General Strategy for Integrated Bioorthogonal Prodrugs: Pt(II)-Triggered Depropargylation Enables Controllable Drug Activation in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.0c01435

Bioorthogonal decaging reactions for controllable drug activation within complex biological systems are highly desirable yet extremely challenging. Herein, we find a new class of Pt(II)-triggered bioorthogonal cleavage reactions in which Pt(II) but not Pt(IV) complexes effectively trigger the cleavage of O/N-propargyl in a variety of ranges of caged molecules under biocompatible conditions. Based on these findings, we propose a general strategy for integrated bioorthogonal prodrugs and accordingly design a prodrug 16, in which a Pt(IV) moiety is covalently connected with an O2-propargyl diazeniumdiolate moiety. It is found that 16 can be specifically reduced by cytoplasmic reductants in human ovarian cancer cells to liberate cisplatin, which subsequently stimulates the cleavage of O2-propargyl to release large amounts of NO in situ, thus generating synergistic and potent tumor suppression activity in vivo. Therefore, Pt(II)-triggered depropargylation and the integration concept might provide a general strategy for broad applicability of bioorthogonal cleavage chemistry in vivo.

Full text of DOI:10.1021/acs.jmedchem.0c01435

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Article
General Strategy for Integrated Bioorthogonal Prodrugs: Pt(II)-
Triggered Depropargylation Enables Controllable Drug Activation In
Vivo
Tao Sun,† Tian Lv,† Jianbing Wu,† Mingchao Zhu, Yue Fei, Jie Zhu, Yihua Zhang,
and Zhangjian Huang*
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ABSTRACT: Bioorthogonal decaging reactions for controllable
drug activation within complex biological systems are highly
desirable yet extremely challenging. Herein, we find a new class of
Pt(II)-triggered bioorthogonal cleavage reactions in which Pt(II)
but not Pt(IV) complexes effectively trigger the cleavage of O/N-
propargyl in a variety of ranges of caged molecules under
biocompatible conditions. Based on these findings, we propose a
general strategy for integrated bioorthogonal prodrugs and
accordingly design a prodrug 16, in which a Pt(IV) moiety is
covalently connected with an O2-propargyl diazeniumdiolate
moiety. It is found that 16 can be specifically reduced by
cytoplasmic reductants in human ovarian cancer cells to liberate
cisplatin, which subsequently stimulates the cleavage of O2-propargyl to release large amounts of NO in situ, thus generating
synergistic and potent tumor suppression activity in vivo. Therefore, Pt(II)-triggered depropargylation and the integration concept
might provide a general strategy for broad applicability of bioorthogonal cleavage chemistry in vivo.
activation, some challenges still remain. Cleavage reagents (B)
and caged active agents (A−C) are usually separated,7−10 and
the reactions between them lack control in terms of location,
limiting broad therapeutic applications in vivo.19,21 There are
published methods including intratumor injection of cleavage
reagents,22 supramolecular self-assembly of cleavage reagents
triggered by enzymes unique in cancer,23 and exosome loading
of ultrathin palladium nanosheets,24 which allow for enrichment
of the cleavage reactions in cancer.
Nevertheless, to the best of our knowledge, direct
modification of cleavage reagents to achieve the desired
selectivity for cancer cells has not been reported yet. To address
the traditional shortcomings of bioorthogonal activation
chemistry, we hypothesize that direct modification of cleavage
reagents (masked-B) could mask its uncaging ability but
selectively recover in a tumor microenvironment (Figure 1B),
and further covalent coupling of the modified cleavage reagents
(masked-B) with the caged active agents (A−C) would provide
INTRODUCTION
■
Bioorthogonal chemistry, which is defined as any facile and
friendly chemical reaction that can occur inside aqueous living
systems,1−4 has an increasing impact on chemical biology and
medicinal chemistry. To date, bioorthogonal chemistry has been
extensively utilized to modulate biomolecules of interest via
“bond formation” or “bond cleavage” reactions.5−8 Some
bioorthogonal bond cleavage reactions, in which cleavage
reagents (B) including transition-metals (Pd, Ru, and Au) and
tetrazines mediate uncaging reactions from caged active groups
(A−C), have been successfully employed to develop bio-
orthogonal cleavage reaction-based prodrugs in the arena of
cancer therapy (Figure 1A).9−19 For instance, gold-triggered
depropargylation from the prodrugs generated floxuridine or
doxorubicin, displaying potent anticancer activity,10 combina-
tion of 5-fluoro-1-propargyl-uracil and Pd(0)-functionalized
resins exhibited strong antiproliferative properties,11 as well as
tetrazine-mediated bond cleavage of 3-isocyanopropyl enabled
the controlled release of doxorubicin and mitomycin, signifi-
cantly inhibiting cancer cell growth.15 Compared with enzyme-
activatable prodrugs, bioorthogonal cleavage reaction-based
prodrugs rely on extrinsic and biocompatible agents to achieve a
superior spatiotemporal drug function, thus minimizing off-
targeting activation and consequent adverse effects.11,20
Received: August 25, 2020
Although much progress has been made in the past few years
in developing bioorthogonal cleavage reactions for prodrug
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Figure 1. (A) Previous work: cleavage reagent (B) triggers the uncaging process of caged active agents (A−C) to release the active group A. (B)
Hypothesis: masked-B could mask the uncaging activity but regenerate the uncaging activity in cancer cells. (C) This work: Pt(II) 1 effectively
catalyzes the depropargylation of 5a, whereas Pt(IV) 3 does not. Based on this finding, an integrated bioorthogonal prodrug 16 is designed and
synthesized for proof-of-concept. (D) Proposed mechanism of 16: as an inert Pt(IV) derivative, 16 could be specifically reduced by cytoplasmic
reductants in cancer cells to release 1 and O2-diazeniumdiolate 14. Then, 1 triggers the bond cleavage of O2-propargyl in 14 to liberate large amounts of
NO in situ, generating selective and synergistic anticancer activity in vivo.
a hitherto-unknown class of integrated bioorthogonal cleavage
reaction-based prodrug (masked-B-A-C, Figure 1B). It is
proposed that masked-B-A-C could be inert, avoiding
intermolecular bioorthogonal reactions. After being selectively
activated in living cancer cells, masked-B-A-C would liberate the
cleavage reagent B and the caged active group A−C, allowing the
former to subsequently trigger the bond cleavage of the latter to
release its active group A in situ. However, currently available
cleavage reagents would not be suitable for structural
modification to mask the uncaging activity. In this regard,
searching for a novel cleavage reagent which could be used to
construct masked-B-A-C is urgently needed for bioorthogonal
chemistry.
we had successfully developed a group of O2-propargylated
diazeniumdiolates as a new class of bioorthogonal cleavage
reaction-based nitric oxide (NO) prodrugs, which can be
effectively uncaged in the presence of Pd(0) within living cells.26
Since Pd and platinum (Pt) belong to the same transition-metal
family, we further hypothesized that Pt may have catalytic
properties similar to that of Pd. Accordingly, we selected some
common Pt compounds including PtCl2, PtCl4, Pt(II)
compounds cisplatin (1) and carboplatin (2), and Pt(IV)
derivatives diamine trichloro hydroxy platinum (3) and
(trimethyl)methylcyclopentadienyl platinum (4) to investigate
the depropargylation efficiency on O-propargyl 4-methylumbel-
liferone (compound 5a)12 by spectrofluorometry- and high-
performance liquid chromatography (HPLC)-based methods.
Interestingly, we found that Pt(II) 1 effectively catalyzed the
Given the extensive applications of the transition-metal
palladium (Pd)-mediated bioorthogonal chemical reactions,
B
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Figure 2. (A) Depropargylation reaction of non-fluorescent 5a to generate highly fluorescent 5 in the presence of equal concentration of Pt
compounds in phosphate-buffered saline (PBS), pH 7.4, containing 5% DMSO, at 37 °C. (B) 1, a representative Pt(II), but not its Pt(IV) complex 3,
effectively triggered the depropargylation reaction of 6a in PBS pH 7.4, containing 5% dimethyl sulfoxide (DMSO), at 37 °C to release NO. (C) 1-
triggered deprotections of O/N-propargyl/allyl/1,2-allene-yl/1,2-butadiene-yl from 4-methylumbelliferone, morpholine diazeniumdiolate, 5-fluoracil,
N-boc-L-tyrosine methyl ester, and 1,2: 3,4-di-O-isopropylidene-D-galactopyranose. (D) Proposed mechanism for Pt(II)-catalyzed depropargylation
reactions.
depropargylation of 5a, whereas Pt(IV) 3 was not able to
catalyze the same reaction (Figure 1C).
