Synthesis and Biological Evaluation of Novel Olean-28,13β-lactams as Potential Antiprostate Cancer Agents

Article abstract of DOI:10.1021/jm5020023

γ-Lactam is an important structural motif in a large number of biologically active natural products and synthetic small pharmaceutical molecules. However, there is currently no effective approach to construct γ-lactam ring directly from natural rigid polycyclic amides. Herein, we report a facile methodology for synthesis of a new group of olean-28,13β-lactams (10a-j) from their corresponding amides, promoted by an easily available reagent 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), through an intramolecular dehydrogenative C-N coupling reaction via a radical ion mechanism. Biological evaluation indicated that the most active lactam 10h displayed potent antiproliferative activity against human cancer cells but 13.84- to 16.92-fold less inhibitory activity on noncancer cells in vitro. In addition, 10h significantly inhibited the growth of implanted prostate cancer in vivo. Furthermore, 10h induced cell cycle arrest and apoptosis and down-regulated the AKT/mTOR signaling in DU-145 cells. Finally, 10h was more stable in rat plasma and human liver microsomes than CDDO-Me and had little hERG channel inhibitory activity. Collectively, 10h may be a potential antiprostate cancer agent for further investigation.

Full text of DOI:10.1021/jm5020023

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Novel Olean-28,13β-lactams
as Potential Antiprostate Cancer Agents
Yong Ai,†,‡,∥ Yang Hu,†,∥ Fenghua Kang,†,‡ Yisheng Lai,†,‡ Yanju Jia,† Zhangjian Huang,*,†,‡
Sixun Peng,†,‡ Hui Ji,*,† Jide Tian,§ and Yihua Zhang*,†,‡
†State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China
‡Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009,
P. R. China
§Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095, United States
S
* Supporting Information
ABSTRACT: γ-Lactam is an important structural motif in a
large number of biologically active natural products and
synthetic small pharmaceutical molecules. However, there is
currently no effective approach to construct γ-lactam ring
directly from natural rigid polycyclic amides. Herein, we report
a facile methodology for synthesis of a new group of olean-
28,13β-lactams (10a−j) from their corresponding amides,
promoted by an easily available reagent 2,3-dichloro-5,6-
dicyanobenzoquinone (DDQ), through an intramolecular
dehydrogenative C−N coupling reaction via a radical ion
mechanism. Biological evaluation indicated that the most
active lactam 10h displayed potent antiproliferative activity
against human cancer cells but 13.84- to 16.92-fold less
inhibitory activity on noncancer cells in vitro. In addition, 10h significantly inhibited the growth of implanted prostate cancer in
vivo. Furthermore, 10h induced cell cycle arrest and apoptosis and down-regulated the AKT/mTOR signaling in DU-145 cells.
Finally, 10h was more stable in rat plasma and human liver microsomes than CDDO-Me and had little hERG channel inhibitory
activity. Collectively, 10h may be a potential antiprostate cancer agent for further investigation.
compound, with potent in vitro and in vivo anticancer activity
comparable to CDDO-Me.11,12
INTRODUCTION
■
Oleanolic acid (OA) is a well-known natural triterpenoid and
has multiple pharmacological activities.1 To improve its potency, a
large number of synthetic OA derivatives (SOADs) have been
prepared, including 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic
acid (CDDO), its ester (CDDO-Me) and amide (CDDO-Im)
(Figure 1), and others.2,3 Compared with OA, these SOADs
γ-Lactam, like γ-lactone, is an important structural motif in a
large number of biologically active natural products13−20 and
synthetic small pharmaceutical molecules for the treatment
of cancer,13,19−21 fungal infection,20 chemotherapy-induced
nausea and vomiting,22 Alzheimer’s disease,23 and human
immunodeficient virus (HIV) infection.24 For example, the
γ-lactam moiety in the inhibitors of histone deacetylase
(HDAC) can fit into the narrow hydrophobic pocket of
HDAC active site better than the larger δ-lactam moiety and
has more potent enzymatic inhibitory and antiproliferative
activities.22 However, there currently is no effective approach to
construct γ-lactam ring directly from natural rigid polycyclic
amides. In this context, we hypothesized that synthesis of novel
olean-28,13β-lactams from their corresponding amides could
generate a new class of SOADs with potent anticancer activity.
To address the hypothesis, lactam compounds 10a−j were
designed and synthesized and their antitumor activities were
evaluated in vitro and in vivo.
Figure 1. Chemical structures of OA and SOADs.
have much stronger anti-inflammatory and antitumor activities
by activating cytoprotective pathways4 and inducing cancer cell
apoptosis.5 CDDO-Me has been tested in clinical trials for
treatment of advanced solid tumors and lymphoid malignan-
cies.6 We have also synthesized SOADs for the intervention of
cancers7−10 and discovered the olean-28,13β-olide, a γ-lactone
Received: December 24, 2014
© XXXX American Chemical Society
A
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a
Scheme 1. Synthetic Route of Olean-28,13β-lactams 10a−j
a
Reagents and conditions: (a) BnCl, K2CO3, DMF, 50 °C, 4 h; (b) Ac2O, pyridine, rt, 4 h; (c) 30% H2O2, HCO2H, CH2Cl2, rt, 24 h; (d) Br2, HBr,
AcOH, 50 °C, 24 h; (e) KOH, MeOH, reflux, 45 min; (f) Jones reagent, acetone, 0 °C → rt, 20 min; (g) HCO2Et, NaOMe, anhydrous CH2Cl2, rt,
2 h; (h) NH2OH·HCl, EtOH, reflux, 1 h; (i) H2, Pd/C, THF, atmospheric pressure, rt, 0.5 h; (j) (COCl)2, anhydrous CH2Cl2, 0 °C → rt, 12 h;
(k) primary amine, TEA, anhydrous CH2Cl2, 0 °C → rt, 12 h; (l) NaOMe, MeOH, Et2O, 0 °C → rt, 1 h; (m) DDQ, anhydrous methylbenzene,
80−110 °C, 8−24 h.
RESULTS AND DISCUSSION
Table 1. Reaction Conditions and Yields of Olean-28,13β-
lactams (10a−j)
■
a
Chemistry. The target compounds 10a−j were synthesized
as depicted in Scheme 1. A bromination reaction of ketone 1,
which was prepared starting from OA as described previously,11
followed by dehydrobromination led to enone 2. The removal
of the acetyl at 3-acyloxy group of 2 by KOH in methanol freed
the 3-hydroxy group, and the resulting compound 3 underwent
a Jones oxidation giving the diketone 4. Formylation at C-2
was performed in the presence of ethyl formate and sodium
methoxide in dichloromethane furnishing an enolate 5, which
was cyclized into isoxazole 6 by treatment with hydroxylamine
hydrochloride in aqueous ethanol. Debenzylation at C-28 of 6
was carried out in tetrahydrofuran using 10% Pd/C as the
catalyst to give free acid 7, which was converted to its acyl
chloride using oxalyl chloride, and without further purifica-
tion the acyl chloride was treated with corresponding primary
amines to yield amides 8a−j. The isoxazole ring in 8a−j was
opened in the presence of sodium methoxide, affording
2-cyanoenolates 9a−j. Finally, the 1,2-double bond and
28,13β-lactam functionalities were generated in a one-pot
procedure via a dehydrogenation reaction mediated by 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in toluene to
provide the target lactams 10a−i.
reaction
isolated
b
entry product
R
conditions
yield, %
1
2
10a
10b
10c
10d
10e
10f
10g
10h
10i
-CH3
80 °C, 4 h
80 °C, 6 h
80 °C, 6 h
80 °C, 8 h
90 °C, 12 h
90 °C, 5 h
90 °C, 10 h
90 °C, 12 h
110 °C, 24 h
reflux, >24 h
75
69
65
60
58
60
55
68
45
0
-CH2CH3
3
-CH2CH2CH3
-CH2(CH2)2CH3
-CH2(CH2)4CH3
-C(CH3)3
4
5
6
7
-CH2CH2OH
-cyclohexyl
8
9
-CH2CH2Ph
-Ph
10
10j
a
All reactions were performed with 2 equiv of DDQ in anhydrous
toluene. Yield of the isolated product based on the reacted substrate.
b
ethyl was much more difficult to be obtained than compound
10b with only an electron-donating ethyl group (110 °C,
24 h, 45% vs 80 °C, 6 h, 69%, respectively). Similarly, when a
cyclohexyl group (10h) was replaced by a phenyl group (10j),
no product was obtained. These results suggest that a smaller
and/or electron-donating substituent may be beneficial for
dehydrogenation to build a 28,13β-lactam ring.
The mechanism underlying the formation of olean-28,13β-
lactams (10a−i) promoted by DDQ may be similar to that of
the generation of olean-28,13β-olides we proposed previ-
ously.11 The 2-cyanoenone functionality is constructed from
It is notable that the yields of the last step reaction were
affected by the steric hindrance and/or electronic effect of the
substituent (R) at the nitrogen atom. As shown in Table 1,
entries 1−5, when R was occupied by bulkier group, the yields
tended to decrease. On the other hand, compound 10i con-
taining an electron-withdrawing phenyl group at 2-position of
B
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Figure 2. A proposed ionic mechanism for the dehydrogenation of ring A of compound 9.
a
Table 2. Reactions of 9h with Various Amounts of DDQ at Different Times
b
b
entry
DDQ (equiv)
time (h)
yield of 11h (%)
yield of 10h (%)
1
2
3
4
5
6
0.5
0.5
1
0.5
12
43
45
0
0
0.5
0.5
12
89
0
1.5
1.5
2
80
10
21
68
65
12
<10
a
b
All reactions were performed with DDQ at 90 °C in anhydrous toluene. Yield of the isolated products.
dehydrogenation on ring A of compound 9 via an ionic
mechanism, and the lactam ring is built from the amide moiety
in the same compound via a radical ion mechanism.
Scheme 2. Olean-28,13β-lactams (10h, 13h, and 15)
Produced from the Corresponding Amides (11h, 8h, and 14)
Promoted by DDQ
The ionic mechanism is shown in Figure 2. DDQ first
abstracts a hydrogen anion from enolate 9 leading to an oxygen
anion of DDQ (DDQH−) and a carbon cation a. Subsequently,
the DDQH− captures a proton from the intermediate a,
generating a 2-cyanoenone product 11 (as shown in Table 2)
and DDQH2.
To clarify this point, the 9h was reacted with various
amounts of DDQ for different time periods. The reaction of 9h
with ≤1 equiv of DDQ for half an hour led to generation of
only compound 11h (Table 2, entries 1−3). However, the
reaction of 9h with >1 equiv of DDQ, particularly with 2 equiv
of DDQ, for longer heating in toluene generated the lactam
10h in a dose- and time-dependent manner (Table 2, entries
4−6). These results indicated that the DDQ-mediated
dehydrogenation of ring A occurred prior to the construction
of the lactam ring and that the subsequent transformation of 11
into 10 proceeded via a distinct mechanism.
On the basis of the above results, we speculate that the
lactam 10h may be produced from the amide 11h by DDQ
promotion. Indeed, when we heated 11h with 1 equiv of DDQ
for several hours, 10h was obtained (Scheme 2). Similarly, the
lactams 13h and 15 could also be produced from the
corresponding amides 8h and 14 under the same conditions,
thus confirming our speculation.
The construction of the lactam ring may proceed via a radical
ion mechanism as depicted in Figure 3. DDQ first traps an
electon from the electron-rich enone on the ring C of 11 to
produce a radical cation b and a radical anion DDQ•−. Sub-
sequent hemolytic cleavage of the C−H bond at the C13
position results in an enol radical cation c. Then the nitrogen
atom with a pair of electrons on the C28-carboxylic amide of c
acts as a nucleophile to attack C13, followed by deprotonation
to form a lactam radical d and DDQH•. The intermediate d is
subsequently oxidized by DDQH• to generate cation e and
DDQH−. Finally, e is deprotonated by DDQH• to provide 10
and DDQH2. According to the mechanism mentioned above,
the substituent R on the nitrogen of amides 9a−j in Scheme 1
may be a rate-limiting factor, and a smaller and/or electron-
donating substituent is beneficial to build a 28,13β-lactam ring.
To verify the proposed radical ion mechanism in the
formation of lactam via an enol as a transient intermediate c
(Figure 3), compound 8h, which could be converted to the
corresponding lactam 13h as mentioned above, was treated
with acetyl chloride in the presence of DDQ. Fortunately, the
C
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Figure 3. A proposed radical ion mechanism for the DDQ-mediated formation of γ-lactam ring of 10a−j.
Table 3. Evidence for an Enol as a Transient Intermediate in
a
the DDQ-Promoted Formation of Olean-28,13β-lactam
8h
AcCl
(equiv)
DDQ
(equiv)
temp
(°C)
isolated yield of
b
entry (equiv)
12, %
1
2
3
4
5
1.0
1.0
1.0
1.0
1.0
10
0
0
90
90
25
90
90
0
Figure 4. Inhibition of DU-145 cell proliferation. DU-145 cells were
treated with, or without, the indicated compounds at 2 μM for 72 h,
and the cell proliferation was measured. The inhibition (%) of each
compound was determined. Data are presented as the mean (%) SD
of each compound from three independent experiments: (∗) P < 0.05
vs CDDO-Me; (∗∗) P < 0.01 vs 11h.
