Novel N-Methylated Cyclodepsipeptide Prodrugs for Targeted Cancer Therapy

Article abstract of DOI:10.1021/acs.jmedchem.0c01387

Coibamide A (1) is a highly N-methylated cyclodepsipeptide with low nanomolar antiproliferative activities against various cancer cell lines. In previous work, we discovered a simplified analogue, [MeAla3-MeAla6]-coibamide (1a), which exhibited the same inhibitory abilities as coibamide A. Herein, to reduce the whole-body toxicity and improve the solubility of 1a, two novel peptide-drug conjugates RGD-SS-CA (2) and RGD-VC-CA (3) were designed, synthesized, and evaluated. Composed of cyclodepsipeptide 1a, a tumor-homing RGD motif, and a conditionally labile linker, the conjugates are expected to release 1a tracelessly in specific tumor microenvironments. Compared with RGD-VC-CA (3), RGD-SS-CA (2) proved to be superior in in vitro drug release and cytotoxicity tests. Notably, intravenous injection of RGD-SS-CA (2) into mice-bearing human tumor xenografts induced significant tumor growth suppression with negligible toxicity. Therefore, as a novel prodrug of the coibamide A analogue, conjugate 2 has great potential for further exploration in cancer drug discovery.

Full text of DOI:10.1021/acs.jmedchem.0c01387

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Article
Novel N‑Methylated Cyclodepsipeptide Prodrugs for Targeted
Cancer Therapy
Chunlei Wu,# Zhehong Cheng,# Danyi Lu, Ke Liu, Yulian Cheng, Pengxin Wang, Yimin Zhou,
Meiqing Li, Ximing Shao, Hongchang Li,* Wu Su,* and Lijing Fang*
Cite This: J. Med. Chem. 2021, 64, 991−1000
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ABSTRACT: Coibamide A (1) is a highly N-methylated cyclodepsipeptide with
low nanomolar antiproliferative activities against various cancer cell lines. In
previous work, we discovered a simplified analogue, [MeAla3-MeAla6]-coibamide
(1a), which exhibited the same inhibitory abilities as coibamide A. Herein, to
reduce the whole-body toxicity and improve the solubility of 1a, two novel
peptide−drug conjugates RGD-SS-CA (2) and RGD-VC-CA (3) were designed,
synthesized, and evaluated. Composed of cyclodepsipeptide 1a, a tumor-homing
RGD motif, and a conditionally labile linker, the conjugates are expected to release
1a tracelessly in specific tumor microenvironments. Compared with RGD-VC-CA
(3), RGD-SS-CA (2) proved to be superior in in vitro drug release and cytotoxicity
tests. Notably, intravenous injection of RGD-SS-CA (2) into mice-bearing human
tumor xenografts induced significant tumor growth suppression with negligible
toxicity. Therefore, as a novel prodrug of the coibamide A analogue, conjugate 2
has great potential for further exploration in cancer drug discovery.
cancer cells but also greatly inhibited tumor growth in vivo.11
As reported by Ishmael et al., coibamide A could cause
different patterns of weight loss at the dosage of 0.3 mg/kg
through intratumoral injection.7 However, we did not observe
obvious weight losses and side effects in the 1a-treated group
at the same dosage by subcutaneous injection, suggesting that
1a might have lower toxicity than 1. In a following study, we
found that the intravenous injection of 1a at the dosage of 1
mg/kg would result in rapid weight loss in the treated mice. In
addition, the solubility of 1a is very poor in a physiological
environment. In view of these limitations, we focused our
efforts on the development of effective targeted delivery
strategies that could be used to temporarily circumvent the
bioactivity and enhance the therapeutic efficiency of coibamide
A and its analogues.
INTRODUCTION
■
Cyclic peptides discovered from natural sources are a
privileged and yet underexploited class of compounds for
drug discovery.1−3 Coibamide A (1), isolated from a marine
filamentous cyanobacterium by McPhail and co-workers, is a
highly N-methylated cyclodepsipeptide.4 Such structural
features endow coibamide A with superior pharmacological
parameters such as metabolic stability, lipophilicity, and
receptor selectivity.5,6 As a potent cancer cell toxin, coibamide
A inhibits the proliferation, migration, and invasion of cancer
cells at subnanomolar concentrations.7 Ishmael et al. have
determined that coibamide A induces a rapid and sustained
autophagic response via an mTOR-independent mechanism
and also induces morphologically and biochemically distinct
forms of cell death according to the cell type.8 Coibamide A
directly binds to Sec61α through a distinct binding mode,
resulting in a broad substrate-nonselective inhibition of the
endoplasmic reticulum (ER) protein import and potent
cytotoxicity against specific cancer cell lines.9 Hence, there
has been considerable interest in the use of coibamide A as a
promising lead agent in cancer drug discovery.