O-depropargylation, we design the first generation of integrated
bioorthogonal prodrug 16, in which the hydroxyl group of 3 and
the hydroxyl group of the O2-propargyl N-methylethanolamine
diazeniumdiolate are connected via a succinic acid moiety
(Figure 1D). We propose that 16 may be metabolically stable
without intermolecular bioorthogonal reactions but could be
specifically reduced by cytoplasmic reductants in cancer cells,
rather than in normal cells, to release 1 and O2-propargyl caged
diazeniumdiolate moiety. The former then catalyzes the bond
cleavage of O-propargyl in the latter to liberate large amounts of
NO in situ, together with 1, generating selective and synergistic
antiproliferative activity against cancer cells, while sparing
normal cells (Figure 1D).
Herein, we first study the chemistry of Pt-derived cleavage
reactions of O/N-propargyl in a range of molecules including a
fluorophore, an anticancer drug 5-fluoracil, tyrosine, NO donor
diazeniumdiolates, and a monosaccharide. The target com-
pound 16, its moieties, and analogue are then synthesized, and
the bioorthogonal cleavage reactions in cell-free solutions are
As it is known, some Pt(IV) complexes, such as ormaplatin,
iproplatin, satraplatin, and so forth,27are kinetically inert
platinum prodrugs under clinical trials and can be converted
to active Pt(II) upon reduction by high concentrations of
cytoplasmic reductants such as ascorbic acid in cancer cells, thus
exhibiting increased toxicity to cancer cells.28−34 In this regard,
Pt(IV) 3 could act as a masked cleavage reagent (masked-B) and
the hydroxyl group could be used for further coupling with the
caged active agents (A−C) to produce a novel class of integrated
bioorthogonal cleavage reaction-based prodrug (masked-B-A-C,
Figure 1B). NO is a potential cancer therapeutic agent and
several kinds of NO donors (diazeniumdiolate, furoxans, etc.)
have been proven to possess potent antiproliferative activity
against cancer cells.35−38 Furthermore, the synergistic effect of
NO and 1 in cancer therapy has been well described.39−41 Given
the concept of integrated bioorthogonal prodrug (masked-B-A-
C) and the findings that Pt (II) 1, but not Pt(IV) 3, can trigger
C
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Scheme 1. Synthetic Route of 16 and 17
investigated. NO-release behaviors, Pt accumulation properties,
and cancer cell growth inhibitory activity are further investigated
in normal and cancer cells as well as in zebrafish.
diazeniumdiolate (6a, 6b), the phenolic hydroxyl group in
tyrosine (8a, 8b), and the hydroxyl group in galactose (9a, 9b),
indicating its extensive catalytic scope (Figure 2C).
Next, we investigated the effects of temperature (Figure S4A),
water (Figure S4B), and pH values (Figure S4C) on the 1-
triggered depropargylation to reveal the underlying mechanism.
It was found that the reaction rate of 1-triggered depropargy-
lation at 37 °C was significantly greater than that at 25 °C, and
water was essential for this reaction. Meanwhile, pH 7.4 and 6.6
were preferred for the depropargylation, superior to the alkaline
condition, suggesting the desired biocompatibility of 1-triggered
depropargylation reaction. Given that hydroxyacetone or 3-
hydroxypropanal was detected in the reaction mixture by liquid
chromatography−mass spectrometry (LC−MS) (Figure S5)
and the mechanism of Pd-triggered depropargylation was
previously reported,9 we proposed one plausible mechanism
of 1-triggered depropargylation. As shown in Figure 2D, 1, as a
four-coordinate complex with unsaturated coordination on Pt
atom, could coordinate with O-propargyl group of compound 5a
in H2O to trigger the bond cleavage reaction the same as Pd,
whereas Pt(IV) 3, a saturated coordination complex, may not be
able to react with propargyl group to trigger the depropargy-
lation reaction.
Synthesis of Integrated Bioorthogonal Prodrug 16
and Its Analogue 17. The synthetic route of target compound
16 and its O2-methyl analogue 17, in which the O2-methyl group
could not be deprotected by Pt(II), is depicted in Scheme 1.
Briefly, the reaction of NO gas (50 psi) with N-methylethanol-
amine (10) in the presence of NaOMe in dry ether offered
diazeniumdiolates sodium salt 11, which was subsequently
treated with propargyl bromide or iodomethane to produce O2-
propargyl 12 and O2-methyl 13, respectively. Then 12 and 13
were individually treated with succinic anhydride to furnish 14
and 15. Finally, the reactions of 14 or 15 with 3 in the presence
of TBTU in dimethylformamide (DMF) generated the target
compounds 16 and 17, respectively.
RESULTS AND DISCUSSION
■
Chemistry of Pt-Derived Cleavage Reactions of O/N-
Propargyl. We first selected some common platinum
compounds including PtCl2, PtCl4, Pt(II) compounds cisplatin
(1) and carboplatin (2), and Pt(IV) derivatives diamine
trichloro hydroxy platinum (3) and (trimethyl) methylcyclo-
pentadienyl platinum (4) to investigate the depropargylation
efficacy on non-fluorescent O-propargyl 4-methylumbelliferone
(compound 5a) to generate highly fluorescent 5 by using
Pd(dba)2 as a positive control via fluorometry (Figure S1) and
HPLC assays (Figure S2). It was found that PtCl2 and PtCl4
effectively triggered the depropargylation of 5a to generate 5,
superior to Pd(dba)2. Interestingly, Pt(II) 1 exhibited more
catalytic activity than 2, whereas Pt(IV) 3 and 4 were not able to
catalyze the depropargylation reaction of 5a (Figure 2A).
Different from the excellent work by Bernardes’s research group
which involved platinum complexes [K2PtCl4 or 1]-triggered
bond-cleavage amide and N-propargyl for drug activation,19 our
parallel work found that Pt(IV) complexes 3 and 4 were not able
to trigger the cleavage of O/N-propargyl. Furthermore, it was
found that 1 effectively triggered the depropargylation of O2-
propargyl-1-(N,N-morpholine)-diazeniumdiolate 6a25,26 to
generate NO (presented as nitrite), while 3 was not able to
trigger this reaction (Figures 2B and S3). To study the scope of
1-triggered cleavage reactions, O-propargyl/allyl/1,2-allene-yl/
1,2-butadiene-yl were respectively introduced to the hydroxyl
group of 5. As shown in Table S1, 1 effectively stimulated the
deprotection of O-propargyl (5a) and 1,2-butadiene-yl (5d)
with converted yields of 5 as 76 and 79%, respectively, superior
to the deprotection of O-allyl (5b) and 1,2-allene-yl (5c). To
further extend the substrates, O/N-propargyl/allyl were
introduced to a range of molecules including morpholine
diazeniumdiolate 6, 5-fluoracil 7, N-boc-L-tyrosine methyl ester
8, and a monosaccharide 1,2:3,4-di-O-isopropylidene-D-galacto-
pyranose 9. Excitingly, 1 effectively triggered the deprotection of
N-propargyl (7a) and allyl (7b) from 5-fluoracil with converted
yields as 66 and 92%, respectively. Importantly, 1 was also able
to deprotect propargyl and allyl from O2 position in morpholine
Degradation and the NO-Release Behaviors of 16 and
Related Compounds in Cell-Free System. The degradation
and the NO-release behaviors of 16 and 17 in blank PBS (pH =
7.4, 37 °C) or in PBS-containing molar equivalent L-
(+)-ascorbic acid were first investigated by using HPLC and
Griess assays, respectively.42 As shown in Figure 3A, both 16 and
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Figure 3. (A) Decomposition kinetics of 16 and 17 (the initial concentration of the test compounds was 500 μM) and (B) NO-release behaviors of 16
and 17 (the initial concentration of the test compounds was 125 μM) in the presence or absence of molar equivalent concentration of L-(+)-ascorbic
acid in PBS (pH = 7.4, 37 °C). Error bars represent SD from three independent experiments. (C) HPLC analysis of ascorbic acid (500 μM)-mediated
degradation of 16 (500 μM) and D) HPLC analysis of ascorbic acid (500 μM)-mediated degradation of 17 (500 μM) in PBS solution containing 5%
DMSO (pH = 7.4, 37 °C).
17 were stable in PBS after incubation for 24 h but rapidly
degraded in the presence of molar equivalent ascorbic acid.
