Design, synthesis and characterization of some bioactive conjugates of curcumin with glycine, glutamic acid, valine and demethylenated piperic acid and study of their antimicrobial and antiproliferative properties

Article abstract of DOI:10.1016/j.ejmech.2007.11.027

The monoesters of curcumin, a symmetric diphenol with valine and glycine have been prepared by a novel solid phase synthesis and its diesters with valine, glutamic acid and demethylenated piperic acid have been prepared by solution phase method. The assessment of their antimicrobial and anticancer (antiproliferative) activities suggested that diesters of curcumin are relatively more active than curcumin itself due to their increased solubility, slow metabolism and better cellular uptake. Furthermore, significant observation was that monoesters of curcumin have even better antimicrobial activity than their corresponding diesters, emphasizing the role of free phenolic group. The conjugate of curcumin with demethylenated piperic acid in which methylenedioxy ring was open also shows enhanced activity than the corresponding piperic acid conjugate, emphasizing the role of free phenolics in the transport or in the binding processes.

Full text of DOI:10.1016/j.ejmech.2007.11.027

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1837e1846
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and characterization of some bioactive conjugates of
curcumin with glycine, glutamic acid, valine and demethylenated piperic
acid and study of their antimicrobial and antiproliferative properties
b
Shiv K. Dubey a, Anuj K. Sharma b, Upma Narain c, Krishna Misra d, , Uttam Pati
*
a Centre for Biotechnology, University of Allahabad, Allahabad 211002, India
b Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
c Kamla Nehru Hospital, Allahabad 211002, India
d Indian Institute of Information Technology, Allahabad 211011, India
Received 10 May 2007; received in revised form 13 November 2007; accepted 22 November 2007
Available online 8 December 2007
Abstract
The monoesters of curcumin, a symmetric diphenol with valine and glycine have been prepared by a novel solid phase synthesis and its
diesters with valine, glutamic acid and demethylenated piperic acid have been prepared by solution phase method. The assessment of their
antimicrobial and anticancer (antiproliferative) activities suggested that diesters of curcumin are relatively more active than curcumin itself
due to their increased solubility, slow metabolism and better cellular uptake. Furthermore, significant observation was that monoesters of cur-
cumin have even better antimicrobial activity than their corresponding diesters, emphasizing the role of free phenolic group. The conjugate of
curcumin with demethylenated piperic acid in which methylenedioxy ring was open also shows enhanced activity than the corresponding piperic
acid conjugate, emphasizing the role of free phenolics in the transport or in the binding processes.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Curcumin; Piperic acid; Valine; Glutamic acid; Antimicrobial; Antiproliferative
1. Introduction
of tumor cells in culture and promote apoptosis through cleav-
age of BID (a Bcl-2 family member protein) [3], cytochrome c
release, down regulation of bcl-2 and activation of caspases [3].
It has been shown to lower blood cholesterol, promote wound
healing, prevent skin wrinkling, inhibit inflammation, suppress
rheumatoid arthritis and inhibit human immunodeficiency
virus replication [4,5]. Curcumin mediates wide variety of ther-
apeutic effects through the regulation of the transcription
factors, nuclear factor kappa B [6e9] and activator protein,
suppression of IKBa kinase and c-jun N-terminal kinase and
inhibition of expression of cyclooxygenase (COX) 2 [10,11],
Cyclin D1, adhesion molecules, matrix metalloproteases [12]
inducible nitric oxide synthase, HER2, epithelial growth factor
(EGF) receptor, bcl-2, bcl-xl, and tumor necrosis factor (TNF)
[13]. Pharmacologically, curcumin is quite safe and doses as
high as 8 g/day have been administered orally to humans
with no side effects.
Curcumin [diferuloylmethane; 1,7-bis-(4-hydroxyl 3-me-
thoxyphenyl)-1,6-heptadiene-3,5-dione; Scheme 1(I)] is the
major pigmentary component of turmeric (Curcuma longa),
a spice commonly used in India, better known as ‘‘Indian solid
gold’’ due to its numerous therapeutic activities, its pharmaco-
logical safety and its colour. Curcumin occurs in turmeric along
with its demethoxy and bis-demethoxy derivatives (curcumi-
noids). Curcumin has been shown to suppress carcinogenesis
of the skin, liver, lung, colon, stomach and breast [1,2]. It has
also been shown to inhibit the proliferation of a wide variety
* Corresponding author. Tel.: þ91 0532 2465462/2467154 (Res); þ91 0532
2922203 (Off); fax: þ91 0532 2461376.
E-mail addresses: krishnamisra@hotmail.com, kkmisra@yahoo.com (K.
Misra).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.027

1838
S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
Glutamic acid enhances the flavor of foods and tobacco
O
O
[24]. It is reported to be useful in muscular dystrophy [25]
and accelerates wound healing and ulcer healing. Glutamine
plays prominent role in circulating amino acid. It plays
a unique role in the transfer of nitrogen from peripheral tissues
to the liver.
Glycine is of major importance. Recently, a glycine gated
chloride channel has been identified in neutrophils that can
attenuate increase in intracellular calcium ions and diminish
oxidant damage mediated by these white blood cells. Thus
glycine may be a novel antioxidant.
H CO
OCH
OH
3
3
I (curcumin)
HO
i) Pyridine / N-Phthaloylglutamoyl
chloride (IIa) / N-Phthaloylvalinoyl
chloride (IIb) / N-Phthaloylglycinoyl
chloride (IIc) / 6h / r.t.
ii) NH : Pyridine (9:1 v/v)
3
O
O
H CO
OCH
3
3
The structureeactivity relationship studies of curcumin
molecule with respect to its multiple biological activities so
far have indicated that the presence of two phenyl rings with
a 7-C linker having b-diketo function (C]O groups as hydro-
gen acceptors and C-4 as hydrogen donor) is the basic neces-
sity [26]. However, unsaturation in the linker (conformational
flexibility) is important for its antitumor/anticancer activity but
not for redox regulatory or apoptotic activities [27,28]. THC
(tetrahydrocurcumin) has even better antioxidant property
than curcumin. The curcumin conjugates reported with modi-
fication at active methylene group or even involving C]O did
not show any enhanced activity. The only alternative was to
use biodegradable (ester) linkage at phenolic hydroxyls.
In our earlier experiments we have observed that conjugates
(phenolic esters) of curcumin with some amino acids show
much better antibacterial, antifungal and antioxidant proper-
ties [14e21], which may be attributed to their better solubility,
enhanced accumulation in the cells, resulting from better
cellular uptake and decreased metabolic rate. Evidently, the
carrier proteins for amino acids, the necessary building blocks
of proteins, must be responsible for dragging the curcumin
molecule along (drug smuggling). Since, for its physiological
activity, specially antioxidant character, presence of free phe-
nolics has been found to be essential, we designed the prepa-
ration of monoesters so that both advantages may be exploited.
