Truncated militarinone fragments identified by total chemical synthesis induce neurite outgrowth

Article abstract of DOI:10.1039/c2md20181j

A group of natural products produced by entomopathogenic fungi has recently been shown to induce neurite outgrowth in the absence of nerve growth factor (NGF) in pheochromocytoma PC-12 cell culture. After completion of a total synthesis program, we continued our efforts in reducing complexity by synthetically preparing truncated analogs. Using this approach we were able to not only significantly simplify the structures but also decrease the minimum concentration needed for neuritogenic activity by a factor of 20. This activity was suppressed in the presence of an ERK1/2 inhibitor suggesting activation of the mitogen activated protein kinase (MAPK) pathway as a potential mode of action of these pyridone alkaloids. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2md20181j

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Truncated militarinone fragments identified by total
chemical synthesis induce neurite outgrowth†
Cite this: Med. Chem. Commun., 2013,
4, 135
*
Fabian Schmid, Henning J. Jessen,‡ Patrick Burch and Karl Gademann
A group of natural products produced by entomopathogenic fungi has recently been shown to induce
neurite outgrowth in the absence of nerve growth factor (NGF) in pheochromocytoma PC-12 cell
culture. After completion of a total synthesis program, we continued our efforts in reducing complexity
by synthetically preparing truncated analogs. Using this approach we were able to not only significantly
simplify the structures but also decrease the minimum concentration needed for neuritogenic activity by
a factor of 20. This activity was suppressed in the presence of an ERK1/2 inhibitor suggesting activation
of the mitogen activated protein kinase (MAPK) pathway as a potential mode of action of these
pyridone alkaloids.
Received 3rd July 2012
Accepted 10th August 2012
DOI: 10.1039/c2md20181j
www.rsc.org/medchemcomm
considered a hallmark of neurodegenerative diseases, by
reconstruction of neuronal networks using small molecules.8
Introduction
The capacity of natural products as powerful modulators of
biological systems has been recognized for centuries, and they
remain a prime source of drug discovery.1 This potential can be
leveraged by organic synthesis and, in particular, total synthesis
of natural products offers the opportunities to obtain powerful
derivatives that are not accessible from the natural product
itself.2 These approaches (sometimes referred to as ‘chemical
editing’ or ‘diverted total synthesis’)3 complement the long-
standing goal of mapping out the pharmacophores of natural
products and lead to successful drugs in the clinic (octreotide
from somatostatin and eribulin from halichondrin).4 Through
such diverted total synthesis efforts, we have shown that the
antitumor polyketide anguinomycin (1) can be truncated and
that fragment 2 retains most of the biological activity with
regard to the inhibition of nuclear export (Fig. 1).5 In this study,
we report on the truncation of pyridone natural products such
as 3 or 4, leading to an optimized fragment of type 5 that
induces neurite outgrowth at lower concentrations.
In the context of our program directed towards the synthesis
of neuritogenic natural products,9 we have shown that different
naturally occurring pyridone alkaloids can be assembled in a
modular way from a bifunctional pyridone core intermediate.9c
Starting from this advanced intermediate, we succeeded in the
stereoselective preparation of the natural products farinosone A
(3), pre-tenellin B, militarinone D, torrubiellone C, putative
natural products and their enantiomers in synthetic form.9c–e
Interestingly, all of these compounds induced neurite
outgrowth in the PC-12 cell line at concentrations of 20 mM. In
addition, the activity was found to be generally independent of
the length of the side chain as well as the absolute conguration
of the stereogenic centers.9c–e Based on these structural insights,
we wanted to (1) evaluate whether the side chain could be
completely truncated while retaining activity and (2) perform
structure activity relationship studies on the biaryl pyridone
system to improve potency.
Various pyridone alkaloids have been isolated from ento-
mopathogenic fungi.6 Representative members of this class
such as farinosone A (3) and militarinone D (4) have been
shown to signicantly induce neurite outgrowth in the absence
of nerve growth factor (NGF).7 This phenotypic observation
could be of interest to target neuritic atrophy, which can be
Results and discussion
Synthesis of the pyridone derivatives
The rst goal consisted of the evaluation of the three different
H-bond donors (denoted by arrows for lead structure 5 in Fig. 1)
on activity. This was achieved by synthesizing pyridones meth-
ylated in the H-bond donor positions following a cross-
University of Basel, Department of Chemistry, St. Johanns-Ring 19, 4056 Basel, coupling/methylation/demethylation
strategy
from
the
Switzerland. E-mail: karl.gademann@unibas.ch; Tel: +41 61 267 11 44
advanced intermediates 7 and 8. This gave access to different
permutations of methylation patterns (Scheme 1), but also
allowed easy introduction of new biaryl scaffolds via a cross-
coupling approach (vide supra).
† Electronic supplementary information (ESI) available: Full characterisation data
and experimental procedures are available. CCDC 888459–888461. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c2md20181j
The synthesis of pyridones with different methyl substitu-
tions began with the preparation of the pyridone core starting
¨
‡ Present address: University of Zurich, Institute of Organic Chemistry,
¨
Winterthurerstrasse 190, 8057 Zurich, Switzerland.
