Synthesis and inhibitory effect of novel glycyrrhetinic acid derivatives on IL-1β-induced prostaglandin E2 production in normal human dermal fibroblasts

Article abstract of DOI:10.1248/cpb.53.1103

Olean-11,13(18)-dien-3β,30-diol dihemiphthalate (3), which was derived from glycyrrhetinic acid (GA), has been reported to produce a potent of anti-inflammatory effect in in vivo assays. Using 3 as a lead compound, we attempted to synthesize some modified compounds which varied in the following; i) the position of a carboxyl group in the phthalate moiety, ii) the number of carboxyls attached to the benzoyl group, iii) conversion of benzene ring to another ring system, iv) the linkage form between the benzene ring and oleanene skeleton at position 3 and/or 30. These were screened for their inhibitory activity against interleukin-1β (IL-1β)-induced prostaglandin E ₂ (PGE₂) production in normal human dermal fibroblasts (NHDF). Although conversion of the ortho-carboxyl group of 3 into the meta-position or the para-position led to an increase in inhibitory activity, the elimination or increase of the carboxyl group resulted in loss of the inhibitory activity. Conversion of the ester bond to the amide bond at position 3 and/or 30 of 3 did not contribute to a significant increase in inhibitory activity. On the other hand, among the derivatives possessing an anthranilic acid moiety at position 30 of 3β-O-acetyl-olean-11,13(18)-dien-30-oic acid (20), 3β-hydroxy-30-nor-olean-11,13(18)-dien-20β-[N-(2-carboxyphenyl)] carboxamide (30) showed the most potent inhibitory activity (IC₅₀ 1.0 μM) in this series.

Full text of DOI:10.1248/cpb.53.1103

September 2005
Chem. Pharm. Bull. 53(9) 1103—1110 (2005)
1103
Synthesis and Inhibitory Effect of Novel Glycyrrhetinic Acid Derivatives
b
on IL-1 -Induced Prostaglandin E2 Production in Normal Human
Dermal Fibroblasts
*
Michiko TSUKAHARA, Takeshi NISHINO, Ikue FURUHASHI, Hideo INOUE, Toshitsugu SATO, and
Hiroatsu MATSUMOTO
Research Laboratory, Minophagen Pharmaceutical Co., Ltd.; 2–2–3 Komatsubara, Zama, Kanagawa 228–0002, Japan.
Received March 7, 2005; accepted May 16, 2005
Olean-11,13(18)-dien-3b,30-diol dihemiphthalate (3), which was derived from glycyrrhetinic acid (GA), has
been reported to produce a potent of anti-inflammatory effect in in vivo assays. Using 3 as a lead compound, we
attempted to synthesize some modified compounds which varied in the following; i) the position of a carboxyl
group in the phthalate moiety, ii) the number of carboxyls attached to the benzoyl group, iii) conversion of ben-
zene ring to another ring system, iv) the linkage form between the benzene ring and oleanene skeleton at position
3 and/or 30. These were screened for their inhibitory activity against interleukin-1b (IL-1b )-induced
prostaglandin E2 (PGE2) production in normal human dermal fibroblasts (NHDF). Although conversion of the
ortho-carboxyl group of 3 into the meta-position or the para-position led to an increase in inhibitory activity,
the elimination or increase of the carboxyl group resulted in loss of the inhibitory activity. Conversion of the
ester bond to the amide bond at position 3 and/or 30 of 3 did not contribute to a significant increase in inhibitory
activity. On the other hand, among the derivatives possessing an anthranilic acid moiety at position 30
of 3b-O-acetyl-olean-11,13(18)-dien-30-oic acid (20), 3b-hydroxy-30-nor-olean-11,13(18)-dien-20b-[N-(2-car-
boxyphenyl)]carboxamide (30) showed the most potent inhibitory activity (IC50 1.0 mM) in this series.
Key words glycyrrhetinic acid derivative; anti-inflammatory; prostaglandin E2; 3b-hydroxy-30-nor-olean-11,13(18)-dien-20b-
[N-(2-carboxyphenyl)]carboxamide; olean-11,13(18)-dien-3b,30-diol dihemiphthalate; normal human dermal fibroblast
Glycyrrhizin (GL), a main saponin component of several 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse
Glycyrrhiza species (G. uralensis, G. glabra, and G. inflata) ear edema,16) arachidonic acid-induced or capsaicin-induced
has been reported to possess various pharmacological activi- mouse ear edema,17,18) and carrageenin-induced rat paw
ties such as anti-inflammation,1) inhibition of prostaglandin edema.19) It was suggested that one of the mechanisms by
E2 (PGE2) production in rat macrophages,2) antiallergic,3) an- which 3 induces anti-inflammatory effects is the inhibition of
tiviral4,5) action, and interferon-g-induction.6) In Japan, a gly- cyclooxygenase or lipoxygenase activities at the inflamma-
cyrrhizin preparation, Stronger-Neo Minophagen C (SNMC), tory site.20) Interestingly, it was found that 3 exhibited anti-
has been used extensively for more than 30 years to treat acetic acid-induced writhing activity15) and an antiulcer ac-
chronic hepatitis.
tivity,21) in addition to the anti-inflammation. In addition, we
Glycyrrhetinic acid (GA), the aglycon of GL, is also have reported that 3 did not inhibit the activity of 11b-HSD-
known to have wide pharmacological effects such as anti-in- 2, which causes an adverse effect of GA.22) These facts sug-
flammation,7,8) antitumor,9) antihepatotoxic activity,10) and in- gest that 3 is promising as a lead compound for a new type of
hibition of the growth of mouse melanoma.11) In 1989, it was anti-inflammatory agent, showing anti-inflammatory action
reported that GA strongly inhibits renal 11b-hydroxysteroid as well as anti-ulcer action, different from conventional non-
dehydrogenase (11b-HSD) in rat,12) which has been regarded steroidal anti-inflammatory drugs (NSAIDs), which are
as a cause of pseudoaldosteronism occasionally induced by mostly ulcerogenic.
the administration of a GL preparation or Carbenoxolone.
Recently, confirmed that normal human dermal fibroblasts
We have synthesized various GA derivatives, such as (NHDF) produce PGE2 in response to IL-1b.23) In fact,
deoxo-glycyrrhetol (1),13) olean-11,13(18)-dien-3b,30-diol NHDF have been used to assess the effect of compounds
(2),14) and their succinate or hemi-phthalate14) forms (Fig. 1), such as Tranilast and Azelastine on PGE2 production.24,25)
in order to enhance the anti-inflammatory activity and sup- However, it is unclear whether 3 and its derivatives have an
press the pseudoaldosteronism due to the inhibition of miner- effect on COX-2 dependent PGE2 production.
alcorticoid metabolism. During the course of this work, 3
In this paper, we synthesized various new compounds de-
was reported to show various anti-inflammatory activities rived from 3 and examined the effect of these compounds on
such as the inhibition of increasing vascular permeability,15) IL-1b-induced PGE2 production in NHDF.
