Anti-AIDS agents 38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives

Article abstract of DOI:10.1021/np990633v

Based on our previous finding that 3-O-acyl-betulinic and -oleanolic acids, especially the 3-O-(3′,3′- dimethyl)-succinyl derivatives (2 and 4), demonstrated potent anti-HIV activity [EC₅₀ < 0.00035 and 0.00086 μM; therapeutic index (TI) > 20 000 and 22 326, respectively], several 3-O-acyl-ursolic acids were prepared and evaluated for anti-HIV activity. Ursolic acid (6) was equipotent (EC₅₀ 4.4 μM) with oleanolic acid (EC₅₀ 3.7 μM), although it was slightly toxic (IC₅₀ 14.3 μM, TI 3.3). 3-O-Diglycoryl-ursolic acid (10) demonstrated relatively potent anti-HIV-activity with an EC₅₀ of 0.31 μM and a TI of 155.5. In contrast, 3-O-(3′,3′-dimethylsuccinyl)-ursolic acid (8), which is analogous to the extremely potent anti-HIV betulinic acid and oleanolic acid derivatives 2 and 4, displayed only weak anti-HIV activity (EC₅₀ 2.1 μM, TI 23.6).

Full text of DOI:10.1021/np990633v

J . Nat. Prod. 2000, 63, 1619-1622
1619
An ti-AIDS Agen ts 38. An ti-HIV Activity of 3-O-Acyl Ur solic Acid Der iva tives1
Yoshiki Kashiwada,*,†, Tsuneatsu Nagao,‡ Ayumi Hashimoto,† Yasumasa Ikeshiro,† Hikaru Okabe,‡
L. Mark Cosentino,§ and Kuo-Hsiung Lee*,
Niigata College of Pharmacy, Kamishin’ei-cho, Niigata 950-2081, J apan, Faculty of Pharmaceutical Sciences,
Fukuoka University, Nanakuma, J onan-ku, Fukuoka 814-0180, J apan, BBI-Biotech Research Laboratories, Perry Parkway,
Gaithersburg, Maryland 20877, and Natural Products Laboratory, School of Pharmacy, University of North Carolina,
Chapel Hill, North Carolina 27599
Received December 22, 1999
Based on our previous finding that 3-O-acyl-betulinic and -oleanolic acids, especially the 3-O-(3′,3′-
dimethyl)-succinyl derivatives (2 and 4), demonstrated potent anti-HIV activity [EC50 < 0.00035 and
0.00086 µM; therapeutic index (TI) > 20 000 and 22 326, respectively], several 3-O-acyl-ursolic acids were
prepared and evaluated for anti-HIV activity. Ursolic acid (6) was equipotent (EC50 4.4 µM) with oleanolic
acid (EC50 3.7 µM), although it was slightly toxic (IC50 14.3 µM, TI 3.3). 3-O-Diglycoryl-ursolic acid (10)
demonstrated relatively potent anti-HIV activity with an EC50 of 0.31 µM and a TI of 155.5. In contrast,
3-O-(3′,3′-dimethylsuccinyl)-ursolic acid (8), which is analogous to the extremely potent anti-HIV betulinic
acid and oleanolic acid derivatives 2 and 4, displayed only weak anti-HIV activity (EC50 2.1 µM, TI 23.6).
