3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives as potent anti-HIV agents

Article abstract of DOI:10.1016/j.bmcl.2004.09.033

3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC₅₀ value of 2 × 10^-5 μM and a TI value of 1.12 × 10⁶. 3-O-(3′,3′- Dimethylsuccinyl)- and 3-O-(3′,3′-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC₅₀ values of 0.0017 and 0.0013 μM, respectively, and TI values of 16,160 and 19,530, respectively. 3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC₅₀ value of 2 × 10^-5 μM and a TI value of 1.12 × 10⁶. 3-O-(3′,3′-Dimethylsuccinyl)- and 3-O-(3′,3′- dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC₅₀ values of 0.0017 and 0.0013 μM, respectively, and TI values of 16,160 and 19,530, respectively.

Full text of DOI:10.1016/j.bmcl.2004.09.033

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5851–5853
3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives
as potent anti-HIV agents
Yoshiki Kashiwada,a,* Michiko Sekiya,a Yasumasa Ikeshiro,a Toshihiro Fujioka,b
Nicole R. Kilgore,c Carl T. Wild,c Graham P. Allawayc and Kuo-Hsiung Leed,*
aFaculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan
bFaculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
cPanacos Pharmaceutical Inc, Perry Parkway, Gaithersburg, MD 20877, USA
dNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
Received 19 August 2004; accepted 15 September 2004
Available online 12 October 2004
Abstract—3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-
dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC50 value of 2 · 10ꢀ5 lM and a TI value of
1.12 · 106. 3-O-(30,30-Dimethylsuccinyl)- and 3-O-(30,30-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV com-
pounds with EC50 values of 0.0017 and 0.0013lM, respectively, and TI values of 16,160 and 19,530, respectively.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Our modification study on betulinic acid (1), which was
identified as an anti-HIV principle [EC50 1.4lM, thera-
peutic index (TI) 9.3] from the leaves of Syzygium
claviflorum,1 has resulted in the design and discovery of
3-O-(30,30-dimethylsuccinyl)-betulinic acid (PA-457, 2).
PA-457 shows extremely potent anti-HIV activity with
a reported EC50 value of <0.00035lM and a therap-
eutic index (TI) value of >20,000.2,3 Moreover,
PA-457 was found to interfere with viral maturation/
budding, the final step in the HIV life cycle, and thus,
has a novel mechanism of action compared with the
current AIDS drugs.4,5 PA-457 is, therefore, the first
compound known to target this step in virus replication
and is undergoing clinical trials.6
H
H
H
COOH
H
COOH
O
H
H
O
HO
O
=
HOOC
betulinic acid (1)
PA-457 (2)
H
H
R1 = R2
3
4
HO
H
O
R2
H
HO
=
HO
=
O
O
R2
R1
H
O
O
O
R1
H
*
Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3993 (K.H.L.); tel./fax: +81 25 269 3140 (Y.K.); e-mail addresses:
kasiwada@niigata-pharm.ac.jp; khlee@unc.edu
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.033

