An efficient synthesis of methyl 2-cyano-3,12-dioxoursol-1,9-dien-28-oate (CDDU-methyl ester): Analogues, biological activities, and comparison with oleanolic acid derivatives

Article abstract of DOI:10.1039/c4ob00679h

An efficient synthesis of methyl 2-cyano-3,12-dioxoursol-1,9-dien-28-oate (CDDU-methyl ester) from commercially available ursolic acid, which features an oxidative ozonolysis-mediated C-ring enone formation, and provides the first access to ursolic acid-derived cyano enone analogues with C-ring activation. These new ursolic acid analogues show potent biological activities, with potency of approximately five-fold less than the corresponding oleanolic acid derivatives.

Full text of DOI:10.1039/c4ob00679h

Organic &
Biomolecular Chemistry
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PAPER
An efficient synthesis of methyl 2-cyano-3,12-
dioxoursol-1,9-dien-28-oate (CDDU-methyl ester):
analogues, biological activities, and comparison
with oleanolic acid derivatives†
Cite this: Org. Biomol. Chem., 2014,
2, 5192
1
a
a
b
b
Liangfeng Fu, Qi-Xian Lin, Karen T. Liby, Michael B. Sporn* and
a
Gordon W. Gribble*
An efficient synthesis of methyl 2-cyano-3,12-dioxoursol-1,9-dien-28-oate (CDDU-methyl ester) from
commercially available ursolic acid, which features an oxidative ozonolysis-mediated C-ring enone for-
mation, and provides the first access to ursolic acid-derived cyano enone analogues with C-ring acti-
vation. These new ursolic acid analogues show potent biological activities, with potency of approximately
five-fold less than the corresponding oleanolic acid derivatives.
Received 31st March 2014,
Accepted 30th May 2014
DOI: 10.1039/c4ob00679h
www.rsc.org/obc
Introduction
The biological importance of naturally occurring pentacyclic
triterpenoids has long been recognized. The annual review of
triterpenoids in Natural Product Reports is ample testimony to
the ubiquitous worldwide distribution of these compounds,
which include squalenes, lanostanes, fusidanes, dammaranes,
euphanes, and others. Within each class is a stunning array of
Fig. 1 Oleanolic acid, ursolic acid, and betulinic acid.
1
structural diversity and a range of biological activity. For
example, many oleanane and ursane triterpenoids, which are
2
derived biosynthetically by cyclization of squalene, have inter-
esting biological, pharmacological, and medicinal activities
not unlike those attributed to retinoids and steroids. These
properties include anti-inflammatory actions, suppression of
tumor promotion, suppression of immunoglobulin synthesis, Fig. 2 CDDO methyl ester and analogues.
protection of the liver against toxic injury, induction of col-
lagen synthesis, and induction of differentiation in leukemia
3
and teratocarcinoma cells.
CDDO-methyl ester (4) (bardoxolone methyl) (Fig. 2), which
5
Oleanolic acid (1), ursolic acid (2), and betulinic acid (3) was developed in our laboratory years ago, has successfully
Fig. 1), probably the most commonly studied triterpenoids, completed a Phase I clinical trial for the treatment of cancer
6
(
exhibit modest biological activity, although 2 has been mar- and a Phase II clinical trial for the treatment of chronic kidney
7
keted in China as an oral drug for the treatment of liver dis- disease in type 2 diabetes patients. During this research we
4
orders in humans.
synthesized a series of CDDO-methyl ester analogues (Fig. 2),
Extensive studies report the use of oleanolic acid as a skel- such as CDDO-ethyl amide (5), CDDO-trifluoroethyl amide (6),
8
eton motif. Indeed, the highly potent oleanolic acid derivative and CDDO-imidazolide (7). Details of their biological pro-
9
–11
perties are described in recent review articles.
Due to the limited structure–activity studies involving
ursolic acid, we have undertaken and now describe an efficient
synthesis of the ursolic acid analogue of CDDO-methyl ester,
CDDU-methyl ester (methyl 2-cyano-3,12-dioxoursol-1,9-dien-
a
Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA.
E-mail: ggribble@dartmouth.edu
b
Department of Pharmacology and Toxicology, Geisel School of Medicine, Hanover,
NH 03755, USA
†
28-oate), along with related derivatives, and describe their
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00679h
biological activities.
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To investigate the anti-inflammatory and cancer chemopre-
ventive properties of these derivatives, we have adopted a pre-
liminary screening assay system that measures inhibition of
nitric oxide (NO) production as induced by interferon-γ (IFN-γ)
in mouse macrophages, and CDDO (8) is 200 000–400 000
times more active than oleanolic acid in this inducible nitric
oxide synthase (iNOS) assay. Also, CDDO at nanomolar concen-
trations suppresses the de novo synthesis of the inflammatory
enzymes iNOS and cyclooxygenase-2 (COX-2) in activated
1
2
macrophages.
Results and discussion
Chemical synthesis
Our rationale for this project is that because ursolic acid is
1
3
often more potent than oleanolic acid, we deemed it impor- Scheme 1 Reported C11–C12 oxidation of oleanolic acid. Reagents:
(
a) m-CPBA, CH
2
Cl
2
; (b) MMPP, acetone; (c) O
3
, CH
2 2
Cl ; (d) DMDO,
tant to synthesize and study the ursolic acid derivatives CDDU-
methyl ester (9), CDDU-ethyl amide (10), CDDU-trifluoroethyl
amide (11), and CDDU-imidazolide (12) (Fig. 3).
CHCl ; (e) Bi(OTf)
3
3
, CH
2
Cl ; (f) Br , AcOH; (g) H
2
2
2
O
2
, AcOH.
Our first goal was to prepare the C-ring enone of ursolic
acid. A number of reagents and reaction conditions are known
to oxidize the C11–C12 alkene of oleanolic acid, including
1
4
15
16
m-CPBA, MMPP, ozone-mediated hydroxy lactonization,
1
7
DMDO-mediated chlorolactonization,
bismuth(III) triflate-
1
8
19
mediated lactonization, and bromolactonization. Oxidation
2
0
2 2
conditions, such as H O –AcOH or m-CPBA-mediated epoxi-
dation and isomerization, ozonolysis have also been deve-
loped for oleanolic acid esters (Scheme 1).
2
1
22
Scheme 2 Reported fluorolactonization on ursolic acid. Reagent:
(a) Selectfluor, dioxane, nitromethane, 80 °C.