Fig. 1. Structures of Glycyrrhetinic Acid (GA) and Its Derivatives
∗ To whom correspondence should be addressed. e-mail: tsukahara@lab.minophagen.co.jp
© 2005 Pharmaceutical Society of Japan

1104
Vol. 53, No. 9
Chart 1
Chart 2
Chemistry
with hydroxylamine hydrochloride in pyridine to give
In regard to the modification of 3, we focused on the fol- dioxime (15) in 69% yield. Then, the reduction of 15 with
lowing parts of 3: i) the position of the carboxyl group in the sodium cyanoborohydride in the presence of titanium(III)
phthalate moiety (4, 5), ii) the number of carboxyls attached chloride and ammonium acetate in methanol afforded a mix-
to the benzoyl group (6—8), iii) conversion of the benzene ture of 3a,30- and 3b,30-diamino forms. Since it was diffi-
ring to other ring systems (9—13), and iv) the linkage form cult to purify the mixture using silica gel column chromatog-
between the benzene ring and oleanene skeleton at position 3 raphy, t-butoxycarbonylation was carried out by a general
and/or 30 (17, 23—31).
method, then the product was purified using silica gel column
As shown in Chart 1, 4 and 5 were obtained by the reac- chromatography. Next, the fraction was subjected to de-t-bu-
tion of 2 with isophthaloyl dichloride or terephthaloyl dichlo- toxycarbonylation with trifluoroacetic acid (TFA) to give
ride in pyridine in 20% and 15% yield, respectively. Esterifi- 3b,30-diamine (16) in 44% overall yield. Compound (19)
cation of 2 with benzoyl chloride, 1,3,5-benzenetricarbonyl was prepared by using a synthetic method of 16 from a 3-oxo
trichloride or pyromellitic dianhydride in pyridine afforded 6, form, which was obtained by selective acetylation of the C-
7 and 8 in 82%, 16% and 35% yield, respectively.
30 hydroxy group in 2 with one equivalent of acetic anhy-
Compounds (9—12), the benzoic acid of which were re- dride, followed by oxidation with Jones reagent, as shown in
placed by another ring system, were obtained in 10—45% Chart 3. Compound (22) was synthesized from 21, which
yield by esterification of 2 with corresponding acid anhy- was prepared from 2029) and ethyl chlorocarbonate, followed
dride in pyridine. Compound (13) was prepared from 2 in by reduction with sodium borohydride30) in THF (Chart 3).
12% yield by activation of 2,5-thiophenedicarboxylic acid Condensation of 16, 19 and 22 with phthalic anhydride in
with 2-chloro-1-methylpyridinium iodide.26,27)
pyridine afforded 17, 23 and 24 in 75%, 75% and 86% yield,
Furthermore, in order to synthesize 17, 23 and 24, which respectively (Charts 2, 4). Furthermore, deacetylation of 23
were altered linkage forms between the oleanene skeleton and 24, followed by their esterification with phthalic anhy-
and benzoic acid, 16, 19 and 22 were prepared by the method dride in pyridine, gave rise to 27 and 28, respectively (Chart
of Sun et al.28) (Charts 2—4). Namely, after deriving 14 4).
(3,30-dioxo form, 50% yield) from 2 with pyridinium
As shown in Chart 5, 29 was obtained in 46% yield by the
chlorochromate (PCC) in dichloromethane, it was reacted condensation of C-30 acyl halide, which was derived from

September 2005
1105
Chart 3
Chart 4
Chart 5
the halogenation of 20 with thionyl chloride in THF, and an- (5) carboxylic compounds led to an increase in the activity
thranilic acid.31) Deacetylation of 29 gave 30 in 99% yield, compared to that of 3. However, the potency remained mod-
and then 30 was esterified with phthalic anhydride in pyri- erate (56ꢀ5% inhibition at 1 mM). Furthermore, elimination
dine to give 31 in 67% yield.
(6) or an increase (7, 8) of the carboxyl group in the benzoic
acid moiety resulted in loss of the inhibitory activity. These
results clearly showed that the number or the position of the
Results and Discussion
All the newly synthesized compounds at a concentration carboxyl group significantly affected the inhibitory activity.
of 1 mM were evaluated for their inhibitory activity on IL- Compounds (9—13), in which the benzene rings in the
1b-induced PGE2 production in NHDF, and the results are benzoyl group were replaced by another ring system, showed
summarized in Table 1. N-(2-Cyclohexyl-oxy-4-nitrophenyl)- more potent activity than that of 3. However, the potencies
methanesulfonamide (NS-398) (100 nM), a cyclooxygenase- were only moderate in 10—12 (31—45% inhibition). Con-
2 (COX-2) inhibitor, completely inhibited PGE2 production version of the ester bond at position 3 and/or 30 to the amide
in response to IL-1b (data not shown), indicating that the IL- bond in 17, 23—28 also failed to get the desired effects,
1b-induced PGE2 is formed by the COX-2 dependent path- while 28 showed higher potency (IC50 2.0 mM). In compari-
way.23) The lead compound (3) showed weak activity son with 17 and 27, 28 was potentiated in its biological activ-
(52ꢀ4% inhibition at 10 mM), whereas the meta- (4) or para- ity by structural modification of the linkage between the

1106
Vol. 53, No. 9
Table 1. Effect of Test Compounds on IL-1b-Induced PGE2 Production in NHDF
Compound
No.
1 mM
Compound
No.
1 mM
IC50 (mM)
ꢁ10
IC50 (mM)
Inhibition (%)
Inhibition (%)
3
3
4
5
6
7
8
9
10
11
12
13
52ꢀ4**,a)
NA
56ꢀ5**
38ꢀ1*
NA
NA
NA
18ꢀ5
45ꢀ12**
33ꢀ7*
31ꢀ2**
16ꢀ15
17
23
24
25
26
27
28
29
30
31
18ꢀ7
NA
16ꢀ7
NA
9ꢀ10
16ꢀ1
39ꢀ10**
52ꢀ8**
54ꢀ8**
32ꢀ11
b)
b)
2.0
1.4
1.0
4.3
b)
b)
b)
NS-398
500 pM
a) The data which was measured in 10 mM.
b) The compound does not inhibit concentration-dependently. ∗ pꢄ0.05, ∗∗ pꢄ0.01 vs. control. NA: not activity, Values represent 0 or less than 0.
(Calcd for C46H55O8: 735.3897).
3b,30-O-Dibenzoyl-olean-11,13(18)-dien (6) To
(220 mg, 0.499 mmol) in pyridine (5 ml) was added benzoyl chloride
(281 mg, 2.00 mmol). After stirring for 4 h at room temperature, MeOH was
added to the reaction mixture. The precipitates were collected by filtration
mother skeleton and the phthalic acid at positions 30 and/or
3.
Compounds (29—31), bearing a different amide linkage
compared with 17 and 23—28, exhibited the most potent ac-
a solution of 2
tivity among the newly synthesized compounds. Particularly, and washed with MeOH to give 6 as a white powder (265 mg, 82% in yield);
mp 231—234 °C; 1H-NMR (500 MHz, CDCl3) d 8.08—8.04 (4H, m),
30 showed the most potent activity in this series (IC50
7.58—7.26 (2H, m), 6.39 (1H, dd, J9,11ꢂ3.1 Hz, J11,12ꢂ11.0 Hz, H-11), 5.57
1.0 mM). In contrast with the relation of the activity of 26 and
(1H, d, H-12), 4.77 (1H, dd, J2a,3ꢂ4.9 Hz, J2b,3ꢂ11.8 Hz, H-3), 4.10 (2H, s,
28, the inhibitory activity of 31 (IC50 4.3 mM), in which ph-
H-30), 1.11, 1.02, 1.00, 0.97, 0.95, 0.93, 0.74 (3Hꢃ7, each s, H-23, H-24,
H-25, H-26, H-27, H-28, H-29); 13C-NMR (125 MHz, C5D5N) d 166.6,
166.3 (CꢂO), 125.9 (C-12), 125.5 (C-11), 81.5 (C-3), 74.7 (C-30); FAB-MS
m/z: 648.4226 (Calcd for C44H56O4: 648.4179).
thalic acid was introduced into position 3 of 30, was de-
creased compared with that of 30.
In this series of the synthetic compounds, the introduction
of anthranilic acid into position 30 is one of the significant
factors for the expression of the activity. Thus, 30 may serve
as a useful lead compound in the search for more powerful
anti-inflammatory agents.
