Design and synthesis of new hybrids from 2-cyano-3,12-dioxooleana- 9-dien-28-oic acid and O2-(2,4-dinitrophenyl) diazeniumdiolate for intervention of drug-resistant lung cancer

Article abstract of DOI:10.1016/j.ejmech.2018.02.062

To search for new drugs for intervention of drug-resistant lung cancer, a series of hybrids 4-15 from 2-cyano-3,12-dioxooleana-9-dien-28-oic acid (CDDO) and O²-(2,4-dinitrophenyl) diazeniumdiolate were designed, synthesized and biologically evaluated. The most active compound 7 produced relatively high levels of nitric oxide (NO) and reactive oxygen species (ROS) in drug-resistant lung cancer A549/Taxol cells which over-express glutathione S-transferase π (GSTπ), and significantly inhibited the cells' proliferation (IC₅₀ = 0.349 ± 0.051 μM), superior to the positive controls CDDO-Me, JS-K and Taxol. The inhibitory activity of 7 could be attenuated by an NO scavenger, ROS scavenger or GSTπ inhibitor. In addition, 7 suppressed the Lon protease expression as well as induced cell apoptosis and cycle arrest in A549/Taxol cells more strongly than CDDO-Me or JS-K. Together, our findings suggest that 7 may be worth studying further for intervention of drug-resistant lung cancer.

Full text of DOI:10.1016/j.ejmech.2018.02.062

Accepted Manuscript
Design and synthesis of new hybrids from 2-cyano-3,12-dioxooleana- 9-dien-28-oic
2
acid and O -(2,4-dinitrophenyl) diazeniumdiolate for intervention of drug-resistant
lung cancer
Fenghua Kang, Yong Ai, Yihua Zhang, Zhangjian Huang
PII:
S0223-5234(18)30199-5
10.1016/j.ejmech.2018.02.062
EJMECH 10239
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 January 2018
Revised Date: 17 February 2018
Accepted Date: 19 February 2018
Please cite this article as: F. Kang, Y. Ai, Y. Zhang, Z. Huang, Design and synthesis of new hybrids
2
from 2-cyano-3,12-dioxooleana- 9-dien-28-oic acid and O -(2,4-dinitrophenyl) diazeniumdiolate for
intervention of drug-resistant lung cancer, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.02.062.
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ACCEPTED MANUSCRIPT
Design and synthesis of new hybrids from 2-cyano-3,12-dioxooleana-
9-dien-28-oic acid and O2-(2,4-dinitrophenyl) diazeniumdiolate for
intervention of drug-resistant lung cancer
Fenghua Kang,a,b,# Yong Ai, a,b,# Yihua Zhang,*,a,b and Zhangjian Huang*,a,b
aState Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P.R. China.
bJiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China
Pharmaceutical University, Nanjing 210009, P.R. China.
# These authors contributed equally to this work.
Graphical Abstract

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Design and synthesis of new hybrids from 2-cyano-3,12-dioxooleana-
9-dien-28-oic acid and O2-(2,4-dinitrophenyl) diazeniumdiolate for
intervention of drug-resistant lung cancer
Fenghua Kang,a,b,# Yong Ai, a,b,# Yihua Zhang,*,a,b and Zhangjian Huang*,a,b
aState Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P.R. China.
bJiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China
Pharmaceutical University, Nanjing 210009, P.R. China.
# These authors contributed equally to this work.
Abstract
To search for new drugs for intervention of drug-resistant lung cancer, a series of
hybrids 4-15 from 2-cyano-3,12-dioxooleana-9-dien-28-oic acid (CDDO) and
O2-(2,4-dinitrophenyl) diazeniumdiolate were designed, synthesized and biologically
evaluated. The most active compound 7 produced relatively high levels of nitric oxide
(NO) and reactive oxygen species (ROS) in drug-resistant lung cancer A549/Taxol
cells which over-express glutathione S-transferase π (GSTπ), and significantly
inhibited the cells’ proliferation (IC50 = 0.349 ± 0.051 µM), superior to the positive
controls CDDO-Me, JS-K and Taxol. The inhibitory activity of 7 could be attenuated
by an NO scavenger, ROS scavenger or GSTπ inhibitor. In addition, 7 suppressed the
Lon protease expression as well as induced cell apoptosis and cycle arrest in
A549/Taxol cells more strongly than CDDO-Me or JS-K. Together, our findings
suggest that 7 may be worth studying further for intervention of drug-resistant lung
cancer.
Keywords: CDDO, O2-(2,4-dinitrophenyl) diazeniumdiolate, nitric oxide, ROS, drug
resistance.

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1. Introduction
It has been reported that the synthetic oleanolic acid derivatives, such as
2-cyano-3,12-dioxooleana-9-dien-28-oic acid (CDDO) and its methyl ester
(CDDO-Me) (Fig. 1) exhibit strong anti-cancer activity by inducing production of
intracellular reactive oxygen species (ROS) in cancer cells [1], and that combination
of CDDO with other anti-cancer agent(s) may synergistically induce oxidative stress
overload [2].
Lon protease (Lon) refers to a highly conserved ATP-dependent serine peptidase
that contributes to protein quality control and stress response pathways by selectively
degrading misfolded, misassembled, or damaged proteins in mitochondria [3]. The
expression of Lon is frequently elevated in cancer cells [3] and down-regulation of
Lon may significantly block the cancer cell proliferation and enhance the sensitivity
of cancer cells to chemotherapeutic agents by promoting apoptosis without significant
toxicity to normal cells [4]. Recently, it has been discovered that CDDO-Me directly
and selectively blocks mitochondrial Lon protease activity through an addition
reaction between CDDO-Me and Lon, ultimately inhibiting proliferation and inducing
apoptosis of the cancer cells [2].
Nitric Oxide (NO) plays an important regulation role in the realm of cancer biology
•-
[5]. High levels of NO tend to interact with ROS, such as superoxide anion (O2 ), to
generate reactive nitrogen species (RNS), such as peroxynitrite anion (ONOO-) [6],
acting as an inducer of cytotoxicity and apoptosis [7] as well as a reversal agent of
multidrug resistance (MDR) by nitration of key protein and alteration of various
redox-sensitive proteins in cancer cells [8,9]. Given its multifunctional biological
effects, NO should be released from NO donors in a controlled manner, preferably in
response to a certain stimulus in cancer cells, thus avoiding off-target effects [10].
O2-(2,4-Dinitrophenyl) diazeniumdiolates, such as PABA/NO and JS-K (Fig. 1),
represent an important class of NO donors as they are able to preferentially release
NO in cancer cells [11], which are promoted by glutathione S-transferase π (GSTπ),

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over-expressed in a large number of cancer cells [12-14].
O
H
O
H
OH
H
O
H
NC
O
NC
O
O
O
H
H
CDDO
CDDO-Me
O
O2N
O
NO2
O2N
NO2
N
O
N
N
N
N
O
O
N
O
O
N
O
N
H
PABA/NO
JS-K
Fig. 1. Chemical structures of CDDO, CDDO-Me, PABA/NO and JS-K.
The above investigations led us to hypothesize that hybridization of
O2-(2,4-dinitrophenyl) diazeniumdiolate moiety with the CDDO scaffold would
provide a new class of NO-donating CDDO derivatives with ROS/RNS inducing and
Lon inhibitory activities against lung cancer resistant cells. To test the hypothesis, we
designed, synthesized the hybrids 4-15 by coupling the carboxyl group of CDDO with
O2-(2,4-dinitrophenyl) diazeniumdiolate moiety through various linkers, and
evaluated the inhibitory activity of these compounds against drug-resistant lung
cancer.
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds is depicted in Scheme 1. The condensation
of diazeniumdiolate sodium salts 1a-f with 1,5-difluoro-2,4-dinitrobenzene in the
mixture of 5% NaHCO3 aqueous and acetone gave arylated diazeniumdiolates 2a-f.

