Spiro[pyrrolidine-3, 3′-oxindoles] and their indoline analogues as new 5-HT6 receptor chemotypes

Article abstract of DOI:10.3390/molecules22122221

Synthetic derivatives of spiro[pyrrolidinyl-3, 3′-oxindole] alkaloids (coerulescine analogues) were investigated as new ligands for aminergic G-protein coupled receptors (GPCRs). The chemical starting point 2′-phenylspiro[indoline-3, 3′-pyrrolidin]-2-one scaffold was identified by virtual fragment screening utilizing ligand- and structure based methods. As a part of the hit-to-lead optimization a structure-activity relationship analysis was performed to explore the differently substituted 2′-phenyl-derivatives, introducing the phenylsulphonyl pharmacophore and examining the corresponding reduced spiro[pyrrolidine-3, 3′-indoline] scaffold. The optimization process led to ligands with submicromolar affinities towards the 5-HT₆ receptor that might serve as viable leads for further optimization.

Full text of DOI:10.3390/molecules22122221

molecules
Article
Spiro[pyrrolidine-3,30-oxindoles] and Their Indoline
Analogues as New 5-HT6 Receptor Chemotypes
1,
Ádám A. Kelemen 1, Grzegorz Satala 2, Andrzej J. Bojarski 2 and György M. Keseru˝
*
1
Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of
Sciences, Magyar tudósok körútja 2, H1117 Budapest, Hungary; kelemen.adam@ttk.mta.hu
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences,
12 Sme˛tna Street, 31-343 Krakow, Poland; grzegorz.satala@gmail.com (G.S.);
andrzej.bojarski@gmail.com (A.J.B.)
2
*
Correspondence: keseru.gyorgy@ttk.mta.hu or gy.keseru@ttk.mta.hu; Tel.: +36-1-382-6821
Received: 1 November 2017; Accepted: 12 December 2017; Published: 14 December 2017
0
Abstract: Synthetic derivatives of spiro[pyrrolidinyl-3,3 -oxindole] alkaloids (coerulescine analogues)
were investigated as new ligands for aminergic G-protein coupled receptors (GPCRs). The chemical
starting point 20-phenylspiro[indoline-3,30-pyrrolidin]-2-one scaffold was identified by virtual
fragment screening utilizing ligand- and structure based methods. As a part of the hit-to-lead
optimization a structure-activity relationship analysis was performed to explore the differently
substituted 20-phenyl-derivatives, introducing the phenylsulphonyl pharmacophore and examining
the corresponding reduced spiro[pyrrolidine-3,30-indoline] scaffold. The optimization process led to
ligands with submicromolar affinities towards the 5-HT6 receptor that might serve as viable leads for
further optimization.
Keywords: oxindole; indoline; coerulescine; 5-HT6R; G-protein coupled receptor
1. Introduction
The recent isolation of naturally occurring and biologically active spiropyrrolidinyl-oxindole
alkaloids initiated a significant research on synthetic derivatives [1], particularly compounds with
the spiro[indoline-3,30-pyrrolidine]-2-one ring system. Spiropyrrolidinyl-oxindole alkaloids were
isolated from different species including Gelsemium sempervirens, Aspidosperma, Mitragyna, Ourouparia,
Rauwolfia, Vinca species [
coerulescine ( ), horsfiline (
Although these compounds have a common tryptamine derived motif, a ChEMBL-analysis [
revealed that no representatives have been ever tested against serotonergic targets. Developing
ligand-(FrAGs) [ ] and structure-based (FrACS) [ ] methods for the identification of aminergic receptor
2]. The most prominent examples are rynchophylline (
1), formosanine (2),
3
4
) and elacomine ( ) that show diverse biological activity (Figure 1).
5
3]
4
5
ligands we identified 6 as a micromolar 5-hydroxytryptamine receptor 6 (5-HT6R) ligand.
The 5-HT6R is a member of the Class A G-protein coupled receptors (aminergic family)
considered to be a current and promising drug target for the treatment of several central nervous
system related indications, such as: cognitive, learning and memory deficits related to Alzheimer’s
disease [6], Parkinson’s disease [7] and schizophrenia [8]. Chemical similarity to the endogenous
agonist serotonin explains the most frequent heteroaromatic ring systems routinely used in 5-HT6R
ligands that include indoles, indolines, indazoles, pyrrolo[2,3-b]pyridines, pyrazolo[1,5-a]pyridines,
[1,2,3]-triazolo[1,5-a]pyrimidines and further 5 + 6 condensed N-heterocycles. Pharmacophore based
approaches on 5-HT6R antagonists are typically focused to the canonical pharmacophore model [9]
(Figure 2), which is defined as having two hydrophobic rings/ring systems connected by a hydrogen
bond acceptor (e.g., sulfonyl, sulfonamide linker). Optionally, a positively ionizable residue is included
in one of the hydrophobic sites of the ligands, typically offering an interacting moiety with the D3.32
Molecules 2017, 22, 2221; doi:10.3390/molecules22122221
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2221
2 of 25
aspartate residue of aminergic GPCR’s as a key factor of aminergic 7TM-receptor activation [10].
A further pharmacophore feature might contain an additional intramolecular hydrogen bond donor
moiety further stabilizing the binding conformation of the ligands [11] (see Figure 2). Selectivity
among other aminergic GPCR’s was shown [12] to be accessible through omitting the positively
ionizable group in the 5-HT6R antagonists. Bis(hetero)arylsulphonyl- and sulfonamide substituents
also contribute to 5-HT6R affinity and selectivity.
Figure 1. Representative natural spiropyrrolidinyl-oxindole alkaloids. The tryptamine scaffold is
shown as red skeleton.
Figure 2. General pharmacophore model of 5-HT6R antagonists. PI: positive ionisable portion; HYD1,2
:
hydrophobic sites; iHBD: intramolecular hydrogen bond donor site; HBA: hydrogen bond acceptor
feature; HSi,j: substituents on the hydrophobic sites.
The tryptamine derived scaffold of
6 and its alignment with the published 5-HT6R
pharmacophore [ ] prompted us to explore the structure-activity relationship around the
9
spiropyrrolidinyl-oxindole core. Here we report the results of our hit-to-lead program, which led to an
identification of promising 5-HT6R ligands of this chemotype.
2. Results and Discussion
2.1. Identification and Early Structure-Activity Data on Spiro[pyrrolidine-3,30-oxindole] Derivatives
Fragment libraries containing a couple hundreds or even thousands of small polar molecules
are routinely used for hit identification at the early stage of drug discovery. Fragment screening
provides diverse chemotypes and significant operational freedom for the further optimization
of promising hits. Inspired by these advantages in a recent study we developed a strategy for
aminergic focused fragment libraries using a sequential filtering methodology applying ligand-

Molecules 2017, 22, 2221
3 of 25
and structure-based scoring functions [
4,
5]. The prospective validation was performed on our
in-house library of 1183 fragments. A physicochemical-property based scoring, followed by docking
the fragments into an ensemble of carefully selected aminergic GPCR X-ray structures (PDB ID:
3PBL, 3RZE, 4IB4, 4IAQ, 3UON, 4MQT, 4LDE, 2RH1, 3NY9). This resulted in a set of 36 top
ranked hit molecules which were measured on an aminergic target not being included in the
original docking ensemble, namely 5-HT6R. We demonstrate the usefulness of the method for
comprehensive aminergic focused screenings. Out of the four hits with low micromolar inhibitory
results, the structurally novel 20-(3-fluorophenyl)spiro[indoline-3,30-pyrrolidin]-2-one (
the spiro[pyrrolidine-3,30-oxindole] scaffold (Table 1), was selected for further optimization.
6) possessing
Table 1. Serotonergic G-protein coupled receptor (GPCR) panel of
four serotonin receptors (Ki values are in µM).
6 as measured in binding assays of
ID
Structure
5-HT1A
5-HT2A
5-HT6
5-HT7
6
16.91
5.05
6.75
8.48
As the next step of exploring the structure-activity relationship substructure search in
the MCULE purchasable database [13] of 5 million compounds resulted in further 887
spiro[pyrrolidine-3,30-oxindole] derivatives. The molecules were prepared by Schrödinger’s
LigPrep [14] creating possible conformers, tautomers and protonation states by default settings.
The ligands were then projected to single precision molecular docking analysis on a nine membered
ensemble of molecular dynamics frames of an 5-HT6R homology model [15] (built using 5-HT2BR
X-ray crystal structure [16] in complex with ergotamine as template (PDB ID: 4IB4)). The docking
poses were filtered in a post-processing step keeping only binding modes forming hydrogen bonds
towards Asp1063.32, Asn2886.55 and optionally Ser1935.43, occupying a primary hydrophobic cleft
defined by Trp2816.48, Phe2846.51 and Phe2856.52. A secondary hydrophobic subpocket was defined
by the pose filtering criteria as following: Val1073.33, Ala1574.56, Leu1604.59, Pro1614.60, Leu1644.63
The satisfactory poses of the molecules were scored to have the best ranking in possibly all of the
.
9 frames using consensus ranking [
our serotonergic panel (Table 2).
5]. Altogether ten compounds (7–16) were purchased and tested in
In spite of the structural diversity, all virtual screening hits were lacking substitution at the
oxindole scaffold. However, compounds and 10 having oxygen-containing substituents in
the 3,4-positions of the 20-phenyl ring showed somewhat improved affinity compared to the initial
compound . The moderate improvement in affinity and the lack of selectivity amongst the closely
7, 9
6
related serotonergic targets have driven us to interpret the results in the context of the known 5-HT6R
pharmacophore patterns [17] (Figure 3). This analysis suggests that introducing a phenylsulfonyl
moiety either to the 1-nitrogen at the oxindole ring or to the 10-nitrogen at the pyrrolidine ring would
be beneficial for both the affinity and selectivity of this chemotype.

Molecules 2017, 22, 2221
4 of 25
Table 2. Serotonergic GPCR panel of derivatives substituted in the 20-phenyl moiety as measured in
binding assays of four serotonin receptors (Ki values are in µM).
ID
Structure
5-HT1A
5-HT2A
5-HT6
5-HT7
7
7.07
5.45
2.26
2.69
8
9
1.74
9.84
2.28
2.69
4.33
8.68
3.18
3.20
4.11
6.20
1.09
8.82
10
11
12.08
49.11
12.74
58.96
12
13
45.67
7.14
1.12
5.77
6.63
7.73
10.45
7.49
14
15
16
7.76
40.50
4.54
23.70
53.89
8.62
9.49
8.98
46.72
not active
16.54
not active

Molecules 2017, 22, 2221
5 of 25
(a)
(b)
Figure 3.
classical pharmacophore; (
(a
) 1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (17) superimposed onto the
0 0
) 1 -(phenylsulfonyl)spiro[indoline-3,3 -pyrrolidin]-2-one (18) fitting to the
b
pharmacophore pattern lacking a positive ionisable moiety.
2.2. Hit-to-Lead Optimization of Spiro[pyrrolidine-3,30-oxindoles]
Detailed elaboration of the spiro[pyrrolidine-3,30-oxindole] scaffold required a viable synthesis
strategy for the preparation of designed analogues. The first, conventional approach is based on an
intramolecular Mannich-reaction used in case of several alkaloids including ( )-horsfiline (4) [18]
±
and Spirotryprostatin B [19]. An alternative approach is the oxidative reaction of tryptolines
induced by tert-butyl hypochlorite, N-bromosuccinimide, N-chlorosuccinimide, sodium tungstate,
lead tetraacetate, or osmium tetroxide [20
of water that finally results in the reorganization of the ring system to spiro[pyrrolidine-3,3 -oxindoles]
(Scheme 1).
,
21]. The reaction is completed by the subsequent elimination
0
Scheme 1. Mechanism of the succinimide assisted oxidative spiro-rearrangement of tryptolines.
Further, sophisticated approaches such as [1,3]-dipolar cycloaddition reactions of azomethine
ylides [22], radical cyclization by AIBN [23], intramolecular Heck reaction [24] and asymmetric
nitroolefination of oxindoles [25] are also available, however, used less frequently.
For the effective exploration of the structure-activity relationship we need a feasible and universal
approach with acceptable functional group tolerance and high variability around the spiro-oxindole
core. Considering the requirements of the early stage optimization program we aimed a synthesis
strategy that
•
•
•
•
uses readily accessible, simple starting materials
applies well-documented, readily available reagents
has key intermediates to access a wide variety of derivatives
is not necessarily stereoselective at this stage of the optimization