1.1
1.1
1.1
1.1
0
10
1
0
trace
21
10
a
All reactions were performed with 2 equiv of TEA and run for 24 h in
anhydrous toluene. Yield of the isolated product based on the reacted
substrate.
b
carcinoma cells), and BEL-7404 (human hepatocellular
carcinoma cells), and human nontumor cell line WPMY-1
(human prostatic stromal myofibroblast cells). The IC50 values
of 10h were from 0.33 to 0.46 μM against all four types of
cancer cells, comparable to CDDO-Me and stronger than
CDDO-Im and 10f. Significantly, 10h had 13.84- to 16.92-fold
less inhibitory activity against nontumor WPMY-1 (IC50 = 6.09
μM) than against all cancer cells tested. Hence, 10h preferably
inhibits the proliferation of cancer cells, superior to CDDO-Me
and CDDO-Im (Table 4).
In Vivo Anticancer Activity of 10h. To evaluate the safety of
10h, adult ICR mice were randomly treated by oral gavage with
a single dose of 10h at 800, 640, 512, 409.6, and 327.8 mg/kg
based on our preliminary study, and the survival of the mice
was monitored up to 14 days after treatment (Table 5). While
treatment with a large dose (800 mg/kg) of 10h killed all
mice, treatment with 327.8 mg/kg did not cause any death or
abnormality in eating, drinking, body weight, or activity
throughout the observation period. As a result, the LD50
value of 10h was 540.7 mg/kg for this strain of mice.
O-acetylated enolate 12h was successfully isolated and
identified in Table 3, suggesting that long-term heating- and
DDQ-promoted enolization of the ketone on ring C of 11 may
be involved in the generation of the lactam ring of 10, which is
in agreement with the radical ion mechanism.
Biological Activity. Assessment of in Vitro Antiprolifer-
ative Activity. Compounds 10a−i and 11h were preliminarily
screened for their inhibitory activitiy against human prostate
carcinoma DU-145 cell proliferation by CCK-8 assay using
CDDO-Me as a positive control. As shown in Figure 4, most of
compounds at 2 μM displayed varying inhibitory activities. 10h
exhibited the most potent inhibitory activity (75%), which was
statistically higher than that of its precursor amide 11h (53%)
and positive control CDDO-Me (63%). Since this study on
small numbers of compounds may have no conclusive data
for structure−activity relationship (SAR), we are interested in
further expanding the compound library to analyze the SAR.
The active compounds 10f and 10h were further assayed for
their antiproliferative activity against four human cancer cell
lines, DU-145 (human prostate cancer cells), H460 (human
non-small-cell lung cancer cells), Bxpc-3 (human pancreatic
To evaluate the in vivo anticancer activity of 10h, male
BALB/c nude mice were inoculated subcutaneously with hu-
man prostate carcinoma DU-145 cells. After the establishment
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Table 4. Antiproliferative Activity of Compounds
a
IC50 (μM)
compd
10h
DU-145
H460
Bxpc-3
BEL-7404
WPMY-1
b
b
b
b
0.39 0.03
0.96 0.08
0.69 0.05
0.53 0.03
0.38 0.03
0.43 0.04
0.42 0.04
0.40 0.02
0.33 0.03
0.66 0.06
1.50 0.12
0.24 0.02
0.46 0.03
1.14 0.10
0.99 0.07
0.81 0.07
6.09 0.53
9.60 0.81
2.09 0.14
1.86 0.11
10f
CDDO-Im
CDDO-Me
a
Data are expressed as mean IC50 SD (μM) of each compound from three independent experiments. IC50 is the drug concentration inhibiting 50%
b
of the cell proliferation. P < 0.01 vs CDDO-Me.
Table 5. Acute Toxicity of 10h in Mice
mouse mortality
a
dose (mg/kg)
no. of mice
1 h
4 h
3 d
4−14 d
total mortality
survival (%) on day 14
LD50 (mg/kg)
800
8
8
8
8
8
1
0
0
0
0
2
0
0
0
0
5
6
3
1
0
0
0
0
0
0
8
6
3
1
0
0
25
640
512
63
540.7
409.6
327.8
88
100
a
The 95% confidence limits: 475.7−614.66 mg/kg.
Figure 5. Inhibitory effects of 10h on the growth of implanted prostatic tumors in mice. Male nude mice were inoculated with DU-145 cells, and
after establishment of solid tumors, the mice were randomly treated with the indicated doses of 10h or vehicle daily, followed by monitoring of the
growth of tumors in mice: (A) dynamic growth of implanted tumors in mice; (B) tumor volumes in mice measured on the last day of the
experiment; (C) tumor weights in mice; (D) body weight changes in mice. Data are expressed as the mean SD from each group of mice (n = 5 per
group): (∗∗) P < 0.01, (∗∗∗) P < 0.001 vs the vehicle-treated control.
of solid tumor, the mice were randomly treated by gavage with
10h and vehicle daily for 21 consecutive days, respectively.
While treatment with 8.5 mg/kg of 10h slightly reduced
the volume of implanted prostatic tumors, treatment with
17 mg/kg of 10h significantly inhibited the growth of
implanted prostatic tumor (P < 0.001 vs the vehicle-treated
control, Figure 5A and Figure 5B). More importantly, the
tumor weights (0.5184 0.1668 g, 0.2786 0.1010 g) in the
mice treated with 10h at 8.5 mg/kg and 17 mg/kg were 30%
and 62% (w/w) less than that in the vehicle-treated controls
that 10h inhibited the growth of implanted tumors in vivo and
its anticancer activity tended to be dose-dependent.
Treatment with 10h Induces Prostatic Cancer Cell Cycling
Arrest and Apoptosis. To understand the mechanisms
underlying the action of 10h, the effects of 10h on prostatic
cancer cell cycling were examined. DU-145 cells were treated
with different concentrations of 10h (0.08 μM, 0.16 μM) and
CDDO-Me (0.26 μM, 0.53 μM) for 24 h and stained with PI,
followed by flow cytometry analysis (Figure 6A). As shown in
Figure 6B, treatment with 10h induced DU-145 cell cycle arrest
at G0/G1 phase and the percentages of DU-145 cells at G0/G1
phase were 68% (0.16 μM 10h) and 68% (0.53 μM CDDO-Me),
respectively, which were significantly higher than that of
(0.7356
0.1992 g, P < 0.01, Figure 5C) while the body
weights of the mice were not significantly changed by treatment
with 10h (Figure 5D). Together, our data clearly demonstrated
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Figure 6. Effects of 10h on cell-cycling of DU-145 cells. Cells were treated with the indicated concentrations of 10h and CDDO-Me for 24 h and
stained with PI, followed by flow cytometry analysis: (A) representative histograms; (B) quantitative analysis. Data are representative histograms and
expressed as the mean SD of each compound from three independent experiments: (∗∗) P < 0.01 vs the vehicle-treated controls.
vehicle-treated controls. As a result, the percentages of prostatic
cancer cells at S phase in 0.16 μM 10h-treated cells or 0.53 μM
CDDO-Me-treated cells were significantly lower than those of
vehicle-treated controls.
activation in DU-145 cells by Western blot assays. As shown in
Figure 8, there was no significant difference in the relative levels
of AKT and mTOR expression in the different groups of cells,
indicating that treatment with either 10h or CDDO-Me did not
alter the levels of AKT and mTOR expression in DU-145 cells.
Interestingly, treatment with CDDO-Me significantly reduced
the relative levels of phosphorylated AKT and mTOR in
DU145 cells, as compared with that in the vehicle-treated
controls. Similarly, treatment with either dose of 10h also
significantly decreased the relative levels of AKT phosphoryla-
tion, and treatment with a higher dose of 10h dramatically
reduced the relative levels of phosphorylated mTOR in
DU-145 cells. Therefore, 10h, like CDDO-Me, inhibited
spontaneous activation of the AKT/mTOR pathway in
DU-145 cells, which might contribute to its pharmacological action
in inhibiting the growth of implanted prostatic cancer in vivo.
The log P, Stability and Caco2 Permeability of 10h. The
log P values of each analogues were analyzed using three
methods reported previously,25−30 and they are shown in
Table 6 and Table S1 in Supporting Information. Furthermore,
we observed that following incubation with 10h or CDDO-Me
in rat plasma at 37 °C for 1 h, 46% of 10h and 32% of CDDO-Me
remained (Table 6), suggesting that 10h may be more stable in
rat plasma than CDDO-Me. Similarly, 10h displayed a longer
half-life than CDDO-Me (39 vs 21 min) in human liver micro-
somes (HLM) (Table 6).
Tumor cells arrested at G0/G1 phase are associated with
high sensitivity to apoptotic triggers. Next, the impact of 10h
on the apoptosis of DU-145 cells was examined by Hoechst
33258 staining. As shown in Figure 7A, the vehicle-treated
prostatic cancer cells (0.1% DMSO) displayed weak homoge-
neous blue in the nuclei while the CDDO-Me (0.53 μM) and
10h (0.08−0.32 μM) treated prostatic cancer cells exhibited
bright chromatin condensation and nuclear fragmentation, a
hallmark of apoptosis. In comparison with that of the vehicle-
treated control DU-145 cells, treatment with 0.08, 0.16, or
0.32 μM 10h, like with 0.53 μM CDDO-Me, significantly
reduced the relative levels of BcL-2 expression, although the
treatment did not alter the relative levels of Bax expression
in DU-145 cells (Figure 7B). As a result, these treatments
obviously increased the ratios of Bax to BcL-2, which was
associated with cell apoptosis. In addition, these treatments also
significantly increased the activity of caspase-3 in DU-145 cells
(Figure 7C). These three independent lines of evidence
indicated that treatment with 10h induced prostatic cancer
cell apoptosis.
Treatment with 10h Inhibits the AKT/mTOR Signaling in
DU-145 Cells. The PI3K/AKT/mTOR pathway is crucial for
the growth of many types of tumors. Finally, we examined the
impact of treatment with 10h on the AKT and mTOR
In addition, we characterized the potential ring-opened prod-
ucts or adduct products by LC−MS/MS following incubation
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Figure 7. Effects of 10h on induction of DU-145 cell apoptosis. DU-145 cells were treated with the indicated concentrations of compound or vehicle
(control) for 24 h. The cells were stained with Hoechst 33258 and examined under a fluorescent microscope. Furthermore, the relative levels of Bax
and BcL-2 as well as the levels of caspase 3 activity in the different groups of cells were determined by Western blot and enzymatic assays,
respectively. Data are representative images and expressed as the mean SD of each group of cells from three separate experiments: (A) fluorescent
staining of tumor cells; (B) Western blot analysis of the levels of Bax and BcL-2 expression; (C) levels of caspase-3 activity; (∗∗) P < 0.01 vs the
vehicle-treated control.
Figure 8. Effects of 10h on the AKT/mTOR activation in DU-145 cells. DU-145 cells were treated with vehicle or the indicated compound for 24 h,
and the relative levels of AKT and mTOR expression and phosphorylation were determined by Western blot assays using β-actin as a control. Data
are representative images and expressed as the mean SD of each group of cells from three separate experiments: (A) Western blot analysis of the
relative levels of AKT and mTOR expression and phosphorylation; (B) quantitative analysis; (∗∗) P < 0.01 vs control.
Table 6. The log P, Rat Plasma Stability, Microsomal Stability, and Caco2 Permeability Data of 10h and CDDO-Me
d
stability
Caco2 (Papp × 10−7 cm/s)
a
b
f
c
e
compd
10h
CDDO-Me
log P
1 h (%)
t1/2 (min)
A to B
B to A
efflux ratio
f
6.7 0.1
5.5 0.3
46 3*
32
39 4*
0.45 0.21
1.42 0.84
0.59 0.17
2.40 1.45
1.31
1.69
3
21
2
a
b
The log P values were calculated using the methods reported previously. Data are expressed as the mean SD from three different programs. Rat
c
d
plasma stability, the percentages of the tested compound remaining after incubations. Half-life (t1/2) in human liver microsomes. Apparent
permeability measured in Caco2 cells. “A to B” indicates the experiment from apical to basolateral, and “B to A” indicates the experiment from
e
f
basolateral to apical. Ratio BA/AB permeability coefficients. (∗) P < 0.05 vs the CDDO-Me.
of 10h with nucleophiles (Cys and GSH), in rat plasma, or
human liver microsomes (HLM) at 37 °C for 1 h. As shown in
Table S2, there were no detectable ring-opened products,
although there were some adduct products following incubation
of 10h with the nucleophiles. These adduct products might
stem from addition of the nucleophiles to ring A of 10h, similar
to that of CDDO-Me under the same conditions. These results
suggest that γ-lactam ring of 10h may be relatively stable in the
rat plasma and HLM.
Next, the Caco2 permeability of 10h was determined by the
Caco2 bidirectional permeability assays. We found that the
permeability data of 10h (Papp,A→B, from apical to basolateral
direction) and (Papp,B→A from basolateral to apical direction)
were 0.45 × 10−7 and 0.59 × 10−7 cm/s, respectively. The efflux
ratio, defined as the ratio of Papp,B→A to Papp,A→B, was 1.31
(Table 6). These data suggest that 10h might not be a substrate
of P-gp transporters, and it may possess a reasonable absorption
rate in human intestine.