However, the corresponding oxidations of ursolic acid are
either rare or unknown. For example, Bag and coworkers
Not surprisingly, we encountered difficulty in the oxidation
of the ursolic acid C-ring. Thus, an initial reaction of ursane
2
reported a selective Br /AcOH-induced bromolactonization of
1
9
oleanolic acid in the presence of ursolic acid. Recently, Csuk
ester 21 with m-CPBA as the oxidant resulted in almost quanti-
and Siewert reported a m-CPBA-mediated oxidative separation
24
tative recovery of 21 (Scheme 3), and classic conditions
1
4b
of oleanolic acid from ursolic acid. Under these conditions,
oleanolic acid was completely converted to the oxidation
product, whereas ursolic acid was recovered nearly quantitat-
ively. The different C-ring reactivity between oleanolic acid and
ursolic acid is presumed to be due to the steric hindrance
imparted by the C19 methyl group in ursolic acid. However,
Salvador, Jing and co-workers describe the introduction of a
employing hydrogen peroxide in acetic acid gave complex mix-
tures. Moreover, neither t-butyl hydroperoxide nor MMPP pro-
vided satisfactory results. Reductive conditions on 21 using
excess borane also failed, yielding partial reduction of both the
C-3 acetate and C-17 methyl ester, but leaving the C11–C12
alkene intact.
To our delight, treatment of 21 with ozone at −40 °C
afforded a mixture of 22 and 23 (4 : 1) (Table 1, entry 3) as an
inseparable mixture, which was evidenced by our proton and
carbon NMR spectra. A temperature study revealed that −78 °C
is optimal (Table 1, entry 4), and these conditions provided 22
and 23 in a ratio of 8 : 1. Without further purification, direct
treatment with pyridinium perbromide in acetonitrile afforded
ring-C enones 24 and 23 in 81% and 10% yield, respectively
2
3
C-12 fluorine in an ursolic acid derivative (Scheme 2).
(
Scheme 3).
Mild base-catalyzed hydrolysis of 24 afforded an alcohol 25
not shown), which was oxidized to the corresponding ketone
(
26 with refluxing iodoxybenzoic acid. A subsequent two-step
selenation/oxidation/elimination protocol gave bis-enone 27 in
good yield. Regioselective iodination using iodine in carbon
tetrachloride and pyridine in the presence of catalytic amount
Fig. 3 CDDU-methyl ester and analogues.
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3 2 2 3 3
Scheme 3 Ozonolysis-mediated C-ring enone formation. Reagent: (a) O , CH Cl , T °C; (b) pyHBr , CH CN, 50 °C.
Table 1 Optimization for ozonolysis-mediated C-ring enone formation
of 22 plus 23
Ratio
(22 : 23)
Yield
(%)
Yield of
22 (%)
Yield of
23 (%)
Entry
T (°C)
1
2
3
4
25
0
−40
−78
2 : 1
2 : 1
4 : 1
8 : 1
84
84
87
91
56
56
70
81
28
28
17
10
Scheme 5 Synthesis of cyano enones 33 and 34. Reagent: (a) K
MeOH; (b) IBX, EtOAc, reflux; (c) LiHMDS, THF, −78 °C to 0 °C; PhSeCl,
THF, −78 °C; (d) H , THF, CH Cl , 0 °C to rt; (e) I , DMAP, pyridine,
CCl , 90 °C; (f) CuCN, DMF, 120 °C.
2 3
CO ,
of dimethylaminopyridine gave iodoenone 28. Final cyanation
using copper(I) cyanide in dimethyl formamide unexpectedly
provided a mixture of the desired cyanoenone 9 and bis-cyano-
enone 29, the latter of which was not observed in our recently
developed CDDO-Me synthesis. The formation of bis-cyanoe-
none 29 may indicate that CDDU-methyl ester (9) is a more
reactive Michael acceptor and will prove more potent than
CDDO-methyl ester (4) (Scheme 4).
2
O
2
2
2
2
4
2
5
In similar fashion, enone 23 was converted in five steps to
cyanoenone 33 and bis-cyanoenone 34 (Scheme 5).
With CDDU-methyl ester (9) in hand, lithium iodide-
mediated demethylation in refluxing pyridine afforded CDDU
(
35) in 81% yield (Scheme 6). Amides 10, 11, 36, and imidazo-
lide 12 were obtained in a two-step sequence from CDDU 35,
via the acid chloride, respectively. Treatment of 36 with tri-
fluoroacetic anhydride and triethylamine in methylene chlor-
ide afforded bis-nitrile 37 in an 89% yield. Finally, direct
treatment of 35 with DDQ in refluxing benzene provided
2
6
lactone 38 in 71% yield (Scheme 6).
Biological evaluation
Scheme 6 Synthesis of CDDU-methyl ester analogues. Reagent: (a) LiI,
pyridine, reflux; (b) (COCl) , DMF, CH Cl ; EtNH ·HCl, Et N, CH Cl
c) (COCl) , DMF, CH Cl ; CF CH NH ·HCl, Et N, CH Cl ; (d) (COCl)
DMF, CH Cl ; imidazole, PhH; (e) (COCl) , DMF, CH Cl ; NH , MeOH; (f)
TFAA, Et N, CH Cl ; (g) DDQ, PhH, reflux.
The anti-inflammatory activity of our ursolic acid cyanoenones
as measured by the inhibition of nitric oxide (NO) production
2
2
2
2
3
2
2
;
(
2
2
2
3
2
2
3
2
2
2
,
2
2
2
2
2
3
3
2
2
in RAW 264.7 macrophages is summarized in Table 2. The
orientation of the ring-C enone is important, as the activity
significantly decreases when this moiety is moved from the
9
1
(11)-en-12-one position in CDDU-methyl ester (9) to the
2(13)-en-11-one position in 33. Similarly, 29 is somewhat
more potent than 34. Notably, the addition of a cyano group at
C1 (29 and 34 compared to 9 and 33) significantly decreases
biological activity, presumably because the C1-cyano group
retards Michael addition with a reactive cysteine or other
nucleophiles on a target protein.
Scheme 4 Synthesis of CDDU-methyl ester (9). Reagent: (a) K
MeOH; (b) IBX, EtOAc, reflux; (c) LiHMDS, THF, −78 °C to 0 °C; PhSeCl,
THF, −78 °C; (d) H , THF, CH Cl , 0 °C to rt; (e) I , DMAP, pyridine,
CCl , 90 °C; (f) CuCN, DMF, 120 °C.
2 3
CO ,
2
O
2
2
2
2
4
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Table 2 Biological potency of triterpenoids with
between ursolic and oleanolic analogues
a
comparison
Conclusions
a
An efficient synthesis of methyl 2-cyano-3,12-dioxoursol-1,9-
dien-28-oate (CDDU-methyl ester) from commercially available
ursolic acid is disclosed, which provides access to ursolic acid-
derived cyanoenone analogues with C-ring enone activation.
The conversion of the C ring C11–C12 alkene to a 9(11)-en-12-
one features an ozonolysis-induced oxidation followed by
enone formation mediated by pyridinium tribromide. Biologi-
cal studies of these ursolic acid analogues display a similar
structure–activity relationship to the corresponding oleanolic
acid analogues, but are less potent by approximately half a log.