Stimuli-responsive peptide−drug conjugates (PDCs), called
“smart” prodrugs, due to their site-specific and effective drug
release characteristics, have been deemed promising agents to
indirectly enhance the therapeutic efficiency and reduce the
severe side effects of highly toxic agents.12,13 Compared with
antibody−drug conjugates (ADCs), “smart” PDCs can be
In previous work, our group accomplished the first total
synthesis of coibamide A and revised two stereochemical
assignments of the originally proposed structure.10 Through
structure−activity relationship (SAR) studies, we discovered a
simplified analogue, [MeAla3-MeAla6]-coibamide A (1a)
(Figure 1), which not only showed nearly the same
antiproliferation activities as coibamide A against the tested
Received: August 24, 2020
Published: January 8, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c01387
© 2021 American Chemical Society
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Figure 1. Structures of coibamide A and its simplified analogue [MeAla3-MeAla6]-coibamide (1a).
easily prepared by conjugating cytotoxic drugs and specific
targeting peptides via conditionally labile linkers, with the
advantages of small size, structural homogeneity, and
penetration deep into tumors.14 The utilization of peptides
allows the tuning of the physicochemical and biological
properties of drugs, in addition to providing targeting ability
and improving hydrophilicity.12 The conditionally labile linkers
responsive to pH,15 redox,16,17 enzymes,18,19 and reactive
oxygen species (ROS),20,21 among others, have been
developed to temporarily mask the toxicity of drugs and
release active molecules under specific tumor microenviron-
ments. In recent years, self-immolating spacers, such as p-
aminobenzyl derivatives, that can be spontaneously eliminated
to yield active molecules using enzyme or glutathione (GSH)
have been utilized in drug delivery and fluorescence-based
probe design.22−25 To connect with p-aminobenzyl derivatives,
functional groups such as amino or hydroxy groups in drugs
are commonly required for carbamate or carbonate formation.
Although coibamide A and its analogue 1a have no such
functional groups, we noticed that the tertiary amine on their
N-terminus could be used to connect with p-aminobenzyl
through quaternary ammonium salt formation. In addition, a
charged quaternary ammonium functional group would
provide better water solubility, decreased aggregation, and
potentially improved therapeutic properties, as discovered by
Pillow et al. for the assembly of ADCs with tertiary and
heteroaryl amines.25 Therefore, we anticipated that stimuli-
responsive peptide−drug conjugates could be constructed by
the reversible attachment of [MeAla3-MeAla6]-coibamide
(1a) to a targeting peptide in a p-aminobenzyl quaternary
ammonium salt (PABQ) form.
Integrin αvβ3 plays a major role in cell−matrix interactions
and in tumorgenesis and is overexpressed in the blood vessels
of solid tumors as well as proliferating tumor endothelial cells
and various tumor cells.26,27 A characteristic feature of αvβ3
integrin is its high binding affinity for the arginyl-glycyl-aspartic
acid (RGD) sequence.28,29 The RGD peptide motif was
designed to be pentacyclic peptides (cyclic RGDyK or RGDfK,
and cRGD) with the superiorities of high stability and
hydrophilicity. They have been widely applied in targeted
cancer imaging and therapy through conjugating with
fluorophores, drugs, or nanomaterials.30−33 Hence, we decided
to use the cRGD peptide as the targeting ligand to construct
the peptide−drug conjugates of 1a, thus endowing the
conjugates with targeting specificity and improving their
water solubility.
In the current work, two novel smart peptide−drug
conjugates RGD-SS-CA (2) and RGD-VC-CA (3) were
designed, synthesized, and evaluated. Composed of a tumor-
homing peptide with the cRGDyK sequence, cyclodepsipep-
tide 1a, and a reduction-cleavable disulfide linker or an
enzyme-responsive dipeptide linker, the conjugates are
expected to release 1a tracelessly in specific tumor micro-
environments with high levels of GSH and cathepsin B,
respectively. Compared with RGD-VC-CA (3), RGD-SS-CA
(2) proved to be superior in in vitro drug release and
cytotoxicity tests. Notably, RGD-SS-CA (2) significantly
suppressed tumor growth in a mouse tumor xenograft model
with negligible toxicity and thus has great potential for further
exploration in cancer drug discovery.
RESULTS AND DISCUSSION
■
Design of RGD-CA Conjugates. Two stimuli-responsive
RGD-CA conjugates, namely RGD-SS-CA (2) and RGD-VC-
CA (3), were designed by using cRGDyK (cyclic RGD) as a
tumor-homing peptide and [MeAla3-MeAla6]-coibamide A
(1a) as the drug. The structure and release mechanism of
conjugates 2 and 3 are shown in Figure 2. For RGD-SS-CA
(2), cyclic RGD and 1a were connected through a reduction-
cleavable disulfide linker with a p-aminobenzyl self-immolative
spacer. The cleavage of the disulfide bond by dithiothreitol
(DTT) or GSH would induce the intramolecular cyclization to
give intermediate 4 with a quaternary ammonium cation in the
benzylic position, which underwent 1,6-elimination of the p-
aminobenzyl group to release the tertiary amine, providing 1a
tracelessly. For RGD-VC-CA (3), an enzyme-responsive
dipeptide connected to the p-aminobenzyl group was used as
the linker, in which the valine−citrulline (Val−Cit, VC)
dipeptide could be cleaved at the C-terminus of Cit upon
exposure to cathepsin B (CTB), a protease upregulated in a
variety of cancers, leading to the release of free 1a through the
same benzylic quaternary ammonium intermediate 4.
