Discovery and structure-activity relationships of spiroindolines as novel inducers of oligodendrocyte progenitor cell differentiation

Article abstract of DOI:10.1016/j.bmc.2020.115348

A novel series of spiroindoline derivatives was discovered for use as inducers of oligodendrocyte progenitor cell (OPC) differentiation, resulting from optimization of screening hit 1. Exploration of structure-activity relationships led to compound 18, which showed improved potency (rOPC EC₅₀ = 0.0032 μM). Furthermore, oral administration of compound 18 significantly decreased clinical severity in an experimental autoimmune encephalomyelitis (EAE) model.

Full text of DOI:10.1016/j.bmc.2020.115348

Journal Pre-proofs
Discovery and structure-activity relationships of spiroindolines as novel in-
ducers of oligodendrocyte progenitor cell differentiation
Katsushi Katayama, Yoshikazu Arai, Kenji Murata, Shoichi Saito, Tsutomu
Nagata, Kouhei Takashima, Ayako Yoshida, Makoto Masumura, Shuichi
Koda, Hiroyuki Okada, Tsuyoshi Muto
PII:
S0968-0896(20)30139-5
https://doi.org/10.1016/j.bmc.2020.115348
BMC 115348
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 November 2019
21 January 2020
27 January 2020
Please cite this article as: K. Katayama, Y. Arai, K. Murata, S. Saito, T. Nagata, K. Takashima, A. Yoshida, M.
Masumura, S. Koda, H. Okada, T. Muto, Discovery and structure-activity relationships of spiroindolines as novel
inducers of oligodendrocyte progenitor cell differentiation, Bioorganic & Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.bmc.2020.115348
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Discovery and structure-activity relationships of spiroindolines as novel inducers of
oligodendrocyte progenitor cell differentiation
Katsushi Katayama†, Yoshikazu Arai†, Kenji Murata†, Shoichi Saito†, Tsutomu Nagata†, Kouhei
Takashima†, Ayako Yoshida†, Makoto Masumura†, Shuichi Koda†, Hiroyuki Okada†, and Tsuyoshi Muto†
Asubio Pharma Co., Ltd., 6-4-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
 Corresponding author. e-mail: katayama.katsushi.ne@daiichisankyo.co.jp
† Present address: Shinagawa R&D Center, Daiichi-Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
ARTICLE INFO
ABSTRACT
Article history:
Received
A novel series of spiroindoline derivatives was discovered for use as inducers of oligodendrocyte
progenitor cell (OPC) differentiation, resulting from optimization of screening hit 1. Exploration of
Received in revised form
Accepted
structure-activity relationships led to compound 18, which showed improved potency (rOPC EC50 =
0.0032 μM). Furthermore, oral administration of compound 18 significantly decreased clinical
Available online
severity in an experimental autoimmune encephalomyelitis (EAE) model.
2019 Elsevier Ltd. All rights reserved.
Keywords:
multiple sclerosis (MS)
myelin
oligodendrocyte precursor cells
spiroindoline
———

1. Introduction
Fig. 1. Chemical structures of reported inducers of OPC differentiation.
O
Multiple sclerosis (MS) is one of the most common chronic,
autoimmune and inflammatory disorders of the central nervous
system (CNS) and affects 2.5 million people worldwide.1 The
onset of MS has been typically observed in individuals aged from
20 to 40 years, with a female to male ratio of 2:1. The pathogenesis
of MS is characterized by acute and chronic demyelination, which
results in a slowing or block of nerve conduction with subsequent
neurologic dysfunction.2 Thus, identification of multiple foci of
demyelination in the CNS is one of the major pathological findings
required to confirm MS diagnosis. Several immunomodulatory
treatments have been developed since the launch of the first drug
for MS in 1993, interferon-β-1b (IFN-β).3 Glatiramer acetate
(GLAT), DNA intercalating drugs such as mitoxantrone,4
spingosine-1-phosphate (S1P) receptor antagonists such as
fingolimod,5 and dimethyl fumarate6 induce anti-inflammatory
pathways via different mechanisms. Additionally, various
monoclonal antibodies, such as natalizumab and ocrelizumab, are
able to inhibit the infiltration of immune cells into the CNS. All of
these MS drugs target the immune system with mechanisms of
action involving general immunosuppression/immunomodulation.
Despite their effectiveness in reducing relapse rates and the
formation of new lesions, these drugs have very limited effects in
preventing the progression of disability. In contrast, progressive
phases of multiple sclerosis are associated with inhibited
differentiation of the progenitor cell population that generates the
mature oligodendrocytes required for remyelination and disease
remission.7 Thus, promoting oligodendrocyte progenitor cell
(OPC) differentiation, remyelination of the CNS, and subsequent
functional recovery of neurons have been proposed to be the new
direction for MS therapy.8 To date, recent studies have shown that
several small molecules, such as triiodothyronine (T3),9
clemastine,10 benztropine,11 sobetirome,12 PPAR agonist,13 8,9-
unsaturated sterols,14 and -opioid receptor agonist,15 have been
reported as inducers of OPC differentiation (Fig. 1).
N
N
H
1
rOPC
logD (pH 7.4) Metabolic stability (h/m)a hERGb
EC50; 0.16 μM
Emax; 246%
2.9 61%/0% 81%
a% original compound remaining after 30 min incubation.
bInhibition at 10 μM.
Fig. 2. Chemical structure, activity and ADMET profile of hit compound 1.
(EC50 = 0.12 μM, Emax =191%) and benztropine (EC50 = 0.27 μM,
Emax = 231%) as evaluated using our in-house functional assay.
Compound 1 has structurally unique spiroindoline core. Although
nitrogen-containing spirocyclic scaffolds are common structural
features in many pharmaceutical candidates and biologically
active alkaloid natural products,18 to the best of our knowledge,
there are only a few reports with showing the pharmacological
activities of spiroindolin-3-one derivatives, e.g. amoenamide C, a
antimicrobial and potent insecticidal activator.19 Furthermore,
compound 1 has high membrane permeability and moderate-to-
high human metabolic stability, but poor mouse metabolic stability
(Fig. 2). Herein, we report on a synthetic development from
compound 1 that aimed to produce compounds with high activity
and a good ADME profile.
2. Results and discussion
2.1. Chemistry
The first of this series, the synthesis of spiro derivatives 1−18,
is described in Scheme 1. Starting from 2-bromo-4-
isopropylaniline 25a−25f, Strecker reaction with N-benzyl-4-
piperidone afforded 26a−26f, respectively. The indolinone
scaffold was then constructed by radical cyclization with 26a−26f,
followed by acidic hydrolysis of the resulting 27a−27f, which
afforded 4 and 28b−28f, respectively.20 As for 28c, the TBS group
was also removed simultaneously under acidic hydrolysis.
Subsequent removal of the benzyl group in compound 4 by
hydrogenolysis afforded 29a, which was converted to the target
compounds 1 and 5−11 by reductive amination with corresponding
aldehydes. Alternatively, with intermediate amine 29a, amidation
with cyclopropylcarboxylic acid afforded 2, and sulfonylation
with cyclopropylsulfonyl chloride afforded 3. Removal of the
benzyl group in compound
Based on these findings, we aimed to create a novel MS
therapeutic agent that produces the differentiation-inducing action
of OPC. To identify novel oligodendrocyte differentiation
inducers with unique scaffolds, we performed an image-based
screen for myelin basic protein (MBP) expression,
a
differentiation marker for oligodendrocytes, using primary rat
OPCs derived from cerebral cortex.16 As a result, compound 1 was
identified as a screening hit with a differentiation-inducing action
against OPC. It was derived from rat neonate (hereafter referred to
as rOPC). Compound 1 showed submicromolar activity (EC50
=
0.16 μM, Emax17 = 246%) against rOPC, which was comparable
to the in vitro activity and efficacy of clemastine
O
H
Me
OH
Me
H
N
N
NH2
O
O
Me
I
I
HO
O
I
Cl
triiodothyronine (T3)
clemastine
benztropine
Cl
Cl
N
O
OH
HO
O
O
N
NH2
sobetirome
PPARδ agonist
O
N
Cl
Cl
F
O
H
N
N
N
N
N
H
HO
H
8,9-unsaturated sterols
-opioid receptor agonist


Scheme 1. Reagents and conditions: (a) N-benzyl-4-piperidone, TMSCN, AcOH, rt, 5 h, 49−98%; (b) AIBN, Bu3SnH, toluene, 120 °C for 15 h, 23−67%; (c) 1 N
Br NC
NH
O
R1
Br
(b)
(a)
(c)
R1
N
R1
R1
N
H
N
N
N
H
NH2
N
H
i
i
i
i
R1 = Pr, 25a
R1 = H, 25b
R1 = Pr, 26a
R1 = Pr, 27a
R1 = H, 27b
R1 = Pr, 4
R1 = H, 26b
R1 = H, 28b
R1 = OH, 28c
R1 = Cl, 28d
R1 = OTBS, 25c
R1 = Cl, 25d
R1 = OTBS, 26c
R1 = Cl, 26d
R1 = OTBS, 27c
R1 = Cl, 27d
t
t
t
t
R1 = Bu, 25e
R1 = Bu, 26e
R1 = Bu, 27e
R1 = Bu, 28e
R1 = CF3, 25f
R1 = CF3, 26f
R1 = CF3, 27f
R1 = CF3, 28f
O
O
(d) for 29a-29c, 29e, 29f
(e) for 29d
O
R1
R1
(d)
NH
N
N
H
N
H
i
R1 = Pr, 29a
R1 = H, 29b
R1 = OH, 29c
R1 = Cl, 29d
R1 = H, 12
R1 = OH, 13
R1 = Cl, 15
(j)
R1 = OMe, 14
R1 = OiPr, 16
(f) for 1, 5-11
(g) for 2
(h) for 3
t
t
R1 = Bu, 29e
R1 = Bu, 17
R1 = CF3, 29f
R1 = CF3, 18
O
t
R2 = Bu, 7
R2 = cyclopropylmethyl, 1
R2 = cyclopropylcarbonyl, 2
R2 = cyclopropylsulfonyl, 3
R2 = phenethyl, 5
N
R2
R2 = cyclobutylmethyl, 8
N
H
R2 = cyclopentylmethyl, 9
R2 = cyclohexylmethyl, 10
R2 = nPr, 6
R2 = tetrahydro-4-pyranylmethyl, 11

