Highly enantioselective catalytic synthesis of neurite growth-promoting secoyohimbanes

Article abstract of DOI:10.1016/j.chembiol.2013.03.011

Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation.

Full text of DOI:10.1016/j.chembiol.2013.03.011

Chemistry & Biology
Brief Communication
Highly Enantioselective Catalytic Synthesis
of Neurite Growth-Promoting Secoyohimbanes
1
1
Andrey P. Antonchick, Sara Lopez-Tosco, Juan Parga,2,6 Sonja Sievers,1 Markus Schu¨ rmann,3 Hans Preut,3
´
Susanne Hoing, Hans R. Scholer,2,5 Jared Sterneckert,2 Daniel Rauh,4 and Herbert Waldmann1,4,
1Abteilung Chemische Biologie, Max-Planck-Institut fu¨ r Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
2
*
¨
¨
2
¨
¨
Max-Planck-Institut fu¨ r Molekulare Biomedizin, Rontgenstrasse 20, 48149 Munster, Germany
3Anorganische Chemie
4Chemische Biologie
¨
¨
Fakultat Chemie, Technische Universitat Dortmund, Otto-Hahn-Strasse 6, 44221 Dortmund, Germany
5
¨
¨
¨
¨
¨
Medizinische Fakultat Munster, Universitat Munster, Domagkstrasse 3, 48149 Munster, Germany
6Present address: Center for Research in Molecular Medicine and Chronic Diseases, University of Santiago de Compostela, Avda. Barcelona,
15782 Santiago de Compostela, Spain
*Correspondence: herbert.waldmann@mpi-dortmund.mpg.de
http://dx.doi.org/10.1016/j.chembiol.2013.03.011
SUMMARY
(Li and Vederas, 2009; Newman and Cragg, 2007; Antonchick
et al., 2010; Bon and Waldmann, 2010; Koch et al., 2005; Kumar
¨
¨
Natural products endowed with neuromodulatory and Waldmann, 2009; Noren-Muller et al., 2006, 2008; Wald-
mann et al., 2008).
With respect to neurotrophic and neuroprotective activity, an
activity and their underlying structural scaffolds
may inspire the synthesis of novel neurotrophic com-
pound classes. The spirocyclic secoyohimbane alka-
loid rhynchophylline is the major component of the
extracts of Uncaria species used in Chinese tradi-
tional medicine for treatment of disorders of the
central nervous system. Based on the structure of
interesting case is the alkaloid rhynchophylline (1). Rhynchophyl-
line is a major component of the extracts of Uncaria species,
which are widely used in traditional Chinese medicine to treat
ailments of the central nervous system. Alkaloids are the active
pharmacological components in Uncaria species and make up
about 0.2% of their dry weight. Rhynchophylline accounts for
rhynchophylline,
a highly enantioselective and
up to 50% of the total amount of alkaloids in these plants and ex-
hibits similar pharmacological activity as the plant extract (Zhou
and Zhou, 2010, 2012).
efficient organocatalyzed synthesis method was
developed that gives access to the tetracyclic se-
coyohimbane scaffold, embodying a quaternary
and three tertiary stereogenic centers in a one-pot
multistep reaction sequence. Investigation of a
collection of the secoyohimbanes in primary rat hip-
pocampal neurons and embryonal stem cell-derived
motor neurons led to discovery of compounds that
promote neurite outgrowth and influence the
complexity of neuronal network formation.
Rhynchophylline (1) and its isomer isorhynchophylline (2)
embody the secoyohimbane scaffold (Figure 1; Shellard and
Lala, 1978; Shellard et al., 1969; Phillipson and Hemingway,
1973a, 1973b). The key structural feature of these molecules is
a complex spiro ring fusion at the three position of the oxindole
core and the one position of an octahydroindolizine. These alka-
loids often occur as pairs of interconvertible isomers (Seaton
et al., 1960; Wenkert et al., 1959), due to isomerization at the
spiro center through Mannich/retro-Mannich reactions (Figure 1).
