Synthesis of CDDO-amino acid-nitric oxide donor trihybrids as potential antitumor agents against both drug-sensitive and drug-resistant colon cancer

Article abstract of DOI:10.1021/jm5019302

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 = 0.2

Full text of DOI:10.1021/jm5019302

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Article
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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Synthesis of CDDOꢀAmino AcidꢀNitric Oxide
Donor Trihybrids as Potential Antitumor Agents
against Both Drugꢀsensitive and Drugꢀresistant
Colon Cancer
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a,b
a,b
a,b,*
a
a,b
Yong Ai, Fenghua Kang, Zhangjian Huang, , Xiaowen Xue, Yisheng Lai, Sixun
a,b
c
a,b*
Peng, Jide Tian, and Yihua Zhang
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing
2
10009, 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 CDDOꢀamino acidꢀNO donor trihybrids (4aꢀq) were designed
and synthesized. Biological evaluation indicated that the most active compound 4c
produced high levels of NO and inhibited the proliferation of drugꢀsensitive (HCTꢀ8, IC50
=
0.294 ꢁM) and drugꢀresistant (HCTꢀ8/5ꢀFU, IC50 = 0.232 ꢁM) colon cancer cells,
which were attenuated by an NO scavenger or typical substrate of PepT1. Furthermore,
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c triggered HCTꢀ8 and HCTꢀ8/5ꢀFU cell apoptosis more strongly than CDDOꢀMe,
inhibited the HIFꢀ1α, Stat3, AKT, ERK signaling, and induced the nitration of Pꢀgp,
MRP1, and BCRP proteins in HCTꢀ8/5ꢀFU cells. Finally, 4c had 4.36~5.53ꢀfold less
inhibitory activity against nonꢀtumor colon epithelialꢀlike cells (CCD841, IC = 1.282
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ꢁM) in vitro, and inhibited the growth of implanted human drugꢀresistant colon cancers
in mice more potently than CDDOꢀMe. Together, 4c is a novel trihybrid with potent
antiꢀtumor activity and may be a promising candidate for the treatment of drugꢀresistant
colon cancer.
INTRODUCTION
Multidrug resistance (MDR) is a leading cause for the failure of antiꢀcancer
1ꢀ3
chemotherapy.
MDR in cancer cells is attributed to many factors, such as
overꢀexpression of the ATPꢀbinding cassette (ABC) transporters, which can export
anticancer drugs out of the cells, leading to low levels of intracellular drugs and
4
consequent drug ineffectiveness. Three major ABC transporters implicated in cancer
MDR are Pꢀglycoprotein (Pꢀgp/ABCB1/MDR1), multidrug resistanceꢀassociated protein
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ꢀ8
1
(MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2). Currently,
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,10
there is no ABC transporter inhibitor available in the clinic. 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
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various physiological and pathophysiological processes. High levels of NO have strong
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antiꢀtumor activity by inducing tumor cell apoptosis, inhibiting tumor metastasis and
sensitizing drugꢀresistant tumor cells to chemotherapy, radiation, and immunotherapy in
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vitro and in vivo.
More importantly, NOꢀdonating antiꢀtumor drugs usually do not
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induce drug resistance in tumor cells. Furoxans, an important class of NO donors, are
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able to produce high levels of NO in vitro, and inhibit the growth of tumors in vivo.
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
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can nitrate the tyrosine residue of many key proteins, including ABC transporters.
We
have also developed a variety of furoxanꢀbased NO donating compounds such as
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oleanolic acid (OA)/furoxan,
anilinopyrimidine/furoxan,
and bifendate/furoxan
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hybrids
with potent antitumor and/or antiꢀMDR activities. Hence, generation of
furoxanꢀbased hybrids is a promising strategy for overcoming the MDR in cancer
therapy.
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OA is a wellꢀknown natural triterpenoid and has multiple pharmacological activities.
To improve its potency, many synthetic OA derivatives (SOADs) have been prepared,
including 2ꢀcyanoꢀ3,12ꢀdioxooleanaꢀ1,9(11)ꢀdienꢀ28ꢀoic acid (CDDO), its ester
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(CDDOꢀMe), amides (CDDOꢀIm), oleanꢀ28,13βꢀolide (Fig. 1), and others.
These
SOADs have strong antiꢀtumor activity by inhibiting tumor cell proliferation and
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inducing cancer cell apoptosis. Recent studies show that OA and its derivative methyl
3,11ꢀdioxooleanꢀ12ꢀenꢀ28ꢀolate (DIOXOL, Fig. 1) have moderate antitumor activity and
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can reverse the Pꢀgpꢀmediated MDR.
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Figure 1. Chemical structures of OA and SOADs.
Peptide transporter 1 (PepT1) is an attractive target for development of antiꢀtumor
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drugs because of its high affinity to its broad substrates. Physiologically, PepT1 is
predominantly expressed in the intestinal epithelium and can effectively transport amino
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acids, peptides, peptideꢀlike drugs, etc. Pathologically, the PepT1 is highly expressed by
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tumor cells, including human colon cancer cells (Cacoꢀ2), pancreatic cancer cells
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(
AsPcꢀ1 and Capanꢀ2), sarcoma cells (HTꢀ1080), prostatic cancer cells (PCꢀ3), lung
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cancer cells (A549), and ovarian adenocarcinoma cisplatinꢀsensitive (2008) and
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resistant cells (C13), and others. However, the low levels of PepT1 are expressed in
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nonꢀtumor tissues.