Furthermore, it was found that with ascorbic acid, 16 was first
degraded to generate O2-propargyl diazeniumdiolate moiety 14
which was gradually exhausted to release up to 44.6 μM of nitrite
anion (an oxidative metabolite of NO) after incubation for 72 h
(Figure 3B,C). In sharp contrast, 17 was reduced by ascorbic
acid to generate O2-methyl diazeniumdiolate moiety 15, while it
was stable without further NO release (Figure 3B,D). Besides,
the degradation and the NO-release behaviors of 14 and 15 in
the presence of molar equivalent 1, 3, 3 + ascorbic acid in PBS
(pH = 7.4, 37 °C) were studied (Figure S6). It was found that
both 1 and 3 + ascorbic acid effectively triggered the degradation
of 14 to liberate a considerable amount of NO, whereas 3 alone
did not catalyze the degradation of 14. Expectedly, neither 1 nor
3 triggered the demethylation of 15. To sum up, these data
preliminarily revealed the proposed bioorthogonal degradation
route of 16, that is, 16 was first reduced by ascorbic acid to
liberate 1 and 14, and then 1 catalyzed the bond cleavage of O2-
propargyl moiety in 14 to produce the diazeniumdiolate anion,
which spontaneously released two molecules of NO.
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Antiproliferative Activity of 16 and Related Com-
pounds Against A2780 and IOSE80 Cells. We then
investigated the antiproliferative activity of Pt(II) 1 and Pt(IV)
3, NO donor moieties 14, 15, 3 + 14 (1:1), and 3 + 15 (1:1), as
well as the target compound 16 and its O2-methyl analogue 17
against human ovarian cancer A2780 cells and human normal
ovarian epithelial IOSE80 cells by MTT assay. As shown in
Table 1, compound 14 exhibited very weak antiproliferative
NO in living cancer cells. Importantly, integrated prodrug 16
exhibited more potent antiproliferative activity (0.23 0.01
μM) against A2780 cells superior to 1 (1.08 0.06 μM), 3 (1.69
0.09 μM) and 3 + 14 (0.81 0.07 μM). Additionally, 16
showed much improved safety to normal epithelial IOSE80 cells
with selective factor (SF: 518) relative to 1 (SF: 7.8) and 3 (SF:
10.9), indicating desirable biocompatibility of the integrated
prodrug. One plausible explanation is that as a derivative of 3, 16
possesses greater steric hindrance and thus more stability in
normal cells. As expected, compared to 16, its O2-methyl
analogue 17, which was not able to be uncaged to liberate NO,
exhibited weaker antiproliferative activity against A2780 cells,
indicating the potential synergistic effect of NO and Pt(II).
NO-Release Behaviors of 16 and Related Compounds
in A2780 and IOSE80 Cells. Next, we examined the NO-
release behaviors of the integrated prodrug 16, its analogue 17,
and the NO donor fragments 14 and 15 in A2780 cells and
IOSE80 cells by using a well-known NO-sensitive fluorophore
probe 4-amino-5-(methylamino)-2′,7′-difluorofluorescein diac-
etate (DAF-FM DA)43 and JS-K (a well-known Glutathione S-
transferase (GST)-activated NO donor prodrug)44 as a positive
control. As shown in Figures 4A and S7A,B, NO donor moieties
14 or 15 alone (0.23 μM, for 8 h) generated very low
fluorescence in both normal epithelial IOSE80 cells and ovarian
cancer A2780 cells as that of the blank control, while the
pretreatment of the same concentration of 3 for 10 h
significantly increased the relative mean fluorescence intensity
(MFI) of 14, but not 15, in cancer A2780 cells rather than in
normal epithelial IOSE80 cells, suggesting that Pt(II)-induced
O2-depropargylation reaction possesses desirable selectivity for
cancer cells. Interestingly, the treatment of 16 (0.23 μM, for 8 h)
generated the highest relative MFI as 25.33 in cancer A2780
cells, superior to 14 plus 3 (15.16) and JS-K (7.75), indicating
that the integrated prodrug could more effectively accumulate in
cancer cells than separated administration, and Pt(II)-induced
O2-cleavage reaction may be more effective than enzymatic O2-
deprotection which is GST enzyme-activity- and concentration-
Table 1. IC50 Values (μM) of the Integrated Prodrug 16 and
a
Its Analogue and Fragments
c
compounds
A2780
IOSE80
SF
1
3
1.080 0.06
1.690 0.09
89.46 4.87
103.9 7.54
0.8100 0.07
23.50 1.38
0.2300 0.01
0.8300 0.05
8.420 0.57
18.47 1.36
186.4 10.27
212.1 12.04
137.6 7.94
164.8 9.15
119.1 7.98
88.28 4.86
7.80
10.9
2.10
2.00
10.0
7.00
518
106
14
15
14 + 3
15 + 3
16
17
b
b
a
Human ovarian cancer A2780 cells and human normal ovarian
epithelial IOSE80 cells were treated with the indicated compounds for
72 h, and the cell viability and IC50 values were determined by MTT
assay. Data were expressed as the mean SD from three individual
b
experiments. The cells were incubated with 3 for 10 h, followed by
being rinsed and resuspended with 14 or 15 under the same
concentrations for 72 h, and the IC50 values were assessed by MTT
c
assay. The SF is defined as the IC50 value against IOSE80 cells/IC50
value against A2780 cells.
activity against both A2780 and IOSE80 cells, while the
pretreatment of 3 for 10 h significantly enhanced the
antiproliferative activity of 14 against A2780 cells (89.46
4.87 μM for 14 alone vs 0.81 0.07 μM for 3 + 14), indicating 3
could enter the cancer cells and be reduced to form 1 to further
trigger the cleavage of O2-propargyl, releasing a large amount of
Figure 4. (A) NO-release behaviors of the test compounds (0.23 μM) in the cells. A2780 and IOSE80 cells were treated with the indicated
concentrations of the test compounds for 8 h, then stained with DAF-FM DA (λex/em = 495/515 nm), and analyzed by fluorescence-activated cell
sorting (FACS). For 3 + 14 and 3 + 15 groups, the cells were first treated with 3 at indicated concentrations for 10 h, followed by being rinsed and
resuspended with the equimolar concentrations of 14 or 15 for additional 8 h, then analyzed as mentioned above. (B) NO released by 16 at different
concentrations (16-L: 0.058, 16-M: 0.115, and 16-H: 0.23 μM) for 8 h in the cells. (C) NO released by 16 (0.058 μM) with different incubation times
(0, 8, 16, 24, 48, and 72 h) in the cells. (D) Effects of NO scavenger carboxy-PTIO (PTIO) on the A2780 cell growth inhibitory activity of 16. A2780
cells were pretreated with PTIO (0, 3, 9, and 27 μM) for 1 h, followed by being rinsed and resuspended with 16 (1 μM) for 24 h, and the cell viability
was determined by MTT assay. Data are expressed as the mean SD from three independent experiments (n = 3): **P < 0.01, *P < 0.05.
F
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Figure 5. (A) Representative confocal microscopy images of A2780 cells and IOSE80 cells. The cells were treated with 16 (30 μM) for 3 h, then
washed and stained with RDC1 (20 μM) for another 4 h (λex/em = 400/565 nm, ZEN 2012 software was used to set as red). (B) Quantification of
fluorescence intensity for the cells in each group after staining with RDC1 for 0 and 3 h. Data are the mean SD from four independent experiments (n
= 4). **P < 0.01. (C) Cellular and nuclei Pt accumulation of 16 and 1 (5 μM) incubated with A2780 (red) and IOSE80 (blue) cells for 24 h. (D)
Platination levels of nuclear DNA following a 24 h incubation of the complexes (5 μM) with A2780 (red) and IOSE80 (blue) cells. Data are the mean
SD from three independent experiments (n = 3): ***P < 0.001, **P < 0.01.
dependent.45 In addition, 16 generated NO in dose- and time-
dependent manners in cancer A2780 cells (Figures 4B,C and
S7C,D). Interestingly, the pretreatment of NO scavenger
carboxy-PTIO partially counteracted the A2780 cell growth
inhibitory activity of 16 in a dose-dependent manner (Figure
4D). These results together with the antiproliferative data above
clearly demonstrated that integrated Pt(IV) derivative 16 was
preferably reduced in cancer cells to generate Pt(II) moiety,
which initiated the O2-cleavage reaction to spontaneously
release NO in situ, generating synergistic and potent
antiproliferative activity against cancer cells, while sparing
normal cells.
