Design, synthesis, and antihepatocellular carcinoma activity of nitric oxide releasing derivatives of oleanolic acid

Article abstract of DOI:10.1021/jm800167u

Novel furoxan-based nitric oxide (NO) releasing derivatives of oleanolic acid (OA) were synthesized for potential therapy of liver cancers. Six compounds produced high levels of NO in human hepatocellular carcinoma (HCC) cells and exhibited strong cytotoxicity selectively against HCC in vitro. Treatment with 8b or 16b significantly inhibited the growth of HCC tumors in vivo. These data provide a proof-in-principle that furoxan/OA hybrids may be used for therapeutic intervention of human liver cancers.

Full text of DOI:10.1021/jm800167u

4834
J. Med. Chem. 2008, 51, 4834–4838
Brief Articles
Design, Synthesis, and Antihepatocellular Carcinoma Activity of Nitric Oxide Releasing
Derivatives of Oleanolic Acid
Li Chen,†,‡ Yihua Zhang,*,† Xiangwen Kong,† Edward Lan,§ Zhangjian Huang,† Sixun Peng,† Daniel L. Kaufman,§ and
Jide Tian*,§
Center of Drug DiscoVery and Department of Phytochemistry, China Pharmaceutical UniVersity, Nanjing 210009, P. R. China, and
Department of Molecular and Medical Pharmacology, UniVersity of CaliforniasLos Angeles, Los Angeles, California 90095
ReceiVed February 18, 2008
Novel furoxan-based nitric oxide (NO) releasing derivatives of oleanolic acid (OA) were synthesized for
potential therapy of liver cancers. Six compounds produced high levels of NO in human hepatocellular
carcinoma (HCC) cells and exhibited strong cytotoxicity selectively against HCC in vitro. Treatment with
8b or 16b significantly inhibited the growth of HCC tumors in vivo. These data provide a proof-in-principle
that furoxan/OA hybrids may be used for therapeutic intervention of human liver cancers.
Chart 1
Introduction
Nitric oxide (NOa) is a key mediator involved in many
physiological and pathological processes.1 High levels of NO
and its metabolic derivatives, the reactive nitrogen species (RNS)
and reactive oxygen species (ROS), can modify functional
proteins by S-nitrosylation, nitration, and disulfide formation,
leading to bioregulation, inactivation, and cytotoxicity, particu-
larly in tumor cells.2 Indeed, some synthesized NO-releasing
compounds have shown cytotoxic activity against human colon
carcinoma cells in vitro, inhibiting the growth and metastasis
of cancers in vivo.3–6 However, the therapeutic effect of those
compounds is limited, possibly because of low levels of NO
release. Furoxans are thermally stable compounds and represent
one class of NO donors that can produce high levels of NO;
however, they have a variety of NO-related bioactivities in
vivo.7–9 To obtain tissue-specific NO-related function, new types
of furoxan/drug hybrids need to be developed.10
Oleanolic acid (OA) is widely found in plants and has been
demonstrated to inhibit hepatitis in humans without apparent
side effects.11,12 Furthermore, OA has been shown to protect
rodent liver from CCl4 and other toxicant-induced hepatotoxicity
and chronic cirrhosis, which is associated with the distribution
and metabolism of OA in the liver and modulation of OA on
cytochrome p450 (CYP) activity.12–15 On the basis of its
therapeutic effects, liver-specific metabolism, and availability,
OA may be an ideal lead compound for the design of novel
furoxan-based NO-releasing compounds that produce high levels
of NO specifically in the liver. We hypothesized that such
furoxan/OA hybrids may have strong cytotoxicity selectively
against HCC, one of the most frequent cancers in the world,16
because of the efficient entry and metabolism of those com-
pounds to produce high levels of NO in HCC cells.