5852
Y. Kashiwada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5851–5853
Various 3,28-di-O-acyl-betulins were also prepared as
PA-457 related derivatives based on the structural simi-
larity of betulin and betulinic acid. 3,28-Di-O-(30,30-
dimethylglutaryl)-betulin (3) and 3-O-(30,30-dime-
thylsuccinyl)-28-O-(200,200-dimethylsuccinyl)-betulin (4)
demonstrated potent anti-HIV activity with EC50 values
of 0.00066 and 0.00087lM, respectively, and TI values
of 21,515 and 42,400, respectively.7,8 In contrast, 28-O-
dimethylsuccinyl-betulins, which were prepared in the
course of the structure elucidation of 3,28-di-O-dime-
thylsuccinyl-betulin isomers, were not potent anti-HIV
agents. These data suggested that a 3-O-acyl group
was essential for potent anti-HIV activity.8 In a contin-
uing structure–activity relationship (SAR) study on PA-
457 derivatives, we have now prepared 3-O-acyl-betulin
and -dihydrobetulin derivatives, and evaluated their
anti-HIV activity.
viously in 3-O-acyl-betulinic acid derivatives.3 After
removing the 28-protective group of the products using
the conditions described above, the desired 30,30-dime-
thylsuccinyl-betulin could be obtained by crystallization
from EtOH. On the other hand, removing the 28-O-tri-
phenylmethyl ether of 3-O-succinyl and 3-O-glutaryl
derivatives with PPTS in CH2Cl2–EtOH yielded mainly
the corresponding ethyl ester derivatives, together with
the desired product. Alternatively, the desired 3-O-succi-
nyl- and 3-O-glutaryl-betulins were obtained by reflux-
ing with ZnCl2 in 95% THF. Initially, we attempted to
prepare the 3-O-acyl-dihydrobetulin derivatives directly
from the 3-O-acyl-betulin 28-O-triphenylmethyl ethers
by catalytic reduction with Pd–C/H2, but the 28-O-tri-
phenylmethyl ether group was not removed under these
conditions. Therefore, the 3-O-acyl-dihydrobetulin
derivatives10 were prepared from the 3-O-acyl-betulin
derivatives by treatment with Pd–C/H2.
The 3-O-acyl-betulin derivatives were prepared as
shown in Scheme 1. Protection of the 28-hydroxyl group
of betulin with triphenylmethyl chloride yielded betulin
28-O-triphenylmethyl ether, whose solution in pyridine
was further treated with an appropriate dicarboxylic
acid in the presence of dimethylamino pyridine at reflux
to give 3-O-acyl-betulin 28-O-triphenylmethyl ether
derivatives. The C-28 protecting group was subse-
quently removed by refluxing with pyridinium p-tolu-
enesulfonate (PPTS) in CH2Cl2–EtOH, and the
product was purified by silica gel chromatography to
give the 3-O-acyl-betulin derivatives. However, although
the reaction of dimethylsuccinic acid and betulin 28-O-
triphenylmethyl ether yielded mainly the 3-O-(30,30-
dimethylsuccinyl)-betulin derivative, the undesired
20,20-dimethyl isomer was also formed, as was seen pre-
Anti-HIV activities for 3-O-acyl-betulin and -dihydro-
betulin derivatives are summarized in Table 1. Among
these derivatives, 3-O-glutaryl-dihydrobetulin (17) dem-
onstrated extremely potent anti-HIV activity in acutely
infected H9 cells with an EC50 value of 2 · 10ꢀ5 lM
and inhibited uninfected H9 cell growth with an IC50
value of 23.59lM. Its calculated TI value was 1.12 ·
106. Compound 17 was more potent than AZT in this
assay, and this analog appears to show the greatest
anti-HIV activity among the betulinic acid, dihydro-
betulinic acid, betulin, and dihydrobetulin derivatives
evaluated thus far. 3-O-(30,30-Dimethylsuccinyl)- and
3-O-(30,30-dimethylglutaryl)-dihydrobetulins (15, 16)
were also potent anti-HIV compounds with EC50 values
of 0.0017 and 0.0013lM, respectively, and TI values
H
H
dicarboxylic acid
O
OH
H
C(C6H5)3
(C6H5)3CCl
H
DMAP
H
H
pyridine
DMAP
DMF
HO
HO
H
H
betulin 28-O-triphenylmethyl ether
betulin
PPTS / CH2Cl2-EtOH
or
H
H
H
O
OH
OH
ZnCl2 / THF
H
C(C6H5)3
H
H
H2/ Pd-C
O
O
O
O
H
H
H
O
O
R
R
R
H
H
H
HO
HO
HO
HO
R=
R=
R=
5
6
10
15
O
O
O
O
O
O
R=
R=
R=
R=
11
16
17
HO
HO
HO
O
O
O
O
O
R=
R=
R=
R=
R=
R=
7
8
9
12
13
14
HO
HO
HO
HO
HO
HO
O
O
O
O
Scheme 1. Preparation of 3-O-acyl-betulin and -dihydrobetulin derivatives.