The composition of the mixture depends on temperature, pH,
and solvent polarity and complicates the study of the biological
profiles of single isomers (Laus, 1998; Laus et al., 1996; Kang
INTRODUCTION
Many neurological diseases are characterized by a progressive et al., 2004; Yuan et al., 2008). The synthesis of natural products
loss of neuronal activity, and novel approaches aimed at restora- containing the secoyohimbane core has been a subject of signif-
tion of neuronal viability and complexity of neuronal networks or icant recent interest (Deiters et al., 2006; Ito et al., 2001; Martin
at prevention of neuronal decline are in high demand. Therefore, and Mortimore, 1990; Stahl et al., 1996; Lerchner and Carreira,
the identification of small molecules with neurotrophic or neuro- 2002, 2006). Inspired by the biological importance of this scaf-
protective properties is of great current interest.
fold, the instability of its isomers, and the lack of efficient synthe-
Frequently, natural products display this kind of activity (Wil- sis methods for the generation of secoyohimbane-inspired
son and Danishefsky, 2006; Carcache et al., 2006; Inoue et al., compound collections, we sought to develop an asymmetric
2007; Rawat et al., 2008; Chen et al., 2009; Jessen et al., 2009; synthesis approach to access nonisomerizable secoyohim-
Schmidt et al., 2009; Jang et al., 2010; Jana et al., 2011; Jessen banes. We report herein a highly enantioselective synthesis
et al., 2011; Cheng et al., 2012; Mehta et al., 2012; Praveen method that gives access to the secoyohimbane scaffold.
Kumar et al., 2012), and their characteristic scaffolds define bio- Our approach employs asymmetric iminium organocatalysis
´
´
logically prevalidated starting points in vast chemical structure (Marques-Lopez et al., 2010; Grondal et al., 2010; Bertelsen
space, inspiring the design and synthesis of compound collec- and Jørgensen, 2009; Melchiorre et al., 2008; Enders et al.,
tions endowed with the same or similar kind of biological activity 2007) as key stereodirecting method, is highly practical, uses
500 Chemistry & Biology 20, 500–509, April 18, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
Figure 1. Isomerization of Secoyohimbane Alkaloids
In nature, rhynchophylline and isorhynchophylline occur as pairs of intercon-
vertible isomers. The isomerization proceeds via a retro-Mannich reaction
involving an open-ring intermediate followed by Mannich reaction.
readily accessible starting materials, and generates the tetracy-
clic core, incorporating one all-carbon quaternary spirocenter
(Bella and Gasperi, 2009; Zhou et al., 2010; Trost and Brennan,
2009; Steven and Overman 2007; Trost and Jiang 2006) and
three tertiary centers in a multistep, one-pot transformation.
While we were exploring this strategy, Wu et al. (2012) and Zhang
et al. (2013) independently investigated a very similar approach.
Motivated by the reasoning of biology-oriented synthesis (Wet-
zel et al., 2011; Du¨ ckert et al., 2012; Wilk et al., 2010; Kaiser
et al., 2008; Lachance et al., 2012; Over et al., 2013) as approach
Figure 2. Planned Synthesis Route to the Secoyohimbane Scaffold
The initial approach based on the use of intermediate 3 for the synthesis of
spirocyclic products was not successful. Desired secoyohimbane scaffold
was obtained using intermediate 7 in key organocatalytic reaction cascade in
the second approach. Im, iminium catalysis.
for the design and synthesis of natural product-inspired com- the attack of the electrophile to the three position. Thus, we
pound collections enriched by molecules with diverse bioac- assumed that the catalytic enantio- and regioselective addition
tivity, we investigated the synthesized compounds in cell-based of 7 to a,b-unsaturated aldehydes would result in the formation
assays monitoring neurite outgrowth from primary hippocampal of hemiaminal (8) (Brandau et al., 2006; Marigo et al., 2006; Fran-
´
neurons and embryonal stem cell (ESC)-derived motor neurons. zen and Fisher, 2009; Hayashi et al., 2009; Valero et al., 2009;
´
The screen identified library members with a pronounced influ- Zhang and Franzen, 2010). On the other hand, the bromine
ence on the complexity of neuronal network formation and polar- served the purpose to enable the diastereoselective conversion
ized cell growth.
