Synthesis and antiproliferative activities of conjugates of paclitaxel and camptothecin with a cyclic cell-penetrating peptide

Article abstract of DOI:10.3390/molecules24071427

Cell-penetrating peptide [WR]₅ has been previously shown to be an efficient molecular transporter for various hydrophilic and hydrophobic molecules. The peptide was synthesized using Fmoc/tBu solid-phase chemistry, and one arginine was replaced with one lysine to enable the conjugation with the anticancer drugs. Paclitaxel (PTX) was functionalized with an esterification reaction at the C2⁰ hydroxyl group of PTX with glutaric anhydride and conjugated with the cyclic peptide [W(WR)₄K(βAla)] in DMF to obtain the peptide-drug conjugate PTX1. Furthermore, camptothecin (CPT) was modified at the C(20)-hydroxyl group through the reaction with triphosgene. Then, it was conjugated with two functionalized cyclic peptides through a formyl linker affording two different conjugates, namely CPT1 and CPT2. All the conjugates showed better water solubility as compared to the parent drug. The cytotoxicity assay of the drugs and their conjugates with the peptides were evaluated in the human breast cancer MCF-7 cell line. PTX inhibited cell proliferation by 39% while the PTX-peptide conjugate inhibited the proliferation by ~18% after 72 h incubation. On the other hand, CPT, CPT1, and CPT2 reduced the cell proliferation by 68%, 39%, and 62%, respectively, in the MCF-7 cell lines at 5 μM concentration after 72 h incubation.

Full text of DOI:10.3390/molecules24071427

molecules
Article
Synthesis and Antiproliferative Activities of
Conjugates of Paclitaxel and Camptothecin with a
Cyclic Cell-Penetrating Peptide
Naglaa Salem El-Sayed 1 , Amir Nasrolahi Shirazi , Muhammad Imran Sajid 1,4,
,2
1,3
Shang Eun Park , Keykavous Parang * and Rakesh Kumar Tiwari 1,*
1
1,
1
Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman
University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA;
nibrahim@chapman.edu (N.S.E.-S.); ashirazi@ketchum.edu (A.N.S.); sajid@chapman.edu (M.I.S.);
park327@mail.chapman.edu (S.E.P.)
Cellulose and Paper Department, National Research Center, Dokki 12622, Cairo, Egypt
Department of Pharmaceutical Sciences, College of Pharmacy, Marshall B. Ketchum University,
2
3
Fullerton, CA 92831, USA
Faculty of Pharmacy, University of Central Punjab, Lahore 54000, Pakistan
4
*
Correspondence: parang@chapman.edu (K.P.); tiwari@chapman.edu (R.K.T.);
Tel.: +1-714-516-+5489 (K.P.); +1-714-516-5483 (R.K.T.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Paula A. C. Gomes
Received: 13 February 2019; Accepted: 6 April 2019; Published: 11 April 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Cell-penetrating peptide [WR] has been previously shown to be an efficient molecular
5
transporter for various hydrophilic and hydrophobic molecules. The peptide was synthesized using
Fmoc/tBu solid-phase chemistry, and one arginine was replaced with one lysine to enable the
conjugation with the anticancer drugs. Paclitaxel (PTX) was functionalized with an esterification
0
reaction at the C2 hydroxyl group of PTX with glutaric anhydride and conjugated with the
cyclic peptide [W(WR) K(βAla)] in DMF to obtain the peptide-drug conjugate PTX1. Furthermore,
4
camptothecin (CPT) was modified at the C(20)-hydroxyl group through the reaction with triphosgene.
Then, it was conjugated with two functionalized cyclic peptides through a formyl linker affording
two different conjugates, namely CPT1 and CPT2. All the conjugates showed better water solubility
as compared to the parent drug. The cytotoxicity assay of the drugs and their conjugates with the
peptides were evaluated in the human breast cancer MCF-7 cell line. PTX inhibited cell proliferation
by 39% while the PTX-peptide conjugate inhibited the proliferation by ~18% after 72 h incubation.
On the other hand, CPT, CPT1, and CPT2 reduced the cell proliferation by 68%, 39%, and 62%,
respectively, in the MCF-7 cell lines at 5 µM concentration after 72 h incubation.
Keywords: cell-penetrating peptide; conjugate; camptothecin; cytotoxicity; MCF-7; paclitaxel;
solubility
1
. Introduction
Intensive efforts have been exerted for the development of an intracellular stimuli-responsive
drug delivery system that is stable under normal physiological conditions (e.g., in blood circulation)
but able to release its payload in the tumor site in a selective and efficient manner [ ]. The tumorigenic
process and microenvironmental characteristics of the tumor tissue have been extensively studied and
used to design and synthesize new targeted molecular therapies [ ]. More precisely, understanding
1
2,3
the tumor microenvironment allows researchers to develop different therapeutic strategies, based on
the numerous differences between the tumor microenvironment and the normal tissues [4–6].