aq. HCl, 100 °C, 2 h, 41−100%; (d) H2, Pd/C, MeOH, rt, 24 h, 73−100%; (e) 1-chloroethyl chloroformate, Et3N, CHCl3, rt to 80 °C, 4 h, 94% (f) R-CHO,
NaBH(OAc)3, AcOH, CH2Cl2, 0 °C to rt, 1 h, 39−94%; (g) cyclopropanecarboxylic acid, EDCI, DMAP, DMF, 0 °C to rt, 18 h, quant.; (h) cyclopropanesulfonyl
chloride, Et3N, CH2Cl2, 0 °C to rt, 1 h, 93%; (i) 4-formyltetrahydropyran, NaBH(OAc)3, AcOH, CH2Cl2, 0 °C to rt, 1 h, 29−99%; (j) R-I, NaH, DMF, 0 °C, 30 min,
17−32%.
28b−28f and subsequent reductive amination with 4-formyltetrahydropyran afforded 12, 13, 15, 17 and 18, respectively. Further, O-
alkylation of 13 with methyl iodide or isopropyl iodide afforded compounds 14, 16, respectively.
The synthesis of compounds 19 and 20 is described in Scheme 2. Starting from commercially-available indanone 30, dialkylation
with N-benzylbischloroethylamine, followed by conversion of the benzyl group to a tetrahydropyranylmethyl group afforded
compound 19. Commercially-available 32 was converted to -hydroxyketone 34 by benzoin condensation using intermediate
cyanohydrin 33, followed by the construction of a benzofuran moiety by SNAr reaction under basic conditions, which afforded 35.
Compound 35 was converted to borate 36 by Miyaura reaction, followed by trifluoromethylation,21 which afforded 37. After
deprotection of the benzyl group of compound 37 by hydrogenolysis, reductive amination with 4-formyltetrahydropyran afforded
compound 20.
O
O
O
O
(a)
(b)
F3C
F3C
F3C
N
N
30
31
19
O
F
OTMS
CN
O
F
OH
(e)
(c)
(d)
Br
Br
Br
Br
H
N
F
32
33
34
O
O
O
(g)
(f) PinB
F3C
N
N
N
O
O
O
36
35
37
O
O
O
(i)
(h)
F3C
F3C
NH
N
O
O
38
20
Scheme 2. Reagents and conditions: (a) N-benzyl-N,N-di(2-chloroethyl)amine
hydrochloride, NaH, DMF, 0 °C to rt, 20 h, 9%; (b) H2, Pd/C, 4-formyltetrahydropyran, MeOH, rt, 24 h, 46%; (c) TMSCN, LiCl, CH2Cl2, 0 °C to rt, 16 h; (d)
33, LDA (1 M in THF solution), THF, −78 °C, 1.5 h followed by N-benzyl-4-piperidone, −78 °C, 3 h, 34% (2 steps); (e) tBuOK, THF, 80 °C, 2 h, 74%; (f)
(PinB)2, KOAc, Pd(dppf)Cl2, DMSO, rt to 110 °C, 1 h, μW, 34%; (g) Togni's reagent, copper(I) thiophene-2-carboxylate, LiOH·H2O, rt to 60 °C, 1 h, μW,
75%; (h) H2, Pd/C, MeOH, rt, 15 h, 89%; (i) 4-formyltetrahydropyran, NaBH(OAc)3, AcOH, CH2Cl2, 0 °C to rt, 1 h, 24%.
The synthesis of compounds 21−24 is described in Scheme 3. Compound 21 was prepared by N-methylation of compound 18.
The reduction of the carbonyl group of 18 with NaBH4 in MeOH afforded compound 22. Further, O-selective methylation of
compound 22 afforded compound 23. Compound 24 was obtained from 22 by reductive removal of the hydroxy group using Et3SiH
and TFA. Further, N-methylation of 24 afforded compound 25.
O
O
O
O
OH
O
(c)
(b)
F3C
F3C
F3C
N
N
N
N
H
N
H
N
H
18
22
23
(d)
(a)
O
O
O
O
(e)
F3C
F3C
F3C
N
N
N
N
N
N
H
24
25
21
Scheme 3. Reagents and conditions: (a) MeI, NaH, DMF, rt, 1 h, 90%; (b) NaBH4, MeOH, 0 °C to rt, 1 h, 73%; (c) MeI, NaH, DMF, 0 °C, 20 min, 34%; (d)
Et3SiH, TFA, CH2Cl2, rt, 1 h, 92%; (e) MeI, NaH, DMF, rt, 5 h, 73%
2.2. SAR study
In order to understand the SAR of this chemical series, the results of cyclopropylmethyl moiety modification of compound 1 are
summarized in Table 1. Replacement of the amine group with an amide (2) or sulfonamide group (3) decreased the activity. This
result indicates that basicity of the piperidine moiety is crucial to the activity of this series of compounds. Replacing the
cyclopropylmethyl group with a benzyl (4) or phenylethyl (5) group resulted in a slight loss in potency versus compound 1.
Compounds with a range of acyclic and cyclic alkyl substituents on the piperidine ring, compounds 6-10, were prepared. The potency
of the compound was highly dependent on the size of the substituent. For example, potency increased as the size of the acyclic group
increased from propyl (6) to iso-butyl (7). Likewise, for cycloalkyl substituents, potency increased with ring size from cyclopropyl to
cyclobutyl (8), while cyclopentyl (9) and cyclohexyl (10) resulted in a slight loss in potency compared to cyclobutyl (8). In addition,
compound 11, with a tetrahydropyranylmethyl group, showed significantly improved potency (EC50 = 0.0046 μM). This result
suggests that the introduction of an oxygen atom is important to the potency.

Table 1. SAR of the substituents on the piperidine ring.
O
N
R
N
H
Compound
R
rOPC EC50 (μM)
1
0.16
2
3
>10
>10
O
S
O
O
4
0.33
5
6
7
0.54
0.55
0.33
8
9
0.016
0.025
10
11
0.037
O
0.0046
Based on the results in Table 1, the substituent on the piperidine ring was fixed to the tetrahydropyranylmethyl group to elucidate
the SAR of the 5-position on the indolinone core (Table 2). Deletion of the isopropyl group (12) led to a significant loss of potency,
and replacement with a hydroxy group (13) provided a completely inactive compound, suggesting a beneficial lipophilic interaction
in this location. On the basis of this finding, we examined the introduction of lipophilic substituents in place of the phenyl ring.
Although the chloro group (14), methoxy group (15) and isopropoxy group (16) had over 10-fold less potency than 11, tert-butyl
group (17) showed similar potency to 11 (EC50= 0.0099 μM). Eventually, the trifluoromethyl group (18) increased potency compared
to that of 11 (EC50= 0.0032 μM).
Table 2. SAR of the substituents on piperidine ring.
O
O
R
N
N
H
Compound
R
iPr
rOPC EC50 (μM)
0.0046
0.55
11
12
13
14
15
16
17
18
H
OH
Cl
>10
0.32
OMe
OiPr
tBu
CF3
0.97
0.059
0.0099
0.0032

Next, we turned our attention to the spiro core (Table 3). Replacing the nitrogen atom of the indolinone core with a carbon atom
(19) or an oxygen atom (20) led to a significant decrease in potency. N-methylation of compound 18 led to a slight loss of potency
(21). Although reduction of the carbonyl group to a hydroxyl group (22) reduced potency compared to compound 18, methylation of
the hydroxy group (23) led to a robust potency (EC50 = 0.0057 μM). Removal of carbonyl group (24) and N-methylation of compound
24 (25) resulted in over 50-fold reductions in potency.
Table 3. SAR of the indolinone core.
O
O
F3C
N
N
H
Compound
R
rOPC EC50 (μM)
O
18
0.0032
N
H
O
19
20
21
22
0.11
O
0.48
O
O
0.0093
0.52
N
OH
N
H
O
23
0.0057
N
H
24
25
0.24
7.1
N
H
N
2.3. Physical Chemical Properties and Pharmacokinetics (PK)
On the basis of a favorable in vitro profile, compound 18 was selected for further evaluation. The metabolic stability of compound
18 in mice was improved as compared to hit 1 (Fig. 3). Compound 18 did not exhibit potent inhibition of cytochrome P450 enzymes
in human live microsomes (CYP3A4, CYP2C9 IC50 > 18 μM) except for CYP2D6 (70% @ 10 μM), and there was no CYP induction
issue.
O
O
F3C
N
N
H
18
rOPC
logD (pH 7.4) Metabolic stability (h/m)a
2.8 35%/68%
hERGb
94%
EC50; 0.0032 μM
Emax; 240%
a% original compound remaining after 30 min incubation.
bInhibition at 10 μM.
Fig. 3. Chemical structure, activity and ADMET profile of compound 18.

A mouse PK study was performed with compound 18, as shown in Table 5. Plasma concentrations were measured after a single
oral dose at 10 mg/kg or single intravenous dose at 1 mg/kg. Although compound 18 showed moderate-to-high plasma clearance (CL:
79.2 mL/min/kg), it displayed high oral bioavailability (F = 116%) and a high brain-to-plasma ratio (25.5 @ 0.5 h). These results
indicate that compound 18 is suitable for in vivo evaluation.
Table 5. Mouse PK profile of compound 18a
CLp (ivb; mL/min/kg)
79.2
31.5
T1/2 (po; h)
F (%)
6.4
Vd (iv; L/kg)
116
25.5 (0.5 h)
50.9 (4 h)
AUClast (poc; ng*h/mL)
Cmax (po; ng/mL)
1775
Kp (brain/plasma)
308
aAbbreviations: CL: plasma clearance; AUC: area under the concentration-time curve; Cmax: peak plasma concentration of drug after administration; T1/2
:
elimination half-life; F: bioavailability; p.o.: per oral; iv: intravenous.
bIV: 1 mg/kg (n = 2, 10% HP-β-CD dissolved in saline/DMSO = 95/5, v/v)
cPO: 10 mg/kg (n = 2, 0.5% HPC dissolved in water)
2.4. Pharmacological evaluation
We evaluated the in vivo therapeutic potential of compound 18 in a mouse EAE model, which is one of the most commonly used
experimental models for the human inflammatory demyelinating disease MS.22 EAE was induced by immunization with myelin
oligodendrocyte glycoprotein peptide (MOG35-55). Oral administration of compound 18 once daily at a dose of 50 mg/kg from 3 days
post-immunization to day 17 significantly reduced the development of clinical symptoms in terms of EAE scores (Fig. 4A, B).
Furthermore, administration of compound 18 delayed disease onset (Fig. 4C). These results indicate that the oral administration of
compound 18 protected against the development of clinical symptoms caused by immunization with myelin oligodendrocyte
glycoprotein (MOG) peptide. Thus, compound 18 might be a promising starting point for further development of optimal
remyelinating agents.