Interestingly, introduction of a cyano substituent at C1 greatly
UA cmpds
IC50 (nM)
OA cmpds
CDDO-Me
IC50 (nM)
4 ± 1.6
9
17 ± 1
380 ± 38
61 ± 7
304 ± 38
54 ± 2
29
33
34
10
11
12
35
36
37
38
CDDO-EA
CDDO-TFEA
CDDO-Im
CDDO
CDDO-CONH
CDDO-CN
5 ± 3
8 ± 4
1 ± 0.4
95 ± 21
7 ± 3
26 ± 4
5 ± 0.2
251 ± 41
48 ± 5
7 ± 0.4
8 ± 2
2
1 ± 0.05
a
IC50 values were determined by serial dilutions of the compounds decreases the biological activity, probably due to steric block-
(range 1 nM–1 µM). All experiments were repeated at least 3 times.
ing of C1 to a Michael addition.
Compounds 9, 10, 11, 12, 35, 36, 37, and 38 are all active at
nanomolar concentrations in this assay (Table 2). Imidazolide
2, nitrile 37, and lactone 38 are the most potent. CDDU-Me
9) is slightly less active than the former three compounds, but
more active than CDDU (35) itself.
Experimental section
Chemistry
1
(
All reactions were performed in a single-neck, round-bottomed
As shown in Table 2, the oleanolic acid analogues are uni- flask fitted with rubber septa under a positive pressure of
formly more potent than the corresponding ursolic acid nitrogen, unless otherwise noted. Organic solutions were con-
derivatives, but with a similar trend. Thus, imidazolides centrated by rotary evaporation below 30 °C. Flash-column
(
(
12 and CDDO-Im), nitriles (37 and CDDO-CN), and a lactone chromatography
38) are the most potent. As the only difference between the (0.04–0.063 mm, 230–400 mesh ASTM) purchased from DAWN
was
performed
using
silica
gel
two scaffolds is the C19 axial methyl substituent for CDDU- RUSSUP Macherey-Nagel Inc. (Bethlehem, PA). Analytical thin-
methyl ester (9) versus C20 equatorial methyl for CDDO-methyl layer chromatography (TLC) was performed using glass backed
ester (Scheme 7), this simple transposition affects the biologi- TLC extra hard layer pre-coated with silica gel (0.25 mm, 60 Å
cal activity by 5–10 fold. This result may be useful for future pore size) impregnated with a fluorescent indicator. TLC plates
studies of structure–activity relationships related to the methyl were visualized by exposure to ultraviolet light (UV) or/and sub-
substituents on oleanolic acid and ursolic acid. Especially mersion in PAA (p-anisaldehyde) or CAM (ceric ammonium
important would seem to be the modification of the gem molybdate) stains followed by brief heating on a hot plate
dimethyl group at C4, since the origin of the biological activity (120 °C, 10–15 s). Commercial solvents and reagents were used
1
is primarily due to the reversible Michael addition mechanism as received. Proton nuclear magnetic spectra ( H NMR) were
at C-1 on ring-A.
recorded at 500 MHz at 24 °C, unless otherwise noted. Chemi-
cal shifts are expressed in parts per million (ppm, δ scale)
downfield from tetramethylsilane and are referenced to
residual protium in the NMR solvent (CHCl , δ 7.26). Data are
3
represented as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet and/or mul-
tiple resonances, br = broad, app = apparent), integration,
coupling constant in Hertz, and assignment. Proton-decoupled
1
3
carbon nuclear magnetic resonance spectra ( C NMR) were
recorded at 500 MHz at 24 °C unless otherwise noted. Chemi-
cal shifts are expressed in parts per million (ppm, δ 77.0).
IR spectra were recorded on a Jasco FT-IR 4100 Series spectro-
−
1
photometer, γmax (cm ) are partially reported. High resolution
mass spectra were acquired from the Mass Spectrometry Lab-
oratory of the University of Illinois (Urbana-Champaign, IL).
Biological evaluation
5
NO assay. RAW 264.7 cells (5 × 10 cells per well) were
plated in 96-well plates. The next day, cells were incubated
Scheme 7 3D models for CDDU-methyl ester and CDDO-methyl ester. with synthetic triterpenoids and 10 ng mL− IFNγ (R & D
1
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systems) for 24 h. NO was measured as nitrite by the Griess acetate (80 mL) was added iodoxybenzoic acid (3.7 mg,
reaction.
Methyl 3-acetoxyl-12-oxoursol-9(11)-en-28-oate (24). To
13.1 mmol, 1.3 equiv.) in one portion. The resulting suspen-
sion was heated to reflux for 24 h. After completion of the reac-
a
stirred solution of ester 21 (500 mg, 0.98 mmol, 1.0 equiv.) in tion, it was cooled in ice bath and was then filtered through
methylene chloride (10 mL) was subjected to ozonolysis at Celite. The resulting filtrate was concentrated and flash
−
78 °C. Upon completion of the reaction, it was allowed to column chromatography over silica gel using hexanes–EtOAc
1
slowly warm to room temperature and kept at room tempera- (2 : 1) gave ketone 26 (4.8 g, 99%) as a yellowish solid. H NMR
ture for 3 h. The solvent was then removed to give crude inse- (500 MHz, CDCl ) δ 5.87 (s, 1H), 3.60 (s, 3H), 2.86 (dd, 1H, J =
3
1
parable reaction mixtures with the desired ketone 22 and 23 11.2 Hz, J
∼8 : 1). The crude mixture was dissolved in acetonitrile (10 mL) 1.35–1.95 (m, 14H), 1.00–1.30 (m, 2H), 1.24 (s, 3H), 1.12 (s,
and pyridinium perbromide (416 mg, 1.30 mmol, 1.3 equiv.) 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.03 (s, 3H), 0.83 (d, 3H, J =
2
= 2.7 Hz), 2.57 (m, 1H), 2.45 (m, 2H), 2.18 (m, 1H),
(
1
3
was added. The resulting mixture was then heated to 50 °C for 6.1 Hz), 0.67 (d, 3H, J = 6.3 Hz); C NMR (500 MHz, CDCl
3
)
1
8 h. After the completion of the reaction, it was allowed to cool δ215.4, 199.9, 177.9, 177.6, 124.2, 51.8, 50.9, 50.5, 49.9, 47.3,
to room temperature and quenched with 20% aqueous sodium 45.3, 42.2, 40.7, 39.4, 39.3, 38.7, 36.8, 36.7, 34.1, 32.0, 31.1,
thiosulfate (20 mL). It was then extracted with methylene chlor- 28.3, 26.5, 24.3, 24.2, 23.8, 21.4, 20.8, 19.7, 19.5, 19.0; IR (solu-
−
1
ide (3 × 20 mL), the combined organic extracts were washed tion, CHCl
3
, cm ): 3019, 2360, 2340, 1698, 1684, 1652, 1558,
with saturated aqueous NaHCO (20 mL), brine (20 mL), and 1540, 1507, 1217, 772, 668; HRMS-ESI (calcd for C H O
3
31 47
4
+
dried over Na
2
SO
4
. Removal of solvent and flash column [M + H] ) 483.3474, found 483.3471.