Synthesis of Conjugates RGD-SS-CA (2) and RGD-VC-
CA (3). The synthetic routes of RGD-SS-CA (2) and RGD-
VC-CA (3) are shown in Scheme 1. Alcohol 5 was obtained
from bis(2-hydroxyethyl) disulfide, and dipeptide 9 was
prepared through the Fmoc-based solid-phase peptide syn-
thesis. For the synthesis of RGD-SS-CA (2), alcohol 5 was
reacted with p-aminobenzyl alcohol through a BTC-mediated
reaction to generate alcohol 6. Treatment of 6 with thionyl
chloride in DCM provided the benzyl chloride intermediate,
which was used directly to react with cyclodepsipeptide 1a,
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Figure 2. Schematic overview of RGD-SS-CA (2) and RGD-VC-CA (3) for stimuli-responsive release.
forming quaternary ammonium salt 7. RGD-SS-CA (2) was
then obtained by an alkyne−azide cycloaddition reaction of
cyclic RGD (8) and compound 7. For the synthesis of RGD-
VC-CA (3), dipeptide 9 was coupled with p-aminobenzyl
alcohol in the presence of DIC/HOAT to yield alcohol 10.
Similarly, as described above, chlorination of 10 with thionyl
chloride followed by the addition of 1a generated the
quaternary ammonium salt 11. Finally, RGD-VC-CA (3) was
prepared by the coupling of 11 bearing a maleimide group and
cyclic RGD (12) bearing a thiol group under mild conditions.
RGD-SS-CA (2) and RGD-VC-CA (3) were purified using
semipreparative RP-HPLC and were validated by HRMS
(Figures S5 and S9).
In Vitro Responsive Cleavage of RGD-SS-CA (2) and
RGD-VC-CA (3). To demonstrate the stimuli-responsive
release of active drug 1a, the two RGD-CA conjugates were
analyzed by RP-HPLC in the presence of DTT and CTB,
respectively. First, the stability of conjugates 2 and 3 was
studied in PBS at 37 °C. There was no obvious degradation
detected under these conditions, with more than 90% of the
conjugates remaining intact after 24 h (Figure S10). Next, a
solution of conjugate 2 (50 μM) was incubated with DTT
(150 μM) in PBS at 37 °C, and the mixture was analyzed at
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Scheme 1. Synthesis of RGD-SS-CA (2) and RGD-VC-CA (3)
different times. As shown in Figure 3A, after incubation with
DTT for 30 min, the peak of 2 (16.07 min) reduced
remarkably, while a new strong peak emerged at 18.08 min,
which was identified to be 1a. From 30 to 90 min, a gradual
decrease of 2 and an increase of 1a were observed. At 90 min,
the peak of 2 disappeared, indicating that this conjugate was
completely cleaved. For RGD-VC-CA (3), upon treatment
with CTB for 4 h, free 1a was also discovered at 18.08 min
(Figure 3B). However, this cleavage reaction was slow since
more than 40% of 3 still existed in the cleavage solution after
incubation for 24 h. These results indicated that RGD-SS-CA
(2) and RGD-VC-CA (3) were able to be cleaved to give free
1a in the presence of DTT and CTB, respectively, but the
CTB-responsive release was much slower than the reduction-
responsive release.
Cytotoxicity Evaluation. The cytotoxicity of RGD-SS-CA
(2), RGD-VC-CA (3), and 1a against the human breast cancer
cell line BT549 and the human non-small-cell lung cancer cell
line A549 was investigated using a standard CCK-8 cell
viability assay. Both of the cell lines are integrin αvβ3
positive.34,35 In our previous study, 1a exhibited high
inhibitory activities (in the nanomolar range) against a number
of cancer cell lines. The cytotoxicity of 1a is no different
between the two cell lines with the IC50 values of 18.0 8.0
nM in BT549 cells and 19.1 8.7 nM in A549 cells after 48 h
incubation (Table 1). The cytotoxicity of the three compounds
ranked from the greatest to the least: 1a > 2 > 3. As shown in
Figure 4A and Table 1, for BT549 cells, the IC50 values of
conjugates 2 (310.7 74.6 nM) and 3 (2.65 0.16 μM) were
decreased 16-fold and 146-fold, respectively, in comparison
with 1a (18.0
8.0 nM). For A549 cells, each conjugate
displayed similar cytotoxicity profiles to the BT549 cells
(Figure 4 and Table 1). Compared with 48 h incubation, the
cytotoxicity of 1a, 2, and 3 increased in both cell lines after 72
h incubation, but the change trend is similar (Figure S11 and
Table S1). In conclusion, the cytotoxicity of 1a was reduced
greatly through the strategy of forming peptide−drug
conjugates. Since the cytotoxicity of 3 was much less than 2,
we inferred that the cleavage of a disulfide linker occurred
more easily and faster in cancer cells compared with the
enzyme-responsive linker, which is consistent with the results
of the in vitro-responsive cleavage of the conjugates.
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Figure 3. Responsive cleavage of RGD-SS-CA (2) and RGD-VC-CA (3) in the presence of DTT and CTB, respectively, analyzed by RP-HPLC.