of the intermediate hemiaminal (8) (after treatment with trifluoro-
acetic acid (TFA) performing a Friedel-Crafts addition followed
by hydrolysis) into the target secoyohimbane scaffold (6).
Gratifyingly, orientating experiments employing 2-bromoin-
dole (7) and cinnamic aldehyde in the presence of an organoca-
talyst indicated that the strategy was indeed viable and that the
RESULTS AND DISCUSSION
Development of an Enantioselective Catalyzed
Synthesis of a Secoyohimbane Library
Initially, we focused on the development of an asymmetric cata- spiroindolinones were formed.
lytic domino Michael-Mannich reaction of racemic oxindole
In order to establish favorable reaction conditions, under
derivative (3) and a,b-unsaturated aldehydes (4) to yield a se- which the desired secoyohimbane-inspired compounds would
coyohimbane (6) (Figure 2). The products (6) would contain an be obtained in preparatively viable yields and with high stereose-
amide function instead of the tertiary amine of the indolizine, lectivity, as model reaction, the addition of nucleophile (7) to
which should prevent isomerization via Mannich/retro-Mannich cinnamic aldehyde in the presence of different organocatalysts
reactions. However, despite extensive examination of various (A–D) in several solvents and in the presence of potassium ace-
reaction conditions, the formation of the target secoyohimbanes tate as additive was optimized. The search for a suitable solvent
(6) was not observed and only cyclic hemiaminals (5) were (Figure 3, entries 1–10) revealed that the best results for 6a with
obtained as stable intermediates. All attempts to convert inter- respect to enantiomeric excess, diastereoselectivity, and yield
mediates (5) to products (6) via formation of an acyliminium ion were obtained in methanol with organocatalyst (A) (Figure 3,
and Mannich reaction in the presence of various Brønsted acids entry 3). We note that the presence of the additive is essential
and bases failed. In the light of this failure, a new approach for the for product formation in all solvents tested. Diphenylprolinol
preparation of the secoyohimbane scaffold needed to be devel- trimethylsilylether (A) gave a high level of enantiocontrol,
oped. We envisioned to use 2-bromoindole derivative (7) (Fig- whereas diphenylprolinol (B) itself did not catalyze the reaction
ure 2; Miyamoto et al., 2006; Miyake et al., 2004) as decisive (Figure 3, entry 8). Jorgensen’s catalyst (C) or MacMillan’s imida-
starting material, in which the 2-bromo substituent would serve zolidinone (D) led to lower levels of enantioselectivity (Figure 3,
a double function. On the one hand, it was meant to serve as a entries 9 and 10). To further optimize the reaction conditions,
protecting group, preventing cyclization to the two position of we varied the additives (Figure 3, entries 11–16). We found that
the indole in 8 (masked by the bromine), and due to that, the for- the addition of caesium acetate slightly improved the yield of
mation of the tetrahydro-b-carboline cannot occur, redirecting product 6a and the enantioselectivity (Figure 3, entry 17). Further
Chemistry & Biology 20, 500–509, April 18, 2013 ª2013 Elsevier Ltd All rights reserved 501

Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
Figure 3. Exploration of Different Reaction
Conditions
aReaction conditions: aldehyde (4) (1.0 equiv),
indole (7) (1.2 equiv), additive, and catalyst
were stirred in solvent at ambient temperature
for the given time. Trifluoroacetic acid was added
at ꢁ60ꢀC, and the reaction mixture was warmed
up to room temperature.
bIsolated yields of 6a after column chromatog-
raphy.
cDetermined by 1H nuclear magnetic resonance
(NMR) analysis.
dDetermined by high-performance liquid chroma-
tography (HPLC) analysis. The ee values for both
spiroindolinones 6a and 60a.
eOpposite enantiomer.
fAt 0ꢀC.