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Article
hERG Channel Inhibition. The human ether-a-go-go-related
gene (hERG) codes the α subunit of a potassium ion channel
(Kv11.1), and inhibition of the channel can result in long QT
syndrome, a potential fetal disorder associated with severe
adverse effect of drugs.31 Accordingly, we tested the potential
inhibition of 10h on the hERG channel activity by the patch-
clamp assay in Chinese hamster ovary (CHO) cells that were
stably expressing the hERG channels. Notably, 10h exhibited
significantly lower hERG inhibitory activity (IC50 > 40 μM)
than the control cisapride (IC50 = 0.04 μM), suggesting that
10h may have little cardiac toxicity (Table 7).
cancer cell cycle arrest at G0/G1 phase and apoptosis in vitro,
increased ratios of Bax to BcL-2 as well as decreased spon-
taneous activation of the AKT/mTOR pathway in DU-145
cells. Finally, 10h was more stable in rat plasma and human
liver microsomes than CDDO-Me and had little hERG channel
inhibitory activity. These findings may provide a proof of
principle that our novel and practical strategy for building of
γ-lactam ring from natural rigid polycyclic amides may have
broad applications in drug discovery and development field and
that olean-28,13β-lactams may be potential chemotherapeutic
agents for the intervention of prostate cancer.
Table 7. Inhibitory Activity of 10h on the hERG Channel by
Patch-Clamp Assay
EXPERIMENTAL PROTOCOLS
■
Chemical Analysis. Melting points were determined on a
Mel-TEMP II melting point apparatus and were uncorrected. Infrared
(IR) spectra (KBr) were recorded on a Nicolet Impact 410 instrument
a
compd
hERG channel inhibition, IC50 (μM)
b
10h
>40*
1
(KBr pellet). H NMR and 13C NMR spectra were recorded with a
cisapride
0.04
Bruker Avance 300 MHz spectrometer at 300 K, using TMS as an
internal standard. MS spectra were recorded on a Mariner mass
spectrometer (ESI) and high resolution mass spectrometry (HRMS)
spectra on an Agilent Technologies LC/MSD TOF instrument.
Analytical and preparative TLC was performed on silica gel (200−300
mesh) GF/UV 254 plates, and the chromatograms were visualized
under UV light at 254 and 365 nm. All solvents were reagent grade
and, when necessary, were purified and dried by standard methods.
Solutions after reactions and extractions were concentrated using a
rotary evaporator operating at a reduced pressure of ∼20 Torr. The
purity of all tested compounds was characterized by HPLC analysis
(LC-10A HPLC system consisting of LC-10ATvp pumps and an
SPD-10Avp UV detector). Individual compounds with a purity of
>95% were used for subsequent experiments (see the Supporting
Information). Oleanolic acid (OA) was commercially available.
Compound 1 was prepared according to the method described
previously.11
a
Experiments were performed using Chinese hamster ovary (CHO)
cells that had been stably transfected with the hERG. CHO cells were
treated with different concentrations of 10h (0.0128, 0.064, 0.32,
1.6, 8, and 40 μM) or the control cisapride (0.00096, 0.0048, 0.024,
0.12, 0.6, and 6 μM), and their whole-cell currents were recorded using
an automated patch-clamp system (Qpatch). Data are expressed as the
IC50 of each compound from three separate experiments. No hERG
inhibition detectable at the highest measured concentration (40 μM).
(∗) P < 0.001 vs cisapride.
b
CYP450 Inhibition of Selective Compounds. Drug-related
adverse effects are a major challenge of drug development32 and
may be attributed to the inhibition or induction of members of
the cytochrome P450 enzyme family.33 To further evaluate the
potential adverse effect of 10h, the inhibition of 10h on five
major CYP450 enzymes was determined in human liver micro-
somes using CDDO-Me as a reference. As shown in Table 8,
both 10h and CDDO-Me had little inhibitory activity against
CYP1A2 and CYP2D6 (IC50 > 100 μM) and medium to high
inhibition on CYP2C19 and CYP3A4 (IC50 = 0.932−19.829 μM).
In addition, 10h had significantly lower inhibition on CYP2C9
(IC50 > 100 μM) than CDDO-Me (IC50 = 8.543 μM).
Benzyl 3β-Acetoxy-12-oxoolean-9(11)-en-28-oate (2). To a
stirred solution of 1 (52.5 g, 87 mmol) in glacial acetic acid (100 mL)
was added dropwise hydrobromic acid (4.4 mL, 38 mmol) at room
temperature. The reaction mixture was then heated to 50 °C, and
bromine (5.6 mL, 104 mmol) was thus added dropwise. The resulting
reaction mixture was kept stirring for another 24 h. After completion
of the reaction monitored by TLC, it was quenched by 20% aqueous
sodium thiosulfate (100 mL) and then diluted with water (1.5 L) and
filtered. The solid was washed with water, dried, and subjected to flash
column chromatography over silica gel using petroleum ether/EtOAc
(9:1) to give a yellowish solid 2 (42 g, 80%). Mp 216−218 °C. IR
(KBr, cm−1): 3410, 2954, 2873, 1733, 1652, 1465, 1385, 1368, 1329,
CONCLUSIONS
■
In summary, we have designed and synthesized a group of
olean-28,13β-lactam compounds 10a−j. This is the first report
on the synthesis of γ-lactams from natural rigid polycyclic
amides, promoted by an easily available reagent DDQ, through
the intramolecular dehydrogenative C−N coupling reaction via
a radical ion mechanism. We found that compound 10h had
potent and selective antiproliferative activity against several
types of human cancer cells in vitro. Furthermore, 10h showed
a relatively lower acute toxicity in mice, and treatment with 10h
significantly inhibited the growth of implanted prostatic cancer
in mice. In addition, treatment with 10h induced prostatic
1
1242. ESI-MS: 603 [M + H]+. H NMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 7.34−7.26 (m, 5H), 5.7 (s, 1H), 5.16 (d, J = 12.6 Hz, 1H),
5.11 (d, J = 12.6 Hz, 1H), 4.46−4.40 (m, 1H), 3.03−3.00 (m, 1H),
2.93 (d, J = 4.5 Hz, 1H), 2.05 (s, 3H), 2.01 (s, 3H), 1.57 (s, 3H), 1.16
(s, 3H), 1.00 (s, 3H), 0.95 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H) ppm.
13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 200.2, 177.8, 177.5,
170.1, 136.4, 128.5, 128.5, 128.4, 128.4, 128.1, 122.8, 79.7, 66.1, 50.3,
49.4, 47.2, 45.2, 41.7, 39.8, 38.2, 36.1, 35.9, 34.6, 33.3, 32.9, 32.8, 31.6,
30.7, 28.0, 28.0, 23.9, 23.9, 23.5, 23.2, 22.8, 21.6, 21.2, 17.8, 16.7 ppm.
Benzyl 3β-Hydroxy-12-oxoolean-9(11)-en-28-oate (3). A
solution of 2 (42 g, 70 mmol) and KOH (39 g, 0.7 mol) in MeOH
Table 8. CYP450 Inhibition by 10h and CDDO-Me
a
human CYP450 inhibition (IC50, μM)
b
c
compd
10h
CDDO-Me
1A2
2C9
2C19
2D6
3A4 (M)
3A4 (T)
d
>100
>100
>100*
8.543
8.107
>100
>100
0.932
4.780
2.535
2.740
19.829
a
b
c
IC50 refers to the concentration of a compound that causes 50% inhibition of the enzyme activity. Midazolam was used as substrate. Testosterone
d
was used as substrate. (∗) P < 0.01 vs CDDO-Me.
H
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Article
(100 mL) was heated under reflux for 45 min. After removal of MeOH
in vacuum, the resulting mixture was acidified with aqueous HCl
(6 M) solution. The aqueous layer was extracted with a mixture of
CH2Cl2 and Et2O (1:2) (three times). The combined organic layers
were washed with saturated sodium hydrogen carbonate solution and
brine and dried over magnesium sulfate. The solvent was removed to
give an amorphous solid 3 (39 g, quantitative), which was used for the
next reaction without further purification. An analytically pure sample
was obtained by recrystallization from methanol. Mp 197−198 °C. IR
(KBr, cm−1): 3506, 2953, 2930, 2867, 1715, 1647, 1587, 1456, 1382,
solution (three times), dried over MgSO4, and filtered. The filtrate
was evaporated in vacuo to give a solid. The solid was subjected to
flash column chromatography (PE/EtOAc = 3:1) to give 6 as an
amorphous solid (25.6 g, 83%). Mp 120−122 °C. IR (KBr, cm−1):
3434, 2947, 2861, 2353, 2312, 1722, 1660, 1581, 1456, 1384. ESI-MS:
584 [M + H]+. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.05 (s,
1H), 7.34−7.26 (m, 5H), 5.8 (s, 1H), 5.16 (d, J = 12.6 Hz, 1H), 5.11
(d, J = 12.6 Hz, 1H), 3.10−3.00 (m, 1H), 2.80 (d, J = 4.5 Hz, 1H),
2.75 (d, J = 15 Hz, 1H), 3.38 (d, J = 15 Hz, 1H), 1.37 (s, 3H), 1.26 (s,
3H), 1.12 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.90 (s,
3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.8, 177.4,
176.0, 172.1, 150.2, 136.3, 128.6, 128.6, 128.5,128.5, 128.1, 124.5,
108.6, 66.2, 49.8, 49.5, 47.2, 45.6, 41.8, 41.3, 36.0, 35.2, 34.6, 33.7,
33.3, 32.9, 31.6, 31.3, 30.7, 28.8, 28.2, 24.6, 23.2, 22.8, 22.8, 21.6, 21.4,
18.3 ppm.
12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oate (7). Com-
pound 6 (2 g, 3.4 mmol) was stirred under H2 in the presence of
10% palladium carbon (0.4 g) in THF for 0.5 h. The mixture was
filtered through a pad of Celite, and the filtrate was dried and
concentrated to offer 7 as a white solid (1.5 g, 91%). Mp 224−226 °C.
IR (KBr, cm−1): 3455, 2939, 2867, 1706, 1659, 1632, 1478, 1454,
1384,1342. ESI-MS: 492 [M − H]−. 1H NMR (300 M Hz, CDCl3, 25
°C, TMS): δ 8.05 (s, 1H), 5.8 (s, 1H), 3.03−3.00 (m, 1H), 2.93 (d, J =
4.5 Hz, 1H), 2.78 (d, J = 15.3 Hz, 1H), 2.40 (d, J = 15.3 Hz, 1H), 1.35
(s, 3H), 1.31 (s, 3H), 1.26 (s, 3H), 1.16 (s, 3H), 1.01 (s, 3H), 0.97 (s,
3H), 0.85 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ
199.8, 183.8, 176.1, 172.1, 150.1, 124.5, 108.5, 49.7, 49.6, 47.1, 45.7,
41.8, 41.3, 35.8, 35.1, 34.5, 33.6, 33.2, 33.0, 31.4, 31.3, 30.6, 28.8, 28.2,
24.5, 23.1, 23.0, 22.6, 21.5, 21.4, 18.2 ppm.
1
1236. ESI-MS m/z: 561 [M + H]+. H NMR (300 M Hz, CDCl3, 25
°C, TMS): δ 7.34−7.26 (m, 5H), 5.7 (s, 1H), 5.16 (d, J = 12.6 Hz,
1H), 5.11 (d, J = 12.6 Hz, 1H), 3.22−3.17 (m, 1H), 3.03−3.00 (m,
1H), 2.25 (d, J = 4.5 Hz, 1H), 1.14 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H),
0.96 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.81 (s, 3H) ppm. 13C NMR
(75 M Hz, CDCl3, 25 °C, TMS): δ 200.4, 178.5, 177.5, 136.3, 128.5,
128.5, 128.4, 128.4, 128.1, 122.6, 77.8, 66.1, 50.2, 49.3, 47.2, 45.3, 41.7,
40.0, 39.3, 36.5, 35.9, 34.5, 33.3, 32.9, 32.8, 31.6, 30.7, 28.1, 28.0, 27.5,
23.8, 23.4, 23.2, 22.8, 21.7, 18.0, 15.7 ppm.
Benzyl 3,12-Dioxoolean-9(11)-en-28-oate (4). To a solution of
3 (39 g, 0.07 mol) in acetone (150 mL) in an ice bath was added Jones
reagent dropwise until the color of the solution changed from green to
pale brown. The mixture was stirred at room temperature for 10 min.
After removal of acetone, water was added to quench the reaction. The
aqueous mixture was extracted with a mixture of CH2Cl2 and Et2O
(1:2) (three times). The combined organic layers were washed with
saturated sodium hydrogen carbonate solution and brine and dried
over magnesium sulfate. The solvent was removed and the crude
product was purified by column chromatography (PE/EtOAc = 6:1)
to obtain 4 (34 g, 88%) as an amorphous solid. Mp 146−147 °C. IR
(KBr, cm−1): 3421, 2946, 2861, 2359, 1723, 1708, 1662, 1599, 1466,
General Procedure for the Preparation of 8a−j. A mixture of
7 (3.0 g, 6.1 mmol) and oxalyl chloride (5 mL) in anhydrous CH2Cl2
(50 mL) was stirred at room temperature overnight. The solvent was
removed in vacuo, and the residue was coevaporated with anhydrous
CH2Cl2 three times, and then used for the next reaction without
further purification.