Table 1. IC50 Values of RGD-SS-CA (2), RGD-VC-CA (3),
and 1a against BT549 and A549 Cells after 48 h of
Treatment
the addition of 2 and coincubation for 48 h. According to the
cytotoxicity results (Figure 4C and Table 1), the IC50 value of
2 was greatly reduced from 261.7 95.5 nM (without GSH-
OEt) to 20.6 7.3 nM, which is almost the same as the IC50
compounds
calcd IC50 values SD (nM)
value of free 1a (19.1
8.7 nM). On the other hand, the
BT549
A549
influence of the cyclic RGD segment, which was generated
during the reductive cleavage of conjugate 2, could be excluded
since cyclic RGD (8) exhibited no cytotoxicity against both
BT549 and A549 cells (Figure 4A,B). Combined, these results
indicate that conjugate 2 has nearly the same potency as 1a
under intracellular reductive conditions.
In Vivo Anticancer Activities. We next examined the
antitumor activity and the toxicity profile of conjugates 2 and 3
in vivo. Before we studied antitumor activities on tumor-
bearing mice, we first compared the solubility of 2 and 3 with
1a, and discovered that 2 and 3 were about 10-fold more
soluble than 1a in 10% DMSO/PBS. We then evaluated the in
vivo toxicity of 1a, 2, and 3. Six-week Balb/c nude male mice
(for each group, n = 3) were treated with various dosages of 1a
and RGD-CA conjugates by intravenous injection every 2 days.
From day 3, we found that 1 mg/kg dosage of 1a caused rapid
weight loss in the treated mice (Figure S12), suggesting that 1a
had high toxicity and was not suitable for in vivo anticancer
evaluation. To our delight, no obvious weight losses were
observed in the group treated with 2 even at a dosage of 5 mg/
1a
18.0 8.0
310.7 74.6
19.1 8.7
a
a
RGD-SS-CA (2)
261.7 95.5
b
20.6 7.3
RGD-VC-CA (3)
2.65 0.16 (μM)
2.72 0.43 (μM)
a
b
Values were measured in the absence of GSH-OEt. Values were
measured in the presence of GSH-OEt. IC50 values are average of
triplicate. SD is the standard deviation of the mean.
Additionally, it is possible that conjugate 3 was located in the
cytoplasm after uptake by cells, where the CTB activity is low.
In contrast, the high concentration of GSH in cancer cell
cytoplasm can explain the higher cytotoxicity of 2.
Considering that conjugate 2 exhibited preferable cytotox-
icity compared to 3, we selected 2 to conduct the subsequent
experiments. The in vitro cytotoxicity of 2 was tested in the
presence of GSH-OEt, which can easily penetrate cell
membranes and thus increase intracellular GSH concentration
through ethyl ester hydrolysis in the cytoplasm.36 A549 cells
were preincubated with GSH-OEt (1 mM) for 2 h, followed by
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Figure 4. (A, B) Cytotoxicity evaluation of RGD-SS-CA (2), RGD-VC-CA (3), and cyclic RGD (8). (C) Cytotoxicity profiles of RGD-SS-CA (2)
in the presence of GSH-OEt (1 mM), compared with 1a. Each experimental point was performed in quadruplicate and each experiment was
repeated three times. The cell viability was measured by standard CCK-8 assay after 48 h of treatment.
Figure 5. In vivo toxicity and anticancer activity of RGD-SS-CA (2) in the mouse tumor xenograft model. In total, 1 × 107 A549 cells were injected
subcutaneously into the right flank of each Balb/c nude male mouse, aged 6 weeks. (A) Weight of mice (average SD) in the 2-treated group
(male, n = 7) and the control group (male, n = 7). (B) Tumor volume (average SD) of the 2-treated group and the control group. (C) Tumor
mass of the 2-treated group and the control group. (D) Photograph of the peeled-off tumor of each group. All statistically significant values were
calculated by one-way ANOVA. (***): P < 0.001. All experiments were performed according to the guideline of the Institutional Ethical
Committee of Animal Experimentation of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences.
kg. Surprisingly, although 3 exhibited less potency in the in
vitro cytotoxicity assay, 5 mg/kg of 3 killed all of the mice after
two injections, and even 2 mg/kg of 3 could cause severe
weight loss. We assume that the release of 1a from conjugate 3
may intensively occur in the critical organs of the mouse,
which can cause severe inflammation or organ failure. Since 2
was about 12-fold less potent than 1a in the in vitro
cytotoxicity assay, a relatively high dosage is needed to reveal
the antitumor activities. Moreover, we also want to evaluate
the toxicity of 2 at a high dosage in a relatively long term.
Therefore, we decided to choose a dosage of 5 mg/kg, equal to
2.7 mg/kg of free 1a, to proceed with the antitumor evaluation
using the A549 tumor xenograft model since the A549 cell line
is more sensitive to conjugate 2. To determine the antitumor
activity in vivo, tumor-bearing male mice (n = 7) were treated
with 5 mg/kg of 2 by intravenous injection every 2 days. As
expected, there were no obvious weight losses or other side
effects observed over 21 days (Figure 5A). Moreover, the
tumor size of the mice treated with 2 remained stable at 50−
150 mm3 without remarkable growth over 3 weeks, whereas
the tumors of the vehicle group (n = 7) continued to grow at a
steady rate. As shown in Figure 5B, the tumor size of the 2-
treated animals was significantly smaller (P < 0.001) than that
of the vehicle control. At the end of treatment, the mice were
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1
49%). H NMR (400 MHz, CDCl3): δ 2.80−2.85 (m, J = 4.8 Hz,
4H), 3.08 (s, 1H), 3.55−3.60 (t, J = 6.8 Hz, 2H), 3.80−3.85 (t, J = 4.5
Hz, 2H) ppm (Figure S1).
sacrificed, and the tumors were peeled off and weighed. The
average tumor mass of the 2-treated group was nearly 4-fold
smaller than that of the vehicle group (Figure 5C,D). These
results demonstrated the significant tumor suppression effect
of 2 with low side effects in the mouse tumor xenograft model.