Molecules 2017, 22, 2221
6 of 25
The oxidative spiro-rearrangement reactions offer a wide variety of oxidative reagents [20,21,26–33]
and the corresponding starting materials are readily accessible through Pictet-Spengler reaction of
tryptamines [34] (Scheme 2).
NH2
glyoxylic acid
monohydrate
H2O, RT, 2h
conc.HCl/H2O
reflux,2h
NH
H
NH
N
N
COOH
N
H
H
H
25
24
23
Scheme 2. Synthesis of tryptoline by Pictet-Spengler condensation.
Following this synthesis strategy, tryptamine (23) was used as a starting material for the synthesis
of the common intermediate tryptoline (25). The Pictet-Spengler condensation reaction was performed
by glyoxylic acid-monohydrate in aqueous medium [35]. The crude acid intermediate 24 was
decarboxylated in refluxing concentrated hydrochloric acid affording the tetrahydro-β-carboline.
The first synthetic route depicted in Scheme 3 starts with the N-acylation of the tryptoline (25).
The application of Cbz (carboxybenzyl) [36] protecting group was necessary for two reasons:
1. achieve the sulfonylation at the oxindol-nitrogen, 2. the spiro-rearrangement reaction is not
occurring in case of unsubstituted pyrido-nitrogen. N-chlorosuccinimide [37] was used in the first
0
experiments as halogenating reagent, providing the Cbz-protected spiro[pyrrolidine-3,3 -oxindole]. The
phenylsulfonylation was performed either by using lithium-hexamethyl disilazane [38] and sodium
hydride [39] as deprotonating agents and acid scavengers. Finally, the deprotection of the Cbz-group
by hydrogenation [40] resulted in the desired 1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one
(17) compound. Following an alternative way, the more basic pyrido-nitrogen of tryptoline 29 might
be first sulfonylated [41] followed by the N-bromosuccinimide assisted spiro-cyclization to 18 [42].
0
Scheme 3. Synthesis of 1- and 1 -phenylsulfonyl derivatives.
Compounds 17 and 18 showed improved selectivity towards the 5-HT6R (Table 3) as compared to
0
the non-sulfonylated 2 -phenyl derivatives, investigated in the first batch (compounds 15
–
24), however,
no improvement in the binding affinity was detected.

Molecules 2017, 22, 2221
7 of 25
Table 3. Serotonergic GPCR panel of the N-phenylsulfonylated derivatives at N0 and N-positions as
measured in binding assays of four serotonin receptors (Ki values are in µM).
ID
Structure
5-HT1A
5-HT2A
5-HT6
5-HT7
17
not active
not active
12.30
58.31
18
not measured
87.35
6.86
not active
Facilitating the potency optimization, we followed the classical 5-HT6R pharmacophore pattern
and tried to remove the polar interacting hydrogen bond acceptor feature in the oxindole ring of
17. For better understanding of the indole, indoline and oxindole-related chemical space of known
5-HT6R ligands we collected all compounds, with at least 0.1
ChEMBL database.
µM activity towards h5-HT6R from the
Substructure filtering by the query of 30 (Figure 4) revealed that altogether 3 examples were found for
oxindoles, as 5-HT6R ligands. All hits belong to the 30-phenylspiro[indoline-3,20-thiazolidine]-2,40-dione
scaffold 31 that was identified by Hostetler et al. [43]. Searching for the 1-(phenylsulfonyl) indoline
scaffold 32, 27 actives were found with the structure 33. Out of the 27 active molecules 4 possess a
positive ionizable moiety built upon the phenyl side of the sulfonyl and all of the remaining examples
are equipped by the classical tryptamine-type amine feature. Searching by the 1-(phenylsulfonyl)indole
34 query resulted in 397 examples with the structure 35. The structures were analyzed based on the
chain-length distance between the 3-carbon of the indole ring and the positive ionizable basic nitrogen
atom, if any. We found 20 examples representing indoles with attached basic nitrogen (all tertiary
amines). Only five compounds were identified with a single methylene linker out of which 2 were
primary amines and 3 were tertiary amines. The largest part of the with structure 34 contains ethylene
linker (201 examples out of 397) of which 123 were tertiary amines, 51 were secondary amines and 19
were primary amines. Much less compounds have 3 atom long linkage, 54 examples were identified,
with 21 tertiary amines and 33 secondary amines and no primary amines. No ligands were found with
basic nitrogen’s being further than 3 atoms from the indole core.
To conclude, we have aimed to synthesize different 1-(phenylsulfonyl)indolines (scaffold 32),
being substituted in the 10-pyrrolidine nitrogen with either basic-nitrogen containing, or lipophilic
groups, presented in Scheme 4—compounds 39a
–
b
. Based on these considerations, first we benzylated [44
]
the tryptoline (25) to afford 36a, further converting it to the corresponding spiro-derivative [45
]
37a. The
reduction of the oxindole oxo-group was performed by applying borane-tetrahydrofuran complex
in refluxing absolutized tetrahydrofuran [46]. It was an important observation, that the reduction
step has to precede the sulfonylation, to avoid the formation of side products when treating the
sulfonamide with the boronic reagent. The phenyl-sulfonylated product 39a was afforded either by
applying LiHMDS [38] or trietylamine/dimethylaminopyridine [47] reagents. The pyridine-4-ylmethyl
derivative (39b) was synthesized based on the same route, however, as a first step, the tryptoline (25)
was treated with 4-chloromethylpyridine [48,49].

Molecules 2017, 22, 2221
8 of 25
O
R
oxindoles
N
S
2/3 examples
O
O
N
N
H
H
31
R
30
R
N
1-(phenylsulfonyl)indolines
N
23/27 examples
N
O
S
O
O
S
O
4/27 examples
33
32
N
R
R
H/R
1-(phenylsulfonyl)indoles
N
N
(H2C)
n
H/R
397 examples
N
O
O
S
S
O
O
35
34
Figure 4. Substructure analysis of ChEMBL 5-HT6R active compounds.
0
Scheme 4. Synthesis of N -substituted and N-phenylsulfonylated indolines.
We also intended to examine the effect of only transforming our initial test compound 17 to the
corresponding reduced indoline, however the debenzylation step of 39a afforded unexpected products
(Scheme 5, 40a and 40b). In case, when methanol was applied as solvent for the debenzylation by
catalytic hydrogenation, a methylated product 40a has formed and respectively, the use of ethanol
afforded an ethyl-alkylated derivative 40b.

Molecules 2017, 22, 2221
9 of 25
0
Scheme 5. Synthesis of N -methyl and ethyl derivatives.
However, both the alkylated derivatives showed some improvement of binding affinity to reach
the low micromolar range, the compound possessing the biggest lipophilic, bulky group 43a has
produced the best, submicromolar binding affinity, with both selectivity against the other closely
related serotonergic targets (Table 4).
Table 4. Serotonergic GPCR panel of N-phenylsulfonyl indolines with different N0-substituents as
measured in binding assays of four serotonin receptors (Ki values are in µM).
ID
Structure
5-HT1A
5-HT2A
5-HT6
5-HT7
N
N
39a
not measured
4.76
0.76
55.76
O
S
O
N
N
N
39b
not active
26.85
6.60
not active
O
S
O
N
N
40a
40b
not measured
not measured
4.52
3.82
1.85
2.42
40.17
O
S
O
N
N
not active
O
S
O
The submicromolar affinity of 39a prompted us investigating the substituent vectors at both the
benzylic and the phenylsulfonyl rings by walking fluorine substituents around. This methodology,
often referred as fluoro-scan has been used effectively for the identification of sites tolerant to
functionalization. In particular, fluoro-scan explores the impact of enhanced lipophilicity, H-bonding

Molecules 2017, 22, 2221
10 of 25
and/or filling a small pocket [50]. In case of the N0-benzylic substitution [13] pattern, 2-, 3- and
4-fluorobenzylchlorides were used as alkylating agents in order to synthesize the corresponding
fluorinated indolines (Scheme 6, 44a–c).
F
F
F
NH
N
N
N
1.) NBS, AcOH
THF/H2O,
BH3/THF
a:2-,b:3-, c:4-fluorobenzylchloride
K2CO3, DMF
0°C, 1.5 h
abs. THF
NH
NH
O
N
H
N
2.) cc. NaHCO3,
RT, 0.5 h
reflux,overnight
RT, overnight
H
43a-c
42a-c
41a-c
25
N
F
a: 2-fluoro
b: 3-fluoro
c: 4-fluoro
PhSO2Cl, TEA
N
O
S
DMAP, DCM
RT, overnight
O
44a-c
0
Scheme 6. Fluoro-scan of the N -benzyl direction.
The substitution of benzylic ring with fluorine caused a minor decrease in affinity, thus has shown,
that growing towards this direction is not beneficial (Table 5). On the other hand, however, some
improvement in the selectivity was observed.
Table 5. Serotonergic GPCR panel of the fluorobenzyl derivatives as measured in binding assays of
three serotonin receptors (Ki values are in µM).
ID
Structure
5-HT1A
5-HT6
5-HT7
F
N
44a
not active
4.53
78.93
N
O
S
O
F
N
44b
44c
not active
not active
1.97
1.77
31.49
15.74
N
O
S
O
N
F
N
O
S
O

Molecules 2017, 22, 2221
11 of 25
The corresponding fluorophenylsulfonyl derivatives 46a
intermediate, previously synthesized (Scheme 7).
–c
were synthesized from compound 38a
Scheme 7. Fluoro-scan of the N-phenylsulfonyl direction.
The alternative growing direction towards the phenylsulfonyl ring (compounds 46a–c) showed
improvement in the affinities (see Table 6), compared to the results of 39a, also retaining the good
selectivity against the 5-HT7 subtype. These data suggest, that position 2 of the phenylsulfonyl
group of 46a is beneficial and therefore it is worth to explore this vector during the forthcoming lead
optimization program.
Table 6. Serotonergic GPCR panel of the fluorophenylsulfonyl derivatives as measured in binding
assays of three serotonin receptors (Ki values are in µM).
ID
Structure
5-HT1A
5-HT6
5-HT7
N
N
46a
not active
0.19
42.57
O
S
O
O
O
F
N
N
46b
not active
0.71
2.45
76.00
56.05
O
S
F
N
N
O
46c
not active
S
F
2.3. Binding Mode Analysis of the Optimized 5-HT6R Ligand
0
We have performed a docking analysis of the 1 -benzyl-1-((2-fluorophenyl)sulfonyl)-spiro[indoline-3,
0
3 -pyrrolidine] (46a) 5-HT6R antagonist using the receptor model reported earlier [15]. Docking of 46a by
Schrödinger—Glide either with, or without applying any constraints (requiring hydrogen bonding