To test this hypothesis, 32 novel furoxan-based NO-releasing
derivatives of OA were designed and synthesized by coupling
phenylsulfonyl- or phenyl-substituted furoxans to the 3- or 28-
position of OA through various chemical linkers. Subsequently,
their anti-HCC activities were evaluated in vitro and in vivo.
Interestingly, some of the derivatives (8a-e, 16b) showed strong
cytotoxicity against HCC cells in vitro, which were associated
with high levels of NO production selectively in HCC cells.
Importantly, treatment with one of the active hybrids 8b or 16b
inhibited the growth of HCC tumors in vivo. We discuss
potential structure-activity relationships among the compounds
and the implication of these findings.
Results and Discussion
Chemistry. A total of seven phenylsulfonyl-substituted
furoxans (1) and four phenyl-substituted furoxans (2) (Chart 1)
were synthesized as described previously.17,18 The preparation
of 3-position derivatives of OA is outlined in Scheme 1. OA
was first modified with ethyl bromide or benzyl bromide in the
presence of Et3N to give the corresponding C-28 esters (3 or 4)
in 65-73% yields. Subsequently, OA, 3, or 4 was reacted with
succinic anhydride in the presence of 4-N,N-dimethylaminopy-
ridine (DMAP) to form 3-O-acyl derivatives (5-7) in 52-60%
yields. Furthermore, 5-7 were esterified with 1 or 2 in the
presence of dicyclohexylcarbodiimide (DCC)/DMAP to generate
target compounds (8a-g, 9b,d, and 10b,c,f,g or 11h-k, 12j,k,
and 13h). Thus, 20 target compounds were generated by
coupling phenylsulfonyl- or phenyl-substituted furoxans to the
3-position of OA.
* To whom correspondence should be addressed. For Y.Z.: phone, +86-
25-83271186; fax, +86-25-86635503; e-mail, zyhtgd@hotmail.com. For
J.T.: phone, 310-206-3350; fax, 310-825-6267; e-mail, jtian@mednet.ucla.edu.
†
Center of Drug Discovery, China Pharmaceutical University.
Department of Phytochemistry, China Pharmaceutical University.
University of CaliforniasLos Angeles.
‡
§
a Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FCS,
fetal calf serum; HCC, human hepatocellular carcinoma; LDH, lactate
dehydrogenase; NO, nitric oxide; OA, oleanolic acid; PE, petroleum ether;
OD, optical density.
10.1021/jm800167u CCC: $40.75
2008 American Chemical Society
Published on Web 07/04/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4835
Scheme 1. Synthesis of Compounds 8-13a
a Reagents and conditions: (i) succinic anhydride, DMAP, dry CH2Cl2, reflux; (ii) DCC/DMAP, dry CH2Cl2, 1 for 8-10, 2 for 11-13. The definitions
of X and Y are indicated in Chart 1.
Scheme 2. Synthesis of Compounds 14-17a
a Reagents and conditions: (i) (CF3CO)2O, toluene, reflux, 4 h, 1 for 14, 2 for 15; (ii) KHCO3, MeOH, room temp. The definitions of X and Y are
indicated in Chart 1.
Table 1. Percentage of HepG2 Cell Deatha
The other 12 target compounds (14a-d,g, 15h,j,k, 16b,c,
compd
1 µM
0.1 µM
0.01 µM
0.001 µM
and 17h,j) were synthesized by modifying the 28-COOH of OA
with phenylsulfonylfuroxan or phenylfuroxan (Scheme 2). Direct
esterification of OA with hydroxyl compound 1 or 2 failed
perhaps because of the large steric hindrance and weak acidity
of the 28-COOH. Alternatively, the carboxyl group of OA was
activated by the mixed anhydride. OA was initially treated with
trifluoroacetic anhydride (TFAA) to form a mixed anhydride
in a quantitative yield, which was subsequently reacted with 1
or 2 to afford 3-O-trifluoroacetyl OA ester 14 or 15 in 63-75%
yields. The trifluoroacetyl groups in 14 and 15 were removed
in diluted KHCO3 solution, without breakdown of other ester
bonds, to produce 16 and 17.