Pt(II) Release, Cellular Accumulation, and Binding to
Nuclear DNA of 16 in A2780 and IOSE80 Cells. We then
used a ratiometric probe RDC1,46 which was reported to be able
to distinguish between Pt(II) 1 and its Pt(IV) derivatives, to
investigate 1 generation after the treatment of 16 in both A2780
and IOSE80 cells. First, 16 (30 μM) was incubated with A2780
and IOSE80 cells for 3 h. The cells were washed and
resuspended with RDC1 (20 μM) for another 4 h. Confocal
microscopy was used to observe the fluorescence intensity of the
cells. As shown in Figure 5A,B, 16 significantly produced 1 in
A2780 cells, whereas a little 1 was generated in normal IOSE80
cells, indicating that the Pt(IV) complex 16 could be preferably
reduced in cancer cells to generate 1. Notably, 1 derived from 16
has two functions: one is as a cleavage reagent to trigger NO
release, and the other is as a cytotoxin via binding DNA. To
identify the spatiotemporal functions of 1 derived from 16, we
studied the cellular Pt uptake, nuclear accumulation, and nuclear
DNA platination of 16 in the cells by using 1 as a control. Briefly,
A2780 and IOSE80 cells were respectively treated with 16 (5
μM) or 1 (5 μM) for 24 h. Then the cell nuclei were separated by
Nuclei EZ Prep kit, and the whole cellular Pt and nuclei Pt
concentrations (expressed as pg Pt/106 cells) were measured by
graphite furnace atomic absorption spectrometry (GF-AAS)
analysis.47−50 Meanwhile, the nuclear DNA was extracted and
purified by the spin column quantification kit (Qiagen, Valencia,
CA), and the nuclear DNA platination levels (expressed as pg
Pt/μg DNA) were measured by using the same assay. As shown
in Figure 5C, 16 exhibited much more improved accumulation
pattern than 1 in whole cells and in nuclei in both cancer and
normal cells. For example, after treatment with 16 for 24 h,
A2780 cells had 763.53 14.81 pg Pt/106 in whole cells and
84.4 1.77 pg Pt/106 cells in nuclei, while A2780 cells had only
98.34 23.00 pg Pt/106 in whole cells and 24.78 1.55 pg Pt/
106 cells in nuclei after treatment with 1 for 24 h. One plausible
reason is that 16 bearing two axial ligands has higher lipophilicity
and thus possesses improved cell penetrating ability relative to 1,
which was approved by the measured LogP value of 16 (−0.89)
compared to the reported LogP value of 1 as −2.25 by the same
method.30,47,51 Importantly, 16 exhibited significant nuclear
DNA platination levels (22.56 pg Pt/μg DNA) superior to that
of 1 (5.18 pg Pt/μg DNA) in A2780 cells (Figure 5D). Overall,
these results demonstrated that 16 effectively penetrated cancer
cells and then was reduced by cytoplasmic reductants to liberate
1 and NO donor moiety. After the cleavage reaction was
triggered by 1, it was able to platinate nuclear DNA, together
with NO, generating selective, synergistic, and potent
antiproliferative activity against cancer cells.
Anticancer Activity and NO-Release Behavior of 16 in
a Zebrafish Embryo Model. Having established that 16 can
generate 1 and release NO, exerting selective and potent
antiproliferation activity against A2780 cells, we aimed to
demonstrate that 16 could be activated in cancer cells in vivo and
inhibit cancer cell growth in a zebrafish embryo model.15,52−55
First, Dil-labeled A2780 cells were microinjected into the yolk
sac of zebrafish embryos to establish the xenograft model. After
24 h, 16 (5% DMSO in water, final concentration 1 μM), blank
solution, and 1 (5% DMSO in water, final concentration 1 μM)
were incubated with zebrafish in a 6-well plate at 37 °C for 72 h.
Images of the zebrafish were acquired at 0 and 72 h after
incubation (Figure 6A). As shown in Figure S8A, all groups were
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Figure 6. (A) Schematic illustration of the assay to evaluate the anticancer activity of 16 in zebrafish implanted with Dil-labeled A2780 cells (λex/em
=
553/570 nm, ZEN 2012 software was used to set as red). (B) Representative confocal microscopy images of the zebrafish for each group at 72 h. (C)
Quantification of fluorescence intensity of the implanted cancer cells for each group at 72 h (n = 8), ***P < 0.001 vs blank control. (D) Schematic
illustration of the dual-imaging assay. (E) Representative confocal microscopy images of the zebrafish for each group at 40 min after the addition of
DAF-FM DA (λex/em = 495/515 nm, ZEN 2012 software was used to set as green). (F) Quantification of the fluorescent intensity in the location of the
implanted cancer cells and backbone of the zebrafish at 40 min after the addition of DAF-FM DA (n = 10), ****P < 0.0001 vs blank control.
successfully implanted with Dil-labeled A2780 cells. The fish in
16 group exhibited significantly lower fluorescence intensity
with an inhibitory rate of 75.03% than those in blank control,
and also superior to 1 with an inhibitory rate as 55.38%,
suggesting potent anticancer activity of 16 in zebrafish (Figure
6B,C). To further confirm that NO release could be specific in
cancer cells, we designed a dual-imaging assay (Figure 6D).
Briefly, zebrafish implanted with Dil-labeled A2780 cells were
incubated with 16 (5% DMSO in water, final concentration 10
μM) in a 6-well plate at 37 °C for 24 h, followed by being
collected, washed, and re-incubated with NO probe DAF-FM
DA (5% DMSO in water, final concentration 5 μM) for
additional 40 min. Images were acquired at 0 h before 16
treatment and 40 min after DAF-FM DA incubation for both
cancer cells and NO (Figure 6D). As shown in Figure S8B, at 0 h
before 16 treatment, the fish had been successfully implanted
with Dil-labeled A2780 cells and did not show any fluorescence
of NO without NO probe. After DAF-FM DA incubation, as
shown in the panel of merge in Figure 6E, the location of NO
overlapped that of the cancer cells, and 16 group exhibited
significantly higher fluorescence of NO in cancer cells (3.24-
fold, P < 0.0001) than the blank control group (Figure 6F).
Interestingly, in the backbone of zebrafish, fluorescence of NO
in 16 group was comparable with that in the blank control group.
These results clearly demonstrated that 16, as an integrated and
inert prodrug, could be stable in vivo after oral administration but
specifically reduced in the implanted human ovarian cancer cells
to liberate 1 (cleavage reagent) and O2-propargyl diazenium-
diolate (caged active group). The former triggered the cleavage
of O2-propargyl in the latter to release a large amount of NO in
situ, generating potent cancer cell growth inhibitory activity in
zebrafish.
CONCLUSIONS
■
In conclusion, to address the shortcomings of current
bioorthogonal cleavage chemistry, we proposed, for the first
time, a strategy of direct modification of cleavage reagents
(masked-B) and integrated bioorthogonal cleavage reaction-
based prodrug (masked-B-A-C). We found a new class of Pt(II)-
triggered bioorthogonal cleavage reactions in which Pt(II) but
not Pt(IV) complexes effectively triggered the cleavage of O/N-
propargyl in various molecules including a fluorophore, NO
donor diazeniumdiolates, an anticancer drug 5-fluorouracil,
tyrosine, and a monosaccharide under biocompatible conditions
with good yields. Based on this interesting discovery, an
integrated bioorthogonal cleavage reaction-based prodrug 16, as
a proof-of-concept, was rationally designed, feasibly synthesized,
and biologically evaluated. This integrated bioorthogonal
prodrug, 16, bearing both a masked cleavage reagent [Pt(IV)
moiety] and a caged active group (O2-propargyl N-methyl-
ethanolamine diazeniumdiolate), efficiently entered cancer cells
and was specifically reduced by cytoplasmic reductants in cancer
cells to release Pt(II) 1 and O2-propargyl-caged diazeniumdio-
late moiety. Depropargylation of the O2 position triggered by 1
generated diazeniudiolates anions, which spontaneously re-
leased an abundance of NO in situ. Additionally, 1 was able to
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2H), 6.15 (s, 1H), 4.76 (d, J = 2.1 Hz, 2H), 2.57 (t, J = 2.4 Hz, 1H), and
2.40 (s, 3H).
platinate nuclear DNA, together with NO, generating selective,
synergistic, and potent antiproliferative activity against cancer
cells. Although Pt(II) is toxic to cells, the depropargylation
reaction selectively occurred in cancer cells and thus was
biocompatible to normal cells. Notably, 16 exhibited more
potent antiproliferative activity against human ovarian cancer
A2780 cells and much lower toxicity to human normal ovarian
epithelial IOSE80 cells than 1, which is a front-line chemo-
therapy for ovarian cancer.56 Significantly, in a xenograft model
in zebrafish, 16 was specifically activated in the implanted A2780
cells to release NO in situ, generating potent cancer inhibitory
activity. 16 possesses potential developability and will be
investigated for anticancer activity in mice and other preclinical
studies, and the corresponding results will be reported in due
course.
Taken together, the fact that Pt(II) 1, but not its Pt(IV)
complex 3, triggers O/N-depropargylation reactions gives us a
chance to realize the strategy of integrated bioorthogonal
cleavage reaction-based prodrugs bearing both a masked
cleavage reagent and caged active group within one small
molecule and possessing controllable drug activation in cancer
cells in vivo. Meanwhile, this integration strategy could be
flexibly expanded to other active molecules of interest. Thus, the
new 1-triggered depropargylation reaction and novel integrated
bioorthogonal chemistry-based prodrug strategy may promote
diverse applications in chemical biology and inspire further
research in bioorthogonal chemistry.
