Synthesis of CDDOâAmino AcidâNitric Oxide Donor Trihybrids as

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Synthesis of CDDO-Amino Acid-Nitric Oxide Donor Trihybrids as Potential Antitumor Agents against Both Drug-sensitive and Drug-resistant Colon Cancer Yong Ai, Fenghua Kang, Zhangjian Huang, Xiaowen Xue, Yisheng Lai, Sixun Peng, Jide Tian, and Yihua Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5019302 • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Medicinal Chemistry

Synthesis of CDDO-Amino Acid-Nitric Oxide Donor Trihybrids as Potential Antitumor Agents against Both Drug-sensitive and Drug-resistant Colon Cancer Yong Ai,a,b Fenghua Kang,a,b Zhangjian Huang,a,b,*, Xiaowen Xue,a Yisheng Lai,a,b Sixun Peng,a,b Jide Tian,c and Yihua Zhanga,b*

a

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing

210009, PR China b

Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China

Pharmaceutical University, Nanjing 210009, PR China c

Department of Molecular and Medical Pharmacology, University of California, Los

Angeles, California 90095, United States

ABSTRACT: Seventeen CDDO-amino acid-NO donor trihybrids (4a-q) were designed and synthesized. Biological evaluation indicated that the most active compound 4c produced high levels of NO and inhibited the proliferation of drug-sensitive (HCT-8, IC50 = 0.294 µM) and drug-resistant (HCT-8/5-FU, IC50 = 0.232 µM) colon cancer cells, which were attenuated by an NO scavenger or typical substrate of PepT1. Furthermore, 1

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4c triggered HCT-8 and HCT-8/5-FU cell apoptosis more strongly than CDDO-Me, inhibited the HIF-1α, Stat3, AKT, ERK signaling, and induced the nitration of P-gp, MRP1, and BCRP proteins in HCT-8/5-FU cells. Finally, 4c had 4.36~5.53-fold less inhibitory activity against non-tumor colon epithelial-like cells (CCD841, IC50 = 1.282 µM) in vitro, and inhibited the growth of implanted human drug-resistant colon cancers in mice more potently than CDDO-Me. Together, 4c is a novel trihybrid with potent anti-tumor activity and may be a promising candidate for the treatment of drug-resistant colon cancer.

INTRODUCTION Multidrug resistance (MDR) is a leading cause for the failure of anti-cancer chemotherapy.1-3 MDR in cancer cells is attributed to many factors, such as over-expression of the ATP-binding cassette (ABC) transporters, which can export anticancer drugs out of the cells, leading to low levels of intracellular drugs and consequent drug ineffectiveness.4 Three major ABC transporters implicated in cancer MDR are P-glycoprotein (P-gp/ABCB1/MDR1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2).5-8 Currently, there is no ABC transporter inhibitor available in the clinic.9,10 Therefore, novel strategies are urgently needed to design and synthesize compounds with potent cytotoxicity against cancer cells with MDR. Nitric oxide (NO) is a signaling and effector molecule, and plays an important role in various physiological and pathophysiological processes.11 High levels of NO have strong 2


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anti-tumor activity by inducing tumor cell apoptosis, inhibiting tumor metastasis and sensitizing drug-resistant tumor cells to chemotherapy, radiation, and immunotherapy in vitro and in vivo.12-15 More importantly, NO-donating anti-tumor drugs usually do not induce drug resistance in tumor cells.16 Furoxans, an important class of NO donors, are able to produce high levels of NO in vitro, and inhibit the growth of tumors in vivo.17,18 Previous studies have shown that doxorubicin/furoxan hybrids can generate high levels of NO in the cancer cells and react with superoxide to produce peroxynitrite anion, which can nitrate the tyrosine residue of many key proteins, including ABC transporters.19,20 We have also developed a variety of furoxan-based NO donating compounds such as oleanolic acid (OA)/furoxan,21 anilinopyrimidine/furoxan,22 and bifendate/furoxan hybrids23,24 with potent antitumor and/or anti-MDR activities. Hence, generation of furoxan-based hybrids is a promising strategy for overcoming the MDR in cancer therapy. OA is a well-known natural triterpenoid and has multiple pharmacological activities.25 To improve its potency, many synthetic OA derivatives (SOADs) have been prepared, including

2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic

acid

(CDDO),

its

ester

(CDDO-Me), amides (CDDO-Im), olean-28,13β-olide (Fig. 1), and others.26-29 These SOADs have strong anti-tumor activity by inhibiting tumor cell proliferation and inducing cancer cell apoptosis.30 Recent studies show that OA and its derivative methyl 3,11-dioxoolean-12-en-28-olate (DIOXOL, Fig. 1) have moderate antitumor activity and can reverse the P-gp-mediated MDR.31,32

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Figure 1. Chemical structures of OA and SOADs.

Peptide transporter 1 (PepT1) is an attractive target for development of anti-tumor drugs because of its high affinity to its broad substrates.33 Physiologically, PepT1 is predominantly expressed in the intestinal epithelium and can effectively transport amino acids, peptides, peptide-like drugs, etc.34 Pathologically, the PepT1 is highly expressed by tumor cells, including human colon cancer cells (Caco-2),35 pancreatic cancer cells (AsPc-1 and Capan-2),36 sarcoma cells (HT-1080),37 prostatic cancer cells (PC-3),38 lung cancer cells (A549),39 and ovarian adenocarcinoma cisplatin-sensitive (2008) and -resistant cells (C13),40 and others. However, the low levels of PepT1 are expressed in non-tumor tissues.35-40 Thus, the differential expression of PepT1 has been utilized for design and synthesis of selective anticancer agents, including the PepT1-targeting dihybrids of amino acid with OA, floxuridine, gemcitabine, cytarabine, and gold (III)-dithiocarbamato, respectively.40-44 In this context, we hypothesized that CDDO-amino acid-NO donor trihybrids could be effectively transported to cancer cells by the PepT1 to exert potent cytotoxicity against both drug-sensitive and drug-resistant colon cancer cells. Accordingly, we synthesized

4


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trihybrids 4a-q by coupling the carboxyl group of CDDO with phenylsulfonyl-substituted furoxan through different amino acids, respectively, and evaluated their bioactivity in vitro and in vivo.

RESULTS AND DISCUSSION Chemistry. The strategies for the synthesis of the target compounds 4a-q are shown in Scheme 1. (3-Phenylsulphonyl-4-hydyoxyalkoxyl)furoxans 2a-e were synthesized through

the

reactions

(butane-1,3-diol,

of

3,4-diphenylsulphonylfuroxan

butane-1,4-diol,

2-butyne-1,4-diol,

with

different

diols

hexane-1,6-diol

or

2,2'-oxydiethanol). The generated 2a-e were reacted with N-protected amino acids (Boc-Glycine, Boc-β-Ala-OH, Boc-L-Pro-OH, Boc-L-Ala-OH, Boc-γ-Aminobutyric acid) respectively hydrochloride

in

the

(EDCI)

presence and

of

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

4-dimethyl-amino-pyridine

(DMAP)

to

afford

the

corresponding esters 3a-q. The Boc group of 3a-q was then removed by trifluoroacetic acid (TFA) to yield amino-containing intermediates, which were directly treated with the acyl chloride of CDDO prepared as reported previously,26,27 in the presence of triethylamine (TEA) to provide the target compounds 4a-q. Their structures were characterized by IR, 1H-NMR, 13C-NMR, MS, and HRMS.

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Scheme 1. Synthetic routes of CDDO-amino acid-NO donor trihybrids 4a-p.a

a

Reagents and conditions: a) Diol, THF, NaH, 0 °C, 15 min.; b) EDCI, DMAP, DCM,

rt, 12 h; c) TFA, DCM, rt, 1 h; d) acyl chloride of CDDO, TEA, DCM, rt, 12 h.

BIOLOGICAL ACTIVITY Assessment of in Vitro Anti-proliferative Activity. Given that the target compounds we designed may be effectively transported to cancer cells by PepT1, we initially assessed the levels of PepT1 expression in drug-sensitive colon cancer HCT-8 and drug-resistant colon cancer HCT-8/5-FU cells as well as in non-transformed colon epithelial-like CCD841 cells by western blot assay.38 The results indicated that high levels of PepT1 were expressed by both HCT-8 and HCT-8/5-FU cells, but only little was detected in CCD841 cells (Fig. S1). 6


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Next, the anti-tumor activity of compounds 4a-q against HCT-8 and HCT-8/5-FU were preliminarily screened by the MTT assay using CDDO-Me as a positive control. Compounds 4a-c, 4p, and 4q (R1 = 2-butyne-1,4-diol), and 4d, 4g, 4j, and 4m (R2 = glycine) at 1 µM inhibited colon cancer proliferation by > 90%, which were higher than that of CDDO-Me (Fig. 2). Other eight compounds 4e, 4f, 4h, 4i, 4k, 4l, 4n, and 4o displayed an anti-tumor potency comparable to CDDO-Me.