1
1383. ESI-MS m/z: 559 [M + H]+. H NMR (300 M Hz, CDCl3,
25 °C, TMS): δ 7.38−7.28 (m, 5H), 5.8 (s, 1H), 5.16 (d, J = 12.6 Hz,
1 H), 5.11 (d, J = 12.6 Hz, 1H), 3.06−3.00 (m, 1H), 2.78 (d, J = 4.5
Hz, 1H), 2.66−2.17 (m, 3H), 1.26 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H),
1.00 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.89 (s, 3H) ppm. 13C NMR
(75 M Hz, CDCl3, 25 °C, TMS): δ 215.7, 199.9, 177.4, 176.6, 136.3,
128.5, 128.5, 128.4, 128.4, 128.2, 124.0, 50.9, 49.5, 47.5, 47.2, 45.4,
41.8, 39.4, 37.0, 35.9, 34.5, 34.2, 33.3, 32.8, 31.9, 31.6, 30.7, 29.7, 28.0,
26.3, 23.8, 23.3, 23.2, 22.8, 21.6, 21.4, 19.1 ppm.
To a solution of the residue (178 mg, 0.35 mmol) obtained above
in anhydrous CH2Cl2 (2 mL) was added a solution of primary amine
(1.5 mmol). The mixture was stirred at room temperature overnight.
The organic layer was separated and then diluted with CH2Cl2
(15 mL). It was washed with saturated sodium hydrogen carbonate
solution and brine and dried over magnesium sulfate. The solvent
was removed and the crude product was purified by flash column
chromatography to obtain 8a−j as an amorphous solid.
Benzyl 2-Hydroxymethylene-3,12-dioxoolean-9(11)-en-28-oate
(5). To a solution of 4 (34 g, 0.06 mol) in dry CH2Cl2 (100 mL) were
added ethyl formate (97%) (18 g, 0.24 mol) and NaOMe (19.5 g,
0.36 mol). The mixture was stirred at room temperature for 2 h. Then
the mixture was diluted with a mixture of CH2Cl2 and Et2O (1:2) and
washed with 5% aqueous HCl solution (three times). The washings
were re-extracted with a mixture of CH2Cl2 and Et2O (1:2), and the
combined organic layers were washed with saturated sodium hydrogen
carbonate solution and brine and dried over magnesium sulfate. The
solvent was removed and the crude product was purified by column
chromatography (PE/EtOAc = 5:1) to obtain 5 (31.5 g, 90%) as an
amorphous solid. Mp 110−114 °C. IR (KBr, cm−1): 3463, 2950, 2869,
1758, 1723, 1661, 1596, 1456, 1384, 1365. ESI-MS: 587 [M + H]+. 1H
NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 14.85 (d, J = 2.4 Hz, 1H),
8.75 (s, 1H), 7.34−7.26 (m, 5H), 5.7 (s, 1H), 5.16 (d, J = 12.6 Hz,
1H), 5.11 (d, J = 12.6 Hz, 1H), 3.03−3.00 (m, 1H), 2.83 (d, J = 4.5
Hz, 1H), 2.61 (d, J = 14.4 Hz, 1H), 2.25 (d, J = 14.4 Hz, 1H), 1.24 (s,
3H), 1.15 (s, 3H), 1.14 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H), 0.94 (s,
3H), 0.85 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ
200.0, 189.8, 188.3, 177.5, 175.7, 136.3, 128.6, 128.6, 128.5, 128.5,
128.2, 124.2, 105.0, 66.2, 49.5, 48.3, 47.2, 45.5, 42.3, 40.5, 39.1, 37.1,
36.0, 34.6, 33.3, 32.9, 31.6, 31.3, 30.7, 29.7, 28.3, 28.1, 23.4, 23.1, 22.8,
21.6, 20.8, 18.9 ppm.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]methylamide
(8a). The title product was obtained in 70% yield as a white powder.
Mp 175−177 °C. IR (KBr, cm−1): 3128, 2971, 2867, 1658, 1526,
1
1400. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H),
5.99−5.95 (m, 1H), 5.89 (s, 1H), 3.05 (d, J = 4.4 Hz, 1H), 2.90−2.76
(m, 5H), 2.44 (d, J = 15.1 Hz, 1H), 1.35 (s, 3H), 1.30 (s, 3H), 1.27 (s,
3H), 1.16 (s, 3H), 1.04 (s, 3H), 1.00 (s, 3H), 0.91 (s, 3H) ppm. 13C
NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 200.2, 177.8, 176.5, 172.0,
150.1, 124.3, 108.4, 49.6, 49.3, 46.6, 45.7, 41.7, 41.2, 36.2, 35.1, 34.6,
34.0, 33.5, 33.2, 32.0, 31.2, 30.6, 28.7, 28.0, 26.5, 24.5, 23.1, 23.1, 22.9,
21.7, 21.3, 18.2. ESI-MS: 529 [M + Na]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]ethylamide
(8b). The title product was obtained in 67% yield as a white powder.
Mp 155−157 °C. IR (KBr, cm−1): 3128, 2971, 2867, 1658, 1526,
1
1400. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H),
5.89−5.87 (m, 2H), 3.28−3.20 (m, 2H), 3.05 (d, J = 4.4 Hz, 1H), 2.87
(d, J = 13.1 Hz, 1H), 2.81 (d, J = 15.2 Hz, 1H), 2.43 (d, J = 15.0 Hz,
1H), 1.35 (s, 3H), 1.30 (s, 3H), 1.27 (s, 3H), 1.16 (s, 3H), 1.04 (s,
3H), 1.01 (s, 3H), 0.91 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3,
25 °C, TMS): δ 199.7, 176.4, 176.1, 171.6, 149.7, 123.9, 108.0, 49.1,
48.9, 46.0, 45.4, 41.3, 40.8, 35.8, 34.7, 34.1, 33.9, 33.6, 33.1, 32.8, 31.5,
30.8, 30.1, 28.3, 27.5, 24.1, 22.8, 22.6, 22.4, 21.3, 20.9, 17.8, 14.7. ESI-
MS: 543 [M + Na]+.
Benzyl 12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oate (6).
To a solution of 5 (31 g, 53 mmol) in EtOH (110 mL) and water
(10 mL) was added hydroxylamine hydrochloride (36.8 g, 530 mmol).
The mixture was heated under reflux for 1 h. The mixture was
concentrated in vacuo, and water (50 mL) was added. The mixture
was extracted with EtOAc (three times). The combined organic layers
were washed with water (three times) and saturated aqueous NaCl
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]-n-propylamide
(8c). The title product was obtained in 88% yield as a white powder.
Mp 159−161 °C. IR (KBr, cm−1): 3128, 2966, 2872, 1660, 1523,
1
1400. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H),
I
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5.94−5.89 (m, 2H), 3.27 (q, J = 6.4, 6.8 Hz, 2H), 3.05 (d, J = 4.2 Hz,
1H), 2.89 (d, J = 12.7 Hz, 1H), 2.81 (d, J = 15.1 Hz, 1H), 2.43 (d, J =
15.0 Hz, 1H), 1.35 (s, 3H), 1.30 (s, 3H), 1.27 (s, 3H), 1.16 (s, 3H),
1.04 (s, 3H), 1.01 (s, 3H), 0.91 (s, 3H) ppm. 13C NMR (75 M Hz,
CDCl3, 25 °C, TMS): δ 199.7, 176.6, 176.1, 171.6, 149.7, 123.9, 108.0,
49.2, 48.9, 46.1, 45.3, 41.3, 40.8, 40.8, 35.8, 34.6, 34.1, 33.7, 33.1, 32.8,
31.5, 30.8, 30.1, 28.3, 27.5, 24.1, 22.8, 22.7, 22.6, 22.5, 21.3, 20.9, 17.8,
11.0. ESI-MS: 557 [M + Na]+.
NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.06 (s, 1H), 7.32−7.18
(m, 5H), 5.92 (t, J = 5.4 Hz, 1H), 5.87 (s, 1H), 3.58−3.49 (m, 2H),
2.97 (d, J = 4.4 Hz, 1H), 2.83−2.76 (m, 4H), 2.42 (d, J = 15.1, 1H),
1.34 (s, 3H), 1.26 (s, 3H), 1.19 (s, 3H), 1.15 (s, 3H), 1.10 (s, 3H),
1.00 (s, 3H), 0.96 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C,
TMS): δ 199.5, 176.6, 175.9, 171.6, 149.7, 149.7, 138.4, 128.2, 128.2,
126.2, 126.2, 123.9, 108.0, 49.1, 48.8, 46.1, 45.3, 41.3, 40.7, 40.3, 35.7,
35.3, 34.6, 34.1, 33.6, 33.1, 32.8, 31.4, 30.7, 30.1, 28.3, 27.4, 24.1, 22.7,
22.6, 22.4, 21.2, 20.9, 17.7. ESI-MS: 619 [M + Na]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]-n-butylamide
(8d). The title product was obtained in 78% yield as a white powder.
Mp 151−153 °C. IR (KBr, cm−1): 3128, 2954, 2871, 1659, 1523,
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]phenylamide
(8j). The title product was obtained in 60% yield as a white powder.
1
Mp 157−159 °C. IR (KBr, cm−1): 3135, 2950, 1655, 1522, 1400. H
1
1400. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H),
NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.05 (s, 1H), 7.49−7.44
(m, 3H), 7.32 (t, J = 7.6, 8.2 Hz, 2H), 7.10 (t, J = 7.3 Hz,1H), 5.88 (s,
1H), 3.16 (d, J = 4.4 Hz, 1H), 3.00 (d, J = 12.7 Hz, 1H), 2.73 (d, J =
15.0, 1H), 2.36 (d, J = 15.0, 1H), 1.33 (s, 3H), 1.29 (s, 3H), 1.25 (s,
3H), 1.13 (s, 3H), 1.16 (s, 3H), 1.04 (s, 3H), 0.96 (s, 3H) ppm. 13C
NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.5, 176.6, 175.2, 171.8,
149.8, 137.4, 128.5, 128.5, 123.9, 123.9, 120.0, 120.0, 108.0, 49.1, 48.8,
47.0, 45.3, 41.3, 40.8, 35.7, 34.6, 34.1, 33.6, 33.2, 33.1, 31.6, 30.7,
30.2, 28.3, 27.4, 24.0, 22.8, 22.7, 22.7, 21.2, 20.9, 17.9. ESI-MS: 591
[M + Na]+, 607 [M + K]+.
General Procedure for the Preparation of the Mixture of
Tautomers (9a−j). To a solution of 8a−j (3.94 mmol) obtained
above in MeOH (20 mL) and Et2O (50 mL) in an ice bath was added
NaOMe (7.25 g, 134 mmol). The mixture was stirred at room
temperature for 45 min and then diluted with a mixture of CH2Cl2 and
Et2O (1:2). It was washed with 5% aqueous HCl solution (three
times), and the acidic washings were reextracted with a mixture of
CH2Cl2 and Et2O (1:2). The combined organic layers were washed
with saturated sodium hydrogen carbonate solution and brine and
dried over magnesium sulfate. The solvent was removed to give a
mixture of tautomers 9a−j (quantitative). This material was used for
the next reaction without further purification.
General Procedure for the Preparation of 10a−j. A mixture of
9a−j (1 mmol) obtained above and DDQ (0.5 g, 2.2 mmol) in dry
methylbenzene (4 mL) was heated to 80−110 °C for 8−24 h. After
insoluble matter was removed by filtration, the filtrate was evaporated
in vacuo to give a solid. The solid was subjected to flash column
chromatography to give 10a−i as an amorphous solid.
Compound 10a. The title compound was obtained in 75% yield as a
white powder. Mp 168−170 °C. IR (KBr, cm−1): 3134, 2971, 2873,
1686, 1614, 1400. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 7.98
(s, 1H), 5.85 (s, 1H), 2.78−2.60 (m, 4H), 1.47 (s, 3H), 1.26 (s, 3H),
1.17 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H), 0.88 (s, 3H), 0.85 (s, 3H)
ppm. 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 198.7, 196.1,
177.2, 168.4, 165.3, 123.4, 114.1, 113.9, 87.0, 49.0, 47.2, 46.0, 45.4,
44.5, 42.1, 41.6, 35.6, 34.1, 33.5, 32.8, 31.6, 31.2, 30.1, 29.2, 27.3, 26.5,
24.3, 22.6, 22.4, 21.3, 21.1, 17.7. ESI-MS: 503 [M + H]+, 525 [M +
Na]+. HRMS: calculated for C32H43N2O3 [M + H]+ 503.3274, found
503.3281.
Compound 10b. The title compound was obtained in 69% yield as a
white powder. Mp 166−168 °C. IR (KBr, cm−1): 3134, 2971, 2873,
1686, 1614, 1400. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.00
(s, 1H), 5.85 (s, 1H), 3.14 (q, J = 7.1, 2H), 2.87 (t, J = 8.8, 1H), 1.45
(s, 3H), 1.28 (s, 3H), 1.25 (s, 3H), 1.15 (s, 3H), 1.04 (s, 3H), 1.01 (s,
3H), 0.99 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ
201.5, 196.0, 165.2, 162.8, 160.8, 120.0, 114.3, 113.2, 85.5, 52.1, 51.4,
45.6, 45.2, 44.9, 39.7, 36.9, 36.7, 34.4, 34.0, 33.5, 33.4, 31.1, 27.7,
25.5, 24.3, 23.6, 23.0, 21.6, 21.1, 20.2, 20.2, 19.0, 15.5. ESI-MS: 517
[M + H]+, 539 [M + Na]+, 555 [M + K]+. HRMS: calculated for
C33H45N2O3 [M + H]+ 517.3430, found 517.3416.
