Side-chain length is important for shogaols in protecting neuronal cells from β-amyloid insult

Article abstract of DOI:10.1016/j.bmcl.2003.12.041

Ten shogaols were synthesized to evaluate the importance of the side-chain length in protecting cells from βA(1-42) insult using PC12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. The compounds cell protectivity against βA insult was demonstrated using 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The efficacy of cell protection from βA insult by these shogaols was shown to improve as the length of the side chain increase.

Full text of DOI:10.1016/j.bmcl.2003.12.041

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1287–1289
Side-chain length is important for shogaols in protecting neuronal
cells from ꢀ-amyloid insult
Darrick S. H. L. Kima,b,* and Jin Yung Kima
aThe Program for Collaborative Research in Pharmaceutical Sciences and the Department of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA
bCurXceL Corporation, The Business and Technology Center, 1291 Cumberland Ave., West Lafayette, IN 47906, USA
Received 2 July 2003; revised 3 December 2003; accepted 9 December 2003
Abstract—Ten shogaols were synthesized to evaluate the importance of the side-chain length in protecting cells from bA(1-42)
insult using PC12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. The compounds cell protectivity against bA
insult was demonstrated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The efficacy of
cell protection from bA insult by these shogaols was shown to improve as the length of the side chain increase.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Gingerols and shogaols found in ginger were shown to
exhibit significant antihepatotoxic actions against CCl4-
and galactosamine-induced cytotoxicity in primary cul-
tured rat hepatocytes.7 The antihepatotoxic activity of
gingerols and shogaols was dependent on the length
of the side chain, with the [7]- and [8]-homologues
eliciting the strongest activity. Oral administration of
[6]-gingerol or [6]-shogaol at doses of 70–140 mg/kg and
iv administration of both compounds at doses of 1.75–
3.7 mg/kg to rats produced an inhibition of spontaneous
motor activity, showed antipyretic and analgesic effects,
and prolonged hexobarbital-induced sleeping time.8,9
[6]-Shogaol was more effective than [6]-gingerol. [6]-
Shogaol was also found to display an intense antitussive
effect in comparison with dihydrocodeine phosphate.
Alzheimer’s disease (AD) is the most common cause of
progressive cognitive dysfunction that affects approxi-
mately four million Americans, causing more than
100,000 deaths each year with a total annual cost
approaching $100 billion.1 b-Amyloid (bA) insult to
neuronal cells was recently hypothesized to be one of
the major potential causes of AD pathology.2,3 Thus,
modulation of bA insult has been speculated to be an
important therapeutic approach to control the onset of
AD. Recently, we reported the discovery of four shogaols
from ginger, Zingiber officinale L. (Zingiberaceae), that
effectively protect IMR-32 human neuroblastoma and
HUVEC cells from bA(25-35) insult.4 The efficacy of
cell protection from bA insult by these shogaols was
suggested to improve as the length of the side chain
increase. Herein, we report synthetic preparation of
shogaols of various lengths and their cell protectivity
from bA insult using PC12 rat pheochromocytoma and
IMR-32 human neuroblastoma cells.
Melting points were determined with a Fisher–Johns
Melting Point apparatus and were uncorrected. 1H
NMR (300 MHz) and 13C NMR (75.6 MHz) spectra
were run on a Bruker DPX-300 spectrometer with TMS
as an internal standard. EI–MS was performed with a
Finnigan MAT 90 instrument. The structures of the
compounds were elucidated using 1H and 13C NMR
and mass spectral analysis.
Ginger is one of the worlds favorite spices, probably
discovered in the tropics of Southeast Asia. Ginger has
benefited humankind as a wonder drug since the beginning
of recorded history.5,6 It is used to treat various kind of
diseases such as common flu, muscle ache, headache, lung
infections, high fever, bacterial and fungal infections, etc.
2. Chemistry
Synthesis of shogaols is shown in Scheme 1. Vanillin (1)
was condensed with acetone in the presence of 75% aqu-
eous NaOH overnight at room temperature, followed by
acidification of the reaction solution using concd HCl to
* Corresponding author. Tel.: +1-765-497-9884; fax: +1-765-463-
7004; e-mail: dkim@curxcel.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.041