Fig. 4. (A) Effect of compound 18 on MOG-induced EAE model. Compound 2
(50 mg/kg, q.d.) or vehicle (0.5% methylcellulose) was orally administered to mice 3 d post-immunization (n = 13 or 14, mean and SEM): EAE clinical score.
(B) Cumulative EAE clinical score, from days 0 to 17. (C) We defined the onset day as the day when the EAE score reached 2 or more (n = 13 or 14), *
indicates p < 0.001 (t-test).
3. Conclusions
We investigated the structure-activity relationship of spiroindolines for activity in OPC differentiation induction. We found that
sidechain replacement of hit compound 1, from the cyclopropylmethyl group to the tetrahydropyranylmethyl group, led to a significant
improvement in potency without increasing lipophilicity. In this transformation, lipophilic efficiency,23 an important metric in drug
discovery programs, increased from 3.9 (for 1) to 5.6 (for 11). Further modification for substituents on the phenyl ring led to
representative compound 18, which showed high oral availability and brain exposure in mice. Furthermore, oral administration of 18
at 50 mg/kg daily resulted in a significant decrease in EAE clinical score in the mouse EAE model. Overall, this novel family of
compounds forms an attractive base for the development of effective new therapies for MS that complement established
immunosuppressive approaches. Further structural optimization of the compound family is currently underway. The results of later
work will be reported in due course.
4. Experimental section
4.1. Chemistry
Proton nuclear magnetic resonance spectra (1H NMR) were recorded on a Brucker Avance III (400 MHz) spectrometer in the indicated
solvent. Chemical shifts (δ) are reported in parts per million relative to the internal standard tetramethylsilane. Abbreviations of
multiplicity are as follows; s: singlet, d; doublet, t: triplet, q: quartet, m: multiplet, br: broad. Data are presented as follows: chemical
shift (multiplicity, integration, coupling constant). Fast atom bombardment (FAB) mass spectra were recorded on a JEOL JMS-700
mass spectrometer. Electrospray ionization (ESI) mass spectra were recorded on an Agilent G1956A MSD spectrometer system.
Chemical reagents and solvents were purchased from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Industries Ltd., Kanto
Kagaku, and Nacalai Tesque and were used without purification. Flash column chromatography was performed using Purif-Pack® SI
30 µm and Purif-Pack® NH 50 µm supplied by Shoko Scientific, or Chromatorex® SI 30 µm and Chromatorex® NH 60 µm supplied
by Fuji Silysia Chemical, or Merck silica gel 60 (230–400 mesh). Conditions of the preparative HPLC purification system were as
follows. Column: YMC-Pack Pro C18 ODS, 4.6 mm × 75 mm, mobile phase: solvent A = MeCN–H2O–trifluoroacetic acid (5:95:0.1),
solvent B = MeCN–trifluoroacetic acid (100:0.09), gradient: 0 to 90% solvent B in solvent A with 26%/min, UV detector: 254 nm,
flow rate: 1.0 mL/min.
4.1.1 N-Benzyl-4-[(2-bromo-4-isopropylphenyl)amino]piperidine
-4-carbonitrile (26a): General procedure A
A solution of 2-bromo-4-isopropylaniline 25a (36 g, 170 mmol) and N-benzyl-4-piperidone (31 mL, 170 mmol) in AcOH (130 mL)
was treated with TMSCN (22 mL, 180 mmol) at room temperature for 5 h. The reaction mixture was neutralized by 8 N aqueous
NaOH (380 mL) at 0 °C, and the mixture was extracted with AcOEt (500 mL). The organic phase was washed with saturated aqueous
NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography
(AcOEt/hexane; 10:90 to 40:60) to afford compound 26a (55 g, 79%) as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.38−7.28
(5H, m), 7.28−7.25 (1H, m), 7.14−7.06 (2H, m), 4.27 (1H, s), 3.56 (2H, s), 2.85−2.72 (3H, m), 2.51 (2H, t, J = 10.7 Hz), 2.36 (2H, d,
J = 13.3 Hz), 2.00 (2H, s), 1.20 (6H, d, J = 6.8 Hz). ESIMS-LR m/z 412, 414 [(M+H)+].
4.1.2 N-Benzyl-4-((2-bromophenyl)amino)piperidine-4-carbonitrile (26b)
Following general procedure A using compound 25b (0.64 mL, 5.8 mmol), N-benzyl-4-piperidone (0.82 mL, 4.6 mmol), TMSCN
(0.94 mL, 7.6 mmol), and AcOH (30 mL), the title compound, 26b (1.8 g, 81%), was obtained as a brown oil. 1H NMR (CDCl3, 400

MHz) δ: 7.48 (1H, dd, J = 8.1, 1.4 Hz), 7.36−7.26 (5H, m), 7.22 (1H, dd, J = 7.4, 1.1 Hz), 7.17 (1H, dd, J = 8.1, 1.4 Hz), 6.73 (1H,
td, J = 7.4, 1.2 Hz), 4.41 (1H, s), 3.56 (2H, s), 2.78−2.72 (2H, m), 2.57−2.48 (2H, m), 2.43−2.35 (2H, m), 2.06-1.97 (2H, m). ESIMS-
LR m/z 370, 372 [(M+H)+].
4.1.3 N-Benzyl-4-((2-bromo-4-(tert-butyldimethylsilyloxy)phenyl)
amino)piperidine-4-carbonitrile (26c)
Step 1. A solution of 4-amino-3-bromo-phenol (1.5 g, 8.0 mmol), imidazole (1.4 g, 21 mmol) in DMF (20 mL) was treated with
TBSCl (1.4 g, 9.3 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. The resulting mixture was partitioned
between AcOEt (100 mL) and H2O (50 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered,
and concentrated in vacuo. The residue was purified by flash chromatography (AcOEt/hexane; 10:90 to 30:70) to afford compound
2-bromo-4-(tert-butyldimethylsilyloxy)aniline 25c (1.4 g, 60%) as a brown oil.
Step 2. Following general procedure A using compound 25c (1.4 g, 4.6 mmol), N-benzyl-4-piperidone (0.82 mL, 4.6 mmol), TMSCN
(0.63 mL, 5.1 mmol), and AcOH (10 mL), the title compound, 26c (2.3 g, 98%), was obtained as a brown oil. 1H NMR (CDCl3, 400
MHz) δ: 7.17−7.08 (5H, m), 6.92 (1H, d, J = 8.8 Hz), 6.85 (1H, d, J = 2.8 Hz), 6.56 (1H, dd, J = 8.8, 2.8 Hz), 3.42 (2H, s), 2.68−2.58
(2H, m), 2.33 (2H, t, J = 10.0 Hz), 2.13 (2H, dd, J = 11.0, 2.5 Hz), 1.88−1.77 (2H, m), 0.80−0.77 (9H, m), 0.01-0.00 (6H, m). ESIMS-
LR m/z 500, 502 [(M+H)+].
4.1.4 N-Benzyl-4-((2-bromo-4-chlorophenyl)amino)piperidine-4-carbonitrile (26d)
Following general procedure A using 2-bromo-4-chloroaniline 25d (3.0 g, 15 mmol), N-benzyl-4-piperidone (2.9 mL, 16 mmol),
1
TMSCN (2.3 mL, 19 mmol), and AcOH (20 mL), the title compound, 26d (3.7 g, 63%), was obtained as a brown amorphous. H
NMR (CDCl3, 400 MHz) δ: 7.49 (1H, d, J = 2.5 Hz), 7.35−7.27 (5H, m), 7.21 (1H, dd, J = 8.8, 2.5 Hz), 7.10 (1H, d, J = 8.8 Hz), 4.37
(1H, s), 3.56 (2H, s), 2.80−2.69 (2H, m), 2.55−2.47 (2H, m), 2.37 (2H, dd, J = 10.9, 2.9 Hz), 1.99 (2H, t, J = 9.8 Hz). ESIMS-LR m/z
404, 406 [(M+H)+].
4.1.5 N-Benzyl-4-((2-bromo-4-tert-butylphenyl)amino)piperidine
-4-carbonitrile (26e)
Following general procedure A using 2-bromo-4-tert-butylaniline 25e (1.8 g, 7.8 mmol), N-benzyl-4-piperidone (2.3 mL, 13 mmol),
1
TMSCN (1.8 mL, 13 mmol), and AcOH (10 mL), the title compound, 26e (3.1 g, 93%), was obtained as a brown amorphous. H
NMR (DMSO-d6, 400 MHz) δ: 7.50 (1H, d, J = 2.3 Hz), 7.36−7.29 (5H, m), 7.27−7.23 (1H, m), 7.08 (1H, d, J = 8.8 Hz), 4.96 (1H,
s), 3.52 (2H, s), 2.74−2.64 (2H, m), 2.39−2.28 (4H, m), 2.03−1.92 (2H, m), 1.24 (9H, s). ESIMS-LR m/z 426, 428 [(M+H)+].
4.1.6 N-Benzyl-4-((2-bromo-3-trifluoromethylphenyl)amino)
piperidine-4-carbonitrile (26f)
Following general procedure A using 3-Amino-2-bromobenzotrifluoride 25f (500 mg, 2.1 mmol), N-benzyl-4-piperidone (0.39 mL,
2.2 mmol), TMSCN (0.61 mL, 4.6 mmol), and AcOH (10 mL), the title compound, 26f (440 mg, 49%), was obtained as a colorless
solid. 1H NMR (CDCl3, 400 MHz): δ: 7.36−7.30 (7H, m), 7.19 (1H, dd, J = 6.3, 3.0 Hz), 4.83 (1H, s), 3.57 (2H, s), 2.83−2.70 (2H,
m), 2.54 (2H, t, J = 9.7 Hz), 2.41 (2H, d, J = 9.7 Hz), 2.08−2.00 (2H, m). ESIMS-LR m/z 438, 440 [(M+H)+].
4.1.7 N-Benzyl-5-isopropylspiro[indoline-2,4'-piperidin]-3-imine (27a) : General procedure B
A suspension of compound 26a (2.0 g, 4.9 mmol) and n-Bu3SnH (1.4 mL, 5.3 mmol) in toluene (40 mL) was treated with AIBN
(0.080 g, 0.049 mmol) at room temperature, and the mixture was stirred at 120 °C for 15 h. The reaction was quenched by saturated
aqueous KF (15 mL) at room temperature. The whole mixture was partitioned between AcOEt (100 mL) and H2O (50 mL), and the
organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified
1
by silica gel column chromatography (MeOH/CHCl3; 0:100 to 14:86) to afford compound 27a (1.1 g, 67%) as a yellow solid. H
NMR (CDCl3, 400 MHz) δ: 7.48 (1H, d, J = 1.5 Hz), 7.39−7.30 (5H, m), 7.28−7.25 (1H, m), 6.83 (1H, d, J = 8.5 Hz), 4.79 (1H, s),
3.59 (2H, s), 2.99 (2H, d, J = 10.8 Hz), 2.90-2.83 (1H, m), 2.20 (2H, t, J = 12.0 Hz), 2.09 (2H, td, J = 12.7, 3.9 Hz), 1.42 (2H, d, J =
12.0 Hz), 1.22 (6H, d, J = 6.8 Hz). ESIMS-LR m/z 334 [(M+H)+].
4.1.8 N-Benzylspiro[indoline-2,4'-piperidin]-3-imine (27b)
Following general procedure B using compound 26b (1.7 g, 4.6 mmol), n-Bu3SnH (1.9 mL, 6.9 mmol), AIBN (0.15 g, 0.92 mmol),
and toluene (40 mL), the title compound, 27b (720 mg, 54%), was obtained as a brown amorphous. 1H NMR (CDCl3, 400 MHz) δ:
7.56 (1H, d, J = 7.5 Hz), 7.41−7.27 (6H, m), 6.85-6.78 (2H, m), 3.64 (2H, s), 3.49 (1H, s), 3.04 (2H, d, J = 10.8 Hz), 2.38−2.21 (2H,
m), 2.03 (2H, td, J = 12.9, 4.0 Hz), 1.60 (2H, d, J = 12.0 Hz). ESIMS-LR m/z 292 [(M+H)+].
4.1.9 N-Benzyl-5-(tert-butyldimethylsilyloxy)spiro[indoline-2,4'-piperidin]-3-imine (27c)
Following general procedure B using compound 26c (600 mg, 1.2 mmol), n-Bu3SnH (0.49 mL, 1.8 mmol), AIBN (40 mg, 0.24 mmol),
and toluene (20 mL) the title compound, 27c (160 mg, 31%), was obtained as a pale yellow amorphous. 1H NMR (CDCl3, 400 MHz)
δ: 7.22−7.09 (5H, m), 6.83 (1H, s), 6.71 (1H, dd, J = 8.5, 2.5 Hz), 6.54 (1H, d, J = 8.5 Hz), 4.16 (1H, s), 3.43 (2H, s), 2.91−2.72 (2H,
m), 2.16−1.96 (2H, m), 1.82 (2H, td, J = 12.9, 3.9 Hz), 1.47−1.28 (2H, m), 0.80 (9H, s), 0.00 (6H, s). ESIMS-LR m/z 422 [(M+H)+].
4.1.10 N-Benzyl-5-chlorospiro[indoline-2,4'-piperidin]-3-imine (27d)

