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SENIOR RESEARCH: THE SYNTHESIS OF DEMETHYLATED CURCUMIN AND
THE POTENTIAL DISAGGREGATION EFFECTS OF CURCUMINOIDS AND
CURCUMIN ANALOGS ON BETA AMYLOID PLAQUES
ERICA LOMBERK
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ABSTRACT: Aggregation of Aβ plaques, has been cited as structural indicator in a number of
neurodegenerative diseases, namely Parkinson’s and Alzheimer’s diseasei. In Alzheimer’s
disease, these aggregations, specifically those of the 1-40 amino chain, have been implicated as
one of the leading neurotoxic structures present in the progression of disease. Available research
has narrowed down the four necessary factors of a compound with successful disaggregation
effects derived from curcuminoids ii.However, while a variety of compounds have been tested in
this manner, no research was available concerning the efficacy of a curcuminoid that was able to
be dissolved in a nontoxic aqueous solution, which would be a useful quality for medical
application. The goal of this research was to successfully synthesize demethylated curcumin,
which, through its chemical properties, should be able to dissolve much more readily in aqueous
solution than its more hydrophobic precursor, curcumin.
The process by which we carried out demethylation involved use of aluminum trichloride
for methoxy group binding and HCl for subsequent acidification. Completion of the entire
synthesis spanned a week and was performed three times, to produce two intermediates, for
backup material, and a finished product. The final compound, although very soluble in acetone,
was partially soluble in water and, therefore, contained successfully demethylated curcumin.
However, due to a shortage of solvent and minimal readings on the NMR, we had to rely
primarily upon identifiers in the HNMR to draw a conclusion.

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Should time and equipment become available in the future, performing a bioassay on the
synthesized compound against the plaque species may afford a more definite conclusion
concerning the efficacy of the dissagregation properties of our final compound.
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INTRODUCTION: Aggregation of Aβ plaques, has been cited as structural indicator in a
number of neurodegenerative diseases, namely Parkinson’s and Alzheimer’s diseaseiii. In
Alzheimer’s disease, these aggregations, specifically those of the 1-40 amino chain, have been
implicated as one of the leading neurotoxic structures present in the progression of disease.
There have been numerous studies that site mutations in the APP (Amyloid Precursor Protein),
PS1 and PS2 (Presinilin 1 & 2) complex and their subsequent processing by BACE (the λ-site
APP cleaving enzyme) as being integral to the production and aggregation of these Aβ plaques in
ADiv.
The self assembly of Aβ sheets into “sticky” fibrillary structures and the discovery of these
aggregates in most post-mortem neural tissue, namely in hippocampus, is a correlative indicator
to AD progressionv. Although there is also support that other neurotoxic species, such as Tau
fibrils, are notable in the progression of AD, the focus of this study will be on the inhibition of
the aggregation of Aβ fibrils.vi
The most notable structures in the inhibition and reversal of aggregation of Aβ peptides include
polyphenols, namely tanninsvii, organic dyes, such as Congo Redviii, and benzothiazoles, such as
those derived from curcuminix . A number of structures have been chosen based upon common
structural indicators cited as being vital for efficient Aβ plaque disaggregation, namely in the 1-
40 regionx. They have been narrowed down to a single compound due to time availability. The
main focus of this study will target those sites responsive to hydroxylated curcumin, which meets

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all of the structural criteriaxi, in addition to its useful ability to dissolve in aqueous solutions. The
disaggregating efficiencies of all structures will be rated against the control structure, curcumin.
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METHODS:
Initial Species (Curcumin) [keto tautomer]:
Fully Synthesized (Demethylated Curcumin) [keto tautomer]:
This method of synthesis was carried out using a demethylation process carried out by A.
Mazumder, et. al. It was carried out in a series of three basic steps, beginning with a combination
of the AlCl3 (1.341g) with curcumin (1.100g), dichloromethane (50mL), and pyridine (3mL), set
in an oil bath and set to reflux overnight. Additional dichloromethane (100mL) was added as
required to keep the material from drying out. After a 24hr reflux, the flask was placed in an ice
bath for 30min.

