Novel synthetic oleanane and ursane triterpenoids with various enone functionalities in ring A as inhibitors of nitric oxide production in mouse macrophages

Article abstract of DOI:10.1021/jm000008j

We initially randomly synthesized about 60 oleanane and ursane triterpenoids as potential anti-inflammatory and cancer chemopreventive agents. Preliminary screening of these derivatives for inhibition of production of nitric oxide induced by interferon-γ in mouse macrophages revealed that 3-oxooleana-1,12-dien-28-oic acid (B-15) showed significant activity (IC₅₀ = 5.6/μM). On the basis of the structure of B-15, 19 novel olean- and urs-12-ene triterpenoids with a 1-en-3-one functionality having a substituent at C-2 in ring A have been designed and synthesized. Among them, 3-oxooleana-1,12-diene derivatives with carboxyl, methoxycarbonyl, and nitrile groups at C-2 showed higher activity than the lead compound B-15. In particular, 2-carboxy-3-oxooleana-1,12-dien-28-oic acid (3) had the highest activity (IC₅₀ = 0.07/μM) in this group of triterpenoids. The potency of 3 was similar to that of hydrocortisone (IC₅₀ = 0.01/μM), although 3 does not act through the glucocorticoid receptor. Interesting structure-activity relationships of these novel synthetic triterpenoids are also discussed.

Full text of DOI:10.1021/jm000008j

1866
J . Med. Chem. 2000, 43, 1866-1877
Novel Syn th etic Olea n a n e a n d Ur sa n e Tr iter p en oid s w ith Va r iou s En on e
F u n ction a lities in Rin g A a s In h ibitor s of Nitr ic Oxid e P r od u ction in Mou se
Ma cr op h a ges†
Tadashi Honda,‡ Gordon W. Gribble,*,‡ Nanjoo Suh,§ Heather J . Finlay,‡,| BarbieAnn V. Rounds,‡ Lothar Bore,‡
Frank G. Favaloro, J r.,‡ Yongping Wang,§ and Michael B. Sporn*,§
Department of Chemistry, Dartmouth College, and Department of Pharmacology and Toxicology, Dartmouth Medical School,
Hanover, New Hampshire 03755
Received J anuary 7, 2000
We initially randomly synthesized about 60 oleanane and ursane triterpenoids as potential
anti-inflammatory and cancer chemopreventive agents. Preliminary screening of these deriva-
tives for inhibition of production of nitric oxide induced by interferon-γ in mouse macrophages
revealed that 3-oxooleana-1,12-dien-28-oic acid (B-15) showed significant activity (IC50 ) 5.6
µM). On the basis of the structure of B-15, 19 novel olean- and urs-12-ene triterpenoids with
a 1-en-3-one functionality having a substituent at C-2 in ring A have been designed and
synthesized. Among them, 3-oxooleana-1,12-diene derivatives with carboxyl, methoxycarbonyl,
and nitrile groups at C-2 showed higher activity than the lead compound B-15. In particular,
2-carboxy-3-oxooleana-1,12-dien-28-oic acid (3) had the highest activity (IC50 ) 0.07 µM) in
this group of triterpenoids. The potency of 3 was similar to that of hydrocortisone (IC50 ) 0.01
µM), although 3 does not act through the glucocorticoid receptor. Interesting structure-activity
relationships of these novel synthetic triterpenoids are also discussed.
In tr od u ction
The high output of nitric oxide (NO) produced by
inducible nitric oxide synthase (iNOS), which is ex-
pressed in activated macrophages, plays an important
role in host defense. However, excessive production of
NO also can destroy functional normal tissues during
acute and chronic inflammation.5 This phenomenon is
also closely related mechanistically to carcinogenesis.6
Thus, inhibitors of NO production in macrophages are
potential anti-inflammatory and cancer chemopreven-
tive drugs. Because oleanolic and ursolic acids are
already known to have weak anti-inflammatory and
anticarcinogenic activity,3a,3b,3e,3f we focused our atten-
tion on therapeutic agents of these diseases. For this
purpose, we have adopted an assay system that meas-
ures inhibition of NO production induced by interferon-γ
(IFN-γ) in mouse macrophages7 as a preliminary screen-
ing assay system. We synthesized various oleanolic and
ursolic acid derivatives and tested them as inhibitors
of NO production. As a result, we have identified a series
of novel olean-12-ene triterpenoids with a 1-en-3-one
functionality having carboxyl, methoxycarbonyl, and
nitrile groups at C-2 in ring A that show significant
inhibitory activity (IC50 ) 0.01-0.1 µM level) against
production of NO induced by IFN-γ in mouse macro-
phages. In particular, 2-carboxy-3-oxooleana-1,12-dien-
28-oic acid (3) had the highest activity (IC50 ) 0.07 µM)
in this group of compounds. The potency of 3 was similar
to that of hydrocortisone (IC50 ) 0.01 µM), although 3
does not act through the glucocorticoid receptor. We
report here the synthesis, inhibitory activity, and
structure-activity relationships of these novel triter-
penoids in detail.
Oleanane and ursane triterpenoids are pentacyclic
compounds with 30 carbon atoms, which are derived
biosynthetically by the cyclization of squalene.1 The
group includes a very large number of naturally occur-
ring members that cover an impressive variety of
functional groups.2 Many compounds of this group are
reported to have interesting biological, pharmacological,
or medicinal activities similar to those of retinoids and
steroids, such as anti-inflammatory activity, suppres-
sion of tumor promotion, suppression of immunoglobulin
synthesis, protection of the liver against toxic injury,
induction of collagen synthesis, and induction of dif-
ferentiation in leukemia or teratocarcinoma cells.3
However, the potency of these triterpenoids is relatively
weak. There are no systematic studies of structure-
activity relationships based on chemical modification of
oleanane and ursane triterpenoids.4 We have therefore
considered that bioassay-directed systematic drug de-
sign and synthesis of derivatives of oleanolic acid (1)
and ursolic acid (2), which are commercially available,
could be of great value in discovering novel structures
with high biological potency.
† Part of this work has been reported in preliminary form: (a)
Honda, T.; Finlay, H. J .; Gribble, G. W.; Suh, N.; Sporn, M. B. 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. (b) Honda, T.; Rounds, B. V.; Bore, L.; Favaloro,
F. G., J r.; Gribble, G. W.; Suh, N.; Wang, Y.; Sporn, M. B. Novel
synthetic oleanane triterpenoids: a series of highly active inhibitors
of nitric oxide production in mouse macrophages. Bioorg. Med. Chem.
Lett. 1999, 9, 3429-3434.
* Address correspondence to either author. For G.W.G.: phone,
603-646-3118; fax, 603-646-3946; e-mail, Grib@dartmouth.edu.
For M.B.S.: phone, 603-650-6557; fax, 603-650-1129; e-mail,
Michael.Sporn@dartmouth.edu.
Ch em istr y
‡ Department of Chemistry.
§ Department of Pharmacology and Toxicology
Discover y of Lea d Com p ou n d . When we started
this project, we had no information about a lead
| Present address: The Bristol-Myers Squibb Pharmaceutical Re-
search Institute, P.O. Box 5400, Princeton, NJ 08543-5400.
10.1021/jm000008j CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/14/2000

Synthetic Oleanane and Ursane Triterpenoids
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1867
Ta ble 1. Preliminary Screening Results of Synthetic Oleanane and Ursane Triterpenoids
inhibition (%)
compd
skeleton
olean-12-ene
urs-12-ene
olean-12-ene
urs-12-ene
olean-12-ene
urs-12-ene
olean-12-ene
urs-12-ene
olean-12-ene
urs-12-ene
olean-12-ene
urs-12-ene
oleanane
oleanane
oleanane
oleanane
oleanane
oleanane
oleanane
oleanane
olean-12-ene
urs-12-ene
oleanane
oleanane
oleanane
oleanane
olean-12-ene
urs-12-ene
olean-12-ene
urs-12-ene
olean-12-ene
C-3
C-12
C-13
C-17
CO2H
at 10 µMb
ref
1
2
A-1
A-2
A-3
A-4
A-5
A-6
â-OH
â-OH
â-OH
â-OH
â-OAc
â-OAc
â-OAc
â-OAc
â-OH
â-OH
â-OAc
â-OAc
â-OAc
â-OAc
â-OAc
â-OAc
â-OH
â-OH
â-OH
â-OAc
R-OH
R-OH
â-OH
â-OH
â-OAc
â-OH
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
H
H
H
H
H
H
H
H
H
H
H
H
R-OH
â-OH
â-OAc
R-OH
R-OH
â-OH
dO
dO
H
H
R-OH
R-OH
12R,13R-epoxy-
R-OH
H
H
H
H
H
H
H
dO
dO
dO
R-Br
dO
H
38
0
0
10
15
10
15
10
15
10
15
28
29
28
29
c
CO2H
CO2Me
CO2Me
CO2Me
CO2Me
CO2H
0
10
15
0
0
0
8
4
0
0
0
0
0
48
20
0
CO2H
A-7
A-8
A-9
CH2OH
CH2OH
CH2OAc
CH2OAc
CO2Me
CO2Me
CH2OAc
CH2OAc
CH2OH
CH2OH
CO2Me
CO2Me
CO2H
A-10
A-11
A-12
A-13
A-14
A-15
A-16
A-17
A-18
A-19
A-20
A-21a
A-22a
A-23
A-24
B-1
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
â-H
â-H
â-H
â-H
â-H
â-H
â-H
â-H
c
c
c
c
c
10
10
30
31
32
32
10
32
10
33
10
15
34
34
c
10
c
c
10
10
9
d
d
14
c
c
0
18
48
21
13
0
22
16
22
24
16
11
21
47
3
37
38
4
0
19
0
CO2H
-O-
-O-
-CH(OH)-
-CO-
CO2Me
CH2OH
CO2H
â-OH
CO2H
CO2Me
CO2Me
CHO
CHO
CO2H
CO2Me
CO2H
CHO
-CO-
-CO-
CO2Me
CO2Me
CO2H
CO2H
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2H
CO2Me
CO2H
CO2H
CO2H
urs-12-ene
oleana-11,13(18)-diene
oleanane
oleanane
oleanane
â-H
â-H
â-H
-O-
B-10
B-11a
B-12a
B-13
B-14
B-15
B-16
C-1a
C-2
oleanane
oleanane
-O-
oleana-1,12-diene
ursa-1,12-diene
oleana-1,12-diene
ursa-1,12-diene
urs-12-ene
olean-12-ene
olean-12-ene
oleana-2,12-diene
oleana-2,12-diene
olean-12-ene
A-ring cleaved olean-12-ene
A-ring cleaved olean-12-ene
A-ring cleaved urs-12-ene
A-ring cleaved olean-12-ene
A-ring cleaved urs-12-ene
A-ring cleaved urs-12-ene
C-ring cleaved oleanane
C-ring cleaved oleanane
C-ring cleaved oleanane
C-ring cleaved oleanane
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
85
41
55
2
0
3
R-Cl
R-Cl
H
C-3
D-1
D-2
c
35
c
H
0
0
D-3a
E-1a
E-2a
E-3a
E-4a
E-5a
E-6a
F -1a
F -2a
F -3a
F -4a
c
21
33
39
22
55
10
52
12
52
28
36
37
37
37
37
37
c
c
c
c
CO2H
CO2H
CH2OAc
CH2OAc
CH2OAc
CO2H
â-OAc
â-OAc
â-OAc
dO