Molecules 2019, 24, 1427; doi:10.3390/molecules24071427
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1427
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Cell-penetrating peptides (CPPs) are positively-charged short peptides with 5–30 amino acids that
are able to penetrate cell membranes and translocate different types of cargos into cells via different
mechanisms such as endocytosis [
and biomedical potential due to their biocompatibility and high level of tissue selectivity through
binding to specific cell receptors [ 12]. However, due to their peptide-based nature, they have poor
7]. Interestingly, some CPPs have shown attractive therapeutic
8–
in vitro and in vivo stability, which may demote their bioactivity [13]. Synthetic amphipathic CPPs
can tolerate the instability of natural CPPs while retaining their ability to permeate the cells and
deliver cargos. Furthermore, the physicochemical profile, self-assembly, receptor recognition, and
stimuli-responsive properties of amphipathic CPPs can be engineered by selecting the proper amino
acid sequences and peptide secondary structures, which are stabilized by hydrophobic–hydrophilic
side chain interactions [14]. Rational functionalization of side chains with acid labile, redox sensitive
bonds (ester bond and disulfide bridge), and tumor homing peptide motif, e.g., IKVAV, PEG, KGGVG,
and RGD, confer remarkable properties to CPPs, such as peptide stability, control over the rate, and
site of cargo release [15–17].
Several studies have revealed the impact of using CPPs as a molecular transporter for many
therapeutic agents, which could bind either through physical loading or via chemical conjugation and
could be released by microenvironmental stimuli, such as the pH, redox, irradiation, or enzyme [18–23].
Mandal et al. [24] reported the synthesis of a series of linear and cyclic CPPs with alternating
tryptophan (W) and arginine (R) amino acids. Mandal et al. study revealed that cyclic peptides [WR]4
and [WR] were efficient in translocating the fluorescently-labeled anti-HIV drug (3TC). Furthermore,
5
[
WR] enhanced the cellular uptake for a number of cell-impermeable phosphopeptides, including
4
0
0
0
0
the fluorescent-labeled F -GpYLPQTV, F -NEpYTARQ, F -AEEEIYGEFEAKKKK, F -PEpYLGLD,
F -pYVNVQN-NH , and F -GpYEEI in human leukemia cells (CCRF-CEM) [25]. Furthermore,
0
0
2
Shirazi et al. employed the cyclic peptide [W(WR) K] as a molecular transporter for the delivery of
4
doxorubicin (Dox) and curcumin (Cur) to different cancer cell lines by their loading in a physical
mixture or via conjugation to the peptide through a hydrolyzable ester bond with glutamate [21,26].
Dox-[W(WR) K] conjugate inhibited the cell proliferation of human leukemia (CCRF-CEM), colorectal
4
carcinoma (HCT-116), ovarian adenocarcinoma (SK-OV-3), and breast carcinoma (MDA-MB-468) cells
in a range of 50% to 79%. Flow cytometry evaluation exhibited a 3.6-fold higher uptake of the conjugate
versus Dox alone in SK-OV-3 cells. Intracellular hydrolysis of Dox from the conjugate was shown
within 72 h incubation. Meanwhile, cellular uptake of Cur by the CCRF-CEM cancer cell line was
improved by 5.7- and 4-fold for the physical mixture and the chemical conjugate, respectively, as
compared to Cur alone. In the meantime, the antiproliferative activity of Cur in the physical mixture
and the chemical conjugate was enhanced by almost 20% and 13%, respectively [21].
Paclitaxel (PTX) is a natural compound isolated from the Pacific Yew tree by Wall and Wani in
1
967 [27]. PTX interacts with β-tubulin located inside of the microtubules that induce the microtubules’
polymerization, causing the arrest of the cell cycle at the G2/M phase and inducing apoptosis in
cancer cells [28]. Camptothecin (CPT) is a natural product isolated from Camptotheca acuminate with
anticancer potency. CPT works by inhibiting type I DNA topoisomerase and inducing apoptosis in
tumor cells [29]. PTX and CPT have shown broad-spectrum cytotoxic potentiality against ovarian,
breast, non-small-cell lung, pancreatic, and brain cancers and Kaposi’s sarcoma [27,30]. Despite the
broad-spectrum antitumor activity of PTX and CPT, their clinical applications are hampered by their
poor aqueous solubility, limited bioavailability, cellular permeability, lack of tumor selectivity, and
serious adverse effects (e.g., myelosuppression and gastrointestinal toxicity) following intravenous
administration [30]. Several efforts have been devoted to the development of active formulations of
PTX and CPT to improve their water solubility and pharmacokinetic, cell membrane permeability, and
tissue selectivity. Nanoparticles, solid lipid nanoparticles, liposomal formulations, hyperbranched
block-copolymers, dendrimers, PEGylated polymeric materials, quantum dots, polymeric micelles,
and PEGylated poly(L-lysine) (PLL) have shown success, and some of these formulations entered
clinical trials [22,30–40].

Molecules 2019, 24, 1427
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In continuation of our efforts to design CPP-drug conjugates based on the CPPs containing
tryptophan and arginine residues, herein, we report the conjugation of PTX and CPT to the [W(RW) K]
4
peptide and compare the antiproliferative activity of the free drugs with the drug-peptide conjugate in
breast cancer cell line MCF-7.