With this intention monoesters of curcumin with glycine and
valine have been prepared and characterized.
A recent joint finding by US, Greek and Chinese workers
[29] has indicated that dimethyl curcumin (DMC), i.e., with
the two free phenolics methylated has better anticancer activ-
ity in vitro, i.e., in cultured human cancer cells. However, it
may be that mechanism of antioxidation and apoptosis is
entirely different. Regarding mechanism of apoptosis by cur-
cumin it is known that it takes place through mitochondrial
disintegration and release of cytochrome c through caspase
activation. However, still more work is required before defi-
nite conclusion can be reached. Free radical formation in
the case of DMC may be through another mechanism involv-
ing tertiary radicals. Keeping in mind the physiological activ-
ities of amino acids, glycine, glutamic acid and valine, the
mono- and diester with both phenolics of curcumin has now
been prepared, characterized and tested for antipathogenic
activities. The diester with demethylenated piperic acid has
been prepared to check the enhancement in bioavailability
due to availability of two free phenolics. These ester linkages
are biodegradable, therefore could get hydrolysed by esterase
OR
R O
1
2
III/ IV/ V
O
O
O
CH C Cl
CH2
CH
C
H2N
III: R =R =
N
1
2
(IIa)
CH2
O
CH2
CH2
C
O
COOH
O
OH
O
O
CH
C
H2N
H2N
IV: R =R =
1
2
N
CH C Cl
CH CH3
CH3
(IIb)
CH CH3
CH3
O
O
O
O
(IIc)
CH
H
C
V: R =R =
N
CH C Cl
1
2
H
O
Scheme 1. Synthesis of 4,40-(di-O-glutamoyl)-curcumin (III), 4,40-(di-O-vali-
noyl) curcumin (IV) and 4,40-(di-O-glycinoyl) curcumin (V).
Numerous studies suggest that Trikatu, an ayurvedic prep-
aration containing black pepper (Piper nigrum), long pepper
(Piper longum) and ginger (Zingiber officinalis), has a bioavail-
ability-enhancing effect [22]. Since curcumin belongs to the
same family as ginger, it has similar enhancing activity [23].
It has been shown that curcumin acts in both ways, i.e., as a an-
tioxidant (antiproliferative) and as a good oxidant and causes
apoptosis of a number of different cancerous cells in vitro
[20,22,23]. We have earlier studied the piperic acid conjugate
of curcumin [19,20]. However, we have now used demethylen-
ated piperic acid for making a conjugate in order to assess the
role of methylenedioxy group and whether free phenolics fur-
ther enhance the activity by providing anchor with the active
site of receptor.
The traditional knowledge that black pepper (piperine) when
mixed with turmeric (curcumin) enhances the activity of latter
several times led us to link the two covalently.
Valine is an essential, naturally occurring non-polar, hydro-
phobic, aliphatic amino acid. During the period of valine
deficiency, all of the other amino acids (and proteins) are
less well absorbed by the GI tract but valine is
1. actively absorbed and used directly by muscle as an en-
ergy source;
2. not processed by the liver before entering the blood stream.

S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
1839
enzymes releasing curcumin at target site and thus these con-
jugates may be acting as prodrugs.
After cooling 0.1 ml of each dilution was added to the test tube
and the final volume was made up to 5.0 ml. The test tubes
were shaken to uniformly mix the inoculums with the broth.
The tubes were incubated at 37 ꢀC for 18 h. Appearance of
any turbidity shows that the compound is not able to inhibit
the growth of bacteria, while no turbidity indicates the inhibi-
tion of microorganism by the sample.
2. Materials and methods
All solvents used were triply distilled and autoclaved prior
to use. Curcumin, glycine, valine, glutamic acid and piperine
were purchased from Merck-Schuchardt, Germany. The
MullereHinton broth, agar and sterile discs were purchased
from Hi Media laboratory Ltd., Mumbai, India.
2.3. In vitro antifungal test
MullereHinton broth and agar have been selected for
testing aerobic and facultative anaerobic bacterial isolates;
for fastidious organisms such as Streptococci and Peptococci,
the agar was supplemented with 5% defibrinated blood. The
microsusceptibility test was standardized at pH 7.4, agare
broth was incubated in an ambient air incubator at 37 ꢀC.
The inoculum was prepared from broth culture that has
been incubated for 4e6 h, when growth was considered in
the logarithmic phase. Amoxyclav was taken as standard anti-
bacterial. Fluconazole was taken as a standard antifungal drug.
The microorganisms were obtained from a city clinic from the
diagnosed patients and were subcultured on Sabouraud dex-
trose agar slant and incubated at 30 ꢀC for 10e12 days. The
sterile discs with 6 mm diameter were further sterilized and
charged with compounds as per requirements. After drying
the discs were stored at 4 ꢀC.
For antifungal testing the compounds were prepared in
acetone at initial concentration of 120 mmol/ml. Aspergillus
fumigatus was obtained from patients. Candida albicans and
Candida parapsilosis were isolated from the patients having
chest infections on Sabouraud dextrose agar without antibi-
otics while Penicillium notatum was obtained from NCL
(National Chemical Laboratory), Pune, India.
A standardized inoculum was prepared by counting micro-
conidia; cultures were grown on Sabouraud dextrose agar for
48 h at 37 ꢀC. Sterile saline solution (0e85%) was added to
the slant and the culture was gently swabbed with cotton
tipped applicator to dislodge conidia from the hyphal mat.
The suspension was transferred to a sterile tube and volume
was adjusted to 5 ml with sterile saline solution. Sabouraud
dextrose agar was poured to depth of 5 mm in 90 mm Petri
dishes and stored at 4 ꢀC. The plates were dried, standardized
suspension was poured and uniformly spread by means of
swab sticks. The excess inoculum was decanted.
2.1. Determination of zone of inhibition by
KirbyeBauer’s method [30]
2.4. Cell culture
The antibacterial susceptibility test was done by deter-
mining the zone of inhibition by Kirby Bauer’s method. The
curcumin conjugates viz. IIIeV, IX, X, XVII and deme-
thylenated piperic acid (XV) were dissolved in acetone to
make a solution of 120 mmol/ml. From this stock solution se-
rial dilutions have been done to 20, 10, 5, 2.5 and 1.25 mmol/
ml in sterile test tubes. Sterilized filter discs were soaked
with some solutions and subsequently dried to remove
excess solvent. Different bacteria viz. Enterobacter cloacae,
Staphylococcus saprophyticus, Micrococci, Klebsiella aerugi-
nosa and Escherichia coli were selected and 1 ml of each bac-
terial broth culture was added in MeH plates and spreaded
with the help of sterile spreader. Charged sterile disks were
placed aseptically over the inoculated plates using the sterile
forceps. All the procedures were done under laminar flow.
