Hippocampal and cortical neuronal growth mediated by the small molecule natural product clovanemagnolol

Article abstract of DOI:10.1016/j.bmcl.2011.06.054

The use of small molecule surrogates of growth factors that directly or indirectly promote growth represents an attractive approach to regenerative medicine. With synthetic access to clovanemagnolol, a small molecule initially isolated from the bark of th

Full text of DOI:10.1016/j.bmcl.2011.06.054

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4808–4812
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hippocampal and cortical neuronal growth mediated by the small
molecule natural product clovanemagnolol
Zin Khaing a, Danby Kang b, Andrew M. Camelio b, Christine E. Schmidt a, Dionicio Siegel b,
⇑
a Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78701, United States
b Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX 78701, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The use of small molecule surrogates of growth factors that directly or indirectly promote growth repre-
sents an attractive approach to regenerative medicine. With synthetic access to clovanemagnolol, a small
molecule initially isolated from the bark of the Bigleaf Magnolia tree, we have examined the small mol-
ecule’s ability to promote growth of embryonic hippocampal and cortical neurons in serum-free medium.
Comparisons with magnolol, a known promoter of growth, reveals that clovanmagnolol is a potent neu-
rotrophic agent, promoting neuronal growth at concentrations of 10 nM. In addition, both clovanemag-
nolol and magnolol promote growth through a biphasic dose response.
Received 7 April 2011
Revised 10 June 2011
Accepted 13 June 2011
Available online 22 June 2011
Keywords:
Natural products
Clovanemagnolol
Growth factors
Neuronal
Ó 2011 Elsevier Ltd. All rights reserved.
Cortical
Hippocampal
Small molecule
Synthesis
Growth factors can significantly enhance the survival and regen-
erative potential of injured neurons either due to injury1–3 or dis-
ease.4–6 These proteins prevent apoptosis, promote the growth and
survival of neurons, and aid in the controlled development of pro-
genitor cells.2,7 Their effective use as therapeutics however has been
problematical due to their pharmacokinetic shortcomings8: proteo-
lytic degradation requires continual infusion and their limited abil-
ity to cross the blood–brain barrier has meant delivery by
intracranial injection, with attendant serious risks to the patient’s
health. Gene therapy-based approaches to inducing production of
neurotrophic factors, using both direct and ex vivo techniques, have
had success in animal models of Parkinson’s disease4 and in regen-
erating axons after spinal cord injury.3,9,10 Ex vivo NGF gene delivery
in clinical trials for Alzheimer’s disease has demonstrated a partial
decrease in degeneration over 2 years.11 Although promising, gene
therapy has yet to overcome the limitations of short-lived effects,
undesirable immune responses, intracranial delivery, and chal-
lenges involving the use of viral vectors.
Figure 1. Neurotrophic natural products, clovanemagnolol (1), caryolanemagnolol
(2), magnolol (3), and honokiol (4).
(2) were isolated from the bark of Magnolina obovata in a screen
for compounds with potential neuroregenerative properties
(Fig. 1).12,13 Two abundant compounds from the same tree bark ex-
tract, magnolol (3) and honokiol (4), have been shown to promote
neuronal growth functioning through the activation of extracellu-
lar signal-related kinases (ERK1/2).14,15 Caryolanemagnolol and
clovanemagnolol are however isolated in much lower quantities
than magnolol (3) and honokiol (4). Specifically, clovanemagnolol
and caryolanemagnolol were isolated in 0.0005% and 0.004% yields,
respectively.12 Although clovanemagnolol and caryolanemagnolol
Using small molecules provides an alternative approach to
regenerative medicine. From a pharmacokinetic perspective, small
molecules possess a pharmacological advantage over the use of
exogenous neurotrophic factors. In this line of inquiry the sesqui-
terpene-neolignans clovanemagnolol (1) and caryolanemagnolol
⇑
Corresponding author.
E-mail address: dsiegel@cm.utexas.edu (D. Siegel).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.054

Z. Khaing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4808–4812
4809
was reported to have similar neurotrophic activity. With promising
preliminary data a more in depth description of the neurotrophic
effects of clovanemagnolol was warranted.