Synthesis of O-Allyl 4-Methylumbelliferone (5b). 4-Methylumbel-
liferone (200 mg), allyl bromide (4.0 equiv), and K2CO3 (4.0 equiv)
were dissolved in acetone (10 mL). The solution was stirred at 60 °C for
12 h. After the reaction mixture was cooled and the solvent was
removed by evaporators, the residue was treated with H2O (15 mL) and
extracted with EA. The organic phase was washed with water, dried over
Na2SO4, and evaporated under vacuum. The crude product was
purified by using flash column chromatography to give compound 5b
(218 mg, 89%). 1H NMR (300 MHz, CDCl3): δ 7.49 (d, J = 8.7 Hz,
1H), 6.90−6.82 (m, 2H), 6.13 (s, 1H), 6.10−5.98 (m, 1H), 5.47−5.32
(m, 2H), 4.60 (d, J = 5.4 Hz, 2H), and 2.39 (s, 3H).
Synthesis of O-1,2-Allene-yl 4-Methylumbelliferone (5c). Com-
pound 5a (200 mg) and t-BuOK (0.5 equiv) were dissolved in 5.0 mL of
dry t-BuOH and stirred at 90 °C for 4 h. The reaction mixture was
concentrated and purified by silica gel column chromatography (1:50,
EA: hexanes) to give the title compound 5c (32 mg, 16%). 1H NMR
(300 MHz, CDCl3): δ 7.07 (d, J = 8.4 Hz, 1H), 6.58−6.49 (m, 3H),
5.85 (s, 1H), 5.36 (d, J = 1.5 Hz, 1H), 5.11 (s, 1H), and 2.09 (s, 3H).
Synthesis of O-1,2-Butadiene-yl 4-Methylumbelliferone (5d).
Compound 5a (200 mg), CuI (0.5 equiv), Cy2NH (1.8 equiv) and
paraformaldehyde (2.5 equiv) were dissolved in dry dioxane (5 mL)
and refluxed for 3 h in a schlenk tube. The reaction mixture was
concentrated and purified by silica gel column chromatography to give
compound 5d (105 mg, 49%). 1H NMR (300 MHz, CDCl3): δ 7.49 (d,
J = 8.7 Hz, 1H), 6.88−6.83 (m, 2H), 6.13 (s, 1H), 5.42−5.33 (m, 1H),
4.92−4.89 (m, 2H), 4.65−4.61 (m, 2H), and 2.39 (s, 3H).
Synthesis of O2-Propargyl-1-(N,N-morpholine)-diazeniumdiolate
(6a). To a solution of N,N-morpholine diazeniumdiolates sodium salts
(200 mg) in DMF (5 mL) at 0 °C under a steady stream of nitrogen was
added dropwise propargyl bromide (1.0 equiv) in DMF (1 mL). The
mixture was allowed to warm to room temperature and stirred
overnight. Then the solvent was removed by an evaporator, and the
obtained residue was treated with H2O (20 mL) and extracted with EA
(3 × 50 mL). The organic layers were combined and washed with brine
(20 mL), dried over Na2SO4, and evaporated under vacuum. The crude
product was purified by flash chromatography on silica gel using EA and
hexane for eluting to give compound 6a (166 mg, 76%). 1H NMR (300
MHz, CDCl3): δ 4.78 (d, J = 2.4 Hz, 2H), 3.84 (t, J = 4.8 Hz, 4H), 3.46
(t, J = 4.8 Hz, 4H), and 2.53 (t, J = 2.4 Hz, 1H).
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents were bought from
commercial suppliers and used as received without further drying or
purification. NO gas was purchased from Nanjing TIANZE GAS Co.,
Ltd. Melting points were determined on a MelTEMP II melting point
1
apparatus. H NMR (300 or 500 MHz) and 13C NMR (75 or 125
MHz) spectra were recorded with Bruker Avance 300 and 500 MHz
spectrometers at 303 K, using tetramethylsilane as an internal standard.
Mass and high-resolution mass spectrometry (HRMS) spectra were
obtained on an Agilent Q-TOF 6520 or Waters Synapt G2-S
spectrometer. Analytical and preparative thin-layer chromatography
was performed on silica gel (200−300 mesh) GF/UV 254 plates, and
the chromatograms were visualized under UV light at 254 nm.
Analytical reversed-phase HPLC (RPLC) was conducted on a
Shimadzu Prominence HPLC system using Innovai ODS-2 column
(5 μm, 100 Å, 150 or 250 × 4.60 mm). Test compounds 14−17 with a
purity of >95% were used for biological experiments.
Synthesis of Diamine Trichloro Hydroxy Platinum (3). Diamine
trichloro hydroxy platinum (3) was synthesized by the reaction of
cisplatin (1) with N-chlorosuccinimide (NCS) as previously
reported.57,58 Briefly, NCS (1.0 equiv), cisplatin (1.0 equiv), and
H2O (20 mL) were added to a 100 mL round-bottom flask, and the
reaction was stirred at room temperature for 5 h in the dark. The solid
residue was removed by centrifugation, and the filtrate was evaporated
to dryness to get a pale-yellow solid that was washed with ethanol and
diethyl ether to furnish 3 as a light yellow solid. Yield 87.6%. 1H NMR
(300 MHz, DMSO-d6): δ 5.38−6.02 (m, 6H). MS (ESI) m/z:
Cl3H7N2OPt, [M + H]+ = 351.9.
Synthesis of O-Propargyl 4-Methylumbelliferone (5a). 4-Methyl-
umbelliferone (200 mg), propargyl bromide (4.0 equiv), and K2CO3
(4.0 equiv) were dissolved in acetone (10 mL). The obtained solution
was stirred at 60 °C for 12 h. After the reaction mixture was cooled and
the solvent was removed by evaporators, the residue was treated with
H2O (15 mL) and extracted with ethyl acetate (EA) (15 mL × 3). The
organic phase was washed with water, dried over Na2SO4, and
evaporated under vacuum. The crude product was purified by using
flash column chromatography to give compound 5a (210 mg, 86%). 1H
NMR (300 MHz, CDCl3): δ 7.52 (d, J = 9.6 Hz, 1H), 6.94−6.91 (m,
Synthesis of O2-Allyl-1-(N,N-morpholine)-diazeniumdiolate (6b).
To a solution of N,N-morpholine diazeniumdiolates sodium salts (200
mg) in DMF (5 mL) at 0 °C under a steady stream of nitrogen was
added dropwise allyl bromide (1.0 equiv) in DMF (1 mL). The mixture
was allowed to warm to room temperature and stirred overnight. Then
the solvent was removed by an evaporator, and the obtained residue was
treated with H2O (20 mL) and extracted with EA (3 × 50 mL). The
organic layers were combined and washed with brine (20 mL), dried
over Na2SO4, and evaporated under vacuum. The crude product was
purified by flash chromatography on silica gel using EA and hexane for
1
eluting to give compound 6b (24 mg, 11%). H NMR (300 MHz,
CDCl3): δ 6.13−5.86 (m, 1H), 5.45−5.23 (m, 2H), 4.69 (d, J = 6.0 Hz,
2H), 3.84 (t, J = 4.8 Hz, 4H), and 3.41 (t, J = 4.8 Hz, 4H).
Synthesis of N-Propargyl 5-Fluorouracil (7a). To a stirred solution
of 5-fluorouracil (200 mg) in anhydrous DMF, K2CO3 (1.0 equiv) was
added and stirred for 30 min. Then propargyl bromide (1.1 equiv) was
added to the mixture and allowed to stir at 50 °C for 12 h. After the
removal of the solvent by evaporating under reduced pressure, the
residue was purified by silica gel column chromatography to give
1
compound 7a (100 mg, 39%). H NMR (300 MHz, DMSO-d6): δ
11.89 (s, 1H), 8.11 (d, J = 6.3 Hz, 1H), 4.46 (s, 2H), and 3.42 (s, 1H).