Molecules 2017, 22, 2221
12 of 25
with both Asn2886.55 and/or Ser1935.43) resulted in consistent docking poses inside the orthosteric
binding pocket (Figure 5).
Figure 5. Binding mode of compound 46a in the orthosteric pocket of 5-HT6R.
The indoline core occupies the primary hydrophobic cavity of the binding pocket (formed by
Trp2816.48, Phe2846.51, Phe2856.52), while the protonated quaternary pyrrolidine-nitrogen is oriented
towards the conserved Asp1063.32, forming a well-established hydrogen bond. The phenyl-sulfonyl
moiety of 46a is sitting at a secondary hydrophobic cleft defined by Val1073.33, Ala1574.56, Leu1604.59
Pro1604.60 and Leu1644.63, positioning the sulfonyl-linker as hydrogen-bond acceptor against Ser1935.43
and Asn2886.55. Interestingly, the fluorine atom as hydrogen bond acceptor is offering a possible polar
interaction with the Asn2886.55 residue, underlining the preference of the ortho-substitution at the
phenyl-sulfonyl ring.
,
3. Conclusions
The structure-activity relationship around the original spiropyrrolidinyl-oxindole hit (6) was first
investigated using the “SAR-by-catalog” approach that resulted in some moderate improvement in
affinity and a low level of selectivity. Therefore, we decided to explore the novel 5-HT6R chemotype by
a more conventional medicinal chemistry strategy. A synthetic tree (summarized in Scheme 8) of the
synthesized oxindoles and indoles was elaborated starting from the core tryptoline intermediate (25).
The removal of the bulky lipophilic site at the 20-position of the pyrrolidine ring and insertion of
the classical phenylsulfonyl part resulted in notable selectivity across the serotoninergic GPCR panel
of 4 receptors. The next optimization step was analyzing the chemistry of known indole, indoline
and oxindole-based 5-HT6R inhibitors in ChEMBL. We have synthesized different 1-(phenylsulfonyl)
indolines, being substituted in the 10-pyrrolidine nitrogen with either basic-nitrogen containing,
or lipophilic groups. As a result, 10-benzyl-1-(phenylsulfonyl) spiro[indoline-3,30-pyrrolidine] (39a
and its corresponding 2-, 3-substituted analogues 46a were identified as 5-HT6R starting points. This
opened a new chemical space for the under-represented spiro[pyrrolidine-3,30-indoline] chemotype.
)
–b

Molecules 2017, 22, 2221
13 of 25
0
0
Scheme 8. Spiro[pyrrolidine-3,3 -oxindoles] and spiro[pyrrolidine-3,3 -indolines] as 5HT6 ligands.
4. Materials and Methods
All chemical reagents used were purchased from commercial chemical suppliers. The NMR
experiments were performed at 500 MHz (1H) on a Varian VNMR SYSTEM spectrometer. Chemical
1
shifts are referenced to the residual solvent signals, 2.50 ppm for H in DMSO-d6 and 7.28 ppm
for 1H in CDCl3. The MS measurements were performed on Shimadzu LCMS2020 LC/MS system.
Flash chromatography was performed using Teledyne ISCO CombiFlash Lumen+ Rf. Purifications
by preparative-HPLC were performed with Hanbon NS4205 Binary high pressure semi-preparative
HPLC. Thin-layer chromatography was performed on TLC Silica Gel 60 F254. High resolution mass
spectrometric measurements were performed using a Q-TOF Premier mass spectrometer (Waters
Corporation, Milford, MA, USA) in positive electrospray ionization mode. Compounds
purchased from Mcule Inc.
7–16 were

Molecules 2017, 22, 2221
14 of 25
4.1. General Procedures for the Synthesis of Spiro[pyrrolidine-3,30-oxindole] and
Spiro[pyrrolidine-3,30-indoline] Derivatives
4.1.1. Procedures to Afford 1-(Phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (17)
Synthesis of Benzyl 2-Oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (27)
To the solution of benzyl 3,4-dihydro-1H-pyrido[3,4-b]indole-2(9H)-carboxylate (26) (918 mg,
3 mmol) in 10 mL tetrahydrofuran was added 10 mL distilled water and triethylamine (0.436 mL,
3.15 mmol, 1.05 equiv.) at room temperature. Afterwards N-chlorosuccinimide (431 mg, 3.24 mmol,
◦
1.08 equiv.) was added at 0 C in portions. The reaction mixture was stirred for 1.5 h at room
temperature. The reaction mixture was diluted with 10 mL brine and was extracted using 2
×
10 mL
ethyl acetate. The combined organic phases were dried over sodium-sulfate, filtered and evaporated
under reduced pressure. The crude material was purified using flash chromatography (gradient:
dichloromethane: methanol 0% to 5% methanol) to give 27 as a yellow solid. Yield: 399 mg (41%).
1H-NMR (500 MHz, DMSO-d6)
δ
ppm 7.42–7.28 (m, 8H, ArH-benzyl, ArH-4, ArH-5, ArH-6), 7.14
(d, J = 7.8 Hz, 1H, ArH-7), 5.11 (s, 2H, benzyl CH2), 3.70–3.53 (m, 4H, pyrrolidine H-20, pyrrolidine
H-50), 2.19–2.05 (m, 2H, H-40). 13C-NMR (125 MHz, DMSO-d6)
ppm 176.9 (C-2), 154.8 (C=O), 142.8
δ
(C-3a), 141.5 (C-7a), 135.9 (benzyl C-1), 128.5 (benzyl C-3, benzyl C-5), 127.9 (benzyl C-4, benzyl C-2,
benzyl C-6), 123.8 (C-4), 122.1 (C-5), 109.3 (C-7), 67.9 (benzyl CH2), 57.7 (C-3), 45.1 (pyrrolidine C-20),
43.3 (pyrrolidine C-50), 34.2 (pyrrolidine C-40). MS (DUIS+) m/z 323 [M + H]+.
Synthesis of Benzyl 2-oxo-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine]-10-carboxylate (28)
(1) Procedure Using Sodium-Hydride
To the solution of benzyl 2-oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (27) (175 mg,
0.542 mmol) in 5 mL anhydrous tetrahydrofuran was added sodium-hydride (60%, 28 mg, 0.705 mmol;
1.3 equiv.) at 0 ◦C. After stirring the reaction mixture at room temperature for 0.5 h, benzenesulfonyl
chloride (0.076 mL, 0.596 mmol, 1.1 equiv.) was added dropwise at 0 ◦C. The reaction mixture was
stirred overnight at room temperature, then it was quenched by 10 mL brine and was extracted using
ethyl acetate (2
×
10 mL). The collected organic phases were dried using sodium sulfate, filtered and
evaporated under reduced pressure. The crude product was purified using flash chromatography
(gradient: hexane: ethyl acetate 0% to 20%) to afford 28 as a colorless solid. Yield: 94 mg (38%).
(2) Procedure Using Lithium-Hexamethyl-Disilazane
To the solution of benzyl 2-oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (27) (161 mg,
0.5 mmol) and benzenesulfonyl chloride (0.076 mL, 0.6 mmol, 1.2 equiv.) in 5 mL anhydrous
tetrahydrofuran was added lithiumhexamethyl-disilazane (1M in THF, 0.85 mL, 0.85 mmol, 1.7 equiv.)
◦
at 0 C and the reaction mixture was stirred at this temperature for 0.5 h. After quenching the reaction
by 10 mL brine, the reaction mixture was extracted using ethyl acetate (2
organic phases were dried over sodium sulfate, filtered and evaporated under reduced pressure.
×
10 mL) and the collected
The crude product was purified using flash chromatography (gradient: hexane: ethyl acetate 0% to
1
20%) to give 28 as a white solid. Yield: 230 mg (99%). H-NMR (500 MHz, DMSO-d6)
δ
ppm 8.02 (d,
J = 7.7 Hz, 2H, ArH-3”, ArH-5”), 7.77 (m, 2H, ArH-4”, ArH-7), 7.67 (m, 2H, phenyl-sulfonyl ArH-2”,
phenyl-sulfonyl ArH-6”), 7.43–7.30 (m, 7H, ArH-4, ArH-6, benzyl ArH-2,3,4,5,6), 7.23 (t, J = 7.4 Hz, 1H,
ArH-5), 5.09 (m, 2H, benzyl CH2), 3.65–3.50 (m, 4H, pyrrolidine H-20, pyrrolidine H-50), 2.25–2.09 (m,
2H, pyrrolidine H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 170.1 (C-2), 154.3 (C=O), 136.2 (benzyl
C-1), 135.5 (C-7a), 134.8 (phenyl-sulfonyl C-1”), 133.9 (C-3a), 133.1 (phenyl-sulfonyl C-4”), 129.2 (C-3”,
C-5”), 128.5 (benzyl C-3, benzyl C-5), 128.0 (C-6, benzyl C-4), 127.5 (benzyl C-2, benzyl C-6), 127.4
(C-2”, C-6”), 123.3 (C-4), 121.8 (C-5), 113.8 (C-7), 67.5 (benzyl CH2), 66.1 (C-3), 46.4 (C-20), 46.1 (C-50),
30.3 (C-40). MS (DUIS+) m/z 463 [M + H]+.

Molecules 2017, 22, 2221
15 of 25
Synthesis of 1-(Phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (17)
To the solution of benzyl 2-oxo-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine]-10-carboxylate
(28) (180 mg, 0.389 mmol) in methanol was added palladium (10%) on charcoal (90 mg, 5 w/w %)
in an autoclave. The reactor was filled with 5 atm hydrogen gas and was stirred for 1 h at room
temperature. The reaction mixture was then filtered on Celite and was evaporated under reduced
pressure. The crude material was purified by flash chromatography (gradient: dichloromethane:
1
methanol 0% to 5% methanol) to give 17 as an orange solid. Yield: 108 mg (85%). H-NMR (500 MHz,
CDCl3)
δ ppm 8.10 (d, J = 8.0 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.91 (d,
J = 8.4 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.65 (t, J = 7.2 Hz, 1H, ArH-7), 7.53 (t, J = 7.2 Hz, 2H,
phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.47 (d, J = 7.2 Hz, 1H, ArH-4), 7.32 (t, J = 8.0
Hz, 1H, ArH-6), 7.19 (t, J = 8.0 Hz, 1H, ArH-5), 3.08 (m, 1H, H-20), 2.93 (m, 1H, pyrrolidine H-20),
2.74–2.69 (m, 2H, pyrrolidine H-50), 2.28–2.23 (m, 1H, pyrrolidine H-40), 2.11–2.04 (m, 1H, pyrrolidine
H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 175.2 (C-2), 166.8 (C-7a), 140.4 (phenyl-sulfonyl
C-1”), 135.2 (C-3a), 133.0 (phenyl-sulfonyl C-4”), 129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”),
128.4 (C-6), 127.1 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 122.5 (C-4), 121.7 (C-5), 109.6 (C-7),
55.8 (C-3), 48.3 (pyrrolidine C-20), 47.2 (pyrrolidine C-50), 33.0 (pyrrolidine C-40). MS (DUIS+) m/z 329
[M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C17H17N2O3S: 329.0960; found: 329.0949.
4.1.2. Synthesis of 10-(Phenylsulfonyl)spiro[indoline-3,30-pyrrolidin]-2-one (18)
To the solution of 2-(phenylsulfonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (29) (312 mg,
1 mmol) in tetrahydrofuran: water (10:10 mL) was added 10 mL glacial acetic acid at 0 ◦C.
N-bromosuccinimide (178 mg, 1 mmol, 1 equiv.) was added slowly in portions at 0 ◦C. The reaction
◦
◦
mixture was stirred for 1.5 h at 0 C. Afterwards the reaction was quenched at 0 C by 10 mL
concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room
temperature. The mixture was extracted using ethyl acetate (2
phases were washed with concentrated sodium-hydrogen carbonate (2
10 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under reduced
pressure. The crude product was purified using flash chromatography (gradient: hexane: ethyl
acetate 0% to 20% ethyl acetate) to afford 18 as a colorless solid. Yield: 180 mg (55%). 1H-NMR
×
10 mL). The combined organic
10 mL) and by brine (2
×
×
(500 MHz, DMSO-d6)
δ ppm 10.47 (s, 1H, NH), 7.87 (d, J = 7.3 Hz, 2H, phenyl-sulfonyl ArH-3”,
ArH-5”), 7.76 (t, J = 7.3 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.67 (t, J = 7.3 Hz, 2H, phenyl-sulfonyl
ArH-2”, phenyl-sulfonyl ArH-6”), 7.15 (t, J = 7.3 Hz, 1H, ArH-4), 6.82 (t, J = 8.5 Hz, 2H, ArH-5,
ArH-6), 6.69 (d, J = 7.3 Hz, 1H, ArH-7), 3.56 (t, J = 6.7 Hz, 1H, pyrrolidine H-20), 3.35 (t, J = 7.3 Hz,
1H, pyrrolidine H-20), 3.35 (d, J = 3.5 Hz, 2H, pyrrolidine H-50), 2.10–2.05 (m, 1H, pyrrolidine H-40),
1.94–1.88 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 181.4 (C-2), 167.0 (C-3a),
141.3 (C-7a), 135.8 (phenyl-sulfonyl C-1”), 133.3 (phenyl-sulfonyl C-4”), 129.5 (phenyl-sulfonyl C-3”,
phenyl-sulfonyl C-5”), 128.3 (C-6), 127.4 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 122.4 (C-4),
121.8 (C-5), 109.6 (C-7), 55.6 (C-3), 47.4 (pyrrolidine C-20, pyrrolidine C-50), 35.8 (pyrrolidine C-40).
MS (DUIS+) m/z 329 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C17H17N2O3S: 329.0960;
found: 329.0957.
4.1.3. Procedures to Afford 10-Benzyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (39a)
To the solution of 10-benzylspiro[indoline-3,30-pyrrolidin]-2-one (37a) (500 mg, 1.7985 mmol)
in 15 mL absolutized tetrahydrofuran was added borane/tetrahydrofuran (1M) complex solution
(4.495 mL, 4.495 mmol, 2.5 equiv.) and the reaction mixture was refluxed overnight. The reaction
was allowed to cool down to room temperature and was quenched using 30 mL concentrated
ammonium-chloride solution and was stirred for 20 min. Afterwards, 20 mL ethyl acetate was
used for extraction. The combined organic phases were dried over sodium-sulfate, filtered and
evaporated under reduced pressure to afford 400 mg 38a, used as a crude product in the further

