C22H33NO5S
424.2152 [M+H]+ , found 424.2147.
Propan-2-yl
2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate (42c)
The title compound was synthesized according to the procedure described for compound 42a from
36c (47% yield).
1
Colorless solid (mp 105-106 ˚C); H NMR (600 MHz, CDCl3) δ ppm 1.23 (d, J = 6.2 Hz, 6H),
1.35-1.62 (m, 8H), 1.69-1.77 (m, 2H), 1.95-2.02 (m, 2H), 2.21-2.30 (m, 1H), 3.18 (s, 3H), 3.25-3.42
(m, 4H), 3.99 (t, J = 6.4 Hz, 2H), 4.86-4.94 (m, 1H), 6.68-6.74 (m, 1H), 6.76-6.80 (m, 1H),
7.82-7.88 (m, 1H); 13C NMR (151 MHz, CDCl3) δ ppm 22.3, 26.7, 28.5, 33.9, 34.0, 36.3, 37.9, 39.6,
40.6, 40.8, 44.2, 68.3, 69.0, 103.3 (d, J = 24.0 Hz), 110.6, 120.1 (d, J = 15.0 Hz), 131.0, 155.4,
160.8 (d, J = 255.3 Hz), 165.2 (d, J = 12.0 Hz); HRMS ESI/APCI Dual m/z calcd for C22H32FNO5S
442.2058 [M+H]+ , found 442.2051.
1-Methylcyclopropyl
2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate (43c)
The title compound was synthesized according to the procedure described for compound 42a from
36c and 1-methylcyclopropyl 4-nitrophenyl carbonate (31% yield).
Colorless solid; 1H NMR (600 MHz, CDCl3) δ ppm 0.59-0.64 (m, 2H), 0.83-0.88 (m, 2H), 1.35-1.60
(m, 11H), 1.70-1.76 (m, 2H), 1.95-2.01 (m, 2H), 2.21-2.30 (m, 1H), 3.19 (s, 3H), 3.21-3.45 (m, 4H),
3.99 (t, J = 6.4 Hz, 2H), 6.68-6.75 (m, 1H), 6.77-6.81 (m, 1H), 7.81-7.89 (m, 1H); 13C NMR (151
MHz, CDCl3) δ ppm 13.0, 21.8, 26.7, 28.5, 33.9, 36.3, 37.9, 39.6, 40.8 (br s), 44.2, 56.3, 69.0, 103.3

(d, J = 24.0 Hz), 110.6, 120.1 (d, J = 15.0 Hz), 131.0, 155.3, 160.8 (d, J = 255.3 Hz), 165.2 (d, J =
12.0 Hz); HRMS ESI/APCI Dual m/z calcd for C23H32FNO5S 454.2058 [M+H]+ , found 454.2051.
Cyclopentyl
2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate (44c)
To a suspension of 36c (0.42 g, 0.92 mmol) in EtOAc (9.0 mL) was added 4 M HCl in EtOAc (9.0
mL). After stirring at room temperature overnight, the mixture was concentrated in vacuo to afford
2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane hydrochloride. To
a
solution of cyclopentanol (16 mg, 0.18 mmol) in THF (0.90 mL) were added triphosgene (19 mg,
0.064 mmol) and Et3N (0.051 mL, 0.37 mmol) under ice cooling. After the mixture being stirred for
30 min, 2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane hydrochloride
(36 mg, 0.092 mmol) was added to the mixture. The mixture was allowed to warm to room
temperature and was stirred overnight. The mixture was then quenched with water and extracted
with CHCl3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography (20-35% EtOAc in hexanes) to afford
44c as a colorless solid (24 mg, 55% yield).
1H NMR (600 MHz, CDCl3) δ ppm 1.35-1.48 (m, 4H), 1.49-1.63 (m, 6H), 1.66-1.77 (m, 6H),
1.79-1.90 (m, 2H), 1.94-2.01 (m, 2H), 2.20-2.31 (m, 1H), 3.18 (s, 3H), 3.24-3.44 (m, 4H), 3.98 (t, J
= 6.4 Hz, 2H), 5.06-5.12 (m, 1H), 6.68-6.74 (m, 1H), 6.76-6.81 (m, 1H), 7.81-7.88 (m, 1H); 13C
NMR (151 MHz, CDCl3) δ ppm 23.7, 26.8, 28.5, 32.9, 34.0, 34.0, 36.4, 38.0, 39.7, 40.7, 40.9, 44.2,
69.0, 77.7, 103.3 (d, J = 27.0 Hz), 110.6, 120.2 (d, J = 18.0 Hz), 131.0, 155.6, 160.8 (d, J = 252.3
Hz), 165.2 (d, J = 9.0 Hz); HRMS ESI/APCI Dual m/z calcd for C24H34FNO5S 468.2214 [M+H]+ ,
found 468.2209.