Thus, the differential expression of PepT1 has been utilized for
design and synthesis of selective anticancer agents, including the PepT1ꢀtargeting
dihybrids of amino acid with OA, floxuridine, gemcitabine, cytarabine, and gold
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(
III)ꢀdithiocarbamato, respectively.
In this context, we hypothesized that CDDOꢀamino acidꢀNO donor trihybrids could be
effectively transported to cancer cells by the PepT1 to exert potent cytotoxicity against
both drugꢀsensitive and drugꢀresistant colon cancer cells. Accordingly, we synthesized
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trihybrids 4aꢀq by coupling the carboxyl group of CDDO with phenylsulfonylꢀsubstituted
furoxan through different amino acids, respectively, and evaluated their bioactivity in
vitro and in vivo.
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RESULTS AND DISCUSSION
Chemistry. The strategies for the synthesis of the target compounds 4aꢀq are shown in
Scheme 1. (3ꢀPhenylsulphonylꢀ4ꢀhydyoxyalkoxyl)furoxans 2aꢀe were synthesized
through the reactions of 3,4ꢀdiphenylsulphonylfuroxan with different diols
(butaneꢀ1,3ꢀdiol,
butaneꢀ1,4ꢀdiol,
2ꢀbutyneꢀ1,4ꢀdiol,
hexaneꢀ1,6ꢀdiol
or
2
,2'ꢀoxydiethanol). The generated 2aꢀe were reacted with Nꢀprotected amino acids
(BocꢀGlycine, BocꢀβꢀAlaꢀOH, BocꢀLꢀProꢀOH, BocꢀLꢀAlaꢀOH, BocꢀγꢀAminobutyric acid)
respectively in the presence of 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀethylcarbodiimide
hydrochloride (EDCI) and 4ꢀdimethylꢀaminoꢀpyridine (DMAP) to afford the
corresponding esters 3aꢀq. The Boc group of 3aꢀq was then removed by trifluoroacetic
acid (TFA) to yield aminoꢀcontaining intermediates, which were directly treated with the
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acyl chloride of CDDO prepared as reported previously,
in the presence of
triethylamine (TEA) to provide the target compounds 4aꢀq. Their structures were
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characterized by IR, HꢀNMR, CꢀNMR, MS, and HRMS.
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Scheme 1. Synthetic routes of CDDOꢀamino acidꢀNO donor trihybrids 4aꢀp.
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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 Antiꢀproliferative 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 drugꢀsensitive colon cancer HCTꢀ8 and drugꢀresistant
colon cancer HCTꢀ8/5ꢀFU cells as well as in nonꢀtransformed colon epithelialꢀlike
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CCD841 cells by western blot assay. The results indicated that high levels of PepT1
were expressed by both HCTꢀ8 and HCTꢀ8/5ꢀFU cells, but only little was detected in
CCD841 cells (Fig. S1).
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Next, the antiꢀtumor activity of compounds 4aꢀq against HCTꢀ8 and HCTꢀ8/5ꢀFU were
preliminarily screened by the MTT assay using CDDOꢀMe as a positive control.
Compounds 4aꢀc, 4p, and 4q (R = 2ꢀbutyneꢀ1,4ꢀdiol), and 4d, 4g, 4j, and 4m (R =
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glycine) at 1 ꢁM inhibited colon cancer proliferation by > 90%, which were higher than
that of CDDOꢀMe (Fig. 2). Other eight compounds 4e, 4f, 4h, 4i, 4k, 4l, 4n, and 4o
displayed an antiꢀtumor potency comparable to CDDOꢀMe.
Figure 2. Compounds 4aꢀq inhibit the proliferation of HCTꢀ8 and HCTꢀ8/5ꢀFU cells.
HCTꢀ8 and HCTꢀ8/5ꢀFU cells were treated with, or without, the indicated compounds at
1
ꢁM 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 CDDOꢀMe.
Subsequently, the nine most potent compounds (4aꢀd, 4g, 4j, 4m, 4p, and 4q) were
further investigated for their antiꢀtumor activity (Table 1). Compound 4c displayed more
potent activity (IC = 0.294 and 0.232 ꢁM) than CDDOꢀMe and 5ꢀFU in HCTꢀ8 and
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HCTꢀ8/5ꢀFU cells. It is notable that CDDOꢀMe had similar antiꢀtumor activities against
both colon cancer and nonꢀtumor colon cells, however, 4c displayed a 4.63~5.53ꢀfold
decrease in its antiꢀproliferative activity on nonꢀtumor CCD841 cells (IC = 1.282 ꢁM),
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suggesting that 4c may be a promising drug candidate for further investigation.
Table 1. The antiꢀproliferative activity of compounds
a
IC (ꢁM)
5
0
Comp.