Following general procedure B using compound 26d (3.9 g, 9.6 mmol), n-Bu3SnH (3.8 mL, 14 mmol), AIBN (0.32 g, 1.9 mmol), and
toluene (30 mL), the title compound, 27d (720 mg, 33%), was obtained as a yellow amorphous. 1H NMR (DMSO-d6, 400 MHz) δ:
9.92 (1H, br s), 7.53 (1H, d, J = 8.0 Hz), 7.37−7.20 (5H, m), 6.71 (1H, s), 6.62 (1H, d, J = 8.0 Hz), 3.54 (2H, s), 2.81 (2H, d, J = 11.5
Hz), 2.28 (2H, t, J = 11.5 Hz), 1.89−1.80 (2H, m), 1.33 (2H, d, J = 12.8 Hz). ESIMS-LR m/z 326 [(M+H)+].
4.1.11 N-Benzyl-5-(tert-butyl)spiro[indoline-2,4'-piperidin]-3-imine (27e)
Following general procedure B using compound 26e (3.1 g, 7.3 mmol), n-Bu3SnH (2.1 mL, 8.0 mmol), AIBN (0.11 g, 0.68 mmol),
and toluene (30 mL), the title compound, 27e (1.3 g, 52%), was obtained as a yellow amorphous. 1H NMR (DMSO-d6, 400 MHz) δ:
7.55 (1H, s), 7.35−7.30 (5H, m), 7.28−7.23 (1H, m), 6.70 (1H, s), 6.68 (1H, d, J = 8.5 Hz), 3.53 (2H, s), 2.79 (2H, d, J = 11.4 Hz),
2.30 (2H, t, J = 11.4 Hz), 1.82 (2H, td, J = 12.5, 3.9 Hz), 1.29 (2H, d, J = 12.5 Hz), 1.24 (9H, s). ESIMS-LR m/z 348 [(M+H)+].
4.1.12 N-Benzyl-5-(trifluoromethyl)spiro[indoline-2,4'-piperidin]-3-imine (27f)
Following general procedure B using compound 26f (4.4 g, 10 mmol), n-Bu3SnH (4.2 mL, 12 mmol), AIBN (0.23 g, 1.4 mmol), and
toluene (30 mL), the title compound, 27f (1.9 g, 53%), was obtained as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.88 (1H, s),
7.65 (1H, dd, J = 8.5, 1.8 Hz), 7.40−7.27 (5H, m), 6.92 (1H, d, J = 8.5 Hz), 5.30 (1H, s), 3.61 (2H, s), 3.02 (2H, d, J = 11.4 Hz), 2.24
(2H, t, J = 11.4 Hz), 2.09 (2H, dt, J = 17.9, 6.3 Hz), 1.60−1.52 (2H, m). ESIMS-LR m/z 360 [(M+H)+].
4.1.13 N-Benzyl-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (4): General procedure C
A solution of compound 27a (0.88 g, 2.6 mmol) in MeOH (5.6 mL) was treated with 1 N aqueous HCl (5.6 mL) at room temperature,
and the mixture was stirred at 100 °C for 2 h, then cooled to room temperature. The resulting mixture was partitioned between CHCl3
(100 mL × 3) and saturated aqueous NaHCO3 (50 mL), and the organic phase was washed with saturated aqueous NaCl, dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was crystalized from Et2O to afford compound 4 (0.69 g, 77%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz) δ: 7.48 (1H, d, J = 1.3 Hz), 7.38−7.29 (5H, m), 7.29−7.25 (1H, m), 6.83 (1H, d, J = 8.5 Hz), 4.79
(1H, s), 3.59 (2H, s), 2.99 (2H, d, J = 11.9 Hz), 2.89−2.83 (1H, m), 2.20 (2H, t, J = 11.9 Hz), 2.09 (2H, td, J = 12.6, 3.8 Hz), 1.42
(1H, d, J = 11.9 Hz), 1.22 (6H, d, J = 7.0 Hz). ESIMS-LR m/z 335 [(M+H)+]; ESIMS-HR calcd for C22H27N2O (M+H)+ 335.2118,
found 335.2129.
4.1.14 N-Benzylspiro[indoline-2,4'-piperidin]-3-one (28b)
Following general procedure C using compound 27b (670 mg, 2.3 mmol), 1 N aqueous HCl (5.0 mL), and MeOH (5.0 mL), the title
compound, 28b (290 mg, 43%), was obtained as a pale yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.61 (1H, dd, J = 7.9, 0.6 Hz),
7.48−7.42 (1H, m), 7.38−7.29 (4H, m), 7.29−7.25 (1H, m), 6.88 (1H, d, J = 7.9 Hz), 6.85−6.81 (1H, m), 4.93 (1H, s), 3.59 (2H, s),
2.99 (2H, dt, J = 12.0, 3.6 Hz), 2.20 (2H, td, J = 12.0, 2.2 Hz), 2.09 (2H, td, J = 12.6, 3.6 Hz), 1.43 (2H, d, J = 11.5 Hz). ESIMS-LR
m/z 293 [(M+H)+].
4.1.15 N-Benzyl-5-hydroxyspiro[indoline-2,4'-piperidin]-3-one (28c)
Following general procedure C using compound 27c (2.5 g, 5.9 mmol), 1 N aqueous HCl (7.0 mL), and MeOH (7.0 mL), the title
compound, 28c (1.5 g, 82%), was obtained as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.38−7.30 (4H, m), 7.29−7.26 (1H, m),
7.09 (1H, dd, J = 8.6, 2.6 Hz), 7.04−7.01 (1H, m), 6.83 (1H, d, J = 8.6 Hz), 4.61 (1H, s), 3.59 (2H, s), 2.99 (2H, d, J = 11.9 Hz), 2.20
(2H, t, J = 11.9 Hz), 2.07 (2H, td, J = 12.6, 3.9 Hz), 1.42 (2H, d, J = 12.6 Hz). ESIMS-LR m/z 309 [(M+H)+].
4.1.16 N-Benzyl-5-chlorospiro[indoline-2,4'-piperidin]-3-one (28d)
Following general procedure C using compound 27d (650 mg, 2.0 mmol), 1 N aqueous HCl (5.0 mL), and MeOH (5.0 mL), the title
compound, 28d (270 mg, 41%), was obtained as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.53 (1H, d, J = 8.1 Hz), 7.38−7.30
(4H, m), 7.29−7.26 (1H, m), 6.88−6.85 (1H, m), 6.78 (1H, dd, J = 8.1, 1.6 Hz), 5.03 (1H, s), 3.58 (2H, s), 2.99 (2H, dt, J = 11.9, 3.6
Hz), 2.18 (2H, td, J = 11.9, 2.3 Hz), 2.07 (2H, td, J = 12.6, 3.6 Hz), 1.43 (2H, d, J = 12.6 Hz). ESIMS-LR m/z 327 [(M+H)+].
4.1.17 N-Benzyl-5-(tert-butyl)spiro[indoline-2,4'-piperidin]-3-one (28e)
Following general procedure C using compound 27e (3.5 g, 10 mmol), 1 N aqueous HCl (10 mL), and MeOH (10 mL), the title
compound, 28e (2.9 g, 83%), was obtained as a yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.57 (1H, dd, J = 8.8, 2.1 Hz), 7.50
(1H, s), 7.37−7.30 (5H, m), 7.29−7.23 (1H, m), 6.86 (1H, dd, J = 8.8, 0.5 Hz), 3.54 (2H, s), 2.85−2.79 (2H, m), 2.29 (2H, td, J = 12.0,
2.3 Hz), 1.76 (2H, td, J = 12.7, 4.1 Hz), 1.28−1.23 (2H, m), 1.24 (9H, s). ESIMS-LR m/z 349 [(M+H)+].
4.1.18 N-Benzyl-5-(trifluoromethyl)spiro[indoline-2,4'-piperidin]-3-one (28f)
Following general procedure C using compound 27f (87 mg, 0.24 mmol), 1 N aqueous HCl (1.0 mL), and MeOH (1.0 mL), the title
1
compound, 28f (68 mg, 78%), was obtained as a pale yellow solid. H NMR (CDCl3, 400 MHz) δ: 7.70 (1H, dd, J = 8.1, 0.8 Hz),
7.38-7.27 (5H, m), 7.12 (1H, d, J = 0.8 Hz), 7.04 (1H, dd, J = 8.1, 0.8 Hz), 5.17 (1H, s), 3.59 (2H, s), 3.00 (2H, dt, J = 12.0, 3.6 Hz),
2.20 (2H, td, J = 12.0, 2.3 Hz), 2.09 (2H, td, J = 12.6, 3.6 Hz), 1.46 (2H, d, J = 12.0 Hz). ESIMS-LR m/z 361 [(M+H)+]; ESIMS-HR
calcd for C20H20F3N2O (M+H)+ 361.1522, found 361.1521.
4.1.19 5-Isopropylspiro[indoline-2,4'-piperidin]-3-one (29a): General procedure D