OA
18
8a
8b
8c
8d
8e
8f
0
0
0
0
0
0
0
0
0
1.70
2.60
2.70
0
0
0
67.41
100.00
98.16
95.63
74.53
0
13.68
93.50
56.35
67.96
13.01
0
10.07
13.34
10.96
17.32
8.51
0
0
0
6.67
11h
14b
16b
0
0
0
0
0
0
89.21
55.11
a Data shown are the average percentages of cell death induced by the
compound at 24 h post-treatment from three independent experiments.
Intragroup variations were less than 10% of the values presented. Treatment
of the cells with the compound for a longer period did not increase its
cytotoxicity. The other 23 synthesized compounds tested showed no
cytotoxicity against HepG2 in our experimental system (data not shown).
All of the compounds generated were further purified and
characterized (see Supporting Information). The success in the
synthesis of these compounds provided a base for the charac-
terization of their anti-HCC activity in vitro and in vivo.
Biological Results. The bioactivities of these compounds
were determined by the lactate dehydrogenase (LDH) assays
(Table 1). While there was no obvious cytotoxicity against HCC
cells for the parent OA at any of the tested concentrations, the
anti-HCC activities of 8a-e and 16b against HepG2 appeared
to be dose-dependent. For example, 8b at a dose of 1 µM
promoted 100% cell death and 0.01 µM of it induced 13.34%
cell death. Similar patterns of cell death induced by those active
compounds were achieved in Hep3b cells (Table 2S, Supporting
Information), and 8b displayed the most potent cytotoxicity
against HepG2 and Hep3b cells. However, 8f, 11h, 14b, and
the other 23 synthesized compounds showed no cytotoxicity
against HepG2 and Hep3b cells in our experimental system (data
not shown). Interestingly, none of the tested compounds under
the same experimental conditions showed any obvious cyto-
toxicity against HeLa, PC-3, MCF-7, and HEK 293 (data not
shown). The strong cytotoxicity against HCC cells, but not other
noncancer and nonliver cancer cell lines tested, suggests that
those compounds may be selectively cytotoxic to HCC cells.

4836 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brief Articles
Table 2. Treatment with 8b or 16b Inhibits the Growth of HCC
Tumors in Vivoa
treatment
tumor size (mm3)
tumor weight (g)
diluent
OA
8b
701 ( 157
668 ( 208
232 ( 77b
289 ( 41c
1.031 ( 0.171
0.961 ( 0.168
0.262 ( 0.082b
0.332 ( 0.074b
16b
a Data shown are the mean ( SD of group mice (n ) 8). b p < 0.01 vs
OA or diluent group, determined by Student t analysis. Experimental and
control groups of mice were tested simultaneously. c p < 0.02 vs OA or
diluent group, determined by Student t analysis. Experimental and control
groups of mice were tested simultaneously.
that the growth of HCC tumors was not significantly affected
by OA treatment. Importantly, the mean size and weight of the
tumors from the mice treated with 8b or 16b were reduced
dramatically. Interestingly, histological analysis of the liver
tissues from those groups of mice showed no morphological
differences (data not shown). Apparently, treatment with 8b or
16b significantly inhibited the growth of HCC tumors but did
not result in an obvious change in the liver structures in mice.
Analysis of structure-activity relationships (SAR) among
these compounds revealed that the 4-phenylsulfonylfuroxan was
crucial for the anti-HCC cytotoxicity of furoxan/OA hybrids
because the phenylsulfonylfuroxan-substituted OA (8a-e and
16b), but not single phenylfuroxan-substituted OA, showed
strong cytotoxicity selectively against HCC cells in vitro.