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Next, substitution of one fluorine atom of 2a or 2b with 2-bomoethylamine
hydrobromide yielded 3a or 3b. Finally, conjugation of 28-COOH in CDDO with 3a
and 3b provided 4 and 5, respectively. In addition, CDDO was treated with oxalyl
chloride and the resulting acylchloride without purification was reacted with
anhydrous piperazine to yield compound 16. The free secondary amine in 16
underwent nucleophilic attack toward 2a-f in the presence of Na2CO3 to furnish 6-11.
Compound 5 or 7 was treated with N-Boc-L-alanine or N-Boc-L-phenyl alanine to
respectively afford the esters 17, 18 or 19, 20, where the Boc group was then removed
with boron trifluoride diethyl etherate to generate target compounds 12-15,
respectively.

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Scheme 1. The synthetic route of the target compounds 4-15. Reagents and conditions:
(i) 5% NaHCO3 aqueous, 1,5-difluoro-2,4-dinitrobenzene, acetone, N2, 0 °C→ r.t.; (ii)
2-Bomoethylamine hydrobromide, Na2CO3, DMF, 4 h; (iii) CDDO, K2CO3, acetone;
(iv) (COCl)2, anhydrous CH2Cl2, 0 °C → r.t., 12 h; (v) Anhydrous piperazine, TEA,
anhydrous CH2Cl2, 0 °C → r.t., 12 h; (vi) 2a-f, Na2CO3, DMF; (vii) N-Boc-protected
L-amino acids, EDCI, DMAP, anhydrous CH2Cl2, 25 °C; (viii) BF3·Et2O, anhydrous

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CH2Cl2, 25 °C.
2.2. Expression of GSTπ in various cell lines
Given that our target compounds bear an O2-(2,4-dinitrophenyl) diazeniumdiolate
moiety which could be preferentially promoted by GSTπ to release NO, we initially
examined the expression of GSTπ in drug-sensitive human lung adenocarcinoma
A549 cells and drug-resistant A549/Taxol cells as well as human lung embryonic
fibroblast MRC-5 cells by western blot assay. As shown in Fig. 2, the levels of GSTπ
in A549 cells were much higher than that in MRC-5 cells but significantly lower than
that in A549/Taxol cells. These results clearly indicated that GSTπ was
lower-expressed in normal lung cells, highly-expressed in drug sensitive lung cancer
cells, while over-expressed in drug-resistant lung cancer cells.
Fig. 2. Expression levels of GSTπ in A549 cells, drug-resistant A549/Taxol cells, as
well as non-cancer MRC-5 cells were measured by western blot assay. Data are
presented as the mean ± SD (n = 3). ***P < 0.001 vs the MRC-5 group, ## P < 0.01 vs
the A549 group.
2.3. Assessment of anti-proliferative activity of the target compounds in drug sensitive
and resistant lung cancer cells
To evaluate the anti-cancer activity of the target compounds, we tested their effects

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on the proliferation of A549 cells and A549/Taxol cells by the MTT assay using
CDDO-Me and JS-K as positive controls. As shown in Fig. 3, all compounds
exhibited more potent cell growth inhibitory activity against A549/Taxol cells with
over-expression of GSTπ than parental sensitive A549 cell with relatively less
expression of GSTπ. Among them, compounds 5, 7, 8, 12 and 14 at 1 µM inhibited
proliferation of A549/Taxol cells by > 50%, which were higher than that of
CDDO-Me, JS-K and the other seven compounds.
Fig. 3. Target compounds inhibit the proliferation of A549/Taxol and A549 cells.
Cancer cell lines were treated with the indicated compounds at 1 µM for 72 h and the
cell proliferation was measured by MTT. The inhibition (%) of each compound was
determined. Data are presented as the means (%) ± SD of each compound from three
independent experiments.
Table 1. Antiproliferative activity of selected compounds.
IC50 (µM)a
Compounds
RFb
A549/Taxol
0.820 ± 0.010
0.349 ± 0.051
0.941 ± 0.050
1.087 ± 0.029
0.697 ± 0.011
A549
1.438 ± 0.116
1.066 ± 0.041
1.078 ± 0.022
1.441 ± 0.094
1.068 ± 0.019
0.6
0.3
0.9
0.8
0.7
5
7
8
12
14

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1.703 ± 0.056
1.987 ± 0.033
3.112 ± 0.469
2.074 ± 0.086
2.313 ± 0.188
0.007 ± 0.073
0.8
0.9
445
CDDO-Me
JS-K
Taxol
aThe inhibitory effects of individual compounds on the proliferation of cancer cells
were determined by the MTT assay. The data are presented as the means ± SD of each
compound from three independent experiments. bResistant factor (RF) was calculated
according to the following equation: RF = IC50 (corresponding resistant cells)/IC50
(parental cells).
Subsequently, the five most potent compounds 5, 7, 8, 12 and 14 were further tested
for their anti-cancer activity by measuring the IC50 values using MTT assay and the
results were summarized in Table 1. We found that these compounds displayed potent
inhibitory activity against both A549/Taxol (IC50 = 0.349 - 1.087 µM) and A549 (IC50
= 1.066 - 1.438 µM) cells, and their antiproliferative activity was more potent than
CDDO-Me (IC50 = 1.703 and 2.313 µM) and JS-K (IC50 = 1.987 and 2.313 µM),
respectively. Compound 7 was the most potent to inhibit A549/Taxol cells with IC50
value of 0.349 ± 0.051 µM, over ten-fold more active than Taxol (IC50 = 3.112 ±
0.469 µM). In contrast, 7 showed approximately 5-fold less toxicity relative to
non-cancer MRC-5 cells (IC50 = 1.604 ± 0.212 µM, more details see Fig. S1 in the
Supporting Information), indicating that 7 preferentially and significantly inhibited
the growth of lung cancer resistant cells in vitro, which may be a promising candidate
for further investigation.
2.4. 7 significantly promoted intracellular ROS accumulation in A549/Taxol cells
To examine ROS produced by 7 in A549/Taxol cells, the cells were treated with
various concentrations of 7 for 24 h in triplicate by using CDDO-Me and JS-K as
positive controls. Dichlorodihydrofluorescein diacetate (DCFH-DA) probe [15] was
then employed to detect the intracellular levels of ROS by measuring the fluorescent

