Synthesis and evaluation of nitric oxide-releasing derivatives of farnesylthiosalicylic acid as anti-tumor agents

Article abstract of DOI:10.1016/j.bmc.2010.03.077

Novel furoxan-based nitric oxide (NO)-releasing derivatives (11a-p) of farnesylthiosalicylic acid (FTA) were synthesized. Compounds 11d, 11f, 11k, and 11m-o displayed anti-tumor activities superior to FTA and sorafenib in most cancer cells tested. Analysis of six compounds revealed that 11d, 11f, 11n, 11o, and 11p, but not 11a that had low anti-tumor activity, produced high levels of NO, associated with their strong anti-tumor activity. Furthermore, the anti-tumor activity of 11f was partially mimicked by the furoxan moiety, but reduced by pre-treatment with hemoglobin. Importantly, treatment with 11f inhibited Ras-related signaling in cancer cells. Apparently, the high anti-tumor activity of 11f was attributed to the synergic effect of high levels of NO production and inhibition of Ras-related signaling in cancer cells. Our findings suggest that the furoxan/FTA hybrids may hold greater promise as therapeutic agents for the intervention of human cancers.

Full text of DOI:10.1016/j.bmc.2010.03.077

Bioorganic & Medicinal Chemistry 18 (2010) 3448–3456
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of nitric oxide-releasing derivatives
of farnesylthiosalicylic acid as anti-tumor agents
a,
a
a
c
Yong Ling a, , Xiaolei Ye b, , Hui Ji b, , Yihua Zhang , Yisheng Lai , Sixun Peng , Jide Tian
*
*
a Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
b Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, PR China
c Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA 90095, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel furoxan-basednitricoxide (NO)-releasing derivatives (11a–p)of farnesylthiosalicylic acid (FTA)were
synthesized. Compounds 11d, 11f, 11k, and 11m–o displayed anti-tumor activities superior to FTA and
sorafenib in most cancer cells tested. Analysis of six compounds revealed that 11d, 11f, 11n, 11o, and
11p, but not 11a that had low anti-tumor activity, produced high levels of NO, associated with their strong
anti-tumor activity. Furthermore, the anti-tumor activity of 11f was partially mimicked by the furoxan moi-
ety, but reduced by pre-treatment with hemoglobin. Importantly, treatment with 11f inhibited Ras-related
signaling in cancer cells. Apparently, the high anti-tumor activity of 11f was attributed to the synergic effect
of high levels of NO production and inhibition of Ras-related signaling in cancer cells. Our findings suggest
that the furoxan/FTA hybrids may hold greater promise as therapeutic agents for the intervention of human
cancers.
Received 15 January 2010
Revised 28 March 2010
Accepted 31 March 2010
Available online 3 April 2010
Keywords:
NO-donor
Farnesylthiosalicylic acid
Furoxans
NO-releasing
Ó 2010 Elsevier Ltd. All rights reserved.
Anti-tumor agents
Cytotoxic activities
Ras-related signaling
1. Introduction
Ras inhibitors with potent anti-tumor activity should be of great
significance.
Ras proteins are membrane-anchored guanine-nucleotide bind-
ing proteins, and function as biological switches that can mediate
signal transduction between G-protein coupled receptors and
downstream events such as MAPK or Akt.1,2 The activation of
MAPK or Akt signaling regulates cell proliferation, migration, dif-
ferentiation, and apoptosis.3–7 Notably, the mutations of the Ras
genes usually result in the constitutive activation of Ras-GTP pro-
teins and overstimulation of downstream signaling, promoting
the development and progression of tumors in human.8 Hence,
the Ras-related signaling events are the therapeutic targets for
intervention of human cancers.
Many efforts to regulate the Ras-related signal pathway have
been made to inhibit cancer progression and some new drugs have
been developed. Farnesylthiosalicylic acid (FTA) is a novel Ras inhib-
itor by dislodging all isoforms of Ras from galectin membrane bind-
ing sites.9,10 FTA has been used for the treatment of a wide range of
malignancies, including pancreatic, and lung cancers.11–13 However,
its therapeutic efficacy is limited. Therefore, development of new
Nitric oxide (NO) is naturally generated from L-arginine by the
action of NO synthase (NOS) and a key signaling molecule involved
in regulating the numerous physiologic and pathologic processes.14
High levels of NO and its metabolic derivatives, the reactive nitro-
gen species (RNS) and reactive oxygen species (ROS), can modify
functional proteins by S-nitrosylation, nitration, and disulfide for-
mation, leading to bio-regulation, inactivation, and cytotoxicity,
particularly in tumor cells.15 Indeed, previous studies have shown
that synthesized NO-releasing compounds have strong cytotoxicity
against human carcinoma cells in vitro and inhibit cancer growth
and metastasis in vivo.16–19 Furoxans are thermally stable com-
pounds and represent an important class of NO-donors, which
can produce high levels of NO in vitro, and inhibit the growth of tu-
mors in vivo.18,20,21 Accordingly, we hypothesized that new types
of synthesized furoxan/FTA hybrids could have Ras inhibitory
activity and produce high levels of NO, leading to potent cytotox-
icity against broad types of cancer cells.
Given that amino acid-coupling compounds can help in deliver-
ing the compounds to tumor cells,22 a total of 16 target compounds
(11a–p) were designed by coupling the carboxyl group of FTA with
phenylsulfonyl-substituted furoxan through various amino acids.
Their anti-tumor activity, NO-releasing ability, and Ras inhibitory
effect were evaluated in vitro. Herein, we report the synthesis
and biological evaluation of the target compounds.
* Corresponding authors. Tel.: +86 25 86021369 (H.J.); tel./fax: +86 25 86635503
(Y.Z.).
E-mail addresses: huijicpu@163.com (H. Ji), zyhtgd@sohu.com, zyhtgd@hot-
mail.com (Y. Zhang).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.077

Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
3449
2.2. Biological evaluation
2. Results and discussion
2.1. Chemistry
The cytotoxicity of individual compounds against human glio-
blastoma cells (U87, U251), human breast cancer cells (MCF-7),
human breast adenocarcinoma cells (MDA-MB-231), and human
gastric cancer cells (SGC-7901) was evaluated by MTT assay using
FTA and sorafenib as positive controls. The IC50 values of individual
compounds against each tumor cell line are presented in Table 1.
Twelve out of 16 compounds displayed cytotoxicity superior to
FTA, some even stronger than that of sorafenib. For example, the
anti-tumor activity of 11n was 50-fold more potent than FTA
The synthetic routes of 11a–p are outlined in Scheme 1. The
substituted furoxans were prepared in a four-step sequence. The
starting material benzenethiol (2) was converted to 2-(phenylthio)
acetic acid (3) by treatment with chloroacetic acid in 97% yield. Com-
pound 3 was oxidized by 30% H2O2 aqueous solution to generate 2-
(phenylsulfonyl) acetic acid (4), and then treated with fuming HNO3
to produce diphenylsulfonylfuroxan (5). Subsequently, 5 was trea-
ted with corresponding diol (butane-1, 3-diol, butane-1, 4-diol,
2-butyne-1, 4-diol or 2,20-oxydiethanol) to form various mono-
phenylsulfonylfuroxans (6a–d) with the yields of 57–73%. Further-
(IC50: 1.07 vs 51.22
lM) and fourfold than sorafenib (IC50: 1.07
vs 4.77 M) against MDA-MB-231 cells. Furthermore, those active
l
hybrids had more potent cytotoxicity against cancer cells than that
of their relevant NO-donors. For example, the IC50 values of 11f,
11g, 11k, 11m–p in MDA-MB-231 cells were much smaller than
more, glycocine(7a), L-phenylalanine(7b), L-alanine(7c)orL-proline
(7d) was treated with di-tert-butyl dicarbonate in the presence of
Et3N and t-BuOH at room temperature to give N-Boc amino acids
(8a–d) in 62–78% yields, respectively. These N-protected amino
acids were reacted with each substituted furoxans (6a–d) in CH2Cl2
in the presence of dicyclohexylcarbodiimide (DCC) and 4-dimethyl-
aminopyridine (DMAP) afforded esters (9a–p) in 57–77% yields. The
N-protected groups of 9a–p were removed by CF3COOH to provide
the intermediates (10a–p), which were directly treated with 1 in
CH2Cl2 in the presence of 1-(3-dimethyl aminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDCI) and DMAP to generate the target
compounds (11a–p) in yields of 46–58%, respectively. The final
products were purified by column chromatography and their struc-
tures were characterized by IR, 1H NMR, MS, and elemental analyses.
that of 6a (15.32
lM), 6b (9.87 lM), 6c (20.48 lM), and 6d
(6.97 M), respectively.
l
Furthermore, we tested whether the levels of NO production by
the indicated compounds in each type of human cancer cells were
associated with their cytotoxicities. MCF-7, MDA-MB-231, U251,
U87, and SGC-7901 cells were exposed to each compound
(100 lM) for varying durations (30–300 min). The levels of
nitrite/nitrate produced in the cell lysates of different types of cells
were characterized using the Griess assay (Fig. 1). As expected,
treatment with FTA resulted in little nitrite/nitrate in any of the
tested cells. In contrast, treatment with a single furoxan/FTA
hybrid promoted comparable levels of nitrite/nitrate production
S
COOH
1
PhO2S
O
PhO2S
O
SO2Ph
ORH
SH
R"
SCH2COOH
SO2CH2COOH
N
N
N
N
O
5
O
3
2
4
6a-d
R"
O
R"
R"
O
N
N
N
N
R'
N
H
R
Boc
Boc
R
O
R'
O
O
N
COOH
N
O
N
H
COOH
O
SO2Ph
R'
R'
O
SO2Ph
7a-d
8a-d
9a-p
10a-p
S
N
O
7-8a. R'= H
R"= H
6a.