A
B
C
D
a
b
c
d
TFE, 2,2,2-trifluorethanol.
unambiguously by means of single-crys-
tal X-ray analysis of compound 6e (Fig-
ure S1 available online).
Mechanistically, the first step of the
developed transformation is a stereo-
e
and regioselective Michael addition of
the 2-bromoindole (7) to the a,b-unsatu-
rated aldehyde, which establishes two
stereocenters (Figure 5). Although 7 con-
tains several nitrogen- and two carbon-
nucleophiles, only addition of the acidic
methylene group to the unsaturated alde-
hyde was observed, which leads to inter-
mediate (9). The stereochemical course
of the organocatalyzed conjugate addi-
tion is in accordance with relevant results
reported for the addition of malonic acid
derivatives to enals (Brandau et al.,
f
´
2006; Marigo et al., 2006; Franzen and
experiments revealed that the catalyst loading could be reduced Fisher, 2009; Hayashi et al., 2009; Valero et al., 2009; Zhang
´
to 10 mol % and that substoichiometric amounts of caesium ac- and Franzen, 2010). Intermediate (9) then undergoes sponta-
etate suffice (Figure 3, entries 18 and 19). The enantioselectivity neous cyclization to form hemiaminal (8). Elimination of water
could be increased from 92% to 95% by lowering the reaction after protonation by the Brønsted acid leads to formation of
temperature to 0ꢀC (Figure 3, entry 20). Further decrease of the iminium intermediate (11), which is in equilibrium with enamide
reaction temperature led to low conversion rates and prolonged (10). The iminium intermediate (11) undergoes an electrophilic
reaction times.
Friedel-Crafts cyclization to yield spirocycle (12), embodying
Notably, an interconversion of single isolated isomers was not an imidoyl bromide, which is hydrolyzed in the presence of
observed upon storage under the reaction conditions for 4 weeks Brønsted acid and water to yield target spiroindolinone (6). Inter-
estingly, Mannich cyclization of cyclic hemiaminal (5), which
at ambient temperature.
With the optimized reaction conditions identified, we investi- could be obtained from 8 by hydrolysis of the imidoyl bromide
gated the substrate scope with regard to various a,b-unsatu- does not occur. In order to rationalize the stereochemical prefer-
rated aldehydes (Figure 4). A broad range of aldehydes with ence in the spirocyclization, we assume that two competing
electron-donating and -withdrawing substituents, para-, meta-, transition states TS1 and TS2 may be passed, in which the indole
and orthosubstituted cinnamaldehydes as well as acroleines attacks the acyl iminium intermediate anti to substituent R (Fig-
substituted with a heterocycle in the b-position can be employed ure S2). In TS1, which leads to the observed configuration of
successfully in the organocatalyzed enantioselective cascade the products, the bromine points away from the ester substitu-
reaction. The secoyohimbane-inspired products were obtained ent. Rotation around the bond connecting C3 of the indole with
in preparatively viable yields and with excellent enantioselectivity the iminium-N would lead to TS2 and yield the isomeric spiro
(88%–96% enantiomeric excess [ee]). The absolute configura- configuration. TS2 is less preferred than TS1, due to unfavorable
tion of the predominantly formed stereoisomer was determined interactions between the bromine and the ester group.
502 Chemistry & Biology 20, 500–509, April 18, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
Figure 4. Results of the Enantioselective
Catalyzed Synthesis of Secoyohimbane-
Inspired Spirocyclic Indolinones
aFor detailed experimental procedures and char-
acterization of the products, see the Supplemental
Information.
bIsolated yields of the single diastereomer after
Entrya
1
R
Time [hr]
20
Yield [%]b drc
ee [%]d
95
column chromatography.
cDetermined by 1H NMR analysis.
dDetermined by HPLC analysis for the major
63
51
55
25
52
55
69
5/1
5/1
2/1
2/1
4a
4b
4c
4d
4e
4f
6a
6b
6c
6d
6e
6f
isomer.