In our continuing search for potential anti-HIV natural
products, betulinic acid (1), isolated from the leaves of
Syzygium claviflorum (Myrtaceae), was identified as an
anti-HIV agent with an EC50 value of 1.4 µM and thera-
peutic index (TI) of 9.3.2 Subsequent modification of betu-
linic acid yielded 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid
(2), which demonstrated extremely potent anti-HIV activity
with an EC50 value of < 0.00035 µM and TI of >20 000.3
The related oleanolic acid (3) was isolated as an anti-HIV
compound from several plants, including Rosa woodsii
(Rosaceae), Prosopis glandulosa (Leguminosae), Phora-
dendron juniperinum (Loranthaceae), Syzygium claviflo-
rum (Myrtaceae), Hyptis capitata (Lamiaceae), and Tern-
stromia gymnanthera (Theaceae), while pomolic acid (5)
was identified as an anti-HIV agent from Pr. glandulosa,
Ph. juniperinum, S. claviflorum, and H. capitata. Both
compounds displayed weak anti-HIV activity, with EC50
values of 3.7 and 3.0 µM, respectively, and TI values of
12.8 and 16.3, respectively.4 As with betulinic acid, similar
modification of oleanolic acid also gave a potent anti-HIV
active derivative, 3-O-(3′,3′-dimethylsuccinyl)-oleanolic acid
(4) (EC50 0.00086 µM; TI 22 400).4
With an EC50 value of 4.4 µM, ursolic acid (6), isolated
from Pr. glandulosa, Ph. juniperinum, S. claviflorum, and
H. capitata, showed a level of anti-HIV activity similar to
that shown by oleanolic acid, although 6 was somewhat
toxic to H9 cells as revealed by the small TI value of 3.3.4
The structures of ursolic acid and oleanolic acid are almost
identical, and differ only in the position of the C-29 methyl
group in the E-ring. Therefore, as with oleanolic acid,
pyridine in the presence of 4-(dimethylamino)pyridine
(DMAP) at reflux. As was found with betulinic acid,3 the
reaction of ursolic acid and 2,2-dimethylsuccinic anhydride
gave a mixture of 2′,2′-dimethylsuccinyl- and 3′,3′-dimeth-
ylsuccinyl-ursolic acids (7 and 8, respectively), in which the
latter isomer was the major product. The mixture was
modification of ursolic acid was expected to yield potent
separated by semipreparative-scale HPLC, and the struc-
anti-HIV derivative(s). Based on these previous findings,
tures of these isomers were assigned by 2D NMR analyses,
we have prepared 3-O-acyl-ursolic acid derivatives and
1
1
including H-13C COSY and long-range H-13C COSY.
evaluated their anti-HIV activity.
3-O-(3′,3′-Dimethylsuccinyl)-ursolic acid (8) was expected
to have potent anti-HIV activity because the corresponding
derivatives of betulinic and oleanolic acids were extremely
potent anti-HIV compounds. However, 8 displayed only
weak anti-HIV activity (EC50 value of 2.1 µM), although it
was less toxic than the parent acid to H9 cells (IC50 49.5
µM). Among all 3-O-acyl-ursolic acid derivatives, 3-O-
diglycoryl-ursolic acid (10) demonstrated relatively potent
anti-HIV activity with an EC50 value of 0.31 µM and a TI
of 155.5. However, 10 was less potent than the correspond-
Resu lts a n d Discu ssion
3-O-Acyl-ursolic acid derivatives were prepared by react-
ing ursolic acid with an anhydride or acid chloride in
* To whom correspondence should be addressed. Tel.: (919) 962-0066.
Fax: (919) 966-3893. E-mail: khlee@email.unc.edu.
† Niigata College of Pharmacy.
‡ Fukuoka University.
§ BBI-Biotech Research Laboratories.
University of North Carolina.
10.1021/np990633v CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/01/2000

1620 J ournal of Natural Products, 2000, Vol. 63, No. 12
Kashiwada et al.
ing betulinic acid derivative (EC50 0.01 µM, TI 1172).3a The
other 3-O-acyl derivatives displayed weak or no anti-HIV
activity, although again they were less toxic than ursolic
acid against H9 cells. In contrast, the 3-oxo-derivative (15)
was toxic; the same result was found with comparable
betulinic and oleanolic acid derivatives. Ursolic acid and
its potassium salt (16) had identical anti-HIV activity,
while the potassium salt of oleanolic acid was more potent
(EC50 0.5 µM, TI 68.6)4 than the weakly active parent acid.
Ta ble 1. Anti-HIV Activity for Betulinic Acid (1), Oleanolic
Acid (3), and Their Derivatives (2 and 4), Pomolic Acid (5), and
Ursolic Acid (6) and Its Derivatives (7-16)
compound
IC50 (µM)a
EC50 (µM)b
TIc
1
2
3
4
5
6
7
8
12.9
7.0
1.4
<0.000 35
3.7
0.000 86
3.0
9.2
>20 000
12.9
22 326
16.3
47.8
19.2
48.9
14.3
30.7
49.5
7
48.2
44.9
30.7
>18.5
>18.0
1.8
4.4
3.3
N.S.d
2.1
23.6
9
N.S.d
0.31
10
11
12
13
14
16
AZT
155.5
2.9
4.8
15.3
6.4
N.S.d
N.S.d
N.S.d
0.045 ( 0.056e
1871
41 667
a
The agent concentration that inhibited H9 cell growth by 50%.
b
The agent concentration that inhibited viral replication in H9
cell by 50%. c In vitro TI ratio: IC50/EC50
.