Then the plates were incubated at 37 ꢀC for 24 h in upright
position. The zone of inhibition was measured by using the
scale.
KB cells and HeLa cells, obtained from NCCS (National
Centre for Cell sciences), Pune, India, were cultured in Dul-
becco’s modified Eagle’s medium (DMEM) (Biological Indus-
tries, Israel) supplemented with 10% heat inactivated fetal calf
serum (Biological Industries, Israel) and IX Penstrep antibiotic
solution (Biological Industries, Israel) and incubated in fully
humidified 5% CO2 incubator at 37 ꢀC.
2.5. MTT assay for cell proliferation
Both the types of cells were seeded at a density 5 ꢁ 103 per
well in 96-well plates (BD Falcon). For measuring the effect
of curcumin and its bioconjugates, the stock (10 mmol) was
diluted in complete media to its final concentrations. After
16 h of seeding old medium is replaced with new curcumin
and its conjugate added media. MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide; SigmaeAldrich,
USA) was added in each well making final concentration of
IX from the stock of 5 mg/ml (10ꢁ) and incubated in 5%
CO2 incubator at 37 ꢀC for further 4 h. After 4 h the medium
was removed and crystals were dissolved in 200 ml DMSO
(SigmaeAldrich, USA). MTT assay was performed after
48 h of transfection, optical density was measured with ELISA
plate reader (Biorad 840) at 570 nm. The comparative results
are shown in Figs. 1 and 2.
2.2. Determination of MIC by the microdilution broth
susceptibility test
Different concentrations (20, 10, 5, 2.5 mmol/ml) of all the
compounds were prepared in sterile dry test tubes to determine
minimum inhibitory concentration (MIC). Nutrient broth was
prepared using MullereHinton broth (M391) and 4.9 ml of
it was taken in each test tube and was sterilized after plugging.

1840
0.75
S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
d ppm ¼ 2.06e2.18 (m, 8H, 30,40-C of glutamate), 3.45 (m,
(III)
(XV)
(X)
(XVII)
(IV)
(I)
2H, CHeNH), 3.70 (s, 6H, eOCH3), 4.46 (s, 2H, C4eH), 4.09
(m, 2H, CHeNH2), 6.53 (d, 2H, C2eH and C6eH), 6.83e
7.11 (m, 6H, AreH), 7.54 (d, 2H, C1eH and C7eH), 11.2 (s,
2H, HOeC]O).
0.7
0.65
0.6
(V)
2.7. 1,7-Bis-(4-O-valinoyl-3-methoxy phenyl) 1,6-
heptadiene-3,5-dione (IV)
0.55
0.5
Curcumin (0.368 g, 1 mmol) was taken in dry pyridine and
mixed with N-phthaloyl-valinoyl chloride (0.53 g, 2 mmol)
and stirred at r.t. for 8 h. After completion of the reaction
as indicated by TLC, the reaction mixture was poured into
crushed ice and repeatedly extracted with ethyl acetate. The
organic layer was concentrated and treated with ammonia/
pyridine (9:1 v/v) to remove phthaloyl group. The organic
layer was concentrated and purified by silica gel column chro-
matography using DCM/methanol gradient. Yield: 45%
(0.254 g); Rf: 0.9 (DCM/MeOH 9:1); UV lmax (MeOH):
295 nm. Anal. Found: C, 65.74; H, 6.71; O, 22.60; N, 4.91.
Calcd. for C31H38O8N2: C, 65.71; H, 6.76; O, 22.59; N,
4.94%. 1H NMR (CDCl3) d ppm ¼ 1.03 (d, 12H, CHe
(CH3)2), 2.65 (m, 2H, 3 0C of valine), 3.70 (s, 6H, eOCH3),
4.09 (s, 2H, C4eH), 4.19 (m, 2H, CHeNH2), 6.53 (d, 2H,
C2eH and C6eH), 6.82e7.13 (m, 6H, AreH), 7.56 (d, 2H,
C1eH and C7eH).
0.45
0.4
0 µm
10 µm
20 µm
30 µm
40 µm
50 µm
100 µm
Concentration of conjugates
Fig. 1. Effect of curcumin bioconjugates on HeLa cancer cell lines (human
origin).
2.6. 1,7-Bis-(4-O-glutamoyl-3-methoxy phenyl)
1,6-heptadiene-3,5-dione (III)
Curcumin (0.368 g, 1 mmol) was taken in dry pyridine and
mixed with N-phthaloyl-glutamoyl chloride (0.55 g, 2 mmol)
and stirred at r.t. for 8 h. After completion of the reaction as
indicated by TLC, the reaction mixture was poured into crushed
ice and repeatedly extracted with ethyl acetate. The organic
layer was concentrated and treated with ammonia/pyridine
(9:1 v/v) to remove phthaloyl group. The organic layer was con-
centrated and purified by silica gel column chromatography us-
ing DCM/methanol gradient. Yield: 40% (0.250 g); Rf: 0.9
(DCM/MeOH 9:1); UV lmax (MeOH): 290 nm. Anal. Found:
C, 59.39; H, 5.40; O, 30.67; N, 4.40. Calcd. for C31H34O12N2:
2.8. 1,7-Bis-(4-O-glycinoyl-3-methoxy phenyl)
1,6-heptadiene-3,5-dione (V)
It was prepared as reported in Ref. [15].
2.9. Demethylenated piperic acid from piperic acid (XV)
1
Piperic acid (0.654 g, 3 mmol) was taken in DCM. PCl5
(0.762 g, 3.5 mmol) dissolved in DCM was added slowly.
The reaction mixture was refluxed for 2 h to give geminal di-
chloride. DCM was evaporated and the residue was taken in
water and again refluxed for 2 h to give catechol. The solution
was cooled and the solid precipitate was collected which was
again washed with cold water and crystallized from ethanol.
