Synthesis of monofunctional curcumin derivatives, clicked curcumin dimer, and a PAMAM dendrimer curcumin conjugate for therapeutic applications

Article abstract of DOI:10.1021/ol702370m

Curcumin, the primary active ingredient in the spice turmeric, was converted to reactive monofunctional derivatives (carboxylic acid/azide/alkyne). The derivatives were employed to produce a 3 + 2 azide-alkyne "clicked" curcumin dimer and a poly(amidoamine) (PAMAM) dendrimer-curcumin conjugate. The monofunctional curcumin derivatives retain biological activity and are efficient for labeling and dissolving amyloid fibrils. The curcumin dimer selectively destroys human neurotumor cells. The synthetic methodology developed affords a general strategy for attaching curcumin to various macromolecular scaffolds.

Full text of DOI:10.1021/ol702370m

ORGANIC
LETTERS
2007
Vol. 9, No. 26
5461-5464
Synthesis of Monofunctional Curcumin
Derivatives, Clicked Curcumin Dimer,
and a PAMAM Dendrimer Curcumin
Conjugate for Therapeutic Applications
Wei Shi,† Sukanta Dolai,† Samar Rizk,† Afshan Hussain,‡ Hussnain Tariq,†
Saadyah Averick,† William L’Amoreaux,‡ Abdeslem El Idrissi,‡
Probal Banerjee,*,† and Krishnaswami Raja*,†
Departments of Chemistry, the Institute for Macromolecular Assemblies and the Center
for Engineered Polymeric Materials, City UniVersity of New York at the College of
Staten Island, 2800 Victory BouleVard, Staten Island, New York 10314
banerjee@mail.csi.cuny.edu; raja@mail.csi.cuny.edu
Received September 28, 2007
ABSTRACT
Curcumin, the primary active ingredient in the spice turmeric, was converted to reactive monofunctional derivatives (carboxylic acid/azide/
alkyne). The derivatives were employed to produce a 3 2 azide alkyne “clicked” curcumin dimer and a poly(amidoamine) (PAMAM) dendrimer−
+
−
curcumin conjugate. The monofunctional curcumin derivatives retain biological activity and are efficient for labeling and dissolving amyloid
fibrils. The curcumin dimer selectively destroys human neurotumor cells. The synthetic methodology developed affords a general strategy for
attaching curcumin to various macromolecular scaffolds.
Curcuma longa commonly referred to as “turmeric” is used
as a spice in South Asian cooking, as a cosmetic, and in the
ancient Ayurvedic system of medicine.1a There has recently
been tremendous interest in curcumin [(1E,6E)-1,7-bis(4-
hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione], the
primary active ingredient in turmeric, because it has been
shown to have antioxidant,1b anticancer,1c anti-inflammatory,1d
and potent anti-Alzheimer’s disease activity.1e Amyloid-â
peptide (Aâ) aggregation is suspected to play an important
role in Alzheimer’s disease (AD);2 amyloid deposits are also
implicated in amyloid heart disease.3 One of the major
limitations of using curcumin as a drug is its poor water and
plasma solubility; even doses as high as 8 g of curcumin
per day administered to human subjects results in an average
peak serum concentration of ∼1.77 µM of curcumin.4 The
† Department of Chemistry.
‡ Institute for Macromolecular Assemblies and the Center for Engineered
Polymeric Materials.
(1) (a) Chattopadhyay, I.; Biswas, K.; Bandyopadhyay, U.; Banerjee, R.
K. Curr. Sci. 2004, 87, 44-53. (b) Agarwal, B. B.; Shishodia, S. Biochem.
Pharmacol. 2006, 71, 1397-1421. (c) Leyon, P. V.; Kuttan, G. J. Exp.
Clin. Cancer Res. 2003, 22, 77-83. (d) Nurfinal, A. N.; Reksohadiprodjo,
M. S.; Timmerman, H.; Jenie, U. A.; Sugiyant, D.; van der Goot, H. Eur.
J. Med. Chem. 1997, 32, 321-328. (e) Yang, F.; Lim, P. L. G. P.; Begum,
A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P.; Kayed,
R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem. 2005, 280,
5892-5901.
(2) Roberson, E. D.; Mucke, L. Science 2006, 314, 781-784.
(3) Waller, B. F. Clin. Cardiol. 1988, 11, 513-517.
10.1021/ol702370m CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/17/2007