Methyl 3,12-dioxoursol-1(2),9(11)-dien-28-oate (27). To
chromatography over silica gel using hexanes–EtOAc (4 : 1 &
a
2
: 1) gave 24 (52 mg, 10%) and 23 (365 mg, 71%) as white stirred solution of ketone 26 (4.8 g, 10.0 mmol, 1.0 equiv.) in
1
solids, respectively. For 24: H NMR (500 MHz, CDCl
H), 4.47 (dd, 1H, J = 11.7 Hz, J = 4.6 Hz), 3.63 (s, 3H), 2.91 (20 mL, 1.0 M solution, 20.0 mmol, 2.0 equiv.); it was kept stir-
dd, 1H, J = 11.5 Hz, J = 3.2 Hz), 2.39 (d, 1H, J = 3.7 Hz), 2.04 ring at −78 °C for 30 min and then allowed to warm to 0 °C
3
) δ 5.87 (s, anhydrous THF (150 mL) at −78 °C was slowly added LHMDS
1
1
2
(
(
1
2
s, 3H), 1.87–2.00 (m, 2H), 1.72–1.80 (m, 4H), 1.36–1.70 (m, and kept at 0 °C for 30 min. It was then cooled back to −78 °C
1
0
5
0H), 1.14–1.29 (m, 2H), 1.21 (s, 3H), 1.12 (s, 3H), 1.08 (s, 3H), and phenylselenium chloride (3.8 g, 20.0 mmol, 2.0 equiv.) in
.90 (s, 3H), 0.88 (s, 3H), 0.83–0.90 (m, 1H), 0.87 (d, 3H, J = anhydrous THF (10 mL) was added slowly. After the com-
13
.6 Hz), 0.73 (d, 3H, J = 6.6 Hz); C NMR (500 MHz, CDCl
3
)
pletion of the reaction, it was quenched with saturated
δ 201.0, 179.3, 178.5, 171.1, 123.3, 80.0, 52.1, 51.2, 50.3, 50.2, aqueous ammonium chloride, and extracted with methylene
4
2
5.4, 42.4, 40.9, 40.1, 39.6, 38.9, 38.3, 37.0, 36.2, 33.0, 31.3, 28.4, chloride (3 × 100 mL). The combined organic extracts were
8.3, 24.6, 24.4, 24.1, 24.0, 21.5, 20.9, 19.9, 19.7, 17.7, 16.9; IR washed with brine and dried over Na SO . The solvent was
, cm ): 3019, 2366, 2340, 1717, 1652, 1558, removed to give crude product, which was used directly for
540, 1520, 1507, 1456, 1217, 772, 669, 463. next step without further purifications. The selenite obtained
Methyl 3-hydroxyl-12-oxoursol-9(11)-en-28-oate (25). To a above was dissolved in a 1 : 1 mixture of THF and methylene
2
4
−1
(solution, CHCl
3
1
stirred solution of enone 24 (7.0 g, 13.3 mmol, 1.0 equiv.) in chloride (100 mL), and 30% aqueous hydrogen peroxide
methanol (100 mL) was added potassium carbonate (7.0 g, (5 mL) was added at 0 °C. The reaction mixture was allowed to
4
0.0 mmol, 3.0 equiv.). The reaction mixture was allowed stir warm to room temperature and stirred until disappearance of
at room temperature for 24 h. After completion of the reaction, starting selenite. The reaction mixture was then diluted with
it was quenched with water (200 mL) and extracted with ethyl acetate (100 mL) and washed with 20% aqueous sodium
methylene chloride (4 × 100 mL). The combined organic thiosulfate (10 mL), brine (10 mL), and dried over Na SO .
2 4
extracts were washed with brine (80 mL) and dried over Removal of solvent and flash column chromatography over
Na SO . Removal of solvent and flash column chromatography silica gel using hexanes–EtOAc (4 : 1 & 2 : 1) gave bis-enone 27
2
4
1
3
over silica gel using hexanes–EtOAc (2 : 1) gave alcohol 25 (3.44 g, 72%) as a yellowish solid. H NMR (500 MHz, CDCl )
5.6 g, 87%) as a white solid. H NMR (500 MHz, CDCl
1
(
3
) δ 5.80 δ 7.32 (d, 1H, J = 10.7 Hz), 6.14 (s, 1H), 5.93 (d, 1H, J =
(
s, 1H), 3.56 (s, 3H), 3.13 (dd, 1H, J = 11.6 Hz, J = 4.5 Hz), 10.3 Hz), 3.66 (s, 3H), 2.95 (dd, 1H, J = 11.2 Hz, J = 3.2 Hz),
1
2
1
2
2
2
1
1
0
1
3
2
3
1
1 2
.83 (dd, 1H, J = 11.4 Hz, J = 3.0 Hz), 2.32 (d, 1H, J = 3.8 Hz), 2.48 (d, 1H, J = 3.7 Hz), 1.89–1.98 (m, 1H), 1.70–1.84 (m, 7H),
.25 (brs, 1H), 1.78–1.91 (m, 2H), 1.37–1.72 (m, 12H), 1.44–1.65 (m, 4H), 1.40 (s, 3H), 1.10–1.29 (m, 3H), 1.20 (s, 3H),
.24–1.35 (m, 2H), 1.03–1.20 (m, 2H), 1.09 (s, 3H), 1.04 (s, 3H), 1.18 (s, 3H), 1.12 (s, 3H), 1.11 (s, 3H), 0.88 (d, 3H, J = 6.1 Hz),
1
3
.01 (s, 3H), 0.95 (s, 3H), 0.79 (d, 3H, J = 6.1 Hz), 0.73 (s, 3H), 0.73 (d, 3H, J = 6.3 Hz); C NMR (500 MHz, CDCl
3
) δ 203.8,
1
3
.65 (d, 3H, J = 6.6 Hz); C NMR (500 MHz, CDCl
3
) δ 201.0, 200.0, 178.4, 173.0, 155.4, 126.5, 124.4, 52.1, 51.4, 50.2, 48.4,
79.9, 178.4, 123.0, 77.9, 52.0, 51.1, 50.1, 45.4, 42.3, 40.8, 40.2, 45.8, 44.9, 42.5, 42.1, 41.1, 39.6, 38.9, 36.9, 32.2, 31.3, 28.5,
9.5, 39.4, 38.8, 36.9, 36.4, 33.0, 31.3, 28.4, 27.6, 24.5, 24.4, 28.0, 27.5, 25.3, 24.0, 21.8, 20.9, 20.0, 19.6, 18.5; IR (solution,
−
1
−1
3.9, 20.9, 19.9, 19.7, 18.0, 15.9; IR (solution, CHCl
019, 2410, 2360, 2340, 1716, 1652, 1558, 1539, 1520, 1507, 1520, 1507, 1216, 770, 668, 451; HRMS-ESI (calcd for C H O
45 4
217, 772, 669, 624, 432.