Yield: 80% (0.494 g); Rf: 0.46 (DCM/MeOH 9.5:0.5); m.p.:
148 ꢀC; UV lmax (MeOH): 340 and 250 nm. Anal. Found:
C, 64.00; H, 4.72; O, 31.10. Calcd. for C11H10O4: C, 64.07;
H, 4.75; O, 31.06. 1H NMR (CDCl3) d ppm ¼ 5.03 (s, 2H, ar-
omatic CeOH), 5.64 (d, 1H, eCH]CHeC]O), 6.38 (d, 1H,
eCH]C), 6.58e7.78 (m, 4H, olefinic and aromatic), 6.73 (d,
1H, AreH), 7.25e7.51 (d, 1H, CH]CH]C]O), 11.2 (s,
1H, HOeC]O).
C, 59.42; H, 5.47; O, 30.64; N, 4.47%. H NMR (CDCl3)
(III)
(XV)
(X)
(XVII)
(IV)
(I)
0.69
0.67
0.65
0.63
0.61
0.59
0.57
0.55
0.53
0.51
0.49
(V)
2.10. Demethylenated piperoyl chloride (XVI)
To demethylenated piperic acid (0.824 g, 4 mmol) was
added redistilled thionyl chloride slowly over a period of
45 min while heating and shaking in a water bath. The reaction
mixture was further refluxed for 30 min and thionyl chloride
was removed at reduced pressure. Yield: 60% (0.537 g).
0 µm
10 µm
20 µm
30 µm
40 µm
50 µm
100 µm
Concentration of conjugates
Fig. 2. Effect of curcumin bioconjugates on KB cancer cell lines (human
origin).

S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
1841
2.11. 1,7-Bis-[4-O-demethylenated piperoyl-
3-methoxyphenyl]-1,6 heptadiene-3,5-dione (XVII)
6. The supernatant was removed and chloroacetic acid
derivatised LCAAeCPG was washed with dry DMF,
methanol followed by ether (2 ꢁ 3 ml). The derivatisation
of chloroacetic acid as linker to LCAAeCPG was con-
firmed by ninhydrin test of the solid support which was
negative indicating no free amino group and was ready
for further synthesis.
7. Sodium salt of curcumin (2 mmol) dissolved in dry pyri-
dine (10 ml) was passed through column repeatedly and
was kept overnight.
Curcumin (0.736 g, 2 mmol) was taken in dry pyridine
and mixed with demethylenated piperoyl chloride (1.008 g,
4.5 mmol) and stirred at r.t. for 6 h. After the completion of
reaction as indicated by TLC, the reaction mixture was poured
onto crushed ice and repeatedly extracted with EtOAc. The
organic layer was concentrated and purified on a silica gel col-
umn using a DCM/methanol gradient. Yield: 45% (0.667 g);
Rf: 0.74 (DCM/MeOH 9.5:0.5); UV lmax (MeOH): 395 and
295 nm. Anal. Found: C, 69.34; H, 4.83; O, 25.80. Calcd for
8. The column was washed with pyridine, methanol followed
by water (2 ꢁ 5 ml). It was again washed with methanol
and ether (2 ꢁ 2 ml).
1
C43H36O12: C, 69.29; H, 4.87; O, 25.78. H NMR (CDCl3)
d ppm ¼ 3.65 (s, 6H, eOCH3), 4.15 (s, 2H, C4eH), 4.65 (d,
2H, O]CeCH2), 5.03 (s, 4H, aromatic CeOH), 5.65 (d, 2H,
eCH]CHeC]O), 6.38 (d, 2H, eCH]C), 6.72e7.80 (m,
8H, olefinic and aromatic), 6.90 (d, 1H, AreH), 7.18e7.48
(d, 2H, CH]CH]C]O), 7.48 (d, 2H, C1eH and C7eH).
9. Phthaloyl glycinoyl chloride (2 mmol) dissolved in pyri-
dine (5 ml) was passed through column with little addition
(5 mg) of DMAP as catalyst and was kept overnight.
10. Column was washed with water (2 ꢁ 2 ml) followed by
methanol (2 ꢁ 2 ml).
11. To the column was added 5 ml solution of ammonia and
pyridine (ammonia/pyridine 9:1) and was kept for 5e
10 min.
2.12. p-Nitrophenyl ester of chloroacetic acid
12. Column was again washed with water (2 ꢁ 2 ml) followed
To chloroacetic acid (0.190 g, 2 mmol) dissolved in dry
dioxane (8 ml), p-nitrophenol (0.280 g, 2 mmol) was added
dropwise. The reaction mixture was made basic by the addi-
tion of 0.5 ml pyridine and 0.5 ml TEA (triethylamine). After
stirring for 10 min dicyclohexyl carbodiimide (DCC) (1.03 g,
5 mmol) was added. The reaction mixture was stirred for 2 h
under nitrogen atmosphere (caution: protection from moisture)
and monitored on TLC. The completion of reaction was
assessed by total consumption of starting reagent. Rf ¼ 0.88
(DCM/MeOH 9.5:0.5).
by methanol (2 ꢁ 2 ml).
13. HI (2 ml) was added to the column and kept for 12 h.
14. The monoester was eluted from the solid support with
methanol along with HI, and methanol was distilled off.
15. The remaining DCM solution was added and it was
washed with aqueous KI (10%) and DCM repeatedly until
all the colours were removed from DCM layer to remove
any I2 and compound was extracted with DCM.
16. The DCM solution was washed with cold 5% aq. sodium
bicarbonate (10 ml) solution followed by water (10 ml) to
remove any HI.
Steps involved in solid phase synthesis of monoesters of
curcumin
17. It crystallized from DCM.
1. suspended dried long chain alkyl amine on CPG resin
(LCAAeCPG) (200 mg, loading 60 mmol/100 mg, 120
2.13. Monoglycinoyl curcumin (IX)
˚
mmol of NH2 gp, pore size 500 A) in dry dimethylforma-
mide (DMF) (2 ml) and loaded on a column.
2. Dissolved chloroacetic acid (0.190 g, 2 mmol) in dry
dioxane (8 ml) containing dry pyridine (0.5 ml) and p-
nitrophenol (0.280 g, 2 mmol) and stirred for 20 min in
a round bottom flask.
Yield: 50% (0.025 g); UV lmax (MeOH): 360 and 295 nm;
Rf: 0.6 (DCM/methanol 9.5:0.5). The pure product was charac-
terized by Anal. Found: C, 64.94; H, 5.41; O, 26.35; N, 3.30.
Calcd. for C23H23O7N: C, 64.93; H, 5.45; O, 26.33, N, 3.29.
1H NMR (CDCl3) d ppm ¼ 3.70 (s, 6H, eOCH3), 4.13 (s,
2H, C4eH), 4.49e4.61 (m, 4H, CH2eNH2), 5.03 (s, 1H, eOH),
6.53 (d, 2H, C2eH and C6eH), 6.85e7.08 (m, 6H, AreH),
7.53 (d, 2H, C1eH and C7eH).