Figure 2. Compounds 4a-q inhibit the proliferation of HCT-8 and HCT-8/5-FU cells. HCT-8 and HCT-8/5-FU cells were treated with, or without, 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 present as the means (%) ± SD of each compound from three independent experiments. *P < 0.05 vs the CDDO-Me.

Subsequently, the nine most potent compounds (4a-d, 4g, 4j, 4m, 4p, and 4q) were further investigated for their anti-tumor activity (Table 1). Compound 4c displayed more potent activity (IC50 = 0.294 and 0.232 µM) than CDDO-Me and 5-FU in HCT-8 and 7



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HCT-8/5-FU cells. It is notable that CDDO-Me had similar anti-tumor activities against both colon cancer and non-tumor colon cells, however, 4c displayed a 4.63~5.53-fold decrease in its anti-proliferative activity on non-tumor CCD841 cells (IC50 = 1.282 µM), suggesting that 4c may be a promising drug candidate for further investigation. Table 1. The anti-proliferative activity of compounds IC50 (µM)a Comp.

a

HCT-8

HCT-8/5-FU

CCD841

4a

0.441 ± 0.081b

0.343 ± 0.044b

1.425 ± 0.154c

4b

0.504 ± 0.098

0.381 ± 0.021b

1.698 ± 0.109c

4c

0.294 ± 0.013b,c

0.232 ± 0.010b,c

1.282 ± 0.095c

4d

0.550 ± 0.067

0.380 ± 0.026b

1.725 ± 0.114c

4g

0.383 ± 0.031b

0.315 ± 0.053b

1.121 ± 0.102c

4j

0.356 ± 0.054b

0.339 ± 0.060b

1.423 ± 0.081c

4m

0.872 ± 0.101

0.562 ± 0.087b

3.129 ± 0.198c

4q

0.370 ± 0.052b

0.276 ± 0.013b

0.958 ± 0.069c

4p

0.379 ± 0.066b

0.462 ± 0.088b

1.472 ± 0.057c

CDDO-Me

0.399 ± 0.047

0.363 ± 0.029

0.316 ± 0.019

5-FU

0.91 ± 0.103

36.638 ± 2.319

-

The compounds were dissolved in DMSO and diluted with culture medium into eight

concentrations (0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 5 µM) of solution for 8


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each compound (final DMSO concentration in medium < 0.4%). The cells (1×104 cells) were treated in triplicate with the indicated concentrations of each compound for 72 h to test their effects on the cell proliferation by the MTT assay. The values of IC50 for each compound were calculated and data are expressed as mean IC50 (µM) ± SD of each compound from three independent experiments. IC50 is the drug concentration inhibiting 50% of the cell proliferation. bP < 0.01 vs. the 5-FU. cP < 0.05 vs. the CDDO-Me.

The Role of NO in the Anti-tumor Activity of 4c. Compound 4c was composed of CDDO moiety (compound 5) and furoxan moiety (2a) (Fig. 3), and their anti-tumor activity against HCT-8 and HCT-8/5-FU cells were also examined. The IC50 values of 4c for HCT-8 (0.294 µM) and HCT-8/5-FU cells (0.232 µM) were significantly less than that of individual moieties, 5 (IC50 = 3.192 and 2.257 µM) and 2a (IC50 = 33.512 and 36.569 µM), and even the combination of 5 and 2a (IC50 = 2.447 and 1.863 µM), respectively. These results suggest that the anti-tumor activity of 4c may be attributed to the synergic effects of CDDO and NO donor moieties as well as PepT1-mediated drug transportation in cancer cells.

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Figure 3. Comparison of anti-cancer activity of trihybrid 4c with that of 5, 2a, and equimolar mixture of 5 and 2a.

To verify the contribution of NO to the anti-tumor activity, the levels of intracellular NO released by the tested compounds were firstly detected. HCT-8, HCT-8/5-FU, and CCD841 cells were treated with 100 µM 4c, 4q, 4f, 4i, 4l, or 5 and the levels of intracellular NO were determined by a Griess assay (Fig. 4A). As expected, treatment with 5 resulted in little NO in both HCT-8 and HCT-8/5-FU cells. In contrast, treatment with individual tested compounds lead to variable levels of NO production in both cancer cell lines. Interestingly, treatment with one of the more active compounds such as 4c or 4q caused higher levels of NO production in the cells, particularly in the drug-resistant HCT-8/5-FU cells, whereas less active compounds 4f, 4i and 4l produced lower levels of 10


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NO. The levels of intracellular NO by different compounds were positively correlated with their anti-tumor activities against HCT-8 and HCT-8/5-FU cells in vitro (R = 0.950, 0.962, respectively, p < 0.05, determined by Pearson’s correlation analysis). These results suggest that the active compounds may have a better ability to penetrate the membrane of HCT-8 and HCT-8/5-FU cells, leading to high levels of NO production in colon cancer cells than the less active compounds. On the other hand, compounds 4c, 4q, 4f, 4i, 4l produced almost negligible amounts of NO in non-tumor colon CCD841 cells, suggesting that PepT1-mediated transport of these compounds may be important for the high levels of NO production and anti-tumor activity of these compounds. These data also explained why 4c had preferable inhibition on the proliferation of colon cancer cells. Furthermore, the effect of NO on the anti-tumor activity of 4c was investigated. HCT-8 and HCT-8/5-FU cells were pretreated with various concentrations of an NO scavenger of carboxy-PTIO for 1 h and then treated with 0.25 µM of 4c. The effects of different treatments on the proliferation of HCT-8 and HCT-8/5-FU cells were determined by the MTT assay (Fig. 4B). Treatment with 4c alone remarkably inhibited the proliferation of HCT-8 and HCT-8/5-FU cells and its inhibitory effects were reduced dose-dependently by pretreatment with different concentrations of carboxy-PTIO. These results suggest that NO produced by 4c may contribute to its inhibition on the proliferation of drug-sensitive and drug-resistant colon cancer cells in vitro.

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Figure 4. Effects of NO produced by the target compounds on colon cell proliferation. (A) Variable levels of NO (present as nitrite) produced by the indicated compounds. HCT-8, HCT-8/5-FU, and CCD841 cells were treated in triplicate with individual compounds at 100 µM for 150 min, 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 < 0.001 vs the CCD841 group, #P < 0.05 vs the HCT-8 group. (B) Effects of NO scavenging, carboxy-PTIO (PTIO) on the antiproliferative effect of 4c. HCT-8, HCT-8/5-FU, and CCD841 cells were pretreated in triplicate with the indicated concentrations of carboxy-PTIO (0, 6.25, 12.5, 25, or 50 µM) for 1 h and treated with 0.25 µM 4c for 24 h. The results are expressed as the percentage of cell growth inhibition relative to control cells without the scavenger. Data are the mean value ± SD obtained from three independent experiments. **P < 0.01, ***P < 0.001 vs the vehicle-treated control.

PepT1-mediated Cell Uptake is Crucial for the Anti-tumor Activity of These 12


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Compounds. To determine the importance of PepT1-mediated update of compound, we tested whether the dipeptide Gly-Sar, a high affinity of substrate for PepT1,38 could modulate the anti-tumor activity of 4c in HCT-8 and HCT-8/5-FU cells. The two cancer cells were treated with different doses of 4c and Gly-Sar and the proliferation of cancer cells were determined using MTT assay. As shown in Figures 5A and 5B, addition of Gly-Sar (0, 0.75 or 1.5 µM) significantly mitigated the inhibitory effects of 4c on the proliferation of HCT-8 and HCT-8/5-FU cells and their regulatory effects tended to be dose-dependent. Furthermore, analysis of the impact of Gly-Sar on the levels of NO production by 4c indicated that pretreatment with Gly-Sar reduced the levels of NO production by 4c in both HCT-8 and HCT-8/5-FU cells in a dose-dependent manner (Fig. 5C). Therefore, the PepT1 on these cancer cells is crucial for the intracellular transportation and anti-tumor activity of 4c.

