Effects of a verbenachalcone derivative on neurite outgrowth, inhibition of caspase induction and gene expression

Article abstract of DOI:10.1016/j.bmcl.2004.12.001

A verbenachalcone derivative was synthesized and shown to protect N2a cells from caspase induction caused by serum starvation and to enhance the effect of NGF on neurite outgrowth in PC12 cells. As an initial investigation of the compound's mechanism(s) o

Full text of DOI:10.1016/j.bmcl.2004.12.001

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1193–1196
Effects of a verbenachalcone derivative on neurite
outgrowth, inhibition of caspase induction and gene expression
Li-An Yeh,a Deppa Padmanaban,a Pei Ho,a Xeuchao Xing,a Patricia Rowley,b
b,c
Lee Jae Morse,b RoderickV. Jensen
and Gregory D. Cunya,*
aLaboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair,
Brigham & Womenꢀs Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
bCenter for Neurologic Diseases, Brigham & Womenꢀs Hospital and Harvard Medical School,
65 Landsdowne Street, Cambridge, MA 02139, USA
cDepartment of Physics, Wesleyan University, Middletown, CT 06459, USA
Received 18 October 2004; revised 15 November 2004; accepted 1 December 2004
Available online 23 December 2004
Abstract—A verbenachalcone derivative was synthesized and shown to protect N2a cells from caspase induction caused by serum
starvation and to enhance the effect of NGF on neurite outgrowth in PC12 cells. As an initial investigation of the compoundꢀs mech-
anism(s) of action, we performed differential gene expression profiling in PC12 cells using oligonucleotide (ꢀ10,000 gene probes)
microarrays. Gene expression patterns were compared in the presence of NGF (2 and 50 ng/mL) and NGF (2 ng/mL) plus the verb-
enachalcone derivative. Ten genes were significantly (P2-fold; p 6 0.05) up-regulated and seven genes were significantly down-regu-
lated in the presence of the compound. These results were independently validated by quantitative real-time PCR for a subset of
genes (cathepsin L, sigma-1 receptor and protein tyrosine phosphatase receptor type R). These genes or their protein products
may represent useful therapeutic targets for treating neurodegeneration, such as Alzheimerꢀs disease.
Ó 2004 Elsevier Ltd. All rights reserved.
Among the suspected causes of neurodegeneration in
Alzheimerꢀs disease (AD) is a decrease in neurotrophic
support, including nerve growth factor (NGF). NGF
belongs to a class of small basic proteins whose effects
are almost exclusively on the central and peripheral ner-
vous systems.1 NGF binds to two classes of cell surface
receptors on cholinergic axon terminals, the high-affinity
TrkA receptor with catalytic tyrosine kinase activity and
the lower-affinity p75 neurotrophin receptor (p75NTR),
followed by retrograde transport to the neuron cell
body. Like other neurotrophins, it serves a vital role in
the development and homeostasis of the nervous system.
Receptor activation can result in an array of cellular
events, including enhanced survival, differentiation, neu-
ritogenesis and neurotransmitter synthesis, and
secretion.2,3
tive role.4,5 However, delivery of exogenous NGF has
failed to demonstrate efficacy in several human clinical
trials.6 In addition, direct in vivo administration of
NGF is hampered by numerous factors, including the
inherent problems frequently associated with peptide
therapeutics (e.g., inadequate blood–brain-barrier pene-
tration and metabolic stability).
Interestingly, NGF protein and mRNA levels remain
either unchanged or are increased in the hippocampus
and cerebral cortex of AD patients and only decreased
in the nucleus basalis.4 However, TrkA receptor
O
OR
A considerable amount of in vitro and in vivo data has
been generated that demonstrates NGFꢀs neuroprotec-
HO
OR
RO
O
OMe
RO
O
*
1 R = H
Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768
8606; e-mail: gcuny@rics.bwh.harvard.edu
2 R = Me
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.001