Compound 10c. The title compound was obtained in 65% yield as a
white powder. Mp 197−199 °C. IR (KBr, cm−1): 3130, 2962, 1755,
1686, 1614, 1400. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 7.95
(s, 1H), 5.86 (s, 1H), 3.10 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 9.2, 1H),
1.47 (s, 3H), 1.25 (s, 3H), 1.15 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H),
0.88 (s, 3H), 0.85 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C,
TMS): δ 196.4, 193.0, 173.1, 165.6, 164.1, 123.5, 115.0, 114.3, 87.0,
48.3, 47.9, 46.7, 45.1, 44.9, 42.9, 42.7, 36.7, 34.4, 34.0, 33.3, 32.8, 31.7,
5.95−5.89 (m, 1H), 5.89 (s, 1H), 3.28 (q, J = 6.4, 6.7 Hz, 2H), 3.06
(d, J = 4.4 Hz, 1H), 2.90 (d, J = 13.0 Hz, 1H), 2.82 (d, J = 15.1 Hz,
1H), 2.43 (d, J = 15.1 Hz, 1H), 1.35 (s, 3H), 1.30 (s, 3H), 1.27 (s,
3H), 1.17 (s, 3H), 1.04 (s, 3H), 1.01 (s, 3H), 0.91 (s, 3H) ppm. 13C
NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 199.7, 176.5, 176.0, 171.6,
149.8, 123.9, 108.0, 49.1, 48.9, 46.1, 45.3, 41.3, 40.8, 38.8, 35.8, 34.6
34.1, 33.7, 33.1, 32.8, 31.5, 31.4, 30.8, 30.1, 28.3, 27.5, 24.1, 22.8, 22.6,
22.5, 21.3, 20.9, 19.7, 17.8, 13.3. ESI-MS: 571 [M + Na]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]-n-hexylamide
(8e). The title product was obtained in 69% yield as a white powder.
Mp 142−144 °C. IR (KBr, cm−1): 3129, 2932, 2860, 1600, 1524,
1
1399. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H),
5.92−5.89 (m, 2H), 3.30 (q, J = 6.0, 7.0 Hz, 2H), 3.06 (d, J = 4.4 Hz,
1H), 2.89 (d, J = 13.1 Hz, 1H), 2.81 (d, J = 15.1 Hz, 1H), 2.43 (d, J =
15.0 Hz, 1H), 1.35 (s, 3H), 1.31 (s, 3H), 1.27 (s, 3H), 1.17 (s, 3H),
1.04 (s, 3H), 1.01 (s, 3H), 0.91 (s, 3H) ppm. 13C NMR (75 M Hz,
CDCl3, 25 °C, TMS): δ 200.0, 176.9, 176.4, 172.0, 150.1, 124.4, 108.4,
49.6, 49.3, 46.5, 45.8, 41.8, 41.2, 39.6, 36.2, 35.1, 34.6, 34.1, 33.5, 33.2,
32.0, 31.4, 31.3, 30.6, 29.8, 28.7, 28.0, 26.6, 24.5, 23.3, 23.1, 23.0, 22.5,
21.7, 21.3, 18.2, 13.9. ESI-MS: 599 [M + Na]+, 615 [M + K]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]-tert-butylamide
(8f). The title product was obtained in 73% yield as a white powder.
Mp 166−168 °C. IR (KBr, cm−1): 3128, 2969, 2871, 1660, 1512,
1400. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H), 5.88
(s, 1H), 5.53 (s, 1H), 3.10 (d, J = 4.3 Hz, 1H), 2.86−2.76 (m, 2H),
2.43 (d, J = 15.0 Hz, 1H), 1.35 (s, 3H), 1.29 (s, 3H), 1.27 (s, 3H), 1.17
(s, 3H), 1.03 (s, 3H), 1.00 (s, 3H), 0.90 (s, 3H) ppm. 13C NMR (75
M Hz, CDCl3, 25 °C, TMS): δ 199.5, 175.8, 175.6, 171.6, 149.7, 124.0,
108.0, 50.3, 49.2, 48.7, 46.2, 45.4, 41.3, 40.7, 35.8, 34.6, 34.2, 33.7,
33.1, 32.8, 31.5, 30.8, 30.1, 28.3, 28.3, 28.3, 28.3, 27.5, 24.0, 22.7, 22.7,
22.6, 21.2, 20.9, 17.8. ESI-MS: 571 [M + Na]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]ethoxylamide
(8g). The title product was obtained in 90% yield as a white powder.
1
Mp 162−164 °C. IR (KBr, cm−1): 3129, 3009, 1655, 1620, 1400. H
NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.08 (s, 1H), 6.55 (m, 1H),
5.89 (s, 1H), 3.72−3.62 (m, 2H), 3.47−3.37 (m, 3H), 3.07−3.00 (m,
1H), 2.94 (d, J = 12.9 Hz, 1H), 2.81 (d, J = 15.0 Hz, 1H), 2.43 (d, J =
15.0 Hz, 1H), 1.35 (s, 3H), 1.32 (s, 3H), 1.27 (s, 3H), 1.17 (s, 3H),
1.04 (s, 3H), 1.00 (s, 3H), 0.91 (s, 3H) ppm. 13C NMR (75 M Hz,
CDCl3, 25 °C, TMS): δ 199.9, 178.4, 176.3, 171.6, 149.7, 123.9, 108.0,
62.4, 49.1, 48.9, 46.2, 45.4, 42.2, 41.3, 40.8, 35.7, 34.7, 34.1, 33.6, 33.1,
32.8, 31.4, 30.8, 30.1, 28.3, 27.5, 24.1, 22.7, 22.6, 22.5, 21.3, 20.9, 17.7.
ESI-MS: 559 [M + Na]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]cyclohexylamide
(8h). The title product was obtained in 88% yield as a white powder.
1
Mp 184−186 °C. IR (KBr, cm−1): 2933, 2856, 1655, 1517, 1385. H
NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H), 5.89 (s, 1H),
5.65 (d, J = 7.9 Hz, 1H), 3.84−3.81 (m, 1H), 3.06 (d, J = 4.2 Hz, 1H),
2.88 (d, J = 13.3 Hz, 1H), 2.81 (d, J = 15.1 Hz, 1H), 2.43 (d, J = 15.1
Hz, 1H), 1.35 (s, 3H), 1.31 (s, 3H), 1.27 (s, 3H), 1.17 (s, 3H), 1.04 (s,
3H), 1.00 (s, 3H), 0.90 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3,
25 °C, TMS): δ 199.6, 175.9, 175.5, 171.6, 149.7, 123.9, 108.0, 49.1,
48.8, 47.5, 45.9, 45.4, 41.3, 40.8, 35.7, 34.6, 34.1, 33.7, 33.1, 32.8, 32.8,
32.7, 31.4, 30.8, 30.1, 28.3, 27.5, 25.0, 24.5, 24.5, 24.1, 22.9, 22.7, 22.5,
21.2, 20.9, 17.8. ESI-MS: 597 [M + Na]+.
1-[12-Oxoisoxazolo[4,5-b]olean-9(11)-en-28-oyl]phenethylamide
(8i). The title product was obtained in 68% yield as a white powder.
1
Mp 151−153 °C. IR (KBr, cm−1): 3132, 2972, 1656, 1520, 1400. H
J
DOI: 10.1021/jm5020023
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
30.7, 28.8, 27.6, 27.3, 25.9, 24.2, 23.8, 23.2, 22.1, 21.5, 20.1, 17.6. ESI-
MS: 531 [M + H]+. HRMS: calculated for C34H47N2O3 [M + H]+
531.3587, found 531.3591.
ESI-MS: 615 [M + Na]+, 631 [M + K]+. HRMS: calculated for
C39H48N2O3Na [M + Na]+ 615.3563, found 615.3572.
Compound 12. A mixture of 8h (50 mg, 0.087 mmol), DDQ
(21.7 mg, 0.096 mmol), acetyl chloride (68 mg, 0.87 mmol), and
triethylamine (17.0 mg, 0.174 mmol) in anhydrous toluene (2 mL)
was reacted for 24 h. After filtration, the filtrate was evaporated
in vacuo to give a residue, which was purified by flash column
chromatography (petroleum ether/EtOAc) to give 12 as an
amorphous solid (16.6 mg, 31%). Mp 98−100 °C. IR (KBr, cm−1):
Compound 10d. The title compound was obtained in 60% yield as a
white powder. Mp 111−113 °C. IR (KBr, cm−1): 3129, 2928, 2857,
1751, 1686, 1400. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 7.99
(s, 1H), 5.84 (s, 1H), 3.08−3.00 (m, 2H), 2.86 (t, J = 9.0 Hz, 1H),
1.48 (s, 3H), 1.25 (s, 3H), 1.14 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H),
0.87 (s, 3H), 0.85 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C,
TMS): δ 200.0, 196.1, 177.2, 164.8, 160.3, 119.5, 113.8, 112.8, 88.6,
51.1, 51.0, 45.1, 44.8, 44.6, 44.4, 39.0, 36.3, 35.9, 33.6, 32.8, 32.6, 32.1,
31.4, 30.6, 29.2, 27.2, 25.8, 25.0, 23.2, 22.3, 20.6, 20.2, 20.1, 19.8, 18.7.
ESI-MS: 545 [M + H]+, 567 [M + Na]+. HRMS: calculated for
C35H49N2O3 [M + H]+ 545.3743, found 545.3755.
Compound 10e. The title compound was obtained in 58% yield as a
white powder. Mp 188−190 °C. IR (KBr, cm−1): 3133, 2930, 2856,
1750, 1686, 1614, 1400. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ
7.95 (s, 1H), 5.84 (s, 1H), 3.06−3.00 (m, 2H), 2.89 (t, J = 9.2, 1H),
1.44 (s, 3H), 1.28 (s, 3H), 1.25 (s, 3H), 1.15 (s, 3H), 1.15 (s, 3H),
1.00 (s, 3H), 0.97 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C,
TMS): δ 201.5, 196.0, 173.1, 165.2, 160.7, 120.0, 114.3, 113.2, 85.6,
52.1, 51.5, 45.6, 45.3, 45.2, 45.0, 36.9, 36.8, 34.5, 34.1, 33.5, 31.7, 31.1,
30.4, 29.7, 27.7, 27.2, 25.5, 24.3, 23.7, 23.0, 22.6, 21.7, 21.2, 20.3, 20.2,
19.0, 14.1. ESI-MS: 573 [M + H]+, 595 [M + Na]+. HRMS: calculated
for C37H53N2O3 [M + H]+ 573.4056, found 573.4072.
Compound 10f. The title compound was obtained in 60% yield as a
white powder. Mp 179−181 °C. IR (KBr, cm−1): 3128, 2927, 2862,
1671, 1521, 1452, 1391. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ
8.10 (s, 1H), 6.04 (s, 1H), 3.36 (d, J = 10.0, 1H), 1.48 (s, 3H), 1.25 (s,
3H), 1.20 (s, 3H), 1.17 (s, 3H), 1.15 (s, 3H), 1.05 (s, 3H), 0.96 (s,
3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 203.5, 196.2,
175.7, 165.3, 163.8, 120.2, 114.5, 113.4, 77.5, 51.1, 50.7, 49.8, 48.5,
45.2, 43.4, 42.6, 41.6, 39.2, 34.0, 33.4, 33.0, 32.0, 31.3, 31.1, 29.9, 29.7,
29.3, 28.5, 26.2, 25.9, 25.2, 24.3, 21.4, 19.5, 14.1. ESI-MS: 567
[M + Na]+, 583 [M + K]+. HRMS: calculated for C35H48N2O3Na
[M + Na]+ 567.3563, found 567.3551.
1
3449, 2931, 2361, 1789, 1638. H NMR (300 M Hz, CDCl3, 25 °C,
TMS): δ 8.04 (s, 1H), 6.41 (d, J = 7.5 Hz, 1H), 5.53 (s, 1H), 3.77−
3.78 (m, 1H), 2.79 (d, J = 15.2, 1H), 2.64−2.59 (m, 1H), 2.45 (d, J =
14.9, 1H), 1.34 (s, 3H), 1.25 (s, 3H), 1.22 (s, 3H), 1.17 (s, 3H), 1.11
(s, 3H), 0.92 (s, 3H), 0.90 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3,
25 °C, TMS): δ 176.3, 172.3, 169.7, 156.5, 149.6, 140.0, 129.2, 115.3,
108.5, 49.9, 48.6, 44.8, 43.1, 42.0, 40.7, 39.8, 34.5, 33.8, 33.6, 32.6,
32.4, 32.4, 32.1, 31.9, 30.2, 30.0, 28.4, 26.4, 25.3, 25.1, 25.0, 24.9, 23.1,
22.6, 20.8, 20.6, 20.2, 18.9,17.9. ESI-MS: 639 [M + Na]+. HRMS:
calculated for C39H56N2O4Na [M + Na]+ 639.4138, found 639.4149.