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Step 1: Combination & Reflux—1st Reaction Step
Step 1: Combination & Reflux—2nd Reaction Step

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Step 1: Combination & Reflux—3rd Reaction Step
Step 2: Acidification & Separation
Upon removing the solution from the ice bath, it was treated with 2M HCl solution (70mL) and
complete acidification was verified with pH strips. The subsequent solution was washed three
times with 10mL apiece of ethyl acetate and the aqueous layer was purified and extracted. The
solution was set to air dry, then dissolved using methanol, transferred to a tared flask, re-dried,
and weighed to determine solid yield before column extraction.

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TLCs were run using the solution to determine the optimal solvent (Rf=0.333) to utilize for the
process of column separation. Solvent determined to be 100% ethyl acetate. Solid was
redissolved in methanol and silica gel was added to the solution before being air-dried over
48hrs. The column was packed and run using ethyl acetate. A series of 105 clean test tubes were
set up on a set of two racks and used to collect the resultant separated material.
TLCs were performed across the samples to determine the presence of desired endproduct in the
compound to the absence of or decrease of the original curcumin signal. When the optimal range
was separated, tubes 25-51, the subsequent material was dried and extracted. The material was
processed using carbon and proton JEOL NMR and processed using the provided software,
Delta. Peaks, integration, and J coupling values were highlighted and utilized to determine the
presence of synthesized product.
Other samples from same range were treated with room temperature and heated (100°C) distilled
water to determine the potential for solubility of the compound.
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RESULTS: (see attached tables and spectra)
 CNMR- 12 definite/6 possible peaks
o Weak signal definition
o 19-21 peaks on curcumin backbone/methylated product
 HNMR- Several overlaps
o 7.5**, 7.2**, 6.7**, 5.6**, 3.7**ppm all species overlapping w/i .05ppmxii
o Most likely combination of seven different species:
 Keto- No demethylation, 1 demethylation, 2 demethylations

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 Enol- No demethylation, 2 1-demethylations, 2 demethylation
 Most stable peak representations for Keto (3) formations
o Splitting of 1.37Hz at 6.996ppm indicative of midrange single-
demethylation
o Any peaks >7.550ppm indicative of full demethylation—One set present
o Too many peak species to represent fully methylated species
o All sets <2.500ppm potential methyl impurities
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DISCUSSION: I was able to perform a successful demethylation of curcumin, although removal
of impurities may assist in clarification via NMR. Mazumderxiii was able to acquire a 75% yield,
while I received back only 41.73% of the original 1.100g curcumin after organic separation. The
liquid material stained glassware and was difficult to fully dissolve and transfer without
significant loss. In future experiments, it may be useful to utilize a transfer method that decreases
the likelihood of loss.
I was also able to determine that demethylation did occur however, until impurity content
can be diminished, the ratio between the mono, di, and non-demethylated species is difficult to
estimate. Through qualitative analysis, the compound was observed to display a mild solubility
for distilled water that increased with temperature.
My proposal indicated that a bioassay of the finished compound and curcumin would be
applied to the BA42 plaques and compared. Both time and resources were unavailable to
perform this portion of the experiment. In future experimentation, it would be useful to
determine the viability of demethylated curcumin’s disaggregation effect with this method.

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Works Cited
Frid, Petrea, Sergey V. Anisimon, and Nataljia Popovic. 2007. “Congo red and protein
aggregation in neurodegenerative diseases”. Brain Research Reviews 53(1):135-160.
Retrieved March 13, 2015
(http://dx.doi.org.proxy.stetson.edu:2048/10.1016/j.brainresrev.2006.08.001).
Grelle, Gerlinde and Albrecht Otto, et. al. 2011. “Black Tea Theaflavins Inhibit Formation of
Toxic Amyloid-β and α-Synuclein Fibrils”. Biochemistry 50(49):10624-10636. Retrieved
March 13, 2015 (http://pubs.acs.org.proxy.stetson.edu:2048/doi/pdf/10.1021/bi2012383).
Irwin, David J, Virginia M. -Y. –Lee, and John Q. Trojanowski. 2013. “Parkinson’s disease
dementia: convergence of α-synuclein, tau and amyloid-β pathologies”. Nature Reviews
Neuroscience 14(9):626-636. Retrieved March 20, 2015
(http://search.ebscohost.com.proxy.stetson.edu:2048/login.aspx?direct=true&db=a9h&A
N=89799123&site=ehost-live).
Mazumder A., N Neamati, S. Sunder, J. Schulz, H.Pertz. E. Eich, Y.Pommier. Curcumin
Analogs with Altered Potencies Against HIV-1 Integrase as Probes for
Biochemical Mechanisms of Drug Addiction. J.Med.Chem (1997) 40:2057-2063.
Patiny, Luc. “HNMR Predict”. NMRDB.org, Institute for Chemical Sciences and Engineering,
EPFL. Retrieved April 4, 2016
( http://www.nmrdb.org/new_predictor/index.shtml?v=v2.46.2)
Pimplikar, Sanjay W., Ralph A. Nixon, et. al. 2010. “Amyloid-Independent Mechanisms in
Alzheimer’s Disease Pathogenesis”. The Journal of Neuroscience 30(45):14946 –14954.
Retrieved February 26, 2015