6b.
6c.
6d.
R=
R=
R=
R=
O
O
O
O
O
R'= H
7-8b.
7-8c.
R"= CH3
R"= CH2Ph
SO2Ph
O
R'= H
R'
R
O
N
O
N
R"
7-8d.
R"
N
R'
=
O
N
11a-p
O
O
O
O
O
O
O
R'= H
R'= H
R'= H
R'= H
R"= CH2Ph
9-11a.
9-11b. R=
9-11i. R=
R"= CH2Ph
R"= CH2Ph
R"= CH3
R=
R'= H
R'= H
R'= H
R'= H
9-11j.
R"= H
R"= H
R"= H
R=
O
O
O
9-11k.
R=
9-11c.
R=
R"= CH2Ph
9-11l. R=
9-11d. R=
O
N
R"
R"= CH3
R'= H
=
=
9-11m. R=
9-11e.
9-11f.
R=
R=
R'
N
N
N
N
R"
R"
O
O
O
O
R'= H
R"= CH3
9-11n.
R=
R'
O
N
R"
N
O
O
9-11g. R=
9-11h. R=
=
9-11o. R=
9-11p. R=
R'
=
R'
O
O
N
R"= CH3
R'= H
R"= H
R'= H
Scheme 1. General method for the synthesis of 11a–p. Reagents and conditions: (a) ClCH2COOH, NaOH (aq), 140 °C, 2 h; (b) 30% H2O2, AcOH, rt, 3 h; (c) fuming HNO3, 90 °C,
4 h; (d) diol, THF, 30% NaOH, rt, 4–8 h; (e) (Boc)2O, t-BuOH, 2.5% NaOH, rt, 6–12 h; (f) 6a–d, DCC, DMAP, CH2Cl2, rt, 6–12 h; (g) CF3COOH, rt, 2 h; (h) 1, EDCI, DMAP, CH2Cl2, rt,
24 h.

3450
Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
Table 1
trite were determined by the MTT and Griess assays, respectively
IC50 values of 11a–p against five human cancer cell lines
(Fig. 2). Treatment with 11f alone greatly inhibited the growth of
MDA-MB-231 cells and treatment with 6a alone mimicked the par-
tial inhibition of 11f on the growth of MDA-MB-231 cells, which
were associated with various levels of nitrate/nitrite. Furthermore,
the inhibitory effects of 11f or 6a on the growth of MDA-MB-231
cells were dramatically reduced by pre-treatment with hemoglo-
bin, which appeared to be dose-dependent. Similar patterns of re-
duced levels of nitrate/nitrite in the cell lysates were observed.
These data clearly demonstrated that high levels of NO production
by the compound contributed to its cytotoxicity against human
cancer cells in vitro.
Finally, we examined whether the furoxan/FTA hybrids could
still retain the Ras inhibitor activity. MDA-MB-231 cells were trea-
ted with vehicle control or different doses of 11f and the expres-
sion and activation of the Ras-related signal events, Akt, ERK, and
Raf were determined by immunoblotting assays (Fig. 3). Obviously,
Compound
In vitro cytotoxicity (IC50, l
M)a
MCF-7
MDA-MB-231
U251
U87
SGC-7901
FTA
Sorafenib
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
46.75
8.83
>50
51.22
4.77
>50
46.91
24.71
>50
53.95
21.07
>50
37.74
10.37
>50
11.91
>50
11.37
>50
11.27
22.53
44.57
>50
>50
11.37
>50
13.93
7.35
7.86
9.11
25.06
6.91
11.14
7.68
7.47
8.68
>50
>50
5.03
>50
6.50
5.49
8.77
6.59
7.26
9.99
7.23
5.05
4.21
3.14
>50
10.78
28.22
11.72
15.32
8.04
6.99
7.95
>50
15.35
>50
14.82
20.38
18.08
9.00
26.36
>50
>50
>50
>50
>50
1.01
>50
7.31
>50
5.18
>50
11m
11n
11o
11p
1.54
1.07
3.16
2.41
7.93
3.56
4.83
8.58
8.47
13.49
12.92
7.34
treatment with 5
lM 11f significantly reduced the expression of
Akt, and ERK in our experimental condition. Importantly, treat-
ment with 11f dramatically inhibited the activation of Akt, ERK,
and Raf. Evidentially, the levels of phosphor-Akt, -ERK, and -Raf
19.50
a
The cytotoxicity of individual compounds against each of the cancer cell lines
was determined by the MTT assay and expressed as the IC50 (a dose achieved 50%
inhibition in the growth of cancer cells cultured.
were almost abolished in the cells treated with 5
lM 11f. However,
treatment with 5 M of FTA did not effectively inhibit the Ras sig-
l
naling (data not shown), similar to a previous report.23 These data
suggest that high levels of NO produced by 11f may contribute to
the low concentration of 11f-mediated inhibition of Ras signal-
ing.24 Given that the Ras-related MAPK and Akt signal pathways
are associated with the growth and anti-apoptosis of many types
of cells the significantly reduced activation of these pathways by
11f should increase the susceptibility of cells to cytotoxic triggers,
contributing to the cytotoxicity of the furoxan/FTA hybrids.
Analysis of structure–activity relationships revealed that 11a–p
with different amino acid linkers had tremendous difference in their
cytotoxicities. The compounds (11g, 11m, 11n, and 11o) linked with
praline had better anticancer activities, while the compounds (11a,
11i, 11j, and 11l) containing phenylalanine had little cytotoxicity.
The cytotoxicity of different hybrids with varying amino acid linkers
was as following sequence: proline>alanine>glycine>phenylala-
nine. The lack of cytotoxicity against human cancer cells may be
due to the low levels of NO production. Evidentially, treatment with
11a that had little cytotoxicity produced significantly lower levels of
NO in MDA-MB-231 cells, as compared with that by high cytotoxic
11f. Furthermore, the low cytotoxicity may also stem from low effi-
cacy of the compounds in entering to the cells and/or loss of the Ras
inhibitor activity. We are interested in further determining the SAR
of furoxan/FTA hybrids, which will provide a new insight into design
of novel anticancer drugs.
in different types of cancer cells while treatment with the tested
furoxan/FTA hybrids produced significantly variable levels of ni-
trate/nitrite in any of the cancer cells tested. Treatment with low
cytotoxic compound 11a produced lower levels of nitrate/nitrite
in all of the tested cells. However, treatment with any of the com-
pounds (11d, 11f, 11n, 11o or 11p) with a high cytotoxicity pro-
duced significantly higher levels of nitrate/nitrite in these cells.
Apparently, the levels of nitrate/nitrite produced by individual
compounds were associated with their cytotoxicities against these
cancer cells in vitro.
To further determine the contribution of NO to the cytotoxicity
of furoxan/FTA hybrids, 11f was selected for testing its cytotoxic-
ity, as compared with its furoxan moiety in the presence or
absence of NO scavenger hemoglobin. MDA-MB-231 cells were
pre-treated with, or without, different concentrations of hemoglo-
bin for 1 h and then treated with the indicated concentrations of
11f or furoxan moiety 6a. The effects of different treatments on
the growth of MDA-MB-231 cells and the production of nitrate/ni-
15
11a
11d
11f
11n
11o
10
3. Conclusion
11p
FTA
5
In summary, we have synthesized 16 novel furoxan-based
NO-releasing derivatives of FTA using various amino acids as
chemical linkers and evaluated their cytotoxicities against human
breast, glioblastoma, and gastric cancer cells in vitro. We found
that most compounds displayed strong cytotoxicity against these
cancer cells, particularly for compounds 11f, 11n, and 11o, which
had a great potency superior to FTA and sorafenib. Furthermore,
the compounds with strong cytotoxicity against human cancer
cells produced high levels of NO in these cancer cells. In addition,
the cytotoxicity of 11f was partially mimicked by furoxan moiety
6a, but reduced by NO scavenger hemoglobin in a dose-dependent
manner. Moreover, compound 11f had strong Ras inhibitory activ-
ity and inhibited the expression and activation of the Ras-related
Akt and Erk signaling in human breast carcinoma cells. Apparently,
the synergic effect of both high levels of NO production and strong
0
1
5
01
U87
-231
79
U2
MCF7
C-
MB
-
SG
MDA
Figure 1. Variable levels of NO were produced by the compounds. Human cancer
cells were treated with each compound at 100 M and the contents of nitrate/
l
nitrite in the cell lysates were determined by Griess assay through the duration of
30–300 min. The individual values were determined by measuring absorbance at
540 nm, and calculated according to the standard curve. Data shown are the mean
value of two experiments for each compound at 240 min post-treatment in
individual types of cells, and similar patterns of nitrate/nitrite production in these
cells were observed at other experimental time points (data not shown).

Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
3451
Figure 2. NO produced by the compound 11f contributed to its cytotoxicity. MDA-MB-231 cells were pretreated with, or without, the indicated concentrations of hemoglobin
for 1 h and treated with 5 or 10 M of 11f or furoxan moiety 6a for 24 h. The cell growth and nitrate/nitrite production were determined by the MTT and Griess assays,
l
respectively. Data are expressed as mean% of inhibition on the growth of MDA-MB-231 cells and mean values of the levels of nitrate/nitrite from three independent
experiments. The intra-group and inter-experimental variations were less than 10% and the cells treated with 1% Triton x-100 were used as positive controls and considered
as 100%. The cells treated with different concentrations of hemoglobin alone did not affect their growth (data not shown).
a JASCO P-1020 polarimeter (cell length: 100 mm). Infrared (IR)
spectra (KBr) were recorded on a Nicolet Impact 410 instrument
(KBr pellet). 1H NMR spectra were recorded with a Bruker Avance
300 MHz spectrometer at 300 K, using TMS as an internal standard.
MS spectra were recorded on a Shimadzu GC–MS 2010 (EI) or a
Mariner Mass Spectrum (ESI). Element analysis was performed
on an Eager 300 instrument. All compounds were routinely
checked by TLC and 1H NMR. TLCs and preparative thin-layer chro-
matography were performed on silica gel GF/UV 254, and the chro-
matograms were conducted on silica gel (200–300 mesh) and
visualized under UV light at 254 and 365 nm. All solvents were re-
agent grade and, when necessary, were purified and dried by stan-
dards methods. Solutions after reactions and extractions were
concentrated using a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Organic solutions were dried over anhy-
drous sodium sulfate. Compounds 1, 2, and 7 were commercially
available. Compounds 3–6 were synthesized according to the re-
ported procedure.26
Figure 3. Effect of 11f on the Ras-related signaling. MDA-MB-231 cells at 1.5 ꢀ 105/
mL were treated with 1 or 5 lM 11f or vehicle control for 8 h. After harvested and
4.1. Synthesis
lyzed, the cell lysates were separated by SDS–PAGE and probed with anti-Akt, anti-
phospho-Akt (Ser473), anti-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-Phos-
pho-Raf (Ser259) and anti-b-actin antibodies (Cell Signaling, Boston), respectively.
The immunoblotting was visualized by HRP-conjugated second antibodies and
enhanced chemiluminescent. Data shown are a representative of two separated
experiments.
4.1.1. Synthesis of N-Boc amino acids (8)
4.1.1.1. 2-(tert-Butoxycarbonylamino) acetic acid (8a). To a
solution of 7a (496 mg, 6.62 mmol) and 2.5% NaOH (12 mL) in
t-BuOH (12 mL) at 0 °C was added Boc2O (2.2 mL, 7.98 mmol) by
slow dropwise. The solution was allowed to warm to room temper-
ature and stirred for 12 h. The solvent was removed under reduced
pressure. The crude residue was dissolved in water and washed with
ether (3 ꢀ 10 mL). The water layer was acidified with 1 M HCl to pH
2, and then extracted with AcOEt (3 ꢀ 12 mL). The organic extracts
were combined, dried with MgSO4, filtered, and concentrated to af-
ford 8a as white solid (904 mg, 78%), mp 88–90 °C [lit.25 88 °C].
Ras inhibitor activity contributed to the potent cytotoxicity of
these furoxan/FTA hybrids. Therefore, our novel findings provide
a proof of principle in design of new NO-releasing furoxan/FTA hy-
brids for the intervention of human cancers. We are interested in
further investigating the effect of these hybrids on the growth of
human cancer cells in vivo and the mechanisms underlying the ac-
tion of these hybrids in inhibition of human tumorigenesis.
4.1.1.2.
(S)-2-(tert-Butoxycarbonylamino) propanoic acid
(8b). Compound 8b was synthesized from 7b (491 mg,
5.52 mmol), 2.5% NaOH (12 mL), and Boc2O (2 mL, 7.25 mmol),
according to the synthetic procedure of 8a in a yield of 74%, white
solid 770 mg, mp 80–83 °C [lit.25 82 °C].
4. Experiment
Melting points were determined on a Mel-TEMP II melting point
apparatus and uncorrected. Optical rotations were measured with

3452
Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
4.1.1.3. (S)-2-(tert-Butoxycarbonylamino)-3-phenyl propanoic
acid (8c). Compound 8c was synthesized from 7c (956 mg,
5.80 mmol), 2.5% NaOH (12 mL), and Boc2O (2 mL, 7.25 mmol),
according to the synthetic procedure of 8a in a yield of 63%, white so-
lid 968 mg, mp 87–89 °C [lit.25 87 °C].
9a in yield 71%, white solid 689 mg, mp 110–112 °C [lit.25
110 °C]. MS (ESI) m/z = 486 [M+1]+.
4.1.2.6. (S)-3-(Phenylsulfonyl)-4-(2,2,6-trimethyl-4,7-dioxo-3,8,
11-trioxa-5-azatridecan-13-yloxy)-1,2,5-oxadiazole-2-oxide (9f).
Compound 9f was synthesized from 8b (378 mg, 2.00 mmol) and 6c
(660 mg, 2.00 mmol) according to the synthetic procedure of 9a in
yield 74%, white solid 741 mg, mp 127–129 °C [lit.26 128 °C]. MS
(ESI) m/z = 502 [M+1]+.
4.1.1.4.(S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxylic acid
(8d). Compound8d was synthesized from 7d (634 mg, 5.52 mmol),
2.5% NaOH (12 mL), and Boc2O (2 mL, 7.25 mmol), according to the
synthetic procedure of 8a in a yield of 62%, white solid 735 mg, mp
135–137 °C [lit.25 136 °C].
4.1.2.7. (S)-4-(4-(1-(tert-Butoxycarbonyl)pyrrolidine-2-carbon-
yloxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (9g).
Compound 9g was synthesized from 8d (430 mg, 2.00 mmol) and
6b (628 mg, 2.00 mmol) according to the synthetic procedure of 9a
in yield 67%, colorless viscous liquid 685 mg, Analytical data for
4.1.2. Synthesis of mono-phenylsulfonylfuroxans N-Boc amino
ester (9)
4.1.2.1. 4-(3-((S)-2-(tert-Butoxycarbonylamino)-3-phenylpropa-
noyloxy) butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(9a). To a solution of 8c (530 mg, 2.00 mmol) in dry CH2Cl2
(40 mL), 6a (628 mg, 2.00 mmol), DCC (415 mg, 2.00 mmol), and
DMAP (317 mg, 2.60 mmol) was added and the mixture was stirred
at room temperature for 12 h. After filtration, the filtrate was evap-
orated to dryness in vacuo, and the crude product was purified by
column chromatography (PE/EtOAc = 10:1–5:1) to yield 9a as a
white solid (864 mg, 77%), mp 120–122 °C. Analytical data for 9a:
9g: ½a 2D6
ꢁ
ꢂ12.7; 1H NMR (CDCl3, 300 MHz): d 8.04 (d, 2H, J = 7.5 Hz,
Ar-H), 7.74 (m, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 4.45 (m, 1H, NCH),
4.22 (m, 4H, 2 ꢀ OCH2), 3.48 (m, 2H, NCH2), 1.94–1.99 (m, 6H,
NCHCH2, OCH2CH2CH2CH2OOC), 1.68 (m, 2H, NCH2CH2), 1.45–1.51
(m, 9H, 3 ꢀ CH3); MS (ESI) m/z = 512 [M+1]+; Anal. Calcd for
C22H29N3O9S: C, 51.65; H, 5.71; N, 8.21. Found: C, 51.26; H, 5.68; N,
7.86.
4.1.2.8. (S)-4-(4-(2-(tert-Butoxycarbonylamino) propanoyloxy)
½
a 2D6
ꢁ
ꢂ3.7; 1H NMR (CDCl3, 300 MHz): d 8.05 (d, 2H, J = 7.8 Hz,
butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(9h).
Ar-H), 7.74 (m, 1H, Ar-H), 7.62 (m, 2H, Ar-H), 7.28 (m, 1H, Ar-H),
7.22 (m, 2H, Ar-H), 7.15 (m, 2H, Ar-H), 5.12 (m, 1H, NCH), 4.51
(m, 1H, COOCH), 4.32 (m, 2H, OCH2CH2) 3.05 (d, 2H, J = 6.0 Hz,
NHCHCH2), 2.07 (m, 2H, COOCHCH2), 1.30–1.41 (m, 12H,
4 ꢀ CH3); MS (ESI) m/z = 562 [M+1]+; Anal. Calcd for C26H31N3O9S:
C, 55.60; H, 5.56; N, 7.48. Found: C, 55.87; H, 5.70; N, 7.23.
Compound 9h was synthesized from 8b (378 mg, 2.00 mmol) and
6b (628 mg, 2.00 mmol) according to the synthetic procedure of 9a
in yield 72%, white solid 698 mg, mp 124–126 °C [lit.26 124 °C]. MS
(ESI) m/z = 486 [M+1]+.