Synthesis of Compound (6). To the solution of BTC (196 mg,
0.66 mmol) in anhydrous DCM (2 mL), compound 5 (359 mg, 2
mmol) and pyridine (242 μL, 3 mmol) in anhydrous DCM (2 mL)
were added at 0 °C. The resulting mixture was stirred at 0 °C for 30
min. Then, the mixture was concentrated in vacuo and the residue
was suspended in 2 mL of anhydrous DCM followed by the addition
of a mixture of p-aminobenzyl alcohol (368.8 mg, 3 mmol) and TEA
(416 μL, 3 mmol) in anhydrous DCM and THF (3 mL, 1:1, v:v). The
reaction mixture was stirred at room temperature for 30 min, then was
diluted with DCM (20 mL), washed twice with 1 M hydrochloric acid
(2 × 10 mL), and finally dried over anhydrous Na2SO4. The organic
phase was concentrated in vacuo to give the crude product, which was
purified by silica gel chromatography (PE:EtOAc = 2:1) to provide
CONCLUSIONS
■
In summary, we have developed two novel peptide−drug
conjugates, RGD-SS-CA (2) and RGD-VC-CA (3), in which
the highly N-methylated cyclodepsipeptide 1a was connected
with cyclic RGD, a tumor-homing peptide, by a reduction-
responsive or an enzyme-responsive linker via the quaternary
ammonium salt formation. The incorporation of cyclic RGD
and stimuli-responsive linkers not only endows the conjugates
with targeting specificity but also improves the solubility of 1a.
Compared with RGD-VC-CA (3), RGD-SS-CA (2) displayed
desirable stimulus-triggered release and higher inhibitory
activities in vitro. Notably, the significant tumor suppression
effect of RGD-SS-CA (2) with negligible toxicity was
demonstrated in the mouse tumor xenograft model. Therefore,
as a novel prodrug of the coibamide A analogue, conjugate 2
has great potential for further exploration in cancer drug
discovery. Moreover, the stimulus-triggered traceless release of
cyclodepsipeptide in specific tumor microenvironments
provides a new approach for further development of coibamide
A and its analogues in targeted cancer therapy.
1
compound 6 (225 mg, 34%) as a yellow oil. H NMR (400 MHz,
CDCl3): δ 2.35 (s, 1H), 2.85−2.89 (t, J = 6.8 Hz, 2H), 2.95−3.0 (t, J
= 4.8 Hz, 2H), 3.57−3.62 (t, J = 6.8 Hz, 2H), 4.39−4.61 (t, J = 4.8
Hz, 2H), 4.60 (s, 2H), 7.13 (s, 1H), 7.26−7.29 (d, J = 8.4 Hz, 2H),
7.34−7.36 (d, J = 8.0 Hz, 2H) ppm (Figure S2). 13C NMR (100
MHz, CDCl3): δ 37.6, 37.8, 50.0, 62.7, 64.8, 118.8, 128.3, 136.2,
137.1, 153.0 ppm (Figure S3).
Synthesis of Compound (7). Step 1. Compound 6 (108 mg,
0.329 mmol) was dissolved in anhydrous DCM (2 mL) and cooled to
0 °C. Thionyl chloride in DCM (1 mol/L, 362 μL, 0.362 mmol) was
added dropwise. Following the addition, the reaction mixture was
held at 0 °C for 30 min and then increased to room temperature.
After successive stirring for 30 min, the mixture was concentrated
under reduced pressure. The residue was dissolved in DCM (10 mL)
and washed with saturated NaHCO3 solution. The organic layer was
collected and dried over anhydrous Na2SO4. After removal of the
solvent under reduced pressure, the residue was dissolved in
anhydrous DCM (2 mL) and used directly without further
purification.
Step 2. To the DCM solution of the product obtained from step 1,
1a (30 mg, 0.024 mmol) and tetrabutylammonium iodide (60 mg,
0.16 mmol) were added, followed by the addition of DIEA (10 μL,
0.057 mmol). The reaction mixture was stirred at room temperature
for 5 h and monitored by RP-HPLC. The mixture was purified by
semipreparative RP-HPLC. After lyophilization, compound 7 was
obtained as a white powder (15 mg, 39%). HRMS (ESI) m/z: calcd.
for C75H112N14O16S2 [M]+ 1537.8521, found 1537.8517 (Figure S4).