2
. Results and Discussion
.1. Synthesis of Cyclic Peptide [(WR) K(βAla)] and Its Functionalization
2
4
Cyclic peptide [W(WR) K(
β
Ala)] contains alternative L-tryptophan (W) and L-arginine (R)
residues and was used in previous studies for the delivery of Cur and Dox to different
cancer cell lines [21 26]. [W(WR) K( Ala)] (P1) was prepared through the Fmoc/tBu
solid-phase chemistry using appropriate Fmoc-protected amino acids and the orthogonally
protected lysine residue with the Dde group [21 26]. The protected cyclic peptide
(W(Boc)R(Pbf)) W(Boc)K( Ala-Boc)] was obtained by the cyclization of the side chain-protected
linear peptide (HOOC-((W(Boc)R(Pbf)) W(Boc)K( Ala-Boc)-NH ) in anhydrous DMF/DCM mixture
4
,
β
4
,
[
β
4
β
4
2
(4:1, v/v) using HOAt/DIC as activating agents. The completion of cyclization was confirmed by
checking the mass of a crude sample taken from the reaction mixture as described in Section 3.2.1.
The cleavage of the protecting group using reagent “R” afforded the cyclic peptide P1 with the
+
exact mass of 1754.0300 corresponding to [M + H ] (see Supplementary Materials). The thiol
functionality was introduced to the cyclic peptide with the coupling of tritylthiopropionic acid in
DMF/DIPEA/HBTU. Completion of the reaction was confirmed by mass spectroscopy. The trityl
protecting group was cleaved from the thiol group using a mixture of TFA/TIS/H O. MALDI
2
−
mass spectroscopy revealed the exact mass at 1842.8826 [M
functionalization was performed using 2-(pyridinyldithio)ethaneamine which reacted with the cyclic
peptide P2. MALDI analysis showed the existence of a single peak at 1917.9098 [M + H] (see
−
H] for P2. The last step of peptide
+
Supplementary Materials) for the final product (P3) as depicted in Scheme 1. P1 was used for the
conjugation with both PTX-2-O-hemiglutarate (Scheme 2) and CPT-20-O-chloroformate (Scheme 3) to
afford PTX-1 and CPT1, respectively. P3 was used for conjugation with CPT-20-O-chloroformate to
afford CPT2 (Scheme 3) as described below.
2
.2. Coupling Hydrophobic Drugs to [W(WR) K(βAla)]
4
Coupling of PTX to [W(WR) K(βAla)]
4
0
PTX is not amenable for coupling unless it is chemically modified. Although PTX has 2 -hydroxyl
and 7-hydroxyl groups which are more susceptible to the chemical modifications. However, the
0
2
-hydroxyl group has superior reactivity than that at the 7-hydroxyl group due to the steric hindrance
at this position [38]. Therefore, PTX was functionalized by its reaction with glutaric anhydride, which
0
was reacted with the hydroxyl group at C2 with the ester linkage. The resulted 2’-O-hemiglutarate was
conjugated with [W(WR) K(βAla)] (P1) through the amide linkage using PyBOP, HOBt, and DIPEA
4
under N at room temperature. The conjugate was purified by RP-HPLC and analyzed by MALDI
2
+
that showed a single peak at 2703.9563 corresponding to [M + H] (see Supplementary Materials) for
the [W(WR) K(βAla-hemiglutarate-2-O-PTX)] conjugate (PTX1), as demonstrated in Scheme 2.
4
Similarly, CPT was conjugated to the [W(WR) K(βAla)] (P1) through an amide bond. Initially, CPT
4
with a C20-chloroformate functional group was prepared by the treatment of CPT with triphosgene
in the presence of DMAP. CPT-20-O-chloroformate was conjugated with P1 through two different
approaches; the first approach was through the pH-labile carbonate bridge, which bound directly to
the free β-alanyl amino group of the cyclic peptide in DMF using a catalytic amount of DIPEA, as
depicted in Scheme 3, to afford carbamate conjugate CPT1. The second approach depends on the
reductive-cleavage for the disulfide bridge and hydrolysis of carbonate linkage (the dual response
approach) in compound CPT2. Compound P3 was reacted with CPT-20-O-chloroformate in the

Molecules 2019, 24, 1427
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presence of DIPEA to afford CPT2. It was expected that in the second approach, the disulfide bridge in
[W(WR) K( Ala)-S-S-CPT] would be cleaved via reduction of the disulfide bridge by the intracellular
(
β
4
glutathione (GSH). Whereas, the concentration of GSH in the tumor tissue is 4 times higher than that
of the normal tissues [40]. Darwish et al. [41] have previously reported the impact of a disulfide bridge
in increasing the cytotoxic activity of Dox conjugated to [C(WR) K] when investigated in HEK-293,
4
HT-1080, and SKOV-3 cells as compared to Dox after 72 h incubation. The mechanism affording the
thiol-containing CPT followed by the carbonate bond cleavage liberating thiazolidinone and CPT and
five-membered thiolactam was demonstrated by Henne et al. and Zhang et al. [39,40].