3 3
, cm ): CHCl , cm ): 3019, 2360, 2340, 1716, 1699, 1652, 1558, 1540,
3
1
+
[M + H] ) 481.3318, found 481.3306.
Methyl 2-iodo-3,12-dioxoursol-1(2),9(11)-dien-28-oate
Methyl 3,12-dioxoursol-9(11)-en-28-oate (26). To a stirred
solution of alcohol 25 (4.9 g, 10.1 mmol, 1.0 equiv.) in ethyl (28). To a stirred solution of bisenone 27 (1.07 g, 2.3 mmol,
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1
.0 equiv.) in a 1 : 1 mixture of pyridine and carbon tetrachlor- 46.2, 46.1, 42.7, 42.1, 39.8, 38.8, 36.8, 31.4, 29.1, 28.8, 26.6,
ide (10 mL) was added dimethylaminopyridine (56 mg, 26.2, 26.1, 24.1, 21.0, 20.9, 20.0, 19.3, 19.2; IR (solution,
−
1
0
3
.46 mmol, 0.2 equiv.) and iodine (1.75 g, 6.9 mmol, CHCl , cm ): 3019, 2929, 2360, 2340, 1732, 1716, 1683, 1668,
3
2
.0 equiv.), and the resulting mixture was heated to 90 °C for 1653, 1558, 1540, 1520, 1507, 1472, 1456, 1217, 772, 669, 473;
+
4 h without light. After the completion of the reaction, the HRMS-ESI (calcd for C H N O [M + H] ) 531.3474, found
3
3
43 2 4
solvent was removed under vacuum, and the residue was 531.3468.
diluted with ethyl acetate (100 mL). The resulting solution was
successively washed with 20% aqueous sodium thiosulfate (3 × (35). To a stirred solution of CDDU-methyl ester (9) (6.0 g,
0 mL), 1 N aqueous HCl (3 × 10 mL), saturated NaHCO 11.9 mmol, 1.0 equiv.) in pyridine (50 mL) was added LiI
10 mL), brine (10 mL), and dried over Na SO . Removal of (16.0 g, 0.12 mol, 10.0 equiv.) and the resulting suspension
2-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-carboxylic
acid
1
3
(
2
4
solvent and flash column chromatography over silica gel using was heated to reflux for 16 h. Additional LiI (3.2 g, 23.8 mmol,
hexanes–EtOAc (4 : 1 & 2 : 1) gave iodo-enone 28 (1.18 g, 87%) 2.0 equiv.) was added and the heating was continued for
1
as a yellowish solid. H NMR (500 MHz, CDCl
6
3
3
) δ 8.11 (s, 1H), another 8 h. After the completion of the reaction, it was cooled
to room temperature, and the solvent was removed by vacuum.
.0 Hz), 2.48 (d, 1H, J = 3.7 Hz), 1.90–1.99 (m, 1H), 1.70–1.87 The reaction was quenched with 2 N HCl (200 mL), and
.11 (s, 1H), 3.66 (s, 3H), 2.95 (dd, 1H, J1 = 11.3 Hz, J2 =
(m, 7H), 1.59–1.65 (m, 1H), 1.44–1.57 (m, 3H), 1.41 (s, 3H), extracted with ethyl acetate (4 × 100 mL). The combined
1
.15–1.26 (m, 3H), 1.18 (s, 3H), 1.22 (s, 3H), 1.17 (s, 3H), 1.13 organic extracts were washed with saturated aqueous NaHCO3
1
3
(s, 3H), 0.89 (d, 3H, J = 6.1 Hz), 0.75 (d, 3H, J = 6.6 Hz);
C
(50 mL), brine (50 mL), and dried over Na
2 4
SO . Removal of
NMR (500 MHz, CDCl
3
) δ 199.8, 197.0, 178.3, 171.5, 163.8, solvent and flash column chromatography over silica gel using
1
3
2
2
24.6, 102.4, 52.2, 51.3, 50.2, 38.3, 36.5, 45.9, 45.4, 42.5, 41.2, hexanes–EtOAc (2 : 1 & 1 : 1) gave acid 35 (4.7 g, 81%) as a
1
9.6, 38.9, 36.9, 31.9, 31.3, 28.6, 28.5, 28.0, 25.3, 24.0, 22.4, yellowish solid. H NMR (500 MHz, CDCl
0.9, 20.0, 19.7, 18.7; IR (solution, CHCl , cm ): 3019, 2360, (s, 1H), 2.95 (dd, 1H, J = 11.0 Hz, J
3 1 2
3
) δ 8.03 (s, 1H), 6.12
= 2.5 Hz), 2.59 (d, 1H, J =
340, 1717, 1652, 1540, 1520, 1507, 1217, 770, 669; HRMS-ESI 3.4 Hz), 1.66–2.00 (m, 9H), 1.43–1.62 (m, 2H), 1.47 (s, 3H),
−1
+
(calcd for C31
H
44IO
4
[M + H] ) 607.2284, found 607.2280.
1.18–1.34 (m, 3H), 1.26 (s, 3H), 1.20 (s, 3H), 1.19 (s, 3H), 1.13
Methyl 2-cyano-3,12-dioxoursol-1(2),9(11)-dien-28-oate (9) (s, 3H), 0.82–0.94 (m, 1H), 0.90 (d, 3H, J = 5.8 Hz), 0.75 (d, 3H,
1
3
and methyl 1,2-dicyano-3,12-dioxoursol-1(2),9(11)-dien-28-oate J = 6.4 Hz); C NMR (500 MHz, CDCl ) δ 199.3, 196.8, 170.0,
3
(29). To a stirred solution of iodoenone 28 (322 mg, 0.5 mmol, 166.3, 124.8, 115.2, 114.6, 51.4, 47.9, 46.0, 45.3, 42.8, 42.6,
1
.0 equiv.) in anhydrous dimethylformamide (5 mL) was 41.2, 39.5, 38.8, 37.0, 31.8, 31.2, 28.5, 27.6, 25.5, 23.8, 21.8,
−1
added copper(I) cyanide (54 mg, 0.6 mmol, 1.2 equiv.), and the 20.9, 20.0, 19.6, 18.4; IR (solution, CHCl , cm ): 3019, 2360,
3
resulting mixture was allowed to heat to 120 °C for 12 h. After 2340, 1716, 1698, 1684, 1652, 1558, 1540, 1520, 1507, 1456,
+
the completion of the reaction, it was cooled to room tempera- 1217, 772, 669; HRMS-ESI (calcd for C31
ture, diluted with ethyl acetate (100 mL). The resulting solu- 492.3114, found 492.3108.