2,2-Dimethylpropyl
2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carboxylate (45c)
The title compound was synthesized according to the procedure described for compound 44c from
36c and 2,2-dimethylpropan-1-ol (51% yield).
Colorless solid; 1H NMR (600 MHz, CDCl3) δ ppm 0.94 (s, 9H), 1.36- 1.63 (m, 8H), 1.69-1.77 (m,
2H), 1.95-2.03 (m, 2H), 2.20-2.31 (m, 1H), 3.18 (s, 3H), 3.31-3.37 (m, 2H), 3.39-3.45 (m, 2H), 3.76
(s, 2H), 3.99 (t, J = 6.6 Hz, 2H), 6.69-6.74 (m, 1H), 6.76-6.81 (m, 1H), 7.82-7.88 (m, 1H); 13C NMR
(151 MHz, CDCl3) δ ppm 26.6, 26.8, 28.5, 31.6, 34.0, 34.0, 36.3 (br), 38.0, 39.7 (br), 40.8, 41.0,
44.2, 69.0, 74.7, 103.3 (d, J = 24.0 Hz), 110.6, 120.2 (d, J = 15.0 Hz), 131.0, 155.9, 160.8 (d, J =
255.0 Hz), 165.2 (d, J = 9.0 Hz); HRMS ESI/APCI Dual m/z calcd for C24H36FNO5S 470.2371
[M+H]+ , found 470.2362.

7-(5-Ethylpyrimidin-2-yl)-2-{3-[4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane (46a)
To a suspension of 36a (0.19 g, 0.43 mmol) in EtOAc (4.0 mL) was added 4 M HCl in EtOAc (4.0
mL). After stirring at room temperature for 5 h, the mixture was concentrated in vacuo. To a solution
of the residue in DMSO (4.0 mL) were added 2-chloro-5-ethylpyrimidine (0.10 mL, 0.87 mmol) and
Cs2CO3 (0.71 g, 2.2 mmol), and the mixture was stirred at 180 ˚C under microwave irradiation for
20 min. The reaction mixture was quenched with water and extracted with EtOAc. The organic layer
was washed with water, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (30-50% EtOAc in hexanes) to afford 46a
as a pale yellow solid (145 mg, 75% yield).
Mp 165-166 ˚C; 1H NMR (300 MHz, CDCl3) δ ppm 1.17 (t, J = 7.6 Hz, 3H), 1.37-1.82 (m, 10H),
1.97-2.09 (m, 2H), 2.20-2.36 (m, 1H), 2.44 (q, J = 7.5 Hz, 2H), 3.03 (s, 3H), 3.60-3.77 (m, 4H), 4.01
(t, J = 6.5 Hz, 2H), 6.96-7.04 (m, 2H), 7.82-7.89 (m, 2H), 8.15 (s, 2H); 13C NMR (151 MHz, CDCl3)
δ ppm 15.7, 22.7, 26.9, 28.6, 34.1, 34.4, 36.4, 38.1, 39.7, 41.0, 41.2, 44.9, 68.5, 115.0, 124.0, 129.6,
132.1, 157.1, 161.0, 163.3; HRMS ESI/APCI Dual m/z calcd for C24H33N3O3S 444.2315 [M+H]+ ,
found 444.2307.
7-(5-ethylpyrimidin-2-yl)-2-{3-[2-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonan
e (46b)
The title compound was synthesized according to the procedure described for compound 46a from
36b (33% yield).
Colorless solid; 1H NMR (200 MHz, CDCl3) δ ppm 1.11-1.23 (m, 3H), 1.35-1.91 (m, 10H),
1.96-2.10 (m, 2H), 2.20-2.37 (m, 1H), 2.38-2.52 (m, 2H), 3.04 (s, 3H), 3.57-3.80 (m, 4H), 4.09 (t, J
= 6.4 Hz, 2H), 6.99-7.13 (m, 1H), 7.56-7.75 (m, 2H), 8.15 (s, 2H); 13C NMR (151 MHz, CDCl3) δ
ppm 15.7, 22.7, 26.8, 28.6, 34.0, 34.4, 36.4, 38.1, 39.7, 41.0, 41.2, 44.8, 69.6, 114.1, 115.6 (d, J =
21.0 Hz), 124.0, 124.7, 132.3 (d, J = 5.7 Hz), 151.9 (d, J = 253.0 Hz), 152.0, 157.1, 161.0; HRMS
ESI/APCI Dual m/z calcd for C24H32FN3O3S 462.2221 [M+H]+ , found 462.2212.
