Synthesis and Evaluation of O 2-Derived Diazeniumdiolates Activatable via Bioorthogonal Chemistry Reactions in Living Cells

Article abstract of DOI:10.1021/acs.orglett.8b00423

A class of O²-alkyl derived diazeniumdiolates 3a-f and 4a-c were designed and synthesized as new bioorthogonal NO precursors, which can be effectively uncaged in the presence of a palladium catalyst via bioorthogonal bond cleavage reactions to generate NO in living cancer cells, eliciting potent antiproliferative activity.

Full text of DOI:10.1021/acs.orglett.8b00423

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis and Evaluation of O2‑Derived Diazeniumdiolates
Activatable via Bioorthogonal Chemistry Reactions in Living Cells
Tian Lv,† Jianbing Wu,† Fenghua Kang,† Tingting Wang,† Boheng Wan,† Jin-Jian Lu,‡ Yihua Zhang,†
and 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
‡State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao,
China
S
* Supporting Information
ABSTRACT: A class of O2-alkyl derived diazeniumdiolates
3a−f and 4a−c were designed and synthesized as new
bioorthogonal NO precursors, which can be effectively
uncaged in the presence of a palladium catalyst via
bioorthogonal bond cleavage reactions to generate NO in
living cancer cells, eliciting potent antiproliferative activity.
ver the past decade, bioorthogonal chemistry has become
Oone main driving force in chemical biology, offering an
unprecedented chance to dissect native biological processes via
chemistry-enabled strategies.1−3 From traditional “bond for-
mation” reactions to “bond cleavage” reactions, bioorthogonal
chemistry has been widely expanded to label, track, manipulate,
or activate biomolecules of interest.4 Recently, the bioorthogonal
“bond cleavage” reactions have triggered great enthusiasm.5 For
example, photodeprotection of the O-nitrobenzyl (ONB) ether
derivatives,6 Cu(I) or palladium (Pd)-mediated depropargyla-
tion and deallylation reactions,7,8 and tetrazine-triggered inverse
electron-demand Diels−Alder (IEDDA) elimination reactions9
were successfully employed to restore or activate proteins or
prodrugs. Notably, the emerging Pd catalysts, which possess
unique catalytic properties and good biocompatibility in living
cells, could overcome the poor penetration capability of light8
Figure 1. (A) Previous work: Bioorthogonal bond cleavage of O/N-
propargyl prodrugs 1a−c mediated by Pd(0) catalyst and the underlying
and cytotoxicity of the Cu(I) ions.5,10 Among all reported
palladium catalysts, Pd(0) catalysts show the best safety11 and
mechanism. (B)This work: Rational for the design of new bioorthogonal
O2-derived diazeniumdiolates 3a−f and 4a−c.
have been successfully used to activate bioorthogonal prodrugs
1a−c (Figure 1A) of 5-fluoroacil,12−14 vorinostat,15 and
gemcitabine,16 respectively.
donor, can spontaneously liberate two molecules of NO under
physiological conditions with a range of half-lives from a few
seconds to several minutes.25 Nevertheless, the O2-derived
diazeniumdiolates could generate stable precursors that can be
enzymatically cleaved in cancer cells26,27 to produce diazenium-
diolate anions and subsequently release NO in situ, exhibiting
potent and selective antiproliferative activity. Some O2-derived
diazeniumdiolates, such as O2-2,4-dinitrobenzene diazeniumdio-
lates28 together with O2-(6-oxocyclohex-1-en-1-yl)methyl dia-
zeniumdiolates29 and O2-indolequinone diazeniumdiolate
(INDQ/NO),30 were reportedly activatable by glutathione S-
transferase (GST)/GSH and NAD(P)H:quinone oxidoreduc-
Nitric oxide (NO) is a widely distributed signaling messenger
that mediates numerous physiological processes, including
vasodilation, neurotransmission, and immune response.17
Presently, NO is considered a potential cancer therapeutic
agent, and several reviews have described the dichotomous role
of NO in cancer biology.18−21 Generally, an optimal concen-
tration window of NO is preferable for cancer proliferation, while
very low intracellular levels of NO induced by nitric oxide
synthase (NOS) inhibition, or high levels of NO released by NO
donors, could produce a cancer cell growth inhibitory effect.22−24
Owing to its numerous biological functions and duality in cancer
biology, the precise release of NO from the NO donors in
location and dose-controlled manners is crucial for NO-based
cancer therapy. Diazeniumdiolates (2), an important class of NO
Received: February 5, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00423
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
