Functionally optimized neuritogenic farinosone C analogs: SAR-study and investigations on their mode of action

Article abstract of DOI:10.1021/ml400435h

Several natural products derived from entomopathogenic fungi have been shown to initiate neuronal differentiation in the rat pheochromocytoma PC12 cell line. After the successful completion of the total synthesis program, the reduction of structural complexity while retaining biological activity was targeted. In this study, farinosone C served as a lead structure and inspired the preparation of small molecules with reduced complexity, of which several were able to induce neurite outgrowth. This allowed for the elaboration of a detailed structure-activity relationship. Investigations on the mode of action utilizing a computational similarity ensemble approach suggested the involvement of the endocannabinoid system as potential target for our analogs and also led to the discovery of four potent new endocannabinoid transport inhibitors.

Full text of DOI:10.1021/ml400435h

Letter
pubs.acs.org/acsmedchemlett
Functionally Optimized Neuritogenic Farinosone C Analogs: SAR-
Study and Investigations on Their Mode of Action
Patrick Burch,†,‡ Andrea Chicca,§ Jurg Gertsch,§ and Karl Gademann†,*
̈
†Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel, Switzerland
‡Swiss Federal Institute of Technology (EPFL), Chemical Synthesis Laboratory (SB-ISIC-LSYNC), 1015 Lausanne, Switzerland
§Institute of Biochemistry and Molecular Medicine, University of Bern, Buhlstrasse 28, CH-3012 Bern, Switzerland
̈
S
* Supporting Information
ABSTRACT: Several natural products derived from entomopathogenic fungi
have been shown to initiate neuronal differentiation in the rat
pheochromocytoma PC12 cell line. After the successful completion of the
total synthesis program, the reduction of structural complexity while retaining
biological activity was targeted. In this study, farinosone C served as a lead
structure and inspired the preparation of small molecules with reduced
complexity, of which several were able to induce neurite outgrowth. This
allowed for the elaboration of a detailed structure−activity relationship.
Investigations on the mode of action utilizing a computational similarity
ensemble approach suggested the involvement of the endocannabinoid
system as potential target for our analogs and also led to the discovery of four
potent new endocannabinoid transport inhibitors.
KEYWORDS: Neurite outgrowth, natural products, endocannabinoid membrane transport, CB1 receptor, SAR, truncated
eurodegenerative disorders are among the leading causes
N
of death in aging societies, and every third person dying
suffers from a type of dementia. To date, approved medication
slows down the progress or reduces the symptoms of
dementia.1 Consequently, neurite-growth-promoting and neu-
roprotective compounds are considered as molecular ap-
proaches for the restoration of brain function.2,3 Several natural
products have been reported to induce neurite outgrowth, such
as withanolide A,4−6 the gentiside family,7,8 gelsemiol9,10 or
jiadifenolide.11 Often, the structural complexity of natural
products is directly associated with their challenging and time-
consuming synthesis.12−14 Therefore, structural simplification
while maintaining or even increasing their respective bioactivity
has emerged as an important strategy in natural products
research (“reduce to the maximum”)15 and is crucial to facilitate
the supply of beneficial small molecules. Wender introduced
the concept of function-orientated synthesis, which is based on
the reduction of molecular complexity by retained function
(bioactivity), resulting in increased step economy.16 Truncation
of complex natural products as exemplified by the modification
of halichondrin B resulted in a drug in clinical use.17,18 In the
area of neuritogenic natural products, we19 and others20 have
recently provided functionally optimized scaffolds for neuronal
differentiation.
Figure 1. Neuritogenic natural products and first analog 4.
Our group accomplished the total synthesis of several
neuritogenic alkaloids originating from entomopathogenic fungi
such as pretenellin (1), farinosone A (2), or farinosone C (3)
and assigned their absolute configuration (Figure 1).9,10,21−23
While the biosynthesis of these compounds is likely to be
related,24,25 their synthesis required at least 14 steps.22 From a
Received: November 5, 2013
Accepted: December 6, 2013
Published: December 6, 2013
© 2013 American Chemical Society
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Letter
very limited structure−activity relationship study (SAR), we
learned that the much simpler L-tyrosinol-propionamide 4 was
able to induce neuronal differentiation, albeit at higher
concentration than the parent analog 3.22 We therefore
aimed to synthesize structurally optimized derivatives of
farinosone C (3), which retain or even display more potent
neurite outgrowth inducing capability. At the same time, this
endeavor would also result in a more defined description of the
pharmacophore. In this communication, we report the results
of this SAR study on farinosone C (3) analogs and perform
preliminary studies on the mechanism of action, identifying the
modulation of the endocannabinoid system as a potential target
pathway.
removal (→ 5), methylation (→ 6), or esterification (→ 7) of
the hydroxyl group might be beneficial for metabolic stability.