A solution of compound 4 (3.3 g, 1.0 mmol) in MeOH (100 mL) was treated with 10% Pd(OH)2/C (0.67 g) and the mixture was
vigorously stirred under H2 atmosphere (balloon pressure) at room temperature for 24 h. The catalyst was filtered off through a Celite
pad and the filtrate was concentrated in vacuo to afford compound 29a (2.5 g, quant.) as a yellow solid. This material was used in the
next reaction without further purification. 1H NMR (DMSO-d6, 400 MHz) δ: 7.55 (1H, s), 7.39 (1H, dd, J = 8.4, 1.9 Hz), 7.24 (1H, d,
J = 1.3 Hz), 6.84 (1H, d, J = 8.5 Hz), 3.17 (1H, s), 3.06−2.97 (2H, m), 2.82 (3H, td, J = 9.7, 4.6 Hz), 1.66 (2H, td, J = 12.7, 4.6 Hz),
1.24−1.17 (2H, m), 1.16 (6H, d, J = 7.0 Hz). ESIMS-LR m/z 245 [(M+H)+].
4.1.20 spiro[indoline-2,4'-piperidin]-3-one (29b)
Following general procedure D using compound 28b (290 mg, 0.99 mmol), 10% Pd(OH)2/C (36 mg) and MeOH (1.75 mL), the title
compound, 29b (180 mg, 90%), was obtained as a yellow solid. ESIMS-LR m/z 203 [(M+H)+].
4.1.21 5-hydroxyspiro[indoline-2,4'-piperidin]-3-one (29c)
Following general procedure D using compound 28c (1.5 g, 0.99 mmol), 10% Pd(OH)2/C (300 mg) and MeOH (20 mL), the title
compound, 29c (810 mg, 76%), was obtained as a yellow solid. ESIMS-LR m/z 219 [(M+H)+].
4.1.22 5-chlorospiro[indoline-2,4'-piperidin]-3-one (29d)
A solution of compound 28d (50 mg, 0.15 mmol), triethylamine (0.064 mL, 0.46 mmol) in CHCl3 (1.5 mL) was treated with 1-
chloroethyl chloroformate (0.033 mL, 0.30 mmol) at room temperature, and the mixture was stirred at 80 °C for 4 h. The reaction was
quenched by MeOH (2.0 mL) at 80 °C for 1 h. The whole mixture was concentrated in vacuo. The residue was purified by silica gel
column chromatography (NH-SiO2, MeOH/CHCl3; 1:99 to 10:90) to afford compound 29d (34 mg, 94%) as a pale yellow solid.
ESIMS-LR m/z 237 [(M+H)+].
4.1.23 5-(tert-butyl)spiro[indoline-2,4'-piperidin]-3-one (29e)
Following general procedure D using compound 28e (2.9 g, 8.4 mmol), 10% Pd(OH)2/C (0.61 g) and MeOH (200 mL), the title
compound, 29e (1.8 g, 80%), was obtained as a yellow solid. ESIMS-LR m/z 259 [(M+H)+].
4.1.24 5-(trifluoromethyl)spiro[indoline-2,4'-piperidin]-3-one (29f)
Following general procedure D using compound 28f (10 mg, 0.028 mmol), 10% Pd(OH)2/C (2.0 mg) and MeOH (0.5 mL), the title
compound, 29f (5.5 mg, 73%), was obtained as a yellow solid. ESIMS-LR m/z 271 [(M+H)+].
4.1.25 N-(Cyclopropylmethyl)-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (1): General procedure E
A solution of compound 29a (60 mg, 0.25 mmol), cyclopropanecarboxaldehyde (24 μL, 0.37 mmol) and AcOH (56 µL, 0.98 mmol)
in CH2Cl2 (2.5 mL) was treated with NaBH(OAc)3 (420 mg, 2.0 mmol) at 0 °C, and the mixture was stirred at room temperature for
1 h. The resulting mixture was partitioned between AcOEt (50 mL) and saturated aqueous NaHCO3 (30 mL), and the organic phase
was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (MeOH/CHCl3; 1:99 to 13:87) to afford compound 1 (61 mg, 83%) as a yellow solid. 1H NMR (CDCl3, 400
MHz) δ: 7.47 (1H, s), 7.36 (1H, dd, J = 8.5, 1.8 Hz), 6.83 (1H, t, J = 8.5 Hz), 4.77 (1H, br s), 3.17 (2H, dt, J = 12.1, 3.8 Hz), 2.92−2.81
(1H, m), 2.35 (2H, d, J = 6.4 Hz), 2.26 (2H, t, J = 11.4 Hz), 2.09 (2H, dt, J = 18.1, 6.4 Hz), 1.50 (2H, d, J = 12.8 Hz), 1.23 (6H, d, J
= 7.0 Hz), 0.98−0.88 (1H, m), 0.55 (2H, ddd, J = 9.2, 4.6, 3.4 Hz), 0.17−0.11 (2H, m). ESIMS-LR m/z 299 [(M+H)+]; ESIMS-HR
calcd for C19H27N2O (M+H)+ 299.2118, found 299.2116.
4.1.26 N-(Cyclopropanecarbonyl)-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (2)
A solution of compound 29a (50 mg, 0.21 mmol), EDCI (78 mg, 0.41 mmol) and DMAP (13 mg, 0.10 mmol) in DMF (2.1 mL) was
treated with cyclopropanecarboxylic acid (0.016 mL, 0.20 mmol) at 0 °C, and the mixture was stirred at room temperature for 18 h.
The resulting mixture was partitioned between AcOEt (50 mL) and H2O (30 mL), and the organic phase was washed with saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography
(MeOH/CHCl3; 1:99 to 10:90) to afford compound 2 (65 mg, quant.) as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.50−7.45 (1H,
m), 7.39 (1H, dd, J = 8.5, 1.9 Hz), 6.86 (1H, d, J = 8.5 Hz), 4.98 (1H, s), 4.55−4.22 (2H, m), 3.51 (1H, br s), 3.19 (1H, br s), 2.92−2.81
(1H, m), 1.99−1.96 (2H, m), 1.79 (1H, ddd, J = 13.5, 7.1, 3.8 Hz), 1.55−1.52 (2H, m), 1.23 (6H, d, J = 6.8 Hz), 1.03−0.97 (2H, m),
0.79 (2H, dd, J = 8.0, 2.8 Hz). ESIMS-LR m/z 313 [(M+H)+]; ESIMS-HR calcd for C19H25N2O2 (M+H)+ 313.1911, found 313.1913.
4.1.27 N-(Cyclopropylsulfonyl)-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (3)
A solution of compound 29a (100 mg, 0.41 mmol), Et3N (110 µL, 0.82 mmol) in CH2Cl2 (2.5 mL) was treated with
cyclopropanesulfonyl chloride (54 µL, 0.53 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The resulting
mixture was partitioned between AcOEt (50 mL) and saturated aqueous NaHCO3 (30 mL), and the organic phase was washed with
saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (Diol, MeOH/CHCl3; 0:100) to afford compound 3 (130 mg, 93%) as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ:
7.47 (1H, d, J = 1.9 Hz), 7.39 (1H, dd, J = 8.4, 1.9 Hz), 6.84 (1H, d, J = 8.4 Hz), 4.59 (1H, s), 3.92−3.86 (2H, m), 3.33−3.27 (2H, m),
2.91−2.84 (1H, m), 2.36 (1H, tt, J = 8.8, 3.5 Hz), 2.08−2.01 (2H, m), 1.68 (2H, ddd, J = 13.8, 6.1, 3.8 Hz), 1.23 (6H, d, J = 6.8 Hz),
1.22−1.18 (2H, m), 1.08−1.02 (2H, m). ESIMS-LR m/z 349 [(M+H)+]; ESIMS-HR calcd for C18H25N2O3S (M+H)+ 349.1580, found
349.1587.

4.1.28 5-Isopropyl-N-phenethylspiro[indoline-2,4'-piperidin]-3-one (5)
Following general procedure E using compound 29a (60 mg, 0.25 mmol), phenylacetaldehyde (40 µL, 0.343 mmol), AcOH (56 µL,
0.98 mmol), NaBH(OAc)3 (420 mg, 2.0 mmol), and CH2Cl2 (2.5 mL), the title compound, 5 (47 mg, 55%), was obtained as a yellow
solid. 1H NMR (CDCl3, 400 MHz) δ: 7.48 (1H, d, J = 1.9 Hz), 7.37 (1H, dd, J = 8.5, 1.9 Hz), 7.33−7.27 (2H, m), 7.25−7.18 (3H, m),
6.84 (1H, d, J = 8.4 Hz), 4.77 (1H, s), 3.10 (2H, d, J = 11.8 Hz), 2.91−2.82 (3H, m), 2.71−2.64 (2H, m), 2.26 (2H, t, J = 11.8 Hz),
2.09 (2H, td, J = 12.7, 4.0 Hz), 1.48 (2H, d, J = 12.7 Hz), 1.23 (6H, d, J = 6.8 Hz). ESIMS-LR m/z 349 [(M+H)+]; ESIMS-HR calcd
for C23H29N2O (M+H)+ 349.2274, found 349.2289.
4.1.29 5-Isopropyl-N-propylspiro[indoline-2,4'-piperidin]-3-one (6)
Following general procedure E using compound 29a (30 mg, 0.12 mmol), propionaldehyde (8.8 µL, 0.12 mmol), AcOH (28 µL, 0.49
mmol), NaBH(OAc)3 (210 mg, 0.99 mmol), and CH2Cl2 (1.2 mL), the title compound, 6 (22 mg, 62%), was obtained as a yellow
solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.47 (1H, s), 7.38 (1H, dd, J = 8.7, 1.8 Hz), 7.23 (1H, d, J = 1.8 Hz), 6.85 (1H, d, J = 8.7
Hz), 2.90−2.77 (3H, m), 2.32 (2H, t, J = 7.3 Hz), 2.24 (2H, t, J = 11.5 Hz), 1.74 (2H, td, J = 12.8, 4.0 Hz), 1.48 (2H, td, J = 14.8, 7.3
Hz), 1.28−1.20 (2H, m), 1.16 (6H, d, J = 7.0 Hz), 0.88 (3H, t, J = 7.4 Hz). ESIMS-LR m/z 287 [(M+H)+]; ESIMS-HR calcd for
C18H27N2O (M+H)+ 287.2118, found 287.2120.
4.1.30 N-Isobutyl-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (7)
Following general procedure E using compound 29a (30 mg, 0.12 mmol), isobutylaldehyde (11 µL, 0.12 mmol), AcOH (28 µL, 0.49
mmol), NaBH(OAc)3 (210 mg, 0.99 mmol), and CH2Cl2 (1.2 mL), the title compound, 7 (27 mg, 73%), was obtained as a yellow
solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.46 (1H, s), 7.38 (1H, dd, J = 8.3, 1.8 Hz), 7.23 (1H, s), 6.85 (1H, d, J = 8.3 Hz), 2.86−2.75
(3H, m), 2.20 (2H, t, J = 11.2 Hz), 2.10 (2H, d, J = 7.3 Hz), 1.85-1.69 (3H, m), 1.22 (2H, d, J = 9.5 Hz), 1.16 (6H, d, J = 7.0 Hz), 0.88
(6H, d, J = 6.5 Hz). ESIMS-LR m/z 301 [(M+H)+]; ESIMS-HR calcd for C19H29N2O (M+H)+ 301.2274, found 301.2264.
4.1.31 N-(Cyclobutylmethyl)-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (8)
Following general procedure E using compound 29a (30 mg, 0.12 mmol), cyclobutanecarbaldehyde (10 mg, 0.12 mmol), AcOH (28
µL, 0.49 mmol), NaBH(OAc)3 (210 mg, 0.99 mmol), and CH2Cl2 (1.2 mL), the title compound, 8 (15 mg, 39%), was obtained as a
yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.43−7.38 (1H, m), 7.34−7.32 (1H, m), 6.88 (1H, d, J = 8.5 Hz), 3.04−2.94 (2H, m),
2.87−2.79 (1H, m), 2.69−2.62 (1H, m), 2.57 (2H, d, J = 7.0 Hz), 2.41 (2H, td, J = 12.2, 2.4 Hz), 2.18−2.07 (2H, m), 2.03−1.88 (3H,
m), 1.88−1.71 (3H, m), 1.39 (2H, d, J = 12.3 Hz), 1.21 (6H, t, J = 6.9 Hz). ESIMS-LR m/z 313 [(M+H)+]; ESIMS-HR calcd for
C20H29N2O (M+H)+ 313.2274, found 313.2273.
4.1.32 N-(Cyclopentylmethyl)-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (9)
Following general procedure E using compound 29a (30 mg, 0.12 mmol), cyclopentanecarbaldehyde (13 µL, 0.12 mmol), AcOH (28
µL, 0.49 mmol), NaBH(OAc)3 (210 mg, 0.99 mmol), and CH2Cl2 (1.2 mL), the title compound, 9 (35 mg, 87%), was obtained as a
yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.46 (1H, s), 7.38 (1H, dd, J = 8.5, 1.8 Hz), 7.23 (1H, s), 6.85 (1H, d, J = 8.5 Hz),
2.91−2.75 (3H, m), 2.30−2.15 (4H, m), 2.09 (1H, td, J = 15.0, 7.4 Hz), 1.80−1.63 (4H, m), 1.60−1.45 (4H, m), 1.27−1.19 (4H, m),
1.16 (6H, d, J = 7.0 Hz). ESIMS-LR m/z 327 [(M+H)+]; ESIMS-HR calcd for C21H31N2O (M+H)+ 327.2431, found 327.2432.
4.1.33 N-(Cyclohexylmethyl)-5-isopropylspiro[indoline-2,4'-piperidin]-3-one (10)
Following general procedure E using compound 29a (150 mg, 0.61 mmol), cyclohexanecarbaldehyde (0.074 mL, 0.62 mmol), AcOH
(0.14 mL, 2.4 mmol), NaBH(OAc)3 (1.0 g, 4.7 mmol), and CH2Cl2 (6.1 mL), the title compound, 10 (200 mg, 94%), was obtained as
a yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.45 (1H, s), 7.38 (1H, dd, J = 8.5, 1.9 Hz), 7.23 (1H, d, J = 1.5 Hz), 6.84 (1H, d,
J = 8.5 Hz), 2.85−2.75 (3H, m), 2.23−2.12 (3H, m), 1.75 (3H, dd, J = 12.8, 3.8 Hz), 1.68 (4H, td, J = 8.2, 6.1 Hz), 1.54−1.46 (1H, m),
1.22 (4H, d, J = 13.1 Hz), 1.16 (6H, d, J = 6.8 Hz), 1.15-1.11 (2H, m), 0.85 (2H, dd, J = 21.1, 11.5 Hz). ESIMS-LR m/z 341 [(M+H)+];
ESIMS-HR calcd for C22H33N2O (M+H)+ 341.2587, found 341.2575.
4.1.34 5-Isopropyl-N-((tetrahydro-2H-pyran-4-yl)methyl)spiro[indoline-2,4'-piperidin]-3-one (11)
Following general procedure E using compound 29a (50 mg, 0.21 mmol), 4-formyltetrahydropyran (47 mg, 0.41 mmol), AcOH (0.047
mL, 0.82 mmol), NaBH(OAc)3 (350 mg, 1.6 mmol), and CH2Cl2 (2.1 mL) the title compound, 11 (57 mg, 81%), was obtained as a
yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ: 7.47 (1H, s), 7.38 (1H, d, J = 8.5 Hz), 7.23 (1H, s), 6.85 (1H, t, J = 8.5 Hz), 3.83 (2H,
d, J = 8.5 Hz), 3.35−3.22 (4H, m), 2.86−2.78 (3H, m), 2.29−2.18 (3H, m), 1.78−1.72 (2H, m), 1.62 (2H, d, J = 11.0 Hz), 1.23 (1H, d,
J = 8.8 Hz), 1.16 (6H, d, J = 6.8 Hz), 1.12 (1H, s). ESIMS-LR m/z 343 [(M+H)+]; ESIMS-HR calcd for C21H31N2O2 (M+H)+ 343.2380,
found 343.2371.
4.1.35 N-(Tetrahydro-2H-pyran-4-ylmethyl)spiro[indoline-2,4'-piperidin]-3-one (12)
Following general procedure E using compound 29b (170 mg, 0.84 mmol), 4-formyltetrahydropyran (190 mg, 1.7 mmol), AcOH
(0.19 mL, 3.3 mmol), NaBH(OAc)3 (1.4 g, 6.6 mmol), and CH2Cl2 (10 mL), the title compound, 12 (72 mg, 29%), was obtained as a
pale yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.61 (1H, d, J = 7.8 Hz), 7.48−7.42 (1H, m), 6.88 (1H, dd, J = 8.3, 0.8 Hz), 6.84−6.79
(1H, m), 5.11 (1H, s), 4.02−3.92 (2H, m), 3.50 (1H, d, J = 6.3 Hz), 3.45−3.34 (2H, m), 2.96 (2H, dt, J = 12.0, 3.3 Hz), 2.25 (2H, d, J