Figure 5. PepT1-mediated cell uptake is important for the anti-tumor activity of 4c. HCT-8 (A) and HCT-8/5-FU (B) cells were pre-treated with, or without, Gly-Sar (0.75 µM or 1.5 µM) for 24 h and treated with the indicated concentrations of 4c for 72 h. The inhibition on the proliferation of cancer cells was determined by MTT. (C) Effects of

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Gly-Saron the levels of NO (present as nitrite) produced by 4c. HCT-8 and HCT-8/5-FU cells were pre-treated with, or without, different concentrations of Gly-Sar (0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 µM) for 150 min and treated with 4c at 150 µM for 150 min. The concentrations of intracellular nitrite were determined by Griess assay. Data are expressed as the means ± SD of each concentration of compounds in individual types of cells from three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the Gly-Sar untreated group.

Induction of Cancer Cell Apoptosis in Vitro. Previous studies have shown that CDDO-Me can induce cancer cell apoptosis by modulating the expression of apoptosis-related regulators and signal events,30 and high levels of NO can induce tumor cell apoptosis.22 Accordingly, the effect of 4c on induction of tumor cell apoptosis was tested. HCT-8 and HCT-8/5-FU cells were treated with variable concentrations of 4c, CDDO-Me, or vehicle (0.4 % DMSO in medium) for 24 h, respectively. 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. Treatment with 4c induced apoptosis in both HCT-8 and HCT-8/5-FU cells in a dose-dependent manner (Fig. 6), and the effects of higher doses of 4c were significantly stronger than that of CDDO-Me in both HCT-8 and HCT-8/5-FU cells.

14


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Figure 6. Compounds 4c and CDDO-Me induce colon cancer cell apoptosis in vitro. HCT-8 (A) and HCT-8/5-FU (B) cells were incubated with the indicated concentrations of 4c or CDDO-Me for 24 h, and the percentages of apoptotic cells were determined by flow cytometry after staining them with Annexin V-FITC and propidium iodide (PI). Data are representative charts or expressed as the means ± SD of the percentages of apoptotic cells from three independent experiments. **P < 0.01 vs the CDDO-Me group.

Effects of 4c on the HIF-1α, Stat3, AKT and ERK Signaling in HCT-8 and HCT-8/5-FU Cells. To gain more insights into the molecular mechanisms underlying the activity of 4c, we examined the regulatory effects of 4c on the HIF-1α and Stat3, AKT, 15



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ERK signaling in HCT-8 and HCT-8/5-FU cells using CDDO-Me as a control. The cells were treated with various concentrations of 4c or CDDO-Me. The expression and activation of HIF-1α, Stat3, AKT, and ERK were determined by western blotting. As shown in Figure 7, treatment with 4c significantly inhibited the HIF-1α expression and Stat3, AKT, and ERK phosphorylation in a dose-dependent manner, although it did not alter the levels of Stat3, AKT, and ERK expression in cancer cells. Similarly, treatment with CDDO-Me also significantly reduced the levels of HIF-1α expression and Stat 3, AKT and ERK phosphorylation in both cell lines and the inhibitory effects of CDDO-Me on the Stat3 phosphorylation and HIF-1α expression in both cell lines were significantly less than that of 4c while the inhibitory effect of CDDO-Me on the ERK phosphorylation in HCT-8/5-FU cells was significantly stronger than that of 4c. Previous studies have shown that aberrant activation of Stat3 and AKT and high levels of HIF-1α expression are associated with the development of human colon cancer45-47 and CDDO-Me can inhibit the proliferation of cancer cells by down-regulating the Stat3 and AKT activation and HIF-1α expression in cancer cells.48-50 Recent studies also show that high levels of ROS directly inhibit the Stat3 and AKT activation.51-53 Accordingly, the strong inhibition of 4c on the Stat3, AKT and ERK activation may reflect the synergistic effect of high levels of ROS and CDDO activity in colon cancer cells. While oxidative stress and related ROS production usually promote the HIF-1α expression, we observed that treatment with 4c significantly reduced the levels of HIF-1α expression in both colon 16


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cancer lines. The reduced levels of HIF-1α expression by 4c may stem from ROS-mediated inactivation of Stat3, which can attenuate the Stat3-up-regulated PI3K/AKT/mTOR activation and downstream HIF-1α expression in colon cancer cells. The precise mechanisms underlying the action of 4c remain to be further investigated.

Figure 7. Effects of 4c and CDDO-Me on the HIF-1α, Stat3, AKT and ERK signaling in HCT-8 and HCT-8/5-FU cells. HCT-8 and HCT-8/5-FU cells were treated with vehicle, or the indicated compound for 24 h and the relative levels of HIF-1α, Stat3, AKT and ERK expression and phosphorylation were determined by western blot assays using β-actin as a control. Data are representative images and expressed as the means ± SD of each group of cells from three separate experiments. A. Western blot analysis of the relative levels of HIF-1α, Stat3, AKT and ERK expression and phosphorylation. B.

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Quantitative analysis. *P < 0.05, **P < 0.01, ***P < 0.001 vs the vehicle-treated control. #

P < 0.05 vs the CDDO-Me group.

Effect of 4c on ABC-transporter Expression. High levels of NO can react with superoxide to produce peroxynitrite anion, which can nitrate the tyrosine residue of many key proteins, including ABC transporters, attenuating MDR.19,20 To further understand the role of 4c in inhibiting drug-resistant colon cancer cell proliferation, the impact of 4c on the levels of Pgp, MRP1 and BCRP expression was determined by western blotting. As shown in Figure 8A, the relative levels of Pgp, MRP1 and BCRP expression in HCT-8/5-FU cells were significantly higher than that in HCT-8 cells. Furthermore, treatment with 4c, but not CDDO-Me, significantly reduced the relative levels of Pgp, MRP1, and BCRP expression in a dose-dependent manner and these inhibitory effects were partially attenuated by pretreatment with the NO scavenger of carboxy-PTIO in HCT-8/5-FU cells. In addition, we also tested the impact of 4c on the nitration of Pgp, MRP1 and BCRP in HCT-8/5-FU cells. HCT-8/5-FU cells were treated with, or without, different concentrations of 4c or CDDO-Me and the nitrated Pgp, MRP1 and BCRP proteins were precipitated by anti-nitrotyrosine antibody. Subsequently, the relative levels of nitrated Pgp, MRP1 and BCRP were determined by western blotting using individual specific antibodies (Fig. 8B). Treatment with 4c, but not CDDO-Me, increased the levels of nitrated Pgp, MRP1 and BCRP in a dose-dependent manner, which was 18


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partially attenuated by pretreatment with carboxy-PTIO in HCT-8/5-FU cells. Apparently, high levels of NO produced by 4c were responsible for the nitration of Pgp, MRP1 and BCRP in HCT-8/5-FU cells.

Figure 8. Nitration of drug efflux pumps by 4c in HCT-8/5-FU cells. (A) Expression of ABC transporters in HCT-8 and HCT-8/5-FU cells. HCT-8 and HCT-8/5-FU cells were treated with, or without, the indicated concentrations of 4c or CDDO-Me for 24 h and the relative levels of Pgp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2 to GAPDH were determined by western blotting. In addition, the nitrated Pgp/ABCB1, MRP1/ABCC1, and

BCRP/ABCG2

in

the

HCT-8/5-FU

cell

lysates

were

precipitated

by

anti-nitrotyrosine antibody, and characterized by western blotting using individual specific antibodies. Data are representative images and expressed as the means ± SD of each group of cells from three separate experiments. (A) Western analysis of the relative 19



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Page 20 of 52

levels of ABC transporter expression. ***P < 0.001 vs the HCT-8 group. (B) Western blot analysis of the levels of nitrated ABC transporters. *P < 0.05, ***P < 0.001 vs the PTIO group.

Safety Profile in Healthy Mice. To evaluate the safety of 4c, the acute toxicity of 4c was examined in healthy mice. Adult ICR mice at either gender were randomly treated by oral gavage with various doses of 4c (dissolved in the vehicle consisting of cremophor-EL/DMSO/PBS (1: 1: 8)),49 based on our preliminary study, and the survival of the mice was monitored up to 14 days post treatment. Interestingly, 4c did not cause any mouse death or abnormality in eating, drinking, body weight, or activity throughout the observation period even when the dose was raised up to 2,000 mg/kg (2.38×103 µmol/kg) (Table 2, data from other doses not shown), indicating a relative safety of 4c in vivo.