Synthesis of N-Allyl 5-Fluorouracil (7b). To a stirred solution of 5-
fluorouracil (200 mg) in anhydrous DMF, K2CO3 (1.0 equiv) was
added and stirred for 30 min. Then allyl bromide (1.1 equiv) was added
to this mixture and allowed to stir at 50 °C for 12 h. After the removal of
the solvent by evaporating under reduced pressure, the residue was
purified by silica gel column chromatography to give compound 7b
(124 mg, 47%). 1H NMR (500 MHz, DMSO-d6): δ 11.78 (s, 1H), 8.01
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(d, J = 11.5 Hz, 1H), 5.94−5.81 (m, 1H), 5.21−5.14 (m, 2H), and 4.24
(d, J = 8.5 Hz, 2H).
under vacuum, and the obtained residue was added into H2O (20 mL)
and extracted with EA (3 × 50 mL). The combined organic layers were
washed with brine (20 mL), dried over Mg2SO4, and evaporated under
vacuum. The crude product was purified by flash chromatography on
silica gel by using EA and hexane for eluting to give target compounds
1226 and 13.59
Synthesis of O-Propargyl N-Boc-L-tyrosine Methyl Ester (8a). N-
Boc-L-tyrosine methyl ester (200 mg), propargyl bromide (4.0 equiv),
and K2CO3 (4.0 equiv) were dissolved in acetone (10 mL). The
solution was stirred at 60 °C for 12 h. After the reaction mixture was
cooled and the solvent was removed by evaporators, the residue was
treated with H2O (15 mL) and extracted with EA. The combined
organic layers were washed with water, dried over Na2SO4, and
evaporated by vacuum. The crude product was purified using flash
O2-Propargyl 1-(N-Methylethanolamine-N-yl)-diazen-1-ium-1,2-
1
diolate (12). Colorless oil, 562 mg, yield 51%. H NMR (ppm, 300
MHz, CDCl3): δ 4.77 (d, J = 2.4 Hz, 2H), 3.74 (t, J = 4.8 Hz, 2H), 3.42
(t, J = 4.8 Hz, 2H), 3.05 (s, 3H), 2.55 (t, J = 2.4 Hz, 1H), and 2.50 (br,
1H). 13C NMR (ppm, 75 MHz, CDCl3): δ 76.33, 60.97, 59.56, 57.05,
and 41.90. MS (ESI) m/z: C6H11N3O3, [M + Na]+ = 196.3.
1
column chromatography to give compound 8a (132 mg, 59%). H
NMR (300 MHz, CDCl3): δ 7.05 (d, J = 8.7 Hz, 2H), δ 6.90 (d, J = 8.7
Hz, 2H), 4.94 (s, 1H), 4.66 (d, J = 2.4 Hz, 2H), 4.54 (d, J = 6.9 Hz, 1H),
3.71 (s, 3H), 3.03 (t, J = 6.0 Hz, 2H), 2.51 (t, J = 2.4 Hz, 1H), and 1.41
(s, 9H).
O2-Methyl 1-(N-Methylethanolamine-N-yl)-diazen-1-ium-1,2-di-
1
olate (13). Light yellow oil, 607 mg, yield 64%. H NMR (ppm, 500
MHz, CDCl3): δ 4.01 (s, 3H), 3.73 (t, J = 4.5 Hz, 2H), 3.38 (t, J = 5.0
Hz, 2H), 3.00 (s, 3H), and 2.32 (s, 1H). 13C NMR (ppm, 75 MHz,
CDCl3): δ 60.64, 59.02, 56.74, and 41.68. ESI-MS m/z: C4H11N3O3,
[M + Na]+ = 172.1.
Synthesis of O-Allyl N-Boc-L-tyrosine Methyl Ester (8b). N-Boc-L-
tyrosine methyl ester (200 mg), allyl bromide (4.0 equiv), and K2CO3
(4.0 equiv) were dissolved in acetone (10 mL). The solution was stirred
at 60 °C for 12 h. After the reaction mixture was cooled and the solvent
was removed by evaporators, the residue was treated with H2O (15 mL)
and extracted with EA. The combined organic layers were washed with
water, dried over Na2SO4, and evaporated by vacuum. The crude
product was purified using flash column chromatography to give
compound 8b (213 mg, 94%). 1H NMR (300 MHz, CDCl3): δ 7.03 (d,
J = 8.4 Hz, 2H), δ 6.84 (d, J = 8.7 Hz, 2H), 6.11−5.98 (m, 1H), 5.40
(dd, J = 1.5 Hz, 1H), 5.27 (dd, J = 1.2 Hz, 1H), 4.94 (s, 1H), 4.51 (d, J =
5.4 Hz, 2H), 3.70 (s, 3H), 3.03 (t, J = 7.8 Hz, 2H), and 1.42 (s, 9H).
Synthesis of O-Propargyl 1,2:3,4-Di-O-isopropylidene-D-galacto-
pyranose (9a). To a stirred solution of 1,2:3,4-di-O-isopropylidene-D-
galactopyranose (100 mg) in DMF (10 mL) at 0 °C was carefully added
NaH (60% in mineral oil, 30 mg). The reaction mixture was stirred for
30 min until hydrogen evolution stopped. Propargyl bromide (66 μL)
was added, and the mixture was stirred at 0 °C for 30 min and then at
room temperature for 12 h. Then the reaction mixture was poured into
brine, and extracted with ether (150 mL × 3). The combined organic
layers were washed with brine, dried over Na2SO4, and concentrated.
The residue was purified by chromatography to give compound 9a (84
mg, 73%). 1H NMR (300 MHz, CDCl3): δ 5.54 (d, J = 5.1 Hz, 1H),
4.60 (dd, J = 2.1, 2.4 Hz, 1H), 4.32−4.20 (m, 4H), 4.02−3.97 (m, 1H),
3.77 (q, J = 4.8 Hz, 1H), 3.67 (q, J = 3.0 Hz, 1H), 2.42 (t, J = 2.4 Hz,
1H), 1.54 (s, 3H), 1.45 (s, 3H), and 1.33 (d, J = 4.2 Hz, 6H).
Synthesis of O-Allyl 1,2:3,4-Di-O-isopropylidene-D-galactopyra-
nose (9b). To a stirred solution of O-propargyl 1,2:3,4-di-O-
isopropylidene-D-galactopyranose (100 mg) in DMF (10 mL) at 0
°C was carefully added NaH (60% in mineral oil, 30 mg). The reaction
mixture was stirred for 30 min until hydrogen evolution stopped. Allyl
bromide (67 μL) was added, and the mixture was stirred at 0 °C for 30
min and then at room temperature for 12 h. Then the reaction mixture
was poured into brine, and extracted with ether (150 mL × 3). The
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated. The residue was purified by chromatography to give
compound 9b (87 mg, 76%). 1H NMR (300 MHz, CDCl3): δ 5.98−
5.85 (m, 1H), 5.53 (d, J = 5.1 Hz, 1H), 5.21 (q, J = 17.1 Hz, 2H), 4.60
(dd, J = 2.1 Hz, 1H), 4.32−4.25 (m, 2H), 4.04 (s, 2H), 3.98 (m, J = 6.0
Hz, 1H), 3.69−3.55 (m, 2H), 1.54 (s, 3H), 1.44 (s, 3H), and 1.33 (d, J =
3.6 Hz, 6H).
General Procedure for the Synthesis of Compound 14 or 15. To a
solution of 12 (700 mg, 4.05 mmol) or 13 (602 mg, 4.03 mmol) in
anhydrous THF (10 mL) was added 4-dimethylaminopyridine (122
mg, 1.0 mmol), and the obtained mixture was allowed to stir at room
temperature for 15 min. A solution of anhydrous THF (5 mL)
containing succinic anhydride (608 mg, 6.08 mmol) was added
dropwise to the reaction mixture, and the obtained mixture was allowed
to reflux overnight. After the reaction, solid residues were removed by
filtration, and the filtrate was concentrated under vacuum. Then, the
resulting residue was added to H2O (50 mL) and extracted with DCM
(6 × 30 mL). The combined organic layers were washed with brine (20
mL), dried with Mg2SO4, filtrated, and evaporated to afford compounds
14 or 15 without further purification.
O2-Propargyl 1-(N-methyl-2-(4-oxo-butanoic acid-4-yl)-oxy-eth-
ylamine-N-yl)-diazen-1-ium-1,2-diolate (14). Light yellow oil, 863
mg, yield 78%. 1H NMR (ppm, 300 MHz, CDCl3): δ 4.78 (d, J = 2.1 Hz,
2H), 4.29 (t, J = 5.4 Hz, 2H), 3.60 (t, J = 5.4 Hz, 2H), 3.07 (s, 3H),
2.70−2.60 (m, 4H), and 2.55 (t, J = 2.4 Hz, 1H). 13C NMR (ppm, 75
MHz, CDCl3): δ 176.86, 171.40, 77.13, 75.76, 60.96, 60.43, 52.26,
40.92, and 28.30. ESI-MS m/z: 296.1, [M + Na]+; HRMS (ESI): calcd
for C10H15N3O6, [M − H]− 272.0888; found, 272.0897, ppm error 3.3.