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coupling,11 possibly due to inactivation of the catalyst by the
bidentate pyridone serving as ligand. Therefore, the SEM pro-
tecting group was introduced, yielding both the N- and O-pro-
tected derivatives 7 and 8 quantitatively as a 3 : 2 mixture.12
Both of these compounds were useful in the course of the
project, amenable to cross-coupling reactions, and therefore no
attempts were made to obtain a single entity. Furthermore, they
could be readily prepared and separated on a gram scale. The N-
protected pyridone
8 was functionalized with either (4-
methoxyphenyl)boronic acid (conditions a) or {4-[(4-methoxy-
benzyl)oxy]phenyl}boronic acid (conditions c) to allow access to
different methyl substitution patterns. For these building
blocks quantitative yields could be obtained using optimized
conditions. The efficiency of the transformations proved to be
highly dependent on the composition of the degassed solvent
mixture with glyme–water–DMF (9 : 1 : 0.5) as the optimal
system. Catalyst screening revealed bis(tri-tert-butylphosphine)
palladium as the most efficient catalyst (complete conversion
aer 1 h at rt, 2% catalyst loading), whereas the PEPPSI-IPr-
catalyst, for example, did not yield the product. As the former
catalyst had to be handled in a glove-box for good results, we
eventually decided to use Pd(PPh3)4 for our transformations,
although at the cost of prolonged reaction times (16 h), higher
temperatures (60 ꢀC) and catalyst loadings (5–10%). Aer
removal of the SEM-group with TBAF under reux, the pyr-
idones 9a and 10 were obtained in quantitative yield. Interest-
ingly, compound 9a was found to crystalize in two polymorphs,
with the ethyl-carboxylate moiety rotated about 180ꢀ in the two
polymorphs (see ESI† for more details). N-Selective methyla-
tions were carried out with methyl iodide and potassium
carbonate, giving rise to the N-methyl pyridones 9c and 11c.
Furthermore, selective cleavage of the C(4)-methoxy ethers with
lithium iodide (LiI) allowed for the construction of pyridone 9b
and N-methyl pyridone 9d. Also the fully deprotected pyridone
11b could be obtained under these conditions. It is interesting
to note that in this series, the rigid biaryl scaffold is decorated
with a variety of H-bond donors and acceptors, thus allowing us
to evaluate the role of each group. Furthermore, ClogP values
shied from ꢁ0.4 for pyridone 11b to 0.5 for methylated pyr-
idone 9c. As will be discussed later in detail, we found that a key
structural feature for neuritogenic activity was represented by
the 40-hydroxy-biaryl unit with the 4-hydroxy group of the pyr-
idone core being less important. Therefore, we focused our
synthetic efforts on the modication of the peripheral phenyl
group and retaining the methyl ether.
Fig. 1 Natural products 1, 3 and 4 and their truncated analogs 2 and 5.
from ethyl cyanoacetate (6) following literature procedures
(Scheme 1).9c,10 As previously shown,9c the pyridone could not be
directly functionalized using the Suzuki–Miyaura cross-
As we had chosen an approach employing cross-coupling
reactions, we were now able to rapidly introduce different aryl
groups derived from commercially available boronic acids and
boronic esters. For this purpose, O-SEM protected pyridine 7 or
N-SEM protected pyridone 8 were subjected to the previously
optimized reaction conditions9c with different building blocks,
resulting in yields of the target compounds ranging from 20%
for electron decient systems to 95% for electron rich systems
(Scheme 2). Aerwards, the SEM protecting group was removed
by either addition of TFA in CH2Cl2 or TBAF in THF, thus
yielding the desired pyridones 12a–h including amine 12f aer
deprotection of 12e with TFA as the TFA salt. The pyridines
Scheme 1 Synthesis of pyridones with different methylation patterns. Reagents
and conditions: (a) (4-methoxyphenyl)boronic acid, K2CO3, 5–10% Pd(PPh3)4,
glyme–H2O–DMF 9 : 1 : 0.5, 60 ꢀC, 16 h; (b) TBAF, THF, 60 ꢀC, 9 h; (c) {4-[(4-
methoxybenzyl)oxy]phenyl}boronic acid, K2CO3, 5–10% Pd(PPh3)4, glyme–H2O–
DMF 9 : 1 : 0.5, 60 ꢀC, 16 h; (d) LiI, THF, reflux; (e) MeI, K2CO3, CH3CN; (f) TFA (5%
in DCM), rt, 30 min.
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compounds, dilution series ranging from 500 nM to 20 mM were
evaluated, which constituted the benchmark concentration
obtained from earlier analyses of the natural products such as
farinosone A (3). To get further insight into the mode of action
of these compounds, the most efficient one was additionally
incubated with PC-12 cells in the presence of 5 mM ERK1/2
inhibitor (extracellular regulated kinase). The results are
summarized in Fig. 3 and 4.
