Total synthesis of calebin-A, preparation of its analogues, and their neuronal cell protectivity against β-amyloid insult

Article abstract of DOI:10.1016/S0960-894X(01)00489-9

A total synthesis of Calebin-A (1), a novel curcuminoid isolated from turmeric (Curcuma longa, Zingiberaceae) that has been demonstrated to protect neuronal cells from β-amyloid insult, was successfully achieved in four steps. Elaborating on this synthetic route, 13 analogues were prepared for a structure-activity relationship (SAR) study. It was found that the parent compound 1 and derivatives 21, 28, and 30 protect PC12 rat pheocromocytoma and IMR-32 human neuroblastoma cells from β-amyloid(25-35) insult. These results suggest that hydroxy group at para-position is most critical for the expression of biological activity.

Full text of DOI:10.1016/S0960-894X(01)00489-9

Bioorganic & MedicinalChemistry Letters 11 (2001) 2541–2543
Total Synthesis of Calebin-A, Preparation of Its Analogues, and
Their Neuronal Cell Protectivity Against ꢀ-Amyloid Insult
Darrick S. H. L. Kim* and Jin Yung Kim
The Program for Collaborative Research in Pharmaceutical Science and the Department of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL, 60612, USA
Received 17 May 2001; accepted 10 July 2001
Abstract—A totalsynthesis of Caelbin-A ( 1), a novelcurcuminoid isoal ted from turmeric ( Curcuma longa, Zingiberaceae) that has
been demonstrated to protect neuronal cells from b-amyloid insult, was successfully achieved in four steps. Elaborating on this
synthetic route, 13 analogues were prepared for a structure–activity relationship (SAR) study. It was found that the parent com-
pound 1 and derivatives 21, 28, and 30 protect PC12 rat pheocromocytoma and IMR-32 human neuroblastoma cells from b-amy-
loid(25–35) insult. These results suggest that hydroxy group at para-position is most criticalfor the expression of biologicalactivity.
# 2001 Elsevier Science Ltd. All rights reserved.
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 totalannualcost of
treatment approaching $100 billion.1 b-Amyloid (bA)
insult to neuronal cells was recently found to be one of
the major causes of AD pathology. Thus, modulation of
bA insult has been speculated to be an important ther-
apeutic approach to controlthe onset of AD.
In the present study, we report the first totalsynthesis of
Calebin-A (1), along with its derivatives for a structure–
activity relationship (SAR) study using PC12 and
IMR32 cells against bA(25–35) insult.
Melting points were determined with a Fisher–Johns
Melting Point apparatus and were uncorrected. UV
spectra were obtained on a Beckman DU-7 spectro-
photometer. 1H NMR (300 MHz) and 13C NMR
(75.6 MHz) spectra were run on a Bruker DPX-300
spectrometer with TMS as an internalstandard. EI-MS
was performed with a Finnigan MAT 90 instrument.
The structures of the compounds were elucidated by
Recently, we discovered a novel curcuminoid, Calebin-
A (1), from turmeric (Curcuma longa L., Zingiberaceae)
that protected PC12 rat pheocromocytoma and normal
human umbilical vein endothelial HUVEC cells from
bA(1–42) insult.2 Turmeric has been used as curry spice
and as a well-known constituent of Indonesian tradi-
1
using H and 13C NMR and mass spectralanayl sis.
3
tionalmedicine.
Total Synthesis of Calebin-A (Scheme 1)
Alcohol functionality of acetol 2 and vanillin 4 was
protected as THP ether using DHP in the presence of
PPTS in THF in quantitative yield.4 THP ether of acetol
3 was reacted with LDA in THF at À78 ꢀC and reacted
with THP ether of vanillin 5 to afford THP ether of b-
hydroxy ketone 6 in 27% yield. THP protection groups
were removed from b-hydroxy ketone THP ether 6 by
stirring in methanolin the presence of PPTS at 50 ꢀC
that also caused the dehydration of b-hydroxylgroup to
afford compound 7 in 42% yield.5 The ferulic acid 8 and
the alcohol 7 were coupled in the presence of dicyclo-
hexylcarbodiimide (DCC), dimethylamino-pyridine
*Corresponding author. Tel.: +1-312-996-1230; fax: +1-312-413-
5894; e-mail: dshlkim@uic.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00489-9