1194
L.-A. Yeh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1193–1196
expression was reduced, while p75NTR receptor expres-
sion levels remain unclear.7 Therefore, AD patho-physio-
logy may in part result from inadequate NGF signal
transduction.8–10 Consequently, molecules that augment
NGF signaling pathways may slow and/or prevent neu-
rodegeneration in patients suffering from AD and other
dementias.
O
O
ii
OEt
iii
OEt
BnO
HO
Br
R
3 R = H
4 R = Br
5
i
R
R
vi
iv
Small organic molecules (MW < 500 amu) that possess
NGF-like properties can generally be classified into
three mechanistic categories: (i) agonists for the TrkA
receptor, (ii) stimulators of NGF synthesis or secretion,
and (iii) enhancers of NGF (i.e., increase the cellular re-
sponse to NGF, but lackintrinsic activity in the absence
of NGF). Recently the natural product verbenachal-
cone, 1, isolated from Verbena littoralis was reported
to enhance NGFꢀs ability to stimulate neurite out-
growths from PC12D cells and to be devoid of this activ-
ity in the absence of the neurotrophin.11
BnO
BnO
O
O
R
OMe
8 R = (CH2)2CO2H
9 R = (CH2)2C(O)NMe(OMe)
R
OMe
v
7 R = (CH2)2CO2Et
O
OMe
RO
OMe
MeO
O
OMe
Herein we describe the synthesis of the verbenachalcone
derivative 2 that has increased hydrophobicity and re-
port its effects on neurite outgrowth, its ability to inhibit
caspase induction caused by serum starvation and the
results of differential gene expression profiling as an ini-
tial investigation of itꢀs mechanism(s) of action.
O
MeO
10 R = Bn
11 R = Ac
2 R = H
vii
viii
Scheme 1. Reagents and conditions: (i) Br2, AcOH, 85%; (ii) BnBr,
EtOH, K2CO3, D, 85%; (iii) 6, 5 mol% (CuOTf)2PhMe, Cs2CO3, py,
110 °C, 78%; (iv) 8 N NaOH, THF/MeOH (2:1), rt, 95%; (v)
MeNHOMeÆHCl, HBTU, DIEA, DCM, 94%; (vi) 1-Li-2,4-(OMe)2Ph,
THF, À78 °C then 1 N HCl, 87%; (vii) 1 atm H2, 10% Pd/C, Ac2O,
NaOAc, benzene, 2 h, 81%; (viii) KOH, MeOH/H2O (2:1), rt, 100%.
The synthesis of 2 began by first converting ethyl 3-(4-
hydroxyphenyl)propionate, 3, to the aryl bromide 4 by
treatment with bromine in acetic acid (Scheme 1).12 Benzyl-
ation of the phenol was accomplished with benzyl
bromide in refluxing ethanol in the presence of potas-
sium carbonate to give 5. Next, the aryl bromide 5
was coupled to ethyl 3-(4-hydroxy-3-methoxyphenyl)-
propionate, 6, which was readily prepared from
commercially available 3-(4-hydroxy-3-methoxyphenyl)-
propionic acid, to give 7 in 78% yield (based on recov-
ered 5) utilizing 5 mol% (CuOTf)2PhMe. The esters
were hydrolyzed with sodium hydroxide in a mixture
of THF and methanol (2:1) to give 8. This material
was converted to its corresponding Weinreb amide 9
utilizing the coupling reagent HBTU in dichloro-
methane (DCM) in the presence of diisopropyl ethyl-
amine (DIEA). Treatment of the amide with 4 equiv of
1-lithio-2,4-dimethoxybenzene (generated in situ from
1-bromo-2,4-dimethoxybenzene and n-BuLi in THF at
À78 °C for 1.5 h) followed by the addition of hydrochlo-
ric acid (1 N) yielded 10. Debenzylation of 10 in the
presence of acetic anhydride, hydrogen (1 atm), 10%
Pd/C, and sodium acetate in benzene gave 11. Interest-
ingly, hydrogenation in the absence of acetic anhydride
resulted in concomitant removal of the benzyl group
and reduction of the ketones to methylene groups. Fi-
nally, hydrolysis of acetate 11 gave 2 in quantitative
yield.
with 2 (Fig. 1). For example, in the presence of NGF
(2 ng/mL) plus 2 (6 lM) a 5-fold increase in the number
of neurites-bearing cells was observed compared with
the NGF (2 ng/mL) control. The compound induced
longer neurite processes in virtually all cells. Compound
2 was devoid of this activity in the absence of NGF.
Increasing the concentration of 2 did not further in-
crease the extent of neurite outgrowth, possibly due to
6
**
5
4
3
2
1
**
**
**
*
0
2 ng/mL
NGF
0.5
1
3
6
9
15
Compound 2 (µM)
Figure 1. Enhancement of NGFꢀs effects for stimulating neurite
outgrowths in PC12 cells with NGF (2 ng/mL) and with NGF (2 ng/
mL) in the presence of 2 (0.5–15 lM) for 48 h, followed by 72 h
incubation in the absence of the compound. The neurite-bearing cell to
total cell ratio was determined and expressed as a fold-change
compared to NGF (2 ng/mL) standard deviation (N = 6). The data
were analyzed by Student unpaired t-test. * Indicates a significant
difference (p < 0.002) versus 2 ng/mL NGF. ** Indicates a significant
difference (p < 0.0008) versus 2 ng/mL NGF.
The ability of 2 to enhance NGFꢀs effects for stimulating
neurite outgrowths in PC12 cells was accessed utilizing a
morphological assay previous reported.13,14 In order to
increase the sensitivity of this assay, PC12 cells were ex-
posed to 2 for 48 h, followed by a 72 h incubation in the
absence of the test compound. A significant dose-depen-
dent enhancement of NGFꢀs effects was demonstrated