Molecules 2017, 22, 2221
16 of 25
steps without purification. To the solution of crude 10-benzylspiro[indoline-3,30-pyrrolidine] (38a
)
(400 mg 1.513 mmol) (in anhydrous dichloromethane was added triethylamine (0.419 mL, 3.03 mmol,
2 equiv.) and N,N-dimethylpyridin-4-amine (10 mg, 0.076 mmol, 0.5 equiv.) at room temperature.
Afterwards benzenesulfonyl chloride (0.203 mL, 1.59 mmol, 1.05 equiv.) was added to the solution at
5 ◦C. The reaction mixture was stirred at room temperature overnight. The reaction was quenched
using 10 mL 10% HCl solution and was extracted by 10 mL dichloromethane. The dichloromethane
phase was collected and dried over sodium-sulfate. The drying agent was filtered-off and the solution
was evaporated under reduced pressure. The crude product was purified by flash chromatography to
1
afford 39a as yellow oil. Yield: 621 mg (85% calculated to 37a). H-NMR (500 MHz, DMSO-d6)
δ ppm
7.73 (d, J = 7.6 Hz, 2H, phenyl-sulfonyl H-2”, phenyl-sulfonyl H-6”), 7.58 (t, J = 7.6 Hz, 1H, H-6), 7.46
(t, J = 7.9 Hz, 3H, phenyl-sulfonyl H-3”, phenyl-sulfonyl H-4”, phenyl-sulfonyl H-5”), 7.31 (t, J = 7.6 Hz
2H, phenyl-sulfonyl H-3”, phenyl-sulfonyl H-5”), 7.27–7.21 (m, 5H, benzyl H-1-5), 7.05 (t, J = 7.6 Hz,
1H, H-5), 3.89 (d, J = 10.9 Hz, 1H, H-2), 3.78 (d, J = 10.9 Hz, 1H, H-2), 3.52 (s, 2H, benzyl CH2), 2.69
,
0
0
0
(m, 1H, pyrrolidine H-2 ), 2.57 (m, 1H, pyrrolidine H-2 ), 2.24 (d, J = 9.1 Hz, 1H, pyrrolidine H-5 ), 2.15
(d, J = 9.2 Hz, 1H, pyrrolidine H-50), 1.91–1.85 (m, 1H, pyrrolidine H-40), 1.78–1.73 (m, 1H, pyrrolidine
H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 141.0 (C-3a), 139.0 (C-7a), 134.2 (benzyl C-1), 129.8
(phenyl-sulfonyl C-1”), 129.8 (phenyl-sulfonyl C-4”), 128.7 (phenyl-sulfonyl C-3”, phenyl-sulfonyl
C-5”), 128.7 (benzyl C-3, benzyl C-5), 127.4 (benzyl C-2, benzyl C-6), 127.4 (C-6), 124.9 (phenyl-sulfonyl
C-2”, phenyl-sulfonyl C-6”, benzyl C-4), 124.2 (C-4), 121.8 (C-5), 114.6 (C-7), 66.7 (pyrrolidine C-20),
0
0
63.5 (benzyl CH2), 59.3 (C-3), 53.3 (pyrrolidine C-5 ), 49.8 (C-4 ). MS (DUIS+) m/z 405 [M + H]+. HRMS
(ESI+): m/z [M + H]+ calcd. for C24H25N2O2S: 405.1637; found: 405.1620.
0
0
4.1.4. Procedures to Afford 1-(Phenylsulfonyl)-1 -(pyridin-4-ylmethyl)spiro[indoline-3,3 -pyrrolidine] (39b
)
Synthesis of 10-(Pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidin]-2-one (37b)
To the solution of the crude 2-(pyridin-4-ylmethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
◦
(
36b) (812 mg) in tetrahydrofuran:water (10:10 mL) was added 10 mL glacial acetic acid at 0 C.
◦
N-bromosuccinimide (549 mg, 3.08◦74 mmol) was added slowly in portions at 0 C.◦The reaction
mixture was stirred for 1.5 h at 0 C. Afterwards the reaction was quenched at 0 C by 20 mL
concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm up to room
temperature. The mixture was extracted using ethyl-acetateethyl acetate (2
organic phases were washed with concentrated sodium-hydrogen carbonate (2
brine (2 10 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated
×
10 mL). The combined
×
10 mL) and by
×
under reduced pressure. The crude product was purified using flash chromatography (gradient:
dichloromethane: methanol 0% to 10% methanol) to afford 37b as a yellow solid. Yield: 293 mg (35%
1
calculated to 25). H-NMR (500 MHz, DMSO-d6)
δ ppm 11.63 (s, 1H, NH), 8.52 (d, J = 5.6 Hz, 2H,
pyridine ArH-2, pyridine ArH-6), 7.59 (d, J = 7.2 Hz, 1H, ArH-4), 7.40 (d, J = 8.0 Hz, 1H, ArH-7), 7.32
(d, J = 5.5 Hz, 2H, pyridine ArH-3, pyridine ArH-5), 7.23 (t, J = 7.6 Hz, 1H, ArH-6), 7.05 (t, J = 7.3 Hz
,
1H, ArH-5), 4.72 (s, 2H, pyridyl-methyl CH2), 3.65 (t, J = 6.7 Hz, 2H, pyrrolidine H-20), 3.31 (m, 2H,
0
0
pyrrolidine H-5 ), 3.02 (t, J = 6.7 Hz, 2H, pyrrolidine H-4 ). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 179.3
(C=O), 148.5 (pyridine C-2, pyridine C-6), 147.2 (pyridine C-1), 141.6 (C-7a), 134.7 (C-3a), 128.1 (C-6),
123.5 (C-4), 122.4 (pyridine C-3, pyridine C-5), 109.7 (C-7), 61.2 (pyridyl-methylene CH2), 60.1 (C-3),
54.4 (pyrrolidine C-20), 53.6 (pyrrolidine C-50), 34.7 (pyrrolidine C-40). MS (DUIS+) m/z 280 [M + H]+.
Synthesis of 1-(Phenylsulfonyl)-10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidine] (39b)
To the solution of 10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidin]-2-one (37b) (289 mg,
1.05 mmol) in 10 mL anhydrousdry tetrahydrofuran was added borane/tetrahydrofuran (1 M) complex
solution (2.63 mL, 2.63 mmol, 2.5 equiv.) and the reaction mixture was refluxed overnight. The reaction
was allowed to cool down to room temperature and was quenched using 15 mL concentrated
ammonium-chloride solution and was stirred for 20 min. Afterwards, 10 mL ethyl acetate was used

Molecules 2017, 22, 2221
17 of 25
for extraction. The combined organic phases were dried over sodium-sulfate, filtered and evaporated
under reduced pressure to afford 292 mg 38b, used as a crude product in the further steps without
purification. To the solution of crude 10-(pyridin-4-ylmethyl)spiro[indoline-3,30-pyrrolidine] (38b
(0.292 mg) in 5 mL anhydrous dichloromethane was added triethylamine (0.307 mL, 2.22 mmol)
and N,N-dimethylpyridin-4-amine (6.8 mg, 0.0555 mmol) at room temperatu◦re. Afterwards
benzenesulfonyl chloride (204 mg, 1.161 mmol) was added to the solution at 5 C. The reaction
mixture was stirred at room temperature overnight. The reaction was quenched using 10 mL 10% HCl
solution and was extracted by 5 mL dichloromethane. The dichloromethane phase was collected and
dried over sodium-sulfate. The drying agent was filtered-off and the solution was evaporated under
)
reduced pressure. The crude product was purified by flash chromatography to afford 39b as yellow
1
solid. Yield: 0.003 mg. H-NMR (500 MHz, DMSO-d6)
δ
ppm 8.50 (d, J = 6.2 Hz, 2H, pyridine ArH-2,
pyridine ArH-6), 7.72 (d, J = 7.9 Hz, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.58–7.53
(m, 3H, pyridine ArH-3, pyridine ArH-5, phenyl-sulfonyl ArH-4”), 7.49 (d, J = 7.8 Hz, 1H, ArH-7), 7.43
(t, J = 8.0 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.28–7.23 (m, 2H, ArH-4, ArH-6),
7.07 (t, J = 7.1 Hz, 1H, ArH-5), 3.90 (d, J = 11.1 Hz, 1H, pyridyl methyl CH2), 3.83 (d, J = 11.1 Hz
,
1H, pyridyl methyl CH2), 3.74 (d, J = 15.2 Hz, 1H, H-2), 3.67 (d, J = 15.2 Hz, 1H, H-2), 2.77 (m, 1H,
pyrrolidine H-20), 2.62 (m, 1H, pyrrolidine H-20), 2.17 (m, 2H, pyrrolidine H-50), 1.95–1.89 (m, 1H,
pyrrolidine H-40), 1.82–1.78 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 148.6
(pyridine C-2, pyridine C-6), 141.0 (pyridine C-1), 143.9 (C-3a), 143.1 (C-7a), 139.5 (phenyl-sulfonyl
C-1”), 134.1 (phenyl-sulfonyl C-4”), 130.0 (phenyl-sulfonyl C-3,” phenyl-sulfonyl C-5”), 128.1 (C-6),
127.3 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 127.6 (C-4), 124.2 (pyridine C-3, pyridine C-5), 124.8
(C-5), 114.1 (C-7), 65.6 (pyrrolidine C-20), 61.5 (benzyl CH2), 60.1 (C-2), 53.0 (C-3), 51.3 (pyrrolidine
C-50), 32.9 (pyrrolidine C-40). MS (DUIS+) m/z 406 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for
C23H24N3O2S: 406.1589; found: 406.1586.
4.1.5. Synthesis of 10-Methyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (40a)
The solution of 10-benzyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (39a) (2 mg,
0.495 mmol) in 5 mL methanol was filled into an autoclave. Palladium (10%) on charcoal (100 mg,
5 w/w %) was added to the solution and reaction was stirred overnight under 5 bar hydrogen
atmosphere at 50 ◦C. The reaction mixture was filtered through a pad of Celite and was evaporated
under reduced pressure. The crude residual was purified by flash chromatography (gradient:
dichloromethane: methanol 0% to 10% methanol) to afford 40a as a colorless solid. Yield: 37 mg (23%).
1H-NMR (500 MHz, DMSO-d6)
δ ppm 7.79 (d, J = 7.5 Hz, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl
ArH-6”), 7.66 (t, J = 7.2 Hz, 1H, phenyl-sulfonyl ArH-4”), 7.56 (t, J = 7.9 Hz, 2H, phenyl-sulfonyl
ArH-3”, phenyl-sulfonyl ArH-5”), 7.48 (d, J = 7.9 Hz, 1H, ArH-7), 7.22 (m, 2H, ArH-4, H-6), 7.03
(t, J = 7.9 Hz, 1H, ArH-5), 3.87 (d, J = 11.8 Hz, 1H, H-2), 3.75 (d, J = 11.8 Hz, 1H, H-2), 2.62 (m,
1H, pyrrolidine H-20), 2.44 (m, 1H, pyrrolidine H-20), 2.24 (d, J = 9.1 Hz, 1H, pyrrolidine H-50), 2.17
(s, 3H, methyl CH3), 2.15 (d, J = 9.1 Hz, 1H, pyrrolidine H-50), 1.85–1.80 (m, 1H, pyrrolidine H-40),
1.74–1.69 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm 143.1 (C-3a, C-7a), 133.1
(phenyl-sulfonyl C-1”, phenyl-sulfonyl C-4”), 129.2 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 127.5
(C-6, phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 124.1 (C-4), 114.5 (C-5), 110.0 (C-7), 69.1 (pyrrolidine
C-20), 60.2 (pyrrolidine C-50), 55.5 (C-3), 52.8 (C-2), 42.3 (pyrrolidine N(10)-methyl), 32.5 (pyrrolidine
C-40). MS (DUIS+) m/z 329 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C18H21N2O2S: 343.1324;
found: 343.1320.
4.1.6. Synthesis of 10-Ethyl-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (40b)
0
0
The solution of 1 -benzyl-1-(phenylsulfonyl)spiro[indoline-3,3 -pyrrolidine] (39a) (100 mg, 0.2475
mmol) in 5 mL methanol was filled into an autoclave. Palladium (10%) on charcoal (500 mg, 5 w/w %)
◦
was added to the solution and reaction was stirred overnight under 5 bar hydrogen atmosphere at 50 C.
The reaction mixture was filtered through a pad of Celite and was evaporated under reduced pressure.