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signals using a fluorescence microplate reader. It was observed that 7
dose-dependently generated ROS, and the amounts of ROS were larger than those
produced by CDDO-Me and JS-K at the same dose in A549/Taxol cells, indicating
that 7 more significantly promoted intracellular ROS accumulation in the cancer
resistant cells relative to CDDO-Me and JS-K (Fig. 4).
Fig. 4. Treatment with 7, CDDO-Me and JS-K (2.4, 4.8 and 9.6 µM) induced the
accumulation of ROS in A549/Taxol after 24 h. Data are expressed as the mean ± SD
of each compound in individual types of cells from three experiments. ***P < 0.001
vs the 7 (9.6 µM) group.
2.5. 7 selectively released high levels of NO in A549/Taxol
Furthermore, we detected intracellular levels of NO released by 7, JS-K and
CDDO-Me in A549/Taxol cells and MRC-5 cells using Griess assay [16], respectively.
As expected, treatment with CDDO-Me resulted in little NO in both A549/Taxol and
MRC-5 cells. In contrast, treatment with 7 led to relatively high levels of NO
production in A549/Taxol, which were larger than that produced by JS-K under the
same conditions. In addition, both 7 and JS-K released small amounts of NO in the

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MRC-5 cells, with less amounts from 7 relative to JS-K (Fig. 5). These results
indicated that 7 selectively released relatively high amounts of NO in A549/Taxol
cells, probably owing to over-expressing GSTπ of the cells.
Fig. 5. Variable levels of NO (present as nitrite) produced by the indicated
compounds in A549/Taxol and MRC-5 cells. Cells were treated in triplicate with
individual compounds at 50 µM for 6 h, and the concentrations of intracellular nitrite
were determined by Griess assay. The individual values were determined by
measuring the absorbance at 540 nm and calculated according to the standard curve.
Data are expressed as the mean ± SD of each compound in individual types of cells
from three experiments. **P < 0.01 and ***P < 0.001 vs the CDDO-Me group, ##P <
0.01 and ###P < 0.001 vs the MRC-5 group.
2.6. GSTπ inhibitor, ROS scavenger or NO scavenger diminished inhibitory activity of
7 against A549/Taxol
To verify the contributions of GSTπ, ROS and NO on the growth inhibitory of 7,
A549/Taxol cells were pretreated with or without GSTπ inhibitor etacrynic acid (EA)
[17], ROS scavenger N-acetylcysteine (NAC) [18] and NO scavenger hemoglobin
(HB) [19] for 1 h and then treated with or without 1 µM of 7 for 72 h using MTT
assay, respectively. The results showed that incubation with EA, HB or NAC did not
affect A549/Taxol cells while treatment with 7 alone remarkably inhibited the

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proliferation of A549/Taxol cells. In sharp contrast, pretreatment with EA, HB or
NAC significantly diminished the inhibitory effects of 7 (Fig. 6). These results
suggest that the high levels of RNS/ROS and GSTπ may be essential for the potent
anti-proliferative activity of 7 against the A549/Taxol cells.
Fig. 6. Effects of GSTπ inhibitor etacrynic acid (EA), ROS scavenger
N-acetylcysteine (NAC) and NO scavenger hemoglobin (HB) on the cell growth
inhibitory activity of 7. A549/Taxol cells were pretreated with or without EA (10 µM),
NAC (10 mM) and HB (20 µM) for 1 h and then treated with or without 1 µM of 7 for
72 h, and the cell inhibition was determined by the MTT assay. Data are the mean ±
SD of the cell inhibition (%) from three independent experiments. ***P < 0.001 vs
the only 7 treated group.
2.7. 7 significantly decreased levels of Lon expression in A549/Taxol cells
Given that 7 may decrease Lon expression in cancer cells, we first examined the Lon
expression in A549, A549/Taxol and MRC-5 cells by Western blot. It was found that
lower levels of Lon were expressed in MRC-5 cells, while elevated expression of Lon
was observed in lung cancer cells, particularly in drug-resistant A549/Taxol cells (Fig.
7A). These results were consistent with the previous report that high levels of Lon are
responsible for the resistance of cancer cells to chemotherapeutics [20]. Significantly,
treatment with 7 reduced the levels of Lon in A549/Taxol cells in a dose-dependent

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manner, and 7 was stronger than the positive controls CDDO-Me and JS-K at the
same concentration (4.8 µM) (Fig. 7B). Previous studies have demonstrated that Lon
protease can be inactivated by peroxynitrite under conditions of elevated oxidative
stress [21], accordingly, decreased levels of Lon expression by treatment with 7 may
be attributed to the synergistic effects of CDDO and O2-(2,4-dinitrophenyl)
diazeniumdiolate which released high levels NO in drug-resistant lung cancer cells.
Fig. 7. A. Lon expression in MRC-5, A549, and A549/Taxol cells. MRC-5, A549,
and A549/Taxol cells were harvested and lyzed. The relative levels of Lon to control
GAPDH in MRC-5, A549, and A549/Taxol cell lysates were determined by Western
blot. B. Effect of 7 on the protein expression of Lon in A549/Taxol cells. Cells were
treated with, or without, the indicated concentrations of 7, CDDO-Me and JS-K for 24
h, and the relative levels of Lon protein to GAPDH in cell lysates were determined by
##
Western blotting. Data are presented as the mean ± SD (n = 3). ***P < 0.001, P <
0.01 and ###P < 0.001.
2.8. 7 induced apoptosis of A549/Taxol cells

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Considering that CDDO-Me is able to induce cancer cell apoptosis by modulating
the expression of apoptosis-related regulators such as Lon [1,2], and that high levels
of NO also induce tumor cell apoptosis [22], the effect of 7 on induction of tumor
cells’ apoptosis was examined and compared with CDDO-Me and JS-K. A549/Taxol
cells were treated with various concentrations of 7, CDDO-Me, JS-K or vehicle for 24
h. The cells were harvested and stained with Annexin V-FITC and propidium iodide
(PI), and the percentages of apoptotic cells were determined by flow cytometry
analysis. It was observed that treatment with 7 induced apoptosis in A549/Taxol cells
in a dose-dependent manner, and was significantly stronger than treatment with
CDDO-Me and JS-K alone at the same doses (Fig. 8). These results suggest that 7
may exert its anti-cancer activity, at least in part, via induction of the cells’ apoptosis.

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Fig. 8. Apoptosis of A549/Taxol cells treated with 7, CDDO-Me, and JS-K for 24 h.
Data are presented as the mean ± SD (n = 3). **P < 0.01 vs the control group, ##P <
0.01.
2.9. 7 arrested cell cycle of A549/Taxol cells
To investigate whether 7 suppressed the cells’ growth by arresting cell cycle, we

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performed the experiment where cell cycle distribution was analyzed by flow
cytometry after staining the DNA with propidium iodide (PI). It was found that
treatment with 7 at 1.2, 2.4 and 4.8 µM for 24 h increased the percentage of cells at
the G2/M phase from 28.3% to 48.3% while decreased G0/G1-phase cells from 61. 3%
to 39.4% in a dose-dependent manner (Fig. 9). Moreover, the blocking effect of 7
were significantly stronger than that of CDDO-Me and JS-K at the same doses
(Representative histograms of CDDO-Me and JS-K were shown on pages S2 in the
Supporting Information). These results indicated that 7 significantly induced
A549/Taxol cell cycle arrest at G0/G1 phase.