HCTꢀ8
HCTꢀ8/5ꢀFU
0.343 ± 0.044
0.381 ± 0.021
CCD841
b
b
b
c
c
c
c
c
c
c
c
c
4
a
0.441 ± 0.081
1.425 ± 0.154
1.698 ± 0.109
1.282 ± 0.095
1.725 ± 0.114
1.121 ± 0.102
1.423 ± 0.081
3.129 ± 0.198
0.958 ± 0.069
1.472 ± 0.057
4b
0.504 ± 0.098
b,c
b,c
4
c
0.294 ± 0.013
0.232 ± 0.010
b
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d
0.550 ± 0.067
0.380 ± 0.026
0.315 ± 0.053
0.339 ± 0.060
0.562 ± 0.087
0.276 ± 0.013
0.462 ± 0.088
b
b
b
b
b
b
4
g
0.383 ± 0.031
0.356 ± 0.054
b
4j
4
m
0.872 ± 0.101
b
4
q
0.370 ± 0.052
0.379 ± 0.066
b
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p
CDDOꢀMe
ꢀFU
0.399 ± 0.047
0.91 ± 0.103
0.363 ± 0.029
36.638 ± 2.319
0.316 ± 0.019
ꢀ
5
a
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 ꢁM) of solution for
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each compound (final DMSO concentration in medium < 0.4%). The cells (1×10 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 IC for each
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compound were calculated and data are expressed as mean IC (µM) ± SD of each
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compound from three independent experiments. IC is the drug concentration inhibiting
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c
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0% of the cell proliferation. P < 0.01 vs. the 5ꢀFU. P < 0.05 vs. the CDDOꢀMe.
The Role of NO in the Antiꢀtumor Activity of 4c. Compound 4c was composed of
CDDO moiety (compound 5) and furoxan moiety (2a) (Fig. 3), and their antiꢀtumor
activity against HCTꢀ8 and HCTꢀ8/5ꢀFU cells were also examined. The IC values of 4c
5
0
for HCTꢀ8 (0.294 ꢁM) and HCTꢀ8/5ꢀFU cells (0.232 ꢁM) were significantly less than
that of individual moieties, 5 (IC = 3.192 and 2.257 ꢁM) and 2a (IC = 33.512 and
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0
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6.569 ꢁM), and even the combination of 5 and 2a (IC50 = 2.447 and 1.863 ꢁM),
respectively. These results suggest that the antiꢀtumor activity of 4c may be attributed to
the synergic effects of CDDO and NO donor moieties as well as PepT1ꢀmediated drug
transportation in cancer cells.
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Figure 3. Comparison of antiꢀcancer activity of trihybrid 4c with that of 5, 2a, and
equimolar mixture of 5 and 2a.
To verify the contribution of NO to the antiꢀtumor activity, the levels of intracellular
NO released by the tested compounds were firstly detected. HCTꢀ8, HCTꢀ8/5ꢀFU, and
CCD841 cells were treated with 100 ꢁM 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 HCTꢀ8 and HCTꢀ8/5ꢀFU 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
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HCTꢀ8/5ꢀFU cells, whereas less active compounds 4f, 4i and 4l produced lower levels of
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NO. The levels of intracellular NO by different compounds were positively correlated
with their antiꢀtumor activities against HCTꢀ8 and HCTꢀ8/5ꢀFU cells in vitro (R = 0.950,
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.962, respectively, p < 0.05, determined by Pearson’s correlation analysis). These results
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suggest that the active compounds may have a better ability to penetrate the membrane of
HCTꢀ8 and HCTꢀ8/5ꢀFU 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 nonꢀtumor colon CCD841 cells, suggesting
that PepT1ꢀmediated transport of these compounds may be important for the high levels
of NO production and antiꢀtumor 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 antiꢀtumor activity of 4c was investigated. HCTꢀ8
and HCTꢀ8/5ꢀFU cells were pretreated with various concentrations of an NO scavenger
of carboxyꢀPTIO for 1 h and then treated with 0.25 ꢁM of 4c. The effects of different
treatments on the proliferation of HCTꢀ8 and HCTꢀ8/5ꢀFU cells were determined by the
MTT assay (Fig. 4B). Treatment with 4c alone remarkably inhibited the proliferation of
HCTꢀ8 and HCTꢀ8/5ꢀFU cells and its inhibitory effects were reduced doseꢀdependently
by pretreatment with different concentrations of carboxyꢀPTIO. These results suggest that
NO produced by 4c may contribute to its inhibition on the proliferation of drugꢀsensitive
and drugꢀresistant 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. HCTꢀ8,
HCTꢀ8/5ꢀFU, and CCD841 cells were treated in triplicate with individual compounds at
1
00 ꢁM 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 HCTꢀ8 group. (B) Effects of NO scavenging,
carboxyꢀPTIO (PTIO) on the antiproliferative effect of 4c. HCTꢀ8, HCTꢀ8/5ꢀFU, and
CCD841 cells were pretreated in triplicate with the indicated concentrations of
carboxyꢀPTIO (0, 6.25, 12.5, 25, or 50 ꢁM) for 1 h and treated with 0.25 ꢁM 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 vehicleꢀtreated control.
PepT1ꢀmediated Cell Uptake is Crucial for the Antiꢀtumor Activity of These
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Compounds. To determine the importance of PepT1ꢀmediated update of compound, we
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tested whether the dipeptide GlyꢀSar, a high affinity of substrate for PepT1, could
modulate the antiꢀtumor activity of 4c in HCTꢀ8 and HCTꢀ8/5ꢀFU cells. The two cancer
cells were treated with different doses of 4c and GlyꢀSar and the proliferation of cancer
cells were determined using MTT assay. As shown in Figures 5A and 5B, addition of
GlyꢀSar (0, 0.75 or 1.5 ꢁM) significantly mitigated the inhibitory effects of 4c on the
proliferation of HCTꢀ8 and HCTꢀ8/5ꢀFU cells and their regulatory effects tended to be
doseꢀdependent. Furthermore, analysis of the impact of GlyꢀSar on the levels of NO
production by 4c indicated that pretreatment with GlyꢀSar reduced the levels of NO
production by 4c in both HCTꢀ8 and HCTꢀ8/5ꢀFU cells in a doseꢀdependent manner (Fig.
