Design, development and synthesis of mixed bioconjugates of piperic acid-glycine, curcumin-glycine/alanine and curcumin-glycine-piperic acid and their antibacterial and antifungal properties

Article abstract of DOI:10.1016/j.bmc.2004.12.057

In the present communication different curcumin bioconjugates viz. 4,4′-di-O-glycinoyl-curcumin, 4,4′-di-O-d-alaninoyl-curcumin, 4,4′-di-O-(glycinoyl-di-N-piperoyl)-curcumin, 4,4′-di-O-piperoyl curcumin, curcumin-4,4′-di-O-β-D-glucopyranoside, 4,4′-di-O-acetyl-curcumin along with piperoyl glycine, have been synthesised and characterised by spectra UV, ¹H NMR and elemental analysis. All the covalent bonds used are biodegradable. This makes these derivatives as potent prodrugs, which can get hydrolysed at the target sites. These bioconjugates were tested in vitro against different bacteria and fungi. The 4,4′-di-O-(glycinoyl-di-N-piperoyl)-curcumin and 4,4′-di-O- acetyl-curcumin are more effective than Cefepime, an antibacterial drug available in market, at the same concentration. The 4,4′-di-O-(glycinoyl- di-N-piperoyl)-curcumin and 4,4′-di-O-piperoyl curcumin had antifungal activity in vitro almost comparable with fluconazole, the most popular antifungal drug. The enhanced activity of these bioconjugates vis-a-vis the parent molecule that is curcumin may be due to improved cellular uptake or reduced metabolism of these bioconjugates resulting in building up of enough concentration inside the infected cells. It opens a new era for exploring suitably designed curcumin bioconjugates as potential antibacterial/antifungal drugs.

Full text of DOI:10.1016/j.bmc.2004.12.057

Bioorganic & Medicinal Chemistry 13 (2005) 1477–1486
Design, development and synthesis of mixed bioconjugates of piperic
acid–glycine, curcumin–glycine/alanine and curcumin–glycine–
piperic acid and their antibacterial and antifungal properties
Satyendra Mishra, Upma Narain, Roli Mishra and Krishna Misra*
Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad 211002, India
Received 4 November 2004; revised 18 December 2004; accepted 19 December 2004
Available online 25 January 2005
Abstract—In the present communication different curcumin bioconjugates viz. 4,40-di-O-glycinoyl-curcumin, 4,40-di-O-D-alaninoyl-
curcumin, 4,40-di-O-(glycinoyl-di-N-piperoyl)-curcumin, 4,40-di-O-piperoyl curcumin, curcumin-4,40-di-O-b-D-glucopyranoside,
1
4,40-di-O-acetyl-curcumin along with piperoyl glycine, have been synthesised and characterised by spectra UV, H NMR and ele-
mental analysis. All the covalent bonds used are biodegradable. This makes these derivatives as potent prodrugs, which can get
hydrolysed at the target sites. These bioconjugates were tested in vitro against different bacteria and fungi. The 4,40-di-O-(glyci-
noyl-di-N-piperoyl)-curcumin and 4,40-di-O-acetyl-curcumin are more effective than Cefepime, an antibacterial drug available in
market, at the same concentration. The 4,40-di-O-(glycinoyl-di-N-piperoyl)-curcumin and 4,40-di-O-piperoyl curcumin had anti-
fungal activity in vitro almost comparable with fluconazole, the most popular antifungal drug. The enhanced activity of these bio-
`
conjugates vis-a-vis the parent molecule that is curcumin may be due to improved cellular uptake or reduced metabolism of these
bioconjugates resulting in building up of enough concentration inside the infected cells. It opens a new era for exploring suitably
designed curcumin bioconjugates as potential antibacterial/antifungal drugs.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tions in the Ames assay and the formation of tumour
in several experimental systems.11–16 As a known antiox-
idant, curcumin exhibits antiproliferative capability and
thus is identified as a potent tool in cancer therapy.17–20
Curcumin has also been reported to decrease total cho-
lesterol and LDL cholesterol level in serum and to in-
crease the beneficial HDL cholesterol level.21 It has
been claimed that curcumin inhibits HIV-I integrase
protein, thus potentially preventing HIV-I from infect-
ing CD-4 and CD-8 cells.22 The other attractive features
of curcumin to explore, as a vulnerary agent is that de-
spite being eaten daily for centuries in Asian countries,
curcumin has not been reported to be toxic.2
Turmeric (Curcuma longa), a common Indian dietary
pigment and spice has been shown to posses a wide
range of therapeutic utilities in the traditional Indian
medicine for external/internal wounds, liver diseases
(particularly jaundice), blood purification, inflamed
joints (rheumatoid arthritis).1–5 Curcumin (1,7-bis
(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione/
diferuloyl methane) the main pigment of turmeric and
its other derivatives have been reported to have antiox-
idant and various biological activities. It has been re-
ported to have antioxidant properties.6–8 In our
previous publications9,10 we have highlighted the medic-
inal importance of curcumin. Turmeric and curcumin
have been shown to inhibit carcinogen-induced muta-
Piperine (1-piperoyl piperidine) an alkaloid present in
Piper nigrum-L (black pepper) has for long been used
widely in Auerveda, an Indian system of medicine.23
Piperine shows important pharmacological activity viz.
antiasthmatic property along with vasak leaves Adho-
toda vasica,24 antifertility,25 CNS depressant and anti-
inflammatory26–28 activities and inhibition of hepatic
monooxygenase and UDP–glucoronyl transeferase.29,30
In addition, piperine has also been shown to enhance
the bioavailability of drugs like spateine, curcumin,
Keywords: Curcumin; Piperine; Bioconjugate; Antibacterial;
Antifungal.
*
Corresponding author. Tel.: + 91 0532 2465462/2467154 (Res); +91
0532 2460816/2461236 (Off); fax: +91 0532 2623221; e-mail
addresses: krishnamisra@hotmail.com; kkmisra@yahoo.com
Present address: Department of Pathology, Kamala Nehru Memorial
Hospital, Allahabad 211002, India.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.057

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S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
barbiturates and oxyphenyl butazone, zoxazolamine,
propanlol and theophylline in animal experi-
ments.21,23,27,30–35
(ii) To decrease the rate of metabolism of curcumin
inside the cell.