Furthermore, the coupling site of OA with furoxans had a great
impact on the cytotoxic potency of the derivatives. The
compounds obtained by coupling 4-phenylsulfonylfuroxan to
the 3-OH of OA were more active than those obtained by
coupling the same furoxan to the 28-COOH (8a vs 14a, 8b vs
14b or 16b, 8c vs 14c or 16c, and 8d vs 14d). Indeed, 5 out of
13 compounds with phenylsulfonylfuroxan to the 3-OH were
bioactive, while only 1 out of 7 derivatives with phenylsulfo-
nylfuroxan to the 28-COOH showed anti-HCC activity. More-
over, the type and length of the linkers, which connected NO
donor moiety to the 3- or 28-position of OA, were important
for compounds’ activities. Target compounds that had aliphatic
linkers showed strong cytotoxicity. For example, 8b and 16b
with a linker bearing a four-carbon aliphatic chain, (CH2)2-
CH(CH3), were highly active. However, those with aromatic
linkers (8f,g) did not show any cytotoxicity. Finally, a free 28-
COOH for compounds with furoxan coupling to the 3-position
of OA appeared to be necessary, evidenced by the fact that all
active compounds (8a-e) in this series had free 28-COOH while
the compounds with corresponding ethyl or benzyl esters (9b,d
and 10b,c) had no cytotoxicity against HCC cells. Similarly, a
free 3-OH was essential for the anti-HCC activity of 28-position
derivatives (16b vs 14b). The striking characteristics may be
required for those active compounds to enter and metabolize
effectively in HCC cells and produce toxic levels of NO and
its derived RNS/ROS, leading to cytotoxicity. However, the
precise SAR of these derivatives and the optimal compounds
with potent anti-HCC activity remain to be further investigated.
Previous study has shown that 18 inhibits liver inflamma-
tion.19 Our previous data demonstrated that nitrate-based deriva-
tives of OA that produced lower levels of NO protected HCC
cells from anti-Fas mediated apoptosis.20 In this study, we
showed that some furoxan-based NO-releasing derivatives of
OA had strong cytotoxicity selectively against HCC cells in
vitro, which was associated with higher levels of NO production
in HCC cells. Treatment with a representative compound 8b or
16b significantly inhibited the growth of HCC tumors inoculated
in vivo. Our data provide a proof-in-principle that NO is a dual
functional regulator for the HCC cells and higher levels of NO
Figure 1. Variable levels of NO produced by furoxan/OA hybrids
selectively in HCC cells. Individual values were obtained by subtracting
the background (diluent treated wells) and calculated according to the
standard curve. Data shown are the mean value of two experiments
for each compound at 240 min post-treatment, and similar patterns of
nitrate/nitrite levels in HCC cells were observed at other experimental
time points (data not shown).
The furoxans can be metabolized predominately by CYP in
liver cells to produce high levels of NO and RNS/ROS.8,9 OA
is mainly metabolized in the liver.11 It is possible that the
selective cytotoxicity of these compounds may stem from high
levels of NO production selectively in the HCC cells. To test
this possibility, HepG2, Hep3b, Hela, PC-3, MCF-7, and Hek
293 cells were exposed to each compound (100 µM) for varying
durations (30-300 min). The levels of nitrite/nitrate in the
lysates of different cell lines were characterized using the nitrite/
nitrate colorimetric assay kit (Figure 1). First, treatment with
OA did not induce a detectable level of nitrite/nitrate in any of
the tested cells. Second, the kinetics of NO release by 8a-e
and 16b were similar to that of the control, a nitrate-based
derivative of ursodeoxycholic acid19 (18, data not shown),
suggesting that NO was slowly released. Furthermore, treatment
with any of the furoxan/OA hybrids tested failed to promote
significantly higher levels of nitrate/nitrite in nonhepatic cells.
Importantly, treatment with 8a-e and 16b, but not 8f, 11h, or
14b, induced high levels of nitrate/nitrite production in HepG2
and Hep3b cells and the levels of nitrate/nitrite were severalfold
higher than that of 18 in HCC cells. Finally, although the levels
of nitrate/nitrite induced by 8a-e and 16b were not significantly
different, they appeared to be positively correlated to anti-HCC
activities of those compounds. Therefore, treatment with those
compounds promoted high levels of NO selectively in HCC
cells in vitro, which may contribute to strong cytotoxicities of
those compounds selectively against HCC in vitro.
