Enantioselective Organocatalytic Synthesis of a Secoyohimbane-Inspired Compound Collection with Neuritogenic Activity

Article abstract of DOI:10.1002/cbic.201700015

Natural products provide evolutionary validated core structures to inspire the synthesis of new compound collections endowed with neurite growth-promoting activity. Rhynchophylline is the major component of Uncaria species, and has been used to treat neurological diseases in Chinese traditional medicine. Based on the structure of this spirocyclic secoyohimbane alkaloid, we developed a highly enantioselective and efficient organocatalyzed synthesis method to provide a tetracyclic secoyohimbane scaffold incorporating a quaternary and three tertiary stereogenic centers, in a one-pot multistep reaction sequence. A compound collection of derived secoyohimbanes was synthesized and expanded by decorating the periphery of the basic scaffold with additional substituents to increase the diversity. Evaluation of the different subcollections of secoyohimbanes for modulation of neurite outgrowth in the SH-SY5Y human cell line led to the discovery of new compounds that promote neurite outgrowth.

Full text of DOI:10.1002/cbic.201700015

Accepted Article
Title: Enantioselective Organocatalytic Synthesis of a Secoyohimbane-
Inspired Compound Collection with Neuritogenic Activity
Authors: Herbert Waldmann, Tim Förster, Sara Lopéz-Tosco, Slava
Ziegler, and Andrey P. Antonchick
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201700015
Link to VoR: http://dx.doi.org/10.1002/cbic.201700015
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ChemBioChem
FULL PAPERS
DOI: 10.1002/cbic.200((will be filled in by the editorial staff)
Enantioselective Organocatalytic Synthesis of a
Secoyohimbane-Inspired Compound Collection
with Neuritogenic Activity
Tim Förster,[a,b] Sara López-Tosco,[a] Slava Ziegler,[a] Andrey P. Antonchick,[a,b] and
Herbert Waldmann*[a,b]
incorporating a quaternary and three tertiary stereogenic centers in a
Natural products provide evolutionary validated core structures
one-pot multistep reaction sequence. A compound collection of
inspiring the synthesis of new compound collections endowed with
derived secoyohimbanes was synthesized and expanded decorating
neurite growth-promoting activity. Rhynchophylline is the major
the periphery of the basic scaffold with additional substituents to
component of Uncaria species which has been used to treat
increase the diversity and the number of the library members.
neurological diseases in Chinese traditional medicine. According to
Evaluation of the different sub-collections of secoyohimbanes for
the structure of this spirocyclic secoyohimbane alkaloid, we
modulation of neurite outgrowth in the SH-SY5Y human cell line led to
developed a highly enantioselective and efficient organocatalyzed
the discovery of new compounds that promote neurite outgrowth.
synthesis method to provide the tetracyclic secoyohimbane scaffold
(Scheme 1). Especially the natural products rhynchophylline and
isorhynchophylline have received a crescent interest recently due
to their wide range of pharmacological activities and applications.
Introduction
For example, rhynchophylline accelerates the inactivation of
Uncaria species have been used in traditional Chinese medicine
voltage-gated K+ (Kv) channels and lowers the activation
for the treatment of disorders of the cardiovascular and the
threshold in mouse neuroblastoma N2A cells.[5] These two natural
central nervous system. The pharmacological activities of this
products also act as noncompetitive antagonists of NMDA
plant (hypotensive, neuroprotective, anticonvulsive, antiepileptic,
receptor channels in Xenopus oocytes and this property may be
antiarrhythmic, anticoagulant, antispasmodic, sedative, etc.)[1] are
useful for the treatment or prevention of neuronal diseases that
related to the presence of active compounds predominantly
involve excess stimulation of NMDA subtype glutamate
indole alkaloids. Especially the neuromodulatory activity plays an
receptors.[6] In addition, rhynchophylline regulates the innate
important role in neuropathological disorders, neuronal damage
immune system via a reduction in the level of superoxide anions,
and regeneration. With the idea that neuritogenic compounds can
activate restoration of damaged neurons, identifying small
JNK phosphorylation, and NF-κB activation.[7] Rhynchophylline
also reduces the release of pro-inflammatory mediators such as
molecules with neurotrophic or neuroprotective properties is of
nitric oxide (NO), prostaglandins E2 (PGE2), monocyte
high interest in neuropharmacological research[2] and natural
chemoattractant protein (MCP-1), tumor necrosis factor-α (TNF-α),
and interleukin-1β (IL-1β) in lipopolysaccharide (LPS)-stimulated
products frequently exhibit this kind of neuroprotective activity.[3]
[a]
Dipl.-Biol. T. Förster+, Dr. S. López-Tosco+, Dr. S. Ziegler, Dr. A.P.