Synthesis of RGD-SS-CA (2). To a solution of compound 7 (28
mg, 0.018 mmol) in THF (1.5 mL), cyclic RGD (8) (15 mg, 0.022
mmol) in water (1.4 mL) was added, followed by the addition of
sodium ascorbate (1.76 M in H2O, 50 μL) and CuSO4·5H2O (0.26 M
in H2O, 50 μL) successively. The reaction mixture was stirred at room
temperature overnight and monitored by RP-HPLC. The mixture was
purified by semipreparative RP-HPLC to yield RGD-SS-CA (2) (12
mg, 30%). HRMS (ESI) m/z: calcd. for C107H167N23O25S2 [M + H]2+
1119.0967, found 1119.09595 (Figure S5).
Synthesis of Compound (9). Commercially available 2-CTC
resin (0.5 g, 1 mmol/g, 0.5 mmol) was preswelled for 20 min in DCM
in a manual solid-phase peptide synthesis vessel (50 mL). The
solution was drained. Anhydrous DMF (6 mL), DIEA (260 μL, 1.5
mmol), and Fmoc-Cit-OH (156 mg, 0.5 mmol) were added to the
resin. The mixture was agitated for 2 h before the unreacted resin was
capped with acetic acid (200 μL). Then, the solvent was drained, and
the resin was washed with DMF (4 × 6 mL). A solution of 20%
piperidine in DMF (6 mL) was added to the resin, and the resulting
suspension was shaken for 5 min. Then, the solution was removed
from the resin. Again, a solution of 20% piperidine in DMF (6 mL)
was added to the resin, and the resulting suspension was shaken for
another 5 min. The solution was drained, and the resin was washed
with DMF (4 × 6 mL) and anhydrous DMF (6 mL). Fmoc-Val-OH
(678.8 mg, 2 mmol) and HATU (760.4 mg, 2 mmol) were dissolved
in anhydrous DMF (3 mL), and DIEA (1 mL, 6 mmol) was added to
the mixture. The solution was stirred for 1 min at room temperature
before it was transferred to the deprotected peptidyl resin. The
EXPERIMENTAL SECTION
■
Materials. Bis(2-hydroxyethyl) disulfide, BTC, p-aminobenzyl
alcohol, TBAI, DIEA, sodium ascorbate, copper sulfate pentahydrate,
DIC, HOAT, HATU, 6-maleimidohexanoic acid, and KI were
purchased from Energy Chemical. TEA, pyridine, methanesulfonyl
chloride, thionyl chloride, anhydrous DCM, anhydrous DMF, and
anhydrous THF were purchased from J&K Scientific. Fmoc-Val-OH,
Fmoc-Cit-OH, and 2-CTC resin were purchased from GL Biochem.
Cyclic RGD (8) and cyclic RGD (12) were purchased from
Synpeptide Co., Ltd.
Characterization Methods. Analytical RP-HPLC was performed
on an Agilent 1260 infinity system equipped with a DAD-UV detector
using an Agilent Poroshell 120, EC-C18 column (4.6 mm × 100 mm,
2.7 μm). The RP-HPLC gradient was started at 10% of B (CH3CN)
and then increased to 100% of B over 20 min (A: 0.1% TFA in water)
with a flow rate of 0.5 mL/min. The purity of the compounds (>95%)
was determined by HPLC. Semipreparative RP-HPLC was performed
on the ULTIMAT 3000 instrument (DIONEX). UV absorbance was
measured using a photodiode array detector at 220 and 254 nm. The
RP-HPLC gradient was started at 10% of B (CH3CN), and then
1
increased to 100% of B over 30 min (A: 0.1% TFA in water). H
NMR (13C NMR) spectra were recorded with a Bruker AV400 at 400
(100) MHz. Chemical shifts are referenced to the signals resulting
from the residual solvent. High-resolution mass spectra were
measured with an ABI Q-star Elite.
Synthesis of Compound (5). Bis(2-hydroxyethyl) disulfide (2.28
g, 15 mmol) and triethylamine (3.06 mL, 22 mmol) were dissolved in
10 mL of anhydrous THF. Methanesulfonyl chloride (1.22 mL, 15.7
mmol) was dissolved in 5 mL of anhydrous THF, and the solution
was added dropwise to the above mixture at 0 °C. The mixture was
stirred at room temperature for 30 min and concentrated in vacuo to
remove the solvent. The residue was suspended in 10 mL of DMF
followed by the addition of sodium azide (2.93 g, 45 mmol). The
slurry was stirred at 60 °C for 6 h. The reaction mixture was diluted
with 80 mL of water and extracted three times with ethyl acetate (3 ×
25 mL). The combined organic layer was collected and dried over
anhydrous Na2SO4. After removal of the solvent under reduced
pressure, the crude product was purified by silica gel chromatography
(PE/EtOAc = 6:1) to give compound 5 as a colorless oil (1.32 g,
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mixture was agitated for 1 h until a negative Kaiser test was observed.
Then, the solvent was drained, and the resin was rinsed with DMF (4
× 6 mL). Followed by the removal of the Fmoc group, 6-
maleimidohexanoic acid (422.4 mg, 2 mmol) was coupled to the
deprotected peptidyl resin using the same method as that used for
Fmoc-Val-OH. Then, the resin was washed with DMF (2 × 6 mL)
and DCM (4 × 6 mL). After the removal of the Fmoc group, a
solution of TFE and HOAc in DCM (TFE/HOAc/DCM = 1:1:8)
was added to the deprotected resin. The resulting mixture was shaken
at room temperature for 12 h. Then, the resin was removed by
filtration through a disposable propylene filter and washed with DCM
(20 mL). The organic solution was concentrated under reduced
pressure, and the residue was purified by semipreparative RP-HPLC.