See also Figure S1 (entry 5, compound 6e).
2
3
4
5
6
7
20
16
16
16
40
20
94
92
95
compounds led to dramatic outgrowth
of the neuronal membrane. Therefore,
they were chosen for more detailed
characterization.
10/1 95
10/1 92
In the subsequent in-depth analysis,
changes in the morphology of neurons
exposed to the 2-naphtyl (6i) and 3-furyl
(6n) derivatives were investigated. Reti-
noic acid (RA) and brain-derived neuro-
trophic factor (BDNF) were used as
positive controls. RA and BDNF bind
to their corresponding receptors, promot-
ing polymerization of the cytoskeleton
preceding the establishment of a more
developed complex neuronal network
(Theus et al., 2006; Clagett-Dame et al.,
2006; Yasuda et al., 2007; Kozisek et al.,
2008). Furthermore, the natural pro-
ducts rhynchophylline (Rhy) and isorhyn-
chophylline (IsoRhy) were included in
the experiment. Rhynchophylline acts
as a noncompetitive antagonist of the
N-methyl-D-aspartate receptor and influ-
ences the modulation of calcium and
potassium ion channels (Kang et al.,
2004; Xu et al., 2012). However, the
exact mechanisms of rhynchophylline ac-
tivity and its targets are not known. The
confocal images acquired after exposure
of the primary cultured rat hippocampal
5/1
5/1
96
89
4g
6g
8
9
40
36
60
62
4h
4i
6h
6i
10/1 95
15/1 95
10
40
61
4j
6j
11
12
20
20
35
46
1.2/1 96
1.5/1 90
4k
4l
6k
6l
13
14
30
40
58
42
4/1
8/1
88
93
4m
4n
6m
6n
Biological Validation of the Secoyohimbane-Inspired
neurons to small molecules and the observed morphological
Library changes were quantitatively analyzed (Figure 6; see also the
In light of the use of Uncaria species for the treatment of neuro- Supplemental Information).
degenerative diseases, we investigated whether the natural
Exposure of primary cultured rat hippocampal neurons to
product-inspired spirocyclic indolinones positively influence rhynchophylline shows a strong effect on the development of
neurite outgrowth from primary rat hippocampal neurons (Dotti neurons. Rhynchophylline increased the complexity of the
et al., 1988). In an initial screen, the major diastereomers (6a– neuronal network formed, since the total neurite length and sin-
6n) were dissolved in DMSO and screened at a final concentra- gle neurite length are higher in comparison to control cells (Fig-
tion of 10 mM for their influence on quantitative membrane ures 6I and 6K). However, in the presence of the natural prod-
outgrowth by monitoring incorporation of a fluorescent dye into uct, the number of neurites remains comparable to treatment
the increasing amount of cell membrane formed. Fluorescence with DMSO (Figure 6J). The formed neurites are longer in com-
intensity is higher with increasing growth and, thereby, formation parison to treatment with the controls, BDNF and RA (Fig-
of neuronal membranes. Analysis of the fluorescent staining re- ure 6J). Furthermore, the length of the axon is similar to
vealed that 2-naphthyl derivative (6i) and 3-furyl derivative (6n) the value recorded for application of the positive control
were most active and exposure of the neuronal cells to these (RA effect) (Figure 6H). This observation suggests that
Chemistry & Biology 20, 500–509, April 18, 2013 ª2013 Elsevier Ltd All rights reserved 503

Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
6n, rhynchophylline. In general, the morphological changes
observed for the primary hippocampal cells after exposure to
the spirocyclic natural product-inspired secoyohimbanes indi-
cate that their mode of action may include modulation of cyto-
skeletal rearrangement processes and proteins involved therein
and possibly an influence on growth cone development (Dent
and Gertler, 2003).