Compound 13h. A mixture of 8h (100 mg, 0.174 mmol) obtained
above and DDQ (39 mg, 0.174 mmol) in dry methylbenzene (4 mL)
was heated to 90 °C for 10 h. After insoluble matter was removed
by filtration, the filtrate was evaporated in vacuo to give a solid. The
solid was subjected to flash column chromatography (petroleum
ether/EtOAc) to give 13h as a white powder (44 mg, 46%). Mp 157−
1
159 °C. IR (KBr, cm−1): 3140, 2931, 2855, 1711, 1674, 1400. H
NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.07 (s, 1H), 6.05 (s, 1H),
3.58−3.46 (m, 2H), 2.85 (d, J = 14.79 Hz, 1H), 2.58 (d, J = 14.79 Hz,
1H), 1.39 (s, 3H), 1.35 (s, 3H), 1.30 (s, 3H), 1.26 (s, 3H), 1.18 (s,
3H), 1.04 (s, 3H), 0.96 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3,
25 °C, TMS): δ 205.0, 175.8, 172.2, 150.2, 126.4, 121.4, 108.6, 83.4,
51.2, 50.9, 48.1, 48.0, 42.0, 41.9, 40.9, 39.1, 35.5, 35.4, 34.0, 33.4, 32.9,
32.8, 32.5, 32.4, 31.1, 30.6, 30.2, 29.7, 28.7, 26.4, 25.5, 24.9, 24.8, 24.2,
24.1, 21.5, 19.4. ESI-MS: 573 [M + H]+, 595 [M + Na]+. HRMS:
calculated for C37H52N2O3Na [M + Na]+ 595.3876, found 595.3857.
Compound 15. A mixture of 14 (100 mg, 0.176 mmol) obtained
above and DDQ (40 mg, 0.176 mmol) in dry methylbenzene (4 mL)
was heated to 90 °C for 5 h. After insoluble matter was removed by
filtration, the filtrate was evaporated in vacuo to give a solid. The solid
was subjected to flash column chromatography (petroleum ether/
EtOAc) to give 15 as a white powder (50 mg, 52%). Mp 134−136 °C.
Compound 10g. The title compound was obtained in 55% yield as a
white powder. Mp 145−147 °C. IR (KBr, cm−1): 3130, 2997, 1673,
1
1608, 1400. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 7.98 (s,
1H), 5.84 (s, 1H), 3.80−3.70 (m, 2H), 3.19 (t, J = 5.2 Hz, 2H), 2.88
(t, J = 8.9 Hz, 1H), 1.45 (s, 3H), 1.28 (s, 3H), 1.25 (s, 3H), 1.15 (s,
3H), 1.15 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H) ppm. 13C NMR (75 M
Hz, CDCl3, 25 °C, TMS): δ 201.4, 195.9, 165.1, 162.8, 160.7, 120.0,
114.3, 113.3, 86.3, 62.3, 52.2, 51.5, 47.0, 45.5, 45.2, 45.0, 37.1, 36.8,
34.4, 34.1, 33.4, 31.1, 29.7, 27.5, 25.5, 24.4, 23.6, 22.9, 21.6, 21.2, 20.3,
20.2, 19.0. ESI-MS: 533 [M + H]+, 555 [M + Na]+, 571 [M + K]+.
HRMS: calculated for C33H44N2O4Na [M + Na]+ 555.3199, found
555. 3213.
Compound 10h. The title compound was obtained in 50% yield as a
white powder. Mp 169−171 °C. IR (KBr, cm−1): 3134, 2931, 2855,
1711, 1674, 1391. 1H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 8.05
(s, 1H), 6.22 (s, 1H), 3.47−3.20 (m, 1H), 2.79 (d, J = 13.1 Hz, 1H),
1.58 (s, 3H), 1.56 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.20 (s, 3H),
0.98 (s, 3H), 0.96 (s, 3H) ppm. 13C NMR (75 M Hz, CDCl3, 25 °C,
TMS): δ 196.4, 193.2, 173.1, 165.6, 162.6, 123.6, 115.0, 114.2, 86.9,
54.8, 47.9, 46.6, 45.1, 44.8, 42.8, 42.7, 42.4, 36.8, 34.8, 34.4, 33.9, 33.3,
32.8, 31.7, 30.5, 28.9, 27.6, 27.3, 25.9, 25.7, 25.2, 25.0, 23.8, 23.2, 22.0,
21.5, 17.6. ESI-MS: 571 [M + H]+, 593 [M + Na]+. HRMS: calculated
for C37H50N2O3Na [M + Na]+ 593.3719, found 593.3734.
1
IR (KBr, cm−1): 3136, 2941, 2840, 1711, 1660, 1398. H NMR (300
M Hz, CDCl3, 25 °C, TMS): δ 5.90 (s, 1H), 4.50−4.45 (m, 1H), 3.41
(d, J = 9.69 Hz, 1H), 1.41 (s, 3H), 1.34 (s, 3H), 1.19 (s, 3H), 1.04 (s,
3H), 0.95 (s, 3H), 0.93 (s, 3H), 0.89 (s, 3H) ppm. 13C NMR (75 M
Hz, CDCl3, 25 °C, TMS): δ 205.2, 176.0, 173.6, 170.8, 119.8, 79.7,
79.7, 52.4, 52.4, 51.0, 50.6, 48.5, 42.7, 42.2, 40.5, 39.3, 38.6, 37.9, 34.1,
33.4, 33.1, 32.3, 31.1, 30.9, 30.3, 28.5, 28.5, 28.5, 27.8, 26.0, 24.3, 24.0,
23.5, 21.2, 19.1, 16.5. ESI-MS: 566 [M + H]+, 588 [M + Na]+. HRMS:
calculated for C36H56NO4 [M + H]+ 566.4209, found 566.4196.
Measurement of Cell Proliferation. The impact of different
compounds on cancer cell proliferation was tested by the CCK-8
method using a cell counting kit-8 (Dojindo Laboratories, Kumamoto,
Japan), according to the manufacturer’s instructions. Briefly, DU-145
(human prostate cancer cells), H460 (human non-small-cell lung
cancer cells), Bxpc-3 (human pancreatic carcinoma cells), BEL-7404
(human hepatocellular carcinoma cells), and nontumor WPMY-1
(human prostatic stromal myofibroblast cells) at 5 × 103 cells/well
were cultured overnight in 10% FCS DMEM in 96-well plates and
treated in triplicate with 0.041−10 μM of each compound for 72 h.
The cells in medium alone were used as controls, and medium alone
was the background. During the last 30 min culture, individual wells
were added with 10 μL of CCK-8 solution and the absorbance of the
supernatants at 450 nm was measured in a microplate reader.
Compound 10i. The title compound was obtained in 45% yield as a
white powder. Mp 127−129 °C. IR (KBr, cm−1): 3134, 2926, 1758,
1
1686, 1400. H NMR (300 M Hz, CDCl3, 25 °C, TMS): δ 7.96 (s,
1H), 7.27−7.01 (m, 5H), 5.83 (s, 1H), 3.39−3.30 (m, 2H), 2.88 (t, J =
8.9 Hz, 1H), 2.76−2.68 (m, 2H), 1.31 (s, 3H), 1.25 (s, 3H), 1.24 (s,
3H), 1.14 (s, 3H), 1.13 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H) ppm. 13C
NMR (75 M Hz, CDCl3, 25 °C, TMS): δ 200.1, 196.8, 176.5, 164.6,
160.4, 128.3, 128.3, 128.2, 127.6, 127.6, 125.3, 119.5, 113.8, 112.7,
88.6, 51.1, 46.6, 44.8, 44.6, 39.0, 35.8, 33.7, 33.4, 32.8, 32.6, 30.6, 29.2,
27.2, 25.8, 25.0, 23.8, 23.0, 22.3, 21.0, 20.7, 20.2, 19.8, 19.5, 18.4.
Acontrol‐background − Atreated‐background
inhibition (%) =
× 100
Acontrol‐background
Acute Toxicity of 10h in Mice. Both genders of ICR mice at
7 weeks of age were purchased from SLACCAS (Shanghai, China) and
K
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Journal of Medicinal Chemistry
Article
were housed in a specific pathogen free facility. The mice were
randomly treated by gavage with a single dose of 800, 640, 512, 409.6,
or 327.8 mg/kg of 10h or vehicle control (n = 8 per group). The mice
were monitored for their behaviors, the amounts of water and food
consumption, body weights, and activity up to 14 days after treatment.
The experimental protocols were evaluated and approved by the Ethics
Committee of the China Pharmaceutical University.
Antitumor Effect of 10h in Mice. Individual male BALB/c nude
mice (6−8 weeks, 18−22 g, SLACCAS) were implanted subcuta-
neously with 2 × 106 DU-145 cells in 0.1 mL of sterile saline on the
right flank of the axillary region to induce solid tumors. When the
tumor reached 100 30 mm3 in size, the mice were then randomly
treated by gavage with the indicated dose of 10h or the same volume
of vehicle (1:1:8 of DMSO/Cremphor EL/PBS) daily for 21 con-
secutive days. The tumor volumes (TV) were measured every 3 days
up to 21 days after initial treatment. The TV was calculated using the
formula
membranes. After being blocked with 5% fat-free dried milk in 10 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20 (TPBS) over-
night at 4 °C, the membranes were incubated with anti-AKT (1:1000,
Cell Signaling Technology, no. 9272), anti-mTOR (FRAP, 1:1000,
SantaCruz Biotechnology, SC-1549), anti-β-actin (1:1000, Boxter,
BM0627), antiphosphorylated AKT (Ser473, 1:1000, Santa Cruz
Biotechnology, SC-7985-R), or anti-phospho-mTOR (Ser2448)
(1:1000, Cell Signaling Technology, no. 2971), anti-Bax (P-19,
1:1000, Santa Cruz Biotechnology, SC-526), anti-Bcl-2 (C-2,1:1000,
Santa Cruz, Biotechnology). The bound antibodies were detected by
HRP-conjugated secondary antibodies, and immune complexes were
visualized using enhanced chemiluminescence reagent from Thermo
Fisher Scientific (Rockford, IL, USA). The relative levels of target
proteins to the control were determined by densimetric analysis using
AlphaEase FC (Alpha Innotech, USA) image analysis software.
log P Calculations. The log P values of each compound were
determined theoretically using three different programs,25 and the
data are presented as the mean log P and standard deviation. These
programs included ChemAxon’s MarvinSketch,26,27 Molinspiration
software,28 and VCCLAB’s ALOGPS software.29,30
length × (width)2
TV =
2
Rat Plasma Stability Assay.34,35 Compound 10h or CDDO-Me
(50 ng/mL) was incubated with rat plasma (pooled, heparinized) at
37 °C for 60 min. The reactions were stopped by precipitation of
plasma proteins with 5 volumes of cold acetonitrile containing an
internal standard, and the concentrations of remaining compound
were analyzed by LC−MS/MS.
Subsequently, the mice were sacrificed and the tumors were dissected
out and weighed. The % tumor growth inhibition was determined as
the tumor growth inhibition
⎛
⎞
⎟
T
C
⎜
TGI (%) = 1 −
× 100
⎝
⎠
Microsomal Stability Assay.36,37 Human liver microsomes
(HLMs) were diluted with potassium phosphate−MgCl2 buffer
(100 mM, pH 7.4) to a final protein concentration of 0.2 mg/mL.
The HLMs were incubated with each compound (5 μM) in the
presence of NADPH system at 37 °C for 0, 5, 10, 15, 30, or 60 min
and quenched with acetonitrile, followed by centrifuged at 4000g for
15 min. The content of each compound in the supernatants was
determined for its half-life (t1/2) by LC−MS/MS analysis.
where T is the mean tumor weight in treated group and C is the mean
tumor weight in vehicle control group at the end point.
Cell Cycle Analysis. DU-145 cells were cultured in 10% FCS
DMEM overnight and treated in triplicate with CDDO-Me (0.26 or
0.53 μM), compound 10h (0.08 or 0.16 μM), or vehicle for 24 h. The
cells were harvested, fixed with 70% ethanol for 2 h, and incubated
with PI/RNase staining buffer (BD Pharmingen) for 15 min at room
temperature. The DNA content in the different groups of cells was
assessed by flow cytometry (FACSCalibur, Becton Dickinson, U.S.)
and analyzed by the software MODFIT.
Hoechst 33258 Assay for Apoptosis. The impact of 10h on
DU-145 cell apoptosis was determined by Hoechst 33258 staining
using the apoptosis Hoechst staining kit (catalog no. C1115, Beyotime
Biotechnology, Jiangsu, China), according to the manufacturer’s proto-
col. Briefly, DU-145 cells were treated with the indicated con-
centrations of 10h and CDDO-Me for 24 h, fixed in 0.5 mL of
methanol for 30 min, and stained with 1 mg/mL of Hoechst 33258,
followed by examining under a fluorescent microscope.