Table 2. Safety of 4c in mice. Dose mg/kg (µmol/kg)

sex

Total mortality (1-14 d)

Day 0

Day 7

Day 14

10

female

0

19.8±0.92

25±1.05

30±1.56

10

male

0

19.5±0.85

26.7±1.89

31.9±1.97

Number of mice

Mouse

Body weighta

2,000 (2.38×103) 2,000 (2.38×103) a

Data shown are the means ± SD of body weights from each group of mice (n = 10).

Antitumor Activity of 4c against the Growth of HCT-8/5-FU Xenograft Tumors in 20


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Mice. To evaluate the in vivo anticancer activity of 4c, BALB/c nude mice were inoculated subcutaneously with drug-resistant colon carcinoma HCT-8/5-FU cells. After the establishment of solid tumor, the mice were randomly treated by gavage with CDDO-Me, 4c at the indicated doses or vehicle (cremophor-EL/DMSO/PBS (1:1:8))49 daily for 21 consecutive days (Fig. 9). It was found that all mice survived and the obvious abnormalities in the liver, kidney, lung and heart in terms of the size, color and weights in the control, 4c or CDDO-Me-treated mice were not observed although the body weights in the control, 4c- and CDDO-Me-treated groups of mice were somewhat reduced (Table 3). In addition, treatment with 15 mg/kg (17.8 µmol/kg) of 4c significantly reduced the volumes of implanted colon tumors, while treatment with 4c at 30 mg/kg (35.7 µmol/kg) further inhibited the growth of the implanted tumors (P < 0.01 vs. the vehicle-treated control, Fig. 9), indicating that the anti-tumor activity of 4c trended to be dose-dependent. Furthermore, the tumor weights (0.48 ± 0.17 g) in the mice treated with 4c at 30 mg/kg (35.7 µmol/kg) were significantly reduced by 60.83 %, as compared with those of the vehicle-treated controls (1.24 ± 0.19 g, P < 0.01, Table 3). More importantly, the anti-tumor activity of 4c at 30 mg/kg (35.7 µmol/kg) was comparable with that of CDDO-Me at 30 mg/kg (59.4 µmol/kg). Therefore, 4c had more potent anti-tumor activity against the growth of implanted human colon tumors than CDDO-Me in mice.

21



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Figure 9. Inhibition of 4c on the growth of implanted drug-resistant colon tumors in mice. Male BALB/c nude mice were inoculated with HCT-8/5-FU cells and after establishment of solid tumors, the mice were randomly treated with the indicated doses of 4c, CDDO-Me or vehicle (cremophor-EL/DMSO/PBS (1:1:8)) daily and the tumor volumes were measured every other day through the observation period in a blinded manner. Data are expressed as the mean ± SD from each group of mice (n = 8 per group). **P < 0.01 vs. the vehicle-treated control.

Table 3. Effects of 4c on the body and tumor weights in micea Dose

Body weight (g)

Inhibitory Tumor weight

Group

mg/kg

ratio day 1

(g)

day 21

(µmol/kg)

(%, w/w)

Control

-

20.8 ± 2.2

18.2 ± 1.3

1.24 ± 0.19

-

CDDO-Me

30 (59.4)

22.4 ± 1.4

16.7 ± 1.2

0.51 ± 0.25b

58.40

4c

15 (17.8)

22.3 ± 1.9

17.5 ± 1.7

0.63 ± 0.25b

49.19

4c

30 (35.7)

21.1 ± 1.5

16.7 ± 1.4

0.48 ± 0.17b

60.83

a

Data shown are the means ± SD of tumor weights in mice and their body weights from

each group of mice (n = 8). bP< 0.01 vs. the vehicle-treated control. 22


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CONCLUSION In summary, a series of trihybrids (4a-q) were designed and synthesized from CDDO, amino acid, and (phenylsulfonyl)furoxan, and were biologically evaluated. The most active compound 4c exhibited strong and preferable anti-tumor activity against both drug-sensitive HCT-8 and drug-resistant HCT-8/5-FU cells. Treatment with 4c induced high levels of NO production in HCT-8 and HCT-8/5-FU cells but not in human non-tumor colon epithelial-like CDD841 cells, and its anti-tumor activity was significantly attenuated by an NO scavenger or a known substrate of PepT1 in a dose-dependent manner. In addition, 4c displayed more potent capacity to induce HCT-8 and HCT-8/5-FU cell apoptosis than CDDO-Me. Furthermore, treatment with 4c inhibited the HIF-1α, Stat 3, AKT, and ERK signaling in HCT-8 and HCT-8/5-FU cells, and reduced the relative levels of P-gp, MRP1, and BCRP by nitrating these cellular drug efflux proteins in HCT-8/5-FU cells. Moreover, 4c was relatively safe to mice and inhibited the growth of implanted human colon tumors more potently than CDDO-Me in mice. Together, these data indicated that 4c, a trihybrid of CDDO, amino acid and NO donor moieties had more potent anti-tumor activity preferably against human colon cancer than CDDO-Me. Our findings may provide a proof of principle for design of such trihybrids like 4c as potent and selective anti-colon cancer agents potentially to circumvent MDR.

23



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EXPERIMENTAL PROTOCOLS Chemical analysis. Melting points were determined on a Mel-TEMP II melting point apparatus and were uncorrected. Infrared (IR) spectra (KBr) were recorded on a Nicolet Impact 410 instrument (KBr pellet). 1H-NMR and 13C-NMR spectra were recorded with a Bruker Avance 300 MHzspectrometer at 300 K, using TMS as an internal standard. MS spectrawere recorded on a Mariner Mass Spectrum (ESI) and high resolution mass spectrometry (HRMS) on Agilent technologies LC/MSD TOF. 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. All solvents were reagent grade and, when necessary, were purified anddried by standard methods. Solutions after reactions and extractions were concentrated using a rotary evaporator operating at a reduced pressure of ca. 20 Torr. The purity of all tested compounds was characterized by HPLC analysis (LC-10A HPLC system consisting of LC-10ATvp pumps and an SPD-10Avp UV detector). Individual compounds with a purity of > 95% were used for subsequent experiments (see the Supporting Information). CDDO, acyl chloride of CDDO, and 2a-e was prepared according to the method described previously.21,26,27 Procedure for the preparation of compound 5 was shown in Figure S2.

General Procedure for the Preparation of Compounds 3a-q. To a solution of Boc-amino

acid

(3.1

mmol)

in

dry

CH2Cl2

(5

mL),

2a-e

(3.0

mmol),

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (6 mmol) and 24


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4-dimethylaminopyridine (DMAP) (0.3 mmol) were added, and the mixture was stirred at room temperature for 6-12 h. The reaction solution was diluted with water and extracted with CH2Cl2. The organic layer was washed with brine, then dried with sodium sulfate, filtered and evaporated in vacuo to give corresponding crude compound 3a-q (93-96%), respectively, which was used in the next without further purification.

General Procedure for the Preparation of Compounds 4a-q. A mixture of 3a-q (2 mmol) and TFA (15 mmol) in anhydrous CH2Cl2 (5 mL) was stirred at room temperature for 0.5 h. The reaction mixture was then cooled to 0 °C, and Et3N (20 mmol), acyl chloride of CDDO (2 mmol) in anhydrous CH2Cl2 (2 mL) was sequentially thus added dropwise, and the mixture was allowed to stir at room temperature for 12 h. The reaction solution was quenched by adding water and extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydroussodium sulfate, filtered, and evaporated under reduced pressure. The resulting crude product was purified by flash chromatography on silica gel column (PE : AcOEt = 7:3-1:1 v/v) to provide the title compounds 4a-q (54-80 %), respectively. Compounds 4a. The title compound was obtained in 65 % yield as a white solid: m.p. 119-121 °C; IR (KBr): 3129, 1729, 1689, 1663, 1615, 1549, 1399 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.09-8.06 (m, 3H, 2 × ArH, C1-H), 7.78-7.66 (m, 3H, 3 × ArH), 5.95 (s, 1H, C11-H), 5.09 (s, 2H, CH2O), 4.81 (s, 2H, CH2O), 4.53-4.50 (m, 1H, NCHCOO), 4.15-4.09 (m, 2H, NCH2), 3.24-3.22 (m, 1H, C13-H), 3.08-3.06 (m, 1H, C18-H), 1.48 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.02 (s, 25



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3H, CH3), 0.88 (s, 3H, CH3), 0.86 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.5, 196.1, 175.4, 171.4, 165.4, 165.2, 157.6, 135.3, 130.4, 129.2, 129.2,128.3, 128.1, 128.1, 123.4, 114.0, 113.9, 83.4, 78.1, 65.0, 61.1, 59.0, 58.1, 51.8, 49.3, 47.9, 47.3, 44.5, 42.0, 41.8, 35.8, 33.6, 32.9, 31.4, 31.2, 31.1, 29.3, 29.2, 28.8, 27.6, 26.1, 25.7, 24.6, 23.4, 22.2. 21.8, 21.0, 17.7; ESI-MS: 881 [M+H]+; HRMS: calculated for C48H56N4O10SNa [M + Na]+ 903.3615, found 903.3635.