5C). Therefore, the PepT1 on these cancer cells is crucial for the intracellular
transportation and antiꢀtumor activity of 4c.
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Figure 5. PepT1ꢀmediated cell uptake is important for the antiꢀtumor activity of 4c.
HCTꢀ8 (A) and HCTꢀ8/5ꢀFU (B) cells were preꢀtreated with, or without, GlyꢀSar (0.75
ꢁM or 1.5 ꢁM) 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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GlyꢀSaron the levels of NO (present as nitrite) produced by 4c. HCTꢀ8 and HCTꢀ8/5ꢀFU
cells were preꢀtreated with, or without, different concentrations of GlyꢀSar (0.75, 1.5, 3, 6,
1
2, 24, 48, 96, and 192 ꢁM) for 150 min and treated with 4c at 150 ꢁM for 150 min. The
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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 GlyꢀSar
untreated group.
Induction of Cancer Cell Apoptosis in Vitro. Previous studies have shown that
CDDOꢀMe can induce cancer cell apoptosis by modulating the expression of
3
0
apoptosisꢀrelated regulators and signal events, and high levels of NO can induce tumor
2
2
cell apoptosis. Accordingly, the effect of 4c on induction of tumor cell apoptosis was
tested. HCTꢀ8 and HCTꢀ8/5ꢀFU cells were treated with variable concentrations of 4c,
CDDOꢀMe, or vehicle (0.4 % DMSO in medium) for 24 h, respectively. The cells were
harvested and stained with Annexin VꢀFITC and propidium iodide (PI), and the
percentages of apoptotic cells were determined by flow cytometry analysis. Treatment
with 4c induced apoptosis in both HCTꢀ8 and HCTꢀ8/5ꢀFU cells in a doseꢀdependent
manner (Fig. 6), and the effects of higher doses of 4c were significantly stronger than that
of CDDOꢀMe in both HCTꢀ8 and HCTꢀ8/5ꢀFU cells.
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Figure 6. Compounds 4c and CDDOꢀMe induce colon cancer cell apoptosis in vitro.
HCTꢀ8 (A) and HCTꢀ8/5ꢀFU (B) cells were incubated with the indicated concentrations
of 4c or CDDOꢀMe for 24 h, and the percentages of apoptotic cells were determined by
flow cytometry after staining them with Annexin VꢀFITC 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 CDDOꢀMe group.
Effects of 4c on the HIFꢀ1α, Stat3, AKT and ERK Signaling in HCTꢀ8 and
HCTꢀ8/5ꢀFU Cells. To gain more insights into the molecular mechanisms underlying the
activity of 4c, we examined the regulatory effects of 4c on the HIFꢀ1α and Stat3, AKT,
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ERK signaling in HCTꢀ8 and HCTꢀ8/5ꢀFU cells using CDDOꢀMe as a control. The cells
were treated with various concentrations of 4c or CDDOꢀMe. The expression and
activation of HIFꢀ1α, Stat3, AKT, and ERK were determined by western blotting. As
shown in Figure 7, treatment with 4c significantly inhibited the HIFꢀ1α expression and
Stat3, AKT, and ERK phosphorylation in a doseꢀdependent manner, although it did not
alter the levels of Stat3, AKT, and ERK expression in cancer cells. Similarly, treatment
with CDDOꢀMe also significantly reduced the levels of HIFꢀ1α expression and Stat 3,
AKT and ERK phosphorylation in both cell lines and the inhibitory effects of CDDOꢀMe
on the Stat3 phosphorylation and HIFꢀ1α expression in both cell lines were significantly
less than that of 4c while the inhibitory effect of CDDOꢀMe on the ERK phosphorylation
in HCTꢀ8/5ꢀFU cells was significantly stronger than that of 4c. Previous studies have
shown that aberrant activation of Stat3 and AKT and high levels of HIFꢀ1α expression
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are associated with the development of human colon cancer
and CDDOꢀMe can
inhibit the proliferation of cancer cells by downꢀregulating the Stat3 and AKT activation
4
8ꢀ50
and HIFꢀ1α expression in cancer cells.
Recent studies also show that high levels of
5
1ꢀ53
ROS directly inhibit the Stat3 and AKT activation.
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 HIFꢀ1α expression, we observed that
treatment with 4c significantly reduced the levels of HIFꢀ1α expression in both colon
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cancer lines. The reduced levels of HIFꢀ1α expression by 4c may stem from
ROSꢀmediated inactivation of Stat3, which can attenuate the Stat3ꢀupꢀregulated
PI3K/AKT/mTOR activation and downstream HIFꢀ1α expression in colon cancer cells.
The precise mechanisms underlying the action of 4c remain to be further investigated.