The results obtained with rat primary hippocampal neurons
were also validated using a different approach, employing
mouse motor neurons derived from ESCs to determine the
total neurite length, the number of branch points and the
maximal length of neurites. Trophic factors, such as BDNF, neu-
rotrophin-4, ciliary neurotrophic factor, and glial cell line-derived
neurotrophic factor (GDNF), have different effects on neurons
in vitro or in vivo, including axonal growth, neurogenesis, and
an increase in survival (Airaksinen and Saarma, 2002; Bothwell,
1995; Zurn et al., 1996), and, as such, have been used as thera-
peutic agents in a wide range of human diseases (Yamamoto
et al., 2001; Schindowski et al., 2008; Henriques et al., 2010;
Masi and Brovedani, 2011). However, with age and in patholog-
ical conditions, the production of neurotrophic factors from
satellite cell or the target organs can be reduced and lead to
increased susceptibility to nerve degeneration and poor
neuronal survival. For that reason, in all of the experiments,
DMSO was considered as a negative control and GDNF as a
positive control to compare with the activity of the molecules
tested, since this factor shows a very potent effect on motor
neurons among these neurotrophic factors. The images
acquired after exposure of ESC-derived motor neurons to small
molecules and the observed morphological changes were
quantitatively analyzed with Cellomics Bioapplication Neurite
Profiling (Figure 7).
In the experiments, p-trifluoromethylphenyl derivative (6b),
p-methoxyphenyl derivative (6f), and 3-furyl derivative (6n),
were identified as hits. In accordance to the neurotrophic hy-
pothesis (Oppenheim, 1989), axonal branches from various
neurons compete for neurotrophic molecules, and the access
to neurotrophic molecules from the target tissue regulates the
number of neurons: the survival of cells with a neurotrophic
deficit is compromised, while those with a correct trophic sup-
port can survive. Small changes in the neurotrophic support
can cause changes in the number of motor neurons obtained
Figure 5. Proposed Mechanism of Organocatalytic Synthesis of the
Secoyohimbane Scaffold
The mechanism of spirocyclic products (6) formation is included in the
sequence of reactions. The key step of the reactions sequence is enantiose-
lective organocatalytic Michael addition to unsaturated aldehydes to form an
intermediate (9). Following cyclization of 9 generates N-acyl-iminium ion (11),
which under the reaction conditions undergoes Mannich reaction to give the
desired products (6). HB, Brønsted acid; HBr, hydrobromic acid.
See also Figure S2.
rhynchophylline induces cytoskeletal rearrangement toward in different wells, with big effect on the neurite branching and
formation of more polarized cells undergoing maturation. The length, especially after 7 days in vitro. Because of that, the values
results support the known neuroprotective activity of rhyncho- of the error bars in the graphs are disproportionately large. Thus,
phylline (Shimada et al., 1999). Interestingly, the spiroisomeric neurite arborization and neuronal survival can be used as an
isorhynchophylline unfavorably impairs the development of indicator of the neurotrophic activity of different molecules.
primary cultured rat hippocampal neurons (Figure 6). To our According to the results in the graphs, compound p-trifluorome-
delight, 3-furyl derivative (6n), which differs in substituent thylphenyl derivative (6b) is the most active in all experiments
0
pattern and absolute configuration at C8a from rhynchophyl- composed of neurite total length, number of branch points,
line, yet clearly has a structure resembling the natural product and neurite maximal length. The most remarkable effect is
and displays an activity profile very similar to the natural alka- shown in neurite maximal length, which means that the cells
loid (Figure 6). Replacement of the 3-furyl group (6n) with the treated with this molecule have the longest neurites. Compound
2-naphtyl group (6i) did not result in a difference in outgrowth 6f is less active, but still presents the same type of activity in all
of the neuronal membrane. However, administration of deriva- experiments. In general, the activity of these spirocyclic indoli-
tive 6i led to the formation of complex neuronal networks, nones are below the values of the powerful trophic factor
which is reflected in a substantial increase of single neurite GDNF; however, they provide good starting points for further
length and lower number of neurites per neuron. This finding structural modifications aimed at increasing their neurotrophic
may reflect a different mode of action of compounds 6i and activity.