N.S. ) no suppression.
d
e This EC50 value represents the mean and standard deviation of
65 EC50 values for AZT.
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
measured on a Yanako micromelting point apparatus and are
uncorrected. Optical rotations were measured on a J ASCO
DIP-1000 or Perkin-Elmer 241 polarimeter. MS were deter-
mined on a J EOL HX-110 spectrometer. 1H and 13C NMR
spectra were measured on J EOL A-400 and J EOL A-500
spectrometers with TMS as an internal standard. Column
chromatography was carried out with Kiesel gel 60 (70-230
mesh, Merck). HPLC was performed on a Waters 600E solvent
delivery system equipped with a Waters R401 differential
refractometer and a Capcell PAK C18 (SG120, 5 µm) (Shiseido)
column (4 mm i.d. × 250 mm and 20 mm i.d. × 250 mm for
analytical and semipreparative scale, respectively).
Gen er a l P r oced u r e for P r ep a r a tion of 3-O-Acyl Ur so-
lic Acid s. A mixture of ursolic acid and appropriate acid
anhydride (2.5-10 molar equiv) and (DMAP) (1 molar equiv)
in pyridine (5-10 mL) was refluxed overnight. The reaction
mixture was diluted with ice water and extracted with CHCl3.
The organic layer was washed with H2O, dried over MgSO4,
and concentrated under reduced pressure. For 9 and 10,
crystallization (diluted MeOH) of the CHCl3-soluble portion
yielded pure samples, whereas for 7 and 8, the reaction of
ursolic acid and 2,2-dimethylsuccinic anhydride gave a crystal-
line mixture, which was purified by semipreparative HPLC
(95% CH3CN-0.2% TFA). Compounds 11-14 were purified
by Si gel column chromatography eluting with C6H6-EtOAc
(1:1, 3:1, and 40:1 for 11, 12, and 13, respectively) and CHCl3
for 14.
Thus, although betulinic, oleanolic, and ursolic acids
share many structural features, the 3-O-acyl-ursolic acid
and oleanolic acid derivatives, except for 4, were generally
much less potent than the corresponding betulinic acid
derivatives. These results suggest that partial structures
in the triterpenoid moiety, together with the structure of
the acyl group, are essential for potent anti-HIV activity.
The structures of these three triterpenoids correlate closely
in the A- through D-rings, but differ significantly in the
E-ring, including the D/E-ring fusion. Betulinic acid has a
five-membered E-ring, an isopropenyl substituent, and a
trans D/E-ring fusion, while oleanolic acid and ursolic acid
contain a six-membered E-ring, two methyl substituents,
and a cis D/E-ring fusion. From these observations, the
structure of the E-ring might play an important role in the
anti-HIV potency. A detailed quantitative structure-
activity relationship analysis is in progress.
3-O-(2′,2′-Dim eth ylsu ccin yl)-u r solic a cid (7): a white
crystalline powder (from MeOH-EtOAc); mp 276-278 °C;
[R]27D +50.0° [c 0.7, MeOH + CHCl3 (1:1)]; 1H NMR, see Table
2; 13C NMR, see Table 3; positive HRFABMS m/z 607.3975
([M + Na]+), C36H56NaO6 requires 607.3974.
3-O-(3′,3′-Dim eth ylsu ccin yl)-u r solic a cid (8): colorless
rods (from MeOH-EtOAc); mp 273-274 °C; [R]26 +50.5° [c
D
0.6, MeOH + CHCl3 (1:1)]; H NMR, see Table 2; 13C NMR,
1
see Table 3; positive HRFABMS m/z 607.3970 ([M + Na]+),
C36H56NaO6 requires 607.3974.
3-O-(3′,3′-Dim eth ylglu ta r yl)-u r solic a cid (9): colorless
needles (from MeOH), mp 230-232 °C; [R]16 +56.9 ° (c 0.58,
D
CHCl3); 1H NMR, see Table 2; 13C NMR, see Table 3; positive
HRFABMS m/z 597.4161 ([M + H]+), C37H57O6 requires
597.4155.