(Boc)
N
O
N
(
Boc)
N
N
(Boc)
O
N
Fmoc Solid-Phase Synthesis
(Boc)
O
(Boc)
O
O
O
H
N
H
H
N
N
NH
NH
2
N
H
N
H
N
H
N
H
N
H
N
(Boc)
O
O
O
O
O
1
. Coupling of Fmoc-AA-OH/ HBTU/DIPEA
O
2
. Deprotection of Fmoc by 20% Piperidine in DMF
NH
NH
2
NH
NH
NH
NH
O
3. Dde removal by 2% hydrazine hydrate in DMF
O
NH
(Pbf)N
(Pbf)N
(Pbf)N
NH
2
(Pbf)N
NH
2
NH
2
2
resin
H-Trp(Boc)-2-chlorotrityl
NH
2
H N
NH(Boc)
2
NH
2
H
2
N
NH
O
NH
NH
H
N
1
2
3
4
5
. AcOH:TFE: DCM ( resin cleavage)
. HOAt/DIC/DMF/DCM (cyclization)
.Final Cleavage by Reagent "R"
. RP-HPLC purification
HO
S
HN
NH
O
O
O
O
HN
HN
NH
NH
NH
HN
. Lyophilization
O
O
N
H
1.
HBTU/DIPEA, /6h
N2
2. TFA/TIS/H2O, 4h
O
NH2
NH2
O
O
O
HN
O
NH
N
H
2
N
NH
NH
H
HN
2
NH
NH
NH
K(beta-Ala)]
P1
3
[
W(RW)
4
NH
NH
H
NH
O
N
H
2
N
NH
2
NH
H
N
2
H N
HN
HN
NH
HN
NH
O
HN
O
H
O
N
H
N
NH
NH2
NH
NH
NH
O
HN
O
HN
O
N
O
O
O
SH
H
O
MeOH/AcOH
NH
NH
HN
O
NH
O
NH
HN
O
HN
O
H
N
HN
O
O
H
2
N
O
NH
O
NH2.HCl
.
S
S
O
NH
HN
H N
NH
N
H
S
S
2
O
HN
HN
N
HN
O
H
O
NH
NH
NH
O
N
HN
NH
2
N
HN
NH
H
HN
NH
2
H N
2
MeOH/AcOH
N
H
HN
NH
P2
S
+
NH
HS
3
Cl
P3
S
N
N
Scheme 1. Synthesis of [W(WR) K(βAla)] and its functionalization with tritylthiopropionic acid and
4
2
-(pyridinyldithio)ethaneamine.
2
.3. Biological Activity
Our previous studies revealed that [W(WR) K(
β
Ala)] did not show any significant cytotoxicity up
4
to 10
µ
M on MDA-MB-468, HCT-116, CCRF-CEM, and SK-OV-3 [21 25 26]. Herein, the antiproliferative
,
,
activity of conjugate was evaluated in the presence of the human breast adenocarcinoma (MCF-7) cells.
Among breast cancer cells, MCF-7 ones were selected to be tested for this assay due to their significant
response to microtubule-targeting drugs [42,43].

Molecules 2019, 24, 1427
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O
O
O
O
O
O
O
O
O
O
NH
O
OH
NH
O
O
OH
O
O
O
O
O
OH
O
OH
H
OH
H
DCM/Pyridine
RT. 48 h
O
O
O
O
O
O
O
O
PTX
O
OH
PTX-2-O
-hemiglutarate
NH2
NH
HN
H N
2
NH
NH
HOBt/DIPEA
PyBOP,
DMF,
NH
H
N
7.5, N , r.t
pH
2
HN
O
NH
O
HN
O
NH
O
H
NH
N
NH
NH2
O
H N
2
NH
N
O
N
H
H
H
N
HN
HN
NH
O
O
O
O
NH
HN
O
H
O
HN
O
N
NH
NH
HN
NH
NH
NH
NH2
O
O
HN
HN
N
H
HN
O
H N
2
NH
HN
O
NH
O
NH2
HN
O
H
NH
O
O
N
O
NH
HN
NH
H N
2
HN
N
H
NH
HN
NH
O
O
NH2
O
HN
4K(b
O
N
H
O
O
[W(WR)
A)]
O
O
OH
O
O
H
O
O
O
HO
O
PTX-1
Scheme 2. Synthesis of PTX-O-hemiglutarate and its conjugation with [W(WR) K(βAla)].
4
Consequently, the concentration of 5
drug-peptide conjugates. Preliminary cytotoxicity investigation against MCF-7 cell lines showed that
both CPT and PTX reduced the cell proliferation of MCF-7 cells at 5 M by ~68% and 39%, respectively,
µM was selected to study the antiproliferation activity of the
µ
after 72 h using MTT assay. We have previously shown that the peptide-drug conjugates containing
doxorubicin and a similar cyclic peptide containing tryptophan and arginine were cleaved by 99% in
the presence of the cancer cells after 72 h of incubation as shown by HPLC analysis [26]. Therefore,
72 h was selected for the incubation of the conjugates in in vitro assays. Further analysis in a shorter
time may be beneficial for comparative studies. Although the cells could be incubated with drugs for
a longer period, the other elements such as sedimentation of the drug would have complicated the
antiproliferative outcomes.