(iii) To enhance the hydrophilicity of the molecule.
(iv) To have a biodegradable linkage in the bioconju-
gate, which could be degraded by the cellular
enzymes and curcumin released at the drug target
site (pro-drug).
The traditional knowledge that piperine when mixed
with curcumin enhances the bioavailability of latter sev-
eral times led us to link the two covalently.21,23 For such
a linking there were two alternatives, (i) to link piperic
acid obtained by hydrolysis of piperine36 to the two free
phenolics of curcumin via ester links or (ii) to link pip-
eric acid to the diglycinoyl curcumin reported earlier
from this laboratory9,10 via two amide links.
In order to achieve these objectives curcumin was cova-
lently linked to amino acids viz. glycine and D-alanine.
Although both are essential amino acids the latter is spe-
cific component of many bacteria and therefore must be
recognised by bacterial cells. The two conjugates, one
with each amino acid, that is, glycine and D-alanine were
prepared involving the two free phenolic groups of cur-
cumin, these ester linkage are biodegradable, giving pro-
drug character to the conjugates and also enhance their
hydrophilicity.
It is well known that curcumin has very poor bioavailabil-
ity,21 cellular uptake is slow and it gets metabolised fast
once inside the cell, requiring repetitive oral doses in order
to achieve significant concentration inside the cells for
therapeutic activity. Moreover, the transportation across
the cellular membrane of the infected cells is also changed,
which necessitates the molecule to be transported to be
well internalised with the changed environment of the cell.
The past decade has seen extensive use of new polymers,
cyclodextrins, liposomes and lipid-based drugs and viral
vectors for targeted delivery of drugs.37–43
During the synthesis the amino functions of glycine and
alanine were protected with phthalic anhydride as N-
phthaloyl glycine and N-phthaloyl alanine and the car-
boxy groups, were activated by treating with thionyl
chloride to get corresponding acid chlorides. The reac-
tion with curcumin in 2:1 molar proportion in pyridine
gave the desired products (Scheme 1).
One of the easily accessible approaches was to make bio-
conjugates of curcumin by covalent attachment with
such ligands, which internalise with cellular environment
of infected cells. The amino acids, for example, glycine
and D-alanine are essential components of bacterial
cells. Glucose and acetic acid being food components
are also well recognised. These components when linked
covalently to curcumin molecule may act as carrier mole-
cules as the cells recognise these molecules and thereby
might increase the intracellular delivery of curcumin.
The conjugates also are likely to be metabolised compar-
atively slowly. Therefore our objective was to synthesise
curcumin bioconjugates with amino acids, piperic acid,
glucose and acetic acid. These curcumin bioconjugates
can serve dual purpose of systemic delivery as well as
act as therapeutic agents against infectious diseases.
The biodegradable bonds used here in (ester amide
and glucoside) are likely to get hydrolysed in the living
system releasing curcumin at the diseased site.
One of the main drawbacks of these synthesis was the
relatively poor yields of the final bioconjugates. In order
to improve the yields we have developed an alternative
approach for these syntheses. The syntheses were carried
out in aqueous medium, that is, aqueous phase synthesis
of curcumin amino acid-bioconjugates especially of (II)
and (III) gave excellent results (yield ꢀ90%).
Piperic acid (V) was obtained by alkaline hydrolysis of
piperine (IV), the alkaloid present in black pepper.36
The –COOH function of piperic acid (V) was esterified
(activated) by reaction with p-nitro phenol by addition
of dicyclohexylcarbodiimide (DCC) in presence of pyr-
idine and triethyl amine to make the solution basic to
get the corresponding activated ester (VI). The activated
piperic acid was then reacted with glycine and 4,40-di-O-
glycinoyl curcumin in 1:1 and 1:2 molar proportions,
respectively, using DCC/DMAP to yield piperoyl gly-
cine (VII) and 4,40-di-O-[N-piperoyl–glycinoyl]-curcu-
min (VIII) (Schemes 2 and 3). This approach that is to
use the p-nitro phenyl ester of acid in place of the corre-
sponding acid chloride or anhydride to get the amide
bond (–CO–NH) is well established strategy in peptide
synthesis. The completion of reaction could be assessed
by precipitation of DHU (dicyclohexylurea).
2. Results and discussion
Curcumin molecule has three functional groups viz. two
phenolic and one active methylene group, which make
excellent sites for any chemical modification including
covalent attachment of biomolecules for synthesising
bioconjugates. In the present work only the phenolic
groups have been used since the C–C bonds with active
methylene group is not biodegradable. The ligands se-
lected were glycine, D-alanine, glucose, acetic acid and
piperic acid. The latter was obtained from the hydrolysis
of the alkaloid piperine. The main objective for prepar-
ing these bioconjugates was:
Curcumin was also directly attached with piperic acid
using piperoyl chloride in 1:2 molar ratio to get the
4,40-di-O-piperoyl curcumin (X).
To synthesise 4,40-di-O-(b-D-glucopyrano)-curcumin
(XIII), penta-O-acetyl-D-glucopyranose (XI) was pre-
pared and taken in dry dichloromethane and saturated
with hydrogen chloride gas with stirring. The solvent
was removed at reduced pressure to obtain the 1-a-
chloro-2,3,4,6-tetra-O-acetyl-glucopyranose (XII). Cur-
(i) To ease the transmembrane passage of curcumin,
in order to build its significant intracellular
concentration.

S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
1479
O
O
H3CO
OCH3
O
H3C C
o
CH3
C
o
O
C
XIV
O
O
H3CO
HO
OCH3
OH
I
O
O
b
a
(i)+(ii)
O
H3CO
RO
OCH3
OR
(i)+(ii)
O
H3CO
RO
X
OCH3
OR
O
C
II / III
CH
O
CH
CH
CH
R =
X:
O
R= - CO-CH2-NH2
II:
III: R = -CO-CH-NH2
CH3
Scheme 1. Synthesis of di-O-glycinoyl curcumin (II); di-O-alaninoyl curcumin (III); (a) i. 10% NaOH/N-phthaloyl glycinoyl chloride/N-phthaloyl
alaninoyl chloride/6 h/0 ꢁC; ii. NH3–pyridine (9:1) v/v; 1 min. Synthesis of 4,40-di-O-piperoyl-curcumin (X): (b) i. pyridine/piperoyl chloride (IX) rt/
6 h. Synthesis of di-acetate derivative (XIV) of curcumin: (c) 10% NaOH/crushed ice/acetic anhydride.
O
O
O
C N
CH
O
O
H3CO
OCH3
O_CO_CH2_NH2
CH
CH
a
CH
_
NH2 CH2_CO_O
a (i) & (ii)
IV
II
O
O
O
HN
OH
+
C
CH
(i) + (ii)
O
CH
CH
H3CO
OCH3
O_CO_CH2_NHR
CH
RHN_CH2_CO_O
O
VIII
b
O
V
O
C
C
CH
O
CH
CH
CH
VIII : R =
CH
O
O
NO2
CH
O
CH
c
CH
O
Scheme 3. Synthesis of 4,40-di-O-[glycinoyl-di-N-piperoyl]-curcumin
(VIII) (a) i. p-nitro phenyl ester of piperic acid (VI); ii. DCC/DMAP.
(i) + (ii) + (iii)
O
VI
COOH
NH CH2
C
CH
O
CH
CH
CH
curcumin was prepared by the acetylation of both the
phenolic functions of curcumin with acetic anhydride
(Scheme 1).
O
VII
Scheme 2. Synthesis of piperic acid (V); p-Nitro phenyl ester of piperic
acid (VI) and piperoyl glycine (VII): (a) Ethanolic KOH (2 N); 2 h
reflux/HCl; (b) i. p-nitrophenol/pyridine/TEA ii. DCC; (c) i. Glycine/
pyridine/(VI), ii. DCC/DMAP.
These derivatives of curcumin viz. 4,40-di-O-(glycinoyl)-
curcumin (II), 4,40-di-O-(D-alaninoyl)-curcumin (III),
4,40-di-O-(glycinoyl-di-N-piperoyl)-curcumin
(VIII),
4,40-di-O-(piperoyl) curcumin (X), curcumin-4,40-di-O-
b-D-glucopyranoside (XIII), 4,40-di-O-acetyl-curcumin
(XIV) and the piperic acid derivative viz. piperoyl gly-
cine (VII), were characterised by spectra UV, 1H
NMR and elemental analysis.
cumin was taken in pyridine and (XII) as colourless syr-
up in pyridine was added dropwise and stirred for 5 h.
The reaction mixture was poured in crushed ice and ex-
tracted with EtOAc (Schemes 4 and 5). The deacetyla-
tion was done using methanolic ammonia (1:1 v/v).
This reaction was followed on TLC and was found to
be complete in 2 h. The di-O-acetyl derivative (XIV) of
The antibacterial activity of curcumin-bioconjugates II,
III, VII, VIII, X, XIII and XIV, was compared with cur-
cumin (I), piperine (IV) and piperic acid (V) itself from

1480
S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
CH2OAc
CH2OH
CH2OAc
O
O
b
O
a
OAc
OH
OAc
Cl
OAc
AcO
OH
OH
(i) + (ii)
(i) + (ii)
HO
OAc
OAc
AcO
XII
XI
Scheme 4. Synthesis of D-glucose-penta acetate (XI) and 1-a-chloro-2,3,4,6 tetraacetyl-D-glucose (XII): (a) i. Dry pyridine/acetic anhydride/0 ꢁC; ii.
DMAP; (b) i. dry dichloroethane/dry HCl/0 ꢁC; ii. Stirred at 0 ꢁC/2.5 h.
O
O
H3CO
HO
OCH3
OH
I
a
O
O
H3CO
O
OCH3
CH2OAc
CH2OAc
O
O
O
b
XIII a
OAc
OAc
OAc
AcO
OAc
OAc
O
O
H3CO
OCH3
O
CH2OH
CH2OH
O
O
O
OH
OH
OH
HO
XIII
OH
OH
Scheme 5. Synthesis of curcumin-4,40-di-b-D-glucopyranoside (XIII): (a) XII/pyridine; (b) NH3–MeOH (1:1 v/v)/2 h at rt.
known micro dilution broth susceptibility test method.
The stock solutions of conjugates viz. II, III, VII, VIII,
X, XIII, XIV along with curcumin (I) piperine (IV) and
piperic acid (V) were prepared in acetone. The stock
solution of each of these bioconjugates was serially di-
luted (20; 10, 5, 2.5, 1.25 lM/mL) and added to Mul-
ler–Hinton broth, after which a standardised bacterial
suspension was added.
good positive results on multiresistant microorganisms.
The most encouraging result was found against P. aeru-
ginosa with VIII and X and E. coli with XIV having
MIC 2.5 lM/mL and compared with one of the best
marketed antibiotic viz. Cefepime, which show MIC
7.0 lM/mL (6.0 lM/mL reported) showing that the di-
O-(glycinoyl-N-di-piperoyl)-curcumin (VIII) and curcu-
min diacetate (XIV) is 2.8 times more effective than
Cefepime at the same concentration.
The lowest concentration of curcumin bioconjugate in
lM/mL that prevented in vitro growth of microorgan-
ism has been represented as MIC (minimum inhibitory
concentration) shown in (Table 2) and correlated with
zone of inhibition (Table 1).
The result of zone of inhibition is also encouraging as
the disc containing 30 lg of Cefepime was purchased
and same amount was loaded on separate disc, the zone
of inhibition of Cefepime was 18 mm while (VIII) and
(XIV) shows zone of inhibition, respectively, 27 and
24 mm. The MIC of II, III, VII and XIII, curcumin
derivative were found between 5 and 10 lm/mL in most
cases as compared to available antibiotics in market
having MIC in between 6 and 10 lM/mL. The piperoyl
curcumin (X) has a significant result against P. aerugin-
osa having MIC 2.5 lM/mL. In comparison to above re-
sults curcumin, piperin and piperic acid were found to
be resistant in most cases. Curcumin shows MIC
20 lM/mL against E. coli and S. aureus. Piperine and
piperic acid show activity at relatively high
concentration.
Susceptibility test in vitro was done on multiresistant
bacteria specially causing secondary infections in human
being, for example, Escherichia coli, Staphylococcus aur-
eus, Pseudomonas aeruginosa, Pseudomonas pyocyanin,
Enterobactor cloacae, Klebsiella aeurogenus, Staphylo-
coccus epidermidis, Staphylococcus saprophyticus, Micro-
coccus, Enterobactor aerogen and Enterococus. These
pathogens were selected for in vitro investigation along
with some other Gram-positive and Gram-negative bac-
teria. The results have been tabulated (Tables 1 and 2).
Each test was performed in triplicate and the MICs re-
ported represent the result of at least two repetitions.
Five bioconjugates (II, VII, VIII, X and XIV) show
The results suggest that positive results with II, III, VII
and VIII may be due to the reason that amino acids gly-

S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
1481
Table 1. Antibacterial activity of curcumin bioconjugates (numerals show size of zone of inhibition in mm) of different dilutions of bioconjugates (20,
10, 5, 2.5 lM/mL) against bacterial strains
Name of bacteria
I
II
III
IV
V
VII
VIII
X
XIII
XIV
E. coli
S. aureus
10
10
—
—
—
—
12
—
—
12
—
10
10, 8
8
—
—
10,9
—
12, 10
10, 8
10, 8
14, 12, 8
14, 12
14, 12
—
24, 22 18, 15
12, 10, 8
15, 12
10, 8
12
10, 8
—
8
—
P. aeruginosa
P. pyocynin
E. cloaca
—
—
16, 13, 8 27, 24, 20, 18 18, 15, 12, 8
12, 10, 8
—
—
12, 8
12
16, 14, 12, 9
15, 13
—
13, 10, 7
14, 12
14, 10
12, 10
11, 9
—
—-
—
14, 12, 9
—
—
K. aeurogenus
S. epidermidis
S. saprophyticus
Micrococcus
E. aerogen
—
11, 9 14, 10, 8
8
10
—
16, 12, 10
—
15, 12
12, 9
12, 8
—
14, 9
11, 8
12
—
—
—
14
14, 12, 10 12, 10, 8
15, 13, 10 14, 10, 8
—
—
—
—
—
—
14, 10, 8
12, 9
—
12, 8
10, 8
10
15, 12, 10
11, 9
—
—
—
—
—
—
11
—
16, 12
—
—
Enterococus
14, 12, 8
—
11
(—) Resistant.
Table 2. Minimum inhibitory concentration (MIC), correlation diagram (in lM/mL) of bioconjugates against bacterial strain
Name of bacteria
I
II
III
IV
V
VII
VIII
X
XIII
XIV
Cefeprime
E. coli
S. aureus
20
20
—
—
—
—
10
—
—
—
20
—
5
20
10
—
5
—
—
20
—
20
10
20
—
—
—
—
—
—
—
—
—
20
20
—
—
—
10
10
—
5
10
10
2.5
2.5
10
—
10
5
10
5
10
10
—
—
—
5
2.5
20
—
—
5
18
20
12
8
P. aeruginosa
P. pyocynin
10
10
20
—
5
2.5
10
20
—
10
10
—
—
—
5
10
10
10
10
5
E. cloacae
—
—
10
5
18
12
8
K. aeurogenus
S. epidermidis
Staphylococcus saprophyticus
Micrococcus
E. aerogen
—
10
20
—
—
20
—
10
10
20
—
5
10
5
5
10
—
5
—
—
—
20
10
—
Enterococus
(—) Resistant.
cine and D-alanine are natural components of bacterial
cell wall. There appears to be very significant inhibition
of growth in case of P. aeruginosa and P. pyocyanin. It
has been reported in literature that piperine enhances
the bioavailability21 and thus the activity of many drugs
as well as herbal preparations is increased. Since, the
diglycinoyl curcumin was found to be relatively more ac-
tive against Gram positive as well as Gram-negative
The many fold enhancement of antibacterial activity of
curcumin-di glycinoyl conjugate (II) on further linking
it covalently with piperic acids (V) is quite significant.
In a physical mixture of two spice components, that is,
curcumin and piperine the bioavailability increases and
latter may be acting as an adjuvant. However, a possible
explanation that can be given is that the covalent linkage
of the two compounds results in extension of the chro-
mophores containing conjugate double bonds, thus
resulting in a better binding site for receptors. However,
more work is required for this verification.
`
bacteria vis-a-vis curcumin, it could be concluded that
covalent linking of one or two glycine units with curcu-
min enhance its bioavailability.9,10 This may be due to
the easy recognition of glycine by the bacterial cells,
which help in better cellular uptake and thereby en-
hances its active concentration inside the cell. Moreover,
the metabolism of these conjugates is also expected to be
slower than curcumin itself. In order to increase its effec-
tiveness, it was condensed further with 2 mol of piperic
acid, via biocompatible amide links. The highly positive
result obtained specially with P. aeruginosa and P. pyo-
cyanin are suggestive of its enhanced bioavailability.
Similarly curcumin linked with piperic acid directly, by
the reaction of curcumin and piperoyl chloride had en-
hanced activity. Thus the most notable result is that this
novel prodrug is relatively more effective towards Gram-
negative bacteria as compared to Gram positive,
whereas the positive result of (VIII) is due to the fact
that piperic acid when linked to curcumin diglycinoyl
conjugate, its activity increases due to itÕs enhanced
bioavailability.
The curcumin diacetate shows best results against E. coli
with MIC (2.5 ml/mL). Acetyl groups are attached with
curcumin by ester bonds, which are biodegradable link-
age, and are easily cleaved under enzymatic conditions
to release curcumin at the site.
However, this approach of combining different active
molecules present in nature, resulting in enhancement
of their bioavailability, are certainly encouraging. These
bioconjugates of curcumin are more hydrophilic than
the parent molecule. The hydrophilic nature of biocon-
jugates may also help in their active transport across
the cellular membrane.
Literature survey however suggests that neither curcu-
min nor any of its bioconjugates have so far been eval-
uated for their antifungal activity. We have now

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S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
evaluated the anti fungal activity of curcumin and its
bioconjugates against specific fungi viz. Aspergillus
fumigatus, Candida krusei GO3, Candida glabrata, Can-
dida albicans (yeast), in vitro. These fungi specially cause
nail infections called ÔonychomycosisÕ. Among the ony-
chomycosis toenail infection is more prevalent than fin-
gernail. These fungus were obtained from a skin clinic
centre from the patients diagnosed for onychomycosis
on potato dextrose agar without the presence of
antibiotics.
sed at the target sites. The moieties selected, e.g.,
glycine, D-alanine, glucose and acetate are such mole-
cules, which are well, recognises by the bacterial/fungal
cells, since these are essential components of these
pathogens, as well as the host cells. Therefore, these syn-
thetic molecules are designed to be internalised with the
cellular components, in order to facilitate the cellular
uptake through receptor mediated endocytosis. D-Ala-
nine is an essential component of many bacterial cell
wall, while in eukaryotic cells it is always present in its
L-form. Piperic acid, an essential component of the alka-
loid piperine has been used, in order to enhance the bio-
availability of curcumin. Thus, the combination of two
active spice ingredients, that is, curcumin and piperine
is likely to enhance the bioavailability and the latter
may act as adjuvant.
From the results shown in Table 3 we can say that cur-
cumin piperic acid (X) 4,40-(di-O-glycinoyl-di-N-pipe-
royl) curcumin (VIII) and piperoyl glycine (VII) show
better antifungal activity specially against C. albicans
(yeast) than curcumin (I), piperine (IV) and piperic acid
(V) itself. The better result is probably due to the fact
that the cellular uptake increases through the fungal cell
wall. Though our work is still in its infancy and includes
limited number of both, that is, causative organism and
bioconjugates nevertheless the significance of work can-
not be ignored. It opens a new era for exploring suitably
designed curcumin bioconjugates as potential antibacte-
rial/antifungal drugs. Exhaustive work based on the
designing and testing the conjugates according to the
molecular organisation of several pathogens is required
before any definite conclusion can be reached.
During the present work we have concluded that the
bioconjugates synthesised from curcumin are more po-
tent than curcumin itself against many common strains
of bacteria as well as fungi. Conventionally, antibiotics
are used to kill bacteria or to inhibit their division with
the intention of preventing replication of bacterial gen-
ome, but over use of antibiotics in human and livestock
has led to the rapid evolution of mutant species of bac-
teria that are resistant to multiple drugs46 and recently
there has been a call for worldwide use of antibiotics
rotation scheme to conquer the problems of resistance.47
The best example is of penicillin, which had been a won-
der antibiotic drug for over half a century. b-Lactamase
present in bacterial cellular fluid hydrolyses the amide
bond of the b-lactam ring of penicillin and cephalospo-
rins, producing acidic derivatives, which have no anti-
bacterial properties, thereby making the bacteria
resistant to that particular antibiotic drug.
The antifungal activity of curcumin bioconjugates was
compared with the standard antifungal drugs like fluco-
nazole, whereby it was found negligible in some cases
(e.g., C. krusei GO3) and better in case of C. albicans
(yeast). This result is quite encouraging since antifungal
drugs are quite rare, and fungal infections are known to
be stubborn.
To develop a new armory of agents active against anti-
biotic-resistant bacteria, nonconventional antibiotics
could be designed that can internalise themselves in
the bacterial cell wall easily. The structures should be de-
void of the b-lactam ring for better efficacy.
The present work suggests that there is need to develop
nonantibiotic drugs,48 which may overcome antibiotic
resistance. Such highly bioactive drugs preferably herbal
preparations with least toxicity (probiotics) may be in
the offing. We have succeeded in our effort to a limited
extent. However, such a study needs exhaustive work,
that is, designing and testing of conjugates according
to the specificity of the concerned pathogenic microor-
ganism specifically its interaction with different host cells
(biodiversity). A little structural variation can cause im-
mense difference in the activity of the drug. This ap-
proach can open new vistas in the chemotherapy of
infective diseases. Since these drugs can internalise
themselves in the bacterial/fungal cell wall easily and
their structure is also devoid of b-lactam ring or other
3. Conclusion
The different derivatives of curcumin have been pre-
pared by covalently linking it with different ligands
through its two phenolic groups. All the covalent bonds
used are biodegradable, that is, hydrolysable with com-
mon enzymes present in living system. This makes these
derivatives as potent prodrugs, which can get hydroly-
Table 3. Antifungal activity of curcumin bioconjugates (zone of inhibition in mm) of bioconjugates against fungal strains
Name of fungi
I
II
III
IV
V
VII
VIII
X
XIII
XIV
Fluconazole
A. fumigatus
—
19
—
15
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
19
—
—
—
24
—
—
—
—
—
—
—
—
—
—
—
—
—
19
15
29
C. krusei GO3
C. glabrata
C. albicans (yeast)
(—) Resistant.
Curcumin (I); 4,40-(di-O-glycinoyl)-curcumin (II); 4,40-(di-O-alaninoyl)-curcumin (III); piperine (IV); piperic acid (V); piperoyl glycine (VII); 4,40-(di-
O-glycinoyl-di-N-piperoyl)-curcumin (VIII); 4,40-(di-O-piperoyl)-curcumin (X); curcumin-4,40-(di-O-b-D-glucopyranoside) (XIII); 4,40-(di-O-acetyl)-
curcumin (XIV).

S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
1483
sensitive chromophores, these can prove to be highly po-
tent b-lactamase resistant drugs.
on TLC (AcOEt–cyclohexane–EtOH: 55:40:5). The r.m.
was treated with NH3–pyridine (9:1 v/v) for 1 min at rt
to remove phthaloyl group. The reaction mixture was
poured in crushed ice and lyophilised to concentrate
the solvent and then the product was extracted with
ethylacetate, evaporated in vaccuo and purification
was done by column chromatography using DCM–
MeOH gradient, Yield 84% (0.448 g). Anal. Found: C,
65.49; H, 5.63; N, 5.20 calcd, for C29H30O8N2; C,
More such bioconjugates may be designed by keeping
one of the components common to either the bacterial
cell wall or bacterial genome conjugated with curcumin,
thereby improving the efficiency to curcumin compre-
hensively at low doses and may prove to be better sys-
temic drug by concentrating mainly on and around
malignant and ill-fated cells by recognizing the bacterial
cells. The results prove once again the efficacy of curcu-
min and its bioconjugates as antibacterial and anti-
fungal agents.
1
65.14; H, 5.65; N, 5.24. H NMR (CDCl3) d = 3.14 (d,
6H, –CH3); 3.81 (s, 6H, –OCH3); 4.12 (s, 2H, C4–H);
4.43–4.57 (m, 2H, 2 ꢁC of alanine); 6.55 (d, 2H, C2–H
and C6–H); 6.83–7.11 (m, 6H, Ar–H), 7.53 (d, 2H, C1–
H and C7–H).
4. Experimental procedures
4.1. General
4.3. Piperic acid from piperine (V)
Piperine (IV) (2.853 g; 0.01 mol) was refluxed with eth-
anolic KOH (2 N, 10 mL) for 2 h. Ethanol was distilled
under reduced pressure, solution cooled in ice salt bath,
the solid potassium salt of piperic acid was suspended in
hot water and acidified with hydrochloric acid, yellow
precipitate was collected, washed with cold water and
recrystallised from ethanol to yield crystals. Yield 88%
(1.91 g); Rf 0.30 (DCM–MeOH: 9.5:0.5); mp 215 ꢁC;
UV kmax (MeOH): 255, 335 nm; Anal. Found: C,
66.05; H, 4.56 calcd, for C12H10O4; C, 66.04; H, 4.61.
1H NMR (CDCl3) d ppm: 5.64 (d, 1H, –CH@CH–
C@O); 5.93 (s, 2H, O–CH2–O); 6.38 (d, 1H, –CH@C);
6.58–7.78 (m, 4H, olefinic and aromatic); 6.73 (d, 1H,
J = 9, Ar–H); 7.25–7.51 (d, 1H, CH@CH@C@O); 11.2
(s, 1H, HO–C@O).
All solvents used were purified and distilled prior to use.
Curcumin, glycine, alanine and piperine were purchased
from Merck-Schuchardt, Germany. UV–visible spectra
were recorded on Hitachi 220S UV–visible spectropho-
tometer. Synthetic grade solvents used were purchased
from Qualigens and were purified and dried prior to
1
use. H NMR were recorded on DRX300 instrument.
The Mueller–Hinton broth, Agar and sterile discs were
purchased from Hi Media Laboratory Ltd., Mumbai,
India. Muller–Hinton broth M391 and agar M173 have
been selected for testing aerobic and facultative anaero-
bic bacterial isolates for fastidious organisms such as
Streptococci and Peptococci the agar was supplemented
with 5% defibrinated blood. The microsusceptibility test
were standardised at pH 7.4, agar-broth were incubated
in an ambient air incubator at 37 ꢁC.
4.4. p-Nitro phenyl ester of piperic acid (VI)
To piperic acid (V) (2.180 g; 10 mmol) dissolved in dry
dioxane (5 mL), p-nitro phenol (1.52 g; 11 mmol) dis-
solved in dioxane was added drop wise. The reaction
mixture was made basic by addition of 0.5 mL pyridine
and 0.5 mL TEA. After stirring for 10 min dicyclohexyl
carbodiimide (DCC) (2.579 g; 12.5 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 as-
sessed by total consumption of starting reagent.
Rf = 0.58 (DCM–MeOH: 9.5:0.5).
The inoculum was prepared from broth culture that has
been incubated for 4–6 h, when growth was considered
in the logarithmic phase. Cefepime (an antibiotic drug)
taken as standard. Fluconazole was taken as a standard
anti fungal drug. The microorganisms were obtained
from a city clinic from the diagnosed patients, specially
a dermatophyte, nondermatophyte and yeast were iso-
lated from the nail of patients diagnosed for onychomy-
cosis on potato-dextrose agar without antibiotic for
testing isolates, these were subcultured on sabourad dex-
trose agar slant and incubated at 30 ꢁC for 10–12 days.
The sterile discs with 6 mm diameter were further steril-
ised and charged with II, III, IV, V, VII, VIII, X, XIII
and XIV as per requirement. After drying the discs, they
were stored at 4 ꢁC.
4.5. Piperoyl glycine (VII)
Glycine (0.15 g; 2 mmol) was dissolved in dry pyridine
(5 mL) was added drop wise to above activated ester
of piperic acid (VI) (0.748 g; 2.2 mmol). The r.m. was
stirred for 10–20 min and DCC (515 mg; 2.5 mmol),
DMAP (12.2 mg; 0.1 mmol) were further added. The
r.m. was stirred for 3 h and monitored on TLC. The pre-
cipitate of dicyclohexyl urea (DCU) started appearing
after 30 min and completed after 3 h. The reaction mix-
ture was filtered to separate DCU and filtrate was evap-
orated to half of its volume and then poured in 2%
sodium bicarbonate solution (to neutralise the residual
acid and to separate excess of p-nitro phenol) and ex-
tracted with dichloromethane (4 · 10 mL). Organic
layer was collected and further washed with distilled
1,7-Bis (4-O-glycinoyl-3-methoxy phenyl)-1,6-heptadi-
ene-3,5-dione (II) was prepared as reported.9
4.2. 1,7-Bis (4-O-D-alaninoyl-3-methoxy phenyl)-1,6-
heptadiene-3,5-dione (III)
Curcumin (I) (368 mg; 1 mmol) was taken in 10%
NaOH and mixed with N-phthaloylalaninoyl chloride
(713 mg; 3 mmol) slowly at 0 ꢁC with constant stirring
at rt for 5 h. The completion of reaction was monitored

1484
S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
water, dried over anhyd Na2SO4 and filtered. The com-
bined dichloromethane layer were concentrated and
crystallised with dry ethanol to get desired product.
Yield 70% (0.192 g); Rf 0.39 (DCM–MeOH:: 9.5:0.5);
mp 232 ꢁC; UV kmax (MeOH): 300, 340 nm.; Anal.
Found: C, 62.05; H, 4.75; N, 5.08 calcd, for
C14H13NO5; C, 61.08; H, 4.76; N, 5.08. 1H NMR
(CDCl3) d ppm: 3.90 (d, 2H, CH2–C@O); 5.70 (d, 1H,
–CH@CH–C@O); 5.86 (t, 1H, CO–NH); 5.96 (s, 2H,
O–CH2–O); 6.72–7.86 (m, 4H, olefenic and aromatic);
6.90 (d, 1H, J = 9, Ar–H); 7.21–7.48 (d, 1H,
CH@CH@C@O).
with EtOAc. The organic layer was concentrated and
purified by silica gel column chromatography using
DCM–methanol gradient. Yield 48% (0.737 g); UV kmax
(MeOH): 230, 295, 390 nm; Rf: 0.87 (MeOH–DCM::
0.5:9.5). 1H NMR (CDCl3) d ppm: 3.65 (s, 6H,
–OCH3); 4.15 (s, 2H, C4–H); 4.65 (d, 2H, O@C–CH2);
5.65 (d, 1H, –CH@CH–C@O); 5.91 (s, 2H, O–CH2–
O); 6.51 (t, 1H, CH2NH@C@O); 6.63 (d, 2H, C2–H
and C6–H); 6.72–7.80 (m, 4H, olefenic and aromatic);
6.82–7.15 (m, 6H, Ar–H); 6.90 (d, 1H, J = 9, Ar–H);
7.18–7.48 (d, 1H, CH@CH–C@O); 7.48 (d, 2H, C1–H
and C7–H).
4.6. 1,7-Bis-[(4-O-glycinoyl-N-piperoyl)-3 methoxy phen-
yl] 1–6-heptadiene-3,5-dione (VIII)
4.9. 1,2,3,4,6-Penta-O-acteyl-a-D-glucopyranose (XI)
This was prepared by standard protocol.44
Di-glycinoyl curcumin (II) (0.482 g; 1 mmol) dissolved
in dry pyridine (5 mL) and solution added drop wise
to the above activated ester (VI) (0.748 mg; 2.2 mmol).
The r.m. was stirred for 10–20 min and DCC (257 mg;
1.25 mmol), DMAP (12.2 mg; 0.1 mmol) were further
added. The r.m. was stirred for 3.5 h and monitored
on TLC. The precipitate of dicyclohexyl urea (DCU)
started appearing after 30 min and completed after 3 h.
The r.m. was filtered to separate DCU and filtrate was
evaporated to half of its volume and then poured in
2% sodium bicarbonate solution (to neutralise the resid-
ual acid and to separate excess of p-nitro phenol) and
extracted with dichloromethane (4 · 10 mL). Organic
layer was collected and further washed with distilled
water, dried over anhyd Na2SO4 and filtered. The com-
bined dichloromethane layer were concentrated and res-
idue crystallised in dry alcohol to get desired product.
Yield 48% (0.423 g); Rf 0.52 (DCM–MeOH:: 9.5:0.5);
UV kmax (MeOH): 240, 298, 340 nm.; Anal. Found: C,
66.59; H, 4.79; N, 3.15 calcd, for C49H42N2O14; C,
66.65; H, 4.79; N, 3.17. 1H NMR (CDCl3) d ppm:
3.65 (s, 6H, –OCH3); 4.15 (s, 2H, C4–H); 4.65 (d, 2H,
O@C–CH2); 5.65 (d, 1H, –CH@CH–C@O); 5.91 (s,
2H, O–CH2–O); 6.51 (t, 1H, CH2NH@C@O); 6.63 (d,
2H, C2–H and C6–H); 6.72–7.80 (m, 4H, olefenic and
aromatic); 6.82–7.15 (m, 6H, Ar–H); 6.90 (d, 1H,
J = 9, Ar–H); 7.18–7.48 (d, 1H, CH@CH–C@O); 7.48
(d, 2H, C1–H and C7–H).
4.10. 1-a-Chloro-2,3,4,6-tetra-O-acetyl-glucopyranose
(XII)
This was prepared from acetylated glucose (XI) (2 g;
5.12 mmol) by dissolving in cold (0 ꢁC) dry dichlorome-
thane (12 mL) and saturating the solution with anhy-
drous hydrogen chloride over 2.5 h at 0 ꢁC. The
solution was removed in vacuo (bath temperature,
25 ꢁC). Colourless syrup was crystallised with diethyl
ether. Yield 68% (1.276 g), Rf 0.66 (DCM–MeOH::
9:1), mp 98–101 ꢁC. 1H NMR (CDCl3) 1.95–2.09
(4 · br s, 12H) acetyl H0s; 4.05–4.10 (d, 2H) H6a0 and
3
H6b0; 4.41–4.46 (m, 1H) H50; 5.00 (dd, J23–13
=
=
3
3
3.9 Hz, J23–33 = 10.6 Hz, 1H) H20; 5.36 (dd, J33–23
3
10.6 Hz, J33–43 = 3.2 Hz, 1H) H30; 5.45–5.48 (m, 1H)
3
H40; d 6.65 (d, J13–23 = 3.9 Hz, 1H) H10.
4.11. 4,40-Di-O-b-D-glucopyranocurcumin (XIII)
Curcumin (I) (0.368 g; 1 mmol) was taken in dry pyr-
idine and the solution of (XII) (0.806 g; 2.2 mmol) in
pyridine was added drop wise in a ice bath. The r.m.
was removed from ice bath and stirred at rt for 3 h.
Reaction was monitored on TLC. After the completion
of reaction mixture was poured in crushed ice and
repeatedly extracted with EtOAc. The organic layer
was concentrated and treated with (6 mL) ammonia–
methanol (1:1 v/v) for 2 h at rt to hydrolyse the acetyl
groups. The reaction was followed on TLC. Poured in
crushed ice and extracted with EtOAc. The organic layer
was concentrated and purified by silica gel column chro-
matography using DCM–MeOH. Yield 46% (0.318 g).
Anal. Found: C, 57.21; H, 5.73 calcd, for C33H40O16;
4.7. Piperoyl chloride (IX)
Piperic acid (V) (2.18 g, 0.01 mol) was heated on water
bath and to it was added redistilled thionyl chloride
(1.09 mL, 0.015 mol) during 45 min, shook the flask
from time to time to ensure mixing. Refluxed for
30 min, protected from moisture and distilled the prod-
uct at reduced pressure. Yield 65% (1.539 g), mp 144–
148 ꢁC.
1
C, 57.21; H, 5.83. H NMR (CDCl3) d ppm = 3.85 (s,
6H, –OCH3); 4.02–4.10 (m, 2H) H6a· and H6b·;
4.29–4.33 (m, 1H) H5·; 5.13 (s, 2H, C4–H); 5.29–5.31
(m, 2H) H3c and H4·; 5.46–5.47 (m, 1H) H2·; 6.48
(d, 2H, C23–H and C6–H), 6.85–7.08 (m, 6H, Ar–H),
6.93 (br s, J13–23 = 1.2 Hz, 1H) H1·; 7.52 (d, 2H, C1–
H and C7–H).
4.8. 1,7-Bis-[(4,40-di-O-piperoyl)-3 methoxy phenyl] 1–6-
heptadiene-3,5-dione (X)
Curcumin (I) (0.736 g; 2 mmol) was taken in dry pyr-
idine and mixed with piperoyl chloride (IX) (1.065 g;
4.5 mmol), stirred at rt for 6 h. After the completion
of reaction as indicated by TLC the reaction mixture
was poured into crushed ice and repeatedly extracted
4.12. 1,7-Bis-4,40-[(di-O-acetyl)-3-methoxy phenyl] 1–6-
heptadiene-3,5-dione (XIV)
To curcumin (I) (0.368 g; 1 mmol) dissolved in 10%
aqueous sodium hydroxide and 7.0 g of crushed ice

S. Mishra et al. / Bioorg. Med. Chem. 13 (2005) 1477–1486
1485
was added followed by 0.18 mL (2.6 mmol) of acetic
anhydride and shook vigorously for 5 min. Stirred for
2 h, and extracted with carbon tetrachloride. Concen-
trated the organic layer in vacuo. Organic layer was
washed with 1% NaHCO3 until effervescence ceased.
Dried the organic layer over Na2SO4. Crystallised from
ally diluted to make effective concentration of 20, 10 and
5.0 lM/mL. The dermatophytes, nondermatophytes and
yeast were isolated from nail chippings from suspected
cases of onychomycosis referred from a skin clinic at
Allahabad. The most prevalent causative organisms
viz. A. fumigatus, C. krusei GO3, C. glabrata, C. albicans
(yeast) were selected. These fungii were isolated from the
nails of patients diagnosed for onychomycosis, on sab-
ourad dextrose agar without antibiotics. For testing iso-
lates were subcultured on subourad dextrose agar slant
and incubated at 30 ꢁC for 10 days. Sterile discs with
6 mm diameter were further sterilised and charged with
compounds (I, II, III, IV, V, VII, VIII, X, XIII and XIV)
and after drying these discs were stored at 4 ꢁC.
1
absolute ethanol. Yield 74% (0.334 g), mp 160 ꢁC. H
NMR (CDCl3) d ppm = 3.75 (s, 6H, –OCH3), 4.10 (s,
2H, C4–H), 4.17 (s, 6H, O–C–O–CH3), 6.45 (d, 2H,
C2–H and C6–H), 6.81–7.17 (m, 6H, Ar–H), 7.44 (d,
2H, C1–H and C7–H).
4.13. To determine zone of inhibition by Kirby–BauerÕs
method9
The antibacterial susceptibility test was done by deter-
mining zone of inhibition by Kirby–BauerÕs method.45
The curcumin bioconjugates, curcumin, curcumin-pip-
eric acid conjugate and piperic acid itself were weighed
and dissolved in acetone to make a solution of concen-
tration 120 lM/mL. From this stock solution serial dilu-
tion have been done to 20, 10, 5, 2.5 and 1.25 lM/mL
with acetone in sterile test tubes. Sterilised filter discs
were dipped in these solutions and subsequently dried
to remove acetone. Mueller–Hinton agar was prepared
and allowed to solidify. One of these discs was kept free
from antibiotic and served as growth control. Twelve
different bacteria were selected viz.—Escherichia coli,
S. aureus, P. aeruginosa, Pseudomonas pyocynin, E. clo-
acae, K. aeurogenus, Staphylococcus epidermidis, S. sap-
rophyticus, Micrococcus, E. aerogen and Enterococus
and 1 mL of each bacterial culture broth were added
in the Mueller–Hinton plates and spread with the help
of sterile spreader. The filter paper discs soaked in above
mentioned dilutions of curcumin, piperic acid and their
conjugates were placed aseptically over the inoculated
plates using sterile forceps. The plates were incubated
at 37 ꢁC for 18 h, in upright position. The zone of inhi-
bition was measured using scale (Table 1).
4.16. Preparation of the inoculum49
A standardised inoculum was prepared by counting
microconidia. Cultures were grown on soluble dextrose
agar for 10 days at 37 ꢁC sterile saline sol (0.85%) was
added to the slant and culture were 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. The resulting suspension was counted with
homocytometer and diluted in sabourad broth to
5 mL. Subourad dextrose agar was poured to depth of
5 mm in 90 mm petridiscs and stored at 4 ꢁC. The plates
were dried; standardised suspension was poured and
uniformly spread by means of swab discs. The excess
inoculum was drained.
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