3-O-Diglyor yl-u r solic a cid (10): colorless needles (from
1
MeOH); mp 263-265 °C; [R]16 +60.1 ° (c 0.45, pyridine); H
D

Anti-HIV 3-O-Acetyl Ursolic Acid Derivatives
J ournal of Natural Products, 2000, Vol. 63, No. 12 1621
Ta ble 2. 1H NMR Data (δ, J in Hz) for Compounds 7-14
compound
proton
H-3
7a
8b
9b
10c
11d
12d
13d
14d
4.76 (dd,
4.76 (dd,
4.5, 11.5)
5.46 (t, 3)
4.75 (dd,
4.5, 11.5)
5.48 (t, 3)
4.63 (dd,
6.5, 9.5)
5.25 (t, 3.5)
2.21 (d, 11.5) 2.63 (d, 12)
0.98 (s)
4.78 (dd,
5, 11)
5.46 (t, 3)
4.74 (dd,
5, 11)
5.48 (t, 3.5)
ca. 2.65e
1.03 (s)
4.73 (dd,
4.5, 11.5)
5.49 (t, 3.5) 5.48 (t, 3.5)
2.65 (d, 12) 2.65 (d, 11.5)
1.04 (s)
4.71 (dd,
4.5, 11)
4.5, 11.5)
5.45 (t, 3.5)
2.62 (d, 11.5) 2.63 (d, 11.5) 2.64 (d, 11.5)
H-12
H-18
H-23
1.04 (s)
1.01 (s)
1.03 (s)
1.02 (s)
1.04 (s)
H-24
0.94 (s)
0.94 (s)
0.93 (s)
0.88 (s)
0.93 (s)
0.90 (s)
0.92 (s)
0.92 (s)
H-25
0.82 (s)
0.81 (s)
0.81 (s)
0.82 (s)
0.83 (s)
0.83 (s)
0.85 (s)
0.85 (s)
H-26
1.02 (s)
0.99 (s)
0.98 (s)
0.90 (s)
0.99 (s)
0.95 (s)
0.95 (s)
0.98 (s)
H-27
1.22 (s)
1.21 (s)
1.23 (s)
1.12 (s)
1.22 (s)
1.24 (s)
1.24 (s)
1.24 (s)
H-29
H-30
1.01 (d, 6)
0.97 (d, 6)
1.00 (d, 6)
0.97 (d, 6)
1.02 (d, 7)
0.96 (d, 6)
0.96 (d, 6)
0.88 (d, 6)
1.01 (d, 6)
0.97 (d, 6)
1.02 (d, 6)
0.98 (d, 6)
1.02 (d, 6)
0.98 (d, 6)
1.02 (d, 6.5)
0.98 (d, 6)
acyl moiety
-CH2-
2.90, 2.94
(d,16)
2.88, 2.95
(d, 15.5)
2.75, 2.82
(d, 14.8)
3.35 (4H, m) 2.89, 2.93
(each 2H, m)
2.24 (2H, m) 2.27 (d, 6.5) 2.24, 2.31
(d, 13)
2.78 (2H, s)
1.38, 1.39
(each 3H, s)
2.64 (4H, m)
-CH3
1.48 (6H, s)
1.54 (6H, s)
0.96 (d, 6.5) 1.09 (9H, s)
a
b
d
Measured at 500 MHz in pyridine-d5. Measured at 300 MHz in pyridine-d5. Measured at 400 MHz in CDCl3 + CD3OD (1:1).
Measured at 400 MHz in pyridine-d5. e The coupling constant could not be determined due to overlap with glutaryl methylene signals.