H
42NO
4
[M + H] )
tion was washed with water (3 × 10 mL), brine (3 × 10 mL), and
dried over Na SO . Removal of solvent and flash column ethyl amide (10). To a stirred solution of acid 35 (400 mg,
chromatography over silica gel using hexanes–EtOAc (4 : 1 & 0.81 mmol, 1.0 equiv.) in methylene chloride (15 mL) was
: 1) gave cyanoenone 9 (142 mg, 53%) and biscyanoenone 29 added oxalyl chloride (0.35 mL, 4.05 mmol, 5.0 equiv.) and
2-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-carboxylic
acid
2
4
2
1
(
(
(
76 mg, 27%) as yellowish solids, respectively. For 9: H NMR anhydrous dimethylformamide (0.11 µL, 2.0 µmol, catalytic)
500 MHz, CDCl ) δ 8.04 (s, 1H), 6.11 (s, 1H), 3.67 (s, 3H), 2.96 slowly at 0 °C, and it was allowed to warm to room temperature
dd, 1H, J
3
1
= 11.5 Hz, J
2
= 2.7 Hz), 2.51 (d, 1H, J = 3.7 Hz), for 2 h. The solvent was removed, and toluene (10 mL) was
1
1
1
0
.91–1.99 (m, 1H), 1.72–1.85 (m, 7H), 1.60–1.68 (m, 1H), added and removed by vacuum, which was repeated for three
.47–1.58 (m, 3H), 1.48 (s, 3H), 1.16–1.30 (m, 2H), 1.26 (s, 3H), times to provide the corresponding acyl chloride. To a stirred
.21 (s, 3H), 1.18 (s, 3H), 1.12 (s, 3H), 0.90 (d, 3H, J = 6.1 Hz), solution of the resulting acyl chloride obtained above in
1
3
.83–0.90 (m, 1H), 0.74 (d, 3H, J = 6.6 Hz); C NMR (500 MHz, methylene chloride (10 mL) was added triethylamine (0.58 mL,
CDCl ) δ 199.3, 196.9, 178.3, 169.8, 166.4, 124.9, 115.1, 114.7, 4.05 mmol, 5.0 equiv.) followed by slow addition of ethylamine
3
5
3
1
1
2.2, 51.4, 50.2, 47.9, 46.0, 45.3, 42.9, 42.6, 41.2, 39.6, 38.9, hydrochloride (340 mg, 4.05 mmol, 5.0 equiv.) at 0 °C. After
6.9, 31.9, 31.3, 28.5, 27.6, 27.5, 25.4, 23.9, 21.8, 20.9, 20.0, the addition, the resulting mixture was allowed to warm to
−
1
9.6, 18.4; IR (solution, CHCl , cm ): 3019, 2360, 2340, 1716, room temperature and kept stirring until the disappearance of
3
652, 1558, 1540, 1520, 1507, 1217, 772, 669, 464; HRMS-ESI acyl chloride. After the completion of the reaction, the solvent
+
(calcd for C32
H
44NO
4
[M + H] ) 506.3270, found 506.3267. For was removed and it was diluted with methylene chloride
1
2
9: H NMR (500 MHz, CDCl ) δ 6.22 (s, 1H), 3.68 (s, 3H), 3.02 (80 mL), washed with 1 N HCl (10 mL), saturated aqueous
3
(
dd, 1H, J
1
2 3 2 4
= 11.6 Hz, J = 2.0 Hz), 2.51 (d, 1H, J = 3.4 Hz), NaHCO (10 mL), brine (10 mL), and dried over Na SO .
1
.74–2.02 (m, 7H), 1.40–1.67 (m, 4H), 1.62 (s, 3H), 1.18–1.34 Removal of solvent and flash column chromatography over
(
0
m, 2H), 1.26 (s, 3H), 1.22 (s, 3H), 1.21 (s, 3H), 1.17 (s, 3H), silica gel using hexanes–EtOAc (2 : 1 & 1 : 1) gave ethyl amide
1
.83–0.97 (m, 1H), 0.90 (d, 3H, J = 5.9 Hz), 0.74 (d, 3H, J = 6.6 10 (418 mg, 99%) as a yellowish solid. H NMR (500 MHz,
13
Hz); C NMR (500 MHz, CDCl
3 3
) δ 198.0, 195.2, 178.3, 166.3, CDCl ) δ 8.49 (s, 1H), 6.36 (s, 1H), 6.16 (t, 1H, J = 5.5 Hz), 3.33
1
46.8, 126.9, 124.6, 113.9, 112.1, 52.3, 51.6, 50.3, 47.3, 46.4, (m, 1H), 3.21 (m, 1H), 2.68 (dd, 1H, J = 10.7 Hz, J = 2.2 Hz),
1
2
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1
.62 (d, 1H, J = 3.4 Hz), 1.60–1.99 (m, 9H), 1.46–1.60 (m, 2H), (500 MHz, CDCl
.44 (s, 3H), 1.05–1.30 (m, 3H), 1.28 (s, 3H), 1.24 (s, 3H), 1.19 11.7 Hz), 2.12 (m, 1H), 1.71–1.92 (m, 4H), 1.60–1.70 (m, 3H),
3
) δ 8.05 (s, 1H), 6.28 (s, 1H), 2.56 (d, 1H, J =
(
s, 3H), 1.10 (s, 3H), 0.85–0.95 (m, 1H), 0.90 (d, 3H, J = 6.1 Hz), 1.57 (s, 3H), 1.50 (s, 3H), 1.39–1.50 (m, 2H), 1.22–1.44 (m, 1H),
1
3
0
1
4
2
3
.68 (d, 3H, J = 6.6 Hz); C NMR (500 MHz, CDCl ) δ 200.5, 1.28 (s, 3H), 1.21 (s, 3H), 1.18 (s, 3H), 1.00–1.10 (m, 1H), 0.95
97.1, 177.4, 171.1, 168.1, 124.9, 115.1, 114.8, 51.0, 49.3, 47.8, (d, 3H, J = 6.3 Hz), 0.82–0.90 (m, 1H), 0.82 (d, 3H, J = 6.4 Hz);
1
3
6.2, 45.3, 43.8, 43.2, 41.2, 39.9, 39.0, 37.9, 34.6, 31.9, 31.4,
3
C NMR (500 MHz, CDCl ) δ 196.6, 192.2, 178.3, 174.3, 165.7,
9.6, 28.2, 27.4, 27.3, 25.8, 23.7, 21.8, 21.0, 19.5, 19.4, 15.2; 123.4, 115.4, 114.5, 87.8, 55.1, 48.1, 46.4, 45.3, 45.1, 43.2, 42.9,
−1
IR (solution, CHCl , cm ): 3019, 2979, 2360, 2340, 1698, 1684, 39.8, 37.5, 32.8, 31.3, 31.2, 30.8, 28.0, 27.6, 26.3, 21.9, 21.8,
1
3
−
1
652, 1636, 1558, 1540, 1520, 1507, 1456, 1217, 771, 669, 444; 20.3, 19.5, 18.6, 17.8; IR (solution, CHCl
3
, cm ): 3019, 2969,
[M + H] ) 519.3587, found 2936, 2360, 2340, 1772, 1715, 1697, 1675, 1652, 1558, 1540,
1520, 1507, 1456, 1215, 909, 749, 669; HRMS-ESI (calcd. for
+
HRMS-ESI (calcd for C33
19.3585.