Compounds 4b. The title compound was obtained in 80 % yield as a white solid: m.p. 102-104 °C; IR (KBr): 3131, 1739, 1685, 1661, 1617, 1549, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.09-8.04 (m, 3H, 2 × ArH, C1-H), 7.79 (t, J = 7.5 Hz, 1H, ArH), 7.65 (t, J = 7.6 Hz, 2H, 2 × ArH), 6.18 (t, J = 5.4 Hz, 1H, CONH), 5.97 (s, 1H, C11-H), 5.11 (s, 2H, CH2O), 4.75 (s, 2H, CH2O), 3.06 (d, J = 4.2 Hz, 1H, C13-H), 2.91-2.87 (m, 1H, C18-H), 2.39 (t, J = 7.1 Hz, 2H, CH2COO), 1.48 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.5, 196.1, 176.8, 171.9, 168.2, 165.3, 157.6, 137.0, 135.3, 130.4, 129.2, 129.2, 128.2, 128.2, 123.5, 114.1, 113.9, 83.4, 78.2, 65.1, 58.1, 51.5, 49.0, 47.2, 46.0, 45.4, 44.5, 42.0, 41.6, 38.5, 35.6, 34.1, 33.6, 32.8, 31.4, 31.2, 30.9, 30.1, 29.2, 27.3, 26.1, 24.3, 22.6, 22.2, 21.2, 21.0, 17.7; ESI-MS: 869 [M+H]+, 891 [M+Na]+; HRMS: calculated for C47H56N4O10SNa [M + Na]+ 891.3615, found 891.3633. 26


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Compounds 4c. The title compound was obtained in 77 % yield as a white solid: m.p. 120-122 °C; IR (KBr): 3129, 1758, 1685, 1660, 1618, 1548, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.15-8.09 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 7.4 Hz, 1H, ArH), 7.64 (t, J = 7.7 Hz, 2H, 2 × ArH), 6.35 (t, J = 5.4 Hz,1H, CONH), 6.00 (s, 1H, C11-H), 5.10 (s, 2H, CH2O), 4.80 (s, 2H, CH2O), 4.13-4.09 (m, 2H, NHCH2COO), 3.14 (d, J = 4.6 Hz, 1H, C13-H), 2.97-2.94 (m, 1H, C18-H), 1.48 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.91 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7, 196.2, 177.3, 169.0, 168.6, 165.6, 157.4, 137.5, 135.3, 129.3, 129.3, 128.1, 128.1, 123.5, 116.4, 114.0, 114.0, 82.9, 78.6, 58.1, 52.0, 49.0, 47.2, 46.1, 45.5, 44.5, 42.1, 41.7, 40.6, 35.5, 34.1, 33.5, 32.7, 31.4, 31.2, 30.1, 29.2, 28.8, 27.1, 26.4, 24.5, 22.6, 21.2, 21.0, 17.7; ESI-MS: 841 [M+H]+, 863 [M+Na]+, 879 [M+K]+; HRMS: calculated for C45H52N4O10SNa [M + Na]+ 863.3302, found 863.3320.

Compounds 4d. The title compound was obtained in 74 % yield as a white solid: m.p. 128-130 °C; IR (KBr): 3131, 1751, 1689, 1662, 1616, 1553, 1398 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.10-8.06 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 6.7 Hz, 1H, ArH), 7.66 (t, J = 6.9 Hz, 2H, 2 × ArH), 6.36 (t, J = 5.4 Hz, 1H, CONH), 5.97 (s, 1H, C11-H), 4.52-4.50 (m, 2H, CH2O), 4.36-4.34 (m, 2H, CH2O), 4.11-4.08 (m, 2H, 27



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NHCH2COO), 3.14 (d, J = 4.2 Hz, 1H, C13-H), 2.98-2.94 (m, 1H, C18-H), 1.46 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm;

13

C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7,

196.2, 177.3, 169.7, 168.5, 165.7, 158.3, 137.3, 135.3,129.3, 129.3, 128.3, 128.3, 123.4, 114.0, 113.9, 110.0, 67.7, 60.9, 49.0, 47.2, 46.0, 45.5, 44.5, 42.0, 41.7, 40.7, 35.5, 34.1, 33.5, 32.7, 31.3, 31.3, 30.1, 29.2, 27.5, 27.1, 26.1, 24.4, 22.6, 22.2, 21.2, 21.0, 17.7; ESI-MS: 831 [M+H]+, 853 [M+Na]+; HRMS: calculated for C44H54N4O10SNa [M + Na]+ 853.3458, found 853.3474.

Compounds 4e. The title compound was obtained in 63 % yield as a white solid: m.p. 116-118 °C; IR (KBr): 3128, 1735, 1685, 1662, 1617, 1553, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07-8.05 (m, 3H, 2 × ArH, C1-H), 7.79 (t, J = 7.5 Hz, 1H, ArH), 7.65 (t, J = 7.9, 7.6 Hz, 2H, 2 × ArH), 6.18 (t, J = 5.4 Hz, 1H, CONH), 5.97 (s, 1H, C11-H), 4.53 (t, J = 6.0 Hz, 2H, CH2O), 4.29 (t, J = 6.0 Hz, 2H, CH2O), 3.41-3.29 (m, 2H, NHCH2), 3.06 (d, J = 4.2 Hz, 1H, C13-H), 2.91-2.87 (m, 1H, C18-H), 2.39 (t, J = 7.1 Hz, 2H, CH2COO), 2.25-2,20 (m, 2H, OCH2CH2CH2O), 1.47 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.0, 196.6, 177.3, 173.2, 168.7, 166.0, 158.9, 137.9, 135.7, 129.7, 129.7, 128.6, 128.6, 124.0, 114.5, 114.5, 110.5, 68.1, 60.5, 49.5, 47.7, 46.5, 45.9, 45.0, 42.6, 42.1, 39.1, 36.0, 34.6, 34.1, 33.3, 31.8, 31.6, 31.6, 28


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30.6, 29.7, 27.9, 27.8, 26.9, 24.8, 24.8, 23.1, 22.9, 21.7, 21.5, 18.2; ESI-MS: 859 [M+H]+, 881 [M+Na]+; HRMS: calculated for C46H58N4O10SNa [M + Na]+ 881.3771, found 881.3787.

Compounds 4f. The title compound was obtained in 58 % yield as a white solid: m.p. 122-124 °C; IR (KBr): 3128, 1743, 1689, 1664, 1613, 1553, 1398 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07-8.04 (m, 3H, 2 × ArH, C1-H), 7.77 (t, J = 7.5 Hz, 1H, ArH), 7.63 (t, J = 7.8 Hz, 2H, 2 × ArH), 5.94 (s, 1H, C11-H), 4.53-4.51 (m, 3H, CH2O, NCHCOO), 4.16-4.06 (m, 2H, CH2O), 3.83-3.81 (m, 2H, NCH2), 3.26-3.22 (m, 1H, C13-H), 3.07-3.05 (m, 1H, C18-H), 1.46 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.88 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7, 196.2, 175.3, 172.2, 168.3, 165.5, 158.3, 137.5, 135.2, 129.2, 129.2, 128.1, 128.1, 123.4, 114.0, 113.9, 110.4, 67.6, 61.3, 60.2, 49.3, 48.0, 47.3, 47.1, 45.5, 44.5, 42.0, 41.7, 35.8, 33.6, 32.9, 31.4, 31.1, 30.9, 30.1, 29.4, 29.2, 28.8, 27.7, 27.1, 26.2, 24.6, 22.9, 22.2, 21.9, 21.0, 17.7; ESI-MS: 871 [M+H]+, 893 [M+Na]+; HRMS: calculated for C47H58N4O10SNa [M + Na]+ 893.3771, found 893.3790.