1288
D. S. H. L. Kim, J. Y. Kim / Bioorg. Med. Chem. Lett. 14 (2004) 1287–1289
Scheme 1. Syntheis of shogaols: (i) acetone, 75% aqueous NaOH overnight followed by concd HCl; (ii) H2, Pd–C, EtOH–HOAc; (iii) DHP, PPTS,
CH2Cl2; (iv) LDA, À78 ꢀC, 3 h followed by addition of alkyl aldehyde; (v) TsOH, benzene.
induce dehydration (overnight at room temperature) to
afford vanillin acetone (2) in 95% yield. The double
bond in vanillin acetone (2) was reduced using Raney
nickel system10 to zingerone (3) in 72% yield. The
phenolic group of zingerone (3) was protected with
4. Bioassay of shogaols against ꢀA insult on PC12
and IMR-32 cells
bA(1-42) was shown to exert direct toxic effects on
neurons and inhibit the neurite outgrowth in vitro in a
dose dependent manner.14,15 Although bA(1-42) did not
appear to cause cell death at concentrations (1–5 mg/
mL), it was found to undermine cell viability as deter-
mined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-
trazolium bromide (MTT) reduction assay.16À18 This
bA(1-42)-induced cell viability reduction was deter-
mined by observing the amount of MTT reduction to
MTT formazan.16À18
THP group(DHP/PPTS/CH Cl2).11 The resulting THP
2
protected zingerone (4) was reacted with lithium diiso-
propylamide (LDA) at À78ꢀC in THF under N2 (2 h) to
generate lithium enolate, which was reacted with various
alkyl aldehydes at À78ꢀC for 2 h, slowly warming up to
room temperature and stirring for an additional 3 h.12,13
The residue from an aqueous work upthat contains b-
hydroxy ketone product (5) was refluxed overnight in
benzene in the presence of TsOH to afford dehydration
product with THP protecting group removed. The reaction
mixture was diluted with EtOAc (100 mL) and washed
with H2O (50 mLÂ3). The water layer was back extracted
using EtOAc (50 mLÂ2) and the organic layers were
combined, dried (MgSO4), filtered, and the solvent was
removed in vacuo. The resulting residue was column
chromatographed over silica gel (230–400 mesh) using
petroleum ether/ ethyl acetate solvent system to afford
the products (6–14) in various yield.
The compounds ability to protect PC12 cells from bA
insult was investigated according to the published
procedure.15,17 For the bioassay, 90 mL of exponentially
growing cells (2000 cells per mL) were plated in
96-well tissue culture plates overnight. Cells were
incubated with bA(1-42) [2.0 mg/mL, prepared from a
stock solution (1.0 mg/mL in DMSO)] and test
compound at various concentrations (25, 5, 1, 0.2, and
0.04 mg/mL) for 24 h. The final DMSO concentration
was less than 1%. The compounds ability to protect
PC12 cells from bA(1-42) insult was determined by
measuring the cells potential to reduce MTT to MTT
formazan against cell treated with 1% DMSO only and
cell treated with 1.0 mg/mL bA(1-42) and 1% DMSO
without the presence of the test compound. After the
incubation of cells in MTT solution (25 mL per well,
1 mg/mL stock solution) for 1 h at 37 ꢀC, 100 mL
Lysing buffer was added and incubated overnight at
37 ꢀC. The optical density of the resulting solutions was
3. Cells
PC12 and IMR-32 cells were obtained from the Amer-
ican Type Culture Collection (Rockville, MD). Cells
were routinely cultured on a polystyrene-coated Corn-
ing tissue culture plate (Corning, New York, NY). Cul-
ture media and supplements were obtained from Life
Technologies (Grand Island, NY). PC12 cells were
grown in high glucose Dulbecco’s Modified Eagle Med-
ium, 10% horse serum, 5% fetal calf serum, and 1%
penicillin/streptomycin. IMR-32 cells were cultured in
Minimum Essential Medium with Earle’s salt with l-
Glutamine, 10% fetal bovine serum, and 1% penicillin/
streptomycin. bA(1-42) was purchased from Bachem
California (Torrance, CA).
colorimetrically determined at 570 nm using
a
microplate reader. Dose–response curves were pre-
pared and the results were expressed as ED50 values
in mM (Table 1). Curcumin (15) was used as a
positive control. Bioassay on IMR-32 cells was similarly
performed.

D. S. H. L. Kim, J. Y. Kim / Bioorg. Med. Chem. Lett. 14 (2004) 1287–1289
1289
5. Results and discussion
Figure 1 shows colorimetrically determined results of
MTT reduction assay on PC12 cells, treated overnight
with test compounds (5.0 mg/mL) in the presence of
bA(1-42) at 5.0 mg/mL, respectively. The results further
confirm that the efficacy of these shogaols in protecting
cells from bA(1-42) insult improves as the side-chain
length increases. The compounds increased cell pro-
tectivity from bA insult with respect to the increasing
side chain length may be due to increased cell perme-
ability of the compounds
In the present study shogaols were prepared in five
steps. These shogaols were shown to protect PC12 and
IMR-32 cells from bA insult (Table 1). Their efficacy to
protect PC12 and IMR-32 cells from bA insult was
shown to improve as the length of the side chain
increase. The best result was obtained with [12]-shogaol
(14). These shogaols did not show any cytotoxicity
against PC12 and IMR-32 cells at concentrations >129
mM. These shogaols were shown to protect cells from
bA(1-42) insult better than curcumin (15) (ED50=17.1–
23.9 mM). Antioxidant property of these compounds19
may be responsible for protecting cells from bA insult.
In conclusion, our results suggest that shogaols may
be potentially valuable natural therapeutic agents for
the treatment of AD patients. We are in a process of
developing these compounds into such therapeutic
agents.
Table 1. Evaluation of zingerone 3, shogaols (6–14), and curcumin
(15) against b-amyloid(1-42) insult toward PC12 and IMR-32 cells
Acknowledgements
We thank PCRPS for the use of NMR and bioassay
facilities.
Compd
PC12
IMR-32
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Figure 1. PC12 cells were treated overnight with test compounds (5
mg/mL) in the presence of bA(1-42) at 5.0 mg/mL, respectively. Cell
viability was colorimetrically determined using MTT reduction assay.
The tests were performed in triplets on three different dates. Data are
meanÆSEM from nine determinations. P<0.05 (student’s t-test).