1868 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Ta ble 1 (Continued)
Honda et al.
inhibition (%)
compd
skeleton
C-3
C-12
C-13
C-17
at 10 µMb
ref
G-1a
olean-12-ene
olean-12-ene
H
H
CO2Me
CO2H
0
51
80
36
37
G-2a
hydrocortisone
a
b
Structure shown below this table. Details of the evaluation method are described in the Experimental Section. c Unknown compound
d
(synthesis and spectral data will be published elsewhere). Unknown compound (synthesis and spectral data are shown in this paper).
Sch em e 1a
a
Reagents: (a) PhSeCl, EtOAc; mCPBA, pyr, EtOAc; (b) LiI, DMF.
compound. Therefore, about 60 oleanolic and ursolic acid
derivatives were initially randomly synthesized. They
are divided into seven categories: 3-hydroxy derivatives,
A; 3-oxo derivatives, B; chloro derivatives, C; dehydroxy-
oleanane derivatives, D; A-ring cleaved derivatives, E;
C-ring cleaved oleanane derivatives, F ; and lactams, G
(see Table 1). In the preliminary screen of these deriva-
tives for inhibition of production of NO induced by IFN-γ
in mouse macrophages, 3-oxooleana-1,12-dien-28-oic
acid (B-15) was found to show significant activity
(inhibition: 85% at 10 µM, IC50 ) 5.6 µM). (See Tables
1 and 2.)
Design a n d Syn th esis of New Der iva tives. When
B-15 is compared with the other derivatives, it has the
following features: first, it is an oleanane; second, it has
a 1-en-3-one functionality in ring A; third, it has a
carboxyl group at C-17. We focused our attention on the
1-en-3-one functionality in ring A among these features.
We therefore designed novel olean- and urs-12-ene
triterpenoids with a 1-en-3-one functionality having a
substituent at C-2 in ring A, 3-19, and novel triter-
penoid-steroid hybrid compounds, 20 and 218 (see
Table 2). The syntheses of these newly designed deriva-
tives and compounds B-13-B-16 are illustrated in
Schemes 1-6.
Ester B-139 was synthesized in 62% yield by intro-
duction of a double bond at C-1 of methyl oleanonate
(B-3)10 with phenylselenenyl chloride (PhSeCl) in ethyl
acetate and sequential addition of pyridine and m-
chloroperbenzoic acid.11,12 Acid B-15 was synthesized in
85% yield by halogenolysis of B-13 with lithium iodide
in N,N-dimethylformamide (DMF).13 Similarly, acid
B-1614 was synthesized in 58% yield via ester B-14 from
methyl ursonate (B-4).15 Epoxide 229 was prepared in
99% yield by epoxidation of B-13 with alkaline hydrogen
peroxide. Treatment of 22 with sodium methoxide16
gave enone 23 (yield, 87%; 98% based on recovered 22).
Diosphenol 24 was synthesized by demethylation of the
methyl enol ether at C-2 of 23 with hydrochloric acid
in acetic acid (yield, 81%). Halogenolysis of 24 gave acid
4 (yield, 18%). Halogenolysis of 23 gave a desired partial
demethylated product 5 in 28% (41% based on recovered

Synthetic Oleanane and Ursane Triterpenoids
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1869
Sch em e 2a
a
Reagents: (a) 30% H2O2, NaOH(aq), THF; (b) NaOMe, MeOH; (c) HCl, AcOH; (d) LiI, DMF.
Sch em e 3a
a
Reagents: (a) HX, AcOH, CHCl3; (b) LiI, DMF.
23) yield.17 Chloride 6 was synthesized in 81% yield
from 22 with hydrogen chloride in acetic acid and
chloroform.18 Halogenolysis of 6 gave chloride 7 in 77%
yield. Similarly, bromides 8 and 9 were prepared from
22 and 8 (yield, 96% and 76%), respectively. Hydroxy-
methylene 2519,20 was prepared in 95% yield by formyl-
ation of B-3 with ethyl formate in the presence of
sodium methoxide in benzene.21 Isoxazole 26 was pre-
pared in 86% yield by condensation of 25 with hydroxyl-
amine.22 Cleavage of the isoxazole moiety of 26 with
sodium methoxide gave nitrile 27 in 99% yield.22 1H
NMR showed that 27 is a mixture of three tautomers
[27a , 27b (2R-cyano), and 27c (2â-cyano)] and that 27a
is the major one in CDCl3. Enone 10 was prepared in
88% yield by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) oxidation of 27 in benzene, although the same
method as for B-13 gave 10 in only 35% yield. Halo-
genolysis of 10 gave acid 11 in 71% (91% based on
recovered 10) yield. Similarly, ursane derivative 12 was
synthesized in 52% yield via 28,20,23 29, and 30 from
B-4. Acid 13 was prepared in 74% yield by halogenolysis
of 12. Enal 14 was prepared from 25 by PhSeCl-
pyridine in methylene chloride and sequential addition
of 30% hydrogen peroxide24 (yield, 71%; 79% based on
recovered 25). Halogenolysis of 14 did not give acid 15
but a complex mixture. Therefore, the synthesis of acid
15 from oleanonic acid (B-1)10 was attempted. Formyl-
ation of B-1 with ethyl formate in the presence of
sodium methoxide in tetrahydrofuran gave 3220 (yield,
45%; 66% based on recovered B-1). Acid 15 was pre-
pared from 32 according to the same method as for 14
(yield, 71%; 84% based on recovered 32). J ones oxidation
of 14 gave acid 16 in 30% (39% based on recovered 14)
yield. Because this yield was not enough to synthesize
derivatives 3 and 17-19 from 16, an alternative route
was adopted. Ester 31 was prepared in 74% (89% based
on recovered B-3) yield from B-3 by Stiles’ reagent
(methoxymagnesium methyl carbonate) in DMF,25 fol-
lowed by methylation with diazomethane. 1H NMR
showed that 31 is the single tautomer in CDCl3 as
depicted in Scheme 5. Enone 17 was prepared from 31
according to the same method as for 14 (yield, 83%; 90%
based on recovered 31). Hydrolysis of 17 with potassium
hydroxide in aqueous methanol gave acid 16 selectively
in 97% yield because the methoxycarbonyl group at C-17
of 17 is sterically hindered. Halogenolysis of 16 gave
dicarboxylic acid 3 in 58% yield. Methylation of 3 with
methanol under acidic conditions gave ester 18 selec-
tively in 78% yield because of the steric hindrance of
the carboxylic acid at C-17 of 3. Amide 19 was prepared
selectively in 96% yield from 17 with saturated am-
monia-methanol.
Biologica l Resu lts a n d Discu ssion
The inhibitory activities [IC50 (µM) value] of com-
pounds B-1, B-13, B-15, B-16, 1-21, and hydrocortisone
(a positive control) on NO production induced by IFN-γ
in mouse macrophages are shown in Table 2. These
derivatives are arranged according to the strength of
Taft’s σ* values26 of substituents at C-2. These results
provide the following interesting structure-activity
relationships:
(1) In the A ring, a 1-en-3-one functionality is impor-
tant for significant activity. The lead compound B-15
is much more potent than the C-3 ketone B-1 and the

1870 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Honda et al.
Sch em e 4a
a
Reagents: (a) HCO2Et, NaOMe, PhH; (b) NH2OH‚HCl, aq EtOH; (c) NaOMe, Et2O, MeOH; (d) DDQ, PhH; (e) LiI, DMF.
Sch em e 5a
a
Reagents: (a) PhSeCl, pyr, CH2Cl2; 30% H2O2, CH2Cl2; (b) J ones; (c) Stiles' reagent, DMF; (d) CH2N2, Et2O, THF; (e) KOH, aq MeOH;
(f) LiI, DMF; (g) H2SO4, MeOH; (h) NH3, MeOH.
Sch em e 6a
a
Reagents: (a) HCO2Et, NaOMe, THF; (b) PhSeCl, pyr, CH2Cl2; 30% H2O2, CH2Cl2.
C-3 alcohol 1 (oleanolic acid). Also, the ursane derivative
B-16 is more potent than the C-3 alcohol 2 (ursolic acid).
(2) A correlation between Taft’s σ* values of substit-
uents at C-2 and biological activity is not observed. This
result shows that the activity does not depend on the
strength of electron-withdrawing effect of a substituent
at C-2.
(3) Carboxyl, methoxycarbonyl, and nitrile groups at
C-2 enhance activity. Compounds 3, 10, 11, 16, and 17
are about 10-100 times more potent than B-15. In
particular, 3 showed the highest activity (IC50 ) 0.07
µM) in this series of compounds. The potency of 3 was
similar to that of hydrocortisone (IC50 ) 0.01 µM).
(4) Hydroxyl, aminocarbonyl, methoxy, chloride, and
bromide groups decrease activity. Compounds 4-9 and
19 are much less potent than B-15.
(5) A formyl group does not confer activity but only
toxicity.
(6) 23,24-Dimethyl groups are important for signifi-

Synthetic Oleanane and Ursane Triterpenoids
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1871
Ta ble 2. Activity of Olean- and Urs-12-ene Triterpenoids with Various 1-En-3-one Functionalities
Taft’s σ*
activityc
IC50 (µM)
compd
skeletona
R1 at C-2
R2 at C-17
value of R1
formula
C31H46O3
C30H44O3‚3/4H2O
C29H40O3‚1/4H2O
C28H38O3‚1/3H2O
C30H44O3
C30H44O4‚1/2H2O
C32H47O4N‚3/4H2O
C31H46O4‚1/2H2O
C33H48O5
C32H46O5
C32H46O5‚1/2H2O
C31H44O5
C32H46O4
C31H44O4‚1/2H2O
C31H45O3Br
analysesb
B-13
B-15
20
21
B-16
5
19
4
17
18
16
3
14
15
8
9
6
O
O
D
D
U
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
U
U
H
H
H
H
H
CO2Me
CO2H
CO2Me
CO2H
CO2H
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
ref 9
C, H
C, H
C, H
ref 14
C, H
C, H, N
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
31
5.6
>40
13
13
27
14
30
OH
CONH2
OMe
CO2Me
CO2Me
CO2H
CO2H
CHO
1.34
1.68
1.81
2.00
2.00
2.08
2.08
2.15
2.15
2.84
2.84
2.96
2.96
3.30
3.30
3.30
3.30
0.9
2.2
0.8
0.07
toxicd
toxicd
>40
7.3
>40
>40
0.7
0.6
5.1
6.2
37
CHO
Br
Br
Cl
Cl
C30H43O3Br‚H2O
C31H45O3Cl
7
C30H43O3Cl‚1/4H2O
C32H45O3N‚1/4H2O
C31H43O3N‚1/2H2O
C32H45O3N‚3/4H2O
C31H43O3N‚H2O
10
11
12
13
B-1
1
CN
CN
CN
CN
C, H, N
C, H, N
C, H, N
C, H, N
ref 10
ref 10
ref 15
oleanonic acid
oleanolic acid
ursolic acid
hydrocortisone
C
C
C
30H46O3
30H48O3
30H48O3
>40
toxice
0.01
2
a
O, 3-oxooleana-1,12-diene; D, 23,24-dinor-3-oxooleana-1,4,12-triene; U, 3-oxoursa-1,12-diene. b C, H, and N analyses were within (0.4%
of the theoretical values. c Details of the evaluation method are described in the Experimental Section. IC50 values of 3 and hydrocortisone
were determined in the range of 0.1 pM-1 µM (10-fold dilutions). The other compounds were assayed in the range of 0.01-40 µM (4-fold
d
dilutions). Values are an average of two separate experiments. Compounds 14 and 15 were toxic to cells above 1 µM and were not active
below 1 µM. e Ursolic acid (2) was toxic to cells above 10 µM and was not active below 10 µM.
cant activity. B-15 is more potent than 23,24-dinor-
olean-1-en-3-one derivative 21.
(7) The oleanane skeleton is more potent than the
ursane skeleton. B-15, 10, and 11 are more potent than
B-16, 12, and 13, respectively.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on
a Thomas-Hoover capillary melting point
apparatus and are uncorrected. Optical rotations were meas-
ured with a J asco DIP-181 digital polarimeter. UV and IR
spectra were recorded on a Hewlett-Packard 8451A UV/VIS
spectrophotometer and a Perkin-Elmer 600 series FTIR spec-
(8) The role of methoxycarbonyl and carboxyl groups
at C-17 is ambiguous. In some analogues, the carboxyl
group is more potent than the methoxycarbonyl group:
acids B-15, 3, 9, and 21 are more potent than esters
B-13, 16, 8, and 20, respectively. For other analogues,
the carboxyl and methoxycarbonyl groups show similar
activity: acids 11 and 13 show similar activity to esters
10 and 12, respectively. Lastly, acid 18 is less potent
than ester 17.
The inhibitory activity of new triterpenoids 3 and 11
was not blocked by the glucocorticoid antagonist, RU-
486,27 which reverses the action of hydrocortisone (see
Figure 1). These data strongly suggest that the actions
of these triterpenoids on the iNOS system are not
mediated by their interaction with the glucocorticoid
receptor.
trophotometer, respectively. H (300 MHz) and 13C (75 MHz)
1
NMR spectra were recorded on a Varian XL-300 Fourier
transform spectrometer. The chemical shifts are reported in
δ (ppm) using the δ 7.27 signal of CHCl3 (1H NMR) and the δ
77.23 signal of CDCl3 (13C NMR) as internal standards. Low-
resolution mass spectra and high-resolution MS data were
obtained on a Micromass 70-VSE unless otherwise stated.
Elemental microanalysis was performed by Atlantic Microlab
Inc. TLC and preparative TLC (prep-TLC) were performed
with Merck precoated TLC plates silica gel 60 F254. Flash
column chromatography was done with Select Scientific silica
gel (230-400 mesh). The standard work up method was as
follows: an organic extract was washed with saturated aque-
ous NaHCO3 solution (three times) followed by saturated
aqueous NaCl solution (three times), then dried over anhy-
drous MgSO4, and filtered. The filtrate was evaporated in
vacuo.
On the basis of these structure-activity relationships,
further lead optimization is in progress. Further biologi-
cal evaluation and studies on the mechanism of action
of 3 are also in progress.
Meth yl 3-Oxoolea n a -1,12-d ien -28-oa te (B-13).9 A solu-
tion of methyl oleanonate (B-3)10 (2.00 g, 4.27 mmol) and
phenylselenenyl chloride (98%) (1.00 g, 5.12 mmol) in EtOAc
(85 mL) was stirred at room temperature for 3 h. To the stirred