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Figure 7. Effects of 4c and CDDOꢀMe on the HIFꢀ1α, Stat3, AKT and ERK signaling in
HCTꢀ8 and HCTꢀ8/5ꢀFU cells. HCTꢀ8 and HCTꢀ8/5ꢀFU cells were treated with vehicle,
or the indicated compound for 24 h and the relative levels of HIFꢀ1α, Stat3, AKT and
ERK expression and phosphorylation were determined by western blot assays using
βꢀactin 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 HIFꢀ1α, 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 vehicleꢀtreated control.
#
P < 0.05 vs the CDDOꢀMe group.
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Effect of 4c on ABCꢀtransporter Expression. High levels of NO can react with
superoxide to produce peroxynitrite anion, which can nitrate the tyrosine residue of many
19,20
key proteins, including ABC transporters, attenuating MDR.
To further understand
the role of 4c in inhibiting drugꢀresistant 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
HCTꢀ8/5ꢀFU cells were significantly higher than that in HCTꢀ8 cells. Furthermore,
treatment with 4c, but not CDDOꢀMe, significantly reduced the relative levels of Pgp,
MRP1, and BCRP expression in a doseꢀdependent manner and these inhibitory effects
were partially attenuated by pretreatment with the NO scavenger of carboxyꢀPTIO in
HCTꢀ8/5ꢀFU cells. In addition, we also tested the impact of 4c on the nitration of Pgp,
MRP1 and BCRP in HCTꢀ8/5ꢀFU cells. HCTꢀ8/5ꢀFU cells were treated with, or without,
different concentrations of 4c or CDDOꢀMe and the nitrated Pgp, MRP1 and BCRP
proteins were precipitated by antiꢀnitrotyrosine 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 CDDOꢀMe, increased
the levels of nitrated Pgp, MRP1 and BCRP in a doseꢀdependent manner, which was
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partially attenuated by pretreatment with carboxyꢀPTIO in HCTꢀ8/5ꢀFU cells. Apparently,
high levels of NO produced by 4c were responsible for the nitration of Pgp, MRP1 and
BCRP in HCTꢀ8/5ꢀFU cells.
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Figure 8. Nitration of drug efflux pumps by 4c in HCTꢀ8/5ꢀFU cells. (A) Expression of
ABC transporters in HCTꢀ8 and HCTꢀ8/5ꢀFU cells. HCTꢀ8 and HCTꢀ8/5ꢀFU cells were
treated with, or without, the indicated concentrations of 4c or CDDOꢀMe 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 HCTꢀ8/5ꢀFU cell lysates were precipitated by
antiꢀnitrotyrosine 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
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levels of ABC transporter expression. ***P < 0.001 vs the HCTꢀ8 group. (B) Western
blot analysis of the levels of nitrated ABC transporters. *P < 0.05, ***P < 0.001 vs the
PTIO group.
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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
4
9
cremophorꢀEL/DMSO/PBS (1: 1: 8)), 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
3
the observation period even when the dose was raised up to 2,000 mg/kg (2.38×10
ꢁ
mol/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
ꢁmol/kg)
Total
mortality
(1ꢀ14 d)
a
Mouse
Body weight
Day 7
Number
of mice
sex
Day 0
Day 14
(
2
,000
1
1
0
0
female
0
0
19.8±0.92
25±1.05
30±1.56
3
(
2.38×10 )
2,000
male
19.5±0.85
26.7±1.89 31.9±1.97
3
(
2.38×10 )
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 HCTꢀ8/5ꢀFU Xenograft Tumors in
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Mice. To evaluate the in vivo anticancer activity of 4c, BALB/c nude mice were
inoculated subcutaneously with drugꢀresistant colon carcinoma HCTꢀ8/5ꢀFU cells. After
the establishment of solid tumor, the mice were randomly treated by gavage with
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4
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CDDOꢀMe, 4c at the indicated doses or vehicle (cremophorꢀEL/DMSO/PBS (1:1:8))
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 CDDOꢀMeꢀtreated mice were not observed although the body weights
in the control, 4cꢀ and CDDOꢀMeꢀtreated groups of mice were somewhat reduced (Table
3
). In addition, treatment with 15 mg/kg (17.8 ꢁmol/kg) of 4c significantly reduced the
volumes of implanted colon tumors, while treatment with 4c at 30 mg/kg (35.7 ꢁmol/kg)
further inhibited the growth of the implanted tumors (P < 0.01 vs. the vehicleꢀtreated
control, Fig. 9), indicating that the antiꢀtumor activity of 4c trended to be doseꢀdependent.
Furthermore, the tumor weights (0.48 ± 0.17 g) in the mice treated with 4c at 30 mg/kg
(35.7 ꢁmol/kg) were significantly reduced by 60.83 %, as compared with those of the
vehicleꢀtreated controls (1.24 ± 0.19 g, P < 0.01, Table 3). More importantly, the
antiꢀtumor activity of 4c at 30 mg/kg (35.7 ꢁmol/kg) was comparable with that of
CDDOꢀMe at 30 mg/kg (59.4 ꢁmol/kg). Therefore, 4c had more potent antiꢀtumor
activity against the growth of implanted human colon tumors than CDDOꢀMe in mice.
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Figure 9. Inhibition of 4c on the growth of implanted drugꢀresistant colon tumors in mice.