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Fig. 9. Effects of 7 on cell cycle of A549/Taxol cells. Cells were treated with the
indicated concentrations of 7, CDDO-Me and JS-K for 24 h and stained with PI,
followed by flow cytometry analysis: (A) representative histograms; (more details of
CDDO-Me and JS-K were shown on pages S2 in the Supporting Information) (B)

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quantitative analysis. Data are representative histograms and expressed as the mean ±
SD of each compound from three independent experiments. **P < 0. 01 vs the control
group, ##P < 0.01.
3. Conclusion
In summary, twelve hybrids from O2-(2,4-dinitrophenyl) diazeniumdiolate and
CDDO were designed and synthesized. The most active hybrid 7 selectively produced
high levels of NO and ROS in drug resistant A549/Taxol cells. In addition, 7
significantly inhibited the proliferation of A549/Taxol cells, superior to CDDO-Me
and JS-K, and over ten-fold more potent than taxol. In contrast, 7 had approximately
5-fold less effect on non-cancer MRC-5 cells relative to A549/Taxol cells, and its
anti-cancer activity was significantly attenuated by a GSTπ inhibitor, ROS scavenger
or NO scavenger. Furthermore, 7 suppressed the Lon expression, induced apoptosis
and cell cycle arrest in A549/Taxol cells more strongly than CDDO-Me and JS-K.
These results suggest that the anti-proliferative activity of 7 could be attributed to the
synergic effects of CDDO and O2-(2,4-dinitrophenyl) diazeniumdiolate moiety,
leading to selective generation of high levels of NO/ROS and enhanced inhibition of
Lon in drug-resistant lung cancer cells. Therefore, our findings provide a proof of
principle in design of new NO-releasing CDDO derivatives for the intervention of
drug-resistant lung cancer.
4. Experimental protocols
4.1. Chemical analysis
All commercially available compounds were used without further purification,
unless otherwise noted. Anhydrous solvents (CH2Cl2) were used as commercially
available. Analytical and preparative TLC was performed on silica gel (200 - 300
mesh) GF/UV 254 plates, and the chromatograms were visualized under UV light at
254 and 365 nm. Melting points of individual compounds were determined on a

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1
Mel-TEMP II melting point apparatus and uncorrected. H-NMR and 13C-NMR
spectra were recorded on a Bruker Avance 300 (1H, 300 MHz; 13C, 75 MHz)
spectrometer at 303 K, using TMS as an internal standard. MS spectra were recorded
with a Mariner mass spectrometer (ESI) and high resolution mass spectrometry
(HRMS) spectra on an Agilent Technologies LC/MSD TOF instrument. Solutions
after reactions and extractions were concentrated using a rotary evaporator operating
at a reduced pressure of ~20 Torr. Individual compounds with a purity of > 95% were
used for biological experiments. CDDO and acyl chloride of CDDO were synthesized
using the reported protocols [23]. Diazeniumdiolate sodium salts 1a-f were prepared
according to the method described previously [13]. O2-(2,4-Dinitrophenyl)
diazeniumdiolates 2a-f were synthesized using the reported methods [12].
4.1.1 General procedure for the preparation of 4 and 5
4.1.1.1 Synthesis of intermediates of 3a and 3b
To a DMF (2.5 mL) solution of 2-bomoethylamine hydrobromide (1.0 mmol) and
2a (1.0 mmol) was added Na2CO3 (2 mmol), and the resulting solution was stirred at
room temperature for 4 h. Then the reaction mixture was diluted with water (15 mL)
and extracted with EtOAc (50 mL × 3). The combined organic layer was washed with
brine, dried with anhydrous sodium sulfate, filtered, and evaporated in vacuum. The
obtained crude product was purified by silica column chromatography (PE/EA = 1:2,
v/v) to afford O2-(2,4-dinitro-5-(2-bromoethylamino)phenyl) 1-(piperidine-1-yl)
diazen-1-ium-1,2-diolate (3a) as yellowish liquid, yield 77%. Synthesis of
O2-(2,4-dinitro-5-(2-bromoethylamino)phenyl) 1-(N-methylethanolamino) diazen-1-
ium-1,2-diolate (3b) was conducted in the similar way with yield 74%.
4.1.1.2 Synthesis of compounds of 4 and 5
The solution of 3a (1.0 mmol) and CDDO (1.0 mmol) in acetone (10 mL) was
added K2CO3 (1.5 mmol). The mixture was then heated to 45 and stirred for 4 h.
The reaction mixture was filtered, and the filtrate was concentrated under reduced
pressure. The resulting residue was purified by flash chromatography (PE/EA = 3:7,

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v/v) to afford the compound 4. Synthesis of 5 were in the similar method.
4.1.1.2.1 Compound 4
O2-(2,4-Dinitro-5-{2-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-oxo-ethyl-a
mino}phenyl) 1-(piperidine-1-yl) diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 68% yield as a yellowish solid: mp: 170 - 172 ; H NMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 9.08 (s, 1H, Ar-H), 8.73 (t, J = 5.1 Hz, 1H, NH-Ar), 8.05 (s,
1H, C1-H), 6.82 (s, 1H, Ar-H), 5.96 (s, 1H, C11-H), 4.47 - 4.44 (m, 2H, OCH2), 3.73 -
3.68 (m, 2H, OCH2), 3.61 (t, J = 5.6 Hz, 4H, 2 × NCH2), 3.07 - 2.97 (m, 1H, C13-H),
2.90 - 2.85 (m, 1H, C18-H), 1.46 (s, 3H, CH3), 1.28 - 1.24 (m, 8H, CH3, 3 × CH2), 1.20
(s, 3H, CH3), 1.16 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.90 (s, 3H,
13
CH3) ppm; C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.0, 196.2, 177.2, 168.0,
165.2, 155.0, 148.0, 127.3, 126.6, 125.8, 123.5, 114.0, 113.9, 97.9, 61.1, 51.3, 49.0,
47.1, 46.8, 45.1, 44.5, 42.0, 41.8, 41.5, 35.1, 33.8, 32.6, 31.1, 30.1, 29.3, 29.0, 28.8,
27.5, 26.4, 26.1, 23.9, 23.8, 22.7, 22.5, 22.1, 21.0, 21.0, 17.6; ESI-MS: 866 [M + Na]+;
HRMS: m/z: Calcd. for C44H57N7O10 [M + Na]+ 866.4167, found 866.4050, ppm error
5.0.
4.1.1.2.2 Compound 5
O2-(2,4-Dinitro-5-{2-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-oxo-ethyl-a
mino}phenyl) 1-(N-methyl ethanolamino) diazen-1-ium-1,2-diolate. The title
compound was obtained in 74% yield as a yellowish solid: mp: 134 - 136 ; 1H NMR
(300 M Hz, CDCl3, 25 °C, TMS): δ 9.07 (s, 1H, Ar-H), 8.69 (s, 1H, NH-Ar), 8.05 (s,
1H, C1-H), 6.88 (s, 1H, Ar-H), 5.97 (s, 1H, C11-H), 4.42 - 4.40 (m, 2H, OCH2), 3.92 -
3.69 (m, 6H, OCH2, 2 × NCH2), 3.41 (s, 3H, NCH3), 2.97 - 2.92 (m, 1H, C13-H), 2.92
- 2.88 (m, 1H, C18-H), 1.46 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.17 (s,
3H, CH3), 1.00 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75
M Hz, CDCl3, 25 °C, TMS): δ 198.9, 196.6, 178.0, 168.9, 165.8, 155.7, 148.6, 127.8,
126.9, 126.2, 123.9, 114.5, 114.5, 98.1, 61.5, 59.7, 55.7, 49.6, 47.6, 47.4, 45.7, 45.0,
42.6, 42.0, 41.9, 40.6, 35.6, 34.3, 33.1, 32.9, 31.5, 30.6, 29.7, 28.0, 26.9, 26.6, 24.4,