1872 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Honda et al.
23.8, 23.5, 23.2, 21.8, 19.1, 18.8, 17.5. EIMS (70 eV) m/z: 466
[M]+ (73), 451 (11), 407 (31), 262 (57), 203 (100). HREIMS:
Calcd for C31H46O3: 466.3447. Found: 466.3446.
Meth yl 3-Oxou r sa -1,12-d ien -28-oa te (B-14). B-14 was
prepared from methyl ursonate (B-4)15 according to the same
method as for B-13 to give an amorphous solid (66%): [R]26
D
+93° (c 0.77, CHCl3). UV (EtOH) λmax (log ꢀ): 232 (3.95) nm.
IR (KBr): 2974, 2935, 2871, 1725, 1669 cm-1 1H NMR
.
(CDCl3): δ 7.06 (1H, d, J ) 10.1 Hz), 5.81 (1H, d, J ) 10.1
Hz), 5.33 (1H, t, J ) 3.8 Hz), 3.63 (3H, s), 2.28 (1H, d, J )
11.5 Hz), 1.17, 1.15 (each 3H, s), 1.10 (6H, s), 0.95 (3H, d, J )
5.4 Hz), 0.87 (3H, d, J ) 6.3 Hz), 0.85 (3H, s). 13C NMR
(CDCl3): δ 205.5, 178.2, 159.5, 139.0, 125.2, 125.0, 53.7, 53.3,
51.7, 48.4, 44.7, 42.6, 41.9, 40.5, 39.5, 39.2, 39.1, 36.8, 33.0,
30.8, 28.2, 28.1, 24.4, 23.7, 23.5, 21.8, 21.4, 19.1, 19.0, 17.7,
17.2. EIMS (70 eV) m/z: 466 [M]+ (14), 406 (12), 262 (74), 203
(100).HREIMS: Calcd for C31H46O3: 466.3447.Found: 466.3442.
3-Oxoolea n a -1,12-d ien -28-oic Acid (B-15). A mixture of
B-13 (100 mg, 0.21 mmol) and LiI (500 mg) in dry DMF (2
mL) was heated under reflux for 6 h. The mixture was acidified
with 5% aqueous HCl solution and then extracted with a
mixture of CH2Cl2 and Et2O (1:2) three times. The extract was
worked up according to the standard method to give a solid
(110 mg). The solid was subjected to flash column chromatog-
raphy [hexanes-EtOAc (5:1) followed by hexanes-EtOAc (2:
1)] to give B-15 as an amorphous solid (82 mg, 85%): [R]26
D
+103° (c 0.45, CHCl3). UV (EtOH) λmax (log ꢀ): 230 (3.75) nm.
IR (KBr): 2941, 2866, 1732, 1695, 1671 cm-1 1H NMR
.
(CDCl3): δ 7.04 (1H, d, J ) 10.2 Hz), 5.81 (1H, d, J ) 10.2
Hz), 5.35 (1H, t, J ) 3.3 Hz), 2.86 (1H, dd, J ) 4.2, 13.4 Hz),
1.16, 1.152, 1.147, 1.07, 0.94, 0.91, 0.84 (each 3H, s). 13C NMR
(CDCl3): δ 205.5, 184.5, 159.2, 144.2, 125.3, 122.1, 53.5, 46.8,
45.8, 44.7, 42.1, 41.9, 41.3, 40.2, 39.7, 34.0, 33.3, 32.6, 32.5,
30.9, 28.0, 27.8, 26.0, 23.7, 23.5, 23.0, 21.8, 19.0, 18.9, 17.7.
EIMS (70 eV) m/z: 452 [M]+ (8.8), 437 (3.8), 406 (6.8), 248
(80), 233 (14), 203 (100). HREIMS: Calcd for
452.3290. Found: 452.3289. Anal. (Table 2).
C30H44O3:
3-Oxou r sa -1,12-d ien -28-oic Acid (B-16).14 B-16 was pre-
pared from B-14 according to the same method as for B-15 to
give an amorphous solid (88%): [R]26 +91° (c 0.84, CHCl3).
D
UV (EtOH) λmax (log ꢀ): 230 (3.99) nm. IR (KBr): 3306, 2973,
1
2930, 2870, 1729, 1695, 1669 cm-1. H NMR (CDCl3): δ 7.07
(1H, d, J ) 10.1 Hz), 5.82 (1H, d, J ) 10.1 Hz), 5.33 (1H, t, J
) 3.7 Hz), 2.24 (1H, d, J ) 11.2 Hz), 1.18, 1.16, 1.11, 1.09 (each
3H, s), 0.96 (3H, d, J ) 6.1 Hz), 0.88 (3H, d, J ) 6.4 Hz), 0.88
(3H, s). 13C NMR (CDCl3): δ 205.5, 183.9, 159.4, 138.8, 125.3,
53.6, 52.9, 48.3, 44.7, 42.5, 41.9, 40.5, 39.6, 39.2, 39.0, 36.8,
32.9, 30.8, 28.2, 28.1, 24.2, 23.7, 23.4, 21.8, 21.3, 19.0, 17.8,
17.2. FABMS (NBA) m/z: 453 [M + H]+ (100) (by a Micromass
ZAB-SE). HRFABMS: Calcd for C30H44O3 + H: 453.3369.
Found: 453.3335 (by a Micromass 70-SE-4F).
F igu r e 1. Blockage by glucocorticoid antagonist RU486 of
hydrocortisone-inhibited NO production but not of triterpenoid
(3 and 11) inhibited NO production in primary mouse mac-
rophages. Macrophage cells were incubated with IFN-γ (20 ng/
mL) together with hydrocortisone or triterpenoids without
RU486 (b); in some cases RU486 (1 µM) was added simulta-
neously to both hydrocortisone- and triterpenoid-treated cell
wells (O). RU486 itself does not interfere with NO production
at the concentration tested.
2-Ca r boxy-3-oxoolea n a -1,12-d ien -28-oic Acid (3). A mix-
ture of 16 (109 mg, 0.21 mmol) and LiI (520 mg) in dry DMF
(1.5 mL) was heated under reflux for 1 h. After 5% aqueous
HCl solution was added, the acidic mixture was extracted with
EtOAc three times. The extract was washed with water (three
times) and saturated aqueous NaCl solution (three times),
dried over anhydrous MgSO4, and filtered. The filtrate was
evaporated in vacuo to give a residue (108 mg). The residue
was subjected to flash column chromatography [CH2Cl2-
MeOH (15:1) followed by CH2Cl2-MeOH (10:1)] to afford 3 as
mixture, saturated aqueous NaHCO3 solution was added. After
most of the aqueous layer was removed, pyridine (844 mg, 10.7
mmol) and m-chloroperbenzoic acid (50-60%) (3.68 g, 10.7
mmol) were added to the organic layer. The mixture was
stirred at room temperature for 1 h. The mixture was washed
with 5% aqueous NaOH solution (three times), saturated
aqueous NH4Cl solution (three times), and saturated aqueous
NaCl solution (three times); dried over anhydrous MgSO4; and
filtered. The filtrate was evaporated in vacuo to give a solid.
The solid was subjected to flash column chromatography
[hexanes-EtOAc (5:1)] to give B-13 as a crystalline solid (1.23
a crystalline solid (61 mg, 58%): mp >250 °C dec; [R]26 +81°
D
(c 0.53, CHCl3). UV (EtOH) λmax (log ꢀ): 234 (3.88) nm. IR
(KBr): 3389, 2943, 2872, 1752, 1696, 1637 cm-1 1H NMR
.
(CDCl3): δ 8.43 (1H, s), 5.37 (1H, t, J ) 3.5 Hz), 2.87 (1H, dd,
J ) 3.8, 13.9 Hz), 1.25, 1.22, 1.18, 1.15, 0.95, 0.93, 0.88 (each
3H, s). 13C NMR (CDCl3): δ 209.0, 183.9, 173.2, 165.2, 144.2,
123.4, 121.7, 52.4, 46.8, 45.7, 45.5, 42.3, 41.4, 41.1, 40.6, 40.4,
34.0, 33.2, 32.5, 32.3, 30.9, 28.4, 27.8, 26.0, 23.7, 23.5, 23.0,
22.0, 19.0, 18.4, 17.8. EIMS (70 eV) m/z: 496 [M]+ (3.0), 478
(3.4), 452 (7.6), 248 (56), 231 (35), 203 (100). HREIMS: Calcd
for C31H44O5: 496.3189. Found: 496.3196. Anal. (Table 2).
g, 62%): mp 159-161 °C; [R]25 +103° (c 0.64, CHCl3). UV
D
(EtOH) λmax (log ꢀ): 230 (3.92) nm. IR (KBr): 2946, 2867, 1728,
1660 cm-1. 1H NMR (CDCl3): δ 7.04 (1H, d, J ) 10.1 Hz), 5.81
(1H, d, J ) 10.1 Hz), 5.36 (1H, t, J ) 3.7 Hz), 3.64, (3H, s),
2.90 (1H, dd, J ) 4.6, 13.9 Hz), 1.17 (3H, s), 1.16 (6H, s), 1.10,
0.94, 0.91, 0.83 (each 3H, s). 13C NMR (CDCl3): δ 205.5, 178.4,
159.3, 144.5, 125.2, 121.9, 53.6, 51.8, 47.0, 45.9, 44.7, 42.2, 42.0,
41.7, 40.3, 39.7, 34.1, 33.3, 32.7, 32.5, 30.9, 28.0, 27.9, 26.0,
2-Hyd r oxy-3-oxoolea n a -1,12-d ien -28-oic Acid (4). 4 was
prepared from 24 according to the same method as for B-15