Molecules 2019, 24, 1427
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O
O
O
N
O
N
1.5 equiv. of DMAP
DCM/N , 2h, RT
2
Cl
O
Cl
O
Cl
Cl
Cl
Cl
O
+
N
HO
N
O
O
O
Cl
O
CPT
CPT-20-O-chloroformate
NH
H
N
NH
O
NH
2
H
2
N
NH
NH
H
N
H
HN
NH
NH
O
N
NH
2
H
2
N
NH
NH
O
HN
O
H
HN
NH
O
N
H
NH
O
N
NH
HN
O
O
O
H
N
HN
O
N
N
NH
O
NH
HN
O
CPT-20-O-chloroformate
O
NH
O
O
O
NH
2
HN
O
NH
O
O
O
NH
O
N
H
H
DMF/ DIPEA, N , 24h, RT
2
O
O
HN
N
O
NH
2
H N
HN
H
O
O
N
N
O
NH
HN
NH
2
H N
NH
H
HN
NH
HN
N
H
NH
NH
NH
2
NH
HN
NH
2
HN
P1
CPT 1
NH
H
NH
H
NH
O
N
NH
N
NH
2
NH
2
H
2
N
NH
H
N
2
NH
H
HN
NH
H
N
HN
NH
N
NH
O
NH
HN
HN
O
O
O
O
CPT-20-O-chloroformate
H
H
O
N
NH
N
NH
O
DIPEA/DMSO, N
, 24h, RT
HN
O
HN
O
2
O
NH
O
NH
O
H
NH
O
NH
O
NH
HN
N
O
S
HN
O
H
H
O
O
S
N
O
O
NH
HN
NH
O
O
2
H N
N
O
NH
HN
NH
HN
H
2
N
S
N
H
HN
S
N
H
NH
2
NH
NH
NH
O
O
NH
NH
NH
2
O
O
NH
2
HN
O
HN
CPT 2
N
P3
N
Scheme 3. Synthesis of CPT-20-O-chloroformate and its conjugation with [W(WR) K(βAla)] and
4
[
(WR) W K(βAla-thiopropionyl-S-S-ethaneamine)] peptide.
4
Antiproliferative results showed that PTX1 inhibited cell proliferation by 18.7%.
The anti-proliferative activity of CPT1 was diminished by 1.9-fold as compared to CPT whereas the
activity of CPT2 was comparable to CPT, since CPT2 reduced the cell viability to 61% as shown in
Figure 1. Since these compounds were peptide-drug conjugates, it was expected that the release of the
parent drug would generate the cytotoxic effect. However, PTX1 was not very cytotoxic presumably
due to the stability of the conjugate. Among CPT conjugates, CPT2 exhibited higher antiproliferative
activity, due to the enhanced release of CPT. We presume that disulfide linker is reduced leading to
the release of active CPT from the conjugate. CPT1 and PTX1 conjugates had less activity than the
corresponding parent analogs since they act as inactive prodrugs and may behave differently on
longer incubation time. The cytotoxicity of PTX and PTX1 was further evaluated in the normal human
embryonic kidney cells (HEK-293) at 5 µM which showed reduced cell proliferation by ~34% and 18%,
respectively, after 72 h using MTT assay, as shown in Figure 2. The lower cytotoxicity of conjugate
PTX1 as compared to PTX in the normal cell lines could be due to slower hydrolysis of PTX from
conjugate PTX1.

Molecules 2019, 24, 1427
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1
20
00
1
80
60
40
20
0
Control
CPT
PTX
CPT1
CPT2
PTX1
Figure 1. The antiproliferative activity of camptothecin (CPT), paclitaxel (PTX), and their conjugates at
5 µM) concentration in MCF-7 cells after 72 h incubation.
(
1
20
00
1
80
60
40
20
0
Control
[W4R4K-ßA]
PTX
PTX1
Figure 2. The antiproliferative activity of PTX and PTX1 at (5
µM) concentration in HEK-298 cell line
after 72 h incubation.
3
. Materials and Methods
3
.1. Materials
All required organic solvents were purchased from Wilkem Scientific (Pawtucket, RI, USA).
The Fmoc-protected building block of amino acids, including Fmoc-Trp(Boc)-OH, Fmoc-Arg
Pbf)-OH, Dde-K(Boc)-OH, Boc- Ala-OH, 3-(tritylthio)propionic acid, and H-Trp(Boc)-2-chlorotrityl
(
β
resin, were purchased from Chem-Impex International Inc. (Wood Dale, IL, USA). Hydroxy-7-
azabenzotriazole (HOAT), 1,3-diisopropylcarbodiimide (DIC), O-(benzotriazole-1-yl)-N,N,N’,N’-
tetramethyluroniumhexafluorophosphate (HBTU), benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP), and hydroxybenzotriazole (HOBt), glutaric anhydride (95%),
triphosgene (99%), 4-dimethylaminopyridine (DMAP, 99%), N,N-diisopropylethylamine (DIPEA),
triethylamine (TEA),
β-mercaptoethylamine hydrochloride (cystamine hydrochloride), and
0
2,2 -dithiodipyridine were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA).