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Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
Figure 6. Neurite Outgrowth-Promoting Activity on Primary Cultured Rat Hippocampal Neurons and Quantification of Morphological
Changes
(A) Morphology of neurons of untreated cells.
(B) Morphology of neurons in the presence of DMSO.
(C) Morphology of neurons treated with BDNF.
(D) Morphology of neurons treated with retinoic acid.
(E) Morphology of neurons treated with 10 mM of 6n.
(F) Morphology of neurons treated with 10 mM of 6i.
(G) Morphology of neurons treated with 10 mM of isorhynchophylline.
(H) Morphology of neurons treated with 10 mM of rhynchophylline.
(I) Total neurite length per cell.
(legend continued on next page)
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Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
Figure 7. Neurite Outgrowth-Promoting Activity and Quantification of Morphological Changes on ESC-Derived Motor Neurons
(A) Morphology of neurons treated with 10 mM of 6b.
(B) Morphology of neurons treated with 10 mM of 6f.
(C) Morphology of neurons treated with 10 mM of 6n.
(D) Morphology of neurons in the presence of DMSO.
(E) Morphology of neurons treated with GDNF.
(F) Neurite total length.
(G) Branch points.
(H) Maximum of neurite length.
Scale bar: 40 mm. Error bars show SD (n = 4).
In conclusion, we developed a highly practical catalytic from simple and readily available starting materials in a one-
method for the synthesis of a collection of complex natural prod- pot multistep reaction sequence. Investigation of this family of
uct-inspired compounds with the basic scaffold of the secoyo- spirocyclic indolinones for neurite outgrowth-promoting activity
himbane alkaloids. The polycyclic heterocycles embodying one employing two different neuronal cell lines revealed remarkable
quaternary and three tertiary stereocenters are obtained with neurotrophic activity and provided insight for further structural
excellent enantioselectivity and with preparatively viable yields modification to arrive at more potent compounds.
(J) Number of neurites per cell.
(K) Length of single neurite per cell.
(L) Axonal length per cell.
Scale bar: 100 mm. CTRL, untreated cells.
See also Figure S3.
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Chemistry & Biology
Catalytic Synthesis of Neurotrophic Compounds
synthesis and cellular evaluation of spirooxindoles inspired by natural prod-
SIGNIFICANCE
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Bella, M., and Gasperi, T. (2009). Organocatalytic formation of quaternary
Many neurological diseases are characterized by a progres-
sive loss of neuronal activity, and novel approaches aimed
at identification of small molecules with neurotrophic
properties are of major importance. Natural products with
neurotrophic activity provide evolutionary validated core
structures for the synthesis of compound classes endowed
with neurite growth-promoting activity. The development of
synthesis methods that make such compound classes
accessible in a very practical, flexible, and enantioselective
manner has high significance. Therefore, a highly effi-
cient diastereo- and enantioselective and organocatalyzed
cascade access to the secoyohimbane scaffold of the
neuromodulatory alkaloid rhynchophylline was developed
that generates four stereocenters in a one-pot multistep re-
action sequence. Investigation of a compound collection
synthesized by means of this method revealed a class of
neurotrophic compounds.
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EXPERIMENTAL PROCEDURES
Cheng, X., Harzdorf, N., Khaing, Z., Kang, D., Camelio, A.M., Shaw, T.,
Schmidt, C.E., and Siegel, D. (2012). Neuronal growth promoting sesquiter-
pene-neolignans; syntheses and biological studies. Org. Biomol. Chem. 10,
383–393.
All products were fully characterized. Spectral data for all compounds are
provided in the Supplemental Information.