4.1.2.9. (S)-4-(4-(2-(tert-Butoxycarbonylamino)-3-phenylpropa-
noyloxy)but-2-ynyloxy)-3-(phenyl sulfonyl)-1,2,5-oxadiazole-2-
oxide (9i). Compound 9i was synthesized from 8c (530 mg,
2.00 mmol) and 6d (620 mg, 2.00 mmol) according to the synthetic
procedure of 9a in yield 68%, colorlessviscous liquid 758 mg. Analyt-
4.1.2.2. 4-(3-(2-(tert-Butoxycarbonylamino) acetoxy) butoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (9b). Compound 9b
was synthesized from 8a (350 mg, 2.00 mmol) and 6a (628 mg,
2.00 mmol) according to the synthetic procedure of 9a in yield
72%, white solid 678 mg, mp 195–197 °C [lit.26 196 °C]. MS (ESI)
m/z = 472 [M+1]+.
ical data for 9i: ½a D25
ꢁ
ꢂ6.6; 1H NMR (CDCl3, 300 MHz): d 8.06 (d, 2H,
J = 8.4 Hz, Ar-H), 7.76 (m, 1H, Ar-H), 7.62 (m, 2H, Ar-H), 7.31 (m,
3H, Ar-H), 7.14 (d, 2H, J = 7.2 Hz, Ar-H), 5.11 (s, 2H, COOCH2), 4.61–
4.95 (m, 3H, CH2OC@N, NCH), 3.11 (m, 2H, NHCH2), 2.07 (m, 2H,
COOCHCH2), 1.41 (m, 9H, 3 ꢀ CH3); MS (ESI) m/z = 558 [M+1]+; Anal.
Calcd for C26H27N3O9S: C, 56.01; H, 4.88; N, 7.54. Found: C, 56.22; H,
5.02; N, 7.31.
4.1.2.3. 4-(4-(2-(tert-Butoxycarbonylamino) acetoxy) butoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (9c). Compound 9c
was synthesized from 8a (350 mg, 2.00 mmol) and 6b (628 mg,
2.00 mmol) according to the synthetic procedure of 9a in yield
74%, white solid 697 mg, mp 60–62 °C. Analytical data for 9c: 1H
NMR (CDCl3, 300 MHz): d 8.04 (d, 2H, J = 7.5 Hz, Ar-H), 7.77 (m,
1H, Ar-H), 7.61 (m, 2H, Ar-H), 5.06 (br s, 1H, –NH–), 4.44 (t, 2H,
J = 6.0 Hz, COOCH2), 4.23 (t, 2H, J = 6.0 Hz, CH2OC@N), 3.92–3.94
(s, 2H, NCH2), 1.95–1.99 (m, 2H, COOCH2CH2CH2), 1.85–1.87 (m,
2H, COOCH2CH2), 1.45 (s, 9H, 3 ꢀ CH3); MS (ESI) m/z = 472
[M+1]+; Anal. Calcd for C19H25N3O9S: C, 48.40; H, 5.34; N, 8.91.
Found: C, 48.22; H, 5.39; N, 8.78.
4.1.2.10. (S)-4-(4-(2-(tert-Butoxycarbonylamino)-3-phenylprop-
anoyloxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(9j). Compound 9j was synthesized from 8c (530 mg, 2.00 mmol)
and 6b (628 mg, 2.00 mmol) according to the synthetic procedure
of 9a in yield 65%, white solid 729 mg, mp 130–132 °C [lit.26
130 °C]. MS (ESI) m/z = 562 [M+1]+.
4.1.2.11. (S)-4-(4-(2-(tert-Butoxycarbonylamino) propanoyloxy)
but-2-ynyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (9k).
Compound9k was synthesized from 8b (378 mg, 2.00 mmol) and 6d
(620 mg, 2.00 mmol) according to the synthetic procedure of 9a in
yield 61%, white solid 587 mg, mp 135–137 °C [lit.26 135 °C]. MS
(ESI) m/z = 482 [M+1]+.
4.1.2.4. 4-(4-(2-(tert-Butoxycarbonylamino) acetoxy) but-2-
ynyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(9d).
Compound 9d was synthesized from 8a (350 mg, 2.00 mmol) and
6d (620 mg, 2.00 mmol) according to the synthetic procedure of 9a
in yield 66%, white solid 812 mg, mp 88–90 °C. Analytical data for
9d: 1H NMR (CDCl3, 300 MHz): d 8.03 (d, 2H, J = 7.5 Hz, Ar-H), 7.88
(m, 1H, Ar-H), 7.73 (m, 2H, Ar-H), 7.28 (br s, 1H, –NH–), 5.22 (s, 2H,
COOCH2), 4.88 (s, 2H, OCH2), 3.70–3.75 (m, 2H, NCH2), 1.38 (m, 9H,
3 ꢀ CH3); MS (ESI) m/z = 468 [M+1]+; Anal. Calcd for C19H21N3O9S:
C, 48.82; H, 4.53; N, 8.99. Found: C, 48.58; H, 4.64; N, 8.81.
4.1.2.12. (S)-4-(6-Benzyl-2,2-dimethyl-4,7-dioxo-3,8,11-trioxa-
5-azatridecan-13-yloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-
oxide (9l). Compound 9l was synthesized from 8c (530 mg,
2.00 mmol) and 6c (660 mg, 2.00 mmol) according to the synthetic
procedure of 9a in yield 67%, white solid 773 mg, mp 107–109 °C
[lit.26 108 °C]. MS (ESI) m/z = 578 [M+1]+.
4.1.2.13. 4-(3-((S)-1-(tert-Butoxycarbonyl) pyrrolidine-2-carbon
yloxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (9m).
Compound 9m was synthesized from 8d (430 mg, 2.00 mmol) and 6a
(628 mg, 2.00 mmol) according to the synthetic procedure of 9a in
4.1.2.5. 4-(3-((S)-2-(tert-Butoxycarbonylamino) propanoyloxy)
butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(9e).
Compound 9e was synthesized from 8b (378 mg, 2.00 mmol) and
6a (628 mg, 2.00 mmol) according to the synthetic procedure of

Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
3453
yield 65%, colorless viscous liquid 664 mg. Analytical data for 9m:
Ar-H), 7.20–7.37 (m, 6H, Ar-H), 5.18 (t, 1H, J = 8.1 Hz, SCH2CH),
5.08 (m, 3H, 2 ꢀ CH2CH@CCH3, NCH), 4.00 (m, 1H, OCH), 3.42 (t,
2H, J = 9.0 Hz, COOCH2CH2), 3.24 (m, 4H, SCH2, CH2Ph), 1.97–2.07
(m, 10H, 2 ꢀ CHCH2CH2CH, OCHCH2), 1.47–1.67 (m, 15H,
4 ꢀ CH@CCH3, CHCH3); MS (ESI) m/z = 802 [M+1]+; Anal. Calcd for
C43H51N3O8S2: C, 64.40; H, 6.41; N, 5.24. Found: C, 63.90; H, 6.36;
N, 5.07.
½
a 2D5
ꢁ
ꢂ20.4; 1H NMR (CDCl3, 300 MHz): d 8.06 (d, 2H, J = 7.5 Hz, Ar-
H), 7.75 (m, 1H, Ar-H), 7.62 (m, 2H, Ar-H), 4.46 (m, 2H, OCH, NCH),
4.23 (m, 2H, OCH2), 3.46 (m, 2H, NCH2), 1.86–1.91 (m, 4H, OCH2CH2,
NCHCH2), 1.50 (m, 2H, NCH2CH2), 1.25–1.45 (m, 12H, 4 ꢀ CH3); MS
(ESI) m/z = 512 [M+1]+; Anal. Calcd for C22H29N3O9S: C, 51.65; H,
5.71; N, 8.21. Found: C, 51.26; H, 6.18; N, 8.03.
4.1.2.14. (S)-4-(4-(1-(tert-Butoxycarbonyl) pyrrolidine-2-car-
bonyloxy)but-2-ynyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2
-oxide (9n). Compound 9n was synthesized from 8d (430 mg,
2.00 mmol) and 6d (620 mg, 2.00 mmol) according to the synthetic
procedure of 9a in yield 57%, colorless viscous liquid 578 mg. Ana-
4.1.3.2. 3-(Phenylsulfonyl)-4-(3-(2-(2-((2E,6E)-3,7,11-trimethy
ldodeca-2,6,10-trienylthio)benzamido) acetoxy) butoxy)-1,2,5-
oxadiazole-2-oxide (11b). Compound 11b was synthesized from
9b (377 mg, 0.80 mmol) and 1 (286 mg, 0.80 mmol) according to
the synthetic procedure of 11a in yield 51%, a white foam
290 mg. Analytical data for 11b: IR (KBr, cmꢂ1): 3424, 2927,
1753, 1629, 1552, 1450, 1371, 1168; 1H NMR (CDCl3, 300 MHz):
d 8.05 (d, 2H, J = 8.1 Hz, Ar-H), 7.70 (m, 1H, Ar-H), 7.58 (m, 2H,
Ar-H), 7.40 (m, 2H, Ar-H), 7.29 (m, 2H, Ar-H), 5.25 (t, 1H,
J = 6.6 Hz, SCH2CH), 5.06 (t, 2H, J = 6.6 Hz, 2 ꢀ CH2CH@CCH3), 4.30
(m, 2H, NCH2), 4.48 (t, 2H, J = 6.0 Hz, CH2O), 4.25 (m, 1H, OCH),
3.54 (d, 2H, J = 7.5 Hz, SCH2), 2.20 (m, 2H, OCHCH2), 1.98–2.05
(m, 8H, 2 ꢀ CHCH2CH2CH), 1.51–1.68 (m, 12H, 4 ꢀ CH@CCH3),
1.39 (d, 3H, J = 6.3 Hz, CHCH3); MS (ESI) m/z = 712 [M+1]+; Anal.