Scheme 1. Synthesis of Monofunctional Curcumin Derivatives
glucuronidation of curcumin in vivo is another possible
in contrast to the previous report, the approach we present
here involves direct one/two-step covalent modification of
curcumin to produce reactive monofunctional derivatives. A
curcumin dimer was synthesized by condensing curcumin
azide and curcumin alkyne using the copper-catalyzed azide-
alkyne “click” reaction.9
Dendritic polymers are arguably the most spectacular
example of a synthetic polyvalent scaffold. Dendrimers have
potential applications in diverse areas ranging from material
science to nanomedicine.10 A cystamine core PAMAM
dendrimer-curcumin conjugate was synthesized, and this
serves as a demonstration of the utility of reactive mono-
functional curcumin derivatives in the synthesis of biomi-
metic polyphenols. Preliminary in vitro biological studies
indicate that the monofunctional derivatives of curcumin
efficiently label and dissolve amyloid fibrils. The curcumin
dimer selectively destroys human neurotumor cells.
The monocarboxylic acid derivative of curcumin 1a was
synthesized by reacting curcumin 1 with glutaric anhydride
in the presence of base in accordance with Scheme 1.
Curcumin monoazide derivative 1b was synthesized by an
amide coupling reaction between curcumin monocarboxylic
acid 1a and an amino-PEG azide using 1,3-dicyclohexyl-
carbodiimide (DCC) at room temperature (Scheme 1). The
monoalkyne derivative of curcumin 1c was synthesized by
etherifying curcumin with propargyl bromide; K2CO3 was
used as a base in DMF at room temperature (Scheme 1).
The etherification of phenols with standard alkyl bromides
requires elevated temperature and extended reaction time.
In contrast, etherification involving curcumin and propargyl
bromide proceeded very efficiently at room temperature;
previous reports with other phenols and propargyl bromide
support this observation.9b,11 The monotriazole-PEG deriva-
tive of curcumin 1d was synthesized by condensing the
monoalkyne derivative of curcumin 1c with azidotriethylene
glycol under the Sharpless click conditions (copper(II) sulfate
and sodium ascorbate). A curcumin dimer was synthesized
reason for the observation of very low plasma levels of the
compound.5 The development of a synthetic methodology
to produce curcumin conjugates with water-soluble polymers
and targeting proteins can potentially enhance the therapeutic
efficacy of curcumin. Higher molecular weight plant polyphe-
nols have been shown to possess amplified physiological
properties such as antioxidant or anticancer properties
compared to their low molecular weight analogues.6 The
chemical synthesis of therapeutically relevant, well-defined
high molecular weight polyphenols is very rare.
In this communication, we present a convenient route to
water-soluble polyvalent curcumin conjugates via the syn-
thesis of novel monofunctional curcumin derivatives in which
one of the phenolic groups of curcumin has been chemically
modified with reactive groups (azide, alkyne, and carboxylic
acid) (Scheme 1). The synthesis of monofunctional curcumin
derivatives affords two advantages: (a) the presence of at
least one free phenolic group is necessary for the biological
activity of many antioxidants such as curcumin;1b (b)
bioconjugation and polymer modifications using monofunc-
tional derivatives produce soluble conjugates in high yields,
whereas bifunctional derivatives would result in insoluble
cross-linked products.7 To the best of our knowledge, this
is the first report describing a general methodology for
preparing reactive monofunctional curcumin derivatives; the
unique carboxylic acid/azide/alkyne groups serve as covalent
functional handles for modifying both synthetic polymers
and proteins. There has been a recent report describing the
multistep synthesis of a monofunctional alkyl fluoride
derivative of curcumin starting from a vanillin derivative,8
(4) Cheng, A. L.; Hsu, C. H.; Lin, J. K.; Hsu, M. M.; Ho, Y. F.; Shen,
T. S.; Ko, J. Y.; Lin, J. T.; Lin, B. R.; Wu, M. S.; Yu, H. S.; Jee, S. H.;
Chen, G. S.; Chen, T. M.; Chen, C. A.; Lai, M. K.; Pu, Y. S.; Pan, M. H.;
Wang, Y. J.; Tsai, C. C.; Hsieh, C. Y. Anticancer Res. 2001, 21, 2895-
2900.
(5) Pfeiffer, E.; Hoehle, S. I.; Walch, S. G.; Riess, A.; Solyom, A. M.;
Metzler, M. J. Agric. Food Chem. 2007, 55, 538-544.
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Org. Lett., Vol. 9, No. 26, 2007