Synthetic Oleanane and Ursane Triterpenoids
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1873
except that the reaction time was 2 h. The reaction mixture
was subjected to flash column chromatography [hexanes-
EtOAc (5:1) followed by hexanes-EtOAc (4:1)] to give 4 as an
amorphous solid (18%): [R]25D +99° (c 0.46, CHCl3). UV (EtOH)
λmax (log ꢀ): 272 (3.71) nm. IR (KBr): 3434, 2938, 1698, 1667,
EtOAc (6:1)] to afford 8 as an amorphous solid (238 mg, 96%):
[R]26D +88° (c 0.51, CHCl3). UV (EtOH) λmax (log ꢀ): 260 (3.69)
nm. IR (KBr): 2943, 2870, 1733, 1691, 1601 cm-1 1H NMR
.
(CDCl3): δ 7.49 (1H, s), 5.35 (1H, t, J ) 3.5 Hz), 3.63 (3H, s),
2.90 (1H, dd, J ) 4.0, 13.8 Hz), 1.20, 1.15 (each 6H, s), 0.94,
0.91, 0.81 (each 3H, s). 13C NMR (CDCl3): δ 197.3, 178.3, 159.5,
144.6, 121.8, 121.5, 53.3, 51.8, 46.9, 46.5, 45.8, 43.1, 42.3, 42.1,
41.7, 40.3, 34.0, 33.3, 32.4, 30.9, 28.7, 27.8, 26.0, 23.8, 23.6,
23.2, 22.3, 19.1, 18.7, 17.5. EIMS (70 eV) m/z: 546 (5.0) and
544 (5.2) [M]+, 262 (8.5), 203 (24), 118 (100), 116 (100).
HREIMS: Calcd for C31H45O3Br: 544.2552. Found: 544.2553.
Anal. (Table 2).
2-Br om o-3-oxoolea n a -1,12-d ien -28-oic Acid (9). 9 was
prepared from 8 according to the same method as for B-15
except that the reaction time was 4 h. The reaction mixture
was subjected to flash column chromatography [hexanes-
EtOAc (4:1) followed by hexanes-EtOAc (3:1)] to give 9 as an
amorphous solid (76%): [R]26D +82° (c 0.31, CHCl3). UV (EtOH)
λmax (log ꢀ): 260 (3.52) nm. IR (KBr): 3434, 2939, 2870, 1727,
1686, 1601 cm-1. 1H NMR (CDCl3): δ 7.49 (1H, s), 5.35 (1H, t,
J ) 3.4 Hz), 2.86 (1H, dd, J ) 4.2, 13.7 Hz), 1.21 (6H, s), 1.16,
1.14, 0.94, 0.92, 0.83 (each 3H, s). 13C NMR (CDCl3): δ 197.2,
184.4, 159.3, 144.3, 121.84, 121.79, 53.3, 46.8, 46.5, 45.8, 43.1,
42.2, 42.0, 41.3, 40.3, 34.0, 33.2, 32.5, 32.4, 30.9, 28.7, 27.8,
26.0, 23.7, 23.5, 23.0, 22.2, 19.1, 18.7, 17.7. EIMS (70 eV) m/z:
532 (13) and 530 (14) [M]+, 285 (5.6), 283 (6.2), 248 (100), 235
(10), 233 (11), 203 (84). HREIMS: Calcd for C30H43O3Br:
530.2396. Found: 530.2383. Anal. (Table 2).
1649 cm-1 1H NMR (CDCl3): δ 6.35 (1H, s), 5.96 (1H, brs),
.
5.34 (1H, t, J ) 3.5 Hz), 2.86 (1H, dd, J ) 3.8, 13.9 Hz), 1.23,
1.22, 1.14, 1.11, 0.94, 0.92, 0.83 (each 3H, s). 13C NMR
(CDCl3): δ 201.2, 184.0, 144.1, 143.9, 128.4, 122.3, 54.0, 46.8,
45.8, 44.1, 43.3, 42.2, 41.3, 40.2, 38.7, 34.0, 33.3, 32.6, 30.9,
27.8, 27.4, 26.1, 23.8, 23.6, 23.0, 22.0, 19.9, 18.9, 17.7. EIMS
(70 eV) m/z: 468 [M]+ (3.2), 248 (13), 203 (23), 149 (42), 84
(100).HREIMS: Calcd for C30H44O4: 468.3240.Found: 468.3222.
Anal. (Table 2).
2-Meth oxy-3-oxoolea n a -1,12-d ien -28-oic Acid (5). A mix-
ture of 23 (230 mg, 0.46 mmol) and LiI (1045 mg) in dry DMF
(3.5 mL) was heated under reflux for 4 h. The reaction mixture
was worked up according to the same method as for B-15 to
give a solid (230 mg). The solid was subjected to flash column
chromatography [hexanes-EtOAc (3:1) followed by hexanes-
EtOAc (2:1), then hexanes-EtOAc (1:1)] to give 24 (35 mg;
16%, 23% based on recovered 23), 23 (74 mg), 4 (27 mg; 12%,
18% based on recovered 23), and 5 as an amorphous solid (63
mg; 28%, 41% based on recovered 23): [R]26 +96° (c 0.29,
D
CHCl3). UV (EtOH) λmax (log ꢀ): 266 (3.84) nm. IR (KBr): 3307,
1
2947, 2862, 1732, 1693, 1622 cm-1. H NMR (CDCl3): δ 5.96
(1H, s), 5.36 (1H, t, J ) 3.3 Hz), 3.56 (3H, s), 2.87 (1H, dd, J
) 4.2, 13.9 Hz), 1.17 (9H, s), 1.11, 0.94, 0.91, 0.84 (each 3H,
s). 13C NMR (CDCl3): δ 200.0, 184.4, 149.1, 144.4, 126.1, 122.1,
55.0, 53.2, 46.8, 45.9, 45.4, 43.3, 42.2, 41.3, 40.2, 38.5, 34.0,
33.3, 32.5, 30.9, 28.6, 27.8, 26.1, 23.8, 23.0, 22.0, 20.4, 19.2,
17.6. EIMS (70 eV) m/z: 482 [M]+ (11), 415 (6.5), 245 (18),
203 (33), 157 (100). HREIMS: Calcd for C31H46O4: 482.3396.
Found: 482.3375. Anal. (Table 2).
Meth yl 2-Cya n o-3-oxoolea n a -1,12-d ien -28-oa te (10). A
solution of 27 (141 mg, 0.28 mmol) and DDQ (98%) (79 mg,
0.34 mmol) in benzene (10 mL) was heated under reflux for 4
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 [benzene-acetone (20:1)] to
Meth yl 2-Ch lor o-3-oxoolea n a -1,12-d ien -28-oa te (6). A
solution of 22 (99 mg, 0.21 mmol) in AcOH including 1 M HCl
(2.5 mL) and CHCl3 (2.5 mL) was stirred at room temperature
overnight. The mixture was diluted with CH2Cl2. After it was
washed with water three times, it was worked up according
to the standard method to give a solid (96 mg). The solid was
subjected to flash column chromatography [hexanes-EtOAc
give a crystalline solid (123 mg, 88%): mp 201-202 °C; [R]26
D
+67° (c 0.53, CHCl3). UV (EtOH) λmax (log ꢀ): 240 (3.65) nm.
IR (KBr): 2945, 2874, 2232, 1724, 1686 cm-1 1H NMR
.
(CDCl3): δ 7.75 (1H, s), 5.36 (1H, t, J ) 3.5 Hz), 3.64 (3H, s),
2.91 (1H, dd, J ) 3.9, 13.9 Hz), 1.22, 1.21, 1.15, 1.14, 0.94,
0.92, 0.83 (each 3H, s). 13C NMR (CDCl3): δ 198.3, 178.3, 170.2,
144.8, 121.1, 115.2, 114.0, 52.8, 51.8, 46.9, 45.8, 45.1, 42.3, 41.7,
41.3, 40.8, 40.5, 34.0, 33.3, 32.4, 32.3, 30.9, 27.9, 27.8, 26.0,
23.8, 23.4, 23.1, 21.8, 18.9, 18.1, 17.6. EIMS (70 eV) m/z: 491
[M]+ (35), 459 (13), 432 (27), 262 (22), 247 (24), 203 (100).
HREIMS: Calcd for C32H45O3N: 491.3399. Found: 491.3391.
Anal. (Table 2).
2-Cya n o-3-oxoolea n a -1,12-d ien -28-oic Acid (11). 11 was
prepared from 10 according to the same method as for B-15
except that the reaction time was 3 h. The reaction mixture
was subjected to flash column chromatography [hexanes-
EtOAc (3:1) followed by hexanes-EtOAc (2:1), then hexanes-
EtOAc (1:1)] to give 11 as an amorphous solid (71%, 91% based
on recovered 10): [R]26D +61° (c 0.66, CHCl3). UV (EtOH) λmax
(log ꢀ): 238 (3.87) nm. IR (KBr): 3387, 2947, 2870, 2233, 1729,
1691, 1609 cm-1. 1H NMR (CDCl3): δ 7.75 (1H, s), 5.35 (1H, t,
J ) 3.3 Hz), 2.86 (1H, dd, J ) 4.0, 13.6 Hz), 1.22, 1.21, 1.15,
1.12, 0.94, 0.92, 0.85 (each 3H, s). 13C NMR (CDCl3): δ 198.2,
184.3, 170.1, 144.4, 121.4, 115.1, 114.1, 52.7, 46.8, 45.7, 45.0,
42.2, 41.3, 40.8, 40.5, 33.9, 33.2, 32.5, 32.2, 30.9, 27.9, 27.7,
25.9, 23.7, 23.4, 22.9, 21.8, 18.9, 18.1, 17.7. EIMS (70 eV) m/z:
477 [M]+ (18), 462 (5.6), 431 (16), 416 (10), 248 (76), 235 (25),
203 (100). HREIMS: Calcd for C31H43O3N: 477.3243. Found:
477.3240. Anal. (Table 2).
(6:1)] to afford 6 as an amorphous solid (84 mg, 81%): [R]26
D
+98° (c 0.26, CHCl3). UV (EtOH) λmax (log ꢀ): 250 (3.91) nm.
1
IR (KBr): 2943, 2866, 1727, 1689 cm-1. H NMR (CDCl3): δ
7.22 (1H, s), 5.34 (1H, t, J ) 3.5 Hz), 3.62 (3H, s), 2.89 (1H,
dd, J ) 4.2, 13.7 Hz), 1.203, 1.197 (each 3H, s), 1.14 (6H, s),
0.93, 0.90, 0.80 (each 3H, s). 13C NMR (CDCl3): δ 197.4, 178.3,
155.0, 144.5, 129.8, 121.5, 53.3, 51.8, 46.9, 46.3, 45.8, 42.2, 42.1,
41.64, 41.57, 40.3, 34.0, 33.3, 32.4, 30.9, 28.4, 27.8, 26.0, 23.8,
23.5, 23.1, 22.1, 19.1, 18.8, 17.5. EIMS (70 eV) m/z: 500 [M]+
(21), 262 (27), 247 (96), 203 (100). HREIMS: Calcd for
C31H45O3Cl: 500.3057. Found: 500.3060. Anal. (Table 2).
2-Ch lor o-3-oxoolea n a -1,12-d ien -28-oic Acid (7). 7 was
prepared from 6 according to the same method as for B-15
except that the reaction time was 4 h. The reaction mixture
was subjected to flash column chromatography [hexanes-
EtOAc (4:1) followed by hexanes-EtOAc (3:1)] to give 7 as an
amorphous solid (77%): [R]26D +88° (c 0.50, CHCl3). UV (EtOH)
λmax (log ꢀ): 252 (3.20) nm. IR (KBr): 3297, 2943, 2870, 1733,
1691, 1601 cm-1. 1H NMR (CDCl3): δ 7.23 (1H, s), 5.35 (1H, t,
J ) 3.3 Hz), 2.86 (1H, dd, J ) 4.3, 13.8 Hz), 1.22, 1.21, 1.16,
1.13, 0.94, 0.92, 0.84 (each 3H, s). 13C NMR (CDCl3): δ 197,4,
184.4, 154.9, 144.3, 129.9, 121.8, 53.2, 46.8, 46.4, 45.8, 42.21,
42.16, 41.6, 41.3, 40.3, 34.0, 33.3, 32.5, 32.4, 30.9, 28.5, 27.8,
26.0, 23.7, 23.5, 23.0, 22.1, 19.0, 18.9, 17.7. EIMS (70 eV) m/z:
486 [M]+ (25), 248 (100), 203 (96). HREIMS: Calcd for
Meth yl 2-Cya n o-3-oxou r sa -1,12-d ien -28-oa te (12). 12
was prepared from 30 according to the same method as for 10
to give an amorphous solid (62%): [R]26D +53° (c 0.35, CHCl3).
UV (EtOH) λmax (log ꢀ): 240 (3.74) nm. IR (KBr): 2973, 2926,
C
30H43O3Cl: 486.2901. Found: 486.2898. Anal. (Table 2).
Met h yl 2-Br om o-3-oxoolea n a -1,12-d ien -28-oa t e (8). A
2870, 2229, 1723, 1686 cm-1 1H NMR (CDCl3): δ 7.77 (1H,
.
solution of 22 (220 mg, 0.46 mmol) in AcOH including 1 M
HBr (4.9 mL) and CHCl3 (6.1 mL) was stirred at room
temperature for 1 h. The mixture was diluted with CH2Cl2.
After it was washed with water three times, it was worked up
according to the standard method to give a solid (260 mg). The
solid was subjected to flash column chromatography [hexanes-
s), 5.33 (1H, t, J ) 3.7 Hz), 3.62 (3H, s), 2.29 (1H, d, J ) 11.2
Hz), 1.23, 1.21, 1.14, 1.11 (each 3H, s), 0.96, 0.88 (each 3H, d,
J ) 6.3 Hz), 0.86 (3H, s). 13C NMR (CDCl3): δ 198.3, 178.1,
170.4, 139.3, 124.2, 115.2, 114.0, 53.2, 52.8, 51.7, 48.3, 45.1,
42.7, 41.2, 40.70, 40.65, 39.1, 39.0, 36.7, 32.6, 30.8, 28.1, 28.0,
24.3, 23.6, 23.4, 21.8, 21.3, 18.9, 18.2, 17.8, 17.2. EIMS (70