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(http://search.ebscohost.com.proxy.stetson.edu:2048/login.aspx?direct=true&db=psyh&
AN=2010-24461-005&site=ehost-live).
Reinke, Ashley and Jason E. Gestwicki. 2007. “Structure –activity Relationships of Amyloid
Beta-aggregation Inhibitors Based on Curcumin: Influence of Linker Length and
Flexibility”. Chemical and Biological Drug Design 70:206-215. Retrieved January 29,
2015
(http://search.ebscohost.com.proxy.stetson.edu:2048/login.aspx?direct=true&db=a9h&A
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Spillantini, Maria Grazia and Michel Goedert. June 2013. “Tau pathology and
neurodegeneration”. Lancet Neurology 12:609-622. Retrieved January 19, 2015
(http://search.ebscohost.com.proxy.stetson.edu:2048/login.aspx?direct=true&db=psyh&
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Sultana, Rukhsana, Debra Boyd-Kimball, et. al. 2006. “Redox proteomics identification of
oxidized proteins in Alzheimer’s disease hippocampus and cerebellum: An approach to
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i Irwin,David J, Virginia M. -Y. –Lee, and John Q. Trojanowski.“Parkinson’s diseasedementia: convergence of α-
synuclein,tau and amyloid-β pathologies”,Nature Reviews Neuroscience, no. 14, issue9 (Sep 2013): (626-36).
ii Reinke, Ashley and Jason E. Gestwicki.2007. “Structure –activity Relationshipsof Amyloid Beta-aggregation
Inhibitors Based on Curcumin:Influence of Linker Length and Flexibility”. Chemical Biology & Drug Design no. 70,
Issue3 (Sep 2007): (206-15)
iii Irwin,(626-636)
iv Pimplikar,Sanjay W.,Ralph A. Nixon, et. al., “Amyloid-Independent Mechanisms in Alzheimer’s Disease
Pathogenesis”. The Journal of Neuroscience no.30, issue45 (Nov 2010):14946 –54.

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v Sultana,Rukhsana,Debra Boyd-Kimball,et. al.2006.“Redox proteomics identification of oxidized proteins in
Alzheimer’s diseasehippocampus and cerebellum: An approach to understand pathological and biochemical
alterations in AD”. Neurobiology of Aging no. 27, Issue12 (Sep 2007):1564-1576.
vi Spillantini,Maria Grazia and Michel Goedert, “Tau pathology and neurodegeneration,” Lancet Neurology no. 12,
Issue6 (Jun 2013):609-622.
vii Grelle, Gerlindeand Albrecht Otto, et. al. “Black Tea Theoflavins InhibitFormation of Toxic Amyloid-β and α-
Synuclein Fibrils”,Biochemistry no. 50, Issue49 (Dec 2011):10624-36.
viii Frid,Petrea, Sergey V. Anisimon,and Nataljia Popovic.“Congo red and protein aggregation in
neurodegenerative diseases”,Brain Research Reviews no. 53, Issue1 (Jan 2007):135-160.
ix Reinke, (206-15)
x Reinke, 209.
xi Reinke, 206-13.
xii Patiny,Luc. “HNMR Predict”. NMRDB.org, Institute for Chemical Sciences and Engineering, EPFL, <
http://www.nmrdb.org/new_predictor/index.shtml?v=v2.46.2>
xiii Mazumder, A, N. Neamati, S. Sunder, J Schulz,H. Pertz, E. Eich.“Curcumin Analogs with Altered Potencies
againstHIV-1 Integrase as Probes for Biochemical Mechanisms of DrugAddiction”, J.Med.Chem no.40 (1997):
2057-2063.