Male BALB/c nude mice were inoculated with HCTꢀ8/5ꢀFU cells and after establishment
of solid tumors, the mice were randomly treated with the indicated doses of 4c,
CDDOꢀMe or vehicle (cremophorꢀEL/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 vehicleꢀtreated control.
a
Table 3. Effects of 4c on the body and tumor weights in mice
Dose
mg/kg
ꢁmol/kg)
ꢀ
Body weight (g)
Inhibitory
ratio
Tumor weight
(g)
Group
day 1
day 21
(
(%, w/w)
ꢀ
Control
20.8 ± 2.2 18.2 ± 1.3
22.4 ± 1.4 16.7 ± 1.2
22.3 ± 1.9 17.5 ± 1.7
21.1 ± 1.5 16.7 ± 1.4
1.24 ± 0.19
b
CDDOꢀMe
30 (59.4)
15 (17.8)
30 (35.7)
0.51 ± 0.25
58.40
49.19
60.83
b
4
4
c
c
0.63 ± 0.25
b
0.48 ± 0.17
a
Data shown are the means ± SD of tumor weights in mice and their body weights from
b
each group of mice (n = 8). P< 0.01 vs. the vehicleꢀtreated control.
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CONCLUSION
In summary, a series of trihybrids (4aꢀq) were designed and synthesized from CDDO,
amino acid, and (phenylsulfonyl)furoxan, and were biologically evaluated. The most
active compound 4c exhibited strong and preferable antiꢀtumor activity against both
drugꢀsensitive HCTꢀ8 and drugꢀresistant HCTꢀ8/5ꢀFU cells. Treatment with 4c induced
high levels of NO production in HCTꢀ8 and HCTꢀ8/5ꢀFU cells but not in human
nonꢀtumor colon epithelialꢀlike CDD841 cells, and its antiꢀtumor activity was
significantly attenuated by an NO scavenger or a known substrate of PepT1 in a
doseꢀdependent manner. In addition, 4c displayed more potent capacity to induce HCTꢀ8
and HCTꢀ8/5ꢀFU cell apoptosis than CDDOꢀMe. Furthermore, treatment with 4c
inhibited the HIFꢀ1α, Stat 3, AKT, and ERK signaling in HCTꢀ8 and HCTꢀ8/5ꢀFU cells,
and reduced the relative levels of Pꢀgp, MRP1, and BCRP by nitrating these cellular drug
efflux proteins in HCTꢀ8/5ꢀFU cells. Moreover, 4c was relatively safe to mice and
inhibited the growth of implanted human colon tumors more potently than CDDOꢀMe in
mice. Together, these data indicated that 4c, a trihybrid of CDDO, amino acid and NO
donor moieties had more potent antiꢀtumor activity preferably against human colon
cancer than CDDOꢀMe. Our findings may provide a proof of principle for design of such
trihybrids like 4c as potent and selective antiꢀcolon cancer agents potentially to
circumvent MDR.
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EXPERIMENTAL PROTOCOLS
Chemical analysis. Melting points were determined on a MelꢀTEMP II melting point
apparatus and were uncorrected. Infrared (IR) spectra (KBr) were recorded on a Nicolet
0
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Impact 410 instrument (KBr pellet). HꢀNMR and CꢀNMR 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 (200ꢀ300 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 (LCꢀ10A HPLC system consisting of LCꢀ10ATvp
pumps and an SPDꢀ10Avp UV detector). Individual compounds with a purity of > 95%
were used for subsequent experiments (see the Supporting Information). CDDO, acyl
chloride of CDDO, and 2aꢀe was prepared according to the method described
2
1,26,27
previously.
Procedure for the preparation of compound 5 was shown in Figure S2.
General Procedure for the Preparation of Compounds 3aꢀq. To a solution of
Bocꢀamino acid (3.1 mmol) in dry CH Cl (5 mL), 2aꢀe (3.0 mmol),
2
2
1
ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀethylcarbodiimide hydrochloride (EDCI) (6 mmol) and
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ꢀdimethylaminopyridine (DMAP) (0.3 mmol) were added, and the mixture was stirred at
room temperature for 6ꢀ12 h. The reaction solution was diluted with water and extracted
with CH Cl . The organic layer was washed with brine, then dried with sodium sulfate,
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filtered and evaporated in vacuo to give corresponding crude compound 3aꢀq (93ꢀ96%),
respectively, which was used in the next without further purification.
General Procedure for the Preparation of Compounds 4aꢀq. A mixture of 3aꢀq (2
mmol) and TFA (15 mmol) in anhydrous CH Cl (5 mL) was stirred at room temperature
2
2
for 0.5 h. The reaction mixture was then cooled to 0 °C, and Et N (20 mmol), acyl
3
chloride of CDDO (2 mmol) in anhydrous CH Cl (2 mL) was sequentially thus added
2
2
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 CH Cl . The organic layer
2
2
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 = 7:3ꢀ1:1 v/v) to provide the title
compounds 4aꢀq (54ꢀ80 %), respectively.
Compounds 4a. The title compound was obtained in 65 % yield as a white solid: m.p.