Typical Procedure for the Organocatalyzed Synthesis
To a solution of the aldehyde (4) (0.4 mmol) and the catalyst (10 mol %, 40 mmol)
in MeOH (0.5 ml) were added CsOAc (60 mol %, 0.24 mmol) and 3-(2-(1H-indol-
3-yl)ethylamino)-3-oxopropanoate (7) (1.2 eq., 0.48 mmol) in MeOH (0.5 ml) at
0ꢀC. The reaction mixture was stirred at 0ꢀC for 20–40 hr, cooled to ꢁ80ꢀC, and
TFA (10 eq) was added. The reaction mixture was warmed to room tempera-
ture, passed through a short plug of silica, and concentrated under reduced
pressure. Purification by column chromatography on silica gel (ethyl acetate/
dichloromethane = 1/5/1/2) yielded the product.
Clagett-Dame, M., McNeill, E.M., and Muley, P.D. (2006). Role of all-trans
retinoic acid in neurite outgrowth and axonal elongation. J. Neurobiol. 66,
739–756.
Deiters, A., Pettersson, M., and Martin, S.F. (2006). General strategy for the
syntheses of corynanthe, tacaman, and oxindole alkaloids. J. Org. Chem.
71, 6547–6561.
Dent, E.W., and Gertler, F.B. (2003). Cytoskeletal dynamics and transport in
growth cone motility and axon guidance. Neuron 40, 209–227.
Dotti, C.G., Sullivan, C.A., and Banker, G.A. (1988). The establishment of
ACCESSION NUMBERS
polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454–1488.
The Cambridge Crystallographic Data Centre accession number for the com-
pound 6e reported in this paper is CCDC 915444.
Du¨ ckert, H., Pries, V., Khedkar, V., Menninger, S., Bruss, H., Bird, A.W.,
Maliga, Z., Brockmeyer, A., Janning, P., Hyman, A., et al. (2012). Natural prod-
uct-inspired cascade synthesis yields modulators of centrosome integrity.
Nat. Chem. Biol. 8, 179–184.
SUPPLEMENTAL INFORMATION
Enders, D., Grondal, C., and Hu¨ ttl, M.R.M. (2007). Asymmetric organocatalytic
Supplemental Information includes three figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
org/10.1016/j.chembiol.2013.03.011.
domino reactions. Angew. Chem. Int. Ed. Engl. 46, 1570–1581.
´
Franzen, J., and Fisher, A. (2009). Asymmetric alkaloid synthesis: a one-pot
organocatalytic reaction to quinolizidine derivatives. Angew. Chem. Int. Ed.
Engl. 48, 787–791.
ACKNOWLEDGMENTS
Grondal, C., Jeanty, M., and Enders, D. (2010). Organocatalytic cascade reac-
tions as a new tool in total synthesis. Nat. Chem. 2, 167–178.
This work was supported by the Max Planck Gesellschaft. The research lead-
ing to these results has received funding from the European Union Seventh
Framework Programme under grant agreement No. HEALTH-F2-2009-
241498 (‘‘EUROSPIN’’ project). S.L.-T. thanks the Spanish ME for a postdoc-
toral fellowship.
Hayashi, Y., Toyoshima, M., Gotoh, H., and Ishikawa, H. (2009).
Diphenylprolinol silyl ether catalysis in an asymmetric formal carbo [3 + 3]
cycloaddition reaction via a domino Michael/Knoevenagel condensation.
Org. Lett. 11, 45–48.
Henriques, A., Pitzer, C., and Schneider, A. (2010). Neurotrophic growth fac-
tors for the treatment of amyotrophic lateral sclerosis: where do we stand?
Front. Neurosci. 4, 32.
Received: January 16, 2013
Revised: March 12, 2013
Accepted: March 19, 2013
Published: April 18, 2013
Inoue, M., Lee, N., Kasuya, S., Sato, T., Hirama, M., Moriyama, M., and
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