A hypothesis for the pharmacophore can be delineated from
these experimental observations. Compounds 9a and 9c did not
display any neuritogenic activity at 20 mM. In contrast,
compounds 11a–c were all active at this concentration. This
underlined the importance of the unprotected para-hydroxy
group on the phenyl moiety. Further evidence was provided by
the inactivity of phenyl- or tert-butyl modied pyridone 12a/c
even at concentrations of 40 mM. In contrast to all these inactive
compounds, the initial hypothesis of whether the complex
polyene side chains of the natural products can be truncated
was corroborated by the activity of the truncated analogs 11a–c,
leading to compounds that were as active as the natural prod-
ucts. Our next aim was to improve this activity.
In order to investigate the role of the phenol moiety, we
investigated the replacement of the OH group by F, NHR or NH2
groups as found in the pyridones 12b, 12e and f, but none of
these modications provided active compounds, even at
concentrations of 40 mM. Another group of compounds inves-
tigated were the pyridines 12g/h; again, these compounds were
found to be inactive, further underlining the importance of the
phenolic OH-group for activity. An interesting result was ach-
ieved with the catechol derived structure 12d. Already in our
rst assays at 20 mM concentration, we observed a convincing
induction of neurite outgrowth as can be seen from the
micrographs shown in Fig. 2.
Scheme 2 Preparation of differently aryl-substituted pyridones. Reagents and
conditions: (a) corresponding boronic acid, K2CO3, 5–10% Pd(PPh3)4, glyme–H2O–
DMF 9 : 1 : 0.5, 60 ꢀC, 16 h; (b) TBAF, THF, 60 ꢀC, 9 h; (c) 2 equiv. TFA, DCM, 5 min;
(d) 50% TFA in DCM, 10 min.
12g/h were obtained in low yields owing to their hydrophilicity
(ClogP ¼ ꢁ1.0), which complicated purications but provided
sufficient material for the bioassays. As initial studies had
shown no decrease in activity for compounds where the polyene
sidechain was replaced by an ethyl carboxylate moiety, no
further modications at the 3-position were performed for this
study. It is also interesting to note that N-methylated 4-hydroxy-
pyridones are intrinsically uorescent compounds emitting
blue light.
Investigation of the neuritogenic properties in the PC-12 assay
All compounds were examined concerning their ability to
induce neurite outgrowth in the PC-12 assay7d,9e,13 (rat pheo-
chromocytoma). Briey, PC-12 cells were grown in DMEM (Dul-
becco’s modied Eagle medium, high glucose + L-glutamine)
containing antibiotics (penicillin, streptomycin) under sterile
conditions. Cells were seeded into collagene coated 24 well
plates (105 cells per well) aer passage through a 21-gauge
needle and aer two days fresh medium containing the
different compounds was added. The cells were incubated for
three more days in a humidied atmosphere at 37 ꢀC (5% CO2)
with the compounds, then xed with formaldehyde, stained
with Giemsa stain and analyzed under a microscope. Pictures
from three to ve randomly chosen areas were taken (see Fig. 2),
the fraction of differentiated cells was calculated with more
than 500 cells in total and the results were analyzed by ANOVA.
Errors are indicated as the standard error of the mean. As
negative and positive controls, cells were incubated with DMSO
(vector ¼ 0.1%) or NGF 7S (nerve growth factor from the murine
submaxillary gland 10 ng mLꢁ1), respectively. For the active
Catechol derived pyridone 12d induced neurite outgrowth
with a comparable efficiency to the natural products farinosone
A (3) and prebassianin B9c at 20 mM. Furthermore, this neu-
ritogenic effect could be observed down to concentrations of
2 mM of 12d. At a concentration of 1 mM an effect could still be
observed, whereas at 500 nM the neuritogenic effect was not
observable anymore. In contrast, prebassianin
B treated
Fig. 3 Neuritogenic activity of the pyridones in the PC-12 assay. Nerve growth
factor (NGF) control: 10 ng mLꢁ1. DMSO control: 0.1%. Incubation period: 3 days.
Number of counted cells: >500. Error bars denote SEM.
Fig. 2 Micrographs of the incubated PC-12 cells: 12d 20 mM (left), NGF positive
control (middle) and DMSO blank (right).
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4-position did not signicantly inuence activity. A 40-hydroxy
substituent was found to be necessary for activity, as none of the
derivatives bearing a functional group other than 40-OH were
found to be active. These optimization efforts resulted in the
catecholate pyridone derivative 12d, which induces neurite
outgrowth at markedly lower concentrations compared to its
natural product progenitors, while cutting the number of
carbon atoms from 26 to 16 and reducing the overall number of
synthetic transformations by 12. As already demonstrated in the
anguinomycin case,5 truncated natural products can offer
advantages of concomitantly increasing biological activity while
reducing structural complexity.
Fig. 4 Neuritogenic activity of 12d in the PC-12 assay. Nerve growth factor
(NGF) control: 10 ng mLꢁ1. DMSO control: 0.1%. Incubation period: 3 days.
Number of counted cells: >500. Error bars denote SEM.
Acknowledgements
K. G. is a European Young Investigator (EURYI). We gratefully
acknowledge nancial support by the DFG (Project JE 572/1-1)
and the SNF (PE002-117136/1) and thank Dr Neuburger for X-ray
analyses.
Fig. 5 Phenyl pyridine 13.
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