Camptothecin (CPT) was purchased from Santa Cruz Biotech, Inc. (Dallas, TX, USA) and Paclitaxel
PTX) was purchased from Euroasias Group Inc. (Woodbridge, NJ, USA).
(

Molecules 2019, 24, 1427
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3
.2. Methods
.2.1. Synthesis of Cyclic Peptide [W(RW) K(βAla)] (P1)
3
4
The linear peptide with protected side chains was prepared using Fmoc/tBu chemistry.
The peptide sequence was assembled on the H-Trp(Boc)-2-chlorotrityl resin (513 mg, 0.78 mmol/g,
.40 mM). After assembling the sequence, the Dde protecting group at the lysine -amino group
was removed by agitating with a solution of hydrazine monohydrate in DMF (2% v/v, 3 25 mL,
0 min). Then, the resin was washed with DMF (1 min 2) and DCM (1 min 2). The peptide
was cleaved from the peptidyl resin using a mixture of acetic acid/trifluoroethanol (TFE)/DCM
1:2:7, v/v/v) by stirring for 2 h at room temperature to afford the linear peptide with protected
side chains. The peptide was collected by filtration and washed with DCM (3 15 mL). The filtrate
0
α
×
1
×
×
(
×
containing the peptide was evaporated under reduced pressure. To remove acetic acid from the
cleaved peptide, the peptide was re-dissolved in 25 mL of DCM and precipitated with 50 mL of hexane,
and the process was repeated four times until the peptide was completely dried and acetic acid was
completely removed. The protected linear peptide was obtained as a solid white powder that was
dried under vacuum overnight. The cyclization of the linear peptide was carried out by dissolving
the peptide in DMF/DCM mixture (250 mL, 4:1 v/v). Then HOAt (223 mg, 1.64 mmol, 4 equiv) and
DIC (290 µL, 1.86 mmol, 4.5 equiv) were added to the peptide solution. The mixture was stirred at
room temperature for 12 h. The completion of the cyclization was confirmed with mass checked by
MALDI-TOF(shown in the Supplementary Materials). After the reaction was completed, the solvents
were removed under reduced pressure using a rotary evaporator. The cleavage of the protecting groups
from the amino acid side chains was carried out by adding reagent “R” containing trifluoroacetic
acid (TFA)/thioanisole/ethanedithiol(EDT)/anisole (90:5:3:2, v/v/v/v, 25 mL) to the cyclic peptide.
The mixture was shaken at room temperature for 6 h, and cold ether was added to precipitate the
crude peptide. The peptide was collected and purified by reversed-phase Hitachi HPLC (L-2455) using
a C18 column (Waters XBridgeTM BEH130 Prep C18 OBDTM 10
µm ODS reversed-phase column)
(
2.1 25 cm) using a gradient system of acetonitrile/water containing 0.1% TFA to yield (~40%)
×
the cyclic peptide [W(WR) K(
1
β
Ala)]. MALDI-TOF (m/z) [C H N O ]: calcd 1753.9331; found
4
88 115 29 11
+
+
755.0300 [M + 2H] , 1792.9736 [M + K] (shown in Supplementary Materials).
3
.2.2. Synthesis of [(WR) WK(βAla-Thiopropionic acid)] (P2) and [(WR) WK(βAla-Thiopropionyl-
4 4
ethaneamine] (P3)
[
W(WR) K(
β
Ala)] peptide (26.30 mg, 0.015 mmol) was dissolved in 1 mL of anhydrous DMF, then
a solution of 3-(tritylthio)propionic acid (6.1 mg, 0.0175 mmol, 1.16 equiv) dissolved in 1 mL of DMF
containing DIPEA (15.7 L, 6 equiv), and HBTU (6.6 mg, 0.0175 mmol) were added to the peptide
4
µ
solution, and the stirring was continued for 6 h under N at room temperature. After the reaction
2
was completed, the solvent was evaporated under reduced pressure until complete dryness. The dry
solid peptide was treated by a mixture of TFA/triisopropyl silane (TIS)/H O (10 mL, 7:2:1, v/v/v) and
2
stirred at room temperature for 4 h to cleave the trityl group. Then the peptide was precipitated with
cold diethyl ether and centrifuged, to yield the cyclic peptide [(WR) WK(
β
Ala-thiopropionic acid)]
4
(
P2) (23.3 mg, 93% yield): MALDI-TOF (m/z) [C H N O S]: calcd 1841.9314; found 1842.8826
91
119 29 12
+
[
M + H] , 1864.8585 [M + Na] (shown in Supplementary Materials).
The peptide [(WR) WK( Ala-thiopropionic acid)] P2 (20 mg, 0.011 mmol) containing free thiol
group was further functionalized by reaction with 2-(pyridinyldithio)-ethaneamine hydrochloride
7.35 mg, 0.033 mmol, 3 equiv) prepared using cystamine and dithiopyridine according to the method
β
4
(
described by Zugates et al. [37] in methanol containing a catalytic amount of acetic acid. The reaction
was stirred overnight at room temperature, and then the peptide (P3) was collected by precipitation
with cold ether and purified by reversed-phase HPLC as mentioned above. The yield was 87.75%
+
(
1
17.55 mg). MALDI-TOF (m/z) [C H N O S ]: calcd 1916.9456; found 1917.9089 [M + H] ,
941.9098 [M + Na] (shown in Supplementary Materials).