3. To it was added dicyclohexyl carbodiimide (DCC)
(1.030 g, 5 mmol). After few minutes dicyclohexylurea be-
gan to precipitate. The reaction mixture was stirred for
2.5 h under nitrogen atmosphere (caution: protection from
moisture) and monitored on TLC. The completion of reac-
tion was assessed by total consumption of starting reagent.
Rf ¼ 0.88 (DCM/MeOH 9.5:0.5).
2.14. Monovalinoyl curcumin (X)
Monovalinoyl curcumin was also prepared by protocol as
stated above. During the synthesis the amino function of valine
was protected with phthalic anhydride as N-phthaloyl glycine
and the carboxy function was activated with thionyl chloride
to get the corresponding acid chlorides. This N-protected
and carboxy activated amino acid was reacted with sodium
salt of curcumin on solid support to give the ester of valine
with curcumin. The phthaloyl group of protected amino acid
4. Removed dicyclohexylurea by filtration and added the
supernatant to LCAAeCPG (200 mg) suspended in
DMF on column.
5. To it was added 0.5 ml of triethylamine (TEA); a bright
yellow colour due to the release of p-nitrophenol devel-
oped immediately. The reaction mixture was kept for
8 h.

1842
S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
was removed using ammonia. HI was used to deblock the
monoester from the solid support by cleaving the ethereal
bond and thus selective esterification of curcumin was accom-
plished. UV lmax (MeOH): 370 and 295 nm; Rf: 0.68 (DCM/
methanol 9.5:0.5). The pure product was characterized by
Anal. Found: C, 66.81; H, 6.20; O, 23.99; N, 2.99. Calcd.
for C26H29O7N: C, 66.80; H, 6.25; O, 23.96; N, 3.0. 1H
NMR (CDCl3) d ppm ¼ 1.03 (d, 6H, CHe(CH3)2), 2.65 (m,
H, 3 ꢀC of valine), 3.70 (s, 6H, eOCH3), 4.09 (s, 2H, C4eH),
4.19 (m, H, CHeNH2), 5.03 (s, 1H, eOH), 6.53 (d, 2H,
C2eH and C6eH), 6.82e7.13 (m, 6H, AreH), 7.56 (d, 2H,
C1eH and C7eH).
During the synthesis of diesters, the eNH2 group of amino
acids (glycine, valine and glutamic acid) was protected as
respective N-phthaloyl derivatives. The carboxyl group was
activated by treating with thionyl chloride to get the corre-
sponding acid chlorides. Curcumin was taken in dry pyridine
and treated with N-phthaloyl chlorides of respective amino
acids in 1:2.5 molar proportion and the mixture was stirred
for 6 h. Phthaloyl group was removed using ammonia and
the product was purified using column chromatography
(Scheme 1).
Synthesis of monoesters of curcumin, a symmetrical diphe-
nolic compound with glycine/valine has been carried out by
anchoring one of its free phenolic group to an insoluble poly-
meric solid support resin (CPGeLCAA) via a 2-carbon linker
by solid phase. However, the protocol of reactions used was
the same as that used in the case of diesters, but due to changed
kinetics of solid phase reactions, the reaction conditions vary
(Scheme 2).
3. Results and discussion
Different curcumin conjugates viz. 4,40-(di-O-glutamoyl)-
curcumin (III), 4,40-(di-O-valinoyl) curcumin (IV), 4,40-
(di-O-glycinoyl) curcumin (V), monoglycinoyl curcumin
(IX), monovalinoyl curcumin (X), 4,40-(di-O-demethylenated
piperoyl) curcumin (XVII) and demethylenated piperic acid
(XV) were prepared and these were tested for their antimicro-
bial activities vis-a-vis curcumin and piperic acid. The mono-
esters of curcumin have been prepared for the first time
which have one phenolic free while the other was attached
with suitable ligand.
One of the phenolic groups of curcumin was attached to
LCAAeCPG (long chain alkyl amine controlled pore glass)
via chloroacetic acid as a linker; the carboxy function of
chloroacetic acid was esterified (activated) by reaction with
p-nitrophenol by the addition of dicyclohexyl carbodiimide
(DCC) in the presence of pyridine and triethylamine to make
the medium basic to get the corresponding activated ester.
Curcumin
CPG-LCAA
activated ester of chloroacetic acid
/ DCC/DMAP
2N aq NaOH
O
O
H3CO
OCH3
H
N
(I)
ONa
CPG-LCA
Cl NaO
O
i. Pyridine/ 8h / r.t.
O
O
H3CO
O
OCH3
ONa
H
N
(VI)
CPG-LCA
i. N-Phthaloyl glycinoyl chloride / N-Phthaloyl
valinoyl chloride DMAP/ overnight/ r.t
O
ii. ammonia: pyridine (9:1 v/v) / 5 min
O
O
H3CO
OCH3
O
H
N
NH2
(VII/ VIII)
CPG-LCA
O
O
O
O
R
HI / 37% / 2 ml
O
O
H3CO
OCH3
(IX / X)
H
O
NH2
N
CH2I +HO
Monoester of curcumin
CPG-LCA
O
R
IX: R=H
CPG--LCAA
= Long chain alkylamine Contreolled pore glass
X: R= CH (CH )
3 2
Scheme 2. Synthesis of monoglycinoyl curcumin (IX)/monovalinoyl curcumin (X) on CPGeLCAA.

S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
1843
The activated ester of chloroacetic acid was reacted with
O
C
amino function of LCAAeCPG using DCC/DMAP to get
the amide bond, a well-established strategy in peptide synthe-
sis. To this was added sodium salt of curcumin, a symmetrical
diphenolic compound, which reacts with chloroacetic acid de-
rivatised LCAAeCPG, such that one phenolic forms the bond
while the other remains free for further synthesis.
O
O
N
CH
CH CH
CH
i. Ethanolic KOH (2N ); 2h reflux / HCl
XI
O
O
O
The N-phthaloyl chlorides of glycine and valine were re-
acted with sodium salt of curcumin on solid support to give
the corresponding monoesters of curcumin. The phthaloyl
group was removed using ammonia. HI was used to deblock
the monoester from the solid support by cleaving the ethereal
bond and thus selective esterification of curcumin with glycine
and valine was accomplished (Scheme 2).
In summary we have developed a novel strategy for the
selective esterification of symmetrical diphenolic compound
like curcumin using solid phase synthesis. This strategy can
be used further for selective synthesis of monoesters of any
symmetrical diphenolic compound. Another advantage of the
procedure is that after deblocking the resin is left activated,
i.e., with a eCOeCH2I group attached to its terminal NH2
group and can be used as such for another cycle.