ꢀ1 1
1
19ꢀ121 °C; IR (KBr): 3129, 1729, 1689, 1663, 1615, 1549, 1399 cm ; H NMR (300 M
Hz, CDCl , 25 °C, TMS): δ 8.09ꢀ8.06 (m, 3H, 2 × ArH, C ꢀH), 7.78ꢀ7.66 (m, 3H, 3 ×
3
1
ArH), 5.95 (s, 1H, C ꢀH), 5.09 (s, 2H, CH O), 4.81 (s, 2H, CH O), 4.53ꢀ4.50 (m, 1H,
1
1
2
2
NCHCOO), 4.15ꢀ4.09 (m, 2H, NCH ), 3.24ꢀ3.22 (m, 1H, C ꢀH), 3.08ꢀ3.06 (m, 1H,
2
13
C ꢀH), 1.48 (s, 3H, CH ), 1.36 (s, 3H, CH ), 1.26 (s, 3H, CH ), 1.16 (s, 3H, CH ), 1.02 (s,
18
3
3
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3
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3
3
H, CH ), 0.88 (s, 3H, CH ), 0.86 (s, 3H, CH ) ppm; C NMR (75 M Hz, CDCl , 25 °C,
3 3 3 3
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,
1
4
28.1, 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,
0
1
2
3
4
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
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8
9
0
4.5, 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; ESIꢀMS: 881 [M+H] ; HRMS: calculated for
+
C H N O SNa [M + Na] 903.3615, found 903.3635.
48
56
4
10
Compounds 4b. The title compound was obtained in 80 % yield as a white solid: m.p.
ꢀ1 1
1
02ꢀ104 °C; IR (KBr): 3131, 1739, 1685, 1661, 1617, 1549, 1400 cm ; H NMR (300 M
Hz, CDCl , 25 °C, TMS): δ 8.09ꢀ8.04 (m, 3H, 2 × ArH, C ꢀH), 7.79 (t, J = 7.5 Hz, 1H,
3
1
ArH), 7.65 (t, J = 7.6 Hz, 2H, 2 × ArH), 6.18 (t, J = 5.4 Hz, 1H, CONH), 5.97 (s, 1H,
C ꢀH), 5.11 (s, 2H, CH O), 4.75 (s, 2H, CH O), 3.06 (d, J = 4.2 Hz, 1H, C ꢀH),
11
2
2
13
2
.91ꢀ2.87 (m, 1H, C ꢀH), 2.39 (t, J = 7.1 Hz, 2H, CH COO), 1.48 (s, 3H, CH ), 1.33 (s,
18 2 3
3H, CH ), 1.25 (s, 3H, CH ), 1.16 (s, 3H, CH ), 1.01 (s, 3H, CH ), 1.00 (s, 3H, CH ),
3
3
3
3
3
13
0
1
.90 (s, 3H, CH ) ppm; C NMR (75 M Hz, CDCl , 25 °C, TMS): δ 198.5, 196.1, 176.8,
3 3
71.9, 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,
3.6, 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;
3
+
+
+
ESIꢀMS: 869 [M+H] , 891 [M+Na] ; HRMS: calculated for C H N O SNa [M + Na]
4
7
56
4
10
891.3615, found 891.3633.
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Compounds 4c. The title compound was obtained in 77 % yield as a white solid: m.p.
ꢀ1
1
1
20ꢀ122 °C; IR (KBr): 3129, 1758, 1685, 1660, 1618, 1548, 1400 cm ; H NMR (300 M
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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60
Hz, CDCl , 25 °C, TMS): δ 8.15ꢀ8.09 (m, 3H, 2 × ArH, C ꢀH), 7.78 (t, J = 7.4 Hz, 1H,
3
1
ArH), 7.64 (t, J = 7.7 Hz, 2H, 2 × ArH), 6.35 (t, J = 5.4 Hz,1H, CONH), 6.00 (s, 1H,
C ꢀH), 5.10 (s, 2H, CH O), 4.80 (s, 2H, CH O), 4.13ꢀ4.09 (m, 2H, NHCH COO), 3.14
11
2
2
2
(
d, J = 4.6 Hz, 1H, C ꢀH), 2.97ꢀ2.94 (m, 1H, C ꢀH), 1.48 (s, 3H, CH ), 1.36 (s, 3H,
13 18 3
CH ), 1.25 (s, 3H, CH ), 1.16 (s, 3H, CH ), 1.02 (s, 3H, CH ), 1.00 (s, 3H, CH ), 0.91 (s,
3
3
3
3
3
13
3
H, CH ) ppm; C NMR (75 M Hz, CDCl , 25 °C, TMS): δ 198.7, 196.2, 177.3, 169.0,
3 3
1
68.6, 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,
+
3
1.4, 31.2, 30.1, 29.2, 28.8, 27.1, 26.4, 24.5, 22.6, 21.2, 21.0, 17.7; ESIꢀMS: 841 [M+H] ,
+
+
+
8
63 [M+Na] , 879 [M+K] ; HRMS: calculated for C H N O SNa [M + Na] 863.3302,
45 52 4 10
found 863.3320.
Compounds 4d. The title compound was obtained in 74 % yield as a white solid: m.p.