7-(5-Ethylpyrimidin-2-yl)-2-{3-[3-fluoro-4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonan
e (46c)
The title compound was synthesized according to the procedure described for compound 46a from
36c (26% yield).
Colorless solid; 1H NMR (200 MHz, CDCl3) δ ppm 1.11-1.23 (m, 3H), 1.34-1.84 (m, 10H),
1.94-2.11 (m, 2H), 2.19-2.38 (m, 1H), 2.38-2.52 (m, 2H), 3.18 (s, 3H), 3.58-3.80 (m, 4H), 4.00 (t, J
= 6.4 Hz, 2H), 6.63-6.84 (m, 2H), 7.78-7.90 (m, 1H), 8.15 (s, 2H); 13C NMR (151 MHz, CDCl3) δ
ppm 15.7, 22.7, 26.8, 28.6, 34.0, 34.4, 36.4, 38.1, 39.7, 41.0, 41.2, 44.2, 69.0, 103.3 (d, J = 24.0 Hz),
110.6, 120.1 (d, J = 15.0 Hz), 124.0, 131.0, 157.1, 160.8 (d, J = 255.0 Hz), 161.0, 165.3 (d, J = 12.0

Hz); HRMS ESI/APCI Dual m/z calcd for C24H32FN3O3S 462.2221 [M+H]+ , found 462.2213.
2-{3-[4-(Methanesulfonyl)phenoxy]propyl}-7-(5-methylpyridin-2-yl)-7-azaspiro[3.5]nonane (47a)
To a suspension of 36a (2.7 g, 6.1 mmol) in EtOAc (31 mL) was added 4 M HCl in EtOAc (31 mL).
After stirring at room temperature overnight, the mixture was concentrated in vacuo. To the resulting
residue were added 1 M NaOH aqueous solution and CHCl3. The organic layer was separated, dried
over
anhydrous
Na2SO4,
filtered,
and
concentrated
in
vacuo
to
afford
2-{3-[4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane (2.0 g).
A suspension of the above crude material (0.10 g, 0.30 mmol), 2-bromo-5-methylpyridine (61 mg,
0.36 mmol), Pd2(dba)3 (14 mg, 0.015 mmol) 2,2’-bis(diphenylphosphino)-1,1’-binaphthyl (BINAP)
(18 mg, 0.030 mmol) and t-BuONa (85 mg, 0.89 mmol) in 1,2-dimethoxyethane (3.0 mL) was
stirred at 60 ˚C for 9 h. The insoluble material was filtered, and the filtrate was concentrated in
vacuo. The residue was purified by silica gel column chromatography (10-40% EtOAc in hexanes)
and solidified with EtOAc and hexanes to afford 47a as a colorless solid (26 mg, 20% yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.38-1.47 (m, 2H), 1.50-1.64 (m, 4H), 1.65-1.81 (m, 4H),
1.96-2.06 (m, 2H), 2.18 (s, 3H), 2.22-2.35 (m, 1H), 3.03 (s, 3H), 3.32-3.39 (m, 2H), 3.41-3.48 (m,
2H), 4.01 (t, J = 6.37 Hz, 2H), 6.57-6.62 (m, 1H), 6.97-7.03 (m, 2H), 7.25-7.31 (m, 1H), 7.81-7.89
(m, 2H), 7.96-8.04 (m, 1H); 13C NMR (151 MHz, CDCl3) δ ppm 17.3, 26.9, 28.6, 34.1, 34.2, 36.2,
38.2, 39.4, 42.8, 43.1, 44.9, 68.5, 107.3, 115.0, 121.6, 129.6, 132.1, 138.3, 147.7, 158.4, 163.3;
HRMS ESI/APCI Dual m/z calcd for C24H32N2O3S 429.2206 [M+H]+ , found 429.2206.
2-{3-[4-(Methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane-7-carbonitrile (48a)
To a solution of 2-{3-[4-(methanesulfonyl)phenoxy]propyl}-7-azaspiro[3.5]nonane (0.10 g, 0.30
mmol) in CHCl3 (3.0 mL) were added water (1.0 mL) and NaHCO3 (50 mg, 0.59 mmol), then CNBr
(36 mg, 0.33 mmol) was added under ice cooling. After stirring overnight at room temperature, the
reaction was quenched with water and extracted with CHCl3. The organic layer was washed with
saturated NaHCO3 aqueous solution and brine, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column chromatography (40-70%
EtOAc in hexanes) to afford 48a as a colorless solid (111 mg, quantitative yield).