1874 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Honda et al.
eV) m/z: 491 [M]+ (38), 431 (35), 262 (46), 249 (82), 203 (65),
84 (100). HREIMS: Calcd for C32H45O3N: 491.3399. Found:
491.3395. Anal. (Table 2).
1722, 1633 cm-1. 1H NMR (CDCl3): δ 8.43 (1H, s), 5.36 (1H, t,
J ) 3.5 Hz), 3.64 (3H, s), 2.90 (1H, dd, J ) 3.9, 13.7 Hz), 1.24,
1.21, 1.19, 1.13, 0.94, 0.91, 0.85 (each 3H, s). 13C NMR
(CDCl3): δ 209.2, 178.4, 173.4, 165.2, 144.5, 123.3, 121.4, 52.4,
51.8, 47.0, 45.7, 45.5, 42.3, 41.7, 41.1, 40.6, 40.4, 34.0, 33.3,
32.4, 32.3, 30.9, 28.3, 27.8, 26.0, 23.8, 23.5, 23.1, 22.0, 19.0,
18.3, 17.7. EIMS (70 eV) m/z: 510 [M]+ (16), 492 (15), 451 (14),
433 (14), 262 (27), 203 (100). HREIMS: Calcd for C32H46O5:
510.3345. Found: 510.3347. Anal. (Table 2).
(2) F r om 17: A solution of 17 (500 mg, 0.95 mmol) in MeOH
(29 mL) and aqueous KOH solution (KOH, 2.9 g; water, 10
mL) was heated under reflux for 15 min. After removal of
MeOH in vacuo, the mixture was acidified with 5% aqueous
HCl solution. It was extracted with EtOAc (three times). The
extract was washed with water and saturated aqueous NaCl
solution (each three times), dried over MgSO4, and filtered.
The filtrate gave 16 as a crystalline solid (470 mg, 97%). It
was used for the next reaction without further purification.
2-Cya n o-3-oxou r sa -1,12-d ien -28-oic Acid (13). 13 was
prepared from 12 according to the same method as for B-15
except that the reaction time was 4 h. The reaction mixture
was subjected to prep-TLC [hexanes-EtOAc (1.5:1)] to give
13 as an amorphous solid (74%): [R]26 +48° (c 0.50, CHCl3).
D
UV (EtOH) λmax (log ꢀ): 238 (3.86) nm. IR (KBr): 3417, 2973,
1
2926, 2870, 2233, 1731, 1689 cm-1. H NMR (CDCl3): δ 7.77
(1H, s), 5.31 (1H, t, J ) 3.2 Hz), 2.24 (1H, d, J ) 11.0 Hz),
1.22, 1.20, 1.12, 1.11 (each 3H, s), 0.95, 0.88 (each 3H, d, J )
5.7 Hz), 0.87 (3H, s). 13C NMR (CDCl3): δ 198.2, 184.2, 170.2,
139.0, 124.4, 115.1, 114.1, 52.8, 52.7, 48.2, 45.0, 42.6, 41.2,
40.68, 40.65, 39.1, 39.0, 36.7, 32.5, 30.7, 28.1, 28.0, 24.1, 23.6,
23.3, 21.8, 21.3, 18.9, 18.2, 17.7, 17.2. EIMS (70 eV) m/z: 477
[M]+ (22), 431 (23), 248 (100), 203 (48). HREIMS: Calcd for
C31H43O3N: 477.3243. Found: 477.3240. Anal. (Table 2).
Meth yl 2-Meth oxyca r bon yl-3-oxoolea n a -1,12-d ien -28-
oa te (17). 17 was prepared from 31 by the similar method as
for 14. The reaction mixture was subjected to flash column
chromatography [hexanes-EtOAc (4:1)] to give 17 as an
Meth yl 2-F or m yl-3-oxoolea n a -1,12-d ien -28-oa te (14). To
a stirred solution of phenylselenenyl chloride (98%) (161 mg,
0.82 mmol) in CH2Cl2 (7.2 mL) was added a solution of pyridine
(75 mg, 0.95 mmol) in CH2Cl2 (1.0 mL) in an ice bath. After
15 min, a solution of 25 (204 mg, 0.41 mmol) in CH2Cl2 (2.0
mL) was added, and the mixture was stirred an additional 1
h. After the mixture was washed with 10% aqueous HCl
solution (3 mL) twice, 30% H2O2 (0.4 mL) was added to the
stirred mixture in the ice bath. After an additional 40 min,
the mixture was worked up according to the standard method
to give a solid (199 mg). The solid was subjected to flash
column chromatography [hexanes-EtOAc (5:1)] to afford 25
(20 mg) and 14 as an amorphous solid (144 mg; 71%, 79%
based on recovered 25): [R]26D +12° (c 0.60, CHCl3). UV (EtOH)
λmax (log ꢀ): 238 (3.85) nm. IR (KBr): 2946, 2867, 1724, 1703,
amorphous solid (83%, 90% based on recovered 31): [R]26
D
+63° (c 0.78, CHCl3). UV (EtOH) λmax (log ꢀ): 230 (3.97) nm.
IR (KBr): 2947, 2866, 1727, 1684, 1624 cm-1 1H NMR
.
(CDCl3): δ 7.73 (1H, s), 5.37 (1H, t, J ) 3.5 Hz), 3.79, 3.64
(each 3H, s), 2.90 (1H, dd, J ) 3.9, 13.7 Hz), 1.16 (6H, s), 1.15,
1.12, 0.94, 0.91, 0.84 (each 3H, s). 13C NMR (CDCl3): δ 201.2,
178.4, 166.0, 164.3, 144.5, 129.2, 121.7, 52.7, 52.4, 51.8, 47.0,
45.9, 45.8, 42.3, 41.8, 41.5, 40.3, 39.5, 34.1, 33.3, 32.4, 32.3,
30.9, 28.7, 27.8, 25.9, 23.8, 23.6, 23.2, 21.5, 19.4, 18.0, 17.5.
EIMS (70 eV) m/z: 524 [M]+ (24), 492 (23), 465 (13), 262 (35),
203 (100). HREIMS: Calcd for C33H48O5: 524.3502. Found:
524.3494. Anal. (Table 2).
1
1673, 1610 cm-1. H NMR (CDCl3): δ 10.00 (1H, s), 7.79 (1H,
s), 5.37 (1H, t, J ) 3.6 Hz), 3.63 (3H, s), 2.90 (1H, dd, J ) 4.2,
13.9 Hz), 1.18, 1.17, 1.16, 1.14, 0.94, 0.91, 0.85 (each 3H, s).
13C NMR (CDCl3): δ 203.7, 190.7, 178.3, 165.2, 144.5, 131.2,
121.6, 52.8, 51.8, 47.0, 45.8, 45.1, 42.3, 41.7, 41.3, 40.5, 39.8,
34.0, 33.3, 32.44, 32.38, 30.9, 28.2, 27.8, 26.0, 23.8, 23.5, 23.2,
21.7, 19.2, 18.2, 17.6. EIMS (70 eV) m/z: 494 [M]+ (95), 435
(87), 262 (40), 203 (100). HREIMS: Calcd for C32H46O4:
494.3396. Found: 494.3398. Anal. (Table 2).
2-Meth oxyca r bon yl-3-oxoolea n a -1,12-d ien -28-oic Acid
(18). A solution of 3 (52 mg, 0.10 mmol) in MeOH (5.2 mL)
containing concentrated H2SO4 (0.15 mL) was heated under
reflux for 30 min. After saturated aqueous NaCl solution was
added to the mixture, it was extracted with EtOAc three times.
The extract was worked up according to the standard method
to give a residue (53 mg). The residue was subjected to flash
column chromatography [hexanes-EtOAc (2:1)] to give 18 as
an amorphous solid (42 mg, 78%): [R]26D +61° (c 0.56, CHCl3).
UV (EtOH) λmax (log ꢀ): 230 (3.83) nm. IR (KBr): 3323, 2947,
2-F or m yl-3-oxoolea n a -1,12-d ien -28-oic Acid (15). 15 was
prepared from 32 according to the same method as for 14. The
reaction mixture was subjected to flash column chromatogra-
phy [hexanes-EtOAc (3:1) followed by hexanes-EtOAc (2:1)]
to give 15 as an amorphous solid (71%, 84% based on recovered
2866, 1733, 1695, 1622 cm-1 1H NMR (CDCl3): δ 7.73 (1H,
.
s), 5.37 (1H, t, J ) 3.4 Hz), 3.79 (3H, s), 2.86 (1H, dd, J ) 4.1,
13.7 Hz), 1.16, 1.15, 1.14, 1.12, 0.94, 0.92, 0.86 (each 3H, s).
13C NMR (CDCl3): δ 201.1, 184.2, 166.0, 164.2, 144.2, 129.2,
122.0, 52.7, 52.4, 46.9, 45.9, 45.8, 42.2, 41.5, 41.4, 40.3, 39.5,
34.0, 33.3, 32.5, 32.3, 30.9, 28.7, 27.8, 26.0, 23.7, 23.6, 23.0,
21.4, 19.4, 18.0, 17.7. EIMS (70 eV) m/z: 510 [M]+ (2.6), 495
(2.0), 478 (2.5), 432 (3.0), 263 (29), 248 (58), 231 (37), 203 (100).
HREIMS: Calcd for C32H46O5: 510.3345. Found: 510.3344.
Anal. (Table 2).
32): [R]26 +26° (c 0.95, CHCl3). UV (EtOH) λmax (log ꢀ): 240
D
(3.82) nm. IR (KBr): 2948, 2866, 1725, 1701, 1674, 1608 cm-1
.
1H NMR (CDCl3): δ 10.00 (1H, s), 7.79 (1H, s), 5.36 (1H, t, J
) 3.3 Hz), 2.86 (1H, dd, J ) 3.8, 13.9 Hz), 1.18, 1.17, 1.15,
1.14, 0.94, 0.92, 0.87 (each 3H, s). 13C NMR (CDCl3): δ 203.7,
190.7, 184.3, 165.0, 144.2, 131.2, 121.8, 52.8, 46.8, 45.7, 45.1,
42.3, 41.4, 41.3, 40.5, 39.8, 34.0, 33.2, 32.5, 32.3, 30.9, 28.2,
27.8, 26.0, 23.7, 23.5, 23.0, 21.6, 19.2, 18.2, 17.8. EIMS (70
eV) m/z: 480 [M]+ (5.5), 434 (3.1), 419 (3.4), 248 (56), 233 (27),
203 (100). HREIMS: Calcd for C31H44O4: 480.3240. Found:
480.3237. Anal. (Table 2).
Met h yl 2-Am in oca r b on yl-3-oxoolea n a -1,12-d ien -28-
oa te (19). A solution of 17 (100 mg, 0.19 mmol) in saturated
ammonia MeOH (10 mL) was kept at room temperature
overnight. The mixture was evaporated in vacuo to give a solid
(108 mg). The solid was subjected to flash column chromatog-
raphy [hexanes-EtOAc (1.5:1)] to give 19 as an amorphous
Meth yl 2-Ca r boxy-3-oxoolea n a -1,12-d ien -28-oa te (16).
(1) F r om 14: To a solution of 14 (357 mg, 0.72 mmol) in
acetone (71 mL) was added J ones reagent (0.5 mL) dropwise
in an ice bath. The mixture was stirred in the ice bath for 20
min. After excess J ones reagent was decomposed with MeOH,
the acetone was evaporated in vacuo. After water was added
to the resultant mixture, the aqueous mixture was extracted
with EtOAc three times. The extract was washed with water
(three times) and saturated aqueous NaCl solution (three
times), dried over anhydrous MgSO4, and filtered. The filtrate
was evaporated in vacuo to give a residue (294 mg). The
residue was subjected to flash column chromatography [hex-
anes-EtOAc (1:1) followed by EtOAc] to afford 14 (89 mg) and
16 as a crystalline solid (109 mg; 30%, 39% based on recovered
solid (94 mg, 96%): [R]26 +77° (c 0.60, CHCl3). UV (EtOH)
D
λmax (log ꢀ): 236 (3.91) nm. IR (KBr): 3413, 2943, 2866, 1727,
1689 cm-1 1H NMR (CDCl3): δ 8.45 (1H, brs), 8.27 (1H, s),
.
5.72 (1H, brs), 5.37 (1H, t, J ) 3.4 Hz), 3.64 (3H, s), 2.90 (1H,
dd, J ) 4.2, 13.9 Hz), 1.17, 1.16, 1.15, 1.14, 0.94, 0.92, 0.84
(each 3H, s). 13C NMR (CDCl3): δ 205.8, 178.4, 169.0, 165.8,
144.3, 121.8, 52.2, 51.8, 47.0, 46.0, 45.7, 42.3, 41.8, 41.2, 40.4,
39.6, 34.1, 33.3, 32.5, 32.3, 30.9, 29.1, 27.8, 26.0, 23.8, 23.6,
23.2, 21.9, 19.4, 18.6, 17.6. EIMS (70 eV) m/z: 509 [M]+ (34),
492 (23), 450 (100), 262 (19), 203 (56). HREIMS: Calcd for
C
32H47O4N: 509.3505. Found: 509.3500. Anal. (Table 2).
Meth yl 1r,2r-Ep oxy-3-oxoolea n -12-en -28-oa te (22).9 To
14): mp 230-231 °C; [R]26 +85° (c 0.61, CHCl3). UV (EtOH)
a solution of B-13 (223 mg, 0.48 mmol) in 2 N aqueous NaOH
D
λmax (log ꢀ): 234 (3.78) nm. IR (KBr): 3436, 2946, 2876, 1756,
solution (1.7 mL) and THF (11 mL) was added a solution of