Molecules 2017, 22, 2221
18 of 25
The crude residual was purified by flash chromatography (gradient: dichloromethane: methanol 0% to
1
10% methanol) to afford 40b as a colorless solid. Yield: 19 mg (22%). H-NMR (500 MHz, DMSO-d6)
δ
ppm 7.78 (m, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.66 (m, 1H, phenyl-sulfonyl
ArH-4”), 7.56 (m, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.50–7.47 (m, 1H, ArH-7),
7.24–7.21 (m, 2H, ArH-4, ArH-6), 7.04 (t, J = 7.6 Hz, 1H, ArH-5), 3.86 (d, J = 10.8 Hz, 1H, H-2), 3.75 (d,
J = 10.8 Hz, 1H, H-2), 2.68 (m, 1H, pyrrolidine H-20), 2.44 (m, 1H, pyrrolidine H-20), 2.34 (q, J = 6.4 Hz,
2H, ethyl CH2), 2.24 (m, 1H, pyrrolidine H-50), 2.11 (q,0J = 8.9 Hz, 1H, pyrrolidine H-50), 1.86–180 (m,
0
1H, pyrrolidine H-4 ), 1.73–167 (m, 1H, pyrrolidine H-4 ), 0.93 (t, J = 7.6 Hz, 3H, ethyl CH3). 13C-NMR
(125 MHz, DMSO-d6) δ ppm 143.3 (C-3a, C-7a), 133.1 (phenyl-sulfonyl C-1”, phenyl-sulfonyl C-4”),
129.5 (phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 127.7 (C-6, phenyl-sulfonyl C-2”, phenyl-sulfonyl
C-6”), 124.1 (C-4), 114.1 (C-5), 110.1 (C-7), 69.1 (pyrrolidine C-20), 60.2 (pyrrolidine C-50), 55.3 (C-3),
52.1 (C-2), 50.0 (pyrrolidine N(10)-ethyl CH2), 32.5 (pyrrolidine C-40), 13.6 (pyrrolidine N(10)-ethyl
CH3). MS (DUIS+) m/z 343 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C19H23N2O2S: 343.1480;
found: 343.1496.
0
0
4.1.7. Procedures to Afford 1 -(2-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,3 -pyrrolidine] (44a
Synthesis of 10-(2-Fluorobenzyl)spiro[indoline-3,30-pyrrolidin]-2-one (42a)
)
To the solution of 4◦1a (1.54 g, 5.5 mmol) in tetrahydrofuran: water (30:30 mL) was added 30 mL
glacial acetic acid at 0 C. N-bromosuccinimide (979 mg, 5.5 mmol, 1 equiv.) was added slowly
◦
◦
in portions at 0 C. The reaction mixture was stirred for 1.5 h at 0 C. Afterwards the reaction
was quenched at 0 ◦C by 40 mL concentrated sodium-carbonate solution and was stirred for 0.5 h,
allowing to warm up to room temperature. The mixture was extracted using ethyl acetate (2 × 20 mL)
The combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 20 mL)
and by brine (2 20 mL). The organic phases were dried over sodium-sulfate, filtered and
.
×
evaporated under reduced pressure. The product 42a was purified by flash chromatography (gradient:
1
dichloromethane: methanol 0% to 10% methanol) Yield: 970 mg (56%). H-NMR (500 MHz, DMSO-d6)
δ
ppm 10.31 (s, 1H, NH), 8.01 (d, J = 8.8 Hz, 1H, benzyl ArH-6), 7.77 (d, J = 7.9 Hz, 1H, ArH-4), 7.65
(t, J = 7.9 Hz, 1H, benzyl ArH-4), 7.47 (t, J = 6.2 Hz, 1H, benzyl ArH-5), 7.24 (t, J = 8.4 Hz, 1H, ArH-6),
7.16 (m, 1H, benzyl ArH-3), 6.94 (t, J = 7.5 Hz, 1H, ArH-5), 6.80 (d, J = 7.5 Hz, 1H, ArH-7), 3.75 (s, 2H,
0
0
benzyl CH2), 3.06 (m, 1H, pyrrolidine H-2 ), 2.76 (d, J = 8.9 Hz, 1H, pyrrolidine H-5 ), 2.69 (d, J = 8.9 Hz
1H, pyrrolidine H-50), 2.61 (q, J = 7.9 Hz, 1H, pyrrolidine H-2’), 2.20–2.15 (m, 1H, pyrrolidine H-40),
1.92–1.87 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, DMSO-d6)
ppm 181.5 (C=O), 178.0 (benzyl
,
δ
C-2), 141.6 (C-7a), 137.0 (C-3a), 135.5 (benzyl C-6), 130.2 (benzyl C-4), 127.6 (C-6), 124.7 (benzyl C-1),
123.9 (benzyl C-5), 123.3 (C-4), 122.3 (C-5), 115.7 (benzyl C-3), 109.6 (C-7), 64.0 (C-3), 53.86 (benzyl CH2),
52.9 (pyrrolidine C-20), 51.7 (pyrrolidine C-50), 37.1 (pyrrolidine C-40). MS (DUIS+) m/z 297 [M + H]+
.
Synthesis of 10-(2-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44a)
Compound 43a was prepared according to the same procedure as 38a, starting from 42a (100 mg,
0.3378 mmol), using borane-tetrahydrofuran (0.6757 mL, 0.6757 mmol, 2.5 equiv.), to afford 10 mg
crude product used in the next step without further purification. Compound 44a was prepared
according to the same procedure as 39a, starting from crude 43a (10 mg, 0.03545 mmol), using
benzylsulfonyl chloride (50 mg, 0.07445 mmol, 1.05 equiv.), triethylamine (0.010 mL, 0.0709 mmol,
2 equiv.) and N,N-dimethylpyridin-4-amine (0.2 mg, 0.0017 mmol, 0.5 equiv.). The product was
purified by preparative-HPLC (gradient: water: acetonitrile 0% to 100% acetonitrile) Yield: 2 mg.
1H-NMR (500 MHz, CDCl3)
δ ppm 8.22 (t, J = 6.7 Hz, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl
ArH-5”), 7.88 (m, 1H, phenyl-sulfonyl ArH-4”), 7.69 (m, 2H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl
ArH-6”), 7.53 (t, J = 6.2 Hz, 1H, benzyl ArH-4), 7.47 (m, 1H, ArH-7), 7.33 (t, J = 7.8 Hz, 1H, benzyl
ArH-5), 7.23 (t, J = 7.8 Hz, 1H, benzyl ArH-6), 7.17–7.08 (m, 2H, ArH-5, ArH-6), 6.73 (d, J = 7.0 Hz, 2H,
0
benzyl ArH-3), 4.22 (m, 2H, H-2), 3.98 (m, 2H, benzyl CH2), 3.75 (m, 1H, pyrrolidine H-2 ), 3.40 (m, 1H,

Molecules 2017, 22, 2221
19 of 25
0
0
0
pyrrolidine H-2 ), 3.14 (m, 1H, pyrrolidine H-5 ), 2.98 (m, 1H, pyrrolidine H-5 ), 2.28 (m, 1H, pyrrolidine
H-40), 1.51 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, CDCl3)
δ ppm 159.3 (benzyl C-2), 144.1
(C-3a), 142.5 (C-7a), 137.3 (phenyl-sulfonyl C-1”), 134.2 (phenyl-sulfonyl C-4”), 130.3 (benzyl C-6), 129.5
(phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 129.2 (benzyl C-4), 128.2 (C-6), 127.6 (phenyl-sulfonyl
C-2”, phenyl-sulfonyl C-6”), 125.8 (benzyl C-1), 125.3 (benzyl C-5), 125.0 (C-4), 121.6 (C-5), 115.7
(benzyl C-3), 113.0 (C-7), 62.3 (C-20), 59.6 (benzyl CH2), 55.8 (C-3), 54.9 (C-50), 53.6 (C-2), 33.1 (C-40).
MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C24H24N2O2FS: 423.1543;
found: 423.1531.
4.1.8. Procedures to Afford 10-(3-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44b)
Synthesis of 10-(3-Fluorobenzyl)spiro[indoline-3,30-pyrrolidin]-2-one (42b)
To the solution of 41b (1.681 g, 6 mmol) in tetrahydrofuran: water (30:30 mL) was added 30 mL
◦
glacial acetic acid at 0 C. N-bromosuccinimide (1.069 g, 6 mmol, 1 equiv.) was added slowly in portions
at 0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was quenched at
0 ◦C by 40 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing to warm
up to room temperature. The mixture was extracted using ethyl acetate (2
organic phases were washed with concentrated sodium-hydrogen carbonate (2
brine (2 20 mL). The organic phases were dried over sodium-sulfate, filtered and evaporated under
×
20 mL). The combined
×
20 mL) and by
×
reduced pressure. The product 42b was purified by flash chromatography (gradient: dichloromethane:
1
methanol 0% to 10% methanol) Yield: 1.31 g (76% calculated to 29). H-NMR (500 MHz, DMSO-d6) δ
ppm 10.31 (s, 1H, NH), 7.38–7.33 (m, 2H, benzyl H-5, benzyl ArH-6), 7.21–7.16 (m, 3H, ArH-4, benzyl
H-2, benzyl ArH-4), 7.04 (m, 1H, ArH-6), 6.98 (t, J = 7.0 Hz, 1H, ArH-5), 6.81 (d, J = 8.0 Hz, 1H, ArH-7),
3.72 (s, 2H, benzyl CH2), 3.07 (m, 1H, pyrrolidine H-20), 2.74 (d, J = 8.8 Hz, 1H, pyrrolidine H-500),
0
0
2.66 (d, J = 8.8 Hz, 1H, pyrrolidine H-2 ), 2.56 (m, 1H, pyrrolidine H-5 ), 2.20 (m, 1H, pyrrolidine H-4 ),
1.90 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, CDCl3)
δ ppm 181.96 (C=O), 161.2 (benzyl C-3),
142.3 (C-7a), 141.4 (benzyl C-1), 130.4 (benzyl C-5), 127.8 (benzyl-C6, C-6), 122.1 (benzyl C-2, benzyl
C-4), 109.4 (C-7), 64.7 (benzyl CH2), 63.9 (C-3), 53.9 (pyrrolidine C-20), 52.7 (C-50), 36.8 (pyrrolidine
C-40). MS (DUIS+) m/z 297 [M + H]+.
Synthesis of 10-(3-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44b)
Compound 43b was prepared according to the same procedure as 38a, starting from 42b (385 mg,
1.3 mmol), using borane-tetrahydrofuran (3.250 mL, 3.25 mmolm 2.5 equiv.), to afford 338 mg crude
product used in the next time without further purification. Compound 44b was prepared according
to the same procedure as 39a, starting from crude 43b (338 mg, 1.197 mmol), using benzylsulfonyl
chloride (221 mg, 1.2579 mmol, 1.05 equiv.), triethylamine (0.331 mL, 2.396 mmol, 2 equiv.) and
N,N-dimethylpyridin-4-amine (7 mg, 0.0599 mmol, 0.5 equiv.). The product was purified by preparative
1
HPLC (gradient: dichloromethane: methanol 0% to 10% methanol) Yield: 11 mg. H-NMR (500 MHz,
CDCl3) δ ppm 7.83 (d, J = 8.2 Hz, 1H, ArH-7), 7.77 (d, J = 7.6 Hz, 1H, benzyl ArH-6), 7.73 (d, J = 7.6
Hz, 1H, ArH-4), 7.64 (d, J = 8.2 Hz, 1H, benzyl ArH-4), 7.58–7.53 (m, 1H, benzyl ArH-5), 7.50–7.39
(m, 3H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-4”, phenyl-sulfonyl ArH-5”), 7.37–7.27 (m, 2H,
phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”), 7.16–7.12 (m, 1H, phenyl-sulfonyl ArH-6), 7.09–7.02
(m, 1H, phenyl-sulfonyl ArH-5), 6.99 (m, 1H, benzyl H-2), 4.23 (m, 1H, H-2), 4.11 (m, 1H, H-2),
3.93–3.71 (m, 2H, benzyl CH2), 3.23 (m, 1H, pyrrolidine H-20), 2.94 (m, 1H, pyrrolidine H-20), 2.83
(m, 1H, pyrrolidine H-50), 2.69 (m, 1H, pyrrolidine H-50), 2.39–2.25 (m, 1H, pyrrolidine H-40), 2.18
(m, 1H, pyrrolidine H-50). 13C-NMR (125 MHz, CDCl3)
δ ppm 163.0 (benzyl C-3), 143.6 (C-3a),
143.5 (C-7a), 137.6 (phenyl-sulfonyl C-1”), 134.2 (phenyl-sulfonyl C-4”), 130.4 (benzyl C-5), 129.5
(phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 129.1 (C-6), 128.0 (phenyl-sulfonyl C-2”, phenyl-sulfonyl
C-6”), 127.7 (benzyl C-6), 125.6 (benzyl C-1), 125.3 (C-4), 124.1 (C-5), 121.8, 115.8 (benzyl C-2), 113.4
(C-7), 113.3 (benzyl C-4), 61.2 (benzyl CH2), 59.9 (pyrrolidine C-20), 55.3 (C-3), 54.8 (pyrrolidine C-50),