L.-A. Yeh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1193–1196
1195
Table 1. Genes significantly up-regulated and down-regulated by 2
compound toxicity. In addition, these experiments illus-
trate that the hydroxyl groups on the terminal aromatic
rings of verbenachacone are not necessary for the
enhancement of NGFꢀs effects on neurite outgrowth.
Gene
Fold + or À p-value
Glycoprotein transmembrane NMB
Transcriptional repressor CREM
Salt-inducible protein kinase
Kruppel-like factor 4
Kinase-deficient TNF-beta
B-cell translocation gene 2
Cathepsin L
+7.15
+3.10
+2.94
+2.91
+2.81
+2.55
+2.38
+2.27
+2.25
+2.06
À2.00
À2.07
À2.07
0.002
0.030
0.006
0.014
0.003
0.025
0.012
0.032
0.007
0.044
0.019
0.043
0.045
0.009
0.017
0.005
0.013
As previously mentioned, neurotrophin signaling is
important for neuron survival. Since 2 has demonstrated
significant enhancement of NGFꢀs effects on neurite out-
growth, its ability to inhibit caspase induction caused by
neurotrophin withdrawl has been evaluated in N2a cells.
The end point of these experiments was caspase-3/7
induction after a 48 h treatment. This time point corre-
sponded to the maximum induction of caspase-3/7.
After 48 h, caspase-3/7 decreased due to cell death. Cells
were grown in the presence of 10% fetal bovine serum, in
the absence of serum, or in the absence of serum but in
the presence of 2 (0.1–15 lM). As can be seen in Figure
2, compound 2 significantly and dose-dependently pro-
tected serum-deprived cells from caspase-3/7 induction
with an EC50 ꢀ 1 lM.15 PDTC, an inhibitor of NF-jB
activation, was utilized as a positive control and had
an EC50 < 5 lM in this model. In addition, 15 lM 2 re-
sulted in 80% inhibition of caspase induction compared
with the serum free control.
Ceruloplasmin
Sigma-1 receptor
Gankyrin homologue
Adenosine A2A receptor
Neuronatin
Interferon regulatory factor 1
Transmembrane superfamily member 4 À2.11
Protein kinase C-binding protein zeta 1 À2.25
Neuroserpin
Protein tyrosine phosphatase
receptor type R (Ptprr)
À2.38
À2.67
treatment of 15 lM 2 plus 2 ng/mL NGF compared with
2 ng/mL of NGF only. The genes significantly altered by
2 are shown in Table 1. Several of the observed gene
changes in response to NGF, such as sigma-1 receptor,17
cathepsin L,18 adenosine A2A receptor,19 neuronatin,20
neuroserpin,21 and protein tyrosine phosphatase recep-
tor type R (Ptprr)22 are in agreement with literature
reports.
Our initial foray into the mechanism(s) of action for 2
utilized a chemical genetics approach. Differential gene
expression profiles of PC12 cells grown in the absence
of NGF, in the presence of NGF (2 and 50 ng/mL)
and in the presence of NGF (2 ng/mL) plus 2 (15 lM)
were compared after 48 h exposure.16 The gene expres-
sion profiles were obtained utilizing Amersham Code-
LinkTM oligonucleotide arrays (ꢀ10,000 genes
represented).
In order to validate the microarray results, quantitative
RT-PCR was performed on a representative subset of
genes (cathepsin L, sigma-1 receptor, and Ptprr). In all
cases the microarray results and the quantitative RT-
PCR results were in close agreement (Table 2). In the
case of the Ptprr gene, exposure to NGF (2 or 50 ng/
mL) did not result in significant changes in expression
compared to untreated controls after 48 h. However,
in the presence of NGF (2 ng/mL) and 2 (15 lM) a sig-
nificant decrease in gene expression was observed as evi-
dent utilizing both methods. Cathepsin L and sigma-1
receptor gene expressions, however, were dose-depen-
dent on NGF. Moreover, a further increase in gene
expression for these two genes was observed in the pres-
ence of a combination of NGF (2 ng/mL) and 2
(15 lM). In fact, the response exceeded that observed
with 50 ng/mL NGF.
The differential gene expression profile of NGF (2 ng/
mL) versus NGF (2 ng/mL) plus 2 (15 lM) showed that
the expression levels of relatively few genes were altered
upon treatment with 2. Only 10 genes (ꢀ0.10%) were sig-
nificantly up-regulated and seven genes (ꢀ0.07%) were
down-regulated by a factor >2 (p < 0.05, N = 2) with
600
500
400
**
***
300
The up-regulation of the sigma-1 receptor is an intrigu-
ing finding. The biochemical and physiological role of
this receptor remains poorly understood. A host of cel-
lular events have been linked to this receptor include
modulation of NGF-induced neurite sprouting, neuro-
transmitter release, and neuronal firing. It is currently
believed that agonism of the sigma-1 receptor results
in the amplification of other concurrent signal transduc-
tion processes.23 In the context of the observations re-
ported herein for 2, up-regulation of the sigma-1
receptor could serve to amplify NGF-signaling path-
ways leading to enhancement of neurite outgrowth.
***
***
200
100
0
PDTC
FBS
SF
0.01 uM 0.1 uM
1 uM
15 uM
Figure 2. Evaluation of 2 for inhibition of caspase induction caused by
serum starvation utilizing N2a cells. Following 48 h cell incubation
with fetal bovine serum (FBS), without serum (SF) or, without serum
but in the presence of various concentrations of 2 (0.1–15 lM) or
PDTC (50 lM), caspase activity was determined (reported as RFU).
Each sample represents the average of six replicates. The data were
analyzed by Student unpaired t-test. *** Indicates a significant
difference (p < 0.0001) versus SF. ** Indicates a significant difference
(p < 0.001) versus SF.
The down-regulation of the Ptprr gene after a 48 h expo-
sure to NGF is another interesting finding. This gene