CH
CH CH
CH
C OH +
HN
PCl / DCM/ Reflux / 2h
5
XII
O
Cl
O
O
CH
CH CH
CH
C OH
Cl
XIII
H O
2
O
O
O
CH
CH CH
CH
OH
C
O
C
Piperic acid was obtained by alkali hydrolysis of piperine
and selective cleavage of methylenedioxy group was accom-
plished by traditional method of halogenation followed by
hydrolysis [31,32]. Halogenation was done with phosphorus
pentachloride to give geminal dichloride and the subsequent
hydrolysis to catechol (Scheme 3).
H O/ Bicarbonate
XIV
2
O
C
HO
HO
OH
CH
CH CH
CH
Curcumin was directly attached to demethylenated piperic
acid [19,20] (Scheme 4). Different conjugates of curcumin
viz. 4,40-(di-O-valinoyl) curcumin, 4,40-(di-O-glutamoyl) cur-
cumin, 4,40-(di-O-demethylenated piperoyl) curcumin and
monoesters of curcumin with glycine and valine were prepared
and characterized.
XV
Scheme 3. Synthesis of demethylenated piperic acid.
Micrococcus and E. cloacae while Amoxyclav, the best
marketed antibiotic, shows MIC of 10 mmol/ml showing that
monovalinoyl curcumin (X) is four times more effective than
Amoxyclav, a marketed drug at the similar concentrations.
3.1. Antibacterial activity
Susceptibility test in vitro was performed on multiresistant
bacteria specially causing secondary infection in human be-
ings, e.g. E. cloacae, S. saprophyticus, Micrococci, K. aerugi-
nosa and E. coli. The results are tabulated in Tables 1 and 2.
The antibacterial activity of curcumin bioconjugates in-
cluding diesters as well as monoesters was compared with
curcumin itself along with demethylenated piperic acid and
its conjugate with curcumin by microdilution broth suscepti-
bility test method. The lowest concentration of curcumin
bioconjugates in mmol/ml that prevented in vitro growth of
microorganism has been represented as correlated with zone
of inhibition Table 1 and MIC (minimum inhibitory concen-
tration) shown in Table 2.
Each test was performed in triplicate and the MICs reported
represent the result of at least two repetitions. Six conjugates
(IIIeV, IX, X, and XVII) show good positive result on multi-
resistant organisms while demethylenated piperic acid (XV)
with methylenedioxy ring open shows better results than pipe-
ric acid (XI). The most encouraging results were obtained in
the case of monoesters (X) having MIC of 2.5 mmol/ml against
O
O
H3CO
HO
OCH3
OH
(I)
i) Pyridine / demethylenated
piperoyl chloride (XVI)
/ 6h / r.t.
O
O
H3CO
RO
OCH3
(XVII)
OR
HO
HO
XVII: R=A
CH CH CH CH
C
O
=A
Scheme 4. Synthesis of curcumin demethylenated piperoyl conjugate (XVII).

1844
S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
Table 1
Antibacterial activity of curcumin bioconjugates (numerals show zone of inhibition in mm) for concentrations (20, 10, 5, 2.5 mmol/ml) of each bioconjugate against
bacterial strains
Name of bacteria
I
III
IV
V
IX
X
XI
XV
XVII
16,12
Micrococci
e
e
e
e
10
18,16
9
16,13
e
14,12
20
20
15,13,10
e
14,12,10
e
26,20,19
18
e
8
15
e
10
18
e
Klebsiella aeruginosa
Staphylococcus saprophyticus
Enterobacter cloacae
Escherichia coli
18
e
11
10
e
e
25
e
e
e
e
12
e
e
12
18,14,12
16
e
13,11
(e) Resistant; curcumin (I); 4,40-(di-O-glutamoyl)-curcumin (III); 4,40-(di-O-valinoyl) curcumin (IV); 4,40-(di-O-glycinoyl) curcumin (V); monoglycinoyl curcu-
min (IX); monovalinoyl curcumin (X); piperic acid (XI); demethylenated piperic acid (XV); 4,40-(di-O-demethylenated piperoyl) curcumin (XVII). Values given
in bold indicate significant inhibition obtained.
The result of zone of inhibition are also encouraging. The
disc containing 20 mmol of Amoxyclav was purchased and
equal amount of conjugates were loaded on separate discs.
The zone of inhibition of Amoxyclav was 20 mm while Mono-
valinoyl curcumin (X) had zone of 26 mm. The conjugates III,
IV, IX and XVII showing MIC of 5 mmol/ml were also com-
parable to Amoxyclav which shows MIC of 10 mmol/ml.
These results suggest that diesters of curcumin have more
antibacterial activity than curcumin itself which may be due
to their increased solubility, better cellular uptake (bioavail-
ability) and slow down of metabolic process due to masking
of free phenolics. However, monoesters have even better activ-
ity than their corresponding diesters since monoesters have
both the advantages, i.e., as a ligand helping in cellular uptake
and a free phenolic for binding at active site. The role of free
phenolics in binding and transport is further clear from the en-
hanced activity of the conjugate having demethylenated pipe-
ric acid in which methylenedioxy ring was open as compared
to that of the corresponding piperic acid.
(1) enhanced metabolic stability due to masking of phenolic
hydroxyl groups and delay in their glucuronide formation
during metabolism,
(2) better cellular uptake due to the transportation of the con-
jugate via the amino acid carrier protein, i.e., drug smug-
gling, and
(3) more solubility of the conjugates because of enhancement
in polar character (hydrophilicity).
Since, the transporter proteins for many amino acids as well
as peptides are known, the monoesters of curcumin with these
amino acids or peptides can act as substrates for the carrier
proteins. ABTOþ is one such example which is a known
substrate for monovalinoyl curcumin and can be used for
characterizing the ester by transporter expression system.
However, these transport proteins for amino acids and peptides
are known to be very promiscuous and can transport amino
acid esters of many other compounds also.
All the covalent bonds synthesized are biodegradable, i.e.,
hydrolysable with common enzymes present in living sys-
tems. This was checked in vitro by treating curcumineglycine
conjugate with chymotrypsin when complete hydrolysis was
observed. This makes these derivatives as potent prodrugs
which can get hydrolysed at the target sites. The amino acids
are known to be transported through carrier proteins associ-
ated with cellular uptake and were linked with curcumin in
order to facilitate the cellular uptake through receptor medi-
ated endocytosis.