tase (DT-diaphorase), respectively, which are overexpressed in
cancer cells. However, current enzymatically triggered O2-
derived diazeniumdiolates mentioned above could inevitably
produce off-target effects because those enzymes are overex-
pressed rather than exclusively expressed in cancer cells.31,32
Inspired by the bioorthogonal chemistry-based prodrug strategy,
especially the Pd(0)-mediated depropargylation and deallylation
reactions, we hypothesized that the modification of O2-site in
diazeniumdiolates with propargyl or allyl moieties, which may be
exclusively cleaved in the presence of Pd(0) catalysts via
bioorthogonal bond cleavage reactions, could produce a new
class of bioorthogonal NO precursors that may have potential
high specificity relative to the existing enzymatically triggered
NO precursors. To test this hypothesis, we designed and
synthesized three new types of O2-derived diazeniumdiolates:
O2-propargyl diazeniumdiolates 3a−e, O2-allyl diazeniumdio-
lates 3f, and O2-(4-alkyloxybenzyl) diazeniumdiolates 4a−c
(Figure 1B). It was proposed that 3a−f can be cleaved from the
O2-propargyl or O2-allyl groups in the presence of Pd(0) catalysts
to produce diazeniumdiolate anion 2 and spontaneously release
NO, while 4a−c can be cleaved from the O-propargyl, O-allyl, or
O-1,2-allenyl groups33 by Pd(0) catalysts to generate O2-(4-
hydroxylbenzyl) diazeniumdiolate intermediates 5, which then
generate diazeniumdiolate anion 2 via a well-known self-
immolative 1,6-elimination reaction34 to spontaneously liberate
NO under physiological conditions. We further investigated their
antiproliferative activity against different cancer cells, the stability
in PBS or plasma, and the capability to release NO in the
presence or absence of a Pd(0) catalyst in living cells.
The subsequent reduction of 5a−b offered alcohols 6a−b, which
were brominated to furnish benzyl bromides 7a−b. Condensa-
tion of 7a−b with 2a resulted in O2-(4-propargyloxybenzyl)
diazeniumdiolate 4a and O2-(4-allyloxybenzyl) diazeniumdiolate
4b, respectively. Interestingly, the reaction of 4a with t-BuOK in
t-BuOH33 successfully furnished O-1,2-allenyl analogue 4c, while
a similar effort on 3a did not produce O2-1,2-allenyl pyrrolidinyl
diazeniumdiolate.
We first investigated the antiproliferative activity of target
compounds 3a−e against human colon carcinoma HCT116
cells, human ovarian OVCAR5 cells, human leukemia HL-60
cells, and human lung cancer A549 cells in the presence or
absence of a Pd(0) catalyst. It was reported that Pd(dba)2, an air-
stable Pd(0) catalyst,35−37 exhibited better safety relative to other
Pd(0) catalysts.10 In our experiment, the treatment of HCT116,
A549, HL-60, and OVCAR5 cells with Pd(dba)2 (10 μM) for 72
h did not affect cell viability (>90%) (Figure S1), suggesting good
biocompatibility to cancer cells. Furthermore, it has been
reported that the 3 h incubation of A549 cells with Pd(dba)2 is
sufficient for intracellular uptake of the catalyst to trigger
depropargylation or deallylation reactions in living cells.8 In these
regards, the four cancer cells were pretreated without or with
Pd(dba)2 (1 μM) for 3 h, rinsed, and then resuspended with 3a−
e (1 μM) for an additional 48 h, and cell survival was determined
by MTT assay. As shown in Figure 2, without the pretreatment of
The synthetic route of the target compounds 3a−f and 4a−c is
depicted in Scheme 1. The reactions of NO gas (50 psi) with the
Scheme 1. Synthetic Route of Compounds 3a−f and 4a−c
Figure 2. Antiproliferative activity of 3a−e against four cancer cells
without or with the pretreatment of Pd(dba)2. A549, HL-60, HCT116,
and OVCAR5 cells were incubated without or with the pretreatment of
Pd(dba)2 (1 μM) for 3 h, followed by being rinsed and resuspended with
3a−e (1 μM) for 48 h, and cell proliferation was assessed by the MTT
assay. Error bars represent SD from three independent experiments.
Pd(dba)2, all test compounds (1 μM) exhibited weak
antiproliferative activity (inhibitory rates ranging from 5.5% to
15.7%) against four cancer cells. In sharp contrast, with the
pretreatment of Pd(dba)2 (1 μM) for 3 h, all test compounds,
especially 3a and 3c, exhibited significantly enhanced anti-
proliferative activity (inhibitory rates ranging from 45.7% to
55.6%), suggesting that Pd(dba)2 could trigger bioorthogonal
cleavage of an O2-propargyl bond to liberate diazeniumdioalte
anion 2, which released NO spontaneously in situ, generating a
cancer cell growth inhibitory effect.