1196
L.-A. Yeh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1193–1196
Table 2. Validation of microarray results by quantitative RT-PCR
Gene
Gene expression (fold change relative to untreated, N = 2)
NGF (2 ng/mL)
—
NGF (50 ng/mL)
—
NGF (2 ng/mL)
+2 (15 lM)
RT-PCR
Micro-array
RT-PCR
Micro-array
RT-PCR
Micro-array
Ptprr
Cathepsin L
Sigma-1 receptor
0.91 0.01
1.50 0.90
1.21 0.01
0.90 0.11
1.77 0.15
1.37 0.11
0.64 0.20
2.31 0.10
1.77 0.11
1.04 0.24
2.75 0.25
2.41 0.11
0.39 0.09
3.40 0.25
2.93 0.11
0.37 0.09
3.88 0.82
3.21 0.24
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encodes for two protein tyrosine phosphatases, PTP-SL,
and PTP-BR7.24 PTP-SL has been shown to down-reg-
ulate the extracellular signal-related protein kinase 5
(ERK5) pathway and to impede translocation of
ERK5 to the nucleus.25 However, endocytosed neuro-
trophin receptors (e.g., TrkA receptor) activated the
ERK5 pathway leading to both enhanced nuclear trans-
location of ERK5 and neuronal survival.26 Thus, the
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In summary, the verbenachalcone derivative 2 enhances
NGFꢀs effects on neurite outgrowths and inhibits cas-
pase induction caused by serum starvation. Differential
gene expression profiles of NGF compared to NGF plus
2 revealed a small subset of genes whose expression was
significantly altered. Further studies are needed to delin-
eate the precise mechanism(s) responsible for these two
observed cellular responses to 2. In addition, further
refinement of the structure–activity relationship of verb-
enachalcone could provide more pharmacologically
tractable molecules enabling in vivo evaluation.
15. (a) Some cell loss (10–20%) was observed in the presence
of 2 (1 and 15 lM), but this did not account for the
significant decrease (50–60%) in caspase induction (see
supplementary data); (b) No direct inhibition of caspase-3/7
enzymatic activity was observed in the presence of 2
(15 lM).
16. (a) Lee, N. H.; Weinstock, K. G.; Kirkness, E. E.; Earle-
Hughes, J. A.; Fuldner, R. A.; Marmaros, S.; Glodek, A.;
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C. M.; Venter, J. C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
8303; (b) A 2.6-fold increase in the number of neurites-
bearing cells was observed in the presence of NGF (2 ng/
mL) plus 2 (15 lM) compared with only NGF (2 ng/mL).
This was similar to the results of 2 (15 lM) in Figure 1.
17. Takemori, H.; Katoh, Y.; Horike, N.; Doi, J.; Okamoto,
M. J. Biol. Chem. 2002, 277, 42334.
Acknowledgements
We thankthe Harvard Center for Neurodegeneration
and Repair (HCNR) for financial support.
18. Liuzzo, J. P.; Petanceska, S. S.; Devi, L. A. Mol. Med.
1999, 5, 334.
Supplementary data
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cology 1997, 36, 1319.
20. Joseph, R.; Tsang, W.; Dou, D.; Nelson, K.; Edvardsen,
K. Brain Res. 1996, 738, 32.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2004.12.001.
21. Parmar, P. K.; Coates, L. C.; Pearson, J. F.; Hill, R. M.;
Birch, N. P. J. Neurochem. 2002, 82, 1406.
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