= 7.3 Hz), 2.17 (2H, td, J = 12.3, 2.4 Hz), 2.05 (2H, td, J = 12.3, 4.0 Hz), 1.83−1.62 (2H, m), 1.43 (2H, d, J = 11.5 Hz), 1.39−1.21
(2H, m). ESIMS-LR m/z 301 [(M+H)+]; ESIMS-HR calcd for C18H25N2O2 (M+H)+ 301.1911, found 301.1913.
4.1.36 5-Hydroxy-N-(tetrahydro-2H-pyran-4-ylmethyl)spiro
[indoline-2,4'-piperidin]-3-one (13)
Following general procedure E using compound 29c (98 mg, 0.45 mmol), 4-formyltetrahydropyran (110 mg, 0.92 mmol), AcOH
(0.10 mL, 1.7 mmol), NaBH(OAc)3 (780 mg, 3.7 mmol), and CH2Cl2 (4.5 mL), the title compound, 13 (84 mg, 59%), was obtained
as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ: 8.98 (1H, s), 7.02 (1H, dd, J = 8.8, 2.6 Hz), 6.96 (1H, s), 6.80 (1H, d, J =
8.8 Hz), 6.72 (1H, d, J = 2.6 Hz), 3.87−3.79 (2H, m), 3.32−3.25 (3H, m), 2.81 (2H, d, J = 11.8 Hz), 2.25−2.14 (4H, m), 1.80-1.67
(3H, m), 1.62 (2H, d, J = 13.1 Hz), 1.20 (2H, d, J = 13.1 Hz), 1.13 (1H, dd, J = 12.7, 4.6 Hz). ESIMS-LR m/z 317 [(M+H)+]; ESIMS-
HR calcd for C18H25N2O3 (M+H)+ 317.1860, found 317.1854.
4.1.37 5-Chloro-1'-(tetrahydro-2H-pyran-4-ylmethyl)spiro[indoline-2,4'-piperidin]-3-one (15)
Following general procedure E using compound 29d (31 mg, 0.13 mmol), 4-formyltetrahydropyran (30 mg, 0.26 mmol), AcOH (0.03
mL, 0.53 mmol), NaBH(OAc)3 (220 mg, 1.0 mmol), and CH2Cl2 (1.3 mL), the title compound, 15 (40 mg, 91%), was obtained as a
pale yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.15 (1H, dd, J = 8.8, 2.8 Hz), 7.05 (1H, d, J = 2.8 Hz), 6.86 (1H, d, J = 8.8 Hz),
4.64 (1H, s), 3.98 (2H, dd, J = 11.3, 3.0 Hz), 3.39 (2H, td, J = 11.7, 1.9 Hz), 2.96 (2H, d, J = 10.8 Hz), 2.25 (2H, d, J = 6.8 Hz), 2.16
(1H, br s), 2.06 (2H, td, J = 12.6, 3.6 Hz), 1.84−1.74 (1H, m), 1.70 (2H, d, J = 13.6 Hz), 1.41 (1H, br s), 1.36−1.21 (4H, m). ESIMS-LR
m/z 335 [(M+H)+]; ESIMS-HR calcd for C18H24ClN2O2 (M+H)+ 335.1521, found 335.1510.
4.1.38 5-(tert-Butyl)-N-(tetrahydro-2H-pyran-4-ylmethyl)spiro[indoline-2,4'-piperidin]-3-one (17)
Following general procedure E using compound 29e (50 mg, 0.19 mmol), 4-formyltetrahydropyran (44 mg, 0.39 mmol), AcOH (0.044
mL, 0.77 mmol), NaBH(OAc)3 (330 mg, 1.5 mmol), and CH2Cl2 (1.9 mL), the title compound, 17 (67 mg, 97%), was obtained as a
pale yellow solid. 1H NMR (CDCl3, 400 MHz): δ: 7.62 (1H, s), 7.56 (1H, d, J = 8.3 Hz), 6.86 (1H, d, J = 8.3 Hz), 4.84 (1H, s), 3.98
(2H, d, J = 6.4 Hz), 3.39 (2H, t, J = 10.9 Hz), 2.96 (2H, d, J = 10.9 Hz), 2.24 (2H, d, J = 6.4 Hz), 2.20−2.01 (4H, m), 1.82−1.63 (3H,
m), 1.42 (2H, d, J = 12.0 Hz), 1.35−1.26 (2H, m) 1.30 (9H, s). ESIMS-LR m/z 357 [(M+H)+]; ESIMS-HR calcd for C22H33N2O2
(M+H)+ 357.2537, found 357.2524.
4.1.39 N-(Tetrahydro-2H-pyran-4-ylmethyl)-5-(trifluoromethyl)spiro[indoline-2,4'-piperidin]-3-one (18)
Following general procedure E using compound 29f (2.0 g, 7.4 mmol), 4-formyltetrahydropyran (1.7 g, 15 mmol), AcOH (1.7 mL,
30 mmol), NaBH(OAc)3 (12 g, 57 mmol), and CH2Cl2 (50 mL), the title compound, 18 (2.7 g, 99%), was obtained as a pale yellow
solid. 1H NMR (CDCl3, 400 MHz) δ: 7.86 (1H, d, J = 9.3 Hz), 7.65 (1H, dd, J = 8.8, 1.8 Hz), 6.95 (1H, d, J = 8.8 Hz), 4.00 (2H, dd,
J = 11.6, 3.4 Hz), 3.41 (2H, td, J = 11.6, 2.0 Hz), 3.29 (2H, t, J = 6.0 Hz), 2.84 (2H, br s), 2.61 (2H, d, J = 6.8 Hz), 2.06−1.91 (5H,
m), 1.80 (2H, d, J = 13.1 Hz), 1.37 (2H, ddd, J = 25.1, 12.0, 4.5 Hz). ESIMS-LR m/z 369 [(M+H)+]; ESIMS-HR calcd for
C19H24F3N2O2 (M+H)+ 369.1784, found 369.1774.
4.1.40 5-Methoxy-N-(tetrahydro-2H-pyran-4-ylmethyl)spiro
[indoline-2,4'-piperidin]-3-one (14)
A suspension of compound 13 (10 mg, 0.032 mmol) and NaH (0.91 mg, 60% dispersion of mineral oil, 0.038 mmol) in DMF (1.0
mL) was treated with MeI (2.0 µL, 0.032 mmol) at 0 °C, and the mixture was stirred at 0 °C for 30 min. The resulting mixture was
partitioned between AcOEt (50 mL) and saturated aqueous NaHCO3 (30 mL), and the organic phase was washed with saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography
(MeOH/CHCl3; 1:99 to 13:87) to afford compound 14 (3.4 mg, 32%) as a pale yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.15 (1H,
dd, J = 8.8, 2.8 Hz), 7.05 (1H, d, J = 2.8 Hz), 6.86 (1H, d, J = 8.8 Hz), 4.64 (1H, s), 3.98 (2H, dd, J = 11.3, 3.0 Hz), 3.78 (3H, s), 3.39
(2H, td, J = 11.7, 1.9 Hz), 2.96 (2H, d, J = 10.8 Hz), 2.25 (2H, d, J = 6.8 Hz), 2.16 (1H, br s), 2.06 (2H, td, J = 12.6, 3.6 Hz), 1.84−1.74
(1H, m), 1.70 (2H, d, J = 13.6 Hz), 1.41 (1H, br s), 1.36−1.21 (4H, m). ESIMS-LR m/z 331 [(M+H)+]; ESIMS-HR calcd for
C19H27N2O3 (M+H)+ 331.2016, found 331.2036.
4.1.41 5-Isopropyloxy-N-(tetrahydro-2H-pyran-4-ylmethyl)spiro[indoline-2,4'-piperidin]-3-one (16)
A suspension of compound 13 (26 mg, 0.082 mmol) and NaH (3.0 mg, 60% dispersion of mineral oil, 0.13 mmol) in DMF (1.0 mL)
was treated with 2-iodopropane (8.2 µL, 0.082 mmol) at 0 °C for 30 min. The resulting mixture was partitioned between AcOEt (50
mL) and saturated aqueous NaHCO3 (30 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (MeOH/CHCl3; 1:99 to 15:85) to
afford compound 16 (4.9 mg, 17%) as a pale yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.12 (1H, dd, J = 8.8, 2.7 Hz), 7.06 (1H, d,
J = 2.7 Hz), 6.84 (1H, d, J = 8.8 Hz), 4.64 (1H, br s), 4.47−4.39 (1H, m), 3.97 (2H, dd, J = 11.6, 2.9 Hz), 3.39 (2H, td, J = 11.6, 1.8
Hz), 2.97 (2H, d, J = 11.0 Hz), 2.26 (2H, d, J = 6.8 Hz), 2.18 (2H, br s), 2.05 (2H, td, J = 12.6, 3.8 Hz), 1.85−1.71 (3H, m), 1.46 (2H,
br s), 1.34−1.28 (2H, m), 1.31 (6H, d, J = 6.0 Hz). ESIMS-LR m/z 359 [(M+H)+]; ESIMS-HR calcd for C21H31N2O3 (M+H)+ 359.2329,
found 359.2345.
4.1.42 N-Benzyl-6-(trifluoromethyl)spiro[indene-2,4'-piperidin]-1(3H)-one (31)