Synthetic Oleanane and Ursane Triterpenoids
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1875
30% H2O2 (1.4 mL) in MeOH (2.8 mL) in an ice bath. The
mixture was stirred at room temperature for 4 h. To the
mixture were added saturated aqueous NaHSO3 and 5%
aqueous NaOH solutions, successively. After removal of THF
and MeOH, the resultant mixture was acidified with 6 M
aqueous HCl solution. The acidic layer was extracted with
CH2Cl2 three times. The extract was worked up according to
the standard method to give 22 as a crystalline solid (228 mg,
99%). This material was used for the next reaction without
further purification. An analytically pure sample was obtained
by recrystallization from MeOH as colorless needles: mp 212-
1588 cm-1. 1H NMR (CDCl3): δ 14.92 (1H, d, J ) 3.1 Hz), 8.58
(1H, d, J ) 3.1 Hz), 5.35 (1H, t, J ) 3.7 Hz), 3.64 (3H, s), 2.90
(1H, dd, J ) 4.2, 13.6 Hz), 2.29 (1H, d, J ) 14.4 Hz), 1.92 (1H,
d, J ) 14.4 Hz), 1.20, 1.16, 1.12, 0.94 (each 3H, s), 0.91 (6H,
s), 0.80 (3H, s). 13C NMR (CDCl3): δ 190.9, 188.6, 178.4, 144.0,
122.3, 106.0, 52.3, 51.8, 47.0, 46.0, 45.9, 42.0, 41.6, 40.3, 39.4,
39.3, 36.5, 34.1, 33.3, 32.5, 32.1, 30.9, 28.6, 27.9, 25.9, 23.8,
23.6, 23.3, 21.1, 19.7, 16.8, 14.7. EIMS (70 eV) m/z: 496 [M]+
(4.4), 437 (23), 262 (38), 233 (20), 203 (100). HREIMS: Calcd
for C32H48O4: 496.3553. Found: 496.3550.
Meth yl Isoxa zolo[4,5-b]olea n -12-en -28-oa te (26). A mix-
ture of 25 (994 mg, 2.0 mmol), hydroxylamine hydrochloride
(1391 mg, 20 mmol) in water (1.8 mL) and EtOH (48 mL) was
heated under reflux for 1 h. After EtOH was removed in vacuo,
EtOAc was added to the resultant mixture. The EtOAc layer
was washed with water (three times) and saturated aqueous
NaCl solution (three times), dried over MgSO4, and filtered.
The filtrate gave a solid (1086 mg). The solid was subjected to
flash column chromatography [hexanes-EtOAc (6:1) followed
by hexanes-EtOAc (5:1)] to give 26 as an amorphous solid
(934 mg, 86%): UV (EtOH) λmax (log ꢀ): 228 (3.65) nm. IR
213 °C; [R]26 +157° (c 0.80, CHCl3). IR (KBr): 2943, 2866,
D
1727, 1699 cm-1. 1H NMR (CDCl3): δ 5.36 (1H, t, J ) 3.3 Hz),
3.64 (3H, s), 3.50 (1H, d, J ) 4.5 Hz), 3.37 (1H, d, J ) 4.5 Hz),
2.90 (1H, dd, J ) 4.2, 13.9 Hz), 1.21, 1.11, 1.01, 0.97, 0.94,
0.92, 0.80 (each 3H, s). 13C NMR (CDCl3): δ 213.0, 178.4, 144.5,
121.8, 64.1, 57.1, 51.8, 47.0, 46.3, 45.9, 45.0, 42.1, 41.7, 40.8,
39.7, 38.8, 34.1, 33.3, 32.5, 32.3, 30.9, 28.2, 28.0, 26.0, 24.0,
23.8, 23.3, 21.1, 19.1, 17.4, 15.1. EIMS (70 eV) m/z: 482 [M]+
(7.7), 422 (13), 262 (31), 249 (11), 203 (100). HREIMS: Calcd
for C31H46O4: 482.3396. Found: 482.3391.
1
(KBr): 2940, 2864, 1725 cm-1. H NMR (CDCl3): δ 7.98 (1H,
Meth yl 2-Meth oxy-3-oxoolea n a -1,12-d ien -28-oa te (23).
A mixture of 22 (300 mg, 0.62 mmol) and Na (360 mg) in
MeOH (36 mL) was heated under reflux for 48 h. After removal
of MeOH in vacuo, the resultant mixture was diluted with
water and then acidified with 6 M aqueous HCl solution. The
aqueous mixture was extracted with a mixture of CH2Cl2 and
Et2O (1:2) three times. The extract was worked up according
to the standard method to give a solid (320 mg). The solid was
subjected to flash column chromatography [hexanes-EtOAc
(4:1)] to afford 22 (31 mg) and 23 as an amorphous solid (270
mg; 87%, 98% based on recovered 22): UV (EtOH) λmax (log
ꢀ): 266 (3.77) nm. IR (KBr): 2946, 2866, 1727, 1682, 1621
s), 5.34 (1H, t, J ) 3.5 Hz), 3.63 (3H, s), 2.89 (1H, dd, J ) 4.4,
13.7 Hz), 2.42 (1H, d, J ) 15.1 Hz), 1.30, 1.21, 1.15, 0.93, 0.90,
0.88, 0.79 (each 3H, s). 13C NMR (CDCl3): δ 178.4, 173.2, 150.4,
144.0, 122.3, 109.0, 53.7, 51.7, 46.9, 46.3, 46.0, 42.0, 41.6, 39.5,
38.9, 35.5, 34.9, 34.0, 33.3, 32.5, 32.1, 30.9, 29.0, 27.9, 25.9,
23.8, 23.5, 23.2, 21.6, 19.0, 16.7, 15.4. EIMS (70 eV) m/z: 493
[M]+ (11), 434 (18), 262 (28), 249 (16), 203 (100). HREIMS:
Calcd for C32H47O3N: 493.3556. Found: 493.3556.
Meth yl 2-Cya n o-3-oxoolea n -12-en -28-oa te (27). To a
stirred solution of 26 (887 mg, 1.80 mmol) in Et2O (50 mL)
and MeOH (25 mL) was added NaOMe (3.2 g) in an ice bath.
The mixture was stirred at room temperature for 1 h. The
mixture was diluted with a mixture of CH2Cl2 and Et2O (1:2)
(50 mL). After the extract was washed with 5% aqueous HCl
solution, it was worked up according to the standard method
to afford 27 as an amorphous solid (879 mg, 99%). This
material was used for the next reaction without further
purification. An analytically pure sample was obtained by flash
column chromatography [hexanes-EtOAc (5:1)] as an amor-
phous solid: UV (EtOH) λmax (log ꢀ): 238 (3.88) nm. IR (KBr):
2946, 2870, 2202, 1724, 1633 cm-1. 1H NMR of major tautomer
27a (CDCl3): δ 6.15 (1H, brs), 5.31 (1H, t, J ) 3.6 Hz), 3.63
(3H, s), 2.88 (1H, dd, J ) 4.0, 13.6 Hz), 2.09 (1H, d, J ) 15.0
Hz), 1.16, 1.13, 1.07, 0.95, 0.93, 0.90, 0.76 (each 3H, s). EIMS
(70 eV) m/z: 493 [M]+ (6.3), 434 (17), 262 (19), 249 (20), 203
(100). HREIMS: Calcd for C32H47O3N: 493.3556. Found:
493.3548.
cm-1 1H NMR (CDCl3): δ 5.96 (1H, s), 5.36 (1H, t, J ) 3.5
.
Hz), 3.64, 3.55 (each 3H, s), 2.90 (1H, dd, J ) 4.1, 13.7 Hz),
1.17 (6H, s), 1.16, 1.13, 0.93, 0.90, 0.81 (each 3H, s). 13C NMR
(CDCl3): δ 200.1, 178.4, 149.0, 144.6, 126.3, 121.9, 54.9, 53.2,
51.8, 47.0, 45.9, 45.4, 43.3, 42.3, 41.7, 40.2, 38.4, 34.0, 33.3,
32.6, 32.5, 30.9, 28.5, 27.8, 26.0, 23.81, 23.76, 23.2, 22.0, 20.4,
19.2, 17.4. EIMS (70 eV) m/z: 496 [M]+ (80), 436 (21), 328 (19),
262 (36), 203 (100). HREIMS: Calcd for C32H48O4: 496.3553.
Found: 496.3544.
Meth yl 2-Hyd r oxy-3-oxoolea n a -1,12-d ien -28-oa te (24).
A suspension of 23 (100 mg, 0.20 mmol) in 3 M aqueous HCl
solution (3 mL) and AcOH (3 mL) was heated under reflux for
5 h. The mixture was neutralized with saturated aqueous
Na2CO3 solution. The mixture was extracted with CH2Cl2 three
times. The extract was worked up according to the standard
method to give a solid (90 mg). The solid was subjected to flash
column chromatography [hexanes-EtOAc (5:1)] to afford 24
as an amorphous solid (78 mg, 81%): UV (EtOH) λmax (log ꢀ):
272 (3.63) nm. IR (KBr): 3426, 2939, 2870, 1725, 1667, 1648
Met h yl 2-Hyd r oxym et h ylen e-3-oxou r s-12-en -28-oa t e
(28).23 28 was prepared from B-4 according to the same method
as for 25 to give an amorphous solid (quantitative). This
material was used for the next reaction without further
purification. An analytically pure sample was obtained by flash
column chromatography [hexanes-EtOAc (7:1)] and subse-
quent recrystallization from MeOH as colorless needles: mp
170-171 °C. UV (EtOH) λmax (log ꢀ): 294 (3.86) nm. IR (KBr):
cm-1 1H NMR (CDCl3): δ 6.35 (1H, s), 5.93 (1H, brs), 5.34
.
(1H, t, J ) 3.5 Hz), 3.63 (3H, s), 2.89 (1H, dd, J ) 4.0, 13.7
Hz), 1.22 (6H, s), 1.13, 1.12, 0.94, 0.91, 0.80 (each 3H, s). 13C
NMR (CDCl3): δ 201.3, 178.4, 144.4, 143.9, 128.4, 122.0, 54.1,
51.8, 47.0, 45.9, 44.1, 43.3, 42.2, 41.6, 40.2, 38.7, 34.1, 33.3,
32.7, 32.5, 30.9, 27.8, 27.4, 26.1, 23.8, 23.6, 23.2, 22.0, 19.8,
18.9, 17.5. EIMS (70 eV) m/z: 482 [M]+ (26), 446 (68), 422 (25),
262 (35), 203 (100). HREIMS: Calcd for C31H46O4: 482.3396.
Found: 482.3387.
3426, 2947, 2921, 2866, 1727, 1637, 1590 cm-1 1H NMR
.
(CDCl3): δ 14.91 (1H, brs), 8.57 (1H, s), 5.31 (1H, t, J ) 3.7
Hz), 3.62 (3H, s), 2.31 (1H, d, J ) 14.4 Hz), 2.27 (1H, d, J )
12.5 Hz), 1.95 (1H, d, J ) 14.4 Hz), 1.19, 1.12, 1.10 (each 3H,
s), 0.96 (3H, d, J ) 6.0 Hz), 0.92 (3H, s), 0.87 (3H, d, J ) 6.6
Hz), 0.81 (3H, s). 13C NMR (CDCl3): δ 191.0, 188.5, 178.2,
138.4, 125.6, 106.0, 53.2, 52.3, 51.7, 48.4, 45.7, 42.4, 40.3, 39.7,
39.5, 39.3, 39.1, 36.8, 36.4, 32.4, 30.9, 28.7, 28.2, 24.4, 23.7,
23.6, 21.4, 21.1, 19.7, 17.2, 17.0, 14.8. EIMS (70 eV) m/z: 496
[M]+ (11), 437 (15), 262 (80), 233 (41), 203 (100). HREIMS:
Calcd for C32H48O4: 496.3553. Found: 496.3547.
Meth yl 2-Hyd r oxym eth ylen e-3-oxoolea n -12-en -28-oa te
(25).19 To a stirred mixture of B-3 (1084 mg, 2.31 mmol) and
ethyl formate (97%) (707 mg, 9.26 mmol) in benzene (12 mL)
was added NaOMe (501 mg, 9.27 mmol). The mixture was
stirred at room temperature for 1 h. After the mixture was
washed with 5% aqueous HCl solution twice, it was worked
up according to the standard method to give 25 as an
amorphous solid (1095 mg, 95%). This material was used for
the next reaction without further purification. An analytically
pure sample was obtained by flash column chromatography
[hexanes-EtOAc (7:1)] and subsequent recrystallization from
MeOH as colorless needles: mp 199-201 °C. UV (EtOH) λmax
(log ꢀ): 296 (3.94) nm. IR (KBr): 3426, 2943, 2862, 1725, 1637,
Meth yl Isoxa zolo[4,5-b]u r s-12-en -28-oa te (29). 29 was
prepared from 28 according to the same method as for 26 to
give an amorphous solid (84%): UV (EtOH) λmax (log ꢀ): 228
(3.70) nm. IR (KBr): 2969, 2922, 2870, 1725 cm-1 1H NMR
.
(CDCl3): δ 7.98 (1H, s), 5.31 (1H, t, J ) 3.4 Hz), 3.62 (3H, s),
2.46 (1H, d, J ) 15.0 Hz), 2.27 (1H, d, J ) 11.1 Hz), 1.31, 1.22,