Antonchick, Prof. Dr. Dr. H. Waldmann
Abteilung Chemische Biologie
Max-Planck-Institut für Molekulare Physiologie
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
Fax: (+)49 231 133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
T. Förster, Dr. A.P. Antonchick, Prof. Dr. H. Waldmann
Chemische Biologie
Fakultät Chemie, Technische Universität Dortmund
Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany
Scheme 1. Isomerization of secoyohimbane alkaloids.
[b]
[+]
The spirocyclic alkaloid rhynchophylline (1) and its isomer
isorhynchophylline (2) are the major components of the extracts
of U. rhynchophylla. They represent the secoyohimbane
scaffold[4] that derives biosynthetically from tryptamine and
secologanine and is characteristic for numerous alkaloids
These authors contributed equally to this work
Supporting information for this article is available on the WWW
under http://www.chembiochem.org
1
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10.1002/cbic.201700015
ChemBioChem
microglia most likely by blocking the activation of NF-κB, ERK
collections, we designed an asymmetric synthesis approach to
and p38 mitogen-activated protein kinases (MAPKs).[8] In addition, obtain nonisomerizable secoyohimbanes. We report herein a
isorhynchophylline suppresses 5-hydroxytryptamine (5-HT2A
)
highly enantioselective synthesis method that provides access to
the secoyohimbane scaffold. The most common approaches to
this type of spiro skeleton consist of the coupling of oxytryptamine
and aldehydes employing a Mannich reaction,[23] and an oxidative
receptor function in the brain in a competitive fashion, whereas
rhynchophylline is less potent, indicating that the configuration of
the isorhynchophylline oxindole is important for its antagonistic
activity.[9]
rearrangement of carboline derivatives into oxindoles[13a, 13e, 13f, 24]
,
but there is a need for new methods enabling the asymmetric
construction of this tetracyclic scaffold. Our approach makes use
of an asymmetric iminium organocatalysis as strategic
stereodirecting method, is very practical, employs easily
accessible starting materials, and creates the tetracyclic core,
including one all-carbon quaternary spirocenter[25] and three
tertiary centers in a multistep, one-pot reaction. While we were
examinating this strategy, Wu et al. and Zhang et al.
independently discovered a very similar approach.[26] In the first
strategy, the authors performed an organocatalytic cascade
reaction blocking the active methylene indole’s α-position by a
methyl or a bromo group enabling the reaction with unsaturated
aldehydes using proline-derived catalysts as the source of
chirality. The second procedure also employed catalysis by
proline derivatives and included an efficient diastereoselective
intramolecular iminium ion spirocyclization/lactamization cascade
sequence of a 2-chlorotryptamine and a conjugate adduct that
was obtained from an asymmetric organocatalytic Michael
addition of dialkyl malonate to α,β-unsaturated aldehydes.
The broad spectrum of pharmaceutical applications,
especially in the neuroprotective field, suggests that these
characteristic scaffolds define biologically prevalidated starting
points in vast chemical structure space, inspiring the design and
synthesis of compound collections endowed with the same or
10]
similar kind of biological activity.[5,
The key structural
characteristic of the secoyohimbanes is a complex spiro ring
fusion at the 3-position of the oxindole core and the 1-position of
an octahydroindolizine (Scheme 1). These alkaloids often occur
as pairs of interconvertible isomers[11], due to isomerization at the
spiro center through Mannich/retro-Mannich reactions involving
an open ring intermediate. Because of that, the composition of
the mixture is influenced by pH, temperature and solvent polarity
and makes the evaluation of the biological profiles of single
isomers difficult.[12]
Results and Discussion
Development of an Enantioselectively Catalyzed Synthesis of
a Secoyohimbane Library
Motivated by the concept of biology-oriented synthesis as
approach for the design and synthesis of natural product-inspired
compound collections enriched in different kinds of
10k, 27]
bioactivities,[10b,
we focused on the development of an
asymmetric catalytic domino reaction of 2-bromoindole derivative
(3) and α,β-unsaturated aldehydes (4) to obtain the
secoyohimbanes (6). Based on previous results the presence of
the bromo substituent is essential to the formation of the desired
product 6 (Scheme 3).[28] This new starting material 3 could have
a double function. First it serves as a protecting group, preventing
cyclization to the 2- position of the indole in 5 and redirecting it to
the 3-position (the tetrahydro-β-carboline cannot be formed). And
secondly, it enables the diastereoselective conversion of the
hemiaminal (5) after addition of trifluoroacetic acid (TFA) into the
desired secoyohimbane (6) incorporating one all-carbon
quaternary spirocenter and three tertiary centers in a multi-step,
one-pot reaction. In addition, secoyohimbane (6) would contain
an amide function instead of the tertiary amine of the indolizine,
which should prevent the isomerisation through Mannich/retro-
Mannich reactions. In planning this approach we assumed that
the first catalytic enantio- and regioselective addition of 3 to
cinnamic aldehydes would lead to the formation of hemiaminal
(5).[29]
Scheme 2. Examples of secoyohimbane alkaloids.