ACCEPTED MANUSCRIPT
23.0, 22.6, 21.6, 21.4, 18.1; ESI-MS: 834 [M + H]+, 856 [M + Na]+; HRMS: m/z:
Calcd. for C42H55N7O11 [M - H]- 832.3960, found 832.3896, ppm error 5.0.
4.1.2 General procedure for the preparation of 6-11
4.1.2.1 Synthesis of intermediates of 16
A mixture of CDDO (6.1 mmol) and oxalyl chloride (5 mL) in anhydrous CH2Cl2
(50 mL) was stirred at room temperature overnight. The solvent was removed in
vacuo, and the residue was coevaporated with anhydrous CH2Cl2 three times, and then
used for the next reaction without further purification.
To a solution of the residue (0.35 mmol) obtained above in anhydrous CH2Cl2 (2
mL) was added a solution of anhydrous piperazine (1.5 mmol). The mixture was
stirred at room temperature overnight. The reaction mixture was then diluted with
CH2Cl2 (15 mL), washed with saturated sodium bicarbonate (NaHCO3) aqueous
solution and brine and dried over magnesium sulfate. The solvent was removed and
the crude product was purified by flash column chromatography (PE/EA = 1:9, v/v) to
obtain 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin (16) as an
1
amorphous solid, yield 68%. mp: 198 - 200 °C; H NMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 8.06 (s, 1H, C1-H), 5.95 (s, 1H, C11-H), 3.70 - 3.68 (m, 4H), 3.36 (s, 1H),
3.09 (d, J = 12.9 Hz, 1H), 2.86 - 2.84 (m, 4H),1.48 (s, 3H), 1.32 (s, 3H), 1.26 (s, 3H),
13
1.17 (s, 3H), 1.02 (s, 3H), 1.01 (s, 3H), 0.91 (s, 3H); C NMR (75 M Hz, CDCl3,
25 °C, TMS): δ 198.8, 196.2, 174.8, 165.5, 165.3, 123.6, 114.0, 112.9, 62.7, 60.2, 49.1,
47.2, 47.0, 45.5, 45.2, 44.5, 42.0, 41.5, 35.6, 33.7, 32.6, 32.6, 32.5, 31.1, 29.8, 29.7,
27.9, 26.4, 26.4, 26.0, 24.0, 23.5, 22.4, 21.3, 21.0, 17.7; ESI-MS: 560 [M + H]+.
4.1.2.2 Synthesis of compounds 6-11
Compounds 6-11 were synthesized using a similar method. A typical procedure can
be described as follows using 6 as the example. To a DMF (2.5 mL) solution of 16
(1.0 mmol) and 2a-f (1.0 mmol) was added Na2CO3 (2 mmol), and the resulting
solution was stirred at room temperature for 4 h. Then the reaction mixture was

ACCEPTED MANUSCRIPT
diluted with water (2 mL) and extracted with EtOAc (50 ml × 3). The combined
organic layer was washed with brine, dried with anhydrous sodium sulfate, filtered,
and evaporated in vacuum. The obtained crude product was purified by silica column
chromatography (PE/EA = 2:3, v/v) to afford compounds 6-11 as yellowish solid.
4.1.2.2.1 Compound 6
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-(piperidine-1-yl) diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 58% yield as a yellowish solid: mp: 168 - 170 °C; HNMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 8.73 (s, 1H, ArH), 8.05 (s, 1H, C1-H), 6.94 (s, 1H, ArH), 5.96
(s, 1H, C11-H), 4.09 - 3.93 (m, 4H, 2 × NCH2), 3.59 (t, J = 5.5 Hz, 4H, 2 × NCH2),
3.35 - 3.25 (m, 5H, 2 × NCH2, C13-H), 3.16 - 3.09 (m, 1H, C18-H), 1.86 -1.81 (m, 6H,
3 × CH2), 1.49 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.17 (s, 3H, CH3),
13
1.03 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.92 (s, 3H, CH3) ppm; C NMR (75 M Hz,
CDCl3, 25 °C, TMS): δ 199.0, 196.5, 175.8, 167.9, 165.9, 165.8, 153.8, 149.8, 133.1,
128.7, 127.2, 124.0, 114.4, 105.6, 51.9, 51.6, 50.5, 49.5, 47.7, 47.6, 45.8, 45.0, 44.9,
42.5, 42.1, 36.1, 34.2, 33.1, 33.0, 31.6, 30.4, 30.4, 29.7, 26.9, 26.5, 24.4, 24.3, 23.9,
23.2, 22.8, 21.7, 21.5, 18.2; ESI-MS: 891 [M + Na]+, 907 [M + K]+;
4.1.2.2.1 Compound 7
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-(N-methyl ethanolamino) diazen-1-ium-1,2-diolate. The title compound
was obtained in 66% yield as a yellowish solid: mp: 152 - 154 ; 1HNMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 8.73 (s, 1H, ArH), 8.07 (s, 1H, C1-H), 6.94 (s, 1H, ArH), 5.96
(s, 1H, C11-H), 3.95 - 3.83 (m, 8H, 4 × NCH2), 3.37 (m, 3H, NCH3), 3.26 - 3.09 (m,
6H, NCH2, OCH2, C13-H, C18-H), 1.48 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.25 (s, 3H,
CH3), 1.16 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.93 (s, 3H, CH3) ppm;
13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.0, 196.1, 175.3, 165.1, 165.1, 153.7,
149.5, 132.5, 126.7, 123.3, 113.9, 113.8, 107.5, 104.8, 59.1, 55.1, 50.0, 49.2, 47.1,
47.0, 45.4, 44.5, 44.5, 42.0, 41.6, 40.1, 35.8, 33.6, 32.6, 32.3, 31.1, 30.0, 29.7, 28.8,