d
Ta ble 3. 13C NMR Data for Compounds 7-14
compound
carbon
7a
8a
9b
10c
11d
12d
13d
14d
1
2
3
4
5
6
7
8
38.3
23.9
80.6
38.1
55.7
18.4
33.4
39.9
47.8
37.1
23.5
125.5
139.3
42.5
28.7
24.9
48.1
53.6
39.5
39.4
31.1
37.4
28.3
17.1
15.5
17.4
23.6
179.9
17.5
21.4
38.4
24.0
81.0
37.9
55.6
18.5
33.4
39.9
47.8
37.1
23.6
125.5
139.3
42.5
28.7
24.9
48.1
53.6
39.5
39.4
31.1
37.5
28.3
17.1
15.5
17.4
23.9
179.9
17.5
21.4
38.4
24.1
80.7
37.8
55.6
18.5
33.4
40.0
47.8
37.1
23.6
122.5
139.3
42.5
28.7
24.9
48.1
53.6
39.5
39.4
32.7
37.5
28.3
17.2
15.5
17.4
23.9
179.9
17.5
21.4
39.5
24.7
84.3
39.0
56.7
19.4
34.2
40.8
48.8
38.1
24.5
126.6
139.6
43.4
29.3
25.5
49.1
54.2
40.4
40.3
31.9
38.1
29.2
17.7
16.6
18.0
24.7
181.8
18.1
22.1
38.3
23.9
80.9
37.9
55.6
18.4
33.3
39.9
47.8
37.0
23.5
125.4
139.2
42.4
28.6
24.8
48.0
53.5
39.4
39.3
31.0
37.4
28.2
17.0
15.4
17.3
23.8
179.8
17.4
21.3
38.3
24.0
80.7
37.9
55.5
18.5
33.4
39.9
47.8
37.1
23.6
125.5
139.3
42.5
28.7
24.9
48.1
53.6
39.5
39.4
31.1
37.4
28.3
17.1
15.5
17.4
23.9
179.9
17.5
21.4
38.3
24.0
80.6
37.9
55.6
18.5
33.4
39.9
47.8
37.1
23.6
125.5
139.3
42.5
28.7
24.9
48.1
53.5
39.5
39.4
31.1
37.5
28.3
17.1
15.5
17.4
17.5
179.9
23.9
30.0
38.4
24.1
80.6
37.8
55.6
18.5
33.4
39.9
47.8
37.1
23.6
125.5
139.3
42.5
28.7
24.9
48.1
53.6
39.5
39.4
31.1
37.5
28.3
17.1
15.5
17.4
17.5
179.9
23.9
21.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
acyl moiety
1′
2′
3′
4′
5′
CH3
173.9
44.8
41.0
171.5
45.3
40.9
172.0
45.9
31.1
45.9
174.5
28.0
172.3
69.3e
174.7
29.9e
30.3e
172.4
173.0
30.0e
21.3
172.6
43.9
26.0
171.9
48.4
30.7
176.6
179.3
69.5e
174.0
34.2e
175.5
25.8
25.9
25.9
26.3
22.4
22.5
29.8
(3C)
a
b
d
Measured at 125 MHz in pyridine-d5. Measured at 75.5 MHz in pyridine-d5. Measured at 100 MHz in CDCl3 + CD3OD (1:1).
Measured at 100 MHz in pyridine-d5. e Assignments may be interchanged in each column.
d
NMR, see Table 2; 13C NMR, see Table 3; negative HRFABMS
NMR, see Table 2; 13C NMR, see Table 3; positive HRFABMS
m/z 593.3815 ([M + Na]+), C35H54NaO6 requires 593.3818.
3-O-Isova la r yl-u r solic a cid (13): colorless needles from
m/z 571.3634 ([M - H]-), C34H51O7 requires 571.3635.
3-O-Su ccin yl-u r solic a cid (11): colorless needles from
1
C6H6, mp 248-249°; [R]23 +37.3° (c 0.23, MeOH); H NMR,
CH3OH, mp 279-281 °C; [R]15 +57.6° (c 0.92, CHCl3); 1H
D
D
see Table 2; 13C NMR, see Table 3; negative HRFABMS m/z
NMR, see Table 2; 13C NMR, see Table 3; positive HRFABMS
m/z 563.4070 ([M + Na]+), C35H56NaO4 requires 563.4076.
3-O-ter t-Bu tyla cetyl-u r solic a cid (14): colorless needles
555.3690 [M - H]-, C34H51O6 requires 555.3685.
3-O-Glu ta r yl-u r solic a cid (12): colorless needles from
CH3OH, mp 212-214 °C; [R]20 +35.9° (c 1.03, pyridine); H
from hexane, mp 265-266 °C; [R]24 +55.8° (c 0.86, CHCl3);
1
D
D

1622 J ournal of Natural Products, 2000, Vol. 63, No. 12
Kashiwada et al.