Next, we selected 3a and HCT116 cells to study the effect of
different concentration ratios of Pd(dba)2/3a on cell growth
inhibitory activity. HCT116 cells were pretreated with different
concentrations of Pd(dba)2 (0.25, 0.5, and 1 μM) for 3 h,
followed by being rinsed and resuspended with 3a (1 μM) for
another 48 h. As shown in Figure S2A, with the pretreatment of
an equivalent concentration of Pd(dba)2 (1 μM), 3a (1 μM)
exhibited the most potent cell growth inhibitory activity against
HCT116 cells. Additionally, the impact of incubation time on the
antiproliferative activity was investigated. HCT116 cells were
pretreated with Pd(dba)2 (1 μM) for 3 h, followed by being
different secondary amines in the presence of NaOMe in ether
offered diazeniumdiolates sodium salts 2a−e, which were
subsequently treated with propargyl bromide to produce the
target compounds 3a−e. Similarly, pyrrolidinyl diazeniumdiolate
2a was treated with allyl bromide to give 3f. Starting from 4-
hydroxy benzaldehyde, the condensation of the hydroxyl group
with propargyl bromide or allyl bromide gave 4-propargyloxy
benzaldehyde 5a and 4-allyloxy benzaldehyde 5b, respectively.
B
DOI: 10.1021/acs.orglett.8b00423
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Organic Letters
Letter
Table 1. IC50 Values (μM) of Compounds 3a and 3c against HCT116, A549, and CCD-841CoN Cells in the Presence or Absence of
a
Pd(dba)2
b
b
3a
3a + Pd
FI
3c
3c + Pd
FI
HCT116
A549
27.2 3.8
33.9 4.6
68.1 4.4
1.4 0.1
1.5 0.1
−
19
23
−
32.6 2.5
25.0 3.9
86.7 5.1
1.3 0.1
1.5 0.1
−
25
17
−
CCD-841CoN
a
Cells were incubated without or with different concentrations of Pd(dba)2 for 3 h, rinsed, and then resuspended with equivalent concentrations of
test compounds for 72 h. Cell viability was assessed by the MTT assay, and IC50 values were calculated accordingly. Data were expressed as the mean
b
SD of each group of cells from three individual experiments. FI (fold increase) is defined as IC50 (compd alone)/IC50 (compd plus Pd(dba)2).
rinsed and resuspended with 3a (1 μM) for 24, 48, and 72 h,
respectively. It was found that the 72 h incubation group
exhibited an improved inhibition rate relative to the 24 and 48 h
groups (Figure S2B). These data indicated that an equivalent
concentration of catalyst to test compound and a 72 h reaction
time could be optimal for a bioorthogonal reaction between 3a
and Pd(dba)2 in HCT116 cells.
pretreatment of Pd(dba)2 did not enhance the inhibitory rate of
6a (Figure S3), suggesting that the [allenyl-Pd(II)] complex may
have little influence on biological activity.
Next, the decomposition rates and NO release kinetics of 3a−
f, 4a−c in the presence of an equivalent concentration of
Pd(dba)2 in PBS (pH = 7.4) were examined by HPLC and Griess
assays,41 respectively (Figure S4). Compound 3a was quickly
degraded (about 49.9% remained at 0.5 h, and 20.6% remained at
24 h) with a large amount of NO generation (up to 58.0% of
theoretical maximum yield at 24 h) (Figure S4A). Importantly,
3a exhibited excellent stability in PBS (pH = 7.4) and bovine
plasma even after incubation for 24 h (Figure S5). Furthermore,
the NO release behaviors of 3a−f and 4a−c in HCT116 cells
without or with the pretreatment of Pd(dba)2 were investigated
by using a well-known NO sensitive fluorophore probe, 4-amino-
5-(methylamino)-2′,7′-difluorofluorescein diacetate (DAF-FM
DA).42 All the compounds alone generated very low fluorescence
in HCT116 cells as in the case of the blank control. In contrast,
with the pretreatment of an equivalent concentration of
Pd(dba)2, they generated significant fluorescence in HCT116
cells. Interestingly, the amounts of NO release in cells from the
compounds were correlated with their IC50’s (Figure 3A and
Accordingly, we determined the IC50 values of 3a and 3c
without or with the pretreatment of Pd(dba)2 against HCT-116
and A549 cells by using the optimized conditions, including an
equivalent concentration of Pd(dba)2 and a 72 h incubation time.