A suspension of 6-(trifluoromethyl)-1-indanone 30 (0.38 mL, 2.5 mmol) and NaH (420 mg, 60% dispersion of mineral oil, 10 mmol)
in DMF (7.0 mL) was treated with N-benzyl-N,N-di(2-chloroethyl)amine hydrochloride (810 mg, 3.0 mmol) at 0 °C, and the mixture
was stirred at room temperature for 20 h. The resulting mixture was partitioned between AcOEt (50 mL) and H2O (30 mL), and the
organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (CHCl3/hexane; 0:100 to 10:90) to afford compound 31 (83 mg, 9.2%) as a pale brown solid.
1H NMR (CDCl3, 400 MHz) δ: 8.02 (1H, s), 7.83 (1H, dd, J = 8.0, 1.5 Hz), 7.58 (1H, d, J = 8.0 Hz), 7.38−7.30 (4H, m), 7.28−7.24
(1H, m), 3.57 (2H, s), 3.09 (2H, s), 2.94 (2H, dt, J = 11.6, 3.2 Hz), 2.18 (2H, td, J = 11.6, 2.0 Hz), 2.08 (2H, td, J = 12.3, 3.2 Hz), 1.39
(2H, d, J = 13.1 Hz). ESIMS-LR m/z 360 [(M+H)+].
4.1.43 N-(Tetrahydro-2H-pyran-4-ylmethyl)-6-(trifluoromethyl)
spiro[indene-2,4'-piperidin]-1(3H)-one (19)
Following general procedure E using compound 31 (38 mg, 0.11 mmol), 4-formyltetrahydropyran (24 mg, 0.21 mmol), 10% Pd/C
(7.6 mg), and MeOH (1.1 mL), the title compound, 19 (18 mg, 46%), was obtained as a colorless solid. 1H NMR (CDCl3, 400 MHz)
δ: 8.03 (1H, s), 7.83 (1H, dd, J = 8.0, 1.3 Hz), 7.58 (1H, d, J = 8.0 Hz), 3.98 (2H, dd, J = 11.6, 4.1 Hz), 3.39 (2H, td, J = 11.6, 2.0
Hz), 3.08 (2H, s), 2.91 (2H, dt, J = 11.6, 3.2 Hz), 2.23 (2H, d, J = 7.0 Hz), 2.19−2.00 (4H, m), 1.83−1.70 (3H, m), 1.39 (2H, d, J =
13.1 Hz), 1.28 (2H, ddd, J = 24.4, 12.2, 4.1 Hz). ESIMS-LR m/z 368 [(M+H)+]; ESIMS-HR calcd for C20H25F3NO2 (M+H)+ 368.1832,
found 368.1834.
4.1.44 2-(5-Bromo-2-fluorophenyl)-2-(trimethylsilyloxy)acetonitrile (33)
A suspension of 5-bromo-2-fluorobenzaldehyde 32 (0.80 g, 3.9 mmol), LiCl (0.033 mg, 0.78 µmol) in CH2Cl2 (15 mL) was treated
with TMSCN (0.49 mL, 3.9 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 h. The resulting mixture was
concentrated in vacuo to afford compound 33 (1.2 g, quant.) as a colorless oil. This material was used to the next reaction without
further purification. 1H NMR (CDCl3, 400 MHz) δ: 7.76 (1H, dd, J = 6.5, 2.5 Hz), 7.50 (1H, dq, J = 8.7, 2.5 Hz), 7.01 (1H, dd, J =
17.9, 8.7 Hz), 5.68 (1H, s), 0.26 (9H, s).
4.1.45 (1-Benzyl-4-hydroxypiperidin-4-yl)(5-bromo-2-fluorophenyl)methanone (34)
A solution of compound 33 (1.0 g, 3.3 mmol) in THF (17 mL) was treated with LDA (1 M in THF solution) (4.7 mL, 4.7 mmol) at
−78 °C for 1.5 h. N-benzyl-4-piperidone (0.84 mL, 4.7 mmol) was slowly added to the mixture at −78 °C for 3 h. The resulting mixture
was partitioned between AcOEt (50 mL) and saturated aqueous NaHCO3 (30 mL), and the organic phase was washed with saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography
(AcOEt/hexane; 20:80 to 70:30) to afford compound 34 (520 mg, 34%) as a brown oil. 1H NMR (CDCl3, 400 MHz) δ: 7.54 (1H, tt, J
= 6.8, 2.3 Hz), 7.44 (1H, dd, J = 5.6, 2.3 Hz), 7.31 (3H, d, J = 4.5 Hz), 7.28−7.22 (2H, m), 7.02 (1H, t, J = 9.0 Hz), 3.54 (2H, s), 3.00
(1H, d, J = 1.5 Hz), 2.78 (2H, d, J = 11.3 Hz), 2.40 (2H, td, J = 11.9, 2.4 Hz), 2.12 (2H, td, J = 12.9, 4.5 Hz), 1.67 (2H, d, J = 11.9
Hz). ESIMS-LR m/z 392, 394 [(M+H)+].
4.1.46 N-Benzyl-5-bromo-3H-spiro[benzofuran-2,4'-piperidin]-3-one (35)
A solution of compound 34 (480 mg, 1.2 mmol) in THF (12 mL) was treated with tBuOK (150 mg, 1.3 mmol) at room temperature,
and the mixture was stirred at 80 °C for 2 h. The resulting mixture was partitioned between AcOEt (50 mL) and saturated aqueous
NaHCO3 (30 mL), and the organic phase was washed was saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (AcOEt/hexane; 20:80 to 50:50) to afford compound 35 (330
mg, 74%) as a yellow oil. 1H NMR (CDCl3, 400 MHz) δ: 7.77 (1H, d, J = 2.2 Hz), 7.68 (1H, dd, J = 8.8, 2.2 Hz), 7.38−7.30 (4H, m),
7.28−7.24 (1H, m), 7.03 (1H, d, J = 8.8 Hz), 3.60 (2H, s), 2.90 (2H, dt, J = 11.5, 3.3 Hz), 2.44 (2H, td, J = 11.9, 2.5 Hz), 2.08 (2H,
dt, J = 18.7, 6.8 Hz), 1.59 (2H, dd, J = 14.2, 2.5 Hz). ESIMS-LR m/z 372, 374 [(M+H)+].
4.1.47 1'-benzyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-one (36)
A suspension of compound 35 (100 mg, 0.27 mmol), bis(pinacolato)diboron (100 mg, 0.40 mmol) and KOAc (79 mg, 0.81 mmol) in
DMSO (2.7 mL) was treated with Pd(dppf)Cl2 (39 mg, 0.053 mmol) at room temperature, and the mixture was stirred at 110 °C for 1
h with MW irradiation, and then the mixture was cooled to room temperature. The resulting mixture was partitioned between AcOEt
(50 mL) and H2O (30 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatography (AcOEt/hexane; 30:70 to 70:30) to afford compound 36 (38
mg, 34%) as a colorless oil. 1H NMR (CDCl3, 400 MHz) δ: 8.16 (1H, d, J = 0.5 Hz), 8.03 (1H, dd, J = 8.5, 1.4 Hz), 7.38−7.30 (4H,
m), 7.28−7.24 (1H, m), 7.09 (1H, dd, J = 8.5, 0.5 Hz), 3.62 (2H, s), 2.95−2.88 (2H, m), 2.47 (2H, td, J = 12.1, 2.2 Hz), 2.14−2.05
(2H, m), 1.59 (2H, d, J = 12.1 Hz), 1.33 (12H, s). ESIMS-LR m/z 420 [(M+H)+].
4.1.48 N-Benzyl-7-(trifluoromethyl)spiro[indoline-2,4'-piperidin]-3-one (37)
Step 2. A suspension of compound 36 (22 mg, 0.052 mmol), 1,10-phenanthroline (1.9 mg, 11 μmol), Copper(I) thiophene-2-
carboxylate (CuTC) (1.0 mg, 5.2 μmol) and LiOH·H2O (4.4 mg, 0.11 mmol) in CH2Cl2 (1.0 mL) was treated with Togni's reagent (1-
trifluoromethyl-3,3-dimethyl-1,2-benziodoxole) (19 mg, 0.058 mmol) at room temperature, and the mixture was stirred at 60 °C for
1 h with MW irradiation, and then the mixture was cooled to room temperature. The resulting mixture was partitioned between AcOEt
(50 mL) and H2O (30 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatography (AcOEt/hexane; 10:90 to 40:60) to afford compound 37 (14