The synthesis of natural products containing the
secoyohimbane core has been a topic of significant recent
interest due to their numerous biological applications.[13] Scheme
2 shows examples of alkaloids that belong to this family of
compounds with interesting bioactivity profiles. For example,
strychnofoline was isolated from the leaves of strychnos
usambarensis[14] and displays high antimitotic activity against
cultures of mouse melanoma B16, Ehrlich ascites- and Hepatom
HW165 tumor cells.[15] Pteropodine is an oxindole alkaloid
component of Uncaria tomentosa that positively modulates the
function of rat muscarinic M1 and 5-HT2 receptors expressed in
Xenopus oocyte.[16] Alstonisine, isolated from Alstonia
macrophylla,[17]
exhibits
antiplasmodial
activity[18]
and
vasorelaxant activity on rat aorta leading to partial mediation of
the NO release from endothelial cells.[19] Chitosenine is a
component of Gardneria multiflora[20] and exhibits short-lived
inhibitory activity of ganglionic transmission in rats and rabbits.[21]
Spirotryprostatin A was isolated from the fermentation broth of
Aspergillus fumigatus and has been shown to completely inhibit
the G2/M progression of mammalian tsFT210 cells.[22]
Motivated by the biological importance of this scaffold, the
instability of its isomers, and the lack of efficient synthesis
methods to synthesize secoyohimbane-inspired compound
2
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10.1002/cbic.201700015
ChemBioChem
temperature due to the low reactivity of some aldehydes that
require longer reaction times that do not improve considerably the
enantioselectivity and yield. Under these reaction conditions also
both enantiomers of the desired secoyohimbanes are obtained
and the enantiomeric excesses are still very good to excellent.
After optimization of the reaction conditions, we investigated
the substrate scope with respect to a wide variety of α,β-
unsaturated aldehydes (Table 1). A broad range of cinnamic
aldehydes can be employed successfully in the enantioselective
cascade reaction. The substitution pattern of these α,β-
unsaturated aldehydes in the organocatalyzed process includes
electron-donating and -withdrawing substituents, para-, meta-,
and ortho-substituted. Substituted acroleines with an heterocycle
in the β-position are also allowed. α,β-Unsaturated aldehydes that
present ortho-substitution or additional bulky groups on the
aromatic rings resulted in lower yields and diastereomeric ratios.
With respect to the electronic effect of functional groups, there is
no clear influence regarding the yield when electron-donating and
-withdrawing substituents are present, but electron-rich
heterocycles like furane and thiophene with their corresponding
Scheme 3. Synthesis approach to the secoyohimbane scaffold.
Gratifyingly, clarifying experiments that used 2-bromoindole
(3) and cinnamic aldehyde together with an organocatalyst
indicated that the strategy was viable and that the
spiroindolinones were formed. With the purpose of establishing
the best reaction conditions to obtain the desired secoyohimbane-
inspired compounds, we optimized the addition of nucleophile (3)
to cinnamic aldehyde in the presence of different organocatalysts
varying the solvent and the additive to be able to run the
reactions in preparatively viable yields and with high
stereoselectivity. Although the best reaction conditions for the
synthesis of (+)-6aa (Table 1) with respect to enantiomeric
excess, diastereoselectivity and yield were obtained in our
previous studies[2] employing 60 mol% of the additive caesium
acetate and 10 mol% of the catalyst diphenylprolinol
trimethylsilylether (A) at 0°C in MeOH, to be more practical we
decided to revert to 120 mol% and 20 mol% respectively at room
substitution
pattern
led
to
lower
yields.
However,
enantioselectivities were not influenced depending on the
electronic effect of the substitution pattern of the rings. Alkyl-
substituted aldehydes are not suitable for the synthesis of target
products. As a result, 48 different examples including both
enantiomers of the secoyohimbane-inspired products were
obtained in preparatively viable yields and with very good to
excellent enantioselectivities (74%–98%). The absolute
configuration
of
the
predominant
stereoisomer
was
unambiguously confirmed by means of single-crystal X-ray
analysis of compound (+)-6ea.
Table 1. Results of the Enantioselective Catalyzed Synthesis of Secoyohimbane-Inspired Spirocyclic Indolinones.