3b,30-O-Bis(3,5-dicarboxybenzenecarbonyl)-olean-11,13(18)-dien (7)
A solution of 2 (440 mg, 0.998 mmol) and 1,3,5-benzenetricarboxylic
trichloride (800 mg, 3.01 mmol) in pyridine (20 ml) was heated for 20 min at
80 °C. To the reaction mixture was added water, then the mixture was ex-
tracted with CHCl3. The CHCl3 layer was washed with 10% HCl, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. To the residue
was added with MeOH, the mixture was filtrated, and then the filtrate was
Experimental
General Experimental Procedures Melting points were taken on a evaporated. The residue was purified on HPLC to give 7 (128 mg, 16% in
1
Yanaco micro melting point apparatus and are uncorrected. 1H- and 13C-
NMR spectra were measured using either a JEOL JNM-A500 (1H: m), 6.53 (1H, bd, J11,12ꢂ10.5 Hz, H-11), 4.89 (1H, dd, J2a,3ꢂ5.6 Hz,
500 MHz; 13C: 125 MHz) or AL-400 (1H: 400 MHz; 13C: 100 MHz) NMR
2b,3ꢂ11.0 Hz, H-3), 4.21, 4.16 (2H, each d, Jgemꢂ10.6 Hz, H-30), 1.19,
yield); mp 266—267 °C; H-NMR (400 MHZ, C4D8O) d 8.88—8.84 (6H,
J
spectrometer, with chemical shifts represented in ppm and TMS used as an 1.10, 1.08, 1.04, 0.99, 0.98, 0.82 (3Hꢃ7, each s, H-23, H-24, H-25, H-26,
internal standard. MS data were obtained on a JEOL JMS-700 or JMS-GC-
MATE.
H-27, H-28, H-29); 13C-NMR (100 MHZ, C4D8O) d 165.3, 165.2, 164.2,
164.0 (CꢂO), 136.1, 134.6, 134.34, 134.27, 133.8, 132.1, 132.0, 131.8,
131.4 (C-13, C-18, Ar), 125.7, 125.5 (C-11, C-12), 81.8 (C-3), 74.7 (C-30);
FAB-MS m/z: 823.3689 (Calcd for C48H55O12: 823.3693).
3b,30-O-Bis(3-carboxybenzenecarbonyl)-olean-11,13(18)-dien (4)
A
solution of 2 (220 mg, 0.499 mmol) and isophthaloyl dichloride (1,3-ben-
zenedicarbonl dichloride) (204 mg, 1.00 mmol) in pyridine (10 ml) was
stirred for 4 h at room temperature. To the reaction mixture was added
3
b,30-O-Bis(2,4,5-tricarboxybenzencarbonyl)-olean-11,13(18)-dien (8)
A solution of 2 (206 mg, 0.468 mmol) and 1,2,4,5-benzentetracarboxylic an-
MeOH. The mixture was filtrated and then the filtrate was evaporated. The hydride (1.02 g, 4.68 mmol) in pyridine (10 ml) was heated for 3 h at 60 °C.
residue was chromatographed on silica gel with CHCl3–MeOH (20 : 1) to
To the reaction mixture was added 10% HCl (20 ml), and this was then
give 4 (60 mg, 20% in yield); mp ꢁ300 °C; H-NMR (500 MHz, C5D5N) d stirred for 1 h. The precipitates were filtered off and washed with a small
1
1
8.73—7.57 (8H, m), 6.61 (1H, dd, J9,11ꢂ2.5 Hz, J11,12ꢂ10.4 Hz, H-11), 5.67
(1H, d, H-12), 5.02 (1H, dd, J2a,3ꢂ4.9 Hz, J2b,3ꢂ11.6 Hz, H-3) 4.31, 4.28 NMR (500 MHz, C5D5N) d 8.92, 8.91, 8.76, 8.73 (1Hꢃ4, each s), 6.56 (1H,
(1Hꢃ2, each d, gemꢂ11.0 Hz, H-30), 2.70, 2.09 (1Hꢃ2, each d,
bd, J9,11ꢂ8.5 Hz, H-11), 5.53 (1H, d, H-12), 5.09 (1H, dd, J2a,3ꢂ4.3 Hz,
2b,3ꢂ11.6 Hz, H-3), 4.36 (2H, dd, Jꢂ11.0 Hz, H-30), 2.74, 2.10 (1Hꢃ2,
amount of MeOH to give 8 (151 mg, 35% in yield); mp 222—225 °C; H-
J
J
gemꢂ14.7 Hz, H-19), 1.17, 1.10, 1.06, 1.01, 1.00, 0.94, 0.80 (3Hꢃ7, each s,
J
H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (125 MHz, C5D5N)
d 168.6, 168.5, 166.0, 165.8 (CꢂO), 126.3 (C-12), 126.0 (C-11), 81.9 (C-3),
75.0 (C-30); FAB-MS m/z: 736.3967 (Calcd for C46H56O8: 736.3975).
each d, Jgemꢂ14.0 Hz, H-19), 1.13, 1.07, 1.02, 1.00, 0.96, 0.83, 0.68 (3Hꢃ7,
each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (125 MHz,
C5D5N) d 169.8, 169.8, 169.8, 169.6, 169.4, 169.2, 168.2, 167.7 (CꢂO),
82.9 (C-3), 76.2 (C-30); FAB-MS m/z: 912.3536 (Calcd for C50H56O16:
912.3568).
3b,30-O-Bis(4-carboxybenzenecarbonyl)-olean-11,13(18)-dien (5)
A
solution of 2 (440 mg, 0.998 mmol) and terephthaloyl dichloride (1,4-ben-
zenedicarbonyl dichloride) (2.0 g, 9.9 mmol) in pyridine (5 ml) was heated
for 10 min at 80 °C. To the reaction mixture was added 10% HCl, and the
mixture was then filtered off. The filtrate was extracted with CHCl3, and the
3
b,30-O-Bis(2-carboxy-1-cyclohexsanecarbonyl)-olean-11,13(18)-dien
(9) A solution of 2 (220 mg, 0.499 mmol) and cyclohexane dicarboxylic
acid anhydride (385 mg, 2.50 mmol) in pyridine (10 ml) was heated for 3 d at
organic layer was concentrated. The residue was chromatographed on silica 80 °C. The reaction mixture was poured into 10% HCl and extracted with
gel with CHCl3–MeOH (20 : 1) to give 5 (113 mg, 15% in yield); mp
CHCl3. The CHCl3 layer was concentrated, then the residue was subjected to
ꢁ300 °C; 1H-NMR (500 MHZ, C5D5N) d 8.57—8.40 (8H, m), 6.38 (1H, dd, silica gel column chromatography and eluted with hexane–acetone (7 : 3) to
J
J
9,11ꢂ2.4 Hz, J11,12ꢂ11.0 Hz, H-11), 5.56 (1H, d, H-12), 4.91 (1H, dd, give 9 (115 mg, 31% in yield); mp 218—219 °C; 1H-NMR (400 MHz,
2a,3ꢂ4.3 Hz, J2b,3ꢂ11.0 Hz, H-3), 4.24 (2H, s, H-30); 13C-NMR (125 MHz,
C4D8O) d 6.42 (1H, dd, J9,11ꢂ2.4 Hz, J11,12ꢂ10.2 Hz, H-11), 5.57 (1H, d, H-
C5D5N) d 168.4, 168.4, 166.0, 165.8 (CꢂO); FAB-MS m/z: 735.3905 12), 4.52 (1H, dd, J2a,3ꢂ5.5 Hz, J2b,3ꢂ11.0 Hz, H-3), 3.79 (2H, s, H-30),

September 2005
1107
Olean-11,13(18)-dien-3,30-dioxime (15)
A
solution of 14 (1.8 g,
2.82—2.76 (1Hꢃ4, m, cyclohexane–CH), 2.47, 1.86 (1Hꢃ2, each d,
4.1 mmol) and hydroxylamine hydrochloride (2.0 g, 29 mmol) in pyridine
(10 ml) was heated for 3 h at 50 °C. After cooling to room temperature, the
reaction mixture was diluted with CHCl3 and washed with 10% HCl. The
CHCl3 layer was dried over anhydrous Na2SO4 and evaporated. The residue
was chromatographed on silica gel with hexane–AcOEt (5 : 1) to give 15
(1.3 g, 69% in yield); mp 272—274 °C; 1H-NMR (400 MHz, C5D5N) d
12.20, 11.92 (1Hꢃ2, s, OH), 7.75 (1H, s, H-30), 6.47 (1H, br d,
J
gemꢂ14.5 Hz, H-19), 1.10, 0.99, 0.94, 0.88, 0.87, 0.81, 0.75 (3Hꢃ7, each s,
H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (100 MHz, C4D8O)
d 174.6, 174.6, 173.3, 173.0 (CꢂO), 137.3, 135.1 (C-13, C-18), 126.4,
126.2 (C-11, C-12), 80.7 (C-3), 74.4 (C-30); FAB-MS m/z: 748.4921 (Calcd
for C46H68O8: 748.4914).