After lyophilization, compound 9 was obtained (173 mg, 73%).
HRMS (ESI) m/z: calcd. for C21H34N5O7 [M + H]+ 468.2453, found
468.2442; calcd. for C21H33N5O7Na [M + Na]+ 490.2272, found
490.2269 (Figure S6).
Institute of Biological Science, Chinese Academy of Sciences, and
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with
high glucose, 10% fetal bovine serum (Gibco, New Zealand), 100 U/
mL penicillin, and 100 μg/mL streptomycin (Hyclone, USA). The
cells were incubated in a humidified cell incubator with 5% CO2 at 37
°C.
Cell Growth Inhibition Assay. In total, 3000−4000 cells were
seeded in flat-bottomed 96-well plates and were preincubated
overnight. Compounds were diluted in a medium to a gradient of
concentration and were used to treat with cells in quadruple for 48 h.
The medium was removed carefully, and the cell proliferation reagent
CCK-8 (10% in fresh medium, 200 μL) was added to each well and
incubated for 1 h at 37 °C. The absorbance at 450 nm was measured
by using a Multiskan GO (Thermo Fisher Scientific) and was directly
proportional to live cell numbers. Each experiment was repeated three
times using four replicates. IC50 values were calculated using
log(inhibitor) versus response fitting by GraphPad Prism 5.0 and
were reported as mean SD from three independent experiments.
Animals. Six-week Balb/c nude male mice were purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd. All
animal studies complied with the principles of care and use of
laboratory animals and were approved by the Institutional Ethical
Committee of Animal Experimentation of Shenzhen Institute of
Advanced Technology, Chinese Academy of Science.
In Vivo Antitumor Evaluation in a Mouse Tumor Xenograft
Model. Six-week Balb/c nude male mice were chosen to establish the
tumor xenograft model. After collection by centrifugation and washing
twice in cold PBS, 1 × 107 A549 cells were suspended in serum-free
DMEM with high glucose and were injected subcutaneously at the
right flank of each mouse. After the tumor size reached ∼50 mm3 (L
× W × 1/2 W), the animals were randomly divided into two groups
(for each group, n = 7). A dosage of 5 mg/kg RGS-SS-CA (2) in 10%
DMSO/PBS (1 mg/mL, 100 μL) was injected intravenously through
the tail vein every 2 days for a total of 10 times. Equal volume of 10%
DMSO/PBS was used as a vehicle control. Tumor size and mouse
weight were measured before each injection. After 10 times of
injections, the mice were sacrificed and the tumors were peeled off
and weighed.
Synthesis of Compound (10). Compound 9 (100 mg, 0.21
mmol), DIC (30 μL, 0.21 mmol), and HOAT (28.6 mg, 0.21 mmol)
were suspended in anhydrous DMF (5 mL). After activation for 1
min, p-aminobenzyl alcohol (28.4 mg, 0.23 mmol) was added. The
reaction mixture was stirred at room temperature until compound 9
was consumed (2−4 h). The mixture was purified by semipreparative
RP-HPLC. After lyophilization, compound 10 was obtained as a
yellow powder (82 mg, 68%). HRMS (ESI) m/z: calcd. for
C28H41N6O7 [M + H]+ 573.3073, found 573.3044; calcd. for
C28H40N6O7Na [M + Na]+ 595.2856, found 595.2849 (Figure S7).
Synthesis of Compound (11). Step 1. To a solution of
compound 10 (42 mg, 0.07 mmol) in anhydrous DMF (0.5 mL)
was added dropwise a solution of thionyl chloride in DCM (1 mol/L,
84 μL, 0.084 mmol) at 0 °C. The reaction mixture was held at 0 °C
for 30 min and then increased to room temperature. After successive
stirring for 30 min, the mixture was concentrated under reduced
pressure and purified by silica gel chromatography (DCM/MeOH =
1
5:1) to afford a yellow solid (18 mg, 44%). The H NMR spectrum
was consistent with the literature.25
Step 2. 1a (10 mg, 0.008 mmol) and KI (11.6 mg, 0.07 mmol)
were added to an anhydrous DMF (0.5 mL) solution of the product
obtained from step 1. The reaction mixture was stirred at 50 °C for 24
h and monitored by RP-HPLC. The mixture was purified by
semipreparative RP-HPLC. After lyophilization, compound 11 was
obtained as a white powder (6 mg, 42%). HRMS (ESI) m/z: calcd.
For C91H146N16O20 [M + H]2+ 891.5444, found 891.5463; calcd. For
C91H145N16O20Na [M + Na]2+ 902.5354, found 902.5357 (Figure
S8).
Synthesis of RGD-VC-CA (3). To a solution of 11 (7 mg, 0.004
mmol) in 30% acetonitrile and aqueous NH4HCO3 buffer (20 mM,
pH 8, 1 mL), cyclic RGD (12) (5.7 mg, 0.008 mmol) was added.