Entry[a]
R1
R2
Cat (R)-A
(+)-6aa[b]
(+)-6ba[b]
(+)-6ca[b]
(+)-6da[b]
(+)-6ea[b]
(+)-6fa[b]
(+)-6ga[b]
(+)-6ha[b]
Yield [%]
63[c]
dr[e]
5/1
ee [%][f]
95
Cat (S)-A
(-)-6aa
(-)-6ba
(-)-6ca
(-)-6da
(-)-6ea
(-)-6fa
Yield [%]
68[d]
dr[e]
ee [%][f]
93
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
3a
3a
3a
3a
3a
3a
3a
3a
H
H
H
H
H
H
H
H
6.6/1
5.8/1
4.7/1
6.0/1
5.5/1
2.1/1
3.3/1
2.9/1
51[c]
5/1
94
64[d]
93
55[c]
2/1
92
60[d]
99
25[c]
2/1
95
57[d]
92
52[c]
10/1
10/1
5/1
95
63[d]
92
55[c]
92
61[d]
89
4g
4h
69[c]
96
(-)-6ga
(-)-6ha
65[d]
92
60[c]
5/1
89
71[d]
89
O
O
9
4i
4j
3a
3a
H
H
(+)-6ia[b]
(+)-6ja[b]
62[c]
61[c]
10/1
15/1
95
95
(-)-6ia
(-)-6ja
62[d]
34[d]
4.6/1
91
92
[g]
10
-
11
4k
3a
H
(+)-6ka[b]
35[c]
1.2/1
96
(-)-6ka
33[d]
1.4/1
98
3
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10.1002/cbic.201700015
ChemBioChem
12
4l
3a
H
(+)-6la[b]
46[c]
1.5/1
90
(-)-6la
43[d]
3.9/1
98
13
14
4m
4n
3a
3a
H
H
(+)-6ma[b]
(+)-6na[b]
58[c]
42[c]
4/1
8/1
88
93
(-)-6ma
(-)-6na
54[d]
45[d]
3.6/1
3.3/1
86
89
15
16
4o
4p
3a
3a
H
H
(+)-6oa
(+)-6pa
43[d]
49[d]
5.6/1
3.0/1
90
92
(-)-6oa
(-)-6pa
58[d]
49[d]
2.6/1
5.3/1
90
94
17
18
4q
4r
3a
3a
H
H
(+)-6qa
(+)-6ra
42[d]
48[d]
2.8/1
2.0/1
79
79
(-)-6qa
(-)-6ra
50[d]
42[d]
2.9/1
2.6/1
74
81
19
4s
4t
3a
3a
H
H
(+)-6sa
(+)-6ta
72[d]
3.2/1
4.0/1
84
85
(-)-6sa
(-)-6ta
64[d]
3.9/1
3.1/1
84
89
20f
49[d]
50[d]
21
4u
3a
H
(+)-6ua
78[d]
5.5/1
89
(-)-6ua
68[d]
4.3/1
89
22
23
4v
3a
3a
H
H
(+)-6va
(+)-6wa
44[d]
78[d]
3.1/1
2.4/1
85
86
(-)-6va
(-)-6wa
53[d]
78[d]
2.7/1
2.5/1
90
87
4w
24
25
4x
4y
3a
7a
H
H
(+)-6xa
(+)-6ya
52[d]
67[d]
3.4/1
3.7/1
91
86
(-)-6xa
(-)-6ya
56[d]
63[d]
2.8/1
3.5/1
90
88
26
4h
3a
Me
(+)-6hb
69[d]
3.9/1
84
(-)-6hb
84[d]
4/1
86
O
O
[g]
[g]
27
28
4i
3a
3a
Me
Me
(+)-6ib
58[d]
69[d]
-
87
88
(-)-6ib
49[d]
65[d]
-
86
86
4n
(+)-6nb
4/1
(-)-6nb
4.7/1
[a] Experimental procedure: aldehyde (4) (1 eq), indole (3) (1.2 eq), catalyst A (20 mol%), CsOAc (120 mol%), in MeOH (0.1 M) at 0°C [b] These experiments
were done with different reaction conditions: aldehyde (4) (1 eq), indole (3) (1.2 eq), catalyst A (10 mol%), CsOAc (60 mol%), in MeOH (0.1 M) at 0°C [c] Isolated
yields of the single diastereomer after column chromatography [d] Calculated yields of the single diastereomer from 1H NMR analysis based on internal standard
[e] Determined by 1H NMR analysis [f] Determined by HPLC analysis for the major isomer [g] These dr[%] could not be determined by 1H NMR analysis.