2542
D. S. H. L. Kim, J. Y. Kim / Bioorg. Med. Chem. Lett. 11 (2001) 2541–2543
(DMAP), and DMAP–HClin CHCl
at room tem-
and 1% penicillin/streptomycin.8 For the bioassay using
bA(25–35), 90 mL of exponentially growing cells (2000
cells per mL) were plated in 96-well tissue culture plates.
bA(25–35), the cytotoxic fragment of bA(1–42),9,10 was
purchased from Bachem California (Torrance, CA,
USA).
3
perature6 to afford product 1 (48%). The H NMR of
the product was identicalto that of the authentic sam-
ple.
1
Synthesis of Calebin-A Derivatives
bA(25–35) was shown to exert direct toxic effects on
neurons and inhibit the neurite outgrowth in vitro in a
dose dependent manner.9,10 Although bA(25–35) did
not appear to cause cell death at low concentration (1–
5 mg/mL), it was found to cause a profound cell damage,
such that, cell viability was undermined. This bA(25–
35)-induced cell viability reduction was determined by
observing the amount of 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) reduction.11,12
The alcohol 7 was coupled with cinnamic acid deriva-
tives 9–18 in the presence of DCC, DMAP, and
DMAP–HClin CHCl at room temperature to afford
3
products 19, 20, 22–27, 29, and 31. Compounds 21, 28,
and 30 were prepared by removing the acetylgroup
from compound 20, 27, and 29, respectively, using
K2CO3/MeOH/H2O system at room temperature
(Scheme 2).
Under our experimentalcondition, bA(25–35) was
found to attenuate the viability of PC12 and IMR-32
cells at ED50=1.0 mg/mL.
Bioassay
The bioassays were performed according to the pub-
lished procedure.2,7 PC12 rat pheochromocytoma and
IMR-32 human neuroblastoma cells were obtained
from the American Type Culture Collection (Rockville,
MD, USA). Cells were routinely cultured on a poly-
styrene-coated Corning tissue culture plate (Corning,
New York, USA). Culture media and supplements were
obtained from Life Technologies (Grand Island, USA).
PC12 cells were grown in high glucose Dulbecco’s
Modified Eagle Medium, 10% horse serum, 5% fetal
calf serum, and 1% penicillin/streptomycin.2,7 IMR-32
cells were cultured in Minimum Esssential Medium with
Earle’s salt with l-glutamine, 10% fetal bovine serum,
The compounds’ ability to protect PC12 and IMR32
cells from bA(25–35) insult was investigated. PC12 cells
(2000 cells per mL) were incubated with bA(25–35)
(1.0 mg/mL, prepared from a stock solution, 1.0 mg/mL
in DMSO) and the test compounds at various con-
centrations (25, 5, 1, 0.2, and 0.04 mg/mL) in a 96-well
culture plate for 24 h. The final DMSO concentration
was less than 1%. The compounds’ ability to protect
PC12 cells from bA(25–35) insult was determined by
measuring the cell’s potential to reduce MTT against
positive control(1% DMSO onyl ) and negative control
[1.0 mg/mL bA(25–35) in 1% DMSO without the pre-
sence of the test compounds]. After an incubation of
cells in MTT solution (25 mL per well, 1 mg/mL stock
solution) for 2 h at 37 ꢀC, 100 mL Lysing buffer was
added and incubated overnight at 37 ꢀC. The optical
density of the resulting solutions was colorimetrically
determined at 570 nm using ELISA microplate reader.
Dose–response curves were prepared and the results
were expressed as ED50 values in mg/mL (Table 1). The
compounds’ ability to protect IMR-32 cells (2000 cells
per mL) from bA(25–35) insult was similarly evaluated
using 1.0 mg/mL of bA(25–35). Curcumin (32) and (Æ)-
a-tocopherol(vitamin E, 33) were used as controlcom-
pounds.
Scheme 1. Synthesis of Calebin-A: (i) DHP, acetol, PPTS, THP; (ii)
THF, DHP, PPTS; (iii) LDA, THF (at À78 ꢀC for 1 h) and then add
compound 5; (iv) PPTS, methanol; (v) compound 8, DMAP, DMAP–
HCl, DCC, CHCl3, overnight.
Discussion
Starting from acetol, Calebin-A (1) was synthesized in
four steps. Using compound 7 as the key intermediate,
Calebin-A derivatives were prepared in good to moder-
ate yield in one step. The low yields in deacetylation of
compounds 20, 27, and 29 are attributed to saponifica-
tion of the ester functionality in the parent structure.
Among the Calebin-A derivatives, only compounds 21,
28, and 30, in addition to the parent compound Cale-
bin-A (1), protected cells from bA(25–35) insult (Table
1). The cell protection from bA(25–35) insult by these
compounds showed that 4-hydroxy group on cinnamate
portion of the compound is important for the com-
pounds’ ability to protect cells from bA(25–35) insult.
The result from compound 28 showed that 3-hydroxy
Scheme 2. Synthesis of Calebin-A analogues.