-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-carboxylic
trifluoroethyl amide (11). To a stirred solution of acid 35
400 mg, 0.81 mmol, 1.0 equiv.) in methylene chloride (15 mL) To a stirred solution of acid 35 (860 mg, 1.75 mmol, 1.0 equiv.)
47 2 3
H N O
5
+
2
acid
C
31
H
40NO
4
[M + H] ) 490.2957, found 490.2961.
2-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-carboxamide (36).
(
was added oxalyl chloride (0.35 mL, 4.05 mmol, 5.0 equiv.) in methylene chloride (15 mL) was added oxalyl chloride
and anhydrous dimethylformamide (0.11 µL, 2.0 µmol, cata- (0.50 mL, 5.72 mmol, 3.3 equiv.) and anhydrous dimethylform-
lytic) slowly at 0 °C, and it was allowed to warm to room temp- amide (0.15 µL, 2.7 µmol, catalytic) slowly at 0 °C, and it was
erature for 2 h. The solvent was removed, and toluene (10 mL) allowed to warm to room temperature for 2 h. The solvent was
was added and removed by vacuum, which was repeated for removed, and toluene (10 mL) was added and removed by
three times to provide the corresponding acyl chloride. To a vacuum, which was repeated for three times to provide the
stirred solution of the resulting acyl chloride obtained above in corresponding acyl chloride. To a stirred solution of the result-
methylene chloride (10 mL) was added triethylamine (0.58 mL, ing acyl chloride obtained above in methylene chloride
4
.05 mmol, 5.0 equiv.) followed by slow addition of trifluoro- (20 mL) was added ammonia (8.75 mL, 1.0 M in MeOH,
ethylamine hydrochloride (340 mg, 4.05 mmol, 5.0 equiv.) at 8.75 mmol, 5.0 equiv.), and the resulting mixture was stirred at
°C. After the addition, the resulting mixture was allowed to room temperature until the disappearance of acyl chloride.
0
warm to room temperature and kept stirring until the dis- After the completion of the reaction, the solvent was removed
appearance of acyl chloride. After the completion of the reac- and flash column chromatography over silica gel using
tion, the solvent was removed and it was diluted with hexanes–EtOAc (1 : 1 & 1 : 2) gave amide 36 (791 mg, 92%)
1
methylene chloride (80 mL), washed with 1 N HCl (10 mL), as a yellowish solid. H NMR (500 MHz, CDCl ) δ 8.06 (s, 1H),
3
3
saturated aqueous NaHCO (10 mL), brine (10 mL), and dried 6.28 (s, 1H), 2.51 (d, 1H, J = 11.7 Hz), 2.10 (m, 1H), 1.69–1.90
over Na SO . Removal of solvent and flash column chromato- (m, 6H), 1.51–1.67 (m, 4H), 1.54 (s, 3H), 1.34–1.50 (m, 2H),
2
4
graphy over silica gel using hexanes–EtOAc (2 : 1 & 1 : 1) gave 1.46 (s, 3H), 1.10–1.30 (m, 2H), 1.23 (s, 3H), 1.17 (s, 3H), 1.15
1
trifluoroethyl amide 11 (387 mg, 83%) as a yellowish solid. H (s, 3H), 0.82–0.92 (m, 1H), 0.91 (d, 3H, J = 6.3 Hz), 0.78 (d, 3H,
1
3
NMR (500 MHz, CDCl
3
) δ 9.14 (s, 1H), 6.92 (t, 1H, J = 6.5 Hz), J = 6.1 Hz); C NMR (500 MHz, CDCl
.74 (t, 1H, J = 5.5 Hz), 4.20 (m, 1H), 3.55 (m, 1H), 2.89 (dd, 171.1, 167.7, 124.9, 115.0, 114.9, 51.2, 49.8, 47.8, 46.2, 45.3,
H, J = 9.9 Hz, J = 2.8 Hz), 2.48 (d, 1H, J = 3.2 Hz), 1.93–2.15 43.9, 43.1, 41.2, 39.9, 39.0, 37.9, 31.9, 31.4, 28.2, 27.5, 25.9,
3
) δ 200.3, 197.0, 180.4,
6
1
1
2
−1
(
m, 1H), 1.69–1.92 (m, 6H), 1.47–1.65 (m, 4H), 1.55 (s, 3H), 23.7, 21.8, 20.9, 20.1, 19.5, 18.4; IR (solution, CHCl
3
, cm ):
1
.05–1.30 (m, 3H), 1.29 (s, 3H), 1.23 (s, 3H), 1.18 (s, 3H), 1.05 3019, 2360, 2340, 1652, 1558, 1540, 1507, 1217, 771, 669, 429;
+
43 2 3
(s, 3H), 0.85–0.95 (m, 1H), 0.87 (d, 3H, J = 6.6 Hz), 0.57 (d, 3H, HRMS-ESI (calcd for C31H N O [M + H] ) 491.3114, found
1
3
J = 6.6 Hz); C NMR (500 MHz, CDCl
3
) δ 200.3, 197.2, 178.2, 491.3105.
1
4
2
71.2, 169.4, 125.0, 124.5 (q), 115.6, 114.4, 51.1, 49.8, 47.7, 2-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-nitrile (37). To a
6.1, 45.3, 43.4, 43.3, 41.1, 40.5 (s), 39.7, 38.7, 37.4, 31.9, 31.3, stirred solution of amide 36 (600 mg, 1.22 mmol, 1.0 equiv.) in
9.6, 27.9, 27.1, 27.0, 25.9, 23.7, 21.8, 20.9, 19.4, 18.3; IR (solu- methylene chloride (40 mL) at 0 °C was added triethylamine
−
1
tion, CHCl , cm ): 3019, 2957, 2869, 2360, 2340, 1652, 1558, (0.44 mL, 3.13 mmol, 2.5 equiv.) and trifluoroacetic anhydride
3
1
539, 1520, 1507, 1465, 1436, 1376, 1364, 1217, 772, 669; (0.26 mL), and the resulting mixture was stirred at 0 °C until
+
HRMS-ESI (calcd for C33
73.3307.