Due to challenges in obtaining significant quantities of clovane-
magnolol from the natural source we developed two syntheses of
clovanemagnolol starting from caryophyllene b-oxide (5). The pos-
tulated pathway for the biosynthesis of clovanemagnolol facili-
tated the assembly of the polycyclic compound. Importantly, the
precursor 5 was readily accessible from caryophyllene, a plant de-
rived compound used in the flavor and fragrance industry.12b,16
Brønsted acid activation of epoxides 5 followed by intramolec-
ular attack of the exocyclic alkene generates the corresponding
bridgehead carbocations (Scheme 1). The cation is aligned with
the transannular bond, enabling a ring expansion/ring contraction
to form a cationic tricycle. The secondary cation is subsequently
trapped by magnolol (3) forming clovanemagnolol. In the struc-
tural elucidation of caryophyllene, Barton et al. were the first to de-
scribe these caryophyllene rearrangement reactions, which
generated clovane and caryolane-based structures.17
Scheme 1. Proposed biosynthesis of clovanemagnolol.
The single step conversion of 5 to clovanemagnolol, following
from the biosynthetic proposal, was achieved using diphenyl phos-
phate and magnolol (3) leading to the formation of clovanemagno-
lol in 10% yield (Eq. 1). Similarly, an acid-mediated rearrangement
of the diasteromeric a-epoxide provided caryolanemagnolol in 15%
yield (Eq. 2).18 Starting from the corresponding epoxides these sin-
gle-step transformations provided rapid access to both natural
products.
Scheme 2. Clovanemagnolol synthesis amenable to analog preparation.
In an effort to provide access derivatives of clovanemagnolol a
second synthesis was developed starting from the recrystallized,
commercial caryophyllene b-oxide (5) (Scheme 2). The carbocyclic
rearrangement was achieved, as before, with diphenyl phosphate
as the Brønsted acid catalyst and using 4-bromophenol as the
nucleophilic trap to provide bromide 6 in 35% yield. Due to an
inability of 2,4-dibromophenol to engage the cation generated dur-
ing the rearrangement, bromination of 6 was required to provide a
handle for the subsequent palladium–catalyzed bond forming
reactions. Preliminary attempts at aryl halogenation proceeded
with concomitant oxidation of the secondary alcohol, generating
were reported to be more effective at inducing growth, the low iso-
lation yields have prohibited additional studies.
Treatment of primary neuronal cell cultures, derived from the
cerebral hemisphere of fetal rat brain, with clovanemagnolol was
reported to lead to neurite outgrowth at 10 to 0.1 l
M.12 In addi-
tion, treatment of cultured neurons with clovanemagnolol at
1 mM resulted in choline acetyltransferase (ChAT) activity being
augmented by 142% relative to control cultures, leading to an
increased biosynthesis of the neurotransmitter acetylcholine.
Similarly, the isomeric natural product caryolanemagnolol (2)

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Z. Khaing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4808–4812
Figure 2. Embryonic hippocampal neurons showed enhanced neurite outgrowth after treatment with Mag and CM. Dissociated E18 mouse hippocampal neurons were
cultured in control (CTR, medium only), DMSO (medium + DMSO), or Mag (0.01 or 1.0
l
M). After 2 days in vitro, 0.01
lM concentration Mag can significantly increase the
neurite length (48%, p <0.02). Treatment with CM also resulted in significant increase in neurite outgrowth at 0.01
lM (63%, p <0.03). However, treatment with higher
concentration of either Mag or CM (1.0
lM) resulted in fewer numbers of neurons bearing significantly shorter neurites for both, 69%, 84%, respectively.
With access to clovanemagnolol (>500 mg) we were able to test
and quantify the small molecule’s effects on different neuronal
Table 1
Neurite outgrowth of embryonic hippocampal neurons after treatment with magnolol
and clovanemagnolol
types. For comparison, we measured the effects of clovanemagno-
lol versus the effects to the related and more extensively studied
compound magnolol. In both hippocampal and cortical neurons
we found clovanemagnolol to be more effective at promoting neu-
ronal growth. Interestingly, both compounds demonstrated bipha-
sic dose profiles.