Molecules 2017, 22, 2221
20 of 25
53.6 (C-2), 33.1 (pyrrolidine C-4’). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd.
for C24H24N2O2SF: 423.1543; found: 423.1558.
0
0
4.1.9. Procedures to Afford 1 -(4-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,3 -pyrrolidine] (44c
Synthesis of 10-(4-Fluorobenzyl)spiro[indoline-3,30-pyrrolidin]-2-one (42c)
)
To the solution of 41c◦(2.361 g, 8.4321 mmol) in tetrahydrofuran: water (30:30 mL) was added 30
mL glacial acetic acid at 0 C. N-bromosuccinimide (1.501 g, 8.4321 mmol, 1 equiv.) was added slowly
in portions at ◦0 ◦C. The reaction mixture was stirred for 1.5 h at 0 ◦C. Afterwards the reaction was
quenched at 0 C by 40 mL concentrated sodium-carbonate solution and was stirred for 0.5 h, allowing
to warm up to room temperature. The mixture was extracted using ethyl acetate (2 × 20 mL). The
combined organic phases were washed with concentrated sodium-hydrogen carbonate (2 × 20 mL
)
and by brine (2
×
20 mL). The organic phases were dried over sodium-sulfate, filtered and
evaporated under reduced pressure. The product 42c was purified by flash chromatography (gradient:
dichloromethane: methanol 0% to 10% methanol) Yield: 320 mg (19% calculated to 25). 1H-NMR
(500 MHz, DMSO-d6) δ ppm 10.30 (s, 1H, NH), 7.41–7.35 (m, 3H, benzyl H-2, benzyl ArH-6, ArH-4),
7.16 (m, 3H, benzyl ArH-3, benzyl ArH-5, ArH-6), 6.96 (t, J = 7.2 Hz, 1H, ArH-5), 6.81 (d, J = 7.4 Hz,
0
1H, ArH-7), 5.73 (s, 2H, benzyl CH2), 3.03 (m, 1H, pyrrolidine H-5 ), 2.73 (d, J = 9.3 Hz, 1H, pyrrolidine
H-20), 2.64 (d, J = 9.3 Hz, 1H, pyrrolidine H-20), 2.58–2.53 (m, 1H, pyrrolidine H-50), 2.21–2.16 (m, 1H,
pyrrolidine H-40), 1.95–1.87 (m, 1H, pyrrolidine H-40) 13C-NMR (125 MHz, DMSO-d6)
δ ppm 179.1
(C=O), 161.9 (benzyl C-4), 141.6 (C-7a), 138.2 (benzyl C-1), 134.6 (C-3a), 130.7 (benzyl C-2, benzyl C-6),
128.0 (C-6), 123.3 (C-4), 122.3 (C-2), 115.4 (benzyl C-3, benzyl C-5), 109.6 (C-7), 64.0 (benzyl CH2), 58.3
(C-3), 54.0 (pyrrolidine C-20), 52.9 (pyrrolidine C-50), 37.0 (pyrrolidine C-40). MS (DUIS+) m/z 297
[M + H]+.
Synthesis of 10-(4-Fluorobenzyl)-1-(phenylsulfonyl)spiro[indoline-3,30-pyrrolidine] (44c)
Compound 43c was prepared according to the same procedure as 38a, starting from 42c (310 mg,
1.0473 mmol), using borane-tetrahydrofuran (2.620 mL, 2.620 mmol, 2.5 equiv.), to afford 461 mg
crude product used in the next step without further purification. Compound 44c was prepared
according to the same procedure as 39a, starting from crude 43c (100 mg, 0.354 mmol), using
benzylsulfonyl chloride (131 mg, 0.745 mmol, 1.05 equiv.), triethylamine (0.099 mL, 0.7092 mmol)
and N,N-dimethylpyridin-4-amine (2 mg, 0.01773 mmol). The product 44c was purified by flash
1
chromatography (gradient: dichloromethane: methanol 0% to 10% methanol) Yield: 10 mg. H-NMR
(500 MHz, CDCl3) δ ppm 7.84–7.77 (m, 2H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.74–7.70
(m, 1H, phenyl-sulfonyl ArH-4”), 7.67–7.57 (m, 3H, phenyl-sulfonyl ArH-2”, phenyl-sulfonyl ArH-6”,
ArH-7), 7.50–7.45 (m, 2H, benzyl ArH-2, benzyl ArH-6), 7.43–7.40 (m, 1H, ArH-4), 7.24–7.20 (m, 1H,
ArH-6), 7.16–7.12 (m, 2H, benzyl ArH-3, benzyl ArH-5), 7.09 (m, 1H, ArH-5), 4.39 (m, 1H, H-2), 4.29
(m, 1H, H-2), 4.18 (m, 1H, benzyl CH2), 413.06 (m, 1H, benzyl CH2), 3.90 (m, 1H, pyrrolidine H-20),
3.70 (m, 1H, pyrrolidine H-20), 3.06 (m, 1H), 2.68 (m, 1H, pyrrolidine H50), 2.33 (m, 1H, pyrrolidine
H-50), 2.33 (m, 1H, pyrrolidine H-40), 1.75 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, CDCl3)
δ
ppm 159.8 (benzyl C-4), 144.1 (C-3a), 142.6 (C-7a), 137.5 (benzyl C-1), 134.2 (phenyl-sulfonyl C-1”,
phenyl-sulfonyl C-4”), 130.7 (benzyl C-2, benzyl C-6), 129.2 (phenyl-sulfonyl C-3”, phenyl-sulfonyl
C-5”), 128.3 (C-6), 127.7 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 125.8 (C-4), 125.2 (C-5), 115.5
(benzyl C-3, benzyl C-5), 113.1 (C-7), 62.4 (pyrrolidine C-20), 59.8 (benzyl CH2), 55.5 (C-3), 55.0
(pyrrolidine C-50), 53.7 (C-2), 33.0 (pyrrolidine C-40). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+):
m/z [M + H]+ calcd. for C24H24N2O2SF: 423.1543; found: 423.1542.
4.1.10. Synthesis of 10-Benzyl-1-((2-fluorophenyl)sulfonyl)spiro[indoline-3,30-pyrrolidine] (46a)
Compound 46a was prepared according to the same procedure as 39a, starting from crude 38a (15
mg, 0.0567 mmol), using 2-fluorobenzylsulfonyl chloride (23 mg, 0.11819 mmol), triethylamine (0.015