acid followed by elimination of water. The iminium intermediate
Mechanistically, the initial step and also the key step of the
reaction sequence is a stereo and regioselective Michael addition
of the 2-bromoindole (3) to the α,β-unsaturated aldehyde (4)
establishing two different stereocenters (Scheme 4). Although 3
can undergo further reactions, since it contains several carbon-
and two nitrogen nucleophiles, only the addition of the acidic
methylene group to the α,β-unsaturated aldehyde was detected,
which leads to intermediate (7). There are several relevant results
reported for the addition of malonic acid derivatives to enals that
corroborate the stereochemical course of the organocatalyzed
(9) suffers an electrophilic Friedel-Crafts cyclization to obtain
spirocycle (10), incorporating an imidoyl bromide, which is
hydrolyzed in the presence of Brønsted acid and water to provide
the desired spiroindolinone (6). Interestingly, Mannich cyclization
of cyclic hemiaminal (11), which could be obtained from 5 by
hydrolysis of the imidoyl bromide, does not take place. To
rationalize the stereochemical preference in the spirocyclization,
we postulate that TS1 and TS2 may be two competing transition
states of this reaction, in which the indole attacks the acyl iminium
intermediate anti to the substituent R1 (Figure 1). In TS1, which
leads to the observed configuration of the products, the bromine
points away from the ester avoiding unfavourable interactions like
the ones that are present in TS2 (less preferred), that come from
rotation around the bond which connects C3 of the indole with the
conjugate addition.[29] Intermediate (7) then experiences
a
spontaneous cyclization to generate hemiaminal (5). The
formation of the iminium intermediate (9), which is in equilibrium
with enamide (8), takes place after protonation by the Brønsted
4
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10.1002/cbic.201700015
ChemBioChem
iminium-N and leads to the spiro isomeric configuration of the
secoyohimbanes.
in different assays. We already introduced diversity in the
substituent of the aldehyde (Table 1, entries 1-25). Another
position in the structure that could be suitable for further
modifications is the nitrogen atom of the oxindole ring. To avoid
competitive reactions in the N-H protection due to similar acidity
in other parts of the scaffold like the C-H of the α-amidoester, we
decided to protect this nitrogen before the formation of the final
product. For that purpose, we followed the observation that
selective methylation of the indole-nitrogen in the tryptamine core
can be achieved with sodium hydride in dimethylformamide.[31] In
contrast, we observed polymethylations and lower yield. However,
it was possible to separate the desired product by column
chromatography obtaining sufficient amounts of N-methylated
tryptamine to synthesize in two more steps the starting material
3b for the cascade reaction (Table 1). With this N-methylated 2-
bromoindol (3b) in hand we synthesized nine new products
employing three different α,β-unsaturated aldehydes (Table 1,
entries 26-28) with similar yield and enantiomeric excess as
compared to the previously synthesized secoyohimbanes
corroborating that the substitution on the nitrogen of the 2-
bromoindol (3) does not affect the described mechanism.
A possible faster alternative to increase the number of
members of the secoyohimbane library avoiding the problem of
polyalkylation of the tryptamine could be the late-stage
modification of the spiroxindoles. To this end, we tried to find a
base suitable for the chemoselective alkylation of the nitrogen in
the oxindole with respect to the C-H of the α-amidoester. Initially,
one equivalent of different sterically hindered bases was
employed to selectively deprotonate the N-H, but in general
double alkylation and incomplete conversion were observed. To
increase the library size double alkylation was further pursued
and potassium tert-butoxide gave the best yields. To corroborate
the relative configuration of the structure, we carried out NOESY
experiments (see the supporting information). The configuration
of new formed quaternary carbon center in the spiroxindole can
be rationalized by shielding of the face in the planar enolate
intermediate, where alkylation occurs from the face opposite to
the R1-group. Following this scenario we synthesized a sub-
library of dialkylated products. Both enantiomers of a variety of
secoyohimbanes were prepared from different aldehydes in larger
scale to evaluate the scope of the reaction with respect to the
alkyl halides (Table 2). The results showed that the reaction
proceeded smoothly with diverse linear alkyl halides with different
substitution patterns (Table 2, entries 1-5 and 8-22), but not with
α-branched (Table 2, entry 6) or long chain alkyl halides (Table 2,
entry 7). In total 37 modified secoyohimbane-inspired compounds
(both enantiomers) were synthesized with moderate to very good
yields.
Scheme 4. Proposed Mechanism for the Organocatalytic Synthesis of the
Secoyohimbane Scaffold.
Figure 1. Proposed transition states in the spirocyclization.
Once we had a collection of secoyohimbane derivatives
synthesized, we decided to expand it exploring the modification of
other parts of the molecule to increase the number of library
members and their diversity for subsequent biological evaluation
Table 2. Results of the Double Alkylation of Secoyohimbane-Inspired Spirocyclic Indolinones.