93
124 30 12 2
+

Molecules 2019, 24, 1427
9 of 13
3
.2.3. Synthesis of Paclitaxel-2-O-Hemiglutarate
Paclitaxel-2-O-hemiglutarate was prepared according to the method described by
Sundaram et al. [38]. In brief, paclitaxel (100 mg, 0.117 mM) and glutaric anhydride (13.70 mg,
.12 mM) were dissolved in 15 mL of DCM, and 10 L of dry pyridine was added to the reaction
0
µ
mixture as a base catalyst. The reaction was stirred under nitrogen for 48 h at room temperature.
The progress of the reaction was monitored by TLC using hexane:ethyl acetate (7:3, v/v). The crude
mixture was purified by silica gel chromatography. The compound was obtained as a white solid
+
(
76 mg, 95% yield). HR-MS (ESI) (m/z) [C H NO ]: calcd 967.3626; found 968.3479 [M + H] .
52
57
17
3
.2.4. General Procedure for the Synthesis of PTX-1 from Coupling of [W(WR) K(βAla)] Peptide with
4
PTX-2-O-Hemiglutarate
PTX-2-O-hemiglutarate (1.2 equiv, 11.6 mg, 1.2 mM), benzotriazol-1-yloxytripyrrolidino-
phosphoniumhexafluorophosphate (PyBOP, 1.5 equiv, 7.81 mg, 0.015 mM), and 1-hydroxybenzo-
triazole (HOBt, 2.5 equiv, 3.38 mg, 0.025 mM) were mixed together in a glass vial under nitrogen
atmosphere using 2 mL of anhydrous DMF to activate the carboxylate group of the hemiglutarate
moiety for 10 min. Then a solution of [W(WR) K(
β
Ala)] peptide (1 equiv, 17.54 mg, 1 mM) in
4
DMF containing DIPEA (3 equiv, 5
µL, 0.03 mM) was added to the activated PTX-2-O-hemiglutarate
mixture dropwise over 10 min. The reaction mixture was stirred for 2–3 h in the dark under N2
gas. The progress of conjugation was monitored by checking the mass of a small sample taken from
the reaction mixture by MALDI-TOF to confirm the formation of the PTX-peptide conjugate. After
the reaction was completed, the crude product was precipitated by adding cold diethyl ether and
centrifuged. The peptide was dried under nitrogen gas, dissolved in acetonitrile/water (50% v/v), and
purified by RP-HPLC affording [W(WR) K(βAla-hemiglutarate-2-O-PTX)] conjugate (PTX-1), which
4
was lyophilized (5.5 mg, 18.9% yield). MALDI-TOF (m/z) [C140H170N O ]: calcd 2703.2852; found
30
27
+
2
703.9563 [M] (shown in Supplementary Materials).
3
.2.5. Synthesis of Camptothecin-20-O-Chloroformate
Camptothecin-20-O-chloroformate was prepared as described by Henne et al. [39]. In brief, CPT
863 mg, 2.5 mM) and DMAP (763 mg, 6.25 mM) were dissolved in 15 mL of dry CH Cl under N
(
2
2
2
atmosphere. Triphosgene (0.250 mg, 0.84 mM) dissolved in 5 mL of dry DCM was added to CPT
◦
solution at 0 C. The mixture was stirred for a further 60 min. Then the reaction mixture was dissolved
in 50 mL of DCM and extracted by addition of 1.0 M HCl solution (2
×
50 mL), and brine (2
×
50 mL).
The organic layer was collected and dried over anhydrous Na SO . DCM was evaporated and used
2
4
without further purification to give a pale-yellow powder (761 mg, 88% yield).
.2.6. Synthesis of CPT1 from Coupling of [W(WR) K(βAla)] Peptide with CPT-20-O-Chloroformate
3
4
[
W(WR) K(
β
Ala)] (1 equiv, 17.54 mg, 1 mM) peptide was dissolved in 1 mL of anhydrous
DMF, and CPT-20-O-chloroformate (1.2 equiv, 4.92 mg, 1.2 mM) dissolved in 5 mL of anhydrous
DCM containing (3 equiv, 5 L, 0.03 mM) of DIPEA was added to the peptide solution,
4
µ
and the reaction was continued for 24 h. The progress of conjugation was monitored by
checking the mass using MALDI-TOF. Once the reaction was completed, the crude product
was precipitated by adding cold diethyl ether and centrifuged, dried under N2. The dried
solid compound was dissolved in acetonitrile/water (50% v/v) and purified by RP-HPLC.
The purified CPT-20-O-formyl-[W(WR) K(
β
Ala)] (CPT1) conjugate was lyophilized (7.56 mg, 33.40%
4
+
yield). MALDI-TOF (m/z) [C109H129N O ]: calcd 2128.0234; found 2128.6907 [M] (shown in
31
16
Supplementary Materials).