Next, we examined the impact of treatment with 8b or 16b
on animal survival and behaviors by acute toxicity assay, and
the growth of HCC tumor in vivo in a mouse model of HCC.
Groups of healthy male Balb/c mice that were treated intrap-
eritoneally (ip) with 8b, 16b, or OA (15, 50, or 100 mg/kg)
appeared healthy throughout a 15-day observation period with
no significant differences in eating, drinking, body weight, and
activity among those groups of mice (data not shown). These
data suggest that administration of these compounds at these
dosages tested did not have adverse effects. Furthermore, the av-
erage size and weight of HCC tumors in the mice that were
inoculated with Hep3b cells and treated with OA were similar
to that in mice treated with control diluent (Table 2), suggesting

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4837
was purified by column chromatography (PE/EtOAc ) 4:1 to 3:1)
to give the title compounds (62-70%).
produced in HCC cells are cytotoxic while lower levels of it
are protective.21,22 Importantly, treatment with a furoxan/OA
hybrid up to 100 mg/kg did not cause any apparent adverse
effects and continual administration of the compound for 35
consecutive days did not result in morphological abnormalities
in mouse liver. Together, our findings suggest that these active
compounds may be promising drug candidates with potent
cytotoxicity selectively against HCC cells and may potentially
be used for therapeutic intervention of liver cancer in the clinic.
The selective cytotoxicity of these compounds against HCC cells
may be associated with the efficient entry and metabolism of
these compounds in HCC cells, but not in other cells tested, to
produce high levels of NO. However, the precise mechanism(s)
underlying the selective effect of these compounds remains to
be further determined.
In summary, a group of novel furoxan-based NO-releasing
derivatives of OA were designed and synthesized by coupling
phenylsulfonyl- or phenyl-substituted furoxans to the 3- or 28-
position of OA through various chemical linkers, respectively.
Compounds 8a-e and 16b exhibited strong cytotoxicity selec-
tively against HCC cells, which appeared to be associated with
high levels of NO production in HCC cells in vitro. Treatment
with 8b or 16b greatly inhibited the growth of HCC tumors
inoculated but did not cause obvious morphological changes in
mouse liver. Our data demonstrate that high levels of NO are
toxic to HCC cells. Our findings provide a base for the design
of novel furoxan/OA hybrids for therapeutic intervention of
human liver cancers.
1-Methyl 3-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-
propyl-3-[(trifluoroacetyl)oxy]olean-12-en-28-oate (14b). The title
compound was obtained starting from OA, TFAA, and 1b. White
solid, 70% yield. Mp 74-76 °C. ESI-MS: 866 [M + NH4]+. IR
(KBr): 2935, 2870, 1779, 1720, 1614, 1550, 1450, 1373, 1163
1
cm-1. H NMR (CDCl3), δ 0.71 (s, 3H, CH3), 0.75 (s, 3H, CH3),
0.87 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.96 (s, 3H, CH3), 1.04 (s,
3H, CH3), 1.12 (s, 3H, CH3), 2.84-2.86 (m, 1H, C18-H),
4.45-4.47 (m, 2H, OCH2), 4.65-4.69 (m, 1H, 3R-H), 5.26-5.27
(m, 1H, OCH), 5.28 (brs, 1H, C12-H), 7.63-7.66 (m, 2H, ArH),
7.74-7.75 (m, 1H, ArH), 8.06-8.09 (m, 2H, ArH). Anal.
(C44H59F3N2O9S) C, H, N.
General Procedure for the Preparation 16b,c and 17h,j.