Entry[a]
R1
R2
(+)-6 ee[%][b]
Prod 6
Yield [%][c]
(-)-6 ee[%][b]
Prod 6
Yield [%][c]
1
2
6aa
6aa
A
B
Me
91
91
(-)-6aaA
(-)-6aaB
55
66
92
92
(+)-6aaA
(+)-6aaB
71
57
Allyl
5
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10.1002/cbic.201700015
ChemBioChem
3
4
5
6
7
8
9
6aa
6aa
6aa
6aa
6aa
6fa
C
D
E
F
Bn
91
91
91
91
91
85
85
85
85
85
83
(+)-6aaC
(+)-6aaD
(+)-6aaE
-
70
64
49
-
92
92
92
92
92
87
87
87
87
87
82
(-)-6aaC
(-)-6aaD
(-)-6aaE
-
64
64
43
-
p-BrBn
iPr
G
A
B
C
D
E
A
CH2(CH2)6CH3
-
-
-
-
Me
(-)-6faA
(-)-6faB
(+)-6faC
(+)-6faD
(+)-6faE
(-)-6saA
86
66
68
73
53
70
(+)-6faA
(+)-6faB
(-)-6faC
(-)-6faD
-
85
65
67
70
-
6fa
Allyl
Bn
10
11
12
13
6fa
6fa
6fa
6sa
p-BrBn
Me
(+)-6saA
73
14
15
16
17
18
19
20
21
22
6sa
6sa
6sa
6sa
6ua
6ua
6ua
6ua
6ua
B
C
D
E
A
B
C
D
E
Allyl
Bn
83
83
83
83
89
89
89
89
89
(-)-6saB
(+)-6saC
(+)-6saD
(+)-6saE
(-)-6uaA
(-)-6uaB
(+)-6uaC
(+)-6uaD
(+)-6uaE
60
71
67
45
72
60
69
61
35
82
82
82
82
87
87
87
87
87
(+)-6saB
(-)-6saC
(-)-6saD
-
63
68
66
-
p-BrBn
Me
(+)-6uaA
(+)-6uaB
(-)-6uaC
(-)-6uaD
-
68
57
71
62
-
Allyl
Bn
p-BrBn
[a] Experimental procedure: secoyohimbane (6) (1 eq), tBuOK (2.1 eq), alkyl halide (2.1 eq) in THF (0.1 M) at RT [b] Determined by HPLC analysis [c] Isolated yields
of the single diastereomer after column chromatography.
starting point for further structural modifications to identify more
potent compounds.
Biological Evaluation of the Secoyohimbane-Inspired
To analyze the potential of the compound collection to
modulate neurite outgrowth we established a cell-based assay
using SH-SY5Y human neuroblastoma cells to detect and
quantify neurite length (Figure 2). These cells can proliferate in
culture for long periods[34] and upon differentiation express
neuronal marker proteins, e.g., neurotrophic receptors,
neuropeptides, microtubule-associated proteins (MAP), tau and
neurofilaments.[35] In addition, differentiated cells display
Compound Collection
Recently,[2] we reported the investigation of fourteen
secoyohimbanes ((+)-6aa-(+)-6na) in two neuronal cell lines
(primary rat hippocampal neurons[32] and embryonal stem cell-
derived mouse motoneurons[33]) for their influence on neurite
outgrowth. To our delight, the synthetic secoyohimbanes
displayed remarkable neurotrophic activity providing a good
6
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10.1002/cbic.201700015
ChemBioChem
structural and biochemical similarity to mature neurons. These
characteristics make them a widely used model for the study of
neuronal processes like differentiation, metabolism, degeneration
and toxicity.[35]
To enhance the neurospecific properties, SH-SY5Y cells were
cultured in
a
medium that contained N2 supplement.[34]
Differentiation to neuron-like cells was induced by addition of 1
µM all-trans retinoic acid (ATRA),[34] which resulted in an intense
network of neurites after 72 hours (Figure 3A). ATRA is the most
commonly used agent to initiate neuronal differentiation in SH-
SY5Y and acts by activation of the PI3K/Akt signaling pathway
and Bcl-2 upregulation[36]. For analysis, cells were stained for
DNA and α-tubulin to visualize nuclei and neurites, respectively.
Automated image acquisition and high-content analysis was
performed using the Neurite Outgrowth application in MetaMorph
(Version 7.7.0.0., Molecular Devices, LLC) that has been used
for various applications in neuroscience[37]
.
Figure 2. Workflow of the phenotypic screening for monitoring neurite outgrowth
modulation in SH-SY5Y cells.
Figure 3. Establishment of a phenotypic screening to monitor neurite outgrowth
in SH-SY5Y cells. (A) Undifferentiated and ATRA-differentiated SH-SY5Y cells.