After being stirred at 0 °C for 2 h, the reaction mixture was purified by
semipreparative RP-HPLC to yield RGD-VC-CA (3) (6.9 mg, 68%).
HRMS (ESI) m/z: calcd. for C121H192N25O29S2 [M + H]2+
1245.19745, found 1245.1964 (Figure S9).
Statistical Analysis. All data are expressed as the mean
SD.
Statistical analyses were performed using one-way analysis of variance
(ANOVA) through GraphPad Prism 5.0 to assess between-group
differences. *P < 0.05 was considered statistically significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01387.
■
sı
NMR and HRMS spectra of the new compounds; RP-
HPLC analysis of compounds 2, 3, and 1a; stability
studies of 2 and 3; and the in vivo toxicity of 2 and 1a
(PDF)
In Vitro Drug Release. RGD-SS-CA (2) in DMSO (2 mM, 13
μL) was diluted to 50 μM with 10% DMSO/PBS (0.5 mL). After the
addition of DTT (15 mM, 5 μL), the mixture was incubated at 37 °C.
At predetermined time points, 30, 60, and 90 min, 50 μL of the
release medium was withdrawn and analyzed by RP-HPLC. RGD-VC-
CA (3) in DMSO (2 mM, 13 μL) was diluted to 50 μM in 10%
DMSO/citrate buffer (100 mM, pH 5.5, cysteine 20 mM, 0.5 mL).
After the addition of a stock solution of CTB (600 U/mL, 15 μL), the
mixture was incubated at 37 °C. At predetermined time points, 4, 12,
and 24 h, 50 μL of the release medium was withdrawn and analyzed
by RP-HPLC. Solutions of compound 2 or 3 in 10% DMSO/PBS (50
μM, 0.5 mL) without DTT and CTB were incubated at 37 °C for
stability studies. At predetermined time points, 0, 4, 8, 12, and 24 h,
50 μL of the solution was withdrawn and analyzed by RP-HPLC.
Each experiment was repeated three times.
Molecular formula strings (XLSX)
AUTHOR INFORMATION
Corresponding Authors
■
Hongchang Li − Guangdong Key Laboratory of
Nanomedicine, Institute of Biomedicine and Biotechnology,
Shenzhen Institute of Advanced Technology, Chinese
Academy of Sciences, Shenzhen, Guangdong 518055, China;
Phone: (+86)755-86585228; Email: hc.li@siat.ac.cn
Wu Su − Guangdong Key Laboratory of Nanomedicine,
Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China;
orcid.org/0000-0001-9958-3434; Phone: (+86)755-
86585203; Email: wu.su@siat.ac.cn
Cell Line and Culture. The human breast cancer cell line BT549
and the human non-small-cell lung cancer cell line A549 were
acquired from the Core Facility of Stem Cell Research, Shanghai
998
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J. Med. Chem. 2021, 64, 991−1000

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Article
Lijing Fang − Guangdong Key Laboratory of Nanomedicine,
Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China; University of
Chinese Academy of Sciences, Beijing 100049, China;
orcid.org/0000-0001-7355-3923; Phone: (+86)755-
86392414; Email: lj.fang@siat.ac.cn
21977111), the Shenzhen Science and Technology Innovation
Commission (Grant Nos. JCYJ20170818153538196 and
JCYJ20170818162642882), and the Natural Science Founda-
tion of Guangdong Province (Grant Nos. 2019A1515012073
and 2018B030308001). The authors appreciate the Peking
University Shenzhen Graduate School for the assistance of
Mass facility.
Authors
ABBREVIATIONS
■
Chunlei Wu − Guangdong Key Laboratory of Nanomedicine,
Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China
Zhehong Cheng − Guangdong Key Laboratory of
Nanomedicine, Institute of Biomedicine and Biotechnology,
Shenzhen Institute of Advanced Technology, Chinese
Academy of Sciences, Shenzhen, Guangdong 518055, China
Danyi Lu − Guangdong Key Laboratory of Nanomedicine,
Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China
BTC, bis(trichloromethyl)carbonate; CH3CN, acetonitrile;
DTT, dithiothreitol; DCM, dichloromethane; DIC, N,N′-
diisopropylcarbodiimide; DIEA, N,N′-diisopropylethylamine;
DMF, N,N′-dimethylformamide; GSH, glutathione; CTB,
cathepsin B; EtOAc, ethyl acetate; HOAc, acetic acid;
HOAt, 1-hydroxy-7-aza-benzotriazole; HATU, 2-(7-azabenzo-
triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-phos-
phate; MeOH, methyl alcohol; PE, petroleum ether; TBAI,
tetrabutylammonium iodide; TFA, trifluoroacetic acid; TFE,
2,2,2-trifluorethanol; TEA, triethylamine; THF, tetrahydrofur-
an
Ke Liu − Guangdong Key Laboratory of Nanomedicine,
Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China
Yulian Cheng − Guangdong Key Laboratory of Nanomedicine,
Institute of Biomedicine and Biotechnology, Shenzhen
Institute of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China
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https://pubs.acs.org/10.1021/acs.jmedchem.0c01387
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ACKNOWLEDGMENTS
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This work was supported by the National Natural Science
Foundation of China (Grant Nos. 21672254, 21778068, and
999
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