Calcd for C36H45N3O8S2: C, 60.74; H, 6.37; N, 5.90. Found: C,
60.41; H, 6.34; N, 5.76.
lytical data for 9n: ½a D24
ꢁ
ꢂ18.9; 1H NMR (CDCl3, 300 MHz): d 8.06
(d, 2H, J = 7.5 Hz, Ar-H), 7.75 (m, 1H, Ar-H), 7.62 (m, 2H, Ar-H),
5.09 (s, 2H, COOCH2), 4.78 (s, 2H, OCH2), 4.46 (m, 1H, NCH), 3.49
(m, 2H, NCH2), 1.98–2.00 (m, 2H, NCHCH2), 1.46 (m, 2H, NCH2CH2),
1.25–1.30 (m, 9H, 3 ꢀ CH3); MS (ESI) m/z = 508 [M+1]+; Anal. Calcd
for C22H25N3O9S: C, 52.06; H, 4.97; N, 8.28. Found: C, 51.83; H,
5.10; N, 8.12.
4.1.2.15. (S)-4-(2-(2-(1-(tert-Butoxycarbonyl) pyrrolidine-2-car-
bonyloxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-
2-oxide (9o). Compound 9o was synthesized from 8d (430 mg,
2.00 mmol) and 6c (660 mg, 2.00 mmol) according to the synthetic
procedure of 9a in yield 61%, colorless viscous liquid 643 mg. Ana-
4.1.3.3. 4-(Phenylsulfonyl)-3-(4-(2-(2-((2E,6E)-3,7,11-trimethy
ldodeca-2,6,10-trienylthio)benzamido) acetoxy)butoxy)-1,2,5-
oxadiazole-2-oxide (11c). Compound 11c was synthesized from
9c (377 mg, 0.80 mmol) and 1 (286 mg, 0.80 mmol) according to
the synthetic procedure of 11a in yield 55%, a white waxy solid
313 mg. Analytical data for 11c: IR (KBr, cmꢂ1): 3259, 2928,
1727, 1621, 1559, 1450, 1163; 1H NMR (CDCl3, 300 MHz): d 8.04
(d, 2H, J = 7.2 Hz, Ar-H), 7.73 (m, 2H, Ar-H), 7.58 (m, 2H, Ar-H),
7.36 (m, 2H, Ar-H), 7.27 (m, 1H, Ar-H), 5.24 (t, 1H, J = 7.5 Hz,
SCH2CH), 5.06 (t, 2H, J = 6.9 Hz, 2 ꢀ CH2CH@CCH3), 4.45 (t, 2H,
J = 6.0 Hz, COOCH2CH2CH2CH2O), 4.28 (m, 4H, NCH2, COOCH2CH2),
3.55 (d, 2H, J = 7.5 Hz, SCH2), 1.99 (m, 10H, 2 ꢀ CHCH2CH2CH,
COOCH2CH2CH2), 1.51–1.69 (m, 14H, 4 ꢀ CH@CCH3, COOCH2CH2);
MS (ESI) m/z = 712 [M+1]+; Anal. Calcd for C36H45N3O8S2: C,
60.74; H, 6.37; N, 5.90. Found: C, 60.50; H, 6.52; N, 5.92.
lytical data for 9i: ½a D25
ꢁ
ꢂ15.0; 1H NMR (CDCl3, 300 MHz): d 8.06 (d,
2H, J = 8.1 Hz, Ar-H), 7.66 (m, 2H, Ar-H), 7.56 (m, 1H, Ar-H), 4.56 (t,
2H, J = 4.5 Hz, COOCH2), 4.25 (m, 1H, NCH), 4.19 (t, 2H, J = 4.5 Hz,
CH2OC@N), 3.91 (t, 2H, J = 4.5 Hz, COOCH2CH2O), 3.79 (t, 2H,
J = 4.5 Hz, OCH2CH2OC@N), 3.50 (m, 2H, NCH2), 1.96 (m, 2H,
NCHCH2), 1.71 (m, 2H, NCH2CH2), 139–1.45 (m, 9H, 3 ꢀ CH3); MS
(ESI) m/z = 528 [M+1]+; Anal. Calcd for C22H29N3O10SꢃCH3COCH3:
C, 51.27; H, 6.02; N, 7.18. Found: C, 51.34; H, 5.98; N, 6.61.
4.1.2.16. 4-(2,2-Dimethyl-4,7-dioxo-3,8,11-trioxa-5-azatridec-
an-13-yloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (9p).
Compound 9p was synthesized from 8a (350 mg, 2.00 mmol) and
6c (660 mg, 2.00 mmol) according to the synthetic procedure of
9a in yield 72%, white solid 701 mg, mp 89–91 °C. 1H NMR (CDCl3,
300 MHz): d 8.03 (d, 2H, J = 7.5 Hz, Ar-H), 7.88 (m, 1H, Ar-H), 7.72
(m, 2H, Ar-H), 7.19 (br s, 1H, –NH–), 4.50 (t, 2H, J = 6.0 Hz,
CH2OC@N), 4.18–4.21 (t, 2H, J = 6.0 Hz, COOCH2), 3.79 (m, 2H,
OCH2CH2OC@N), 3.63–3.71 (m, 4H, COOCH2CH2, NCH2), 1.37 (m,
9H, 3 ꢀ CH3); MS (ESI) m/z = 488 [M+1]+; Anal. Calcd for
C19H25N3O10S: C, 46.81; H, 5.17; N, 8.62. Found: C, 46.55; H,
5.24; N, 8.76.
4.1.3.4. 3-(Phenylsulfonyl)-4-(4-(2-(2-((2E,6E)-3,7,11-trimethyl
dodeca-2,6,10-trienylthio)benzamido) acetoxy) but-2-ynyloxy)-
1,2,5-oxadiazole-2-oxide (11d). Compound 11d was synthesized
from 9d (374 mg, 0.80 mmol) and 1 (286 mg, 0.80 mmol) accord-
ing to the synthetic procedure of 11a in yield 47%, colorless viscous
liquid 266 mg. Analytical data for 11d: IR (KBr, cmꢂ1): 3418, 2928,
1758, 1617, 1548, 1451, 1361, 1169; 1H NMR (CDCl3-d6): d 8.06 (d,
2H, J = 7.5 Hz, Ar-H), 7.74 (m, 2H, Ar-H), 7.61 (t, 2H, J = 7.5 Hz, Ar-
H), 7.30 (m, 3H, Ar-H), 5.24 (m, H, SCH2CH), 5.06 (m, 4H,
2 ꢀ CH2CH2CH@CCH3, CH2O), 4.86 (s, 2H, COOCH2), 4.31 (d, 2H,
J = 5.4 Hz, NCH2), 3.55 (d, H, J = 7.8 Hz, SCH2), 1.98–2.12 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.42–1.67 (m, 12H, 4 ꢀ CH3); MS (ESI) m/
z = 708 [M+1]+; Anal. Calcd for C36H41N3O8S2: C, 61.08; H, 5.84;
N, 5.94. Found: C, 60.76; H, 5.81; N, 5.87.
4.1.3. Synthesis of furoxan/FTA hybrids (11)
4.1.3.1. 4-(3-((S)-3-Phenyl-2-(2-((2E,6E)-3,7,11-trimethyldod eca
-2,6,10-trienylthio)benzamido) propanoyloxy)butoxy)-3-(phen-
ylsulfonyl)-1,2,5-oxadiazole-2-oxide (11a).
A solution of 9a
(449 mg, 0.80 mmol) and CF3COOH (1 mL) in dry CH2Cl2 (4 mL)
was stirred at room temperature for 2 h. The solvent was removed
under reduced pressure. The crude residue was dissolved in 15 mL
dichloromethane and 2 mL Et3N was slowly added to the solvent,
and stirred at room temperature for 30 min. The salts were removed
by filtration over a pad of Celite. The filtrate was mixed with 1
(286 mg, 0.80 mmol), EDCI (153 mg, 0.8 mmol), DMAP (97 mg,
0.80 mmol). The mixture was stirred at room temperature for 24 h.
After filtration, the filtrate was evaporated to dryness in vacuo, and
the crude product was purified by column chromatography (PE/
EtOAc = 6:1–3:1) to yield 11a as a white waxy solid (333 mg, 52%).
4.1.3.5. 3-(Phenylsulfonyl)-4-(3-((S)-2-(2-((2E,6E)-3,7,11-trim-
eth yldodeca-2,6,10-trienylthio)benzamido) propanoyloxy)but-
oxy)-1,2,5-oxadiazole-2-oxide (11e). Compound 11e was
synthesized from 9e (388 mg, 0.80 mmol) and
1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
58%, a white foam 336 mg. Analytical data for 11e: ½a D25
ꢂ8.1; IR
ꢁ
(KBr, cmꢂ1): 3297, 2924, 1737, 1633, 1555, 1450, 1356, 1168; 1H
NMR (CDCl3-d6): d 8.06 (d, 2H, J = 7.8 Hz, Ar-H), 7.71 (m, 2H, Ar-
H), 7.59 (m, 2H, Ar-H), 7.51 (m, 1H, Ar-H), 7.38 (m, 2H, Ar-H),
5.18 (m, 2H, SCH2CH, NCH), 5.08 (m, 2H, 2 ꢀ CH2CH@CCH3), 4.74
Analytical data for 11a: ½a D26
ꢁ
ꢂ8.6; IR (KBr, cmꢂ1): 3397, 2928,
1736, 1641, 1614, 1553, 1450, 1361, 1169; 1H NMR (CDCl3-d6): d
8.04 (d, 2H, J = 7.5 Hz, Ar-H), 7.86 (m, 1H, Ar-H), 7.52–7.67 (m, 5H,

3454
Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
(m, 1H, OCH), 4.46 (t, 2H, J = 6.0 Hz, OCH2CH2), 3.54 (d, 2H,
J = 7.5 Hz, SCH2), 2.17 (m, 2H, OCH2CH2), 1.98 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.59–1.67 (m, 12H, 4 ꢀ CH@CCH3), 1.51 (d,
2H, J = 7.2 Hz, NCHCH3), 1.38 (d, 2H, J = 6.0 Hz, OCHCH3); MS
(ESI) m/z = 726 [M+1]+; Anal. Calcd for C37H47N3O8S2ꢃH2O: C,
59.74; H, 6.64; N, 5.65. Found: C, 59.65; H, 6.68; N, 5.23.