3b,30-O-Bis(2-carboxy-1-cyclohexenecarbonyl)-olean-11,13(18)-dien
(10) To a solution of 2 (440 mg, 0.998 mmol) in pyridine (15 ml) was
added 1-cyclohexene-1,2-dicarboxylic anhydride (1.50 g, 9.86 mmol). After
stirring for 24 h at 120 °C, the mixture was poured into ice water and ex-
tracted with CHCl3. The CHCl3 layer was washed with H2O, 10% HCl and
aqueous NaHCO3, dried over anhydrous Na2SO4 and concentrated. The
residue was chromatographed on silica gel with CHCl3–MeOH (20 : 1) to
give 10 (35 mg, 10% in yield); mp 141—142 °C; 1H-NMR (400 MHz,
CD3OD) d 6.41 (1H, dd, J9,11ꢂ2.7 Hz, J11,12ꢂ10.5 Hz, H-11), 5.59 (1H, d,
H-12), 4.56 (1H, dd, J2a,3ꢂ6.6 Hz, J2b,3ꢂ9.8 Hz, H-3), 3.88 (2H, s, H-30),
2.82—2.76 (1Hꢃ4, m, cyclohexane–CH), 2.47, 1.86 (1Hꢃ2, each d,
J
J
11,12ꢂ10.2 Hz, H-11), 5.62 (1H, d, H-12), 3.40 (1H, m, H-2a), 2.75 (1H, d,
gemꢂ14.4 Hz, H-19), 2.49 (1H, m, H-2b), 2.24 (1H, d, H-19), 1.38, 1.15,
1.11, 1.10, 1.02, 1.02, 0.80 (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27,
H-28, H-29); 13C-NMR (100 MHz, C5D5N) d 164.0 (C-3), 158.3 (C-30),
136.2, 135.0 (C-13, C-18), 126.3, 125.8 (C-11, C-12); FAB-MS m/z:
467.3676 (Calcd for C30H47O2N2: 467.3638).
Olean-11,13(18)-dien-3b,30-diamine (16) Sodium cyanoborohydride
(1.4 g, 22 mmol) was added to a methanol solution of 15 (1.0 g, 2.1 mmol)
and ammonium acetate (1.8 g, 23 mmol). The solution was chilled in ice
water, and 15% aqueous titanium trichloride (5.8 ml) was added dropwise
over 20 min. The mixture was stirred at room temperature for 12 h and ad-
justed with 2 N sodium hydroxide to pH 10. The aqueous solution was ex-
tracted with CHCl3, and the CHCl3 layer was washed with distilled water
and concentrated under reduced pressure to give a mixture of 3a,30- and
3b,30-diamine (880 mg). To the mixture in CH2Cl2 (15 ml) was added
Boc2O (517 mg, 2.4 mmol). The reaction mixture was stirred at room tem-
perature for 2 h and then diluted with CHCl3. The solution was washed with
water, dried over anhydrous Na2SO4 and concentrated. The residue was
chromatographed on silica gel with hexane–AcOEt (50 : 1→50 : 2→
50 : 3→50 : 4) to give 3b,30-N,Nꢅ-di(tert-butoxycarbonyl)-11,13(18)-dien
(254 mg, 50% in yield); 1H-NMR (CDCl3) d 6.35 (1H, dd, J9,11ꢂ3.1 Hz,
J
gemꢂ14.1 Hz, H-19), 1.10, 0.99, 0.96, 0.91, 0.87, 0.83, 0.75 (3Hꢃ7, each s,
H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (400 MHz, CD3OD)
d 171.9, 171.7, 170.3, 170.2 (CꢂO), 137.4, 135.7 (C-13, C-18), 137.4,
136.4, 136.0, 135.2 (cyclohexene-1,2), 83.2 (C-3), 76.2 (C-30); FAB-MS
m/z: 744.4629 (Calcd for C46H64O8: 744.4601).
3b,30-O-Bis(2-carboxy-4-cyclohexenecarbonyl)-olean-11,13(18)-dien
(11) To a solution of 2 (220 mg, 0.499 mol) in pyridine (20 ml) was added
cis-1,2,3,6-tetrahydrophthalic anhydride (760 mg, 5.00 mmol). The solution
was stirred overnight at 120 °C, then the solvent was evaporated. The residue
was dissolved with CHCl3, washed with H2O, and concentrated. The residue
was purified by silica gel column chromatography using hexane–acetone
(8 : 2) to give 11 (140 mg, 38% in yield); mp 205—206 °C; 1H-NMR
(400 MHz, CD3OD) d 6.40 (1H, d, J11,12ꢂ10.5 Hz, H-11), 5.66, (4H, br s,
J11,12ꢂ10.4 Hz, H-11), 5.53 (1H, d, H-12), 4.64 (1H, t, J30,NꢅHꢂ6.1 Hz, NꢅH),
4.40 (1H, d, J3,NHꢂ10.4 Hz, NH), 3.30 (1H, ddd, J2a,3ꢂ9.9 Hz, J2b,3ꢂ3.5 Hz,
H-3), 2.98 (t, 2H, H-30), 1.45, 1.44 (9Hꢃ2, each s, t-Bu), 1.10, 0.96, 0.92,
0.86, 0.75, 0.73, 0.70 (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28,
H-29); 13C-NMR (CDCl3) d 156.3, 155.8 (CꢂOꢃ2), 136.5, 134.2 (C-13, C-
18) 125.7, 125.5 (C-11, C-12), 79.0, 78.9 (C-3, C-30). Following the addi-
tion of TFA (0.5 ml) to 3b,30-N,Nꢅ-di(tert-butoxycarbonyl)-11,13(18)-dien
(484 mg), the mixture was stirred at room temperature for 15 min. The reac-
tion mixture was adjusted with 1 N sodium hydroxide to pH 10. The aqueous
solution was extracted with CHCl3, and the organic layer was washed with
distilled water and concentrated to give 16 (290 mg, 87%); mp 184—186 °C;
1H-NMR (400 MHz, CD3OD) d 6.41 (1H, dd, J9,11ꢂ2.9 Hz, J11,12ꢂ10.5 Hz,
H-11), 5.57 (1H, d, H-12), 3.31 (3H, m, H-3, H-30), 1.09, 0.98, 0.96, 0.90,
0.77, 0.75, 0.74 (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-
29); 13C-NMR (100 MHz, CD3OD) d 138.03, 135.3 (C-13, C-18), 126.6,
126.5 (C-11, C-12), 60.7, 56.9 (C-3, C-30); FAB-MS m/z: 439.4060 (Calcd
for C30H51N2: 439.4052).
cyclohexene–CH), 5.58 (1H, d, H-12), 4.51 (1H, dd,
J2a,3ꢂ5.6 Hz,
J
2b,3ꢂ11.0 Hz, H-3), 3.85 (2H, m, H-30), 1.10, 0.99, 0.95, 0.89, 0.87, 0.81,
0.74 (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR
(100 MHz, CD3OD) d 176.8, 175.1, 174.7 (CꢂO), 137.4, 135.7 (C-13, C-
18), 126.7, 126.34, 126.29, 126.10, 126.07 (C-11, C-12, cyclohexene–CH),
82.6 (C-3), 75.6 (C-30); FAB-MS m/z: 744.4565 (Calcd for C46H56O8:
744.4601).