Molecules 2017, 22, 2221
21 of 25
mL, 0.1188 mmol, 2.1 equiv.) and N,N-dimethylpyridin-4-amine (0.7 mg, 0.006 mmol, 0.1 equiv.).
The product was purified by flash chromatography (gradient: dichloromethane: methanol 0%
to 10% methanol) Yield: 6.5 mg. 1H-NMR (500 MHz, CDCl3)
δ ppm 8.00 (t, J = 6.7 Hz, 1H,
phenyl-sulfonyl ArH-4”), 7.53 (m, 3H, phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-6”, ArH-7),
7.41–7.34 (m, 5H, benzyl ArH-2, benzyl ArH-3, benzyl ArH-5, benzyl ArH-6, ArH-4), 7.27 (t, J =
8.1 Hz, 1H, phenyl-sulfonyl ArH-5”), 7.17 (t, J = 8.1 Hz, 1H, benzyl ArH-4), 7.10 (t, J = 9.4 Hz,
1H, ArH-6), 7.04 (t, J = 7.6 Hz, 1H, ArH-5), 4.16–4.07 (m, 5H, benzyl CH2, H-2, pyrrolidine H-20),
3.34 (m, 1H, pyrrolidine H-20), 3.17 (m, 1H, pyrrolidine H-50), 3.05 (m, 1H, pyrrolidine H-50), 2.36
(m, 1H, pyrrolidine H-40), 2.14 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, DMSO-d6)
δ ppm
155.6 (phenyl-sulfonyl C-2”), 149.7 (C-3a), 142.8 (C-7a), 133.2 (benzyl C-1), 131.8 (phenyl-sulfonyl
C-4”), 131.0 (phenyl-sulfonyl C-6”), 129.6 (benzyl C-3, benzyl C-5), 128.4 (benzyl C-2, benzyl C-6),
124.2 (phenyl-sulfonyl C-5”), 122.9 (benzyl C-4), 122.5 (C-4), 121.0 (C-5), 120.4 (phenyl-sulfonyl C-1”),
116.8 (phenyl-sulfonyl C-3”), 111.3 (C-7), 63.2 (pyrrolidine C-20), 62.6 (benzyl CH2), 55.1 (C-3), 52.5
(pyrrolidine C-50), 49.2 (C-2), 37.1 (pyrrolidine C-40). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+):
m/z [M + H]+ calcd. for C24H24N2O2SF: 423.1543; found: 423.1550.
4.1.11. Synthesis of 10-Benzyl-1-((3-fluorophenyl)sulfonyl)spiro[indoline-3,30-pyrrolidine] (46b)
Compound 45b was prepared according to the same procedure as 39a, starting from crude 38a (15
mg, 0.0567 mmol), using 3-fluorobenzylsulfonyl chloride (23 mg, 0.1188 mmol, 2.1 equiv.), triethylamine
(0.015 mL, 0.1188 mmol, 2.1 equiv.) and N,N-dimethylpyridin-4-amine (0.7 mg, 0.006 mmol, 0.1 equiv.).
The product was purified by flash chromatography (gradient: dichloromethane: methanol 0% to 10%
1
methanol) Yield: 5.5 mg. H-NMR (500 MHz, CDCl3)
δ
ppm 8.25 (m, 1H, phenyl-sulfonyl ArH-2”),
7.65–7.58 (m, 2H, phenyl-sulfonyl ArH-5”, phenyl-sulfonyl ArH-6”), 7.48–7.38 (m, 6H, ArH-7, benzyl
ArH-2–6), 7.29–7.25 (m, 2H, ArH-4, ArH-6), 7.21 (m, 1H, phenyl-sulfonyl ArH-4”), 7.08 (t, J = 7.6 Hz,
1H, ArH-5), 4.16 (d, J = 12.7 Hz, 1H, benzyl CH2), 4.09 (d, J = 12.7 Hz, 1H, benzyl CH2), 3.99 (d, J = 10.9
Hz, 1H, H-2), 3.85 (d, J = 10.9 Hz, 1H, H-2), 3.33–3.24 (m, 2H, pyrrolidine H-20), 3.12 (d, J = 11.6 Hz,
1H, pyrrolidine H-50), 3.00 (d, J = 11.6 Hz, 1H, pyrrolidine H-50), 2.26–2.20 (m, 1H, pyrrolidine H-40),
2.05–2.00 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz, CDCl3)
δ ppm 158.6 (phenyl-sulfonyl C-3”),
146.8 (C-3a), 145.4 (C-7a), 137.5 (benzyl C-1), 130.5 (phenyl-sulfonyl C-1”), 129.7 (phenyl-sulfonyl C-5”),
129.0 (benzyl C-3, benzyl C-5), 128.7 (benzyl C-2, benzyl C-6), 124.5 (C-6), 122.8 (benzyl C-4), 122.7
(phenyl-sulfonyl C-6”), 121.7 (C-4), 120.2 (C-5), 117.7 (phenyl-sulfonyl C-4”), 114.3 (phenyl-sulfonyl
C-2”), 109.5 (C-7), 63.0 (pyrrolidine C-20), 62.6 (benzyl CH2), 55.4 (C-3), 52.7 (pyrrolidine C-50), 49.2
(C-2), 37.9 (pyrrolidine C-40). MS (DUIS+) m/z 423 [M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for
C24H24N2O2SF: 423.1543; found: 423.1547.
4.1.12. Synthesis of 10-Benzyl-1-((4-fluorophenyl)sulfonyl)spiro[indoline-3,30-pyrrolidine] (46c)
Compound 46c was prepared according to the same procedure as 39a, starting from crude
38a (15 mg, 0.0567 mmol), using 4-fluorobenzylsulfonyl chloride (23 mg, 0.1182 mmol, 2.1 equiv.),
triethylamine (0.015 mL, 0.1188 mmol, 2.1 equiv.) and N,N-dimethylpyridin-4-amine (0.7 mg,
0.006 mmol, 0.1 equiv.). The product was purified by flash chromatography (gradient: dichloromethane:
methanol 0% to 10% methanol) Yield: 7.7 mg. 1H-NMR (500 MHz, CDCl3)
δ ppm 7.80 (m, 2H,
phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.63 (d, J = 7.9 Hz, 1H, ArH-7), 7.47 (m, 2H,
benzyl H-3, benzyl ArH-5), 7.40 (m, 3H, benzyl ArH-2, benzyl ArH-4, benzyl ArH-6), 7.28 (m, 2H,
phenyl-sulfonyl ArH-3”, phenyl-sulfonyl ArH-5”), 7.09–7.05 (m, 3H, ArH-4, ArH-5, ArH-6), 4.10 (d,
J = 12.3 Hz, 1H, H-2), 4.02–3.96 (m, 2H, benzyl CH2), 3.84 (d, J = 11.3 Hz, 1H, H-2), 3.25 (m, 1H,
0
0
0
pyrrolidine H-2 ), 3.16 (m, 1H, pyrrolidine H-2 ), 2.94 (m, 1H, pyrrolidine H-5 ), 2.89 (m, 1H, pyrrolidine
H-50), 2.24–2.18 (m, 1H, pyrrolidine H-40), 2.03–1.99 (m, 1H, pyrrolidine H-40). 13C-NMR (125 MHz,
CDCl3)
δ ppm 163.7 (phenyl-sulfonyl C-4”), 152.7 (C-3a), 150.6 (C-7a), 147.0 (benzyl C-1), 129.6
(phenyl-sulfonyl C-1”), 129.5 (phenyl-sulfonyl C-2”, phenyl-sulfonyl C-6”), 129.2 (benzyl C-3, benzyl
C-5), 129.1 (benzyl C-2, benzyl C-6), 128.8 (C-6), 128.6 (benzyl C-4), 125.6 (C-4), 124.4 (C-5), 122.8

Molecules 2017, 22, 2221
22 of 25
(phenyl-sulfonyl C-3”, phenyl-sulfonyl C-5”), 116.1 (C-7), 63.1 (pyrrolidine C-20), 62.4 (benzyl CH2),
54.1 (C-3), 52.8 (pyrrolidine C-50), 49.1 (pyrrolidine C-20), 36.6 (pyrrolidine C-40). MS (DUIS+) m/z 423
[M + H]+. HRMS (ESI+): m/z [M + H]+ calcd. for C24H24N2O2SF: 423.1543; found: 423.1563.
4.2. General Procedure for the Serotonergic Screening Assays
4.2.1. Cell Culture and Preparation of Cell Membranes
HEK293 cells with stable expression of human serotonin 5-HT1A, 5-HT6 or 5-HT7b receptors
(all prepared with the use of Lipofectamine 2000) or CHO-K1 cells with plasmid containing the
◦
sequence coding for the human serotonin 5-HT2A receptor (PerkinElmer) were maintained at 37 C in a
humidified atmosphere with 5% CO2 and were grown in Dulbeco’s Modifier Eagle Medium containing
10% dialysed fetal bovine serum and 500 µg/mL G418 sulphate. For membranes preparations, cells
◦
were subcultured in 150 cm2 flasks, grown to 90% confluence, washed twice with prewarmed to 37 C
phosphate buffered saline (PBS) and were pelleted by centrifugation (200 g) in PBS containing 0.1 mM
EDTA and 1 mM dithiothreitol. Prior to membrane preparations pellets were stored at −80 ◦C.
4.2.2. Radioligand Binding Assays
Cell pellets were thawed and homogenized in 10 volumes of assay buffer using an Ultra Turrax
◦
tissue homogenizer and centrifuged twice at 35,000 g for 20 min at 4 C, with incubation for 15 min at
◦
37 C in between. The composition of the assay buffers was as follows: for 5-HT1AR: 50 mM Tris–HCl,
0.1 mM EDTA, 4 mM MgCl2, 10
µM pargyline and 0.1% ascorbate; for 5-HT2AR: 50 mM Tris–HCl, 0.1
mM EDTA, 4 mM MgCl2 and 0.1% ascorbate; for 5-HT6R: 50 mM Tris–HCl, 0.5 mM EDTA and 4 mM
MgCl2, for 5-HT7bR: 50 mM Tris–HCl, 4 mM MgCl2, 10
were incubated in a total volume of 200
µ
M pargyline and 0.1% ascorbate. All assays
◦
µ
L in 96-well microtiter plates for 1 h at 37 C, except for
5-HT1AR and 5-HT2AR which were incubated at room temperature for 1 h and 1.5 h respectively.
The process of equilibration is terminated by rapid filtration through Unifilter plates with a 96-well
cell harvester and radioactivity retained on the filters was quantified on a Microbeta plate reader
(PerkinElmer).
For displacement studies the assay samples contained as radioligands: 2.5 nM [3H]-8-OH-DPAT
(5002.4 GBq/mmol) for 5-HT1AR; 1 nM [3H]-Ketanserin (1975.8 GBq/mmol) for 5-HT2AR; 2 nM
[3H]-LSD (3093.2 GBq/mmol) for 5-HT6R or 0.8 nM [3H]-5-CT (1450.4 GBq/mmol) for 5-HT7R.
Non-specific binding is defined with 10
µM of 5-HT in 5-HT1AR and 5-HT7R binding experiments,
whereas 10 M of chlorpromazine or 10
µ
µM of methiothepine were used in 5-HT2AR and 5-HT6R
assays, respectively. Each compound was tested in triplicate at 7–8 concentrations (10−11–10−4 M).
The inhibition constants (Ki) were calculated from the Cheng-Prusoff equation [47]. Results were
expressed as means of at least three separate experiments (SD ≤ 24%).
4.2.3. Docking Procedure
The homology model of h-5HT6R [15] was prepared for docking experiment my Schrödinger’s
Protein Preparation Wizard by default settings for protomer-state optimization and restrained
minimization by OPLS_2005 force field. Single precision docking was performed with Schrödinger’s
Glide by default settings. The ligand (46a) was prepared for docking by Schrödinger’s LigPrep,
generating possible ionization states (at pH range of 7.0
Constrained docking was set to two required interactions (at least 1 match): hydrogen bond formed
with Asn2886.55 and/or hydrogen bond formed with Ser1935.43
±
2.0), tautomers and stereoisomers.
.
Acknowledgments: The authors are grateful to László Drahos and Ágnes Gömöry (RCNS, Hungary) for the exact
mass (high-resolution MS) measurements and we are thankful for Aaron Keeley (RCNS, Hungary) for carefully
reading the manuscript and for Stefan Mordalski (Institute of Pharmacology, Poland) for helpful discussions.
The support of the National Brain Research Program (KTIA_NAP_13-1-2013-0001) is acknowledged.