50%, a white foam 319 mg. Analytical data for 11i: ½a D25
ꢂ7.6; IR
ꢁ
(KBr, cmꢂ1): 3267, 1744, 1649, 1617, 1545, 1450, 1360, 1168; 1H
NMR (CDCl3, 300 MHz): d 8.06 (d, 2H, J = 7.2 Hz, Ar-H), 7.77 (m,
2H, Ar-H), 7.64 (m, 1H, Ar-H), 7.59 (m, 2H, Ar-H), 7.38 (m, 2H,
Ar-H), 7.26 (m, 5H, Ar-H), 5.17 (t, 1H, J = 6.9 Hz, SCH2CH), 5.05
(m, 5H, 2 ꢀ CH2CH@CCH3, COOCH2, NCH), 4.82 (m, 2H, OCH2),
3.45 (m, 2H, SCH2), 3.20–3.37 (m, 2H, CHCH2), 1.96 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.47–1.57 (m, 12H, 4 ꢀ CH@CCH3); MS (ESI)
m/z = 798 [M+1]+; Anal. Calcd for C43H47N3O8S2: C, 64.72; H,
5.94; N, 5.27. Found: C, 64.55; H, 5.89; N, 5.20.
4.1.3.6. 3-(Phenylsulfonyl)-4-(2-(2-((S)-2-(2-((2E,6E)-3,7,11-trim
ethyldodeca-2,6,10-trienylthio)benzamido) propanoyloxy)eth-
oxy)ethoxy)-1,2,5-oxadiazole-2-oxide (11f). Compound 11f
was synthesized from 9f (401 mg, 0.80 mmol) and 1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
4.1.3.10. 3-(4-((S)-3-Phenyl-2-(2-((2E,6E)-3,7,11-trimethyldode
ca-2,6,10-trienylthio)benzamido) propanoyloxy)butoxy)-4-(phe
nylsulfonyl)-1,2,5-oxadiazole-2-oxide (11j). Compound 11j was
51%, a white foam 302 mg. Analytical data for 11f: ½a D15
ꢂ19.2; IR
ꢁ
(KBr, cmꢂ1): 3421, 2928, 1739, 1640, 1614, 1554, 1452, 1356,
1168; 1H NMR (CDCl3, 300 MHz): d 8.04 (d, 2H, J = 7.8 Hz, Ar-H),
7.70 (m, 2H, Ar-H), 7.59 (m, 3H, Ar-H), 7.34 (m, 2H, Ar-H), 5.27
(t, 1H, J = 7.2 Hz, SCH2CH), 5.05 (m, 2H, 2 ꢀ CH2CH@CCH3), 4.84
(m, 1H, NCH), 4.54 (m, 2H, COOCH2CH2OCH2CH2O), 4.38 (m, 2H,
COOCH2CH2), 3.93 (m, 2H, COOCH2CH2OCH2), 3.83 (t, 2H,
J = 4.2 Hz, COOCH2CH2), 3.54 (d, 2H, J = 7.8 Hz, SCH2), 2.01 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.52–1.67 (m, 12H, 4 ꢀ CH@CCH3), 1.38 (d,
3H, J = 7.5 Hz, NCHCH3); MS (ESI) m/z = 742 [M+1]+; Anal. Calcd
for C37H47N3O9S2: C, 59.90; H, 6.39; N, 5.66. Found: C, 59.90; H,
6.52; N, 5.32.
synthesized from 9j (449 mg, 0.80 mmol) and
1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
55%, a white waxy solid 352 mg. Analytical data for 11j: ½a D24
ꢁ
ꢂ3.8; IR (KBr, cmꢂ1): 3301, 2926, 1731, 1644, 1555, 1448, 1373,
1166; 1H NMR (CDCl3-d6): d 8.03 (d, 2H, J = 7.5 Hz, Ar-H), 7.74 (t,
1H, J = 7.2 Hz, Ar-H), 7.61 (m, 3H, Ar-H), 7.51 (d, 1H, J = 7.8 Hz,
Ar-H), 7.37 (m, 2H, Ar-H), 7.22–7.33 (m, 5H, Ar-H), 5.19 (t, 1H,
J = 7.8 Hz, SCH2CH), 5.06 (m, 3H, 2 ꢀ CH2CH@CCH3, NCH), 4.39 (t,
2H, J = 6.0 Hz, COOCH2CH2), 4.23 (m, 2H, CH2CH2O), 3.25 (d, 2H,
J = 6.0 Hz, SCH2), 1.97–2.01 (m, 8H, 2 ꢀ CHCH2CH2CH), 1.81 (m,
4H, OCH2CH2CH2CH2CH2O), 1.57–1.59 (m, 12H, 4 ꢀ CH3); MS
(ESI) m/z = 802 [M+1]+; Anal. Calcd for C43H51N3O8S2: C, 64.40; H,
6.41; N, 5.24. Found: C, 64.30; H, 6.39; N, 5.16.
4.1.3.7. 3-(Phenylsulfonyl)-4-(4-((S)-1-(2-((2E,6E)-3,7,11-trimet
hyldodeca-2,6,10-trienylthio)benzoyl) pyrrolidine-2-carbonyl-
oxy)butoxy)-1,2,5-oxadiazole-2-oxide (11g). Compound 11g
was synthesized from 9g (409 mg, 0.80 mmol) and 1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
4.1.3.11. 3-(Phenylsulfonyl)-4-(4-((S)-2-(2-((2E,6E)-3,7,11-trim
ethyldodeca-2,6,10-trienylthio)benzamido) propanoyloxy)but-
2-ynyloxy)-1,2,5-oxadiazole-2-oxide (11k). Compound 11k was
synthesized from 9k (385 mg, 0.80 mmol) and 1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
49%, colorless viscous liquid 283 mg. Analytical data for 11k:
49%, colorless viscous liquid 294 mg. Analytical data for 11g: ½a D24
ꢁ
ꢂ14.5; IR (KBr, cmꢂ1): 3437, 2926, 1743, 1619, 1552, 1449, 1365,
1169; 1H NMR (CDCl3, 300 MHz): d 8.04 (d, 2H, J = 7.5 Hz, Ar-H),
7.75 (m, 1H, Ar-H), 7.60 (m, 2H, Ar-H), 7.34 (m, 1H, Ar-H), 7.32
(m, 2H, Ar-H), 7.20 (m, 1H, Ar-H), 5.27 (t, 1H, J = 7.5 Hz, SCH2CH),
5.05 (t, 2H, J = 5.1 Hz, 2 ꢀ CH2CH@CCH3), 4.67 (m, 1H, NCH), 4.47
(t, 2H, J = 6.0 Hz, COOCH2CH2CH2CH2O), 4.29 (m, 2H, COOCH2CH2),
3.57 (d, 2H, J = 7.8 Hz, SCH2), 3.35 (m, 1H, NCH2), 2.02 (m, 10H,
CH2CHCOO, 2 ꢀ CHCH2CH2CH), 1.90 (m, 2H, COOCH2CH2CH2),
1.53–1.67 (m, 16H, 4 ꢀ CH@CCH3, COOCH2CH2, NCH2CH2); MS
(ESI) m/z = 752 [M+1]+; Anal. Calcd for C39H49N3O8S2: C, 62.29; H,
6.57; N, 5.59. Found: C, 62.04; H, 6.61; N, 5.44.
½
a 1D5
ꢁ
ꢂ15.8; IR (KBr, cmꢂ1): 3421, 2926, 1749, 1622, 1548, 1451,
1362, 1169; 1H NMR (CDCl3, 300 MHz): d 8.06 (d, 2H, J = 7.8 Hz,
Ar-H), 7.72 (m, 2H, Ar-H), 7.61 (m, 3H, Ar-H, NH), 7.44 (m, 1H,
Ar-H), 7.33 (m, 1H, Ar-H), 7.29 (m, 1H, Ar-H), 5.24 (t, 1H,
J = 7.5 Hz, SCH2CH), 5.05–5.13 (m, 4H, 2 ꢀ CH2CH@CCH3, COOCH2),
4.90 (m, 3H, NCH, CH2O), 3.54 (d, 2H, J = 7.8 Hz, SCH2), 1.98 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.50–1.69 (m, 15H, 4 ꢀ CH@CCH3, NCHCH3);
MS (ESI) m/z = 722 [M+1]+; Anal. Calcd for C37H43N3O8S2: C,
61.56; H, 6.00; N, 5.82. Found: C, 61.42; H, 6.19; N, 5.73.