1H NMR (600 MHz, CDCl3) δ ppm 1.38-1.44 (m, 2H), 1.55-1.62 (m, 4H), 1.68-1.76 (m, 4H),
1.96-2.02 (m, 2H), 2.21-2.31 (m, 1H), 3.03 (s, 3H), 3.06-3.11 (m, 2H), 3.14-3.19 (m, 2H), 4.00 (t, J
= 6.4 Hz, 2H), 6.96-7.02 (m, 2H), 7.83-7.88 (m, 2H).
2-{3-[4-(Methanesulfonyl)phenoxy]propyl}-7-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]-7-azaspiro[3.5
]nonane (49a)
To
a
solution of 48a (0.11 g, 0.32 mmol) in THF (1.0 mL) were added

N'-hydroxy-2-methylpropanimidamide (39 mg, 0.38 mmol) and 1 M ZnCl2 diethyl ether solution
(0.38 mL, 0.38 mmol). After stirring at room temperature for 30 min, the precipitate was collected
by filtration. The resulting solid was dissolved in EtOH (2.0 mL) and concentrated HCl solution (2.0
mL). After stirring under reflux for 3 h, the mixture was quenched with saturated NaHCO3 aqueous
solution under ice cooling and extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (35-50% EtOAc in hexanes) and solidified with EtOAc and hexanes to
afford 49a as a colorless solid (53 mg, 37% yield).
1
Mp 124-125 ˚C; H NMR (600 MHz, CDCl3) δ ppm 1.28 (d, J = 7.0 Hz, 6H), 1.41-1.48 (m, 2H),
1.54-1.63 (m, 4H), 1.68-1.78 (m, 4H), 2.00-2.06 (m, 2H), 2.23-2.36 (m, 1H), 2.83-2.92 (m, 1H), 3.03
(s, 3H), 3.44-3.48 (m, 2H), 3.51-3.57 (m, 2H), 4.01 (t, J = 6.4 Hz, 2H), 6.97-7.02 (m, 2H), 7.83-7.88
(m, 2H); 13C NMR (151 MHz, CDCl3) δ ppm 20.5, 26.9, 27.1, 28.6, 33.5, 34.0, 35.7, 37.8, 38.9, 43.0,
43.2, 44.9, 68.4, 114.9, 129.6, 132.2, 163.3, 171.0, 175.8; HRMS ESI/APCI Dual m/z calcd for
C23H33N3O4S 448.2265 [M+H]+ , found 448.2249.
Propan-2-yl 2-(3-ethoxy-3-oxopropyl)-7-azaspiro[3.5]nonane-7-carboxylate (50)
To a suspension of 32 (5.7 g, 18 mmol) in EtOAc (88 mL) was added 4 M HCl in EtOAc (88 mL).
After stirring at room temperature for 1.5 h, the mixture was concentrated in vacuo. The resulting
residue was neutralized with saturated aqueous NaHCO3 solution and extracted with EtOAc and
with CHCl3. The organic layers were combined, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. To an ice cooled solution of the residue in CHCl3 (90 mL) were added Et3N
(4.9 mL, 35 mmol) and a solution of isopropyl chloroformate (2.4 mL, 21 mmol) in CHCl3 (24 mL),
and the mixture was stirred at room temperature for 4 h. The reaction was quenched with water and
extracted with CHCl3. The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column chromatography (15-25%
EtOAc in hexanes) to afford 50 as a colorless solid (5.5 g, quantitative yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.15-1.30 (m, 9H), 1.31-1.61 (m, 8H), 1.66-1.78 (m, 2H),
1.89-2.01 (m, 2H), 2.16-2.28 (m, 3H), 3.23-3.41 (m, 4H), 4.12 (q, J = 7.1 Hz, 2H), 4.82-4.96 (m,
1H); MS ESI/APCI Dual m/z 312 [M+H]+, 334 [M+Na]+.
Propan-2-yl 2-(3-hydroxypropyl)-7-azaspiro[3.5]nonane-7-carboxylate (51)
The title compound was synthesized according to the procedure described for compound 34 from
50 (91%yield).
1H NMR (300 MHz, CDCl3) δ ppm 1.23 (d, J = 6.4 Hz, 6H), 1.31-1.62 (m, 10H), 1.88-2.02 (m, 2H),
2.12-2.31 (m, 1H), 3.15-3.43 (m, 4H), 3.55-3.68 (m, 2H), 4.80-4.97 (m, 1H); MS ESI/APCI Dual
m/z 270 [M+H]+, 292 [M+Na]+.