Y. Kashiwada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5851–5853
5853
Table 1. Anti-HIV activities9 for betulinic acid (1), PA-457 (2), 3,28-
di-O-acyl-betulin derivatives (3, 4), and 3-O-acyl-betulin (10–14) and
-dihydrobetulin (15–17) derivatives
2. Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Chen,
C. H.; Garrett, P. E.; Lee, K. H. J. Med. Chem. 1996, 39,
1016.
TIc
a
b
3. Hashimoto, F.; Kashiwada, Y.; Cosentino, L. M.; Chen,
C. H.; Garrett, P. E.; Lee, K. H. Bioorg. Med. Chem. 1997,
5, 2133.
Compd
IC50
EC50
1
12.9
7.0
1.4
9.2
2
<0.00035
0.00066
0.00087
0.0056
0.0044
0.0662
N.S.d
>20,000
21,515
42,400
5974
4. Kanamoto, T.; Kashiwada, Y.; Kanbara, K.; Gotoh, K.;
Yoshimori, M.; Goto, T.; Sano, K.; Nakashima, H.
Antimicrob. Agents Chemother. 2001, 45, 1225.
5. Li, F.; Goila-Gaur, R.; Salzwedel, K.; Kilgore, N. R.;
Reddick, M.; Matallana, C.; Castillo, A.; Zoumplis, D.;
Martin, D. E.; Orenstein, J. M.; Allaway, G. P.; Freed, E.
O.; Wild, C. T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
13555.
6. Panacos Pharmaceuticals Inc., announcement, 2004.
7. Sun, I. S.; Wang, H. K.; Kashiwada, Y.; Shen, J. K.;
Cosentino, L. M.; Chen, C. H.; Yang, L. M.; Lee, K. H.
J. Med. Chem. 1988, 41, 4648.
3
14.2
36.9
4
10
11
12
13
14
15
16
17
AZT
33.46
28.9
6568
670
44.36
25.98
>44.75
26.99
25.69
23.59
1873
—
0.0246
0.0017
0.0013
2 · 10ꢀ5
0.0062
>1819
16,160
19,530
1.12 · 106
302,096
a Concentration (lM) that is toxic to 50% of mock-infected H9 cells.
b Concentration (lM) that inhibits HIV-1 replication by 50%.
8. Kashiwada, Y.; Chiyo, J.; Ikeshiro, Y.; Nagao, T.; Okabe,
H.; Cosentino, L. M.; Fowke, K.; Lee, K. H. Bioorg. Med.
Chem. Lett. 2001, 11, 183.
9. The HIV-1 (IIIB isolate) growth inhibition assay in the H9
cells was performed according to the procedure described
in Refs. 1–3 and 7.
c TI (therapeutic index) is defined by IC50/EC50
.
d No suppression at concentrations up to 100lg/mL.
10. All new compounds gave satisfactory analytical and
spectroscopic data. Selected spectroscopic data for 3-O-
glutaryl dihydrobetulin (17): Colorless needles (from
MeOH); mp 230–231ꢁC; [a]D ꢀ6.6 (c 0.47, pyridine);
1H NMR (500MHz, pyridine-d5) d: 0.83, 0.89 (each 3H,
d, J = 6.7Hz, CH3-29 and 30), 0.81, 0.90, 0.91, 0.97, 1.01
(each 3H, s, CH3-23, 24, 25, 26, 27), 2.23 (2H, m, glutaryl
H2-30), 2.59–2.66 (4H, m, glutaryl H2-20 and 40), 3.60,
4.06 (each 1H, d, J = 10.5Hz, H2-28), 4.74 (1H, dd,
J = 4.5, 11.5Hz, H-3); positive FABHRMS m/z 581.4174
([M+Na]+), C35H58O5Na requires 581.4182. 3-O-(30,30-
dimethylglutaryl)-dihydrobetulin (16): Colorless needles
(from MeOH); mp 224–225ꢁC; [a]D ꢀ12.5 (c 0.4,
of 16,160 and 19,530, respectively. 3-O-Acyl-betulin
derivatives (10, 11, 12) also showed relatively potent
anti-HIV activities with EC50 values ranging from
0.0044 to 0.0662lM and TI values ranging from 670
to 6568, but they were less active than the corresponding
dihydrobetulin derivatives. Comparison of the anti-HIV
activities for 3-O-acyl-betulin and -dihydrobetulin deri-
vatives suggested that hydrogenation of the betulin D29
double bond increased anti-HIV potency. 3-O-Diglycol-
yl-betulin (14) also displayed significant anti-HIV activ-
ity, which might be improved by hydrogenation.
1
pyridine); H NMR (500MHz, pyridine-d5) d: 0.83, 0.90
(each 3H, d, J = 7.0Hz, CH3-29 and 30), 0.81, 0.93, 0.95,
0.97, 1.00 (each 3H, s, CH3-23, 24, 25, 26, 27), 1.37, 1.38
(each 3H, s, dimethylglutaryl CH3), 2.75, 2.81 (each 1H,
d, J = 14Hz, dimethylglutaryl H2-20), 2.69 (2H, s,
dimethylglutaryl H2-40), 3.60, 4.05 (each 1H, d,
J = 11Hz, H2-28), 4.75 (1H, dd, J = 4.5, 11.5Hz, H-3);
positive FABHRMS m/z 609.4502 ([M+Na]+),
C37H62O5Na requires 609.4495. 3-O-(30,30-dimethylsucci-
nyl)-dihydrobetulin (15): Colorless needles (from
MeOH); mp 273–274ꢁC; [a]D ꢀ15.6 (c 0.5, pyridine);
1H NMR (pyridine-d5): 0.84, 0.90 (each 3H, d,
J = 7.0Hz, CH3-29 and 30), 0.79, 0.95, 0.97, 0.97, 0.99
(each 3H, s, CH3-23, 24, 25, 26, 27), 1.54 (6H, s,
dimethylsuccinyl CH3), 2.88, 2.95 (each 1H, d,
J = 15.5Hz, dimethylsuccinyl H2-20), 3.59, 4.04 (each
1H, d, J = 10.5Hz, H2-28), 4.76 (1H, dd, J = 4.5, 11.8Hz,
H-3); Positive FABHRMS m/z 595.4326 ([M+Na]+),
C36H60O5Na requires 595.4338.
Acknowledgements
The authors wish to thank Mr. H. Hanazono (Fukuoka
University) for measurement of mass spectra. This
investigation was supported by a grant from the Promo-
tion and Mutual Aid Corporation for Private schools
of Japan (Y.K.), as well as Grant AI-33066 from the
National Institute of Allergies and Infectious Diseases
(K.H.L.).
References and notes
1. Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Cosentino,
L. M.; Ballas, L. M.; Jiang, J. B.; Janzen, W. P.; Chen, I.
S.; Lee, K. H. J. Nat. Prod. 1994, 57, 243.