O2-Methyl 1-(N-methyl-2-(4-oxo-butanoic acid-4-yl)-oxy-ethyl-
amine-N-yl)-diazen-1-ium-1,2-diolate (15). Light yellow oil, 725 mg,
yield 72%. 1H NMR (ppm, 500 MHz, CDCl3): δ 4.26 (t, J = 5.5 Hz,
2H), 4.00 (s, 3H), 3.53 (t, J = 5.5 Hz, 2H), 3.01 (s, 3H), and 2.68−2.60
(m, 4H). 13C NMR (ppm, 125 MHz, CDCl3): δ 177.36, 177.83, 61.44,
61.02, 52.90, 41.76, and 28.74. ESI-MS m/z: 272.1, [M + Na]+; HRMS
(ESI): calcd for C8H15N3O6, [M − H]− 248.0888; found, 248.0892,
ppm error 1.6.
General Procedure for the Synthesis of Compound 16 or 17. To a
mixture of 14 (217 mg, 0.79 mmol) or 15 (200 mg, 0.80 mmol) and
Et3N (86.6 mg, 0.86 mmol) in anhydrous DMF (5 mL) was added 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU, 265 mg, 0.83 mmol), and the reaction mixture was stirred at
room temperature for 30 min. Then diamine trichloro hydroxy
platinum (3) (200 mg, 0.57 mmol) was added, and the mixture was
stirred in the dark for 12 h. The reaction mixture was distilled under
vacuum to remove DMF. The obtained residue was purified by silica gel
chromatography by using DCM and methanol for eluting to obtain the
final products 16 and 17.
Synthesis of 1-(N-Methylethanolamine-N-yl)-diazen-1-ium-1,2-
diolate Sodium Salt (11). To a solution of N-methylethanolamine (10,
10.0 g) in ether (150 mL) in a Teflon bottle was added a mixture of
sodium methoxide (1.05 equiv) in methanol (20 mL). The bottle was
sealed, flushed with nitrogen (five times), and charged with NO gas (50
psi). After 24 h, a white precipitate was collected by filtration and
washed with copious amounts of ether to produce compound 11, which
was used without further purification.
General Procedure for the Synthesis of Compounds 12 and 13.
Compound 11 (1.0 g, 6.37 mmol) was suspended in 5 mL of DMF at 0
°C under a steady stream of nitrogen, to which was added propargyl
bromide (757.78 mg, 6.37 mmol) or iodomethane (904.16 mg, 6.37
mmol). The reaction mixture was allowed to warm to room
temperature and stirred overnight. Then the solvent was removed
O2-Propargyl 1-(N-Methyl-2-(4-oxo-butanoic acid diamine tri-
chloro hydroxyl platinum ester-4-yl)-oxy-ethylamine-N-yl)-diazen-
1-ium-1,2-diolate (16). Light yellow solid, 84 °C (decomposition), 97
mg, yield 28%. 1H NMR (ppm, 300 MHz, DMSO-d6): δ 6.28−6.03 (m,
6H), 4.82 (d, J = 2.4 Hz, 2H), 4.16 (t, J = 5.4 Hz, 2H), 3.58 (t, J = 2.4
Hz, 2H), 2.98 (s, 3H), and 2.49−2.42 (m, 5H). 13C NMR (ppm, 75
MHz, DMSO-d6): δ 178.90, 172.01, 78.75, 60.54, 60.39, 51.83, 40.52,
30.94, and 29.80. HRMS (ESI): calcd for C10H20Cl3N5O6Pt, [M + H]+
607.0200; found, 607.0191, ppm error −1.5.
O2-Methyl 1-(N-Methyl-2-(4-oxo-butanoic acid diamine trichloro
hydroxyl platinum ester-4-yl)-oxy-ethylamine-N-yl)-diazen-1-ium-
1,2-diolate (17). Light yellow solid, 103 °C (decomposition), 134 mg,
yield 37.4%. 1H NMR (ppm, 500 MHz, DMSO-d6): δ 6.28−6.03 (m,
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6H), 4.15 (t, J = 5.0 Hz, 2H), 3.90 (s, 3H), 3.52 (t, J = 5.0 Hz, 2H), 2.95
(s, 3H), and 2.52−2.44 (m, 4H). 13C NMR (ppm, 125 MHz, DMSO-
d6): δ 182.01, 175.12, 63.69, 63.57, 55.06, 43.86, 34.06, and 32.93.
HRMS (ESI): calcd for C8H20Cl3N5O6Pt, [M + H]+ 583.0200; found,
583.0191, ppm error −1.5.
Decaging of Compound 9a. Compound 9a (29.8 mg) and 1 (30.0
mg) were stirred in PBS (10 mL, pH 7.4) containing 5% DMSO and 5%
DMF at 37 °C for 48 h. After the reaction mixture was cooled and the
solvent was removed by evaporators, the residue was purified by flash
chromatography to afford compound 9 (7.54 mg, 29%). 1H NMR (300
MHz, CDCl3): δ 5.57 (d, J = 5.1 Hz, 1H), 4.61 (dd, J = 2.1, 2.4 Hz, 1H),
4.34−4.26 (m, 2H), 3.85 (t, J = 7.5 Hz, 2H), 3.75 (q, J = 6.6 Hz, 1H),
1.53 (s, 3H), 1.45 (s, 3H), and 1.33 (s, 6H).
Decaging of Compound 9b. Compound 9b (30.0 mg) and 1 (30.0
mg) were stirred in PBS (10 mL, pH 7.4) containing 5% DMSO and 5%
DMF at 37 °C for 48 h. After the reaction mixture was cooled and the
solvent was removed by evaporators, the residue was purified by flash
chromatography to afford compound 9 (4.68 mg, 18%).
MTT Assay. Human ovarian cancer A2780 cells and human normal
ovarian epithelial IOSE80 cells were purchased from American type
culture collection. The cells were planked in a 96-well plate with a
concentration of 104 cells/well and cultured in 37 °C under 5% CO2
until 90% cells fused. Then the cells were incubated in serum-free RPMI
1640 or McCoy’s 5A culture medium for 2 h for synchronization.
Subsequently, the supernatant was discarded, and RPMI 1640 or
McCoy’s 5A medium with or without different concentrations of 3 were
added and incubated for 10 h. After incubation, the supernatant was
discarded and cells were rinsed with PBS three times. Then cells were
treated with test compounds for 72 h. Four hours before the end of
incubation, MTT solution (5 mg/mL, 20 μL) was added to each well.
After incubation for 4 h, the supernatant of each well was discarded and
150 μL of DMSO was added. The cells were oscillated for 10 min in a
cell oscillation apparatus, and OD570 was determined by enzyme
labeling after the crystal was fully dissolved. The cell viability was
expressed as a percentage of OD570. IC50 values were calculated by
GraphPad Prism 6.0.
Measurement of NO-Release Behavior of Related Com-
pounds in Living Cells. For groups of 14 and 15, A2780 or IOSE80
cells in 96-well plates were treated without or with 3 for 10 h, followed
by being rinsed with PBS three times. Subsequently, the cells were
resuspended with equivalent concentration of test compounds (14 or
15) for additional 8 h. While for other groups, A2780 or IOSE80 cells in
96-well plates were treated with the test compounds at indicated
concentrations for indicated times. The cells were then collected and
resuspended with DAF-FM DA (1.5 mL, 5 μM) at 37 °C for 20 min in
the dark. The cells were rinsed three times with PBS and then analyzed
by FACS. NO production was measured with the flow cytometer with
excitation and emission wavelengths of 495 and 515 nm, respectively.
BD Accuri C6 flow cytometer was used, and 10,000 cells was gated for
each sample.
Synthesis of RDC1. RDC1 was synthesized from 7-(diethylamino)-
coumarin-3-carboxylic acid and Rho-6G as per previously reported
procedures.46 1H NMR (300 MHz, CDCl3): δ 7.89 (d, J = 8.5 Hz, 2H),
7.33 (t, J = 9.0 Hz, 3H), 6.98 (d, J = 7.2 Hz, 1H), 6.67−6.53 (m, 3H),
6.49 (s, 1H), 6.33 (s, 2H), 4.49 (t, J = 6.1 Hz, 2H), 4.25 (s, 4H), 3.80 (d,
J = 6.2 Hz, 4H), 3.44 (q, J = 7.2 Hz, 6H), 3.20 (q, J = 7.2 Hz, 4H), 1.96
(s, 6H), and 1.32−1.25 (m, 12H). ESI-MS m/z: 877.5, [M + H]+.
Decaging of Compound 5a. A solution of the compounds 5a (500
μM) and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 5 according to the
previously established standard curve of concentrations-peak areas. The
conversion ratio from 5a to 5 was 76%.
Decaging of Compound 5b. A solution of the compounds 5b (500
μM) and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 5 according to the
previously established standard curve of concentration-peak areas. The
conversion ratio from 5b to 5 was 13%.