Effects of clovanemagnolol and magnolol on hippocampal neurons
in vitro is concentration dependent: Dissociated embryonic hippo-
campal neurons were cultured in vitro in the presence of control,
DMSO, or varying amounts of magnolol or clovanemagnolol
(Fig. 2). After 2 days in vitro, we found that treatment with either
Treatment
Average neurite length
per neuron ( m)
Percent change
from control
l
Control
DMSO
176.59 27.0
168.73 21.5
–
À4.5
50.1
23.9
À69.0
62.8
26.5
À83.9
Mag (0.01
Mag (0.1
Mag (1
l
l
M)
M)
M)
265.07 67.6
218.76 40.7
54.73 17.34
l
CM (0.01 M)
CM (0.1 M)
CM (1 M)
l
287.55 82.8
223.38 49.9
28.51 10.9
l
l
magnolol (0.01
p <0.03) was able to significantly enhance the neurite outgrowth
of neurons compared to control cultures. At 0.1 M concentration,
lM; p <0.02) or clovanemagnolol (0.01 lM;
l
the corresponding ketone of alcohol 6. Due to the unavoidable side
reaction, protection of the secondary alcohol of 6 as the corre-
sponding acetate was required. Following acylation, bromination
was accomplished using bromine and sodium acetate in acetic acid
at 60 °C forming dibromide 7 in 74% yield over the two-step se-
quence. Sequential Suzuki reactions were envisioned to form the
allyl appendage as well as the biaryl bond. A key issue was the
selectivity of the first Suzuki reaction. In the majority of allylation
reactions examined, cross coupling led to unavoidable diallylation
of 7. However, with close monitoring of the reaction by TLC com-
pound 9 could be isolated in 70% yield. A second Suzuki reaction
between bromide 9 and pinacol ester 10 (prepared in three steps
from 4-allylanisole, Supplementary data) using cross coupling con-
ditions developed by Fu et al. formed the biaryl bond, generating
11 in 74% yield.19 Removal of both the acetate and carbamoyl
groups was achieved in the last step with lithium aluminum
hydride, providing clovanemagnolol (1) in 73% yield.
the addition of either molecule resulted in a modest enhancement
in neurite outgrowth but this did not reach statistical significance.
At micromolar concentrations, treatment with either compound
resulted in a significant decrease in the average neurite outgrowth
(69.45% and 84.08% reduction for magnolol and clovanemagnolol,
respectively). Therefore, we found that both magnolol and clovane-
magnolol have neurite outgrowth enhancing properties in embry-
onic hippocampal neurons in a concentration dependent manner
(Table 1).
Effects of clovanemagnolol and magnolol on cortical neurons
in vitro is concentration dependent: Dissociated embryonic cortical
neurons were treated with either magnolol or clovanemagnolol
( Fig. 3). At lower concentrations clovanemagnolol’s effects on
growth were shown to be greater than magnolol. After 2 days
in vitro we found that treatment with magnolol resulted in en-
hanced neurite outgrowth at both 0.01 and 0.1
lM concentrations,
although only treatment at 0.1 M reached statistical significance
l

Z. Khaing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4808–4812
4811
Figure 3. Embryonic cortical neurons showed enhanced neurite outgrowth after treatment with Mag and CM. Dissociated E18 mouse hippocampal neurons were cultured in
control (CTR, medium only), DMSO (medium + DMSO), or Mag (0.01 or 1.0 M). After 2 days in vitro, 0.1 M concentration Mag can significantly increase the neurite length
(56%, p <0.03). Treatment with CM also resulted in significant increase in neurite outgrowth at 0.01 M (44%, p <0.01). However, treatment with higher concentration of either
M) resulted in fewer numbers of neurons bearing significantly shorter neurites (75%, 80%, respectively).
l
l
l
Mag or CM (1.0
l
was removed. The residue was diluted with hexane (50 mL) and
washed with phosphate buffer (pH 7.4, 2 Â 10 mL) and 1 M NaOH
(3 Â 10 mL (to recover the magnolol)). The organic layer was
washed with brine (10 mL) and dried over Na2SO4 and then concen-
trated. The residue was purified by silica gel chromatography (hex-
Table 2
Neurite outgrowth of embryonic cortical neurons after treatment with magnolol and
clovanemagnolol
Treatment
Average neurite length
per neuron ( m)
Percent change
from control
l
ane/EtOAc = 85:15 as eluent) to yield clovanemagnolol (2)
colorless oil (12 mg, 9.5% yield).
a
Control
DMSO
352.71 54.40
347.21 82.82
–
À1.6
Cell culture and isolation of primary cells: Primary neuronal cells
were obtained from E16–18 mouse embryos. Pregnant mice were
sacrificed using isoflurane asphyxiation and embryos were col-
lected in 1Â Hank’s buffered salt solution (HBSS) with gentamycin.