4.1.3.8. 4-(Phenylsulfonyl)-3-(4-((S)-2-(2-((2E,6E)-3,7,11-trimet
hyldodeca-2,6,10-trienylthio)benzamido) propanoyloxy)but-
oxy)-1,2,5-oxadiazole-2-oxide (11h). Compound 11h was syn-
thesized from 9h (388 mg, 0.80 mmol) and 1 (286 mg, 0.80 mmol)
according to the synthetic procedure of 11a in yield 53%, a white
4.1.3.12. 4-(2-(2-((S)-3-Phenyl-2-(2-((2E,6E)-3,7,11-trimethyldo
deca-2,6,10-trienylthio)benzamido) propanoyloxy)ethoxy)eth-
oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (11l). Com-
pound 11l was synthesized from 9l (462 mg, 0.80 mmol) and 1
(286 mg, 0.80 mmol) according to the synthetic procedure of 11a
in yield 52%, a white waxy solid 340 mg. Analytical data for 11l:
foam 307 mg. Analytical data for 11h: ½a D24
ꢁ
ꢂ3.9; IR (KBr, cmꢂ1):
3262, 2927, 1730, 1625, 1555, 1450, 1372, 1167; 1H NMR (CDCl3,
300 MHz): d 8.04 (d, 2H, J = 7.5 Hz, Ar-H), 7.69 (m, 2H, Ar-H),
7.60 (m, 2H, Ar-H), 7.54 (d, 1H, J = 6.6 Hz, Ar-H), 7.41 (d, 1H,
J = 7.8 Hz, Ar-H), 7.29 (m, 1H, Ar-H), 5.24 (t, 1H, J = 7.5 Hz, SCH2CH),
5.05 (t, 2H, J = 6.9 Hz, 2 ꢀ CH2CH@CCH3), 4.79 (m, 1H, NCH), 4.44 (t,
2H, J = 6.0 Hz, COOCH2CH2CH2CH2O), 4.26 (t, 2H, J = 6.0 Hz,
COOCH2CH2), 3.54 (d, 2H, J = 7.8 Hz, SCH2), 1.99 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.88 (m, 2H, COOCH2CH2CH2), 1.51–1.69 (m,
17H, 4 ꢀ CH@CCH3, COOCH2CH2, NCHCH3); MS (ESI) m/z = 726
[M+1]+; Anal. Calcd for C37H47N3O8S2: C, 61.22; H, 6.53; N, 5.79.
Found: C, 61.02; H, 6.80; N, 5.50.
½
a 2D6
ꢁ
ꢂ2.5; IR (KBr, cmꢂ1): 3286, 2923, 1737, 1642, 1612, 1552,
1453, 1355, 1157; 1H NMR (CDCl3, 300 MHz): d 8.03 (d, 2H,
J = 7.5 Hz, Ar-H), 7.71 (m, 2H, Ar-H), 7.57 (m, 3H, Ar-H), 7.48 (m,
1H, Ar-H), 7.26–7.41 (m, 6H, Ar-H), 5.16–5.22 (m, 3H,
3 ꢀ CH2CH@CCH3), 4.52 (t, 2H, J = 4.5 Hz, COOCH2), 4.27 (m, 3H,
NCH, OCH2), 3.89–3.96 (m, 4H, OCH2CH2OCH2), 3.21–3.36 (m, 4H,
SCH2, CHCH2), 2.01 (m, 8H, 2 ꢀ CHCH2CH2CH), 1.47–1.67 (m,
12H, 4 ꢀ CH@CCH3); MS (ESI) m/z = 818 [M+1]+; Anal. Calcd for
C43H51N3O9S2: C, 63.14; H, 6.28; N, 5.14. Found: C, 62.88; H,
6.35; N, 5.18.
4.1.3.9. 4-(4-((S)-3-Phenyl-2-(2-((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trienylthio)benzamido) propanoyloxy)but-2-ynyloxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (11i). Compound 11i
was synthesized from 9i (446 mg, 0.80 mmol) and 1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
4.1.3.13. 3-(Phenylsulfonyl)-4-(3-((S)-1-(2-((2E,6E)-3,7,11-trim-
ethyldodeca-2,6,10-trienylthio)benzoyl) pyrrolidine-2-carbonyl-
oxy)butoxy)-1,2,5-oxadiazole-2-oxide (11m). Compound 11m
was synthesized from 9m (409 mg, 0.80 mmol) and 1 (286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield

Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
3455
51%, colorless viscous liquid 306 mg. Analytical data for 11m: ½a D25
ꢁ
4.2. Biology
ꢂ18.6; IR (KBr, cmꢂ1): 3326, 2927, 1743, 1626, 1552, 1449, 1368,
1169; 1H NMR (CDCl3, 300 MHz): d 8.07 (d, 2H, J = 7.5 Hz, Ar-H),
7.71 (m, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 7.34 (m, 2H, Ar-H), 7.22
(m, 2H, Ar-H), 5.22 (t, 1H, J = 6.3 Hz, SCH2CH), 5.08 (t, 2H,
J = 5.1 Hz, 2 ꢀ CH2CH@CCH3), 4.67 (m, 1H, NCH), 4.65–4.50 (m,
2H, NCH, OCH), 3.80 (m, 2H, OCH2), 3.52 (d, 2H, J = 7.2 Hz, SCH2),
3.30 (m, 2H, NCH2), 2.29 (m, 2H, OCHCH2), 1.98 (m, 8H,
2 ꢀ CHCH2CH2CH), 1.52–1.67 (m, 16H, NCHCH2, 4 ꢀ CH@CCH3,
NCH2CH2), 1.24 (d, 3H, J = 6.3 Hz, CHCH3); MS (ESI) m/z = 752
[M+1]+; Anal. Calcd for C39H49N3O8S2ꢃH2O: C, 60.84; H, 6.68; N,
5.46. Found: C, 60.67; H, 6.50; N, 5.68.
4.2.1. Cytotoxicity assay
The cytotoxic activity of individual compounds in vitro against
several cancer cell lines was measured by MTT assay. Human breast
carcinoma (MDA-MB-231, MCF-7), gastric carcinoma (SGC-7901),
lung carcinoma (A-549) cells at 5–10 ꢀ 103 cells/well were cultured
in RPMI-1640 medium (Hyclone, USA) overnight and treated in trip-
licate with different concentrations of individual compounds for
48 h. During the last 4 h culture, the cells were exposed to MTT
(1 mg/mL) and the resulting formazan crystals were dissolved in
200 lL DMSO and measured at 570 nm with reference wavelength
at 595 nm on a microplate reader (Thermo, USA). The unmanipu-
latedcells wereused asnegativecontrolswhilethecells treatedwith
1% Triton were used as positive controls. The inhibition rate of indi-
vidual compounds was calculated by (the OD value of negative-
experiments/the OD values of negative controls) ꢀ 100%. The con-
centration required for 50% inhibition (IC50) was calculated using
the software of cell viability.
4.1.3.14. 3-(Phenylsulfonyl)-4-(4-((S)-1-(2-((2E,6E)-3,7,11-trim-
ethyldodeca-2,6,10-trienylthio)benzoyl) pyrrolidine-2-carbon
yloxy)but-2-ynyloxy)-1,2,5-oxadiazole-2-oxide
(11n). Com-
pound 11n was synthesized from 9n (406 mg, 0.80 mmol) and 1
(286 mg, 0.80 mmol) according to the synthetic procedure of 11a
in yield 46%, colorless viscous liquid 275 mg. Analytical data for
11n: ½a 2D5
ꢁ
ꢂ30.3; IR (KBr, cmꢂ1): 3439, 2926, 1752, 1618, 1548,
1450, 1362, 1169; 1H NMR (CDCl3, 300 MHz): d 8.07 (d, 2H,
J = 7.2 Hz, Ar-H), 7.74 (m, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 7.37 (m,
2H, Ar-H), 7.28 (m, 2H, Ar-H), 5.24 (t, 1H, J = 7.5 Hz, SCH2CH),
5.08 (m, 4H, 2 ꢀ CH2CH@CCH3, COOCH2), 4.82 (m, 2H, CH2O),
4.71 (t, 1H, J = 3.9 Hz, NCH), 3.57 (d, 2H, J = 7.5 Hz, SCH2), 3.30
(m, 2H, NCH2), 2.29 (m, 2H, OCHCH2), 1.98 (m, 10H,
2 ꢀ CHCH2CH2CH, NCHCH2), 1.58–1.75 (m, 14H, 4 ꢀ CH@CCH3,
NCH2CH2); MS (ESI) m/z = 748 [M+1]+; Anal. Calcd for
C39H45N3O8S2: C, 62.63; H, 6.06; N, 5.62. Found: C, 62.45; H,
6.15; N, 5.57.
4.2.2. Nitrate/nitrite measurement in vitro
Thelevels of nitrate/nitriteformed from individual compoundsin
the cells were determined by the colorimetric assay using the ni-
trate/nitrite colorimetric assay kit (Beyotime, China), according to
the manufacturer’s instructions. Briefly, MDA-MB-231 cells
(5 ꢀ 106/well) were treated in triplicate with 100
lM of one of the
compounds (11a, 11d, 11f, 11n–p, FTA) for 24 h. The cells were
harvested lyzed. The cell lysates were mixed with Griess for
30–300 min, followed by measuring at 540 nm. The cells treated
with diluent were used as negative controls for the background
levels of nitrate/nitrite production, while with sodium nitrate at
different concentrations was used as positive controls for the
standard curve.