ACCEPTED MANUSCRIPT
27.8, 26.3, 25.8, 24.0, 23.6, 22.5, 21.6, 21.0, 17.6; ESI-MS: 881 [M + Na]+, 897 [M +
K]+; HRMS: m/z: Calcd. for C44H58N8O10 [M - H]- 857.4276, found 857.4209, ppm
error 5.0.
4.1.2.2.3 Compound 8
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-
yl}phenyl) 1-(morpholine-1-yl) diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 60% yield as a yellowish solid: mp: 158 - 160 ; HNMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 8.73 (s, 1H, ArH), 8.05 (s, 1H, C1-H), 6.93 (s, 1H, ArH), 5.96
(s, 1H, C11-H), 4.03 - 3.97 (m, 8H, 4 × NCH2), 3.61 (t, J = 4.7 Hz, 4H, 2 × OCH2),
3.30 (s, 4H, 2 × NCH2), 3.16 - 3.12 (m, 2H, C13-H, C18-H), 1.48 (s, 3H, CH3), 1.31 (s,
3H, CH3), 1.25 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.04 (s, 3H, CH3), 1.02 (s, 3H, CH3),
13
0.92 (s, 3H, CH3) ppm; C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.4, 196.0,
175.4, 167.3, 165.1, 153.0, 149.0, 133.0, 126.8, 123.5, 119.5, 114.0, 113.8, 105.3,
65.0, 50.6, 50.0, 49.0, 47.2, 47.1, 45.3, 44.5, 44.4, 44.2, 42.0, 41.7, 35.6, 33.7, 32.5,
32.4, 31.1, 29.9, 29.8, 29.1, 28.8, 27.9, 26.7, 26.1, 23.9, 23.4, 22.5, 21.2, 21.0, 17.7;
ESI-MS: 893 [M + Na]+; HRMS: m/z: Calcd. for C45H58N8O10 [M + Na]+ 893.4276,
found 893.4193, ppm error 5.0.
4.1.2.2.4 Compound 9
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-(N,N-diethylamino) diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 58% yield as a yellowish solid: mp: 162 - 164 ; HNMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 8.76 (s, 1H, ArH), 8.06 (s, 1H, C1-H), 6.98 (s, 1H, ArH), 5.99
(s, 1H, C11-H), 4.02 - 3.87 (m, 4H, 2 × NCH2), 3.56 - 3.49 (m, 4H, 2 × NCH2), 3.35 -
3.22 (m, 5H, 2 × NCH2, C13-H), 3.16 - 3.12 (m, 1H, C18-H), 1.49 (s, 3H, CH3), 1.32 (s,
3H, CH3), 1.27 - 1.25 (m, 9H, 2 × CH3, CH3), 1.17 (s, 3H, CH3), 1.03 (s, 3H, CH3),
1.01 (s, 3H, CH3), 0.92 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS):
δ 199.0, 196.1, 175.8, 167.9, 165.7, 153.8, 149.7, 133.2, 128.7, 127.3, 123.9, 114.6,
114.4, 105.4, 60.3, 50.5, 49.5, 47.8, 47.6, 47.3, 45.8, 45.0, 44.9, 42.5, 42.1, 36.1, 34.2,

ACCEPTED MANUSCRIPT
33.1, 33.0, 31.6, 30.4, 30.4, 29.6, 28.4, 26.9, 26.5, 24.4, 24.0, 22.9, 21.8, 21.5, 18.2,
14.2, 14.1, 11.6; ESI-MS: 879 [M + Na]+; HRMS: m/z: Calcd. for C45H60N8O9 [M +
Na]+ 879.4483, found 879.4395, ppm error 5.0.
4.1.2.2.5 Compound 10
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-(4-methylpiperazine-1-yl) diazen-1-ium-1,2-diolate. The title compound
was obtained in 61% yield as a yellowish solid: mp: 167 - 169 ; 1HNMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 8.73 (s, 1H, ArH), 8.01 (s, 1H, C1-H), 6.91 (s, 1H, ArH), 5.96
(s, 1H, C11-H), 4.03 - 3.98 (m, 4H, 2 × NCH2), 3.67 (s, 4H, 2 × NCH2), 3.35 - 3.30 (m,
5H, 2 × NCH2, C13-H), 3.16 - 3.09 (m, 1H, C18-H), 2.71 (t, J = 4.5 Hz, 4H, 2 × NCH2),
2.40 (s, 3H, NCH3), 1.49 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.17 (s,
3H, CH3), 1.03 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.92 (s, 3H, CH3) ppm; 13C NMR (75
M Hz, CDCl3, 25 °C, TMS): δ 199.0, 196.5, 175.9, 167.9, 165.6, 153.6, 149.7, 133.2,
128.8, 127.2, 124.1, 114.5, 114.4, 105.5, 53.2, 50.5, 50.4, 49.5, 47.8, 47.6, 45.8, 45.4,
45.0, 44.9, 42.5, 42.1, 36.2, 36.1, 34.2, 33.1, 32.9, 31.6, 30.4, 30.4, 29.7, 28.4, 26.9,
26.5, 24.5, 24.0, 22.8, 21.8, 21.6, 18.2; ESI-MS: 884 [M + H]+, 906 [M + Na]+, 922
[M + K]+; HRMS: m/z: Calcd. for C46H61N9O9 [M + Na]+ 906.4592, found 906.4506,
ppm error 5.0.
4.1.2.2.6 Compound 11.
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-(pyrrolidine-1-yl) diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 58% yield as a yellowish solid: mp: 174 - 176 ; HNMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 8.67 (s, 1H, ArH), 8.01 (s, 1H, C1-H), 6.94 (s, 1H, ArH), 5.93
(s, 1H, C11-H), 4.05 - 3.85 (m, 4H, 2 × NCH2), 3.74 (s, 4H, 2 × NCH2), 3.37 - 3.25 (m,
5H, 2 × NCH2, C13-H), 3.14 - 3.10 (m, 1H, C18-H), 2.08 - 2.06 (m, 4H, 2 × CH2), 1.47
(s, 3H, CH3), 1.32 (s, 3H, CH3), 1.21 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s, 3H,
13
CH3), 1.00 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; C NMR (75 M Hz, CDCl3, 25 °C,
TMS): δ 199.2, 196.5, 175.8, 165.9, 161.0, 154.2, 150.0, 136.6, 132.4, 127.4, 124.0,

ACCEPTED MANUSCRIPT
114.5, 107.4, 104.2, 68.2, 50.6, 50.4, 49.5, 47.7, 47.6, 45.8, 45.0, 44.8, 42.5, 42.2,
36.0, 34.2, 34.2, 33.1, 31.6, 31.2, 30.9, 30.4, 28.4, 26.9, 26.5, 24.4, 23.9, 23.5, 22.7,
21.7, 21.6, 19.1, 18.2; ESI-MS: 877 [M + Na]+, 893 [M +K]+; HRMS: m/z: Calcd. for
C45H58N8O9 [M + Na]+ 877.4321, found 877.4236, ppm error 5.0.
4.1.3 General procedure for the preparation of 12-15
4.1.3.1 Synthesis of intermediates of 17, 18 and 19, 20
5
(3.0 mmol), N-Boc-protected L-amino acids (N-Boc-L-alanine and
N-Boc-L-phenyl alanine, 3.1 mmol), EDCI (6.0 mmol) and DMAP (0.3 mmol) were
added in anhydrous CH2Cl2 (5 mL) and stirred for 6 - 12 h at room temperature. The
reaction mixture was then diluted with CH2Cl2, washed with saturated sodium
bicarbonate (NaHCO3) aqueous solution, brine, dried with anhydrous sodium sulfate,
filtered, and evaporated in vacuum. The obtained crude product was purified by silica
column chromatography to afford compounds 17 or 18 as yellowish solid.
Compounds 19 and 20 were synthesized using a similar procedure starting from 7.
4.1.3.1.1 Compound 17
O2-(2,4-Dinitro-5-{2-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-oxo-ethyl-a
mino}phenyl)
1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)proionyl-1-
oxo]ethylamino} diazen-1-ium-1,2-diolate. The title compound was obtained in 84%
1
yield as a yellowish solid: mp: 114 - 116 ; H NMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 9.10 (s, 1H, Ar-H), 8.72 (t, J = 4.7 Hz, 1H, NH-Ar), 8.03 (s, 1H, C1-H), 6.87
(s, 1H, Ar-H), 5.96 (s, 1H, C11-H), 4.44 - 4.41 (m, 4H, OCH2), 4.32 - 4.22 (m, 1H,
COCH), 3.92 - 3.91 (m, 2H, NCH2), 3.71 - 3.70 (m, 2H, NCH2), 3.36 (s, 3H, NCH3),
3.03 - 2.99 (m, 1H, C13-H), 2.91 - 2.89 (m, 1H, C18-H), 1.46 (s, 3H, CH3), 1.43 (s, 9H,
3 × CH3), 1.25 (s, 3H, CH3), 1.19 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.00 (s, 3H, CH3),
0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS):
δ197.8, 195.9, 177.1, 167.8, 165.0, 155.0, 147.8, 127.4, 127.3, 126.5, 125.9, 123.4,
114.0, 113.7, 97.8, 61.0, 51.8, 51.7, 49.1, 48.8, 46.9, 45.1, 44.5, 42.0, 41.7, 41.6, 40.2,