(B) Concentration-dependent influence of BDNF and DMSO on ATRA-
differentiated SH-SY5Y after 96 hours treatment (C) Mean neurite outgrowth
per cell. Cells were fixed, permeabilized and stained for DNA (blue) and
Since brain-derived neurotrophic factor (BDNF) is known to
increase neurite outgrowth[38], cells were exposed to BDNF to
assess a suitable time point to monitor neurite outgrowth upon
compound addition. Treatment with 200 ng/ml BDNF led to a 2-
fold increase in mean neurite outgrowth per cell after 96 hours
(Figure 3B and C).
microtubules (green) using DAPI and an anti-α-tubulin antibody and
a
secondary antibody coupled to Alexa 488. Scale bar: 50 µm. Data are mean
values ± s.d. of single data points (n = 27 - 54). ATRA: 1 µM
Additionally, treatment of ATRA-differentiated SH-SY5Y cells
with 1 % DMSO led to the formation of relatively shorter neurites
after 96 hours (Figure 3B and C), which is in line with neurite
We subjected 87 compounds of the secoyohimbanes library to
the neurite outgrowth assay to analyze their potential to modulate
neurite outgrowth in SH-SY5Y. 200 ng/ml BDNF was employed
as a positive control (Figure S1). Two enantiomer pairs enhanced
neurite outgrowth in SH-SY5Y at 1 and 10 µM, respectively
(Figure 4). (+)-6ra increased the relative mean neurite outgrowth
by 42% at a concentration of 1 µM and proved to be most potent
among the assayed small molecules (Figure 4A and B).
Treatment with 10 µM (+)-6ra resulted in cell death (Figure S2).
The respective enantiomer (-)-6ra increased neurite outgrowth up
to 47% at 10 µM, while lower concentrations had no significant
influence on neuritogenesis (Figure 4A and C). Compound (+)-
6qa enhanced the neurite outgrowth by 61% at 10 µM, whereas
the corresponding enantiomer (-)-6qa had a weaker effect and
increased the relative outgrowth by only 20% at 10 µM (Figure 4A
and C). (+)-6qa and (-)-6qa did not show significant activity at 1
retraction at high concentrations of DMSO[39]
.
µM. Both neuroactive enantiomer pairs contain
a 2-furyl
substituent at R1, coupled to 2-chlorophenyl- or 2,3-
dichlorophenyl group respectively (Table 1, entries 17, 18).
Compounds embodying other substituents at this position as well
7
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700015
ChemBioChem
as methyl at R2 did not influence neurite outgrowth in SH-SY5Y.
Likewise, double alkylated compounds did not modulate the
relative neurite outgrowth in comparison to cells treated with
0.1 % DMSO.
Experimental Section
All products were fully characterized. Spectral data for all compounds
are provided in the Supporting Information.
Typical Procedure for the Organocatalyzed Synthesis. To a
solution of the aldehyde (4) (0.4 mmol) and the catalyst (20 mol %, 80
mmol) in MeOH (0.5 mL) were added CsOAc (120 mol %, 0.48 mmol)
and 3-(2-(1H-indol-3-yl)ethylamino)-3-oxopropanoate (3a) (1.2 eq.,
0.48 mmol) in MeOH (0.5 ml) at RT. The reaction mixture was stirred
at RT for 16 hr, cooled to -80°C, and TFA (10 eq) was added. The
reaction mixture was warmed to room temperature and concentrated
under reduced pressure. Purification by column chromatography on
silica gel (AcOEt / DCM = 1/5/1/2) yielded the product.
The Uncaria alkaloids rhynchophylline and isorhynchophylline
did not alter neurite outgrowth in SH-SY5Y (Figure 4A and D).
Rhynchophylline is even known to protect cells against NGF-
induced outgrowth in Dawley rat neurons [40]
.
Typical Procedure for the Double Alkylation Process. A solution
of secoyohimbane (6) (0.18 mmol) at RT in anhydrous THF (2 mL)
was added tBuOK (0.38 mmol, 2.1 eq) and alkyl halide (0.38 mmol,
2.1 eq). The mixture was stirred for 16h. After that time, it was
quenched by addition of saturated aqueous NH4Cl. The resulting
mixture was extracted with DCM and the organic layers were dried
with Na2SO4 and concentrated. Purification by column
chromatography on silica gel (AcOEt/DCM = 1/9/1/2) yielded the
product.
Cell culture
SH-SY5Y cells were obtained from DSMZ (Germany) and cultured in
Dulbecco’s modified Eagle’s medium with L-glutamine (DMEM, PAN
Biotech, Germany) supplemented with 10% fetal bovine serum (FBS,
Life Technologies, USA), sodium pyruvate, non-essential amino acids,
100 µg/ml streptomycin and 100 U/ml penicillin (PAN Biotech,
Germany) at 37°C and 5% CO2 in humidified atmosphere.