Compound 14 or 15 was dissolved in MeOH, and their pH was
adjusted slowly with diluted KHCO3 solution to pH 8-9. The
solution was kept stirring at room temperature for 1 week and
concentrated under reduced pressure. The residue was diluted with
water and extracted with EtOAc. The obtained organic layer was
washed with water and saturated NaCl solution, dried, and
evaporated. The crude product was purified by column chroma-
tography (PE/EtOAc ) 4:1 to 3:1) to give the title compounds
(92-98%).
1-Methyl 3-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-
propyl-3-hydroxyolean-12-en-28-oate (16b). The title compound
was obtained starting from 14b. White solid, 92% yield. Mp 86-88
°C. ESI-MS: 775 [M + Na]+. IR (KBr): 3438, 2947, 2869, 1720,
1616, 1551, 1451, 1367, 1170 cm-1. 1H NMR (CDCl3), δ 0.66 (s,
3H, CH3), 0.73 (s, 6H, 2 × CH3), 0.87 (s, 6H, 2 × CH3), 0.93 (s,
3H, CH3), 1.11 (s, 3H, CH3), 2.79-2.81 (m, 1H, C18-H),
3.14-3.19 (m, 1H, 3R-H), 4.14-4.46 (m, 2H, OCH2), 5.07-5.08
(m, 1H, OCH), 5.24-5.25 (brs, 1H, C12-H), 7.59-7.64 (m, 2H,
ArH), 7.71-7.73 (m, 1H, ArH), 8.03-8.06 (m, 2H, ArH). Anal.
(C42H60N2O8S) C, H, N.
Biological Experiments. Cytotoxicity Assay in Vitro. The
cytotoxicity of individual compounds screened was determined by
the LDH assay using the cytotoxicity detection kit (Roche)
according to the manufacturer’s instructions. Briefly, human liver
cancer cell lines (HepG2 and Hep3b), human cervical cancer cell
line (HeLa), human prostate cancer cell line (PC-3), human breast
cancer cell line (MCF-7), and human kidney epithelial cell line
(HEK 293) were obtained from ATCC and were cultured in 10%
fetal calf serum (FCS) Dulbecco’s modified Eagle’s medium
(DMEM). The cells (104/well) were in triplicate exposed to varying
concentrations of individual compound (diluted from the stock in
DMSO) in 2% FCS phenol red free DMEM for 24-72 h. The cells
exposed to the same concentration of DMSO in DMEM were used
as negative controls (spontaneous releasing of LDH), and the cells
exposed to 1% Triton X-100 in DMEM were used as positive
controls (maximum releasing of LDH). A portion of the cell
supernatant (50 µL) was harvested from each well, and LDH levels
were determined by LDH assays at optical density (OD, 490/650).
The percent specific cytotoxicity of each compound was determined
based on [(OD experiments) - (OD negative controls)]/[(OD pos-
itive controls) - (OD negative controls)] × 100.
Experimental Section
General. Melting points were determined using a capillary
apparatus (RDCSY-I) and are reported directly. All of the com-
pounds synthesized were purified by column chromatography (CC)
on silica gel 60 (200-300 mesh) and thin-layer chromatography
(TLC) on silica gel 60 F254 plates (250 µm; Qingdao Ocean
Chemical Company, China). Subsequently, they were routinely
analyzed by IR (Shimadzu FTIR-8400S), 1H NMR (Bruker ACF-
300Q, 300 MHz), MS (Hewlett-Packard 1100 LC/MSD spectrom-
eter), and elemental analysis (Elementar Vario EL III instrument).
General Procedure for the Preparation of 8a-g, 9b,d, 10b,c,f,g,
11h-k, 12j,k, and 13h. A solution of 5, 6, or 7 (0.42mmol), 1 or
2 (0.42mmol), DCC (86.5 mg, 0.42mmol), and DMAP (5.1 mg,
0.042mmol) in dry CH2Cl2 (20 mL) was stirred at room temperature
for 4-10 h. After filtration, the filtrate was evaporated to dryness
in vacuo, and the crude product was purified by column chroma-
tography (petroleum ether (PE)/EtOAc ) 4:1 to 3:1) to yield the
compounds (39-72%).