Compounds 4g. The title compound was obtained in 70 % yield as a white solid: m.p. 110-112 °C; IR (KBr): 3130, 1744, 1685, 1662, 1616, 1549, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.13-8.04 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 7.0 Hz, 1H, 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ArH), 7.64 (t, J = 7.7 Hz, 2H, 2 × ArH), 6.53 (t, J = 5.4 Hz, 1H, CONH), 6.03 (s, 1H, C11-H), 4.46 (t, J = 5.3 Hz, 2H, CH2O), 4.24 (t, J = 6.1 Hz, 2H, CH2O), 4.13-4.08 (m, 2H, NHCH2COO), 3.17 (d, J = 3.9 Hz, 1H, C13-H), 2.99-2.95 (m, 1H, C18-H), 1.47 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm;

13


196.3, 177.3, 169.8, 168.5, 165.8, 158.4, 137.4, 135.3, 129.2, 129.2, 128.0, 128.0, 123.4, 114.1, 113.8, 110.1, 70.4, 63.9, 48.9, 47.1, 46.0, 45.4, 44.5, 42.1, 41.7, 40.1, 35.5, 34.1, 33.5, 32.7, 31.3, 31.1, 30.1, 29.2, 27.1, 26.1, 24.6, 24.5, 24.4, 22.6, 22.6, 21.2, 21.0, 17.7; ESI-MS: 845 [M+H]+, 867 [M+Na]+; HRMS: calculated for C45H56N4O10SNa [M + Na]+ 867.3615, found 867.3630.

Compounds 4h. The title compound was obtained in 74 % yield as a white solid: m.p. 137-139 °C; IR (KBr): 3129, 1738, 1685, 1658, 1618, 1553, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.11-8.05 (m, 3H, 2 × ArH, C1-H), 7.79 (t, J = 7.5 Hz, 1H, ArH), 7.65 (t, J = 7.9 Hz, 2H, 2 × ArH), 6.32 (t, J = 5.4 Hz, 1H, CONH), 6.00 (s, 1H, C11-H), 4.47 (t, J = 6.0 Hz, 2H, CH2O), 4.19 (t, J = 6.2 Hz, 2H, CH2O), 3.33 (q, J = 6.0 Hz, 2H, NHCH2CH2), 3.07 (d, J = 4.3 Hz, 1H, C13-H), 2.93-2.89 (m, 1H, C18-H), 2.39 (t, J = 7.2 Hz, 2H, CH2COO), 1.48 (s, 3H, CH3), 1.33, (s, 3H, CH3) 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7, 196.2, 176.9, 173.0, 168.3, 165.5, 158.4, 137.3, 30


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135.2, 130.4, 129.2, 129.2, 128.0, 128.0, 123.5, 113.9, 113.9, 70.5, 63.3, 49.0, 47.1, 45.9, 45.4, 44.5, 42.1, 41.6, 38.7, 35.5, 34.1, 33.5, 32.8, 31.2, 31.1, 30.1, 29.2, 27.2, 26.4, 26.1, 24.7, 24.5, 24.2, 22.6, 22.4, 21.2, 21.0, 18.7, 17.7; ESI-MS: 873 [M+H]+; HRMS: calculated for C47H60N4O10SNa [M + Na]+ 895.4084, found 895.4102.

Compounds 4i. The title compound was obtained in 54 % yield as a white solid: m.p. 115-117 °C; IR (KBr): 3130, 1737, 1688, 1658, 1614, 1546, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.06-8.04 (m, 3H, 2 × ArH, C1-H), 7.77 (t, J = 7.3 Hz, 1H, ArH), 7.64 (t, J = 7.3 Hz, 2H, 2 × ArH), 5.94 (s, 1H, C11-H), 4-52-4.44 (m, 3H, CH2O, NCHCOO), 4.21-4.19 (m, 2H, CH2O), 3.83-3.80 (m, 2H, NCH2), 3.26-3.22 (m, 1H, C13-H), 3.13-3.10 (m, 1H, C18-H), 1.47 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.88 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.6, 196.2, 175.2, 172.3, 168.2, 165.5, 158.4, 137.5, 135.2, 129.2, 129.2, 128.0, 128.0, 123.4, 113.9, 113.9, 110.0, 65.0, 63.4, 61.3, 49.3, 47.9, 47.2, 47.0, 45.5, 44.5, 42.0, 41.7, 35.8, 33.6, 32.9, 31.4, 31.2, 30.1, 29.4, 29.2, 28.8, 27.5, 27.1, 26.1, 25.7, 24.6, 23.9, 23.4, 22.2, 21.8, 21.0, 17.7; ESI-MS: 885 [M+H]+, 907 [M+Na]+; HRMS: calculated for C48H60N4O10SNa [M + Na]+ 907.3928, found 907.3943.

Compounds 4j. The title compound was obtained in 72 % yield as a white solid: m.p. 109-111 °C; IR (KBr): 3129, 1747, 1689, 1662, 1616, 1553, 1400 cm-1; 1H NMR (300 M 31



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Hz, CDCl3, 25 °C, TMS): δ 8.07-8.04 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 7.5 Hz, 1H, ArH), 7.63 (t, J = 7.9, 7.6 Hz, 2H, 2 × ArH), 6.41 (t, J = 5.4 Hz, 1H, CONH), 6.00 (s, 1H, C11-H), 4.43 (t, J = 6.4 Hz, 2H, CH2O), 4.16 (t, J = 6.5 Hz, 2H, CH2O), 4.05 (t, J = 6.7 Hz, 2H, NHCH2COO), 3.16 (d, J = 4.2 Hz, 1H, C13-H), 2.99-2.94 (m, 1H, C18-H), 1.48 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7, 196.2, 177.2, 169.8, 168.5, 165.6, 158.5, 137.6, 135.2, 129.2, 129.2, 128.0, 128.0, 123.5, 114.0, 113.9, 110.0, 70.9, 64.6, 49.0, 47.2, 46.0, 45.4, 44.5, 42.1, 41.7, 40.7, 35.5, 34.1, 33.5, 32.7, 31.3, 31.2, 30.1, 29.2, 28.8, 27.9, 27.8, 27.1, 26.1, 24.9, 24.8, 24.4, 22.6, 21.2, 21.0, 17.7; ESI-MS: 873 [M+H]+, 895 [M+Na]+; HRMS: calculated for C47H60N4O10SNa [M + Na]+ 895.3928, found 895.3946.

Compounds 4k. The title compound was obtained in 67 % yield as a white solid: m.p. 108-110 °C; IR (KBr): 3128, 1729, 1685, 1662, 1616, 1553, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07-8.05 (m, 3H, 2 × ArH, C1-H), 7.80 (t, J = 9.3 Hz, 1H, ArH), 7.63 (t, J = 8.0, 7.5 Hz, 2H, 2 × ArH), 6.16 (t, J = 5.6, 1H, CONH), 5.97 (s, 1H, C11-H), 4.43 (t, J = 6.4 Hz, 2H, CH2O), 4.11 (t, J = 6.6 Hz, 2H, CH2O), 3.35-3.28 (m, 2H, NHCH2CH2), 3.07 (t, J = 4.3 Hz, 1H, C13-H), 2.92-2.87 (m, 1H, C18-H), 2.37 (t, J = 7.1 Hz, 2H, CH2COO), 1.48 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.90 (s, 3H, CH3) ppm; 13C NMR (75M Hz, 32


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CDCl3, 25 °C, TMS): δ 198.6, 196.2, 176.8, 173.0, 168.1, 165.6, 158.5, 137.5, 135.2, 129.2, 129.2, 128.0, 128.0, 123.5, 114.0, 113.9, 110.0, 70.9, 63.9, 48.9, 47.1, 45.9, 45.4, 44.5, 42.1, 41.6, 38.7, 35.5, 34.1, 33.5, 32.8, 31.3, 31.2, 31.1, 30.1, 29.2, 27.9, 27.8, 27.3, 26.0, 25.0, 24.8, 24.3, 24.2, 22.6, 22.5, 21.2, 21.0, 17.7; ESI-MS: 901 [M+H]+, 923 [M+Na]+; HRMS: calculated for C49H64N4O10SNa [M + Na]+ 923.4241, found 923.4258.