Determination of neurite outgrowth
8 x 104 cells were seeded per well in triplicates in black 96-well plates
with clear bottom (Corning, USA). Differentiation was induced by
replacing the medium with freshly prepared DMEM:Nutrient Mixture
F12 (DMEM:F12, 1:1, PAN Biotech, Germany) containing 3% N2
supplement and 1 µM all-trans retinoic acid (Sigma-Aldrich, Germany)
for 72 hours. N2 supplement was prepared according to a published
protocol and contained 5 µg/ml insulin, 20 nM progesterone, 30 nM
Figure 4. Influence of secoyohimbane-inspired small molecules on neurite
outgrowth in SH-SY5Y cells. Upon ATRA-induced differentiation, SH-SY5Y cells
were incubated with the compounds at a concentration of 1 µM for (+)-6ra or 10
µM of all others for 96 h. Cells were fixed, permeabilized and stained for DNA
(blue) and microtubules (green) using DAPI and an anti-α-tubulin antibody and
a secondary antibody coupled to Alexa 488. Representative images (A) and the
mean neurite outgrowth per cell (B-D) are shown. Scale bar: 50 µm. Data are
mean values ± s.d. of single data points (n = 35 – 75). Statistical significance
was analyzed by two-sample t-test (**: p < 0.01; ***: p < 0.001; ****: p < 0.0001).
sodium selenite, 100 µM putrescine and 100 µg/ml apo-transferrin[34]
.
Upon differentiation, cells were incubated with the compounds in
DMEM:F12 with N2 supplement for 96 hours. 48 hours after
compound addition, the medium was replaced by fresh DMEM:F12
with N2 supplement and the respective compound. 200 ng/ml BDNF
was used as a positive control for neurite outgrowth stimulation.
Upon compound treatment, cells were fixed using 3.7% formaldehyde
and permeabilized with 0.1% Triton X-100 in one step for 30 minutes.
Unspecific binding sites were blocked using 2% bovine serum
albumin (BSA, Sigma-Aldrich, Germany) prior to staining of the cells
for DNA and microtubules with 4’, 6’-diamidino-2-phenylindole (DAPI,
Sigma-Aldrich, Germany) and an anti-α-tubulin antibody (Abcam, UK)
and a secondary antibody coupled to Alexa® 488 (Life Technologies,
USA), respectively. Images were acquired on an automated
microscope (Axiovert M200, Zeiss, Germany). High-content analysis
of the raw data was performed with the Neurite Outgrowth tool in the
MetaMorph® software (Molecular Devices, USA). Statistical analysis
was performed using two-sample t-test.
Conclusion
We developed a highly practical catalytic method for the
synthesis of
a library of complex natural product-inspired
compounds with the basic scaffold of the secoyohimbane
alkaloids. The polycyclic heterocycles incorporating one
quaternary and three tertiary stereocenters were obtained with
excellent enantioselectivity and in preparatively viable yields from
simple and readily accessible starting materials in a one pot
multistep reaction process. It was possible to introduce in the
secoyohimbane structure two new alkyl substituents generating a
new family of more complex and diverse spirocyclic indolinones
at retained high enantioselectivity. Exploration of this family of
spiroxindoles for neurite outgrowth-promoting activity revealed
two enantiomer pairs significantly enhancing neurite outgrowth in
SH-SY5Y cells. These results expand the neuroactive properties
of spiroxindoles by neurite outgrowth modulation and provide a
suitable starting point to further investigate the neuritogenic
potential of secoyohimbane alkaloids and analogues thereof.
Acknowledgements
This work was supported by the Max Planck Gesellschaft and the
European Research Council under the European Union's Seventh
Framework Programme (FP7/2007-2013) / ERC Grant agreement
n°268309. The research leading to these results has received
funding from the European Union Seventh Framework
8
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10.1002/cbic.201700015
ChemBioChem
Programme under grant agreement No. HEALTH-F2-2009-
241498 (‘‘EUROSPIN’’ project). S.L.-T. thanks the Spanish ME
and Max-Planck Society for a postdoctoral fellowship.
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Keywords: Alkaloids · asymmetric synthesis ·
enantioselectivity · iminium organocatalysis · neurite
outgrowth
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10.1002/cbic.201700015
ChemBioChem
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FULL PAPERS
Neuritogenic Secoyohimbanes: A
library of spirocyclic indolinones
derived from the neuroprotective
alkaloid rhynchophylline was
Tim Förster, Sara López-Tosco, Slava
Ziegler, Andrey P. Antonchick and
Herbert Waldmann*
synthesized employing a highly
efficient enantioselective and
organocatalyzed cascade process.
Evaluation of the developed
compound collection showed a family
of neurotrophic compounds.
Page No. – Page No.
Enantioselective Organocatalytic
Synthesis of a Secoyohimbane-
Inspired Compound Collection with
Neuritogenic Activity
11
This article is protected by copyright. All rights reserved.