3-{[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadia-
zol-3-yl]oxy}propoxy)-4-oxobutanoyl]oxy}olean-12-en-28-oic Acid
(8b). The title compound was obtained starting from 5 and 1b.
White solid, 48.8% yield. Mp 140-142 °C. ESI-MS: 875 [M +
Na]+. IR (KBr): 3398, 2943, 2879, 1731, 1694, 1621, 1556, 1370,
1
1155 cm-1. H NMR (CDCl3), δ 0.73 (s, 3H, CH3), 0.77 (s, 3H,
CH3), 0.81 (s, 6H, 2 × CH3), 0.89 (s, 3H, CH3), 0.91 (s, 3H, CH3),
1.11 (s, 3H, CH3), 2.59 (s, 4H, 2 × COCH2), 2.77-2.83 (m, 1H,
C18-H), 4.39 (brs, 1H, 3R-H), 4.43 (t, 2H, OCH2, J ) 6 Hz),
5.12-5.19 (m, 1H, OCH), 5.26 (brs, 1H, C12-H), 7.58-7.63 (m,
2H, ArH), 7.70-7.75 (m, 1H, ArH), 8.05 (d, 2H, ArH, J ) 8 Hz).
Anal. (C46H64N2O11S) C, H, N.
Nitrate/Nitrite Measurement in Vitro. The levels of nitrate and
nitrite produced by the compounds were determined by the
colorimetric assay using the nitrate/nitrite colorimetric assay kit
(Cayman Chemical) according to the manufacturer’s instructions.
Briefly, HepG2, Hep3b, Hela, PC-3, MCF-7, and Hek 293 cells (5
× 106/well) were treated with 100 µM of one of the compounds
(8a-f, 11h, 14b, 16b, 18, or OA) for varying periods (30-300
min). Subsequently, the cells were harvested and their cell lysates
were prepared. Following microfuge ultrafiltration, a 40 µL sample
was used for analysis at OD 540. The cells treated with diluent
were used as negative controls for the background levels of nitrate/
nitrite production, and nitrate at different concentrations was used
as positive controls for the standard curve.
General Procedure for the Preparation of 14a-d,g and 15h,j,k.
OA (456 mg, 1mmol) dissolved in toluene was mixed with TFAA
(0.82 mL, 5.87mmol) by stirring at room temperature for 10 min,
followed by the addition of 1 or 2 (1mmol) to the reaction mixture
and allowing the mixture to reflux for an additional 4 h with stirring.
After cooling to room temperature, the mixture was gradually
neutralized with 10% NaOH, and the organic layer was washed
with water and saturated NaCl solution sequentially, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product
Acute Toxic Assay in Vivo. Groups of male Balb/c mice (n )
8 per group, Jackson Laboratory) were treated ip with 8b, 16b, or

4838 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brief Articles
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HCC Model and Treatment in Vivo. Groups of male NOD/
scid mice at 6-8 weeks of age (Taconic) were inoculated
subcutaneously (sc) with 106 Hep3b cells and then injected ip with
about 100 µL of 8b, 16b, OA (15 mg/kg) or the diluent daily for
35 consecutive days. The mice were sacrificed, and their tumor
size and weight were measured by an external caliper or a balance
in a blinded fashion.
Acknowledgment. The authors thank Drs. Louis Ignarro and
Jon M. Fukuto for their advice. This work was partially
supported by a grant from the National Institutes of Health (NIH)
(Grant CA116846 to J.T.).
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Supporting Information Available: General synthetic proce-
dures; spectroscopic data of target compounds except for 8b, 14b,
and 16b; elemental analysis results of all target compounds; and
data of cytotoxicity against Hep3b cells. This material is available
free of charge via the Internet at http://pubs.acs.org.
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