Caspase-3 Activity Assay. Caspase-3 activity was assayed using a
caspase-3 activity kit (catalog no. C1115, Beyotime Institute of
Biotechnology, Haimen, Jiangsu, China), according to the manufactur-
ere’s protocol. This assay is based on the ability of caspase-3 to catalyze
acetyl-Asp-Glu-Val-Asp p-nitroanilide into the yellow formazan
product p-nitroaniline. DU-145 cells were treated with individual
compounds, as described above, and harvested. After being washed
with cold PBS, the cells were lyzed in lysis buffer (100 μL per 2 × 106
cells) on ice for 15 min and then centrifuged at 18 000g at 4 °C for
10 min. The cell lysates were tested for their caspase-3 activity by
mixing 10 μL of individual cell lysates with 10 μL caspase-3 substrate
(Ac-DEVD-pNA, 2 mM) in 80 μL of reaction buffer in triplicate at
37 °C for 2 h. The caspase-3 activity in individual samples was
calculated based on the absorbance at 405 nm and was expressed as
the relative percentage of enzyme activity to the control.
Caco2 Permeability Assay.38−42 Caco2 cells between passages
20 and 40 were cultured at 37 °C in a 5% CO2 and 90% relative
humidity environment. After 21 days of culture, the integrity of the cell
monolayer was verified by measuring the trans-epithelial electrical
resistance (TEER). The drug transport from the apical side to the
basolateral side (A−B) or from the basolateral side to the apical side
was measured under the same conditions. The monolayer of cells was
treated in triplicate with 0.5 μM of the tested drug in the well with the
appropriate pH (pH 6.8 for apical side and pH 7.4 for basolateral side)
at 37 °C for 30 min. Samples were collected from the donor and
receiver sides at 0 and 5 min postincubation. The concentrations of
the drug were determined by LC−MS/MS. The apparent permeability
values (Papp) were calculated according to the equation,
dQ /dt
AC0
P
=
app
where dQ/dt is the slope of the cumulative amount transported during
the study period, A is the area of the insets, and C0 is the starting
concentration. For bidirectional permeability, the efflux ratio (ER) was
defined as Papp(B→A)/Papp(A→B), where A → B is apical to basolateral
transport and B → A is basolateral to apical transport. A high efflux
ratio (>3) indicates that a compound may be a substrate for P-gp or
other active transport systems.
hERG Channel Inhibition Assay.43−48 Chinese hamster ovary
(CHO) cells stably expressing the hERG channel were cultured in
DMEM/F-12, GlutaMAX with 10% fetal bovine serum, 100 units/mL
penicillin, 100 μg/mL streptomycin, 1% Geneticin, and 10 μM HEPES
at 37 °C in a humidified atmosphere of 5% CO2. When the cells
reached 60−80% confluency, the cells were harvested and placed on
the Qstirrer in serum-free media for ∼30 min.
Western Blotting Assay. DU-145 cells (5 × 105/well) were
cultured in 10% FCS Ham’s F-12K medium in six-well plates
overnight, and the cells were treated in duplicate with vehicle alone or
the indicated compounds for 24 h. The cells were harvested and lysed
in lysis buffer [50 mM Tris-HCl, pH 8, 5 mM EGTA, 150 mM NaCl,
0.5% Nonidet P-40, 0.5% Triton X-100], followed by centrifuging
at 14 000g for 10 min at 4 °C. After quantification of protein con-
centrations using the BCA assay (Pierce BCA protein assay kit, prod-
uct no. 23225), the cell lysate samples (50 μg/lane) were separated on
10−14% SDS−polyacrylamide gel and transferred to nitrocellulose
The extracellular Ringer’s solution (2 mM CaCl2, 1 mM MgCl2,
10 mM HEPES, 4 mM KCl, 145 mM NaCl, 10 mM glucose, pH 7.40,
adjusted with NaOH, 305 mOsm) and intracellular Ringer’s solution
(5.374 mM CaCl2, 1.75 mM MgCl2, 120 mM KCl, 10 mM HEPES,
5 mM EGTA, 4 mM Na-ATP, pH 7.25, adjusted with KOH,
L
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Journal of Medicinal Chemistry
Article
290 mOsm) containing 4 mM Na2-ATP were used for measuring the
whole-cell currents using a QPatch system (Sophion) in response to
continuously executed voltage protocols, according to the manufac-
turer’s recommendations. Upon onset of the voltage protocol, the cells
were maintained at a holding potential (Vh) of −80 mV, clamped
briefly to −50 mV (50 ms), depolarized to 20 mV for 5000 ms, and
finally repolarized to −50 mV for 5000 ms, at which potential, the peak
outward tail current was measured. Finally, the voltage returned to
Vh for 3100 ms. Thus, voltage protocols were repeated every 15 s.
For each cell, extracellular solution was applied prior to increasing
concentrations of the tested compound.
Inhibition of Hepatic CYP Enzymes. Inhibition of each drug on
metabolizing CYP enzymes was determined using recombinant human
CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and
CYP3A4).49−52 Individual CYP enzymes reacted with their substrates
at a final concentration approximating their Km (phenacetin, 60 μM;
diclofenac, 6 μM; smephenytoin, 20 μM; dextromethorphan, 5 μM;
midazolam, 8 μM; testosterone, 50 μM) in the presence or absence of
nine concentrations of each compound up to 1000 nM in duplicate
for 1 h. The reactions were quenched with acetonitrile, and after being
centrifuged, the content of probe metabolites in the supernatants was
determined by LC−MS/MS. Amounts of metabolites produced were
normalized to vehicle controls, and IC50 values of each compound for
individual CYP enzymes were calculated using GraphPad Prism 5.0.
between the start of the Q wave and end of the T wave on an
electrocardiogram; CYP, cytochrome P450 enzyme; HEPES,
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; EGTA,
ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid
REFERENCES
■
(1) Sporn, M. B.; Liby, K. T.; Yore, M. M.; Fu, L.; Lopchuk, J. M.;
Gribble, G. W. New synthetic triterpenoids: potent agents for
prevention and treatment of tissue injury caused by inflammatory
and oxidative stress. J. Nat. Prod. 2011, 74, 537−545.
(2) Honda, T.; Rounds, B. V.; Bore, L.; Finlay, H. J., Jr.; Favaloro, F.
G.; Suh, N.; Wang, Y.; Sporn, M. B.; Gribble, G. W. Synthetic oleanane
and ursane triterpenoids with modified rings A and C: a series of
highly active inhibitors of nitric oxide production in mouse
macrophages. J. Med. Chem. 2000, 43, 4233−4246.
(3) Honda, T.; Honda, Y., Jr.; Favaloro, F. G.; Gribble, G. W.; Suh,
N.; Place, A. E.; Rendi, M. H.; Sporn, M. B. A novel
dicyanotriterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oni-
trile, active at picomolar concentrations for inhibition of nitric oxide
production. Bioorg. Med. Chem. Lett. 2002, 12, 1027−1030.
(4) Liby, K. T.; Yore, M. M.; Sporn, M. B. Triterpenoids and
rexinoids as multifunctional agents for the prevention and treatment of
cancer. Nat. Rev. Cancer 2007, 7, 357−369.
(5) Liby, K. T.; Sporn, M. B. Synthetic oleanane triterpenoids:
multifunctional drugs with a broad range of applications for prevention
and treatment of chronic disease. Pharmacol. Rev. 2012, 64, 972−1003.
(6) Hong, D. S.; Kurzrock, R.; Supko, J. G.; He, X.; Naing, A.;
Wheler, J.; Lawrence, D.; Eder, J. P.; Meyer, C. J.; Ferguson, D. A.;
Mier, J.; Konopleva, M.; Konoplev, S.; Andreeff, M.; Kufe, D.; Lazarus,
H.; Shapiro, G. I.; Dezube, B. J. A phase I first-in-human trial of
bardoxolone methyl in patients with advanced solid tumors and
lymphomas. Clin. Cancer Res. 2012, 18, 3396−3406.
(7) Chen, L.; Zhang, Y.; Kong, X.; Lan, E.; Huang, Z.; Peng, S.;
Kaufman, D. L.; Tian, J. Design, synthesis, and antihepatocellular
carcinoma activity of nitric oxide releasing derivatives of oleanolic acid.
J. Med. Chem. 2008, 51, 4834−4838.
(8) Huang, Z.; Zhang, Y.; Zhao, L.; Jing, Y.; Lai, Y.; Zhang, L.; Guo,
Q.; Yuan, S.; Zhang, J.; Chen, L.; Peng, S.; Tian, J. Synthesis and
antihuman hepatocellular carcinoma activity of new nitric oxide-
releasing glycosyl derivatives of oleanolic acid. Org. Biomol. Chem.
2010, 8, 632−639.
(9) Huang, Z.; Fu, J.; Liu, L.; Sun, Y.; Lai, Y.; Ji, H.; Knaus, E. E.;
Tian, J.; Zhang, Y. Glycosylated diazeniumdiolate-based oleanolic acid
derivatives: Synthesis, in vitro and in vivo biological evaluation as
antihuman hepatocellular carcinoma agents. Org. Biomol. Chem. 2012,
10, 3882−3891.
(10) Fu, J.; Liu, L.; Huang, Z.; Lai, Y.; Ji, H.; Peng, S.; Tian, J.; Zhang,
Y. Hybrid molecule from O2-(2,4-dinitrophenyl)diazeniumdiolate and
oleanolic acid: a GSTπ activated NO prodrug with selective anti-
human hepatocellular carcinoma activity and improved stability. J.
Med. Chem. 2013, 56, 4641−4655.
(11) Ding, Y.; Huang, Z.; Yin, J.; Lai, Y.; Zhang, S.; Zhang, Z.; Fang,
L.; Peng, S.; Zhang, Y. DDQ-promoted dehydrogenation from natural
rigid polycyclic acids or flexible alkyl acids to generate lactones by a
radical ion mechanism. Chem. Commun. 2011, 47, 9495−9497.
(12) Zhang, Z.; Zhang, C.; Ding, Y.; Zhao, Q.; Yang, L.; Ling, J.; Liu,
L.; Ji, H.; Zhang, Y. The activation of p38 and JNK by ROS, contribute
to OLO-2-mediated intrinsic apoptosis in human hepatocellular
carcinoma cells. Food Chem. Toxicol. 2014, 63, 38−47.
ASSOCIATED CONTENT
* Supporting Information
■
S
Compound purity measured by HPLC, log P of selective com-
pounds, possible metabolic products of 10h and CDDO-Me, and
a csv file containing molecular formula strings. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/jm5020023.
AUTHOR INFORMATION
Corresponding Authors
■
*Z.H.: phone, +86-25-83271072; e-mail, cpudahuang@163.com.
*H.J.: phone, +86-25-86021369; fax, +86-25-86635503; e-mail,
huijicpu@163.com.
*Y.Z.: phone, +86-25-83271015; fax, +86-25-83271015; e-mail,
zyhtgd@163.com.
Author Contributions
∥Y.A. and Y.H. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by grants from the National Natural
Science Foundation of China (Grants 81273378, 21372261,
81202408, and 21472244) and the Project Program of State
Key Laboratory of Natural Medicines, China Pharmaceutical
University (Grants ZJ12020 and SKLNMZZCX201403).
ABBREVIATIONS USED
■
CDDO-Me, methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate;
CDDO-MA, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-
methylamide; CDDO-Im, 1-[2-cyano-3,12-dioxooleana-1,9(11)-
dien-28-oyl]imidazole; PI3K, phosphoinositide 3-kinase; AKT,
protein kinase B, a serine/threonine protein kinase; mTOR,
mammalian target of rapamycin; PBS, phosphate buffered saline;
SD, standard deviation; HPLC, high-performance liquid
chromatography; DMSO, dimethyl sulfoxide; PE, petroleum
ether; LD50, dose that is lethal in 50% of test subjects; TMS,
tetramethylsilane; UV, ultraviolet; w/w, weight per unit weight
(weight-to-weight ratio); Bn, benzyl; QT, the length of time
(13) Fenical, W.; Jensen, P. R.; Palladino, M. A.; Lam, K. S.; Lloyd, G.
K.; Potts, B. C. Discovery and development of the anticancer agent
salinosporamide A (NPI-0052). Bioorg. Med. Chem. 2009, 17, 2175−
2180.
(14) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Salinosporamide A: a highly cytotoxic
proteasome inhibitor from a novel microbial source, a marine-
M
DOI: 10.1021/jm5020023
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
bacterium of the new genus Salinospora. Angew. Chem., Int. Ed. 2003,
42, 355−357.
(15) Wilson, M. C.; Nam, S.-J.; Gulder, T. A. M.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W.; Moore, B. S. Structure and biosynthesis of
the marine Streptomycete Ansamycin Ansalactam A and its distinctive
branched chain polyketide extender unit. J. Am. Chem. Soc. 2011, 133,
1971−1977.
(16) Okazaki, Y.; Ishizuka, A.; Ishihara, A.; Nishioka, T.; Iwamura, H.
New dimeric compounds of avenanthramide phytoalexin in oats. J.
Org. Chem. 2007, 72, 3830−3839.
(17) Guntern, A.; Ioset, J. R.; Queiroz, E. F.; Sandor, P.; Foggin, C.
M.; Hostettmann, K. Heliotropamide, a novel oxopyrrolidine-3-
carboxamide from Heliotropium ovalifolium. J. Nat. Prod. 2003, 66,
1550−1553.