H
44
N
2
F
3
O
3
[M + H] ) 573.3304, found the disappearance of starting material. After the completion of
the reaction, it was quenched with saturated aqueous NaHCO3
5
2
-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-carboxyl-13-oxyl- (20 mL), and extracted with methylene chloride (3 × 20 mL).
actone (38). To a stirred solution of acid 35 (250 mg, The combined organic extracts were washed with brine
.51 mmol, 1.0 equiv.) in benzene (15 mL) was added DDQ (10 mL) and dried over Na SO . Removal of solvent and flash
0
2
4
(136 mg, 0.61 mmol, 1.2 equiv.) and the resulting mixture was column chromatography over silica gel using hexanes–EtOAc
heated to reflux for 24 h. After the completion of the reaction, (4 : 1 & 2 : 1) gave the desired product 37 (407 mg, 89%) as a
1
it was cooled to room temperature and filtered through Celite. white solid. H NMR (500 MHz, CDCl ) δ 8.06 (s, 1H), 6.16 (s,
3
1 2
Removal of solvent and flash column chromatography over 1H), 3.17 (d, 1H, J = 3.7 Hz), 2.72 (dd, 1H, J = 11.2 Hz, J = 3.2
silica gel using hexanes–EtOAc (4 : 1 & 2 : 1) gave the desired Hz), 1.95–2.18 (m, 4H), 1.74–1.86 (m, 3H), 1.55–1.66 (m, 3H),
1
product 38 (175.5 mg, 71%) as a reddish solid. H NMR 1.53 (s, 3H), 1.34–1.50 (m, 2H), 1.45 (s, 3H), 1.27 (s, 3H),
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1
5
.16–1.26 (m, 2H), 1.20 (s, 3H), 1.12 (s, 3H), 0.92 (d, 3H, J =
3 T. Honda, H. J. Finlay, G. W. Gribble, N. Suh and
M. B. Sporn, New enone derivatives of oleanolic acid and
ursolic acid as inhibitors of nitric oxide production in
mouse macrophages, Bioorg. Med. Chem. Lett., 1997, 7,
1623–1628.
4 M. B. Sporn, K. T. Liby, M. M. Yore, L. Fu,
J. M. Lopchuk and G. W. Gribble, New synthetic triter-
penoids: potent agents for prevention and treatment of
tissue injury caused by inflammatory and oxidative
stress, J. Nat. Prod., 2011, 74, 537–545; and references
therein.
5 T. Honda, B. V. Rounds, L. Bore, H. J. Finlay, F. G. Favaloro
Jr., N. Suh, Y. Wang, M. B. Sporn and G. W. Gribble, Syn-
thetic oleanane and ursane triterpenoids with modified
ring A and C: a series of highly active inhibitors of nitric
oxide production in mouse macrophages, J. Med. Chem.,
2000, 43, 4233–4246.
6 D. S. Hong, R. Kurzrock, J. G. Supko, X. He, A. Naing,
J. Wheler, D. Lawrence, J. P. Eder, C. J. Meyer,
D. A. Ferguson, J. Mier, M. Konopleva, S. Konoplev,
M. Andreeff, D. Kufe, H. Lazarus, G. I. Shapiro and
B. J. Dezube, A phase I first-in-human trial of bardoxolone
methyl in patients with advanced solid tumors and lym-
phomas, Clin. Cancer Res., 2012, 18, 3396–3406.
13
.6 Hz), 0.82–0.92 (m, 1H), 0.76 (d, 3H, J = 6.6 Hz); C NMR
) δ 198.1, 196.8, 170.4, 166.1, 125.0, 124.8,
(500 MHz, CDCl
3
1
3
1
1
1
15.2, 114.6, 51.5, 48.0, 46.1, 45.3, 44.3, 43.0, 41.5, 41.2, 39.0,
8.7, 36.7, 32.0, 30.6, 28.8, 27.6, 27.5, 25.8, 25.6, 21.8, 20.6,
−
1
9.8, 19.6, 18.4; IR (solution, CHCl
3
, cm ): 3019, 2360, 2340,
697, 1684, 1662, 1653, 1558, 1540, 1520, 1507, 1473, 1456,
217, 909, 772, 669, 450; HRMS-ESI (calcd for C H N O
41 2 2
3
1
+
[
M + H] ) 473.3168, found 473.3165.
-Cyano-3,12-dioxoursol-1(2),9(11)-dien-28-carboxylic
imidazolide (12). To a stirred solution of acid 35 (460 mg,
.0 mmol, 1.0 equiv.) in methylene chloride (20 mL) was
2
acid
1
added oxalyl chloride (0.50 mL, 5.8 mmol, 5.8 equiv.) and
anhydrous dimethylformamide (0.15 µL, 2.0 µmol, catalytic)
slowly at 0 °C, and it was allowed to warm to room temperature
for 2 h. The solvent was removed, and toluene (10 mL) was
added and removed by vacuum, which was repeated for three
times to provide the corresponding acyl chloride. To a stirred
solution of the resulting acyl chloride obtained above in
benzene (10 mL) was added imidazole (350 mg, 5.0 mmol,
5
.0 equiv.) and the resulting mixture was stirred at room temp-
erature until the disappearance of acyl chloride. After the com-
pletion of the reaction, removal of solvent and flash column
chromatography over silica gel using hexanes–EtOAc (4 : 1 &
2
: 1) gave imidazolide 12 (458 mg, 90%) as a yellowish solid.
7 P. E. Pergola, P. Raskin, R. D. Toto, C. J. Meyer, J. W. Huff,
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1
H NMR (500 MHz, CDCl ) δ 8.34 (s, 1H), 8.05 (s, 1H), 7.66 (s,
3
1
1
0
H), 7.06 (s, 1H), 6.10 (s, 1H), 3.27 (d, 1H, J = 9.8 Hz), 2.43 (s,
H), 0.98–2.06 (m, 11H), 1.44 (s, 3H), 1.23 (s, 3H), 1.16 (s, 9H),
.96 (d, 3H, J = 5.1 Hz), 0.78 (d, 3H, J = 5.9 Hz); C NMR
) δ 197.9, 196.8, 175.5, 170.2, 166.3, 137.8,
13
(500 MHz, CDCl
3
1
4
2
3
1
30.6, 124.6, 117.9, 115.1, 114.6, 52.6, 51.1, 47.9, 46.0, 45.2,
3.1, 42.9, 41.3, 39.9, 38.8, 35.8, 31.7, 31.0, 28.1, 27.5, 27.5,
5.4, 21.8, 20.8, 20.0, 19.7, 18.3; IR (solution, CHCl , cm ):
3
019, 2360, 2340, 1716, 1698, 1652, 1558, 1540, 1520, 1507,
214, 909, 773, 669, 438; HRMS-ESI (calcd for C H N O
44 3 3
−1
3
4
+
[M + H] ) 542.3383, found 542.3377.
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terpenoids: multifunctional drugs with a broad range of
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