1876 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Honda et al.
1.10 (each 3H, s), 0.96 (3H, d, J ) 6.3 Hz), 0.90 (3H, s), 0.88
(3H, d, J ) 6.3 Hz), 0.81 (3H, s). 13C NMR (CDCl3): δ 178.2,
173.2, 150.4, 138.4, 125.5, 109.1, 53.7, 53.2, 51.7, 48.3, 46.3,
42.3, 39.7, 39.3, 39.1, 38.8, 36.8, 35.8, 34.9, 32.4, 30.9, 29.1,
28.3, 24.4, 23.7, 23.5, 21.6, 21.4, 19.0, 17.2, 16.9, 15.6. EIMS
(70 eV) m/z: 493 [M]+ (9.1), 434 (20), 262 (65), 249 (33), 203
(100). HREIMS: Calcd for C32H47O3N: 493.3556. Found:
493.3547.
(Cambridge, MA). All other chemicals were purchased from
Sigma Chemical Co. (St. Louis, MO). Inhibitory test com-
pounds were dissolved in DMSO before addition to cell
cultures; final concentrations of DMSO were 0.1% or less.
Controls with DMSO alone were run in all cases.
2. Cell Cu ltu r e. To obtain primary macrophages, female
CD-1 mice, 5-10 weeks of age (Charles River Breeding
Laboratories, Wilmington, MA), were injected intraperitoneally
with 2 mL of 4% thioglycollate broth (Difco Laboratories,
Detroit, MI). Four days after injection, peritoneal macrophages
were harvested and processed according to Nathan’s procedure.7b
Cells were seeded in 96-well plates at 2 × 105 cells/well and
incubated for 48 h with 20 ng/mL IFN-γ in the presence or
absence of inhibitory test compounds.
3. Mea su r em en t of NO P r od u ction in Mou se Ma c-
r op h a ges. Nitrite accumulation was used as an indicator of
NO production in the medium and was assayed by the Griess
reaction.7a Griess reagent (100 µL) was added to 100 µL of each
supernatant from IFN-γ or inhibitory test compound-treated
cells in triplicate. The protein determination was performed
by Bradford protein assay. The plates were read at 550 nm
against a standard curve of sodium nitrite.
Meth yl 2-Cya n o-3-oxou r s-12-en -28-oa te (30). 30 was
prepared from 29 according to the same method as for 27 to
give an amorphous solid (quantitative). This material was used
for the next reaction without further purification. An analyti-
cally pure sample was obtained by flash column chromatog-
raphy [hexanes-EtOAc (5:1)] as an amorphous solid: UV
(EtOH) λmax (log ꢀ): 238 (3.93) nm. IR (KBr): 2947, 2870, 2203,
1
1724, 1631 cm-1. H NMR of major tautomer 30a (CDCl3): δ
5.92 (1H, brs), 5.28 (1H, t, J ) 3.5 Hz), 3.61 (3H, s), 2.26 (1H,
d, J ) 11.0 Hz), 2.13 (1H, d, J ) 15.0 Hz), 1.16, 1.13, 1.08,
1.07, 0.96 (each 3H, s), 0.95, 0.77 (each 3H, d, J ) 6.3 Hz).
EIMS (70 eV) m/z: 493 [M]+ (6.8), 434 (19), 262 (62), 249 (44),
203 (100). HREIMS: Calcd for C32H47O3N: 493.3556. Found:
493.3558.
Meth yl 3-Hydr oxy-2-m eth oxycar bon ylolean a-2,12-dien -
28-oa te (31). A mixture of B-3 (2.0 g, 4.27 mmol) and 1.8 M
DMF solution of methoxymagnesium methyl carbonate (Stiles’
reagent) (20 mL, 36 mmol) was heated under reflux for 2 h
while a slow stream of N2 was bubbled through the mixture
with a pipet. To the mixture were added 5% aqueous HCl
solution and EtOAc. The aqueous layer was extracted with
EtOAc (three times). The combined organic layers were washed
with water (three times) and saturated aqueous NaCl solution
(three times), dried over MgSO4, and filtered. The filtrate was
evaporated in vacuo to give a solid (2.26 g). To a solution of
the solid in THF (30 mL) was added excessive amount of
ethereal diazomethane. The mixture was kept at room tem-
perature for 10 min. The mixture was evaporated in vacuo to
give a solid (2.38 g). The solid was subjected to flash column
chromatography [hexanes-EtOAc (7:1)] to give B-3 (330 mg)
and 31 as crystals (1.66 g; 74%, 89% based on recovered B-3):
mp 160-162 °C. UV (EtOH) λmax (log ꢀ): 262 (4.01) nm. IR
Ack n ow led gm en t. We thank Drs. Carl Nathan and
Qiao-wen Xie for expert advice on the preparation of
macrophages and the nitric oxide assay. We also thank
Dr. Steven Mullen (University of Illinois) for the mass
spectra and Professor David A. Evans and Mr. Brett D.
Allison (Harvard University) for the optical rotation
measurements. This investigation was supported by
funds from NIH Grant 1 R01-CA78814; the Norris
Cotton Cancer Center; U.S. Department of Defense
Grants DAMD17-96-1-6163, DAMD17-98-1-8604, and
DAMD17-99-1-9168; the Oliver and J ennie Donaldson
Charitable Trust; the National Foundation for Cancer
Research; and a Zenith Award from the Alzheimer’s
Association. M.B.S. is an Oscar M. Cohn Professor,
F.G.F. is an Oscar M. Cohn Scholar, and Y.W. is a
Howard Hughes Medical Institute Predoctoral Fellow.
1
(KBr): 2948, 2858, 1737, 1660, 1615 cm-1. H NMR (CDCl3):
δ 12.51 (1H, s), 5.33 (1H, t, J ) 3.7 Hz), 3.74, 3.63 (each 3H,
s), 2.89 (1H, dd, J ) 4.2, 13.9 Hz), 2.35 (1H, d, J ) 15.7 Hz),
1.18, 1.14, 1.10, 0.94 (each 3H, s), 0.91 (6H, s), 0.78 (3H, s).
13C NMR (CDCl3): δ 178.5, 177.9, 174.2, 143.8, 122.6, 94.3,
52.5, 51.8, 51.7, 47.0, 46.13, 46.09, 42.0, 41.7, 39.4, 38.6, 38.4,
35.7, 34.1, 33.3, 32.6, 32.1, 31.0, 28.8, 27.9, 26.0, 23.8, 23.6,
23.3, 20.4, 19.8, 16.8, 15.1. EIMS (70 eV) m/z: 526 [M]+ (0.6),
494 (5.6), 479 (2.5), 466 (1.6), 435 (13), 262 (28), 203 (100).
HREIMS: Calcd for C33H50O5: 526.3658. Found: 526.3658.
2-Hydr oxym eth ylen e-3-oxoolean -12-en -28-oic Acid (32).
To a stirred mixture of oleanonic acid (B-1)10 (540 mg, 1.19
mmol) and ethyl formate (97%) (357 mg, 4.66 mmol) in THF
(12 mL) was added NaOMe (258 mg, 4.78 mmol). The mixture
was stirred at room temperature overnight. The mixture was
acidified with 10% aqueous HCl solution. The mixture was
extracted with EtOAc three times. The extract was worked
up according to the standard method to give a solid (600 mg).
The solid was subjected to flash column chromatography
[hexanes-EtOAc (5:1) followed by hexanes-EtOAc (4:1)] to
afford B-1 (168 mg) and 32 as a crystalline solid (260 mg; 45%,
66% based on recovered B-1): mp 200-203 °C dec. UV (EtOH)
λmax (log ꢀ): 292 (3.93) nm. IR (KBr): 2946, 2654, 1732, 1694,
1644, 1587 cm-1. 1H NMR (CDCl3): δ 14.91 (1H, brs), 8.59 (1H,
s), 5.34 (1H, t, J ) 3.5 Hz), 2.86 (1H, dd, J ) 4.5, 13.9 Hz),
2.29 (1H, d, J ) 14.6 Hz), 1.93 (1H, d, J ) 14.6 Hz), 1.19, 1.16,
1.10, 0.94, 0.92, 0.91, 0.82 (each 3H, s). 13C NMR (CDCl3): δ
190.7, 188.8, 184.7, 143.8, 122.6, 105.9, 52.2, 46.8, 46.0, 45.9,
41.9, 41.2, 40.2, 39.34, 39.30, 36.5, 34.0, 33.3, 32.6, 32.0, 30.9,
28.6, 27.8, 25.9, 23.7, 23.5, 23.1, 21.0, 19.6, 17.0, 14.6. EIMS
(70 eV) m/z: 482 [M]+ (1.8), 438 (2.7), 436 (3.6), 248 (77), 203
(100).HREIMS: Calcd for C31H46O4: 482.3396.Found: 482.3392.
Refer en ces
(1) (a) Connolly, J . D.; Overton, K. H. The Triterpenoids. In
Chemistry of Terpenes and Terpenoids; Newman, A. A., Ed.;
Academic Press: New York, 1972; pp 207-287. (b) Nakanishi,
K.; Ito, S. Biosynthesis of oleanene and ursene triterpenes in
tissue culture of Isodon japonicus. In Natural Products Chem-
istry, Vol. 3; Nakanishi, K., Goto, T., Ito, S., Natori, S., Nozoe,
S., Eds.; Kodansha: Tokyo, 1983; pp 185-187.
(2) Devon, T. K.; Scott, A. I. The Terpenes. In Handbook of Naturally
Occurring Compounds, Vol. 2; Academic Press: New York, 1972;
pp 281-384.
(3) (a) Nishino, H.; Nishino, A.; Takayasu, J .; Hasegawa, T.;
Iwashima, A.; Hirabayashi, K.; Iwata, S.; Shibata, S. Inhibition
of the tumor-promoting action of 12-O-tetradecanoylphorbol-13-
acetate by some oleanane-type triterpenoid compounds. Cancer
Res. 1988, 48, 5210-5215. (b) Hirota, M.; Mori, T.; Yoshio, M.;
Iriye, R. Suppression of tumor promoter-induced inflammation
of mouse ear by ursolic acid and 4,4-dimethylcholestane deriva-
tives. Agric. Biol. Chem. 1990, 54, 1073-1075. (c) Yu, L.; Ma,
R.; Wang, Y.; Nishino, H.; Takayasu, J .; He, W.; Chang, M.;
Zhen, J .; Liu, W.; Fan, S. Potent anti-tumorigenic effect of
tubeimoside 1 isolated from the bulb of Bolbostemma panicu-
latum (Maxim) Franquet. Int. J . Cancer 1992, 50, 635-638. (d)
Umehara, K.; Takagi, R.; Kuroyanagi, M.; Ueno, A.; Taki, T.;
Chen, Y.-J . Studies on differentiation-inducing activities of
terpenes. Chem. Pharm. Bull. 1992, 40, 401-405. (e) Singh, G.
B.; Singh, S.; Bani, S.; Gupta, B. D.; Banerjee, S. K. Anti-
inflammatory activity of oleanolic acid in rats and mice. J .
Pharm. Pharmacol. 1992, 44, 456-458. (f) Huang, M.-T.; Ho,
C.-T.; Wang, Z. Y.; Ferrao, T.; Lou, Y.-R.; Stauber, K.; Ma, W.;
Georgiadis, C.; Laskin, J . D.; Conney, A. H. Inhibition of skin
tumorigenesis by rosemary and its constituents carnosol and
ursolic acid. Cancer Res. 1994, 54, 701-708. (g) Liu, J .; Liu, Y.;
Mao, Q.; Klaassen, C. D. The effects of 10 triterpenoid com-
pounds on experimental liver injury in mice. Fundam. Appl.
Toxicol. 1994, 22, 34-40. (h) Liu, J . Pharmacology of oleanolic
acid and ursolic acid. J . Ethnopharmacol. 1995, 49, 57-68 and
references therein.
Eva lu a tion Meth od s. 1. Rea gen ts. Recombinant mouse
IFN-γ (LPS content, <10 pg/mL) was purchased from Genzyme