(18) Omura, S.; Fujimoto, T.; Otoguro, K.; Matsuzaki, K.; Moriguchi,
R.; Tanaka, H.; Sasaki, Y. Lactacystin, a novel microbial metabolite,
induces neuritogenesis of neuroblastoma cells. J. Antibiot. 1991, 44,
113−116.
(19) Fenteany, G.; Standaert, R. F.; Lane, W. S.; Choi, S.; Corey, E.
J.; Schreiber, S. L. Inhibition of proteasome activities and subunit-
specific amino-terminal threonine modification by lactacystin. Science
1995, 268, 726−731.
(20) Manam, R. R.; Teisan, S.; White, D. J.; Nicholson, B.; Grodberg,
J.; Neuteboom, S. T. C.; Lam, K. S.; Mosca, D. A.; Lloyd, G. K.; Potts,
B. C. M. Lajollamycin , a nitro-tetraene spiro-β-lactone-γ-lactam
antibiotic from the marine actinomycete Streptomyces nodosus. J. Nat.
Prod. 2005, 68, 240−243.
(21) Choi, E.; Lee, C.; Cho, M.; Seo, J. J.; Yang, J. S.; Oh, S. J.; Lee,
K.; Park, S. K.; Kim, H. M.; Kwon, H. J.; Han, G. Property-based
optimization of hydroxamate-based γ-lactam HDAC inhibitors to
improve their metabolic stability and pharmacokinetic profiles. J. Med.
Chem. 2012, 55, 10766−10770.
(31) Fermini, B.; Fossa, A. A. The impact of drug-induced QT
interval prolongation on drug discovery and development. Nat. Rev.
Drug Discovery 2003, 2, 439−447.
(32) Kola, I.; Landis, J. Can the pharmaceutical industry reduce
attrition rates? Nat. Rev. Drug Discovery 2004, 3, 711−715.
(33) Guengerich, F. P. Cytochrome p450 and chemical toxicology.
Chem. Res. Toxicol. 2008, 21, 70−83.
(34) Zhu, W.; Hu, Q.; Hanke, N.; van Koppen, C. J.; Hartmann, R.
W. Potent 11β-hydroxylase inhibitors with inverse metabolic stability
in human plasma and hepatic S9 fractions to promote wound healing.
J. Med. Chem. 2014, 57, 7811−7817.
(35) Emmerich, J.; Hu, Q.; Hanke, N.; Hartmann, R. W. Cushing’s
syndrome: development of highly potent and selective CYP11B1
inhibitors of the (pyridylmethyl)pyridine type. J. Med. Chem. 2013, 56,
6022−6032.
(36) Lin, X.; Chen, W.; Qiu, Z.; Guo, L.; Zhu, W.; Li, W.; Wang, Z.;
Zhang, W.; Zhang, Z.; Rong, Y.; Zhang, M.; Yu, L.; Zhong, S.; Zhao,
R.; Wu, X.; Wong, J. C.; Tang, G. Design and synthesis of orally
bioavailable aminopyrrolidinone histone deacetylase 6 inhibitors. J.
Med. Chem. 2015, 58, 2809−2820.
(37) Lee, S.; Lim, D.; Lee, E.; Lee, N.; Lee, H. G.; Cechetto, J.;
Liuzzi, M.; Freitas-Junior, L. H.; Song, J. S.; Bae, M. A.; Oh, S.; Ayong,
L.; Park, S. B. Discovery of carbohybrid-based 2-aminopyrimidine
analogues as a new class of rapid-acting antimalarial agents using
image-based cytological profiling assay. J. Med. Chem. 2014, 57, 7425−
7434.
(38) Zheng, Y.; Benet, L. Z.; Okochi, H.; Chen, X. pH Dependent
but not P-gp dependent bidirectional transport study of s-propranolol:
The importance of passive diffusion. Pharm. Res. [Online early access].
DOI: 10.1007/s11095-015-1640-3. Published Online: February 19,
2015.
(39) Duan, H.; Ning, M.; Zou, Q.; Ye, Y.; Feng, Y.; Zhang, L.; Leng,
Y.; Shen, J. Discovery of intestinal targeted TGR5 agonists for the
treatment of type 2 diabetes. J. Med. Chem. 2015, 58, 3315−3328.
(22) Duffy, R. A.; Morgan, C.; Naylor, R.; Higgins, G. A.; Varty, G.
B.; Lachowicz, J. E.; Parker, E. M. Rolapitant (SCH 619734): A potent,
selective and orally active neurokinin NK1 receptor antagonist with
centrally-mediated antiemetic effects in ferrets. Pharmacol., Biochem.
Behav. 2012, 102, 95−100.
́
(40) Cinelli, M. A.; Li, H.; Chreifi, G.; Martasek, P.; Roman, L. J.;
Poulos, T. L.; Silverman, R. B. Simplified 2-aminoquinoline-based
scaffold for potent and selective neuronal nitric oxide synthase
inhibition. J. Med. Chem. 2014, 57, 1513−1530.
(41) Plowright, A. T.; Nilsson, K.; Antonsson, M.; Amin, K.;
Broddefalk, J.; Jensen, J.; Lehmann, A.; Jin, S.; St-Onge, S.;
Tomaszewski, M. J.; Tremblay, M.; Walpole, C.; Wei, Z.; Yang, H.;
Ulander, J. Discovery of agonists of cannabinoid receptor 1 with
restricted central nervous system penetration aimed for treatment of
gastroesophageal reflux disease. J. Med. Chem. 2013, 56, 220−240.
(42) Over, B.; McCarren, P.; Artursson, P.; Foley, M.; Giordanetto,
(23) Yang, Z. Q.; Shu, Y.; Ma, L.; Wittmann, M.; Ray, W. J.; Seager,
M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau,
M. T.; Kuduk, S. D. Discovery of naphthyl-fused 5-membered lactams
as a new class of M1 positive allosteric modulators. ACS Med. Chem.
Lett. 2014, 5, 604−608.
(24) Kazmierski, W. M.; Andrews, W.; Furfine, E.; Spaltenstein, A.;
Wright, L. Discovery of potent pyrrolidone-based HIV-1 protease
inhibitors with enhanced drug-like properties. Bioorg. Med. Chem. Lett.
2004, 14, 5689−5692.
F.; Gronberg, G.; Hilgendorf, C.; Lee, M. D.; Matsson, P.; Muncipinto,
̈
G.; Pellisson, M.; Perry, M. W., 4th; Svensson, R.; Duvall, J. R.;
Kihlberg, J. Impact of stereospecific intramolecular hydrogen bonding
on cell permeability and physicochemical properties. J. Med. Chem.
2014, 57, 2746−2754.
(25) Aksenov, A. V.; Smirnov, A. N.; Magedov, I. V.; Reisenauer, M.
R.; Aksenov, N. A.; Aksenova, I. V.; Pendleton, A. L.; Nguyen, G.;
Johnston, R. K.; Rubin, M.; De Carvalho, A.; Kiss, R.; Mathieu, V.;
Lefranc, F.; Correa, J.; Cavazos, D. A.; Brenner, A. J.; Bryan, B. A.;
Rogelj, S.; Kornienko, A.; Frolova, L. V. Activity of 2-aryl-2-(3-
indolyl)acetohydroxamates against drug-resistant cancer cells. J. Med.
Chem. 2015, 58, 2206−2220.
(43) Kutchinsky, J.; Friis, S.; Asmild, M.; Taboryski, R.; Pedersen, S.;
Vestergaard, R. K.; Jacobsen, R. B.; Krzywkowski, K.; Schrøder, R. L.;
Ljungstrøm, T.; Helix, N.; Sørensen, C. B.; Bech, M.; Willumsen, N. J.
́
Characterization of potassium channel modulators with QPatch
automated patch-clamp technology: system characteristics and
performance. Assay Drug Dev. Technol. 2003, 1, 685−693.
(44) Zhou, P. Z.; Babcock, J.; Liu, L. Q.; Li, M.; Gao, Z. B. Activation
of human ether-a-go-go related gene (hERG) potassium channels by
small molecules. Acta Pharmacol. Sin. 2011, 32, 781−788.
(45) Lucas, M. C.; Bhagirath, N.; Chiao, E.; Goldstein, D. M.;
Hermann, J. C.; Hsu, P. Y.; Kirchner, S.; Kennedy-Smith, J. J.;
Kuglstatter, A.; Lukacs, C.; Menke, J.; Niu, L.; Padilla, F.; Peng, Y.;
Polonchuk, L.; Railkar, A.; Slade, M.; Soth, M.; Xu, D.; Yadava, P.; Yee,
C.; Zhou, M.; Liao, C. Using ovality to predict nonmutagenic, orally
efficacious pyridazine amides as cell specific spleen tyrosine kinase
inhibitors. J. Med. Chem. 2014, 57, 2683−2691.
(46) Brodney, M. A.; Beck, E. M.; Butler, C. R.; Barreiro, G.;
Johnson, E. F.; Riddell, D.; Parris, K.; Nolan, C. E.; Fan, Y.; Atchison,
K.; Gonzales, C.; Robshaw, A. E.; Doran, S. D.; Bundesmann, M. W.;
(26) MarvinSketch: Marvin, version 6.3.0; ChemAxon: Budapest,
Hungary, 2014; http://www.chemaxon.com.
(27) Klopman, G.; Li, J.-Y.; Wang, S.; Dimayuga, M. Computer
automated log P calculations based on an extended group contribution
approach. J. Chem. Inf. Comput. Sci. 1994, 34, 752−781.
(28) Interactive log P calculator. Molinspiration Cheminformatics.
www.molinspiration.com, 2014.
(29) Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.;
Livingstone, D.; Ertl, P.; Palyulin, V. A.; Radchenko, E. V.; Zefirov,
N. S.; Makarenko, A. S.; Tanchuk, V. Y.; Prokopenko, V. V. Virtual
computational chemistry laboratorydesign and description. J.
Comput.-Aided Mol. Des. 2005, 19, 453−463.
(30) ALOGPS; VCCLAB. Virtual Computational Chemistry
Laboratory. http://www.vcclab.org, 2005.
N
DOI: 10.1021/jm5020023
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Buzon, L.; Dutra, J.; Henegar, K.; LaChapelle, E.; Hou, X.; Rogers, B.
N.; Pandit, J.; Lira, R.; Martinez-Alsina, L.; Mikochik, P.; Murray, J. C.;
Ogilvie, K.; Price, L.; Sakya, S. M.; Yu, A.; Zhang, Y.; O’Neill, B. T.
Utilizing structures of CYP2D6 and BACE1 complexes to reduce risk
of drug-drug interactions with a novel series of centrally efficacious
BACE1 inhibitors. J. Med. Chem. 2015, 58, 3223−3252.
(47) Díaz, J. L.; Christmann, U.; Fernan
́
dez, A.; Torrens, A.; Port, A.;
́
Pascual, R.; Alvarez, I.; Burgueno, J.; Monroy, X.; Montero, A.; Balada,
̃
A.; Vela, J. M.; Almansa, C. Synthesis and structure-activity
relationship study of a new series of selective σ(1) receptor ligands
for the treatment of pain: 4-aminotriazoles. J. Med. Chem. 2015, 58,
2441−2451.
(48) Cho, Y. S.; Whitehead, L.; Li, J.; Chen, C. H.; Jiang, L.; Vogtle,
̈
M.; Francotte, E.; Richert, P.; Wagner, T.; Traebert, M.; Lu, Q.; Cao,
X.; Dumotier, B.; Fejzo, J.; Rajan, S.; Wang, P.; Yan-Neale, Y.; Shao,
W.; Atadja, P.; Shultz, M. Conformational refinement of hydroxamate-
based histone deacetylase inhibitors and exploration of 3-piperidin-3-
ylindole analogues of dacinostat (LAQ824). J. Med. Chem. 2010, 53,
2952−2963.
(49) Li, T.; Li, N.; Guo, Q.; Ji, H.; Zhao, D.; Xie, S.; Li, X.; Qiu, Z.;
Han, D.; Chen, X.; You, Q. Inhibitory effects of wogonin on catalytic
activity of cytochrome P450 enzyme in human liver microsomes. Eur.
J. Drug Metab. Pharmacokinet. 2011, 36, 249−256.
(50) Yang, S.; Qiu, Z.; Zhang, Q.; Chen, J.; Chen, X. Inhibitory
effects of calf thymus DNA on metabolism activity of CYP450 enzyme
in human liver microsomes. Drug Metab. Pharmacokinet. 2014, 29,
475−481.
(51) Spicer, T. P.; Jiang, J.; Taylor, A. B.; Choi, J. Y.; Hart, P. J.;
Roush, W. R.; Fields, G. B.; Hodder, P. S.; Minond, D. Character-
ization of selective exosite-binding inhibitors of matrix metal-
loproteinase 13 that prevent articular cartilage degradation in vitro. J.
Med. Chem. 2014, 57, 9598−9611.
(52) Ling, C.; Fu, L.; Gao, S.; Chu, W.; Wang, H.; Huang, Y.; Chen,
X.; Yang, Y. Design, synthesis, and structure-activity relationship
studies of novel thioether pleuromutilin derivatives as potent
antibacterial agents. J. Med. Chem. 2014, 57, 4772−4795.
O
DOI: 10.1021/jm5020023
J. Med. Chem. XXXX, XXX, XXX−XXX