3.2. Antifungal activity of curcumin bioconjugates
We have also evaluated the antifungal activity of curcumin
and its bioconjugates against A. fumigatus, C. albicans (yeast),
C. parapsilosis and P. notatum. The antifungal activity of cur-
cumin bioconjugates was compared with the standard drug flu-
conazole where it was found comparable to C. albicans (yeast
form). From the results shown in Table 3 we can infer that
monoglycinoyl curcumin (IX) shows better activity against
A. fumigatus, C. albicans (yeast), C. parasilosis and P. nota-
tum, than its corresponding diester 4,40-(di-O-glycinoyl) cur-
cumin (V) vis-a-vis curcumin (I). Similarly demethylenated
piperic acid (XV) shows better results than piperic acid (XI)
in the case of C. albicans emphasizing the role of free phenolic
groups as we have seen in antibacterial activity. The results are
significant as fungal diseases are stubborn to most of the anti-
fungal drugs.
The enhancement in antimicrobial activity of these curcu-
min bioconjugates may be due to
Table 2
MIC (minimum inhibitory concentration) correlation diagram (in mmol/ml) of
bioconjugates against bacterial strains
Name of bacteria
I
III IV V IX X XI XV XVII Amoxyclav
Micrococci
Klebsiella aeruginosa R 20
e
5
10 10 R 2.5 e 10 10
10
12
10
R
10
R
R
5
5
20
R
R 20
20 20
Staphylococcus
saprophyticus
e
R
R
R
3.3. Anticancer activity of curcumin bioconjugates on
human cancer cell lines, HeLa (cervical cancer) and KB
(oral cancer)
Enterobacter cloacae
Escherichia coli
e R
20 R
5
5
R
R
20 20 10
2.5 e 10
R
5
10
10
10
R
(R) Resistant; curcumin (I); 4,40-(di-O-glutamoyl)-curcumin (III); 4,40-(di-O-
valinoyl) curcumin (IV); 4,40-(di-O-glycinoyl) curcumin (V); monoglycinoyl
curcumin (IX); monovalinoyl curcumin (X); piperic acid (XI); demethylen-
ated piperic acid (XV); 4,40-(di-O-demethylenated piperoyl) curcumin
(XVII). Values given in bold indicate significant inhibition obtained.
Antiproliferative property of curcumin is very well known.
In traditional medicine also turmeric is not given to pregnant
woman, as it may have detrimental effect on growth of fetus.
Recent evidences suggest that the antiproliferative property is

S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
1845
Table 3
Antifungal activity of curcumin bioconjugates (numerals show zone of inhibition in mm) in concentrations (20, 10, 5, 2.5 mmol/ml) of each bioconjugate against
fungal strains (fluconazole concentration was 20 mmol/ml)
Name of fungi
I
III
IV
V
IX
X
XI
XV
XVII
Fluconazole
Aspergillus fumigatus
Candida albicans
Candida parapsilosis
Penicillium notatum
e
15
e
e
e
e
e
e
e
e
e
e
e
e
e
e
25, 20
20, 18
14
e
e
e
12
e
e
e
e
e
16
e
e
e
16
e
e
e
29
20
e
20, 16
(e) Resistant; curcumin (I); 4,40-(di-O-glutamoyl)-curcumin (III); 4,40-(di-O-valinoyl) curcumin (IV); 4,40-(di-O-glycinoyl) curcumin (V); monoglycinoyl curcu-
min (IX); monovalinoyl curcumin (X); piperic acid (XI); demethylenated piperic acid (XV); 4,40-(di-O-demethylenated piperoyl) curcumin (XVII). Values given
in bold indicate significant inhibition obtained.
due to its ability to induce apoptosis. However, the molecular
mechanism through which curcumin induces apoptosis is not
fully understood.
to suitable ligands which get internalized with the cellular en-
vironment can enhance their therapeutic activity multiple
times. Use of biodegradable linkages for attaching the ligands
makes these conjugate ‘‘prodrugs’’, i.e., a significant concen-
tration can be built up inside the infected cells and then can
get hydrolysed to release the active component at the target
site. However, still the toxicity of these bioconjugates has to
be tested in vivo.
How curcumin conjugates produce therapeutic effects is
not fully understood, but they are probably mediated in part
through the antioxidant and anti-inflammatory actions of cur-
cumin. It is quite likely that curcumin mediates its effects
through other mechanisms as well. Over a dozen different
cellular proteins and enzymes have been identified to which
curcumin binds.
However, curcumin does have apoptotic effect on cancer-
ous cells through mitochondria and the process occurs through
activation of different caspases. Here, we have assessed the
anticancerous properties of curcumin bioconjugates against
human cancer cell lines, HeLa (Fig. 1) and KB (Fig. 2).
In the present study curcumin diglutamoyl derivative was
found to be more potent against cancer cell lines, HeLa and
KB, than other derivatives, more over the monoester of curcu-
min with valine and the corresponding diester showed almost
similar results in HeLa cells. The demethylenated piperic acid
shows better results than the curcuminepiperic acid conjugate
at higher concentration in HeLa cells.
In the case of KB cells similar pattern was observed show-
ing highest activity in the case of curcumin diglutamoyl deriv-
ative while monoester of curcumin with valine shows less
activity than the corresponding diester of valine with curcumin.
Generation of free radicals has been shown to be responsi-
ble for cell death. In several apoptotic models, increased gen-
eration of ROS was described as an early event; in addition
enhanced ROS formation and impairment of the cellular anti-
oxidant mechanism may also lead to cellular apoptosis.
We have also found that diesters show better anticancerous
property than their corresponding monoesters, and best result
was seen in diglutamoyl curcumin (III), divalinoyl curcumin
(IV) and diglycinoyl curcumin (V) esters. However, it may
be that apoptosis induction is due to the activation of caspases
which is facilitated due to accumulation and better stability of
diglutamic curcumin and other diesters of curcumin. However,
further experiments are needed to confirm the hypothesis.
The better results in the case of curcumin bioconjugates are
probably due to their better solubility, enhanced cellular
uptake through carrier proteins and decreased metabolic deg-
radation. Though our work is still in its infancy our results
cannot be ignored. It opens a new era for exploring suitably
designed curcumin bioconjugates as potential antibacterial,
antifungal and antiproliferative drugs. Exhaustive work based
on the designing and testing of the conjugates according to the
molecular organization of several pathogens and cell lines is
required before any definite conclusion can be reached. How-
ever, further studies are needed to evaluate the in vivo activity
and selectivity of the compounds presented.
A large number of studies unequivocally identified the
numerous pharmaceutical actions of curcumin, its acceptance
as a ‘wonder compound’ is slowly forthcoming. Curcumin has
a plethora of beneficial effects and certainly qualifies for seri-
ous consideration as a pharmaceutical/nutraceutical/phyto-
ceutical agent.