4.1.3.15. 3-(Phenylsulfonyl)-4-(2-(2-((S)-1-(2-((2E,6E)-3,7,11-tri
methyldodeca-2,6,10-trienylthio)benzoyl) pyrrolidine-2-carbon
yloxy)ethoxy)ethoxy)-1,2,5-oxadiazole-2-oxide
(11o). Com-
pound 11o was synthesized from 9o (422 mg, 0.80 mmol) and 1
(286 mg, 0.80 mmol) according to the synthetic procedure of 11a
in yield 48%, colorless viscous liquid 295 mg. Analytical data for
4.2.3. Western blot
The Ras inhibitor activity of 11f was determined by Western
blot assay. MDA-MB-231 cells at 1.5 ꢀ 105/mL were treated with
11o: ½a 2D5
ꢁ
ꢂ19.1; IR (KBr, cmꢂ1): 3434, 2917, 1744, 1628, 1552,
1 or 5
lM 11f or vehicle control for 8 h. After harvested and lyzed,
1448, 1360, 1170; 1H NMR (CDCl3, 300 MHz): d 8.04 (d, 2H,
J = 8.1 Hz, Ar-H), 7.73 (m, 2H, Ar-H), 7.61 (m, 2H, Ar-H), 7.23–7.39
(m, 3H, Ar-H), 5.27 (t, 1H, J = 6.3 Hz, SCH2CH), 5.08 (t, 2H,
J = 5.1 Hz, 2 ꢀ CH2CH@CCH3), 4.57 (t, 2H, J = 4.5 Hz, COOCH2), 4.39
(t, 3H, J = 4.5 Hz, CH2OCN), 4.11 (m, 1H, NCH), 3.93 (t, 2H,
J = 4.5 Hz, COOCH2CH2O), 3.83 (t, 2H, J = 4.5 Hz, COOCH2OCH2),
3.55 (d, 2H, J = 7.5 Hz, SCH2), 3.33 (m, 2H, NCH2), 1.96–2.04 (m,
10H, NCHCH2, 2 ꢀ CHCH2CH2CH), 1.52–1.67 (m, 14H, 4 ꢀ CH3,
NCH2CH2); MS (ESI) m/z = 768 [M+1]+; Anal. Calcd for C39H49N3O9S2:
C, 61.00; H, 6.43; N, 5.47. Found: C, 60.63; H, 6.47; N, 5.42.
the cell lysates (50 lg/lane) were separated by SDS–PAGE (12% gel)
and transferred onto nitrocellulose membranes. After blocked with
5% fat-free milk, the target proteins were probed with anti-Akt,
anti-phospho-Akt (Ser473), anti-ERK, anti-phospho-ERK (Thr202/
Tyr204), anti-Phospho-Raf (Ser259), and anti-b-actin antibodies
(Cell Signaling, Boston), respectively. The bound antibodies were
detected by HRP-conjugated second antibodies and visualized
using the enhanced chemiluminescent reagent.
References and notes
1. Maity, A.; Pore, N.; Lee, J.; Solomon, D.; O’Rourke, D. M. Cancer Res. 2000, 60, 5879.
2. Porfiri, E.; McCormick, F. J. Biol. Chem. 1996, 271, 5871.
3. Boguski, M. S.; McCormick, F. Nature 1993, 366, 643.
4. Goldberg, L.; Kloog, Y. Cancer Res. 2006, 66, 11709.
5. Joneson, T.; White, M. A.; Wigler, M. H.; Bar-Sagi, D. Science 1996, 271, 810.
6. Walsh, A. B.; Bar-Sagi, D. J. Biol. Chem. 2001, 276, 15609.
7. Downward, J. Curr. Opin. Genet. Dev. 1998, 8, 49.
8. Bos, J. L. Cancer Res. 1989, 49, 4682.
9. Elad-Sfadia, G.; Haklai, R.; Ballan, E.; Gabius, H. J.; Kloog, Y. J. Biol. Chem. 2002,
277, 37169.
10. Rotblat, B.; Niv, H.; Andre, S.; Kaltner, H.; Gabius, H. J.; Kloog, Y. Cancer Res.
2004, 64, 3112.
11. Haklai, R.; Elad-Sfadia, G.; Egozi, Y.; Kloog, Y. Cancer Chemother. Pharmacol.
2008, 61, 89.
12. Tsimberidou, A. M.; Rudek, M. A.; Hong, D.; Ng, C. S.; Blair, J.; Goldsweig, H.;
Kurzrock, R. Cancer Chemother. Pharmacol. 2009. doi:10.1007/s00280-009-
1027-4.
13. Zundelevich, A.; Elad-Sfadia, G.; Haklai, R.; Kloog, Y. Mol. Cancer Ther. 2007, 6,
1765.
14. Ignarro, L. J. Biochem. Pharmacol. 1991, 41, 485.
15. Fukuto, J. M.; Wink, D. A. Met. Ions Biol. Syst. 1999, 36, 547.
4.1.3.16. 3-(Phenylsulfonyl)-4-(2-(2-(2-(2-((2E,6E)-3,7,11-trimet
hyldodeca-2,6,10-trienylthio)benzamido) acetoxy)ethoxy)eth-
oxy)-1,2,5-oxadiazole-2-oxide (11p). Compound 11p was syn-
thesized from 9p (390 mg, 0.80 mmol) and
1
(286 mg,
0.80 mmol) according to the synthetic procedure of 11a in yield
53%, a white waxy solid 308 mg. Analytical data for 11p: IR (KBr,
cmꢂ1): 3422, 2927, 1749, 1628, 1553, 1449, 1362, 1168; 1H NMR
(CDCl3, 300 MHz): d 8.05 (d, 2H, J = 8.1 Hz, Ar-H), 7.73 (m, 2H,
Ar-H), 7.62 (m, 2H, Ar-H), 7.39–7.42 (m, 4H, Ar-H), 5.27 (t, 1H,
J = 6.3 Hz, SCH2CH), 5.08 (t, 2H, J = 5.4 Hz, 2 ꢀ CH2CH@CCH3), 4.56
(t, 2H, J = 4.5 Hz, COOCH2), 4.40 (t, 3H, J = 4.5 Hz, CH2OCN), 4.31
(m, 2H, NCH2), 3.93 (t, 2H, J = 4.5 Hz, COOCH2CH2O), 3.84 (t, 2H,
J = 4.5 Hz, COOCH2OCH2), 3.54 (d, 2H, J = 7.5 Hz, SCH2), 2.01 (m,
8H, 2 ꢀ CHCH2CH2CH), 1.47–1.67 (m, 12H, 4 ꢀ CH3); MS (ESI) m/
z = 728 [M+1]+; Anal. Calcd for C37H47N3O9S2: C, 59.40; H, 6.23;
N, 5.77. Found: C, 59.22; H, 6.32; N, 5.59.

3456
Y. Ling et al. / Bioorg. Med. Chem. 18 (2010) 3448–3456
16. Millet, A.; Bettaieb, A.; Renaud, F.; Prevotat, L.; Hammann, A.; Solary, E.;
Mignotte, B.; Jeannin, J. F. Gastroenterology 2002, 123, 235.
17. Postovit, L. M.; Adams, M. A.; Lash, G. E.; Heaton, J. P.; Graham, C. H. Int. J.
Cancer 2004, 108, 47.
18. Chen, L.; Zhang, Y.; Kong, X.; Lan, E.; Huang, Z.; Peng, S.; Kaufman, D. L.; Tian, J.
J. Med. Chem. 2008, 51, 4834.
21. Moharram, S.; Zhou, A.; Wiebe, L. I.; Knaus, E. E. J. Med. Chem. 2004, 47, 1840.
22. Killian, D. M.; Hermeling, S.; Chikhale, P. J. Drug Deliv. 2007, 14, 25.
23. Yue, W.; Wang, J.; Li, Y.; Fan, P.; Santen, R. J. Int. J. Cancer 2005, 117, 746.
24. Marra, M.; Lombardi, A.; Agostinelli, E.; Giuberti, G.; Zappavigna, S.; Tempera,
G.; Vitale, G.; Bifulco, M.; Abbruzzese, A.; Caraglia, M. Biochim. Bioph. Acta 2008,
1783, 2269.
19. Dhar, A.; Brindley, J. M.; Stark, C.; Citro, M. L.; Keefer, L. K.; Colburn, N. H. Mol.
Cancer Ther. 2003, 2, 1285.
25. Azuse, I.; Tamura, M.; Kinomura, K.; Okai, H.; Kouge, K.; Hamatsu, F.; Koizumi,
T. Bull. Chem. Soc. Jpn. 1989, 62, 3103.
20. Maksimovic-Ivanic, D.; Mijatovic, S.; Harhaji, L.; Miljkovic, D.; Dabideen, D.;
Fan Cheng, K.; Mangano, K.; Malaponte, G.; Al-Abed, Y.; Libra, M.; Garotta, G.;
Nicoletti, F.; Stosic-Grujicic, S. Mol. Cancer Ther. 2008, 7, 510.
26. Kong, X.; Zhang, Y.; Dai, L.; Lai, Y.; Ji, H.; Peng, S. Chin. Chem. Lett. 2008, 19, 149.