However, all these modifications led to a complete loss of
bioactivity, which suggested that this functional group is
essential for activity. The role of the primary hydroxyl group
was investigated next. Again, methylation and esterification
were not tolerated, as the corresponding compounds 8 and 9
were found to be inactive, as was the doubly methylated
compound 10. Interestingly, however, oxidation to the
carboxylic acid 11 and derivatization to its methyl ester analog
12 led to biologically active compounds. We then investigated
the influence of the stereogenic center: formation of the D-
tyrosinol-propionamide 13 or complete removal of the CH2OH
side chain in 14 resulted in inactivity, showing that the presence
of the hydroxymethylene moiety in the (S)-configuration is
mandatory for neuritogenic activity in the PC12 cell assay. The
close catechol analog of 14, dopaminyl propanoate (15), caused
cytotoxicity at our standard concentration of 50 μM and was
inactive at 5 μM. The amide moiety was also shown to be
essential, as the secondary amine 16 was inactive. Modifications
of the amide only tolerated a planar aromatic substituent (→
17); the more bulky isobutyramide 18 and pivalamide analogs
19 displayed no significant activity. With these results in hand,
we turned our attention toward the alkyl chain, the part of
farinosone C (3) that has synthetically been the most
demanding. The very bulky triazole-adamantyl derivative 20
was not active; however, the dimer 21 of L-tyrosinol-
propionamide 4 showed significant bioactivity. Finally, we
were interested in the role of the acidic terminus of farinosone
C (3). It appears that a polar terminus is required for the long
chain aliphatic compounds, as the apolar amide 22 revealed no
activity. Terminal acid 23 and the terminal alcohol 24 showed
good activity; the latter compound was even able to induce cell
differentiation at 10 μM concentration, thus rendering the triol
24 an even more potent compound than the parent natural
product farinosone C (3, Figure 3). This interesting result
demonstrated that the synthetically challenging side chain of
the parent natural product can be replaced by an unbranched
and fully saturated alkyl chain.
We prepared a collection of farinosone C (3) derivatives
(Table 1; for details see Supporting Information) and profiled
Table 1. Synthesized Farinosone C Analogs 4−24
Many natural products with neuritogenic or neuritotrophic
properties have been reported.4−11 However, the underlying
biological pathways involved in neuronal cell differentiation,
which are influenced by such compounds, are only partially
understood. We therefore decided to investigate the molecular
targets of the synthetic derivatives prepared that display
neuritogenic properties. Computational approaches by struc-
tural similarity searching30 hinted at fatty acid amides such as
OMDM-2 (25) and OMDM-4 (26), which bind to the
cannabinoid CB1 receptors in the low micromolar range. These
compounds serve as aromatic structural analogs for the
endogenous cannabinoid N-arachidonoylethanolamine (AEA)
27 (Figure 4).31−33
̀
Figadere and co-workers demonstrated that fatty acid amide
derivatives of tryptamine are also able to induce neuronal
differentiation.34 Activation of the CB1 receptor was reported to
induce neuronal differentiation through a complex signaling
network in Neuro-2A cells35,36 and to restore the neurite
outgrowth in hyperglycemic PC12 cells.37 Based on these
structural precedents for both CB1 binding and its role as
neuritogenic target, we prepared a second collection of
compounds (Table 2) by structurally expanding the hepta-
methylene scaffold of compounds 22−24 to obtain longer alkyl
chains with no or partial unsaturation.
their neuritogenic properties (Figure 2) using a modified
procedure of the well established PC12 cell assay that can serve
as a simplified model system for NGF-mediated, neuronal
differentiation.21,22,26−28 First, the role of the phenolic OH
group was evaluated. Since phenols are known to be easily
oxidized in an enzymatic environment,29 we envisioned that the
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Figure 2. Neuritogenic activity of farinosone C (3) and its simplified analogs (4−24) in the PC12 assay. All values were determined at 50 μM, except
the following: a, 20 μM; b, 5 μM; c, 10 μM. Positive control: nerve growth factor (NGF), 20 ng mL−1. Solvent control: DMSO (0.1%). Incubation
period: 2 days. Values are reported as mean (unpaired t test; confidence interval 95%; significance: *** = P < 0.0001, n = 3, mean SEM).
Compounds 28−35 and OMDM-2 (25) were first screened
for neurite outgrowth at 10 μM concentration. We noticed that
all compounds bearing a secondary amine function (28−31)
were not tolerated by PC12 cells, as they induced cytotoxicity.
After reduction of the concentration by one order of
magnitude, neither toxicity nor neuritogenic activity could be
observed for these amines (Figure 5). Among the amide
Figure 3. Representative images of differentiated PC12 cells: left, 24,
10 μM; right, DMSO vehicle control. Arrows indicate differentiated
PC12 cells.