Molecules 2019, 24, 1427
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3
.2.7. Synthesis of CPT2 from Coupling of [(WR) WK(βAla-thiopropionyl-S-S-ethaneamine)] with
4
CPT-20-O-chloroformate
CPT-20-O-chloroformate (1.2 equiv, 4.92 mg, 1.2 mM) was dissolved in 5 mL of anhydrous DCM
containing DIPEA (3 equiv, 5 L, 0.03 mM). The peptide (1 equiv, 17.54 mg, 0.1 mM) was dissolved
in 1 mL of anhydrous DMSO and added dropwise over 10 min to CPT-20-O-chloroformate solution.
µ
The reaction mixture was stirred for 24 h at room temperature under N , and the reaction progress
2
was monitored using MALDI-TOF to check the formation of CPT-peptide conjugate. Once the reaction
was completed, the crude product was precipitated by adding cold diethyl ether, centrifuged, and
dried under N . The dried solid compound was dissolved in acetonitrile/water (50%, v/v) and
2
purified by RP-HPLC (6.21 mg, 25.8% yield). The purified [W(WR) K(βAla—formyl-O-20-CPT)]
4
(
2
CPT2) conjugate was lyophilized. MALDI-TOF (m/z) [C114H138N O S ]: calcd 2291.0359; found
291.9724 [M] (shown in Supplementary Materials).
32
17 2
+
3
.2.8. Cell Culture
Human breast cancer cell line (MCF-7, ATCC# HTB-22™) cells and human embryonic kidney
cells (HEK-293, ATCC# CRL-1573) were purchased from the American Type Culture Collection
2
(
Manassas, VA, USA). All MCF-7 cells were cultured using 75 cm cell culture flasks. A complete
®
™
Eagle’s minimum essential medium (EMEM) (ATCC 30-2003 ) containing fetal bovine serum (FBS,
1
0
0%), and penicillin-streptomycin solution (1%, penicillin (10,000 units) and streptomycin (10 mg in
◦
.9% NaCl) were used under the atmosphere of CO (5%) and air (95%) at 37 C.
2
3
.2.9. Antiproliferative Assay
The comparative antiproliferative assay was conducted using the MTS cell viability method to
evaluate the potency of the conjugates to inhibit the proliferation of MCF-7 breast cancer cells and
HEK-293 normal embryonic kidney cells. The cells were seeded (5000/0.1 mL) in each well using
a 96-well plate. The MCF-7 cells were treated with PTX and CPT drugs and their corresponding
conjugates in the medium. The final concentrations of all the compounds were adjusted to be 5 µM.
Cells were incubated with the treatments for 4 h. Then, the treatments were removed and replaced by
the fresh medium and incubated for a further 72 h. A similar treatment was followed for the HEK-293
cell line with PTX1, PTX, and cyclic peptide [W(WR) K(βAla)] at 5 µM. The CellTiter 96 aqueous
4
solution (Promega, Madison, WI, USA) was used to measure the cell viability based on the fluorescence
intensity of them at 490 nm. Here, SpectraMax M2 microplate spectrophotometer was employed for
the assay. The cell viability was calculated relatively based on the cell survival as [(OD value of cells
treated with the test mixture of compounds)
cells) − (OD value of culture medium)] × 100%.
−
(OD value of culture medium)]/[(OD value of control
4
. Conclusion
A cyclic cell-penetrating peptide [W(WR) K(
β
Ala)] was synthesized and used as a molecular
4
cargo for hydrophobic anticancer drugs CPT and PTX. The drugs, CPT and PTX, were functionalized
with a linker and conjugated with the peptide. The conjugates showed better water solubility due to
their attachment to [W(WR) K(βAla)] which promises improved bioavailability and pharmacokinetic
4
profiles. The antiproliferative activities of the peptide-drug conjugates were less than the free
hydrophobic drugs in MCF-7 after 72 h incubation, which suggests the prodrug formation of CPT and
PTX. The CPT conjugate named [W(WR) K-(
β
Ala)]-S-S-CPT (CPT2) was found to be more cytotoxic
4
when compared with [W(WR) K-(
βAla)]-CPT (CPT1), suggesting the fast release of CPT analog in this
4
conjugate. Future studies will be conducted to explore the potency of these conjugates on different cell
lines and to understand the actual mechanism for the release of PTX and CPT from the peptide and
improving the conjugation strategy of the drugs to the peptide.

Molecules 2019, 24, 1427
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Supplementary Materials: The MALDI spectra of selected compounds are provided.
Author Contributions: R.K.T. and K.P. planned and designed the experiments; N.S.E.-S. and M.I.S. synthesized
peptides, the drug modifications, and conjugation; A.N.S. and S.E.P. performed the cell assays; N.S.E.-S., K.P., and
R.K.T. contributed reagents/materials/analysis tools; N.S.E.-S., K.P., and R.K.T. wrote the manuscript.
Acknowledgments: The authors greatly acknowledge financial support for this research from the Chapman
University School of Pharmacy.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available for short time from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(