Synthetic Oleanane and Ursane Triterpenoids
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1877
(4) After we started this project, the following systematic studies
of structure-activity relationships based on chemical modifica-
tion of oleanane triterpenoids were reported: (a) Sakurawi, K.;
Yasuda, F.; Tozyo, T.; Nakamura, M.; Sato, T.; Kikuchi, J .; Terui,
Y.; Ikenishi, Y.; Iwata, T.; Takahashi, K.; Konoike, T.; Mihara,
S.; Fujimoto, M. Endothelin receptor antagonist triterpenoid,
myriceric acid A, isolated from Myrica cerifera, and structure
activity relationships of its derivatives. Chem. Pharm. Bull.
1996, 44, 343-351. (b) Kashiwada, Y.; Wang, H.-K.; Nagao, T.;
Kitanaka, S.; Yasuda, I.; Fujioka, T.; Yamagishi, T.; Cosentino,
L. M.; Kozuka, M.; Okabe, H.; Ikeshiro, Y.; Hu, C.-Q.; Yeh, E.;
Lee, K.-H. Anti-AIDS agents. 30. Anti-HIV activity of oleanolic
acid, pomolic acid, and structurally related triterpenoids. J . Nat.
Prod. 1998, 61, 1090-1095.
(5) (a) Moncada, S.; Palmer, R. M. J .; Higgs, E. A. Nitric oxide:
physiology, pathophysiology, and pharmacology. Pharmacol. Rev.
1991, 43, 109-141. (b) Nathan, C. F.; Xie, Q.-W. Regulation of
biosynthesis of nitric oxide. J . Biol. Chem. 1994, 269, 13725-
13728. (c) Anggard, E. Nitric oxide: mediator, murderer, and
medicine. Lancet 1994, 343, 1199-1206. (d) Nathan, C. F.; Xie,
Q.-W. Nitric oxide synthases: roles, tolls, and controls. Cell 1994,
78, 915-918.
(6) (a) Sporn, M. B.; Roberts, A. B. Peptide growth factors and
inflammation, tissue repair, and cancer. J . Clin. Invest. 1986,
78, 329-332. (b) Ohshima, H.; Bartsch, H. Chronic infections
and inflammatory processes as cancer risk factors: possible role
of nitric oxide in carcinogenesis. Mutat. Res. 1994, 305, 253-
264.
(7) (a) Ding, A.; Nathan, C. F.; Graycar, J .; Derynck, R.; Stuehr, D.
J .; Srimal, S. Macrophage deactivating factor and transforming
growth factors-â1, -â2, and -â3 inhibit induction of macrophage
nitrogen oxide synthesis by IFN-γ. J . Immunol. 1990, 145, 940-
944. (b) Bogdan, C.; Paik, J .; Vodovotz, Y.; Nathan, C. Contrast-
ing mechanisms for suppression of macrophage cytokine release
by transforming growth factor-â and interleukin-10. J . Biol.
Chem. 1992, 267, 23301-23308.
(19) Zaprutko, L. Triterpenoids. Part 9. [1] Structure elucidation of
a new sodium dichromate oxidation product of methyl oleanolate
and some of its derivatives. Pol. J . Chem. 1995, 69, 1003-1012.
(20) 1H and 13C NMR of 25, 28, and 32 in CDCl3 showed that they
are the single tautomer as depicted in Schemes 4 and 6 (see
Experimental Section).
(21) Clinton, R. O.; Manson, A. J .; Stonner, F. W.; Neumann, H. C.;
Christiansen, R. G.; Clarke, R. L.; Ackerman, J . H.; Page, D. F.;
Dean, J . W.; Dickinson W. B.; Carabateas, C. Steroidal[3,2-c]-
pyrazoles. II. Androstanes, 19-norandrostanes and their unsat-
urated analogs. J . Am. Chem. Soc. 1961, 83, 1478-1491.
(22) J ohnson, W. S.; Shelberg, W. E. A plan for distinguishing
between some five- and six-membered ring ketones. J . Am.
Chem. Soc. 1945, 67, 1745-1754.
(23) Glen, A. T.; Lawrie, W.; McLean, J .; Younes, M. E.-G. Triter-
penoid constituents of rose-bay willow-herb. J . Chem. Soc. (C)
1967, 510-515.
(24) Liotta, D.; Barnum, C.; Puleo, R.; Zima, G.; Bayer, C.; Kezar,
H. S., III. A simple method for the efficient synthesis of
unsaturated â-dicarbonyl compounds. J . Org. Chem. 1981, 46,
2920-2923.
(25) Finkbeiner, H. L.; Stiles, M. Chelation as a driving force in
organic reactions. IV. Synthesis of R-nitro acids by control of the
carboxylation-decarboxylation equilibrium. J . Am. Chem. Soc.
1963, 85, 616-622.
(26) Albert, A. Table and discussion: electronic effects in molecules
(Hammett and Taft sigma values). Selective Toxicity, 7th ed.;
Chapman and Hall: London, 1985; pp 644-649.
(27) Gagne, D.; Pons, M.; Philibert, D. RU38486: a potent antigluco-
corticoid in vitro and in vivo. J . Steroid Biochem. 1985, 23, 247-
251.
(28) Narayanan, C. R.; Natu, A. A. Synthesis of some bridged
triterpene ethers. J . Org. Chem. 1974, 39, 2639-2641 and
references therein.
(29) Younes, M. E.-G. Chemical examination of local plants. XIV.
triterpenoids from the leaves of Egyptian Callistemon lanceola-
tus. Aust. J . Chem. 1975, 28, 221-224.
(30) Huneck, S. Triterpene-IV. Die Triterpensa¨uren des Balsams von
Liquidambar orientalis Miller. Tetrahedron 1963, 19, 479-482.
(31) Huneck, S.; Snatzke, G. Triterpene, IX. U¨ ber die Triterpene aus
der Rinde von Sambucus nigra L. und die Darstellung von 3-epi-
Ursolsa¨ure. Chem. Ber. 1965, 98, 120-125.
(32) Kitagawa, I.; Kitazawa, K.; Yoshioka, I. Photochemical trans-
formation leading to eupteleogenin-I. Introduction of epoxy-
lactone system. Tetrahedron 1972, 28, 907-921.
(33) Kuwada, S.; Matsukawa, T. Ursolic acid. II. Oxidation of ursolic
acid and its derivatives with chromic acid. J . Pharm. Soc. J pn.
(Yakugaku Zasshi) 1933, 53, 593-612.
(34) Sahu, N. P.; Mahato, S. B.; Chakravarti, R. N. Effect of Raney
nickel on some dihydroxy triterpenoids in high boiling solvents.
J . Indian Chem. Soc. 1973, 50, 771-773.
(35) Djerassi, C.; Thomas, D. B.; Livingston, A. L.; Thompson, C. R.
Terpenoids. XXXI. The structure and stereochemistry of medi-
cagenic acid. J . Am. Chem. Soc. 1957, 79, 5292-5297.
(36) Sundararamaiah, T.; Ramraj, S. K.; Rao, K. L.; Bai, V. V.
Synthesis of A-aza triterpenes-I: A-aza triterpenes from methyl
oleanonate, methyl betulonate and lupenone. J . Indian Chem.
Soc. 1976, 53, 664-665.
(37) Finlay, H. J .; Honda, T.; Gribble, G. W.; Danielpour, D.; Benoit,
N. E.; Suh, N.; Williams, C.; Sporn, M. B. Novel A-ring cleaved
analogs of oleanolic and ursolic acids which affect growth
regulation in NRP.152 prostate cells. Bioorg. Med. Chem. Lett.
1997, 7, 1769-1772.
(8) Synthesis of 20 and 21 was already reported: Honda, T.; Gribble,
G. W. Design and synthesis of 23,24-dinoroleanolic acid deriva-
tives, novel triterpenoid-steroid hybrid molecules. J . Org. Chem.
1998, 63, 4846-4849.
(9) Brieskorn, C. H.; Seifert, M. Methylgruppen-Umlagerungen an
Triterpenoiden, 2. Mitt. Aromatisierung des Ringes A. Arch.
Pharm. 1982, 315, 846-851.
(10) Simonsen, J .; Ross, W. C. J . Oleanolic acid. In The Terpenes;
Cambridge University: Cambridge, 1957; Vol. 5, pp 221-285.
(11) Sharpless, K. B.; Lauer, R. F.; Teranishi, A. Y. Electrophilic and
nucleophilic organoselenium reagents. New routes to R,â-
unsaturated carbonyl compounds. J . Am. Chem. Soc. 1973, 95,
6137-6139.
(12) DDQ oxidation of B-3 in benzene (reflux) did not give B-13 at
all.
(13) Dean, P. D. G. Halogenolysis of methyl glycyrrhetate with
lithium iodide-dimethylformamide. J . Chem. Soc. (C) 1965, 6655.
(14) Begum, S.; Adil, Q.; Siddiqui, B. S.; Siddiqui, S. Synthesis of
2â-hydroxyursolic acid and other ursane analogues from ursonic
acid. Aust. J . Chem. 1993, 46, 1067-1071.
(15) Simonsen, J .; Ross, W. C. J . Ursolic acid. In The Terpenes;
Cambridge University: Cambridge, 1957; Vol. 5, pp 114-135.
(16) Kurata, Y.; Hirota, H.; Honda, T.; Takahashi, T. Preparation of
a tricyclic A-ring analog of quassin. Chem. Pharm. Bull. 1987,
35, 837-840.
(17) In addition to 5, these conditions also gave the completely
demethylated product 4 (yield, 12%; 18% based on recovered 23)
and the other partially demethylated product 24 (yield, 16%; 23%
based on recovered 23) from 23.
(18) Shaw, J . I.; Stevenson, R. 4-Bromo- and 4-chloro-cholest-4-en-
3-one. J . Chem. Soc. 1955, 3549-3551.
J M000008J