Decaging of Compound 5c. A solution of the compounds 5c (500
μM) and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 5 according to the
previously established standard curve of concentration-peak areas. The
conversion ratio from 5c to 5 was 12%.
Decaging of Compound 5d. A solution of compounds 5d (500 μM)
and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentrations of generating 5 according to
the previously established standard curve of concentration-peak areas.
The conversion ratio from 5d to 5 was 79%.
Decaging of Compound 6a. A solution of compounds 6a (125 μM)
and 1 (125 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. At 24 h, the concentration of NO (presented as
−
NO2 ) was measured by using nitrite colorimetric kit. The conversion
ratio from 6a to NO was 41%.
Decaging of Compound 6b. A solution of compounds 6b (125 μM)
and 1 (125 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. At 24 h, the concentration of NO (presented as
−
NO2 ) was measured by using nitrite colorimetric kit. The conversion
ratio from 6b to NO was 8.7%.
Cellular Accumulation of Platinum. The cellular accumulation
of platinum was investigated as per the reported method. A2780 or
IOSE80 cells (2.5 × 106) were seeded in 75 cm2 flasks in growth
medium (20 mL). After overnight incubation, the medium was replaced
and the cells were treated with 16 or 1 at the concentration of 5 μM for
24 h. Cell monolayers were washed twice with cold PBS, harvested, and
counted. Cell nuclei were isolated by means of the nuclei isolation kit
Nuclei EZ Prep (Sigma-Aldrich, St. Louis, MO). Then samples were
subjected to three freezing/thawing cycles at −80 °C and then
vigorously vortexed. The samples were treated with highly pure nitric
acid (Pt: ≤0.01 μg kg−1, Sigma Chemical, St. Louis, MO) and
transferred into a microwave Teflon vessel. Subsequently, samples were
submitted to standard procedures using a speed wave MWS-3 Berghof
instrument (Eningen, Germany). After cooling, each mineralized
sample was analyzed for platinum by using a Varian AA Duo graphite
furnace atomic absorption spectrometer (Varian, Palo Alto, CA; USA)
at the wavelength of 324.7 nm.
Measurement of Water−Octanol Partition Coefficient
(LogPo/w) for 16. We used the shake flask method to determine
the partition coefficient (P) of 16. Octanol was saturated with a PBS
buffer by shaking the two-phase mixture overnight. And then 50 μM 16
was added to a mixture of octanol-saturated water (5 mL) and water-
saturated octanol (5 mL) in a 15 mL tube. The tube was shaken at room
temperature in the dark for 6 h. The two phases were separated by
centrifugation, and the 16 concentrations in these two phases were
Decaging of Compound 7a. A solution of compounds 7a (500 μM)
and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO were
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 7 according to the
previously established standard curve of concentration-peak areas. The
conversion ratio from 7a to 7 was 66%.
Decaging of Compound 7b. A solution of compounds 7b (500 μM)
and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 7 according to the
previously established standard curve of concentration-peak areas. The
conversion ratio from 7b to 7 was 92%.
Decaging of Compound 8a. A solution of compounds 8a (500 μM)
and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 8 according to the
previously established standard curve of concentration-peak areas. The
conversion ratio from 8a to 8 was 18%.
Decaging of Compound 8b. A solution of compounds 8b (500 μM)
and 1 (500 μM) in PBS (5 mL, pH 7.4) containing 5% DMSO was
incubated at 37 °C. An aliquot (400 μL) at 24 h was subjected to HPLC
analysis to determine the concentration of generating 8 according to the
previously established standard curve of concentrations-peak areas. The
conversion ratio from 8b to 8 was 13%.
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J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
determined by HPLC. The LogPo/w was expressed as a mean of three
independent experiments.
Tian Lv − State Key Laboratory of Natural Medicines, Jiangsu Key
Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu Key
Laboratory of Drug Screening, China Pharmaceutical University,
Nanjing 210009, P. R. China
Jianbing Wu − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases,
Jiangsu Key Laboratory of Drug Screening, China
DNA Platination. A2780 or IOSE80 cells (3 × 106) were seeded in
10 cm Petri dishes in 10 mL of culture medium. Subsequently, cells
were treated with 16 or 1 at the concentration of 5 μM for 24 h. DNA
was extracted and purified by a commercial spin column quantification
kit (Qiagen, Valencia, CA). Only highly purified samples (A260/A230
≅ 1.8 and A280/A260 ≅ 2.0) were included for analysis to avoid any
artifacts. The samples were completely dried and redissolved in 200 μL
of Milli-Q water (18.2 MΩ) for at least 20 min at 65 °C in a shaking
thermo-mixer, mineralized and analyzed for total Pt content by GF-
AAS as described above.
Pharmaceutical University, Nanjing 210009, P. R. China;
orcid.org/0000-0003-2725-9859
Mingchao Zhu − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P. R. China
Yue Fei − State Key Laboratory of Natural Medicines, Jiangsu Key
Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu
Key Laboratory of Drug Screening, China Pharmaceutical
University, Nanjing 210009, P. R. China
Jie Zhu − State Key Laboratory of Natural Medicines, Jiangsu Key
Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu
Key Laboratory of Drug Screening, China Pharmaceutical
University, Nanjing 210009, P. R. China
Yihua Zhang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P. R. China;
orcid.org/0000-0003-2378-7064
Establishment of Zebrafish Embryo Ovarian Cancer Model.
First, the ovarian cancer cells A2780 were collected in DMEM (with
10% fetal bovine serum), then A2780 and 10 μg/mL CM-DiI solution
(5% DMSO/water) and were incubated together until fluorescence was
observed (∼15 min), and the cell density was adjusted to 2 × 107 cells/
mL. The labeled cells were microinjected into the yolk space of 2 dpf
zebrafish juveniles within 2 h. The number of cells was controlled to
about 400 per fish. The zebrafish were cultivated at 37 °C for 24 h;
totally 40−60 fish with successful transplantation were selected under
fluorescence microscopy for further assays. All animal experiments and
animal care were conducted in accordance with the guidelines of the
Provision and General Recommendation of Chinese Experimental
Animals in China. The experimental protocols were approved by the
Animal Research and Care Committee of China Pharmaceutical
University [SYXK (SU) 2016-0011].
ASSOCIATED CONTENT
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01435
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01435.
Author Contributions
†T.S., T.L., and J.W. contributed equally to this work.
The depropargylation efficacy catalyzed by various Pt
complexes (Figures S1 and S2), the degradation of
compound 6a and NO release from 6a in the presence of 1
or 3 (Figure S3), 1-mediated cleavage reactions of various
substrates (Table S1), the effects of temperatures, water,
and pH values on the 1-triggered depropargylation
(Figure S4), the LC−MS spectrum of the reaction
mixture of 1 with 5a (Figure S5), the decomposition
kinetics of 14 and 15 in different conditions measured by
HPLC (Figure S6), NO released from the test
compounds in A2780 and IOSE80 cells (Figure S7), the
representative confocal microscopy images of established
zebrafish models (Figure S8), 1H NMR, 13C NMR,
HRMS, and HPLC spectra for compounds 14−17 (PDF)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (nos. 81822041, 21977116, 81673305 and
81773573), National Science & Technology Major Project “Key
New Drug Creation and Manufacturing Program” (number:
2018ZX09711002-006-013), the Funding of Double First-Rate
Discipline Construction (CPU2018GY06 and
CPU2018GY18), open project of State Key Laboratory of
Natural Medicines, nos. SKLNMZZCX201824 and
SKLNMZZ202029, and State Key Laboratory of Pathogenesis,
Prevention and Treatment of High Incidence Diseases in
Central Asia Fund (SKL-HIDCA-2018-1). We also thank
Nanjing Xinjia Medical Technology Corporation for assistance
with the zebrafish assays. The authors thank Dr. Binghe Wang
from Georgia State University for manuscript editing.
Molecular formula strings for target compounds 14−17
and the calculated IC50 values against A2780 and IOSE80
cells (CSV)
AUTHOR INFORMATION
■
Corresponding Author
ABBREVIATIONS
■
Zhangjian Huang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases,
Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, P. R. China;
orcid.org/0000-0001-6409-8535;
NO, nitric oxide; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazo-
line-1-oxyl 3-oxide; DAF-FM DA, 4-amino-5-(methylamino)-
2′,7′-difluoro-fluorescein diacetate; PBS, phosphate buffered
solution; NCS, N-chlorosuccinimide; EA, ethyl acetate; FACS,
fluorescence-activated cell sorting; TBTU, 2-(1H-benzotria-
zole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; DMF,
N,N-dimethylformamide; DMAP, 4-dimethylaminopyridine
Email: zhangjianhuang@cpu.edu.cn
Authors
Tao Sun − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu
Key Laboratory of Drug Screening, China Pharmaceutical
University, Nanjing 210009, P. R. China
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