ꢀ1 1
128ꢀ130 °C; IR (KBr): 3131, 1751, 1689, 1662, 1616, 1553, 1398 cm ; H NMR (300 M
Hz, CDCl , 25 °C, TMS): δ 8.10ꢀ8.06 (m, 3H, 2 × ArH, C ꢀH), 7.78 (t, J = 6.7 Hz, 1H,
3
1
ArH), 7.66 (t, J = 6.9 Hz, 2H, 2 × ArH), 6.36 (t, J = 5.4 Hz, 1H, CONH), 5.97 (s, 1H,
C ꢀH), 4.52ꢀ4.50 (m, 2H, CH O), 4.36ꢀ4.34 (m, 2H, CH O), 4.11ꢀ4.08 (m, 2H,
11
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NHCH COO), 3.14 (d, J = 4.2 Hz, 1H, C ꢀH), 2.98ꢀ2.94 (m, 1H, C ꢀH), 1.46 (s, 3H,
2
13
18
CH ), 1.35 (s, 3H, CH ), 1.26 (s, 3H, CH ), 1.16 (s, 3H, CH ), 1.02 (s, 3H, CH ), 0.99 (s,
3
3
3
3
3
13
3
1
H, CH ), 0.90 (s, 3H, CH ) ppm; C NMR (75 M Hz, CDCl , 25 °C, TMS): δ 198.7,
3 3 3
0
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2
3
4
5
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7
8
9
0
1
2
3
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7
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4
5
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0
96.2, 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,
3.5, 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;
3
+
+
+
ESIꢀMS: 831 [M+H] , 853 [M+Na] ; HRMS: calculated for C H N O SNa [M + Na]
4
4
54
4
10
853.3458, found 853.3474.
Compounds 4e. The title compound was obtained in 63 % yield as a white solid: m.p.
ꢀ1 1
116ꢀ118 °C; IR (KBr): 3128, 1735, 1685, 1662, 1617, 1553, 1400 cm ; H NMR (300 M
Hz, CDCl , 25 °C, TMS): δ 8.07ꢀ8.05 (m, 3H, 2 × ArH, C ꢀH), 7.79 (t, J = 7.5 Hz, 1H,
3
1
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,
C ꢀH), 4.53 (t, J = 6.0 Hz, 2H, CH O), 4.29 (t, J = 6.0 Hz, 2H, CH O), 3.41ꢀ3.29 (m, 2H,
11
2
2
NHCH ), 3.06 (d, J = 4.2 Hz, 1H, C ꢀH), 2.91ꢀ2.87 (m, 1H, C ꢀH), 2.39 (t, J = 7.1 Hz,
2
13
18
2
H, CH COO), 2.25ꢀ2,20 (m, 2H, OCH CH CH O), 1.47 (s, 3H, CH ), 1.33 (s, 3H, CH ),
2 2 2 2 3 3
1.25 (s, 3H, CH ), 1.16 (s, 3H, CH ), 1.01 (s, 3H, CH ), 0.99 (s, 3H, CH ), 0.90 (s, 3H,
3
3
3
3
13
CH ) ppm; C NMR (75 M Hz, CDCl , 25 °C, TMS): δ 199.0, 196.6, 177.3, 173.2, 168.7,
3
3
1
66.0, 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,
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+
3
0.6, 29.7, 27.9, 27.8, 26.9, 24.8, 24.8, 23.1, 22.9, 21.7, 21.5, 18.2; ESIꢀMS: 859 [M+H] ,
+
+
881 [M+Na] ; HRMS: calculated for C H N O SNa [M + Na] 881.3771, found
4
6
58
4
10
8
81.3787.
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11
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17
18
19
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22
23
24
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Compounds 4f. The title compound was obtained in 58 % yield as a white solid: m.p.
ꢀ1
1
1
22ꢀ124 °C; IR (KBr): 3128, 1743, 1689, 1664, 1613, 1553, 1398 cm ; H NMR (300 M
Hz, CDCl , 25 °C, TMS): δ 8.07ꢀ8.04 (m, 3H, 2 × ArH, C ꢀH), 7.77 (t, J = 7.5 Hz, 1H,
3
1
ArH), 7.63 (t, J = 7.8 Hz, 2H, 2 × ArH), 5.94 (s, 1H, C ꢀH), 4.53ꢀ4.51 (m, 3H, CH O,
1
1
2
NCHCOO), 4.16ꢀ4.06 (m, 2H, CH O), 3.83ꢀ3.81 (m, 2H, NCH ), 3.26ꢀ3.22 (m, 1H,
2
2
C ꢀH), 3.07ꢀ3.05 (m, 1H, C ꢀH), 1.46 (s, 3H, CH ), 1.34 (s, 3H, CH ), 1.26 (s, 3H, CH ),
13
18
3
3
3
1
3
1.15 (s, 3H, CH ), 1.01 (s, 3H, CH ), 0.91 (s, 3H, CH ), 0.88 (s, 3H, CH ) ppm; C NMR
3
3
3
3
(
75 M Hz, CDCl , 25 °C, TMS): δ 198.7, 196.2, 175.3, 172.2, 168.3, 165.5, 158.3, 137.5,
3
1
35.2, 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,
+
+
2
7.7, 27.1, 26.2, 24.6, 22.9, 22.2, 21.9, 21.0, 17.7; ESIꢀMS: 871 [M+H] , 893 [M+Na] ;
+
HRMS: calculated for C H N O SNa [M + Na] 893.3771, found 893.3790.
4
7
58
4
10
Compounds 4g. The title compound was obtained in 70 % yield as a white solid: m.p.
ꢀ1
1
1
10ꢀ112 °C; IR (KBr): 3130, 1744, 1685, 1662, 1616, 1549, 1400 cm ; H NMR (300 M
Hz, CDCl , 25 °C, TMS): δ 8.13ꢀ8.04 (m, 3H, 2 × ArH, C ꢀH), 7.78 (t, J = 7.0 Hz, 1H,
3
1
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