After removing the meninges, cortical tissue or hippocampi were
dissected away from the surrounding tissue. Samples were treated
with 0.25% Trypsin/EDTA (Sigma–Aldrich) for 20 min and were
mechanically dissociated using fire polished Pasteur pipettes. Cells
Mag. (0.01
l
M)
M)
M)
424.10 24.19
548.78 123.98
57.75 27.37
20.2
55.6
À74.6
44.2
22.7
À80.7
Mag. (0.1
Mag. (1
l
l
CM (0.01
CM (0.1
CM (1
l
l
M)
M)
M)
508.45 87.01
432.85 64.49
68.21 22.23
l
(p <0.03). Treatment with clovanemagnolol resulted in a significant
increase in neurite outgrowth at 0.01 M (p <0.01), whereas treat-
ment at 0.1 M resulted in a slight increase in average neurite
length (p >0.05) (Table 2). At higher concentration (1 M), the
addition of both compounds resulted in significant reduction for
both magnolol and clovanemagnolol treated cells (p <0.001) com-
pared to neurons in the control culture. Therefore, we found that,
similar to hippocampal neurons, treatment with either magnolol
or clovanemagnolol has neurite outgrowth enhancing properties
for embryonic cortical neurons, and this effect is concentration
dependent.
Synthesis of clovanemagnolol (1): To a solution of recrystallized
b-caryophyllene b-oxide 5 (60 mg, 0.259 mmol, 1 equiv) and mag-
nolol (3) (103 mg, 0.388 mmol, 1.5 equiv) in dichloromethane
(2 mL) was added a solution of diphenylphosphate (32.4 mg,
0.129 mmol, 0.5 equiv) in dichloromethane (1 mL) over 3 min. The
orange solution was stirred at 38 °C for 2.5 h and then the solvent
were plated on 12 mm round glass coverslips coated with poly-
D-
l
lysine (20 g/ml) at a density of 30,000 cells per coverslip in a
l
l
DMEM/F12 media supplemented with N2 in the absence of serum.
All cultures were grown at standard condition with 37 °C in 5%
CO2. Cells were fed every 3 days by replacing half of the media with
fresh medium.
l
Immunostaining: After 2–4 days in culture, cells were fixed with
2% paraformaldehyde at room temperature for 20 min, and incu-
bated in blocking buffer (3% normal goat serum + 0.3% Triton
X-100 in PBS) for 1 h at room temperature. Primary antibody
against betaIII tubulin (1:1000, Abcam) was used to visualize neu-
rons. Samples were incubated with primary antibody in blocking
buffer overnight at 4 °C. Samples were washed with PBS and incu-
bated with goat anti-mouse IgG conjugated to AlexaFlour 488
(Invitrogen). Samples were counter stained with DAPI (1:1000, Sig-
ma–Aldrich) to visualize all nuclei, and mounted onto glass slides
using Fluoromount G (SouthernBiotech, Birmingham, Alabama).

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Pyzalski, R.; Taylor, M. D.; Carvey, P.; Ling, Z.; Trono, D.; Hantraye, P.; Deglon,
N.; Aebischer, P. Science 2000, 290, 767.
5. Mattson, M. P. Brain Res. 2000, 886, 47.
Three to five random areas (covering 30% of the total surface area
of the 12 mm glass coverslip) were recorded using an Olympus
IX70 (Olympus America, Melville, NY) inverted microscope and
the images were captured with an Optronics MagnaFire (Goleta,
CA) digital color camera. Acquired images were analyzed using Im-
ageJ, and composite figures were produced using Adobe Photoshop
CS4.
Analysis of neurite outgrowth: To determine the average neurite
length, samples were imaged at 3–5 random areas (covering over
30% of the glass coverslip). Using the ImageJ Plug-in ‘‘Neurite
Tracer’’ program automated quantification of neurite growth was
determined as previously described.20
6. Tuszynski, M. H. Lancet Neurol. 2002, 1, 51.
7. (a) Rangasamy, S. B.; Soderstrom, K.; Bakay, R. A.; Kordower, J. H. Prog. Brain
Res. 2010, 184, 237; (b) Bennet, M. R.; Gibson, W. G.; Lemon, G. Auton. Neurosci.
2002, 95, 1.
8. (a) Seiger, A.; Norddberg, A.; von Holst, H.; Backman, L.; Ebendal, T.; Alafuzoff,
I.; Amberla, K.; Hartvig, P.; Lilja, A. Behav. Brain Res. 1993, 57, 255; (b) Backman,
C.; Rose, G. M.; Hoffer, B. J.; Henrgy, M. A.; Bartus, R. T.; Friden, P.; Granholm, A.
C. J. Neurosci. 1996, 16, 5437; (c) O’Leary, P. D.; Hughes, R. A. J. Neurochem. 1998,
70, 1712; (d) Aebischer, P.; Lilja, A. Trends Neurosci. 2001, 24, 533.