D. S. H. L. Kim, J. Y. Kim / Bioorg. Med. Chem. Lett. 11 (2001) 2541–2543
2543
Table 1. Evaluation of Calebin-A analogues against b-amyloid(25–
35) insult toward PC12 and IMR-32 cells
cytotoxic at concentrations >25 mg/mL. These results
clearly demonstrated that compounds 1 and 21 warrant
further investigation for the development as potentially
valuable agents to treat AD patients. We are in the
process of developing these compounds into therapeutic
agents that may be used to treat AD.
Compound
ED50 (mg/mL)a
PC12
IMR32
1
1.0Æ0.3
npb
np
1.4Æ1.1
np
np
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
0.7Æ0.4
1.2Æ0.6
Acknowledgements
np
np
np
np
We thank PCRPS for the use of NMR and bioassay
facilities.
np
np
np
np
np
np
np
np
13.2Æ0.9
15.8Æ1.2
References and Notes
np
7.3Æ0.5
np
np
7.9Æ0.6
np
1. Garber, K. Technol. Rev. 2001, 104, 70.
7.1Æ0.4
7.4Æ0.7
2. Park, S. Y.; Kim, D. S. H. L. J. Nat. Prod. 2001, in press.
3. Nurfina, A. N.; Reksohadiprodjo, M. S.; Timmerman, H.;
Jenie, U. A.; Sugiyanto, D.; van der Goot, H. Eur. J. Med.
Chem. 1997, 32, 321.
4. Miyashita, N.; Yoshikoshi, A.; Grieco, P. A. J. Org. Chem.
1977, 42, 3772.
5. Artico, M.; Santo, R. D.; Costi, R.; Novellino, E.; Greco,
G.; Massa, S.; Tramontano, E.; Marongiu, M. E.; Montis,
A. D.; Colla, P. L. J. Med. Chem. 1998, 41, 3948.
6. Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394.
7. Kim, D. S. H. L.; Park, S. Y.; Kim, J. Y. Neurosci. Lett.
2001, 303, 57.
8. Hirayama, S.; Bujo, H.; Yamazaki, H.; Kanaki, T.; Taka-
hashi, K.; Kobayashi, J.; Schneider, W. J.; Saito, Y. Biochem.
Biophys. Res. Commun. 2000, 275, 365.
np
np
The tests were performed in triplets on three different dates. Data are
meanÆSEM from nine determinations. P<0.05 (Student’s t-test).
aED50 represents the sample concentration that is required to achieve
50% cell viability, a mid-point between the positive control values and
the negative controlvaul es.
bnp denotes no protection from bA(25–35) insult.
group is less effective than 4-hydroxy group in protect-
ing cells from bA(25–35) insult. The result from com-
pound 30 suggested that 3-hydroxy group may be
hindering the efficacy of 4-hydroxy group, probably
through hydrogen bonding between them. Evidently, 3-
methoxylgroup is not criticalfor the expression of the
biological activity. Under the same experimental condi-
tion, vitamin E (33) did not protect cells from bA(25–
35) insult at concentrations >25 mg/mL, while curcumin
(32) protected cells at ED50=7 mg/mL.2,7 It is note-
worthy that compounds 1, 21, 28, and 30 were not
9. Pike, C. J.; Walencewicz, A. J.; Glabe, C. G.; Cotman,
C. W. Brain Res. 1991, 563, 311.
10. Pollack, S. J.; Sadler, I. I. J.; Hawtin, S. R.; Tailor, V. J.;
Shearman, M. S. Neurosci. Lett. 1995, 184, 113.
11. Mosmann, T. J. Immunol. Meth. 1983, 65, 55.
12. Shearman, M. S.; Ragan, C. I.; Iversen, L. L. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 1470.