3b,30-O-Bis(2-carboxyl-1-cyclopentenecarbonyl)-olean-11,13(18)-dien
(12) A solution of 2 (220 mg, 0.499 mmol) and 1-cyclopentene-1,2-dicar-
boxylic anhydride (600 mg, 4.34 mmol) in pyridine (10 ml) was heated for
4 h at 80 °C. The reaction mixture was poured into ice water and extracted
with CHCl3. The CHCl3 layer was washed with H2O, 10% HCl and aqueous
NaHCO3, dried over anhydrous Na2SO4, and concentrated. The residue was
recrystallized from hexane–AcOEt (1 : 1) and filtered off. The crystals were
washed with a small amount of MeOH to give 12 (157 mg, 45% in yield);
mp 163—165 °C; 1H-NMR (400 MHz, C5D5N) d 6.60 (1H, dd, J9,11ꢂ2.4 Hz,
3b,30-Bis[(2-carboxyphenyl)carboxamido]-olean-11,13(18)-dien (17)
To a solution of 16 (290 mg, 0.661 mmol) in pyridine (10 ml) was added ph-
thalic anhydride (900 mg, 6.08 mmol). After being stireed for 3 h at 80 °C,
the reaction mixture was diluted with CHCl3, then the CHCl3 layer was
washed with 10% HCl, dried over anhydrous Na2SO4, and concentrated. To
the residue was added MeOH, then the precipitates were filtered off to give
J
J
11,12ꢂ10.5 Hz, H-11), 5.54 (1H, d, H-12), 4.93 (1H, dd, J2a,3ꢂ4.6 Hz,
2b,3ꢂ11.5 Hz, H-3), 4.21, 4.15 (2H, each d, Jgemꢂ10.6 Hz, H-30), 2.90—
2.78 (12H, m, cyclopenten–CH2), 1.09, 1.09, 1.01, 1.00, 0.98, 0.84, 0.72
(3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR
(100 MHz, C5D5N) d 168.9, 168.6, 165.8, 165.7 (CꢂO), 144.3, 143.0,
137.3, 136.4, 136.5, 134.5 (C-13, C-18, cyclopentene–CH), 125.9, 125.8 (C-
11, C-12), 81.5 (C-3), 74.9 (C-30); FAB-MS m/z: 716.4282 (Calcd for
C44H60O8: 716.4288).
3b,30-O-Bis(5-carboxythiophenecarbonyl)-olean-11,13(18)-dien (13)
A solution of 2 (220 mg, 0.499 mmol), 2,5-thiophenedicarboxylic acid
(430 mg, 2.50 mmol), 2-chloro-1-methylpyridinium iodide (638 mg,
2.50 mmol) and tri-n-butylamine (463 mg, 2.50 mmol) in toluene (20 ml)
was heated for 2 d at 120 °C. The reaction mixture was concentrated, then
the residue was subjected to silica gel column chromatography and eluted
with CHCl3 to give 13 (43 mg, 12% in yield); mp ꢁ300 °C; 1H-NMR
(500 MHz, C5D5N) d 7.95—7.90 (4H, m, thiophene–CH), 6.54 (1H, dd, H-
11), 4.90 (1H, dd, H-3), 4.21 (2H, s, H-30); FAB-MS m/z 748.3145 (Calcd
for C42H52O8S: 748.3104).
1
17 (362 mg, 75% in yield); mp 212 °C; H-NMR (400 MHz, C5D5N) d 9.10
(1H, t, J30,NHꢂ6.1 Hz, NꢅH), 8.55 (1H, d, J3,NHꢂ9.2 Hz, NH), 8.18—7.36
(8H, m), 6.59 (1H, bd, J11,12ꢂ10.4 Hz, H-11), 5.51 (1H, d, H-12), 4.42 (1H,
ddd,
J2a,3ꢂ4.3 Hz, J2b,3ꢂ12.2 Hz, H-3), 3.76, 3.66 (1Hꢃ2, each d,
J
gemꢂ10.7 Hz, H-30), 1.39, 1.14, 1.13, 1.05, 1.02, 0.88, 0.75, (3Hꢃ7, each s,
H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (100 MHz, C5D5N)
d 170.7, 170.2, 169.8, 169.7 (CꢂO); FAB-MS m/z: 735.4416 (Calcd for
C46H59O6N2: 735.4373).
30-O-Acetyl-olean-11,13(18)-dien-3b-ol (18) To a solution of 2 (2.0 g,
4.5 mmol) in pyridine (10 ml) was added acetic anhydride (0.86 ml) at 0 °C,
then stirring was continued for 6 h. The reaction mixture was poured into ice
water and extracted with CHCl3. The CHCl3 layer was washed with water,
10% HCl and aqueous NaHCO3, dried over anhydrous Na2SO4, and concen-
trated. The residue was chromatographed on silica gel with hexane–AcOEt
(20 : 1→10 : 1→5 : 1) to give 18 (1.2 g, 54% in yield); mp 185—186 °C; 1H-
NMR (400 MHz, CDCl3) d 6.34 (1H, dd, J9,11ꢂ2.9 Hz, J11,12ꢂ10.5 Hz, H-
11), 5.55 (1H, d, H-12), 3.85, 3.81 (1Hꢃ2, each d, Jgemꢂ10.7 Hz, H-30),
3.24 (1H, dd, J2a,3ꢂ5.1, J2b,3ꢂ11.2 Hz, H-3), 2.08 (3H, s, Ac), 1.07, 0.99,
0.96, 0.89, 0.81, 0.78, 0.71, (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27,
H-28, H-29); 13C-NMR (100 MHz, CDCl3) d 171.2 (CꢂO), 136.0, 134.4
(C-13, C-18), 125.9, 125.4 (C-11, C-12), 79.0 (C-30), 74.4 (C-3); FAB-MS
m/z: 482.3758 (Calcd for C32H50O3: 482.3760).
3-Oxo-olean-11,13(18)-dien-30-al (14) To a solution of 2 (2.00 g,
4.55 mmol) in CH2Cl2 (21 ml) was added PCC (2.96 g, 13.7 mmol). After
being stireed overnight at room temperature, the mixture was diluted with
Et2O and filtered through a Florisil column. The eluate was concentrated
under a vacuum, then the residue was chromatographed on silica gel with
hexane–AcOEt (9 : 1) to give 14 (1.0 g, 50% in yield); mp 206—208 °C; 1H-
NMR (400 MHz, CDCl3) d 9.51 (1H, s, H-30), 6.34 (1H, dd, J9,11ꢂ2.9 Hz,
J
11,12ꢂ10.5, H-11), 5.57 (1H, d, H-12); 13C-NMR (100 MHz, CDCl3) d
217.2 (C-3), 205.9 (C-30), 135.5, 135.4 (C-13, C-18), 126.0, 125.4 (C-11,
C-12); EI-MS m/z: 436.3342 (Calcd for C30H44O2: 436.3341).