Figure 5. Neuritogenic activity of OMDM-2 (25) and fatty acid
derived analogs. Values were determined at a: 10 μM, b: 1 μM, NGF
control: 20 ng mL−1. DMSO control: 0.1%. Incubation period: 2 days.
Values are reported as means
SEM (Unpaired t test, confidence
interval 95%, significance: *** = P < 0.0001, n = 3).
Figure 4. Chemical structure of the endocannabinoid AEA (27), two
endocannabinoid-like CB1 ligands (25 and 26) and the endocanna-
binoid transport inhibitor UCM707 (36).
bearing amphiphiles (25, 32−35), only 34 (hereafter referred
as BSL34) showed a significant activity at 10 μM (Figure 5),
which suggests that the degree of unsaturation of the alkyl chain
plays a crucial role. Also the length of the alkyl chain is of
importance; 24 has been identified as the most potent
compound, whereas triol amide 33 is inactive. Interestingly,
they only differ in the length of the alkyl chain (C7 versus
C14).
Table 2. Fatty Acid Amide Derived Analogs 28−35
Next, we investigated the binding affinities for the
compounds at CB receptors using membrane preparations
from CHO cells stably transfected with human CB receptors.
Most of the compounds showed weak binding to CB1 and CB2
receptors at the screening concentration of 1 μM (Supporting
Information Figure 1A and 1B). Nonetheless, we noticed that
29 and, more importantly, the neuritogenic omega-6 fatty acid
amide derivative BSL34 selectively bind to the CB1 receptor
with moderate potency (Ki values of 530 and 810 nM,
respectively (Figure 6)).
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Table 3. [3H]AEA transport inhibition parameters. EC50
values and maximal effect were determined from
concentration-dependent curves performed in U937 human
leukemic monocyte lymphoma cells as previously described
(n = 3-5).41
one did to some extent. This SAR study unearthed seven
neuritogenic molecules (10, 11, 17, 21, 23, 24, BSL34), of
which two, the triol 24 and the fatty acid derivative BSL34,
possessed a superior neurotrophin-like function than the
natural product 3 itself, with a much reduced molecular
complexity. Both can be obtained from cheap commercial
starting materials in one step, and supply is therefore ensured.
Our data also suggest the involvement of the endocannabinoid
system in neuronal differentiation induced by these classes of
compounds. The previously reported44 CB1 receptor-induced
neuritogenic effect was not reproduced in our hands, as the
selective agonist O-689 did not lead to any significant neuronal
differentiation and the BSL34-induced effect was not blocked
by the selective CB1 receptor antagonists AM251 or
rimonabant. The experimental conditions of the PC12 assay
have a significant impact on the read-out. Indeed, HU-210 was
shown to restore the neurite outgrowth in hyperglycemic cells
to a degree comparable with normal cells in a CB1 receptor-
dependent mechanism, while contrasting data were reported for
normoglycemic cells. CB1 receptor activation was shown either
to trigger44 or to impair neurite outgrowth.45 In addition,
different studies describe a variable CB1 receptor expression in
PC12 cells. The receptor was either found37 or not found46,47
on the plasma membrane of undifferentiated PC12 cells. Others
reported CB1 receptor expression only in NGF-differentiated
PC12 cells.45 Possible reasons for apparent discrepancies
between these and our findings might relate to the relative
expression level of CB1 receptors and thus point toward a CB1
receptor-independent effect. However, as BSL34 showed a
potent EMT inhibition, we assume that the resulting changes in
the local concentration of endocannabinoids, for example, AEA
and 2-AG, could affect cell differentiation. EMT is involved in
the bidirectional trafficking of endocannabinoids across the
plasma membrane,41 and its inhibition leads to a different
compartmentalization of AEA (27) and 2-AG. AEA (27) has
several other targets beyond the plasma membrane located
CB1/2 receptors, such as TRPV1 channels, intracellular CB1
receptors, and nuclear PPARs. Some of those receptors are
involved in neuronal differentiation. For example, PPAR-γ
activation was shown to induce neurite outgrowth in SH-SY5Y
human neuroblastoma cells,48 while TRPV1 is involved in
maintaining the [Ca2+] homeostasis which is primarily involved
during the development and differentiation of the nervous
system. TRPV1 expression and activity was found to be
increased in SH-SY5Y upon neuronal differentiation.49 TRPV1
was also shown to be functionally expressed in PC12 cells,50
which also are able to synthesize AEA (27).51 Therefore,
TRPV1 might be one of the candidates for the AEA-induced
CB1 receptor-indpendent targets of neurite outgrowth shown in
Figure 6. CB1 binding properties of compounds 29 and BSL34. The
assay was performed as described before38 by incubating for 90 min at
30 °C CHO-hCB1 membranes with different concentrations of tested
compounds (or the positive control, WIN55,212-2) in the presence of
0.5 nM [3H]-CP55940 (mean SD; n = 2−3).