Molecules 2017, 22, 2221
23 of 25
Author Contributions: G.M.K. and A.J.B. conceived the study; Á.A.K. performed the syntheses, G.S. performed
the in vitro measurements.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Russel, J.S. Oxindoles and spirocyclic variations: Strategies for C3 functionalization. Top. Heterocycl. Chem.
2011, 26, 397–432. [CrossRef]
2.
3.
4.
Bindra, J.S. Oxindole alkaloids. Alkaloids Chem. Physiol. 1973, 14, 83–121. [CrossRef]
ChEMBL. Available online: Https://www.ebi.ac.uk/chembl/ (accessed on 12 October 2017).
Kelemen, Á.A.; Ferenczy, G.G.; Keseru, G.M. A desirability function-based scoring scheme for selecting
fragment-like class A aminergic GPCR ligands. J. Comput. Aided Mol. Des. 2015, 29, 59–66. [CrossRef]
[PubMed]
5.
Kelemen, Á.A.; Kiss, R.; Ferenczy, G.G.; Kovács, L.; Flachner, B.; Lorincz, Z.; Keseru, G.M. Structure-based
consensus scoring scheme for selecting class A aminergic GPCR fragments. J. Chem. Inf. Model. 2016, 56,
412–422. [CrossRef] [PubMed]
6.
7.
Upton, N.; Chuang, T.T.; Hunter, A.J.; Virley, D.J. 5-HT6 receptor antagonists as novel cognitive enhancing
agents for Alzheimer’s disease. Neurotherapeutics 2008, 5, 458–469. [CrossRef] [PubMed]
Messina, D.; Annesi, G.; Serra, P.; Nicoletti, G.; Pasqua, A.; Annesi, F.; Tomaino, C.; Cirò-Candiano, I.C.;
Carrideo, S.; Caracciolo, M.; et al. Association of the 5-HT6 receptor gene polymorphism C267T with
Parkinson’s disease. Neurology 2002, 58, 828–829. [CrossRef] [PubMed]
8.
9.
Codony, X.; Vela, J.M.; Ramírez, M.J. 5-HT6 receptor and cognition. Curr. Opin. Pharmacol. 2011, 11, 94–100.
[CrossRef] [PubMed]
Holenz, J.; Mercè, R.; Díaz, J.L.; Guitart, X.; Codony, X.; Dordal, A.; Romero, G.; Torrens, A.; Mas, J.;
Andaluz, B.; et al. Medicinal chemistry driven approaches toward novel and selective serotonin 5-HT6
receptor ligands. J. Med. Chem. 2005, 48, 1781–1795. [CrossRef] [PubMed]
10. Kobilka, B.K. G protein coupled receptor structure and activation. Biochim. Biophys. Acta Biomembr. 2007
1768, 794–807. [CrossRef] [PubMed]
,
11. Ivachtchenko, A.V. Sulfonyl-containing modulators of serotonin 5-HT6 receptors and their pharmacophore
models. Russ. Chem. Rev. 2014, 83, 439–473. [CrossRef]
12. Staron´, J.; Warszycki, D.; Kalinowska-Tłus´cik, J.; Satała, G.; Bojarski, A.J. Rational design of 5-HT6R ligands
using a bioisosteric strategy: Synthesis, biological evaluation and molecular modelling. RSC Adv. 2015, 5,
25806–25815. [CrossRef]
13. Mcule. Available online: https://mcule.com/ (accessed on 12 October 2017).
14. Small-Molecule Drug Discovery Suite 2017-3; Schrödinger, LLC: New York, NY, USA, 2017.
15. Vass, M.; Jójárt, B.; Bogár, F.; Paragi, G.; Keseru, G.M.; Tarcsay, Á. Dynamics and structural determinants
of ligand recognition of the 5-HT6 receptor. J. Comput. Aided Mol. Des. 2015, 29, 1137–1149. [CrossRef]
[PubMed]
16. Wacker, D.; Wang, C.; Katritch, V.; Han, G.W.; Huang, X.-P.; Vardy, E.; McCorvy, J.D.; Jiang, Y.; Chu, M.;
Siu, F.Y.; et al. Structural features for functional selectivity at serotonin receptors. Science 2013, 340, 615–619.
[CrossRef] [PubMed]
17. Kelemen, Á.A.; Mordalski, S.; Bojarski, A.J.; Keseru˝, G.M. Computational modeling of drugs for Alzheimer’s
disease: Design of serotonin 5-HT6 antagonists. In Computational Modeling of Drugs against Alzheimer’s Disease;
Humana Press: New York, NY, USA, 2017; Volume 132, pp. 419–461. ISBN 978-1-4939-7403-0.
18. Laronze, J.-Y.; Bascop, S.I.; Sápi, J.; Levy, J. On the synthesis of the oxindole alkaloid: (
Heterocycles 1994, 38. [CrossRef]
±
)-Horsfiline.
19. Overman, L.E.; Rosen, M.D. Total synthesis of ( )-spirotryprostatin B and three stereoisomers. Angew. Chem.
−
Int. Ed. 2000, 39, 4596–4599. [CrossRef]
20. Finch, N.; Taylor, W.I. The conversion of tetrahydro-β-carboline alkaloids into oxindoles. The structures and
partial syntheses of mitraphylline and rhyn cophylline. J. Am. Chem. Soc. 1962, 84, 1318–1320. [CrossRef]
21. Wang, H.; Ganesan, A. A biomimetic total synthesis of ( )-spirotryprostatin B and related studies. J. Org. Chem.
−
2000, 65, 4685–4693. [CrossRef] [PubMed]

Molecules 2017, 22, 2221
24 of 25
22. Sebahar, P.R.; Williams, R.M. The asymmetric total synthesis of (+)- and (
−
)-spirotryprostatin B. J. Am.
Chem. Soc. 2000, 122, 5666–5667. [CrossRef]
23. Jones, K.; Wilkinson, J. A total synthesis of horsfiline via aryl radical cyclisation. J. Chem. Soc. Chem. Commun.
1992, 5, 1767–1769. [CrossRef]
24. Abelman, M.M.; Oh, T.; Overman, L.E. Intramolecular alkene arylations for rapid assembly of polycyclic
systems containing quaternary centers. A new synthesis of spirooxindoles and other fused and bridged ring
systems. J. Org. Chem. 1987, 52, 4130–4133. [CrossRef]
25. Fuji, K.; Node, M.; Nagasawa, H.; Naniwa, Y.; Terada, S. Asymmetric Induction via addition-elimination
process: Nitroolefination of α-substituted lactones. J. Am. Chem. Soc. 1986, 108, 3855–3856. [CrossRef]
26. Stahl, R.; Borschberg, H.-J. A Reinvestigation of the oxidative rearrangement of yohimbane-type alkaloids.
Part A. Formation of pseudoindoxyl (=1,2-dihydro-3H-indol-3-one) derivatives. Helv. Chim. Acta 1994, 77,
1331–1345. [CrossRef]
27. Shavel, J.; Zinnes, H.; Oxindole Alkaloids, I. Oxidative-rearrangement of indole alkaloids to their oxindole
analogs. J. Am. Chem. Soc. 1962, 84, 1320–1321. [CrossRef]
28. Martin, S.F.; Mortimore, M. New methods for the synthesis of oxindole alkaloids. Total syntheses of
isopteropodine and pteropodine. Tetrahedron Lett. 1990, 31, 4557–4560. [CrossRef]
29. Takayama, H.; Seki, N.; Kitajima, M.; Aimi, N.; Seki, H.; Sakai, S.-I. Indoloquinolizidine and yohimbine
were converted to the corresponding Na-methoxyindoles and Na-methoxyoxindoles by the oxidation of the
dihydroindole derivatives with H2O2 and sodium tungstate. Heterocycles 1992, 33, 121–125. [CrossRef]
30. Yu, P.; Cook, J.M. Diastereospecific synthesis of ketooxindoles. Potential intermediates for the synthesis
of alstonisine as well as for voachalotine related oxindole alkaloids. Tetrahedron Lett. 1997, 38, 8799–8802.
[CrossRef]
31. Pellegrini, C.; Strässler, C.; Weber, M.; Borschberg, H.J. Synthesis of the oxindole alkaloid ( )-horsfiline.
−
Tetrahedron Asymmetry 1994, 5, 1979–1992. [CrossRef]
32. Edmondson, S.D.; Danishefsky, S.J. The total synthesis of spirotryprostatin A. Angew. Chem. Int. Ed. 1998, 37,
1138–1140. [CrossRef]
33. Edmondson, S.; Danishefsky, S.J.; Sepp-Lorenzino, L.; Rosen, N. Total synthesis of spirotryprostatin A,
leading to the discovery of some biologically promising analogues. J. Am. Chem. Soc. 1999, 121, 2147–2155.
[CrossRef]
34. Pictet, A.; Spengler, T. Über die Bildung von Isochinolin Derivaten durch Einwirkung von Methylal auf
Phenylethylamin, Phenylalanin und Tyrosin. Eur. J. Inorg. Chem. 1911, 44, 2030–2036. [CrossRef]
35. Zhao, Y.; Yu, S.; Sun, W.; Liu, L.; Lu, J.; McEachern, D.; Shargary, S.; Bernard, D.; Li, X.; Zhao, T.; et al.
A potent small-molecule inhibitor of the MDM2-p53 interaction (MI-888) achieved complete and durable
tumor regression in mice. J. Med. Chem. 2013, 56, 5553–5561. [CrossRef] [PubMed]
36. Kath, J.C.; Tom, N.J.; Cox, E.D.; Bhattacharya, S.K. Heteroaromatic Bicyclic Derivatives Useful as Anticancer
Agents. U.S. Patent 6,867,201 B2, 22 August 2002.
37. Powell, N.A.; Kohrt, J.T.; Filipski, K.J.; Kaufman, M.; Sheehan, D.; Edmunds, J.E.; Delaney, A.; Wang, Y.;
Bourbonais, F.; Lee, D.Y.; et al. Novel and selective spiroindoline-based inhibitors of sky kinase. Bioorg. Med.
Chem. Lett. 2012, 22, 190–193. [CrossRef] [PubMed]
38. Li, C.; Chan, C.; Heimann, A.C.; Danishefsky, S.J. On the rearrangement of an azaspiroindolenine to a
precursor to phalarine: Mechanistic insights. Angew. Chem. Int. Ed. 2007, 46, 1444–1447. [CrossRef]
[PubMed]
39. Ottoni, O.; Cruz, R.; Krammer, N.H. Regioselective nitration of 3-acetylindole and its N-acyl and N-sulfonyl
derivatives. Tetrahedron Lett. 1999, 40, 1117–1120. [CrossRef]
40. Lakshmaiah, G.; Kawabata, T.; Shang, M.; Fuji, K. Total synthesis of ( )-horsfiline via asymmetric
−
nitroolefination. J. Org. Chem. 1999, 64, 1699–1704. [CrossRef] [PubMed]
41. Aurora Fine Chemicals Inc. Available online: http://www.aurorafinechemicals.com/ (accessed on 22 April 2017).
42. Mitchell, I.S.; Blake, J.F.; Xu, R.; Kallan, N.C.; Xiao, D.; Spencer, K.L.; Bencsik, J.R.; Liang, J.; Safina, B.;
Zhang, B.; et al. Hydroxylated and Methoxylated Cyclopenta [D] Pyrimidines as Akt Protein Kinase
Inhibitors. Patent No. CA26,56,622 A1, 5 July 2007.
43. Hostetler, G.; Dunn, D.; McKenna, B.A.; Kopec, K.; Chatterjee, S. 1-Thia-4,7-diaza-spiro[4.4]nonane-3,6-dione:
A structural motif for 5-hydroxytryptamine 6 receptor antagonism. Chem. Biol. Drug Des. 2014, 83, 149–153.
[CrossRef] [PubMed]

Molecules 2017, 22, 2221
25 of 25
44. Protiva, M.; Vejdeˇlek, Z.J.; Jílek, J.O.; Macek, K. Synthetische modelle hypotensiv wirksamer alkaloide
V. Einige weitere derivate des tryptamins und 1,2,3,4-tetrahydronorharmans. Collect. Czechoslov. Chem.
Commun. 1959, 24, 3978–3987. [CrossRef]
45. Bell, S.E.V.; Brown, R.F.C.; Eastwood, F.W.; Horvath, J.M. An approach to some spiro oxindole alkaloids
through cycloaddition reactions of 3-methylideneindolin-2-one. Aust. J. Chem. 2000, 53. [CrossRef]
46. Lim, Y.-H.; Guo, Z.; Ali, A.; Edmondson, S.D.; Liu, W.; Gallo-Ettienne, G.V.; Wu, H.; Gao, Y.-D.;
Stamford, A.M.; Yu, Y.; et al. Factor XIa Inhibitors. Patent No. WO201,5164,308, 29 October 2015.
47. Allen, J.; Brasseur, D.M.; De Bruin, B.; Denoux, M.; Pérard, S.; Philippe, N.; Roy, S.N. The use of biocatalysis
in the synthesis of labelled compounds. J. Label. Compd. Radiopharm. 2007, 50, 342–346. [CrossRef]
48. Manda, S.; Khan, S.I.; Jain, S.K.; Mohammed, S.; Tekwani, B.L.; Khan, I.A.; Vishwakarma, R.A.; Bharate, S.B.
Synthesis, antileishmanial and antitrypanosomal activities of N-substituted tetrahydro-β-carbolines.
Bioorg. Med. Chem. Lett. 2014, 24, 3247–3250. [CrossRef] [PubMed]
49. Sevrin, M.; Morel, C.; Menin, J.; George, P. 2-(Methyl(4-piperidinyl))-1,2,3,4-tetrahydro-9H-pyrido(3,4-b
Indole Derivatives, Their Preparation and Therapeutical Use. Patent No. EP0,302,788 B1, 5 February 1992.
50. Shah, P.; Westwell, A.D. The role of fluorine in medicinal chemistry. J. Enzym. Inhib. Med. Chem. 2007, 22,
527–540. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).