mg, 75%) as a brown oil. 1H NMR (CDCl3, 400 MHz) δ: 7.98−7.94 (1H, m), 7.87−7.81 (1H, m), 7.39−7.29 (4H, m), 7.29−7.24 (1H,
m), 7.22 (1H, d, J = 8.8 Hz), 3.61 (2H, d, J = 5.5 Hz), 2.91 (2H, dq, J = 15.2, 3.9 Hz), 2.45 (2H, ddd, J = 22.3, 11.7, 2.6 Hz), 2.10
(2H, ddd, J = 26.2, 13.8, 4.6 Hz), 1.67-1.55 (2H, m). ESIMS-LR m/z 362 [(M+H)+].
4.1.49 5-(Trifluoromethyl)-3H-spiro[benzofuran-2,4'-piperidin]-3-one (38)
A solution of compound 37 (21 mg, 0.058 mmol) in MeOH (1.0 mL) was treated with 10% Pd/C (5.0 mg) and the mixture was
vigorously stirred under H2 atmosphere (balloon pressure) at room temperature for 15 h. The catalyst was filtered off through a Celite
pad, and the filtrate was concentrated in vacuo to afford compound 38 (14 mg, 89%) as a colorless solid. This material was used in
the next reaction without further purification. ESIMS-LR m/z 272 [(M+H)+].
4.1.50 N-((Tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)
-3H-spiro[benzofuran-2,4'-piperidin]-3-one (20)
Following general procedure A using compound 38 (9.0 mg, 0.033 mmol), 4-formyltetrahydropyran (12 mg, 0.11 mmol), AcOH (12
µL, 0.21 mmol), NaBH(OAc)3 (88 mg, 0.42 mmol), and CH2Cl2 (1.0 mL), the title compound, 20 (4.6 mg, 24%), was obtained as a
colorless solid. 1H NMR (CDCl3, 400 MHz) δ: 7.96 (1H, s), 7.85 (1H, dd, J = 8.8, 1.9 Hz), 7.23 (1H, d, J = 8.8 Hz), 3.98 (2H, dd, J
= 11.5, 2.8 Hz), 3.40 (2H, td, J = 11.5, 2.0 Hz), 2.91−2.85 (2H, m), 2.42 (2H, td, J = 11.9, 2.6 Hz), 2.29 (2H, d, J = 7.3 Hz), 2.08 (2H,
td, J = 13.0, 4.5 Hz), 1.84−1.73 (1H, m), 1.71 (2H, d, J = 14.3 Hz), 1.62 (2H, dd, J = 14.3, 2.5 Hz), 1.31-1.26 (2H, m). ESIMS-LR
m/z 370 [(M+H)+]; ESIMS-HR calcd for C19H23F3NO3 (M+H)+ 370.1625, found 370.1622.
4.1.51 1-Methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)spiro[indoline-2,4'-piperidin]-3-one (21)
A suspension of compound 18 (2.9 g, 7.8 mmol) and NaH (0.93 g, 60% dispersion of mineral oil, 23 mmol) in DMF (10 mL) was
treated with MeI (1.5 mL, 23 mmol) at 0 °C for 1 h. The resulting mixture was partitioned between AcOEt (100 mL) and saturated
aqueous NaHCO3 (50 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatography (MeOH/CHCl3; 1:99 to 10:90) to afford compound 21 (2.7
g, 90%) as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ: 7.80 (1H, d, J = 0.8 Hz), 7.64 (1H, dd, J = 8.6, 1.8 Hz), 6.76 (1H, d, J = 8.6
Hz), 3.98 (2H, dd, J = 10.7, 3.4 Hz), 3.40 (2H, td, J = 11.9, 1.9 Hz), 2.97 (3H, s), 2.90 (2H, td, J = 11.9, 2.3 Hz), 2.73 (2H, d, J = 11.3
Hz), 2.35 (2H, d, J = 7.0 Hz), 2.03 (2H, td, J = 12.5, 4.3 Hz), 1.85−1.73 (1H, m), 1.70 (2H, d, J = 13.3 Hz), 1.50 (2H, dd, J = 12.5,
1.5 Hz), 1.30 (2H, ddd, J = 24.7, 12.0, 4.5 Hz). ESIMS-LR m/z 383 [(M+H)+]; ESIMS-HR calcd for C20H26F3N2O2 (M+H)+ 383.1941,
found 383.1949.
4.1.52 N-((Tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)
spiro[indoline-2,4'-piperidin]-3-ol (22)
A solution of compound 18 (300 mg, 0.81 mmol) in MeOH (5.0 mL) was treated with NaBH4 (150 mg, 4.1 mmol) at 0 °C, and the
mixture was stirred at room temperature for 1 h. The resulting mixture was partitioned between AcOEt (50 mL) and saturated aqueous
NaHCO3 (30 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (MeOH/CHCl3; 1:99 to 15:85) to afford compound 22 (220
mg, 73%) as a colorless solid. 1H NMR (CDCl3, 400 MHz) δ: 7.54 (1H, s), 7.38 (1H, d, J = 8.3 Hz), 6.64 (1H, d, J = 8.3 Hz), 4.77
(1H, s), 4.28 (1H, s), 3.97 (2H, dd, J = 10.9, 3.4 Hz), 3.39 (2H, td, J = 11.7, 1.8 Hz), 2.66−2.55 (2H, m), 2.37 (2H, t, J = 8.9 Hz),
2.14−2.05 (1H, m), 1.80−1.69 (5H, m), 1.61−1.53 (2H, m), 1.27 (2H, ddd, J = 24.5, 12.2, 4.0 Hz). ESIMS-LR m/z 371 [(M+H)+];
ESIMS-HR calcd for C19H26F3N2O2 (M+H)+ 371.1941, found 371.1926.
4.1.53 3-Methoxy-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)spiro[indoline-2,4'-piperidine] (23)
A suspension of compound 22 (30 mg, 0.081 mmol) and NaH (5.8 mg, 60% dispersion of mineral oil, 0.24 mmol) in DMF (1.0 mL)
was treated with MeI (7.6 µL, 0.12 mmol) at 0 °C for 20 min. The resulting mixture was partitioned between AcOEt (50 mL) and
saturated aqueous NaHCO3 (30 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by silica gel column chromatography (NH-SiO2, AcOEt/hexane; 30:70 to 70:30) to
afford compound 23 (11 mg, 34%) as a colorless solid. 1H NMR (CDCl3, 400 MHz) δ: 7.49 (1H, s), 7.39 (1H, dd, J = 8.3, 1.3 Hz),
6.66 (1H, d, J = 8.3 Hz), 4.32 (1H, s), 3.97 (2H, dd, J = 11.7, 2.6 Hz), 3.42 (3H, s), 3.37 (2H, dd, J = 11.7, 2.1 Hz), 2.62−2.49 (2H,
m), 2.44−2.32 (2H, m), 2.22 (2H, d, J = 7.0 Hz), 1.82−1.72 (2H, m), 1.71−1.68 (3H, m), 1.33−1.23 (4H, m). ESIMS-LR m/z 385
[(M+H)+]; ESIMS-HR calcd for C20H28F3N2O2 (M+H)+ 385.2097, found 385.2079.
4.1.54 N-((Tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)
spiro[indoline-2,4'-piperidine] (24)
A solution of compound 22 (50 mg, 0.14 mmol), Et3SiH (0.11 mL, 0.68 mmol) in CH2Cl2 (2.7 mL) was treated with TFA (0.10 mL,
1.3 mmol) at room temperature for 1 h. The resulting mixture was partitioned between AcOEt (50 mL) and saturated aqueous NaHCO3
(30 mL), and the organic phase was washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (MeOH/CHCl3; 1:99 to 13:87) to afford compound 24 (44 mg, 92%) as a
colorless solid. 1H NMR (CDCl3, 400 MHz) δ: 7.26-7.23 (2H, m), 6.53 (1H, d, J = 8.8 Hz), 3.96 (2H, dd, J = 10.8, 3.5 Hz), 3.38 (2H,
td, J = 11.7, 1.8 Hz), 2.89 (2H, s), 2.53−2.43 (2H, m), 2.43−2.34 (2H, m), 2.21 (2H, d, J = 7.0 Hz), 1.82−1.63 (7H, m), 1.33-1.21 (2H,
m). ESIMS-LR m/z 355 [(M+H)+]; ESIMS-HR calcd for C19H26F3N2O (M+H)+ 355.1992, found 355.1996.

4.1.55 1-Methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)spiro[indoline-2,4'-piperidine] (25)
A suspension of compound 24 (30 mg, 0.085 mmol) and NaH (6.8 mg, 60% dispersion of mineral oil, 0.17 mmol) in DMF (1.0 mL)
was treated with MeI (7.9 µL, 0.13 mmol) at 0 °C, and the mixture was stirred at room temperature for 5 h. The resulting mixture was
partitioned between AcOEt (50 mL) and saturated aqueous NaHCO3 (30 mL), and the organic phase was washed with saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography
(NH-SiO2, AcOEt/hexane; 10:90 to 50:50) to afford compound 25 (23 mg, 73%) as a colorless oil. 1H NMR ((DMSO-d6, 400 MHz)
δ: 7.29 (1H, d, J = 8.3 Hz), 7.24 (1H, s), 6.40 (1H, d, J = 8.3 Hz), 3.82 (2H, dd, J = 11.3, 2.5 Hz), 3.28 (2H, td, J = 11.7, 1.8 Hz), 2.90
(2H, s), 2.80 (2H, d, J = 11.3 Hz), 2.69 (3H, s), 2.15 (2H, d, J = 7.3 Hz), 1.99 (2H, t, J = 11.3 Hz), 1.87 (2H, td, J = 12.4, 4.0 Hz),
1.73 (1H, tdd, J = 14.8, 7.3, 3.6 Hz), 1.60 (2H, dd, J = 13.1, 1.8 Hz), 1.37 (2H, d, J = 11.8 Hz), 1.11 (2H, ddd, J = 24.8, 11.8, 4.4 Hz).
ESIMS-LR m/z 369 [(M+H)+]; ESIMS-HR calcd for C20H28F3N2O (M+H)+ 369.2148, found 369.2136.
4.2. In vitro and in vivo evaluations
4.2.1. Animals
Prenatal/postnatal Wister rats were purchased from JAPAN SLC, Inc. In vivo experiments were performed on female C57BL/6 mice
aged 7 weeks. All of the experimental protocols for animal studies were approved by the committee for ethics in animal experiments
of Asubio Pharma Co., Ltd.
4.2.2 OPC differentiation assay
Neonatal rats (P4-6) were sacrificed by decapitation. The brains were dissected and the cerebral cortices were collected. Cell
dissociation was performed by using a papain dissociation system (Worthington, PDS2). Dissociated cells were seeded on a 10 cm
dish and incubated at 36 °C with 5% CO2. Half the volume of the medium was removed and the same volume of fresh medium was
added at 6 DIV (days in vitro). At 8 DIV, cells were detached and re-seeded on a 96-well plate. DMSO-dissolved compounds were
added to the medium at 10 DIV. Cells were fixed with paraformaldehyde at 13 DIV, and immunostained with anti-MBP and anti-
OLIG2 primary antibodies (Santa Cruz Biotechnology, sc-13914 and sc-48817, respectively) and Alexa488- and Alexa555-
conjugated secondary antibodies. The MBP-positive area and the number of OLIG2-positive cells were analyzed with OperaLX
(Perkin Elmer). The medium used in this study consisted of DMEM/F-12 (Thermo Fisher Scientific, 11320-033), StemPro Neural
supplement (Thermo Fisher Scientific, A1050801), Anti-Anti (Thermo Fisher Scientific, 15240-096), 10 ng/mL bFGF (Peprotech,
100-18B) and 10–30 ng/mL PDGF-AA (R&D Systems, 221-AA). All the dishes and plates were coated with polyetheleneimine and
laminin.
4.2.3 In vivo evaluation (induction of EAE and grading of disabilities)
Induction of EAE, grading of disabilities, and compound treatment female C57BL/6 mice (7 weeks old) were subcutaneously
immunized with 100 µg of myelin oligodendrocyte glycoprotein (MOG) peptide (amino acids 35–55) emulsified with incomplete
Freund's adjuvant containing 5 mg/ml of Mycobacterium tuberculosis (Day 0), followed by an interperitoneal administration of 200
ng of pertussis toxoid on Days 0 and 2. The animals were examined for disabilities until Day 17 with clinical grading as follows: 0,
no signs; 1, limp tail; 2, ataxia and/or paresis of hindlimbs; 3, paralysis of hindlimbs and/or paresis of forelimbs; 4, tetraparalysis; 5,
moribund or death. From Day 3, mice were orally administered compound 18 once per day. The control mice were administered with
vehicle alone (0.5% methylcellulose).
Acknowledgments
We thank Dr. Tomohiro Honda for conducting pharmacokinetic evaluation. The authors are grateful to Dr. Masashi Hasegawa for
helpful discussions on medicinal chemistry and manuscript preparation.
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Graphical Abstract
Discovery and structure-activity
relationships of spiroindolines as novel
inducers of oligodendrocyte progenitor cell
differentiation
Leave this area blank for abstract info.
Katsushi Katayama*†, Yoshikazu Arai†, Kenji Murata†, Shoichi Saito†, Tsutomu Nagata†, Kouhei
Takashima†, Ayako Yoshida†, Makoto Masumura†, Shuichi Koda†, Hiroyuki Okada† and Tsuyoshi Muto†
O
O
O
F3C
optimization
N
N
N
N
H
H
1
18
rOPC: 0.16 μM
metabolic stability (%) h/m: 61/0
rOPC: 0.0032 μM
metabolic stability (%) h/m: 35/68
Declaration of interests
☒ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☒The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: