Cyclic peptide conjugate of curcumin and doxorubicin as an anticancer agent

Article abstract of DOI:10.1016/j.tetlet.2017.10.065

The hydrophobicity of curcumin creates hurdle towards its use in the anticancer therapy. Herein, we synthesized a curcumin-doxorubicin conjugated cyclic peptide scaffold to improve the solubility of curcumin and create a conjugate containing two anticance

Full text of DOI:10.1016/j.tetlet.2017.10.065

Accepted Manuscript
Cyclic peptide conjugate of curcumin and doxorubicin as an anticancer agent
Shaban Darwish, Saghar Mozaffari, Keykavous Parang, Rakesh Tiwari
PII:
S0040-4039(17)31369-2
https://doi.org/10.1016/j.tetlet.2017.10.065
TETL 49427
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Reference:
Tetrahedron Letters
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24 October 2017
26 October 2017
Please cite this article as: Darwish, S., Mozaffari, S., Parang, K., Tiwari, R., Cyclic peptide conjugate of curcumin
and doxorubicin as an anticancer agent, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.2017.10.065
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Cyclic peptide conjugate of curcumin and doxorubicin
as an anticancer agent
Shaban Darwish, Saghar Mozaffari, Keykavous Parang and Rakesh Tiwari

1
Tetrahedron Letters
journal homepage: www.els evier.com
Cyclic peptide conjugate of curcumin and doxorubicin as an anticancer agent
Shaban Darwisha,b, Saghar Mozaffaria, Keykavous Paranga,*, and Rakesh Tiwaria,*
aCenter for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane
Rinker Health Science Campus, Irvine, California 92618, United States.
bOrganometallic and Organometalloid Chemistry Department, National Research Centre, El Bohouth st., Dokki, Giza, Egypt
ARTICLE INFO
ABSTRACT
Article history:
Received
The hydrophobicity of curcumin creates hurdle towards its use in the anticancer therapy. Herein,
we synthesized a curcumin-doxorubicin conjugated cyclic peptide scaffold to improve the
solubility of curcumin and create a conjugate containing two anticancer agents. A solid-phase
Fmoc/tBu solid phase methodology was used to synthesize a cell-penetrating nuclear targeting
peptide with free thiol and amine groups, which was coupled with the activated doxorubicin
(Dox) and curcumin, affording Dox-peptide-curcumin conjugate (DPCC) (10). The
antiproliferative activity of the conjugate was evaluated in human leukemia carcinoma cell
(CCRF-CEM), human ovarian carcinoma cell (SKOV-3), and normal kidney cell line (LLCPK).
Cyclic peptide-doxorubicin conjugate (7) and DPCC (10) did not inhibit the proliferation of
normal kidney LLCPK cells after 72 h incubation, but were cytotoxic in CCRF-CEM (73% and
41%, respectively) and SKOV-3 (55% and 30%, respectively) cells under similar conditions,
suggesting selectivity of these compounds towards cancer cells while Dox was cytotoxic (60-
Received in revised form
Accepted
Available online
Keywords:
Antiproliferative activity
Combination therapy
Curcumin
Cyclic peptide
Doxorubicin
79%) in all three cell lines under similar conditions.
All rights reserved
2009 Elsevier Ltd.
.
1. Introduction
will be beneficial to improve the physiochemical property of
curcumin for the therapeutic applications.
Cancer is a leading cause of death worldwide; nearly 1 in 6
deaths is reported due to cancer.1 There was 14.1 million new
cancer cases and 8.2 million cancer deaths in 2012. This global
burden is expected to increase to 21.7 million new cases and 13
million cancer deaths by 2030.2 Thus, there is an urgent need to
develop new drugs and/or use new strategies to combat cancer.
Over the last decades, several chemical compounds either
naturally or synthetically grabbed scientists' attentions for their
anticancer activity; however, the situation is far from satisfactory,
due to the toxicity related to nonspecific accumulation or their
hydrophobicity, which are considered critical challenges for
clinical applications. Therefore, developing a strategy to improve
the physicochemical property of existing drugs will be required.
Doxorubicin (Dox), which belongs to anthracycline antibiotic
family, extracted from the bacteria Streptomyces pecetius,18 is
one of the most vital antitumor compounds. Dox is highly
effective in the treatment of various types of cancer including
breast, acute lymphocytic leukemia, bladder, and prostate
cancer.19-22 It has a unique mechanism of action through
inhibition topoisomerase II, which interfere with DNA through
insertion, inducing cancer cell apoptosis.23 Similar to curcumin,
Dox has limited clinical use, due to its pharmacokinetic
properties, such as short half-life and a high cumulative dose,
leading to dose-independent side effects.24
Furthermore, the conventional single treatment can lead to the
development of drug resistance over the course of therapy.25
Recently, combinational chemotherapy has emerged as one of the
strategies that have gained attention to surmount the associated
problems with the effectiveness of cancer treatment by reducing
side effects and overcoming drug resistance, thereby achieving
synergistic therapeutic efficacy.26,27 Additionally, the treatment
success hinges on appropriate control over the timing, extent, and
location of dosing. In this regard, drug delivery system
techniques play an important role towards increasing the efficacy
of the drug by delivering it to where it is needed, at the correct
dose and extended release over a desired period.28-30
Curcumin (diferuloylmethane) is a hydrophobic polyphenol
compound derived from Curcuma Longa (turmeric), has attracted
attention as a promising natural product.3 An extensive research
indicated that curcumin possesses several chemopreventive and
pharmacological activities,4,5 including potent antioxidant6 and
antitumor properties7 with extremely superior safety profiles.8-10
Its anticancer activities are mediated through the inhibition of
NF-ĸB activity occurring through the down-regulation of IKK.11
Another study conducted by Gopinath et al. has provided
evidence that curcumin decreases protein damage by quenching
free radicals.12 Moreover, it can improve the cytotoxicity of some
other anticancer molecules against different cancer cells.13 In
spite of the promising biological effects of curcumin at the
preclinical level, it fails clinically because of its poor
bioavailability,14,15 rapid metabolism, and excretion.16,17 Thus, it
Our group has reported the use of the cyclic amphiphilic
peptide as a drug delivery system.31-33 Moreover, the amphiphilic
peptide was expected to increase the solubility of hydrophobic
molecules such as curcumin.34 Dox is a hydrophilic anticancer

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Tetrahedron Letters
agent. Cyclic [W(RW)4]-Dox significantly inhibited the
proliferation of human leukemia (CCRF-CEM) and breast
carcinoma (MDA-MD-468) cells and showed 3.6–fold higher
cellular uptake than Dox alone in SKOV-3 cells.33 We
Scheme 1. Synthesis of cyclic amphiphilic peptide [C(WR)4K2(β-A)] (4).
hypothesized that physicochemical and/or biological properties
of curcumin could be improved by using an amphiphilic cyclic
peptide-Dox conjugate. Here we report the synthesis and
evaluation of an anticancer agent using a cyclic amphipathic
peptide.
reagents, N,N’-diisopropylcarbodiimide (DIC) and 1-hydroxyl-7-
azabenzotriazole (HOAt), followed by side chain deprotection
generating the targeted amphiphilic cyclic peptide [C(WR)4K2(β-
A)] (4) (Scheme 1). The chemical structure of the conjugate
confirmed by MALDI-TOF/TOF MS, showing the peak 1883
Da, corresponding to [M + H]+.
2. Results and discussion
2.1. Chemistry
We have previously reported that cyclic peptides containing
alternative tryptophan and arginine residues act as a molecular
transporter.31,34 Here, a trifunctional cyclic peptide was prepared
through conjugation of two anticancer drugs with a cell-
penetrating cyclic peptide in multiple steps to improve the
physicochemical properties and biological activities.
First, amphiphilic cyclic peptide [C(WR)4K2(β-A)] was
synthesized using Fmoc/tBu solid phase peptide chemistry.31
Fmoc and side chain-protected amino acids, mainly tryptophan,
arginine, cysteine, lysine, and β-alanine were assembled on
preloaded Trp(Boc)-2-chlorotrityl resin (1) to generate side
chain-protected linear peptide (2). Piperidine (20% v/v) in DMF
was used to remove Fmoc group at N-terminal, followed by the
cleavage of the protected peptide from the resin with agitation in
a mild acidic solution (dichloromethane (DCM), trifluoroethanol
(TFE), and acetic acid) to generate the linear peptide with a free
carboxylic group (3).35 Under diluted and anhydrous conditions,
the linear protected peptide (3) was cyclized using coupling

3
Scheme 2. Synthesis of Dox-SS-Pyridyl (6) and Dox-cyclic
Cyclic peptide [C(WR)4K2(β-A)] was used for conjugation
with Dox through cysteine and using a disulfide bond. The
disulfide linkage is relatively stable in extracellular
compartments as compared to intracellular tumor fluid, where it
gets hydrolyzed due to elevated glutathione (GSH)
concentration.36-39 Therefore, Dox was modified with sulfhydryl
group and activated using dithiodipyridine to generate Dox-SS-
Pyridyl compound (6) (Scheme 2). The modified Dox (6) was
conjugated with the thiol group on a cysteine residue of the
cyclic peptide (4) under aqueous conditions affording Dox-SS-
cyclic peptide (7) (Scheme 2). MALDI spectra showed a distinct
peak at 2529 Da corresponding to [M + 4]+, in addition to two
fragments one related to extrusion tetracyclic anthracycline
aglycone at 2131 Da and other for protonated cysteinyl peptide at
1887 Da, which may be due to specific fragmentation of disulfide
bond under MALDI-MS.40
peptide conjugate (7).
tetracyclic anthracycline aglycone and protonated cysteinyl
peptide as described above previously. (Scheme 3).
2.2. The cytotoxicity assay of the synthetic compounds
The cytotoxicity of all compounds was tested in two different
cancer cell lines, human leukemia carcinoma cells (CCRF-CEM),
human ovarian carcinoma cells (SKOV-3), in addition to normal
kidney cell line (LLCPK), using MTS assay with concentration 5
µM and incubation time 24 h (Figure 1)and 72 h (Figure 2).
Curcumin did not inhibit the proliferation of CCRF-CEM cells
as expected because of low solubility and precipitation in cell
culture media. Cyclic peptide (4), Dox-attached cyclic peptide
(7), and DPCC (10) showed less cytotoxicity than Dox after 24 h
incubation. Dox-attached cyclic peptide (7) reduced the cell
proliferation by 73% after 72 h, but it was still less cytotoxic than
Dox alone (79%). The non-covalent physical mixture between
Dox, the cyclic peptide, and curcumin reduced the cell
proliferation by 78% and 84%, slightly higher than that of Dox
(77% and 79%) after incubation 24 h and 72 h, respectively.
Moreover, the physical mixture of Dox and the cyclic peptide
exhibited slightly more inhibition when compared with Dox
alone, showing 78% versus 77% after 24 h, and 82% versus 79%
after 72 h incubation.
Curcumin (8) was modified with glutaric anhydride41 using a
ring opening reaction of through a nucleophilic attack of one of
the phenolic group under on anhydride carbonyl group under a
basic condition, forming a stable ester linkage containing a free
carboxylic group as shown in Scheme 3. The synthesis of
monofunctional curcumin (9) permits the presence of one free
phenolic group which is found important for the antioxidant
activity of the molecule. The presence of glutarate chain
decreases the steric hindrance for conjugation with the cyclic
peptide- attached Dox (7).
In ovarian carcinoma cell line (SKOV-3), Dox-attached cyclic
peptide (7) was found to be more cytotoxic as compared to
compound DPCC (10) and reduced the cell proliferation by 55%
versus 30%, but still less active than Dox (62%) after 72 h
incubation. The physical mixing of Dox, [C(WR)4K2(β-A)], and
curcumin reduced the cell proliferation by 34% and 80%, a
slightly higher than Dox (22% and 62%) after 24 h and 72 h
incubation, respectively. Similarly, the physical mixture of Dox
and the peptide showed slightly higher inhibition when compared
with Dox (32% versus 22%) after 24 h incubation (Figures 1 and
2).
Dox-peptide-curcumin conjugate (DPCC) 10 was synthesized
by amide coupling reaction between the amine group of the
doxorubicin-cyclic peptide (7) and the monocarboxylic acid
group of curcumin compound (9) using phosphonium
hexafluorophosphate (PyBOP) and 1-hydroxybenzotriazole
derivatives (HOBt) under basic medium at room temperature for
three hours.42 The targeted compound was analyzed by MALDI,
showing the peak at 2993 Da corresponding to [M + 4]+. In
addition to two fragments 2597 and 2351 Da related to extrusion
Scheme 3. Synthesis of curcumin monoglutarate (9) and Dox-Cyclic Peptide-curcumin Conjugate (10).

4
Tetrahedron Letters
10 exhibited inhibition of proliferation of CCRF-CEM (73% and
In normal kidney cell line, cyclic peptide (4) was not
41%, respectively) and SKOV-3 (55% and 30%, respectively)
cells under similar conditions, suggesting selectivity of these
compounds towards cancer cells. The data demonstrate selective
cytotoxic effect by DPCC (10) and cyclic peptide-Dox conjugate
7 in cancer cells.
cytotoxic, but Dox-SS-cyclic peptide (7) and Dox-cyclic peptide-
curcumin (10) exhibited less than 37% and 31% cytotoxicity,
respectively, after 24 h. On the other hand, the physical mixture
of the cyclic peptide, curcumin, and Dox showed cytotoxicity
(50%) comparable to Dox (50%) after 24 h incubation but was
less toxic than Dox (55% vs 60%) after 72 h incubation.
Regarding the physical mixture between the cyclic peptide and
Dox, it showed slightly higher toxicity than Dox (51% versus
50%) after 24 h incubation but was slightly less toxic than Dox
(58% versus 60%) after 72 h incubation (Figures 1 and 2).
Conclusions
A synthetic methodology was developed for the synthesis of a
conjugate containing an amphipathic cyclic peptide, Dox, and
curcumin. The cyclic amphiphilic peptide containing lysine and
cysteine residues was synthesized using Fmoc/tBu solid phase
methodology. Both curcumin and Dox were modified by the
reaction of glutaric anhydride and 2-iminothiolane, respectively.
Dox-cyclic peptide (7) was reacted with curcumin monoglutarate
to Dox-cyclic peptide-curcumin, which was characterized by
MALDI-TOF. The purity of the compounds was confirmed by
analytical HPLC. The antiproliferative assay of the synthesized
compounds showed that Dox-cyclic peptide conjugate exhibited
a significantly higher cytotoxicity than the corresponding DPCC
(10) but still less than Dox in human leukemia and human
ovarian carcinoma cells. The physical mixture of Dox,
[C(WR)4K2(β-A)] and curcumin showed a considerable activity
higher than Dox after 24 h and 72 h incubation. Overall, DPCC
(10) and compound (7) were found to be more selective towards
cancer cells vs normal cells used after 72 h incubation.
120
LLCPK
SKOV-3
CCRF-CEM
100
80
60
40
20
0
Figure 1. Comparison of cytotoxicity between Dox (5 µM), curcumin (5
µM), and cyclic peptide (5 µM), Dox-SS-cyclic peptide (5 µM), DPCC (5
µM), and the corresponding physical mixture Dox (5 µM), cyclic peptide (5
µM), and curcumin (5 µM) against human leukemia, ovarian carcinoma cells
and normal kidney cell after 24 h.
Acknowledgments
The authors greatly acknowledge financial support for this
research from Chapman University School of Pharmacy and
Egyptian Cultural Affairs and Mission Sector, Egypt.
140
120
Supplementary Material
The experimental details, HPLC, NMR, and Mass spectra of
synthesized compound can be found in the supplementary file.
100
LLCPK
SKOV-3
80
CCRF-CEM
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60
40
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Highlights
• Doxorubicin and curcumin were conjugated
with a cell-penetrating peptide.
• Doxorubicin-peptide-curcumin conjugate
(DPCC) was synthesized.
•
DPCC was found to be more cytotoxic
against cancer cells versus normal cells.