ACCEPTED MANUSCRIPT
35.1, 33.8, 32.6, 32.4, 31.1, 31.0, 30.1, 29.1, 27.8, 27.7, 26.5, 26.1, 23.9, 22.4, 22.1,
21.0, 20.9, 17.6; ESI-MS: 1027 [M + Na]+, 1043 [M + K]+.
4.1.3.1.2 Compound 18
O2-(2,4-Dinitro-5-{2-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-oxo-ethyl-a
mino}phenyl)
1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)-3-phenyl
propionyl-1-oxo]ethylamino} diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 82% yield as a yellowish solid: mp: 102 - 104 ; H NMR (300 M Hz,
CDCl3, 25 °C, TMS): δ 9.08 (s, 1H, Ar-H), 8.70 (t, J = 4.9 Hz, 1H, NH-Ar), 8.03 (s,
1H, C1-H), 7.32 - 7.24 (m, 3H, 3 × ArH), 7.16 - 7.14 (m, 2H, 2 × ArH), 6.84 (s, 1H,
Ar-H), 5.96 (s, 1H, C11-H), 4.39 - 4.31 (m, 4H, OCH2), 4.16 - 4.09 (m, 1H, COCH),
3.83 - 3.82 (m, 2H, NCH2), 3.66 - 3.65 (m, 2H, NCH2), 3.28 (s, 3H, NCH3), 3.11 -
3.04 (m, 2H, CH2Ar), 3.02 - 2.95 (m, 1H, C13-H), 2.91 - 2.85 (m, 1H, C18-H), 1.45 (s,
3H, CH3), 1.40 (s, 9H, 3 × CH3), 1.25 (s, 3H, CH3), 1.19 (s, 3H, CH3), 1.16 (s, 3H,
13
CH3), 1.00 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; C NMR (75 M
Hz, CDCl3, 25 °C, TMS): δ197.9, 196.0, 177.3, 167.7, 165.1, 155.1, 147.9, 135.3,
128.7, 128.2, 127.3, 126.7, 126.6, 125.8, 123.5, 114.0, 113.8, 97.7, 61.3, 61,2, 54.1,
51.7, 49.0, 48.6, 47.1, 46.8, 45.2, 44.4, 42.0, 41.6, 41.6, 40.3, 37.7, 35.0, 33.8, 33.4,
32.6, 32.3, 31.0, 31.0, 30.9, 30.0, 29.6, 29.2, 28.8, 27.8, 27.6, 26.4, 26.1, 25.1, 24.4,
23.8, 22.4, 22.1, 21.0, 17.5; ESI-MS: 1103 [M + Na]+, 1119 [M + K]+.
4.1.3.1.3 Compound 19
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl)
1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)proionyl-1-
oxo]ethylamino} diazen-ium-1,2-diolate. The title compound was obtained in 78%
1
yield as a yellowish solid: mp: 134 - 136 ; HNMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 8.73 (s, 1H, ArH), 8.07 (s, 1H, C1-H), 6.96 (s, 1H, ArH), 5.98 (s, 1H, C11-H),
4.46 - 4.42 (m, 2H, CH2O), 4.02 - 3.88 (m, 8H, 4 × NCH2), 3.35 (s, 3H, NCH3), 3.34 -
3.33 (m, 3H, COCH, NCH2), 3.19 - 3.12 (m, 2H, C13-H, C18-H), 1.49 (s, 3H, CH3),
1.42 (s, 9H, 3 × CH3), 1.32 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.01 (s,

ACCEPTED MANUSCRIPT
13
3H, CH3), 1.00 (s, 3H, CH3), 0.92 (s, 3H, CH3) ppm; C NMR (75 M Hz, CDCl3,
25 °C, TMS): δ 199.0, 196.4, 176.0, 165.7, 153.9, 149.6, 137.9, 134.2, 133.0, 129.8,
129.5, 128.6, 127.4, 114.5, 114.4, 105.2, 61.6, 58.1, 52.5, 50.5, 49.5, 49.1, 47.8, 45.8,
45.0, 42.5, 42.1, 41.0, 34.2, 33.9, 33.1, 31.6, 30.4, 29.7, 29.3, 28.3, 26.9, 26.5, 25.6,
24.9, 24.4, 23.9, 21.6, 18.2, 15.6, 11.5; ESI-MS: 1052 [M + Na]+, 1068 [M + K]+.
4.1.3.1.4 Compound 20
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)-3-phenyl propionyl-1-
oxo]ethylamino) diazen-1-ium-1,2-diolate. The title compound was obtained in 80%
1
yield as a yellowish solid: mp: 121 - 123 ; HNMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 8.74 (s, 1H, ArH), 8.06 (s, 1H, C1-H), 7.32 - 7.21 (m, 3H, 3 × ArH), 7.17 -
7.15 (m, 2H, 2 × ArH), 6.96 (s, 1H, ArH), 5.96 (s, 1H, C11-H), 4.16 - .09 (m, 2H,
CH2O), 4.04 - 3.70 (m, 8H, 4 × NCH2), 3.54 - 3.42 (m, 2H, CH2Ar), 3.31 - 3.28 (m,
3H, COCH, NCH2), 3. 25 (s, 3H, NCH3), 3.06 - 3.04 (m, 2H, C13-H, C18-H), 1.48 (s,
3H, CH3), 1.39 (s, 9H, 3 × CH3), 1.31 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H,
13
CH3), 1.03 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.92 (s, 3H, CH3) ppm; C NMR (75 M
Hz, CDCl3, 25 °C, TMS): δ 199.0, 196.6, 176.0, 175.6, 167.8, 165.6, 149.5, 148.5,
135.9, 129.2, 129.2, 129.1, 128.7, 127.3, 127.2, 124.0, 114.4, 114.3, 105.6, 61.8, 54.9,
52.4, 50.5, 49.3, 47.8, 47.5, 45.8, 45.0, 42.4, 42.0, 40.8, 38.2, 36.0, 34.3, 33.9, 33.1,
31.6, 30.3, 29.5, 28.3, 26.9, 26.5, 25.6, 24.9, 24.4, 23.8, 21.7, 21.4, 18.2; ESI-MS:
1128 [M + Na]+, 1144 [M + K]+.
4.1.3.2 Synthesis of compounds 12-15
Compounds 12-15 were synthesized using a similar method. A typical procedure
can be described as follows using 17 as the example. A mixture of 17 (1.0 mmol) and
boron trifluoride diethyl etherate (2 mL) in anhydrous CH2Cl2 (5 mL) was stirred at
room temperature (1.0 mmol) for 1 - 2 h. The reaction mixture was then quenched
with saturated sodium bicarbonate (NaHCO3) aqueous solution, diluted with CH2Cl2,
washed with saturated sodium bicarbonate aqueous solution, brine, dried with