by reacting curcumin monoalkyne derivative 1c with cur-
cumin monoazide derivative 1b using copper(II) sulfate and
sodium ascorbate (Scheme 1). The curcumin dimer 1e has
two curcumin moieties connected via a triazole link and a
PEG spacer; the dimer has two phenolic groups like the
parent molecule 1. The structures of all the curcumin
1
derivatives 1a, 1b, 1c, 1d, and 1e were confirmed by H
NMR, 13C NMR, and mass spectrometry (MS) (see Sup-
porting Information). The carboxylic acid group of 1a can
be conjugated to proteins, biopolymers, and synthetic
polymers; the azide 1b and the alkyne derivative 1c could
be attached to modified proteins and polymers via the click
bioconjugation reaction. It should be noted that monofunc-
tional curcumin derivatives are necessary to produce soluble
conjugates.
A Generation 4 cystamine core poly(amidoamine) den-
drimer, with amine surface groups from Dendritic Nano-
technologies was coupled to curcumin monocarboxylic acid
1a using DCC, N-hydroxysuccinimide (NHSu), and TEA in
DMF to produce the conjugate 2a (Scheme 2). The product
Figure 1. (A) FPLC of G4 cystamine core poly(amidoamine)
dendrimer-curcumin conjugate sample 2a; using a Hi-Prep 26/10
desalting column, 0.1 M Na2CO3 solution (pH ) 9) was used as
the running buffer. (B) Aqueous solution of the PAMAM den-
drimer-curcumin conjugate 2a.
Curcumin binds and dissolves amyloid fibrils very
effectively.1e Preliminary studies were carried out to assess
whether the monofunctional curcumin derivatives retained
the ability to bind and dissolve amyloid fibrils in vitro.
Human heart tissue containing intercellular amyloid was
stained with Congo Red12 according to the “Benhold’s”
protocol (control sample) or with curcumin monocarboxylic
acid 1a and imaged using a polarized light microscope; the
images indicate that 1a labels amyloid fibrils very effectively
(Figure 2b). Curcumin and its derivatives have the advantage
Scheme 2. Synthesis of the Dendrimer-Curcumin Conjugate
was dialyzed extensively using a 3500 MWCO membrane
and further purified by a Sephadex LH20 size exclusion
column. The number of 1a units attached per dendrimer was
estimated to be 37 from 1H NMR by comparing the intensity
of Ar-H (δ ) 6.31-7.41 ppm) from the curcumin compo-
nent with (-CONH-CH2-CH2-NHCO-) protons from the
dendrimer component at δ ) 3.11-3.15 ppm. The cur-
cumin-dendrimer conjugate 2a is freely soluble in water,
and the aqueous solution has the characteristic color of
curcumin (Figure 1B). The conjugate was analyzed via fast
protein liquid chromatography (FPLC), using 0.1 M Na2-
CO3 (pH ) 9) solution as the running buffer. The peak which
eluted at the same volume as the dendrimer (275 nm) with
strong absorbance at 430 nm arising from curcumin indicated
that curcumin was indeed conjugated to the dendrimer
(Figure 1A). Appropriate control experiments with unmodi-
fied PAMAM dendrimer and a purified noncovalent mixture
of the dendrimer with 1a were also performed (see Support-
ing Information). It is anticipated that the conjugate 2a will
behave differently from a small molecule in vivo and exhibit
the EPR (enhanced permeability and retention) effect. The
conjugate 2a can be reduced to form two dendrons each
possessing a single thiol group which could then be attached
to targeting peptides/proteins to deliver plasma-soluble
curcumin for several potential therapeutic applications.1b-d
Figure 2. Human heart tissue containing intercellular amyloid
stained using (a) 0.014 M Congo Red (control sample) and (b) 50
nM curcumin monocarboxylic acid 1a imaged using a polarized
light microscope under cross polarizers. (c) Transmission electron
micrograph of amyloid fibrils produced by incubating Aâ 1-40
for 6 days at 37 °C. (d) No fibrils are detected by TEM when
amyloid fibrils produced by incubating Aâ 1-40 for 3 days were
further incubated with 1a (8 µM) for 3 days at 37 °C. The final
Aâ 1-40 concentration is the same in both c and d. The scale bar
for the TEM images is 500 nm.
that they can effectively label fibrils at a much lower
(6) Ihara, N.; Schmitz, S.; Kurisawa, M.; Chung, J. E.; Uyama, H.;
Kobayashi, S. Biomacromolecules 2004, 5, 1633-1636.
concentration than Congo Red: 50 nM of 1a compared to
Org. Lett., Vol. 9, No. 26, 2007
5463