1108
30-O-Acetyl-olean-11,13(18)-dien-3b-amine (19) To a solution of 18
Vol. 53, No. 9
1
(634 mg, 48% in yield); H-NMR (CDCl3) d: 9.50 (1H, s), 6.30 (1H, dd,
Jꢂ10.5, 2.9 Hz), 5.57 (1H, d, Jꢂ10.5 Hz), 4.52 (1H, dd, Jꢂ5.5, 2.8 Hz),
2.47 (1H, d, Jꢂ12.4 Hz), 2.05, 1.09, 0.98, 0.97, 0.92, 0.87, 0.86, 0.72
(3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29). A solution of
3-O-acetyl-olean-11,13(18)-dien-30-al (634 mg, 1.32 mmol) and hydroxy-
lamine hydrochloride (320 mg, 4.60 mmol) in pyridine (5 ml) was heated for
3 h at 50 °C. After being cooled to room temperature, the reaction mixture
was diluted with CHCl3 and washed with 10% HCl. The CHCl3 layer was
dried over anhydrous Na2SO4 and evaporated. The residue was chro-
matographed on silica gel with hexane–AcOEt (5 : 1) to give 3-O-acetyl-
olean-11,13(18)-dien-30-oxime (648 mg, 99% in yield). Sodium cyanoboro-
hydride (466 mg, 7.42 mmol) was added to a methanol solution of 3-O-
acetyl-olean-11,13(18)-dien-30-oxime (680 mg, 1.97 mmol) and ammonium
acetate (600 mg, 7.78 mmol). The solution was chilled in ice water, and 15%
aqueous titanium trichloride (2 ml) was added dropwise over 20 min. The
mixture was stirred at room temperature for 12 h and adjusted with 2 N
sodium hydroxide to pH 10. The aqueous solution was extracted with
CHCl3, and the CHCl3 layer was washed with distilled water and concen-
trated under reduced pressure to give 22 (542 mg, 82% in yield); mp 117—
(105 mg, 0.218 mmol) in THF (5 ml) was added dropwise Jones reagent (5
drops), and then the mixture was stirred at room temperature for 15 min. The
reaction mixture was filtered, then the filtrate was poured into ice water and
extracted with CHCl3. The CHCl3 layer was washed with water, dried over
anhydrous Na2SO4, and concentrated. The residue was chromatographed on
silica gel with hexane–AcOEt (20 : 1→10 : 1) to give 30-O-acetyl-olean-
1
11,13(18)-dien-3-one (86 mg, 81% in yield); H-NMR (CDCl3) d 6.38 (1H,
dd, J9,11ꢂ3.1 Hz, J11,12ꢂ10.4 Hz, H-11), 5.54 (1H, d, H-12), 3.85, 3.82
(1Hꢃ2, each d, Jgemꢂ10.4 Hz, H-30), 2.08 (3H, s, Ac), 1.10, 1.08, 1.04,
1.01, 0.98, 0.81, 0.75, (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28,
H-29); 13C-NMR (CDCl3) d 217.4 (C-3), 171.3 (CꢂO), 136.7, 134.1 (C-13,
C-18), 125.8, 125.3 (C-11, C-12), 74.3 (C-30). A solution of 30-O-acetyl-
olean-11,13(18)-dien-3-one (229 mg, 0.476 mmol) and hydroxylamine hy-
drochloride (87 mg, 1.25 mmol) in pyridine (2 ml) was heated for 3 h at
50 °C. After being cooled to room temperature, the reaction mixture was di-
luted with CHCl3 and washed with 10% HCl. The CHCl3 layer was dried
over anhydrous Na2SO4 and evaporated. The residue was chromatographed
on silica gel with hexane–AcOEt (5 : 1) to give 30-O-acetyl-olean-
118 °C; 1H-NMR (400 MHz, CDCl3)
d
6.36 (1H, dd,
11,12ꢂ10.6 Hz, H-11), 5.52 (1H, d, H-12), 4.52 (1H, dd, J2a,3ꢂ5.7 Hz,
2b,3ꢂ10.6 Hz, H-3), 2.48 (2H, s, H-30), 2.33 (1H, d, Jgemꢂ13.9 Hz, H-19),
J9,11ꢂ2.6 Hz,
1
11,13(18)-dien-3-oxime (109 mg, 47% in yield); H-NMR (CDCl3) d 6.36
J
J
(1H, d, J11,12ꢂ10.4 Hz, H-11), 5.55 (1H, d, H-12), 3.85, 3.82 (1Hꢃ2, each d,
Jgemꢂ10.4 Hz, H-30), 3.11 (1H, m, H-2a), 2.40 (1H, d, Jgemꢂ14.0 Hz, H-19),
2.05 (3H, s, Ac), 1.06, 0.96, 0.92, 0.87, 0.85, 0.75, 0.71 (3Hꢃ7, each s, H-
23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (100 MHz, CDCl3) d
170.8 (CꢂO), 137.0, 133.7 (C-13, C-18), 125.5, 125.3 (C-11, C-12), 80.8
(C-3); FAB-MS m/z: 482.4042 (Calcd for C32H52O2N: 482.3998).
2.29 (1H, m, H-2b), 2.09 (3H, s, Ac), 1.18, 1.07, 1.07, 1.00, 0.95, 0.81,
0.74, (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-
NMR (CDCl3) d 171.3 (CꢂO), 166.7 (C-3), 136.4, 134.3 (C-13, C-18),
125.7, 125.6 (C-11, C-12), 74.4 (C-30). Sodium cyanoborohydride (660 mg,
10.5 mmol) was added to a methanol solution of 30-O-acetyl-olean-
11,13(18)-dien-3-oxime (475 mg, 0.462 mmol) and ammonium acetate
(841 mg, 10.9 mmol). The solution was chilled in ice water, and 15% aque-
ous titanium trichloride (2.7 ml) was added dropwise over 20 min. The mix-
ture was stirred at room temperature for 12 h and adjusted with 2 N sodium
hydroxide to pH 10. The aqueous solution was extracted with CHCl3, and
the CHCl3 layer was washed with distilled water and concentrated under re-
duced pressure to give the mixture 30-O-acetyl-olean-11,13(18)-dien-3-
amine (373 mg). The mixture of 30-O-acetyl-olean-11,13(18)-dien-3-amine
(690 mg, 1.43 mmol) was dissolved in CH2Cl2 (20 ml), then Boc2O (500 mg,
2.29 mmol) was added to the mixture. The reaction mixture was stirred at
room temperature for 12 h and then concentrated. The residue was chro-
matographed on silica gel with hexane–AcOEt (25 : 2) to give 30-O-acetyl-
3b-N-(tert-butoxycarbonyl)-olean-11,13(18)-dien (500 mg, 59%).
30-O-Acetyl-3b-(2-carboxyphenyl)carboxamido-olean-11,13(18)-dien
(23) To a solution of compound 19 (236 mg, 0.490 mmol) in pyridine
(10 ml) was added phthalic anhydride (900 mg, 6.08 mmol). After being
stirred for 3 h at 80 °C, the reaction mixture was diluted with CHCl3, and the
CHCl3 layer was washed with 10% HCl, dried over anhydrous Na2SO4, and
concentrated. To the residue was added MeOH, then the precipitate was fil-
1
tered off to give 23 (362 mg, 75% in yield); mp 239—239.5 °C; H-NMR
(500 MHz, C5D5N) d 8.56 (1H, d, Jꢂ9.8 Hz, NH), 8.16 (1H, d, Jꢂ7.3 Hz),
7.75 (1H, d, Jꢂ7.3 Hz), 7.46 (1H, t, Jꢂ7.3 Hz), 7.37 (1H, t, Jꢂ7.3 Hz), 6.48
(1H, bd, J11,12ꢂ10.4 Hz, H-11), 5.61 (1H, d, H-12), 4.01, 3.98 (1Hꢃ2, each
d, Jgemꢂ10.5 Hz, H-30), 2.52 (1H, d, Jgemꢂ14.0 Hz, H-19), 2.12 (3H, s, Ac),
1.41, 1.09, 1.07, 1.03, 0.92, 0.90, 0.80 (3Hꢃ7, each s, H-23, H-24, H-25, H-
26, H-27, H-28, H-29); 13C-NMR (125 MHz, C5D5N) d 170.9, 170.2, 169.7
(CꢂO), 141.1, 136.4, 134.9, 131.8, 131.4, 130.3, 128.9, 128.5, 125.9, 123.9
(Ar, C-11, C-12, C-13, C-18), 74.3 (C-30); FAB-MS m/z: 630.4207 (Calcd
for C40H56O5N: 630.4158).