9. Taylor, L.; Jones, L.; Tuszynski, M. H.; Blesch, A. J. Neurosci. 2006, 26, 9713.
10. Tuszynski, M. H.; Grill, R.; Jones, L. L.; Brant, A.; Blesch, A.; Low, K.; Lacroix, S.;
Lu, P. Exp. Neurol. 2003, 181, 47.
11. Tuszynski, M. H.; Thal, L.; Pay, M.; Salmon, D. P.; U, H. S.; Bakay, R.; Patel, P.;
Blesch, A.; Vahlsing, H. L.; Ho, G.; Tong, G.; Potkin, S. G.; Fallon, J.; Hansen, L.;
Mufson, E. J.; Kordower, J. H.; Gall, C.; Conner, J. Nat. Med. 2005, 11, 551.
12. (a) Fukuyama, Y.; Otoshi, Y.; Kodama, M. Tetrahedron Lett. 1990, 31, 4477; (b)
Fukuyama, Y.; Otoshi, Y.; Miyoshi, K.; Nakamura, K.; Kodama, M.; Nagasawa,
M.; Hasegawa, T.; Okazaki, H.; Sugawara, M. Tetrahedron 1992, 48, 377.
13. (a) Heymann, H.; Tezuka, Y.; Kikuchi, T.; Supriyatna, S. Chem. Pharm. Bull. 1994,
42, 138; (b) Heymann, H.; Tezuka, Y.; Kikuchi, T.; Supriyatna, S. Chem. Pharm.
Bull. 1994, 42, 941; (c) Matsuda, H.; Kageura, T.; Oda, M.; Morikawa, T.;
Sakamoto, Y.; Yoshikawa, M. Chem. Pharm. Bull. 2001, 49, 716; (d) Wang, J.-D.;
Zhang, W.; Li, Z. Y.; Xiang, W.-S.; Guo, Y.-W.; Krohn, K. Phytochemistry 2007, 68,
2426; (e) Zhu, Y.; Zhao, Y.; Huang, G.-D.; Wu, W.-S. Helv. Chim. Acta. 2008, 91,
1894.
Acknowledgments
The authors are grateful for the financial support of the Robert
A. Welch Foundation (F-1694), The Texas Institute for Drug and
Diagnostic Development, and The University of Texas at Austin.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.054.
14. Zhai, H.; Nakade, K.; Oda, M.; Mitsumoto, Y.; Akagi, M.; Sakurai, J.; Fukuyama,
Y. Eur. J. Pharmacol. 2005, 516, 112.
15. Hsu, Y.-F.; Lee, T.-S.; Lin, S.-Y.; Hsu, S.-P.; Juan, S.-H.; Hsu, Y.-H.; Zhong, W.-B.;
Lee, W.-S. Mol. Carcinog. 2007, 46, 275.
16. (a) Fujita, M.; Itokawa, H.; Sashida, Y. Yakugaku Zasshi 1973, 93, 415; (b)
Sashida, Y.; Itokawa, H.; Akita, Y.; Fujita, M. Yakugaku Zasshi 1976, 96, 218.
17. (a) Aebi, A.; Barton, D. H. R.; Lindsey, A. S. J. Chem. Soc. 1953, 3124; (b) Aebi, A.;
Barton, D. H. R.; Burgstahler, A. W.; Lindsey, A. S. J. Chem. Soc. 1954, 4659.
18. Cheng, X.; Harzdorf, N. L.; Shaw, T.; Siegel, D. Org. Lett. 2010, 12, 1304.
19. (a) Fu, G. C. Acc. Chem. Res. 2008, 41, 1555; (b) Littke, A. F.; Dai, C.; Fu, G. C. J. Am.
Chem. Soc. 2000, 122, 4020.
References and notes
1. Tobias, C. A.; Shumsky, J. S.; Shibata, M.; Tuszynski, M. H.; Fischer, I.; Tessler, A.;
Murray, M. Exp. Neurol. 2003, 184, 97.
2. Tuszynski, M. H. Expert. Opin. Biol. Ther. 2003, 3, 815.
3. Gros, T.; Sakamoto, J. S.; Blesch, A.; Havton, L. A.; Tuszynski, M. H. Biomaterials
2010, 31, 6719.
20. Pool, M.; Thiemann, J.; Bar-Or, A.; Fournier, A. E. J. Neurosci. Methods 2008, 168,
134.
4. Kordower, J. H.; Emborg, M. E.; Bloch, J.; Ma, S. Y.; Chu, Y.; Leventhal, L.;
McBride, J.; Chen, E. Y.; Palfi, S.; Roitberg, B. Z.; Brown, W. D.; Holden, J. E.;