4. Conclusion
Acknowledgement
The advantage with herbal drugs is that their target proteins
are already known; moreover, these are not toxic to living sys-
tems. Therefore, it is advantageous to start with a food compo-
nent, which can well internalize with the cellular environment,
and subsequently modify it chemically to suit the requirement.
This is what we have achieved in the present work, i.e., assess-
ment of bioconjugates of curcumin with appropriate biomole-
cules which can help in transporting these molecules to their
specific targets for their therapeutic importance. Chemical
modification of the herbal products, specially their attachment
One of the authors (S.K.D.) wishes to thank UGC, India for
providing financial assistance by a scholarship during the
course of this study.
References
[1] C. Ramachandran, H.B. Fonseca, P. Jhabvala, E.A. Escalon, S.J. Melnick,
Cancer Lett. 184 (2002) 1e6.
[2] K. Mehta, P. Pantazis, T. McQueen, B.B. Aggarwal, Anti-Cancer Drugs 8
(1997) 470e481.

1846
S.K. Dubey et al. / European Journal of Medicinal Chemistry 43 (2008) 1837e1846
[3] R.J. Anto, A. Mukhopadhyay, K. Denning, B.B. Aggarwal, Carcinogen-
esis 23 (1) (2002) 143e150.
[20] S. Mishra, N. Kapoor, A.A. Mubarak, B.V.V. Pardharasaradhi, L.A. Kumari,
A. Khar, K. Misra, Free Radical Biol. Med. 38 (2005) 1353e1360.
[21] N. Kapoor, A.K. Sharma, V. Dwivedi, A. Kumar, K. Misra, U. Pati,
Cancer Lett. 248 (2007) 245e250.
[4] Z. Sui, R. Salto, J. Li, C. Craik, P.R. Ortiz de Montellano, Bioorg. Med.
Chem. 1 (6) (1993) 415e422.
[5] S. Barthelemy, L. Vergnes, M. Moynier, D. Guyot, S. Labidalle,
E. Bahraoui, Res. Virol. 149 (1) (1998) 43e52.
[22] R.K. Johri, U. Zutshi, J. Ethnopharmacol. 37 (2) (1992) 85e91.
[23] G. Shoba, D. Joy, T. Joseph, M. Majeed, R. Rajendran, P.S. Srinivas,
Planta Med. 64 (4) (1998) 353e356.
[6] A. Mazumder, N. Neamati, S. Sunder, J. Schulz, H. Pertz, E. Eich,
Y. Pommier, J. Med. Chem. 40 (19) (1997) 3057e3063.
[7] A.C. Bharti, N. Donato, S. Singh, B.B. Aggarwal, Blood 101 (3) (2003)
1053e1062.
[24] P.J. Reeds, D.G. Burrin, B. Stoll, F. Jahoor, J. Nutr. 130 (4) (2000)
978e982.
[25] S. Okumoto, L.L. Looger, K.D. Micheva, Proc. Natl. Acad. Sci. U.S.A.
102 (24) (2005) 8740e8745.
[8] A.S. Baldwin, J. Clin. Invest. 107 (3) (2001) 241e246.
[9] H.L. Pahl, Oncogene 18 (49) (1999) 6853e6866.
[10] S. Shishodia, P. Potdar, C.G. Gairola, B.B. Aggarwal, Carcinogenesis 24
(7) (2003) 1269e1279.
[26] Shishir Shishodia, Krishna Misra, Bharat B. Aggarwal, Turmeric as cure
(cumin): promises, problems, and solutions as an invited review for the
book, in: Young-Joon Surh, Lester Packer (Eds.), Dietary Modulation
of Cell Signaling, CRC Press, 2007.
[11] R.E. Harris, G.A. Alshafie, H. Abou-Issa, K. Seibert, Cancer Res. 60 (8)
(2000) 2101e2103.
[12] R. Mohan, J. Sivak, P. Ashton, L.A. Russo, B.Q. Pham, N. Kasahara,
M.B. Raizman, M.E. Fini, J. Biol. Chem. 275 (14) (2000) 10405e10412.
[13] M.F. Iademarco, J.L. Barks, D.C. Dean, J. Clin. Invest. 95 (1) (1995)
264e271.
[27] H. Ohtsu, Z. Xiao, J. Ishida, M. Nagai, H.K. Wang, H. Itokawa, C.Y. Su,
C. Shih, T. Chiang, E. Chang, Y. Lee, M.Y. Tsai, C. Chang, K.H. Lee,
Antitumor agents. 217. Curcumin analogues as novel androgen receptor
antagonists with potential as anti-prostate cancer agents, J. Med. Chem.
45 (23) (2002) 5037e5042.
[14] S. Kumar, K.K. Dubey, S. Tripathi, M. Fuji, K. Misra, Nucleic Acids
Symp. Ser. 44 (2000) 52e53.
[28] L. Lin, Q. Shi, A.K. Nyarko, K.F. Bastow, C.C. Wu, C.Y. Su, C.C. Shih,
K.H. Lee, Antitumor agents. 250. Design and synthesis of new curcumin
analogues as potential anti-prostate cancer agents, J. Med. Chem. 49 (13)
(2006) 3963e3972.
[15] S. Kumar, U. Narain, S. Tripathi, K. Misra, Bioconjugate Chem. 12 (4)
(2001) 464e469.
[16] S. Kumar, A. Misra, S. Tripathi, K. Misra, Nucleic Acids Symp. Ser.
(2001) 137e138.
[29] C. Tomovakopoulous, K. Dimas, Z.D. Sofianos, S. Hatziantoniou,
Z. Han, Z.L. Liu, J.H. Wyche, P. Pomtazis, Clin. Cancer Res. 13 (4)
(2007) 1269e1277.
[30] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turk, Am. J. Pathol. 45 (1966)
493.
[17] S. Kumar, V. Shukla, A. Misra, S. Tripathi, K. Misra, Indian J. Biotech-
nol. 1 (2002) 158e163.
[18] S. Mishra, S. Tripathi, K. Misra, Nucleic Acids Res. Suppl. 2 (2002)
2777e2778.
[19] S. Mishra, U. Narain, R. Mishra, K. Misra, Bioorg. Med. Chem. 13 (5)
(2005) 1477e1486.
[31] J.S. Buck, Zimmermann, in: A.H. Blatt (Ed.), Organic Synthesis, vol. 2,
John Wiley and Sons, Inc., New York, 1943, pp. 549e550.
[32] G.L. Trammeli, Tetrahedron Lett. 18 (1978) 1525e1528.