A mixture of 30-O-acetyl-3b-N-(tert-butoxycarbonyl)-olean-11,13(18)-
dien (500 mg) and TFA (1 ml) was stirred at room temperature for 15 min.
The reaction mixture was adjusted with 1 N sodium hydroxide to pH 10. The
aqueous solution was extracted with CHCl3, then the CHCl3 layer was
washed with distilled water and concentrated to give 19 (373 mg, 92% in
yield); mp 134—135 °C; 1H-NMR (400 MHz, CD3OD) d 6.37 (1H, dd,
3b-O-Acetyl-30-(2-carboxyphenyl)carboxamido-olean-11,13(18)-dien
(24) 24 (201 mg, 86%) was obtained from 22 (180 mg) according to the
1
above procedure; mp 236—237 °C; H-NMR (500 MHz, CDCl3) d 7.88—
7.71 (4H, m), 6.33 (1H, dd, J9,11ꢂ2.4 Hz, J11,12ꢂ10.4 Hz, H-11), 5.50 (1H, d,
H-12), 4.51 (1H, dd, J2a,3ꢂ5.5 Hz, J2b,3ꢂ10.4 Hz, H-3), 3.56 (2H, s, H-30),
2.05 (3H, s, Ac), 1.04, 0.94, 0.91, 0.86, 0.85, 0.83, 0.70 (3Hꢃ7, each s, H-
23, H-24, H-25, H-26, H-27, H-28, H-29); FAB-MS m/z: 630.4161 (Calcd
for C40H56NO5: 630.4158).
J9,11ꢂ2.9 Hz, J11,12ꢂ10.5 Hz, H-11), 5.60 (1H, d, H-12), 3.83 (2H, s, H-30),
2.45 (1H, Jgemꢂ12.0 Hz, H-19), 2.37 (1H, J2a,3ꢂ4.8 Hz, J2b,3ꢂ11.3 Hz, H-
3), 2.07 (3H, s, Ac), 1.10, 0.98, 0.96, 0.91, 0.82, 0.76, 0.74 (3Hꢃ7, each s,
H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR (100 MHz, CD3OD)
d 172.9 (CꢂO), 137.2, 135.8 (C-13, C-18), 126.9, 126.4 (C-11, C-12), 75.4
(C-3); FAB-MS m/z: 482.3975 (Calcd for C32H52O2N: 482.3975).
3b-(2-Carboxyphenyl)carboxamido-olean-11,13(18)-dien-30-ol
(25)
To a solution of 23 (204 mg, 0.324 mmol) in MeOH (5 ml) was added
NaOMe (18 mg, 0.33 mmol). After being stirred overnight at room tempera-
ture, the reaction mixture was neutralized with Amberlite IR-120B, and the
resin was removed by filtration. The filtrate was concentrated to give 25
3b-O-Acetyl-olean-11,13(18)-dien-30-ol (21) To
a solution of 20
(2.4 g, 4.8 mmol) in THF (20 ml) was added ethyl chlorocarbonate (2.3 ml)
and triethylamine (3.3 ml) at ꢆ5 °C. The reaction mixture was stirred for 2 h
at ꢆ5 °C and filtrated. To the filtrate was added sodium borohydride (1.0 g,
15.9 mmol) in water (3 ml), and the mixture was stirred for 30 min at ꢆ5 °C.
The reaction mixture was evaporated, then the residue was dissolved in
CHCl3. The solution was washed with water, dried over anhydrous Na2SO4,
and concentrated. To the residue was added MeOH, then the precipitates
were filtrated off to give 21 (2.0 g, 85% in yield); mp 214—216 °C; 1H-
NMR (400 MHz, CDCl3) d 6.36 (1H, dd, J9,11ꢂ2.8 Hz, J11,12ꢂ10.6 Hz, H-
11), 5.52 (1H, d, H-12), 4.52 (1H, dd, J2a,3ꢂ2.7 Hz, J2b,3ꢂ5.4 Hz, H-3), 3.36
(2H, s, H-30), 2.37 (1H, d, Jgemꢂ13.9 Hz, H-19), 2.05 (3H, s, Ac), 1.07,
0.96, 0.92, 0.87, 0.85, 0.78, 0.71 (3Hꢃ7, each s, H-23, H-24, H-25, H-26,
H-27, H-28, H-29); 13C-NMR (100 MHz, CDCl3) d 170.8 (CꢂO) 136.6,
133.9 (C-13, C-18), 125.5, 125.3 (C-11, C-12), 80.8 (C-3), 74.1 (C-30);
FAB-MS m/z: 482.3741 (Calcd for C32H50O3: 482.3760).
1
(152 mg, 80% in yield); mp 214—216 °C; H-NMR (500 MHz, C5D5N) d
8.98 (1H, d, Jꢂ7.3 Hz, NH), 8.17 (1H, d, Jꢂ6.7 Hz), 7.89 (1H, d, Jꢂ7.3 Hz),
7.42 (1H, t, Jꢂ7.3 Hz), 7.37 (1H, t, Jꢂ6.7 Hz), 6.59 (1H, br d, J11,12ꢂ9.2 Hz,
H-11), 5.58 (1H, d, H-12), 4.40 (1H, m, H-3), 3.65 (2H, s, H-30), 2.71, 2.19
(1Hꢃ2, each d, Jgemꢂ14.3 Hz, H-19), 1.35, 1.16, 1.10, 1.034, 1.026, 0.89,
0.80 (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); FAB-MS
m/z: 588.4066 (Calcd for C38H54O4N: 588.4053).
30-(2-Carboxyphenyl)carboxamido-olean-11,13(18)-dien-3b-ol
(26)
26 (159 mg, 75%) was obtained from 24 (171 mg), according to the above
procedure. mp 178—179 °C; 1H-NMR (400 MHz, CDCl3) d 7.99 (1H, d,
Jꢂ7.8 Hz, NH), 7.53—7.46 (3H, m), 6.53 (1H, t, Jꢂ6.3 Hz), 6.33 (1H, dd,
J9,11ꢂ2.7 Hz, J11,12ꢂ10.7 Hz), 5.53 (1H, d, H-12), 3.28—3.24 (2H, m, H-30),
2.41 (1H, d, Jgemꢂ13.9 Hz, H-19), 1.06, 0.98, 0.96, 0.87, 0.83, 0.77, 0.70
(3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C-NMR
(100 MHz, CDCl3) d 170.8, 169.1 (CꢂO), 137.1, 136.1, 134.4, 131.9,
131.3, 130.0, 127.6, 125.9, 125.3 (C-11, C-12, C-13, C-18, Ar), 79.0, 77.2
(C-3, C-30); FAB-MS m/z: 588.4048 (Calcd for C38H54NO4: 588.4053).