ACCEPTED MANUSCRIPT
anhydrous sodium sulfate, filtered, and evaporated in vacuum. The obtained crude
product was purified by silica column chromatography to afford compounds 12 as
yellowish solid. Compounds 13-15 were synthesized using a similar procedure
starting from 18, 19 or 20, respectively.
4.1.3.2.1 Compound 12
O2-(2,4-Dinitro-5-{2-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-oxo-ethyl-a
mino}phenyl)
1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)proionyl-1-
oxo]ethylamino} diazen-1-ium-1,2-diolate. The title compound was obtained in 87%
1
yield as a yellowish solid: mp: 153 - 155 ; H NMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 9.07 (s, 1H, ArH), 8.74 (t, J = 5.3 Hz, 1H, NH-Ar), 8.08 (s, 1H, C1-H), 6.89
(s, 1H, ArH), 5.98 (s, 1H, C11-H), 4.42 - 4.40 (m, 4H, 2 × CH2O), 3.97 - 3.94 (m, 2H,
NCH2), 3.74 - 3.72 (m, 2H, NCH2), 3.64 - 3.57 (m, 1H, COCH), 3.38 (s, 3H, NCH3),
3.02 - 2.97 (m, 1H, C13-H), 2.92 - 2.89 (m, 1H, C18-H), 2.30 ( br s, 2H, NH2), 1.47 (s,
3H, CH3), 1.25 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.00 (s, 3H, CH3),
0.97 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS):
δ 198.6, 196.6, 177.9, 175.9, 168.5, 165.9, 155.4, 148.5, 127.9, 126.9, 126.3, 124.0,
114.5, 114.5, 98.2, 61.5, 61.2, 52.2, 50.0, 49.5, 47.6, 47.3, 45.6, 45.0, 42.5, 42.0, 40.4,
35.6, 34.3, 33.1, 32.9, 31.6, 31.5, 30.6, 29.3, 28.1, 26.9, 26.6, 24.4, 22.9, 22.6, 21.5,
21.4, 20.5, 18.1; ESI-MS: 905 [M + H]+, 927 [M + Na]+; HRMS: m/z: Calcd. for
C45H60N8O12 [M + H]+ 905.4331, found 905.4392, ppm error 5.0.
4.1.3.2.2 Compound 13
O2-(2,4-Dinitro-5-{2-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-oxo-ethyl-a
mino}phenyl)
1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)-3-phenyl
propionyl-1-oxo]ethylamino) diazen-1-ium-1,2-diolate. The title compound was
1
obtained in 85% yield as a yellowish solid: mp: 151 - 153 ; H NMR (300 MHz,
CDCl3, 25 °C, TMS): δ 9.08 (s, 1H, ArH), 8.72 (t, J = 5.3 Hz, 1H, NH-Ar), 8.04 (s,
1H, C1-H), 7.27 - 7.17 (m, 5H, 5 × ArH), 6.86 (s, 1H, ArH), 5.96 (s, 1H, C11-H), 4.45
- 4.32 (m, 4H, 2 × CH2O), 3.92 - 3.85 (m, 2H, NCH2), 3.78 - 3.65 (m, 3H, NCH2,

ACCEPTED MANUSCRIPT
COCH), 3.29 (s, 3H, NCH3), 3.10 - 3.03 (m, 3H, CH2Ar, C13-H), 2.90 - 2.89 (m, 1H,
C18-H), 1.45 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.20 (s, 3H, CH3), 1.16 (s, 3H, CH3),
13
1.05 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; C NMR (75 M Hz,
CDCl3, 25 °C, TMS): δ 198.6, 196.7, 177.9, 174.6, 168.5, 165.7, 158.8, 148.5, 129.9,
129.2, 128.7, 127.9, 127.0, 126.4, 124.9, 124.0, 123.6, 116.1, 113.6, 110.8, 98.2, 61.5,
61.2, 55.9, 52.2, 49.5, 47.7, 47.3, 45.6, 45.0, 42.0, 41.1, 40.5, 35.6, 34.3, 33.1, 32.9,
31.5, 30.6, 29.7, 29.3, 28.0, 27.2, 27.0, 26.6, 24.4, 22.9, 22.7, 21.5, 21.4, 18.1;
ESI-MS: 981 [M + H]+, 1003 [M + Na]+; HRMS: m/z: Calcd. for C51H64N8O12 [M +
Na]+ 1003.4644, found 1003.4526, ppm error 5.0.
4.1.3.2.3 Compound 14
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl)
1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)proionyl-1-
oxo]ethylamino} diazen-1-ium-1,2-diolate. The title compound was obtained in 80%
1
yield as a yellowish solid: mp: 144 - 146 ; H NMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 8.72 (s, 1H, ArH), 8.09 (s, 1H, C1-H), 6.94 (s, 1H, ArH), 5.97 (s, 1H, C11-H),
4.44 - 4.41 (m, 2H, CH2O), 3.98 - 3.85 (m, 5H, 2 × CONCH2, CHNH2), 3.41 - 3.25
(m, 9H, NCH3, 3 × NCH2), 3.16 - 3.10 (m, 2H, C13-H, C18-H), 1.49 (s, 3H, CH3), 1.32
(s, 3H, CH3), 1.25 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.03 (s, 3H, CH3), 1.01 (s, 3H,
13
CH3), 0.92 (s, 3H, CH3) ppm; C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.2,
196.7, 175.9, 165.9, 153.8, 149.8, 132.8, 132.3, 130.9, 128.9, 128.3, 127.4, 124.0,
114.5, 105.1, 71.8, 61.3, 52.3, 52.2, 50.4, 49.9, 49.5, 47.7, 45.7, 45.0, 42.5, 42.1, 40.7,
40.5, 36.1, 34.2, 33.1, 31.6, 30.4, 29.7, 28.4, 27.7, 26.9, 24.5, 24.4, 23.9, 22.7, 21.8,
21.6, 20.3, 19.2, 18.2; ESI-MS: 930 [M + H]+; HRMS: m/z: Calcd. for C47H63N9O11
[M + H]+ 930.4647, found 930.4735, ppm error 5.0.
4.1.3.2.4 Compound 15
O2-(2,4-Dinitro-5-{4-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]piperazin-1-y
l}phenyl) 1-{N-methyl-2-[2-(S)-(tert-butoxycarbonylamino)-3-phenyl propionyl-1-
oxo]ethylamino) diazen-1-ium-1,2-diolate. The title compound was obtained in 82%