1H NMR, see Table 2; 13C NMR, see Table 3; positive HR-
FABMS m/z 577.4227 ([M + Na]+), C36H58NaO4 requires
577.4233.
infected cells. After a 4-h incubation at 37 °C and 5% CO2,
both cell populations were washed three times with fresh
medium and then added to the appropriate wells of a 24-well
plate containing the various concentrations of the test drug
or culture medium (positive-infected control/negative-control
drug). In addition, AZT was also assayed during each experi-
ment as a positive-control drug. The plates were incubated at
37 °C and 5% CO2 for 4 days. Cell-free supernatants were
collected on day 4 and tested by an in-house p24 antigen
ELISA assay. p24 Antigen is a core protein of HIV and,
therefore, is an indirect measure of virus present in the
supernatants. Toxicity was determined by performing cell
counts via a Coulter counter on the mock-infected cells, which
had either received culture medium (no toxicity) or test sample
or AZT. If a test sample had suppressive capability and was
Oxid a tion of Ur solic Acid (15). A mixture of ursolic acid
(6) (145 mg) and pyridinium chlorochromate (150 mg) in CH2-
Cl2 (45 mL) was stirred at room temperature for 25 h. The
solid was filtered using Celite and discarded, and the filtrate
was evaporated. The reaction product was purified by Si gel
column chromatography using CHCl3 as eluent to give ursolic
acid (3-oxo-urs-12-en-28-oic acid) (15) (95 mg): colorless prisms
(from n-hexane-EtOAc), mp 282-285 °C; [R]25D +85.7° (c 0.90,
MeOH); HREIMS m/z 454.3429 [M]+, C30H46O3 requires
454.3447.
P r ep a r a tion of P ota ssiu m Sa lts of Ur solic Acid (16).
A solution of ursolic acid (95 mg) was treated with 2% KOH
in Me2CO-H2O (1:1) at room temperature for 30 min. After
removal of Me2CO by evaporation, the resulting aqueous
solution was passed through an MCI-gel CHP20P column, and
washed with H2O to remove excess KOH. Subsequent elution
with MeOH gave potassium ursoate (16) (85 mg) as a white
powder; mp 255-258 °C; FABMS m/z 495 [M + H]+, 533
[M + K]+.
not toxic, its effects are reported in the following terms: IC50
,
the concentration of test sample that was toxic to 50% of the
mock-infected cells; EC50, the concentration of the test sample
that was able to suppress HIV replication by 50%; and TI, the
ratio of IC50 to EC50
.
Ack n ow led gm en t. This investigation was supported by
the Scientific Grant from the Ministry of Education, Science,
Sports, and Culture of J apan and the Promotion and Mutual
Aid Corporation for Private Schools (Y.K.), as well as Grant
AI-33066 from the National Institute of Allergies and Infec-
tious Diseases (K.H.L.).
An ti-HIV Assa y. The T cell line, H9, was maintained in
continuous culture with complete medium (RPMI 1640 with
10% fetal calf serum supplemented with L-glutamine) at 5%
CO2 and 37 °C. Aliquots of this cell line were used in
experiments only when in log-phase of growth.
Test samples were first dissolved in dimethyl sulfoxide. The
following are the final drug concentrations routinely used for
screening: 100, 20, 4, and 0.8 µg/mL. For active agents,
additional dilutions are prepared for subsequent testing so that
an accurate EC50 value (see definition below) could be achieved.
As the test samples were being prepared, an aliquot of H9
cells was infected with HIV-1 (IIIB isolate), while another
aliquot was mock-infected with complete medium. The mock-
infected cells were used for toxicity determinations (IC50, see
definition below). The stock virus used for these studies
typically had a TCID50 value of 104 infectious units (IU)/mL.
The appropriate amount of virus for a multiplicity of infection
between 0.1 and 0.01 IU/cell was added to the first aliquot of
cells. The other aliquot of cells received only culture medium
and was then incubated under identical conditions to the HIV-
Refer en ces a n d Notes
(1) For part 37, see: Xie, L.; Takeuchi, Y.; Cosentino, L. M.; Lee, K. H.
J . Med. Chem. 1999, 42, 2662-2672.
(2) Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Cosentino, L. M.; Ballas,
L. M.; J iang, J . B.; J anzen, W. P.; Chen, I. S.; Lee, K. H. J . Nat. Prod.
1994, 57, 243-247.
(3) (a) Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Chen, C. H.;
Garrett, P. E.; Lee, K. H. J . Med. Chem. 1996, 39, 1016-1017. (b)
Hashimoto, F.; Kashiwada, Y.; Cosentino, L. M.; Chen, C. H.; Garrett,
P. E.; Lee, K. H. Bioorg. Med. Chem. 1997, 5, 2133-2143.
(4) 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. J . Nat. Prod. 1998, 61,
1090-1095.
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