As summarized in Table 1, 3a and 3c exhibited weak
antiproliferative activity against HCT116 and A549 cells
(IC50’s ranging from 25.0 to 33.9 μM), which may be due to
the potential thiol−yne click reactions in living cells.38,39 In
contrast, with the pretreatment of Pd(dba)2, 3a and 3c elicited
much improved antiproliferative activity against the cells (IC50’s
ranging from 1.3 to 1.5 μM) with fold increase (FI) values
ranging from 17 to 25. Additionally, the cell growth inhibitory
effects of 3a and 3c on human normal colonic epithelial CCD
841 CoN cells in the absence of Pd(dba)2 were examined, and
the IC50 values were 68.1 and 86.7 μM, respectively. These
results demonstrated that 3a and 3c exhibited much lower
antiproliferative activity to normal cells than cancer cells in the
absence of the Pd(dba)2 catalyst, whereas they produced potent
antiproliferative activity against cancer cells with the pretreat-
ment of Pd(dba)2. Meanwhile, in the LDH assay (Table S1), 3a
exhibited similar IC50 values to those in MTT assay against
HCT116 cells in the presence or absence of Pd(dba)2. Next, the
antiproliferative activity of two fragments of 3a after Pd(dba)2
mediated biorthogonal reaction, i.e., diazeniumdiolate 2a and
hydroxyacetone, was examined by MTT assay with JS-K as a
positive control (Table S2). Expectedly, JS-K (IC50 1.2 μM)
displayed potent antiproliferative activity, whereas hydroxyace-
tone (IC50 259.9 μM) showed weak activity. Interestingly, both
2a (IC50 155.0 μM) and 2a plus hydroxyacetone (IC50 120.1
μM) exhibited weak activity. One plausible reason is that the
poor cell membrane penetrability and short half-life of 2a in cell
culture medium resulted in fast NO release in the medium rather
than the cells.40 On the basis of the former results, IC50 values of
the analogue compounds 3f and 4a−c against HCT116 cells
were further examined by MTT assay (Table S3). Expectedly, all
the test compounds elicited more significant antiproliferative
activity in the presence of Pd(dba)2 (IC50’s ranging from 1.5 to
7.0 μM) than in the absence of Pd(dba)2 (26.9−32.7 μM).
Notably, the reaction of Pd(dba)2 with the propargyl moiety
could form an [allenyl-Pd(II)] complex (Figure 1A), which is a
good electrophile and may react with some biomolecules.
Accordingly, we studied the cell growth inhibitory effect of (4-
(prop-2-yn-1-yloxy)phenyl)methanol (6a) which has a prop-
argyl group without NO production. It was found that 6a showed
a weak inhibitory rate (7.29% at 1 μM for 72 h), and the
Figure 3. NO release behavior of 3a−f, 4a−c (A) and 3a (B) in
HCT116 cells. HCT116 cells were treated without or with Pd(dba)2 at
the indicated concentrations for 3 h, followed by the equivalent
concentrations of test compound for 8 h, and then stained with DAF-
FM DA and analyzed by fluorescence-activated cell sorting (FACS).
Data are presented as means SD (n = 3). ***P < 0.001 vs control
group, ###P < 0.001.
Figure S6A). For example, 3a with an IC50 of 1.4 μM exhibited a
relative mean fluorescence intensity (MFI) of 9.70, whereas 4a
with an IC50 of 7.0 μM exhibited a relative MIF of 4.26.
Moreover, 3a generated significant fluorescence in a dose-
dependent manner in HCT116 cells (Figure 3B and Figure S6B),
suggesting Pd(dba)2 could effectively induce bioorthogonal
cleavage of 3a and produce a diazeniumdiolate anion, which
released NO spontaneously in living cells.
Herein, we report, for the first time, a new class of O2-alkyl
derived diazeniumdiolates 3a−f and 4a−c, which can be uncaged
by a biocompatible Pd(0) catalyst (Pd(dba)2) via bioorthogonal
bond cleavage reactions to liberate NO in living cells. O2-
Propargyl pyrrolidinyl diazeniumdiolate 3a is metabolically
C
DOI: 10.1021/acs.orglett.8b00423
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Organic Letters
Letter
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.8b00423.
■
S
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: zhangjianhuang@cpu.edu.cn.
ORCID
Jin-Jian Lu: 0000-0001-6703-3120
Yihua Zhang: 0000-0003-2378-7064
Zhangjian Huang: 0000-0001-6409-8535
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Natural
Science Foundation of China (Nos. 81673305 and 81773573)
and Jiangsu Province Funds for Distinguished Young Scientists
(BK20160033). Part of the work was supported by the College
Students Innovation Project for the R&D of Novel Drugs
(201710316236).
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