0.014 M for Congo Red. The ability of 1a to dissolve
amyloid aggregates (fibrils) was also evaluated. In a typical
experiment, amyloid fibrils were formed by incubating Aâ
1-40 peptide. Either curcumin monocarboxylic acid 1a or
control buffer was added to the fibrils followed by visualiza-
tion using transmission electron microscopy (TEM). A
network of fibrils was observed in the TEM image of the
control amyloid fibril sample (Figure 2c), whereas the fibrils
were absent in the sample treated with 1a (Figure 2d). This
observation was further supported by UV spectroscopy (see
Supporting Information). These experiments indicate that the
curcumin derivatives are promising candidates for the
dissolution of amyloid fibrils.
assay was performed on healthy control neuronal cells at
500 µM concentration; curcumin induced considerable apo-
ptosis, whereas the dimer 1e induced only marginal apoptosis
(Raja and Banerjee, unpublished results).
In conclusion, novel monofunctional curcumin derivatives
containing reactive azide, alkyne, and carboxylic acid groups
were synthesized. These derivatives were further employed
to synthesize a triazole-PEG derivative, a “clicked” curcumin
dimer, and a water-soluble PAMAM dendrimer-curcumin
conjugate. The ability of the curcumin derivatives to stain
and dissolve amyloid fibrils in vitro was evaluated. The
selectivity of the curcumin dimer in destroying human
neurotumor cells over curcumin suggests that it is a promis-
ing drug candidate. The biological activity studies of 2a in
vitro and in vivo are currently underway. The curcumin
derivatives are currently being attached to various macro-
molecular scaffolds including synthetic polymers and target-
ing proteins to produce biomimetic polyphenols with po-
tentially amplified biological activity.
The ability of the curcumin derivatives to eliminate
SHSY5Y metastatic human neurotumor cells was evaluated
using a caspase-3 activation assay. The dimer 1e was found
to be the most selective derivative. As can be seen from
Figure 3, both curcumin and 1e show a concentration--
Acknowledgment. We would like to thank Dr. Hsin
Wang from the College of Staten Island (CSI NMR facility)
and CSI undergraduates Alexia Berliner, Francoise Sidimie,
and Solomon Gulyamov. We also thank PSC-CUNY (69535-
00-38) for funding.
Supporting Information Available: Experimental pro-
cedures and characterization of synthesized compounds,
FPLC chromatograms, UV spectra of amyloid samples. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL702370M
Figure 3. Caspase-3 activity in SHSY5Y metastatic human
neurotumor cells in response to curcumin and curcumin dimer.
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dependent caspase activation; at 500 µM, curcumin is slightly
better than the dimer in inducing cell apoptosis. The same
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Diego, CA, 1996; Chapter 15, p 607.
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