3b-(2-Carboxyphenyl)carboxamido-30-O-phthaloyl-olean-11,13(18)-
dien (27) To a solution of 25 (102 mg, 0.211 mmol) in pyridine (5 ml) was
3b-O-Acetyl-olean-11,13(18)-dien-30-amine (22) To a solution of 2
(1.3 g, 2.9 mmol) in CH2Cl2 (20 ml) was added PCC (1.6 g, 7.4 mmol). After
being stirred for overnight at room temperature, the mixture was diluted
with Et2O and filtered through a Florisl column. The eluate was concentrated
under a vacuum, then the residue was chromatographed on silica gel
with hexane–AcOEt (9 : 1) to give 3-O-acetyl-olean-11,13(18)-dien-30-al

September 2005
1109
added phthalic anhydride (120 mg, 0.810 mmol). After being stirred
overnight at 80 °C, the reaction mixture was diluted with CHCl3, and the
CHCl3 layer was washed with 10% HCl, dried over anhydrous Na2SO4, and 10% fetal calf serum (FCS) and 60 mg/ml kanamycin.
broblasts (NHDF) from the foreskin were purchased from Kurabo (Osaka,
Japan) and cultured in a-minimum essential medium (a-MEM) containing
concentrated. To the residue was added MeOH, then the precipitates were
Assay for Cytokines, PGE2 Production NHDF (1—5ꢃ104 cells/
filtered off to give 27 (95 mg, 75% in yield); mp 185—187 °C; 1H-NMR 0.25 ml/well) were seeded in 48 well plates (BD Biosciences) and cultured
(CDCl3) d 7.90—7.44 (8H, m), 6.34 (1H, br d, J11,12ꢂ10.4 Hz, H-11), 6.03
in a-MEM supplemented with 10% FCS. When cells grew to confluency,
(1H, d, J3,NHꢂ7.9 Hz, NH), 5.51 (1H, d, H-12), 4.09 (2H, s, H-30), 4.00 (1H, the medium was replaced with 0.25 ml a-MEM containing 0.5% FCS, and
dd, J2a,3ꢂ4.3 Hz, J2b,3ꢂ12.2 Hz, H-3), 1.05, 1.03, 0.98, 0.87, 0.86, 0.81, cells were kept for 48 h. These cells were then incubated with 1 ng/ml IL-1b
0.69, (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29); 13C- in the presence or absence of various agents for 24 h. The culture super-
NMR (CDCl3) d 171.4, 170.2, 169.2, 168.3 (CꢂO); FAB-MS m/z: 734.4064 natants were collected and kept at ꢆ80 °C until being for assay. PGE2 (de-
(Calcd for C46H56NO7: 734.4057).
tection limit: Ca 12 pg/ml) in the culture supernatant was measured by an
30-(2-Carboxyphenyl)carboxamido-3b-O-phthaloyl-olean-11,13(18)- enzyme linked immunosorbent assay (ELISA) kit (Cayman Chemical Co.).
dien (28) 28 (13 mg, 12%) was obtained from 26 (85 mg) according to the
above procedure. mp 167—168 °C; 1H-NMR (500 MHz, C5D5N) d 9.07
(1H, t, Jꢂ6.4 Hz, NH), 8.16—7.36 (8H, m), 6.59 (1H, dd, J9,11ꢂ2.4 Hz,
The effect of test agents on the release of soluble mediators in response to
IL-1b was expressed as percent inhibition in comparison with the control.
Statistical Analysis All results are expressed as the meanꢀS.E.M. The
statistical significance of difference between the control and test groups was
J11,12ꢂ11.0 Hz, H-11), 5.46 (1H, d, H-12), 5.08 (1H, dd, J2a,3ꢂ4.6 Hz,
J2b,3ꢂ11.9 Hz, H-3), 3.77—3.65 (2H, m, H-30), 1.13, 1.13, 1.12, 1.04, 0.97, determined using the Tukey-Kramer multiple comparisons test after one-way
0.83, 0.73 (3Hꢃ7, each s, H-23, H-24, H-25, H-26, H-27, H-28, H-29);
FAB-MS m/z: 734.4049 (Calcd for C46H56NO7: 734.4057).
analysis of variance (ANOVA). Appropriate groups were compared by Stu-
dent’s t-test. IC50 values were calculated with PRISM (Graphpad Software,
3b-O-Acetyl-30-nor-olean-11,13(18)-dien-20b-[N-(2-carboxyphenyl)]- Inc.).
carboxamide (29) To a solution of 20 (2.5 g, 5.0 mmol) in CH2Cl2 (20 ml)
was added SOCl2 (3 ml). After being stireed for 3 h at 70 °C, the reaction
Acknowledgements We thank Prof. Tadahiro Takeda (Kyoritsu College
of Pharmacy) for the mass spectral measurements. We also thank Dr. Shoji
mixture was concentrated. To the residue was added THF (25 ml), an-
thranilic acid (1.03 g, 7.51 mmol) and triethylamine (0.5 ml), then the mix- Shibata for helpful discussions during the preparation of this manuscript.
ture was stirred overnight at 70 °C. The reaction mixture was diluted with
CHCl3, and the CHCl3 layer was washed with 5% HCl, dried over anhydrous References and Notes
Na2SO4, and concentrated. The residue was chromatographed on silica gel
with CHCl3 to give 29 (1.4 g, 46% in yield); mp 252—254 °C; 1H-NMR
(400 MHz, CDCl3) d 11.21 (1H, s, NH), 8.82 (1H, d, Jꢂ8.3 Hz), 8.13 (1H,
dd, Jꢂ7.9, 1.6 Hz), 7.60 (1H, dt, Jꢂ5.3, 2.6 Hz), 7.09 (1H, t, Jꢂ7.6 Hz),
6.43 (1H, dd, J9,11ꢂ3.0 Hz, J11,12ꢂ10.4 Hz, H-11), 5.57 (1H, d, H-12), 4.52
(1H, dd, Jꢂ5.4, 2.7 Hz, H-3), 2.84, 2.34 (1Hꢃ2, each d, Jgemꢂ14.4 Hz, H-
19), 2.06 (3H, s, Ac), 1.22, 1.14, 0.99, 0.90, 0.87, 0.85, 0.73 (3Hꢃ7, each s,
H-23, H-24, H-25, H-26, H-27, H28, H-29); 13C-NMR (100 MHz, CDCl3) d
177.88 (C-30), 171.84, 170.94 (CꢂO), 142.42, 135.51, 135.32, 135.18,
131.62, 126.20, 125.23, 122.23, 120.38, 113.71 (C-13, C-18 C-11, C-12,
Ar), 80.84 (C-3); FAB-MS m/z: 616.4002 (Calcd for C39H54O5N: 616.4002).
3b-Hyroxy-30-nor-olean-11,13(18)-dien-20b-[N-(2-carboxyphenyl)]-
carboxamide (30) To a solution of 29 (94 mg, 0.15 mmol) in MeOH
(20 ml) was added KOH (92 mg). After being stireed overnight at 70 °C, the
reaction mixture was neutralized with Amberlite IR-120B, then the precipi-
tates were dissolved with CHCl3, and the resin was removed from the solu-
tion. The solution was evaporated, then the residue was recrystallized from
MeOH to give 30 (86 mg, 99% in yield); mp 286—288 °C; 1H-NMR
(400 MHz, C5D5N) d 12.51 (1H, s, NH), 12.51 (1H, s), 9.34 (1H, d,
Jꢂ8.5 Hz), 8.50 (1H, d, Jꢂ7.8 Hz), 7.60 (1H, t, Jꢂ6.7 Hz), 7.17 (1H, t,
Jꢂ7.6 Hz), 6.65 (1H, dd, J9,11ꢂ2.4 Hz, J11,12ꢂ10.5 Hz, H-11), 5.67 (1H, d,
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