To further clarify the role of the CB1 receptor in our system,
we incubated PC12 cells with the CB1 selective agonist O-689
at 100 nM and with two CB1 antagonists, rimonabant (1 μM
and 100 nM) and AM251 (3 μM and 100 nM).39,40 Despite
multiple attempts under manifold conditions, neuronal differ-
entiation could not be achieved by O-689 treatment nor were
the selective CB1 antagonists able to suppress the BSL34-
induced neuronal differentiation, suggesting that CB1 is not
directly involved in our PC12 neurite outgrowth assay. The
endocannabinoid system included several proteins involved in
the biosynthesis, degradation, and trafficking of the two main
endogenous ligands of CB receptors, AEA (27), and 2-
arachidonoyl glycerol (2-AG). Intra- and extracellular levels of
2-AG and AEA (27) are under control of degrading enzymes,
intracellular carriers, and the putative endocannabinoid
membrane transporter (EMT). Modulation of those targets’
function leads to a change in the levels of AEA (27) and 2-AG,
thus raising indirect CB receptor activation.41 We have
therefore evaluated the impact of our compounds on the
activity of those targets. Most of the compounds tested at 1 μM
showed a weak inhibition (20−25%) of fatty acid amide
hydrolase (FAAH), the main enzyme involved in AEA
hydrolysis (Supporting Information Figure 2). The main
enzymes involved in 2-AG hydrolysis (monoacylglycerol lipase,
MAGL, α,β-hydrolase-6 and -12, ABHD-6 and -12) were not
significantly inhibited at the same concentration (Supporting
Information Figure 3A and 3B). AEA analogs (28−35) could
also modulate the trafficking of endocannabinoids across the
plasma membrane. Gratifyingly, this hypothesis was corrobo-
rated by the discovery of four fatty acid amides as excellent
EMT inhibitors. Four of the tested compounds inhibited AEA
(27) cellular uptake with submicromolar EC50 values. The most
potent compound, BSL34, inhibited the EMT activity with one
order of magnitude more than UCM707 (36), a commercially
available benchmark inhibitor (Table 3 and Supporting
Information Figure 4).41−43
With the synthesized collection of farinosone C analogs
reported herein, we were able to elucidate the structure
requirements for activity derived from the parent natural
product 3. It was demonstrated, for example, that the branched
and unsatured side chain can by simplified or truncated. The
phenolic hydroxyl group allowed no alteration, but the primary
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ASSOCIATED CONTENT
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Complete experimental details and supporting figures. This
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: karl.gademann@unibas.ch. Phone: +41 (0)61 267 11
44.
Funding
(17) Towle, M. J.; Salvato, K. A.; Budrow, J.; Wels, B. F.; Kuznetsov,
G.; Aalfs, K. K.; Welsh, S.; Zheng, W.; Seletsky, B. M.; Palme, M. H.;
Habgood, G. J.; Singer, L. A.; Dipietro, L. V.; Wang, Y.; Chen, J. J.;
Quincy, D. A.; Davis, A.; Yoshimatsu, K.; Kishi, Y.; Yu, M. J.;
Littlefield, B. A. In Vitro and In Vivo Anticancer Activities of Synthetic
Macrocyclic Ketone Analogues of Halichondrin B. Cancer Res. 2001,
61, 1013−1021.
Financial support from the Swiss National Science Foundation
(SNF) (200021-144028), the University of Bern Forschungs-
stiftung, and COST CM0804 is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
(18) Aicher, T. D.; Buszek, K. R.; Fang, F. G.; Forsyth, C. J.; Jung, S.
H.; Kishi, Y.; Matelich, M. C.; Scola, P. M.; Spero, D. M.; Yoon, S. K.
Total Synthesis of Halichondrin B and Norhalichondrin B. J. Am.
Chem. Soc. 1992, 114, 3162−3164.
ACKNOWLEDGMENTS
■
The authors thank Dr. Henning J. Jessen (University of Zurich,
CH) and Dr. Linda D. Simmler (Vanderbilt University,
Nashville, US) for their technical support with the biological
experiments. Dr. Jennifer Zampese (University of Basel, CH)
and Dr. Rosario Scopelliti (EPF Lausanne, CH) are acknowl-
edged for the X-ray diffraction analysis.
(19) Schmid, F.; Jessen, H. J.; Burch, P.; Gademann, K. Truncated
militarinone fragments identified by total chemical synthesis induce
neurite outgrowth. MedChemComm 2013, 4, 135−139.
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■
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