Compounds 4l. The title compound was obtained in 63 % yield as a white solid: m.p. 120-122 °C; IR (KBr): 3129, 1738, 1685, 1663, 1613, 1553, 1399 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07-8.04 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 7.5 Hz, 1H, ArH), 7.63 (t, J = 7.9 Hz, 2H, 2 × ArH), 5.93 (s, 1H, C11-H), 4.52-4.50 (m, 1H, NCHCOO), 4.43 (t, J = 6.4 Hz, 2H, CH2O), 4.13 (t, J = 6.4 Hz, 2H, CH2O), 3.83-3.80 (m, 2H, NCH2), 3.26-3.21 (m, 1H, C13-H), 3.13-3.10 (m, 1H, C18-H), 1.47 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.85 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7, 196.2, 175.1, 172.3, 168.0, 165.5, 158.5, 137.6, 135.2, 129.2, 129.2, 128.0, 128.0, 123.4, 114.0, 113.9, 110.2, 70.9, 64.1, 61.3, 49.2, 47.9, 47.2, 47.0, 45.5, 44.5, 42.0, 41.8, 35.7, 33.7, 32.9, 31.2, 31.1, 30.1, 29.3, 29.2, 28.0, 27.8, 27.6, 27.1, 26.1, 25.7, 24.9, 24.8, 24.5, 23.3, 22.2, 21.7, 21.0, 17.7; ESI-MS: 913 [M+H]+, 935 [M+Na]+; HRMS: calculated for C50H64N4O10SNa [M + Na]+ 935.4241, found 935.4261.

33



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Compounds 4m. The title compound was obtained in 66 % yield as a white solid: m.p. 118-120 °C; IR (KBr): 3128, 1747, 1691, 1658, 1620, 1552, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.08-8.06 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 7.4 Hz, 1H, ArH), 7.64 (t, J = 7.8, 7.3 Hz, 2H, 2 × ArH), 6.36 (t, J = 5.4 Hz, 1H, CONH), 5.96 (s, 1H, C11-H), 4.58-4.56 (m, 2H, CH2O), 4.35-4.33 (m, 2H, CH2O), 4.11-4.09 (m, 2H, NHCH2COO), 3.92-3.90 (m, 2H, CH2O), 3.81-3.79 (m, 2H, CH2O), 3.14 (d, J = 4.2 Hz, 1H, C13-H), 2.98-2.94 (m, 1H, C18-H), 1.47 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.18 (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): δ 198.5, 196.8, 177.2, 169.7, 168.2, 165.4, 157.4, 137.5, 135.2, 129.2, 129.2, 128.1, 128.1, 123.5, 114.0, 113.9, 110.0, 70.0, 68.7, 67.8, 63.4, 48.9, 47.2, 46.0, 45.4, 44.5, 42.0, 41.7, 40.6, 35.5, 33.5, 32.7, 31.3, 31.2, 30.1, 29.2, 28.8, 27.2, 26.1, 24.4, 22.7, 22.6, 21.2, 21.0, 17.7; ESI-MS: 863 [M+Na]+; HRMS: calculated for C45H56N4O11SNa [M + Na]+ 883.3564, found 883.3583.

Compounds 4n. The title compound was obtained in 59 % yield as a white solid: m.p. 126-128 °C; IR (KBr): 3128, 1729, 1685, 1662, 1611, 1555, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.08-8.06 (m, 3H, 2 × ArH, C1-H), 7.78 (t, J = 7.1 Hz, 1H, ArH), 7.64 (t, J = 7.7 Hz, 2H, 2 × ArH), 6.19 (t, J = 5.5 Hz, 1H, CONH), 5.96 (s, 1H, C11-H), 4.58 (t, J = 4.1 Hz, 2H, CH2O), 4.29 (t, J = 4.4 Hz, 2H, CH2O), 3.92 (t, J = 4.3 Hz, 2H, CH2O), 3.80 (t, J = 4.7 Hz, 2H, CH2O), 3.32-3.28 (m, 2H, NHCH2CH2), 3.08 (d, 34


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J = 4.2 Hz, 1H, C13-H), 2.92-2.88 (m, 1H, C18-H), 2.40 (t, J = 6.9 Hz, 2H, CH2COO), 1.47 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.89 (s, 3H, CH3) ppm;

13

C NMR (75 M Hz, CDCl3, 25 °C,

TMS): δ 198.5, 196.1, 176.8, 172.9, 168.1, 165.4, 158.4, 137.4, 135.2, 129.2, 129.2, 128.1, 128.1, 127.4, 123.5, 114.0, 113.9, 70.0, 68.9, 67.8, 63.0, 48.9, 47.2, 45.9, 45.4, 44.5, 42.0, 41.6, 38.6, 35.5, 34.1, 33.6, 32.8, 31.3, 31.2, 31.2, 30.1, 29.2, 27.3, 26.1, 24.3, 24.2, 22.6, 22.5, 21.2, 21.0, 17.7; ESI-MS: 889 [M+H]+, 911 [M+Na]+; HRMS: calculated for C47H60N4O11SNa [M + Na]+ 911.3877, found 911.3898.

Compounds 4o. The title compound was obtained in 63 % yield as a white solid: m.p. 122-124 °C; IR (KBr): 3129, 1738, 1691, 1661, 1616, 1549, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.08-8.06 (m, 3H, 2 × ArH, C1-H), 7.77 (t, J = 7.5 Hz, 1H, ArH), 7.64 (t, J = 7.4 Hz, 2H, 2 × ArH), 5.93 (s, 1H, C11-H), 4.61-4.57 (m, 3H, NCHCOO, CH2O), 4.32-4.27 (m, 2H, CH2O), 3.96-3.90 (m, 2H, CH2O), 3.81-3.79 (m, 4H, CH2O, NCH2), 3.15-3.13 (m, 1H, C13-H), 3.09-3.06 (m, 1H, C18-H), 1.44 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.91 (s, 3H, CH3) ppm;

13


196.1, 175.2, 172.2, 168.0, 165.4, 158.4, 137.5, 135.2, 129.2, 129.2, 128.1, 128.1, 127.4, 123.4, 114.0, 113.9, 70.0, 68.9, 67.8, 63.2, 61.2, 49.3, 47.9, 47.3, 47.0, 45.5, 44.5, 42.0, 41.8, 35.7, 33.7, 32.9, 31.2, 31.1, 30.1, 29.3, 29.2, 27.6, 27.1, 26.1, 25.7, 24.5, 23.4, 21.8, 35



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21.5, 21.0, 17.7; ESI-MS: 901 [M+H]+, 923 [M+Na]+; HRMS: calculated for C48H60N4O11SNa [M + Na]+ 923.3877, found 923.3893.

Compounds 4p. The title compound was obtained in 73 % yield as a white solid: m.p. 132-134 °C; IR (KBr): 3130, 1744, 1691, 1662, 1616, 1549, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.09-8.05 (m, 3H, 2 × ArH, C1-H), 7.79-7.62 (m, 3H, 3 × ArH), 6.37-6.36 (m, 1H, CONH), 5.94 (s, 1H, C11-H), 5.12 (s, 2H, CH2O), 4.75 (s, 2H, CH2O), 3.58-3.56 (m, 2H, NHCH2CH2), 3.04-3.05 (m, 1H, C13-H), 2.91 (m, 1H, C18-H), 2.60-2.61 (m, 2H, CH2COO), 1.48 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.88 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.9, 196.6, 177.3, 171.6, 168.6, 165.8, 158.4, 135.8, 131.0, 129.7, 129.7, 128.8, 128.7, 128.7, 123.9, 114.5, 114.4, 83.7, 78.9, 58.6, 52.1, 49.4, 47.7, 46.4, 45.9, 45.0, 42.5, 42.1, 36.0, 35.1, 34.6, 33.9, 33.2, 31.8, 31.7, 30.6, 29.7, 27.7, 27.0, 26.6, 24.7, 23.1, 23.0, 21.7, 21.6, 18.2; ESI-MS: 877 [M+Na]+; HRMS: calculated for C46H54N4O10SNa [M + Na]+877.3458, found 877.3473.

Compounds 4q. The title compound was obtained in 68 % yield as a white solid: m.p. 129-131 °C; IR (KBr): 3129, 1747, 1689, 1662, 1616, 1549, 1400 cm-1; 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.09-8.06 (m, 3H, 2 × ArH, C1-H), 7.78-7.64 (m, 3H, 3 × ArH), 6.22-6.19 (m, 1H, CONH), 5.99 (s, 1H, C11-H), 5.10 (s, 2H, CH2O), 4.81 (s, 2H, 36


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CH2O), 4.63-4.61 (m, 1H, NHCH(CH3)COO), 3.14-3.08 (m, 1H, C13-H), 2.94-2.91 (m, 1H, C18-H), 1.49 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.18 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.88 (s, 3H, CH3) ppm; 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.0, 196.6, 177.0, 172.5, 168.7, 165.9, 157.0, 135.8, 130.9, 129.7, 129.7, 128.8, 128.7, 128.7, 124.0, 114.5, 114.4, 83.4, 79.0, 65.8, 58.5, 52.7, 49.5, 47.7, 46.4, 45.9, 45.0, 42.5, 42.1, 36.0, 35.9, 34.6, 34.0, 33.2, 31.9, 31.8, 30.6, 29.7, 27.6, 27.0, 26.6, 24.7, 23.1, 21.7, 21.6, 18.2; ESI-MS: 877 [M+Na]+; HRMS: calculated for C46H54N4O10SNa [M + Na]+877.3458, found 877.3481.

MTT Assay. The effects of different trihybrids and CDDO-Me on the proliferation of human colon cancer and non-tumor cells were investigated by the MTT assay. The compounds were dissolved in DMSO and diluted with culture medium into eight concentrations (0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 5 µM) of solution for each compound (final DMSO concentration in medium < 0.4 %). HCT-8, HCT-8/5-FU, or non-tumor CCD841 cells (1.0 × 104 cells/well) were cultured in 10% fetal bovine serum (FBS) DMEM medium (Invitrogen) as the complete medium in 96-well cell plates overnight and treated in triplicate with, or without, different concentrations of individual compounds for 72 hours. During the last 4 h culture, the cells were exposed to MTT (5 mg/mL, Signma), and the resulting formazan crystals were dissolved in 150 µL of DMSO and measured using an microplate reader (Tecan) at 570 nm. Inhibition rate (%) = 37



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[(Acontrol−Atreated)/Acontrol] × 100%.

Nitrite Measurement in Vitro. The levels of intracellular NO generated by individual compounds were determined by the colorimetric assay using the nitrite colorimetric assay kit (Beyotime, China), according to the manufacturer’s instructions. Briefly, HCT-8, or HCT-8/5FU, or CCD841 cells (1 × 106/well) were treated with 100 µM or 150 µM of each compound for 150 min in the presence or absence of different concentrations of a PpeT1 substrate of Gly-Sar. Furthermore, some experiments were performed after pre-treatment of cells with different concentrations of a NO scavenger of PITO for 150 min. Subsequently, the cells were harvested, and their cell lysates were prepared and then mixed with Griess reagent for 10 min at 37 °C, followed by measurement at 540 nm by an microplate reader. The cells treated with 0.4 % DMSO in medium were used as negative controls for the background levels of nitrite production, while sodium nitrite at different concentrations was prepared as the positive control for the establishment of a standard curve.

Flow Cytometry Analysis of Apoptotic Cells. HCT-8 and HCT-8/5-FU cells (1 × 105/well) were cultured in complete medium in six-well plates for 24 h and treated in duplicate with different concentrations of 4c or CDDO-Me for 24 h. The control cells wre treated with vehicle (0.4 % DMSO in complete medium). The cells were harvested, 38


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washed and stained with propidium iodide (PI), and FITC-Annexin-V in the dark for 15 min using the FITC-AnnexinV Kit (BD PharMingen). The percentages of apoptotic cells were determined by flow cytometry on an FACS Calibur flow cytometer (Becton Dickinson).

Western Blotting Analysis. HCT-8 or HCT-8/5-FU (1 x 106 cells/well) were cultured in 6 cm dishes for 24 h and were treated in duplicate with 0.25, 0.5, or 1 µM 4c or 1 µM CDDO-Me in the presence of absence of PTIO for 24 h. The cells were harvested and lysed. After protein quantification using the BCA protein assay kit (Beyotime, China), the cell lysates (20 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE, 7.5% gel) and transferred onto polyvinylidenedifluoride (PVDF) membranes. After the membranes were blocked in 5% fat-free milk for 1 h, the target proteins were probed with anti-Pgp, anti-MRP1, anti-BCRP, anti-HIF-1α, anti-AKT, anti-phospho-AKT (Ser473), anti-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-Stat3, anti-phoshpo-Stat3, anti-GAPDH, and anti-β-actin antibodies (Cell Signaling, Boston, MA). The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated second antibodies and visualized by the enhanced chemiluminescent reagent. The relative levels of interesting protein expression and phosphorylation were determined by densitometric scanning using Image J software. In addition, the relative levels of PepT1, Pgp, MRP1 and BCRP expression in the unmanipulated cells were also 39



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determined by western blotting using specific antibodies. Analysis of Nitrated Drug Transporters. Some HCT-8/5-FU cell lysates were incubated with anti-nitrotyrosine antibody (Cambridge, MA, USA) and protein G microbeads (Cambridge, MA, USA) overnight at 4ºC with gently shocking. After centrifugation, the microbeads were washed extensively and the immune precipitated nitrolized proteins were eluted. Subsequently, the contents of nitrated Pgp, MRP1 and BCRP were characterized by western blotting using anti-Pgp, anti-MRP1 and anti-BCRP, respectively.

Acute toxicity of 4c in mice. Both genders of ICR mice at 7 weeks of age were purchased from Slac Laboratory Animal Co, of China Academy of Science (SLACCAS, Shanghai, China), and were housed in a specific pathogen free facility. The mice were randomly treated by oral gavage with a single dose of 4c at 500, 1000, 1500, and 2000 mg/kg (0.60×103, 1.19×103, 1.79×103, and 2.38×103 µmol/kg), respectively. The mice were monitored for their survival, behaviors, the amounts of water and food consumption, body weights and activity up to 14 days post treatment. The experimental protocols were evaluated and approved by the Ethics Committee of the China Pharmaceutical University.

Mouse Tumor Xenograft Model. Male athymic BALB/c nude mice at 3-5 weeks old were obtained from SLACCAS and housed in an individual ventilated cage under 40


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controlled environmental conditions in an AAALAC-accredited facility. The mice at 6-8 weeks old were inoculated subcutaneously with HCT-8/5-FU cells (2 × 106/mouse) in the right flank. After establishment of solid tumors at an average volume of 180-200 mm3 (9-11 days post-implantation), the animals were randomly treated by gavage with vehicle alone (0.2 mL) consisting of cremophor-EL/DMSO/PBS (1:1:8),49 15 mg/kg (17.8 µmol/kg) or 30 mg/kg (35.7 µmol/kg) 4c, or 30 mg/kg (59.4 µmol/kg) CDDO-Me in vehicle daily for 21 consecutive days. Tumor volumes were measured every other day using a vernier caliper and calculated using the following formula: tumor volume (mm3) = W2 (L/2), where W = width and L = length (mm).

ASSOCIATED CONTENT

Supporting Information. Preparative procedures and spectral data for compound 5 and HPLC spectra for the purities of compounds 4a-q. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors: *Tel: +86-25-83271015, Fax: +86-25-83271015, E-mail: [email protected] (Y. Zhang); Tel: +86-25-83271072, E-mail:[email protected] (Z. Huang).

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ACKNOWLEGEMENTS This study was supported by grants from the National Natural Science Foundation of China (No. 81273378, No. 21372261, No. 81202408, and No. 21472244).

ABBREVIATIONS USED

CDDO-Me,

methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate;

CDDO-Im,

1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole; SD, standard deviation; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; PE, petroleum ether; TMS, tetramethylsilane; UV, ultraviolet; w/w, weight per unit weight (weight- to -weight ratio); 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO); BOC, tert-butoxycarbonyl; ESI, electrospray ionization; AKT (protein B),

a

serine/threonine

protein

kinase; mTOR,

mammalian

kinase

target

of

rapamycin; ERK, extracellular regulated kinase; PI3K, phosphoinositide 3-kinase; Stat3, signal transducer and activator of transcription 3; HIF-1α, human hypoxia inducible factor 1 alpha; etc., and so forth; ROS, reactive oxygen species; rt, room temperature; MTT, 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide; 5-FU, 5-fluoro2,4(1H, 3H)-pyrimidinedione.

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